Expression Of Beta Chain Research Articles

C8B in Complement and Coagulation Cascades Signaling Pathway is a predictor for Survival in HBV-Related Hepatocellular Carcinoma Patients.

ObjectiveThe role of the complement and coagulation cascades signaling pathway in the pathogenesis of cancers remains uncertain. This study aimed to investigate the associations between enriched differentially expressed genes (DEGs) in this pathway and hepatitis B virus (HBV)-related hepatocellular carcinoma (HCC) patients.Materials and MethodsClinical and gene expression data of the Gene Expression Omnibus (GEO) series profile GSE14520 were downloaded. The “Limma” package was used to screen the DEGs and the “clusterProfiler” package was used to identify the complement and coagulation cascades pathway and enriched significant genes. Cox regression analysis, the Kaplan–Meier method, and the nomogram model were used to address the correlations between significantly enriched DEGs in the complement and coagulation cascades pathway and HCC survival.ResultsA total of 220 HBV-related HCC patients were enrolled in this study. The complement and coagulation cascades pathway was significantly enriched by 37 DEGs (p-value < 0.05 and adjusted p-value < 0.05). Complement 8 beta chain (C8B) expression levels had protective effects on overall survival (OS) and recurrence-free survival (RFS) in HBV-related HCC patients. High levels of C8B contributed to favorable OS and RFS in this population (both p < 0.01), even after adjustment of clinicopathological characteristics including tumor node metastasis (TNM) staging, Barcelona Clinic liver cancer (BCLC) staging, gender, and fibrinogen beta chain (FGB) expression (all p < 0.05).ConclusionC8B in the complement and coagulation cascades signaling pathway serves as a predictive candidate for survival in HBV-related HCC patients.

The First Functional HLA-a*01:01-Restricted EBV-LMP2-Specific T-Cell Receptors for TCR Gene Therapy of Patients with EBV-Associated Type II/III Malignancies

Epstein Barr virus (EBV) is associated with the development of a broad range of malignancies, including Burkitt's lymphoma, Hodgkin and non-Hodgkin lymphomas, post-transplant lymphoproliferative disorder (PTLD), nasopharyngeal carcinoma and gastric carcinoma. Differential expression of immunogenic antigens (e.g. EBV Nuclear Antigen (EBNA2-6) and Latent membrane proteins (LMPs)) is seen at the different latent phases of the viral infection. Although many EBV-associated lymphomas only express weakly immunogenic EBV antigens (e.g. EBNA1 and BARF1), lymphomas with type II or III latency express LMP1 and LMP2. Growth of such lymphomas can be curbed using adoptively transferred EBV-LMP1/2-specific T cells. Surprisingly, T cells recognizing EBV-derived peptides in the common HLA allele A*01:01 have not been found. In addition, an HLA-A*01:01-associated increased risk for EBV+ Hodgkin lymphomas and infectious mononucleosis has been reported, suggesting that HLA-A*01:01-restricted EBV-specific T cells may be absent or present at very low frequencies in these patients. A need thus exists for HLA-A*01:01-restricted EBV-specific T-cell products, especially directed against EBV-LMP1/2. Based on MHC class I peptide predictions, HLA-A*01:01-binding peptides derived from different immunogenic EBV antigens were identified and tetramer complexes were synthesized (EBNA3A-YTDHQTTPT, EBNA3A-FLQRTDLSY, BZLF1-FTPDPYQVPF, LMP2-ESEERPPTPY, LMP2-LTEWGSGNRTY). HLA-A*01:01-restricted EBV-specific T cells were present at very low frequencies in total PBMCs from all 6 donors. After sorting using flow cytometry, only EBV-LMP2-ESE specific T cells could be expanded (for 5/6 donors), yielding pure tetramer+ CD8 T-cell populations. Four out of 5 isolated T-cell populations exhibited intermediate to high avidity recognition of HLA-A*01:01-transduced TAP2-deficient T2 cells, loaded with EBV-LMP2-ESE peptide. This specific LMP2-derived peptide showed to be functionally processed, presented and recognized by EBV-LMP2-ESE-specific T cells when using HLA-A*01:01/LMP2-transduced K562 cells. To assess the suitability for TCR gene-therapy, the TCRs from the 4 functional T-cell populations were sequenced and cloned into a retroviral vector. Surprisingly, all 4 EBV-LMP2-ESE-specific T-cell populations used the TRBV6-2 gene for TCR beta-chain expression. Additionally, for TCRalpha-chain expression these populations used either TRAV12 or TRAV30. These TCRs contained small differences in the CDR3 region. Despite differences in tetramer binding, all TCRs were functional when transduced into primary CD8 T cells, CD4 T cells, and CD8 negative TCR knock-out Jurkat cells, implying CD8 independent recognition. Finally, recognition of HLA-A*01:01+ EBV-LCLs demonstrated the potential of these EBV-LMP2-ESE-specific TCRs to recognize naturally occurring endogenous LMP2. In conclusion, we isolated and validated the first functional HLA-A*01:01-restricted EBV-LMP2-specific T-cell populations and TCRs, which can be used for adoptive transfer or retro/lentiviral TCR gene therapy to treat EBV-associated type II/III lymphomas, EBV+ malignancies of epithelial origin and PTLDs. Disclosures No relevant conflicts of interest to declare.

