Eh GSH) and cysteine (Eh CyS) were measured. Angiotensin converting enzyme (ACE) levels and activity were measured. On univariate analysis, HFpEF was associated with male sex (p = 0.04), higher body mass index (BMI) (p = 0.003), less oxidized Eh CyS (p = 0.001), lower DROMs (p = 0.02), and lower IsoP (p = 0.03). Higher BMI  and less oxidized Eh CyS  maintained associations with HFpEF on multivariate analysis. Though ACE levels were higher in early HFpEF , ACE activity was similar to that in controls. HFpEF is not associated with significant systemic RAS activation or oxidative stress. This may explain the failure of RAS inhibitors to alter outcomes in HFpEF.Animal models have suggested a role of renin-angiotensin system (RAS) activation and subsequent cardiac oxidation in heart failure with preserved ejection fraction (HFpEF). Nevertheless, RAS blockade has failed to show efficacy in treatment of HFpEF. We evaluated the role of RAS activation and subsequent systemic oxidation in HFpEF. Oxidative stress markers were compared in 50 subjects with and without early HFpEF. Derivatives of reactive oxidative metabolites (DROMs), F2-isoprostanes (IsoPs), and ratios of oxidized to reduced glutathione ( Heart failure with preserved ejection fraction (HFpEF) accounts for up to 50% of heart failure (HF) cases , 2. The It has been shown that nitric oxide (NO) and nitric oxide synthase (NOS) have a role in cardiac relaxation, with a reduction in cardiac NO contributing to DD , 6. The Eh GSH. Cysteine (CyS) comprises the major extracellular thiol, and, along with oxidized cysteine (CySS), it represents another measurable redox couple, Eh CyS [Several convenient methods are available to measure oxidative stress in blood. Glutathione (GSH) is a major soluble intracellular peptide that eliminates peroxides and other oxidants . GSH and Eh CyS . Derivat Eh CyS . F2-isop Eh CyS . Recentl Eh CyS . Additio Eh CyS .2∙−). Overall, these are thought to lead to a reduction in NO bioavailability and a defect in myocardial relaxation [Preclinical studies have proposed that RAS and subsequent oxidation play a role in pathogenesis of DD in HFpEF. The cardiovascular effects of Ang II are believed to be because of its activation of NADPH oxidase . Ang II laxation . Neverthlaxation –11. Thislaxation , 21.To help explain this paradox, we tested whether systemic RAS activation and associated oxidative stress were present in patients with DD in early HFpEF.NCT00142194). Cases and controls were matched for age in decades, smoking history, and diabetes mellitus, all known confounders in oxidative stress measurements. The protocol was approved by the Emory University Institutional Review Board. A written informed consent for participation in the study was obtained from all subjects.In a cross-sectional, case-control study, 50 subjects with NYHA functional Class I-II HF symptoms and echocardiographic evidence of HFpEF, as defined by preserved LV ejection fraction (EF) of >50% and abnormal echocardiographic LV relaxation pattern on pulsed-wave and tissue Doppler, and matched controls were recruited from the outpatient clinics and hospital at the Atlanta Veterans Affairs Medical Center and Emory University Hospital from July 2006 to February 2008  > 180/100 mmHg on medications, concurrent illness resulting in life expectancy <1 year, and illicit drug or alcohol abuse.Demographic and clinical data were collected by review of medical records and physical examination upon enrollment. A qualifying standard 2D echocardiogram with Doppler examination was obtained at entry into the study. A single blood draw in a nonfasting state was obtained between 8:30 AM and 5:00 PM. Blood samples were collected from the antecubital vein and, for thiol measures, were immediately transferred to a microcentrifuge tube with 0.5 mL preservative solution of 100 mmol/L serine borate (pH 8.5), containing (per mL) 0.5 mg sodium heparin, 1 mg bathophenanthroline disulfonate sodium salt, and 2 mg iodoacetic acid. This was done to minimize autoxidation and hemolysis . SamplesE) and late atrial contraction (A) wave velocities were measured; the proportion of E/A waves was calculated. Mitral annular velocities were measured in the A4C plane at the septal and lateral mitral leaflet insertion sites using PW tissue Doppler imaging (TDI) [E/e′) and annular early/annular late velocities (e′/a′) were calculated per standard guidelines. Isovolumic relaxation time and deceleration time were recorded. An independent cardiologist interpreted the studies using standard protocols [All echocardiographic studies were performed with a GE System Vivid 7 Echocardiogram with the patient in left lateral position. Standard echocardiographic views were obtained per protocol. Left ventricular ejection fraction was calculated using the biplane modified Simpson rule. Cardiac inflow velocities were obtained by pulsed-wave (PW) Doppler analysis performed in the apical-4 chamber (A4C) plane. Peak early filling (ng (TDI) . The prorotocols .Eh GSH) and cysteine (Eh CyS) in plasma (thiol ratios) [Eh) of thiol/disulfide pools were calculated using Nernst equation:Eo is the standard potential for redox couple, R is the gas constant, T is the absolute temperature, n is the number of electrons transferred, and F is Faraday constant. Eo used for glutathione and cysteine redox couples was −264 mV and −250 mV, respectively. Less negative Eh numbers implied a more oxidized state.Markers used to measure systemic oxidative stress were the same as those we have characterized previously : redox p ratios) , DROMs [ ratios) , and Iso ratios) . The samFor measurement of IsoPs, plasma samples were acidified and a deuterated standard was added. This was followed by C-18 and Silica Sep-Pak extraction . IsoPs w μL) was diluted 1 : 5 parts with phosphate buffered saline (PBS) and incubated at 37°C with 200 μL of substrate for 2 hours. ACE activity was determined fluorometrically with two different substrates, Hip-His-Leu  and Z-Phe-His-Leu , and expressed as mU/mL [ACE activity and protein levels were analyzed in 31 (15 cases and 16 controls) subjects not taking any form of RAS inhibitor, since these are known to alter the measures –27. Hepaas mU/mL , 29. Levas mU/mL –27.Western blot analysis was used to measure extracellular copper-zinc superoxide dismutase (ec-SOD) and the copper-delivering protein, ceruloplasmin (Cp), expression in representative samples from both groups. Briefly, plasma ec-SOD or Cp was concentrated by concanavalin-A sepharose chromatography, and protein expression was examined by immunoblotting with antibody against ec-SOD or ceruloplasmin  , 29.α-level of 0.05 [t-test for continuous variables and Chi-square/Fisher exact test for categorical variables. Nonparametric tests were used for variables with skewed distribution. Mean  levels of oxidative stress and ACE markers in cases and controls were compared using t-test for normally distributed variables and NPAR1WAY procedure (SAS software 9.1) for variables with skewed distribution. All variables significant on univariate analysis were entered into multiple logistic regression models to calculate adjusted odds ratios.Statistical analyses were performed using SAS software 9.1 . Sample size was based on a 0.90 power to detect the same difference that we observed in the least sensitive measure of oxidative stress in our previous study using a two-tailed  of 0.05 . Baselinp = 0.96), smoking (p = 1.00), and diabetes mellitus (p = 0.77). The mean age of cases and controls was 64.8 ± 10.8 years (range: 45–83 years) and 65.0 ± 11.3 years (range: 43–88 years), respectively.We enrolled 50 patients with and without echocardiographic evidence of DD in HFpEF. The groups were well matched for known confounders in measurement of oxidative stress markers, including age (model 1) using all demographic and clinical parameters as predictive variables  in patients with early DD . There was no significant difference in Eh GSH , a less sensitive measure of plasma redox state [p = 0.03) and DROMs  were significantly lower in cases. The association between a more reduced Eh CyS and DD was maintained on a multivariate analysis (model 2) using gender, BMI, and oxidative stress measures as predictive variables .ox state . IsoPs lp = 0.1). ACE protein levels were only marginally higher in patients with early DD .ACE activity, determined with HHL as substrate, demonstrated a mild but statistically insignificant increase in cases compared to controls   . Since eIn this study, we found no evidence of significant RAS activation or systemic oxidative stress in cases compared to matched controls. This is consistent with a lack of effect of RAS inhibitors and antioxidants in DD –12, 30. There is a growing evidence that ROS signaling is compartmentalized . It is pAlternatively, the lack of RAS activation and systemic oxidation may imply that other factors might be responsible for the onset of DD in HFpEF. Obesity has been associated with DD and may be one such factor , 36. Ourα, and thiobarbituric acid reactive substances (TBARS) in eighteen HFpEF patients and 14 controls. The authors concluded that HFpEF exhibits an elevation in a broad spectrum of biomarkers indicative of an inflammatory and a prooxidative state [Few recent studies have found an association of systemic oxidative stress with HFpEF. In their study –45, Vitive state . Of noteve state . Our stuve state –12.There are limitations to our study. Foremost, despite consistency with previous clinical trials, it is possible that our study did not have sufficient power to detect associations between RAS or oxidative stress and HFpEF. On the other hand, a similar sized study easily detected a difference in oxidative stress markers in patients with and without AF . Blood sIn conclusion, we did not find evidence of systemic RAS activation or oxidative stress in patients with early HFpEF. This finding is consistent with the lack of efficacy of RAS inhibitors in the treatment of HFpEF. The lack of RAS activation and systemic oxidation seems to differentiate HFrEF from HFpEF. This suggests different mechanisms in genesis or propagation of these two forms of HF, which would explain the difference in benefit with treatment modalities.


