TNF G-308A, in sudden infant death syndrome (SIDS) infants, SIDS and control parents, and ethnic groups with different incidences of SIDS; (2) the effects of two risk factors for SIDS, cigarette smoke and virus infection, on TNF-α responses; and (3) to assess effects of genotype, cigarette smoke, and gender on TNF-α responses to bacterial toxins identified in SIDS infants. TNF G-308A genotypes were determined by real-time polymerase chain reaction for SIDS infants from Australia, Germany, and Hungary; parents of SIDS infants and their controls; and populations with high , medium (Caucasian), and low (Bangladeshi) SIDS incidences. Leukocytes from Caucasian donors were stimulated in vitro with endotoxin or toxic shock syndrome toxin-1 (TSST-1). TNF-α responses were measured by L929 bioassay (IU/ml) and assessed in relation to genotype, smoking status, and gender. There was a significantly higher proportion of the minor allele AA genotype among Australian SIDS infants  compared to 3/62 (4.8%) controls (p = 0.03). There were no significant differences in TNF-α responses by TNF G-308A genotypes when assessed in relation to smoking status or gender. Given the rarity of the TNF G-308A A allele in Caucasian populations, the finding that 24% of the Australian SIDS infants tested had this genotype requires further investigation and cautious interpretation. Although non-smokers with the AA genotype had higher TNFα responses to both TSST-1 and endotoxin, there were too few subjects with this rare allele to obtain statistically valid results. No effects of genotype, smoking, or gender were observed for TNF-α responses to these toxins.Dysregulation of the inflammatory responses has been suggested to contribute to the events leading to sudden infant deaths. Our objectives were (1) to analyze a single nucleotide polymorphism (SNP) associated with high levels of tumor necrosis factor-α (TNF-α) responses,  TNF G-308A, has been associated with severe responses to infection. Cytokine gene polymorphisms can significantly affect the level of these substances produced in response to infection  is a key pro-inflammatory cytokine which could play a role in several of the mechanisms proposed to explain sudden infant death syndrome (SIDS). In relation to the anaphylaxis hypothesis, TNF-α is stored in mast cell granules and is released on antigen stimulation. TNF-α also induces fever and affects respiration  and can nfection . In vitresponses  and indiesponses . The molesponses .TNF G-308A SNP indicate the AA genotype carried an increased risk of death from cerebral malaria , moderate (Caucasian), or high  incidences of SIDS.Reports on the  malaria , and dea malaria . Two stu malaria , 12. BecStaphylococcus aureus  to assess effects of virus infection and cigarette smoke on TNF-α responses; and (3) to assess the effect of gender, TNF G-308A, and cigarette smoke on responses to bacterial antigens (LPS and TSST-1) identified in SIDS infants.The objectives of the study were (1) to analyze the distribution of Approval for the study was obtained from the Lothian Health Ethics Committee (UK), Hunter Area Research Ethics Committee  and the University of Newcastle Human Research Ethics Committee .n = 34) and Australia (n = 60) and a comparison group with no family history of sudden infant deaths . Paraffin-fixed samples of tissue from SIDS infants were obtained from Australia (n = 25), Hungary (n = 18), and Germany (n = 46). Stored frozen blood samples from Aboriginal Australians (n = 117) and buccal epithelial cells from Bangladeshi donors (n = 32) were used as sources of DNA for comparisons among ethnic groups. The methods for extraction of DNA from the samples have been described previously  genotype was determined by a commercial allelic discrimination polymerase chain reaction (PCR) assay (Assay ID: C___7514879_10) (PE Applied Biosystems). Primers and probes were provided in a 20× assay mix, sequences and concentrations of which are unknown.Each PCR reaction contained 10 ng of sample DNA, 1× Assay mix, and 1× TaqMan Universal PCR Master Mix (PE Applied Biosystems) made up to a final volume of 5 μl with sterilized MilliQ water. PCR was performed using the ABI PRISM 7900HT sequence detection system (PE Applied Biosystems) at the following thermal cycling conditions: 50°C for 2 min; 95°C for 10 min; 92°C for 15 s; and 60°C for 1 min, for 40 cycles.2) test or Fisher’s Exact test, if appropriate, was used to assess the distribution of TNF G-308A in SIDS infants, parents of SIDS infants, and between ethnic groups. Genotype distribution from the Hardy–Weinberg equilibrium (HWE) was assessed using the χ2 test.Data were analyzed using the statistical software package Statistics/Data Analysis™ (STATA) Version 8.0 . The Chi-square (χTumor necrosis factor-α responses of the THP-1 human monocytic cell line were measured as previously described in the model system to assess the effects of surrogates for infection (IFN-γ) and exposure to CSE on cells with a uniform genetic background .in vitro cytokine stimulation assays and plasma was collected for the assessment of cotinine for evidence of exposure to cigarette smoke, a confounding variable for altered cytokine responses. TNF G-308A genotype was determined as described above. Donors with detectable levels of cotinine were excluded from the analysis. Only ARCBS donor samples that were cleared for infectious agents were received. Leukocytes were collected and stored as described previously  have been described previously. Buffy coats from 14 male and 14 female donors, aged 20–55 years, were purchased from the Australian red cross blood service (ARCBS) . Ethical permission was obtained from University of Newcastle Human Research Ethics Committee (H-229–0606) and ARCBS Ethics Committee (07-11NSW-07) for the purchase and use of human buffy coats for the purposes of the study. PBMC were collected from each donor for eviously .n = 34) and a comparison group, individuals unrelated to the families and who had no family history of SIDS (n = 59). DNA was extracted from the leukocytes and screened for the TNF G-308A alleles as described above. The leukocytes were assessed for inflammatory responses to TSST-1 and LPS as described previously. All samples were coded and tested without knowing the sex or smoking status of the donors. Leukocytes (1 × 106 cells ml−1) were stimulated in vitro with 0.01 μg ml−1 or 1 μg ml−1Escherichia coli LPS or 0.5 μg ml−1 TSST-1  for 24 h. Cell culture conditions have been described previously  collected from British parents of SIDS infants  and German  (p < 0.01) SIDS infants.The majority of each SIDS group possessed the GG genotype; however, Australian SIDS infants had a significant increase in proportion with the AA genotype  compared to the Hungarian  (p = 0.02). Only 3/62 (5%) of controls had the AA genotype compared with 24% of SIDS infants. No significant differences were detected between the distribution of TNF G-308A genotype for the combined SIDS infant group compared to the Caucasian controls (p = 0.41).The distribution of allele frequencies for the Australian control population differed significantly from that observed for Australian SIDS infants . Parents of SIDS infants recruited from Britain had a genotype distribution similar to their control population; the majority of individuals had the GA genotype (p = 0.89).The distribution of TNF G-308A varied significantly among individuals from different ethnic groups. The majority of both British and Australian control populations possessed the GG genotype with fewer than 5% of individuals possessing the AA genotype.The distribution of p = 0.01) and Aboriginal Australians (p < 0.01). More than 90% of the Bangladeshi and Aboriginal Australian populations possessed the GG genotype, and 0% had the AA genotype.As there were no differences between the allele frequencies for British and Australian control populations, these data were combined for further comparison with the Bangladeshi and Aboriginal Australian populations. The distribution of allele frequencies differed significantly between the Caucasian group and both Bangladeshis (−1 (low) or 10 ng ml−1 (high) concentrations. Priming of THP-1 cells with INF-γ resulted in dose-dependent enhancement of TNF-α responses to LPS (50 ng ml−1) . As observed with THP-1 cells, CSE did not elicit TNF-α from PBMC. In contrast to the results obtained with THP-1 cells, the high dose of CSE significantly reduced TNF-α responses to LPS (p < 0.0001) .  compared with responses from cells of male donors (4621 pg ml−1) if both INF-γ and CSE were used to pre-treat cells prior to exposure to endotoxin (p = 0.014). There were no significant differences in TNF-α responses associated with genotypes GG (n = 15), GA (n = 10) or AA (n = 3) under any of the conditions tested. For donors with the GG genotype, there were higher responses to LPS from cells of female donors (n = 9) (1674 pg ml−1) than from cells of male donors (n = 6) (989 pg ml−1) (p = 0.03). While the responses from cells of females with the GA genotype (n = 2) were approximately twice that of those from cells of male donors (n = 8), the differences were not significant (p > 0.05). There were only three donors with the AA genotype and all were female.When assessed by gender of the donor, there were no significant differences between: TNF-α responses to LPS, INF-γ, or CSE; or combinations of LPS with CSE or LPS with INF-γ. There was a significant difference in TNF-α responses of cells from female donors (6510 pg mlTNF G-308A for smokers (n = 38) or non-smokers (n = 60). For the GG and GA genotypes, the geometric mean TNF-α responses were below 10 IU ml−1. For the three donors with the AA genotype, there was one smoker and two non-smokers. The responses of the smoker donor with the AA genotype were similar to those of the GG and GA donors (<10 IU ml−1). The response of the two donors who were not smokers was nearly 20 IU ml−1 for responses to TSST-1 and over 50 IU ml−1 for responses to the higher dose of LPS. There were not enough donors with the AA genotype for statistical analyses.This experiment utilized cells from the UK donors. There were no significant differences in the median TNF-α response to TSST-1 or LPS associated with different genotypes of TNF G-308A genotype AA in Australian SIDS infants. Given the rarity of the TNF G-308A A allele in the Caucasian population, it was an unusual observation that 24% of the Australian SIDS infants tested in this study were homozygous. The