Hemoglobin Expression and Function in Human Corneal Epithelium

The corneal epithelium forms the outer layer of the cornea. It provides mechanical protection, prevents fluid loss and forms a barrier to invasive pathogens. Though hemoglobin expression has been extensively studied in erythroid cells, recent work in multiple cell systems has documented hemoglobin expression in cells of non-erythroid origin. However, the function of hemoglobin in non-erythroid cells has yet to be established. The hypothesis that hemoglobin is expressed in corneal epithelium and that it functions to protect against oxidative stress will be examined in the present work. Hemoglobin expression and function was examined by immunocytochemistry and western blot analysis. Native human corneal explants and an immortalized human corneal epithelial cell line were examined. Expression of hemoglobin beta and delta chains was demonstrated at the protein level in both native and cell culture preparations. Hexamethylene bisacetamide is a known inducer of hemoglobin beta chain expression in murine erythroleukemia cells. HMBA did function to increase beta chain expression in cultured corneal epithelium as well. Beta chain immunolocalization was primarily cytoplasmic, while the delta chain localized to both cytoplasmic and membrane domains. Oxidative stress, from hydrogen peroxide exposure, was shown to upregulate delta chain expression. In conclusion, hemoglobin chains are expressed in corneal epithelium and could function to protect against oxidative stress. This is relevant given that exposure to light and high oxygen tensions render the cornea particularly susceptible to oxidative damage.

Hemoglobin Expression and Function in Human Corneal Epithelium

The corneal epithelium forms the outer layer of the cornea. It provides mechanical protection, prevents fluid loss and forms a barrier to invasive pathogens. Though hemoglobin expression has been extensively studied in erythroid cells, recent work in multiple cell systems has documented hemoglobin expression in cells of non-erythroid origin. However, the function of hemoglobin in non-erythroid cells has yet to be established. The hypothesis that hemoglobin is expressed in corneal epithelium and that it functions to protect against oxidative stress will be examined in the present work. Hemoglobin expression and function was examined by immunocytochemistry and western blot analysis. Native human corneal explants and an immortalized human corneal epithelial cell line were examined. Expression of hemoglobin beta and delta chains was demonstrated at the protein level in both native and cell culture preparations. Hexamethylene bisacetamide is a known inducer of hemoglobin beta chain expression in murine erythroleukemia cells. HMBA did function to increase beta chain expression in cultured corneal epithelium as well. Beta chain immunolocalization was primarily cytoplasmic, while the delta chain localized to both cytoplasmic and membrane domains. Oxidative stress, from hydrogen peroxide exposure, was shown to upregulate delta chain expression. In conclusion, hemoglobin chains are expressed in corneal epithelium and could function to protect against oxidative stress. This is relevant given that exposure to light and high oxygen tensions render the cornea particularly susceptible to oxidative damage.

