Receptor Beta-subunit Research Articles

Adiponectin, Leptin, and Insulin-Pathway Receptors as Endometrial Cancer Subtyping Markers

Developing a system of molecular subtyping for endometrial tumors might improve insight into disease etiology and clinical prediction of patient outcomes. High body mass index (BMI) has been implicated in development of endometrial cancer through hormonal pathways and might influence tumor expression of biomarkers involved in BMI-sensitive pathways. We evaluated whether endometrial tumor expression of 7 markers from BMI-sensitive pathways of insulin resistance could effectively characterize molecular subtypes: adiponectin receptor 1, adiponectin receptor 2, leptin receptor, insulin receptor (beta subunit), insulin receptor substrate 1, insulin-like growth factor 1 receptor, and insulin-like growth factor 2 receptor. Using endometrial carcinoma tissue specimens from a case-only prospective sample of 360 women from the Nurses' Health Study, we scored categorical immunohistochemical measurements of protein expression for each marker. Logistic regression was used to estimate associations between endometrial cancer risk factors, especially BMI, and tumor marker expression. Proportional hazard modeling was performed to estimate associations between marker expression and time to all-cause mortality as well as time to endometrial cancer-specific mortality. No association was observed between BMI and tumor expression of any marker. No marker was associated with time to either all-cause mortality or endometrial cancer-specific mortality in models with or without standard clinical predictors of patient mortality (tumor stage, grade, and histologic type). It did not appear that any of the markers evaluated here could be used effectively to define molecular subtypes of endometrial cancer.

Association analysis of the GABRB3 promoter variant and susceptibility to autism spectrum disorder

ObjectiveSeveral previous linkage and association studies and an expression analysis have suggested the gamma-aminobutyric acid type A receptor beta3 subunit (GABRB3) gene as an important candidate gene for autism. In addition, polymorphisms in the promoter region of GABRB3 might modulate the expression of this gene. In order to investigate the underlying mechanism of the role of GABRB3 in autism susceptibility, we designed a case-control study to analyze the association of the rs4906902 single nucleotide polymorphism (SNP) in the promoter region of the GABRB3 gene with autism spectrum disorder (ASD) in Iranian patients. Materials & methodsThe rs4906902 polymorphism was genotyped using the polymerase chain reaction- restriction fragment length polymorphism (PCR-RFLP) technique. The frequency of the risk allele and its association with disease was examined in 518 patients and 472 control individuals. ResultThe results demonstrated that the genotype frequencies were in Hardy-Weinberg equilibrium in both the case and the control groups. The results shown that neither allelic frequencies nor genotypic distribution of the rs4906902 was significantly different between autism patients and healthy controls. In conclusionIt seems that the GABRB3 gene may influence the Autism susceptibility via a different SNPs in this gene. Also, another independent mechanisms like epigenetic effects should not be ignored when we want to explore the link between GABRB3 and ASD.

Role of ash2 (absent, small, or homeotic)-like and Jumonji domain-containing protein 3 on histone methylation of interferon-gamma gene and their associations with vascular damage of Kawasaki disease

Objective: To investigate the impacts of ash2 (absent, small, or homeotic)-like (Ash2L) and Jumonji domain-containing protein 3 (Jmjd3) on histone methylation of interferon-gamma(IFN-γ) gene and association with vascular damage of Kawasaki disease (KD) in acute phase. Methods: This study was performed among 36 children with KD in acute phase (KD group) and 28 age-matched health children (control group), who were treated or underwent physical examination in our hospital between February 2015 and June 2016. Patients were further divided into KD groups with or without coronary artery lesions (KD-CAL(+) , 16 cases; KD-CAL(-), 20 cases). All KD patients were treated with intravenous immunoglobulin. The proportion of type 1 helper T(Th1) cells and protein levels of IFN-γ, T-box expressed in T cells(T-bet), phosphorylated signal transducer and activator of transcription 1(pSTAT1) and phosphorylated signal transducer and activator of transcription 4(pSTAT4) were analyzed by flow cytometry.Chromatin immunoprecipitation was performed to determine histone methylation (histone H3 tri-methyl K4(H3K4me3), histone H3 tri-methyl K27(H3K27me3)) and binding levels of Ash2L, Jmjd3 and Ezh2 associated with IFN-γ in CD4(+) T cells. Quantitative real-time PCR was used to determine mRNA levels of IFN-γ, interferon γ receptor 1(IFN-γR1), interferon γ receptor 2(IFN-γR2), interleukin 12 receptor subunit beta 1(IL-12Rβ1), interleukin 12 receptor subunit beta 2(IL-12Rβ2), interleukin 18 receptor subunit beta α(IL-18Rα), interleukin 18 receptor subunit beta β(IL-18Rβ), tumor necrosis factor receptor 1(TNFR1), toll-like receptor 4(TLR4), receptor interacting serine/threonine kinase 1(RIP-1) and myeloid differentiation primary response gene 88(MyD88) in CD4(+) T cells. Plasma concentrations of IFN-γ, interleukin 12(IL-12), interleukin 18(IL-18) and tumor necrosis factor α(TNF-α) were measured by enzyme-linked Immunosorbent assay. Results: (1)The proportion of Th1 and its protein level of IFN-γ were significantly higher in KD group than those in control group and higher in KD-CAL(+) group than in KD-CAL(-) group (all P<0.05), and lower after treatment than before treatment (all P<0.05). (2)Compared with control group, mRNA level of IFN-γ and IFN-γ-associating H3K4me3 was increased, while level of IFN-γ associating H3K27me3 in CD4(+) T cells was reduced in KD group (all P<0.05), which resulted in a higher rate of H3K4me3/H3K27me3 (P<0.05) in KD group, which was positively correlated with IFN-γ mRNA in KD group(r=0.55, P<0.05). Similar results were found between KD-CAL(+) group and KD-CAL(-) group (all P<0.05). Level of IFN-γ associating H3K27me3 was increased, and mRNA level of IFN-γ and IFN-γ associating H3K4me3 was decreased after treatment than before treatment (all P<0.05). (3)Expression of T-bet protein and binding levels of Ash2L and Jmjd3 with IFN-γ gene were significantly higher in KD group than those in control group(all P<0.05), higher in KD-CAL(+) group than those in KD-CAL(-) group (all P<0.05). These parameters were significantly lower after treatment than before treatment (all P<0.05). Binding level of Ezh2 with IFN-γ gene was similar among various groups (all P>0.05). (4)In comparison with control or after treatment, surface receptors(IFN-γR1/2, IL-12Rβ1/2, IL-18Rα/β, TNFR1 and TLR4) and its downstream molecules(pSTAT1, pSTAT4, RIP(1) and MyD88) in CD4(+) T cells, and plasma concentrations of inflammatory cytokines(IFN-γ, IL-12, IL-18 and TNF-α) were found to be higher in KD group(all P<0.05). These parameters were also higher in KD-CAL(+) group than in KD-CAL(-) group (all P<0.05). Conclusion: Aberrant histone methylation of IFN-γ associating H3K4me3 and H3K27me3 caused by over-binding of Ash2L and Jmjd3 might be involved in immune dysfunction and vascular damage in KD in the acute phase.

