The chiropractic profession in Norway has increased five-fold in the last two decades. As there is no academic graduate program in Norway, all chiropractors have been trained outside of Norway, in either Europe, America or Australia. This might have given Norwegian chiropractors heterogenic characteristics concerning practice routines and clinical settings. However, little is known about what characterizes this profession and how it compares to other chiropractic professions in Europe. The aim of this survey was to describe major characteristics of the chiropractic profession in Norway.Two surveys were distributed to all 530 registered chiropractors in Norway in 2011. One survey was for all chiropractors (Survey 1) and the other for clinic owners (Survey 2). Results have been reported as tables and as approximate percentages in the text for ease of reading.Response rates were 61%  and 71% . More than two-thirds of the chiropractors in Norway had been in practice for under a decade. Only one in four chiropractors worked in solo practice and the majority shared premises with at least one colleague, typically at least one physiotherapist and one additional health practitioner. Today, only one in five clinics possessed radiologic equipment and one in ten had access to diagnostic ultrasound equipment. The majority of the chiropractors reported to apply mainly similar treatment modalities. More than 90% reported to use manipulation techniques on most patients, with soft tissue techniques and exercise modalities being almost as common. More than 3/4 of the profession reported that their clinical practice was in accordance with available clinical guidelines and about one third were positive about participating in future clinical research.The Norwegian chiropractic profession is relatively young and members report being satisfied with their work conditions. There is a clear difference from the earlier practice pattern in that intra- and inter-professional collaboration is more common and it is considered desirable. The profession seems to follow the modern trends in evidence-based practice by using X-rays more sparingly than previously, adhering to guidelines and being positive about research. Chiropractors all over the world practise under different legislations and circumstances. In Norway, the first chiropractor started his practice in 1922 [Legislation for chiropractors in Norway was introduced in 1989 . BetweenToday (2014), the chiropractic profession consists of approximately 650 individuals, of which the national association organizes more than 90%. The ratio of chiropractor/number of inhabitants is very high in Norway and according to records from the European Chiropractic Union. Norway and Lichtenstein are the European countries with the highest accessibility of chiropractors. In fact, the number of authorized chiropractors in Norway has increased five -fold in the last two decades. In addition, it is estimated that approximately 300 young Norwegians are enrolled in chiropractic graduate programs in various parts of the world. Hence, in 2020 the profession is estimated to consist of at least 1000 individuals.This newly acquired favourable status as an officially recognized health profession is probably one of the reasons for the strong recruitment of chiropractic graduates in recent decades. However, this high number may seem puzzling, as there is no academic undergraduate institution for chiropractors in Norway. Although the high costs related to graduate programs abroad might diminish the interest for this type of education, this is probably offset by the fact that the Norwegian state offers beneficial educational loans and funding for foreign education and has a firm tradition of supporting and facilitating international education.Easy access to care is obviously a good thing. However, the diversity of educational institutions that these chiropractors have attended could also result in an unacceptable heterogeneity of the profession. The fact that all chiropractors have a foreign educational background may result in a less successful integration of the profession into the Norwegian health care system. This might be due to both cultural and language differences between the country where the training was received and the country of practice, resulting in variations in practice routines and clinical assessment procedures.However, little is known about the Norwegian chiropractic profession. Previous publications have reported mainly demographic data –12. ModeOther concerns could be that an overproduction of chiropractors may lead to some negative consequences, such as a tendency towards over-servicing, unsuitable “additional” procedures, reduced income and even unemployment. In order to learn more about current practice of Norwegian chiropractors two surveys were carried out: one aimed at all chiropractors in Norway and the other one for the clinic owners. The aim of this report is to describe major characteristics of the chiropractic profession in Norway, specifically to describe their clinical settings and practice routines, satisfaction with work and to study evidence of inter-professional collaboration, using data from these two surveys. In addition, the aim is to describe attitudes towards research participation and clinical guidelines.An invitation was sent to all 530 members of the Norwegian Chiropractors’ Association with a registered e-mail address in May 2011. The invitation contained a description of the two surveys and consent to participate to be signed. All chiropractors were asked to complete Survey 1 regarding practice routines. Clinic owners were asked also to answer Survey 2 regarding clinic settings and clinic routines.Reminders were sent weekly until the end of data collection in August 2011. The invitation and reminders were administered by secretaries at the Norwegian Chiropractors’ Association.These two surveys were largely based on two similar surveys of Danish chiropractors conducted in 2010, designed by researchers at the Nordic Institute of Clinical Chiropractic and Biomechanics . Permission to use this survey instrument was given by the administration of NIKKB.Additional questions added to the Danish survey included some questions concerning compliance to the present national clinical guidelines for low back pain in Norway, and opinions on and attitudes to practice routines.Data were collected in the open online survey database “Lime-survey”. As this was done anonymously, it was not possible to identify or compare responders and non-responders. The survey software did not permit more than one survey response. As the survey was anonymous and no track of participants was possible, repeated reminders to participate in the survey were sent regardless of previous participation.As no personal data were collected, and all data collected anonymously, approval from the Regional Committee for Medical and Health Research Ethics was not needed.Analysis was performed using the statistical software Analyze-It®. Obvious errors were amended manually when the correct information could be rationally extrapolated. Some responses were only partially completed. Such missing data have been labeled “no-responders” in the results section.The response rates for Survey 1  and Survey 2 (clinic owners) were 61%(N = 320/530) and 71%(N = 217/306), respectively.To determine the representativeness of the two study samples, comparisons were made between the responders and all members of the Norwegian Chiropractors’ Association in regard to sex and birthdate for Survey 1 and in regard to geographical distribution for Survey 2. In Survey 1 the representation of male responders was 64%, compared to 65% in the target population. With regard to age, there was a slight over-representation among the responders in Survey 1 of those below 40 years of age; but overall, all age groups were well represented. All counties were represented among the responders similar to the target population.Results from both surveys have been reported below, grouped under separate topics and not by survey, in such a way that the profile of the chiropractic profession can best be described. In order to make the reading and comprehension easier, percentages have often been rounded off to the nearest “larger” figure in the text; i.e. 32.2% would be reported as “1/3” or as “about thirty percent” and 18.7% as “1/5” or “about 20 percent”. Exact estimates are available from the authors on request.Among the chiropractors who responded to the survey, 64% were male with the median and mean age of 36 and 38 years respectively and with 70% being below 40 years of age. Almost 4/5 had been in practice for less than 15 years, and only 8% had been in practice for more than 25 years (1% non-responders). The majority were trained in Europe; approximately 40% in the UK and 20% in Denmark; the others in North America (30%) or in Australia (10%) (2% non-responders).None reported to have a PhD degree, although 2% reported to be involved in PhD programs. In addition, close to 50% reported to have some kind of additional higher education background: 7% at Master’s level (primarily a Master’s degree in Sports) and 18% at Bachelor level. The most frequently reported professional backgrounds were physiotherapy (5%) and nursing (1%) (53% non-responders).Almost all chiropractors (97%) worked in private practice settings (3% non-responders). More than half of the clinics were located in an office building, adapted to wheelchair users, with access to an elevator, and in a building with other health practitioners, whereas only 20% practised in small, separate buildings (6% non-responders).The majority (61%) had more than 4 treatment rooms in the clinic and therefore often practised together with other health practitioners (1% non-responders). In contrast, about one in four practised in solo practices. About 3/4 of the clinics consisted of more than one chiropractor; almost half of the clinics included at least one physiotherapist and one additional health practitioner , but only 10% of the clinics reported to have a general practitioner linked to the clinic (0% non-responders).About 1/3 of the clinics had access to large, separate exercise facilities or smaller exercise equipment (2/3), but imaging facilities were not common, as only 1/5 had their own X-ray equipment and 12% had diagnostic ultrasound. A mere 1% had magnetic resonance imaging facilities within their clinic setting (6% non-responders).Among all the chiropractors, one third reported the total weekly workload to be 30–37 hours and another third reported it to be 38 – 45 hours. A further 1/5 reported to work for more than 45 hours per week (3% non-responders). As 38 hours per week is considered full time office hours in Norway, this means that about half of chiropractors reported to work more than full time), whereas less than 10% worked for less than 25 hours per week. The majority of chiropractors (60%) reported to attend to 15 – 20 patients per day. Eighty percent reported spending 30–49 minutes on a first consultation and 60% spent 15–20 minutes on a regular consultation (2% non-responders).The most common treatment modalities, i.e. those used on more than 50% of patients were reported to be: manipulation (97%), soft tissue techniques (80%) and instructions on home exercises (67%). More detailed information is provided in Table In addition to instruction on home exercises, half of the chiropractors reported to refer patients to physiotherapists either within or outside the clinic. However, only one in five clinics offered some type of group exercise.The use of X-rays was infrequent, either through referral to hospitals or private centres or (less commonly) within the clinics themselves. However, only 21% of the clinics reported to use radiological imaging on a daily basis. The questions in the survey allowed only one answer per category which might give rise to some under-reporting. For instance, it could be that chiropractors with X-ray imaging facilities in the clinic were still referring patients for more complex imaging at hospitals.Full spine radiological imaging on a frequent basis (every day or week) was reported by almost 10%, either performed in their own clinic or by referral. The majority of chiropractors (62%) never referred for or reported taking full spine X-ray at all. Referral for MR imaging of patients was reported by more than three-quarters to occur at least every month. Referrals for ultrasound imaging were not as common as for X-ray and MR imaging .One in five reported to have interdisciplinary meetings at least every month. The most common health personnel to be engaged in such meetings were physiotherapists and massage therapists, in contrast to regular (monthly) meetings with GPs, which was reported to occur by only 20% of these (24% non-responders).With regard to referrals, 3/4 of the chiropractors reported to refer patients to physiotherapists every month  and half of all chiropractors referred patients to orthopaedic specialists, 1/3 to neurologists, and 1/6 to rheumatologists, at least every month . Communications to other health personnel were either written or verbal and for most occurred less than 5 times per week . Chiropractors reported to engage infrequently with certain health personnel such as nutritional therapists and manual therapists. The vast majority (86%) reported the desire to have stronger and better cooperation with other health professions (6% non-responders).About half of the chiropractors reported both that less than 10% of their patients were referred from general practitioners and other health personnel. Almost 4/5 reported that less than 10% of their patients were below 10 years of age. In addition, as many as 3/5 reported that between 10-50% of their patients had problems unrelated to the spine .The majority (80%) of chiropractors reported that they practised in accordance with the Norwegian clinical guidelines for low back pain . When paOnly 5% were involved in Master’s programs and 4% were engaged in various research projects. However, almost 1/3 reported to be willing to participate in clinical research projects depending on the demands on their resources (e.g. extra time in relation to data collection) and compensation (economic or merit). Ten percent reported to be positive about participating in research-related projects regardless the circumstances .Almost all chiropractors (90%) reported to be content with their choice of profession. Furthermore, the greater majority expressed that they had sufficient professional challenges and felt that their income reflected their workload. Nevertheless, more than half were uncertain or negative with regard to working conditions in the future .We performed two web-based surveys among registered chiropractors in Norway, members of the national chiropractic association. The results from these studies showed that the majority of Norwegian chiropractors are young and that they graduated relatively recently from a European school. Almost all work in private practice, with several health practitioners in the clinic and most of them work full time. Chiropractors’ treatment modalities vary to some extent, but in general, manipulation, soft tissue techniques and exercise advice are most commonly used. Although interdisciplinary cooperation is common with regard to communication, this applies to some health professions more than others, such as physiotherapists (most common) versus medical specialists (least common). Chiropractors appear to recognize, mainly agree with and practice according to a recent Norwegian guideline for back problems . They haThe two surveys had a response rate of 61% and 71%, respectively. This raises some questions as to how representative the responses were in relation to all chiropractors in Norway. However, when demographic data from responders were compared with all the members of the Norwegian Chiropractors’ Association (NCA) , it wasOne of the limitations of these two surveys was that no preparatory pilot study was performed. A pilot study may have revealed some weaknesses in the questionnaire allowing for the creation of a more user-friendly survey instrument, which in turn could have resulted in a lower rate of no response in some of the more complex questions. However, the major parts of the two surveys were based on a previous Danish survey , designeIn addition, one of the major limitations of the survey was the design itself. Data were primarily self-reported and not based on actual counts in patient files or appointment books, nor were they collected with clinical monitoring procedures. The result of this is that there was probably a higher response rate than there would have been with a more cumbersome data collection technique, but some information provided by the chiropractors would be subjective rather than clearly objective. This lack of precise information made it suitable to report the results in a general way, rather than reporting specific and detailed conclusions.Some questions had quite a few non-responders. Nevertheless, the proportion of these was always taken into account in the analyses.In the past, chiropractors worked almost exclusively as solo practitioners and in private practice. Today, more practitioners work in groups, together with other chiropractors and/or with other health practitioners ,17. ThisDespite the high density of chiropractors in Norway, as compared to other countries, only a few chiropractors reported working part time and none of them were registered as being unemployed. This aspect needs to be closely monitored in the near future as it is anticipated that the number of practitioners will increase considerably in the future.A wide variety of treatment modalities was reported among the chiropractors. Three major approaches were noted to be used by the majority of the chiropractors. These were: manipulation techniques, soft tissue techniques (mainly trigger point and stretching) and instruction/advice on exercise. This is not surprising as these findings probably best describe the activities of a chiropractor –23. TechThree decades ago almost three-quarters of all new patients presenting with LBP were assessed with radiological procedures in Norway , often aIt is encouraging to see that some chiropractors have started using diagnostic ultrasound, which presents no harm  to the patient. Further, it allows for dynamic examinations of muscles and tendons, allowing perhaps for an increased scope of practice.Recent guidelines advise a multidisciplinary approach to the management of long-lasting musculoskeletal problems ,25. The Interdisciplinary communication and meetings were typically found to occur on a weekly basis, however most frequently with physiotherapists and massage therapists. Communication with the patients’ GPs is probably less than optimal, as patient-reports are actually mandatory (by legislation ) to the Chiropractors in Norway have the right to formally refer patients for medical specialist assessment and to physiotherapists for rehabilitation services. Our results indicate that this generally takes place on a monthly basis, though with wide variation in the frequency. As this legislation was introduced in 2006 , it mighPrior to 2006, patients required a referral from a general practitioner in order to be eligible for reimbursement for chiropractic treatment. According to a survey in 1992, at that time, one third of all chiropractic patients were referred from a general practitioner . As thisMost chiropractors reported to be familiar with the Norwegian clinical guidelines of low back pain, though almost 1/4 failed to answer this question. When they were questioned on specific items from this guideline, they agreed with most of them. Some disagreement would be acceptable though, as different guidelines also diverge in their conclusions and only a few key elements can be clearly synthesized .There have been practising chiropractors for about a century in both the USA and many European countries. The European Chiropractic Union, an umbrella organization for chiropractic associations in Europe, is actually more than 75 years old . DespiteThere are several demographic and practice-related differences among the chiropractic professions in Europe. For instance, most countries, including Norway, are still dominated by male chiropractors (between 60-80%) as has been the case for more than 20 years in most European countries . In contThe Norwegian chiropractic profession is relatively young and practitioners report to be satisfied with their work conditions. There is a clear difference from the earlier practice pattern in that intra- and inter-professional collaboration is more common and it is considered desirable. The profession seems to follow the modern trends in evidence-based practice by using X-rays more sparingly than previously, adhering to guidelines and being positive towards research.


In the continuing search for new cerebrospinal fluid (CSF) biomarkers for Alzheimer’s disease (AD), reasonable candidates are the secretase enzymes involved in the processing of the amyloid precursor protein (APP), as well as the large proteolytic cleavage fragments sAPPα and sAPPβ. The enzymatic activities of some of these secretases, such as BACE1 and TACE, have been investigated as potential AD biomarkers, and it has been assumed that these activities present in human CSF result from the soluble truncated forms of the membrane-bound enzymes. However, we and others recently identified soluble forms of BACE1 and APP in CSF containing the intracellular domains, as well as the multi-pass transmembrane presenilin-1 (PS1) and other subunits of γ-secretase. We also review recent findings that suggest that most of these soluble transmembrane proteins could display self-association properties based on hydrophobic and/or ionic interactions leading to the formation of heteromeric complexes. The oligomerization state of these potential new biomarkers needs to be taken into consideration for assessing their real potential as CSF biomarkers for AD by adequate molecular tools. Alzheimer’s disease (AD) is an age-related neurodegenerative disorder recognized as the most common cause of dementia among the elderly. The pathologic characteristics of AD are neurodegeneration and proteinaceous deposits, including extracellular plaques composed mostly of β-amyloid peptides (Aβ) and intracellular tangles of the microtubule-associated protein tau abnormally hyperphosphorylated (P-tau). Both pathological effectors, Aβ and P-tau, can be monitored in cerebrospinal fluid (CSF). In late-onset AD, concentrations of tau and P-tau in CSF are increased and probably reflect neuronal damage, but levels of Aβ peptides are decreased. These changes can be measured in CSF before the onset of any other symptoms, and, therefore, they can be used as a diagnostic marker for the disease . AlthouAccording to the amyloid hypothesis, accumulation of Aβ in the brain, resulting from an imbalance between production and clearance, is the primary influence driving AD pathogenesis . The Aβ The predisposition for self-association of Aβ42 determines that while Aβ42 content is increased in the AD brain, its levels in CSF are decreased presumably due to its increasing deposition in brain tissue  proposedIn accordance with the mentioned challenges, there is a need to identify additional β-amyloid-related markers of AD. Reasonable candidates are proteins, such as secretases, involved in the pathological processing of APP, and the large proteolytic cleavage fragments sAPPα and sAPPβ. Since most of these secretases are transmembrane proteins, their assessments in CSF were not considered until recent years. The purpose of this article is to review recent evidence about the presence of secretase components in CSF and their potential as AD biomarkers. In addition, we summarize our recent findings about the presence of soluble full-length APP (sAPPf) in CSF and their oligomerization into heteromers. Our studies demonstrated that sAPP heteromers contribute to the estimation of sAPPα and sAPPβ levels, which needs to be taken into consideration for their assessment by ELISA. The suitability of applying adequate molecular tools for the assessment in CSF of hydrophobic proteins and soluble heteromeric aggregates is absolutely necessary to evaluate their potential as biomarkers.The processing of APP begins with the action of either α-secretase or β-secretase, initiating mandatory pathways. The initial shedding by α-secretase or β-secretase releases large soluble proteolytic cleavage fragments of APP, sAPPα and sAPPβ, respectively, both present in human CSF , 12. SinIn conclusion, an optimal approach to quantify sAPPα and sAPPβ in CSF has been based on ELISA determinations, but the presence of heteromeric complexes of sAPP obligate adjusting protocols. Moreover, the characterization of a soluble transmembrane protein might be hindered by the difficulty in distinguishing it from the truncated species generated by cleavage of the transmembrane protein. The existence of different sAPP isoforms, generated from alternative exon splicing , adds coThe major neuronal β-secretase has been identified as beta-site APP cleaving enzyme 1 , thoughFurthermore, similarly to APP, BACE1 occurred as a dimer in human brain tissue , 42. Thein vivo  family, ADAM10, ADAM17 (TACE), and ADAM9 have been proposed as α-secretases , and othin vivo , 47. To in vivo  and othein vivo . Howeverin vivo  and plasin vivo , 52 fromin vivo . The synin vivo ; thus, iMoreover, ADAM proteases, similarly to BACE1, are type I transmembrane proteins, but also include secreted isoforms . Indeed,post-mortem CSF, where artifacts are likely to appear. APH1 and PEN2, but not nicastrin, co-exist within these CSF–PS1 complexes. We were unable to detect γ-secretase activity in human CSF, and presumed that CSF–PS1 complexes may result from non-specific aggregation of these transmembrane proteins with large numbers of hydrophobic regions. PS1 aggregates have previously been described as temperature-sensitive (post-mortem samples (disease at term) display higher levels of PS1 than those present in non-demented control cases, particularly the stable complexes resolved by sucrose density gradients. Lumbar CSF samples from probable AD cases (early stages of the disease) display only significant differences in the proportion of the PS1 stable complex, but not in total levels (γ-Secretase is an intramembrane protease complex composed of presenilin-1 (PS1), nicastrin, APH1 (anterior pharynx-defective 1), and PEN2 (presenilin enhancer 2) . Since mensitive ; similarl levels . The amoBecause CSF is in direct contact with the extracellular space of the central nervous system, biochemical changes in the brain could potentially be reflected in CSF. It is expected that potential AD biomarkers involved in AD pathogenesis will mirror AD progression. However, to date, no single biomarker has reached expectations. Several models of CSF secretion have been proposed –63, but Our understanding of the potential roles for APP, BACE1, ADAM proteins, PS1, and other related proteins in CSF is lacking, but of interest in order to design adequate quantification strategies to assess their real potential as biomarkers for AD. Ultimately, it is anticipated that a combination of CSF biomarkers might serve for early diagnosis, but also for assessing disease progression and especially the efficiency of secretase inhibitors during the course of clinical trials. In this review, we presented evidence that most of the proteins related with APP processing are measurable in CSF. More investigation should focus on the possibility of monitoring soluble forms of APP and secretase components, and to evaluate the progress and feasibility of developing molecular tools for these potential new CSF biomarkers for AD.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.