Proteomic analysis of Paracoccidioides brasiliensis complex isolates: Correlation of the levels of differentially expressed proteins with in vivo virulence

BackgroundParacoccidioidomycosis (PCM) is a systemic mycosis commonly found in Latin America that is caused by distinct species of Paracoccidioides genus: Paracoccidioides brasiliensis complex (S1, PS2, PS3 and PS4) and Paracoccidioides lutzii. Its pathobiology has been recently explored by different approaches to clarify the mechanisms of host-pathogen interactions underpinning PCM. The diversity of clinical forms of this disease has been attributed to both host- and fungus-related factors.Methodology/Principal findingsFor better understanding of the molecular underpinnings of host-fungus interactions, we evaluated in vivo virulence of nine Paracoccidioides brasiliensis complex isolates and correlated it to protein expression profiles obtained by two-dimensional gel electrophoresis. Based on the recovery of viable fungi from mouse organs, the isolates were classified as those having low, moderate, or high virulence. Highly virulent isolates overexpressed proteins related to adhesion process and stress response, probably indicating important roles of those fungal proteins in regulating the colonization capacity, survival, and ability to escape host immune system reaction. Moreover, highly virulent isolates exhibited enhanced expression of glycolytic pathway enzymes concomitantly with repressed expression of succinyl-CoA ligase beta chain, a protein related to the tricarboxylic acid cycle.Conclusions/SignificanceOur findings may point to the mechanisms used by highly virulent P. brasiliensis isolates to withstand host immune reactions and to adapt to transient iron availability as strategies to survive and overcome stress conditions inside the host.

Correlation between hepatic oxidative damage and clinical severity and mitochondrial gene sequencing results in biliary atresia

To assess the level of hepatic oxidative damage and its correlation with clinical severity in biliary atresia (BA), and to understand BA mitochondrial gene sequencing. Forty-eight BA patients and 28 control subjects (20 hepatoblastoma and 8 cholestasis patients) were enrolled. Hepatic oxidative damage was assessed by the expression of oxidation and antioxidant genes, and the correlation between oxidative damage and BA incidence, liver inflammation, and fibrosis was evaluated. Moreover, 8-hydroxyguanine (8-OHdG), mitochondrial DNA (mtDNA) copy number, and mitochondrial gene sequences were determined to evaluate oxidative mtDNA damage in BA. The expression of oxidation gene cytochrome b-245 beta chain (CYBB) in BA was significantly increased and patients with a higher CYBB expression had the higher risk of BA incidence, liver inflammation, and cirrhosis. However, the expression of antioxidant genes was significantly decreased, and glutathione S-transferase alpha 1 (GSTA1) negatively correlated with BA incidence and cirrhosis. When GSTA1 mRNA expression was <0.5487, the sensitivity was 80.85% and the specificity was 80% for BA diagnosis. Moreover, 8-OHdG was increased, whereas mtDNA copy number was significantly decreased in BA. Using mitochondrial gene sequencing, 10 mutation sites were identified, and one family showed a maternal inheritance in genetic loci 15 326. In BA, oxidative damage positively correlated with BA incidence, liver inflammation, and cirrhosis. GSTA1 could be a novel diagnostic indicator. Genetic loci 15 326 could be a maternal genetic mutation site. Taken together, antioxidation therapy after Kasai surgery might have great potential in relieving liver inflammation and fibrosis in BA patients.

MiRNA-125b-5p Suppresses Hypothyroidism Development by Targeting Signal Transducer and Activator of Transcription 3.

BackgroundA deficiency of maternal thyroid hormones (THs) during pregnancy has severe impacts on fetal brain development. Neural stem cells (NSCs) are major targets of THs and provided a powerful model to explore the underlying mechanism of THs during brain development. Although miRNA-125 might be associated with the NSCs differentiation, the relationship between miR-125 and hypothyroidism (HypoT) development remains unclear.Material/MethodsIn our study, we screened a differentially expressed gene miR-125b-5p from brain between euthyroid (EuT) and HypoT rats. In vitro, we employed anion exchange resin to remove THs to stimulate HypoT. QRT-PCR and Western blot were used to examine the expression of signal transducer and activator of transcription 3 (Stat3). The relationship between miR-125b-5p and Stat3 was detected via a dual-luciferase assay.ResultsQRT-PCR results showed that the level of miR-125b-5p in HypoT rat brains was significantly suppressed, suggesting some relationship between miR-125b-5p and HypoT. In C17.2, miR-125b-5p promoted cell differentiation into neurons by regulating the expression of tubulin beta chain 3 (TUBB3) and glial fibrillary acid protein (GFAP). QRT-PCR and Western blot results revealed that miR-125b-5p mimic modulated the contents of total Stat3 and p-Stat3. A dual-luciferase assay showed that miR-125b-5p negatively regulated the expression of Stat3 by binding with the first site in 3′ UTR of Stat3.ConclusionsThese results revealed Stat3 is a new target of miR-125b-5p and revealed the mechanism of miR-125b-5p suppressing HypoT development. These findings provide a new target for HypoT therapy.

