Increased Expression of Long Noncoding RNA LOC100506314 in T cells from Patients with Nonsegmental Vitiligo and Its Contribution to Vitiligo Pathogenesis.

This study aimed to identify the abnormal expression of long noncoding RNAs (lncRNAs) in T cells from patients with vitiligo and to investigate their functional roles in the immune system. Using microarray analysis, the expression levels of RNA transcripts in T cells from patients with vitiligo and controls were compared. We identified several genes and validated their expression levels in T cells from 41 vitiligo patients and 41 controls. The biological functions of the lncRNAs were studied in a transfection study using an RNA pull-down assay, followed by proteomic analysis and western blotting. The expression levels of 134 genes were significantly increased, and those of 142 genes were significantly decreased in T cells from vitiligo patients. After validation, six genes had increased expression, and three genes had decreased expression in T cells from patients with vitiligo. T-cell expression of LOC100506314 was increased in vitiligo, especially CD4+, but not CD8+ T cells. The expression levels of LOC100506314 in CD4+ T cells was positively and significantly associated with the severity of vitiligo. LOC100506314 was bound to the signal transducer and activator of transcription 3 (STAT3) and macrophage migration inhibitory factor (MIF). Enhanced expression of LOC100506314 inhibited the phosphorylation of STAT3, protein kinase B (AKT), and extracellular signal-regulated protein kinases (ERK), as well as the levels of nuclear protein of p65 and the expression of IL-6 and IL-17 in Jurkat cells and T cells from patients with vitiligo. In conclusion, this study showed that the expression of LOC100506314 was elevated in CD4+ T cells from patients with vitiligo and associated the severity of vitiligo. LOC100506314 interacted with STAT3 and MIF and inhibited IL-6 and IL-17 expression by suppressing the STAT3, nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB), AKT, and ERK pathways. Enhanced expression of LOC100506314 in T cells may be a potential treatment strategy for vitiligo.

Brain-Derived Neurotrophic Factor Suppressed Proinflammatory Cytokines Secretion and Enhanced MicroRNA(miR)-3168 Expression in Macrophages.

We investigated the role of brain-derived neurotrophic factor (BDNF) and its signaling pathway in the proinflammatory cytokines production of macrophages. The effects of different concentrations of BDNF on proinflammatory cytokines expression and secretion in U937 cell-differentiated macrophages, and human monocyte-derived macrophages were analyzed using enzyme-linked immunosorbent assay and real-time polymerase chain reaction. The CRISPR-Cas9 system was used to knockout p75 neurotrophin receptor (p75NTR), one of the BDNF receptors. Next-generation sequencing (NGS) was conducted to search for BDNF-regulated microRNA. A very low concentration of BDNF (1 ng/mL) could suppress the secretion of interleukin (IL)-1ß, tumor necrosis factor (TNF)-α, and IL-6 in lipopolysaccharide (LPS)-stimulated macrophages but did not change their mRNA expression. BDNF suppressed IL-1ß and IL-6 secretion in human monocyte-derived macrophages. In U937 cells, BDNF suppressed the phosphorylation of JNK and c-Jun. The p75NTR knockout strongly suppressed IL-1ß, IL-6, and TNF-α secretion in macrophages and LPS-stimulated macrophages. BDNF regulated the expression of miR-3168 with Ras-related protein Rab-11A as its target. In conclusion, BDNF suppressed proinflammatory cytokines secretion in macrophages and inhibited the phosphorylation of JNK. Knockout of p75NTR suppressed proinflammatory cytokines expression and secretion. BDNF upregulated the expression of miR-3168. The inhibition of p75NTR could be a potential strategy to control inflammation.

HLA-B*27 Heavy Chain Homo-Oligomers Promote the Cytotoxicity of NK Cells via Activation of PI3K/AKT Signaling.