Modulation of defensive reactivity by GLRB allelic variation: converging evidence from an intermediate phenotype approach

Representing a phylogenetically old and very basic mechanism of inhibitory neurotransmission, glycine receptors have been implicated in the modulation of behavioral components underlying defensive responding toward threat. As one of the first findings being confirmed by genome-wide association studies for the phenotype of panic disorder and agoraphobia, allelic variation in a gene coding for the glycine receptor beta subunit (GLRB) has recently been associated with increased neural fear network activation and enhanced acoustic startle reflexes. On the basis of two independent healthy control samples, we here aimed to further explore the functional significance of the GLRB genotype (rs7688285) by employing an intermediate phenotype approach. We focused on the phenotype of defensive system reactivity across the levels of brain function, structure, and physiology. Converging evidence across both samples was found for increased neurofunctional activation in the (anterior) insular cortex in GLRB risk allele carriers and altered fear conditioning as a function of genotype. The robustness of GLRB effects is demonstrated by consistent findings across different experimental fear conditioning paradigms and recording sites. Altogether, findings provide translational evidence for glycine neurotransmission as a modulator of the brain’s evolutionary old dynamic defensive system and provide further support for a strong, biologically plausible candidate intermediate phenotype of defensive reactivity. As such, glycine-dependent neurotransmission may open up new avenues for mechanistic research on the etiopathogenesis of fear and anxiety disorders.

Role of insulin receptors in myocardial ischaemia-reperfusion injury during cardiopulmonary bypass.

Ischaemia-reperfusion injury after cardiac bypass causes cardiac dysfunction and tissue damage. Insulin resistance during reperfusion contributes to the dysfunction, but the role of changes in myocardial insulin receptors during this period has not been determined yet. Twelve mongrel dogs underwent cardiac bypass, 6 for 30 min and 6 for 120 min. Blood samples were taken from the coronary artery and coronary sinus and tissue samples from the apex of the left ventricle before bypass and 15, 45, and 75 min after termination of bypass surgery and initiation of reperfusion. Plasma glucose and insulin, the Insulin Resistance Index, myocardial glycogen, insulin receptor alpha and beta subunits, and total insulin mRNA were measured in these samples. Plasma glucose and insulin and the insulin resistance index all increased significantly during the reperfusion period. A significant decrease in myocardial glycogen occurred at the same time. The alpha subunits of the insulin receptor were seen on the myocyte surface and the beta subunits mostly in the cytoplasm.The expression of both subunits and total insulin mRNA decreased in a similar manner after termination of bypass surgery. All parameters gradually returned toward pre-bypass values as the post-bypass period progressed. And at all post-bypass time points, the 120-min bypass group showed a significantly greater effect from ischaemia than the 30-min bypass group. A decrease in insulin receptor expression is a cause of post-bypass insulin resistance, and this decrease is initiated at the mRNA level. Increased insulin resistance leads to excessive reliance on myocardial glycogen as an energy source and a deficit in energy substrates that contributes to cardiac dysfunction.

