Immune Response-related Gene Expression Research Articles

Granular analysis of individual immune-related gene expression in a randomized phase I/II study of the oncolytic adenovirus, ONCOS-102, in combination with pemetrexed/cisplatin (P/C) in patients (pts) with unresectable malignant pleural mesothelioma (MPM).

e20536 Background: ONCOS-102 is an oncolytic adenovirus expressing granulocyte-macrophage colony stimulating factor (GM-CSF; Ad5/3-D24-GMCSF) that stimulates immune responses and modulates the tumor microenvironment, both as a monotherapy and in combination with programmed cell death protein 1 (PD-1) blockade. We present granular analyses of individual immune-related gene expression in response to ONCOS-102 in a randomized phase I/II study in MPM. Methods: Following a safety run-in (n = 6), 25 pts were randomized to receive ONCOS-102 (3 x 1011 virus particles in 2.5 mL) intratumorally under CT or US guidance on Days 1, 4, 8, 36, 78 and 120, plus six cycles of P/C starting on Day 22 (ONCOS+P/C), or six cycles of P/C only (control). Both treatment-naïve (1L) and previously treated patients were enrolled. Survival was followed up to 30 months. Tumor biopsies were collected at baseline and Day 36, and immunofluorescence for immune cell subsets, RNASeq and qPCR were performed. Results: In the pooled population (n = 31), median overall survival (OS) was 16.6 vs 18.3 months for ONCOS-102 + P/C vs control arms ( P> 0.05). In 1L patients (n = 17) median OS was 20.3 vs 13.5 months ( P> 0.05), and 30-month OS rate was 34.1% vs 0. A significant increase in tumour-infiltrating immune cells was observed (baseline to Day 36) with ONCOS-102 + P/C (n = 20) but not control (n = 11) using immunofluorescence and transcriptome analysis. CD8+, GranzymeB+ CD8+ cells, and CD8+:regulatory T-cell and M1:M2 macrophage ratios increased in ONCOS-102-treated pts (P≤0.016). A difference in tumor immune-infiltration was observed in ONCOS-102 + P/C pts stratified by survival at Month 18: while deregulation of complement pathways and inflammatory response was seen at baseline, at Day 36 adaptive immune response and T-cell activation were observed in pts who were alive but not in those deceased. Additionally, GM-CSF protein, likely derived from ONCOS-102, was transiently present in higher concentration at Days 4 and 8 in 18-month survivors vs deceased pts. Based on a previously described predictive inflammatory gene signature for treatment with checkpoint inhibitors in mesothelioma, a binomial model assessing baseline transcriptome levels demonstrated 89% accuracy in predicting 18-month survival. Conclusions: ONCOS-102 caused increased T-cell infiltration and upregulated immune response-related gene expression in tumor lesions. A trend for prolonged OS was observed in the 1L setting, only. Consistent with observations in PD-1 resistant melanoma, ONCOS-102-induced immune response in the tumor is hallmarked by a high prevalence of cytotoxic T-cells and depolarization of macrophages, which may support addition of checkpoint inhibitors to the combination of ONCOS-102 plus P/C in MPM. Clinical trial information: NCT02879669 .

Transcriptome-based network analysis related to M2-like tumor-associated macrophage infiltration identified VARS1 as a potential target for improving melanoma immunotherapy efficacy

