CD4 Transcription Research Articles

Gene Expression Quantification of Toll like Receptors 2, 4 and Co-molecules in Human Glioblastoma Cell Line (U87-MG): Toward a New In vitro Model of Inflammation.

Pattern recognition receptors (PRRs) are the main part in the innate immune response. Human glioblastoma cell line (U87-MG) is an established adherent cell line model of this common cancer; due to genetic variations between individuals it is likely more suitable for investigating molecular aspects of innate immunity. Therefore, we undertook a novel characterization of the immune phenotype of U87-MG toward establishing a base for future researches. In this study, U87-MG cells where cultured in a normal condition, to investigate levels of toll-like receptor 2 (TLR2), TLR4, myeloid differentiation factor-88 (MyD88) and CD14 transcripts expression in these cells. Both RT-PCR and qPCR were applied to detect and quantify the expression levels of these genes in U87-MG cells and compare them to their levels in the peripheral blood mononuclear cells (PBMC) of healthy individuals, as a common reference. Expression level of TLR2 and TLR4 are not significantly different in U87-MG cells in comparison to PBMC. Also, expression levels of MyD88 and CD14 in U87-MG cells are significantly lower than their levels in PBMC. Furthermore, expression levels of MyD88 and CD14 in both PBMC and U87-MG are significantly lower than TLR2 and TLR4 transcripts. The data reveal expression of TLR4, CD14, MyD88 and TLR2 genes in U87-MG cell line, for the first time. Expression detection of these genes in human glioblastoma cell line might have a potential for diagnosis of inflammatory mechanisms in immune mediated disorders of in vitro models of human brain inflammatory disease.

Down-regulation of CD14 transcripts in human glioblastoma cell line U87 MG.

Pattern recognition receptors (PRRs) are the main sensors of pathogen and danger signals in innate immunity of which Toll Like Receptors (TLRs) are the most studied ones. The contribution of PRRs in cerebral inflammation induced by microbial infection, tissue damage and cancer has not extensively been addressed so far. Glioma is the most common tumor of the central nervous system and glioblastomas are the most common and most malignant primary brain tumors. The objectives of the present study were to investigate the expression of several PRRs including TLR2, TLR4, MyD88 and CD14 transcripts in human glioblastoma cell line U87 MG and compare their expression level with peripheral blood mononuclear cells (PBMC) obtained from healthy individuals. Touchdown PCR (TD-PCR) and Real-time quantitative PCR (qPCR) were applied to detect and quantify the expression level of TLR2, TLR4, MyD88 and CD14 transcript in U87 MG cell line and (PBMC) of healthy individuals. According to our results, human glioblastoma cell line U87 MG expresses TLR2, TLR4, MyD88 and CD14 transcripts in TD-PCR. Moreover, the quantification of the expression of these genes revealed a highly significant down-regulation of CD14 and a slight up-regulation of TLR2 transcripts as compared to PBMC of healthy individuals. The lower expression level of CD14 in human glioblastoma cell line, might have a potential implication for CD14 mediated cerebral pathology.

Abstract 3078: CD44 mRNA stability is regulated by RNA binding protein HuR in ovarian cancer cells

Abstract CD44, a major hyaluronic acid (HA) receptor, is involved in cell migration, metastasis and tumor progression. While in normal ovarian epithelium CD44 is not expressed, ovarian carcinoma cells express multiple CD44 isoforms as the tumor progresses and eventually CD44 expression is abrogated in advanced ovarian cancer metastatic sites. We as well as others have observed that the levels of CD44 mRNA transcript not always correspond to the CD44 protein expression levels. This observation suggests that CD44 is post-transcriptionally regulated. To test this hypothesis we first analyzed the CD44 3’ untranslated region (3’ UTR) sequence and found numerous AU-rich elements (ARE) in this region. Some of these sequences are known HuR binding site consensus sequences. HuR is an RNA binding protein that is known to stabilize transcripts. HuR is expressed in the nucleus and is transported to the cytoplasm where it functions as an mRNA stabilizer. We performed mRNA half-life experiments that showed a two-fold reduction in the stability of CD44 transcript in siHuR transfected ovarian cancer cells. Consistent with this result, treatment of these cells with inhibitors of DNA methylation and histone deacetylation (i.e., 5-azacytidine and Trichostatin A, respectively) known to diminish the presence of HuR in the cytoplasm, also decreased the stability of CD44 mRNA. Western blot analysis showed the expected reduction in cytoplasmic HuR as well as down-regulation in CD44 protein expression. A direct interaction of HuR with the CD44 3’ UTR was detected by biotin pulldown assays. These results indicate that HuR posttranscriptionally stabilizes the expression of CD44 in the cytoplasm. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3078. doi:10.1158/1538-7445.AM2011-3078