Antiproliferation effect of the uremic toxin para‑cresol on endothelial progenitor cells is related to its antioxidant activity.

Endothelial dysfunction and impaired endothelial regenerative capacity are key contributors to the high incidence of cardiovascular disease in patients with chronic kidney disease (CKD). Uremic toxins are associated with this pathogenesis. Previous studies have revealed that a uremic toxin, para-cresol (p-cresol), exerts an antiproliferation effect on human endothelial progenitor cells (EPCs), but the mechanism remains unclear. In the present study, reactive oxygen species (ROS) were confirmed to function as signaling molecules that regulate growth factor-dependent EPC proliferation. EPCs were treated with p-cresol for 72 h, using a concentration range typically found in CKD patients. ROS production was analyzed by fluorescence microscopy and flow cytometry, and protein expression levels of nicotinamide adenine dinucleotide phosphate oxidase, a major source of ROS, were analyzed by western blot analysis. mRNA expression levels of antioxidant genes were assessed by reverse transcription-quantitative polymerase chain reaction analysis. The results revealed that p-cresol partially inhibits ROS production, and this effect may be associated with a significant reduction in cytochrome b-245 alpha and beta chain expression in EPCs. An increase of glutathione peroxidase 4 mRNA expression was also detected. In conclusion, the present study revealed that the antiproliferation effect of p-cresol on EPCs might act via its antioxidant activity. The results of the present study may facilitate understanding of uremic toxin toxicity on the cardiovascular system.

A novel therapeutic target for peripheral nerve injury-related diseases: aminoacyl-tRNA synthetases

Aminoacyl-tRNA synthetases (AminoARSs) are essential enzymes that perform the first step of protein synthesis. Beyond their original roles, AminoARSs possess non-canonical functions, such as cell cycle regulation and signal transduction. Therefore, AminoARSs represent a powerful pharmaceutical target if their non-canonical functions can be controlled. Using AminoARSs-specific primers, we screened mRNA expression in the spinal cord dorsal horn of rats with peripheral nerve injury created by sciatic nerve axotomy. Of 20 AminoARSs, we found that phenylalanyl-tRNA synthetase beta chain (FARSB), isoleucyl-tRNA synthetase (IARS) and methionyl-tRNA synthetase (MARS) mRNA expression was increased in spinal dorsal horn neurons on the injured side, but not in glial cells. These findings suggest the possibility that FARSB, IARS and MARS, as a neurotransmitter, may transfer abnormal sensory signals after peripheral nerve damage and become a new target for drug treatment.

Multiple STAT3 Mutations In Different Lymphocyte Clones Of Large Granular Lymphocytic Leukemia Patients