This study aims to evaluate the impact of colony stimulating factor-1 (CSF-1) expression on recurrence and survival of patients with clear-cell renal cell carcinoma (ccRCC) following surgery.We retrospectively enrolled 267 patients  with ccRCC undergoing nephrectomy at a single institution. Clinicopathologic features, cancer-specific survival (CSS) and recurrence-free survival (RFS) were recorded. CSF-1 levels were assessed by immunohistochemistry in tumor tissues. Kaplan-Meier method was applied to compare survival curves. Cox regression models were used to analyze the impact of prognostic factors on CSS and RFS. Concordance index (C-index) was calculated to assess predictive accuracy.In both cohorts, CSF-1 expression positively correlated with advanced Fuhrman grade and necrosis. High CSF-1 expression indicated poor survival and early recurrence of ccRCC patients after surgery, especially those with advanced TNM stage disease. Multivariate Cox regression analysis showed CSF-1 expression was an independent unfavorable prognostic factor for recurrence and survival. The predictive accuracy of the University of California Los Angeles Integrated Staging System (UISS) was significantly improved when CSF-1 expression was incorporated.High CSF-1 expression is a potential adverse prognostic biomarker for recurrence and survival of ccRCC patients after nephrectomy. Renal cell carcinoma (RCC) accounts for approximately 3% of all adult malignancies, representing the seventh most common cancer in men and the ninth most common cancer in women. Based on current guidelines, surgery remains the only curative treatment option in patients with localized renal cell carcinoma (RCC) -3. HowevColony stimulating factor-1 (CSF-1), also known as macrophage colony-stimulating factor (M-CSF), is the primary cytokine that regulates the proliferation and differentiation of monocytes and macrophages . CSF-1 iIn this study, we analyzed CSF-1 expression by immunohistochemistry in ccRCC tumor tissues and its association with clinicopathologic characteristics and patient outcome. We further evaluated whether this parameter could add additional prognostic information to well-established pathologic factors and prognostic models.A total of 267 patients diagnosed with clear-cell RCC (ccRCC) at Zhongshan Hospital  were retrospectively included in the study. We enrolled a training cohort of 195 consecutive patients undergoing nephrectomy between January 2003 and December 2004. For validation, we also enrolled 72 consecutive patients who experienced surgery in 2001. This study was approved by the Ethics Committee of Zhongshan Hospital, Fudan University. Informed consent was obtained from each patient. For each patient, the following clinicopathologic information was collected: age, gender, tumor size, TNM stage, Fuhrman grade, presence of histologic tumor necrosis, and eastern cooperative oncology group performance status (ECOG-PS). Patients were staged using radiographic reports and postoperative pathological data, and were reassigned according to 2010 AJCC TNM classification. None of the patients received neoadjuvant treatment. Patients who died within 30 days of surgery or before discharge were excluded from the study. CSS was calculated from the date of surgery to the date of death or last follow-up, and RFS was calculated from the date of surgery to the date of recurrence or last follow-up. Patients with metastatic disease were not included in the analyses using RFS as the endpoint.Patients with localized RCC were treated with radical or partial nephrectomy, and patients with metastatic RCC were treated with cytoreductive nephrectomy followed by interferon-α-based immunotherapy. After surgery, patients were evaluated with physical examination, laboratory studies, chest imaging, and abdominal ultrasound or CT scan every six months for the first two years and annually thereafter. Survival status was updated in October 2013. Median follow-up was 103 months  in the training cohort and median follow-up was 72 months  in the validation cohort.P value” approach was used to obtain the cutoff providing the most optimal separation between the groups of patients in the training cohort related to their CSS by X-tile software. The validation cohort was separated into CSF-1-low patients and CSF-1-high patients with the same cutoff value.Tumor samples were reviewed histologically using hematoxylin and eosin staining, and then we marked representative areas more centrally on the paraffin blocks away from hemorrhagic and necrotic areas. Duplicate 1.0-mm tissue cores from two different areas were used to construct the TMA. Primary antibody against human CSF-1  was used in the procedure. The specificity of the antibody was confirmed by western blot using RCC cell lines. Tissue samples processed similarly, except for the omission of the primary antibody, were used as negative controls in immunohistochemistry. The immunostaining was evaluated by two pathologists (L. Chen and Q. Fu) without the knowledge of patient outcome. A semi-quantitative immunohistochemistry score on a scale of 0–300 was calculated for each sample by multiplying the staining intensity  and the percentage of cells (0–100%) at each intensity level . For eacMedCalc 12.7.0. and Stata 12.0. were used to perform statistical analyses. Correlations between immunohistochemical variables and clinicopathologic characteristics were analyzed with χ2 and t tests. Kaplan-Meier method with log-rank test was applied to compare survival curves. All statistical tests were two sided and performed at a significance level of 0.05. Cox regression models were used to analyze the impact of prognostic factors on RFS and CSS. The predictive accuracy of various Cox regression models was quantified by Harrell's concordance index (C-index), which ranges from 0.5 (no predictive power) to 1 (perfect prediction).We analyzed a total of 267 patients with ccRCC, 195 in the training cohort and 72 in the validation cohort  and tumor necrosis .CSF-1 positive staining mainly appeared in the cytoplasm of tumor cells. Representative CSF-1 immunohistochemical images of low expression (score = 15) and high expression score = 240) have been shown in Figure 40 have bP = 0.003 and P = 0.005, respectively). We next evaluated the independent prognostic value of CSF-1 expression using Cox regression analysis  and RFS  for patients with ccRCC after surgery in both cohorts. We further performed a subgroup analysis by TNM stage. The prognostic value of CSF-1 expression was restricted to patients with TNM stage III/IV disease  was first performed to compare predictive accuracy of the prognostic models. For RFS Figure A and B, P = 0.004). However, the C-index of the SSIGN was slightly increased from 0.710 to 0.718 after the addition of CSF-1, which failed to reach statistical significance (P = 0.393). Similarly for CSS, the C-index of the UISS was improved from 0.708 to 0.742 (P = 0.001) when CSF-1 expression was supplemented, whereas the C-index of the SSIGN was merely increased from 0.753 to 0.764 (P = 0.231) after the incorporation of CSF-1. We further calculated the C-indices with respect to predictive models within TNM stage I/II and III/IV disease, respectively, and the predictive accuracy of the UISS and SSIGN were significantly improved when CSF-1 expression was added only for CSS in TNM stage III/IV subgroup  and potential clinical use, CSF-1 expression measured as a continuous variable was dichotomized into low and high groups at the cost of great information loss. Furthermore, determination of cases with CSF-1 expression near cutoff value could be difficult because a difference of 20–40 in the semiquantitative immunohistochemistry assessment could be quite subjective, especially in the clinical setting. Fourthly, functional studies are needed to elucidate the biological mechanisms involved in this association.In conclusion, the present study demonstrated that CSF-1 expression is an independent adverse prognostic biomarker for recurrence and survival of patients with ccRCC after nephrectomy. Incorporating CSF-1 expression into the UISS prognostic model could significantly improve its predictive accuracy.