115 MOLECULAR CHARACTERIZATION OF RENAL CELL CARCINOMA AND ONCOCYTOMA

You have accessJournal of UrologyKidney Cancer: Basic Research1 Apr 2011115 MOLECULAR CHARACTERIZATION OF RENAL CELL CARCINOMA AND ONCOCYTOMA Andrew Chi, Jeffrey Gahan, Miguel Gosalbez, Samir Shirodkar, Vincent Bird, Mark Soloway, and Vinata Lokeshwar Andrew ChiAndrew Chi Miami, FL More articles by this author , Jeffrey GahanJeffrey Gahan Miami, FL More articles by this author , Miguel GosalbezMiguel Gosalbez Miami, FL More articles by this author , Samir ShirodkarSamir Shirodkar Miami, FL More articles by this author , Vincent BirdVincent Bird Gainsville, FL More articles by this author , Mark SolowayMark Soloway Miami, FL More articles by this author , and Vinata LokeshwarVinata Lokeshwar Miami, FL More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2011.02.181AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES Advanced imaging techniques allow better detection of small renal masses (<4cm). However, 16–28% of these small tumors have benign pathology. About 50% of these benign tumors are oncocytomas, which may be radiologically similar to renal cell carcinoma (RCC). Percutaneous biopsy can differentiate between oncocytoma and renal cell carcinoma; however, it relies on the presence of histopathological architecture to yield a definitive diagnosis. In this study we performed molecular characterization of oncocytomas, chromophobe and RCC by analyzing the expression of 11 genes (or their splice variants) associated with tumor growth, metastasis and angiogenesis. METHODS Tumor and matched normal tissues were collected from 86 renal masses undergoing nephrectomy (Tissue: clear cell carcinoma (RCC) 73, oncocytoma (ONC) 5, chromophobe 5, sarcoma 3; Stage T1 (a=b), 40; T2, 14; T3, 21; T4, 6; Grade 1, 5; Grade 2, 32; Grade 3, 25; Grade 4, 19). RNA isolated from tissues was subjected to real time RT-PCR for 11 markers: Hyaluronic acid receptors (CD44-standard form, RHAMM); hyaluronidases (HYAL, HYAL4) and HA-synthase (HAS2), chemokine receptors (CXCR4, CXCR7); chemokines (IL-8, SDF1-v1, v2, v3). The transcript levels were normalized to TATA binding protein. Statistical analysis was performed using Mann-Whitney U-test and logistic regression. RESULTS All markers except IL-8 and HYAL4 showed differential expression in normal and tumor tissues. Specifically, RHAMM, CD44, SDF1-v3, and CXCR7 transcript levels were 3-5-fold elevated, and HAS2 and SDF1-v1 levels were 2.5-fold down regulated in tumors when compared to normal tissues (P < 0.0001). CD44 (P = 0.019; t-ratio = 2.4) and RHAMM (p = 0.0022; t-ratio = 3.15) levels correlated with tumor grade; none of the marker levels correlated with tumor stage. The levels of all markers were elevated in RCC when compared to ONC. RHAMM (RCC: 38+/−68; ONC: 4.3+/−2.5; P = 0.014), CD44 (RCC: 690+/−1027; ONC: 49+/−2; p=0.002), CXCR4 (3363+/−5219; ONC: 787+/−971; P=0.003), and CXCR7 (1023+/−1178; ONC: 236+/−299; P=0.02) transcript levels were significantly elevated in RCC when compared to ONC tumors. Additionally, the transcript levels of all the markers were not significantly different when comparing RCC and chromophobe tumors (P > 0.05). CONCLUSIONS CD44, RHAMM CXCR7, CXCR4, and SDF1-splice variants are associated with the pathophysiology of kidney cancer. Moreover, differential expression of RHAMM, CD44, CXCR4 and CXCR7 can potentially distinguish between malignant and benign renal masses. © 2011 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 185Issue 4SApril 2011Page: e48-e49 Advertisement Copyright & Permissions© 2011 by American Urological Association Education and Research, Inc.MetricsAuthor Information Andrew Chi Miami, FL More articles by this author Jeffrey Gahan Miami, FL More articles by this author Miguel Gosalbez Miami, FL More articles by this author Samir Shirodkar Miami, FL More articles by this author Vincent Bird Gainsville, FL More articles by this author Mark Soloway Miami, FL More articles by this author Vinata Lokeshwar Miami, FL More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

