Transcriptional regulation of intermolecular Ca2+ signaling in hibernating ground squirrel cardiomyocytes: The myocardin-junctophilin axis.

The contraction of heart cells is controlled by the intermolecular signaling between L-type Ca2+ channels (LCCs) and ryanodine receptors (RyRs), and the nanodistance between them depends on the interaction between junctophilin-2 (JPH2) in the sarcoplasmic reticulum (SR) and caveolin-3 (CAV3) in the transversal tubule (TT). In heart failure, decreased expression of JPH2 compromises LCC-RyR communication leading to deficient blood-pumping power. In the present study, we found that JPH2 and CAV3 transcription was concurrently regulated by serum response factor (SRF) and myocardin. In cardiomyocytes from torpid ground squirrels, compared with those from euthermic counterparts, myocardin expression was up-regulated, which boosted both JPH2 and CAV3 expression. Transmission electron microscopic imaging showed that the physical coupling between TTs and SRs was tightened during hibernation and after myocardin overexpression. Confocal Ca2+ imaging under the whole-cell patch clamp condition revealed that these changes enhanced the efficiency of LCC-RyR intermolecular signaling and fully compensated the adaptive down-regulation of LCCs, maintaining the power of heart contraction while avoiding the risk of calcium overload during hibernation. Our finding not only revealed an essential molecular mechanism underlying the survival of hibernating mammals, but also demonstrated a "reverse model of heart failure" at the molecular level, suggesting a strategy for treating heart diseases.

Plasma EBF1 as a Novel Biomarker for Postmenopausal Osteoporosis.

Postmenopausal osteoporosis (OPO) is one of the most common types of primary osteoporosis. There is currently lack of a plasma biomarker for sensitive and early diagnosis of OPO. Here we aimed to explore the potential of early B cell factor 1 (EBF1) as a new plasma biomarker of OPO. Quantitative real-time PCR was used to measure the plasma EBF1 levels. Absorptiometry markers, such as lumbar spine (LS) bone mineral density (BMD) and LS T score were obtained after X-ray scans. Biochemical analyses used to measure osteopontin (OPN), ß-isomerized C-terminal telopeptides and total N-terminal procollagen of type-I collagen levels of patients with osteopenia (OPE, n = 81), osteoporosis (OPO, n = 98) as well as healthy subjects (NC, n = 110). Quantitative real-time PCR was used to measure the plasma levels of PAX5 and GSTP1, which are target genes of EBF1. EBF1 was downregulated in OPO patients. Levels of EBF1 were positively correlated to clinicopathological characteristics, including LS BMD and LS T scores, and negatively correlated to OPN and total N-terminal procollagen of type-I collagen levels. Increased PAX5 and GSTP1 levels also demonstrated strong correlations with higher EBF1, LS BMD and LS T score. Anti-osteoporotic treatment resulted in significant upregulation of EBF1, PAX5 and GSTP1 at 6 mo after treatment. Our study suggests that plasma EBF1 is a potential biomarker for diagnosing and assessing treatment outcome of OPO.

Overexpression of metastasis-associated in colon cancer 1 in retinoblastoma.

INTRODUCTION: Metastasis-associated in colon cancer 1 (MACC1), one of the prognostic markers for colonic and other tumours was noted to be overexpressed in retinoblastoma (Rb) Y79 cancer stem cells. This prompted us to evaluate its expression in primary Rb tumour and serum samples with clinicopathologic correlation. The interacting partner, c-MET was also evaluated in primary tumour tissues to explore the activation of MACC1 signaling. METHODOLOGY: This study was done following institutional review board approval from participating institutes. Semiquantitative gene expression for MACC1 was evaluated using formalin-fixed paraffin-embedded sections and unfixed tumour samples from primary Rb cases (n = 44). Immunolocalization for MACC1 was assessed in primary Rb tumours (n = 22), bone marrow aspirates with metastasis (n = 3), and c-MET expression was also assessed in Rb tumours (n = 17). Serum MACC1 levels were analysed using enzyme-linked immunosorbent assay in samples collected from Rb patients undergoing enucleation (n = 31), Rb patients with proven clinical metastasis (n = 3), and compared to appropriate controls. Clinicopathologic correlation of MACC1 expression was analysed using the medical records with specific reference to histologic risk factors (HRF) for metastasis and differentiation. RESULTS: High expression of MACC1 gene was noted in all the tumour samples (n = 44), more so in cases with versus without HRF (p < 0.0001). In cases with HRF, MACC1 and c-MET showed diffuse nuclear and cytoplasmic staining whereas it was predominantly cytoplasmic in cases without HRF. Mean immunoreactivity score of MACC1 and c-MET tissue immunolocalization revealed that cases with HRF showed significantly higher expression compared to cases without HRF (p < 0.05). Unlike the findings in colonic tumours, serum levels of MACC1 were lower in patients compared to normal controls. CONCLUSION: Overexpression of MACC1 and c-MET in retinoblastoma tissues, specifically those with risk factors for metastasis, suggests its role in proliferation and possibly in invasion. However, the current data do not support it to be a clinical prognostic marker in retinoblastoma tumours. The inverse serum expression is an intriguing finding, which warrants further studies especially in retinoblastoma.

