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the vast majority of human thyroid cancers are differentiated and pathological examination reveals that most of these are papillary thyroid cancers (ptc). the long - term prognosis and recommended treatment for patients with ptc are dependent on the stage of disease. papillary thyroid microcarcinoma (ptmc) are small thyroid tumors (1 cm in diameter) that belong to the low - risk well - differentiated ptc, which are probably of little clinical significance, and do not affect patient survival (arem., 1999 ; lloyd., 2004 ; pazaitou - panayiotou., 2007 ; shaha., 2007). it is important to distinguish between ptmc in a clinically recognized malignant thyroid micro nodule and an incidental (asymptomatic) ptmc found after thyroidectomy performed for other indications (e.g., benign thyroid diseases) or during thyroid ultrasound. incidental ptmc has an outstandingly good prognosis and there is nearly no risk of recurrence or death from ptmc (sugitani and fujimoto, 1999 ; barbaro. however, ptmc may be associated with lymph node metastases at presentation and/or neck loco - regional recurrences during follow - up (pazaitou - panayiotou., 2007). papillary thyroid microcarcinomas may be categorized as : (i) ptmc found at autopsy or incidentally at histology, (ii) ptmc found incidentally during thyroid or neck ultrasound and diagnosed before surgery by cytology on thyroid fine needle aspiration (fna) material, and (iii) clinical ptmc (i.e., tumors whose presenting symptoms were loco - regional or distant metastases). ptmcs are found in otherwise normal thyroids or in multinodular goiters (mngs), and they account for nearly 50% of new cases of ptc (ptc accounts for over 80% of all human thyroid cancers ; leenhardt., 2004 ; davies and welch, 2006 ; xing, 2009). papillary thyroid microcarcinomas are also referred to as small, tiny, or minute carcinomas. some authors suggest that ptmc found at histology should be called occult papillary tumors instead of carcinomas to reflect their benignity. the use of ultrasound (us) in the assessment of thyroid disease has greatly increased the detection of thyroid micro nodules not detected at clinical examination (ezzat., 1994 ; papini., 2011). prospective studies have undertaken systematic evaluation of thyroid nodules incidentally discovered by us and have correlated size and the us and color - doppler (cd) findings with the prevalence of cancer and its pathologic staging (leenhardt., 1999). several recent studies have suggested that us - detected ptmc (figure 1) is a different disease entity from ptmc detected at autopsy, based on the histological findings and on the existence of some cases with poor prognosis (sugitani and fujimoto, 1999 ; yamashita., 1999 ; papini., 2002 ; chow. 2006). the vast majority of non - palpable thyroid nodules identified by us and color - doppler display a hypoechoic pattern, irregular margins, microcalcifications, and intranodular vascularity indicating risk for malignancy, which can be confirmed by cytological evaluation of fna material. due to the non - negligible prevalence of extra - capsular growth and nodal metastasis, us - guided fna should be performed on all 815 mm hypoechoic nodules with irregular margins, intranodular vascular spots, or microcalcifications. non - palpable thyroid lesions without us risk factors should be followed up after 612 months by repeating clinical and us evaluation (papini., 2002). (a b) papillary thyroid microcarcinoma (ptmc) nodule (5.8 mm) with hypoechoic pattern, microcalcifications, well - defined margins, and intrinsic vascularity. (c d) ptmc nodule (6.3 mm) with isoechoic or mixed echo texture, cystic elements, irregular margins, hypovascularity, and coarse or peripheral calcifications [also taller than wider in (d) ]. (e h) histological features of 2 mm encapsulated ptmc with classical - type features in a multinodular goiter [arrows, (e g) ] : papillary structure with fibrovascular stalk, nuclear grooves, and nuclear pseudo inclusions (h). the tumor cells had pale eosinophilic cytoplasm with the characteristic vesicular to ground glass appearing nuclei (h) [magnification : (e) 20, (f) 40, (g) 100, and (f) 1000 ]. treatment of ptmc has been addressed in both european thyroid association (eta ; pacini., 2006) and american thyroid association (ata ; cooper., 2009) guidelines. when ptmc is diagnosed preoperatively, routine total, or near - total thyroidectomy is the main treatment to eradicate multifocal disease and decrease overall recurrence rate. ata guidelines for patients with differentiated thyroid cancer state that the benefit of radioiodine treatment appears to be restricted to patients with larger tumors (> 1.5 cm) or residual disease after surgery. there is no evidence that there is any benefit of radioiodine treatment in lower risk patients (defined by the following criteria : ptmc with no extension beyond the thyroid capsule, unifocal tumor, no aggressive histologic subtypes, no extra thyroidal extension or angioinvasion, no local or distant metastases, complete resection of macroscopic tumor, and no i uptake outside the thyroid bed on the post - therapeutic whole - body scan if i was administered ; jonklaas., 2006 ; cooper., 2009). instead, the recommendation for radioactive iodine is supported by a study (sakorafas., 2007) that followed 27 of 380 (7.1%) patients diagnosed with incidental ptmc (mean diameter 4.4 mm) following thyroid surgery for benign thyroid disease (20 patients with mng, six patients with follicular adenoma, and one patient with nodular hyperplasia ; sakorafas., patients with cytological preoperative diagnosis of thyroid malignancy were excluded from this study. in 11 patients (40.7%), the tumor was multifocal and in about half of them tumor foci were found in both thyroid lobes. in two patients, all patients with incidental ptmc received suppression therapy and 20 underwent adjuvant radioactive iodine therapy at a dose of 80100 mci based mainly on the presence of multifocal ptmc and infiltration of the thyroid capsule. follow - up (mean 4.56 years, range 112 years) was completed in 25 patients ; all of these were alive and disease - free. the authors of this study concluded that ptmc is not an uncommon incidental finding after surgery for benign thyroid lesions and that therefore the possibility of ptmc (in particular multifocal incidental ptmc) should be considered in the management of patients with benign thyroid disease. also, total / near - total thyroidectomy should be considered in patients with apparently benign nodular thyroid disease who exhibit risk factors including findings from history and clinical examination (i.e., development of hoarseness, progressive dysphagia or shortness of breath, rapid growth of the thyroid nodule, especially if observed under thyroid hormone suppressive therapy, or presence of cervical lymphadenopathy) or suspicious findings on preoperative imaging evaluation. importantly, whether individual tumor foci in patients with multifocal ptc or ptmc are clonally related or they arise independently is still controversial. while shattuck. (2005) demonstrated that individual tumor foci in patients with multifocal ptc often arise as independent tumors, mccarthy. thus, intrathyroid metastasis may play an important role in the spread of ptc and ptmc (mccarthy., 2006), but the origin of multifocal tumors is unclear. remarkably, some studies (baudin., 1998 ; chow., 2003a) have reported that the number and the size of tumor foci in ptmcs correlated with lymph node metastases at clinical presentation and with recurrence, suggesting that those pathologic features may represent relevant prognostic indicators in patients with ptmc. in fact, ptmc multifocality can be associated with high frequency of bilateral involvement regardless of tumor size (baudin., 1998). even when there is extended disease, the prognosis of patients with ptmcs is excellent, and the majority of studies report a mortality rate between 0 and 0.4% and a recurrence rate between 1.4 and 7.3% (pazaitou - panayiotou., 2007). however, there is some controversy regarding clinical outcome and treatment of ptmc, as evidenced in the following selected studies : ito. (2003) followed 162 patients with lesions ranging from 3 to 10 mm. in 58 of these they found no change in 60.3% of these cases, a decrease in 12.1%, and, in two patients, no identifiable cancer in two consecutive ultrasounds. (2004) also reported that ptmcs 7 mm were more likely to show lateral neck metastasis, suggesting that detailed us examination for lateral metastasis is necessary in patients with a tumor measuring 7 mm. (1992) evaluated 535 patients with ptmc with a median tumor size of 8 mm. two patients (0.4%) died, and the 20-year tumor recurrence rate was 6%. one group consisted of 52 patients diagnosed with incidental ptmc following thyroidectomy for benign thyroid disease. the second group included 191 patients who underwent thyroidectomy because fna biopsy of a thyroid nodule and/or clinical evaluation was diagnostic or suspicious for malignancy. no significant differences in clinical and histo - pathological characteristics were observed between the two groups. mean ptmc diameter was 0.55 0.26 cm in the incidental group and 0.56 0.27 cm in the suspected cancer group. a total of 34 patients had neck node and/or distant metastases at the time of diagnosis. distant metastases were only significantly observed in patients with ptmc 8 mm. thirty - two patients with ptmc with a diameter 5 mm, and two with ptmc with a diameter < 5 mm had lymph node metastases (roti., 2006). lymph nodes and distant metastases from thyroid cancers with diameters of 815 mm have been also described in other studies (lin., 1997 ; sugitani., 1998 ; 2006) reported only four patients (1.7%) with recurrent or persistent disease, and no ptmc - related mortality. similarly, an excellent outcome for ptmc has been described by other investigators (bramley and harrison, 1996 ; noguchi., 1996 ; rodriguez., 1997 ; 2004), whereas other studies found persistent or recurrent disease in 614.4% of patients with ptmc (hay., 1992 ; chow., 2003b ; pellegriti., 2004). one major issue with ptmc is the threshold value of tumor diameter and whether the outcome for slightly larger thyroid tumors (i.e., 1.11.5 cm in diameter) is similar to ptmc. 2004) investigated predictors of relapse in ptmc vs. ptc between 1.1 and 1.5 cm. the authors performed a retrospective study of 299 patients with ptc treated and followed up between 1975 and 2001. near - total or total thyroidectomy was performed in 292 patients, and lobectomy in seven patients. this study indicated that a high proportion of ptc 1.5 cm carry one or more risk factors at clinical presentation, including bilateral foci. in particular, authors found that approximately 20% of small (1.5 cm) ptcs had extra - thyroid invasion and/or bilateral foci. however, 43 (14.4%) patients still had persisting / recurrent disease at the last follow - up visit. ptc were subdivided into three groups according to their size (diameter : < 0.5, 0.61.0, and 1.11.5 cm) and a progressively increasing frequency of signs of tumor aggressiveness (multifocality, bilaterality, extrathyroidal invasion, and lymph node involvement) which correlated with increasing tumor size. this was particularly evident for ptc greater than 1.0 cm vs. ptc less 1.0 cm in diameter (pellegriti. presence or absence of risk factors / aggressiveness features are important indicators in planning thyroid surgery for ptmc (e.g., lobectomy alone vs. total thyroidectomy with central compartment neck dissection). previous studies have shown considerable variability in the prevalence of aggressive features and but significant differences between ptmc and ptc in the prevalence of aggressive features. this was addressed in a recent study (yun., 2010) that found that out of 87 patients (preoperative retrospective study), 44 (51%) had extra thyroidal extension, and 27 (31%) had central lymph node metastasis. positron emission tomography (pet)/computed tomography (ct) showed discernible fluorodeoxyglucose (fdg) uptake in 46 ptmcs (53%). fdg positivity of ptmcs was the only variable that correlated with both extra thyroidal extension and central lymph node metastasis ; there was a significant difference in the prevalence of both extra thyroidal extension (70 vs. 29%) and central lymph node metastasis (41 vs. 19.5%) between fdg - positive and -negative groups. the authors concluded that visual fdg positivity in ptmcs is a potential risk factor that may be useful for preoperative risk stratification. however, this study lacked long - term follow - up of correlation of fdg positivity in ptmcs with disease relapse rate, thus prospective studies are needed to assess the long - term benefit, cost effectiveness, sensitivity, and specificity of fdg - pet in patients with ptmc. 2004) have reported that ptmc can be associated with medullary thyroid cancer (mtc). twenty - seven of 196 (13.8%) mtc cases showed an association with ptc, and 21 of 190 (11%) mtc showed an association with incidental ptmc. this percentage is higher than that reported in the literature on the association of ptmc with graves disease (gd ; 2.8%4.5% ; schwartz., 1989 ; mazzaferri, 1990 ; hori., 1995 ; merchant., 2002) or with mng (3% ; pelizzo., the authors excluded the possibility that this association was caused by an increase in the general frequency of ptmc. furthermore, although it was not possible to completely exclude a shared pathogenic event as the cause of both mtc and ptmc, the molecular analysis of ret gene alterations did not show any common mutation between these two types of thyroid cancers. the clinical behavior of mtc does not seem to be influenced by the presence or specific therapeutic treatment of a concomitant ptmc (biscolla. thyroidectomy is the treatment of choice in patients with ptc followed by radioactive iodine ablation, in case of a tumor size greater than 1.0 cm or the presence of lymph node or distant metastasis, to achieve negative i whole - body scan and undetectable thyroglobulin (tg) levels during follow - up. post - surgical radioiodine treatment in the case of patients with ptmc (tumor size less or equal to 1 cm in diameter) is clearly indicated in the presence of high risk factors, but can be avoided in patients with low - risk (unifocal tumor, no extra thyroidal extension, no tall cell variant, columnar cell, diffuse sclerosing, and solid / trabecular variants, and no lymph node, or distant metastases ; falvo., 2003 ; the search for molecular targets for ptc has focused primarily on the ret (tyrosine kinase receptor)/ras / braf / mapk (mitogen - activated extracellular signal regulated kinase, i.e., erk1/2) kinase signaling pathway as the oncogenic event in ptc progression (xing, 2007 ; nucera., 2010, 2011a, b ; knauf., 2011 ; ringel, 2011). the major genetic alterations so far described in ptc are translocations in the ret gene (the estimated prevalence is highly variable between different studies, from 3 to 92%, and depends on the experimental methodology used) and the v600e point mutation in the braf gene (nikiforova., 2003 ; xing, 2007 ; marotta., 2011), which occurs in about 50% of ptc. both of these genetic alterations trigger the erk1/2 pathway, which causes abnormal cell proliferation, adhesion, migration, and invasion (melillo., 2005 ; nucera., 2010, 2011b ; a high prevalence (about 4552%) of ret / ptc translocations has been reported in ptmcs (tallini., 1998 ; corvi., 2001), suggesting that the activation of this oncogene (ret / ptc) plays a role in the early stage in ptmc development. the braf mutation appears to occur early in ptc development, based on evidence that it is also harbored in ptmc (sedliarou., 2008 ; nucera., 2009) and plays an important role in cell proliferation by regulating cyclins (e.g., cyclins d1 and p27 ; motti., 2007 ; xing, 2007 ; salerno., 2010 ; nucera., although the overall prevalence of the braf mutation in ptc worldwide is relatively high (xing, 2007), the prevalence of this mutation in ptmc is generally much lower in many parts of the world (lupi., 2007 ; ugolini. 2008) and as low as 18% in ptmc less than 5 mm in diameter (ugolini., 2007). excluding the areas in korea where the braf mutation in ptmc is high (xing, 2007), the overall frequency of the braf mutation in ptmc is around 30% (xing, 2007). (2009) studied the clinico - pathological characteristics and the braf mutational status of 64 cases of ptmcs. braf - positive ptmcs exhibited significantly more features of aggressiveness (advanced disease stages, extra thyroidal extension, and nodal metastasis) than ptmcs without the v600e mutation, indicating that braf may be a marker of aggressiveness and tumor progression from ptmc to ptc these data indicate that the braf mutation might be a molecular marker of tumor invasiveness and, moreover, that this relationship is independent of tumor size (e.g., greater than 1.1 cm vs. less than 1 cm). braf triggers a cascade that leads to human papillary thyroid microcarcinoma (ptmc) proliferation. phospho - mek1/2 induces hyperphosphorylation of erk1/2 which translocates into the nucleus, triggering cell cycle progression, and abnormal cell proliferation by up - regulating cyclins (e.g., cyclin d1) crucial for the checkpoint machinery in g1s phases and inhibiting anti - cell cycle cyclins (e.g., p27). up - regulation of cyclins (e.g., cyclin d1) leads to hyper - proliferation of papillary thyroid microcarcinoma cells and increase in papillae size. (2011) analyzed a group of aggressive ptmc selected based on the presence of lymph node metastasis or tumor recurrence and compared with a group of non - aggressive ptmc. the groups were matched for age, sex, and tumor size, but with no extra thyroidal spread (significantly more prevalent in the aggressive group). importantly, these authors detected braf in 77% of aggressive and 32% of non - aggressive ptmcs, suggesting that the v600e mutation may be a marker of invasiveness and, together with histo - pathologic features of aggressiveness, may allow clinical risk stratification of ptmcs. the braf mutation has been correlated with multifocality pathologic features in thyroid cancers (xing, 2007). as is the case for ptc, the importance of the distinction between multifocal independent primary (ip) ptmc and ptmc with intrathyroid metastasis is unclear. incidental ptmc can be multifocal (lin., 2008) and associated with lymph node metastases and increased neck lymph nodes recurrence after surgical treatment. lin. (2008) found that the incidence of lymph node metastasis for multifocal ptmc was 42.9%, similar to previous studies (david., 1992 ; rodriguez., 1997 ; baudin., 1998 ; lin. also found that a high percentage (75%) of ptmc with intrathyroid metastasis were positive for lymph node metastasis, while no patients with multifocal ip ptmc had metastatic disease. this suggests that it is important to separate ptmc with intrathyroid metastasis from multifocal ip tumors. in this study, about 50% of multifocal ptmcs showed intrathyroidal metastasis ; the molecular profile of this group of ptmcs was characterized by loss of heterozygosities (lohs) at chromosomes 1p36, 18q21, and 22q13, and/or braf mutation, suggesting that both different chromosomal losses and point mutations in the braf gene could be important pathological events in the origin of multifocal ptmc. in summary, there is good evidence that braf - positive ptmc are more likely to have a poor prognosis and therefore, it seems reasonable to be more aggressive in treating patients with ptmc carrying the braf mutation. soares and sobrinho - simoes (2011) recently suggested that genetic screening for the braf mutation might also help assess risk stratification and manage patients with ptmc. ptmc without braf may be conservatively managed unless the presence of other markers of poor prognosis indicate a more aggressive therapeutic approach (xing, 2009). since most ptmc progress very slowly while others show more aggressive behavior, it would be clinically relevant to determine a gene signature that can predict tumor aggressiveness. few studies have focused on the molecular pathways that underlie the patho - biology of ptmc. kim. (2010) recently performed oligonucleotide array analysis of ptmc and found that cell adhesion molecules were up - regulated in ptmc. in addition, they found no differences in gene expression between ptmc and ptc, suggesting that some ptmc may not be occult indolent thyroid cancers but are an earlier stage of ptc. (2008) found that ptmc with extra thyroidal extension and multifocality exhibited significant expression of s100a4 and that its expression predicted lymph node metastasis. s100a4 (garrett., 2006) is a member of the s100 family of calcium - binding proteins, which includes metastasin, fibroblast - specific protein, pel-98, 18a2, capl, and calvasculin. in vitro and in vivo studies in rodents have provided evidence that s100a4 is directly involved in tumor progression and metastasis, and promotes angiogenesis. therefore, preoperative evaluation of the expression of s100a4 in cytological specimens should be helpful in guiding therapy for patients with ptmcs. s100a4 immunoreactivity may predict lymph node metastasis in ptmc and might therefore be useful as an immunohistochemical marker to distinguish between more aggressive ptmc and clinically indolent ptmc. therefore, novel therapies are needed to improve disease outcome for patients with these cancers. studies based on preclinical models of targeted therapies highlight the importance of individualized genomic profiling to guide patient selection for inclusion in clinical trials. some drugs that target the braf oncoprotein kinase have recently begun clinical trials in patients with melanoma. selective pharmacologic targeting of braf may prove effective for treating patients with ptc harboring this mutation. for example, plx4720 and plx4032 are novel orally available selective small molecule inhibitors specifically designed to insert into the atp - binding site and trap oncogenic braf in an inactive conformation (tsai., 2008 ; bollag., these compounds inhibit braf kinase activity in melanoma, thyroid cancer, and colorectal cancer cells (tsai., 2008 ; nucera., plx4032 (vemurafenib) induced complete or partial tumor regression in 81% of patients enrolled for phase i ii clinical trial who had melanoma with the braf mutation (flaherty., 2010). in addition, it significantly improved rates of overall survival and progression - free survival (74%) in phase iii clinical trials in braf - positive melanoma patients (chapman., 2011). the effect of plx4720 in a preclinical model of metastatic human thyroid cancer suggests that these inhibitors might be effective for treating patients with braf - positive thyroid cancers that are refractory to conventional therapy (nucera., 2010). interestingly, overexpression of angiogenic signaling cascade pathways has been described in human ptc, and preclinical models have shown that inhibition of key molecules (i.e., protein kinases) in these pathways can have anti - tumor effects (gild., 2011). some of these kinase inhibitors have now been tested in clinical trials, with modest results. for example, sorafenib was designed as a c - raf inhibitor ; however, it has been reported to target other kinases, including vascular endothelial growth factor receptors (vegfr) and braf, therefore, it has been classified as non - selective inhibitor of braf. sorafenib has been assessed clinically in patients with braf - positive or genetically unknown advanced melanoma and did not show any benefit in those clinical trials (hauschild., 2009 ; ott., 2010 ; caronia., 2011). additionally, in phase i ii clinical trials, only 15% of patients with metastatic ptc showed a partial response to sorafenib (kloos. although only a small percentage of all ptmc are metastatic, 77% of braf - positive ptmc show features associated with aggressiveness (i.e., extra thyroidal extension ; niemeier., 2011). for these tumors, targeted therapies based on selective inhibitors of braf could be effective in the near future. the management and treatment of malignant thyroid micro nodules (i.e., ptmc) can be a challenge for physicians. most ptmc are indolent and have an excellent prognosis ; however, a subgroup shows an aggressive biological and clinical behavior similar to ptc. while additional robust prospective studies are required, there is now a body of evidence suggesting that braf - positive ptmcs show aggressive behavior, whereas braf - negative ptmcs have a good prognosis. this suggests that it will be valuable to consider the braf mutation as a prognostic marker of ptmc aggressiveness and to undertake prospective studies with systematic screening for the braf mutation and long - term follow - up to validate this marker of tumor aggressiveness. although, additional biological studies are needed to address definitively whether ptmc is an indolent cancer or an early stage of ptc, current clinical and molecular data suggest that patients with ptmcs that harbor the braf mutation and exhibit aggressive clinical there is sufficient promising preclinical experimental data to suggest that physicians could begin to test such targeted treatments now, preferably in the context of a prospective clinical trial following patients with aggressive braf - expressing ptmc. the authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. | most human thyroid cancers are differentiated papillary carcinomas (ptc). papillary thyroid microcarcinomas (ptmc) are tumors that measure 1 cm or less. this class of small tumors has proven to be a very common clinical entity in endocrine diseases. ptmc may be present in 3040% of human autopsies and is often identified incidentally in a thyroid removed for benign clinical nodules. although ptmc usually has an excellent long - term prognosis, it can metastasize to neck lymph nodes ; however deaths related to this type of thyroid tumor are very rare. few data exist on molecular pathways that play a role in ptmc development ; however, two molecules have been shown to be associated with aggressive ptmc. s100a4 (calcium - binding protein), which plays a role in angiogenesis, extracellular matrix remodeling, and tumor microenvironment, is over - expressed in metastatic ptmc. in addition, the brafv600e mutation, the most common genetic alteration in ptc, is present in many ptmc with extra thyroidal extension and lymph node metastasis. importantly, recently developed selective [e.g., plx4720, plx4032 (vemurafenib, also called rg7204) ] or non - selective (e.g., sorafenib) inhibitors of brafv600e may be an effective treatment for patients with brafv600e - expressing ptmcs with aggressive clinical pathologic features. here, we summarize the clinical outcome, cancer genetics, and molecular mechanisms of ptmc. |
the sit - to - stand (sts) movement is a movement people frequently perform as they change from a sitting to standing position, and the ability of an individual to rise from a chair has been considered an important indicator of an elderly person s functional independence ; if impaired, it increases the risk of falling1. among community - dwelling older adults, one in three experience at least one fall each year, with more than 30% requiring medical treatment after falling2. compared with young adults, healthy people 65 years of age or older exhibit moderate to high age - associated reductions in strength and functional limitations for tasks such as the sts task3. additionally, particularly in the frontal plane, older adults have greater difficulty maintaining and recovering postural stability caused by weak lower extremity muscles4. a previous study examined the functional role of hip adduction (add) and abduction (abd) during squat exercise. coqueiro.5 reported increased activity of the medial and lateral portions of the quadriceps during squats with isometric hip add. felcio.6 reported the squat exercise was associated with hip add producing higher activation of the vastus medialis oblique muscle and an increase in the activity of the gluteus medius muscle. hip abd during the squat exercise stimulated activation of the gluteus medius and produced higher activity in the vastus lateralis longus muscle. pereira.7 showed that displacement from 60 to 90 knee flexion increased myoelectric activity during a squat with hip abd. although the sts movement is necessary for older adults to live independently, no study of the sts task with hip add and abd has been performed. furthermore, in contrast to hip and knee muscle activity, relatively few studies have documented ankle muscle activity, such as the activities of the tibialis anterior (ta) and soleus (sol) muscles during the sts task. therefore, in the present study we compared the activation of the ankle joint muscles during the sts movement with hip add and hip abd in elderly females. sixteen healthy elderly subjects (67.75 1.61 years of age, range, 6570 years) participated in this study. the mean height and weight of the subjects were 153.5 6.61 cm and 54.46 7.22 kg, respectively. subjects were included in the study if they were able to follow directions, had no lower - extremity amputations, and had no restrictions on lower - extremity movement or weight bearing ordered by a physician. the study participants had no muscular pathology or gait or balance disorders, no chronic deficiencies associated with a neurological, rheumatologic or orthopedic affections ; and no chronic or acute illness leading to an inflammatory syndrome. this study was approved by the inje university faculty of health science human ethics committee, and all subjects provided written informed consent prior to participating in the study. the activities of the dominant lower extremity muscles were measured using a wireless electromyography (emg) system (delsys, inc., boston, ma, usa) with surface electrodes fixed at an interelectrode distance of 10 mm. emg surface electrodes were placed over muscle bellies of the dominant side tibialis anterior (ta) and soleus (sol) muscles. for normalization, maximal emg signals were acquired during a maximum voluntary isometric contraction (mvic) maneuver, which was performed for 5 s. the emg data expressed the entire sts task as a percentage of the mvic. the starting position for the subjects was standardized. sitting posture and movement pattern during the sts task were explained to the subjects before measurement. in a sitting posture, the subjects were positioned on an adjustable height chair without armrests or a backrest. the subjects were barefoot, placed both their legs symmetrically with their feet shoulder - width apart, and stretched their trunk in a straight line. the seat height for each subject was 100% of the lower leg length (lll) measured from the center of the knee joint to the floor with the subject standing barefoot. to minimize any influence of the upper extremities, subjects were instructed to cross their arms lightly against their chest to minimize movement during observation. they then performed a total of nine sts trials, including three trials each for hip add, hip abd and natural sts (n) tasks, with the ordering of the conditions randomized. subjects were instructed to face forward and stand up from the seat at their own speed. the emg data during the sts tasks were collected from frontal and transversal planes with the participants feet apart and hips in a neutral position. the sts tasks with the mivc of hip add were performed in the same position as for n, a supportive position with the legs bent at the medial femoral epicondyle level. the sts tasks with hip abd were performed in the same position as for n but with the mivc of hip abd resisted by a nonelastic band, adjustable with velcro, positioned at the lateral femoral epicondyle level. following the sts movement, the subjects were instructed to stand still for 5 s. subjects were allowed to practice the sts movement before the actual data collection started. due to the sts start and finish times were determined with a marker attached on the right acromion. for the purpose of analysis, the sts movement was divided into a pre - thigh - off (to) phase and a post - to phase with respect to chair seat support. to assess differences in emg activity in the sts testing, standard descriptive statistics were performed, and the data are reported as means standard deviation., chicago il, usa) with the level of statistical significance set at p 0.05 ; add, 10.17 8.06%, abd, 8.98 4.69%). in the post - to phase, the normalized emg data of the ta muscle showed a significant increase (p < 0.05) during the sts task with hip add (25.27 19.48%) compared with hip abd (20.58 15.53%). additionally, the normalized emg data of the sol muscle increased significantly (p < 0.05) when the sts task was performed with hip add (26.55 9.14%) compared with hip abd (23.50 9.11%). in the elderly, a postural control system deficit is caused by greatly decreasing muscle strength and power. this leads to deterioration of the ability to rise from a chair and can become a critical limiting factor for quality of life3. the purpose of this study was to compare the activation of the ta and sol muscles during the sts movement with hip adduction and hip abduction in elderly females. in the pre - to phase, the normalized emg data of the ta muscle increased significantly when the sts task was performed with hip add compared with hip abd. in the post - to phase, the normalized emg data of the ta muscle showed a significant increase during the sts task with hip add compared with hip abd. additionally, the normalized emg data of the sol muscle increased significantly when the sts task was performed with hip add compared with hip abd. in the present study, we examined muscle activity during the sts task with hip add and abd movement to create baseline data regarding ankle stability in healthy elderly subjects. sts motion requires optimal neuromuscular coordination and postural adjustments to control movement changes and to prevent loss of balance8. postural adjustments necessary for task modification or in altered environmental conditions must be controlled appropriately to maintain postural balance9. in the present study, the muscle activities of the ta and sol were investigated during sts tasks under hip add and abd conditions. the emg activities in the ta and sol were greater during the sts task with hip add compared with hip abd movement. the results showed that in the elderly performing the sts task with hip add, the ta and sol muscles were recruited more to maintain body stability. moreover, muscle co - contraction has been observed as a task - independent strategy used to stiffen the joint and enhance stability10. our results suggest that older adults may employ greater coactivation of the uniarticular ta and sol muscles during the sts movement with hip add in the post - to phase, which may increase ankle stiffness and improve balance. therefore, the sts movement with hip add poses a greater challenge for balance control, indicating that certain elderly individuals have difficulty in executing an abrupt adjustment in their dynamic postural stability during the sts movement, resulting in an increased risk of falling. | [purpose ] the purpose of this study was to compare the activation of the tibialis anterior (ta) and soleus (sol) muscles during the sit - to - stand (sts) task with hip adduction and hip abduction in elderly females. [subjects ] we recruited 16 healthy elderly females with no pain in the knee joint and no other orthopedic problems of the lower limbs. [methods ] the activities of the dominant lower extremity muscles were measured using a wireless electromyography (emg) system. subjects then performed a total of nine sts trials, including three trials each for hip adduction, hip abduction, and natural sts tasks. [results ] in the pre- thigh - off (to) phase, the normalized emg data of the ta muscle increased significantly when the sts task was performed with hip adduction compared with hip abduction. in the post - to phase, the normalized emg data of the ta muscle showed a significant increase during the sts task with hip adduction compared with hip abduction. additionally, the normalized emg data of the sol muscle increased significantly when the sts task was performed with hip adduction compared with hip abduction. [conclusion ] therefore, the sts movement with hip adduction poses a greater challenge for balance control, indicating that certain elderly individuals would have difficulty in executing an abrupt adjustment in their dynamic postural stability during the sts movement. |
there were 60 registered hiv - positive patients in greenland, most of whom lived in the 2 towns of nuuk and sisimiut (fig. hiv patients living in nuuk are treated at the central queen ingrid 's hospital in nuuk, while in the districts hiv treatment and control are guided from queen ingrid 's hospital, but administered by unspecialized local health personnel. all supervision of hiv patients and initiation of haart are performed by one of the authors (kl). patients living in nuuk are controlled in the outpatient clinic every 34 months, and in the districts at least twice a year, the latter mainly due to logistic reasons with sample shipping. blood samples for hiv - rna and cd4 cell counts are sent by courier to denmark. plasma samples are analysed for hiv - rna at the department of virology, statens serum institut, copenhagen, and cd4 cell count at rigshospitalet, copenhagen. in greenland, plasma is assayed for genotypic resistance in all patients at the time of hiv diagnosis and in case of hiv - rna > 1000 copies per ml. all hiv patients in greenland including data on viral load, cd4 counts, etc. are registered in a central database www.infcarehiv.dk. in this study virologic failure was defined by 2 repeated hiv rna>200 copies per ml (17). to collect data on living conditions and quality of life, all known hiv - positive patients were asked to fill in a questionnaire during a routine outpatient visit in the study period 20082009. most patients were interviewed or guided by a greenlandic speaking doctor (mr) not responsible for the clinical care of the patients. the questionnaire was a modification of a questionnaire used for a study of 1,212 hiv - positive danes (18) and comprised questions regarding living conditions and life quality issues. the modification included questions about inuit ethnicity and specific greenlandic socio - demographic issues. inuit was defined by both parents being born in greenland, mixed when one of the parents was born in greenland and one elsewhere or of unknown origin, and as non - inuit if none of the parents were known to be born in greenland. adherence was defined as being seen in the outpatient clinic at least twice a year, and when on haart having taken the last dose of medicine within 24 hours and not having skipped a dose within the last 4 days. the relative risk of hiv progression measured by high viral load and low cd4 count for non - adherence versus adherence was calculated as the proportion of non - adherent patients with such markers divided by the proportion of adherent patients using a binomial log - linear regression model in proc genmod in sas v9.2. the relative risks of non - adherence according to a wide range of living conditions and quality of life markers were calculated in a similar way. when having cells with 0 observations, no rr was estimated and the differences in proportions were evaluated by an exact pearson chi - square test in proc freq in sas. from these univariate analyses a multivariate analysis was carried out by including significant variables (p<0.05) in a single model and then using backward elimination until all remaining factors had reached significance. the investigation was scientifically and ethically approved by the commission for scientific research in greenland. patients were informed that their responses remained confidential and would have no consequences for their treatment. the relative risk of hiv progression measured by high viral load and low cd4 count for non - adherence versus adherence was calculated as the proportion of non - adherent patients with such markers divided by the proportion of adherent patients using a binomial log - linear regression model in proc genmod in sas v9.2. the relative risks of non - adherence according to a wide range of living conditions and quality of life markers were calculated in a similar way. when having cells with 0 observations, no rr was estimated and the differences in proportions were evaluated by an exact pearson chi - square test in proc freq in sas. from these univariate analyses a multivariate analysis was carried out by including significant variables (p<0.05) in a single model and then using backward elimination until all remaining factors had reached significance. the investigation was scientifically and ethically approved by the commission for scientific research in greenland. written informed consent was obtained from all participants. patients were informed that their responses remained confidential and would have no consequences for their treatment. forty - six (77%) patients participated in the study, 17 women and 29 men aged 2374 years with a median of 56 years. virus transmission route was by heterosexual activity in 37 patients (80%) and by men having sex with men (msm) activity in 8 (17%). the patients had their hiv diagnosis established between 1 and 18 years, median 7 years, prior to the interview. thirty - seven patients were infected in greenland, the remaining 9 abroad, mostly in denmark. forty - four of the patients were born in greenland. by the definition used, 43 were inuit, 1 non - inuit and 2 of mixed origin. forty - one of the patients received treatment with haart, and 5 were untreated. twenty - five of the participating patients lived in nuuk, and 21 in the districts (fig. 1). of the non - participating 14 patients, 3 women and 11 men (median age at diagnosis 55 years, range 3164 years), 3 refused to participate, 3 lived in remote areas, 1 was in jail and 2 failed to attend appointments. furthermore 5 registered hiv - positive patients had refused treatment and control in the clinic. these 14 patients had been tested hiv - positive between 2 and 13 years (median 7 years) prior to the study. thirty - one patients (67%) were found to be adherent according to definition. twenty - nine (71%) of those treated with haart stated that they had taken at least 90% of the prescribed drugs within the last 30 days and none of them had missed a dose within the last 4 days. there was a significantly higher rate of sufficient virologic response among adherent patients on haart than among non - adherent on haart, and more adherent patients had cd4 cell counts exceeding 350 cells per l (table i). although overall only 73% had sufficient virologic suppression, almost all (97%) adherent patients had viral loads < 200 copies per ml in contrast to only 17% of non - adherent. association between adherence and virologic response to haart and cd4 count among 46 hiv patients from greenland 2008 to 2009 41 patients on haart. there was a significant difference in age distribution (p=0.003) with adherent patients being relatively older (median 58 years, range 4574) than non - adherent patients (median 49 years, range 2360) (fig. adherence was significantly better among patients living in nuuk than among patients living in the districts outside of nuuk. fifty percent of those infected through msm activity were non - adherent compared with 27% of heterosexually infected, but the numbers are small and the difference is not significant (table ii). there was no difference with regard to duration of hiv or of treatment with haart or having an aids - defining event, and no difference with regard to education or financial situation. regarding quality of life (table iii) there was no difference between the 2 groups as to self - perception of health, having a steady partner, or living alone and no difference with regard to having friends or feeling lonely. there was no difference between the 2 groups with regard to sexual activity or satisfaction, but unsafe sex was significantly more common among non - adherent patients (table iii). alcohol and tobacco use did not differ significantly among the 2 groups although both showed a trend for association to non - adherence. it is remarkable that 13 (28%) of the patients abused hash, 3 of them daily (table iii). association between adherence and socio - demographic and clinical data among 46 hiv patients from greenland 2008 to 2009 note : there may be missing values when the patients refused to answer the question. adjusted for other significant factors. due to convergence problems /empty cells, no rrs are presented and the p - values presented are from an exact pearson test. due to correlation between the variables occupation and financial support, occupation was chosen to be included in the multivariate analysis because financial support is a subset and thus excludes otherwise valid observations. association between adherence and life style and quality of life indicators among 46 hiv patients from greenland 2008 to 2009 note : there may be missing values when the patients refused to answer the question. due to few answers not included in the multivariate model, but adjusted for age and place of living. by multivariate analysis, young age and living in a district as opposed to nuuk were found to be the 2 factors independently associated with poor adherence (table ii). due to few answers unsafe sex this study is, to our knowledge, the first study among hiv patients in an arctic population of living conditions and quality of life and their association with adherence and treatment outcome. the hiv population in greenland differs from most other hiv populations throughout the world, as most patients are relatively old at the time of diagnosis, reflecting late infection rather than late diagnosis (5), and as most are infected heterosexually. as the main part are unemployed they are relatively poor with 76% in our study reporting an income less than 20,000 usd per year compared with 64% of the greenland population as a whole (1). there are no universally accepted criteria of adherence in hiv patients, and different criteria have been used (13,1921). we used a combination of adherence to appointments and to medication, as both are crucial for optimal infection control. according to these criteria although the results are not directly comparable with other studies (10,13,22), the fraction of non - adherent hiv patients in greenland seemed relatively high especially when considering that 8% of the registered hiv population had refused to participate in the hiv treatment program and had skipped appointments for years. one patient who asserted to be adherent had a viral load of more than 80,000 copies per ml despite full viral sensitivity to his treatment, which questions the validity of his statement. even so there was such a strong association between adherence and disease control with adherent persons having substantially lower viral loads and higher cd4 counts than non - adherent patients that it, like elsewhere in the world, underlines the importance of adherence. we found that only 2 factors were independently associated with adherence, old age and living in the capital nuuk. studies from other parts of the world have indicated that the health care provider 's level of knowledge, experience and skills are of great importance for patient adherence, as is the level of support and encouragement (14). in agreement with those results we found that living in a district outside of nuuk was associated with poor adherence. while the staff situation may differ between the district hospitals, in general patients from the districts have no direct contact with hiv - committed personnel. this emphasizes the importance of having dedicated and skilled health staff members to take care of this patient group. the finding that adherence was highest among elderly people on age pension is supported by results from other studies showing that adherence improves with age of the patient (19,21). related to the relatively high age, a large fraction of the patients were sexually inactive and many of them had no sexual needs, but sexual activity was relatively high among the young patients with poor adherence. although not significant, we found a trend towards higher adherence with higher income. in accordance with this it has been shown that low educational levels are associated with poorer hiv outcome (20). although treatment is free of charge the finding may indicate that financial situation influences compliance. in contrast to others we found no association with hiv history such as duration of hiv or of haart treatment or having an aids - defining event (11). most patients in the present study lived alone without a steady partner, and although most of them reported to have friends and only few felt lonely, it is possible that a higher degree of support from relatives could be helpful. alcohol abuse has previously been reported to be common among hiv patients in greenland (5). the present study did not confirm that since only 13% of the patients had a weekly alcohol intake while, although not legalized, hash abuse was relatively common. however, there was an insignificant trend of use of alcohol being associated with poor adherence. it should, however, be considered that some of the characteristics associated with poor adherence, e.g. low income and alcohol abuse, could actually be results of low compliance and thus progression in hiv disease rather than the opposite. this study is cross - sectional and does not reveal causal associations. to determine such causal relationships longitudinal studies with knowledge of pre - infectious status are needed. poor drug - adherence is associated with increased morbidity and mortality (1113) and the low compliance among relatively young patients warrants a special effort for this group. directly observed therapy (dot) has been promoted by the who to improve adherence to tuberculosis programs and has also been suggested for hiv treatment. however, systematic reviews of randomized trials showed no benefit of this strategy, neither towards tuberculosis (23) nor towards hiv (24). it is possible that earlier initiation of haart with newer simplified drug regimens can improve adherence in vulnerable patient groups (25). in greenland especially young homosexual men with a high level of sexual activity could be a target for such strategy, and we would thereby not only improve treatment outcome, but also reduce hiv transmission (26). poor adherence and consequently inferior treatment outcome are common among greenlandic hiv patients, especially in the younger age groups and among patients living in areas outside of the capital nuuk. the results indicate that close attention should be paid to these groups of hiv patients to improve adherence and reduce hiv - related morbidity and mortality. | objectivesdespite a high level of sexually transmitted infections, hiv incidence has remained quite stable in greenland with 56 new cases per year (approximately 10 per 100,000). however, disease control is suboptimal and mortality is relatively high. the aim of the present study was to determine associations between adherence to treatment and treatment outcome, living conditions and quality of life among hiv patients in greenland.material and methodscross - sectional questionnaire - based cohort study of hiv patients in greenland during 20082009. data regarding treatment, viral load, cd4 count, etc. were obtained from a central hiv-database.resultsforty-six persons, 17 women and 29 men, of the 60 registered hiv - positive patients (77%) were included. eighty percent were heterosexually infected and 17% by men having sex with men (msm) activity. median age at the time of diagnosis was 48 years (range 2063). eighty - nine percent received highly active antiretroviral therapy (haart). sixty - seven percent were adherent as defined by a combination of adherence to appointments and to treatment. ninety - seven percent of adherent and 17% non - adherent patients on haart had hiv - rna less than 200 copies per ml (rr=24.2, p<0.0001). poor adherence was associated with younger age (< 50 years) (adjusted rr=7.95, p=0.005) and living in remote areas with no direct contact with skilled personnel (adjusted rr=6.75, p=0.01). unsafe sex was also more frequent among non - adherent patients (rr=4.12, p=0.026), but due to few answers this topic was not included in the multivariate model.conclusionthe hiv population in greenland is peculiar since most patients are heterosexually infected and middle - aged at diagnosis. a relatively poor adherence and consequently inferior treatment outcome is related to young age and living in remote areas. |
the genus aspergillus is a group of filamentous fungi found in the atmosphere and is often the blue green mould found on the bread. a. fumigatus and a. flavus are the most commonly isolated spores of the disease process aspergillosis. inhalation of aspergillus spores can lead to colonization in the upper and lower respiratory tract with allergic response or invasive destruction. a. fumigatus is the species mainly held responsible for the infection of the paranasal sinuses. but a. flavus is considered most destructive in paranasal sinuses because of its potent toxins. this fungus contaminates the paranasal sinuses by two routes. in the first or the aerogenic route the spores are inhaled directly into the antrum where they multiply best in the anaerobic medium. the second route involves an iatrogenic model where spores are introduced into antrum via an oroantral communication formed due to root canal perforation or dental extraction.[68 ] once the spores are introduced they act as opportunistic pathogens and colonize the maxillary sinus, particularly when condition that decrease sinus ventilation such as bacterial sinusitis already exist aspergillosis of paranasal sinuses is commonly seen in normal healthy individual. this condition may present as mycetoma, or occasionally as an invasive form of the disease.[1012 ] in patients with history of asthma and recurrent nasal polyps, an allergic aspergillosis may occur.[1315 ] however it presents as invasive and fulminant variant in cancer patients with impaired host defense, disease and treatment - induced leucopenia, long - term use of antibiotics or corticosteroids. this article is reported because of the rarity of the invasive variant occurring in immunocompetent host. a 47-year - old female reported to the outpatient department at government dental college, rohtak, with complaint of pain and foul discharge from upper left region of the jaw, distortion of left eye and nasal stuffiness for last three months.. left infraorbital prominence was lacking with eyeball pushed upward, prominent lower sclera and difficulty in eye movement. intraorally there was a sinus at the site of healed socket in the left maxillary canine region. the overlying mucosa was normal and the adjacent teeth were free of caries or any other periodontal pathology [figure 1 ]. photograph showing intraoral sinus at the site of extracted left maxillary canine paranasal sinus view showed destruction of left infraorbital margin along with increased radiodensity of left maxillary sinus [figure 2 ]. ct scan showed prominent and radiodense left maxillary sinus and thinned out infraorbital plate [figure 3 ]. routine blood investigations showed leucocytosis with selective eosinophilia total leucocyte count was 9900/ mm, polymorphs : 62, lymphocytes : 28%, eosinophils : 8%, and monocytes : 2%. pns view showing increased radiodensity of left maxillary sinus and destruction of left infraorbital margin ct scan showing hyperdense mass in left maxillary sinus and thinning of infra orbital margin histopathology of h and e stained specimen revealed chronic granulation tissue with faint hyphae [figure 4 ]. methanamine silver staining revealed septate hyphae with branching at 45 degree angle, suggestive of aspergillosis [figure 5 ]. photomicrograph showing scattered multinucleated giant cells, some langhans type in granuloma formations with lymphocytic infiltration (h and e, 25) photomicrograph of aspergillum organism in granulomas showing typical branching septate hyphae (methenamine silver 25) the patient was treated with oral itraconazole 100 mg twice daily for 3 months and was followed biannually. at follow up the patient remains free of clinical disease. initially hora in 1965 classified the infection of nasal and paranasal sinuses as non - invasive and invasive. this classification was based mainly on the fact whether bone has been involved or not. sarti and lucenten gave four clinical variants of aspergillus in 1988 as allergic aspergillus sinusitis, non - invasive type, invasive type and fulminant type. rowe jones in 1994 classified aspergillosis into three chief variants : invasive, non - invasive and non - invasive destructive type. non - invasive type is further classified into aspergilloma, fungal ball, mycetoma (usually affecting one sinus) or allergic aspergillus sinusitis (involving more than one sinus). invasive type represents true fungal tissue invasion that can be either slow progressive and destructive (non - fulminant) or highly aggressive and lethal (fulminant). solitary aspergillosis of the maxillary sinus occurs almost exclusively in otherwise healthy patients and has no tendency to recur after complete removal of the mycotic masses (aspergilloma, mycetoma, fungal ball). invasive and fulminant types are common in immunocompromised patients, whose inherited response to pathogen predispose to the progressive infiltrating disease. the invasive lesion of the aspergillosis comprises of a chronic inflammatory granulomatous reaction that includes giant cells and a large amounts of septate mycelial filament. the invasiveness of fungus becomes most lethal once the hyphae enter blood vessels, where thrombi are formed, precipitating embolism and necrosis. in addition to immunocompromised status, occupations like milling and farming are also the predisposing factors for aspergillosis. a. flavus is most destructive in paranasal sinuses and oral cavity because of its potent toxin producing abilities. paranasal aspergillosis can manifest as localized disease or destructive and invasive and even extend up to intracranial structures or oral cavity causing palatal perforation. the symptoms of chronic, sometimes acutely exacerbating sinusitis, progress over a month or even years. nasal obstruction and rhinorrhea may also develop with the late development of ocular and neurological signs due to local compression or direct invasion. the clinical picture of paranasal aspergillosis can therefore be similar to that of malignant disease, although chronic sinusitis with osteomyelitis, mucormycosis and inverted papilloma must also be considered. the aspergillus hyphae invade local blood vessels, leading to ischemic tissue necrosis and bony destruction. only biopsy with or without culture of fungus is confirmatory. grossly, the infective tissue exhibits yellowish, brown, grey or black color, cheesy in consistency containing dirty or muddy material. radiographic changes in non - invasive mycetoma include the presence of radiodense foci in association with homogeneous opacification of the sinus. in addition, it is found to affect only one sinus at a time. radiographically invasive and fulminant in present case, there was partial destruction of inferior wall of the orbit and left maxillary sinus on pns. histopathologically, invasive lesions are made up of chronic granulomatous reaction and are similar to sarcoidosis, midline lethal granuloma or foreign body granuloma. if langhans type giant cells are seen then pre - existing tb granuloma has to be ruled out. to see hyphae clearly selective special staining with pas or methanamine silver they appear as septate hyphae with branching at 45 angles and are about 2 - 4 micrometer in diameter. this fungus can be differentiated from mucormycosis where broader non - septate hyphae with dichomatous branching at 90 angle are observed. as culture may be negative even after employing sabouraud agar, demonstration of hyphae in tissue sections are more reliable and conclusive but species can not be confirmed. management of aspergillosis mycetoma requires the removal of the mycotic mass while the restoration of mucociliary drainage and sinus ventilation is simultaneously ensured, sincetreatment of invasive aspergillosis consists of antifungal drugs with concomitant surgery. in allergic aspergillosis, surgical debridement and aeration of the antrum with or without use of systemic steroids is advocated. | aspergillosis is a common opportunistic fungal infection affecting the nose and paranasal sinuses. the disease presents in various forms ranging from non - invasive to invasive, destructive and allergic types. we report here a rare case of invasive aspergillosis in an immunocompetent host with the literature review. |
disorders of vitamin d metabolism are frequent in chronic kidney disease (ckd) and represent a crucial physiopathological mechanism of ckd and mineral bone disorders. vitamin d deficiency is a highly prevalent abnormality in ckd patients,1,2 as well as the reduction of the active form, calcitriol 1,25-hydroxyvitamin d. sun exposure is crucial to meet the requirements for vitamin d. thus, factors such as climate, location, aging, lifestyle, and skin pigmentation may affect the endogenous production of vitamin d. however, dietary intake of cholecalciferol (from animal sources) or ergocalciferol (from plant sources) plays a role and represents nearly 20% of the 25-hydroxyvitamin d supply. it has been suggested that renal diets targeted to reducing dietary phosphorus intake can contribute to the development of hypovitaminosis d in ckd patients, especially in dialysis patients.3 vitamin d deficiency is defined as 25-hydroxyvitamin d serum levels lower than 20 ng / ml, whereas vitamin d insufficiency is defined as 25-hydroxyvitamin d serum levels ranging 2130 ng / ml.4 vitamin d deficiency causes bone and mineral abnormalities and may also play a role in several diseases including infection, cardiovascular diseases, malignancy, insulin resistance, diabetes, autoimmune disease, and impaired physical functioning.4 in ckd patients, vitamin d deficiency is associated with poorer outcomes.5,6 the prevalence of vitamin d insufficiency / deficiency status is similar in people without or with ckd who do not require dialysis.7 major determinants of vitamin d status include age, sex, body mass index, obesity, and diabetes.8 instead, the level of residual renal function is not a direct major determinant of vitamin d status.7 however, certain conditions associated with ckd such as protein losses or inadequate food intake can predispose ckd patients to severe hypovitaminosis d. guidelines recommend to treat hypovitaminosis d and suggest an optimal 25-hydroxyvitamin d serum levels > 30 ng / ml, even if this cutoff is not based on strong evidence. in recent years, vitamin d supplementation practices are increased significantly and have achieved greater serum 25-hydroxyvitamin d levels.9,10 in the present study, we examined the prevalence of vitamin d deficiency in ckd patients living in our county, and the correlates for vitamin d insufficiency / deficiency including dietary regimens. the effects of a once - a - week cholecalciferol supplementation schedule in vitamin d depleted ckd patients have been examined, as well. this population - based, prospective study includes ckd outpatients, free from dialysis, on tertiary care. patients were included if aged > 18 years, if were in stable clinical conditions, and if in ckd stages 24. exclusion criteria were malignancy, acute illness, hospital admission in the previous 3 months, systemic inflammatory diseases, hepatic insufficiency, inflammatory bowel disease, pancreatitis, hypercalcemia, hyperphoshatemia, and parathyroid hormone (pth) serum levels > 500 pg / ml. kidney transplant recipients or patients taking any vitamin d product (calcitriol, calcifediol, paracalcitol, ergocalciferol, and cholecalciferol) were excluded as well. we finally included 405 prevalent outpatients : 260 males (age 68.312.3 years) and 145 females (age 68.913.0 years). pth, calcium, phosphorus, serum albumin, lipid panel as well as serum creatinine, urea, electrolytes, uric acid, and hemoglobin were determined in overnight fasting blood samples. a subgroup of 100 consecutive patients with hypovitaminosis d (0.3 g / day, 25-hydroxyvitamin d serum levels were inversely related to urine protein excretion (r=0.315, p<0.05). on multiple regression analysis, only age (r=0.014) and pth levels (r=0.060) were independently associated with 25-hydroxyvitamin d levels (r=0.15) (table 3). in the 100 ckd patients receiving oral supplementation of cholecalciferol (10,000 iu / week), we observed a twofold increase in 25-hydroxyvitamin d serum levels (table 4) and the prevalence of 25-hydroxyvitamin d deficiency lowered from 76% to 33% of patients. a mild but significant reduction of pth level occurred despite a mild reduction of egfr. although serum 25-hydroxyvitamin d nearly doubled in all the ckd stages, a significant reduction of pth occurred in stage 3b patients (13966 vs 11757 pg / ml, p<0.05) only. the present study confirms the high prevalence of 25-hydroxyvitamin d depletion in a cohort of ckd patients living in a temperate climate, namely tuscany (italy). these data are well in agreement with others from different regions and reported in the paper by cuppari and garcia - lopes.11 in most of the published reports, 25-hydroxyvitamin d serum levels were lower than 20 ng / ml, so the prevalence of 25-hydroxyvitamin d deficiency or insufficiency ranged from 70% to nearly 100%.11 a significantly higher average level of 25-hydroxyvitamin d has been reported in ckd patients living in southern italy : this is well in accordance with the more sunny climate of sicily than tuscany.5 patients older than 65 years showed a very high prevalence of hypovitaminosis d, approaching 90%. hypovitaminosis d was also associated with higher charlson comorbidity index, a finding that is in keeping with the well - known relationship between hypovitaminosis d and morbidity / mortality risk. as already reported, hypovitaminosis d was independent from the different stages of ckd. at variance with the literature, moreover, proteinuric patients can develop vitamin d deficiency by urinary loss of vitamin d - binding protein, which binds most of the serum calcifediol. this is of a real concern since hypovitaminosis d was associated with an increased risk of kidney disease progression and mortality.12,13 in our series, we found a relationship between hematocrit or hemoglobin and 25-hydroxyvitamin d. these findings are in agreement with other authors who reported that in the early ckd 25-hydroxyvitamin d and 1,25-hydroxyvitamin d deficiencies were independently associated with low hemoglobin levels.14 however, the reasons accounting for this relationship are not yet known, as it is not proven the favorable effect of vitamin d supplementation in improving anemia in ckd patients.15 renal diets consist of low - protein, low - phosphate regimens including avoidance of dairy products and yolk (for the very high phosphate content) and limitation of meat and fish. hence these diets supply lower amount of vitamin d than mixed free - choice diets. nevertheless, we did not find any association between renal diet regimens and 25-hydroxyvitamin d deficiency. namely, the prevalence of hypovitaminosis d or 25-hydroxyvitamin d serum levels did not differ between patients on low - protein, low - phosphorus diets16,17 and those on free - choice diets. this is not surprising, since dietary supply represents a minor component for determining vitamin d status. the 25-hydroxyvitamin d serum levels were higher when blood collection was taken in summer, to confirm the seasonal variability of calcifediol (figure 1). this is also the reason why the effect of cholecalciferol administration was assessed at 12 months and not at 3 or 6 months. multivariate analysis showed that age and pth explained 1.5% and 4.5%, respectively, of 25-hydroxyvitamin d variance. evidence exists that not only the reduction of calcitriol, but also lower levels of calcifediol can contribute to the development of secondary hyperparathyroidism. therefore, vitamin d repletion using ergocalciferol or cholecalciferol is recommended to obtain adequate vitamin d status in ckd.11 k - digo guidelines suggest that vitamin d deficiency and insufficiency in patients with ckd stages 35d should be corrected as it occurs in the general population. however, this recommendation is based on low quality of evidence (2c stage).3 then, supplementation of cholecalciferol we proposed was addressed to patients with hypovitaminosis d and the effects were measured at 12-month follow - up, to avoid the changes linked to the potential changes of lifestyle and sun exposure related to seasonal - climate changes. in our treated patients, serum levels of 25-hydroxyvitamin d almost doubled, together with a mild decrease of pth despite a decrease of renal function. this suggests that cholecalciferol supplementation may be effective and safe in the early - stage ckd patients with hypovitaminosis d. as already discussed, the inverse relationship between pth and 25-hydroxyvitamin d is well known, and it occurs in the earlier stages of ckd, ie, when more chances of 1,25-hydroxyvitamin d synthesis happen.11 to confirm, studies of ergocalciferol or cholecalciferol supplementation have demonstrated pth reductions (even if quite modest) that are more likely to occur in stage 3 ckd, while in stages 45 ckd this beneficial effect was not observed.1820 the attenuation of secondary hyperparathyroidism by ergocalciferol or cholecalciferol was not confirmed in other series.21,22 given that the majority of ckd patients, independently on the stage, have a condition of hypovitaminosis d, the cholecalciferol administration may safely contribute to ameliorate calcium phosphate abnormalities in ckd. oral cholecalciferol at the dosage of 10,000 iu once - a - week should be recommended as a regular practice in ckd care management even when determinations of 25-hydroxyvitamin d serum levels are not feasible. major limitation of the study consists in the observational, single - center, retrospective design. conversely, the number of patients is quite larger than other published studies, and it allows analysis of the different ckd stages too. although the cholecalciferol supplementation was performed with a nonrandomized retrospective design, the 12-month follow - up was longer than in other studies and may prevent the bias due to seasonal variability. in summary, hypovitaminosis d is very prevalent in ckd patients in italy, and it is similar to other locations. pth serum levels and age, but not renal function, are the major correlates of hypovitaminosis d. implementation of renal diets, including low - protein diets, is not associated with higher risk of vitamin d depletion. oral cholecalciferol supplementation should be recommended as a regular practice in ckd patients, also when 25-hydroxyvitamin d determination is not available or feasible. | this study investigated the factors associated with hypovitaminosis d, in a cohort of 405 prevalent patients with chronic kidney disease (ckd) stages 24, living in italy and followed - up in tertiary care. the effect of cholecalciferol 10,000 iu once - a - week for 12 months was evaluated in a subgroup of 100 consecutive patients with hypovitaminosis d. vitamin d deficiency was observed in 269 patients (66.4%) whereas vitamin d insufficiency was found in 67 patients (16.5%). in diabetic patients, 25-hydroxyvitamin d deficiency was detected in 80% of cases. in patients older than 65 years, the prevalence of hypovitaminosis d was 89%. in the univariate analysis, 25-hydroxyvitamin d was negatively related to age, parathyroid hormone (pth), proteinuria, and charlson index, while a positive relationship has emerged with hemoglobin level. on multiple regression analysis, only age and pth levels were independently associated with 25-hydroxyvitamin d levels. no relationship emerged between vitamin d deficiency and renal function. serum levels of 25-hydroxyvitamin d or prevalence of hypovitaminosis d did not differ between patients on a free - choice diet and on a renal diet, including low - protein, low - phosphorus regimens. twelve - month oral cholecalciferol administration increased 25-hydroxyvitamin d and reduced pth serum levels. in summary, hypovitaminosis d is very prevalent in ckd patients (83%) in italy, and it is similar to other locations. pth serum levels and age, but not renal function, are the major correlates of hypovitaminosis d. implementation of renal diets is not associated with higher risk of vitamin d depletion. oral cholecalciferol administration increased 25-hydroxyvitamin d and mildly reduced pth serum levels. oral cholecalciferol supplementation should be recommended as a regular practice in ckd patients, also when serum 25-hydroxyvitamin d determination is not available or feasible. |
sturge weber syndrome (sws) has been included in the group of phakomatoses that includes neurofibromatosis, klippel trenaunay syndrome, tuberous sclerosis, and von hippel lindau syndrome.1 sws, also known as encephalotrigeminal angiomatosis, is a condition that includes leptomemeningeal hemangioma, facial angiomatosis or nevus flammeus (also called port - wine stain [pws ]), and ocular pathological changes.25 the incidence of sws is ~ 1:50,000 infants, with no significant difference between males and females.6 no hereditary pattern or predisposition has been shown, and no malignant transformation has been demonstrated.7,8 several genes in the 17p1p13 region, which are known to be involved in sws, are also known to be linked to rare abnormalities and syndromes, such as retinitis pigmentosa, cerebral astrocytoma, subglottic stenosis, klippel trenaunay weber syndrome, and phakomatosis pigmentovascularis.913 the embryologic basis of sws has been reported to be related to an impaired development of the cell precursors in the neural crest during the first embryological trimester, leading to the characteristic malformations observed in the central nervous system, skin, and eyes.3 happle suggested that somatic mosaic mutations involving the skin cause sporadic or scattered birth defects, and the phase of embryonic development during which this occurs is fundamental.14 shirley recently published a ground - breaking study where a mutation in the gnaq gene was identified, which leads to stimulation of cell proliferation and inhibition of apoptosis.15 the authors found that the mutation is associated with both sporadic pwss and sws.15 diagnosis is easily performed when the classical clinical signs of sws are present, consisting of unilateral facial pws along the first branch of the trigeminal nerve, hemiatrophy, progressive seizures, contralateral hemiparesis, mental deficiencies, hemianopia, and ipsilateral glaucoma.16 however, waelchli showed that the pws distribution may in fact follow the embryonic vasculature distribution of the face, rather than the trigeminal nerve.17 neuroim - aging techniques help to perform the diagnosis, showing gyriform calcifications that engage the parietal and occipital lobes, leptomeningeal angiomatosis, and astrogliosis in the brain.18 the diagnosis of sws is based on the presence of at least two of the three manifestations of the classic triad (leptomeningeal angioma, pws, and ocular abnormalities). however, clinical findings of sws may vary, showing variable neural signs and symptoms with the absence of or varying ocular involvement.19 according to the clinical manifestation, sws is classified into four types : 1) presence of brain and facial angioma, with or without glaucoma, 2) pws without brain involvement, with or without glaucoma, 3) isolated brain angioma, usually without glaucoma, and 4) type 1 associated with systemic manifestation such as tuberous sclerosis (table 1).20 approximately 50% of sws patients show pathologic ocular changes, usually ipsilateral to the pws, involving the eyelid, anterior chamber, cornea, choroid, and retina.39,13,16,1921 the presence of pws may involve the eyelid and trigger pathological alterations in the ocular blood flow. the bulbar conjunctiva (frequently at the limbus) may show diffused or localized area of pinkish discoloration related to increased conjunctival vascularization. episcleral vessel dilatation can be observed in approximately half of sws patients.21 several anterior chamber changes have been described in sws including vascular formations in the trabecular mesh - work near the scleral spur surrounded by large homogeneous extracellular matrix, association of the endothelial layer lining of schlemm s canal with the basal lamina, and presence of several villi and giant vacuoles within the endothelial cells that appear as transcellular channels.22 these anatomical alterations justify the frequent development of glaucoma in 30%70% of sws patients.23,24 glaucoma in sws patients shows a bimodal peak of age development : an early - onset (congenital) form affecting ~ 60% of patients and a later - onset form during childhood and adolescence (40% of cases).23 glaucoma in sws is unilateral and is associated with the presence of an ipsilateral pws,24 which most frequently involves both the eyelids (72%) and sometimes only the upper eyelid (21%).13 the most frequent form of glaucoma in sws patients is open - angle glaucoma leading to progressive visual field loss. however, acute glaucoma attack due to chamber angle closure has also been described.2527 congenital glaucoma is frequently associated with corneal changes (25%), including haze, megalocornea, and buphthalmos.21 other anterior segment alterations have been described in sws patients including iris heterochromia and cataract.19 the posterior segment of the eye is also involved with hemangiomas of the choroid (20%70% of cases).28 choroidal hemangiomas can be clinically divided into localized and diffuse forms, but it is the diffuse form that typically occurs in sws patients.19,21 clinically, ophthalmoscopy shows a bright red or red orange color appearance of the fundus, related to the increase of well - formed choroidal vessels, while hemangiomas appear as diffuse or localized areas with a dark red color and a tomato ketchup appearance.13 the presence of choroidal hemangiomas is frequently asymptomatic ; however, the choroid may become significantly thickened,29 and an increased risk of glaucoma development has been reported in patients with choroidal hemangioma.30 alteration of the choroidal vessels may lead to severe retinal complications and potentially to visual loss. subretinal hemorrhage, retinal degeneration, retinal serous detachment, photoreceptor degeneration, cystoid macular edema, macular serous detachment, tortuous retinal vessels, and optic disc coloboma have been described in sws patients.21,31 routine slit lamp examination is sufficient to detect most of the alterations typically observed in sws patients ; however, if young children can not cooperate and there is concern for visually threatening diseases such as glaucoma, examination under general anesthesia may be recquired. anterior segment alterations include eyelids, conjunctival and episcleral hemangiomas, and corneal changes related to congenital glaucoma. fundus examination by indirect ophthalmoscopy or biomicroscopy shows the typical features of choroidal hemangioma as diffuse or rarely localized areas of flat or elevated dark retina, and frequently highlights the difference in fundus color with respect to the fellow eye. clinicians can rely on more than one parameter for diagnosing and monitoring glaucoma. in very young patients, parameters such as axial gonioscopy is a simple and useful examination to detect iridocorneal angle malformation and to orient the diagnosis toward the type of glaucoma. intraocular pressure (iop) measurement, optic nerve evaluation, as well as visual field testing represent the main procedures to diagnose and monitor glaucoma progression. the optic nerve head can be observed with optical coherence tomography, which is a non - invasive and relatively rapid method of evaluation in children who have difficulty in cooperating due to their young age and/or intellectual difficulty linked to diseases or syndromes such as the sws.32,33 visual field analysis is fundamental in evaluating glaucoma progression, but this examination can be challenging to perform in children, although with increasing age and learning more reliable results can be obtained.3437 several instrumental examinations may also improve the diagnosis and monitoring of choroidal hemangioma and retinal complications, especially in the presence of anterior segment opacity, such as in congenital glaucoma. ultrasonography allows to detect choroidal alterations and to evaluate their extension, characteristics, and echogenicity. indocyanine green angiography can add useful information on the extension and intralesional vascularity of the choroid as well as the potential detection of arterovenous shunts. currently, the gold standard examination to characterize choroidal and retinal morphology is enhanced depth imaging spectral domain optical coherence tomography, which allows the quantification of choroidal thickness as well as the morphology and the caliber of dilated choroidal vessels.29,38 magnetic resonance imaging may also be useful to detect broader thickening of the eyes as a consequence of diffuse choroidal hemangioma. it is crucial to plan complete ophthalmic examinations in the follow - up of sws patients in order to avoid visual function loss, frequently related to progressive glaucoma, and also to detect the onset of retinal complications such as exudative detachment. several new hypotheses and factors have been formulated on the pathogenic mechanisms leading to the development of glaucoma, and several hypotheses have been formulated to explain the pathogenic mechanisms leading to the development of glaucoma associated with rare conditions.3941 the main theories on the pathogenesis of glaucoma in sws include the following : a mechanical mechanism related to congenital malformation of the anterior chamber angle leading to increased resistance to aqueous humor outflow : in this case, the iris can also not have the flat anterior insertion characteristic of the congenital form;24an increase in episcleral venous pressure (evp) due to arteriovenous shunts into the episcleral hemangioma:29 this theory is based on the observation of a normal angle structure, blood within schlemm s canal, and more severe glaucoma;4244fluid hypersecretion by either the ciliary body or the choroidal hemangioma;45 andabnormal hemodynamics of the episclera and the anterior chamber angle due to premature aging of the trabecular meshwork schlemm s canal complex, as observed in sws later - onset glaucoma.46 a mechanical mechanism related to congenital malformation of the anterior chamber angle leading to increased resistance to aqueous humor outflow : in this case, the iris can also not have the flat anterior insertion characteristic of the congenital form;24 an increase in episcleral venous pressure (evp) due to arteriovenous shunts into the episcleral hemangioma:29 this theory is based on the observation of a normal angle structure, blood within schlemm s canal, and more severe glaucoma;4244 fluid hypersecretion by either the ciliary body or the choroidal hemangioma;45 and abnormal hemodynamics of the episclera and the anterior chamber angle due to premature aging of the trabecular meshwork schlemm s canal complex, as observed in sws later - onset glaucoma.46 in sws patients, an increase in evp, as compared to normal eyes, has been reported in glaucoma - affected eyes.42,47 however, no significant difference in evp was observed between glaucoma - affected and normal eyes in sws patients, suggesting that elevated evp is only a risk factor for glaucoma onset.47 retrobulbar blood flow was also impaired in sws patients, suggesting an increased risk to develop glaucoma with aging. however, no significant difference in the arterial retrobulbar blood flow was observed between the glaucomatous and the normal fellow normal.48,49 glaucoma pathogenesis in sws patients is complex, including different interlinked mechanisms that change their role with aging. indeed, in congenital and early - onset glaucoma forms, angle malformations play a crucial role. in late - onset glaucoma, an increase in evp, probably related to progressive hypertrophy and dilatation of the episcleral veins as reported for pws,50 has a major pathogenic role. a few cases of acute glaucoma attack due to angle closure have been described in sws. maruyama reported a case of a patient with sws who developed acute glaucoma associated with posterior scleritis, edema of the ciliary body, ciliochoroidal effusion, and anterior rotation of the ciliary body.25 in this case, it was hypothesized that the ciliary body effusion induced angle closure by moving the iridolenticular diaphragm forward, which led to acute glaucoma attack. this mechanism of glaucoma induction has also been described following the use of the drug topiramate, which is used for seizures that can occur in sws patients with leptomeningeal involvement.27 angle - closure glaucoma in sws has also been described as related to pupil block with or without ectopia lentis and pigment dispersion.51,52 in these cases, ultrasound biomicroscopy may represent a precious tool to identify and characterize the pathogenic mechanism of acute glaucoma attack.53 the main goal of glaucoma treatment is to control iop and to avoid progressive optic nerve damage and visual field loss. both medical and surgical approaches have been performed to halt glaucoma progression in sws patients (table 2). due to the rare nature of the disease, only small case series topical antiglaucoma drugs seem to be less efficacious in sws patients with congenital glaucoma, while they represent first - line therapy for patients with late - onset glaucoma.44 ong showed that latanoprost eye drops, as adjunctive therapy, were effective in controlling glaucoma in 50% of 14 patients with sws at 1 year of follow - up.54 in line with this, yang demonstrated that latanoprost treatment induced a significant pressure decrease in 33% (two eyes) of sws glaucoma patients.55 latanoprost acts by increasing uveoscleral outflow, theoretically bypassing the obstacle to the passage of aqueous humor due to the increase in evp. however, long - term use of prostaglandins in sws patients should be carefully evaluated since one of the potential side effects of these drugs is uveal effusion. only a few case reports describe the use of other anti - glaucoma drugs in patients with sws, suggesting that treatment with beta - blockers and carbonic anhydrase inhibitors is effective in the absence of buphthalmos.56 wygnanski - jaffe used oral propanolol for treating glaucoma in sws patients.57 systemic propranolol is effective in inducing reduction of hemangiomas of the skin, orbit, larynx, and eyes.58,59 however, systemic propanolol was ineffective in three of four children with sws - related glaucoma.57 a hypothesis to justify the low efficacy of antiglaucoma drugs in controlling sws - related glaucoma is that most of these drugs do not affect evp, highlighting the need for novel antiglaucoma medications specifically targeting epv. frequently, medical treatment in sws patients is not sufficient to guarantee a good long - term control of glaucoma ; therefore, surgical procedures are commonly performed in patients under 2 years of age. goniotomy and trabeculotomy represent the most appropriate surgical procedures to overcome the malformed anterior chamber angle under 4 years of age. however, saltzmann demonstrated that trabeculotomy fails to control congenital glaucoma in all patients with sws.60 these authors suggested to consider alternative surgical procedures and closer postoperative monitoring due to this significant relative risk of failure.60 one of the possible explanations of surgery failures in sws children is that goniosurgery does not affect evp, suggesting that pathogenic mechanisms other than the simple angle malformation influence glaucoma progression. other surgical approaches include filtering procedures such as trabeculectomy, posterior lip sclerotomy,42 and trabeculotomy trabeculectomy,61,62 which bypass the alterated trabecular meshwork schlemm s canal complex, creating an alternative outflow passage for the aqueous humor, independent to the distal episcleral veins. however, filtering procedures have been associated with severe complications such as expulsive choroidal hemorrhage, bleeding, prolonged flat anterior chamber, and high risk of bleb failure.46,63 intraoperative use of antimitotic agents such as mitomycin does not improve the outcome.64 therefore, combined procedures such as trabeculotomy trabeculectomy have been suggested as the first - line approach in infants and children.61,65 alternative surgical procedures include nonpenetrating sclerectomy, valve drainage implants, and ciliodestructive procedures in adults.66,67 nonpenetrating sclerotomy has been shown to have an efficacy similar to trabeculectomy in controlling sws - related glaucoma, with a lower rate of complications.67 however, in sws patients, the presence of episcleral hemangioma and angle malformations make this procedure extremely difficult and increase the rate of failure. valve implant is also a successful method for iop management.68 in sws, the ahmed - type valve was shown to induce long - term decrease of iop by improving aqueous humor outflow.69 on the other hand, the use of the molteno tube showed an unfavorable outcome due to an elevated complication rate in children with sws.70 two studies reported that ex - press valve was successfully implanted in children with sws as an elective surgical procedure,71 and in the other case, 10 days prior to performing trabeculectomy.72 on the other hand, the use of the ex - press glaucoma implant in another child with sws was complicated by choroidal detachment, which resolved with medical therapy. lastly, it is worth mentioning that cyclocryotherapy has been used successfully in patients with sws and buphthalmos in combination with topical antiglaucoma therapy73 or trabeculectomy.74 nevertheless, cyclocryotherapy can be burdened by severe complications such as hypotony and phthisis bulbi.74 unfortunately, sometimes, even with medical and/or surgical treatment glaucoma leads to severe visual field damage, visual acuity loss, and optic nerve atrophy ; only rehabilitation methods can help in improving the quality of life of these patients.7577 better esthetic results are achieved when treating the lesions localized on the central forehead rather than the lesions on the central face. to obtain better results, pws should be treated early since there is a tendency to thickening and nodular transformation with aging.78 laser treatment should be superficial in order to avoid potential complications related to a decrease of brain venous outflow through pws vessels, potentially leading to cerebral venous deterioration, choroidal vessel dilatation, retinal exudative detachment, and increase in iop.7 therefore, deep photocoagulation and debulking surgery should be avoided in the treatment of pws.7 as previously described, visual loss in sws patients is mainly related to glaucoma development, but choroidal hemangiomas may also lead to visual impairment thorough exudative retinal detachment and macular edema. the treatment of exudative hemangioma complications is aimed at destructing the tumor or at least at decreasing tumor leakage.79 several treatments have been proposed including photocoagulation, photodynamic therapy (pdt), external beam radiotherapy, brachytherapy, and anti - vascular endothelial growth factor (vegf). the scopes of these methods are to reduce tumor size, induce vessel atrophy, close leaking vessels, and induce inhibition of vegf to ultimately diminish subretinal fluid, which causes increased retinal and macular thickness as observed with optical coherence tomography.8082 confluent photocoagulation causing destruction of the tumor has been considered as the most effective treatment in reducing leakage.83 however, several complications can be observed following intense photocoagulative treatment, leading to the proposal of an alternative, less invasive laser procedure, such as grid treatment, that is safer but has a higher recurrence of subretinal fluid accumulation.84,85 pdt is theoretically the most indicated procedure to treat choroidal hemangiomas, inducing vessel atrophy and reducing leakage.66 however, currently, only a few cases of diffuse choroidal hemangioma have been treated with pdt, probably due to the risk of inducing scarring and pigmentary changes of the fovea.86 in case of diffuse choroidal hemangioma complicated by serous retinal detachment, an alternative treatment is external beam radiotherapy.87,88 clinical improvement has been observed months following the first application but recurrence frequently occurs. repeated applications should be carefully evaluated since they can induce several complications such as cataract, neuropathy, and radiation retinopathy.87 exudative retinal detachment related to choroidal hemangiomas has also been successfully treated using brachytherapy with cobalt-60 and ruthenium-106.8992 intravitreal injection of pegaptanib (an anti - vegf) has also been described in one sws patient with exudative retinal detachment with satisfactory results.93,94 the most frequent form is congenital glaucoma, but it can also occur in children and adults, making careful ophthalmic follow - up of sws patients mandatory. indeed, glaucoma secondary to sws is a challenging disease due to its early development and poor response to standard medical treatment. surgery is frequently required to obtain long - term control of iop in order to avoid visual function loss. however, several severe complications related to surgical procedures have been described in these patients, including choroidal effusion, expulsive hemorrhage, and exudative retinal detachment. moreover, in sws, the surgical success rate is the lowest among secondary glaucomas, since surgical failure, uncontrolled iop, and low vision outcomes have been frequently reported.45,60,61 to date, despite the wide range of available medical and surgical approaches to treat both the congenital and late - onset forms of glaucoma, the development of this ocular complication still represents the worst prognostic factor for vision loss in sws patients. choroidal hemangiomas are present in 20%70% of cases, and the choroid may become significantly thickened and can be associated with an increased risk of glaucoma development.29 choroidal hemangiomas may be asymptomatic ; however, severe forms may lead to visual impairment thorough exudative retinal detachment and macular edema. the treatment of exudative complications is aimed at destructing the tumor or at least at decreasing tumor leakage. | sturge weber syndrome has been included in the group of phakomatoses that is characterized by hamartomas involving the brain, skin, and eyes. the characteristic facial port - wine stain, involving the first branch of the trigeminal nerve and the embryonic vasculature distribution in this area, leads to several ocular complications of the anterior segment and can involve the eyelids and conjunctiva. the posterior segment of the eyes is also affected with diffuse choroidal hemangiomas. however, the most frequent ocular comorbidity is glaucoma with a prevalence rate ranging from 30%70%. glaucoma is related to anterior chamber malformations, high episcleral venous pressure (evp), and changes in ocular hemodynamics. glaucoma can be diagnosed at birth, but the disease can also develop during childhood and in adults. the management of glaucoma in sturge weber syndrome patients is particularly challenging because of early onset, frequently associated severe visual field impairment at the time of diagnosis, and unresponsiveness to standard treatment. several surgical approaches have been proposed, but long - term prognosis for both intraocular pressure control and visual function remains unsatisfactory in these patients. choroidal hemangiomas may also lead to visual impairment thorough exudative retinal detachment and macular edema. treatment of exudative hemangioma complications is aimed at destructing the tumor or decreasing tumor leakage. |
a 60-year - old man was presented to an orthopedician in the operating theatre as a case of difficult urinary catheterization. the patient was going for a lengthy orthopedic operation that required a drained urinary bladder beforehand. the urologist was called and he found the patient anesthetized, having circumcised penis and a pin - hole external urethral meatus (eum) at the corona penis level. the tight meatus was negotiated first with a lubricated 4-french (fr) bougie urethral dilator followed by subsequent sized dilators up to 14- fr caliber when a proper foley catheter was introduced easily into the bladder. the patient was carefully interviewed to reveal that circumcision was performed neontally as a religious rite, and he has no history of urologic diseases or trouble voiding, as this was the first time for him to meet a urologist. the patient, who is unaware of his condition, is a father of 12 children and never underwent urinary catheterization. hypospadias is a highly prevalent congenital anomaly of the male genitalia. in comprehensive review, sorensen (1953) credited rennes in 1831 with reporting a prevalence of hypospadias of 1 in 300 recruits and also reported the same figure for live male births in denmark. there is a plethora of hypospadias cases of different degrees of severity repaired in childhood that resulted in different psychological, social, and sexual outcomes. psychosexual effects of hypospadias repair are endured in adulthood although affected men maintain satisfaction in their sexual life. it is unusual in the sense that the elderly man should have had his hypospadias deformity repaired decades ago in order to pass urine freely and to impregnate his spouse with a wider, and properly located meatus. the tight meatus in this case was severe, hardly admitting a lubricated 4-fr bougie dilator. litvak., reported that the meatus in children younger than one year accepts a lubricated 5-fr feeding tube. they also reported that, in children aging 1 - 6 years, an 8-fr feeding tube could pass without difficulty. the social and sexual life of adults operated for hypospadias during childhood has been studied by a few authors. a few publications reported patient and partner dissatisfaction with the appearance of genitalia, as sexual dissatisfaction is often attributed to penile size and curvature. noted that self - reported strength of libido was slightly better for controls compared to patients with hypospadias, but without a statistically significant difference. noted that only about 10% of both patients and controls reported that their libido was low. problems reported include weak or dribbling ejaculation, having to milk out ejaculate after orgasm, quantity of semen passing after intercourse, anejaculation with or without orgasm. liu. observed that the rates of ejaculation problems in the distal and proximal groups were 19.5% (8/41) and 48.6% (17/30), respectively [5, 6 ]. the erectile problems in repaired hypospadias may be attributed to surgically correctable and non - correctable causes. more commonly encountered correctable causes include persistent chordee, penile torsion, fistula formation, acquired meatal stenosis, and inadequate cosmetic outcome. similarly, in unrepaired cases, as the eum is distal enough to deposit the seminal fluid high in the posterior vagina with no difficulty, fertility is supposed to be preserved. furthermore, aho. found that men who had hypospadias during childhood were less likely to live with a partner, and that they had fewer children (0.8 vs. 1.1). figure 1 extremely tight external meatus in a fairly asymptomatic 60 yrs, old man with distal hypospadias. the literature is scant about the urinary, social, sexual, and fertility aspects of unrepaired hypospadias cases. to our knowledge, no other case has been reported for a circumcised uncorrected hypospadias with extremely tight eum in a the finding in this treatise supports the previously published data surrounding the unaffected sexual and fertility aspects of patients having distal hypospadias, and limits repair to cosmetic reasons. | hypospadias is a highly prevalent congenital anomaly. english articles, indexed in pubmed, published the long - term sexual and reproductive outcome following hypospadias repair. almost all repairable cases of hypospadias are operated in childhood. although distal hypospadias does not interfere with fertility, it is worthy reporting a case of an elderly man, who fathers 12 children and has no urologic complaint, presented with unrepaired coronal hypospadias and severely tight external urethral meatus. |
phospholipidosis was first believed to be observed by nelson and fitzhugh in 1948,(1) when they reported the accumulation of foam macrophages in rats after long - term treatment with chloroquine. it has been observed since then that numerous cationic amphiphilic drugs can induce phospholipidosis in several cell types and that it can be characterized by the excess accumulation of phospholipids. electron microscopy is the most reliable method of identifying whether a compound has induced phospholipidosis, by the presence of lamellar inclusion bodies.(5) however, it may also be identified by light microscopy in which cells appear vacuolated and contain foamy macrophages.(6) the observation of compound - induced phospholipidosis in the drug development process is considered manageable as the effect often only occurs at very high doses, many times that of the intended therapeutic dose.(7) there is at present no strong evidence that that the condition is harmful to human health, and it is reversible once treatment is terminated, the drug is expelled from the cell and phospholipid levels return to normal.(8) this process can take weeks, however, and in some cases has been reported to last several months. it can be especially important in the context of the nervous system, where phospholipids may disrupt cell signaling in neurons and could possibly be linked to several genetic diseases such as niemannpick disease. the occurrence of phospholipidosis in the drug development process therefore can cause delays as more tests need to be carried out to satisfy regulatory bodies. it is also possible that the occurrence may sometimes stop the drug development process altogether. a recent minireview(6) shows that the method by which compounds induce phospholipidosis is still not well understood and indeed suggests that the underlying mechanism is not exactly the same for each compound. the most common mechanism is the inhibition of lysosomal phospholipase activity leading to the accumulation of several classes of phospholipids ; it has also been shown, however, that an increase in synthesis of acidic phospholipids may occur leading to the redirection of phospholipid synthesis. the application of an in silico model for predicting phospholipidosis to produce an accurate and fast method could be of great use to the pharmaceutical industry, where early screening is of great importance. indeed, this has already been recognized with many early attempts at doing just this. first attempts at producing such a model by ploemen.(13) used pka and clogp. here ploemen. suggested that a compound would be phospholipidosis - inducing (ppl+) provided that pka > 8 and clogp > 1 and also that the inequality, eq 1, is satisfied. another simple model was suggested by tomizawa.,(5) in which a modification to the ploemen model involved replacing pka with nc, the sum of the charge of all dissociable functional groups in a molecule. this ploemen model was further tested by pelletier.(7) with a larger data set, and it was shown that improvements in the rules could be made to produce better predictivity. the inclusion of additional descriptors and the use of a bayesian model also produced even better predictivity than that of the modified ploemen model. kruhlak.(14) used two commercially available software packages, mc4pc and mdl - qsar, to produce predictive models for phospholipidosis and on a 10-fold cross - validation test produced { 76% positive, 78% negative predictivity } and { 65% positive, 87% negative predictivity }, respectively. all these approaches are based on relatively simple models, and as phospholipidosis is clearly a complex effect, applying state of the art machine learning techniques could therefore produce even better predictive results. ivanciuc(15) produced a comprehensive list of state of the art machine learning techniques and their ability to predict phospholipidosis, using the nonproprietary data from the pelletier data set ; the models were tested on a 10-fold cross validation. the best model for prediction was a support vector machine(16) with an rbf kernel and = 0.01, producing on the validation fold 97% accuracy and 0.94 matthews correlation coefficient. here we propose testing the machine learning models on a larger data set than the pelletier one. it was mainly created from a combination of two data sets producing a total of 185 compounds, of which 102 were positive for phospholipidosis (ppl+) and 83 were negative (ppl). the literature mined data from the pelletier data set were used as the basis for our data set of 117 compounds, and this was supplemented by data taken from kruhlak.,(14) consisting of compounds compiled from 12 other sources. the kruhlak. data include compounds marked as negative solely due to the absence of a reported positive result, and these compounds, which may in fact be untested, were excluded from our database to reduce the possibility of erroneous data. this means that all of the 83 ppl compounds were reported negative by electron microscopy. out of the 102 ppl+ compounds, 34 of these compounds are reported positive by the presence of foamy macrophages or vacuolations and the remaining 68 of these compounds were confirmed by electron microscopy. the phospholipidosis - inducing compounds have been observed to act on a variety of species including humans, rats, mice, dogs, rabbits, hamsters and monkeys as well as a variety of tissue types including lungs, kidney and liver. a full breakdown of the positive compounds in the pelletier data set is shown in table 1 of pelletier.(7) negative compounds from the pelletier data set included druglike molecules searched from the literature, contrasting with the kruhlak data included in our data set, in which the negative compounds were all drugs. as reported by perez,(17) a good measure of the dissimilarity is the average value of the dissimilarity of the members of the set : where t(i, j) is the tanimoto coefficient. here a value of 1 would represent a very diverse set and a value of 0 would be a set of very similar compounds. the value obtained for our data set was calculated as 0.833 and therefore suggests that we have a reasonable diversity of compounds contained within our data. the structures of the compounds were stored in smiles(18) format, and missing smiles strings were obtained from pubchem (http://pubchem.ncbi.nlm.nih.gov) using the name of the compound as a search term. the smiles for each molecule was converted into sdf format and was standardized using tools from chemaxon.(19) the standardization involved removing fragments, rearomatizing, removing explicit hydrogens and cleaning the 2d structure. two different sets of descriptors, e - dragon(20) and circular fingerprints,(21) were calculated for the data set. pka, clogp and (clogp) + (pka) were also calculated using chemaxon tools, and these were added as descriptors to both of the original sets of descriptors. this leads to a total of 1,669 descriptors for e - dragon and 320 for circular fingerprints. a final descriptor set was created by combining both of the different descriptor sets, creating a total of 1986 descriptors. a stratified 10-fold cross validation was used for each run : 8 folds were used for training, one was used for internal validation and one was used as a test set. stratification was used to maintain the proportion of positive and negative compounds in the folds. the test and validation folds were cyclically rotated so that each fold was used once each as a test set and as an internal validation set. this internal validation was used to tune parameters without causing bias in the prediction of the test fold. feature selection was then performed on the training data using the weka(22) function svmattributeeval with default parameters to select the top 50 features. attributes are ranked by the square of the weight assigned by svm.(23) we compare two different machine learning algorithms and their power to predict phospholipidosis ; the r(24) implementation of random forest(25) and the r implementation of support vector machines (svm)(16) which uses libsvm.(26) the number of trees in random forest was set to 1000. the generalization error for forests converges as the number of trees in the forest become large.(25) we can therefore simply choose a large number of trees, and as our data set is small, our computation time is not limiting. the variable, mtry, which controls the number of features selected at random at each node, of which the feature providing the best split is chosen, was varied from 1 to 50 with a step size of 1. svm was run with an rbf kernel meaning that two parameters needed tuning, and c. these were varied by (2, 2,..., 2, 2) and (2, 2,..., 2, 2) respectively. its value can range from 1 to 1, where 1 is a perfect anticorrelation, 0 is the equivalent of random guessing and 1 is a perfect correlation. it is used here for comparison between the two algorithms as it is arguably the best single valued metric that describes the confusion matrix of a binary classification problem. it can be an especially useful measure where classes are unbalanced as can be seen in eq 3 ; it takes into account not only true positives (tp) and true negatives (tn) but also false positives (fp) and false negatives (fn). accuracy, the percentage of correctly predicted instances, is not a good measure of prediction quality for unbalanced classes. for example, when a problem has a very skewed proportion of two classes, predicting each instance to belong to the larger class will lead to a high percentage accuracy. this prediction is, however, not very useful or informative, especially if one is interested in predicting membership of the smaller class correctly. indeed, even if either the predicted or real data contain no members of one class, causing one of the four sums in the denominator to go to zero, one can show that the correct limiting value of the mcc is still zero(28) and pragmatically one can specify a minimum value of one for the denominator.(29) hence the uninformative prediction of the same class for every instance is seen to be no more useful or informative than random guessing. ten independent runs of both methods were used for each fold, and the average mcc on both the validation and test sets for each of the parameters was calculated. initially it was seen that a single 10-fold cross - validation run could give large deviations across each individual fold. therefore in order to get a more realistic value of performance, we repeated the 10-fold cross validation 10 times with different fold definitions. we report here in tables 13 the average mcc and the standard deviation for each of the 10-fold cross validations performed for the three different descriptor sets used. the parameters are chosen so that the maximum mcc value is obtained for the internal validation. this is done by calculating the mcc of the internal validation for all tested parameters over each of the 10 repeated runs of each fold. this value is then averaged to give a value for the performance on that fold. this is done for all of the 10 folds, in each of the 10 different 10-fold cross validations. these mcc values are then averaged over their respective 10 folds, and the value is summed for each parameter across the 10 different definitions of folds. the parameter which produced the highest averaged mcc value over the different fold definitions was selected. we report the averaged mcc value over each of the 10 repeated 10-fold validations and 10-fold tests, for that parameter. we also report the standard deviation across the 10 folds for each of the fold definitions, t. (mcc) is the standard deviation of the mcc of the test set, across the 10 different fold definitions. highlighted in bold is the highest mcc on the test fold for each fold definition. similarly, t represents the standard deviation for the test set. highlighted in bold is the highest mcc on the test fold for each fold definition. similarly, t represents the standard deviation of the test set. highlighted in bold is the highest mcc on the test fold for each fold definition. random forest produces the best result with an averaged mcc of 0.532 across the 10 separate 10-folds. svm only produces a better result on one of the different fold definitions and produces an averaged mcc of 0.485. random forest is also more reliable, producing a smaller averaged standard deviation on the test folds, t, of 0.193 compared to that of svm. standard deviations for different fold definitions range from 0.133 to 0.257 for random forest and from 0.182 to 0.293 for svm. for the cfp descriptors, svm produces the best result overall with an averaged mcc of 0.650 compared to an mcc of 0.619 for random forest. the standard deviations for the averaged mcc for each fold definition ((mcc)) are much lower for both svm and random forest, with values 0.031 and 0.033 respectively. for here we see again random forest producing a higher averaged mcc value on the test set compared to that of svm. again the best mtry parameter selected is 4 and random forest produces more reliable results with a t = 0.178. the standard deviation of the average mcc across each fold definition is small for both random forest and svm, with values 0.034 and 0.058. overall the most predictive method is an svm model with = 0.011 and c = 0.841 using cfp descriptors. the use of machine learning algorithms and a more sophisticated descriptor set can lead to improved prediction. while it is hard to distinguish which had the larger effect, it has been shown that using more sophisticated descriptors than simple dumb descriptors leads to an increase in predictivity.(30) it has also been shown that both random forest and svm do significantly better than simpler methods such as linear trees. using a repeated 10-fold cross validation with 10 different definitions allows for a more reliable result to be obtained. this can be seen by the small deviation in results across different fold definitions ((mcc)). another option could have been to select the folds so that dissimilar compounds appear in each. while this produces a good test for the algorithm, it may not be similar to how the algorithms would be used for a real world problem. often these techniques will be trained on all possible data available, therefore when a new molecule is tested it will not always be highly dissimilar to those molecules in the training set. when artificially selecting folds it can be the case that the test set has a larger amount of unseen molecules than when used in the real world, and hence an artificially lower mcc value could occur. a stratified cross validation, in which compounds are selected randomly while maintaining their class proportions, can still give the variation in folds necessary to test the algorithm. this can be seen from the large standard deviation across individual folds (t). these large variations across individual folds can suggest that certain molecules are particularly difficult to predict. a confidence index was derived which can be used as a guide to which molecules were hard to predict. for the cfp data set we calculated the proportion of times the majority prediction was made for each compound over all the runs. this produces a value between 0.5, suggesting that over the runs both classes were predicted equally, and 1.0, for which over the runs only one class was predicted for this compound. a table of the compounds and their respective indexes for both svm and random forest is included in the supporting information. in order to validate that our model was not overfitting, we investigated y - scrambling(33) of the data. the data were initially split into a training set and a test set (60%/40%) using a weighted random sampling. in the training set the class column was permuted randomly, and then the same procedure as before was followed. 50 features were selected using svmattributeeval with default parameters, and then svm with = 0.011 and c = 0.841 and random forest with mtry = 4 were trained on the data. the mcc and the fraction of correct predictions, acc, are shown in figure 1 for random forest and svm respectively. the model was also trained without the initial scrambling following the same procedure with 10 repeats, and the results are shown as red stars. it can be seen that our methods do produce both higher mcc and higher acc, fraction of correct predictions, than those with y - scrambling, suggesting that information is gained in the process of learning. the results from the y - scrambled models are statistically in line with those expected on the basis of purely random prediction. we also see a good correlation between mcc and acc, which is as expected for a reasonably balanced data set. the best result for scrambling the mean of the scrambled data for both svm and random forest lies around the (0.5,0.0) point as expected, and all points lie within 2 standard deviations. with a larger data set and therefore test set we would expect a reduction in the deviation of the mcc and acc values for scrambling. (a) svm (= 0.0110, c = 0.841) results for y - scrambling. the mcc is plotted against the accuracy, acc (the fraction of correct predictions). red stars show our best model run on this split of the unscrambled data which is repeated 10 times. for svm (b) random forest (mtry = 4) results for y - scrambling. here random forest produces 3 distinct confusion matrices for the 10 runs on the unscrambled data. table 4 shows the top 10 ranked features across all of the runs for the combination of descriptors data set. out of the top 10, 8 features are selected from cfp, and 2 are selected from the e - dragon descriptors which are the sophisticated descriptors : 3d - morse signal 23/unweighted (mor23u) and the ghoseviswanadhanwendoloski antihypertensive - like index at 50% (hypertens-50). for the top 50 features selected for the combination data set, 25 were on average chosen as cfp descriptors and 25 were chosen as e - dragon. despite the use of feature selection, as can be seen from the results, using a larger number of descriptors can lead to a less predictive model. surprisingly, as they have been suggested as important descriptors in previous work, pka and clogp do not appear in the top 10 selected features. looking at the top 50 features for the cfp data set, however, both clogp (top) and pka (third) appear. this could be the main reason why we see a large improvement with cfp, as these descriptors seem important and are picked out more easily with the smaller number of descriptors to choose from. this also gives a good justification for believing that just using a large number of features, and hoping that feature selection will pick the most descriptive features out, is a bad approach to machine learning. the rank is determined from the feature selection performed using svmattributeeval on the training folds. here mor23u relates to 3d - morse signal 23/unweighted and hypertens-50 relates to ghoseviswanadhanwendoloski antihypertensive - like index at 50%. we have made numerous attempts to repeat ivanciuc s study(15) of the pelletier database. structures were downloaded using the cas numbers supplied by pelletier.(7) using pubchem (http://pubchem.ncbi.nlm.nih.gov). e - dragon(20) descriptors were calculated from these structures once any fragments had been removed. we have used the same feature selection method from weka,(22) svmattributeeval with default parameters, to choose the top 50 e - dragon descriptors, just as ivanciuc did. we have implemented a workflow which is effectively identical to that with which dr. ovidiu ivanciuc kindly supplied us, using weka to build an svm model with a gaussian radial basis function kernel. as previously discussed, this involves the use of two parameters and c. we chose the same parameters as ivanciuc reports and chose c = 100 just as he did (private communication). we randomly split the pelletier database into 10 folds for cross validation, and repeated this splitting 10 times, so that we have carried out 10 independent 10-fold cross validations on the 117-molecule data set. this is a significant difference compared with the accuracy of 0.970 and mcc of 0.944 reported by ivanciuc, but is in line with what we report for our own models. to ensure that our own results can be reproduced, we supply as supporting information our database containing structures in smiles format and the necessary scripts used to run our models. as can be seen from the results reported for our models on the smaller 117 molecule data set, an increase in the size of the data set can cause a difference in predictivity. one possible reason for the large deviation across folds is the relatively small number of molecules in each fold (18 in the full data set and only 12 in the smaller one) and hence in the test set. a small number of molecules implies that the mcc will vary to a much greater extent when a single molecule s prediction is changed. therefore despite the 50% increase in size of our full 185 molecule data set, an individual molecule can still have a large effect on the results. figure 1 shows that, with a small test set, in this case 74 molecules, there is a greater chance of a random y - scrambled model predicting reasonably well. hence, with a larger data set more reliable measures of performance can be calculated. a further future increase in the size of the available data sets for phospholipidosis would also hopefully cause an increase in the size of chemical space sampled and allow for more general rules to be learned. we have used svm and random forest to generate predictive models for phospholipidosis inducing potential. svm produces the best predictive model using cfp descriptors giving an average mcc of 0.650 in a 10-fold cross validation. indeed, we obtain universally better results with cfp than either with e - dragon descriptors or with a combination of the two. the results of the y - scrambling tests confirm that our models have actually learned and that their success is not due to chance correlations. a relatively large deviation occurs between individual folds in each set of 10, suggesting that some individual molecules could be hard to predict. however, the deviation is small between the averaged results from the different partitions of the whole data set into folds. this suggests that we have a reliable value for the mcc describing the overall predictivity of our models. we find lower mcc values for the larger 185 molecule data set than for the 117 molecule pelletier data set. this suggests, for more reliable and robust predictivity, the need for a much larger publicly available database of phospholipidosis inducing potential. | phospholipidosis is an adverse effect caused by numerous cationic amphiphilic drugs and can affect many cell types. it is characterized by the excess accumulation of phospholipids and is most reliably identified by electron microscopy of cells revealing the presence of lamellar inclusion bodies. the development of phospholipidosis can cause a delay in the drug development process, and the importance of computational approaches to the problem has been well documented. previous work on predictive methods for phospholipidosis showed that state of the art machine learning methods produced the best results. here we extend this work by looking at a larger data set mined from the literature. we find that circular fingerprints lead to better models than either e - dragon descriptors or a combination of the two. we also observe very similar performance in general between random forest and support vector machine models. |
the advancement of visual techniques implemented in perinatal medical care enables surgeons and anesthesiologists to perform airway management and to detect, monitor, and surgically treat life - threatening anomalies of the fetal tracheo - bronchial tree [111 ]. thus, the tracheo - bronchial angles in the human fetus are of increasing relevance in perinatal medicine to determine both normal and pathological criteria adapted to anatomical particularities of the fetal tracheo - bronchial tree [1113 ]. the angle of tracheal bifurcation refers to both the interbronchial angle, measured between the central axes of the right and left main bronchi, and the subcarinal angle, measured along the inferior borders of the 2 main bronchi [1316 ]. as presented in helical ct images, the interbronchial angle is somewhat wider than the subcarinal one. the tracheal bifurcation angle was found to vary from 40 to 99, on average 6065 [1821 ]. interestingly enough, in indian population the subcarinal angle was considerably smaller, and averaged 53.54 in fetuses with crl of 131200 mm, 62.98 in neonates, 58.58 in children aged 110 years, and 54.56 in teenagers. since the deviation of the left main bronchus from the vertical is considerably greater than that of the right main bronchus, the left bronchial angle is much larger than the right one. to date, the radiographic and anatomical literature has mainly focused on the tracheal bifurcation angle in neonates, children, and adults [12,15,16,1822 ], and to some extent on the fetus, mainly based on very subjective methods with the use of protractors and goniometers. another serious limitation of most radiographic studies is the lack of standardization with respect to the phase of respiration, type of projection, degree of rotation, or posture. in fact, the angle of tracheal bifurcation tends to decrease by up to 9 between inspiration and expiration alone. the present autopsy study is the first in the medical literature to highlight the fetal tracheo - bronchial angles with the help of objective and repeatable digital image analysis, using an adequate program of nis - elements br 3.0 (nikon). to supplement the missing information on the fetal tracheo - bronchial angles, in this study we aimed to investigate : values of the right and left bronchial angles, and their sum as the interbronchial angle at varying gestational ages ; and possible both male - female and age - related differences between the studied parameters the study sample consisted of 73 human fetuses of both sexes (39 males and 34 females) at the age of 1425 weeks of gestation (table 1), which had been derived from spontaneous abortions and stillbirths, and never intubated. in no case was the cause of fetal death related to congenital cardiovascular or laryngeo - tracheal anomalies. the series included fetuses, which were only the outcome of causes of placental insufficiency. the research was consistent with the rules of the research ethics committee of our university (kb 189/2011). after opening the thorax through sternotomy, the heart and its great vessels were dissected with the use of microsurgical instruments, and then removed from the mediastinum. thus, the trachea and main bronchi with no conspicuous anomalies were visualized in the posterior mediastinum. in every fetus, the trachea and 2 main bronchi, in situ with a millimeter scale, were placed perpendicularly to the optical lens axis, recorded using a nikon d200 camera, and digitalized to tiff images. afterward, digital pictures of the trachea and main bronchi (figure 1) were subjected to digital image analysis, using an adequate computer program (nis - elements br 3.0, nikon). first, in each picture, the following 3 borderlines were consistently reproduced, as follows : the central axis of the right main bronchus, the central axis of the left main bronchus, and the vertical passing through the inferior point of tracheal bifurcation. after that, the 3 variables of tracheal bifurcation angle were precisely defined and assessed. right bronchial angle (in degrees), computed between the central axis of the right main bronchus and the vertical passing through the inferior point of tracheal bifurcation ; left bronchial angle (in degrees), computed between the central axis of the left main bronchus and the vertical passing through the inferior point of tracheal bifurcation ; and interbronchial (tracheal bifurcation) angle (in degrees), computed as the angle of intersection between the 2 central axes of the right and left main bronchi. we did not evaluate the subcarinal angle because of its irregular outlines. in a continuous attempt to minimize measurement and observer bias, the 3 repeated semi - automatic measurements were taken for each variable and averaged to minimize intra - observer variation. the differences between the repeated measurements, as the intra - observer variation, were evaluated by one - way anova test for paired data and post - hoc bonferroni test. the data obtained were checked for normality of distribution using the kolmogorov - smirnov test and for homogeneity of variance with the use of levene s test. as the first step in the statistical analysis, the t - test was used to examine the influence of sex on the values of the parameters studied. since the fetuses were not equally distributed with relation to gestational age, to examine sex differences we tested possible differences between the following 9 age groups : 1416, 17, 18, 19, 20, 21, 22, 23, and 2425 weeks. next, we tested possible sex differences for the whole sample, without taking into account age. to examine the relationship between values of every tracheo - bronchial angle and fetal age, the kruskal - wallis test was used. the values of 3 tracheo - bronchial angles were plotted against fetal age in weeks to establish their growth dynamics. we attempted to introduce both linear and nonlinear regression analysis to derive the possible curve of best fit for each angle studied against gestational age. no statistically significant differences were found in the evaluation of intra - observer reproducibility of the 3 tracheo - bronchial angles (p>0.05, the one - way anova test for paired data, and post - hoc bonferroni test). obviously, inter - observer variability was not assessed, since all measurements had been done by 1 observer. the morphometric values obtained were characterized by normality of distribution (the kolmogorov - smirnov test) and homogeneity of variance (levene s test). as a consequence, all the quantitative variables were expressed as the mean standard deviation. the statistical analysis showed no male - female differences for the 3 tracheo - bronchial angles (p>0.05, t - test), so the numerical data without regard to sex are presented in table 2. the statistical analysis showed that values of the 3 tracheo - bronchial angles evolved independently of fetal age (p>0.05, kruskal - wallis test). the right bronchial angle (figure 2) ranged from 11.4 to 41.8, with its overall mean of 26.97.0. throughout analyzed period, the values of left bronchial angle (figure 3) varied from 24.8 to 64.8, and averaged 46.28.0. as a consequence, the interbronchial angle totalled from 36.2 to 96.6. the overall mean of the interbronchial angle averaged 73.112.7 (figure 4). the regression curves of best fit for every tracheo - bronchial angle against fetal age could not be modelled because of great inter - individual variability of their values at varying gestational ages. instead, figure 5 summarizes only the developmental trend of the 3 tracheo - bronchial angles during the analyzed period. the present study describes a cross - sectional interpretation of the 3 tracheo - bronchial angles based on the evidence from 73 fetuses at ages of 1425 weeks. thus, it is rather a populational perspective than a true representation of growth in itself. the main limitation of this study is a relatively narrow gestational age, ranging from 14 to 25 weeks. apart from this, our results suffer from lack of inter - observer variability because all numerical data have been controlled by 1 researcher. fortunately, the adequate digital program of nis - elements br 3.0 (nikon) appears to compensate for that disadvantage due to precise semi - automatic evaluation of the 3 tracheo - bronchial angles in digital pictures. the medical literature on the tracheal bifurcation angle offers many discrepancies between reported values with relation to sex- and age - dependent differences. as reported by karabulut, the 4 tracheo - bronchial angles (right and left bronchial, interbronchial and subcarinal) were significantly greater in females than males. similarly, according to kamel., the subcarinal angle in adults ranged from 36 to 121 in men, and from 47 to 115 in women, with overall means of 7620, and 8120 respectively, without any correlation with age. this remains contradictory to most of the previous studies, which reported the tracheal bifurcation angle to be independent of sex [12,13,1820,25 ], and to our findings in the material under examination. to our knowledge, the significantly greater tracheo - bronchial angles in females may partly have resulted from obesity of the examined women, positively correlated with the tracheal bifurcation angle. most authors have agreed that, irrespective of sex and age, the right bronchial angle was smaller than the left one [9,12,13,15,16,1921,25,26 ]. according to harjeet., in the 3 groups of fetuses with crl of 61130 mm, 131200 mm, and 201270 mm, the left bronchial angles were consequently wider than the right ones, and averaged 32.176.98 vs. 17.377.77, 33.798.57 vs. 19.758.73, and 34.743.67 vs. 20.541.86, respectively. in the material under examination, the right and left bronchial angles correlated with the literature data, ranging from 11.4 to 41.8, with the mean of 26.97.0 on the right, and from 24.8 to 64.8 with the mean of 46.28.0 on the left, with no effect of age or sex. as reported by jit and jit, in neonates, infants, and children, the right bronchial angle was always smaller than the left. however, tahir. noticed the right main bronchus deviates significantly less from the vertical on chest radiograms in children over 3 years when compared to younger children (25 vs. 28). this may have been influenced by a greater proportion of upright chest radiograms in older children and a greater proportion of supine chest radiograms in younger children. a radiographic study by harjeet. revealed an insignificant increase in the subcarinal angle with age from 49.5312.10 in fetuses with crl of 61130 mm, through 53.5416.17 in fetuses with crl of 131200 mm, up to 55.273.66 in fetuses with crl of 201270 mm. although in the present study the interbronchial angle attained much larger values than the afore - mentioned subcarinal angle, varying widely from 36.2 to 96.6 to average 73.112.7, there were no statistical differences with age, in keeping with karabulut s helical ct studies. of note, in the present study the values of fetal tracheo - bronchial angles were unpredictable in particular individuals. in fact, regression curves of best fit for every tracheo - bronchial angle against fetal age could not be computed. in patients aged 1785 years, the mean interbronchial and subcarinal angles averaged 7713 (range 49109) and 7316 (range 34107). furthermore, with relation to the subcarinal angle, the interbronchial angle was greater, smaller, or equal in 67%, 28%, and 5% of individuals, respectively. after presenting the morphometric analysis of tracheo - bronchial angles the subcarinal and interbronchial angles were found to correlate positively with the size of the left atrium of the heart. the tracheal bifurcation angle may be widened due to cardiac disease (left atrial enlargement, cardiomegaly, and pericardial effusion) and mediastinal abnormalities (subcarinal masses). however, the angle of tracheal bifurcation may be reduced after pulmonary lobectomy and lobar collapse. furthermore, there was a weak inverse correlation between the shape of thorax and the tracheal bifurcation angle. thus, as the thorax was shorter and wider, the angle of tracheal bifurcation increased. both major anatomical and clinical textbooks support that inhaled foreign bodies are more likely (70%) to enter the right main bronchus than the left because the former is wider and steeper, slightly deviating from the midline of the trachea [12,13,16,1921,30 ]. some authors emphasized the greater airflow through the right lung, and the left position of the carina.. stated that in 66% of children, especially under age 1 year, the carina was positioned to the left of the midline of the trachea. furthermore, kamel. found the carina to be situated on the left in up to 81.4% of individuals. the left - sided carina is reported to increase the catchment area of the right main bronchus. the tracheo - bronchial angles change independently of sex and fetal age. the left bronchial angle is wider than the right. values of the 3 tracheo - bronchial angles are unpredictable because their regression curves of best fit with relation to fetal age can not be modelled. the 2 bronchial angles and the interbronchial angle are of great relevance in the location of inhaled foreign bodies, and in the diagnosis cardiac diseases and mediastinal abnormalities. | backgroundboth the advancement of visual techniques and intensive progress in perinatal medicine result in performing airway management in the fetus and neonate affected by life - threatening malformations. this study aimed to examine the 3 tracheo - bronchial angles, including the right and left bronchial angles, and the interbronchial angle, in the fetus at various gestational ages.material/methodsusing methods of anatomical dissection, digital image analysis with an adequate program (nis - elements br 3.0, nikon), and statistics, values of the two bronchial angles and their sum as the interbronchial angle were semi - automatically measured in 73 human fetuses at the age of 1425 weeks, derived from spontaneous abortions and stillbirths.resultsno male - female differences between the parameters studied were found. the 3 fetal tracheo - bronchial angles were found to be independent of age. the right bronchial angle ranged from 11.4 to 41.8, and averaged 26.97.0 for the whole analyzed sample. the values of left bronchial angle varied from 24.8 to 64.8, with the overall mean of 46.28.0. as a consequence, the interbronchial angle totalled 36.296.6, and averaged 73.112.7.conclusionsthe tracheo - bronchial angles change independently of sex and fetal age. the left bronchial angle is wider than the right one. values of the 3 tracheo - bronchial angles are unpredictable since their regression curves of best fit with relation to fetal age can not be modelled. both of the 2 bronchial angles and the interbronchial angle are of great relevance in the location of inhaled foreign bodies, and in the diagnosis cardiac diseases and mediastinal abnormalities. |
mesenchymal breast neoplasms are rare. sarcoma is a heterogeneous neoplasm arising from mesenchymal cells. breast sarcomas are rare neoplasms derived from non- epithelial elements of the gland and represent 6 of 13 indicates that necrotizing fasciitis should be seriously considered. in this case, a score of 8 was allocated for serum sodium of 126 mmol / l, haemoglobin of 6.6 g / dl and c - reactive protein of 306.7 mg / l. her other biochemical markers were normal, including serum creatinine of 68 mol / l, plasma glucose of 8.8 mmol / l and a leukocyte count of 11.5 10/l. a provisional diagnosis of necrotizing fasciitis was made, and the patient was referred to the breast surgeons. the histopathology demonstrated that the breast was partially replaced by a partly cystic and partly solid necrotic lesion. a microscopic examination showed an atypical undifferentiated spindle cell lesion with a fascicular architecture, pleomorphism and marked mitotic activity with areas of ulceration and necrosis (figs 1 and 2). the initial set of immunostains performed showed that tumour cells were negative for cytokeratins (fig. 3), s100 and lca. the tumour cells showed diffuse and strong vimentin positivity (fig. further immunocytochemical labelling showed weak expression of cd99, patchy strong expression of cd10 and patchy weak expression of smooth muscle actin. cytokeratins (cam 5.2) and ema were expressed and there was no tumour - specific expression of cd34, desmin or myogenin. fluorescent in - situ hybridization analysis failed to demonstrate evidence of a t(x:18) translocation using the lys - syt break apart probe. the diagnosis of a grade 3 undifferentiated spindle cell sarcoma was made and although the tumour was reported to have a clear resection margin (5 mm) in most areas, the presence of necrosis and granulation tissue reaching the deep margin made the status of the margin uncertain. figure 1:photomicrograph 4 h&e ; spindle cell tumour with areas of haemorrhage and necrosis. figure 2:photomicrograph 40 h&e ; pleomorphic spindle cells in fascicles with several mitoses. computed tomography scan demonstrated left axillary lymphadenopathy and a small left - sided basal effusion. following discussion of the case at the multidisciplinary meeting, radical completion mastectomy was recommended. reconstruction was with a split thickness skin graft by the plastic surgery team prior to her radiotherapy treatment (fig. necrotizing fasciitis is a highly lethal infection that causes rapidly spreading necrosis of fascia and subcutaneous tissues, sometimes involving muscle and skin. treatment is with haemodynamic support of sepsis, broad spectrum antibiotics, intravenous immunoglobulin and extensive debridement. following debridement in this patient, sections from the specimen demonstrated an undifferentiated spindle cell sarcoma with areas of haemorrhage and necrosis, contrary to the initial presentation of presumed necrotizing fasciitis. the treatment for breast sarcomas is planned by a multidisciplinary team and follows the treatment model of sarcomas in other locations. localized tumours should be treated by complete excision, while mastectomy should be performed for more sizeable tumours. adequate surgical excision seems to be one of the most important prognostic factors along with the tumour 's diameter and grade. axillary dissection is unnecessary for breast sarcomas, as they rarely metastasize via the lymphatic system. while chemotherapy is the mainstay of treatment for advanced systemic disease, radiotherapy has a role in lymphatic metastasis and preventing loco - regional recurrence. the reported 5-year survival in patients with breast sarcomas varies between 40 and 91% in different studies. | breast sarcomas are rare neoplasms arising from the few epithelial elements of the gland. it represents much < 1% of all breast cancer. of the heterogeneous group of sarcomas, the more common subtypes include spindle cell sarcoma. the main risk factor for the development of breast sarcomas is previous radiation therapy following breast - conservation surgery for breast cancer or non - hodgkin 's lymphoma. we report on an idiopathic presentation of spindle cell sarcoma in an otherwise healthy middle - aged woman. an emphasis is made on the rare occurrence of lymphatic metastasis. we discuss our recommended management strategy with particular reference to the benefit of multidisciplinary team decision - making. |
address = administration of drotrecogin alfa (activated) in early stage severe sepsis ; apache = acute physiology and chronic health evaluation ; drotaa = drotrecogin alfa (activated) ; prowess = recombinant human activated protein c worldwide evaluation of severe sepsis. jf was involved with the conception and design of the study, acquisition, analysis and interpretation of data, and wrote the first draft of the manuscript. na was involved with the conception and design of the study, analysis and interpretation of data, and critical revision of the manuscript for important intellectual content. mm was involved with the interpretation of data and critical revision of the manuscript for important intellectual content. relative risk (rr) and 95% confidence intervals (95% ci) for 28-day mortality in each study. n / n = number of deaths at 28 days divided by the total number of patients randomly assigned to drotrecogin alfa (activated) or placebo. for the phase ii trial, only patients who were randomized to the same dose and duration of drotrecogin alfa (activated) used in the recombinant human activated protein c worldwide evaluation of severe sepsis (prowess) and administration of drotrecogin alfa (activated) in early stage severe sepsis (address) trials are included. results are classified by low - risk and high - risk subgroups from the recombinant human activated protein c worldwide evaluation of severe sepsis (prowess) and administration of drotrecogin alfa (activated) in early stage severe sepsis (address) trials, defined by either acute physiology and chronic health evaluation (apache ii) score (less than 25, and 25 or more) or organ failure (single and multiple). weight refers to the contribution of each study to the overall pooled estimate of treatment effect for each low - risk and high - risk subgroup. the weight of each study is calculated as the inverse of the variance of the natural logarithm of its relative risk. the size of the symbol denoting each point estimate approximates the weighting of each study to each pooled effect measure. each pooled effect measure is calculated with the use of a random - effects model. n / n = number of deaths at 28 days divided by the total number of patients in each particular subgroup randomly assigned to drotrecogin alfa (activated) or placebo. the numbers of patients and deaths at 28 days in each subgroup were estimated from data provided in references, and for the prowess trial, and in references and for the address trial. | two international multicentre randomised controlled trials of drotrecogin alfa (activated) (drotaa), the recombinant human activated protein c worldwide evaluation of severe sepsis (prowess) and administration of drotrecogin alfa (activated) in early stage severe sepsis (address) trials, have produced inconsistent results. when 28-day mortality data from these trials for patients with severe sepsis and at high risk of death are pooled using a standard random - effects meta - analysis technique, there is no statistically significant survival benefit (for patients with acute physiology and chronic health evaluation (apache ii) scores of 25 or more), or a borderline significant benefit (for patients with multi - organ failure). we argue that two important methodological issues might explain the disparate results between the two trials. these issues centre on early trial stopping, which exaggerates treatment effects, and reliance on subgroup analyses, which for drotaa yields inconsistent results across different definitions of high risk. these concerns call into question the effectiveness of drotaa in any patients with severe sepsis. consequently, further randomised trials of this agent in prospectively defined high - risk patients are required to clarify its role in the management of severe sepsis. |
globus pharyngeus, the sensation of something stuck in the throat, has been noted since the time of hippocrates. purcell first used the term globus hystericus in the early 18th century. in 1968, malcomson suggested the term globus pharyngeus as a more accurate description since not all patients with globus were either hysterical or female. typically, globus is relieved by ingestion of solids or liquids and tends to be worse on dry swallows. globus may be associated with throat irritation, soreness, dryness, catarrh, or constant throat clearing. it forms a large part of ent practice and may account for about 4% of referrals to our outpatient clinics. the prevalence is much higher in the general population as most people may not present to hospital with it. a recent study by ali and wilson found that up to 78% of patients presenting to non - ent clinics had had globus - type symptoms. despite the high prevalence in the community, the aetiology of globus remains unclear and highly controversial. it is slowly being accepted that it may be multifactorial and that when it occurs in isolation it rarely hides any sinister pathology. most of the recent work has suggested several mechanisms in isolation or not uncommonly in combination are to blame for the manifestation of globus pharyngeus ; these include psychological factors, gastro - esophageal reflux (gor), pharyngeal dysmotility, hypertonic upper oesophageal sphincter (uos), and local anatomic abnormalities [611 ]. as its earlier name, globus hystericus, suggests, there has been a long history of links between globus and psychological factors. it is the fourth most discriminating symptom of a somatisation disorder after vomiting, aphonia, and painful extremities. as most of the globus patients are quite rightly referred to ent surgeons rather than to psychiatrists, a psychogenic basis must always be borne in mind. gale. in a detailed medical and psychological examinations including assessment with the minnesota multiphasic personality inventory (mmpi) of 4240 us this globus group scored higher in nine out of ten of the mmpi clinical scales. they concluded that in men there is a significant link to depression and somatization disorder and as a result other related treatable psychopathology should be investigated. when comparing globus patients with other ent patients (as a control group) found that globus patients had had more severe life events in the year and less confiding relationships than controls. the link between gor and globus has been a matter of controversy for over forty years. they found that 66.6% of the nonreflux globus group and 80% of the gor globus group had significant episodes of reflux (based on ph monitoring). in direct contrast, chen. in a similar study found no evidence of reflux in globus patients based on ambulatory ph monitoring. anandasabapathy and jaffin using multichannel intraluminal impedance and ph monitoring (mii - ph) have suggested that globus may also be due to nonacid (nar) reflux. as mii - ph can detect reflux episodes independent of acid changes, it is allegedly more accurate at picking up proximal reflux. based on porcine models, pepsin has been shown to increase the levels of laryngeal protective proteins and thus explain the nar link. even though low ph is needed to activate pepsin, its stability means that it may be activated intracellularly or when the larynx is later exposed to acid. hypertonicity of the uos has been suggested as a cause of globus, but several studies have yielded conflicting results. it has long been recognised that the uos pressure profile is asymmetrical, especially when using multilumen catheters. therefore, uos pressure measurements obtained using circumferential transducers are regarded as being more reflective of true intraluminal pressure. sun. looked at twenty - four healthy volunteers and thirty - two patients with globus and found uos pressure to be normal in most of the globus patients and could not suggest it as a possible aetiological factor. interestingly they found that videofluoroscopic evidence of pharyngeal dysfunction especially laryngeal penetration had a strong association with globus., however, showed that perfusion of hcl into the distal oesophagus was related to a sensation of globus associated with a rise in uos pressure. this rise in pressure was independent of the detection of a rise in ph in the hypopharynx. recently there have been reports of very subtle changes in anatomy that when rectified have given relief of globus. the definition of a retroverted epiglottis is if the tip touches the tongue base when the tongue is protruded. ulug and ulubil have presented a case of corniculate cartilage subluxation presenting with globus. other postulated causes include eagles syndrome (calcified stylohyoid ligament), impalpable thyroid nodules, cervical osteophytes, lingual tonsils, or prominent greater cornu of the hyoid. gastric inlet patches have also been aetiologically linked to globus [19, 20 ]. these are congenital islands of ectopic gastric mucosa found in the cervical oesophagus. with the incidence of gastric inlet patch being quite common (3.6%) have gone further to suggest that it is h. pylori infection of the inlet patch that causes altered cervical perception and hence globus. what is worrying about this is that these patches have been associated with both squamous cell carcinomas and adenocarcinomas of the upper oesophagus [19, 22 ]. more interestingly though shiomi. looked at the mucus in the epipharynx of patients with globus and compared it with that from healthy volunteers, they found that there were significantly increased concentrations of fucose and sialic acid (the main determinants of mucus viscosity) in the mucus of those with globus as compared to normal subjects. lastly, though there is no evidence to suggest this, some ent surgeons believe that globus may simply be a local sensory abnormality just like tinnitus. as with all our patients, the key is in taking a proper history. pointers that would suggest sinister underlying pathology would include dysphagia, aspiration, regurgitation, weight loss, voice change, and pain. the presence of overt symptoms of gor should be noted. the head and neck should be thoroughly examined. this should include transnasal fibre - optic laryngoscopy (fol) or if available transnasal flexible laryngooesophagoscopy (tno). any further investigation should be based on the findings at history and examination. in ent departments in the uk, contrast swallows are the most popular radiological investigations used to investigate globus, with some departments historically using them to screen patients for upper aerodigestive tract malignancy [23, 24 ]. they have been favoured because they are safe (compared to rigid endoscopy), quick, and believed to increase diagnostic yield. one of the authors (rph) retrospectively reviewed a series of 1275 patients that had barium swallows. six hundred and ninety - nine patients had globus and 451 of these patients had globus without sinister symptoms. in these patients, barium swallows did not show any sinister pathology. another review of barium swallows by hajioff and lowe looked at 2854 barium swallows from two centres, and of the 2011 patients that presented with globus, none had a worrying abnormality on barium swallow. only one retrospective case series has found an association between isolated globus and hypopharyngeal cancer. two cases out of twenty - three cases were retrospectively found to have malignancies (a piriform fossa and postcricoid tumour). more recent and larger studies have failed to make a similar association. in the light of the previously mentioned we do not recommend barium swallows routinely for globus. the diagnostic yield for malignancy is poor though it may reassure the patient. the main drawback of this is that flexible oesophagoscopy often requires sedation, while rigid endoscopy requires a general anaesthetic and carries a small but significant risk of perforation. lorenz. carried out flexible endoscopies on patients that had been referred by ent for further investigation of globus, and all of the patients had had a normal outpatient ent examination and barium swallow. 62.7% of the patients were found to have pathology that could possibly have caused their globus though no sinister pathology was noted. similarly, nagano. in their study found a 36.5% incidence of benign oesophageal pathology in patients with globus on flexible endoscopy, but again no malignancies were identified. the most common recorded anomalies were cricopharyngeal spasm (4.8%) and reflux (4.4%). no tumours were found, and they concluded that rigid endoscopy played a limited role in the investigation of globus. it combines the main advantages of both conventional flexible and rigid oesophagoscopy with none of the major disadvantages. there is total examination of the upper digestive tract down to the stomach with the ability to take biopsies at the same time. it has been shown to be safe with a high patient satisfaction rate. though tno is not yet routinely available in the uk, we think that it is the ideal investigation for those ent surgeons who want a relatively safe, cheap, and quick way of visualising the upper digestive tract especially the hypopharynx and postcricoid regions. where tno is available, we eagerly await studies comparing the diagnostic yield of tno to that of rigid oesophagoscopy. despite the controversies, a large number of uk ent surgeons believe that reflux plays a role in globus. many of us do not use any scores or indices in our assessment of patients with globus. the reflux symptom index and the reflux finding score are not particularly valid diagnostic tools when used in globus patients. the glasgow edinburgh throat score (gets) has been validated for use in globus but is not widely used. because of the benign nature of globus, we rarely ever ask for ph or impedance studies in our patients. where there is uncertainty about the aetiology there will be uncertainty about the management. if patients have overt signs or symptoms suggestive of reflux in addition to globus, we would treat them aggressively with a proton pump inhibitor (ppi) twice daily and a reflux suppressant for at least 4 months. a study from the cleveland clinic using a regimen similar to ours has been found to be effective in controlling the symptoms of laryngopharyngeal reflux (lpr). most of the ent surgeons in the uk seem to be prescribing sub - optimal doses of ppis. in cases where there is globus but with no evidence of gor two recent meta - analyses of the role of ppis in reflux related laryngeal disease have shown little or no benefit over placebo [36, 37 ]. they both recommend that more studies are required to define the subgroup of patients that will benefit from ppis. a few trials have shown that globus symptom scores do improve after a course of speech therapy [38, 39 ]. what is not clear from these studies is whether there is a specific effect from speech therapy or if improvement is due to increased reassurance. hypnotically assisted relaxation (har) therapy has also been reported in a recent case series to improve globus sensation regardless of the cause. manometric uos readings in the patients showed no change before and after har. in cases where there are anatomical anomalies, the trend seems to be excision of the offending local structure, most often some part of the cartilaginous framework of the larynx [9, 10 ]. these results have to be viewed with caution as the numbers are small with short follow - up intervals. we must also remember to assess the whole patient and make referrals to the psychiatrists where it is indicated. therefore in most cases of globus, if the history and examination of the patient suggest no sinister pathology, then reassurance is often enough. rowley showed that at 7 years about 55% of patients were asymptomatic and none had developed an upper aerodigestive tract malignancy. at the present, we do not recommend any further radiologic or endoscopic examination for the patient with isolated globus. a complete head and neck examination including fibreoptic laryngoscopy is more than adequate to confidently discharge the classic globus pharyngeus patients. the introduction of tno in one stop globus clinics has meant that with appropriate training otolaryngologists can nowadays and in selected cases complete a thorough upper aerodigestive tract examination, thus avoiding the need for any other investigations such as barium swallows or oesophagoscopies under general anaesthesia. overinvestigating these patients can often add unnecessary stress to a group of patients who already seem to have higher levels of depression, anxiety, and other somatic concerns. in fact the authors believe that both barium swallow and panendoscopy under ga are things of the past and should not form part of the standard globus assessment. more research needs to be carried out into the aetiology, treatment, and long - term prognosis of persistent globus. | globus pharyngeus is a common ent condition. this paper reviews the current evidence on globus and gives a rational guide to the management of patients with globus. the aetiology of globus is still unclear though most ent surgeons believe that reflux whether acidic or not plays a significant role. though proton pump inhibitors are used extensively in practice, there is little evidence to support their efficacy. most patients with globus can be discharged after simple office investigations. the role of pepsin - induced laryngeal injury is an exciting concept that needs further study. given the benign nature of globus pharyngeus, in most cases, reassurance rather than treatment or extensive investigation with rigid oesophagoscopy or contrast swallows is all that is needed. we need more research into the aetiology of globus. |
metastatic cardiac involvement is reported in the literature highly variable, ranging from 2.3 to 18.3% in different reported studies. tumors can spread to the heart through direct extension of extra cardiac neoplasm, by bloodstream or lymphatic system diffusion and by intracavitary invasion through both the superior and inferior vena cava or pulmonary vessels. lung cancer is one of the most common metastatic neoplasms to the heart, usually associated with malignant pericardial effusion. the presence of cardiac involvement is often only accidentally detected, prognosis is poor and therapeutic options limited. non - invasive imaging methods are available for evaluation of suspected cardiovascular complications of malignancy. in may 2012, a 52-year - old caucasian woman, with no significant past cardiovascular history and risk factors, was admitted to the department of medical oncology because a computed tomography (ct) scan, performed 1 month ago for worsening cough and fever, revealed a heterologous neoformation occluding the bronchial branch of the upper lobe, with extension at the parietal pleura, involvement of periaortic and precarenal lymph nodes, and another hypodense formation of about 2 cm with hyperdense rib in correspondence of the right supraspinatus muscle. bronchoscopy also, with bioptic sample of the mass, confirmed the diagnosis of bronchial spinocellular carcinoma, p63 +, thyroid transcription factor-1 (ttf-1). on physical examination, were found two painful and non - mobile swellings localized in the right forearm and in the right scapula compatible with repetitive lesions, but not appreciable superficial pathological lymph nodes. the overall clinical condition of the patient were discrete and karnofsky performance status (kps) of 80/90. this clinical aspect refers to advanced clinical stage iv (ct3, cn2, and m1) with a poor prognosis, therefore the patient was not considered candidable for surgery, but it was considered appropriate to perform a chemotherapy scheme with carboplatin area under the time - concentration curve (auc) 6 g1 + paclitaxel 175 mg / m g1, every 21 days, and, in reason of the symptoms, also a radiotherapy treatment. in june, after the second cycle of chemotherapy treatment, the patient was admitted to emergency care unit for syncope associated with sphincters incontinence and nosebleed. electrocardiographic examination (ecg) revealed a sustained ventricular tachycardia (vt) ; the sinus rhythm was restored firstly with electrical shock of 150 j and then with amyodarone intra - venous infusion. the patient was discharge with clopidrogrel 75 mg / day, ramipril 5 mg / day, and bisoprololo 2.5 mg / day. a ct evaluation showed a progression of the disease with infiltration of the chest wall, mediastinal pleura and heart chambers [figure 1 ]. it was decide to administer a second line of chemotherapy scheme with vinorelbine 25 mg / mq g1, g8, and g15 every 21 days, started in july. the arrows show the infiltration of left ventricular lateral wall and apex after 1 week of beginning the treatment, the patient presents chest pain and she was referred to the cardiology department. at clinical examination she was eupnoic, with a normal physical examination ; ecg revealed sinus tachycardia but with st segment elevation in leads di and avl [figure 2a ] and poor progression of r wave in leads v1-v4 [figure 2b ]. two- and three - dimensional transthoracic echocardiography (2d/3d tte) showed myocardial involvement with infiltration of the anterolateral left ventricular (lv) wall [figure 2c ] by the side of mass extending from the epicardial to the endocardial layer, with apical hypokinesia and reduction of the lv ejection fraction (lvef, 45%) ; 3d global longitudinal strain [figure 2d ], obtained from the triplane cine - loops using the automated functional imaging (afi) modality, was reduced (average 11.9%) especially at posterior wall ; there was mild mitral insufficiency, but was absent pericardial effusion. (a and b) electrocardiographic examination (ecg) : st segment elevation in di and avl leads (arrows) and r - wave poor progression v1-v4 leads. (c) two- and three - dimensional transthoracic echocardiography (2d/3d tte) : anterolateral left ventricular wall and apex infiltration. (d) a lesser 3d global longitudinal strain at posterior (post), lateral (lat), and aterior (ant) wall under the impression of acute coronary syndrome we initially intent to perform emergency coronary angiography. however, the cardio - oncology team resolved to not perform the invasive cardiac procedure or coronary ct in view of the progressive decline in global performance status, of the serially followed cardiac enzyme levels within their normal range and also because the coronary ct do not take advantages for diagnostic approach in this clinical contest, the patient was already exposed to elevated ionizing radiation and, in order to evaluate the progression of disease, she could be exposed to other radiation. it was decide to start the third - line of erlotinib, an oral epidermal growth factor receptor tyrosine kinase inhibitor (egfr - tki),150 mg / die, reduced to 100 mg daily after 1 month because of severe skin toxicity. three months from the beginning of erlotinib, the ecg revealed a sinus rhythm with depth of negative t wave in leads di and avl [figure 3a ] and poor progression of r wave in leads v1 and v3 [figure 3b ], but without st elevation. at this time 2d/3d - tte showed a mild regression of the lv lateral wall infiltration [figure 3c ] and a slightly improvement of lvef (50%). global and regional strain [figure 3d ] increased (average 14.6%) ; the akinesia of the ventricular wall was unchanged. a ct scan showed partial remission of the primary lung lesion, of intracavitary and intramyocardial mass and an assessment of the size and morphology of the cardiac chambers [figure 4 ]. we subsequently performed egfr mutation analysis that showed single - point substitution mutation l858r in exon 21, specific types of activating mutations that confer sensitivity to egfr - tkis. (a and b) negative t wave in di and avl leads (arrows), but without st elevation and r - wave poor progression v1-v3 leads. (c) 2d/3d tte : mild regression of the left ventricle lateral wall infiltration. (d) recovery of 3d global longitudinal strain thorax ct scan at 3 months from the beginning of chemotherapy. lung cancer is one of the most common metastatic neoplasms to the heart, usually associated with malignant pericardial effusion. clinical manifestations are extremely variable ; some common presentations of cardiac metastases include atrial and ventricular tachyarrhythmia or premature beats, conduction disorders and blocks, syncope and sudden death as result of lv metastatic infiltration causing episodes of vt or lv outflow tract dynamic obstruction, myocardial dysfunction, angina pectoris, and myocardial infarction as result of coronary embolism, but also of invasion or compression of a coronary artery. when a cardiac metastasis is diagnosed antemortem, signs and symptoms of the primary cancer are usually the presenting features, the presence of cardiac involvement is often only accidentally detected, prognosis is poor, and therapeutic options limited. our patient also exhibited findings suggestive of myocardial ischemia with a previous episode of vt, but without pericardial effusion. the explanations for the ecg findings are the probably occlusion of the circumflex epicardial artery by the neoplasm infiltration, tumor microembolization occluding minor branches of myocardial vessels, expression of myocardial lesions currents, or coronary artery disease (cad) causing myocardial infarction. we could not exclude the presence of cad because the patient did not undergo coronary angiography mainly for ethical reasons. however, considering the clinical context and the temporal duration of ecg alterations, the presence of cad was very unlikely. according to lim, previous vt episode might also be related to focal myocardial ischemia, but we rather think that it is mainly due to spatial heterogeneity of refractoriness or localized conduction delay caused by direct invasion mass. after treatment with erlotinib, the patient showed a significant clinical improvement and we observed a reduction in lung carcinoma and myocardial involvement. we conclude that the noninvasive strategy in this context was more suitable for patient s quality life. noninvasive imaging methods are useful for evaluation of suspected cardiovascular complications of malignancy and to guide proper management of these patients. | a 52-year - old woman with a lung carcinoma metastatic to bone was admitted to the cardiology department for acute chest pain after 1 week of the oncologic therapy. electrocardiographic examination (ecg) revealed ischemic picture with st - t wave abnormalities in di and avl leads and poor progression of r wave in v1-v4 leads. two- and three - dimensional transthoracic echocardiography (2d/3d tte) showed myocardial involvement with infiltration of the anterolateral left ventricular (lv) wall from the epicardial to the endocardial layer, apical hypokinesia, lv ejection fraction (lvef) and global 3d longitudinal strain reduction, but was absent pericardial effusion. three months from the beginning of erlotinib, the patient showed a significant reduction in myocardial involvement with no ecg - st elevation. echo showed a mild regression of the wall infiltration and a slightly improvement of lvef and strain. a computed tomography (ct) scan showed partial remission of the primary lung lesion, intracavitary and intramyocardial mass. |
c - reactive protein (crp) is an inflammatory marker that raises in inflammation that occurs due to smoking. people with increased levels of crp are on higher risk to develop obesity, diabetes, cardiovascular diseases, and hypertension. it has been experienced that the smoker males had increased levels of serum crp as compared to nonsmokers. it has been observed that people with altered blood pressure and obesity has affected vascular endothelial function ; it occurs due to hypomagnesaemia and increase in plasma levels of crp in cellular processes. healthy smokers have more insulin resistance which is a characteristics of metabolic syndrome as compared to nonsmokers. it is also seen that they have also increased glucose levels, decreased high density lipoproteins (hdl) cholesterol, and much more very low - density lipoproteins (vldl) cholesterol. since these variables are not direct measures of magnesium levels in smokers even then they clearly show that smoking decreases cellular magnesium concentration. the purpose of present study was to find out the inverse relation between serum levels of crp and magnesium in smokers. systemic acute phase reaction occurs due to inflammation, different studies calculated the level of circulating crp which shows the severity of inflammation. the major initiator, interlukin-6, released by hepatocyte increases the synthesis of crp in acute phase reactants. in this case - control study, we took 192 healthy subjects including 96 smokers and 96 nonsmokers (age range 20 - 40 years). a written consent was obtained from every subject who was involved in our study before taking blood sample. the serum levels for crp and magnesium were determined by standard methods using nycocard and diasys kits, respectively. we included all the patients, who were smokers but healthy, having no any associated diseases like chronic obstructive pulmonary disease (copd), obesity, angina, or myocardial infarction. spirometry was not done, and we have ruled out any pathology concerned with lungs by history and symptoms, like they have no cough, tuberculosis (tb), or any associated diseases. statistical analysis was restricted by the number of cases over a period of 18 months, but sample size was not determined. comparisons obtained in present study were presented as mean standard deviation (sd). comparisons of mean values between smokers and nonsmokers were done by using student 's t - test. statistical analysis was restricted by the number of cases over a period of 18 months, but sample size was not determined. comparisons obtained in present study were presented as mean standard deviation (sd). comparisons of mean values between smokers and nonsmokers were done by using student 's t - test. table 1 shows the inverse relationship between serum crp and magnesium levels in smokers and nonsmokers. the data shows that the mean serum crp concentration in smokers (14.62 0.16 mg / l) was significantly higher (p < 0.001) [figures 1 and 2 ]. comparison of serum c - reactive protein and magnesium levels between smokers and nonsmokers correlation between serum c - reactive protein (crp) and magnesium (mg) in smokers correlation between serum c - reactive protein (crp) and magnesium in nonsmokers similarly, the mean serum magnesium concentration in nonsmokers (2.52 0.18 mg / l) was significantly higher (p < 0.001). surprisingly, serum magnesium levels in all smokers were found to be less than 1.8 mg / dl, while serum crp levels higher than 10 mg / l. on the contrary, all nonsmokers had their serum magnesium concentration greater than 2.0 mg / dl and serum crp concentrations less than 7.5 mg / l. the crp to magnesium ratio in serum samples of smokers ranged between 0.73 and 5.0 as against 0.12 and 0.29 in nonsmokers, and it was significantly higher (p < 0.001) in smokers as compared to nonsmokers [table 2 ]. this difference in the range of ratio clearly shows that decreased serum magnesium levels are independently related to high crp levels in smokers [figure 3 ]. comparison for c - reactive protein (crp) to magnesium (mg) ratio between smokers and nonsmokers correlation between mean levels of serum c - reactive protein (crp) and magnesium (mg) in smokers vs nonsmokers table 3 shows the analysis of two variables, that is, mg and crp are highly negatively related with each other, which means that when one variable increases other will decrease and vice versa, the analysis also shows the strength of relation between two variables. the analysis also shows the strength of the relation between two variables. in this table, the strength is (0.764), which shows that both variables are strongly negatively related and the results are significant at p < 0.001. pearson correlation between serum c - reactive protein (crp) and magnesium levels in smokers and nonsmokers table 4 shows the normal range of body mass index (bmi) in both cases and control ; hence, there is no obesity seen in both the groups.table 4comparison of age and body mass index (bmi) between healthy adult male smokers and nonsmokers comparison of age and body mass index (bmi) between healthy adult male smokers and nonsmokers in our study we have found that there is significantly increased level of serum crp, which is supported by das., observed the early outcomes of magnesium deficiency in rats during inflammatory response ; similarly, the present study also shows the deficiency of magnesium in inflammatory response. the finding of the present study that smokers have increased level of crp with decreased level of magnesium is fully supported by khand. guerrero - romero., found that activated state of immune cells is the result of hypomagnesemia in nondiabetic, nonhypertensive, obese subjects ; this also supports the present study., that individuals with low intake of magnesium were more likely to have increased crps. the present study clearly demonstrated that smoking significantly decreases serum magnesium concentration and increases serum crp concentration resulting in an inverse relationship between serum crp and magnesium concentrations in smokers only., that magnesium intake is inversely related with the crps in plasma for the prevalence of metabolic syndrome. according to another study of mendenhall., smoking, cardiovascular disease, and bmi have a great association with crps and we also found similar findings in our results with smoking., justified our study in way that crp have an association with the bmi, cardiovascular diseases, and smoking ; this study favors our results, but does not explain the inverse relationship between magnesium and crps. | background : smoking plays a key role in increasing the inflammatory marker c - reactive protein (crp).aims : to examine inverse correlation between crp and magnesium levels in smokers and nonsmokers.materials and methods : a total of 192 healthy adult male subjects were included in the present study, out of which 96 were smokers and the remaining 96 were nonsmokers having age range from 20 to 40 years, and all the subjects belonged to district matyari of hyderabad. serum crp was measured by nycocard standard kit method and magnesium levels by diasys standard kit method in smokers and nonsmokers.results:the levels of serum crp in smokers (14.62 0.16 mg / l) is high as compared to nonsmokers (4.81 0.38 mg / l), which is highly significant (p < 0.001). however, inverse results were seen for serum magnesium levels which were significantly higher (p < 0.001) in nonsmokers (2.52 0.18 mg / l) as compared to the smokers (1.09 0.38 mg / dl). a significant (p < 0.001) inverse relationship between serum crp and magnesium concentrations were seen in smokers.conclusion:this result shows that smoking increases serum crp, an inflammatory marker parallel to decrease in serum magnesium levels in smokers having 20 - 40 years of age. |
a 73 year old man, 158 cm and weighing 52.8 kg, was scheduled for an emergency repair of a type - a aortic dissection and concomitant coronary arterial bypass grafting. hypertension and a transient ischemic infarct (tia) that occurred 3 years ago were under conservative treatment. he had undergone aortic valve replacement surgery for severe aortic stenosis 26 years earlier, and also had had percutaneous coronary stents inserted in the left circumflex artery and right coronary artery 10 years earlier for coronary stenosis. in a preoperative review of his chest computed tomography, dissection from the sinotubular junction of the ascending aorta to the aortic arch with a large intimal defect was observed. stenotic lesions requiring concomitant coronary arterial bypass grafting were discovered at the initial coronary aorta ct scan. although a coronary angiogram can be a risky procedure in an aortic dissection patient, his vital signs were stable and it was necessary to evaluate a more accurate range of the stenotic lesions. 50% left circumflex artery occlusion and total occlusion of the right coronary artery was confirmed with a coronary angiogram. duplex sonography in the carotid artery showed 400,000 platelets / mm) and leukocytosis (> 120,000/mm) suggested that he had polycythemia vera. in the operating room, monitoring included a five - lead electrocardiogram with continuous st segment analysis, pulse oximetry, non - invasive blood pressure, arterial blood pressure with left radial arterial catheter, bispectral index, and a cerebral oximetry. initial vital signs, checked in the operating room, were stable with a blood pressure of 130/66 mmhg and a heart rate of 85 bpm. anesthesia was induced and maintained with a continuous infusion of propofol, remifentanil, and cisatracurium. following endotracheal intubation, transesophageal echocardiography was performed to evaluate cardiac function and to guide the hemodynamic treatment. a triple - lumen catheter with introducer (advanced venous access, edward lifescience, irvine, ca, usa) was placed in the right internal jugular vein by a sono - guided technique, and a pulmonary artery catheter (swan - ganz catheter, edward lifescience, irvine, ca, usa) was also inserted. intraoperative baseline arterial blood gas analysis showed a hematocrit level of 61% and a hemoglobin level of 20.6 g / dl. to perform acute normovolemic hemodilution (anh), 1.5 units (650 ml) of whole blood was drawn and replaced with 650 ml of 6% hydroxyethylstarch solution (he s, volulyte, fresenius kabi, deutchland gmbh, friedberg, germany) for 30 minutes. during the procedure, his hemodynamic condition was stable, and following the procedure, the hematocrit level was 50%. we also performed rotational thromboelastometry (rotem, pentapharm gmbh, munich, germany) analysis, and the results were within the near - normal ranges with the exception of a hypercoagulable state in some results (table 1). the patient underwent a bentall operation, hemi - arch replacement, and coronary artery bypass grafting under cardiopulmonary bypass. during the hemi - arch replacement, there was deep hypothermic circulatory arrest for 41 minutes and during that period, icepacks were applied around the patient 's head, 500 mg of intravenous pentothal sodium was injected, and cerebral oximetry values were monitored continuously (fig. 1). while attempting to wean from the cardiopulmonary bypass, his hematocrit level was 43% (hemoglobin 14.5 g / dl) and in the rotem analysis, the results of extem showed normal values. the results of fibtem showed a slightly higher amplitude 20 minutes after the start of assay (a20), and maximal clot formation (mcf) in the upper normal range indicating a hypercoagulable state. at the end of surgical wound closure following protamine reversal, surgeons complained that there was a lot of microvascular bleeding, inconsistent with our rotem analysis. even though the hematocrit was 29% and our transfusion guideline was not satisfied, two units of packed rbcs, 10 units of cryoprecipitate, 10 units of platelets, and two units of fresh frozen plasma were transfused. the rotem analysis at the end of the surgery were within normal ranges. during the whole operation period, the values of cerebral oximetry on both hemispheres were shown to be above 60%, and for fluid management during the operation, we used crystalloid, with the exception of 650 ml colloid for anh. following transfer to the intensive care unit, his mental recovery was delayed until postoperative day 1, and the postoperative brain mri showed an acute ischemic embolic infarct in the right basal ganglia. after starting heparinization, his neurological problems resolved fully, he was discharged from the hospital on postoperative day 13 with no neurological sequelae and without any postoperative bleeding problems. polycythemia vera is a chronic myeloproliferative disorder characterized by excessive red blood cell production, and is more common in men, with a prevalence of 4 - 16/million. in patients with polycythemia vera, increased circulating red blood cells are associated with hyperviscosity of the blood, which can lead to an increased risk of thrombotic complications such as cerebrovascular accidents, myocardial infarction, or peripheral vascular events. moreover, approximately half of the patients with polycythemia vera also have proliferation of all blood cell lines, causing thrombocytosis and leukocytosis. in a previous survey relating to postoperative outcomes in patients with polycythemia vera, an increased risk of both vascular occlusive (7.7%) and hemorrhagic (7.3%) complications abnormal platelet function such as decreased platelet factor 3, reduced platelet adhesiveness, and abnormal platelet aggregation, may in fact be associated with a paradoxical bleeding risk. in order to be positively diagnosed with polycythemia vera, two major criteria and one minor criterion or the first major criterion together with two minor criteria are required. major criteria include 1) hemoglobin > 18.5 g / dl in men, > 16.5 g / dl in women, 2) presence of the jak2 v617f or similar mutation. minor criteria include 1) bone marrow biopsy showing myeloproliferation, 2) serum erythropoietin level below the normal range, and 3) endogenous erythroid colony formation in vitro. with polycythemia vera having bleeding tendency, it is necessary to distinguish this condition from secondary polycythemia. it seems likely that this patient suffered from secondary polycythemia, because he had chronic hypoxia. in our case, primary polycythemia vera was suggested from the patient 's preoperative hemoglobin and hematocrit levels of 20.4 g / dl and 61.8%, respectively, and from the results of his blood tests such as thrombocytosis (> 400,000 platelets / mm), leukocytosis (> 120,000/mm), positive jak gene mutation, and a low level of serum erythropoietin. several perioperative pretreatments can be considered to prevent perioperative thromboembolic complications in patients with polycythemia vera, such as the use of low - dose heparin, a course of weekly phlebotomy to reduce hematocrit to below 45%, or intraoperative acute normovolemic hemodilution, extracting blood that is simultaneously replaced by colloids until the hb is reduced to 14.5 g / dl with a hematocrit level of 45%. in this case, as the patient had an emergency operation, anh was the only available technique. our target hematocrit level after anh (50%) was slightly higher than recommended, due to the patient having concomitant ischemic heart disease and several risk factors for post - procedural bleeding, such as the use of a large dose of heparin for the cardiopulmonary bypass, re - sternotomy, and deep hypothermic circulatory arrest that is usually associated with decreased coagulation factor activation, platelet dysfunction, and excessive fibrinolysis resulting in a bleeding diathesis. anh extracted volume (650 ml) was calculated as followed v = ebv (ho - hf) / hav, initial hematocrit (ho, this patient ; 60%), minimum allowable hematocrit (hf, this patient ; 50%), average hematocrit (hav, this patient ; 55%), estimated blood volume (ebv, this patient ; 50 kg 70 ml / kg), volume (v). there have been a few reports of intraoperative anh in polycythemic patients undergoing cardiovascular surgery with or without cardiopulmonary bypass. reported a case of off - pump coronary artery bypass grafting in a polycythemic patient having preoperative hemoglobin of 20.1 g / dl and hematocrit of 60.3%. they performed anh through the venous sheath (650 ml of 6% he s colloid was used as replacement fluid after 2 units of blood) and hemoglobin was reduced to 16 g / dl. reported a polycythemic patient with preoperative hematocrit of 51% undergoing aortic dissection repair, and intraoperative anh was performed to reduce the hematocrit level to 44%. although anh should be applied with caution in patients with ischemic heart disease, some previous studies reported its protective effects in patients undergoing coronary arterial bypass grafting. licker. demonstrated that anh has cardio - protective effects by lowering blood viscosity in patients undergoing coronary artery bypass surgery with aortic cross clamping. lowered blood viscosity can improve coronary arterial perfusion, cardiac output, higher oxygen extraction ratios and oxygen delivery to the underperfused myocardial area. recently, we have been reluctant to use he s colloid in cpb, depending on the operation, due to raised concerns about the increasing bleeding risk. however, he s colloid was used at a relatively low risk in hypercoagulable status of pv. the thromboelastographic pattern in polycythemia vera shows minute changes in reaction time and maximal amplitude and a significant increase in the alpha angle, indicating a hypercoagulable state. in our case, we performed rotem analysis three times during the operation, and the majority of the results were near - normal and close to baseline (after anh) showing a hypercoagulable state (prolongated clotting time, clot formation time) consistent with previous reports. however, the patient suffered from microvascular bleeding of the surgical field after weaning from cardiopulmonary bypass, which is inconsistent with our rotem analysis. this may suggest that although the amounts of fibrinogen and platelets are increased in a polycythemia vera patient, platelet function may have been deficient, and thus associated with paradoxical bleeding. our limitation was the platelet function test, as the platelet count was not evaluated during the operation or postoperatively. the actual cause of the event could not be determined, however there were several related risk factors for postoperative cerebral accidents, such as the use of cardiopulmonary bypass, deep hypothermic circulatory arrest, and the presence of polycythemia. fortunately, the patient was discharged from the hospital with no apparent neurological sequelae, and there has been no further event during the postoperative follow - up period. in conclusion, thus, the results of conventional coagulation tests or rotem analysis could be used carefully with consideration of the actual clinical situation in a patient with polycythemia vera. | polycythemia vera is a chronic progressive myeloproliferative disease characterized by increased circulating red blood cells, and the hyperviscosity of the blood can lead to an increased risk of arterial thrombosis. in a previous survey regarding postoperative outcomes in polycythemia vera patients, an increased risk of both vascular occlusive and hemorrhagic complications have been reported. aortic surgery involving cardiopulmonary bypass may be associated with the development of a coagulopathy, and as a result, the occurrence of thrombotic complications should be avoided after coronary anastomosis. thus, optimizing the hemostatic balance is an important concern for anesthesiologists. however, only a few cases of anesthetic management in polycythemia vera patients undergoing concomitant aorta and coronary arterial bypass surgery have ever been reported. here, we experience a polycythemia vera patient who underwent an emergency repair of a type - a aortic dissection and concomitant coronary artery bypass grafting, and report this case with a review of the relevant literature. |
leprosy is a disease that shares some characteristics with multiple skin disorders ; the early stage of morphea is one of those disorders with not only clinical but also histopathological similarity. through the history, the true diagnosis of leprosy has always been a challenge, not only for the multiple diseases that shares some clinical features but also because the similarity of a few in the histopatological aspect. we report in here a case of morphea showing very similar characteristics in the clinical and histopatological findings with paucibacillary leprosy, we also discuss the different aspects between the two entities and finally we focus in an atypical pattern of infiltration in morphea. a 24 yr - old male presented to our dermatology department complaining of hypochromic plaques in his abdomen and neck. in november of 2009, he consulted for a solitary oval hypochromic plaque with reddish - edge and smooth surface in the abdomen with absent of hair inside the plaque [figure 1 ]. oval hypocromic plaque with lilac - edge and smooth surface in the abdomen with absent of hair inside the plaque. notice the scar of the first biopsy done from the edge of the lesion a biopsy was performed in the edge of the lesion under suspicion of paucibacillary leprosy. perineurovascular and intersticial infiltrate predominantly lymphocytic, with linear arrays between the collagen bundles (h and e, 100) the patient was diagnosed with paucibacillary leprosy and treatment was established. the patient received treatment for five months with rifampicin 600 mg / monthly and dapsone 100 mg / daily without improvement. in the fifth month of treatment the patient developed a satellite lesion with similar characteristics and another plaque with the same morphology in the neck, reason that made him consult again. on physical examination the main lesion was an oval indurated hypochromic plaque with reddish - edge and smooth surface in the abdomen with some hairless areas inside the plaque and no alteration of sensibility. in the upper part of the main lesion there was a smaller hypocromic plaque with lilac - colored edge and no hair inside, did not have alteration of sensibility either ; in the neck there was another hypochromic plaque with light erythematous edge and scaly surface with alteration of sensibility. a new biopsy from the center of the initial plaque was performed suspecting localized morphea. the result showed subepidermic hyalinized collagen with loss of epidermal rete ridge pattern and some dilated superficial venules in the superficial dermis ; there was atrophy of the adnexal structures. in the middle dermis there was a lymphocytic infiltrate with linear arrays between the thickened collagen bundles that enclose some ducts and eccrine glands [figure 3 ]. there is a hyalinized superficial dermis with thickened collangen bundles in the mid and deep reticular dermis ; notice the absence of adnexal structures (h and e, 100) those findings in the histopatological exam confirmed the diagnosis of morphea. morpheae is a disorder of unknown cause in which there is localized sclerosis of the skin. the etiology appears to be involved with the fibroblastic cells in which alterations in the grow factors (platelet - derived growth factor) and receptor expression (tgf-) have been reported in in - vitro studies. those alterations appear to lead to increased connective tissue growth factor (ctgf) gene expression and finally fibrosis. immunological cytokines, auto - immune, trauma, immobility, radiotherapy, hormonal and infection etiology mainly with borrelia burgdorferi also have been reported. after some months they become thickened and and a characteristic lilac - colored edge develops. biopsies done in the periphery of the lesion will show markedly lymphocytic and histiocytic inflammatory infiltrate scattered in the middle dermis. in the lower part of the dermis and subcutaneous tissue, it begins to appear as broadening of the collagen bundles with diminished interbundle spaces. when the disease progresses, the inflammatory infiltrate slowly starts to disappear and is replaced by hyalinized connective tissue. the atrophic adnexal structures diminish in number until they disappear.[69 ] the sweat glands generally are located in the middle of the sclerotic collagen bundles in the middle and deep dermis. in brazil, leprosy is a frequent infectious disease and is first impression differential diagnosis in several diseases. clinically patients with leprosy can be in two opposite sides, the paucibacillary (1 - 5 lesions) or the multibacillary (more the 5 lesions) subtype, reflective of the host immune response ; the paucibacillary subtype is characterized by a predominantly th1 cell - mediated immune response and the multibacillary subtype is characterized by a predominantly th2 humoral response. the initial indeterminate lesions consist of hypopigmented macules with not well defined borders, generally without involving hair growth and nerve function. in the histopathological findings tuberculoid lesions present as a plaque that is frequently solitary, with central hypopigmentation and raised erythematosus edge, the surface is sometimes scaly hairless and with alteration of sensibility. in the histopathological exam it presents with non - caseating granulomas composed of epitheliod cells, lymphocytes and langhans cells. in our case the initial histopathological findings showing non- specific perineurovascular infiltrate directed to the diagnosis of paucibacillary leprosy. in the literature review there was no evidence of such pattern of infiltration in morphea but with the lack of response to the treatment and the appearance of new lesions the diagnosis of morphea became more likely. it was interesting to note the localization of the lymphocytic infiltrate around the nerve and the linear disposition of the lymphocytes that havent been reported until now. we suggest this could be another pattern of infiltration in morphea and there should be new studies to confirm this theory. the perineural infiltration with linear array of lymphocytes in the histopathological examination could be an initial pattern of morphea. | clinically and histopathologically paucibacillary leprosy shows similar features with initial morphea. in this case we report a 24 yr - old male patient who presented to our dermatology department with diagnosed paucibacillary leprosy by his local dermatologist, and confirmed by perineurovascular lymphocytic infiltrate in the histopathological exam. on physical examination we found new plaque lesions that were suggestive of morphea with alteration of sensitivity. a new biopsy was performed showing sclerotic superficial dermis with thickening of the collagen bundles in deep dermis and linear arrays lymphocytic infiltrate between the collagen bundles that confirm the diagnosis of morphea. |
emerging multi - drug resistant organisms are major contributors to morbidity and mortality following burn injury. pseudomonas aeruginosa infections are common following burns, often presenting as wound infections [24 ]. current prophylactic measures intended to prevent these infections in burn patients consist of isolation and use of strict sterile techniques combined with rapid surgical wound closure. although these prevention efforts can be effective under ideal circumstances, they often fail due to insufficient nursing, anesthesia, and critical care management resources, insufficient donor sites for immediate wound closure, or spread of infection from the surrounding environment during the initial first aid provided. treatment of pseudomonas wound infections, along with the frequently associated shock and respiratory failure, can involve complex fluid and ventilator management, definitive surgical debridement, and advanced wound closure technologies. the use of antimicrobial agents such as colistin (polymyxin e), tailored to navigate the most recent antibiotic resistance pattern, has been useful. clearly, new innovations for the prevention and treatment of pseudomonas wound infections in burn patients are needed. ongoing basic research efforts have made strides in dissecting the pathogenesis of pseudomonas infections using various models of infections in plants, flies, and mice [610 ]. p. aeruginosa coordinates the expression of multiple virulence factors using a complex regulatory communication system, namely, quorum sensing (qs), which has been shown to control approximately 10% of p. aeruginosa genes [11, 12 ]. qs is a cell - density - dependent phenomenon based on the release of low - molecular weight molecules that coordinate gene expression in a cell population [11, 13, 14 ]. in response to population density, p. aeruginosa produces and secretes such molecules, some of which act as specific chemical signals that control the production of virulence factors mediating acute infection. a decade ago, we identified the qs transcriptional regulator mvfr (multiple virulence factors regulator). the mvfr protein controls the expression of most of p. aeruginosa 's acute virulence - associated factors as well as the biosynthesis of more than 60 excreted anthranilic acid (aa) low - molecular weight derivatives, most of which belong to the 4-hydroxy-2-alkylquinolines (haqs) family [12, 13, 16 ]. haq expression is subject to positive autoregulation, with two haqs, namely, 4-hydroxy-2-heptylquinoline (hhq) and 3,4-dihydroxy-2-heptylquinoline (pqs), functioning as mvfr ligands. these haqs, in concert with mvfr, positively regulate the transcription of the pqsabcd and phnab operons, which encode haq biosynthetic enzymes of these molecules [9, 15, 19 ]. pqs and hhq are produced in vitro and in vivo in acute models of infection [9, 11, 15, 19 ]. we showed previously that pqs is more potent than hhq in inducing mvfr binding to the pqsa promoter as well as inducing pqsa - e transcription in pqsa::pqsh p. aeruginosa mutant. the relative potency of hhq versus pqs in inducing mvfr ligand - binding domain precipitation suggests that these molecules induce different mvfr conformational changes ; a difference in conformation effects could subsequently results in different binding affinities to the same mvfr target promoters, and differences in mvfr - regulated gene transcription. because haqs govern and dictate infection course, they may be of use as indicators of infection stage. moreover, interfering with these molecules therefore, here, we examined for the first time whether clinically important pseudomonas specimens, derived from an active burn wound infection in a human patient, produce and excrete detectable levels haqs in situ. permission for use of excess tissue and wound drainage material for the purposes of this study and permission for medical record review were obtained from the hospital institutional review board. the samples were obtained from a discarded resected infected burn wound on the patient right leg. three types of specimen were collected : (i) one containing necrotic muscle with fat ; (ii) one wet and oozing sample with pus and liquefied fat mixed together with some saline solution from the operative field ; finally (iii) drainage liquid collected out from the wound. wound biopsies were performed for the purposes of diagnosis and therapy at the time of surgery in the course of the patient 's treatment. the biopsy specimens were fixed in formalin, blocked in paraffin, and stained with hematoxylin and eosin. wound drainage and resected tissues in excess of the amount required for the biopsy were used in this analysis. tissues were weighed, cut into small pieces, and then homogenized in 50% methanol. whenever appropriate, liquid was mixed 1 : 1 with 50% methanol. the quantification of haqs was performed by liquid chromatography / mass spectrometry (lc / ms) as described elsewhere, using hhq - d4 and pqs - d4 as internal standards. positive electrospray was employed in mrm mode with 2 10 mtorr argon and as the collision gas and 30 v of energy to quantify haqs by lc / ms. the following ion transitions were used : hhq 244>159, hhq - d4 248>163, hqno 260>159, pqs 260>175, and pqs - d4 264>179. he sustained burns to 58% of his total body surface area (tbsa), including partial thickness burns to his face, both arms, back, and buttocks, with full thickness injury to his right lower leg and medial left thigh. his initial treatment at two different hospitals in honduras included right lower extremity fasciotomies and multiple dressing changes. he was transferred to the shriners boston burns hospital 9 days after sustaining the original injury. upon admission to shriners hospital, the patient was awake and alert but mildly febrile and tachycardic. his lower extremity wounds were grossly infected with deep necrotic wounds to the right lower extremity where eschar had been partially resected. biopsies showed diffuse inflammatory infiltrate and intramuscular rod - shaped bacteria (figure 1(a)). cultures from the right lower extremity revealed three p. aeruginosa isolates, a pan - resistant acinetobacter baumannii, klebsiella pneumoniae, clostridium species, and candida krusei. initially, the pseudomonas was susceptible to aztreonam, ciprofloxacin, levofloxacin, piperacillin / sulbactam, colistin ; and it was resistant to amikacin, cefepime, gentamycin, meropenem, ticarcillin, tobramycin, and imipenem. pseudomonas specimens from the central line tip that the patient came in with were also grown in culture. the patient was treated initially with vancomycin and meropenem as well as topical mafenide acetate. is characterized by the presence of a diffuse inflammatory infiltrate related to muscle necrosis (figure 1(a)), together with intracellular rode shaped bacteria (figure 1(b) + inset). bacteria - induced muscle necrosis and the bacteria 's ability to invade muscle cells are two phenotypic characteristics that strongly rely on the coordinated expression of several bacterial virulence factors, many of which are under the control of mvfr. we thus sought to determine the major mvfr ligands that are produced during burn wound infection in humans. as shown in figures 1(c) and 1(d), we detected hhq, pqs, and hqno in whole muscle tissue as well as in drainage liquid. this result corroborates the ratios of these molecules that we measured previously in muscle biopsies from burned and p. aeruginosa infected mice, and are in sharp contrast to ratios measured in bacterial cell cultures [12, 13, 21 ], indicating that the in vitro bacterial culture setting does not reflect accurately the in vivo burn wound situation. thus, in agreement with our previous results in burned and infected animals, we found that hhq is produced at high levels in human burn wounds and not fully converted into pqs. pqs is converted from its precursor hhq by the enzyme pqsh, whose activity is regulated by the homoserine lactone regulator lasr. the low pqs levels observed in both mouse and human infected tissues might be due to reduced pqsh activity in vivo. nevertheless, we have shown that pqs is fully dispensable in that pqsh - mutant cells, which produce hhq but completely lack pqs, display normal mvfr - dependent gene expression and virulence in burned and infected mice. the findings presented here show for the first time that haqs are produced during acute p. aeruginosa infections in human burn wounds. moreover, these findings demonstrate that haqs cell - to - cell communication molecules can be measured directly from an infected patient and that the ratio between the various haq molecule types resembles that found in burned and infected mice but not that produced in bacterial cell cultures. these results validate the use of burn mouse models in studies of qs - dependent p. aeruginosa acute infection. the fact that these qs low - molecular - weight molecules are detected also in drainage liquid collected from surgical sites is of the utmost importance for several reasons : (i) it demonstrates that these small molecules can affect large area of wounds and that in humans the bacteria can communicate beyond their immediate surroundings ; (ii) it indicates that these molecules might affect other pathogens in polymicrobial infections [23, 24 ], such as in the presently reported case ; (iii) it supports the hypothesis that targeting virulence and qs with selective anti - qs inhibitors may diminish infections caused by multi - drug resistant bacteria [10, 25 ]. the ability to accurately follow bacterial pathogenesis is an important prerequisite for the development of antivirulence agents aimed at controlling the course of infection. in this context, qs is a suitable drug target for new antimicrobials because it controls bacterial pathogenesis and it is evolutionarily conserved among pathogenic bacteria [26, 27 ]. pharmacological targeting of nonessential functions such as virulence factor production may decrease the emergence of resistance since bacterial survival is not directly challenged or subjected to selection pressure. for instance, we showed that pharmacologic inhibition of the mvfr regulon greatly reduced p. aeruginosa virulence in mice. in conclusion, here we demonstrated that qs signaling molecules can be measured directly from an infected patient and that the ratio between the various haq molecule types resembles that found in burned and infected mice, demonstrating the importance of animal models in the evaluation of the efficacy of new drugs. these results also strengthen the validity of the hypothesis that qs inhibition may be an excellent target to prevent or halt multi - drug resistant p. aeruginosa infections. | pseudomonas aeruginosa has developed a complex cell - to - cell communication system that relies on low - molecular weight excreted molecules to control the production of its virulence factors. we previously characterized the transcriptional regulator mvfr, that controls a major network of acute virulence functions in p. aeruginosa through the control of its ligands, the 4-hydroxy-2-alkylquinolines (haqs)4-hydroxy-2-heptylquinoline (hhq) and 3,4-dihydroxy-2-heptylquinoline (pqs). though hhq and pqs are produced in infected animals, their ratios differ from those in bacterial cultures. because these molecules are critical for the potency of activation of acute virulence functions, here we investigated whether they are also produced during human p. aeruginosa acute wound infection and whether their ratio is similar to that observed in p. aeruginosa - infected mice. we found that a clinically relevant p. aeruginosa isolate produced detectable levels of haqs with ratios of hhq and pqs that were similar to those produced in burned and infected animals, and not resembling ratios in bacterial cultures. these molecules could be isolated from wound tissue as well as from drainage liquid. these results demonstrate for the first time that haqs can be isolated and quantified from acute human wound infection sites and validate the relevance of previous studies conducted in mammalian models of infection. |