Toll-like receptor 4 (TLR4) expression in human and murine pancreatic beta-cells affects cell viability and insulin homeostasis

BackgroundToll-like receptor 4 (TLR4) is widely recognized as an essential element in the triggering of innate immunity, binding pathogen-associated molecules such as Lipopolysaccharide (LPS), and in initiating a cascade of pro-inflammatory events. Evidence for TLR4 expression in non-immune cells, including pancreatic β-cells, has been shown, but, the functional role of TLR4 in the physiology of human pancreatic β-cells is still to be clearly established. We investigated whether TLR4 is present in β-cells purified from freshly isolated human islets and confirmed the results using MIN6 mouse insulinoma cells, by analyzing the effects of TLR4 expression on cell viability and insulin homeostasis.ResultsCD11b positive macrophages were practically absent from isolated human islets obtained from non-diabetic brain-dead donors, and TLR4 mRNA and cell surface expression were restricted to β-cells. A significant loss of cell viability was observed in these β-cells indicating a possible relationship with TLR4 expression. Monitoring gene expression in β-cells exposed for 48h to the prototypical TLR4 ligand LPS showed a concentration-dependent increase in TLR4 and CD14 transcripts and decreased insulin content and secretion. TLR4-positive MIN6 cells were also LPS-responsive, increasing TLR4 and CD14 mRNA levels and decreasing cell viability and insulin content.ConclusionsTaken together, our data indicate a novel function for TLR4 as a molecule capable of altering homeostasis of pancreatic β-cells.

The expression of CD8α discriminates distinct T cell subsets in teleost fish

CD8, belonging to the TCR complex, is the main marker molecule of CTLs. Although CD8 genes have been detected in many fish species, the analysis of teleost CD8 + cells has been limited because of the lack of antibodies. Using newly established mAbs against rainbow trout CD8α, we found high ratios of CD8α + cells in trout thymus, gill and intestine, but relatively low abundance in pronephros, spleen and blood. Accordingly, tissue sections revealed many CD8α + cells in thymus, numerous intra- and subepithelial CD8α + cells in intestine and gill and few scattered CD8α + cells in spleen and pronephros. In secondary lymphoid tissues, CD8α + lymphocytes, which did not react with anti-thrombocyte or anti-IgM mAbs, expressed CD8α, CD8β and TCRα, while Ig and CD4 transcripts were found in CD8α − lymphocytes. In contrast, considerable CD4 expression in CD8α + thymocytes suggests the presence of double-positive early T cells. Highly expressed TCRγ, LAG3 and CTLA4 in CD8α + lymphocytes imply that they constitute a heterogeneous population different from found in non-mucosal tissues. PHA stimulation resulted in an up-regulation of CTL effector genes ( perforin, granulysin and IFN-γ) in CD8α + pronephrocytes, while both Th1 ( IFN-γ) and Th2 ( IL-4/ 13A) cytokines were up-regulated in CD8α − pronephrocytes. Although the basic characteristics of CD8α + lymphocytes seem similar in teleost and mammals, features such as the low proportion of teleost CD8α + lymphocytes in blood and their high abundance in respiratory tissue reveal a unique dynamics and distribution.