Long Non-Coding RNA MACC1-AS1 Is Involved in Distant Recurrence of Hepatocellular Carcinoma After Surgical Resection.

BACKGROUND We explored the role of MACC1-AS1 in hepatocellular carcinoma (HCC). MATERIAL AND METHODS Measurement of preoperative plasma levels of MACC1-AS1 was performed by qPCR, and the comparison between the HCC and Control group was performed by unpaired t test. The overexpression of TGF-ß1 in SNU-182 and SNU-398 cells was confirmed by qPCR. RESULTS MACC1-AS1 was overexpressed in HCC patients. In comparison to pretreatment level, distant recurrence (DR) was accompanied by increased levels of MACC1-AS1 in plasma, but this phenomenon was not observed in cases of local recurrence (LR) or non-recurrence (NR). In HCC cells, MACC1-AS1 positively regulated the expression of TGF-ß1. MACC1-AS1 overexpression resulted in increased invasion and migration rates of HCC cells, while siRNA silencing resulted in reduced rates. Moreover, TGF-ß1 overexpression reduced the effects of MACC1-AS1 siRNA silencing. CONCLUSIONS MACC1-AS1 is involved in the distant recurrence of HCC, and its actions are possibly mediated by TGF-ß1.

Significance of plasma MACC1 levels on the prognostic stratification in patients with colorectal cancer.

The clinical significance of metastasis-associated in colon cancer-1 (MACC1) has been investigated but the relevance of peripheral MACC1 levels was rather limited. Herein, our data revealed that plasma MACC1 levels in 117 colorectal cancer patients (CRC) were dramatically higher than that in normal controls (P < 0.001), and with a strong discrimination power between the two groups (AUC = 0.960, P < 0.001). Moreover, MACC1 is an independent prognostic factor for CRC patients. When clinical parameters stratified by MACC1low and MACC1high , MACC1 levels exhibited further significant predictive value. Summary, plasma MACC1 levels could be a useful prognostic and diagnostic biomarker, and could improve the prognostic value of traditional prognosticators for colorectal cancer patients.

A novel HBx genotype serves as a preoperative predictor and fails to activate the JAK1/STATs pathway in hepatocellular carcinoma.

BACKGROUND & AIMS: Genetic variability in the hepatitis B virus X gene (HBx) is frequently observed and is associated with hepatocellular carcinoma (HCC) progression. However, a genotype classification based on the full-length HBx sequence and the impact of genotypes on hepatitis B virus (HBV)-related HCC prognosis remain unclear. We therefore aimed to perform this genotype classification and assess its clinical impact. METHODS: We classified the genotypes of the full-length HBx gene through sequencing and a cluster analysis of HBx DNA from a cohort of patients with HBV-related HCC, which served as the primary cohort (n = 284). Two independent HBV-related HCC cohorts, a validation cohort (n = 171) and a serum cohort (n = 168), were used to verify the results. Protein microarray assay analysis was performed to explore the underlying mechanism. RESULTS: In the primary cohort, the HBx DNA was classified into 3 genotypes: HBx-EHBH1, HBx-EHBH2, and HBx-EHBH3. HBx-EHBH2 (HBx-E2) indicated better recurrence-free survival and overall survival for patients with HCC. HBx-E2 was significantly correlated with the absence of liver cirrhosis, a small tumor size, a solitary tumor, complete encapsulation and Barcelona Clinic Liver Cancer (BCLC) stage A-0 tumors. Additionally, HBx-E2 served as a significant prognostic factor for patients with BCLC stage B HCC after hepatectomy. Mechanistically, HBx-E2 is unable to promote proliferation in HCC cells and normal hepatocytes. It also fails to activate the Janus kinase 1 (JAK1)/signal transducer and activator of transcription 3 (STAT3)/STAT5 pathway. CONCLUSION: Our study identifies a novel HBx genotype that is unable to promote the proliferation of HCC cells and suggests a potential marker to preoperatively predict the prognosis of patients with BCLC stage B, HBV-associated, HCC. LAY SUMMARY: We classified a novel genotype of the full-length hepatitis B virus X gene (HBx), HBx-E2. This genotype was identified in tumor and nontumor tissues from patients with hepatitis B virus-related hepatocellular carcinoma. HBx-E2 could preoperatively predict the prognosis of patients with intermediate stage hepatocellular carcinoma, after resection.

