Impact of beta-2 microglobulin expression on the survival of glioma patients via modulating the tumor immune microenvironment.

AIMS: High immune cell infiltration in gliomas establishes an immunosuppressive tumor microenvironment, which in turn promotes resistance to immunotherapy. Hence, it is important to identify novel targets associated with high immune cell infiltration in gliomas. Our previous study showed that serum levels of beta-2 microglobulin (B2M) in lower-grade glioma patients were lower than those in glioblastoma patients. In the present study, we focused on exploring the roles of B2M in glioma immune infiltration. METHODS: A large cohort of patients with gliomas from the TCGA, CGGA, and Gravendeel databases was included to explore differential expression patterns and potential roles of B2M in gliomas. A total of 103 glioma tissue samples were collected to determine the distributions of B2M protein levels by immunofluorescent assays. Kaplan-Meier survival analysis and meta-analysis were used for survival analysis. GO(Gene-ontology) enrichment analysis, co-expression analysis, KEGG(Kyoto Encyclopedia of Genes and Genomes) pathway analysis, and immune infiltration analysis were performed to explore roles and related mechanisms of B2M in glioma. RESULTS: We found that both B2M mRNA and protein levels were abnormally upregulated in glioma samples compared with those from normal brain tissue. B2M expression was correlated with tumor grade and was downregulated in IDH1 mutant samples. Furthermore, B2M was a moderately sensitive indicator for predicting the mesenchymal molecular subtype of gliomas. Interestingly, glioma patients with lower B2M expression had remarkably longer survival times than those with higher B2M expression. Moreover, meta-analysis showed that B2M was an independent predictive marker in glioma patients. The results of GO enrichment analysis revealed that B2M contributed to immune cell infiltration in glioma patients. In addition, results of KEGG pathway analysis and co-expression analysis suggested that B2M may mediate glioma immune infiltration via chemokines. CONCLUSIONS: We conclude that B2M levels are critical for the survival times of glioma patients, at least in part due to mediating high immune infiltration.

ß2-microglobulin has a different regulatory molecular mechanism between ER+ and ER- breast cancer with HER2.

BACKGROUND: Previous studies have demonstrated that ß2-microglobulin (ß2M) promotes the growth and survival of a variety of cancer cells and has different regulatory effects on the expression of Bcl-2 and HER2 in HER2- breast cancer cells. However, ß2M-mediated signaling in ER+ and ER- breast cancer with HER2- remains unclear. METHODS: ß2M expression vector and siRNA were transfected into two types of HER2- breast cancer cells, and the possible relevant signaling molecules were subsequently analyzed by real-time PCR and western blotting. These signaling molecules were also analyzed by real-time PCR and immunohistochemistry (IHC) in two types of HER2- breast cancer tissues, and the associations between ß2M and these signaling molecules were assessed using Spearman's correlation analysis. RESULTS: ß2M silencing downregulated p-SGK1/SGK1 levels and Bcl-2 expression, and ß2M overexpression downregulated p-CREB/CREB and significantly upregulated p-SGK1/SGK1 levels and Bcl-2 expression, and both resulting processes did not affect HER2, HIF-1α, VEGF, and ERK signaling in ER+ breast cancer cells with HER2-. ß2M silencing upregulated p-CREB/CREB and VEGF protein and significantly downregulated p-ERK/ERK levels, and ß2M overexpression downregulated p-CREB/CREB and VEGF, significantly upregulated p-ERK/ERK levels, and both resulting processes did not affect HIF-1α and SGK1 signaling in ER- breast cancer cells with HER2-. ß2M expression was positively correlated with p-CREB, p-SGK1, and Bcl-2 expression and had no correlation with HIF-1α, VEGF, and p-ERK1/2, whereas p-SGK1 exhibited a significantly positive correlation with Bcl-2 expression in cancer tissues of patients with luminal A breast cancer, which coincide with the results obtained from the same molecular types of breast cancer cells except CREB signaling. However, ß2M expression did not show a significant correlation with HIF-1α, p-CREB, VEGF, p-SGK1, p-ERK1/2, and Bcl-2 expression in cancer tissues of patients with basal-like breast cancer, which was discordant with the results obtained from the same molecular types of breast cancer cells. CONCLUSIONS: ß2M has a different molecular regulatory mechanism between ER+ and ER- breast cancer with HER2-, and it may promote tumor survival through the SGK1/Bcl-2 signaling pathway in ER+ breast cancer with HER2- and has no regulatory effects on ER- breast cancer with HER2-.

