Extended co-expression of inhibitory receptors by human CD8 T-cells depending on differentiation, antigen-specificity and anatomical localization.

Inhibitory receptors mediate CD8 T-cell hyporesponsiveness against cancer and infectious diseases. PD-1 and CTLA-4 have been extensively studied, and blocking antibodies have already shown clinical benefit for cancer patients. Only little is known on extended co-expression of inhibitory receptors and their ligands. Here we analyzed the expression of eight inhibitory receptors by tumor-antigen specific CD8 T-cells. We found that the majority of effector T-cells simultaneously expressed four or more of the inhibitory receptors BTLA, TIM-3, LAG-3, KRLG-1, 2B4, CD160, PD-1 and CTLA-4. There were major differences depending on antigen-specificity, differentiation and anatomical localization of T-cells. On the other hand, naive T-cells were only single or double positive for BTLA and TIM-3. Extended co-expression is likely relevant for effector T-cells, as we found expression of multiple ligands in metastatic lesions of melanoma patients. Together, our data suggest that naive T-cells are primarily regulated by BTLA and TIM-3, whereas effector cells interact via larger numbers of inhibitory receptors. Blocking multiple inhibitory receptors simultaneously or sequentially may improve T-cell based therapies, but further studies are necessary to clarify the role of each receptor-ligand pair.

Zinc-alpha 2-glycoprotein gene expression in adipose tissue is related with insulin resistance and lipolytic genes in morbidly obese patients.

OBJECTIVE Zinc-α(2) glycoprotein (ZAG) stimulates lipid loss by adipocytes and may be involved in the regulation of adipose tissue metabolism. However, to date no studies have been made in the most extreme of obesity. The aims of this study are to analyze ZAG expression levels in adipose tissue from morbidly obese patients, and their relationship with lipogenic and lipolytic genes and with insulin resistance (IR). METHODS mRNA expression levels of PPARγ, IRS-1, IRS-2, lipogenic and lipolytic genes and ZAG were quantified in visceral (VAT) and subcutaneous adipose tissue (SAT) of 25 nondiabetic morbidly obese patients, 11 with low IR and 14 with high IR. Plasma ZAG was also analyzed. RESULTS The morbidly obese patients with low IR had a higher VAT ZAG expression as compared with the patients with high IR (p = 0.023). In the patients with low IR, the VAT ZAG expression was greater than that in SAT (p = 0.009). ZAG expression correlated between SAT and VAT (r = 0.709, p<0.001). VAT ZAG expression was mainly predicted by insulin, HOMA-IR, plasma adiponectin and expression of adiponectin and ACSS2. SAT ZAG expression was only predicted by expression of ATGL. CONCLUSIONS ZAG could be involved in modulating lipid metabolism in adipose tissue and is associated with insulin resistance. These findings suggest that ZAG may be a useful target in obesity and related disorders, such as diabetes.

Expression of non-TLR pattern recognition receptors in the spleen of BALB/c mice infected with Plasmodium yoelii and Plasmodium chabaudi chabaudi AS

The spleen plays a crucial role in the development of immunity to malaria, but the role of pattern recognition receptors (PRRs) in splenic effector cells during malaria infection is poorly understood. In the present study, we analysed the expression of selected PRRs in splenic effector cells from BALB/c mice infected with the lethal and non-lethal Plasmodium yoelii strains 17XL and 17X, respectively, and the non-lethal Plasmodium chabaudi chabaudi AS strain. The results of these experiments showed fewer significant changes in the expression of PRRs in AS-infected mice than in 17X and 17XL-infected mice. Mannose receptor C type 2 (MRC2) expression increased with parasitemia, whereas Toll-like receptors and sialoadhesin (Sn) decreased in mice infected with P. chabaudi AS. In contrast, MRC type 1 (MRC1), MRC2 and EGF-like module containing mucin-like hormone receptor-like sequence 1 (F4/80) expression decreased with parasitemia in mice infected with 17X, whereas MRC1 an MRC2 increased and F4/80 decreased in mice infected with 17XL. Furthermore, macrophage receptor with collagenous structure and CD68 declined rapidly after initial parasitemia. SIGNR1 and Sn expression demonstrated minor variations in the spleens of mice infected with either strain. Notably, macrophage scavenger receptor (Msr1) and dendritic cell-associated C-type lectin 2 expression increased at both the transcript and protein levels in 17XL-infected mice with 50% parasitemia. Furthermore, the increased lethality of 17X infection in Msr1 -/- mice demonstrated a protective role for Msr1. Our results suggest a dual role for these receptors in parasite clearance and protection in 17X infection and lethality in 17XL infection.