Our aim was to assess the impact of increase in homeostasis model assessment of insulin resistance (HOMA-IR) on the development of type 2 diabetes in Japanese individuals with impaired insulin secretion (IIS). This study included 2,209 participants aged 30–69 without diabetes at baseline who underwent comprehensive medical check-ups between April 2006 and March 2007 at Saku Central Hospital. Participants were classified into eight groups according to the combination of baseline IIS status (non-IIS and IIS) and category of HOMA-IR change between the baseline and follow-up examinations . Type 2 diabetes was determined from fasting and 2 h post-load plasma glucose concentrations at the follow-up examination between April 2009 and March 2011. At baseline, 669 individuals (30.3%) were classified as having IIS. At follow-up, 74 individuals developed type 2 diabetes. After adjusting for confounding factors including baseline HOMA-IR values, the multivariable-adjusted odds ratios  for type 2 diabetes in the non-IIS with a decrease (mean change in HOMA-IR: −0.47), non-IIS with a moderate increase (mean change in HOMA-IR: 0.28), non-IIS with a large increase (mean change in HOMA-IR: 0.83), IIS with a decrease (mean change in HOMA-IR: −0.36), IIS with no change/small increase (mean change in HOMA-IR: 0.08), IIS with a moderate increase (mean change in HOMA-IR: 0.27), and IIS with a large increase (mean change in HOMA-IR: 0.73) groups, relative to the non-IIS with no change/small increase (mean change in HOMA-IR: 0.08) group were 0.23 , 1.22 , 2.01 , 1.37 , 3.60 , 5.24 , and 7.01 , respectively. Moderate and large increases in HOMA-IR had a strong impact on the development of type 2 diabetes among individuals with IIS in this Japanese population. The incidence of type 2 diabetes is significantly increasing in Asian countries We recently reported on the impact of IIS and IR on the incidence of type 2 diabetes in a Japanese population The Saku study included community residents who underwent comprehensive medical check-ups for the prevention and early detection of various diseases, including diabetes, cardiovascular disease and cancer, at Saku Central Hospital, one of the core hospitals in the Nagano prefecture, located in Saku city, Japan. The full details of this study have been previously described The study protocol was in accordance with the Declaration of Helsinki and was approved by the Ethics Committee of Saku Central Hospital. Written informed consent was obtained from each participant.2). Waist circumference was measured around the abdomen at the level of the navel at the late expiratory phase using a tape measure. Body fat percentage was evaluated by the bioelectric impedance method with an automatic scale. Blood pressure was measured by trained nurses using an automatic sphygmomanometer, with the individual in the sitting position after resting for at least 5 min. Each check-up also included standard questionnaires on demographic characteristics, medical history, family history and health-related habits. Alcohol consumption (ethanol) was categorised as 0 g/week, 1–139 g/week, or ≥140 g/week, and exercise was categorised as 0 min/week, 1–119 min/week, or ≥120 min/week.All participants were fasted overnight (12 h), and had a standard 75 g oral glucose tolerance tests (OGTTs) in the morning. Blood samples were obtained at 0 (fasting), 30, 60 and 120 min, with PG measured on all four occasions and serum insulin concentrations measured at 0 and 30 min in the clinical laboratory of Saku Central Hospital. Serum insulin concentrations were measured using a chemiluminescence enzyme immunoassay . Blood glucose, high density lipoprotein cholesterol, and serum triacylglycerol concentrations were measured by enzymatic methods. Weight, height, waist circumference and body fat percentage were measured in the morning during the fasting state. BMI was calculated as the weight (kg) divided by the height squared (m30 [pmol/l]−Insulin0 [pmol/l])/(Glucose30 [mmol/l]−Glucose0 [mmol/l]) 0 (µU/ml) Glucose0 (mmol/l)/22.5 Figure S1 in Type 2 diabetes was determined using the 1999 World Health Organization criteria χ2 tests for dichotomous and categorical data.Differences in baseline characteristics among the eight groups were determined by: analysis of covariance with adjustments for age and sex for normally distributed continuous data; Kruskal-Wallis H tests for non-normally distributed continuous data; and Logistic regression analysis was used to estimate the multivariable-adjusted odds ratios (ORs) and 95% confidence intervals (CIs) for the development of type 2 diabetes among the eight groups. The non-IIS with no change/small increase in HOMA-IR group was used as the reference group. Age, sex, and follow-up years (3 or 4 years) were included in model 1; and all factors in model 1 plus family history of diabetes (yes or no), current smoking (yes or no), alcohol consumption , exercise , baseline HOMA-IR, FPG, and 2 h PG were included in model 2. Moreover, we added eight individuals who received medical treatment for diabetes between the baseline and follow-up examinations. In these 2,217 individuals, data were adjusted for all factors in model 2.In order to assess the impact of severe IIS and HOMA-IR increases on the development of type 2 diabetes, baseline IIS status was classified into stage 1 (insulinogenic index 35.5–51.7 pmol/mmol) and stage 2 (insulinogenic index 2.8–35.4 pmol/mmol) using the median of the insulinogenic index (35.5 pmol/mmol). Participants were classified into 12 groups according to the combination of baseline IIS status  and category of ΔHOMA-IR . Logistic regression analysis was used to estimate the multivariable-adjusted ORs and 95% CIs for the development of type 2 diabetes among these 12 groups. The non-IIS with no change/small increase in HOMA-IR group was used as the reference group. Data were adjusted for age, sex, follow-up years, family history of diabetes, current smoking, alcohol consumption, exercise, baseline HOMA-IR, FPG, and 2 h PG.Additionally, in order to confirm the risk factors for an increase in HOMA-IR in the non-IIS and IIS groups, multivariable-adjusted ORs and 95% CIs for moderate and large increases in HOMA-IR according to major risk factors for diabetes were calculated using multinomial logistic regression analysis in the non-IIS and IIS groups.p values are two-tailed; values <0.05 were considered statistically significant.All data were analysed using SPSS statistical software . All reported 2, mean HOMA-IR 1.16 (1.26 in the non-IIS group and 0.91 in the IIS group), and median insulinogenic index 74.9 pmol/mmol (99.7 pmol/mmol in the non-IIS group and 35.5 pmol/mmol in the IIS group). At baseline, 669 individuals (30.3%) were classified as having IIS. The mean age of the 2,209 participants was 55.5 years, mean BMI 23.1 kg/mAt follow-up examination, 74 individuals  developed type 2 diabetes. Because the youngest of these individuals was 40 years old at baseline examination, all incident cases were assumed to be type 2 diabetes. Table S1 in This community-based cohort study clearly shows the relationship of HOMA-IR increases on the development of type 2 diabetes in Japanese individuals with IIS. Our main finding was that, after adjusting for confounding factors including baseline HOMA-IR values, the ORs for type 2 diabetes were 5.24 in the IIS with a moderate increase group and 7.01 in the IIS with a large increase group when compared with the non-IIS with no change/small increase group. Additionally, when IIS was classified into stage 1 (insulinogenic index 35.5–51.7 pmol/mmol) and stage 2 (insulinogenic index 2.8–35.4 pmol/mmol), the ORs for type 2 diabetes were 3.93 in the stage 1 IIS with a large increase group, 5.07 in the stage 2 IIS with no change/small increase group, 9.19 in the stage 2 IIS with a moderate increase group, and 15.00 in the stage 2 IIS with a large increase group, relative to the non-IIS with no change/small increase group.Our findings indicate that moderate and large increases in HOMA-IR had a strong impact on the development of type 2 diabetes among individuals with IIS in this Japanese population, regardless of baseline HOMA-IR, FPG, and 2 h PG. On the other hand, moderate and large increases in HOMA-IR did not increase the risk for type 2 diabetes in those without IIS. Each mean HOMA-IR increment in the IIS with a moderate increase group (0.27) and a large increase group (0.73) was similar to that in the non-IIS with a moderate increase group (0.28) and a large increase group (0.83). Therefore, it is thought that those with IIS cannot compensate for moderate and large increases in HOMA-IR  because of their low insulin-secreting ability. When we calculated the value corresponding to the maximum Youden index (sensitivity + specificity − 1) using receiver operating characteristic curves analysis, the optimal cut-off point for ΔHOMA-IR was 0.24 in the IIS group (data not shown). It is necessary for those with IIS to be more careful about increases of IR. On the other hand, it is thought that non-IIS individuals with moderate and large increases in HOMA-IR might have delayed onset of diabetes compared with those with IIS. It has been reported that the development of diabetes following the development of IR can take several years The association between moderate and large increases in HOMA-IR and the development of type 2 diabetes was strengthened in the stage 2 IIS group, i.e., severe IIS. The impact of an increase in HOMA-IR on the development of type 2 diabetes in Japanese individuals would differ depending on the degree of insulin-secreting ability. Therefore, both the assessment of insulin-secreting ability in individuals without diabetes and the stricter prevention of IR increases in those with low insulin-secreting ability will be necessary for the prevention of type 2 diabetes in Japanese individuals. Regarding the prevention of IR increases, weight gain over a short period of time was associated with increased IR development regardless of baseline BMI status KCNQ1 and KCNJ15 variants have been reported to affect the development of type 2 diabetes by impairing beta cell function The risk for type 2 diabetes increased in the stage 2 IIS group  with no change/small increase in HOMA-IR. Insulin secretion in Japanese individuals is reported to be less than half that of whites Of the 300 individuals with IIS for whom we recorded a decrease in HOMA-IR, 17 (5.7%) developed type 2 diabetes despite the decrease in their HOMA-IR. At baseline, 12 (70.6%) of these 17 individuals had pre-diabetes, and 6 (35.3%) had a family history of diabetes (data not shown). Additionally, the mean HOMA-IR of these 17 individuals was 1.52, and the median insulinogenic index was 22.4 pmol/mmol (data not shown). It is thought that these 17 individuals with IIS with a decrease in HOMA-IR developed type 2 diabetes as a result of their baseline status, especially the high proportion of pre-diabetes, HOMA-IR value, and low insulin secretion.The strengths of the present study include its community-based cohort, which consisted of residents of many cities throughout Nagano prefecture. Furthermore, we screened all participants for type 2 diabetes by the 75 g OGTT. The 12 h overnight fast before the OGTT was managed by hospitalising participants the day before the test. This study, however, also had several limitations. First, the estimates of insulin secretion and IR were made using calculations based on the OGTT, not by the ‘gold standard’ test, i.e., the glucose-clamp technique. However, the glucose-clamp technique is not feasible in large-scale epidemiological studies, and we believe that proxy measures are reliable in large datasets. Second, estimation of IR by calculating the HOMA-IR, which primarily reflects hepatic IR In conclusion, moderate and large increases in HOMA-IR had a strong impact on the development of type 2 diabetes regardless of baseline HOMA-IR values among individuals with IIS in this Japanese population. The National Nutrition Survey, a nationwide survey in Japan, has reported an increase in the number of overweight Japanese men during the past 20–30 years because of changes in eating habits and decline in physical activity File S1Combined supporting information file. Table S1. ORs for the development of type 2 diabetes according to baseline IIS status and another category of ΔHOMA-IR . Note: IIS, impaired insulin secretion; HOMA-IR, homeostasis model assessment of insulin resistance; OR, odds ratio; CI, confidence interval. Non-IIS, insulinogenic index >51.7 pmol/mmol (40.0 µU/mg); IIS, insulinogenic index ≤51.7 pmol/mmol (40.0 µU/mg). Δ = follow-up examination minus baseline examination. ΔHOMA-IR was analyzed by analysis of covariance with adjustments for age and sex, and is shown as age- and sex-adjusted mean . Model 1 was adjusted for age, sex, and follow-up years (3 or 4 years). Model 2 was adjusted for all factors in model 1 plus family history of diabetes (yes or no), current smoking (yes or no), alcohol consumption , exercise , baseline HOMA-IR, fasting plasma glucose, and 2 h post-load plasma glucose. Figure S1. Definition of IIS and category of HOMA-IR change. Note: IIS, impaired insulin secretion; HOMA-IR, homeostasis model assessment of insulin resistance. Figure S2. HOMA-IR (A-1 and A-2), BMI (B-1 and B-2), and waist circumference (C-1 and C-2) at baseline and follow-up examinations among individuals who developed type 2 diabetes  and individuals who maintained normal glucose regulation  in the non-IIS and IIS groups. Note: IIS, impaired insulin secretion; HOMA-IR, homeostasis model assessment of insulin resistance; BMI, body mass index. Data are shown as age- and sex-adjusted means . Incident cases in the non-IIS group: n = 22; controls in the non-IIS group: n = 1,518; incident cases in the IIS group: n = 52; controls in the IIS group: n = 617.(PDF)Click here for additional data file.