RationaleThe M2-like tumor-associated macrophages (TAMs) are independent prognostic factors in melanoma.MethodsWe performed weighted gene co-expression network analysis (WGCNA) to identify the module most correlated with M2-like TAMs. The Cancer Genome Atlas (TCGA) patients were classified into two clusters that differed based on prognosis and biological function, with consensus clustering. A prognostic model was established based on the differentially expressed genes (DEGs) of the two clusters. We investigated the difference in immune cell infiltration and immune response-related gene expression between the high and low risk score groups.ResultsThe risk score was defined as an independent prognostic value in melanoma. VARS1 was a hub gene in the M2-like macrophage-associated WGCNA module that the DepMap portal demonstrated was necessary for melanoma growth. Overexpressing VARS1 in vitro increased melanoma cell migration and invasion, while downregulating VARS1 had the opposite result. VARS1 overexpression promoted M2 macrophage polarization and increased TGF-β1 concentrations in tumor cell supernatant in vitro. VARS1 expression was inversely correlated with immune-related signaling pathways and the expression of several immune checkpoint genes. In addition, the VARS1 expression level helped predict the response to anti-PD-1 immunotherapy. Pan-cancer analysis demonstrated that VARS1 expression negatively correlated with CD8 T cell infiltration and the immune response-related pathways in most cancers.ConclusionWe established an M2-like TAM-related prognostic model for melanoma and explored the role of VARS1 in melanoma progression, M2 macrophage polarization, and the development of immunotherapy resistance.

Effects of hyperosmotic stress on the intestinal microbiota, transcriptome, and immune function of mandarin fish (Siniperca chuatsi)

As a specific ecological factor of the aquatic environment, salinity can significantly affect intestinal and intestinal microbial homeostasis, which play essential roles in maintaining host health and antibacterial function. To evaluate the effects of saline-alkali water on host health and immune response, in this study, the effects of hyperosmotic stress on intestinal functions, including the intestinal microecosystem, intestinal immune response-related gene expression and intestinal antioxidant capacity, were investigated in mandarin fish (Siniperca chuatsi) after 45 days of exposure to freshwater (0 ppt salinity) or 3 ppt salinity, 6 ppt salinity, 9 ppt salinity, and 12 ppt salinity water environments. The results revealed that the intestinal microbial community composition could be altered and reshaped by hyperosmotic stress. Notably, the relative abundances of the probiotic bacterium Lactobacillus, the pathogenic bacterium Vibrio, and the metabolism-related bacteria Cetobacterium and Gluconobacter in the 6 ppt salinity group was higher than those in other groups, whereas no significant difference was observed among five groups. The analysis of intestinal transcriptome revealed several differentially expressed genes (DEGs) associated with several immune-related DEGs related to proinflammatory cytokines, anti-inflammatory cytokines, antioxidant defense, antimicrobial effects, and cell apoptosis. According to an analysis of intestinal physiological properties, the activity of alkaline phosphatase (AKP) and acid phosphatase (ACP) was significantly increased by hyperosmotic stress, especially in the 6 ppt salinity group. The present study provides essential insights into the influences of saline-alkali water on the health status and immune functions of mandarin fish; additionally, it provides an important foundation for further studies on the effects of complex relationship between intestinal flora and complex host environment on host disease resistance.

Enhanced Programmed Cell Death Protein 1/Programmed Cell Death Ligand 1 Expression Induced by Severe Influenza A Virus Infection Impairs Host's Antiviral Response.

Emerging research shows that the Programmed Cell Death Protein 1/Programmed Cell Death Ligand 1(PD-1/PD-L1) pathway modulates the antiviral response following influenza A virus (IAV) infection, and there is a need to understand further the role of the PD-1/PD-L1 signaling pathway in IAV infection. BALB/c mice were infected with different types of IAV to establish models of varying degrees of infection (mild and severe). The mice were pretreated with or without a PD-1 antagonist to evaluate the role of the PD-1/PD-L1 pathway in IAV infection. The general activity, degree of weight change, viral titer, pathological damage, protein expression, transcriptome level, and cytokine expression were evaluated in the mice. IAV infection, especially severe infection, induced expression of PD-1 and PD-L1 in the lungs and spleen of the mice at 6 days postinfection. Moreover, the expression level was positively correlated with the degree of pathological damage in the lung. PD-1 antagonists can alleviate weight loss in severely infected mice, reduce the viral load and pathological damage, enhance immune response-related gene expression, and induce the most robust responses of interferon-gamma without inducing an obvious Th1/Th17 response. The PD-1/PD-L1 signaling pathway induced by severe IAV infection seriously impairs the host's antiviral response; thus, blocking this signaling pathway may promote IAV recovery.