The level of ascorbate peroxidase is enhanced in benznidazole-resistant populations of Trypanosoma cruzi and its expression is modulated by stress generated by hydrogen peroxide

Ascorbate peroxidases (APX) are class I heme-containing enzymes that convert hydrogen peroxide into water molecules. The gene encoding APX has been characterized in 11 strains of Trypanosoma cruzi that are sensitive or resistant to benznidazole (BZ). Bioinformatic analysis revealed the presence of two complete copies of the T. cruzi APX (TcAPX) gene in the genome of the parasite, while karyotype analysis showed that the gene was present in the 2.000-kb chromosome of all of the strains analyzed. The sequence of TcAPX exhibited greater levels of similarity to those of orthologous enzymes from Leishmania spp than to APXs from the higher plant Arabidopsis thaliana. Northern blot and real-time reverse transcriptase polymerase chain reaction (RT-PCR) analyses revealed no significant differences in TcAPX mRNA levels between the T. cruzi strains analyzed. On the other hand, Western blots showed that the expression levels of TcAPX protein were, respectively, two and three-fold higher in T. cruzi populations with in vitro induced (17 LER) and in vivo selected (BZR) resistance to BZ, in comparison with their corresponding susceptible counterparts. Moreover, the two BZ-resistant populations exhibited higher tolerances to exogenous hydrogen peroxide than their susceptible counterparts and showed TcAPX levels that increased in a dose-dependent manner following exposure to 100 and 200 µM hydrogen peroxide.

An evaluation of p16INK4a expression in cervical intraepithelial neoplasia specimens, including women with HIV-1

Although several studies have evaluated the role of p16INK4a as a diagnostic marker of cervical intraepithelial neoplasia (CIN) and its association with disease progression, studies regarding the role of p16INK4a in human immunodeficiency virus (HIV)-infected patients remain scarce. The present study was designed to determine the potential utility of p16INK4a as a diagnostic marker for CIN and invasive cervical cancer in HIV-positive and negative cervical specimens. An immunohistochemical analysis of p16INK4a was performed in 326 cervical tissue microarray specimens. Performance indicators were calculated and compared using receiving operating characteristics curve (ROC)/area under the curve. In HIV-1-negative women, the percentage of cells that was positive for p16INK4a expression was significantly correlated with the severity of CIN (p < 0.0001). A ROC curve with a cut-off value of 55.28% resulted in a sensitivity of 89%, a specificity of 81%, a positive predictive value of 91% and a negative predictive value of 78%. HIV-seropositive women exhibited decreased expression of p16INK4a in CIN2-3 specimens compared with HIV-negative specimens (p = 0.031). The ROC data underscore the potential utility of p16INK4a under defined conditions as a diagnostic marker for CIN 2-3 staging and invasive cervical cancer. HIV-1 infection, however, is associated with relatively reduced p16INK4a expression in CIN 2-3.

Expression and localization of PPARs in the rat ovary during follicular development and the periovulatory period.

PPARs are a family of nuclear hormone receptors involved in various processes that could influence ovarian function. We investigated the cellular localization and expression of PPARs during follicular development in ovarian tissue collected from rats 0, 6, 12, 24, and 48 h post-PMSG. A second group of animals received human CG (hCG) 48 h post-PMSG. Their ovaries were removed 0, 4, 8, 12, and 24 h post-hCG to study the periovulatory period. mRNAs corresponding to the PPAR isotypes (alpha, delta, and gamma) were localized by in situ hybridization. Changes in the levels of mRNA for the PPARs were determined by ribonuclease protection assays. PPAR gamma mRNA was localized primarily to granulosa cells, and levels of expression did not change during follicular development. Four hours post-hCG, levels of mRNA for PPAR gamma decreased (P &lt; 0.05) but not uniformly in all follicles. At 24 h post-hCG, levels of PPAR gamma mRNA were reduced 64%, but some follicles maintained high expression. In contrast, mRNAs for PPAR alpha and delta were located primarily in theca and stroma, and their levels did not change during the intervals studied. To investigate the physiologic significance of PPAR gamma in the ovary, granulosa cells from PMSG-primed rats were cultured for 48 h with prostaglandin J(2) (PGJ(2)) and ciglitazone, PPAR gamma activators. Both compounds increased progesterone and E2 secretion (P &lt; 0.05). These data suggest that PPAR gamma is involved in follicular development, has a negative influence on the luteinization of granulosa cells, and/or regulates the periovulatory shift in steroid production. The more general and steady expression of PPARs alpha and delta indicate that they may play a role in basal ovarian function.