Major Histocompatibility Complex Class II and Programmed Death Ligand 1 Expression Predict Outcome After Programmed Death 1 Blockade in Classic Hodgkin Lymphoma.

Purpose Hodgkin Reed-Sternberg (HRS) cells evade antitumor immunity by multiple means, including gains of 9p24.1/ CD274(PD-L1)/ PDCD1LG2(PD-L2) and perturbed antigen presentation. Programmed death 1 (PD-1) receptor blockade is active in classic Hodgkin lymphoma (cHL) despite reported deficiencies of major histocompatibility complex (MHC) class I expression on HRS cells. Herein, we assess bases of sensitivity to PD-1 blockade in patients with relapsed/refractory cHL who were treated with nivolumab (anti-PD-1) in the CheckMate 205 trial. Methods HRS cells from archival tumor biopsies were evaluated for 9p24.1 alterations by fluorescence in situ hybridization and for expression of PD ligand 1 (PD-L1) and the antigen presentation pathway components-ß2-microglobulin, MHC class I, and MHC class II-by immunohistochemistry. These parameters were correlated with clinical responses and progression-free survival (PFS) after PD-1 blockade. Results Patients with higher-level 9p24.1 copy gain and increased PD-L1 expression on HRS cells had superior PFS. HRS cell expression of ß2-microglobulin/MHC class I was not predictive for complete remission or PFS after nivolumab therapy. In contrast, HRS cell expression of MHC class II was predictive for complete remission. In patients with a > 12-month interval between myeloablative autologous stem-cell transplantation and nivolumab therapy, HRS cell expression of MHC class II was associated with prolonged PFS. Conclusion Genetically driven PD-L1 expression and MHC class II positivity on HRS cells are potential predictors of favorable outcome after PD-1 blockade. In cHL, clinical responses to nivolumab were not dependent on HRS cell expression of MHC class I.

Expression pattern of immunosurveillance-related antigen in adult T cell leukaemia/lymphoma.

AIMS: Adult T cell leukaemia/lymphoma (ATLL) is an aggressive malignancy with a poor prognosis. Human leucocyte antigen (HLA) and ß2 microglobulin (ß2M) serve as key molecules in tumour immunity, and their expression is reduced frequently in tumour cells. Programmed cell death (PD)-1/PD-ligand1 (PD-L1) interactions play a role in escape of tumour cells from T cell immunity. Therefore, this study aimed to determine the clinicopathological relevance of HLA and ß2M expressions in ATLL cells and PD-L1 expression in lymphoma or stromal cells and predict the overall survival of patients with ATLL. METHODS AND RESULTS: We analysed a total of 123 biopsy samples from patients newly diagnosed with ATLL by using immunohistochemical analysis. Of the patients enrolled, 91 (74%) were positive for HLA (in cell membrane, 60 patients), 89 (72%) were positive for ß2M (in cell membrane, 54 patients) and 48 (39%) were positive for both HLA and ß2M in the cell membrane (HLAm+ ß2Mm+ ). No significant clinical differences other than prognosis were found between the HLAm+ ß2Mm+ group and the other groups. Immunophenotypical evaluation revealed significantly higher rates of CD30-positive lymphoma cells (P = 0.003) and PD-L1-positive stromal cells in microenvironments (miPD-L1high ) (P = 0.011) of the HLAm+ ß2Mm+ group than in the other groups. The HLAm+ ß2Mm+ group had a significantly better prognosis that the other groups (P = 0.0096), and patients showing HLAm+ ß2Mm+ with miPD-L1high had the most favourable prognosis among all groups. CONCLUSIONS: The membranous expression of HLA and ß2M is likely to reflect the immune response and would be useful to predict prognosis before starting ATLL therapy.

Selection of suitable endogenous reference genes for qPCR in kidney and hypothalamus of rats under testosterone influence.