apoptosis, necrosis, necroptosis, autophagy, and pyroptosis are different modalities of cell death programs that play important roles in regulation of the immune system. failure in cell death program leads to either increased numbers of lymphocytes, increased number of infected phagocytic cells, autoimmune diseases, or the inability to dampen immune response and malignancy. different cellular stimuli, for example, tnf, fas ligand, trail ligand, double - stranded rna (dsrna), interferon- (ifn-), atp depletion, ischemia - reperfusion injury, and pathogens have been shown to induce cell death and consequently the release of the of endogenous cell death - derived products [24 ]. these chemically distinct microbial and endogenous products are named as pathogen - associated molecular pattern (pamps), damage - associated molecular pattern (damps), cell - death associated molecular patterns (cdamps), and alarmins [25 ]. once released from dead and dying cells, they acquire adjuvant activity and cooperate with the cytokines : il-1, il-, il- 33, tnf-, trail, ifns, il-8, and il-12 and antibiotic peptides in augmenting innate response [25 ]. they are expressed in different immune and nonimmune cell types and are localized in the nucleus and cytoplasm, for example : high - mobility group box 1 (hmgb1), il-1 (nucleus), s100 proteins, atp, and uric acid (cytoplasm), heat shock proteins (exosomes), formyl peptides, mdna (mitochondria) and heparan sulphate, and hyaluronan fragments (extracellular matrix). these mediators can act via different plasma membrane and intracellular recognition receptors and are part of host 's normal protective response to tissue injury, sterile inflammation, and infection according to the danger model proposed by polly matzinger in 1994. it is still uncertain to what extent the many and overlapping functions of cytokines, microbial and damage- and cell death - associated molecular patterns, impact on the molecular switcher of the genetic program specifying th17 and regulatory t cells (tregs) and thereby impairing the th1 or th1 cell response. overproduction of cytokines, pamps, damps, and alarmins appears to restrict the growth of pathogens by fueling a potent immune response. however, in some situations, this response can be detrimental to the host and may contribute to autoimmune and autoinflammatory diseases and cancer. nonetheless, in some context, this could be a mechanism to prevent the initial proliferation of autoreactive cells, thus preventing autoimmune disease. along the acute and chronic inflammation is commonly observed cycles of proliferation and death of immune cells as well as the secretion of growth factors with survival and suppressor activity that exert regulatory effects on a relatively larger number of cell types in an autocrine and paracrine manner. the proinflammatory mediator catabolism by the parenchymal / stromal cells reverts back the tissue to noninflammatory phenotype, which is called the resolution of inflammation. therefore, futures studies to measure the impact of cell death subtypes and their products on the innate and adaptive response in the context of pathophysiological processes, such as acute and chronic inflammation, will be critical for designing new strategies to control inflammatory response in many diseases. here, we will present an update on recent findings showing that various damps / cdamps and alarmins act as direct mediators of the inflammation and have great impact on the outcome on the inflammatory response. the inflammatory reaction during the innate immune response is the first - line host - defense to pathogens such as bacteria, fungi, parasites, and virus. the majority of pathogens can be detected by conserved and unique structural microbial components such as polysaccharides and polynucleotides that differ little from one pathogen to another but are not found in the host. the immune cells recognize such molecules referred to as pamps (or inducers) through one or more the pattern - recognition receptors (prrs). these receptors (or sensors) contain a ligand - sensing region referred to as leucine - rich repeats (lrrs). the phagocytic leukocytes, endothelial and mucosal epithelial cells and antigen - presenting cells, and various targeted tissues express prrs message and proteins during the inflammatory response. prr families include the family of the toll - like receptors (tlrs), the nucleotide - binding domain leucine - rich repeat - containing receptors (nlrs), c - type lectins (ctls), rna - sensing rig - like helicases (rlhs), and rage and dna sensors [1013 ]. the same repertoire of prrs can recognize damps originating from dying cells. the prrs share some molecular features and signaling pathways which are essential for their crosstalk and intracellular signaling that lead to activation of transcription of mediators and their receptors. the mediators include inflammatory cytokines (tnf, il-1, il-1, and il-6), chemokines (ccl2 and cxcl8), bioactive amines (histamine), lipid mediators from arachidonic acid (prostaglandins and leukotrienes), and products of proteolytic cascades, such as bradykinin and complement component 5a. these mediators are in general short living molecules and act on target tissues, including local blood vessels, to induce vasodilation, extravasation of neutrophils, and leakage of plasma into the infected tissue. the acute inflammatory response is finished once the triggering insult is eliminated, the infection is cleared, and damaged tissue is repaired. many pamps and damps released in the inflammatory environment play activate roles in the subjacent processes that control and resolve the inflammation and promote repair and regeneration of tissues, promoting the reestablishment of the host homeostasis. the final resolution of inflammation is an active and highly regulated process orchestrated by specialized proresolving mediators derived from poly - unsaturated fatty acids [9, 14 ]. next, we will present a short update on important aspects of prrs family member and their signaling cascades. tlrs are expressed either on plasma membrane (tlr1, tlr2, tlr4, tlr5, and tlr6) or on endosomal membranes of endoplasmic reticulum (tlr3, tlr7, and tlr9). each tlr specifically interacts with a rather diverse plethora of ligands, including bacterial flagellins, single strand (ss) rna, double strand (ds) rna, peptidoglycans, and imidazoquinoline compounds. the intracellular adaptors and transducers involved in their signaling cascades have been described in detail elsewhere [5, 12 ]. after binding, the tlr intracellular tir domain interacts with myd88, tirap / mal, or trif, which are proteins sharing a similar tir domain. upon stimulation, myd88, via its death domain, interacts with the death domain of serine / threonine kinase (irak) family. irak1 and irak4 are activated by phosphorylation, leading to the dissociation of irak1 from the receptor complex. irak1 in turn interacts with the tumor necrosis factor receptor - associated factor (traf) family member traf6 and with tak1 (transforming growth factor--activated kinase). the activation of tak1 leads to the formation of the ib kinase and nf-b essential modulator complex (ikk / nemo). the proteasome - mediated degradation of ib is required for nf-b heterodimer (p50/rela) activation and its translocation to the nucleus. the nf-b then mediates the transcription of genes for cytokines such as il-1 and, il-6, il-8, tnf-, il-12, il-15, ifn- and ifn-, cyclooxygenases (cox1 and cox2), inducible nitric oxide synthase (inos), chemokines, e - selectins, angiogenic factors, matrix metalloproteases, and genes for initiation of an adaptive immune response such as cd80, cd86, and cd40. the anti - cell death genes such as c - flip, c - iap-1, c - iap-2, a20, sod2 (superoxide dismutase 2), and bcl - xl are also induced by nf-b. tlr3 and tlr4 and tlr7 and tlr9 also activate irf3 (interferon regulatory factor 3) and irf7, respectively, leading to the production of ifn- and - in a cell - type - specific manner [5, 12 ]. the interleukin receptors il-1r and il-18r contain three extracellular immunoglobulin domains and one intracellular toll / il-1r homology (tir) domain. interacts with the death domain of serine / threonine kinase (irak) family and, in turn, with nf-b essential modulator complex (ikk / nemo), leading to nf - kb activation and production of inflammatory mediators. the nucleotide - binding domain and leucine - rich repeat - containing receptors (nlrs) consist of more than 23 members. the 14 neuronal apoptosis inhibitor proteins (nalps or nlrps), ice protease activating factor (ipaf), nod1 (or nlrc1) and nod2 (or nlrc2) subfamily of cytoplasmic proteins share a central nucleotide - binding and oligomerization domain (nacht) and differ in their n - terminal domains [10, 11, 18, 19 ]. nod1 is constitutively expressed in a wide variety of cell types of both haematopoietic and non - haematopoietic origin, whereas nod2 is predominantly expressed in monocytes, dentritic cells, paneth cells, and intestinal epithelial cells. they can recognize peptides derived from bacterial gram - positive and -negative wall peptidoglycans, -d - glutamyl - meso - diaminopimelic acid (ie - dap), and muramyl dipeptide (mdp). both nod1 and nod2 transduce signals through the adapter protein receptor - interacting protein 2 kinase (ripk-2), also known as rick, which leads to nf-b downstream signaling and induction of proinflammatory cytokines. nod2 is crucial to maintain microbial balance and its mutations increase the development of chronic inflammatory diseases. nlrp1, 2, and 3 molecules harbor a central nucleotide - binding and oligomerization domain (nacht), pyrin domain (pyd), acidic transactivating domain, or baculoviral inhibition of apoptosis protein repeat domain (bir) and caspase recruitment domain (card) [10, 11, 16 ]. the pyd and card domains of the nlr / pyhin receptors and the pyd and card domains of caspase-1 promote the interaction with an adapter molecular referred to as the apoptosis - associated speck - like protein (asc). asc speck, which provides a platform for the activation of caspase-1 [10, 11, 19 ]. once activated, caspase-1 promotes the cleavage of the il-1 precursor as well as the il-18 precursor into active cytokines which are then released from secretory lysosomes or via cellular leakage. caspase-1 activation can help in tissue repair and release of many proteins without sequence signal such as il-1. caspase-1 cleaves il-33, but in this case, il-33 is inactivated [16, 17 ]. danger signals such as uric acid crystals, basic calcium pyrophosphate dihydrate (bcp) crystals, hyaluronan, elevated extracellular glucose, and fibrillar amyloid- peptide. moreover, various bacteria that secrete pore - forming toxins, such as s. pneumoniae, which produces pneumolysin, and b. anthracis, which produces anthrolysin o, as well as, the pore - forming channels such as nigericin, maitotoxin, and aerolysin are known to promote the cellular acidification and release of k, thereby inducing the assembly of nalp3 inflammasome and caspase-1 activation [10, 11 ]. it has been proposed that binding of atp molecules to p2x7 purinergic receptor gated ion channel results in the recruitment and opening of a pannexin-1 membrane pore and intracellular k+ efflux, leading to activation of nrlp3 inflammasome. thioredoxin - interacting protein (txnip) interacts and promotes the inhibition of thioredoxins (trx1 and 2), which are redox cytosolic (trx1) and mitochondrial (trx2) proteins capable of reducing thiols, thereby controlling damage induced by ros. after an increase in ros, txnip is released from oxidized trx1 and in turn binds to nlrp3, which is essential for the nlrp3 inflammasome activation. notably, inflammasome activation is negatively regulated by mitophagy / autophagy confirming that inflammasome senses mitochondrial dysfunction. the c - type lectin - like receptor (clr) is another family of transmembrane - associated innate immune recognition receptors. examples of ligands for clrs include the spliceosome - associated protein 130 (sap130) and filamentous actin. clr contains the c - type lectin - like domain (ctld), a conserved structural motif arranged as two protein loops stabilized by two disulfide bridges at the base of each loop [12, 13 ]. the lectin activity of clrs is mediated by conserved carbohydrate - recognition domains (crds), which contain four ca binding sites, and by an epn (glu - pro - asn) and qpd (gln - pro - asp) motifs, which confer specificity for mannose- and galactose - based ligands, respectively. clrs are distinguished from c - type lectin receptors in that clrs have a major role in cell activation through their cytoplasmic signaling domain, or they acquire signaling features by association with other receptors, such as the tlr or fc gamma receptor (fcr)- chain that has a receptor tyrosine - based activation motif (itam) within the cytoplasmic domain. clrs family members include dectin-1 (dendritic cell - associated c - type lectin-1), dectin-2, macrophage - inducible c - type lectin (mincle), the dendritic cell - specific icam3-grabbing nonintegrin (dc - sign), and dc nk lectin group receptor-1 (dngr-1). dngr-1 is essential for mhc class i cross - presentation of dead - cell associated antigens, whereas mincle recognizes sap-130. dectin-1 elicits a signaling cascade that begins with the tyrosine phosphorylation of the ita, subsequently recruiting and activating the tyrosine kinase syk. syk induces production of ros that act as microbicidal agents and contribute to the activation of the nalp3 inflammasome, leading to the production of il-1. syk also recruits and activates card9/bcl10 that in turn activates the canonical p65/p50 pathway. dectin also activates the p38, erk, and jnk cascades, as well as nfat, all of which regulate gene transcription in cooperation with nf-b. dectin-1-syk signaling induces dc maturation and secretion of cytokines, including il-2, il-10, il-6, tnf-, and il-23, rendering dcs fully competent to direct priming of cd4 + t helper cells, cd8 + cytotoxic t cells, and antibody responses. the (rig)-i - like receptors (rlrs) is comprised of retinoic acid inducible gene 1 (rig - i), melanoma differentiation associated gene 1 (mda5), and laboratory of genetics and physiology 2 (lgp2) member [13, 22 ]. these cytoplasmic card module - containing rna helicase proteins function as intracellular sensors of rna viruses. rlrs mediate type i and type iii ifn expression through an adapter molecule, ifn- promoter stimulator-1 (ips-1), and subsequent activation of irf3 and nf-b signal transduction pathways. the first identified cytosolic dna sensor, named dna - dependent activator of ifn - regulatory factors (dai), binds cytosolic dsdna and leads to the production of type i ifns [22, 23 ]. the endoplasmic reticulum- (er-) resident transmembrane protein stimulator of ifn genes (sting) functions as an essential signaling adaptor that coordinates the cytosolic detection of dna to the tbk1-irf3 signaling axis. sting is also known to recognize conserved products of microbial metabolism such as cyclic di - gmp, a universal bacterial second messenger released by microbial pathogens such as listeria monocytogenes. absent in melanoma 2 (aim2) is a member of the pyrin and hin domain (pyhin) family of cytosolic dna receptor. aim2 forms an inflammasome with asc triggering caspase 1 activation and the subsequent of production of il-1 and il-18. dna of various origins such as poly(da : dt), plasmidic dna and dna from the bacterium l. monocytogenes have been shown to activate aim2. upon activation, aim2 interacts with asc, leading to the cleavage of caspase-1 and the secretion of il-1 and il-18 [22, 23 ]. it is becoming clear that these cytosolic dna / rna receptors play a major role in autoimmunity diseases more than in cell - death - induced acute inflammation. rage is a ~4755 kda protein, originally discovered as a receptor for advanced glycation end products (age). it is a multiligand receptor of the immunoglobulin superfamily that plays a key role in immune response and in the resolution of inflammation, tissue homeostasis, and repair / regeneration after acute injury [2426 ]. it is expressed on monocytes, macrophages, t cells, dcs, smooth muscle cells, immature myofibers, endothelial cells, embryonic neurons and tumor cells. rage contains a single variable (v) domain containing two n glycosylation sites, followed by two constant (c1 and c2) domains, a transmembrane segment, and a short cytoplasmic tail necessary for ligand - induced signal transduction [24, 26 ]. rage needs to associate with adaptor proteins for intracellular signaling pathways which lead to the activation of nf-b, ap-1, creb, stat3, and nfat transcription factors and thereby the inflammatory response and/or cell proliferation, survival, differentiation, and motility in a cell - specific manner. rage acts as receptor to hmgb1 and s100 proteins and its interactions mediate nf-b - dependent production of the cytokines tnf, il-1, and il-6 and upregulation of the intercellular adhesion molecule 1 and the vascular cell adhesion molecule 1 on the surface of endothelial cells [27, 28 ]. on the other hand, new interest in rage comes from studies showing its ability to induce nervous system repair and cardiac muscle regeneration, which may depend on the local concentrations of its ligands. figure 1 shows an integrative overview of intracellular pathways by il-1r, tlrs, nlrs and clrs, as reviewed in this section. the killing of cells is one of the most primitive host - defense techniques against intracellular infection. the cells dye by apoptosis, a physiological and regulated cell death process which is intrinsically tolerogenic (noninflammatory), or by necrosis, a pathological regulated cell death, which is inherently immunogenic and elicits an inflammatory reaction [1, 3, 29 ]. pyroptosis, autophagy, and immunogenic cell death are other distinct processes recognized at morphological and biochemical levels. genetic dissection in many organism and animal models provided us with knowledge of the early and late morphological and biochemical events of cell death programs. we will summarize our current understating of the multiple intracellular signal pathways and the consequences of subtypes of cell death in physiological and pathological processes. the complex cellular morphology known as apoptosis can be confidently recognized by a series of morphological changes at electron microscopy level. apoptosis is characterized by cell shrinkage, membrane blebbing, condensation and margination of nuclear chromatin, degradation of dna into nucleosomal units, and formation of apoptotic bodies (figure 2). however, the hallmark of an apoptotic process is its dependence on caspase activation [1, 29 ]. cells undergo apoptosis in response to extrinsic or intrinsic pathways which are regulated by various antiapoptotic and proapoptotic proteins [1, 30 ]. the extrinsic pathway is mediated by the tumor necrosis factor receptor (tnfr) superfamily. the interaction of the tnfr-1 with either fadd or pro - caspase-8 and -10, via both death domain (dd) and death effector domain (ded) triggers the apoptotic signaling cascade, whereas the interaction with negative regulator cflip (fadd - like il-1-converting enzyme - inhibitory protein) will block this apoptotic signaling cascade, leading to cellular survival and nf-b - mediated proinflammatory response (figure 4). the tnfr1 complex i comprises the adaptor protein tnfr1-associated death domain protein (tradd), the death domain - containing protein kinase receptor - interacting protein 1 (ripk1), and several ubiquitin e3 ligases, including tnfr - associated factor 2 (traf2) and cellular inhibitor of apoptosis protein 1 (ciap1). the tnfr1 complex ii comprises the adaptor fas - associated death domain protein (fadd), caspase-8, and ripk1. activation of caspase-8 or -10 within either tnfr complex i or ii propagates the activation of effector caspases-3, -6, and -7, which then cause cellular destruction without mitochondria participation (known as type i extrinsic pathway). apoptotic cell death caused by mitochondrial dysfunction involves a rapid collapse of inner membrane potential, alterations of ions gradients due to loss or accumulation of metabolites and ions at different mitochondrial compartments, and the release of cytochrome c. these mitochondrial events seem to be either directly or indirectly regulated by the oligomerization and outer membrane permeabilization activity of bax and bak when bh3 ligands engage multiple bcl-2-like relatives, thereby promoting their activation. bax and bak promote apoptosis by perturbing the permeability of the mitochondrial outer membrane (referred to as momp) and facilitating the release of cytochrome c, a cofactor for activation of caspase-9 that, in turn, activates the effector caspases-3, -6, and -7. in addition, several studies support the involvement of a putative mitochondrial permeability transition pore complex (ptpc) that regulates the inner membrane permeabilization in apoptosis. the mitochondrial outer membrane ruptured could permeate the release of cytochrome c, but this remains controversial. in vivo, apoptotic cells maintain their plasma membrane integrity and are rapidly phagocytosed in the absence of an inflammatory response. apoptotic cells expose ecto - crt, phosphatidylserine, hsp70, hsp90, opsonins, thrombospondin, hmgb1, and other molecules that serve to eat - me signals for professional apcs, monocytes and macrophages recognition, and engulfment. the uptake of apoptotic cells by macrophages promotes cell growth and wound healing through the release of vascular endothelial growth factor (vegf) and transforming growth factor- (tgf-), respectively. this controlled process of apoptotic cell clearance is accompanied by suppressive effects on most immune cells under the effects of tgf-1 and pge2. necrotic death occurs quickly as a consequence of extreme physicochemical stress, such as heat, acidification, osmotic shock, mechanical stress, and freeze - thawing of cells. therefore, this cell death has been described as uncontrolled and accidental necrosis and is characterized by loss of plasma membrane integrity and cellular collapse (figure 3). in necroptosis, there is no massive caspase activation. necrotic cell death promotes inflammation and caspases exert a critical role as positive and negative regulators of inflammation induced by necroptosis. the inactivation of caspases proteolytic activity by z - vad - fmk, a pan - inhibitor z - vad - fmk, strongly sensitizes cell to tnf-, trail, and fas - induced necroptosis [29, 3739 ]. necroptosis is inhibited by necrostatin-1, a small molecule inhibitor of ripk1 (the receptor interacting protein kinase 1) that contains a death domain at the carboxyl terminus ; thus, it is recruited to the tnf receptor 1 (tnfr-1). smac mimetics are peptide antagonists of ciap-1 (inhibitor of apoptosis), ciap-2, and xiap that can further enhance tnf - induced necrosis. the necrotic cell death occurs upon the assembly of a large, signal - induced multiprotein complex named ripoptosome that contains caspase-8, fadd, ripk1, ripk3, and mlkl (mixed lineage kinase domain - like, which then initiates the extrinsic necroptosis pathway. the rhim domain of ripk1 and ripk3 can form filamentous amyloid structures that are important for mediating necroptosis. caspase-8 inhibition blocks the cleavage of ripk1 and ripk3 allowing ripk1 to phosphorylate ripk3 and thereby the assembly of ripk1-ripk3 necrosome. this complex initiates the intrinsic necroptosis pathway with the participation of pgam5l and pgam5s (figure 4). these two protein phosphatases cause the activation of dynamin - related protein 1 (drp1) and its translocation to the mitochondria. in mammalian cells, mitochondrial fusion is regulated by mitofusin-1 and -2 (mfn-1/2) and optic atrophy 1 (opa1), whereas mitochondrial fission is controlled by a dynamin - related protein 1 (drp1). along necrosis process, drp1 associated with its mitochondrial anchors fis1 (mitochondrial fission protein 1) and mff (mitochondrial fission factor) to induce mitochondrial fragmentation ; however, cytochrome c is not released as it occurs in the apoptosis intrinsic pathway. thus both mitochondrial fission and fusion proteins appear to modulate necroptosis through activities that are distinct from their roles in mitochondrial dynamics. activation of (ripk1)-ripk3 necrosome initiates the cascade of phosphorylation of several downstream target proteins including phospholipase a2, the proteases calpains and cathepsins, the cytoplasmatic noxa1/nadph oxidase complex, and the mitochondrial complex i, thereby leading to excessive ros production, atp depletion, and opening of the mitochondrial permeability transition pores. these events are accompanied by prolonged jnk activation and ripk3-induced stimulation of glycolysis, glycogenolysis, and glutaminolysis as well as the stimulation of krebs cycle [38, 39, 43 ]. in the physiological condition, mice deficient in fadd or caspase-8 die during embryogenesis ; however, mice with triple deletion of fadd, caspase-8, and ripk3 are viable [39, 40, 44, 45 ]. therefore, fadd and caspase-8 act as prosurvival factors that suppress the deleterious effects of necrosis by promoting the cleavage and inactivation of ripk1 and ripk3. programmed necrotic cell death occurs under various pathological processes such as ischemic brain injury, myocardial infarction, organ transplantation, and virus replication and is accompanied by strong inflammatory response. studies on fadd - tnfr1 and fadd - myd88 deficiency revealed that both tnf and tlr signaling partially contribute to progression of inflammation [39, 45, 46 ]. studies on mouse models of the tnf - induced systemic inflammatory response syndrome (sirs) and clp - induced peritoneal sepsis have shown that multiple organ failure and animal mortality are driven by both ripk1 and ripk3-dependent necroptosis [29, 47 ]. many human diseases are driven by activation of sterile inflammatory response, including ischemia - reperfusion injury, alzheimer 's disease, atherosclerosis, and toxic insults to liver and lung. necrotic cells release their cellular contents from organelles and nucleus (rna, dna, and nucleotides) as well as universal damps such as il-1, hmgb1, atp, uric acid, and hsps that recruit and activate neutrophil, dcs, and macrophages, thereby promoting a highly inflammatory process [29, 34 ]. immunogenic cell death (icd) is a special type of cancer cell death elicited by some classes of anticancer chemotherapeutics, including oxaliplatin, mitoxantrone, bortezomib, and radiation and photodynamic therapy. the cell death - associated products released by these dying cells attract circulating dendritic cells (dcs) and other antigen - presenting cells (apcs). the uptake of dead cell - derived antigens by dcs and, consequently, the cytotoxic responses by effector t cells and nk cells contribute at least in part to success of therapy. dead cells expose several proinflammatory signals including crt on the plasma membrane and the release of atp, hmgb1, hsp70, and hsp90. large - scale clinical studies are been conducted to determine the prognostic value of icd induced by current approved chemotherapy to different types of cancers [49, 50 ]. the activation of inflammasome containing intracellular engulfed bacterial and viral molecules induces pyroptosis which is a proinflammatory and cell death program dependent on caspase-1 activation [51, 52 ]. it has been considered as a cell death modality with morphological and biochemical features of necrosis and apoptosis [51, 52 ]. the mechanism, characteristics, and outcome of caspase 1-dependent cell death are distinct from apoptosis. along the pyroptotic program, a connected interplay of biochemical and morphological events causes the formation of pores (1 - 2 nm) in the plasma membrane, which leads to potassium efflux, water influx, cell swelling, and rupture of plasma membrane and release of intracellular contents. it has been shown that during pyroptosis, caspase-1 and likely caspase-7 (21) act together in the proteolytic digestion of several types of proteins including chaperone hsp-90, -actin, ataxin-3, hnrnp - a2, and the glycolysis enzymes glyceraldehyde-3-phosphate dehydrogenase, enolase, pyruvate kinase, among others proteins. host cell death by pyroptosis contributes to the control of microbial infection such as salmonella, shigella, listeria, pseudomonas, francisella, and legionella [51, 52 ]. more studies are needed to identify which caspase-1 substrates have the ability to induce pyroptosis features in these pathologies. most cells infected by bacteria, such as shigella and legionella, viruses, and protozoa undergo autophagy. autophagy is a cellular process involved in aberrant proteins and damaged organelles degradation by hydrolases into the lysosomes. autophagy plays a role in a wide variety of normal physiological processes including energy metabolism, organelle turnover, growth regulation, aging, and cellular self - digestion during starvation and hormone deprivation. along this process, organelles such as the mitochondria, endoplasmic reticulum, and protein aggregates are first enwrapped in double membrane vesicles, named autophagosomes, which deliver their content to endosomes and late to lysosomes. the formation of autophagic vacuoles is mediated up to 30 autophagy - related proteins codified by atg genes, which were first identified in yeast. the transition from diffuse cytosolic to punctuate pattern of the lipidated form of lc3 (atg8) is used as one of the most reliable autophagy markers. notably, the membrane trafficking events required for autophagy also participate in pathogen delivery into the lysosome and into endosomal compartments containing the toll - like receptors, such as tlr3, 7, 9, and 10. the formation of these complexes leads to activation of type i interferon signaling as well as delivery of endogenously synthesized viral antigens to mhc - ii - processing and -loading compartments. therefore, autophagic cells are likely to instigate the biochemical events leading to innate and adaptive responses. autophagy has not been considered as modality of cell death ; nonetheless, many stimuli that activate apoptosis induce autophagy, whereas signals that inhibit apoptosis inhibit autophagy. the pan caspase inhibitor z - vad - fmk inhibits caspases but also blocks lysosomal cathepsins and hence cell death by autophagy. antiapoptotic proteins, such as bcl-2 family members, bind to and inhibit beclin (atg 6), and proapoptotic factors, such as bh3-only proteins, disrupt this inhibitory interaction and thereby activate autophagy or vise - versa. autophagy is triggered by ros derived from either the mitochondrial electron transport chain or napdh oxidases. autophagy of damaged mitochondria limits ros - modulated caspase-1 activation and seems to negatively regulate pyroptosis. mitophagy is a specialized form of autophagy in which mitochondria are specifically targeted for degradation at the autophagolysosome. it is becoming increasingly clear that endoplasmic reticulum- (er-) stress induces autophagy (boland., 2013). calcium depletion, oxidative damage, and energy depletion cause er stress, leading to the unfold protein response (upr) via three major transmembrane proteins : pancreatic er kinase- (pkr-) like er kinase (perk), activating transcription factor-6 (atf6), and inositol - requiring enzyme 1 (ire1). atf6 controls the synthesis of the genes encoding er - associated protein degradation (erad), whereas perk suppresses protein synthesis by phosphorylating eukaryotic initiation factor 2 (eif2). activated ire1 activates the transcription factor x - box binding protein 1 (xbp-1). if severe enough, each of these stimuli can result in cell death ; ire1 facilitates apoptosis by recruiting ask1 and jnk (c - jun n - terminal kinase). therefore, there are many lines of evidence connecting upr, cell death and magnitude, and duration of inflammatory process. reactive oxygen species (ros) and nitrogen species (rns) regulate a wide variety of signaling pathways including anti - inflammatory responses and adaptation to hypoxia. ros / rns can cause damage to all biomolecules (proteins, lipids, and dna) and ultimately lead to cell death. reactive oxygen species are produced mainly by two sources : transmembrane nadph oxidase (nox family) and the mitochondrial electron transport chain (etc) macromolecular complexes. oxidation of sulfur alters chemical reactivity and metal binding properties of proteins, and it can serve as a molecular switch to control protein structure and function. redox - active cysteine residues in protein are subject to more than one kind of modification, which include disulfide, glutathionyl, nitrosyl, or sulfenic acid modification [61, 62 ]. s - glutathionylation is the formation of disulfide between the cysteine of glutathione and the cysteine moiety of a protein, also known as protein - mixed disulfide or pssg. the cytotoxic potential of ros is controlled by various mitochondrial, cytosolic, and peroxisomal antioxidant systems. there are two different intracellular redox compartments into cells, the endoplasmic reticulum and peroxisomes / mitochondria that consisted of highly oxidizing organelles, as opposite, the cytoplasm and nucleus which are very decreasing compartments due to the presence of the thioredoxin peroxidase - thioredoxin reductase and glutathione peroxidase - glutathione reductase systems. the normal extracellular environment is highly oxidizing, and following types of injuries such as ischemia, o2 deprivation and infarction, the release oxidoreductases and thiols affect the composition and properties of extracellular environment and this may sustain and prolong the inflammatory reaction. the critical role of the redox state of the injured tissue in the regulation of cytokines and various cell death - associated molecules has been previously proposed. specific antibodies and chemical are available for detection s - glutathionylation (cys - ssg), s - nitrosylation (cys - sno), sulfonated cysteine (cys - so3), and sulfenic acid modified proteins. these tools have been used in proteomic and immunochemical methods for monitor global changes in cysteine oxidation of proteins in diverse normal and pathological conditions. one study has identified the transition cysteine to sulfenic acid in over 175 proteins bearing cysteine modification [61, 64 ]. in the list there are important cell death - associated molecules with known role in the innate and adaptive immune system, including chaperones belonging to hsp60, 70, 75, and 90 kda families, calnexin (cnx), and calreticulin (crt). the functional consequences of oxidative cysteine modifications of important cdamps / damps and alarmins in the context of inflammation are described below. hmgb1 is a chromatin component of 27 kda that is structurally composed of three different domains : two homologous dna - binding sequences entitled box a and box b and a highly, negatively charged c terminus. it is involved in v(d)j recombination by acting as a cofactor of the rag 1 and 2 complex [65, 66 ]. hmgb1 lacks a classic signal sequence, although there is homology to a nuclear localization sequence that may participate in the nuclear functions of the protein. administration of hmgb1 to mice mediates the development of fever, anorexia, and sickness behavior. acting as cytokine, hmgb1 transduces signals and coordinates cellular activities through (rage), tlr2, tlr4, tim-3, chemokine cxc receptor (cxcr)-4, and cd24-siglec g/10. addition of hmgb1 to monocyte and macrophages stimulates the synthesis of tnf-, il-1, il-1, il-1ra, il-6, il-8, macrophage - inflammatory protein-1 (mip-1), and mip-1 but not the synthesis of il-10 or il-12. hmgb1 can interact with ssdna, lps, il-1, and nucleosomes and amplifies tlr - mediated inflammatory responses possibly by binding to tlr2, tlr4, tlr9, il-1r, and rage. hmgb1 can induce dc maturation as evidenced by increased cd83, cd54, cd80, cd40, cd58, and mhc class ii expression [48, 66 ]. hmgb1 is also a proliferative signal for both human cd4 + and cd8 + t - cells. confirming these important roles of hmgb1, hmgb1-deficient mice die at as early as e15 day as result of hypoglycaemia, reduced autophagy, and lack of dc - induced inflammatory response, critically important for survival in the neonatal period [65, 66 ]. hmgb1 has a role in the pathogenesis of a variety of sterile inflammatory conditions including rheumatoid arthritis, lupus erythematosus, and sjgren syndrome, trauma and hemorrhagic shock, and ischemia - reperfusion injury of the liver, heart, kidney, and brain [65, 66 ]. hmgb1 adopts different redox states under oxidative stress and the oxidation serves as a feedback mechanism to control the proinflammatory activity of hmgb1 in vivo. hmgb1 contains three conserved cysteines, which are sensitive to oxidation : cys23, cys45, and cys106. for instance, if all cysteines in the hmgb1 protein are reduced, it binds to cxcr4 and acts as chemoattractant. hmgb1 linked with disulfide binds to tlr-4 and induces proinflammatory cytokines, whereas further cysteine oxidation to sulfonates by ros abrogates both activities. on the other hand, reduced hmgb1 binds to rage, induces beclin-1-dependent autophagy, and promotes resistance to chemotherapeutic agents or ionizing radiation, while oxidized hmgb1 increases the cytotoxicity of certain chemotherapeutics via induction of apoptosis. furthermore, various reports have indicated that hmgb1 is involved in tumor tissue invasion and metastasis by recruiting macrophages and endothelial cell precursors. within cells, hsps act as chaperone and exposed to membranes, ecto - hsp70 and hsp90 are important inducers of the immunogenicity of stressed and dying cells. extracellular hsp70, hsp90, and gp96 are peptide carriers, inducers of cytokines, and stimulants for immune cells during stress. these proteins, for instance, have been described to bind to tlr4 and to cd14, which are lipopolysaccharide membrane protein receptors. hsps induce the maturation of dendritic cells and present peptide molecules to antigen presenting cells (apcs), thus linking the innate immune and adaptive immune systems. on the other hand, hsp peptides can be a route to induction of regulatory t cells (treg) which inhibit reactive t cells, as well as, il-10, thereby contributing to anti - inflammatory response. hsp70 has anti - inflammatory properties including downregulating inflammatory cytokine production, increasing cell and tissue tolerance of cytokine - induced cytotoxicity. one report has shown that the binding of peptides is more pronounced for hsp70 than for hsc70 and is accompanied with a gradual change in secondary structure under oxidative conditions. hsp70 and hsp90 contain cysteine residues in which oxidation induces change to a conformation with high chaperone activity. it is not known what happens to these hsps immune functions once owing this modification if, for example, this leads to an anti - inflammatory or proinflammatory phenotype. crt is a 46-kda ca binding er - resident proteins that participate in ca storage in the lumen of the er [71, 72 ]. the role of crt in ca homeostasis is critical since crt - deficient mice die at e14.5 embryonic age. crt, gp96 (grp94 or endoplasmin), protein disulfide isomerases (pdis), immunoglobulin - heavy - chain - binding protein (bip / grp78), and gpr78 are also er folding factors (or chaperones), aiding newly synthesized er proteins in proper folding. some folding factors, such as erp57 and pdi, are oxidoreductases that catalyze proper disulfide bond formation in protein substrates to aid in their folding. crt and cnx (calnexin) interact with the erp57 to promote disulfide bond isomerization in bound unfolded glycoproteins. therefore, oxidized calreticulin itself might drive protein folding in er by promoting disulfide formation. under apoptotic stress, crt associates with phosphatidylserine (ps) in a ca dependent manner and the crt / ps complex is recognized by a variety of receptors and adaptor molecules and function as most notable eat - me signal of apoptotic cells [34, 48 ]. crt binds to cd91/lrp1 to trigger macrophages to secrete cytokines, stimulates dendritic cells to express antigen presenting costimulatory molecules, and is involved in wound healing. the mechanism by which crt is released in extracellular space is only speculative ; change in ph and/or calcium levels may be involved in the release of crt from the er. crt is sensitive to sulfenic acid modification and it can affect both its function and its subcellular location, a hypothesis that deserves to be evaluated. there are over 24 homologous intracellular s100 proteins, which are characterized by calcium - binding ef hand motifs, low molecular weights, ability to form homodimers, heterodimers and oligomers, and tissue - specific expression. binding of ca and binding of zn are known to induce major conformational changes in s100 proteins, leading to an exposure of hydrophobic patches that interact with protein targets involved mainly in the cytoskeleton and cell proliferation. s100 proteins can bind to g - protein - coupled receptors, scavenger receptors, or heparan sulfate proteoglycans and n - glycans. s100a7 (psoriasin) is overexpressed in inflammatory skin diseases and is induced in keratinocytes by il-17 and il-22 and flagellin via tlr7. s100a8 and s100a9 are present in neutrophils, monocytes, and myeloid progenitors and can be induced in keratinocytes during inflammation. the s100a8 and s100a9 form a heterocomplex (known as calprotectin) that bind to carboxylated glycans and rage, leading to intracellular nf-b activation and thereby the expression of cytokines that act as growth factors for tissue repair and regeneration [75, 76 ]. s100a8/a9 and s100a12 induce prothrombotic and proinflammatory responses in endothelial cells including induction of thrombospondin, chemokines, and adhesion molecules. human s100a8/s100a9 is chemotactic for neutrophils and influences migration of other cell types, including myeloid - derived suppressor. s100a8/s100a9 induces proinflammatory cytokine tnf-, il-1, il-6, and il-8 in macrophages via nf-b activation. s100a8/a9 and s100a12 are elevated early in tissues and serum immune pathological conditions associated with inflammation such as arthritis, inflammatory bowel disease, vasculitis, multiple sclerosis, psoriasis, and cystic fibrosis and are considered suitable biomarkers of inflammation. s100b - rage is one important cellular signaling to severe pulmonary infection by aspergillus fumigatus. the sustained activation of the s100b - rage signaling is also involved in the hypoxia - induced inflammation in cystic fibrosis. it has been reported that s - nitrosylation and s - glutathionylation regulate the activity of some representative s100 proteins. another study showed that s - glutathionylation of s100a1 at the single cys-85 leads to a 10-fold increase in the affinity of the n- and c - loops of the protein for ca binding. human s100a8 is chemotactic for neutrophils and the activity may depend on its oxidation state. s - nitrosylated s100a8 reduces mast cell activation and mast cell - mediated leukocyte adhesion and transmigration in the microcirculation in vivo. s100a8/s100a9 can induce pro- and anti - inflammatory actions which are linked to their oxidation states [75, 76 ]). the il-1 family (il-1f) comprises 11 members and the most studied are il-1 and il-1. il-1 is a potent pyrogen that induce leukocyte tissue migration and expression multiple cytokines and chemokines. interleukin-1 receptor antagonist (il-1ra) is a specific inhibitor of il-1 and il-1. the gene deletion studies showed that mice deficient for il-1 and il-1 as well as for caspase-1, il-6, and tnf- and tnfr1 are viable and do not develop spontaneous diseases. interleukin-1 is the most power danger signal released upon necrosis that exerts effects on both innate and adaptive immunity [2, 83 ]. il-1 is a chromatin - associate cytokine and is highly dynamic in the nucleus of living cells. during apoptosis, intracellular il-1 concentrates in dense nuclear foci and is not released along with cytoplasmic contents. il-1 precursor is a signal peptideless protein ; it is not readily secreted and only released from cells undergoing necrosis. il-1 binds to 1 il-1 receptor (il-1r1) leading to multiple proinflammatory effects including cytokine secretion, neutrophil recruitment, and upregulation of major histocompatibility complex (mhc) and costimulatory molecules on antigen presenting cells. the influx of neutrophils towards necrotic dendritic cell - derived products from a sterile inflammation (in the absence of pathogens) is mediated through il-1. the il-1 precursor is released by hypoxic cells and incites an inflammatory response by recruiting myeloid cells into the area. il-1 stimulates the production of chemokines cxcl1 and cxcl2, which are involved in the neutrophil recruitment. the neutrophil migration along the inflammation to sterile necrotic cell death depends on both il-1 and il-1. cd11b+ macrophages are required to produce il-1 and bone - marrow - derived cells are required to produce il-1. il-1 is processed by caspase-1 in nlrp3 inflammasome and also leukocytes serine proteases and calpains. il-33, a il-1 family member, is secreted by necrotic cells independent of caspase-1 and caspase-8 or calpain, but it is inactivated by caspase-1. it is interesting that il-1, il-33, and hmgb-1 act as dna - binding cytokines allowing the access of several transcription factors, including steroid hormone receptors, p53/p73 complexes and recombinases for genomic dna repair and modification. therefore, it seems like that these cytokines act as dna damage sensor during stress and prevent nuclear breakdown and release of inflammatory dna and histones from nucleosomes ; to date only limited information is available about these mechanisms. this short review has highlighted the intricately and connected extrinsic and intrinsic molecular pathways and alternative ways to induce cell death programs named apoptosis, necrosis / necroptosis, and pyroptosis. autophagy can modulate these programs by limited control of damage organelles such as mitochondria. in vivo studies, in particular, in response to a variety of stress conditions and chemical insults, have furthered the knowledge and acceptance that cell death plays a multifaceted modulatory role in both innate and adaptive immune responses. the immunogenic cell death has emerged as cancer cell death modality stimulated by certain chemotherapeutic regime that exerts antineoplastic effects by eliciting a novel or reestablishing a preexisting antitumor immune response. the sterile necrotic cell death is a cell death modality in the absence of pathogen that elicits an innate immune - mediated acute inflammation. dying and death cells release in extracellular milieu an increasing number of nuclear-, cytosol-, and mitochondria molecules that stimulate an inflammatory response. among them, hmgb1, crt, hsps, and il-1 are the most relevant to date. these cell death - associated molecules trigger their inflammatory signaling pathways by binding to tlrs, nlrs, ctls, rlhs, and other membrane and intracellular receptors present in immune and nonimmune cells. these leads to activation of the inflammasomes and nf-b transcription factor, which in turn promote the synthesis and release of proinflammatory cytokines and other mediators connecting cell death and magnitude and duration of inflammatory reactions. caspases act as anti - inflammatory enzymes serving to limit the potentially proinflammatory consequences of cell death. a major goal in future studies overproduction and release of the self - molecules by stressed, dying, and dead cells influence the development and outcome of diverse range of diseases, including atherosclerosis, inflammatory bowel disease, diabetes, obesity, and inflammatory neurodegenerative disorders. similarly, the overproduction and release nonprotein thiols and thiol - regulating enzymatic systems as well as reactive oxygen and nitrogen species exert important positive and negative control in diseases - associated inflammation in part by modulating the cell death - associated factor immunogenic activities. even though antioxidants do not hold much potential to treat inflammatory diseases, many new small peptides and compounds for control cell death are under development and although there is much information about the diverse, sometimes pleiotropic effects of mediators of inflammation, future studies will define the interplay and cooperative role of cytokines, chemokines, lipid messengers, and cell death - derived mediators in cell survival, inflammation, or cell death. elucidating the critical nodes in the cell death signaling pathways will help us to design and develop new therapeutic strategies for acute inflammation and inflammatory disorders. | apoptosis, necroptosis, and pyroptosis are different cellular death programs characterized in organs and tissues as consequence of microbes infection, cell stress, injury, and chemotherapeutics exposure. dying and death cells release a variety of self - proteins and bioactive chemicals originated from cytosol, nucleus, endoplasmic reticulum, and mitochondria. these endogenous factors are named cell death - associated molecular - pattern (cdamp), damage - associated molecular - pattern (damp) molecules, and alarmins. some of them cooperate or act as important initial or delayed inflammatory mediators upon binding to diverse membrane and cytosolic receptors coupled to signaling pathways for the activation of the inflammasome platforms and nf-b multiprotein complexes. current studies show that the nonprotein thiols and thiol - regulating enzymes as well as highly diffusible prooxidant reactive oxygen and nitrogen species released together in extracellular inflammatory milieu play essential role in controlling pro- and anti - inflammatory activities of cdamp / damp and alarmins. here, we provide an overview of these emerging concepts and mechanisms of triggering and maintenance of tissue inflammation under massive death of cells. |
the first published description of using a recombinant virus to deliver vaccine antigens from another infectious agent was the vaccinia virus recombinant engineered to express hepatitis b surface antigen in animal cells, which upon immunization in chimpanzees, induced protective immune response against hepatitis b challenge. since then, numerous breakthroughs have been made in the fields of genetics and molecular biology, and consequently, various recombinant viral vectors were created and evaluated for vaccine and immunotherapeutic applications. arguably, the preeminent rationale that has been driving the continual development of viral - vectored vaccines and therapeutics is the promising immunogenicity that they offer. generally, the recombinant antigens that are delivered either as dna plasmid or subunit protein are reasonably safe in contrast, replicating viral vectors are often highly immunogenic, but they also carry the risk of recombination, reversion to virulence, and subsequently, pathogenesis. the search for an optimal medium has driven substantial efforts in the development of recombinant viral vectors with the strategy aimed at optimizing immunogenicity and safety. in this review, we have described some of the on - going preclinical and clinical studies to utilize various recombinant viral vectors as platforms to deliver vaccine antigens and as immunotherapeutic agents to specifically target and kill cancer cells. adenoviruses (ads) have been extensively studied for their potential usage in gene therapy applications. owing to years of research, which has established a foundation for the use of this linear double - stranded dna virus as a vector for vaccine delivery, major advantages of using ad as a vaccine platform include their ability to infect broad range of hosts and to induce high levels of transgene expression without the potential of viral genes being integrated into the host genome. importantly, due to their ability to grow in high titers in cell culture, ads can be manufactured safely and inexpensively. adenoviral vectors can inherently stimulate innate immune responses via toll - like receptor - dependent and toll - like receptor - independent pathways. moreover, given that ads infect dendritic cells (dcs), consequential up - regulation of co - stimulatory molecules accompanied by increased cytokine and chemokine production by the infected dcs can contribute to more effective antigen presentation to the immune cells. such inherent ability of adenoviral vectors to stimulate innate immune response in such multi - faceted fashion and to ameliorate the process of antigen presentation can produce adjuvant - like effect thereby promoting the development of potent humoral and cell - mediated immune responses against the vaccine antigen which would otherwise be less immunogenic. furthermore, as ads also possess tropism for epithelial cells, adenoviral vectors can be administered to target both mucosal and systemic immunity. to that end, various recombinant adenoviral vectors have been engineered and tested for vaccine applications for wide range of diseases including cancer, human immunodeficiency virus (hiv), and malaria [3 - 5 ]. adenoviral vectors can be generated in two different forms : replication - defective or replication - competent. replication - defective adenoviral vectors can be rendered by deletion of the e1 genes, which is essential for replication. usually, replication - defective adenoviral vectors lack e3 genes as well in order to create more space for foreign gene inserts. thereafter, an expression cassette with desired transgene under the control of a strong exogenous promoter can be inserted. the e1 genes remain intact, and, as a consequence, these vectors possess limited capacity for foreign gene inserts compared to replication - defective vectors. however, their " dose sparing " effect can virtually offset such limitation as the optimal dosages of replication - competent ad - vectored vaccines ; based on the safe doses of license wild type ad4 and ad7 vaccines, are 2 to 3 logs lower than those of replication - defective ad5-based vaccines currently being tested in clinical trials. also, as replication - competent ad - vectors mimic the natural viral infection, a potent adjuvant effect can be exerted due to the inherent stimulation of various elements of innate and adaptive immunity. rts, s, a leading current malaria subunit vaccine candidate created by fusing a portion of the plasmodium falciparum - derived circumsporozoite (cs) protein to the hepatitis b surface antigen, protected 56% of the vaccinees from naturally occurring malaria infection. this protection efficacy was well below the levels achieved by other vaccines currently being used for other infectious diseases and fell short of the threshold which must be reached to eradicate malaria. hence, in order to further improve the efficacy of malaria vaccines, the use of viral vaccine platform has been considered, and the adenoviral vector - based vaccine candidates have shown compelling anti - malaria immune responses [5,7 - 9 ]. for example, administration of a recombinant ad5 expressing p. falciparum cs protein (ad5pfcs), in the absence of any additional adjuvant, produced comparable cs protein - specific humoral and cell - mediated immune responses as the leading adjuvanted malaria subunit vaccine, rts, s / as01b. also, in mouse models, administration of recombinant ad5 expressing p. yoelii cs protein (ad5pycs) conferred cs protein - specific cytotoxic t lymphocyte (ctl)-mediated protection from intravenous p. yoelii challenge [8 - 10 ]. induction of potent cd8 t cell response has been linked to the protection against pre - erythrocytic plasmodium sporozoites challenge. however, ad5-based malaria vaccine candidates have been shown to be capable of inducing humoral responses against erythrocytic stage antigens. previous reports indicated that recombinant ad5-based vaccines expressing blood stage antigen, such as the apical membrane antigen-1 or the merozoite surface protein-1, induced vaccine antigen - specific antibody titers equivalent to adjuvanted protein subunit vaccines. the use of recombinant adenoviral vectors have been explored in the area of hiv vaccine development as well. noticeably, the effectiveness of the recombinant ad5-based vaccine, expressing hiv-1 gag, pol, and nef genes as target antigens, was evaluated in a non - human primate model. the authors of this study reported that administration of this novel hiv vaccine induced cell - mediated immune response against each transgene product despite the presence of existing ad5 immunity. however, in human clinical trials that followed, recombinant ad5 expressing hiv-1 gag, pol, and nef antigens failed to offer protection and were found to be associated with an increased risk of hiv-1 acquisition in men with pre - existing ad5 immunity. the question of which factors contributed to the observed failure of this ad5-based hiv vaccine still remains to be answered. interestingly, however, the vaccine appeared to have increased immune pressure on gag, pol, and nef protein epitopes following hiv-1 infection, suggesting that there indeed was a vaccine - mediated immunologic impact on infecting viruses. the major hurdle to overcome this recombinant ad5 vaccine seems to be the inherent capacity of hiv-1 to evade vaccine - elicited cd8 t cell responses. there after, more progress has been made in the development of recombinant ad - based vaccines for hiv-1. for example, genetical recombination of alternative ad serotype vectors to express hiv-1 antigens and prime - boost regimens using heterologous ad serotype vectors have been attempted and will be evaluated in clinical trials. ads also have been received much attention as a potential anti - cancer agent. oncolytic ads can be engineered to express tumor - specific antigens for prophylactic or therapeutic anti - tumor treatments. moreover, such recombinant oncolytic ads can be specifically designed to target and replicate in cancer cells but not in normal cells. for example, the onyx-015 is a novel adenoviral cancer treatment platform which is the first replication - selective oncolytic virus tested in humans for cancer therapy. onyx-015 has been engineered to lack the gene that encodes the p53-inactivating protein, e1b. onyx-015 can specifically replicate within tumor cells lacking a functional p53 and kill them, whereas normal cells, which are subject to p53-mediated cell cycle arrest, are not effected. onyx-015 has been extensively tested in human clinical trials for treatment of various forms of cancers such as prostate, ovarian, colorectal, head and neck, hepatocellular, and pancreatic carcinomas [19 - 22 ]. encouragingly, intratumoral administration of onyx-015 to treat recurrent head and neck cancer seemed to be well tolerated in recipients during phase i and ii clinical trials. despite its safety, however, the observed antitumor efficacy of onyx-015 in other clinical trials was relatively low [24 - 26 ]. hence, in order to amend this critical problem, alternative approaches, such as development of cytokine / co - stimulatory molecule - expressing or chemokine - expressing oncolytic ads, are being evaluated in attempts to improve the antitumor efficacy of this oncolytic adenoviral therapy. alphaviruses are single - stranded positive - sense rna viruses that replicate in the cytoplasm of infected cells, which, as is true for most positive - sense rna viruses, conveniently eliminates the concern for potential integration of viral genes into the host genome. notable alphaviruses that are currently being evaluated as potential vaccine delivery platforms are venezuelan equine encephalitis virus (vee), sindbis virus (sin), semliki forest virus (sfv), and vee - sin chimeras. alphaviral vaccine vectors have been explored in myriad of vaccine applications for cancers, hiv, and human parainfluenza virus [30 - 32 ]. generally, alphaviral vectors are designed with the deletion of genes encoding structural proteins, and the advantages of such approach can be enumerated. first, absence of the structural genes enables the insertion of a foreign transgene sized up to ~5 kb. second, less viral vector components translates to less vector - specific immunity, which could potentially curtail the vaccine - specific immunogenicity upon repeated administration of alphavirus - vectored vaccines. also, elimination of structural gene products circumvents safety concerns as it allows for cytoplasmic replication of the vector rna but renders defective viral particle formation. such alphavirus vectors are known as " replicons ", and alphavirus replicons have been shown to efficiently elicit antigen - specific immune responses in several of animal models [32 - 36 ]. importantly, alphavirus vectors target antigen presenting cells, such as dendritic cells, in the draining lymph nodes, which leads to the efficient generation of antigen - specific immune responses. also, alphavirus vectors can create a proper environment for the cross - priming of vaccine antigen by inducing apoptosis in some cells. in addition, vaccine immunity can be further enhanced by the alphavirus vector itself. within target cells, replication of the vector rna occurs via double - stranded rna intermediate, which can be detected by the memvers of the rig - i - like receptor famil in the cytoplasm, and this engagement leads to the activation of the interferon cascade and innate immunity. vee is pathogenic in humans, but sin is not. in order to circumvent the safety concerns, vee / sin chimeras have been developed and investigated. in a vee / sin chimera, such chimeric vector expressing hiv-1 p55 has been shown to induce potent vaccine antigen - specific immune responses as vee or sin vector itself in mice. also, vee / sin chimera - elicited immune responses were more robust than those elicited by sin / vee chimera containing sin replicon component and vee packaging components. another study reported that vee / sin chimera was able to induce potent systemic and mucosal immune responses to an inserted hiv envelope glycoprotein in rhesus macaques. further, prime - boost strategy involving priming with vee / sin chimera encoding hiv (env) and siv (gag) genes and boosting with recombinant hiv envelope glycoprotein (gp140) elicited both neutralizing antibodies and cell - mediated immune responses in rhesus macaques and demonstrated significant decrease in acute viremia following intravenous challenge with the chimeric simian - human immunodeficiency virus, r5-tropic shiv (sf162p4), which allows the assessment of anti - hiv-1 envelop - specific immunity in primates by mimicking natural hiv-1 infection in humans, suggesting that the potential of chimeric vee / sin vectors should be further explored in hiv vaccine research. alphaviral vectors also have been assessed for their applications as anticancer agents either to prophylactically / therapeutically induced antitumor immunity or as oncolytic agents that stimulate mechanisms (e.g., apoptosis) to specifically kill tumor cells. for example, efficacy of recombinant alphaviruses as anticancer agents has been tested in a limited number of preclinical tumor models. the first tumor model study performed with alphaviral vector was the p815 mastocytoma model, where p815 tumor antigen - expressing recombinant sfv (rsfv) was constructed and administered in mice. the results from this study indicated that rsfv immunized mice induced strong antigen - specific ctl response and were protected from p815 tumor challenge. furthermore, recombinant sfv has been evaluated in a human melanoma antigen (mage-3) model, where mice were immunized with the recombinant sfv to deliver viral rna that encodes mage-3. the results from this study demonstrated that recombinant sfv could elicit human mage-3-specific humoral and ctl responses. although encouraging, the caveat of these studies investigating the potential use of sfv vectors as anticancer agents is that the studies have been performed in mouse models with well - known tumor antigens. therefore, the safety and efficacy of using alphaviral vectors as anticancer agents remain to be further investigated. meanwhile, only one clinical trial has been conducted to evaluate the induction of immune responses against tumor associated antigens using alphaviral vectors. this trial was an open - label, dose - escalation phase i / ii study conducted by alphavax (research triangle park, nc, usa) to assess the safety and immunogenicity of recombinant vee expressing carcinoembryonic antigen (cea) in subjects with advanced or metastatic cea - expressing malignancies (http://www.alphavax.com/docs/pr/release_54.pdf). the vaccination was well - tolerated in recipients, and this alphaviral vector awaits to be more thoroughly evaluated in larger clinical studies. moreover, a previous study described immunogenicity and protective efficacy of an alphavirus replicon particle vaccine against parainfluenza virus type 3 (piv3). in this study, chimeric vee / sin vector was engineered to express the piv3 hn glycoprotein and tested in mouse and hamster models. the results indicated that chimeric vee / sin - hn were immunogenic in mice and hamsters, and induced neutralizing antibody responses that protected hamsters from intranasal piv3 challenge. due to the success of vaccinia virus - vectored vaccines in the eradication of smallpox, poxviruses have been widely evaluated for their use in vaccine applications. advantageously, poxvirus genome is very large ; mammalian poxviruses possess a genome of approximately 130 kb, and avian poxvirus genome is even larger at approximately 300 kb. such large genome size enables the insertion of more than 10 kb of foreign dna without compromising the infectivity or other essential viral functions. unlike other dna viruses, poxviruses have their own transcription machinery, viral dna - dependent rna polymerase and post - transcriptional modifying enzymes, allowing self - sufficient cytoplasmic replication. this is important since it eliminates the concerns regarding potential mutation in the host genome that can be caused by the integration of viral genome into the host dna. moreover, inserted transgene products can be expressed at high levels, resulting in potent cellular immune responses. as such, recombinant poxviruses numerous early studies have been conducted with recombinant vaccinia virus expressing various tumor antigens, such as the extracellular domain of the rat neu oncogene - encoded protein, p185, epithelial tumor antigen, cea, polyomavirus - specific tumor - specific antigens, and early bovine papillomavirus proteins with positive results. subsequently, the cdna containing the full coding sequence of cea was isolated from a human colon tumor cell library and inserted into the vaccinia genome, resulting in a creation of replication competent recombinant vaccinia virus which directs cell - surface expression of cea in infected cells. this recombinant vaccinia expressing cea (rv - cea) was the first tumor antigen - expressing poxviral vector tested in humans. several phase i clinical trials conducted with rv - cea indicated that rv - cea administration via different routes was well tolerated. however, the efficacy of anti - tumor immune responses generated upon rv - cea administration varied between differential study settings [51 - 53 ]. the current platform designed to express a tumor antigen such as prostate - specific antigen (psa) or cea on a poxviral vector expressing multiple human t - cell co - stimulatory molecules (b7.1, lfa-3, and intracellular adhesion molecule-1), named tricom, demonstrated promising prospect in both pre - clinical and clinical studies. such incorporation of tumor antigen transgenes into the tricom platform led to the development of rv / f - psa - tricom (prostvac - v / f) and rv / f - muc1-cea - tricom (panvac - v / f) which are currently being evaluated in a phase iii clinical trial in metastatic castration - resistant prostate cancer and in advanced pancreatic cancer, respectively. the use of poxviral vectors have been extensively investigated in the field of hiv vaccines as well. however, due to the unsettling concept of administering replicating viral vector in immune - compromised individuals, safer, non - replicating or attenuated poxviral vectors in hiv vaccine applications have received much spotlight. based on the efficiency of transgene expression and the safety records, three of the most promising non - replicating poxviral vectors for human use are the attenuated modified vaccinia virus ankara (mva), the nyvac, and the alvac strains. mva was rendered replication - deficient by loss of approximately 15% of its original genome resulting from repetitive passaging in chick embryo fibroblasts. nyvac strain, derived from the copenhagen strain of vaccinia, was rendered replication - defective by deletion of 18 different open reading frames from the original viral genome. alvac is a canarypoxviral vector that does not replicate in human cells with further attenuation induced via over 200 passages in chicken embryo fibroblasts. the strategy to utilize replication - defective poxviral recombinants for the delivery of hiv vaccine antigens dates back decades, but a strong interest in developing poxvirus - based hiv vaccines has been rekindled by the results of rv-144 phase iii clinical trial in thailand. the results indicated that administration of four priming injections of alvac - hiv (vcp1521) in combination with two booster immunizations of a recombinant gp120 subunit vaccine (aidsvax b / e) produced 31% efficacy in preventing infection. however, the results also demonstrated that this vaccination regimen had little effects on viremia levels or cd4 t - cell count in the recipients who were later diagnosed with hiv-1 infection. the vaccine efficacy observed in this study was, indeed, modest. however, the significance of this study to be underscored is that it has shown, for the first time, hiv vaccine is capable of preventing hiv infection. recently, a series of phase i and ii clinical trials have been conducted with mva- or nyvac - based hiv vaccines, showing that poxvirus - based hiv vaccines are highly immunogenic in dna - prime / poxviral vector - boost immunization regimens [57 - 59 ]. for example, a recombinant mva vector was generated to express an antigen consisting of a linear construct of cd4 t - cell, cd8 t - cell, and b - cell epitopes from the pre - erythrocytic stage p. falciparum fused to the thrombospondin - related adhesion protein. this candidate vaccine was found to be safe, immunogenic, and partially protective against experimental malaria challenge in adults. recombinant mva has also been investigated as a vector to deliver the immunodominant mycobacterium tuberculosis antigen 85a to both naive volunteers and bacille calmette - gurin (bcg)-immunized volunteers. the results demonstrated that heterologous prime - boost immunization regimen substantially increased the levels of antigen - specific cellular immunity as bcg - primed recipients had memory cd4 t - cell response that was twofold higher than the responses observed in the bcg - naive group and tenfold higher than the control group given bcg alone. overall these results indicate that booster immunization with mva85a following bcg priming may be a practical strategy to enhance and prolong antimycobacterial immunity in bcg - vaccinated individuals. in this review, we have described few of the viral vectors that have been investigated for their application as the vaccine antigen carriers or anticancer agents. the use of replicating or non - replicating viruses as a platform to deliver vaccine antigen / transgene or as an oncolytic agent comes with challenges. the major challenge to consider is the likely induction of vector - specific immunity which subsequently impairs the ability of viral vectors to elicit proper immune responses against the antigen / transgene being carried or to infect and kill target tumor cells. however, this problem can be averted by adopting appropriate immunization strategies such as the use of viruses that do not circulate in humans and/or the use of different virus serotypes for prime and boost immunizations. there might be an array of choices as to which viral vectors should be chosen for a specific vaccine application, and the proper viral vector selection contributes largely to the success of the vaccine. in choosing a viral vector for vaccine development, for example, the target population for hiv vaccine consists of potential recipients who are already hiv - positive and, therefore, immune - suppressed, requiring extra caution in selecting the pertinent viral vector. also, in regards to vaccine development, appropriate vector selection entails a thorough knowledge of the infectious agent for which the viral - vectored vaccine is being developed. understanding the mode of transmission, tropism, mechanisms of infection and replication, and the immune responses elicited by the host to control the infection during the course of the disease may provide an essential information regarding the type and magnitude of immune response desired and must be considered in choosing the germane viral vector. moreover, the size of the transgene inserts that can be stably accommodated within a viral vector varies by the virus. for example, for expression of a large or multiple gene products, a vector with a large capacity for foreign gene inserts should be used, rather than multiple recombinants. practical aspects should also be considered in viral vector selection. as vaccines or immunotherapeutic agents must be distributed for global use, a strategy for the adequate storage of the recombinant vectors must be considered. moreover, sophisticated equipment for vaccine / therapeutic administration, and trained medical professionals are not always available in many developing countries. as such, viral vectors that are physically stable and do not require cold storage are preferred. further, capacity to scale up the production of the viral - vectored vaccines or immunotherapeutic agents to meet the increasing demands of millions of people worldwide is also an important consideration. the development of viral - vectored vaccines and immunotherapeutic agents for clinical applications faces abundant challenges in both the scientific and economical aspects. however, recombinant viral vectors have demonstrated promising results in animal and humans in various fields of research. accordingly, many viral - vectored vaccine and immunotherapeutic agents are currently being evaluated in human clinical trials, and many other candidates are waiting to be tested in advanced clinical studies. although the additional challenges lie ahead, the prospect of viral vector usage in vaccine and anti - cancer applications looks promising. | traditional approach of inactivated or live - attenuated vaccine immunization has resulted in impressive success in the reduction and control of infectious disease outbreaks. however, many pathogens remain less amenable to deal with the traditional vaccine strategies, and more appropriate vaccine strategy is in need. recent discoveries that led to increased understanding of viral molecular biology and genetics has rendered the used of viruses as vaccine platforms and as potential anti - cancer agents. due to their ability to effectively induce both humoral and cell - mediated immune responses, viral vectors are deemed as an attractive alternative to the traditional platforms to deliver vaccine antigens as well as to specifically target and kill tumor cells. with potential targets ranging from cancers to a vast number of infectious diseases, the benefits resulting from successful application of viral vectors to prevent and treat human diseases can be immense. |
regulation of protein translation is a critical step of the gene expression process, which allows cellular adaptation during stress conditions by rapidly reprograming the proteome output without the requirement for changes in rna synthesis. in conditions, such as heat shock, hypoxia, endoplasmic reticulum (er) stress, and apoptosis, an immediate change in protein levels is required, stressing the importance of translational regulation, responsible for rapid adaptation to physiological conditions. however, there is an emerging body of evidence that shows a limited correlation between the transcriptome and the corresponding proteome, suggesting that when it comes to translation, not all transcripts are treated equally. epidermal growth factor (egf) treatment of serum - starved hela cells resulted in only 4.8% differentially expressed genes (degs), where a deg represents a significant change in both the transcriptome and translatome in the same direction (homodirectionally). in opposition, the 95.2% of uncoupled degs represent a significant change in either the transcriptome or translatome or an inverse relationship between the transcriptome and translatome. using parallel genome - scale measurements of mrna and corresponding protein levels and half - lives, mrnas were found to explain 40% of the variability in protein levels, with translation efficiency being the best predictor of protein levels in mouse fibroblasts. accordingly, translation control is considered to play a central role in eukaryotic gene expression. as new evidence is being uncovered, scientists have now started to appreciate the critical role of mrna translation in tumor progression. in a wide range of cancer types, inappropriate translation of oncogenes, tumor suppressors, and eukaryotic translation initiation factors is a critical process in cancer cell proliferation [46 ]. even during times of stress, when global levels of protein synthesis are reduced, these transcripts encode prosurvival proteins that are translated by alternative (noncanonical) mechanisms [1, 7, 8 ]. studies continue to provide new knowledge with regard to the developmental causes and possible novel treatments of various types of cancers [47, 9 ]. significantly, much of this information can be tied back to the paradigm of translational regulation and its critical contribution to our understanding of cancer etiology. in this review, we will first discuss the mechanism of canonical translation initiation, followed by noncanonical mechanisms that utilize rna sequence features including upstream open reading frames (uorfs) and internal ribosome entry site- (ires-) mediated translation mechanisms, the role of eukaryotic initiation factors in noncanonical translation, and their significance in cancer progression. in addition to uorfs- and ires - mediated translation regulation, other noncanonical translation mechanisms exist, such as gamma interferon - activated inhibitor of translation (gait) complex, 5 terminal oligopyrimidine (5 top) elements, and au - rich elements (ares) [10, 11 ]. these mechanisms are out of the scope of this review article. canonical translation initiation. eukaryotic cap - dependent translation initiation includes the recognition and recruitment of mrna onto the small ribosomal (40s) subunit, followed by ribosomal scanning in a 53 direction. subsequently, the 60s large ribosomal subunit is recruited, forming the 80s initiation complex. at this stage, an initiator methionyl - trna (met - trnai) is in the ribosomal peptidyl (p) site at the mrna start codon [1, 13 ]. canonical initiation is a complex process utilizing more than 25 proteins, including a minimum of twelve eukaryotic initiation factors (eifs). the rate of initiation varies between different mrnas and is influenced by accessibility to the methylated guanosine cap structure (mg cap) at the 5 terminus of the mrna, the length and secondary structure of the 5 untranslated region (utr), the sequence and secondary structure surrounding the start codon, and the poly(a) tail [15, 16 ]. initiation begins with the assembly of eif4f complex comprising eif4e, eif4 g, and eif4a onto the 5mg cap (figure 1). eif4e binds to the mg cap, which then interacts with the multidomain scaffold protein eif4 g and the atp - dependent rna helicase eif4a. the ternary complex (eif2-gtp - met - trnai) associated with a 40s ribosomal subunit is then recruited to the 5 end of the mrna via a critical link to eif4 g (captured at the cap via eif4e) mediated by eif3, forming the 43s preinitiation complex. eif1 and eif1a assist in stimulating recruitment of the ternary complex, as well as acting synergistically to promote continuous ribosomal scanning for aug start codons. this 43s preinitiation complex then scans the 5 utr of the mrna, with the help of eif4a, until an initiation codon in optimal context is recognized. eif5 and eif5b then mediate subsequent hydrolysis of gtp to release the bound initiation factors from the 48s complex, leaving the start codon in the ribosomal p - site with the met - trnai and allowing 60s ribosomal subunit to bind [1, 19 ]. the now competent 80s initiation complex then proceeds to translation elongation (figure 1). noncanonical translation initiation. during stress conditions, such as hypoxia, nutrient deprivation, or endoplasmic reticulum (er) stress, alternative mechanisms that are mediated by cis - acting sequences in specific mrna subsets, such as uorf and ires, drive the translation of stress response mrnas [1, 68, 18, 20 ]. studies have revealed that a decent portion of human transcripts is known to contain uorfs (upstream of the initiation codon of the coding region) that function as translation or mrna stability regulators. recent ribosome profiling data reveals that uorfs can exist in an out - of - frame relative to the main coding sequence. however, an overlap can also occur between uorfs and the coding sequence, in which alternative translation of an upstream in - frame start codon of a gene can possibly produce an extended protein product. the mechanism of uorf - mediated translation functions primarily during eif2 phosphorylation conditions and enhances the expression of proteins involved in cell - cycle regulation and apoptosis. typical examples of uorf - mediated translation regulation include general control nonderepressible 4 (gcn4), the yeast transcriptional activator, and activating transcription factor 4 (atf4) in mammals. normally, translation initiation occurs from the start codons located in the 5 utr of mrna which leads to the translation of small uorfs. additionally, reinitiation of terminating ribosomes will typically not occur on the downstream cistron ; thus, the translation of the main coding sequence is inhibited in these conditions. however, during stress conditions eif2 phosphorylation attenuates translation of uorf sequences and allows the translation of main coding sequence. eif2 phosphorylation and reduced availability of the eif2-gtp - trnai ternary complex favor translation reinitiation at the gcn4p coding region (in yeast), subsequently resulting in activation of numerous genes [25, 26 ]. gcn4p activates these target genes by binding to them and functioning as a transcription factor. the complexity of rna structure in the 5 utr also plays a crucial role in uorf - mediated translation. for example, translation of -site app - cleaving enzyme 1 (bace1), which is implicated in alzheimer 's disease (ad) progression, is regulated through uorfs. however, high gc content and complexity of the rna secondary structure are also crucial decisive factors for uorf - mediated translation of bace1. additionally, recent genome - level studies indicate that rna secondary structure is negatively correlated with uorf translational efficiency as upstream (relative to uorfs) structures restrict or even arrest ribosomal preinitiation complex (pic), whereas structures downstream of uorfs enhance translation initiation of coding sequences. ireses are rna sequence elements that were initially discovered in the 5 leader sequences of poliovirus and encephalomyocarditis virus genomic rna that lack the 5 cap structure but nonetheless are efficiently translated in the host cell [18, 29 ]. the viral ires elements comprise secondary and tertiary structures that play a role in direct interactions with the translation initiation machinery. mutations in viral ireses such as hepatitis c virus (hcv), classical swine fever virus (csfv), and cricket paralysis virus (crpv) affect the secondary and tertiary rna structures and render these ireses inactive. these viral ireses are classified based on structural and sequence similarities, as well as their requirement for eifs and other protein factors for translation initiation [19, 20 ]. picornavirus ires elements are the examples of types i and ii ireses which require eif4 g, eif4a, and eif3 to assemble 48s initiation complex. type iii viral ireses require eif4 g, with hcv ires being an exception ; hcv ires interacts with eif3 for recruitment of the 40s ribosomal subunit in close proximity to the start codon, circumventing the requirement for the 5 cap structure [1, 19 ]. although ires - mediated translation operates independently of many canonical initiation factors, it requires rna - binding proteins known as ires trans - acting factors (itafs). many cellular mrnas are known to comprise ires elements, but they do not share structural or sequence similarities, unlike their viral counterparts [20, 31 ]. however, similar to viral ireses, cellular ireses participate in multiple interactions with the canonical initiation factors and itafs to recruit the ribosome [1, 20 ]. in fact, despite sequence and structural dissimilarities, cellular ireses are reported to share critical itafs. ires elements have been identified in mrnas encoding stress response proteins (pro- and antiapoptotic), such as x - linked inhibitor of apoptosis (xiap), cellular inhibitor of apoptosis 1 (ciap1), b cell lymphoma extralarge (bcl - xl), bcl-2, bag-1, apoptotic protease - activating factor 1 (apaf-1), p53, l - myc, n - myc, c - myc, and death - associated protein 5 (dap5) [1, 8, 3234 ]. translation initiation switches from cap - dependent to ires - dependent mode during stress conditions such as hypoxia, vascular lesions, serum deprivation, -irradiation, apoptosis, growth arrest, and angiogenesis. this shift is attributed to eif2 phosphorylation, eif4e - bp dephosphorylation, and eif4 g cleavage, any of which can inhibit canonical translation initiation [33, 36 ]. although the cellular ires elements are activated under stress conditions, these ireses differ in their requirement for eifs. for example, the l - myc ires requires the eif4f complex and interaction of both poly(a) tail binding protein (pabp) and eif3 with eif4 g for translation. on the other hand, partial silencing assays (using the knockdown plasmid, psilencer31 (si31), and hippuristanol (eif4a inhibitor) treatment) have demonstrated that c- and n - myc ireses can function only in presence of the c - terminal domain of eif4gi, eif4a, and eif3 ; this ires does not require full - length eif4gi or pabp. er stress caused by the accumulation of unfolded proteins triggers the unfolded protein response (upr) which in turn modulates both transcription and translation of key regulators (e.g., atf4) of the cellular stress response. a recent study has shown that an alternatively spliced variant of the previously discussed human atf4 transcript (variant v1) is translated by an ires - mediated mechanism. this variant also has a unique 5 leader sequence and is found to be less abundant than other variants in numerous human tissues. interestingly, these variants were shown to be translated by different mechanisms by using luciferase reporters and modifying 5 leader sequences with stem - loop insertions. chan. found high gc content in the long and highly structured 5 leader region of v1 in comparison to v2, suggesting a possibility of ires - mediated translation. additional tests involving inhibition of critical canonical eifs (e.g., eif4g1) and eif2 phosphorylation indeed confirmed the ires - mediated translation of v1 during upr. during amino acid starvation, viral infection, or endoplasmic reticulum stress, several kinases are activated that induce eif2 phosphorylation (figure 2). this phosphorylation, in turn, decreases eif2-gtp - trnai ternary complex activity, resulting in suppression of cap - dependent translation. however, several viral and cellular mrnas are insensitive to this mode of translation inhibition. this suggests that mrnas can employ alternative factors or mechanisms to recruit the eif2-gtp - trnai ternary complex. examples of cellular ireses unaffected by eif2 phosphorylation are xiap, c - myc, cationic amino acid transporter-1 (cat-1), and n - myc. the exact mechanism by which the consequences of eif2 phosphorylation are avoided by the hcv ires is not known. however, some ireses (csfv and hcv) employ eif5b, an orthologue of prokaryotic if2, during conditions of increased eif2 phosphorylation. the eif5b - eif3 mediated mechanism involves eif3 stimulating trnai binding to the 40s subunit (in the ires/40s complex) in an eif5b - dependent manner, which allows for the formation of the 48s initiation complex and, subsequently, the translation - competent 80s ribosome. like if2, eif5b binds and delivers initiator trna during translation initiation on these ireses [8, 30 ]. we have recently found that the xiap ires uses a similar mechanism during eif2 phosphorylation conditions. this finding suggests that eif5b - dependent activation of ires - mediated xiap mrna translation would act as a critical prosurvival switch in cells under stress. moreover, a recent publication from our lab suggests that the xiap ires does not require eif4 g, eif4e, and eif4a for initiation complex formation. the inhibition of eif4a activity by hippuristanol or pateamine a treatment did not affect the ability of xiap ires to form initiation complexes, suggesting eif4a is not required for ires - mediated translation of xiap. additionally, eif3 and pabp bind synergistically with in vitro - transcribed, uncapped, and poly(a)-tailed xiap ires rna and recruit ribosomes near the start codon (figure 3). although cellular ireses do not share structural similarities in studies conducted thus far, the secondary structure is indeed important as we have found it to be required for efficient recruitment of eif3 and the ribosome. a recent study suggests that eif3 subunit d plays a key role in alternative mechanisms of translation initiation by a noncanonical mechanism of cap recognition. specifically, the study classifies this method as a novel, eif4e - independent mechanism of translation initiation. found that the eif3d subunit alone provided rnase protection to the mrna of the early response transcription factor c - jun by directly interacting with a mature, methylated 5 cap structure. the specificity of eif3d binding to only a subset of capped mrnas is suggested to occur through an rna gate domain that regulates this novel cap - binding function of eif3d. in the larger context of eif3 specialized translation pathways, eif3d seems to play a critical role as a cap - binding protein that helps cells regulate protein synthesis even during times when the eif3f complex (required for canonical translation) is inactivated or inhibited. mitochondria play an important role in the intrinsic pathway of apoptosis, which is regulated by the bcl-2 family of proteins. for example, bax and bac proteins activate mitochondria - dependent apoptosis, whereas bcl - xl and bcl-2 proteins inhibit apoptosis. during apoptosis, the phosphorylation levels of eif4e and 4e - bp1 are influenced by sgp-2 which, in turn, affects the bcl - xl ires - dependent translation by regulating the stability of eif4f complex. overexpression of some protooncogenic proteins (bcl - xl and c - myc), as implicated in numerous cancer types, could be a result of elevated levels of eif4f. dap5 (p97), eif4g1, and eif4g2 comprise the eif4 g family and function in the formation of the translation initiation complex. dap5 domains share homology with the central and c - terminal region of eif4 g that interacts with eif4a, eif3, mnk1, and eif2 but not eif4e. the interaction of dap5 with eif2 and eif4ai facilitates the ires - mediated translation of various cellular mrnas, including those encoding pro- and antiapoptotic proteins, such as c - myc, bcl2, apaf1, xiap c - iap1, and dap5 itself [42, 43 ]. moreover, the cleavage of dap5 by caspase generates a smaller fragment, p86, which also facilitates the ires - mediated translation of various cellular mrnas. the cleavage of eif4gi during apoptosis yields three major cleavage products : n - fag (n - terminal fragment of apoptotic cleavage of eif4 g), m - fag (middle - fag), and c - fag (c - terminal fag) ; the cleavage causes disassembly of the eif4f complex as the fragments no longer retain the ability to bind eif4e, eif4a, and eif3. surprisingly, eif4 g m - fag alone supports the ires - mediated translation of mrnas, including p97/dap5, xiap, and c - iap1. during angiogenesis, ires expression of the potent angiogenic factor, vascular endothelial growth factor (vegf), is regulated by its interaction with eif4e and eif4 g. the direct interaction of eif4g1 with vegf ires enhances its translation during breast cancer progression. knockdown of eif4gi using rna interference in a chick chorioallantoic membrane system resulted in decreased vegf protein levels and a reduction in angiogenesis. this reduction was specific to ires - mediated translation, as silencing of eif4gi had no drastic effect on global rates of protein synthesis in normoxic conditions. the depletion of eif4gi attenuated global mrna translation rates, suggesting a bigger requirement of eif4gi during hypoxic conditions. the c - terminus of eif4gi stimulates cap - independent translation initiation at the 5 utr of c - myc and vegf. under hypoxic conditions, vegf, fgf-2, bcl-2, and hypoxia - inducible factor 1 (hif1) are all overexpressed due to upregulation of ires - mediated translation [46, 47 ]. this selective translation is mediated by the overexpression of eif4e - bps and eif4 g and is particularly advantageous for cancer cells as vegf, fgf-2, hif1, and bcl-2 are all significant factors in promoting tumor growth and survival [45, 48 ]. the itafs are protein factors that interact specifically with the ires based on the sequence and structure of the mrna and modulate ires - mediated translation. itafs can act as a molecular chaperone or modify the structure of rna to facilitate direct recruitment of the eukaryotic initiation factors and the ribosome to form 48s initiation complex. some of the well - characterized itafs are human antigen r (hur), la autoantigen, programed cell death 4 (pdcd4), polypyrimidine tract binding (ptb) protein, heterogeneous nuclear ribonucleoprotein a1 (hnrnpa1), hnrnac1/c2 upstream of nras (unr), nuclear factor 45 (nf45), insulin - like growth factor 2-binding protein 1 (igf2bp1), y - box protein 1 (yb1), and poly(c) binding protein (pcbp) [20, 49, 50 ]. the levels, activity, and localization of these itafs are regulated by various signaling pathways, which in turn regulate the ires - mediated translation. there are two classes of itafs : one that facilitates while the other represses ires - dependent translation. for example, nf45 promotes while hnrnpa1 inhibits the ires - mediated translation of xiap [51, 52 ]. hur directly interacts with the xiap and bcl - xl ires and modulates their translation. hur interacts with the xiap ires through the rna recognition motifs, rrm1 and rrm2, to stimulate protein synthesis. on the contrary, interaction with the bcl - xl ires decreases translation and enhances the membrane integrity of mitochondrial promoting cell survival [53, 54 ]. la autoantigen as part of rnp complex enhances xiap ires - mediated translation as demonstrated by in vitro and in vivo assays. pdcd4 had been considered the general translation inhibitor which sequesters eif4a and inhibits its helicase activity. however, it is shown that the direct interaction of pdcd4 with the xiap ires is required to inhibit the ires - mediated translation of xiap. pdcd4 in the absence of activated s6k2 directly binds to the xiap and bcl - xl ires and blocks 48s preinitiation complex formation. the activation of s6k2 by the fibroblast growth factor 2 (fgf2) results in phosphorylation of pdcd4 and subsequent removal by the proteasomal pathway, which in turn upregulates ires - mediated translation of xiap and bcl - xl. ptb protein forms ribonucleoprotein complexes with psf, hnrnpl, and other proteins to regulate the gene expression, stability, and localization of mrna during apoptosis. when trail activates apoptosis, ptb protein forms complex with psf, ybx1, nono / p54nrb, hnrnpa2/b1, hnrnpc1/c2, and ddx3x and regulate the ires activity of mrnas involved in apoptosis. the interactions could occur in the nucleus prior to splicing and in the cytoplasm aiding recruitment of the ribosome. cytoplasmic translocation of ptb protein leads to increase in traf1, p53, and p47 mrna expression. specific mutation of cytosine to thymidine observed in domain 2 of c - myc ires derived from multiple myeloma cell lines demonstrated enhanced interactions with ptb protein and y - box binding protein 1. this increase in protein binding is correlated with an elevated ires activity of c - myc mrna in multiple myeloma. in human melanoma, single nucleotide polymorphism at the ptb protein binding site present on p53 5 utr decreased ires activity, emphasizing the importance of ptb protein as an itaf [5862 ]. overexpression of hnrnpc activates xiap ires activity with no effect on cap - dependent translation. also, hnrnpc1/c2 interaction with the p53 ires is critical for mrna expression and thus affects transcription of proapoptotic mrna [64, 65 ]. moreover, hnrnpc interacts with the heptameric uridine sequence in the c - myc ires and enhances c - myc expression only during g2/m phase of cell cycle. hnrnpa1, which is part of hnrnp family of proteins, regulates expression of bcl - xl and xiap mrna. phosphorylation at the rrm1 domain of hnrnpa1 by s6k2 selectively promotes association of bcl - xl or xiap with hnrnpa1 and exports the rna - protein complex into the cytoplasm. the suppression is relieved by sumoylation of the rrm2 domain of hnrnpa1, resulting in the decreased affinity of hnrnpa1 to protein and translocation into the nucleus. unr acts as either a positive or a negative regulator of apoptosis depending on the cell type. deleting unr in the embryonic stem cells resulted in decreased expression of p53, gadd45 g, and caspase-3, impairing apoptosis signaling pathway in response to gamma irradiation, whereas when unr expression was partially silenced, induction of apoptosis was observed. unr interaction with the apaf1 ires opens the stem - loop structure of ires enabling ptb protein binding. ptb proten and unr act synergistically as chaperones to create a single - stranded region for the recruitment of 48s complex. besides the mentioned positive regulator, itafs such as hnrnpc1 and nucleolin can negatively regulate p53 expression. in conclusion, the levels and localization of itafs in the cell are critical for regulation of gene expression. during stress conditions, proteins are modified mostly by phosphorylation reactions that trigger nuclear to cytoplasmic localization and modulate ires - mediated translation. eif2 is a heterotrimeric protein, composed of,, and subunits. eif2 is a required element of the ternary complex that delivers met - trnai to the 40s ribosomal p - site in translation initiation. eif2 exists in a gdp- or gtp - bound configuration, which has a critical role in translational control during stress. during translation initiation, eif2 bound gtp hydrolysis is induced by eif5, releasing eif2-gdp in the inactive form. eif2b catalyzes the exchange of gdp to gtp which is necessary for reformation of the ternary complex. under stress conditions such as an excess of unfolded proteins in the endoplasmic reticulum, or amino acid starvation, subunit of eif2 is phosphorylated at serine 51 by one of four members of the eif2 kinase family [72, 73 ]. inhibition of active eif2-gtp regeneration results in decreased ternary complex thus inhibiting overall translation (figure 2). furthermore, eif2 phosphorylation has a role in suppression of tumorigenesis, demonstrated by the ability of protein kinase rna - activated (pkr) to promote malignant transformation of nih 3t3 cells [70, 74 ]. the transformation mechanism inhibits eif2 phosphorylation by decreasing the activity of upstream target pkr, potentially through the formation of inactive pkr heterodimers [70, 74 ]. decreased levels of phosphorylated eif2 were found in osteosarcoma tumors, while increased pkr levels and associated phosphorylated eif2 levels were correlated with tumor cell differentiation [74, 75 ]. expression levels of phosphorylated eif2 serve as a marker for determining the prognosis of non - small lung cancer (nsclc) patients. er stress is closely associated with solid tumor progression, having implications in cancer proliferation and apoptosis. downstream mediators and targets of er stress include activating transcription factor 6, inositol - requiring enzyme 1 (ire1), and protein kinase rna - like er kinase (perk) which is an upstream activator of eif2 phosphorylation [77, 78 ]. er stress is induced in chronic myeloid leukemia (cml), which activates perk and eif2 phosphorylation. phosphorylation of eif2 supports cml progression with a prosurvival role, shown in the inhibition of perk, which prevents eif2 phosphorylation. this, in turn, allows sensitization of cml cells to imatinib and decreases their proliferative abilities. insulin - like growth factor binding protein-5 (igfbp-5) and protein family member igfbp-3 upregulate expression of growth arrest and dna damage - inducible protein 34 (gadd34), which assembles an eif2 dephosphorylation complex, enabling regeneration of active eif2, critical to ternary complex formation. gadd34 's dephosphorylation activity is required to recommence protein synthesis after periods of global translation inhibition. however, translation of uorf - containing, prooncogenic protein atf4 is upregulated by eif2 phosphorylation, promoting osteogenesis and osteoblast differentiation. in ovarian cancer cells, autophagy and activation of the perk / eif2 pathway attenuate and in contrast, protein obg - like atpase 1 (ola1) inhibits protein synthesis and promotes integrated stress response without utilizing eif2 phosphorylation. ola1 is a gtpase that binds to eif2, preventing ternary complex formation. in vivo, ola1-knockdown inhibits the mainly prosurvival integrated stress response (isr) pathway in cancer cells, which is responsible for restoring cellular homeostasis in response to physiological changes as well as intrinsic stresses such as er stress [82, 83 ]. inhibition of the isr pathway results in attenuated ccaat - enhancer - binding protein homologous protein (chop) levels and promotion of tumor growth and metastasis through cell proliferation. chop expression is triggered by unfolded protein accumulation in the endoplasmic reticulum (er). chop induces apoptosis during prolonged stress or stress response malfunction, through the formation of heterodimers with other c / ebp family members. besides phosphorylation of eif2, increased levels of eif2 expression are detected in tumor samples in bronchioloalveolar carcinomas of the lung, hodgkin 's lymphoma, gastrointestinal carcinomas, malignant melanoma, and melanocytic neoplasms [70, 85 ]. eif2 levels were highly expressed, along with eif4e in the germinal centers of reactive follicles when examined in several types of non - hodgkin 's lymphoma. comparably, eif2 and eif4e were present in the nuclei and cytoplasm of brain tumor cells, with a higher concentration of eif2 in the nuclei of gastrointestinal cancer tumor cells [87, 88 ]. expressions of eif2 may relate to abnormal protein synthesis, furthering its role in tumorigenesis. as the main effector of both global translation and translation of specific subsets of mrnas, eif2 continually shows potential for cancer therapeutics and treatments. eukaryotic initiation factor 3 (eif3) is a 13-subunit complex of 800 kilodaltons, required for translation initiation through interactions with the 40s ribosomal subunit, mrna, and other eifs necessary for the formation of competent translation initiation complexes [19, 114 ]. eif3, along with eif1a and plausibly eif5, associates with the ternary complex and the 40s ribosomal subunit to form the 43s preinitiation complex. eif3 enhances the stability of 43s preinitiation complex through eif3-eif4 g interaction [1, 19, 89 ]. primarily, eif3j regulates eif3 interaction with the mrna - binding cleft on the 40s subunit by inhibiting mrna entry and confirming met - trnai is present in the p - site [115117 ]. interestingly, beyond the protein synthesis related functions of the eif3 complex, dysregulation of eif3 subunits has been implicated in several types of cancers [70, 90 ]. variations in the levels and activity of eif3 subunits are a result of upstream signaling molecules such as protein kinases, involving phosphorylation of eif3 subunits. for example, the mammalian target of rapamycin complex 1 (mtorc1), a key protein kinase involved in the regulation of protein synthesis, facilitates interaction between paip1 and eif3. the eif3 g subunit directly interacts with paip1 in an rna - independent manner, which enhances paip1-mediated translation stimulation in vivo [111, 118 ]. stimulation of mtorc1 (e.g., by amino acids) phosphorylates s6k, which ultimately stimulates interaction between eif3 and paip1. this paip1-eif3 interaction is also proposed to stabilize the conformation of circularized mrna by stimulating the eif4g - pabp interaction [111, 118 ]. moreover, mtor inhibition by rapamycin and pp242 (inhibitor of mtorc1 and mtorc2) significantly decreased s6k1 phosphorylation and subsequently diminished this paip1-eif3 interaction [111, 118 ]. evidence for the direct association of mtor and s6k1 with eif3 points to probable effects on translation. specifically, studies have shown that the eif3 complex, as found on the translation preinitiation complex (eif3-pic), functions as a scaffolding structure which is associated with mtor by mitogen / hormone stimulation, whereas s6k1 dissociates from this complex upon similar stimulation. immunoprecipitation experiments in the immortalized human embryonic kidney cells (hek293e / t) have shown that s6k1 associates with eif3b, eif3c, eif3e, and eif3f and mtor coimmunoprecipitates with eif3c [94, 111 ]. t cells are derived from the original hek293e cell lineage and have been modified to allow for transient transfection of vectors containing the sv40 origin of replication. interestingly, in nutrient - deprived conditions, the s6k1-eif3 association is observed, whereas the addition of amino acids diminishes this direct interaction. notably, with insulin treatment, s6k1 dissociates from the eif3-pic, whereas mtor is associated with this complex. this insulin stimulation results in an increase in cap - dependent translation, suggesting the mtor - eif3-pic association and the subsequent series of phosphorylation events are critical for efficient protein synthesis. in addition to the critical function of the eif3 complex in translation initiation, many eif3 subunits have been shown to be involved in a diverse set of cellular processes including apoptosis, oncogenesis, and cellular growth and proliferation [89, 100, 109, 114, 120122 ]. upregulation of eif3a, eif3b, eif3c, eif3d, eif3e, eif3h, and eif3i along with reduced levels of eif3e and eif3f has been observed in several cancers (table 1). most notably, increased levels of the largest eif3 subunit, eif3a, have been found in breast, cervix, esophagus, lung, and stomach cancers [89, 90 ]. the mechanism by which upregulation of eif3a promotes the malignant phenotype in lung cancer (h1299) and breast cancer (mcf7) cells involves enhancing dna synthesis for maintaining cell proliferation. during the s phase of the cell - cycle, eif3a upregulates the translational expression of ribonucleotide reductase m2, which in turn maintains high levels of dna synthesis [89, 91 ]. when eif3a is downregulated by using antisense eif3a cdna, ribonucleotide reductase m2 levels (and, subsequently, dna synthesis) are significantly reduced. furthermore, independently downregulating ribonucleotide reductase m2 expression levels has shown to reduce the extent of malignancy in human cancer cells. these findings delineate the mechanism of how high expression levels of eif3a maintain cell proliferation in cancer cells. additionally, eif3a is also known to enhance phagocytosis during apoptosis by facilitating the association between apoptotic cells and macrophages. overexpression of eif3b has been observed in breast, bladder, and prostate cancers ; however, the specific mechanism through which upregulated eif3b promotes the cancer state is still unclear. increased eif3c transcript levels have been found in human testicular seminomas as well as increased eif3c gene expression in colon cancer cells. interestingly, eif3c also directly binds to the neurofibromatosis 2 (nf2) tumor suppressor protein, schwannomin, in sts26 t schwannoma cells. schwannomin is thought to employ its tumor suppressive functions by binding eif3c and inhibiting eif3c - mediated cell proliferation, possibly due to the role of eif3c during protein translation initiation. additionally, in meningiomas, which have significantly reduced levels of schwannomin, eif3c is upregulated, suggesting a role of eif3c in tumor growth and proliferation. furthermore, overexpression of eif3c or eif3h resulted in the enhanced translation of cell proliferation mrnas encoding growth - regulating cyclin d1, c - myc, fibroblast growth factor 2, and ornithine decarboxylase (odc). this enhancement of translation rates for these proteins and subsequent production of malignant phenotypes may not be a direct consequence of the overexpression of a single eif3 subunit since enhanced levels of other eif3 subunits (a, b, c, f, h, and j) were also noted. thus, the overexpression of eif3a, eif3b, or eif3c subunits stimulates the expression of other eif3 subunits that further supports the translational components necessary for faster cancer cell growth. like other eif3 subunits, eif3d is also involved in protein synthesis and has been shown to be upregulated in gastric cancer and mesothelioma [70, 104 ]. studies using lentivirus - mediated rna interference to knockdown eif3d in the colon (hct116) and non - small cell lung cancer cells (nsclc a549 and 95d) showed significantly reduced cell proliferation (induced apoptosis) and inhibited colony formation due to induced cell - cycle arrest in the g2/m phases. notably, in hct116 cells, eif3d knockdown resulted in phosphorylation of ampk, bad, pras40, sapk (stress - activated protein kinase)/jnk, gsk3, and parp [poly(adp - ribose) polymerase ] cleavage. phosphorylation of bad, a proapoptotic protein, induces apoptosis while phosphorylation of gsk3 promotes reovirus - induced apoptosis. furthermore, parp cleavage is typically used as a signal for apoptosis induction. eif3d knockdown in nsclc cells resulted in decreased phosphorylation and thus inhibition of akt, hsp27, and sapk / jnk (involved in cellular growth and cancer progression pathways). this data supports the crucial role of eif3d in cell proliferation and cancer growth [104, 105 ]. in breast cancer cells, reduction of eif3e expression by rnai induces emt (epithelial - to - mesenchymal transition), suggesting a role of eif3e in breast cancer metastasis. this study and others [108, 124 ] suggest that eif3e normally functions as a tumor suppressor since the reduction of its expression results in enhanced mrna stability and expression of the transcription factors and emt regulators, snail1 and zeb2. this suggests that the loss of eif3e directly results in cancer progression and metastasis as emt is induced in breast cancer cells. conversely, another report has shown that eif3e functions as an oncogene. in this study, the knockdown of eif3e using rnai in u2os and mda - mb-231 resulted in a reduction in protein levels of bcl - xl (antiapoptotic protein) and urokinase - type plasminogen activator (plau) but an increase in mad2l1 (mitotic checkpoint component). overabundant bcl - xl protein levels are associated with chemoresistance in cancers while plau functions in promoting metastasis in tumors. interestingly, bcl - xl mrna associates directly with the eif3 complex in an eif3e - dependent manner as determined by rna ip. following eif3e knockdown, the specific changes in protein levels of the mentioned eif3e targets, without any changes in global protein synthesis, suggest that eif3e specifically regulates translation of mrnas involved in tumorigenesis. furthermore, eif3e gene silencing using sirna in glioblastoma results in cell - cycle arrest in the g1 phase, decreases cell proliferation, and induces both caspase - dependent and caspase - independent apoptosis. furthermore, a recent study suggests the role of eif3d - eif3e module within the eif3 complex that regulates the translation of specific mrnas involved in maintaining metabolic pathways that are likely disrupted in cancer cells. critical components of the mitochondrial electron transport chain were downregulated in both yeast and mammalian cells (nontumorigenic : mcf-10a and nontumorigenic : mcf7) that were eif3e - depleted using sirna, whereas glucose metabolism and amino acid biosynthesis processes were upregulated. their findings suggest that depletion of eif3e triggers a metabolic switch that increases dependence on glycolysis, as respiratory deficiencies alongside increased sensitivity to oxidative stress are also observed when eif3d is depleted in addition to eif3e knockdown. essentially, this data suggests that the novel function of eif3d - eif3e in maintaining mitochondrial respiration components and serving to adjust metabolic pathways may help us better understand how the cancer - promoting properties of the eif3 complex emerge. unlike eif3e, eif3f is consistently shown to function as a tumor suppressor in pancreatic cancer. endogenous levels of both eif3f mrna and protein levels are reduced in pancreatic cancer cells, whereas eif3f - overexpressing nih3t3 cells have shown reduced cell proliferation and induced apoptosis. likewise, eif3f knockdown in normal human pancreatic epithelial cells has shown an increase in cell proliferation and increased resistance to apoptosis. by utilizing a bicistronic luciferase report system, it was shown that eif3f normally inhibits both cap - dependent and cap - independent (i.e., ires) mechanisms of translation initiation. furthermore, one mechanism of translation inhibition likely involves eif3f - mediated rrna degradation by a direct eif3f - hnrnp k (rna - binding protein) interaction. specifically, when eif3f is present, it binds to hnrnp k, preventing it from binding to rrna, which subsequently is degraded, and the translation is reduced. in cancer cells, the loss of eif3f results in an increased binding of hnrnp k to rrna, reducing rrna degradation, and possibly favoring oncogenesis through increased translation. these findings suggest that eif3f functions as a negative regulator of cell growth due to the naturally reduced levels of eif3f contributing to cancer development and overexpression resulting in apoptosis. despite the vast quantity of correlative evidences between eif3 subunit expression levels and observed cancer phenotypes, it has been difficult to establish a clear mechanism of how cancer progression is directly affected by eif3 expression. understanding the interactions of the eif3 subunits with antiapoptotic proteins, cellular growth and proliferation proteins, and oncogenic proteins may provide a better understanding of how eif3 subunits contribute to supporting or inhibiting the oncogenic state. a functional mechanism will most likely involve aspects of translation initiation, preinitiation complex formation, and other processes, such as the recruitment of the preinitiation complex to the cellular mrna and ribosome scanning, all of which are mediated to some degree by a subset of eif3 subunits. additionally, studies depicting eif3 as both an activator (for the protooncogene c - jun) and repressor (for the negative regulator of cell proliferation btg1) of cap - dependent translation, mediated by binding to specific rna structural elements, illustrate the diversity of functions carried out by the eif3 complex through interactions with proteins and nucleic acids. future studies will need to focus on determining the signaling pathways that are involved in regulation of eif3 and the consequences of eif3-directed therapeutics for human cancers. this information will assist in the development of novel therapeutics that target eif3 subunits in order to treat cancers and possibly other human diseases. eif4e the interaction with eif4 g is inhibited by binding of eif4e - binding protein (4e - bp) to eif4e. eif4 g and 4e - bp are known to compete for a common binding site on eif4e. the eif4e and 4e - bp interaction is highly regulated via a phosphorylation reaction and acts as a primary target for hindering translation initiation during stress conditions. activation of the mtor pathway phosphorylates 4e - bp and enhances cap - binding efficiency of eif4e. in contrast, inhibition of the mtor pathway by amino acid starvation dephosphorylates 4e - bp, resulting in increased association between 4e - bp and eif4e, thus repressing cap - dependent translation. overexpression of 4e - bp leads to a decrease in the mrna and protein levels of cyclin d1 and an increase in p27, a cell - cycle regulatory protein that promotes cell - cycle arrest in breast cancer cell lines. it localizes to the nuclear bodies by interacting with the eif4e - transporter protein (4e - t). in the nuclear bodies, it remains associated with promyelocytic leukemia proteins that share a common binding site with 4e - t and 4e - bp. thus, an increase in 4e - bp may affect the mrna transport function of eif4e from the nucleus to the cytoplasm, resulting in differential expression of cyclin d1. also, dysregulation of 4e - bp phosphorylation is correlated with poor prognosis in lung cancer, breast cancer, melanoma, cervical carcinoma, and astrocytoma [131, 132 ]. eif4e is phosphorylated at ser209 by p38 mitogen - activated protein kinase (mapk). p38 mapk phosphorylates mnk, a serine / threonine kinase, and enables mnk interaction with eif4 g and phosphorylation of eif4e. induction with transforming growth factor (tgf) increases eif4e phosphorylation alongside mesenchymal markers such as n - cadherin, fibronectin, and vimentin, as a result of the noncanonical signaling pathway. as a downstream target of p38, this eventually leads to the upregulation of snail and matrix metalloproteinase 3, promoting cell invasion and metastasis. phosphorylation of eif4e is not an absolute requirement for translation but is observed to increase the rate of translation initiation. genome - wide studies of translating mrna have indicated that eif4e phosphorylation is necessary for synthesizing proteins essential for tumorigenesis, and the levels of eif4e are critical for antiapoptotic protein expression [135137 ]. in nasopharyngeal carcinoma, the latent membrane protein 1 enhances transcription of many oncogenes such as vegf, c - myc, and matrix metalloproteinases (mmp). eif4e promoter activity is enhanced by c - myc and, as a feedback mechanism, eif4e increases translation of these oncogenes. further, overexpression of eif4e leads to increased expression of a subset of proteins, influencing angiogenesis and tumor progression (vegf and fibroblast growth factor-2 (fgf-2)), growth stimulation (platelet - derived growth factor), prosurvival (bcl-2 and bcl - xl), cell - cycle progression (c - myc, cyclin d1, and ornithine decarboxylase), epithelial - to - mesenchymal transition (snail and mmp), and invasion (integrin 1) [134, 137, 139142 ]. besides the role of eif4e as a cap - binding protein, it is reported to stimulate the helicase activity of eif4a and aid translation of mrnas comprising long, structured 5 utrs. a subset of transcripts harbors a 12-nucleotide motif (cgg)4 called the g - quadruplex in their 5 utrs. some examples of mrnas including such structures are vegf, bcl-2, and nras. these mrnas are sensitive to silvestrol, a drug that inhibits the helicase activity of eif4a, thus indicating the role of eif4a, discrete from mtor - dependent translation regulation [144146 ]. ribosome footprinting and polysome profiling experiments have detected more than 250 genes, including myc, mdm2, cdk6, and afr6 that are affected by silvestrol treatment making it a promising drug to treat cancer [146, 147 ]. in the case of breast cancer mucin 1 forms a complex with egfr and activates the pi3 k - akt - mtor pathway. akt and p70 s6k along with activation of mek - erk signaling promote ubiquitination and degradation of pdcd4. this favors translation, as the ma-3 protein binding domain of pdcd4 is known to interact with eif4a n - terminal domain and inhibit its helicase activity. activation of the helicase activity stimulates the autoinductive pathway and increases levels of oncogenic proteins [56, 148, 150 ]. further, pdcd4 inhibits eif4a - eif4 g interaction by interacting with eif4a as well as eif4 g. colocalization of eif4a and pdcd4 in the cytoplasm inhibits eif4a helicase activity [151, 152 ]. eif4a also interacts with the paip1 domain, containing sequence similarity to eif4 g, and promotes circularization of the mrna, affecting protein expression involved in apoptosis. as mentioned earlier, eif4 g interacts with eif4e, eif4a, and eif3 to form the preinitiation complex. eif4 g consists of two binding sites for eif4a and a single binding site for eif4e, eif3, pabp, mnk1, and rna, which interact independently of each other. consequently, it increases the cap - binding activity of eif4f and joining of the 60s ribosomal subunit to the 40s subunit [155157 ]. interaction of eif4 g with eif4e enhances eif4e - mediated cap recognition and the cap - binding activity of eif4f complex. therefore, hoxa9 is displaced from eif4e in the cytoplasm to initiate translation. during translation, eif4 g and eif3 interactions bring the ternary complex onto the mrna and stabilize the 43s preinitiation complex. these interactions are mediated by the 3c, 3d, and 3e subunits of eif3 complex. activation of mtor by insulin, amino acids, or growth factors influences the direct interaction between mtor and eif3 and increases eif4g - eif3 interaction. eif5 is a 49 kda protein in mammals and 46 kda in saccharomyces cerevisiae [158, 159 ]. eif5 interacts with the 40s initiation complex to mediate hydrolysis of eif2-bound gtp. this step is critical to initiation complex formation, as the release of multiple eifs including eif3, eif4e, and eif2-gdp is necessary for the recruitment of 60s ribosomal subunit. in higher eukaryotes including humans, eif5 has a 9-residue c - terminal tail that can bind to the eif5b - ctd subdomain. this is opposed to the binding of the eif1a c - terminus to eif5b - ctd, which is a relatively weak interaction in both humans and yeast. the interaction between eif1a and eif5b coordinates recruitment and release of one another in s. cerevisiae. however, in humans, eif1a / eif5b interaction facilitates subunit joining, and recruitment is coordinated separately through eif5/eif5b interaction. eif5 is a downstream target of casein kinase 2 (ck2), which phosphorylates eif5 at the major sites : ser389 and ser390. ck2 has a significant role in cell proliferation, and both ck2 and eif5 have critical roles in cell - cycle control and progression. the ck2 enzymatic activity also increases and phosphorylates eif5, 3 hours after serum stimulation of cell - cycle arrested (g0) human embryonic kidney hek-293 cells and normal human fetal lung fibroblasts tig-7 cells, suggesting a role for ck2 and eif5 in promoting cell proliferation. in tig-7 cells, eif5 was associated with ck2. when ck2 levels were highest and when eif5 mutants were unable to be phosphorylated by ck2, there was a decrease in growth rate, mature translation initiation complex formation, and expression of cell - cycle - regulated proteins. nuclear ck2 (catalytic subunit) localization is a sign of poor prognosis in prostate cancer and gastric carcinoma [163165 ]. ck2s phosphorylation targets include deleted in breast cancer 1 (dbc1), eif5, and endothelin - converting enzyme-1c (ece-1c), which promote cancer cell invasion and progression [163165 ]. depletion of eif5 in s. cerevisiae resulted in the inhibition of cell growth and a decrease in the rate of in vivo protein synthesis. in yeast, eif5 is able to mimic the effect of eif2 phosphorylation, acting as a translational inhibitor and promoting translation of prooncogenic protein gcn4 (yeast equivalent of atf4) [166, 167 ]. when overexpressed in s. cerevisiae, eif5 increases the levels of an eif2/eif5 complex, which prevents eif2b interaction and subsequently prevents ternary complex formation. human 5mp1 protein was found to interact with human eif2s subunit, similarly to eif5, eif2b, and kra. furthermore, in vitro, eif2 demonstrated mutually exclusive interactions with eif5 and 5mp1, suggesting 5mp1 as a competitive inhibitor of eif5. 5mp promotes expression of gadd34 (a downstream target of atf4) in tribolium castaneum. further, 5mp binds eif2 to inhibit general translation and when overexpressed, it promotes atf4 expression in fibrosarcoma [166, 169 ]. atf4 is expressed in hypoxic- and nutrient - deprived tumor regions, with functions in development, promoting metabolic homeostasis, and cancer cell proliferation. eif5 demonstrates critical roles in cell - cycle regulation and cell proliferation with specific oncogenic protein interactions. eif5a is a 17 kda protein that is activated by posttranslational hypusination and functions to mediate cell proliferation, apoptosis, and inflammatory response [171, 172 ]. hypusination is unique to eif5a and is a posttranslational enzymatic modification that involves two sequential enzymes and the substrate spermidine. deoxyhypusine synthase (dhs) catalyzes nad - dependent cleavage and transfer of an aminobutyl moiety of spermidine to the -amino group of a conserved lysine of eif5a. the resulting intermediate residue, deoxyhypusine, is hydroxylated by deoxyhypusine hydroxylase (dohh), yielding a hypusine residue, and an active eif5a. new treatments for chronic myeloid leukemia utilize hypusination inhibitors to deactivate eif5a, creating a target in response to imatinib resistance towards the bcr - abl tyrosine kinase. inhibition of eif5a results in an antiproliferative effect on bcr - abl positive- and negative - leukemia cell lines in vitro. eif5a is overexpressed in murine pancreatic intraepithelial neoplasia (panin) and in human pancreatic ductal adenocarcinoma (pdac). pharmacological inhibitors n(1)-guanyl-1,7,-diamineoheptane (gc7) and ciclopirox olamine (cpx) are able to inhibit dhs and dohh, respectively, which further inhibits eif5a hypusination and results in eif5a genetic knockdown. genetic knockdown of eif5a inhibited pdac cell growth in vitro and orthotopic tumor formation in vivo, potentially through pseudopodium - enriched atypical kinase 1 (peak1), which is essential to pdac tumor growth, metastasis, and gemcitabine resistance. in melan - a (a murine melanocyte cell line) and tm5 (a murine melanoma cell line derived from melan - a), gc7 was used to inhibit eif5a. more pronounced dna fragmentation was observed in tm5 cells and decreased viability was observed in both cell lines. additionally, treatment with gc7 was tested on melanoma growth in c57bl/6 mice and found to inhibit further tumor growth, although it did not induce volume reduction of established tumors. eif5a 's isoform eif5a2 is upregulated in various cancer types including hepatocellular carcinoma, ovarian carcinoma, and colorectal carcinoma (crc) [176178 ]. eif5a2 overexpression in cancer cells is correlated to prognosis factors of tumor metastasis and venous infiltration [176, 177 ]. in ovarian carcinoma, overexpression of eif5a2 was detected in 7% cystadenomas, 30% borderline tumors, and 53% invasive carcinomas, as opposed to normal expression in normal ovaries. in crc, eif5a2 upregulates metastasis - associated protein 1 (mta1) by increasing the enrichment of regulator gene c - myc on mta1s promoter. this both increases cell motility and invasion in vitro and results in tumor metastasis, inducing epithelial - mesenchymal transition and further promoting crc aggressiveness. in hepatocellular carcinoma cells in vivo, eif5a2 suppression attenuates tumorigenic properties [176, 177 ]. eif5as unique activation by hypusination creates a desirable mechanism of regulation and knockdown within various cancer lines. eif5b is a 175 kda protein that promotes 60s ribosome subunit joining and pre-40s subunit proofreading and can indirectly support the trna - meti association with the ribosome in translation initiation [179181 ]. in serum - starved thp1 cells, elevated eif5b levels resulted in increased eif5b complexes with trna - meti, as well as increased phosphorylation of eif2. these eif5b complexes are formed during times of attenuated global translation, instead of promoting translation of specific stress - related mrnas. eif5b is antagonistic of g0 and g0-like states, with eif5b overexpression promoting maturation and cell death. eif5b depletion is associated with increased phosphor - cdc2, which is a marker for immaturity. this suggests a critical role for eif5b in the regulation of cell - cycle transitions. eif5b interacts with dead - box rna helicase vasa (vas), with reduction of vas - eif5b interaction in drosophila causing female sterility, reduced gurken (grk) protein levels, nearly complete loss of germ cell formation, and reduction of somatic posterior patterning. eif5b interaction with vas is necessary for early drosophila development through the progression of oogenesis and pole plasm assembly, suggesting eif5b as a potential method to regulate vas and subsequently grk. in poliovirus (pv) and coxsackie b virus (cbv) infection of cultured cells, eif5b is proteolytically cleaved, suggesting that eif5b cleavage is involved in the translation inhibition within enterovirus - infected cells. infection of enteroviruses pv and cbv begins with inhibition of host cell translation, followed by ires - directed enterovirus protein synthesis, with eif5b cleavage beginning at 3 hours after infection, during the attenuation of host cell protein synthesis [183, 184 ]. eif5b, eif5, and eif5a all contain links into hallmark stages and mechanisms of cancer proliferation, and manipulation of these proteins offers a potential regulation point of gene expression regulation. mechanisms utilizing elements of protein translation, specifically in the rate - limiting step of initiation, offer potential methods to diagnose and treat cancer. translational regulation is capable of efficiently altering specific protein levels in physiological stress conditions that are typical in cancer. translation is mediated by eukaryotic initiation factors (eifs), which have varying roles in regulating the rate of initiation as described in this review. the critical role of eif2 phosphorylation has been classified in the integrated stress response, with the requirement of eif4f complex for cap - binding and efficient translation through eif4e and eif4 g. other mechanisms include eif3 subunit interactions with s6k1 and mtor and eif5a 's necessary activation through posttranslational hypusination, important to the mediation of cell proliferation, apoptosis, and inflammatory response. varying levels of eifs in various cancer lines and stages, along with the mechanistic background, enforce the use of eifs to regulate gene expression in cancer. during cellular stress induced by cancer, noncanonical translation utilizing uorf elements or ires elements drives translation of specific stress response and adaptation proteins. proteins such as atf4 and gcn4 have critical roles in the integrated stress response and help determine whether cell proliferation ensues. the eifs required for ires - dependent translation are specific to the mrna in question, with a subset of cellular ireses not requiring eif4 g and eif4a, while l - myc requires the full eif4f complex and pabp. ires - dependent translation, which specifically favors about 10 percent of cellular mrnas under cellular stress, offers potential gene regulation targets, including various eifs and itafs with critical implications for cancer treatment. the mechanisms underlying both canonical and noncanonical translation and the proteins responsible for the processes, are critical in treating the dysregulated gene expression in cancer. however, these studies are often limited in terms of what one can extract from them, since the observed phenotypical changes in nearly all levels of complexity, starting from the cellular level to the whole organism level, tend to be complicated by several other protein - protein and possibly protein - nucleic acid interactions. future studies will need to continue building on the present data by taking mechanistic approaches in elucidating the signaling pathways, transcriptomic, translatomic, and proteomic profiles of patient samples and model systems to determine the appropriate methodology to target the function of specific regulators (e.g., proteins like initiation factors) to produce effective novel therapeutics that have intrinsically high specificity. currently, there are several drugs and antisense oligonucleotides being tested against the initiation factors to increase mortality of cancer cells. initiation factors, being a common element across various types of translation, hold great potential to be tested for rna - based therapeutics as well as chemical compound - based therapeutics to treat cancer. | protein synthesis can be segmented into distinct phases comprising mrna translation initiation, elongation, and termination. translation initiation is a highly regulated and rate - limiting step of protein synthesis that requires more than 12 eukaryotic initiation factors (eifs). extensive evidence shows that the transcriptome and corresponding proteome do not invariably correlate with each other in a variety of contexts. in particular, translation of mrnas specific to angiogenesis, tumor development, and apoptosis is altered during physiological and pathophysiological stress conditions. in cancer cells, the expression and functions of eifs are hampered, resulting in the inhibition of global translation and enhancement of translation of subsets of mrnas by alternative mechanisms. a precise understanding of mechanisms involving eukaryotic initiation factors leading to differential protein expression can help us to design better strategies to diagnose and treat cancer. the high spatial and temporal resolution of translation control can have an immediate effect on the microenvironment of the cell in comparison with changes in transcription. the dysregulation of mrna translation mechanisms is increasingly being exploited as a target to treat cancer. in this review, we will focus on this context by describing both canonical and noncanonical roles of eifs, which alter mrna translation. |
medullary thyroid carcinoma (mtc) is a neoplasia of parafollicular cells or of thyroid c - cells, which represents approximately 4% of thyroid malignant tumors. they can be part of multiple endocrine neoplasia (men) type 2, 2a or 2b, or occur as a familial mtc or other inherited forms. molecular studies to identify genetic mutation responsible for mtc started in 1970 and, in 1993 and ret proto - oncogene was identified as the responsible for occurrence of this neoplasia. among various substances produced by c cells the most important one is calcitonin that is used for detection, post - operative follow - up and evaluation of individuals at risk of developing mtc. prognosis of mtc is related to the cure of the disease at the moment of the diagnosis, and this disease is usually diagnosed when there is already a metastatic involvement. it is estimated that 0.4 to 1.37% of patients with thyroid nodules have mtc. as an attempt to increase the number of diagnoses at an earlier stage, in 1994, the routine use of calcitonin in the evaluation of thyroid nodules was proposed. since then the role of serum calcitonin in the evaluation of thyroid nodules has been widely discussed and there is still no consensus about the administration of blood calcitonin measurement in the initial evaluation of all thyroid nodules. problems related to : cost - benefit, lab evaluation methods, false positives and low prevalence of medullary thyroid carcinoma are supported by authors who consider it unnecessary to include calcitonin in the evaluation of a thyroid nodule. a higher expectation of cure for a neoplasia that usually has a late diagnosis, and whose prognosis can be altered by its early detection, is the main reason that justifies serum calcitonin levels measurement as a screening test for mtc in all thyroid solid or cystic - solid nodules. studies that have analyzed the role of calcitonin measurement in the investigation of thyroid nodules applied a strategy that has used levels of basal calcitonin followed by a stimulation test for calcitonin (usually pentagastrin) in cases with high serum calcitonin levels, for a more specific confirmation. high calcitonin levels can be detected in other thyroid and extra - thyroid pathologies, neoplastic or not. the stimulation test seems to provide a more specific diagnosis, but false positive results may also occur. a 32-year - old, female, white, brazilian patient has sought medical advice with an endocrinologist as she noticed a lump in the cervical region. thyroid palpation indicated an enlargement of the gland of about three to four times with irregularities in the lower left third and a palpable nodule in the lower right third. ultrasound evidenced two nodules in the left lobe (0.6 0.9 and 1.0 1.0 cm), the lower third of the last one demonstrated hyperechoic and hypovascularized region. a solid nodule was also found next to the lower pole of the left lobe with no continuity to it, of 1 cm the diameter. normal levels of serum tsh and free t4, total and ionic serum calcium, inorganic phosphorus and chlorine were showed, and anti - thyroglobulin and anti - thyroperoxidase antibodies were reactants. calcitonemia was 10 pg / ml (normal levels= 40 pg / ml). recently, karanikas., have reported a prevalence of 25% of increase in serum basal calcitonin levels in patients with hashimoto thyroiditis, indicating a significant relation between nonnodular thyroid disease and high levels of serum calcitonin. this author proposed measurement of calcitonin not only in patients with thyroid nodules but also in patients with ht, what we do not agree. irrespective of the results from the discussion this proposal may cause, the occurrence of ht is a relevant factor to hypercalcitonemia by not all established mechanisms, and may lead to false positive results when measurement of calcitonin, basal or stimulated, is performed as a screening method. one hypothesis to hypercalcitonemia is that c cells response may be neoplastic or physiological. in ht, as well as in other situations related to the increment in serum calcitonin levels, either a physiological or a reactive response seems to occur, different from the response observed in hcc related to mtc. in these situations, basal calcitonin may be high, but rates after stimulation are usually lower than the ones observed in cases of mtc. in the second case, the patient also indicated high serum calcitonin levels, in two evaluations, using immunoradiometric assay from kits available. stimulation test using calcium evidenced high rates, suggesting medullar neoplasia (890 pg / ml). however, anatomopathologic diagnosis evidenced adenomatous goiter in eleven nodules, papillary thyroid microcarcinoma in one nodule and diffuse c cells hyperplasia (cch). we believe, in this case, that this huge increment is a result of c cells hyperplasia. hcc is a known reason for increases in levels of stimulated and basal calcitonin, what frequently makes it difficult to differentiate this situation from cases like medullar microcarcinoma and mtc itself. gibelin considers that in cases when preoperative rates of stimulated calcitonin range between 30 and 100 pg / ml it is difficult to predict the presence of hcc or of medullar microcarcinoma, but he considers that rates higher than 100 pg / ml are strong evidences of mtc. differentiated thyroid tumors may cause increase in serum calcitonin, similar to the one observed in nonneoplastic diseases therefore much lower than that evidenced in mtc cases. the main concern here is the rate of increase in calcitonin after the stimulation test. there is no established rate that can biochemically differentiate c cells hyperplasia from mtc. as an attempt to answer this question and to determine a biochemical rate that could differentiate c cells hyperplasia from mtc, scheuba., demonstrated levels of basal calcitonin higher than 64 pg / ml or a rate of stimulated calcitonin higher than 560 pg / ml in 31 of 38 patients that underwent a thyroidectomy with histological diagnosis of medullar carcinoma, assuming that these rates are a prediction of medullar carcinoma in 81% of the cases. they also observed that patients with stimulated calcitonin lower than 129 pg / ml had only hyperplasia of c cells and not medullar carcinoma. among patients with medullar carcinoma, the ones who indicated basal calcitonin lower than 22 pg / ml did not have positive lymph nodes. iacobone., proposed, in 2002, cut - off rates for basal and after stimulation calcitonin, indicating that basal rates higher than 30 pg / ml and post - stimulation rates higher than 200 pg / ml have 61.3% sensitivity, 100% specificity and 100% positive prediction value. in that study, cases diagnosed as mtc, rates of basal calcitonin ranged between 145 and 205 pg / ml. routine use of serum calcitonin followed by a confirming stimulation test in the cases of high basal levels is the most recommended approach, due to inefficiency of fna and lack of better data on the role of ultrasound for mtc. questions concerning cost - benefit and the possibility of false positives may limit this recommendation, but it is clear that this approach increases the number of diagnosis of mtc. although there still are many questions regarding administration of calcitonin, this is a valid, factual and relatively simple method that allows the preoperative diagnosis of mtc. one should be careful when there is evidence of a condition different from mtc that is known to increase calcitonin or when there is a slight increase in the levels of calcitonin, basal or after stimulation. subsequent studies should be carried out to establish appropriate rates that could separate benign from malignant diseases, in order to reduce possible errors. | the role of serum calcitonin as part of the evaluation of thyroid nodules has been widely discussed in literature. however there still is no consensus of measurement of calcitonin in the initial evaluation of a patient with thyroid nodule. problems concerning cost - benefit, lab methods, false positive and low prevalence of medullary thyroid carcinoma (mtc) are factors that limit this approach. we have illustrated two cases where serum calcitonin was used in the evaluation of thyroid nodule and rates proved to be high. a stimulation test was performed, using calcium as secretagogue, and calcitonin hyper - stimulation was confirmed, but anatomopathologic examination did not evidence medullar neoplasia. anatomopathologic diagnosis detected hashimoto thyroiditis in one case and adenomatous goiter plus an occult papillary thyroid carcinoma in the other one. recommendation for routine use of serum calcitonin in the initial diagnostic evaluation of a thyroid nodule, followed by a confirming stimulation test if basal serum calcitonin is showed to be high, is the most currently recommended approach, but questions concerning cost - benefit and possibility of diagnosis error make the validity of this recommendation discussible. |
although healthcare is not only medical - care, the part played by medical services is an essential determinant of overall health of a population. all public systems look for an organizational structure on how to channel 710% of the national income into healthcare services. however, most healthcare systems fall under one of two broad categories (table 1). despite the expansion of medical knowledge and the use of increasingly sophisticated technology and training, healthcare systems in most countries of the world are considered to be in crisis as most of them are underperforming systems [24 ]. broad categories of current healthcare systems in spite of the apparent public health well - being in libya, the libyan health system is a low responsive, inefficient and underperforming system which lacks goals and/or smart (specific, measurable, achievable, realistic, time specific) objectives. many of the improvements that occurred in the last few decades were due to public actions from outside the health sector rather than clear vision from within the healthcare system. these public measures include education particularly of females, food subsidy policies, and increasing purchasing power. lessons from better performing healthcare systems around the world are needed in our efforts to reorganize and improve our healthcare services. in a who report evaluating healthcare systems of different nations (2000), the french health care system (fhcs) ranked first among 191 member countries surveyed. france was described as having a technically efficient and generous healthcare system that provides the best overall health care. aspects examined in this evaluation included universal coverage, equity, distribution of costs, responsiveness of healthcare providers, patient satisfaction, patient and provider freedoms, and the health and longevity of the population. according to the same report, the best health system in the near and middle - east region was oman (8 position), while saudi arabia, united arab emirates, and morocco were in the 26, 27 and 29 positions respectively. another credit to the fhcs came from the european health consumer powerhouse. in their 2006 report, the fhcs ranked first among the eu member states according to the euro health consumer index (ehci). these include aspects as patient rights, information, waiting times for treatment, outcomes, generosity and pharmaceuticals. in different ehci reports, top performing countries are those which have a long tradition of plurality in healthcare financing and provisions, i.e. with a consumer choice between different insurance providers, who in turn do not discriminate between providers who are private for - profit, non - profit or public. these countries have adopted a bismarckian healthcare system. there is relatively a large gap of points of these countries to the first beveridge country, which is in sixth place. although france performed relatively less well (third and fifth position) in later reports, very subtle changes in single scores modify the internal order of the five top countries. canada, libya and most of the healthcare systems in our part of the world follow the british system which has been a notoriously mediocre performer since the start of the ehci. libya ranked 87th in the who report, canada places 23rd out of 30 in the latest edition of the ehci. canada spends more money to achieve worse results than any other country in the lowest quartile of the ehci. these achievements make the fhcs an interesting case and a practical model on how to organize many of the essential aspects of modern national health services that we have discussed in our previous works [5, 8 ]. it plays a direct role in the funding and provision of health care and regulates the relations between funding institutions, health professionals and patients (table 2). responsibility of the state in the french national health system the population in france is considered to be in good health. they consult their doctors more often, are admitted to hospital more often, and purchase more prescription drugs. the french had structured their system to be an ideal synthesis of solidarity and liberalism. although the state imposes strong price control policies on the entire health sector, there is no public perception in france that health services are rationed to patients. the system lies between britain 's nationalized health service, where there is too much rationing, and the united states competitive the fhcs is planned and organized in relation to the needs and expectations of the population. it has a strong commitment to a universal, obligatory, and solidarity method of health care delivery. there are many protective mechanisms built in so that cost sharing does not prevent people from receiving necessary care. it ensures equitable geographical coverage and an efficient interaction between the different players, i.e. hospitals (public and private), private practitioners, medical auxiliaries, the pharmaceutical industry, etc. there are no waiting lists for elective procedures and patients need not seek pre - authorizations. in line with the principle of social solidarity both citizens and non - citizen residents contribute according to their means and obtain services according to their needs. each person with social insurance benefits from a protection that he finances according to his resources against the risk and consequences of illness independently of his age or state of health. in some instances, the statutory health insurance system provides 100% coverage, as in the case of perinatal care and costs related to industrial injury and long - term illnesses, where social security pays the whole cost of life - long essential medications as insulin. some pharmacists use digital information systems to deduct sums reimbursed by social security. in the same manner, many are also linked to certain complementary insurance schemes. to insure equity, patients are exempt from payment when (1) expenditures exceed approximately $ 100, (2) hospital stays exceed 30 days, (3) patients suffer from serious, debilitating, or chronic illness, or (4) if income falls below a certain threshold, thereby qualifying them for free supplementary coverage or a complementary state - funded healthcare. the fhcs provides a high level of resources and a higher volume of service provided which covers prescriptions of homeopathic products, thermal cures, nursing home care, cash benefits, and to a lesser extent, dental and vision care. to assure access to health services, the national health insurance (nhi) in the fhcs covers fees for transport of patients to health care facilities. they cover private taxis for alternative interventions such as physiotherapy. for certain medical treatments such as non - routine dental care, contact lenses, non - standard lenses for glasses, certain laboratory and radiology tests, physiotherapy and speech therapy, and thermal and therapeutic treatments, one must obtain proper approval from social security. the card contains their social security number and covers all members of their family. through these cards, people are linked to a computerized system containing all medical records and vital information such as the card - holder 's blood type and allergies. the fhcs is based on a single nhi fund, which is part of france 's extensive social security system. initially modeled after the german sickness fund fostered by chancellor bismarck, the fhcs had evolved into a blend of bismarck and beveridge, public and private, centralization and decentralization [10, 11 ]. in contrast to germany where numerous sickness funds are regional or tied to professions, all residents in france are automatically enrolled with an insurance fund based on their occupational status. in addition, all residents can also subscribe to supplementary health insurance to cover other benefits not covered by nhi. health insurance is funded by contributions based on professional earnings (51.1%) and a tax levied on all income (including investment income) known as the general social contribution (34.6%). for professional earnings, remaining sources include special taxes on automobiles, tobacco, alcohol, and a specific tax on the pharmaceutical industry. for several years health expenditure france 's health expenditure in 2006 was 120.2 milliard euros, while the deficit was 6 milliard euros. official figures published in july, 2006, put french health care expenditure at 11.14% of gdp. this is third worldwide after usa and switzerland (uk < 8%, canada < 10%, germany < 11%, and us 14.6%). this figure represents a per capita health expenditure of 3,038. health expenditure in 2008 the health budget in libya during the year 2000 was 500 million libyan dinars (ld). this represented a per capita health expenditure of 121 ld, among which 45 ld were for medical supplies and drugs. the payment system is dominated by solo - based, fee - for service private practice for ambulatory care and public hospitals for acute institutional care. subscribing to health insurance is compulsory ; one may not opt out. for ambulatory care, all health insurance plans operate on the traditional indemnity - model reimbursement for services rendered. the entire population thus has health insurance coverage, generally on a work - related basis. in fact, patients could choose what physician to see, while physicians had almost unfettered freedom to prescribe tests and treatments. the fhcs offers a great deal of tolerance for organizational diversity, whether complementary, competitive, or both. this tolerance justifies the coexistence of public and private hospitals and both office - based private practice and public ambulatory care. service chiefs in public hospitals have the right to use a small portion of their beds for private patients. the tolerance justifies also the coexistence of multiple statutory health insurance plans, complementary private health insurance coverage, and significant cost sharing directly by patients. social security refunds 70% of the cost of an office visit to the treating physician and most specialists. patients are required to participate in health expenditures, with the aim of fostering consumer responsibility toward the cost of health care. unlike in the uk, both private and public practices are not free at the point of delivery in france. even if subscribed to social security, when consulting a physician or specialist, one has to first pay the full tariff and would only be reimbursed after - ward in full or in part by the patient 's complementary and /or private insurance company. the current fhcs is a product of a long history of development of its health establishments. the maison royale de sant (currently hpital saint - louis) was the first hospital in the modern - day sense. it forms now part of the current assistance public hospitals in paris (ap - hp), an important part of french teaching university hospitals (chu). these chus are among the best hospitals not only in france but in the world. french hospitals have significantly contributed to modern medicine as rene laennec 's invention of the stethoscope and louis pasteur whose work has saved millions of lives (table 3). major land - marks of contribution by french hospitals in modern medicine france boasts a higher number of hospital beds in proportion to its population than most european and other countries (8.7 per 1,000 compared with 7.6 in spain and italy, 6.9 in the uk and 3.7 in libya). these include hospital centers or short - stay hospitals (hpital de court sjour), medium stay centers (centre de moyen sjour) and long term treatment centers (centre et unit de long sjour). hospital centers include general hospitals, the ap - hp, specialist hospitals and regional centers (centre hospitalier rgional / chr or centre hospitalier universitaire / chu when associated with a university). medium stay hospitals contain facilities for convalescence, occupational and physical therapy, and recuperative treatment for drug and alcohol abuse and mental illness. long - term treatment centers are for those who are unable to care for themselves without assistance and include psychiatric hospitals, nursing homes for the aged and hospitals for children. private health care institutions include non - profit establishments as the red cross hospitals and for - profit establishments (private clinics). other health facilities include mother and child welfare services (pmi, services de protection maternelle et infantile) that are set up by the departments to provide regular check - ups for pregnant women and infants. their activities are neither industrial nor commercial and they may be municipal, departmental, interdepartmental or national in status. france 's private sector is the largest in europe and is accessible to all insured patients. proprietary hospitals are reimbursed on a negotiated per diem basis (with supplementary fees for specific services) and public hospitals (including private non - profit hospitals working in partnership with them) are paid on the basis of annual global budgets negotiated every year between hospitals, regional agencies, and the ministry of health. private hospitals and clinics that are non - contractual may also have an agreement with social security. both public and private hospitals will henceforth be funded through diagnosis - related payments, which are progressively being put in place. private practitioners are remunerated on a fee - for - service basis and are allowed to combine private practice with salaried work. many physicians work in private practice and also have the opportunity to work part - time in public hospitals. a number of agencies are set up to improve health safety and prevention, exercising some of the responsibilities of the state (table 4). agence national d'accrditation et dvaluation en sant has a responsibility to promote health care evaluation, to prepare hospital accreditation procedures, and to establish medical practice guidelines. it also sets up regional hospital agencies with authority to coordinate public and private hospitals and allocate their budgets. structures set up to improve health safety and prevention as part of the responsibilities of the state in the french national health system six percent (10.5 billion) of health expenditure was devoted to prevention in 2002. half of this was earmarked for disease prevention, one - quarter for screening and testing, and one quarter to cover risk factors. currently, the main strategic public health priorities include cancer, the environment, some rare diseases such as alzheimer 's, violence, abuse, risky behaviour, addictive behaviour, chronic illnesses, and quality of life. based on current expertise and resources, these strategic plans are broken down into 100 achievable targets. the french institute for public health surveillance (institut de veille sanitaire, invs) is a national organisation responsible for surveillance and alert in all domains of public health. this is comparable to centre of disease control in atlanta - usa (cdc). it prospectively detects sudden and/or gradual changes in risk factors that might modify or alter the health of the population, or certain groups at risk. it alerts the health ministry of any threat to the health of population whether infectious, environmental, occupational hazards and/or chronic diseases and injuries. hr include medical professions (general practitioners, specialists, dental surgeons, pharmacists etc.), and the paramedical professions (nurses, nursing auxiliaries, physiotherapists, laboratory technicians, x - ray operators, etc.). nurses, nursing auxiliaries, clerical and technical staff account for about three - quarters of all health sector staff. there are around three doctors per 1,000 persons in france (compared with 1.2 in libya, 1.7 in the u.k. university hospital doctors have dual status as civil servants due to their teaching and research responsibilities, and state hospital sector employees due to their medical work. numerous clauses system fixes the number of medical and dental students to be admitted to medical schools each year (5,700 medical students in 2004). a dictate from the code of public health obligates health professionals to attend a minimum number of continuous education hours. these are mandatory and are paid by the establishment and considered part of the work days. france is one of the world 's largest consumers of drugs and it is the biggest in europe. prescription drug purchases account for a larger proportion of outpatient health care consumption than its equivalent in libya. france is also europe 's largest consumer of sleeping pills, tranquillizers and anti - depressants. although pharmacies are private undertakings, they must comply with government demographic norms which determine where they may operate. the fhcs has reasonably good outcomes and/or good quality and is in the top of international health systems in its generosity. in spite of its success, it is one of the most costly in europe yet its health professionals are amongst the worst paid. its real challenge is how to bring costs under control without jeopardizing aspects that make the system so popular, such as quality of care, freedom of choice, and equality of access. these include how to be more evidence - based, more cost - effective, efficient, and more quality - oriented. solutions should be sought as to how to obtain new funds from those able to pay, how to move health insurance financing away from payroll and wage levies since this hampers employers ' willingness to hire ; and how to create a governing council of the health insurance funds with more authority and responsibility. measures such as shared, computerized medical records for each patient, and instituting care teams for patients with chronic illnesses should lead to better coordination of care with less duplication of services. other measures that are being implemented are developing and enforcing more practice guidelines, creating a sense of responsibility in both groups of professionals and patients, more attention paid to prevention, decreasing hospitalization rates, decreasing costs of medical prescriptions, using more generic medications and negotiating lower prices for medications and other health products. other weak points are related to methods for introducing new drugs to the market with regard to reimbursement, and providing more general information to the patients. a series of measures were taken in the last decade to modernize health care and decrease the ever rising costs in health expenditure. the hospital - plan 2007 and its extension hospital - plan 2012 are other tools that are expected to promote modernization of the hospital sector (with the introduction of public - private cross subsidies) and the widespread use of diagnosis - related payments. this is expected to allow for an increased comparison of volumes of activity and greater transparency of public and private sector costs. earnings hopefully would be issued based on hospital activities that determine the expenditure and not the inverse. hospitals are also strongly encouraged to gain more autonomy through local governance that involves establishment of a cluster - based organization in order to foster synergies and economies of scale and ensure a comprehensive continuum of care for patients. the plans were extended to form focus groups and activities that are based on economical reasons. examples include grouping different medical disciplines such as cardiology, nephrology, oncology, pediatrics, etc together. these shared interest groups are allotted management departments (personnel, investment, contracting of objectives). recently, based on a senate report, the french president announced 16 other measures in 2008 to make public hospitals more efficient and better managed. the plan included measures to further increase the power of hospital directors and gives public hospitals a real chance to compete with private clinics. currently public hospitals have to wait two years to acquire new equipment while private clinics wait for only six months. the measures aim to reorganize the geographical map too so that they would be more responsive to the health needs of a certain territory and be able to make rational management and dispensing decisions. measures promised financial aid for public establishments that agree to form a joint - community of hospitals. hospitals in a geographic area should coordinate together forming a hospital of reference and essential basic services and cooperation with local hospitals. private clinics would be invited to accept emergencies, to have continuous services, and accept the most underprivileged patients, in order to also be eligible for financial support from the state. cutting edge equipment would be used in research for cancer, genetic diseases, geriatric illnesses such as alzheimer 's and parkinsonism. in these hospitals, private partners are called to participate in modernization efforts under the principle of ppp which stands for public - private - partnership. these are contracts where a private operator takes the responsibility of investing in a public establishment in exchange for a lease or franchise of 20 to 30 years. canada health systems spend a higher portion of gdp, but rank very low in the who report and ehci respectively. the fhcs, which is a leader in most international evaluations, has been successful in insuring universal coverage while maintaining a sustainable pluralistic delivery system that limits perceptions of health care rationing and restrictions on patient choice, with public - private partnership. reforms in fhcs were accomplished in incremental stages in 1928, with big extensions in 1945, 1961, 1966, 1978, 2000, 2007, and lately in 2008. the result has been excellent as shown by patient satisfaction scores, high volume output and general wellbeing of the population. | all public systems look for the best organizational structure to funnel part of their national income into healthcare services. appropriate policies may differ widely across country settings. most healthcare systems fall under one of two broad categories, either bismark or beveridge systems. there is no simple ideal model for the organization of health services, but most healthcare systems that follow the beveridge healthcare model are poor performers. the libyan health system is a low responsive, inefficient and underperforming system that lacks goals and/or smart. (specific, measurable, achievable, realistic, time specific) objectives. a look at different organization models in the world would reinforce efforts to reorganize and improve the performance of the libyan national healthcare services. the french health care system (fhcs) ranked first according to the who and the european health consumer powerhouse. the fhcs was described to have a technically efficient, generous healthcare system that provides the best overall health care. this makes the fhcs a practical model of organization having many of the essential aspects of a modern national health service. in this review, we describe the main features of the fhcs, current challenges and future trends with particular attention paid to aspects that could be of importance to the libyan healthcare system. |
intravenous catheters and those related devices used to gain access to the veins for the purpose of infusing medications or solutions have evolved significantly over the past three decades. early concerns over needle safety for healthcare workers led to the creation of products that provide needle - free access. while these products did eliminate the risk of accidental needle injury for the clinician, some needleless products raised new issues for the patient ; namely, a noted increase in the occurrence of catheter associated bloodstream infections (cabsi) and central line associated bloodstream infections (clabsi) [13 ]. risk factors for infection include poor adherence to aseptic technique, needleless connector (nc) design variations, and inconsistent health care staff education and training [13 ]. nc are used on virtually all intravascular devices in the usa ; they provide an easy access point for syringe or tubing attachment and have now become the central access point for all connections. yet, despite providing some level of safety, concerns over infection related to nc contamination exist. surface design, gaps around valve closure surface, segmented fluid pathway with dead space, differing internal mechanisms, clear or obscured visibility, variable blood reflux, clamping sequences, and different flushing instructions, depending on the type of nc, all play a part in the level of risk associated with the device. before the advent of nc, clinicians had an intuitive understanding that prior to penetrating the septum with the needle the septum required disinfection. initially, needleless split septum access points used a blunt needle - looking type cannula. as a result split septum access devices continue to be recommended as a lower risk option for needleless connection ; however, they have lost popularity because they require multiple parts and pieces for access and allow direct needle access through the septum / diaphragm leading many facilities to switch to luer access devices. with the changes to the access point using direct luer connection through the nc, the intuitive sense to disinfect the surface prior to access is lost ; many clinicians fail to realize the consequences of this breech in aseptic technique [46 ]. colonization of catheter hubs and nc, with subsequent bacterial ingress into the catheter lumen, is considered the cause of 50% of postinsertion catheter - related infections [37 ]. disinfection of the exposed surface of the nc is necessary to avoid contamination and subsequent intraluminal biofilm formation and protect patients from infection. the results of the groundbreaking keystone initiative demonstrated the effect of five measures, known as the central line bundle, on the improvement of outcomes during insertion of central venous catheters [64, 105 ]. consistent application of the bundle, with compliance verified during the insertion procedure (checklist), has reduced insertion related clabsi by more than 44% in the usa. however, despite the successes of the insertion bundle, full compliance more than seven years later is still lacking, with reported compliance rates at one institution ranging from 0.0% at the beginning of the intervention to 37.1% (139/375), according to the jeong study, with similar results in other institutions [65, 66, 106, 107 ]. even in institution where full compliance of the bundle exists, disinfection of the nc access site was not included in the insertion related central line bundle. the goal of any effective infection prevention program is zero clabsis. to reach the goal of zero, consideration must be given for the pathogenesis of catheter related infections and an investigation into current human factors of catheter management preventing achievement of this goal. while many experts agree that application of the insertion bundle is one of the best ways to prevent insertion - related infection, the bundle does not address nc, aseptic access, or any postinsertion catheter usage issues. a pennsylvania study reported that 71.7% (468/653) of central line infections occurred five days or more after insertion and may have been directly related to use and care of intravascular devices [60, 108110 ]. contamination of the catheter directly through the catheter hub has been confirmed through published studies [12, 13, 111113 ]. these studies found that bacteria identified on external hub surfaces were also present in biofilm sampled from random locations within the needless connector. research performed at one institution revealed that patient skin flora was not the source of catheter related bloodstream infections in any of their cases ; all infections in this study originated from the catheter hub [6, 113 ]. infections later in the life of the catheter develop from improper catheter manipulation, failure to perform hand hygiene, inadequate time to clean nc, inadequate training, and poor access and exit site management [2, 67, 110, 112 ]. disinfection of a catheter hub prior to flushing or prior to the administration of medications is required for all aseptic access, yet in the karchmer study, 31% of clinicians did not even attempt to disinfect, even when under active observation [1, 64, 88, 114 ]. in a study by lee the disinfection compliance by clinicians prior to nc access was measured at only 10%. this common break in aseptic technique sets the stage for biofilm formation within nc and catheters and increases the potential for delayed infection of both central and peripheral catheters [14, 60, 68, 112, 116 ]. the results of the pennsylvania patient safety advisory report and independent biofilm sampling of nc suggest that more attention is needed for aseptic access and maintenance practices. according to the epic3 evidence - based guidelines for preventing healthcare infections, disinfection is defined as the use of chemical or physical methods to reduce the number of pathogenic microorganisms on surfaces to a level at which they are not able to cause harm, but which does not usually destroy spores. these guidelines further state that disinfection methods used in combination with cleaning blood or other debris off the surface as disinfectants have limited ability to penetrate organic material. the association for professionals in infection control (apic) defines disinfection as a process to eliminate microorganisms accomplished with the use of liquid chemicals or pasteurizing ; process works best by having proper contact time and dilution of disinfection agent. recommendations from the centers for disease control, the agency for healthcare research and quality, the society for healthcare epidemiology of america, and the infusion nurses society [11, 118 ] state that nc should be consistently and thoroughly disinfected using mechanical friction with 70% alcohol, alcoholic chlorhexidine, or povidone iodine prior to each access of an intravascular device and listed in evidence as a category 1a. the goal of this review is to assess current literature related to disinfection of nc to establish recommendations that promote aseptic access, reducing infection risk for the patient. the purpose of this systematic review was to evaluate the supporting evidence for disinfection practices of nc, catheter hub, stopcock, and side ports that reduce the transfer of microorganisms through intravascular device access. this report is based on an electronic systematic literature search and review of published materials from pubmed, medline, scopus, ovid, jstor, cinahl, cochrane, athens, and sciencedirect by cross - referencing these key terms for years 1977december 2014. high level evidence from rcts that tested cause and effect relationships between different disinfection approaches for nc and patient infection was initially sought. since no rcts were found, lower level evidence including clinical and in vitro (laboratory) studies was reviewed, as long as these included reporting of quantitative data. broad mesh search term disinfection and needleless connector combinations were used with additional keywords listed below : disinfection, antiseptic, alcohol, chlorhexidine, and anti - infective agents, intravenous, intravascular, and vascular access, hub, catheter hub, scrub the hub, intravenous connector, nc, luer activated device, and mechanical valve, aseptic practices, contamination, and compliance, education, staff education, and medical education, infection, infection prevention, catheter related infections, clabsi, bloodstream infections, bacteremia, sepsis, and cross - infection, catheter maintenance and line care, insertion and bundle, intravenous technology, catheter cap, access port, disinfecting cap, antimicrobial cap, hub protection cap, and port protector, infection prevention guidelines and recommendations. disinfection, antiseptic, alcohol, chlorhexidine, and anti - infective agents, intravenous, intravascular, and vascular access, hub, catheter hub, scrub the hub, intravenous connector, nc, luer activated device, and mechanical valve, aseptic practices, contamination, and compliance, education, staff education, and medical education, infection, infection prevention, catheter related infections, clabsi, bloodstream infections, bacteremia, sepsis, and cross - infection, catheter maintenance and line care, insertion and bundle, intravenous technology, catheter cap, access port, disinfecting cap, antimicrobial cap, hub protection cap, and port protector, infection prevention guidelines and recommendations. manufacturers websites of disinfection ports and two manufacturers (excelsior medical, neptune, nj ; ivera medical corporation, san diego, ca) were contacted directly requesting all published materials and posters on disinfection products. there were no identified formal published systematic reviews of the effectiveness of nc disinfection practices, indicating a knowledge gap in this area. initial selection process and critique was performed by one researcher (nm), with evidence rating performed by two researchers independently (nm and jf), with any disagreement in quality rating resolved by discussion. criteria checklist for inclusion was any nc disinfection publications and abstracts that fit subcategories for disinfection, hub contamination / infection prevention, education / compliance, surveys, and guidelines / recommendations for disinfection. inclusion criteria consisted of publications meeting search terms and topic requirements under sub groupings : disinfection agents used on intravascular device surfaces including studies and reviews of nc and infection prevention, sources of contamination through intravascular devices, education and compliance for infection prevention, guidelines and recommendations for infection prevention with disinfection. disinfection agents used on intravascular device surfaces including studies and reviews of nc and infection prevention, sources of contamination through intravascular devices, education and compliance for infection prevention, guidelines and recommendations for infection prevention with disinfection. exclusion criteria werenonresearch papers, studies of adult, pediatric, or neonatal increasingly important role patients not inclusive of intravascular device disinfection practices, primary populations outside acute care, publications not translated into english, studies prior to 1984. studies of adult, pediatric, or neonatal increasingly important role patients not inclusive of intravascular device disinfection practices, primary populations outside acute care, publications not translated into english, studies prior to 1984. after initial review 259 articles did not meet eligibility requirements and were removed. included studies consisted of 140 publications dealing with disinfection / catheter hub were graded according to the strength of the study. the study results and ratings of the literature are included in tables 15 and figure 1, with recommendations are represented in table 6. a catheter is inserted into a vein or artery to provide a pathway for the administration of medications or solutions necessary to improve a patient 's health or condition. because catheters provide an open conduit into the vasculature, a nc is attached, via luer threaded connection, to the integrated hub end of the catheter establishing a closed system. studies reflecting benefits of closed systems with nc have trended toward demonstration of protection for catheter and hub colonization [4, 119 ]. in a prospective controlled study by rosenthal and maki and multicenter prospective cohort by rangel - frausto., open systems compared to closed systems resulted in major reductions in catheter related infections. nc used as a closed system must be weighed with consideration for potential negative factors of design features, poor aseptic practices, and lack of disinfection that all contribute to risk of infection [2, 121, 122 ]. any puncture through the protective skin barrier creates a portal for bacteria to enter the body. recognized routes of catheter contamination are classified as either extraluminal or intraluminal and include (a) migration of microorganisms from the skin at the insertion site (considered the source in short term infections), (b) catheter hub contamination, (c) hematogenous seeding from another infection source in the body, and (d) direct contamination from an infusate [8, 108, 123 ]. after insertion of a catheter, introduction of microorganisms occurs primarily from two routes : the skin / insertion track or through the lumen of the catheter [15, 124127 ]. the greatest risk for contamination of the catheter after insertion is the access hub with 3345% (402/900) contaminated in normal patient use [6, 15, 128132 ]. in early studies by sitges - serra colonization of the catheter hub was considered the primary pathogenesis of catheter associated infection [15, 113 ]. linares and colleagues reported 14 episodes of sepsis (70% of total catheter related septic events) resulted from hub - related contamination [127, 133 ]. moro reported hub colonization in only 3.5% (21/607), but found that this group was responsible for severe systemic infections more frequently. studies indicate that, during periods of nonuse, colony forming units (cfu) are present on access hubs in numbers ranging from 15 to 1000 cfu, representing quantities sufficient to cause contamination, biofilm formation, and potentially bacteremia if not sufficiently disinfected prior to access [3, 5, 12, 99, 110, 111, 134143 ]. as demonstrated by multiple studies, infections are drastically lower or eliminated by disinfecting or covering the access hub with an antimicrobial cap [14, 1620, 113, 127, 144146 ]. hub contamination plays an increasingly important role with infection risk the longer the catheter is in place. intraluminal contamination and subsequent colonization become more prominent with longer dwell times [110, 147 ]. perez and associates found 59% (42/75) of one group of nc colonized with biofilm and salzman found that 71% (20/28) of catheter related infections originated in the catheter hub presumably from contamination [15, 21, 22, 148 ]. clearly hub contamination is a causative element in catheter related infections and one that demonstrates the necessity for effective hub disinfection prior to access [110, 113, 127, 133, 144, 149 ]. disinfection points to gain access to intravenous or intravascular devices may include tubing side ports, direct catheter connections, stopcocks with needle free caps, nc of various types (split septum, mechanical valves, positive pressure valves, zero, or neutral connectors), traditional silicone septum, or other forms of access integrated with the catheter or tubing. any intravascular access point with a surface open to the environment requires disinfection prior to use, as it acts as the immediate portal of entry for intraluminal contaminants [23, 99, 113, 127, 133, 144, 150152 ]. needle free devices constitute more than 80% of access devices, are recommended by centers for disease control for all tubing / catheter access, and are now more common than traditional covered septal access ports which allowed needles to pass easily through the silicone or rubber covered access [8, 153 ]. primary areas of focus for disinfection of access sites are the point where the sterile syringe or tubing contacts the site, as in the top septum surface, and the threads or side surfaces [7, 143, 154 ]. manufacturers are required to include instructions for device use and disinfection recommendations with each product to guide in the correct and safe usage of the nc. effective disinfection of a nc is influenced by several factors including : ability to clean the nc surface, the amount and position of grooves or gaps present, and the roughness or smoothness of the septum [1, 3, 7, 69, 150, 154156 ]. all nc consist of a septum, a fluid pathway and a mechanism for activation ; the design, space, volume, and human factors all affect how easy a product is to use and disinfect, and may also act as contributors to the potential risk of catheter associated bloodstream infection [3, 69, 150, 155, 157, 158 ]. nc have gaps of differing widths between the septal seal and the housing which may allow ingress of microorganisms [7, 23, 99, 143 ]. adequate cleaning or need for additional cleaning of the septal access site may be based on the specific design features of the individual nc [2, 4, 5, 7, 70, 111, 129, 153, 159161 ]. new products or technology should be transitioned into a healthcare facility only after a complete evaluation of both the research and the performance of the product to determine the impact of the change on patient outcomes [71, 97, 98, 149, 160, 162166 ]. recommendations from both the centers for disease control and the infusion nurses society state clinician should minimize contamination risk by disinfection the access ports of the add - on device using friction with an appropriate disinfectant (70% alcohol, chlorhexidine, povidone iodine, and iodophors) prior to any access [8, 11, 24, 167, 168 ]. the 70% isopropyl alcohol wipe is most commonly used to disinfect the access surface of nc and has been proven effective or ineffective at disinfection times from 5 seconds to 60 seconds [14, 19, 2427, 131, 151, 169177 ]. the major biocide effect of alcohol occurs while wet and immediately after drying, allowing for dehydration of bacterial cells, whereas alcoholic chlorhexidine is most effective during the drying process, where it enters the cell to cause destruction providing ongoing antimicrobial effect [150, 172 ]. the disinfecting action of chlorhexidine in combination with alcohol, allowing for both immediate and sustained action ; has proven to be more effective than either agent alone [21, 131, 168, 172, 176, 178180 ]. faster drying time with alcohol makes it superior to other disinfection agents and provides an advantage to chlorhexidine when used in combination. effectiveness of 70% alcohol disinfection is variable based on application techniques and characteristics of nc surface and design, leading some researchers to conclude that complete disinfection of microorganisms on some nc surfaces may not be achievable. in the menyhay prospective in vitro study, 20 (67%) of 30 nc disinfected with 70% alcohol resulted in transmission of contaminants (44225,000 cfu) yet 60 tested with barrier caps (containing 2% chlorhexidine and 70% alcohol) showed only one (1.6%) with transmission of contaminants. in both the kaler laboratory and the ruschman randomized experimental design studies, using a 15 second and 60 second scrub respectively, disinfection with 70% alcohol eliminated all microorganisms [173, 181 ]. kaler performed laboratory testing on contaminated nc with a small sample using 70% alcohol and alcoholic chlorhexidine and found both to be effective for hub disinfection. rupp demonstrated 5 second alcohol disinfection was effective ; this was in direct contrast with the smith study where contact time of 10/12/15 seconds was deemed adequate, but 5 and 8 seconds were not as effective to prevent bacterial transfer [170, 171 ]. simmons and colleagues found 3/10/15 seconds significantly decreased the bacterial load in an in vitro laboratory study, with some level of bacteria remaining during all duration levels tested ; disinfection failed to completely eliminate contaminants. more studies are needed to provide efficacy for optimal time necessary to eliminate surface contaminants. research of macias and associates with 2% chlorhexidine in 70% isopropyl alcohol on skin proved an added substantive effect, even against freshly introduced organisms, for up to 24 hours, establishing this agent as a superior disinfecting agent when longer action is needed, in comparison with single agents of 70% isopropyl alcohol, 10% povidone iodine and 10% sodium hypochlorite. alcoholic chlorhexidine performed consistently well or better than other disinfection agents in multiple studies [21, 24, 131, 168, 172, 179, 182 ]. in the research by hong., a 5 second scrub with alcoholic chlorhexidine fully disinfected nc surfaces treated with pseudomonas aeruginosa. in the most recent epic3 united kingdom report of evidence - based guidelines, recommendations by expert consensus include a 15 second cleansing with alcoholic chlorhexidine prior to and after each access. in actual practice disinfection prior to access is expected, while cleansing after each access is rarely done. disinfection research includes various forms of passive antimicrobial hub protection with 70% alcohol caps (swabcap, excelsior medical, neptune, nj ; curos port protector, ivera medical, san diego, ca ;, effectiv - cap, hospira, lake forest, il ; dualcap, catheter connections, salt lake city, ut), iodinated alcohol hub, povidone iodine gauze and specialty covers, and combination chlorhexidine / alcohol caps [12, 14, 18, 19, 23, 2543, 146, 178, 183192 ]. in a randomized prospective trial by pittiruti, 46 catheters received a 70% alcohol port protector with no detected clabsis over 707 catheter days, colonization in two catheters and no contaminated blood cultures. of note the pittiruti study resulted in reductions of clabsis in the port protector / disinfection cap group and the control group, with improvements attributed to both the disinfection caps and educational efforts. these disinfection caps applied and left in place provide active mechanical friction along with longer contact time creating a physical and chemical barrier between the lumen and the environment. as a progressive clabsi intervention posa at st. joseph mercy health system implemented an insertion bundle, chlorhexidine bathing, a maintenance bundle, chlorhexidine dressing for central catheters, and educational programs, however it was not until implementing the 70% alcohol disinfection cap that their rates of clabsi fell to zero and remained from 2011 to the end of 2012 the disinfection cap placed on all access ports eliminates human factor issues requiring clinicians to remember to carry the necessary disinfection supplies to the bedside or even to remember to perform the act of disinfection before each access. an in vivo hospital study by devries gave nurses a choice to use either this single use cleansing cap or a disinfection cap to leave on the nc access site, clinicians preferred the longer lasting disinfection cap. in another retrospective study, schears noted a predisinfection cap clabsi rate of 1.682/1000 catheter days and a clabsi rate of 0.6461/1000 catheter days after implementing disinfection caps, representing a statistically significant 61% reduction in clabsi. in wright 's study at northshore university healthsystem, a four university hospital system, the intervention with 70% alcohol disinfection caps reported clabsi rates declining from 1.42/1000 catheter days (16/11,540) to 0.69 (13/18,972) with a 95% confidence interval, based on 799 enrolled patients, representing a statistically significant decrease. another alcoholic hub protector study by sweet. included 472 patients and 3005 catheter days and showed a decrease in overall clabsis from 2.3 to 0.3/1000 catheter days and a picc clabsi reduction from 2.3 to 0, a statistically significant change, with an 85.2% compliance rate. stango and associates reported a 50% reduction in clabsis and a savings of $ 464,440 per year after alcoholic cap implementation. numerous studies have demonstrated consistent clinical effectiveness of 70% alcohol caps alone in studies and abstracts graded c or d [18, 26, 27, 31, 33, 41, 43, 178, 189, 191194 ]. alcoholic chlorhexidine caps are also effective in preventing contamination and completely disinfecting nc access surfaces (combination of alcoholic chlorhexidine in cap form is not commercially available in the usa) [2, 12, 19, 178, 179 ]. another engineered solution for hub cleaning involves a 70% alcohol foam cap (site scrub, bard access, salt lake city, ut) designed for use as an access site cleansing cap for single use, then discarded. the holroyd in vitro study at university of florida compared the single use of this cleansing cap with 70% alcohol to traditional 70% alcohol wipes. when the cleansing cap was used on stopcocks holroyd found contamination and increased cfu. this study found 70% alcohol wipes and this alcohol cleansing cap were both effective on the surface of nc and catheter hubs. other groups also used this single use cleansing cap in combination with other 70% alcohol disinfecting caps designed to be left in place until the next access [14, 177 ]. since the advent of nc as access hubs for the administration of medications or fluids, there has been a need to verify compliance with disinfection practices prior to access. during the period of needle usage for catheter access, nurses and doctors intuitively knew the necessity of disinfecting the access septum prior to inserting a needle. with nc, these questions arise : is disinfection always performed prior to access ? is disinfection performed in an effective manner ? disinfection practices with alcohol or alcoholic chlorhexidine that include adequate contact disinfection time are effective if performed at all. according to a recent publication by ryder, an issue was raised regarding whether failure to disinfect is considered a medical error and if so, is this omission considered negligence ? catheter associated infections are a significant safety issue, and contamination caused by lack of aseptic technique is preventable. once contamination occurs, bacteria attach to the inner lumen of the catheter, begin to grow and form biofilm, making successful eradication extremely difficult [6, 2830, 113, 127, 133, 140142, 144, 195 ]. joint commission now requires hospitals in the usa to protect patients by having a standard and measurable protocol for hub / access site disinfection [61, 62, 196 ]. measurement of compliance with hub disinfection is challenging, requiring direct observation of the action unless disinfection caps / ports are used on all nc hubs as a form of verification. passive disinfection through hub protectors / disinfection caps have differing designs and colors, leading to easy recognition and validation of compliance with usage. reimbursement structures in the usa that now promote pay for performance and penalize poor outcomes will assist in driving these passive safety strategies that aid in monitoring and improving compliance with disinfection. while policies for disinfection of access devices are a first step, methods to validate actual practice and patient safety must be integrated into hospital culture. the central line bundle checklist is used as evidence to demonstrate compliance with safety practices during insertion, but the aspect of day to day management is not addressed in the bundle. care and management of catheters takes up more than 99% of the dwell time of a catheter compared to the one hour or less for catheter insertion. consistent hand hygiene and gloving performed prior to any procedure or even touching of a catheter helps reduce bacterial transfer. application of alcoholic chlorhexidine to disinfect skin for central line insertions and now, more and more with peripheral catheter insertions, is helping to reduce bacterial ingress to the bloodstream. maximum sterile barriers also reduce contamination during the insertion process so that overall, clabsi rates occurring in the first few days of insertion continue to fall. even with the success of the central line bundle on clabsi reductions, a majority of hospitals remain well above zero for infections. access and maintenance activities with the catheters may be to blame. when clabsis occur well after the 96 hour mark, contamination of the catheter through the nc is likely the culprit. from the evidence presented, nc and catheter hubs are a primary source of bacterial contamination, and subsequent transmission of contamination into the catheter lumen [6, 30, 113, 127, 133, 142, 144 ]. just one omission of scrubbing the hub prior to access permits bacterial entry, attachment and biofilm formation that allow the bacteria to strengthen prior to release into the bloodstream. preventing this form of contamination requires teaching and constant reinforcement of the required practice of regular and consistent disinfection prior to every access. verification of compliance with hub disinfection by clinicians requires direct observation of the action unless disinfection hub protectors are used, providing a form of passive immediate visual verification. more and more studies are demonstrating lack of compliance with hub disinfection despite educational initiatives and better disinfection agents. disinfection methods that incorporate prolonged duration of contact with an antiseptic agent to significantly decrease the level of surface bacteria present may provide a solution to the problem of hub contamination and variation in nc designs. various studies provide statements regarding conformity or lack of conformity concerning disinfection practices, attributing noncompliance to a lack of universal protocols, excessive workloads (e.g., when clinicians become busy, they are less likely to comply), or just forgetting to bring alcohol wipes to the bedside [54, 64, 72, 8993, 188 ]. the smith study on behavioral intention indicated a negative correlation between performing optimal disinfection with increasing age of clinicians and more years of experience. clearly there are human factors working against disinfection of hubs prior to access requiring engineered solutions such as passive disinfecting cap strips hanging on intravenous pump poles, supply dispensers of alcohol wipes at the bedside, or on the intravenous pump to ensure greater, even 100% compliance with disinfection each and every time [91, 170 ]. monitoring and validation of hub disinfection compliance is necessary to determine if other measures are needed such as disinfection caps / port protectors. in an evaluation of 5877 physicians, nurses and technicians, jardim. documented compliance with hub disinfection 38.7% of the time, leaving more than 61% of accesses without disinfection, leading to possible contamination and biofilm growth. platace. evaluated clinician hands during invasive procedures demonstrating 100% of the 48 nurses sampled exceeded acceptable levels of microorganisms, with the potential to contaminate and cause bloodstream infection. studies show there is a need for clear recommendations and practices that prevent transmission of contaminants through nc [68, 90 ]. targeting of education for providers responsible for cvad insertion and care for identifying appropriate indications, performing insertion with the central line bundle, performing surveillance of clabsi and scrubbing the hub with an appropriate antiseptic are category ia recommendations by ahrq as critical components of a comprehensive clabsi prevention program. this systematic review highlights the lack of available high quality research in this area that tests the cause and effect relationship between nc disinfection practices and patient infection outcomes. it also asks the question what are we basing our clinical practice guidelines on for disinfection of nc ? absence of high quality rct evidence required authors to include any clinical observational and cohort studies and laboratory studies. overall, the evidence base for the effectiveness of various disinfection strategies is low level, resulting in recommendations compiled from the available publications. the strength of this review is that it includes all relevant, currently available pieces of evidence ; however there remains a high level of uncertainty in the estimates of effectiveness of various decontamination techniques, and these are highly likely to change with the publication of new studies in the literature. studies to date have a risk of unintentional bias due to the lack of randomization and control groups / strategies, in addition to small sample sizes and retrospective study designs. randomized controlled studies are needed to rigorously evaluate the efficacy of disinfection practices and antiseptic hub protectors in preventing patient infection. adequately large randomized controlled trials are urgently needed to establish high quality evidence of the efficacy of various disinfection practices to prevent infection. randomized controlled trials are needed to identify if risk reduction differs with the type of antiseptic, for example, 70% alcohol versus 70% alcohol and chlorhexidine, or with differing concentrations of chlorhexidine in their efficacy for disinfection nc. research may also validate the substantive effect of alcoholic chlorhexidine on nc and its continued antimicrobial activity on these surfaces, potentially establishing a reduced cleaning frequency or duration for nc. study considerations for passive disinfection coupled with prefilled flush syringes could demonstrate drastic reduction of hub contamination and intraluminal biofilm colonization, but ultimately patient infection outcomes are needed. research that replicates solid studies provides a stronger foundation for evidence - based practice and should be encouraged. translational research is growing, providing clinical implications that directly apply to bedside practices [14, 5759, 67, 70, 7375 ]. nc disinfection is an excellent subject for efficacy studies utilizing comparative research for various disinfection approaches identifying relative reductions in patient infection risk., professor of nursing with griffith university, states it well we are belatedly realising that to eliminate these complications (infections) we must conduct research, implement evidence - based interventions and reduce the clinical practice variations that lead to their occurrence. research and study are necessary as an integral part of professional practice, providing a means to direct clinical activity and to share with rising young clinicians long after we are gone. more and more studies reveal lack of compliance with disinfection of access ports prior to and after access, despite educational initiatives, and better disinfection agents [1, 26, 27, 38, 54, 57, 64, 67, 69, 72, 74, 78, 83, 8893, 188, 198205 ]. rather than creating devices such as the ultraviolet c port to eradicate contamination within the hub, the goal should be to eliminate surface pathogens before entering the nc or catheter. passive disinfection caps reduce guess work, provide clinicians with a point of use solution, and reduce contamination. it is critical for healthcare facilities and clinicians to take responsibility for compliance with aseptic technique for nc disinfection, to monitor compliance regularly, to involve frontline staff in solutions, and to facilitate education that promotes understanding of the consequences of failure to comply with the standard of care for access site disinfection. | background. needleless connectors (nc) are used on virtually all intravascular devices, providing an easy access point for infusion connection. colonization of nc is considered the cause of 50% of postinsertion catheter - related infections. breaks in aseptic technique, from failure to disinfect, result in contamination and subsequent biofilm formation within nc and catheters increasing the potential for infection of central and peripheral catheters. methods. this systematic review evaluated 140 studies and 34 abstracts on nc disinfection practices, the impact of hub contamination on infection, and measures of education and compliance. results. the greatest risk for contamination of the catheter after insertion is the nc with 3345% contaminated, and compliance with disinfection as low as 10%. the optimal technique or disinfection time has not been identified, although scrubbing with 70% alcohol for 560 seconds is recommended. studies have reported statistically significant results in infection reduction when passive alcohol disinfection caps are used (4886% reduction). clinical implications. it is critical for healthcare facilities and clinicians to take responsibility for compliance with basic principles of asepsis compliance, to involve frontline staff in strategies, to facilitate education that promotes understanding of the consequences of failure, and to comply with the standard of care for hub disinfection. |
tear lysozyme is a high molecular weight, long chain glycolytic enzyme secreted by the lachrymal gland. among the tear proteins identified, lysozyme constitutes around 20%40% of the total tear protein (farris 1985) and its concentration in the tear film is higher than in any other fluid of the body (fleming 1922). this protein has the capacity to dissolve gram - negative bacteria walls by the enzymatic digestion of mucopolysaccharides (milder 1987). due to this bactericidal action, lysozyme has been considered as one of the essential elements of the protective tear film barrier against ocular infection (mackie and seal 1976). the importance of this lachrymal component has contributed to develop methods for its detection and measurement, as well as to correlate its concentration with ocular pathologies. several studies have shown a decrease in the concentration of lysozyme in patients with keratoconjunctivitis sicca (van bijsterveld 1969 ; mackie 1984 ; montero 1990), suggesting that a drop off in tear lysozyme levels may constitute an important parameter to detect a malfunctioning lachrymal gland (klaeger 1999). recently, the presence of a new family of compounds in the tear film have been described : the diadenosine polyphosphates (pintor, carracedo 2002). these naturally occurring dinucleotide compounds exhibit both intracellular and extracellular physiological actions, these including vasoactive properties, neuromodulatory regulation of neurotransmitter release or intracellular modulation of ion channels (mclennan 2000 ; hoyle 2002). formed by two adenosine molecules joined by a variable phosphate chain, they are abbreviated as apna (n = 27, where n describes the number of phosphates). the activity of these nucleotides on ocular tissues is being investigated, and it is known that they act through p2 receptors to modulate intraocular pressure in rabbits (pintor, peral, pelez 2002) ; ap4a and utp improve the rate of wound healing in the cornea of new zealand white rabbits (pintor, bautista 2004) and also, ap4a, ap5a, and ap6a, can stimulate tear secretion after single - dose topical application in rabbits (pintor, peral, hoyle 2002). in order to investigate the physiological role of nucleotides onto the tear film, lysozyme levels in new zealand white rabbits tears have been measured after a topical application of the mentioned substances. the methodological approach used to get the lysozyme levels has been the agar diffusion method described by van bijsterveld (1974). the analysis of the data suggests an increase in tear lysozyme levels with the application of the tested nucleotides. twelve male new zealand white rabbits from granja cunicula san bernardo (navarra, spain) weighing 2.02.5 kg were kept in individual cages with free access to food and water and subjected to regular cycles of light and dark (12 hours each). the new zealand white rabbits were 6 months old and the slit lamp exam evidenced no ocular pathology or alteration that could affect the tear secretion. all the experiments were performed according to association for research in vision and ophthalmology (arvo) statement for the use of animals in ophthalmic and vision research and in accordance with the european communities council directive (86/609/eec). three nucleotides were tested along this work : utp from amersham biosciences, inc., (piscataway, nj) ; ap4a from sigma chemical (st louis, mo) ; up4u (diquafosol or formerly ins365) kindly provided by inspire pharmaceuticals (durham, nc, usa). the p2 antagonists employed were pyridoxalphosphate-6-azophenyl-2, 4-disulfonic acid (ppads), suramin and reactive blue-2 (rb-2) were purchased from sigma / rbi (natick ma). for single dose experiments, all the nucleotides were applied in a concentration of 100 m in a volume of 10 l in the tested eye. the p2 receptor antagonists ppads, suramin and rb-2 were instilled in concentrations of 100 m (10 l), 30 min before the application of any of the nucleotides. the utp, ap4a and up4u nucleotides were tested twice in the whole sample (n = 24 each agonist). the p2 receptor antagonists were experienced once for each agonist in the whole sample (n = 12 for each agonist). the diffusion in agar method was employed to measure the lysozyme levels in rabbit tears. callibration curves as well as all the lysozyme measurements were performed as indicated by van bijsterveld (1974) and mackie and seal (1976). briefly, the protein amount was obtained by measuring the inhibitory halos around a whatman n1 paper disc of 5 mm in diameter. the paper discs were placed with clamps in the upper bulbar conjunctiva to avoid mucus strands. when the tears soaked the discs ; they were removed and put on petri dishes with agar medium where micrococus lisodeikticus was grown. the petri dishes were then incubated at 37 c for 24 hours and finally the zones of lysis of micrococus lisodeikticus were measured. lysozyme standard curves for its quantification was performed with known concentrations of hel (hen egg lysozyme). for the mentioned curve, the 5 mm paper discs were weighed before and after being soaked by rabbit tears in order to estimate the equivalent volume of lysozyme solution to apply on the calibration paper discs. the difference in weight was the equivalent to 5 l of lysozyme solution, so that volume was applied to estimate the standard curve. concentrations between 0.1 mg / ml and 1.0 mg / ml of standard hen lysozyme were applied on the discs in a final volume of 5 l. after two control measurements, 10 l of the studied compound was topically instilled in one eye (taking the contralateral eye as a control), and the tear samples were collected every hour for five hours. for measuring the diameter of the halos, petri dishes were scanned and analyzed by the computer program imagej (v.1.37, nih, usa). briefly, scanned images of the inhibition halos were transformed into 8-bit black and white images and further transformed into a binary image prior to the corresponding calculation. with this standardized method, data were analyzed using the paired t - test and significance was set at p < 0.05. twelve male new zealand white rabbits from granja cunicula san bernardo (navarra, spain) weighing 2.02.5 kg were kept in individual cages with free access to food and water and subjected to regular cycles of light and dark (12 hours each). the new zealand white rabbits were 6 months old and the slit lamp exam evidenced no ocular pathology or alteration that could affect the tear secretion. all the experiments were performed according to association for research in vision and ophthalmology (arvo) statement for the use of animals in ophthalmic and vision research and in accordance with the european communities council directive (86/609/eec). three nucleotides were tested along this work : utp from amersham biosciences, inc., (piscataway, nj) ; ap4a from sigma chemical (st louis, mo) ; up4u (diquafosol or formerly ins365) kindly provided by inspire pharmaceuticals (durham, nc, usa). the p2 antagonists employed were pyridoxalphosphate-6-azophenyl-2, 4-disulfonic acid (ppads), suramin and reactive blue-2 (rb-2) were purchased from sigma / rbi (natick ma). for single dose experiments, all the nucleotides were applied in a concentration of 100 m in a volume of 10 l in the tested eye. the p2 receptor antagonists ppads, suramin and rb-2 were instilled in concentrations of 100 m (10 l), 30 min before the application of any of the nucleotides. the utp, ap4a and up4u nucleotides were tested twice in the whole sample (n = 24 each agonist). the p2 receptor antagonists were experienced once for each agonist in the whole sample (n = 12 for each agonist). the diffusion in agar method was employed to measure the lysozyme levels in rabbit tears. callibration curves as well as all the lysozyme measurements were performed as indicated by van bijsterveld (1974) and mackie and seal (1976). briefly, the protein amount was obtained by measuring the inhibitory halos around a whatman n1 paper disc of 5 mm in diameter. the paper discs were placed with clamps in the upper bulbar conjunctiva to avoid mucus strands. when the tears soaked the discs ; they were removed and put on petri dishes with agar medium where micrococus lisodeikticus was grown. the petri dishes were then incubated at 37 c for 24 hours and finally the zones of lysis of micrococus lisodeikticus were measured. lysozyme standard curves for its quantification was performed with known concentrations of hel (hen egg lysozyme). for the mentioned curve, the 5 mm paper discs were weighed before and after being soaked by rabbit tears in order to estimate the equivalent volume of lysozyme solution to apply on the calibration paper discs. the difference in weight was the equivalent to 5 l of lysozyme solution, so that volume was applied to estimate the standard curve. concentrations between 0.1 mg / ml and 1.0 mg / ml of standard hen lysozyme were applied on the discs in a final volume of 5 l. after two control measurements, 10 l of the studied compound was topically instilled in one eye (taking the contralateral eye as a control), and the tear samples were collected every hour for five hours. for measuring the diameter of the halos, petri dishes were scanned and analyzed by the computer program imagej (v.1.37, nih, usa). briefly, scanned images of the inhibition halos were transformed into 8-bit black and white images and further transformed into a binary image prior to the corresponding calculation. with this standardized method, data were analyzed using the paired t - test and significance was set at p < 0.05. to determine whether these nucleotides were able to modify rabbit tear lysozyme levels at intervals after the nucleotide topical application, single doses of utp, ap4a, up4u were applied at 100 m (at a final volume of 10 l) (figure 1). the results for the time - course of the single - dose application compared with the basal curve for lysozyme is represented in figure 2. it is possible to see that all the three substances increase the concentration of lysozyme in the rabbit tears, and that the maximal effect is obtained two hours after the instillation of the corresponding nucleotide. this effect remained for at least 3 more hours, then returned to basal levels (figure 2). a representative experiment showing the inhibitory halos around whatman n1 paper discs in the moment of maximal effect is presented in figure 3. these halos represent the zones of lysis of micrococcus lysodeikticus due to the action of lysozyme. the first disc shows the basal diameter of lysis for a control rabbit (basal lysozyme concentration present in tears, figure 3a). the others showed a maximal increased diameter of lysis due to the action of the different nucleotides on the tear lysozyme levels after their topical application (figure 3a). a representative value to compare effects among nucleotides can be obtained if we represent the mean of lysozyme concentration obtained for the period between 2 and 5 hours plateau for any of the substances versus basal concentration. as it is shown in figure 3b, it is possible to see that the mean values for all the nucleotides showed a significant increase in lysozyme concentrations over the basal level. the increase in lysozyme concentration after the nucleotides application was 93 % for ap4a, 119% for up4u and 67% for utp (figure 3b). the activity of the substances on tear lysozyme levels suggests the activation of p2 nucleotide receptors. to confirm this, the effects of three non - selective nucleotide receptor antagonists on lysozyme levels were studied : ppads, suramin and rb-2. diadenosine tetraphosphate effect on lysozyme production was significantly reversed by the antagonist ppads, returning the lysozyme concentration to control values. neither of the other two, rb-2 or suramin was able to modify the effect triggered by ap4a. up4u was antagonized by ppads and suramin, the reduction being 42% and 52% for ppads and suramin respectively. moreover, in the case of rb-2, there was a potentiation of utp effect when compared with the effect of this nucleotide alone, showing an increase of almost 25% above the lysozyme concentration obtained when utp is applied alone. this study shows for the first time the physiological effect of ap4a, up4u and utp on the tear lysozyme levels in new zealand white rabbits. all the tested nucleotides showed an increase in the lysozyme concentrations for a single - dose application, which was measurable for several hours after the topical application of the compounds. since lysozyme is one of the main proteins involved in the protective tear film barrier against ocular infection, together with lactoferrin, an increase in their levels may, in general, enhance the bactericidal action of tears, preserving the health of the ocular surface. the presence of nucleotides and dinucleotides in the tear is suggesting their involvement in several relevant physiological processes of the ocular surface. we have added to the list of biological actions of nucleotides and dinucleotides the ability of these compounds to increase the tear concentrations of lysozyme. it is moreover relevant the fact of having important changes in the concentrations of this enzyme when up4u or utp are topically applied. these two nucleotides have not been described in tears but they behave similarly to ap4a, suggesting that all may activate the same p2y purinoceptor subtype. in this sense, the three tested nucleotides have been claimed to be quite selective purinergic receptors (lazarowski 1995 ; for the p2y2 mundasad 2001). nonetheless, the results obtained by means of the non - selective p2 receptor antagonists depict a different pattern depending on the tested nucleotide. in the case of ap4a the only effective antagonist was ppads while in the case of up4u apart from ppads, suramin was also able to partially reverse the dinucleotide action. one may expect both nucleotides to stimulate the same receptor or group of receptors (see the similarities in the structure showed in figure 1). taking into account our results, it seems that ap4a and up4u share some p2y receptor activation but in the case of up4u it may activate more than one, since its increase in lysozyme is partially blocked by suramin while ap4a effect is not. this may be at least in part justified by the fact of the presence of several p2y receptor in the lachrymal gland and cornea (cowlen 2003 ; pintor, bautista 2004 ; pintor, sanchez - nogueiro 2004), and also by the fact that up4u is known to activate at least p2y2 and p2y4 receptors (brunschweiger and muller 2006). it is noteworthy the effect the antagonists of p2 receptors display on the utp effect. although one should expect that rb-2 might block the effect of utp, the observed behavior of this antagonist shows the opposite effect. reactive blue 2 has been described as a very potent inhibitor of all plasma membrane - bound ntpdases as reported by many authors (mateo 1996, 1997). since rb-2 inhibits ecto - enzymes and does not antagonize p2y receptors it is reasonable to see a potentiation of the utp effect since it will not be degraded as fast as when it is applied alone., it has been described a decrease in lysozyme levels with age (mackie and seal 1976) as well as in other pathological states running with a drop off in tear secretion like keratoconjunctivitis sicca or sicca syndrome (mackie and seal 1984). therefore, in these cases, it may be good to incorporate any of the indicated nucleotides as a new compound as a pharmacological treatment to reinforce the barrier against opportunistic infections. recently, apart from the bactericidal role, tragoulias (2005) and millar (2006) have suggested that lysozyme contributes to lower the surface tension of the tear film. in this case, an increase in lysozyme levels could contribute to stablizing the tear film, thus avoiding an excessive evaporation and the resulting problems on the ocular surface. in summary, we have shown that the p2y receptor agonists, ap4a, up4u and utp, are able to increase the tear concentrations of lysozyme. due to the importance of this protein, these nucleotides may be used to fortify the tear film barrier by increasing a naturally occurring defender against some ocular surface infections. | the present work studies the effects of topical application of nucleotides on rabbit tear lysozyme levels. lysozyme values were determined by the diffusion in agar method described by van bijsterveld in 1974, and the protein amount was obtained by measuring the inhibitory halos around a whatman n1 paper disc of 5 mm in diameter. the tested nucleotides were utp, ap4a and up4u. these compounds were topically instilled in a single - dose in one eye (with the contralateral eye as a control) and the lysozyme halos were measured along 5 hours. the obtained results showed an increase in the lysozyme concentrations of 67%, 93%, and 119% for utp, ap4a, and up4u, respectively, over the basal levels of lysozyme. for this reason, we suggest these molecules as a potential treatment for the reinforcement of the tear film barrier against ocular infection. |