Evaluation of SUMO1 and POU2AF1 in whole blood from rheumatoid arthritis patients and at risk relatives.

Rheumatoid arthritis (RA) is a systemic autoimmune disorder characterized by chronic and symmetrical inflammation of synovial tissue with subsequent joint destruction. SUMO1 is an important regulator of apoptosis through non-canonical mechanism in synovial fibroblasts, and POU2AF1 is a known B-cell transcriptional co-activator. The specific objective of this study was to measure the expression of SUMO1 and POU2AF1 on first-degree relatives of patients with RA and also in the preclinical and clinical stages of RA and describe their possible role in RA physiopathology. Blood samples were collected from ACPA+, ACPA-, early and established RA subjects recruited. ACPAs and CarP autoantibodies were determined by ELISA Eurodiagnostica CCplus kit according to previously described protocols. RNA was isolated from blood samples; the purity as integrity was determined. Gene expression analysis was made by RT-qPCR using specific primers for SUMO1 and POU2AF1 mRNAs; relative expression was determined according to the 2-ΔΔct method procedure. Significant differences in the expression of both, SUMO1 and POU2AF1 were identified when comparing arthritis versus healthy or ACPA+ individuals, suggesting that the down regulation of such genes starts after the onset of symptoms in RA patients. Also, a significant correlation was identified for POU2AF1 and disease progression whit a downward trend for those with established RA. The implications of such gene down regulation are discussed in the context of RA physiopathology.

Exacerbation of oxygen-glucose deprivation-induced blood-brain barrier disruption: potential pathogenic role of interleukin-9 in ischemic stroke.

Interleukin (IL)-9 exerts a variety of functions in autoimmune diseases. However, its role in ischemic brain injury remains unknown. The present study explored the biological effects of IL-9 in ischemic stroke (IS). We recruited 42 patients newly diagnosed with IS and 22 age- and sex-matched healthy controls. The expression levels of IL-9 and percentages of IL-9-producing T cells, including CD3+CD4+IL-9+ and CD3+CD8+IL-9+ cells, were determined in peripheral blood mononuclear cells (PBMCs) obtained from patients and control individuals. We also investigated the effects of IL-9 on the blood-brain barrier (BBB) following oxygen-glucose deprivation (OGD) and the potential downstream signaling pathways. We found that patients with IS had higher IL-9 expression levels and increased percentages of IL-9-producing T cells in their PBMCs. The percentages of CD3+CD4+IL-9+ and CD3+CD8+IL-9+ T cells were positively correlated with the severity of illness. In in vitro experiments using bEnd.3 cells, exogenously administered IL-9 exacerbated the loss of tight junction proteins (TJPs) in cells subjected to OGD plus reoxygenation (RO). This effect was mediated via activation of IL-9 receptors, which increased the level of endothelial nitric oxide synthase (eNOS), as well as through up-regulated phosphorylation of signal transducer and activator of transcription 1 and 3 and down-regulated phosphorylated protein kinase B/phosphorylated phosphatidylinositol 3-kinase signaling. These results indicate that IL-9 has a destructive effect on the BBB following OGD, at least in part by inducing eNOS production, and raise the possibility of targetting IL-9 for therapeutic intervention in IS.

Interferon and interferon-inducible gene activation in patients with type 1 diabetes.