CD4 and CD8 T Cell Immune Activation during Chronic HIV Infection: Roles of Homeostasis, HIV, Type I IFN, and IL-7

Immune activation plays an important role in the pathogenesis of HIV disease. Although the causes are not fully understood, the forces that lead to immune dysfunction differ for CD4 and CD8 T cells. In this study, we report that the molecular pathways that drive immune activation during chronic HIV infection are influenced by differences in the homeostatic regulation of the CD4 and CD8 T cell pools. Proliferation of CD4 T cells is controlled more tightly by CD4 T cell numbers than is CD8 T cell proliferation. This difference reflects the importance of maintaining a polyclonal CD4 T cell pool in host surveillance. Both pools of T cells were found to be driven by viral load and its associated state of inflammation. In the setting of HIV-induced lymphopenia, naive CD4 T cells were recruited mainly into the proliferating pool in response to CD4 T cell depletion, whereas naive CD8 T cell proliferation was driven mainly by levels of HIV RNA. RNA analysis revealed increased expression of genes associated with type I IFN and common γ chain cytokine signaling in CD4 T cell subsets and only type I IFN-associated genes in CD8 T cell subsets. In vitro studies demonstrated enhanced STAT1 phosphorylation in response to IFN-α and increased expression of the IFNAR1 transcripts in naive and memory CD4 T cells compared with that observed in CD8 T cells. CD4 T cell subsets also showed enhanced STAT1 phosphorylation in response to exogenous IL-7.

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CD28 costimulation regulates FOXP3 in a RelA/NF‐κB‐dependent mechanism

The molecular mechanisms whereby CD28 alone or associated with TCR can regulate FOXP3 expression are not understood, although the importance of CD28 as a pivotal regulator of CD4(+) CD25(+) FOXP3(+) T cells is well recognized. We previously demonstrated that unique CD28-induced, NF-κB-dependent signals were sufficient to activate FOXP3 transcription in human CD4(+) CD25(-) T cells; however, the exact mechanisms are currently unknown. In this study, we have identified novel κB-binding sites on FOXP3 gene and demonstrated that CD28 signals mediated FOXP3 trans activation by nuclear translocation of RelA/NF-κB and not of c-Rel. The occupancy of FOXP3 κB-binding sites by RelA dimers that correlated with histone acetylation and recruitment of Pol II were required both to initiate FOXP3 transcription and to control the promoter occupancy by NFAT. Interestingly, knockdown of RelA in CD4(+) CD25(-) T cells stimulated through TCR and CD28 significantly affected FOXP3 expression, confirming that also the transcriptional activation of FOXP3 gene by TCR in the presence of CD28-costimulatory signals is RelA-dependent. In conclusion, these data suggest a new mechanism by which FOXP3 is activated and supports the critical role of CD28 in the regulation of peripheral tolerance.

IRF4 regulates IL-10 gene expression in CD4 + T cells through differential nuclear translocation

CD4 + T cells play critical roles in the generation of protective immunity against a variety of pathogens. The main two types of effector CD4 + T cells, Th1 and Th2 are characterized by their ability to produce signature cytokines. Among them, IL-10 is a multi-functional cytokine that plays a crucial role in maintaining the balance between immunity and tolerance. Although IL-10 is produced by both differentiated primary Th1 and Th2 cells, Th2 cells produce much higher levels of IL-10 upon stimulation. However, little information is available on the molecular mechanisms of IL-10 gene regulation at the transcriptional level. Interferon regulatory factor IRF4 is a member of the IRF family of transcription factors and plays critical roles in the development of CD4 + T cells into Th2 cells. In this present study, we elucidate the underlying mechanism of IRF4 mediated IL-10 gene transcription in primary CD4 + T cells. Th2 specific binding of IRF4 to the IRF4 responsive elements in IL-10 locus potentiated IL-10 expression in Th2 cells. Knockdown of IRF4 by siRNA decreased IL-10 expression level in Th2 cells. Nuclear translocation of IRF4 was much higher in Th2 cells upon stimulation, which contribute to maintain IL-10 high phenotype of Th2 cells. Collectively, our results suggest that stimulation driven quantitative differences of IRF4 in the nucleus and its binding to IL-10 regulatory elements are crucial mechanisms to induce IL-10 high gene expression in Th2 cells.