MicroRNA expression profiling in Imatinib-resistant Chronic Myeloid Leukemia patients without clinically significant ABL1-mutations.

The development of Imatinib Mesylate (IM), the first specific inhibitor of BCR-ABL1, has had a major impact in patients with Chronic Myeloid Leukemia (CML), establishing IM as the standard therapy for CML. Despite the clinical success obtained with the use of IM, primary resistance to IM and molecular evidence of persistent disease has been observed in 20-25% of IM treated patients. The existence of second generation TK inhibitors, which are effective in patients with IM resistance, makes identification of predictors of resistance to IM an important goal in CML. In this study, we have identified a group of 19 miRNAs that may predict clinical resistance to IM in patients with newly diagnosed CML.

Real-time recording of circadian liver gene expression in freely moving mice reveals the phase-setting behavior of hepatocyte clocks.

The mammalian circadian timing system consists of a master pacemaker in the suprachiasmatic nucleus (SCN) in the hypothalamus, which is thought to set the phase of slave oscillators in virtually all body cells. However, due to the lack of appropriate in vivo recording technologies, it has been difficult to study how the SCN synchronizes oscillators in peripheral tissues. Here we describe the real-time recording of bioluminescence emitted by hepatocytes expressing circadian luciferase reporter genes in freely moving mice. The technology employs a device dubbed RT-Biolumicorder, which consists of a cylindrical cage with reflecting conical walls that channel photons toward a photomultiplier tube. The monitoring of circadian liver gene expression revealed that hepatocyte oscillators of SCN-lesioned mice synchronized more rapidly to feeding cycles than hepatocyte clocks of intact mice. Hence, the SCN uses signaling pathways that counteract those of feeding rhythms when their phase is in conflict with its own phase.

Differential expression of triiodothyronine receptors in schwannoma and neurofibroma: role of Schwann cell-axon interaction.

Regulation of gene expression in Schwann cells may be determined, at least in part, by the interaction of these cells with axons. Two peripheral nerve tumors, neurofibroma and schwannoma, represent good tools for studying Schwann cell activity in the presence or absence of axon action. In the present work we studied the expression of triiodothyronine receptors (T3R) by Schwann cells in these two tumors and also in adult normal sciatic nerve. Confirming the results of the histological examination, immunostaining of the neurofilaments showed the presence of fascicles or scattered axons in all neurofibroma sections studied. In these neurofibromas, Schwann cells did not express T3R immunoreactivity. Furthermore, in adult normal sciatic nerve, Schwann cells which ensheathed axons were devoid of any T3R expression. In contrast, in schwannoma, the complete absence of axons was demonstrated by the lack of neurofilament immunostaining. Here, Schwann cells deprived of axonal interaction displayed clear T3R immunoreactivity. In schwannoma cell cultures, Schwann cells continued to express T3R, even in cultures treated with medium that had been conditioned with rat sensory neurons. On the basis of these results, we suggest that, beside the possible regulatory mechanisms for T3R, the synthesis of T3R is regulated, at least in part, by Schwann cell-axon interaction.

CpG-ODN-induced sustained expression of BTLA mediating selective inhibition of human B cells.

BTLA (B- and T-lymphocyte attenuator) is a prominent co-receptor that is structurally and functionally related to CTLA-4 and PD-1. In T cells, BTLA inhibits TCR-mediated activation. In B cells, roles and functions of BTLA are still poorly understood and have never been studied in the context of B cells activated by CpG via TLR9. In this study, we evaluated the expression of BTLA depending on activation and differentiation of human B cell subsets in peripheral blood and lymph nodes. Stimulation with CpG upregulated BTLA, but not its ligand: herpes virus entry mediator (HVEM), on B cells in vitro and sustained its expression in vivo in melanoma patients after vaccination. Upon ligation with HVEM, BTLA inhibited CpG-mediated B cell functions (proliferation, cytokine production, and upregulation of co-stimulatory molecules), which was reversed by blocking BTLA/HVEM interactions. Interestingly, chemokine secretion (IL-8 and MIP1β) was not affected by BTLA/HVEM ligation, suggesting that BTLA-mediated inhibition is selective for some but not all B cell functions. We conclude that BTLA is an important immune checkpoint for B cells, as similarly known for T cells.