BackgroundLarge granular lymphocytic (LGL) leukemia is a clonal disease of mature cytotoxic T- or natural killer (NK)-cells, which was recently characterized by gain-of-function somatic STAT3 mutations in 40-70% of patients. Most of the T-LGL leukemia patients harbor one major Vbeta clone corresponding even up to 90% of total CD8+ T-cell population. Interestingly though, in a small proportion of T-LGL leukemia patients we have detected multiple mutations in the STAT3 gene suggesting the presence of subclones. Here, we aimed to study the clonal architecture and mutation spectrum of expanded lymphocytes with deep sequencing method and to follow the clones during immunosuppressive treatment. MethodsDNA samples from 228 LGL leukemia patients were available for STAT3 mutation analysis. Additional flow cytometry-based sorting was done from 12 STAT3 mutation-positive patients, 6 of which had multiple STAT3 mutations in peripheral blood mononuclear cells (PBMNCs). First, frozen live PBMNCs were sorted into CD4+, CD8+ T-cell, and NK-cell fractions using antibodies for CD3, CD4, CD8, and CD16/56. Then CD8+ T-cell population was further sorted into clonal/non-clonal cells based on the flow cytometry analysis of T-cell receptor beta chain expression (Vbeta). STAT3 exon 21 was sequenced using Illumina Miseq platform with coverage aim over 10,000. The data was analyzed using an in-house bioinformatics pipeline: mutations were considered to be true if variant allele frequency (VAF) was over 1%, and false-positives were filtered out by comparing the VAF with calculated error rate of the amplicon. ResultsIn total, 12/228 patients had multiple STAT3 mutations (16% of all STAT3 mutation positive cases). In all studied patients with multiple mutations (Table 1, 4 cases presented), the VAF was 30-50% in the purified major Vbeta clone suggesting that the whole population belonged to the same clone harboring a heterozygous STAT3 mutation (the clone size can be estimated to be twice the VAF). In addition to the major clone, STAT3 mutations were also discovered in smaller Vbeta expansions and in some cases in the non-clonal CD8+ population (Table 1). Interestingly, one patient diagnosed with T-LGL leukemia did not have STAT3 mutations in the major Vbeta expansion (65% of CD8+ cells) but harbored a D661Y mutation with 38% VAF in the NK-cell fraction (Patient 5 in Table 1).The follow-up samples during the treatment were available from 4 patients. In patients 1 and 2 (Table 1), the size of the clone was unchanged during 32 and 37 months follow-up despite of the treatment with methotrexate and cyclophosphamide. In patient 3 (Table 1), the Vb7.1+ clone carrying Y640F mutation decreased from 10% to 3% of CD8+ cells, whereas the Vb5.1+ clone (D661Y) was unchanged during the methotrexate treatment. In patient 5 complete remission was achieved with cyclophosphamide treatment and that was accompanied with the disappearance of D661Y-mutated NK-cells.Table 1Vbeta results and variant allele frequencies (VAFs) of STAT3-mutations. The size of the mutated clone is approximately 2xVAF in the case of heterozygous mutation.Pat.NoVbeta (% of CD8+)PB MNCCD3+CD8+Vb+CD3+CD8+VbnegCD3negCD16/56+1Vb13.6 (96%)D661Y 38%, D661V 1%D661Y 47%D661V 17%N/A2Vb3 (90%)Y640F 42%, I659L 1%Y640F 49%I659L 20%N/A3Vb7.1 (13%) Vb5.1 (3%)Y640F 1%, D661Y 2%, D661V 2%Vb7.1: Y640F 35%Vb5.1: D661Y 33%Vb7.1neg: D661Y 6%, D661V 3%, N647Y 5%Vb5.1neg: Y640F 1%, D661V 4%, N647Y 2%No mutations4Vb16 (50%) Vb13.6 (11%)D661V 6%Vb16: D661V 41%Vb13.6: D661Y 11%Vb16neg: D661Y 4%Vb13.6neg: D661V 26%N/A5Vb20 (65%)D661Y 6%No mutationsNo mutationsD661Y 38% DiscussionOur preliminary results provide evidence that the STAT3 mutations are not only restricted to the significantly expanded lymphocyte clone in LGL leukemia patients, but they can also be found in smaller subclones mimicking the situation in acute leukemia. The actual cause of the mutations is unknown, but the results suggest the presence of a strong initial immune activation, which predisposes existing lymphocyte clones to somatic mutagenesis during cell proliferation. Considering the effects of treatment on STAT3-mutated clones, the only complete remission seen was connected to the disappearance of the mutated clone, which warrants STAT3-inhibitor trials in the future. Disclosures:Porkka:BMS: Consultancy, Research Funding, Speakers Bureau; Novartis: Consultancy, Research Funding, Speakers Bureau. Maciejewski:NIH: Research Funding; Aplastic anemia&MDS International Foundation: Research Funding. Mustjoki:Novartis: Consultancy, Speakers Bureau; BMS: Consultancy, Speakers Bureau.