Maternal Influences on the Transmission of Leukocyte Gene Expression Profiles in Population Samples from Brisbane, Australia

Two gene expression profiling studies designed to identify maternal influences on development of the neonate immune system and to address the population structure of the leukocyte transcriptome were carried out in Brisbane, Australia. In the first study, a comparison of 19 leukocyte samples obtained from mothers in the last three weeks of pregnancy with 37 umbilical cord blood samples documented differential expression of 7,382 probes at a false discovery rate of 1%, representing approximately half of the expressed transcriptome. An even larger component of the variation involving 8,432 probes, notably enriched for Vitamin E and methotrexate-responsive genes, distinguished two sets of individuals, with perfect transmission of the two profile types between each of 16 mother-child pairs in the study. A minor profile of variation was found to distinguish the gene expression profiles of obese mothers and children of gestational diabetic mothers from those of children born to obese mothers. The second study was of adult leukocyte profiles from a cross-section of Red Cross blood donors sampled throughout Brisbane. The first two axes in this study are related to the third and fourth axes of variation in the first study and also reflect variation in the abundance of CD4 and CD8 transcripts. One of the profiles associated with the third axis is largely excluded from samples from the central portion of the city. Despite enrichment of insulin signaling and aspects of central metabolism among the differentially expressed genes, there was little correlation between leukocyte expression profiles and body mass index overall. Our data is consistent with the notion that maternal health and cytokine milieu directly impact gene expression in fetal tissues, but that there is likely to be a complex interplay between cultural, genetic, and other environmental factors in the programming of gene expression in leukocytes of newborn children.

Molecular cloning and expression of orange-spotted grouper ( Epinephelus coioides) CD8α and CD8β genes

T-cell surface glycoprotein CD8 consists of two distinguished chains, termed α and β chains, and functions as a co-receptor for the T-cell receptor by binding to MHC class I proteins. In this study we report the cloning and identification of both CD8α and CD8β genes from orange-spotted grouper ( Epinephelus coioides). The predicted grouper CD8α and CD8β proteins were structurally similar to other fish especially to those of Pleuronectiformes. Real-time RT-PCR revealed that the CD8 mRNA was much higher in the thymus than in other immune organs, and the expression level were very low in stomach, liver, and brain. During embryonic development of the grouper, the highest CD8 transcripts were detected in the multi-cell stage, followed by muscle burl stage, which suggested that the multi-cell stage may be critical in CD8 transcript synthesis. Moreover, CD8 mRNA levels were examined in lymphocytes at different time treated with lipopolysaccharide (LPS), polyriboinosinic polyribocytidylic acid (PolyI:C), phytohemagglutinin (PHA), and concanavalin A (ConA). The result showed that the CD8 mRNA levels were significantly affected in time-dependent manner by PolyI:C, PHA, and ConA, but not by LPS.

Intestinal T cells of Dicentrarchus labrax (L.): Gene expression and functional studies

Cellular and molecular data have evidenced a gut-associated lymphoid tissue in a variety of teleost species, abundantly containing T cells, whose origin, selection and functions are still unclear. This study reports CD4, CD8-α, MHCI-α , MHCII-β , rag-1 and TCR-β gene transcription along the intestine (anterior, middle and posterior segments) and in the thymus of one year-old Dicentrarchus labrax (L.). Real-time PCR findings depicted a main role of the thymus in T-cell development, but also rag-1 and CD8-α transcripts are detected in the intestine, having significant expression in the posterior segment. In the whole intestine TCR-β and CD8-α exceeded CD4 transcripts. RNA ISH confirmed these data and detailed that mucosal CD8-α + cells were especially numerous in the epithelium and in aggregates in the lamina propria. Regional differences in T-cell-specific gene expressions are first described in the intestine of a bony fish. High non-specific cytotoxic activity against xenogeneic and allogeneic cells was found in lymphocytes purified from the intestinal mucosa, providing further insight into their local defence roles.