Transcriptome analysis in rhesus macaques infected with hepatitis E virus genotype 1/3 infections and genotype 1 re-infection.

Hepatitis E virus (HEV) genotype 1 (gt1) and gt3 infections have distinct epidemiologic characteristics and genotype-specific molecular mechanisms of pathogenesis are not well characterized. Previously, we showed differences in immune response-related gene expression profiles of HEV gt1 and gt3 infections using qPCR. We hypothesize that HEV gt1 and gt3 infections induce transcriptome modifications contributing to disease pathogenesis. RNAseq analysis was performed using liver biopsy samples of naïve (baseline), HEV gt1, or gt3-infected rhesus macaques, and nine anti-HEV positive rhesus macaques re-inoculated with HEV gt1. All 10 primary HEV gt1/gt3 infected animals exhibited the typical course of acute viral hepatitis and cleared the infection between 27 to 67 days after inoculation. Viremic stages of HEV infection were defined as early, peak, and decline based on HEV RNA titers in daily stool specimens. During early, peak, and decline phases of infection, HEV gt1 induced 415, 417, and 1769 differentially expressed genes, respectively, and 310, 678, and 388 genes were differentially expressed by HEV gt3, respectively (fold change ≥ 2.0, p-value ≤ 0.05). In the HEV gt1 infection, genes related to metabolic pathways were differentially expressed during the three phases of infection. In contrast, oxidative reduction (early phase), immune responses (peak phase), and T cell cytokine production (decline phase) were found to be regulated during HEV gt3 infection. In addition, FoxO and MAPK signaling pathways were differentially regulated in re-infected and protected animals against HEV gt1 reinfection, respectively. Significant differences of hepatic gene regulation exist between HEV gt1 and gt3 infections. These findings reveal a new link between molecular pathogenesis and epidemiological characteristics seen in HEV gt1 and gt3 infections.

Comprehensive Analysis of the Safety Profile of a Single-Stranded RNA Nano-Structure Adjuvant.

Adjuvants enhance the efficacy of vaccines by stimulating immune response-related gene expression and pathways. Although some adjuvants have been approved for commercial use in human vaccines (e.g., Alum, MF59, and AS03), they might elicit adverse side effects, such as autoimmune diseases. Recently, we developed a novel single-stranded RNA (ssRNA) nano-structure adjuvant, which can stimulate both Th1 and Th2 responses. In this study, we evaluated the safety and toxicological profiles of this ssRNA nano-structure adjuvant in vitro and in vivo. Mice were intramuscularly immunized with the ssRNA nano-structure adjuvant three times, once every 2 weeks. The results indicate no significant differences in hematological and serum biochemistry parameters between the ssRNA-treated groups and the control group. From a histopathological perspective, no evidence of tissue damage was found in any group. The levels of IgE and anti-nuclear antibodies, which are markers of autoimmune disease, were not different between the ssRNA-treated groups and the control group. The findings of this study suggest that the ssRNA nano-structure can be used as a safe adjuvant to increase vaccine efficacies.

Lymph node-fibroblastic reticular cells regulate differentiation of CD4+ T cells through CD25

Abstract Lymph node-fibroblastic reticular cells (LN-FRCs) provide functional structure to LNs and play important roles in interactions between T cells and antigen-presenting cells. However, the direct impacts of LN-FRCs on naïve CD4+ T cell differentiation have not been explored. Here, we show that FRCs in T cell zones of LNs express CD25 (IL-2 receptor α chain (IL-2Rα) of the IL-2R hetero-trimer). LN-FRCs trans-presented IL-2 to naïve CD4+ T cells through CD25, thereby facilitating early IL-2-mediated signaling. CD25-deficient LN-FRCs attenuated STAT5 phosphorylation of naïve CD4+ T cells during CD4+ T cell lineage differentiation, promoted T helper 17 (Th17) cell differentiation, and induced Th17 immune response-related gene expression. In an experimental autoimmune encephalomyelitis (EAE) disease model, Th17 cell-mediated EAE severity was elevated in mice lacking CD25 in LN-FRCs. Therefore, our results suggest that CD25 expression in LN-FRCs regulates CD4+ T cell differentiation by modulating early IL-2 signaling of neighboring naive CD4+ T cells, influencing immune responses.