number of infants assessed in this study was small; therefore, data should be interpreted with caution. In the Norwegian study, 3% of SIDS infants, 5% of borderline SIDS, and 3% of infants who died of infection had the AA genotype; while none of the controls had this SNP genotype  or low (UK Bangladeshi) incidences of SIDS had similar genetic profiles.While the distribution of If the finding of a significant increase in the AA genotype associated with high TNF-α reflects a risk factor for SIDS, the findings need to be assessed in relation to the various hypotheses proposed to explain the physiological events leading to SIDS.Long QT interval is reported to be an important risk factor for SIDS based on a prospective study of a large group of infants . The resDysregulation of glucose metabolism was proposed as one mechanism triggering the physiological events leading to SIDS . Acute hThe A allele has been associated with increased production of TNF-α , 28. TheTNF G-308A genotype on TNF-α responses to TSST-1 or LPS in the different populations. Although the responses of the non-smokers with the AA genotype were higher than those observed for the other genotypes, there were too few donors with this genotype for statistical analyses. These studies need to be repeated with more samples from individuals with the rare genotype.Despite finding interactions with smoking and cytokine gene polymorphisms in our previous studies , this stIn the experiments with cells from healthy Australian blood donors which were controlled for exposure to cigarette smoke and infection, females generally had higher TNF-α responses than males . When asTNF G-308A genotype results indicated that for the Australian population, infants with the AA genotype might be at increased risk of SIDS. The study needs to be expanded and investigations need to include thorough investigation for microorganisms and their toxins as well as investigation of the genetics and cytokine responses. While the effects of genotype on TNF-α responses remain unclear in the models tested, the effects of INF-γ are unequivocal for both THP-1 cells and PBMC. The results indicate that virus infection might enhance TNF-α responses to minor bacterial infection. While exposure to cigarette smoke is a risk factor for SIDS, it does not appear to enhance TNF-α responses to LPS. For the PBMC studies, CSE reduced significantly the enhanced responses of IFN-γ primed cells to LPS.In conclusion, the Each of the authors made substantial contributions to the conception, design, analyses, and interpretations of the work. They assisted in preparing the article, critically assessed the final version and agree to be accountable for the accuracy and integrity of the work.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


The partogram is the most effective tool to use to monitor the progress of labour and complications associated with labour.The main objective of this study was to assess the knowledge level of midwives on the effective use of the partogram in monitoring the progress of labour in the Tamale Metropolis of Ghana.This study was a cross-sectional descriptive study conducted on 140 midwives selected using random sampling technique from March to July, 2011.The study revealed that all respondents had knowledge of the supposed use of the partogram. However, the study also identified inadequate knowledge on the proper use of the tool (as observation showed that some of the partogram sheets were inadequately filled). Inadequate knowledge, inadequate staffing (1.0 % of respondents) and extra workload on the few midwives (2.6 %) were some of the factors that militated against the effective use of the partogram. There were enough partogram sheets in the health facilities under study as 91.4 % of respondents had access to the partogram.Midwives in Ghana know about the supposed use of partogram as a monitoring tool for labour; however inadequate knowledge on the proper use of the tool and inadequate staffing militated against effective use. We recommended constant refresher training on the use of the partogram and also training of more midwives to enhance effective utilisation of the partogram. A considerable number of women suffer complications from labour and some of these complications result in maternal and infant mortality. According to recent global estimates, about 289, 000 women die annually from pregnancy related complication . Most ofThe partogram is a sigmoid curve tool that can be used to assess the progress of labour and to identify when intervention is necessary: It is a graphical record of cervical dilatation in centimetres against duration of labour in hours. Studies have shown that using the partogram can be highly effective in reducing complications from prolonged labour for the mother  and for the new-born , . In addiThe United Nations noted that the use of the Partogram ensured that, there is a comprehensive monitoring of the progress of labour as well as maternal and foetal wellbeing. This makes it possible to address in a timely manner the complications that arise . Using tGhana adopted the use of the WHO Partogram in labour wards throughout the country in 1990 with the intention of improving maternal health and reducing maternal and perinatal mortality . This waAlthough a number of studies have been conducted in the area of maternal and infant mortality, leading to the implementation of several interventions aimed at reducing the trends, several maternal and perinatal mortalities still occur as a result of obstructed labour. It is in this regard that, it is important to collect relevant data on knowledge and attitudes of midwives towards the effective use of the partogram. This study also sought to find out the factors that influence the effective use of the Partogram by midwives. It also provided a better understanding on the importance of using the Partogram effectively to monitor labour.The goal of this study was to assess the knowledge level and attitudes of midwives towards the effective use of the partogram in monitoring the progress of labour in Tamale Metropolis.The study design adopted is a descriptive study design that assessed the level of knowledge and implementation on the use of the partogram by midwives. The specific study design adopted was cross-sectional study.The total number of midwives within the Tamale Metropolis was two hundred and fifteen (215). The study was done among one hundred and forty midwives from three main hospitals in the Metropolis. The hospitals included; Tamale Teaching hospital, West Hospital, and Tamale Central hospital. Midwives were chosen because they are those who monitor labour and conduct deliveries for pregnant women in the hospitals. The partogram is a tool that helps them to assess whether labour is progressing normally or not, so that any necessary intervention could be taken.A sample of one hundred and forty midwives was selected using random sampling technique with a 95 % confidence interval and 5 % reliability. There was one hundred percent response rate to the questionnaire. Sample size was calculated using the formula by Glenn D. Israel.The study was mainly conducted using a structured questionnaire, however, interviews and direct observation of respondents provided validation of the data. A structured questionnaire was designed and administered to each respondent. The questionnaire included questions on socio-demographic characteristics, knowledge on the partogram, factors that influence the effective use of the partogram, availability of the partogram in the health facilities and number of deliveries conducted using the partogram.Data collected was entered into Microsoft Excel 2007 spread sheet and imported to SPSS  version 16 for analysis. Results were presented with simple statistical tools.In order to test for the validity and reliability of the research’s data collection methods, a pre-test of the questionnaire was carried out among five midwives of the Bolgatanga Municipal hospital. Their responses with regard to the questions asked enabled the researcher to re-phrase the questions’ sentences/language to enhance clarity and precision.Introductory letters with information sheets of the study was submitted to the Research Unit of the Tamale Teaching hospital, West and Central hospitals following which permission was granted for the study to be conducted.This study included midwives within the Tamale Teaching Hospital, West Hospital and Central Hospital. A total of one hundred and forty (140) midwives completed the research questionnaire. The results of the study indicate that, the majority of the midwives (54.3 %) were between the ages of 51–60 years whiles the least number of respondents were those between 20 and 30 years (8.6 %). The number of respondents who have been working for the past 21–30 years were 30, representing 21.4 % and those who have worked for 31 years and above were 14 (10.0 %). It was revealed that a considerable number of midwives (53.6 %) have access to in-service training on the use of the partogram once every six months whiles 20.7 % do so once in a year. However, 19 midwives out of the 140 (13.6 %) had not gone for in-service training during the period of their practice.Findings on the knowledge of midwives about the use of the partogram, indicated that all respondents have heard about the tool. Majority (76.4 %) further indicated that the partogram is a tool used to monitor the progress of labour while 15.7 % indicated that the partogram could both be used to monitor labour and to check maternal and foetal conditions. In addition to what was already mentioned, 7.9 % added that it could also be used to check for any abnormalities in the foetus. This study revealed that only 40.0 % of midwives responded correctly when they were to determine when to start plotting on the partogram. The remaining 60.0 % had varied responses that were not consistent with the time to start plotting on the partogram.X was used in contrast to 4.3 % who were of the view that it was plotted with the symbol O. Also, 92.9 % of the respondents demonstrated that, descent of the foetal head was plotted using the symbol O, whereas 7.1 % did not know what symbol was used.An analysis of the knowledge of midwives about symbols provided on the questionnaire of this study about cervical dilatation indicated that 95.7 % of the respondents agreed that the symbol There was varied access to the partogram as 91.4 % of the midwives had access to