Real-time quantitative PCR (qPCR) is the most reliable and accurate technique for analyses of gene expression. Endogenous reference genes are being used to normalize qPCR data even though their expression may vary under different conditions and in different tissues. Nonetheless, verification of expression of reference genes in selected studied tissue is essential in order to accurately assess the level of expression of target genes of interest. Therefore, in this study, we attempted to examine six commonly used reference genes in order to identify the gene being expressed most constantly under the influence of testosterone in the kidneys and hypothalamus. The reference genes include glyceraldehyde-3-phosphate dehydrogenase (GAPDH), actin beta (ACTB), beta-2 microglobulin (B2m), hypoxanthine phosphoribosyltransferase 1 (HPRT), peptidylprolylisomerase A (Ppia) and hydroxymethylbilane synthase (Hmbs). The cycle threshold (Ct) value for each gene was determined and data obtained were analyzed using the software programs NormFinder, geNorm, BestKeeper, and rank aggregation. Results showed that Hmbs and Ppia genes were the most stably expressed in the hypothalamus. Meanwhile, in kidneys, Hmbs and GAPDH appeared to be the most constant genes. In conclusion, variations in expression levels of reference genes occur in kidneys and hypothalamus under similar conditions; thus, it is important to verify reference gene levels in these tissues prior to commencing any studies.

Characterization of MHC Class I and ß-2-Microglobulin Expression in Pediatric Solid Malignancies to Guide Selection of Immune-Based Therapeutic Trials.

BACKGROUND: Over 10,000 US children are diagnosed with cancer yearly. Though outcomes have improved by optimizing conventional therapies, recent immunotherapeutic successes in adult cancers are emerging. Cytotoxic T lymphocytes (CTLs) are the primary executioners of adaptive antitumor immunity and require antigenic presentation in the context of major histocompatibility complex (MHC) class I and the associated ß-2-microglobulin (B2M). Loss of MHC I expression is a common immune escape mechanism in adult malignancies, but pediatric cancers have not been thoroughly characterized. The essential nature of MHC I expression in CTL-mediated cell death may dictate the success of immunotherapies, which rely on eliciting an adaptive response. PROCEDURE: We queried pediatric tumor microarray databases for MHC I and B2M gene expression. We detected MHC I in pediatric tumor cell lines by flow cytometry and characterized MHC I and B2M expression in patient samples by immunohistochemistry. To determine whether therapeutic approaches might enhance MHC I expression in selected models in vitro, we tested effects of exposure to IFN-γ and histone deacetylase inhibitors. RESULTS: Pediatric tumors overall, as well as samples within select individual tumor subtypes, exhibit wide ranges of MHC I and B2M gene and protein expression. For most cell lines tested, MHC I was inducible in vitro. CONCLUSIONS: MHC I and B2M expression vary among pediatric tumor types and should be evaluated as potential biomarkers, which might identify patients most likely to benefit from MHC I dependent immunotherapies. Modulation of MHC I expression may be a promising mechanism for enhancing MHC I dependent immunotherapeutic efficacy.

Toll-like receptor 4-induced inflammatory responses contribute to the tumor-associated macrophages formation and infiltration in patients with diffuse large B-cell lymphoma.

To evaluate the expression of tumor-associated macrophages (TAMs) and Toll-like receptor 4 (TLR4) in diffuse large B-cell lymphoma (DLBCL) and their correlation with patient clinical characteristics, we detected using immunohistochemistry in 81 specimens of patients with DLBCL. The correlation between protein expression levels and clinical parameters, as well as the association between CD68 and TLR4 were analyzed. The number of CD68 TAMs was closely related to ß2-microglobulin (P = .028 and P < .05), whereas there was no significant correlation between the number of CD68 TAMs and other clinical factors. Toll-like receptor 4 was related to tumor size and peripheral blood lymphocyte to monocyte ratio. The Spearman correlation coefficient indicated a significant positive correlation between CD68 TAMs and TLR4 expression (r = 0.240; P = .038, P = .05). These results, on one hand, indicated that TLR4-induced inflammatory responses may affect TAM infiltration and accumulation, and that TAMs and TLR4 may interact to play important roles in DLBCL microenvironment regulating the tumor growth, but, on the other hand demonstrated that both of TAMs and TLR4 had not only one side on DLBCL growth.

Lifespan of mice and primates correlates with immunoproteasome expression.