Sufficient secreted TNF-α produced also activated IL-1β and IL-8 expression in wt HaCaT cells. Importantly, stimulated expression of IL-1β and IL-8 in HaCaT-TNF-α were blocked by Quercetin, a flavanol shown to possess anti-TNF-α activities. This novel in vitro cell model provides an efficient tool to investigate the dual signaling of TNF-α. Importantly, this model provides an effective, fast, and simple screening for compounds with anti-TNF-α activities for chronic inflammatory disease therapies.Tumor necrosis factor alpha (TNF-α) is a pro-inflammatory cytokine important in normal and pathological biological processes. Newly synthesized pro-TNF-α is expressed on the plasma membrane and cleaved to release soluble TNF-α protein: both are biologically active. Secreted TNF-α signals through TNF receptors and the membrane-bound TNF-α acts by cell contact-dependent signaling. Anti-TNF-α antibodies have been used effectively for treatment of chronic inflammation, however with adverse side effects. Thus, there is a need for new anti-TNF-α small molecule compounds. Anti-TNF-α activity assays involve treatment of keratinocytes with exogenous TNF-α before or after anti-TNF-α incubation. However, this model fails to address the dual signaling of TNF-α. Here we describe a Doxycycline (Dox)-inducible TNF-α (HaCaT-TNF-α) expression system in keratinocytes. Using this  Chlamydia pneumonia, has been shown to increase coronary heart disease risk [Inflammation is an essential innate immunity response that is crucial to combat pathogens. However, dysregulated and untimely inflammation contributes to several chronic inflammatory diseases such as psoriasis, atopic dermatitis, rheumatoid arthritis, coronary heart diseases, Crohn’s disease and cancer –3. For ease risk . Local iase risk , Given tase risk , 7. NeveThe inflammation process involves intricate networking and interactions between immune cells and pro-inflammatory mediators such as Interleukin (IL)-1, IL-6, nuclear factor kappa-light chain enhancer of activated B cell (NF-κB) and tumor necrosis factor alpha (TNF-α) . TNF-α iPathophysiological conditions of psoriasis involves hyperproliferation of keratinocytes in combination with abnormal differentiation of the epidermis layer and infiltration of inflammatory cytokines secreted from activated T cells or antigen presenting cells from the dermis and epidermis cell layers. TNF-α is one of the most important molecules involved the induction and progression of psoriasis and stimulates the production of inflammatory cytokines such as IL-6, IL-8 and granulocyte macrophage colony stimulating factor (GM-CSF) resulting in an accumulation of inflammatory cells in the dermis and epidermis layers of psoriatic lesions . BlockagTo minimize immunological and other adverse effects caused by anti-TNF-α Mabs an alternative approach, with potentially fewer immune-mediated side effects, is the use of small chemical molecules acting as TNF-α antagonists by blocking TNF-α activities. These TNF-α antagonists may be used as a complementary treatment for chronic inflammatory diseases. There are several advantages of developing small-molecular TNF-α antagonists including, targeting of specific signaling pathways, high tissue penetration, ease of use and economical manufacturing .Synthesis and secretion of TNF-α involves a two-step process. Newly synthesized pro-TNF-α is expressed on the plasma membrane and cleaved by TNF-α converting enzyme or (TACE or ADAM-17) to release a soluble TNF-α protein , 21. Botin vitro cell-based model used for anti-TNF-α activity screening in keratinocytes (HaCaT cells) involves treating cells with recombinant purified TNF-α before or after treatment with chemical compounds or extracts [in vitro cell models are limited. In many chronic inflammatory diseases, such as psoriasis, rheumatoid arthritis and inflammatory bowel diseases, cells themselves express both membrane bound and secreted TNF-α, suggesting TNF-α exerts its biological actions in these cells through the dual action of both forms of TNF-α (membrane bound and secreted). Addition of exogenous TNF-α or the secreted form of TNF-α activates TNF-α receptor-mediated signaling, nevertheless there is no evidence to suggest that contact-dependent signaling mediated by membrane bound TNF-α is affected. Therefore, anti-TNF-α activities assayed by current in vitro cell models may lack an important signaling component mediated by membrane bound TNF-α.However, the common and distinct biological functions of membrane-bound and secreted TNF-α remains to be determined. The current extracts –29. Howein vitro cell-based model for the identification of novel small-molecule TNF-α antagonists, we constructed inducible TNF-α keratinocyte (HaCaT) cell lines that mimic expression of endogenous TNF-α from activated keratinocytes in vivo, allowing evaluation of both membrane-bound and secreted components of TNF-α signaling. We show that TNF-α is induced by doxycycline (Dox) in a dose-dependent manner and demonstrate that ectopic expression of TNF-α in keratinocytes produces comparable TNF-α-mediated responses. Importantly, our results demonstrate that these responses are attenuated by a known TNF-α blocker in this newly developed cell model, similar to those of keratinocytes treated with exogenous TNF-α. This new in vitro cell model provides an efficient system to explore TNF-α downstream signaling events and inflammatory responses. Importantly it provides a fast and convenient way to screen, identify and evaluate anti-TNF-α small molecules.To provide an alternative and more effective 2. All cultures were routinely tested and were mycoplasma-free.Human embryonic kidney (HEK293T) cells were obtained from American Type Culture Collection (ATCC) and used for lentiviral production. HEK293T were cultured in Dulbecco's modification of Eagle's medium  supplemented with 10% fetal bovine serum  and 1% penicillin streptomycin (PenStrep) . HaCaT cells, immortalized human epidermal keratinocytes , were puTo construct the tetracycline (Tet)-inducible vector TNF-α, a pHAGE-TNF-α encoding TNF-α was synthesized. The hTNF-α cDNA was PCR amplified from pMD18-T-hTNF-α cDNA  using a TNF-α specific forward primer (5’-GAT CGC GGC CGC GAC ACC ATG AGC ACT GAA AGC ATG ATC-3’) and a TNF-α specific reverse primer (5’-GAT CGG CGC GCC AGG GCA ATG ATC CCA AAG T-3) containing restriction sites for NotI and AscI respectively. Cycling conditions were as follows: an initial denaturing step , amplification 30 cycles of 45 sec, denaturation at 98°C, 45 sec of annealing at 60°C, 50 sec of extension 72°C and final extension step  using a Thermal Cycler . The PCR products were separated by electrophoresis on a 1% agarose gel and visualized by ethidium bromide staining. The resulting PCR products were further purified using QIAquick gel extraction kit  according to the manufacturer's instructions. PCR products were digested with NotI/AscI  and inserted into NotI/AscI digested pENTR/D-TOPO  to generate pENTR/D-TNF-α. cDNA was then cloned into the attR1 and attR2 sites of pHAGE-Dest, ). Constitutive expression of the neomycin resistance gene is driven by the ubiquitin C (Ubc) promotor  using th5 cells in 6-well tissue culture plates to achieve 50–60% confluency per well and incubated for 24 hr. Cells were treated with Opti-MEM I medium  mixed with packaging plasmid (psPAX2), envelope plasmid (pMD2G), DNA construct (pHAGE-TNF-α or pHAGE-GFP) and X-tremeGENE HP DNA transfection reagent . psPAX2 and pMD2G plasmids were gifts from Didier Trono . The ratio of lentiviral DNA construct: psPAX2: pMD2G was 1:2:2 and the ratio of the DNA construct: XtremeGENE HP DNA transfection reagent was 1:3.5. Cells were incubated with DNAXtremeGENE mixture for 48 hr at 37°C, 5% CO2. Medium containing viral particles were collected through a 0.45 μM sterile filter PVDF membrane  and stored at -80°C [To generate lentiviral particles, 6-well tissue culture plates coated with 1 mL of 0.1mg/mL poly-D-lysine solution  were incubated for least 4 hr at room temperature (RT). Plates were washed four times with 1X Phosphate buffer saline (PBS) , PBS was removed and plates air-dried under sterile conditions. HEK293T cells were seeded at 1x10at -80°C .5 cells in 35 x 10 mm cell culture dishes and incubated 24 hr to achieve 50–60% confluency at 37°C and 5% CO2. Cells were treated with 1 mL of medium containing virus pHAGE-TNF-α viral particle mixed with 8 μg/mL of polybrene transfection reagent [HaCaT cells were seeded at 1x10 reagent   were added to HaCaT-TNF-α cells to induce the expression of TNF-α proteins. TNF-α protein expression in cell models was detected by western blotting as previously described , 34. Bri2, 1 mM EDTA, 1 mM EGTA) containing protease inhibitor cocktail  added immediately and solution placed on ice. Cells were harvested by scraping with a cell scraper to collect cell lysate, and transferred to 1.5 ml tubes and rotated end-over-end at 4°C for 30 min. Lysates were centrifuged at 720 x g at 4°C for 5 min and the supernatant was transferred to a new 1.5 ml tube and centrifuged, 10,000 x g at 4°C for 10 min. The supernatant containing cytosolic and membrane fraction was transferred to a new 1.5 ml tube and centrifuged in an ultracentrifuge, 100,000 x g at 4°C for 1 hr before carefully transferring the supernatant, cytosolic fraction, to a new 1.5 ml tube. The pellet was washed and resuspended