Microbial Disease Spectrum Linked to a Novel IL-12Rβ1 N-Terminal Signal Peptide Stop-Gain Homozygous Mutation with Paradoxical Receptor Cell-Surface Expression

Patients with Mendelian Susceptibility to Mycobacterial Diseases (MSMD) exhibit variable vulnerability to infections by mycobacteria and other intramacrophagic bacteria (e.g., Salmonella and Klebsiella) and fungi (e.g., Histoplasma, Candida, Paracoccidioides, Coccidioides, and Cryptococcus). The hallmark of MSMD is the inherited impaired production of interferon gamma (IFN-γ) or the lack of response to it. Mutations in the interleukin (IL)-12 receptor subunit beta 1 (IL12RB1) gene accounts for 38% of cases of MSMD. Most IL12RB1 pathogenic allele mutations, including ten known stop-gain variants, cause IL-12Rβ1 complete deficiency (immunodeficiency-30, IMD30) by knocking out receptor cell-surface expression. IL12RB1 loss-of-function genotypes impair both IL-12 and IL-23 responses. Here, we assess the health effects of a rare, novel IL12RB1 stop-gain homozygous genotype with paradoxical IL-12Rβ1 cell-surface expression. We appraise four MSMD children from three unrelated Brazilian kindreds by clinical consultation, medical records, and genetic and immunologic studies. The clinical spectrum narrowed down to Bacillus Calmette-Guerin (BCG) vaccine-related suppurative adenitis in all patients with one death, and recrudescence in two, histoplasmosis, and recurrence in one patient, extraintestinal salmonellosis in one child, and cutaneous vasculitis in another. In three patients, we established the homozygous Trp7Ter predicted loss-of-function inherited genotype and inferred it from the heterozygote parents of the fourth case. The Trp7Ter mutation maps to the predicted IL-12Rβ1 N-terminal signal peptide sequence. BCG- or phytohemagglutinin-blasts from the three patients have reduced cell-surface expression of IL-12Rβ1 with impaired production of IFN-γ and IL-17A. Screening of 227 unrelated healthy subjects from the same geographic region revealed one heterozygous genotype (allele frequency 0.0022) vs. one in over 841,883 public genome/exomes. We also show that the carriers bear European ancestry-informative alleles and share the extended CACCAGTCCGG IL12RB1 haplotype that occurs worldwide with a frequency of 8.4%. We conclude that the novel IL12RB1 N-terminal signal peptide stop-gain loss-of-function homozygous genotype confers IL-12Rβ1 deficiency with varying severity and early-onset age through diminished cell-surface expression of an impaired IL-12Rβ1 polypeptide. We firmly recommend attending to warning signs of IMD30 in children who are HIV-1 negative with a history of adverse effects to the BCG vaccine and presenting with recurrent Histoplasma spp. and extraintestinal Salmonella spp. infections.

The influence of chronic IL-6 exposure, in vivo, on rat Achilles tendon extracellular matrix

When compared to placebo, acetaminophen (APAP) reduces tendon stiffness and collagen cross-linking. APAP also enhances the exercise-induced increase in peritendinous levels of IL-6. Elevated levels of IL-6 are associated with tendinopathy, thus we hypothesized that chronic, elevated peritendinous IL-6 would alter tendon extracellular matrix (ECM). IL-6 (∼3000pgml−1) was injected (3dwk−1 for 8-wks) into the Achilles peritendinous region of male Wistar rats (n=16) with the opposite leg serving as a sham. Fractional synthesis rates (FSR) were determined using deuterium oxide. Collagen (hydroxyproline) and hydroxylysl pyridinoline (HP) cross-linking were analyzed by HPLC. ECM and IL-6 related genes were evaluated using qRT-PCR. Relative to sham, collagen (Col) 1a1 but not Col3a1 expression was suppressed (47%) in tendons exposed to IL-6 (p<0.05). Lysyl oxidase (LOX) and MMP-1 expression were also reduced (37%) in IL-6 treated tendons (p<0.05). Relative to sham the expression of MMP-2, -3, -9, and TIMP-1 were not altered by IL-6 treatment (p>0.05). Interleukin-6 receptor subunit beta precursor (IL6st) was lower (16%) in IL-6 treated tendons when compared to sham (p<0.05). Suppressor of cytokine signaling 3 (Socs3), signal transducer and activator of transcription 3 (STAT3), and protein inhibitor of activated STAT 1 (Pias1) were not altered by IL-6 exposure (p>0.05). Neither collagen nor cross-linking content were altered by IL-6 (p>0.05). Additionally, IL-6 treatment did not alter tendon FSR. Chronic treatment with physiologically relevant levels of IL-6 suppresses expression of Col1a1 and LOX while also altering expression of select MMPs but does not alter Achilles tendon collagen synthesis.

Mutations in Interleukin-10 Receptor and Clinical Phenotypes in Patients with Very Early Onset Inflammatory Bowel Disease: A Chinese VEO-IBD Collaboration Group Survey.

