A Chromogenic In Situ Hybridization (CISH) Assay for Detection of HBV RNA, DNA, and cccDNA in Liver Tissue.

Hepatitis B virus (HBV) developed highly intricates mechanisms exploiting host resources for its multiplication within a constrained genetic coding capacity. With the aid of a series of classical analytical methods such as ultrafiltration, and Southern and Northern blots, a general framework of HBV life cycle has been established. However, this picture still lacks many key histological contexts which involves pathophysiological changes of hepatocytes, non-parenchymal cells, infiltrated leukocytes, and associated extracellular matrix. Here, we describe a CISH protocol modified from the ViewRNA assay that allows direct visualization of HBV RNA, DNA, and cccDNA in liver tissue of chronic hepatitis B patients. By coupling it with immunohistochemistry and other histological stains, much richer information regarding the HBV-induced pathological changes can be harvested.

Chromogenic LMO2 mRNA ISH Expression Correlates with LMO2 Protein and Gene Expression and Captures Their Survival Impact in Diffuse Large B-Cell Lymphoma, NOS.

BACKGROUND: LMO2 is a relevant gene involved in B-cell ontogeny and a survival predictor of aggressive large B-cell lymphomas (aLBCL). Most studies assessing LMO2 mRNA expression have relied on microarray platforms or qRT-PCR methods, overlooking tissue morphology. In this study, we evaluate LMO2 RNA expression by chromogenic in situ hybridization (CISH) in normal tissue and in a series of 82 aLBCL. METHODS: LMO2 CISH was performed in formalin-fixed paraffin-embedded tissues, scored by three different methods, and correlated with a transcriptome panel. RESULTS: We obtained statistically significant results correlating the methods of evaluation with LMO2 protein expression and gene expression results. Normal tonsil tissue showed high levels of LMO2, particularly within the light zone of the germinal center. Conversely, in aLBCL, a notable reduction in LMO2 expression was noted, remarkably in cases carrying MYC rearrangements. Furthermore, significant results were obtained through overall survival and Cox regression survival analysis, incorporating International Prognostic Index data alongside LMO2 expression levels. CONCLUSIONS: We show a reliable method to identify LMO2 mRNA expression by CISH, effectively capturing many of the reported biologic features of LMO2.

Enhancing the utility of chromosome 6 and 8 testing in uveal melanoma biopsies.

BACKGROUND: The aim of this study was to evaluate the significance of testing the gain of chromosome 8 and the gain of chromosome 6 as prognostic markers in histopathological samples of enucleated eyes in with uveal melanoma. METHODS: This is a retrospective study of 54 enucleated eyes. The status of chromosomes 3, 8 and 6 was tested by CISH, and FISH was used in a few samples. A follow-up for the detection of metastases was conducted in all patients. The statistical significance of chromosomal abnormalities as a prognostic factor for the development of metastases was determined. RESULTS: The study group consists of 54 patients (average age 63 years), 28 men (51.9%) Monosomy 3 together with gain of chromosome 8 was found in 10 samples (18.5%). Both chromosomal abnormalities were detected in 6 (11%) patients. No chromosomal abnormality in 3 or 8 was detected in 21 (38.9%) patients. Abnormalities of chromosome 6 were present in 6 (11%) patients. Progression free survival after 5 years was 33.3% (95% CI 0.0; 83.3) in these patients. CONCLUSIONS: Our findings indicate a correlation between progression-free survival and the presence of changes in chromosome 3 and e 8 in uveal melanomas. The results underline the necessity of testing for both chromosomal aberrations.

High-Risk HPV CISH Detection in Cervical Biopsies with Weak and/or Focal p16 Immunohistochemical Positivity.