sulfur mustard (2,2-dichloroethylsulfide ; sm) is a bifunctional alkylating and vesicant agent with the highest military significance. after the first world war, iraq 's military usage of sm against iranian military and civilians as well as their own kurdish civilians (19801988) was the most extensive usage of this agent. the outcome of this genocide was over 100,000 chemical warfare victims that have been grouped into three categories of injuries based on affecting dose and time (mild, moderate, and severe). one - third of these victims are still suffering from sm delayed effects which present an abrupt onset within a period of anywhere from 1 to 40 years after initial exposure [1, 2 ]. medical effects of exposure to sm include ocular, skin, and respiratory system damage as the three main areas of concern [13 ] ; furthermore several studies indicate agitation impression of sm in gastrointestinal, hematological, neuropsychiatric, and immune system effects and skin cancer increasing [6, 7 ]. patients with delayed ocular manifestations of sm suffer from itching, photophobia, limbal ischemia, corneal neovascularization, conjunctival, thinning, and irregularity, chronic blepharitis, corneal degeneration, recurring epithelial lesions, meibomian gland dysfunction, and decreased vision [810 ]. it is considerable that there are significant similarities with known pathological effects of radiation - induced damages like skin erythema, mucositis, nausea, and diarrhea for acute effects and fibrosis, organ dysfunction, necrosis, and vascular damages for late effects which are the consequences of an imperfect tissue remodeling [11, 12 ]. the most considered mechanisms include sm - induced dna alkylation damage, poly(adp - ribose)polymerase activation, calcium signaling and calmodulin, nitric oxide signaling and oxidative stress, inflammatory reactions, matrix - metalloproteinase activity, immunologic effects, neurotrophic effects, and limbal stem cell deficiency. although the phenomena considered sounds to be similar to the main known mechanism involved in radiation - induced late effects (inflammation, oxidative damages, fibrosis, vascular alterations, and cellular depletion) [11, 16, 17 ], the underlying pathogenesis and molecular basis of sm acute and delayed effects are still a controversial issue. ocular surface damages are primarily due to local contact to sm but its systemic toxicity may lead to some serum protein changes that may continually and gradually exacerbate the ocular surface injuries. here we have analyzed serum proteome of patients against healthy control groups, by 2de followed with maldi tof / tof mass spectrometry to identify molecular basis of delayed ocular symptoms induced by severe sm exposure. furthermore, we have evaluated several - sample processing approach in order to overcome serum proteomic major challenges such as wide dynamic range of protein concentration and presence of high abundance proteins like igg and albumin. following institutional ethical approval and written informed consent, blood samples were obtained from ten victims with known pathology of sm severe ocular effects and ten controls by venipuncture into red top tubes (without coagulant activator). all severe victims were man with average age of 54 4.2 in which nearly 28 4.6 years have passed since injury. some of them were treated corneal transplant surgery and none have any significant pulmonary and skin complications. the blood was centrifuged (for 15 minutes at 1200 g in room temperature) immediately to prevent secretion interference of blood cells during clotting. obtained plasma was allowed to clot naturally at 4c less than 60 minutes to prevent protease activities. then the serum was separated from coagulation factors after centrifugation at 15000 g at 4c for 15 minutes. finally, the serum samples then delipidated by centrifuging at 14000 rpm at room temperature for 15 min and stored in 80c. 25 l of human serum was precipitated by addition of 100 l of 10% tca / acetone solution and was mixed by gentle vortexing. the mixture was incubated at 20c for 90 minutes and then centrifuged at 15000 g and 4c for 15 minutes. then 1 ml of ice - cold acetone (20c) was added to wash the precipitate and supernatant was separated. the acetone containing supernatant was removed and the precipitate was dried in air a few minutes. one volume serum sample (25 l) was mixed with four - volume methanol. simultaneously by vortex one - volume chloroform and then three - volume double distillate water (ddw) were added and then centrifuged at 14000 g for 1 min and aqueous layer was removed. again with vortexing four - volume methanol was added and centrifuged at 14000 g for 2 min. the pellet was washed by cold acetone (20c), air - dried for a few minutes, then resolubilized in sample buffer, and concentrated. one - volume serum sample was mixed with four volumes cold acetone (20c), incubated for 120 min in 20c, and centrifuged at 15000 g for 15 min. the pellet was washed by cold acetone, air - dried for a few minutes, and concentrated after resolubilization in sample buffer. one volume of serum was mixed with two volumes of ddw and 0.023 gr ammonium sulfate was added gradually. incubated about 30 min at room temperature and then centrifuged at 12000 rpm for 10 min. the pellet was washed by cold acetone and air - dried for a few minutes and concentrated after resolubilization in sample buffer. the acn precipitation was carried out as reported by merrell. with minor modification. they were immediately vortexing for 5 sec vigorously, incubated for 30 min at room temperature, and then centrifuged at 12000 rpm for 10 min. upper solution was incubated by cold acetone for 60 min and centrifuged as above. one volume of serum sample was performed by proteomine protein enrichment kit as manual protocol in four steps : column preparation, binding, washing, and elution. elution step remained acidic solution, so it was adjusted to ph : 7.4 by pbs buffer ; then in order to remove ionic contaminant adjusted to 1 ml by cold acetone and incubated for 90 min. after centrifugation the pellet was air - dried and resolubilized in sample buffer and concentrated. precipitated proteins obtained from any of the above methods were separately dissolved in sample buffer (sodium dodecyl sulfate (sds) : 2% w / v and dithiothreitol (dtt) : 1.25% w / v) and their concentration was determined by uv absorbance at 280 nm (nanodrop 2000c, thermo scientific, nanodrop product, wilmington, de, usa). then 80 g proteins were diluted to 300 l by rehydration buffer (urea : 7 m, thiourea : 2 m, chaps : 4% w / v, ampholyte 3/10 : 2% w / v, dtt : 100 mm). the rehydration stage was performed in 17 cm ipg nonlinear ph : 310 strips (bio - rad, hercules, ca, usa) about 16 h actively (constant voltage : 50 v). immediately isoelectric focusing was performed according to the program : (i) up to 150 v for 90 min rapidly (desalting) ; (ii) focused at 2000 v for 180 min linearly ; (iii) up to 10000 v for 240 min linearly ; (iv) finally, maintained at 10000 v for a total of 60000 v - h rapidly. during isoelectric focusing (ief) we use wicks before desalting (anode and cathode : ddw) and after desalting (anode : ddw, cathode : dtt (20 mm)) step. then ief strips were equilibrated for 20 minutes in both equilibration buffer i (urea : 6 m, tris - hcl : 1.5 m ph : 8.8, sds : 2% w / v, glycerol : 20%, dtt : 2.5% w / v) and equilibration buffer ii (urea : 6 m, tris - hcl : 1.5 m ph : 8.8, sds : 2% w / v, glycerol : 20%, iodoacetamide : 4% w / v), respectively. the second dimension was performed on 11% sds polyacrylamide gels at constant current ((i) 16 ma / gel for 30 min ; (ii) 24 ma / gel) in a protean ii xi 2-d cell (bio - rad, hercules, ca, usa). after protein fixation for overnight with methanol containing acetic acid and formaldehyde, the gel was stained according to ms - compatible silver nitrate procedure according to yan.. after staining gels were scanned by gs-800 calibrated densitometer (bio - rad, hercules, ca, usa), the scanned gels were analyzed with samespots progenesis version 3.3 (and 4.1) (nonlinear dynamics, durham nc, usa). the relative intensities of spots were used for comparison between chemical warfare victims and control groups. for a comparison of protein peak intensity differences between two groups, analysis of variance (anova) was performed. the software provides some powerful multivariate statistics analysis including principal component analysis (pca), correlation analysis, power analysis, and q - values (false discovery rate adjusted p - values) to make reliable biomarker detection and explore data trends. also maximum of coefficients of variance (cv%) compared between two groups according to the spots normalized volume to explore specific groups of spots the selected spots were separated from gel manually and ms spectrometry analysis was done at the university of york source bioscience lifesciences, england. then identification of protein query was performed by using the mascot program searches within ncbinr 20101130 database according to the following parameters : peptide mass tolerance, 100 ppm ; ms / ms ion mass tolerance, 0.5 da ; allowing up to one missed cleavage ; variable modifications considered were methionine oxidation and cysteine carboxy - amido - methylation. equal amount of depleted serum of each groups was pooled and then separated by 11% one - dimensional sds - page and transferred to nitrocellulose membranes in a transblot electrophoresis transfer cell (bio - rad, hercules, ca, usa). western blot analyses were performed by using rabbit polyclonal antibodies against tf (diluted 1 : 1000, abcam) and a1at (diluted 1 : 500, sigma - aldrich). the membranes were blotted with a horseradish peroxidase - conjugated goat anti - rabbit igg secondary antibody (diluted 1 : 20000, sigma - aldrich) for 1 h and then detected with enhanced chemiluminescence reagents for 1 min. among precipitation methods, 10% tca / acetone indicates better 2d pattern according to the removal of albumin and igg (table 1). also this precipitation method in comparison with proteominer kit shows more spot numbers while proteominer kit removes albumin and igg almost completely, leading to nonspecific removal of some proteins and minor spot number (figure 1). the quality of grouping was evaluated by pca plot for spots with anova p value 0.08, among selected spots with anova p value 0.8. eventually, result of multivariate statistics leads to selection of twenty spots with both anova p value < 0.05 and power 0.8. in final stage thirteen spots (except keratin 1) were identified by using maldi tof / tof mass spectrometry and search within ncbi database with mascot (table 2). to confirm the mass spectrometry obtained data, we performed western blot analyses of two proteins tf and a1at as shown in figure 4(c). the immunoblot analysis also showed decreased a1at and tf isoforms in the pooled serum of severe patient compared with healthy control group. according to mentioned approach we identified thirteen proteins including albumin, keratin (except keratin 1), and hp isoforms as well as immunoglobulin kappa chain that were upregulated in patients plus two downregulated proteins including tf and a1at. 's procedure eventuates the best quality of two - dimensional gel profile in comparison with relevant available precipitation protocols. in addition, further modification of chen 's method indicated more protein spots as well as less streaking in 2de profile than proteominer protein enrichment kit (figure 1). tca / acetone precipitation has shown good potential for minimizing protein degradation by proteases and removal of contaminant such as salts. hp is a plasma 2-glyco - protein that belongs to the family of acute phase proteins. the hp-1 and hp-2 have the same chain but they differ in their chain isoforms. there are several well - known roles for hp, but the more significant of them is as hemoglobin (hb) binding protein. iron derived from hb can catalyze a series of oxidative reaction that is able to participate in organic and inorganic oxygen radical reactions and promoting the accumulation of hydroxyl radicals according to fenton and heber - weiss reactions [28, 29 ]. also reactive oxygen and nitrogen spaces seem to contribute as key elements in the radiation - induced late effects which could promote the fibrogenesis through contribution in the transforming growth factor- (tgf) signaling pathway activation [11, 17 ]. it is notable that the complexes of hp-2hb have a higher half - life in the circulation than hp-1hb complexes, causing increased accumulation of hb in the plasma and the tissues, particularly in the proximal tubule of the kidney, resulting in vascular damages. hp plays a double - edge role. while its expression is elevated to prevent oxidative stress of hb derived iron via stabilizing heme in the heme pocket of hb, increasing of hp-2 genotype due to longer half - life of its hp - hb complexes in circulation leads to increased accumulation of hb and its derived iron in the plasma and the tissues. also the allele frequencies of hp and their association in diseases are reviewed in detail by carter and worwood. in addition, they found hp isoforms in bronchoalveolar lavage fluid of sm exposed patients were significantly elevated in moderate and severe lung disease patients compared to mild and healthy controls. de kleijn. have shown that arterial expressed hp stimulates angiogenesis, tissue regeneration, and cell migration through inducing accumulation of gelatin by inhibition gelatinases such as mmp-2 and mmp-9. these collagen deposition and extracellular matrix accumulation have been also due to activation of tgf signaling pathway after radiation exposure. from this point of view our finding could elevate the hp and mmp roles in delayed sm ocular pathology which are also previously discussed by sardasht - iran cohort studies in long - term pulmonary complication induced by sm [39, 40 ]. in our study tf is a plasma glycoprotein which has the capacity to bind to two free atoms of ferric iron (fe) in biological fluid. tf represents a protective mechanism against the presence of free iron in the plasma, which could be extremely toxic. the increasing level of serum tf leads to iron deficiency anemia while decreasing level of that leads to iron overload disorders [31, 43, 44 ].. therefore reduction of tf level as we found could lead to imbalances in free iron homeostasis effects including free radical production, mutation, and also cell damages. furthermore, tf deficiency anemia could induce expression of transcription factors such as hypoxia induced factors due to lack of oxygen. this tissue hypoxia could seemingly be one of the mechanisms perpetuating the fibrogenic response in radiation - induced late effects due to vascular damage and tissue remodeling [11, 17 ]. this protein also revealed downregulation in our study (figure 4(b)) while yarmohammadi. have reported significantly higher amount of salivary a1at in salivary of sulfur mustard exposed patients 20 years after the exposure. a1at like hp is an acute phase glycoprotein and prototypic member of the serpin family [48, 49 ]. it has a wide spectrum of antiprotease activity and inhibits several serine proteases such as neutrophil elastase, cathepsin - g, and proteinase-3. it thought to play an important role in limiting host tissue injury by proteases at sites of inflammation. several studies have shown that a1at specifically inhibits the activity of caspase-3 [5153 ]. the inherited a1at deficiency exhibits an increased susceptibility to chronic inflammatory conditions, including chronic obstructive lung disease, liver diseases, and occasionally, systemic vasculitis, and necrotizing panniculitis. here, it appears that lack of a1at leads to protease induced tissue damages which cooperate with mmp activity eventuating cell apoptosis which could promote in association with free iron toxicity [52, 53, 55 ]. findings that a1at enhances the synthesis of both transferrin receptor and ferritin revealed a role of a1at in iron metabolism. here it would be considered maybe that a1at reduction affects incompetency of tf receptor and ferritin syntheses and lead to hp over expression due to cytokines (tnf-) stimulation effects [48, 56, 57 ]. our goal was to find a molecular basis for sulfur mustard delayed ocular symptoms and eventually treatment of patients. finally, our finding elevated important effects of free iron induced oxidative stresses which are supplemented with protease activity (especially mmp) in development of delayed eye symptoms which both indirectly could uphold cell depletion supposition as bases for delayed ocular symptoms. although the rules of all mentioned proteins are not specified here completely, delayed ocular injuries may be partially explained by these protein changes. also it can be inferred that changes in serum constituents are due to systemic side effects of sm rather than its local ocular side effects. we hope that our results have revealed some furtive aspects of sm exposed delayed ocular symptoms that will be promoted by future studies. | objective. sulfur mustard (sm) is a highly reactive alkylating agent which produces ocular, respiratory, and skin damages. eyes are the most sensitive organ to sm due to high intrinsic metabolic and rapid turnover rate of corneal epithelium and aqueous - mucous interfaces of the cornea and conjunctiva. here we investigate underlying molecular mechanism of sm exposure delayed effects which is still a controversial issue after about 30 years. materials and methods. following ethical approval, we have analyzed serum proteome of ten severe sm exposed male patients with delayed eye symptoms with two - dimensional electrophoresis followed by matrix - assisted laser desorption / ionization time - of - flight / time - of - flight mass spectrometry. the western blotting was used to confirm the proteins that have been identified. results. we have identified thirteen proteins including albumin, haptoglobin, and keratin isoforms as well as immunoglobulin kappa chain which showed upregulation while transferrin and alpha 1 antitrypsin revealed downregulation in these patients in comparison with healthy control group. conclusions. our results elevated participation of free iron circulatory imbalance and local matrix - metalloproteinase activity in development of delayed ocular symptoms induced by sm. it demonstrates that sm induced systemic toxicity leads to some serum protein changes that continually and gradually exacerbate the ocular surface injuries. |
diabetes mellitus is a group of metabolic diseases characterized by hyperglycemia resulting from defects in insulin secretion, insulin action, or both. there are three major types of diabetes : type 1 diabetes, type 2 diabetes, and gestational diabetes. type 1 diabetes mellitus (t1 dm) is produced by the destruction of cells of the pancreas. this leads to insulin deficiency, which can be mild initially, but develops quickly towards a complete lack of the hormone. the velocity of -cells destruction is variable and in case of infants it usually progresses rapidly while this loss is somewhat slower in case of adults. a predisposition to develop t1 dm is passed through generations in families, but the inheritance pattern is unknown. moreover, there are also environmental factors related to the probability of developing t1 dm that are still unclear. generally, people with t1 dm present with acute symptoms of diabetes and markedly elevated blood glucose levels.1 the pathophysiology of type 2 diabetes mellitus (t2 dm) combines defects that lead finally to hyperglycemia. first of all, there is insulin resistance produced in the striated muscle (also called peripheral insulin) and insulin resistance produced in the liver (also called central insulin resistance). however, since the cells fail to maintain sufficient hormone quantity to offset insulin resistance, hyperglycemia arises, which indicates failure in insulin secretion. this form of diabetes accounts for 90%95% of those patients with diabetes, but many cases remain underdiagnosed until complications appear. at the mild stage however, as this type of diabetes is progressive and chronic, it requires more intensive therapy as the disease progresses. eventually, patients need to take medication to keep blood glucose at normal levels.1 diabetes mellitus is a worldwide problem, which accounted for 1.3 million lives in 2008.2 this chronic illness requires continuing medical care and patient self - management education. in addition to the consequences of abnormal glucose metabolism, diabetes mellitus can lead to long - term complications (cardiovascular, peripheral vascular, ocular, neurologic, and renal).3,4 in europe, there are about 60 million people with diabetes, approximately 10.3% of men and 9.6% of women aged 25 years and over. across countries, germany, spain, and italy were respectively on the second, fourth, and fifth position regarding the highest numbers of people with diabetes in europe in 2013.5 diabetes mellitus has been shown to be an expensive condition, costing the european union over 50 billion per year.6,7 the impact of diabetes can be measured by using the concept of burden of disease (bod), which is used to assess and compare the relative burden of different conditions by quantifying health loss due to the disease and injury that remains after treatment, rehabilitation, or prevention efforts of the health system and society in general.8 the bod can be measured by cost, morbidity, and mortality. in the last few decades, this burden has been increasingly measured across nations for comparability purposes.912 disability - adjusted life years (dalys)13 are a measure that combines the concept of potential years of life lost due to premature death and years of healthy life lost in states of less than full health (disability states);14 hence, dalys reflect the burden of a condition and facilitate comparisons of different (in theory all) types of health conditions or health outcomes. in particular, dalys may be used to include the burden caused by disability and chronic diseases in cost - effectiveness studies.15 compared to quality - adjusted life years (qalys), dalys permit comparisons across different interventions or health areas to understand the effectiveness of a portfolio of interventions by using a standardized set of disability weights.16 the objective of this study was to carry out a literature review to collect data on epidemiology, life expectancy (le), mortality, quality of life (qol), and disability for daly calculation on diabetes for france, germany, italy, spain, and the uk. the data were used to calculate the number of dalys for the total patient population with diabetes for the countries of interested by sex and age group and to compare between countries for 2010. the daly is a health gap measure that combines information on fatal and nonfatal health outcomes ; hence, the daly measures the health loss. the basic assumption of dalys is that similar events would be treated equally in all populations to ensure comparability ; hence, individual characteristics are restricted to age and sex. this research used the hybrid daly (h - daly) approach to calculate the dalys for comparisons.17,18 h - dalys are calculated by adding incidence of years of life lost (ylls) to prevalence of years lived with disability (ylds), and thus quantifies both the bod occurring during the reference period and the burden accrued into the future. the hybrid method is a true period - measure because all the data needed to calculate h - dalys can be measured in the time period. the yll is the number of years that death occurred earlier than the age the person was expected to die if he / she had not suffered from the disorder causing death. yll was calculated as follows : (1)yll = delwith de being the number of deaths related to diabetes as the primary cause in a certain time period and l being the residual le at each year of age8 (standard le when being in a certain age category). the yld is the number of years due to disability and is calculated by multiplying the years lost with a disability weight reflecting the severity of the condition. yld is calculated as follows : (2)yldpervx = pxdwxwith px being the prevalent cases and dwx being the disability weight. from the prevalence and the population data, the prevalent cases with diabetes per age category are calculated as the outcome of prevalence multiplied by population (total), which are calculated per country, age group, and sex. then, the daly formula is as follows : (3)dalys = yldsprevx+ylls in this study, since the incidence of diabetes was not available to calculate the ylds component of the daly, an incidence - based method was not feasible. nonetheless, the data available on prevalence for diabetes of different age categories were available for france, germany, italy, spain, and the uk. moreover, the discounting process is not applicable in this context since this study is a static picture of the burden of diabetes in 2010. finally, no age - weighting factor was applied to dalys, which is similar to the last version of the global bod by murray, which also did not apply any age weights.19 the h - daly avoids both the polio and elvis problems. the polio problem refers to the fact that the incidence is captured at a certain point in time and if there are no new cases in the next year, the burden might be underestimated. the elvis problem refers to the counterfactual assessment that overestimates the prevalence since some cases could be diagnosed many years ago. moreover, only ylls were calculated from an incidence perspective, which makes it easy to choose death as the event and connect all ylls to the moment of death. the hybrid methods consistently combine the projection of the future duration of disability (incidence part) and the knowledge of deaths that occurred prior to the time period in question (prevalence part). the demographic data on the population size specified by age and gender were collected for every country from each national statistics database.2024 similar to population size data, le (which is not related to any specific disease) by age group, and sex were collected for the five countries from their national statistics. the epidemiological data with reference to prevalence were obtained from each national statistics database, except for france (ricci)25 and germany (heidemann).26 prevalence data for the uk included region - specific data for england,27 scotland,28 wales,29 and northern ireland.30 to be able to compare the uk with other countries of interest, uniform age categories for the communities within the uk were defined. for the age group 014 years, prevalence data only included t1 dm, as no data on type 2 diabetes for this age category were available. it is assumed that 97% of the population in this age group was suffering from t1 dm. the demographic data on deaths caused by diabetes from france,31 germany,32 italy,33 spain,34 and the uk35 were obtained. diabetes mortality rates were obtained by using the number of deaths due to diabetes relative to the total population. figures 1 and 2 contain estimations of mortality due to diabetes for men and women, respectively. in case of men s mortality differences across countries are clear : the uk had the lowest mortality rate while france is the second best - off ; the mortality rates for france were two times higher than the uk rates. at the age range 75 + years, germany and spain had very similar mortality rates. by far the highest value was for italy, where more than 250 out of 100,000 men aged 75 years and over died from diabetes in 2010. for women (figure 2), diabetes mortality was quite low for the younger age ranges but there was a steep increment from 6574 years to the 75 + years age range for france, germany, italy and spain. for instance in italy, the mortality rate went from 40 to 263 per 100,000 women. these rates were used to obtain the yll by using the average age of death per age group and le for every age group. the full formula used for yll calculation was derived from the world health organization (who) and fox - rushby and hanson.10,12 furthermore, for this study, the disability weights for the treated population were 0.033. it is assumed that 2.4% of the adult population 15 years old and over are not treated for their diabetes based on estimates by hill ; the disability weight applied for untreated diabetics was 0.012.36 these disability weights were obtained from the global bod study.9 for children between 014 years old, it is assumed that this population was 100% treated since they almost always uniquely suffer from t1 dm. for the sensitivity analysis, a disability weight of 0.07 was used for yld calculation based on stouthard and mathers.37,38 the demographic data on the population size specified by age and gender were collected for every country from each national statistics database.2024 similar to population size data, le (which is not related to any specific disease) by age group, and sex were collected for the five countries from their national statistics. the epidemiological data with reference to prevalence were obtained from each national statistics database, except for france (ricci)25 and germany (heidemann).26 prevalence data for the uk included region - specific data for england,27 scotland,28 wales,29 and northern ireland.30 to be able to compare the uk with other countries of interest, uniform age categories for the communities within the uk were defined. for the age group 014 years, prevalence data only included t1 dm, as no data on type 2 diabetes for this age category were available. it is assumed that 97% of the population in this age group was suffering from t1 dm. the demographic data on deaths caused by diabetes from france,31 germany,32 italy,33 spain,34 and the uk35 were obtained. diabetes mortality rates were obtained by using the number of deaths due to diabetes relative to the total population. figures 1 and 2 contain estimations of mortality due to diabetes for men and women, respectively. in case of men s mortality differences across countries are clear : the uk had the lowest mortality rate while france is the second best - off ; the mortality rates for france were two times higher than the uk rates. at the age range 75 + years, germany and spain had very similar mortality rates. by far the highest value was for italy, where more than 250 out of 100,000 men aged 75 years and over died from diabetes in 2010. for women (figure 2), diabetes mortality was quite low for the younger age ranges but there was a steep increment from 6574 years to the 75 + years age range for france, germany, italy and spain. for instance in italy, the mortality rate went from 40 to 263 per 100,000 women. these rates were used to obtain the yll by using the average age of death per age group and le for every age group. the full formula used for yll calculation was derived from the world health organization (who) and fox - rushby and hanson.10,12 furthermore, for this study, the disability weights for the treated population were 0.033. it is assumed that 2.4% of the adult population 15 years old and over are not treated for their diabetes based on estimates by hill ; the disability weight applied for untreated diabetics was 0.012.36 these disability weights were obtained from the global bod study.9 for children between 014 years old, it is assumed that this population was 100% treated since they almost always uniquely suffer from t1 dm. for the sensitivity analysis, a disability weight of 0.07 was used for yld calculation based on stouthard and mathers.37,38 detailed results of yll, yld, and daly for 2010 are presented in table 1. estimates for france, germany, italy, spain, and the uk were calculated as a rate per 1,000 inhabitants (inh) in order to compare results between countries. overall, the relative burden of diabetes was shown to be the highest for germany and italy : their estimates were 5.9 and 5.8 dalys per 1,000 inh, respectively. on the contrary, the uk had the lowest value of total dalys lost, at 2.94 dalys per 1,000 inh. in between these extremes, the negative impact of diabetes in france and spain caused 3.70 and 4.36 dalys per 1,000 inh, respectively. as has been mentioned, each component of the daly reflects the health losses associated with diabetes. the yll is the difference between the expected age at death (le) and actual age at death (mortality due to diabetes). regardless of sex, estimates of yll were greater than yld for france, germany, italy, and spain ; in contrast, ylds were greater than ylls for the uk. the differences were driven by the values of yll for both sexes. for instance, for men, although ylds values could be considered that follow a similar pattern, the uk has one - third of germany s yll. indeed, the difference between the highest and the lowest yld was 0.49, while the disparity in yll values was 2.49 points. this suggests that there is a higher burden caused by mortality due to diabetes for france, germany, italy, and spain, whereas in the uk, the burden due to disability was higher. differences in daly values indicate that the burden of diabetes was higher for women compared to men in germany, italy, and spain in 2010. conversely, dalys in france and the uk were higher for men, suggesting a higher burden of diabetes for men compared to women in these two countries of interest. the total number of dalys per age group for men and women are shown in figures 3 and 4, respectively. diabetes dalys lost for men were very low for the younger age ranges, for all countries. however, in the uk, for those aged 3544 years, the burden increased substantially, while diabetes dalys remained low for the rest of these countries of interest. diabetes was shown to consistently increase the burden of the disease across all age groups in france, germany, italy, and spain with the exception of the uk, whose burden did not start to expand again until the 5564 years age group upwards (figure 3). diabetes dalys for women were very low for the younger age ranges in 2010 (figure 4). the impact of diabetes continued quite constantly until the 4554 years age group, after which diabetes burden became notable. although increments of dalys lost for the 4554 and 6574 years age groups were modest, diabetes burden increased heavily in the older age groups, for all five countries. for all five countries, dalys due to diabetes of the elderly population (6574 and 75 + years) were divergent across countries for both men and women, which suggests that burden of the disease is quite different across countries for the oldest groups. indeed, the uk s values were almost one - third of the dalys in germany. the increasing number of dalys along with older ages might be explained due to the increasing prevalence and mortality of diabetes in older age groups (figures 1 and 2). thus, it is straightforward to distinguish different patterns : italy, germany, and spain had a steep increment in diabetes burden as age increases, while the uk was shown to have the mildest burden, and france was in between the two trends. in the sensitivity analysis, the disability weight used was 0.07 for the treated and untreated diabetics;25 the results are shown in table 2. this fact is reflected in the dalys estimates : the loss of dalys due to diabetes was higher for all countries in comparison with the base case. the ranking is unaltered : germany has the highest daly loss, italy is the second worse - off, the third position was held by spain, and the fourth and fifth positions were held by france and the uk, respectively. despite the unaltered burden ranking, germany also has the biggest difference if we compared estimates of the sensitivity analysis and the base case (2.87 daly per 1,000 inh). the second biggest difference was for the uk, which had 2.17 dalys extra per 1,000 inh. the smallest difference was for france, with a difference of only 1.6 daly lost per 1,000 inh. in the sensitivity analysis, the disability weight used was 0.07 for the treated and untreated diabetics;25 the results are shown in table 2. this fact is reflected in the dalys estimates : the loss of dalys due to diabetes was higher for all countries in comparison with the base case. the ranking is unaltered : germany has the highest daly loss, italy is the second worse - off, the third position was held by spain, and the fourth and fifth positions were held by france and the uk, respectively. despite the unaltered burden ranking, germany also has the biggest difference if we compared estimates of the sensitivity analysis and the base case (2.87 daly per 1,000 inh). the second biggest difference was for the uk, which had 2.17 dalys extra per 1,000 inh. the smallest difference was for france, with a difference of only 1.6 daly lost per 1,000 inh. the present study provides a reliable image of the burden of diabetes for france, germany, italy, spain, and the uk in 2010 according to the methodology by the who.19,20 we provide estimates on diabetes itself, not based on the comorbidities due to diabetes. the approach used presents a broad insight of the burden of this condition because it makes comparison between countries feasible. using the h - daly method, with incidence ylls and prevalence ylds, dalys due to the major part of the burden was caused by premature mortality in france, germany, italy, and spain. however, disability due to diabetes was shown to be the primary contributor to the burden for the uk. a possible explanation for the lowest daly due to diabetes in the uk could be that the age - specific mortality has been decreasing, with considerably fewer numbers of deaths due to diabetes from the age of 65 years in men and women compared to the mortality rates of the other countries of interest. further, given that ylds are quite similar across countries, the results suggest that diabetes is better managed in the uk, avoiding fatal events associated with this condition. given these results, to determine in which country the burden of diabetes was the largest, dalys seemed necessary since the burden is not mainly driven by epidemiological frequency (mortality). additionally, it was possible to compare the burden of diabetes in men and women. overall, men had a higher number of dalys (46.46 per 1,000) compared to women (35.52 per 1,000) despite the steep increase in the number of dalys in women from 75 + years. regarding the methodological aspects, h - dalys have the advantage of avoiding both the polio and elvis problems, and only ylls were calculated from an incidence perspective, which makes it easy to choose death as the event and connect all ylls to the moment of death.20 all the data needed to calculate h - dalys can be measured at the time in question, so these dalys are a true period measure prevalence data were not directly available for all districts from one national statistics database in the uk. therefore, prevalence data were taken from different sources and adjusted to the relevant age categories. in case prevalence information was missing for an age category, it was estimated based on the mean prevalence of an equivalent age category from another country of interest. secondly, disability weights were equal for all age categories and for both sexes as no direct qol data were available to obtain individual disability weights that could have affected ylds. in general, diabetes might be perceived differently at different ages,39 which could vary the estimates. therefore, either different disability weights should be applied for each age range or age weighting should be introduced. both alternatives are controversial, and so far, there is no agreement of consistent estimates of potential weights (murray and acharya, 1996 ; anand and hanson, 1997). moreover, in this study, a sensitivity analysis was performed with alternative disability weights, which was shown to vary results significantly, and estimates indicate a greater burden of diabetes for all countries included in this analysis. thirdly, the assumptions of this study are consistent with hill, whose estimates suggested that 2.4% of diabetics in the us are not being treated.36 we were aware that this percentage might be different due to region - specific characteristics though. the estimates of dalys from this study for treated and untreated patients are pooled in a single number and further research is needed in order to estimate more specific dalys, since diabetes is a heterogeneous disease. results of this study suggest that mortality due to diabetes has been postponed until older ages in most cases. while the burden of diabetes due to premature death was still significant in 2010, it is also important to remark the substantial burden of diabetes whose medical consequences lead to disability problems. therefore, greater allocation of resources to prevent diabetes or to reduce its impact on qol may be necessary in the upcoming years. this study estimated the burden of diabetes for france, germany, italy, spain, and the uk in 2010 by using the h - daly method. the results show that germany has the highest number of total dalys lost, with a rate of 5.9 for 2010 in their population due to diabetes, while the uk has the lowest number of dalys due to diabetes. with reference to the male population, diabetes health loss estimates show that germany has the highest number of dalys, while the uk had the lowest number of dalys. these results are equivalent for the female population : germany had the biggest burden measured by 9.32 dalys due to diabetes and the uk had 4.72 dalys, which were also the lowest compared to the other countries of interest. furthermore, dalys lost to diabetes for men and women are relatively lower at younger ages and significantly increase from the 3544 years age group in all the populations of the countries of interest. this pattern may be explained by the increasing prevalence and mortality of diabetes among the older age groups. it is worth mentioning that for all countries except for the uk, a significant increase in dalys lost to diabetes in women is observed for the 75 years and over population, which may lead to a higher burden of diabetes for the national health system due to a higher likelihood of elderly women in developing comorbidities at these ages. dalys have been increasingly cited as a powerful tool to estimate the burden of different conditions and these results could be useful for the policy making process, since dalys seem to be a necessary measure to determine which country has the largest burden of diabetes. however, as for other measures, dalys should not be used as a cookbook recipe to implement public policies. | aimsto compare the burden of disease (bod) attributable to diabetes expressed in disability - adjusted life years (dalys) for five european countries in 2010.methodsdalys lost to diabetes as the sum of years of life lost and years lived with disability were estimated by sex and age using country - specific epidemiological data and global disability weights. data from various secondary sources were combined to estimate health loss due to diabetes for france, germany, italy, spain, and the uk. national statistical databases were used and if necessary, community studies were used to derive the prevalence of diabetes by sex and age group, which were weighted proportionately for a national population burden of disease estimate. all identified data were adapted to the global burden of disease methodology (2010) to calculate the burden attributable to diabetes. no age weighting and discounting was applied. sensitivity to different sources of variation was examined. germany and italy lost the largest number of dalys due to diabetes, with 5.9 and 5.8 per 1,000 inhabitants, respectively, followed by spain (4.4), france (3.7), and the uk (2.9). the highest burden was caused by mortality due to diabetes, with the exception of the uk, for which the burden due to disability of diabetes was higher. mean dalys lost were higher for women in germany, italy, and spain and shown to increase with age for all countries. sensitivity analysis in variation in disability weights and uncertainty in epidemiological data were shown to have effects on dalys lost.conclusionin spite of data limitations, the estimates reported here show that daly loss due to diabetes imposes a substantial burden on countries. cross - national variation in disease epidemiology was the largest source of variation in the burden of diabetes between countries. |
vitiligo persists to evade the researchers and patients in spite of the considerable advancement in basic understanding of the disease process and the treatment protocol over the years. when the disease becomes refractory to conventional therapy, transplantation techniques are the only options left to replenish the lost melanocytes. various surgical modalities and transplantation techniques have evolved during last few decades.[110 ] but, till date none of the medical or surgical therapeutic choices could assure guaranteed success in all the cases. this is primarily because of the obscure etiopathogenesis and elusive activity profile of the disease itself. not only with medical therapy but also with any of the surgical modus operandi deployed to achieve accomplishment, any endeavor of defining norms or principles for selection is based on one single criterion, i.e. stability of the disease. it is taken as the most important parameter before opting for any transplantation technique to treat vitiligo. even after recognizing the significance of stability and after three decades of experience in vitiligo surgery, it is quite surprising to note the little consensus regarding the optimal required period of stability. the complete lack of unanimity can be glaring in some instances [table 1 ]. in one study, the minimal period of stability as a prerequisite for grafting was mentioned to be as little a period as 4 months. while on the other side of the spectrum in another study, it was taken as 3 years. minimum period of stability in different studies other variable figures like 6 months, 1 year and 2 years can easily be obtained from some other studies as well.[1216 ] even same author has considered different period of stability in different articles. unfortunately, none of these observations were based on any concrete logic / authentic proof. even the 2 year time frame used to confirm whether lesions are progressive or stable has been a matter of divergence of opinion. the iadvl taskforce recommendation to embark on vitiligo surgery after 1 year of inactivity, a recent article has also mentioned this ambiguity and cumbersome compromise. due to a dearth of a better alternative, this capricious criteria of minimal period of stability still holds good before opting for surgical procedure. the first and foremost goal for vitiligo surgery is to induce repigmentation in depigmented part of skin. but due to some inexplicable reason, simultaneous depigmentation and repigmentation have been noticed in different areas of the same patient. variable results were obtained over the donor site and recipient site of the same patient. depigmentation of grafts was documented in herpes labialis - induced lip leucoderma.[2127 ] concurrent donor site repigmentation and depigmentation of grafts at the recipient site or donor site depigmentation with complete repigmentation of the recipient area with pigment growing out from each graft have also been noticed [figures 1 and 2 ]. depigmentation of grafts in the recipient area of stable vitiligo (shown with arrow) repigmentation of the donor site in the same patient it was suggested to observe the donor site as well, to rule out koebnerization as a side effect of surgical manipulation while commenting on the stability status of the patient by minigraft test. simultaneous perigraft spread of pigment in one area and spread of vitiligo in another area can be found in the same patient [figures 3 and 4 ]. perigraft spread of pigment (shown with arrow) simultaneous spread of disease over thumb and knee (shown with starburst sign) parallel concurrence of surgical repigmentation from the grafts and increase in size of an existing lesion in the same anatomical location was also observed [figures 57 ]. minigrafting on dorsum of foot, first session (shown with blue arrow) second session, perigraft repigmentation from grafts of previous session (shown with yellow arrow) appreciable repigmentation (yellow arrow). reactivation of disease on the same anatomical site.(shown with red starburst) this area based variable status of stability is obviously not related to the conventional refractory behavior of vitiligo in some typical so - called resistant anatomical sites such as palm, sole, lips, nipples, areola, glans penis, and bony prominences. neither is this related to the type of vitiligo such as unilateral (segmental / focal) or bilateral (symmetric, vulgaris, or generalized). before any surgical intervention or even just to have an idea about which way the disease is progressing, evaluation of stability status of vitiligo is an essential prerequisite. clinically, this can be judged by three simple indicators : historykoebner phenomenontest grafting these were elaborated and to assess the activity status of the disease some benchmark guidelines have been proposed by falabella. those are : lack of progression of old lesions within the past 2 years (in unilateral vitiligo may be shorter, and in bilateral vitiligo, stability establishes after several years);no new lesions developing within the same period;absence of a recent koebner phenomenon (either from history or experimentally produced);repigmentation of depigmented areas by medical treatment or sometimes spontaneous repigmentation;a positive mini - grafting test ; andlack of koebnerization at donor site. lack of progression of old lesions within the past 2 years (in unilateral vitiligo may be shorter, and in bilateral vitiligo, stability establishes after several years) ; no new lesions developing within the same period ; absence of a recent koebner phenomenon (either from history or experimentally produced) ; repigmentation of depigmented areas by medical treatment or sometimes spontaneous repigmentation ; a positive mini - grafting test ; and lack of koebnerization at donor site. on the backdrop of a pervasive incongruity about the minimal period of stability, an attempt was made for the first time by falabella in 1995 to fathom stability before surgery by introducing the minigrafting test. the objective of this test vowed to serve several purposes : establishing the stability of the depigmenting process ; determine a means by which patients could be selected;identifying patients who may respond to pigment cell transplantation;to anticipate the response to surgical repair. determine a means by which patients could be selected ; identifying patients who may respond to pigment cell transplantation ; to anticipate the response to surgical repair. in the original suggested procedure, a few grafts (1.0 - 1.2 mm) were placed in the center of the depigmented lesion to be scrutinized. after removal of the tape, the area was exposed to sunlight for 15 min daily for a period of 3 months. the test was considered positive if unequivocal repigmentation takes place beyond 1 mm from the border of the implanted grafts. on the other hand, if less than 1 mm or no repigmentation was observed, the test was considered as negative. in some of the biggest series, this evaluation has been termed as test grafting (tg) and found this to be a more reliable exercise than the unjustified dependence on period of stability alone. over the years, test has been vindicated and acknowledged as a powerful tool for detecting stable vitiligo, which anticipates the repigmentation success in vitiligo when surgery becomes a therapeutic option. behl expressed some reservation while commenting on falabella 's work on mini grafting and claimed better results with thin thiersch grafting. in a rejoinder falabella reiterated his faith in miniature punch grafting and countered with his sets of reasons and logics in favor of punch grafting. but, simultaneously some valid logic of skepticism was flowing parallel to this recognition. in the original article, one of the opinions was to take the outcome of the donor site as well into consideration, concurrently along with observing the behavior of the minigrafts in the recipient site. that will give a more comprehensive idea about the status of stability. but, doubts were registered over overdependence on test grafting per se as that attaches a quite a few fallacies. tg positivity does not necessarily guarantee a favorable outcome in all cases even after perfect graft take up.encouraging results were obtained even after grafting of a few tg negative cases.simultaneous donor site repigmentation and depigmentation of grafts at the recipient site or donor site depigmentation with complete repigmentation of the recipient area are not hard to come by. tg positivity does not necessarily guarantee a favorable outcome in all cases even after perfect graft take up. simultaneous donor site repigmentation and depigmentation of grafts at the recipient site or donor site depigmentation with complete repigmentation of the recipient area are not hard to come by. it seems we have to wait until the method of identification and isolation of skin - homing melanocyte - specific cytotoxic t lymphocytes is available. none other than falabella himself expressed his doubts about the comprehensiveness of minigraft test. he observed that even when the minigrafting test is positive, depigmenting activity may still be present and prevent satisfactory repigmentation. more recently, njoo explored the association between the experimentally induced koebner phenomenon (kp e) and the kobner phenomenon by history (kp - h), disease activity, and therapeutic responsiveness in vitiligo vulgaris. in the article, they proposed to measure disease activity on a 6-point scale from 1 to + 4 (vitiligo disease activity [vida ] score) and therapy - induced repigmentation grade. the authors have observed and concluded the following : the vida score did not always predict a positive kp - ethe kp - e may function well as a clinical factor to assess present disease activity and may also predict the responsiveness to fluticasone propionate plus uv - a therapy but not to uv - b (311 nm) therapy. the vida score did not always predict a positive kp - e the kp - e may function well as a clinical factor to assess present disease activity and may also predict the responsiveness to fluticasone propionate plus uv - a therapy but not to uv - b (311 nm) therapy. it this context, it can be concluded that overdependence on kp or tg may be sometimes misleading in this enigmatic disease. because what these two reveal is the apparent clinical stability only and that may not be the true reflection of stability status of the disease at the cellular level. repigmentation has been successfully induced in previous graft failure cases under nb - uvb (311 nm) phototherapy. it was also proposed that nb - uvb might have played the pivotal role of stabilizing the disease by promoting apoptosis of the perilesional and circulating melanocyte - specific cytotoxic cd 8 cells. the observation of spontaneous repigmentation of non - grafted vitiligo patches [figures 810 ] points towards a possible release of fresh cytokines from the donor skin while stimulating the vitiliginous patches and hair follicles of the grafted sites may have played some role at the distant sites by local absorption.[3638 ] another theory was the immunogenic mechanism which was originally responsible for the development of vitiligo may have lost its antigenicity due to the autologous grafts. punch grafting on medial aspect of left leg spontaneous repigmentation of medial aspect of right leg punch grafting on right leg (shown with blue arrow). spontaneous repigmentation over left leg (shown with yellow arrow) successful split thickness skin graft (stsg) in pg failure cases further complicates the picture. that means, to qualify as stable, the repigmentated status must be maintained for a period of not less than 1 year. the exact pathomechanism of stability is as vague as the pathogenesis of the disease itself. the incompletely understood mechanism may involve many assortments of theories and hypothesis or may be a combination various factors. the only definite and certain fact regarding the pathogenesis of vitiligo is the absence of melanocytes in the lesion. so, it can be deducted that destruction of melanocytes is the keystone for unfolding of the disease process. classically, there have been three hypotheses to elucidate vitiligo : the neural hypothesis, the self - destruct hypothesis, and the immune hypothesis of which the last one is gaining grounds. most of the patients with generalized vitiligo were found to have circulating antibodies to cell surface antigens on normal human melanocytes ; these antibodies were cytotoxic to normal melanocytes and to melanoma cells in tissue culture. perilesional and circulating t - cells strongly suggest that melanocyte - specific cytotoxic t - cells are involved. repigmentation has been successfully induced in previous graft failure cases under nb - uvb (311 nm) phototherapy. in 1986, grimes first reported a reduction in the number of lymphocytes and helper t cells and an increase in number of natural killer cells in vitiligo patients. she also noticed decreased helper / suppressor ratios, particularly among patients with vitiligo of less than a year 's duration and among those whose serum assayed positive for autoantibodies. but damelio in 1990 came up with just the reverse observation. in a study of 22 patients with vitiligo stable for a year or more demonstrated high helper t - cell levels and high helper / suppressor ratios (also in 23 first - degree relatives) compared to controls. he also reported loss of circadian rhythm of helper t cells in patients with active diseases and no loss of circadian rhythm of suppressor t cell. an attempt was made by hann in 1994 to compare active and inactive lesion of vitiligo by immunohistochemical methods. he documented that the percentage of b cells was higher in those with disease of recent onset (under a year) than in controls (those with later onset disease). he also noticed an increase in number of cd4 lymphocytes (especially in active lesions), but not cd8 lymphocytes, in marginal skin of active lesions. as per his observation, epidermal icam-1 was expressed in active but not in stable epidermal lesions. in the subsequent year, hann detected decreased helper t - cell levels with a reversed helper / suppressor ratio in 1995. in the margins of lesions, badri found increases in cd3 +, cd4 +, and cd8 + t cells ; many were activated and expressed the cutaneous lymphocyte - associated antigen (heca-452 +) typical of infiltrating t cells ; these changes were most marked within 0.6 mm of the lesional edge. after that, some very interesting observations by wankowicz - kalinska have opened a new horizon in the basic understanding of the pathogenesis of the disease. the detection of melanocyte - reactive cytotoxic t cells in the peripheral blood of vitiligo patients and the observed correlation between perilesional t - cell infiltration and melanocyte loss in situ suggest the important role of cellular autoimmunity in the pathogenesis of this disease. t cells have been isolated from both perilesional and nonlesional skin biopsies of vitiligo patients, then cloned and their profile of cytokine production analyzed. perilesional t - cell clones (tcc) derived from patients with vitiligo revealed a predominant type-1-like cytokine secretion profile, but the degree of type-1 polarization in uninvolved skin - derived tcc correlated with the process of microscopically observed melanocyte destruction in situ. furthermore, cd8 + tcc derived from two patients also were analyzed for reactivity against autologous melanocytes. the antimelanocyte cytotoxic reactivity was observed among cd8 + tcc isolated from perilesional biopsies of patients with vitiligo. the theory of melanocytorrhagy and apoptosis as one of the primary defects underlying melanocyte loss was discussed in another article. this theory proposes that non - segmental vitiligo is a primary melanocytorrhagic disorder with altered melanocyte responses to friction and possibly other types of stress, inducing their detachment and subsequent transepidermal loss. very recently, a group of worker while determining the markers of the stability of vitiligo cd 8, cd 45 ro, and fox p3) has observed that percentage of cd8 and cd 45 ro in lesional skin biopsy may provide objective indication of lesion / area based stability. they have also observed that a higher percentage of cytotoxic cd8 cells is associated with failure of melanocyte transplantation. so, where do we stand ? it is obvious that before any attempt to predict the outcome of any vitiligo surgery, along with the clinical stability, assessment of cellular stability is of paramount importance and the cellular parameters should also be taken into consideration. when these ultarstructural investigation facilities are now available, an attempt should be made to clearly fathom stability, not merely only on clinical ground but along with electron microscopy and histoenzymological analysis of the perilesional and nonlesional skin of vitiligo patients. probably some growth factors which are responsible for both mitogenic and melanogenic stimulation of melanocytes should also be taken into account. some serological test(s) could guide us to measure these growth factors. before embarking upon any surgical alternative no preconceived the understanding of the area - based variable status and limited period of inertia or activity might necessitate test grafting at various sites at different time interval. till those unexplored areas are charted and the cellular parameters come into existence, we should not refrain the surgical option rather we should continue surgical intervention based on logical application of our clinical knowledge. | stability is taken as the most important parameter before opting for any transplantation technique to treat vitiligo. but, simultaneous donor site repigmentation and depigmentation of grafts at the recipient site has been noted. similarly donor site depigmentation with complete repigmentation of the recipient area with pigment growing out from each graft has been observed. successful repigmentation after regrafting in previous punch failure cases has also been reported. koebner 's phenomenon from history (kp - h) and test grafting were the only available indicators to assess stability. it is quite ironic to note that even after four decades of experience in vitiligo surgery, there seems to be little consensus among workers regarding the optimal required period of stability. moreover, the exact concept of stability in vitiligo is itself still not transparent and defined beyond doubt overdependence on kph or tg may be sometimes misleading in vitiligo. these two reveal the apparent clinical stability only and that may not be the true reflection of stability status of the disease at the molecular level. antimelanocyte cytotoxic reactivity was observed among cd8 + tcc isolated from perilesional biopsies of patients with vitiligo. an attempt should be made to clearly fathom and define stability, not merely only on clinical ground but along with electron microscopy and histoenzymological analysis of the perilesional and nonlesional skin of vitiligo patients. probably some growth factors which are responsible for both mitogenic and melanogenic stimulation of melanocytes should also be taken into account. some serological test(s) could guide us to measure these growth factors. |
mastocytosis encompasses a group of rare disorders characterized by clonal proliferation of mast cells (mc) in cutaneous and/or one or more extracutaneous visceral tissue. based on the tissue affected, mastocytosis can be divided into cutaneous mastocytosis, systemic mastocytosis (sm), and unifocal mc tumor. the clinical course and prognosis of patients with sm range from indolent to aggressive. the 2008 world health organization (who) classification categorized a patient with sm into 4 distinct subtypes.1 in this classification, coexistence of sm and myeloid or lymphoid hematologic neoplasm is designated as sm with associated clonal hematologic non - mc lineage disease. the name of this category has been recently shortened to systemic mastocytosis with an associated hematological neoplasm (sm - ahn).2 this category represents the second most frequent subtype, comprising approximately 30% to 40% of all cases of sm.1,3 all subtypes of hematologic neoplasms have been previously reported within the context of sm - ahn. myeloid disorders comprise 80% to 90% of the ahn, including myelodysplastic syndrome, myeloproliferative neoplasms, myelodysplastic / myeloproliferative neoplasms, and acute myeloid leukemia (aml). however, lymphoproliferative neoplasms are much less commonly implicated accounting for about 10% to 20%, with few reported cases referring to non - hodgkin lymphoma, chronic lymphocytic leukemia / small lymphocytic lymphoma, hairy cell leukemia, multiple myeloma, and acute lymphoblastic leukemia.47 based on cytogenetic abnormalities, t(8;21)(runx1-runx1t1)-positive aml is the most common subtype of sm - aml.8 the histologic and cytologic features of sm may be masked by the associated malignancy. this is especially true when the aggregates of atypical mc infiltrate are subtle, small, or infrequently present within the biopsy specimen and obscured by the non - mc proliferation., we report a case of aml with inv(16) in whom a coexisting mastocytosis was not detectable at initial diagnosis because of an extensive involvement by aml blasts, obviously masking the compact mc infiltrate. a 30-year old nepalese man presented to emergency department with 2 weeks history of fever, abdominal pain, and fatigue. physical examination revealed fever, pallor, jaundice, and hepatosplenomegaly. peripheral blood analysis revealed moderate normochromic normocytic anemia (hemoglobin, 9 g / dl ; normal, 1317 g / dl), severe thrombocytopenia (platelets, 25 10/l ; normal, 150400 10/l), and marked leukocytosis of 164 10/l (normal, 410 10/l). peripheral blood smear showed many blast cells with left shift and increased monocytic cells with a differential count of neutrophils 7%, lymphocytes 3%, eosinophils 1%, monocytes 16%, promyelocytes 8%, promonocytes 23%, and blasts 42%. the blasts were medium to large in size with fine chromatin and prominent nucleoli, some with irregular / convoluted nuclear contour (figure 1a). initial bone marrow (bm) aspirate stained with wright stain was hypercellular with 39% blasts, 13% promonocytes (blasts equivalent), and increased monocytes (17%). maturing myeloid cells comprised (28%) severe dysplastic features and included 2% eosinophils (figure 1b). bone marrow biopsy showed markedly hypercellular bm with diffuse infiltration by blasts (figure 1c), positive for cd34, lysozyme, myeloperoxidase, and cd68, with partial positivity for cd117. there were prominent eosinophilic cells, scattered and in small groups with marked suppression of normal trilineage hemopoiesis. multicolor flow cytometry (fcm) analysis was performed on bm aspirate using cd45-gating strategy to identify the immunophenotype of the blasts. acute leukemia panel of 28 antibodies in a 4-color combination (fitc / pe / ecd / pc5 fluorescent conjugates) was used, as follows : (1) cd34/cd117/cd45/cd19, (2) cd14/cd13/cd45/cd64, (3) hla - dr / cd7/cd45/cd5, (4) cd34/cd33/cd45/cd56, (5) cd19/cd10/cd45/cd3, (6) cd15/cd33/cd45/cd2, (7) cd9/cd19/cd45/cd4, (8) cd20/cd10/cd19/cd45, (9) cmpo / ccd79a / ccd3/scd45, (10)tdt / scd19/scd3/scd45, (11) cd36/cd11c / cd45/cd11b, and (12) cd41/glycophorin a / cd45/cd61(pc7). flow cytometry analysis revealed approximately 56% myeloid blasts with moderate cd45 and expressing cd34, cmpo, cd33, cd13, and cd9, and the majority was positive for cd117 and hla - dr, with partial expression of cd15 (on approximately 35% of the cells) (figure 2). the blasts were negative for cd56, cd14, cd64, cd11b, cd11c, cd36, cd61, cd41, glycophorin a, tdt, and b - cell and t - cell markers. monocytic cells increased (23%) expressing cd64, and the majority was positive for cd14. cells in the granulocytic gate comprised approximately 17% showing loss of cd10 expression (features of dysmaturation). the morphology and fcm findings were consistent with the diagnosis of aml with increased monocytic cells. cytogenetic analysis by fluorescent in situ hybridization (fish) using a probe for cbfb (bar) gene revealed an abnormal hybridization signal pattern, indicating rearrangement in 56% of the cells. this was confirmed by conventional karyotype which showed inv(16)(p13.1q22), concluding the diagnosis of aml with inv(16)(p13.1;q22) cbfb : myh11 (figure 3). patient initial laboratory workup revealed elevated total bilirubin of 82.6 mol / l (normal, 320 mol / l), and high liver enzymes, with alanine transaminase (alt) of 187 iu / l (normal, 040 iu / l), aspartate transaminase (ast) of 87 iu / l (normal, 037 iu / l), and alkaline phosphatase of 378 iu / l (normal, 40150 iu / l). renal function tests were normal with creatinine of 94 mol / l (normal, 70115 mol / l). hepatitis serology screening showed hepatitis e virus igg and igm with evidence of past hepatitis b virus infection, for which lamivudine was given before starting chemotherapy. magnetic resonance imaging of the liver and magnetic resonance cholangiopancreatography done to exclude biliary obstruction revealed hepatomegaly with no focal lesion, multiple porta hepatis, peripancreatic, mesenteric, and para - aortic lymph nodes with no evidence of biliary obstruction. all septic workup and the patient was started on cytarabine (100 mg / m) for 7 days. the patient had persistent fever during the neutropenia phase despite treatment with broad - spectrum antimicrobial and antifungal drugs. miliary tuberculosis (tb) was suspected, for which liver biopsy was performed and was consistent with cholestatic hepatitis with the absence of granulomas or leukemic infiltration. the patient had no clinical findings, suggesting central nervous system (cns) involvement ; however, intrathecal chemotherapy was planned upfront as the patient was considered to be at high risk due to cns involvement. examination of cerebrospinal fluid was delayed as the patient was critically ill ; it was done just before the first consolidation and revealed few blasts confirmed by fcm analysis. the patient was treated with triple intrathecal chemotherapy twice weekly for total of 8 doses. bone marrow examination post first chemotherapy cycle showed cellular aspirate with approximately 19% blasts, and bm biopsy showed interstitially increased primitive cells. immunohistochemical stains showed increased cd34 positivity roughly estimated at 20% to 30% and overall increased positivity for cd68 with focal collections of cd117-positive cells interpreted as persistence of the leukemic process. after recovery of neutropenia, the patient was started on second induction 3 + 7 protocol. therapeutic pleural tapping was done and showed white blood count of 1000/l with 65% lymphocytes, no blast cells, glucose of 6.2 mmol / l, protein of 50.6 g / l, lactate dehydrogenase of 104 iu / l, ph 7.5, and negative for acid - fast bacilli and tb by polymerase chain reaction (pcr). few days later, fever subsided, jaundice resolved, and the patient s condition improved. evaluation of bm after second induction showed cellular bm with 3% blasts, indicating remission. however, the bm aspirate showed the presence of morphologically atypical mc comprising approximately 2% of the total cells, mostly with irregular nuclear contour (kidney - shaped, bilobed, or multilobed nuclei), pale cytoplasm with cytoplasmic hypogranulation or irregular metachromatic granule distribution, some with fine nuclear chromatin (promastocyte morphology), together with some cells showing eccentric nucleus or spindle shape (figure 4a and b). examination of the bm biopsy revealed multiple perivascular and randomly distributed focal collections of mc (in clusters of > 15 cells) with some interstitially increased mc with spindle forms and some with lobed nucleus. these findings established the diagnosis of sm with aml. when reexamining the bm at diagnosis, mc were not impressive in the aspirate ; however, re - evaluation of the marrow biopsy with the aid of tryptase and cd25 immunostain revealed many scattered and multiple dense clusters of cells positive for cd117, tryptase, and cd25, indicating that mastocytosis was present from the start but masked by the extensive infiltration by blasts. g / l), and allele - specific oligonucleotide polymerase chain reaction (aso - pcr) for kit asp816val gene mutation was performed on peripheral blood and proved to be negative. first consolidation with high - dose cytarabine 3 g / m on days 1, 3, and 5 along with idarubicin 12 mg / m was given. subsequently, the patient received 2 further consolidation courses with high - dose cytarabine that was complicated with febrile neutropenia with enterobacter cloacae bacteremia on third cycle that required intensive care admission and antimicrobial treatment with meropenem. bone marrow evaluation after completion of therapy revealed no increase in blast cells, but there was persistence of mastocytosis. as there was a high risk of relapse, allogeneic stem cell transplant was planned. however, it was not done as the patient traveled back to his home country. world health organization classification of tumors of hematopoietic and lymphoid tissue (2008 edition) recognizes 4 major subcategories for systemic mastocytosis (sm) : indolent sm (ism) with little or no evidence of organ dysfunction, aggressive sm (asm) with the presence of disease - related organopathy, sm associated with a clonal hematologic non - mc lineage disease (sm - ahn), and mast cell leukemia (mcl) with the presence of 20% mc in bm aspirate.1,2 the who defines 1 major criterion and 4 minor criteria for the diagnosis of sm. the major criterion is detection of multifocal, dense infiltrates of mc (15 mc in aggregates) in an adequate bm biopsy specimen and/or other extracutaneous organ(s). the minor criteria are as follows : (1) greater than 25% of mc (in the bm or other extracutaneous organ biopsy specimens) are spindle shaped or have atypical morphology, or greater than 25% of the mc in the bm aspirate smear are immature or atypical ; (2) an activating point mutation at codon 816 of kit in bm, blood, or other extracutaneous organs is detected ; (3) cd2 and/or cd25, in addition to normal mc markers, are expressed on mc in the bm, blood, or other extracutaneous organs ; and (4) serum total tryptase levels persistently exceed 20 ng / ml. the presence of 1 major and 1 minor criterion or 3 minor criteria is required for the diagnosis of systemic mastocytosis. the fourth minor criterion involving elevation of serum total tryptase levels is excluded from the diagnostic criteria in cases of sm - ahn.1 the diagnosis of sm - ahn is established when who criteria for sm and a distinct hematologic non - mc lineage disease are met. in the case reported here, the criteria for the diagnosis of aml with inv(16) were fulfilled and the diagnosis of sm was established based on the presence of multifocal, dense infiltrates of mc in bm biopsy (major criterion) and 2 minor criteria, including morphologically abnormal mc that exhibit an aberrant immunophenotype (cd25 expression). therefore, the serum tryptase level in our case was elevated (38.5 g / l). the determination of serum tryptase levels is in principle a good diagnostic and differential diagnostic parameter. elevated serum tryptase levels, however, are not pathognomonic for sm, as elevated levels can also be detected in approximately 40% of patients with aml9 and in a variable number of cases with myelodysplastic syndrome (mds).10 in sm - ahn, the associated clonal hematological non - mc lineage disorder may be diagnosed before, simultaneously with, or after the diagnosis of sm. the diagnosis of sm - ahn in the bm may be difficult and the diagnosis of sm may be missed / masked at the time of diagnosis, mainly due to the tendency of mc to localize within stroma of bm particles and the compact mc infiltrates in the marrow biopsy may be obscured by the associated hematological neoplasm.11 this case represents a diagnostic challenge as it appeared to be a classical straightforward case of aml with inv(16) without evidence of mastocytosis at the time of initial diagnosis. however, following aml - directed chemotherapy, the presence of multiple perivascular and randomly distributed focal collections of mc positive for tryptase, cd117, and cd25 was unveiled with the reduction in blast cells that established the diagnosis of sm. morphologically, the neoplastic mc have been classified into 3 subtypes : metachromatic blast and atypical mc types i and ii.12 metachromatic blasts demonstrate blast - like nuclear morphology with several metachromatic granules. atypical mc type i shows elongated cytoplasmic projections (spindle form), oval eccentric nuclei, and hypogranulation, whereas atypical mc type ii typically has bilobed or polylobed nuclei. in our case, there was enhanced proliferation predominantly of atypical mc type ii with kidney - shaped, bilobed, or multilobed nuclei with cytoplasmic hypogranulation or irregular metachromatic granule distribution, some with fine nuclear chromatin (promastocyte), together with some atypical mc of type i (figure 4a and b). tryptase (cytoplasmic), cd117 (membranous and cytoplasmic), and naphthol as - d chloroacetate esterase are expressed in normal and neoplastic mc. in addition, neoplastic mc show aberrant expression of cd2 and/or cd25.12 in our case, the abnormal mc expressed cd25 but not cd2. cd25 is considered more sensitive and specific for detection of neoplastic mc by both fcm and immunohistochemistry, whereas cd2 expression is inconsistent and mostly weak or absent.13 moreover, it is reported that cd2 expression was observed significantly less often in sm - ahn than in pure sm patients for unclear reason.7 the most commonly associated hematologic malignancies are aml, mds, chronic myeloproliferative disorders, and myelodysplastic / myeloproliferative neoplasms.5 most of the published cases of sm associated with aml cover the spectrum of the french - american - british classification of aml, with a majority representing either the m2 or m5 subtype.4 based on cytogenetic abnormalities, aml with t(8;21) (q22;q22) ; runx1-runx1t1 is the most common subtype of sm - aml.11,14 however, case reports on sm with aml with inv(16)(p13.1;1q22) ; cbf :myh11 seem to be extremely rare. on literature review and up to our knowledge, this is the first comprehensive case report of aml with inv(16) presented with concurrent hidden systemic mastocytosis. the diagnostic workup for systemic mastocytosis includes molecular analysis for kit d816v mutation. in the patient reported here, the test for asp816val c - kit mutation using aso - pcr performed outside clinical laboratory was negative (sensitivity of the assay, 0.01% mutated alleles). as the aso - pcr used for this patient was a qualitative pcr with analytic sensitivity of 0.01%, false - negative results caused by low number of neoplastic cells that is below the detection limit can not be entirely ruled out. kit encodes for a receptor tyrosine kinase (tk) present on the cell surface of hematopoietic cells as well as other cell types. activating kit d816v mutation is found in greater than 80% of cases with mastocytosis.15 kit mutations are frequently detectable as well in a group of patients with core - factor binding leukemias. it is reported in approximately 22% of aml with t(8;21) and in 30% of aml with inv(16) cases. in such cases, exon 17 d816v is the most common mutation found.16 detection of kit mutations in patients with t(8;21) or inv(16)/t(16;16) is indicative of a worse prognosis.17 whether patients with aml and kit d816v mutation have coexisting occult sm is still a controversial issue. a study by kristensen suggested that sm is uncommon in cbf leukemias positive for kit d816v mutation. their study examined the frequency of sm in 20 cases of cbf aml ; 4 of 13 cases of aml with t(8;21) and 4 of 7 cases of aml with inv(16) examined carried the d816v mutation assessed by real - time pcr of peripheral blood mononuclear cells. none of the 8 positive cases had evidence of sm by immunohistochemistry for cd117 and tryptase. johnson reported sm in 4 (10%) of 40 patients with aml with t(8;21). kit d816v mutation was identified in 3 of these 4 patients. in keeping with the latter finding, fritsche - polanz found high frequency of concomitant mastocytosis and reported sm in 7 of 7 d816v - positive non - cbf aml cases. in patients with sm - ahn, a clonal relationship between the mc and the associated hematologic non - mc lineage disorder (ahn) component has been sought using kit and other mutations as markers of clonality. pullarkat examined 1 case of sm - aml with t(8;21) using fish technique and demonstrated that mc also possessed the runx1-runx1t1 translocation. moreover, bae demonstrated the presence of d816v kit mutation using reverse transcription polymerase chain reaction and runx1-runx1t1 expression by using fish in both cell lineages in a case of sm - aml with t(8;21). these studies had demonstrated evidence that neoplastic mc and myeloid leukemic blasts are likely to develop from common hematopoietic progenitors. however, sotlar and colleagues using laser capture microdissection showed that kit d816v mutations are not only present in mc but are also variably present in the cells of the ahn component. the frequency of kit d816v mutation, however, is dependent on the type of anh. these findings challenge the concept that the sm and ahn components in patients with sm - ahn arise uniformly from a precommitted neoplastic progenitor (stem) cell harboring a kit and suggest that the sm - ahn category proposed is highly heterogeneous.6 in cases of sm - ahn, the hematologic disorder must be treated, whereas management options for sm include observation alone, symptom management, supportive measures, and cytoreductive therapy for mc debulking in the setting of aggressive, advanced, or treatment - refractory disease. histamine h1 receptor antagonists and histamine h2 receptor antagonists are used to control mediator - related symptoms. interferon alfa (ifn-) as first - line cytoreductive treatment has activity in all sm subtypes and has been shown to improve dermatological, hematologic, gastrointestinal, and systemic symptoms associated with histamine release. cladribine (2-cda) is used as a first - line treatment in cases where rapid mc debulking is indicated or in symptomatic patients who are refractory or intolerant to ifn-. in recent years, various tk inhibitors have also been used for sm. the rare sm cases that harbor an imatinib - sensitive kit mutation or those that are kitd816 unmutated may be appropriate candidates for imatinib treatment.22,23 new tk inhibitors, such as midostaurin, target kit mutants that are reported to act against aggressive systemic mastocytosis and mc leukemia.24 allogeneic hemopoietic stem cell transplantation has been reported to induce continuous remission in a subset of patients with aggressive sm and mc leukemia.25 an open - label, phase 2 study with midostaurin (multikinase inhibitor) targeting kit d816v showed efficacy in the treatment of patients with advance systemic mastocytosis, including cases of sm - ahn.26 in our case, and after discussion with a multidisciplinary team, the specific treatment for aml (appropriate for age and performance status of the patient) was given. specific treatment for mastocytosis was not necessary as there were no definite symptoms related to mc mediator release. allogeneic stem cell transplant was planned as there was high risk of relapse. in conclusion, this report indicates that the diagnosis of sm with associated clonal non - mc neoplasm may be very challenging in some cases. the diagnosis can be easily missed as aggregates of mc may be overlooked at the time of initial diagnosis of aml and that the concomitant diagnosis of sm with aml requires comprehensive morphologic and immunophenotypic evaluation for detection of abnormal mc proliferation. based on our experience in this case, we recommend routine screening for the presence of hidden mastocytosis in cases of aml at initial diagnosis by including mc tryptase in the immunohistochemistry panel, particularly in cases of cbf aml. monitoring of larger number of aml patients with sm may help to assess whether patients prognosis is truly affected by the presence of concurrent mc disease and whether specific customized treatment may benefit those patients. | systemic mastocytosis (sm) is a condition associated with clonal neoplastic proliferation of mast cells. in up to 40% of systemic mastocytosis cases, an associated clonal hematological disease of non mast cell lineage, such as acute myeloid leukemia (aml), is diagnosed before, simultaneously with, or after the diagnosis of sm. herein, we report a case of a 30-year - old man diagnosed with aml with inv(16) (p13;q22) cbfb : myh11. associated mastocytosis was not noted at diagnosis and was only detected in the bone marrow at time of remission after successful chemotherapy. the diagnosis of mastocytosis was based on the demonstration of a multifocal dense mast cell infiltrate in the marrow biopsy with aberrant immunophenotype, with coexpression of tryptase, cd117, and cd25. the mast cells showed atypical morphology mostly with irregular nuclear contour, bilobed or multilobed nuclei with cytoplasmic hypogranulation or irregular metachromatic granule distribution, and some cells with eccentric nucleus or spindle shape. reexamination of the pretherapeutic bone marrow with immunostain for tryptase and cd25 revealed that mastocytosis was present from the start but masked by extensive blast proliferation. this case indicates that mast cell infiltrates are sometimes underappreciated at the original diagnosis of aml with inv(16) and that the concurrent diagnosis of sm with aml requires a high index of suspicion supported with comprehensive morphologic and immunohistochemical evaluation for a neoplastic mast cell proliferation. |
as environmental health professionals, we are dedicated to controlling and protecting our environment to minimize the factors that contribute to human disease, as well as enhancing human health and well - being. while the historical gains in public health are in large part due to environmental health efforts, many challenges lay ahead us in the twenty - first century. environmental health threats stemming from emerging diseases, climate change, population growth, and globalization (eg, food and chemical products), among others, will present major challenges to public health. environmental health professionals will be a critical part of the future public health workforce because they have far - reaching impacts on large segments of the population.1 efforts have been ongoing before and during the twenty - first century to enhance the environmental public health workforce.2 in 2003, the centers for disease control and prevention, with contributions from many stakeholders, published a national strategy to revitalize environmental public health services.3 since then, progress toward the strategy s goals has been laudable and discussed.4,5 yet, some issues appear insurmountable and have been highlighted elsewhere.6,7 more recently, important discussions have focused on the quantity and quality of environmental health professionals.1,8 we agree wholeheartedly with these authors and would like to focus our discussion on the specific issue of educating future environmental health professionals. but first, we must distinguish between training and education, terms that are frequently used interchangeably. according to the business dictionary, training is an organized activity aimed at imparting information and/or instructions to improve the recipient s performance or to help him or her attain a required level of knowledge or skill, whereas education is the wealth of knowledge acquired by an individual after studying particular subject matters or experiencing life lessons that provide an understanding of something.9 looking at these definitions in another way, in the words of robert h. essenhigh, training is the know how and education is the know why.10 obviously, in environmental health practice, the know how is of utmost importance, but this is not enough. the know why is important for many reasons ; foremost among them is to understand the underlying theories and basis for a procedure or standard. without this depth of understanding and knowledge, an educated professional is capable of developing solutions to problems that do not fall into the categories of the procedural or routine. in most cases, an individual with a newly minted degree in environmental health is not ready to walk into a job without some level of training or internship. however, in the long run, the properly educated individual will be more likely to excel in his or her profession and to offer innovative solutions to unprecedented problems. as we discussed earlier, many unprecedented problems will be encountered in the future of environmental health. most parents with college - age children have first - hand knowledge of issues such as rising tuition costs and increasing student debts. but there are many other issues associated with our institutions of higher education that will culminate in a crisis. these issues have been succinctly discussed and illustrated in the cnn documentary ivory tower and in a two - part series of articles by futurist thomas frey.11,12 table 1 lists some of the issues highlighted by the media and scholars with regard to the crisis in higher education. environmental health science (ehs) in fact, we contend that ehs academic programs while essential to the quantity and quality of the future environmental health workforce are at risk of becoming greatly scaled back or eliminated in the looming crisis. if thomas frey s predictions are correct, half of our colleges will collapse over the next 15 years.12 even if this dire prediction is overly pessimistic, there will certainly be a loss of degree programs at colleges and universities across the country, including ehs - degree programs. as academicians, we have experienced severe budget cuts in the past five years and participated in discussions aimed at eliminating courses and even entire programs that have enrollments below a certain threshold. unfortunately, many of the ehs academic courses fall below this threshold, and ehs academic programs have been discontinued in several universities. presumably, some of the reasons are student perceptions of lower wages, fewer training opportunities, and poorly defined career paths, as highlighted by resnick.7 on the other hand, students may not be aware of the career opportunities for ehs - degree candidates within the private sector. yet another reason may be competition with degree programs that are specialized or complementary to ehs : students can earn degrees with majors in food science and safety, industrial hygiene, toxicology, epidemiology, general public health, and a number of other related majors. many of these complementary academic programs are rivals to ehs academic programs. research is urgently needed to systematically examine the reasons for low enrollments in ehs academic programs. nonetheless, we believe that ehs academic programs will close at several institutions, and among the surviving programs, course offerings in environmental health will be significantly curtailed, perhaps resulting in the loss of accreditation status. in any case, limited course offerings will not provide an individual student with a well - rounded background in environmental health and he or she will not be exposed to the many possible career paths in environmental health. consequently, graduates will be less prepared to assume leadership positions that require coordination and integration of multidisciplinary approaches to environmental health problems. the academic programs in ehs that flourish will likely be affiliated with academic programs having a larger infrastructure. case in point, accredited schools of public health are required to have an environmental health component. unfortunately, schools of public health are not accessible to all students, and course catalogs may not be sufficient to offer undergraduate and/or graduate majors in environmental health. another possibility is that ehs academic programs will splinter into several specialties or complementary majors, again likely affiliated with academic programs having a larger infrastructure. since the passage of the food safety modernization act of 2011, for example, many food science departments and schools of agriculture have developed curricula to educate and train experts in food safety. similarly, schools of engineering have specialty courses to educate engineering students in certain aspects of environmental health such as water and wastewater. biology and chemistry departments sometimes offer environmental courses, albeit many of these courses are peripherally relevant to environmental public health. those of us who have practical experience in environmental health understand that problems do not confine themselves to a single specialty. as an example, food safety problems are frequently interwoven with issues related to pest control, water quality, infectious diseases, and chemical safety and toxicology. similarly, occupational health problems may involve exposures that require knowledge of infection control and biological safety. the challenge of developing effective environmental precautions and isolation procedures for ebola patients is a recently publicized example. while the issues listed in table 1 potentially impact all academic programs, several of them have special significance for ehs academic programs. foremost, large student debts will place a disproportionate burden on government employees such as environmental health professionals. another significant issue affecting ehs academic programs is the poor science preparation of students ; degree majors in ehs require a solid foundation in the basic sciences. if ehs academic programs are curtailed, then it will be also more difficult to attract and retain instructors with environmental health experience. the rise of online courses and degree programs poses special challenges for ehs academic programs ; many environmental health courses require hands - on laboratories and learning experiences to develop competency. the foremost goal of any ehs academic program is to offer excellent curricula with highly qualified faculty and motivated students. it is also important to realistically examine the opportunities and threats to ehs academic programs particularly in an era of unprecedented changes in our institutions of higher learning. following a sabbatical exchange, dr. charles hart shared his experiences from visiting several ehs academic programs in canada.13 it was especially interesting to note that the canadian institute of public health inspectors has direct control over both professional certification and accreditation of ehs academic programs. unlike canada, there is no single entity or system in the us that is responsible for both professional certification and ehs academic accreditation. hart, the canadian ehs academic programs are more narrowly focused on educating public health inspectors ; the ehs academic programs in the us have a broader focus that include other employment opportunities, such as positions in the private sector and environmental resource agencies. in the absence of a single structure, cooperative agreements between the national environmental health association (neha), the association of environmental health academic programs (aehap), the national environmental health science and protection accreditation council, and federal and state agencies are paramount to producing environmental health professionals. in this context, the statement attributed to professor larry gordon at the 2000 meeting of the environmental health competency project is worthy of repeating : environmental health leaders [need ] to take an active role not just in defining competencies but also in strengthening the organizations, funding, and standards that produce environmental health practitioners.2 environmental health leaders [need ] to take an active role not just in defining competencies but also in strengthening the organizations, funding, and standards that produce environmental health practitioners.2 whatever happens in the near- and far - term future, a few predictions can be made with some degree of certainty. first, environmental health issues will become more complex in the future, which will necessitate a well - educated and well - trained environmental health workforce. public health authorities need to develop policies that will help educate and train the next generation of environmental health professionals. second, without revolutionary innovation, rising tuition costs and student debts will discourage many qualified candidates from pursuing an education in environmental health. in turn, this will likely result in the reduction and/or loss of ehs programs at universities. strong consideration should be given to student debt forgiveness and/or scholarships for students majoring in environmental health, perhaps with a contractual obligation to work in government service after graduation. third, interconnected technologies will continue to increase online enrollments, but the format and structure of online courses will need to be rigorously reviewed using competency - based outcome metrics especially for environmental health students. organizations such as neha and aehap should develop criteria to evaluate, certify, and/or accredit online courses and degree programs. these criteria should be measureable and competency based ; procedures to ensure academic integrity should also be included in the criteria. finally, it is the responsibility of all pubic officials to develop and implement succession plans for workforce development and turnover. senior and retiring environmental health professionals especially those in influential positions have a special obligation to develop comprehensive succession plans. environmental health profoundly affects the health and well - being of the entire public, and environmental health leadership is needed now more than ever. | future environmental health problems will require a new generation of educated and trained professionals. efforts to enhance the environmental public health workforce have been promoted by several organizations. while progress has been measured by these organizations, many environmental health academic programs are experiencing budget reductions and lower enrollments. one of the reasons for this trend is the so - called higher education crisis. we argue that training is not equivalent to education in the environmental health sciences, albeit the two terms are often used interchangeably. organizations involved with the education, training, and credentialing of environmental health professionals must work together to ensure the viability and effectiveness of environmental health academic programs. |
strychnine is a bitter, white, powder alkaloid derived from the seeds of the tree strychnos nux - vomica. it has had an interesting past, initially having been introduced in the 16th century as a rodenticide, and until recently it was used as a respiratory, circulatory and digestive stimulant. it is no longer used in any pharmaceutical products, but is still used as a rodenticide. strychnine is also found as an adulterant in street drugs such as amphetamines, heroin and cocaine. strychnine poisoning is uncommon, and in most severe cases, the patient dies before reaching hospital. the management of strychnine poisoning is well documented, but there is less information available about the elimination kinetics of strychnine. we report the toxicokinetics of strychnine in a patient who survived a deliberate strychnine ingestion. a 42-year - old man with no significant past medical history presented approximately 1 hour after ingestion of a bottle of wine together with some " white powder " from his garden shed (this was later confirmed to be strychnine). he was able to walk in to the emergency department, but he was agitated and ataxic, in keeping with his ethanol intake. within a few minutes of his arrival, his condition rapidly deteriorated and he developed a marked tremor and muscular spasms and shortly after this had a respiratory and secondary cardiac arrest. at this stage he was transferred to the intensive care unit (icu) and the national poisons information service (london) was contacted for further advice on management. he continued to have marked muscle spasms and so was paralysed with 0.1 mg / kg pancuronium given intravenously. postarrest, his blood pressure was 85/40 mmhg, heart rate 96 beats / min and temperature 38.2c. arterial blood gases showed a severe metabolic acidosis (ph6.51, pao2 9.5 kpa, paco2 2.6 kpa, hco3 3.7 mmol / l, base excess [be ] -18) and he was given 3 mmol / kg 8.4% sodium bicarbonate ; his metabolic acidosis improved over the next 8 hours (ph7.26, paco2 5.35 kpa, pao2 13.4 kpa, hco3 18 mmol / l, be -9). he remained hypotensive despite fluid resuscitation and over the first 24 hours he required norepinephrine to maintain his blood pressure (maximum dose 900 g / h). his temperature rose to 39.9c on day2 but settled after simple cooling measures and rehydration. iu / l, (although there was no evidence of myoglobinuria) and his creatinine peaked at 194 mol / l on day 2. he was extubated on day3, initially with some persisting twitching and muscular spasms, requiring boluses of midazolam and diazepam and an alfentanil infusion for analgesia. by day5 eight serum samples were obtained over the first 5 days and subsequently analysed on a hewlett - packard 6890 gas chromatograph (stockport, cheshire, uk) equipped with a nitrogen phosphorous detector for strychnine concentrations. the patient 's initial concentration at 1.5 hours after ingestion was 4.73 mg / l, falling to 0.38 mg / l and to 0 at 74 hours and 100 hours postingestion, respectively. the data conformed to a monoexponential elimination curve and the calculated elimination half - life was 12 hours. strychnine poisoning is an uncommon but potentially fatal poisoning and most patients die before reaching hospital. in the case he was initially asymptomatic but rapidly developed severe muscle spasms leading to respiratory arrest. with meticulous supportive care in the icu he made a complete recovery. this poison is a competitive antagonist of the inhibitory neurotransmitter glycine at receptors in the spinal cord, brain stem and higher centres. the classical features of strychnine poisoning occur from 15 to 30 minutes after ingestion and include heightened awareness, muscular spasms and twitches and hypersensitivity to stimuli. in large ingestions, these can progress to painful generalised convulsions, during and after which the patient retains consciousness. the cause of death is usually respiratory arrest secondary to respiratory muscle spasms, although prolonged muscular spasm can lead to hyperthermia, rhabdomyolysis and associated renal failure due to myoglobinuria. severe metabolic acidosis can occur due to increased lactate levels following repeated muscular activity, although the lactate is rapidly removed once the muscular spasms have been controlled. prompt recognition of poisoning and initiation of treatment are required to prevent deterioration and death. control of convulsions and muscular spasms requires either high doses of benzodiazepines, such as 1 mg / kg diazepam, or, in resistant cases, paralysis with neuromuscular blockers such as pancuronium. in addition, meticulous supportive care is required, and careful monitoring and observation for complications such as acute renal failure. clinical effects of strychnine ingestion have been seen at doses as small as 2 mg in a child and 20 mg in an adult. survival after ingestion of large amounts has been reported : 480 mg (24 mg / kg) in a child and 3750 mg in an adult. death from strychnine ingestion has been reported in adults after doses as low as 5 to 10 mg. since severe, life - threatening features have been reported after the ingestion of small amounts and patients can deteriorate rapidly, strychnine ingestion should be regarded as potentially fatal and the patient should be admitted to an icu for close observation and appropriate treatment. in this case, as with many other reported cases, both the concentration of strychnine in the preparation ingested and the amount taken were unknown. the serum strychnine concentration of 4.73 mg / l at 1.5 hours postingestion is the highest reported concentration that a patient has survived. in previous reports, concentrations of 2.1 mg / l at 3 hours and 3.8 mg / l at 0.5 hours caused death. the potential fatal serum concentration of strychnine is not known and clinical features rather than the serum concentration should guide management. at a postmortem, less than 4 hours after an intentional ingestion of strychnine, a serum concentration of just 0.33 mg / l was found. a postmortem study showed that a strychnine concentration of 3.32 mg / l in the blood of the inferior vena cava was associated with concentrations of 11.4 mg / l, 2.42 mg / kg, 2.32 mg / kg and 98.6 mg / kg in bile, brain, skeletal muscle and liver, respectively. of the 50 reported cases of strychnine poisoning in the past 36 years, only two nonfatal cases have included data on serum strychnine concentrations. both of these patients had much lower concentrations (1.6 mg / l at 4 hours after ingestion and 2.17 mg / l at 6 hours after ingestion, respectively) than that found in our patient. strychnine is rapidly absorbed from the gastrointestinal tract, with symptoms occurring within 10 to 20 minutes of ingestion. it is also absorbed through other mucous membranes, as in poisonings when strychnine has been mistaken for cocaine. strychnine has been shown in animal studies to be metabolised in the liver by cytochrome p-450 microsomal enzymes, but 130% is excreted unchanged in the urine. the proportion of strychnine excreted unchanged appears to be smaller when larger amounts are ingested. in our patient, elimination of strychnine obeyed first - order kinetics with a half - life of 12 hours. only three other papers have reported on the elimination kinetics of strychnine, and these provide conflicting views of its toxicokinetics. edmunds and colleagues were the first authors to describe the elimination of strychnine and theirs is the only other report on the kinetics of strychnine in a patient who survived. they reported a 42-year - old man who ingested an unknown quantity of strychnine and developed severe muscle cramps and convulsions and had a respiratory arrest 35 minutes after ingestion. in that case, the elimination of strychnine obeyed first - order kinetics with a half - life of 10 hours, based on 19 blood samples collected over a 53-hour period, the first of which was taken at 4 hours after ingestion. palatnick. also reported first - order kinetics with a half - life of 16 hours, based on 18 serum concentrations over a 51-hour period in a 34-year - old man after ingestion of 125 ml of a 2% strychnine solution. heiser. reported zero - order kinetics in a fatal case in a 51-year - old man who presented after ingesting 4.8 g of strychnine. his serum concentrations were measured in only five samples, ranging from 3.5 mg / l at 0.5 hours after ingestion to < 0.1 mg / l at 43 hours after ingestion. the differences in the kinetics of strychnine found in our patient from the kinetics in the above three cases almost certainly reflect the influence of clinical parameters such as different periods of hypotension, metabolic acidosis and renal impairment, all of which would influence hepatic metabolism and renal elimination of strychnine. however, the calculated half - life of 12 hours in our patient is comparable to the half - lives previously reported (10 hours and 16 hours), suggesting that the true elimination half - life of strychnine lies between 10 and 16 hours. we describe a case of severe strychnine poisoning with a favourable outcome in which the strychnine half - life was 12 hours. the patient presented early, his symptoms were recognised and meticulous supportive care was instituted without delay. all of these factors were probably important in preventing what would otherwise have been a fatal ingestion of strychnine. strychnine overdose is rare but potentially fatal with prompt recognition of the characteristic features and rapid supportive care, survival is possible ingestion of small amounts and/or low plasma strychnine concentrations are associated with fatality strychnine has a toxicokinetic half - life in overdose of 12 hours elimination of strychnine in our patient obeyed firstorder kinetics, as has previously been reported | introductionstrychnine poisoning is uncommon, and in most severe cases, the patient dies before reaching hospital. the management of strychnine poisoning is well documented, although there are few data on the kinetics of elimination of strychnine after overdose.case reporta 42-year - old man presented shortly after ingestion of an unknown quantity of strychnine powder. after a respiratory arrest, with intensive supportive management requiring admission to an intensive care unit, he survived. eight serum samples were taken over the first 5 days and analysed subsequently for strychnine concentrations.resultsthe initial concentration at 1.5 hours after ingestion was 4.73 mg / l, falling to 0.38 mg / l at 74 hours postingestion. serum concentrations followed a monoexponential elimination curve with a calculated elimination half - life of 12 hours.discussion and conclusionstrychnine poisoning presents with classical features, and with early diagnosis and supportive management, the patient can survive. the initial serum concentration of 4.73 mg / l is the highest reported concentration in a patient who has survived. previous reports of the elimination half - life have suggested it is between 10 and 16 hours, which conforms to the elimination data in our case. |
one fourth of the population lives in the capital, nuuk, while the rest live in 16 towns and several settlements (the districts) scattered around the island. most persons (86%) are native greenlanders ; the rest are caucasians, mostly danes. the laboratory at queen ingrids hospital in nuuk is the only microbiologic laboratory in greenland. from laboratory files, we identified all bacterial isolates from invasive disease cases reported from january 1, 1995, though december 31, 2004. demographic information about patients was obtained from the civil registration system of greenland (6). the study was approved by the commission for scientific research in greenland and reported to the danish data protection agency. positive blood cultures were detected by using manual readings of blood culture bottles from statens serum institut, copenhagen, denmark ; beginning in 1999, the bactec / alert system (organon teknika, turnhout, belgium) was used. in this system, 2830 ml of blood positive samples are characterized by gram stain and cultured on blood agar supplemented with 5% defibrinated horse blood. antigen / antibody tests are carried out for s. pneumoniae, n. meningitidis, and h. influenzae (the last 2 only on cerebrospinal fluid). serotyping and group typing of s. pneumoniae and h. influenzae (quellung method) and n. meningitidis (latex agglutination method) are performed at statens serum institut. in total, 281 unique bacterial isolates from 254 episodes of invasive disease among 242 patients were identified : 72% of isolates in blood, 18% in cerebrospinal fluid, and 10% in other samples. of the 254 episodes, 47% occurred among patients from the districts and 53% in patients from nuuk. in 53% of episodes among patients from the districts, there were no differences in age, sex, ethnicity, underlying conditions, or mortality rates between district and nuuk patients, but greenlandic patients were younger (median 46 years, 25%75% quartiles 1759 years) than danish patients (median 57 years, 25%75% quartiles 4462 years) (p = 0.07). twenty different bacterial species or groups were identified (22 isolates could not be characterized by species) ; the most numerous were s. pneumoniae, staphylococci, and e. coli (table). for most bacteria, the invasive disease incidence varied by age in a u - shaped fashion (figure 1). most n. meningitidis cases (73%) occurred in children < 10 years of age, the 4 cases of h. influenzae group b infection occurred in children < 1 year of age, and the youngest case - patient with gbs was 12 years of age. per 100,000 population. most numerous serotypes were 12f (9) and 22f (5) ; 41 isolates not were serotyped. # two isolates in group b, 4 in group c, and 9 not grouped. s. bacillus sp. (1), corynebacterium (1), clostridium perfringens (2), listeria monocytogenes (1), and propionebacterium sp. incidence of invasive bacterial disease did not differ according to patient sex but did differ markedly according to ethnicity and region. the age - adjusted rate ratio was 1.8 (95% confidence interval [ci ] 1.12.9) for greenlanders compared with danes and 3.5 (95% ci 2.74.4) for persons living in nuuk compared with persons living in the districts, a rate ratio observed for almost all bacterial species. the overall incidence of invasive bacterial isolates increased during the study period from 17.9/100,000 in 1995 to 79/100,000 in 2004 ; the most marked increase occurred in 19971998, when incidence almost doubled. the increase occurred mainly in blood culture samples ; cerebrospinal fluid samples remained constant over time. the increase in isolates from blood cultures occurred equally in nuuk and in the districts and for most bacteria, although the incidence of n. meningitidis remained constant (figure 2). h. influenzae group b isolates were not identified after 1998 (childhood vaccination was introduced in 1997) ; the 5 gas isolates first appeared in 20012004. s. pneumoniae was the most commonly involved, confirming the importance of this pathogen in native populations in the arctic (13). however, the second and third most frequent invasive bacteria were e. coli and s. aureus, a finding which, to our knowledge, had not been described before in greenland. in contrast, the 5 bacteria included in the circumpolar surveillance system accounted for only 47% of microorganisms (5). surveillance of these 5 bacteria is relevant, though, as they are all serious, potentially fatal diseases of infants or children and are associated with some form of preventive action (either vaccines or prophylaxis). s. aureus and e. coli are not amenable to simple specific public health actions, so surveillance for these infections is less useful. evaluation of outcome and risk factors for s. aureus and e. coli in arctic areas may lead to potential preventive or clinical care options. worldwide, microbiologic causes of bacteremia have shifted from gram - negative organisms being the most common in the 1970s to gram - positive organisms with coagulase - negative staphylococci, s. aureus, and enterococci being the most frequent in the last part of the 20th century (8). likewise, we found gram - positive bacteria in 67% of invasive disease cases and gram - negative bacteria (mostly enterobacteriaceae) in 33% and increasing incidence of both gram - positive and gram - negative bacteria over the study period. the overall incidence of invasive bacterial disease was 3.5-fold higher among nuuk patients than among district patients. most likely this finding represents sampling bias ; microorganisms sent from the districts are less likely to survive than samples taken in nuuk, and district doctors may be less likely to submit samples than nuuk doctors, given the longer time to receive test results. however, we can not confirm this explanation because of lack of information on the total number of submitted samples. furthermore, the 39% higher average income in nuuk compared with the rest of greenland makes a nuuk / district invasive disease incidence rate ratio of 3.5:1 less likely (7). the difference suggests that the true invasive disease rates in greenland may be much higher than those found in this study or reported to ics. a clear recommendation from this study is to develop a more accessible, rapid, and reliable diagnostic system for districts outside of nuuk. the lower age - adjusted invasive bacterial disease incidence in danes than greenlanders may be explained by the healthy worker effect. danes in general come to greenland to work and belong to higher social classes and live in better housing conditions than the general population of greenland, factors that may reduce the risk for infection (9). hence, although genetic factors may account for some of the difference, the difference is probably confounded by environmental and social factors. the increase in invasive bacterial isolates during the study period most likely represents changes in submission or detection practices. the increase occurred almost exclusively in blood cultures, and a new blood culture detection system was introduced in 1999. | invasive bacterial disease occurs frequently among native populations in the arctic. although a variety of bacteria are involved in invasive bacterial disease in greenland, streptococcus pneumoniae, escherichia coli, staphylococcus aureus, and other staphylococci are responsible for most cases (69%) ; incidence varies according to region and ethnicity. |
transverse vaginal septum is a rare anomaly that results from incomplete fusion of urogenital sinus and the vaginal parts of the mullerian duct. this septum is usually found in the upper and mid - vagina but can be located at any level in the vagina and varies in thickness too. clinical manifestations depend on whether it is complete or partial septum. in case of complete septum, it results in hematocolpos and hematometra due to accumulation of menstrual blood resulting in distension of the structures above the septum postpuberty. these patients present with symptoms of lower abdominal cyclic pain with ultrasonic findings of hematocolpometra. occasionally, a palpable mass is found in the lower abdomen (hematometra). in case of incomplete septum, patients complain of cryptomenorrhea, dysmenorrhea, and dyspareunia due to limited egress of menstrual blood. cervical atresia / dysgenesis is still a rare uterovaginal anomaly that can be congenital or acquired leading to fertility problems. having said about two rare anomalies, here, we present the case of a 16-year - old girl with cyclic abdominal pain who had the rare combination of transverse vaginal septum and cervical dysgenesis with severe anemia and amenorrhea. resection of the septum with end - to - end anastomosis is the first surgical management of transverse vaginal septum, which might lead to scarring, and development of contractures. custom - made prosthetic stents can be used to prevent these postsurgical contractures and maintain the patency of the vagina and cervix. this case report deals with interdisciplinary role of conservative surgical management and custom - made prosthetic appliances in the management of transverse vaginal septum and cervical atresia at an early stage. a 16-year - old girl reported with cyclical abdomen pain for past 4 months with amenorrhea. minimal pubic and axillary hair growth (tanner ii) with normal breast development was observed. height and weight were not appropriate for the age, and no tenderness or mass on palpation was observed. the history of never menstruated was obtained during discussion with the patient 's mother. ultrasonographic (usg) studies showed that the liver, kidneys, spleen, ureters, and urinary bladder were all normal and uterus and ovaries of normal size, with hemoglobin of 7.1 g / dl. the patient was diagnosed with transverse vaginal septum with probable cervical dysgenesis with the help of usg [figure 1 ]. surgery was planned with the vagina being atretic ; dissection was done in the loose areolar space between the bladder and rectum toward the cervix. then, canalization of the cervix was done through the identified dimple using mosquito artery forceps and with the help of abdominal ultrasound probe as a guide. a 4 cm long, 2.5 cm diameter hollow prosthetic stent (acrylic) was subjected to low - steam sterilization before placing and sutured to the cervical wall to hold the cannula in place. this hollow stent helped in flushing of the blood during the menstrual period along with maintaining the patency of the cervix. ultrasonographic showing hematocolpos ethical clearance and patient consent were obtained for publishing this case report in the journal. a 20 ml syringe and suction tip the syringe was modified according to the specification of 30 mm long and suction tip was cut to 10 mm. the putty impression material was used to occupy the mold space, to obtain a hollow cylinder configuration. modeling wax was used for fabrication of the wax pattern over the putty [figures 3 and 4 ]. the wax pattern along with the putty mold was flasked and processed with heat cure acrylic resin followed by finishing and polishing [figure 5 ]. modified 20 ml syringe and suction tip putty mold and wax pattern fabricated flasking followed by dewaxing of the mold processed vaginal stent placed in the cervix a 20 ml syringe and suction tip were used as the base model [figure 2 ]. the syringe was modified according to the specification of 30 mm long and suction tip was cut to 10 mm. the putty impression material was used to occupy the mold space, to obtain a hollow cylinder configuration. modeling wax was used for fabrication of the wax pattern over the putty [figures 3 and 4 ]. the wax pattern along with the putty mold was flasked and processed with heat cure acrylic resin followed by finishing and polishing [figure 5 ]. modified 20 ml syringe and suction tip putty mold and wax pattern fabricated flasking followed by dewaxing of the mold processed vaginal stent placed in the cervix transverse vaginal septum is a rare occurrence. when it coexists with cervical dysgenesis, it is even rarer. transverse vaginal septum is found between the upper one - third (mullerian origin) and lower two - third of the vaginal canal (urogenital sinus origin). it is categorized as class ii congenital uterovaginal anomaly under the rock and adam 's classification. patients with transverse vaginal septum experience pain, retrograde menses, difficulty during sexual intercourse and childbearing, and delivery and are exposed to high risk of infection. after surgical correction of the septum is done, end - to - end anastomosis of the vagina is required. when transverse septum is diagnosed, generally a major portion of the vagina is missing, making it difficult for the anastomosis of the upper and lower segments, postpuberty. to overcome this difficulty, z - plasty and simpler flap methods have been advocated. however, neither of the methods were used in this case. a simple excision and dissection through loose tissue the process of re - anastomosis becomes easy due to distension of the upper vagina with menstrual blood, which acts as tissue distender ; however, in cases where this is not the situation, the use of a vaginal dilator is advised to thin the septum and ease re - anastomosis. the dimension of the canal was measured and a custom - made hollow prosthesis (dilator) was fabricated. after 2 weeks, the dilator was removed under general anesthesia to make the procedure pain - free and for proper evaluation, and new one was fabricated with which the patient was trained to use until re - anastomoses was achieved to satisfactory level. the main advantages of this technique are (a) the patency of the canal was maintained, (b) the hollow in the stent acted as a vessel for collecting the blood and provided a path for its removal, thereby eliminating the risk of canal closure reducing the risk of infection due to pooling of blood. detailed medical history, clinical examination, and pelvic ultrasound are the ways and means to diagnose the conditions of transverse vaginal septum and cervical dysgenesis / atresia. diagnosis of cervical obstruction should be made as early as possible so that aggressive surgical procedures such as hysterectomy can be avoided. this conservative surgical management was made possible with the emerging trends in reproductive technology and laparoscopic techniques and was perhaps going to be the first - line treatment option with the expertise in laparoscopic surgery along with prosthetic management. where no specific funding was sought, department of prosthodontics, sri ramachandra university, porur, chennai-116, fund was used for fabrication of the prosthesis, and we kindly acknowledge the department for helping us in the case. where no specific funding was sought, department of prosthodontics, sri ramachandra university, porur, chennai-116, fund was used for fabrication of the prosthesis, and we kindly acknowledge the department for helping us in the case. | transverse vaginal septum is a rare anomaly that results from incomplete fusion of urogenital sinus, and the vaginal parts of the mullerian duct. cervical atresia / dysgenesis is still a rare uterovaginal anomaly that can be congenital or acquired leading to fertility problems. transverse vaginal septum when it coexists with cervical dysgenesis, it becomes a rare combination where management becomes highly complex. this case report deals with interdisciplinary role of conservative surgical management and custom made prosthetic appliances in the management of transverse vaginal septum and cervical atresia at an early stage and perhaps this goes to be the first - line treatment option with the expertise in laparoscopic surgery along with prosthetic management. |
chronic subdural hematoma (csdh) is one of the most common types of intracranial hemorrhage and is a well - known traumatic disease caused by tearing of bridging veins after minor head injury.38) however, some patients develop csdh from causes other than head injury, such as brain surgery, neovascularization of the hematoma capsule, or coagulation factors.710) in addition, some factors, such as old age, alcoholism, coagulopathy, neurological status at admission, hematoma density, and irrigation, are reported to be correlated with outcome.49121821) however, these results are still controversial, and the association between head trauma and outcome of csdh has not yet been fully elucidated. therefore, the purpose of our study was to examine the prognostic factors for the outcome in patients with csdh who had undergone burr hole drainage procedures and to determine the association between outcome and traumatic head injury. between january 2008 and december 2015, 284 patients with csdh were admitted and treated in a single tertiary hospital. this study was approved by the institutional review board, and informed consent was waived. inclusion criteria were as follows : patients must have undergone a burr hole drainage procedure for treatment of symptoms caused by mass effects of csdh, and the follow - up assessment must have occurred more than 1 month after the initial operation. eighteen patients with no follow - up history and 28 patients who had not undergone burr hole drainage procedures were excluded. factors shown to relate to prognosis and recurrence in previous studies were analyzed, including sex, age, diabetic status, hypertension, end - stage renal disease (esrd), history of stroke, alcoholism, use of anticoagulant and antiplatelet agents, and previous head injury. patients were considered to have hypertension when oral hypertensive medications were prescribed to control blood pressure. esrd was defined as kidney failure (glomerular filtration rate < 15 ml / min/1.73 m) requiring permanent renal replacement therapy, while stroke was defined as intracranial hemorrhage due to cerebral vascular conditions and/or cerebral infarction. alcoholism was defined as consumption of more than 14 standard drinks per week or 4 drinks per day, and head trauma was defined as the occurrence of head injury within 3 months prior to hospital admission. patients who had complained of symptoms such as headache, general weakness, and/or neurological deficits were diagnosed with csdh using computerized tomography (ct) scans of the brain. when csdh was observed on brain ct scan, several parameters were assessed and documented : hematoma thickness, midline shifting based on pineal gland, hematoma density (classified as high, iso, low, or mixed based on brain parenchymal density), and location of hematoma (unilateral versus bilateral).17) in addition, patient status was evaluated and classified according to the modified rankin scale (mrs) and glasgow coma scale (gcs). the mass effects of the hematoma were assessed, and procedures were performed under general anesthesia at the discretion of the surgeon as follows : either a single burr hole or two burr holes were trephined, and the site was either irrigated with saline or not irrigated. a silicone tube was inserted into the subdural space to allow for 1 to 3 days of drainage. if sufficient drainage had occurred at the time of the postoperative ct scan and/or the symptoms had improved, the silicone tube was removed. brain ct scans were performed at 1 month and 3 months post - operation through our outpatient department. recurrence was defined as increasing hematoma thickness, based on brain ct scan at discharge, and worsening symptoms. in addition, good outcome at the final follow - up was assessed using the same method. baseline characteristics were assessed and compared between the two groups (no head injury and head injury) using the chi - square test, fisher s exact test, linear by linear test, and student s t - test. to assess good outcome and recurrence, univariate analysis of clinical, radiological, and neurological findings and operative technique was performed using a chi - square test, fisher s exact test, and a student s t - test. variables with a p - value of < 0.2 in univariate analysis were then chosen for multivariate analysis using a logistic regression model. the result of head injury was included in multivariate analysis regardless of the result of univariate analysis, as this was the variable of interest in this study. in all analyses, between january 2008 and december 2015, 284 patients with csdh were admitted and treated in a single tertiary hospital. this study was approved by the institutional review board, and informed consent was waived. inclusion criteria were as follows : patients must have undergone a burr hole drainage procedure for treatment of symptoms caused by mass effects of csdh, and the follow - up assessment must have occurred more than 1 month after the initial operation. eighteen patients with no follow - up history and 28 patients who had not undergone burr hole drainage procedures were excluded. factors shown to relate to prognosis and recurrence in previous studies were analyzed, including sex, age, diabetic status, hypertension, end - stage renal disease (esrd), history of stroke, alcoholism, use of anticoagulant and antiplatelet agents, and previous head injury. patients were considered to have hypertension when oral hypertensive medications were prescribed to control blood pressure. esrd was defined as kidney failure (glomerular filtration rate < 15 ml / min/1.73 m) requiring permanent renal replacement therapy, while stroke was defined as intracranial hemorrhage due to cerebral vascular conditions and/or cerebral infarction. alcoholism was defined as consumption of more than 14 standard drinks per week or 4 drinks per day, and head trauma was defined as the occurrence of head injury within 3 months prior to hospital admission. patients who had complained of symptoms such as headache, general weakness, and/or neurological deficits were diagnosed with csdh using computerized tomography (ct) scans of the brain. when csdh was observed on brain ct scan, several parameters were assessed and documented : hematoma thickness, midline shifting based on pineal gland, hematoma density (classified as high, iso, low, or mixed based on brain parenchymal density), and location of hematoma (unilateral versus bilateral).17) in addition, patient status was evaluated and classified according to the modified rankin scale (mrs) and glasgow coma scale (gcs). the mass effects of the hematoma were assessed, and procedures were performed under general anesthesia at the discretion of the surgeon as follows : either a single burr hole or two burr holes were trephined, and the site was either irrigated with saline or not irrigated. a silicone tube was inserted into the subdural space to allow for 1 to 3 days of drainage. if sufficient drainage had occurred at the time of the postoperative ct scan and/or the symptoms had improved, the silicone tube was removed. brain ct scans were performed at 1 month and 3 months post - operation through our outpatient department. recurrence was defined as increasing hematoma thickness, based on brain ct scan at discharge, and worsening symptoms. in addition, good outcome at the final follow - up was assessed using the same method. baseline characteristics were assessed and compared between the two groups (no head injury and head injury) using the chi - square test, fisher s exact test, linear by linear test, and student s t - test. to assess good outcome and recurrence, univariate analysis of clinical, radiological, and neurological findings and operative technique was performed using a chi - square test, fisher s exact test, and a student s t - test. variables with a p - value of < 0.2 in univariate analysis were then chosen for multivariate analysis using a logistic regression model. the result of head injury was included in multivariate analysis regardless of the result of univariate analysis, as this was the variable of interest in this study. in all analyses, among the 238 patients who had undergone burr hole drainage procedures, 127 (53.4%) patients were included in the head trauma group and compared with the no head trauma group. with regard to baseline characteristics, no factor was significantly different between the two groups (table 1). at admission, 114 (47.8%) patients had headache, 4 (1.7%) patients had dizziness, 9 (3.8%) patients had dysarthria or aphasia, 2 (0.8%) patients had numbness, 12 (5.0%) patients had gait disturbance or weakness in both legs, 27 (11.3%) patients had decreasing mentality, 68 (28.6%) patients had hemiparesis, and 2 (0.8%) patients had seizure. after the burr hole drainage procedure, mrs score improved from 1.931.27 at admission to 1.061.05 at discharge and 0.741.05 at the final follow - up. mechanisms of head trauma included traffic accident total 37 (29.1%), car 15 (11.8%), motorcycle 11 (8.7%), bicycle 9 (7.1%), cultivator 2 (1.6%), slip down 53 (41.7%), fall down 14 (11.0%), head collision 18 (14.2%), assault 3 (2.4%), and sports head injury 2 (1.6%). in addition, the mean period from accident was 5.762.61 weeks. the longest period was traffic accident (7.113.95 weeks), and the shortest period was sports head injury (3.00 weeks) (table 2). one hundred thirty - three (55.9%) patients showed good outcome at discharge, and 171 (71.8%) patients showed good outcome at the final follow - up. none of the factors was significantly correlated with outcome at discharge. however, some factors correlated with outcome at the final follow - up. diabetes (p=0.024) was statistically significant in univariate analysis, but in multivariate analysis, only history of head injury (p=0.033, odds ratio [or ] 0.511, 95% confidence interval [ci ] 0.277 - 0.946) was significantly correlated with outcome (table 3). three patients (1.3%) expired after burr hole drainage procedure, one had pneumonia associated with decreasing mentality, one had peritonitis with unknown origin, and one had sudden cardiac arrest. additional complications included post - operative infection in one patient and epidural hematoma related to burr hole drainage in another (figure 1). recurrence occurred in 20 (8.4%) cases in the total group : 9 (45%) patients in the no head injury group and 11 (55%) patients in the head injury group, and there was no significant difference between them. although there were no significant factors in the univariate analysis, in multivariate analysis of recurrence, right side hematoma (p=0.026, or 0.234, 95% ci 0.065 - 0.839) alone was statistically significant (table 4). some recent studies have reported on the diagnosis of minor head injury, but currently, no established optimal guidelines exist. previously, in cases of minor head injury, the status of the damaged brain was evaluated solely using brain ct, but more recently, magnetic resonance imaging (mri) and/or diffuse tensor imaging (dti) techniques for the evaluation of white matter damage and/or axonal injury have been studied.21516) in our study, because the standard protocol for evaluating csdh and minor head trauma used only brain ct, it is possible that this procedure was not sensitive enough to determine the true extent of damage. for this reason, although history of head trauma was not correlated with outcome at discharge, it may be correlated with poor outcome at the final follow - up. recently, some reports have supported the use of mri and/or dti for evaluation of minor head trauma.1319) as such, patients with csdh should be considered candidates for these evaluation methods. many studies report that gcs score at admission is strongly correlated with outcome. in a study by amirjamshidi.1), lower initial gcs and higher glasgow outcome scale scores upon discharge were associated with negative outcomes. in our study, gcs score at admission was 14.181.81 in the total cohort, and scores of the head trauma group (14.251.69) and the no head trauma group (14.101.94) were not significantly different. in addition, gcs score at admission was not correlated with good outcome at discharge or good outcome at the final follow - up. because good outcome was defined as improvement of mrs score from admission to discharge or the final follow - up. if not the initial state is corrected, it is considered that the initial state will have a too significant impact on the result. old age has also been considered a strong risk factor for csdh outcome due to age - related brain atrophy. fukuhara.5) reported that brain surface elasticity is an important factor in csdh. ro.14) reported correlation of old age and poor outcome due to brain expansion. in our study, age was not significantly correlated with outcome at discharge and the final follow - up. however, there were three mortality cases (1.3%), and these patients were elderly (more than 70 years old). although we observed no association with burr hole drainage procedure directly, conditions associated with aging may contribute to poor outcome. investigation of factors relating to recurrence of csdh revealed that only location of the hematoma in the right hemisphere significant correlated with low recurrence. although it was not statistically significant, location of the hematoma in the left hemisphere was also more strongly correlated with low recurrence than bilateral location of the hematoma. to confirm these results, subgroups divided by hematoma location were analyzed. among 210 patients (right / left : 119/91) with unilateral hematoma, 115 patients (54.8%, left / right : 61 [51.3%]/54 [59.3% ]) received irrigation during the operation, and among 28 patients with bilateral hematoma, 7 patients (25.0%) received irrigation. although irrigation was not correlated with outcome in multivariate analysis in our study, irrigation may have some effects on recurrence. many studies have shown correlation between recurrence of csdh and irrigation.61120) to determine the important effects of irrigation on recurrence, well - designed and larger prospective studies are necessary. therefore, it may be subject to selective bias and compounding effects. to prevent these problems, second, to evaluate brain damage after minor head trauma, mri and dti were not performed. to determine whether other lesions affected outcome, a prospective study using mri and/or dti would be necessary. third, mechanisms of head trauma and time periods following accidents were studied, and we found that larger vector mechanisms, such as traffic accidents, were associated with longer periods of time before csdh occurrence. thus, we predict that mechanisms with larger vectors should be observed for longer periods than minor vector mechanisms. because this was outside the scope of our study, further investigation is needed. in conclusion, history of head trauma is correlated with poor outcome at long - term follow - up in csdh patients following burr hole drainage. therefore, csdh patients with history of head injury are susceptible to poor outcome, warranting more careful evaluation and treatment after burr hole drainage. in addition, further study is needed to investigate the correlation between irrigation and recurrence of csdh following burr hole drainage. | objectiveour study examined the prognostic factors involved in the outcome of patients with chronic subdural hematoma (csdh) who had undergone burr hole drainage procedures, and investigated the association between outcome and traumatic head injury. in addition, we explored factors related to recurrence.methodsthis study enrolled 238 patients with csdh who had undergone burr hole drainage. patients with history of head injury were categorized into the head trauma group and were compared with the no head trauma group. outcome was considered good when modified rankin scale scores improved from admission to discharge and the final follow-up.resultsamong 238 patients, 127 (53.4%) were included in the head trauma group. one hundred thirty - three (55.9%) patients demonstrated good outcome at discharge, and 171 (71.8%) patients demonstrated good outcome at the final follow - up. none of the factors examined was significantly correlated with good outcome at discharge. however, only history of head injury (p=0.033, odds ratio 0.511, 95% confidence interval 0.277 - 0.946) was significantly correlated with poor outcome at long - term follow - up. recurrence occurred in 20 (8.4%) cases in the total cohort and 11 (55%) patients in the head trauma group.conclusionhistory of head trauma is correlated with poor outcome at long - term follow - up in csdh patients having undergone burr hole drainage. therefore, csdh patients with history of head injury are susceptible to poor outcome, warranting more careful evaluation and treatment after burr hole drainage. |
molecular imaging is defined as the in vivo measurement of biological processes at the cellular and molecular levels.24 - 26 molecular - imaging - based visualization of in vivo pathophysiologic processes could provide information regarding specific molecular alterations underlying the disease status of individual patients in real time.27 - 29 by providing molecular information unobtainable using conventional anatomy - based imaging modalities, molecular imaging would allow 1) earlier detection of diseases, 2) precise discrimination of the stable versus unstable disease status, and 3) both diagnostic and therapeutic quantitative monitoring of disease progression.30 - 32 the various modalities of molecular imaging (ct / mri / fluorescent optical imaging / positron - emission tomography, pet / single - photon - emission ct / spect / ultrasound) have their own specific advantages and disadvantages that are related to how the images are generated.21,32 the application of fluorescent proteins or fluorochromes to the life sciences during the last 2 decades has considerably advanced basic and translational biological research, and these advances are now beginning to affect clinical practice.29,33,34 fluorescence involves the absorption of light at characteristic wavelengths, and the emission of the stored energy at longer wavelengths.35 the advantages of fluorescence as a molecular imaging modality include picomolar molecular sensitivity, absence of ionizing radiation, the possibility of using it in many modalities with different scales, and relatively low cost. however, poor tissue penetration capability is a major obstacle to overcome.36,37 the attenuation of light by tissue is lowest in the near - infrared (nir, 700 - 900 nm) region, and imaging in this region offers 1) markedly less photon absorption by blood hemoglobin, lipid, and water, allowing light to penetrate centimeters into the body ; and 2) substantially reduced tissue autofluorescence, enabling higher sensitivity detection of targeted nir fluorescent (nirf) molecular imaging agents against a low background.31,36,38 this technology can potentially be combined with a non - invasive optical tomography system, intra - operative nirf imaging system, or fluorescence - sensing catheter - based system.39 jaffer. recently demonstrated that a nirf - sensing catheter based on a clinical coronary artery guidewire could detect in vivo cathepsin b (catb) protease activity in rabbit vessels the size of human coronary arteries in real time. nirf imaging requires a much smaller dose of fluorescence probes to detect molecules of interest - nanomoles of fluorochromes can be detected, compared to micromoles for mri or millimoles for ct.31,32 the relatively high spatial resolution (typically less than 1 mm) of the catheter - based imaging system is another advantage of fluorescence imaging.39 scatter reduces the spatial resolution of non - invasive fluorescence molecular tomography (fmt) relative to using an endoscopic imaging device (-1 mm), and is within the range of resolution provided by spect and pet.36 studies have demonstrated that the formation of a thrombus due to rupture of unstable atherosclerotic plaques, followed by thrombotic or embolic occlusion of an artery, is the leading cause of stroke, accounting for up to 80% of cases of ischemic stroke in some autopsy series.10,40 there is a pressing need for tools to identify these vulnerable plaques, and thereby identify patients and lesions at high risk for vascular events, so that risk - altering treatments might be offered to improve clinical outcomes. according to the current practice guidelines and consensus, a carotid lesion is likely to cause ischemic stroke when stenosis of over 60 or 70% is detected by angiography.41 however, it has become clear that many strokes are attributable to plaques in the arteries with stenosis of 50% or less, highlighting the importance of plaque ruptures as a causative mechanism.42 - 44 rupture - prone vulnerable plaques are not well identified by conventional measures of stenosis.45 - 47 ultrasonic characterization of plaques as heterogeneous or of low echodensity on carotid duplex ultrasonography has been regarded by some as suitable for detecting unstable plaques, but firm conclusions await further studies.48,49 plaque size and morphology (fig. 1) are poor substitutes for the molecular events that shaped them, and the measurement of underlying molecular states provides the best hope for determining the propensity to cause complications. for asymptomatic patients with significant carotid stenosis, the number needed to treat to prevent one stroke from any cause at two years is 67, which could increase to 111 to prevent one large - artery stroke at 2 years, since not all strokes originate from a narrowed internal carotid artery.50 for symptomatic patients, endarterectomy is beneficial in the long term even in the presence of a contralateral occlusion and increased perioperative risk.51,52 however, from a patient 's standpoint, opting for a procedure with a 3 - 6% immediate perioperative risk to reduce a future stroke chance by 6 - 15% at 5 years in both asymptomatic and symptomatic cases can be daunting.53 this illustrates the difficulty of weighing the risks and benefits in treating an individual patient.51 characteristic features of vulnerable atherosclerotic plaques are accumulation of inflammatory cells inside the plaque (fig. 2), formation of a large lipid core, and thinning of the overlying fibrous cap.5 heavily clustered macrophages in and around the shoulder region of the cap would secrete matrix - disorganizing proteolytic enzymes such as cathepsins and matrix metalloproteinases (mmps), which could render atherosclerotic plaques prone to rupture.6,54 thus, inflammatory protease activity could be regarded as a hallmark of the plaque vulnerability. in fact, using the affymetrix gene chip to profile genes expressed in stable and unstable atherosclerotic plaques revealed that the expressions of catb, cathepsin s, and mmp-9 were up - regulated in unstable atherosclerotic plaques.55 taken together, these data suggest that imaging protease activity in vivo could allow identification of vulnerable plaques. the weissleder group and others have developed protease - sensing nirf molecular imaging agents.56 - 60 unlike " always - on " probes such as nonspecific nirf probes [e.g., indocyanine green (icg) ] or targeted nirf probes relying on affinity ligands, the protease - sensing nirf probes are " optically silent at injection " because of intra - molecular autoquenching between closely spaced fluorochromes. enzyme - specific protease - mediated cleavage spatially separates fluorochromes so that they become dequenched and brightly fluorescent. multiple nir fluorochromes positioned on a polylysine backbone can be activated by catb.34 the gelatinase substrate sequence ggprqitag has been used to synthesize a nirf probe that can be activated by mmp-2/9.58 animal studies using these activatable probes have shown that in vivo and ex vivo nirf imaging could visualize the protease activity in the aortic or carotid atheromata of apoe knock - out mice (figs. 3 and 4).58,59,61,62 immunohistochemical analyses demonstrated that the nirf signal precisely reflected the spatial distribution of the inflammatory protease activities. in addition, the protease immunoreactivity co - localized with mac-3 or cd-68 positive macrophages. despite the high expectations for and rapidly increasing interest in molecular imaging techniques, there are significant challenges in translating the advances from the laboratory to the atherosclerosis clinic. as a step toward clinical translation, we recently demonstrated that catb nirf imaging could reliably reflect the anti - atherosclerotic effect of atorvastatin and glucosamine as well as the pro - atherosclerotic effect of a high cholesterol diet (fig. we also revealed that plaque populations were heterogeneous within individuals, with some plaques showing high and others lower catb - related signals.61 notably, these differences in signal intensity could not be predicted by visual inspection of the plaques. in every case where imaging predicted an inflammatory component in the plaque, histological studies confirmed it to be the case.61 regarding translation steps from animals to humans, catheter - based fluorescence imaging systems are likely to be available in the atherosclerosis clinic. in addition, it should be remembered that since nir photons can potentially penetrate > 5 centimeters into the body, noninvasive fmt systems may eventually detectnirf signals from human carotid atheromata.36,37 moreover, the protease - sensing probe is expected to enter clinical trials in 2010.31 other molecular imaging biomarkers that reflect plaque vulnerability include annexin for apoptosis,63,64 integrins for angiogenesis,65 and inflammatory cells such as macrophages.66 - 68 to summarize, molecular optical imaging could contribute to stroke prevention by detecting protease activity in atherosclerotic plaques in vivo ; it would thereby serve as a powerful tool for evaluating vascular inflammation, for determining individualized therapeutic strategies including pre - operative planning, and for monitoring the effects of therapeutic interventions. fibrinolytic therapy, which is the only fda - approved treatment for acute cerebral infarction, inevitably imposes a hemorrhagic risk.10,13,14 delayed or intra - arterial thrombolysis is associated with higher complication rates, and enormous effort has been expended to empirically define the risks and benefits of thrombolysis at different times after vascular occlusion.14 predicting the thrombolytic resistance and hemorrhagic risk in individual patients would be extremely helpful when deciding whether or not to perform thrombolytic therapy in individual patients. as a first step toward imaging thrombolytic resistance in vivo, we and others chose to devise and characterize a molecular imaging agent to probe for the activity of factor xiii (fxiii) enzyme, a thrombin activated transglutaminase.69 - 71 the fxiii probe contains nir fluorochromes attached to the peptide sequence nqeqvspltllk, which is specifically recognized by the coagulation enzyme as its substrate. the normal function of activated fxiii (fxiiia) enzyme is to stabilize a thrombus by cross - linking fibrin fibers - thus increasing the tensile strength - so as to covalently bind molecules that impede plasmin activity, such as 2-antiplasmin.72 imaging this coagulation enzyme activity may be useful to staging the thrombotic disease when determining the appropriate therapy by clarifying the acute - versus - chronic state of the clot, which is difficult or impossible to achieve with anatomic imaging only. when the probe was injected intravenously into mice with ferric - chloride - induced thrombi in the femoral artery, the fxiiia - related nirf signal intensity was proportional to the age of the thrombi.71 the activity of fxiiia enzyme is expected to change far more rapidly than the clot volume, and may serve as an early predictor of the success or failure of a therapeutic regimen.73 in this context, we investigated whether fxiiia imaging could reflect treatment effects. to study the effect of heparin in a mouse model of cerebral venous thrombosis, heparin was administered immediately before thrombus induction and the fxiii probe was injected 3 hours later.70 the fxiiia - related nirf signal, captured through a cranial window using an intravital fluorescence microscope (fig. 5), was significantly weaker in the heparin - pretreated group than in the untreated group. notably, the therapeutic response to the anticoagulation treatment could be monitored in real time. we are currently studying if fxiiia imaging can predict thrombolytic resistance in an arterial stroke model. thrombolytic resistance is likely multifactorial in origin, and hence other imaging or plasma biomarkers of thrombolytic resistance merit further investigations, such as plasminogen activator inhibitor-1, 2-antiplasmin, and factor v.74,75 the future combination of molecular optical imaging with fluorescence - sensing intra - arterial catheter systems is expected to be suitable for clinical application, with molecular diagnosis guiding therapy with fibrinolytic agents or thrombin inhibitors and their successor drugs in acute ischemic stroke. this approach may allow discrimination of thrombi that would be resistant to chemical thrombolytic agents. early triage to mechanical recanalization and clot retrieval methods would be rational when chemical thrombolysis is expected to fail, if patients could be triaged appropriately to these treatments (fig. intra - operative molecular imaging may assist neurosurgeons who are performing procedures such as post - stroke decompression surgery or revascularization operations to visualize cerebral perfusion and penumbra in real time. woitzik and colleagues76 introduced intraoperative cerebral videoangiography, an imaging system integrated into the surgical microscope, which allowed 1) real - time imaging of the cortical blood flow in patients undergoing decompressive craniectomy and 2) cortical perfusion areas to be stratified into ischemic core and penumbral areas. they used icg, which binds tightly to plasma proteins and becomes confined to the vascular system ; its short half - life and emission at a nir wavelength (835 nm) make it an advantageous contrast agent for in vivo angiography. a cerebral blood flow index (the ratio of the difference in fluorescence intensity and rise time) was calculated to determine flow maps that were used to assign a significant portion of tissue (15 - 37%) to the penumbra,76 which is a potential target to maximize benefits from the decompressive craniectomy, which in turn reduces intracranial pressure and improves cerebral blood flow.77 clinically, in vivo retinal angiography with icg has long been used to visualize neovascularization and hemorrhage in retinal and choroidal tissues.78 in a mouse model with a cranial window, we showed that cerebral icg - based angiography could reveal permeability of the blood - brain barrier by sensing icg leakage from cerebral vessels due to thrombosis - induced venous hypertension in real time in vivo.70 the technique was recently used to evaluate cortical microvascularization to compensate for a reduced cerebral blood flow in moyamoya disease.79 icg - based angiography demonstrated that the microvascular density and diameter were significantly elevated in patients with moyamoya disease. in addition to the cerebral perfusion status, the penumbral area could be defined by various physiological parameters including decreased ph due to increased intracellular lactate accumulation.80 - 82 ph - activatable molecular optical imaging probes conjugated to a cancer - targeting monoclonal antibody were developed recently.83 the probes consist of a boron - dipyrromethene (bodipy) as a fluorophore and a dialkylated aniline that renders the fluorophore fluorescent at different acidic phs. bodipy dye with a nonprotonated form of dialkylated aniline is nonfluorescent, whilst the dye with a protonated form of aniline is highly fluorescent. conjugation of the probe to a cancer - specific monoclonal antibody, trastuzumab, leads to recognition and internalization by her2-positive cancer cells via the endosomal - lysosomal degradation pathway. an acidic ph in the lysosome then turns on the fluorophore, and hence the probe offers tumor specificity with a minimal background signal. it should be noted that the imaging agent is not an " always - on " probe like conventional targeted probes. instead, it becomes fluorescent under specific conditions, which improves the specificity and sensitivity as in the case of protease activatable probes. more importantly, since the proton pump is required to maintain the acidic condition in the lysosome, only viable cancer cells can be visualized by the probe. this kind of ph - sensing probe may be useful in delineating cerebral penumbra and infarcted tissues. molecular imaging researchers are actively pursuing 1) the development of novel molecular imaging strategies, particularly multi - modal molecular imaging, 2) innovations in molecular imaging - based theranostics, and 3) bench - to - bedside translation of associated cutting - edge technologies. cardiovascular and neurovascular molecular optical imaging researchers pursue similar aims. multi - modal molecular imaging probes are detectable by optical, mri, and nuclear approaches simultaneously, which provides both high molecular sensitivity and high anatomical resolution as shown by kircher.84 combined mr and optical imaging. nahrendorf. labeled dextranated and dtpa - modified magnetofluorescent nanoparticles with cu to yield a macrophage - targeted trimodality reporter for combined mri, pet, and optical imaging.67 combining diagnostic molecular imaging with therapeutic (theranostic) approaches would allow diseases to be detected at an early stage, drug delivery to desired targets to be monitored, quantification of the therapeutic efficacy in an individual patient in vivo, and the treatment for the patient to be tailored based on the imaging results. the development and utilization of protease - mediated photodynamic agents could be regarded as a theranostic approach.85 the agents are nontoxic in their native state, but they become fluorescent and produce singlet oxygen on protease conversion ; after being delivered and activated in and around the macrophages infiltrating vulnerable plaques, intravenously injected theranostic agents are expected to not only visualize rupture - prone plaques but also stabilize them as a photodynamic therapy while avoiding systemic phototoxicity. healthcare in the 21 century is evolving to be personalized, predictive, and preventive. molecular imaging provides a fundamentally new type of information that allows a timely and precise assessment of pathophysiologic states at the cellular and molecular levels, thereby complementing that available from anatomically based imaging modalities. most importantly, well - designed translational animal studies and human trials will be key to establishing the use of molecular imaging tools in clinical stroke management.86 | molecular imaging is a novel technology to visualize biological processes at the cellular and molecular levels, which is reshaping both biomedical research and clinical practice. by providing molecular information to supplement and augment conventional anatomy - based imaging, molecular imaging is expected to allow 1) the earlier detection of diseases, 2) precise evaluation of disease stages, and 3) both diagnostic and therapeutic monitoring of disease progression in a quantitative manner. in this brief review, we present our view on the prospects of molecular optical imaging in the field of stroke practice, focusing on the imaging vulnerability of atherosclerotic plaques, thrombolytic resistance, real - time cerebral perfusion, and penumbra. |
with a mortality rate > 40%, management of multi - trauma patients presenting with exsanguinating pelvic fractures is a therapeutic challenge. mortality within the first 24 h is primarily due to complications of the bleeding itself, while associated coexisting cerebral injuries, thromboembolism, and multiple organ failure account for most of the mortality afterward. these patients present with the life - threatening hemodynamic instability due to massive pelvic bleeding from the presacral vessels damaged by the unstable pelvic fractures. this massive intrapelvic bleeding is difficult to control, and traditional treatment is aimed at decreasing the retroperitoneal bleeding by the containment of the retroperitoneal hematoma using techniques for reducing the pelvic volume and stabilizing the pelvic fractures, together with emergent angiographic catheterization and arterial embolization of retroperitoneal bleeders. despite widespread use of these approaches, mortality rates remain high (> 40%), and concerns have been raised about the effectiveness of these life - saving efforts to contain the retroperitoneal hematoma and bleeding, including pelvic clamps, external fixation, or strapping maneuvers to reduce the pelvic volume and stabilize the pelvic fractures. traditional methods that attempt to control bleeding in an indirect fashion, such as external pelvic fixation and arterial embolization, are time - consuming (30 - 90 min) and effective to varying degrees in 40 - 50% of cases at best, and may not be available in all hospital settings. furthermore, it is difficult to contain bleeding into the pelvic retroperitoneal space even when stabilization of the pelvis has been successfully achieved, since this space is continuous with the abdominal retroperitoneal space as well as with the buttocks and thighs through the greater sciatic notch. in contrast, extra - peritoneal pressure packing (eppp) enables immediate and direct application of pressure upon bleeding wounds and vessels, leading to the successful cessation of life - threatening exsanguination. most of the retroperitoneal bleeding comes from the pelvic venous plexus and from the bone itself and not from the arterial plexus. the additive effect of angiographic catheterization and arterial embolization is limited to the bleeding iliac arteries and does nothing to stanch the flow from the venous retroperitoneal bleeders, which is the major cause of death. emergent surgical access to the presacral vessels and direct application of pressure packing onto the retroperitoneal bleeding vessels via eppp has gained popularity following reports of its effectiveness and advantages over earlier approaches. typically eppp is employed in conjunction with the application of external fixation with the concept that the external fixation will counteract the forces associated with packing and insertion of multiple abdominal pads into the pelvis which increases the pelvic volume. it has not, however, been conclusively established whether eppp is effective as an immediate stand - alone surgical treatment for multi - trauma patients, that is, carried out without external pelvic fixation or emergent angiography. in this study, eppp was employed as a first - line sole surgical treatment without external fixator application in exsanguinating unstable patients. the aim of this study is to describe the survival rates associated with prompt employment of the eppp technique, as a stand - alone surgical treatment for multi - trauma patients and the clinical and physiological outcomes. a retrospective cohort study of prospectively collected data of all patients treated for traumatic pelvic fractures at our medical center between the years 2005 and 2011 that was approved by our institutional review board. multi - trauma patients who were exsanguinating from their pelvic fractures which were treated by the eppp technique were prospectively followed for clinical and physiological outcomes and survival rates. inclusion criteria for a patient to be considered for emergent stand - alone eppp included : hemodynamic instability in multi - trauma patients (systolic blood pressure 15 (the median iss score was 29, range : 17 - 38). patient characteristics despite multiple transfusions of packed rbc units and life - saving resuscitation, the mean systolic blood pressure was 62.66 mmhg before eppp and increased to 113.33 mmhg, after eppp. all patients were therefore in shock class 3 - 4 (according to the atls guidelines). the mean number of rbc packs decreased significantly following eppp (3.45 rbc units) compared with the rate before the procedure (12 rbc units). none succumbed due to ongoing hemorrhage, but rather from other major associated injuries, including intracranial hemorrhage, and chest contusions with cardiac arrhythmias [tables 2 and 4 ]. two patients were died within 1-day of undergoing eppp and the three patients were died 4 days later with intracranial hemorrhage being the presumed cause of death. comparison between survivors and nonsurvivors apart from survival and mortality, not all parameters were available for all patients ; therefore the number of patients with available data is indicated in parenthesis in the tables. all the patients had other associated injuries (urinary bladder rupture, fractures of long bones, and intracranial bleeding, etc.,) motor vehicle accidents accounted for 60% of the injuries, and accidental falls from heights accounted for the remaining 40% [table 3 ]. all study patients were involved in high - energy injuries, with an iss score > 15 (the median iss score was 29, range : 17 - 38). despite multiple transfusions of packed rbc units and life - saving resuscitation, the mean systolic blood pressure was 62.66 mmhg before eppp and increased to 113.33 mmhg, after eppp. all patients were therefore in shock class 3 - 4 (according to the atls guidelines). the mean number of rbc packs decreased significantly following eppp (3.45 rbc units) compared with the rate before the procedure (12 rbc units). none succumbed due to ongoing hemorrhage, but rather from other major associated injuries, including intracranial hemorrhage, and chest contusions with cardiac arrhythmias [tables 2 and 4 ]. two patients were died within 1-day of undergoing eppp and the three patients were died 4 days later with intracranial hemorrhage being the presumed cause of death. comparison between survivors and nonsurvivors apart from survival and mortality, not all parameters were available for all patients ; therefore the number of patients with available data is indicated in parenthesis in the tables. management of trauma patients with bleeding pelvic fractures depends on their hemodynamic stability upon arrival. in the majority of cases, bleedings stops after standard initial resuscitation. however, there is a subgroup of patients that present with the life - threatening retroperitoneal bleeding which does not respond to resuscitation efforts. thus, the indication for eppp employment was continuous life - threatening deterioration of hemodynamic status despite repeated resuscitative efforts in an exsanguinating patient with a known unstable pelvic fracture. the reported mortality rate of patients who present with massive pelvic bleeding associated with unstable pelvic fractures reportedly exceeds 40%. in the current study, eppp was promptly instituted as a stand - alone initial surgical procedure in the initial phases of resuscitation due to life - threatening hemodynamic deterioration in patients that were refractory to multiple blood transfusions and resuscitation and despite external pelvic binders. as such, external pelvic fixators were not applied, nor were these patients emergently taken to the angiography suite upon arrival to the hospital. abandoning the use of emergent angiography and application of external fixation became our practice after experiencing patient deaths in the angiography suite and realizing that it is both time - consuming and in our experience a less effective means, to stop the major cause of bleeding that occurs from pelvic veins. eppp was shown to be very effective in stopping both bleeding pelvic presacral veins and arteries and can be achieved quickly by orthopedic and general surgeons who are readily available at all times, as opposed to the need to await for the angiography team to arrive and set up. the concept of employing eppp as the initial and sole surgical therapeutic management is based on a paradigm shift in treatment rationale aimed at damage control. when bleeding is severe, and resuscitation efforts do not stabilize the patients hemodynamic status, it seems that one should direct all attention toward obtaining immediate surgical access to the bleeding presacral vessels and control of these venous and arterial bleeders via direct pressure packing. this is in contrast to the tradition of employing indirect trials to contain the bleeding via external pelvic stabilization or angiography with arterial embolization that do not fully succeed in stopping the venous presacral bleeding, that is, considered the major cause of bleeding and of hemodynamic instability. this change in treatment sequence using eppp first during the initial resuscitation while abandoning angiography and pelvic external fixation for a later stage brought about a dramatic change in patient survival and outcomes whereby initial resuscitation was successful and hemodynamic stabilization was achieved in all patients (i.e., none died from exsanguination). this 100% initial resuscitation success rate was followed by a 79% long - term survival rate and eventual discharge from rehabilitation. previous reports on eppp recommended that the pelvis should be fixed before packing is performed. as mentioned, in this study external fixation was not used in the initial treatment and interestingly, the 79% survival rate with eppp reported here (11 of 14 patients) is similar to that reported by ertel., who described a 71% survival rate in 10 of 14 patients with hemorrhagic shock after retroperitoneal pelvic packing and application of a c - clamp. noteworthy, the addition of the external pelvic fixation reportedly does not seem to make a difference in patient survival. the results of the current study concur, and demonstrate that immediate external fixation of the pelvis is not needed in order to control the bleeding. initial pelvic stabilization relies on the misconception that the pelvis is a closed contained space and that minimizing the volume of the fractured pelvis would, therefore, initiate a tamponade effect. however, intraoperative observation refutes this notion by showing that despite the use of external fixators, the bleeding retroperitoneal hematoma continues to expand into the abdominal retroperitoneal space above the pelvis, and through the sciatic notch into the gluteal space below the pelvis. emergent angiographic embolization has been advocated in the control of life - threatening pelvic hemorrhage. however, the angiographic procedure may be time - consuming, with reported procedure times of 30 - 90 min, the severely hemodynamically unstable patient may not be able to sustain life for that long a period of time. furthermore, angiographic services may not be available in all hospital settings at all times and may require awaiting the arrival of the angiography trained invasive radiology specialist. in contrast, eppp can be accomplished efficiently and quickly (medium time of 30 min in our hands). finally, huittinen and sltis showed that most of the bleeding comes from the presacral venous plexus and from the bone, whereupon angiographic arterial embolization would do nothing to stop the major cause of death, which is bleeding from the venous and intraosseous bleeding vessels. our protocol presented here [figure 3 ] emphasizes that the use of angiography should be reserved for later stages of treatment, that is, only after the patient 's hemodynamic and life - threatening conditions are addressed and stabilized by more immediate resuscitative measures. in other words, using damage control concepts, the multiple pads inserted during the immediate eppp stop major bleeding by pressing directly upon all vessels and allow stabilizing the patients physiological status. then, typically after 2 - 3 days of treatment in intensive care he / she can safely be transported to the angiography suite in an orderly scheduled manner. our working scheme delegates the use of angiography to serving as an adjunct to intensive care treatment, only after the hemodynamically stable patient can be safely transported to the angiography suite. at this point, with the patient 's hemodynamic status well controlled, and with the angiography team ready and in place, arterial angiographic catheterization access through the femoral artery is initiated before the pfannenlstiel wound is re - opened and the abdominal pads of the eppp are removed in a controlled manner. this allows both fluoroscopic visualizations by the angiographer and direct visualization of the now thrombosed bleeding vessels in the surgical wound by the surgeon. if any remaining arterial bleeding resumes after the pressure packing is removed, it can now be easily addressed by the angiography team that already has their catheters in place. limitations of this study are apparent and include a small patient group (fortunately these devastating injuries are rare) with only historic controls. not all the physiological data was recorded in the patients files due to lack of reporting by the trauma teams at the time of emergent life - saving resuscitative efforts, therefore some of the data includes < 14 patients as is noted in the tables where applicable. it is an invasive, open procedure done as a last resort life - saving procedure in extreme and emergent situations and carries a risk of infection. in our study, the infection rate was 35% (5/14), similar to a rate of 33% reported by ttterman. all infections were aggressively treated with intravenous antibiotics as well as by repetitive irrigation and debridement with daily repacking as needed until the wounds were clean and could be closed with vacuum drains. none of the patients succumbed to infection, and none of them sustained an intra - abdominal abscess. another disadvantage of eppp is the need for repetitive surgical procedures for bandage exchanges and repacking, typically after 24 - 48 h, and until the wounds are clean and can be closed. as in any surgical procedure strict adherence to technique is mandatory, emphasis must be placed on correctly directing the packing deep down into the pelvis and onto the presacral vessels while avoiding pressure packing onto the more lateral and superficial femoral arteries so as to maintain blood flow to the lower extremities. ertel., reported that 30% of the patients that were treated with pelvic packing developed abdominal compartment syndrome. fortunately, none of the patients developed this complication, since the eppp procedure is extra - peritoneal and does not violate the abdominal compartment. the implementation of eppp as part of the initial resuscitative treatment of life - threatening hemodynamically unstable multi - trauma patients with unstable pelvic fractures in our trauma center led to excellent early survival rates. eppp effectively stabilized all patients that were refractory to initial resuscitative measures, and eliminated the early mortality associated with massive pelvic bleeding. eppp was shown to be effective as a stand - alone surgical procedure while abandoning the use of emergent angiographic arterial embolization or early external pelvic fixation. eppp enables a unique advantage of directly compressing the life - threatening retroperitoneal bleeders by applying direct pressure and causing a tamponade effect, effectively stanching both venous and arterial pelvic blood flow. | purpose : traditional maneuvers aim to decrease retroperitoneal bleeding in hemodynamically unstable multi - trauma patients with unstable pelvic fractures, are reportedly successful in approximately only 50%. the life - saving effect of extra - peritoneal pressure packing (eppp) is based on direct compression and control of both venous and arterial retroperitoneal bleeders. this study describes the safety and efficacy of emergent eppp employment, as a stand - alone surgical treatment, that is, carried out without external pelvic fixation or emergent angiography.materials and methods : a retrospective chart review of all hemodynamic unstable, multi - trauma patients with mechanically unstable pelvic fractures treated by the eppp technique at our medical center between the years 2005 and 2011. survival rates, clinical, and physiological outcomes were followed prospectively.results:twenty-five of the 181 pelvic fracture patients had biomechanically unstable fractures that required surgical fixation. fourteen of those 25 patients had deteriorating hemodynamic instability from massive pelvic bleeding which was resistant to resuscitation, and they underwent eppp, as a stand - alone treatment. the procedure successfully achieved hemodynamic stability in all 14 patients and obviated the early mortality associated with massive pelvic bleeding. three of these patients eventually succumbed to their multiple injuries.conclusion:implementation of eppp improved all measured physiological outcome parameters and survival rates of hemodynamically unstable multi - trauma patients with unstable pelvic fractures in our trauma center. it provided the unique advantage of directly compressing the life - threatening retroperitoneal bleeders by applying direct pressure and causing a tamponade effect to stanch venous and arterial pelvic blood flow and obviate the early mortality associated with massive pelvic bleeding. |
l. amylovorus cp1563 was isolated from human fecal specimens and stocked in our culture collection library. it was screened as a strain with high potential by luciferase assays for ppar ligand activity (9). here, the strain was cultured in an original food - grade medium (table 1) for 24 h at 37c under a partially anaerobic atmosphere. at the start of the culture, the ph of the medium was adjusted to 6.8, and thereafter, the ph was controlled at 5.2 by the addition of sodium hydroxide. the resulting bacterial pellet was harvested by filtration with a ceramic filter, washed with sterilized water, and then filtered again. the resulting bacterial powder was fragmented by a fs-4 jet mill (seishin enterprise co., ltd., tokyo, japan) under the pressure of 0.64 mpa at a feed speed of 0.5 kg / h. the degree of fragmentation was evaluated as the ratio of the average long dimension of the fragmented bacterial cell preparation against that of the original bacterial cells. the quality of the fragmented bacterial preparation was checked by standard analysis methods (table 2). composition of the medium for cp1563 culture the ph value of the medium was adjusted to 6.8 before sterilization, and after inoculation, the ph was controlled to approximately 5.2. results of general analysis of fragmented cp1563 powder all analyses were performed at general foundation japan food research laboratories (tokyo, japan). test beverages were prepared by blending powdered skim milk, citrate, flavors, sweeteners, soybean polysaccharide, food emulsifier, and 200 mg of the fragmented cp1563 powder in water, followed by carbonation, pasteurization, and packaging into 500 ml bottles. the placebo beverage was prepared using the same formula and procedure as for the test beverage, but it did not contain cp1563 powder. the test beverage provided a total of 83.7 kj (20 kcal / day), 1.0 g of protein, 4.5 g of carbohydrates, 0 g of fat, and 128 mg of sodium, whereas the placebo beverage provided a total of 83.7 kj (20.0 kcal), 1.0 g of protein, 4.5 g of carbohydrates, 0 g of fat, and 128 mg of sodium. the subjects were recruited from a pool of volunteers from the clinical support corporation (sapporo, japan). the subjects were all healthy adult women and men who underwent a medical examination within a month before the trial at the medical corporation hokubukai utsukushigaoka hospital, which is affiliated with the clinical support corporation. the selection criteria included healthy men and women classified as having obesity class i according to the guidelines of the japanese society for the study of obesity, having a bmi (in kg / m) of 25 or more but less than 30, and not receiving treatment for any lifestyle - related diseases (14). the exclusion criteria included the following : allergy to cow 's milk and soy bean ; use of medications and health foods affecting lipid metabolism ; designation of unsuitable by the medical doctor in charge of the study ; a history of severe disorders ; a history of gastrointestinal tract surgery ; pregnancy or breastfeeding ; withdrawal of more than 400 ml of whole blood or blood components within the 4 months prior to the study and/or more than 200 ml of whole blood within the 2 months prior to this study ; extremely irregular dietary habits ; alternative work schedule or employment in the midnight shift ; and smoking and high alcohol intake. allocation to the test or placebo group was concealed from the investigator who enrolled the subjects, the nurses, and the medical doctor in charge of the study. this study was approved by the institutional review board of the medical corporation hokubukai utsukushigaoka hospital according to ethical principles and an experimental plan based on the declaration of helsinki. prior to the trial, the medical doctor (kt) provided the subjects with a full explanation of the purpose and methodology of the study. this study followed a randomized, double - blinded, placebo - controlled design, and the experimental periods were divided into 2 weeks of observation before treatment, 12 weeks of treatment, and 4 weeks of observation after treatment. a concealed study coordinator (it) randomly and blindly assigned the 200 subjects into two groups of 100 that were matched according to age, gender, bmi, and abdominal fat area. volunteers in one group (n=100) received the test beverages (a 500 ml bottle of active beverage per volunteer per day : test group), and those in the other group (n=100) received one placebo beverage per day (a 500 ml bottle of the beverage per volunteer per day : placebo group). the subjects were also instructed to assess their health condition and maintain healthy living practices, including diet and exercise. the sample size was set to 100 individuals per group to detect changes in the whole body fat area at week 12 of the treatment using a ct scanner between the test and placebo groups at a p - value of 0.05, with 80% power using analysis of covariance (ancova) based on information from a preliminary dose - finding study (unpublished information). a dropout rate of 10% was expected ; thus, 200 participants were recruited. at baseline (week 0), every 4 weeks (weeks 4 and 8), at the end of the intervention (week 12), and at the end of the post - observation period (week 16), we performed the following anthropometric measurements at the hospital : body weight, bmi, hip and waist circumferences, body fat percentage, systolic and diastolic blood pressures, pulse rate, body temperature, subcutaneous fat thickness (arm and back), blood sampling, and urinalysis. tokyo, japan) scans and dual bioelectrical impedance analysis (hds2000, omron healthcare co., ltd., kyoto, japan) were conducted to measure the abdominal visceral and subcutaneous fat areas at weeks 0, 4, 8, 12, and 16 and at weeks 0 and 12, respectively. height was only measured at the screening examination, and the bmi was calculated based on this measurement. each participant maintained a daily record of test or placebo beverage consumption and diet, exercise, and physical condition, including the presence of any subjective symptoms during the trial. the participants also maintained a detailed record of diet and pedometer measurements for 3 consecutive days before each visit : at the start of treatment (week 0) and at weeks 4, 8, 12, and 16. a managerial dietitian analyzed the dietary records to determine the intake of total energy, protein, carbohydrate, fat, total fiber, magnesium, calcium, potassium, and sodium using excel eiyokun ver. tokyo, japan). blood examination and urinalysis were performed at sapporo clinical laboratory inc. the following biochemical and hematological parameters were measured : total protein, albumin, total bilirubin, aspartate aminotransferase (ast), alanine aminotransferase (alt), lactate dehydrogenase (ld), alkaline phosphatase (alp), -glutamyl transpeptidase (-gtp), total cholesterol, hdl cholesterol, ldl cholesterol, triglyceride (tg), uric acid, blood urea nitrogen (bun), creatinine, sodium, potassium, chloride, calcium, glucose, adiponectin, white blood cells, red blood cells, hemoglobin, hematocrit, and platelets. protein, glucose, urobilinogen, bilirubin, ketone bodies, occult blood, ph, and density were determined through urinalysis. the participants were required to fast for at least 12 h prior to the test dates. medical examinations and inquiries were performed, and a medical doctor (kt) assessed the subjective symptoms at each measurement. statistical analyses were performed using jmp software (version 11 ; sas institute japan ltd., tokyo, japan) and spss software (version 20 ; ibm spss japan, tokyo, japan). for comparisons between the groups, ancova for repeated measures was primarily used to assess the time course of treatment. because of the wide ranges of the initial values of these parameters, ancova for repeated measures was the first choice for analyzing the data to not only minimize type ii errors but also ensure that the overall probability of type i errors was less than 0.05. multivariate analysis of variance (manova) for repeated measures was also applied to compare the variation of the mean values with time, where necessary, in cases for which the mean value was considered to be the representative value. ancova with baseline values as the covariate and student 's t - test were used as post hoc tests. compliance with the test or placebo beverage was assessed, and participants with 80% consumption of the beverages were considered eligible. l. amylovorus cp1563 was isolated from human fecal specimens and stocked in our culture collection library. it was screened as a strain with high potential by luciferase assays for ppar ligand activity (9). here, the strain was cultured in an original food - grade medium (table 1) for 24 h at 37c under a partially anaerobic atmosphere. at the start of the culture, the ph of the medium was adjusted to 6.8, and thereafter, the ph was controlled at 5.2 by the addition of sodium hydroxide. the resulting bacterial pellet was harvested by filtration with a ceramic filter, washed with sterilized water, and then filtered again. the resulting bacterial powder was fragmented by a fs-4 jet mill (seishin enterprise co., ltd., tokyo, japan) under the pressure of 0.64 mpa at a feed speed of 0.5 kg / h. the degree of fragmentation was evaluated as the ratio of the average long dimension of the fragmented bacterial cell preparation against that of the original bacterial cells. the quality of the fragmented bacterial preparation was checked by standard analysis methods (table 2). composition of the medium for cp1563 culture the ph value of the medium was adjusted to 6.8 before sterilization, and after inoculation, the ph was controlled to approximately 5.2. results of general analysis of fragmented cp1563 powder all analyses were performed at general foundation japan food research laboratories (tokyo, japan). test beverages were prepared by blending powdered skim milk, citrate, flavors, sweeteners, soybean polysaccharide, food emulsifier, and 200 mg of the fragmented cp1563 powder in water, followed by carbonation, pasteurization, and packaging into 500 ml bottles. the placebo beverage was prepared using the same formula and procedure as for the test beverage, but it did not contain cp1563 powder. the test beverage provided a total of 83.7 kj (20 kcal / day), 1.0 g of protein, 4.5 g of carbohydrates, 0 g of fat, and 128 mg of sodium, whereas the placebo beverage provided a total of 83.7 kj (20.0 kcal), 1.0 g of protein, 4.5 g of carbohydrates, 0 g of fat, and 128 mg of sodium. the subjects were recruited from a pool of volunteers from the clinical support corporation (sapporo, japan). the subjects were all healthy adult women and men who underwent a medical examination within a month before the trial at the medical corporation hokubukai utsukushigaoka hospital, which is affiliated with the clinical support corporation. the selection criteria included healthy men and women classified as having obesity class i according to the guidelines of the japanese society for the study of obesity, having a bmi (in kg / m) of 25 or more but less than 30, and not receiving treatment for any lifestyle - related diseases (14). the exclusion criteria included the following : allergy to cow 's milk and soy bean ; use of medications and health foods affecting lipid metabolism ; designation of unsuitable by the medical doctor in charge of the study ; a history of severe disorders ; a history of gastrointestinal tract surgery ; pregnancy or breastfeeding ; withdrawal of more than 400 ml of whole blood or blood components within the 4 months prior to the study and/or more than 200 ml of whole blood within the 2 months prior to this study ; extremely irregular dietary habits ; alternative work schedule or employment in the midnight shift ; and smoking and high alcohol intake. allocation to the test or placebo group was concealed from the investigator who enrolled the subjects, the nurses, and the medical doctor in charge of the study. this study was approved by the institutional review board of the medical corporation hokubukai utsukushigaoka hospital according to ethical principles and an experimental plan based on the declaration of helsinki. prior to the trial, the medical doctor (kt) provided the subjects with a full explanation of the purpose and methodology of the study. this study followed a randomized, double - blinded, placebo - controlled design, and the experimental periods were divided into 2 weeks of observation before treatment, 12 weeks of treatment, and 4 weeks of observation after treatment. a concealed study coordinator (it) randomly and blindly assigned the 200 subjects into two groups of 100 that were matched according to age, gender, bmi, and abdominal fat area. volunteers in one group (n=100) received the test beverages (a 500 ml bottle of active beverage per volunteer per day : test group), and those in the other group (n=100) received one placebo beverage per day (a 500 ml bottle of the beverage per volunteer per day : placebo group). the subjects were also instructed to assess their health condition and maintain healthy living practices, including diet and exercise. the sample size was set to 100 individuals per group to detect changes in the whole body fat area at week 12 of the treatment using a ct scanner between the test and placebo groups at a p - value of 0.05, with 80% power using analysis of covariance (ancova) based on information from a preliminary dose - finding study (unpublished information). at baseline (week 0), every 4 weeks (weeks 4 and 8), at the end of the intervention (week 12), and at the end of the post - observation period (week 16), we performed the following anthropometric measurements at the hospital : body weight, bmi, hip and waist circumferences, body fat percentage, systolic and diastolic blood pressures, pulse rate, body temperature, subcutaneous fat thickness (arm and back), blood sampling, and urinalysis. abdominal computed tomography (somatic emotion 16 excel, siemens japan k.k., tokyo, japan) scans and dual bioelectrical impedance analysis (hds2000, omron healthcare co., ltd., kyoto, japan) were conducted to measure the abdominal visceral and subcutaneous fat areas at weeks 0, 4, 8, 12, and 16 and at weeks 0 and 12, respectively. height was only measured at the screening examination, and the bmi was calculated based on this measurement. each participant maintained a daily record of test or placebo beverage consumption and diet, exercise, and physical condition, including the presence of any subjective symptoms during the trial. the participants also maintained a detailed record of diet and pedometer measurements for 3 consecutive days before each visit : at the start of treatment (week 0) and at weeks 4, 8, 12, and 16. a managerial dietitian analyzed the dietary records to determine the intake of total energy, protein, carbohydrate, fat, total fiber, magnesium, calcium, potassium, and sodium using excel eiyokun ver. tokyo, japan). blood examination and urinalysis were performed at sapporo clinical laboratory inc. the following biochemical and hematological parameters were measured : total protein, albumin, total bilirubin, aspartate aminotransferase (ast), alanine aminotransferase (alt), lactate dehydrogenase (ld), alkaline phosphatase (alp), -glutamyl transpeptidase (-gtp), total cholesterol, hdl cholesterol, ldl cholesterol, triglyceride (tg), uric acid, blood urea nitrogen (bun), creatinine, sodium, potassium, chloride, calcium, glucose, adiponectin, white blood cells, red blood cells, hemoglobin, hematocrit, and platelets. protein, glucose, urobilinogen, bilirubin, ketone bodies, occult blood, ph, and density were determined through urinalysis. the participants were required to fast for at least 12 h prior to the test dates. medical examinations and inquiries were performed, and a medical doctor (kt) assessed the subjective symptoms at each measurement. statistical analyses were performed using jmp software (version 11 ; sas institute japan ltd., tokyo, japan) and spss software (version 20 ; ibm spss japan, tokyo, japan). for comparisons between the groups, because of the wide ranges of the initial values of these parameters, ancova for repeated measures was the first choice for analyzing the data to not only minimize type ii errors but also ensure that the overall probability of type i errors was less than 0.05. multivariate analysis of variance (manova) for repeated measures was also applied to compare the variation of the mean values with time, where necessary, in cases for which the mean value was considered to be the representative value. ancova with baseline values as the covariate and student 's t - test were used as post hoc tests. compliance with the test or placebo beverage was assessed, and participants with 80% consumption of the beverages were considered eligible. approximately 21 kg of dried cp1563 cells were harvested from the culture medium of 4,000 l. the fragmentation process was successfully completed as indicated by an average long dimension of less than 70% of that of the native bacteria (9). the results of a standard method analysis of the fragmented bacterial preparation are shown in table 2. the quality was satisfactory for a food material for the test beverages. in total, 200 subjects were enrolled, and 197 participants completed the present study. three individuals discontinued participation in the study because of an inability to comply with the time constraint (n=1) or an extended business trip (n=2). during the study, adverse effects, such as abnormal values of hepatic or renal function, cutaneous symptoms, nausea, abdominal discomfort, and edema caused by ppar or ppar synthetic agonists, although transient diarrhea (four subjects in the placebo group and two subjects in the test group) and constipation (two subjects in the placebo group and one subject in the test group) were observed, the doctor determined that the symptoms were not associated with the intake of the supplementary beverages. no signs of imbalance in the occurrence rates of unwellness between the test and placebo groups were recorded. no significant differences were observed between the test and placebo groups regarding gender, age, bmi, or abdominal fat area (p>0.05). this study achieved relatively high compliance : the frequencies of test or placebo beverage consumption were 99.850.59 and 99.840.59, respectively. the baseline demographic, anthropometric, and clinical characteristics of the participants were comparable in both groups (table 3). table 4 shows the daily energy, protein, fat, and carbohydrate intake based on body weight. daily energy, protein, fat, and carbohydrate intake based on weight measured every 4 weeksa no significant difference was observed between groups (manova was used for repeated measures analysis). time - dependent changes in bmi, body fat percentage, visceral fat area, and whole body fat area are shown in fig. 2 (p=0.524, p<0.001, p<0.001, and p<0.001, respectively). in stratified analyses, the subjects whose visceral fat exceeded 100 cm showed a significantly larger reduction in the test group than in the placebo group in whole body fat area (p<0.001, data not shown). however, the participants in the other subgroup with a low baseline value (visceral fat<100 cm) also showed a significant reduction in the body fat area in the test group (p<0.05 [test for efficacy ratio ], p=0.031 ; 390.194.75 cm at the start [meansem ] to 381.894.70 cm at 12 weeks on cp1563 [meansem ]), but those in the placebo group did not. another stratified analysis of the participants in the subgroup with a high baseline value (visceral fat 100 cm) exhibited a larger reduction in visceral fat area (fig. a significant difference was observed between the placebo and test groups in men (table 5 ; p=0.035). in women, a reduction in the body weight regarding waist circumference, a significant interaction between the factor group and time was observed (p<0.001), and the graph showed a significant difference between the placebo and test groups at 12 weeks in the measurement item, as shown in fig. 4a. similarly, in terms of hip circumference, a significant difference between the placebo and test groups was noted (fig. (a) bmi, (b) body fat percentage, (c) visceral fat area, and (d) whole body fat area. changes in bmi and fat area based on ct scans from baseline in obese class i participants during the 12-week period of consumption in the test group (closed circles ; n=100) and the placebo group (open circles ; n=100). the participants ingested one bottle (500 ml) of carbonated beverage containing fragmented cp1563 or the same amount of carbonated placebo beverage daily for 12 weeks. after the treatment was initiated, the whole and visceral fat areas were measured at weeks 4, 8, 12 and 16. subgroup analysis was performed after the groups were divided into two subgroups using 100 cm of visceral fat area as the cutoff value. statistical significance was determined according to the interaction of the factor grouptime using ancova for repeated measures. time - dependent changes in visceral fat area (cm) in subjects with higher values (100 cm) at baseline. visceral fat areas were measured at weeks 4, 8, 12 and 16 in subjects with higher initial values at baseline. the solid line is the regression line, and the dotted line shows the 95% confidence intervals of the population mean values. statistical significance was determined according to the interaction of the factor grouptime using ancova for repeated measures. changes in (a) waist, and (b) hip circumferences at week 12. changes in anthropometric measurements during the course of the study bmi, body mass index ; ancova for repeated measures was used to analyze the changes in these measures during the course of the study. systolic blood pressure did not show a significant change ; however, diastolic blood pressure was affected by the constant ingestion of fragmented cp1563 (table 5 ; p<0.001). the regular intake of fragmented cp1563 significantly affected the triglyceride and total and ldl - cholesterol levels in the plasma samples (fig. 5 ; p<0.001, p<0.001, and p<0.001, respectively). larger reductions of these lipid compositions were observed in the test group than in the placebo group at week 12, particularly in men (table 6). there was no major change in the hdl - cholesterol level, but a significant difference between the placebo and test groups was observed, particularly in men. changes in biological markers in the blood after 12 weeks of ingestion : triglycerides, total cholesterol, and ldl - cholesterol. statistical significance was determined according to the interaction of the factor grouptime using ancova for repeated measures. changes in serum metabolic variables during the course of the study t - c, total cholesterol ; init., initial ; hdl - c, hdl - cholesterol ; ldl - c, ldl - cholesterol ; tg, triglyceride ; glc, glucose ; ins, insulin ; homa - ir, homeostasis model assessment - insulin resistance ; ua, uric acid. statistical analysis with ancova was used for repeated measures, except for the items fasting insulin and homa - ir. the fasting insulin concentration was measured at only 2 time points : before and after ingestion ; therefore, ancova was used for these variables. blood glucose, insulin, and homeostasis model assessment - insulin resistance (homa - ir) were affected by the ingestion of fragmented cp1563 (fig. 6, table 6), and significant differences between the placebo and test groups were observed in these parameters (p<0.001, p=0.004, and p<0.001, respectively). however, glycosylated hemoglobin (hba1c) levels were not significantly different between the two groups (data not shown). total bilirubin was significantly decreased in the test group during the early stage of the time course, especially in women (table 7 ; p<0.001). these differences were relatively well shown in the participants who had high baseline values for these parameters. changes in the concentrations of chemical substance in the blood after 12 weeks of ingestion : glucose, insulin, bilirubin and uric acid. the solid line is the regression line, and the dotted line shows the 95% confidence intervals of the population mean values. statistical significance was determined according to the interaction of the factor grouptime using ancova for repeated measures. changes in hematological and serum biochemical variables during the course of the study wbc, white blood cell ; rbc, red blood cell ; alt, alanine aminotransferase ; ast, aspartate aminotransferase ; -gtp, -glutamyl transpeptidase ; ld, lactate dehydrogenase ; tp, total protein ; bun, blood urea nitrogen ; ua, uric acid ; cre, creatinine ; t - bil, total bilirubin ; na, sodium ; k, potassium ; cl, chloride. bun, sodium, and chloride exhibited significant reductions in the test group compared with the placebo group (table 7, fig. red blood cells, hemoglobin, and hematocrit showed significant increases in the test group compared with the placebo group (table 7 and fig. 8 ; p<0.001, p<0.001, and p<0.001, respectively). in contrast, the white blood cell count was significantly lower in the test group than in placebo group (table 7 ; p<0.001). alt, ast, and ld exhibited significant reductions in the test group compared with those in the placebo group (table 7 ; p<0.001, p<0.001, and p<0.001, respectively). in contrast, -gtp was increased to a greater extent in the test group than in the placebo group (table 7 ; p<0.001). these changes in blood test values were, however, within the normal ranges, and even the results obtained for individual subjects failed to reveal changes that might develop into abnormal ranges (data not shown). changes in the plasma concentrations of chemical elements throughout the ingestion period : (a) sodium, (b) chloride and (c) potassium. statistical significance was determined according to the interaction of the factor grouptime using ancova for repeated measures. changes in red blood cell counts throughout the ingestion period : (a) erythrocyte, (b) hemoglobin concentration and (c) hematocrit concentration. statistical significance was determined according to the interaction of the factor grouptime using ancova for repeated measures. after cp1563 treatment, decreases in the number of white blood cells (wbcs) and the concentrations of ast, alt, ld, bun, uric acid, creatinine, sodium, and chloride ; and increases in -gtp and potassium were observed (table 7). in addition, no statistically significant changes in any of the other markers, except those mentioned above, were recorded. no abnormal changes were observed in urinalysis during or after intervention in the test and placebo groups (data not shown). although two subjects were positive for uric protein and uric glucose during the pre - intake period, no abnormal changes were observed between the intake and post - intake periods. the baseline demographic, anthropometric, and clinical characteristics of the participants were comparable in both groups (table 3). table 4 shows the daily energy, protein, fat, and carbohydrate intake based on body weight. daily energy, protein, fat, and carbohydrate intake based on weight measured every 4 weeksa no significant difference was observed between groups (manova was used for repeated measures analysis). time - dependent changes in bmi, body fat percentage, visceral fat area, and whole body fat area are shown in fig. 2 (p=0.524, p<0.001, p<0.001, and p<0.001, respectively). in stratified analyses, the subjects whose visceral fat exceeded 100 cm showed a significantly larger reduction in the test group than in the placebo group in whole body fat area (p<0.001, data not shown). however, the participants in the other subgroup with a low baseline value (visceral fat<100 cm) also showed a significant reduction in the body fat area in the test group (p<0.05 [test for efficacy ratio ], p=0.031 ; 390.194.75 cm at the start [meansem ] to 381.894.70 cm at 12 weeks on cp1563 [meansem ]), but those in the placebo group did not. another stratified analysis of the participants in the subgroup with a high baseline value (visceral fat 100 cm) exhibited a larger reduction in visceral fat area (fig. a significant difference was observed between the placebo and test groups in men (table 5 ; p=0.035). in women, a reduction in the body weight was observed in the test group (table 5 ; p=0.051). regarding waist circumference, a significant interaction between the factor group and time was observed (p<0.001), and the graph showed a significant difference between the placebo and test groups at 12 weeks in the measurement item, as shown in fig. 4a. similarly, in terms of hip circumference, a significant difference between the placebo and test groups was noted (fig. (a) bmi, (b) body fat percentage, (c) visceral fat area, and (d) whole body fat area. changes in bmi and fat area based on ct scans from baseline in obese class i participants during the 12-week period of consumption in the test group (closed circles ; n=100) and the placebo group (open circles ; n=100). the participants ingested one bottle (500 ml) of carbonated beverage containing fragmented cp1563 or the same amount of carbonated placebo beverage daily for 12 weeks. after the treatment was initiated, the whole and visceral fat areas were measured at weeks 4, 8, 12 and 16. subgroup analysis was performed after the groups were divided into two subgroups using 100 cm of visceral fat area as the cutoff value. statistical significance was determined according to the interaction of the factor grouptime using ancova for repeated measures. time - dependent changes in visceral fat area (cm) in subjects with higher values (100 cm) at baseline. visceral fat areas were measured at weeks 4, 8, 12 and 16 in subjects with higher initial values at baseline. the solid line is the regression line, and the dotted line shows the 95% confidence intervals of the population mean values. statistical significance was determined according to the interaction of the factor grouptime using ancova for repeated measures. changes in (a) waist, and (b) hip circumferences at week 12. changes in anthropometric measurements during the course of the study bmi, body mass index ; ancova for repeated measures was used to analyze the changes in these measures during the course of the study. systolic blood pressure did not show a significant change ; however, diastolic blood pressure was affected by the constant ingestion of fragmented cp1563 (table 5 ; p<0.001). the regular intake of fragmented cp1563 significantly affected the triglyceride and total and ldl - cholesterol levels in the plasma samples (fig. 5 ; p<0.001, p<0.001, and p<0.001, respectively). larger reductions of these lipid compositions were observed in the test group than in the placebo group at week 12, particularly in men (table 6). there was no major change in the hdl - cholesterol level, but a significant difference between the placebo and test groups was observed, particularly in men. changes in biological markers in the blood after 12 weeks of ingestion : triglycerides, total cholesterol, and ldl - cholesterol. statistical significance was determined according to the interaction of the factor grouptime using ancova for repeated measures. changes in serum metabolic variables during the course of the study t - c, total cholesterol ; init., initial ; hdl - c, hdl - cholesterol ; ldl - c, ldl - cholesterol ; tg, triglyceride ; glc, glucose ; ins, insulin ; homa - ir, homeostasis model assessment - insulin resistance ; ua, uric acid. statistical analysis with ancova was used for repeated measures, except for the items fasting insulin and homa - ir. the fasting insulin concentration was measured at only 2 time points : before and after ingestion ; therefore, ancova was used for these variables. blood glucose, insulin, and homeostasis model assessment - insulin resistance (homa - ir) were affected by the ingestion of fragmented cp1563 (fig. 6, table 6), and significant differences between the placebo and test groups were observed in these parameters (p<0.001, p=0.004, and p<0.001, respectively). however, glycosylated hemoglobin (hba1c) levels were not significantly different between the two groups (data not shown). total bilirubin was significantly decreased in the test group during the early stage of the time course, especially in women (table 7 ; p<0.001). these differences were relatively well shown in the participants who had high baseline values for these parameters. changes in the concentrations of chemical substance in the blood after 12 weeks of ingestion : glucose, insulin, bilirubin and uric acid. the solid line is the regression line, and the dotted line shows the 95% confidence intervals of the population mean values. statistical significance was determined according to the interaction of the factor grouptime using ancova for repeated measures. changes in hematological and serum biochemical variables during the course of the study wbc, white blood cell ; rbc, red blood cell ; alt, alanine aminotransferase ; ast, aspartate aminotransferase ; -gtp, -glutamyl transpeptidase ; ld, lactate dehydrogenase ; tp, total protein ; bun, blood urea nitrogen ; ua, uric acid ; cre, creatinine ; t - bil, total bilirubin ; na, sodium ; k, potassium ; cl, chloride. bun, sodium, and chloride exhibited significant reductions in the test group compared with the placebo group (table 7, fig. red blood cells, hemoglobin, and hematocrit showed significant increases in the test group compared with the placebo group (table 7 and fig. in contrast, the white blood cell count was significantly lower in the test group than in placebo group (table 7 ; p<0.001). alt, ast, and ld exhibited significant reductions in the test group compared with those in the placebo group (table 7 ; p<0.001, p<0.001, and p<0.001, respectively). in contrast, -gtp was increased to a greater extent in the test group than in the placebo group (table 7 ; p<0.001). these changes in blood test values were, however, within the normal ranges, and even the results obtained for individual subjects failed to reveal changes that might develop into abnormal ranges (data not shown). changes in the plasma concentrations of chemical elements throughout the ingestion period : (a) sodium, (b) chloride and (c) potassium. statistical significance was determined according to the interaction of the factor grouptime using ancova for repeated measures. changes in red blood cell counts throughout the ingestion period : (a) erythrocyte, (b) hemoglobin concentration and (c) hematocrit concentration. closed circles indicate the test group, and open circles indicate the placebo group. statistical significance was determined according to the interaction of the factor grouptime using ancova for repeated measures. after cp1563 treatment, decreases in the number of white blood cells (wbcs) and the concentrations of ast, alt, ld, bun, uric acid, creatinine, sodium, and chloride ; and increases in -gtp and potassium were observed (table 7). however, these changes were within the normal ranges. in addition, no statistically significant changes in any of the other markers, except those mentioned above, were recorded. no abnormal changes were observed in urinalysis during or after intervention in the test and placebo groups (data not shown). although two subjects were positive for uric protein and uric glucose during the pre - intake period, no abnormal changes were observed between the intake and post - intake periods. in the present study, we demonstrated that non - viable, that is, paraprobiotic, lactic acid bacteria consumption has a beneficial body fat - lowering effect on humans and affects lipid, glucose, and uric acid metabolism. regarding the relationship between fat metabolism and the administration of lactic acid - producing bacteria, the effects of bifidobacterium breve b-3 (15), lactobacillus gasseri sbt2055 (16), and other bacteria on abdominal adiposity our previous study has evaluated the ppar/ activation potential of organic extracts from various lactic acid bacteria in vitro, and the highest activity was observed in l. amylovorus cp1563 (9). furthermore, the anti - dyslipidemic and anti - obesity effects of fragmented cp1563 have been recorded in high - fat diet - induced obese mice, and the fragmentation of cp1563 is required for its ppar-agonistic activity in vivo. in the present study, we demonstrated the effect of 12 weeks of ingestion of fragmented cp1563 on lipid metabolism, including decreased body fat, plasma triglycerides, and ldl - cholesterol in pre - obese and mildly obese participants. all 200 participants were confirmed to meet the criteria for investigating food for specified health use (foshu) for somatic fat reduction, as verified by the control doctor (kt). significant reductions in plasma glucose, insulin, homa - ir, and uric acid were observed in the test groups (fig. 6 and table 6). the parameters used to diagnose metabolic syndrome included waist circumstance, which represents visceral fat area ; insulin resistance ; serum / plasma triglycerides and hdl - cholesterol ; blood pressure ; and fasting blood glucose. in the present study, the body fat and visceral fat areas ; insulin resistance ; and levels of plasma triglycerides, hdl - cholesterol, insulin, homa - ir, and fasting blood glucose were significantly improved in the test group compared with the placebo group (tables 5 and 6). the results also demonstrated that a longer ingestion period is desirable to obtain health - promoting benefits. the expected changes only begin to occur during ingestion periods of at least 3 months. regarding the average initial visceral fat area, the initial value obtained in the present study was lower than those in other studies with similar study designs : 108.135.8 (cm) in the present study compared with 114118 (cm) in other studies (16, 17). in addition, the initial range of visceral fat area values of the participants was wider than previously reported in other studies. the initial value of the visceral fat area was inversely correlated with the reduction in visceral fat area after 12 weeks of intervention in the test group compared with the placebo group (fig. 3), suggesting that a reduction in visceral fat area is expected when the initial value falls within a higher range. however, the whole body fat area in the participants with low visceral fat (< 100 cm) was also significantly reduced in the test group but not in the same subgroup of the placebo group. this fact may suggest that fragmented cp1563 cells work not only in the patients classified as obese class i but also in individuals with moderate levels of fat. these results suggest that the constant intake of beverages containing fragmented cp1563 could improve metabolic syndrome, although further clinical studies are needed to determine the effects of longer ingestion periods. these findings indicate that fragmented l. amylovorus cp1563 has the same benefits as synthetic ppar, ppar, or ppar/ agonists. furthermore, uric acid was significantly decreased in the test group compared with the placebo group (fig. the synthetic ppar agonist, fenofibrate, was shown to significantly decrease the concentration of serum uric acid in healthy male subjects (18). the ppar agonist property of fragmented cp1563 likely decreased the plasma uric acid levels in the present study. the serum level of uric acid is positively correlated with the frequency of metabolic syndrome, and metabolic syndrome is associated with a high incidence of hyperuricemia (19). indeed, the use of glitazones leads to weight gain, edema, bone fractures, and heart failure, which limits the use of these drugs in diabetic patients with high lipid levels (10). in the present study, no adverse effects attributed to the administration of the fragmented cp1563 food - derived ppar agonists, such as fragmented cp1563, are expected to be relatively safe compared with ppar agonist drugs ; and dietary approaches utilizing food ingredients with ppar and ppar dual - activating properties have become important for preventing metabolic disorders. in addition, the average reduction in visceral fat after intervention in the present study was relatively small compared with those observed in previous studies (16, 17). the test beverages were consumed from september to december (autumn to winter), a period when people tend to gain weight easily. it has been reported that body weight tends to decrease in summer and increase in winter (20, 21) ; therefore, in the present study, the visceral fat in the test group was relatively unlikely to decrease (fig. 2c). another limitation is the lack of information concerning the daily food consumption and calorie intake. we did not ensure strict dietary regulations or supply the participants with study diets in the present study ; instead, the participants maintained a detailed record of diet and pedometer measurements for 3 consecutive days prior to each visit. analysis of the records revealed that the calorie intake and nutrient composition were similar between the placebo and test groups (table 4). however, other detailed information concerning daily dietary records was absent, and the daily calorie intake and nutrition might be altered during the intervention period, although the participants were instructed to maintain healthy living habits, including diet and exercise. indeed, approximately one - fifth of all study participants have been shown to engage in underreporting, whereas half of the study participants have been shown to demonstrate over reporting, which is a common phenomenon in the assessment of habitual dietary intake (22, 23). the results of the present study suggest that fragmented cp1563 is a novel foodstuff that regulates lipid and glucose metabolism. several food - derived natural compounds with ppar and/or ppar dual - activating properties have been reported (24). for example, docosahexaenoic acid (dha) and eicosapentaenoic acid (epa) are ppar agonists, and they are representative of foods that affect lipid metabolism, as supported by abundant evidence from clinical studies (25). the dosages used in relevant studies range from 0.185 to 9 g / day and are much higher than the dosage of fragmented cp1563 used in the present study, suggesting that lower dosages of cp1563 could exhibit ppar agonist activity equivalent to the dha / epa activity. alternatively, ppar and ppar dual activation could be the reason underlying the lower effective dosage of fragmented cp1563 (9). we isolated ppar/ agonists from cp1563 to clarify the significance of the ligands roles in metabolism. the series of information of the working materials, their working mechanisms, their absorbency indices, and their structures will soon be published. in lipid metabolism, a close relationship between the composition of gut microbiota and obesity might exist, although conflicting research findings have been reported (26). in the present study, we did not investigate changes in the gut microbiota during the intervention period according to cp1563 treatment, although much attention has been paid to gut microbes. if changes in gut microbiota are related to obesity, then non - viable fragmented cp1563 might affect the composition. indeed, in the case of l. gasseri cp2305, the consumption of beverages containing this strain led to some changes in the gut microbiota (27, 28). thus, an undefined component(s) of cp1563 might directly or indirectly influence the composition of the gut microbiota and subsequently improve glucose and fat metabolism. in conclusion, the daily consumption of beverages containing fragmented cp1563 for 12 weeks by obese class i subjects improves anthropometric measurements and markers related to lipid and glucose metabolism without any adverse effects. although further clinical trials and investigations of the mechanisms of action are needed, the results of the present study suggest that the consumption of foods containing fragmented cp1563 ameliorates obesity and prevents metabolic syndrome and complications. fn, yi, ka, ds, na, ts, and ya performed experiments and analyzed the data. fn, yi, ka, ds, na, ts, ya, and sf are employees of asahi group holdings, ltd. none of the other authors have any conflicts of interest. | backgroundpreviously, we showed that fragmented lactobacillus amylovorus cp1563 (cp1563) functions as a dual agonist of peroxisome proliferator - activated receptor and in vitro and in vivo.objectivehere, we examined the safety and effect of cp1563 ingestion on body fat in obese class i participants in a double - blinded, placebo - controlled, randomized clinical trial (rct).designin the rct, 200 participants with a body mass index (bmi) of 2530 kg / m2 consumed test beverages with or without 200 mg of cp1563 daily for 12 weeks. in total, 197 subjects completed the study without any adverse effects.resultsbody fat percentage, whole body fat, and visceral fat were significantly decreased in the test group compared with the placebo group (p<0.001, p<0.001, and p<0.001, respectively). triglycerides, total cholesterol, ldl - cholesterol, and diastolic blood pressure showed significant reductions in the test group compared with the placebo group (p<0.001, p<0.001, p<0.001, and p<0.001, respectively). additionally, significant differences in the changes in blood glucose, insulin, homeostasis model assessment - insulin resistance (homa - ir), and uric acid were observed between the two groups (p<0.001, p=0.004, p<0.001, and p<0.001, respectively). improvements in anthropometric measurements and markers were observed in obese class i subjects in the test group.conclusionsdaily consumption of beverages containing fragmented cp1563 for 12 weeks by obese class i subjects improved anthropometric measurements and markers related to lipid and glucose metabolism without any adverse effects. these results suggest that the consumption of foods containing fragmented cp1563 reduces body fat and prevents metabolic syndrome. |