with SF buffer and centrifuged, 100,000 x g at 4°C for 1 hr. The supernatant was removed and the remaining membrane fraction pellet resuspended in RIPA buffer  containing protease inhibitor cocktail . Twenty micrograms of protein per sample was loaded onto 12% SDS-PAGE and analyzed by Western Blot as previously described. GAPDH was used as a loading control for cytosolic protein contamination.Subcellular fractionation and isolation of the membrane-rich fraction was carried out by differential centrifugation as previously described . BrieflyDox-induced TNF-α was secreted from HaCaT-TNF-α cell cultures and supernatants and analyzed after 24, 48, 72, and 96 hrs. TNF-α was measured by ELISA assays  as recommended by the manufacturer. Briefly, the ELISA plate was coated with 1 μg/mL capture antibody (antigen-affinity purified rabbit anti-hTNF-α and 2.5mg D-mannitol) for 24 hr at RT. After 24 hr the plate was washed with wash buffer (0.05% Tween-20 in PBS) four times and then blocked with blocking buffer (1% BSA in PBS) for 1 hr RT. After blocking, the plate was washed with wash buffer. Human TNF-α standard was diluted to concentrations 0–2 ng/mL in diluent (0.05% Tween-20 in 0.1% BSA-PBS) and standards and samples were added to each well in triplicate for 2 hr at RT. The ELISA plate was washed four times and 0.5 μg/mL detection antibody (biotinylated antigen-affinity purified rabbit anti-hTNF-α + 2.5mg D-mannitol) added for 2 hr and washed four times. Avidin-HRP conjugate (1:2000) was added and incubated for 30 min and then washed four times. Finally, ABTS substrate was added into each well and color development monitored with an ELISA plate reader  at 405 nm. The plate was monitored at 5 min intervals for approximately 30 min. For quantitative analysis a standard curve was generated for each assay using serially diluted hTNF-α protein standard concentration from 0–2 ng/mL for a total of 8 data points.HaCaT-TNF-α and–GFP cells were seeded in DMEM supplemented with 10% FBS and 1% PenStrep at 2,000 cells per well in a 96-well tissue culture plate and incubated for 24 hr to achieve 50–60% confluence. Next day, cells were induced with increasing concentration of Dox  for 24, 48, 72 and 96 hrs. In some cases, cells were treated with 10 ng/mL TNF-α for 24, 48, 72 and 96 hrs. Following treatments, 20 ul per well of 50 mg/mL MTT -2,5-diphenyltetrazolium bromide) solution  were added to cells and incubated for 4 hr until purple precipitate was visible. Precipitates were dissolved with DMSO, centrifuged at 1000g for 10 min and were transferred to a new 96-well plate and measured using a microplate spectrophotometer  absorbance 550 nm. All experiments were carried out in quadruplets and data was calculated from three independent experiments as mean ± SEM. Results are presented as % cell viability as compared to the control value of each experiment.-ΔΔCt method [Total RNA was extracted from HaCaT-TNF-α cell lines using Genezol reagent . cDNA was synthesized from 1 μg of total RNA using Accupower RT Premix  with oligo (dT) 18 primer following the manufacturer's protocol. Quantitative real-time PCR for IL-1β, IL-6, IL-8, NF-κB1, KRT16, FOSL1, MMP9 and GAPDH was performed by using Green Star PCR Master Mix  and gene-specific primers, as listed in t method .Results were expressed as the mean ± standard error of means (S.E.M.) of at least three independent experiments. Differences between the control and variable conditions were determined by a two-tailed Student’s t-tests and one way ANOVA and results were considered to be significant at P < 0.05 or P < 0.01.in vitro TNF-α producing keratinocyte model, we constructed an in vitro cell model to study TNF-α function in HaCaT cells. HaCaT, an immortalize and spontaneously transformed human keratinocyte cell line [To develop a novel ell line , was choell line –40. In tell line . Human TTo test whether TNF-α protein was induced by Dox treatment, HaCaT-TNF-α and -GFP control cells were treated with increasing concentrations of Dox for 48 hr. Cells were harvested and analyzed for TNF-α expression by western blotting analysis using a TNF-α specific monoclonal antibody as described in Materials and Methods. Dox treatment (3 μg/mL) did not induce TNF-α expression in HaCaT-GFP cells . HoweverPrevious studies showed that newly synthesized TNF-α is expressed on plasma membranes and subsequently cleaved by TNF-α converting enzyme releasing soluble TNF-α proteins into the cell growth medium , 21. To To determine how Dox-induced TNF-α affected keratinocyte cell viability, HaCaT-GFP (control) and HaCaT-TNF-α cells were treated with increasing doses of Dox (1–3 μg/mL) or recombinant TNF-α (10 ng/mL) for 24–96 hrs. Percent cell viability was determined by MTT assays as described in Materials and Methods. In the control HaCaT-GFP cells TNF-α treatment significantly decreased cell viability  whilst tTNF-α promotes and maintains chronic inflammation conditions such as psoriasis through induction of several pro-inflammatory cytokines . This prTNF-α is initially synthesized as pro-TNF-α, expressed on the plasma membrane and cleaved by ADAM-17 before releasing the soluble TNF-α protein , 23. We In addition to producing secreted soluble TNF-α as shown in We next examined whether the HaCaT-TNF-α cell model could be used to assess the anti-TNF-α activity of a chemical compound Quercetin. Quercetin is a flavonoid that can be found in several types of fruits, vegetables, leaves and grains and has been shown to possess anti-psoriatic and anti-inflammatory activities , 41 To din vitro models used to study TNF-α and anti-TNF-α activities involve treating cells with exogenous recombinant TNF-α and examining changes associated with the TNF-α treatment [Dysregulation or untimely inflammation can lead to many diseases such as Alzheimer’s, cancer cardiovascular, arthritis, inflammatory bowel diseases, and psoriasis , 5. TNF-reatment –29. Whilreatment , 42. Thein vitro cell models [To develop a new and improved model to investigate TNF-α and anti-TNF-α activities that would enable examination of TNF-α dual signaling from both membrane-associated and cleaved TNF-α, we took advantage of lentiviral technology and constructed epidermal keratinocytes (HaCaT cells)  expressil models –29. Compin vitro cell models that use only exogenous TNF-α to activate TNF-α signaling.How TNF-α mediated cell signaling in keratinocytes is unclear. A previous study showed that TNF-α upregulated its own mRNA through an autocrine mechanism but soluble TNF-α was undetected in the conditioned medium. Autocrine upregulated TNF-α mRNA levels declined sharply upon removal of TNF-α . MembranIn addition to inflammation, keratinocyte hyperproliferation and abnormal differentiation are often observed in psoriatic skin lesions , 46. Howin vitro cell models. Furthermore, this inducible HaCaT-TNF-α lentivirus system can be applied to any cell model, is not confined to one specific cell line. Therefore, an important application for this system, inducible TNF-α can be used to further investigate the dual cell signaling pathways in different inflammatory disease states. A future application for Dox-inducible TNF-α model is the analyzes of TNF-α activities and screening of small molecule compounds for anti-TNF-α activities: providing an effective, fast, and simple in vitro screening for compounds with anti-TNF-α activities for chronic inflammatory disease therapies.We propose that this newly developed HaCaT-TNF-α cell model, whereby expression of TNF-α can be controlled by variable Dox concentrations, can be used as an alternative or a complementary cell system to the current


What is the basis of the human capacity for language: Is language shaped only by sensorimotor constraints and experience, or are some aspects of language universal, abstract, and potentially amodal? The set of papers assembled in this collection represent state of the art research on this age-old set of questions.To gauge the universality of language structure and its abstraction, the first group of papers examines the grammatical organization of mature languages across modalities. The papers by Baus et al.  and GuelFurther insights into grammar and its origins are presented in papers on the genesis of sign languages in Deaf communities and in individual homesigners . Given the poverty of linguistic input available to these individuals, and the fact that the manual modality affords iconic depiction, we might expect emerging sign languages to be overwhelmingly iconic. But the role of iconicity is actually far more constrained and nuanced than one might have presumed.prior to the arguably more iconic organization of morphosyntax . Moving to another example, this time a sign language that is growing up in Nicaragua, Kocab et al.  vs. associationist accounts of spoken language into the domain of sign language—what computational mechanisms are used by signers to support linguistic productivity? The papers by Caselli and Cohen-Goldberg , on one Our review has so far highlighted commonalities across different language modalities and different levels of experience. But the effects of modality and experience are undeniable and significant—the papers by Supalla et al.  and EmmoThe final four papers in this volume consider the development of sign languages and their evolution. Morgan  argues tThe 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.