There is large variation in lifespan among different species, and there is evidence that modulation of proteasome function may contribute to longevity determination. Comparative biology provides a powerful tool for identifying genes and pathways that control the rate of aging. Here, we evaluated skin-derived fibroblasts and demonstrate that among primate species, longevity correlated with an elevation in proteasomal activity as well as immunoproteasome expression at both the mRNA and protein levels. Immunoproteasome enhancement occurred with a concurrent increase in other elements involved in MHC class I antigen presentation, including ß-2 microglobulin, (TAP1), and TAP2. Fibroblasts from long-lived primates also appeared more responsive to IFN-γ than cells from short-lived primate species, and this increase in IFN-γ responsiveness correlated with elevated expression of the IFN-γ receptor protein IFNGR2. Elevation of immunoproteasome and proteasome activity was also observed in the livers of long-lived Snell dwarf mice and in mice exposed to drugs that have been shown to extend lifespan, including rapamycin, 17-α-estradiol, and nordihydroguaiaretic acid. This work suggests that augmented immunoproteasome function may contribute to lifespan differences in mice and among primate species.

Anti-ß2-microglobulin monoclonal antibodies overcome bortezomib resistance in multiple myeloma by inhibiting autophagy.

Our previous studies showed that anti-ß2M monoclonal antibodies (mAbs) have strong and direct apoptotic effects on multiple myeloma (MM) cells, suggesting that anti-ß2M mAbs might be developed as a novel therapeutic agent. In this study, we investigated the anti-MM effects of combination treatment with anti-ß2M mAbs and bortezomib (BTZ). Our results showed that anti-ß2M mAbs enhanced BTZ-induced apoptosis of MM cell lines and primary MM cells. Combination treatment could also induce apoptosis of BTZ-resistant MM cells, and the enhanced effect depended on the surface expression of ß2M on MM cells. BTZ up-regulated the expression of autophagy proteins, whereas combination with anti-ß2M mAbs inhibited autophagy. Sequence analysis of the promoter region of beclin 1 identified 3 putative NF-κB-binding sites from -615 to -789 bp. BTZ treatment increased, whereas combination with anti-ß2M mAbs reduced, NF-κB transcription activities in MM cells, and combination treatment inhibited NF-κB p65 binding to the beclin 1 promoter. Furthermore, anti-ß2M mAbs and BTZ combination treatment had anti-MM activities in an established MM mouse model. Thus, our studies provide new insight and support for the clinical development of an anti-ß2M mAb and BTZ combination treatment to overcome BTZ drug resistance and improve MM patient survival.

[Optimization of Prokaryotic Expression Conditions of Human ß2-microglobulin in E. Coli and Its Purification].

To obtain recombinant human ß2-microglobulin (rhß2M) with properties of good solubility and high purity from E. coli, prokaryotic expression conditions were optimized and protein purification was performed in this study. After testing the effect of different IPTG concentrations, temperatures and induction times on the production of rhß2M, the optimum expression conditions were determined, i. e. joining IPTG to final concentration being 0.8 mmol/L and inducing time 6 h and at temperature of 25 degrees C. Under the optimum induction conditions, the ratio of soluble rhß2M to soluble bacterial protein was 63.7%. After purified by Ni Sepharose 6 Fast Flow, the purity of rhß2M achieved a greater value of 95%. Western blot analysis revealed that rhß2M possessed the antigen property that specifically interacted with anti-ß2M antibody.

Increased expression of human leucocyte antigen class I free heavy chains on monocytes of patients with spondyloarthritis and cells transfected with HLA-B27.

Human leucocyte antigen (HLA)-B27 expression is correlated with spondyloarthritis (SpA), but its role in disease pathogenesis remains unclear. The aim of the study was to determine whether HLA-B27 free heavy chain (FHC) contributes to SpA pathogenesis. Flow cytometry was used to analyse the FHC expression on CD3+ and CD14+ cells in the peripheral blood (PB) and synovial fluid (SF) from SpA patients, healthy controls, and rheumatoid arthritis (RA) patients. Human monocytic U937 cell lines stably expressing enhanced green fluorescence protein (EGFP)/HLA-B27, EGFP/HLA-A2 or EGFP alone were created to further investigate the relation between HLA-B27 and FHC expression. The relative FHC level on CD14+ PB cells was significantly higher in SpA patients than in controls, but lower than on the SF cells of SpA patients. No significant correlation was found for relative FHC expression with HLA-B27 or ß2-microglobulin expression. HLA-B27-transfected U937 cells expressed higher FHC levels than either EGFP/HLA-A2- or EGFP-transfected cells. HLA class I FHC expression was significantly increased on monocytes of SpA patients and HLA-B27-transfected cells, implying that FHC, perhaps mostly derived from HLA-B27, plays an important role in SpA pathogenesis.