Type 1 diabetes (T1D) is an autoimmune disease that is thought to be triggered by environmental factors in genetically susceptible individuals. Enteroviruses have been mentioned as the most probable induction component of the disease. Nevertheless, the literature is controversial regarding the association of T1D with viral infection and first-line antiviral defence components, for example type I interferons (IFNs). Our aim was to test the hypothesis that an abnormality in IFN-stimulated gene patterns may cause a failure in immunological tolerance and, thereby, initiate T1D as an autoimmune disorder. We studied material from 64 T1D and 36 control subjects, divided into two age groups: <10 years and ≥10 years old. Using a relative gene expression method, we observed a lower expression of interferon-induced helicase 1 (IFIH1) and other type I IFN-induced genes in the blood cells of T1D subjects, especially subjects under 10 years old, in spite of their higher IFN levels as measured by the pSTAT1-inducing capacity of their sera. Likewise, freshly purified CpG-stimulated cells from T1D patients showed significantly lower upregulation of IFN-induced genes, that is IFIH1 and CXCL10, compared to cells from the control group. The identified dysregulation in the IFN-α-induced antiviral response in T1D patients, especially in early childhood, could be one of the factors affecting T1D development.

Serum Zta antibody of Epstein-Barr virus exerts potential function in the diagnosis of nasopharyngeal cancer.

The diagnosis of nasopharyngeal cancer (NPC) remains a clinical challenge. Many studies have assessed the diagnostic potential of Zta antibody of the Epstein-Barr virus (EBV) in NPC patients but with controversial results. This study aims to summarize the overall diagnostic performance of EBV Zta antibody in NPC. Based on a comprehensive search of the Pubmed and Embase, Web of Science, Chinese National Knowledge Infrastructure (CNKI), Wanfang Databases and China Citation Databases, we identified outcome data from all articles estimating diagnostic accuracy of EBV Zta antibody for NPC. A summary estimation for sensitivity, specificity, and other diagnostic indexes were pooled using a bivariate model. The overall measure of accuracy was calculated using summary receiver operating characteristic curve and the area under curve (AUC) was calculated. According to our inclusion criteria, 17 studies with 11,822 subjects (1,645 NPC cases, 10,177 controls) were included. The summary estimates were: sensitivity 0.87 (95 % confidence interval [CI] = 0.86-0.89), specificity 0.94 (95 % CI = 0.93-0.94), positive likelihood ratio 8.05 (95 % CI = 5.59-11.59), negative likelihood ratio 0.16 (95 % CI = 0.12-0.21), diagnostic odds ratio 52.93 (95 % CI = 29.95-93.56), the AUC and Q* index were 0.9352 and 0.8714, respectively. In conclusion, serum EBV Zta had a better diagnostic performance for NPC. Further studies should be performed to confirm our findings.

Upregulation of IL-23 expression in patients with chronic hepatitis B is mediated by the HBx/ERK/NF-κB pathway.

IL-23 is a newly discovered proinflammatory cytokine that contributes to the maintenance and expansion of Th17 cells. IL-23 has recently been identified as playing a critical role in a number of chronic inflammatory diseases. However, the regulatory mechanism of IL-23 in chronic hepatitis B (CHB) remains largely unknown. The aims of this study were to detect the expression of IL-23 in CHB patients and to explore the molecular mechanism of hepatitis B virus (HBV)-induced IL-23 expression. Serum levels and hepatic expression of IL-23 were significantly upregulated in CHB patients. A positive correlation was found between IL-23 expression and the histological activity index score, HBV DNA load, and serum alanine aminotransferase and aspartate aminotransferase levels. HBx protein increased IL-23 expression in a dose-dependent manner. It also aided in the nuclear translocation of NF-κB, which directly bound to the promoters of IL-23 subunits p19 and p40 to facilitate their transcription. NF-κB inhibitors blocked the effect of HBx on IL-23 induction, and NF-κB subunits p65 and p50 increased the augmented IL-23 expression. Inhibition of ERK1/2 activation and transfection with ERK dominant-negative plasmid significantly blocked the HBx-induced IL-23 expression. Furthermore, PI3K and Ras-MEK-MAPK inhibitors significantly decreased the ERK1/2 activation and IL-23 expression. Thus, we report a new molecular mechanism for HBV-induced IL-23 expression, which involves the activation of the ERK/NF-κB pathway by HBx, leading to the transactivation of the IL-23 p19 and p40 promoters.