Front. Plant Sci. 7:88. doi: 10.3389/fpls.2016.00088). I wholeheartedly agree with Schubert that far too many reviews are getting published. Many are what I call “catalog” reviews, and any good Ph.D student worth his salt can, with the help of PubMed, make a compilation of the recent (last 5 years) literature on a particular topic within days. Reviews of this kind seldom draw attention to areas of conflict, rarely provide insight into technical difficulties, and often fail to point to challenges for future research.I read with interest Ingo Schubert's short article on the inflationary number and value of review papers , they should make the effort to read and evaluate the original papers they cite rather than simply cite what was previously cited by others. Above all, they need to accomodate the reader's desire to be directed to controversies, but also to take a standpoint on them. These may seem banal criteria, but they remain frequently unfulfilled. The essence of a good review is not so much as to highlight individual papers but to critically evaluate all the relevant papers on a particular subject.Would like to draw attention to an alternative to a high profile review to focus interest on controversial topical issues. Essentially this is an open debate with the conflicting parties putting their case in writing for the scientific public to judge for themselves whose arguments are the more convincing. I have tried this out twice, in both cases with an extremely positive feedback from reviewers/editors. My first attempt, over 20 years ago, was to put the spotlight on the auxin binding protein (ABP; Hertel, More recently, I managed to persuade three experienced colleagues to voice their opinions on vesicles vs. tubes as a means of transport between the endoplasmic reticulum and the Golgi apparatus (Robinson et al., The author confirms being the sole contributor of this work and approved it for publication.The author declares 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 treatment of traumatic brain injury (TBI) represents an unmet medical need, as no effective pharmacological treatment currently exists. The development of such a treatment requires a fundamental understanding of the pathophysiological mechanisms that underpin the sequelae resulting from TBI, particularly the ensuing neuronal cell death and cognitive impairments. Tumor necrosis factor-alpha (TNF-α) is a cytokine that is a master regulator of systemic and neuroinflammatory processes. TNF-α levels are reported to become rapidly elevated post TBI and, potentially, can lead to secondary neuronal damage.To elucidate the role of TNF-α in TBI, particularly as a drug target, the present study evaluated (i) time-dependent TNF-α levels and (ii) markers of apoptosis and gliosis within the brain and related these to behavioral measures of ‘well being’ and cognition in a mouse closed head 50 g weight drop mild TBI (mTBI) model in the presence and absence of post-treatment with an experimental TNF-α synthesis inhibitor, 3,6′-dithiothalidomide.mTBI elevated brain TNF-α levels, which peaked at 12 h post injury and returned to baseline by 18 h. This was accompanied by a neuronal loss and an increase in astrocyte number  and glial fibrillary acidic protein (GFAP) immunostaining), as well as an elevation in the apoptotic death marker BH3-interacting domain death agonist (BID) at 72 h. Selective impairments in measures of cognition, evaluated by novel object recognition and passive avoidance paradigms - without changes in well being, were evident at 7 days after injury. A single systemic treatment with the TNF-α synthesis inhibitor 3,6′-dithiothalidomide 1 h post injury prevented the mTBI-induced TNF-α elevation and fully ameliorated the neuronal loss (NeuN), elevations in astrocyte number (GFAP) and BID, and cognitive impairments. Cognitive impairments evident at 7 days after injury were prevented by treatment as late as 12 h post mTBI but were not reversed when treatment was delayed until 18 h.These results implicate that TNF-α in mTBI induced secondary brain damage and indicate that pharmacologically limiting the generation of TNF-α post mTBI may mitigate such damage, defining a time-dependent window of up to 12 h to achieve this reversal. Traumatic brain injury (TBI) is a common cause of morbidity and mortality across both the civilian and military populations, with a reported worldwide annual incidence of some ten million cases . Indeed,mTBI-associated brain damage can be subdivided into two phases: an initial primary phase that is immediate and results from the mechanical force(s) applied to the skull and brain at the time of impact, potentially inducing shearing and compression of neuronal and vascular tissue that results in brain contusion, axonal injury, blood vessel rupture, and hemorrhage. This is followed by an extended second phase that involves cascades of biological processes initiated at the time of injury that may persist over subsequent days, weeks, and possibly months, consequent to ischemia, neuroinflammation, glutamate toxicity, altered blood-brain barrier permeability, oxidative stress, astrocyte reactivity, cellular dysfunction, and apoptosis -22. As sIt is widely recognized that inflammatory cytokines, chemokines, and growth factors play significant roles in the pathophysiology of TBI. Albeit that initiation of an inflammatory response can be essential to promote neuroreparative mechanisms in response to a physiological insult -28, if tInhibiting the generation of TNF-α may thus reinforce its role in mTBI and define its value as a potential treatment target, as it is considered a master regulator of the inflammatory response. Sudden and substantial rises in TNF-α can induce a diverse array of cell death processes, including NF-kB activation, apoptosis, and necrosis . In addiad libitum access to food and water, and kept on a 12:12 h light/dark cycle. Lighting during the light phase remained constant, and all experimental manipulations were undertaken during this light phase of the cycle. A minimum number of animals were included into studies, and all efforts were made to minimize potential suffering. Each animal was used for only a single experiment, and all experimental procedures and housing conditions were approved by the Institutional Animal Care and Use Committee of Tel Aviv University (M-10-006).Male ICR mice (30 to 40 g of weight and 6 to 8 weeks of age) were bred and raised within the vivarium of Tel Aviv University, Israel, originally derived from breeding pairs purchased from HSD Jerusalem, Israel. They were housed four to six per cage, maintained at a constant 22 ± 1°C, had Mice were subjected to mTBI using a weight drop device that has previously been described -46. MiceSynthesis of 3,6′-dithiothalidomide  was achieved by a published synthetic route , and chen = 4 to 5 per time). The right cortex was immediately frozen in liquid nitrogen and homogenized with appropriate protease inhibitors . The samples were then quantified for TNF-α levels by ELISA assay .To verify the occurrence of TNF-α elevation in our mTBI model and define its time dependence, mice were subjected to mTBI and brains were removed at specific times thereafter  were evaluated 30 min before 3,6′-dithiothalidomide administration and at 1 and 4 h following mTBI or sham procedure.v/v) between animals.Anxiety-like behavior and motor activity were evaluated by elevated plus maze. The maze was elevated 60 cm above the floor level and comprised of 4 arms (30 × 5 × 15 cm) along which mice could walk that formed a ‘+’ shape . Two conTwo behavioral paradigms were evaluated: Y-maze and novel object recognition (NOR).v/v).Spatial memory was evaluated by Y-maze, as initially described by Dellu and colleagues , and is v/v) between each trial. Object exploration (defined as rearing on the object or sniffing it at a distance of less than 2 cm and/or nose touching it) was recorded and discrimination of recognition novelty was determined as a preference index  or across measurement times (30 min before injury and 1 and 4 h post-mTBI/injection)  (data not shown).Subjectively and in accord with prior studies , mice suF = 0.791, NS] , respectively (data not shown). Likewise, no differences were evident between any groups in relation to motor skills evaluated at 72 h and 7 days post-injury [F = 1.13 NS] , respectively (data not shown). Together these result indicate that mice were healthy and that neither mTBI nor 3,6′-dithiothalidomide impacted their well-being.The elevated plus maze was used to examine anxiety-like behavior and motor activity. No differences were found between any groups in anxiety-like behavior at 72 h and 7 days post-injury . LSD post hoc analyses confirmed that the 12-h mTBI group was significantly different from all other groups (p < 0.0001). Levels were elevated to 132.9 pg/ml at 12 h versus a baseline value of 53.4 pg/ml. In animals subjected to mTBI and administered 3,6′-dithiothalidomide 1 h post injury, the elevated TNF-α 12 h post injury response was ameliorated. Specifically, mice treated with 3,6′-dithiothalidomide post injury had similar brain TNF-α levels as the sham group, 67.0 and 53.4 pg/ml, respectively, F = 14.579, p < 0.0001, Figure post hoc analyses confirmed that the mTBI 12-h group was significantly different from all other groups (p < 0.0001).As illustrated in Figure F3,3 = 30.52F = 6.462, p < 0.01] .When evaluated by Y-maze at 7 days post procedure, vehicle-treated mTBI-challenged mice demonstrated a significant impairment in spatial memory, as compared to sham control animals. This mTBI-induced deficit was ameliorated by a single dose of 3,6′-dithiothalidomide administered either 1 or 12 h post injury. However, when 3,6′-dithiothalidomide administration was withheld until 18 h, mice displayed impairment and, together with the mTBI vehicle group, their preference index was significantly reduced compared to sham controls . LSD post hoc analyses established that the mTBI + vehicle and the mTBI + 18 h 3,6′-dithiothalidomide groups were significantly different from all other groups (p < 0.05).As illustrated in Figure Together these results extend the work of Baratz and colleagues  and defiTo evaluate the impact of mTBI at the cellular level, particularly in relation to the described amelioration