In cervical biopsies, for diagnosis of Human Papilloma Virus (HPV) related conditions, the immunohistochemical staining for p16 has a diagnostic value only if diffusely and strongly positive, pattern named "block-like". "Weak and/or focal (w/f) p16 expression" is commonly considered nonspecific. In our previous study, we demonstrated the presence of high-risk HPV (hrHPV) DNA by LiPa method in biopsies showing w/f p16 positivity. The aim of the present study was to investigate the presence of hrHPV-DNA by CISH in the areas showing w/f p16 expression. We assessed the presence of hrHPV16, 18, 31, 33, 51 by CISH in a group of 20 cervical biopsies showing w/f p16 expression, some with increased Ki67, and in 10 cases of block-like expression, employed as control. The immunohistochemical p16 expression was also assessed by digital pathology. hrHPV-CISH nuclear positivity was encountered in 12/20 cases of w/f p16 expression (60%). Different patterns of nuclear positivity were identified, classified as punctate, diffuse and mixed, with different epithelial distributions. Our results, albeit in a limited casuistry, show the presence of HPV in an integrated status highlighted by CISH in w/f p16 positive cases. This could suggest the necessity of a careful follow-up of the patients with "weak" and/or "focal" immunohistochemical patterns of p16, mainly in cases of increased Ki67 cell proliferation index, supplemented with molecular biology examinations.

BMSCs-derived Exosome CISH Alleviates Myocardial Infarction by Inactivating the NF-κB Pathway to Stimulate Macrophage M2 Polarization.

Current myocardial infarction (MI) treatments are suboptimal, necessitating deeper pathogenesis understanding of MI. This research explored how exosomes (Exo) derived from bone marrow mesenchymal stem cells (BMSCs) contribute to MI mitigation and their therapeutic potential. Isolated BMSCs was identified by microscope, flow cytometry, alizarin red and oil red O staining. Exo were identified by TEM, NTA and western blot. HE staining, masson staining, and cardiac function parameters were used to assess the cardiac function in MI mice. TUNEL staining, western blot and qRT-PCR were used to detect apoptosis, inflammatory factors and M1/M2 markers. The NF-κB pathway activation was detected through western blot assays. Immunofluorescence, qRT-PCR, western blot, and flow cytometry were employed to evaluate macrophage polarization. MI mice showed cardiac injury, increased apoptosis and inflammation, while BMSCs-Exo treatment alleviated these effects. In MI mice, the macrophage M1 polarization was increased and the NF-κB pathway was activated, whereas BMSCs-Exo treatment reversed these changes. Furthermore, CISH expression was reduced in MI mice, but was elevated with BMSCs-Exo treatment. In vitro, LPS shifted RAW264.7 cells to M1 phenotype and activated the NF-κB pathway, yet BMSCs-Exo shifted them to M2 phenotype and inhibited the NF-κB pathway. Mechanistically, BMSCs-Exo induced macrophage M2 polarization by transmitting CISH to inhibit NF-κB activation. BMSCs-Exo mitigates MI by transmitting CISH to inhibit the NF-κB pathway, promoting macrophages to M2 type. This implies BMSCs-Exo could be a useful treatment for MI, and CISH could be a potential therapy target.

CSF1 expression in xanthogranulomatous epithelial tumor/keratin-positive giant cell-rich tumor.

"Xanthogranulomatous epithelial tumor" (XGET) and "keratin-positive giant cell-rich soft tissue tumor" (KPGCT), two recently described mesenchymal neoplasms, likely represent different aspects of a single entity. Both tumors are composed of only a small minority of tumor cells surrounded by large numbers of non-neoplastic inflammatory cells and histiocytes, suggesting production of a paracrine factor with resulting "landscape effect," as seen in tenosynovial giant cell tumor. Recent evidence suggests that the paracrine factor in XGET/KPGCT may be CSF1, as in tenosynovial giant cell tumor. We hypothesized that CSF1 is overexpressed in XGET/KPGCT. To test our hypothesis, we performed quantitative real time PCR (qPCR) for CSF1 expression and CSF1 RNAscope chromogenic in situ hybridization (CISH) on 6 cases of XGET/KPGCT. All cases were positive with CSF1 CISH and showed increased expression of CSF1 by qPCR. Our findings provide additional evidence that the CSF1/CSF1R pathway is involved in the pathogenesis of XGET/KPGCT. These findings suggest a possible role for CSF1R inhibition in the treatment of unresectable or metastatic XGET/KPGCT.