Can HIV infection be eradicated through use of potent antiviral agents?

This review focuses on recent advances in HIV research and therapy that seek to eradicate persistent HIV in patients on suppressive therapy. The source of persistent HIV in patients on suppressive therapy is debated. Recent studies of treatment intensification have produced varied results: no reduction in low-level plasma viremia indicating the source of persistent viremia is long-lived HIV-infected cells that release HIV when activated and increase in episomal HIV DNA indicating active replication persists in some infected individuals on suppressive therapy. In addition, clonal HIV sequences found in plasma from patients on long-term suppressive therapy are rarely found in CD4+ memory T cells. These results indicate that persistent viremia may arise from several different sources. Recent studies emphasize the complexity of HIV latency. Current strategies for HIV eradication focus on compounds that activate viral transcription in memory CD4+ T cells by many routes, including inhibiting histone deacetylation and activating nuclear factor kappa B. Several compounds and combinations of these compounds appear to induce the expression of integrated HIV in different latency models. The eradication of HIV requires the elimination of persistent HIV during suppressive therapy. Recent studies have focused on the source of persistent viremia, mechanisms of intracellular HIV latency, and reactivation of latent HIV. It remains to be seen whether alternative treatment strategies may be required to eradicate HIV.

Effect of cytotoxic T lymphocyte‐associated molecule 4 1661 gene polymorphism on its expression and transcription in ulcerative colitis

Our aim was to investigate the expression of cytotoxic T lymphocyte-associated molecule 4 (CTLA-4) in ulcerative colitis (UC) and to evaluate the effect of CTLA-4 gene -1661A/G polymorphism on CTLA-4 expression and transcription. A total of 20 UC patients and 22 healthy controls matched by age and sex were enrolled at Zhongnan Hospital of Wuhan University in central China. The CTLA-4 -1661A/G polymorphism was genotyped by the polymerase chain reaction-restriction fragment length polymorphism method. A Western blot analysis was performed to determine the full length CTLA-4 (flCTLA-4) protein expression in the peripheral blood of the UC patients. Serum-soluble CTLA-4 (sCTLA-4) levels were measured by enzyme-linked immunosorbent assay. CTLA-4-1661G mutant promoter transcription function was analyzed by site-directed PCR-based mutagenesis. CTLA-4 protein expression on CD4(+) T cells in UC patients was lower than that in the healthy controls (P < 0.001) while serum sCTLA-4 in the UC patients was significantly higher than that in the healthy controls (P < 0.001). No correlation was found between flCTLA-4 and sCTLA-4 expression levels and the -1661 A/G polymorphism of the CTLA-4 gene. Meanwhile, CTLA-4 -1661 allele A had no significant impact on the promoter activity compared with allele G (P > 0.05). CTLA-4 expressions were aberrant in UC patients compared with the healthy controls. CTLA-4 -1661A/G polymorphism had no significant impact on CTLA-4 expression and transcription in the peripheral CD4 T cells of UC patients.

Expression of α-defensin 1-3 in T cells from severe cutaneous drug-induced hypersensitivity reactions