Background and Objectives: Ankylosing spondylitis (AS) is a chronic inflammatory disease and is highly linked with the expression of the human leukocytic antigen-B*27 (HLA-B*27) genotype. HLA-B*27 heavy chain (B*27-HC) has an innate characteristic to slowly fold, resulting in the accumulation of the misfolded B*27-HC and the formation of homo-oligomeric B*27-HC molecules. The homo-oligomeric B*27-HC can act as a ligand of KIR3DL2. Interaction of the homo-oligomeric B*27-HC molecules with KIR3DL2 will trigger the survival and activation of KIR3DL2-positive NK cells. However, the effects of homo-oligomeric B*27-HC molecules associated with KIR3DL2 on the cytotoxic activity of NK cells and their cytokine expressions remain unknown. Materials and Methods: HLA-B*-2704-HC was overexpressed in the HMy2.C1R (C1R) cell line. Western blotting and quantitative RT-PCR were used to analyze the protein expression and cytokine expression, respectively, when C1R-B*-2704 cells that overexpress B*2704-HC were co-cultured with NK-92MI cells. Flow cytometry was used to analyze the cytotoxicity mediated by NK-92MI cells. Results: Our results revealed that NK-92MI cells up-regulated the expression of perforin and enhanced the cytotoxic activity via augmentation of PI3K/AKT signaling after co-culturing with C1R-B*2704 cells. Suppression of the dimerized B*27-HC formation or treatment with an inhibitor of PI3K, LY294002, or with an anti-B*27-HC monoclonal antibody can reduce the perforin expression of NK-92MI after co-culturing with C1R-B*-2704. Co-culturing with C1R-B*-2704 cells suppressed the TNF-α and IL6 expressions of NK-92MI cells. Conclusion: Stimulation of NK cell-mediated cytotoxicity by homo-oligomeric B*27-HC molecules may contribute to the pathogenesis of AS.

Increased Serum Levels of Brain-Derived Neurotrophic Factor Contribute to Inflammatory Responses in Patients with Rheumatoid Arthritis.

The aim of this study is to investigate the role of brain-derived neurotrophic factor (BDNF) in the inflammatory responses in patients with rheumatoid arthritis (RA). Serum levels of BDNF and the precursor form of BDNF (proBDNF) from 625 RA patients and 40 controls were analyzed using enzyme-linked immunosorbent assay. Effects of BDNF on the mitogen-activated protein kinase pathway were analyzed by Western blotting. Microarray analysis was conducted to search BDNF regulated gene expression in Jurkat cells, and the differentially expressed genes were validated using T cells from patients with RA and controls. Serum BDNF levels were significantly elevated in patients with RA compared with the controls. Low serum BDNF levels were found in RA patients with anxiety or receiving biologics treatment. BDNF (20 ng/mL) enhanced the phosphorylation of ERK, JNK, and c-Jun, but suppressed the phosphorylation of p38, whereas BDNF (200 ng/mL) enhanced the phosphorylation of ERK and p38. After validation, the expression of CAMK2A, MASP2, GNG13, and MUC5AC, regulated by BDNF and one of its receptors, NGFR, was increased in RA T cells. BDNF increased the IL-2, IL-17, and IFN-γ expression in Jurkat cells and IL-2 and IFN-γ secretion in activated peripheral blood mononuclear cells.

Identification of phostensin in association with Eps 15 homology domain-containing protein 1 (EHD1) and EHD4.

Phostensin (PTS) encoded by KIAA1949 is a protein phosphatase 1 (PP1)-binding protein. In order to explore the cellular functions of PTS, we have searched PTS-binding proteins by using co-immunoprecipitation in combination with shotgun proteomics. Here, we report two novel PTS-binding proteins, Eps 15 homology domain-containing protein 1 (EHD1) and EHD4. PTS associated with EHD proteins was also observed in GST pull-down assays. Immunofluorescence microscopy demonstrated that the complex was co-localized at the endocytic vesicles. EHD proteins have been known to play a critical role in regulation of endocytic transport. Overexpression of PTS-ß can attenuate the endocytic trafficking of transferrin.