of cognitive deficits imparted by lowering TNF-α generation, immunohistochemical analyses were undertaken at 72 h post injury. These focused on two key brain areas ipsilateral to injury: the cerebral cortex, as the area closest to impact, and the dentate gyrus, a region of the hippocampal formation considered to contribute to the formation of new episodic memory ,53, the F = 7.198, p < 0.005, Figure F = 5.641, p < 0.05, Figure Post hoc analyses revealed that the mTBI alone group was different from all other groups (p < 0.05) in both brain regions and was reduced by 42.5% and 22.3% versus sham values in cortex and dentate gyrus, respectively. Correlated with this was an elevation in apoptotic cell number, as revealed from BID staining in the cortex [F = 23.067, p < 0.0001, Figure F = 6.301, p < 0.05, Figure post hoc analyses demonstrated that the mTBI group was different from all other groups . In addition and illustrated in Figures F = 37.641, p < 0.0001, Figure F = 13.284, p < 0.001, Figure F = 1.009, NS, Figure F = 2.251, NS, Figure Illustrated in Figures F,13 = 7.1In conclusion, the early administration (1 h post injury) of a single dose of the TNF-α synthesis inhibitor 3,6′-dithiothalidomide inhibited cellular changes induced by mTBI in two key brain regions evaluated, cerebral cortex and dentate gyrus.TNF-α has been implicated in the pathogenesis of a wide number of neurological disorders that develop both acutely, as in TBI and stroke, and chronically, as in Alzheimer’s disease and Parkinson’s disease -42,55,56In light of (i) the high incidence of mTBI ; (ii) the increased risk of dementia resulting from mTBI, particularly in the older people ; (iii) tTNF-α is a well-characterized protein that regulates numerous cellular processes, including inflammation and cell death as well as cellular differentiation and survival, by binding to and activation of two cognate receptors: TNF-α receptor 1 (TNFR1) (p55) and TNFR2 (p75) -31,61.TNFR1 is expressed ubiquitously, including neurons, astrocytes, and microglia throughout the brain. With its intracellular death domain, it contributes to neuronal dysfunction and death and primarily is activated by soluble TNF-α . TNFR2, To define the relationship between the mTBI-induced elevation in TNF-α and cognitive impairment evident 7 days later, 3,6′-dithiothalidomide was administered 1, 12, and 18 h following mTBI, extending our initial concentration-dependent studies of the compound in this same mTBI model . NotablyTo evaluate the basis of the mTBI-induced cognitive impairment, brain regions ipsilateral to the side of injury were evaluated at 72 h, as this time coincides with the substantial occurrence of markers of neuronal apoptosis ,82. AsseTaken together, these studies support an important role for TNF-α in neuroinflammation and the modulation of neuronal function and viability across a broad range of neurological disorders. Consequent to the availability of both biological and small molecular weight TNF-α inhibitors in preclinical and clinical research, there is growing evidence that whereas physiological TNF-α levels are critical in normal brain physiology ,38,55, eThe single dose of 3,6′-dithiothalidomide used in the current study (28 mg/kg in mouse) compares favorably with former studies of thalidomide (20 to 300 mg/kg) and equates to a dose of 150 mg in a 65 kg human (2.3 mg/kg), following normalization of body surface area in accord with FDA guidelines . Prior cIn summary, our studies suggest that the administration of a TNF-α synthesis inhibitor, 3,6′-dithiothalidomide, within the initial 12-h window of a mTBi event, may be therapeutically valuable at a time when elevated TNF-α interacts with TNFR1 to drive the development of neuroinflammation, neuronal dysfunction, and apoptosis. But such a therapeutic strategy should best be acute to allow later potentially beneficial actions of TNF-α mediated via TNFR2. Our results, together with other studies ,74-78,97This study implicates TNF-α in the delayed neuronal cell death and gliosis that occurs within the brain following mTBI, which leads to cognitive deficits. It additionally indicates that pharmacologically limiting the elevation of TNF-α within 12 h of the mTBI event markedly reduces such secondary damage and leads to improved cognitive outcome measures. Such a window provides an opportunity for translational studies in mTBI that is more difficult to define for other neurological disorders . Buildin


In conjunction with the development of a bidirectional scattering metrology project, a large number of papers pertaining to the theory and measurement of bidirectional scattering from optical surfaces were collected and categorized. This collection includes papers that deal with various aspects of the bidirectional scattering distribution function (BSDF), its measurement, interpretation, use, and implications. Each paper is classified in one or more subject categories on the basis of its technical content. The subject categories are included just to serve as a key to the most salient characteristics of each paper cited. Because of the interest in this field, this bibliography is being published as a service to the public. The bidirectional scattering distribution function (BSDF) radiometrically characterizes the scatter of optical radiation from a surface as a function of the angular positions of the incident and scattered beams. By definition, it is the ratio of the scattered radiance to the incident irradiance: the unit is inverse steradian. The term bidirectional reflectance distribution function (BRDF) is used when specifically referring to reflected scatter. Likewise, bidirectional transmittance distribution function (BTDF) refers to scatter transmitted through a material.The bidirectional characterization of elastic scatter from surfaces is a property that is required for the evaluation of elements contained within larger systems that need minimal or controlled scattered light. The need for this information is readily seen in applications such as ring laser gyroscopes and telescopes. This type of information is also requisite for characterization of materials intended for use in temperature control where thermal radiation must be modelled or in imaging applications where stray light must be suppressed. It may also be used to assist the acceptance/rejection process in optical manufacturing settings.The present state of most of the facilities measuring this quantity needs to be upgraded to support new and more stringent requirements as well as recent strides in the production of high-quality optics.There is a lack of uniformity throughout the community for physical standards which can ascertain the accuracy of BSDF measurements (paper 91). The NIST Bidirectional Scattering Metrology Project is currently developing an instrument which will later serve to develop standard reference materials as well as a standard measurement technique.In conjunction with the development of this BSDF instrument, a large number of papers pertaining to the theory and measurement of bidirectional scattering from optical surfaces were collected and categorized. This collection includes papers that deal with various aspects of the BSDF, its measurement, interpretation, use, and implications.Each paper is classified in one or more subject categories on the basis of its technical content. The subject categories are included just to serve as a key to the most salient characteristics of each paper cited. In the literature, there is a bibliography (paper 117) of papers published prior to 1975 that relate to scattering from surfaces. Building upon that bibliography, this bibliography includes papers related to BSDF published since that time to the present. Neither the category classification list nor the list of papers is complete. There were two selection criteria used to determine whether a paper should be included in this bibliography: the paper was used during certain phases of the NIST project development and/or it was regarded as relevant and important to the field. However, inclusion or omission from the list does not necessarily imply endorsement or reproval, respectively. This systematized bibliography is sufficiently extensive to be of significant help to workers in the field, and particularly to those just beginning to work in it. For further breadth of information, the reader is suggested to review the conference proceedings from the Society of Photo-Optical Instrumentation Engineers (SPIE) that focus on scattering from surfaces; some papers from each of these are cited here.As an aid to identifying papers related to a certain field, each of the papers is listed under each category in which it has been classified. Due to the wide interdisciplinary nature of optical scattering metrology, some topics had to be grouped together so that the list would not become unmanageable.  