Interleukin-10 (IL10) signaling plays an important role in the pathogenesis of very early onset inflammatory bowel disease (VEO-IBD) in children. However, little is known about the role of the IL10 axis in children with VEO-IBD in China. The Chinese VEO-IBD Collaboration Group was created to collect clinical and genetic data from patients deficient in IL10 and the IL10 receptor. High-throughput sequencing was performed to identify mutations in these genes. We identified 32 compound heterozygous mutations and 9 homozygous mutations in IL10 receptor subunit alpha and 1 homozygous mutation in IL10 receptor subunit beta. Among these mutations, 10 novel mutations were identified, and 6 pathogenic mutations had been previously described. In patients with IL10 receptor subunit alpha mutations, c.301C>T (p.R101RW) and c.537 G>A (p.T179T) were the most common mutations. For 88.1% of the patients, the initial symptom was diarrhea, with a time of onset of 10.4 ± 8.0 days. Oral ulcers were the first symptom in 23.8% of the patients, with a time of onset of 9.7 ± 2.8 days. Extraintestinal manifestations included perianal abscesses (22/42), perianal fistulas (23/42), oral ulcers (20/42), and recurrent eczema (15/42). Twelve patients underwent enterostomy. These patients also had lower average Z scores in height-for-age and weight-for-age. Various treatment strategies were used, including fecal microbiota transplantation; however, only hematopoietic stem cell transplantation was efficacious. This study identified genotypes and phenotypes of Chinese VEO-IBD infants with IL10 receptor mutations. Our study expands the current knowledge on the involvement of the IL10 axis in patients with VEO-IBD.

Comprehensive chromosome screening and gene expression analysis from the same biopsy in human preimplantation embryos.

STUDY QUESTIONCan simultaneous comprehensive chromosome screening (CCS) and gene expression analysis be performed on the same biopsy of preimplantation human embryos?SUMMARY ANSWERFor the first time, CCS and reliable gene expression analysis have been performed on the same human preimplantation embryo biopsy.WHAT IS KNOWN ALREADYA single trophectoderm (TE) biopsy is routinely used for many IVF programs offering CCS for selection of only chromosomally normal embryos for transfer. Although the gene expression profiling of human preimplantation embryos has been described, to date no protocol allows for simultaneous CCS and gene expression profiling from a single TE biopsy.STUDY DESIGN, SIZE AND DURATIONThis is a proof of concept and validation study structured in two phases. In Phase 1, cell lines were subjected to a novel protocol for combined CCS and gene expression analysis so as to validate the accuracy and reliability of the proposed protocol. In Phase 2, 20 donated human blastocysts were biopsied and processed with the proposed protocol in order to obtain an accurate CCS result and characterize their gene expression profiles using the same starting material.PARTICIPANTS/MATERIALS, SETTING AND METHODA novel protocol coupling quantitative real-time PCR-based CCS and gene expression analysis using RT-PCR was designed for this study. Phase 1: six-cell aliquots of well-characterized fibroblast cell lines (GM00323, 46,XY and GM04435, 48,XY,+16,+21) were subjected to the proposed protocol. CCS results were compared with the known karyotypes for consistency, and gene expression levels were compared with levels of purified RNA from same cell lines for validation of reliable gene expression profiling. Phase 2: four biopsies were performed on 20 frozen human blastocysts previously diagnosed as trisomy 21 (10 embryos) and monosomy 21 (10 embryos) by CCS. All samples were processed with the proposed protocol and re-evaluated for concordance with the original CCS result. Their gene expression profiles were characterized and differential gene expression among embryos and early embryonic cell lineages was also evaluated.MAIN RESULTS AND THE ROLE OF CHANCECCS results from cell lines showed 100% consistency with their known karyotypes. ΔΔCt values of differential gene expression of four selected target genes from the cell lines GM4435 and GM0323 were comparable between six-cell aliquots and purified RNA (Collagen type I alpha-1 (COL1A1), P = 0.54; Fibroblast growth factor-5 (FGF5), P = 0.11; Laminin subunit beta-1 (LAMB1), P = 1.00 and Atlastin-1 (ATL1), P = 0.23). With respect to human blastocysts, 92% consistency was reported after comparing embryonic CCS results with previous diagnosis. A total of 30 genes from a human stem cell pluripotency panel were selected to evaluate gene expression in human embryos. Correlation coefficients of expression profiles from biopsies of the same embryo (r = 0.96 ± 0.03 (standard deviation), n = 45) were significantly higher than when biopsies from unrelated embryos were evaluated (r = 0.93 ± 0.03, n = 945) (P < 0.0001). Growth differentiation factor 3 (GDF3) was found to be significantly up-regulated in the inner cell mass (ICM), whereas Caudal type homebox protein-2 (CDX2), Laminin subunit alpha-1 (LAMA1) and DNA methyltransferase 3-beta (DNMT3B) showed down-regulation in ICM compared with TE. Trisomy 21 embryos showed significant up-regulation of markers of cell differentiation (Cadherin-5 (CDH5) and Laminin subunit gamma-1 (LAMC1)), whereas monosomy 21 blastocysts showed higher expression of genes reported to be expressed in undifferentiated cells (Gamma-Aminobutyric Acid Type-A Receptor Beta3 Subunit (GABRB3) and GDF3).LARGE SCALE DATAN/ALIMITATIONS, REASONS FOR CAUTIONGene expression profiles of chromosomally normal embryos were not assessed due to restrictive access to euploid embryos for research. Nonetheless, the profile of blastocysts with single aneuploidies was characterized and compared. Only 30 target genes were analyzed for gene expression in this study. Increasing the number of target genes will provide a more comprehensive transcriptomic signature and reveal potential pathways paramount for embryonic competence and correct development.WIDER IMPLICATIONS OF THE FINDINGSThis is the first time that CCS and gene expression analysis have been performed on the same human preimplantation embryo biopsy. Further optimization of this protocol with other CCS platforms and inclusion of more target genes will provide innumerable research and clinical applications, such as discovery of biomarkers for embryonic reproductive potential and characterization of the transcriptomic signatures of embryos, potentially allowing for further embryo selection prior to embryo transfer and therefore improving outcomes.STUDY FUNDING AND COMPETING INTERESTSThis study was funded by the Foundation for Embryonic Competence, Basking Ridge, NJ, USA. No conflicts of interests declared.