TUSC2 Immunogene Therapy Synergizes with Anti-PD-1 through Enhanced Proliferation and Infiltration of Natural Killer Cells in Syngeneic Kras-Mutant Mouse Lung Cancer Models.

Expression of the multikinase inhibitor encoded by the tumor suppressor gene TUSC2 (also known as FUS1) is lost or decreased in non-small cell lung carcinoma (NSCLC). TUSC2 delivered systemically by nanovesicles has mediated tumor regression in clinical trials. Because of the role of TUSC2 in regulating immune cells, we assessed TUSC2 efficacy on antitumor immune responses alone and in combination with anti-PD-1 in two Kras-mutant syngeneic mouse lung cancer models. TUSC2 alone significantly reduced tumor growth and prolonged survival compared with anti-PD-1. When combined, this effect was significantly enhanced, and correlated with a pronounced increases in circulating and splenic natural killer (NK) cells and CD8+ T cells, and a decrease in regulatory T cells (Tregs), myeloid-derived suppressor cells (MDSCs), and T-cell checkpoint receptors PD-1, CTLA-4, and TIM-3. TUSC2 combined with anti-PD-1 induced tumor infiltrating more than NK and CD8+ T cells and fewer MDSCs and Tregs than each agent alone, both in subcutaneous tumor and in lung metastases. NK-cell depletion abrogated the antitumor effect and Th1-mediated immune response of this combination, indicating that NK cells mediate TUSC2/anti-PD-1 synergy. Release of IL15 and IL18 cytokines and expression of the IL15Rα chain and IL18R1 were associated with NK-cell activation by TUSC2. Immune response-related gene expression in the tumor microenvironment was altered by combination treatment. These data provide a rationale for immunogene therapy combined with immune checkpoint blockade in the treatment of NSCLC. Cancer Immunol Res; 6(2); 163-77. ©2018 AACR.

TUSC2 Enhances Sensitivity to Anti-PD1 in Kras Mutant Syngeneic Mouse Lung Cancer Through NK Cells

Expression of the tumor suppressor gene TUSC2, a specific multikinase inhibitor, is lost or decreased in several cancer types, including NSCLC. We assessed TUSC2 effects on antitumor activity and immune response alone and in combination with anti-PD1 in two Kras mutant syngeneic mouse lung cancer models. TUSC2 alone significantly reduced tumor growth and survival compared with anti-PD1. When combined, this effect was significantly enhanced. Immune profile analysis showed a pronounced increase of circulating and splenic NK cells, an increase of CD8+ T lymphocytes, and a decrease in the levels of Tregs, MDSCs, and T cell checkpoint receptors PD1, CTLA-4, and TIM-3. TUSC2 alone increased serum levels of IFNɣ, IL15 and IL18. Immunohistochemistry revealed higher tumor infiltration of activated NK and CD8+ T cells by TUSC2 + anti-PD1 than each agent alone. NK depletion abrogated the antitumor effect of this combination, indicating that NK cells are important mediators of TUSC2/anti-PD1 synergy. NanoString analysis showed that this combination significantly altered immune response related gene expression in the tumor microenvironment. These preclinical data provide a rationale for immunogene therapy combined with immune checkpoint inhibitors in the treatment of lung NSCLC.

Improvement of Therapeutic Efficacy of Oral Immunotherapy in Combination with Regulatory T Cell-Inducer Kakkonto in a Murine Food Allergy Model.