IL-6 induces haptoglobin expression through activating STAT3 in human head and neck cancer.

OBJECTIVE: Haptoglobin (Hp) is an acute-phase protein secreted by the liver; its concentration increases rapidly during infection, inflammation, and tumor formation. It has been reported that the level of Hp α alleles is altered in the serum of patients with head and neck squamous cell carcinoma (HNSCC), and the cellular level of Hp is strongly associated with the recurrence rate of HNSCC in patients. In the present study, the regulated mechanism of Hp expression was explored. MATERIALS AND METHODS: We first identified the genetic polymorphism of Hp by PCR. The expression of Hp isoforms was determined through Western Blotting analysis. With the JAK specific inhibitors, the clear regulation mechanism was explored. RESULTS: We observed that Hp exhibited variant polymorphisms in different cells. We found that interleukin-6 (IL-6) induced the expressions of Hp α2 in FaDu cells, and Hp α1 in SCC4 cells. Furthermore, the phosphorylated level of STAT3 was elevated with IL-6 treatment. Janus-associated kinase 2 (JAK-2) inhibitor, WP1066, reduced the phosphorylation of STAT3 after IL-6 induction, leading to the downregulation of Hp expression. CONCLUSIONS: The expression of Hp was increased via IL-6 induction through the activation of the transcription factor STAT3 in HNSCC cells.

The Essential Role of Peptidylarginine Deiminases 2 for Cytokines Secretion, Apoptosis, and Cell Adhesion in Macrophage.

OBJECTIVE: The study aims to investigate the functional roles of peptidylarginine deiminase 2 (PADI2) in macrophages. METHODS: The clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated protein-9 nuclease (Cas9) system was used to knockout PADI2 in U937 cells. U937 cells were introduced to differentiate macrophages and were stimulated with lipopolysaccharides (LPS). The protein expression of PADI2, PADI4, and citrullinated proteins were analyzed by Western blotting. The mRNA and protein levels of interleukin 1 beta (IL-1ß), IL-6, and tumor necrosis factor-alpha (TNF-α) were analyzed using RT-PCR and ELISA, respectively. Cell apoptosis was analyzed using flow cytometry. Cell adhesion assay was performed using a commercially available fibrinogen-coated plate. RESULTS: PADI2 knockout could markedly suppress the PADI2 protein expression, but not the PADI4 protein expression. PADI2 knockout decreased the protein levels of citrullinated nuclear factor κB (NF-κB) p65, but not those of citrullinated histone 3, resulting in the decreased mRNA expression levels of IL-1ß and TNF-α in the U937 cells and IL-1ß and IL-6 in the differentiated macrophages and the macrophages stimulated with LPS. The cytokines levels of IL-1ß, IL-6, and TNF-α were all dramatically decreased in the PADI2 knockout group compared with in the controls. PADI2 knockout prevented macrophages apoptosis via the decreased caspase-3, caspase-2, and caspase-9 activation. PADI2 knockout also impaired macrophages adhesion capacity through the decreased protein levels of focal adhesion kinase (FAK), phospho-FAK, paxillin, phospho-paxillin, and p21-activated kinase 1. CONCLUSION: This study showed that PADI2 could promote IL-1ß, IL-6, and TNF-α production in macrophages, promote macrophage apoptosis through caspase-3, caspase-2, and caspase-9 activation and enhance cell adhesion via FAK, paxillin, and PAK1. Therefore, targeting PADI2 could be used as a novel strategy for controlling inflammation caused by macrophages.

Conformational Characterization of Native and L17A/F19A-Substituted Dutch-Type ß-Amyloid Peptides.