Cytotoxic T cells seem to be the main effector cells in Stevens-Johnson syndrome/toxic epidermal necrolysis (SJS/TEN). However, recent data support a role of the innate immune system in the etiopathology of drug-induced cutaneous reactions. In this study, we analyzed the expression of α-defensins 1-3 in mononuclear cells from patients with SJS/TEN, drug-induced maculopapular exanthema (MPE), and healthy donors. DEFA1A3 gene expression was analyzed by quantitative and end-point RT-PCR. Intracellular flow cytometry, immunofluorescence and immunohistochemistry were carried out to verify α-defensin 1-3 protein expression in mononuclear cells from peripheral blood and skin infiltrates. α-Defensin 1-3 concentration was evaluated in plasma and blister fluid samples by ELISA. We herein describe DEFA1A3 gene expression in peripheral blood mononuclear cells (PBMCs) from patients with drug-induced cutaneous diseases. Gene expression analysis unveiled transcription in CD4 and CD8 peripheral blood T cells. Protein expression was confirmed by intracellular flow cytometry in mononuclear cells from the patients, including monocytes, NK cells, and T cells from peripheral blood and blister fluid. Further analysis of protein content by flow cytometry revealed higher protein levels in CD56(+) CD3(+) lymphocytes from patients with SJS/TEN when compared to MPE and healthy donors. Immunohistological analysis was used to confirm expression in dermal infiltrates. α-Defensin levels were estimated by ELISA to be 3- to 175-fold higher in blister fluid when compared to simultaneously drawn plasma samples. Upregulation of innate immune molecules such as α-defensins 1-3 in T cells from patients with SJS/TEN may be involved in the etiopathology of these life-threatening diseases induced by medications.

Enhanced Mammary Progesterone Receptor-A Isoform Activity in the Promotion of Mammary Tumor Progression by Dietary Soy in Rats

Dietary contribution to breast cancer risk, recurrence, and progression remains incompletely understood. Increased consumption of soy and soy isoflavones is associated with reduced mammary cancer susceptibility in women and in rodent models of carcinogenesis. In rats treated with N-methyl-N-nitrosourea, dietary intake of soy protein isolate (SPI) reduced mammary tumor occurrence but increased incidence of more invasive tumors in tumored rats, relative to the control diet casein. Here we evaluated whether mammary tumor progression in tumor-bearing rats lifetime exposed to SPI is associated with deregulated progesterone receptor (PR) isoform expression. In histologically normal mammary glands of rats with invasive ductal carcinoma lesions, PR-A protein levels were higher for SPI- than casein-fed rats, whereas PR-B was undetectable for both groups. Increased mammary PR-A expression was associated with higher transforming growth factor-β1, stanniocalcin-1, and CD44 transcript levels; lower E-cadherin and estrogen receptor-α expression; and reduced apoptotic status in ductal epithelium. Serum progesterone (ng/ml) (CAS: 25.94 ± 3.81; SPI: 13.19 ± 2.32) and estradiol (pg/ml) (CAS: 27.9 ± 4.49; SPI: 68.48 ± 23.87) levels differed with diet. However, sera from rats of both diet groups displayed comparable mammosphere-forming efficiency in human MCF-7 cells. Thus, soy-rich diets may influence the development of more aggressive tumors by enhancing PR-A-dependent signaling in premalignant breast tissues.

Fyn and CD70 Expression in CD4+ T Cells from Patients with Systemic Lupus Erythematosus

CD4+ T cells from patients with systemic lupus erythematosus (SLE) display defective function that contributes to abnormal activation of B cells and autoantibody production. We compared the transcript and protein levels of Fyn and CD70 in CD4+ T cells from patients with SLE (n = 41) and healthy individuals (n = 34). The CD4+ T cells were isolated by positive biomagnetic separation technique. The quantitative analysis of messenger RNA was performed by reverse transcription and real-time quantitative PCR. The protein contents in the CD4+ T cells were determined by Western blotting analysis. We observed significantly higher levels of Fyn (p = 0.03) and CD70 (p = 0.029) transcripts in SLE CD4+ T cells than in controls. There was a significant increase in CD70 protein levels (p < 0.0001), but not Fyn protein levels (p = 0.081) in CD4+ T cells from patients with SLE compared to healthy individuals. In the group with high disease activity [SLE Disease Activity Index (SLEDAI) >/= 9], we observed a significantly higher Fyn protein content than in controls (p = 0.030). There was no correlation between Fyn and CD70 protein levels in SLE CD4+ T cells and disease activity as expressed in the SLEDAI scale. We confirmed previous observations of higher expression of CD70 in CD4+ T cells from patients with SLE. Our findings suggest that increased Fyn protein content in CD4+ T cells can be associated with high SLE disease activity.