the partogram. Careful observational method used during the study identified that some of the partogram sheets were inadequately or improperly filled. The 8.6 % of the midwives that answered no to the usage of the partogram in their health facilities gave the following reasons for their response: unavailability of the partogram sheets (3.4 %), inadequate number of staff (1.0 %), too much workload on midwives (2.8 %), and inadequate knowledge on the use of the partogram (1.4 %). Majority (72.1 %) of the midwives acknowledged, there were enough partogram sheets in their facilities ranging between 21 and 500 sheets.The age distribution of midwives in the Tamale metropolis support the view that most of the midwives are, ageing and will soon retire especially when they approach sixty (60) years which is the retirement age for service workers in Ghana. This age distribution also means that, some midwives were trained before the implementation of the partogram as an international standard tool and are most likely not to have adequate knowledge about the partogram. Until this declaration, training of midwives did not mandatorily incorporate the use of the partogram. This necessitates continues in-service training of midwives on the use of the partogram as a refresher means for effective implementation of the tool in health facilities. Without this in-service training as espoused by some midwives, the use of the partogram in health facilities is bound to have some challenges.African reproductive health journal reported in 2008 that, the partogram is made up of the following components: progress of labour, foetal condition and maternal condition. It is therefore a tool that is used to monitor these parameters during labour . MidwiveX in the centre of the partogram, at the intersection of the horizontal line whiles descent of the foetal head is measured by abdominal palpation and expressed in number of finger widths (Fifths of the head) above the pelvic brim. It is recorded in the central part of the partogram with an O. The specific identification of these symbols is important for smooth communication among the health care team members. However, most of the midwives included in this study could not identify the recordings on the partogram according to this guidelines stipulated by the WHO. Based on this reasons of not effectively using the tool as well as the inability to plot readings on the partogram, it can be deduced that the partogram is not effectively used in most health facilities in the Metropolis as reported in similar studies This study also identified three factors that influenced the utilisation of the partogram; non availability of the sheets, time consuming nature of the partogram and inadequate staffing. These findings agreed to the results of the study in Kenya where staff shortages, lack of knowledge especially on interpretation of findings, negative attitudes, conflict between providers as to their roles in filling the partogram, were among factors that contributed to poor usage of the partogram [Most midwives in this study could not identify the specific time to commence recording on the partogram. According to the journal of the African Reproductive Health, the active phase of labour begins when the cervix is 4 cm dilated, a point where plotting on the partogram should begin. On the World Health Organisation (WHO) partogram, cervical dilatation is measured by the diameter in centimetres. This is recorded with an artogram .n = 196), concluded that although the partogram was available for most eligible deliveries in three hospitals studied, ‘the overall’ proportion of implementation was only 58 % [A study in Tanzania (nly 58 % . The resResponses given by interviewed midwives concerning the benefits derived from the use of the partogram during labour, coupled with other suggestions they made as to how to ensure the effective use of the partograam in all health facilities in the Metropolis, provided enough proof that they were fully aware of its importance. With 91.4 % of midwives having access to the partogram, makes it cost effective.Midwives in the Tamale Metropolis generally have high access to partogram sheets as was revealed in the study that access to the partogram did not pose as a challenge in using it. However the results of the study further revealed an inadequate knowledge on how to plot information on the partogram, particularly the symbols used. Generally the study showed ineffective usage of the partogram by midwives within the metropolis.Based on the findings of the study we recommend training and employment of more midwives and continuous in-service training on the use of partogram as well as constant monitoring and supervision of health professionals particularly midwives to ensure effective use of the partogram.


TNF mRNA and protein secretion in castration resistant prostate cancer (C4-2) cells, but not in macrophage-like (THP1) or myofibroblast-like (WPMY1) cells. Androgen deprivation therapy (ADT) induced autocrine CCL2 expression in C4-2 (as well as a murine CRPC cell line), while exogenous TNF induced CCL2 in THP1 and WPMY1. TNF was most potent in myofibroblast cultures, suggesting ADT induces CCL2 via paracrine interactions within the tumor microenvironment. A soluble TNF receptor (etanercept) blocked enzalutamide-induced CCL2 protein secretion and mRNA, implying dependence on secreted TNF. A small molecule inhibitor of CCR2 (the CCL2 receptor) significantly reduced TNF induced migration, while etanercept inhibited enzalutamide-induced migration and invasion of C4-2. Analysis of human prostate cancers suggests that a TNF-CCL2 paracrine loop is induced in response to ADT and might account for some forms of prostate cancer therapy resistance.The dramatic responses tumors display to targeted therapies are limited by acquired or pre-existing mechanisms of therapy resistance. We recently discovered that androgen receptor blockade by the anti-androgen enzalutamide paradoxically enhanced metastasis and that these pro-metastatic effects were mediated by the chemoattractant CCL2. CCL2 is regulated by TNF, which is negatively regulated by androgen signaling. Thus, we asked if TNF mediates the pro-metastatic effects of enzalutamide. We found that androgen withdrawal or enzalutamide induced  The concept of ‘oncogene addiction’  has givein vitro as well as distant metastases in orthotopic tumor models of CRPC [TNF mRNA in rodent prostatic stroma. Promoter analysis has shown that CCL2 is regulated by TNF via NFκB [We (SY and CC) recently discovered that androgen deprivation therapy (ADT) unexpectedly enhances the metastatic phenotype of castration resistant prostate cancer (CRPC), even as these therapies inhibit tumor growth –13. Spec of CRPC . This ma of CRPC , 12, a c of CRPC , 15. Mon of CRPC . In addi of CRPC , 12, 16. of CRPC . Specifivia NFκB . Indeed,via NFκB  as well via NFκB –22, and via NFκB . Given tin vivo context of PCa. C4-2, a sub-line of the human androgen-dependent LNCaP prostate cancer cell line, derived by selecting for growth as a xenograft in a castrated athymic nude mouse [To address the role of TNF in metastasis following androgen deprivation, we initially employed three cell lines, representing CRPC (C4-2), prostate stromal myofibroblasts (WPMY-1) and tumor associated macrophages (THP-1), either alone, in co-culture or via conditioned media, to simulate the de mouse –26, is ade mouse  is derivde mouse , indicatde mouse .TNF gene by the AR. However, we cannot exclude effects on mRNA stability. Neither DHT nor enzalutamide affected TNF expression in the TAM-like THP-1 or stromal myofibroblast-like WPMY-1 cell lines. Similarly, in the rat stroma-derived PS-1 cells, there was no change in TNF expression in response to enzalutamide (data not shown). The anti-androgen bicalutamide and the synthetic androgen R1881 had analogous effects to enzalutamide and DHT, respectively (Following treatment with dihydrotestosterone (DHT), TNF secretion was reduced in C4-2 Figure . Conversectively . SimilarCCL2 mRNA levels  in C4-2 cells. Etanercept also blocked CCL2 secretion, but not the mRNA level, in WPMY-1 myofibroblasts , enzaluAs noted, we have previously demonstrated that enzalutamide induces a metastatic phenotype in both cell culture and mouse models of CRPC . To deteAR mRNA expression levels are increased in both data sets  that displays a castration-resistant cancer phenotype, including the ability to grow in castrate hosts, and enhanced expression of cancer stem cell genes. A similar selection process may have been at work in the development of C4-2 and PTEN-CaP8 from the respective parental cell lines LNCaP and PTEN-P8 [−/lo (progenitor) cells, and contributes to the aggressive tumor phenotype of this cancer population, which expands following ADT [Observations that TNF can transcriptionally regulate the CL2 gene , that NFCL2 gene –38, and CL2 gene –13, led CL2 gene . CastratCL2 gene  and downCL2 gene , but in CL2 gene , blockinCL2 gene . The indCL2 gene –13, suggCL2 gene , 44. Fire et al.  found th PTEN-P8 , 25, 31.wing ADT .CCL2 mRNA and CCL2 protein in macrophage- and myofibroblast-like cells, suggesting paracrine regulation of CCL2 production  that depend on CCL2 signaling. Again, exogenous cytokine addition or conditioned media – both mimicking possible paracrine effects within the microenvironment - demonstrated a functional role for TNF in the paracrine loop controlling metastasis in response to enzalutamide  PCa tumors showed CCL2 levels correlated with Gleason score and tumor stage , analysiCL2 mRNA . Since tCL2 mRNA , but notCL2 mRNA , as wellCL2 mRNA . Taken tWe therefore propose that CCL2 primarily acts via a therapy-induced paracrine loop, such as the one we document in cell culture models, at the site of the primary tumor but not at the site(s) of metastases. Another way to view this is that the process of metastasis interrupts the paracrine loop (TNF®CCL2) by relocating tumor cells away from the original tumor microenvironment. This further suggests that therapies directed against CCL2 or TNF might be effective only during a narrow window of time; that is, only if administered in conjunction with the initial course of androgen deprivation therapy given to patients still possessing a primary tumor, prior to the onset of CRPC and metastases. Thus, our mechanistic observations may explain the recent failure of CCL2 directed monoclonal therapy in PCa  since thThe following reagents were used: DHT , enzalutamide , etanercept , R1881 , CCR2 antagonist BMS-741672 , and TNF .The C4-2 cell line was from Dr. Jer-Tsong Hsieh, UT Southwestern Medical Center, Dallas, TX. LNCaP, THP1, WPMY-1, PTEN-P8 and PTEN-CaP8 cell lines were from the American Type Culture Collection . C4-2, WPMY-1, THP1 and LNCaP, were cultured in RPMI-1640 media supplemented with 10% FCS and 1% penicillin/streptomycin. For THP1, the media also contained 10 mM HEPES and 2 mM glutamine; LNCaP culture media also contained 1 mM sodium pyruvate. PTEN-P8 and PTEN-CaP8 were cultured in DMEM supplemented with 10% FCS and 1% penicillin/streptomycin.5/ml and supplemented with enzalutamide, DHT or etanercept as indicated. For TNF treatment, cells were cultured at 4 × 104/ml. Cells were seeded at 5 × 104/ml for C4-2/WPMY-1 co-cultures and 1 × 105/ml for C4-2/THP1 co-cultures. TNF and CCL2 were quantitated with ELISA kits , per the manufacturer's instructions.Cells were cultured at a density of 1 × 10in vitro transwell assays  were performed to assess metastatic phenotypes. For migration assays, cells were serum starved for 24 h, trypsinized if required, washed and resuspended in serum-free media. Approximately 1 × 105 cells were placed in the upper chamber of a 24-transwell plate . Matrigel coated chambers  was added to wells used for invasion assays. Conditioned medium was prepared by diluting culture supernatants with an equal amount of fresh media plus 5% FCS. For C4-2 cells, after 22 h incubation, chambers were fixed and stained with 1% toluidine blue for manual counting of photomicrographs under the microscope . For THP-1 cells, after 16 h incubation, media was collected and cells counted by Coulter counter. Counts were normalized to vehicle control.Two ® Green qPCR Master Mix  in a StepOnePlus instrument . PCR primers, spanning at least one intron, were: TNF-forward: 5′-CCTCTCTCTAATCAGCCCTCTG-3′; TNF-reverse: 5′-GAGGACCTGGGAGTA-GATGAG-3′; CCL2-forward: 5′-CAGCCAGATGCAATCAATG-CC-3′; CCL2-reverse: 5′-TGGAATCCTGAACCCACTTCT-3′; β-actin-forward: 5′-CATGTAC-GTTGCTATCCAGGC-3′ and β-actin reverse: 5′-CTCCTTAATGTCACGCACGAT-3′. Standard curves were generated using linearized plasmids encoding the corresponding cDNAs. β-Actin was used to normalize target gene expression. At least three biological replicates were analyzed (unless otherwise indicated), and at least three trials were conducted for each sample.Cells were cultured at the same densities used for the ELISA assays. Total RNA was extracted and reverse transcribed using pdN15 with Superscript III reverse transcription kit  to generate single-strand cDNA. PCR was performed using SYBR3 cells/well. Cells were cultured in complete medium supplemented with 100 ng/ml TNF for 72 h. Viability was measured by WST-1 assay, as per the manufacturer .C4-2 cells at 50%-75% confluence were trypsinized and seeded into 96-well plates at 4.5 × 10http://www.oncomine.org). For statistical analysis individual data values were extracted from the Oncomine database. Homology regions of microarray probes were determined using UCSC genome browser (http://genome.ucsc.edu).Plots of gene expression levels were obtained from Oncomine . Differences between two means was assessed by Student's unpaired


We compute classical trajectories of Ultracold neutrons (UCNs) in a superconducting Ioffe-type magnetic trap using a symplectic integration method. We find that the computed escape time for a particular set of initial conditions (momentum and position) does not generally stabilize as the time step parameter is reduced unless the escape time is short (less than approximately 10 s). For energy intervals where more than half of the escape times computed for UCN realizations are numerically well determined, we predict the median escape time as a function of the midpoint of the interval. There are at present several Ultracold neutron (UCN) experiments that seek to measure the neutron lifetime using magnetic confinement techniques (see these proceedings). Experiments are underway and are being proposed that utilize permanent magnets, superconducting magnets, and a combination of the two to create magnetic field geometries that can be used to spatially confine UCNs. Such techniques are advantageous because they minimize (or eliminate) any interactions that a UCN has with the material walls of the container, thus dramatically reducing (or removing) the largest systematic error that has plagued UCN lifetime experiments to date. Reducing the uncertainty of a neutron lifetime measurement beyond roughly a factor of two  is a challenge that requires a new approach and/or substantial increases in the number of available UCNs. Since magnetic trapping techniques offer great promise to meet this challenge, they are very popular.One key systematic effect that arises when using these new magnetic trapping techniques is marginal trapping; neutrons with a total energy greater than the trap depth may be temporarily contained in the trap. For example, a neutron in a circular orbit (with its plane perpendicular to the trap axis) can gradually turn into an elliptical orbit (due to angular momentum nonconservation in an angular dependent field) and thus reach a greater distance from the origin. If the initial energy of this neutron is larger than the trap depth, the neutron can then strike the container wall and be ejected from the trap before it decays. Consequently, the neutron lifetime estimated from data consisting of a mixture of permanently trapped and marginally trapped neutrons will be systematically shorter than the actual lifetime of the neutron.In this work, as part of the UCN lifetime experiment presently underway at National Institute of Standards and Technology (NIST) –3, we prTo determine the optimal lowered trap depth for removal of practically all of the marginally trapped UCNs, one must understand the escape time distribution of UCNs. In this work, we simulate realizations of UCNs and compute their escape times assuming that the trajectories are classical. The escape time is defined to be the time it takes the UCN to first cross the boundary of a cylindrical detection volume. As a caveat, this model for escape simplifies quantum mechanical aspects of neutron interactions. In a later work, we shall develop a more realistic model for neutron escape that accounts for such effects as described in ref. . For eacOur primary result is that the computed escape time for a particular set of initial conditions (momentum and position) does not always stabilize. In general, for the cases studied here, computed escape time stabilizes for cases where the neutron promptly escapes (less than approximately 10 s). For longer escape times, stability is generally rare. We interpret the observed instability of the computed escape time as a manifestation of chaotic behavior. For such trajectories, slight differences in computed trajectories due to numerical noise (which depends on the time step size) can be magnified dramatically at advanced times. For more discussion of this sensitivity in related problems, see refs.  and [10]4He. By creation of a single phonon in the superfluid, a cold neutron with wavelength near 0.89 nm (speed of approximately 400 m/s) is scattered to a state of near rest. .) The resulting UCNs are confined in a potential field V (x) produced by the interaction of the magnetic moment of a neutron and a spatially varying magnetic fieldB is the magnetic field, |B(x)| is the magnitude of B, and µ is the magnetic moment of the neutron 10]. In oVmax and the minimum potential on the boundary is Vmin = 0.573 Vmax . We determine the model parameters by the method of ordinary least squares and their associated 1-sigma random uncertainties by a nonparametric bootstrap resampling scheme [N data pairs zi =  i = 1, …, N, where xi is the ith energy bin midpoint and and yi is the ith median escape time. We draw N realizations of a random integer that is uniformly distributed between 1 and N. Denote this string of random integers as . Our bootstrap replication of the observed data is 4 bootstrap replications of the data.If more than half of the computed escape times are well determined numerically for given energy interval, the median is well determined for that interval. Thus, we can still extract useful information from the computed escape times even though some may not be well determined. We bin computed escape time data from simulated realizations of UCNs according to energy, and empirically model the logarithm of the median escape time for each bin as a function of the bin midpoint, g scheme –19. The E > 0.8 Vmax, the estimated parameters are For 0.655 We modeled the trajectories of UCNs in a magnetic trap classically and predicted the trajectory of the neutron by integrating the laws of motion using a symplectic integration scheme. In our approach, we modeled the potential produced by the spatially varying field in the trap using a spline interpolation scheme. Since this interpolation scheme is an approximate method, the computed escape times may differ from those computed by use of an exact model for the potential. Since each component of the magnetic field in the trap satisfies Laplace’s equation, it might be possible to develop a special function expansion approximation for the potential. As a caveat, the escape time distribution for UCNs in an actual experiment may be affected by mechanical vibrations and slight instabilities in the magnetic field. In this work, we did not include these possible effects.We defined the escape time of a neutron to be the first time that the classical neutron trajectory crosses the boundary of the trapping volume. The trapping volume boundary is the union of the endcaps and the surface of a cylinder. For all cases considered, we computed numerically stable escape times for trajectories that promptly escaped the trap in about 10 s or less. However, in general, for longer escape times, stability is rare. How to interpret a computed escape for a particular time step for such unstable cases is not clear. Other researchers ,10 have We also predicted the median escape time for simulated UCNs based on escape time data at energies where the median was well determined. The accuracy of this model for lower energies is an open issue. In future work, we plan to study a more realistic model for neutron escape that accounts for additional quantum effects. In this more realistic model, neutrons would escape or scatter off the boundary according to stochastic laws.