Abstract A64: Mast cell expressed FcεR beta subunit (MS4A2) is prognostic in lung adenocarcinoma

Abstract Background: The context of immune cell infiltrates within the tumor is associated with cancer prognosis. The type of immune cells and their activation state can provide mediators that either promote or inhibit tumor growth. A growing body of evidence suggest the presence of innate immune cells play a role in shaping early tumor growth. In attempts to gain insight into the immune networks that regulate tumorigenesis, we used genome wide expression datasets of patients with resected early stage non-small cell lung cancer (NSCLC) to identify immune-related genes associated with patient survival. Methods: Gene expression analysis was conducted on microarray datasets from 128 early-stage NSCLC adenocarcinoma resected tumor samples. Limiting analysis to immune-related genes, we identified a minimum gene set that is prognostic for lung adenocarcinoma and validated the gene signature in additional published microarray NSCLC datasets. Using pathway analysis and immunohistochemistry (IHC), we identified a role for mast cell-expressed IgE receptor beta subunit, MS4A2, in lung adenocarcinoma progression. Results: From an immune-related gene list, we identified a minimal gene signature that was able to separate patients into high or low-risk survival groups. This gene signature was shown to be prognostic for NSCLC adenocarcinoma in nine validation datasets (n=1264, hazard ratio 2.07, 95% confidence interval 1.69-2.53, p&amp;lt;0.0001). Pathway analysis of differentially expressed genes within high or low-risk survival subgroups reveals gene ontologies and immune signatures that are enriched for IgE receptor signaling and mast cells in patients with favourable prognosis. Univariate analysis shows that the IgE receptor complex genes, as well as mast cell specific protease, mast cell carboxypeptidase A, are highly prognostic for survival. We are currently using IHC of matched patient samples to validate receptor localization. Conclusion: By limiting gene expression analysis to immune-related genes, we identified a minimal gene set that indicate an important role for IgE receptor signaling and mast cells in favourable lung cancer prognosis. Our data highlights the importance of innate immune cells in shaping lung cancer development. Citation Format: Dalam Ly, Chang-Qi Zhu, Michael Cabanero, Ming-Sound Tsao, Li Zhang. Mast cell expressed FcεR beta subunit (MS4A2) is prognostic in lung adenocarcinoma. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2016 Oct 20-23; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2017;5(3 Suppl):Abstract nr A64.

Tumor Necrosis Factor Inhibitors May Improve Glycemic Control in Patients Rheumatoid Arthritis and Concomitant Type 2 Diabetes Mellitus

Insulin resistance is a key feature of obesity, metabolic syndrome, and Type 2 Diabetes Mellitus (T2DM). Inflammation and insulin resistance are closely linked with each other. I. Tumor necrosis factor-α (TNF-α) has been found to impair the insulin sensitivity and promote insulin resistance through multiple actions on the insulin sensitive tissues. Inflammatory cytokines such as TNF, Interleukin (IL)-6, IL-1 and IL-8 may inhibit insulin signaling.1 Hotamisligil et al2 did the pioneering work in 1993 confirming the link between TNF-α and insulin resistance in mice. Animal studies have confirmed that the TNF-α interferes with phosphorylation cascades of the insulin receptor beta subunit and insulin receptor substrate-1, thereby altering the transmembrane signaling that is essential for insulin action in various insulin sensitive tissues.3-5 Also TNF-α causes depletion of GLUT 4, the insulin sensitive glucose transporter in adipocytes and muscles.2,6,7 A intravenous administration of a recombinant TNF-α receptor antibody resulted in improvement in insulin sensitivity2 and dramatic reductions in plasma insulin, glucose, and non-esterified fatty acid levels5 in obese, as compared with lean rats

Protein and glycomic plasma markers for early detection of adenoma and colon cancer

ObjectiveTo discover and confirm blood-based colon cancer early-detection markers.DesignWe created a high-density antibody microarray to detect differences in protein levels in plasma from individuals diagnosed with colon cancer <3 years...

Distinctive expression of interleukin-23 receptor subunits on human Th17 and γδ T cells.

The interleukin-23 (IL-23) pathway, T helper 17 (Th17) cells and γδ T cells, which respond to IL-23, have major pro-inflammatory roles. We have used unique IL-23 receptor (IL-23R) subunit-specific monoclonal antibodies, X67 and X68, and IL-12 receptor beta-1 subunit (IL-12Rβ1) expression levels to evaluate the IL-23R complex on CD4 αβ TCR Th17 cells and on γδ T cells. Both IL-23R and IL-12Rβ1 subunits constitute the functional IL-23R. Expression of the IL-23R subunit by cultured Th17 cells was heterogeneous. Th17 cells expressed consistent high levels of the IL-12Rβ1 subunit, which appeared a better predictor of responsiveness to IL-23 than the expression of the IL-23R subunit. Moreover, sorting memory CD4 T cells by high IL-12Rβ1 expression selectively enriched cells committed to IL-17 production from the blood. IL-23R expression was also observed on freshly isolated and cultured γδ T cells and the cultured γδ T cells were not responsive to IL-23.