Some mutations which occur in the α/ß-discordant region (resides 15 to 23) of ß-amyloid peptide (Aß) lead to familial Alzheimer's disease (FAD). In vitro studies have shown that these genetic mutations could accelerate Aß aggregation. We recently showed that mutations in this region could alter the structural propensity, resulting in a different aggregative propensity of Aß. Whether these genetic mutations display similar effects remains largely unknown. Here, we characterized the structural propensity and aggregation kinetics of Dutch-type Aß40 (Aß40(E22Q)) and its L17A/F19A-substituted mutant (Aß40(L17A/F19A/E22Q)) using circular dichroism spectroscopy, nuclear magnetic spectroscopy, and thioflavin T fluorescence assay. In comparison with wild-type Aß40, we found that Dutch-type mutation, unlike Artic-type mutation (E22G), does not reduce the α-helical propensity of the α/ß-discordant region in sodium dodecyl sulfate micellar solution. Moreover, we found that Aß40(L17A/F19A/E22Q) displays a higher α-helical propensity of the α/ß-discordant region and a slower aggregation rate than Aß40(E22Q), suggesting that the inhibition of aggregation might be via increasing the α-helical propensity of the α/ß-discordant region, similar to that observed in wild-type and Artic-type Aß40. Taken together, Dutch-type and Artic-type mutations adopt different mechanisms to promote Aß aggregation, however, the L17A/F19A mutation could increase the α-helical propensities of both Dutch-type and Artic-type Aß40 and inhibit their aggregation.

Targeted Delivery of the HLA-B∗27-Binding Peptide into the Endoplasmic Reticulum Suppresses the IL-23/IL-17 Axis of Immune Cells in Spondylarthritis.

Ankylosing spondylitis (AS) is highly associated with the expression of human leukocyte antigen-B27 (HLA-B∗27). HLA-B∗27 heavy chain (B27-HC) has an intrinsic propensity to fold slowly, leading to the accumulation of the misfolded B27-HC in the endoplasmic reticulum (ER) and formation of the HLA-B∗27 HC homodimer, (B27-HC)2, by a disulfide linkage at Cys-67. (B27-HC)2 displayed on the cell surface can act as a ligand of the killer-cell Ig-like receptor (KIR3DL2). (B27-HC)2 binds to KIR3DL2 of NK and Th17 cells and activates both cells, resulting in the activation of the IL-23/IL-17 axis to launch the inflammatory reaction in AS patients. However, activation of the IL-23/IL-17 axis originally derived from the HLA-B∗27 misfolding in the ER needs to be characterized. In this study, we delivered two HLA-B∗27-binding peptides, KRGILTLKY and SRYWAIRTR, into the ER by using a tat-derived peptide (GRKKRRQRRR)-His6-ubiquitin (THU) vehicle. Both peptides are derived from the human actin and nucleoprotein of influenza virus, respectively. Our results demonstrated that targeted delivery of both HLA-B∗27-binding peptides into the ER can promote the HLA-B∗27 folding, decrease the levels of (B27-HC)2, and suppress the activation of the IL-23/IL-17 axis in response to lipopolysaccharide. Our findings can provide a new therapeutic strategy in AS.

Increased gene expression of TGF-ß in peripheral blood mononuclear cells from renal transplant patients with polyomavirus BK viremia.

We aimed to investigate the roles of cytokines during polyomavirus BK (BKV) reactivation in renal transplant patients. Forty-eight renal allograft recipients were enrolled, and their sera BKV viral load and mRNA expression levels of cytokines in peripheral blood mononuclear cells were measured by real-time polymerase chain reaction. Patient's age and gene expression levels of interleukin (IL)-2 (10.04 ± 2.63 vs. 8.70 ± 2.40, p = 0.049) and transforming growth factor (TGF)-ß (12.58 ± 2.59 vs. 10.89 ± 1.91, p = 0.015) were significantly higher in BKV viremia (+) renal transplant patients. Multivariate logistic regression analysis revealed that age and mRNA expression levels of TGF-ß, but not IL-2, significantly correlated with the presence of BKV viremia. Sera BKV viral loads showed a positive correlation with patient age and the levels of TGF-ß and IL-6 mRNA. After adjusting for age and sex in the regression model, both age and TGF-ß mRNA levels maintained a significant positive association with sera BKV viral loads. Serum TGF-ß concentration tended to be higher in BKV viremia (+) patients (p = 0.079). In conclusion, expression levels of TGF-ß were found to correlate with both BKV viremia positivity and sera BKV viral loads in renal transplant patients.