To assess the efficacy of midazolam for anxiety control in third molar extraction surgery.Electronic retrievals were conducted in Medline , the Cochrane Central Register of Controlled Trials (CENTRAL) , Embase (via OVID 1974-2013.12), and the System for Information on Grey Literature in Europe (SIGLE). The bibliographies of relevant clinical trials were also checked. Randomized controlled trials satisfying the inclusion criteria were evaluated, with data extraction done independently by two well-trained investigators. Disagreements were resolved by discussion or by consultation with a third member of the review team.Ten studies were included, but meta-analysis could not be conducted because of the significant differences among articles. All but one article demonstrated that midazolam could relieve anxiety. One article demonstrated that propofol offered superior anxiolysis, with more rapid recovery than with midazolam. Compared with lorazepam and diazepam, midazolam did not distinctly dominate in its sedative effect, but was safer. Two articles used midazolam in multidrug intravenous sedation and proved it to be more effective than midazolam alone.It was found, by comparison and analysis, that midazolam might be effective for use for anxiety control during third molar extraction and can be safely administered by a dedicated staff member. It can also be used with other drugs to obtain better sedative effects, but the patient’s respiratory function must be monitored closely, because multidrug sedation is also more risky. Dentistry and anxiety have always been inextricably linked, particularly with regard to third molar extractions, which provide a relatively intense surgical stimulus. Anxiety toward dental procedures varies from a suppressed fear of pain to a phobia. Patients may also have symptoms such as sweating, tremors, arrhythmias, and vasovagal reactions, which may make treatment difficult or even impossible. Thus, sedative measures have attracted serious attention among dental practitioners and researchers. “Minimal sedation” or “anxiolysis” is a drug-induced condition in which patients respond normally to verbal commands, while the cardiovascular systems and spontaneous breathing are unaffected. Cognitive function and coordination may be impaired .Midazolam, as a benzodiazepine, has both anxiolytic and amnesic properties. It has become increasingly popular as an anesthetic modality for managing the apprehensive dental patient –4. In adThough some patients require sedation to make tooth extraction more tolerable, the precise effects of midazolam remain unclear, due to incremental increases in anxiety. Therefore, we reviewed the selected articles to determine the efficacy and safety of midazolam used in anxious patients during third molar extraction and compared them with other regimens of sedative drugs to determine the incidence of common adverse drug reactions.This study was approved by the Ethics Committee of the Sichuan University, Chengdu, China. Two reviewers independently assessed the title and abstract of each retrieved citation for eligibility according to the following inclusion criteria: (1) Studies had to include dentally anxious outpatients who had undergone third molar extraction, regardless of gender or race; (2) published studies, including grey literature, had to be randomized, double-blind, and refer to midazolam’s effect on dental anxiety compared with that of placebo or other anti-anxiety agents; (3) and studies had to have outcome indices, including the patients’ anxiety levels. Studies were excluded if they met the following criteria: (1) Descriptive research such as reviews, case reports, and clinical observations were excluded, as were basic experiments; and (2) studies including patients younger than 18 years old were excluded. For all potentially eligible citations, we retrieved the full-text article, which was assessed independently by two reviewers. Disagreements were resolved by discussion or by consultation with a third member of the review team.Two reviewers independently searched for all publications using electronic databases, including the Cochrane Central Register of Controlled Trials (CENTRAL) , Medline (via PubMed 1950–2012.12), Embase (via OVID 1974–2012.12), and SIGLE (System for Information on Grey Literature in Europe). The following search terms were used to search Medline and CENTRAL: “midazolam”, “tooth extraction”, “tooth extractions”, “anxiety”, “third molar”, “third molars”,”wisdom tooth”, and “wisdom teeth”. These terms were modified to search Embase and other databases as required. The search strategy was combined with the highly sensitive strategy developed by the Cochrane Collaboration for identifying RCTs in Medline: sensitivity maximizing. We also searched the reference lists of the identified studies.We produced a form for data extraction including population, study design, treatment, and efficacy assessment for each study . QualitiThe Effect Size (ES) is calculated as the ratio of the mean difference between treatment and placebo relative to the standard deviation (SD) of that difference:ES = Mean of difference between treatment and placebo/SD of difference between treatment and placebo.t-tests. Unlike t-test statistics, however, ES aims to estimate a population parameter, so it is not affected by sample size. The larger the ES, the more effective the treatment.The ES is defined as small (0.2), medium (0.5), or large (0.8)  and is cWe identified 10 randomized clinical trials (RCTs) that form the basis of this review. Nine were double-blinded RCTs, and one was a partially blinded RCT. From 6 of these 10 articles, data comparing midazolam with placebo were retrieved. Information about midazolam compared with other sedative drugs  could also be retrieved. These sedative drugs were given to patients intravenously or orally. To assess patients’ anxiety levels, investigators in these trials used different questionnaires, scales, and salivary cortisol measurements. A particularly important type of diversity existed in the comparisons being made by the primary studies, so meta-analysis was not possible .Except for one study, all others showed that midazolam had a statistically significant difference in anxiety relief when compared with placebo. After analysis, we found numerous shortcomings in research design in the only article in which contradictory results were reported , and thip = 0.010) [p = 0.02) at 10 minutes post-administration, with greatest improvement in the diazepam group. There were no statistically significant differences among the three groups on arrival in the recovery room. Compared with the other two groups, patients in the diazepam group reported pain on injection [In a single-blind controlled study  of patie= 0.010) . Both tenjection .Two double-blind controlled studies ,15 examiThird molar extraction is a comparatively complicated procedure and commonly causes discomfort and anxiety for patients. Today, however, with the advances in dental research, people prefer, and have come to expect, more comfortable therapeutic procedures. Therefore, dental practitioners worked to find ways to relieve patient anxiety during surgery and identified the drug benzodiazepine, which has both anxiolytic and amnesic properties . One of Of the 10 articles included in our systematic review, only one concluded that midazolam was restricted to decreased anxiety during surgery . This arAnalysis of data from these documents also showed that diazepam had the best sedative effect when compared with that of midazolam and lorazepam. However, diazepam often caused pain on injection and venous thrombosis; furthermore, it prolonged anti-anxiety because of its active metabolites with long plasma half-lives . When weFurther, the studies revealed that benzodiazepine, opioid, and ultra-short-acting barbiturate combinations had sedative effects superior to those obtained with midazolam alone but with increased risk, most notably associated with significant respiratory depression. To guarantee patient safety, we should continuously monitor the patient’s respiratory function, coupled with the practitioner’s experience and training, along with the equipment and drugs necessary to manage this complication. Now recent research verified that capnography  can provMany previous investigations revealed the amnesic effects of midazolam. After review of our included literature, we found that although amnesia following the use of midazolam may be well documented by picture or object recall, incomplete amnesia was reported for operations. Thus, the effect of midazolam on the patient’s ability to forget pictures, objects, or a list of words cannot be directly related to clinical events . It has The most important factor, which made meta-analyses impossible in our review, is that many anxiety scales were used. Data acquired from these scales could be either continuous variables or categorical variables, so it was difficult for us to merge them. In addition, some of the scales surveyed anxiety levels from the patients’ perspective, and some through the perspective of observers. Neither patients nor observers can accurately measure anxiety levels because of the influence of subjective factors. Two included studies ,17 introOur review had shortcomings, since meta-analyses could not be performed because of the severe heterogeneity of the articles studied. We will continue to pay close attention to further research about the anxiolytic effect of midazolam and update our results. Finally, we call on more researchers to focus on unifying the methods of measuring anxiety and to search for more accurate and quantitative methods.The ideal sedative drug, which we believe to be suitable for use as a local anesthetic, should be reliable for anti-anxiety and have minimal effects on circulation, respiration, and recovery. The selected studies provided evidence that midazolam is efficacious for anxiety control. To ensure that the anxiolytic process is safe, we must evaluate the general condition of patients. Midazolam may come into wider use not only as an anxiolytic drug in third molar extraction surgery, monitoring of respiratory function using capnography may further increase safety during midazolam use as a sedative-hypnotic.S1 PRISMA Checklist(PDF)Click here for additional data file.


RBP4) adipokine. This study aims to determine if genetic polymorphisms in RBP4 are associated with the risk of coronary artery disease (CAD) in Chinese patients. RBP4 polymorphisms were analyzed by high resolution melting (HRM) analysis in a case-control study of 392 unrelated CAD patients and 368 controls from China. The Gensini score was used to determine the severity of CAD. The genotypic and allelic frequencies of RBP4 single-nucleotide polymorphisms were evaluated for associations with CAD and severity of disease. The A allele frequency was significantly higher in CAD case groups compared to control groups (16.7% vs. 8.8%) at the RBP4 rs7094671 locus. Compared to the G allele, this allele was associated with a higher risk of CAD (OR = 2.07 (1.50–2.84)). Polymorphisms at rs7094671 were found to associate with CAD using either a dominant or recessive model . Adjusting for sex, history of smoking, serum TC, TG, LDL-c, and HDL-c, the risk of CAD for carriers remained significantly higher in both dominant and recessive models . However, this SNP was not significantly associated with severity of CAD using angiographic scores in multivariable linear regression models (p = 0.373). The RBP4 rs7094671 SNP is associated with CAD; however, our results do not indicate that this locus is associated with clinical severity of CAD or the extent of coronary lesions.Insulin resistance and obesity is influenced by the retinol binding protein 4 ( Coronary artery disease (CAD) is polygenic and the leading cause of morbidity and mortality worldwide . TraditiRBP4), as a contributor to insulin resistance [RBP4 expression and the development of insulin resistance. Conversely, when GLUT4 was overexpressed in adipocytes, RBP4 expression decreased and insulin sensitivity improved. The connection between RBP4 and insulin resistance was more specifically demonstrated with RBP4 transgenic mice and by administering recombinant protein to wild-type mice [RBP4 levels in the plasma of subjects with insulin resistance and a positive correlation between RBP4 expression and the magnitude of insulin resistance [RBP4 is causally involved in the origin of cardio-metabolic diseases [RBP4 level that was accompanied by decreases in levels of systemic inflammation and insulin resistance [In 2005, a study on the genetics of glucose transport protein 4 (GLUT4) identified a new type of adipokine, retinol-binding protein 4  risk factors including, metabolic syndrome [RBP4 levels have been positively associated with carotid intima-media thickness, metabolic complications, atherosclerosis [RBP4 and lower GLUT4 levels in epicardial and subcutaneous adipose tissue [RBP4 is associated with a three-fold increased risk of CAD in women [RBP4 may play a role in the development of CAD. The aim of this study was to determine whether there is an association between CAD and RBP4 genetic polymorphisms in a Chinese population. To test this hypothesis, we examined the association between SNPs within the RBP4 locus and angiographically-defined CAD severity. These results may be helpful for predicting the development of CAD and for implementing early-stage intervention strategies.In addition to its effects on insulin resistance, positive associations have also been documented between syndrome ,12,13, osyndrome , dyslipisyndrome , and infsyndrome . Moreoveclerosis . CAD is e tissue . Recentlin women . Therefovs. 