Mycobacterium avium subsp. paratuberculosis (MAP). While multiple single farm studies of MAP dynamics were reported, there is not large scale meta-analysis of both natural and experimental infections. Large difference in shedding patterns between experimentally and naturally infected cows were observed. Experimental infections are thus probably driven by different pathological mechanisms. For further evaluations of shedding patterns only natural infections were used. Within such infections, the transition to high shedding was studied as a proxy to the development of a clinical disease. The majority of studied cows never developed high shedding levels. Those that do, typically never reduced their shedding level to low or no shedding. Cows that eventually became high shedders showed a pattern of continuous shedding. In contrast, cows with an intermittent shedding pattern had a low probability to ever become high shedders. In addition, cows that start shedding at a younger age (less than three years of age) have a lower hazard of becoming high shedders compared to cows starting to shed at an older age. These data suggest the presence of three categories of immune control. Cows that are intermittent shedders have the infection process under control (no progressive infection). Cows that start shedding persistently at a young age partially control the infection, but eventually will be high shedders (slow progressive infection), while cows that start shedding persistently at an older age cannot effectively control the infection and become high shedders rapidly.The objective of this paper is to study shedding patterns of cows infected with  Incubation periods and sub-clinical periods of chronic infectious diseases are quite variable in duration, with some individuals becoming ill shortly after infection and some individuals surviving months or years without illness (e.g. Human Immuno-deficiency Virus (HIV) infections , Herpes Mycobacterium avium subspecies paratuberculosis (MAP). The infection with MAP in cattle often, but not exclusively, occurs in young animals and will lead to long-term persistent infections. In a proportion of MAP infected animals, eventually clinical disease will develop. Clinical signs include chronic diarrhea, weight loss and decrease in milk productivity [An important mycobacterial infection in farm animals is caused by uctivity -12. Evenuctivity . For theMycobacterium tuberculosis cases, progression may be triggered in a portion of infected individuals by secondary exposure [The idea of elite controllers, infected individuals with pathogen loads below the detection threshold, have been reported for HIV ,14 and mexposure . Previouexposure .In this study, we evaluate infection progression in animals followed over the course of their adult lives. ELISA, fecal culture and tissue culture data are available for different subsets of animals in this study. Our objectives include assessing whether all animals are on the same progression pathway, identifying markers for progression, and evaluating whether environment (farm) influenced rate of progression.A)Can infected cows be divided into two groups: cows that are prone to become high shedders of MAP bacteria and cows unlikely to become high shedders?B)Given the division into groups; early markers for classification are required. Moreover, in the group with a high shedding probability (high probability of progression to high shedding), is it possible to identify markers for progression. Such Markers can be crucial for management of the infection on dairy farms.C)Finally, we address the difference in infection progression and shedding patterns in animals after natural versus experimental exposure.Specifically, we plan to address three main issues in the following sections:The analysis of MAP within host dynamics relies on different aspects of host immunology and pathogen distribution within hosts. Each of those aspects suffers from different limitations and caveats. While these tests are quite specific, none of the pre-mortem tests are extremely sensitive. Infectious contribution of individual animals can be assessed by fecal culture or PCR. Following death of animals, tissue samples can be used to confirm whether animals are infected with much higher sensitivity than tests available in living animals. However, due to the limited sensitivity of diagnostic tests for MAP infection prior to the emergence of clinical signs, infectious progression patterns are difficult to assess. We here combine a large number of parallel measurements in natural infections to analyze the relationship between shedding patterns and disease progression.Data for this study were gathered from 3 longitudinal field studies, one longitudinal follow up in an experimentally aggregated population and multiple experimental infection trials.Field study 1 comprised three dairy farms  in the Northeastern US . AnimalsField study 2 followed animals on a single dairy farm with approximately 100 lactating Guernsey breed cattle in Pennsylvania (USA) for a period of 20 years during an intervention program. Details of farm size, MAP prevalence and study design are available in previously published work . AnimalsField study 3 followed animals on 17 Dutch dairy farms  during a national monitoring program over the course of 3.5 years. Animals were tested by ELISA and fecal culture at 6 month intervals .Naturally exposed animals were aggregated at a research facility for population 4. These animals were either from known infected dams or from known highly infectious environments. Animals were tested by fecal culture at three to six month intervals as well as by immune assays (ELISA) from the time of entrance into the facility. In this population, CFUs were determined on a semi-quantitative scale of +, ++ and +++, representing 1–10, 11–100, and 100+ CFU/sample tube (which is approximately of the order of CFU per gram). This scale was interpreted as 5, 50 and 100+ CFU per animal. For a more detailed description of these cows, see the paper by Karcher et al. .Experimentally infected calves (N = 20) were followed monthly from time of infection to slaughter by fecal culture, ELISA test and either ELISPOT or (other) IFN-g related tests were performed to evaluate infection status. For a detailed description see Langelaar .Throughout this study, only fecal shedding patterns are described. ELISA data were collected but not further analysed in this study. ELISA data are further analysed in a companion paper .Shedding patterns from experimentally infected animals were compared to shedding patterns from longitudinally followed naturally infected animals . In mostData from all naturally and experimentally infected animals was aggregated into a single database and analyzed via Matlab v2011a . Hazard and survival probabilities were computed using a Cox proportional hazards model, where the events where either first shedding or first high shedding and censoring was based on the absence of future observations (where we assume these cows were removed from the sample).Survival and hazard curves of high shedding were evaluated by Kaplan-Meier survival analysis  with eitNon Parametric methods were used, since the age at first high shedding and the interval between initial and high shedding deviate from a normal distribution. Throughout this paper, shedding of 50+ cfu per gram of faeces is considered high shedding. Low shedders are shedding 1–49 cfu per gram of faeces. When 4 levels of infection were provided , only +++ was considered high shedding.Experimental infections are often used to study MAP development and shedding patterns. However, such infections can present substantially different conditions for both environment and infections. These differences can influence the shedding patterns. We compared shedding patterns among experimentally infected and naturally infected animals to evaluate whether experimentally infected animals were representative of natural infections. Shedding patterns were obtained from multiple sources. In order to standardize the different data-sets, we used three shedding levels, as detailed in methods. Using this division, animals were evaluated by the number of changes in shedding state over all available time points. Transitions between non-shedding and shedding states were summed for a total number of “switches” of shedding state. For natural infections in which animals were not followed from birth, animals which were high shedders at their first sampling time point were only included in the analysis if the first sample was taken within their first year in the lactating herd. As all animals would be left-censored at this time point, results should be consistent across study years. Animals which transitioned from non-shedding to high shedding within one sampling interval were assumed to have reached high shedding at the midpoint between the two sampling time points and were considered as continuous individuals. Note that such a scheme can lead to some underestimation of the variance .Survival analysis was performed using the R survival toolbox. The single parameter survival plots was based on the surv and survfit functions implementing a Cox model. The analysis of the farm effect was based on a mixed effect cox model as implemented by coxme ,26, and The shedding patterns of natural and experimentally induced infections differ in both the fraction of cows becoming high shedders and by the