The abundance of memory-like T cells depends on TCR/MHC interactions (P1442)

Abstract To investigate impact of TCR transgenic chains in formation of activation surface phenotype of T lymphocytes, we have generated mouse strain expressing transgenic beta-chain of TCR 1D1 (belonging to Vbeta6 family) on genetical background B10.D2(R101). Genomic sequence encoding TCR 1D1 beta-chain was isolated from memory T-hybridoma specific to Kb molecule. In thymus of transgenic mice accumulation of CD4-CD8-CD3- T lymphocytes takes place on the rate exceeding by several times the number of these cells in wild type animals. In transgenic animals about 70-80% of peripheral T lymphocytes express Vbeta6, whereas the percent of these cells does not exceed 5-7% in wild type animals. The ratio of peripheral CD4 and CD8 T lymphocytes stayed unchanged in transgenic mice. The percent of T cells with naive phenotype CD44-CD62L+ was significantly increased, whereas the levels of effector memory CD44+CD62L- and central memory CD44+CD62L+ T lymphocytes were markedly decreased. T lymphocytes expressing endogenous beta-chains mostly had the phenotype of memory-like T cells. Expression of 1D1 beta-chain was associated with naïve phenotype and lowered MLR-responses. These results suggest that appearance of memory-like T cells depends on interactions between TCRs and endogenous MHC/peptide complexes, whereas abundances of naive and memory-like T cells are regulated by competition of naive T cells for restricted number of appropriate MHC/peptide complexes in lymphoid tissue.

Expression of TCR trangenic beta-chain leads to contraction of T lymphocytes pools with surface phenotypes of effector and central memory cells.

Expression of TCR trangenic beta-chain leads to contraction of T lymphocytes pools with surface phenotypes of effector and central memory cells.

Marek's Disease Virus Influences the Expression of Genes Associated with IFN-γ-Inducible MHC Class II Expression

Chickens infected with Marek's disease virus (MDV) become lifelong carriers regardless of their susceptibility to clinical disease. Therefore various viral immune-evasive mechanisms must play a role in MDV-host interactions. MDV has previously been shown to influence the expression of major histocompatibility complex (MHC) class II molecules. However, little is known about the underlying mechanisms of this phenomenon. In the present study, we studied the effect of MDV infection on the expression of several genes associated with IFN-gamma-inducible MHC class II expression at 4, 7, 14, and 21 days post-infection (dpi). There was a significant (p <or= 0.05) downregulation of MHC class II beta chain expression throughout the experiment, while other components of the MHC class II heterotrimer (i.e., alpha chain and the invariant chain) were significantly downregulated only at 4 and 21 dpi. Furthermore, the expression of components of the IFN-gamma-receptor complex was significantly downregulated at 4 dpi. In contrast, a number of other IFN-gamma-signaling molecules, including signal transducer and activator of transcription 1 (STAT1), interferon-responsive factor 1 (IRF-1), and class II transactivator (CIITA) were significantly upregulated at most time points. The results of this study shed light on the possible mechanisms by which MDV may evade host immunosurveillance.

S100A7 (psoriasin) is induced by the proinflammatory cytokines oncostatin-M and interleukin-6 in human breast cancer

S100A7 promotes aggressive features in breast cancer, although regulation of its expression is poorly understood. As S100A7 associates with inflammation in skin and breast tissue, we hypothesized that inflammatory cytokines may regulate S100A7 in breast cancer. We therefore examined the effects of several cytokines, among which oncostatin-M (OSM) and the related cytokine, interleukin (IL)-6, showed the most significant effects on S100A7 expression in breast tumor cells in vitro. Both cytokines consistently induced S100A7 expression in three cell lines (MCF7, T47D and MDA-MB-468) in a dose- and time-dependent manner. Induction of S100A7 was inhibited by blockade of STAT3, phosphatidylinositol 3 kinase (PI3K) and ERK1/2 signaling and small interference RNA (siRNA)-mediated knockdown of S100A7 eliminated the promigratory effects of OSM treatment. S100A7 mRNA levels in a case-control cohort of breast tumors (n=20) were significantly associated with expression of the OSM receptor beta (OSMRbeta) chain (P=0.0098). This association was confirmed using publicly available microarray data from an independent breast tumor cohort (n=201, P=0.0005) and a correlation between S100A7 and poor patient survival was observed specifically in cases with high OSMRbeta expression (HR=2.35; P=0.0396; n=85). We conclude that inflammatory cytokines can regulate S100A7 expression and that S100A7 may mediate some of their effects in breast cancer.

TCR beta feedback signals inhibit the coupling of recombinationally accessible V beta 14 segments with DJ beta complexes.