Spatial Localization of Eubacterial 16S rRNA in Early Pregnancy Placenta and Decidua.

Bacteria derived from the maternal circulation have been suggested to seed the human placenta during pregnancy leading to development of an intrinsic placental microbiome; however, other data indicates these bacteria are artifactual contaminants. Limited research on the localization of bacteria in human placental tissue is available, which may help differentiate resident placental bacteria from contaminants. This study spatially localizes bacteria in situ in normal late first to early second trimester human placenta by 16S rRNA chromogenic in situ hybridization and demonstrates patterns consistent with both contaminants and intraparenchymal signals. These results suggest that placental microbiome studies may benefit from spatial strategies that can exclude surface contamination.

Cish knockout mice exhibit similar outcomes to malaria infection despite altered hematopoietic responses.

The Cytokine-inducible Src homology 2 domain-containing (CISH) protein is a negative feedback regulator induced by cytokines that play key roles in immunity and erythropoiesis. Single nucleotide polymorphisms (SNPs) in the human CISH gene have been associated with increased susceptibility to severe malaria disease. To directly assess how CISH might influence outcomes in the BALB/c model of malaria anemia, CISH knockout (Cish-/-) mice on this background were infected with Plasmodium berghei and their hematopoietic responses, cytokine production and ability to succumb to severe malaria disease evaluated. Despite basal erythrocytic disruption, upon P. berghei infection, the Cish -/- mice were better able to maintain peripheral blood cell counts, hemoglobin levels and a steady-state pattern of erythroid differentiation compared to wild-type (Cish+/+) mice. Ablation of CISH, however, did not influence the outcome of acute malaria infections in either the BALB/c model or the alternative C57BL/6 model of experimental cerebral malaria, with the kinetics of infection, parasite load, weight loss and cytokine responses being similar between Cish+/+ and Cish-/- mice, and both genotypes succumbed to experimental cerebral malaria within a comparable timeframe.

Role of Cytokine-Inducible SH2 Domain-Containing (CISH) Protein in the Regulation of Erythropoiesis.

The cytokine-inducible SH2 domain-containing (CISH) protein was the first member of the suppressor of cytokine signaling (SOCS) family of negative feedback regulators discovered, being identified in vitro as an inducible inhibitor of erythropoietin (EPO) signaling. However, understanding of the physiological role played by CISH in erythropoiesis has remained limited. To directly assess the function of CISH in this context, mice deficient in CISH were characterized with respect to developmental, steady-state, and EPO-induced erythropoiesis. CISH was strongly expressed in the fetal liver, but CISH knockout (KO) mice showed only minor disruption of primitive erythropoiesis. However, adults exhibited mild macrocytic anemia coincident with subtle perturbation particularly of bone marrow erythropoiesis, with EPO-induced erythropoiesis blunted in the bone marrow of KO mice but enhanced in the spleen. Cish was expressed basally in the bone marrow with induction following EPO stimulation in bone marrow and spleen. Overall, this study indicates that CISH participates in the control of both basal and EPO-induced erythropoiesis in vivo.

Immunohistochemical Detection of the Expressed BRCA1 and BRCA2 Proteins in Microenvironment of Malignant Breast Cancerous Tissues Infected with Human Mammary Tumor Virus.