laboratory testing is a highly complex process, and although laboratory services are relatively safe, they are not as safe as they could or should be. clinical laboratories have long focused their attention on quality control methods and quality assessment programs dealing with the analytical aspects of testing. however, a growing body of evidence accumulated in recent decades demonstrates that quality in clinical laboratories can not be assured by merely focusing on purely analytical aspects.15 more recent surveys of errors in laboratory medicine have concluded that mistakes occur more frequently before (preanalytical) and after (postanalytical) the test is performed. the risk of errors due to analytical problems has been significantly reduced over time, but there is evidence that carelessness may still have a serious impact on reporting. mistakes made during the entire testing process are referred to as laboratory errors, although these may be due to haste or poor instruction and communication on the part of personnel, actions taken by others involved in the testing process (eg, physicians, nurses, phlebotomists, and laboratory technicians), or a poorly designed process, all of which are beyond the control of the laboratory. constant turnover of staff in the emergency department and use of travelling nurses present a difficult challenge in terms of maintaining good practice. further, there is evidence that laboratory information is often only partially utilized.68 point of care testing (poct) is a modern approach that could help to resolve some health care problems because it is centered on the needs and satisfaction of patients, particularly if laboratory departments have to provide a lot of test answers in a short space of time. this methodology is defined as medical testing at or near the site of patient care, to support timely, safe, and effective acute care (cardiac, metabolic, coagulation, respiratory distress). however, this strategy must be of the same standard as that in the central laboratory, and necessitates the involvement of more personnel from specialties outside the laboratory.9 in the changing landscape of the health care system, hospitals are becoming increasingly involved in the treatment of acute disease, and clinical laboratories have to increase their efficiency. therefore, laboratory professionals, who are required to reduce costs, simplify processes, and decrease staff numbers, have considered the possibility of incorporating emergency testing into their routine work as a result of the improved productivity and flexibility of laboratory automation. in many situations, it is faster to perform both routine and emergency testing, with priority given to emergency testing, thereby simplifying laboratory processes and improving turnaround time. however, if the turnaround time does not satisfy physicians in the emergency department, the laboratory may need to take action to reduce the time interval between receiving requests and providing results. this improves clinical decision - making and patient management. the creation of large core laboratories as the centerpiece of pathology consortiums will increase the demand for poct unless transport of specimens and information technology facilities are radically improved.10 this paper describes the most frequent and risky analytical errors, especially in the preanalytical phase, occurring at the children s hospital in trieste, northeast italy, related to the laboratory and emergency departments. the use of poct as a modern pathology service involves information and instruction management about the importance to compare the test results with an external quality control. the purpose of this research was to demonstrate that meetings and audit processes involving sanitary personnel from both the laboratory and emergency departments can improve the performance of poct and decrease the number of preanalytical laboratory errors. this work was conducted between january 2012 and june 2013, and involved sanitary personnel from three medical units (neonatology, resuscitation, delivery room) and technicians in the laboratory department. studying this period of time, the preanalytical errors of tests sent to the laboratory department were examined. in the same period the emergency department was provided with three new poct and the related external quality control of the analysis. three meetings were held with physicians, nurses, and laboratory technicians, to highlight two problems, ie, preanalytical errors and analytical methodologies used for poct. for poct in the emergency department, we used a hematology analyzer (abacus junior 30, radiometer, milan, italy) to determine hemoglobin, hematocrit, mean corpuscular hemoglobin, mean corpuscular hemoglobin concentration, mean corpuscular volume, mean platelet volume, plateletcrit, platelets, red blood cells, red cell distribution, and white blood cells, and two blood gas instruments (abl 800 flex, radiometer, milan, italy) to analyze pco2, ph, po2, calcium, chloride, glucose, lactate, potassium, and sodium. riqas reports from randox laboratories ltd (crumlin, uk) were used for external quality control. the test was used to test for a statistically significant difference between the number of preanalytical errors made at the beginning and end of the study. all members of the nursing staff signed a consent form to confirm their voluntary participation in the study, and accepted the methodology of the course, ie, objectives, teaching and learning methods, and expected outcome. the core laboratory director provided weekly external quality control reports for poct, and a review of preanalytical errors was undertaken. after 6 months, the laboratory director held the first meeting with sanitary personnel of the emergency and laboratory departments, to explain the problems identified and the methodology needed to solve these problems. at the end of the study, the results were forwarded to the medical units and key points were discussed (tables 14). of 800 samples examined in the laboratory department in january 2012, we identified 64 preanalytical errors (8.0%), classified as hemolysis, insufficient sampling, late arrival, not conforming to medical protocol, and without diagnosis ; however, in june 2013, of 800 samples examined, only 17 preanalytical errors (2.1%) were identified, representing a significant decrease (p<0.05, test). during the study, there was an improvement in external quality control for poct from 3 or 2 standard deviations or more to one standard deviation for all parameters. in the emergency department setting, time is an important factor in the assessment, diagnosis, and stabilization of patients, and every effort should be made to save time when collecting laboratory specimens. nurses often collect blood from an existing intravenous catheter to avoid a second venepuncture, thereby increasing efficiency and decreasing patient discomfort ; however, increased hemolysis rates have been attributed to the practice of obtaining blood specimens in this way. data collection points demonstrated that drawing blood through an intravenous catheter was associated with hemolysis more frequently than using an intravenous catheter with a connected syringe. the principal problems identified were to do with transport of specimens, deviation from diagnostic protocols, and software programs used in the laboratory. other observations noted by sanitary personnel included the finite staffing resources available in the emergency department and the high workload of staff caring for disabled pediatric and small for date neonatology patients, which can reduce the efficiency of certain procedures. such problems were often due to poor communication, and action should be taken by all sanitary personnel involved in the testing (physicians, nurses, and phlebotomists in the laboratory and emergency departments) and transport process. in the collaborative approach to ensuring overall quality, the risk of errors and mistakes in laboratory testing must be minimized to guarantee the total quality of a laboratory service.1116 health personnel in the emergency department can affect tests in the laboratory, but nurses and doctors must be educated, given specific instructions, and complete courses on poct. the improvement in external quality control seen in this study demonstrates that nursing staff in the emergency department can provide more efficient patient care. their frequent execution, almost weekly, making sure data diagnostic process, can facilitate clinical decision, leading to a decrease in specimen recollection rates. it is important to improve specimen acceptability results in quicker laboratory results, to physicians who can have available the patient test report sooner ; this positively affects patient outcomes in the emergency department. the study shows only a decrease in errors, but it does not ensure in general that appropriate therapies were given. poct is not the solution to preanalytical nursing errors, but is important with regard to the needs of patients.17,18 in the past, laboratory testing in italy has been performed by physicians and laboratory technicians in a central laboratory, with some testing performed by satellite laboratories separate from the central laboratory, but still involving laboratory personnel. attitudes concerning laboratory testing have changed significantly in the last few years and the economic problem of personnel cost imposes, in a modern approach, that technology poct can be used by nurses at or near the site of patient care monitoring the patient without hospital admission. maintaining the status quo (a central laboratory and other satellite laboratories) is not a good strategy for survival in laboratory medicine today.1922 high - quality biological samples are needed, but are not sufficient alone for the quality of laboratory results. the total quality of a test is determined by control of analytical and preanalytical processes and by cooperation between laboratory technicians and other health care professionals, especially nurses. this is essential for reducing errors and for better analytical performance. training protocols must be established and all potential operators must achieve an adequate level of competence. the content of the training program (including temperature, time, and modality of transportation to avoid contamination) and assessment of knowledge / skill levels should be documented in a training manual with a job description, that includes sample requirements, specimen collection, sample preparation, stability of reagents, and calibration of instruments.23,24 international guidelines and recommendations, along with procedures for quality and performance control, must also be approved and used for poct in the emergency department. laboratory medicine, like all health care, takes place in a dynamic, rapidly changing environment. as the standard of care changes, technology develops, and the economics of health care continue to change, the technology of poct will be challenged to find new strategy. it means this approach to affront the clinical needs of the standard of care will be more used in the future. by understanding and considering poct in the next future this concept may become aware of best way to meet the needs of medical care at residence (ie, management of coagulation therapy).2527 recognition of normal and abnormal results, as well as an understanding of the multidisciplinary action needed in the event of an abnormal result are essential.2830 faster is not always better, and in our experience poct should be restricted to measurement of vital functions requiring an immediate response, eg, blood glucose, hemoglobin, and electrolytes (sodium, potassium, ionized calcium).31 the authors do not recommend introduction of poct in the emergency department to solve the problem of inappropriate samples and inadequate transport to the laboratory department. however, collaboration and instruction of all sanitary personnel involved is essential to deal with preanalytical errors, performance of poct, and external quality control, to maintain patient safety, and to improve risk management. the lack of studies like this can carry two other considerations, ie, the limits of a very expensive technology and the difficult in applying it, not only in the hospital but also at place of residence.32 further studies and experience are necessary to resolve these questions. | involvement of health personnel in a medical audit can reduce the number of errors in laboratory medicine. the checked control of point of care testing (poct) could be an answer to developing a better medical service in the emergency department and decreasing the time taken to report tests. the performance of sanitary personnel from different disciplines was studied over an 18-month period in a children s hospital. clinical errors in the emergency and laboratory departments were monitored by : nursing instruction using specific courses, poct, and external quality control ; improvement of test results and procedural accuracy ; and reduction of hemolyzed and nonprotocol - conforming samples sent to the laboratory department. in january 2012, point of care testing (poct) was instituted in three medical units (neonatology, resuscitation, delivery room) at the children s hospital in trieste, northeast italy, for analysis of hematochemical samples. in the same period, during the months of january 2012 and june 2013, 1,600 samples sent to central laboratory and their related preanalytical errors were examined for accuracy. external quality control for poct was also monitored in the emergency department ; three meetings were held with physicians, nurses, and laboratory technicians to highlight problems, ie, preanalytical errors and analytical methodologies associated with poct. during the study, there was an improvement in external quality control for poct from 3 or 2 standard deviations or more to one standard deviation for all parameters. of 800 samples examined in the laboratory in january 2012, we identified 64 preanalytical errors (8.0%) ; in june 2013, there were 17 preanalytical errors (2.1%), representing a significant decrease (p<0.05, 2 test). multidisciplinary management and clinical audit can be used as tools to detect errors caused by organizational problems outside the laboratory and improve clinical and economic outcomes. |
although most of these measurements were based on manual inspection and intervention, with the advent of fluorescence microscopy, many studies also involved quantitative imaging of living cells either using video or ccd cameras (inoue, 1981 ; allen and allen, 1983). in the early years of live - cell microscopy, methods for segmentation and tracking of cells (berg and brown, 1972 ; berns and berns, 1982) were rapidly developed and adapted from other areas. nowadays, techniques for fully automated analysis and time space visualization of time series from living cells involve either segmentation and tracking of individual structures, or continuous motion estimation (for an overview, see fig. 1). for tracking a large number of small particles that move individually and independently from each other, single particle tracking approaches are most appropriate (qian., 1991). once the images have been acquired by microscopy and preprocessed to improve the signal - to - noise ratio, they can be directly visualized by methods like volume rendering. for multiple objects in motion, single particle tracking, in which a particle is tracked over different time - steps, is the most direct method used. surface rendering is obtained after segmentation of contours in each individual section and gives rise to volumetric measurements such as volume and surface area. measurements of concentration changes for segmented areas in frap or fluorescence loss in photobleaching experiments give rise to estimates of kinetic parameters such as diffusion and binding coefficients. the estimation of flow of gray values is an approach to quantify mobility in continuous space. all these processes lead to accurate estimates of quantitative parameters. for the determination of more complex movement, two independent approaches were initially developed, but recently have been merged. optical flow (mitiche and bouthemy, 1996) methods image registration (terzopoulos., 1991 ; lavallee and szeliski, 1995) aims at identifying and allocating certain objects in the real world as they appear in an internal computer model. the main application of image registration in cell biology is the automated correction of rotational and translational movements over time (rigid transformation). this allows the identification of local dynamics, in particular when the movement is a result of the superposition of two or more independent dynamics. registration also helps to identify global movements when local changes are artifacts and should be neglected. the basic principle of single particle tracking is to find for each object in a given time frame its corresponding object in the next frame. the correspondence is based on object features, nearest neighbor information, or other inter - object relationships. object features can be dynamic criteria such as displacement and acceleration of an object as well as area / volume or mean gray value of the object. optical flow has been defined as the motion flow (i.e., the motion vector field) that is derived from two consecutive images in a time series (jhne, 2002). however, due to high levels of noise, this assumption is usually distorted, and standard region - based matching techniques give unsatisfactory results (anandan, 1989). a more reliable tracking approach involves fuzzy logic - based analysis of the tracking parameters (tvarusko., 1999). (apprehend and allocate) certain objects in the real world as they appear in an internal computer model. initially, only rigid transformations were used to superimpose the images, whereas nowadays, research is focused on the integration of local deformations. a parametric image registration algorithm specifies the parameters of a transformation in a way that physically corresponding points at two consecutive time steps are brought together as close as possible. such algorithms have been broadly studied in medical imaging and cell biology (maintz and viergever, 1998 ; bornfleth., 1999). although one class of algorithms operates on previously extracted surface points (lavallee and szeliski, 1995), other algorithms register the images directly based on the gray - value changes. nonrigid deformations, i.e., transformations others than rotation and translation, present an active body of research in computer vision. nonrigid approaches differ with respect to the underlying motion model (terzopoulos., 1991 ; szeliski, 1996). most commonly, a cost or error function is defined and an optimization method is chosen that iteratively adjusts the parameters until an optimum has been achieved. other approaches extract specific features (e.g., correspondence between points) that serve as a basis for directly calculating the model parameters (arun., 1987 ; rohr, 1997). computer vision is a discipline that focuses on information extraction from the output of optical sensors, and on the representation of this information in an internal computer model (faugeras, 1993). a computer vision framework for detecting and tracking diffraction images of linear structures in differential interference contrast microscopy was developed for measuring deflections of clamped microtubules with a freely moving second end (danuser., 2000). based on measurements of thermal fluctuations further, prior knowledge based on geometric and dynamic models of the scene can lead to restoration of information beyond the resolution limit of an imaging system (danuser, 2001). this super - resolution concept was illustrated by the stereo reconstruction of a micropipette moving in close proximity to a stationary target object. complex dynamic processes in cells should ideally be studied in three spatial dimensions over time. thereby, large and complex data sets typically consisting of 5,00010,000 single images are generated. such data are virtually impossible to interpret without computational tools for visual inspection in space and time. typically, 3-d images have been represented as stereoscopic pairs or as anaglyphs by pixel shift method (white, 1995). displaying time series as movies is still a widely used method for visual interpretation. for fast - moving objects such as trafficking vesicles imaged with high time resolution, time - lapse movies are very informative. however, for much slower nuclear processes or for processes with mixed kinetics that need to be observed over a longer period of time, the total number of time points for imaging are limited due to the photo toxicity of the light exposure during in vivo observation (konig., 1996). therefore, an interpolation between consecutive time steps is required to reconstruct intermediate time steps. as a side effect, additional information about the continuous development of the observed processes between the imaged time steps (subpixel resolution in time) is achieved, and quantitative information can be derived (see next section). although early studies explored 4-d data sets by simply browsing through an image gallery and highlighting interactively selected structures (thomas., 1996), two commonly used rendering algorithms for displaying 3-d structures are volume rendering and surface rendering (chen., 1995 ; fig. 1). volume rendering is a technique for visualizing complex 3-d data sets without explicit definition of surface geometry. the classification step assigns a level of opacity, contrast, and color to each voxel in the 3-d volume (e.g., wright., then, shading techniques are used to simulate both the object surface characteristics and the position and orientation of surfaces with respect to light sources and the observer. the colored, semitransparent volume a ray is cast into the volume through each grid point on the projection plane. as the ray progresses through the volume, it computes the color and opacity at evenly spaced sample locations, and finally yields a single pixel color. although volume rendering techniques provide a satisfactory display of biological structures, this method is limited to pure visualization and does not deliver quantitative information. in addition, the high anisotropy typical for live - cell imaging with low z - resolution limits the quality of this visualization technique. these limitations are overcome by surface - rendering techniques, where the object surface is represented by polygons. the polygonal surface is displayed by projecting all the polygons onto a plane that is perpendicular to a selected viewing direction. the most commonly used method to triangulate the 3-d surface is the marching cube algorithm (cline., 1988). the 3-d structure is defined by a threshold value throughout the data set, constructing an isosurface. the drawback of this method is that the surface of many biological structures can not be defined using a single intensity value, resulting in loss of relevant information. a great advantage of the combination of segmentation and surface reconstruction is the immediate access to quantitative information that corresponds to visual data (eils., 1996 ; these approaches were designed to deal particularly with the high degree of anisotropy typical for 4-d live - cell recordings and to directly estimate quantitative parameters, e.g., the gray values in the segmented area of corresponding images can be measured to determine the amount and concentration of fluorescently labeled proteins in the segmented cellular compartments. measuring concentration changes by frap and fluorescence loss in photobleaching have become standard methods to evaluate diffusion, binding, and trafficking in live cells (for review see phair and misteli, 2001). these methods give direct access to kinetic parameters such as the diffusion coefficient of molecules (axelrod. 2000) or exchange rates of molecules between different compartments (hirschberg., 1998 ; phair and misteli, 2001). in combination with motion estimation techniques, parameters such as the velocity of the mass center for individual objects or for each point on the object surface can be readily accessed. further, local parameters such as acceleration, tension, or bending (bookstein, 1989) can be estimated. during motion estimation, global quantities are estimated such as the parameters of rotation and translation (germain., 1999). the evolution of these eigenvalues can be used to characterize and analyze the observed motion. statistical analysis of velocity histograms can be applied to compute peak velocities corresponding to the most frequently occurring velocity values (uttenweiler., 2000). an alternative technique for statistical analysis is the confinement tree analysis of the intensity image (mattes., 2001). for different threshold levels, objects (confiners) are segmented in the image. calculated for different levels, besides the estimation of global quantitative values (e.g., the global homogeneity of the motion), this approach allows the analysis and comparison of movements. a challenge for future work is to better understand the biomechanical behavior of cellular structures, e.g., cellular membranes, by fitting a biophysical model to the data an approach already successfully implemented in various fields of medical image analysis (ferrant., 2001). in vivo images of gfp - tagged proteins combined with computational imaging has revealed the dynamic organization of various nuclear subcompartments in the interphase nucleus. live - cell microscopy images of labeled pre - mrna splicing factors were examined for evidence of regulated dynamics by computational segmentation and tracking (eils., 2000). it was shown that the velocity and morphology of speckles, as well as budding events, were related to transcriptional activity. 1997) undergoes slow diffusional motion, and that this movement is confined to relatively small regions in the nucleus. importantly, the constraint on diffusional motion is regulated throughout the cell cycle (heun.. a long - standing question has been whether nuclear compartments can also undergo directed, energy - dependent movements, thereby providing a potential mechanism of regulated gene expression. computational imaging revealed that several nuclear subcompartments do undergo directional transport dependent on metabolic energy (calapez., 2002 ; muratani., 2002 ; platani., 2002). the role of dynamic tension in actin polymerization in motile cells was investigated by analyzing polarized light images of the flow of the actin network and the motion of actin bundles and filopodia in crawling neurons (oldenbourg., 2000). in a study on nuclear envelope breakdown, quantification and visualization of four - channel images with labeled chromatin, lamin - b receptor, nucleoporin, and tubulin (beaudouin., 2002) revealed that piercing of the nuclear envelope by spindle microtubules was the mechanism responsible for forming the initial hole during nuclear envelope breakdown. to further investigate stresses on the nuclear envelope during breakdown stresses detected during hole formation were compared for the different grid vertices with respect to the positions of the hole, thus providing information about localized stresses during the tearing process (mattes., 2001). conversely, the effect of stress on the morphology of cells has been measured using an experimental approach of imposing known stresses on cells in solid - state culture. changes in height, width, volume, and surface area of the cell are measured from 3-d confocal microscopy images, helping to understand the mechano - transduction response (guilak, 1995). the positioning of chromosomes during the cell cycle was investigated in live mammalian cells with a combined experimental and computational approach. in contrast to the random behavior predicted by a computer model of chromosome dynamics, a striking order of chromosomes was observed throughout mitosis (gerlich., 2003). further, strong similarities between daughter and mother cells were found for mitotic single chromosome positioning. these results support the existence of an active mechanism that transmits chromosomal positions from one cell generation to the next. computational imaging has been proven to be a powerful and integral part of cell biology. computational imaging provides an important building block for the description of biological phenomena on a quantitative level, which is a prerequisite for mathematical models of dynamic structures and processes in the cell. in combination with models of biochemical processes and regulatory networks, computational imaging as part of the emerging field of systems biology (kitano, 2002) will lead to the identification of novel principles of cellular regulation derived from the huge amount of experimental data that are currently generated. | microscopy of cells has changed dramatically since its early days in the mid - seventeenth century. image analysis has concurrently evolved from measurements of hand drawings and still photographs to computational methods that (semi-) automatically quantify objects, distances, concentrations, and velocities of cells and subcellular structures. today 's imaging technologies generate a wealth of data that requires visualization and multi - dimensional and quantitative image analysis as prerequisites to turning qualitative data into quantitative values. such quantitative data provide the basis for mathematical modeling of protein kinetics and biochemical signaling networks that, in turn, open the way toward a quantitative view of cell biology. here, we will review technologies for analyzing and reconstructing dynamic structures and processes in the living cell. we will present live - cell studies that would have been impossible without computational imaging. these applications illustrate the potential of computational imaging to enhance our knowledge of the dynamics of cellular structures and processes. |
it has been estimated that 16.8% of live births across the world in 2013 were in women who had some form of hyperglycemia in pregnancy. in india, gestational diabetes mellitus (gdm) has been estimated to affect over 5 million women. gdm poses serious health consequences for mother and the baby both in the short and in the long - term. in addition, this risk starts even at maternal glucose levels below those traditionally considered as diagnostic of gdm. however, treatment of maternal hyperglycemia has been shown to reduce this risk almost to the level seen in women without gdm. although most women with gdm usually return to the normoglycemic state shortly after childbirth, they still have 7 times higher risk of developing type 2 diabetes (t2 dm) in future. accurate and timely diagnosis of gdm, therefore, provides a window of opportunity for intervention to reduce the growing burden of t2 dm. prioritizing postpartum care and continued follow - up will help to prevent / delay the onset of t2 dm. this is particularly relevant in india, which already suffers from a massive burden of t2 dm. though the importance of recognizing and managing gdm is now well - accepted, there are no universally accepted criteria for the screening and diagnosis of this condition. despite several guidelines laid down by various organizations, some parts of the world follow risk - based screening whereas others follow universal screening. there is also no consensus on the diagnostic test to be used and the glucose cut - offs to be applied. use of different criteria makes the accurate estimation of prevalence of gdm difficult and raises the possibility of over- and under - diagnosis of the condition. in addition, in india, care of women with gdm is carried out by a variety of healthcare professionals (hcp). they face unique challenges in their interactions with pregnant women, leading them to favor one diagnostic approach over the other, even though such decisions may not be based on sound scientific evidence. we, therefore, attempted to understand the perceptions and practices of two important categories of hcps involved in the care of gdm in india (physicians / diabetologists / endocrinologists and obstetricians / gynecologists [ob / gyns ]), regarding diagnosis, management, and follow - up of gdm. the results from this study will help to gauge the different screening methods and diagnostic criteria employed in india for the diagnosis and management of gdm, and thus understand the gaps and highlight target areas for improvement with regard to provision of care for women with gdm. the present study aims to obtain information on existing practices for the diagnosis and management of gdm among physicians / diabetologists / endocrinologists and ob / gyns from different parts of india. the present study aims to obtain information on existing practices for the diagnosis and management of gdm among physicians / diabetologists / endocrinologists and ob / gyns from different parts of india. a nationwide survey on the practice patterns with respect to diagnosis and management of gdm was carried out covering physicians / diabetologists / endocrinologists and ob / gyns from 24 states of india. data collection involved two methods : self - completed questionnaires and an online web - based software. questionnaires were handed over directly to the doctors to be filled on the spot and returned. the online questionnaire was filled through customized web - based software called wings survey created by the madras diabetes research foundation. the frontend software was developed using microsoft asp.net, and the data collected was stored in the microsoft sql server 2000 database. the questionnaire addressed different screening techniques employed ; gdm diagnostic guidelines and diagnostic cut - offs based on blood glucose levels ; management and follow - up, pharmacotherapy and postpartum follow - up. several of the questions allowed multiple responses leading to the reported percentage adding up to more than 100%. a total of 3841 doctors (2020 physicians / diabetologists / endocrinologists and 1821 ob / gyns) participated in the survey. the survey covered 24 states of india including the national capital territory of delhi and two union territories, chandigarh, and puducherry. figure 1 shows the state wise percentage distribution of physicians / diabetologists / endocrinologists and ob / gyns, who participated in the survey. percentage of respondents in different states of india more than half of the doctors who participated in the survey practiced in private clinics and hospitals whereas the rest worked in multispecialty hospitals and government hospitals [table 1 ]. type of institution where the doctors practised the vast majority of the ob / gyns (84.9%) screened all pregnant women for gdm, i.e., universal screening while 14.5% preferred to do only risk - based screening. the rest (0.6%) reported that they do not screen for gdm in pregnant women [figure 2 ]. universal versus risk based screening most of the ob / gyns performed screening for gdm in the first trimester (18.8% at booking and 49% between 8 and 20 weeks). the screening was performed between 20 and 28 weeks by 40% and after 28 weeks by 2.8%. this question allowed multiple responses, and hence, the percentages reported are more than 100. among the 1634 (89.7%) ob / gyns who responded to this question, 600 (36.7%) reported using the diabetes in pregnancy study group india (dipsi) criteria, 403 (24.7%), the world health organization (who) 1999 criteria, 389 (23.8%), the international association for diabetes and pregnancy study groups (iadpsg) criteria, and 242 (14.8%), the american diabetes association (ada) 2-step method (50 g glucose challenge test followed by 100 g 3 h glucose tolerance test with the cut - offs proposed by carpenter and coustan or the national diabetes data group). among the physicians / diabetologists / endocrinologists, 1903 (94.2%) responded to this question, out of whom 560 (29.4%) reported using the dipsi criteria, 428 (22.5%) the who 1999 criteria, 364 (19.1%), the iadpsg criteria, and 551 (29%) the ada criteria. however, as shown in table 2, responses to subsequent questions on type of blood sample collected, the glucose load used, and the cut - offs as per the criteria revealed that 54.9% ob / gyns and 54.7% diabetologists / endocrinologists did not correctly follow any of the criteria. screening criteria for gestational diabetes mellitus followed by the obstetricians / gynecologists and physicians / diabetologists / endocrinologists when women required pharmacological treatment to manage their gdm, 1019 (50.4%) of diabetologists / endocrinologists said they used insulin for all women with gdm, 758 (37.5%) said they used insulin only for some, and 243 (12.1%) reported using no insulin at all. among those who preferred using oral hypoglycemic agents, metformin was used by 1087 (53.8%) of diabetologists / endocrinologists, sulfonylureas by 72 (3.6%), and a combination of the two by 247 (12.2%) whereas 591 (29.3%) reported no use of oral hypoglycemic agents (ohas) and 23 (1.1%) reported using other ohas such as alpha - glucose inhibitors. the majority of ob / gyns (78.3%) routinely delivered women with gdm before 38 weeks gestation whereas 10.9% waited beyond 38 weeks and 10.8% up to 40 weeks. fifty - six percent of physicians / diabetologists / endocrinologists and 71.6% ob / gyns said they advised women with gdm to undergo oral glucose tolerance testing (ogtt) after delivery. ogtt was advised within 6 weeks of delivery by 42.4% of diabetologists / endocrinologists and 44.2% of ob / gyns, and between 6 and 2 weeks after delivery by 48% of diabetologists / endocrinologists and 49.4% of ob / gyns. more than half of the doctors who participated in the survey practiced in private clinics and hospitals whereas the rest worked in multispecialty hospitals and government hospitals [table 1 ]. the vast majority of the ob / gyns (84.9%) screened all pregnant women for gdm, i.e., universal screening while 14.5% preferred to do only risk - based screening. the rest (0.6%) reported that they do not screen for gdm in pregnant women [figure 2 ]. most of the ob / gyns performed screening for gdm in the first trimester (18.8% at booking and 49% between 8 and 20 weeks). the screening was performed between 20 and 28 weeks by 40% and after 28 weeks by 2.8%. this question allowed multiple responses, and hence, the percentages reported are more than 100. among the 1634 (89.7%) ob / gyns who responded to this question, 600 (36.7%) reported using the diabetes in pregnancy study group india (dipsi) criteria, 403 (24.7%), the world health organization (who) 1999 criteria, 389 (23.8%), the international association for diabetes and pregnancy study groups (iadpsg) criteria, and 242 (14.8%), the american diabetes association (ada) 2-step method (50 g glucose challenge test followed by 100 g 3 h glucose tolerance test with the cut - offs proposed by carpenter and coustan or the national diabetes data group). among the physicians / diabetologists / endocrinologists, 1903 (94.2%) responded to this question, out of whom 560 (29.4%) reported using the dipsi criteria, 428 (22.5%) the who 1999 criteria, 364 (19.1%), the iadpsg criteria, and 551 (29%) the ada criteria. however, as shown in table 2, responses to subsequent questions on type of blood sample collected, the glucose load used, and the cut - offs as per the criteria revealed that 54.9% ob / gyns and 54.7% diabetologists / endocrinologists did not correctly follow any of the criteria. screening criteria for gestational diabetes mellitus followed by the obstetricians / gynecologists and physicians / diabetologists / endocrinologists when women required pharmacological treatment to manage their gdm, 1019 (50.4%) of diabetologists / endocrinologists said they used insulin for all women with gdm, 758 (37.5%) said they used insulin only for some, and 243 (12.1%) reported using no insulin at all. among those who preferred using oral hypoglycemic agents, metformin was used by 1087 (53.8%) of diabetologists / endocrinologists, sulfonylureas by 72 (3.6%), and a combination of the two by 247 (12.2%) whereas 591 (29.3%) reported no use of oral hypoglycemic agents (ohas) and 23 (1.1%) reported using other ohas such as alpha - glucose inhibitors. the majority of ob / gyns (78.3%) routinely delivered women with gdm before 38 weeks gestation whereas 10.9% waited beyond 38 weeks and 10.8% up to 40 weeks. fifty - six percent of physicians / diabetologists / endocrinologists and 71.6% ob / gyns said they advised women with gdm to undergo oral glucose tolerance testing (ogtt) after delivery. ogtt was advised within 6 weeks of delivery by 42.4% of diabetologists / endocrinologists and 44.2% of ob / gyns, and between 6 and 2 weeks after delivery by 48% of diabetologists / endocrinologists and 49.4% of ob / gyns. it is evident from our survey that the majority of doctors in india prefer universal screening for gestational diabetes. this is consistent with findings from an online survey conducted by divakar and manyonda in 2011 among physicians in india and is in line with the dipsi guidelines, which favor universal screening as well as with many of the international guidelines, which recommend that all women of high - risk ethnicity be screened for gdm. as the prevalence of gdm is almost 11-fold higher in indian women when compared to their caucasian counterparts, universal screening is an essential tool in india to ensure that no case of gdm or preexisting diabetes is missed out. it is heartening to note that the majority of ob / gyns screen their antenatal patients for diabetes in the first trimester itself. delaying screening till the second trimester carries the risk of missing preexisting (pregestational) diabetes, especially in a population such as india, where the background prevalence of t2 dm is high. it would, however, be ideal if all pregnant women (as opposed to 18.8% in the present survey) were to be screened at the first booking visit itself as recommended by the guidelines. the multiplicity of available criteria for the diagnosis of gdm, and frequent changes in recommendations made by international organizations, have led to confusion among hcps as to the best screening test to be used for gdm. this is reflected in the results of our survey where a variety of guidelines were reported to be used by ob / gyns and physicians / diabetologists / endocrinologists for diagnosing gdm. what is even more worrisome is that the majority of hcps applied these criteria incorrectly, raising the possibility of over- and under - diagnosis of gdm in india. for example, 36.7% ob / gyns and 29.4% physicians / diabetologists / endocrinologists said they used the dipsi criteria, but in reality, only 12.7% and 3.8%, respectively, used the dipsi criteria. our results highlight the need for creating greater awareness among hcps regarding the currently accepted guidelines for the diagnosis of gdm so that the majority of women with gdm are accurately identified and appropriately managed. insulin is the first - line pharmacologic therapy for gdm ; however, there is some evidence that ohas such as metformin are safe in pregnancy. decision - making with respect to the initiation of insulin is often driven by patient preference. data from our survey shows that more than half of the physicians / diabetologists / endocrinologists preferred to use insulin for women with gdm. when oha were preferred, 53.8% reported the use of metformin, 3.6% used sulfonylurea alone, and 12.2% used both. it is a matter of some concern that 1.1% of all physicians / diabetologists / endocrinologists reported using other ohas during pregnancy (none of which have been approved for use in this setting). there is little consensus regarding the preferred method and timing of delivery in women with gestational diabetes, which is usually based on expert opinion. in the absence of any maternal or fetal complications, ob / gyns routinely deliver at 40 weeks of gestation. however, our survey reports that majority of the ob / gyns deliver their patients before 38 weeks, presumably to minimize the risk of sudden intra uterine death during the 3 trimester, which has been shown to occur despite intensive fetal surveillance. they are also at risk of development of gdm at an earlier stage during subsequent pregnancies. the ada recommends that women with gdm should undergo screening for t2 dm with ogtt, 6 weeks after delivery. although guidelines for postpartum care are established, data from our survey reports that not all diabetologists / endocrinologists and ob / gyns advise postpartum ogtt. a possible explanation could be that many physicians do not prioritize these guidelines in practice or may view another health practitioner as responsible for the follow - up. omitting this important piece of advice will lead to the majority of women with gdm remaining unaware of their glycemic status postdelivery, and thereby running the risk of entering another pregnancy with undiagnosed diabetes, as well as accumulating prolonged duration of unrecognized, uncontrolled hyperglycemia. both physicians / diabetologists / endocrinologists as well as ob / gyns have an enormous responsibility for providing proper care and management for women with gdm. this large survey conducted all over india, covering 24 states reveals that more than half of the diabetologists / endocrinologists and ob / gyns in india do not follow any of the recommended guidelines for the diagnosis of gdm possibly due to lack of awareness about these guidelines. this emphasizes the need for increased awareness about screening and diagnosis of gdm both among physicians and ob / gyns. proper educational intervention to address these gaps will help doctors to understand their role in promoting better pregnancy outcomes. | aim : to obtain information on existing practices in the diagnosis and management of gestational diabetes mellitus (gdm) among physicians / diabetologists / endocrinologists and obstetricians / gynecologists (ob / gyns) in india.methods:details regarding diagnostic criteria used, screening methods, management strategies, and the postpartum follow - up of gdm were obtained from physicians / diabetologists / endocrinologists and ob / gyns across 24 states of india using online / in - person surveys using a structured questionnaire.results:a total of 3841 doctors participated in the survey of whom 68.6% worked in private clinics. majority of ob / gyns (84.9%) preferred universal screening for gdm, and screening in the first trimester was performed by 67% of them. among the ob / gyns, 600 (36.7%) reported using the nonfasting 2 h criteria for diagnosing gdm whereas 560 (29.4%) of the diabetologists / endocrinologists reported using the same. however, further questioning on the type of blood sample collected and the glucose load used revealed that, in reality, only 208 (12.7%) and 72 (3.8%), respectively, used these criteria properly. the survey also revealed that the international association of diabetes and pregnancy study groups criteria was followed properly by 299 (18.3%) of ob / gyns and 376 (19.7%) of physicians / diabetologists / endocrinologists. postpartum oral glucose tolerance testing was advised by 56% of diabetologists and 71.6% of ob / gyns.conclusion : more than half of the physicians / diabetologists / endocrinologists and ob / gyns in india do not follow any of the recommended guidelines for the diagnosis of gdm. this emphasizes the need for increased awareness about screening and diagnosis of gdm both among physicians / diabetologists / endocrinologists and ob / gyns in india. |
in oral cavity, mechanical stress was generated in many situations, such as mastication, functional / parafunctional habits, orthodontic treatment, and occlusal trauma. occlusal force plays a pivotal role in the regulation of periodontium homeostasis [13 ]. the mechanical force in the range of physiological condition was involved in the maintaining of the periodontium system. however, the force exceeding physiological limitation could lead to pathological change, such as periodontal ligament (pdl) space widening, periodontium destruction, and alveolar bone resorption [5, 6 ]. several lines of evidence demonstrated the effect of mechanical stress on cellular response, including periodontal ligament cells (pdls) [4, 7 ]. it has been illustrated both in vitro and in vivo that mechanical stress influenced pdl behavior. several techniques were employed to investigate the effect of mechanical stress in vitro, for example, shear stress, cyclic tensile stress [9, 10 ], and static compressive stress. the previous data showed that pdl responded to mechanical stress by releasing atp [12, 13 ], increasing intracellular calcium [11, 14 ], changing actin filament organization, and upregulating of several cytokines or growth factor, including insulin - like growth factor-1 (igf-1) [1618 ]. igf-1 plays a role in various cellular activities, including survival, proliferation, and differentiation [2026 ]. igf-1 is involved in several kinds of cells and tissues while igf-2 plays an important role mainly during prenatal development. it has been illustrated that human pdl expressed the igf1 receptor, implying the ability to igf-1 stimulation. previous report showed that igf-1 enhanced human periodontal ligament cells (hpdls) survival by inducing antiapoptotic molecules and downregulating proapoptotic molecules. it was noted that the application of orthodontic force on rat teeth resulted in the upregulated igf-1 release in pdl in vivo [16, 18, 28 ], though the molecular mechanism, by which mechanical stress stimulates igf-1 expression, is yet unclear. therefore, the present study aimed to investigate molecular signaling mechanism of intermittent mechanical stress on the igf-1 expression in human pdls. furthermore, the influence of hypoxia on the intermittent mechanical stress regulated igf-1 expression was examined. cell culture medium was purchased from gibco brl (brl, carlsbad, ca, usa). culture dishes and plastic tubes were purchased from corning (corning, ny, usa). cyclohexylamine, genistein, monensin, tgf- receptor i inhibitor (sb431542), and recombinant human tgf-1 (rhtgf-1) were purchased from sigma - aldrich (st. louis, mo, usa). p38 mapk inhibitor (sb203580) was purchased from calbiochem (merck chemicals, gibbstown, nj, usa). the tgf-1 antibody was purchased from r&d systems inc. all protocol was approved by the ethics committee of the faculty of dentistry, chulalongkorn university. third molars and premolars extracted for orthodontic reasons at the faculty of dentistry, chulalongkorn university, were collected for cell isolation. the periodontal tissue was obtained from middle third of teeth 's root and the tissue was cultured in standard medium (dulbecco 's modified eagle 's medium (dmem) containing 10% fetal bovine serum with 1% l - glutamine and 1% ab / am) and incubated at 37c in a humidified atmosphere of 5% co2 in air. after the cells migrated from the tissue and became confluent, they were detached with 0.25% trypsin - edta and subcultured at a 1 : 3 ratio. in each experiment, cells from at least 3 donors were used. cells were incubated with cocl2 at 150 or 300 m for 30 min prior to applying intermittent mechanical stress. a cell compressive force loading apparatus (thai patent i d : 1401006767) was designed and constructed to mechanically stimulate cells in a culture plate. cells were seeded in 6-well culture plates at a density of 3 10 cells per well overnight. the cells were starved with serum - free culture medium for 4 h before loading force. compressive force generator v2.5 software was used to set times, loading type, and the amount of force. in brief, the loading cycle was set to press for 1 s and to unpress for 2 s to yield a loading cycle approximately 1/3 hertz and the force amount 1.5 g / cm. in some experiments, sb203580 (3.5 m), cocl2 (150 m), cyclohexylamine (10 m), genistein (92.5 m), monensin (100 m), sb431542 (10 m), rhtgf-1 (2 ng / ml), or tgf-1 antibody (5 g / ml) was added in the culture condition. hpdls were seeded in 6-well plates at a density of 3 10 cells per well for applying the force and 24-well plates at density of 5 10 cells per well for being treated with cocl2. subsequently cells were starved with serum - free media 4 h before treatment. at 24 h, hpdls were incubated with 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide solution for 30 min. formazan crystals were solubilized in dmso / glycine buffer solution (0.1 m glycine/0.1 m sodium chloride ph10). the solution was further measured for an absorbance at 570 nm in a microplate reader (elx800, biotek, usa). after 24 h, total cellular rna was extracted with trizol reagent (molecular research center, cincinnati, ohio, usa) according to the manufacturer 's instructions. rna was quantified using a nanodrop 2000 spectrophotometer (thermo scientific, wilmington, de, usa). one microgram of rna sample was converted to cdna by improm - ii (promega, madison, wi, usa). subsequently, the real - time pcr reaction was using a lightcycler instrument (roche diagnostics, usa) with the lightcycler 480sybr green i master kit according to the manufacturer 's specifications. the oligonucleotide sequences were as follows : igf-1 (nm000618.3), forward 5-catgcctgctcagaagggta-3, reverse 5-gcctctgatccttgaggtga-3 ; 18s (nr003286.2), forward 5-ggcgtcccccaacttctta-3, reverse 5-gggcatcacagacctgttatt-3. radioimmunoprecipitation assay (ripa) supplemented with protease inhibitors was used to extract cellular protein. the amount of protein was measured by a bca protein assay kit (pierce, rockford, il). whole cell lysate and condition medium were collected at 80c for measuring the level of protein. elisa was used for measuring the protein level according to the manuals of elisa kits (quantikine immunoassay r&d systems). the absorbance of elisa reaction product was measured at od 450 nm using microplate reader (biotek, elx800, usa). statistical analyses were performed for two independent samples using the student t - test for two - group comparisons. a one - way analysis of variance (anova) followed by turkey 's post hoc analysis (spss, chicago, il, usa) was employed for three or more group comparisons. the p value less than 0.05 was considered as statistically significant. we began by investigating the effect of intermittent mechanical stress on hpdls viability and morphology using a microscope at 100x magnification. hpdls morphology was similar in all groups (see supplementary figure 1c in supplementary material available online at http://dx.doi.org/10.1155/2015/369874) and mechanical stress did not affect the hpdls viability (supplementary figures 1a and 1b). next, we investigated the effect of intermittent mechanical stress on igf-1 expression in hpdls at different time points (figure 1). there was no significant difference in igf-1 expression at 2 h, 4 h, or 8 h between the intermittent mechanical stress - treated group and the control group. however, the igf-1 mrna levels were significantly increased at 24 h after exposing to mechanical stress. thus, these results demonstrated intermittent mechanical stress - induced igf-1 expression in hpdls at 24 h. we started to pretreat hpdls with sb203580 which is p38 mapk inhibitor prior to applying the force. our results demonstrated that p38 mapk inhibitor failed to block intermittent mechanical stress - induced igf-1 expression in hpdls (supplementary figure 2). the results showed that cycloheximide pretreatment inhibited the intermittent compressive force - induced igf-1 mrna expression. further, the mechanical force - induced igf-1 expression was also inhibited by the monensin, a protein transport inhibitor (figure 2(b)). these results imply that the intermittent mechanical stress required the release of intermediate protein to induce igf-1 expression. the intracellular mechanism was further identified using genistein, a tyrosine kinase inhibitor (figure 2(c)). corresponding to the effect of cycloheximide and monensin, genistein abolished the intermittent mechanical stress - induced transcription of igf-1. taken together, we concluded that intermittent mechanical stress required intermediate protein related to tyrosine kinase to induce igf-1 expression in hpdls. as described above, the genistein inhibition blocked the intermittent mechanical stress - induced igf-1 expression. thus, sb431542 (tgf- receptor type i (tri) inhibitor) was chosen to clarify mechanism (figure 3(a)). the result demonstrated that sb431542 completely suppressed intermittent mechanical stress - induced igf-1 mrna expression. to confirm the tgf-1 role in this phenomenon, the neutralizing antibody against tgf-1 correspondingly with sb431542 treatment, the neutralizing antibody against tgf-1 reduced the igf-1 transcription under intermittent mechanical stress stimulation (figure 3(b)). finally, the addition of exogenous rhtgf-1 resulted in the upregulation of igf-1 mrna levels at 24 h (figure 3(c)) however, to determine intermittent mechanical stress - induced igf-1 expression through tgf-1 protein secretion, we collected the cell culture medium from intermittent mechanical stress - treated group (cms) as well as the control group (cmc) and transferred it to another set of unstimulated hpdls for 24 h. surprisingly, igf-1 expression in those cells incubated with cms - treated group and cmc - treated group did not differ (figure 3(d)). thus, we further measured the protein levels of tgf-1 in both condition mediums and found that tgf-1 protein levels in cms did not differ from cmc (data not shown). however, the whole cell lysate from intermittent mechanical stress - treated group expressed significantly higher tgf-1 protein levels than the control group (figure 3(e)). such evidence may imply intermittent mechanical stress - induced tgf-1 protein to activate igf-1 expression in hpdls. the results showed that cocl2 did not significantly affect igf-1 expression in normal culture (figure 4(a)). however, cocl2 significantly inhibited igf-1 expression upon the intermittent stress treatment in a cocl2 dose - dependent manner (figure 4(a)). therefore, we hypothesized that cocl2 affect the intracellular signaling of tgf-1 in order to induce igf-1 expression in hpdls. however, in pathological condition (i.e., periodontal disease), the physiological force may lead to tissue destruction [30, 31 ]. igf-1 is an important growth factor regulating cell proliferation and differentiation in hpdls [23, 24 ]. further, the hypoxic mimic condition using cocl2 could attenuate the intermittent compressive stress - induced igf-1 expression, implying that occlusal force may not induce igf-1 expression in deep periodontal pocket, where it was considered as hypoxic microenvironment. the suggested model of intermittent mechanical stress - induced igf-1 expression in hpdls igf-1 plays an important role in bone growth and development [32, 33 ] and promotes cell proliferation and osteogenic differentiation in hpdls [20, 24 ]. in addition, the in vitro study demonstrated that igf-1 is a growth factor that responds early to mechanical stress. in the in vivo orthodontic tooth movement model, the orthodontic force or occlusal stimuli thus, the present study was the first report which demonstrated that the intermittent mechanical stress promoted igf-1 expression by hpdls through tgf-1 pathway. the present study showed that the intermittent compressive stress enhanced the increase of tgf-1 protein expression in cell lysate and the addition of rhtgf-1 resulted in the upregulation of igf-1 expression similar to those treated with the intermittent mechanical stress. correspondingly, it was previously demonstrated that the tgf-1 treatment significantly increased igf-1 expression in dose- and time - dependent manner in human marrow stromal osteoblast precursor cells. it was also shown that single - dose administration of tgf-1 promoted the osteogenic maker expression via the expression of igf-1 since the knockdown of insulin receptor substrate 1 could attenuate the tgf-1-induced osteogenic marker expression. however, it should be noted that the repeat - dose of tgf-1 led to the inhibition of igf-1 expression and subsequently caused the suppression of osteogenic differentiation in hpdls, human mesenchymal stem cells, and murine preosteoblast (mc3t3-e1 cells). moreover, tgf- inhibited migration in c2cl2 skeletal muscle satellite cell and p19 embryonal carcinoma cell via decreasing igf-1. collectively, several lines of evidence indicated the close relationship between tgf-1 and igf-1 in a positive or negative regulator depending on cell types. the influence of hypoxia can be found in inflamed tissue including periodontitis. the hypoxic condition is associated with imbalance between elevating the oxygen demand from inflammatory cells penetration and inadequate oxygen supply by poor perfusion [26, 37, 38 ]. in periodontitis, the hpdls respond to hypoxia by increasing the inflammatory mediator [39, 40 ] and enhancement of alveolar bone loss [41, 42 ]. therefore, both intermittent mechanical stress and hypoxia are contributing factors to periodontal disease progression, leading us to investigate the effect of combining those two factors on hpdls. in this study, artificial hypoxic agent, cocl2, abolished the intermittent mechanical stress - induced igf-1 expression in hpdls. this condition represents the clinical situation, where the physiological force was loaded on periodontitis ' teeth. therefore, this data assumed that hypoxia attenuated the intermittent mechanical stress - induced osteogenic differentiation through decrease in igf-1 expression in hpdls. recently, it was demonstrated that the cyclic tensile stress under hypoxic condition regulated proliferation and osteogenic differentiation in hpdls via mapk pathway. thus, this information could imply that the type, amount, and direction of force may play an important role in the hpdls ' response under hypoxic condition. cocl2, an inducer of hypoxia, is well known and commonly employed to establish the physical hypoxic - like condition in cell culture [4346 ]. our previous study reported that cocl2 could stabilize hypoxia - inducible factor-1 alpha (hif-1), a key transcription factor for hypoxic condition in hpdls. in contrast to some lines of evidence, there were some aspects of detailed different mechanisms between hypoxia and cocl2 [51, 52 ]. thus, the results from the present study may not be directly implied to those of physical hypoxia setting. further experiment is required to fully investigate the role of hypoxia on the intermittent compressive stress - induced igf-1 expression in hpdls. the present study showed that hypoxic mimic condition attenuated the intermittent mechanical stress - induced igf-1 expression in hpdls. corresponding to previous studies, physical hypoxia and cocl2 attenuated igf-1 expression via the suppression of runx2 and the induction of c / ebp in rat osteoblasts. runx2 could bind to the upstream element in igf-1 gene promoter and regulated igf-1 expression. further, in systemic investigation, the serum igf levels were decreased in acute respiratory distress patients, which were a hypoxia state. on the contrary further, in the present study, we demonstrated that cocl2 inhibited rhtgf-1-induced igf-1 expression in hpdls. taken together, further study to evaluate the mechanism of hypoxic mimic condition on the inhibition of tgf-1-induced igf-1 expression in hpdls is necessitated. in conclusion, our results indicated the intermittent mechanical stress - induced igf-1 expression via tgf-1 signaling pathway in hpdls. | mechanical force was shown to promote igf-1 expression in periodontal ligament both in vitro and in vivo. though the mechanism of this effect has not yet been proved, here we investigated the molecular mechanism of intermittent mechanical stress on igf-1 expression. in addition, the role of hypoxia on the intermittent compressive stress on igf-1 expression was also examined. in this study, human periodontal ligament cells (hpdls) were stimulated with intermittent mechanical stress for 24 hours. igf-1 expression was examined by real - time polymerase chain reaction. chemical inhibitors were used to determine molecular mechanisms of these effects. for hypoxic mimic condition, the cocl2 supplementation was employed. the results showed that intermittent mechanical stress dramatically increased igf-1 expression at 24 h. the pretreatment with tgf- receptor i or tgf-1 antibody could inhibit the intermittent mechanical stress - induced igf-1 expression. moreover, the upregulation of tgf-1 proteins was detected in intermittent mechanical stress treated group. correspondingly, the igf-1 expression was upregulated upon being treated with recombinant human tgf-1. further, the hypoxic mimic condition attenuated the intermittent mechanical stress and rhtgf-1-induced igf-1 expression. in summary, this study suggests intermittent mechanical stress - induced igf-1 expression in hpdls through tgf-1 and this phenomenon could be inhibited in hypoxic mimic condition. |
in the mammalian eye, the image - forming pathway transmits highly sensitive color, temporal, and spatial light - dependent information from rod and cone photoreceptors through an intermediate retinal network of bipolar, amacrine, and horizontal cells which convey light - dependent responses via conventional retinal ganglion cells (rcgs) to the lateral geniculate nucleus (lgn). this pathway contributes to conscious vision. the second pathway, the non image - forming pathway, is an ancestral pathway that enabled species (including photosensitive microorganisms and invertebrates1) to sense light and irradiance levels as a key means for organism response and survival. once image - forming vision evolved, many scientists believed that it replaced the ancestral visual system. however, mammals, including people and rodents, that had lost their image - forming visual system (rod and cone photoreceptor blind) still synchronized their circadian rhythm with the light cycle and exhibited a pupillary light reflex, raising the question of a third photoreceptor in the eye.25 when melanopsin was shown to be an active photopigment expressed in a small subclass of rgcs, the search for this third photoreceptor ended and a new area of study opened up for enlightenment.610 melanopsin containing intrinsically photosensitive retinal ganglion cells (iprgcs) are unusual photoreceptors. they receive photic information from rods and cones via bipolar cells but contribute an additional photic dimension via melanopsin. five classes, m1 through m5, of iprgcs that differ by morphology, dendritic localization, melanopsin content, electrophysiological profiles, and projections, have been characterized to date in rodents, although only m1 and m2 classes have been identified in non - human primates.1117 some distinguishing features of iprgcs include their use of a gq/11 and plc / ip3 signaling pathway in contrast to the transducin g- protein and cyclic gmp pathway used by rod and cone opsins,1722 and their ability to show sustained firing under saturating and continuous light exposure.13,2325 this non image - forming pathway is classically thought to inform unconscious vision that photoentrains the circadian cycle, controls the pupil light reflex, and regulates activity levels (masking) and sleep, which has been extensively reviewed elsewhere.1,17,2630 this review will focus on recently illuminated functions and applications of this pathway, some of which blur the lines between conscious and subconscious vision, and the retinal neurons that act as gateways to this system, the iprgc. recent experiments have blurred the distinction between image - forming and non image - forming visual pathways. the ability of rodless, coneless mice to perform pattern discrimination suggested that iprgcs contribute to image - forming vision.31 this was substantiated by anatomical findings in melanopsin reporter mouse lines that the m3/m4/m5 subclasses of iprgcs project directly to the lgn,31 a region dedicated to image - forming vision. a direct lgn projection encoding color and irradiance had previously been observed in non - human primates16 but not in other species. subsequent electrophysiological evidence estimated that 40% of lgn - cortical cells receive melanopsin - specific signals in rodents.32 closer examination identified the mouse m4 iprgcs, which express low levels of melanopsin and were not previously well characterized, as a major input to the lgn.15 these m4 cells were further identified as the well - known alpha on ganglion cells capable of contrast detection.15 tiger salamander retinae also have on ganglion cells that are intrinsically photosensitive, indicating evolutionary conservation.33 their importance in contrast detection was demonstrated when mice lacking melanopsin showed decreased contrast sensitivity which was further decreased in mice lacking the m2-m5 classes of iprgcs.34 these recent findings, that one of the iprgc subclasses previously considered to contribute only to the non image - forming visual pathway, is actually a well - known component of the image - forming pathway and truly highlights that surprising and revolutionary knowledge is still being discovered. the melanopsin phototransduction system is evolutionarily older than the rod and cone systems. given its ancient origin and key role in light - dependent survival, it is not surprising that melanopsin is expressed prenatally and that iprgcs are the first photoresponsive cells of the mammalian retina3540 and are capable of detecting light embryonically.41 more iprgcs are initially generated than survive and programmed apoptosis sculpts the mature rodent retina to form a photoreceptive net which samples the entire retina with very little overlap.35,39,4245 the development of the mammalian retina proceeds such that rod and cone photoreceptors integrate with pre - established iprgcs while forming their separate image - forming pathway. disruption of bax - mediated apoptosis in a mutant mouse line results in clusters of iprgcs that are capable of photoentraining circadian rhythm via melanopsin but incapable of mediating photoentrainment using rod and cone signaling.43 iprgcs also play a key role in the light - dependent modeling of the visual system. during development, waves of coordinated activity sweep across the retina to direct wiring of the retina to central targets. melanopsin - dependent photoreception increases the duration of these activity bursts ; mice lacking melanopsin have decreased segregation of ipsi- and contralateral lgn projections.46 the consequences of this small decrease (approximately 5%) in crossover remains unclear. for comparison, approximately 90% of retinal fibers project contralaterally (compared to the normal 55%), and is associated with nystagmus, strabismus, and amblyopia in people.47 while iprgcs are the first photoreceptors to develop, they have a longer period of proliferation48 and may be some of the last of the retinal neurons to die during the course of an organism s lifetime. rod and cone photoreceptors are highly susceptible to degeneration, and diseases that affect rods and cones are the leading cause of blindness. rgcs also degenerate in diseases that cause increased pressure including glaucoma and ischemia, vascular disorders like diabetes, neurodegenerative diseases such as alzheimer s disease and parkinson s, as well as optic atrophy among others. in rodent models of many of these conditions and in human glaucoma, iprgcs appear to be resistant4954 but not necessarily immune to degeneration.55,56 this suggests protective factors may be expressed in iprgcs compared to non - melanopsin rgcs. lastly, their long - term survival has made them a candidate for gene therapy. not only are they more easily transfected by virus compared to cells in the outer retina, they are also likely to be healthier in a disease state.57,58 the intrinsic circadian clock calculates an approximate 24 hour light / dark cycle.59 normal fluctuations in day length and phase delays from events like daylight savings require the ability to photoentrain this cycle,60,61 a function of iprgcs.62,63 axon collaterals within the retina allow intraretinal signaling between iprgcs and dopamine - producing amacrine cells. melanopsin is required for feedback control to maintain retinal dopamine levels high at night and low during the day.64 this nighttime high level of dopamine increases gap junction coupling of rod - rod and rod - cone photoreceptors.65,66 coupling of rod photoreceptor both decreases noise and increases the effective receptive field, possibly allowing increased contrast detection to occur under scotopic or mesopic light conditions.65,67 by directly regulating the levels of dopamine, iprgcs influence detection of dim objects at night68,69 but also are able to see.70 melanopsin is also required for enhanced cone photoreceptor responses during mid - day, with photoreceptor coupling to other retinal neurons the proposed mechanism for modulation of visual processing.71 together, these animal studies show how iprgcs contribute to shaping responses of the image - forming pathways. the pupillary light reflex is an important protective mechanism that regulates the amount of light reaching the retina. the circuit begins with light detection via rhodopsin, cone opsin, and melanopsin.9,72,73 iprgcs convey this irradiance information bilaterally,62,63,74 specifically with m1 iprgcs signaling the shell of the olivary pretectal nucleus.48,75 the preganglionic parasympathetic neurons in the edinger - westphal nucleus and the ciliary ganglia complete the circuit controlling the sphincter muscles of the iris.76 the pupillary light reflex consists of an initial fast constriction phase that is dominated by rod and cone photoreceptors, an escape phase characterized by some loss of constriction and a final sustained phase attributed to melanopsin.25,77 consequently, spectral sensitivity of the rod, cone, and iprgc photopigments, as well as the different phases of the pupillary light reflex, can be used to assess the functional state of the three photoreceptors in rodents, dogs, or humans.7881 such differential diagnoses for rod or cone dystrophies and rgc damage are starting to emerge.8190 it is often said that the eye is the window to the brain. this is particularly true with pupil responses that have been leveraged to assess the pupillary light reflex reflex loop, making it an ideal clinical diagnostic tool for concussion, stroke, and numerous neurological conditions.85 alterations in the ipsilateral and contralateral (consensual) responses can indicate sites of damage. furthermore, unilateral stimulation of temporal retinal hemisphere results in greater constriction of the ipsilateral iris compared to consensual response in the contralateral iris.91 the segregation of axons at the optic chiasm and subsequent innervation was suggested as a basis for this difference. an alternative mechanism identified in rodents, dogs, cats, and rabbits (but not in non - human primates) may also contribute to this difference as light - dependent constriction of an isolated iris muscle was recently shown to be melanopsin - dependent based on spectral sensitivity, immunohistochemistry, a melanopsin promoter - drive fluorescent tag, mrna analyses, and loss of the effect in the absence of melanopsin.92 this may be mediated by melanopsin - expressing cells in the iris or by projections from iprgcs that synapse in the iris.93 both of these possibilities are intriguing as they represent identification of a novel cell type that expresses melanopsin or a novel projection of iprgcs, respectively. nocifensive behaviors are protective behaviors associated with noxious stimuli. for bright light stimuli, these behaviors may include blinking, squinting, pupil constriction, and avoiding light. a direct role for melanopsin in light avoidance was shown in mouse pups. at a developmental stage in which only iprgcs are photoreceptive, pups turn away from blue light, a response lacking in melanopsin - deficient mice.94 this response was accompanied by ultrasonic vocalizations, which are used by rodents to communicate threats or danger, and activation of the posterior thalamus (implicated in migraine - related pain95) and the central amygdala (associated with nociception96). a direct role for iprgcs in light aversion was also shown for adult mice. innate light aversion, which is revealed by prior environmental and light exposure, is decreased in mice lacking iprgcs but not rod and cone photoreceptors.97,98 the involvement of iprgcs in clinical models of photoallodynia (ocular or headache pain initiated or modulated by normal light levels) is likely dependent on the etiology of photoallodynia. in a mouse model of dry eye damage caused by corneal application of a common preservative in ophthalmic solutions, light aversion is dependent on iprgcs.99 by contrast, iprgcs are not required in a mouse model of nitroglycerin- induced migraine.99 other studies, however, have identified visually blind (lacking rod and cone photoreceptors) people that still experience photoallodynia, strongly suggesting that iprgcs mediate this function in both migraine and non - migraine conditions.95,100 furthermore, supporting evidence from rodents shows that light directly or indirectly modulates migraine - related chemo- and mechano - sensitive dura neurons, which terminate in close apposition to iprgc projections in a thalamic region mediating pain.95 light has also been shown to increase activation in the trigeminal nucleus devoted to pain perception,101,102 although the photoreceptors required for this are unknown. the identification of a direct retinal - thalamic pulvinar tract may provide an anatomical basis for photoallodynia in people,103 however an interesting twist came with recent findings showing that c1c3 cervical nerves are also capable of evoking periorbital pain in migraine patients.104 together, these data highlight how little is known about the connection between light and pain. the relationship between light and anxiety in rodents is well established.105 open and brightly lit spaces are typically characterized as dangerous environments, making a light / dark box exploration assay a good measure for anxiety and anxiolytic drugs.106,107 avoidance of light in a light / dark box is melanopsin-108,109 and iprgc - dependent98 however anxiety from novel environments increases the level of light aversion,98,110 indicating that iprgcs mediate both innate and anxiety - induced light aversion. the aversive capacity of light is also observed in mice in pavlovian, associative conditioning to a noxious stimulus.111,112 normal and melanopsin - deficient mice showed enhanced learning in pavlovian fear conditioning, while mice lacking rod and cone photoreceptors did not. this effect was not accompanied by increased anxiety,112 suggesting an additional role for light in memory modulation (also, see below). together, this suggests a complex role for light in anxiety and enhanced responses to fear memory. a biological basis for the role of melanopsin in mood and depression was established when human mutations in melanopsin were linked to seasonal affective disorder (sad).113 sad is associated with physiological, behavioral, and mood changes that are evident during winter months with a shorter photoperiod.114 a link between light cycle, iprgcs and mood was also shown in mice. a short photoperiod was associated with depressive behaviors including increased anhedonia in a sucrose preference test and increased hopelessness in a forced swim test, effects which were reversed with antidepressants.115 the same shortened photoperiod also affected hippocampal - dependent spatial learning and memory, with a concurrent decrease in hippocampal - based synaptic plasticity,115 the neural basis thought to underlie learning and memory.116 mice lacking m1 iprgcs were protected from the effect of this shortened photoperiod on the forced swim test, and hippocampal memory and plasticity, suggesting that iprgcs are required for regulation of mood / depressive state associated with circadian rhythm disturbances and learning deficits. whether this role of iprgcs is important for light - enhanced fear conditioning described above is unclear since iprgcs were not tested and the memory task used was hippocampus - independent. light reaches essentially every corner of the brain to influences neural responses, either directly or indirectly. specific neuroanatomical circuits define both the rod / cone photoreceptor dependent image - forming pathway and the iprgc dependent non image- forming pathway. the latter pathway has projections to multiple regions with the potential to influence mood, pain, cognition, addiction, sleep, circadian rhythm, pupil responses, and even vision.117 the identification of melanopsin as an active photopigment, and iprgcs as unique photoreceptors has provided mechanistic explanations for the effect of light on many biological functions. the effect of these irradiation detectors on additional light - modulated functions remains to be elucidated. | mammalian vision consists of the classic image - forming pathway involving rod and cone photoreceptors interacting through a neural network within the retina before sending signals to the brain, and a non image - forming pathway that uses a photosensitive cell employing an alternative and evolutionary ancient phototransduction system and a direct connection to various centers in the brain. intrinsically photosensitive retinal ganglion cells (iprgcs) contain the photopigment melanopsin, which is independently capable of photon detection while also receiving synaptic input from rod and cone photoreceptors via bipolar cells. these cells are the retinal sentry for subconscious visual processing that controls circadian photoentrainment and the pupillary light reflex. classified as irradiance detectors, recent investigations have led to expanding roles for this specific cell type and its own neural pathways, some of which are blurring the boundaries between image - forming and non image - forming visual processes. |
pulmonary hypertension (ph) in adults with sickle cell disease (scd) is associated with early mortality 1, and other complications, including renal insufficiency, leg ulcers, abnormal liver function, systemic hypertension, avascular necrosis, seizures, and cerebrovascular events 2. right heart catheterization (rhc) is the gold standard to confirm the diagnosis of ph and hemodynamic stratification to determine treatment and followup. the hemodynamic characteristics of ph, particularly pulmonary artery pressure (pap) 3, and tricuspid regurgitation velocity (trv) 1, have been shown to be independent predictors of mortality in adults with scd. since scd is a systemic vascular disease 4, it is likely that scd subjects with ph have alterations in microvascular hemodynamics in multiple organ systems. previous studies have reported conjunctival microvascular hemodynamics in scd subjects 5, 6. however, to the best of our knowledge, there have been no studies evaluating conjunctival microcirculatory hemodynamics in scd subjects with or without ph. in this study, we report the conjunctival and pulmonary hemodynamic properties in scd subjects with and without ph. prior to subject enrollment, the research study was explained to the subjects, and informed consents were obtained according to the tenets of the declaration of helsinki. two scd (ss genotype) subjects, one with and one without ph, participated in the study. ten agesimilar africanamerican subjects (age range : 3268 years) without scd or history of cerebrovascular, hypertension or ocular diseases participated as healthy control subjects. systolic and diastolic blood pressures were measured three times at the time of conjunctival imaging, and the averaged values were reported. imaging of the conjunctival microcirculation was performed with our previously described and validated optical imaging system (eyeflow) 6, 7, 8, 9, 10, 11. briefly, the imaging system comprised of a slit lamp biomicroscope and a digital chargedcoupled device camera, was used to acquire image sequences of red blood cell movement within the conjunctiva microcirculation. several image sequences were analyzed to provide the measures of diameter (d) and axial blood velocity (v) of conjunctival venules, as previously described 9. mean values of d and v were calculated from one randomly selected eye in each subject. based on data in control subjects, 95% normal confidence intervals (ci) were calculated for conjunctival d and v. in scd subjects, data from rhc, doppler echocardiography, and laboratory tests were obtained from medical records. pulmonary hemodynamic data including systolic, diastolic, and mean pap (spap, dpap, mpap), pulmonary capillary wedge pressure (pcwp), and cardiac output were obtained from rhc, and trv was documented from doppler echocardiography. diagnosis of ph was based on a mean pulmonary artery pressure (mpap) greater than 25 mm hg 12. exercise capacity was assessed using a 6 minute walk distance (6mwd) test (normal range : 380782 m) 14. the scd subject with ph was categorized according to the world health organization (who) clinical classification of ph 15. table 3 reports conjunctival hemodynamic properties of control and scd subjects. in control subjects, the 95% cis of mean conjunctival d and v were 1723 m (n = 10) and 0.390.59 mm / sec, respectively. demographic and clinical characteristics of healthy control subjects and sickle cell disease subjects with and without pulmonary hypertension (ph). systolic (sbp) and diastolic (dbp) blood pressures were available in seven healthy control subjects pulmonary hemodynamic properties in sickle cell disease subjects with and without pulmonary hypertension (ph) spap, systolic pulmonary artery pressure ; dpap, diastolic pulmonary artery pressure ; mpap, mean pulmonary artery pressure ; pcwp, pulmonary capillary wedge pressure ; trv, tricuspid regurgitation velocity. conjunctival hemodynamic properties in healthy control subjects and sickle cell disease subjects with and without pulmonary hypertension (ph). data in healthy control subjects were reported as 95% confidence intervals case 1 was a 41yearold male with scd and controlled systemic hypertension, cholelithiasis, priapism, and acute chest syndrome during childhood. the subject was on hydroxyurea treatment, and had not received a blood transfusion within 2 months prior to conjunctival imaging. the echocardiography data obtained 40 days before conjunctival imaging demonstrated mild pulmonary valve stenosis, with right ventricle systolic pressure of 54 mm hg. since pulmonary stenosis may confound pulmonary artery hemodynamics, diagnostic rhc was performed for further evaluation of ph, which revealed a normal mpap of 15 mm hg. during the 2month time interval between conjunctival imaging and rhc, the subject had two clinical visits, which indicated an unchanged clinical status and medication list. mean conjunctival d and v measurements were 19 4 m (n = 16 venules) and 0.52 0.22 mm / sec, respectively. both conjunctival d and v measurements were within the normal 95% cis based on data in control subjects. case 2 was a 50yearold male with scd and past medical history of ph, controlled systemic hypertension and diabetes, renal insufficiency, chronic leg ulcer, and gout. this subject was not receiving hydroxyurea treatment and did not have a blood transfusion within 2 months of conjunctival imaging. the echocardiography data obtained 11 months before conjunctival imaging showed an increased trv of 3.63 m / sec. the initial 6mwd test performed 10 months before conjunctival imaging was 244 m and lower than normal. the oxygen saturation was measured to be 87% at rest and 88% during activity, and therefore the subject was prescribed supplemental oxygen of 2 l / min at rest and 3 l / min during activity. rhc revealed postcapillary ph with an elevated mpap of 35 mm hg and pulmonary capillary wedge pressure of 23 mm hg. the subject was categorized as group 1 of the who clinical classification of ph (pulmonary arterial hypertension). during the 7month time interval between conjunctival imaging and rhc, the subject had six followup clinical visits with one echocardiography which demonstrated persistent increase in trv and pap with negligible improvements in exercise capacity, and shortness of breath. mean conjunctival d and v measurements were 17 4 m (n = 8 venules) and 0.19 0.05 mm / sec, respectively. conjunctival d was within the normal 95% ci in control subjects, while conjunctival v was lower than normal 95% ci limit. ph is a prognostic factor in scd and is associated with early mortality 1 and other complications 2. rhc is the gold standard for the diagnosis and monitoring of ph, but it is invasive and carries a small, but present risk of mortality and other complications. in contrast, conjunctival microvascular imaging is a noninvasive and noncontact technique to assess hemodynamic properties of the microcirculation. conjunctival microvascular and hemodynamic abnormalities including abnormal vessel diameter, vessel tortuosity, microaneurysms, and decreased blood velocity have been documented in scd subjects 5, 6. decreased conjunctival blood velocity has been associated with scd complications such as stroke 16 and retinopathy 7. in this study, we reported conjunctival blood velocity in two scd subjects with and without ph. the mean conjunctival v in the scd subject with ph (case 2) was far below the lower limit of the 95% ci established in healthy control subjects, indicating the potential of conjunctival imaging to detect hemodynamic abnormalities in scd subjects with ph. the exact mechanism of decreased conjunctival v in the scd subject with ph is not clear. although scd is associated with microvascular hemodynamics abnormalities which vary based on pathophysiology of the disease complications, the substantially lower conjunctival v observed in case 2 is likely attributed to the combination of ph and scd. one possible explanation could be higher microvascular network resistance due to increased adherence of red and white blood cells, and platelet to the endothelium and to each other resulting from hypoxia in the scd subjects with ph 17. interestingly, the conjunctival d was within the normal range in the scd subject with ph in this study despite lower than normal conjunctival v. this finding may suggest a diminished vasodilatory response to hypoxia in this scd subject due to vasculopathy 18, increased consumption of nitric oxide, or decreased activity of soluble guanylate cyclase and other downstream messengers in vascular smooth muscle 19. future studies in a larger population are required to investigate the relationships between severity of ph and microvascular velocity in scd subjects. conjunctival v measurements in healthy control subjects in this study were lower than values reported in previous studies 20, 21, which may be attributed to differences in ethnicity of the study populations and measurement techniques. furthermore, decreased dbp in scd subjects as compared to healthy control subjects is consistent with previous studies 22, 23, 24. this may be attributed to a progressive renal tubular defect in scd subjects, which begins in early childhood ages 23, 24 assessment of conjunctival microvascular hemodynamics may improve understanding of the pathophysiology of pulmonary hypertension in sickle cell disease. | key clinical messageconjunctival microvascular hemodynamic alterations were reported for the first time in sickle cell subjects with and without pulmonary hypertension. assessment of the conjunctival microcirculation using noninvasive imaging may improve understanding of microvascular hemodynamic alterations that occur due to pulmonary hypertension in sickle cell disease. |
red blood cell (rbc) transfusions are a mainstay of treatment in the management of patients with sickle cell disease (scd). indications for acute and chronic transfusion have expanded, resulting in increased numbers of patients receiving multiple transfusions. overall, there is a paucity of clinical trials directly addressing the use of red blood cell transfusions, and published prospective studies have focused primarily on patients with hemoglobin ss and not the other genotypes. all of the published randomized control trials (rcts) have enrolled patients with hemoglobin ss. a rct with a limited number of subjects demonstrated no benefit for prophylactic transfusion in pregnancy compared to transfusion at a target hemoglobin level in terms of maternal and perinatal complications. the stroke prevention trial in sickle cell anemia (stop) demonstrated efficacy of chronic transfusion compared to conventional therapy for the primary prevention of stroke in children at increased risk based on transcranial doppler (tcd) velocity [4, 5 ]. demonstrated a reduction in the rate of alloimmunization from 3% to 0.5% per unit and a reduction in hemolytic transfusion reactions by 90% with the use of limited phenotypic rbc matching in the stop trial. recently, results from a rct comparing preoperative transfusion use and no transfusion in patients with hemoglobin ss demonstrated increased complications for patients undergoing low- or moderate - risk procedures not receiving transfusions. guidelines based on review of literature, consensus or expert opinion pertaining to a broader range of clinical indications for transfusion have been published [812 ]. uptake of recommendations from the published literature into clinical practice has not been examined. in a survey of blood bank management of sickle cell patients at comprehensive sickle cell centers in the usa, a lack of consensus on transfusion practices for scd was observed. the study focused on transfusion service physicians and laboratory directors and did not address preferences of hematologists. the purpose of this study was to examine transfusion practices by hematologists / oncologists in the clinical management of patients with scd. a comprehensive literature search was performed to identify articles addressing transfusion management in scd and to construct items, both demographic and clinical, for inclusion in the survey. a draft survey was generated with input from a health service researcher with expertise in physician surveys, a transfusion medicine specialist, and hematologists with expertise in scd. the survey was refined with the input from a group of academic hematologists in other regions of the united states with particular interest in scd. face and content validity were evaluated by pilot testing the survey with current fellows and previous trainees of the university of florida hematology / oncology fellowship program in practice locations outside of florida. feedback was obtained and revisions were implemented accordingly, with additional input from two other healthcare provider survey experts. the survey and an accompanying cover letter were submitted and approved by the institutional review board. the self - administered mail questionnaire used in this survey contained thirty - two (32) items. items addressed include educational background, clinical practice setting, professional practice characteristics of physicians, their scd patient populations, and transfusion practices (see survey, appendix). we asked about the availability of information on transfusion recommendations for scd patients, attendance at continuing education programs, and utilization of the national institutes of health (nih) sickle cell disease management monograph available at the time of the survey. selected items were multiple choice, while others were open ended or had an open - ended option attached. the survey included 4 clinical vignettes, where physicians were asked to choose their single most likely treatment recommendation. rbc product modification choices were indicated as leukocyte reduced, irradiated, washed, sickle cell negative, and other products. availability and frequency of automated exchange transfusion and transfusion recommendations for acute and chronic complications of scd were also probed. there were 6 acute transfusion indications and 10 chronic transfusion indications, where practitioners were asked to rank responses using a likert scale, using the following choices : always, sometimes, rarely, or never. the survey was mailed to hematologists / oncologists (h / os) in the state of florida from fall 2005 through spring 2006. a list of respondents was compiled from membership directories of the american society of hematology (ash), the american society of clinical oncology (asco), and the florida association of pediatric tumor programs (faptp). surveys were distributed to physicians in three sequential mailings, with telephone and email follow - up for nonresponders., cary, nc, usa) statistical software was used for all statistical analyses. frequencies and percentages were calculated for categorical data, with means and standard deviations calculated for numerical data. chi - square and fisher 's exact tests were used to test relationships between bivariate categorical data. one hundred fifty - two h / os completed the survey out of 471 requested providers (32% response rate). the study 's response rate may be deflated because not all practitioners contacted were involved in the treatment or transfusion of scd patients in their practices. there was no ability to determine the reason for the nonresponse and whether this was related to area of practice or another factor. data extraction from medicaid claims revealed that only 264 practitioners in florida provided rbc transfusions to scd patients during the time period of the survey (abraham hartzema, unpublished data). the h / os were asked to select pediatric or adult - oriented and academic or nonacademic as a practice setting. eighty - two percent of the adult - oriented practice respondents were in a non - academic setting compared to 51% of the pediatric practice respondents. ninety - three percent of the non - academic adult - oriented practitioners were greater than four years from the completion of their fellowship in comparison to the 86% of non - academic pediatric practitioners. seventy - seven percent of the adult academic respondents and 69% of pediatric academic respondents were greater than four years postfellowship training. thus, overall the majority of respondents completed fellowship training 4 or more years prior to the survey and were in a non - academic practice setting. scd practice pattern information is presented in table 1 comparing pediatric and adult - oriented practitioners. adult - oriented practice respondents reported having fewer scd patients than pediatric h / os. when comparing academic and non - academic practice respondents, 49% of the academic respondents had 51 or more scd patients in comparison to only 17% of the non - academic practice respondents. the highest numbers of scd patients with stroke were noted in the pediatric practices with 87% having six or more patients. fifty - four percent of the non - academic practice respondents had no stroke patients in comparison to 24% of the academic practice respondents. the majority of pediatric practice respondents (95%) had three or more patients receiving hu, with 40% having 16 or more patients receiving hu. in contrast, 70% of the adult practice respondents had two or fewer patients on hu, with 32% having no patients receiving hu. sixty - two percent of academic practice respondents had three or more patients receiving hu, while 57% of the non - academic practice respondents had two or fewer patients receiving hu. most of the pediatric practice respondents (63%) routinely request limited or extended phenotype matching in comparison to only 30% of the adult practice respondents. automated exchange transfusion was available on an emergent basis in the majority of pediatric (94%) and adult - oriented practices (70%). we asked respondents how often specific rbc products were requested for a sickle cell patient excluding the bone marrow transplantation setting. overall, respondents always requested leukocyte reduced products (84%). a minority of practitioners requested sickle negative rbcs all the time. requests for irradiated products we asked respondents what clinical indications they would consider for acute (episodic) transfusion in patients with scd. other acute indications were specified by respondents as write - in answers and included aplastic crisis, bone marrow transplant, severe symptomatic anemia, multiorgan system failure, hepatic sequestration, hospitalization for infection in a nonacute pain crisis, pregnancy with and without complications, nonhealing ulcers, and hypoxia with pneumonia. overall 79% of the respondents thought that acute transfusion was always indicated for acute stroke. always transfuse for acute priapism. in addressing the pre - operative setting with general anesthesia, interestingly, 9% of respondents thought transfusion was always and 41% sometimes indicated for acute painful episodes. the majority of respondents (72%) indicated always or sometimes for vitreoretinal surgery. statistically significant differences among pediatric and adult - oriented practice respondents in reference to acute transfusion indications were identified. the following differences were observed for indications, where there is greater consensus in the literature [812, 14 ]. pediatricians rarely considered acute painful episodes an indication for transfusion in comparison to adult practice respondents (78% versus 26%) (p < 0.0001). in contrast, 53% of adult practice respondents sometimes considered acute painful episode as an indication for transfusion compared to 3% of pediatric practice respondents (p < 0.0001). pediatric practice respondents were more likely to report an acute stroke as an indication for acute transfusion with 95% reporting always in comparison to 73% of adult practice respondents (p < 0.005). general anesthesia was always an indication for transfusion in 73% of pediatric practices compared to 41% of adult practices (p = 0.003). other indications specified by respondents as written in answers included abnormal tcd, bone marrow failure, a fall in reticulocyte count, and viral infection. we queried transfusion preferences for primary stroke prevention, but the survey question did not specifically address patients with hemoglobin ss or whether patients had a high risk tcd result. therefore, it was not possible to discern the preferences of the respondents pertaining to primary stroke prevention, and thus the data are not shown. prevention of stroke recurrence was always an indication for chronic transfusion in 95% of pediatric practice respondents, compared to only 25% of the adult practice respondents (p < 0.0001). a history of acs was always an indication for chronic transfusion in 11% of all respondents. a history of acs was sometimes an indication for chronic transfusion in 68% of pediatric and 28% of adult practice respondents (the occurrence of recurrent debilitating painful episodes was always indicated by a minority of respondents. recurrent debilitating pain was sometimes an indication for chronic transfusion for 60% of pediatric and 33% of adult - oriented practice respondents (p = 0.005) there was a spectrum of responses pertaining to transfusion for uncomplicated pregnancy with 56% of respondents selecting never or rarely. although the majority (71%) of the pediatric respondents selected n / a, there was no statistically significant difference between pediatric and adult practice respondents. there was no statistically significant difference between pediatric and adult practice respondents for congestive heart failure, pulmonary hypertension, nonhealing ulcers, or recurrent priapism, where the majority indicated sometimes or rarely. the clinical vignettes were based on commonly encountered scenarios involving the transfusion management of scd patients (see table 2). the vignettes revealed statistically significant differences between pediatric and adult choices (figure 4). in case 1, the vast majority of pediatric respondents indicated simple transfusion to a hematocrit of 30% for an adolescent undergoing cholecystectomy. although representing a minority of the adult - oriented respondents, there were responses opting for no transfusion (17%) or an exchange transfusion (16%), respectively. a marginal number of adult practice respondents (10%) indicated the less desirable option of a higher target hematocrit (hct) level, whereas none of the pediatric respondents opted for this choice. in case 2, regarding splenic sequestration, the majority of pediatric respondents selected simple transfusion, which is appropriate given the low hemoglobin level ; however, 40% of the adult - oriented practice respondents indicated exchange transfusion. the majority of respondents indicated utilizing exchange transfusion with an appropriate target hct level for case 3 with progressive acs [14, 15 ]. case 4 addressed duration of chronic transfusions in a patient with stroke occurring in childhood who is now an adult. continuing simple transfusions was the mainstay consideration by both pediatric and adult respondents at 48% and 36%, respectively. while both groups also gave consideration to the option of discontinuing transfusion, all pediatric practice respondents selecting discontinuation of transfusions would prescribe hu therapy (32% of responses) for this young adult, whereas simple discontinuation of transfusions alone was selected by 8% of adult respondents. there was a statistically significant difference between adult and pediatric practice respondents with respect to the use of transfusion guidelines and the use of the nih management of scd monograph. when asked if their practice used specific transfusion guidelines, 60% of pediatric practice respondents answered yes compared to only 8% of adult - oriented practice respondents (p < 0.0001). fifty - five percent of pediatric practice respondents reported use of the nih monograph compared to 26% of adult - oriented practice respondents (p = 0.0012). eighty - two percent of pediatric - oriented and 63% of the adult - oriented practice respondents indicated availability. interestingly through a closer examination, academic adult and pediatric practice respondents both had 88% availability, whereas their non - academic counterparts accounted for the observed differences. seventy - one percent of the pediatric and nineteen percent of the adult - oriented practice respondents had attended at least one conference or presentation on the management of scd (not specifying transfusion as a topic) in the two - year period of time prior to the survey ; there was also a significant difference comparing academic to non - academic practices (61 versus 24%), respectively, (p < 0.0001). importantly, in response to an open - ended question about materials that would be helpful, 35% of physicians who requested materials wanted specific transfusion guidelines for the management of scd. the findings of the survey provide insights into transfusion practices across a spectrum of clinical practice settings. the results of the survey indicate variation in the transfusion management of scd among florida h / os. although the non - academic and adult - oriented practices are largest in number, the academic and pediatric practices have the most scd patients and more readily use resources available for scd management. however, comparison of consensus - based recommendations and transfusion practices of respondents revealed variability between recommended and typical practice patterns. there are factors at the patient, provider, and health care delivery system levels which contribute to medical decisions concerning transfusion not captured by this type of survey. phenotype matching and blood product selection are important aspects of transfusion management of patients with scd. in a survey of 1182 north american laboratories, the majority did not determine the red cell antigen phenotype of nonalloimmunized scd patients beyond abo and d. in the assessment of blood banks associated with institutions previously designated as comprehensive sickle cell centers, more than 90 percent of institutions provided c, e, and kell routinely. in this study, the majority of respondents did not routinely request phenotypically matched rbcs for scd patients until the patient demonstrated a new antibody. of note, pediatric practice respondents did request up - front matching more frequently than the adult - oriented practices. the majority of respondents used leukocyte reduced products, consistent with current recommendations [6, 11 ]. there was variability in the request of sickle negative products, which was consistent with previous survey results of the blood bank practices. however, a survey of blood bank centers associated with previously designated comprehensive sickle cell centers revealed 66% of respondents always provided sickle cell trait - negative rbcs. the indications for washed and irradiated rbc product are limited to specific situations such as previous anaphylactic transfusion reactions or bone marrow transplant recipients, respectively. the survey question was specifically indicated to address choices in an scd patient who had not had a bone marrow transplant. this response rate is in contrast to the survey of blood banks, where irradiated and washed rbcs were rarely or never indicated by the majority of institutions. the discrepancies found in this portion of the survey highlight specific provider issues, which can be addressed in providing appropriate blood products for scd patients. a small rct previously demonstrated no benefit of prophylactic transfusion in pregnant patients with hemoglobin ss in terms of maternal and perinatal complications. in concordance with these findings published recommendations do not identify acute painful episodes without complications as an indication for transfusion [10, 12, 14 ]. in our survey, 9% of the sample indicated this was always an indication, whereas the combination of rarely and never constituted 50% of the responses. there is a clear opportunity to provide practitioners with information on when blood transfusions would not be an indicated intervention, as acute painful episodes are a common complication of scd. cholecystectomy is the most commonly performed surgery in patients with scd. in the clinical vignette case 1, the majority of adult and pediatric respondents indicated conservative transfusion management of laparoscopic cholecystectomy with an appropriate end point hematocrit level [14, 15 ]. variance in the responses for adult - oriented practitioners compared to the pediatric practice respondents was similarly reflected in the more generic preoperative clinical scenario queried in the survey section on acute transfusion indications. a contemporary survey of north american members of the society for pediatric anesthesia using clinical vignettes showed preference for preoperative transfusion for patients with increasing sickle - cell - related severity and invasiveness of procedures, but there was a considerable variability of responses. the results of a recent rct showing the benefit of preoperative transfusion compared to no transfusion in patients undergoing cholecystectomy and other moderate risk procedures was not available at the time of the current or previously mentioned survey. the majority (95%) of respondents selected to use transfusion always or sometimes for acs which is consistent with the findings of physicians participating in the national acute chest syndrome study. there are limited data addressing transfusion use in vitreoretinal surgery and most of the reports are dated. it is not surprising that responses for this clinical indication were varied. in summary, for most of the acute indications, no clinical trial data are available to provide strong recommendations. rct data support chronic transfusions for the primary prevention of stroke in high risk children [4, 5 ]. unfortunately, our survey question did not explicitly indicate a prerequisite for high risk tcd results, and we were unable to infer how the sample frame would have handled this indication. ninety - five percent of pediatric practice respondents selected always, in comparison to 25% of adult - oriented practice respondents. multiple factors may account for this difference and may include familiarity with the apparent risk for event recurrence beyond childhood years and the lack of availability for an oral iron chelator at the time of the survey. furthermore, chronic transfusion practices in the adult scd population may pose logistical difficulties for the physician and the patient. patients with higher baseline hemoglobin / hematocrit levels pose challenges when transfusions are indicated to reduce the fraction of hb s containing cells. limited data are available to draw upon in addressing the transfusion of patients with hb sc or hbs / beta+ thalassemia. in the clinical vignette case 3, for a patient with hb sc presenting with acs, the majority of respondents selected exchange transfusion. also in the survey, the majority of respondents indicated having emergent availability of apheresis to perform a red cell exchange. although no clinical trial has examined simple techniques compared to exchange techniques in acs, there is literature supporting effectiveness of red cell exchange [20, 23, and 24 ]. a published guideline for treatment of acs using a clinical severity score has incorporated both techniques. in this particular case, the high hematocrit level would preclude using simple transfusion, selected by approximately 20% of the respondents. the last clinical vignette addressed a typical scenario pertaining to patients aging out of pediatric practices. interestingly, there were just as many physicians that opted to discontinue transfusions and begin hu as there were to continue transfusions. at the time of the survey, use of hu for this indication was derived from limited observational data published on pediatric patients with previous strokes. the stroke with transfusion changing to hydroxyurea (switch) trial was designed to address this issue in children ; at the time of the survey, there was no prospective clinical study data to inform decision making. although we attempted to specifically contact only h / os prescribing transfusions to scd patients, our sample size of 474 represents all physicians listed in the directories and not actually the number of h / os managing scd patients with transfusions. generalization of the results is also limited by the fact that only one state was sampled and practice can vary by state and country based on perceived standard of care and available resources [26, 27 ]. this survey provided a cross - sectional evaluation of scd transfusion practices during 2005 - 2006. however, at the time of the survey, deferasirox was just undergoing food and drug administration approval ; therefore, deferoxamine was the only readily available iron chelating agent in the usa followup surveys will be informative and necessary pertaining to chronic transfusion with the availability of an effective oral iron chelator. our study indicates variability in the incorporation of literature - based approaches when comparing pediatric and adult - oriented practices. in addition, routine use of local transfusion guidelines and availability of exchange transfusion on an emergent basis were more widespread for pediatric providers. many points of reference for scd treatment are considered controversial with the lack of extensive high - grade evidence and therefore differences in actual practice should be anticipated. availability of comprehensive recommendations on the transfusion management of scd in a user - friendly format would provide an opportunity to eliminate potentially unnecessary transfusions, enhance the proportion of patients who benefit from chronic transfusion, and reduce transfusion complications by making the most effective clinical decisions. the national heart, lung, and blood institute (nhlbi) is currently in the process of producing clinical guidelines for patients with sickle cell disease using rigorous methodology including systematic review of the literature and grading of evidence and recommendations. there will be an opportunity to reexamine h / o practices in florida after those guidelines are available. there remains a clear need for prospective clinical trials addressing blood transfusion in patients with scd. | the purpose of this study was to characterize transfusion practices in the management of sickle cell disease and to identify factors attributing to differences in prescribing practices among florida hematologists / oncologists. a cross - sectional study was performed in 2005 - 2006 utilizing a mail survey. the survey instrument addressed practice characteristics, sickle cell patient populations, transfusion settings, indications and techniques, red blood cell phenotype specifications / modifications, use of practice guidelines, and educational resource utilization. one hundred fifty two physicians (75% adult - oriented, 25% pediatric) completed the survey. non - academic practice settings (78 %) were the primary location. pediatric practices had a larger percentage of patients with overt strokes, and receiving hydroxyurea therapy than adult - oriented practices. the majority of survey respondents did not request limited phenotypically matched red blood cells on a routine basis. the majority of pediatric practices (60%) had individually defined transfusion practice guidelines in contrast to 8% of adult - oriented practices. there were statistically significant differences for pediatric and adult - oriented practices in managing certain acute and chronic transfusion indications. analysis of clinical vignette data revealed variation among hematologists / oncologists in the transfusion management of common clinical scenarios. the study underscores the need for the development and dissemination of comprehensive sickle cell transfusion guidelines and protocols. |
gram - positive infections account for up to 50% of cases of severe sepsis in the modern intensive care unit. the complex processes by which gram - positive organisms cause sepsis are poorly understood by comparison with gram - negative sepsis. much interest is currently focused on the role of certain protein exotoxins synthesized by staphylococcus aureus and streptococcus pyogenes, which share the immunological property of being super - antigens. superantigens are characterized by the ability to bypass normal major histocompatibility complex (mhc)-restricted, intracellular, antigen processing and presentation. through direct binding to the mhc class ii molecule and the t cell receptor, at sites away from those involved in conventional antigen binding, superantigens activate up to 50% of the whole t cell repertoire rather than the 1% fraction stimulated by conventional antigens. such toxins are, however, believed to have a role in causation of two classic superantigen - mediated diseases, staphylococcal and streptococcal toxic shock syndromes, that kill 5000 americans per annum. they may also contribute to the pathogenesis of other forms of gram - positive shock. in addition to intensive care support and antimicrobial therapy, various adjunctive treatments for toxic shock syndrome have been evaluated both in vitro and in clinical trials (table 1). nevertheless, the treatment of toxic shock syndrome, like that of conventional forms of sepsis, remains suboptimal and the disease is still associated with mortality in the region of 50% and associated with considerable morbidity. they identified a dodecapeptide that is highly conserved among different bacterial superantigens and lies in a region of the super - antigen molecule away from sites involved with either mhc class ii or t cell receptor interaction. several modified forms of this peptide acted as antagonists to a range of bacterial superantigens against which they were tested in vitro. one dodecapeptide, which was a particularly effective antagonist, was administered to mice challenged with bolus doses of bacterial superantigen. arad speculate that the mechanism of inhibition may involve co - stimulatory pathways of t cell activation. interestingly, protection against subsequent challenges, at 3-weekly intervals, improved with each challenge. this improved protection correlated with antibody production against the whole challenge superantigen, while antibody against the dodecapeptide was not detected. this finding is in keeping with the previously observed correlation between lack of antibody against streptococcal pyrogenic exotoxin a and development of invasive s. pyogenes infection. one consequence of the cytokine storm induced by superantigens may be to disrupt the development of antibody - mediated immunity. by switching off superantigenicity, the dodecapeptide may be allowing normal antibody production to occur. although the prospect of drugs to switch off superantigenicity is exciting, there have been many false dawns in the field of sepsis research. all laboratory animals are intrinsically resistant to the effects of bacterial superantigens. the mouse model, while being one of the best established systems for studying toxic shock, requires far higher doses of superantigen than are needed to induce shock in humans, and prior ' sensitization ' of the animal with the hepatotoxin d - galactosamine. we have recently demonstrated, in a mouse model of invasive streptococcal infection, that other properties of these toxins may be more important than their superantigenicity and, paradoxically, such effects may in fact be advantageous to the host. furthermore, administration of bolus doses of superantigen probably does not reflect the pattern of toxin production in clinical cases. certain findings of the report by arad are at odds with our current understanding of bacterial superantigens. the study found that animals protected from one super - antigen in an initial challenge were cross - protected against different superantigens in subsequent challenges. this effect was observed for toxins as dissimilar as staphylococcal exotoxin b and toxic shock syndrome toxin 1, which have only 6% sequence homology. this is hard to understand in terms of neutralizing antibody since no cross - reactivity between toxic shock syndrome toxin 1 and other superantigens has been demonstrated in serological or neutralization assays. the paper by arad is the first published report of superantigen antagonist peptides. encouragingly, at least one other group is making progress in the same area, and have demonstrated a protective effect not only against bolus doses of superantigen, but also in a model of co - challenge with endotoxin. further studies to address the mode of action of these peptides, particularly in super - antigen - sensitive animal models (sriskandan, manuscript submitted), are necessary before speculation about clinical trials is warranted. specific approaches to treatment of toxic shock syndromes spea, streptococcal pyrogenic exotoxin a ; spec, streptococcal pyrogenic exotoxin c. | the production of superantigenic exotoxins by gram positive bacteria underlies the pathology of toxic shock syndrome. future treatment strategies for superantigen - mediated diseases are likely to be directed at blocking the three - way interaction between superantigen, t cell receptor and major histocompatibility class ii molecule, which inititates an excessive and disordered inflammatory response. in this article, we review the first published data to address one such strategy in the context of other recognised and experimental treatments. |
this novel ttf system is an fda approved device that delivers intermediate frequency, low intensity alternating electric field directly to the brain for the treatment of recurrent glioblastomas. it is considered a fourth treatment modality for the treatment of cancer in addition to surgery, radiation and chemotherapy. during preclinical experimentation, it was shown that the exposure of cancer cells to the tumor treating fields or ttfields resulted in disruption of cell division and subsequently apoptosis. glioblastomas are the most common type of glioma and also the most aggressive. in the newly diagnosed setting, a standard treatment approach consists of concurrent radiation and temozolomide followed by adjuvant temozolomide for 6 months. a recently completed phase iii clinical trial showed a significant prolongation of median overall survival for patients treated with concurrent and adjuvant radiation with temozolomide as opposed to radiation alone. this trial led to the adoption of this protocol as a new standard of care for the management of newly diagnosed glioblastoma. there is no standard approach for the treatment of recurrent glioblastoma ; however, there are two fda approved treatment modalities, namely bevacizumab and the novel ttf system. bevacizumab, a monoclonal antibody directed against the vegf protein results in the blockage of the vegf protein / receptor interaction. the ttf system works through an entirely different mechanism, that is through delivery of continuous alternating electric fields that results in inhibition of cell division and apoptosis. despite all available treatment modalities, here we describe a novel approach for the treatment of recurrent glioblastoma using the delivery of both ttfields as well as the simultaneous bevacizumab infusion. the hope is that the combination approach would prove superior to monotherapy but this remains to be verified in a large scale clinical trial. the electromagnetic basis for the ttf system in order to understand the antimitotic effects of electric field - based treatment for gbm (tumor treating fields therapy or ttf therapy) this theory was formulated by michael faraday in the 1800s and states that a source charge is surrounded by an electromagnetic field. this can exert a force on a test charge that is placed within that field. the electric field can be either uniform or non - uniform. in a uniform electric field, this can be represented by parallel lines of force. in a non - uniform electric field, the field intensity is non - uniform and varies from one end of the field to the other. this in turn can be represented by converging or diverging lines of force, where converging lines of force represent the area of higher field intensity and vice versa. a test charge will move towards the area of higher field intensity within that field. on the other hand, an electric field the source charge in a constant electric field will remain the same while that same charge will oscillate / alternate between positive and negative in a time - varying field as a function of time. the direction of movement of a test charge in an electric field depends on several parameters. an electric charge is either positive or negative while a dipole is positive on one end and negative on the other. the ttf system delivers an alternating electric field and therefore both charges and dipoles move or rotate in the direction of the opposite charge and higher field intensity. during the formation of the daughter cells in telophase, the morphology of the cells results in a non - uniform electric field and a field gradient leading to dielectrophoresis. dielectrophoresis is defined as the migration of uncharged particles towards the position of maximal field strength in a non - uniform field. the mechanism of the anti - mitotic effects of tumor treating - fields the idea of using ttfields (tumor treating fields) for the treatment of cancer was originally conceptualized by professor yoram palti. palti theorized that mitotic activity of cancer cells would be disrupted by applying properly tuned electric fields. the hypothesis was subsequently tested in various cancer cell cultures where it was demonstrated that electric fields disrupted the polymerization of tubulin subunits and therefore prevented the formation of mitotic spindles necessary for cell division. for example, in an in vitro high - grade glioma model, the optimal ttfield frequency shown to exert the maximal cell kill without excessive tissue stimulation or heating was determined to be 200 khz. the application of low frequency (< 1 khz) electric fields is known to result in biological tissue stimulation through membrane depolarization. as the frequency increases well above 1 khz, the stimulatory effect greatly diminishes since the membranes hyperpolarization and depolarization cycles are integrated and the net effect becomes closer to nil. at significantly higher frequencies (mhz range), this concept has been applied in clinical practice in applications such as diathermy and radiofrequency tumor ablation. the optimal effect was also dependent on the field intensity where fields in the rage of 1 - 3 v / cm were most effective without causing tissue heating. in addition, since the fields applied were of intermediate frequency (200 khz in the case of glioma cells) they did not result in biological membrane stimulation. the application of low - intensity (1 - 3 v / cm), intermediate frequency (200 khz) tumor treating fields to cells undergoing mitosis therefore resulted in the alignment of the highly charged tubulin subunits in the direction of higher field intensity, in this case towards the cells cleavage furrow. this resulted in disruption of mitosis, the formation of plasma membrane blebs and ultimately apoptotic cell death (see video portion of manuscript). kirson and colleagues also showed that the maximal effects were observed when the field was applied roughly along the same direction as the cells undergoing mitosis. fields applied in that manner and on a continuous basis for at least 24 hr were shown to result in arrest of cell proliferation and destruction of cells undergoing mitosis. using these preclinical data, the current method of applying the ttf system arrays is such that two sequential field directions are applied to the tumor to optimize cell kill rate. as such, the arrays layout is planned using the tumor mri data to achieve the maximal desired biological activity. mechanism of action of bevacizumab and rationale for combining with electric fields for treating rgbm bevacizumab is a humanized monoclonal antibody that targets the vegf molecule and prevents its interaction with the vegf receptor. it received us food and drug administration (fda) approval in 2009 for the treatment of recurrent glioblastoma based on two phase ii, open - label, non - comparative studies. in the brain study, the objective response rate was 28% (24/85), with a median duration of response of 5.6 months. the pfs-6 rate with single - agent bevacizumab was 42.6% (95% ci, 29.6%55.5%), and the median os was 9.2 months (95% ci, 8.210.7 months). the second study (nci 06-c-0064e) the objective response rate was 19.6% (11/56 ; 95% ci, 10.9%31.3%). the median pfs was 16 weeks (95% ci, 1226 weeks), the pfs-6 rate was 29% (95% ci, 18%48%), and the median os was 31 weeks (95% ci, 2154 weeks). in summary, the two studies found that when compared with historical controls, the use of bevacizumab was associated with higher progression - free survival rates and disease response rates. on the other hand, there is no strong evidence to indicate that bevacizumab can prolong median overall survival when used as an upfront treatment for newly diagnosed gbm patients. bevacizumab had been tried in combination with several chemotherapeutic agents in the past. a retrospective review of recurrent gbm patients treated with a bevacizumab containing regimen and subsequently treated with a different bevacizumab containing regimen after progression concluded that there is no benefit with continuation of bevacizumab following tumor progression. furthermore, despite the favorable radiographic response based on reduction in the enhancing disease seen after bevacizumab treatment, a recent study concluded that non - enhancing disease progression is common after bevacizumab treatment and may be associated with worse outcomes. several preclinical and early clinical data indicate that the combination of tumor treating fields with chemotherapeutic agents maybe more effective (and potentially synergistic) than chemotherapy alone. for example, a study assessed the effects of ttfields alone or in combination with various chemotherapies (paclitaxel, doxorubicin, cyclophosphamide and dacarbazine) on human breast carcinoma (mda - mb-231) and human glioma (u-118) cell lines. the same study examined the effects of ttfields in combination with these chemotherapeutic agents in an animal tumor model and in a pilot clinical trial in recurrent and newly diagnosed gbm patients. the study concluded that the sensitivity to chemotherapeutic treatment was increased by 1 - 3 orders of magnitude by the addition of ttfields. in a pilot clinical trial involving patients with newly - diagnosed and recurrent gbm, the combination approach resulted in a significantly improved pfs and os (progression free survival of 155 weeks and overall survival of 39 + months) compared with historical controls. on the other hand, a large phase iii trial comparing ttf therapy to physicians choice chemotherapy in the treatment of rgbm (ef-11) showed that both treatment approaches resulted in similar survival outcomes while ttf therapy afforded a better side effect profile compared with chemotherapy. given that both bevacizumab and ttf therapy have shown activity and are currently fda approved albeit as monotherapy for rgbm, we hypothesized that the combination of the two treatment modalities may afford an advantage over the use of either agent alone. one hypothesis of why the combination bevacizumab with chemotherapy may offer little advantage in terms of patient overall survival is the dependence of chemotherapy on compromise of the blood brain barrier. when bevacizumab corrects the blood brain barrier, it also affects the ability of chemotherapy to reach the tumor effectively. ttf therapy as a physical modality is hypothesized not to be dependent on the blood brain barrier for it efficacy. it is unclear whether the contraindications for the combination approach are similarly those of the individual treatment modalities when employed as monotherapy or if there are additional precautions with the combination approach. from our limited experience with this novel approach, it remains to be seen in a large scale clinical trial whether this approach will provide any additional advantage (overall survival or progression - free survival) over the currently available treatment protocols. currently, there is a large unmet need for developing effective treatment approaches for rgbm as its prognosis remains dismal despite all available treatment modalities. this approach will need to be assessed in a large scale clinical trial to determine if it can address this unmet need for this unfortunate patient population. note : this protocol consists of two parts : the detailed methodology for applying the ttf system and the methodology for bevacizumab infusion. patients eligible for ttf therapy are those 22 years of age or older with recurrent glioblastoma, typically after surgical, surgery and radiation options have been exhausted but should have received at least one prior chemotherapy regimen. contraindications to the use of ttf therapy include sensitivity to conductive hydrogels, the presence of an active implanted medical device (deep brain stimulators, spinal cord stimulators, vagus nerve stimulators, pacemakers, defibrillators, and programmable shunts), a skull defect or bullet fragment(s). while the ttf therapy is generally well tolerated, the most typical device - related adverse event is skin redness and occasionally skin breakdown due to the prolonged contact of the transducer arrays to the skin surface. to minimize this potential side effect, the arrays are typically changed twice a week and if a skin reaction is observed, the arrays can be moved away from the site of skin redness or inflammation. these skin reactions can also be treated by topical steroids and/or topical antibiotics if needed. note : the system consists of the following components : an electric field generator (the device), a connection cable and box, a portable battery(s), a charger for portable batteries, a plug - in power supply, and a set of transducer arrays (4) (figure 1). assembling the system note : in order for the electric field generator to deliver the electric fields to the tumor mass, the components have to be interconnected in the following manner : charge the battery(s) using the battery charger by connecting its power cord to a standard wall outlet and switch on the power button on the back of the charger. note : a battery rack can hold up to 3 batteries at a time.use a charged battery to power the electric field generator or device, which in turn delivers the electric fields to the transducer arrays.plug the battery in through its connecter to a socket labeled dc in on the front panel of the device. ensure that the arrows on the battery connector are facing up towards the dc in sign while plugging in the connector.turn on the power button after the battery is plugged into the device.connect each of the four transducer array connectors, using the color coded plugs, to the matching color - coded socket on the connection cable box. note : for example, the transducer array connector with the red ring is plugged into the red socket (labeled n1) on the connection cable box (see figure 1).connect the coiled connection cable to the box on one end and the device to the other end.connect the battery to the device to provide the necessary power for the device to generate the alternating electric fields.connect the device to the connection cable and box using the color coded sockets that match color coded cables attached to the transducer arrays. charge the battery(s) using the battery charger by connecting its power cord to a standard wall outlet and switch on the power button on the back of the charger. note : a battery rack can hold up to 3 batteries at a time. use a charged battery to power the electric field generator or device, which in turn delivers the electric fields to the transducer arrays. plug the battery in through its connecter to a socket labeled dc in on the front panel of the device. ensure that the arrows on the battery connector are facing up towards the dc in sign while plugging in the connector. connect each of the four transducer array connectors, using the color coded plugs, to the matching color - coded socket on the connection cable box. note : for example, the transducer array connector with the red ring is plugged into the red socket (labeled n1) on the connection cable box (see figure 1). connect the coiled connection cable to the box on one end and the device to the other end. connect the battery to the device to provide the necessary power for the device to generate the alternating electric fields. connect the device to the connection cable and box using the color coded sockets that match color coded cables attached to the transducer arrays. applying the transducer arrays to the scalp note : an over - the - counter hydrocortisone (steroid) cream may be used if the scalp is irritated.lay out the transducer arrays according to a pre - determined specific array layout on a patients clean and shaven scalp. note : the array layout is calculated using special software that utilizes head and tumor measurements from the patients magnetic resonance imaging (mri) in order to generate the optimal electric field intensity at the site of the tumor to achieve maximal results.peel off the envelopes and remove the arrays then apply to the scalp according to specific array layout color scheme. note : the transducer array locations and colors are : front (blue), back (red), right (yellow), left (white) according to where they are placed on the scalp. note : transducer arrays are supplied in clear sterile envelopes.snap the plugs into the connector.place the color - coded plugs on the transducer array connectors of the four transducer arrays. note : these colors match the color coding of the transducer array placement diagram that has been generated for each patient with the help of special software (1.2.2).plug the array cables into the color - matched connection cable and box as mentioned above (see section 1.1).turn on the power switch on the device once the arrays are in place and the system is fully connected.switch on the ttf field button and the system is now ready for electric field delivery. note : there is no standard duration for continuation of ttf therapy, however, in the author s opinion ttf therapy can be continued until the time of clinical and/or radiographic progression of the disease or development of unacceptable toxicity. to achieve an optimal response, ttf therapy should be used on a continuous basis for a minimum of 18 hr a day. treatment duration of less than 18 hr per day has been associated with suboptimal response. a monthly compliance log can be downloaded from the device software by a novocure technician. note : an over - the - counter hydrocortisone (steroid) cream may be used if the scalp is irritated. lay out the transducer arrays according to a pre - determined specific array layout on a patients clean and shaven scalp. note : the array layout is calculated using special software that utilizes head and tumor measurements from the patients magnetic resonance imaging (mri) in order to generate the optimal electric field intensity at the site of the tumor to achieve maximal results. peel off the envelopes and remove the arrays then apply to the scalp according to specific array layout color scheme. note : the transducer array locations and colors are : front (blue), back (red), right (yellow), left (white) according to where they are placed on the scalp. note : transducer arrays are supplied in clear sterile envelopes. snap the plugs into the connector. place the color - coded plugs on the transducer array connectors of the four transducer arrays. note : these colors match the color coding of the transducer array placement diagram that has been generated for each patient with the help of special software (1.2.2). plug the array cables into the color - matched connection cable and box as mentioned above (see section 1.1). turn on the power switch on the device once the arrays are in place and the system is fully connected. switch on the ttf field button and the system is now ready for electric field delivery. note : there is no standard duration for continuation of ttf therapy, however, in the author s opinion ttf therapy can be continued until the time of clinical and/or radiographic progression of the disease or development of unacceptable toxicity. to achieve an optimal response, ttf therapy should be used on a continuous basis for a minimum of 18 hr a day. treatment duration of less than 18 hr per day has been associated with suboptimal response. a monthly compliance log can be downloaded from the device software by a novocure technician. note : bevacizumab is a humanized monoclonal antibody targeted against the vascular endothelial growth factor (vegf) protein. by blocking the interaction of vegf with its receptor, typically bevacizumab is administered as an iv infusion (typically over 30 min) every 2 weeks. however, the ttf device is designed to be used on a continuous basis or at least for 18 hr per day in order to achieve optimal outcomes. note : the exclusion criteria for bevacizumab include recent intracranial hemorrhage, recent stroke or mi (less than 1 year), major surgery within 4 weeks, uncontrolled hypertension, pregnancy or lactation and imaging showing minimal or no contrast enhancing disease. also, caution must be exercised in patients with renal disease including proteinuria, bleeding disorders, history of dvt, uncontrolled angina, cardiac arrhythmias, congestive heart failure, prior chest wall radiation, prior anthracycline exposure, patients on anticoagulants, recent (less than 6 months) arterial thromboembolic events and other serious medical illness. prescreen patients with baseline testing to include complete blood count with differential, renal and liver function testing, serum electrolytes, urine dipstick for protein and pre and post infusion blood pressure measurement. administer the bevacizumab infusion once a patient is deemed an appropriate candidate using the standard dose of 10 mg / kg body weight and start the ttf system simultaneously with the bevacizumab infusion. note : give the initial infusion over 60 min and mix in 100 ml of normal saline. provided that no complications develop, bevacizumab can be given over 30 min on subsequent infusions. monitor the patient for infusion reactions such as fever, chills, pruritis, rash or angioedema. if these occur stop the bevacizumab infusion, and treat the reactions using standard approaches based on their degree of severity. note : other complications of bevacizumab treatment that require dose modification or discontinuation include proteinuria and hypertension, both of which have been observed in clinical trials with bevacizumab. if acute hypertension (a rise of 20 mmhg or more in diastolic blood pressure or a blood pressure of 150/100 mmhg above normal baseline values) occurs, withhold bevacizumab. if the blood pressure subsequently returns to normal within 1h following the holding of the infusion, restart the infusion at the initial rate. if sustained hypertension occurs then hypertension modification agents such as calcium channel blockers, ace inhibitors or diuretics (hydrochlorothiazide). if proteinuria (2, 3 or 4on urine dipstick) occurs then perform a 24 hr urine collection for protein. if the 24 hr urine protein reveals a protein level of less than 2 g/24 hr then administer bevacizumab at full strength. for levels between 2 - 4 g/24 hr, administer bevacizumab at 5 mg / kg body weight (the standard dose). discontinue bevacizumab for protein levels of more than 4 g/24 hr. perform gadolinium - dtpa enhanced magnetic resonance imaging once every 8 - 12 weeks to assess for tumor response. note : a flair sequence is also particularly helpful especially while administering bevacizumab, since occasionally the contrast enhanced component may diminish while the flair signal change may show signs of progression. assess the radiographic change of the glioblastoma using the newly published revised assessment in neurooncology (rano) criteria (modified from the older macdonald criteria). note : the system consists of the following components : an electric field generator (the device), a connection cable and box, a portable battery(s), a charger for portable batteries, a plug - in power supply, and a set of transducer arrays (4) (figure 1). assembling the system note : in order for the electric field generator to deliver the electric fields to the tumor mass, the components have to be interconnected in the following manner : charge the battery(s) using the battery charger by connecting its power cord to a standard wall outlet and switch on the power button on the back of the charger. note : a battery rack can hold up to 3 batteries at a time.use a charged battery to power the electric field generator or device, which in turn delivers the electric fields to the transducer arrays.plug the battery in through its connecter to a socket labeled dc in on the front panel of the device. ensure that the arrows on the battery connector are facing up towards the dc in sign while plugging in the connector.turn on the power button after the battery is plugged into the device.connect each of the four transducer array connectors, using the color coded plugs, to the matching color - coded socket on the connection cable box. note : for example, the transducer array connector with the red ring is plugged into the red socket (labeled n1) on the connection cable box (see figure 1).connect the coiled connection cable to the box on one end and the device to the other end.connect the battery to the device to provide the necessary power for the device to generate the alternating electric fields.connect the device to the connection cable and box using the color coded sockets that match color coded cables attached to the transducer arrays. charge the battery(s) using the battery charger by connecting its power cord to a standard wall outlet and switch on the power button on the back of the charger. note : a battery rack can hold up to 3 batteries at a time. use a charged battery to power the electric field generator or device, which in turn delivers the electric fields to the transducer arrays. plug the battery in through its connecter to a socket labeled dc in on the front panel of the device. ensure that the arrows on the battery connector are facing up towards the dc in sign while plugging in the connector. connect each of the four transducer array connectors, using the color coded plugs, to the matching color - coded socket on the connection cable box. note : for example, the transducer array connector with the red ring is plugged into the red socket (labeled n1) on the connection cable box (see figure 1). connect the coiled connection cable to the box on one end and the device to the other end. connect the battery to the device to provide the necessary power for the device to generate the alternating electric fields. connect the device to the connection cable and box using the color coded sockets that match color coded cables attached to the transducer arrays. applying the transducer arrays to the scalp ensure no stubble remains and wipe the scalp with 70% medical grade alcohol. note : an over - the - counter hydrocortisone (steroid) cream may be used if the scalp is irritated.lay out the transducer arrays according to a pre - determined specific array layout on a patients clean and shaven scalp. note : the array layout is calculated using special software that utilizes head and tumor measurements from the patients magnetic resonance imaging (mri) in order to generate the optimal electric field intensity at the site of the tumor to achieve maximal results.peel off the envelopes and remove the arrays then apply to the scalp according to specific array layout color scheme. note : the transducer array locations and colors are : front (blue), back (red), right (yellow), left (white) according to where they are placed on the scalp. note : transducer arrays are supplied in clear sterile envelopes.snap the plugs into the connector.place the color - coded plugs on the transducer array connectors of the four transducer arrays. note : these colors match the color coding of the transducer array placement diagram that has been generated for each patient with the help of special software (1.2.2).plug the array cables into the color - matched connection cable and box as mentioned above (see section 1.1).turn on the power switch on the device once the arrays are in place and the system is fully connected.switch on the ttf field button and the system is now ready for electric field delivery. note : there is no standard duration for continuation of ttf therapy, however, in the author s opinion ttf therapy can be continued until the time of clinical and/or radiographic progression of the disease or development of unacceptable toxicity. to achieve an optimal response, ttf therapy should be used on a continuous basis for a minimum of 18 hr a day. treatment duration of less than 18 hr per day has been associated with suboptimal response. a monthly compliance log can be downloaded from the device software by a novocure technician. note : an over - the - counter hydrocortisone (steroid) cream may be used if the scalp is irritated. lay out the transducer arrays according to a pre - determined specific array layout on a patients clean and shaven scalp. note : the array layout is calculated using special software that utilizes head and tumor measurements from the patients magnetic resonance imaging (mri) in order to generate the optimal electric field intensity at the site of the tumor to achieve maximal results. peel off the envelopes and remove the arrays then apply to the scalp according to specific array layout color scheme. note : the transducer array locations and colors are : front (blue), back (red), right (yellow), left (white) according to where they are placed on the scalp. note : transducer arrays are supplied in clear sterile envelopes. snap the plugs into the connector. place the color - coded plugs on the transducer array connectors of the four transducer arrays. note : these colors match the color coding of the transducer array placement diagram that has been generated for each patient with the help of special software (1.2.2). plug the array cables into the color - matched connection cable and box as mentioned above (see section 1.1). turn on the power switch on the device once the arrays are in place and the system is fully connected. switch on the ttf field button and the system is now ready for electric field delivery. note : there is no standard duration for continuation of ttf therapy, however, in the author s opinion ttf therapy can be continued until the time of clinical and/or radiographic progression of the disease or development of unacceptable toxicity. to achieve an optimal response, ttf therapy should be used on a continuous basis for a minimum of 18 hr a day. treatment duration of less than 18 hr per day has been associated with suboptimal response. a monthly compliance log can be downloaded from the device software by a novocure technician. note : bevacizumab is a humanized monoclonal antibody targeted against the vascular endothelial growth factor (vegf) protein. by blocking the interaction of vegf with its receptor, typically bevacizumab is administered as an iv infusion (typically over 30 min) every 2 weeks. however, the ttf device is designed to be used on a continuous basis or at least for 18 hr per day in order to achieve optimal outcomes. note : the exclusion criteria for bevacizumab include recent intracranial hemorrhage, recent stroke or mi (less than 1 year), major surgery within 4 weeks, uncontrolled hypertension, pregnancy or lactation and imaging showing minimal or no contrast enhancing disease. also, caution must be exercised in patients with renal disease including proteinuria, bleeding disorders, history of dvt, uncontrolled angina, cardiac arrhythmias, congestive heart failure, prior chest wall radiation, prior anthracycline exposure, patients on anticoagulants, recent (less than 6 months) arterial thromboembolic events and other serious medical illness. prescreen patients with baseline testing to include complete blood count with differential, renal and liver function testing, serum electrolytes, urine dipstick for protein and pre and post infusion blood pressure measurement. administer the bevacizumab infusion once a patient is deemed an appropriate candidate using the standard dose of 10 mg / kg body weight and start the ttf system simultaneously with the bevacizumab infusion. note : give the initial infusion over 60 min and mix in 100 ml of normal saline. provided that no complications develop, bevacizumab can be given over 30 min on subsequent infusions. monitor the patient for infusion reactions such as fever, chills, pruritis, rash or angioedema. if these occur stop the bevacizumab infusion, and treat the reactions using standard approaches based on their degree of severity. note : other complications of bevacizumab treatment that require dose modification or discontinuation include proteinuria and hypertension, both of which have been observed in clinical trials with bevacizumab. if acute hypertension (a rise of 20 mmhg or more in diastolic blood pressure or a blood pressure of 150/100 mmhg above normal baseline values) occurs, withhold bevacizumab. if the blood pressure subsequently returns to normal within 1h following the holding of the infusion, restart the infusion at the initial rate. if sustained hypertension occurs then hypertension modification agents such as calcium channel blockers, ace inhibitors or diuretics (hydrochlorothiazide). if proteinuria (2, 3 or 4on urine dipstick) occurs then perform a 24 hr urine collection for protein. if the 24 hr urine protein reveals a protein level of less than 2 g/24 hr then administer bevacizumab at full strength. for levels between 2 - 4 g/24 hr, administer bevacizumab at 5 mg / kg body weight (the standard dose). discontinue bevacizumab for protein levels of more than 4 g/24 hr. perform gadolinium - dtpa enhanced magnetic resonance imaging once every 8 - 12 weeks to assess for tumor response. note : a flair sequence is also particularly helpful especially while administering bevacizumab, since occasionally the contrast enhanced component may diminish while the flair signal change may show signs of progression. assess the radiographic change of the glioblastoma using the newly published revised assessment in neurooncology (rano) criteria (modified from the older macdonald criteria). the above protocol was used to treat 2 patients with recurrent glioblastoma who have failed conventional surgical, radiation and chemotherapy treatments. patient was a 56 year old female who was initially treated with standard chemo - radiation according to the stupp protocol. at the time of disease progression, she elected to pursue dose - dense temozolomide according to the protocol used in the previously reported rescue study. her disease remained stable for several months on dose dense temozolomide but subsequently developed a second radiographic progression in addition to myelosuppression (grade 3 thrombocytopenia). she then decided to pursue the above protocol with the knowledge that the combination approach represents an off - label use of bevacizumab and novottf therapy, since both are fda approved as monotherapy in the recurrent setting. she was using ttf therapy on average 10 - 12 hr a day but later became progressively agitated and was pulling on the device cables. interestingly, both the enhancing (as represented by the mr post - gadolinium) and the non - enhancing disease (mr flair sequence) showed radiographic regression (see figure 2). when avastin is administered as monotherapy for recurrent gbm, it is not unusual to observe a reduction in the enhancing component of the tumor while simultaneously observing progression of the flair signal change. the exact mechanism underlying this tumor response is unclear however it is postulated that bevacizumab through reducing blood vessel leakiness and permeability may result in a decrease in gadolinium extravasation without affecting the non - enhancing disease. whether this combination approach resulted in a true reduction of both the enhancing and non enhancing components of the tumor remains to be verified in future large scale studies. patient had developed chest pain and shortness of breath (per her daughter 's account) immediately prior to her death and therefore (although not proven through investigations) we felt that the cause of death was a catastrophic pulmonary embolus rather than directly related to her underlying gbm. as noted earlier, bevacizumab treatment is associated with an increased risk of pulmonary embolism. (a) the transducer arrays are shown as they are applied to the scalp (1) with the array cables connected to the color coded sockets on the connection cable and box (2). the box in turn is connected to the electric field generator with the battery as the power supply (3). pretreatment axial mr image post gadolinium - dtpa administration (a), coronal post gadolinium - dtpa (b), and flair (c) in a 57 year old female with recurrent gbm. post - treatment mr scans (1 month after concurrent treatment) showing significant reduction of both enhancing, axial (d) and coronal (e) as well as no - enhancing (f) flair signal change. this involves the use of continuous low intensity, intermediate frequency electric field treatment with the ttf system in combination with the antiangiogenic agent, bevacizumab. although each of those treatment modalities is fda approved as monotherapy, given the dismal prognosis of gbm at the time of recurrence, combination approaches may prove superior to monotherapy, but this remains a subject of ongoing clinical trials. this approach has never been reported in the literature, however a clinical trial is currently examining whether this combination would result in better median overall survival in patients who relapsed after standard chemoradiation. for example, the device power indicator light may fail to switch on despite turning ' on ' the device.. the first step would be to replace the dead battery with a fully charged spare. otherwise, the device should be turned ' off ' and the technical support helpline contacted. a cable may be detached from the transducer arrays, its connection cable or the device itself. this may indicate too much physical force on the cable or may indicate a faulty connection with the system components. replacing the transducer arrays may fix the problem but if problem not fixed then once again the ttf therapy should be switched ' off ' and the manufacturer contacted. the device also has a built in alarm system that indicates a problem with the device or its mode of operation. if the device alarm is activated this may indicate a low battery, loose or disconnected cable, blockage of the device side vents, poor transducer array contact or simply device malfunction. troubleshooting should therefore include replacing the battery (especially if the low battery lights are lit), or if low battery lights are not lit then all connections should be checked to ensure there are no loose connections, side vents should be checked for blockages and transducer arrays also should be checked to ensure they are properly applied as well. side effects from the application of the transducer arrays to the scalp may also arise and include itching, redness or less commonly blisters. in case of itching or redness, a 0.1% hydrocortisone cream may be applied and the arrays shifted by of an inch away from the site of itching or redness. blisters on the other hand may indicate an infection and may require antibiotic treatment. limitations of the technique : the ttf system has been recently approved by the fda for the treatment of recurrent gbm in patients 22 years and older. those include use of an implanted device such as a deep brain or spinal cord or vagus nerve stimulators, programmable shunts, defibrillators and pacemaker devices. also, the use of the ttf system is contraindicated in patients with a skull defect and/or bullet fragments. sensitivity to conductive hydrogels such as those used with electroencephalogram stickers is a contraindication for use. apart from the rather limited scope for use, the application of alternating electric fields is not a known cure for recurrent gbm although the 2- and 3-year survival rates rates were 8% (95% ci : 4%13%) and 4% (95% ci : 1%8%) versus 5% (95% ci, 3%10%) and 1% (95% ci, 0%3%), for ttf versus active control. its use has been shown to be equivalent to physicians choice chemotherapy but with lesser side effects after failure of standard approaches such as surgery radiation and first line chemotherapy regimens. as the therapy continues to be refined in the future, alternating electric fields may prove efficacious in the front line treatment of glioblastoma. significance with respect to existing methods : as noted above, the ttf therapy is currently fda approved for recurrent gbm and not for the front - line treatment for newly diagnosed gbm. the standard of care for the treatment of newly diagnosed gbm currently is the stupp protocol, which involves the administration of concurrent radiation (60 gy over 6 weeks) together with temozolomide chemotherapy followed by adjuvant temozolomide. despite the significant prolongation of median overall survival of this treatment regimen, gbm is still a highly resistant disease to all forms of available treatment modalities and remains uniformly fatal. the addition of a fourth treatment modality, namely the alternating electric fields to the armamentarium for the treatment of gbm is much needed at this point. future studies using a variety of combinations and permutations of ttf therapy with the other currently approved treatment modalities may prove highly beneficial. critical steps within the protocol : the proper operation of the ttf system is critical for successful treatment. the steps described in the protocol section should be followed accurately but it should be noted that the fda mandates appropriate training through the device manufacturer for healthcare providers who intend to use the device. the critical steps include charging the battery appropriately, assembling the components correctly as noted in the protocol section, properly shaving and cleaning the scalp, applying conductive hydrogel and finally applying the transducer arrays per the treatment layout to the shaved scalp. this helps ensure the proper conduction of the alternating electric fields to their intended brain region. after ensuring proper assembly of the ttf system and application of transducer arrays, the power button is turned on and finally the ttfield button is turned on to start treatment. the use of ttf therapy in the treatment of gbm, both in the recurrent and newly - diagnosed setting is likely to be refined in the future. because of its unique mechanism of action and favorable safety profile, it is likely that ttf therapy in combination with a variety of chemotherapeutic as well as molecularly - targeted agents will be employed in future clinical trials. currently there is an ongoing trial testing the safety and efficacy of ttf therapy in combination with adjuvant temozolomide in patients 18 years and older with newly diagnosed gbm (ef-14). a positive outcome from this trial may result in widening the indication of ttf therapy to include newly diagnosed gbm patients. in addition, the ttf therapy will also be tested for a variety of other cancers including metastatic brain disease. hopefully, as the techniques are refined, these approaches will have a significant impact on such a uniformly fatal disease. this study was supported by a grant from novocure limited to cover the publication costs. | a novel device that employs ttf therapy has recently been developed and is currently in use for the treatment of recurrent glioblastoma (rgbm). it was fda approved in april 2011 for the treatment of patients 22 years or older with rgbm. the device delivers alternating electric fields and is programmed to ensure maximal tumor cell kill1.glioblastoma is the most common type of glioma and has an estimated incidence of approximately 10,000 new cases per year in the united states alone2. this tumor is particularly resistant to treatment and is uniformly fatal especially in the recurrent setting3 - 5. prior to the approval of the ttf system, the only fda approved treatment for rgbm was bevacizumab6. bevacizumab is a humanized monoclonal antibody targeted against the vascular endothelial growth factor (vegf) protein that drives tumor angiogenesis7. by blocking the vegf pathway, bevacizumab can result in a significant radiographic response (pseudoresponse), improve progression free survival and reduce corticosteroid requirements in rgbm patients8,9. bevacizumab however failed to prolong overall survival in a recent phase iii trial26. a pivotal phase iii trial (ef-11) demonstrated comparable overall survival between physicians choice chemotherapy and ttf therapy but better quality of life were observed in the ttf arm10.there is currently an unmet need to develop novel approaches designed to prolong overall survival and/or improve quality of life in this unfortunate patient population. one appealing approach would be to combine the two currently approved treatment modalities namely bevacizumab and ttf therapy. these two treatments are currently approved as monotherapy11,12, but their combination has never been evaluated in a clinical trial. we have developed an approach for combining those two treatment modalities and treated 2 rgbm patients. here we describe a detailed methodology outlining this novel treatment protocol and present representative data from one of the treated patients. |
endometriosis is a common gynaecological condition characterised by the presence of endometrial glands and stroma at extrauterine sites. ectopic endometrial implants have been reported in nearly every tissue or organ, including the skin. of the reported cases of cutaneous endometriosis, over 70% the case of primary cutaneous endometriosis described here remains a rare entity with a reported incidence of 0.51% of all extragenital endometrial ectopia. in december 2012, a 31-year - old caucasian woman presented to the dermatology outpatient clinic with a 1-month history of a persistent, raised lesion in the umbilicus. the patient had noted swelling and one episode of spontaneous frank bleeding from the lesion. there was no associated cyclical pain at the lesion site. at the time of examination, a single firm 0.5-cm black - coloured papule was seen within the umbilicus at the 3 o'clock position. no discharge or underlying umbilical hernia was noted. the patient had a history of 2 previous melanomas : the first in 2003 on the left abdomen and the second on the right buttock in 2006. given the anatomical location, a skin punch biopsy of the umbilical lesion was performed. during dermatology review, 1 month later, it was noted that 3 distinct skin - coloured papules had appeared next to the initial lesion (fig. histological examination of the punch biopsy revealed a lesion in the superficial dermis comprising a single dilated glandular structure, surrounded by cellular endometrial - type stroma (fig. the gland was lined by a single layer of columnar cells, a few of which had apical cilia. subsequent gynaecological evaluation revealed a 3-year history of dysmenorrhoea. a provisional diagnosis of coexistent pelvic endometriosis was made. a diagnostic and therapeutic laparoscopic examination was advised if the patient remained unable to conceive over the following 6 months. less than 30% of cutaneous endometriosis appears in the absence of a prior surgical history and is termed spontaneous (primary) cutaneous endometriosis. of these cases, umbilical endometriosis characteristically occurs in women of reproductive age, who present with an umbilical nodule at the site of a prior surgical incision associated with cyclical pain at the lesion site. however, the inconsistent appearance and rarity of this phenomenon means it is often clinically misdiagnosed. endometriomas under the skin have been described as red, blue, black or flesh - coloured. classic associated symptoms include cyclical pain, discharge, bleeding or swelling of the lesion correlated with the menstrual cycle. the most widely accepted pathogenesis of secondary endometriosis is the iatrogenic implantation of endometrial cells as a result of surgery, commonly laparoscopic procedures. however, the pathogenesis of primary endometriosis remains uncertain. theories suggested include the implantation of cells through sanguineous or lymphatic spread, or differentiation from coelomic pluripotent cells in the skin. the heterogeneity in the clinical presentation of cutaneous umbilical endometriosis reflects the breadth of differential diagnoses that may mimic the condition (table 1). malignancies, in particular melanoma and umbilical metastasis of visceral carcinoma (sister mary joseph nodule), must be considered. distinctive dermatoscopic, ultrasonographic and magnetic resonance imaging findings have been described ; however, histopathological examination remains the gold standard. hormonal therapy in the form of gonadotropin - releasing hormone agonists, oral contraceptives and danazol may be given preoperatively to reduce the size of lesions and ameliorate symptoms consistent with pelvic endometriosis. subsequent gynaecological evaluation for pelvic endometriosis is recommended for all patients. around 15% of patients diagnosis of the umbilical skin lesion facilitated the provisional diagnosis and management of underlying symptomatic pelvic endometriosis. of note, the patient in this case study has a strong history of melanoma. recent data have reported a statistically significant association between endometriosis and the occurrence of melanoma. further studies are required to examine the possible association of cutaneous endometriosis and the development of melanoma. in summary, nevertheless, it should be considered in the differential diagnosis when examining any umbilical lesion, even in absence of pathognomonic cyclical symptoms. | cutaneous endometriosis that arises de novo, without a prior history of surgery, is a rare phenomenon. the clinical diagnosis of cutaneous endometriosis remains challenging due to the variable clinical appearance and symptoms of the condition, and therefore must be considered in the differential diagnosis of any umbilical lesion. we report a 31-year - old woman who presented with spontaneous cutaneous endometriosis of the umbilicus. |
since the first modified rna ribonucleic acid was found 60 years ago, 150 known rna modifications have been reported. emerging evidences suggest that rna modifications are critical components of the gene regulatory landscape and are involved in a variety of biological processes in the post - transcriptional level, such as protein translation and localization, mrna splicing, affecting ribosome biogenesis, mediating antibiotic resistance, and stem cell pluripotency. however, many aspects of rna modifications remain unknown. therefore, detecting the positions of rna modifications plays an essential role for understanding their molecular mechanisms and functions. the advent of next - generation sequencing technologies has allowed investigation of rna modifications on a genome - wide scale.9, 10, 11, 12, 13, 14, 15 for example, the n - methyladenosine (ma),9, 10 n - methyladenosine (ma), and 5-methylcytosine (mc) maps are available for the human transcriptome. although these experimental methods played active roles in promoting the research progress on understanding the biological functions and the identification of rna modifications, they are still labor - intensive. as excellent complements to experimental techniques, some computational methods (based on the high - resolution experimental data) have been developed to identify rna modifications.7, 16, 17, 18, 19, 20, 21 reminiscent of the regulation of gene expression by histone modifications, it is also possible to mediate biological functions in a collective way by combining different kinds of rna modifications. unfortunately, to the best of our knowledge, no computational tool is available for dealing with a system that simultaneously contains several different kinds of rna modifications. actually, this kind of multi - modification systems may contain much more interesting things worthy of exploration. in view of this, the present study was initiated in an attempt to fill such a void by establishing a seamless package or platform that can be used to analyze a biological system that simultaneously contains the three well known types of rna modifications : ma, ma, and mc (figure 1). by incorporating collective effects of nucleotides into pseknc,22, 23 a seamless platform called irna - psecoll has been developed for identifying the occurrence sites of different rna modifications. it has been observed by the most rigorous cross - validation, the jackknife test, that the success rates achieved by the new predictor are quite high for the three different types of rna modification sites, respectively (table 1). because it is the first platform predictor ever developed for simultaneously identifying three different types of rna modification sites based on its sequence information alone, it is not possible to demonstrate its power by a comparison with its counterparts because there is no such a counterpart yet for exactly the same purpose. nevertheless, as we can see from table 1, all the scores are quite high, particularly for the overall accuracy (acc) and mathew s correlation coefficient (mcc). as it is, the graphical approach is a useful vehicle for studying complicated biological systems because it can provide intuitive insights, as demonstrated by a series of previous studies.25, 26, 27, 28, 29, 30, 31, 32, 33, 34 therefore, it would be instructive and illuminative to give an intuitive illustration for the current study as well. to realize this, the graph of receiver operating characteristic (roc)35, 36 was adopted as shown in figure 2, where the roc curves for the current method in identifying ma, ma, and mc modifications were given, respectively. the best possible prediction method would yield a point with the coordinate (0, 1) representing 100% sensitivity and 0 false - positive rate or 100% specificity. therefore, the (0, 1) point is also called a perfect classification. a completely random guess would give a point along a diagonal from the point (0, 0) to (1, 1). the area under the roc curve, also called auroc, is used to indicate the performance quality of the classifier : the value 0.5 of auroc is equivalent to random prediction while 1 of auroc represents a perfect one. the auroc for the case of ma, ma, or mc is 0.998, 0.849, or 0.911, respectively, indicating that the proposed platform is quite promising, holding very high potential to become a useful high throughput tool for genome analyses. inspired by a series of recent publications,20, 21, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53 papers published with a publicly accessible web server will significantly enhance their impacts ; this is particularly true for those papers aimed at developing novel prediction methods. accordingly, the web server for the current platform has been established. moreover, for the convenience of the scientific community, a user guide is given in the supplemental materials and methods. according to the chou s five - step guidelines that have been followed by many investigators in a series of recent publications,21, 39, 41, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 56, 57, 58, 59, 60, 61, 62, 63 to develop a new prediction method that not only can be easily used by most experimental scientists but also can inspire theoretical scientists to develop many other relevant prediction methods, we should make the following five procedures very clear : (1) how to construct or select a valid benchmark dataset to train and test the prediction model, (2) how to represent a biological sequence sample with a mathematical formulation or vector that is really correlated with the target concerned, (3) how to introduce or develop a powerful engine (or algorithm) to run the prediction model, (4) how to properly perform the cross - validation tests to objectively evaluate the anticipated accuracy, and (5) how to design a user - friendly web server to make it easy for people to get their desired results. below, we elaborate the five procedures in establishing the new predictor. owing to the fast development of high - throughput experimental techniques, the experimentally confirmed ma, ma, and mc modification data is available for the human genome.9, 10, 13, 15 by mapping the experimental data to the human genome, the sequence samples with statistical significance were obtained for the three kinds of rna modification sites as well. for facilitating the formulation, let us use the following scheme to represent a potential rna modification - site - containing sample(equation 1)r()=nn(1)n2n1n+1n+2n+(1)n+,where the symbol denotes the single nucleic acid code a (adenine) or c (cytosine), the subscript is an integer, n represents the -th upstream nucleotide from the center, the n+ represents the -th downstream nucleotide, and so forth. the (2+1) -tuple rna sample, r(), can be further classified into the following two categories:(equation 2)r(){r+(),ifitscentercanbeof2-o - methylationr(),otherwise, where r+() denotes a true modification segment with a or c at its center, r() denotes a false modification segment with a or c at its center, and the symbol means a member of in the set theory. in literature, the benchmark dataset usually consists of a training dataset and a testing dataset : the former is for the use of training a model, while the latter for testing the model. however, as elucidated in a comprehensive review, there is no need to artificially separate a benchmark dataset into the aforementioned two parts if the prediction model is examined by the jackknife test or subsampling (k - fold) cross - validation, because the outcome thus obtained is actually from a combination of many different independent dataset tests. thus, the benchmark datasets for the current study can be further formulated as(equation 3){s(m1a)=s+(m1a)s(m1a),when=as(m6a)=s+(m6a)s(m6a),when=as(m5c)=s+(m5c)s(m5c),when=c, where the positive subset s+(m1a) only contains those rna samples that can have m1a modification, and the negative subset s(m1a) only contains those rna samples that can not have m1a modification, while u denotes the symbol of union in the set theory, and so forth. the benchmark datasets were derived from the rna sequences in human genome that have the experimentally confirmed ma, ma, and mc modification sites.9, 10, 13, 15 the detailed procedures to construct the benchmark dataset are as follows. first, as done in chou, by sliding the (2+1) -tuple nucleotide window (figure 3) along each of the aforementioned rna sequences, only those rna segments with = a or c at the center were collected. second, if the upstream or downstream in a rna sequence was less than or greater than l where l is the length of the rna sequence concerned, the lacking code was filled with the same code of its nearest neighbor. third, the rna segment samples thus obtained were put into the positive subset s+(m1a), s+(m6a), or s+(m5c) if their centers were experimentally annotated as the ma, ma, or mc sites ; otherwise, into the corresponding negative subset s(m1a), s(m6a), or s(m5c). fourth, to reduce redundancy and bias, none of the included rna segments had pairwise sequence identity with any other in a same subset. by strictly following the above procedures, we obtained an array of benchmark datasets with different values and hence different lengths of rna samples (2+1) as well (see equation 1), as illustrated below(equation 4)s(){23nucleotides, when=1125nucleotides, when=1227nucleotides, when=1339nucleotides, when=1941nucleotides, when=2043nucleotides, when=21,where the symbol means it was observed via preliminary tests as well as many reports19, 43, 66 that when =20 (i.e., the rna samples formed by 41 nucleotides [nt ]), the corresponding results were most promising. by doing so, we obtained 6,366, 1,130 and 120 sequence samples for the positive subsets s+(m1a), s+(m6a), and s+(m5c), respectively. the numbers of samples thus obtained in the corresponding negative subsets are much greater, and hence the benchmark datasets would be very imbalanced. using such highly skewed benchmark dataset to train predictors would lead to the outcome that many positive cases might be mispredicted as negative ones.42, 44, 56, 67 to balance out the size between the positive subset and the negative subset, we randomly picked out 6,366, 1,130, and 120 from the corresponding negative samples to for the negative subsets s(m1a), s(m6a), and s(m5c), respectively, as done in chen. and feng. finally, the detailed rna sequence samples thus obtained for the benchmark dataset s=20(m1a), s=20(m6a), and s=20(m5c) are given in the supplemental materials and methods, which can also be directly downloaded from http://lin.uestc.edu.cn/server/irna-psecoll/dataset.htm. one of the most challenging problems in computational biology today is how to formulate a biological sequence with a vector that can reflect its key pattern important for the function or mechanism concerned. the importance of such a challenge is due to the fact that nearly all the existing machine - learning algorithms were developed to handle vector rather than sequence samples, as elucidated in a review article. unfortunately, a vector defined in a discrete model may lose many important sequence pattern features. to deal with such a problem for protein / peptide sequences, the pseudo amino acid composition (pseaac)68, 69, 70, 71, 72 was developed. ever since it was introduced, the concept of pseaac has penetrated into nearly all the areas of computational proteomics (see a long list of references cited in two review papers55, 73). inspired by the concept of pseaac and encouraged by its great successes, the pseudo nucleotide composition (pseknc)22, 74, 75, 76 was proposed and has been increasingly used in various fields of genome analysis.20, 21, 23, 37, 39, 40, 42, 43, 51, 52, 53, 58, 59, 60, 77, 78, 79, 80, 81, 82, 83, 84, 85 with both pseaac and pseknc being increasingly and widely used, it is highly desired to design a seamless package that can generate various modes of pseaac and pseknc according to users needs for protein / peptide and dna / rna sequences, respectively. this was exactly the driving force of establishing the web server called pse - in - one and what it is about. the general form of pseknc for an rna sequence sample is given by(equation 5)r=[12u]t, where t is a transpose operator, while the subscript an integer and its value as well as the components u (u=1,2,,) will depend on how to extract the desired features from the rna sequence sample. in order to make equation 4 able to reflect both the local feature of its individual constituent nucleotides and that of their collective effect, let us define the components in equation 4 from the following two different approaches. rna consists of four types of nucleotides : a (adenosine), c (cytidine), g (guanosine), and u (uridine). they can be classified into three different categories (table 1) : (1) from the angle of ring number, a and g have two rings, whereas c and u only one ; (2) from the chemical functionality, a and c belong to amino group, while g and u to keto group ; and (3) from the angle of hydrogen bonding, c and g can be bonded to each other with three hydrogen bonds, but a and u with only two (figure 4). all these properties would have different impacts to rna s low - frequency internal motion87, 88 and its biological function89, 90, 91. to reflect the aforementioned features, let us denote the i - th nucleotide of equation 1 by92, 93(equation 6)ni=(xi, yi, zi),where xi, yi, and zi refer to the attributes of (1) ring structure, (2) functional group, and (3) hydrogen bonding in table 2, respectively. accordingly, the nucleotide a can be formulated as (1, 1, 1), c as (0, 1, 0), g as (1, 0, 0), and u as (0, 0, 1) ; or generally we have(equation 7)xi={1,ifni{a, g}0,ifni{c, u};yi={1,ifni{a, c}0,ifni{g, u};zi={1,ifni{a, u}0,ifni{c, g}. there are some methods to reflect the coupling of a biological sequence or the collective effect of its constituent elements, such as the conditional probability approach, degenerate kmer strategy, and g - gap dipeptide mode. in this study, we would like to use a different approach ; i.e., consider the occurrence frequency of a nucleotide not only for its local site but also for its distribution along the sequence of an rna sample, as defined by the following equation(equation 8)di=1lij=1f(nj),where di is the density of the nucleotide ni at the site i of a rna sequence, li the length of the sliding substring concerned, denotes each of the site locations counted in the substring, and(equation 9)f(nj)={1,ifnj = thenucleotideconcerned0,otherwise. the density of a at the sequence position 1, 2, 3, 4, 5, or 6 is 0=0/1, 0.5=1/2, 0.331/3, 0.25=1/4, 0.20=1/5, or 0.16=1/6, respectively ; that of c is 1=1/1, 0=0/2, 0.662/3, 0.5=2/4, 0.4=2/5 or 0.5=3/6, respectively ; and so forth. by combing (equation 6), (equation 9), the i - th nucleotide of equation 1 can be uniquely defined by a set of four variables ; i.e.,(equation 10)ni=(xi, yi, zi, di). for example, the rna sequence cacguc can be expressed by the following five sets of digital numbers : (0, 1, 0, 1), (1, 1, 1, 0.5), (0, 1, 0, 0.66), (1, 0, 0, 0.25), (0, 0, 1, 0.2), and (0, 1, 0, 0.5). submitting these numbers into equation 5, we have(equation 11)r(cacguc)=[01011110.50100.661000.250010.20100.5]t, meaning that the 6-nt nucleotide example can be defined by a 64=24 -d (dimensional) pseknc vector. accordingly, all the samples in the current benchmark datasets (supplemental materials and methods) can be formulated with a 414=164 -d vector. the prediction was operated by svm (support vector machine), which has been widely used in various areas of bioinformatics and computational biology.20, 40, 42, 59, 67, 77, 78, 79, 80, 81, 95, 96, 97, 98, 99, 100, 101, 102, 103 its basic idea has been elaborated in the aforementioned the papers, and there is no need to repeat it here. in the current study, the libsvm package 3.18 was used to implement svm, which can be downloaded for free from http://www.csie.ntu.edu.tw/cjlin / libsvm/. the svm algorithm contains two uncertain quantities : one is the regularization parameter c and the other is the kernel width parameter. they were optimized via an optimization procedure using the grid search approach as described by(equation 12){25c215withstepc=221525withstep=21,where c and represent the step gaps for c and, respectively. for those readers who are interested in knowing more about svm, see chou and cai and cai. or a monograph where a brief introduction or detailed description were given, respectively. the platform predictor obtained via the aforementioned procedures is called irna - psecoll, where i stands for identify, pse for pseudo component approach, and coll for collective effects of nucleotides. quality control is a very important process in industries ; it is even more important for a predictor. to deal with this problem, we need to address the following two issues : (1) what standard or metrics should we adopt to measure the predictor s quality, and (2) what test process or method we should take to calculate the metrics. the current prediction is belonging to the category called binary classification widely existing in genome analyses. to measure the prediction quality of this kind, a set of four metrics are usually used in literature : (1) sensitivity or sn, (2) specificity or sp, (3) overall accuracy or acc, and (4) mathew s correlation coefficient or mcc. unfortunately, their formulations were directly taken from mathematical literature and difficult to be understood by most biological scientists. fortunately, using the symbols introduced by chou in studying signal peptides, xu. and chen. have derived a new set of metrics that is equivalent to the old one but much more intuitive and easier to be understood by most biologists, as given below to address this, we need to consider two issues : one is what metrics should be used to reflect the predictor s success rates ; the other is what test method should be adopted to derive the metrics rates. to quantitatively evaluate the quality of a binary classification predictor, they are : (1) acc for the predictor s overall accuracy ; (2) mcc for its stability ; (3) sn for its sensitivity ; and (4) sp for its specificity. unfortunately, the conventional formulations for the four metrics are not quite intuitive, and most biologists have difficulty understanding them, particularly the stability of mcc. fortunately, as elaborated in yu. and chen., by using the chou s symbols and derivation in studying signal peptides, the conventional metrics can be converted into a set of four intuitive equations, as formulated below:(equation 13){sn=1n+n+0sn1sp=1n+n0sp1acc==1n++n+n++n0acc1mcc=1(n+n++n+n)(1+n+n+n+)(1+n+n+n)1mcc1,where n+ represents the total number of positive samples investigated, n+ is the number of positive samples incorrectly predicted to be the negative, n is the total number of negative samples investigated, and n+ is the number of the negative samples incorrectly predicted to be the positive. with the metrics of equation 13, the meanings of sn, sp, acc, and mcc have become crystal clear as discussed and used in a series of follow - up studies for many different areas.20, 21, 38, 40, 42, 44, 45, 46, 47, 48, 49, 56, 57, 61, 67, 80, 82, 84, 97, 99, 112, 113, 114, 115 it is instructive to point out that more multi - label sequence samples have been emerging in system biology and medicine.49, 116, 117, 118, 119 to deal with this kind of multi - label system, a much more sophisticated set of metrics is needed as elaborated in chou. these methods include : (1) an independent dataset test, (2) a subsampling (or k - fold cross - validation) test, and (3) the jackknife test. however, as elucidated in chou in the above three choices, the jackknife test has been demonstrated to be the least arbitrary that can always yield a unique outcome for a given benchmark dataset. therefore, the jackknife test has been widely recognized and increasingly adopted by researchers to analyze the quality of various predictors.83, 103, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131 in view of this, we also used the jackknife test to examine the quality of the current prediction method. the jackknife test can exclude the memory effect because both the training dataset and testing dataset in a jackknife system are actually open, and each sample will be, in turn, moved between the two. the arbitrariness problem intrinsic to the independent dataset and subsampling tests no longer exists, because the outcome derived via the jackknife test for a predictor is always the same on a given benchmark dataset. w.c., h.l., and k.- c.c. conceived and designed the study, h.l., and k.- c.c. performed the analysis and wrote the paper. | there are many different types of rna modifications, which are essential for numerous biological processes. knowledge about the occurrence sites of rna modifications in its sequence is a key for in - depth understanding of their biological functions and mechanism. unfortunately, it is both time - consuming and laborious to determine these sites purely by experiments alone. although some computational methods were developed in this regard, each one could only be used to deal with some type of modification individually. to our knowledge, no method has thus far been developed that can identify the occurrence sites for several different types of rna modifications with one seamless package or platform. to address such a challenge, a novel platform called irna - psecoll has been developed. it was formed by incorporating both the individual and collective features of the sequence elements into the general pseudo k - tuple nucleotide composition (pseknc) of rna via the chemicophysical properties and density distribution of its constituent nucleotides. rigorous cross - validations have indicated that the anticipated success rates achieved by the proposed platform are quite high. to maximize the convenience for most experimental biologists, the platform s web - server has been provided at http://lin.uestc.edu.cn/server/irna-psecoll along with a step - by - step user guide that will allow users to easily achieve their desired results without the need to go through the mathematical details involved in this paper. |
missing permanent teeth and non - compliance of the patients can pose anchorage problems during treatment. to eliminate these problems, intraoral skeletal anchorage systems, such as palatal implants1, retromolar implants,2 onplants,3 zygomatic ligatures,4 minis - crews59 and miniplates10,11 have been introduced to clinical use. currently, miniscrews and miniplates are being widely used because of their small size and superiority over endossous implants due to the fact that they can be immediately loaded. miniplates basically have the same features with the plates used in maxillofacial surgery. unlike these miniplates, orthodontic miniplate s one end is fixed to the cortical bone and the other end has attachments to engage orthodontic auxiliaries. umemori,11 and jenner and fitzpatrick12 were the first ones to use maxillofacial bone plates for skeletal anchorage in orthodontics. since then, mini - plate design variations have been introduced like mpi (tasarimmed, istanbul, turkey),10 bollard mini plate(surgitec, bruges, belgium),13 c - tube miniplate (kls martin, umkirch, germany),14 and surgitec zygoma anchor miniplate (surgitec, bruges, belgium).15 with the aid of miniplates, intrusion of single or groups of teeth, alignment of severe crowding, correction of severe class ii malocclusion, and management of anterior open bite have been accomplished succesfully.10,11,14,16 on the other hand, besides these successful case reports clinical studies regarding miniplates had reported high failure rates of 7 8.6%. inflammation around the neck and the forces effecting on the stability of the fixation screws are shown to be the reasons for these failures.17,18 veziroglu that, inadequate design and non - homogeneous force distribution along the anchorage system can cause stress directly effecting on the screws and may impair screw stability. they also added that, mobile plates can irritate the surrounding tissues and may be the reason for initiating or aggravating the inflammation around the neck of the miniplate piercing the oral mucosa. a design that prevents the stress acting non - homogeneously on the fixation screws can be a solution to avoid mobilization of screws, and consequently inflammation that may arose due to mobilized screws. with the use of three dimensional finite element method (fem), load transfer of orthodontic forces from the screws to the bone and the stress distribution around these can be effectively evaluated.17, 20, 21 thus, the aims of this fem study were to (1) design a new miniplate structure featuring spikes placed on the surface facing the cortical bone, referred as spiky miniplate, and (2) compare with the use of fem the force distribution along the conventional miniplate - screw system and the newly designed spiky miniplates inserted to the cortical bone. this study was carried out by yeditepe university faculty of dentistry and ay tasarim ltd.. nextengine (nextengine inc. santa monica, california 90401 usa) laser scanner was used for three - dimensional scanning and rhinoceros 4.0 (3670 woodland park ave., seattle, wa 98103 usa) three - dimensional software modeling and algor fempro (algor, inc. 150 beta drive pittsburgh, pa 15238 - 2932 usa) softwares were used for analysis. the characteristics (elasticity modulus and poisson s ratio)22 of the materials are given in table 1. cortical bone thickness was assumed as 1.5 mm in the models.23,24 the screws used in the study had a diameter of 2 mm and a length of 5 mm. the dimensions of the miniplates are given at figure 1a, b. the interface between the screw and the host bone was assumed to be fully bonded, consistent with screw - material interfaces. the spikes had a length of 0.7 mm and a base diameter of 0.6 mm and are assumed to be fully penetrated into the bone. the miniplate and cancellous bone were subtructed from the cortical plate using boolean method.25 this enabled the transfer of force in finite element analysis software. the models were fixed at the lower and lateral surfaces to keep fixation sites away from the experimental region. 200 g force was applied at the same point to the miniplate towards the same direction (figure 1a). new spiky miniplates and the conventional plate tested in the study were modelled by using rhinoceros 4.0 software. the three models obtained were as follows (figure 2) : conventional miniplate design with two screw holesnewly designed miniplate with four spikes with two screw holesnewly designed miniplate with four spikes with single screw hole conventional miniplate design with two screw holes newly designed miniplate with four spikes with two screw holes newly designed miniplate with four spikes with single screw hole the maximum stress values created on the miniplates and screws are given in tables 2 and 3 and the data for bone are given in table 4. several terms had been used throughout the manuscript for the parts of the screws and spikes according to their localization. for the two - holed miniplates, the term near screw has been used for the screw that is adjacent to the lever arm. the term far screw was used for the distant screw. similarly, for the spikes the terms near spikes and far spikes had been used. in all of the miniplates the most increased level of stresses were seen at the neck of the miniplates and the point where the force was applied (table 2, figure 3a c). the maximum stress values for one - holed spiky miniplate were located around all of the spikes ; whereas for the two - holed spiky miniplate, they were located at the near spikes. the highest stress value recorded for the spikes was 43.58 mpa for the one - holed spiky miniplate. when the highest stress values around the screw holes were evaluated, similar stress values were observed (table 3). in all the screws, except the far screw of the two - holed spiky miniplate, the highest stress level was recorded at the neck (figure 4). the highest stress value was 13.32 mpa at the near screw of the two - holed conventional miniplate (table 2, figure 4a). for the two holed spiky miniplate, almost no stress was observed at the far screw (figure 4b). the data related to the cortical bone that is in contact with the fixation screws are given in table 4. the maximum stress recorded for the bone was around the near screw in the two - holed conventional miniplate (figure 5a, 6a). for the two - holed spiky miniplate, the highest stress was found at the region where the near spikes entered the bone (figure 5b, 6b). for the one - holed spiky miniplate, at the cortical bone level, the highest stress level was found at the region where the spikes entered the bone (figure 5c, 6c). the maximum tension and compression stresses seen at the cortical bone around the near screw at the two - holed conventional miniplate were 1.51 and 1.34 mpa, respectively (table 4). in the present study, two newly designed mini - plates were tested with three - dimensional fem. however, three - dimensional fem studies provide the following advantages for the researchers working orthodontic force systems.19 simulate oral structures in vitrosimulate the orthodontic force system applied clinicallyanalyze the response of the bone and the materials usedseveral experimental designs can be simulated for numerous times keeping the properties of the materials unchangednewly designed appliances can be tested invitro and non - invasively before clinical use simulate oral structures in vitro simulate the orthodontic force system applied clinically analyze the response of the bone and the materials used several experimental designs can be simulated for numerous times keeping the properties of the materials unchanged newly designed appliances can be tested invitro and non - invasively before clinical use the three - dimensional fem was preferred because of the advantages described above prior to clinical application. 200 g force that is usually used in clinical practice was applied to the miniplates.26 as a control, a conventional miniplate that is routinely used in practice was chosen. the results of this study revealed remarkable difference in the stress distribution at the cortical bone that is in contact with the fixation screws between the conventional and the newly designed miniplates. also the fixation screws received almost half of the stress values for the new mini - plates. when the two - holed spiky miniplate and two - holed conventional miniplate were compared, the highest tension and compression stress found at the cortical bone around the near screw at the new spiky miniplate was almost half of the conventional one (table 4). as for the far screw of the spiky miniplate, there was almost no stress at the cortical bone. similarly the stress level at the body of the screws also showed substantial decrease when spiky miniplate was used which had almost no stress at the far screw (figure 4b). comparison of the one- and two - holed spiky miniplates revealed the same amount of tension and compression stresses of 0.63 and 0.65 mpa and 0.62 and 0.80 mpa, respectively (table 4). when the one - holed spiky miniplate and two - holed conventional miniplate was compared, even though one - holed spiky miniplate had only one screw, the maximum stress around the screw was half of the conventional one (table 4). when the data for the screw material was compared, the single screw at the one - holed spiky miniplate was found to carry half of the stress of the near screw in the two holed conventional miniplate had (table 2, figure 4). the failures of miniplates can generally be attributed to two major reasons ; stress directly affecting the screws and inflammation.17,18 stability of the miniplates is directly affected by the stability of the fixation screws. applied forces to the mini - plates are transmitted to the screws which create stresses especially on the near screw that may impair the screw stability.19 with the conventional plates, load of the orthodontic forces are directly transferred from the plate to screws, whereas with the new ones, spikes act as an barrier before the load reaches the screws. non - homogenous stress distribution on the fixation screws is not the only reason for the failures. the reason for the screw loosening is not clear, but choi reported that it might also be due to insertion technique, force level, force duration, patient s oral hygiene or thickness of cortical bones. haug reported that the stability of the miniplates can be improved by increasing the number of the fixation screws. however, with respect to failure rates, no significant statistical difference was found between the plates with different number of screws.17 as a result of the present fem study, remarkably lower stresses on the fixation screws between the conventional miniplates and the new ones have been observed. also, in time, resorption can occur at the bone around the spikes, and the stresses around the screws may increase. in vivo studies are necessary to investigate possible histological changes to ensure the safety and the stability of the newly designed miniplates. cortical bone thickness is one of the major factors for the success rates of the miniscrews. in this study, an average of 1,5 mm cortical bone thickness was modeled.21,22 when the average thicknesses for the cortical bone was considered, a length of 0.7 mm for the spikes was estimated to be safe in order not to protrude from the cortical bone. if the cortical bone thickness is thin like in vertical - growing patients, then the success rates of miniscrews may be lower than for the average or horizontal - growing patients.28 in the light of this knowledge it becomes obvious that maximum support from the cortical bone should be obtained. spikes on the newly designed miniplates decrease the stress on the screws providing more homogeneous support from the cortical bone. the advantage of one holed new miniplate is that it can be used with a smaller incision. if there is enough keratinized gingival height, one holed miniplate can be applied with the use of a punch to excise the soft tissue. thus, the miniplate can be applied with very small surgery with no incision or sutures. this study reported the newly designed mini - plates featuring spikes placed on the surface facing the cortical bone. the fem study revealed that the new miniplates are highly efficient in reducing stress on the fixation screws. | objectives : non - homogeneous force distribution along the miniplates and the screws is an unsolved question for skeletal anchorage in orthodontics. to overcome this issue, a miniplate structure was designed featuring spikes placed on the surface facing the cortical bone. the aim of this study was to examine and compare the force distribution of the newly designed plate - screw systems with the conventional one.methods:a model of bone surface with 1.5 mm cortical thickness, along with the two newly designed miniplates and a standard miniplate - screw were simulated on the three - dimensional model. 200 g experimental force was applied to the tip of the miniplates and the consequential effects on the screws and cortical bone was evaluated using three - dimensional finite element method.results:as a result of this finite element study, remarkably lower stresses were observed on the screws and the cortical bone around the screws with the newly designed miniplate when compared with the conventional one.conclusion:the newly designed miniplate that has spikes was found effective in reducing the stress on and around the screws and the force was distributed more equivalently. |