Oral immunotherapy (OIT) has been considered a promising approach for food allergies (FAs). However, the current OIT strategy is limited in terms of the long-term efficacy and safety. We have previously demonstrated that kakkonto, a traditional Japanese herbal medicine, suppresses the occurrence of allergic symptoms in a murine model of ovalbumin (OVA)-induced FA, which is attributed to the induction of the Foxp3+ CD4+ regulatory T cells. In this study, we established an OIT model using the FA mice with already established allergic symptoms and determined whether kakkonto could improve the efficacy of OIT. The OIT method consisted of initially administrating a very small amount of OVA and slowly increasing the amount. Allergic symptoms decreased in the OIT-treated FA mice. OIT significantly downregulated Th2 immune response-related gene expression in the FA mouse colon, and decreased the level of mouse mast cell protease-1, a marker of mast cell degranulation in the FA mouse plasma. Moreover, the concomitant use of kakkonto significantly enhanced the effectiveness of OIT on the allergic symptoms, and the combination therapy further suppressed the Th2 immune responses and the mast cell degranulation. In addition, OIT significantly increased the population of Foxp3+ CD4+ regulatory T cells in the FA mouse colon, and this population was further increased by OIT in combination with kakkonto. Furthermore, the combined therapy with kakkonto reduced the expression of RA-degrading enzyme CYP26B1 mRNA in the FA mouse colon. These findings indicated that the combination of OIT with kakkonto represents a promising approach for FA treatment.

The role of HPV RNA transcription, immune response-related gene expression and disruptive TP53 mutations in diagnostic and prognostic profiling of head and neck cancer.

Stratification of head and neck squamous cell carcinomas (HNSCC) based on HPV16 DNA and RNA status, gene expression patterns, and mutated candidate genes may facilitate patient treatment decision. We characterize head and neck squamous cell carcinomas (HNSCC) with different HPV16 DNA and RNA (E6*I) status from 290 consecutively recruited patients by gene expression profiling and targeted sequencing of 50 genes. We show that tumors with transcriptionally inactive HPV16 (DNA+ RNA-) are similar to HPV-negative (DNA-) tumors regarding gene expression and frequency of TP53 mutations (47%, 8/17 and 43%, 72/167, respectively). We also find that an immune response-related gene expression cluster is associated with lymph node metastasis, independent of HPV16 status and that disruptive TP53 mutations are associated with lymph node metastasis in HPV16 DNA- tumors. We validate each of these associations in another large data set. Four gene expression clusters which we identify differ moderately but significantly in overall survival. Our findings underscore the importance of measuring the HPV16 RNA (E6*I) and TP53-mutation status for patient stratification and identify associations of an immune response-related gene expression cluster and TP53 mutations with lymph node metastasis in HNSCC.

Differential Level of Host Gene Expression Associated with Nucleopolyhedrovirus Infection in Silkworm Races of Bombyx mori

The variation in the level of immune response related gene expression in silkworm, Bombyx mori following infection with Bombyx mori nucleopolyhedrovirus (BmNPV) was analyzed at different time intervals. The occlusion bodies of BmNPV orally inoculated to the two most divergent silkworm races viz., Sarupat (resistant to BmNPV infection) and CSR2 (susceptible to BmNPV infection) were subjected to oral BmNPV inoculation. The expression profile of gp 41 gene of BmNPV in the Sarupat and CSR2 races revealed that the virus could invade the midguts of both susceptible and resistant races. However, its multiplication was significantly less in the midgut of resistant race, while, in the susceptible race, the viral multiplication reached maximum level within 12 h. These findings indicate that potential host genes are involved in the inhibition of viral multiplication within larval midgut. The immune response genes arylphorin, cathepsin B, gloverin, lebocin, serpin, Hsp 19.9, Hsp 20.1, Hsp 20.4, Hsp 20.8, Hsp 21.4, Hsp 23.7, Hsp 40, Hsp 70, Hsp90 revealed differential level of expression on NPV infection. The gloverin, serpin, Hsp 23.7 and Hsp 40 genes are significantly up-regulated in the resistant race after NPV infection. The early up-regulation of these genes suggests that these genes could play an important role in baculovirus resistance in the silkworm, B. mori.