OBJECTIVE: The objective of the study was to examine the prevalence of HMTV infection, its associations with breast malignant tissues, and the expression of BRCA1 and BRCA2 proteins. METHODS: One hundred archival breast tissues, 40 biopsies from female patients with breast cancer (BC), and 20 healthy breast tissues from the control group were used in the study. Immunohistochemical analysis was conducted to detect the expressed BRCA1 and BRCA2 proteins. Digoxigenin-labeled HMTV probes were used in chromogenic in situ hybridization for the identification of HMTV in breast tumor tissues. The complementary sequence sites of the HMTV probe sequences were stained by NBT/BCIP as blue signals. RESULTS: There were 12 out of 40 (30%) benign breast tumorous tissues and 14 out of 40 (35%) BC tissues, while healthy control breast tissues were 10% (2 out of 20 tissues). Positive immunohistochemical (IHC) reactions for BRCA2 protein were observed in 12 out of 40 BC tissues (30.0%), 25% of benign breast tumorous tissues, and 5% of the control group. A significant (p < 0.05) statistical difference in the percentages of HMTV in the studied groups was found. CONCLUSION: HMTV might contribute to the development of subsets of benign and malignant breast tumors. The observed rates of defective or mutated BRCA1 and BRCA2 genes in healthy tissues indicate a role in the development of breast tumors.

The Role of Cytokine-Inducible SH2 Domain-Containing Protein (CISH) in the Regulation of Basal and Cytokine-Mediated Myelopoiesis.

Cytokine-inducible SH2 domain-containing protein (CISH) is a member of the suppressor of cytokine signaling (SOCS) family of negative feedback regulators shown to play crucial roles in lymphoid cell development and function as well as appetite regulation. It has also been implicated in the control of signaling downstream of the receptors for the cytokines granulocyte/macrophage colony-stimulating factor (GM-CSF) and granulocyte colony-stimulating factor (G-CSF) in myeloid cells. To investigate the physiological role of CISH in myelopoiesis, mice deficient in CISH were analyzed basally and in response to administration of these cytokines. CISH knockout (KO) mice possessed basally elevated neutrophils in the blood, bone marrow, and spleen compared to wild-type (WT) mice. During GM-CSF-induced myelopoiesis, the frequency of neutrophils, myeloid dendritic cells (DCs), and CFU-M in the bone marrow was higher in the KO, as were the neutrophils and CFU-G in the spleen. In contrast, no differences were observed between KO and WT mice during G-CSF-induced myelopoiesis apart from an elevated frequency of CFU-G and CFU-M in the spleen. This work has identified a role for CISH in the negative regulation of granulopoiesis, including that mediated by GM-CSF.

Intestinal Epithelial Cell-specific Deletion of Cytokine-inducible SH2-containing Protein Alleviates Experimental Colitis in Ageing Mice.

BACKGROUND AND AIMS: The incidence of inflammatory bowel disease [IBD] in the elderly has increased in recent years. However, the mechanisms underlying the ageing-related IBD susceptibility remain elusive. Cytokine-inducible SH2-containing protein [CISH] is involved in regulating metabolism, the expansion of intestinal tuft cells and type-2 innate lymphoid cells, and ageing-related airway inflammation. Here, we investigated the role of CISH in ageing-related colitis susceptibility. METHODS: CISH and phosphorylated signal transducer and activator of transcription-3 [p-STAT3] levels were evaluated in the colons of ageing mice and older ulcerative colitis [UC] patients. Mice with intestinal epithelial cell-specific knockout of Cish [CishΔIEC] and Cish-floxed mice were administered dextran sodium sulphate [DSS] or trinitrobenzene sulphonic acid [TNBS] to induce colitis. Colonic tissues were analysed in quantitative real-time polymerase chain reaction, immunoblotting, immunohistochemical, and histological staining experiments. Differentially expressed genes from colonic epithelia were analysed by RNA sequencing. RESULTS: Ageing increased the severity of DSS-induced colitis and the expression of colonic epithelial CISH in mice. CishΔIEC prevented DSS- or TNBS-induced colitis in middle-aged mice but not in young mice. RNA-sequencing analysis revealed that CishΔIEC significantly suppressed DSS-induced oxidative stress and proinflammatory responses. During ageing in the CCD841 cell model, knockdown of CISH decreased ageing-induced oxidative stress and proinflammatory responses, whereas these effects were compromised by knocking down or inhibiting STAT3. The increase in CISH expression was higher in the colonic mucosa of older patients with UC than in that of healthy controls. CONCLUSIONS: CISH might be a proinflammatory regulator in ageing; therefore, targeted therapy against CISH may provide a novel strategy for treating ageing-related IBD.