29.6%) and be hypertensive (50.5% vs. 32.0%) compared to the control group.The demographic profile of subjects at the time of blood collection was comparable between the CAD and control groups . Subjectp = 0.09; control group, p = 0.25). This indicated that the case and control groups were representative of the population and had no selection bias. However, the genotype and allele frequencies at the rs13376835 locus did not conform to the Hardy-Weinberg equilibrium. There was no significant difference between the CAD and control groups in the allele or genotype frequencies at the rs12265684 locus (p > 0.05) (p < 0.001) but not in allele frequency between the CAD and control groups (p = 0.27). Significant differences were also found between groups at the rs7094671 locus for both genotype (p = 0.001) and C/A allele (p = 0.001) frequencies. For example, the frequency of the A allele was significantly higher in the case group than in the control group (16.7% vs. 8.8%). Therefore, we can conclude that the A allele at rs7094671 is associated with a higher risk of CAD compared to the G allele (OR = 2.07 (1.50–2.84) (We estimated linkage disequilibrium (LD) among the three variants by using SHESIS software  see Fig. The SNP > 0.05) . At the 50–2.84) .Compared with the G allele at the rs7094671 locus, a significant increased risk of CAD was associated with the A allele using both dominant and regression models . Multivariate logistic regression was used to further analyze the data by adjusting for gender, sex, history of smoking, serum TC, serum TG, LDL-c levels, and HDL-c levels. After making these adjustments, there was still a significant difference between rs7094671 polymorphic genotypes related to CAD. The risk of CAD for A allele carriers remained high in both dominant and recessive models  .p = 0.373)  .RBP4 and CAD. We found that subjects in the CAD patient group were more likely to be homozygous for allele A at the RBP4 rs7094671 locus compared to a control group. In addition, the frequency of the A allele is higher in the CAD group than the control group. According to these results, we conclude that the homozygous AA genotype at the RBP4 rs7094671 locus is associated with an increased risk of CAD in Chinese populations. While there are records of a GG genotype at the rs13376835 locus in other Chinese population studies, we did not identify any in this study. The sample size and the geographical factors of the subjects in the study may explain why this genotype was not found. The subjects in this study were predominantly from southwestern China, whereas other studies have been based on subject groups from different geographical locations.In this study, three SNPs were examined in 760 Chinese subjects to reveal any associations between the gene encoding RBP4 gene is located on chromosome 10 (10q23–q24) near a region linked to increased fasting glucose levels in European Caucasians [RBP4 is expressed in mature adipocytes. RBP4 is a retinol (vitamin A) transport protein, and recently Yang et al. found that it can also contribute to systemic insulin resistance [RBP4 levels in serum and adipose tissue strongly correlate with subclinical inflammation and pro-inflammatory cytokine levels [RBP4 impairs insulin signaling in adipocytes indirectly by inducing pro-inflammatory cytokine production from macrophages through Toll-like receptor 4 (TLR4) and c-Jun N-terminal protein kinase (JNK) pathways [RBP4/retinol complex stimulates JAK2/STAT5 signaling and expression of the suppressor of cytokine signaling 3, which is implicated in insulin resistance [RBP4 levels cause insulin resistance, in part by activating adipose tissue APCs that induce Th1 polarization and inflammation, and RBP4-induced activation of macrophages and antigen presentation to CD4T Cells are partially dependent on JNK signaling, resulting in increased levels of TNF and IFN-γ and further increase inflammation [RBP4 was also found to induce in vitro inflammation in endothelial cells, by stimulating expression of pro-inflammatory molecules, such as vascular cell adhesion molecule 1 (VCAM-1), E-selectin, intercellular adhesion molecule 1 (ICAM-1), monocyte chemoattractant protein 1 (MCP-1), and interleukin-6 (IL-6) [RBP4 levels independently predicted early endothelial dysfunction, linking adipose tissue inflammation and subclinical atherosclerosis [RBP4 expression can directly contribute to endothelial inflammation and may influence the development or progression of vascular inflammation during cardiovascular disease and diabetes. RBP4 has been recently identified as an HDL associated protein; it is demonstrated that in patients with acute coronary syndrome [RBP4 levels promote the development of atherosclerosis. In clinical studies, RBP4 serum concentrations have been shown to be associated with increased levels of systolic blood pressure. Increased serum RBP4 levels positively correlated with body mass index (BMI) in obese non-diabetic and diabetic subjects. Several RBP4 gene variants have been identified that associate with the accumulation of adipose tissue, insulin resistance, and T2D [RBP4 associated with CAD.The ucasians  and typeucasians . The genme 10 10q–q24 nearpathways . In addisistance . Increasammation . RBP4 wa6 (IL-6) . RBP4 leclerosis . A recenclerosis . Its levclerosis . These ssyndrome . It may syndrome . These f and T2D . This stFrom July 2006 to July 2014, 392 patients (ages 51.9 ± 12.6 years for women and 54.1 ± 9.2 years for men) admitted to West China Hospital were enrolled the study following a coronary angiography. The diagnosis of CAD was established by coronary angiography: >70% luminal stenosis of one major coronary artery or >50% of the left main coronary artery was considered positive for CAD. The control group consisted of 368 individuals who had no significant coronary obstruction by coronary angiography . Patients were excluded if they had diabetes mellitus (DM), active inflammatory conditions, an autoimmune disease, malignancies, a known hematological disorder, or used immunosuppressive drugs. Medical history, physical examinations, and clinical laboratory tests were assessed prior to enrolment. Hypertension was defined as SBP ≥140 mmHg or DBP ≥90 mmHg, or the patient was on antihypertensive medication. DM was diagnosed according to one of the following criteria: (1) a history of diagnosed DM and treatment with medications, diet, and/or exercise; (2) non-fasting blood glucose of >11.1 mmol/L; and (3) current use of hypoglycemic therapy to control DM. The study was approved by the Ethics Committee of West China Hospital of Sichuan University. All patients signed an informed consent prior to their inclusion in the study.We utilized Gensini’s scoring system : angiogrRBP4 genetic locus. Common variants  ≥ 0.2) with r2 ≥ 0.8 in the Hap Map CHB reference panel were selected. We selected three SNPs from the intron of RBP4: rs12265684, rs7094671, and rs13376835. Genotyping was performed by high-resolution melt (HRM) analysis, and the primer sequences used are described in 2, 1.2 μL Master Mix . Thermo cycling for PCR included one denaturation cycle at 95 °C for 10 min followed by 55 cycles consisting of denaturation at 95 °C for 15 s, annealing at 61 °C for 20 s and extension at 72 °C for 30 s. After the amplification phase, PCR products were denatured at 95 °C for 1 min and cooled to 40 °C for 1 min to form double-stranded DNA. Then, the HRM was performed by gradually increasing the temperature from 65 to 95 °C at a rate of 0.01 °C/s. Melting of the PCR products was monitored by plotting the change in fluorescence that occurred when a double-stranded DNA binding dye was released. The collected data were analyzed using the LightCycler 480 II Gene Scanning software v2.0 .Patient blood samples (approximately 5 mL) were drawn directly from the arterial sheath before diagnostic angiography. Genomic DNA was extracted from whole-blood specimens using a commercially available DNA extraction kit  according to the manufacturer’s instructions. We utilized the Hap Map database  to identContinuous variables are expressed as the mean ± standard deviation (SD), and categorical variables are reported as counts and percentages. To test for differences between groups, analysis of variance (ANOVA) and chi-squared tests were used for continuous and categorical variables, respectively. Logistic regression multivariate analyses were performed to evaluate the odds ratio (OR) for CAD after adjusting for other conventional risk factors. Variables were tested for normality with Kolmogorov-Smirnov statistics and  transformed for purposes of parametric analyses. Reverse log-transformation was applied to obtain clinically interpretable values. Linear regression models were constructed to test the additive effect of the SNP on CAD phenotypes including severity.RBP4 rs7094671 locus is associated with CAD in the Han Chinese population. However, the rs7094671 SNP at this RBP4 risk locus is not associated with the severity of CAD in a population undergoing coronary angiography.In conclusion, this study demonstrates that A/A genotype at the