Immune response-related gene expression profile of a novel molluscan IκB protein member from Manila clam (Ruditapes philippinarum)

Mollusks lack an adaptive immune system and rely solely on the innate immune response. The nuclear factor-kappa B (NF-κB) signaling pathway is one of the most important components of the innate immune system and its activity is regulated by physical interaction with the inhibitor of NF-κB (IκB) protein. The manila clam, Ruditapes philippinarum (Rp), is a key species of the world's aquaculture industry, and recent pathogenic threats, such as the Gram-negative lipopolysaccharide (LPS)-expressing Vibrio tapetis bacteria, have produced severe adverse economic impacts. Here, we describe identification, characterization and immune responses of novel IκB (Rp-IκB) in the manila clam. The Rp-IκB cDNA is comprised of a 1,032bp open reading frame, which encodes 343 amino acid residues and has a predicted molecular mass of 38kDa. The Rp-IκB protein exhibits typical structural features of IκB family members, including the IκB degradation motif, PEST sequence, and six ankyrin repeats. Phylogenetic analysis showed that manila clam and other known molluscan IκB proteins grouped together in the invertebrate cluster. Analysis of the tissue expression distribution revealed that Rp-IκB was ubiquitously expressed. However, immune challenge with V. tapetis and purified LPS endotoxin induced significant up-regulation of Rp-IκB expression in gill and hemocytes. These results indicated that Rp-IκB may play an important role in manila clam defense against bacterial infection.

Increased Microglia/Macrophage Gene Expression in a Subset of Adult and Pediatric Astrocytomas

Glioblastoma (GBM) is a highly malignant brain tumor with a dismal prognosis. Gene expression profiling of GBM has revealed clinically relevant tumor subtypes, and this provides exciting opportunities to better understand disease pathogenesis. Results from an increasing number of studies demonstrate a role for the immune response in cancer progression, yet it is unclear how the immune response differs across tumor subtypes and how it affects outcome. Utilizing gene expression data from The Cancer Genome Atlas Project and the Gene Expression Omnibus database, we demonstrate an enrichment of immune response-related gene expression in the mesenchymal subtype of adult GBM (n = 173) and pediatric high-grade gliomas (n = 53). In an independent cohort of pediatric astrocytomas (n = 24) from UCSF, we stratified tumors into subtypes and confirmed these findings. Using novel immune cell-specific gene signatures we demonstrate selective enrichment of microglia/macrophage-related genes in adult and pediatric GBM tumors of the mesenchymal subtype. Furthermore, immunostaining of adult GBM tumors showed significantly higher cell numbers of microglia/macrophages in mesenchymal versus non-mesenchymal tumors (p = 0.04). Interestingly, adult GBM tumors with the shortest survival had significant enrichment of microglia/macrophage-related genes but this was not true for pediatric GBMs. Consistent with an association with poor outcome, immune response-related genes were highly represented in an adult poor prognosis gene signature, with the expression of genes related to macrophage recruitment and activation being most strongly associated with survival (p<0.05) using CoxBoost multivariate modeling. Using a microglia/macrophage high gene signature derived from quantification of tumor-infiltrating cells in adult GBM, we identified enrichment of genes characteristic of CD4 T cells, granulocytes, and microglia/macrophages (n = 573). These studies support a role for the immune response, particularly the microglia/macrophage response, in the biology of an important subset of GBM. Identification of this subset may be important for future therapeutic stratification.

Worsening of Obesity and Metabolic Status Yields Similar Molecular Adaptations in Human Subcutaneous and Visceral Adipose Tissue: Decreased Metabolism and Increased Immune Response