dynamics of each cow. In natural infections, becoming a high shedder is a rare and terminal event. Only 7% of the cows ever become high shedders, but once they become high shedders, over 95% of them are culled or die within a year  in our data are ever observed to reach high shedding levels. A larger number of animals have occasional and intermittent low level shedding (15%) Table p < 1.e-10, Chi Square test, 20 degree of freedom).To disqualify the possibility that intermittent shedders are an artefact of test sensitivity in a population of mostly infected animals, positive tests per individual relative to total tests per individual was plotted for intermittent shedders Figure A. If intTo test whether intermittent shedding is evidence for a containment of the MAP infection, we separated the populations into cows that have at least one switch  and cows that never switched (cows that shed but never went down to no shedding). We denote these populations as intermittent (I) and “continuous shedders” (CS).p < 0.01), suggesting that most of intermittently shedding cows are truly containing the MAP infection and will probably never be high shedding, despite the persistence of MAP infection. When intermittent shedders are compared to continuous shedders, using the G-rho family of Harrington and Fleming, a highly significant difference between the survival of intermittent and continuous shedders is obtained (p < 1.e-10). Similarly, when the analysis is repeated to include the farm effect (22 different farms), using a mixed effect cox model (as implemented by the R coxme), a difference between intermittent and continuous shedders is still obtained . The overall probability per year of becoming high shedder is less 0.0003 + −0.0001 per day (2 standard deviations) per day for intermittent cows, while more than 0.0006+/−0.0001 per day for continuous shedders. Note that the absolute chance per cow is 0.0001 per day, since the majority of cows are neither intermittent nor continuous shedders.We tested the fraction of I and CS cows that never reach high shedding Figure B and theA simple test would be two consecutive samples. Cows shedding once at a low level have a 25% of ever becoming high shedders. Cows shedding twice at low levels have a 48% chance of becoming high shedders, while cows shedding once at a low level and not shedding at the next sample (six month later) have a less than 6% chance of ever becoming high shedders.Beyond, intermittence, age also affects future shedding patterns. The hazard is slowly changing as a function of the cow age Figure A, beyondp < 1.e-8). A difference of 3 years in the initiation of shedding leads to a reduction of 5 months in the time to high shedding.Following the initiation of shedding, the time it takes to reach high shedding may be affected by the age at first shedding. A clear negative correlation was observed between the age at first shedding and the duration of time until high shedding Figure B (R = −0p < 0.05), but no difference between the two older age groups (3–5 and above 5 years) in the time until high shedding . There was a significant difference between the young infected cows and the other two groups (p < 0.05). Note that even at the first shedding point, shedding can be high, thus the fraction of non-high shedders does not start from 1 at time 0.Finally, we compared the effect of switching on the time from low shedding to high shedding in young and adult cows. Since there was no difference between medium and old cows, they were combined into a single group. As mentioned above, intermittent cows have a low probability of becoming high-shedders. They thus have a low fraction of high shedders even two years after the first measured positive time point. There is no significant effect of the age of initial shedding on the survival of intermittent animals, suggesting that these animals were able to control the disease. In the continuous shedders, the age of at first shedding has a crucial effect. Cows that start shedding at a young age survive for a much longer time than cows shedding for the first time at and advanced age Figure D , it should be considered a high risk animal for the herd. If it stops shedding at some point, then it is highly probable that it will never start shedding again and very likely not progress to high shedding and clinical disease. More precisely, if the animal is faecal culture positive at two subsequent examinations at a 6-month interval, it will become a high shedder with a high probability. If within the same interval, the animal goes back to no shedding; it will have a low probability of becoming a high shedder. This distinction in containing the infection and infection progression is only true for natural infections. Shedding levels in experimental infections fluctuate strongly and these fluctuations are not predictive for future high shedding as almost all experimentally infected cows will become high shedders eventually. Moreover, naturally infected cow that become high shedders, practically never stop being high shedders. Experimental infections are often the opposite with many cows fluctuating from high to low shedding and back.These considerations strongly suggest that natural and experimental infection follow two different paths to disease. Natural infections are typically dormant until an external or internal event or characteristic drives a change in shedding pattern. When such an event happens, either the immune system controls the infection, as is the case for intermittently shedding cows, or it fails to control it, as is the case for continuous shedders. Experimental infections are characterized by a much more complex pathogenesis with different stages of an active infection and eventual progression to high shedding in virtually all cows.Some of the intermittent shedders may never be infected, and the observed shedding may be a “pass through phenomenon”. In such a case, we would expect them not to become high shedders. This cannot explain all the intermittent shedding, since intermittent shedders have a higher probability of becoming high shedders than cows never shedding.The probability to become a shedder or a high shedder is not strongly affected by the age of the cow, beyond an initial period of two years, which may be affected by our sampling method. However, the duration of time a cow will be shedding MAP before it reaches high shedding decreases with the age of the cows at first shedding. When shedding starts later in life, the time until high shedding is significantly reduced. The simplest explanation for this observation would be a weakening of the immune response with age.These observations taken together shed a clear light on the dynamics of MAP infections. Natural, likely low dose, MAP infections leads to a dormant infection status. The probability to move from this dormant stage to low shedding and eventual high shedding and the duration of the high shedding phase are to be pre-determined by the quality of the immune response. In contrast, high dose experimental challenge infections lead to a rapid transition to an active infectious state, with rapid fluctuations in the amount of free bacteria.The presented analysis miss the first stage of the infection, since most cows here were not sampled during the first two years of life. It is generally assumed that most naturally infected animals will be infected in their first year of life. Thus, possible correlation between the early shedding properties or properties of the early immune response and the chance to become high shedders were not observed in these data. Another important limitation of the current analysis is the relative low sampling rate  in the natural infections datasets. As a consequence, some rapid fluctuations may in principle be missed. However, if such fluctuations are frequent, we would have observed their signature across the very large number of cows in our study.A)We showed that infected cows can be divided into two groups: cows that are prone to become abundant shedders of MAP bacteria and cows that control the infection.B)We showed that given MAP infection, the only early marker predicting high shedding that can be detected are the shedding patterns. However, we were not able to find other cow-specific markers for progression to high shedding; suggesting that in the susceptible group transition to high shedding may be a stochastic event .C)Finally, we addressed the difference between experimentally and naturally infected cows. We showed that most naturally infected animals show a pattern of low and intermittent shedding and never progress to high shedding. In small subset natural infections show a long period of no shedding, followed by a short period of high shedding. Experimental infections on the other hand have much more complex shedding patterns with rapid progression and significant fluctuations in shedding levels.Specifically, we have addressed the three main issues outlined in the introduction and:The key finding from this study is the identification of two distinct shedding patterns in naturally infections with MAP in dairy cattle. A very large proportion of MAP infected cows appears to be able to contain the infection and not show any signs of pathology during infection. A much smaller proportion of cows show progression to high shedding and likely clinical disease. Early identification of these animals would be of value in infection control programs.