Induction of rainbow trout MH class I and accessory proteins by viral haemorrhagic septicaemia virus.

Major histocompatibility (MH) class I receptors are glycoproteins which play a critical role during responses to intracellular pathogens by presenting endogenous peptides to cytotoxic T cell lymphocytes (CD8+). To date, little is known about MH class I regulation at the protein level during viral infections in fish. In this study, we characterised the MH class I pathway response to polyinosinic-polycytidylic acid (poly I:C) and upon infection with viral haemorrhagic septicemia virus (VHSV) genotype IVa using the rainbow trout monocyte/macrophage cell line RTS11. A 14-day challenge with VHSV IVa at 14°C demonstrated enhanced expression of the class I heavy chain, ß2 microglobulin (ß2M) and tapasin, while the expression of other accessory molecules ERp57 and calreticulin remained unchanged. However, when infection occurred at 2°C no change in expression levels of any of these molecules was observed. ß2M accumulated in the media of RTS11 over time, however the ß2M concentrations were 2 fold higher in cultures infected with VHSV 14 days post infection. Strikingly, when cells were maintained at 2°C the secretion of ß2M was significantly reduced in both infected and non-infected cultures. These results indicate that VHSV infection alters the kinetics of ß2M release as well as the expression of MH class I and suggests that cellular immunity against VHSV can be compromised at low temperatures which may increase host susceptibility to this virus during the winter.

The similar expression pattern of MHC class I molecules in human and mouse cerebellar cortex.

The major histocompatibility complex (MHC) class I molecules are considered to be important in the immune system. However, the results reported in the past decade indicate that they also play important roles in the central nervous system. Here we examined the expression of MHC I and ß2-microglobulin (ß2m) in human and mouse cerebellar cortex. The results show that MHC I molecules are expressed both in human and mouse cerebellar cortex during brain development. The expression of H-2K(b)/D(b) is gradually increased with the development of mouse cerebellar cortex, but finally decreased to a very low level. Similarly, the expression of HLA-B/C genes is increased in developing human cerebellar cortex, but decreased after birth. The spatial and temporal expression of ß2m overlaps mostly with that of HLA-B/C molecules, and they are co-expressed in Purkinje cells. Our findings provide a fundamental basis to reveal the functions of neuronal MHC class I molecules in the development of human cerebellum.

Ridostin induces transcription of a wide spectrum of interferon genes in human cells.

The effects of Ridostin on the transcription of IFN family genes in human fibroblasts and lymphocytes were studied by quantitative real-time PCR. The degree of gene induction by Ridostin was most pronounced in fibroblasts, and was significantly higher than the induction by Kagocel: transcription of IFN-ß, oligoadenylate synthetase, and double-stranded RNA-dependent protein kinase genes increased by about 2000, 100, and 20 times, respectively. In lymphocytes, Ridostin also activated a wide variety of IFN family genes, including genes of IFN-ß, IFN-γ, and IFN-dependent enzymes, but this induction was less pronounced than in the fibroblasts. It was shown that gene response in lymphocyte from a child with cancer is reduced in comparison with that of adult healthy participant. Ridostin, and even more so Reaferon up-regulated activities of ß-actin, glycerophosphate dehydrogenase, and ß2-microglobulin genes, thus making impossible or limiting their use as constitutive stable reference genes (standards) in PCR-assays of IFN and their inductors.

Both the cis-trans equilibrium and isomerization dynamics of a single proline amide modulate ß2-microglobulin amyloid assembly.

The human protein ß2-microglobulin (ß2m) aggregates as amyloid fibrils in patients undergoing long-term hemodialysis. Isomerization of Pro32 from its native cis to a nonnative trans conformation is thought to trigger ß2m misfolding and subsequent amyloid assembly. To examine this hypothesis, we systematically varied the free-energy profile of proline cis-trans isomerization by replacing Pro32 with a series of 4-fluoroprolines via total chemical synthesis. We show that ß2m's stability, (un)folding, and aggregation properties are all influenced by the rate and equilibrium of Pro32 cis-trans isomerization. As anticipated, the ß2m monomer was either stabilized or destabilized by respective incorporation of (2S,4S)-fluoroproline, which favors the native cis amide bond, or the stereoisomeric (2S,4R)-fluoroproline, which disfavors this conformation. However, substitution of Pro32 with 4,4-difluoroproline, which has nearly the same cis-trans preference as proline but an enhanced isomerization rate, caused pronounced destabilization of the protein and increased oligomerization at neutral pH. More remarkably, these subtle alterations in chemical composition--incorporation of one or two fluorine atoms into a single proline residue in the 99 amino acid long protein--modulated the aggregation properties of ß2m, inducing the formation of polymorphically distinct amyloid fibrils. These results highlight the importance of conformational dynamics for molecular assembly of an amyloid cross-ß structure and provide insights into mechanistic aspects of Pro32 cis-trans isomerism in ß2m aggregation.