furchgott and zawadzki described the obligatory role of the endothelial cells during relaxations of the isolated rabbit aorta to acetylcholine and demonstrated the transfer of a vasodilator substance termed endothelium - derived relaxing factor. this factor was identified as nitric oxide (no) and became a major player in physiology, pharmacology, and pathophysiology. over the years, however, it became obvious that no could not explain all endothelium - dependent responses, and that endothelium - dependent relaxations can be caused by hyperpolarizations of the underlying vascular smooth muscle cells that are not due to endothelium - derived no but rather to endothelium - derived hyperpolarizing factors (edhfs). another important aspect of endothelium - dependent control of vasomotor tone emerged with the demonstrations that endothelial cells can produce contracting factors, particularly vasoconstrictor prostanoids [4, 5 ]. it has thus become obvious that no single factor or mechanism is responsible for all endothelium - dependent responses, and that their modulation by age and disease leads to endothelial dysfunction, which has become a hallmark of vascular disease and a predictor of major cardiovascular events. this brief review presents some recent information concerning the multiplicity of endothelium - dependent signals and their possible contribution to the control of vascular tone in health and its derangement in hypertension. a common denominator in hypertension, regardless of its etiology (essential, renovascular, malignant, or preeclamptic), is endothelial dysfunction, which involves reduced production, decreased bioavailability, and/or impaired cellular effects of no. therefore, various potential therapeutic targets have been identified all along the l - arginine no - synthase soluble guanylyl cyclase pathway (fig. 1). 1nitric oxide (no) synthase, lipoxygenase (lo), and cytochrome p450 monooxygenase (cyp) pathways and responses mediated by endothelium - derived hyperpolarizing factors (edhfs) : potential sites of therapeutic intervention for hypertension. the circled numbers indicate potential sites of intervention : 1, l - arginine supplementation. 2, inhibition of protein arginine n - methyltransferase type i (prmt - i) to prevent the formation of asymmetric dimethyl - l - arginine (adma). 3, increased expression and/or activity of dimethylarginine dimethylaminohydrolase-2 (ddah-2) to facilitate adma catabolism. 4, inhibition of arginase-2 to prevent l - arginine metabolism. 5, increased expression and/or activity of endothelial nitric oxide synthase (enos). 6, design of drugs that evoke endothelium - dependent relaxations. 7, enhanced expression and/or activity of guanosine triphosphate cyclohydrolase (gtpch), the rate - limiting enzyme for tetrahydrobiopterin (bh4) synthesis, or direct supplementation with bh4 or its precursor sepiapterin. 8, enhanced expression and/or activity of dihydrofolate reductase (dhfr), involved in bh4 regeneration. 9, scavengers of reactive oxygen species (ros), antioxidants. 10, inhibition of the activity and/or expression of enzymes that generate ros, such as nad(p)h oxidases (nox), cyclooxygenases (cox), lipoxygenases (lox), or cytochrome p450 monooxygenases (p450). 11, enhanced expression and/or activity of enzymes that metabolize ros, such as superoxide dismutase (sod) or catalase (or, alternatively, synthesis of mimetics). 15, inhibition of soluble epoxide hydrolase (seh) to suppress degradation of epoxyeicosatrienoic acids (eets). 16, opening calcium - activated potassium channels of small, intermediate, or large conductance (skca, ikca, bkca). aa arachidonic acid ; bh2dihydrobiopterin ; cat-1cationic amino acid transporters ; cav voltage - activated calcium channel ; cgmp cyclic guanosine monophosphate ; dhets dihydroxyeicosatrienoic acids, ec endothelial cell ; fad flavin adenine dinucleotide ; fmn flavin mononucleotide ; heeta hydroxy - epoxyeicosatrienoic acid ; 12-hete12-hydroxyeicosatetraenoic acid ; kir inward rectifying potassium channel ; megj myoendothelial gap junction ; o2superoxide anion ; onoo peroxynitrite ; pkg protein kinase g ; theta, trihydroxyeicosatrienoic acid ; vsmc vascular smooth muscle cell nitric oxide (no) synthase, lipoxygenase (lo), and cytochrome p450 monooxygenase (cyp) pathways and responses mediated by endothelium - derived hyperpolarizing factors (edhfs) : potential sites of therapeutic intervention for hypertension. the circled numbers indicate potential sites of intervention : 1, l - arginine supplementation. 2, inhibition of protein arginine n - methyltransferase type i (prmt - i) to prevent the formation of asymmetric dimethyl - l - arginine (adma). 3, increased expression and/or activity of dimethylarginine dimethylaminohydrolase-2 (ddah-2) to facilitate adma catabolism. 4, inhibition of arginase-2 to prevent l - arginine metabolism. 5, increased expression and/or activity of endothelial nitric oxide synthase (enos). 6, design of drugs that evoke endothelium - dependent relaxations. 7, enhanced expression and/or activity of guanosine triphosphate cyclohydrolase (gtpch), the rate - limiting enzyme for tetrahydrobiopterin (bh4) synthesis, or direct supplementation with bh4 or its precursor sepiapterin. 8, enhanced expression and/or activity of dihydrofolate reductase (dhfr), involved in bh4 regeneration. 9, scavengers of reactive oxygen species (ros), antioxidants. 10, inhibition of the activity and/or expression of enzymes that generate ros, such as nad(p)h oxidases (nox), cyclooxygenases (cox), lipoxygenases (lox), or cytochrome p450 monooxygenases (p450). 11, enhanced expression and/or activity of enzymes that metabolize ros, such as superoxide dismutase (sod) or catalase (or, alternatively, synthesis of mimetics). 15, inhibition of soluble epoxide hydrolase (seh) to suppress degradation of epoxyeicosatrienoic acids (eets). 16, opening calcium - activated potassium channels of small, intermediate, or large conductance (skca, ikca, bkca). cav voltage - activated calcium channel ; cgmp cyclic guanosine monophosphate ; dhets dihydroxyeicosatrienoic acids, ec endothelial cell ; fad flavin adenine dinucleotide ; fmn flavin mononucleotide ; heeta hydroxy - epoxyeicosatrienoic acid ; 12-hete12-hydroxyeicosatetraenoic acid ; kir inward rectifying potassium channel ; megj myoendothelial gap junction ; o2superoxide anion ; onoo peroxynitrite ; pkg protein kinase g ; theta, trihydroxyeicosatrienoic acid ; vsmc vascular smooth muscle cell when l - arginine is deficient, endothelial no synthase (enos) can generate both superoxide anions and no, leading to the detrimental production of peroxynitrite. it is still a matter of debate whether l - arginine deficiency occurs in vivo to limit the production of no by enos, but l - arginine supplementation improves endothelial dysfunction in hypercholesterolemia and hypertension. in addition, endogenous analogues such as asymmetric dimethyl - l - arginine (adma) can compete with l - arginine for its specific membrane transporter and also directly for access to enos, where adma acts as an inhibitor. the plasma concentration of adma represents an independent predictor for all causes of cardiovascular mortality. free dimethylarginines are the products of proteolytic degradation of arginine - methylated proteins by protein arginine n - methyltransferase type i (prmt - i). in endothelial cells, adma is metabolized mainly by dimethylarginine dimethylaminohydrolase-2 (ddah-2). during angiotensin ii administration and oxidative stress, the observed elevation in adma levels is associated with an increase in the activity of prmt and a decrease in the activity of ddah. silencing the ddah-2 gene impairs endothelium - dependent relaxation and no production. therefore, the inhibition of prmt - i and the activation or enhanced expression of ddah-2 could be beneficial in treating cardiovascular disease. endothelial cells express arginases (with arginase-2 being the predominant isoform), which metabolize l - arginine to l - ornithine and urea. arginase-2 competes with enos for substrate, and its expression and activity are enhanced in cardiovascular diseases, perhaps because of increased oxidative stress. in animal models, inhibition and gene deletion of arginase-2 improve endothelium - dependent relaxations and the vascular production of no, prevent the development of hypertension, and decrease the generation of endothelial reactive oxygen species (ros) and the formation of atherosclerotic plaques. arginase-2 may therefore represent a promising novel therapeutic target that could reverse vascular dysfunction in hypertension. reduced expression of enos could be responsible for decreased no production, but in most situations where endothelial dysfunction is encountered, the expression of enos is increased paradoxically, most likely because oxidative stress generates hydrogen peroxide, which increases the expression of the enzyme. endothelial dysfunction associated with this increased expression of enos shows that the ability to generate no is reduced or its bioavailability is decreased. the reduction in no generation can be attributed to enos uncoupling, whereby the enzyme itself is a source of superoxide anions and a cause of endothelial dysfunction. tetrahydrobiopterin (bh4) is an essential cofactor that critically controls the assembly and activity of enos. decreased endothelial levels of bh4 are responsible not only for a reduction in no production but also for the uncoupling of enos. bh4 is synthesized de novo from guanosine triphosphate (gtp) following a series of enzymatic reactions, whereby gtp - cyclohydrolase i (gtpch - i) is the first and rate - limiting step. an alternative pathway for bh4 synthesis, the salvage pathway, involves the formation of sepiapterin and its subsequent reduction in dihydrobiopterin (bh2), which is further reduced by dihydrofolate reductase (dhfr) to form bh4. following oxidation, bh4 can be regenerated by either dhfr or dihydropteridine reductase. increased vascular homocysteine is a risk factor for atherosclerosis ; it leads to endothelial dysfunction, and some of its effects may be mediated by inhibition of bh4 de novo synthesis. direct supplementation with bh4 or its precursor, sepiapterin, improves endothelial function in animals, in smokers, and in patients with hypertension, diabetes, hypercholesterolemia, or coronary disease. enhancing the expression or the activity of gtpch - i (for de novo bh4 synthesis) or of dhfr (for bh4 regeneration) may prevent the occurrence of endothelial dysfunction. alternative strategies include supplementation with folic acid, which enhances the binding affinity of bh4 to nos, stabilizes bh4 chemically, and stimulates dhfr. preventing peroxynitrite formation (and therefore bh4 oxidation) or facilitating the recycling of bh4 regeneration with vitamin c can also be considered. inhibition or deletion of enos however, apoe mice overexpressing enos develop larger atherosclerotic lesions than control apoe mice. again, this paradox is explained best by the peroxynitrite - dependent uncoupling of enos and the subsequent production of superoxide anions. however, enos uncoupling can be prevented if the upregulation of enos is associated with an increased availability of essential cofactors such as bh4. compounds such as ave9488 and ave3085, which enhance enos promoter activity, possess such a coordinated activity and validate the concept that enhanced transcriptional expression of enos can be beneficial in preventing cardiovascular diseases [11 ]. besides preventing enos uncoupling, other therapeutic strategies can be designed to restore proper no levels. these include drugs that stimulate the release of no by endothelial enos, no donor drugs, antioxidant compounds, drugs that boost the antioxidant defense mechanisms, and inhibitors of enzymes involved in the generation of ros. some antihypertensive drugs for instance, nebivolol, various dihydropyridines, angiotensin - converting enzyme (ace) inhibitors, angiotensin (at1) receptor blockers (arbs), and possibly renin inhibitors can directly or indirectly stimulate the release of endothelial no. additionally, chronic treatments with ace inhibitors, arbs, or renin inhibitors increase enos expression. although chronic treatment with ace inhibitors or arbs consistently improves endothelial function in animal models of hypertension, variable effects have been reported in hypertensive patients, possibly because of the multifactorial etiology of essential hypertension or differences in the experimental clinical protocols. besides uncoupled enos, nad(p)h oxidase is a predominant source of excess ros in the vascular wall in essential hypertension. thus, inactivation of superoxide anions with superoxide dismutase mimetics or scavenging of ros with antioxidants seems an obvious way of increasing no bioavailability. however, large clinical trials of chronic antioxidant therapy in hypertensive patients have not produced major reductions in arterial blood pressure and did not improve the associated morbidity and mortality, with the possible exception of chronic intake of polyphenols (present in red wine, fruit, and vegetables). synthetic inhibitors of nad(p)h oxidase have been proposed as an alternative, but it is still uncertain which isoforms of nad(p)h should be inhibited, and the available inhibitors are neither specific nor potent enough, and they have poor pharmacokinetic properties [15 ]. activation of the renin - angiotensin system with subsequent stimulation of at1 receptors is a major stimulus for nad(p)h oxidase and production of ros in both animal models and in hypertensive patients. induced hypertension and markedly reduces the associated endothelial generation of superoxide anions. in rat models of hypertension, ace inhibitors and arbs decrease the generation of superoxide anions, diminish the amplitude of endothelium - dependent contractions, and restore the amplitude of both no - mediated and edhf - mediated endothelium - dependent relaxations. in addition to lowering lipids, statins also decrease the expression of nad(p)h subunits and increase enos expression, improving the balance between no and ros. to date, the clinical use of no donor drugs is limited by the development of nitrate tolerance, a complex multifactorial phenomenon associated with the generation of oxidative stress and endothelial dysfunction. new chemical classes of no donors, some of which can spontaneously release no, have been synthesized and may have a therapeutic interest beyond the treatment of coronary disease and heart failure. earlier compounds such as nicorandil (an organic nitrate that opens potassium channels) and nipradilol (a nonselective adrenoceptor blocker with no - releasing properties) are still predominantly prescribed for angina and glaucoma, respectively. new hybrid compounds including no - releasing antiadrenergic drugs, no - releasing dihydropyridines, no - releasing statins, no - releasing arbs, and no - releasing ace inhibitors are of therapeutic interest for treating hypertension and associated end - organ damage [19 ]. the regulation of nitric oxide bioactivity can also be achieved by modulating soluble guanylyl cyclase, the main physiological target of no, or the half - life of the signaling molecule, cgmp, which is readily hydrolyzed by phosphodiesterases. two different classes of compounds stimulators and activators interact with soluble guanylyl cyclase [20, 21 ]. the former stimulate the enzyme in an no - independent but heme - dependent manner ; acting as allosteric modulators, they enhance no - dependent cgmp production. by contrast, activators of soluble guanylyl cyclase target the enzyme when its heme is oxidized and no longer responsive to no. both activators and stimulators of soluble guanylyl cyclase are potent vasodilators and inhibitors of platelet aggregation. the stimulators are currently undergoing clinical trials in acute decompensated heart failure and peripheral artery occlusive disease, and the activators are being evaluated in pulmonary hypertension. potential therapeutic indications of these new compounds include hypertension, myocardial ischemia, erectile dysfunction, atherosclerosis, and thrombosis [20, 21 ]. phosphodiesterase 5 (pde-5) was the first identified selective cgmp esterase in the cardiovascular system and is the major isoform involved in the hydrolysis of the cgmp pools generated by the activation of soluble guanylyl cyclase. specific pde-5 inhibitors are currently prescribed for erectile dysfunction but, in the future, their therapeutic indications may also include pulmonary hypertension, heart failure, and essential hypertension. edhf - mediated responses can provide a vasodilator reserve in hypertension and can compensate, at least temporarily, for endothelial dysfunction due to compromised synthesis or availability of no. however, reduced endothelium - dependent hyperpolarizations also can contribute to arterial dysfunction in animal and human cardiovascular disease, especially hypertension and diabetes. in any case, whether edhf - mediated responses are diminished or conserved, it is still possible to pharmacologically stimulate the endothelial cells in order to inhibit the underlying vascular smooth muscle, thereby improving vasodilator responses and lowering arterial blood pressure in experimental and human hypertension or reversing endothelial dysfunction in diabetes [23, 24, 25 ]. there are presumably a variety of structurally very distinct factors or pathways that can produce edhf - mediated dilatations. indeed, residual no, products of the metabolism of arachidonic acid (including prostacyclin), hydrogen peroxide, potassium ions, and c - natriuretic peptide all have been proposed as edhfs. in addition, a nonchemical edhf - signaling pathway involves transfer of negative charges from the endothelium to the smooth muscle via gap junctions. in the case of edhf pathways involving k ions and electrical gap - junctional myoendothelial coupling, activation of endothelial calcium - activated potassium (kca) channels initiates the dilator response in many preparations by triggering hyperpolarization and k release (fig. 1). the in vivo relevance of endothelial kca channels is indicated by the severely impaired edhf - mediated responses and the elevated arterial blood pressure in mice deficient of endothelial intermediate - conductance channels (ikca, kca3.1) and small - conductance channels (skca, kca2.3) [24, 26 ]. moreover, selective openers of endothelial ikca and skca, ns-309 and ska-31, may be of therapeutic use in cardiovascular disease because they have been shown to improve endothelial function in type 2 diabetes and to lower blood pressure in mice with angiotensin ii induced hypertension. hydrogen peroxide, another putative edhf, may amplify this ikca- and skca - driven edhf system by facilitating inositol - trisphosphate (ip3)-mediated ca release and thus enhancing endothelial ca signaling [3, 28 ], thereby further stimulating the two channels. however, the well - known harmful effects of hydrogen peroxide make it questionable whether pharmacologic enhancement of its production could be a strategy to lower arterial blood pressure. of the products of the metabolism of arachidonic acid, eicosanoids generated by lipoxygenase (lo) and cytochrome - p450 (cyp) monooxygenase pathways are the most likely candidates as diffusible endothelium - derived relaxing factors [30 ]. lo and cyp pathways are complex and yield many metabolites with diverse effects on vascular tone. the mechanisms by which lo - derived and cyp - derived eicosanoids produce vasodilatation include the activation of potassium channels (ca - activated large - conductance [bkca ] channels and small - conductance [skca - like ] channels) leading to hyperpolarization, closure of l - type ca channels, and thus relaxation (fig. 1). because of this ability of lo - derived and cyp - derived eicosanoids to both hyperpolarize and relax smooth muscle, their vasodilator activity has been linked to edhf - mediated responses in several vascular beds [3, 30 ]. the endothelium expresses the three major lo isoforms : 5-lo, 12-lo, and 15-lo. the products of 12-lo and 15-lo cause dilatation, whereas leukotrienes generated by 5-lo are associated with vasoconstriction [30 ]. the 12-lo product, 12(s)-hete, produces vasodilatation by activating bkca channels and thus hyperpolarizing the vascular smooth muscle cells, whereas heeta and theta, generated by 15-lo, open skca - like channels [30 ]. the role in vivo of these lo products as endothelium - derived relaxing factors is uncertain. pharmacologic inhibition with bw755c or genetic knockdown of 12/15-lo had no effect on arterial blood pressure [30 ], but gene transfer of human 15-lo-1 enhanced the hypotension caused by acetylcholine [30 ]. in contrast, lo inhibitors reduce angiotensin - ii induced hypertension [30 ], and inhibition of 12/15-lo by baicalein and derivatives has beneficial effects in experimental ischemic stroke [30 ]. considering the blood pressure lowering effects and cerebrovascular protective effects of lo inhibitors on one hand, and the beneficial effects on arterial blood pressure of 15-lo overexpression on the other hand, it is difficult to decide on the potential benefit of stimulating the production of lo - derived endothelium - derived relaxing factors in hypertension. with respect to inflammatory responses and atherosclerosis, the polymorphisms in the human 12/15-lo gene (alox15) lead to either enhanced enzyme activity with a lowered risk of coronary artery disease or to impaired enzyme activity with enhanced risk. endothelial cells express cyps of the 4a, 2c and 2j subfamilies [3, 30 ]. cyp4a -hydrolase produces the vasoconstrictor 20-hete, but cyp2c and cypj2 epoxygenases generate epoxyeicosatrienoic acids (eets)5,6-eet, 11,12-eet, or 14,15- of which produce vasodilatation in certain human arteries [3, 30 ]. like other arachidonic acid metabolites, their vasodilator action is closely related to edhf - mediated arteriolar responses because they stimulate bk channels and cause hyperpolarizations followed by relaxation. moreover, they also facilitate endothelial ca signaling and likely augment no synthesis in an autocrine fashion. in addition, 5,6 eet, 11,12-eet, and 14,15-eet, as well as their degradation products, dihydroxyeicosatrienoic acids (dhets), may produce vasodilatation in a strictly no - dependent fashion. this would be explained if eets / dhets stimulate no formation, either by causing enos phosphorylation or by enhancing ca signaling following activation of cation channels of the transient receptor potential gene family (trpv4) [24, 29 ]. whether these vascular trpv4 channels can be treated with drugs, the cyp pathway must be relevant also for hypertension : clinical data have demonstrated the ability of cyp products to compensate at least in part for the loss of no - mediated dilator responses in patients with essential hypertension, and soluble epoxide hydrolase (seh), an inhibitor of the eet degrading enzyme, exerts blood pressure lowering effects in experimental hypertension involving the activation of the renin - angiotensin system. this blood pressure lowering effect was not seen after inhibition of no synthesis by l - arginine - methylester (l - name), however, thus further showing that augmented eet levels favor no formation, which by itself could lower arterial blood pressure. a specific inhibitor of seh, ar9281, has been tested in clinical trials for antihypertensive efficacy and for the treatment of type 2 diabetes but has failed because of lack of efficacy. nevertheless, data from animal studies show that seh inhibition or gene deletion could be effective in pulmonary hypertension or atherosclerosis and may have protective effects on brain, heart, and kidneys [25, 34 ]. the relevance of cyps and seh for cardiovascular disease is supported further by the identification of polymorphisms (causing reduced enzyme activities), which have been associated although not consistently with a higher risk for cardiovascular disease, hypertension, and type 2 diabetes. considering bk channels as putative targets of the products of both cyp and 15-lo, it is noteworthy that gain of function polymorphisms in bk - subunit genes decrease the risk of hypertension, whereas loss of function polymorphisms increase the risk. with aging and in various cardiovascular diseases particularly essential (spontaneous) hypertension endothelial dysfunction is importantly due to the release of endothelium - derived contracting factors (edcfs) that counteract the effect of endothelium - derived vasodilators. in healthy blood vessels, the major edcfs that contribute to endothelial dysfunction in hypertension appear to be products of the metabolism of arachidonic acid. this conclusion is based mainly on data obtained in arteries of the spontaneously hypertensive rat (shr), although it is supported by indirect evidence in humans [4, 5, 39 ]. upon activation of the endothelial cell and the resulting increase in cytosolic caconcentration, arachidonic acid is released from cell membrane phospholipids, whereby phospholipase a2 (pla2), in both calcium - dependent and calcium - independent forms, contributes to the process. actually, the activation of calcium - dependent pla2 explains the calcium dependency of endothelium - dependent contractions [40, 41 ]. procedures that reduce the increase in endothelial ca concentration, such as the administration of vitamin d3, curtail the occurrence of endothelium - dependent contractions. the released arachidonic acid is then metabolized by cyclooxygenases (cox), an enzyme with two major isoforms : cox-1 is expressed constitutively and is usually abundant in all animal and human endothelial cells, whereas endothelial cox-2 is induced mainly during inflammatory responses in nearly all animals. in humans, although cox-2 is the predominant contributor to the systemic generation of prostacyclin, endothelial cox-1 is also a major source of prostaglandins in both healthy and diseased blood vessels [4346 ]. in arteries of the shr, there is a marked increase in the mrna expression and protein presence of cox-1, which underlies the augmented release of edcfs. the formed unstable endoperoxide (prostaglandin h2, pgh2) is metabolized further into prostaglandin d2, e2, \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ { { \hbox{f}}_2}_\alpha $ $ \end{document }, i2 (prostacyclin), and thromboxane a2 by specific synthases. depending on the blood vessel studied and the agonist used to activate the endothelial cells, different prostaglandins can contribute to endothelium - dependent contractions. in the shr aorta, pgh2 and prostacyclin appear to play the dominant role, mainly because of the abundance in the endothelial cells of prostacyclin synthase, compared with the other specific synthases, resulting in the overwhelming production and release of prostacyclin [43, 47 ]. the contribution of prostacyclin to edcf - mediated responses may seem paradoxical, as one intuitively would expect the prostanoid to contribute instead to endothelium - dependent relaxations. specific prostacyclin ip receptors are predominantly coupled to the gs - adenylyl - cyclase - cyclic adenosine monophosphate (camp)-pka pathway, and their activation results in vasodilatation. a characteristic of the shr (and of aged normotensive rats) is that their vascular smooth muscle cells have lost responsiveness to ip receptor activation [4, 39 ]. when they reach their target, prostaglandins interact preferentially, but not exclusively, with specific seven transmembrane domains, g - protein - coupled receptors (dp, ep, fp, ip, and tp receptors). of all the prostanoid receptors leading to activation of vascular smooth muscle cells, tp receptors appear to be solely responsible for endothelium - dependent contractions. this conclusion is based on numerous observations that tp receptor antagonists abolish edcf - mediated responses [39, 43, 45, 47 ]. thromboxane a2 is by far the preferential physiological ligand of tp receptors, but endoperoxides (pgh2) and higher concentrations of the other prostaglandins (including prostacyclin, especially in vascular smooth muscle lacking ip receptor sensitivity), as well as isoprostanes, activate this receptor with a varying range of potency. hence, at least in the shr, endoperoxides and prostacyclin play a key role as edcfs [39, 43 ]. to judge from data obtained in the shr aorta, the tp receptors in this strain are hyperresponsive to endogenous vasoconstrictor prostanoids early in the hypertensive process. this hyperresponsiveness, the ip receptor insensitivity, and the larger release of endoperoxides and prostacyclin then explain the preponderance of endothelium - dependent contractions and the resulting endothelial dysfunction in the blood vessels (fig. 2). 2endothelium - dependent effects of acetylcholine in aorta of wistar kyoto (wky) rats and spontaneously hypertensive rats (shr). left, endothelium - dependent relaxations in normotensive wky rats. right, cyclooxygenase - dependent, endothelium - dependent contractions to acetylcholine in shr aorta. a23187calcium ionophore ; aa arachidonic acid ; ac adenylyl cyclase ; atp adenosine triphosphate ; camp cyclic adenosine monophosphate ; cav voltage - activated calcium channel ; cgmp cyclic guanosine monophosphate ; cox1cyclooxygenase 1 ; enos endothelial nitric oxide synthase ; gtp guanosine triphosphate ; ip pgi2 receptor ; m muscarinic receptor ; o2superoxide anion ; pgh2endoperoxide ; pgi2prostacyclin ; pgis prostacyclin synthase ; pla2phospholipase a2 ; p2y putinergic receptor y2 ; sgc soluble guanylyl cyclase ; sr sarcoplasmic reticulum ; tp tp receptor ; txa2thromboxane a2. (from fltou and vanhoutte. used with permission of the american physiological society.) endothelium - dependent effects of acetylcholine in aorta of wistar kyoto (wky) rats and spontaneously hypertensive rats (shr). left, endothelium - dependent relaxations in normotensive wky rats. right, cyclooxygenase - dependent, endothelium - dependent contractions to acetylcholine in shr aorta. a23187calcium ionophore ; aa arachidonic acid ; ac adenylyl cyclase ; atp adenosine triphosphate ; camp cyclic adenosine monophosphate ; cav voltage - activated calcium channel ; cgmp cyclic guanosine monophosphate ; cox1cyclooxygenase 1 ; enos endothelial nitric oxide synthase ; gtp guanosine triphosphate ; ip pgi2 receptor ; m muscarinic receptor ; o2superoxide anion ; pgh2endoperoxide ; pgi2prostacyclin ; pgis prostacyclin synthase ; pla2phospholipase a2 ; p2y putinergic receptor y2 ; sgc soluble guanylyl cyclase ; sr sarcoplasmic reticulum ; tp tp receptor ; txa2thromboxane a2. (from fltou and vanhoutte. used with permission of the american physiological society.) tp receptor antagonists given in vivo evoke no change or only minor changes in arterial blood pressure, but they limit the endothelial dysfunction associated with hypertension, diabetes, and atherosclerosis ; they are potent antithrombotic agents ; and they reduce vascular inflammation. therefore, tp receptor antagonists possess a unique potential to treat cardiovascular complications, exerting therapeutic benefits beyond those associated with cox inhibition only. actually, the addition of tp antagonism to cox-2 inhibition activity may improve the cardiovascular risk profile of cox-2 inhibitors. prostaglandin\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ { { \mathbf{f}}_{\mathbf{2}}}_{\mathbf{\alpha } } $ $ \end{document } is also a potent vasoconstrictor ; when administered in mice, it increases arterial blood pressure. in the hamster aorta. genetic deletion of the fp receptor, its preferential receptor, reduces blood pressure, and it delays the occurrence of atherosclerosis in low - density - lipoprotein (ldl) receptor deficient mice. blockade of fp receptors may offer a new approach to treating hypertension and its associated vascular complications. endothelial dysfunction, a hallmark of the hypertensive blood vessel wall, probably reflects the premature aging of the intima exposed to the chronic increase in arterial blood pressure. it is caused by complex changes in the balance between endothelium - dependent vasodilator and vasoconstrictor signals. the release of no and the occurrence of endothelium - dependent hyperpolarizations can be endangered. as a result, the production of vasoconstrictor prostanoids (mainly endoperoxides and prostacyclin) is unleashed. augmenting the bioavailability of no, favoring the occurrence of edhf - mediated responses, and preventing the release or action of edcfs all can contribute to restoring proper endothelial function and thus can delay the appearance of vascular complications resulting from the hypertensive process. | endothelial cells regulate vascular tone by releasing various contracting and relaxing factors including nitric oxide (no), arachidonic acid metabolites (derived from cyclooxygenases, lipoxygenases, and cytochrome p450 monooxygenases), reactive oxygen species, and vasoactive peptides. additionally, another pathway associated with the hyperpolarization of the underlying smooth muscle cells plays a predominant role in resistance arteries. endothelial dysfunction is a multifaceted disorder, which has been associated with hypertension of diverse etiologies, involving not only alterations of the l - arginine no - synthase soluble guanylyl cyclase pathway but also reduced endothelium - dependent hyperpolarizations and enhanced production of contracting factors, particularly vasoconstrictor prostanoids. this brief review highlights these different endothelial pathways as potential drug targets for novel treatments in hypertension and the associated endothelial dysfunction and end - organ damage. |
the term liver function tests (lft) is commonly used in clinical practice when referring to liver enzymes. however this term is misleading since these tests primarily assess liver injury rather than liver function. in addition, this term also implies that these tests are solely of hepatic origin, but in fact, they are not always specific for the liver. the serum aminotransaminases, alanine transaminase (alt, formally serum glutamic - pyruvic transaminase (sgpt)) and aspartate transaminase (ast, formally serum glutamic - oxaloacetic transaminase (sgot)), are frequently obtained in primary care for screening and diagnosis of liver diseases and have therefore led to increased number of asymptomatic patients with mild hypertransaminasemia (i.e., less than five times the upper limit of normal). a population - based survey in the united states found that hypertransaminasemia was present in 8.9 percent of the population. although the majority of these individuals will have benign conditions, a subgroup will have underlying significant liver diseases that require further evaluation and therapeutic intervention. an audit of primary care practices found that these abnormalities are often not adequately investigated, missing an important chance of identifying treatable chronic liver disease. given the importance and frequency of this issue, the primary care physician should develop a rational approach to the management of mild hypertransaminasemia. there are several published guidelines for the evaluation of asymptomatic hypertransaminasemia [1, 4, 5 ], which are mainly based on expert opinions, and a very limited number of clinical studies. understanding the basic disease processes that cause mildly hypertransaminasemia and the epidemiology of each disease can help guide the clinical evaluation and efficient use of laboratory testing. we searched pubmed using the following keywords : elevated, liver tests, liver enzymes, transaminase, and aminotransferase. the articles were excluded from our paper if they did not pertain to the topic of hypertransaminasemia, or if they were in a language other than english. the rest of our recommendations are based on data from few retrospective studies, case series (level b), or expert consensus guidelines (level c). the serums alt and ast are the most reliable and sensitive indicators of hepatocellular injury. alt, which is present mainly in the cytosol of liver and much less in the other tissues, is the most specific for liver disease, whereas ast, which has cytosolic and mitochondrial forms, is less specific for liver disease as it is found, in addition to the liver, in the heart, skeletal muscle, kidneys, brain, pancreas, lungs, leukocytes, and erythrocytes. although levels of alt and ast can be extremely elevated (greater than 15 times the upper limit of normal (uln) in cases of acute liver injury related to drugs, toxins, ischemia, and hepatitis), elevations less than 5 times the uln are much more common in primary care practice. although one study suggested that the majority of asymptomatic individuals (88%) with mild alt elevations do not have identifiable causes, a scandinavian study of 151 consecutive patients with mild to moderate elevations (42300 iu / l) of alt levels for at least 6 months revealed that identifiable causes of liver disease were more common. diagnoses included nafld in 42%, chronic hcv in 15.3%, presumed alcoholic liver disease in 8% ; autoimmune hepatitis, primary biliary cirrhosis, and alpha1 antitrypsin deficiency were much less common. both of these studies were conducted in the era of less sensitive hcv testing and therefore the true prevalence of hcv infection could not be ascertained. the ast / alt ratio can suggest certain disease patterns. in most of the liver diseases (acute or chronic), the ratio is less than or equal to 1 such as nonalcoholic fatty liver disease (nafld) and viral hepatitis (b or c), whereas, an ast / alt ratio greater than 2 characteristically is present in alcoholic hepatitis and a ratio greater than 4 suggests wilson 's disease. in addition, smaller increases in the ratio to values greater than 1.0 suggest the presence of cirrhosis in different causes of nonalcoholic liver diseases such as nafld, hepatitis b, and hepatitis c. therefore, a ratio greater than 1.0 in a cirrhotic patient is not necessarily indicative of alcoholic liver disease. furthermore, while these ratios are suggestive of certain conditions, there is too much overlap between groups to rely on them exclusively when making a diagnosis. in addition, aminotransferase levels may be normal in a subset of liver disease despite advanced histologic features. in other words there is a lack of correlation between the level of transaminases and severity of the histologic damage in patients with chronic hepatitis such as nonalcoholic steatohepatitis and hepatitis c [13, 14 ]. alt is commonly used in clinical practice as a screening, diagnostic, and monitoring test for liver diseases. however, accepted definitions and uniform measurements of the normal range have been an unsettled issue. therefore, alt level, which differentiates between asymptomatic persons who have liver disease and those who do not, is still uncertain. two recent reports from united states showed a wide variation in alt uln values across laboratories [16, 17 ]. some of this variation may be attributed to the use of different instruments or methods to perform the measurement or to differences in reference populations used to define the uln. the reference ranges for routine laboratory tests are determined based on 2 methods, either on values obtained from healthy individuals or on health outcomes. the former, which is the commonly used method, involves selecting apparently healthy individuals and setting the reference limits (upper and lower ends of the normal range) to include the central 95% of values for the test, whereas, the use of health outcome - based reference ranges requires that there is a high degree of standardization across different labs, and a precise relationship exists between adverse health outcome and a discrete level of the lab value. when using the central 95% of alt values of the normal population, 5% will have abnormal alt (2.5% are above and 2.5% are below the normal range) which means that 2.5% of normal individual will have abnormally high results. iu / l in women) has been established in 1980s when it was introduced as a surrogate marker for screening of hepatitis c among blood donors and before the implementation of anti - hcv testing and restrictive behavioral criteria for donor selection. this explains why alt uln is different between labs as those individuals were only apparently healthy, and have other reasons for increased alt. other factors also known to cause increased alt include medications, herbal supplements, and excessive ethanol ingestion. in a study of blood donors conducted in milan, italy, which included individuals at lowest risk of liver disease (no hepatitis b or c, not overweight, alcohol abuse or taking any medications, and those with normal levels of cholesterol, triglycerides, or glucose), the uln was 30 in addition, a similar result for men was obtained in a south korean outcome study of more than 90,000 men followed for liver related mortality over 8 years, where cut - off value of 30 this new uln was shown to increase sensitivity for detection of hepatitis c virus among blood donors from 40% to 61% with very slight decline in specificity from 97.6% to 95.5%. using the old uln of alt, 50% of the individuals with increased alt had no obvious cause for liver injury, but most of them (85%) had evidence of steatosis on liver ultrasound. therefore, it is clear that the current different uln used by different labs is insensitive to the presence of liver disease and does not detect limits at which risk of liver mortality seems to increase. in addition, this wide variation in levels considered abnormal between different labs leads to differences in the recognition of liver diseases and decision to treat hepatitis b virus. in the other hand, using the new lower uln might lead to labeling a large number of the populations as having liver injury when the cost of evaluation would be high and its utility is still uncertain. healthy range of alt of the target population including using similar lab methods and excluding individuals with risk factors for liver diseases. we, the authors of this review, commonly use 30 iu / l for men and 19 iu / l for women as a reference for uln of alt when dealing with patients at high risk for liver diseases. while alt is useful as an initial test in detecting liver disease, emerging data highlight its potential value as a measure of overall health and survival. there are several observational studies which have shown a strong relationship between alt activity and liver and all - cause mortality. the strongest is a population - based study from korea which included a cohort of 142,055 participants of ages between 35 and 59 years followed up to 10 years, when death certificates were used to determine survival and causes of death. in this study, the risk of death from liver disease started to increase at alt value above 20 iu / l. moreover, increased alt has been associated with the risk of death from all - cause or cardiovascular disease with risk begining to increase even at level below current uln [21, 23, 24 ]. while mortality risk may be due to unknown liver disease, it may partly be explained by the presence of metabolic syndrome in patients with nafld, in addition to alcohol consumption, which are linked to nonliver health risks. the clinical significance of mild hypertransaminasemia must be interpreted in the context of the clinical presentation. in general, symptomatic patients (i.e. with signs or symptoms of chronic liver disease or evidence of hepatic decompensation e.g. ascites, encephalopathy, coagulopathy, or portal hypertension) should be evaluated and treated in a more expeditious manner than asymptomatic patients with normal physical exam. unfortunately, there are no data available on the cost - effectiveness of evaluating patients with asymptomatic mild hypertransaminasemia, nor on the natural history of the potential liver disease in these patients. however, given the high prevalence of this abnormality in the primary care settings and the significant costs of an extensive evaluation, rational stepwise approach should be guided by the pretest probability of the underlying liver disease, the pattern of abnormalities, and suggestive features obtained from the history and physical examination. the following sections will provide this approach based on the published guidelines [1, 4, 5 ] (figure 1). a detailed history and physical examination are essential for the initial evaluation to determine whether the liver injury is acute or chronic (defined as 6 months), the underlying cause, and associated comorbidities. important considerations include : the presence of any accompanying symptoms such as abdominal pain, fever, and weight loss or symptoms of liver dysfunction such as jaundice, confusion, ascites, and leg swelling;the exposure to any medication including prescription and over - the - counter medications as well as herbal therapies;occupational exposure to other hepatotoxins and alcohol consumption;risk factors for viral hepatitis including possible parenteral exposures including transfusions, intravenous and intranasal drug use, tattoos, and sexual activity;family history of liver disease or other autoimmune disorders. the presence of any accompanying symptoms such as abdominal pain, fever, and weight loss or symptoms of liver dysfunction such as jaundice, confusion, ascites, and leg swelling ; the exposure to any medication including prescription and over - the - counter medications as well as herbal therapies ; occupational exposure to other hepatotoxins and alcohol consumption ; risk factors for viral hepatitis including possible parenteral exposures including transfusions, intravenous and intranasal drug use, tattoos, and sexual activity ; family history of liver disease or other autoimmune disorders. the physical examination should include assessment for signs of metabolic syndrome, a risk factor for nafld, such as hypertension, obesity (body mass index, bmi, and waist circumference), dyslipidemia (corneal urcus and xanthomas), and insulin resistance (acanthosis nigricans). in addition, patients should be examined for findings suggesting the presence of liver disease, including the assessment for stigmata of chronic liver disease (e.g., spider nevi, palmar erythema, gynecomastia, etc.), splenomegaly (suggestive of portal hypertension), and ascites. if the history and physical examination suggest a particular diagnosis, a targeted testing should follow. on the other hand, more than 30% of individuals with initially mild hypertransaminasemia will have normal levels on retesting. therefore, if alt elevation is less than 2 times normal and if the history and physical examination do not suggest an etiology or a laboratory error is strongly suspected, it is reasonable to repeat the test in four weeks [1, 4 ]. however, it should be cautioned that hepatitis c may present with fluctuating elevated liver enzymes levels. in addition, a period of effective lifestyle changes can be initiated including complete abstinence from alcohol, control of diabetes and hyperlipidemia, weight loss in overweight patients, and stopping or changing potentially hepatotoxic medications and supplements (figure 1). such lifestyle changes directly impact several of the causes of mild hypertransaminasemia and may be all that is needed to correct the abnormalities (table 1). additional laboratory tests for common causes of liver injury should be obtained when history and physical examination show no obvious cause (table 1). these conditions were found to be responsible for mild hypertransaminasemia in only 31% of patients. the majority of the patients (69%) had unexplained elevations, but there were strong associations with markers of metabolic syndrome and therefore may represent nafld. this observation was also confirmed in a previous study which showed that nafld was responsible of at least 80% of asymptomatic hypertransaminasemia after other causes were ruled out. the measurement of a complete blood count with platelet count, coagulation profile, and albumin should be considered if liver dysfunction is suspected. if the etiology was not reached despite the above workups, observation with lifestyle changes should be undertaken for up to 6 months. if hypertransaminasemia persists or worsens, the patient should be reevaluated and a further diagnostic testing should be performed, if necessary. the next step should include testing for nonhepatic causes, including muscles and thyroid diseases, as well as rare causes like celiac disease, based on the clinical scenario (table 1). in addition, rare liver diseases, including wilson 's disease, alpha-1 antitrypsin deficiency, and autoimmune hepatitis, should be considered at this stage (table 1). consultation with a gastroenterologist or hepatologist should be considered for the following groups of patients : patients with unexplained hypertransaminasemia on two occasions, a minimum of 6 months apart despite life style changes;symptoms or signs of liver decompensation (stigmata of chronic liver diseases, ascites, and hepatic encephalopathy);evidence of liver dysfunction (high bilirubin, low albumin, and prolonged pt or inr);evidence of liver disease where treatment beyond the withdrawal of implicated agent (alcohol or drugs) is warranted, for example, hepatitis b, hepatitis c, autoimmune hepatitis, wilson 's disease, hemochromatosis, and nafld. patients with unexplained hypertransaminasemia on two occasions, a minimum of 6 months apart despite life style changes ; symptoms or signs of liver decompensation (stigmata of chronic liver diseases, ascites, and hepatic encephalopathy) ; evidence of liver dysfunction (high bilirubin, low albumin, and prolonged pt or inr) ; evidence of liver disease where treatment beyond the withdrawal of implicated agent (alcohol or drugs) is warranted, for example, hepatitis b, hepatitis c, autoimmune hepatitis, wilson 's disease, hemochromatosis, and nafld. in general, liver biopsy has three major roles : to make a firm diagnosis (or exclude a diagnosis of any serious or significant liver disease), for assessment of prognosis (disease staging), and/or to assist in making therapeutic management decisions. in cases of mild hypertransaminasemia, it is often considered in patients in whom all noninvasive tests were negative or in patients in whom a specific liver disease has been considered but has not yet been confirmed, for example, wilson 's disease and alpha-1 antitrypsin deficiency. while it remains less likely that the biopsy will provide a diagnosis or lead to changes in management, it is often reassuring for the patient and clinician to know that there is no serious disorder. unfortunately, there are no data available on the cost - effectiveness of evaluating such patients. if the history and physical examination do not suggest a cause, a stepwise approach should be initiated based on the pretest probability of the underlying liver disease. patients with an abnormal albumin or prothrombin time or with evidence of chronic liver disease and/or hepatic decompensation should typically have more expeditious evaluations preferably by a specialist. referral to a specialist is also recommended if unexplained asymptomatic hypertransaminasemia remains elevated for six months or more. | the liver enzymes, alanine transaminase (alt) or aspartate transaminase (ast), are commonly used in clinical practice as screening as well as diagnostic tests for liver diseases. alt is more specific for liver injury than ast and has been shown to be a good predictor of liver related and all - cause mortality. asymptomatic mild hypertransaminasemia (i.e., less than five times normal) is a common finding in primary care and this could be attributed to serious underlying condition or has transient and benign cause. unfortunately, there are no good literatures available on the cost - effectiveness of evaluating patients with asymptomatic mild hypertransaminasemia. however, if the history and physical examination do not suggest a clear cause, a stepwise approach should be initiated based on pretest probability of the underlying liver disease. nonalcoholic fatty liver disease is becoming the most common cause of mild hypertransaminasemia worldwide. other causes include alcohol abuse, medications, and hepatitis b and c. less common causes include hemochromatosis, 1-antitrypsin deficiency, autoimmune hepatitis, and wilson 's disease. nonhepatic causes such as celiac disease, thyroid, and muscle disorders should be considered in the differential diagnosis. referral to a specialist and a possible liver biopsy should be considered if persistent hypertransaminasemia for six months or more of unclear etiology. |
introduced by cowley in the 1970s, the golden hour concept has since governed planning and preparedness in trauma systems development as a major source of morbidity and mortality to the population that contributes to the economy, i.e., people less than 45 years of age, trauma is a significant problem that needs continuous systems improvement. an integrated trauma system improves outcomes for patients of trauma, but the exact parameters that determine effectiveness have yet to be conclusively defined. the relationship between shortened pre - hospital time and improved survival exists, but is most consistently demonstrated when comparing urban and rural locals. no data exists that specifically quantifies the relationship between pre - hospital time and outcomes within purely urban areas, and especially in patients sustaining penetrating trauma. these patients are most likely to benefit from expedited transport if definitive surgical management is required. current literature has failed to study the association between pre - hospital transport time and survival for penetrating trauma patients. the studies that have examined the urban setting have yielded contradictory conclusions ; sloan and petri in chicago evaluated blunt and penetrating trauma patients and did not find a relationship between transport time and outcome. pepe in houston assessed hypotensive patients with penetrating trauma and was unable to demonstrate any relationship. gervin and feero, on the other hand, demonstrated that shorter pre - hospital time improved survival. the contradictory results may be secondary to the heterogeneity of the trauma patient population at different locations. trauma centers in many high - volume locations do not have significant number of penetrating trauma patients. this begs further investigation to define the influence of pre - hospital time factors on trauma outcomes in urban environments and in particular, in patients with penetrating trauma. our study explores the impact of pre - hospital times on penetrating trauma outcomes within urban centers of illinois. we hypothesized that there would be a relationship between total pre - hospital time and outcomes in patients with penetrating thoracic trauma, an injury that is potentially lethal without emergent intervention. since injury severity is strongly associated with mortality, we specifically studied the patients at highest risk, hypotensive patients who would be most likely to have hemorrhage that would benefit from rapid surgical control. this registry contains data from all traumatically injured patients brought to the 67 level i and level ii trauma centers in illinois. the data are entered by a registrar at each trauma center and uploaded to the illinois department of public health, who maintains the data set and website. not included in this registry are patients who are declared dead - on - scene and not transported to a trauma center and patients not treated at or transferred to a level i or level ii trauma center. the emergency medical system (ems) in illinois is a tiered system with paramedics trained in both basic life support and advanced life support techniques. the chicago committee on trauma works with the state of illinois and pre - hospital providers to ensure safe, standardized care and undergoes rigorous quality improvement scrutiny. the illinois state trauma registry (istr) includes approximately 45,000 records per year. to limit our study to urban trauma patients, we selected patients of all ages injured in the major urban areas of chicago, east st. louis, and springfield (more than 2/3 of cases annually). because our question of interest was the effect of transport time on mortality, we excluded all transfer patients, as well as walk - in and self - transport patients (10% of cases annually). we identified patients with penetrating injuries using information regarding the primary mechanism of injury recorded in the database (approximately 2,500 cases per year) and then classified those patients with thoracic trauma using icd-9 cm codes. the final list of icd-9 codes was limited to penetrating thoracic injuries (codes 860 - 862, approximately 150 cases per year), consistent with the center for medicaid and medicare services (cms) designations. the pre - hospital vital sign data were poorly conserved with fifty percent missing ; for that reason, hypotension was defined as a systolic blood pressure (sbp) less than 90 mmhg on the first reading in the emergency department. total pre - hospital time consisted of response time, scene time, and transport time. each of these variables was evaluated separately and in aggregate as total pre - hospital time. student 's t - test was used as the test statistic for the comparisons of mean pre - hospital time between cohorts. the injury severity score (iss) was used as an anatomic marker of injury severity and was highly conserved in the data set (< 3% missing data). a two - sample test of proportions and pearson 's chi - squared test were used to compare survival among patients with iss < 16 and 16 and total pre - hospital time. logistic regression statistics were performed to determine the independent effect of transport time on mortality when controlling for age, race, and severity of injury for both hypotensive patients with penetrating thoracic injuries (hpti) and non - hypotensive patients with penetrating thoracic injuries (npti). for the models, transport times were categorized into 15 min intervals to determine the independent effect of successively longer transport times. a total of 908 patients with penetrating thoracic injuries (pti)were evaluated during the study period. one - hundred forty - three patients (16%) were hypotensive on presentation, with a systolic blood pressure less than 90mmhg (hypotensive pti group = hpti), while 765 patients (84%) had systolic blood pressures greater than 90 mmhg (normotensive pti group = npti). the mean age was approximately 27 years (age range 11 - 78, 99% between ages 15 and 55) and the majority of patients were african american, male and uninsured [table 1 ]. thirty percent (230) of npti patients had an iss greater than 16, while 55% (79) of hpti patients had an iss greater than 16 (p < 0.001). the time for paramedics to get to the scene (mean response time, 4.5 min vs. 5.4, p = 0.05), mean transport time (8.9 min vs. 11.2, p = 0.02) and total pre - hospital time (25.67 vs. 29.47, p = 0.02) were significantly shorter for hpti patients with an iss 16 compared to hpti patients with iss < 16. however, mean scene times did not differ (p = 0.64) [table 2 ]. injury severity and transport times, bivariate statistics crude mortality was higher for hpti patients compared with npti patients (73% vs. 12%, p < 0.001). hypotensive patients with penetrating thoracic injuries (hpti) patients showed increased mortality with increased total pre - hospital time [figure 1 ]. the odds ratio (or) of death for hpti patients, similarly, increased with increasing total pre - hospital time [table 3, figure 2 ]. for instance, if pre - hospital time was greater than 40 min, then the or was increased to 13 (p < 0.001). table 3 reports the regression results for hpti patients. non - hypotensive patients with penetrating thoracic injuries (npti) regression results are not included, but the covariates for age, race, gender, and iss were of similar direction and magnitude. conversely, npti patients with longer total pre - hospital times exhibited decreased mortality (or 0.93, 95% ci 0.83 - 0.99, p < 0.001). death ratefor non - hypotensive patients with penetrating thoracic injuries and hypotensive patients with penetrating thoracic injuries patients grouped by pre - hospital time adjusted risk of death for hpti patients by pre - hospital time rr of death for hypotensive patients with penetrating thoracic injuries vs. non - hypotensive patients with penetrating thoracic injuries patients grouped by pre - hospital time of the survivors, the majority of patients, in both npti and hpti groups, were discharged directly to home. there were no significant differences in the discharge destination for ntpi and hpti patients [table 4 ]. the study investigates association between transport time and outcome for urbanvictims of penetrating thoracic trauma. the analysis indicates that more severely injured patients may survive devastating injuries with rapid transit : the sicker the patient, the quicker they should be in the trauma bay. a significantly positive finding of this study was that patients with higher injury severity experienced shorter pre - hospital transport time. this finding in our study is not unique ; newgard demonstrated this in a multi - institutional study mixed population of blunt and penetrating trauma. given the significant correlation between pre - hospital time and mortality for hpti patients, this is clearly a success of the urban trauma system. the association of transport time with mortality has been well described in rural trauma systems, but never clearly described in the urban setting, with a homogeneous population of patients. this study is one of the first to show a consistent association between pre - hospital time and survival in an urban trauma system with uniform professional pre - hospital ems care. variations in transport times might be due to travel distances to the nearest trauma center from incident scenes, traffic congestion or road conditions, or weather conditions. it is possible with new global positioning systems (gps) data that incorporates traffic congestion and travel distances, pre - hospital times may be shortened. some rural trauma systems have augmented their pre - hospital services with aeromedical transporter provided increased trauma training for first responders to decrease the impact of longer transport times on injury mortality. these options might be employed to a greater degree in urban trauma systems as well. however, comprehensive changes in a system that is working well for most patients may not be indicated or cost - effective. it is important to note that our findings are limited to a small portion of all trauma patients : in our database, pti patients comprised approximately 2% of all trauma patients and hpti patients comprised only 0.4% of all trauma patients. it is likely that many, less severely injured will not experience a survival benefit associated with decreased pre - hospital time ; as notably seen in the npti group, in whom survival was not adversely affected by increased transport time, and was actually improved. it is important to recognize that the majority of the overall trauma patient population with penetrating thoracic injuries did not benefit from shorter transport times. in addition, pre - hospital data were missing up to 50% of the time, including sbp. though no systematic changes have occurred in urban pre - hospital transport during that time, additional years of data should be accessed when available to validate these results. despite these limitations, these results suggest that careful planning to optimize transport time - encompassing hospital capacity and existing resources, traffic patterns, and trauma incident densities - may be beneficial in areas with a high burden of penetrating trauma. in conclusion, we have shown that minimizing pre - hospital times does in fact impact survival in patients that are hypotensive victims of penetrating thoracic trauma. hpti patients in an optimally designed trauma system should be delivered to definitive care expeditiously. | background : achieving definitive care within the golden hour by minimizing response times is a consistent goal of regional trauma systems. this study hypothesizes that in urban level i trauma centers, shorter pre - hospital times would predict outcomes in penetrating thoracic injuries.materials and methods : a retrospective cohort study was performed using a statewide trauma registry for the years 1999 - 2003. total pre - hospital times were measured for urban victims of penetrating thoracic trauma. crude and adjusted mortality rates were compared by pre - hospital time using stata statistical software.results:during the study period, 908 patients presented to the hospital after penetrating thoracic trauma, with 79% surviving. patients with higher injury severity scores (iss) were transported more quickly. injury severity scores (iss) 16 and emergency department (ed) hypotension (systolic blood pressure, sbp < 90) strongly predicted mortality (p < 0.05 for each). in a logistic regression model including age, race, and iss, longer transport times for hypotensive patients were associated with higher mortality rates (all p values < 0.05). this was seen most significantly when comparing patient transport times 0 - 15 min and 46 - 60 min (p < 0.001).conclusion : in victims of penetrating thoracic trauma, more severely injured patients arrive at urban trauma centers sooner. mortality is strongly predicted by injury severity, although shorter pre - hospital times are associated with improved survival. these results suggest that careful planning to optimize transport time - encompassing hospital capacity and existing resources, traffic patterns, and trauma incident densities may be beneficial in areas with a high burden of penetrating trauma. |
this retrospective study included 40 dogs that were diagnosed with biliary sludge and gallbladder mucoceles, and had their gallbladder contents collected during surgery or autopsy at the university of miyazaki veterinary teaching hospital, miyazaki, japan, between may 2012 and july 2016. component analysis was performed using 43 samples of gallbladder contents from 40 dogs diagnosed with biliary sludge or gallbladder mucoceles for three samples, the same animal was re - examined over the course of follow - up. samples that were diagnosed by autopsy were from dogs that either died of natural causes or were euthanized for reasons other than gallbladder diseases. euthanization was conducted as below : first, dogs were administered with pentobarbital sodium (50 mg / kg, iv ; somnopentyl, kyoritsu seiyaku corp., five min later, potassium chloride (2 meq / kg, iv ; kcl corrective injection 1 meq / ml, otsuka pharmaceutical factory, inc. biliary sludge and gallbladder mucoceles were diagnosed by gross examination during surgery or autopsy. biliary sludge was diagnosed when the gallbladder contents presented a sludge - like appearance. gallbladder mucoceles were diagnosed when the gallbladder appeared to be completely filled with a jelly - like substance (figs. biliary sludge presents as hyperechogenic accumulated matter in the direction of gravity on ultrasonography images mild (a), moderate (b) and severe (c). gallbladder contents in moderate biliary sludge (d) have a black sand - like appearance, while those in severe biliary sludge (e) have a black sludge - like appearance. biliary sludge was classified according to its rate of retention within the gallbladder as mild (25%), moderate (2575%) or severe (75%). and 2fig.. however, gallbladder contents in gallbladder mucoceles present varied appearances ; in the present study, while gallbladder contents in a few mucoceles presented a black and jelly - like appearance (c), those in other mucoceles presented as an indurated, jelly - like material radiating to the margin (d).). biliary sludge was classified according to severity based on the content retention rate relative to size of the gallbladder determined by ultrasonography, as follows : mild (25% retention), moderate (2575% retention) or severe (75% retention ; fig. biliary sludge presents as hyperechogenic accumulated matter in the direction of gravity on ultrasonography images mild (a), moderate (b) and severe (c). gallbladder contents in moderate biliary sludge (d) have a black sand - like appearance, while those in severe biliary sludge (e) have a black sludge - like appearance. biliary sludge was classified according to its rate of retention within the gallbladder as mild (25%), moderate (2575%) or severe (75%). ultrasonography images and gallbladder content findings of gallbladder mucoceles. pattern within the gallbladder on abdominal ultrasonography images (a and b). however, gallbladder contents in gallbladder mucoceles present varied appearances ; in the present study, while gallbladder contents in a few mucoceles presented a black and jelly - like appearance (c), those in other mucoceles presented as an indurated, jelly - like material radiating to the margin (d). of the 40 dogs included in the present study, 18 (21 samples) exhibited biliary sludge, and 22 (22 samples) exhibited gallbladder mucoceles. dogs included in this study were of the following breeds : miniature dachshund, chihuahua and toy poodle (n=6, each) ; miniature schnauzer (n=4) ; pug and mixed breed (n=3, each) ; american cocker spaniel, shetland sheepdog, yorkshire terrier, beagle, french bulldog and doberman (n=2, each) and pomeranian, shiba inu and papillon (n=1, each). the median age of the 40 dogs (43 samples) included in the present study (male, 5 ; castrated male, 11 ; female, 7 ; and spayed female, 20) was 9 years (range, 115 years, including one dog of unknown age), and the median weight was 5.2 kg (range, 1.929.0 kg). all dogs were examined with the owners consent having been obtained prior to surgical treatment (cholecystectomy, 25 dogs and gallbladder irrigation, 15 dogs) or autopsy (3 dogs). gallbladder irrigation was performed by making a small incision on the gallbladder wall, followed by aspiration of contents in the lumen of the gallbladder and irrigation and cleaning up of the inside of the gallbladder with saline. for component analysis, 43 canine gallbladder content samples with bile (biliary sludge, 21 and gallbladder mucoceles, 22) were first dried well and then prepared as potassium bromide (kbr) pellets for evaluation by ir spectroscopy (ir spectroscopy ft / ir-410, jasco corp., tokyo, japan.). in a previous study on the ir spectroscopy of ovary - derived mucins therefore, mucins derived from swine stomach (swine mucin ; purity 80%, wako pure chemical industries, ltd., tokyo, japan.) were used to establish the reference ir spectrum for mucins in this study. the ir spectra of biliary sludge and gallbladder mucoceles contents were compared with that of swine mucin in order to determine the composition of the former. of the 43 canine gallbladder content samples, 41 (biliary sludge, 20 and gallbladder mucoceles, 21) were evaluated by bacterial culture. the animals were administered antimicrobials after collection of gallbladder contents, but not within 12 hr prior to collection. bacterial cultures of gallbladder contents were established by aseptically swabbing the inside of the gallbladder with a sterile cotton swab and inoculating the samples onto bacterial media for aerobic and anaerobic culture, followed by bacterial identification. as determined by ultrasonographic findings, there was no mild case, moderate was 10 cases, with all samples exhibiting biliary sludge retention rates 50%, and severe was 11 cases. the ir spectrum of swine mucin corresponded to proteins, with amide bands near 1,650 and 1,550 cm, and polysaccharides, with spectral characteristics of sugar chains (glycosylation bands) near 1,070 and 1,050 cm(fig. swine mucin exhibits characteristic absorption bands of proteins (amide bands near 1,650 and 1,550 cm ; indicated by arrowheads) and sugar chains (glycosylation bands near 1,070 and 1,050 cm ; indicated by arrows).). this ir spectrum was used as the reference for identification of mucins in gallbladder contents. swine mucin exhibits characteristic absorption bands of proteins (amide bands near 1,650 and 1,550 cm ; indicated by arrowheads) and sugar chains (glycosylation bands near 1,070 and 1,050 cm ; indicated by arrows). of the 21 samples of gallbladder contents of dogs with biliary sludge, 20 (95.2%)ten samples each of moderate and severe biliary sludge exhibited ir spectra coinciding with amide and glycosylation bands similar to those observed with swine mucin and were thus identified as being composed of mucins (fig. ir spectra of swine mucin (a ; dotted line) and severe (b ; solid line) and moderate (c ; solid line) biliary sludge. ir spectra of severe biliary sludge that was identified as proteins (d ; solid line). moderate (b) and severe (c) biliary sludge both exhibited amide bands (near 1,650 and 1,550 cm ; indicated by arrowheads) and glycosylation bands (near 1,070 and 1,050 cm ; indicated by arrows), with the exception of one sample (d) of severe biliary sludge, where only amide bands were observed. ir spectra of only representative samples are presented in b and c ; b (n=9) and c (n=10) exhibited similar spectra.). the ir spectrum of the one remaining sample (4.8%), which was from a dog with severe biliary sludge, did not exhibit clear glycosylation bands and instead presented only amide bands ; this sample was, therefore, identified as being composed of proteins (fig. ir spectra of swine mucin (a ; dotted line) and severe (b ; solid line) and moderate (c ; solid line) biliary sludge. ir spectra of severe biliary sludge that was identified as proteins (d ; solid line). moderate (b) and severe (c) biliary sludge both exhibited amide bands (near 1,650 and 1,550 cm ; indicated by arrowheads) and glycosylation bands (near 1,070 and 1,050 cm ; indicated by arrows), with the exception of one sample (d) of severe biliary sludge, where only amide bands were observed. ir spectra of only representative samples are presented in b and c ; b (n=9) and c (n=10) exhibited similar spectra. all 22 samples of gallbladder contents of dogs with gallbladder mucoceles exhibited ir spectra coinciding with amide and glycosylation bands similar to those observed with swine mucin and were thus identified as mucins (fig. 5fig. ir spectra of swine mucin (a ; dotted line) and gallbladder mucoceles (b ; solid line). gallbladder mucoceles (b) exhibited amide bands (near 1,650 and 1,550 cm ; indicated by arrowheads) and glycosylation bands (near 1,070 and 1,050 cm ; indicated by arrows). ir spectrum of only a representative sample is presented in b. the gallbladder contents of all gallbladder mucoceles (n=21) exhibited similar spectra.). the ir spectra of biliary sludge and gallbladder mucoceles contents were found to be similar. ir spectra of swine mucin (a ; dotted line) and gallbladder mucoceles (b ; solid line). gallbladder mucoceles (b) exhibited amide bands (near 1,650 and 1,550 cm ; indicated by arrowheads) and glycosylation bands (near 1,070 and 1,050 cm ; indicated by arrows). ir spectrum of only a representative sample is presented in b. the gallbladder contents of all gallbladder mucoceles (n=21) exhibited similar spectra. three of the dogs with biliary sludge received gallbladder irrigation, followed by 12 years of follow - up, at the end of which, progressively accumulated gallbladder contents were again collected by gallbladder irrigation. of the three dogs, one underwent cholecystectomy for extrahepatic bile duct obstruction due to re - accumulation of gallbladder contents about 1 year and 8 months after the second gallbladder irrigation. unlike the other two dogs, the gallbladder contents of this dog at the time of the cholecystectomy had clearly transitioned from sludge - like to jelly - like appearance. therefore, the dog was diagnosed with a gallbladder mucocele rather than biliary sludge based on the findings of analysis of gallbladder contents from the third collection (fig. ultrasonography findings at 1 year and 7 months after first collection (a) and 10 days after second collection (b) indicated biliary sludge. gallbladder contents of both first and second collections were similar and determined to be biliary sludge (c). ultrasonography findings at 1 year and 8 months after second collection when extrahepatic duct obstruction occurred (d). gallbladder content findings at third collection indicated a gallbladder mucocele (f). for treatment of biliary sludge, the inside of the gallbladder was cleaned with saline, and all of the sludge - like content (c) was collected (b). however, biliary sludge recurred and accumulated progressively (d). during the interval of 1 year and 7 months between the initial collection of biliary sludge and second collection of gallbladder contents, the dog exhibited no clinical symptoms of extrahepatic bile duct obstruction, and its recovery was uneventful. however, upon recurrence of biliary sludge, the patient received additional follow - up for 1 year and 8 months. to resolve the extrahepatic bile duct obstruction observed during follow - up (d), the contents of the excised gallbladder had changed in appearance from sludge (c) to jelly - like consistency (f), leading to the diagnosis of gallbladder mucocele.). the gallbladder contents of this dog exhibited nearly similar ir spectra at all three collections and were thus identified each time as being composed of mucins (fig. 7fig. (b) collections, both of which were determined to be biliary sludge. ir spectra of gallbladder contents from the third collection (c), which was determined to be from a gallbladder mucocele. gallbladder contents from the first, second and third collections exhibited similar ir spectra ; both amide bands (near 1,650 and 1,550 cm ; indicated by arrowheads) and glycosylation bands (near 1,070 and 1,050 cm ; indicated by arrows) were observed, and the contents were identified as mucins.). the gallbladder contents of the remaining two dogs presented a sludge - like appearance at the first and second collections, based on which the dogs were diagnosed with biliary sludge. the gallbladder contents were identified as being composed of mucins based on their ir spectroscopic findings. ultrasonography and gallbladder content ultrasonography findings at 1 year and 7 months after first collection (a) and 10 days after second collection (b) indicated biliary sludge. gallbladder contents of both first and second collections were similar and determined to be biliary sludge (c). ultrasonography findings at 1 year and 8 months after second collection when extrahepatic duct obstruction occurred (d). gallbladder content findings at third collection indicated a gallbladder mucocele (f). for treatment of biliary sludge, the inside of the gallbladder was cleaned with saline, and all of the sludge - like content (c) was collected (b). however, biliary sludge recurred and accumulated progressively (d). during the interval of 1 year and 7 months between the initial collection of biliary sludge and second collection of gallbladder contents, the dog exhibited no clinical symptoms of extrahepatic bile duct obstruction, and its recovery was uneventful. however, upon recurrence of biliary sludge, the patient received additional follow - up for 1 year and 8 months. to resolve the extrahepatic bile duct obstruction observed during follow - up (d), the patient underwent cholecystectomy (e). the contents of the excised gallbladder had changed in appearance from sludge (c) to jelly - like consistency (f), leading to the diagnosis of gallbladder mucocele. second (b) collections, both of which were determined to be biliary sludge. ir spectra of gallbladder contents from the third collection (c), which was determined to be from a gallbladder mucocele. gallbladder contents from the first, second and third collections exhibited similar ir spectra ; both amide bands (near 1,650 and 1,550 cm ; indicated by arrowheads) and glycosylation bands (near 1,070 and 1,050 cm ; indicated by arrows) were observed, and the contents were identified as mucins. the infective bacterial species were identified to be escherichia coli and serratia marcescens in one sample each. of the 21 gallbladder mucocele samples, 3 (14.3%) exhibited bacterial infection. the infective bacterial species were identified to be escherichia coli in two samples and enterococcus spp. in one sample. as determined by ultrasonographic findings, there was no mild case, moderate was 10 cases, with all samples exhibiting biliary sludge retention rates 50%, and severe was 11 cases. the ir spectrum of swine mucin corresponded to proteins, with amide bands near 1,650 and 1,550 cm, and polysaccharides, with spectral characteristics of sugar chains (glycosylation bands) near 1,070 and 1,050 cm(fig. swine mucin exhibits characteristic absorption bands of proteins (amide bands near 1,650 and 1,550 cm ; indicated by arrowheads) and sugar chains (glycosylation bands near 1,070 and 1,050 cm ; indicated by arrows).). this ir spectrum was used as the reference for identification of mucins in gallbladder contents. swine mucin exhibits characteristic absorption bands of proteins (amide bands near 1,650 and 1,550 cm ; indicated by arrowheads) and sugar chains (glycosylation bands near 1,070 and 1,050 cm ; indicated by arrows). of the 21 samples of gallbladder contents of dogs with biliary sludge, 20 (95.2%)ten samples each of moderate and severe biliary sludge exhibited ir spectra coinciding with amide and glycosylation bands similar to those observed with swine mucin and were thus identified as being composed of mucins (fig. ir spectra of swine mucin (a ; dotted line) and severe (b ; solid line) and moderate (c ; solid line) biliary sludge. ir spectra of severe biliary sludge that was identified as proteins (d ; solid line). moderate (b) and severe (c) biliary sludge both exhibited amide bands (near 1,650 and 1,550 cm ; indicated by arrowheads) and glycosylation bands (near 1,070 and 1,050 cm ; indicated by arrows), with the exception of one sample (d) of severe biliary sludge, where only amide bands were observed. ir spectra of only representative samples are presented in b and c ; b (n=9) and c (n=10) exhibited similar spectra.). the ir spectrum of the one remaining sample (4.8%), which was from a dog with severe biliary sludge, did not exhibit clear glycosylation bands and instead presented only amide bands ; this sample was, therefore, identified as being composed of proteins (fig. ir spectra of swine mucin (a ; dotted line) and severe (b ; solid line) and moderate (c ; solid line) biliary sludge. ir spectra of severe biliary sludge that was identified as proteins (d ; solid line). moderate (b) and severe (c) biliary sludge both exhibited amide bands (near 1,650 and 1,550 cm ; indicated by arrowheads) and glycosylation bands (near 1,070 and 1,050 cm ; indicated by arrows), with the exception of one sample (d) of severe biliary sludge, where only amide bands were observed. ir spectra of only representative samples are presented in b and c ; b (n=9) and c (n=10) exhibited similar spectra. all 22 samples of gallbladder contents of dogs with gallbladder mucoceles exhibited ir spectra coinciding with amide and glycosylation bands similar to those observed with swine mucin and were thus identified as mucins (fig. ir spectra of swine mucin (a ; dotted line) and gallbladder mucoceles (b ; solid line). gallbladder mucoceles (b) exhibited amide bands (near 1,650 and 1,550 cm ; indicated by arrowheads) and glycosylation bands (near 1,070 and 1,050 cm ; indicated by arrows). ir spectrum of only a representative sample is presented in b. the gallbladder contents of all gallbladder mucoceles (n=21) exhibited similar spectra.). the ir spectra of biliary sludge and gallbladder mucoceles contents were found to be similar. ir spectra of swine mucin (a ; dotted line) and gallbladder mucoceles (b ; solid line). gallbladder mucoceles (b) exhibited amide bands (near 1,650 and 1,550 cm ; indicated by arrowheads) and glycosylation bands (near 1,070 and 1,050 cm ; indicated by arrows). ir spectrum of only a representative sample is presented in b. the gallbladder contents of all gallbladder mucoceles (n=21) exhibited similar spectra. three of the dogs with biliary sludge received gallbladder irrigation, followed by 12 years of follow - up, at the end of which, progressively accumulated gallbladder contents were again collected by gallbladder irrigation. of the three dogs, one underwent cholecystectomy for extrahepatic bile duct obstruction due to re - accumulation of gallbladder contents about 1 year and 8 months after the second gallbladder irrigation. unlike the other two dogs, the gallbladder contents of this dog at the time of the cholecystectomy had clearly transitioned from sludge - like to jelly - like appearance. therefore, the dog was diagnosed with a gallbladder mucocele rather than biliary sludge based on the findings of analysis of gallbladder contents from the third collection (fig. ultrasonography findings at 1 year and 7 months after first collection (a) and 10 days after second collection (b) indicated biliary sludge. gallbladder contents of both first and second collections were similar and determined to be biliary sludge (c). ultrasonography findings at 1 year and 8 months after second collection when extrahepatic duct obstruction occurred (d). gallbladder content findings at third collection indicated a gallbladder mucocele (f). for treatment of biliary sludge, the inside of the gallbladder was cleaned with saline, and all of the sludge - like content (c) was collected (b). however, biliary sludge recurred and accumulated progressively (d). during the interval of 1 year and 7 months between the initial collection of biliary sludge and second collection of gallbladder contents, the dog exhibited no clinical symptoms of extrahepatic bile duct obstruction, and its recovery was uneventful. however, upon recurrence of biliary sludge, the patient received additional follow - up for 1 year and 8 months. to resolve the extrahepatic bile duct obstruction observed during follow - up (d), the contents of the excised gallbladder had changed in appearance from sludge (c) to jelly - like consistency (f), leading to the diagnosis of gallbladder mucocele.). the gallbladder contents of this dog exhibited nearly similar ir spectra at all three collections and were thus identified each time as being composed of mucins (fig. 7fig. (b) collections, both of which were determined to be biliary sludge. ir spectra of gallbladder contents from the third collection (c), which was determined to be from a gallbladder mucocele. gallbladder contents from the first, second and third collections exhibited similar ir spectra ; both amide bands (near 1,650 and 1,550 cm ; indicated by arrowheads) and glycosylation bands (near 1,070 and 1,050 cm ; indicated by arrows) were observed, and the contents were identified as mucins.). the gallbladder contents of the remaining two dogs presented a sludge - like appearance at the first and second collections, based on which the dogs were diagnosed with biliary sludge. the gallbladder contents were identified as being composed of mucins based on their ir spectroscopic findings. ultrasonography findings at 1 year and 7 months after first collection (a) and 10 days after second collection (b) indicated biliary sludge. gallbladder contents of both first and second collections were similar and determined to be biliary sludge (c). ultrasonography findings at 1 year and 8 months after second collection when extrahepatic duct obstruction occurred (d). gallbladder content findings at third collection indicated a gallbladder mucocele (f). for treatment of biliary sludge, the inside of the gallbladder was cleaned with saline, and all of the sludge - like content (c) was collected (b). however, biliary sludge recurred and accumulated progressively (d). during the interval of 1 year and 7 months between the initial collection of biliary sludge and second collection of gallbladder contents, the dog exhibited no clinical symptoms of extrahepatic bile duct obstruction, and its recovery was uneventful. however, upon recurrence of biliary sludge, the patient received additional follow - up for 1 year and 8 months. to resolve the extrahepatic bile duct obstruction observed during follow - up (d), the contents of the excised gallbladder had changed in appearance from sludge (c) to jelly - like consistency (f), leading to the diagnosis of gallbladder mucocele. ir spectra of gallbladder contents from the first (a) and second (b) collections, both of which were determined to be biliary sludge. ir spectra of gallbladder contents from the third collection (c), which was determined to be from a gallbladder mucocele. gallbladder contents from the first, second and third collections exhibited similar ir spectra ; both amide bands (near 1,650 and 1,550 cm ; indicated by arrowheads) and glycosylation bands (near 1,070 and 1,050 cm ; indicated by arrows) were observed, and the contents were identified as mucins. the infective bacterial species were identified to be escherichia coli and serratia marcescens in one sample each. of the 21 gallbladder mucocele samples, 3 (14.3%) exhibited bacterial infection. the infective bacterial species were identified to be escherichia coli in two samples and enterococcus spp. in one sample. canine gallbladder diseases especially gallbladder mucoceles are caused by excessive mucin secretion. however, to our knowledge, there are no reports regarding component analysis of actual contents from gallbladder mucoceles. a previous study performed ir spectroscopic analysis of human ovary - derived mucins with the ir spectrum of bovine submaxillary gland - derived mucins as reference. therefore, in the present study, the ir spectrum of swine mucin was employed as the reference for comparative evaluation of ir spectra of biliary sludge and gallbladder mucocele samples. nearly all of the gallbladder content samples of dogs with biliary sludge (95.2%) exhibited similar ir spectra as swine mucin, irrespective of the severity of biliary sludge, which ranged from moderate (5075% biliary sludge) to severe (fig. 4). in case of gallbladder mucocele contents, all samples exhibited similar ir spectra as swine mucin (fig. 5). these findings indicated that the principal components of gallbladder contents in both biliary sludge and gallbladder mucoceles were mucins, which suggested the possibility that mucins were involved in the pathogenesis of not only gallbladder mucoceles but also biliary sludge. 7, it was observed that there were some changes between biliary sludge (a and b) and gallbladder mucocele (c). in particular, these changes were observed near 1,460, 1,375 and 1,055 cm in fig. 7 (c). these were characteristic ir spectra showing cholesterol. it was suggested that these ir spectra were possible to have been affected by cholesterol because cholesterol was contained in the bile. therefore, it was possible that amount of cholesterol in the gallbladder content was increased with varying from biliary sludge to gallbladder mucocele in this case. although these characteristic ir spectra showing cholesterol were observed in some of gallbladder mucocele cases, not in all gallbladder mucocele cases. therefore, since all gallbladder mucocele samples in this study showed amide bands and glycosylation bands, it was considered that the principal component of gallbladder mucocele was mucin. in present study, the gallbladder contents of one of the dogs with biliary sludge were identified as being composed of proteins (fig. the gallbladder in this case had presented extensive bleeding upon histopathological examination, which indicated the possibility that gallbladder bleeding affected the composition of the biliary sludge contents. in order to verify these hypothesis, the ir spectrum of a clot of peripheral venous blood from a healthy dog was compared with that of the gallbladder contents in this case. the ir spectrum of the blood clot exhibited amide bands but no glycosylation bands, and therefore, the blood clot was identified as being composed of proteins. the ir spectrum of the proteinaceous biliary sludge sample was found to be similar to that of the blood clot (fig. ir spectra of swine mucin (a ; dotted line) and a blood clot from a healthy dog (b ; dashed line). gallbladder contents of biliary sludge determined as being composed of proteins (c ; solid line). the blood clot exhibited only amide bands in its ir spectrum and was, therefore, determined as being composed of proteins. the ir spectra of the blood clot and the biliary sludge determined as being composed of proteins were similar. arrowheads indicate amide bands (near 1,650 and 1,550 cm) ; arrows indicate glycosylation bands (near 1,070 and 1,050 cm).), which may be attributable to the mixing of biliary sludge with plasma proteins from blood because of gallbladder bleeding. these findings suggest that, in cases where histopathological findings reveal gallbladder bleeding, the composition of gallbladder contents might be different from mucin. ir spectra of swine mucin (a ; dotted line) and a blood clot from a healthy dog (b ; dashed line). gallbladder contents of biliary sludge determined as being composed of proteins (c ; solid line). the blood clot exhibited only amide bands in its ir spectrum and was, therefore, determined as being composed of proteins. the ir spectra of the blood clot and the biliary sludge determined as being composed of proteins were similar. arrowheads indicate amide bands (near 1,650 and 1,550 cm) ; arrows indicate glycosylation bands (near 1,070 and 1,050 cm). both biliary sludge and gallbladder mucocele samples exhibited a low incidence of bacterial infection (10.0 and 14.3%, respectively). a previous study reported a 13.5% rate of bacterial infection in gallbladder mucoceles, which corresponds with the findings of the present study. several bacterial species identified in gallbladder infections are among those that constitute normal intestinal flora. therefore, it is possible that the cases of gallbladder infection observed in the present study were due to ascending infection from the duodenum. the findings of component analysis of gallbladder contents from canine biliary sludge and gallbladder mucoceles in the present study have demonstrated the previously unreported fact that mucins are the principle components of gallbladder content in both pathophysiologies. there were no differences in gallbladder composition among biliary sludge samples of various severities. additionally, the low rates of bacterial infection in both biliary sludge and gallbladder mucocele samples indicated that these two disease differ in pathophysiology from gallstones. from the results of this study, it was considered that the pathophysiology of canine biliary sludge may be occurred by progressive accumulation of mucin in the gallbladder. in one of the dogs included in the present study, gallbladder disease progressed from biliary sludge to gallbladder mucocele (fig. these findings suggest not only that biliary sludge and gallbladder mucoceles share a pathophysiology involving the progressive accumulation of mucins, but also that biliary sludge possibly represents the stage preceding gallbladder mucoceles. however, the factors responsible for the transition of gallbladder contents from sludge to jelly - like appearance in the present study are not clear. as a criterion for differentiating between the sludge and jelly - like appearances of gallbladder contents, in the present study, a resected gallbladder with contents that were semisolid, low in liquid content and appeared molded to the gallbladder was diagnosed as a gallbladder mucocele. the water content of gallbladder contents might be one of the factors responsible for the transition of gallbladder contents from sludge to jelly - like appearance. all of gallbladder contents in this study were dried with bile and analyzed by ir spectroscopy. generally, the components contained in bile are bile acid, cholesterol, lecithin, bilirubin and more, and do not contain volatile components. therefore, these analyses by ir spectroscopy were carried out in gallbladder contents which contained liquid components, and it was considered that only the water content of gallbladder contents was changed by drying. moisture in bile is removed by the actions of the na k and cl hco3 pumps of gallbladder epithelial cells [19, 21 ] and / or based on reports in mice aquaporins, which are channels responsible for transporting water into the body [18, 24 ]. these transport channels have been reported to possess the ability to concentrate bile or manipulate the composition of gallbladder bile. therefore, abnormal functioning of bile moisture absorption mechanisms might be involved in the pathophysiology of canine biliary sludge and gallbladder mucoceles. in this study, since the ir spectroscopy was a qualitative analysis method, it is necessary to quantitatively analyze components of gallbladder content, such as amount of mucin and/or cholesterol, in the future study. it is not known whether biliary sludge is similar to gallbladder mucoceles in terms of involvement of excessive mucin secretion from the gallbladder mucosal epithelium. therefore, future studies on histopathological evaluation of gallbladder mucosal epithelium have been planned to investigate whether excessive mucin secretion is involved in the pathophysiology of biliary sludge. additionally, in the case of gallbladder content transition with histological findings of severe gallbladder bleeding observed in the present study, it is necessary to histopathologically investigate the gallbladder and its principal components in detail. to conclude, the present findings have demonstrated that the principle components of both biliary sludge and gallbladder mucoceles are mucins. these findings suggest the possibility that the two diseases are not independent of each other, but in fact, represent one continuous disease. further studies involving extensive data collection of gallbladder contents from biliary sludge and gallbladder mucocele, and histopathological evaluation of these gallbladder will be conducted to elucidate the pathophysiologies of gallbladder mucoceles and biliary sludge. | the pathophysiology of canine gallbladder diseases, including biliary sludge, gallbladder mucoceles and gallstones, is poorly understood. this study aimed to evaluate the component of gallbladder contents and bacterial infection of the gallbladder in order to elucidate the pathophysiology of biliary sludge and gallbladder mucoceles. a total of 43 samples of canine gallbladder contents (biliary sludge, 21 and gallbladder mucoceles, 22) were subjected to component analysis by infrared spectroscopy, and the resultant infrared spectra were compared with that of swine mucin. of the 43 samples, 41 were also evaluated by aerobic and anaerobic bacterial culture. the contents of 20 (95.2%) biliary sludge and 22 (100%) gallbladder mucocele samples exhibited similar infrared spectra as swine mucin. although biliary sludge and gallbladder mucocele contents exhibited similar infrared spectra, one sample of biliary sludge (4.8%) was determined to be composed of proteins. the rate of bacterial infection of the gallbladder was 10.0% for biliary sludge and 14.3% for gallbladder mucoceles. almost all of the identified bacterial species were intestinal flora. these results indicate that the principal components of gallbladder contents in both gallbladder mucoceles and biliary sludge are mucins and that both pathophysiologies exhibit low rates of bacterial infection of the gallbladder. therefore, it is possible that gallbladder mucoceles and biliary sludge have the same pathophysiology, and, rather than being independent diseases, they could possibly represent a continuous disease. thus, biliary sludge could be considered as the stage preceding the appearance of gallbladder mucoceles. |
the state of lowered metabolism during hibernation in mammals is a showcase of cell preservation strategies for muscle, bone, and the circulatory and innate immune systems. hibernating mammals tolerate extremes in organ perfusion, oxygen saturation, temperature, immobilization, and calorie intake - which in combination would be lethal to humans. hibernators may therefore act as reverse translational models for human health and disease. in most hibernating mammals, torpor phases are interrupted by euthermic arousal phases (reviewed in : 1). during torpor, metabolism is severely depressed but hibernation also involves the inhibition of thermogenesis, leading to a considerable decrease in body temperature (tb). the induction of torpor begins with lowering of the metabolic rate, followed by hypothermia as the tb drifts downward (reviewed in : 1 - 3). various degrees of cold torpor have been observed over a phylogenetically wide range of mammals and is most drastic in bats and rodents. the champion of all torpid mammals is the hibernating arctic ground squirrel (urocitellus parryii, previously called spermophilus parryii), which exhibits body temperatures as low as -2.9c 4. in a recent paper by bouma. although there are few data from this field, one of the most striking phenomena is the reduced number of circulating leukocytes found in all hibernating mammals studies so far. 5, finding leukopenia in hibernating mammals, were all conducted on small species, including the european hamster (cricetus cricetus), european hedgehog (erinaceus europeaus), european ground squirrel (spermophilus citellus), arctic ground squirrel (urocitellus parryii), and the thirteen - lined ground squirrel (ictidomys tridecemlineatus, previously called spermophilus tridecemlineatus) ; all species in which tb drops drastically during hibernation. the authors 5 raised the question of whether the reduced number of circulating leukocytes in mammalian hibernators is due to torpor per se or secondarily to the low tb. most recently, the same authors showed that, at least in small species (syrian and djungarian hamsters), tb during hibernation indeed controls leukopenia 6. the definition of hibernation as a temporary physiological state, characterized by a controlled lowering of the metabolic rate, as well as a dramatic drop in tb 1, does not strictly apply to the brown bear. instead, the bear has been classified as a hibernator based on the length of the torpid period (5 - 7 months in scandinavia) 7 - 9. however, in a recent study on black bears (ursus americanus), it was shown that bears are true hibernators ; involving processes of both metabolic and thermal regulation 10. whereas the bear 's body temperature remains relatively stable during hibernation with a less dramatic drop in comparison to other hibernators (from 37c in summer to 33 - 30c in winter) 10, 11, the absolute metabolic rate in the bear is substantially reduced by 75%, and it takes several weeks after emergence for metabolic rate to return to normal levels 10. the slight tb lowering in hibernating black bears is uncoupled from metabolic suppression 10, and, most recently, it was demonstrated that the expression of a number of genes involved in regulating the metabolism is adjusted during winter hibernation 12. by this, the bear offers a unique opportunity to further elucidate whether immune depression during hibernation is primarily caused by a lowered tb or might be intrinsic to torpor per se. in the present study, we examined immune cell counts in peripheral blood of 13 free - ranging scandinavian brown bears during hibernation and again during the active period in summer. the results were compared to data from the literature available on different species on immune function during hibernation. blood samples were collected from 13 free - ranging brown bears, (7 females, 6 males ; age 2 (n=4), 3 (n=8) and 4 (n=1) years old), during hibernation (february / march 2010 or 2011) and again from the same bears during their active period in summer (june 2010 or 2011). the bears were immobilized in the den during february / march with combination of tiletamine - zolazepam, medetomidine and ketamine and from a helicopter during june by darting with a combination of tiletamine - zolazepam and medetomidine 13, 14., bod, norway), with a manufacturer reported accuracy of 0.1c, was used. all bears were clinically examined by a wildlife veterinarian, and all animals were apparently healthy with no signs of infection. none of the bears were pregnant, and since bears do not have menstruation or any other bleeding during the ovulatory cycle, we did not exclude female bears. the study was approved by the swedish ethical committee on animal research (c212/9). white blood cell (wbc) count and the number of different leukocytes were determined at the accredited clinical chemical laboratory at rebro university hospital, sweden, by a fully automated hematology analyzer (xe-5000, sysmex corporation, kobe, japan). blood cells were differentiated by the instruments using standardized wbc differential analysis settings by applying a combination of forward scatter, side scatter and fluorescence of nucleic acid material using highly specific polymethine dyes. the leukocyte differential counts were confirmed manually, by the same lab technician for all samples, with a microscope at the haematology and chemistry laboratory, university animal hospital, swedish university of agricultural sciences, uppsala, sweden. a web of science search was performed to find studies on hibernators in which both white blood cell counts and tb data were available. this search identified ten studies, including 9 different species6, 16 - 22 ; european hamster (cricetus cricetus), arctic ground squirrel (urocitellus parryii), woodchuck (marmota monax), 13-lined ground squirrels (ictidomys tridecemlineatus), syrian hamster (mesocricetus auratus), european ground squirrel (spermophilus cirellus), northern leopard frog (rana pipiens), painted turtle (chrysemys picta) and grass snake (natrix natrix). for the later study, as the grass snake is an ectotherm, we made a search on meteorological websites. the average temperature of grass snakes, corresponding to the month the study was conducted, was assumed as tb. including the present study, 10 species were included in the comparative analysis. the effects of hibernation on innate immune cells were analyzed by using mixed linear models with time as the repeated measure (hibernation vs. active), and bears and year of capture as random effects. further analyses were performed by adjusting on sex, age and body masses to check for possible confounding factors on cell counts. the relationship between leukopenia (expressed as a percentage of the active period) and the drop in tb during hibernation two - sided p - values are reported and significance was set to < 0.05 (spps version 16). blood samples were collected from 13 free - ranging brown bears, (7 females, 6 males ; age 2 (n=4), 3 (n=8) and 4 (n=1) years old), during hibernation (february / march 2010 or 2011) and again from the same bears during their active period in summer (june 2010 or 2011). the bears were immobilized in the den during february / march with combination of tiletamine - zolazepam, medetomidine and ketamine and from a helicopter during june by darting with a combination of tiletamine - zolazepam and medetomidine 13, 14., bod, norway), with a manufacturer reported accuracy of 0.1c, was used. all bears were clinically examined by a wildlife veterinarian, and all animals were apparently healthy with no signs of infection. none of the bears were pregnant, and since bears do not have menstruation or any other bleeding during the ovulatory cycle, we did not exclude female bears. the study was approved by the swedish ethical committee on animal research (c212/9). white blood cell (wbc) count and the number of different leukocytes were determined at the accredited clinical chemical laboratory at rebro university hospital, sweden, by a fully automated hematology analyzer (xe-5000, sysmex corporation, kobe, japan). blood cells were differentiated by the instruments using standardized wbc differential analysis settings by applying a combination of forward scatter, side scatter and fluorescence of nucleic acid material using highly specific polymethine dyes. the leukocyte differential counts were confirmed manually, by the same lab technician for all samples, with a microscope at the haematology and chemistry laboratory, university animal hospital, swedish university of agricultural sciences, uppsala, sweden. a web of science search was performed to find studies on hibernators in which both white blood cell counts and tb data were available. this search identified ten studies, including 9 different species6, 16 - 22 ; european hamster (cricetus cricetus), arctic ground squirrel (urocitellus parryii), woodchuck (marmota monax), 13-lined ground squirrels (ictidomys tridecemlineatus), syrian hamster (mesocricetus auratus), european ground squirrel (spermophilus cirellus), northern leopard frog (rana pipiens), painted turtle (chrysemys picta) and grass snake (natrix natrix). for the later study, no data on tb was given in the reference 22. as the grass snake is an ectotherm, we made a search on meteorological websites. the average temperature of grass snakes, corresponding to the month the study was conducted, was assumed as tb. including the present study, 10 species were included in the comparative analysis. the effects of hibernation on innate immune cells were analyzed by using mixed linear models with time as the repeated measure (hibernation vs. active), and bears and year of capture as random effects. further analyses were performed by adjusting on sex, age and body masses to check for possible confounding factors on cell counts. the relationship between leukopenia (expressed as a percentage of the active period) and the drop in tb during hibernation was checked by simple regression analysis. two - sided p - values are reported and significance was set to < 0.05 (spps version 16). blood samples were collected from all 13 bears during hibernation and again in summer. during winter, the rectal temperature, recorded with a digital thermometer, was 33.5 1.1 (32.2 - 35.4) c and during summer 39.6 0.8 (38.8 - 40.9) c (figure 1a). rectal temperatures seen during summer captures were typical for bears immobilized during the active period 23. bears had significantly lower counts of peripheral blood leukocytes during hibernation compared to the active period (4.5 1.1 vs. 7.4 2.4 x10/l, p = 0.001 ; figure 1b). these data are in agreement with a previous study showing lower leukocyte counts in captive brown bears during denning compared to non - hibernating animals 11. in addition, we found that the bears had significantly fewer neutrophils (3.0 0.7 vs. 5.7 2.4 x10/l, p = 0.001 ; figure 1c) and monocytes (0.35 0.08 vs. 0.61 0.4, p = 0.039 ; figure 1d) during hibernation. these findings are consistent with the results for small hibernating animals 5. on the other hand, we could not detect any change in lymphocyte counts between hibernation and active periods (1.1 0.4 vs. 1.1 0.4 x10/l, p = 0.599 ; data not shown). in one of the bears, wbc was 21.9 x10/l during hibernation, which is 4 - 5 times higher compared to the other bears (figure 1). neutrophils, monocytes and lymphocytes were slightly elevated (4.3, 1.1 and 2.0 x 10/l, respectively) compared to the other bears (figure 1). however, the number of eosinophils was extremely high in this bear compared to the other bears (14.9 vs. 0.22 0.11 x10/l). c - reactive protein (crp) was not elevated (0.085 vs. 0.046 0.04 mg / the leukocyte count of this bear was similar to the other bears when examined in the summer. still, based on the haematology analyses, this bear was excluded from the study. our data support the view that the innate immune system is suppressed during hibernation. as discussed by bouma. 5, this could affect the defence towards infections, as illustrated by the massive death of hibernating bats caused by a fungus 24 that has an optimal growth temperature of 12.5 - 15.8c but grows well at 3 - 6c 25. interestingly, it has been suggested that one reason why animals arouse periodically from torpor is to reactivate a dormant immune system to combat pathogens that enter the body during the torpor bouts 26. although arousals in bears show very different patterns as compared to small - bodied hibernators 10, further investigation is required to find out whether or not hibernating bears exhibit increased cell counts upon arousal from torpor. in contrast to the extreme decrease in peripheral blood lymphocytes in small hibernating animals 5, 6, our data suggest that cells of the adaptive immune system are not affected in bears during hibernation. however, given that many acquired immune responses are stimulated by innate cells, there may still be an effect on the functioning of this immunological aspect. during arousal bouts in small hibernating animals, there is a rapid (within a few hours) reappearance of retained lymphocytes probably from the gut and spleen to the blood 6, 27, 28. hibernating black bears do not show spontaneous arousals to normothermic levels of tb 10, as do small hibernators. instead, they change position on a daily basis and display multiday cycles of tb between 30 - 36c 10. still, bears can be aroused relatively easily, and one could speculate that the short awaken period before we anesthetized the bears could have influenced the number of lymphocytes. however, this period of time was very short (approx. 15 min), and, in addition, the number of circulation lymphocytes during arousal found in small hibernators is still only half of the number found in active animals during summer 29, suggesting that this was not a major factor influencing our results. another explanation of depressed neutrophils and monocytes during torpor could be that longer - lived lymphocytes would remain in circulation longer than the short - lived neutrophils. on the other hand, this explanation requires a decreased rate of granulopoiesis or a shorter life span of granulocytes. if bear leukocytes are sequestered in peripheral tissue during hibernation as found in small hibernators 6, 27, 28, this phenomenon might explain why monocytes are depressed with torpor due to their ability to leave the circulation. also, further studies should investigate the hibernating effect of lymphocytes in sub - groups to see if there are different patterns between, for instance, b- and t - lymphocytes, and if this could explain the numbers of lymphocytes found in hibernating vs. active bears. although the length of time and body temperature history during hibernation could play a role in determining the leukocyte profile at the particular time of sampling, this is unlikely based on work done in ground squirrels 19 that found that the numbers of leukocytes decreased by 90% within 24 hours of torpor and remained unchanged during the remainder of the torpor - period. although this indicates that the length of time hibernating prior to sampling may not have an impact on the results, further studies including the temperature history (using biologging techniques) would be necessary to determine if these results in ground squirrels apply to the brown bear. our study thus supports the concept that hibernation has significant effects on the immune system. whether or not the hibernation - induced leukopenia is a general phenomenon of hibernation, independently of body mass, resulting from a direct body temperature effect, is an important question from an evolutionary point of view. in small hibernators, the number of circulating white blood cells drops dramatically when the body temperature decreases to 5c, and it was thus recently argued that the hibernation induced immune depression is a temperature - dependent mechanism 6. even more recently, metabolic suppression in bears during hibernation was suggested to be independent of tb 10 ; indicating that processes of hibernation may occur in a temperature - independent matter. the direct corollary is to question whether the hibernation - induced leukopenia is independent of tb when body size is large or whether the temperature effect is a general feature of hibernation. the between - species - comparison we performed shows that, across taxa, the depression of the immune function appears indeed as a direct function of the temperature reached during torpor (figure 2). the intercept is slightly off zero, indicating that we can not rule out the contribution of other mechanisms. however, given the strength of the relationship with tb explaining 83% of the relationship, it is likely that any alternative mechanisms are of modest importance, but further studies on large animals, such as different bear species, are needed. taken together, our results suggest that the drop in tb, reached during the torpor bout, is the main drive of the process of leukopenia observed during hibernation and this may be a general mechanism in hibernators. we think that similar approaches are needed to investigate the relationship between metabolic depression and hypothermia. indeed, we find it hard to explain how such an independency is possible given the first law of thermodynamics. we rather think that further studies are clearly needed to look at regional / organ body temperature regulations. in conclusion the present study supports the hypothesis that the reduced number of white blood cells during hibernation is a temperature - dependent phenomenon conserved across evolution. future studies are needed to understand the mechanisms regulating the innate immune system during hibernation.. one explanation could be that the suppression in immune function, as a function of body temperature, is proportional to the decrease in virulence of any pathogens present in the hibernator. whether this is due to energy benefits of a reduced immune function 6 or other mechanisms, our findings of a hibernation - associated drop in white blood cells in brown bears could inspire research in human pathophysiological states and increase the understanding of hibernation physiology in general and aspects of ecological immunology. high numbers of neutrophils are associated with disorders, including ischemic heart disease 30, complications of metabolic syndrome 31 and hypertension 32. patients with coronary artery disease display dysregulated neutrophils 33, 34, and activated neutrophils play a role in morbid obesity 35, rheumatoid arthritis 36 and bronchial asthma 37. if the regulatory mechanisms behind white blood cell lowering in bears could be worked out, this might have therapeutic potential. understanding the mechanisms of specific physiological alterations involved in hibernation is of relevance for pharmacologically induced suspended animation - a therapeutic option in patients with acute severe cerebral or cardiovascular disease 6. acquisition of data : sahdo, evans, blanc, frbert. analysis and interpretation of data : sahdo, evans, arnemo, blanc, frbert, srndahl. preparation of manuscript : sahdo, evans, arnemo, blanc, frbert, srndahl. all authors were involved in revising the manuscript for important intellectual content and approved the final version. | background : hibernation involves periods of severely depressed metabolism (torpor) and decreases in body temperature (tb). small arctic mammals (< 5 kg), in which tb generally drop drastically, display leukopenia during hibernation. this raised the question of whether the decreased leukocyte counts in mammalian hibernators is due to torpor per se or is secondary to low tb. the present study examined immune cell counts in brown bears (ursus arctos), where torpor is only associated with shallow decreases in tb. the results were compared across hibernator species for which immune and tb data were available.methods and results : the white blood cell counts were determined by flow cytometry in 13 bears captured in the field both during summer and winter over 2 years time. tb dropped from 39.60.8 to 33.51.1c during hibernation. blood neutrophils and monocytes were lower during hibernation than during the active period (47%, p= 0.001 ; 43%, p=0.039, respectively), whereas no change in lymphocyte counts was detected (p=0.599). further, combining our data and those from 10 studies on 9 hibernating species suggested that the decline in tb explained the decrease in innate immune cells (r2=0.83, p<0.0001).conclusions : bears have fewer innate immune cells in circulation during hibernation, which may represent a suppressed innate immune system. across species comparison suggests that, both in small and large hibernators, tb is the main driver of immune function regulation during winter dormancy. the lack of a difference in lymphocyte counts in this context requires further investigations. |
it was discovered in 1839 by christian friedrich schonbein when he noticed the emergence of a pungent gas with an electric smell. the word ozone is derived from greek word ozein that means odor. since then it has been the field of research and clinical application in medicine and dentistry. the first medical application of ozone was traced back to 1870 by dr. c. lender, who used it for purifying blood in test tubes. ozone is a triatomic gaseous molecule, consisting of three oxygen atoms, showed its efficacy in the management of various pathologies in the field of medicine and dentistry. ozone, which is used for medical purposes, is a gas mixture comprised of 9599.95% oxygen and 0.055% pure ozone. due to proven therapeutic advantages of ozone review of literature revealed few studies indicating the use of medical grade ozone in the management of oral lesions and conditions. most of them used gaseous form which is produced by specially designed ozone generator, is very expensive and need to be implicated immediately over the lesion. for this, other method of ozone application is developed where it is used in solution form. more viscous solutions like olive oil is used for the better shelf life of the medication. therefore, we conducted a study to evaluate the efficacy of ozonized olive oil in the treatment of oral lesions and conditions, and to evaluate the frequency and duration of the ozonized olive oil application required to manage the oral lesions and conditions. a longitudinal study was conducted on the patients attending the outpatient department of oral medicine and radiology, swami devi dyal hospital and dental college, panchkula. the patients with following oral lesions and conditions were included in the study and followed up for 1 year [table 1 ]. oral lesions and conditions included in the study the diagnosis of all these lesions was based on the clinical sign and symptoms and chairside diagnostic methods such as exfoliative cytology. all the subjects with above - mentioned conditions were explained about the conditions and educated about the ozone therapy. patients with conditions like pregnancy / lactating mother, history of systemic disease with surgical and nonsurgical therapy 6 months prior to the study, history of antibiotic, chemotherapeutic treatment, myocardial infarction, allergy to ozone, alcohol intoxication, hyperthyroidism, and severe anemia were excluded. a total of 50 patients were included in the study and followed up for 6 months. proper record of the clinical data (signs and symptoms, clinical photographs of the lesions, etc.) was maintained at every visit for each patient. ozonized olive oil manufactured by ozone forum india, bisleri, mumbai, india, was used in the study and prescribed to the patient. topical application of ozonized olive oil was done for all the patients included in the study until the lesion cured. patients were asked to rinse the mouth with distilled water. the part of the oral mucosa with the lesion was isolated and ozonized olive oil was applied topically over the lesion with the help of the sterile cotton plug or gloved finger. the viscous oil was massaged over the area for 1 min [figure 1 ]. the patient was advised not to have anything to eat or drink for h. the application was done twice daily till the time lesion subsides, to a maximum of 6 months duration. topical application of ozonized oil all the patients were recalled weekly after the regression of lesion for a maximum of 6 months. at every recall visit, patient 's signs and symptoms such as burning sensation were evaluated and noted in the records. all the patients were recalled weekly after the regression of lesion for a maximum of 6 months. at every recall visit, patient 's signs and symptoms such as burning sensation were evaluated and noted in the records. all the patients with oral candidiasis, angular cheilitis, aphthous ulcers, and herpes labialis showed 100% cure with variable duration of treatment interval 2.1, 2.3, 1.5, and 2.2 days respectively [table 2 and figures 2 - 4 ]. results of ozone therapy preoperative picture of a case of pseudo membranous candidiasis postoperative picture of a case of pseudo membranous candidiasis graphical presentation of the treatment outcomes. blue : number of patients, red : time in days, green : standard deviation all the five patients of oral lichen planus showed improvement in the signs and symptoms with dramatic reduction in their burning sensation to no burning sensation in 4.6 days (mean) [table 2 ]. its molecular weight is 47, 98 g / mol and thermodynamically highly unstable compound that dependent on system conditions like temperature and pressure, decompose to pure oxygen with a short half life. ozone is 1.6 fold denser and 10 fold more soluble in water (49.0 ml in 100 ml water at 0c) than oxygen. although ozone is not a radical molecule, it is the third most potent oxidant (e_5 12.076 v) after fluorine and persulfate. ozone is an unstable gas that can not be stored and should be used at once because it has a half - life of 40 min at 20c. ozone is naturally produced by the photodissociation of molecular oxygen into activated oxygen atoms, which then react with further oxygen molecules. this transient radical anion rapidly becomes protonated, generating hydrogen trioxide, which, in turn, decomposes to an, even more, powerful oxidant, the hydroxyl radical. it is the fundamental form of oxygen that occurs naturally as a result of ultraviolet energy or lightning, causing a temporary recombination of oxygen atoms into groups of three. synthetic ozone can be produced by 3 different systems : ultraviolet system produces in low concentrations of ozone used in esthetics, saunas, air purificationcold plasma system used in air and water purificationcorona discharge system produces a high concentration of ozone. ultraviolet system produces in low concentrations of ozone used in esthetics, saunas, air purification cold plasma system used in air and water purification corona discharge system produces a high concentration of ozone. presently, there are nine methods of ozone therapy in medical practice namely direct intra - arterial and intravenous application, rectal insufflations, intramuscular injections, major and minor autohemotherapy, ozonated water, intra - articular injection, ozone bagging, ozonated oil, and inhalation of ozone. topical preparations have no adverse effects, so ozonated olive oil was used in the present study. in the clinical setting, ozone gas has a high oxidation potential and is 1.5 times greater than chloride when used as an antimicrobial agent against bacteria, viruses, fungi, and protozoa. such features justify the current interest in its application in medicine and dentistry and have been indicated for the treatment of 260 different pathologies. ozone is an inactivated, trivalent (o3) form of oxygen (o). ozone breaks down into two atoms of regular oxygen by giving up an atom of singlet oxygen over a period of 2030 min. ozone is considered one of the most potent oxidants in nature, but the mechanism of its therapeutic action is unclear. some of the possible explanations for this include the generation of peroxides by ozonolysis with unsaturated fatty acids in cell membranes, activation or generation of reactive oxygen species which function as physiological enhancers of various biological processes (including increased production of adenosine triphosphate), and increased expression of intracellular enzymes with antioxidant activity. it has been reported that exposure to ozone results in a change in the level of a variety of biological factors, e.g., cytokines (interferon c, tumor necrosis factors a, transforming growth factor b and interleukin-8), acute phase reactants and adhesion molecules from the integrin family such as cd11b. other reports suggest increased motility and adhesion of peripheral blood polymorphonuclear cells to epithelial cell lines after exposure to ozone. similarly, major autohemotherapy - induced leukocytosis and enhanced phagocytic activity of polymorphonuclear cells have been reported. the present study showed 100% cure rates in coated tongue, recurrent aphthous stomatitis, angular cheilitis, herpes patients, and improvements in the oral lichen planus patients can be due to above said mechanism. no patient included in the study showed adverse effects or toxicity, showing the safety margins of the topical agent. within the limitation of the study of a small number of cases, the healing of the lesions showed faster rates compared to the other conventional treatments, depicting the higher efficacy of the topical ozone therapy. the main limitation of the study was small sample size because it was a clinical trial, so a multicentered study with larger sample size is invited. ozone is an inactivated, trivalent (o3) form of oxygen (o). ozone breaks down into two atoms of regular oxygen by giving up an atom of singlet oxygen over a period of 2030 min. ozone is considered one of the most potent oxidants in nature, but the mechanism of its therapeutic action is unclear. some of the possible explanations for this include the generation of peroxides by ozonolysis with unsaturated fatty acids in cell membranes, activation or generation of reactive oxygen species which function as physiological enhancers of various biological processes (including increased production of adenosine triphosphate), and increased expression of intracellular enzymes with antioxidant activity. it has been reported that exposure to ozone results in a change in the level of a variety of biological factors, e.g., cytokines (interferon c, tumor necrosis factors a, transforming growth factor b and interleukin-8), acute phase reactants and adhesion molecules from the integrin family such as cd11b. other reports suggest increased motility and adhesion of peripheral blood polymorphonuclear cells to epithelial cell lines after exposure to ozone. similarly, major autohemotherapy - induced leukocytosis and enhanced phagocytic activity of polymorphonuclear cells have been reported. the present study showed 100% cure rates in coated tongue, recurrent aphthous stomatitis, angular cheilitis, herpes patients, and improvements in the oral lichen planus patients can be due to above said mechanism. no patient included in the study showed adverse effects or toxicity, showing the safety margins of the topical agent. within the limitation of the study of a small number of cases, the healing of the lesions showed faster rates compared to the other conventional treatments, depicting the higher efficacy of the topical ozone therapy. the main limitation of the study was small sample size because it was a clinical trial, so a multicentered study with larger sample size is invited. ozone therapy in the present stage is reducing caries activity, improving periodontal health, and also improving the healing time of oral lesions and wounds. ozone therapy is bound to revolutionize the way dentistry is going to be practiced in the future. current data on the usage of ozone therapy as therapeutic options for various oral lesions and conditions lacks sufficient data and therapeutic advantage over available conventional therapeutic modalities. | the oral cavity is an open ecosystem that shows a dynamic balance between the entrance of microorganisms (bacterial, viral or fungal), colonization modalities, nutritional balance, and host defenses against their removal. the oral lesions including aphthous ulcerations, herpes labialis, oral candidiasis, oral lichen planus, and angular cheilitis some of the common entities encountered in the clinical practice. a variety of treatment options is available in the literature for all of these lesions and conditions. topical ozone therapy is a minimally invasive technique that can be used for these conditions without any side effects.aim and objectives : to evaluate the efficacy of ozonized olive oil in the treatment of oral lesions and conditions.materials and methods : a longitudinal study was carried out on 50 patients (aphthous ulcerations, herpes labialis, oral candidiasis, oral lichen planus, and angular cheilitis). the ozonized olive oil was applied twice daily until the lesion regresses for a maximum of 6 months.results:all the lesions regress in patients with aphthous ulcerations, herpes labialis, oral candidiasis and angular cheilitis or showed improvement in the signs and symptoms in oral lichen planus patients. no toxicity or side effect was observed in any of the patients.conclusion:ozone therapy though requires a gaseous form to be more effective, but topical form can also bring out the positive results without any toxicity or side effect. hence, it can be considered as a minimally invasive therapy for the oral infective and immunological conditions. |
the cast index (ci, a ratio of anteroposterior to lateral internal diameters of the cast at the fracture site) is a simple, reliable marker of quality of molding and a ci of > 0.8 correlates with increased risk of redisplacement. we hypothesize that an acceptable ci is more difficult to achieve and does not predict outcome in fractures of the proximal forearm. seventynine cases of pediatric forearm fractures initially treated by manipulation alone over a year were included in this retrospective radiographic analysis. all fractures were divided as either proximal or distal half forearm based on the location of the radius fracture. ci was higher in proximal half forearm fractures (0.83 vs. 0.76, p = 0.006), nonetheless these fractures did not re - displace more than distal fractures. we found that in proximal half forearm fractures it is difficult to achieve a ci of 0.5 were grouped as proximal. these measurements were made from the proximal radioulnar joint proximally to the wrist joint distally. x - ray of forearm with wrist joint lateral and anteroposterior views showing calculation of cast index (ci). ci = a / b, a = internal anteroposterior diameter of cast excluding padding, b = internal mediolateral diameter of cast excluding padding x - ray of forearm with wrist and elbow joint lateral view showing calculation of fracture position. fracture position = x/(x + y), x = length of distal fragment, y = length of proximal fragment data were analyzed using microsoft excel 2010 software (redmond, wa 98052 - 7329, usa). the primary variable of interest was the fracture position and the main outcomes were ci and remanipulation due to redisplacement. paired, two - tailed student 's t - tests were performed to analyze statistical differences between means in the groups of continuous data. for categorical data, seventy - nine cases (47 males and 32 females) between ages 2 and 15 years (mean age 8.6 years) were included in the study after exclusions. thirteen children had incomplete image series (either of the initial reduction or subsequent x - rays) and two children were followed up elsewhere and were therefore excluded. the mean fracture position was 0.26 (range 0.030.71) that is, the distal radius fragment was just over a quarter of the total bone length. five out of the 79 cases were subsequently remanipulated and/or fixed percutaneously due to redisplacement. all patients were followed up a week after manipulation and then every 12 weeks until union. median followup time was 2 weeks and maximum time to union was 11 weeks after manipulation. the pearson 's product - moment correlation coefficient was used to assess correlation between the repeat measurements and the original measurements. both analyses showed that inter and intraobserver agreements were very high and that ci could be reproduced reliably [table 1 ]. of the five patients that required reoperation, the average redisplacement was 18.4 (range 726), whereas in the non reoperated group, the average final angulation was 4.7 (range 018). all of the reoperated fractures were distal half (position 0.090.40) and there were no data to suggest any causal link between fracture position and ci and remanipulation risk. between the two groups there were not any significant differences between the gender, age and incidence of ulna fracture. the initial displacement of the fractures was also not significantly different [table 2 ]. comparison between reoperated and non reoperated fractures table 3 analyses ci and fracture position when patients are grouped by their age (15 years, 610 years and 1015 years). the only significant difference is that older children (1015 years) are more likely to have more distal fractures forearm fractures compared to younger age groups. analysis of ci and fracture position limited by age table 4 summarizes the results when fractures are grouped as either proximal or distal half of the forearm. there is a significant difference in the two sets of ci between proximal half and distal half forearm fractures. patients with proximal half forearm fractures were older than those with distal half fractures and were more likely to have a concurrent ulna fracture. however, the fact that proximal half forearm fractures are more likely to have a concurrent ulna fracture did not result in more redisplacement in this group. in the same two groups, there is no significant difference between reangulation of fractures, other fracture characteristics or patient demographics. closed reduction and further stabilization of forearm fractures with percutaneous k - wires or elastic nails are acceptable methods for fixation of fractures at high risk of displacement.5 in our hospital, k - wire fixation / elastic nail fixation is reserved for fractures that are unstable after manipulation. the position of the elbow during casting was previously thought to be important in maintaining immobilization of the fracture site. however, more recent research has shown that it does not affect the final outcome. the same has been shown for positioning the wrist in pronation / neutral / supination.1213 despite this, uniform cast molding and positioning techniques were used throughout this study. significant risk factors for loss of reduction following manipulation of forearm fractures can be divided into fracture related, surgeon related and patient related.8 the most important of these factors are the initial displacement of the fracture, near anatomical reduction and a close fitting cast. previous studies have also correlated an increased risk of redisplacement in combined radius and ulna fractures ; however, our results did not show this.13 the most important factors for adequate application of a plaster cast are thin and uniform padding and good molding that achieves adequate three - point fixation.14 numerous previous studies have validated the use of cast indices as predictors of redisplacement, both in distal radius fractures and both bone fractures.8 a previous study also found that teaching the use of ci and padding index to orthopedic surgeons significantly improved their accuracy of assessing the risk of redisplacement of forearm (radius fracture with or without ulna fracture, proximal and distal) fractures in children.15 clearly, these indices need to be used in association with patient and fracture characteristics in clinical assessment. previous studies have used ci for proximal and distal forearm fractures, not just limited to distal forearm fractures.10 however, previous authors have not studied whether it is easier or more difficult to achieve an ideal ci in more proximal forearm fractures as compared to distal fractures. we therefore analyzed our data to examine the difference in cast indices and displacement as forearm fractures become more proximal. our data suggest that for proximal forearm fractures, it is more difficult to achieve a ci of < 0.8. nevertheless, this did not cause significant loss of reduction and thus these fractures did not have to be re - manipulated [table 4 ]. this correlates with our hypothesis that an acceptable ci is more difficult to achieve in proximal half forearm fractures. there is more soft tissue present in the proximal forearm compared with the distal forearm and therefore a cast that is more elliptical in cross section is less likely. however, a less elliptical proximal forearm cast (i.e., one with a higher ci) may still provide adequate three point fixation. an assumption that has been made in this study is that a higher ci in the proximal forearm is due to increased soft tissue mass and not due to other factors such as inadequate molding in the proximal forearm. the mean cast indices of the two groups of patients (proximal half and distal half forearm fractures) are significantly different. furthermore, table 4 demonstrates that the two sets of fractures are not significantly different demographically or in the type of fracture. it may be argued that the difference in cast indices between the two groups is due to inadequate molding of the proximal forearm casts, however, as these fractures did not significantly displace we feel that proper molding techniques were applied throughout. inter and intra observer errors were low suggesting that calculation of the ci from radiographs can be reproduced reliably by different observers or after an amount of time has elapsed. we also realize that our number of reoperated fractures is small (5/79 fractures) and that gives a statistically weaker calculation. however, it still stands that a low ci is difficult to achieve in the proximal forearm and that none of the proximal fractures had to be re - operated despite a higher ci. cast index remains a useful clinical tool to rapidly assess cast molding following closed reduction of distal forearm fractures and to predict redisplacement of distal forearm fractures as highlighted in multiple previous studies.9101115 its use in proximal half forearm fractures should be discouraged, however, as the shape of the proximal forearm makes it difficult to achieve an acceptable ci of < 0.8 despite adequate molding and a higher ci in the proximal forearm does not predict the risk of redisplacement or re - manipulation. | background : many pediatric forearm fractures can be treated in plaster following closed reduction. the cast index (ci, a ratio of anteroposterior to lateral internal diameters of the cast at the fracture site) is a simple, reliable marker of quality of molding and a ci of > 0.8 correlates with increased risk of redisplacement. previously, ci has been applied to all forearm fractures. we hypothesize that an acceptable ci is more difficult to achieve and does not predict outcome in fractures of the proximal forearm.materials and methods : seventynine cases of pediatric forearm fractures initially treated by manipulation alone over a year were included in this retrospective radiographic analysis. the ci was calculated from the post manipulation radiographs. all fractures were divided as either proximal or distal half forearm based on the location of the radius fracture. subsequent radiographs were reviewed to assess redisplacement and reoperation.results:the mean ci was 0.77. remanipulation was required in five cases (6%), all distal half fractures mean ci 0.79. ci was higher in proximal half forearm fractures (0.83 vs. 0.76, p = 0.006), nonetheless these fractures did not re - displace more than distal fractures.conclusion:cast index is useful in predicting redisplacement of manipulated distal forearm fractures. we found that in proximal half forearm fractures it is difficult to achieve a ci of < 0.8, but increased ci does not predict loss of position in these fractures. we therefore discourage the use of ci in proximal half forearm fractures. |
multifocal motor neuropathy (mmn) and chronic inflammatory demyelinating polyradiculoneuropathy (cidp) are acquired immune - mediated peripheral neuropathies (pn). mmn is a pure motor neuropathy syndrome usually beginning in one or both hands and principally affecting the upper extremities, characterized by a chronic or stepwise progressive asymmetrical limb weakness and muscle atrophy. cidp is an immune - mediated peripheral neuropathy that may cause weakness, paralysis, and/or impairment in both motor and sensory functions, usually affecting both sides of the body (symmetrical). the neurophysiological hallmark of mmn is conduction blocks (cb) outside the usual sites of nerve compression [1, 3 ], while cidp typical features are cb, slowed motor, and sensory nerves conduction velocities and prolonged distal latencies. in addition, both diseases may present a variable extent of axonal loss [5, 6 ], which has been attributed both to recurrent demyelinating insults and intrinsic pathogenic features, especially in the case of mmn. neuromuscular ultrasound (us) is a noninvasive, painless, and radiation - free complementary imaging technique for the diagnostic work - up of pn [7, 8 ]. focal nerve enlargements can be observed in the majority of mmn patients and generalized nerve enlargements can be observed in cidp patients, interestingly showing alterations also in limbs without signs of neurophysiological dysfunction [911 ]. however, the correlation between us, neurophysiological findings, and functional disability is still partially controversial [1214 ]. some authors have found an association between disease duration and the extent of nerve enlargement, while others have suggested a specific us pattern in relation to different mechanisms of injury : demyelinating insults might result in swollen, enlarged, and hypoechoic nerves, while axonal damage may be characterized by hyperechoic atrophic bundles of fascicles. in addition, a variable combination of axonal and demyelinating insults may also coexist, resulting in hyperechoic and hypertrophic nerves. most of literature data have been collected on cidp patients, while a few sonographic - clinical - electrophysiological studies have been currently reported in mmn [9, 11, 1618 ]. the aim of this study is to analyze us findings in patients with cidp and mmn at different functional disability, in order to correlate us qualitative and quantitative measures with clinical and neurophysiological features. this cross - sectional observational study includes 22 cidp (5 females ; 17 males) and 10 mmn (4 females ; 6 males) patients recruited from the neuromuscular unit of turin university hospital between may 2014 and may 2015 and 70 healthy controls (43 men and 27 women). patients were evaluated by means of a clinical, us, electrophysiological assessment, and a structured clinical interview. all subjects fulfilled the european federation of neurological society / peripheral nerve society (efns / pns) criteria for cidp or mmn [3, 19 ] and, at the time of us examination, were receiving a monthly treatment with intravenous immunoglobulin (ivig) (1 - 2 g / kg / month) for at least 6 months. written informed consent and local ethical committee (aou san giovanni battista di torino) approval were obtained. the authors acted in accordance with the ethical standards laid down in the 1964 declaration of helsinki. a complete neurological examination was performed by means of the inflammatory neuropathy cause and treatment (incat) disability scale, the medical research council (mrc) score in 8 muscle groups bilaterally (shoulder abduction, elbow flexion, wrist flexion, first dorsal interosseous, hip flexion, knee extension, and ankle flexion / extension), and the overall neuropathy limitation scale (onls). according to the incat disability scale, upper limb activities were scored as no / minimal impairment (not affected), moderate impairment (affected but not prevented), or severe impairment (prevented), while the evaluation of walking difficulties was based on the use of aids : no / minimal impairment (moderate gait impairment, independent or with unilateral support) ; severe impairment (severe gait impairment, bilateral supports or wheelchair). the us assessment was performed by means of a sonosite m - turbo ultrasound machine equipped with a broadband linear transducer (frequency band 615 mhz). the us scan was performed the same day of the neurological assessment by an evaluator (michela rosso), who was blinded to the clinical and neurophysiological data. the following quantitative and qualitative us parameters were collected for the median, ulnar, peroneal, tibial, and sural nerves bilaterally. csa was measured by tracing the nerve just inside the hyperechoic rims with the ellipse method when applicable (when the nerve in the transverse scan had an elliptical or roundish shape) or alternatively tracing the nerve shape (when the nerve had an irregular shape). csa was evaluated for each nerve at predetermined sites : median nerve was evaluated at wrist (entrance of carpal tunnel), middle third of the forearm, elbow (before penetrating the pronator teres muscle next to the brachial artery), and middle third of the arm (middle of the distance between medial epicondyle and axillary fossa) ; ulnar nerve at wrist (guyon 's canal), middle third of the forearm, elbow (between medial epicondyle and olecranon), and middle third of the arm (middle of the distance between medial epicondyle and axillary fossa) ; peroneal nerve at the fibular head and popliteal fossa ; tibial nerve at popliteal fossa and at medial malleolus before its division into plantar nerves (ankle) ; sural nerve at the ankle. maximal csa (csa) enlargement was recorded for each nerve ; median and ulnar nerves were scanned along the entire viewable tract, from the wrist to the middle third of the arm. abnormal csa values at entrapment sites (wrist, elbow, and fibular head) were excluded to avoid confounding local neuropathies. intranerve csa variability. intranerve csa variability was calculated as the ratio between csa / csa, for each nerve (available for median and ulnar nerves). side - to - side intranerve variability was calculated as the side - to - side ratio of the intranerve csa variability. qualitative analysis of nerve fascicles. nerves were classified as abnormal if 3 fascicles showed a cross - sectional area 2 mm, regardless of the csa value. normative us reference values were obtained by the assessment of healthy controls (table 1), considering the upper threshold of the normality range (ut) to be the average value + 2 standard deviations. then, in order to compare the csa of different nerves taking into account their relative normative values, a normalized csa (csa) was calculated by dividing the csa of each nerve to the corresponding ut value (csa = csa / csa). nerve conduction studies were performed by means of a keypoint (natus medical incorporated, san carlos, ca, usa) electromyography (emg) machine by evaluators blinded to the us study, assessing the bilateral peroneal, tibial, ulnar, and median motor nerves and the bilateral sural, median, and ulnar sensory nerves. nerve conduction velocities (cv), compound muscle action potentials (cmap), and sensory action potentials (snap) were collected and compared to the normality cut - off values of our laboratory ; all patients were checked for skin temperature with a probe on the emg machine. if needed, the body temperature was maintained above + 34c by means of an infrared lamp. cb was defined in accordance with the efns / pns criteria [3, 19 ], excluding possible sites of entrapment (wrist, elbow, and fibular head) to avoid confounding focal neuropathies. moreover, neurophysiological alterations of nerve segments were stratified in predominantly myelin damage or axonal damage in accordance with the classification already proposed by di pasquale.. descriptive statistics (mean, standard deviation, and range) were used for continuous variables. mann - whitney and cramer 's v tests were used for comparison between patients with different disease severity and neurophysiological alterations and between cidp and mmn patients. spearman 's rho, kendall 's tau - b, and pearson 's correlations were used to estimate correlations between clinical, us, and electrophysiological characteristics, while a linear regression model was applied to estimate the influence of age, disease and treatment duration, and ivig doses on csa values. bonferroni 's correction for multiple comparisons was applied to avoid statistical biases of repeated testing effects. the average csa values in bilaterally measured nerves were obtained pooling together data of the two sides. however, in order to take into account the asymmetrical involvement typical of mmn, we also considered the side - to - side intranerve variability, calculated by dividing the intranerve variability of the most affected side with the intranerve variability of the less affected side. complete clinical, us, and neurophysiological data were available for 22 cidp and 10 mmn patients with similar age (62.7 13.8 versus 55.1 14.9 years old ; p : 0.119) and disease duration (81.5 60.0 versus 87.3 46.6 months ; p : 0.734). us data of 70 healthy controls (58.4 16.1 years old ; range 3082) with normal clinical and neurophysiological assessments were used as normative reference values (table 1). according to the incat disability score (table 2), 7/32 subjects required a bilateral support / wheelchair (cidp = 18% ; mmn = 30%) ; 17/32 required a unilateral support (cidp = 64% ; mmn = 30%) ; and 8/32 did not show any significant impairment of gait (cidp = 18% ; mmn = 40%). the upper limbs score showed that 9/32 subjects had a severe impairment in daily living activities (cidp = 23% ; mmn = 40%) ; 12/32 reported a moderate impairment (cidp = 41% ; mmn = 30%) ; and 11/32 did not report any significant impairment (cidp = 36% ; mmn = 30%). no differences were observed between cidp and mmn patients at the incat (p : 0.519), onls (p : 0.724), and mrc (p : 0.327) scores (table 2). a total of 320 nerves (220 cidp and 100 mmn) were evaluated by means of nerve conduction studies and us assessments : neurophysiological alterations were found in 78.0% of cidp nerve segments (predominant myelin damage = 41.6% ; predominant axonal damage = 36.4%) and in 62.5% of mmn nerve segments (predominant myelin damage = 35.0% ; predominant axonal damage = 27.5%). quantitative and/or qualitative us alterations were observed in 43.2% (95/220) of cipd nerve segments and in 40.0% (40/100) of mmn nerve segments. in both cases these alterations were found more frequently in nerves with predominantly myelin versus axonal damage (cidp = 74.6% versus 25% [p : 0.001 ] ; mmn = 78.3% versus 20% [p : 0.010 ]). us abnormal features were additionally observed in 14.4% of cidp and 10.1% of mmn nerve segments without significant neurophysiological alterations. as shown in figure 1(a) and table 3, cidp and mmn patients with intermediate functional disability (gait disturbance that might require a unilateral support) showed higher csa values than patients with no / minimal gait difficulties (p : 0.001 for cidp and p : 0.002 for mmn) or higher functional disability (p : 0.041 for cidp and p : 0.034 for mmn). moreover, higher csa values were observed (figure 1(c)) in nerves with demyelinating features versus axonal damage (p : 0.048 for cidp and p : 0.049 for mmn). the quantitative us analyses showed higher csa in cidp than in mmn patients in peroneal nerve (16.81 3.01 mm versus 13.60 2.27 mm ; p : 0.024), tibial nerve (23.46 2.23 mm versus 18.64 2.66 mm ; p : 0.027), and sural nerve (3.56 0.31 mm versus 2.60 0.49 mm ; p : 0.047). the qualitative us analyses revealed abnormal fascicles in 40% of mmn versus 22.7% of cidp peroneal nerve segments (p : 0.171) and in 35% of mmn versus 15.9% of cidp tibial nerve segments (p : 0.087). additionally, a significant correlation was found between abnormal nerve fascicles and cb (peroneal nerve : tau = 0.411 [p : 0.015 ] in cidp and tau = 0.302 [p : 0.046 ] in mmn ; tibial nerve : tau = 0.365 [p : 0.042 ] in cidp and tau = 0.282 [p : 0.048 ] in mmn) and between abnormal nerve fascicles and csa values, both in cidp (peroneal nerve : rho = 0.329 [p : 0.033 ] ; tibial nerve : rho = 0.296 [p : 0.049 ]) and in mmn (peroneal nerve : rho = 0.229 [p : 0.046 ] ; tibial nerve : rho = 0.454 [p : 0.044 ]). median and ulnar nerves csa were significantly higher in patients with moderate impairment compared to subjects with either a more severe functional impairment (p : 0.037 for cidp and p : 0.047 for mmn) or milder disability (p : 0.042 for cidp and p : 0.037 for mmn) (table 4, figures 1(b) and 2). the quantitative us analyses showed higher csa in cidp than in mmn patients in median nerve (18.70 2.30 versus 14.85 2.58 ; p : 0.042) and ulnar nerve (13.27 2.64 versus 10.75 2.23 ; p : 0.040), while the side - to - side intranerve variability was higher in mmn (median nerve : 1.9 0.6 versus 1.5 0.6 [p : 0.035 ] ; ulnar nerve : 1.8 0.4 versus 1.4 0.4 [p : 0.007 ]) (table 4). the correlation between abnormal fascicles and cb was also confirmed for the upper limbs (median nerve : tau = 0.310 [p : 0.046 ] in cidp and tau = 0.213 [p : 0.045 ] in mmn ; ulnar nerve : tau = 0.260 [p : 0.049 ] in cidp and tau = 0.271 [p : 0.048 ] in mmn), as was for the correlation between altered fascicles and csa, both in cidp (median nerve : rho = 0.315 [p : 0.037 ] ; ulnar nerve : rho = 0.447 [p : 0.002 ]) and in mmn subjects (median nerve : rho = 0.331 [p : 0.043 ] ; ulnar nerve : rho = 0.564 [p : 0.001 ]). qualitative us analyses showed an inverse pattern compared to that observed at the lower limbs, with a moderately higher prevalence of altered fascicles in cidp than in mmn nerve segments (median nerve : 38.6% versus 15% [p : 0.059 ] ; ulnar nerve : 36.4% versus 20% [p : 0.194 ]). as observed in the lower limbs, the csa values were higher in nerve segments with predominantly demyelinating features versus axonal damage (p : 0.001 for cidp and p : 0.049 for mmn) (figure 1(d)). there was a direct correlation between axonal damage and gait impairment at the lower limbs (cidp : r = 0.456 [p : 0.002 ] ; mmn : r = 0.450 [p : 0.036 ]) and between axonal damage and functional disability at the upper limbs (cidp : r = 0.402 [p : 0.001 ] ; mmn : r = 0.325 [p : 0.047 ]). no linear correlations were observed between us data and incat score (cidp : r = 0.121 [p : 0.110 ] ; mmn : r = 0.239 [p = 0.190 ]) or between us data and onls score (cidp : r = 0.053 [p = 0.415 ] ; mmn : r : 0.211 [p : 0.140 ]), while an inverse correlation was observed in cidp patients between mrc scores of muscles innervated by median and ulnar nerves and the corresponding intranerve variability (table 5). csa values were not influenced by age (cidp : = 0.141 [p : 0.456 ] ; mmn : = 0.205 [p : 0.437 ]), disease duration (cidp : = 0.005 [p : 0.954 ] ; mmn : = 0.195 [p : 0.305 ]), treatment duration (cidp : = 0.027 [p : 0.749 ] ; mmn : = 0.295 [p : 0.247 ]), or ivig dose (cidp : = 0.023 [p : 0.809 ] ; mmn : = 0.186 [p : 0.663 ]). this study reports the peripheral nerve us findings of 32 cidp and mmn patients at different functional disabilities. lower csa values were associated with more severe clinical alterations and/or axonal damage, while higher csa values were associated with intermediate functional disability (clinical alterations without loss of functionality) and/or demyelinating damage. these data are in accordance with the findings reported by di pasquale., who observed that nerve segments characterized by predominantly myelin damage had greater csa than nerves with predominantly axonal damage and normal nerves, which virtually overlapped. in addition, we found some differential aspects between mmn and cidp : greater side - to - side intranerve variability was observed in mmn, in line with the pattern of heterogeneous and multifocal involvement characteristic of the disease ; patients with cidp showed higher csa values, potentially indicating more prominent demyelinating processes ; qualitative us analyses revealed a different distribution of abnormal fascicles in the upper and lower limbs, with more prominent us alterations in district affected by predominantly demyelinating damage (frequently associated with a less marked functional impairment) compared to district affected by secondary axonal degeneration. the majority of literature data reported increased csa values in cidp, with a possible association between intranerve variability and functional scores [23, 24 ]. less data are available for mmn, where asymmetric and focal csa enlargements have also been reported in nerves without neurophysiological alterations, suggesting disease processes that could extend beyond the sensitivity of standard diagnostic techniques [9, 11, 1618 ]. several complex phenomena, such as segmental demyelination, proliferation of schwann cells in response to repeated inflammatory insults, onion bulbs formation, and a variable degree of axonal loss might underlie these us findings [22, 25, 26 ]. however, their correlation with the mechanisms of nerve damage and repair still remains to be clarified. our data support the complementary role of us in the assessment of cidp and mmn, suggesting a different pattern in nerves with demyelinating versus axonal damage and in cidp versus mmn patients, in possible relationship with the different pathological processes involved. previous studies reported a correlation between disease duration and csa values [14, 22, 27 ], while in our heterogeneous sample of patients we observed a we speculate that different disease phases might be associated with different us patterns, with an initial / intermediate phase of inflammation and myelin damage, characterized by increased csa and enlarged swollen fascicles and a late phase of severe axonal degeneration, characterized by small atrophic fascicles, reduced csa and greater functional impairment. other factors, such as ivig pharmacological treatment and/or individual inflammatory response, might also be implicated in the morphological modifications of peripheral nerves. however, the similar therapeutic regimen (ivig) administered to our patients did not allow a post hoc analysis of treatment effects on csa values. finally, patients with cidp, characterized by more prominent inflammatory and demyelinating features, might display greater nerve enlargement compared to mmn or to peripheral neuropathies characterized by primary axonal degeneration (i.e., chronic idiopathic axonal polyneuropathy). taken together, these findings suggest variable applications for us in the field of immune - mediated peripheral neuropathies, ranging from the more accurate clinicopathophysiologic phenotyping to the early detection of morphological changes associated with critical disease milestones. in addition innovative us score, such as the bochum ultrasound score, will likely allow a more accurate differentiation between cidp and other acquired or inherited peripheral neuropathies. our findings suggest that cidp and mmn patients with an intermediate functional disability may present more pronounced quantitative and qualitative us alterations than patients with higher disability. moreover, some differential aspects can be recognized in cidp versus mmn and greater us alterations might be observed in nerve segments with demyelinating versus axonal damage. the strength of our findings is partially limited by the relatively small sample size and the lack of serial prospective follow - up assessments. in addition, two aspects should be considered in the interpretation of data : (a) the u - shaped relationship between us findings and functional impairment, which might result in a similar us pattern in patients with either minimal or severe disability ; (b) the association of csa reduction with two different factors (axonal damage and functional impairment), indicating the need of further prospective studies to analyze which of the two features primarily correlates with nerve size reduction. | objective. this cross - sectional study analyzes the pattern of ultrasound peripheral nerve alterations in patients with chronic inflammatory demyelinating polyradiculoneuropathy (cidp) and multifocal motor neuropathy (mmn) at different stages of functional disability. material and methods. 22 cidp and 10 mmn patients and a group of 70 healthy controls were evaluated with an ultrasound scan of the median, ulnar, peroneal, tibial, and sural nerves. results were correlated with clinical disability scales and nerve conduction studies. results. patients with intermediate functional impairment showed relatively larger cross - sectional areas than subjects with either a milder (p < 0.05) or more severe impairment (p < 0.05), both in cidp and in mmn. in addition, mmn was associated with greater side - to - side intranerve variability (p < 0.05), while higher cross - sectional areas were observed in cidp (p < 0.05) and in nerve segments with predominantly demyelinating features (p < 0.05). higher csa values were observed in nerves with demyelinating features versus axonal damage (p < 0.05 for cidp ; p < 0.05 for mmn). discussion and conclusions. greater extent of quantitative and qualitative us alterations was observed in patients at intermediate versus higher functional disability and in nerves with demyelinating versus axonal damage. cidp and mmn showed differential us aspects, with greater side - to - side intranerve variability in mmn and higher cross - sectional areas in cidp. |
causes of anisocoria include compression or destruction of the third cranial nerve by increased intracranial pressure (icp) from tumour, thrombus, oedema, aneurysm or haemorrhage. cases of anisocoria have rarely been reported in the postoperative intensive care unit (icu) setting. we report a case of anisocoria in icu that was in all likelihood linked to the vulnerability of brain with old traumatic brain injury (tbi) to regional increase in icp secondary to general anaesthesia and intraoperative fluid resuscitation. a 45-year - old male with severe acute pancreatitis (sap) was referred to the icu with acute physiology and chronic health evaluation (apache ii) score 20 and intra - abdominal hypertension. about 5 years back, he had had a tbi (marshal grade iii) with significant intracranial bleed. it was a focal lesion and he had a complete clinical recovery with conservative management. after surgical necrosectomy performed in the sixth week of sap, there was fresh bleeding from the drains requiring an urgent re - exploration. his haemoglobin dropped from 10 gm% to 6 gm% within few hours, and his prothrombin time was 6 s prolonged, with a platelet count of 60,000/cmm. the intraoperative period was uneventful from the point of adverse events like hypoxia, hyperventilation, severe hypotension (mean blood pressure < 60) or hypertension. however, he required significant volume resuscitation (about 5 l in 2 h) comprising of 1.5 l of crystalloid solution, 0.5 l of colloid solution, 4 units of packed red blood cells (prbc), 4 units of fresh frozen plasma (ffp), 10 units of random donor platelet and 10 units of cryoprecipitates, to maintain haemostasis and haemodynamics. soon after shifting him back to the icu a negative pilocarpine test ruled out adie syndrome and he did not exhibit features of horner 's syndrome. absence of any appreciable localizing sign was suggestive of a nonvascular major intracranial event as the cause of acute anisocoria. in the background of existing coagulopathy, but, an urgent computed tomography scan revealed an old, healed haemorrhage in the left fronto - temporal region as sequelae to the past tbi and no new organic lesion or bleed [figure 1 ]. soon thereafter, interventions were started to decrease icp and 1 g / kg intravenous bolus mannitol and 20 mg frusemide were administered. repeat doses were given every 4-hourly for 24 h and he was nursed in the head - up position. within 12 h of initiating measures for icp reduction, hypertension and bradycardia resolved and his left pupil started to decrease in size and reaction to light was restored. after normalization of pupillary size and response to light, mannitol and frusemide were stopped. on day 3 of the event, he was fully conscious and his trachea was extubated. anisocoria during general anaesthesia has been reported mostly during induction of anaesthesia on account of parasympathetic dominance by anaesthetic drugs such as thiopental, midazolam, isoflurane and vecuronium induced autonomic tone disturbance.[37 ] lazar. reported that midazolam can cause a brief ischemic event in the left cerebral hemisphere in patients with an associated abnormality of the left side of the brain. although thiopental, midazolam, isoflurane and vecuronium were administered in our patient, anisocoria appeared during recovery from general anaesthesia and not during the induction period. the triad of acute onset hypertension, sinus bradycardia and anisocoria is suggestive of regional increase of icp. easy reversibility of the triad after a short period of mannitol and frusemide therapy also justifies the transient rise in regional icp as cause of anisocoria in our case. haemodynamically, volume resuscitation of 5 l in a short period of 2 h was well tolerated by the patient and the central venous pressure (cvp) never went beyond 10 mmhg. following tbi, an increased cvp occurring with aggressive crystalloid resuscitation may contribute to the loss of brain compliance and the development of intracranial hypertension. in our case, this was probably not the underlying mechanism. rather, tbi has been reported to give rise to latent occult cryptic vascular lesions. we postulate that an old tbi may render a brain vulnerable to a regional increase in icp during active fluid resuscitation, which may also get influenced by the autonomic tone disturbance secondary to drugs like thiopental, isoflurane and vecuronium by the following mechanisms : clinically latent occult cryptic vascular lesions may persist for an unidentified period after tbi.these cryptic vascular lesions may have much autoregulatory dysfunction.such lesions may cause regional elevation of icp during fluid resuscitation with or without general anaesthetics. clinically latent occult cryptic such lesions may cause regional elevation of icp during fluid resuscitation with or without general anaesthetics. although anisocoria has been considered as a benign event in a critical care patient, our case suggests that anisocoria in a subset of the population may actually be an impending neurologic emergency. authors think it appropriate to believe that one should exert extra caution about the rapidity of fluid resuscitation in the management of a case of old tbi. also, it may be advised that fluid resuscitation in such patients is accompanied by concomitant pupillary examination. | anisocoria is an uncommon entity in general postoperative intensive care. we present a case of a 45-year - old man suffering from severe acute pancreatitis with a past history of traumatic brain injury (tbi), who developed hypertension, bradycardia and anisocoria soon after re - exploration surgery under general anaesthesia. computed tomography showed no new lesion. measures directed towards reducing intracranial pressure resulted in amelioration in about 12h. the possible role of old tbi in the causation of anisocoria during general anaesthesia and resuscitation has been explored in this report. |
the explosion in the knowledge of cancer genetics of the past 25 years has totally changed our approach to cancer treatment. but, as is true of many political and cultural revolutions, the pace of progress is maddeningly ponderous. although recent relaxations of the grips of smoking and hormone replacement therapy has led to a welcome decline in cancer incidence, millions more lives will be lost before we truly understand or have the tools to provide effective therapy for this vastly complex group of diseases. despite the long road ahead, investigators, clinicians and patients share a mounting confidence that new therapeutic research will ultimately be successful. through oncogenetics, we will be able to document the drivers of an individual cancer and delineate the gain - of - function mutations that give rise to growth - promoting proteins that in turn induce oncogene ' addiction ', in which a cancer is dependent on such proteins. we will also determine the loss - of - function mutations that deprive cancer cells of the proteins that direct dna repair and/or provide directions to the cell death pathway. armed with such oncogenetic data, we will match the validated mutations of a particular cancer to an appropriate and scientifically determined targeted drug array. this great challenge, one akin to president kennedy 's thrust to the moon, is definitely possible. here, we provide the background to our optimism and also describe some of the roadblocks that obstruct the path of progress. modern treatment of invasive cancers that have extended beyond the reach of the surgeon 's knife or the radiation therapist 's beam began in the post world war ii era when sidney farber and his colleagues introduced aminopterin to induce remissions in childhood leukemia. farber was committed to sequential application of single agents and urged an extensive public and private antibiotic and anti - cancer drug screening program to produce many active compounds that had in common the induction of injuries to cell dna or the process of cell division. these were eventually used in various combinations with the intent of achieving selective toxicity and minimizing resistance. after 50 years of almost entirely empirical clinical trials, combination chemotherapy moved the prognosis of standard childhood leukemia from invariably fatal to an 85% cure rate. clear - cut benefit was established in breast, head and neck, ovarian, testicular and colon cancer. but very little progress was made in lung, prostate, liver, pancreas or brain cancer, and the toxicities of the treatments remain considerable - normal cells as well as cancer cells are badly injured in the process. although surgery, radiation therapy and combination chemotherapy have made an impact on the disease, early and late side effects are significant. towards the end of the 20th century it became clear that the efficacy of this ' carpet - bombing ' treatment of cancer had reached a plateau. during this period of trial and error some critically important concepts of cancer pathophysiology have been realized. chromosomes in the leukemias and lymphomas may appear normal but frequently show translocations and deletions that provide clues to the relatively few genes that are responsible for such tumors. in contrast, the chromosomes in epithelial cancer cells are almost always broadly and heavily damaged. the chromosome wreckage includes massive deletions, amplifications and rearrangements, as well as point mutations and translocations, and can be observed in very early stages of the growth of such tumors. the latter observation suggests that several mutated genes in an individual cancer may be responsible for the malignant state and that many more are mere accidents of the chromosome breakage. the art of oncogenetics lies in validating the relatively few significant mutations and differentiating them from the many innocuous byproducts of the chromosome breakdown that characterizes the epithelial cancer process. put simply, the epithelial cancer problem can be understood and effectively treated only by documentation of the truly oncogenic results of widespread dna damage. this demonstrates the critical importance of advances in the detection of cancer - causing genes. the role of cancer genetics as a discipline that would probably lead to more effective targeted treatment was highlighted by the discovery of a gain - of - function abl kinase gene that produced hyperactive abl kinase, a unique driver of early stage chronic myelogenous leukemia. in fact the successful interruption of unbridled abl kinase activity by imatinib ushered in a new era of ' smart drug ' treatment of cancer. in this new era, three major paths of progress have been and continue to be explored. the first has used patient - derived cancers, transgenic mice and transfected cells, dna and rna (including microrna) array technology and gene sequencing to establish potential genetic drivers of the initiation and maintenance of cancer cell growth. the second approach has focused on the failure of human and murine cancer cells either to repair their damaged dna or, without dna repair, to plunge fatally into a metabolic death pathway. both failures contribute to the malignant process, particularly in the common human epithelial cancers and also in certain lymphomas. a third and more recent approach has examined the organ environment of cancer and has revealed that the stromal fibroblasts surrounding cancer cells, such as ductal carcinomas, exude signals that break down the myoepithelial cell barrier that would otherwise confine the cancer cells to the duct [15 - 17 ]. furthermore, certain cancers, such as neurofibromas that arise as a result of homozygous loss - of - function mutations of a tumor suppressor gene (nf1), do not become actual tumors unless their surrounding stromal cells lack one of the two copies of the gene. in all three of these areas of oncogenetic research, drugs and antibodies have been sought that would either block gain - of - function proteins or replace key loss - of - function proteins. a considerable effort has also been expended to produce murine models of epithelial cancers so as to hasten the development of effective therapies. while this extensive basic research has been in progress, clinical scientists have been exploring the many ' smart drugs ' that have come off the assembly lines of pharmaceutical companies. what have been the results of all of this effort ? among the first waves of research have been discoveries of the genetic drivers of common and uncommon cancers. breast cancers, whether acquired or (rarely) inherited, are prime examples. despite vast chromosome damage and multiple mutations, most breast cancers are largely maintained by overexpression or possibly (and contested) amplification of the estrogen and progesterone receptor genes. simple estrogen receptor blockade or inhibition of estrogen synthesis combined with limited surgery and radiation therapy and ordinary combination chemotherapy can cure up to 80% of these cancers if they are diagnosed before widespread disease has occurred. in approximately 20% of cases a different mutation is responsible for the malignant state. in those less common cases the cause is amplification of her2-neu, a receptor kinase gene that expresses a subtype of an epidermal growth factor receptor. such her2-neu - positive cases have, until recently, been burdened by a very poor prognosis. however, recent clinical studies have demonstrated that early infusions of traztuzumab, a monoclonal antibody directed against the receptor, combined with standard treatment markedly improves the outlook for these heretofore unfortunate patients. the third major subtype of breast cancer, representing about 15% of cases, includes the so - called basal - like or triple - negative tumors that lack estrogen and progesterone receptors and her2-neu and have not been amenable to targeted therapy. recently, however, advantage has been taken of the resemblance of these tumors to those of the rare brca1 mutation carriers who have a high rate of inherited breast cancer. brca1 and brca2 are dna repair genes ; they thus provide protection from dna cross - linking agents, such as radiation or cisplatinum. basal - like tumors are therefore sensitive to cisplatinum and poly adp ribose polymerase (parp) inhibitors, an excellent example of how inquiries into cancer genetics improve cancer therapy. diffuse gastrointestinal stromal tumor, a rare and uniformly fatal sarcoma that, in its advanced stages, also shows severe chromosomal damage, can be obliterated by imatinib. resistance generally (but not always) occurs when the drug is used as a single agent, but the dramatic effect of imatinib proves that such tumors become dependent on or addicted to mutated tyrosine kinases (in this case kit or platelet derived growth factor receptor). the dramatic therapeutic results provide a remarkable example of the value of a single drug that can interrupt more than one of the gain - of - function proteins that drive such tumors. given that there are over 500 protein kinases in the human ' kinome ', major efforts are now in place to define their roles in the hundreds of fatal cancers. slowly but surely, incriminating evidence is being gathered that implicates previously unsuspected kinase mutations in various cancers. in addition to kinase drivers, the genes of other growth promoters, including transcription factors such as myc or signaling proteins such as wnt, have been shown to be mutated or amplified in many different cancers. indeed, mutations of kinases seem to be relatively uncommon causes of cancer, although they are the subjects of recent excitement. finally, loss - of - function mutations of dna repair genes prevent the repair of cancer - inducing genes, and the frequently observed loss or inactivation of genes such as p53 and mdm2 reduces the capacity of injured cancer cells to quit the cellular scene by means of apoptosis. this leads to cancer - cell immortality and failure of cancer chemotherapy and radiotherapy to achieve cancer cell death. in an entirely novel approach to drug development, peptides ' stapled ' by fatty acids have been used as effective experimental drugs that replace such missing proteins. as dna sequencing and array technology advances, investigators are churning out vast amounts of genetic information about common cancers, such as colon, breast, prostate, pancreas, lung and glioblastoma [32 - 36 ], and about unusual cancers, such as mesothelioma. recently, the entire dna sequence of a single case of acute myelogenous leukemia that had a normal karyotype was published. in addition to two previously described genetic abnormalities, six previously unappreciated mutations were observed. it is likely that only a few of the detected dna variations that emerge from complete dna sequencing will actually prove to be responsible for the tumors. the immense task is to sort through them and define them and then develop the drugs to either block or replace them. then we must face the pernicious problems of genetic or epigenetic mutational heterogeneity among the cells in a single tumor and of continued mutation. if we have the appropriate drugs, can we kill the cancer cells before they mutate again to develop new drivers that were not present before our new therapy was launched ? we need to develop the tools that will allow us to define tumor genetic and epigenetic heterogeneity. even when we solve those diagnostic problems (and we will), we and our patients face yet another barrier. it is already very clear that the new ' smart drug ' era does not imply that we will be successful very often with single agents. cancer cells with extra - labile dna will mutate to circumvent smart drugs very easily. this means that we must treat patients with combinations of drugs that block multiple metabolic pathways. toxicity may become a very severe problem as we force patients down that route. despite all these caveats, we are on the verge of understanding the biology of cancer, and with that understanding will come the drugs that will help us to beat it down. we may not actually cure all or even many of our previously unmanageable patients, but we will convert such cancers from killers to chronic smoldering illnesses that can be endured. our goal will be to provide cancer sufferers with a fulfilling life. this objective has been achieved in many cases of aids. it will take years of hard work, but we owe that commitment to the cancer patients who rely on us for a better future. we thank kornelia polyak md, phd, and kimberly stegmaier md, phd, for their critiques of the manuscript. | cancer is the most common acquired genetic disease. great progress has been made in documenting the genetic abnormalities that cause the disease, and in the future each tumor will be subjected to genetic analysis and the appropriate combination of drugs selected. although there are serious technological and cost hurdles to surmount and resistance and continued mutation will be a constant problem, the way is clear to rational therapy. |
androgen insensitivity gives rise to a wide spectrum of disorders in man, the most severe being complete sex reversal, to milder forms of pais associated with ambiguous or underdeveloped genitalia, or even milder forms causing only male infertility in otherwise healthy males. often mutations in the androgen receptor (ar) are involved which interfere with ligand binding, dna binding, or increase or decrease intramolecular interactions between ar domains. where no mutations are identified in the ar mutations in ar coregulators may be implicated in failure to activate or repress androgen - regulated target genes. although there are a number of mouse models available to study impaired ar function in vivo, the signalling networks are too complex to dissect without using simpler cell models. the aim of this study was to develop a cell model for the study of ar signalling in the urogenital tract. in turn male genital development relevant murine cell lines pc1 (proximal caput epithelial cells from mouse epididymus) and mfvd (mesenchymal fetal vas deferens cells) were genetically modified to express a tagged wild - type ar to test the system. the modifications allow purification of multiprotein complexes associated with ar under native conditions and analysis of the copurified protein complexes by mass spectrometry. the data was analysed using readily available bioinformatics software : the pathway mining tool of david bioinformatics resources [6, 7 ] and the gene group functional profiling tool of g : profiler. by focussing solely on known ar coregulators, we were able (as a proof of principle) to uncover differences in the proteome of proliferating and nonproliferating epithelial pc1 cells. the n - tap was designed by modifying the c - terminal tandem affinity tag (c - tap) from fernndez., 2009. the hat tag was amplified, using the c - terminal tag as a template and pcr primers (figure 1(a)) designed to add the tev protease cleavage site with the forward primer and the glycine - alanine repeat and an additional noti - cloning site with the reverse primer. the hindiii - noti fragment was then inserted into the polylinker region of p3xflag - cmv-10 (sigma) in frame with 3xflag (figure 1(b)). the mouse androgen receptor cdna clone (gift from professor jan trapman, department of pathology, erasmus mc / jni rotterdam) was modified by replacing the start methionine atg with an noti site - by - site directed mutagenesis (stratagene) and introducing the 2.787 kb noti - bhi full length mouse cdna into the n - tap - cmv-10 vector (figure 1(c)). the n - tap - mar fusion construct was confirmed by sequencing. in total 10 cos-1 or hela cells / well were seeded into 12-well tissue culture plates in dmem, containing 10% charcoal - stripped serum. cells were transiently transfected using fugene (roche) or lipofectamine 2000 (invitrogen) with 25 ng ar or n - tap - mar, 500 ng of pgre - luciferase and 25 ng ptk - rl according to manufacturer 's instructions. 1216 hours after transfection, the medium was replaced with dmem, containing 10% charcoal - stripped serum + or 10 nm dihydrotestosterone (dht ; sigma). 24 h later, cells were harvested and lysed in 25 mm glycine (ph 7.8), 15 mm mgso4, 4 mm egta, 1% triton 100 and 1 mm dithiothreitol. luciferase assays were performed with reagents from nanolight technology and the ratio of luciferin : renillaluciferase activity was measured using a turner td-20/20 luminometer. androgen responsive cell lines pc1 and mfvd from the mouse urogenital tract served as control cell lines in their nonmodified state. the pc 1 cell line was a gift from araki. and the mfvd cell line a gift from umar.. the pc1 cell line is an epididymal cell line immortalized with sv 40 large t - antigen and has been characterized in great detail regarding morphology, epithelial and epididymus specific gene expression, and androgen responsiveness. also the mfvd cell line is immortalized by expression of a temperature sensitive sv 40 large t - antigen but of mesenchymal origin. both cell lines were continuously cultured under conditions given elsewhere [4, 5 ] in the presence of 5 nm mibolerone, a synthetic androgen. the control cell lines pc1 and mfvd were transfected with the scai (unique site in the bacterial ampicillin resistance) linearised n - tap - mar vector using lipofectamine 2000 (invitrogen). after 48 hours, transfected cells were replated in dilutions from 1010 cells/14 cm diameter dish and g418 resistant clones were selected with 750 g / ml g418 (pc1) and 250 ug / ml g418 (mfvd). single colonies coming up were picked with cloning rings, grown up and tested for n - tap - mar expression by western blot analysis and immunocytochemistry using the flag m2 antibody (sigma) and the ar - n20 antibody (santa cruz). pc1 cells, derived from the mouse proximal caput epididymus and immortalized by expression of a temperature sensitive sv 40 large t - antigen, were continuously cultured at 33c, the permissive temperature of large t in the presence of 5 nm mibolerone. at physiological temperature (37c), cell growth is partly inhibited and t - antigen - expressing cells can survive. large t is however degraded after prolonged exposure of the cells to the nonpermissive temperature (39c), then significant cell death occurs and the cells do not recover. to prevent cell death but still have an inhibitory effect on proliferation early passage pc1 and p17 cells were cultured to near confluence at 33c and kept then for 1 week at 37c in growth medium containing 5 nm mibolerone before preparing cytoplasmic and nuclear extracts of the still healthy looking cells. for extracts made from proliferating pc1 and p17 cells, the cells were grown at 33c in growth medium containing 5 nm mibolerone. similar amounts of cell pellets from proliferating and nonproliferating cells were processed for protein extraction. cells from subconfluent cultures were washed, trypsinized, and pelleted by centrifugation and washed 3x in cold pbs. for whole cell lysates, cells were lysed in sds loading buffer (0.03125 m tris ph 6.8, 5% glycerol, 0.001% bromphenol blue 3, 1% sds, 2.5% -mercaptoethanol). samples were subjected to sodium dodecyl sulphate - polyacrylamide gel electrophoresis (sds - page), transferred to polyvinylidene difluoride membrane, blocked and probed in phosphate - buffered saline (150 mm nacl, 3 mm kcl, 10 mm phosphate salts (mono and dibasic) (ph 7.3), 0.05% (vol / vol) tween 20) containing 10% nonfat dry milk. primary antibodies were used at recommended dilutions and horseradish peroxidase - conjugated secondary antibodies (dako) and ecl plus blotting detection reagents (amersham) or supersignal west femto (thermo scientific) were used as described in the manufacturer 's instructions. src-1 (128e7) and ctnnb1 (9587) antibodies were purchased from neb, flag m2 (f3165) from sigma, and n20-ar (sc-816), nr3c1 (sc-8992), smarcc1 (sc-9748), actb (sc-81178) from santa cruz. the protein extraction protocol is a modified version of a chromatin extraction protocol for co - purification of histones by saade., 2009. cells were grown on 14 cm diameter dishes to confluence (10 plates pc1 or 20 plates p17 gave about 700 l cell volume), washed 3x with prewarmed pbs, trypsinised with 1 ml trypsin - edta (sigma)/plate 5 minutes 37c, inactivated with medium, pooled in 50 ml falcon, spun1200 rpm 5 mins rt, and washed 3x with cold (4c) pbs. cells were transferred to a 2 ml microfuge tube and pelleted by centrifugation at 6500 rpm 2 - 3 minutes at 4c. pellets (2 200 l) were resuspended in 2 1.8 ml hypotonic buffer (10 mm tris ph 7.5, 10 mm kcl, 1.5 mm mgcl, 0.1% triton, 4.5 mm -mercaptoethanol, protease inhibitor cocktail (roche), 5 nm mibolerone, 1 mm pefabloc (roche), and phosstop (roche)) by pipetting up and down and vortexing vigorously on highest setting for 15 seconds followed by 45 minute incubation on ice vortexing every 10 minutes. nuclei were spun down at 4c 13.000 rpm for 5 minutes, the supernatant, called here cytosol fraction, transferred to a clean tube and nacl was added to 15 mm. coupling of the magnetic beads (dynabeads m-270 epoxy from invitrogen) to the flag m2 antibody (sigma f3165) was performed according to manufacturer 's instructions. nuclei were taken up in sucrose buffer (0.34 m sucrose, 10 mm tris ph8, 3 mm mgcl2, 1 mm cacl2, 150 mm nacl, 1 mm dtt, protease inhibitor cocktail (roche), 5 nm mibolerone, 1 mm pefabloc (roche), phosstop (roche)) 150 l/100 l cell pellet and resuspended. 0.3 units micrococcal nuclease from sigma (0.1 u/l in 10 mm tris/0.1 mm cacl2 ph8) for 100 l nuclear extract were added and incubated for 30 min at 37c. extracts were then diluted with 1 volume of sucrose buffer and sonicated 6 10 seconds on ice with 10 sec bursts and 10 sec cool downs alternating. extracts were finally loaded onto flag m2-coupled magnetic beads and rotated for at least 1 hour at 4c in cold room. supernatants were kept as flow through and aliquots before loading on to the beads as nuclear input. after the incubation period, beads loaded with cytosol preparations were washed 3x with hypotonic wash buffer (10 mm tris ph 7.5, 10 mm kcl, 1.5 mm mgcl, 0.1% triton, 4.5 mm -mercaptoethanol, protease inhibitor cocktail (roche), 5 nm mibolerone, 1 mm pefabloc (roche) and phosstop (roche), 0.5% np40, 0.05% sodium deoxycholate, 0.005% sds, 15 mm nacl) or sucrose buffer (nuclear preps) followed by 3 washes with tev buffer (0.05 m tris ph 8, 1 mm dtt, 0.5 mm edta) supplemented with protease inhibitor cocktail (roche), mibolerone (5 nm), and phosphatase inhibitors (phosstop and pefabloc (roche), and nacl to15 mm nacl in cytosolic and 150 mm nacl in nuclear tev buffer. the beads were finally taken up in 100 ul tev buffer and bound protein complexes were eluted with 10 units of tev protease (gst - tag) (tevp us biological) at rt for 1 hour. colloidal coomassie stained protein bands (colloidal blue staining kit, invitrogen) were excised out of a 5% page after separation in 7 slices covering a size spectrum of 48 kda to the top of the gel. gel slices were briefly washed in milliq water and kept in water at 80c until submitted to cambridge proteomics services where all lc - ms / ms experiments were performed using an eksigent nanolc-1d plus (eksigent technologies, dublin, ca) hplc system and an ltq orbitrap velos mass spectrometer (thermofisher, waltham, ma). separation of peptides was performed by reverse - phase chromatography used at a flow rate of 300 nl / min and an lc - packings (dionex, sunnyvale, ca) pepmap 100 column (c18, 75 m i.d. peptides were loaded onto a precolumn (dionex acclaim pepmap 100 c18, 5 m particle size, 100a, 300 m i.d. 5 mm) from the autosampler with 0.1% formic acid for 5 minutes at a flow rate of 10 l / min. after this period, the valve was switched to allow elution of peptides from the precolumn onto the analytical column. solvent a was water + 0.1% formic acid and solvent b was acetonitrile + 0.1% formic acid. the lc eluant was sprayed into the mass spectrometer by means of a new objective nanospray source. all m / z values of eluting ions were measured in an orbitrap velos mass analyzer, set at a resolution of 30000. data dependent scans (top 20) were employed to automatically isolate and generate fragment ions by collision - induced dissociation in the linear ion trap, resulting in the generation of ms / ms spectra. briefly, all ms / ms data were converted to mgf (text) files. these files were then submitted to the mascot search algorithm (matrix science, london uk) and searched against uniprot mouse database, using a fixed modification of carbamidomethyl and variable modifications of oxidation (m). stable mfvd and pc1 clones expressing n - tap - mar were developed as described in section 2. total protein was extracted from several clones and analyzed for n - tap - mar expression by western blot using flag and ar - specific antibodies (data not shown). a second criteria was the nuclear localization of n - tap - mar, which was examined in several clones by immunofluorescence using flag and ar - specific antibodies. one mfvd cell line (m7) and one pc1 cell line (p17) were selected on the basis that they expressed similar levels of stably integrated n - tap - mar and endogenous wild - type ar (wt ar). both were predominantly located in the nucleus when grown in medium supplemented with 5 nm mibolerone (figure 2(a)). the transactivation properties of n - tap - mar were tested in cos cells by cotransfection of a gre reporter construct, with expression levels confirmed by western blot (figures 2(b) and 2(c)). levels of expression are stable, allowing considerable grow up of cells. different protein purification protocols for producing nuclear and cytosol cell extracts were tested. commercially available purification kits and a modified version of a chromatin purification protocol (flag - antibody capture) were compared and the latter was chosen. flag - antibody capture gave a good recovery of n - tap - mar when preparing extracts from m7 (figure 3(a)) or p17 cells (figure 3(b)). the known coregulators src-1 and ctnnb1 were copurified in p17 extracts but not in m7 extracts. attempts to optimize his - tag purification conditions were unsuccessful ; nickel affinity purification of the pc1 (n - tap - mar negative control) chromatin extracts gave high background with unspecific protein binding to the nickel resin. the cell lines proliferate at 33c with growth driven by temperature sensitive sv40 t - antigen ; and proliferation stops at 37c. figure 3(c) illustrates a preparative gel of tev protease - eluted proteins purified from the nuclei of p17 cells cultured at 33c and 37c. the gel slices were selected for lc- ms / ms, and data processed using the mascot search engine at the cambridge centre for proteomics. the results from the lc - ms / ms search are portrayed as peptide matches and grouped as protein hits using a simple parsimony algorithm (http://www.matrixscience.com/). only those ions scores that exceed a significance threshold of 0.05 (1 in a 20 chance of being a false positive) contributed to the score. this would translate into 1500 peptides falling within the mass tolerance window to have a score of 45. known ar - interacting proteins (http://androgendb.mcgill.ca) were identified among the protein hits and are listed in table 1 along with their respective peptide scores. in this first mass spec analysis, we identified 1196 ar associated proteins in flag purified extract from proliferating cells and 1456 from nonproliferating cells. of those 882 were common between those two groups, 314 were only picked up in flag purifications from proliferating cells and 574 were only picked up in flag purifications from nonproliferating cells. functional profiling was carried out using web tools david [6, 7 ] and g : profiler, providing functional enrichments in the form of pathways, biological processes, molecular functions, metabolic functions, cellular localization, protein - protein interactions, and shared transcription factor binding sites. only pathways and biological processes which received the highest scores are utilized. 37cthe gene list of known ar - interacting proteins identified in the nuclear flag purifications of the 37c samples (37 only + common, table 1) was accepted as 65 david i d 's using david bioinformatics resources [6, 7 ]. goterm_bp_fat gives the highest score to the biological process (bp) regulation of transcription with 40 contributing genes (table 2). listed 11 genes : ncor2, smarca4, ar, psmc3, prkdc, kdm1a, trp53, and ep300 common to both temperatures and med1, nf1, and sp1 specific to the 37c samples (table 4). when analyzing the gene list for pathways, 10 genes are components of the kegg pathway : pathways in cancer : ep300, ar, ctnnb1, dapk3, hsp90, pias1, rb1, stat3, hdac1, and trp53. of these 10 genes only stat3, rb1, and dapk3 have inhibitory function in this pathway. 6 of those 10 genes (ep300, ar, ctnnb1, hsp90, rb1, and trp53) have been associated with prostate cancer (table 3).the biocarta chart revealed overrepresented pathways : telomeres, telomerase, cellular aging, and immortality, with 5 genes (xrcc5, xrcc6, hsp90, rb1, and trp53) involved. another overrepresented pathway chromatin remodeling by hswi / snf atp - dependent complexes involves genes smarca4, actb, nr3c1, nf1, arid1a. a third overrepresented biocarta pathway is control of gene expression by vitamin d receptor with ep300, smarca4, med1, ncoa2, and arid1a involved (table 3). the gene list of known ar - interacting proteins identified in the nuclear flag purifications of the 37c samples (37 only + common, table 1) was accepted as 65 david i d 's using david bioinformatics resources [6, 7 ]. goterm_bp_fat gives the highest score to the biological process (bp) regulation of transcription with 40 contributing genes (table 2). another high scoring biological process chordate embryonic development listed 11 genes : ncor2, smarca4, ar, psmc3, prkdc, kdm1a, trp53, and ep300 common to both temperatures and med1, nf1, and sp1 specific to the 37c samples (table 4). when analyzing the gene list for pathways, 10 genes are components of the kegg pathway : pathways in cancer : ep300, ar, ctnnb1, dapk3, hsp90, pias1, rb1, stat3, hdac1, and trp53. of these 10 genes only stat3, 6 of those 10 genes (ep300, ar, ctnnb1, hsp90, rb1, and trp53) have been associated with prostate cancer (table 3). the biocarta chart revealed overrepresented pathways : telomeres, telomerase, cellular aging, and immortality, with 5 genes (xrcc5, xrcc6, hsp90, rb1, and trp53) involved. another overrepresented pathway chromatin remodeling by hswi / snf atp - dependent complexes involves genes smarca4, actb, nr3c1, nf1, arid1a. a third overrepresented biocarta pathway is control of gene expression by vitamin d receptor with ep300, smarca4, med1, ncoa2, and arid1a involved (table 3). 33cthe gene list of known ar - interacting proteins identified in the nuclear flag purifications of the 33c samples (33 only + common, table 1) was accepted as 56 david i d 's using david bioinformatics resources [6, 7 ]. arid1b was not detected as david i d and therefore not included in the analysis. goterm_bp_fat gives the highest score to the biological process (bp) regulation of transcription with 34 contributing genes (table 2). looking at overrepresented pathways only 7 ar - interacting proteins are components of the kegg pathway pathways in cancer : ep300, ar, ctnnb1, hsp90, stat3, hdac1, and trp53. proteins rb1, dapk3, and pias1 are not present in the 33c samples (table 3). following on from that, only 5 of those genes have been associated with prostate cancer (ep300, ar, ctnnb1, hsp90, and trp53) and the inhibitory function of rb1 is missing (table 3).again the biocarta chart brings up as overrepresented pathways : telomeres, telomerase, cellular aging, and immortality, but here with only 4 genes being involved (xrcc5, xrcc6, hsp90, and trp53) and rb1 missing. chromatin remodeling by hswi / snf atp - dependent complexes has nr3c1 and nf1 missing and gained 3 new components with arid1b, smarcc1, and smarcd1. the pathway control of gene expression by vitamin d receptor has now 5 components present ep300, smarca4, but not med1 and ncoa2 anymore. the gene list of known ar - interacting proteins identified in the nuclear flag purifications of the 33c samples (33 only + common, table 1) was accepted as 56 david i d 's using david bioinformatics resources [6, 7 ]. arid1b was not detected as david i d and therefore not included in the analysis. goterm_bp_fat gives the highest score to the biological process (bp) regulation of transcription with 34 contributing genes (table 2). looking at overrepresented pathways only 7 ar - interacting proteins are components of the kegg pathway pathways in cancer : ep300, ar, ctnnb1, hsp90, stat3, hdac1, and trp53. proteins rb1, dapk3, and pias1 are not present in the 33c samples (table 3). following on from that, only 5 of those genes have been associated with prostate cancer (ep300, ar, ctnnb1, hsp90, and trp53) and the inhibitory function of rb1 is missing (table 3). again the biocarta chart brings up as overrepresented pathways : telomeres, telomerase, cellular aging, and immortality, but here with only 4 genes being involved (xrcc5, xrcc6, hsp90, and trp53) and rb1 missing. chromatin remodeling by hswi / snf atp - dependent complexes has nr3c1 and nf1 missing and gained 3 new components with arid1b, smarcc1, and smarcd1. the pathway control of gene expression by vitamin d receptor has now 5 components present ep300, smarca4, but not med1 and ncoa2 anymore. identical gene lists submitted to david were also submitted to the gene ontology online tool g : profiler for functional characterization. regulation of transcription was not a high scoring biological process on this occasion, whereas gene expression scored high at both temperatures, with many genes overlapping in both categories (table 2). also here the kegg pathway components for prostate cancer are revealed for both temperatures, but only 4 (hsp90, ar, ctnnb1, and trp53) as shown in table 3. 37ckegg pathway : pathways in cancer is only detected in the 37c samples, with 9 components represented (ep300, stat3, hdac1, hsp90, ar, ctnnb1, trp53, dapk3, and pias1). the gene ontology subgroups for biological processes (bps), anatomical structure morphogenesis, and embryo development are overrepresented in the 37c samples and overlapping components are involved (table 4). embryo development components are ncor2, ar, ctnnb1, kdm1a, prkdc, sp1, med1, smarca4, hdac1, trp53, tgfb1l, psmc3, and nf1. anatomical structure morphogenesis components are ncor2, ar, ctnnb1, actb, prkdc, sp1, med1, smarca4, hdac1, trp53, tgfb1l, gsn, nf1, gata3, stat3, nr3c1, and stat3. comparison of these 2 groups suggests a more specific function in morphogenesis during embryo development for nr3c1, gata3, and stat3 (strong evidence) gsn and actb (weak evidence). kegg pathway : pathways in cancer is only detected in the 37c samples, with 9 components represented (ep300, stat3, hdac1, hsp90, ar, ctnnb1, trp53, dapk3, and pias1). the gene ontology subgroups for biological processes (bps), anatomical structure morphogenesis, and embryo development are overrepresented in the 37c samples and overlapping components are involved (table 4). embryo development components are ncor2, ar, ctnnb1, kdm1a, prkdc, sp1, med1, smarca4, hdac1, trp53, tgfb1l, psmc3, and nf1. components are ncor2, ar, ctnnb1, actb, prkdc, sp1, med1, smarca4, hdac1, trp53, tgfb1l, gsn, nf1, gata3, stat3, nr3c1, and stat3. comparison of these 2 groups suggests a more specific function in morphogenesis during embryo development for nr3c1, gata3, and stat3 (strong evidence) gsn and actb (weak evidence). 33camong cellular component profiling, the nbaf complex is solely detected in the 33c samples with smrcc1, smarcd1, smrca4, and arid1a (not listed in tables). these proteins are also components of the swi / snf chromatin remodelling complex. among cellular component profiling, the nbaf complex is solely detected in the 33c samples with smrcc1, smarcd1, smrca4, and arid1a (not listed in tables). the observation that some ar - interacting proteins were only detectable in proliferating cells and others only in nonproliferating cells (table 1) could simply mean that the quality of the cell extracts varies and much less protein is present in one of the extracts. another reason might be that expression levels of those proteins alter during cell proliferation, and less or more protein is available for interaction with ar. a third option is that the binding affinity of ar to the interacting proteins changes with the proliferation status of the cells. to address these questions, we carried out western blots, probing those ar - interacting proteins which were differentially expressed (figure 4). no major differences in expression were observed when extracts were probed with ar - n20 and actb control antibodies, but differences were found elsewhere. nr3c1 is expressed at much higher levels in nonproliferating versus proliferating p17 cells, and expression levels of smarcc1 were slightly lower in nonproliferating (37c) pc1/p17 when compared to proliferating cells (33c). the difference in the amounts of ar copurified smarcc1 in proliferating versus nonproliferating is not as striking as it is for nr3c1, and the amounts at low expression the proliferation status of p17 cells seems to affect smarcc1 and nr3c1 expression levels per se as smarcc1 expression is up and nr3c1 expression is down in proliferating cells. however, the loss of nr3c1 expression in proliferating cells is unlikely to account for all the loss in ar binding observed here. similar loss of smarcc1 expression in nonproliferating cells does not account for all the loss in ar binding detected. therefore, expression levels of coregulators and binding affinity to ar may contribute to the differences observed in the 33c and 37c coregulator profiles. the n - tap - mar itself appears to increase gr expression levels by at least 3 fold under nonproliferating conditions comparing gr expression in pc1 and p17 input samples (figure 4). we have developed the epithelial and mesenchymal mouse cell lines p17 and m7 for copurification of ar - associated protein complexes under native conditions. the two cell lineages are derived from the proximal caput epididymus (p17) and the mesenchyme of the fetal vas deferens (m7) of the mouse. the decision to carry out preparative scale n - tap - mar purification on pc1 cells rather than mfvd cells was based on the observation that ctnnb1 and src-1 could not be copurified from mfvd cells with our extraction protocol, whereas in p17 copurifications both coactivators were easily detected. another advantage of choosing the pc1 cell line was the more abundant ar expression and the rapid growth compared to the mesenchymal cells. one confluent dish of pc1 gave about 510 times as much cell pellet than 1 confluent dish of mfvd. to demonstrate proof of principle we used the newly developed purification protocol to detect differences in ar cofactor binding in cells grown under proliferating (33c) and nonproliferating (37c) conditions. as expected, many of the copurified proteins confirm the role of the ar in chromatin remodelling machinery, transcriptional complexes and associated with the cytoskeleton. the purification protocol we use is relatively crude and results in enrichment of 200500 bp dna fragments after a micrococcal nuclease digestion. no size fractionation step is included, which could allow isolation of larger chromatin fractions, especially heterochromatic fractions which are not degraded by micrococcal nuclease. this fraction could contribute to a background of unspecific binding, which is difficult to control for as this might not occur in the pc1 control sample, where the anchor in form of the n - tap - mar is missing. on the other hand, the purification protocol is selecting for stable interactions, because no cross - linking step is included in our purification procedure. we have not tested whether novel interacting proteins identified with this approach are indeed associated with ar or whether they are just contaminants. these potential interacting proteins could be novel ar - interacting proteins, but could also bind unspecific to the flag - m2-coupled magnetic beads. unspecific binding to the flag antibody - coupled magnetic beads is estimated at 5% based on the recovery of protein from the flag purification of the pc1 control sample. this would mean that 1 out of 20 identified proteins is not part of the ar - associated complex. unfortunately we were not able to reduce this background with an additional his purification step, because the flag purified and protease (tev) eluted fraction was not able to specifically bind nickel resin, probably caused by complex components covering up the his tag. in this study we concentrate only on a small proportion of all the potential ar - binding partners identified, representing the already known ar - interacting proteins. we show that our approach has the potential to differentiate between proteins which preferably form part of the ar complexes in proliferating or nonproliferating conditions, and those proteins where interaction is independent of the proliferation status of the cells. common pathways enriched for or overrepresented in proliferating and nonproliferating ar flag purifications were the biological processes transcriptional regulation and gene expression. considering that the androgen receptor is classified as a transcription factor, scoring surprisingly high were biocarta pathways not involving ar at all such as chromatin remodelling by hswi / snf atp - dependent complexes and control of gene expression by vitamin d receptor (vdr) (figure 5), which involves the winac chromatin - remodelling complex. both winac and swi / snf complexes the requirement of the baf complexes has been shown in vitro for ligand - dependent transactivation by nuclear hormone receptors, such as vitamin d3 receptor, retinoid x receptor, and peroxisome proliferator - activated receptor ppar-. it has also been shown in vivo for reconstitution of glucocorticoid - receptor-(nr3c1-) dependent transcription, chromatin remodelling on interferon and virus inducible genes and in neural development with a subunit switch in the npbaf (neural progenitors - specific) chromatin remodelling complex, essential for the transition from neural stem / progenitors to postmitotic neurons but has never been associated with ar. also the vitamin d3 receptor has not been identified as ar - interacting protein neither in our purification (data not shown) nor by others. it is however possible that ar transactivation might be stimulated by components of the winac complex. the vdr and the ar could therefore compete for shared coregulators, which would explain the observation that ar stimulation by androgens suppresses vdr [20, 21 ], while ar downregulation by sirna stimulates vdr levels in lncap cells. winac complex components might be potential coplayers in ar transactivation, which could be tested with sirna cotransfection experiments in our cell line. g - profiler, which does not offer a tool such as the biocarta, also identified baf components as being overrepresented. another overrepresented pathway is the prostate cancer pathway, which is picked up by both bioinformatics tools. g - profiler does not identify ep300 as a gene involved in prostate cancer as it is done by david (table 3 bottom). also the kegg cancer pathway is picked up by both bioinformatics tools : g - profiler identifies the same list of genes among the known ar - interacting proteins as, only that rb1 is not included in the g - profiler gene list (table 3 bottom). for male genital development and pais relevant biological processes are embryo development, which is here represented with 13 genes identified by g - profiler and 11 identified by david. of those 10 are overlapping (table 4). the embryonic genes identified by david are restricted to chordate development and do not include ctnnb1, hdac1 and tgfbi1, although ctnnb1 and hdac1 knock outs in mouse have been shown to result in developmental phenotypes [22, 23 ]. a role of ep300 in patterning and development was suggested by studies in mice in which ep300 expression was disrupted. both bioinformatic tools david and g - profiler complemented each other in this study in identifying androgen receptor regulated pathways and biological processes. in future we aim to replace the endogenous ar with n - tap - mar. we will hopefully be able to apply the flag purification protocol tested in this study to identify differences in the proteome caused by the respective ar mutation. the protein purification approach taken here and shown to identify differences in co - factor recruitment of ar under proliferating and nonproliferating conditions is encouraging to undertake further experiments aiming to identify specific interaction protein profiles for ar mutants associated with pais. | partial androgen insensitivity syndrome (pais) is associated with impaired male genital development and can be transmitted through mutations in the androgen receptor (ar). the aim of this study is to develop a cell model suitable for studying the impact ar mutations might have on ar interacting proteins. for this purpose, male genital development relevant mouse cell lines were genetically modified to express a tagged version of wild - type ar, allowing copurification of multiprotein complexes under native conditions followed by mass spectrometry. we report 57 known wild - type ar - interacting proteins identified in cells grown under proliferating and 65 under nonproliferating conditions. of those, 47 were common to both samples suggesting different ar protein complex components in proliferating and proliferation - inhibited cells from the mouse proximal caput epididymus. these preliminary results now allow future studies to focus on replacing wild - type ar with mutant ar to uncover differences in protein interactions caused by ar mutations involved in pais. |
intraoperative blood loss is one the most commonly used parameters to assess the safety of surgical procedures. available data regarding laparoscopic radical prostatectomy (lrp) indicates unambiguously that the surgery is safe, also in terms of blood loss [1, 2 ]. what is more, reduced blood loss remains one of crucial advantage of lrp when compared with open radical prostatectomy [3, 4, 5 ]. many investigators confirmed all facts mentioned above since 1997 when schuessler performed the first lrp in history. 250 ml is reported in recent studies enrolling a large series of patients. however, traditionally we base blood loss on blood volume collected intraoperatively in the suction device bottles. we already know that both anesthesiologists and urologists tend to underestimate blood loss during open retropubic prostatectomy and that the underestimation is relatively important. the little accuracy of visual estimation of blood loss had been clearly proven by gynecologists and obstetricians [9, 10, 11 ]. in this setting, we decided to answer the question whether intraoperative blood loss during lrp assessed clinically corresponds with changes in blood cell count (bcc). the aim of this study was to analyze changes in bcc resulting from lrp and to compare them with clinical blood loss estimation. there were 71 men submitted to extraperitoneal lrp due to organ confined prostate cancer in our institution from september 2009 to march 2011. based on a surgeon 's subjective assessment reported in their surgical protocol, we isolated the group of 61 men with clinically minimal intraoperative blood loss, which was defined as loss of less then 200 ml. for the final analysis we included 60 men with full data regarding surgery and laboratory findings in perioperative period. mean age of the cohort was 62.8 years. we performed retrospective analysis of medical documentation with special interest in laboratory findings. according to standard management with patients submitted to lrp in our institution, all the group had blood tests performed a day before (test 1) and 6 hours after the surgery (test 2). we registered results of all bcc parameters, creatinine serum concentration and estimated glomerular filtration rate (egfr) at these two time intervals. an automated hematology analyzer was used for bcc and the colorimetric jaffe reaction for creatinine serum concentration measurement. estimated gfr was calculated using the mdrd (modification of diet in renal disease) formula. paired t - test was used for comparing results of tests 1 and 2 for each parameter. normal distribution was confirmed by the shapiro - walk test while the equality of variances was assessed using levene 's test before all the t - tests were performed. the differences were considered to be statistically significant when the p - value was lower than 0.02. there were 71 men submitted to extraperitoneal lrp due to organ confined prostate cancer in our institution from september 2009 to march 2011. based on a surgeon 's subjective assessment reported in their surgical protocol, we isolated the group of 61 men with clinically minimal intraoperative blood loss, which was defined as loss of less then 200 ml. for the final analysis we included 60 men with full data regarding surgery and laboratory findings in perioperative period. mean age of the cohort was 62.8 years. we performed retrospective analysis of medical documentation with special interest in laboratory findings. according to standard management with patients submitted to lrp in our institution, all the group had blood tests performed a day before (test 1) and 6 hours after the surgery (test 2). we registered results of all bcc parameters, creatinine serum concentration and estimated glomerular filtration rate (egfr) at these two time intervals. an automated hematology analyzer was used for bcc and the colorimetric jaffe reaction for creatinine serum concentration measurement. estimated gfr was calculated using the mdrd (modification of diet in renal disease) formula. paired t - test was used for comparing results of tests 1 and 2 for each parameter. normal distribution was confirmed by the shapiro - walk test while the equality of variances was assessed using levene 's test before all the t - tests were performed. the differences were considered to be statistically significant when the p - value was lower than 0.02. we noticed statistically and clinically significant differences regarding all bcc parameters measured at the two time intervals. the measurement results are expressed as mean values and standard deviations rbc red blood cells ; hb hemoglobin concentration ; hct hematocrit ; wbc white blood cells ; pct platelet crit simultaneously, renal function, assessed by creatinine serum concentration measurement and egfr calculation, was stable. the measurement results are expressed as mean values and standard deviations creat creatinine serum concentration ; egfr estimated glomerular filtration rate our retrospective investigation was aimed at critical analysis of the value of clinical assessment of blood loss during lrp. to the best of our knowledge, such an analysis has never been done before, while its clinical importance remains obvious. based on previous studies we can calculate the effect of blood loss on hemoglobin concentration. using the same formulas taking the results of a study performed by davies., we may assess the real blood loss in our group of patients to be as high as 8 ml / kg, hence 600 ml in a typical 75 kg male prostate cancer patient. at the same time, clinically assessed blood loss was calculated by the surgeon to be not more than 200 ml. our study shows discrepancies between clinical estimation and laboratory results using the laparoscopic approach, which occurred to be much more significant. while the differences in open prostate surgery do not exceed 30%, we noticed differences of 300% in our group. finally, it is also worth remembering that laboratory tests are imperfect. showed that when assessing blood loss based on a decrease in hemoglobin concentration, we can underestimate it by more than 30%. it means that real blood loss in our group of patients might even exceed 800 ml, hence we found a four - fold clinical underestimation of blood loss. we can summarize that clinical and laboratory methods of blood loss estimation are neither practical nor reliable. this important statement is supported by schorn 's critical review of methods of blood loss measurement. the reason for such important differences between clinical and laboratory estimation of blood loss during lrp should be indicated. in general, bleeding during laparoscopic procedures may be reduced following insufflation of the appropriate cavity and the associated tamponade effect [1518 ]. in this setting, venous bleeding is more likely to occur shortly after the procedure than during the surgery. we suppose that visual underestimation of blood loss may result from early postoperative formation of perivesical, perineal, or obturator hematomas, while direct measurement of blood loss does not consider it. first, it is its retrospective character, which is supported by archival medical documentation. this fact may affect results, however, obtained results are clearly unambiguous. whether we under- or overestimate the significance of the problem second, being a pilot study, this analysis covers only 60 cases of lrp with clinically minimal blood loss. based on our preliminary results, it may be justified to collect the data prospectively in order to finally confirm our findings. the accuracy of clinical estimation of blood loss during lrp is low. while clinically insignificant, blood loss seems to be as high as approx. bcc seems to be a more accurate method of intraoperative blood loss estimation compared to measurement of blood volume collected intraoperatively in the suction device bottles. | introductionthe traditional assessment of blood loss during laparoscopic radical prostatectomy (lrp) is based on the blood volume collected intraoperatively in the suction device bottles. while this method is not perfect, analysis of changes in blood cell count (bcc) resulting from lrp is advisable.material and methods71 men were submitted to lrp due to prostate cancer in our institution over an 18-month time period. from this group, we isolated 60 men with clinically minimal intraoperative blood loss (< 200 ml) and included them into the study. mean age of the cohort was 62.8 years. we performed standard bcc on the day before and 6 hours after the surgery. at the same time points, we measured creatinine serum concentration and calculated egfr to avoid the data misinterpretation resulting from impaired renal function in the postoperative period.resultsstatistically and clinically significant differences regarding all bcc parameters measured pre- and postoperatively were observed. the number of red blood cells, hemoglobin concentration, and hematocrit diminished by 17.5% (4.68t / l vs. 3.86t / l, p < 0.02), 17.0% (8.93 mmol / l vs. 7.41 mmol / l, p < 0.02), and 17.9% (0.429 vs. 0.352, p < 0.02), respectively. simultaneously, renal function was stable with no significant change in egfr (82.9 ml / min/1.73 m^2 vs. 79.09 ml / min/1.73 m^2, p = 0.28).conclusionsstandard lrp brings on a significant blood loss. while clinically insignificant, this blood loss seems to be as high as approx. 600 ml based on laboratory findings. bcc seems to be a more accurate method of intraoperative blood loss estimation compared to measurement of blood volume collected intraoperatively in the suction device bottles. |