Context: It is not known whether biological differences reported between sc adipose tissue (SAT) and visceral adipose tissue (VAT) depots underlie the pathogenicity of visceral fat. Objective: We compared SAT and VAT gene expression according to obesity, visceral fat accumulation, insulin resistance, and presence of the metabolic syndrome. Design: Subjects were assigned into four groups (lean, overweight, obese, and obese with metabolic syndrome). Setting: Subjects were recruited at a university hospital. Patients: Thirty-two women were included. Main Outcome Measures: Anthropometric measurements, euglycemic-hyperinsulinemic clamps, blood analyses, and computed tomography scans were performed, and paired samples of SAT and VAT were obtained for DNA microarray-based gene expression profiling. Results: Considering the two fat depots together, 1125 genes were more and 1025 genes were less expressed in lean compared with metabolic syndrome subjects. Functional annotation clustering showed, from lean to metabolic syndrome subjects, progressive down-regulation of metabolic pathways including branched-chain amino acid, fatty acid, carbohydrate, and mitochondrial energy metabolism and up-regulation of immune response genes involved in toll-like receptor, TNF, nuclear factor-κB, and apoptosis pathways. Metabolism and immune response genes showed an opposite correlation with fat mass, fat distribution, or insulin resistance indices. These associations were similar in SAT and VAT, although about 1000 genes showed differential expression between SAT and VAT. Conclusions: The increase in adiposity and the worsening of metabolic status are associated with a coordinated down-regulation of metabolism-related and up-regulation of immune response-related gene expression. Molecular adaptations in SAT prove as discriminating as those in VAT.

Worsening of Obesity and Metabolic Status Yields Similar Molecular Adaptations in Human Subcutaneous and Visceral Adipose Tissue: Decreased Metabolism and Increased Immune Response

It is not known whether biological differences reported between sc adipose tissue (SAT) and visceral adipose tissue (VAT) depots underlie the pathogenicity of visceral fat. We compared SAT and VAT gene expression according to obesity, visceral fat accumulation, insulin resistance, and presence of the metabolic syndrome. Subjects were assigned into four groups (lean, overweight, obese, and obese with metabolic syndrome). Subjects were recruited at a university hospital. Thirty-two women were included. Anthropometric measurements, euglycemic-hyperinsulinemic clamps, blood analyses, and computed tomography scans were performed, and paired samples of SAT and VAT were obtained for DNA microarray-based gene expression profiling. Considering the two fat depots together, 1125 genes were more and 1025 genes were less expressed in lean compared with metabolic syndrome subjects. Functional annotation clustering showed, from lean to metabolic syndrome subjects, progressive down-regulation of metabolic pathways including branched-chain amino acid, fatty acid, carbohydrate, and mitochondrial energy metabolism and up-regulation of immune response genes involved in toll-like receptor, TNF, nuclear factor-κB, and apoptosis pathways. Metabolism and immune response genes showed an opposite correlation with fat mass, fat distribution, or insulin resistance indices. These associations were similar in SAT and VAT, although about 1000 genes showed differential expression between SAT and VAT. The increase in adiposity and the worsening of metabolic status are associated with a coordinated down-regulation of metabolism-related and up-regulation of immune response-related gene expression. Molecular adaptations in SAT prove as discriminating as those in VAT.

Structural organization and expression of the gaegurin 4 gene of Rana rugosa

Gaegurin 4 (GGN4) is a member of the antimicrobial peptide subfamily isolated from the skin of Rana rugosa. We cloned gDNA encoding GGN4 to study its gene organization and regulation of expression. The GGN4 gene occurs in single copy in the R. rugosa genome and contains a single intron of about 3.4 kb. The transcription start site is located 68 bases upstream of the translation initiation codon. The GGN4 gene was expressed both in Xenopus kidney epithelial cells (A6) and in Xenopus oocytes using the chloramphenicol acetyltransferase reporter gene system. The 5′ flanking region of the GGN4 gene contains a dl binding site that is known to regulate acute phase immune response related gene expression in mammals and insects. The dl protein bound specifically to the GGN4 gene promoter region. Mutants that serially delete the 5′ flanking region show that removal of the dl binding site inhibited GGN4 gene expression in both A6 cells and Xenopus oocytes. From these results, we propose that expression of the GGN4 gene may be regulated by the region containing the dl element which plays a key role in the regulation of antimicrobial peptide genes in Drosophila and mammals.