Prenatal testosterone exposure decreases colocalization of insulin receptors in kisspeptin/neurokinin B/dynorphin and agouti-related peptide neurons of the adult ewe.

Insulin serves as a link between the metabolic and reproductive systems, communicating energy availability to the hypothalamus and enabling reproductive mechanisms. Adult Suffolk ewes prenatally exposed to testosterone (T) display an array of reproductive and metabolic dysfunctions similar to those seen in women with polycystic ovarian syndrome (PCOS), including insulin resistance. Moreover, prenatal T treatment alters neuropeptide expression in KNDy (co-expressing kisspeptin, neurokinin B/dynorphin) and agouti-related peptide (AgRP) neurons in the arcuate nucleus, two populations that play key roles in the control of reproduction and metabolism, respectively. In this study, we determined whether prenatal T treatment also altered insulin receptors in KNDy and AgRP neurons, as well as in preoptic area (POA) kisspeptin, pro-opiomelanocortin (POMC), and gonadotropin-releasing hormone (GnRH) neurons of the adult sheep brain. Immunofluorescent detection of the beta subunit of insulin receptor (IRβ) revealed that KNDy, AgRP and POMC neurons, but not GnRH or POA kisspeptin neurons, colocalize IRβ in control females. Moreover, prenatal T treatment decreased the percentage of KNDy and AgRP neurons that colocalized IRβ, consistent with reduced insulin sensitivity. Administration of the anti-androgen drug, Flutamide, during prenatal T treatment, prevented the reduction in IRβ colocalization in AgRP, but not in KNDy neurons, suggesting that these effects are programmed by androgenic and oestrogenic actions, respectively. These findings provide novel insight into the effects of prenatal T treatment on hypothalamic insulin sensitivity and raise the possibility that decreased insulin receptors, specifically within KNDy and AgRP neurons, may contribute to the PCOS-like phenotype of this animal model.

Characterization of Receptor-Associated Protein Complex Assembly in Interleukin (IL)-2- and IL-15-Activated T-Cell Lines.

It remains a paradox that IL-2 and IL-15 can differentially modulate the immune response using the same signaling receptors. We have previously dissected the phosphotyrosine-driven signaling cascades triggered by both cytokines in Kit225 T-cells, unveiling subtle differences that may contribute to their functional dichotomy. In this study, we aimed to decipher the receptor complex assembly in IL-2- and IL-15-activated T-lymphocytes that is highly orchestrated by site-specific phosphorylation events. Comparing the cytokine-induced interactome of the interleukin receptor beta and gamma subunits shared by the two cytokines, we defined the components of the early IL-2 and IL-15 receptor-associated complex discovering novel constituents. Additionally, phosphopeptide-directed analysis allowed us to detect several cytokine-dependent and -independent phosphorylation events within the activated receptor complex including novel phosphorylated sites located in the cytoplasmic region of IL-2 receptor β subunit (IL-2Rβ). We proved that the distinct phosphorylations induced by the cytokines serve for recruiting different types of effectors to the initial receptor/ligand complex. Overall, our study sheds new light into the initial molecular events triggered by IL-2 and IL-15 and constitutes a further step toward a better understanding of the early signaling aspects of the two closely related cytokines in T-lymphocytes.

Modulation of the Expression of the GABAA Receptor β1 and β3 Subunits by Pretreatment with Quercetin in the KA Model of Epilepsy in Mice: -The Effect of Quercetin on GABAA Receptor Beta Subunits.

Objectives:Quercetin is a flavonoid and an important dietary constituent of fruits and vegetables. In recent years, several pharmacological activities of quercetin, such as its neuroprotective activity and, more specifically, its anti-convulsant effects in animal models of epilepsy, have been reported. This study evaluated the role of quercetin pretreatment on gene expression of γ-amino butyric acid type A (GABAA) receptor beta subunits in kainic acid (KA)-induced seizures in mice.Methods:The animals were divided into four groups: one saline group, one group in which seizures were induced by using KA (10 mg/kg) without quercetin pretreatment and two groups pretreated with quercetin (50 and 100 mg/kg) prior to seizures being induced by using KA. Next, the messenger ribonucleic acid (mRNA) levels of the GABAA receptor β subunits in the hippocampus of each animal were assessed at 2 hours and 7 days after KA administration. Quantitative real-time polymerase chain reaction (RT-PCR) assay was used to detect mRNA content in hippocampal tissues.Results:Pretreatments with quercetin at doses of 50 and 100 mg/kg prevented significant increases in the mRNA levels of the β1 and the β3 subunits of the GABAA receptor at 2 hours after KA injection. Pretreatment with quercetin (100 mg/kg) significantly inhibited β1 and β3 gene expression in the hippocampus at 7 days after KA injection. But, this inhibitory effect of quercetin at 50 mg/kg on the mRNA levels of the β3 subunit of the GABAA receptor was not observed at 7 days after KA administration.Conclusion:These results suggest that quercetin (100 mg/kg) modulates the expression of the GABAA receptor β1 and β3 subunits in the KA model of epilepsy, most likely to prevent compensatory responses. This may be related to the narrow therapeutic dose range for the anticonvulsant activities of quercetin.