TNFRSF1A gene, encoding tumor necrosis factor receptor 1 (TNF-R1), are associated with the autosomal dominant autoinflammatory disorder, called TNF receptor associated periodic syndrome (TRAPS). TRAPS is clinically characterized by recurrent episodes of long-lasting fever and systemic inflammation. A novel mutation  in the TNFRSF1A gene at residue 88 of the mature protein was recently identified in our laboratory in an adult TRAPS patient. The aim of this study was to functionally characterize this novel TNFRSF1A mutation evaluating its effects on the TNF-R1-associated signaling pathways, firstly NF-κB, under particular conditions and comparing the results with suitable control mutations.Mutations in the TNFRSF1A mutants. Peripheral blood mononuclear cells (PBMCs) were instead isolated from two TRAPS patients carrying S59P and R92Q mutations and from five healthy subjects. Both transfected HEK-293 and PBMCs were stimulated with tumor necrosis factor (TNF) or interleukin 1β (IL-1β) to evaluate the expression of TNF-R1, the activation of TNF-R1-associated downstream pathways and the pro-inflammatory cytokines by means of immunofluorescent assay, array-based technique, immunoblotting and immunometric assay, respectively.HEK-293 cell line was transfected with pCMV6-AC construct expressing wild-type (WT) or c.262 T > C (S59P), c.362G > A (R92Q), c.236C > T (T50M) in vitro by the enhancement of p-IκB-α and p65 nuclear subunit of NF-κB expression in all mutants in the presence of TNF or IL-1β stimulation. These in vitro results correlated with patients’ data from PBMCs. Concerning the pro-inflammatory cytokines secretion, mainly IL-1β induced a significant and persistent enhancement of IL-6 and IL-8 in PBMCs carrying the S59P mutation.TNF induced cytoplasmic accumulation of TNF-R1 in all mutant cells. Furthermore, all mutants presented a particular set of active TNF-R1 downstream pathways. S59P constitutively activated IL-1β, MAPK and SRC/JAK/STAT3 pathways and inhibited apoptosis. Also, NF-κB pathway involvement was demonstrated The novel S59P mutation leads to defective cellular trafficking and to constitutive activation of TNF-R1. This mutation also determines constitutive activation of the IL-1R pathway, inhibition of apoptosis and enhanced and persistent NF-κB activation and cytokine secretion in response to IL-1β stimulation.The online version of this article (doi:10.1186/s13075-015-0604-7) contains supplementary material, which is available to authorized users. TNFRSF1A gene located on chromosome 12p13 and encodes the 55 kDa receptor for tumor necrosis factor receptor 1 (TNF-R1). The disease is clinically characterized by periodic fever, abdominal pain, skin lesions, conjunctivitis, myalgia and pericarditis. Disease onset generally occurs in early childhood but it can present in adults, and inflammatory attacks can last several weeks. The most severe complication is AA-type serum amyloidosis, which can lead to renal impairment and failure [Tumor necrosis factor receptor-associated periodic syndrome  is the second most common inherited autosomal dominant autoinflammatory disease [The TNF-R1 presents an extracellular domain involved in ligand binding, a transmembrane domain involved in receptor solubilization and an intracellular region where death domains (DD) are involved in signal transduction ,6. The bTNFRSF1A variations, such as R92Q, are associated with milder clinical manifestations, suggesting that in these cases other genetic and/or environmental factors can play a role in the disease’s pathogenesis [TNFRSF1A mutations involving the cysteine residues  are less frequent (34%) than those with a low penetrance (76%), the latter being mainly represented by the T50M (15.8%) and R92Q (28.9%) variants [Almost all known mutations associated with TRAPS are characterized by the replacement of amino acids in the extracellular domain of the TNF-R1. More than 100 mutations are registered in the INFEVERS database , which aogenesis -26. The variants .TNFRSF1A gene  in an adult TRAPS patient. Clinically, the patient had fever, leukocytosis, recurrent bronchopneumonia, palpebral ptosis, episodes of arthralgia, myalgia and intermittent erythematosus skin lesions of the limbs and trunk. His serum immunoglobulin D (IgD), immunoglobulin A (IgA) and serum amyloid A protein (SAA) levels were elevated. While traditional therapy with the TNF inhibitor etanercept was unsuccessful in this patient, alternative therapy with the interleukin-1 receptor antagonist (IL-1Ra) anakinra promptly induced disease remission. The identified new genetic variant results in a proline for serine amino acid substitution (S59P or p.Ser88Pro) at residue 88, localized in the extracellular domain of the mature protein. This variant might be included in the above-described category of mutations that introduce proline residues. The distinctive cyclic structure of proline’s side chain gives it an exceptional conformational rigidity, interfering in peptide bond formation with other amino acids and affecting the secondary structure of the protein [We recently identified a novel mutation in the  protein .Overall, the molecular pathogenesis of TRAPS is not yet completely understood. The change in secondary structure might cause defective TNF-R1 trafficking, altered ligand binding affinity, reduced activation-induced shedding and impaired cell signaling. Based on these premises, several hypotheses have been formulated: the shedding hypothesis ,29, the Since the patient carrying the S59P mutation was responsive to IL-1Ra but not to anti-TNF therapy, the aim of this study was to verify whether this mutation modifies the cell signaling, in particular in the NF-κB pathway, after TNF and IL-1β stimulation.TNFRSF1A cDNA ready for transfection. Three new constructs containing three mutant TNFRSF1A - cDNA (c.262 T > C (S59P); c.362G > A (R92Q) and c.236C > T (T50M)) were obtained using site-direct mutagenesis, and the following mutagenic primers were used: TNFRFS59P: 5′AGTGTGAGAGCGGCCCCTTCACCGCTTCAG3′; TNFRFR92Q: 5′ CTTCTTGCACAGTGGACCAGGACACCGTGTGTGGCTG 3′; TNFRRT50M: 5′ CTCACACTCCCTGCAGTCCATATCCTGCCCCGGGCCTGG 3′.We purchased the vector pCMV6-AC  containing wild-type (WT) TNFRSF1A coding region to ensure that only the desired mutations were introduced. Plasmid isolation from bacterial culture was carried out using the PureYield Plasmid Maxiprep System .All the products were sequenced along the full length of the Human embryonic kidney (HEK-293) cell line was used for the transfection. This cell line expressed moderate levels of endogenous TNF receptors and exhibited high transfection efficiency. A total of 4 μg of plasmid were incubated with 10 μL Lipofectamine™ 2000  in 500 μL serum and antibiotic-free DMEM  for 20 minutes at 25°C. The reagent was then directly added to each cell culture well  in a 6-well plate format. After 6 hours, media were replaced with fresh serum-supplemented media, and the cells were maintained at 37°C for the subsequent 24 hours. To obtain stable transformed cells, they were treated with 0.8 mg/mL Geneticin® (G418)  for 15 days.TNFRSF1A gene were studied as healthy controls. At the time the study was carried out, the TRAPS patients were on anakinra and infliximab therapy, respectively, and did not present inflammatory symptoms. The study protocol was approved by our institutional review board  and the patients gave their fully informed written consent to participate in the study. The clinical characteristics of the enrolled TRAPS patients are detailed in Table Two TRAPS patients were referred to the Rheumatology Unit of University of Padova and were studied: one carrying the S59P mutation and the other the R92Q mutation. Moreover, 5 subjects  without any variants in the According to our medical file, which includes 300 Italian patients with a clinical suspect of TRAPS, none carried the T50M mutation.et al. [TNFRSF1A gene was performed using the automatic sequencer 3130ABI PRISM Genetic Analyzer . Chromatograms were analyzed with Chromas Lite 2.01 software .Genomic DNA was extracted from whole blood using the standard method . After amplification by polymerase chain reaction with primers as described by D’Osualdo et al. , sequenc3 test tubes and was processed within 2 hours of sampling to isolate mononuclear cells by gradient centrifugation . Peripheral blood mononuclear cells (PBMCs) were suspended in culture medium  supplemented with 0.1% gentamicin, 10% FCS and 1% glutamine; Invitrogen, San Giuliano Milanese, Italy) and incubated at 37°C for 24 and 72 hours prior to experimental manipulation to prevent any experimental anomalies arising as a consequence of therapy.Blood (20 mL) was collected with EDTA KTNFRSF1A were seeded onto slides (3 × 105) and maintained in culture for 24 hours. Cells were stimulated with or without TNF (60 ng/mL)  for 1 hour to induce TNF receptor expression. They were then washed in PBS 1X and fixed in 4% formaldehyde fixative  for 10 minutes at room temperature.HEK-293 transfected with WT or mutant 4Cl (50 mM) for 10 minutes and with Triton X-100 (0.1%) for 5 minutes. The cells were then washed in PBS 1X and incubated with PBS with BSA (1%) for 45 minutes. The first primary antibody was added to the cells and incubated for one hour at room temperature in the dark. Cells were then washed three to four times in PBS 1X. The secondary antibody was added to the cells and incubated for one hour at room temperature in the dark. Confocal microscopy was performed using a Radiance 2000 confocal laser scanning microscope .Dual indirect staining was performed using primary monoclonal mouse antibody anti-human CRI/TNF-R1-PE (R&D Systems Inc.) Minneapolis, Minnesota, USA and the appropriate Alexa Fluor 488-conjugated secondary antibody . The cells were washed in PBS 1X, incubated with NHReverse phase protein array (RPPA) analysis was performed using a procedure previously optimized and validated . BrieflyTNFRSF1A HEK-293 and PBMCs were seeded (5 × 106) onto Petri dishes (diameter: 10 cm) and maintained