in the human male, testosterone (t) and estradiol (e2) are the main circulating sex steroids acting on bone tissue. the first is produced from the leydig cells in the testis, while the latter derives from the aromatization of the androgens by means of the enzymatic complex of aromatase. aromatase is a cytochrome p450 enzyme encoded by the cyp19a1 gene that plays a key role in estrogen biosynthesis : it catalyzes the conversion of -androstenedione into estrone and that of t into e2 [2, 3 ]. aromatase is widely expressed in a large number of tissues such as testis (sertoli and leydig cells), ovary (granulosa cells and luteal corpus), brain (including hypothalamus), hair follicles, and fibroblasts [2, 3 ]. adipose tissue also expresses aromatase and it constitutes an important source of estrogens, especially in men [14 ]. in men, in fact, e2 is mainly produced by the testis and secondarily by adipose tissue [24 ]. biological actions of estrogens are mediated by their receptor (er) that belongs to the nuclear receptors family, and, to date, two different ers have been identified : er- and er-. a further nongenomic pathway of estrogen action has been described probably involving a plasma membrane interaction of the er [6, 7 ]. animal [8, 9 ] and human [10, 11 ] models of male congenital estrogen deficiency offered a new scenario useful for better understanding estrogen effects on male bone as well as several organs and tissues in men [1214 ]. all these physiological actions of estrogens in men remained overlooked for a long time (see for review). in the past, estrogen was also erroneously considered indispensable for blastocyst implantation and congenital estrogen defects are supposed to be incompatible with life [10, 11 ]. the discovery of the first cases of congenital estrogen defects in humans allowed understanding that aromatase deficiency is due to mutations of the gene coding for the aromatase enzyme complex, which leads to lack of both estrogen synthesis and action, while estrogen resistance is due to mutations of the genes coding for estrogen receptors and leads to resistance to estrogen actions even in presence of circulating estrogens [10, 11 ]. this review will focus on the role of estrogens on human male bone according to all skeletal physiological events that occur in vivo in different life stages in men. the rise of t and e2 in men at puberty progressively exposes bone to sex steroids, thus allowing them to act on the growing skeleton. sex steroids modify the way through which immature bone develops in terms of size, structure, bone mineral density (bmd), and proportions till the achievement of final skeletal maturation. after the achievement of peak bone mass, estrogens continue to influence bone remodeling in adulthood, the decline of circulating e2 being directly correlated with bone loss from adult to aging life. very low levels of estrogens circulate in the blood even in male children during infancy, but their real physiological significance is not known. in prepubertal boys with a genital tanner stage 1, serum e2 measured with the gold standard liquid chromatography tandem mass spectrometry (lc / ms / ms) starts to increase ranging from 0.51.0 to about 1.9 pg / ml in healthy controls and obese boys, respectively [17, 18 ]. considering the developing skeleton to not be under the effects of estrogens before puberty in male fetuses and children is a good simplification, even though this is a poorly investigated field of research (table 1). the initial activation of the hypothalamic - pituitary - gonadal axis in male children resulting in a progressive, slow increase of sex steroids, including estrogens, characterizes the peripubertal period [1719 ]. bone exposure to low concentrations of estrogens leads to well - known estrogen - dependent bone changes in males [20, 21 ]. the growth of long bones occurs at the growth plate, a thin layer of cartilage that separates the epiphysis from the metaphysis. the growth plate consists of three distinct layers of resting (stem cell - like), proliferative and hypertrophic chondrocytes [22, 23 ]. the concept that sex steroids promote epiphyseal growth and maturation during puberty in both sexes was a well - known issue in endocrinology since the beginning of the last century [2426 ]. this classical endocrinological theory was based on a well - distinct action of estrogen from androgen on bone. in fact, it was believed that the former leads to growth plate maturation only in women, while the latter leads to growth plate maturation only in men [28, 29 ]. as a matter of fact, the failure of epiphyseal closure and osteoporosis observed in adult men with congenital hypogonadotropic hypogonadism or with childhood onset severe t deficiency were both traditionally ascribed to insufficient bone exposure to androgen at puberty [30, 31 ]. notwithstanding evidence on estrogen actions on bone maturation in men had become available since the 1980s [3234 ] ; this viewpoint lasted since the beginning of the 1990s when the idea that estrogen is the main sex steroid involved in male bone maturation started to advance thanks to the description of the first cases of congenital defects of estrogen synthesis or action [3640 ]. the clinical phenotype presented by the unique male patient described to date with estrogen resistance was very close to that of men with aromatase deficiency [37, 40 ]. it is characterized by tall stature, a history of continuous linear growth into adulthood, unfused epiphyses, progressive genu valgum, eunuchoid proportion of the skeleton, delayed bone age, and osteoporosis. in 1997, carani. demonstrated that transdermal e2 replacement is effective in obtaining complete epiphyseal closure, final skeletal maturation, the arrest of growth in height, the increase in bmd, and peak bone mass [10, 38 ]. this result was subsequently replicated by many other authors in all aromatase - deficient men described so far [4046 ]. on the other hand, six months of treatment with high doses of t, given before the diagnosis of aromatase deficiency in an attempt to arrest continuous linear growth, had no effects on bone age in this patient [10, 38 ]. besides, e2 treatment was not effective in the estrogen - resistant man, as expected. all these findings suggest that epiphyseal closure is an estrogen - dependent phenomenon even in males and that androgens by themselves are not effective to ensure a normal skeletal development during late pubertal stages [10, 11, 2123 ] (table 1). later on, these findings opened the way to studies investigating the role of estrogens on bone growth and maturation not only in the context of congenital estrogen defects but also in normal boys. serum e2 was found to increase simultaneously with t levels during puberty in boys and to correlate directly with chronological and skeletal age, height, weight, and pubertal stages, thus confirming the crucial role of estrogens on bone physiology at puberty even in the male [19, 21 ] (table 1). in particular, estrogens seem to have a dose - dependent effect on growth plates [21, 47 ] : actually low doses of e2 stimulate ulnar growth in boys, while higher doses lead to an inhibition of this process of growth. recently, circulating serum e2 measured by lc / ms / ms resulted directly related to both the genital tanner stage and the skeletal maturation in pubertal boys. in addition, serum e2 was significantly higher and bone age more advanced in obese boys compared with healthy boys at the same pubertal stage. the excess of adipose tissue in obese boys probably accounts for increased aromatization of androgens into estrogens and for the advancement of bone age due to the higher amounts of circulating estrogens [18, 48 ]. this result is in line with the well - known gender difference in the progression of skeletal maturation, which is more rapid in women than in men and parallels gender differences in the way serum e2 increases throughout puberty [19, 20, 33, 35 ]. at the beginning of puberty, when circulating e2 is low, the prevailing effect of e2 consists in the promotion of chondrocytes proliferation within the growth plate, resulting in growth plate lengthening and accelerated bone elongation (figure 1) (table 1). this corresponds to the increase of height velocity occurring during pubertal growth spurt that is postulated to be under estrogen control (table 1) [21, 40, 47 ]. as puberty goes on, the rise in serum t ensures high e2 circulating levels, typical of late puberty ; e2 inhibits chondrocyte proliferation and stimulate chondrocyte differentiation, thus inducing the progressive ossification of the growth plate and its final disappearance (figure 1). at present, the amount of e2 required for shifting from the increase in length of the growth plate to its growth deceleration and final closure of the growth plate line is not known in detail. data available in literature clearly show that no difference in serum t is present between men with idiopathic hypogonadotropic hypogonadism with fused epiphyses compared to those with unfused epiphyses, but no data are available in literature on serum estrogens in these rare conditions. this implies that androgen is not involved in the process of shifting from growth plate elongation to progressive growth plate thinning and final disappearance (table 1). based on the poor compliance of an aromatase - deficient man, we tried to develop a dose - response relationship between serum e2 and radiological changes of the long bones in terms of bone age. due to patient 's poor compliance, serum e2 remained below 20 pg / ml for a long time without any change of bone age and growth plate appearance at x - ray. the closure of the epiphyses was obtained only several months later when e2 rose above 20 pg / ml and the patient was taking the right dose of transdermal e2. this suggests that serum e2 above 20 pg / ml is necessary for epiphyseal cartilage fusion and that only in the case of severe estrogen deficiency the epiphyses remain still open despite the advancement of the chronological age (figure 2). the same results can be deduced from a recent study comparing sex steroids, pubertal stage, and skeletal maturation between obese and lean boys. if boys at the end of puberty (with a genital tanner stage 5) are considered, bone age (of about 18 years on average) was greatly advanced and consistent with fused epiphyses in obese boys with a mean serum e2 clearly above 20 pg / ml (median 34.8 pg / ml, min max : 25.641.1 pg / ml), while bone age (of about 16 years on average) was less advanced and consistent with still unfused epiphyses in lean boys with a mean serum e2 below 20 pg / ml (median 15.7 pg / ml, min max : 13.221.0 pg / ml). the molecular mechanism through which estrogens act on the growth plate in vivo is still not known in detail, but recently advance on this issue has been reached [22, 23 ]. several convincing evidence suggests that the growth plate width progressively decreases as a consequence of a process of senescence involving the chondrocytes, mainly in the resting zone. how this senescence occurs and progresses is not known, but several mechanisms such as apoptosis, autophagy, chondrocytes differentiation into osteoblasts, and hypoxia have been proposed and are currently object of undergoing investigation by basic scientists. certainly, estrogens exert a strong effect on one or more of these pathways finally resulting in the promotion of chondrocytes involution and in the assurance of final epiphyseal closure followed by growth arrest [2123 ]. what is evident is that the number of both chondrocytes and progenitor cells progressively decreases in the resting zone of the growth plate and that estrogens accelerate this process, especially when they reach a critical level. both er- and er- are expressed by human epiphyseal chondrocytes [51, 52 ] ; moreover, the membranous - g - protein - coupled estrogen receptor, namely, gpr30, is also expressed in the hypertrophic zone of human growth plate. furthermore, the fact that aromatase is also expressed by chondrocytes which are able to produce estrogens [5456 ] implies that both circulating and locally produced estrogens are able to exert their actions within the growth plate via the activation of all the available estrogen transduction signaling pathways [22, 23 ]. low levels of estrogens, similar to those locally produced in vivo, are able to promote chondrocytes proliferation and to protect them from cell death in vitro. this mechanism might explain why low circulating and/or locally produced estrogens enhance longitudinal growth during early puberty. conversely, the expression of the estrogen receptor gpr30 decreases dramatically during pubertal progression in humans when circulating estrogens reach the highest values typical of late puberty and longitudinal growth decelerates up to cessation. gpr30 is a good candidate for explaining estrogen actions on growth plate since knock - out mice in which this receptor is disrupted do not respond to estrogen in terms of longitudinal growth deceleration and cessation. er-, rather than er-, seems to be mainly involved in the induction of growth plate fusion in response to supraphysiological e2 exposure. however, the complex interaction between estrogens and their receptors within the growth plate remains to be elucidated in detail. in the unique man with estrogen resistance, epiphyseal fusion did not occur at the expected time, despite high circulating e2 and normally functioning er-. besides, cultured cells obtained from the bone biopsy of this patient did not respond to estrogen exposure, differently from wild type cells [61, 62 ]. this issue is further complicated by the possible crosstalk between er- and er-. theoretically, in fact, it is possible that the residual truncated n - terminal fragment of the disrupted er- may have acted as a negative inhibitor of er- in this patient [62, 63 ], thus accounting for the very delayed epiphyseal closure in this estrogen - resistant man reached at the age of 35.5 years. other hormones are good candidates for explaining growth plate proliferation, longitudinal bone growth, and growth plate involution. among them, growth hormone (gh) and insulin - like growth factor-1 (igf-1) exert an anabolic effect on bone and are necessary for longitudinal bone growth and growth acceleration during infancy and at the time of the growth spurt, respectively. the role of gh and igf-1 on longitudinal bone growth and growth spurt could be even indirect through the well - known ability of estrogens to enhance gh and igf-1 secretion, an event that occurs during late puberty and that concurs to accelerate growth during the pubertal spurt [10, 11, 35, 64 ] (figure 1). however, longitudinal bone growth might occur and progress also independently from the gh / igf-1 status but at a lower rate since men with aromatase deficiency continue to slowly increase their stature during adulthood, despite severe gh deficiency. accordingly, gh response to ghrh plus arginine in four patients with aromatase deficiency was significantly lower than that in normal subjects, both before and after transdermal e2 replacement therapy with e2. in particular, e2 replacement did not restore normal gh secretion and igf-1 that remained significantly lower than normal age - matched controls. the fact that estrogen replacement treatment was effective on epiphyseal closure and growth arrest in all patients with aromatase deficiency, despite insufficient gh and igf-1 production, implies that gh and igf-1 do not play a major role in the process of growth plate closure in humans. these data suggest that a tall stature (higher than the genetic target) may be reached despite the coexistence of gh deficiency in these patients. even though we have no data about the gh - igf1 axis in these patients during their childhood and puberty, they were able to increase their stature during the first period of e2 treatment, soon before epiphyseal closure. besides, when aromatase inhibitors are administered at puberty in order to increase final height, usually a reduction of gh and igf1 levels is observed but a benefit on final height is even obtained thanks to the epiphyseal fusion blockade and a longer time available for growth [66, 67 ] (figure 2). all these data suggest that both longitudinal bone growth and a slow progressive increase in height during adulthood are possible even in presence of circulating gh and igf-1 lower than normal on condition that epiphyseal growth plates remains open. this condition might lead to the development of tall stature. outside the context of these rare clinical conditions that help to better know how sex steroids act on the growing skeleton in healthy children and boys, gh and igf-1 remain very important physiological determinants of growth during infancy and puberty since they ensure bone elongation and a normal height velocity. accordingly, gh deficiency represents one of the most important causes of growth retardation and (if untreated) of final short stature. indeed, r - hgh replacement treatment is effective in restoring a normal height velocity in children and boys with gh deficiency. in men, estrogen at puberty modulates both growth and the increase in stature in a fascinating way that allows accelerating the growth of the appendicular skeleton for a brief period only characterized by low but detectable serum e2 (early puberty)while preserving, at the same time, harmonic skeletal proportions. accordingly, the further increase of serum estrogens triggers both epiphyseal closure and cessation of growth during mid - to - late puberty, thus avoiding the development of an altered ratio between the appendicular and the axial skeleton. even though androgens alone are not able to induce bone maturation, they exert direct and indirect anabolic action on bone before, during, and after puberty (figure 1). in particular, during puberty, androgens probably promote the continuous linear growth, especially at the level of long bones as substantiated by the expression of androgen receptors within the human growth plate [23, 68 ] and the promotion of growth sustained by androgens through the elongation of the growth plate, at least in rats (figure 1). disproportional growth of long bones leads to eunuchoid proportions of the skeleton characterized by the prevailing length of arms and legs over the spine. usually the eunuchoid skeleton is defined by the finding of an abnormal upper to lower segment ratio (1) [40, 47, 70 ]. men with estrogen deficiency have a prolonged time available for linear growth thanks to a still open growth plate and they exhibit eunuchoid body proportions (figure 1) [40, 47 ], which worsen if they are not treated with exogenous estrogens. conversely, if the onset of e2 treatment starts at the proper time, during early puberty, body proportions are unaffected in males with aromatase. this evidence highlights the concept that estrogen rather than androgen is necessary also for a harmonic skeletal growth (figure 1) (table 2). as a matter of fact, normal skeletal proportions are found in patients affected by complete androgens insensitivity syndrome (cais) where normal - to - high e2 serum levels allow epiphyseal closure at the right timing, despite the absence of androgen action [71, 72 ]. the adult height of patients with cais usually corresponds, in fact, to both the calculated target and mean height of men rather than females standard. all clinical conditions that lead to severe t deficiency before the completion of puberty and that are characterized by eunuchoid proportions of the skeleton share the same mechanism of severe estrogen deficiency secondary to hypogonadism (figure 1). thus, relative severe estrogen deficiency secondary to insufficient androgen production leads to eunuchoid body proportions in prepubertal male hypogonadism [70, 73 ], 17,20-lyase deficiency and combined 17-hydroxylase and 17,20-lyase deficiency [40, 47 ]. the peak of bone mass (bone growth and bmd increase during childhood and young adulthood) determines the total amount of bone mineralized tissue available during adulthood till aging [74, 75 ]. once the peak of bone mass is achieved during puberty and early adulthood (before the age of about 30 years), the bone becomes unable to reach further significant bone mass accrual. thus, the bone mass remains stable or decreases on the basis of the balance between bone formation and resorption [74, 75 ]. the achievement of peak bone mass is prompted by the rise in sex steroids at puberty. even this process was previously supposed to be under the control of estrogens in females and of androgens in males [10, 11, 76 ]. all the events that can negatively interfere with the achievement of peak bone mass predispose to the development of osteoporosis later in life in both sexes [74, 75 ]. among them, physiological or pathological conditions that determine sex steroid deficiency result in impaired peak of bone mass, such as in the case of prepubertal hypogonadism or delayed puberty in boys and anorexia nervosa in girls [30, 31, 75 ]. probably androgens alone are not sufficient for the achievement of a normal bmd and an optimal peak of bone mass since severe osteoporosis was reported in all young adults with estrogen resistance or aromatase deficiency [3840 ]. the importance of estrogens for the acquisition of peak bone mass during puberty is evident by the results obtained from both human and mice models of estrogen deficiency (table 1). several mice models of estrogen deficiency have been generated and all confirmed that even in rodent estrogens mediate most of the actions exerted on bone by androgens [8, 77, 78 ]. in particular, the knock - out of the er- in male mice leads to the increase of trabecular bone, the reduction of cortical bone, and the decrease of longitudinal bone growth, while the knock - out of the er- does not impact cortical and trabecular bone. all these data reinforce the importance of er- in male bone homeostasis [78, 79 ]. studies performed on animal models, however, do not allow transposing all the results to human male bone physiology due to substantial differences in sex steroid and bone physiology among species. in rodent, in fact, circulating estrogens are very low and often undetectable so that the intracrine role of estrogens prevails over that of serum e2. furthermore, in rodent, sex steroids do not bind sex hormone binding globulin, and finally the process of bone maturation is different in rodent due to the absence of epiphyseal cartilage [77, 78 ]. the idea that estrogen is the main sex steroid involved in the acquisition of peak bone mass has been confirmed by other several data available in literature from rare models of sex steroids deficiency and from studies on pubertal boys. in men with cais, the peak of bone mass is only in part reduced, with intermediate values in - between those of male and female subjects, while men with aromatase excess syndrome display an increased bmd at the end of puberty due to high circulating estrogens throughout puberty. in addition, outside the context of rare syndromes of sex steroids deficiency, several other studies involving pubertal boys clearly demonstrate that peak bone mass is under the control of estrogen even in men (table 1) [17, 18, 48, 8183 ]. during puberty, e2 leads to the increase of bone mass mainly by increasing bmd, especially at the level of cortical bone whereas t contributes to increase the bone size, a phenomenon that is mainly mediated by the mechanical load exerted on bone by the increasing muscle mass. as far as bone geometry is concerned, e2 seems to be negatively associated to endosteal circumference and seems to positively influence the increase of cortical thickness both at the level of radius and tibia. thus, both estrogens and androgens seem to be necessary for normal bone mass accrual during puberty. androgens limit endosteal expansion and estrogens ensure adequate periosteal bone expansion (figure 3). the final result is a bone size greater than that of the female counterpart. compared to females, male bone has, in fact, a larger cortical portion due to greater periosteal apposition and a larger endosteal circumference due to reduced endosteal apposition [18, 75, 77 ] (figure 3). as a result of all these events, final peak bone mass is determined by the increase of bmd during puberty and early adulthood plus the remaining more slow bone accrual that continues till the 3rd decade of life and accounts for about 20% of peak bone mass. in order to maintain a biomechanically efficient bone, the skeleton needs to continuously remodel and repair the microcracks that develop both in the trabecular and cortical bone during lifetime [75, 83 ]. this process of remodeling occurs in basic multicellular units (bmus) which include osteoclasts, osteoblasts, and osteocytes that act altogether by coupling bone resorption and bone formation. the balance between bone formation and bone resorption determines the maintenance (if the two processes balance out the amount of bone mass) or the loss (in the case of resorption higher than formation) of bone mass in men [74, 84 ]. sex steroids exert a direct action on the bmus and can regulate, at least in part, bone remodeling [8385 ]. while this effect was traditionally ascribed to estrogens in females, in the last 20 years, several studies progressively have disclosed the same outcome in men [62, 74, 75, 8385 ]. evidence from basic research had already demonstrated a direct estrogen action on bone cells and more data, especially on biomolecular mechanisms of action, have been progressively obtained till now [84, 85 ]. in particular, estrogens inhibit the apoptosis of osteocytes both in trabecular and cortical bone [84, 86 ]. they reduce bone resorption by means of both direct and indirect effects on osteoclasts and act on osteoblasts, by inhibiting their apoptosis [84, 87 ]. in general, estrogen regulates bone remodeling by (i) inhibiting the activation of bone remodeling and the initiation of new bmus ; (ii) reducing the number and activity of osteoclasts (i.e., inhibition of their differentiation and promotion of apoptosis) and bone resorption ; and (iii) increasing the number and the activity of osteoblasts (i.e., promotion of their commitment and differentiation and inhibition of apoptosis) and bone formation [77, 79, 84, 88 ]. for all these reasons, bone loss in man is mainly related to relative estrogen deficiency [74, 77, 84, 88, 89 ], while androgens have a minor role [74, 75, 83, 84 ]. accordingly, the net effect of androgens per se on bone mass in vivo is quite poor. dht, for example, is not able to increase bmd in patients with benign prostate hyperplasia. in these patients, lumbar bmd decreases of about 1.5% after 24 months of treatment as a result of the net compensation between the poor anabolic effects of high circulating dht and its prevalent negative effect on bmd. the latter is due to the inhibition of gonadotropin secretion and the reduction of sera t and e2, with a reduced estrogen action on bone. several studies investigated the relationship between estrogen and bmd in men through different types of study design and all unequivocally demonstrate that estrogen action on the male bone is more determinant than androgen action [74, 75, 77, 83, 84, 89 ]. serum e2 and relative estrogen deficiency resulted associated with altered bone turnover markers, bmd [9298 ], and fracture risk [99102 ]. most of these findings were confirmed by large epidemiological longitudinal studies [92, 94, 96101 ]. altogether, these studies provide evidence that e2 is a better predictor of male bone health than t. in addition, e2 administered to male to female transsexuals [103105 ] or to men with prostate cancer significantly increases bmd, despite endogenous serum t suppression. otherwise, aromatase inhibitors lead to alterations of bone turnover markers and impairment of bmd. finally, genetic studies revealed an association between ers [109, 110 ] or aromatase enzyme [111, 112 ] polymorphisms with decreased bmd in men. exogenous e2 acts in a dose - dependent fashion on male bone since it restores a normal bmd in aromatase - deficient men given at a dose that ensures stable serum e2 levels within the normal male range [40, 113, 114 ]. a daily dose of e2 lower than 20 g is usually unable to keep serum e2 within the normal range in these patients. the result is bmd worsening or failure in restoring a normal bmd in men previously treated with higher doses and in naive patients, respectively. similarly, in adult men without genetic diseases involving estrogen pathways, e2 seems to be protective for bone but only when serum levels are above a critical threshold. in cohort studies, this threshold has been settled in - between 15 and 20 pg / ml. by studying the effects of estrogen treatment in an aromatase - deficient man, this threshold has been more precisely determined as being around 16 pg / ml (figure 2). this value has been also confirmed by studies on fracture risk in older men (figure 2). recently khosla. stated that a serum e2 level at least above 25 pg / ml is certainly protective for bone in men (figure 2). all these data suggest that serum e2 levels of 20 pg / ml or above are needed for optimal skeletal maturation and achievement of optimal peak bone mass. this threshold is very close to that required also for the epiphyseal closure (figure 2) [45, 116 ]. the concept that estrogens act on bone only when a specific amount is reached is well established, whereas the precise threshold value remains to be settled. differences in study design and overall in methods used for estrogen assays might explain the discrepancy between values obtained from different studies. in the future, the wide use of the gold standard methods (lc / ms / ms) for the measurement of estrogen in serum will provide more reliable information on the exact threshold value : the latter will probably fall within the suggested range of 1525 pg / ml (figure 2). furthermore, future studies will be of help in disclosing if individual, genetic differences in estrogen sensitivity might influence the amount of estrogens needed for a full estrogen action on bone. in conclusion, clinical and basic research demonstrate that e2 is the main sex steroid required for bone homeostasis in men [74, 77, 83, 84, 88, 89 ]. bone size exhibits evident gender differences that are ascribed mainly to sex steroids actions on bone [83, 117, 118 ]. estrogen controls the final length of long bones by acting on epiphyseal closure (see the paragraph above for details). this could be once again the effect of the more rapid increase of estrogens at puberty in women which is responsible for an anticipated epiphyseal closure and growth cessation [17, 18, 48 ]. actually, bone mass and strength are greater in men than in women ; probably these differences are due to different length and bone structure among the two sexes (figure 3). bone size, in fact, is larger in men than in women mostly as a consequence of a wider width of bone, especially of its cortical portion (figure 3) [75, 77, 115, 118, 119 ]. the enlargement of the periosteum involves the appendicular skeleton, and it mainly occurs from puberty to the 3rd decade of life. in men, this process leads to continuous increase of the cortical thickness (figure 3) [119121 ]. conversely, in females, this process ceases earlier and does not continue during young adulthood [119121 ]. in addition, the very high amount of estrogen in women is responsible mainly for endosteal bone formation. the final result is that adult females have smaller cortical bone portion and a shorter endosteal circumference than males (figure 3) [75, 77, 115121 ]. it has been suggested that the different levels of circulating androgens between the two sexes can explain this sexual dimorphism in bone structure. as the final pathways of sex steroids actions in bone are the same in the two sexes, this sexual dimorphism in bone size probably comes from indirect actions of sex steroids on tissues different from bone. with this in view, a possible role of t in establishing bone size can be explained by sex differences in muscle mass which start to appear at the time of puberty. muscle mass is androgen - dependent and greater muscle mass can exert greater mechanical action on bone, thus resulting in increased bone size and bone mass. obese boys, for example, have larger muscle size and larger bones in the legs if compared with lean boys at the same pubertal stage. furthermore, when t is administered to ovariectomized female - to - male transsexuals, both muscle and skeletal mass change and the final musculoskeletal system resembles that of the male counterpart. the old view postulated that androgen stimulates periosteal expansion in men, whereas estrogen inhibits periosteal apposition in women. the finding of significant periosteal expansion prompted by estrogen treatment in a boy with aromatase deficiency suggests a more complex cross - talk between these two hormones within the bone periosteal surface. estrogens might exert a permissive action on androgens, thus facilitating and promoting their anabolic effect on the periosteum ; this event does not take place or is minimized when estrogens are lacking or are below the normal range (figure 3). in fact, bone size has a female appearance in xy patients with cais [126, 127 ]. accordingly, vandenput and colleagues studied the role of the androgen receptor in the skeletal homeostasis of androgen - resistant, testicular feminized, male mice and observed that bone size and bone formation at the periosteal surface depend also on a functional androgen receptor. the mechanism consists in a permissive estrogen role on androgens and seems to operate also on trabecular bone homeostasis. this permissive estrogen action, however, needs to be confirmed and better clarified on a molecular point of view. in conclusion, the role of each sex steroid on bone size and on gender difference is a complex phenomenon that involves also other endocrine systems like the gh / igf-1 and that needs still to be better clarified. the progressive decline of t that usually occurs with advancing age might result in a corresponding decrease of circulating estrogens in men [74, 98, 102, 129 ]. relative estrogen deficiency in elderly men with low t is common and has been clearly demonstrated in most of the longitudinal studies on sex steroids in older men [98, 101, 102, 129 ]. there is, however, the possibility that both androgens and estrogens do not decline with advancing age, especially in older men in a good health status. a mild - to - severe reduction of circulating t in older men who develop age - related hypogonadism is directly responsible for bone loss, but the concomitant decrease of circulating e2 has a main impact on bone health [14, 129 ]. in older men, the 7085% of the decrease in bmd related to sex steroids decline is imputable to estrogen deficiency while only the remaining 1530% is imputable to androgens [74, 84, 91 ]. relative estrogen deficiency causes a decrease of both trabecular [83, 88, 89, 98101 ] and cortical bone in older men, leading to a bone structure that strongly resembles that of young aromatase - deficient men [74, 132 ]. bone size, in fact, decreases at least in part as an effect of contraction of the cortical portion of the bone [75, 77, 116, 117 ]. however, the main changes related to aging that occur in male bone regard the bone structure. in particular, cortical porosity increases with age [75, 77, 131 ] while bmd decreases [75, 77, 83, 84 ]. bmd changes are due to the reduction of trabecular bone volume that is sustained by the thinning of the trabeculae rather than a reduction of their number, the latter being a mechanism involved in female bone aging [75, 77, 83, 84 ]. endocortical resorption is less pronounced than in women but substantially contributes to decreasing cortical cross - sectional area and consequently bone strength (figure 3) [74, 75, 77, 83, 84, 88, 89, 98101, 131 ]. to what extent the decrease in bmd due to relative estrogen deficiency contributes to the incidence of fractures in aging men with late - onset hypogonadism is still not completely clear. men with congenital estrogen deficiency were classically considered at risk to develop fracture, but only recently a history of pathological fractures of the forearm after minimal trauma has been observed in a man with aromatase deficiency. long - term outcome concerning fractures in those rare male patients remains still not available. data obtained from older men demonstrate that e2 is inversely associated with bmd [9298 ] and seems to predict fractures better than t [99102 ]. however, not all the older men with low serum t develop relative estrogen deficiency [98, 102 ] and probably only men with concomitant low serum e2 are at high risk of fracture. individual differences in both aromatase activity and expression probably might explain why in the presence of low serum t only a subgroup of men with late - onset hypogonadism has relative estrogen deficiency (table 2). however, well designed studies aimed at exploring this hypothesis are still lacking. among men with low serum t, those with concomitant estrogen deficiency should be considered to be at high risk of osteoporotic fractures (table 2), especially when serum e2 is below 25 pg / ml (figure 2). accordingly, muscle mass reduction that is related to androgen depletion worsens bmd and increases the risk of falling in elderly men [115, 133 ]. in conclusion, elderly male might present relative estrogen deficiency that may be implied in several age - related conditions that can affect and worsen quality of life, including bone loss. even though now the crucial role of estrogen on bone homeostasis is well established many uncertainties still remain to be clarified both in the field of basic and clinical research as well as in the field of translational endocrinology. estrogen effect on male skeleton during fetal life and childhood has not yet been investigated while preliminary data during pubertal development are becoming available [1618, 48 ] (table 1). furthermore, data on the effects of estrogen deprivation on the growing skeleton in men with congenital estrogen deficiency are scanty. the exact molecular mechanism of estrogen action on the process of skeletal maturation remains to be established in details. we still do not know whether estrogens are directly involved in epiphyseal closure or whether their effect is mediated by a more complex hormonal network (endocrine and/or paracrine) including cytokines and growth factors. the lack of animal models useful to study the pathophysiology of the growth plate complicates the advancements on this issue. the physiology of growth plate and the process of ossification of long bones in rodents are very different from those of humans [22, 23, 60 ]. the rabbit is closer to human but presents several significant differences [22, 23, 4951 ]. while the role of circulating estrogens on bone maturation and accrual has been clarified, the contribution of intracrine estrogen production is unknown. furthermore, the other endocrine systems and growth factors recruited by estrogen and having an important role within the bone remains to be identified. the effects on bone of selective estrogen receptor modulators (serms) remain doubtful due to conflicting results available in the literature. raloxifene, a serm with a proven estrogen agonist action on bone with estrogen antagonist actions on other tissues, was not effective in inducing epiphyseal closure in an aromatase - deficient man after 24 months of treatment. conversely, tamoxifen, another serm with agonist action on bone, induces permanent growth arrest through the apoptosis of chondrocytes in rats. raloxifene, however, is able to increase bmd both in aromatase - deficient men and in men with prostate cancer [136, 137 ]. about 20 years of research in the field of estrogen on bone did not lead to significant changes in clinical practice. therapeutic strategies aiming to target estrogens for inducing changes in the male bone include aromatase inhibitors for the treatment of short stature in children and adolescents and estrogen - like compound for osteoporosis in adult and elderly men. none of these strategies, however, reached the expected results and nowadays they remain confined to the clinical research area without any real impact and extensive use in clinical practice. aromatase inhibitors are effective in increasing final height [67, 138 ] but the results in terms of centimeters reached are less than those expected if the epiphyseal closure is completely blocked. aromatase inhibitors decelerate the advancement of bone age [67, 138 ], but the latter is not completely blocked as it happens in aromatase - deficient men [67, 138 ]. probably even the most potent 3rd generation aromatase inhibitors are not able to completely block the enzyme or, alternatively, after a first period of successful blockade, a phenomenon of escape might occur since serum e2 tends to progressively increase even though at a lower rate than in the placebo group. this probably also accounts for the lack of reported undesired effects, such as osteoporosis, which would be expected in the case of complete blockade of the enzyme. recently, however, hero. reported vertebral deformities in boys treated with letrozole. it is not clear whether severe prepubertal and pubertal estrogen deficiency might lead to skeletal deformities. sporadic skeletal deformities (i.e., kyphosis and bilateral femoral osteonecrosis) have been observed in men with estrogen deficiency, but the cause - effect relationship with estrogen deprivation needs to be confirmed. finally, further data from clinical trials are required in order to obtain final data on safety for the use of aromatase inhibitors in boys since theoretically more adverse events are expected to occur [141, 142 ]. the most critical issue in clinical practice is the poor reliability of the commercially available assays. their accuracy and reproducibility are insufficient especially in the low range of serum e2 typical of men [40, 74, 143 ]. the development of new techniques that are considered to be the gold standard, such as lc / ms / ms, is providing precise standard methodologies useful for serum estrogen measurement. in the last years, these new methodologies are becoming even more widespread among clinical laboratories since they could be cost and time saving, especially in laboratories that perform a great number of assays per day. at present, serum e2 is not currently part of the work - up used for the clinical diagnosis and management of male osteoporosis. several authors, however, are going to introduce this biochemical test in the clinic when the lab outcomes are accurate. thus, we will be able to improve our knowledge about the real critical amount of circulating estrogens required to ensure bone health. osteoporosis still remains an overlooked and undermanaged disease in older men [74, 144 ], while all the advancements in the field of estrogen deficiency and bone did not result in practical, significant changes in the approach to male osteoporosis. all these uncertainties reflect the wide differences among physicians approaching estrogen deficiency in the context of osteoporosis in older men [74, 144 ] as well as all the difficulties in identifying a way to physiologically increase serum e2 without concomitantly impairing androgen production. the only practicable way remains t therapy that is able to restore both normal sera t and e2 with consequent beneficial effects on bone in the presence of a normal functioning enzyme. however, t replacement treatment should be considered only after careful evaluation of potential benefits and disadvantages since it could be harmful especially in older men who are not in a good health status [145147 ]. it should be remarked that t treatment is currently not a treatment of choice for male osteoporosis. several evidence support the view that estrogens are the main sex steroids involved in processes such as bone maturation, bone mass accrual, and epiphyseal closure in men. estrogen actions on bone, especially on bone maturation, remained quite unaltered among both gender and species during evolution, thus suggesting a high degree of conservative functions for estrogen that are also confirmed by the high degree of homologies of the aromatase enzyme. conversely, estrogen actions on other tissues and organs are determinant in ensuring gender differences (e.g., primary and secondary sexual characteristics) and in promoting sexual divergence between the two sexes during evolution. the existence of a threshold level for serum e2 that is necessary for ensuring skeletal maturation and adequate bone size and bmd confirms how complex the way estrogen acts on bone in men is. all these evidence contribute to make the issue of estrogen action on bone a fascinating one in the field of both basic and experimental research and encourage researches in order to find new strategies for the management and treatment of bone diseases related to estrogen deficiency. | before the characterization of human and animal models of estrogen deficiency, estrogen action was confined in the context of the female bone. these interesting models uncovered a wide spectrum of unexpected estrogen actions on bone in males, allowing the formulation of an estrogen - centric theory useful to explain how sex steroids act on bone in men. most of the principal physiological events that take place in the developing and mature male bone are now considered to be under the control of estrogen. estrogen determines the acceleration of bone elongation at puberty, epiphyseal closure, harmonic skeletal proportions, the achievement of peak bone mass, and the maintenance of bone mass. furthermore, it seems to crosstalk with androgen even in the determination of bone size, a more androgen - dependent phenomenon. at puberty, epiphyseal closure and growth arrest occur when a critical number of estrogens is reached. the same mechanism based on a critical threshold of serum estradiol seems to operate in men during adulthood for bone mass maintenance via the modulation of bone formation and resorption in men. this threshold should be better identified in - between the ranges of 15 and 25 pg / ml. future basic and clinical research will optimize strategies for the management of bone diseases related to estrogen deficiency in men. |
autoimmune hepatitis (aih) is a chronic inflammatory disease of unknown etiology characterized by the presence of circulating autoantibodies, hypergammaglobulinemia, necroinflammatory changes on hepatic histology, and a dramatic response to immunosuppressive therapy. earliest descriptions include those by amberg in 1942 and leber in 1950 describing a form of chronic liver disease prevalent among young women and characterized by an excessive increase in serum protein and gamma - globulins. in 1951, kunkel. termed the condition hypergammaglobulinemic chronic hepatitis. since then, it has been known by various names including chronic active hepatitis, chronic aggressive hepatitis, plasma cell hepatitis, and autoimmune chronic active hepatitis. cowling and mackay coined the term lupoid hepatitis after they noted the association of this entity with autoimmune syndromes and the le cell phenomenon. the disease is rare with a mean incidence of 1 - 2 per 100,000 and a point prevalence of 1117 per 100,000 [5, 6 ]. although more frequently seen in young women (sex ratio 3.6 : 1), it can affect children and adults of all ages and ethnicities [7, 8 ]. a minority of patients may present with acute liver failure and need liver transplantation, but for the majority, the prognosis of aih is good and mostly determined by response to corticosteroid therapy. in general, long - term survival and average life expectancy are excellent and estimated to be comparable to the normal population. type i aih is characterized by the presence of antinuclear antibody (ana), anti - smooth muscle antibody (sma), or both and constitutes 80% of aih cases. about 25% have cirrhosis at presentation, and association with other autoimmune diseases is common (celiac disease, ulcerative colitis, autoimmune thyroid disease) [10, 11 ]. type 2 aih is characterized by the presence of anti - liver kidney microsomal (lkm) 1 and/or anti - lkm3 and/or anti - liver cytosol 1 (lc1) [12, 13 ] antibodies. most patients are children, acute severe presentation can occur, and progression to cirrhosis commonly ensues. in patients who are negative for conventional antibodies and aih is strongly suspected, additional tests can be done including perinuclear antineutrophil cytoplasmic antibodies (panca), actin (anti - actin), soluble liver antigen (anti - sla), asialoglycoprotein receptor (anti - asgpr), chromatin, and liver cytosol type 1 (anti - lc1). in our experience, 1015% patients do not have either ana, sma, or anti - lkm1 at presentation, but 25% of these will have detectable conventional antibodies later in their course. another 1020% of the seronegative patients at presentation will have panca or anti - sla. although the exact etiopathogenesis is unknown, aih, like many autoimmune diseases, is thought to be caused by environmental triggers and failure of immune tolerance mechanisms in a genetically susceptible host. these triggers may be of viral or drug etiology, but most cases have an unknown trigger. triggers may share epitopes that resemble self - antigens, and molecular mimicry between foreign antigens and self - antigens is the most frequently proposed initiating mechanism in type 2 aih where the autoantigen is known. repeated exposures to the triggering antigen, in turn, may trigger autoreactive organ - specific responses. aih is a complex polygenic disease and different populations may have different genetic and environmental triggers and genetic association varies in study populations. the human leukocyte antigen (hla) genes on chromosome 6 are the most commonly described association with aih. hla may be associated with age at presentation, disease severity, and response to therapy. how the hla genes predispose to disease is not exactly known but is likely due to their role in autoreactive t cell selection and autoantigenic peptide presentation. different susceptibility alleles like hla dr1 0301, dr1 0401, dr1 0404, and dr1 0405 share a common motif, namely, amino acids lleqkr or lleqrr at position 67 - 72 of class ii hla, whereas the resistant alleles dr11501 encodes ileqar [20, 21 ]. in contrast, hla - dr1 1501, encodes for the ileqar motif and is associated with protection from aih. substitution of a lysine or arginine to alanine at position 71 is postulated to change the polarity and charge of the peptide binding groove of the major histocompatibility complex thereby influencing autoantigenic peptide presentation. however, these associations are not absolute and significant geographic differences exist, for example, in japan dr2 (drb1 1501) is a weak susceptibility rather than a resistance allele and in south american children drb1 1301 is a strong susceptibility allele.. a form of aih can be seen in 20% of patients with autoimmune polyendocrinopathy - candidiasis - ectodermal dystrophy (apeced) syndrome. apeced is a monogenic, autosomal recessive disorder characterized by hypoparathyroidism, adrenal insufficiency, and chronic mucocutaneous candidiasis. apeced is caused by mutations in a transcription factor relevant to immune tolerance called aire (autoimmune regulator) on chromosome 21q223. aire is expressed in medullary epithelial and dendritic cells within the thymus and regulates clonal deletion of autoreactive t cells. the liver autoantigens associated with apeced are cytochrome p450 (cyp) 1a2, cyp2a6, and cyp2d6 [2426 ]. this is the only syndrome involving aih that exhibits a mendelian pattern of inheritance, and genetic testing and counseling for the patient and family members are warranted. the liver is part of the lymphoid system with the normal lymphocyte population mainly residing in the portal tracts. aih is an inflammatory disorder of the liver involving multiple components of the immune system including t cells, b cells, and cytokines. hepatocytes isolated from aih patients are coated with immunoglobulins and are susceptible to antibody - dependent cellular cytotoxicity (adcc) when exposed to autologous mononuclear cells bearing fc receptors. cyp2d6, an important cytoplasmic enzyme is targeted by anti - lkm1 antibodies and plays a crucial role in liver damage. mice immunized with plasmid containing human cyp2d6 antigenic region and human formiminotransferase cyclodeaminase (another autoantigen), have established a murine model for autoimmune hepatitis type 2. these mice develop autoantibodies, elevation in transaminases, along with portal and periportal inflammatory infiltrate. another model using adenovirus vector containing human cyp2d6 infection of cyp2d6 transgenic mice had focal hepatocyte necrosis and hepatic fibrosis. these models will aid the development of more therapeutic options in the management of autoimmune hepatitis. studies have demonstrated presence of cytotoxic cells in both t and non - t cell compartment of peripheral blood from aih patients. this cytotoxic activity is higher in patients with active disease but seen in only 40% of patients in remission. patients with aih have a ten fold higher frequency of liver - specific t cells compared to normal subjects. in patients with predisposing hla allele dr1 0701, cd4 t cells are able to recognize autoantigen cyp2d6 and secrete interferon-. cd8 t cells have cytotoxic capability, are capable of secreting ifn-, and their responses correlate with disease activity. defects in numbers and function of regulatory cells (t regs) have been demonstrated in aih. t regulatory cells normally control or limit immune responses by acting as immunoregulators, preventing the proliferation and effector function of autoreactive t cells. in patients with aih, the number of t regs is decreased more so at disease presentation than at drug - induced remission. their level correlates inversely with levels of anti - sla and anti - lkm-1 autoantibody titers. longhi. in their study demonstrated that tregs generated under cyp2d6-specific conditions and cocultured with semimature dendritic cells are highly effective at controlling autoreactive t cells, thus providing a potential tool for immunotherapy in type 2 aih. t regs may, therefore, be an attractive therapeutic target, but more studies are needed to elucidate this better. several drugs have also been implicated as triggers for aih including infliximab, minocycline, atorvastatin, diclofenac, isoniazid, -methyldopa, nitrofurantoin, and propylthiouracil and hepatitis a vaccine. herbal agents such as black cohosh and dai - saiko - to have been proposed to induced aih. the exact reason for drug - induced aih is not known but may be due to hepatotoxic effect of these chemicals, upregulation of proteins expression (p450s, immunoregulatory proteins), or related to the drug acting as a hapten by modifying the hepatic protein, making them immunogenic. drug - induced aih may improve after discontinuation of offending agent, thus initial observation is warranted. viruses such as hepatitis a, b, or c, in addition to measles have been implicated as triggers for aih. ana and sma can occur in diverse causes of acute and chronic hepatitis including alcoholic, nonalcoholic fatty liver disease, and viral hepatitis. they are usually low titer, background reactivities that should not alter diagnosis or management. if clinical concern for autoimmune hepatitis exists, antiactin antibodies can be checked as they increase the specificity of sma testing for diagnosing aih. anti - lkm1 has been found in as many as 10% of patients with chronic hepatitis c and is different from the anti - lkm1 found in classic autoimmune hepatitis. molecular mimicry at the b - cell level between a structural motif of cyp2d6 and hcv proteins could explain the production of anti - lkm1 antibodies in hcv - infected patients. women constitute at least 70% of cases, and 50% are younger than 40 years ; however, age at onset may range from infancy to the elderly. forty percent of patients may present with an acute onset, but the presentation of severe fulminant hepatic failure is rare. presenting symptoms may include fatigue, lethargy, malaise, arthralgia of small joints, anorexia, nausea, abdominal pain, and dark urine. clinical manifestations may vary by ethnicity ; the presentation is acute and icteric in alaskan native patients, cholestatic in aboriginal north american, african, asian, and arab patients, mild in japanese patients, but severe and rapidly progressive in somali patients. physical examination may be normal, but may also reveal hepatomegaly, splenomegaly, jaundice, and stigmata of chronic liver disease. findings such as acne, hirsutism, obesity, and amenorrhea in young women are rarely seen. other autoimmune diseases such as hashimoto thyroiditis, type 1 diabetes, rheumatoid arthritis, systemic lupus erythematosus, ulcerative colitis / crohn 's disease, and celiac disease can be seen in 20% of patients. this subtype is associated with higher serum levels of igg, aih severity scores, and, more importantly, an excellent response to prednisone therapy for induction and maintenance of remission. positive igg4 staining is suggested by the authors as a surrogate marker for the subtype of aih that may respond well to corticosteroid therapy alone. whether this is a form of classic aih or a distinct entity awaits a more extensive description of its clinical and immunohistological features. the diagnosis of aih requires the presence of characteristic clinical features and exclusion of other chronic liver conditions, such as viral hepatitis, drug - induced hepatitis, fatty liver disease, alcohol related liver disease, wilson 's disease, alpha 1 antitrypsin deficiency, or hemochromatosis. laboratory studies typically show elevation of aspartate aminotransferase (ast) and alanine aminotransferase (alt) levels, but levels are generally < 500 u / l, but on rare occasions can range between 5001000 some patients may have high conjugated bilirubin and alkaline phosphatase necessitating exclusion of extrahepatic biliary obstruction, cholestatic forms of viral hepatitis, drug - induced disease, primary biliary cirrhosis (pbc), and primary sclerosing cholangitis (psc). the alkaline phosphatase rarely exceeds 4 x normal and generally remains < 2 times normal. another characteristic laboratory feature of aih is hypergammaglobulinemia, with a selective increase in igg, which is 1.23.0 times higher than the upper level of normal. it should be noted that hla typing has not been endorsed as a diagnostic or prognostic tool. the characteristic circulating autoantibodies seen in aih include ana, sma, and (lkm-1) autoantibodies. a list of the important autoantibodies and their autoantigenic targets is summarized in table 2. they are helpful in diagnosis as well as for classification of aih into type 1 and type 2 diseases. the reader is referred to excellent reviews for description of methodology, sensitivity, and assay performance [27, 39, 64 ]. except for panca, which is readily available and can be positive in 5090% of type i aih, only antiactin can be easily in measured in some laboratories. recently, antibodies to cyclic citrullinated peptides (ccp) have been described in 911% of patients with aih in absence of rheumatoid arthritis. antimitochondrial antibodies are sometimes present in patients with aih and an overlap syndrome of aih and pbc should be considered in these patients. a diagnostic system was proposed by the international autoimmune hepatitis group (iaihg) in 1993 and subsequently updated in 1999 [66, 67 ]. in 2008, they proposed a simplified set of diagnostic criteria to facilitate early recognition and initiation of adequate immunosuppressive treatment. these included the presence of specific autoantibodies (ana, sma, lkm antibody, sla antibody) in moderate to high titers, hypergammaglobulinemia, typical histological pattern on liver biopsy, and exclusion of viral hepatitis. these criteria have a lower sensitivity (85% versus 100%) but higher specificity (99% versus 93%) than the original criteria and are good at identifying patients with all typical characteristics of a classic case of aih [69, 70 ]. however, miyake. showed that 30% of males, 23% of patients with acute clinical presentation, and 46% patients negative for ana were not diagnosed with aih by simplified criteria even though they met the original criteria. therefore, it fails to adequately identify cases with atypical features which is an important point to keep in mind. the histologic hallmark of aih is a lymphoplasmacytic periportal infiltrate invading the limiting plate, also called piecemeal necrosis or it causes distortion of the hepatic lobule and the appearance of regenerative nodules, resulting in cirrhosis. many patients with acute presentation may have chronic features on liver biopsy indicating a subclinical phase of disease and several patients with mild clinical disease may have advanced fibrosis on biopsy. of important note is the fact that the fibrosis and even cirrhosis in aih is reversible to a significant degree with immunosuppressive therapy unlike in other chronic liver diseases. there are no specific imaging techniques to confirm the diagnosis of autoimmune hepatitis. in adults with both aih and ibd, cholangiographic changes suggestive of psc are present in up to 44% patients and may affect therapy and prognosis. in children with aih, autoimmune sclerosing cholangitis can be present with or without inflammatory bowel disease. histologic presence of bridging or multilobular necrosis is associated with progression to cirrhosis in 82% cases and a 5-year mortality of 45% in untreated patients. in asymptomatic patients with inactive cirrhosis (defined as no or limited inflammation) patients without cirrhosis who undergo treatment have a 1020 year survival probability more than 80%, similar to the general population. retrospective analysis of patients with mild disease has demonstrated the possibility of long - term survival without treatment, but very careful follow - up is required. untreated patients may, rarely, recover spontaneously, but improvement is less common than treated patients, and long - term survival is lower. aih can have unpredictable and varying disease activity and ultimately the majority of patients with active inflammation will warrant therapy. indications for treatment are listed in table 3 and are based on the presence and severity of hepatic inflammation. the indications are also reflective of risk factors for disease progression as severely abnormal liver enzyme elevation, incapacitating symptoms, histological presence of interface hepatitis, bridging necrosis, or multiacinar collapse portend a worse prognosis without treatment. prednisone alone (60 mg daily with taper down to 20 mg daily in 4 weeks) or at a lower dose (30 mg with taper down to 10 mg daily in 4 weeks) in combination with azathioprine (50 mg daily) is the most effective treatment regimen studied in randomized clinical trials. histologic improvement lags behind clinical and laboratory resolution by 3 to 8 months, and therapy should be continued for at least 36 months beyond this point of improvement. treatment is often maintained for at least 2 years before withdrawal of drug therapy is considered. the end points for treatment include remission, treatment failure, incomplete response, or development of drug toxicity. prednisone is used alone in patients with severe cytopenias, active malignancy, pregnant or contemplating pregnancy, and those with complete thiomethylpurine transferase (tpmt) enzyme deficiency. combination therapy is associated with lesser side effects and is preferred when treatment is expected to be more than 6 months and in patients at risk of side effects including postmenopausal women, brittle diabetics, labile hypertensive, and osteoporotic patients. therapy may span over several years and hence treatment side effects must be taken into consideration. corticosteroids can cause weight gain, central obesity, moon facies, prominent supraclavicular fat pad, acne, bruising, cutaneous striae, cataracts, glaucoma, peptic ulcers, deterioration of hypertension and diabetic control. long - term side effects include increased risk of fractures secondary to osteoporosis and avascular necrosis of bone. patients with brittle diabetes, severe osteoporosis, vertebral compression fractures, psychosis, obesity, and uncontrolled hypertension should be carefully evaluated for a treatment benefit before starting corticosteroids. if severity of disease necessitates corticosteroid therapy, adequate measures should be instituted to control the comorbid conditions. in patients with mild disease or relative contraindications to prednisone, budesonide 3 mg tid (in place of prednisone) is an option to reduce overall treatment side effects with no impairment of efficacy [77, 78 ]. its benefits are derived from the 90% first pass metabolism which results in less steroid - induced side effects while maintaining efficacy. alternative regimens must be considered in several circumstances : after treatment failure with prednisone (60 mg daily) or prednisone (30 mg daily) and azathioprine (150 mg daily), incomplete response to conventional therapy, or intolerance to conventional therapy. mycophenolate mofetil (2 g daily) has shown improvement in 3984% patients who were unable to tolerate azathioprine but use was limited by side effects (nausea, vomiting, rash, pancreatitis, diarrhea, cytopenia) [7981 ]. there are studies demonstrating benefit to the use of cyclosporine (in conjunction with prednisone) for patients refractory to standard therapy. in addition, a report suggests some benefit to tacrolimus, but has not been evaluated in randomized clinical trials. ursodeoxycholic acid has been studied in randomized trials and unfortunately was not found to be of benefit. relapse is characterized by an increase in the serum aminotransferase levels to at least threefold normal. relapse occurs in 50% to 86% of patients, most often during the first 6 months after the termination of therapy (50%). the first relapse after drug withdrawal should be retreated with a combination of prednisone plus azathioprine at the same treatment regimen as with the initial course of therapy and then tapered to monotherapy with either azathioprine (2 mg / kg daily) as a long - term maintenance therapy or indefinite low - dose prednisone (10 mg daily) in patients intolerant of azathioprine. gradual withdrawal from maintenance therapy may be attempted again after at least 24 months of treatment and continued normal serum ast or alt level only after careful benefit risk evaluation in patients who had previously relapsed. aih is the underlying cause for 4%6% cases of liver transplants done in the western world [85, 86 ]. it usually results from a failure to diagnose and treat aih as an etiology of cirrhosis, inadequate response or intolerance to immunosuppressive therapy, or noncompliance with treatment. treatment failure requiring transplant is more often associated with the hla genotype drb1 0301. liver transplantation should be considered in patients with aih and acute liver failure, decompensated cirrhosis with a meld score 15, or hepatocellular carcinoma meeting criteria for transplantation. a combination of prednisone and a calcineurin inhibitor (tacrolimus more frequently than cyclosporine) is the most common immunosuppression regimen after lt. despite this, aih can recur in transplanted livers or occur de novo in liver transplants done for non - aih conditions, but discussion of this is beyond the scope of this review article. the greatest risk is prematurity, but fetal mortality has been reported to be as high as 21%. maternal antibodies to sla and extractable nuclear antigens (ro / ssa) have been associated with a more complicated pregnancy. azathioprine is an fda category d drug and safety in pregnancy has not been well established in human studies. although increased number of birth defects have not been reported in neonates of women receiving this treatment and no adverse consequences of breast feeding have been noted by treated mothers [91, 92 ], congenital malformations have been reported in pregnant mice, and, thus there is a potential risk for teratogenicity. this justifies exercising caution when using in pregnancy, thus the mainstay of treatment in pregnancy is prednisone at as low dose as possible. aih commonly exacerbates following delivery, therefore therapy must be resumed (if stopped) or increased 2 weeks prior to anticipated delivery and continued in the postpartum period. women with advanced cirrhosis and portal hypertension are at high risk for variceal hemorrhage during pregnancy. aih is associated with chronic inflammation that may proceed to cirrhosis and end - stage liver disease which also puts aih patients at risk of developing hcc. however, unlike other cohorts of cirrhotic patients, the majority of patients with aih respond well to immunosuppression and in those whom enter a sustained remission, the potential exists to retain a near normal life expectancy. however, the interactions of disease activity, response to treatment, and other factors in relation to the risk of hcc development in aih are unknown. although the development of hcc in patients with aih and cirrhosis is considered a rare occurrence, the true incidence remains unknown due to the paucity of published data addressing this issue. a large prospectively obtained cohort at a single center (n = 243) determined a rate of hcc development of 1.1% per year, with equal proportions among men and women. the median duration from time of confirmed cirrhosis to not surprisingly, hcc was found to occur more frequently in patients with cirrhosis at presentation (9.3% versus 3.4%, p =.048) or history of variceal bleed as the index presentation of aih (20% versus 5.3%, p =.003). median survival in patients whose hcc was diagnosed on surveillance was higher (19 months versus 2 months) compared with patients presenting symptomatically (p =.042). the majority of patients develop hcc after having cirrhosis for an average of 9 years, and although the incidence of hcc is less common than in other chronic liver diseases, the risk may be sufficient to undertake surveillance in all patients with cirrhosis with aih who are candidates to undergo curative therapies. autoimmune hepatitis is one of the few liver diseases with excellent response to therapy. on the other hand, it still remains a liver disease with many unanswered questions, particularly in respect to its etiology and pathogenesis. there is significant heterogeneity in its presentation that may mask its identity, affect its clinical behavior, and confound its management. it may start with a fulminant course, and the diagnosis should not be overlooked when dealing with patients with acute liver failure. alternatively, it may behave as a slowly progressing disease, and it is still controversial whether those patients need immunosuppressive treatment at all. there is no prescribed minimum or maximum duration of treatment. over the last decade, remarkable progress has been made in understanding and clarifying the areas of diagnosis with introduction of classification criteria, and broadening therapeutic options, with trial of several new medications like budesonide and mycophenolate mofetil, and more in the pipeline. management, however, still faces several other important issues, such as in children, the elderly, in males, and during the preconception period, pregnancy, and lactation. a key to successful management is thinking of it, recognizing the nonclassical presentations, and individualizing therapy. | autoimmune hepatitis (aih) is a chronic inflammatory disorder characterized by periportal inflammation, elevated immunoglobulins, autoantibodies, and a dramatic response to immunosuppression. an environmental agent is hypothesized to trigger an immune - mediated attack directed against liver antigens in genetically predisposed individuals. a plethora of clinical presentations can be seen ranging from chronic indolent disease to fulminant hepatic failure, and diagnosis requires exclusion of other causes of liver disease. corticosteroid therapy must be instituted early and modified in an individualized fashion. treatment decisions are often complicated by the diverse clinical manifestations, uncertainty about natural history, evolving ideas about treatment end points, and a multitude of alternative immunosuppressive agents. achieving normal liver tests and tissue is the ideal treatment end point, but needs to be weighed against the risk of side effects. decompensated patients may benefit from early liver transplantation. long - term prognosis is excellent with early and aggressive initiation of therapy. our paper discusses aih, giving a detailed overview of its clinical presentation, risk factors, immunopathogenesis, up - to - date diagnostic criteria, current updates in therapy with a brief discussion of aih in pregnancy, and long - term implications for cirrhosis and hepatocellular carcinoma in aih patients. |
walking is the most common form of human motion, and is composed of cyclically switching single - support phases. moreover, the trunks ability to balance load is a very important element in this switching motion1. the trunk needs to maintain the body s center of gravity within the base of support, and is an important part of motor control required for individuals to perform various tasks in daily life1. normal sensory feedback and an ability for integrated higher center control are required to continuously maintain the body s center of gravity within the base of support in the body. maintenance of balance requires a complex process requiring visual and vestibular organs and proper somatosensory response2. among the sensory organs necessary for maintaining balance when vision is blocked, there is an increased reliance on the vestibular, proprioceptive, and tactile senses. hence blindfolding can facilitate these senses and improve balance and gait abilities2. in recent years, exercises such as the swiss ball exercise, proprioceptive neuromuscular facilitation, virtual reality training, and horse riding simulation have been conducted to improve balance and gait for stroke or polio patients. horse riding simulation exercise is a feedback exercise designed to correct postural changes following the movements of a mechanical horse. this differs from most other exercises performed for balance improvement because it is a feed forward exercise that can predict movements3. the horse riding simulation is an exercise of the whole body, which is performed using muscles and joints. it can recover the ability to walk and can improve joint range of movements, sense of balance, coordination, muscular strength, and endurance for patients with reduced physical functions4. a number of studies have been conducted to examine the effects of horse riding simulation exercise on trunk muscle activity. however, studies on the effect of this treatment on balance and gait are limited. therefore, this study was conducted to identify the effects of horse riding simulation with blindfolding on the balance and gait of healthy individuals. thirty subjects who participated in our experiment were randomly divided into an experimental group (n=15) and a control group (n=15). those subjects were included who had not undergone orthopedic surgery in the lower limbs, not taken medicine due to neurological problems, and did not have any musculoskeletal back injury. their average ages, heights, and weights were 21.32 1.04 and 20.13 1.57 years, 169.32 10.01 and 171.24 9.64 cm, and 67.45 12.31 and 66.61 16.32 kg in the experimental and control groups, respectively. information on the study was provided to all of the subjects prior to their participation and written informed consent was obtained according to the ethical standards of the declaration of helsinki. the subjects in the experimental group covered their eyes using a blindfold, climbed onto a horse riding simulator, and performed the horse riding simulation exercise. the subjects in the control group performed the horse riding simulator exercise in the same manner without wearing a blindfold. all of the subjects performed the 20 minutes long exercise once a day, five times a week, over a four - week period. the horse riding exercise machine (joba, eu7805, panasonic, japan) creates a figure - eight movement using five axes, and creates three - dimensional movements (front and back, left and right, up and down) similar to the movements of a live horse3. the machine is designed to enable the rider to experience various movements similar to the movements of a real horse based on a built - in program in the joba simulator. the subjects were instructed to maintain a correct sitting posture after seating themselves, and to hold on to the handle to prevent a fall3. they were also continuously instructed by the therapist to make postural corrections while sitting for the correct alignment of the trunk. the speed of the horse riding exercise machine was gradually increased up to phase 3, according to the adaptability of each subject3, 4. a balance measuring device (good balance, metitur, finland) was used for the quantitative measurement of the ability to balance after the horse riding simulation exercise treatment. in order to measure balance, the subjects were instructed to step onto a triangular platform and maintain a symmetrical standing posture by placing their legs apart at shoulder width. the participants head movements were minimized by guiding them to gaze at a fixed forward point. the center of pressure (cop) was measured for 30 seconds while each subject was standing, eyes open, with each arm placed comfortably by the hip joint. after three repeated measurements, their average was calculated. a gait measuring analyzer (gait rite, technologies ltd. it can measure the velocity, step length, stride length, single support, double support, cadence, functional ambulation profile (fap), step time, and cycle time. each subject was verbally instructed to walk as usual, while starting from a point 2 m away from an ambulatory mat to help measure the natural gait. the subjects were instructed to lift their heads and look straight forward, and then walk barefoot while naturally moving their upper limbs. each subject performed this task three times, and the corresponding data were collected. the rater reliability was r=0.90, and the interclass correlation coefficient for all of the gait measurements at comfortable gait speeds was substantially high at r=0.965. data analysis was performed using the spss software package version 18.0 (spss inc.,, differences in the general characteristics of the experimental and control groups were compared using independent t - tests and tests. comparisons of variables before and after training within each group were made using paired sample t - tests. comparisons of pre- and post - test differences in variables between the experimental and control groups were performed using the independent t - test. an effect size 0.8, a large difference. statistical analysis was performed at a 95% confidence level, and p values<0.05 were considered statistically significant6. thirty subjects completed this experiment. balance and gait were measured for the experimental and control groups before and after intervention (table 1table 1.comparison of change in characteristics of the experimental group and control group with values presented as mean (standard deviation)eg (n=15)cg (n=15)pre - testpost - testpre - testpost - testbalancestatic balance (score)78.3 (6.2)84.4 (7.2)77.1 (9.7)81.3 (8.6)dynamic balance (score)76.1 (8.7)81.6 (6.3)73.6 (9.5)76.6 (7.5)gaitvelocity (m / s)87.3 (6.7)92.4 (5.8)82.4 (5.4)86.3 (6.9)single support (%) 40.3 (7.9)44.8 (8.50)41.6 (7.5)43.8 (9.1)cadence (steps / min)96.7 (13.6)103.7 (14.86)91.7 (14.8)96.5 (15.2)eg : experimental group ; cg : control group. the experimental group showed significant improvement in static balance, dynamic balance, velocity, and cadence compared to the pre - intervention results (p<0.05). in addition, the control group showed significant improvement in static and dynamic balance, single support, and cadence compared to the pre - intervention results (p<0.05). significant differences in the post - training gains in static and dynamic balance and cadence were observed between experimental and control groups (p<0.05). both the experimental and control groups showed a big gain in static balance (effect size=0.87) and cadence (effect size=1.31). this study was undertaken to determine the effects of horse riding simulation exercise with blindfolding on balance and gait of healthy individuals. the experimental group, which was additionally blindfolded, showed statistically significant improvements in balance and cadence compared to the control group. a previous study reported that horse riding simulator treatment improved symmetrical weight - bearing in a standing posture, and improved balance through the maintenance of correct posture7. a horse riding simulation exercise generates 50 to 100 three - dimensional physical movements (front and back, left and right, up and down) due to the movements of the horse riding exercise machine. this exercise can produce effects similar to walking, activate the trunk muscles to continuously maintain balance, and help correct posture8. another study reported that the horse riding simulator normalized the trunk muscle activity and developed equilibrium reactions, to help improve balance in the trunk9. this concurs with the present study, in which we found that the horse riding simulation exercise improved balance and gait. the present study showed improvements in both static and dynamic balance following horse riding simulation exercise with blindfolding. this may be because the subjects have an increased reliance on the vestibular organs and somatosensory system. this then improves the subjects motor control, enabling proper maintenance of physical alignment and the spontaneous control of movement3. in addition, the improvement in gait found in the study may be from the improved stability and coordination in the trunk. this may have occurred through motor learning of the trunk according to the changes in the base of support. this result suggests that a horse riding simulator can improve balance and gait. as the horse riding simulation exercise generates higher levels of self - motivation and interest when compared to other exercises, we recommend the clinical application of this exercise for patients with impaired balance4. first, as healthy individuals were used, additional studies should be conducted involving patients with functional disorders. second, the intervention period was short and no long term follow - up was performed. | [purpose ] the study was conducted to determine the effect of horse riding simulation combined with blindfolding on healthy individuals balance and gait. [subjects and methods ] thirty subjects were randomly divided into an experimental group (n=15) and a control group (n=15). the subjects in the experimental group covered their eyes using a blindfold, climbed onto a horse riding simulator, and performed the horse riding simulation exercise. the control group took part in the horse riding exercises without a blindfold. all of the subjects performed the 20 minutes long exercise once a day, five times a week, over a four - week period. [results ] the experimental group showed significant improvement in static balance, dynamic balance, velocity, and cadence compared to pre - intervention measurements. in addition, the control group showed significant improvement in static balance, dynamic balance, single support, and cadence compared to pre - intervention measurements. significant differences in post - training gains in static balance, dynamic balance, and cadence were observed between the experimental group and the control group. [conclusion ] subjects that performed horse riding simulation exercise after blindfolding showed significant improvements in balance and cadence compared to the control group. |
a and v patterns describe horizontal strabismus that is vertically incomitant and is characterized by a substantial change in the horizontal deviation from the midline position in upgaze as compared to downgaze. v patterns [14 ]. a and v patterns are relatively frequent in patients with infantile - onset strabismus. a pattern is present when a horizontal deviation shows a more convergent (less divergent) alignment in upgaze compared with downgaze. pattern describes a horizontal deviation that is more convergent (less divergent) in downgaze compared with upgaze. the a pattern is considered clinically significant when the difference in the measurement between upgaze and downgaze, each approximately 25 from the primary position, is at least 10 prism diopters (). pattern is considered clinically significant when the difference is at least 15. other patterns include y where exodeviation is present only in upgaze, lambda () where exodeviation is present only in downgaze, or x where exodeviation is present in downgaze and upgaze compared to the primary position. this review will focus on a and v pattern strabismus. several theories have been proposed to describe the pathophysiology of a and v pattern strabismus. initially it was believed that pattern strabismus is the manifestation of the dysfunction of horizontal and vertical recti muscles [5, 6 ]. however, since elevation in adduction is frequently associated with the v pattern and depression in adduction with the a pattern strabismus, it is now believed that the a or the report of the national eye institute sponsored workshop a classification of eye movement abnormalities and strabismus (cemas)superior oblique overaction is referred to as overdepression in adduction while inferior oblique overaction is referred to as overelevation in adduction. thus, if the superior oblique is overacting and inferior oblique is underacting it will result in relative divergence in downgaze compared to upgaze, resulting in the a pattern. however, a and v pattern strabismus may be seen without any clinically apparent oblique dysfunction. the etiology of isolated extraocular muscle (eom) overaction or underaction is unknown ; several hypothetical constructs have been proposed. broadly, there are two likely sources contributing to pattern strabismus peripheral and central. the peripheral mechanism suggests mechanical orbital factors induce a gaze position - dependent ocular misalignment resulting in pattern strabismus. in contrast, the central mechanism proposes the role of abnormal neurophysiology as a cause of pattern strabismus. as is often the case, it is possible that pattern strabismus is multifactorial and that it could represent a common phenotype with a combination of central and peripheral etiologies. increased frequency of the a and v pattern strabismus has been associated with craniofacial dysmorphisms such as upslanted and downslanted palpebral fissures, plagiocephaly, and hydrocephalus [811 ]. in such cases the strabismus could be related to anomalies in the orientation of superior oblique and inferior oblique muscles, leading to a misdirected muscle force. with the advent of advanced orbital imaging techniques, it was proposed that oblique muscle dysfunction is more a description of the appearance of eye misalignment rather than an actual mechanism of pattern strabismus. high - resolution orbital magnetic resonance imaging (mri) has demonstrated that, unlike their names, the human recti muscles do not follow straight paths from origins to insertions. in eccentric gaze, the rectus muscle paths are inflected in the anterior orbit. the pulleys have been shown to serve as a functional origin of the recti muscles. the pulleys are comprised of annular condensations of the posterior tenon capsule, composed of collagen and elastin, and stiffened by smooth muscle [13, 14 ]. the orbital layer inserts on its respective pulley thus moving the pulley posteriorly during contractions. recti muscle pulleys minimize the sideslip relative to the orbit of posterior muscle paths during globe rotation and determine the effective pulling direction of each rectus muscle. examination of the mechanical properties of the connective tissues defined as pulley bands by van den bedem in fresh human specimens obtained during exenteration of orbits with no strabismus has shown that the pulleys are unsuited for stabilization of rectus eom paths [1517 ]. however, several other studies using in vivo mri in humans have shown that orbitally coupled connective tissues are responsible for rectus eom resistance to sideslips and for sharp eom path inflections [13, 1821 ]. this is in corroboration with finding and persistence of eom path inflections even after globe enucleation and little alteration of posterior rectus muscle paths after large surgical transpositions. in addition, neural recordings in non - human primates suggest that the orbitally stabilized pulleys can change eom pulling direction as eye position changes [24, 25 ]. thus, there is ample evidence from in vivo imaging, histology, and neurophysiological experiments to support the view that the pulleys can exhibit large shifts and stabilize the posterior eom rectus paths. the question, then, is whether abnormalities in eom pulleys can cause pattern strabismus. displacement of the pulley location, perpendicular to a muscle 's plane of action, for example, vertical displacement of horizontal rectus muscle pulley or horizontal displacement of vertical rectus muscle pulley, can cause incomitant strabismus [26, 27 ]. heterotopic misplacement of pulleys can produce the clinical appearance of oblique dysfunction, particularly in patients with orbital dysmorphism or connective tissue disorders [2830 ]. although there is a high prevalence of craniofacial anomalies in patients with furthermore, orbital mri and histologic studies of naturally pattern strabismic non - human primates did not show differences in horizontal recti cross - sectional areas, orbital pulleys, muscle plane paths, innervation densities, or cytoarchitecture compared to the control animals. these animals had neuroanatomical deficits evident as fewer horizontal binocular connections in visual area v1 (striate cortex). abnormal neural connections could cause pattern strabismus by altering the direction of the eom pull. the comparison group was age - matched controls who maintained fusion postoperatively. about 43% of patients with consecutive esotropia, versus only 5% of controls, developed a or consequently, the authors suggested that loss of fusion leads to a torsional drift, analogous to exotropic drift, seen in the patients with monocular sensory deprivation. as a result of the torsional drift the pulling direction of the recti muscles v pattern strabismus, a tonic torsional shift of the eye with counterclockwise torsion in the right eye and clockwise torsion in the left alters the action of the superior rectus as partial abductor and inferior rectus as partial adductor (figure 2(a)). thus pathologically altered force vectors of the vertical recti cause an exoshift in upgaze and an esoshift in downgaze, causing the v pattern. by the same mechanism, the medial recti would also function as partial elevators and lateral recti as partial depressors causing vertical deviation on side gaze mimicking inferior oblique overaction (elevation in adduction) (figure 2(b)). in contrast to this theory a prospective study had reported abnormal torsion on fundus examination years before adduction in elevation (inferior oblique overaction) was seen in infantile esotropia. hence the role of static torsion in the etiology of pattern strabismus remains a matter of debate. recent studies in non - human primates have suggested the possibility of abnormal supranuclear circuits [3537 ]. newly born macaques reared with alternate monocular occlusion (amo) during the critical periods of visual development (first 46 months of life) develop large horizontal misalignment with a or behaviorally, these animal models show inappropriate cross - axis movement of the nonviewing eye in the plane orthogonal to the desired eye - movement trajectory. for example, during a desired horizontal task, the nonviewing eye in addition to horizontal movement also has a simultaneous vertical component, causing the covered eye to move in an oblique trajectory. it was proposed that such cross - coupled movements cause ocular misalignment in a gaze position - dependent fashion, as seen in pattern strabismus. one putative source of the cross - coupled response could be the altered direction of the recti pull either from the abnormal static torsion or pathologies in the peripheral eye plant. if this were the case, there should be an evidence of some neural correlate of the horizontal cross - coupled response in the activity of the vertical motoneurons and vice versa. however, the animal models of pattern strabismus provide evidence that vertical motoneurons fire in response to vertical eye movements and vertical cross - axis component, and horizontal motoneurons fire in response to horizontal eye movements and horizontal cross - axis component [36, 37 ]. it was subsequently proposed that the cross - coupled responses could be secondary to cross - talk between the horizontal and vertical eye movement areas. anatomical studies have shown that vertical and torsional eye movement structures project to medial rectus motoneurons. it is possible that such projections could be the source of cross - coupled eye movements. the caveat of this hypothesis is that it is based upon the animal model reared by alternate monocular occlusion. these animals develop strabismus but do not have the classic oculomotor deficits like nasotemporal asymmetry of optokinetic nystagmus (okn) or latent nystagmus (ln) that is often seen in patients with infantile strabismus. latent nystagmus has also been studied in animals reared with binocular lid suture without tarsal plate and those reared with prism glasses. animals reared with alternate monocular occlusion (amo) have good visual acuity from each eye but the binocularity is completely disrupted. in contrast, animals reared with binocular lid suture without tarsal plate have thin translucent eyelids which allow diffuse luminance to the retina but prevent spatial vision. animals reared with prism goggles have binocular image noncorrespondence thus preventing a normal binocular visual experience but retaining some form of vision from each eye. mustari and colleagues have shown that nasotemporal optokinetic nystagmus (okn) asymmetry and latent nystagmus (ln) in monkeys reared with binocular deprivation are due to defects in nucleus of optic tract (not). hoffmann and stone have shown that at birth not receives direct input from contralateral retina. ipsilateral eye inputs from striate and extrastriate visual cortical areas develop with normal binocular visual experience [41, 42 ]. the not units have been shown to have an absence of binocular cells when animals have early onset strabismus [43, 44 ]. neuroanatomically, it has been shown that monkeys reared with amo have an absence of binocular connections between ocular dominance columns of opposite ocularity in the striate cortex whereas other motion processing areas including extrastriate visual cortex - medial temporal area (mt) and subcortical area - not have preserved binocular cells. thus, these animals do not develop latent nystagmus. on the other hand, monkeys with some form vision from each eye such as those reared with binocular lid suture without tarsal plate, the not is mainly driven by the contralateral eye with a paucity of cells that respond to both eyes resulting in okn asymmetry and ln. monkeys reared with prism goggles develop strabismus, ln, and nasotemporal asymmetry. the severity of strabismus, ln, and okn asymmetry increases with increasing duration of binocular image noncorrespondence induced by prism goggles [47, 48 ]. the prism goggles reared animals have been shown to have a loss of binocular connections between ocular dominance columns of opposite ocularity in visual area v1. this in turn is passed on to extrastriate visual motion processing areas, medial temporal cortex and medial superior temporal cortex (mt and mst). the extrastriate cortex areas project to downstream areas including not and other brainstem areas. hence it would be interesting to investigate whether the cross - coupled responses seen in pattern strabismic monkeys reared with amo are also seen in animals reared with binocular lid suture without tarsal plate (media opacity model, e.g., congenital cataracts) or those with prism goggles (infantile onset strabismus model) which allows some form of vision from both eyes. in this hypothesis, a pattern strabismus was viewed as a special form of skew deviation that is seen in patients with brainstem or cerebellar disease. the authors suggested that in a pattern strabismus, bilateral brainstem lesions decrease the otolith equivalent of the anterior semicircular canal input. hence there would be activation of the inferior rectus and superior oblique but inhibition of the inferior oblique and superior rectus muscles causing intorsion. imbalance between the tone of superior and inferior obliques and relative overaction of superior oblique in response to premotor inputs naturally predicts increased ocular tilt responses during head tilt causing otolith stimulation. absence of such responses in a pattern strabismus led to the prediction that the a pattern is a special form of skew deviation. while novel, it was based on data from patients who had other neurological deficits including neural tube defects, spinabifida, or hydrocephalus. such coexisting neurological deficits are not always present in patients with a pattern strabismus. the etiology of pattern strabismus, particularly the role of torsion, is the key determinant in the choice of surgery and potential for favorable outcome. for example, surgical treatment for patients with overacting obliques involves weakening of the appropriate obliques, whereas for those without clinically apparent oblique dysfunction it involves horizontal recti transposition to collapse the pattern. it has been shown that horizontal recti transposition collapses the pattern but results in increased objective torsion. therefore, if ocular torsion drives a and v pattern strabismus then the surgery that increases the torsion should worsen the pattern strabismus. kushner has reported that rectus muscle transposition surgery for pattern strabismus can cause abnormal ocular torsion, and transposition to address torsion may cause pattern strabismus. thus, these surgical results suggest that abnormal ocular torsion is less likely to be a cause of pattern strabismus. in addition, results of strabismus surgery with similar dosing vary in patients with pattern versus nonpattern exotropia. it has been reported that the postoperative drift in patients with pattern intermittent exotropia was consistently less than in comitant exotropia. the drift was less if the pattern persisted postoperatively and if the exotropia was undercorrected. this suggests differing underlying causes of pattern versus nonpattern exotropia [55, 56 ]. late - onset pattern strabismus or pattern strabismus in patients with craniofacial anomalies could be due to orbital pulley instability or abnormal static torsion. | vertically incomitant pattern strabismus comprises 50% of infantile horizontal strabismus. the oblique muscle dysfunction has been associated with pattern strabismus. high - resolution orbit imaging and contemporary neurophysiology studies in non - human primate models of strabismus have shed light into the mechanisms of pattern strabismus. in this review, we will examine our current understanding of etiologies of pattern strabismus. speculated pathophysiology includes oblique muscle dysfunction, loss of fusion with altered recti muscle pull, displacements and instability in connective tissue pulleys of the recti muscles, vestibular hypofunction, and abnormal neural connections. orbital mechanical factors, such as abnormal pulleys, were reported as a cause of pattern strabismus in patients with craniofacial anomalies, connective tissue disorders, and late - onset strabismus. in contrast, abnormal neural connections could be responsible for the development of a pattern in infantile - onset strabismus. pattern strabismus is likely multifactorial. understanding the mechanisms of pattern strabismus is pivotal to determine an appropriate surgical treatment strategy for these patients. |
although the etiology of ulcerative colitis (uc) remains unknown, a systemic immune disorder might be involved in its development. patients with uc are treated with anti - inflammatory or immunosuppressive treatments, including 5-aminosalicylic acid (5-asa), corticosteroids, immunomodulators or biologics. however, surgery for uc is often performed in patients with a flare - up and refractory disease unresponsive to medical therapy. it has been reported that female sex appears to be a risk factor for frequent uc flare - ups, and the disease is often exacerbated with flare - ups during pregnancy [1, 2 ]. medical treatments are restricted when flare - ups occur during pregnancy due to concern about fetal safety. according to statements by the united states food and drug administration (fda), 5-asa, corticosteroids and cytapheresis (cap) have been proven safe and are recommended treatments during pregnancy. with immunomodulators and biologics, although thiopurine is not recommended for treatment during pregnancy and thiopurines belong to fda pregnancy category d, some studies have reported no evidence for an increased risk in pregnancy except in the period before birth [3, 4, 5, 6 ]. on the other hand, biologics and tacrolimus have been given a b category, which means that studies in animals do not show fetal risks, but there have been no controlled studies in humans, or adverse effects have been observed in animal studies, but these have not been confirmed in controlled studies. although there are reports of successful treatment of uc with immunomodulators, biologics and cap during pregnancy, one should consider surgical treatment for severe uc when medical treatment fails to induce remission [3, 4, 5, 8, 9 ]. dozois. previously reported 38 cases of uc managed with surgery during pregnancy. surgical intervention coincided with cesarean section or delivery in 16 patients, with the pregnancy continuing after surgery in the remaining 22 cases. these surgical interventions, which included total colectomy, subtotal colectomy, ileostomy alone or ileostomy and colostomy, were performed according to each patient 's condition. therefore, the strategy of surgical procedure during pregnancy remains unclear. in this study, two cases of severe uc during pregnancy unresponsive to medical therapy are described, and the standard surgical procedure for severe uc during pregnancy is considered with regard to both fetal and maternal safety. a diagnosis of left - sided uc was made on the basis of colonoscopic findings and confirmed by the typical histological features of biopsy samples (fig. she presented with moderate colitis, having frequent loose, bloody stools less than 10 times a day, mild anemia, abdominal pain that was not severe and low - grade fever. she was initially started on oral corticosteroid therapy at 0.5 mg / kg / day of prednisolone (20 mg / day) with 90 mg / kg / day of 5-asa (3,600 mg of mesalazine daily). despite 8 days of this treatment, her condition was not cured, and she required increased doses of prednisolone intravenously (1.5 mg / kg / day). however, she was referred to our hospital for further treatment 24 days after onset of uc at 22 weeks and 0 days of her pregnancy because her symptoms had worsened, with frequent bloody stools and abdominal pain. although she was treated with high - dose prednisolone and intensive cap that was performed once every 2 days in our institution, she had moderately severe colitis, with frequent loose stools more than 10 times per day, abdominal cramps, high - grade fever and a high c - reactive protein value of 20.4 mg / dl on laboratory blood testing. abdominal ultrasound examination at 34 days from the onset of uc (at 23 weeks and 3 days of pregnancy) showed a thin wall with dilatation of the colonic lumen, consistent with megacolon (fig. urgent surgery was indicated for the colitis that was unresponsive to medical therapy with suspected toxic megacolon. since the fetus showed no growth abnormalities and even though it was at a gestational age of 23 weeks, a cesarean section was performed before the colectomy. although the neonate required treatment for retinopathy of prematurity in the neonatal intensive care unit, both the baby and mother improved without any postoperative complications. the patient finally underwent restorative proctocolectomy with ileal pouch anal anastomosis 8 months after the initial surgery. a 28-year - old woman who had a 2-year history of left - sided uc and was treated with 60 mg / kg / day of 5-asa (2,400 mg of mesalazine daily) was referred to our hospital for a flare - up of symptoms with increasing loose bloody stools at 11 weeks and 1 day of pregnancy. she was started on corticosteroid therapy at a dose of 1.0 mg / kg / day of prednisolone with 80 mg / kg / day of 5-asa and cap. since her symptoms did not change during the 3 weeks of initial treatment, the next step involved 5 mg / kg of infliximab. after 7 days of infliximab administration, she complained of abdominal distension, cramps and increased loose stools. although laboratory blood tests showed no evidence of sepsis, the white blood cell count increased to 23,130/l and the hemoglobin count decreased from 8.3 to 6.4 g / dl within 1 day. urgent surgery was performed at 15 weeks of pregnancy because she had refractory disease unresponsive to medical therapy, with suspected megacolon and bowel obstruction. total colectomy with hartmann 's procedure and end ileostomy was performed as initial surgery without interruption of the pregnancy (fig. at present, the standard surgical procedure for uc is restorative proctocolectomy with ileal pouch reconstruction. although emergent surgery that includes only colectomy without manipulation of the rectum can be performed for patients with penetration, toxic megacolon or fulminant disease, urgent surgery that includes total proctocolectomy with pouch reconstruction could be selected for patients with refractory disease depending on the presence of systemic sepsis. the most important issue, regardless of pregnancy, is to determine promptly whether emergent / urgent surgical intervention is required based on the severity of the colitis and the general maternal condition. namely, it should be recognized that delayed surgery can result in sepsis, which can adversely affect both the mother and the fetus. furthermore, surgery and the severity of uc should never be the determining factors for discontinuing pregnancy. in general, in non - pregnant patients, urgent surgery for refractory disease unresponsive to medical therapy as in the two cases presented could include two - stage restorative proctocolectomy with diverting ileostomy. however, we should consider some fetal and maternal issues during pregnancy before surgery. the size of the uterus and its effect on the surgical procedure should be considered. generally, during the first trimester, the uterus rises out of the pelvic cavity and reaches the level of the umbilicus by week 20. proctectomy without touching the uterus to avoid stimulating the baby is impossible even in other rectal surgery and pouch surgery. therefore, total colectomy with ileostomy should be performed as initial surgery in a staged approach, and ileal pouch reconstruction should be performed a sufficient time after delivery. it has been reported that reducing the uterus to normal size, as before pregnancy, takes at least 46 weeks. thus, secondary surgery for restorative proctocolectomy is desirable at that time. although we recommended the creation of a mucous fistula with colectomy rather than hartmann 's procedure due to its safety for pelvic abscess in a previous report, the presence of a mucous fistula is a contraindication in pregnant patients who are continuing pregnancy after colectomy because of uterine and fetal growth after surgery. we should consider whether cesarean section should be done or whether the pregnancy should be continued before surgery. generally, cesarean section should be considered when there are fetal or maternal issues, such as previous cesarean section, dystocia, breech presentation or fetal distress, as mentioned above. the indication for cesarean section or abortion can not be determined based on uc severity. therefore, cesarean section to allow for proctectomy and pouch reconstruction should not be considered before urgent / emergent colectomy. furthermore, although abortion is legal up to 24 weeks of pregnancy in the united kingdom or up to 22 weeks of pregnancy in japan, abortion should not be considered in anticipation of later proctocolectomy even in early pregnancy within the above weeks when no fetal abnormalities are found. recently, premature babies have been defined as those born before 37 weeks of gestational age. extremely premature infants are those born before 32 weeks, and moderately premature infants are those born between 32 and 37 weeks. high - level neonatal intensive care units have been shown to provide benefits for both extremely premature and moderately premature infants. it has been reported that more than 99% of mildly premature infants at 3338 weeks are expected to survive today. however, morbidity can be increased up to approximately 10%, and mild or severe morbidity can reach over 50% in extremely premature infants born at 2330 weeks of gestation. given this, it is clearly better for the infant to avoid an early birth as much as possible, and cesarean section should generally be avoided until at least 26 weeks of pregnancy. however, cesarean section prior to colectomy should be considered with severe colitis after 26 weeks of pregnancy because the severe uc could easily result in maternal morbidity. it is important to perform cesarean section and colectomy emergently at any time during pregnancy when the maternal and fetal conditions deteriorate and sepsis with perforative peritonitis develops to at least save the mother 's life. in conclusion, regardless of pregnancy, the need for emergent / urgent surgical intervention should be determined promptly based on the severity of colitis and the general maternal condition. surgery and the severity of uc should never be the determining factors for discontinuing pregnancy. the pregnancy can be continued for as long as possible without fetal and maternal problems in surgery for severe uc. | refractory ulcerative colitis (uc) that does not respond to medical therapy often requires surgery even during pregnancy. although surgical cases of uc during pregnancy were reported previously, the standard surgical strategy for both colitis and pregnancy was unclear. herein, fetal and maternal safety as well as the strategy for this unusual surgical procedure during pregnancy in patients with uc are considered. a 28-year - old woman was diagnosed with left - sided moderate uc at 12 weeks of pregnancy ; toxic megacolon was suspected, and surgery was required. although the baby 's gestational age was 23 weeks and 3 days, a cesarean section was performed before the colectomy. in a next case, a 28-year - old woman had a 2-year history of left - sided uc. her colitis flared up at 11 weeks of pregnancy. colectomy was performed because her colitis was unresponsive to conservative therapy, and the pregnancy was continued, with a transvaginal delivery at 36 weeks. in patients with uc, the need for surgery should be determined promptly based on disease severity, whether or not the patient is pregnant. the need for surgery should not be affected by pregnancy. the pregnancy should be continued for as long as possible when there are no fetal and maternal complications. both cesarean section and colectomy should be performed independently if necessary. |
ultrasound guided fine - needle aspiration (usfna) is accepted as the least invasive and most accurate method of identifying the nature of thyroid nodules (1, 2). during the procedure, the goal is to obtain the most cellular specimen that represents the target nodule. published literature revealed substantial variability in usfna specimen cellularity, ranged from 66.4% to 96.6% depending on different specimen obtaining techniques (2, 3). during aspiration, color doppler sonography (cds) is usually used to identify the perinodular and intranodular vessels to provide a safe access site and reduce the amount of blood in the aspirate as possible (1). the guidelines for aspiration of thyroid nodules have been published and vascular patterns of thyroid nodules were extensively studied (4). however, the question of placing the needle tip according to the vascular pattern is yet to be answered. this study was designed to assess whether the hypervascular or hypovascular site of a solid thyroid nodule would reveal more cellular aspirates when usfna is performed under cds guidance. the study group consisted of patients with thyroid nodules referred to bursa state hospital radiology department for usfna. in our institution, institutional review board approved the study protocol and informed consents were obtained from all patients enrolled in the study. between january 2011 and june 2011, 23 consecutive patients (19 females, 4 males ; mean age of 49.7, 28 - 71) referred for usfna with solid nodules greater than 20 mm and clear distinction between hypervascular and hypovascular parts were included in the study prospectively. cystic and degenerated / necrotic nodules all patients were examined with gray scale thyroid us followed by cds by the same investigator (u.o.) with 10 years of relevant experience. the us unit uses logic 400 pro (ge medical systems, kyunggi - do, korea) equipped with 6 - 9 mhz linear transducer. the size of each nodule was recorded based on its largest diameter. for all nodules, with freehand technique and under direct cds visualization, a 25-gauge needle attached to a 20-ml plastic syringe was placed in the targeted part ; the syringe plunger was pulled back by 5 ml. for hypovascular aspirate, the needle tip was located at least 10 mm to the nearest vessel and as central as possible to the nodule. the second pass was from the hypervascular site and the needle tip was placed in the most hypervascular part of the nodule as close as possible to the blood vessels whilst avoiding intravascular sampling. single pass was used for each sample (hypo- and hypervascular) and the first pass was from the hypovascular site to avoid potential hemorrhage from adjacent vessels of the hypervascular site. when the needle was placed in the desired site, aspiration was made until material reached the hub and the needle tip was kept in place during aspiration without moving in the tissue to avoid any trauma or hemorrhage. all samples examined by the same pathologist (s.a.) with 12 years of thyroid cytology experience blinded to patient data. the smears were air - dried, because may - grnwald - giemsa (mgg) stain was preferred for detailed analysis of cytoplasm and colloid. smears were evaluated using the cytological features of smear cellularity, groups or single cells, background features and classified as follows ; score 1 : the number of cell groups less than 5 : inadequate specimen, score 2 : the number of thyrocyte groups between 5 to 10, score 3 : specimens with more than 10 cell groups. endothelial cells were also noted as randomly scattered single endothelial cells = 1 and groups of endothelial cells = 2. the rate of adequate sampling between hypo- and hypervascular regions was calculated by mcnemar test and the results were further statistically evaluated with weighted kappa and power analysis. twenty - three nodules (hypoechoic : 9, isoechoic : 2, hyperechoic : 4, heterogeneous and calcified : 8) in 23 patients (mean nodule diameter : 35.8 mm (20 - 100 mm) were aspirated. nineteen had benign cytology and 2 (9.5%) had malignant cells indicative of papillary carcinoma. in two cases, the specimens did not contain adequate number of cells for pathologic assessment (table 1). adequate sampling was limited to 65% (15/23) of cases when only hypervascular site was used (figure 1) and 83% (19/23) of cases when only hypovascular site was used. when both sites were evaluated together, overall adequate sampling was 91% (21/23). in hypovascular group 17% (4/23) of aspirates were inadequate compared to 35% (8/23) in hypervascular group. the adequate sampling rate between hypo- and hypervascular regions was not statistically significant according to mcnemar test (p = 0.289) (table 1). however, the calculated power of this data was 36% (alpha error 0.05), which was low, limited by the number of patients. adding a sample from hypervascular site to hypovascular aspirate increased the adequate sampling by 8%. for pathological score, the weighted kappa for adequate sampling from hypovascular and hypervascular aspirates was calculated as 0,101 p = 0.473. thus, when the pathological score of one aspirate was high, the score of the same nodule tends to be low on the other aspirate and vice versa. as a result, when both sites were evaluated together, the number of nodules with cell groups more than 10 increased to 16 patients. the highest pathological score was achieved when both hypo- and hypervascular site aspirates were evaluated together (table 2). in hypovascular aspirates, groups of endothelial cells were observed in 17% (4/23) of the cases, whereas in hypervascular aspirates, groups of endothelial cells were observed in 52% (12/23) of the cases. the higher number of endothelial cell groups in hypervascular group did not affect the cytology scores. weighted kappa = 0.101, p = 0.473. score 1 : the number of cell groups less than 5, inadequate specimen. thyroid nodule vascularity can be easily detected with cds while performing usfna, demonstrating the relative position of needle tip to the vessels. our study showed that when the first aspirate is taken from the hypovascular site of a solitary nodule, adding a second pass from the hypervascular site would provide more cell groups for the pathological assessment. therefore, the aspirates from the hypovascular and hypervascular sites of solid nodules are complementary and should be performed consecutively. in addition, increased number of endothelial cells in the hypervascular site aspirates does not decrease the cytology score of the nodule. the biopsy guidelines recommend sampling the solid and viable parts of the tumor instead of cystic, necrotic or more sclerotic parts that may yield inadequate biopsy material. for example, in the diagnosis of bone tumors, a diligent search for more solid regions or areas of enhancement at mr imaging or ct is considered vital in directing the biopsy to reach viable regions harboring diagnostic tissue (5). in patients with prostate disease, choosing the hypervascular site may preclude a positive result that yields carcinoma or inflammation (6). this raises the question of whether sampling from the hypervascular part of a thyroid nodule would yield better cytological results. colleagues suggested the use of color doppler mapping of the nodule immediately before needle aspiration to delineate perinodular flow and larger blood vessels within the nodule to avoid injury during the aspiration biopsy and reduce the amount of blood in the aspirate (1). in this study, nodules larger than 2 cm were included to demonstrate the hypo- and hypervascular sites separately. it has been reported that the lowest rate of specimen adequacy was observed among nodules larger than 3 cm due to increased vascularity and the larger size of blood vessels, with resultant bloodstaining of the material (3). in accordance with their findings, we observed that if the aspirate is solely from the hypervascular part of a large nodule, adequate sampling is limited to 65%. however, intravascular sampling and blood staining may be avoided using cds guidance. however, in nodules larger than 2 cm, if the first aspirate is taken from hypovascular site of the nodule, preferring the hypervascular site for the second pass would yield 8% increase in adequate sampling. preferring hypo- or hypervascular sites for aspiration did not yield statistically significant difference for adequate sampling. thus, we suggest that, instead of avoiding hypervascular sites whilst sampling, cds guidance should be used to avoid intravascular sampling since this approach may reveal better complementary results. the overall incidence of cancer in patients with thyroid nodules selected for fine needle aspiration is approximately 9.2%-13.0 (7). in this series, 9.5% of the nodules were malignant, probably because only large and solid nodules were investigated in the study. this is a limitation since there are various different us features proven suspicious for malignancy (2, 7). larger series including smaller nodules as well as various nodules with us features suggestive of malignancy should be tested to verify the results of this study. in addition, a different study with additional techniques to take better samples like two consecutive passes from suspicious and/or vascular sites with control groups should be conducted. the sample size was also limited with insufficient power to demonstrate the superiority of one site to the other and the results of this study should be verified with larger series. in conclusion, cds may improve aspiration results by enabling determination of the appropriate site. the aspirates from hypovascular and hypervascular sites of solid nodules are complementary and should be performed consecutively for obtaining higher number of cell groups. | background : the goal of ultrasound guided fine - needle aspiration (usfna) is to obtain most cellular specimen that represents the nodule. however, there is substantial variability in specimen cellularity depending on the obtaining techniques. while performing usfna, it is not clear whether the needle tip should be placed at hypovascular or hypervascular site of the nodule to obtain more cells for cytological analysis.objectives:the aim of the study was to assess whether usfna of the hypovascular or hypervascular site of a thyroid nodule would reveal more cells for cytological analysis.patients and methods : twenty - three consecutive patients with solid thyroid nodules larger than 2 cm were aspirated under color doppler sonography guidance. first pass was from the hypovascular site and the second pass from the hypervascular site. the aspirates were scored from 1 - 3 by cytologist according to number of cell groups.results:when only hypervascular site was used, adequate sampling was limited to 65% of the cases. when both sites were evaluated together, overall adequate sampling was 91%. adding a sample from the hypervascular site to hypovascular aspirate increased the adequate sampling by 8%. the highest pathological score was achieved when both hypo- and hypervascular site aspirates were evaluated together.conclusions:the aspirates from the hypovascular and hypervascular sites of solid nodules are complementary and should be performed consecutively. |
glucose production (gp) is regulated by gluconeogenesis and glycogenolysis. in type 2 diabetes, elevation of gp an elevation of plasma gluconeogenic substrate precursors such as free fatty acids (ffa) or lactate is also seen in type 2 diabetes [25 ]. these findings led to the working hypothesis that elevation of gluconeogenic substrate precursors increases gluconeogenesis and gp. the findings as a whole indicate that during the pancreatic clamp, when gluco - regulatory hormones and glucose levels are maintained at basal, intravenous (i.v.) infusion of ffa or lactate increases hepatic gluconeogensis but, contrary to the hypothesis, not gp because of a compensatory inhibition of glycogenolysis [69 ]. the underlying nutrient - sensing mechanisms that inhibit glycogenolysis and counteract the direct effects of circulating ffa or lactate on hepatic gluconeogenesis remain to be explored. in this study, we began to assess the underlying nutrient - sensing mechanisms that counteract the direct effect of circulating lactate on hepatic gluconeogenesis in vivo. the lipid - sensing mechanisms that counteract the direct effect of circulating ffa on hepatic gluconeogenesis have recently been studied. it is demonstrated that the inhibition of hepatic glycogenolysis induced by circulating ffa to restrain gp is mediated by hypothalamic lipid - sensing mechanisms in rodents. the hypothalamic lipid - sensing mechanisms to restrain glycogenolysis and gp are disrupted in diet - induced insulin resistance and obesity, leading to hyperglycaemia in response to systemic lipid infusions. this is in line with previous reports indicating that in patients with metabolic stress conditions such as type 2 diabetes, reciprocal changes in glycogenolysis do not compensate for changes in gluconeogenesis when plasma ffa concentrations are experimentally manipulated. unlike the lipid - sensing mechanisms in the body, even in metabolic stress patients with hyperglycaemia, an elevation of circulating lactate still does not increase gp because of an inhibition of glycogenolysis. consistent with this, in contrast to hypothalamic lipid - sensing mechanisms, activation of central lactate metabolism via direct delivery of lactate into the hypothalamus lowers gp in normal and in early onset of diabetic and obese rodents [14, 15 ]. on the basis of these observations, we postulate that (i) the hypothalamic - sensing mechanism of circulating lactate that regulate gp is intact in normal, diabetic and obese individuals, and (ii) these hypothalamic nutrient - sensing mechanisms are designed to counteract the direct stimulatory effect of circulating lactate on hepatic gluconeogenesis to maintain glucose homeostasis. given that the ability of the hypothalamus to sense circulating lactate to regulate gp and maintain glucose homeostasis is yet to be evaluated, this will be the first study to address our working hypothesis in normal rodents. to examine whether the hypothalamus senses circulating lactate to regulate gp and maintain glucose homeostasis in vivo, we infused i.v. l - lactate to elevate plasma lactate levels in the presence or absence of direct inhibition of brain lactate - sensing mechanisms. to directly inhibit central / hypothalamic lactate - sensing mechanisms, we independently infused (i) lactate dehydrogenase inhibitor oxamate (oxa) into the third cerebral ventricle (intracerebroventricular i.c.v.), (ii) i.c.v. atp - sensitive potassium (katp) channel blocker glibenclamide (gli) or (iii) oxa directly into the mediobasal hypothalamus (mbh) (fig. 1a and b) that has been previously described to prevent the inhibitory effects of central lactate administration on gp. tracer - dilution methodology and the pancreatic clamp technique were used to assess the effect of i.v. and i.c.v./mbh administrations on whole body glucose kinetics independent of changes in circulating gluco - regulatory hormones. based on this cross - discipline experimental approach, we provided the first evidence, to our knowledge, that direct inhibition of central / hypothalamic lactate - sensing mechanisms by three independent methods increased gp and disrupted glucose homeostasis in response to systemic lactate elevations in vivo. the hypothalamic metabolism of lactate to pyruvate and the subsequent activation of the katp channels are required to maintain glucose homeostasis in response to systemic lactate elevations. somatostatin (srif). during the final 30 min. of the clamps, (c) intravenous lactate infusion elevated plasma lactate levels compared to control (saline, sal) by 2- to 2.5-fold. p < 0.001 versus sal. (d) plasma glucose, (e) plasma insulin, (f) plasma adiponectin and glucagon levels were comparable in all groups during the clamps. adult 8-week - old male sprague dawley rats (250280 g) were obtained from charles river laboratories (montreal, qc, canada) and maintained on a standard light - dark cycle with access to rat chow and water ad libitum. rats underwent stereotaxic surgery to insert single catheters in the third cerebral ventricle (i.c.v.) or bilateral catheters into the mbh 2 weeks before the experiments in vivo as previously described [14, 16, 17 ]. one week later, catheters were placed in the internal jugular vein and the carotid artery for infusion and sampling during the clamp procedures as previously described [14, 16, 18 ]. recovery from surgery was monitored by measuring daily food intake and weight gain for 45 days after surgery. the study animal protocol was approved by the institutional animal care and use committee of the university health network. all the rats were restricted to 20 g of food the night before the experiments to ensure the same nutritional status. to groups of conscious unrestrained rats, we infused i.v. sodium l - lactate (100 mol / kg min., ph 7.0) to elevate plasma lactate concentrations by 2- to 2.5-fold as seen in exercise for 4 hrs (fig. infusions consisted of the (i) lactate dehydrogenase inhibitor oxa (dissolved in artificial cerebrospinal fluid [acsf ] to 50 mm [5 l / hr ] and was first given as an i.c.v. bolus [3 l ]), (ii) katp channel blocker gli (dissolved in 5% dmso to 100 m ; 5 l / hr) or (iii) vehicle (5% dmso or acsf ; 5 l / hr) (fig. mbh infusions consisted of (i) oxa (50 mm ; 0.33 l / hr and was first given as a mbh bolus [0.33 l ]) or (ii) vehicle (acsf ; 0.33 l / hr) (fig. oxa or gli and mbh oxa administered at these concentrations alone do not affect glucose kinetics but were sufficient to abolish the gp - lowering effect of direct brain lactate administrations. saline (sal) or l - lactate infusion and a primed - continuous infusion of [3-h]-glucose (perkin elmer, woodbridge, on, canada ; 40 ci bolus ; 0.4 ci / min) were initiated at 120 min. and maintained throughout the study to assess the rate of gp and glucose uptake based on the tracer - dilution methodology. (steady - state basal period), the rate of gp (mg / kg min.) and plasma glucose levels (mm) were 11.9 0.5 and 8.3 0.1 (i.c.v. lactate), 12.6 0.2 and 8.2 0.2 (mbh vehicle + i.v. lactate) and 12.2 1.2 and 8.9 0.6 (mbh oxa + i.v. lactate). a pancreatic clamp was performed in the final 2 hrs of the study starting at 240 min ; a continuous infusion of insulin (1.5 mu / kg min.) and somatostatin (3 g / kg min.) was administered, and a variable infusion of a 25% glucose solution was started and periodically adjusted to maintain the plasma glucose concentration at 8 mm. saline) were performed, and the gp during the clamps in both groups (gp : i.c.v. plasma samples for the determination of lactate, insulin, adiponectin and glucagon concentrations were obtained at 10-min. somatostatin. in the presence of systemic lactate elevation, direct inhibition of lactate metabolism within the mediobasal hypothalamus (mbh) via mbh administration of oxamate (compared to control) (b) decreased exogenous glucose infusion rate and (c) increased gp during the final 30 min. 0.001 versus lactate with mbh vehicle. central sensing mechanisms of circulating lactate regulate glucose production (gp). in the presence of systemic elevation of lactate, direct inhibition of central lactate metabolism via i.c.v. administration of lactate dehydrogenase inhibitor oxamate (oxa) (compared to control) led to a marked (a) decrease in exogenous glucose infusion rate and (b) increase in gp during the final 30 min. of the clamps. administration of blocker glibenclamide (a) decreased exogenous glucose infusion rate and (b) increased gp in response to systemic lactate infusions. plasma lactate concentrations were determined by a kit in accordance with manufacturer s instructions (sigma diagnostics. plasma glucose concentrations were measured by the glucose oxidase method (glucose analyzer, analox instruments, lunenberg, ma, usa). statistical analysis was done by anova or unpaired student s t - test as appropriate. adult 8-week - old male sprague dawley rats (250280 g) were obtained from charles river laboratories (montreal, qc, canada) and maintained on a standard light - dark cycle with access to rat chow and water ad libitum. rats underwent stereotaxic surgery to insert single catheters in the third cerebral ventricle (i.c.v.) or bilateral catheters into the mbh 2 weeks before the experiments in vivo as previously described [14, 16, 17 ]. one week later, catheters were placed in the internal jugular vein and the carotid artery for infusion and sampling during the clamp procedures as previously described [14, 16, 18 ]. recovery from surgery was monitored by measuring daily food intake and weight gain for 45 days after surgery. the study animal protocol was approved by the institutional animal care and use committee of the university health network. all the rats were restricted to 20 g of food the night before the experiments to ensure the same nutritional status. to groups of conscious unrestrained rats, we infused i.v. ph 7.0) to elevate plasma lactate concentrations by 2- to 2.5-fold as seen in exercise for 4 hrs (fig infusions consisted of the (i) lactate dehydrogenase inhibitor oxa (dissolved in artificial cerebrospinal fluid [acsf ] to 50 mm [5 l / hr ] and was first given as an i.c.v. bolus [3 l ]), (ii) katp channel blocker gli (dissolved in 5% dmso to 100 m ; 5 l / hr) or (iii) vehicle (5% dmso or acsf ; 5 l / hr) (fig. mbh infusions consisted of (i) oxa (50 mm ; 0.33 l / hr and was first given as a mbh bolus [0.33 l ]) or (ii) vehicle (acsf ; 0.33 l / hr) (fig. oxa or gli and mbh oxa administered at these concentrations alone do not affect glucose kinetics but were sufficient to abolish the gp - lowering effect of direct brain lactate administrations. saline (sal) or l - lactate infusion and a primed - continuous infusion of [3-h]-glucose (perkin elmer, woodbridge, on, canada ; 40 ci bolus ; 0.4 ci / min) were initiated at 120 min. and maintained throughout the study to assess the rate of gp and glucose uptake based on the tracer - dilution methodology. (steady - state basal period), the rate of gp (mg / kg min.) and plasma glucose levels (mm) were 11.9 0.5 and 8.3 0.1 (i.c.v. lactate), 12.6 0.2 and 8.2 0.2 (mbh vehicle + i.v. lactate) and 12.2 1.2 and 8.9 0.6 (mbh oxa + i.v. lactate). a pancreatic clamp was performed in the final 2 hrs of the study starting at 240 min ; a continuous infusion of insulin (1.5 mu / kg min.) and somatostatin (3 g / kg min.) was administered, and a variable infusion of a 25% glucose solution was started and periodically adjusted to maintain the plasma glucose concentration at 8 mm. saline) were performed, and the gp during the clamps in both groups (gp : i.c.v. plasma samples for the determination of lactate, insulin, adiponectin and glucagon concentrations were obtained at 10-min. somatostatin. in the presence of systemic lactate elevation, direct inhibition of lactate metabolism within the mediobasal hypothalamus (mbh) via mbh administration of oxamate (compared to control) (b) decreased exogenous glucose infusion rate and (c) increased gp during the final 30 min. 0.001 versus lactate with mbh vehicle. central sensing mechanisms of circulating lactate regulate glucose production (gp). in the presence of systemic elevation of lactate, direct inhibition of central lactate metabolism via i.c.v. administration of lactate dehydrogenase inhibitor oxamate (oxa) (compared to control) led to a marked (a) decrease in exogenous glucose infusion rate and (b) increase in gp during the final 30 min. administration of blocker glibenclamide (a) decreased exogenous glucose infusion rate and (b) increased gp in response to systemic lactate infusions. plasma lactate concentrations were determined by a kit in accordance with manufacturer s instructions (sigma diagnostics. plasma glucose concentrations were measured by the glucose oxidase method (glucose analyzer, analox instruments, lunenberg, ma, usa). statistical analysis was done by anova or unpaired student s t - test as appropriate. here we first examined whether selectively blocking the central effects of lactate through the i.c.v. administration of lactate dehydrogenase inhibitor oxa or katp channel blocker gli is sufficient to alter the metabolic response of i.v.. systemic infusion of lactate at 100 mol / kg min. for 4 hrs elevated plasma lactate by 2- to 2.5-fold compared to i.v. 1c). in the final 2 hrs of the lactate elevation, we performed pancreatic - insulin clamps with tracer dilution methodology to assess the effects of i.v. lactate on glucose kinetics independent of changes in plasma gluco - regulatory hormones (fig. lactate infusion did not increase gp during the pancreatic clamps when plasma glucose (fig. 1e and f) were maintained near basal levels. to assess whether central sensing mechanisms restrain plasma lactate from elevating gp, we administered i.c.v. f), central oxa administration decreased exogenous glucose infusion rate required to maintain euglycaemia (fig. this decrease in glucose infusion rate was due entirely to an elevation of gp (fig. together, these data indicate that direct inhibition of central lactate metabolism disrupted glucose homeostasis and increased gp in the presence of systemic lactate elevations. to complement the function findings obtained with i.c.v. katp channels blocker gli to alternatively negate central lactate effects and examine whether central sensing mechanisms restrain plasma lactate to increase gp. similar to the effects of i.c.v. oxa, central gli administration decreased the exogenous glucose infusion rate required to maintain euglycaemia (fig. 2a) in the presence of similar degree of plasma lactate elevation as observed in the other groups (fig. the drop in glucose infusion rate was caused by an increase in gp (fig. these findings indicate that direct inhibition of central katp channels disrupted glucose homeostasis in response to systemic lactate elevation. to investigate the central neuroanatomical localization of the cns - sensing mechanisms of circulating lactate on gp, we negated the lactate - sensing mechanisms specifically within the mbh. lactate infusions (100 mol / kg min.) with the administration of oxa (50 mm) bilaterally into the mbh (fig., mbh oxa administration decreased glucose infusion rate required to maintain euglycaemia in the same degree of plasma lactate elevation (fig. again, the decrease in glucose infusion rate was due to increased gp (fig. 3d). thus, direct inhibition of lactate metabolism within the mbh disrupted glucose homeostasis and increased gp in the presence of systemic lactate infusions. it remains to be assessed whether the hypothalamic restraining effect on gp in response to circulating lactate is mediated by an inhibition of hepatic glycogenolysis and/or gluconeogenesis. the hypothalamus has been recently demonstrated to detect a rise in both hormones and nutrients to regulate peripheral metabolic processes [9, 15, 1929 ]. specifically, direct activation of either (i) insulin / leptin signalling pathways [19, 2125, 28, 29 ] or (ii) lipid / lactate metabolism [9, 15, 30 ] in the hypothalamus regulates gp. furthermore, the demonstration of the ability of the hypothalamus to sense circulating lipids to lower hepatic glycogenolysis and restrain gp (9) has added to the existing knowledge of nutrient - sensing mechanisms in the body. these findings, at least in rodents, suggest that the hypothalamus senses circulating lipids to counteract the direct stimulatory effects of lipids on hepatic gluconeogenesis. more importantly, the hypothalamic lipid - sensing mechanisms are disrupted in the early onset of diet - induced obese rodents, leading to a rise in gp and glucose levels induced by circulating lipids. these observations, if extended to human beings, could in part begin to address the inability of the body to compensate for a lipid - induced increase in gluconeogenesis in type 2 diabetes. in direct contrast to lipid - sensing mechanisms, the body has the ability to inhibit hepatic glycogenolysis and restrain gp to compensate for the circulating lactate - induced increase in hepatic gluconeogenesis in both normal individuals and metabolic stress individuals with hyperglycaemia [8, 12 ]. in an attempt to elucidate the underlying mechanisms of lactate - sensing that are responsible for this metabolic restraining effect on gp, we assessed whether the hypothalamus senses an elevation of plasma lactate by 2- to 2.5-fold for 4 hrs to restrain gp and maintain glucose homeostasis in normal rodents. lactate infusion on glucose metabolism during pancreatic clamps when gluco - regulatory hormones (fig. consistent with previous reports in human beings, an elevation of circulating gluconeogenic substrate precursor lactate did not increase gp. we postulated that the hypothalamic lactate - sensing mechanisms are responsible for this glucose homeostatic control. if this is true, direct inhibition of cns / hypothalamic lactate - sensing mechanisms should disrupt glucose homeostasis and lead to an elevation of gp in the presence of systemic lactate infusion. to test this hypothesis, we directly inhibited cns / hypothalamic lactate - sensing mechanisms that regulate gp by three independent approaches. during the pancreatic clamps, all three experimental interventions in the brain (comparing to control) led to a marked increase in gp in response to systemic lactate infusion. together, these data indicated that hypothalamic sensing mechanisms restrain systemic lactate to increase gp and is required to maintain glucose homeostasis. this is the first report that demonstrates the ability of the hypothalamus to sense circulating lactate to restrain gp and maintain glucose homeostasis in normal rodents. in light of the fact that (i) direct central lactate administration lowers gp in normal and early onset of diabetic and obese rodents [14, 15 ], (ii) an elevation of circulating lactate does not increase gp in normal individuals and in patients with hyperglycaemia [8, 12 ], we postulate that the hypothalamic nutrient sensing mechanisms of circulating lactate are intact in at least the early onset of diabetes and obesity. however, future studies are required to address this hypothesis. in summary, our data suggest that therapeutic strategies designed to activate cns lactate - sensing mechanisms by physiological route could eventually be proven useful to maintain glucose homeostasis in vivo. | emerging studies indicate that hypothalamic hormonal signalling pathways and nutrient metabolism regulate glucose homeostasis in rodents. although hypothalamic lactate - sensing mechanisms have been described to lower glucose production (gp), it is currently unknown whether the hypothalamus senses lactate in the blood circulation to regulate gp and maintain glucose homeostasis in vivo. to examine whether hypothalamic sensing of circulating lactate is required to regulate gp, we infused intravenous (i.v.) lactate in the absence or presence of inhibition of central / hypothalamic lactate - sensing mechanisms in normal rodents. inhibition of central / hypothalamic lactate - sensing mechanisms was achieved by three independent approaches. tracer - dilution methodology in combination with the pancreatic clamp technique was used to assess the effect of i.v. and central / hypothalamic administrations on glucose metabolism in vivo. in the presence of physiologically relevant increases in the levels of plasma lactate, inhibition of central lactate - sensing mechanisms by lactate dehydrogenase inhibitor oxamate (oxa) or atp - sensitive potassium channels blocker glibenclamide increased gp. furthermore, direct administration of oxa into the mediobasal hypothalamus increased gp in the presence of similar elevation of circulating lactate. together, these data indicate that hypothalamic sensing of circulating lactate regulates gp and is required to maintain glucose homeostasis. |
advances in the treatment of locally advanced lung cancer had no impact on overall 5-year survival rates from this disease that remains only of 15%. although the rates of lung cancer mortality have started to decrease in countries where smoking habits have been modified, the projections are not optimistic because of the recent surge in tobacco consumption among young people. in addition, even if smoking habits could be modified significantly, the long lag time between peak of tobacco consumption and the development of lung cancer will assure a long life for this epidemic. the role of tobacco smoking in the aetiology of lung cancer has been widely evaluated. many compounds present in the smoke of cigarettes, such as the polycyclic aromatic hydrocarbons (pah), induce dna adducts after metabolic activation. unrepaired dna adducts can cause mutations, including mutational hot spots in p53 tumour suppressor gene, and lead to unregulated cell growth and cancer. increased dna adduct levels have been suggested to be predictive of lung cancer risk, reflecting both the environmental exposure to carcinogens than individual susceptibility [58 ]. it has been hypothesized that interindividual difference in lung cancer risk may be due to differences in dna repair. in support of this hypothesis, different studies have indicated that dna variation in dna repair genes may influence cancer susceptibility. our group has conducted a number of studies that have shown associations between dna polymorphisms in dna repair genes, mainly in xrcc1 (x - ray repair cross complementing) and xrcc3 genes, cancer, and/or dna adducts [1014 ] and unpublished results. thus, we have decided to extend our analysis to the individuals included in a lung cancer case - control study. in this study, we have analyzed the levels of dna adducts and dna polymorphisms in two dna repair genes, for example, xrcc1 arg194trp and xrcc1 arg399gln, and xrcc3 thr241met polymorphisms, representing the base excision repair (ber) and the double - strand breaks repair (dsb) pathways, in lung cancer cases in respect to individuals with benign lung disease and to healthy controls. then, we decided to investigate the combination of the variant allele / s of xrcc1 arg194trp and arg399gln polymorphisms with the wild type allele of xrcc3 thr241met. the analysis of the effects of different combinations of dna repair polymorphisms on dna adducts has been done under the assumption that the combination of polymorphisms can have additive or more than additive effects on dna adduct formation. peripheral blood samples were collected, after written informed consent to participate in the present study, from 34 nonsmall cell lung cancer patients (26 males and 8 females, mean age 63.4 years) and from 30 (22 males and 8 females, mean age 63.5 years) subjects with benign lung diseases admitted to the national cancer institute and san martino hospital, genoa, italy. 40 controls (25 males and 15 females, mean age 63.4 years) were recruited from a group of blood donors. lung cancer cases were asked to participate in the study after the diagnosis, but before radio and chemotherapy. the group of benign lung diseases was formed by subjects affected by chronic obstructive pulmonary disease (copd), asthma, and pneumoconiosis. a standard questionnaire was administered to all volunteers by personal interview at the time of blood collection. smoking status was defined as smoker, within the last year, former smoker, at least one year before diagnosis, and nonsmoker. peripheral blood lymphocytes (pbls) were separated from 5 ml freshly collected whole blood by centrifugation on a ficoll gradient. pbl dna was extracted and purified using a method that requires rna and protein digestion and extraction with organic solvents. pbl dna adducts were analysed using the nuclease p1 modification of the p - postlabelling technique. dna samples (15 g) were digested with micrococcal nuclease (32.17 mu) and spleen phosphodiesterase (21.6 mu). hydrolized dna was treated with nuclease p1 (110 mu) for 30. the nuclease p1 resistant dna samples were then labelled by incubation with 25 ci of carrier - free [-p]atp (3000 ci / mm) and t4-polynucleotide kinase (112.5 mu). the obtained p - labelled samples were analysed using 1.0 m sodium phosphate, ph 6.8. dna adduct resolution was achieved using 4.0 m lithium formate, 7.5 m urea, ph 3.5 and 0.65 m lithium chloride, 0.45 m tris base, 7.7 m urea, ph 8.0. detection and quantification of pbl dna adducts and normal nucleotides (nn) were obtained by storage phosphor imaging techniques employing intensifying screens. after background subtraction, the levels of dna adducts were expressed such as relative adduct labelling (ral) = screen pixel in adducted nucleotides / screen pixel in nn. polymerase chain reaction followed by enzymatic digestion was used for the genotyping of xrcc1 arg194trp and arg399gln, and xrcc3 thr241met. logistic regression analysis was carried out to calculate odds ratios (ors) adjusted for different covariates (i.e., age, sex, smoking, and dna polymorphisms, as appropriate) categorizing dna adduct levels by ral median value (above / below 0.1 dna adducts per 10 nn). a multiple regression analysis has also been performed grouping individuals according to the number of at risk alleles. genotype and allele frequencies were calculated by counting, and genotype distributions were in hardy - weinberg equilibrium. genotype frequencies were, respectively : xrcc1 arg194arg = 88.8%, arg194trp = 11.2%, xrcc1 arg399arg = 38.5%, arg399gln = 49%, gln399gln = 12.5%, xrcc3 thr241thr = 40%, thr241met = 41%, and met241met = 19%, in keeping with those reported previously. the highest levels of dna adducts were detected in individuals that reported to smoke for more than 40 years. a slightly increment of dna damage was observed in benign lung disease and lung cancer patients in respect to controls. multiple regression analysis shows an increased frequency of pbl dna adducts in smokers for more than 40 years (or = 5.28, 95% confidence interval (c.i.) 1.0027.72, p =.049). a significant trend with increasing number of smoked cigarettes was found (p for trend <.05). after the previous cited adjustments, no differences were observed comparing controls with benign lung diseases and lung cancer patients. when the associations of dna adducts with dna polymorphisms were considered, a null association with xrcc1 arg194trp and arg388gln polymorphisms was found (or = 4.08, 95% c.i. 0.7721.48, p =.098 and or = 1.32, 95% c.i. 0.345.18 ; p =.689, resp.). conversely, a statistically significant inverse effect was observed with xrcc3 thr241met polymorphism (or = 0.17, 95% c.i. 0.050.61, then, we investigated the combination of the variant allele / s of xrcc1 arg194trp and arg399gln polymorphisms with the wild type allele of xrcc3 thr241met. the choice was based on the different association of xrcc1 and xrcc3 polymorphisms with lung cancer risk, for example, positive for xrcc1 arg194trp and arg399gln and negative for xrcc3 met241met [9, 16, 17 ]. the analysis of the effect of different combinations of dna repair single nucleotide polymorphisms on dna adducts has been performed under the assumption that the combination of different polymorphisms can have additive or more than additive effects. when the study population was categorized in base to the number of risk alleles, the levels of dna adducts were statistically significantly increased in individuals bearing three - four risk alleles (or=4.1 95% c.i. a significant association with smoking was noticed in smokers for more than 40 years carrying 3 - 4 risk alleles (or=36.38, 95% c.i. a not statistically significant increment of lung cancer risk was observed in the same group (or = 4.54, 95% c.i. 0.3362.93, a significant trend with increasing the number of risk alleles was also observed (p for trend <.05). many studies have explored the influence of smoking on the levels of dna adducts in nucleated blood cells in order to identify an early and sensitive biomarker of effective intake of tobacco carcinogens. in our study, we have analyzed pbls as surrogate and more accessible tissues than bronchial biopsies, and we compared the levels of dna adducts in patients with lung cancer in respect to those with benign lung diseases and controls. our aim was to evaluate whether the levels of dna adducts were associated with benign or malignant lung chronic diseases. the question of the utility of pbls as a valid surrogate for a specific organ like lung, representing the events occurring in the target tissue, is still open. nevertheless, some studies have indicated that the use of pbls such as a biological marker, may help in the identification of subjects at elevated risk [68 ]. pbls are considered suitable to monitor environmental and occupational carcinogen exposure and to estimate the burden of dna adducts in respiratory tissue [5, 2022 ]. in fact, increased amount of pbl dna adducts have been found among subjects heavily exposed to air pollution. the relationship with target tissue dna adducts may vary between type of carcinogen and target tissue although significant correlations have been seen between the levels of dna damage in pbls and bronchial mucosa. our findings show that the levels of dna adducts of smokers were higher than those of former and nonsmokers. our finding shows that pbl dna adduct may reflect exposures to carcinogens, such as those contained in tobacco smoke better than other surrogate tissues, such as leukocyte dna adducts. this is probably due to methodical differences in the p - dna postlabelling protocol applied from research laboratories. next results show that the effect of smoking on dna damage was more marked in the subjects that reported to smoke for more than 40 years. although detailed information on smoking history, for example, number of pack of cigarettes smoked per years was missing, our findings support the hypothesis that the formation of dna adducts is significantly influenced by chronic carcinogen exposure. furthermore, when study population was subgrouped for the number of risk alleles, a significant association with smoking was observed in the subjects carrying three or more risk alleles who reported to smoke for more than 40 years. conversely, no effect of smoking was observed in smokers bearing one or less risk allele. the contribution of duration of exposure to cigarette smoke has important implications for both research studies and prevention strategies. it has been shown that the age at first exposure and duration are associated influences to the levels of dna adducts. smoking during adolescence has been shown to produce physiological changes leading to increased persistence of dna adducts less, and subjects who begin smoking very early in life tend to be heavy smokers. two large epidemiological studies demonstrated that duration is more important than intensity of cigarette smoking in predicting lung cancer risk [24, 25 ]. herein reported results support in part this hypothesis, thus shedding light on the mechanisms involved in the aetiology of smoking related cancers. it is likely that a plateau of the formation of dna adducts is reached at these time points. in fact, the persistence of dna adducts in pbls is less than one decade, which is the maximum lifespan for long - living lymphocytes. our next results show that dna adduct levels were comparable in individuals with benign lung disease or with lung cancer and in controls. however, when specific combinations of variant alleles were investigated, a not significant increased lung cancer risk was observed in individuals bearing the same number of risk alleles. in a meta - analysis of cancer and bulky dna adducts, dna damage has been reported to be predictive of lung cancer, particularly in smokers. in veglia 's meta - analysis, smokers presented a significant difference between lung cancer cases and controls, with patients having 83% higher amount of dna adducts than controls. we know that the interpretation of the meta - analysis is limited by the fact that in case - control studies, the level of biomarker may reflect the presence of cancer disease rather than its aetiology. however, an exception is represented by three cohort studies, in which dna adducts have been found to be prospectively predictive of lung cancer outcome [68 ]. the importance of these studies is based on the fact that biomarker measurement in pbls collected several years before cancer onset ruled out the possibility that the higher levels of dna damage were reflecting metabolic changes associated with cancer. no increment of dna adducts has been found in subjects with different benign bronchial pathologies, such as copd, asthma, and pneumoconiosis, characterized by important inflammatory processes in respect to controls. however, such inflammatory phenomena can influence dna adduct levels in lung target cells by increasing the biologically effective dose of pah. this hypothesis is consistent with a previous case - control study where the levels of dna adducts in individuals with inflammatory diseases were significantly higher than those of controls. dna damage primarily reflects exposures to carcinogens but is modulated by inherited and acquired susceptibilities. age, gender, and life - style and dietary habits have been reported to influence levels of dna adducts [5, 29, 30 ]. dna adducts may be also influenced by the individual 's ability to remove dna adducts undergoing from interindividual variability. although the main pathway for removal of bulky dna adducts is nucleotide excision repair, it has been shown that ber and dsb repair mechanisms may participate in bulky dna adduct repair, supporting the association of xrcc1 and xrcc3 polymorphisms with such kind of dna damage [32, 33 ]. in this study positive nonstatistically significant associations with xrcc1 arg194trp and gln399gln genotypes were found whereas an inverse significant association was detected in xrcc3 met241met carriers. our findings are in keeping with previous studies showing that the variant alleles of the xrcc1 arg399gln polymorphism is associated with dna adducts [10, 12, 13 ]. a recent pooled analysis has shown a protective effect conferred by xrrc3 carriers against lung cancer. however, other studies have reported higher levels of dna damage in individuals with xrcc1 arg194arg and xrcc3 met241met genotypes [12, 33 ]. our results suggest that case - control studies are more indicative for the determination of genetic susceptibility than cross - sectional studies. to study the effect of different combinations of dna repair single nucleotide polymorphisms on dna adducts, we have investigated the combination of the variant allele / s of xrcc1 arg194trp and arg399gln polymorphisms with the wild type allele of xrcc3 thr241met. this was based on the different association of xrcc1 and xrcc3 polymorphisms with lung cancer risk, for example, positive for xrcc1 arg194trp and arg399gln and negative for xrcc3 thr241met [9, 16, 17, 34 ]. our findings show that the combination of different polymorphisms can have additive effects on the levels of dna adducts. in fact, when the number of risk alleles was analyzed, dna adducts were higher in individuals carrying three - four risk alleles. furthermore, when our population was categorized in base to the number of risk alleles, the association between smoking and lung cancer risk tended to be present in the same individuals bearing three - four risk alleles. although statistical significances were seen in our analyses, our study is underpowered, and larger studies are needed to confirm the associations between dna polymorphisms, cancer, and dna adducts. furthermore, a previous report has shown that smoking is strong harmful factor that can eliminate the effect of dna polymorphisms of dna repair genes on lung cancer susceptibility. smoking could lead to cancer due to its toxic effect regardless of whether individuals have polymorphisms with low repair proficiency. thus, the examination of the effect of different combinations of dna polymorphisms for the prediction of lung cancer susceptibility could be more useful in nonsmokers exposed to relatively minor environmental factors. the results of the present study support the utilisation of pbls as surrogate and more accessible tissues than bronchial biopsies. in fact, we have observed a stronger effect of smoking on dna adducts of 40 years smokers. when study population was subgrouped for number of risk alleles, the association with smoking was concentrated in carriers of 3 - 4 risk alleles that reported to have smoked for more than 40 years. a nonsignificant increased lung cancer risk was observed in individuals bearing the same number of risk alleles. our results suggest that analysis of risk alleles can predict the interindividual variation in dna adduct levels observed in smokers and lung cancer cases. | smoke constituents can induce dna adducts that cause mutations and lead to lung cancer. we have analyzed dna adducts and polymorphisms in two dna repair genes, for example, xrcc1 arg194trp and arg399gln genes and xrcc3 thr241met gene, in 34 lung cancer cases in respect to 30 subjects with benign lung cancer disease and 40 healthy controls. when the study population was categorized in base to the number of risk alleles, adducts were significantly increased in individuals bearing 3 - 4 risk alleles (or = 4.1 95% c.i. 1.2813.09, p =.009). a significant association with smoking was noticed in smokers for more than 40 years carrying 3 - 4 risk alleles (or = 36.38, 95% c.i. 1.171132.84, p =.040). a not statistically significant increment of lung cancer risk was observed in the same group (or = 4.54, 95% c.i. 0.3362.93, p =.259). our results suggest that the analysis of the number of risk alleles predicts the interindividual variation in dna adducts of smokers and lung cancer cases. |
unfortunately, in many countries around the world, including ghana, water has become a scarce commodity1 as only a small proportion of the populace has access to treated water. alternative sources of water such as rainwater and ground water have become major sources of drinking water for people living in new settlements and some residents who do not have access to treated water in ghana. the need to assess the quality of water from some of these alternative sources has become imperative because they have a direct effect on the health of individuals.2 contaminants such as bacteria, viruses, heavy metals, nitrates and salt have polluted water supplies as a result of inadequate treatment and disposal of waste from humans and livestock, industrial discharges, and over - use of limited water resources.3 even if no sources of anthropogenic contamination exist there is potential for natural levels of metals and other chemicals to be harmful to human health. this was highlighted in bangladesh where natural levels of arsenic in groundwater were found to be causing harmful effects on the population.4 unfortunately, this problem arose because the groundwater was extracted for drinking without a detailed chemical investigation. the natural water analyses for physical and chemical properties including trace element contents are very important for public health studies.5 these studies are also a main part of pollution studies in the environment. in addition, coliform enters water supplies from the direct disposal of waste into streams or lakes or from runoff from wooded areas, pastures, feedlots, septic tanks, and sewage plants into streams or groundwater. coliform can also enter an individual house via backflow of water from a contaminated source, carbon filters, or leaking well caps that allow dirt and dead organisms to fall into the water.6 the presence of escherichia (e) coli in drinking water denotes that the water has been focally contaminated and therefore presents a potential health risk to households that use them untreated.7 research conducted in ghana8 indicated that 77% of filtered underground water samples sold as sachet water that were analyzed contained infective stages of pathogenic parasitic organisms. 14/27 (51.2%), cryptosporidium parvum 17/27 (63.0%), cyclospora cayetenens 16/27 (59.3%), sarcocystis sp. 18/27 (66.7%), rotifers 5/27 (18.5%), and charcoat leyden crystals 12/27 (evidence of allergies or parasitic infection) (44.4%). a total of 29.6% of the samples contained at least one type of parasite, 14.8% contained at least 2 types of parasites, 25.9% contained at least three types of parasites, and 29.6% contained four types of parasites. this has grim public health implications as the organisms identified can cause water related diseases that have serious complications in children and adults particularly immunocompromised individuals. during this study, some physicochemical properties and biological parameters of untreated water from hand - dug wells were determined and evaluated. the purpose of this study was to assess the quality of water from hand - dug wells in the kumasi metropolis, ghana. two water samples were collected each of ten different hand - dug wells in various residences in kumasi. ph of the water samples was measured on - site with a suntex sp-707 (taipei, taiwan) portable ph meter. all the water samples were collected in duplicate and stored in ice in the laboratory until analysis were completed within 14 days. the determinations of the other physicochemical properties of the water samples were performed on the same day of sampling. test on samples for bacteria was conducted within 6 hours of sampling while that for anions was within 14 days. a photometric method was used for the determination of fe, mn, no3, no2, so4, po4, sio2 and f. analytical water test tablets prescribed for palintest photometer 5000 (wagtech, thatcham. each sample was analysed for fe, mn, no3, no2, so4, po4, sio2 and f using procedures outlined in the palintest photometer method for the examination of water. other analyses such as the determination of total hardness, mg and ca concentrations, were done by complexometric titration using edta. total dissolved solids and electrical conductivity were determined by a means of a multifunctional wtw cond. standard methods for the determination of total coliform and fecal coliform9,10 (brenner, 1993 and apha, 1995) were employed. two water samples were collected each of ten different hand - dug wells in various residences in kumasi. ph of the water samples was measured on - site with a suntex sp-707 (taipei, taiwan) portable ph meter. all the water samples were collected in duplicate and stored in ice in the laboratory until analysis were completed within 14 days. the determinations of the other physicochemical properties of the water samples were performed on the same day of sampling. test on samples for bacteria was conducted within 6 hours of sampling while that for anions was within 14 days. a photometric method was used for the determination of fe, mn, no3, no2, so4, po4, sio2 and f. analytical water test tablets prescribed for palintest photometer 5000 (wagtech, thatcham. each sample was analysed for fe, mn, no3, no2, so4, po4, sio2 and f using procedures outlined in the palintest photometer method for the examination of water. other analyses such as the determination of total hardness, mg and ca concentrations, were done by complexometric titration using edta. total dissolved solids and electrical conductivity were determined by a means of a multifunctional wtw cond. standard methods for the determination of total coliform and fecal coliform9,10 (brenner, 1993 and apha, 1995) were employed. the mean (average) values for ph, conductivity, total dissolved solids, hardness, alkalinity and cations determined in the hand dug well water samples are shown in table 1. the mean (average) values of ph of the samples ranged from 6.3 to 7.7. ph values lower than 6.5 are considered too acidic for human consumption and can cause health problems such as acidosis. the ph values greater than 8.5 are considered to be too alkaline for human consumption. the who permissible limit for electrical conductivity (ec) of water is 300 s / cm. conductivity values of the samples ranged from 46 to 282 s / cm. these values are below the who permissible limit. total hardness ranged from 8.0 to 103 mg / l and alkalinity from 20 to 80 mg / l. all the samples had the total hardness and alkalinity concentrations below the who permissible limits. the concentrations of trace metals iron and manganese and bulk metal calcium and magnesium ions in the drinking water samples are also presented in table 1. iron was below the minimum detection limit of 0.01 mg / l in all the samples except bhd, which generated a result of 1.2 mg / l. this may have resulted from the materials used in the construction of the well and the type of soil in which the well has been constructed. manganese (mn) concentrations in the samples ranged from below detection limit to 0.018 mg / l. calcium (ca) and magnesium (mg) concentrations ranged from 0.09 to 24.80 and 0.01 to 11.80 respectively. the levels of iron (fe) and manganese (mn) in most cases were below the limits permitted by who in drinking water (table 1) and usepa.11,12 though these trace metals are needed by the body to satisfy its nutritional requirements, only minute quantities are required as high doses lead to health hazards which are sometimes lethal. calcium (ca) and magnesium (mg) however are needed by the body in much larger quantities and its lack in the human system will lead to adverse health effects. the mean (average) values of cl concentration in the water samples ranged from 0.229.4 these values are below the who quality standard for drinking water of 250 mg / l. phosphate (po4) ranged from 0.33 to 9.30 fluoride (f) varied from 0.20 to 0.80 mg / l. the minimum value (0.20 mg / l) was observed in two samples, bhe and pm among all the samples analyzed. the maximum concentration (0.80 mg / l) of f was observed in bb. permissible limit for f concentration is 1.01.5 mg / l according to.12 fluoride (f) has a significant mitigating effect against dental caries if the concentration is approximately1 however, continuing consumption of higher concentrations of 4 mg / l or more can cause dental fluorosis and in extreme cases even skeletal fluorosis.13 nitrate and nitrite in the investigated samples ranged from below detection (mdl : 0.001) to 0.968 mg and below detection (mdl : 0.001) to 0.063 respectively. no3 and no2 are considered to be non - cumulative toxins.14 high concentrations of no3 and no2 may give rise to potential health risks such as methmoglobinemia or blue - baby - syndrome particularly in pregnant women and bottle - fed infants respectively,15 no3 at elevated concentrations is also known to result in cyanosis in infants. the range of sulfate (so4) in the samples was 3.0 to 37.0 mg / l, the values recorded for nitrate, nitrite and sulfate were all below the who permissible limits as shown in table 2. total coliform and e. coli (table 3) were below detection in all the samples. work done on the assessment and comparison of microbial quality of drinking water in chikwawa, malawi revealed that though the microbiological analysis of borehole abstracted water did not reveal the presence of either total coliform or escherichia coli at mdl of 20 mpn per 100 ml. the results of all water samples taken from every drinking water storage container from the study area were positive for total.16 groundwater is a relatively safe source of potable water compared with other unprotected water sources e.g. rivers, springs, rainwater. water samples taken directly from hand - dug wells in this study contained levels of coliform below the mdl of the mpn technique. these results are similar to the findings of a study undertaken in chikwawa,16 which found levels of fecal coliform below the mdl of 20 mpn/100 ml in water samples taken from 27 boreholes. in this study, the concentrations of the investigated anions and trace metal ions in the water samples from hand dug wells from the kumasi metropolis in the ashanti region of ghana were found to be acceptable according to the guidelines for drinking water provided by the world health organization (who). the quality of groundwater supplied by the wells was satisfactory with fecal indicator bacteria below the mdl of 20 mpn 100 ml. the water therefore may, according to who standards be safely used as drinking water. however since contamination after collection, during transportation and storage is increasingly being recognized as an issue of public health importance,17,18 it may require treatment such as boiling or treatment with hypochlorite solution since that will kill most microbial parasites before drinking. further research on other communities in this region for the assessment of the quality of drinking water is required as levels of contaminants may vary due to different soil types, water chemistry and different human activities. intensification of education and implementation of regulations on safe drinking water by the ghana standards board, the ghana epa and district environmental units and other state enforcements agencies will go along way to reduce incidences of water pollution and the associated water borne diseases. | this study focused upon the determination of physicochemical and microbial properties, including metals, selected anions and coliform bacteria in drinking water samples from hand - dug wells in the kumasi metropolis of the republic of ghana. the purpose was to assess the quality of water from these sources. ten different water samples were taken from different parts of kumasi, the capital of the ashanti region of ghana and analyzed for physicochemical parameters including ph, electrical conductivity, total dissolved solids, alkalinity total hardness and coliform bacteria. metals and anions analyzed were ca, mg, fe, mn, no3, no2, so42, po42, f and cl. bacteria analysed were total coliform and escherichia coli.the data showed variation of the investigated parameters in samples as follows : ph, 6.300.70 ; conductivity (ec), 46682 s / cm ; po43, 0.6776.00 mg / l ; f, 0.200.80 mg / l ; no3, 00.968 mg / l ; no2, 00.063 mg / l ; so42, 3.007.0 mg / l ; fe, 01.2 mg / l ; mn, 00.018 mg / l. total coliform and escherichia coli were below the minimum detection limit (mdl) of 20 mpn per 100 ml in all the samples. the concentrations of most of the investigated parameters in the drinking water samples from ashanti region were within the permissible limits of the world health organization drinking water quality guidelines. |
achalasia is a chronic esophageal motility disorder associated with esophageal retention of foods and fluids, bacterial overgrowth, and impaired clearance of regurgitated gastric contents. these factors usually lead to chronic inflammation of the esophageal mucosa, which potentially increases the risk of hyperplasia, dysplasia, and esophageal cancer [2, 3 ]. esophageal squamous cell carcinomas in achalasia patients have been investigated previously. in a large cohort followup study, wychulis. analyzed 1,318 patients and found a 7-fold increased risk of esophageal squamous cell carcinomas in achalasia patients compared to the general population. despite some contradictory data [57 ], achalasia is generally accepted as a condition associated with an increased risk for developing esophageal squamous cell carcinoma [8, 9 ]. chromoendoscopy with lugol 's staining remains the gold standard technique for detecting superficial esophageal squamous cell carcinoma [10, 11 ]. although lugol 's staining is a simple and low - cost method, instillation of its solution may lead to complications, such as hypersensitivity to iodine, laryngitis, and pneumonitis, as well as frequent painful sensations and nausea [1215 ]. kondo. demonstrated a significant reduction in retrosternal discomfort with the use of sodium thiosulfate. an alternative technique such as narrow - band imaging for optical staining would be desirable, especially because it is a simpler technique and has no known complications. narrow - band imaging technology may be useful for detecting squamous cell carcinomas of the pharynx and esophagus. muto. and yoshida. observed morphological pattern changes in intrapapillary capillary loops. these patterns can be useful for diagnosing squamous cell carcinomas and even to predict lesion extension. various reports of early - stage pharyngeal and esophageal squamous cell carcinomas identified with narrow - band imaging technology can be found in the literature. without using imaging magnification, muto. diagnosed a superficial squamous cell carcinoma of the pharynx, which appeared as a small and well - defined brownish area. watanabe., who also used narrow - band imaging without magnification, found six pharyngeal squamous cell carcinomas, and goda. found an esophageal squamous cell carcinoma that was not identified by conventional endoscopy (obscure lesion). recently, our group demonstrated that narrow - band imaging performs as well as lugol 's chromoendoscopy for the detection of esophageal squamous cell carcinoma in patients with head and neck cancer. the aim of this study was to compare narrow - band imaging technology with lugol 's staining during endoscopic examination of the esophagus for the detection of high - grade intraepithelial neoplasias and superficial squamous cell carcinomas in patients with esophageal achalasia. this was a cross - sectional study, and esophageal mucosal examination was analyzed in a sequential approach divided into three phases. the first phase was with white light, the second phase was with narrow - band imaging, and the final phase was with lugol 's staining. at the end of each phase, the patients were submitted to an endoscopic procedure, and it was performed under conscious sedation with midazolam and fentanyl. only one senior endoscopist (edson ide) performed all of the endoscopic procedures. at the first phase, if there were lesions, they were mapped using the anterior, posterior, right, and left esophageal walls and the distance up to anterior incisors as references. the second phase involved the use of narrow - band imaging assessment of the esophageal mucosa. at this moment, mucosa, which is green independent of changes in surface or vascular texture. in the third phase, lugol 's staining was performed by spraying 20cc of a 2% lugol 's solution at esophageal mucosa. after the staining, white - colored areas were suspected of being neoplasia, in contrast with brown or brownish normal areas. the operator took biopsies of every suspicious lesion detected by any phase of the study. biopsies were only performed after lugol 's staining was completed, and, after mucosal examination and biopsy, a volume of 20cc of a 0.5% sodium thiosulfate solution was instilled to remove lugol 's solution in order to reduce spasm and pain. complications due to lugol 's solution were registered including laryngitis, chemical pneumonitis, hypersensitivity, and anaphylactic shock. the size and macroscopic shape of the lesions were evaluated according to the paris classification for superficial esophageal lesions. topography was divided into cervical (up to 5 cm of cricopharyngeus muscle), thoracic, and abdominal esophagus. from october 2006 to february 2007, 48 consecutive patients with achalasia were referred to the gastrointestinal endoscopy unit of a tertiary academic center for early esophageal cancer detection. these patients usually participate in a surveillance protocol, which consists of upper gastrointestinal endoscopy with associated esophageal lugol 's staining every 3 years. inclusion criteria were indications to take part in the surveillance protocol for patients with achalasia, despite any treatment (e.g., pharmacologic, endoscopic dilation, and surgical cardiomyotomy) they had undergone previously. exclusion criteria were as follows : clinical conditions that prevented upper gastrointestinal endoscopy examination or lugol 's staining ; previous history of allergic reaction to iodine ; esophageal stasis that could not be cleared by endoscopic procedures ; and endoscopic detection of an ulcerated, infiltrative, or stenotic lesion. this study was approved by the ethics committee of the university of so paulo medical school. an exera ii evis 180 gif180 videogastroscope (olympus, tokyo, japan) with high resolution (1,080 dpi), 1.5-fold magnification, and narrow - band imaging technology was used. histology was performed by a senior pathologist, who was aware of the endoscopic suspicion of esophageal squamous cell carcinoma. the lesions were classified in accordance with the revised vienna classification. in the absence of lamina propria invasion, noninvasive neoplastic lesions were divided into two groups based on the degree of intraepithelial neoplasia : low grade and high grade. high - grade dysplasia, intraepithelial carcinoma, and carcinoma in situ were considered equivalent entities. whenever the lamina propria of the mucosa was invaded, the lesion was referred to as intramucosal carcinoma. in this study, only findings of high - grade intraepithelial neoplasia (carcinoma in situ) and intramucosal carcinoma of squamous cells were considered true positives for esophageal squamous cell carcinoma. values and 95% confidence intervals were calculated for sensitivity, specificity, positive predictive value, negative predictive value, accuracy, and positive and negative likelihood ratios. of the 48 patients enrolled, five were excluded : one because he had an advanced malignant esophageal lesion that was easily detected by conventional endoscopy, three because they had esophageal stasis, and one because of a prior history of allergy to iodine. of the remaining 43 patients, the 43 patients underwent all stages of the protocol of investigation for this study (figure 1). narrow - band imaging and lugol 's staining found seven and nine suspected lesions, respectively. conventional endoscopy revealed one superficial lesion with a flat morphology (0-iib according to paris classification), which was also detected by narrow - band imaging and lugol 's staining (figure 2). this lesion proved to be an esophageal neoplasia (squamous cell carcinoma in situ), sized 15 mm in diameter, and was located in the thoracic esophagus. of the seven lesions found by narrow - band imaging, histopathology revealed that one was normal mucosa, five were esophagitides, and one was squamous cell carcinoma in situ. of the nine lesions found by lugol 's staining, histopathology revealed that eight were esophagitides and one was squamous cell carcinoma in situ. the performance of narrow - band imaging was similar to that obtained by lugol 's staining. sensitivity and negative predictive value were 100% for both methods, and the specificity was 85.7% (75.1%96.3%) for narrow - band imaging and 81% (69.1%92.8%) for lugol 's staining. diagnostic performances for conventional endoscopic examinations, narrow - band imaging, and lugol 's staining are presented in table 2. in the lugol 's staining group the present study selected patients with achalasia as an increased - risk group for esophageal squamous cell carcinoma. these patients usually present with delayed esophageal emptying, and they report worsening of these symptoms due to development of an obstructive tumor at late stages. without surveillance, esophageal carcinoma is usually diagnosed in advanced stages with poor prognosis. the lugol 's staining technique is based on the presence of large amounts of glycogen in the squamous epithelium, which stains intensely with iodine ; in contrast, dysplastic and carcinoma cells contain little or no glycogen, which results in no staining [15, 2527 ]. thus, upper gastrointestinal endoscopy with lugol 's staining is still considered the best method for the diagnosis and delimitation of superficial esophageal squamous cell carcinoma [2729 ]. however, lugol 's solution irritates the mucosa and may lead to retrosternal chest pain and discomfort, because of its alcoholic nature. its utilization is limited by other factors, namely, hypersensitivity to iodine and the risk of chemical esophagitis, laryngitis, and bronchopneumonia. several authors have reported necrosis and injury to the esophageal and gastric mucosa caused by hypersensitivity to lugol 's solution [30, 31 ]. furthermore, esophageal chromoendoscopy with lugol 's staining significantly increases the length of the examination period. narrow - band imaging enhances the visualization of superficial capillaries, as well as mucosal surface structure, and has an effect similar to that of chromoendoscopy. moreover, narrow - band imaging does not have the limitations of lugol 's staining chromoendoscopy and could be considered as a potential alternative method for the detection of esophageal squamous cell carcinoma. few studies have evaluated the capacity of narrow - band imaging without magnification to detect esophageal squamous cell carcinoma. watanabe. found that narrow - band imaging was more likely 2 folds than conventional white - light evaluation to detect pharyngeal squamous cell carcinoma. in a multicenter study that compared narrow - band imaging with conventional white - light evaluation, when lugol 's staining chromoendoscopy was compared with narrow - band imaging with image magnification, the sensitivity was the same (92.3%), but narrow - band imaging had a higher specificity (91.7% versus 72.2%). in our study, we compared lugol 's staining with narrow - band imaging technology without magnification. many medical centers do not have the resources for magnification ; therefore, the aim of this study was to determine whether narrow - band imaging alone would suffice to detect small and superficial neoplasias of the esophagus. narrow - band imaging and lugol 's staining identified one esophageal neoplasia that was also detected by conventional white - light examination. both methods had 100% sensitivity and negative predictive value. although narrow - band imaging without magnification had a higher specificity for detecting early squamous cell neoplasias in the esophagus, it was similar to lugol 's staining : 85.7% (75.1%96.3%) and 81% (69.1%92.8%), respectively. in a study of patients with head and neck squamous cell carcinomas employing the same methodology, ide. observed similar results when they compared narrow - band imaging without magnification with lugol 's staining. takenaka. [33, 34 ] found that the sensitivity of narrow - band imaging for detecting esophageal squamous cell carcinoma and high - grade intraepithelial neoplasia was 90.9% (58.7%99.8%), the specificity was 95.4% (90.398.3%), and the accuracy was 95.1% (90.1%98.0%). narrow - band imaging without magnification and lugol 's staining had equivalent performances ; this indicates that narrow - band imaging is a potential surveillance method for patients with esophageal achalasia. a sequential approach was adopted in which the standard endoscopy, narrow - band imaging, and the lugol 's staining were employed by the same operator in the same patient. this setting might have affected the results since the operator possessed prior information after each phase of endoscopic procedure. furthermore, this methodology was used in similar studies [31, 33, 34 ]. in conclusion, the results obtained with narrow - band imaging technology without magnification were comparable with those obtained with lugol 's staining for the screening of esophageal squamous cell carcinoma in patients with achalasia. although narrow - band imaging does not have the risks and technical difficulties associated with lugol 's staining, larger multicenter studies are necessary in order to analyze the cost and benefits of this technology and to determine whether narrow - band imaging could replace lugol 's staining for screening of early - stage esophageal squamous cell carcinoma. | chromoendoscopy with lugol 's staining remains the gold standard technique for detecting superficial scc. an alternative technique, such as narrow - band imaging (nbi), for optical staining would be desirable, since nbi is a simpler technique and has no known complications. in this study, we compare nbi without magnification and chromoendoscopy with lugol 's staining for detecting high - grade dysplasia and intramucosal esophageal squamous cell carcinoma (scc) in patients with achalasia. this was a prospective observational study of 43 patients with achalasia referred to the gastrointestinal endoscopy unit of the hospital of clinics, so paulo, university medical school, brazil, from october 2006 to february 2007. conventional examinations with white light, nbi, and lugol staining were consecutively performed, and the suspected lesions were mapped, recorded, and sent for biopsy. the results of the three methods were compared regarding sensitivity, specificity, accuracy, positive predictive value, negative predictive value, positive likelihood value, and negative likelihood value. of the 43 patients, one was diagnosed with esophageal squamous cell carcinoma, and it was detected by all of the methods. nbi technology without magnification has high sensitivity and negative predictive value for detecting superficial esophageal squamous cell carcinoma, and it has comparable results with those obtained with lugol 's staining. |
adrenocortical carcinoma (acc) is a very rare entity in the pediatric population, and is rarely associated with virilizing symptoms. the gland is grossly enlarged and the phenotypic features overlap with an adrenocortical adenoma, which need to be evaluated carefully, so as to distinguish it from the latter, as complete surgical excision is curative in an adenoma unlike in acc which may need adjuvant therapy and follow - up. a two - year - old male child presented with features of virilization, which included growth of facial and pubic hair, deepening of voice, and penile growth for five months [figure 1 ]. the features of cushing syndrome (moon face, pot belly, skin stria) were not present. ultrasonography showed a complex echogenic mass in the left paravertebral region. the child 's testosterone and dehydroepiandrostenidione levels were elevated and the serum cortisol level was within normal limits [table 1 ]. a provisional diagnosis of adrenocortical carcinoma was made, an adrenalectomy was done, and a histopathological diagnosis of adrenocortical carcinoma was made. two - year - old male child with features of virilization (pubic hair and penile growth) preoperative and postoperative (after two months) hormonal profile grossly, the adrenalectomy specimen measured 11 9 7 cm and the weighed 240 g. the external surface was unremarkable. [figure 2 ] cut section of tumor showing reddish brown areas with areas of hemorrhage. a narrow strip of normal adrenal was seen compressed to the periphery microscopy revealed large cells with mild - to - moderate pleomorphism. the cells were arranged in sheets and trabaculae [figure 3 ] ; > 75% of the cells had abundant eosinophilic cytoplasm. a majority of the tumor had a diffuse architecture, interspersed with areas of hemorrhage ; there was no evidence of atypical mitosis or vascular and capsular invasion. (h and e 100) photomicrograph showing cells having abundant eosinophilic cytoplasm and hyperchromatic nucleus with one - to - two nucleoli. (h and e 400) based on the weight, size of the tumor, and microscopic features, and considering the criteria proposed by weiss, a diagnosis of adrenocortical carcinoma was made. after the operation, the manifestations of virilization disappeared gradually, except for the lower voice. grossly, the adrenalectomy specimen measured 11 9 7 cm and the weighed 240 g. the external surface was unremarkable. [figure 2 ] cut section of tumor showing reddish brown areas with areas of hemorrhage. a narrow strip of normal adrenal was seen compressed to the periphery microscopy revealed large cells with mild - to - moderate pleomorphism. the cells were arranged in sheets and trabaculae [figure 3 ] ; > 75% of the cells had abundant eosinophilic cytoplasm. a majority of the tumor had a diffuse architecture, interspersed with areas of hemorrhage ; there was no evidence of atypical mitosis or vascular and capsular invasion. (h and e 100) photomicrograph showing cells having abundant eosinophilic cytoplasm and hyperchromatic nucleus with one - to - two nucleoli. (h and e 400) based on the weight, size of the tumor, and microscopic features, and considering the criteria proposed by weiss, a diagnosis of adrenocortical carcinoma was made. after the operation, the manifestations of virilization disappeared gradually, except for the lower voice. acc and adrenocortical adenoma are the two ends of a spectrum that need to be differentiated. these carcinomas are also seen with increased frequency in children with the li - fraumeni syndrome, beckwith - wiedermann syndrome, and congenital adrenal hyperplasia. a slight preponderance in the female population has been noted. acc can be associated with cushing 's syndrome and minerlocorticoid production, although > 75% do not have any hormone overproduction. grossly, carcinomas, as compared to adenomas, are large and weigh more than 100 g. it has been stated that any adrenal tumor weighing more than 250 g, age > 3.5 years at diagnosis, and associated with symptoms of marked hormone overproduction, is to be considered malignant with poor prognosis. pure virilizing carcinomas, in general, appear to have a better prognosis than other adrenal carcinomas. in acc, the cut section of the gland is pink to yellow tan, with areas of hemorrhage and necrosis. microscopically, the tumor has different patterns of growth and as the distinction between benign and malignant neoplasms is difficult, the weiss criteria, which require histopathological findings, are employed to distinguish the benign from the malignant tumors. this classification is based on the mitotic index, confluent necrosis, atypical mitosis, and nuclear grade, and a tumor is rendered as malignant when the score is three or more than three. our case has a score of 3, although the weiss criteria is not useful in the pediatric population. hence, in our case, considering the weight and size of the gland, coupled with the microscopy, elevated hormonal levels, and the clinical symptoms, a diagnosis of acc was made. in our case, good prognostic factors were, weight less than 250 g, age less than 3.5 years, and a pure virilizing form of carcinoma. all adrenocortical carcinomas are highly malignant tumors, and hence, they need to be resected. mitotane, an insecticide derivative that produces adrenocortical necrosis, has been extensively used in adults with acc, but its efficacy in children who highlight a case of virilizing adrenocortical carcinoma, a very aggressive tumor, which is very rare in the pediatric age group and requires surgery, is not known. in this communication | adrenocortical carcinomas are rare tumors with an incidence of one to two cases per million population and are still more rarer in the pediatric age group. adrenocortical carcinomas can be functional or may be unassociated with syndromes of hormone overproduction. it is very important to differentiate an adrenocortical adenoma from a carcinoma, as both share a large number of phenotypic features, and assess their prognosis, as adrenocortical carcinoma may need an adjuvant therapy. in this communication, we describe the case of a two - year - old boy, who presented with iso - sexual precocious puberty, having features of virilization, which included growth of facial and pubic hair, deepening of voice, and penile growth. |
the study was conducted in a wintering population of tits in wytham woods, u.k. 1). 1018 nest - boxes suitable for great tits are installed at this site, with the vast majority of great tits breeding in boxes. individuals are trapped as nestlings and breeding adults at nest - boxes and fitted with both a british trust for ornithology metal leg ring and a plastic leg ring containing a uniquely identifiable passive integrated transponder (pit) tag (ib technology, aylesbury, u.k.). there is a further mist - netting effort over autumn and winter to tag individuals immigrating into the population, and we estimate that over 90% of individuals were pit - tagged at the time of the study. in this population, great tits form loose fission - fusion flocks of unrelated individuals in autumn and winter. flocks congregate at patchy food sources, and can be observed at bird feeders fitted with pit - tag detecting antennae. experiments were conducted in eight sub - populations within wytham woods that had relatively little short - term between - area movement of individuals (extended data fig. the experimental apparatus consisted of an opaque plastic box with a perch positioned in front of a door that could be slid to either side with the bill to gain access to a feeder concealed behind. the left side of the door was colored blue and the right side red, with a raised front section on the door to allow an easier grip. the concealed feeder contained approximately 500 live mealworms and was refilled up to twice daily. 5), and as live mealworms were used, solvers typically extracted one worm and then carried it away from the puzzle - box to kill and eat it (confirmed with video observations) ; supplementary video 1 - 2. each puzzle - box was surrounded by a 11 m cage with a 55 cm mesh that gave unlimited access to small birds, but prevented access by large non - target species such as corvids or squirrels. a freely accessible bird feeder filled with peanut granules was also provided in the cage, at approximately 1 m from the puzzle - box. peanut granules are a much less preferred food source (extended data fig. 5). each peanut feeder had two access points fitted with rfid antenna and data - logging hardware. this feeder was used to attract the original demonstrator to the location, and to record the identity of individuals that did not contact the puzzle - box. all puzzle - boxes contained a printed circuit board (pcb) and motor, and were powered by a 12v sealed battery. the perch also functioned as an rfid antenna that registered the visit duration (time to nearest second) and identity of the visiting individual. solve was recorded if the door was opened during an individual visit to the device, with the side direction also noted. if a solution occurred without an accompanying identified individual, this was recorded as unidentified solve. if further individuals visited before this happened, then a scrounge was recorded, as they were assumed to have taken food from the open door (confirmed from video observations). the door reset immediately after two individuals were registered scrounging, preventing more than two possible scrounging events per solve (supplementary video 2). two males were captured from each sub - population (11 adults, 5 juveniles) to act as demonstrators, either by removal from roosting boxes on sunday night, or by mist - netting at a sunflower - seed feeder on monday morning. they were transferred to individual cages in indoor captive facilities, and over four days each pair of birds was subjected to one of three training regimes using step - wise shaping, either : (i) given no training and left in the cage with ab lib food (control) ; (ii) trained to solve the novel puzzle - box by pushing the blue side of the door to the right (option b) ; or (iii) trained to solve the novel puzzle - box by pushing the red side of the door to the left (option a). with the exception of control areas, which were clustered in the south of the woodland to avoid cross - contamination, sub - populations were randomly assigned to a training regime, with both demonstrators from a single sub - population trained on the same technique. during training, the demonstrators were initially exposed to an open puzzle - box baited with mealworms, which was then gradually closed over the course of four days until the subjects were reliably re - opening it. the birds were released back at the site of capture in each respective sub - population ; puzzle - boxes at which both options were available and equally rewarding were installed at three sites 250 m apart on the following sunday night (extended data fig. these puzzle - boxes were run over a four - week period at each site, continuously operating from monday to friday and then removed on saturday and sunday, for a total of 20 days of data collection. four replicates were conducted in the first year of data collection (december 2012-february 2013 ; c1 - 2, t1, t3). at three of these replicates (c1, t1, t3) puzzle - boxes were simultaneously re - installed at the same locations for 5 days of further data collection in december 2013. no additional demonstrators were trained, and no individual had contact with the puzzle - box in the 9 months between the two data collection periods. this second exposure aimed to test the long - term stability of social learning at the sub - population level. they were run prior to the second year of data - collection for the cultural diffusion experiment in order to exclude the possibility that dispersing individuals from new replicates could be re - introducing the novel behaviour. an additional four replicates were then conducted from december 2013 - february 2014 in new sub - populations, using the same initial protocol (c3, t2, t4, t5). the local population size for each replicate was defined as comprising all individuals in a replicate that had been recorded at least once at either : (i) the puzzle - box, (ii) the nearby peanut feeder, or (iii) the nearest network - logger feeders (operated saturday - sunday, see below), during the experimental period (i.e. from the weekend following the release of the demonstrators, to the weekend after the 20 day of operation of the puzzle - boxes). persistence trial in the following year, the local population was defined just as (i) all individuals observed at the puzzle - box or (ii) nearby peanut feeder, so that areas were comparable. to analyse the results of the initial experiment we first compared control replicates and treatment replicates, using welch two - sided t - tests, and by fitting linear and sigmoidal models to the data, with the best model ascertained by difference in aic values. if individuals were using social information when learning about the puzzle - box, then we expected that there would be a difference between areas seeded with a trained demonstrator (treatment) and those without (control). replicates were thus compared in terms of latency to first solve (seconds from beginning of the experimental period, excluding demonstrator), and the total number of solutions. secondly, we compared the total number of solutions in the two different experimental treatments. here if a more complex form of social learning than local enhancement to the feeding site was occurring, then we expected a consistent bias towards the seeded variant in the different treatments. to analyse the change in individual and population preferences for option a or b over time, we used a generalised estimating equation model (gee) where the dependent variable was the proportion of solutions using the seeded technique on each day of data collection, and the explanatory variables were the individuals and replicate, weighted by the overall number of solutions per day. the seeded technique (a / b) was initially also included as an explanatory variable, but was not significant (coefficient se = 0.130.22, p = 0.55). three individual variables were included in a gee model ; sex, age and natal origin. sex was determined at capture using plumage coloration, age was either determined from breeding records or plumage coloration, and individuals were classed as immigrants if they had dispersed into the study site, and locally - born if they had been ringed as a nestling in the study site. only age was significant (coefficient se = 0.920.20, p < 0.001), and was included in the final model (sex : coefficient se = 0.380.22, p = 0.08 ; natal origin : coefficient se = 0.380.22, p = 0.08). if population - level conformity was partly the result of a conformist transmission bias at first acquisition we would expect a sigmoidal relationship between population - level frequency of the variant and adoption probability, with adoption of the majority variant disproportionately more likely than its absolute frequency. by contrast, copying the last individual observed, or random copying, should yield a linear relationship, with probability of adopting option a / b roughly equal to its proportion in the overall population. to investigate this, we isolated all individuals first observed solutions in all experimental replicates, and compared the option choice to the proportion of all previous solves as option a observed in the individual s group at that site. group length was set at 245 sec, which was the average group length observed using gaussian mixture models on temporal patterns of flocking (see below) at network - logging sunflower feeders. both linear and sigmoidal models were then fitted to the data, with the best model ascertained by difference in aic values. we further examined the subset of individuals that moved between sub - populations (n=40). this subset included all individuals recorded in more than one experimental replicate, whether within the season (n=16), or between seasons (n=24). no individual was observed in more than two replicates, and this analysis did not include individuals in the persistence trial. a preference for option a / b at each location was defined as more than 75% of all solves for either option a / b in that replicate. finally, in order to analyse the change in within - individual bias towards option a / b between the initial experiment and the second - year persistence trials, we used a general linear model where the dependent variable was the number of solves as the seeded variant over the total number of solves for each individual observed in both years. explanatory variables were treatment type and year, with individual identity as a random effect. sunflower bird - feeding stations were deployed at 65 locations around wytham woods on an approximate 250250 m square grid, as part of long - term research into social - network structure in tits (see). each station had two access points, each fitted with rfid antennae and data logging hardware. feeding stations automatically opened from dawn to dusk on saturday and sunday, scanning for pit - tags every 16 of a second. this study used the data from the eight nearest locations to each set of puzzle - boxes, for 10 dates within and surrounding the cultural diffusion experiment (the standard logging protocol runs from september - february in wytham woods). great tits were detecting visiting feeding stations and individually identified by their pit - tags. we then applied a gaussian mixture model to the spatiotemporal data stream to detect distinct clusters of visits. this method locates high - density periods of feeding activity, isolating flocks of feeding birds without imposing artificial assumptions about group boundaries. a gambit of the group approach was used with a simple - ratio index to calculate social associations, where individual association strengths (network edges) were scaled between 0 (never observed foraging together in the same group) to 1 (always observed in the same group, never observed apart). while a single co - occurrence may not be meaningful, our automated data collection method resulted in thousands of repeated group sampling events, allowing social ties between individuals to be built up from multiple observations of co - occurrences over time and across spatial locations. networks contained 123 (t1), 137 (t2), 154 (t3), 95 (t4) and 110 (t5) nodes ; average edge strength was 0.09 (t1), 0.05 (t2), 0.08 (t3), 0.07 (t4) and 0.07 (t5). to test whether networks contained significantly preferred and avoided relationships, we ran permutation tests on the grouping data, controlling for group size and the number of observations, restricting swaps within days and sites. we tested whether observed patterns of associations were non - random by comparing the coefficient of variance in the observed network to the coefficient of variance in the randomised networks. social networks for all replicates significantly differed from random, even at local scales (t1 : p<0.0001 ; t2 : p=0.0005 ; t3 : p<0.0001 ; t4 : p=0.0002 ; t5 : p=0.0002) finally, we used network - based approaches to ask whether the behaviour was socially transmitted through foraging associations. network - based diffusion analysis (nbda) tests for social learning by assuming that if social transmission is occurring, then the spread of trait acquisition should follow patterns of relationships between individuals, with transmission rate linearly proportional to association strength. we used nbda r code v.1.2, with the time of each individual s first solution (seconds since the beginning of the experiment) entered into the continuous time of acquisition analysis function. individuals that solved, but that did not appear in the social network (i.e. had not been recorded in the standardised weekend logging) were excluded from the analysis. the effects of three individual level variables were also incorporated into the analysis : sex, age, and natal origin. all combinations of nbda provided in the nbda r code v1.2 were run with social transmission rate allowed to vary for each replicate. the study was conducted in a wintering population of tits in wytham woods, u.k. 1). 1018 nest - boxes suitable for great tits are installed at this site, with the vast majority of great tits breeding in boxes. individuals are trapped as nestlings and breeding adults at nest - boxes and fitted with both a british trust for ornithology metal leg ring and a plastic leg ring containing a uniquely identifiable passive integrated transponder (pit) tag (ib technology, aylesbury, u.k.). there is a further mist - netting effort over autumn and winter to tag individuals immigrating into the population, and we estimate that over 90% of individuals were pit - tagged at the time of the study. in this population, great tits form loose fission - fusion flocks of unrelated individuals in autumn and winter. flocks congregate at patchy food sources, and can be observed at bird feeders fitted with pit - tag detecting antennae. experiments were conducted in eight sub - populations within wytham woods that had relatively little short - term between - area movement of individuals (extended data fig. the experimental apparatus consisted of an opaque plastic box with a perch positioned in front of a door that could be slid to either side with the bill to gain access to a feeder concealed behind. the left side of the door was colored blue and the right side red, with a raised front section on the door to allow an easier grip. the concealed feeder contained approximately 500 live mealworms and was refilled up to twice daily. 5), and as live mealworms were used, solvers typically extracted one worm and then carried it away from the puzzle - box to kill and eat it (confirmed with video observations) ; supplementary video 1 - 2. each puzzle - box was surrounded by a 11 m cage with a 55 cm mesh that gave unlimited access to small birds, but prevented access by large non - target species such as corvids or squirrels. a freely accessible bird feeder filled with peanut granules was also provided in the cage, at approximately 1 m from the puzzle - box. each peanut feeder had two access points fitted with rfid antenna and data - logging hardware. this feeder was used to attract the original demonstrator to the location, and to record the identity of individuals that did not contact the puzzle - box. all puzzle - boxes contained a printed circuit board (pcb) and motor, and were powered by a 12v sealed battery. the perch also functioned as an rfid antenna that registered the visit duration (time to nearest second) and identity of the visiting individual. solve was recorded if the door was opened during an individual visit to the device, with the side direction also noted. if a solution occurred without an accompanying identified individual, this was recorded as unidentified solve. if further individuals visited before this happened, then a scrounge was recorded, as they were assumed to have taken food from the open door (confirmed from video observations). the door reset immediately after two individuals were registered scrounging, preventing more than two possible scrounging events per solve (supplementary video 2). two males were captured from each sub - population (11 adults, 5 juveniles) to act as demonstrators, either by removal from roosting boxes on sunday night, or by mist - netting at a sunflower - seed feeder on monday morning. they were transferred to individual cages in indoor captive facilities, and over four days each pair of birds was subjected to one of three training regimes using step - wise shaping, either : (i) given no training and left in the cage with ab lib food (control) ; (ii) trained to solve the novel puzzle - box by pushing the blue side of the door to the right (option b) ; or (iii) trained to solve the novel puzzle - box by pushing the red side of the door to the left (option a). with the exception of control areas, which were clustered in the south of the woodland to avoid cross - contamination, sub - populations were randomly assigned to a training regime, with both demonstrators from a single sub - population trained on the same technique. during training, the demonstrators were initially exposed to an open puzzle - box baited with mealworms, which was then gradually closed over the course of four days until the subjects were reliably re - opening it. the birds were released back at the site of capture in each respective sub - population ; puzzle - boxes at which both options were available and equally rewarding were installed at three sites 250 m apart on the following sunday night (extended data fig. these puzzle - boxes were run over a four - week period at each site, continuously operating from monday to friday and then removed on saturday and sunday, for a total of 20 days of data collection. four replicates were conducted in the first year of data collection (december 2012-february 2013 ; c1 - 2, t1, t3). at three of these replicates (c1, t1, t3) puzzle - boxes were simultaneously re - installed at the same locations for 5 days of further data collection in december 2013. no additional demonstrators were trained, and no individual had contact with the puzzle - box in the 9 months between the two data collection periods. this second exposure aimed to test the long - term stability of social learning at the sub - population level. they were run prior to the second year of data - collection for the cultural diffusion experiment in order to exclude the possibility that dispersing individuals from new replicates could be re - introducing the novel behaviour. an additional four replicates were then conducted from december 2013 - february 2014 in new sub - populations, using the same initial protocol (c3, t2, t4, t5). the local population size for each replicate was defined as comprising all individuals in a replicate that had been recorded at least once at either : (i) the puzzle - box, (ii) the nearby peanut feeder, or (iii) the nearest network - logger feeders (operated saturday - sunday, see below), during the experimental period (i.e. from the weekend following the release of the demonstrators, to the weekend after the 20 day of operation of the puzzle - boxes). persistence trial in the following year, the local population was defined just as (i) all individuals observed at the puzzle - box or (ii) nearby peanut feeder, so that areas were comparable. to analyse the results of the initial experiment we first compared control replicates and treatment replicates, using welch two - sided t - tests, and by fitting linear and sigmoidal models to the data, with the best model ascertained by difference in aic values. if individuals were using social information when learning about the puzzle - box, then we expected that there would be a difference between areas seeded with a trained demonstrator (treatment) and those without (control). replicates were thus compared in terms of latency to first solve (seconds from beginning of the experimental period, excluding demonstrator), and the total number of solutions. secondly, we compared the total number of solutions in the two different experimental treatments. here if a more complex form of social learning than local enhancement to the feeding site was occurring, then we expected a consistent bias towards the seeded variant in the different treatments. to analyse the change in individual and population preferences for option a or b over time, we used a generalised estimating equation model (gee) where the dependent variable was the proportion of solutions using the seeded technique on each day of data collection, and the explanatory variables were the individuals and replicate, weighted by the overall number of solutions per day. the seeded technique (a / b) was initially also included as an explanatory variable, but was not significant (coefficient se = 0.130.22, p = 0.55). three individual variables were included in a gee model ; sex, age and natal origin. sex was determined at capture using plumage coloration, age was either determined from breeding records or plumage coloration, and individuals were classed as immigrants if they had dispersed into the study site, and locally - born if they had been ringed as a nestling in the study site. only age was significant (coefficient se = 0.920.20, p < 0.001), and was included in the final model (sex : coefficient se = 0.380.22, p = 0.08 ; natal origin : coefficient se = 0.380.22, p = 0.08). if population - level conformity was partly the result of a conformist transmission bias at first acquisition we would expect a sigmoidal relationship between population - level frequency of the variant and adoption probability, with adoption of the majority variant disproportionately more likely than its absolute frequency. by contrast, copying the last individual observed, or random copying, should yield a linear relationship, with probability of adopting option a / b roughly equal to its proportion in the overall population. to investigate this, we isolated all individuals first observed solutions in all experimental replicates, and compared the option choice to the proportion of all previous solves as option a observed in the individual s group at that site. group length was set at 245 sec, which was the average group length observed using gaussian mixture models on temporal patterns of flocking (see below) at network - logging sunflower feeders. both linear and sigmoidal models were then fitted to the data, with the best model ascertained by difference in aic values. we further examined the subset of individuals that moved between sub - populations (n=40). this subset included all individuals recorded in more than one experimental replicate, whether within the season (n=16), or between seasons (n=24). no individual was observed in more than two replicates, and this analysis did not include individuals in the persistence trial. a preference for option a / b at each location was defined as more than 75% of all solves for either option a / b in that replicate. finally, in order to analyse the change in within - individual bias towards option a / b between the initial experiment and the second - year persistence trials, we used a general linear model where the dependent variable was the number of solves as the seeded variant over the total number of solves for each individual observed in both years. explanatory variables were treatment type and year, with individual identity as a random effect. sunflower bird - feeding stations were deployed at 65 locations around wytham woods on an approximate 250250 m square grid, as part of long - term research into social - network structure in tits (see). each station had two access points, each fitted with rfid antennae and data logging hardware. feeding stations automatically opened from dawn to dusk on saturday and sunday, scanning for pit - tags every 16 of a second. this study used the data from the eight nearest locations to each set of puzzle - boxes, for 10 dates within and surrounding the cultural diffusion experiment (the standard logging protocol runs from september - february in wytham woods). great tits were detecting visiting feeding stations and individually identified by their pit - tags. we then applied a gaussian mixture model to the spatiotemporal data stream to detect distinct clusters of visits. this method locates high - density periods of feeding activity, isolating flocks of feeding birds without imposing artificial assumptions about group boundaries. a gambit of the group approach was used with a simple - ratio index to calculate social associations, where individual association strengths (network edges) were scaled between 0 (never observed foraging together in the same group) to 1 (always observed in the same group, never observed apart). while a single co - occurrence may not be meaningful, our automated data collection method resulted in thousands of repeated group sampling events, allowing social ties between individuals to be built up from multiple observations of co - occurrences over time and across spatial locations. networks contained 123 (t1), 137 (t2), 154 (t3), 95 (t4) and 110 (t5) nodes ; average edge strength was 0.09 (t1), 0.05 (t2), 0.08 (t3), 0.07 (t4) and 0.07 (t5). to test whether networks contained significantly preferred and avoided relationships, we ran permutation tests on the grouping data, controlling for group size and the number of observations, restricting swaps within days and sites. we tested whether observed patterns of associations were non - random by comparing the coefficient of variance in the observed network to the coefficient of variance in the randomised networks. social networks for all replicates significantly differed from random, even at local scales (t1 : p<0.0001 ; t2 : p=0.0005 ; t3 : p<0.0001 ; t4 : p=0.0002 ; t5 : p=0.0002) finally, we used network - based approaches to ask whether the behaviour was socially transmitted through foraging associations. network - based diffusion analysis (nbda) tests for social learning by assuming that if social transmission is occurring, then the spread of trait acquisition should follow patterns of relationships between individuals, with transmission rate linearly proportional to association strength. we used nbda r code v.1.2, with the time of each individual s first solution (seconds since the beginning of the experiment) entered into the continuous time of acquisition analysis function. individuals that solved, but that did not appear in the social network (i.e. had not been recorded in the standardised weekend logging) were excluded from the analysis. the effects of three individual level variables were also incorporated into the analysis : sex, age, and natal origin. all combinations of nbda provided in the nbda r code v1.2 were run with social transmission rate allowed to vary for each replicate. total area of wytham woods is 385 ha ; location and size of the separate woodland areas within this are labeled on the map. (d) indicates locations where trained demonstrators were caught from and released to. a, feeding station (shut), with sunflower - feeder, rfid antennae, and data - logging hardware. stations are approximately 250 m apart and open simultaneously dawn - dusk on saturday and sunday over winter. c, grouping events are inferred from the temporal data stream gained from feeding stations, with individuals assigned to grouping events in a bipartite network. d, repeated co - occurrences are used to create social networks (adapted from psorakis. red nodes are individuals that acquired the novel behaviour after 20 days of exposure, black nodes are nave individuals and yellow nodes are trained demonstrators. networks are heavily thresholded to only show links above the average edge strength for each replicate (t1 - 5 : 0.09, 0.05, 0.08 0.07, 0.07). a, social network for t1 replicate (n=123). e, network for t5 replicate (n=110). only individuals that performed both options are included, and individuals that moved between replicates are excluded. lines are running proportions of seeded variant for each individual over its last 10 visits. a, t1 (option a), n=30 ; b, t2 (option a), n=10 ; c, t3 (option b), n=19 ; d, t4 (option b), n=4 ; e, t5 (option b), n=15. birds were presented with a freely available mix of 40 mealworms, peanut granules and sunflower seeds for 1 hr on 2 days over 1 week at 6 sites (3 sites in t4 and t2). trials were conducted 2 weeks after the end of the main experiment, in march 2014. food choice was identified from video camera footage, and the trial was halted when all of one prey item was taken. only great tits were included, but birds could not be individually identified. birds clearly preferred the live mealworms to either peanut granules or sunflower seeds. | in human societies, cultural norms arise when behaviours are transmitted with high - fidelity social learning through social networks1. however a paucity of experimental studies has meant that there is no comparable understanding of the process by which socially transmitted behaviours may spread and persist in animal populations2,3. here, we introduce alternative novel foraging techniques into replicated wild sub - populations of great tits (parus major), and employ automated tracking to map the diffusion, establishment and long - term persistence of seeded behaviours. we further use social network analysis to examine social factors influencing diffusion dynamics. from just two trained birds in each sub - population, information spread rapidly through social network ties to reach an average of 75% of individuals, with 508 knowledgeable individuals performing 58,975 solutions. sub - populations were heavily biased towards the technique originally introduced, resulting in established local arbitrary traditions that were stable over two generations, despite high population turnover. finally, we demonstrate a strong effect of social conformity, with individuals disproportionately adopting the most frequent local variant when first learning, but then also continuing to favour social over personal information by matching their technique to the majority variant. cultural conformity is thought to be a key factor in the evolution of complex culture in humans4 - 7. in providing the first experimental demonstration of conformity in a wild non - primate, and of cultural norms in foraging techniques in any wild animal, our results suggest a much wider evolutionary occurrence of such apparently complex cultural behaviour. |