The MRC1/CD68 Ratio Is Positively Associated with Adipose Tissue Lipogenesis and with Muscle Mitochondrial Gene Expression in Humans.

BACKGROUND Alternative macrophages (M2) express the cluster differentiation (CD) 206 (MCR1) at high levels. Decreased M2 in adipose tissue is known to be associated with obesity and inflammation-related metabolic disturbances. Here we aimed to investigate MCR1 relative to CD68 (total macrophages) gene expression in association with adipogenic and mitochondrial genes, which were measured in human visceral [VWAT, n = 147] and subcutaneous adipose tissue [SWAT, n = 76] and in rectus abdominis muscle (n = 23). The effects of surgery-induced weight loss were also longitudinally evaluated (n = 6). RESULTS MCR1 and CD68 gene expression levels were similar in VWAT and SWAT. A higher proportion of CD206 relative to total CD68 was present in subjects with less body fat and lower fasting glucose concentrations. The ratio MCR1/CD68was positively associated with IRS1gene expression and with the expression of lipogenic genes such as ACACA, FASN and THRSP, even after adjusting for BMI. The ratio MCR1/CD68 in SWAT increased significantly after the surgery-induced weight loss (+44.7%; p = 0.005) in parallel to the expression of adipogenic genes. In addition, SWAT MCR1/CD68ratio was significantly associated with muscle mitochondrial gene expression (PPARGC1A, TFAM and MT-CO3). AT CD206 was confirmed by immunohistochemistry to be specific of macrophages, especially abundant in crown-like structures. CONCLUSION A decreased ratio MCR1/CD68 is linked to adipose tissue and muscle mitochondrial dysfunction at least at the level of expression of adipogenic and mitochondrial genes.

Expression of the transcription factor NFAT2 in human neutrophils: IgE-dependent, Ca2+- and calcineurin-mediated NFAT2 activation.

NFAT (nuclear factors of activated T cells) proteins constitute a family of transcription factors involved in mediating signal transduction. The presence of NFAT isoforms has been described in all cell types of the immune system, with the exception of neutrophils. In the present work we report for the first time the expression in human neutrophils of NFAT2 mRNA and protein. We also report that specific antigens were able to promote NFAT2 protein translocation to the nucleus, an effect that was mimicked by the treatment of neutrophils with anti-immunoglobulin E (anti-IgE) or anti-Fcepsilon-receptor antibodies. Antigens, anti-IgE and anti-FcepsilonRs also increased Ca2+ release and the intracellular activity of calcineurin, which was able to interact physically with NFAT2, in parallel to eliciting an enhanced NFAT2 DNA-binding activity. In addition, specific chemical inhibitors of the NFAT pathway, such as cyclosporin A and VIVIT peptide, abolished antigen and anti-IgE-induced cyclooxygenase-2 (COX2) gene upregulation and prostaglandin (PGE(2)) release, suggesting that this process is through NFAT. Our results provide evidence that NFAT2 is constitutively expressed in human neutrophils, and after IgE-dependent activation operates as a transcription factor in the modulation of genes, such as COX2, during allergic inflammation.

Differential expression of the costimulatory molecules CD86, CD28, CD152 and PD-1 correlates with the host-parasite outcome in leprosy

Leprosy is a spectral disease exhibiting two polar sides, namely, lepromatous leprosy (LL) characterised by impaired T-cell responses and tuberculoid leprosy in which T-cell responses are strong. Proper T-cell activation requires signalling through costimulatory molecules expressed by antigen presenting cells and their ligands on T-cells. We studied the influence of costimulatory molecules on the immune responses of subjects along the leprosy spectrum. The expression of the costimulatory molecules was evaluated in in vitro-stimulated peripheral blood mononuclear cells of lepromatous and tuberculoid patients and healthy exposed individuals (contacts). We show that LL patients have defective monocyte CD86 expression, which likely contributes to the impairment of the antigen presentation process and to patients anergy. Accordingly, CD86 but not CD80 blockade inhibited the lymphoproliferative response to Mycobacterium leprae. Consistent with the LL anergy, there was reduced expression of the positive signalling costimulatory molecules CD28 and CD86 on the T-cells in these patients. In contrast, tuberculoid leprosy patients displayed increased expression of the negative signalling molecules CD152 and programmed death-1 (PD-1), which represents a probable means of modulating an exacerbated immune response and avoiding immunopathology. Notably, the contacts exhibited proper CD86 and CD28 expression but not exacerbated CD152 or PD-1 expression, suggesting that they tend to develop a balanced immunity without requiring immunosuppressive costimulatory signalling.