The subject categories are coded and used as follows:The optical design of a system determines the aberrations that will be present – assuming accurate alignment–and their magnitudes. The calculation of these aberrations and their effect on the measurement of BSDF are addressed in the following papers: 3,96,102,159,160.Many different instruments are described in the literature. A sampling of these apparatus overviews is contained in the following papers: 4,12,31,41,51,52,63,65,66,76,77,79,83,84,87,89,90,93,94,95,103,111,112,132,113,118,120,122,128,129,136,137,138,140,145,146,151,159,160,161,166,168,169.Techniques for calibration and/or error analysis include reference methods and absolute methods. Specific details concerning calibration and the philosophy of calibration are discussed in these papers: 23,33,34,83,89,108,145,158.The use of laser sources in BSDF instruments is very common. Coherence becomes a critical property of these sources in diffuse BSDF metrology. The following papers address the property as it pertains to radiometry in general and to BSDF metrology in particular: 3,8,37,39,40,49,59,61,63,73,80,97,98,101,125,126.Design criteria and plans or layouts of components or subsystems of BSDF instruments are separated out and described in detail. These subsystems include source, sample manipulation, receiver, attenuation, apertures, etc: 3,10,11,21,22,27,28,29,31,35,41,51,52,60,66,68,69,71,76,85,86,87,89,90,93,94,95,108,112,132,118,119,124,128,130,136,141,142,156,157,159,160,162,163,166,168,170.Truncation of optical beams by apertures cause diffraction and thereby affect the instrument signature of BSDF instruments. This effect is discussed in the following papers: 3,49,69,70,72,89,96,139,159,160.These articles include experimental results in the form of tabulated data or graphs from a variety of different types of measurements. Some of these include data from actual BSDF scans on particular material samples while others give interpretive results highlighting instrument capabilities. Still other papers give profilometry data that yield topographic information. The particular type of experimental data given within each paper should be obvious from the title of the paper: 4,5,7,9,13,15,16,17,18,20,22,24,25,26,28,29,30,32,35,36,38,40,41,42,44,46,48,52,53,54,57,59,64,67,73,76,78,79,83,88,91,92,94,99,100,101,103,104,109,110,111,132,113,114,115,119,120,121,122,123,127,128,129,130,131,133,134,135,138,139,140,142,143,145,147,149,151,152,154,158,161,165,167,168,169,170.The background measurement of the noise equivalent BSDF, or the instrument signature, limits an instrument to the measurement of samples that have a BSDF larger than the NEBSDF. Specific instrument profiles or signatures are displayed in some of the following papers. The other papers address general concerns in obtaining and improving the signature of an instrument: 4,12,36,76,77,85,86,90,120,124,130,136,137,139,141,151,159,160,162,164,166,168,171.Particular optical materials and/or coatings are identified and experimental BSDF data are given for each of these in the following papers: 2,7,9,13,16,17,22,24,25,28,29,32,35,42,48,52,53,64,67,76,78,83,92,94,99,100,104,109,110,113,114,115,121,122,123,129,130,131,133,135,138,145,149158,162,165,Polarization control, theory, and associated problems are examined: 5,6,23,24,25,59,79,95,144,147,167.Various types of mechanical and optical surface profiling techniques are described and/or compared against optical scattering predictions of surface finish : 1,2,17,20,30,39,43,44,46,54,56,57,59,65,88,91,100,109,111,134,137,140,150,151.The following papers describe techniques used for, or problems in, referencing BSDF measurements, thereby assigning a level of confidence to the accuracy of the measurement: 33,53,83,91,92,108,130,145,158,164.Control, or suppression, of geometrically stray light is examined in this group of papers. Included in these are some papers which evaluate baffling materials with BRDF data: 18,22,27,28,29,42,50,58,60,69,71,78,87,90,113,123,146,149,156,157,163,170.Below is a list of papers that are outstanding in their fields and provide comprehensive coverage of a well-defined topic within BSDF metrology: 19,24,45,46,65,74,76,77,81,82,89,91,92,105,106,107,108,117,130,140,148,171.Among the theoretical questions explored throughout these papers are: basic definition of BSDF, scattering theory of surfaces, mathematical treatment of surfaces, subsurface contributions to scatter, inverse scattering problem, and scaling of BSDF with respect to angle of incidence and wave-length: 1,3,5,6,7,8,12,14,15,16,17,18,20,23,26,37,38,39,40,43,44,45,46,47,49,55,56,57,58,59,61,62,69,70,71,72,74,75,77,79,80,81,82,90,94,96,97,98,101,103,105,106,107,108,109,110,111,116,120,124,125,126,129,131,134,139,144,146,147,148,150,152,153,154,155,156,157,161,162,167,170.Surface roughness and sample isotropy, homogeneity, and cleanliness are all topographic contributions to optical scatter. These topics as well as some subsurface contributions are treated in the following papers: 1,2,4,9,14,15,16,17,18,19,20,30,38,39,40,43,44,45,46,47,54,55,56,57,62,74,75,88,100,109,110,111,116,120,121,125,127,134,135,139,142,144,147,150,152,153,154,155,161,162,167,169.Issues related to the bidirectional transmittance distribution function, BTDF, are considered in these papers: 52,103,122,126.


Bone regeneration involves a series of events in a coordinated manner, including recruitment of mesenchymal stem cells, induction of immune response, inflammatory activity and vascular ingrowth. The microenvironment of bone regeneration is hypoxic. Low oxygen tension (hypoxia) promotes the upregulation of several signaling molecules. The primary mediating factor is the hypoxia-inducible factor-1 (HIF-1). Hypoxia stimulates the expression of a variety of cytokines from inflammatory cells, fibroblasts, endothelial cells, and osteoblasts. TNF-α is a key proinflammatory cytokine. The molecular events involved in osteoblast dysfunction under hypoxia are not fully understood. This study determined the effects of hypoxia on TNF-α in osteoblasts, and molecular mechanisms were explored. We observed that hypoxia induced TNF-α expression in a time-dependent manner in osteoblasts. Experiments using a potent HIF-1α activator DFO demonstrated that hypoxia-induced TNF-α was mediated by HIF-1-α. In addition, this study showed that hypoxia activated cyclooxygenase-2 (COX2) expression along with TNF-α. Inhibition experiments using COX2 inhibitor N398 indicated that COX2 was involved in hypoxia-mediated TNF-α expression, and this observation was further confirmed by Small interfering RNA against COX2. On the other hand, TNF-α didn’t lead to the activation of COX2 expression. We conclude that COX2 is involved in hypoxia-induced TNF-α expression in osteoblast. Bone formation is a highly regulated process that takes place during embryonic development, growth, remodeling and fracture repairFracture healing is a complex and sequential set of events to restore bone fracture to pre-fracture condition. This is a recapitulation of molecular mechanism that controls bone formation during development and makes facture healing unique as a regenerative process than repair. Bone regeneration and fracture healing depend on different parameters present locally and systemically including growth factors, hormones, pH, oxygen tensions, immune response, etc. As in many repairs or regenerative process in the body, facture repair begins with the induction of an immune response. A hematoma is formed resulting in an inflammatory process. This is brought about by cytokines. Cytokines, such as interleukins and tumor necrosis-factor-alpha (TNF-α) secreted by inflammatory cells, have a chemotactic effect on other inflammatory cells and on the recruitment of MSCs, usually in the first three days after the factureAngiogenesis and osteogenesis are coupled each otherLow oxygen tension or hypoxia is a pathophysiological component of many human diseases such as cancer, heart attack and stroke. Recent evidence suggests that hypoxia also plays an important role in skeletal development and cell differentiation5The molecular events involved in osteoblast dysfunction under hypoxia are not fully understood. Of particular importance in the genesis of inflammatory events are the immunomodulators referred to as cytokines2) or hypoxia (1%O2) condition under a humidified hypoxia incubator. Total RNA was purified 48 hr following culture in the presence or absence of hypoxia. As shown in To explore the effect of hypoxia on expression of TNF-α in osteoblasts, we used quantitative real-time RT-PCR to examine the changes of gene expressions under hypoxia. MC3T3 osteoblastic cells were cultured and maintained in normoxic  condition under a humidified hypoxia incubator. Real-time RT-PCR was performed to analyze gene expression levels. As shown in To further examine the effect of COX2 on TNF-α expression induced by hypoxia, we used siRNA to knockdown COX2 expression in MC3T3 cells. MC3T3 cells were transfected by siRNA against COX2 and cultured in hypoxia  quantitative RT-PCR results showed that COX2 gene was upregulated along with TNF-α under hypoxia; 2) the treatment of COX2 inhibitor N398 abolished the activation of TNF-α expression induced by hypoxia; 3) the inhibition of COX2 by siRNA in osteoblasts led to the expression decrease of TNF-α gene under hypoxia. Therefore, the loss-of-function experiments demonstrated a role for COX2 in hypoxia-induced TNF-α gene expression. However, our study cannot rule out other possible mechanisms of the effect of hypoxia/HIF-1α on TNF-α expression. The preponderance of evidence suggests that hypoxia results in up-regulation of several molecular pathways involved in the fracture repair cascade. The influence of hypoxia/HIF-1α on the bone regenerative process will continue to be explored.In summary, hypoxia/HIF-1α activates the expression of TNF-α through controlling COX2 expression. While further studies need to address the role of COX2 regulation by hypoxia/HIF-1α, these early stage studies revealed one mechanism through which hypoxia activates TNF-α expression in osteoblasts. Elucidation of HIF-1α activation of TNF-α will help to better understand the molecular mechanism of HIF-1α effect on osteoblasts.2 humidified incubator. Cells were trypsinized and plated before transfection. In hypoxia experiments, MC3T3 cells were maintained in Alpha Minimum Essential Medium, and cultured in normoxic (20%O2) or hypoxia (1%O2) condition incubator with 5%CO2 and the balanced N2 before harvest conclusionMC3T3 osteoblastic cells (ATCC) were cultured in Alpha Minimum Essential Medium with ribonucleosides, deoxyribonucleosides, 2mM L-glutamine and 1mM sodium pyruvate (GIBCO) and supplemented with 10% FBS and penicillin plus streptomycin as previously describedT (∆∆CT) method as described by the manufacturer. Target quantity is normalized to endogenous control and relative to a calibrator, and is calculated using formula: Target amount = 2−∆∆CT.Total RNA was isolated from MC3T3 osteoblasts with TRIzol reagent (Invitrogen) followed by RNeasy mini kit (Qiagen) as previously describedProtein was isolated by acetone precipitation from the cell lysates as previously described2 incubator for 24 hr before harvest.MC3T3 cells were transfected by siRNA with Lipofectamine 2000 as previously describedAll experiments were repeated a minimum of 3 times. Data was reported as the mean ± standard deviation (S.D.). Comparisons were made between groups by Student’s t test with p < 0.05 being considered as statistically significant.How to cite this article: Xing, Y. et al. Cox2 is involved in hypoxia-induced TNF-α expression in osteoblast. Sci. Rep.5, 10020; doi: 10.1038/srep10020 (2015).