Increased Serum Levels of Anti-Carbamylated 78-kDa Glucose-Regulated Protein Antibody in Patients with Rheumatoid Arthritis.

The objective of this study was to investigate the presence and titer of anti-carbamylated 78-kDa glucose-regulated protein (anti-CarGRP78) antibody in serum from controls, and patients with rheumatoid arthritis (RA), systemic lupus erythematosus (SLE) and primary Sjögren syndrome (pSS). Thirty-three RA patients, 20 SLE patients, 20 pSS patients, and 20 controls were enrolled from our outpatient clinic. GRP78 was cloned and carbamylated. Serum titers of anti- cyclic citrullinated peptides (anti-CCP), anti-GRP78, and anti-CarGRP78 were measured with an enzyme-linked immunosorbent assay. No differences in serum titers of anti-GRP78 antibody in patients with RA, SLE, or pSS compared with the controls were observed. Serum levels of anti-carGRP78 antibody in patients with RA, but not SLE or pSS, were significantly higher compared with the controls (OD405 0.15 ± 0.08 versus 0.11 ± 0.03, p = 0.033). There was a positive correlation between the serum levels of anti-GRP78 antibody, but not anti-CarGRP78 antibody, with the levels of anti-CCP antibody in patients with RA. Both anti-GRP78 and anti-carGRP78 antibodies failed to correlate with C-reactive protein levels in patients with RA. In conclusion, we demonstrated the presence of anti-CarGRP78 antibody in patients with RA. In addition, the serum titer of anti-CarGRP78 antibody was significantly elevated in patients with RA compared with the controls. Anti-CarGRP78 antibody could also be detected in patients with SLE or pSS.

Aberrant T cell expression of Ca2+ influx-regulated miRNAs in patients with systemic lupus erythematosus promotes lupus pathogenesis.

OBJECTIVE: The aim of this study was to investigate the pathogenic role of calcium (Ca(2+)) influx-regulated microRNAs (miRNAs) in T cells from patients with SLE. METHODS: Expression profiles of 270 human miRNAs in Jurkat cells co-cultured with or without ionomycin were analysed by real-time PCR. Differential expression of miRNAs in T cell samples from 28 patients with SLE (SLE T cells) and 20 healthy controls were investigated using western blot analysis of proteins expressed by respective miRNA target transcripts. Transfection studies were conducted to investigate miRNA-specific biological functions. RESULTS: Initial analysis revealed differential expression of nine miRNAs in Jurkat cells after co-culture with ionomycin. Of these, miR-524-5p and miR-449b were overexpressed in SLE T cells. Levels of expressed miR-524-5p showed a significant direct correlation with the SLEDAI. Transfection of Jurkat cells with miR-524-5p mimic suppressed Jagged-1 and Hes-1 protein expression. Likewise, expression of both Jagged-1 and Hes-1 proteins were diminished in SLE T cells. Upon activation of Jurkat cells transfected with miR-524-5p mimic, production of IFN-γ increased but the apoptotic rate was unaffected. CONCLUSION: In SLE T cells, miR-524-5p and miR-449b (both regulated by Ca(2+) influx) were overexpressed. Moreover, increased miR-524-5p expression, as shown by patients with SLE, directly paralleled disease activity (SLEDAI). Transfection of miR-524-5p also enhanced IFN-γ production in activated Jurkat cells.

Combined effect of individual and neighbourhood socioeconomic status on mortality of rheumatoid arthritis patients under universal health care coverage system.