in a laboratory culture for 24 hours. Cells were incubated with and without TNF (6 ng/mL) or with IL-1β (1 ng/mL)  for 10 minutes to activate signaling pathways. Following stimulation, the Petri dishes were immediately transferred to an ice bath, and the cells were washed twice with cold PBS, resuspended in 100 μL of cold lysis buffer ). Lysates were centrifuged for 10 minutes at 14,000 rpm at 4°C and the supernatants were collected. The pellets were resuspended in a nuclear extraction buffer mix  and proceeded to generate nuclear extracts, as described in the manufacturer’s instructions. Total cytosolic and nuclear proteins were measured using the Bio-Rad Protein Assay Kit .WT or mutant 32) and anti-phospho-NF-κB subunit p65 (Ser536)  and anti-β-actin ) diluted 1:5,000 (β-actin) or 1:3,000  in blocking buffer. The blots were washed three times in PBS-T for 15 minutes and incubated for 1 hour with alkaline phosphatase-conjugated anti-rabbit or anti-mouse secondary antibodies . The blots were washed three times in PBS-T for 15 minutes and developed with the ECL Advance Western Blot Detection Kit .For each sample, 40 μg proteins were electrophoresed using a 4-12% NuPAGE® Novex Bis-Tris SDS-PAGE Gel or 3-8% NuPAGE® Novex Tris-Acetate SDS-PAGE Gel , and were electrophoretically transferred to nitrocellulose membrane  using iBlot® Dry Blotting System . The membranes were incubated for 1 hour in blocking buffer (5% low-fat powder milk resuspended in PBS-T (PBS with 0.1% Tween-20)). They were then incubated overnight with the primary antibodies (anti-phospho IkB-α (SerTNFRSF1A and PBMCs cultured for 24 and 72 hours were incubated at 200 × 103/mL in 24-well plates in culture medium. The cells were stimulated with and without TNF (6 ng/mL) or IL-1β (1 ng/mL) or lipopolysaccharide ((LPS) 1 μg/mL) as positive controls (PC) for four hours. Supernatants were collected, centrifuged to remove cells and debris and stored at −80°C. IL-1β, IL-6, IL-8 and TNF cytokine analysis was performed using an immunometric assay .Cells transfected with WT or mutant Two sets of data were analyzed using an independent sample t test. The data of more than two groups were analyzed using one-way analysis of variance (ANOVA) and Bonferroni’s test for pairwise comparisons. The statistical software Package for the Social Sciences (SPSS version 9) was used. All experimental data were obtained from a series of independent experiments (n = 3).TNFRSF1A sequencing, this patient was demonstrated to carry in heterozygosis a mutation which has never been previously described  and p65 component (p-NF-κB (Ser536)), were assessed by Western blot analysis in WT, S59P, R92Q and T50M HEK-293, and in PBMCs obtained from TRAPS patients and controls.We then ascertained in more depth whether a different response to TNF and IL-1β in terms of activation or inhibition of the NF-κB pathway, depends on pathways , and tha32) in WT cells, while the opposite was found when they were added to S59P and T50M mutant cells. In R92Q cells, little if any variation in IκB-α phosphorylation was seen. The Western blot analysis of the p65 component in isolated nuclei (p-NF-κB Ser536) showed that TNF and IL-1β did not activate p65 in WT HEK-293 cells. By contrast, this component was phosphorylated by TNF in all mutants, and by IL-1β in S59P and R92Q, but not T50M mutants. Confirming RPPA findings, p65 was constitutively activated in the T50M HEK-293, and this activation was sustained by TNF and partly counteracted by IL-1β.Figure 32) and of nuclear p65 component (p-NF-κB Ser536) in control and TRAPS PBMCs kept in culture for 24 hours unstimulated or stimulated with TNF and IL-1β. In basal conditions IκB-α phosphorylation was higher in TRAPS with respect to control PBMCs. In both control and TRAPS patients TNF reduced IκB-α phosphorylation. IL-1b reduced the phosphorylation of IkB-a in control PBMCs, while enhnacing it in TRAPS PBMCs. In the same experimental conditions, p65 activity in isolated nuclei was constitutively elevated only in S59P TRAPS PBMCs. Both TNF and IL-1β induced the p65 phosphorylation in controls and TRAPS PBMCs.Figure We then examined if NF-κB activation might be functionally linked to the secretion of inflammatory cytokines. Levels of IL-8 were measured in WT, S59P, R92Q and T50M HEK-293 supernatants. Levels of IL-1β, IL-6, IL-8 and TNF were measured in PBMCs supernatants. Cells were incubated in fresh media with or without TNF (6 ng/mL), IL-1β (1 ng/mL) or LPS (1 μg/mL) as a positive control for 4 hours.P <0.0001; t = −89.2, P <0.0001; t = −8.6, P <0.001)  and R92Q , not in T50M  mutants, with respect to WT HEK-293 cells.Non-stimulated S59P, R92Q and T50M HEK-293 released higher levels of IL-8 compared to WT cells , IL-6 , IL-8  and TNF  secretion. Both TNF and IL-1β induced significantly higher IL-8 release in the control with respect to the TRAPS PBMCs .In all PBMCs  and R92Q  TRAPS PBMCs. LPS maintained its stimulatory effect at 72 hours on IL-6 , IL-8  and TNF  secretion. TNF-stimulated IL-8 secretion was significantly higher in the S59P and R92Q TRAPS than in the control PBMCs . IL-1β stimulus maintained both IL-6 and IL-8 secretion higher in the S59P and R92Q TRAPS than in the control PBMCs  .In a 72-hour culture , and others which are reported less frequently (pericarditis and pneumonia) [TNFRSF1A gene was performed in this patient after about 18 years of recurrent episodes of bronchopneumonia, fever and other systemic signs of inflammation with an incomplete response to steroid treatment. This long lag time between disease onset and diagnosis depends on the lag time between disease onset and TRAPS first description [We identified a novel eumonia) . SequencAny amino acid substitution in the ectodomain of TNF-R1 might affect its physiology, including membranal expression levels, three-dimensional receptor folding and stimulus-dependent or independent activation. The novel S59P mutation determines the presence in the TNF-R1 of one more proline residue, which might interfere with three-dimensional receptor folding, since the distinctive cyclic structure of proline’s side chain gives it an exceptional conformational rigidity and might cause a bend in the receptor’s secondary amino acid structure . These sTo understand the functional features of this novel S59P mutation, we set out to compare it with two frequent mutations that are well known in the literature: the R92Q and T50M mutations ,38,44,45in vitro model and only the noteworthy results were examined using the patients’ cells. We first focused on the expression of TNF-R1 by immunofluorescence and found that this receptor is homogeneously distributed in the cytosol of WT cells. S59P and R92Q mutations did not significantly modify this pattern, while T50M caused an enhanced cytosolic TNF-R1 expression with a granular appearance. These results are supported by previous data from Rebelo et al. [As it is difficult to study the pathophysiologic mechanisms of TRAPS in the patients themselves, we employed an o et al. , who shoWe then verified whether the exposure of cells to the TNF-R1 cognate ligand TNF exerts a mutation-dependent effect on receptor expression. In WT, S59P and R92Q mutant cells, TNF caused an enforced expression of the TNF-R1 mainly in the cytosol but also at the cell membrane, while in T50M mutant cells TNF induced an enforced expression of cytoplasmic aggregates. It might be suggested that S59P, similarly to the R92Q mutation, does not alter TNF-R1 trafficking from the cytoplasm to the surface membrane, while the T50M mutation causes a misfolded protein that overloads, leading to the formation of aggregates in the cytosol ,45.The pivotal role of an altered TNF receptor in TRAPS pathophysiology supports traditional therapy with etanercept, a fusion molecule of TNF-R2/FcIg ,25,36, oTNFRSF1A mutant differently affects the pro-inflammatory signaling pathways downstream of TNF-R1: the T50M mutant activates TNF-R1 pro-inflammatory signaling pathways, the novel identified S59P mutant activates IL-1 signaling and the R92Q mutant inhibits, not activates, TRADD-dependent signaling. A common feature of all studied TNFRSF1A mutants was the constitutive inhibition of apoptosis by increased inhibition of BCL-2 and BAD. Considering that the molecular pathogenetic mechanisms proposed for TRAPS include defective autophagy, ROS production, defective apoptosis and enhanced activation of NF-κB, our results suggest that a defective apoptosis is a common feature of different TNFRSF1A mutations, and that their differences in causing more or less severe forms of disease are probably related to their ability in sustaining ligand-dependent and independent NF-κB activation and inflammation.By studying all the above TNF-R1 signaling pathways and the IL-1 pathway in WT and mutant cells with a comprehensive RPPA approach, we demonstrated that in the presence of T50M, not S59P or R92Q mutations, TNF-R1, NF-κB and caspase 1 are constitutively activated. In agreement with the hypothesis that a possible association between TNF-R1 mutation and IL-1 signaling exists, constitutively activated IRAK and TAK proteins, involved in the IL-1 signaling pathway, were observed in cells carrying the S59P mutation. In R92Q cells we observed an attenuation of NF-κB, c-Jun, ERK, Akt, and inflammasome pathways and an increase in suppressor of cytokine signaling (SOCS) protein 3, which attenuates the intensity and/or duration of a cell’s response to a diverse range of extracellular stimuli by suppressing the signal transduction processes. These findings indicate that any TNFRSF1A cells. This phenomenon could be explained by the combined TNF-independent autoactivation of the mutated receptor and by the TNF-dependent activation of the WT receptor, since both receptors are present in these heterozygous cells. The IκB-α downstream signaling involves the p65 subunit, which plays an important role in protecting from apoptosis and innate immunity [et al. on T50K [et al. on T50M [TNFRSF1A cells and, using Western blot analysis