Analysis of single-molecule ruptures further demonstrates that TNF strengthens sub-cellular single rupture events at short cell-surface contact times. Hence, our results provide quantitative evidence for the significant impact of TNF-induced inside-out signaling in the T-lymphocyte initial adhesion machinery.Integrin-mediated leukocyte adhesion to endothelial cells is a crucial step in immunity against pathogens. Whereas the outside-in signaling pathway in response to the pro-inflammatory cytokine tumour necrosis factor (TNF) has already been studied in detail, little knowledge exists about a supposed TNF-mediated inside-out signaling pathway. In contrast to the outside-in signaling pathway, which relies on the TNF-induced upregulation of surface molecules on endothelium, inside-out signaling should also be present in an endothelium-free environment. Using single-cell force spectroscopy, we show here that stimulating Jurkat cells with TNF significantly reinforces their adhesion to fibronectin in a biomimetic  Lβ2 on neutrophils and α4β1 on T cells and ICAM-1, and VCAM-1 on endothelial cellsImmunity against pathogens critically depends on the ability of leukocytes to rapidly transmigrate from the bloodstream into inflamed tissue and to the site of infection. Diapedesis occurs via a series of steps that require the integrin-mediated adhesion of leukocytes to endothelial cells of the blood vesselet al. have reported that TNF can bind to the integrin ligand fibronectin and, as part of this complex, augment the β1 integrin-mediated adhesiveness of activated CD4-positive T cells1213Integrins naturally adopt a low-affinity, inactive conformation that needs to be converted to an active, high-affinity conformation for efficient cell adhesion. This conversion can either occur by binding to external ligands  or by interaction of the cytoplasmic domains of integrin chains with intracellular signaling cascades triggered by foreign antigens, chemokines or proinflammatory cytokines Single-cell force spectroscopy (SCFS) has evolved in recent years as an excellent quantitative method to characterize cell-cell and cell-surface adhesion16181920In the study presented here, we employed SCFS and quantified the impact of TNF stimulation on lymphocyte adhesion in an endothelium-free, biomimetic fibronectin environment. The excellent force resolution of SCFS allowed us to study TNF-induced changes in lymphocyte adhesion both at the sub-cellular and the cellular level. Our results demonstrate that TNF enhances very initial cell-fibronectin contacts by reinforcing the forces associated with single rupture events and by increasing the number of bonds formed. This result provides quantitative evidence for the existence of an inside-out signaling mechanism involved in the TNF-mediated activation of lymphocyte adhesion.in vivo endothelial cell layer, we carried out in vitro adhesion experiments on surfaces coated with fibronectin. Fibronectin is a ligand known to bind many different integrin types262728104β1 and α5β1 integrins are fibronectin-binding integrins and can modulate their activation31324 compared to α5 integrins  were evaluated in order to learn about time-dependent effects of TNF stimulation. As shown in 34To further investigate if the TNF-induced reinforcement of Jurkat adhesion observed for the cell detachment forces can be explained by changes in molecular binding processes, we carried out a detailed analysis of single ruptures in force-distance curves as a function of TNF stimulation 4 integrin-mediated adhesion and for integrin α4 clusteringWe have used single-cell force spectroscopy (SCFS) for proving the existence of a long-hypothesized TNF-induced inside-out-signaling mechanism that reinforces integrin-mediated Jurkat cell adhesion. In order to separate effects caused by inside-out signaling from those induced by outside-in signaling, we investigated the forces required to detach single cells from fibronectin-coated surfaces in an endothelium-free act time . This rede novo protein expression to occur (any effect on protein expression caused by the 25 min TNF treatment will be detected as background in the measurements) and there is, to the best of our knowledge, no study reporting an enhanced level of α4β1 expression due to TNF stimulation. Therefore we attribute the effects that we have observed to the stabilization of single rupture events, which is presumably to a significant extent due to a switching of integrins from an inactive to active state, i.e. TNF induces an increase of binding affinity. An additional effect might be that binding avidity, i.e. the number of binding competent molecules that are available in the membrane to be accessed by the binding partnerBy further analyzing the force-distance curves in detail, we found two mechanisms that are presumably the main reasons for the observed TNF-induced reinforcement of initial cell adhesion, i.e. at the shortest contact times: Then, (i) the number of rupture events required to completely detach a cell from the surface is increased in TNF-stimulated cells, and (ii) the forces associated with single rupture events are reinforced by TNF-stimulation . Note, t4 integrins has recently also been reported for leukocyte adhesion under shear stressvβ3 integrins in focal adhesionsInterestingly, we have determined a large increase of the forces associated with the last rupture events with increasing cell-surface contact time . TNF has4243In all our experiments, we consistently observe that cell detachment forces increase with contact time, while at the same time the effect of TNF-induced adhesion reinforcement becomes less relevant with increasing contact. This increase in cell detachment force with contact time is a typical result and can on the one hand be due to an increase in the number of bonds until a constant cell adhesion area, and hence binding equilibrium, is reached. On the other hand, it can be due to a stabilization of the ligand-receptor complex47An important strategy to investigate if the mechanical coupling of molecular bonds involved in cell adhesion is influenced by certain experimental conditions is the comparison of the cell detachment forces and the work of detachment36392β1 mediated inside-out signaling pathway within a short amount of timeAs an important response to extracellular stimuli, Bouaouina and co-workers found that the phosphorylation of p38 in floating neutrophils can be detected after 10 min with TNF incubation, and the level was reduced after 60 minin vivo by a combined effect of inside-out and outside-in signaling. Integrins are directly activated by TNF, their binding to fibronectin is facilitated and bonds are reinforced, leading in turn to an increase in the number of bonds between the lymphocyte and the fibronectin layer situated on top of the endothelial cells. As shown in the inset of 1213The observed very strong effect of TNF on initial cell adhesion events suggests that TNF rather strengthens short-term binding events, such as single molecules and possibly the formation of initial adhesion clusters, e.g. in lipid rafts. Integrin transport in lipid rafts has previously been shown for a different integrin type in T lymphocytesIn conclusion, we provide clear evidence for the reinforcement of lymphocyte adhesion by TNF inside-out signaling, thereby highlighting a novel function of TNF in the regulation of immunological adhesion of T lymphocytes and pointing to yet another, so far undiscovered, role of TNF in T cell-mediated adaptive immunity.2, and about 90% humidity. Cells were passaged twice a week and were maintained up to passage 10. For stimulation with TNF, cells were incubated with TNF (100 ng/ml)  in culture medium at 37 °C for 25 min prior to an experiment.Jurkat E6-1 cells were originally obtained from the ATCC and cultured in Click’s/ RPMI 1640 medium  containing 10% v/v fetal bovine serum , 100 μg/ml penicillin and streptomycin (Biochrom) at 37 °C, 5% w/v CO2. To remove excess proteins, the fibronectin surface was washed several times with 1x PBS.For preparing the biomimetic adhesion environment, a coverslip was cleaned with 70% v/v ethanol and glued into a Petri dish  with biocompatible glue . Human fibronectin  was physisorbed on the coverslip at 4 °C overnight at a concentration of 15 μg/cmCell adhesion forces were measured using atomic force microscopy (AFM). In detail, a NanoWizard 3 Bioscience with piezo range up to 15 μm  was used for the shortest cell-surface contact time and a CellHesion 200  with 100 μm piezo travel length was used for longer cell-surface contact times  in order to make sure that the cell detachment length can fully be reached with the piezo. Tipless cantilevers  were rinsed once with acetone before calibrating their spring constants in a fluid chamber  filled with 1 × PBS. Cantilevers B, C, and D with nominal spring constants between 10 and 30 mN/m were chosen. Calibration was carried out at room temperature according to the thermal noise method with correction for hydrodynamic effects using standard routines implemented in the JPK AFM software, leading to averaged calibrated spring constants from 14 to 46 mN/m. Calibrated cantilevers were incubated in biotin-BSA  overnight at 37 °C in a Petri dish wrapped with parafilm. Afterwards, they were incubated with streptavidin  for 10 minutes at room temperature, and then incubated 10 minutes in biotin-concanavalin A  at room temperature22For the adhesion force experiments, 2 ml serum-free RPMI 1640 medium  supplemented with 100 μg/ml penicillin/streptomycin  were added into the Petri dish containing the fibronectin surface. All experiments were carried out at approximately 36 °C in a heated fluid chamber . The functionalized cantilever was approached to the surface with the AFM and cantilever sensitivity was measured. For attaching a living cell to the free end of the cantilever, a droplet of cell suspension was pipetted into TNF-free medium in the Petri dish far away from the fibronectin-coated coverslip. Then (lag time ≈ 20 s), the cantilever was pressed to a cell for several tens of seconds to guarantee successful attachment. After cell attachment, the cantilever was lifted upwards and moved above the fibronectin-coated coverslip. The cell was allowed to relax at the cantilever for about 2 minutes before cell adhesion forces were measured for the first time. This procedure was exactly the same for all experimental situations. In the cell adhesion force experiments, the cell was approached to the surface at a speed of 3 μm/s until a maximum contact force of 500 pN was reached before it was retracted at a speed of 3 μm/s. During cantilever retraction the deflection of the cantilever was recorded. All experiments were carried out in closed-loop and constant height mode, i.e. the cantilever position was kept at a constant height after reaching the maximum contact force. We optically confirmed  that the cell did not change its position during the experiment in order to exclude data from loosely attached cells. 11–15 cells were used for experiments in each experimental situation (i.e. for each contact time and with/without TNF treatment) and for each cell at least 15 force-distance curves were recorded. Hence, the maximum timespan between adding the cells to the liquid chamber until finishing the experiment was about 5–10 min. Force-distance curves were analyzed with a commercial data processing software , where the detection of single rupture events is based on a previously published algorithmThe probability density distributions of the logarithmic rupture position and the slope  were calHow to cite this article: Li, Q. et al. Reinforcement of integrin-mediated T-Lymphocyte adhesion by TNF-induced Inside-out Signaling. Sci. Rep.6, 30452; doi: 10.1038/srep30452 (2016).