BACKGROUND: The National Health Insurance program in Taiwan is a public insurance system for the entire population of Taiwan initiated since March 1995. However, the association of socioeconomic status (SES) and prognosis of rheumatoid arthritis (RA) patients under this program has not been identified. OBJECTIVES: Using the National Health Insurance Research Database in Taiwan, we aimed to examine the combined effect of individual and neighbourhood SES on the mortality rates of RA patients under a universal health care coverage system. MEASURES: A study population included patients with RA from 2004 to 2008. The primary end point was the 5-year overall mortality rate. Individual SES was categorized into low, moderate and high levels based on the income-related insurance payment amount. Neighbourhood SES was defined by household income and neighbourhoods were grouped as an 'advantaged' area or a 'disadvantaged' area. The Cox proportional hazards regression model was used to compare outcomes between different SES categories. A two-sided P value < 0.05 was considered statistically significant. RESULTS: Medical data of 23900 RA patients from 2004 to 2008 were reviewed. Analysis of the combined effect of individual SES and neighbourhood SES revealed that 5-year mortality rates were worse among RA patients with a low individual SES compared to those with a high SES (P < 0.001). In the Cox proportional hazards regression model, RA patients with low individual SES in disadvantaged neighbourhoods incurred the highest risk of mortality (Hazard ratio = 1.64; 95% confidence interval, 1.26-2.13, P < 0.001). CONCLUSIONS: RA patients with a low SES have a higher overall mortality rate than those with a higher SES, even with a universal health care system. It is crucial that more public policy and health care efforts be put into alleviating the health disadvantages, besides providing treatment payment coverage.

Sulfasalazine Treatment Suppresses the Formation of HLA-B27 Heavy Chain Homodimer in Patients with Ankylosing Spondylitis.

Human leukocytic antigen-B27 heavy chain (HLA-B27 HC) has the tendency to fold slowly, in turn gradually forming a homodimer, (B27-HC)2 via a disulfide linkage to activate killer cells and T-helper 17 cells and inducing endoplasmic reticulum (ER) stress to trigger the IL-23/IL-17 axis for pro-inflammatory reactions. All these consequences lead to the pathogenesis of ankylosing spondylitis (AS). Sulfasalazine (SSA) is a common medication used for treatment of patients with AS. However, the effects of SSA treatment on (B27-HC)2 formation and on suppression of IL-23/IL-17 axis of AS patients remain to be determined. In the current study, we examine the (B27-HC)2 of peripheral blood mononuclear cells (PBMC), the mean grade of sarcoiliitis and lumbar spine Bath Ankylosing Spondylitis Radiology Index (BASRI) scores of 23 AS patients. The results indicated that AS patients without (B27-HC)2 on PBMC showed the lower levels of mean grade of sarcoiliitis and the lumbar spine BASRI scores. In addition, after treatment with SSA for four months, the levels of (B27-HC)2 on PBMCs were significantly reduced. Cytokines mRNA levels, including TNFα, IL-17A, IL-17F and IFNγ, were also significantly down-regulated in PBMCs. However, SSA treatment did not affect the levels of IL-23 and IL-23R mRNAs.

Characterization of the recognition specificity of BH2, a monoclonal antibody prepared against the HLA-B27 heavy chain.

BH2, a monoclonal antibody prepared against the denatured human leukocytic antigen-B27 heavy chain (HLA-B27 HC), can immunoprecipitate the misfolded HLA-B27 HC complexed with Bip in the endoplasmic reticulum and recognize the homodimerized HLA-B27 HC that is often observed on the cell membrane of patients suffered from ankylosing spondylitis (AS). However, the recognition specificity of BH2 toward the other molecules of HLA-B type and toward the different types of HLA molecules remained uncharacterized. In this study, we carried out the HLA-typing by using the Luminex Technology to characterize the recognition specificity of BH2 and analyzed the binding domain of HLA-B27 HC by BH2. Our results indicated that BH2 preferably binds to molecules of HLA-B and -C rather than HLA-A and the binding site is located within the α2 domain of HLA-B27 HC.