Chronic IFN-γ production in mice induces anemia by reducing erythrocyte life span and inhibiting erythropoiesis through an IRF-1/PU.1 axis.

Anemia of chronic disease is a complication accompanying many inflammatory diseases. The proinflammatory cytokine IFN-γ has been implicated in this form of anemia, but the underlying mechanism remains unclear. Here we describe a novel mouse model for anemia of chronic disease, in which enhanced CD27-mediated costimulation strongly increases the formation of IFN-γ-producing effector T cells, leading to a progressive anemia. We demonstrate that the anemia in these mice is fully dependent on IFN-γ and that this cytokine reduces both the life span and the formation of red blood cells. Molecular analysis revealed that IFN-γ induces expression of the transcription factors of interferon regulatory factor-1 (IRF-1) and PU.1 in both murine and human erythroid precursors. We found that, on IFN-γ stimulation, IRF-1 binds to the promoter of SPI.1 (PU.1) and induces PU.1 expression, leading to inhibition of erythropoiesis. Notably, down-regulation of either IRF-1 or PU.1 expression is sufficient to overcome IFN-γ-induced inhibition of erythropoiesis. These findings reveal a molecular mechanism by which chronic exposure to IFN-γ induces anemia.

Serum methylation levels of TAC1. SEPT9 and EYA4 as diagnostic markers for early colorectal cancers: a pilot study.

OBJECTIVE: To identify methylated genes in serum with diagnostic potentials for early colorectal cancer (CRC). METHODS: Serum methylation levels of up to 12 genes were measured in two sets of serum samples with the second set from 26 stage I CRC patients and 26 age/gender-matched controls. RESULTS: Serum methylation levels of TAC1, SEPT9, and EYA4 were significant discriminants between stage I CRC and healthy controls. Combination of TAC1 and SEPT9 rendered 73.1% sensitivity with 92.3% specificity. CONCLUSION: Serum methylation levels of TAC1. SEPT9 and EYA4 may be useful biomarkers for early detection of CRC though a validation study is necessary.

Expression dynamics of the immune mediators ARG1, TBET, CIITA, IL10 and TGFB1 in chronic myeloid leukaemia patients during the first year of imatinib therapy.

The use of tyrosine kinase inhibitors seems to restore the broadly compromised immune system described in chronic myeloid leukaemia (CML) patients at diagnosis leading to a re-activation of the effector-mediated immune surveillance. Here, we describe the expression dynamics of immune factors during the first year on imatinib therapy. Gene expression was evaluated in 132 peripheral blood samples from 79 CML patients, including 34 who were serially followed. An aliquot of the stored sample used to monitor BCR-ABL1 levels was retro-transcribed to cDNA and gene expression was quantified by real-time PCR. An elevated expression of ARG1 was observed at diagnosis, while TBET, CIITA, IL10 and TGFB1 were significantly decreased. Once on therapy, each gene displayed a particular behaviour. ARG1 normalized to control levels at 3 months only in optimal molecular responders and was identified as the major contributor to the difference among patients. TBET reached normal levels after 12 months in optimal responders and non-responders, regardless the Th1-response previously associated, and CIITA continued downregulated. IL10 and TGFB1 achieved normal levels early at 3 months in both groups, afterwards IL10 was sustained while TGFB1 was slightly increased after 1 year in responders. Our findings are in agreement with an immune re-activation after imatinib initiation; however, some immune mediators may require a longer exposition. The follow-up of novel and reliable biomarkers, such as ARG1, one of the principal mechanisms of myeloid-derived-suppressor cells to inhibit immune system, may be useful to deepen the characterization of early responder patients.

Clinical significance of CDX2-positive circulating tumour cells in colorectal cancer patients.