Vitamin A status affects the Body Mass Gain and Hepatic Gene Expression in Rats fed a High‐Fat Diet

IntroductionVitamin A (VA) plays a role in variety of physiological functions from development to metabolic homeostasis. High‐fat (HF) diet has been shown to cause the development of metabolic diseases such as obesity and type II diabetes. We hypothesize that VA status may affect the gain of body mass in rats fed a HF diet and regulate the expression levels of genes involved in hepatic glucose and fatty acid metabolism.MethodsSprague Dawley (SD) rats at weaning were fed either a HF‐VA sufficient (HF‐VAS) or HF‐VA deficient (HF‐VAD) diet for 8 weeks. The HF‐VAS and HF‐VAD diets are isocaloric. Their body mass and food intake were recorded once a week. The expression levels of hepatic genes for glucose and fatty acid metabolism, and insulin signaling pathway were determined via immunoblotting.ResultsFrom week 6, body mass of HF‐VAD rats was significantly lower than that of VAS group. The total food intake was similar between VAD and VAS groups. The liver mass and white adipose mass of HF‐VAD group were also significant smaller than that of HF‐VAS group. The hepatic expression level of Cytochrome P450 26A1 protein in HF‐VAS group is higher than that in HF‐VAD group, suggesting reduced retinoic acid production. For glucose metabolism, the HF‐VAD rats had higher glycogen synthase 3α (GSK‐3α) and lower GSK‐3β expression levels than the HF‐VAS rats did. The expression levels of glucokinase and cytosolic form of phosphoenoylpyruvate carboxykinase in HF‐VAS group are higher than that in HF‐VAD group. Proteins levels of acetyl‐CoA carboxylase (ACC), phospho‐ACC, fatty acid synthase, and ATP citrate lyase in fatty acid synthesis, are similar between two groups. The levels of insulin receptor beta subunit and insulin receptor substrate 1 are increased in HF‐HF‐VAD rats, while that of insulin receptor substrate 2 are decreased. The expression level of mitogen‐activated protein kinase of HF‐VAD group was higher than that of HF‐VAS group.ConclusionsVA status reduces the body mass gain in rats fed a HF diet. In addition, the expression levels of genes for glucose metabolism and insulin signaling pathway are also altered by the development of VA deficiency in these rats. These results support our previous observations that VA status and its metabolism affect the hepatic expression levels of insulin‐regulated genes in rats fed the diets with normal fat content. More studies are needed to determine the underlying mechanisms for these changes.Support or Funding InformationInternal funding

Abstract B015: Antitumor activity of the CD122-biased immunostimulatory cytokine combined with immune checkpoint blockade requires innate and adaptive immunity

Abstract Background: While immune checkpoint blockade is a promising therapeutic approach, combination with agents that modulate complementary pathways may improve responses. Interleukin-2 (IL-2) immunotherapy leads to long-term responses in a small percentage of cancer patients, but systemic toxicity limits its use. In addition, IL-2 expands T regulatory cells, antagonizing antitumor immunity and resulting in a poorer clinical outcome. NKTR-214 is a novel CD122-biased immunostimulatory cytokine which combines biased activation of the IL-2R beta receptor subunit, greatly favoring activation of effector over regulatory T cells, with improved pharmacokinetics and tolerability compared to Proleukin in non-human models. Here we examine the efficacy and mechanism of NKTR-214 combined with anti-CTLA-4 in murine tumor models. Methods: Mice bearing subcutaneous EMT6 mammary tumors were treated with NKTR-214 q9d, murine anti-CTLA-4 or anti-PD-1 twice-weekly, or both in combination. Immune cell profiling was assessed by flow cytometry following treatment. CD8 or NK cells were depleted in vivo by serial anti-CD8 or anti-asialo-GM1 antibody injections, respectively. Antitumor memory and specificity was assessed in complete responders by challenging with EMT6 or CT26 colon carcinoma implants with no additional treatment. Results: While NKTR-214 and anti-CTLA-4 were not as efficacious individually, their combination was synergistic and well-tolerated with 83% of test animals tumor-free. Combination treatment increased NK and memory effector CD8 cells in both tumor and spleen. Antitumor immunity by the combination was durable and specific as 70% and 100% of mice remained tumor-free after challenge with a second and third EMT6 implant, but not after a subsequent CT26 implant. NKTR-214 combined with anti-PD-1 also proved synergistic with 40% of animals remaining tumor free following treatment. In vivo depletion of either CD8 effector or NK cells abrogated efficacy suggesting both contribute to the response. Conclusions: The mechanism of NKTR-214 antitumor immunity is complementary to checkpoint inhibition. Favorable pharmacokinetics of NKTR-214 allows sustained tumor exposure and dosing schedules commensurate with other therapies. This new therapeutic combination of NKTR-214 and checkpoint inhibition may similarly enable durable responses in humans. Citation Format: John L. Langowski, Seema S. Kantak, Rhoneil Pena, Yolanda Kirksey, Murali Addepalli, Steve Lee, Ute Hoch, Deborah H. Charych, Stephen K. Doberstein. Antitumor activity of the CD122-biased immunostimulatory cytokine combined with immune checkpoint blockade requires innate and adaptive immunity. [abstract]. In: Proceedings of the CRI-CIMT-EATI-AACR Inaugural International Cancer Immunotherapy Conference: Translating Science into Survival; September 16-19, 2015; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(1 Suppl):Abstract nr B015.