In vitro and in vivo experiments demonstrated that the increased MDSC induced by tumor-derived G-CSF is involved in the development of chemoresistance. The depletion of MDSC via splenectomy or the administration of anti-Gr-1 antibody sensitized G-CSF-producing cervical cancer to cisplatin. In conclusion, tumor G-CSF expression is an indicator of an extremely poor prognosis in cervical cancer patients that are treated with chemotherapy. Combining MDSC-targeting treatments with current standard chemotherapies might have therapeutic efficacy as a treatment for G-CSF-producing cervical cancer.Granulocyte-colony stimulating factor (G-CSF) producing malignant tumor has been reported to occur in various organs, and has been associated with poor clinical outcome. The aim of this study is to investigate the significance of tumor G-CSF expression in the chemosensitivity of uterine cervical cancer. The clinical data of recurrent or advanced cervical cancer patients who were treated with platinum-based chemotherapy were analyzed. Clinical samples, cervical cancer cell lines, and a mouse model of cervical cancer were employed to examine the mechanisms responsible for the development of chemoresistance in G-CSF-producing cervical cancer, focusing on myeloid-derived suppressor cells (MDSC). As a result, the tumor G-CSF expression was significantly associated with increased MDSC frequencies and compromised survival.  Cervical cancer, which has an annual global incidence of 530,000 new cases, is the second most common type of cancer affecting women worldwideChemotherapy is the main treatment for patients with recurrent or advanced cervical cancer, except for those who are amendable to surgical resection or salvage radiotherapy. Based on phase III trials conducted in the past few decades, platinum-based combination chemotherapies including cisplatin/paclitaxel and carboplatin/paclitaxel have become the standard regimens for recurrent or advanced cervical cancer33Tumor-related leukocytosis (TRL) is a paraneoplastic syndrome that is occasionally encountered in patients with malignant tumors (either at diagnosis or during the course of the disease), especially in those with advanced-stage disease69in vitro and in vivo experimental models we proposed novel treatment strategies for overcoming the chemoresistance of G-CSF-producing cervical cancer.In the current study, we examined the prognostic significance of tumor G-CSF expression in patients with recurrent or metastatic cervical cancer that had been treated with platinum-based chemotherapy. Moreover, after investigating the underlying causative mechanism in A total of 82 cervical cancer patients who had been treated with platinum-based chemotherapy were included in the current study. Clinicopathological characteristics of these patients are shown in in vitro and in vivo  frequencies in their bone marrow, blood, spleens, and tumors compared with the ME180-control-derived tumor-bearing mice - Signal transducer and activator of transcription 3 (Stat3) pathwayWe next investigated the mechanism by which MDSC impair the efficacy of chemotherapy with focuses on the tumor-angiogenesis. As an unregulated tumor-angiogenesis is known to increase the interstitial fluid pressure and reduces the chemosensitivity of human malignancies including uterine cervical cancer14Collectively, these results suggest that tumor-derived G-CSF is involved in the cisplatin resistance in cervical cancer by inducing the production of MDSC, prolonging their survival, and increasing their activities against the cancer cells and the cancer microenvironment .+ CD33+ HLA-DR− cells. In contrast, we only detected very low numbers of MDSC in the blood of the healthy donors and the cervical cancer patients with normal serum G-CSF levels . Consistent with the findings obtained in mice, the peripheral blood of the cervical cancer patients with elevated serum G-CSF levels contained significantly higher numbers of MDSC; i.e., CD11bF levels .in vivo. As shown, splenectomy  and related transfectants. As Stone et al. reported HPV positive cervical cancer cell lines expressed significantly higher levels of cytokines than HPV negative cellsWe also found that treatment with anti-Gr-1-neutralizing antibodies enhanced the sensitivity of G-CSF-producing cervical cancer to cisplatin . In precG-CSF is widely used during chemotherapy to reduce the risk of chemotherapy-induced neutropenia and facilitate the delivery of maximally effective doses of cytotoxic anti-cancer agents. According to previous investigations, the use of G-CSF does not have a negative impact on the survival of patients with lung cancerIn conclusion, we have shown that tumor G-CSF expression is an independent poor prognostic factor in cervical cancer patients that are treated with platinum-based chemotherapy. Moreover, the increased accumulation of MDSC in tumors induced by tumor-derived G-CSF is responsible for the development of cisplatin resistance in G-CSF-producing cervical cancer. We intend to prospectively evaluate our clinical findings and mechanistic hypothesis in a collaborative multi-institutional investigation involving G-CSF-producing cervical cancer patients in future.Permission to proceed with the data acquisition and analysis was obtained from Osaka University Hospital’s institutional review board (IRB). A list of patients who were treated with platinum-based chemotherapy for recurrent or advanced cervical cancer at Osaka University Hospital from April 1996 to March 2011 was generated from our institutional tumor registry, and their clinical data were retrospectively analyzed. Cervical tumor tissue and blood samples were also collected and archived according to protocols approved by the IRB of Osaka University Hospital. All experiments were performed in accordance with guidelines and regulations approved by the IRB of Osaka University Hospital. Appropriate informed consent was obtained from each patient.PFS was measured as the time from the diagnosis of recurrence or advanced disease to disease progression. OS was defined as the time from the diagnosis of recurrence or advanced disease to death or the last observation. The treatment-free interval was defined as the time from the end of the primary treatment to the detection of recurrence.The response to treatment was assessed according to the Response Evaluation Criteria in Solid Tumors after every 3 cycles of each regimen. A CR was defined as the disappearance of all target and non-target lesions and the absence of new lesions on two consecutive assessments performed at least 4 weeks apart. A PR was defined as at least a 30% reduction in the sum of the longest dimensions of the target lesions on two consecutive assessments performed at least 4 weeks apart. Progressive disease was defined as a 20% increase in the sum of the longest dimensions of the target lesions or the development of new lesions. Stable disease implies that none of the above applies. The response rate was defined as the percentage of complete and partial responders.705) and β-actin  were used for the Western blotting analysis. A neutralizing antibody against Gr-1 (RB6-8C5) was purchased from BioXCell . A neutralizing antibody against G-CSF and recombinant human G-CSF were kindly provided by Kyowa Hakko Kirin, Co., Ltd. . G-418 was purchased from Life Technology . Cisplatin was purchased from Sigma .The following labeled monoclonal antibodies were used for the staining experiments: anti-human antibodies: V450-conjugated anti-CD33  and APC-conjugated anti-HLA-DR ; anti-human/mouse antibodies: FITC-conjugated anti-CD11b ; anti-mouse antibodies: PE-conjugated anti-Gr-1 , APC-conjugated anti-Ly6G, and PE-conjugated anti-Ly6C . Antibodies against the G-CSF receptor (G-CSFR), Stat3, phospho-Stat3  supplemented with 10% FCS.The expression vector for the mouse G-CSF gene (pCAmG-CSF) and the empty vector (pCAZ 2) used in this study, which were described previously34The G-CSF concentrations of the serum samples were determined by enzyme-linked immunosorbent assay (ELISA) using Quantikine assay system for human G-CSF  according to the manufacturer’s protocol.RNA was extracted from cells using Trizol . The resultant total RNA (1 μg) was used to synthesize cDNA with ReverTraAce qPCR RT Master Mix . PCR was performed using Taq PCR master mix  and specific primers. Amplification was performed using a Takara PCR personal-type thermal cycler . The PCR primers were purchased from Life Technologies . The sequences of the primers used were as follows: β-actin: forward primer, 5′-CGTGACATTAAGGAGAAGCTGTG-3′ and reverse primer, 5′-GCTCAGGAGGAGCAATGATCTTGA-3′; G-CSF: forward primer, 5′-TGAGTGTGCCACCTACAAGC-3′ and reverse primer, 5′-GACACCTCCAGGAAGCTCTG-3′; G-CSFR: forward primer, 5′-ACAAGCCGCAGCGTGGAGAAG-3′ and reverse primer, 5′-TTCTGAAGGCAGGTGGAAGGTG-3′.Real-time RT-PCR was performed using SYBR Green PCR master mix  on a Step One Plus sequence detection system . Relative mRNA expression fold values were determined using standard deltaCt calculations. The PCR primers for GAPDH were purchased from Invitrogen , and those for Bv8 were purchased from Eurofins Operon . The sequences of the primers used were as follows: GAPDH: forward primer, 5′-CCCTCAAGATTGTCAGCAATGC-3′ and reverse primer, 5′-GTCCTCAGTGTAGCCCAGGAT-3′; Bv8: forward primer, 5′-GCATGACAGGAGTCATCATTTT-3′ and reverse primer, 5′-AAATGGCAGGATATCAGGAAA-3′.The cells were lysed for 10 minutes at 4 °C. Equal amounts of protein were separated by SDS-PAGE and transferred to PVDF membranes. Western blot analyses were conducted using various specific primary antibodies. The resultant immunoblots were visualized with horseradish peroxidase (HRP)–coupled immunoglobulins using an enhanced chemiluminescence Western blotting system .Tumor samples were fixed in 10% neutral buffered formalin, embedded in paraffin, sectioned, and processed for immunohistochemical staining. The primary antibody used was anti-G-CSF polyclonal antibody (N-20) . The surrounding non-neoplastic stroma served as an internal negative control for each slide, as reported previously6The MTS assay was used to analyze the effects of cisplatin. Cervical cancer cells were plated in 96-well plates and exposed to cisplatin at different concentrations in the presence of 10% FCS. After 72 hours’ incubation, the number of surviving cells was assessed by determining the absorbance of the dissolved formazan product at 490 nm, as described by the manufacturer . Cell viability is expressed as follows: absorbance of the experimental group/ absorbance of the control group.in vivo anti-tumor activity of cisplatin and anti-Gr-1-neutralizing antibody five- to seven-week-old BALB/c nude mice were subcutaneously inoculated with 5 × 106 ME180-control or ME180-G-CSF cells in 100 μL of phosphate-buffered saline (PBS). Cisplatin was administered weekly at a dose of 4 mg/kg after the tumors reached about 50 mm3 in volume. Anti-Gr-1 antibody was given from the first day of cisplatin administration at a dose of 200 μg/mouse every 48 hours. Both cisplatin and anti-Gr-1 antibody were administered intraperitoneally. The splenectomy procedures were performed as reported previouslyAll of the procedures involving animals and their care were approved by the animal care and usage committee of Osaka University, in accordance with institutional and NIH guidelines. To examine the + Gr-1+ cells was >95%MDSC were isolated from single-cell preparations of mouse splenocytes using the MDSC isolation kit (mouse) and an MS column , according to the manufacturer’s instructions. The purity of the isolated cell populations was determined by flow cytometry, and the frequency of CD11bTo assess the lifespan of the MDSC, cells were stained with propidium iodide and annexin V using the annexin V-FITC apoptosis detection kit , according to the manufacturer’s instructions. Fluorescence data were collected using flow cytometry.Single cell suspensions were prepared from mouse bone marrow, blood, spleen, and tumor specimens. RBCs were removed using ammonium chloride lysis buffer. To prepare human samples, peripheral blood mononuclear cells (PBMC) were separated by gradient centrifugation using Lymphoprep . Then, cells were filtered through 40 μm nylon strainers, incubated with antibodies, and analyzed by flow cytometry. Flow cytometric data were acquired on FACSCanto II flow cytometer and analyzed using FACSDiva software . Cells that had been incubated with irrelevant isotype-matched antibodies and unstained cells served as controls.A 96-well plate was coated with 1 μg/well anti-CD3e antibody . CD8 positive T cells were purified from the spleen of Balb/c mice using T cell isolation columns  according to manufacturer’s instructions. To determine the impact of MDSC on T cell proliferation, purified MDSC from the spleen of a G-CSF treated mouse were co-cultured with T cells. Cell proliferation was assayed using a cell proliferation ELISA BrdU kit .t test or Wilcoxon rank sum test. Frequency counts and proportions were compared between groups using the chi-square test or a two-tailed Fisher’s exact test, as appropriate. We compared the Kaplan–Meier curves for each subgroup with the log rank test. Cox proportional hazards regression analysis with stepwise variable selection was performed to identify significant independent predictors of OS. P-values of <0.05 were considered statistically significant. All analyses were performed with SAS version 9.1 for Windows .Continuous data were compared between groups using the Student’s How to cite this article: Kawano, M. et al. The significance of G-CSF expression and myeloid-derived suppressor cells in the chemoresistance of uterine cervical cancer. Sci. Rep.5, 18217; doi: 10.1038/srep18217 (2015).