Ag receptor allelic exclusion is thought to occur through monoallelic initiation and subsequent feedback inhibition of recombinational accessibility. However, our previous analysis of mice containing a V(D)J recombination reporter inserted into Vbeta14 (Vbeta14(Rep)) indicated that Vbeta14 chromatin accessibility is biallelic. To determine whether Vbeta14 recombinational accessibility is subject to feedback inhibition, we analyzed TCRbeta rearrangements in Vbeta14(Rep) mice containing a preassembled in-frame transgenic Vbeta8.2Dbeta1Jbeta1.1 or an endogenous Vbeta14Dbeta1Jbeta1.4 rearrangement on the homologous chromosome. Expression of either preassembled VbetaDJbetaC beta-chain accelerated thymocyte development because of enhanced cellular selection, demonstrating that the rate-limiting step in early alphabeta T cell development is the assembly of an in-frame VbetaDJbeta rearrangement. Expression of these preassembled VbetaDJbeta rearrangements inhibited endogenous Vbeta14-to-DJbeta rearrangements as expected. However, in contrast to results predicted by the accepted model of TCRbeta feedback inhibition, we found that expression of these preassembled TCR beta-chains did not downregulate recombinational accessibility of Vbeta14 chromatin. Our findings suggest that TCRbeta-mediated feedback inhibition of Vbeta14 rearrangements depends on inherent properties of Vbeta14, Dbeta, and Jbeta recombination signal sequences.

An Essential Role of the RNA Editing Enzyme ADAR1 in T Cell Development.

Abstract 917Post-transcriptional regulation such as RNA editing in hematopoiesis and lymphopoiesis is poorly understood. ADAR1 (adenosine deaminase acting on RNA-1) is a RNA editing enzyme essential for embryonic development. Disruption of the ADAR1 gene was shown to cause defective embryonic hematopoiesis (Wang Q et al, Science 2000). Moreover, we have recently obtained direct evidence for the preferential effect of ADAR1 deletion on adult hematopoietic progenitor cells as opposed to the more primitive cells via a RNA-editing dependent mechanism by different conditional gene deletion strategies (Xufeng R et al PNAS 2009, in press). To further determine the role of ADAR1 in T cell development, we generated a mouse model in which ADAR1 was deleted specifically in T lymphocytes by interbreeding ADAR1lox/lox mice with Lck-Cre transgenic mice. In our current study, we report that ADAR1 is essential for T cell differentiation at the late progenitor stage in the thymus, coincident with T cell receptor-α/β expression. In ADAR1lox/loxLck-Cre mice, mature T cells decreased dramatically in peripheral blood, spleen and lymph nodes in comparison to littermate controls. In the thymus, the production of CD4+/CD8+ double positive cells was severely impaired and massive cell death was observed in pre-T cell populations. Within the pro-T cells, ADAR1 deletion resulted in a significant decrease of late progenitor cells but not early progenitor subsets. In both pro-T and pre-T cell stages, defective T cell development preferentially occurred in the beta chain positive cells, but was not apparent in gamma/delta T cells. Our data demonstrated an indispensable role of ADAR1 in early T cell differentiation that correlated with T cell receptor beta chain expression, thereby indicating that RNA editing by ADAR1 is an essential event in T cell development. Disclosures:No relevant conflicts of interest to declare.

Impact of Melatonin and Molecular Clockwork Components on the Expression of Thyrotropin Beta Chain (Tshb) and the Tsh Receptor in the Mouse Pars Tuberalis

Photoperiodic regulation of reproduction in birds and mammals involves thyrotropin beta chain (TSHb) which is secreted from the pars tuberalis (PT) and controls the expression of deiodinase typ 2 (Dio2) and 3 (Dio3) in the ependymal cell layer of the infundibular recess (EC) via TSH receptors (TSHR). To analyze the impact of melatonin and the molecular clockwork on the expression of Tshb and Tshr we investigated melatonin proficient C3H wildtype (WT), melatonin receptor 1 (MT1) and mPER1-deficient mice. Expression of Tshb and TSHb immunoreactivity in PT were low during day and high during night in WT, high during day and low during night in mPER1-deficient and equally high during day and night in MT1-deficient mice. Melatonin injections into WT acutely suppressed Tshb expression. Transcription assays showed that mPER1 significantly reduced the CLOCK:BMAL1-induced transcription of the Tshb. Tshr levels in PT were low during day and high during night in WT and mPER1-deficient mice and equally low in MT1-deficient mice. Tshr expression in the EC did not show a day/night variation. Melatonin injections into WT acutely induced Tshr expression in PT, but not in EC. TSH stimulation of hypothalamic slice cultures of WT induced pCREB in PT and EC and Dio2 in the EC. Our data suggest that 1) Tshb expression in PT is controlled by melatonin and the molecular clockwork and 2) melatonin activates Tshr expression in PT, but not in EC. They also confirm the functional importance of TSHR in the PT and EC.