oxatomide is an antihistaminic drug that has been reported to have applications in the treatment of a number of different types of allergic and other hypersensitivity reactions. these include but are not restricted to the symptomatic treatment of allergic rhinitis and chronic urticaria, the classical nasal and ocular symptoms associated with hay fever, the reduction of the severity of the erythema, and pruritus in cases of chronic urticaria [1, 2 ]. although oxatomide has been extensively investigated and approved for the use in the treatment of a broad range of diseases, it still possesses bioavailability limitations and poor dissolution properties that restrict its full clinical use. one of the major current challenges of the pharmaceutical industry is related to strategies that improve the water solubility of drugs [3, 4 ]. low solubility can cause low bioavailability or give rise to large fluctuations in the fraction absorbed in humans that can often not be compensated by a high permeability. furthermore, low solubility may be associated with stability problems and difficulties in developing an acceptable formulation. by improving the drug release profile of these drugs, it is possible to enhance their bioavailability and reduce side effects [69 ]. cyclodextrins are a family of cyclic oligosaccharides that are composed of -1, 4-linked glucopyranose subunits. these macrocyclic carbohydrates with lipophilic central cavities and hydrophilic outer surfaces can form complexes with and solubilize many normally water - insoluble compounds. the lipophilic cavity of cyclodextrin molecules provides a microenvironment into which appropriately sized nonpolar moieties can enter to form inclusion complexes. currently, -cyclodextrin is the most common, the most accessible, the lowest - priced, and generally the most useful cyclodextrin in pharmaceutical formulations [12, 13 ]. the purpose of study was to improve oxatomide water solubility by preparation of inclusion complex with -cyclodextrin and evaluate the influence of complex formation on the physicochemical properties and bioavailability of the drug. hydroxy propyl methyl cellulose (hpmc) and polyvinyl pyrrolidone k15 (pvp - k15) fluka, chemie, usa. an excess of oxatomide was added to screw - capped vials containing aqueous solutions of various concentrations of -cyclodextrin. after 2 days, aliquots were withdrawn and filtered using 0.45 m pore size filter. the filtrate was suitably diluted and analyzed spectrophotometrically at the wavelength of 226 nm. the same procedure was repeated with cyclodextrin aqueous solution containing either 0.25% w / v pvp - k15 or 0.1% w / v hpmc. oxatomide -cyclodextrin solid complex was prepared with equimolar ratio of both oxatomide and -cyclodextrin by using five different techniques as follows. the technique simply relies on the idea that both oxatomide and -cyclodextrin were properly blended together in a ceramic mortar using pestle. then, oxatomide was added slowly, and the combined mixture was kneaded with the addition of few drops of distilled water. the dough mass was pressed and stretched with the hand fingers, folded over, and rotated through 90, repeatedly. this process was continued for additional 15 min until the dough is elastic and smooth and then the obtained dough mass was left to dry under fume hood at room temperature for 24 hrs. coevaporation technique. simply it consists of the simultaneous deposition of the oxatomide and -cyclodextrin under vacuum conditions. this is simply done by dissolving weighed amount of oxatomide in 10 ml of 60% methanol solution in a glass beaker. in another glass beaker the contents of the two beakers are then mixed together, and the obtained solution is put in sonicator for 25 min at room temperature to obtain a clear solution. the resultant solution is dried by being subjected to evaporation using bchi rotavapor (r200v800, bchi labortechnik, switzerland) at 70c under vacuum (55 mbar). spray drying was performed in a bchi mini spray drier (b 290, bchi labortechnik, switzerland). this is simply done by dissolving weighed amount of oxatomide in 10 ml 60% methanol solution, in 100 ml glass beaker. in another 100 ml glass beaker the contents of the two beakers were then mixed together and sonicated at room temperature for 25 min to obtain and ensure a clear solution. the inlet adjusted to a flow rate of 880 ml / h and at temperature 120c. the nitrogen gas adapted to a flow rate of 357 nl / h u, and outlet temperature was 85c. freeze - drying technique. freeze - drying process was typically carried out in heto power dry ll 3000 freeze dryer (thermo electron corporation, czech republic) at 50c, under vacuum (0.128 mbar). briefly, in a 100 ml glass beaker, oxatomide was dissolved in 10 ml 60% methanol solution. in another 100 ml glass beaker, the contents of the two beakers are then mixed together, and the obtained solution is put in sonicator at room temperature for 25 min to ensure that a clear solution is obtained. the resultant solution is then frozen by placing at 30c freezer for at least 24 hrs to ensure that the solution was completely frozen. then, frozen solution was kept for 24 hrs into the vacuum chamber at 50c where the freeze - drying process takes place. oxatomide -cyclodextrin solid complexes were prepared with a molar ratio of 1 : 1 in addition to 21% of water soluble polymer such as pvp - k15, hpmc, or peg 6000 using coevaporation, spray - drying, and freeze - drying techniques. the oxatomide--cyclodextrin complex obtained from each technique ir spectra of oxatomide, -cyclodextrin, and the prepared oxatomide -cyclodextrin solid complexes were obtained at room temperature using an infrared spectrometer (ir100/ir200, thermo nicolet corp. the scanning range was 400 to 4000 cm, and the resolution was 4 cm. the thermal properties of oxatomide, -cyclodextrin, and the prepared oxatomide -cyclodextrin solid complexes were characterized using shimadzu differential scanning calorimeter, dsc60, japan. the measurements were carried out at a heating rate of 10c / min. in order to provide the same thermal history, each sample (1.2 to 1.8 mg) was heated from room temperature to 200c and rapidly cooled down to room temperature, then the dsc scan was recorded by heating from 30 to 200c. the measurements were carried out at conditions : ni - filtered cuk radiation, voltage 50 kv, current 30 ma, scanning speed 1 (2)/min, and investigating the samples in the 2 range 030. the dissolution studies of oxatomide powder and the prepared oxatomide -cyclodextrin solid complexes were performed according to the usp xxiii rotating basket method. the stirring speed was 50 rpm, and the temperature was maintained at 37 0.5c. at predetermined times intervals samples of 5 ml were withdrawn by using syringe filter (0.45 m millipore filter) and analyzed spectrophotometrically for oxatomide concentration at max 277 nm. the study was performed to assess the bioavailability of the suggested formula (oxatomide--cyclodextrin - pvpk15) selected on the basis of the dissolution studies in comparison with the reference oxatomide commercial product (tinset 30 mg tablets). the study was approved by the animal ethics committee of faculty of pharmacy, helwan university. sixteen new zealand white male rabbits (15 weeks old and weight 2.53 kg) were used in the study. the rabbits were divided randomly into two groups ; each was of eight rabbits and all rabbits were fasted overnight for 12 hrs with free access to water. on the day of experiment, each group received a single oral dose equivalent to 5 mg / kg oxatomide from each formulation by intragastric feeding tube. the doses of both products were suspended in a solution of 4.6% glycerin, 87.6% polyethylene glycol 400, and 7.8% distilled water. the blood samples were withdrawn from central ear artery 1 hr before the drug administration and after administration at specific time points (0.5, 1, 2, 4, 6, 8, 10, 12, 24, 36, and 48 hrs). the blood samples were centrifuged at 3,000 rpm for 10 min, and the plasma was transferred to separate glass tubes to be kept frozen until analysis. prior to analysis of plasma samples, aliquots of plasma (0.5 ml) spiked with 75 ng / ml flunarizine hydrochloride (internal standard) were vortexed for two min with (100 l 1 m) sodium hydroxide solution (100 l 1 m) and 5.0 ml ethyl ether. the samples were centrifuged at 2000 rpm for 10 min at room temperature. the upper organic layer was transferred to another centrifuge tube and evaporated to dryness under a gentle stream of nitrogen gas in a water bath at 40c. the residues were then redissolved in 200 l mobile phase under vortex and centrifuged at 18000 rpm for 8 min. aliquots of 5 l of the supernatant were injected into the liquid chromatography mass spectrometry (lc - ms system). plasma samples were analyzed for oxatomide concentration by using a validated lc - ms assay with some modifications. briefly, lc - ms analysis was performed on an agilent 1100 series lc / msd chromatographic system (agilent, usa) consisting of a water hplc system equipped with an on - line solvent degasser, binary solvent delivery system, autosampler, and an agilent technologies single quadrupole mass spectrometer with an electrospray ionization (esi) interface. the ms detector has electrospray capability with a mass range of m / z 503000 and a mass accuracy of 0.1 amu. a liquid chromatographic separation was achieved on a phenomenex c18 (250 2.0 mm i.d.) the mobile phase consisting of 85% methanol and 15% (v / v) aqueous ammonium acetate solution (10 mm, ph 4.0) was pumped at an isocratic flow rate of 0.2 ml / min. the [m+h ], m / z 427.10, for oxatomide and [m+h ], m / z 405.05, for flunarizine hydrochloride were selected as detecting ions, respectively. the ms operating conditions were optimized as follows : nebulizer gas rate, 1.5 l / min ; cdl temperature, 250c ; block temperature, 200c ; probe voltage, + 4.5 kv.. the lower limit of quantification was 5 ng / ml. the standard calibration curve for oxatomide was linear (correlation coefficients were > 0.9975) over the studied concentration range (5200 ng / ml). data acquisition and processing were accomplished using envirolab version 5 for the lc - ms system. the elimination rate constant (kel) was estimated by least square regression of plasma concentration - time data points in the terminal log - linear region of the curves. the area under the plasma concentration - time curve from zero to the last measurable plasma concentration at time t (auc0t) was calculated using linear trapezoidal rule. the area under the curve from zero to infinity, auc0, was calculated as auc0 = (auc0t) + ct / kel, where ct is the last measured concentration at the time t. peak plasma concentration (cmax) and the time to peak concentration (tmax) were obtained directly from the individual plasma concentration versus time curve. in order to compare the results student 's t - test (spss program ; version 12.0) was used. the phase solubility studies for oxatomide with -cyclodextrin were carried out according to the method described by higuchi and connors. the phase solubility diagrams of oxatomide in different concentration of -cyclodextrin in distilled water, 0.1% hpmc solution, and 0.25% pvp solution are presented in figure 1. the phase solubility profile of oxatomide -cyclodextrin complex showed a typical bs - type solubility curve, where the initial ascending portion is followed by a plateau region and then a decrease in total oxatomide solubility accompanied by precipitation of a microcrystalline complex. the values of ks were calculated according to the equation of higuchi and connors and from the initial straight line portion of the solubility diagrams by assuming that a 1 : 1 complex was initially formed according to the following equation : (1)ks = slope/[s0(1slope) ], where ks is the stability constant for the complex and s0 is the solubility of oxatomide in absence of -cyclodextrin. the ks value of oxatomide -cyclodextrin inclusion complex was found to be [1443 m ] in water and increased to [2027 m ] and [1388 m ] in presence of pvp - k15 and hpmc, respectively. ir spectra of oxatomide and its inclusion complexes with -cyclodextrin prepared by different methods are presented in figure 2. oxatomide is characterized by the absorption of the carbonyl (c = o) group at 1702 cm ; in the spectra of the inclusion complex, this band was shifted towards higher frequencies at 1727 cm in case of coevaporated solid complex and to lower frequencies at 1682 cm in spray - dried solid complex and decreased in intensity in freeze - dried complex. the absorption of n h group in oxatomide at 3172 cm was shifted toward lower frequencies at 29362924 cm in all types of solid complexes. as spectral changes always concern c oh, ch2, and ch groups of the -cd, it should be suggested that the host - guest interactions are dominated by hydrogen bonds among the above - mentioned groups. the thermal behavior of oxatomide -cyclodextrin inclusion complexes was studied using dsc to confirm the formation of the solid complexes. when guest molecules are incorporated in the cyclodextrin cavity or in the crystal lattice, their melting, boiling, and sublimation points usually shift to a different temperature or disappear within the temperature range at which the cyclodextrin lattice is decomposed. the dsc thermogram of -cyclodextrin shows a very broad endothermic peak around 95c corresponding to the release of water molecules. the thermogram of the physical mixture showed the melting endothermic peak characteristic of pure oxatomide and the broad peak corresponding to the dehydration of -cd as if this thermogram was the superposition of those components, indicating the absence of interaction between oxatomide and -cyclodextrin in such mixture. thermograms of oxatomide -cyclodextrin complexes prepared by kneading displayed a broad endothermic at 71.80c due to the dehydration of the complex, and the peak at 160c can still reflect the presence of a few drug crystals in the preparation. however, this thermal effect appeared more broadened and reduced in intensity, which suggests some drug - cyclodextrin interaction. in addition to changes in the broad peak of -cyclodextrin dehydration provided a further indication of the existence of inclusion complexes in these systems. however, the kneading and physical methods do not provide complete inclusion, and oxatomide was dispersed in the free state between inclusion complexes. the complete disappearance of the endothermic peak of pure oxatomide was observed in thermograms of freeze - dried, spray - dried, and coevaporated complexes and is attributed to the formation of an amorphous solid product or inclusion of the drug inside the -cyclodextrin cavity or both. the x - ray diffraction patterns of pure oxatomide, -cyclodextrin, and their physical and kneaded mixture, coevaporated, freeze - dried, and spray - dried inclusion complexes are represented in figure 4. the diffractograms of oxatomide and -cyclodextrin exhibited a series of intense peaks which are indicative of their crystallinity. the differences in the diffraction patterns of physical mixture, coevaporated, kneaded, freeze - dried, and spray - dried complexes from each isolated constituent seem to indicate the formation of a new solid phase as a result of formation of inclusion complexes which is in good agreement with dsc studies. furthermore, a reduction in peak height in diffraction pattern of the binary systems that was observed in all complexes indicated also a reduction in crystallinity. as illustrated in figure 5, it is clear that all inclusion complexes prepared by coevaporation, spray drying, and freeze drying exhibited higher dissolution characteristics than kneaded mixture, physical mixture, and pure oxatomide in distilled water. the percentages of drug release after 5 min (q5%), 90 min (q90%), % dissolution efficiency (% de) at 5 min (% de5 min), and 60 min (% de60 min) were determined and presented in table 1. the % de was calculated as the percent ratio of area under the dissolution curve up to time t (5 and 60 min), to that of the area of the rectangle described by 100% dissolution at the same time. regarding q5, the inclusion complexes prepared by spray drying, freeze drying, and coevaporation (figure 5) showed a prompt drug release compared to pure oxatomide (p 0.05) when analyzed student 's t - test. the individual auc0 values for oxatomide--cyclodextrin - pvpk15 complex were compared to those for the commercial product to determine the relative bioavailability. the mean relative bioavailability of oxatomide--cyclodextrin - pvpk15 complex to the commercial product was 173.15 16.53 %. this result indicated that 73.15% increase in the oral bioavailability of oxatomide was achieved by the complex formulation. although oxatomide -cyclodextrin complex improvesthe bioavailability of oxatomide in terms of extent of absorption from the gi tract, it did not change the rate of drug absorption in terms of tmax and achieved significantly lower cmax values in comparison with the commercial drug product. comparing the values of elimination rat constants (kel) for oxatomide--cyclodextrin - pvpk15 complex with those for commercial product reveals a marked effect of complexation on increasing the duration of action of oxatomide. the results reported here suggest that inclusion complex of oxatomide with -cyclodextrin prepared by coevaporation in presence of pvp - k15 results not only is an enhancement of the oxatomide dissolution rate but also improvesthe bioavailability of oxatomide in terms of extent of absorption from the gi tract and increases the duration of action of oxatomide. | the objective of this study was to evaluate the influence of oxatomide -cyclodextrin inclusion complex on the physicochemical properties and bioavailability of the drug. oxatomide -cyclodextrin solid complex was prepared with equimolar ratio of both oxatomide and -cyclodextrin in presence or absence of water soluble polymers using different techniques. the coevaporated complex prepared in presence of pvp - k15 showed a prompt drug release and significantly increased % dissolution efficiency (p < 0.05) compared to the pure oxatomide. moreover, the results of bioavailability evaluation of this complex in rabbits compared to commercial drug product indicated a 73.15% increase in the oral bioavailability of oxatomide. in conclusion, inclusion complex of oxatomide with -cyclodextrin prepared by coevaporation in presence of pvp - k15 not only results in an enhancement of the oxatomide dissolution rate but also improves the bioavailability of oxatomide. |
in january 2015, two sisters aged four (sibling 1) and six (sibling 2) with asymptomatic, consanguineous (first cousin) parents were referred to our ophthalmology department for evaluation of bilateral high astigmatism. both siblings suffered since birth from poor vision in both eyes and photophobia since birth. on ophthalmological examination, autorefractometer measurements for sibling 1 and sibling 2 were od 3.75 2 179/os 3.75 2.75 177 and od 2.25 7.75 166/os 3.75 5.75 174, respectively. reliable visual acuity measurements could not be obtained in sibling 1, whereas best - corrected visual acuity for sibling 2 was 20/32. blue sclera and mild globular protrusion of the cornea were observed bilaterally in both siblings. slit - lamp examination of the cases revealed that both corneas were globular in shape with peripheral corneal thinning. corneal topography and pachymetry maps were obtained using a scheimpflug imaging (pentacam, oculus optikgerte gmbh, wetzlar, germany) [fig. 1 ]. maximal keratometry (kmax) indices for sibling 1 and sibling 2 were 52.9 d (od)/53.5 d (os) and 54.4 d (od)/55.4 d (os), respectively. the corneal thickness values for sibling 1 and sibling 2 were 241 (od)/291 (os) and 286 (od)/305 (os), respectively. corneal topographic images of the left eye of sibling 1 showing limbus - to - limbus corneal thinning the two siblings had in common the features of keratoglobus, blue sclera, atypical faces, hearing loss, and hypermobile joints [fig. 2 ]. therefore, they were referred to the genetic department for systemic evaluation. on general assessment both siblings had flat midfaces, large eyes, frontal bossing, mild hypertelorism, calvarial thickening, and absent frontal sinuses, suggesting a diagnosis of marshall syndrome [fig. 3 ]. these siblings had also mandibular hypoplasia, hyperextensible joints, and metaphyseal widening of long bones, suggesting a diagnosis of stickler syndrome. otoacoustic emission test was performed to evaluate hearing loss and was found abnormal in both siblings. other detailed clinical characteristics of marshall syndrome, stickler syndrome, and our cases are presented in table 1. we tentatively diagnosed the sisters as having an overlapping marshall / stickler phenotype based on clinical and radiological findings. frontal and profile views of sibling 1 (top) and 2 (bottom) absence of frontal sinus is seen in x - ray graphy of sibling 2 clinical features of marshall syndrome, stickler syndrome, and overlapping phenotype of our cases written informed consent was taken for photography and genetic analysis. we could study only the exon 49 and exon 52 regions of the col11a1 gene, and the results were negative. protective eyeglasses were prescribed for both siblings, and avoidance of contact sports was highly recommended owing to the high risk of perforation. to our knowledge, this is the first report describing congenital keratoglobus with blue sclera in two siblings with overlapping marshall - stickler phenotype. in the past, it is proposed that the existing phenotypic overlap suggests that marshall and stickler syndromes are probably allelic expressions of the same locus. although jun. demonstrated the expression of the col11a1 gene in human donor corneas, corneal disease associated with marshall - stickler syndrome due to col11a1 mutation has not yet been reported. the altered collagen due to col11a1 mutation may weaken collagen fibers in the cornea, which could play a role in the development of keratoglobus in these cases. furthermore, thinning and breakdown of the collagen is the main histological finding in cases of blue sclera. in practice, there are difficulties in obtaining whole genome analysis due to size and complexity of the col11a1 gene and also to the high cost of these investigations. only the exon 49 and exon 52 regions of the col11a1 gene were identified and found to be mutation negative. furthermore, we could not screen the col2a1 gene in relation to some forms of stickler syndrome. khalifa. pointed out that dominant and recessive mutations in the col11a1 gene cause marshall and stickler syndromes. in this report, our cases were the offspring of unaffected consanguineous parents, suggesting autosomal recessive inheritance. our cases also showed bilateral involvement. in the literature, keratoglobus with blue sclera was reported in association with connective tissue disorders. these disorders are ehlers - danlos type vi, marfan syndrome, rubinstein taybi syndrome, and osteogenesis imperfecta. until now, keratoglobus with blue sclera has not been reported in association with marshall / stickler phenotype. as a connective tissue disorder, marshall - stickler syndrome might be in the differential diagnosis of keratoglobus with blue sclera. 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. | we aimed to describe congenital keratoglobus with blue sclera in two siblings with overlapping marshall / stickler phenotype. two sisters (ages four and six) with bilateral high astigmatism were evaluated by slit - lamp microscopy. corneal topography and pachymetry maps were also obtained. slit - lamp examination revealed that both corneas were globular in shape with peripheral corneal thinning. pachymetry maps showed diffuse corneal thinning. two siblings had in common the features of keratoglobus, blue sclera, atypical face, hearing loss, and hypermobile joints. we tentatively diagnosed the sisters as having an overlapping marshall - stickler phenotype based on clinical and radiological findings. marshall - stickler syndrome may exist in the differential diagnosis of keratoglobus with blue sclera. |
andropause, or more accurately, testosterone deficiency syndrome (tds) is one of the new issues which can affect the quality of life in older men. it is a natural phenomenon that occurs with age in men and is diagnosed by clinical manifestation and a decrease in serum testosterone levels ; it affects the performance of many systems of the body from the head to toe. in an attempt to alleviate the scarcity of descriptive epidemiology of androgen deficiency, the massachusetts male aging study (mmas) reported a crude incidence rate of 12.3 per 1000 person - year, leading to the prevalence of 481000 new cases of andropause per year among american men aged 40 to 69 years old. this age - related decline starts in the third decade of life and progresses slowly. in men, this process shows a significant inter - individual variability in the age of the onset and speed and depth of the decline. various terms used to describe this phenomenon include male menopause, male climacteric, viropause, androgen deficiency in aging males (adam), partial androgen deficiency in aging male (padam), and recently late - onset hypogonadism (loh), symptomatic late - onset hypogonadism (sloh), or more accurately, testosterone deficiency syndrome (tds). testosterone is the principle androgen hormone responsible for the primary and secondary male sex characteristics ; thus, the lowered testosterone levels will lead to alteration in sexual and psychological function and body composition. these changes comprise mood swing, depression, poor concentration and memory, anxiety and irritability, sleep problem, loss of libido, erectile dysfunction, reduced muscle mass and strength, loss of body hair, hot flushes, excessive sweating, lack of motivation and decline in general well - being. results of some studies showed that there was a relationship between geriatric syndrome such as osteoporosis, cardiovascular disease and andropause. although andropause can be diagnosed through symptoms but not sufficiently, for accurate results laboratory tests should be performed to determine the decrease in testosterone levels. from different experimental values of testosterone, bioavailable or free testosterone level treatment of andropause is compromised of changes in lifestyle, exercise, diet and testosterone replacement therapy. testosterone replacement therapy is indicated to restore testosterone levels to normal and correct the features of androgen deficiency. with this increase in aged male population and life span in iran and focus on quality of life, andropause will become a major health issue that needs to be addressed in order to prevent disability and morbidity. in addition, andropause is a new phenomenon in scientific research which has recently attracted the experts attention but despite its importance for the early diagnosis and treatment, little information is available in the books and journals about the issue. community health nurses, as people who work in different levels of society, must participate actively in the three levels of prevention. one of the most important roles of nurses is to assess the training need of the community for health promotion. so primary care physicians, as the first - line health care providers, are probably the best ones to evaluate the andropause syndrome in aged clients and with early detection and timely treatment they can reduce the cost of health care and complications of andropause. in addition, physicians will be able to transfer their information about andropause to other health care professionals, specially nurses to increase their knowledge and attitude and then health promotion. the results of studies done in different parts of the world indicate that the level of awareness and knowledge about andropause was low in health care professionals such as primary care physicians but in iran correct data about this are not available. also, in the study done to assess the impact of socio - demographic factors on knowledge and attitude towards andropause among health professionals in ile - ife, nigeria, results showed that 23% of the participants looked with optimism toward andropause. generally, the pcps knowledge of andropause was satisfactory but there were clear gaps as to the depth of knowledge and understanding of the control and treatment of this condition. overall, the first step in health education is to analyze the knowledge and attitude of the subjects to determine the next steps in planning and implementation of training programs about andropause, improve men s health, and promote knowledge and attitude of gps ; this study was conducted to reach this goal. this is a cross - sectional study that began in february 2014 and the data collection was performed within 4 months. the study subjects consisted of all male gps working in hospitals, health centers, offices and clinics affiliated to shiraz university of medical sciences. research has shown that general practitioners as the first level of health care provider are socially and professionally in the best position to assess the andropause period. with regard to the purpose and the type of this study and consideration of assumptions including estimates of error of 5% and 95% confidence, the following formula was used to calculate the sample size of the this study. due to the large population of general practitioners in shiraz and with regard to the fact that validity and reliability of our questionnaire was not reported in previous studies, for each question thus, a total of 215 to 430 people could participate in the study but for more accurate results a sample size of 402 practitioners was considered. generally, there are 31 public and private hospitals in shiraz ; among them, 10 public and 8 private hospitals were randomly selected. then in each hospital, according to the list of general practitioners working in the center, samples were selected randomly. inclusion criteria were willingness to participate in the study, filling out the consent form and at least 2 years of experience in practice. the exclusion criteria included being a student in the residency period and trained in andropause. the instrument used was a self - administered questionnaire developed based on the literature review and sources available on andropause ; it was designed to find out the level of knowledge and attitude about andropause. the first part was about demographic characteristics of the participants, and the second and third sections focused on the respondents knowledge and attitude of andropause, respectively. 27 questions were about knowledge scored based on 1 point for each correct answer and a total score of 76. the scores between 55 - 76 demonstrated good knowledge, those between 31 and 54 represented moderate knowledge and those between 30 and below indicated poor knowledge. 15 items were about attitude scored based on 3-point likert scale with a maximum score of 3, minimum score of 1 and a total score of 45 for men over 40 years and 36 for men under 40 years of age (the difference in scores is due to the fact that 3 questions were only for men over 40 years). in order to determine the content validity of the questionnaire, they were investigated by 10 faculty members of nursing and midwifery, urologists and statisticians in shiraz. also, to determine the face validity of the questionnaire, experts comments were used. the results showed that the content and face validity of the questionnaire were appropriate. in order to assess the reliability, a pilot study was done ; the reliability was confirmed by cronbach s alpha of 0.723 for the part on knowledge and.68 for the part on attitude. then, the study was done at a broader level, the questionnaires were filled out by 402 general physicians and cronbach s alpha was re - calculated. the reliability was confirmed by cronbach s alpha of.895 for the part on knowledge and.839 for the part on attitude, shown by increasing the number of the samples as well as increased level of reliability. after determining the validity and reliability of the questionnaire, the researcher attended the workplace of the gps to fill out the questionnaires. in order to increase the participants confidence, explanations were provided about the purpose of the study, voluntary participation and completion of the informed consent. after the questionnaires were distributed among the volunteers, the complete questionnaires were collected as soon as possible, preferably on the same day or in the following days. also, the data obtained from the questionnaires were analyzed using spss 18. according to the type of variables, pearson correlation, anova and descriptive statistics were used. of 402 gps participating in the study, 69.60% were family doctors and the rest were general practitioners. most of the participants were in the age group 40 - 49 years (37.81) and the lowest percentage of participants was in the age group 60 years and above (3.73). most of the participants were employed in the public sector (49%) and the lowest percentage of participants was employed in both private and public sectors (4.97%). in terms of work experience of the participants, most of gps had a work experience about 10 - 14 years (25.62 %) and the minority had a work experience of over 20 years (12.43%). the lowest average scores were related to the premature andropause (1.851.13) and the highest mean scores were related to treatment and control of andropause (10.154.48). the overall mean score of total knowledge was 29.4312.69 out of 76, indicating the poor knowledge of gps about andropause. table 1 shows the means and standard deviations of different dimension of gp s knowledge about andropause. if the physicians knowledge scores, based on the number of correct answers, are divided by 3 categories, 55.2% of the physicians had low level of knowledge, 41.3% moderate, and only 3.5% had a high level of knowledge. of the 402 gps participating in this study, 48.8% had heard of andropause and among them familiarity with different terms used to describe the condition of low testosterone in men was widely varying. the most recognized terms were male climacteric (31.4%), adam (23.46%), andropause (23.46), tds (14.07%), padam (6.13%) and sloh with 3.24%, respectively (figure 1). mean and standard deviation of general physicians knowledge frequency distribution of hearing the different terms. androgen deficiency in aging males ; partial androgen deficiency in aging male ; symptomatic late - onset hypogonadism ; testosterone deficiency syndrome of 48.8% of the subjects who had heard of andropause, the main source of information was the colleagues, friends (13.4%) followed by continuing medical education (10.7%), advertisements in health media (9.5%), print media (9.2%), and major medical meeting (6.2%). the results of analysis of variance (anova) showed that there was a significant relationship between occupational status and knowledge about andropause (p<0.001) ; it means that gps employed in the public sector gained higher score of knowledge. the mean score of the gps attitude about andropause was 35.164.48 out of 45, reflecting a positive attitude toward andropause. both age groups, younger than 40 and over 40 years, obtained high scores which reflects positive attitude about andropause (table 2). mean and standard deviation of general physicians attitude in addition, there was a statistically significant relationship between attitude and demographic characteristics (p<0.05) ; it means that with an increase in professional history, in married persons, in age group older than 40 years and in participants employed in both the private and public sectors, the andropause attitude grew better. table 3 shows meansd of general physicians attitude according to demographic characteristics and the relationship between mean score of attitude and characteristic. meansd of general physicians attitude according to demographic characteristics and the relationship between mean score of attitude and characteristics using anova pearson correlation coefficient showed that there was no relationship between the mean scores of knowledge and attitude toward andropause (p=0.548). this means that by increasing the level of knowledge, attitudes did not rise and vice versa. this was a cross - sectional study performed to assess the level of the knowledge and attitude about andropause among gps of shiraz in 2014. the results showed a low level of knowledge about andropause among gps (the overall mean score of total knowledge was 29.4312.69 out of 76). in this regard, several studies have obtained similar results. in the study done to investigate the level of knowledge, attitudes and practices of health personnel in larestan regarding andropause, the result showed that the mean scores of knowledge, attitudes and practice in relation to andropause among nursing staff were significantly lower in contrast with those of practitioners. the mean scores of knowledge and practice between nursing staff and practitioners were significantly different (p<0.001) but those of attitude did not differ between the two groups (p=0.84). results of the study conducted to assess the awareness and perception of androgen deficiency of aged males (adam) among men in osogbo, nigeria indicated that a large percentage of men had poor awareness about andropause. among those who had good awareness about this condition another study was done in north india to assess the awareness of men about andropause ; the results showed that their information about this condition and its treatment is low. another study conducted to investigate the awareness and knowledge of andropause among chinese males in hong kong showed poor knowledge with the mean knowledge score of 5.94. also, in a study performed in victoria, bc, canada to determine the level of knowledge of general practitioners about andropause, it was found that a large number of participants had heard the term andropause (96.3%). of the physicians who completed the survey, 92.6% agreed that men experience sometimes similar to women s menopause when they age. of 402 gps participating in this study, 48.8% had heard of andropause and among them the most recognized term was male climacteric (31.4%). in this study a very high percentage of physicians had heard of male climacteric (97.6%) and andropause (96.3%). colleagues and friends (13.4%) were the major source of andropause information among the gps. this finding was different from the results in canada, reporting that continuing medical education was the primary sources of information. some studies have reported a general lack of knowledge about andropause by both male population and health care providers, especially male gps ; however, gps are the first level of health care provider and in our country they play the role of family physicians and in contact with patients who are frequently consulted, but they are not prepared academically to make the right decision there was a significant relationship between occupational status and knowledge about andropause (p<0.001). this finding was different from the results in nigeria reporting that occupational status of the respondents do not have significant effects on knowledge about andropause (p=0.415). the mean score of the gps attitude about andropause was 35.16424.48082 out of 45 ; this reflects a positive attitude toward andropause. in this regard the results of this study showed that the practitioners attitude about andropause was high (84.2 percent) ; they believed that andropause significantly affects the quality of life. a study conducted in netherlands found similar results. in this study, they indicated that seven key domains (energy, emotional, social, social emotional, mental functioning, physical functioning and sexual functioning) should be considered when assessing the impact of andropause. they found that understanding the impact of low testosterone levels on qol is critical to diagnosis and effective treatment of andropause. but this finding contradicted the results of the study done in changzhi city which was investigation of male residents understanding and attitude toward andropause syndrome. it was shown that they had relatively negative attitudes regarding andropause ; this could be due to differences in the study subjects and their demographic backgrounds in these two studies. a major concern is that many men do not accept the concept of male menopause. a high proportion of men inadvertently deny the presence of these changes, especially sexually related ones, and have negative attitude toward andropause. in the past however, with advancement of science and numerous investigations in this field, gp s knowledge is increasing and with increased knowledge, more positive attitude results. a positive attitude for physicians is very important because strengthening the attitude and proper training of physicians by improving the quality of their practice, diagnosis and treatment reduces the complications of andropause in the community, influences the attitude of other men, and assists them in creating a positive attitude because more exposure and discussion about andropause can help them to accept this condition. there was a statistically significant relationship between attitude and demographic characteristics (p<0.05) ; that is, with an increase in professional history, in married persons, in age group older than 40 years and in participants employed in both private and public sectors, the andropause attitude grew better. in this regard, a study done in jakarta investigated the proportion and acceptance of andropause symptoms among the elderly men, finding similar results which showed that marriage status can affect the acceptance of andropause (p=0.031). in addition, in a study done in iran similar results were found in this regard. this study showed a statistically significant positive correlation between professional records and attitude about andropause, i.e. the subjects with higher professional records had a better attitude towards andropause (p=0.04). but the result of the study done in nigeria showed no significant relationship between the mean score of attitude and age of the participants. there was no statistically significant relationship between the mean score of knowledge and that of attitude about andropause (p=0.548). limitation of this study include lack of cooperation of some gps due to high workload and distribution of study subjects in different parts of the city, but through explaining the importance of andropause and by the help of the researcher they were resolved almost. the overall results showed that gps have a low level of knowledge and positive attitude toward andropause ; thus, there is a need for improved continuing medical education programmers to give gps the skills to diagnose and manage andropause. recommendations from the results of this study consist of planning for courses and educational interventions during the service for gps about andropause, holding integration programs of health care professionals and the media working together to educate the men, who are the target population, about the probability of men experiencing andropause, establishing special clinics for andropause in health centers and holding internal and external meetings to exchange information and use the experience of others about andropause. | background : andropause in men refers to the clinical and biochemical syndrome associated with advanced age and characterized by a deficiency in serum testosterone levels. with the increase in aging male population and life span in iran and focus on quality of life, andropause will become a major health issue that needs to be addressed in order to prevent disability. the results of some research have shown that there is still low level of knowledge and attitude toward andropause among health professionals. this study aimed at assessing the level of knowledge and attitude of general physicians regarding andropause in 2014.methods:this cross - sectional study was carried out on 402 general physicians in shiraz. a researcher - made questionnaire was developed for assessing the level of knowledge and attitude of general physicians about andropause. spss 18 was used to analyze the data, and descriptive statistics, anova and pearson correlation were applied for data analysis.results:the mean score of knowledge and attitude about andropause was 29.4 out of 76 and 35.1 out of 45, respectively. the findings showed a poor level of knowledge and positive attitude toward andropause among general physicians. there was a significant relationship between occupational status and knowledge about andropause (p<0.001). there was a statistically significant relationship between attitude and demographic characteristics (p<0.05). the correlation between knowledge and attitude toward andropause was not statistically significant (p=0.548).conclusion : the findings of the present study indicate the need for designing educational interventions to improve the knowledge and attitude of andropause among general physicians. |
the latest advances in ultrasonic measurement techniques have helped indentify that 20~30% women have polycystic ovaries (pco). the incidence of pco is up to 34% for those women diagnosed by reproductive specialists. a subpopulation of these pco cases is ovulatory pco, in which women do not display any typical symptom of the polycystic ovary syndrome (pcos) although they do have pco. some ovulatory pco patients reportedly show mild endocrine abnormalities including high levels of luteinizing hormone (lh) or androgen as well as insulin resistance, which are similar to those experienced by pcos patients [46 ]. however, a recent study demonstrates that isolated pco is an age - dependent, normal finding among ovulatory women that has no pathologic or clinical significance. to date, numerous studies have extensively investigated the in vitro fertilization and embryo transfer (ivf - et) in pcos patients [813 ], whereas little information is available for ivf - et in ovulatory pco patients [1417 ]. the aim of the present study was to directly address this deficiency in the literature and compare the ovulation and treatment of ivf - et among patients suffering from pcos, ovulatory pco, or normal ovaries with other complications (no). patients receiving ivf or intracytoplasmic sperm injection (icsi) treatment at the reproductive medicine center of the first affiliated hospital, sun yat - sen university, between january and september 2010, were selected for analysis. patient information was documented in details, including age, weight, menstruation history, pelvic ultrasound examination results, and basal hormone levels. patients with endometriosis or any other endocrine complications such as thyroid dysfunction, hyperprolactinemia, cushing syndrome, or atypical congenital adrenal hyperplasia were excluded from the study. based on the diagnosis, patients were divided into three groups : pcos (122 patients, 122 cycles), ovulatory pco (208 patients, 208 cycles), and normal ovaries (660 patients, 660 cycles). there were 122 pcos patients diagnosed according to the rotterdam standard who had a total of 122 ivf / icsi cycles, oligo - ovulation was defined as a menstrual cycle of longer than 35 days ; hyperandrogenism was diagnosed with either clinical or biochemical profiles ; pco was defined as the presence of 12 or more follicles in each ovary measuring 29 mm in diameter, or increased ovarian volume larger than 10 ml. moreover, 208 ovulatory pco patients had 208 ivf / icsi cycles, which were diagnosed with a regular menstrual cycle (2135 days), no clinical or biochemical profiles of hyperandrogenism and the presence of 12 or more follicles with a diameter of 29 mm in either side of the ovary, and/or total ovary volume 10 ml. in addition, 660 patients with normal ovaries had a total of 660 ivf / icsi cycles were recruited as controls, they all exhibited normal ovarian morphology and regular menstrual cycles. causes of infertility for these patients include tubal blockage, pelvic adhesions, and/or male factor.. briefly stated, long - acting gonadotropin - releasing hormone agonist 1.0 mg or 1.3 mg was intramuscularly administered in the midluteal phase (gnrh - a, diphereline, france). following pituitary downregulation, patients were administered gonadotropin (gn, gonal - f, switzerland) from the 3th-5th day of the menstrual cycle. the initial gn dose was determined by a variety of factors, including age, number of ovarian follicles, basal fsh levels, and history of ovarian response. during the ovarian stimulation, transvaginal ultrasound and serum sex hormone levels were monitored to evaluate the development of ovarian follicles, and the gn dose was adjusted based on the intensity of ovarian response. when more than (and including) 2 ovarian follicles with the diameter 18 mm, or more than (and including) 3 ovarian follicles with the diameter 17 mm, were detected, patients were injected with 10,000 iu human chorionic gonadotropin (hcg, switzerland) to trigger oocyte maturation. oocytes were collected about 36 hours later. the fertilization protocol (regular ivf or icsi) was determined by the condition of semen on the same day as the oocyte extraction. embryo transplantation was determined by the number of oocytes, the estradiol (e2) level, and the patient conditions. no more than three embryos were implanted into the uterine cavity three days after the oocyte extraction. if the hcg test was positive, ultrasonic examination was conducted two weeks later to determine the clinical pregnancy. a blood test was performed on the 2nd-5th day of the menstrual cycle before the treatment to determine the levels of fsh, lh, e2, and testosterone (t). the pcos and ovulatory pco patients received transvaginal ultrasound before the ivf treatment to examine the pelvic conditions. all ovulatory pco patients and some of the pcos patients exhibited the symptoms of polycystic ovary. during treatment, patient information was documented, including age, body mass index (bmi), duration of gn administration, total gn dose, e2 levels on the hcg day, endometrial thickness on the hcg day, numbers of collected oocytes, fertilization rates, oocyte cleavage rates, numbers of available embryos and transferred embryos, hcg positive rates, numbers of gestational sacs, incidence of spontaneous abortion, incidence of ovarian hyperstimulation syndrome (ohss), and incidence of cycle cancellation. the ohss classification was based on the expert consensus on diagnosis and treatment of polycystic ovarian syndrome standard set by the endocrinology group of the obstetrics and gynecology society, chinese medical association (cma). the group differences were analyzed by analysis of variance (anova) or rank test for quantitative data and chi - square test for qualitative data. compared to the no patients, patients with pcos or ovulatory pco were younger (p < 0.01). the pcos patients exhibited the highest bmi as well as basal lh and t levels (p < 0.01), whereas the no patients exhibited the highest basal fsh levels (p < 0.01) (table 1). compared to the no patients, patients with pcos or ovulatory pco exhibited a significantly shorter stimulation duration and lower gn dose but showed much higher e2 levels on the hcg treatment day and more achieved oocytes (p < 0.01) (table 2). the fertilization rate of the pcos patients was significantly lower than the other two groups. the pcos and ovulatory pco patients exhibited similar lower cleavage rates, but more available embryos. we did not observe any significant differences in the numbers of transplanted embryos, pregnancy rates, clinical pregnancy rates, or implantation rates among the three groups. the pcos patients exhibited a higher miscarriage rate (17.5%), although the difference was not significant. the incidence of moderate to severe ohss was 15.8% and 11.1% for the pcos and ovulatory pco patients, respectively, which was significantly higher than that of the no patients (5.4%, p < 0.01) (table 3). besides moderate to severe ohss, cycle cancellation was also a notable adverse outcome of the ivf treatment. cycle cancellation could be classified into three types : (1) the cycle was canceled during the process of ovarian stimulation because of poor ovarian response ; (2) the embryo transfer was canceled due to excessive numbers of oocyte achieved, high e2 levels on the hcg treatment day, and/or showing symptoms of overstimulation, including abdominal distension and apparent pelvic fluid ; (3) other complications, including oocyte collection failure, abnormalities in the oocyte fertilization and/or cleavage, poor embryo quality, and/or endometrial factors. in the pcos, ovulatory pco, and no patients, the rates of cycle cancellation due to poor ovarian response were 1.6% (2/122), 0% (0/208), and 2.7% (16/660), respectively ; the rates of embryo transfer cancellation were 18.0% (22/122), 22.1% (46/208), and 4.9% (32/660), respectively ; the rates of other causes were 1.6% (2/122), 1.0% (2/208), and 2.4% (16/660), respectively. thus, the overall rates of cycle cancellation for these three groups were 21.2% (26/122), 23.1% (48/208), and 10.0% (64/660), respectively. our results suggested that compared to the no patients, the pcos and ovulatory pco patients exhibited significantly higher rates of embryo transfer cancellation and overall cycle cancellation (p < 0.05). contrary to some reports [46 ], in the current study, we found that bmi and levels of lh and androgen were similar between the ovulatory pco and no patients, which were significantly higher than those in the pcos patients. insulin - sensitizing agent metformin which has been examined as a cotreatment during ivf in women with pcos, brought an increase in pregnancy rate and a significant reduction in rates of ohss, however, for women with pco but no other manifestations of pcos, metformin cotreatment before and during ivf did not bring any positive effect in clinical pregnancy, live birth, or severe ohss. the most likely explanation is that women with pco may be less insulin resistance compared with women with pcos. while some women with pco to share some endocrinological abnormalities with those with pcos, the difference between these studies may be attributed to variations in categorizing patients. according to the widely used rotterdam diagnostic criteria for pcos, thus, pco and pcos are not necessarily related to each other. in this study, the ovulatory pco patients did not have the endocrine and metabolic abnormalities manifested by the pcos patients, indicating that polycystic ovary might only represent some normal variation in the ovarian morphology. all patients in the present study were treated with standard protocol of long - term gnrh - a administration in midluteal phase, which was commonly used for infertility women including pcos patients. in addition to its well - known advantages such as easy to operate and satisfactory pregnancy rates, for pcos patients, gnrh - a can effectively reduce the lh / androgen level and inhibit the inflammatory factors, thus may improve the quality of the oocytes and embryos as well as endometrial receptivity. compared with the pcos patients treated with oral contraceptive pill [ocp ] alone, patients treated with ocp plus long - term gnrh agonist exhibit lower hormone levels and better amelioration of clinical symptoms [810 ]. in addition, the fertilization rates and pregnancy rates of gnrh - a - treated pco patients were similar to those of the non - pco patients. therefore, based on the current findings, clinical experience, as well as our findings in the present study, we propose that gnrh - a long - term protocol is suitable strategy for the pcos patients treated with ivf. our study showed that both the ovulatory pco and pcos patients displayed high - ovarian responsiveness, which was consistent with previous research. compared to the no patients, the ovulatory pco and pcos patients exhibited reduced duration of ovarian stimulation and total gn dose, as well as increased e2 levels and number of collected oocytes. previous studies have suggested that compared to other infertility patients, pcos patients exhibited a higher degree of ovary vascularization during the process of ovarian stimulation, which paralleled with vascular endothelial growth factor [vegf ] levels in the serum and follicular fluid. the ovary vascularization and vegf levels were positively correlated with the e2 levels and numbers of oocytes achieved [11, 22 ]. coffler. suggest that in pcos patients, follicular granulosa cells can show two types of response to fsh : (1) pcos patients show remarkably higher e2 levels in response to fsh above the threshold dose ; (2) pcos patients treated with a single fsh stimulation exhibit much faster rise and decay of the e2 levels, which were much different from the response of the control patients. however, serum fsh levels of both groups were similar, a phenomenon which is probably explained by the fact that most follicular granulosa cells have either apoptosed or can not effectively respond to fsh. based on previous and current findings, we conclude that both pcos and ovulatory pco patients are sensitive to gn stimulation, thus it is difficult to determine an appropriate gn dose for each individual patient. in our study, although we gradually reduced the gn dose to avoid such overstimulation, the follicle degeneration and atresia were still observed in some patients, which were also reported by coffler.. compared to the no patients, the pcos but not the ovulatory pco patients showed lower fertilization rates, whereas both groups exhibited a significantly lower oocyte cleavage rates and more available embryos. thus, we further investigated whether the lower fertilization rates of the pcos patients were due to abnormal oocyte morphology or changes in cytosolic factors which could affect the quality of the oocytes. previous studies report that the morphology of the cytoplasm and extra cellular matrix of the oocytes and embryos were similar among the pcos, pco, and no patients, indicating that the affected fertilization and early embryonic development of the pcos and/or pco patients are not because of intrinsic abnormalities of the oocytes. endocrine disorders and internal oocyte abnormalities can also result in low rates of oocyte fertilization and cleavage. for instance, high lh levels and insulin resistance can cause the malfunction of follicular granulosa cells and the abnormal expression of gdf-9, both of which markedly reduce the quality of the embryos [3, 14, 25 ]. our results also demonstrated that the ovulatory pco and pcos patients exhibited similar rates of pregnancy, clinical pregnancy, and implantation. although the higher average age of the no patients might be a confounding factor for the analysis, we still considered that the pregnancy rates of the ovulatory pco and pcos patients were satisfactory. our observations were consistent with previous studies by esmailzadeh., kim., swanton., and esinler.. in esmailzadeh., the fertilization and pregnancy rates of the ovulatory pco patients treated with gnrh - a long - term protocol were similar to those of the non - pco patients. similar outcomes (duration of stimulation, dose of gn used, e2 level on hcg day, number of retrieved oocytes, rates of implantation, clinical pregnancy and miscarriage, any incidence of severe ohss) were observed in patients with pcos and sonographic pco - only for ivf - et treatment. live birth rates are similar among women with pco (38%), pcos (37%), and normal ovaries (40%). severe ohss rates were significantly higher in women with pco (12.6%) and pcos (15.4%) compared to those with normal ovaries (2.7%). in the study of esinler., the satisfactory pregnancy rates of the ovulatory pco and pcos patients were due to sufficient numbers of collected oocytes, fertilized oocytes, and transferred high - quality embryos. pcos patients exhibited the highest abortion rate, although no significant difference was detected, which could be explained by small sample size and age variation. whether this high risk of abortion is related to the quality of the oocytes remains unclear. the risk may as well be caused by other factors than the oocytes. for instance, central obesity patients exhibit high lh levels and insulin resistance which can cause abortion, whereas administration of gnrh - a to these patients can effectively reduce the risk of abortion. wang. postulated that the relatively high abortion rates in the pcos patients were due to combined effects of obesity, treatment for infertility, and causes of infertility. after correction for these factors, the abortion rates in the pcos patients were comparable to those in the controls, indicating that the intrinsic factors of pcos contributed little to the abortion issue. in addition, the high e2 levels induced by ovary stimulation might increase the risk for abortion by lowering the endometrial capacity. however, both the viewpoints need further research for verification. in summary, a subpopulation of polycystic ovaries cases are called ovulatory pco patients, who do not display high levels of androgen and have regular menstrual cycles although they do have pco. unlike the pcos patients, they have almost normal endocrine and metabolic characteristics and their pco may be a normal variation of ovary morphology. when treated with ivf - et, ovulatory pco and pcos patients similarly manifest high ovarian responsiveness, relatively good ivf pregnancy rates, and incidence of adverse outcomes, including high ohss risk, cycle cancellation rates and abortion rates (although the differences were not statistically significant). | aim. to compare the basic endocrine profile and outcomes of in vitro fertilization (ivf) in women with polycystic ovary syndrome (pcos), ovulatory polycystic ovaries (pco), or normal ovaries (no). methods. the basic clinical features and in vitro fertilization and embryo transfer outcome in patients receiving ivf or intracytoplasmic sperm injection (icsi) were retrospectively analyzed. results. the body mass index, basal luteinizing hormone, and testosterone levels were significantly lower in patients with ovulatory pco compared to those in patients with pcos. the pcos patients exhibited the shortest duration of ovarian stimulation and lowest dose of gonadotropin, followed by the ovulatory pco and no patients. the ovulatory pco and pcos patients showed similar levels of e2 on the human chorionic gonadotropin treatment day and numbers of oocytes, which were both significantly higher than those of the no patients. the fertilization rate of the pcos patients was significantly lower than the other two groups. compared to no patients, the cleavage rate was lower in both pcos and ovulatory pco patients, however, the number of available embryos was significantly more in these two groups. the incidence of the moderate to severe ovarian hyperstimulation syndrome (ohss) was markedly higher in the pcos and ovulatory pco patients. conclusion. ovulatory pco patients do not express similar endocrine abnormalities as pcos patients. although the fertilization rate and cleavage rate were relatively low in pcos patients, ultimately, all the three groups showed similar transferred embryo numbers, clinical pregnancy rates, and implantation rates. since the incidence of ohss was much higher in the pcos and ovulatory pco patients, we should take more care of these patients and try to prevent severe ohss. |
development leading to the spread of malaria has been located in terms of disease and political economy. sheldon watts and ira klein have linked the increase and spread of malaria in colonial india to the rapid commercialisation of agriculture, deforestation, expansion of the railways, construction of embankments, as well as urbanisation, consequent on the monetisation of the colonial indian economy in the nineteenth century.1 borrowing from twentieth - century nationalist critiques, historians have identified british development policies as the cause of malaria in colonial bengal, but have not critically analysed the medical debates around malaria and development in colonial bengal.2 klein has studied the relationship between ecology, environmentalism and malaria in colonial india and has argued that there was a relative decrease in malaria mortality in bengal in the mid - twenties, and attributed it to the rise in immunity in the surviving populations in the worst - affected districts of bengal. the links between malaria and development are, however, tortuous : randall packard has pointed out that anti - malarial policy and the development that followed in the transvaal benefited large plantation owners and marginalised both small white farmers and african peasants.3 less debated have been the trajectories of malaria research, anti - malarial policy, and the role of the colonial state and private entrepreneurship in formulating and directing anti - malarial programmes. it is important to do so because malaria research was conducted at important sites of colonial economy, such as plantations, port cities, and cantonments, where malaria threatened economic or military activities. sustained anti - malarial initiatives were undertaken in the rubber estates in malaya, in the coffee plantations of ceylon, the large tea plantations of assam, and at the eastern frontier during the second world war.4 the liverpool school of tropical medicine was supported almost exclusively by entrepreneurial patronage from planters and miners in southeast asia and west africa. this paper explores the incentives for medical research in a tropical colony by analysing malaria research in one such area, the tea plantations of north bengal. the site of research assumed significance as a primary factor both in the identification of the causation of disease, as well as in the economic concerns of colonialism. yet, this focus on specific localities in terms of research and economic activities did not extend to sustained implementation of anti - malarial policies at those very sites. historians have established that in the nineteenth century, british indian medical officers believed that local conditions in india contributed to a unique disease zone, to the extent that many rejected bacteriological explanations for cholera.5 the logic of location transcended miasmatic theories of fever and was appropriated within the framework of tropical medicine, including malaria research in the twentieth century and informed anti - malarial policy. the explorations of the relationships between vector control and transmission among the mosquito vector, the parasite, the environment, and the behaviour of human carriers in particular, privileged malaria studies based at the sites of the occurrence of the disease in the field, a term used by scientists and anthropologists alike. research on trypanosomiasis in africa also appropriated a complexity of variables including ecology, insect vector, parasite, and human and animal infection in the inter - war years.6 the emphasis on environment and its links with malaria was retained long after the discovery of the insect vector of the malaria parasite, and medical research linked fevers to localities in fresh terms. swellengrebel in 1911 to link carrier anopheline species with specific habitats, enriched the cognitive connection between ecology and malaria research.7 in india, this cognitive link had particular resonance, as british indian sanitary theories had long favoured the uniqueness of location in medical discourse, particularly with respect to cholera.8 the logic of as this paper will show, malaria research in the darjeeling hills was imbued with the multiple and ambiguous identifications of the local. yet, paradoxically perhaps, malaria research was an international project. in the years following ronald ross s discovery of the insect - vector factor in malaria (1897), carefully selected sites became locations for intensive anti - malarial activity in many parts of the world. in italy, a generation of malariologists established research and conducted anti - malarial sanitation.9 after the first world war, the league of nation s fledgling health committee conducted a series of malaria surveys in several mediterranean and baltic countries, formerly theatres of the war, where malaria mortality had been enormous. within the british empire, malaria researchers focused intensely on specific sites, especially those that were of strategic or economic interest in africa, india, ceylon, and malaya between 1900 and 1930. the london and liverpool schools of tropical medicine provided institutional support to many researchers who travelled to many parts of the tropical world, and who exchanged research and debated anti - malarial policies and strategy vigorously in international conferences and medical journals. malaria research in india was a part of the international research agenda, often borrowing from, as well as contributing to contemporary international debates. sinton compiled a bibliography of malaria research in india, he filled two hundred pages with more than two thousand items from scientific and medical journals.10 as we shall see, every local anti - malaria project in india borrowed ideas from contemporary international research and attempted to utilise them in specific local contexts. christophers, the malariologist who was most influential in conducting research, benefited from the work of angelo celli in the roman campagna, as well as from robert koch s immunity studies on malaria. within tropical colonies, malcolm watson s ideas on the flying radius of the anopheles, from swellengrebel s work on species sanitation, targeting the malaria parasite carriers, reinforced control programmes in india and ceylon. this juxtaposition between international research and the influence of local human and environmental factors was the main characteristic of malaria research in colonial india. the focus of medical research on malaria was not surprising, for after cholera, it was the principal cause of mortality in colonial india. it was either constantly prevalent or periodically erupted in epidemic form : in the nineteenth century and between the 1890s and 1921, it probably took twenty million lives.11 in the nineteenth century, bengal suffered particularly : malaria depopulated entire districts, and state public health policies, and to an extent, popular political movements engaged with its effects.12 in the nineteenth century, british indian medical discourse had relegated malaria - ridden indians to racial and civilisational degeneracy, beyond the reforming momentum of british imperialism.13 subsequent to the discovery and acceptance of the insect - vector transmission of the disease by ronald ross, incremental research in tropical medicine privileged selected sites for research on the control of malaria. both local specificities and international exchange and co - operation were integral aspects of malaria research, and this paper argues that the paradox of local and international priorities were never resolved in malaria research. the tea plantations of north bengal were privileged sites of economic activity in colonial india. large - scale tea plantations were established from 1856 in terai and duars, which were located in the foothills of the himalayan darjeeling. in 1900, the combined tea production of darjeeling, terai, and duars exceeded a quarter of india s annual tea export and employed over two hundred thousand workers.14 the plantations were reclaimed from forests, reed jungles, and miles of swampy land that had a reputation for malarial fevers. duars and terai were well known to travellers and soldiers as dank and febrile territories. in 1866, when the vicereine lady canning stopped in the terai on her way to darjeeling, she contracted a fever from which she never recovered.15 arthur story, a physician employed in the duars plantation remarked : talk about darkest africa, i think darkest hindustan is pretty nearly as bad.16 with the expansion of the plantation economy, the region was transformed both ecologically and demographically. the plantations cleared forests and introduced the flow of immigrant labour, as well as foreign flora. the labourers were recruited from the impoverished santhal parganas and the chotanagpur districts of western bengal through agents (sardars), who themselves worked in supervisory positions in the plantations. in north bengal, unlike in the older established tea plantations in neighbouring assam, there was no indentured labour : the labourers were legally free to move. in fact, their mobility depended on their ability to repay loans they acquired from the planters or their agents to pay for living expenses. each community lived in bamboo and thatch huts (coolie lines), next to its sardars. the tea plantations were generally flanked by villages (bastis), settled by tenant cultivators who worked in the plantations in the peak season. each tea estate also housed a few clerical staff, as well as two to three assistant managers and the manager, whose bungalow was located at the centre of each estate. the size of the labouring population within a tea estate varied from three - to - four hundred to two - to - three thousand in the larger estates. while ross s discovery appeared to promise the elimination of anopheline mosquitoes, the dynamic aspects of the locality itself challenged the presumptions of malaria research. in 1902, the government of india invited the malaria committee of the royal society to india. stephens, who had initially investigated malaria along the amazon and was a fellow of the royal society, s.r. christophers, who later became the director of the malaria bureau of india, and c. w.w. daniels representing the colonial office.17 their indian tour included the capital city of calcutta, the duars, and the agriculturally important punjab. their findings suggested that although anophelines were responsible for the transmission of malaria, the degree of infection at a given locality was not directly proportional to the total number of anophelines there. in calcutta, for instance, they found more anophelines than in the worst fever districts of africa.18 yet none were found to be infected with malaria.19 whereas in the duars, there were fewer anophelines but the incidence of malaria was very high.20 the report highlighted this factor of locality in the phenomenon of anophenilism without malaria, concluding that specific breeding places and species of anopheline carriers determined the extent of malarial infection in a locality.21 therefore, the first study on malaria in the tea plantations emphasised the importance of locality in the transmission of the disease, and the duars region assumed special significance to malaria research. an early experiment at anti - malarial sanitation was conducted between 1902 and 1909 at mian mir, a military cantonment near lahore in the punjab. the study was conducted by the malaria committee of the royal society, facilitated by the government of india and supervised by stephens and christophers. the conclusions at mian mir were responsible for the retreat of anti - malarial sanitation programmes in india through government initiatives for some time.22 in official policy and medical discourse, attention shifted towards the human body as the site of prevention. sheldon watts has pointed out that christophers, as the chief malariologist in india until the mid - thirties, kept the focus of anti - malarial policy in india away from the canals and irrigated rice fields, and instead concentrated on a quinine policy.23 watts argued that direction away from anti - malarial sanitation and towards quinine prophylaxis was motivated by the need to sustain the irrigation policy of the government in british india, particularly the canals in the punjab, from which private investors in england earned rich dividends and the government of india reaped the benefits of substantial agricultural revenue. the irrigation canals were the basis of the agrarian economy of the british punjab, which was commercialised and greatly expanded under british rule. two - fifths of the army in colonial india was recruited from the rural peasantry of the punjab : the loyalty of this army, especially after the revolt of 1857, was linked to the prosperity of the agrarian economy.24 moreover, as ira klein has pointed out, watts ignored the various studies, especially by bentley, which drew links between ecological degradation and the incidence of malarial fever in colonial bengal.25 but there was more to the evolving discourse of malaria and the practices of anti- malarial sanitation in india than a simple dichotomy between the sanitarian approach and the scientific one of malarial prophylaxis through the use of quinine. ross, who battled the indian medical service (ims) establishment to focus more on anti - malarial sanitation had also argued that, before practical results can be reasonably looked for, however, we must find precisely (a) what species of indian mosquito do and do not carry malaria ? (b) what are the habits of dangerous varieties?26 therefore, there was no question of indiscriminate implementation of anti - malarial sanitation, even by its strongest advocate. it required detailed research into the habits and breeding places of anopheline mosquitoes, and therefore the question of locality remained. between 1902 and 1930, malaria research all over the world, as well as in india, demonstrated the enormous variety of types, as well as breeding habits of anopheles, which differed from one terrain to another, making the question of locality a crucial one. by 1913, still convinced that the destruction of parasite - carrying anopheles was possible, ross presented the successes of the campaigns towards total extermination of malaria in the panama canal, in ismailia in egypt (near the suez canal), and in the malay straits. he did not contemplate large - scale prevention in rural areas of either africa or india.27 years later, malcolm watson would point out that ross had insisted that he was misrepresented and that he had never thought and certainly never stated that it would be possible to exterminate mosquitoes throughout africa, for instance always referred to large towns.28 in india, christophers and his colleagues, such as s.p. sinton, were the most outspoken partisans of quinine prophylaxis as the best mode to control malaria. the strategy of quinisation was complicated : authorities disagreed over whether it could be best used as prophylaxis or as a curative the supply was far less than the demand for quinine.29 the dutch monopoly over almost ninety percent of the cinchona produced in the world created problems of access. but the quinisation approach was combined with sporadic implementation of anti - malarial sanitation as well as segregation which were all attempted, in various degrees, in the tea plantations.30 british indian malariologists borrowed theories of transmission and control of malaria from the research conducted in italy two decades previously, including identifying the human factor in transmission, the use of quinine prophylaxis, and also construction of mechanical screening. christophers and bentley s work in the duars opened a new possibility in identifying the causation and the control of the disease : the collective body of labour congregation in the plantations. in 1906, the british planters in duars petitioned for a commission to investigate malaria and blackwater fevers in their locality. bentley to investigate malaria and blackwater fever in the region.31 their report linked malaria with the structures of the plantation economy and located the disease in the community of newly immigrant labourers, rather than on local miasmatic influence. this transformed the question of location. referring to researches on immunity conducted by robert koch and by angelo celli (in the roman campagna), they asserted universal links between malaria with congregations of a labouring population : we may say that in our researches on malaria we have for some time recognised the almost constant association of labour camps with severe malaria. it is not the soil disturbance, we believe, but the occurrence of labour camp conditions, or what we shall call for convenience of description the tropical aggregation of labour, in association with these enterprises which has given them their evil reputation.32 we may say that in our researches on malaria we have for some time recognised the almost constant association of labour camps with severe malaria. it is not the soil disturbance, we believe, but the occurrence of labour camp conditions, or what we shall call for convenience of description the tropical aggregation of labour, in association with these enterprises which has given them their evil reputation.32 the tropical aggregation of labour or the human factor in the transmission of malaria marked a shift in the significance of location in malaria research. although the human factor in malaria that informed the thesis of tropical aggregation of labour was previously described by koch and celli, it had particular resonance for immunity studies in industrial sites in tropical countries. the study linked, for the first time in india, epidemiological explanation of malaria to the influx of immigrant labour and the factor of non - immune migration at any industrial site : ports, jute mills, tea gardens, and railway construction sites.33 the movement of infected workers made them mobile reservoirs of malaria.34 the thesis of the tropical aggregation of labour gained credence through reiteration in national and international forums. in 1929, the league of nations malaria committee carried out an inspection tour of india at the invitation of the government of india. the factor of non - immune immigration at industrial sites was cited as a major cause of the spread of malaria, particularly mines, plantations, and ports.35 in 1923, christophers, then director of the malaria bureau of india, wrote a report on the prevalence of malarial fever in the bengal iron company s mines at singhbhum.36 he made similar recommendations to those he made in the duars in 1908, such as the screening of european bungalows, segregation, and the use of quinine as prophylaxis.37 by this time, the concept of the tropical aggregation of labour was entrenched. christophers further refined the theory : that settled workers acquired an immunity to malaria that was not racially inherited but acquired through repeated infections suffered by the newly immigrant labourers and by newborn children and infants. therefore, even if the spleen rate of a certain area showed a high endemicity of malaria, the adult population was not unduly affected, as they had gone through the process of acquiring immunity through repeated malarial infections. industrial and plantation sites with immigrant workers acquired new significance as locations of malarial fever. the focus on the body as the location of disease relocated the links between malaria, race, and immunity. christophers and bentley denied that race was significant in the causation of malaria.38 nevertheless, in 1923, christophers referred to certain susceptible races such as the european staff and the skilled labour (mostly hindus and muslims). the unskilled mine workers were indigenous, largely aboriginal and were fairly immune to malaria, which came after a period of acute infestation of malaria for about two years a process he compared to the salting of animals in trypanosomiasis.39 in 1926, when the government of bengal reported its findings on anti - malarial measures in a tea estate in duars, it sketched out possibilities of infection very similar to the conclusions reached in the mining areas of bihar : if a mixed population of men, women and children who were susceptible to malaria were introduced into such an area, there would in the first instance be an explosive outbreak of malaria amongst the new comers in a vigorous race, there would be a rally in the individual against the parasite and gradually a tolerance or relative immunity would be developed a time would come when the only persons not possessing a relative immunity would be newly born children. these would all be intensely affected and would suffer from continuous fever until they either died or gradually acquired a relative tolerance.40 if a mixed population of men, women and children who were susceptible to malaria were introduced into such an area, there would in the first instance be an explosive outbreak of malaria amongst the new comers in a vigorous race, there would be a rally in the individual against the parasite and gradually a tolerance or relative immunity would be developed a time would come when the only persons not possessing a relative immunity would be newly born children. these would all be intensely affected and would suffer from continuous fever until they either died or gradually acquired a relative tolerance.40 such a conception of immunity from malarial infection that posited distinctions between vigorous and other races led to the conclusion that certain communities were more likely to acquire immunity than others : for instance, the government of bengal report of 1926 pointed out there are certainly racial differences. santals, for instance, seem to get immune more quickly than nepalese, who would appear to possess small powers of immunity production.41 although there was some ambivalence about it, the assumption of immunity or partial immunity of certain races to malaria continued to inform british indian medical discourse.42 this was different from the earlier nineteenth - century ideas of racial immunity to fever. in the mid - nineteenth century, david rennie, in his account of the bhutan campaign of 1865, mentioned that the meches, the pre - colonial inhabitants of the region, were peculiarly immune to fevers.43 by linking racial immunity to acclimatisation in a specific locality, rennie conceived of immunity as both racial as well as locational. this was a general conception among contemporary medical practitioners and colonial ethnologists, such as e.t. dalton.44 this particular link between racial immunity, location, and fever became irrelevant in the twentieth century, by which time the indigenous meches were pushed out of the region.45 in the twentieth century, malaria research retained both racial and locational categories and re - articulated them in the bodies of infected immigrant races. when patrick hehir wrote malaria in india in 1927, he emphasised that dark - skinned races, living in malarious regions, possess a relative immunity to malarial infection..46 he quoted koch to argue that the acquired immunity occurred relatively rapidly in cases where quinine was not used. this emphasis on acquired immunity from malaria reinforced arguments against the adoption of more expensive quinine prophylaxis as well as anti - malarial sanitation in the plantations. immunity studies in malaria, developed out of the theory of tropical aggregation of labour, acquired economic as well as epidemiological implications in the tea plantations. in 1927, when ronald ross visited duars, the chairman of the planters association wanted research on immunity to malaria : i have often felt that it should be possible to create this immunity by some artificial means and thereby hasten on what nature now does so slowly.47 at the same time, the idea of the local continued to appear in multifarious forms. along with the congregative body of the labourers, malariologists linked transmission to local ecological factors. at an institutional level, malaria research in india was initially conducted at the central research institute and was sponsored by the indian research fund association, (irfa) set up in 1911. this research was subsidised partly by the government of india, and published the indian journal of medical research.48 the irfa also solicited private subscription. besides army cantonments (such as mian mir), anti - malarial sanitation was attempted at mines and plantations. the success or failure of any anti - mosquito campaign was understood with reference to local ecological conditions. the imperial conference on malaria in shimla in 1909, led to the formation of a central malaria committee to direct the course of anti - malarial operations in different provinces.49 at the conference, j.t.w. leslie, the sanitary commissioner, pointed out that the drainage committee of bengal had found that local ecological factors determined the extent of malaria.50 officials concluded that we have little exact knowledge of the distribution of malaria in the country, of the local conditions which favour it, and of the best means to render these causes inoperative.51 this concept of local knowledge or this notion of locality was an ambiguous scientific category : identified simultaneously with the vector, the land, and demography. yet, local knowledge was a powerful motif for colonial governance. in colonial india, it became the basis of colonial rule : in terms of the knowledge of local languages, demography, caste, and economic geography.52 for the colonial government, any move for sanitary reform had to be based on local knowledge : [we ] must recognise the diversity of local conditions in a country which includes numerous communities, castes and creeds and which exhibits almost every variety of climate, temperature, humidity and level of sub - soil water, from the deltas of bengal with their steamy atmosphere and dense lush vegetation to the burnt brown hills of the north - west frontier. 53 [we ] must recognise the diversity of local conditions in a country which includes numerous communities, castes and creeds and which exhibits almost every variety of climate, temperature, humidity and level of sub - soil water, from the deltas of bengal with their steamy atmosphere and dense lush vegetation to the burnt brown hills of the north - west frontier. 53 this emphasis on locality and local knowledge was such a fundamental element in colonial administration in india that it continued to inform medical research and practice. this administrative prerogative informed british indian epidemiological theories, which continued to emphasise the agency of local factors in the causation of disease. the tenacity with which the indian medical establishment perceived india s disease terrain as both distinctive and esoteric, requiring experience and familiarity to be medically understood, demonstrates the persistence of the rhetoric of the local.54 the insect - vector theory and the subsequent research on malaria reinforced the idea of the importance of local disease factors, such as the diversity in the anopheline species and the variables in their breeding. medical professionals in britain also made the link between local ecology and epidemics in the inter - war years. mendelsohn has pointed out, in the inter - war years medical scientists working on bacteriological epidemiology in germany and britain borrowed increasingly from older traditions of epidemiology, as well as using new mathematical models to formulate what he described as holistic and non - reductionist explanations of epidemics.55 helen tilley has claimed that research on trypanosomiasis in africa also appropriated a complexity of variables among ecology, insect vector, parasite, and human and animal infection in the inter - war years. although malaria research in colonial india both borrowed from and informed the intellectual paradigms of malaria causation internationally, it was in some senses a particularly colonial story. the significance of local environment was a prerogative of colonial rule that had informed british ideas of health, european and native bodies, and colonial policy from the eighteenth century.56 in the nineteenth century, this informed the medical discourse of the anti - contagionists.57 in colonial india, the recurring alignment of local factors in disease causation through divergent and multiple meanings, including ecological factors, human agency, and the cultural practices of the disease - stricken, had preceded the holistic medicine of inter - war britain. the constitution of the local differed : it could be located in ecological, cultural, or economic factors. for instance, in the duars, while planters and officials emphasised the cultural and political factors in the implementation of anti - malarial programmes, medical experts generally focused on the heterogeneity of ecological and environmental factors. so far as malariologists were concerned, the focus of the local shifted to the human factor following the work of christophers. the colonial government believed that the responsibility for the lack of sanitary reform in india could be attributed to the apathy, fatalism, and resentment of interference of the uneducated masses.58 besides reinforcing the paternalism of colonial administration, this engendered a gradualist view of sanitary reforms acquired systematically from the knowledge of the local conditions, linking indigenous people with topography, often used as interchangeable categories. the multiplicity of local conditions involved in malaria control in the tea plantations was demonstrated in the anti - malarial sanitation project at the meenglas tea estate in duars. it was owned by the managing agency of duncan brothers limited, a british company. the experiment lasted eight years, beginning in 1917.59 the aim of the experiment was to prevent the breeding of carrier anophelines in a terrain where the land was cut up by several seasonal streams (jhoras). in the duars, the seasonality of the streams depended on the terrain : for instance, the ones on the slopes remained dry for most of the year, while the ones closer to the plains were perennial.60 other ecological characteristics of the area were the proximity of jungles, rice fields, and three fast - flowing rivers. the area under the experiment was three - quarter - mile radius chosen carefully based on the views of malcolm watson, whose successful policies in malaya had shown that the flight of anophelines did not exceed half a mile. however, the local factors of duars soon became apparent to the surveyors. the three most dangerous local carriers were identified as a. maculatus, a. listoni and a. culicifacies. unlike the malayan experience, here the anophelines that inhabited the nearby forests, principally the a. aitkeni, were found to be harmless.61 the experiment demonstrated that the underground drainage of streams controlled the breeding of anophelines. it demonstrated that the spleen index of the children and malarial fever could be reduced for a limited period within the controlled area.62 in 1925, bentley, who was now director of public health for the government of bengal and who initiated the scheme, reported on the efficiency of sub - soil drainage in reducing the death rate and the fever index.63 however, the average overall death rate in the tea estate did not decline during the eight years of the experiment. the spleen index remained almost static from 1923 to 1927.64 the final report concluded, as regards the actual reduction of malaria, this is a point on which it is very difficult to form an exact opinion, mostly owing to the factor of shifting population.65 the indifferent success rate here highlighted the complex political, economic, and environmental factors in anti - malarial sanitation in tea plantations. the planters retained the system of free labour because it exempted the plantations from government regulations, such as in neighbouring assam, where contractual labour had to be examined by medical officials for fitness. the labourers living in the bastis of the north bengal tea gardens were used in peak times but the management did not assume responsibility for them. the relationship between the tea garden and the basti was one of dependence, suspicion, and even hostility. tea production relied on the seasonal labour from the bastis, yet every epidemic disease in any plantation was rumoured to have originated first from the bastis. in managerial discourse, the tea garden was the enclave that protected the health of its labourers, as well as the pristine and primitive nature of their cultures. yet, the economy of production demanded a labour force that would work in the peak seasons and settle outside, to relieve the plantations from welfare measures for them. the duars plantations refused to finance large - scale anti - malarial sanitation except for spending a little on spraying kerosene in their streams.66 these measures did not prove adequate for the control of malaria.67 there was a second local factor crucial to anopheline control in meenglas : the existence of rice fields close to the plantations and often within them. most plantations allotted a part of their estates to the labourers where they grew rice and vegetables. the allotments were made to labourers on the condition of work, to keep wages low. they served as instruments of control because the labourers had no tenants rights here.68 this arrangement severely inhibited malaria control. rice cultivation should not be allowed.69 the contradictions of malaria control within the plantation economy were fundamental and could not be resolved within the existing administrative and economic structure. when anophelines of all varieties were caught at the meenglas tea estate it was found that the three dangerous carriers, a. maculatus, a. minimus, and a. culcifacies inhabited the cleared and cultivated areas. a survey of the ecology of malaria in the forested areas of bengal revealed that forests did not breed anopheline carriers. the clearing of forests and the introduction of tea bushes and rice fields did away with harmless anophelines such as a. aitkeni and a. barbirostri, which were then replaced by the carriers.70 malaria was, in other words, endemic to human habitation and livelihood. this conclusion, from the meenglas experiment and from other contemporary studies, such as those of bentley on the links between embankments and malaria, led to the conceptual linking of malaria with modernity and development.71 it also contributed to the nationalist critique of british policies in india.72 paddy fields were held to be the cause of malarial fever in the twentieth century, not just in the duars, but in most of rural india. this was emblematic of the paradoxical relationship between modernisation and malaria in india, where railways and rice fields, the consequence of commercialised agriculture, were seen to cause the disease and yet were impossible to obliterate from the modern indian landscape. bentley acknowledged the link between rice fields and malarial fever.73 his contribution to the debate was to implement the italian concept of bonificazione- which according to him embodied measures designed for a double purpose, viz. to improve agriculture and improve in western bengal, he advocated anti - malarial sanitation not through drainage, but through further inundation through irrigation. therefore, he avoided making a direct criticism of the development policies of the government and instead suggested a solution that would lead to more, rather than less, investment in irrigation. this strengthened the rationale of the developmental projects of the colonial government.75 therefore, the case for malaria control was laid to rest with the emphasis on rice cultivation. this was ironic, for it had been the call of tropical medicine to render a great gift to the human race, to penetrate impenetrable tropical jungles and make them habitable.76 tropical medicine identified this paradox but failed to resolve it. almost two decades previously, in a continuation of the acrimonious debate on the failure of the anti - malarial measures at mian mir, ross had alleged both the lack of adequate data and the faulty application of scientific knowledge in the mian mir operations. he was convinced that anophelines could be destroyed in any given area, and what remained to be accomplished was to accommodate local variables.77 in meenglas, the malaria surveys were conducted and the entomological studies and the spleen index of labourers in neighbouring tea estates examined. the experiment at meenglas (and a similar experiment at the mining sites of singaran and topsi in bengal) demonstrated that eradication of malaria would not be possible in hyper - endemic areas.78 the experiment confirmed that while the idea of locality in disease control remained problematic, in administrative terms the category of the local assumed fresh significance. the public health department utilised the lessons of meenglas to negate the possibility of drainage operations at any cultivated site in the intensely malarial lower bengal. in twentieth century india, medical research and the newly established institutes of public health received financial aid from private entrepreneurs, as well as the government. the calcutta school of tropical medicine was instituted in 1921 and was funded with donations from the government of india, the bengal government and the local indian elite.79 it also received subscriptions from british - dominated jute, tea, and mining industries in eastern india.80 this patronage occasionally defined its activities. initially the calcutta school began research on kala azar in the tea plantations of assam. when the planters realised that the government was to fund a separate research programme on the same disease, they proposed to divert the school s research to malaria surveys.81 in assam, the total production of tea was more than two and a half times that of northern bengal, involving a heavier cost of labour. but the planters of north bengal invited the government to aid research highlighting the unique conditions of the region that they believed were ideal for the study of malaria and other obscure tropical diseases.82 they discussed funding for a research institute in the duars, but realising that it would cost around 35,000 to 50,000 rupees annually, opted for a more economical alternative : a malarial survey by an expert from the school, much like the assam survey.83 in 1926, c.k. strickland, a malariologist of the calcutta school conducted a preliminary survey in the duars, as in assam.84 the arrangement between the calcutta school and the dooars planters association resulted in several malaria surveys of darjeeling and the terai. until the advent of ddt, the tea plantations continued to be sites of malarial research, providing ideal most studies located the infectivity of anophelines in specific breeding conditions : there was some anti - malarial drainage in one tea estate in darjeeling.85 others studied comparative causes of epidemics of malaria in hill stations, such as shillong (assam) and kurseong (darjeeling).86 the terai planters association also funded a survey through the agency of the calcutta school of tropical medicine.87 despite obligatory visits to the location, malaria research remained unconnected with any real action on the ground. the logic of location in malaria research was fulfilled within the surveys undertaken in the sites. in 19267, when ross visited the area, he was invited to the annual meeting of the duars planters association. with typical candour, he pointed out the futility of endless surveys without action ; i now ask what does the dooars intend to do?88 he received no commitment from the planters. in his preface to the league of nations malaria commission report, christophers conceded that anti - malarial initiatives rarely proceeded from surveys to preventive operations in the tea estates in india : at present, after a survey and recommendations, nothing very much often follows, largely because it is then left to the manager of such estate to do what he can, whereas the proper course would be to engage a suitable man to reside on the area and see to the carrying out of whatever was possible.89 at present, after a survey and recommendations, nothing very much often follows, largely because it is then left to the manager of such estate to do what he can, whereas the proper course would be to engage a suitable man to reside on the area and see to the carrying out of whatever was possible.89 why this lack of antimalarial action within the region by the planters ? in administrative terms, the main hindrance to the efforts on the part of the tea estates to engage in anti - malarial operations was the ad hoc nature in which they operated and the short term incentives involved, where the managers of the estate were personally responsible for the finances of the tea estate. any long - term investment in a tea estate would detract from immediate profits and targets and thereby from the commission received by them. strickland pointed this out in his report while suggesting that the managers not be made responsible for the anti - malarial sanitation work.90 some initiatives were undertaken from england as well. in 1930, the ross institute in putney, which was supported partly by the india tea association in london, opened a branch in shillong, in north - eastern india. however, the drainage measures undertaken in india under the supervision of the ross institute were concentrated in the tea estates of assam, where the large managing agencies had contiguous territories and several tea gardens under their control.91 the duars and darjeeling regions remained overlooked despite the shortage of quinine during the second world war, which aggravated the problem of malaria in the plantations of north bengal.92 mortality from fever remained the single largest cause of death in the plantations.93 the emphasis on productivity and possibilities for entrepreneurial patronage did not translate into sustained anti - malarial sanitation in the duars and terai plantations. firstly, assam was geographically more distant from the labour recruiting districts of western bengal and bihar. recruitment of labour was expensive in assam compared to duars and terai, so it made better sense to make arrangements for the healthcare of the working population of the assam plantations. this did not necessarily mean welfare of the labourers, as the assam plantations had a darker reputation for coercion and ill - treatment of workers by the planters and sardars and much of the funds were usurped by them.94 secondly, the average acreage of tea estates in assam was larger than in north bengal, and the gardens there were owned in contiguous territories by british managing agencies, all of whom were members of the british - dominated indian tea association. in the duars and terai, on the other hand, the estates were smaller in size, about a quarter of which were under indian management. the indian planters felt excluded from and resented the british - dominated indian tea association. moreover, several indian planters owned jotes, tenanted agricultural land for rice cultivation often next to the tea estates, which would have been adversely affected by antimalarial drainage operations. finally, since the tea estates in north bengal recruited casual basti labour, the very system depended on labourers who would cultivate rice in the neighbouring paddy fields and work in the plantations as well. therefore, although public health officials and medical experts from calcutta and london pointed out the advantages of investment in anti - malarial sanitation, the structure of the plantation economy in duars and terai inhibited any large - scale investment in anti - malarial sanitation. the logic of location emerged within malaria research as an attempt to accommodate local variability in the causation of the disease. however, this also exposed the tension between scientific research, entrepreneurship, and local realities. the darjeeling foothills and the plains of terai and duars were the sites of studies in tropical medicine conducted from london and calcutta. these led to the identification of causation of malaria in industrial locations all over india, with the factor of non - immune immigration and the tropical aggregation of labour. at the same time, these local sites were exposed to the confluence of metropolitan scientific ideas. the malaria control experiment that was started at the meenglas tea estate borrowed from the knowledge of anophelines and preventive measures developed by ronald ross (the anopheline count per head of the population factor in infectivity) and malcolm watson (subsoil drainage), and sought to re - examine their theses in that locality. thus, the tracts of duars and darjeeling terai were at once connected to the entire tropical world and its diseases and to the world of metropolitan and colonial tropical medicine. in doing so, although malariologists failed to specify links between locality and disease in clear terms, and the ambiguities of the local in terms of ecology, demography, and race confounded anti malarial policy, the logic of location continued to be the focus of such surveys. colonial realities informed, complicated, and challenged the inadequacies of the contemporary medical theories of the metropolis. twentieth - century medical research is seen to have become inclusive and holistic as it ventured from the laboratories to the field.95 however, as this paper has shown, within scientific research the incorporation of the problematic of location or of the field did not necessarily lead to incorporating the problems of the localities. contemporary research on conditions, such as the hospitability of the different terrains to particular sub - species of the vector anophelines, merged seamlessly with concerns over the peculiarities of labouring populations within the tea plantations and outside them, at the bastis all framed in a set of local conditions. so far as the workers were concerned, the planters generally claimed that quinine prophylaxis could not be administered effectively because the workers were resistant to it. they also insisted that the local government should not interfere with the management of disease within the plantations. close knowledge of the labourers and their customs, and the knowledge of local conditions that was the staple of management discourse on the tea estates (at least until a communist - led labour movement dented it substantially after 1946), was presumed to have entitled them to be the guardians of labour welfare. travers, after the visit of the royal commission on labour to the tea estates is representative of their gradualist and paternalist approaches to reform in health infrastructure within the plantations : many of their racial and religious customs tend to impede the work of health improvement and welfare, and therefore it is of great importance that all measures for their uplift in any direction should be under the control and direction of persons who really know and understand the customs, traditions and habits of these aboriginal people.96 many of their racial and religious customs tend to impede the work of health improvement and welfare, and therefore it is of great importance that all measures for their uplift in any direction should be under the control and direction of persons who really know and understand the customs, traditions and habits of these aboriginal people.96 on the other hand, the idea of species sanitation in tropical medicine provided a new dimension to the colonial understanding of the local, in terms of yet the experts, planters, and local administrators also formed collaborations for conducting malarial experiments in the plantations. the planters association at duars highlighted the unique local conditions when asking the government to fund malaria research in the duars. this appeal was partly rhetorical : the planters associations generally sought to shift pecuniary responsibility for any research or sanitary works to the government. nevertheless, the distinctiveness of the region and the knowledge of local conditions gathered through colonial and managerial discourses did contribute to the knowledge of tropical medicine. the sub - himalayan tea plantations were an important site of exploration for new ideas and experimentation in methods of anopheline control. at the same time, the political economy of the plantations highlighted a complex set of factors that inhibited both anti - malarial sanitation as well as systematic and extensive use of quinine prophylaxis within the tea plantations. some european medical officers within the plantations made isolated attempts at destruction of the anopheline breeding sites within a decade of the discovery of the mosquitovector transmission by ross.97 some managers made similar attempts in the 1920s and 1930s.98 however, the region itself did not experience any sustained anti - malarial measures and malaria continued to kill the labouring population of the tea gardens. the logistics and structure of the plantation economy could not accommodate any enduring system of malarial prevention. | this article explores the scientific and entrepreneurial incentives for malaria research in the tea plantations of north bengal in colonial india. in the process it highlights how the logic of location emerged as the central trope through which medical experts, as well as colonial administrators and planters, defined malaria research in the region. the paper argues that the local emerged as both a prerequisite of colonial governance as well as a significant component of malaria research in the field. despite the ambiguities that such a project entailed, tropical medicine was enriched from a diverse understanding of local ecology, habitation, and structural modes of production. nevertheless, the locality itself did not benefit from anti - malarial policy undertaken either by medical experts or the colonial state. this article suggests that there was a disjuncture between tropical medicine and its field that could not be accommodated within the colonial plantation system. |
the relative incidence of primary cutaneous lymphoma variants has been widely described in european and north american anglo - saxon countries while little is known regarding the rate of secondary cutaneous and primary oral lymphomas. by contrast, no such studies have been conducted thus far in mexico. the relative incidence of primary cutaneous lymphoma variants has been widely described in european and north american anglo - saxon countries while little is known regarding the rate of secondary cutaneous and primary oral lymphomas. by contrast, no such studies have been conducted thus far in mexico. classification of lymphomas, particularly of primary cutaneous lymphomas (pcl), has dramatically evolved over the last decades due to advances in the characterization of their clinical, morphological, immunophenotypic, and molecular features. pcl are lymphomas occurring solely in the skin after a complete staging evaluation has ruled out extracutaneous involvement, differing in biological behavior from systemic lymphomas with secondary cutaneous dissemination (secondary cutaneous lymphomas [scl ]). the 2005-world health organization / european organization for the treatment and research of cancer (who / eortc) classification for pcl includes 13 specific entities, subcategorized into those of indolent, intermediate, or aggressive clinical behavior. these comprise a heterogeneous group of clonal neoplasms with protean manifestations, arising from skin - homing t-/natural killer (nk)-, b- or precursor - lymphocytes which albeit their rarity, represent the second most common form of extranodal lymphomas (19%), following those of the gastrointestinal tract. their reported incidence among european countries is 1:100,000 persons / year, whereas recent studies in the usa suggest an incidence as high as 10.7:100,000 persons / year, with increased prevalence among non - hispanic whites. in contrast to nodal lymphomas, where b - cell lymphomas (bcl) outnumber t - cell lymphomas (tcl), pcl are mainly derived from t - cells (65%80%) with primary cutaneous - bcl (pc - bcl) in the minority (20%29%). among well - defined pc - tcl, mycosis fungoides / sezary syndrome (mf / ss) and cd30 + lymphoproliferative disorders (lymphomatoid papulosis [lyp ] and anaplastic large cell lymphoma [alcl ]) embody most cases ; 55% of pc - bcl fall under the indolent category (follicle center bcl and marginal zone lymphoma), and approximately 40% represent intermediate lymphomas of the diffuse large bcl (dlbcl) variant. oral cavity lymphomas are scarcer than pcl, with scant reports and lack of consensus regarding a specific classification for primary oral lymphomas (pol). the incidence and clinical - histopathological features of non - mf / ss pcl and pol in latin america, and particularly mexico, is largely unknown. in this paper, we reviewed oral and cutaneous lymphomas (primary and secondary) other than mf / ss, which presented to our institution over a 24-year period and recategorized them according to the 2005-who / eortc and 2016-who classifications. to the best of our knowledge, this paper represents the first mexican series from a single referral center using such classification systems. finally, we compare our findings to those deriving from foreign cohorts and analyze gross geographical differences. skin and oral biopsies diagnosed as oral or non - mf / ss cutaneous lymphoma over a 24-year period (19882012) were retrieved from the pathology archive of instituto nacional de ciencias mdicas y nutricin salvador zubirn, after approval by the institutional committee for medical ethics. only cases with sufficient tissue available for adequate assessment of morphological and immunophenotypic features clinical information was obtained through medical records and phone interviews when necessary for data confirmation. formalin - fixed paraffin - embedded tissue (ffpet) sections were stained with hematoxylin and eosin. immunophenotyping with a comprehensive panel of stains was performed under standard procedures on ffpet, using commercially purchased primary antibodies according to the diagnostic possibilities for each case, concurring with the 2005-who / eortc and the 2016-who classification criteria. in situ hybridization (ish) for epstein barr virus (ebv) encoding region (eber) and kappa / lambda immunoglobulin light chains were performed when deemed necessary in a case - by - case basis. skin lymphomas were typified as pcl if no lymph node or visceral involvement was present at diagnosis, after performing a complete staging evaluation (physical examination, head to pelvis computed tomography, and bone marrow biopsy). twenty - four (30%) fulfilled inclusion criteria for proper recategorization ; of these, 18 (75%) were skin - limited (pcl), 4 (16%) represented scl [tables 1 and 2 ], and 2 (8%) pol [table 3 ]. clinical and immunohistochemical features of non - mycosis fungoides / szary syndrome primary cutaneous lymphomas clinical and immunohistochemical features of lymphomas from the oral cavity clinical and immunohistochemical features of secondary cutaneous lymphomas non - mf / ss pc - tcl outnumbered pc - bcl (11 cases, 61% vs. 7, 39%). non - mf / ss pc - tcl were categorized as follows : three cd30 + lymphoproliferative disorders (two lyp and one pc - alcl), one subcutaneous panniculitis - like tcl (spltcl), two pc - nk / tcl, one hydroa vacciniforme - like lymphoproliferative disorder (hvllpd), one aggressive epidermotropic cd8 + tcl (pc - aecd8 + tcl), one gamma / delta tcl (pc - gd - tcl), and two peripheral tcl, not otherwise specified (pc - ptcl - nos). pc - bcl were primarily of follicle center cell origin : four cases (57%) of follicle center lymphoma (pc - fcl), three cases (43%) of pc - dlbcl further subdivided into two of leg type variety and one ebv+ dlbcl nos. age range at diagnosis was 1967 years (mean 44 years), similar in all subgroups of non - mf / ss pcl. these arose primarily in a single anatomic region (13/18, 72%) such as trunk (4/13), extremities (4/13), head (3/13), or genital skin (2/13). two regions were involved in 4/18 cases (22%) : trunk and extremities (2), trunk and head (1), head and extremities (1). only one case, corresponding to hvllpd, presented with skin lesions extending to all body segments (except palms and soles), accentuated on sun - exposed areas. most non - mf / ss pcl had more than one clinical morphology (12/18, 66%) as red or red - violaceous nodules (11) and/or infiltrated plaques (8), commonly developing secondary ulceration (6). less frequent morphologies were recurring waxing and waning papules (in two patients with lyp), facial edema, hemorrhagic vesicles, small crusted ulcers and smallpox - like scars (in hvllpd), flaccid bullae progressing to large ulcers (in a patient with pc - aecd8 + tcl), and ulcers without preceding nodules or plaques (in a patient with dlbcl - nos). histopathologic and immunohistochemical features primary cutaneous cd30 + lymphoproliferative disorders both lyp cases showed a perivascular wedge - shaped dermal infiltrate with scant to moderate epitheliotropism (epidermotropism and folliculotropism). clusters of large atypical lymphocytes were evident amidst a background of inflammatory cells (small lymphocytes, eosinophils, and few neutrophils). large lymphocytes comprised 60 months), manifesting initially as vulvar and perianal ulcers, and recurring postradiotherapy and chemotherapy as necrotic ulcers involving one earlobe. while ebv+ mucocutaneous ulcer was considered as an alternative diagnosis, the young age of the patient, absence of immunosuppression, presence of extragenital ulcers, and finally, conspicuous evidence of angiodestruction and pleomorphism strongly favored the diagnosis of pc - nk / tcl. similarly, a case of pc - gd - tcl presented with topographic restriction to genital skin before progressing into disseminated disease. finally, a case of pc - aecd8 + tcl was atypical in its clinical similarity with pemphigus vulgaris given the presence of flaccid bullae although the neoplasm behaved in the classical aggressive fashion with the patient perishing few weeks after diagnosis. in contrast to pcl, the clinical and histopathological features of scl remain poorly understood with very few studies from northeast asia and the usa. in the largest retrospective review from korea, scl consisted of mature nk / tcl (56%), mature bcl (35%), immature hematopoietic malignancies (8%), and hl (1%). in our very small cohort, half of scl were mature nk / tcl (1 en - nk / tcl, 1 ptcl - nos), with one case each of mature bcl (dlbcl) and hl. while the small number of scl in our study impede meaningful comparisons with previous series, it suggests a closer distribution trend with asian registries as opposed to population - based studies from the usa, where sc - mature bcl significantly outnumber sc - mature nk / tcl. as for scl, extranodal lymphomas of the oral cavity have been scantly reported due to their rarity. in the few retrospective studies available, the leading histopathological subtypes are malt lymphoma, followed by dlbcl in one cohort, and plasmablastic lymphoma, dlbcl, and ptcl - nos in decreasing order in another. in our series, one case corresponded to malt lymphoma and the other to ptcl - nos, the rarest pol variant reported. no cases of plasmablastic lymphoma were observed though albeit our institution being a major referral center for hiv patients. this could relate to a selection bias in the hiv+ population at our hospital however since most have undetectable viral loads due to successful antiretroviral treatment. in summary, albeit limitations in our study include the inherent biases of institutional series as opposed to population - based studies, and the inability to recategorize the totality of our cohort, our results support the utility of the 2005-who / eortc and 2016-who classification systems, even when dealing with clinically atypical cases, and the notion of geographical variances in the rate of lymphomas. in particular, we report that some of the disparity may arise from viral - induced lymphomas which might show partial geographical restriction. the differential rates of oral and cutaneous lymphomas in our population may hold closer similarities to registries from asian populations than to those from europe and usa, supporting the concept of individual and/or racial susceptibility. further, the relative frequencies of cutaneous and oral lymphomas in the first mexican cohort hold closer similarities to asian registries than from those of europe and usainternational geographical disparities in the rate of cutaneous lymphomas may arise from viral - induced lymphomas which might show partial geographical restriction. the relative frequencies of cutaneous and oral lymphomas in the first mexican cohort hold closer similarities to asian registries than from those of europe and usa international geographical disparities in the rate of cutaneous lymphomas may arise from viral - induced lymphomas which might show partial geographical restriction. the relative frequencies of cutaneous and oral lymphomas in the first mexican cohort hold closer similarities to asian registries than from those of europe and usainternational geographical disparities in the rate of cutaneous lymphomas may arise from viral - induced lymphomas which might show partial geographical restriction. the relative frequencies of cutaneous and oral lymphomas in the first mexican cohort hold closer similarities to asian registries than from those of europe and usa international geographical disparities in the rate of cutaneous lymphomas may arise from viral - induced lymphomas which might show partial geographical restriction. the relative frequencies of cutaneous and oral lymphomas in the first mexican cohort hold closer similarities to asian registries than from those of europe and usainternational geographical disparities in the rate of cutaneous lymphomas may arise from viral - induced lymphomas which might show partial geographical restriction. the relative frequencies of cutaneous and oral lymphomas in the first mexican cohort hold closer similarities to asian registries than from those of europe and usa international geographical disparities in the rate of cutaneous lymphomas may arise from viral - induced lymphomas which might show partial geographical restriction. | background : nonmycosis fungoides / szary syndrome (non - mf / ss) primary cutaneous lymphomas (pcl) are currently categorized under the 2005-world health organization / european organization for research and treatment of cancer (who - eortc) classification for pcl. these differ in behavior from secondary cutaneous lymphomas (scl) and to lymphomas limited to the oral cavity (primary oral lymphomas [pol ]) both categorized under the 2016-who classification for lymphoid neoplasms.aims:this study aims to report the first series of non - mf / ss pcl, scl, and pol in a mexican cohort, examine the applicability of current classification systems and compare our findings with those from foreign cohorts.materials and methods : eighteen non - mf / ss pcl, four scl, and two pol with available tissue for morphology and immunophenotypic assessment were reclassified according to the 2005-who / eortc and 2016-who classifications.results:non-mf/ss pcls were primarily of t - cell origin (61%) where cd30 + lymphoproliferative disorders predominated, followed by epstein barr virus - induced lymphomas, and peripheral t - cell lymphomas, not otherwise specified. primary cutaneous b - cell lymphomas (bcl) were primarily of follicle center cell origin followed by postgerminal lymphomas of the diffuse large bcl variety.conclusions:most non - mf / ss pcl, scl, and pol can be adequately categorized according to the 2005-who / eortc and 2016-who classification systems, even when dealing with clinically atypical cases. the relative frequencies in our cohort hold closer similarities to asian registries than from those of europe / usa, supporting the concept of individual and/or racial susceptibility, and the notion of geographical variances in the rate of lymphomas. in particular, such disparity may arise from viral - induced lymphomas which might show partial geographical restriction. |
aesthetics of the orofacial region are very important aspects of human life. they might affect the quality of a patient 's life. among the most important goals of dental care is helping patients in their attempts to reach an acceptable level of satisfaction with their oral cavity and dentition. dentofacial conditions and status affect patients ' appearance, performance, and function and shape patients ' satisfaction with their dentition [2, 3 ]. dental disease may influence the capacity to enjoy life, live comfortably, experience relationships, be successful in employment, and possess a positive self - image. variable oral situations as pain, speech, chewing ability, taste, and aesthetics affect various aspects of life quality as well as patient satisfaction with teeth [3, 5 ]. dental professionals need an accurate perception of how patients feel about their teeth and the impact this has on their daily living. consequently, dental care providers should be aware of the dental needs of patients, how patients feel about their teeth, and the impact this has on their satisfaction and daily living. therefore, satisfaction and sociopsychological dimensions should be assessed whenever dental needs are assessed [3, 6 ]. these include presence of fillings and tooth colour, position, alignment, shape, and number [713 ]. higher levels of satisfaction with appearance, better quality of life, and better psychological condition were associated with adequate dental aesthetics and dental treatments that improve dental aesthetics [1420 ]. aesthetic dental treatments including crowns, bleaching, orthodontic treatment, and tooth - coloured restorations are often desired by patients who seek better aesthetics [9, 13, 21, 22 ]. the aim of this study was to investigate participants ' satisfaction with the appearance of their teeth and what are the desired treatments that patients seek to improve dental appearance. in total, 220 university students (127 males and 93 females) were recruited into the study. their age ranged from 18 to 27 years old (mean age = 21.4 1.5 years). to be included in the study, recruited participants had to be 18 years old or older in order to understand and score the questionnaires, and had no medical disease or condition (including mental problems, cognitive disturbances, and psychological disorders) that might affect their ability to comprehend, understand, and/or score the questionnaires, and received no dental treatment for the last 6 months. the study was approved by the research committee, faculty of dentistry, al jouf university, ksa. each participant was given a brief explanation of the study, and an informed consent was obtained from each subject before being recruited into the study. a structured questionnaire was used to assess patients ' satisfaction with their appearance and what treatment they desire to improve their aesthetics (table 1). it included items regarding sociodemographic data (such as age, gender, specialty of studies, and level of study) and items that tackle patient satisfaction with their teeth in general, tooth colour, tooth alignment, and tooth position. also, the questionnaire included items that inquired about presence of caries, tooth - coloured fillings, and tooth fractures. furthermore, the questionnaire included items that attempted to identify whether participants desire treatment to improve their appearance including orthodontics, bleaching, dental crowns, tooth - coloured fillings, and prosthetic rehabilitations and dentures. the questionnaire was modified for the purpose of this study by including a visual analogue scale for the participants to score the level of their satisfaction with their appearance in general. the vas scale ranged from zero to ten where zero means the least satisfaction with appearance and ten means the maximum satisfaction with appearance. the questionnaire was administered to the participants, and the process of completing the questionnaire was supervised by the investigator. each participant was provided with a full explanation of the questionnaire as well as the method of scoring it. the english version of the questionnaire was translated into arabic language by three expert and fluent bilingual individuals and then backtranslated into english by another three individuals who were fluent in arabic and english. fifty undergraduate dental students at al jouf university were asked to score the english format of the questionnaire and then they were asked to score the translated arabic version. the answers of the two formats of the questionnaire were compared using the t - test, and no statistical significant differences were found. then, the final version of the questionnaire was distributed to the participants in the main study sample. the data were analyzed using the spss computer software (statistical package for the social sciences, version 19.0, spss inc. then, the association between the variables was analyzed using the pearson correlation test, while the anova test was used to compare satisfied and dissatisfied patients. in total, 220 participants (127 males and 93 females) were recruited into the study. their age ranged from 18 to 27 years old (mean age = 21.4 1.5 years). specialty of participants ' field of study included medicine, dentistry, medical sciences and nursing, pharmacy, engineering, science, education, and preparatory year of study (n = 40, 23, 36, 22, 14, 24, 58, and 3 participants, resp.). using vas scale, the total satisfaction score of participants ranged from 0 to 10 (mean score = 6.8 2.3). ten (4.5%) participants were totally not satisfied with their appearance and scored 0 on the vas scale, while 19 (8.6%) participants were totally satisfied with their appearance and scored 10 on the vas scale, and 51 (23.2%) participants scored 5 or less on the vas scale (table 2). table 3 presents the distribution of the study sample by their answers to the questionnaire items. half of the participants were dissatisfied with the appearance of their teeth, and 65.9% were dissatisfied with the colour of their teeth. also, 25% of the participants felt that they had crowded teeth, 41.8% felt that their teeth were poorly aligned, and 17.7% felt that they had protruding teeth. furthermore, 15.5% of the participants reported that they had caries, 16.4% had nonaesthetic fillings, and 23.2% had fractures in their anterior teeth (table 3). correlations between the answers of the questionnaire items and each of age, gender, and specialty showed that younger participants had more fractures in anterior teeth (item 8, p =.014, r =.165) and more dental caries in anterior teeth (item 6, p =.02, r =.156). also, males had less nonaesthetic fillings in anterior teeth (item 7, p =.012, r =.169) and had less desire to undergo denture treatment in order to improve the appearance of their teeth (p =.034, r =.143). in addition, specialty of participants had significant relationship with the participants feel that their teeth were crowded (p =.017, r =.161), and participants desire to undergo dental crowning to improve the appearance of their teeth (p =.009, r =.175). medicine and dentistry students had less feelings of having crowded teeth and less desire to undergo dental crowning treatment than education, science, and engineering students. also, medicine and dentistry students had higher satisfaction scores on vas scale than education, science, and engineering students (p =.046, r =.134). the lower satisfaction scores on vas scale were associated with less satisfaction with appearance of teeth (items 1, p =.000, r =.262), feeling that teeth were poorly aligned (item 4, p =.002, r =.212), having dental caries in anterior teeth (item 6, p =.000, r =.250), having nonaesthetic fillings in anterior teeth (item 7, p =.000, r =.242), and having fractures in anterior teeth (item 8, p =.001, r =.221). also, lower satisfaction scores on vas scale were associated with higher participants ' desire to improve the appearance of their teeth by undergoing the following treatments : orthodontic treatment (p =.003, r =.202), tooth whitening (p =.035, r =.143), dental crowning (p =.004, r =.193), tooth - coloured fillings (p =.007, r =.183), and denture prosthesis (p =.012, r =.169). also, participants ' satisfaction with appearance of teeth (item 1) was associated with more satisfaction with tooth colour (items 2, p =.000, r =.355), less feelings that teeth were poorly aligned (item 4, p =.000, r =.258), and having less fractures in anterior teeth (item 8, p =.038, r =.140). also, participants ' satisfaction with appearance of teeth (item 1) was associated with less desire to improve the appearance of their teeth by undergoing the following treatments : orthodontic treatment (p =.007, r =.182), dental crowning (p =.005, r =.188), and denture prosthesis (p =.038, r =.140). participants ' satisfaction with the colour of their teeth (item 2) was associated with less desire to undergo tooth - whitening (p =.000, r =.334) and tooth - coloured fillings (p =.039, r =.139) to improve the appearance of their teeth. participants ' feeling of having crowded teeth (item 3) was associated with more desire to undergo orthodontic treatment (p =.000, r =.242) and denture prosthesis (p =.000, r =.280) to improve the appearance of their teeth. participants ' feeling that teeth were poorly aligned (item 4) was associated with more desire to undergo orthodontic treatment (p =.000, r =.393), dental crowning (p =.018, r =.160), and denture prosthesis (p =.031, r =.146) to improve the appearance of their teeth. participants ' feeling that their teeth were protruding (item 5) was associated with more desire to undergo orthodontic treatment (p =.000, r =.305), dental crowning (p =.019, r =.158), and denture prosthesis (p =.003, r =.196) to improve the appearance of their teeth. having dental caries (item 6) was associated with more desire to undergo tooth - coloured filling (p =.039, r =.139) and denture prosthesis (p =.007, r =.182) to improve the appearance of their teeth. having nonaesthetic fillings in anterior teeth (item 7) was associated with more desire to undergo orthodontic treatment (p =.021, r =.156), tooth whitening (p =.024, r =.152), dental crowning (p =.005, r =.188), and tooth - coloured filling (p =.000, r =.247) to improve the appearance of their teeth. having fractures in anterior teeth (item 8) was associated with more desire to undergo orthodontic treatment (p =.006, r =.185), dental crowning (p =.009, r =.175), and denture prosthesis (p =.006, r =.183) to improve the appearance of their teeth. table 4 presents distribution of satisfied (n = 110) and dissatisfied (n = 110) participants by the mean vas scale score of satisfaction and participants ' sociodemographic factors. participants who answered yes for the item are you satisfied with the general appearance of your teeth ? scored significantly higher values on vas scale than those whose answer was no (p =.000) (table 4). however, no significant differences were found between the two groups in terms of gender, age, and specialty (p >.05) (table 4). table 5 presents the distribution of satisfied (n = 110) and dissatisfied (n = 110) participants by their answers to the questionnaire items. participants who answered yes for the item are you satisfied with the general appearance of your teeth ? were significantly more satisfied with the colour of their teeth, felt that their teeth were better aligned, and had less fractures in their front teeth than those whose answer was no (p =.000,.000, and.038, resp.) however, no significant differences were found between the two groups in terms of feeling of tooth crowding, feeling of tooth protrusion, having caries in front teeth, and having nonaesthetic fillings in their front teeth (p >.05) (table 5). this study investigated satisfaction with appearance, dental appearance, and desire for treatment to improve dental appearance. previous studies in different populations showed different levels of satisfaction among the studied samples, for example, 47.2% in malaysia, 57.3% in turkey, 65% in palestine, 65.5% in jordan, and 76% in uk. this could be attributed to the use of different measures to evaluate satisfaction, cultural factors, religion, and racial factors as well as to that dental appearance is affected by individual characteristics, compliance, or unrealistic expectations [7, 13, 15, 21 ]. other studies reported higher levels of satisfaction with tooth colour [9, 11, 13 ]. this could be attributed to the difference in sample size and used measures to evaluate satisfaction, psychological factors, and religious and sociocultural factors. this finding supports the idea that satisfaction with tooth colour strongly impacts on satisfaction with dental appearance [9, 13 ]. furthermore, tooth whitening was found to be the most desired treatment by participants in order to improve tooth appearance, and this further supports the impact of tooth colour on satisfaction with dental appearance. satisfaction with dental appearance was significantly related to the feeling of presence of poor tooth alignment and presence of fractured anterior teeth. this could be due to that poor tooth alignment and anterior tooth fractures change the appearance of teeth causing them to be less attractive. this was supported by the finding that satisfaction with dental appearance had significant relation to the desire for orthodontic, dental crowning, and denture treatments in order to improve the appearance of teeth. this concurs the results of previous studies [13, 15, 21, 25 ]. however, it disagrees with the results of previous studies that found more satisfaction with appearance among older subjects [11, 20, 24 ]. again, this could be attributed to the use of different measures to evaluate satisfaction and also to that dental appearance is affected by individual characteristics as well as cultural, religious, or racial factors [7, 13, 15, 21 ]. this is similar to the findings reported by previous studies [11, 15, 20, 21 ]. however, the results disagree with the findings of previous studies that found less satisfaction with dental appearance among females [9, 13, 23 ]. different samples, different measuring techniques, individual characteristic, and psychology, as well as cultural, religious, and racial back grounds might explain this controversy regarding the relationship between gender and satisfaction with dental appearance and tooth colour. in this study, satisfaction with general appearance (identified by vas scale) had a significant relation to satisfaction with teeth. this concurs the idea that satisfaction with teeth and tooth colour contributes to the overall satisfaction with appearance [10, 15, 20 ]. this was further supported by the findings of this study where satisfaction with general appearance was found to have a significant relation with participants ' desire for all types of treatments that could improve the appearance of teeth. in contrast, other studies found no relation between satisfaction with dental appearance and satisfaction with general appearance [21, 26 ]. cultural, social, psychological, economic, or religious factors in different populations might affect satisfaction with the appearance of teeth. further studies are required to identify the potential effects of such factors in this regard. high levels of dissatisfaction with tooth appearance and tooth colour were reported in this study. higher levels of dissatisfaction with tooth colour contributed to the increased dissatisfaction with appearance of teeth. dissatisfaction with tooth appearance, colour, alignment, and condition were significantly related to the high desire for treatments that improve dental aesthetics. | objective. to identify participants ' satisfaction with appearance and the desired treatment to improve aesthetics. materials and methods. 220 participants (127 males and 93 females, mean age = 21.4 1.5 years) were recruited into the study. a structured questionnaire was used to assess patients ' satisfaction with appearance and what treatment they desire to improve aesthetics. participants scored the level of satisfaction with appearance using visual analogue scale. results. the vas mean score of satisfaction with general appearance was 6.8 2.3. half participants were dissatisfied with tooth appearance and 65.9% were dissatisfied with tooth colour. higher vas scores were associated with higher desire for all treatments that improve tooth appearance (p <.05). dissatisfaction with tooth appearance increased with increased dissatisfaction with teeth colour, feeling of poor tooth alignment, presence of fractured anterior teeth, and increased desire for orthodontic, crowns, and dentures treatments (p <.05). dissatisfaction with tooth colour was associated with increased desire for tooth whitening and tooth coloured fillings (p <.05). conclusions. participants had high levels of dissatisfaction with tooth appearance and tooth colour. dissatisfaction with tooth colour contributed to the increased dissatisfaction with tooth appearance. dissatisfaction with tooth appearance, colour, alignment, and condition was significantly related to high desire for aesthetic treatments. |
health is a common theme in most cultures and is a fundamental human right without distinction of race, religion, and political belief, economic and social condition. oral health is a standard of health of the oral and related tissues that enables an individual to eat, speak and socialize without active disease, discomfort or embarrassment and contributes to the general well being. it is concerned with maintaining the health of craniofacial complex, the teeth and gums, as well as the tissue of the face and head that surrounds the mouth. oral diseases are major health problem, especially in children, owing to their high prevalence and incidence in all the regions of the world. at the global level, prevalence rates and pattern of oral disease have changed considerably over the past two decades. increasing levels of dental caries among children are observed in some developing countries, especially for those countries where community and school based preventive oral care programs are not established. health education is an important tool of public health and an effective primary preventive method. research has revealed the efficacy of health education in controlling plaque and thereby dental diseases. health education programs are not isolated events but educational aspects of any curative, preventive and promotional health activity. health education for the school age child is a specialized field within the broad discipline of education. no evaluation study of oral health education program for school children have been reported till date in himachal pradesh. hence, the present study has been undertaken to evaluate the impact of oral health education on the status of plaque, dental caries and gingival health among 12 and 15 years old children attending government school in shimla city. a total of 306 children of both genders studying at government senior secondary school sanjauli, shimla aged 12 and 15 years were examined on the first visit. the study was conducted over a period of 4 months from may 2010 to august 2010. prior to the conduct of the study, the purpose was clearly explained to the school authorities and written permission was obtained. consent was obtained from the parents and teachers of the students. a one time verbal consent inclusion criteria : all the children aged 12 and 15 years who were present in the school on the day of examination and were willing to participate in the study. exclusion criteria : those children under antibiotic therapy within 30 days of the initial examination, undergone oral prophylaxis and those who were absent on the day of examination were excluded from the study. at the initial visit, each subject was interviewed using a specially designed questionnaire and examined for plaque, gingival and caries status by using silness and loe plaque index, loe and silness gingival index and who modified dmft index. a trained recorder was made to sit close to the examiner so that instructions could be heard easily. and kappa statistics showed higher degree conformity with the observations (kappa co - efficient = 0.9). after collecting the base line data, oral health education was imparted to children, which included instructions on the importance of maintenance of oral hygiene, use of appropriate oral hygiene aids and demonstration of the method of tooth brushing. bass method of tooth brushing was taught as it is the easiest and most effective technique for the children to learn. oral health education was supplemented with the use of teaching resources such as chalk and blackboards, models, posters and charts. daily, twice tooth brushing was emphasized for prevention of bad breath and tooth decay. to evaluate the effectiveness of the educational program, the presence of plaque, gingivitis and dental caries was assessed at the end of 3 months. chicago) to find out the differences between plaque scores, gingival and caries status before and after the education program. paired t - test was used to evaluate the changes in the plaque and gingival status at baseline and after 3 months. wilcoxon signed rank sum test was used to find the difference in mean caries status (dt) of the subjects before and after dental health education. a total of 306 school children were examined at the baselines and 276 were examined after 3 month period at the time of follow - up. 12 years, 146 school children were examined out of which 79 (54.1%) were males and 67 (45.9%) were females. among 130, 15 years old children 71 (54.6%) and 59 (45.4%) were males and females, respectively. majority of school children 257 (93.1%) were using toothbrush, with once a day history of tooth brushing 220 (79.7%). two hundred and thirty six (91.8%) practiced a faulty horizontal pattern of cleansing with brush, whereas 21 (8.2%) employed the vertical method of tooth brushing [table 1 ]. significant reduction was observed for plaque and gingival score (p > 0.05) among 12 and 15 years subjects irrespective of gender [tables 2 and 3 ]. distribution of study subjects according to different variables plaque score of the study subjects at baseline and 3 months after oral health education mean gingival score of study subjects at baseline and 3 months after oral health education mean caries status of study subjects at baseline and 3 months after oral health education the present study was targeted at school going children because of the ease of accessibility. school forms an environment that provides considerable number of individuals of the same age and strata. pretest questionnaire was used to access the oral hygiene practices and visit to a dentist. since the analysis of the questionnaire revealed poor dental attendance and inadequate oral hygiene habits, the oral health education emphasizing on proper method of tooth brushing, the importance of oral hygiene and regular dental checkup was provided. in 15 years age group, no significant difference was found among female study subjects, whereas the reduction in the plaque scores was significant among male subjects. reason for the less improvement of oral hygiene among 12 years old study subjects may be because of failure of younger study subjects in complying with the instruction given during oral health education program. reduction in plaque and gingival scores may be due to change in tooth brushing frequency and adoption of appropriate tooth - brushing technique after oral health education. older study subjects, particularly female subjects showed better understanding of oral health education program. ajith krishnan,. have found similar significant reduction in mean plaque scores after oral health education in their studies. similar results were obtained in the studies, which revealed an improvement in silness and loe index, which has been used in the present study also. however, contrary to the results of this study, franklin,. and significant reduction in the gingival scores was observed in the study depicting the transient improvement of gingival health. similar results were observed in the study conducted by shenoy,. and petersen,. reduction in the mean caries status of the study subjects was insignificant, which is in agreement with the results of the studies. the study revealed that short term oral health education program may be useful in improving oral hygiene and gingival health but not effective in improving caries status. reinforced oral health education may improve oral hygiene and gingival health to a significant extent, but may prove inadequate in the long run if low cost oral hygiene aids are not made available to the general population, which is an uphill task. such programs should be conducted annually in the schools with the provision of oral hygiene aids at concessional rates. in the present study, parental participation was not included, which is otherwise essential for the achievement of long term benefits of the enhancement of the program for implementation during their stay at home. long term value of the improvement need to be confirmed by further studies because improved oral hygiene in children may exist only during the program or a short period thereafter. a chapter on importance of oro - dental health should be included in the syllabus. coordinating efforts should be made between school personnel, health professionals and parents to ensure long - term benefits of such programs. | background : health education for the school age child is a specialized field within the broad discipline of education. oral health education program are educational aspects of any curative, preventive and promotional health activity.aim:the study has been undertaken to evaluate the impact of oral health education on the status of plaque, gingival health and dental caries among 12 and 15 years old children attending government school in shimla city.materials and methods : two hundred and seventy six school children participated in the study. the study was conducted over a period of 4 months from may 2010 to august 2010 in government senior secondary school, sanjauli. plaque, gingival and caries status was assessed by using silness and loe plaque index, loe and silness gingival index and who modified dmft index, respectively. data was analyzed using the software spss version 15. paired t - test and wilcoxon signed rank sum test were used appropriately for statistical comparisons. p value 0.05 was considered statistically significant.results:overall mean plaque score and gingival score decreased significantly after oral health education irrespective of gender. however, decrease in plaque score among 15 years old female children and gingival scores among 12 and 15 years old female subjects was not significant. difference in mean caries status was statistically insignificant among all the subjects.conclusion:short term oral health education program may be useful in improving oral hygiene and gingival health. coordinating efforts should be enhanced between school personnel, parents and health professionals to ensure long - term benefits of such program. |
however, all these therapies are associated with a potential risk of damaging the overlying retina. we report a case of circumscribed choroidal hemangioma (cch) in a 59-year - old man refractory to laser treatment. visual acuity was 20/200 and a serous macular detachment was present. the cch was treated with oral propanolol, whereupon visual acuity improved to 20/20 and the macular detachment resolved without systemic or local adverse effects. propanolol is a -blocker commonly used in cardiology that may induce endothelium vasoconstriction and inhibit endothelial proliferation. it has been shown to be effective in infantile facial hemangiomas, and proved safe and effective for the cch in our patient. there are two types of choroidal hemangiomas : circumscribed choroidal hemangioma (cch) and diffuse choroidal hemangioma (dch). cch is a rare benign, commonly asymptomatic vascular tumor which is usually diagnosed in adulthood when it becomes symptomatic with visual acuity decline, visual field defect or metamorphopsia due to exudative retinal detachment. cch appears as an orange choroidal mass with indistinct borders, usually within the macular area. in contrast, dch is usually evident at birth and typically occurs as part of neuro - oculocutaneous hemangiomatosis (sturge - weber syndrome), but it frequently does not become manifest until adolescence. dch appears as a diffuse orange choroidal thickening. despite this, focal regions of excessively thickened choroid may simulate cch. choice of treatment for choroidal hemangiomas is based on tumor location, presence of subretinal fluid, extent of symptoms, and potential for visual recovery. when vision loss occurs, different treatment options include : photodynamic therapy (pdt), transpupillary thermotherapy, vascular endothelial growth factor (vegf), laser photocoagulation, episcleral plaque radiotherapy, cryotherapy, external beam radiotherapy, and stereotactic radiotherapy. despite their efficacy, inducing tumor atrophy, all these treatments are associated with the potential risk of damaging the overlying retina. a new treatment has emerged for infantile hemangiomas : systemic propanolol. it seems to be safe and effective in orbital, eyelid and facial hemangiomas in children. a 59-year - old man with arterial hypertension (150/95 mm hg) was seen in our department because of visual acuity (va) decline and metamorphopsia of his left eye (os) for 5 weeks. we performed laser photocoagulation (the patient refused treatment with pdt) and 3 months later va improved to 20/100 but foveal serous detachment persisted (fig. 2). thereafter we decided to begin treatment with oral propanolol daily (120 mg). one month later, va improved to 20/20 and serous foveal detachment disappeared (fig. 3). in addition, his arterial hypertension was under control (125/80 mm hg). after 8 months of follow - up, both va (20/20) and the macular thickness remained stable. their physiopathology is still poorly understood ; nonetheless, vegf and basic fibroblast growth factor (bfgf) have been involved in infantile hemangiomas growth. increased levels of these factors have been detected in cutaneous hemangiomas and vegf - receptors have been evidenced in proliferative infantile hemangiomas. in addition, it has been shown that hypoxia - induced factors important for postnatal vasculogenesis like hypoxia - inducible factor 1 (hif-1), hif-2 and vegf are up - regulated in children with proliferative infantile hemangiomas, leading to vegf expression by endothelial cells [4, 5, 6, 7 ]. propranolol is a nonselective -adrenergic receptor blocker, and the adrenergic system is the major regulator of cardiac and vascular function. capillary endothelial cells express 2-adrenergic receptors [7, 8 ] which modulate the release of nitric oxide, causing endothelium - dependent vasodilatation. in addition, -adrenergic receptor stimulation can induce modifications of signal transduction pathways of angiogenic factors such as vegf or bfgf, enhancing neoangiogenesis in ischemic events [7, 8, 9 ]. moreover, a study of the use of -adrenergic receptor blockers for the treatment of cardiac hypertrophy revealed that carvedilol reversed levels of hif-1 and vegf to baseline values. therefore propanolol may induce endothelium vasoconstriction, decrease expression of bfgf, hif-1 and vegf through the raf mitogen - activated protein kinase pathway [4, 8 ] and inhibit endothelial proliferation. laser photocoagulation induces resolution of subretinal fluid, exudative retinal detachment and tumor regression ; however, subretinal fluid has a high tendency to recur, and 40% of patients require additional treatment. radiotherapy is effective in the majority of cases, but it is associated with significant treatment morbidity. transpupillary therapy may avoid some of the limitations of laser photocoagulation (especially the failure to induce tumor regression) while avoiding complications of radiotherapy. verteporfin is an important vaso - occlusive treatment, and pdt may destroy the tumor without damaging the overlying retina and its vascularization. if the tumor or subretinal fluid persist further, pdt re - treatments can be performed, but repeated treatments and over - treatment or overlapping pdt spots may result in delayed choroidal atrophy and fibrosis, limiting the visual outcomes [2, 13 ]. moreover, some authors suggest that it is preferable to avoid treatments within the fovea in order to decrease fibrosis and retinal pigment epithelium (rpe) changes. long - term visual prognosis for patients with cch is guarded secondary to vision loss induced by subretinal fluid, macular edema, foveal distortion, and treatment side effects, like choroidal atrophy, fibrosis and rpe changes. nowadays, eyelid and orbital infantile hemangiomas can be treated successfully with oral and topical -blockers [14, 15 ]. this may entail a new treatment for refractory hemangiomas, foveal hemangiomas, or hemangiomas the treatment of which implies high risk of scarring or rpe changes, like tumors that need pdt retreatment or hemangiomas with thin retinal detachments. we hypothesized that this treatment would be useful in our patient, who showed no response to laser treatment, refused pdt, and presented poor va. although the treatment with -blockers has been effective and safe in our patient, we are not able to determine whether laser treatment could have modified the final outcome. the good visual results after treatment with oral -blockers may open a new treatment research line for cch. | introductionseveral therapeutic approaches have been developed to treat choroidal hemangioma. however, all these therapies are associated with a potential risk of damaging the overlying retina.case reportwe report a case of circumscribed choroidal hemangioma (cch) in a 59-year - old man refractory to laser treatment. visual acuity was 20/200 and a serous macular detachment was present. the cch was treated with oral propanolol, whereupon visual acuity improved to 20/20 and the macular detachment resolved without systemic or local adverse effects.discussionpropanolol is a -blocker commonly used in cardiology that may induce endothelium vasoconstriction and inhibit endothelial proliferation. it has been shown to be effective in infantile facial hemangiomas, and proved safe and effective for the cch in our patient. further studies are needed to confirm our observation. |
the effects of the phosphorylation of serine 273 on the protein structure of ppar were studied by comparing the structures of phosphorylated serine 273 and the wild type, with a particular focus on the dbd of the receptor complex, using md simulations. although a detailed study on dbd dynamical properties under perturbing stimuli and its relation to dr1-binding specificity is not the subject of this paper, a simple comparison of time - averaged molecular motions can indicate regions of altered stability. indeed, serine 273 phosphorylation affects the stability of rxr, mostly in the zinc - finger helix which is in contact with dna 's major groove (figure 2a). the general change in the structure of dbd - rxr is relatively small : 0.5 of averaged atomic displacement (root - mean s.d.) (figure 2b). nevertheless, the whole domain became less stable, as shown in differences in the root mean square fluctuation profiles (figure 2c), with the most remarkable fluctuation occurring in the helical region of one of the zinc fingers. this indicates that phosphorylation introduces changes in the dna - binding domain, shifting the protein population towards a state which could contribute to an increase in disease - related gene expression. the lbd of ppar is not affected on a large scale ; despite some differences, both profiles are following a similar trend (figure 2d). to study the effects of modified ppar gene regulation, we reconstructed an interaction network consisting of 235 degs, 152 of which were upregulated in the phosphorylated state (wild type) and 48 of which were upregulated in the mutated state (s273a mutant). subsequently, we searched a sub - network that makes an important contribution to the stability of the global network. we then selected the top 10 statistically significant motif types consisting of 2, 3, 4 or 5 nodes using a z - value - based ranking, all having a p - value < 0.01 (only 10 of 1000 randomly generated networks contained motifs at a higher frequency). we examined the expression profile of the genes in each motif to ensure that the expression profile was consistent in all members of the identified motif : among the top ranked 2-, 3-, 4- or 5-node motifs that exhibited multi - stable behavior, we identified a number of 3- and 4-node motifs that were consistent with expression values (figure 3). we thus identified 39 genes involved in 55 switches forming a single cluster, which we call the core ' network (figure 4). computation of the stability of the core network cluster reveals two attractors, in which all genes are either in an on or an off state, which exactly match the expression pattern. the two most commonly found motifs are positive forward loop motifs with off / off / off changed to the on / on / on states during the course of the disease. consistent with this, the in silico perturbation of some nodes of the core cluster trigger a transition from the off to the on attractor, but the opposite transition (from on to off) is not triggered by the perturbation of any node in the core. in all, 34 nodes in the cluster are capable of triggering the transition from off to on when they are perturbed. in contrast, five nodes (col1a1 (collagen type i-1), col1a2 (collagen type i-2), kruppel - like factor 5 (klf5), perforin-1 and runt - related transcription factor 1) constitute a set of genes that potentially could be in the on state, taking part in different processes without leading to the activation of the cluster as a consequence. we can see in figure 5 that perturbation of klf5, involved in a motif with early growth response 1 (egr1), does not trigger the transition of the cluster from off to on (see supplementary figure 1 for all perturbation experiments). furthermore, we compared properties of genes in the core network with remaining genes in the global network (supplementary table 1). specificity of inter - gene interactions may be reflected in the network modularity. using the newman girvan algorithm, we identified 11 clusters of which clusters 1 and 2 are mainly occupied by core genes. to identify inter - cluster connectors, we calculated the participation coefficient that on average was significantly different when comparing nodes in the core and global networks. the median participation in the core and global networks was 0.45 and 0, respectively ; the distributions in the two groups differed significantly (mann whitney the betweenness centrality of a gene is a centrality measure that is proportional to the number of shortest paths between genes in the network that go through the gene in question. high betweenness centrality corresponds to high level of inter - node communication, and is therefore an appropriate measure for highlighting which genes link different molecular processes and pathways. median betweenness centrality in the core and global networks are 243 and 0, respectively ; the distributions in the two groups differ significantly (mann whitney wilcoxon w=1430, n1=44, n2=191, p - value=3.65e-14). we identified a group of genes, which act as potential hubs and exist in the core network (having a betweenness centrality score 0.10 in the core network, as well as 2900 in the global network, being the top four ranking genes in both cases). the genes with the highest betweenness centrality were hif1a (hypoxia - inducible factor 1-subunit), egr1, stat1 (signal transducers and activators of transcription 1) and cxcl12 (chemokine (c x c motif) ligand 12) and they are all overexpressed in the case of phosphorylated ppar (the putative disease state). we examined these genes in more detail with regard to their association with adipogenesis, for which ppar is the master regulator under any condition, to confirm that our core network is consistent with experimental studies. we found that this was indeed the case : the role of the transcription factor hif1a in adipocyte differentiation has been described previously, egr1 functions as a transcriptional regulator the expression of which is rapidly induced during the differentiation of murine 3t3-l1 adipocytes, stat1 also acts as a transcription activator, which is rapidly activated in the 3t3-l1 adipocyte cell culture model, and cxcl12 is a chemokine, which demonstrates a significant increase in expression in differentiating 3t3-l1 preadipocytes. the metabolic syndrome is a common disease and is associated with increased incidence of type ii diabetes mellitus and cardiovascular mortality. its treatment is designed to target the different components of the syndrome (such as high triglyceride, cholesterol and glucose levels and high blood pressure). thus, the discovery of ppar as a master regulator of adipogenesis, the decreased activity of which was also shown to be linked to insulin resistance, was very important. it was assumed that increasing ppar activity would potentially represent a unifying strategy to target the metabolic syndrome as an entity. unfortunately, unwanted side effects such as weight gain, heart failure and even osteoporosis, as well as discrepant results between genetic and pharmacological modulations of ppar, outbalance the observed clinical benefits, which mostly rely on improved blood sugar control and lipid metabolism. ppar still represents a desirable drug target, but research over the last decade showed that its biological function with regard to adipogenesis is a balanced and thrifty response. aspects such as co - factor recruitment and function and/or post - translational modifications have to be taken into consideration, before more specific / distinct new approaches to modify ppar action can lead to new compounds. ppar expression is also influenced by gata - binding protein 2 (gata2), a transcription factor specifically expressed in the stromal vascular fraction of the adipose tissue, which is also where preadipocytes are located. the transition from preadipocyte to adipocyte is blocked, at least in part, by gata2 's suppression of ppar ; insulin has downstream effects on the kinase v - akt murine thymoma viral oncogene homolog 1 (akt) which phosphorylates gata2, impairing gata2 's translocation to the nucleus, leading to a reduced suppression of ppar in the nucleus, thus promoting adipogenesis. in the insulin - resistant state, the phosphorylation of gata2 by akt is disturbed and consequently adipogenesis regulation is dysregulated. ppar-driven adipogenesis is reduced, whereas the remaining preadipocytes start expressing pro - inflammatory cytokines, an observation contributing to the understanding of the puzzling phenomenon of improved metabolic outcome of adipogenesis - promoting ppar agonists. gata2 was not among the degs in our study, but this is not unexpected as gata2 's activity is regulated through phosphorylation. metalloproteinase inhibitor 3 (timp3) is a member of our core network and was originally linked to sorsby 's fundus dystrophy, which is an autosomal dominant macular degeneration disorder. this gene has been linked to a number of other diseases including, more recently, the metabolic syndrome, in particular with insulin resistance, hepatic inflammation, dyslipidemia and atherosclerosis in diabetic subjects. a possible mechanism of how ppar and timp3 are connected may be that timp3 expression influences c / ebp (ccaat / enhancer - binding protein beta) expression, which in turn induces c / ebp (ccaat / enhancer - binding protein alpha) and ppar during adipocyte differentiation. post - translational modification can be seen as perturbations of the environment in which a protein exists. the effect of such a post - translational modification on a protein is a good situation to examine the hypothesis of the population shift of molecules, which proposes that the external stimuli stabilize and therefore populate pre - existing, although previously non - overrepresented molecule states. in biological terms, a population of protein conformers may be shifted towards malfunctioning, diseased - related conformation(s), that is, the sustained perturbation of protein structure is a potential trigger for changes of molecular networks associated with specific disease phenotypes. choi. studied the role of ppar in the context of post - translational modification, that is, the phosphorylation of serine 273 in the lbd of ppar ; a number of genes were identified the differential expression of which is caused by a change in the conformation of the pparrxr dimer when serine 273 is phosphorylated. this modification was shown to influence gene expression of disease (obesity, insulin resistance)-relevant genes independently from the general receptor transcriptional activity. to study the effects of perturbation on a structural level, we performed a md simulation to examine the impact of phosphorylation on the pparrxr dimer, demonstrating that phosphorylated ppar destabilizes the pparrxr dimer, which may be a mechanism explaining the altered transcriptional regulation of ppar target genes. interestingly, phosphorylation at another residue (ser112) modulates ppar activity with positive effects on glucose and lipid metabolism in vivo possibly by decreasing its affinity for endogenous ligands. furthermore, we analyzed perturbations of biological networks associated with changes in ppar, by examining the degs comparing wild - type phosphorylated ppar with mutant non - phosphorylated ppar. a network was constructed connecting these degs using the biomedical literature - based resnet mammalian database. therefore, it can be expected that there will be little bias introduced by orthologs from exotic species, that is, species distant to mouse in the phylogenetic tree. we found an enrichment of bi - stable switches in wild - type phosphorylated state containing genes that significantly change their expression levels in an adipogenesis - dependent manner. of particular interest were the genes with the highest betweenness centrality : hif1a, egr1, stat1 and cxcl12. these genes are all overexpressed in the putative disease state and all have been previously described to have a role in adipogenesis. betweenness centrality is a measure of the influence that a node has over the spread of the information through the network, and our finding that the hub genes with the highest levels of betweenness centrality consist of three transcriptional regulators and one chemokine is consistent with them functioning as information propagators in the network. the bi - stable motifs we identified assemble an interconnected core network of genes, which have two steady - stable states corresponding to gene functionality states. this stable core network gives robustness to the cellular network by keeping the system in the non - phosphorylated putative healthy state ; however, once the system is pulled out of this state, it stabilizes the network in a phosphorylated putative disease - related state. the therapeutic ligand present in the intact structure of the pparrxrdna complex (pdb i d : 3dzy) was replaced with docosahexanoic acid, which is a natural ligand of ppar. md simulations were performed with gromacs and served as a tool to generate perturbed states of proteins. implicit solvent generalized born simulations with the amber99sb - ildn force field were carried out for wild - type and mutant proteins : energy minimizations were performed and the production runs were 50 ns in length. for our analysis, we extracted degs from the results of gene expression analysis experiments by choi., in which ppar-null mouse embryonic fibroblasts were transfected with wild - type ppar (phosphorylated) or the s273a ppar mutant (not phosphorylated). the cutoffs for selecting the degs were uncorrected p - values 0.008 and corrected false discovery rate p - values 0.15. subsequently, the resnet mammalian database from ariadne genomics (http://www.ariadnegenomics.com/) was used to construct an interaction network of 235 degs with directed interactions (the global ' network). this database includes biological relationships and associations, which have been extracted from the biomedical literature using ariadne 's medscan technology. medscan processes sentences from pubmed abstracts and produces a set of regularized logical structures representing the meaning of each sentence. the resnet mammalian database stores information harvested from the entire pubmed, including more than 715 000 relations for 106 139 proteins, 1220 small molecules, 2175 cellular processes and 3930 diseases. motif detection was performed using the fanmod algorithm for the global network and limited to motifs consisting of 2, 3, 4 or 5 nodes each. each resultant topology was analyzed in comparison with 1000 separately randomized versions of the initial network. using both the original and the randomized versions, z - scores and p - values the z - score of a motif is the original frequency minus the random frequency divided by s.d. the p - value of a motif is the number of random networks in which it occurred more often than in the original network, divided by the total number of random networks. of the bi - stable switch motifs we identified, we also examined the expression profile of the genes in each motif to ensure that the expression profile was consistent in all motif members. we constructed a network (the core ' network) consisting of 39 degs that occur in bi - stable motifs, which are significantly overrepresented in the global network and the genes of which are in the stable state, matching the experimental expression values. to compute the attractors of the core network the program converts the network into a continuous dynamical system based on ordinary differential equations. in the absence of detailed kinetic parameters, the program interpolates a sigmoid curve between the states completely on and completely off for each node. squad first calculates the steady states found in a discrete dynamical system (boolean model) and then uses these states as a guide to localize the steady states in the continuous model. each perturbation is a single pulse that changes the state of the node from 0 to 1 in the off attractor or from 1 to 0 in the on attractor as initial states of the system. the degs of the core and global networks were divided into groups by topological modularization using the newman girvan algorithm. participation (p) of a node in intra - modular communication is calculated as follows : where nm = number of modules, ks = number of connections with module s, ki = total degree of module k. analysis of betweenness centrality was calculated with igraph library in r comparing the core and global networks. the participation coefficient and betweenness centrality results for the core and global network groups were compared with the mann we also examined these genes in more detail with regard to their association with adipogenesis, for which ppar is the master regulator under any condition, to confirm that our core network is consistent with experimental studies. the therapeutic ligand present in the intact structure of the pparrxrdna complex (pdb i d : 3dzy) was replaced with docosahexanoic acid, which is a natural ligand of ppar. md simulations were performed with gromacs and served as a tool to generate perturbed states of proteins. implicit solvent generalized born simulations with the amber99sb - ildn force field were carried out for wild - type and mutant proteins : energy minimizations were performed and the production runs were 50 ns in length. for our analysis, we extracted degs from the results of gene expression analysis experiments by choi., in which ppar-null mouse embryonic fibroblasts were transfected with wild - type ppar (phosphorylated) or the s273a ppar mutant (not phosphorylated). the cutoffs for selecting the degs were uncorrected p - values 0.008 and corrected false discovery rate p - values 0.15. subsequently, the resnet mammalian database from ariadne genomics (http://www.ariadnegenomics.com/) was used to construct an interaction network of 235 degs with directed interactions (the global ' network). this database includes biological relationships and associations, which have been extracted from the biomedical literature using ariadne 's medscan technology. medscan processes sentences from pubmed abstracts and produces a set of regularized logical structures representing the meaning of each sentence. the resnet mammalian database stores information harvested from the entire pubmed, including more than 715 000 relations for 106 139 proteins, 1220 small molecules, 2175 cellular processes and 3930 diseases. motif detection was performed using the fanmod algorithm for the global network and limited to motifs consisting of 2, 3, 4 or 5 nodes each. each resultant topology was analyzed in comparison with 1000 separately randomized versions of the initial network. using both the original and the randomized versions, z - scores and p - values the z - score of a motif is the original frequency minus the random frequency divided by s.d. the p - value of a motif is the number of random networks in which it occurred more often than in the original network, divided by the total number of random networks. of the bi - stable switch motifs we identified, we also examined the expression profile of the genes in each motif to ensure that the expression profile was consistent in all motif members. we constructed a network (the core ' network) consisting of 39 degs that occur in bi - stable motifs, which are significantly overrepresented in the global network and the genes of which are in the stable state, matching the experimental expression values. to compute the attractors of the core network the program converts the network into a continuous dynamical system based on ordinary differential equations. in the absence of detailed kinetic parameters, the program interpolates a sigmoid curve between the states completely on and completely off for each node. squad first calculates the steady states found in a discrete dynamical system (boolean model) and then uses these states as a guide to localize the steady states in the continuous model. each perturbation is a single pulse that changes the state of the node from 0 to 1 in the off attractor or from 1 to 0 in the on attractor as initial states of the system. the degs of the core and global networks were divided into groups by topological modularization using the newman girvan algorithm. participation (p) of a node in intra - modular communication is calculated as follows : where nm = number of modules, ks = number of connections with module s, ki = total degree of module k. analysis of betweenness centrality was calculated with igraph library in r comparing the core and global networks. the participation coefficient and betweenness centrality results for the core and global network groups were compared with the mann we also examined these genes in more detail with regard to their association with adipogenesis, for which ppar is the master regulator under any condition, to confirm that our core network is consistent with experimental studies. | peroxisome proliferator - activated receptor gamma (ppar) is a key regulator of adipocyte differentiation and has an important role in metabolic syndrome. phosphorylation of the receptor 's ligand - binding domain at serine 273 has been shown to change the expression of a large number of genes implicated in obesity. the difference in gene expression seen when comparing wild - type phosphorylated with mutant non - phosphorylated ppar may have important consequences for the cellular molecular network, the state of which can be shifted from the healthy to a stable diseased state. we found that a group of differentially expressed genes are involved in bi - stable switches and form a core network, the state of which changes with disease progression. these findings support the idea that bi - stable switches may be a mechanism for locking the core gene network into a diseased state and for efficiently propagating perturbations to more distant regions of the network. a structural analysis of the pparrxr dimer complex supports the hypothesis of a major structural change between the two states, and this may represent an important mechanism leading to the differential expression observed in the core network. |
adolescence is a period of profound changes in the brain structure with a complex interplay between biological, psychological, and social factors. mental health problems commonly emerge in adolescence and many adolescents have enduring disorders rather than simply a transient teenage emotional turmoil. individuals who develop psychosis often experience a prodromal phase (also known as at - risk mental state), which typically involves changes in perception, behavior, cognition, mood, and physiology (yung and mcgorry, 1996, mcgorry., 2001). this phase is ambiguous because of the non - specificity of symptoms that are commonly observed during development in adolescence, and the low predictive power in identifying individuals who make a transition to psychosis (mcgorry and killackey, 2002). structured instruments, such as structured interview for prodromal syndromes (sips ; mcglashan., 2001), scale of prodromal symptoms (sops ; miller., 1999), the bonn scale for the assessment of basic symptoms (bsabs ; gross., 1987), the personal assessment and crisis evaluation (pace ; phillips., 2002), and the comprehensive assessment of at risk mental states (caarms ; yung., 2004), can help better characterize the prodromal phase although these lack predictive value. in recent years, better characterization of the prodrome phase of the disorder and improved prediction algorithms have shown to be effective in informing the timing and management process of early intervention (cadenhead., 2010). (2004) conducted a 12-month follow - up on individuals with ultra high - risk, proposing a four - or - more algorithm for risk identification, including poor functioning, long duration of symptoms, high levels of depression, and reduced attention. the algorithm showed a positive predictive value of 80%, with heightened specificity and sensitivity values (yung., 2004). in the north american prodrome longitudinal study (napls) cohort, cannon. (2008) followed up 291 treatment - seeking patients with prodromal symptoms at regular intervals for up to 2.5 years. the study showed conversion rates to be 35% at follow - up, with baseline characteristics predictive of psychosis to include a genetic predisposition of schizophrenia alongside reduced overall functioning, heightened levels of abnormal thought content, greater psychosocial impairment, and a history of substance abuse (cannon., 2008). similar findings have been reported in the european prediction of psychosis (epos) study (ruhrmann., 2010). however, there is a paucity of biomarkers for this phase with good predictive value either for course or treatment response. although the identification of biomarkers for schizophrenia is in its early stages, some attempts have been made to discover biomarkers in eos (taurines. there is limited evidence that management and symptom specific treatment of help - seeking prodromal patients may delay or prevent the onset of psychosis ; however the uncertainty of course prediction adds to the risk of stigmatization and heightened anxiety in individuals and their families. additionally, the eos cases tend to be insidious, non - episodic, and typically result in rapid deterioration ; providing a limited chance to study and intervene during this window. the identification of biomarkers would improve the ability to intervene during the prodromal period or earlier. the search for clinically relevant biomarkers is a challenging task in a heterogeneous disorder like schizophrenia but studying eos provides a unique opportunity as the phenotype is relatively homogenous, associated with less risk of secondary influences from disease associated alterations of environment (e.g., marijuana, smoking, hospital admissions, etc.) and typically shows more salient genetic loading (vyas. for example, a neurocognitive deficits in schizophrenia is considered a core feature of the illness (meta - analysis, heinrichs and zakzanis, 1998 ; heaton., 2001 ; keefe and fenton, 2007) and studies on ultra high - risk cohorts suggest that impairments in olfactory identification and spatial working memory (measures targeting the dorsolateral prefrontal cortex and cortical physiological processes), may have a strong predictive value for conversion to psychosis (brewer., 2006). neurophysiological measures such as electroencephalography, event - related potentials, prepulse inhibition, and mismatch negativity, also show promise as potential biomarkers (javitt. 2012) but further work to elucidate the relationship of such measures with specific clinical expression (e.g., negative symptoms, cognitive functioning) is needed. neuroimaging studies have shown relatively less predictive value despite consistent reports of progressive structural brain abnormalities associated with schizophrenia (gogtay., 2004 ; rapoport and gogtay, 2011), and non - psychotic siblings of cos patients (gogtay., 2003 ; greenstein., 2011). some studies have suggested that high - risk individuals that go on to become psychotic show less gray matter volume in the right medial temporal, lateral temporal, inferior frontal cortex, and in the cingulate cortex bilaterally, while individuals who do not develop psychosis show changes restricted to the cerebellum (pantelis., 2003). there is a relative dearth of evidence - based studies of antipsychotic (both typical and atypical) efficacy in eos, partly because of the rarity of the population and partly because it is difficult to do treatment trials in children with severe illness (gogtay and rapoport, 2008). taken together, these studies suggest that although first - generation antipsychotics (fgas) improve positive symptomatology, they elicit significant extra pyramidal side effects, tardive dyskinesia, and prolactin elevations (pool., 1976 ; realmuto., 1984 ; spencer., 1992 ; findling.,, second - generation antipsychotics (sga, or atypical antipsychotics) have become the mainstay of therapy in the treatment of eos, because of their potential for lower propensity to induce extrapyramidal symptoms and reduced risk of tardive dyskinesia (madaan., 2008 ; however the cochrane review identified 6 (clinical trials) studies with a total of 256 children and adolescents, to examine the effects of antipsychotic medication for eos (kennedy., 2007). the authors concluded that there was limited data that supported one antipsychotic medication over another for the treatment of eos. there was no superiority of sgas over fgas, given the evidence showing small differences in effect size for alleviating positive and negative symptoms. furthermore, an 8-week, government - funded, randomized double - blind trial on eos entitled treatment of early - onset schizophrenia spectrum (teoss) showed that sgas, risperidone and olanzapine, were not superior to fga, molindone, in symptom improvement. risperidone and olanzapine were associated with high degrees of weight gain (risperidone additionally showed elevated prolactin concentration) in comparison with molindone, while individuals prescribed to molindone showed akathisia (sikich., 2008). clozapine remains the gold standard treatment for schizophrenia, and has been shown to have a more favorable profile of clinical response compared with haloperidol and olanzapine in treatment - refractory eos (spencer., 1992 ; mozes., 1994 ; towbin., 1994 ; kumra., 1996, 2008 ; mcevoy., however, clozapine remains as the last resort choice limited by its significant side effect profile on the hematopoietic system (agranulocytosis), cardiovascular system (myocarditis), central nervous system (seizures, akinesia, myoclonic jerks), and liver function, along with other side effects such as severe movement disorders, hypersalivation, hyperglycemia, diabetes, and weight gain, which are particularly problematic for children and young adults (connor., 2001 ; vyas., 2011b). clearly, there is an ongoing debate about the efficacy of atypical medications accounting for the long - term side effects profile, and therefore there is a pressing need for larger randomized control trials (rcts), to delineate the best available antipsychotic agents, and provide a platform for novel drug discovery. to date, there are no published rcts of psychosocial treatments for children with schizophrenia. however, the adult literature has supported adjunct psychosocial and individualized psychological treatments (eack., 2009 ; review, vyas., 2012). a review concluded that psychosocial therapies (cognitive behavioral therapy, cbt), family intervention, social skills training, and cognitive remediation) are effective adjuncts to pharmacological interventions in adults with schizophrenia (mcgurk., 2007 ; patterson and leewenkamp, 2008). for example, cbt addresses dysfunctional beliefs, coping strategies, tuning of cognitive abilities, and behavior modification, by linking and then re - evaluating thoughts and feelings about the presentation of clinical symptoms, which in turn aims to improve the mental states of patients. cognitive enhancement therapy has also shown to be effective in improving neurocognitive functioning in outpatients with eos or schizoaffective disorder (eack., 2009). indeed, family therapy, psycho - education, and social skills training have also shown improvement in clinical symptoms of newly diagnosed patients with schizophrenia (petersen., 2005). a recently published multicenter rct was conducted on young people with an early initial prodromal state of psychosis, to investigate the effectiveness of integrated psychological intervention (including cbt, group skills training, cognitive remediation therapy, and multifamily psycho - education) and supportive counseling on prevention of psychosis (bechdolf., 2012). the results showed that the integrated psychological intervention was more effective in delaying the onset of psychosis over a 24-month follow - up period (bechdolf., 2012). these studies suggest that psychotherapeutic interventions may thus be an important platform to alleviate psychotic relapse, re - admission, and medication compliance ; systematic validation of such measures however is warranted. early onset schizophrenia is a rare, severe, and treatment - refractory form of the adult - onset illness. although antipsychotic treatment, in addition to psychotherapeutic interventions, provides some symptom relief, there are a very high percentage of residual psychotic symptoms and cognitive deficits. existing medication treatments do not result in adequate response and the side effects in children remain daunting. hence, there is a dire need for early characterization of symptoms and biomarkers, better understanding of the pathophysiology and progression of the illness, and exploring novel and outside the box treatment options such as transcranial magnetic stimulation (tms ; tanaka and watanabe, 2009), or transcranial direct current stimulation (tdcs) trials, which are well tolerated in pediatric populations (mattai., 2011 ; vercammen., 2011) non - invasive neurostimulation techniques such as these have been shown to ameliorate cognition and negative symptoms in schizophrenia (levkovitz., 2011 ; minzenberg and carter, 2012), features commonly reported in early onset cases (vyas., 2011a). however, new treatment strategies should be informed by advancing knowledge from neurochemical and neuroanatomic studies, which may provide more specific targets in the brain. recent advances in neuroimaging methodologies, particularly those that provide a window into brain functioning and circuitry, may provide a blueprint for identification of novel biomarkers for schizophrenia. for instance, resting - state and task orientated functional mri or magnetoencephalography (meg) analyses show abnormal brain synchrony and neural networks in schizophrenia (reite., 1999 ; banaschewski and brandeis, 2007 ; ford., 2007 ; brookes 2011). an ongoing meg study from our nimh cos cohort showed abnormal oscillatory patterns in cos patients compared to healthy controls (n. s. vyas, unpublished data). treatment strategies could be ideally designed (e.g., regionally specific neuromodulation using tdcs) to normalize these abnormal brain circuitries or evaluate efficacy of new compounds. research and implementation of novel treatments coupled with advances in genome - wide microarray technology may lead to the identification of genes that are relevant not only in the pathophysiology of schizophrenia, but also in providing an insight into treatment response, or course prediction. the authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. | early onset schizophrenia (onset before adulthood) is a rare, severe, and chronic form of schizophrenia. the clinical presentation of schizophrenia at this unusually early age of onset has been associated with premorbid developmental abnormalities, poor response to neuroleptic treatment, greater admission rates, and poor prognosis. this is a brief, condensed review of current treatment strategies for the early onset population highlighting the need for novel treatment strategies for these generally treatment - refractory cases. based on the current literature, second - generation antipsychotics remain the mainstay of treatment, although current medications provide suboptimal response at best. based on the adult literature, combining antipsychotic treatment with psychotherapeutic intervention may be a more comprehensive treatment strategy. indeed, early detection, identification of relevant biomarkers, coupled with advancing knowledge of the neurochemical and neuroanatomic pathways may help design informed and novel treatment strategies. |
a seventeen - year - old pregnant woman with a mesencephalic tumor and vp drainage was admitted to the obstetric department to be prepared for elective c - section. anamnestically, and from the available medical data, we discovered that the patient had the mesencephalon tumor since her 9th year. during her childhood, vp drainage was implanted, because of the brain tumor, and an attempt of a stereotactic biopsy was performed. the stereotactic biopsy was unsuccessful and was not repeated due to the improvement of the patient 's medical condition and the lack of evidence that the illness was progressing. since then, the patient has been under regular annual neurosurgical control. on the most recent magnet resonance imaging (mr), performed one month before her pregnancy, the dimensions of the tumor were : axial 27.7 27.2 mm, coronal 28.9 19.8, sagittal 27.1 23. 2 mm (fig. we assume that vp drainage worked well because the patient had no symptoms of increased intracranial pressure. in the 27 week there was a urinary - infection that was treated with antibiotics, and the patient also developed gestational diabetes. the patient was scheduled for elective c - section because of her prime disease (mesencephalic tumor), breech position of the baby, gestational diabetes and expected weight of the baby of more than 4 kg. under the assumption that the vp drainage worked well, we decided to perform the operation under spinal anesthesia because of expected difficult airway management. the patient had a mallampati score iv, edematous soft tissue of the face and mouth and a high body mass index (bmi) 38. coagulation parameters were good : prothrombin time 10 s, platelets count 386 10/l, and blood pressure was 150/100 mmhg. the patient signed the informed consent and was prepared for a regional anesthesia with intravenous prehydration. spinal block was performed with a pencil point spinal needle 27 g, at the l3-l4 intervertebral space, with hyperbaric bupivacaine 8 mg plus fentanyl 15 g. we used the manufacturer 's dose recommendation (from 10 - 20 mg), though with a reduction because of the substitution of fentanyl. the patient was hemodynamically stabile during the anesthesia, systolic blood pressure was between 120 and 150 mmhg, pulse was 80 - 120/min, and oxygen saturation was 100%. the operative procedure was uneventful ; a female child was born, weighing 4,050 g, and 50 cm in length. the postoperative period was also uneventful, so the mother and child were discharged from the hospital on the 8 day after birth. while referring to the past literature, we found a series of case reports about newly detected hydrocephalus or brain tumor during pregnancy [3 - 8 ]. today, it is presumed that vp drainage on a pregnant patient with hydrocephalus does not affect the mode of labor : vaginal or caesarean section. c - section should be reserved only for patients with a malfunction of the shunt or with obstetric indications. epidural or spinal analgesia and anesthesia are also allowed, but a certain amount of attention must be given. increased intra - abdominal pressure during the pregnancy can lead to disturbance of liquor drainage and eventually result in a malfunction of the shunt. according to wisoff, there is an increase of the amount of liquor content and venous distension, which compromises compliance and leads to symptoms of increased intracranial pressure. the risk of vp drainage malfunction is minimal because the valve is unidirectional and intra - abdominal pressure during labor is only intermittently increased. the approach taken with pregnant patients with a cerebral tumor should be multidisciplinary, involving a neurosurgeon, obstetrician, neonatologist and anesthesiologist. individual case management is advised according to the surgical and neuroanesthetic requirements and to the gestational age. therapeutic options include : neurosurgery performed while maintaining fetus in utero in early pregnancyc - section before neurosurgical operationc - section followed by neurosurgical operation neurosurgery performed while maintaining fetus in utero in early pregnancy c - section before neurosurgical operation c - section followed by neurosurgical operation there are no general recommendations for delivery with these patients so the decision lies on the recommendation of the neurosurgeon for each patient. although there are some mentioned cases of vaginal delivery, most of the patients with a brain tumor are scheduled for c - section. pregnancy itself causes hormonal imbalance with fluid retention, a more turbulent response to stress, and some repercussions to the central nervous system (cns). a brain tumor causes shifting of neurovascular structures and the surrounding oedema. in combination with pregnancy, especially during the vaginal (natural) delivery where a rise in intracranial pressure is present, there is a greater risk of fatal complications such as brain incarceration or haemorrhage of the tumor. anesthesia for patients with a brain tumor must be treated very delicately and the anesthesiologist must decide whether to use general or regional anesthesia. general anesthesia comes with the danger of aspiration and an insufficient depth of anesthesia can cause a rise in intracranial pressure (icp). regional anesthesia, e.g. spinal anesthesia, regional anesthesia may be appropriate when delivery is performed subsequent to successful and uncomplicated neurosurgery. also, while most of the anesthesiologists chose general anesthesia for c - section, nowadays there are several case reports of regional anesthesia for patients with brain tumors [12 - 14 ]. with our patient since the patient had no symptoms of her prime disease during the pregnancy, and no signs of raised icp, we treated her as capable for regional (spinal) anesthesia. the patient 's medical records reassured us that her brain tumor was stationary and the functionality of the vp drainage was evaluated on the grounds of lack of symptoms for raised icp. according to the available literature, this is the first case of a pregnant woman with cerebral tumor and vp drainage who underwent successful delivery with c - section under spinal anesthesia. from our experience and referring to the past literature the following conclusions were drawn : vp drainage in a pregnant patient with hydrocephalus does not affect the mode of labor : vaginal or caesarean sectionepidural or spinal analgesia and anesthesia are also allowed in patients with vp drainagedelivery method for patients with brain tumors and/or vp drainage should be determined by consensus between the obstetrician and neurosurgeonregional anesthesia is safe for patients who have a brain tumor and implanted vp - drainage that works properly vp drainage in a pregnant patient with hydrocephalus does not affect the mode of labor : vaginal or caesarean section epidural or spinal analgesia and anesthesia are also allowed in patients with vp drainage delivery method for patients with brain tumors and/or vp drainage should be determined by consensus between the obstetrician and neurosurgeon regional anesthesia is safe for patients who have a brain tumor and implanted vp - drainage that works properly | a seventeen - year - old pregnant woman with a mesencephalic tumor and ventriculoperitoneal (vp) drainage was admitted to the hospital at full term pregnancy to give birth. elective cesarean section was performed because of her prime disease (mesencephalic tumor), breech position of the baby, gestational diabetes and expected weight of the baby of more than 4 kg. the operation was performed under spinal anesthesia. spinal block was performed smoothly, using a pencil point spinal needle 27 g, at the l3-l4 intervertebral space, with hyperbaric bupivacaine 8 mg plus fentanyl 15 g. sensory block th 5 was reached within 5 minutes. the patient was hemodynamically stabile during the anesthesia and the procedure was uneventful. the woman developed no neurologic symptoms, and a healthy female child was born. this is the first case of a pregnant woman with a cerebral tumor and vp drainage on whom a successful delivery was performed with c - section under spinal anesthesia. |