Abstract A95: NKTR-214, a novel CD122-biased immunostimulatory cytokine, does not cause hypotension in non-human primates despite greater cytokine exposure than achieved with high dose Interleukin-2 (HD-IL-2)

Abstract Background: IL-2 was the first cancer immunotherapy approved, producing durable responses in ∼10% of patients treated for metastatic melanoma and renal cancer. However, IL-2 has poor pharmacokinetics, is both an activator and suppressor of the immune system, and is associated with severe cardiovascular side effects of hypotension and vascular leak syndrome, limiting widespread use. NKTR-214 is a novel immunotherapeutic consisting of IL-2 conjugated at a defined region within the protein to 4-6 polyethylene glycol (PEG) chains. Once administered, PEG chains slowly release to generate active IL-2 conjugates biased towards activation of CD122, the IL-2 receptor beta subunit (IL-2Rβ), highly expressed on memory effector CD8+ T cells (CD8T). In the tumor microenvironment, NKTR-214 preferentially expands CD8T over regulatory T cells (Treg), thus tipping the balance in favor of immune activation with a CD8T/Treg &amp;gt;400. In syngeneic mouse tumor models, NKTR-214 demonstrated robust single agent efficacy, synergized with checkpoint inhibitor antibodies, and protected against tumor rechallenge. Here we describe the cardiovascular safety of NKTR-214 in monkeys. Methods: Cardiovascular function was assessed in telemeterized male monkeys after single IV bolus injections of 0.01, 0.03, and 0.1 mg/kg using a Latin square design with a 14-day washout period between doses. Telemetry recordings were collected from three critical 24 hr time periods post-dose. Arterial blood pressure was collected continuously, averaged every 5 min, and reported every 0.25 hour. Inflammatory cytokines (TNFα and IFNγ), immune system activation markers (sCD25, lymphocytes), and IL-2 conjugate exposure were analyzed by ELISA in blood collected by venipuncture at multiple timepoints post dose. Results: There were no ECG changes that were attributed to NKTR-214 treatment at doses up to 0.1 mg/kg/dose. sCD25 and total lymphocyte counts increased from baseline in a dose-responsive manner, producing a 24-fold and 4-fold increase in sCD25 and lymphocytes respectively at 0.1 mg/kg. Continuous monitoring of blood pressure for up to 104 hrs post-dose indicated the absence of hypotension at all dose levels. The inflammatory cytokines TNFα and IFNγ remained undetectable, suggesting no-associated cytokine release, despite the robust immune activation. Exposure to active IL-2 conjugate was high and sustained, exceeding that achieved with the HD-IL-2 regimen in patients. Conclusion: The results support the safety and tolerability of NKTR-214 at doses expected to activate the immune system and support the development of this agent for patients with intractable cancers. Citation Format: Chunmei Ji, Ute Hoch, Deborah H. Charych, Theresa D. Sweeney. NKTR-214, a novel CD122-biased immunostimulatory cytokine, does not cause hypotension in non-human primates despite greater cytokine exposure than achieved with high dose Interleukin-2 (HD-IL-2). [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr A95.

Abstract 46: Cellular Cholesterol Homeostasis is Altered in Murine Models of Rheumatoid Arthritis and is Linked to Enhanced Myelopoiesis

Rheumatoid arthritis (RA) is associated with a ~2-fold elevated risk of morbidity and mortality from atherosclerotic cardiovascular disease (CVD) compared with the general population. Atherosclerosis in RA patients tends to be more aggressive and therefore more challenging to treat. Identifying CVD in these patients is difficult as traditional CVD risk factors, such as changes in plasma lipid profiles (i.e. elevated LDL, decreased HDL) are not always observed, underscoring the need for better understanding of the reasons contributing to the enhanced atherosclerosis in RA patients. People with RA often have monocytosis and neutrophilia, which we hypothesize to plays causal roles in atherosclerosis. Two mouse models of RA were used, K/BxN serum transfer and collagen induced arthritis (CIA). Flow cytometry was used to quantify the abundance of leukocyte and stem cell subsets. BODIPY-cholesterol was employed to determine the membrane cholesterol status of the various cells. Prominent monocytosis and neutrophilia due to an expansion and increased proliferation of the haematopoietic stem and multipotential progenitor cells (HSPCs) in the bone marrow (BM) was observed in both models of RA. There was also an increase in the mobilisation of HSPCs into the circulation, which homed to the spleen, resulting in extramedullary haematopoiesis. Interestingly, key cholesterol efflux genes, Abca1, Abcg1 and Apoe were down regulated in the BM HSPCs isolated from the K/BxN mice, resulting in increased cell membrane cholesterol levels. We also observed an increase in the expression of the common beta subunit of the interlukin-3 receptor on the HSPCs and the M-CSF receptor in myeloid progenitor cells, likely explaining their increased proliferation and skewing to the myeloid lineage. Moreover, blood monocytes and neutrophils had increased membrane cholesterol content, independent of changes in plasma cholesterol levels. These data suggest that while plasma cholesterol levels may not be increased in RA, cellular cholesterol homeostasis might be increased. We hypothesize that this, together with enhanced monocyte production, underlies the increased risk of CVD in RA.