Differential Recognition of CD1d-α-Galactosyl Ceramide by the Vβ8.2 and Vβ7 Semi-invariant NKT T Cell Receptors

The semi-invariant natural killer T cell receptor (NKT TCR) recognizes CD1d-lipid antigens. Although the TCR alpha chain is typically invariant, the beta chain expression is more diverse, where three V beta chains are commonly expressed in mice. We report the structures of V alpha 14-V beta 8.2 and V alpha 14-V beta 7 NKT TCRs in complex with CD1d-alpha-galactosylceramide (alpha-GalCer) and the 2.5 A structure of the human NKT TCR-CD1d-alpha-GalCer complex. Both V beta 8.2 and V beta 7 NKT TCRs and the human NKT TCR ligated CD1d-alpha-GalCer in a similar manner, highlighting the evolutionarily conserved interaction. However, differences within the V beta domains of the V beta 8.2 and V beta 7 NKT TCR-CD1d complexes resulted in altered TCR beta-CD1d-mediated contacts and modulated recognition mediated by the invariant alpha chain. Mutagenesis studies revealed the differing contributions of V beta 8.2 and V beta 7 residues within the CDR2 beta loop in mediating contacts with CD1d. Collectively we provide a structural basis for the differential NKT TCR V beta usage in NKT cells.

Primary Cutaneous Peripheral T-Cell Lymphoma With Aberrant Coexpression of CD20: Case Report and Review of the Literature

We report an unusual case of primary cutaneous T-cell lymphoma in a 45-year-old male showing expression of the B-cell marker CD20. The lesion presented as a large plaque on the right shin and rapidly developed into additional lesions on the right lower leg and right anterior chest. Despite therapy, bone involvement was discovered at 1 year after initial presentation. Punch biopsy of the right leg revealed a diffuse, atypical dermal lymphocytic infiltrate with papillary edema and no epidermotropism. The atypical cells strongly expressed the T-cell markers CD3, CD2, and CD43, with additional expression of cytotoxic T-cell markers CD8, cytotoxic granule-associated RNA binding protein (TIA-1), and granzyme B and loss of CD7 by immunohistochemistry (IHC). In addition, the atypical cells with the above phenotype exhibited moderate expression of CD20 by IHC. Coexpression of CD8 and CD20 in the atypical cells was confirmed by a dual-labeled IHC stain. Clonal rearrangements of the T-cell receptor gamma- and beta-chain genes were detected by polymerase chain reaction; however, there was no expression of T-cell receptor beta-chain identified by IHC. In conclusion, we present a case a of CD8-positive primary cutaneous T-cell lymphoma with CD20 expression, exhibiting aggressive behavior.

Niacin: An old drug rejuvenated

Niacin has long been used in the treatment of dyslipidemia and cardiovascular disease. Recent research on niacin has been focused on understanding the mechanism of action of niacin and preparation of safer niacin formulations. New findings indicate that niacin does the following: 1) it inhibits hepatic diacylglycerol acyltransferase 2, resulting in inhibition of triglyceride synthesis and decreased apolipoprotein B-containing lipoproteins; 2) it decreases the surface expression of hepatic adenosine triphosphate synthase beta-chain, leading to decreased holoparticle high-density lipoprotein catabolism and increased high-density lipoprotein levels; and 3) it increases redox potential in arterial endothelial cells, resulting in inhibition of redox-sensitive genes. Flushing, an adverse effect of niacin, results from niacin receptor GPR109A-mediated production of prostaglandin D2 and E2 via DP1 and EP2/4 receptors. DP1 receptor antagonist (laropiprant) attenuates the niacin flush. A reformulated preparation of extended-release niacin (Niaspan; Abbott, Abbott Park, IL) lowers flushing compared with an older Niaspan formulation. These advancements in niacin research have rejuvenated its use for the treatment of dyslipidemia and cardiovascular disease.