pain affects all aspects of a person 's life including sleep, work, leisure and relationships. it impacts on the lives of sufferers by causing limitations in daily life and this in turn imposes a huge burden on society, due to the high direct costs of treatment and indirect costs from lost productivity [1 - 4 ]. approaches to the measurement of pain include verbal and numeric self - rating scales, behavioural observation scales, and physiologic responses. because of complex nature of the experience of pain and its subjective characteristic, self - reported measures of oral health quality of life relate altered functions and symptoms to social and psychological well - being. however, different oral conditions may affect functioning and physical and emotional problems in different ways and therefore disease specific instruments may be invaluable in investigating the impact of these conditions on individuals. brazilian studies about disabilities caused by orofacial pain use rdc / tmd (research diagnostic criteria for temporomandibular disorders) axis ii, oidp (oral impact daily performance), ohip (oral health impact profile), ghq-12 (general health questionnaire) and brazilian version of mcgill pain questionnaire. although these tools were not specifically developed to assess orofacial pain related disability, they all concluded that oro - facial pain imposes a huge burden on the daily life of sufferers [7 - 9 ]. the manchester orofacial pain disability scale (mopds) construction and validation has been previously demonstrated. this tool has been shown to be robust in measuring orofacial pain related disability and had good construct validity. with the increasing number of international research projects, the need to adapt measures of health status to use the language of origin has grown rapidly. most questionnaires were developed in english - speaking countries, but even within those countries, researchers should consider translating these for immigrants, especially when their exclusion may lead to systematic bias in studies of health care related quality of life. recommend cross - cultural adaptation in addition to simple translation for questionnaires of health status that are going to be used in a different language and culture from which they were originally developed. this paper therefore aims to adapt the manchester orofacial pain disability scale to a portuguese language version through a formal translation / back - translation process and summarize available data about its psychometric properties. specific objectives were to determine the internal consistency and reliability of the translated scale and to validate its use for measuring orofacial pain specific disability in brazilian patients. the guidelines for translation and cross - cultural adaptation process (beaton. and wild. were used following the stages above : stage i : initial translation two independent translations from english to portuguese were made by two bilingual translators whose mother tongue is the portuguese language in order to accurately reflect the nuances of the language. both translators had different profiles or backgrounds - one with academic vinculum (with theme knowledge) and the second one a clinical dentist. stage ii : synthesis of the translations a synthesis of these translations was first conducted (producing one common translation) by the authors of the research and two non - native english language teachers. stage iii : back translation working from the synthesis of the translated versions and totally blind to the original version, two different non - native english language teachers without dental knowledge translated the questionnaire back to the english language. stage iv : expert committee an expert committee reviewed the pre - final version : the researchers, two other non - native english language teachers and one native english speaking professor. stage v : test of the prefinal version this field test of the new questionnaire was used in 8 patients from the target setting. each subject completed the questionnaire and was interviewed to probe what he or she believed to be the meaning of each questionnaire item and its response. in this stage these patients suggested to exclude the question " i am irritable, angry and easily frustrated ", justifying that its items are repeated in other questions. the second question suggested to exclude was " i have lost earnings " because its semantic equivalence with the item " i have had to take time off work ". stage vi : submission of documentation to the coordinating committee for appraisal of the adaptation process the final stage in the adaptation process was the submission of all the reports to a committee formed by the authors of the research and another portuguese speaking professor. as related in the item above this committee decided to remove the questions " i am irritable, angry and easily frustrated " and " i have lost earnings ". the brazil - mopds was applied in fifty patients with symptoms of orofacial pain who consulted at the tmj and occlusion clinic of the prosthetic department of so paulo university school of dentistry, brazil. all patients were recruited before any type of treatment, clinical consultation or other information about orofacial pain was collected and all of approached patients agreed to participate. initially they were 75 but only 50 concluded the steps of the designed study (figure 1). the brasil - mopds was administered twice by an interviewer (15 - 20 day interval) and once by a second independent interviewer. the brazilian version of the short form oral health impact profile (ohip-14) questionnaire and the visual analogue scale (vas) were applied on the same day. ohip-14 score were based on its seven dimensions (physiological discomfort, pain, physiologic inability, physical inability, function limitation, disability and social inability) and scored from 0 - 4 based on a likert scale : 0 - never, 1 - rarely, 2 - sometimes, 3 - frequently and 4 - always. this value was then multiplied by the weight of each question to give a score ranging from 0 (less oral health impact on daily profile) to 28 points (more impact). all participants have read and signed informed consent form. the use of human subjects in this study has been reviewed and approved by university of so paulo dentistry school ethics committee. this study was conduced from august 1, 2008 to march 1, 2009). data were tabulated and analyzed in stata 10 and for all tests it were used confidence interval of 95%. internal consistency was examined by cronbach. in order to find the correlation between each question and the overall outcome of the test, the spearman correlation coefficient (scc) was performed, using data from the first interview (main test). psychometric properties : reliability was estimated by assessing the internal consistency (indicated by cronbach) and reproducibility (test - retest). the statistical value of cronbach was also calculated for each excluded question from the brazil - mopds as the objective of statistical investigation was to numerically represent the interests of uniformity or trend in the responses within each item of the questionnaire. reliability was checked by the application of brazil - mopds on two occasions by the same rater, called internal reliability. external reliability was the comparison of the answers obtained by the first and the second evaluator. for these analysis interclass correlation coefficient (icc) and bland - altman test were used. the samples were distributed by gender and age. most of the participants were women (86%) with the average age of 40.7 (sd 14.03) years old (table 1). the answers of the questionnaire obtained by the first rate (first and second interview) and the second rate are described in table 2. the correlation between each question, the whole result and cronbach is shown in table 3. answers distribution by the three interviews n = number of participants. correlation between questions and whole brazil - mopds and cronbach if the question were excluded pearson correlation, question - whole result. reproducibility (test - retest) : interobserver correlation data obtained in questionnaire administration indicated an excellent agreement with icc = 0.924 (ci 0.46 - 0.98) (figure 2). in order to evaluate graphically the agreement or discrepancy between the sums of numerical responses given by patients questioned on the same day by two different interviewers, the results of applications of brazil - mopds were plotted in a bland - altman plot (figure 3). bland - altman plot with inter observer reproducibility. this enabled us to recognize the magnitude of variation between the responses as well as the existence of systematic bias between the two interviewers. the value of the arithmetic mean was equal to - 0.48 (ci 1.08 - 0.12), showing a strong correlation. data obtained in the intra - observer correlation in the administration of the questionnaire, also showed an excellent agreement with icc = 0.982 (ci 0.967 - 0.997) (p < 0.001) (figure 3. criterion validity was established by comparing the data of the scale with the results of the ohip-14, vas and the three brazil - mopds interviews (table 4). the correlation between the scores of brazil - mopds and ohip-14 was high, r = 0.857 (ci 0.765 - 0.915) (p < 0.001) and the correlation of scores of brazil - mopds with vas was also strong - r = 0.758 (ci 0.615 - 0.852) (p < 0.001). we have successfully adapted the mopds into the portuguese language. not only were we able to translate it, but also validate and culturally adapt it. previous instruments have not been specifically designed to measure the impact associated with orofacial pain. orofacial pain has not only biological but also psychological and social effects on patients. psychosocial effects are difficult to objectively measure and certainly deserve attention in the evaluation of therapeutic measures for chronic orofacial pain. in fact they may be the only measures to assess patient improvement particularly where there is no underlying pathology to explain reported symptoms [17 - 21 ]. further, indicators of oral health related quality of life are often used to complement clinical data in cross - sectional and longitudinal studies. borges. estimated the prevalence of dental and gingival pain and associated factors among 16,126 young brazilians (15 - 19 years) who participated in the epidemiological survey of oral health (2002 - 2003) in brazil for six months. through simple and multiple poisson regression there was a high prevalence of dental and gingival pain : 35.6% (ci : 34.8 - 36.4). the increased prevalence of pain was associated with female public school students, low income and education gap. adolescents with high levels of caries and dental calculus also reported a higher prevalence of dental pain. the successful translation and cross - cultural adaptation of the mopds, which had its development based on people who had just this specific condition will therefore be extremely valuable to measure the specific effects of orofacial pain in brazilian populations. other questionnaires like gohai (geriatric oral health assessment index, rdc / tmd, ohip, mcgill pain questionnaire and oidp have been translated and validated for use in brazilian researches [24 - 29 ]. however, these instruments have not been specifically designed to measure the impact associated with orofacial pain. although some english terms do not have equivalent descriptors in portuguese, trans - cultural adjustments proved to be a valid and efficient alternative to overcome this difficulty, which can be confirmed in the application of the final version of the questionnaire. adaptation to local culture is essential for a correct evaluation of the process of pain. therefore the brazil - mopds was obtained from translation and cultural adaptation as performed according to current and internationally accepted guidelines. it showed excellent reproducibility, validity and practicality application with high cronbach 's scores, and good correlation co - efficient. the bland - altmann test, associated with the icc was used for providing more information than the use of a test alone. one of the main advantages of this method, in which differences between the scores of the first and second evaluation are plotted on the mean values is to allow the recognition of both the amplitude of variation as to the existence of systematic bias between the two interviews it is necessary at least 50 individuals for the use of this method. however, our study was conducted on patients referred into a tertiary setting who may represent the most severe and intractable cases of orofacial pain. further research is needed in establishing the validity of the brazil - mopds in population settings and also its responsiveness as a measurement of treatment outcomes in clinical trials of chronic orofacial pain. we envisage that it will be an important tool for such patients where improvement can not be assessed in terms of structural improvement (elimination of infection or removal of diseased tissue) as there is no underlying cause for reported symptoms. rather, measurements of disability before and after treatment may provide important information on treatment outcomes and therefore influence further management of the patient. the data showed that the process of translation and cross - cultural adaptation of manchester orofacial pain disability scale was successful and that brazil orofacial pain disability scale seems to be a valid and reliable instrument for describing pain - related impact among patients with symptoms of orofacial pain. the study was funded by the author and school of dentistry of university of so paulo, brazil. vishal aggarwal is funded by a clinician scientist award issued by the nihr - grant number cs/2008/08/001. the views expressed in this publication are those of the author(s) and not necessarily those of the nhs, the national institute for health research or the department of health uk. vishal aggarwal also has a substantive part - time appointment as a senior lecturer in academic general dental practice at queen mary 's university london, uk. no external funding, apart from the support of the authors ' institution, was available for this study. | abstractobjectivesthe purpose of the present study was to translate and perform a cross - cultural adaptation of manchester orofacial pain disability scale to the portuguese language.material and methodsa synthesis of two independent translations done by bilingual translators whose mother tongue was the portuguese language began the process of translation. from the synthesis of the translated version and totally blind to the original version, two different non - native english language teachers without dental knowledge translated the questionnaire back to english. the pre - final version was done by an expert committee : the researchers, two other non - native english language teachers and one native english language speaker. the new questionnaire was then piloted among 8 patients from the target setting that were interviewed to probe it on their perceived meaning of each question. the manchester orofacial pain disability scale (mopds) thus translated was called brasil - mopds and was validated in 50 patients with orofacial pain from tmj and occlusion clinic ambulatory of so paulo university school of dentistry. the brasil - mopds was administered twice by an interviewer (15 - 20 day interval) and once by a second independent interviewer. the brazilian version of the short form oral health impact profile (ohip-14) questionnaire and the visual analogue pain scale (vas) were applied on the same day.resultsinternal consistency (cronbach 's = 0.9), inter - observer (icc = 0.92) and intra - observer (icc = 0.98) correlations presented high scores. validity of brasil - mopds compared to ohip-14 (r = 0.85) and vas (r = 0.75) shown high correlations.conclusionsbrasil-mopds was successfully translated and adapted to be applied to brazilian patients, with satisfactory internal and external reliability. |
immune checkpoint inhibition with anti - ctla-4 blockade (e.g., ipilimumab) and anti - pd-1 antibodies (e.g., nivolumab) has improved the poor prognosis of unresectable malignant melanoma [1, 2 ]. these newly approved medications induce many types of immune - related adverse events (iraes), including pneumonitis, colitis, hepatitis, and endocrinopathies such as hypophysitis and thyroiditis. although iraes induced by ipilimumab have occasionally been reviewed in the literature, studies on nivolumab are also gradually being described. a 52-year - old male with lung and liver metastasis of malignant melanoma was put on nivolumab therapy (3 mg / kg every 3 weeks). after approximately 34 weeks of nivolumab administration (fig. 1), laboratory tests revealed a sudden elevation of liver enzymes (ast / alt) to 1,225/824 u / l (grade 4). computed tomography detected the exacerbation of liver metastasis, identifying a lesion that was slightly larger than the nondiffuse, localized lesion that had been observed 3 months earlier (fig. 2). therefore, we suspected hepatitis associated with nivolumab because this drug has a low probability of causing serious liver damage to localized lesions, even when they are large. the patient stopped taking nivolumab and was treated with 70 mg / day (1.0 mg / kg / day) of systemic corticosteroids. his ast / alt level promptly improved to 112/389 u / l within 3 days. the median onset of ipilimumab - related hepatitis is approximately 812 weeks after initial treatment. therefore, the interval from nivolumab initiation to the occurrence of hepatitis was much longer than that reported in previous cases. hepatitis induced by nivolumab was also observed at 712 weeks in a pooled analysis of a nivolumab phase iii trial. however, the incidence of grade 23 nivolumab - induced hepatitis was approximately 1% in a phase iii clinical study. in that study, the liver function test improved to grade 1 within 415 days of initiation of corticosteroid treatment. the liver dysfunction caused by long - term nivolumab therapy in this case is noteworthy. although many diseases induce liver dysfunction, in this case, we definitively excluded other causes of hepatitis such as viral infection (e.g., hbv, hcv, hsv, cmv, and ebv), medication other than nivolumab, and other forms of autoimmune hepatitis. to manage hepatitis associated with nivolumab, these possibilities must be excluded as causes of the disease. the recommended initial treatment of hepatitis in the risk evaluation and mitigation strategy (rems) is to administer systemic corticosteroids (12 mg / kg / day of prednisone). if the symptoms continue after 35 days, alternative immunosuppressive therapy, such as 500 mg of oral mycophenolate mofetil every 12 h, must be considered. in this case, because the laboratory test showed an ast / alt level > 20 the upper limit of normal, we considered systemic corticosteroids to be the most appropriate treatment. fortunately, the ast / alt level rapidly improved to grade 12 in 3 days, and modification was not necessary. it is very important to note that severe hepatitis can occur despite long - term nivolumab therapy. in this case, it was possible to quickly reach a correct diagnosis because we were aware of the possibility of drug - induced hepatitis, even though the period of occurrence of nivolumab - induced hepatitis had passed. therefore, we believe that this report may be very informative when treating hepatitis as an irae of nivolumab. in addition, to our knowledge, this patient exhibited the longest time between nivolumab administration and the onset of hepatitis that has been reported to date. | immune checkpoint inhibitors have drastically changed in the treatment of many kinds of malignancies, especially malignant melanoma. the focus of the recent experiments has not only been on their efficacy but also immune - related adverse events (iraes). we report a case of fulminant hepatitis due to nivolumab. in this case, the patient had undergone long - term nivolumab therapy. he did not complain of any symptoms but his liver enzyme levels were extremely elevated (grade 4). we promptly decided to start oral corticosteroids in the patient. his liver function rapidly improved. the dose of corticosteroids was gradually reduced. our case demonstrates that sudden onset fulminant hepatitis can occur despite the safe use of long - term nivolumab therapy. the irae can improve rapidly with proper corticosteroid treatment. this report will be useful for the physicians who always use immune checkpoint inhibitors. |
families play an essential role in maintaining children s mental, social, and physical health. the family provides the first and the most important social context for human development. during the normal development of every child almost all children will have problems during their development and this, in compatibility with the accompanying changes, stress, and conflicts, can cause behavioral, emotional, and learning problems. most behavioral problems in children reflect the complex individual situations among family members, particularly the parents. in other words, the child s behavioral problems are due to the damaged relationships of the family members with each other and are associated with the incorrect training methods of parents and their defective interactions with their children. the purpose of a parenting style is to help parents educate their children and reflect their attitudes toward their children and at the same time, execute the rules and regulations enacted by them. family, as a primary context, provides the necessary resources and opportunities for the healthy development of children (1). appropriate parenting skills are the key variables that predict children s positive outcomes in the first and middle years of life (1). the purpose of positive parenting styles is to shape the mental character and strengthen the competence of the child. the importance of these issues has led researchers to discuss parenting styles and offer a variety of methods (2) among which we can refer to the theories of ericsson, baumrind, and young. baumrind proposed three styles of parenting based on the two features of requesting (this refers to the attributes of control, monitoring, and expected mature behavior from the child) and being responsive (including support, love, and acceptance of the child) (3). in the authoritative style, parental demands are met at high levels (4) and these demands are reasonable (5). parents allow their children to comment and enjoy independence and freedom of thought, and a warm and cordial relationship exists between the child and the parents at a high level (2). they allow their children to express their ideas, and provide the grounds for their future progress. this style of parenting can lead to increased self - regulation, compliance, and obtaining a college education (6). nevertheless, parents who practice the authoritarian parenting style have a rigid set of rules and illustrate heartlessness, lack of attention to the developmental needs of the child, low emotional support, and strict discipline (6). this style is associated with features such as reduced admission and high control that create underlying problems such as weak social skills, low self - esteem, and aggressiveness, and will prevent them from becoming highly educated individuals (1). acceptance, high responsiveness, relaxation in social attitudes, discipline, and customs (7), and less control from parents cause underlying problems such as aggression, low self - control, negligence, emotional problems, school dropout, and tendency toward drugs and crime ; however, they also result in high confidence (1). in this parenting style, abundant love substitutes punishment by parents, both of which, in turn, may be devastating (8). young suggests a subset of schemas that are called primary dysfunctional schemas (9). the schema - focused approach places the main emphasis on understanding the deepest levels of cognition, which is primary dysfunctional schemas, rather on automatic thoughts and underlying assumptions. models that are focused on schemes define primary dysfunctional schemas as inclusive and extensive subjects with regard to personal and individual relationships with others that are created in childhood and last throughout a person s life and may be extended with a degree of inefficiency (9). primary dysfunctional schemas are fundamentally implicit and unconscious contexts that are retained by the individual. primary dysfunctional schemas are used as a model for processing experiences throughout their lives as well as for behaviors, thoughts, feelings, and their relationships with other people. in contrast to the underlying assumptions, primary dysfunctional schemas are often unconditional, and therefore, very inflexible. essentially, primary dysfunctional schemas are authentic representations of early childhood unpleasant experiences (9). according to young, the primary dysfunctional schemas are among the main causes of pathology generated through interpersonal experiences with close people such as parents (9). when primary dysfunctional schemas are activated due to events, the levels of emotion created directly and indirectly lead to various forms of psychologic distress including depression, anxiety, loneliness, anorexia nervosa, conflicts, and interpersonal relationship problems (9). they stated that these schemas are divided according to five unsatisfied emotional needs called schema areas (10). the people, whose schemas take place in this area, can not interact in a secure and satisfying attachment with others. the schemas of this area include abandonment / instability, mistrust / abuse, emotional deprivation, defectiveness / shame, and social isolation / alienation. the person s expectation from himself / herself and environmental interaction with her / his tangible abilities consist of separation, survival, and functioning independently, or to work successfully. the schemas of this area include dependence / incompetence, vulnerability to harm or illness, enmeshment / undeveloped self, and failure. the schemas of this area include entitlement / grandiosity and insufficient self - control / discipline. these individuals prioritize the satisfying of the needs of others in order to receive emotional support, maintain ongoing relationships, and avoid revenge. the schemas of this area include subjugation, self - sacrifice, and approval - seeking / recognition - seeking., individuals place extreme emphasis on rejection of feelings and impulses in order to act according to their inflexible and internal rules even at the cost of losing joy and peace of mind. the schemas of this area include negativity / pessimism, emotional inhibition, unrelenting standards / hypercriticalness, and punitiveness (10). young and brown argued that an individual s unique experiences in childhood contribute to and influence the development of a distinct set of core beliefs about themselves and others, which they called early maladaptive schemas (11). young believes that any childhood experience can have an effect on the formation of early maladaptive schemas (12). the hypothesis of this research was whether baumrind s parenting styles (evolutionary root) were predictors of early maladaptive schemas. in this descriptive - correlational study, samples were selected through cluster random sampling method. a total of 357 male and female students of islamic azad university, urmia branch, iran, were selected using the morgan table during 2013 - 2014. due to the purpose and nature of this research, the best way to gather the needed information was to complete an inventory ; therefore, two inventories were used in this study. the early maladaptive schema inventory by young and brown was designed to measure early maladaptive schemas (11). the early maladaptive schema inventory - short form (sq - sf) was created because of its briefness ; however, it is used as an instrument to measure primary maladaptive schemas. the sq - sf includes 75 items of the 205 items in the original form. these 75 items questioned 15 early maladaptive schemas of emotional deprivation (sentences 1 to 5), abandonment (sentences 6 to 10), mistrust / abuse (sentences 11 to 15), social isolation (sentences 16 to 20), defectiveness / shame (sentences 21 to 25), failure (sentences 26 to 30), dependence / incompetence (sentences 31 to 35), vulnerability to harm or illness (sentences 36 to 40), enmeshment (sentences 41 to 45), subjugation (sentences 46 to 50), self - sacrifice (sentences 51 to 55), emotional inhibition (sentences 56 to 60), unrelenting standards (sentences 61 to 65), entitlement (sentences 66 to 70), and insufficient self - control / self - discipline (sentences 71 to 75). each one of these 75 scales of the sq - sf is graded on a six - point scale as follows : 1) totally wrong about me ; 2) almost wrong about me ; 3) slightly more true to false ; 4) almost true ; 5) truer about me / truer than me ; and 6) fully described me. the higher scores on each item indicate the presence of a wide range of early maladaptive schemas in the answerer (13). the reliability and validity of this instrument have been demonstrated in several studies (14). the farsi version of this inventory was standardized in the university of tehran, iran, by divandari. therefore, the internal consistency obtained using cronbach s alpha was 0.97 in the female population and 0.98 in the male population (15). gunty and buri designed the baumrind s parenting questionnaire based on the parental authority theory. it contains 30 questions with three scales (authoritative, authoritarian, and permissive), and each scale has ten items (16). each item is rated based on the likert scale from totally agree to totally disagree (5 scales) and is scored from zero to four (17). gunty and buri (16) reported the reliability of this tool in mothers and fathers as respectively 0.81 and 0.77 for permissive style, 0.86 and 0.85 for authoritarian style, and 0.78 and 0.88 for authoritative style. they also used the discriminant validity method for questionnaire validity that obtained the following results : authoritarian mother has a reverse association with permissive mother (-0.38) and rational authority of mother (-0.48), also authoritarian father has a reverse association with permissive father (0.50) and rational authority of father (-0.52) (16). this questionnaire has been translated into farsi and was used by esfandiari (18). he reported a retest reliability coefficient of 0.69, 0.77, and 0.73 for permissive, authoritarian, and authoritative styles, respectively. data analysis was performed using spss 18 for windows (spss inc, chicago, illinois, the united states) and regression analysis. since regression analysis was used in the present research, the aim was to make use of one or more variables to predict the criterion of one or more of the predictor variables. the early maladaptive schema inventory by young and brown was designed to measure early maladaptive schemas (11). the early maladaptive schema inventory - short form (sq - sf) was created because of its briefness ; however, it is used as an instrument to measure primary maladaptive schemas. the sq - sf includes 75 items of the 205 items in the original form. these 75 items questioned 15 early maladaptive schemas of emotional deprivation (sentences 1 to 5), abandonment (sentences 6 to 10), mistrust / abuse (sentences 11 to 15), social isolation (sentences 16 to 20), defectiveness / shame (sentences 21 to 25), failure (sentences 26 to 30), dependence / incompetence (sentences 31 to 35), vulnerability to harm or illness (sentences 36 to 40), enmeshment (sentences 41 to 45), subjugation (sentences 46 to 50), self - sacrifice (sentences 51 to 55), emotional inhibition (sentences 56 to 60), unrelenting standards (sentences 61 to 65), entitlement (sentences 66 to 70), and insufficient self - control / self - discipline (sentences 71 to 75). each one of these 75 scales of the sq - sf is graded on a six - point scale as follows : 1) totally wrong about me ; 2) almost wrong about me ; 3) slightly more true to false ; 4) almost true ; 5) truer about me / truer than me ; and 6) fully described me. the higher scores on each item indicate the presence of a wide range of early maladaptive schemas in the answerer (13). the reliability and validity of this instrument have been demonstrated in several studies (14). the farsi version of this inventory was standardized in the university of tehran, iran, by divandari. therefore, the internal consistency obtained using cronbach s alpha was 0.97 in the female population and 0.98 in the male population (15). gunty and buri designed the baumrind s parenting questionnaire based on the parental authority theory. it contains 30 questions with three scales (authoritative, authoritarian, and permissive), and each scale has ten items (16). each item is rated based on the likert scale from totally agree to totally disagree (5 scales) and is scored from zero to four (17). gunty and buri (16) reported the reliability of this tool in mothers and fathers as respectively 0.81 and 0.77 for permissive style, 0.86 and 0.85 for authoritarian style, and 0.78 and 0.88 for authoritative style. they also used the discriminant validity method for questionnaire validity that obtained the following results : authoritarian mother has a reverse association with permissive mother (-0.38) and rational authority of mother (-0.48), also authoritarian father has a reverse association with permissive father (0.50) and rational authority of father (-0.52) (16). this questionnaire has been translated into farsi and was used by esfandiari (18). he reported a retest reliability coefficient of 0.69, 0.77, and 0.73 for permissive, authoritarian, and authoritative styles, respectively. data analysis was performed using spss 18 for windows (spss inc, chicago, illinois, the united states) and regression analysis. since regression analysis was used in the present research, the aim was to make use of one or more variables to predict the criterion of one or more of the predictor variables. the mean standard deviations of the descriptive findings of 357 samples are illustrated in table 1. according to table 1, the best parenting style was authoritarian, and most schemes belong to the fields of rejection and disconnection, impaired autonomy, and over - vigilance, respectively. according to table 2, based on the significance level of > 0.05, permissive parenting style could not explain early maladaptive schemas in the areas of rejection and disconnection, impaired autonomy and performance, impaired limits, other - direction, and over - vigilance and inhibition. however, authoritarian parenting style could positively predict the early maladaptive schemas in the area of rejection / disconnection. this approach could explain 1.3% of the variance of early maladaptive schemas in the area of rejection / disconnection that could also positively predict early maladaptive schemas in the area of other - direction. baumrind s authoritarian parenting style could explain 3.4% of the variance of early maladaptive schemas in the area of other - direction, but not for impaired performance and over - vigilance / inhibition. finally, baumrind s authoritative parenting style could negatively predict early maladaptive schemas in the area of rejection or disconnection. thus, this approach could explain 2.6% of the variance of early maladaptive schemas in the area of rejection and disconnection, and negatively explain other - direction. baumrind s authoritative parenting style could explain 3.4% of the variance of early maladaptive schemas in the area of other - direction, but not in impaired autonomy and performance, impaired limits, and over - vigilance / inhibition. results showed that some of baumrind s parenting styles are predictors of early maladaptive schemas. in addition, the results revealed that baumrind s authoritative parenting style is a negative predictor of schemas in the area of rejection or disconnection. to explain these results using young s approach, we can say that parents with baumrind s authoritative parenting style have some features such as being warm and friendly, allowing children to express their own opinions, and being reasonable in expressing their demands. hence, the schemas of this area created by rejection, abuse, and unstable, cold, and heartless manners are not effective parenting styles. on the other hand, the authoritative style is a negative predictor of schemas in the area of other - direction. as a result, the schemas of this area for parenting styles, which parents need to prioritize, did not meet the children s demands. another finding of this study was that the authoritarian parenting style is a good predictor of schemas in the areas of other - direction and rejection / disconnection. to explain these results, we can say that parents with authoritarian parenting style provide a base for schemas in the area of rejection / disconnection, the area that does not consider the child s need for love and safety ; thus, their families are unstable, cold, ostracized, and isolated. the results show that parenting style is a predictor of schemas in the area of other - direction. furthermore, we can say that because the authoritarian style possesses the characteristics of low acceptance and lack of attention to the emotional needs of the child, it causes a schema in the area of other - direction that emanates from neglecting children s needs (10). the results of this study have been confirmed by the studies by young. (10), gunty and buri (16), esmali (19), and shahamat. (20). in conclusion, conducting a research on people who have different educational levels, live in various regions, and have different cultural expressions is recommended. the limitations of this study include the use of a self - report tool (questionnaire) and a student sample ; therefore, the results are not applicable to other communities. recommendations are made to improve the quality of future research in the area of association of attachment styles and early maladaptive schema with parenting styles. | background : families play an essential role in maintaining children s mental, social, and physical health. the family provides the first and the most important social context for human development.objectives:the present study aimed to predict early maladaptive schemas using baumrind s parenting styles (root development).patients and methods : a total of 357 undergraduate students of islamic azad university, urmia branch, iran, were selected through random cluster sampling during 2013 and 2014. the students were assessed using the schema questionnaire - short form (sq - sf) and the baumrind s parenting styles inventories.results:the result of regression analysis showed that baumrind s parenting styles are significant predictors of early maladaptive schemas (p < 0.001).conclusions : the authoritative parenting style has some features such as showing high levels of warmth or encouraging kids to express their own possibly divergent opinions. the authoritarian parenting style, however, possesses traits such as heartlessness, impassiveness, strictness, and lack of attention to the children s developmental needs, which is not acceptable. |
a 17-year - old female patient, resident of south india, presented with the complaints of sudden onset of the decrease in vision with gradual worsening in the left eye for the last 3 months duration. the patient was diagnosed with p. falciparum malaria about a week prior to the onset of the ophthalmic symptoms with sudden onset of chills and rigor with high grade fever, and peripheral blood film showing p. falciparum trophozoites and ring forms. there was no history of convulsions, seizures or unconsciousness during or preceding the treatment. the patient recovered from the fever within 10 days, with a gradual onset of decreased vision in the left eye. during the treatment, an ophthalmic referral was taken, which showed features suggesting neovascularization and vitreous hemorrhage. at presentation to us, the visual acuity in the right eye was 20/20, n6, and in the left eye was 20/1200, n36. the dilated fundus examination of the right eye revealed a normal fundus [fig. 1 ]. 2 ] were as follows : the media was clear, with a small amount of vitreous hemorrhage inferiorly. the major retinal arcade veins showed a severe obliteration of various degrees at the posterior pole as well as the periphery, with visible small neovascular fronds along the superior arcade vessels. color fundus picture of the right eye showing a normal posterior pole color fundus picture of the left eye showing a pale optic disc, with a few exudates along superior arcade vessels, and over macula. the major retinal arcade veins showing a patchy and severe obliteration of various degrees at the posterior pole, with visible small neovascular fronds along the superior arcade vessels (black arrows), and a small subhyaloid hemorrhage along the inferior arcade (white arrow). the superior arcade veins are dilated and tortuous as compared with the inferior veins the fundus fluoresceine angiogram of the right eye showed a normal study. the left eye, however [fig. 3 ] showed large areas of capillary drop out in the superior, temporal and inferior retina, various degrees of retinal vessel obliteration as well as multiple neovascular fronds along the superior arcade vessels. the vessel wall staining was evident, as was the capillary obliteration of the foveal avascular zone. the optical coherence tomography (oct) in the right eye showed a normal foveal contour with a central subfield thickness of 171 microns. 4 ] showed a schitic retina superior to the macula, with a few cystic spaces inferiorly, and subhyaloid hemorrhage with a central subfield thickness of 67 microns. the fundus fluoresceine angiogram of the left eye with following prominent findings : (a) the late arterio - venous phase of the posterior pole with obliteration of capillaries at the foveal avascular zone with capillary drop out areas (hypofluorescent) and neovascular fronds (hyperfluorescent) seen superiorly ; (b and c) large areas of capillary drop out in the superior, temporal and inferior retina, various degrees of retinal vessel obliteration as well as multiple neovascular fronds along the superior arcade vessels ; while (d) late leakage from the neovascular fronds superiorly, with generalized vascular staining optical coherence tomography of the left eye showing a schitic retina superior to the macula with a few cystic spaces, and a central subfield thickness of 67 microns the patient was advised for scatter laser photocoagulation in the left eye. she was followed over a period of 2 years, with slow but complete resolution of the proliferative changes in the left eye [fig. 5 ], with resultant fibro - vascula proliferation causing intermittent vitreous hemorrhage, which cleared spontaneously. the vision at the last follow - up in the left eye was 20/400, n36. a color fundus montage picture of the left eye at the last follow - up showing a pale disc, well ablated retina, with regressed neovascular fronds and sclerosed retinal vessels superiorly malaria is a mosquito borne infectious disease caused by parasitic protozoa of the genus plasmodium. the retinopathy seen in association with severe malaria is well - described in african sub - continent. p. falciparum has been found to be associated with retinopathy in malaria in almost all the cases. actually the term malaria retinopathy has been used in association with severe malaria caused by p. falciparum. cerebral malaria is typically diagnosed based on the clinical signs of fever and deranged cerebral dysfunction (impaired consciousness, convulsions, focal neurological deficit, or psychosis) with a recent history of travel to the endemic area. diagnosis of malaria is confirmed with the blood films for asexual forms of the malarial parasite, and computed tomography scan, followed by lumbar puncture (to exclude other causes like viral encephalitis or meningitis). since our patient was diagnosed as malaria based on the peripheral blood films, with no other neurological signs, further investigations were not deemed necessary by the primary physician. malaria retinopathy has been described to have four distinct components, namely patchy retinal whitening, focal changes in vessel wall coloration, retinal hemorrhages, and papilledema. so characteristic are the first two findings, that they have been characterized to diagnose malaria in a comatose patient, even if the peripheral blood smear is negative for malarial parasites. fundus changes in non - cm have also been studied by various investigators in pediatric and adult populations. even though, some or all the retinal findings may be noted in noncm, retinal whitening and hemorrhages have been found to be particularly associated with the cm. since the severe retinopathy has been described mainly in patients with cm so far, the hypothesized patho - physiology has also been described accordingly in both the organ systems. obstruction of microvasculature by sequestration of erythrocytes has been thought to play a central role, both in cerebral and retinal manifestations. the physical obstruction by these rigid cytoadherent parasitized erythrocytes is compounded by reduced red cell deformability and adhesive forces between infected erythrocytes (autoagglutination) and between infected and uninfected erythrocytes (rosetting). impaired retinal vessel perfusion was demonstrated in vivo by fluorescein angiography in 28 of 34 children with cm in a study in malawi. these patients similarly showed large areas of capillary drop outs as in our case study, but with no evidence of proliferative retinopathy secondary to these changes. authors hypothesize that the perfusion was impaired in the retinal circulation to such an extent so as to produce large areas of capillary nonperfusion, resulting in the formation of new blood vessels. the treatment in the form of 360 scatter laser photocoagulation resulted in reduced ischemic load and regression of the neovascularization, thus stabilizing the retinopathy. the present case report underlines the importance of having early ophthalmic examination and possible intervention in all patients of malaria, even if the central nervous system is not affected. this case appears to be the first in the literature describing the presence of unilateral proliferative retinopathy, in a patient with severe non - cm caused by p. falciparum, being described in indian population. | the retinopathy in association with malaria fever described so far includes retinal hemorrhages, vessel changes, retinal discoloration / whitening and papilledema. malaria retinopathy has been mostly described in severe cases, associated with plasmodium falciparum, correlating the patho - physiology of retinal and cerebral manifestations. we report an unusual case of proliferative retinopathy as a manifestation of malaria fever, caused by p. falciparum with no cerebral involvement. the patient had features of unilateral retinal vascular occlusion with proliferative changes and vitreous hemorrhage. to the best of our knowledge, such a case has never been reported so far in the literature. this report highlights the possible occurrence of severe proliferative changes associated with malaria fever, which if diagnosed early can prevent possible blindness. |
the management of critically ill patients with a suspected invasive fungal infection based on predefined clinical and microbiological criteria or the punctuation of a score may be a valid approach when the definitive diagnosis is feasible only in a small proportion of patients. this is what vandewoude and colleagues propose with their retrospective analysis of all patients who had aspergillus spp. fungal infections have increased in intensive care units (icus) over the past decades. although less common than candidiasis, aspergillosis is more likely to result in a life - threatening infection. thus, neutropenic patients, and those who receive long - lasting corticosteroid treatments, are at high risk for invasive aspergillosis. this organism grows on a wide variety of organic material and the conidia are easily aerosolised. although exposure is universal, invasive infection occurs almost entirely in immuno - suppressed individuals. outbreaks have been described in bone marrow transplantation, solid organ transplant recipients and leukaemia patients in association with hospital construction and/or ventilation system contamination with aspergillus. indeed, multiple organ failure and prolonged stays in the icu are associated with a complex decrease in immune functions, deactivation of macrophages and altered cellular response. this is usually not feasible given the special circumstances of critically ill ventilated patients. likewise, screening the blood for galactomannan may be very valuable in neutropenic patients but its usefulness in icu patients is limited. before new antifungal agents were available, mortality of critically ill patients with invasive aspergillosis was nearly 100%. currently, the therapeutic armoury has significantly improved with the introduction of new azoles (i.e., voriconazole) and the echinocandins (i.e., caspofungin), a new class of drugs with a novel target. many problems contribute to the lack of confident and timely diagnosis of invasive aspergillosis in critically ill patients. on one hand, the early administration of antifungal agents may be life - saving, but clinicians must also bear in mind the problems and costs associated with needless treatments derived from the overinterpretation of the potential clinical significance of isolates of aspergillus spp. in respiratory samples. propose a clinical algorithm based on the criteria defined by an international conference on the diagnosis of aspergillosis in immunocompromised patients. with this approach, approximately 50% of the patients were diagnosed with invasive aspergillosis and in the other 50% the isolation was considered colonization. unfortunately, histology was available only in a small proportion of patients (one - fourth of patients with presumed diagnosis of infection and one - tenth of the patients with the diagnosis of colonization). at first glance, these results seem very hopeful, although positive and negative predictive values can not be calculated with these figures. the diagnostic accuracy of this algorithm can be improved. many authors have documented that invasive aspergillosis can occur in certain types of ' non - immunocompromised ' critically ill patients. three high risk groups stand out for invasive aspergillosis : chronic obstructive pulmonary disease, prolonged multiple dysfunction syndrome in the situation of immunoparalysis, and severe hepatic failure. these underlying conditions are not included in the proposed criteria and they should be added to the list. this may avoid the misclassification of these high risk patients if semiquantitative culture of bronchoalveolar lavage was not positive, a criterion not universally accepted. moreover, a high resolution ct scan is nowadays mandatory and a normal portable chest x - ray may lead to an erroneous classification. the significance of a positive respiratory culture for aspergillus spp. in a non - immunodepressed patient causes the clinician great uncertainty and doubt. nowadays, the isolation of aspergillus spp. in a critically ill patient is not an exceptional curiosity. definitions proposed by the european organisation for the research and treatment of cancer were not designed to guide clinical practice. critical care physicians need a helpful instrument to decide in which circumstances antifungal therapy should be initiated early, given the high mortality of this infection but the availability of new and active agents. obviously, this and other strategies need to be validated in large cohorts of critically ill patients before they can be recommended. this is an urgent task because we do not expect to have at our disposal a precise microbiological test in the near future. | the clinical relevance of recovering aspergillus species in intensive care unit patients is unknown. diagnosis of invasive pulmonary aspergillosis is extremely difficult because there are no specific tests sensitive enough to detect it. the rapidly fatal prognosis of this infection without treatment justifies early antifungal therapy. a clinical algorithm may aid clinicians to manage critically ill patients from whose respiratory specimens aspergillus spp. have been isolated. this new tool needs to be validated in a large cohort of patients before it can be recommended. |
by using a previously described dna restriction analysis procedure (7), we studied 76 archived adenovirus isolates collected among influenzalike - illness surveillance sites across iowa from 1992 to 2002. among the 76 isolates, 40 (53%) were ad7d2, and 6 (8%) were ad7h (figure). the first ad7d2 specimen was isolated in march 1994 from a child living in south - central iowa. the first ad7h specimen was isolated in november 1993 from a child living in north - central iowa. number of adenovirus (ad) isolates collected in iowa during influenzalike - illness surveillance by genome type and year. ad7d2 caused illness among patients in iowa ranging in age from 3 months to 49 years. although the clinical details are sparse, a number of patients were thought to have influenza or were diagnosed with respiratory distress syndrome. at least 4 children from an ad7d2 october 2000 epidemic at a long - term care facility in des moines, iowa, died. ad7d2 became increasingly more prevalent across iowa, displacing ad7b, the predominant genome type circulating in the united states since the early 1970s (8). in 2002, data suggest that ad7d2 supplanted all other ad7 genome types (9 of 9 ad7 isolates were a7d2) (figure). ad7d2 was first detected in israel in 1992 ; beginning in 1995, it was associated with epidemics of unusually severe respiratory disease with high fevers among children in japan (9,10). ad7h was first detected in south america in 1986 ; since then it has supplanted the previous most prevalent genome type, ad7c, in chile, uruguay, argentina, and possibly other countries (11). ad7h has caused pediatric respiratory epidemics, and infected children had longer hospitalizations, had higher temperatures, and required more supplemental oxygen (12). in at least 1 study, up to 94% of adenovirus deaths were attributed to ad7h (11). whether these strains are truly more virulent or whether they better evade the host s immune system is a matter for future study. what does seem to be clear is that a simple mutation (ad7d2) (9) or recombination (ad7h) (13) may generate new adenovirus strains that could result in more epidemics and higher death rates. the number of immunocompromised patients in the united states is increasing, and they, in addition to young children, may be at increased risk for severe disease from emergent adenovirus strains. developing molecular typing strategies for emerging ad strains seems prudent, as does improving local and national surveillance for adenovirus illness. considering adenovirus to be a potential nosocomial pathogen seems wise, and researchers should seek to identify effective antiviral therapy for outbreak interventions. these actions will help public health officials better understand the changing epidemiology of adenovirus infections. because of increased adenovirus morbidity (14,15), the u.s. department of defense recently contracted to again produce ad4 and ad7 vaccines for military trainees. if civilian populations were identified to be at high risk for serious ad4 or ad7 disease, they might also benefit from these vaccines. | we evaluated 76 adenovirus type 7 (ad7) isolates collected in iowa from 1992 to 2002 and found that genome type ad7d2 became increasingly prevalent. by 2002, it had supplanted all other ad7 genome types. the association of ad7d2 with severe illness and death calls for heightened public health concern. |
on the basis of mechanism - driven reaction design, a pd - catalyzed nucleophilic fluorination of aryl bromides and iodides has been developed. the method exhibits a broad substrate scope, especially with respect to nitrogen - containing heteroaryl bromides, and proceeds with minimal formation of the corresponding reduction products. a facilitated ligand modification process was shown to be critical to the success of the reaction. |
|
details of the alphatocopherol, betacarotene cancer prevention (atbc) study have been described.8 male caucasian 5069 year old smokers from southwestern finland (n = 29,133) were recruited from 1985 to 1988. they were assigned to one of the following intervention groups based on a 2 2 factorial trial design : (i) tocopherol (dltocopherylacetate, 50 mg / day), (ii) carotene (20 mg / day), (iii) both supplements or (iv) placebo. participants took the study capsules for 58 years (median 6.1 years) through april 30, 1993 or until they left the trial. questionnaires about general risk factors, smoking and medical history as well as a validated foodfrequency questionnaire were completed at entry, height and weight were measured and serum was collected (after an overnight fast) protected from light and stored at 70 c until assayed. written informed consent was obtained from all participants and the trial was approved by institutional review boards at both the finnish national public health institute and the us national cancer institute. the present analysis includes 200 confirmed cases of incident prostate adenocarcinoma (icd9 185) matched 1:1 with controls on age (1 year) and date of baseline blood collection (30 days). these cases and controls were independent of our original study,7 having no overlap. to enable us to examine aggressive cases separately and to examine associations stratifying by time between blood collection and diagnosis, cases were randomly selected such that 25 nonaggressive and 25 aggressive cases were sampled from each of four periods between blood collection and diagnosis : 20 (5%) missing values, 626 identified compounds remained for analysis. based on existing literature, metabolites were categorized as eight mutually exclusive chemical classes (amino acids, carbohydrates, cofactors and vitamins, energy metabolites, lipids, nucleotides, peptides and xenobiotics). the median and interquartile range of the cv% across the metabolites was 9% (420%). these cvs are similar to those previously observed for blood samples analyzed by the same laboratory.11 serum total prostate specific antigen (psa) concentration was measured in the clinical chemistry laboratory of dr. each batch included blinded duplicate qc samples, with the cvs ranging from 5 to 10%. to standardize for batch variability, the signal strength of each metabolite was divided by the median value of participants in a given batch ; these normalized levels were then logtransformed and missing values were assigned the minimum nonmissing value. in addition to the matching factors, the following variables were evaluated as potential confounders : trial treatment group, family history of prostate cancer, history of benign prostatic hyperplasia (bph), physical activity, body mass index, smoking (cigarettes per day), serum total and hdl cholesterol, serum retinol and serum tocopherol. inclusion of any of these variables in the model did not change the effect of any metabolite by 10% or more. subgroup analyses were conducted based on trial tocopherol and carotene supplementation (yes / no for each), smoking intensity (split at 20 cigarettes daily) and bmi, serum total and hdl cholesterol (all based on medians). the threshold for statistical significance in our primary analysis is p = 0.00008, based on a bonferroni correction for 626 tests ; the threshold is 0.003 for the most significant metabolites in the main analysis, based on correction for 170 tests. it should be noted that this is a highly conservative (i.e., stringent) threshold because of intercorrelation among many of the metabolites. key findings of this analysis were metaanalyzed with data from our initial study in this cohort7 using a random effects modeling approach. we evaluated the association between chemical classes of metabolites and prostate cancer using gene set analysis (gsa) as described in detail by efron and tibshirani,12 a potentially more robust and powerful test than the gene set enrichment analysis described by subramanian.13 letting { z 1,, z s}be the z values from testing the s metabolites in a pathway, gsa evaluates the statistic max (z, z), where z (z) is the average of all positive (negative) values and calculates the pvalues by 10,000 permutations. all analyses were performed using sas version 9.1.3 (sas institute, cary, n.c.), with the exception of the gsa, which was conducted using r ; all statistical tests were 2sided. details of the alphatocopherol, betacarotene cancer prevention (atbc) study have been described.8 male caucasian 5069 year old smokers from southwestern finland (n = 29,133) were recruited from 1985 to 1988. they were assigned to one of the following intervention groups based on a 2 2 factorial trial design : (i) tocopherol (dltocopherylacetate, 50 mg / day), (ii) carotene (20 mg / day), (iii) both supplements or (iv) placebo. participants took the study capsules for 58 years (median 6.1 years) through april 30, 1993 or until they left the trial. questionnaires about general risk factors, smoking and medical history as well as a validated foodfrequency questionnaire were completed at entry, height and weight were measured and serum was collected (after an overnight fast) protected from light and stored at 70 c until assayed. written informed consent was obtained from all participants and the trial was approved by institutional review boards at both the finnish national public health institute and the us national cancer institute. the present analysis includes 200 confirmed cases of incident prostate adenocarcinoma (icd9 185) matched 1:1 with controls on age (1 year) and date of baseline blood collection (30 days). these cases and controls were independent of our original study,7 having no overlap. to enable us to examine aggressive cases separately and to examine associations stratifying by time between blood collection and diagnosis, cases were randomly selected such that 25 nonaggressive and 25 aggressive cases were sampled from each of four periods between blood collection and diagnosis : 20 (5%) missing values, 626 identified compounds remained for analysis. based on existing literature, metabolites were categorized as eight mutually exclusive chemical classes (amino acids, carbohydrates, cofactors and vitamins, energy metabolites, lipids, nucleotides, peptides and xenobiotics). the median and interquartile range of the cv% across the metabolites was 9% (420%). these cvs are similar to those previously observed for blood samples analyzed by the same laboratory.11 serum total prostate specific antigen (psa) concentration was measured in the clinical chemistry laboratory of dr. each batch included blinded duplicate qc samples, with the cvs ranging from 5 to 10%. to standardize for batch variability, the signal strength of each metabolite was divided by the median value of participants in a given batch ; these normalized levels were then logtransformed and missing values were assigned the minimum nonmissing value. in addition to the matching factors, the following variables were evaluated as potential confounders : trial treatment group, family history of prostate cancer, history of benign prostatic hyperplasia (bph), physical activity, body mass index, smoking (cigarettes per day), serum total and hdl cholesterol, serum retinol and serum tocopherol. inclusion of any of these variables in the model did not change the effect of any metabolite by 10% or more. subgroup analyses were conducted based on trial tocopherol and carotene supplementation (yes / no for each), smoking intensity (split at 20 cigarettes daily) and bmi, serum total and hdl cholesterol (all based on medians). the threshold for statistical significance in our primary analysis is p = 0.00008, based on a bonferroni correction for 626 tests ; the threshold is 0.003 for the most significant metabolites in the main analysis, based on correction for 170 tests. it should be noted that this is a highly conservative (i.e., stringent) threshold because of intercorrelation among many of the metabolites. key findings of this analysis were metaanalyzed with data from our initial study in this cohort7 using a random effects modeling approach. we evaluated the association between chemical classes of metabolites and prostate cancer using gene set analysis (gsa) as described in detail by efron and tibshirani,12 a potentially more robust and powerful test than the gene set enrichment analysis described by subramanian.13 letting { z 1,, z s}be the z values from testing the s metabolites in a pathway, gsa evaluates the maxmean statistic max (z, z), where z (z) is the average of all positive (negative) values and calculates the pvalues by 10,000 permutations. all analyses were performed using sas version 9.1.3 (sas institute, cary, n.c.), with the exception of the gsa, which was conducted using r ; all statistical tests were 2sided. all characteristics are from the baseline questionnaire except family history, which was collected during followup and is available for 264 men in this analysis. serum metabolites associated with risk of total, nonaggressive and aggressive prostate cancer at a p < 0.05 level of statistical significance are shown sorted by chemical class and then by p values in tables 2, 3, 4 (none achieved p = 0.00008 level of significance). although not found for all cases combined or for nonaggressive disease (tables 2 and 3, respectively), we observed strong risk associations between the energy and lipid metabolite chemical classes and aggressive cancer, with both being statistically significantly overrepresented among our top metabolites (table 4, energy p = 0.018, lipids p = 0.041). with the exception of erucoylsphingomyelin (or = 1.53) and trimethylamine oxide (tmao ; or 1.36), all other lipids were inversely related to aggressive prostate cancer (table 4). inositol1phosphate showed the strongest association (or = 0.56, 95% ci0.39 0.81, p = 0.002), although glycerophospholipids (e.g., oleoyllinoleoylglycerophosphoinositol and 1stearoylglycerophosphoglycerol) and fatty acids (e.g., stearate and docosadienoate) were heavily represented. the lipid signal for overall prostate cancer was weaker (chemical class test, p = 0.27 ; table 2). the data in tables 2, 3, 4 were not materially altered by adjustment for serum total psa, total cholesterol or retinol (data not shown). mutual adjustment for each other metabolite led to relatively little overall attenuation of the ors, although some attenuation was evident when adjusting within chemical classes (e.g., within lipids, stearate did attenuate the ors for many of the other fatty acids), while for some metabolites, associations were strengthened (data not shown). higher positive correlations were seen among fatty acids and glycerophospholipids and psa was weakly correlated with the metabolites. serum metabolites associated with overall prostate cancer (p < 0.05) the odds ratio per 1 standard deviation increase in metabolite level. serum metabolites associated with nonaggressive prostate cancer (p < 0.05) the odds ratio per 1 standard deviation increase in metabolite level. the odds ratio per 1 standard deviation increase in metabolite level. of the metabolites most strongly associated with prostate cancer in our initial study,7 the association between alphaketoglutarate and aggressive prostate cancer replicated in the present data (or = 0.69, 95% ci0.51 0.94, p = 0.02, table 4). another tca cycle (i.e., citric acid or krebs cycle) metabolite, citrate, was similarly but independently associated with aggressive cancer risk (or = 0.69 ; table 4). even though we identified a large number of glycerophospholipids and long chain fatty acids (lcfa) among the top metabolites, 1stearoylglycerol and glycerol (i.e., the two other top signals from our prior study7) specifically were not associated with prostate cancer risk in the present analysis (1stearoylglycerol : or = 1.03, 95% ci = 0.841.26, p = 0.79 ; glycerol : or = 0.96, 95% ci = 0.781.19, p = 0.71), with no difference based on disease aggressiveness (data not shown). metaanalysis with our prior data also showed nonstatistically significant inverse associations for overall prostate cancer : 1stearoylglycerol or = 0.61, 95% ci = 0.211.79, p = 0.37 ; glycerol or = 0.77, 95% ci = 0.451.31, p = 0.33. stratifying our cases and their controls by the median time from serum collection to diagnosis revealed more abundant glycerophospholipid, sphingolipid and steroid hormone metabolites inversely associated with aggressive prostate cancer diagnosed within 10 years of serum collection, whereas medium chain fatty acid (mcfa), lcfa and polyunsaturated fatty acid (pufa) metabolites were prominently and inversely associated in serum sampled from cases 1020 years prior to diagnosis (data not shown). we observed no statistically significant interaction between any of the 34 metabolites associated with aggressive prostate cancer at p < 0.05 and the following factors : the atbc trial tocopherol or carotene supplementation, bmi, cigarettes per day and serum total or hdl cholesterol. we found other notable, biologically relevant metabolite signals for prostate cancer. for aggressive disease, elevated thyroxine and tmao 95% ci = 1.08 2.54, p = 0.021 ; and or = 1.36, 95% ci = 1.02 1.81, p = 0.039. metabolites associated with nonaggressive cancers were nucleotides (2deoxyuridine and adenosine 5monophosphate (amp) : or = 1.81, ci = 1.15 2.85, p = 0.010 ; and, or = 1.50, ci = 1.03 2.16, p = 0.032, respectively ; p = 0.03 for nucleotide class), two steroid hormones (11dehydrocorticosterone and 21hydroxypregnenolone monosulfate : or = 0.62, ci = 0.44 0.88, p = 0.0069 ; and or = 1.46, ci = 1.03 2.06, p = 0.034, respectively) and two tobacco metabolites (cotinine and hydroxycotinine : or = 0.59, ci = 0.38 0.90, p = 0.014 ; and, or = 0.67, ci = 0.47 0.96, p = 0.029, respectively). it should also be noted that we did not observe a positive risk association for the branchedchain amino acids leucine, isoleucine and valine (data not shown), as was recently reported for pancreatic cancer risk.14 the present metabolomics analysis reveals associations between aggressive prostate cancer and a large number serum glycerophospholipids, lcfas, pufas and tca cycle compounds, consistent with our recent preliminary data.7 specifically, we replicated the original inverse association with alphaketoglutarate (metaanalysis of the two sets reveals alphaketoglutarate as the top metabolite associated with aggressive disease : or = 0.62, 95% ci = 0.46 0.82, p = 0.0009) and identified several additional biologically relevant diacylglycerophospholipids. these findings were independent of bph and serum total psa, as evidenced by the low psametabolite correlations and the lack of metabolite association attenuation upon model adjustment for psa or bph. other than our initial investigation,7 this may be the only report of a prospective metabolomics profiling study of prostate cancer risk having long followup. crosssectional clinical studies recently reviewed5, 6 have primarily focused on metabolomic profiles of tumor tissue (primaries and metastases ; biopsies and prostatectomies) and urine in men with prostate cancer. although a large number of metabolites were detected in relation to disease aggressiveness, metastases and biochemical recurrence, evidence to support the associations with increased glycerophospholipids, cholines and sarcosine and decreased citrate and polyamines is more preponderant (albeit, with inconsistent findings).6 these data do in fact indicate that metabolite associations vary by cancer aggressiveness, mostly defined by stage at diagnosis, and we might expect the serologic molecular patterns for aggressive and advanced disease to be more similar to prostate cancer cases profiled at the time of diagnosis than to early, nonaggressive cancers. for example, we did identify several glycerophospholipids and citrate as being inversely associated with aggressive disease. studies of serial prospective metabolomic profiles leading up to clinical diagnosis will be useful in this regard. the strong association signals for lipids and tca cycle members are consistent with current knowledge from abundant basic research regarding cancer cell metabolic changes related to energy, biosynthetic capacity and cell signaling.15, 16, 17, 18 that rapidly proliferating malignant cells derive much of their energy requirements from cytosolic anaerobic glycolysis, possibly in response to hypoxic intratumor conditions, was originally proposed by warburg several decades ago.19 more recently, laboratory studies support some degree of functional tca cycle and electron transportoxidative phosphorylation production of atp in malignancies.20 fatty acids from de novo biosynthesis or through monoacylglycerol or diacylglycerol lipase activity reported to be overexpressed in aggressive but not nonaggressive prostate cancers15, 17, 21, 22provide a key molecular source of acetylcoa (beyond pyruvate from glycolysis) through successive twocarbon betaoxidation in mitochondria.16, 17 this is facilitated by carnitine palmitoyl transferase1 transport of fatty acids into the mitochondrial matrix, including specifically in prostate cancer.23 acetylcoa then enters the tca cycle via citrate synthase and oxaloacetic acid to yield citrate. these additional cellular perturbations of fatty acid metabolism involving lcfas, acetylcoa and citrate provide energy and other macromolecules for malignant cell proliferation and tumor anabolic requirements and they potentially account for the lower lipid metabolite profiles in the prostate cancer cases. beyond energy and anabolic requirements, altered lipid regulation also supports membrane macromolecular species needed during rapid cell proliferation, including for lipid rafts and other key signaling constituents.24, 25 consistent with this, we identified a large number of glycerophospholipids (including monoacyl and diacyl glycerophosphoinositols, glycerophosphoethanolamines and glycerophosphocholines), lcfa, pufa and erucoylsphingomyelin among the top metabolites associated with aggressive prostate cancer, with the phosphorylated derivative of myoinositol, inositol1phosphate or i1p, ranking highest. i1p is a member of the phosphatidylinositide family of compounds that have become increasingly recognized as integral plasma membrane, protein phosphorylation and second messenger signaling components.26 i1p and related ip species such as ip4,5 (i.e., phosphorylated at carbons 4 and 5 of the inositol ring) are functionally related to the numerous diacylglycerophospoinositol lipid metabolites we found associated with aggressive prostate cancer. these compounds serve as substrates for plasma membrane phospholipase c that produces, (i) ip1,4,5 which is released into the cytosol where it mobilizes ionized calcium from endoplasmic reticulum and (ii) diacylglycerol, which remains membraneassociated to transactivate protein kinase c.15, 18, 27 triggered by plasma membrane hormone receptor binding and related to prostate cancer aggressiveness, both ip1,4,5 and diacylglycerol can therefore serve as second messengers for signal transduction through protein phosphorylation.21, 27, 28 similar in association strength to our finding for alphaketoglutarate, but independent of it, we found lower risk of aggressive prostate cancer in men with higher serum citrate status. citrate is concentrated in the normal prostate peripheral zone glandular epithelium owing to high zinc ion concentration inhibition of mitochondrial aconitase conversion of citrate to isocitrate.29 in turn, citrate is secreted in prostatic fluid in high concentration, a process referred to as net citrate production,29 possibly to maintain a lower semen ph or provide an energy source for sperm motility or oocyte fertilization. bph tissue also exhibits elevated citrate content.30, 31 by contrast, malignant prostate cells exhibit low zinc and low citrate concentrations, apparently the result of a biochemical shift to citrate oxidation (along with alphaketoglutarate) through the tca cycle to provide greater atp production for tumor cell anabolism and proliferation. such a metabolic transformation of malignant prostate cells could account for our finding of lower citrate status in aggressive cases versus noncases and be reflective of the higher tumor energy requirements. alternatively, tumor cell conversion of alphaketoglutarate to citrate through reductive carboxylation as a result of isocitrate dehydrogenase isoforms and nadp+/nadph, with subsequent acetylcoa formation and fatty acid synthesis, has also been suggested in the setting of mitochondrial dysregulation.32 citrate and alphaketoglutarate might also be metabolized to biosynthetic precursors for fatty acids and sterols (citrate) and glutamate (alphaketoglutarate). it is also relevant and of interest that recently developed prostate cancer magnetic resonance spectroscopic metabolic imaging includes targeting decreased citrate as well as increased choline, creatine, polyamines and myoinositol.30, 31, 33 beyond the strong signals we detected for lipid and energy metabolites, other specific metabolites appeared associated with prostate cancer, including tmao and thyroxine for aggressive disease. the choline derivative tmao has been linked to atherosclerosis and cardiovascular disease,34 and most recently to elevated colorectal cancer risk.35 given the role of intestinal microbiota in human tmao production and the potential for local luminal epithelial effects, the higher risk for prostate cancer suggested here deserves further study. thyroid hormone status has been previously associated with prostate cancer, including in a recent study from the atbc cohort showing lower risk for hypothyroid men.36 the positive associations between some nucleotides, including amp and nonaggressive disease (chemical class pvalue = 0.03) require reevaluation in other studies, particularly given the number of known metabolites examined. that the branchedchain amino acids did not show positive associations with prostate cancer similar to those observed for pancreatic cancer14 supports the likelihood that organ site differences in metabolomic changes reflect underlying tissuespecific biological changes that should be further investigated. our study has several important strengths, primary among which are : (i) having up to two decades between blood sampling and prostate cancer diagnosis, (ii) analysis of serum collected after an overnight fast and (iii) complete, population registrybased case ascertainment. long followup affords us the opportunity to examine reverse causality and possible temporal changes in circulating metabolites ranging from up to 20 years before clinical diagnosis to within 10 years. qualitative differences in lipid subclasses based on time from blood collection to diagnosis were detected (e.g., fatty acids vs. glyceropholipids) and these patterns may provide biologically based leads regarding transformations during prostate cancer development and progression. fasting serum should in theory reduce metabolite variation related to recent food consumption and may offer a snapshot of basal metabolomic profiles less influenced by recent food / beverage consumption. prostate cancers were for the most part diagnosed clinically and not from population psa screening. study limitations include the relatively homogeneous population (i.e., finnish smokers of five or more cigarettes daily who were of caucasian descent) and modest sample size. whether our findings for cotinine and hydroxycotinine in nonaggressive prostate cancer were influenced by metabolite changes related to tobacco smoking is possible but unlikely given that all cases and controls were smokers at the time of serum collection and those compounds are but two of several known tobaccorelated metabolites.37 further, our results were unchanged when adjusting for cigarettes smoked per day and were similar for lighter and heavier smokers, suggesting that cigarette smoking did not strongly influence the results. given that we identified 626 known biochemicals, the findings for some of the metabolites may be due to chance. how the numerous metabolites identified in the present study might biologically impact prostate cancer risk or relate to known biochemical changes during its development and progression the specificity and subpathway interrelatedness of the metabolites we identified are compelling in light of wellestablished metabolic changes in prostate malignancies, however. to our knowledge, alphaketoglutarate, citrate, i1p and most of the other metabolites we found associated with aggressive prostate cancer have not been similarly examined in epidemiologic studies to date. lipidlowering statin use is associated with lower risk of aggressive prostate cancer38, 39 and a recent metaanalysis of cohorts with an average followup of five years found that lower plasma / serum stearic acid and higher eicosapentaenoic and docosapentaenoic acid concentrations, were related to higher risk (with no other fatty acid or phospholipid associations).40 the casecontrol differences in the circulating metabolites we observed were present up to 20 years before clinical diagnosis. given the widely accepted pathobiology of latent, slowgrowing, subclinical prostate cancers that is supported by autopsy studies41 and registry analyses,42 our findings for aggressive disease could be interpreted as reflecting the impact of metabolic changes in larger and disseminated malignancies on circulating metabolites. alternatively, greater availability of lipid and energy metabolites in circulation resulting from lifestyle exposures or genetic predisposition may inhibit the initiation or growth of prostate cancers. it remains unclear whether the magnitude of the differences we observed in metabolite signals between cases and controls would be sufficient to affect prostate cancer growth or initiation. additional basic and clinical research should address the questions raised by these data. in conclusion, our prospective study data indicate that several circulating glycerophospholipid, fatty acid, energy and related metabolites are inversely associated with aggressive prostate cancer up to 20 years prior to diagnosis. the findings may provide valuable and timely prospective human validation of substantial prior basic and clinical research relevant to lipid and energy metabolic transformations during prostate carcinogenesis. further replication in other prospective studies, including, where possible, metabolomic profiling of serial prediagnostic blood samples and in prostate tissue (tumor and adjacent normal), will be informative. | despite decades of concerted epidemiological research, relatively little is known about the etiology of prostate cancer. as genomewide association studies have identified numerous genetic variants, so metabolomic profiling of blood and other tissues represents an agnostic, broadspectrum approach for examining potential metabolic biomarkers of prostate cancer risk. to this end, we conducted a prospective analysis of prostate cancer within the alphatocopherol, betacarotene cancer prevention study cohort based on 200 cases (100 aggressive) and 200 controls (age and blood collection datematched) with fasting serum collected up to 20 years prior to case diagnoses. ultrahigh performance liquid chromatography / mass spectroscopy and gas chromatography / mass spectroscopy identified 626 compounds detected in > 95% of the men and the odds ratio per 1standard deviation increase in logmetabolite levels and risk were estimated using conditional logistic regression. we observed strong inverse associations between energy and lipid metabolites and aggressive cancer (p = 0.018 and p = 0.041, respectively, for chemical class overrepresentation). inositol1phosphate showed the strongest association (or = 0.56, 95% ci = 0.390.81, p = 0.002) and glycerophospholipids and fatty acids were heavily represented ; e.g., oleoyllinoleoylglycerophosphoinositol (or = 0.64, p = 0.004), 1stearoylglycerophosphoglycerol (or=0.65, p = 0.025), stearate (or=0.65, p = 0.010) and docosadienoate (or = 0.66, p = 0.014). both alphaketoglutarate and citrate were associated with aggressive disease risk (or = 0.69, 95% ci = 0.510.94, p = 0.02 ; or = 0.69, 95% ci = 0.500.95, p = 0.02), as were elevated thyroxine and trimethylamine oxide (or = 1.65, 95% ci = 1.082.54, p = 0.021 ; and or = 1.36, 95% ci = 1.021.81, p = 0.039). serum psa adjustment did not alter the findings. our data reveal several metabolomic leads that may have pathophysiological relevance to prostate carcinogenesis and should be examined through additional research. |
a stepwise model of development, from well - differentiated precursors to poorly differentiated progressed hepatocellular carcinomas (hccs), is well established by evidences accumulated in the past three decades. in 1995 and 2009, international reproducible criteria for the diagnosis of nodular lesions of hepatocarcinogenesis were developed by the remarkable endeavors of the east and the west pathologists. at present, the criteria involve not only pathologic but also radiologic features, especially hemodynamic findings. for example, progressed hccs are defined as radiologically hypervascular lesions without portovenous supply which histologically appear moderately or poorly differentiated ; early hccs and dysplastic nodules are defined as iso- or hypovascular lesions with portovenous supply on radiological images which appear well differentiated on histology [1, 2 ]. thus, an intimate knowledge of the relations between tumor hemodynamics and hepatocarcinogenesis would be useful for the management of carcinogenic nodules as well as for the better understanding of multistep models of hepatocarcinogenesis. this review focuses on radiologic hemodynamic features of carcinogenic hepatocyte nodules arising from cirrhotic livers, that is, dysplastic nodules, early hccs, and progressed hccs, paying close attention to radio - pathological correlations and outcomes of the nodules. on histology, the blood circulation is not kinetically depicted, but tumor blood inflows can still be analyzed through the quantification of the inflow vessels with morphometric technique. the pathology of inflows of carcinogenic hepatocyte nodules has been morphometrically evaluated by two methods : counting the number of inflows vessels and measuring the luminal area of them. there are three types of inflow vessels of carcinogenic hepatocyte nodules portal vein, hepatic artery, and abnormal artery which are termed unpaired or nontriadal arteries (figures 1 and 2) [310 ]. the former two ones, portal vein and hepatic artery, are accompanied by bile ducts running within the portal tracts, whereas the last, unpaired artery, is not accompanied by bile ducts or portal veins independently running outside of portal tracts. it is now considered that the unpaired arteries are new vessels developed through neovascularization during hepatocarcinogenesis [310 ]. the inflow vessels in the carcinogenic nodules of the liver have been histomorphometrically quantified by counting the number of vessels in the unit area : vessel density. during dedifferentiation of the nodules, portovenous densities of the nodules the portovenous densities within dysplastic nodules are lower than those within the liver [3, 9].the arterial densities of low grade dysplastic nodules are almost as high as those of the surrounding livers ; those of high grade dysplastic nodules are also almost as high as those of the livers ; those of progressed hccs are much higher than those of the livers. the proportions of the numbers of unpaired arteries to those of total arteries increase during the dedifferentiation. measuring luminal areas of the vessels is more recommendable than counting their number although the former is much more laborious than the latter, because luminal areas reflect angiography more accurately than numbers. the portovenous luminal areas of low grade dysplastic nodules per unit area are as large as those of the surrounding livers ; those of high grade dysplastic nodules are smaller than those of the livers ; those of progressed hccs are almost null. the arterial luminal areas of low grade dysplastic nodules per unit area are slightly smaller than those of the livers ; those of high grade dysplastic nodules are smaller than those of the livers ; those of progressed hccs are much larger than those of the livers. the proportions of unpaired arteries to total arteries of low grade dysplastic nodules in luminal area are small, those of high grade dysplastic nodules are moderate, and those of progressed hccs are almost 100%. to summarize, during the dedifferentiation, the portovenous areas of carcinogenic hepatocyte nodules monotonically decrease whereas the arterial areas bitonically change, first decrease because hepatic arterial areas decrease and then increase because unpaired arterial areas increase (figure 3). hepatocyte nodules with smaller portovenous areas and the same arterial areas to the surrounding livers are just on the final stage of high grade dysplastic nodules transiting to progressed hccs. tumor blood inflows, both portovenous and arterial, can be evaluated with imaging techniques. there are two techniques for evaluation of tumor portovenous inflows : doppler sonography and ct during arterial portography (ctap). with doppler sonography, inflows in some large portovenous branches in tumors may be visualized, but portovenous perfusions can not be evaluated. with ctap, tumor portovenous perfusions can be evaluated not only qualitatively but semiquantitatively [1315 ]. for example, we can tell that a portovenous perfusion of a nodule is null or reduced. in addition, we can identify the perfusion difference between nodules and the surrounding livers. thus, ctap should be applied in patients with carcinogenic hepatocyte nodules as principal technique to confirm the portovenous inflows of the nodules. there are six imaging techniques for evaluation of tumor arterial inflows : doppler sonography as noncontrast imaging [11, 16 ], contrast sonography with perfluorobutane microbubbles [1719 ], dynamic contrast - enhanced ct and dynamic contrast - enhanced mr imaging as intravenous contrast enhanced imaging, sonography with carbon dioxide microbubbles, and ct during hepatic arteriography (ctha) as intra - arterial contrast - enhanced imaging. among these techniques, ctha is the best suited to the understanding of the complex hemodynamic interactions in an organ with a dual blood inflow since it depicts both the lesions and the livers free from portovenous enhancement and also avoids improper scan delays that occasionally occur in dynamic contrast - enhanced ct or mr imaging. imaging techniques illustrate the relations, mentioned in section 2.1, between tumor blood inflows and histological grades in carcinogenic hepatocyte nodules [14, 20, 21 ]. the attenuations of the nodules on ctap decrease during dedifferentiation of the nodules : low grade dysplastic nodules show isoattenuating ; high grade dysplastic nodules and early hccs show isoattenuating or slightly hypoattenuating ; progressed hccs show hypoattenuating(figures 4 and 5). the attenuations of the nodules on ctha first decrease and then increase : low grade dysplastic nodules show isoattenuating or slightly hypoattenuating ; high grade dysplastic nodules and early hccs show hypoattenuating or isoattenuating ; progressed hccs exclusively show hyperattenuating (figures 4 and 5). it is noteworthy that high grade dysplastic nodules and/or early hccs harboring progressed hccs in nodule - in - nodule fashion are demonstrated on ctap as isoattenuating or slightly hypoattenuating nodules carrying internal definite hypoattenuating foci, but as hypo- or isoattenuating nodules containing hyperattenuating nodules on ctha (figure 6). tumor arterial inflows via unpaired arteries can not be differentiated from those via hepatic arteries by radiological imaging. however, when an isointense nodule on ctha is depicted as a hypointense nodule on ctap, the nodule can be interpreted as receiving arterial inflows via unpaired arteries and on the final stage of dysplastic nodule just before progressed hcc (figure 3). although ctap and ctha are rather invasive, they are applied as principal techniques for diagnoses of carcinogenic hepatocyte nodules because intravenous contrast enhanced ct is relatively insensitive not only for detection but also for the characterization of these nodules [22, 23 ]. it is reported that seven dysplastic nodules were categorized as hypoattenuating or isoattenuating on arterial phase ct, but another dysplastic nodule was hyperattenuating ; therefore, incorrectly categorized as progressed hcc ; 31 progressed hccs were hyperattenuating and correctly categorized but one progressed hcc was incorrectly categorized as dysplastic nodule. evaluation of arterial inflows of dysplastic nodules by dynamic contrast enhanced mr imaging is often difficult because dysplastic nodules are generally hyperintense on noncontrast t1-weighted images [2426 ]. however, a progressed hcc harbored by dysplastic nodule is depicted as a hyperintense nodule within a hypointense or isointense nodule on arterial phase images. the outcomes of the carcinogenic hepatocyte nodules can be predicted when both portovenous and arterial inflows are evaluated by ctap and ctha, respectively,. no nodules which are isoattenuating on both ctap and on ctha became progressed hccs ; approximately 30% of the nodules showing slightly hypoattenuating on both ctap and ctha became progressed hccs after two years. on the other hand, approximately 90% of the nodules carrying hypoattenuating foci on ctap and hyperattenuating foci on ctha became entirely hypervascular progressed hccs after two years (figure 7). reduced portal blood flow in the nodule on ctap is one of the most important predictors for development of progressed hcc (figure 8). on histology, tumor blood outflows can be speculated through the connections between the vessels within the tumors (capillarized intratumoral sinusoids) and those in the surrounding livers. there are three types of growth patterns in hccs, namely, replacing growth (figure 9), compressing growth without capsule (figure 10), and that with capsule (figure 11). replacing growth is seen both in hypovascular early hccs and in some well differentiated progressed hccs. hypovascular early hcc with replacing growth carry more hepatic veins in and around themselves connected with intratumoral blood sinusoids than hypervascular progressed hccs. nodules with compressing growth can be classified into two types : nodules without capsule and those with capsule. the former type carries intranodular capillarized sinusoids connected directly to the surrounding hepatic sinusoids and partly to extranodular portal veins. the latter carries intranodular capillarized sinusoids connected to portal venules within the capsule but not directly to the surrounding hepatic sinusoids. intra- or perinodular hepatic venules decrease in accordance with the grade of malignancy of the nodules and usually no intratumoral hepatic venules are observed in encapsulated hccs. in poorly differentiated hccs with invasive growth to the surrounding liver, these connections there are five imaging techniques for evaluations of tumor blood outflow : single - level dynamic ctha [29, 30 ], biphasic ctha, contrast sonography with perfluorobutane microbubbles, intravenous dynamic contrast enhanced ct, and intravenous dynamic contrast enhanced mr imaging [26, 33, 34 ]. single - level dynamic ctha, the first technique which demonstrated the tumor blood outflow ever, is the principal technique kinetically depicting both inflow and outflow at one time with the highest temporal resolution. images can be obtained with 30- to 40-second continuous acquisition without table increment under intrahepatic arterial injection of a small amount of contrast material, for example, 10 ml of 300370 mgi / ml at a rate of 1.0 ml / sec. biphasic ctha is the second technique for evaluations of tumor blood outflow, which covers the whole liver and multiple lesions. images are obtained with two whole - liver acquisitions at following delays : 10 seconds after start of intrahepatic arterial injection of contrast material of 30 ml of 300370 mgi / ml at a rate of 1.0 ml / sec for first scan (early phase ctha) and 60 seconds after start of injection for second scan (late phase ctha). we can evaluate the arterial inflows with the first scan and the sequential outflows with the second scan. with the remaining three techniques, contrast sonography with perfluorobutane microbubbles, intravenous dynamic contrast enhanced ct, and mr imaging, we can also estimate the outflows, but the reproducibilities and accuracies of the evaluations are much reduced compared with the former two techniques. radiological findings of tumor blood outflows of the carcinogenic hepatocyte nodules depend on the channels of blood drainage : hepatic veins, sinusoid, or portal veins. a nodule shows isoattenuation or slight hypoattenuation on ctap and early phase ctha and does not show corona enhancement on late phase ctha when its main drainage channel is hepatic vein. a nodule shows hypoattenuation on ctap, hyperattenuation on early phase ctha, and thin corona enhancement (2 mm or less in thickness) on late phase ctha when its main drainage channel is sinusoid. a nodule shows hypoattenuation on ctap, hyperattenuation on early phase ctha, and thick corona enhancement (more than 2 mm in thickness) on late phase ctha when its main drainage channel is portal vein. nodules with thick corona enhancement on late phase ctha commonly show compressing growth with fibrous capsule (figure 12). nodules with thin corona enhancement on late phase may show compressing growth without fibrous capsule (figure 13). nodules without corona enhancement on late phase ctha, which appear slightly hypoattenuating on both ctap and early phase ctha, show replacing growth (figure 14)., the nodules show replacing growth with indistinct margin at first, then expansile growth without fibrous capsule and finally expansile growth with fibrous capsule. thus, radiologic evaluations of tumor blood outflows of carcinogenic hepatocyte nodules, especially corona enhancement on late phase ctha, demonstrate their differentiations as well as their growth pattern. hemodynamics of carcinogenic hepatocyte nodules depicted with imaging techniques, especially with ctap and ctha, are closely related to differentiations, growth patterns, and outcomes of the nodules. hyperattenuating nodules with thick corona enhancement on ctha showing hypoattenuation on ctap are encapsulated progressed hccs. hypoattenuating or isoattenuating (= invisible) nodules carrying small hyperattenuating areas on ctha are early hccs or high grade dysplastic nodules containing tiny progressed hccs, 90% of which would become wholly progressed hccs in two years. isoattenuating nodules on ctha showing hypoattenuation on ctap are early hccs or the final stage of high grade dysplastic nodule just before progressed hcc, 90% of which would become progressed hccs in two years. hypoattenuating nodules on ctha and ctap are high grade dysplastic nodules, 30% of which would become progressed hccs in two years. isoattenuating nodules on ctha and ctap detected by other imaging techniques, such as mr imaging, are low grade dysplastic nodule which will seldom change into progressed hccs. these guides will be helpful in drawing up therapeutic strategies for hepatocyte nodules arising in cirrhosis. | tumor hemodynamics of carcinogenic hepatocytes nodules, that is, low grade dysplastic nodules, high grade dysplastic nodules, early hepatocellular carcinomas (hccs), and progressed hccs, change during multistep dedifferentiation of the nodules. morphometric analyses of inflow vessels of these nodules indicate that the portal veins of carcinogenic hepatocyte nodules monotonically decrease whereas the arteries bitonically change, first decrease and then increase. findings on imaging techniques depicting these changes in tumor blood inflows, especially intra - arterial contrast - enhanced computed tomography, closely related not only to the histological differentiation of the nodules but also to the outcomes of the nodules. histological analyses of connections between the vessels within the tumors and those in the surrounding livers and findings on imaging techniques indicate that drainage vessels of hcc change from hepatic veins to hepatic sinusoids and then to portal veins during multistep hepatocarcinogenesis. understanding of tumor hemodynamics through radio - pathological correlations will be helpful in drawing up therapeutic strategies for carcinogenic hepatocyte nodules arising in cirrhosis. |
the cornea is part of the optical system of the eye, and its condition is directly related to the quality of eyesight. measurement of central corneal thickness (cct) and endothelial cell parameters is important when undertaking a functional and morphologic evaluation of the cornea for diagnostic purposes or before various surgical interventions. due to the increasing popularity of correction of refractive defects by excimer laser, cct has come to have higher prognostic significance for determination of the success of surgery and probable post - surgical complications. surgery in the case of a cornea that is too thin may result in a very serious postoperative complication, ie, corneal ectasia. it has been assumed that the optimal cct for refractive surgery is more than 500 m ; however, opinions regarding the impact of cct in anticipating treatment of refractive defects (ie, which cct value is too low to perform surgery safely) are not consistent and studies of patients with relatively thin cct indicate no excess risk for postoperative complications.1,2 it should be remembered that intraocular pressure depends on corneal thickness ; a thick cornea may explain a falsely elevated intraocular pressure reading and, conversely, falsely low intraocular pressure is measured in eyes with thin corneas.35 this should be taken into consideration in order to avoid both overdiagnosis and underdiagnosis of glaucoma and ocular hypertension. factors thought to affect cct include race, age, sex, anthropometric parameters, drugs, time of day, blink rate, and type of measuring equipment used., none of these factors alone is able to predict cct. standards must be established in order to compare results obtained in different population groups. since cct varies in these populations, it is very important to verify the results and determine the limits of normal values.68 further, corneal parameters differ according to patient age.613 awareness of these ethnicity - related and age - related physiologic changes enables us to assess the influence of disease and surgical procedures more accurately. the purpose of this study was to estimate the mean cct in the lithuanian population and compare cct between subjects according to age group and identify correlations between cct, age, and sex. this was a prospective study performed between march 2009 and december 2010, and enrolled lithuanian citizens visiting primary health care institutions in the cities and regions of vilnius, kaunas, klaipda, iauliai, panevys, teliai, taurag, utena, alytus, and marijampol. all measurements were taken by the same examiner, who visited the above - mentioned health care institutions during the study period. after obtaining informed consent, 1,650 adult caucasians comprising 688 (41.7%) men and 962 (58.3%) women of lithuanian origin were examined. participants had a standardized interview about any previous or current ocular disease, previous eye trauma or surgery, wearing of contact lenses, and comorbidity, eg, diabetes or other chronic disease that may influence cct. cct was measured using an ultrasound contact pachymeter (quantel medical, france), applying one drop of 0.5% proxymetacaine (alcon - couvreur, puurs, belgium) for local anesthesia. the pachymeter automatically recorded five cct measurements and the mean value was taken as the result of the test. exclusion criteria were history of corneal degeneration or ulceration, anterior segment surgical procedures, glaucoma, diabetes mellitus, current conjunctivitis, or wearing of contact lenses. the subjects were stratified into seven groups on the basis of age as follows : 1829 years, 3039 years, 4049 years, 5059 years, 6069 years, 7079 years, and 8089 years (table 1). the sex distribution of the participants was similar ; however, there were more women in the age groups of 1829, 4049, 5059, and 6069 years. the study protocol followed the guidelines of the declaration of helsinki and was approved by the local ethics committee. continuous variables were evaluated using the mean and standard deviation. when the distribution was normal, the mean differences between independent samples for the two groups were assessed using the student s two - sided t - test, and the paired student s t - test was used to compare means of dependent samples. means of more than two groups were compared using one - way analysis of variance. a p - value less than 0.05 was considered to be statistically significant. continuous variables were evaluated using the mean and standard deviation. when the distribution was normal, the mean differences between independent samples for the two groups were assessed using the student s two - sided t - test, and the paired student s t - test was used to compare means of dependent samples. means of more than two groups were compared using one - way analysis of variance. a p - value less than 0.05 was considered to be statistically significant. the average age of the study population was 55.419.2 years (53.618.8 years for women and 57.319.5 years for men). mean cct for the overall patient population was 544.630.5 (range 654449) m in the right eye (figure 1), 545.230.5 (range 650451) m in the left eye (figure 2), and 544.630.5 m in both eyes. the average cct for both eyes was 545.026.4 m in men and 544.433.2 m in women. there was no statistically significant difference in cct between the eyes (p>0.05), so the right eye was selected for further investigation. the average cct for the right eye in men was 545.025.6 (range 468645) m and for women was 544.433.5 (range 449654) m. no statistically significant difference was found between the sexes (p>0.05) ; however, there was a significant sex difference in one age group, ie, men aged 1829 years had higher cct than women (p 60 years, see table 3). corneas were thinner in people over 60 years of age when compared with the other age groups (p 609 m (percentile > 97, extremely thick cornea). more details on percentiles for cct are shown in table 3. the purpose of this study was to estimate mean cct in adult lithuanians and identify any correlations between cct, age, and sex. there are reasonable data showing differences in cct between various ethnic groups.14,15 the mean cct in our study (544.6 m) was similar to that of 545.2 m reported for the caucasian population by nemesure and within the limits for mean cct reported by other studies, ie, 520579 m.6,1618 the corneas of african americans have been reported to be thinner and those of caucasians and mongoloids as thicker.6,9,14,15,1923 thin corneas were identified in mongoloids residing in mongolia (504.5 m) and india (520.7 m), whereas mongoloids living in singapore and the people s republic of china had cct values similar to those of caucasians (541.2 m and 556.2 m, respectively).9,14,15,19 some of the available data show that cct correlates significantly with sex while others do not. however, a statistically significant difference was found between young men and women aged 1829 years, with cct being higher in men. leskul did not find any correlation between these parameters in their study in thailand, in which 467 subjects aged 1260 years were examined.24 hashemi examined 800 eyes and also did not find any relationship between cct and sex.25 eballe examined 970 eyes in their study and reported finding no statistically significant difference in cct between men and women.8 findings of other studies indicate that corneas are thicker in men than in women. in the beijing eye study conducted in 2008 in the people s republic of china, xu examined 3,251 participants and found that corneas were thicker in men than in women.26 suzuki examined 2,848 men and 4,465 women and came to the same conclusion (finding a mean cct of 521 m in men and 514 m in women).27 similar results have been published elsewhere.2830 there is no clear answer as to why women have thinner corneas than men. it has been shown established that cct is lowest in women at the beginning of the menstrual cycle and highest at the end and during ovulation.31 further, keskin determined that cct was significantly decreased in postmenopausal women compared with their premenopausal counterparts.32 we observed a weak correlation between cct and age. some authors did not define any dependence between these parameters ; others, however, refer to a decrease in cct with age.7,9,33 kamiya examined 204 eyes from 204 healthy subjects, and reported that biomechanical data for the cornea change during the course of the lifetime, but could not identify significant changes in age - related cct or intraocular pressure.34 mercieca reported that the cornea becomes thinner with age and mean cct is lower in women than in men.10 the reikjavik eye study published in 2002, in which 925 healthy people aged 50 years and older were examined, showed that cct does not depend on age.4 prasad reached the same conclusion after measuring corneal parameters in 5,158 subjects.35 thinning of the cornea at a rate of 37 m per decade has been observed in older age in some ethnic groups. a study in 1998 with 1,242 participants aged 1087 years showed that cct decreased by 56 m for each decade of life.9 eballe reported that cct decreases by 4.2 m for each 10 years of life, with the thickest corneas found in subjects younger than 20 years.8 aghaian indicated that cct decreased by 3 m over a decade, and brandt reported a 6.3 m decrease over the same period.14,36 our data show that cct is decreasing at a rate of 28 m per decade in lithuanians over 40 years of age. leskul examined 467 subjects aged 1260 years of age and found that young people had higher cct.24 a similar conclusion was reached in the liwan eye study reported by wang.11 wangsupadilok also identified corneal thinning over the lifetime after examining subjects aged 1896 years.37 pfeiffer, who reported the european glaucoma prevention study, found that younger persons had thicker corneas.12 narayanaswamy who examined 1,132 individuals, reported that biomechanical features of the cornea change with the age, ie, there is a reduction in corneal resistance factor and corneal hysteresis.13 the impact of age on corneal thickness can explained in a number of different ways. referring to theory based on histologic studies, the corneas of older people are thinner because of a reduction in keratocyte density and possible destruction of collagen fibers, and senior individuals are exposed to environmental factors for a longer period of time, which might influence corneal structure.38 hasemian demonstrated that corneal endothelial cell density decreases in persons until 60 years of age ; however, the volume of these cells increases.39 as yet there is no consensus among scientists as to whether the corneal endothelium and cct are interdependent.40,41 it has been established that cct is lower in children of african american origin (same as in adults) in comparison with caucasian children.42,43 it has also been demonstrated that cct is lower in children born prematurely than in those born at term.42,44 lim examined 271 children of various ages and did not found any interdependence between cct and the axial length of the eye or refractive defects.45 hikoya examined 169 children aged 118 years and established that cct in japanese children is lower than in caucasian children, but higher than in children of african american origin.46 osmera who examined 124 subjects from 7 to 17 years of age, reported that cct and intraocular pressure values in czech children and adults are very similar to those in adults.47 in a study involving 106 children younger than 18 years, dai determined that corneas in children of african american origin were thinner than in caucasians.48 further, a survey conducted in singapore showed that chinese children had thicker corneas than malay or indian children.49 classification of cct according to our nomogram could be used in clinical practice to evaluate a patient s cct in context of the rest of the population, ie, percentile 97, severely thick cornea. our study shows that the mean cct of the adult lithuanian population is similar to the corneal thicknesses reported for other caucasian populations, and was within the limits of the mean cct defined in similar studies. a difference in cct between the sexes was observed only in the youngest age group, indicating that young men have a higher cct than women. a weak negative relationship between cct and age this age dependence was stronger in men (a moderate negative correlation) than in women (a weak negative correlation). these data will help to evaluate cct in our patients in a more practical and accurate manner. | backgroundthe purpose of this study was to estimate mean central corneal thickness (cct) and determine whether there are any correlations between cct, age, and sex in the adult lithuanian population.methodsa total of 1,650 caucasians of lithuanian origin (aged 1889 years) comprising 688 (41.7%) men and 962 (58.3%) women were examined. subjects were stratified by age into seven groups. cct was measured using ultrasonic pachymetry. correlations between cct, age, and sex were sought.resultsmean (standard deviation) cct for both eyes was 544.630.5 m. mean cct was 545.230.5 m in the left eye and 544.630.5 m in the right eye, and was 545.025.6 m in men and 544.433.5 m in women. mean cct was 550.835.7 m in subjects aged 1829 years, 557.527.6 m in those aged 3039 years, 551.331.4 m in those aged 5059 years, 544.031.4 m in those aged 5059 years, 544.231.6 m in those aged 6079 years, 535.127.8 m in those aged 7079 years, and 530.116.8 m in those aged 8089 years. no statistically significant difference in cct was found between the sexes (p>0.05). however, there was a significant difference in subjects aged 1829 years ; men had higher cct than women (p<0.05). a statistically significant negative correlation was found between cct and age (r=-0.263, p<0.05) that was stronger in men (r=-0.406, p<0.05) than in women (r=-0.118, p<0.05).conclusionthe mean cct in adult lithuanians was 544.630.5 m, of the left eye 545.230.5 m and of the right 544.630.5 m. cct of the right eye was equal to the cct of both eyes. mean cct was 545.025.6 m in men and 544.433.5 m in women. young men tended to have higher cct than women. cct decreases over the lifetime, meaning that older people have thinner corneas. cct s dependence on age is stronger in men. |
chronic pelvic pain (cpp) is estimated to affect more than 9 million women in the united states. up to 40% of laparoscopies and 10% to 12% of all hysterectomies in addition to lost productivity and decreased quality of life, the diagnosis and treatment of cpp consumes nearly $ 3 billion of health care expenditures annually. the management of cpp is challenging due to the numerous possible differential diagnoses and contributing factors associated with this condition. possible differential diagnoses include endometriosis, endosalpingiosis, pelvic adhesions, ovarian remnant syndrome, interstitial cystitis (ic), adenomyosis, and uterine leiomyomatas. these conditions may present with similar symptoms, and one or more may exist concomitantly in a given patient. endometriosis is one of the more prevalent gynecologic diagnoses among women with cpp, affecting more than half of those patients who receive a diagnosis for their cpp. symptoms include dyspareunia, cyclic perimenstrual low abdominal pelvic pain, symptom flares after sexual intimacy, and irritative voiding in the case of urinary tract involvement. a definitive diagnosis of endometriosis requires visual confirmation of the lesion during laparoscopy, and histologic confirmation of the presence of both ectopic endometrial glands and stroma. interstitial cystitis, or pelvic pain of bladder origin, is another disorder that may be associated with cpp. the cause of ic is unclear, but it is thought to be multifactorial and progressive, involving bladder epithelial dysfunction, mast cell activation, and bladder sensory nerve upregulation (figure 1). estimates of the prevalence of ic in the united states range from 10 to 510/100 000 cases. recent evidence suggests that this condition may, in fact, be much more prevalent than current estimates. the symptoms of ic include urinary urgency / frequency and/or pelvic pain in the absence of urinary tract infection. some patients may present with only urologic symptoms ; conversely, 15% of patients present with chronic pain and no urologic symptoms. patients with early ic may have only 1 or 2 symptoms, whereas at later stages, the full spectrum of symptoms may emerge and bladder capacity may be reduced. recent data suggest that outcomes may be improved when ic is detected and treated at earlier stages. the potassium sensitivity test (pst) was developed by parsons to indicate abnormal permeability of the bladder epithelium, which is thought to play a central role in the pathophysiology of ic. use of the pst has been validated in a number of studies, and may be used to help confirm the diagnosis of ic, particularly at earlier stages of the disease. in a previously published retrospective review, we diagnosed biopsy - confirmed active endometriosis in 81% of patients with ic who had undergone concurrent laparos - copy and cystoscopy with hydrodistention in evaluation for cpp. those findings suggested that these 2 conditions often exist concomitantly, and that the diagnosis of one condition should not preclude evaluation for the other. the objective of this study was to incorporate the use of the pst along with cystoscopy / hydrodistention and laparoscopy to prospectively determine the prevalence of ic and endometriosis in patients with cpp. this prospective evaluation included 178 women aged 18 to 60 years who presented from october 2000 to november 2002 at the midwest regional center for chronic pelvic pain and bladder control. subjects were selected for this study based on the presence of abdominal / pelvic, uterine, uterine sacral, and bladder / anterior vaginal wall tenderness during a physical examination. symptoms of cpp had persisted for more than 6 months and included dyspareunia, dysmenorrhea, and low abdominal pelvic pain with or without irritative voiding symptoms, such as urinary frequency, urgency, nocturia, hesitancy, and sensation of incomplete emptying. all patients were tested for bladder potassium sensitivity using the pst as previously described by parsons, and all patients underwent concurrent laparoscopic, cystoscopic, and bladder hydrodistention evaluations. a diagnosis of endometriosis the criteria used for a diagnosis of endometriosis included the pathologist - confirmed presence of ectopic glands and stroma. bladder hydrodistention was conducted using sterile water with 80 cm to 100 cm of passive hydrostatic pressure being applied. the bladder was distended for a minimum of 2 minutes, and then the water was slowly drained. the submucosal petechial hemorrhages, or glomerulations, were observed as the bladder emptied, or during a second filling of the bladder. the diagnosis of ic was based on the national institutes of health national institute of diabetes and digestive and kidney diseases (nih - niddk) guidelines, including the presence of 10 glomerulations per quadrant in 3 of the 4 quadrants of the bladder cavity and the presence of terminal hematuria. all patients in this study presented with both bladder base and uterine tenderness on physical examination. overall, 145 (81%) of the 178 study subjects with cpp had irritative urinary symptoms suggestive of overactive bladder. of the 178 patients evaluated, 159 (89%) exhibited cystoscopic findings of ic, 134 (75%) had a diagnosis of endometriosis based on histologic evaluation, and 115 (65%) were diagnosed with both conditions. in addition, 19 (11%) patients had endometriosis with no diagnosis of ic, and 44 (25%) had confirmed ic without endometriosis. the 159 patients with ic met the nihniddk guidelines for ic with the presence of glomerulations and hematuria. the diagnosis of ic was confirmed by experienced urologists who were involved in clinical research for treatment of ic. cpp = chronic pelvic pain ; em = endometriosis ; ic = interstitial cystitis ; pst = potassium sensitivity test. glomerulations and hematuria according to the national institutes of health national institute of diabetes and digestive and kidney diseases guidelines. presence of pathologist - confirmed presence of ec - topic gland stroma. among the total study population of 178 patients, a positive pst was found in 146 (82%) patients. of these 146 patients, 114 (78%) had irritative voiding symptoms. in addition, cystoscopic evidence of ic was found in 140 (96%) patients and, based on laparoscopic findings, endometriosis was demonstrated in 105 (72%). in the original subgroup of 159 patients with cystoscopic evidence of ic, 140 (88%) showed increased potassium sensitivity based on the pst, and 129 (81%) had urologic symptoms of urinary urgency and frequency. cystoscopic evidence of ic was found in 19 (12%) patients who had a negative pst, and 15 of those patients presented with irritative voiding symptoms. all patients in this study presented with both bladder base and uterine tenderness on physical examination. overall, 145 (81%) of the 178 study subjects with cpp had irritative urinary symptoms suggestive of overactive bladder. diagnosis and pst results for patients in this study are shown in figure 2. of the 178 patients evaluated, 159 (89%) exhibited cystoscopic findings of ic, 134 (75%) had a diagnosis of endometriosis based on histologic evaluation, and 115 (65%) were diagnosed with both conditions. in addition, 19 (11%) patients had endometriosis with no diagnosis of ic, and 44 (25%) had confirmed ic without endometriosis. the 159 patients with ic met the nihniddk guidelines for ic with the presence of glomerulations and hematuria. the diagnosis of ic was confirmed by experienced urologists who were involved in clinical research for treatment of ic. cpp = chronic pelvic pain ; em = endometriosis ; ic = interstitial cystitis ; pst = potassium sensitivity test. glomerulations and hematuria according to the national institutes of health national institute of diabetes and digestive and kidney diseases guidelines. among the total study population of 178 patients, a positive pst was found in 146 (82%) patients. of these 146 patients, 114 (78%) had irritative voiding symptoms. in addition, cystoscopic evidence of ic was found in 140 (96%) patients and, based on laparoscopic findings, endometriosis was demonstrated in 105 (72%). in the original subgroup of 159 patients with cystoscopic evidence of ic, 140 (88%) showed increased potassium sensitivity based on the pst, and 129 (81%) had urologic symptoms of urinary urgency and frequency. cystoscopic evidence of ic was found in 19 (12%) patients who had a negative pst, and 15 of those patients presented with irritative voiding symptoms. in this study, we have shown that endometriosis and ic often exist concomitantly in patients with cpp who present with uterine and bladder tenderness. among the 134 patients with endometriosis, conversely, among the 159 patients with ic, 72% were also found to have endometriosis. we have therefore prospectively confirmed our earlier published results that ic and endometriosis have a high rate of overlap in this group of patients. as the patients in this study were selected for both uterine and bladder tenderness, the rate of overlap between gynecologic and urologic diagnoses is likely to be higher than in the general population of patients with cpp. however, these results emphasize the need for a careful physical examination and symptom history in patients with cpp, and the need to perform concurrent cystoscopy with hydrodistention and laparos - copy. if cystoscopy had been performed only in patients with irritative voiding symptoms / overactive bladder, a diagnosis of ic would have been missed in approximately 20% of patients. other studies have also found that ic is frequently diagnosed in patients with cpp, and that it overlaps to a high degree with other diagnoses. in one study of 45 patients from general gynecologic practices who were scheduled to undergo laparoscopy for cpp, 17 (38%) were found to have ic based on the presence of irritative voiding symptoms and positive cystoscopic findings. in that study, 7 (41%) women diagnosed with ic also had endometriosis. endometriosis was also diagnosed in 14 (50%) women who did not have ic. notably, 30 (67%) of the women in that study had clinical symptoms of urgency and frequency or nocturia, and 10 women who were ic - negative had at least 10 glomerulations per quadrant noted in only 2 quadrants of the bladder. these results suggest that some women may have had early ic that did not meet the stringent niddk criteria for a diagnosis of ic. a questionnaire - based study evaluated the prevalence of concomitant disease in individuals with diagnosed ic. in this study in which patients with ic were not selected for cpp or gynecologic symptoms, the prevalence of endometriosis was approximately 17%. by comparison, the prevalence of endometriosis in the general population is estimated to be 1% to 7%. in this survey, patients with ic were also found to be substantially more likely than the general population to have various autoimmune disorders, such as inflammatory bowel disease (100-fold greater risk) and systemic lupus erythematosus (30- to 50-fold greater risk), although these conditions were still relatively rare compared with other concomitant illnesses. the cause of both ic and endometriosis is thought to include an immune component, suggesting a possible pathophysiologic basis for the increased risk of one condition with the other. in this study, we have shown that the pst is reasonably good at detecting the presence of ic : 96% (140) of patients with a positive pst had cystoscopic findings compatible with ic. previous studies have also reported that the rate of false positives is very low with the pst. these results indicate that a positive pst may be sufficient to indicate a diagnosis of ic. however, in our study, 12% (19) of women with cystoscopically confirmed ic had a negative pst, suggesting that, in the case of a negative pst, cystoscopy should still be performed. simple conservative methods are available for treating ic, including diet, pelvic floor physical therapy, and medications like hydroxyzine and pentosan polysulfate sodium (elmiron, ortho - mcneil pharmaceutical, inc, raritan, nj). recent published articles have indicated the efficacy of oral contraceptives in treating ic and cpp. many thought - leaders believe that the treatment of women with cpp has often been ineffective because the underlying cause is actually urologic rather than gynecologic. therefore, it is reasonable to conclude that ineffective management of cpp and treatment failures may be the result of missed diagnoses of ic. this study demonstrates that patients with cpp may have ic, endometriosis, or both conditions concomitantly. it is therefore imperative to evaluate the patient for both conditions, even when the diagnosis of one has been confirmed. when possible, cystoscopy with hydrodistention should be performed concurrently with laparoscopy to eliminate the need for a second procedure with the patient under general anesthesia and to facilitate the rapid detection and treatment of ic. a negative pst is not sufficient to rule out the presence of ic and should be followed up with cystos - copy. in the evaluation of the female with cpp, consideration should be given to the bladder as a source of pain early in the diagnostic workup. | objective : to determine the prevalence of interstitial cystitis and endometriosis in patients with chronic pelvic pain.methods:a prospective analysis was conducted in 178 women with cpp who presented with bladder base / anterior vaginal wall and/or uterine tenderness, with or without irritative voiding symptoms. the potassium sensitivity test was used to assess bladder epithelial dysfunction. patients were evaluated with concurrent laparoscopy and cystoscopy with hydrodistention.results:laparoscopic findings among the 178 patients with chronic pelvic pain supported a diagnosis of endometriosis in 134 (75%) patients, and cystoscopy confirmed a diagnosis of interstitial cystitis in 159 (89%) patients. both interstitial cystitis and endometriosis were diagnosed in 115 patients (65%). the potassium sensitivity test was positive in 146 (82%) patients, with 140 (96%) of these patients diagnosed with interstitial cystitis and 105 (72%) with endometriosis.conclusions:results of this prospective study show that interstitial cystitis and endometriosis may frequently coexist in patients with chronic pelvic pain. a positive potassium sensitivity test accurately predicted the presence of interstitial cystitis in 96% of these patients with chronic pelvic pain, as confirmed by cystoscopic hydrodistention. it is necessary to consider the diagnosis of endometriosis and interstitial cystitis concurrently in the evaluation of patients with chronic pelvic pain to avoid unnecessary delay in identifying either condition. |
acute right ventricular (rv) failure is associated with significant morbidity and mortality, which may develop as a consequence of acute myocardial infarction, pulmonary embolism, myocarditis, ventricular septal defect or post - cardiotomy [2, 3 ]. despite optimal medical management the rv may exhibit a greater capacity for rapid recovery compared with the left ventricle. recent studies suggests that most of the patients recover sufficient function to allow explantation of the impella recovery right direct (irrd) or right peripheral was reported. herein we report a case of successful implantation of the irrd in a patient with acute rv failure after previous aortic valve (av) replacement despite cabg, maximal medical therapy, intra - aortic balloon counterpulsation (iabp) and extracorporeal membrane oxygenation (ecmo). a 58-year - old woman was admitted in our division due to severe av stenosis with a mean transaortic gradient of 53 mmhg. on the second postoperative day the patient referred a sharply chest pain, fatigue, vomitus and sweating. the coronaro - angiography demonstrated a normal visualization of the left coronary and absence of visualization of the right coronary artery. iabp was placed immediately and an emergent off - pump coronary revascularization consisting in a vein graft to the right coronary artery was performed. five hours later, the hemodynamic was unstable, and symptoms of tamponade were present, despite maximum medical therapy and iabp. the patients was sent into the operating room. even with an open sternum, the rv insufficiency was not improved. ecmo was implanted and with an open sternum the patient was sent into the icu. then we decided to support the rv with irrd (abiomed, danvers, ma). purse string suture was made around the right atrial appendage and the inflow cage was introduced to the right atrium and fixed. in the pulmonary artery (pa) a purse string suture was made in the middle of distance between the pulmonary valve and division of pulmonary branch. the tip of the cannula (outflow tract) was inserted into the pa (fig. the pump was started with slow rotational speed and flow was adjusted to 3.5 l / min. the thorax was left open because of a large edema of the rv and to sustain this we used a plastic spacer in both margin of the sternum. to cover the wound we employed povidone the activated clotting time was maintained between 180 and 200 s. daily the sternal wound was revised. starting from the second day the pump flow was reduced half litre every day controlling rv function by transesophageal echocardiography and monitoring hemodynamic conditions. after 9 days of irrd the rv was recovered. the decision to remove the device 4). we examinated the inner surface of the graft after removal and a thin epithelized layer of the inner surface was found without any thrombus formation (fig. four days later the patient was extubated and 16 days after irrd explanted the patient was discharged in good clinical conditions. figure 1:schematic presentation of the impella right direct implantation.figure 2:the impella right direct implanted.figure 3:removal of the outflow tip of the device.figure 4:removal of the inflow tip of the device.figure 5:there are no thrombus inside the device.figure 6:the outflow tip of the impella device.figure 7:the inflow tip of the device.figure 8:after removal of the device. acute rv failure is an increasingly common clinical problem and despite optimal medical management, some patients fail to improve and require implantation of a rv assist device, which improves end - organ perfusion and provides an opportunity for reversal of multisystem organ failure. schmidt. first described its use in 2003, employing such a device in 8 patients and later christiansen. subsequently, the device has been used post - cardiotomy, post - transplant, and post left ventricular assist device implantation. we have described the employment of the irrd in a patient with rv failure due to myocarcial infarction post av replacement, unresponsive to cabg. there is growing evidence to suggest that the irrd can be successfully used for the treatment of refractory acute rv failure. the reasons why we choose this device were : (i) easy to insert, (ii) accommodates patients of all size (flow rate up to 5.5 l), (iii) minimal anticoagulation, (iv) minimal hemodilution, (v) minimal destruction of blood or plasma components, (vi) possibility of sternal closure and (vii) reduction of myocardial workload and oxygen consumption. important aspects of blood pumps are lifetime, costs, adaptability to diverse applications and patient requirements, rapid and easy deployment, thrombogenicity, flow characteristics and blood damage. the major advantages of the irrd are its small size, the simple design, the low energy requirements, and the avoidance of a priming volume. these figures lead to a reliable pump function without technical failures and may result in a reduced number of transfusion requirements which is reported to be excessively high in patients with ecmo support. flow and pressure characteristics, shear stress, blood exposure times to artificial surfaces and the size of the pump are important factors contributing to hemolysis. some studies demonstrated beneficial effects of pulsatile flow, others could not confirm these results. this might be due to release of endothelium - derived relaxing factor by rhythmic stimulation of endothelial cells through oscillating changes in vessel wall shear stress. this effect of pulsatile flow does not appear to improve pulmonary gas exchange, peak inspiratory pressure, mean pa pressure, oxygenation capacity and development of pulmonary edema. it seems to be justified to support the rv with non - pulsatile flow devices, especially when support duration is limited to a few days as in patients with post - cardiotomy failure. the main disadvantage of the irrd is that implantation requires a sternotomy and direct cannulation of the right atrium and pa. this increases the risk of bleeding and infection and necessitates an additional operation for device explantation. these risks are not insignificant, considering that patients requiring implantation of an rv assist device are frequently critically ill with multisystem organ failure and may be coagulopathic due to hepatic congestion and/or the presence of anti - platelet medications. the impella right peripheral is an alternative to irrd which allows the percutaneous implantation of the device. the margey. used this device for temporary rv mechanical circulatory support in a similar patient presenting with an inferoposterior st - segment elevation myocardial infarction, who despite revascularization, optimized medical therapy, iabp, remained in profound cardiogenic shock. we have described the clinical outcome in our patient with refractory acute rv failure supported with the irrd. our findings suggest that implantation of these devices is clinically feasible, associated with hemodynamic improvement, and facilitate successful bridge - to - recovery in most patients with post - cardiotomy rv failure due to myocardial infarction unresponsive to cabg, maximal medical therapy, iabp and ecmo. the authors declare that there is no conflict of interest regarding the publication of this article. | abstracta 58-year - old woman underwent aortic valve replacement. on the second postoperative day the patient referred a sharply chest pain, and an emergent coronary angiography revealed total occlusion of the right coronary artery. an intra - aortic ballon pump was placed and the patient underwent emergent off - pump coronary revascularization of the right coronary artery. five hours later, due to unstable hemodynamic the extracorporeal membrane oxygenation was implanted without improvement of the right ventricular (rv) function. then we decided to implant the impella right direct (rd). after 9 days of impella s insertion the rv was recovered and the device was successfully explanted. after 16 days of impella explanted the patient was discharged. this case suggest that implantation of impella rd is clinically feasible, associated with hemodynamic improvement, and facilitate successful bridge - to - recovery in patients with post - cardiotomy rv failure due to myocardial infarction unresponsive to coronary artery bypass grafting, maximal medical therapy, contrapulsation and extracorporeal membrane oxygenation. |
gain- and loss - of - function experiments have shown laminar - specific effects on local cortical processing (beltramo., 2013, olsen., 2012), but how the layers work together remains unclear. the synchrony of action potential (ap) firing across cortical layers is thought to be a fundamental aspect of translaminar processing and is determined by the strength, sign and timing of the underlying synaptic input. here, we investigate the synaptic mechanisms of cortical synchrony between excitatory neurons in layers 2/3 and 5 in behaving mice. measuring translaminar membrane potential (vm) synchrony and linking it to sensory processing and behavior require simultaneous vm recordings from different layers in awake animals. however, the vast majority of vm recordings of cortical neurons in behaving animals have been made from superficial layers (bennett., 2013, poulet and petersen, 2008, poulet., 2012, these studies have shown that internally generated, spontaneous network activity dominates the vm of cortical neurons across cortical regions and is correlated with the behavioral and arousal state. large - amplitude, slow fluctuations are highly correlated between neighboring layer 2/3 (l2/3) neurons in resting animals but are abolished during movement, resulting in a desynchronized or active cortical state (harris and thiele, 2011, poulet and petersen, 2008). the active state may result from arousal - related effects associated with movement and has been linked to a modulation in sensory responsiveness (crochet and petersen, 2006, otazu., 2009, pinto., 2013,, 2013, reimer., 2014, schneider., 2014,, 2015, zhou., 2014), adaptation (castro - alamancos, 2004), and even perception itself (bennett., 2013, mcginley., 2015). few studies have examined the vm activity of deeper layer cortical neurons in behaving animals (mcginley., 2015, schiemann., 2015). extracellular recordings, however, have shown higher spontaneous and sensory - evoked firing rates in deeper layer neurons (de kock., 2007, oconnor., 2010) and, intriguingly, that sensory - evoked and spontaneous spiking have different temporal structures across layers (sakata and harris, 2009). the rodent forepaw somatosensory system is a relevant and accessible model system to investigate cortical sensory processing during behavior. the forepaw has five digits (figure 1a) that can be used to grasp and manipulate objects as well as discriminate somatosensory stimuli (milenkovic., 2014). we made whole - cell recordings from primary forepaw somatosensory cortex l2/3 and l5 excitatory neurons in awake mice to compare the synchrony and integration of external (sensory) and internal (movement - evoked and spontaneous) synaptic input. our data highlight layer - specific membrane properties that underlie differences in ap firing and show that translaminar vm synchrony is dependent both on the behavioral state and the source of synaptic input. to investigate the intrinsic membrane properties of l2/3 and l5 excitatory neurons, we made blind whole - cell patch clamp recordings targeted to the digit 3 cortical representation in primary somatosensory forepaw cortex (s1) in awake mice. mice were head - restrained and had their right forepaw tethered to the platform (figure 1b). the tips of the digits 2 and 4 overhung the edge of the platform while the tip of digit 3 was positioned on a flat, circular head of a combined movement sensor and force - feedback tactile stimulating arm. the stimulating arm head was held in contact with the glabrous skin of digit 3 throughout all recordings. whole - cell recordings from 17 l2/3 neurons were made at subpial depths between 121 and 384 m (245.30 17.91 m) and from 28 l5 neurons between 538 and 823 m (649.43 14.28 m) (figure 1c). all neurons had evoked regular - spiking firing patterns during current injection, and a subset (l2/3 = 4/17 and l5 = 15/28 neurons) was confirmed by post hoc biocytin staining to be excitatory pyramidal neurons. we first examined the intrinsic membrane properties soon after break - in during quiet wakefulness. l5 neurons generated more aps in response to equivalent current injection amplitudes than l2/3 neurons (figures 1d and 1e). the increased excitability may be due to the higher resting vm value of l5 neurons during quiet wakefulness (l2/3mean = 56.92 1.21 mv, n = 12 cells versus l5mean = 50.70 0.65 mv, n = 19 cells ; p 60% of the vm range between the most hyperpolarized and depolarized values for at least 100 ms. plotting the duration and vm of isolated sdes across seven dual recordings as a heatmap (figures 4b and s4) revealed a range of durations with a mean of 300 ms (figure s1b for single and dual recordings, l2/3 = 329.06 27.55 ms, n = 13 cells, l5 = 266.94 11.15 ms, normally a sde in one neuron was accompanied by a sde in the simultaneously recorded neuron, but a minority of small - amplitude, short - duration sdes were observed in one layer only (figure s5), likely accounting for the slightly shorter durations of sdes in l5. in those sdes with a measureable vm onset in both layers, plotting the trial - by - trial onset times (figure 4c ; supplemental experimental procedures), the population averaged vm (figure 4d) and population spiking rates (figure 4e) revealed that l5 sdes started earlier than those in l2/3 (figure 4f ; l5 leading l2/3 = 9.07 2.19 ms, n = 7 pairs, p = 0.031). in contrast, the hyperpolarizing offset time was not significantly different across layers when sdes were triggered on l2/3 onset (figures 4a and 4f). sdes thus have an earlier onset in higher - firing l5 neurons, supporting the proposal that l5 neurons are important drivers of supragranular slow network activity (beltramo., 2013, sanchez - vives and mccormick, 2000). is the laminar - specific synaptic input observed during ap firing (figure 3) and spontaneous activity (figure 4) a general feature of subthreshold processing in cortical neurons, or are there other sources of more synchronized synaptic input ? movement triggers an active, desynchronized cortical state, which can be driven by thalamic input (poulet. to examine the fast dynamics of movement related synaptic input across layers, we next analyzed the vm dynamics at m onset (detected by thresholding the first derivative of the digit movement signal ; supplemental experimental procedures). averaging the vm showed that m onset is accompanied by simultaneous depolarizing synaptic input to l2/3 and l5 neurons (figures 5a and 5b). close inspection of the log - scale digit movement trace indicated that the depolarization in both layers was tightly coupled to the tiny initial movements of the digits and did not appear before movement. to quantify the timing of the depolarization, we performed cross - correlation analysis between the averaged digit movement and the averaged vm response at m onset. the peak of the cross - correlation showed no significant time lag (figure 5b), suggesting an internal, non - sensory origin for synaptic input at movement onset. analysis of 2-s epochs of vm activity showed an overall reduction in the sd of the vm during extended m periods (figure 2), but how fast this occurs was unclear. we therefore quantified the variance of the l2/3 and l5 subthreshold activity around all recorded m onsets, including short - duration movements (figures 5e and 5f). in all cells across both layers, we observed a rapid and robust reduction in variance within the first 200 ms of movement onset. thus, early movement - evoked input acts to reduce subthreshold variability simultaneously across cortical layers. the reduction in variance, however, does not result in synchronous ap firing. instead, l5 neurons showed higher firing rates after movement onset than l2/3 neurons (figures 5c and 5d). thus, unlike the laminar - specific vm dynamics during sdes and spontaneous aps, movement onset triggers simultaneous vm depolarization across layers, which results in an increase in firing only in l5 neurons. thalamo - cortical axons are unequally distributed across the cortical layers, with some l5 neurons receiving direct thalamic input (bureau. we therefore next examined whether there is laminar specificity in sensory processing across layers in behaving animals. we delivered brief (2 ms), light (10 mn) tactile stimuli via the stimulating / sensing arm to digit 3 during single and dual whole - cell recordings. the sensing arm was in constant contact with the glabrous skin of digit 3. while the first spike latency was similar for both behavioral conditions and layers (figure s6b), the amplitude of the vm response was strongly dependent on the behavioral state (figures 6a6d). in q periods, stimuli evoked a large - amplitude depolarizing response in both layers, whereas the amplitude of the response was reduced during m (figures 6c and 6e ; l2/3amp q = 10.80 0.65 mv, m = 4.84 1.24 mv, n = 10 cells, p = 0.037 ; l5amp q = 6.19 0.69 mv, m = 2.41 0.60 mv, n = 20 cells, p = 0.001 ; q l2/3amp versus l5amp p 10 hz (figure 6f). future work targeting whole - cell recordings to within - layer excitatory neuron subtypes in sensory cortex will be of great importance. cortical sensory responses are modulated by behavioral state in different sensory systems (bennett., 2013, crochet and petersen, 2006, niell and stryker, 2010, otazu., 2009, schneider., 2014, zhou., we report a reduction in subthreshold response amplitude in l2/3 neurons to brief tactile stimuli during movement. we go on to show that l5 neurons also have a reduced subthreshold response during movement (figure 6). despite this, the numbers of evoked aps remained the same in both layers during quiet and moving periods. in both layers, subthreshold responses were reduced in amplitude as the baseline vm values became more depolarized and exhibited a reversal potential more hyperpolarized than ap threshold. interestingly, in the cells with higher sensory - evoked firing rates, the sensory reversal potential was closer to threshold. together, this suggests that strong, local gabaergic inhibition plays a significant role in clamping the subthreshold sensory response below ap threshold and regulating ap firing during behavior in both l2/3 (crochet., 2011) and l5 neurons. synchronous activity in cortical networks is thought to be fundamental to sensory processing and perception. prior work has shown that spontaneous vm activity in l2/3 neurons during resting states is more correlated than in activated states in behaving or attentive animals (okun. neurons showed large - amplitude, highly correlated fluctuations of the vm during resting periods and an active state with low sd, as well as a reduction in slow fluctuation amplitude and translaminar vm synchrony (figures 2 and 3). however, close inspection of the fine timing of subthreshold inputs evoked at different time points revealed differences in the timing of synaptic input across layers. spontaneous aps were driven by cell - specific, depolarizing inputs (figure 3). in contrast, movement- (figure 5) and sensory - driven (figure 6) synaptic input had a similar timing across layers. interestingly, slow subthreshold fluctuations in resting mice had an earlier onset in l5, reminiscent of the earlier timing of upstate onsets in deeper layers observed in anesthetized and sleeping animals (chauvette., 2010, sakata and harris, 2009), cortical slices (sanchez - vives and mccormick, 2000), and extracellular recordings in awake rats (sakata and harris, 2009). thus, the type of input (spontaneous, sensory, or movement evoked) determines the timing of synaptic input across layers. one possibility is that they result from differences in the wiring supporting spontaneous, sensory, and motor events. in support of this suggestion, a recent anatomical study showed laminar differences in local and long - range cortico - cortical inputs to s1 excitatory neurons (denardo., 2015). l2/3 neurons receive proportionally more input from distant l2/3 neurons than l5 neurons, which receive a greater proportion from distant l5 neurons. moreover, locally, l2/3 neurons receive a greater proportion of inhibitory inputs than l5 neurons. luczak., 2007) and slow fluctuations in awake resting animals (ferezou., 2007) are thought to propagate as waves of activity across cortex supported by long - range cortico - cortical connections. thus, a later onset in l2/3 neurons could result from a combination of lower firing rates, increased inhibitory input, and laminar - specific cortico - cortical wiring. in contrast, because sensory thalamic neurons are driven both by sensory stimulation and movement (poulet., 2012), we suggest that punctate thalamic input drives the synchronized synaptic input following tactile stimuli or movement onset. subthreshold sensory responses are correlated not only with the behavioral and cortical state but also with the behavioral outcome. both l2/3 and l5 neurons showed a larger - amplitude subthreshold sensory response in trials that lead to short - latency forepaw movements (qm trials) compared to trials with no movement before or after the stimulus (qq trials). in l5 neurons, a difference was also observed in the pre - stimulus vm, with qm trials having a more hyperpolarized value than qq trials. this suggests that the link between subthreshold response amplitude and behavioral output is due to the prestimulus cortical state rather than delivery of different amplitude stimuli. our findings resemble recent vm recordings in auditory cortex during an auditory discrimination go / no - go task, where neurons from l4 and l5 showed more hyperpolarized vm value in hit trials than in false - positive trials in mice performing an auditory perception task (mcginley., 2015). future experiments could address whether this is the result of a higher signal to noise ratio in a phase of low network activity resulting in an enhanced probability of signal transmission to downstream motor centers. during evoked movement trials, neurons in both layers showed a prominent late depolarization (figure 7), with l5 neurons also showing an increase in late spiking. because forepaw movements necessarily occurred soon after tactile stimulation in qm trials, it was difficult to assess whether the late activity is causally related to the perception of the stimulus (sachidhanandam., 2013), an intrinsic part of the transformation of sensory input to motor output and/or the start of the active cortical state associated with attention or arousal. examining mice trained to delay sensory - triggered movements will help link late activity to perception and movement. we observed higher background firing rates in l5 neurons but no difference in the numbers of additional, sensory - evoked spikes across layers, suggesting that l2/3 and l5 have distinct sensory coding strategies (sakata and harris, 2009). the fine timing differences of synaptic input may be important in the processing of dynamic sensory stimuli and for changes of synaptic strength under spike - time - dependent plasticity rules. fast, laminar - specific manipulations of synchronized activity in trained mice are now required to define the causal role of translaminar synchrony in perception. all experiments were approved by the berlin animal ethics committee and carried out in accordance with european animal welfare law. head - restrained 6- to 9-week - old c57bl6j mice were paw - tethered, and digit movements were monitored by a force - feedback sensing and stimulating arm. the sensing arm ending was a 4.7-mm - diameter flat disk with one edge pressed up against the glabrous skin of digit 3. blind whole - cell patch - clamp recordings were made from primary somatosensory forepaw cortex located by intrinsic optical imaging. all data were statistically analyzed using non - parametric tests, paired data with a wilcoxon signed rank test and unpaired data with a wilcoxon rank sum test. | summarythe synchronized activity of six layers of cortical neurons is critical for sensory perception and the control of voluntary behavior, but little is known about the synaptic mechanisms of cortical synchrony across layers in behaving animals. we made single and dual whole - cell recordings from the primary somatosensory forepaw cortex in awake mice and show that l2/3 and l5 excitatory neurons have layer - specific intrinsic properties and membrane potential dynamics that shape laminar - specific firing rates and subthreshold synchrony. first, while sensory and movement - evoked synaptic input was tightly correlated across layers, spontaneous action potentials and slow spontaneous subthreshold fluctuations had laminar - specific timing ; second, longer duration forepaw movement was associated with a decorrelation of subthreshold activity ; third, spontaneous and sensory - evoked forepaw movements were signaled more strongly by l5 than l2/3 neurons. together, our data suggest that the degree of translaminar synchrony is dependent upon the origin (sensory, spontaneous, and movement) of the synaptic input. |
with the advancement of dentistry and the growing interest in the developments of ideal materials to replace lost dental tissue, resin - based composites have been widely used to restore posterior teeth. concerns about aesthetics, the content of mercury in amalgam restorations and the possibility of more conservative restorations, using the minimally invasive technique, has been essential in the selection of these materials in stress - bearing areas. however, some intrinsic characteristics from resin composite, for example, polymerization shrinkage and elastic modulus different of dental structure can produce several problems including postoperative sensitivity, secondary caries, pulp inflammation, tooth or restoration fractures, and marginal defects of the composite with the adjacent tooth structure. the failure of such treatment is a public health problem which increases the cost of dental treatment and decreases dental health. frequently, a range of experimental resins is launched on the market aiming to better physical and chemical properties, and consequently, an increase in the longevity of restorations. thus, variations in the form and amount of inorganic particles in the polymeric matrix, in the photoinitiator system, handling, indications, among other factors, are increasingly frequent. however, the characteristics and effects of these composites are not yet fully elucidated, especially in the short term (early failure) and associated a clinical performance, making it difficult to choose the ideal restorative material. the measure of success of a restorative treatment appears to be strongly associated to its longevity. although laboratory tests can provide relevant information on the restoration longevity, the long - term performance of these systems still depends on clinical evaluations. however, clinical studies require a more effective approach and greater precision in the experimental design due to several variables which can influence the results such as : difficulty of patient compliance, difficulty of evaluating the restorations longevity as an objective factor (operator, patient, and material), and difficulty to acquire a heterogenic group such as that present in clinical routine. hence, several studies have used selective groups (e.g., dental students). however, these groups are not truly representative because they have distinguished prevention and hygiene practices. therefore, all factors observed in several studies served as a base to the development of this study. the purpose of this randomized and double - blind study was to assess the clinical performance of two composites for posterior tooth, in class i and ii restorations, using public health service system, during baseline, after 6-, 12-, and 24-months. the null hypothesis tested was that there is no significant difference in clinical performance between z250 and p60 resin systems when using in posterior teeth. a total of seventy restorations were placed in molar and premolars in thirty patients (14 females and 16 males ; 1840 years). the following parameters were set for sample size calculation : the expected proportion was 0.8, the relative standard error was of 10%, the power of the test was calculated to be 80% (beta error), and the two - tailed alpha error of 5% was 2-sided. based on these data, a sample size of 25 subjects was found to be necessary. taking into consideration a possible loss during the study, a 20% increase in sample size was set totalizing 30 subjects. all patients received dental treatment in a public health service system, allowing the formation of a heterogeneous sample, during 24-months. the following criteria were used to select subjects to the study : good oral hygiene, low caries index, no periodontal disease neither parafunctional habits, the patients should have at least two posterior teeth (molars and/or premolars) with initial carious lesions to be treated, and/or teeth with unsatisfactory restorations which needed to be replaced. patients committed to attend the annual recall by participating in the study, and they were divided into two study groups according to the tested materials [table 1 ]. the randomization process that has allocated the cavities and materials were performed by software spss - statistical package for social science, software version 12, spss inc., furthermore, aiming to secure a blind study, patients and examiners were unaware of any data from the randomization process. groups, composition and lot of materials used in the study restorative procedures were carried out by an experienced clinician, who was submitted to a calibration process that consisted of performing 10 repeated restorations of each material under direct supervision of the project 's coordinator. the operator 's questions were addressed, and consensus was obtained during the calibration session. teeth were carefully cleaned with pumice / water slurry, rinsed with water, and air - dried. amalgam and/or resin composite restorations considered unsatisfactory and cavity preparation were carried out with 245 carbide burs (jet burs, beavers dental division of sybron, canada). all internal angles were rounded, and dislodged enamel prisms at gingival wall were removed with a gingival margin trimmer. the criteria used in the evaluation of unsatisfactory restorations were restoration failure, dentin or base was exposed, contact point was missing (present periodontal inflammation), color and/or translucency beyond the normal range of tooth colors, marginal staining penetration in the direction of the pulp, marginal gap exhibiting the enamel dentin junction, fractured, loose filling that is missing partly or completely, and presence of caries (associated with the filling). every detail of cavity preparation such as cavity depth, enamel presence at the cervical margin, sclerotic dentin, possible pulp exposition, fissures and defects in the enamel, and cavity extension were recorded. therefore, four teeth were covered with calcium hydroxide (dycal dentsply ind e. com. petrpolis, rj, brazil) and resin - modified glass ionomer cement (vitrebond ; 3 m dental products, st. paul, mn, usa) since the thickness of the remaining dentin was less than approximately 0.5 mm. enamel was etched for 30 s and dentin for 15 s with 37% phosphoric acid gel (scotchbond etchant ; 3 m dental products, st. paul, mn, usa), washed for 30 s, and gently dried with air jet. paul, mn, usa) and the resins composite were applied to the dental substrates in accordance with the manufacturer 's instructions. to provide the largest free surface area, each increment was light - cured for 40 s using a halogen curing light (curing light xl 3000, 3 m dental products, st. paul, mn, usa), with minimum light intensity of 600 mw / cm and checked by a light - curing meter (hilux dental curing light meter, benlioglu dental inc., turkey). class ii cavities, the proximal wall, was reconstructed to transform into a class i cavity, with the aid of polyester strip matrix (k - dent quimidrol, joinville, santa catarina, brazil) and dental interproximal wedges (tdv dental ltda, pomerode, santa catarina, brazil). resin insertion in the occlusal box was performed in cone - shaped increments according to progressive waxing technique. the restorations were immediately finished by removing excess material, whereas the finishing and polishing were performed after 1 week, using a 12-fluted tungsten carbide burs (jet burs, beavers dental division of sybron, canada), the enhance polishing system (dentsply, rio de janeiro, brazil) and sof - lex polishing discs (3 m dental products, st. the restorations were evaluated by two calibrated examiners, and they were unaware of which material had been used. the examiners were calibrated before the baseline evaluation using picture slides representing each condition to be assessed in the study, and any disagreements during evaluation were resolved by consensus among the examiners. modified us public health service (usphs) criteria [table 2 ] were used to evaluate marginal discoloration, marginal integrity, surface texture, wear, postoperative sensitivity, and recurrent caries at baseline, after 6-, 12-, and 24-months. the data collection form used at baseline and the restorations were classified and demonstrated by scores : alpha ideal clinical situation, bravo clinically acceptable, and charlie clinically unacceptable situation. modified - usphs rating criteria interexaminer reliability was assessed using kappa index (0.90). comparisons of ratings for restorations and between baseline and follow - up examinations were analyzed by fisher 's and mcnemar 's chi - square tests for each category. the value p 0.05 was set as the standard value considered to demonstrate statistically significant differences. the statistical analyses were carried out with spss 16.0 (spss inc., chicago, il, usa). a total of seventy restorations were placed in molar and premolars in thirty patients (14 females and 16 males ; 1840 years). the following parameters were set for sample size calculation : the expected proportion was 0.8, the relative standard error was of 10%, the power of the test was calculated to be 80% (beta error), and the two - tailed alpha error of 5% was 2-sided. based on these data, a sample size of 25 subjects was found to be necessary. taking into consideration a possible loss during the study, a 20% increase in sample size was set totalizing 30 subjects. all patients received dental treatment in a public health service system, allowing the formation of a heterogeneous sample, during 24-months. the following criteria were used to select subjects to the study : good oral hygiene, low caries index, no periodontal disease neither parafunctional habits, the patients should have at least two posterior teeth (molars and/or premolars) with initial carious lesions to be treated, and/or teeth with unsatisfactory restorations which needed to be replaced. patients committed to attend the annual recall by participating in the study, and they were divided into two study groups according to the tested materials [table 1 ]. the randomization process that has allocated the cavities and materials were performed by software spss - statistical package for social science, software version 12, spss inc., furthermore, aiming to secure a blind study, patients and examiners were unaware of any data from the randomization process. restorative procedures were carried out by an experienced clinician, who was submitted to a calibration process that consisted of performing 10 repeated restorations of each material under direct supervision of the project 's coordinator. the operator 's questions were addressed, and consensus was obtained during the calibration session. teeth were carefully cleaned with pumice / water slurry, rinsed with water, and air - dried. amalgam and/or resin composite restorations considered unsatisfactory and cavity preparation were carried out with 245 carbide burs (jet burs, beavers dental division of sybron, canada). all internal angles were rounded, and dislodged enamel prisms at gingival wall were removed with a gingival margin trimmer. the criteria used in the evaluation of unsatisfactory restorations were restoration failure, dentin or base was exposed, contact point was missing (present periodontal inflammation), color and/or translucency beyond the normal range of tooth colors, marginal staining penetration in the direction of the pulp, marginal gap exhibiting the enamel dentin junction, fractured, loose filling that is missing partly or completely, and presence of caries (associated with the filling). every detail of cavity preparation such as cavity depth, enamel presence at the cervical margin, sclerotic dentin, possible pulp exposition, fissures and defects in the enamel, and cavity extension were recorded. therefore, four teeth were covered with calcium hydroxide (dycal dentsply ind e. com. ltda., petrpolis, rj, brazil) and resin - modified glass ionomer cement (vitrebond ; 3 m dental products, st. paul, mn, usa) since the thickness of the remaining dentin was less than approximately 0.5 mm. enamel was etched for 30 s and dentin for 15 s with 37% phosphoric acid gel (scotchbond etchant ; 3 m dental products, st. paul, mn, usa), washed for 30 s, and gently dried with air jet. paul, mn, usa) and the resins composite were applied to the dental substrates in accordance with the manufacturer 's instructions. to provide the largest free surface area, each increment was light - cured for 40 s using a halogen curing light (curing light xl 3000, 3 m dental products, st. paul, mn, usa), with minimum light intensity of 600 mw / cm and checked by a light - curing meter (hilux dental curing light meter, benlioglu dental inc., turkey). class ii cavities, the proximal wall, was reconstructed to transform into a class i cavity, with the aid of polyester strip matrix (k - dent quimidrol, joinville, santa catarina, brazil) and dental interproximal wedges (tdv dental ltda, pomerode, santa catarina, brazil). resin insertion in the occlusal box was performed in cone - shaped increments according to progressive waxing technique. the restorations were immediately finished by removing excess material, whereas the finishing and polishing were performed after 1 week, using a 12-fluted tungsten carbide burs (jet burs, beavers dental division of sybron, canada), the enhance polishing system (dentsply, rio de janeiro, brazil) and sof - lex polishing discs (3 m dental products, st. the restorations were evaluated by two calibrated examiners, and they were unaware of which material had been used. the examiners were calibrated before the baseline evaluation using picture slides representing each condition to be assessed in the study, and any disagreements during evaluation were resolved by consensus among the examiners. modified us public health service (usphs) criteria [table 2 ] were used to evaluate marginal discoloration, marginal integrity, surface texture, wear, postoperative sensitivity, and recurrent caries at baseline, after 6-, 12-, and 24-months. the data collection form used at baseline and the restorations were classified and demonstrated by scores : alpha ideal clinical situation, bravo clinically acceptable, and charlie clinically unacceptable situation. comparisons of ratings for restorations and between baseline and follow - up examinations were analyzed by fisher 's and mcnemar 's chi - square tests for each category. the value p 0.05 was set as the standard value considered to demonstrate statistically significant differences. the sample (30 subjects, 14 females and 16 males, 1840 years), the number of restorations (35 per material), the distribution of restoration (maximum of three pairs in the same patient), the relationship between class i and class ii (1:2) followed american dental association guidelines for new material testing. the distribution of restorations and recall characteristics for both tested groups are described in table 3. the returns showed a collaboration rate of patients of 96.6% during 6 months, and after 24 months, 28 patients were examined, representing 93.3%. distribution of restorations and recall characteristics for both tested groups the results of this study revealed an excellent quality of resin composite restorations, which showed no cases of nonacceptable restorations in any aspect. no statistically significant differences between the two materials were found at 2 years with respect to evaluated criteria. the only criterion that presented a numerical, but not statistically, difference was marginal integrity. thus, given the experimental conditions, our results demonstrated no material - time - dependency. evaluation of the materials at baseline, 6, 12 and 24 months (alfa score percentage) clinical evaluation of restorative procedures requires (a) choices of clinically relevant criteria, (b) assessment using simple nominal scales, (c) calibration of evaluators, (d) two independent evaluations, and (e) nonparametric statistical analysis that recognizes the patient (and not the restoration) as the independent variable. furthermore, usphs evaluates only the degree of deviation from ideal restoration. however, there is still no complete and accurate method that can evaluate all these numerous variables. problems in tooth restoration interface such as marginal integrity, marginal stain, and secondary caries are associated to materials physics and mechanics properties - elasticity modulus, thermal expansion coefficient, and curing shrinkage - and overall to enamel marginal finishing. a low elasticity modulus determines a greater material deformation when force is applied, damaging adhesive bonding due to fatigue in tooth restoration interface. tooth and restorative materials show different thermal expansion coefficient ; therefore, temperature change that takes place in oral cavity leads to stress in adhesive interface breaking hybridization bonding. marginal integrity criteria showed distinct values ; nevertheless, there was no significant statistically difference. both materials showed a slight degradation along the time, noticed through the changing in restoration scores from alpha to bravo along the 6 and 24 months. in the first 6 months, group i (single bond and filtek z250) showed 100% alpha score, after 12 months 97.1% and 2.9% bravo, and after 24 months showed 88.6% alpha score and 11.4% bravo score. group ii (single bond and filtek p60) showed after 6 months 6% bravo score, and after 12 and 24 months 91.4% to alpha score and 8.6% to bravo score. this could probably be due to small fracture of the cavosurface margin and material. according to marginal discoloration, both materials showed similar score, showing after 12 months 100% alpha score and after 24 months 97.1% alpha score. concerning secondary caries both materials showed identical clinical behavior after 24 months which was 100% alpha score. those results matched the findings from trkn and oguz, leinfelder, cenci. analyzing the results for postoperative sensitivity, it was noticed that both materials (group i and group ii) showed 100% alpha score in all evaluated periods. all cavity margins had enamel present which increases significantly the restorative adhesive system sealing capacity, decreasing possibility of marginal microleakage, and its consequences such as postoperative sensitivity. nevertheless, there was a numerical difference (percentage) to marginal integrity in group i, which is presented in table 4. the resins composite are both indicated for posterior restorations but differ in terms of handling characteristics. however, the lack of difference occurred due to similarities in the chemical composition of the composites used [table 1 ]. p60 is filled to 61% by volume with zirconia / silica filler particles having a size range of from 0.01 to 3.5 m, contain a greater number of smaller particles and the total load is approximately to 84% for weight. z250 is filled to 60% by volume with zirconia / silica particles having a size range of 0.013.5 m and an average size of 0.6 m, contains also a greater number of smaller particles and the total load is approximately to 78% for weight. since materials were distributed likewise in the same patient, the variable patient factor can not be used as a cause of greater wear. it was observed no wear after 12 months, only notice after 24 months, which matched the results from trkn and oguz, gianordoli neto. that happened because the improving of composites physic properties. new formulations decreasing tremendously the average size of particles have been developed over the last decade. besides that, the incremental technique favored better clinical results, once lower increments decreased inner porosity and improved composite conversion rate, which reduced the observed wear. the polishing consists of making the restoration surface look as enamel. after evaluation of superficial texture in different periods (baseline, 6, 12, and 24 months), filtek z250 (group i) restorations showed 100% after 12 months and 94.3% alpha score after 24 months and filtek p60 (group ii) showed after the same period 97.1% and 94.3%. both restorative adhesive systems used are hybrid and have as inorganic phase extremely small silica and zircon particles. it is associated to resin matrix with components (triethylene glycol dimethacrylate) increasing the molecular bonding and a higher conversion rate of double linking during curing process, which leads to a great resistant organic matrix. according to anusavise and leinfelder, decreasing size, changing composition, and increasing quantity of particles made nowadays composites 1015 times superior than the previous ones in wear resistance and surface texture maintenance. in accordance with the results of the present study all restorations after 24 months appeared to be clinically acceptable but are important to follow - up these restorations for a longer period to analyze the clinical performance of the resin composite systems. long - term clinical trials are certainly needed because they remain the ultimate way to collect scientific evidence on the clinical effectiveness of restorative treatments. on the basis of the results and despite the limitations of this study, it seems reasonable to conclude that all direct resin composite restorations were clinically satisfactory and no significant differences were found among them over an evaluation period of two years. | background : clinical evaluations as fundamental method to prove the efficiency of restorative materials.aim:this study evaluated the clinical performance of restorative systems during 2 years of clinical service.materials and methods : this study assessed the clinical performance of restorative systems (filtek z250 and p60), during 2 years of clinical service, using the us public health service system. the randomized and double - blind study comprising thirty volunteers. the restorations were evaluated at baseline, 6, 12, and 24 months. it was used the following criteria : marginal discoloration (md), marginal integrity (mi), superficial texture (st), wear (w), postoperative sensitivity (ps) and recurrent caries (rc).results : statistic analysis was performed using fisher 's and mcnemar 's exact tests and pearsons 's chi - square in a significance level of 5%. the results at baseline and 24 months for group i were : md 100, 100% ; mi 100, 88.6% ; st 100, 94.3% ; w 100, 94.3% ; ps 100, 100% ; rc 100, 100%, of alpha scores ; group ii : md 100, 97.1% ; mi 100, 91.4% ; st 100, 94.3% ; w 100, 91.4% ; ps 100, 100% ; rc 100, 100%, of alpha scores. it was observed no statistical difference in the evaluated criteria and period.conclusions:after 24 months of evaluation, both restorative systems exhibited acceptable clinical performance. |
although ligands having oxygen and nitrogen as donor atoms are by far the most extensively studied, interest in sulfur donor chelating agents has grown over the years and the number of chemical studies in this area has increased considerably. interest in complexes of these ligand systems now covers several areas, ranging from general considerations of the effect of sulfur and electron delocalization in transition metal complexes to potential biological activity and practical application [24 ]. the aim of the present work is to synthesize new thiocarbohydrazone ligand, and to study its coordination behavior with co(ii), ni(ii), cu(ii), and zn(ii) ions. n, n-thiocarbohydrazide condenses easily with two molecules of carbonyl compounds viz., isatin, on the nh2 and nh2 hydrazine amino groups to produce the desired ligand shown in figure 1. the tautomerism in this ligand and also the well - known tendency of oxygen and sulfur donors to act as bridging sites [5, 6 ] allows various structural possibilities for the corresponding metal complexes. a considerable number of metal complexes are now known to possess antitumor activity. there are reports that sulphur - containing ligands and platinum complexes of sulphur - containing amino acids were found to have inhibitory action against tumors. however, since the class of sulphur - containing compounds such as thiosemicarbazones, an structural analogue of the thiocarbohydrazone, have been reported to have anticancer activity owing to the specific and unique properties of their metal chelate, it is suggested that the present compounds, being similar in structure and character, may be acting by virtue of their chelating properties at the cellular level thereby exerting their anticancer activity. hence, it was considered worthwhile to subject the presently studied complexes for evaluation of their anticancer activity. the antimicrobial properties of metals have been recognized for centuries and have represented some of the most fundamental breakthroughs in medicinal history. many studies stressed the role of metal ions in important biological processes, whereas the inorganic pharmacology started to be an important field with more than 25 inorganic compounds, being used in therapy as antibacterial, antiviral, and anticancer drugs [12, 13 ]. kirschner. have suggested that the transfer of the metal ion from the ligand to the cancer - associated viruses was an important mechanism for designing new anticancer therapies. the inverse process, that is, coordinating a metal ion from an important biomolecule, such as, for instance, a zinc finger protein, has recently been used to design novel antiviral therapies, targeted against human immunodeficiency (hiv) and human papilloma virus (hpv) infections. we have already drawn attention to the strong relationship between metals or their complexes [1618 ], and antibacterial, antitumour, and anticancer activities. a number of in vivo studies have indicated that biologically active compounds become more bacteriostatic and carcinostatic upon chelation. the metal content of the complexes were estimated after decomposition with mixture of hcl and hclo4 by gravimetric method (copper and nickel) and edta titration method (cobalt and zinc). magnetic susceptibility of complexes were measured at room temperature on a faraday balance using hg[co(scn)4 ] as a calibrant. electronic spectra were recorded using varian cary 50 bio uv - visible spectrophotometer in dmso. the ir spectra of ligand and its complexes were recorded as kbr pellets in the region 4000400 cm on nicolet 170 sx ft - ir spectrometer. the h - nmr spectra of ligand and its zinc (ii) complex were recorded in dmso - d6 on bruker 300 mhz spectrometer using tms as an internal standard. the cyclic voltammetry experiments were carried out with a three - electrode apparatus using a chi1110a electrochemical analyzer (usa). the epr spectra of copper (ii) complexes were recorded at room temperature on varian e-4 x - band spectrometer using tcne as g - marker. conductivity measurements were made on 10 m solutions of complexes in dmso using elico - cm82 conductivity bridge provided with a cell having cell constant 0.51. a solution of isatin 5.8 g (0.04 mol) in ethanol (30 ml) was added drop wise to a refluxing solution of thiocarbohydrazide 2.1 g (0.02 mol) in the same solvent (40 ml). few drops of glacial acetic acid were added to the reaction mixture and was refluxed for 2 hours. at the end of the reaction, monitored by tlc (n - hexane / ethyl acetate 2:3), a yellow - orange powder was filtered and washed with warm ethanol and then with diethyl ether. the complex was prepared by the addition of ethanolic solution of metal (ii) chloride or aqueous ethanolic solution of metal (ii) acetates (0.003 mol) with constant stirring to the corresponding amount of the ligand (0.003 mol) in the same solvent. the product was filtered off, washed several times with ethanol, and dried in vacuum over p2o5 (scheme 2). the brine shrimp lethality test was used to predict the presence of cytotoxic activity. the brine shrimp eggs were hatched in artificial sea water at room temperature under constant aeration for 48 hours. after hatching, 10 larvae were placed in a vial containing 5 ml of artificial sea water. a drop of dry yeast suspension (3 mg in 5 ml sea water) was added to each vial as food for shrimps. test compounds in different concentrations (10, 100, and 1000 ppm) were added to the vials before making up the final volume to 5 ml with sea water. the experiments were done in triplicate and mean of three readings was taken as final result. after 24 hours, the survivors were counted using a 3x magnifying glass, and the percent deaths and lc50 (lethal concentration for half the population) values wear calculated by using finney computer program. ehrlich ascitic carcinoma (eac), to induce cancer in animal model (mice) were obtained from (amala cancer research center, amala nagar, kerala, india.) the cells were maintained as ascites tumor in swiss albino mice by intraperitoneal inoculation of 1106 viable cells. six - to - eight week old female swiss albino mice (255 g body weight) were selected from (central animal facility, manipal academy of higher education, manipal, karnataka, india.) the animals were acclimatized to the experimental room having temperature 232c, controlled humidity conditions, and 12:12 hour light and dark cycle. the mice were housed in sterile polypropylene cages containing sterile paddy husk as bedding material with a maximum of 4 animals in each cage. the mice were fed on autoclaved standard mice food pellets (hindustan lever) and had access to water ad libitum. the animal experiments were performed according to the rules and regulations of the institutional animal ethics committee (iaec). the compounds (dose 50 mg / kg), suspended in 4 % gum acacia (gummy exudates from the bark of acacia senegal, an inactive substance, which forms a mucilage with water) were administrated intraperitoneally, daily once for 5 days from day 10 posttumor inoculation in a volume of 0.1 ml/10 g mouse. the eac cells that were collected from the animal peritoneum by aspiration were washed repeatedly with phosphate buffered saline (pbs) to free it from blood. the viability of the cells was checked in a haemocytometer. the cells (1106 in 0.1 ml pbs) were incubated in clean sterile tubes with the test compounds (0.01 ml, 150 g / ml in dimethyl sulfoxide (dmso)) for 3 hours at 37c, keeping the final volume at 0.9 ml. the volume of dmso was pegged below 0.1 % of the total volume. the live (without stain) and dead (with blue stain) cells were counted using haemocytometer and percent cell death was calculated using the formula : (1)%cytotoxicity=100(tdeadcdead)/ttot, where tdead is the number of dead cells in the treated group, cdead is that in the control group, and ttot is the total number of dead and live cells in the test compound treated group. antitumor activity of the compounds was determined using ehrlich ascites carcinoma (eac) tumor model in mice. ((a) normal mice for hematological studies, (b) tumor - bearing mice, (c) tumor - bearing mice treated with one dose of cisplatin, (d) tumor - bearing mice groups treated with compounds for 5 days.). the ascitic carcinoma - bearing mice (donor) were used for the study, 15 days after tumor transplantation. the ascitic fluid tumor viability was determined by tryphan blue exclusion test and cells were counted using haemocytometer. the ascitic fluid was suitably diluted in normal saline to get a concentration of 10 cells / ml of tumor cell suspension. the mice were weighed on the day of tumor inoculation and then once in three days thereafter. the compounds were administered from tenth day for 5 days intraperitoneally. after the administration of last dose followed by 18-hour fasting, six mice from each group were sacrificed for the study of antitumor activity and hematological parameters. the remaining animals in each of the groups were kept to check the mean survival time (mst) of the tumor - bearing hosts. antitumor effects of compounds were assessed by observation of following parameters. upon weighing the animal on the day of inoculation and after once in three days in the postinoculation period, the percentage increase in weight was calculated using the formula : % increase in weight = [(animal weight on respective day / animal weight on day-0) 1 ] 100. total number of days an animal survived from the day of tumor inoculation was counted. the percentage increase in lifespan (% ils) was calculated using the formula : ils (%) = [(mean survival time of treated group / mean survival time of control group) 1]100. in order to detect the influence of compounds on the hematological status of eac - bearing mice, comparison was made amongst groups of mice for each compound on the fourteenth day after transplantation. blood was drawn from each mouse from retro orbital under ether anesthesia and the white blood cell (wbc) total count, differential leukocyte counts, red blood cell (rbc) total count, and hemoglobin content parameters were evaluated. results were analyzed by one - way anova by scheffe 's posthoc test using spss computer package. antibacterial activity of test compounds was assessed against bacillus cirroflgellosus by cup - plate method. the definite volumes of peptone (0.5 %), yeast extract (0.15 %), beef extract (0.15 %), sodium chloride (0.35 %), dipotassium phosphate (0.36 %), and potassium dihydrogen phosphate (0.13 %) were dissolved in distilled water and the ph was adjusted to 7.2. one day prior to these tests, inoculation of above bacterial cultures was made in the inoculation medium as described above and incubated at 37c for 1824 hours. base - layer medium was prepared by dissolving definite volumes of peptone (0.6 %), yeast extract (0.3 %), beef extract (0.13 %), and agar (2.5 %) in distilled water. the ph of this medium was also adjusted to 7.2 and sterilized by autoclaving at 15 p.s.i. for 20 minutes. each test compounds (5 mg) was dissolved in dimethylformamide (5 ml) to give a solution of 1000 g / ml. out of this base - layer was obtained by pouring about 1015 ml of base - layer medium into each sterilized petridishes and were allowed to attain room temperature. this solid layer after attaining room temperature is called base - layer. over - night grown sub - cultures of bacteria were mixed with seed layer medium and immediately poured into petridishes containing the base - layer and then allowed to attain room temperature. the cups were made by scooping out nutrient agar with a sterile cork borer. to these cups, solutions of test compounds (0.1 ml) were added using sterile pipettes and these plates were subsequently incubated at 37c for 36 hours. the zone of inhibitions, if any, was measured in mm for the particular compound. norfloxacin was used as positive - control and solvent - control was also used to know the activity of the solvent. fungicidal activity of test compounds was assessed against aspergillus niger and candida albicans by cup - plate method. inoculation medium was prepared by dissolving definite volumes of peptone (1.0 %), yeast extract (0.6 %), sodium chloride (0.5 %), potassium dihydrogen phosphate (0.3 %), and glucose (1.0 %) in distilled water. the ph of the medium was adjusted to 6.0 and sterilized at 15 p.s.i. for 20 minutes. one day before testing, inoculation of fungi, were made in the inoculation medium and incubated at 37c for 1824 hours. the definite volumes of peptone (4.0 %), yeast extract (0.6 %), sodium chloride (0.5 %), potassium dihydrogen phosphate (0.3 %), glucose (1.0 %), and agar (2.5 %) were dissolved in distilled water. the ph of the medium was adjusted to 6.0 and sterilized by autoclaving at 15 p.s.i. for 20 minutes. the definite volumes of peptone (4.0 %), yeast extract (0.6 %), sodium chloride (0.5 %), potassium dihydrogen phosphate (0.3 %), glucose (1.0 %) and agar (2.5 %) were dissolved in distilled water. the ph of medium was adjusted to 6.0 and was sterilized separately by autoclaving at 15 p.s.i. for 20 minutes. the method of testing for antifungal activity is the same as that adopted for assessing antibacterial activity. grisofulvin was used as a positive control and solvent content was also used to know the activity of the solvent. the ligand was synthesized by acid catalysed condensation of thiocarbohydrazide with the isatin in ethanol (scheme 1) ; the condensation proceeds, as usual, selectively on the carbonyl in position 3 in the isatin ring. the ligand is characterized by means of ir, h nmr spectroscopy, and elemental analysis. in the h nmr spectrum of the ligand there is only one set of signals for the aromatic protons, while the nh of isatin and nh of hydrazone give rise to two different singlets in the 1114 ppm range. the presence of the nh groups is confirmed in the ir spectra by a broad peak around 3200 cm ; the c = o groups absorb near 1690 cm. the reactions between the metal(ii) chlorides / acetates and the ligand (1:1) lead to formation of complexes, (scheme 1). the growth of single crystals of these complexes for x - ray studies is very difficult owing to their amorphous nature and we were unsuccessful in our attempts to do so. the elemental analyses of these complexes reveal 1:1 ligands to metal stoichiometry in case of cu(ii) and ni(ii). copper forms the complexes of the type [cu(l)cl]2h2o with cu(ii) chloride and [cu(l)oac]2h2o in case of cu(ii) acetate. nickel forms [ni(l)cl ] type complex with ni(ii) chloride and [ni(l)oac ] with acetate. both cobalt and zinc chloride and acetates form the same complexes with the 2:1 ligand to metal stoichiometry, with general formula, [m(l2) ]. the complexes are found to be soluble in dimethylformamide, dimethylsulphoxide, and acetonitrile but insoluble in common organic solvents such as ethanol, methanol, benzene, acetone, and so forth. the composition and coordination geometry of these complexes has been established on the following experimental observations. the molar conductance values in dimethylsulphoxide fall in the expected range (1032 cmmol) of nonelectrolytes. the complexes were analyzed for metal, nitrogen, carbon, hydrogen and chloride. the analytical, conductivity, and magnetic moment data of the complexes are summarized in table 1. the ir spectral data of the ligand n, n-bis(isatin)thiocarbohydrazone shows a sharp absorption band around 3200 cm attributed to the presence of nh group and a very strong band near 1690 cm assigned to (c = o) stretching vibration. the band at 1619 cm is assigned to the azomethine (c = n) stretching. the ir spectral assignment of metal complexes was aided by comparison with the vibration frequencies of the free ligand. the broad band that appears in the range of 31603210 cm is assigned to the stretching vibration of ring (nh). there is only one band in the v(c = o) region and it does not differ significantly from the band in the ligand ; suggesting that the carbonyl groups are weakly involved in the coordination. although there is coordination of nitrogen atom of the azomethine group to the central metal atom, we could not appreciably conclude from the ir data, because of remaining uncoordinated azomethine group that absorbs at a just lower frequency near 1614 - 1615 cm as compared to their ligand. the band corresponding to the stretching vibration of the c = s group appears at 12001194 cm in the ligand. the absence of this band in the ir spectra of the metal complexes can be explained by the tautomerism of the c = s group with one of the imino groups to form the csh and the coordination of sulphur after deprotonation. the band that appears in the range of 690750 cm is thus assigned to the (cs) in the ir spectra of the metal complexes. the ir spectra of the complexes derived from copper and nickel acetate, show an absorption bands in the region 16651650 cm which is assigned to 8 (co) antisymmtric stretching of acetate group and another in the region 12971258 cm and which can be assigned to 3 (co) symmetric stretching vibration of acetate. difference 83 which is around 407493 cm indicates the unidentate coordination of the acetate group. the complexes show a broad band around 3400 cm, which is assigned to the (h2o) absorption. the (mn) and (mo) stretching vibrations are observed at about 490 and 455 cm, respectively, in the spectra of the complexes. the h nmr spectrum of the ligand shows only one set of signals for the aromatic protons around 6.937.60 ppm, while the nh of isatin and nh of hydrazone give rise to two different singlets in the 1114 ppm range. the h nmr spectrum of the zn(ii) complex is less informative, the presence of the hydrazonic proton is confirmed by a peak at about 13 ppm, which is slightly shifted to down field after complexation. peak due to ring nh and remaining signals of the ligand remain substantially unchanged upon complexation. electronic spectral data of the ligand and their transition metal complexes were recorded in dmf solutions. in the electronic spectrum of the ligand, three prominent absorption bands at 266, 382, and 670 nm were characterized. the band at 266 nm corresponds to the transition of the c = s group. the band at 382 nm corresponds to the transition of azomethine group and the band at 670 nm corresponds to the n transition from the amide oxygen of the isatin moiety to the azomethine group. in case of complexes, the bands appeared in the almost same position as they were appeared in ligand. followed by these, the bands displayed at 485, 485 and 495 nm in case of cu(ii), ni(ii) and co(ii) complexes respectively are assigned to ligand to metal charge transfer transitions (lmct). apart from these, the bands around 899, 780 nm in case of cu(ii) and ni(ii) complexes were assigned as d - d transition bands. the electrochemical behavior of copper(ii) complex has been investigated in dmso containing 0.1 m tetraethyl ammonium chloride supporting electrolyte, using glassy carbon working electrode and ag / agcl, pt electrodes as reference and counter electrodes, respectively. the complex involves single redox step corresponding to cu(ii)cu(i), quasireversible electrode process at epc = 0.659 v and the associated anodic peak at epa = 0.504 v suggesting the tetrahedral environment of the copper ion. the magnetic moments of the complexes were recorded at room temperature and the observed magnetic moment value for the co(ii) complex is 4.98 bm, which is in the range of 4.4 to 5.5 bm observed for the octahedral co(ii) complexes. the ni(ii) complexes derived from chloride and acetate salts exhibit the magnetic moment values 3.55 and 2.92 bm suggesting tetrahedral environment around metal ion. the value of 1.91 bm for cu(ii) chloride and 1.89 bm for cu(ii) acetate complex suggests the four coordinated tetrahedral complexes, which further supported by their electronic spectral data and cv studies. x - band esr spectra of the polycrystalline cu(ii) complex were recorded at room temperature. the esr spectra of the mononuclear copper chloride and acetate complexes show a giso value in the range of 2.066 and 2.092 respectively. the brine shrimp lethality bioassay has been chosen to assess the in vitro cytotoxic effects of the compounds, as it is an inexpensive, reliable, and quick method for the purpose. all the tested compounds showed considerable cytotoxic activity in the brine shrimp lethality bioassay. the compounds were tested using the short - term in vitro cytotoxicity towards eac cells as a preliminary screening technique of tryphan blue exclusion method (cell viability test) for their cytotoxic potential. this test is based on the principle that living cell membrane has the ability to prevent the entry of dye. hence, they remain unstained and can be easily distinguished from dead cells, which take the dye. results of the short - term in vitro cytotoxicity of the compounds are shown in table 3. all the compounds were found to be cytotoxic and produced 50% cell death at or below a concentration 19.1 g / ml. at 50 g / ml concentration, the standard (cisplatin) showed 98 % cell death. at 50 g / ml concentration, the [cu(l)oac]2h2o, [cu(l)cl]2h2o showed more than 80 % cell death. all the compounds were found to have considerable cytotoxicity in the cell viability test. cisplatin administration (on tenth postinoculation day) significantly (p 50). on the other hand, all the compounds significantly prolonged the mean survival times. the influence of all the compounds on % ils was more than 25 %. by convention, a 25 % increase in lifespan is considered as possible anticancer activity of a test compound. tumor induction significantly (p<.05) increased the total number of wbc by almost 4 times. all the compounds significantly (p<.05) reversed the tumor - induced rise in total counts of wbc. however, they were not as efficacious as cisplatin in reversing the tumor - induced total counts. on differential counts, tumor - induction caused a significant reduction in lymphocyte and a significant (p<.05) increase in neutrophil counts. this was significantly (p<.05) reversed towards normal by cisplatin and the test compounds. tumor induction caused significant decrease in rbc and hb almost to the half of the normal animals. this was significantly (p<.05) reversed towards normal by cisplatin and the test compounds. in the light of interesting antimicrobial activities of the coordination complexes, the ligands and their corresponding complexes were screened for antifungal and antibacterial activity against aspergillus niger, candida albicans, and bacillus cirroflagellosus, respectively, by the cup plate method using nutrient agar. the radial growth of the colony was recorded on completion of the incubation and the mean diameter for each complex at a single concentration was recorded. the average percentage inhibition of the fungicidal growth medium compared using the vincent equation : i=100(ct)/c, where i= percentage inhibition, t = average diameter of the fungal and bacterial growth on the tested plates, and c= average diameter of the growth on the control plates. the screening data of the inhibition of the fungi and bacteria are given in table 8. from the data, it is clear that the free ligand n, n-bis(isatin)thiocarbohydrazone is moderately active against b. cirroflagellosus where as highly active against fungi a. niger and c. albicans. on complexation, there is a notable enhancement of both antibacterial as well as antifungal activity. the promising results were observed for the co(ii) complex against both the fungi. the higher fungi toxicity exhibited by the complexes may be ascribed to the fact that the metal complexes are more susceptible to fungal cells than bacterial cells. from the elemental analysis, molar conductivity, uv - visible, magnetic, ir and h nmr spectral data it was possible to determine the type of coordination of the ligand in their metal complexes. in all the complexes, only one part of the ligand is coordinated to the metal ion resulting mononuclear complexes. the ligand coordinates essentially through the carbonyl oxygen of the isatin fragment, the nitrogen atom of the azomethine group and sulfur atom after deprotonation to give five membered rings. the compounds showed considerable cytotoxic activity in the trypan blue exclusion method. in the in vivo cancer model (ehrlich ascitic carcinoma model), the compounds significantly (p<.05) reversed the tumor - induced changes in the parameters monitored viz., percentage increase in body weight, percentage increase in lifespan, tumor - viable count, and hematological parameters (total and dlc of wbc, total rbc, and hemoglobin count). these effects were almost comparable to cisplatin the standard drug used in the study. the compounds, however, were found to have good effect in prolonging the lifespan (ils) as compared to standard drug cisplatin. based on the data of the present study, it is very difficult to suggest the possible mechanism of these compounds for anticancer effects. the compounds tested in our present study have shown promising cytotoxic activity when screened using the in vitro method and at the same time were shown to have good activity when tested using the ehrlich ascites carcinoma model. though it is very difficult to conclude anything at this stage, it can be assumed that after testing against various other cancer models and at different doses these compounds may prove to be safer drugs for tomorrow. further, the promising results were observed for the antimicrobial screening especially for the co(ii) complex against both the fungi and what may be attributed to the fact that the metal complexes are potentially active against fungal cells than bacterial cells. | the synthesis, structure, electrochemistry, and biological studies of co(ii), ni(ii), cu(ii), and zn(ii) complexes of thiocarbohydrazone ligand are described. the ligand is synthesized starting from thiocarbohydrazide and isatin. it is evident from the ir data that in all the complexes, only one part of the ligand is coordinated to the metal ion resulting mononuclear complexes. the ligand coordinates essentially through the carbonyl oxygen of the isatin fragment, the nitrogen atom of the azomethine group, and sulfur atom after deprotonation to give five membered rings. h1 nmr spectrum of the ligand shows only one set of signals for the aromatic protons, while the nh of isatin and nh of hydrazone give rise to two different singlets in the 1114 ppm range. the formulations, [cu(l)cl]2h2o, [cu(l)(ch3coo)]2h2o, [ni(l)cl ], [ni(l)(ch3coo) ], [co(l2) ], and [zn(l2)]2h2o are in accordance with elemental analyses, physical, and spectroscopic measurements. the complexes are soluble in organic solvents. molar conductance values in dmf indicate the nonelectrolytic nature of the complexes. copper complex displays quasireversible cyclic voltametric responses with ep near 0.659 v and 0.504 v vs ag / agcl at the scan rate of 0.1 v / s. copper(ii) complexes show a single line epr signals. for the observed magnetic moment and electronic spectral data possible explanation has been discussed. from all the available data, the probable structures for the complexes have been proposed. the compounds synthesized in present study have shown promising cytotoxic activity when screened using the in vitro method and at the same time were shown to have good activity when tested using the ehrlich ascites carcinoma (eac) model. the antimicrobial screening showed that the cobalt complex possesses enhanced antimicrobial activity towards fungi. |
allergen - specific immunotherapy in the treatment of ige - mediated allergy has been used for longer than a century ; yet, its major form, subcutaneous immunotherapy (scit) has not become a widely accepted routine treatment for allergy. patients will often suffer from severe symptoms and allergic comorbidities before consulting with an allergist or considering immunotherapy. subcutaneous injections for immunotherapy are believed to be tedious and unlikely to lead to sustained improvement. standardization, safety, and efficacy concerns, along with the inconvenience of injections and frequent office visits, keep the vast majority of allergic patients from receiving scit. recruitment to immunotherapy is poor : less than 5% of all allergic patients receive immunotherapy. compliance is even poorer : among adult patients who agree to undergo scit, adherence is disappointing with more than two thirds dropping out within a year of initiation. one tenth of scit candidates fail to show up for their first injection. in some countries, the scope of scit has been curtailed substantially by administrative decisions. at the same time, a wealth of evidence in literature and clinical practice supports the safety, efficacy, feasibility, compliance, and economic profile of sublingual immunotherapy (slit) [37 ]. in the united states, however, slit remains uncommon and is only offered by few practices with special interest in this method. with this study, we sought to evaluate the subjective symptom responses of patients treated with multiantigen slit. the information provided with the present study may lead to better appreciation of the potential of slit and may foster the design of large - scale, multicenter studies for its full appraisal. subjects were recruited from patients of allergy associates of la crosse, a single specialty practice that has been offering slit for 41 years. the study was approved by the mayo clinic health system franciscan healthcare - la crosse, institutional review board. all subjects were diagnosed with allergic rhinoconjunctivitis on the basis of their history and positive skin test results. skin test positivity was assessed by obtaining a response greater than the negative control and greater than two thirds of the histamine control using intradermal dilution testing (idt). antigens selected for testing were determined by a self - administered patient history questionnaire and initial consultation with their physician. figure 1 also shows that the patient population was affected by one or more comorbid allergic condition upon arrival for their first appointment. skin test panels included 1530 antigens representing dust mite, weed, tree, grass, and fungal allergens typical of the northern midwest (see table 1). the number of allergen extracts varied by patient, as the number of offending allergens ranged from six to 24 with the mean of 15.15. round one patient enrollment occurred from july through december and round three visits occurred from january through november, thus crossing multiple peak pollen seasons and limiting the influence of allergen season bias. sublingual immunotherapy based on skin test reactivity was initiated according to the la crosse method practice protocol. a capital aspect of slit, at least as practiced in the united states, is the adjustment of the treating dose to skin reactivity. for this purpose the purpose of such dilution is to adjust dose to skin reactivity under the premise that adverse reactions (including local reactions) define a level of tolerance. for many patients, skin test reactivity does not necessarily reflect the degree of sensitization. a negative skin test, however, and minimal / absent late - phase responses do establish a de facto threshold of tolerance. dosing for each patient was tied to skin test results for each individual antigen and adjusted over the course of treatment (see figure 2). with ongoing treatment, the need to regularly adjust the degree of testing (and dosing) to the long - term effects of immunotherapy is dictated by the fact that, over time, skin test reactivity tends to decline with immunotherapy. thus, initiation of slit at a strength corresponding to the highest dilution that produced a near - negative skin test establishes a safe threshold of tolerance ; thereafter, upward titration of immunotherapy doses against declining skin reactivity is used for safe build - up and unnecessary local or systemic reactions. a skin test of greater than 7 mm using dilution number 7 correlates with the highest level of reactivity. thus, the lowest dose administered of the offending allergen is dilution number 7. as skin test reactivity improves, doses are escalated to the next allergen dilution until the patient has reached dilution number 1 for his / her different allergens. the starting dose of each individual antigen is titrated based on skin test (or in vitro specific ige testing) level of reactivity, (see tables 2 and 3). sublingual immunotherapy with multiantigen treatment addresses multiple allergies that are specific to each individual patient. each bottle consisted of a 90-day supply that was individually prepared for the patient using greer laboratory and alk - abello extracts and compounded in the allergy associates of la crosse clinical laboratory. over a 1000-fold range of antigen dilutions have been reported to produce clinical improvement with slit suggesting that a straight dose - effect does not exist. other variables such as dosing frequency, extract quality, and length of treatment also need to be considered. numerous studies have observed and suggested a limited capacity of the sublingual mucosa and have shown clinical improvement with lower, but more frequent doses [1113 ]. patients were advised to take their sublingual immunotherapy drops three times daily. given that slit is retained in the sublingua for up to 48 hours, administering two to three doses per day is reasonably expected to secure unbroken allergen - exposure and overlap generously with antigen uptake by the dendritic cells and migration to lymphoid organs. this disease - specific validated questionnaire was developed by professor elizabeth juniper and has been used extensively throughout the world in a large number of clinical trials. new clinic patients were asked to complete the rqlq at their first visit before the onset of sublingual immunotherapy treatment and two subsequent follow - up visits at three- to six - month intervals. the full - version rqlq encompasses 28 questions in seven domains (activity limitations, sleep problems, non - nose / eye symptoms, practical problems, nose symptoms, eye symptoms, and emotional function). patients were asked to recall their experiences during the previous seven day period and to give their responses on a 0- to 6-point scale (none of the time to all of the time). a total rqlq score is also calculated by adding the scores of the individual domains together. this study was a prospective analysis that compiled and compared collected rqlq data from patients undergoing sublingual immunotherapy. collected data for each patient were compared to that particular patient 's baseline data and two subsequent patient visits for changes in each rqlq parameter as well as total rqlq score. timing of follow - up for visit two ranged from 1.23 months to 10.94 months, with a mean follow - up time of 4.1 months. follow - up for visit three ranged from 2.82 months to 17.94 months with a mean follow - up time of 7.06 months. paired rqlq data were available for 51 patients who were skin tested and started on slit. participants were comprised of 13 males and 38 females, with ages ranging from 22 to 63, and a mean age at initiation of slit of 45.8 years. paired rqlq results revealed statistically significant (p < 0.05) improvement in six of seven domains evaluated by the rqlq after four months of treatment. improvements were seen in the activities, nonnose / eye symptoms, practical problems, nasal symptoms, eye symptoms, and emotional categories. furthermore, the total rqlq for the whole cohort declined significantly (p < 0.5) from 126.02 to 74.96 within the first four months of treatment (see figure 3). although just shy of achieving statistical significance, participants that adhered to three times daily dosing of sublingual immunotherapy showed better improvement in rqlq scores than participants who were suboptimally compliant. advised compliance to treatment declined from round two to three, which may have affected round two to round three overall rqlq scores (see figure 4). further studies with larger study populations are needed to validate treatment compliance and multiple versus single daily dosing. a number of studies have demonstrated statistically significant effects on allergic rhinoconjunctivitis and asthma symptoms in slit [1520 ]. since no two studies used the same protocol, the value of their meta - analysis is questionable. they all used single - allergen monotherapy to evaluate efficacy, an approach which does not reflect the sensitization status of patients with allergic rhinoconjunctivitis, and may in fact have led to undertreatment and subsequent under - appreciation of the efficacy of slit. the number of slit (or even scit) efficacy studies employing multiple allergens in the treatment regimen is so surprisingly small that their low number and insufficient data on efficacy have been addressed unfavorably. they were not followed by subsequent studies that would have established a continuity of approach which might have made up, to some extent, for methodological defects. significantly, the ultimate end - point, which is improvement of symptoms, was not assessed by a validated instrument, developed by an independent party, such as the rqlq. to our knowledge, a modified, shortened version of the rqlq, the mini - rqlq, has been used in one study employing slit for multiple allergens but this study relied on retrospective selection of subjects and only enrolled fifteen patients, thus raising significant questions as to both its power and freedom of bias. our study is the first prospective study of slit efficacy, employing multiple allergen extracts for treatment, a protocol for slit which has been applied for 41 years, a validated questionnaire, and a number of subjects large enough to satisfy power requirements. in the present study, slit, as formulated by the la crosse method protocol, is effective in reducing symptoms and improving quality of life after four months of treatment (see table 4). this improvement was most prominent in activity, non - nose / eye symptoms, nasal symptoms, and emotional domains. improvement in the sleep domain of the rqlq was also observed, but did not reach statistical significance. this improvement was sustained and demonstrated again at 1012 months of treatment. given the high compliance rates with the la crosse method slit, it is expected that the improvement achieved is likely to be sustained and possibly expanded with ongoing treatment beyond the first year. sneezing and irritability, two parameters, which in a previous slit efficacy study employing the mini - rqlq were found unaffected, are demonstrated to decline in the course of the first four months of slit. the mechanism underlying slit has been reviewed. although not fully delineated, it appears that a systemic alteration of the th1/th2 balance is effected in slit by the promotion of tolerogenic t - cell clones. interaction of dendritic cells with nave t - cells is necessary for this change to occur. production of tgf-, il-10, and possibly other regulatory cytokines appears to be critical. ongoing changes may in some cases be reflected in skin reactivity as well as in specific igg and ige production changes. the protocol used in the present study may be well suited to effect these changes. it can be summarized in three cardinal points : (i) initial and thereafter regular titration of treating slit doses against skin reactivity and symptom response with skin reactivity meant as a biphasic response whose late phase reactions are also taken into account ; (ii) frequent administration of slit doses to secure continuous, maximal, and uninterrupted saturation of the sublingual dendritic cells ' potential for phagocytosis and migration, that is, three doses per 24 hours ; and (iii) maintenance of allergens in high glycerin solutions in order to prevent decay and suppress proteolytic activity. in summary, this study represented a preliminary attempt to investigate the effectiveness of multiantigen slit in a complex patient base. experience with this protocol over the years has been rewarding and has shown clinical benefit with a wide variety of allergic conditions including advanced respiratory disease in adults with mold allergy, asthma prevention in pediatric patients, and contact allergies including nickel and poison ivy while maintaining a remarkable paucity of adverse reactions of any significance. the present study underscores the efficacy of slit ; however, we recognize that the absence of a placebo group limits the interpretation of results. given the large number of patients currently treated and high rates of compliance, multicenter, controlled studies are needed of greater magnitude and expanded scope to include morbidities and associations such as recurrent / chronic sinusitis, atopic dermatitis, gastroesophageal reflux, and migraines. sustained suppression of symptoms after eventual completion of slit will also need to be studied. statistically significant reduction of symptoms and improvement of quality of life are demonstrated during the initial four month period of slit. these data support the efficacy of slit and need to be followed by controlled trials to evaluate the efficacy of this method. | due to its excellent safety profile, ease of administration, and economic considerations, sublingual immunotherapy (slit) is becoming a preferred form of allergen specific immunotherapy. the efficacy of slit is still debated. the purpose of this act of practice trial is to evaluate quality of life outcomes in patients treated with slit. fifty one patients with allergic rhinoconjunctivitis demonstrated by skin testing completed the rhinoconjunctivitis quality of life questionnaire (rqlq) at initiation, at four months and at 1012 months of slit. significant improvement (p < 0.05) on six of seven domain categories of the rqlq questionnaire was noted. total rqlq scores also showed significant improvement. this study supports slit as a modality effective in controlling allergic symptoms. |
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