Effects of calcitonin gene-related peptide on the immune privilege of human hair follicles.

The hair follicle is a widely available and instructive miniature organ in the human body that experiences major histocompatibility complex (MHC) class I dependent immune privilege (IP). There are various regulation factors that act on the generation, maintenance, and collapse of hair follicle IP. Neuropeptides such as calcitonin gene-related peptide (CGRP) are created in many organs, including skin, and display various immune regulation effects. The purpose of this study was to investigate the phenotypic effect of CGRP on the hair follicle's IP. First, we used interferon-γ (IFN-γ) to generate ectopic MHC antigen expression model in cultured human hair follicles as previously described. Then, we examined the effects of CGRP on the regulation of ectopic MHC antigen expression in cultured human hair follicles using reverse transcriptase-polymerase chain reaction (RT-PCR) and immunohistochemical staining techniques. IFN-γ (75 IU/ml) induced ectopic MHC expression. CGRP down-regulated INF-γ-induced ectopic MHC class I mRNA expression. These down-regulated effects were especially evident in 10(-8)M. In addition, CGRP also suppressed the staining intensity related to the expression of MHC class I and MHC class I-pathway related molecules (ß2-microglobulin), but had no effect on MHC class II antigen expression. Taken together, these results indicate that CGRP might be an important regulatory factor for IP maintenance and restoration of IP via suppression of MHC class I antigen.

Reprogramming of the MHC-I and its regulation by NFκB in human-induced pluripotent stem cells.

The immunogenicity of human pluripotent stem cells plays a major role in their potential use in the clinic. We show that, during their reprogramming, human-induced pluripotent stem (iPS) cells downregulate expression of human leukocyte antigen (HLA)-A/B/C and ß2 microglobulin (ß2M), the two components of major histocompatibility complex-I (MHC-I). MHC-I expression in iPS cells can be restored by differentiation or treatment with interferon-gamma (IFNγ). To analyze the molecular mechanisms that regulate the expression of the MHC-I molecules in human iPS cells, we searched for correlation between the expression of HLA-A/B/C and ß2M and the expression of transcription factors that bind to the promoter of these genes. Our results show a significant positive correlation between MHC-I expression and expression of the nuclear factors, nuclear factor kappa B 1 (NFκB1) and RelA, at the levels of RNA, protein and was confirmed by chromatin binding. Concordantly, we detected robust levels of NFκB1 and RelA proteins in the nucleus of somatic cells but not in the iPS cell derived from them. Overexpression of NFκB1 and RelA in undifferentiated pluripotent stem cells led to induction in expression of MHC-I, whereas silencing NFκB1 and RelA by small hairpin RNA decreased the expression of ß2M after IFNγ treatment. Our data point to the critical role of NFκB proteins in regulating the MHC-I expression in human pluripotent stem cells.

Beta 2 microglobulin and the risk for cardiovascular events in patients with asymptomatic carotid atherosclerosis.

BACKGROUND AND PURPOSE: Atherosclerosis is a chronic inflammatory disease. Ongoing inflammation is associated with elevated levels of beta 2 microglobulin (B2M). We investigated B2M levels in a large cohort of patients with carotid atherosclerosis for the occurrence of major adverse cardiovascular events. METHODS: One thousand five of 1286 consecutive, neurologically asymptomatic patients with carotid atherosclerosis were followed for a median of 3 years (interquartile range, 2.5 to 3.5) for the occurrence of major adverse cardiovascular events, a composite of myocardial infarction, percutaneous coronary intervention, coronary bypass graft, stroke, and death. RESULTS: We recorded 359 major cardiovascular events in 271 (27%) patients. B2M was significantly associated with the occurrence of major adverse cardiovascular events. With increasing quartiles of B2M, the adjusted hazard ratios were 1.19 (95% CI, 0.81 to 1.73), 1.51 (95% CI, 1.05 to 2.18), and 1.88 (95% CI, 1.26 to 2.79) compared with the lowest quartile, respectively (P<0.001). Adjusted hazard ratios for the occurrence of death, myocardial infarction, and stroke for increasing quartiles of B2M were 1.25 (95% CI, 0.92 to 1.70), 1.52 (95% CI, 1.12 to 2.06), and 1.62 (95% CI, 1.16 to 2.67) compared with the lowest quartile, respectively (P<0.001). Through statistical estimation of improvement in risk stratification, addition of B2M to baseline risk factors improved the risk stratification for major cardiovascular events, at least as much as high-sensitivity C-reactive protein or even better. CONCLUSIONS: B2M was independently and significantly associated with adverse cardiovascular outcome in patients with prevalent asymptomatic carotid atherosclerosis.