Identification of the high molecular weight isoform of phostensin.

Phostensin is encoded by KIAA1949. 5'-RACEanalysis has been used to identify the translation start site of phostensin mRNA, indicating that it encodes 165 amino acids with an apparent molecular weight of 26 kDa on SDS-PAGE. This low-molecular-weight phostensin is present in human peripheral blood mononuclear cells and many leukemic cell lines. Phostensin is a protein phosphatase-1(PP1) binding protein. It also contains one actin-binding motif at its C-terminal region and binds to the pointed ends of actin filaments, modulating actin dynamics. In the current study, a high-molecular-weight phostensin is identified by using immunoprecipitationin combination with a proteomic approach. This new species of phostensin is also encoded by KIAA1949 and consists of 613 amino acids with an apparent molecular weight of 110 kDa on SDS-PAGE. The low-molecular-weight and high-molecular-weight phostensins were named as phostensin-α and phostensin-ß, respectively. Although phostensin-α is the C-terminal region of phostensin-ß, it is not degraded from phostensin-ß. Phostensin-ß is capable of associating with PP1 and actin filaments, and is present in many cell lines.

Anti-citrullinated protein antibodies activated ERK1/2 and JNK mitogen-activated protein kinases via binding to surface-expressed citrullinated GRP78 on mononuclear cells.

In a previous study, we found that anti-citrullinated protein antibodies (ACPAs) enhance nuclear factor (NF)-κB activity and tumor necrosis factor (TNF)-α production by normal human peripheral blood mononuclear cells (PBMCs) and U937 cells via binding to surface-expressed citrullinated glucose-regulated protein 78 (cit-GRP78). However, the downstream signaling pathways remain unclear after binding. In the present study, we firstly measured the effects of different kinase inhibitors on ACPA-mediated TNF-α production from normal PBMCs and monocytes. Then, the native and phosphorylated mitogen-activated protein kinases (MAPKs) were detected in ACPA-activated U937 cells by Western blotting. We also explored the role of the phosphoinositide 3-kinase (PI3K)-Akt pathway in activating IκB kinase alpha (IKK-α) in ACPA-stimulated U937 cells. Finally, we measured the amount of cit-GRP78 from PBMC membrane extracts in RA patients and controls. We found that MAPK and Akt inhibitors, but not PI3K inhibitor, remarkably suppressed ACPA-mediated TNF-α production. Interestingly, ACPAs selectively activated extracellular signal-regulated kinase 1/2 (ERK1/2) and c-jun N-terminal kinase (JNK), but not p38 MAPK, in U937 cells. This activation was suppressed by cit-GRP78, but not GRP78. The JNK activation further enhanced the phosphorylation of Akt and IKK-α. The expression of cit-GRP78 on cell membrane was higher in RA than normal PBMCs. Taken together; these results suggest that through binding to surface, over-expressed cit-GRP78 on RA PBMCs, ACPAs selectively activate ERK1/2 and JNK signaling pathways to enhance IKK-α phosphorylation, which leads to the activation of NF-κB and the production of TNF-α .

SOCS-1 promoter methylation and treatment response in chronic hepatitis C patients receiving pegylated-interferon/ribavirin.