Different human retroviruses, such as Human Immunodeficiency Virus (HIV) and Human T-cell Lymphotropic Virus (HTLV), can cause neurologic infection. However, a definitive diagnosis may be hampered by several factors. Quantification of the viral or proviral load in cerebrospinal fluid (CSF) may be helpful in the diagnosis of nervous system disorders due to retroviral infection and may influence the treatment approach. The present work discusses retrovirus infection and neurologic impairment, as well as the usefulness of the determination of the HIV and HTLV proviral or viral load in cerebrospinal fluid in cases of neurologic disorder, in light of recent advances in this field. This study also discusses the different molecular techniques for quantifying the proviral load  that are currently available. Retroviridae family comprises enveloped RNA viruses that can infect several hosts. The viral particle presents the enzymes reverse transcriptase and integrase. Thus, after the reverse transcription, the proviral DNA can be integrated into host cell genome [The l genome . In thisl genome –5. Humanl genome –12. Howel genome , 13–16. l genome –22.In the current study, we discuss the usefulness of HIV and HTLV-1 quantification in CSF among patients with neurologic disorders due to retrovirus infection, on the basis of the recent knowledge achieved in this field.HTLV-1 can successfully infect the central nervous system (CNS). Although the great majority of HTLV-1 infected individuals remain asymptomatic throughout their lifetime, about 5 % may present a chronic, incapacitating neurologic disorder , 23, 24.Regarding HIV infection, severe neurocognitive conditions, usually resulting in death, have been detected in HIV-infected individuals since the beginning of the HIV epidemic , 26. ThiAccording to WHO guidelines for HAM/TSP diagnosis, a patient can be classified under definitive HAM/TSP in the presence of chronic progressive spastic paraparesis associated with antibodies detection in both blood and CSF. Some individuals, however, do not present antibodies in CSF or do not present the classical symptoms of disease, despite the presence of antibodies in both compartments. Such individuals are classified under probable HAM/TSP . MoreoveThe detection of HTLV-1 antibodies is done by screening (ELISA) and confirmatory tests (Western blot). Some patients can present indeterminate or discordant Western blot results , 33. TheRegarding the PVL in CSF, patients with HAM/TSP were found to present a higher PVL compared with asymptomatic HTLV-1–infected individuals , 21, 37.Therefore, the quantification of the PVL in CSF may be a good marker for HAM/TSP diagnosis, mainly when associated with other tests, such as HTLV-1 AI.The determination of the HIV viral load in blood is widely used to assess the disease progression and the response to antiretroviral therapy. As previously mentioned, HIV can infect the CNS, and HAND can be observed even in patients with adequate control of viral replication in plasma , 22, 39.HIV presents a high mutation rate, which can result in the coexistence of viral quasispecies within a host. These quasispecies evolve as a result of selection pressures, such as those imposed by antiviral therapy, and can be restricted to cells or tissues  . PreviouThus, several studies have shown that HIV can escape in CSF despite viral suppression in blood , 22, 43.These data highlight the importance of the quantification of the proviral or viral load in CSF among HIV-infected patients presenting neurologic disturbance and/or drug resistance. In such cases, genotyping tests of CSF virus are extremely important. In fact, some studies have reported successful genotyping in CSF in cases in which it was not possible to apply genotyping in blood .Previous studies have reported the effect of antiretroviral treatment on the HIV proviral load. In particular, the proviral load in CSF was shown to decrease after the implementation of a therapeutic regimen with drugs with improved CNS penetration .Indeed, the drug concentration in the CNS seems to be crucial for viral control in the nervous system. A recent study tested two different therapeutic regimens with distinct concentrations of darunavir. Only two patients presented detectable viral genome in CSF, and those two had the lower concentration of the drug in CSF despite the prescribed dosage of orally administered drug, confirming that CSF assessment is an important issue .Some drugs used as monotherapy, such as lopinavir/ritonavir and zidovudine, had a significant effect on decreasing CSF replication. In contrast, didanosine and saquinavir were not able to control viral replication in CSF. Abacavir was also tested as an adjunctive therapy in patients presenting HAD, with no effect in CSF HIV RNA. In this context, a CNS penetration-effectiveness (CPE) score was proposed. According to the CPE, the antiretrovirals are scored from 1 to 4 (with 4 indicating the most neuro-effective drug). This is determined based on the drug characteristics, pharmacokinetic features, and pharmacodynamic properties. The composite CPE can be calculated by summing single drug scores to obtain the treatment score. Usually, a higher CPE score is associated with a lower viral load in CSF , 48.Another important point associated with the successful control of viral replication in CSF is related to viral resistance. Thus, the therapeutic regimen should be modified according to the results obtained by genotypic resistance testing in CSF samples.Regarding HTLV infection, the specific therapy for HAM/TSP remains very disappointing, and symptomatic treatment is still the most commonly used therapy . There aCurrently, quantitative real-time polymerase chain reaction (qPCR) and reverse transcription (RT)-qPCR are the standard and most commonly used techniques to determine the proviral or viral load , 49, 50.In this context, a novel technique, droplet digital PCR (ddPCR), can also be used for viral or proviral load quantification. The advantage of this technique is that it does not require a standard curve. In fact, ddPCR allows the direct absolute quantification of a target gene, which can positively influence the accuracy of the PVL quantification. In a recent study, ddPCR presented low intra- and inter-assay variability in the determination of the HTLV-1 PVL in CSF of infected patients. The same study concluded that, regarding the HTLV-1 proviral load in peripheral blood mononuclear cells (PBMCs), the inter-assay variability of ddPCR was lower compared with qPCR . It is iRegarding HIV infection, because some individuals can present viral replication suppressed below the detection limit of the available diagnostic methods , some auRegardless of the chosen technique, prior validation is essential, that is, before implementation in CSF routine analysis. Cross-validation by multiple laboratories is indicated.The determination of the viral or proviral load in CSF of HTLV-1– and HIV-infected individuals with neurologic disease may be a good marker for diagnosis, considering the proximity of the lesion and the local infection. The association of PVL measurement with other tests, such as PVL in blood and intrathecal synthesis of specific antibodies analysis in HTLV infection and HIV genotyping, might bring an important contribution to clinicians. In some cases, it can make the difference for a conclusive diagnosis or even to therapeutic approach. However, prior validation of molecular assays before implementation in laboratorial routine is essential, mainly regarding the CSF examination. Many laboratories use the same methodology applied in blood, forgetting that CSF is a body fluid with different constituents that may interfere in laboratory tests. Even greater emphasis should be given to HTLV, which, being a neglected disease, does not have approved commercial kits for use in CSF testing; thus, “in-house” tests are mandatorily used. In this case, the international standardization of tests would be beneficial for the acquisition of reliable and comparable results that would make possible a better clinical interpretation of the laboratory findings of patients.


Further CSF analyses, including polymerase chain reaction (PCR) and detection of intrathecal synthesis of antibodies, showed a VZV infection. Clinical and CSF follow-up examinations proved the successful antiviral treatment. In conclusion, even young immunocompetent patients without rash might present with VZV meningitis. CSF examination is a key procedure in the diagnosis of CNS infections but in rare cases the standard values cell count and total protein might misleadingly indicate a bacterial infection. Thus, virological analyses should be considered even when a bacterial infection is suspected.Meningitis caused by varicella zoster virus (VZV) is rare in healthy population. Predominantly immunocompromised patients are affected by reactivation of this virus with primary clinical features of rash and neurological symptoms. Here we report a young otherwise healthy man diagnosed with a VZV meningitis without rash. He complained of acute headache, nausea, and vomiting. The clinical examination did not show any neurological deficits or rash. Cerebrospinal fluid (CSF) analysis revealed a high leukocyte cell count of 1720 cells/ Infections with neurotropic herpes viruses ) are frequent in humans. These viruses persist within cranial nerves, dorsal roots, and autonomic ganglia causing latent infections with the ability of reactivation . ReactivAcute infection or VZV reactivation affects predominantly older individuals and/or immunocompromised patients –3, 9. CNAn 18-year-old man experienced severe occipital headache accompanied by nausea and vomiting. All symptoms started immediately after a backward roll doing judo exercise. Treatment with paracetamol, acetylsalicylic acid, and metamizole did not show beneficial effects. Thus, he presented to our emergency room nine days after the first symptoms.The physical examination was normal. Particularly, stiff neck as a typical sign of meningitis was not found and he did not show any rash. He had never been sick before and did not take any medications regularly. Furthermore, he did not smoke or drink alcohol excessively.Subarachnoid hemorrhage or dissection of cerebral arteries was first considered. Magnetic resonance imaging (MRI) scan of the brain showed no abnormalities such as bleeding, infarction, or malignancy. Additional MR-angiography and ultrasound examination of carotid, vertebral, and intracranial arteries revealed no vascular alterations. Measurements of body temperature and blood pressure as well as laboratory urine and blood examinations , anti-nuclear antibodies, anti-neutrophil cytoplasmic antibodies, anti-cardiolipin antibodies, angiotensin-converting enzyme, and amount of vitamins B12, B6, and B1) showed normal results.μL and an elevated total CSF protein of 1460 mg/L. Furthermore, slightly elevated CSF lactate concentration of 3.0 mmol/L and a slightly reduced CSF glucose concentration of 42 mg/dL were found. Considering a bacterial infection, intravenous treatment with ceftriaxone (2 g/day) and ampicillin (15 g/day) was applied.CSF analysis revealed a high leukocyte cell count of 1720 cells/Cytologic analysis of the CSF revealed 3% plasma cells. The remaining cells were predominantly lymphocytes with normal morphology. Further CSF abnormalities included elevated albumin quotient (QAlb) indicating a moderate blood-CSF-barrier-dysfunction . Intrath Borrelia burgdorferi, Treponema pallidum, Mycobacterium tuberculosis, toxoplasmosis, Candida, and Cryptococcus neoformans showed negative results. Virological examinations presented negative results for herpes simplex virus, cytomegalovirus, Epstein-Barr virus, enteroviruses, tick-borne encephalitis, human immunodeficiency virus, and hepatitides B, C, and A. Quantitative real-time PCR analysis [Microbiological analyses did not detect any bacterial infection. Further analyses foranalysis  of the Canalysis  of VZV iμL was still found. Plasma cells were not found and lymphocytes and monocytes showed normal morphology. VZV-PCR was negative but a persisting intrathecal IgG production to VZV (specific antibody index of 21.5) indicated a preceding VZV infection. The patient felt well without any symptoms. He practiced judo again four times per week.Eleven days after intravenous therapy, the patient was discharged feeling well. Follow-up CSF examination was performed 23 days after the end of antiviral treatment. A slight pleocytosis with 19 cells/Here we present a young previously healthy man with a VZV meningitis without rash. This case is extraordinary because the clinical presentation was unusual for a patient with meningitis and the initial CSF findings with very high pleocytosis and elevated total CSF protein initially misleadingly suggested a bacterial infection. Interestingly, further CSF examinations detected a VZV infection.μL, mild to moderately elevated total protein, and normal lactate levels [μL, 132/μL, 286/μL, and 293/μL were found and the cell counts ranged from 15 to 840 cells/μL [μL) that has been described for this group of patients. In addition, total protein and lactate concentration were elevated leading to a misleading diagnosis of bacterial meningitis.Our case underlines the importance of specialised CSF diagnostics in acute neurological emergency situations. CSF examination is generally considered a key procedure in the diagnosis of CNS infections . Using se levels –18. In pcells/μL –18. In oIn conclusion, even young and previously healthy patients without clinical features of dermal irritation such as rash might present with VZV meningitis. We highlight the importance of considering VZV as a possible cause for meningitis even in previously healthy young patients and the recommended diagnostic lumbar puncture. Detailed CSF diagnostic procedures including PCR and detection of intrathecal synthesis of antiviral antibodies  should be considered even though CSF cell count and total protein seem to indicate a bacterial infection.