BACKGROUND: Our recent work has shown the feasibility of using a refined immunomagnetic enrichment (IE) assay to detect cytokeratin 20-positive circulating tumour cells (CK20 pCTCs) in colorectal cancer (CRC) patients. We attempted to improve the sensitivity for CRC by detecting another intestinal-type differentiation marker, CDX2 pCTCs, using the same methodology. METHODS: CDX2 pCTCs were detected in patients with CRC, colorectal adenoma (CAD), benign colorectal diseases (BCD), other common cancers (OCC) and normal subjects (NS). Statistical analysis was used to correlate CDX2 pCTCs to the clinicohistopathological factors, recurrence, metastasis and survival after follow-up for 42 months in CRC patients. RESULTS: CDX2 pCTCs were detected in 81% CRC patients (73 out of 90, median number=21.5 CTCs), 7.5% CAD patients (3 out of 40), 0% patients with BCD (0 out of 90), 2.5% patients with OCC (2 out of 80) and 0% NS (0 out of 40). Furthermore, statistical analysis showed that CDX2 pCTC numbers were associated with tumour- node-metastasis stage and lymph node status. Using the median CDX2 pCTC numbers as the cutoff points, stratified groups of CRC patients had significant differences in their recurrence and survival. CONCLUSIONS: This study showed that the refined IE assay can detect CDX2 pCTCs with high sensitivity and that CDX2 pCTCs can generate clinically important information for CRC patients.

EBF1-Correlated Long Non-coding RNA Transcript Levels in 3rd Trimester Maternal Blood and Risk of Spontaneous Preterm Birth.

Biomarkers associated with spontaneous preterm birth (sPTB) before labor onset could aid in prediction, triage, and stratification for testing interventions. In this study we examined maternal blood EBF1-correlated long non-coding RNAs (lncRNAs) in relation to sPTB. We retrieved all lncRNA transcripts from a public gene expression dataset (GSE59491) derived from maternal blood in trimesters 2 and 3 from a Canadian cohort with a matched set of sPTB (n = 51) and term births (n = 106). LncRNA transcripts differentially expressed (limma moderated t-tests) in sPTB vs. term were tested for correlations (Pearson) with EBF1 mRNA levels in the same blood samples. Using logistic regression, EBF1-correlated lncRNAs were divided into tertiles and assessed in relation to odds of sPTB. Two lncRNA transcripts in the 3rd trimester maternal blood were differentially expressed between sPTB and term births (all p < 0.001 and FDR < 0.250) and positively and negatively correlated with EBF1 mRNA levels. They were as follows: (1) LINC00094 r = 0.196 (95% CI: 0.039 to 0.344), p = 0.015, and BH adjusted p = 0.022 and (2) LINC00870 r = - 0.303 (95% CI: - 0.441 to - 0.152), p < 0.001, and BH adjusted p < 0.001. As compared with term births, sPTBs were more likely to be in the highest tertile of LINC00870 (odds ratio (OR) = 4.08 (95% CI 1.60, 10.40), p = 0.003) and the lowest tertile of LINC00094 (OR = 5.16 (95% CI 1.96, 13.61), p < 0.001). Two sPTB-associated EBF1-correlated lncRNAs (LINC00870 and LINC00094) had multiple potential enhancers containing EBF1 binding site(s). Our current findings, along with previous reports linking EBF1 and sPTB, motivate additional research on the EBF1 gene-related gene expression and regulation in relation to sPTB within other cohorts and within laboratory-based models.

circRNA circ-MYBL2 is a novel tumor suppressor and potential biomarker in multiple myeloma.

Currently, multiple myeloma (MM) is still an incurable disease. Deciphering its pathogenesis will bring new targets for clinical diagnosis and treatment. In the present study, we identified a MM-associated circular RNA (circRNA), circ-MYBL2, which was dramatically decreased in MM tissue and serum samples in comparison to normal samples. Low circ-MYBL2 level was closely correlated with high clinical stage and unfavorable outcome, and serum circ-MYBL2 had excellent accuracy in diagnosing MM. Exogenous circ-MYBL2 expression notably repressed MM cell viability, DNA synthesis and cell cycle progression. Further exploration revealed that circ-MYBL2 exerted the tumor-inhibiting effect by affecting the phosphorylation level of its linear isoform, in which circ-MYBL2 facilitated the binding of Cyclin F to MYBL2, dampening MYBL2 phosphorylation and activation, thereby inhibiting the transcription of a number of well-known proliferation-related oncogenes. Importantly, overexpression of circ-MYBL2 significantly reduced the tumor size of subcutaneous xenografts in nude mice. Taken together, our data unveil a regulatory mechanism linking circ-MYBL2 and its host gene mediated by Cyclin F, providing a potential diagnostic, prognostic and therapeutic target for MM patients.