Urinary epidermal growth factor, monocyte chemotactic protein-1, and ß2-microglobulin in children with ureteropelvic junction obstruction.

BACKGROUND/PURPOSE: We demonstrated down-regulation of epidermal growth factor (EGF) and up-regulation of monocyte chemotactic protein-1 (MCP-1) in the renal parenchyma in children who underwent pyeloplasty for ureteropelvic junction obstruction (UPJO). These findings were paralleled by urinary levels of EGF and MCP-1 before and after surgery. The aim of this study is to evaluate the urinary excretion of these cytokines and ß2-microglobulin (ß2M) in children with urine flow impairment at the ureteropelvic junction or who underwent pyeloplasty. METHODS: Seventy-six patients with UPJO and 30 normal children (CTRL) were enrolled in the study. The UPJO patients were divided into obstructive (12), functional (36), and operated (28). Epidermal growth factor, MCP-1, and ß2M urinary levels were measured by enzyme-linked immunosorbent assay and normalized to urine creatinine. RESULTS: Urinary ß2M and MCP-1 increased significantly in the UPJO groups compared with the CTRL and significantly improved in the operated group. The obstructive group displayed reduced EGF excretion compared with the CTRL group. The urinary (u)EGF/uMCP-1, and uEGF/uß2M ratios significantly decreased in both untreated groups. In the operated group, these ratios improved significantly. CONCLUSIONS: The present study substantiates the role of urinary EGF, MCP-1, and ß2M as markers of tubulointerstitial damage in human obstructive nephropathy. Furthermore, it suggests that surgical intervention is effective in the management of children with UPJO.

Ontogeny and tissue-specific expression of innate immune related genes in rohu, Labeo rohita (Hamilton).

The innate immune response in fish represents an early and rapid defense against pathogens. The present study aims at looking into ontogeny of innate immune system in the teleost, Labeo rohita using RT-PCR based approach. Total RNA extracted from unfertilized and fertilized eggs, and hatchlings (hatched at 28 ± 2 °C) at 0, 1, 3, 6, 12, 24 h, and 3, 7, 16, 21, 31 days post-fertilization were subjected to RT-PCR using self-designed or earlier published primers to amplify some innate immune relevant genes (lysozyme C, lysozyme G, beta-2 microglobulin, toll-like receptor 22-like and transferrin). The constitutive expression of ß-actin was detected in unfertilized eggs and further developmental stages. Transferrin and TLR22-like mRNA transcripts were detected by RT-PCR from 6 h post-fertilization to 31 day post-fertilization, whereas ß-2 microglobulin transcripts were detected only from 7 day post-fertilization onwards. Lysozyme C mRNA transcripts were detected from 24 h post-fertilization to 31 day post-fertilization. Lysozyme G mRNA transcripts were detected early from unfertilized egg stage onwards. Similarly, tissues viz. intestine, heart, ovary, gill, spleen, muscle, liver, brain, skin, anterior kidney, posterior kidney, and blood collected from juveniles of rohu were subjected to detection of all above mentioned gene transcripts by RT-PCR. ß2-microglobulin mRNA transcript was expressed in all tissues. Lysozyme C mRNA expression is confined to blood and posterior kidney only whereas lysozyme G mRNA is expressed in all tissues. TLR22-like mRNA is expressed in all tissues except ovary and liver whereas transferrin mRNA transcript is detected only in liver. Finally, all these information taken are likely to shed light on the ontogeny of innate immunity in L. rohita, which offers new insights to developmental biology when compared to higher vertebrates and also helpful in the development of preventive measures against problems concerning infectious diseases.