Potential biomarkers to aid diagnosis and therapy need to be identified for Amyotrophic Lateral Sclerosis, a progressive motor neuronal degenerative disorder. The present study was designed to identify the factor(s) which are differentially expressed in the cerebrospinal fluid (CSF) of patients with sporadic amyotrophic lateral sclerosis , and could be associated with the pathogenesis of this disease.Quantitative mass spectrometry of ALS-CSF and control-CSF  samples showed upregulation of 31 proteins in the ALS-CSF, amongst which a ten-fold increase in the levels of chitotriosidase-1 (CHIT-1) was seen compared to the controls. A seventeen-fold increase in the CHIT-1 levels was detected by ELISA, while a ten-fold elevated enzyme activity was also observed. Both these results confirmed the finding of LC-MS/MS. CHIT-1 was found to be expressed by the Iba-1 immunopositive microglia.Elevated CHIT-1 levels in the ALS-CSF suggest a definitive role for the enzyme in the disease pathogenesis. Its synthesis and release from microglia into the CSF may be an aligned event of neurodegeneration. Thus, high levels of CHIT-1 signify enhanced microglial activity which may exacerbate the process of neurodegeneration. In view of the multifold increase observed in ALS-CSF, it can serve as a potential CSF biomarker for the diagnosis of SALS. Selective loss of cortical and spinal motor neurons is the characteristic feature of Amyotrophic Lateral Sclerosis (ALS), an adult onset progressive fatal neurodegenerative disorder. Factors predisposing the most prominent form of this multifactorial disease viz. sporadic ALS (SALS) remain obscure due to the difficulties in developing animal models. Therefore, development of novel therapeutics is also severely hampered. This is also largely attributed to the lack of a ‘biomarker’ which can be objectively measured as an indicator of pathogenic processes and/or pharmacologic response to therapeutic interventions . The disin-vitro) and intrathecal injection of the same into neonatal rats (in-vivo) induced degenerative changes in motor neurons and showed the involvement of astrocytes [We have earlier shown that exposure of embryonic rat spinal cord cultures to ALS-CSF  in ALS-CSF through proteomic analysis. Quantitative mass spectrometric analysis of ALS-CSF compared to age-matched controls showed upregulation of 31 proteins, amongst which Chitotriosidase-1 (CHIT-1) showed more than 10 fold increase. The biological significance of CHIT-1 expression is intriguing in view of the absence of its natural substrate chitin in human brain. Earlier studies have shown an increase in CSF CHIT-1 levels in multiple sclerosis (MS) and Alzheimer’s disease (AD) ,17.The toxicity of the ALS-CSF samples was confirmed prior to proteomic analysis. Exposure of NSC-34 cells to the CSF obtained from ALS patients (ALS-CSF) resulted in a dramatic decrease in their viability when compared to the control groups; i.e. the cells treated with CSF from normal individuals (N-CSF) or without CSF (NC) . In the ALS-CSF, the level of CHIT-1 ranged between 5000 – 54,000 pg/ml, with a mean of 17570 ± 4883 pg/ml (n = 16). The mean increase in the ALS-CSF was approximately 17 fold  while osteopontin levels showed 1.9-fold increase in ALS-CSF compared to the control CSF   whereas in the ALS-CSF it was 0.0809 – 4.1658 μmol/min/μl . Thus the patient CSF samples revealed approximately a 10-fold higher enzymatic activity  (Tables in-vitro (DIV), the cultures were placed on incubated orbital shaker at 200 rpm for 3–4 hrs. The supernatant containing microglia was centrifuged at 1500 rpm for 5 min, and the pelleted cells were seeded onto poly-l-lysine coated coverslips at the density of 4.4×104 cells/ml. The cultures were maintained in DMEM with 10% FBS (GIBCO-BRL). The cells were then exposed to ALS- CSF or allowed to propagate under normal conditions.We followed a modified protocol to establish pure microglial cultures from P0 Wistar rat pups . BrieflyNSC-34 cell line  routinely maintained in DMEM with 10% FBS  was usedNSC-34 cells seeded into 96 well plates (500 cells/well) were exposed to 10% v/v N-CSF or ALS-CSF on 5th DIV for 48 hrs, and then subjected to MTT assay -2,5-diphenyltetrazolium bromide) and LDH assay .CSF samples from controls (n = 10) and ALS patients (n = 10) were pooled individually and used for iTRAQ based quantitative proteomic comparison by mass spectrometry Figure . SamplesProtein estimation was carried out using Lowry’s method. 200 μg protein each from controls and ALS was used for proteomic comparison. Sample normalization was based on total protein amount which was further verified by SDS-PAGE.Individual depleted extract (125 μg) was treated with 2% SDS and reducing agent  and alkylated with 1 μl of cysteine blocking agent for 10 min at RT. Proteins were digested overnight at 37°C using sequencing grade trypsin at 1:20 (w/w) ratio. Thereafter, peptides from N-CSF and ALS-CSF were labeled using iTRAQ reagents yielding reporter ions of m/z114 and 115 respectively, for 2 hrs at RT. The reaction was quenched by adding 100 μl of water and the peptides from both N-CSF and ALS-CSF were pooled and fractionated using strong cation exchange (SCX) chromatography. The labeled samples were distributed into 19 fractions using SCX. The fractions were desalted using C18 zip tips, dried and reconstituted in 10 μl of 0.1% trifluoroacetic acid before mass spectrometry analysis .LC-MS/MS analysis of iTRAQ-labeled peptides was carried out on an LTQ-Orbitrap Velos mass spectrometer  interfaced with Agilent’s 1200 series nanoflow liquid chromatography system . Each sample was loaded on to the enrichment column  at a flow rate of 3 μl/min and then resolved on an analytical column  at a flow rate of 300 nl/min using a linear gradient of 10% - 40% solvent B (90% acetonitrile in 0.1% TFA) over a period of 70 min. The total run time per sample was 115 min. The resolved peptides from analytical column were delivered to LTQ Orbitrap Velos mass spectrometer through an emitter tip . LC-MS/MS data was acquired in a data dependent manner in FT- FT mode. MS spectra were acquired with a mass range of m/z 350 to 1800. Twenty most abundant precursor ions were selected for fragmentation from each MS scan. Data was acquired at MS resolution of 60,000 (m/z 400) and MS/MS resolution of 15,000. Precursor ion fragmentation was carried out using higher energy collision (HCD) mode with normalized collision energy of 41%. Monoisotopic precursor selection was enabled and the precursor ions that were selected for fragmentation were dynamically excluded for 30 sec .The MS data was analyzed using the Proteome Discoverer software . The data was searched against human protein database (NCBI RefSeq 45) along with known contaminants using SEQUEST and MASCOT search algorithms. The parameters used for data analysis included trypsin as a protease , iTRAQ labeling at N-terminus and lysine residues, and cysteine modification by methyl methanethiosulfonate (MMTS) as fixed modifications and oxidation of methionine as a variable modification. The precursor ion mass error tolerance was set to 20 ppm and product ion mass error tolerance was set to 0.1 Da. The peptide data was extracted using 1% FDR as a threshold. Relative abundance of proteins between N-CSF and ALS-CSF was determined by Proteome Discoverer based on difference in the peak intensity of reporter ions in the MS/MS spectra of each peptide that was ultimately used for quantifying the corresponding protein.N-CSF (n = 13) and ALS-CSF samples (n = 16) were subjected to ELISA based analysis using commercially available kits for CHIT-1 : N-CSF: undiluted; ALS-CSF 1:20), CHI3L1 , CHI3L2  and Osteopontin . The protocol was followed as per the manufacturer’s instructions.2CO3 -NaHCO3 buffer (pH 10.7). The fluorescence was recorded at 365 nm excitation/450 nm emissions  and measured as micromoles of substrate hydrolysed/min/l.4-methylumbelliferyl-β - d N, N’, N” –triacetylchitotriose (Sigma-Aldrich USA) was used as a substrate to assay the enzyme activity. CSF (2.5 g protein) was added to 150 μl of 22 μmol solution prepared in 0.5 M citrate-phosphate buffer (pH 5.2). Following incubation for 15 min at 37°C, the reaction was stopped using 100 μl of 0.5 mol NaFixed primary microglial cultures were stained with rabbit polyclonal anti-CHIT-1 antibody  for 24 hr after blocking with 3% bovine serum albumin (BSA)  and detected using FITC-conjugated anti-rabbit secondary antibody . The cultures were further incubated with a goat polyclonal anti Iba-1 antibody  for 24 hr and detected with CY3-conjugated anti-goat antibody . The staining was viewed using confocal microscopy . The emission frequencies were segregated to avoid non-specific overlap of labelling .t test or one way ANOVA as applicable, followed by Tukey’s post hoc tests.The data was statistically assessed for significance by either Student’s SALS: Sporadic amyotrophic lateral sclerosis; ALS-CSF: Cerebrospinal fluid from amyotrophic lateral sclerosis patients; N-CSF: CSF from patients undergoing orthopaedic surgery; CHIT-1: Chitotriosidase-1; LC-MS/MS: Liquid chromatography-tandem mass spectrometry; Iba-1: Ionised calcium binding adapter molecule-1; GFAP: Glial fibrillary acidic protein; NSC-34: Spinal cord motor neurons fused with neuroblastoma; iTRAQ: Isobaric tags for relative and absolute quantitation; MARS-14: Agilent’s multiple affinity removal system.The authors declare that they have no competing interests.AMV collected samples, performed the experiments, analysed and wrote the manuscript. AS performed the experiments, analysed and wrote the manuscript. PM carried out microglial experiments, analysed and wrote the manuscript. VK analysed and wrote the manuscript. HHC supervised mass spectrometry, analysed and wrote the manuscript. TNS facilitated obtaining control CSF samples and critically evaluated manuscript. SB designed and supervised the experiments and critically reviewed the manuscript. NA enrolled patients with ALS, performed clinical evaluations and provided ALS-CSF. PAA designed, performed experiments, analyzed and wrote the manuscript. TRR conceptualized the project, obtained funding, supervised the study and critically reviewed the manuscript. All the authors read and approved the final manuscript.List of proteins identified in ALS-CSF. Description of Data: A list of the peptides and their fold changes of the 819 proteins identified in ALS-CSF using SEQUEST and Mascot.Click here for fileUp-regulated proteins in ALS-CSF. Description of data: Table showing 31 up-regulated proteins with more than 1.5-fold increase in ALS-CSF compared to normal CSF.Click here for fileProteins down-regulated in ALS-CSF. Description of Data: List of 17 down-regulated proteins which showed a decrease of 0.5 fold or more, in ALS-CSF samples.Click here for file