ELISA test for p63 antibodies in chronic ulcerative stomatitis.

OBJECTIVE: To develop a novel test for chronic ulcerative stomatitis (CUS), a chronic immunologically mediated condition that produces oral ulcerations. Current diagnostic methods require expensive and technically demanding in situ immunofluorescence (IF) studies. DESIGN: An Enzyme-Linked ImmunoSorbent Assay (ELISA) was prepared and tested with serum samples from patients with CUS and negative controls. MATERIALS AND METHODS: The N-terminal portion of the CUS autoantigen, DeltaNp63alpha, was produced as a purified recombinant protein and used to coat ELISA plates. Sera from 25 patients with CUS and 16 negative controls were analyzed for reactive antibodies. The optimal cut-offs for positive and negative samples were determined. MAIN OUTCOME MEASURES: The optimal cut-off of 0.236 resulted in a sensitivity and specificity of the ELISA of 0.80 and 0.75, respectively (exact 95% confidence intervals, P-value of <0.001). RESULTS: The ELISA developed in this study provides a novel and reliable diagnostic assessment to distinguish CUS from other oral ulcerative diseases. CONCLUSIONS: Immunoassay will allow the true incidence and prevalence of CUS to be determined in future studies. When combined with clinical correlations, the ELISA results will facilitate the evaluation of the prognostic utility of antibody titers and allow correlation with treatment responses in individual CUS cases.

Differential levels of Alpha-1-inhibitor III, Immunoglobulin heavy chain variable region, and Hypertrophied skeletal muscle protein GTF3 in rat mammary tumorigenesis.

Early detection of breast cancer can be best facilitated by the development of precancerous markers. Serum proteins being the sensitive signatures, can be the ideal choice. We previously demonstrated the reduced levels of two serum proteins at a very early stage of tumorigenesis in a breast cancer model, developed in Wistar rats by 7,12-dimethylbenz[a]anthracene (DMBA) administration. Here we report the dysregulation of three more proteins in the serum collected at another early stage (15 weeks) of tumorigenesis in the same model. The proteins were identified (as Alpha-1-inhibitor III (Mug1), Immunoglobulin heavy chain variable region (IGHV), and Hypertrophied skeletal muscle protein GTF3) by MALDI-TOF MS after the screening and fingerprinting of serum samples by one-dimensional (1D) and two-dimensional (2D) electrophoresis respectively. Relative expression analysis of corresponding genes was also carried out, and the results were found as supporting the proteomic findings. In addition, the candidate proteins of the study and their corresponding ribonucleic acids (RNAs) were subjected to homology modelling and docking (using softwares like MODELLER, 3dRNA, Autodock4.0, and GROMACS etc), which revealed the binding sites for carcinogen (DMBA) and its nature of interaction with proteins and RNAs. Moreover, the network analysis by GeneMANIA unraveled the protein/gene functional network in which Mug1, IGHV, and GTF3 are involved. Based on the significant protein and gene expression alterations in early tumorigenesis, these proteins may prove very effective in search for biomarkers for the early detection of mammary cancer. Further, these proteins can also be tried as targets for chemotherapy.

Anti-hepatitis B virus X protein in sera is one of the markers of development of liver cirrhosis and liver cancer mediated by HBV.

Hepatitis B virus X protein (HBx) plays a crucial role in the development of hepatocellular carcinoma (HCC). However, the significance of circulating antibody to hepatitis B virus X antigen (anti-HBx) in sera remains unclear. In the present study, we examined the titers of anti-HBx (IgG) in the sera from 173 patients with chronic hepatitis B (CHB), 106 liver cirrhosis (LC), and 61 HCC by enzyme-linked immunosorbent assay (ELISA), respectively. Our data showed that the positive rates of anti-HBx were higher in sera of LC (40.6%) and HCC (34.4%) than those of CHB (10.4%), P < .05. In all 40 patients with anti-HBx+ out of 340 patients, 39 (97.5%) were HBsAg/HBeAg/anti-HBc+ and 1 (2.5%) was anti-HBs+ (P < .01), suggesting that anti-HBx in sera is a marker of HBV replication rather than a protective antibody. Thus, our findings reveal that circulating anti-HBx in sera is one of the markers of development of LC and HCC mediated by HBV.