Programmed Death-1 Antibody Blocks Therapeutic Effects of T-Regulatory Cells in Cockroach Antigen-Induced Allergic Asthma

We recently reported that the adoptive transfer of T-regulatory cells (Tregs) isolated from lung and spleen tissue of green fluorescent protein-transgenic mice reversed airway hyperresponsiveness and airway inflammation. Because Programmed Death-1 (PD-1) is a pivotal receptor regulating effector T-cell activation by Tregs, we evaluated whether PD-1 is involved in the therapeutic effect of naturally occurring Tregs (NTregs) and inducible Tregs (iTregs) in cockroach (CRA)-sensitized and challenged mice. The CD4(+)CD25(+) NTregs and CD4(+)CD25(-) iTregs isolated from the lungs and spleens of BALB/c mice were adoptively transferred into CRA-sensitized and CRA-challenged mice with and without anti-PD-1 antibody (100 μg/mice). The CD4(+)CD25(+) T cells in the lung were phenotyped after adoptive transfer. Concentrations of IL-4, IL-5, IL-10, IFN-γ, and IL-13 in bronchoalveolar lavage fluid (BALF) were measured using ELISA. The NTregs and iTregs from either lung or spleen tissue reversed airway hyperresponsiveness for at least 4 wk. However, the therapeutic effect was blocked by administering the anti-PD-1 antibody. The administration of Tregs-recipient mice with anti-PD-1 antibody significantly decreased cytotoxic T-lymphocyte antigen-4 expression, with low concentrations of Forkhead-winged transcriptional factor box 3 (Foxp3) mRNA transcripts in lung CD4(+)CD25(+) T cells. These mice had substantially higher concentrations of BALF IL-4, IL-5, and IL-13, but significantly decreased levels of BALF IL-10. Adoptive therapy recipients without the anti-PD-1 antibody exhibited high levels of CTLA-4 expression and Foxp3 transcripts in lung CD4(+)CD25(+) T cells, with a significant decrease in BALF IL-4, IL-5, and IL-13 concentrations and a substantial increase in BALF IL-10 concentrations. These data suggest that the reversal of airway hyperresponsiveness and airway inflammation by Tregs is mediated in part by PD-1, because other costimulatory molecules (e.g., inducible costimulatory molecule [ICOS] or CTLA-4) have been shown to play a role in Treg-mediated suppression.

Transcriptional Activation of the Interleukin-21 Gene and Its Receptor Gene by Human T-cell Leukemia Virus Type 1 Tax in Human T-cells

At the incipient stages of the development of adult T-cell leukemia, T-cells infected with human T-cell leukemia virus type 1 (HTLV-1) suffer disregulation in cell growth caused by aberrant expression of host genes by the HTLV-1 transactivator protein Tax (Tax1). Tax1-mediated growth promotion is thought to result from, at least in part, up-regulation of genes for growth factors and their receptors that induce T-cell growth. In the present study, we demonstrate that Tax1 transactivates the interleukin-21 (IL-21) and its receptor (IL-21R) genes in human T-cells. Introduction of Tax1 via recombinant adenoviruses induced expression of endogenous IL-21 and IL-21R. Isolated promoters of the IL-21 and IL-21R genes were activated by Tax1 in reporter assays, which further revealed that there were at least two Tax1-responsive elements in either the IL-21 promoter or the IL-21R promoter. Chromatin immunoprecipitation assay and gel mobility shift assay exhibited that the IL-21 promoter elements bound transcription factors AP-1 and NF-kappaB, and the IL-21R promoter elements were associated with AP-1 and interferon regulatory factor. Collectively, Tax1-dependent activation of these transcriptional factors presumably contributes to expression of the IL-21 gene and its receptor gene. The related virus HTLV-2 with Tax2 similar to Tax1 is known not to be pathogenic. Tax2 exhibited little, if any, or no induction of the IL-21 transcription in CD4+ T-cells, in contrast to Tax1. The study suggests insights into cytokine-dependent aberrant growth of HTLV-1-infected T-cells and the molecular basis of different pathogenicity between HTLV-1 and HTLV-2.