PURPOSE: Suppressor of cytokine signaling-1 (SOCS-1) is a negative regulator of the Janus kinase/signal transducer and activation of transcription pathway. The purpose of this study was to investigate the relationship between methylation of SOCS-1 and sustained virologic response (SVR) in chronic hepatitis C (CHC) patients treated with pegylated interferon (PEG-IFN)-alpha and ribavirin (RBV). METHODS: In total, 106 CHC patients treated with PEG-IFN-alpha and RBV were included. Serum samples were obtained at baseline (P), end of treatment (EOT), and 6 months post treatment (F6). Methylation status of the promoter region of SOCS-1 was examined by quantitative methylation specific PCR (qMSP). RESULTS: Median baseline methylation level of SOCS-1 was -0.95 log10 copies/mL, which increased to 0.57 log10 copies/mL at EOT and then returned to -0.57 log10 copies/mL at F6 (baseline vs EOT, P < 0.001; EOT vs F6, P < 0.001). The overall SVR was 75.5%. Univariate analysis indicated that SVR was significantly associated with genotype, baseline HCV RNA, body mass index (BMI) and higher EOT SOCS-1 methylation. Multivariate analysis confirmed that the SVR was significantly associated with genotype (OR: 13.40, 95% CI: 1.73-103.58, P = 0.013), baseline HCV RNA (OR: 0.19, 95% CI: 0.06-0.59, P = 0.004), BMI (OR: 0.73, 95% CI: 0.56-0.96, P = 0.022), and EOT SOCS-1 methylation (OR: 1.71, 95% CI: 1.11-2.62, P = 0.014). CONCLUSION: CHC patients with significantly higher SOCS-1 methylation at the end of treatment had better SVRs. The role of SOCS-1 methylation in affecting treatment response deserves further investigation.

Renal dysfunction by BK virus infection is correlated with activated T cell level in renal transplantation.

BACKGROUND: BK virus (BKV) is known to be associated with nephropathy. Here, we investigated the relationships between BKV levels, T-cell activation, and kidney function in kidney transplant recipients. MATERIALS AND METHODS: In renal transplant patients and controls, urine BKV levels were detected by quantitative real-time PCR, and the percentage of activated T lymphocytes in blood was determined by flow cytometry. The correlations between viral load, activated T cell percentage, and renal function were determined. RESULTS: Urine BKV viral loads and the activated T cell percentage were significantly elevated in transplant recipients. Correlational analysis indicated that transplant recipients that had BKV levels of more than 10(6) copies/mL and an activated T lymphocyte percentage of less than 20% were likely to have poor renal function. CONCLUSIONS: Urine BKV levels and the percentage of activated T lymphocytes can be used as clinical indices to optimize the dosage of immunosuppressive drugs.

The role of bacterial exopolysaccharides (EPS) in the synthesis of antimicrobial silver nanomaterials: A state-of-the-art review.

The emergence of multi-drug resistant (MDR) pathogens poses a significant global health concern due to the failure of conventional medical treatment. As a result, the development of several metallic (Ag, Au, Zn, Ti, etc.) nanoparticles, has gained prominence as an alternative to conventional antimicrobial therapies. Among these, green-synthesized silver nanoparticles (AgNPs) have gained significant attention due to their notable efficiency and broad spectrum of antimicrobial activity. Bacterial exopolysaccharides (EPS) have recently emerged as a promising biological substrate for the green synthesis of AgNPs. EPS possess polyanionic functional groups (hydroxyl, carboxylic, sulfate, and phosphate) that effectively reduce and stabilize AgNPs. EPS-mediated AgNPs exhibit a wide range of antimicrobial activity against various pathogenic microbes, including Gram-positive and Gram-negative bacteria, as well as fungi. The extraction and purification of bacterial EPS play a vital role in obtaining high-quality and -quantity EPS for industrial applications. This study focuses on the comprehensive methodology of EPS extraction and purification, encompassing screening, fermentation optimization, pretreatment, protein elimination, precipitation, and purification. The review specifically highlights the utilization of bacterial EPS-mediated AgNPs, covering EPS extraction, the synthesis mechanism of green EPS-mediated AgNPs, their characterization, and their potential applications as antimicrobial agents against pathogens. These EPS-mediated AgNPs offer numerous advantages, including biocompatibility, biodegradability, non-toxicity, and eco-friendliness, making them a promising alternative to traditional antimicrobials and opening new avenues in nanotechnology-based approaches to combat microbial infections.