Expression of CISH, an Inhibitor of NK Cell Function, Increases in Association with Ovarian Cancer Development and Progression.

Epithelial ovarian cancer (OVCA), a fatal malignancy of women, disseminates locally. Although NK cells mount immune responses against OVCA, tumors inhibit NK cells, and the mechanism is not well understood. Cytokines stimulate NK cells; however, chronic stimulation exhausts them and induces expression of cytokine-inducible SH2-containing protein (CISH). Tumors produce anti-inflammatory cytokine interleukin (IL)-10 which may induce NK cell exhaustion. The goal of this study was to examine if CISH expression in NK cells increases during OVCA development and to determine the mechanism(s) of OVCA-induced CISH expression in NK cells. Normal ovaries (n = 7) were used for CISH, IL-10 and GRP78 expression. In tumor ovaries, CISH was examined in early and late stages (n = 14 each, all subtypes) while IL-10 and GRP78 expression were examined in early and late stage HGSC (n = 5 each). Compared to normal, the population of CISH-expressing NK cells increased and the intensity of IL-10 and GRP78 expression was significantly higher in OVCA (p < 0.05). CISH expression was positively correlated with IL-10 expression (r = 0.52, r = 0.65, p < 0.05 at early and late stages, respectively) while IL-10 expression was positively correlated with GRP78 expression (r = 0.43, r = 0.52, p < 0.05, respectively). These results suggest that OVCA development and progression are associated with increased CISH expression by NK cells which is correlated with tumor-induced persistent cellular stress.

Disruption of CISH promotes the antitumor activity of human T cells and decreases PD-1 expression levels.

Tumor cells and the immunosuppressive tumor microenvironment suppress the antitumor activity of T cells through immune checkpoints, including the PD-L1/PD-1 axis. Cytokine-inducible SH2-containing protein (CISH), a member of the suppressor of cytokine signaling (SOCS) family, inhibits JAK-STAT and T cell receptor (TCR) signaling in T and natural killer (NK) cells. However, its role in the regulation of immune checkpoints in T cells remains unclear. In this study, we ablated CISH in T cells with CRISPR-Cas9 and found that the sensitivity of T cells to TCR and cytokine stimulation was increased. In addition, chimeric antigen receptor T cells with CISH deficiency exhibited longer survival and higher cytokine secretion and antitumor activity. Notably, PD-1 expression was decreased in activated CISH-deficient T cells in vitro and in vivo. The level of FBXO38, a ubiquitination-regulating protein that reduces PD-1 expression, was elevated in activated T cells after CISH ablation. Hence, this study reveals a mechanism by which CISH promotes PD-1 expression by suppressing the expression of FBXO38 and proposes a new strategy for augmenting the therapeutic effect of CAR-T cells by inhibiting CISH.

[Enhanced Natural Killers with CISH and B2M Gene Knockouts Reveal Increased Cytotoxicity in Glioblastoma Primary Cultures].

In an experimental study using the CRISPR/Cas9 system, "enhanced" NK cell lines with knockout of CISH, the gene for the CIS protein (a negative regulator of NK cytotoxicity), as well as two lines with a knocked-out ß2-microglobulin gene, which provides membrane exposure of MHC class I, were obtained from two parental lines of human natural killers (YT wild type and YT-VAV1^(+) overexpressing the VAV1 cytotoxicity enhancing protein). The knockout efficiency was determined by real-time PCR as well as by flow cytometry with specific antibodies. The resulting CISH^(-/-) or B2M^(-/-) knockout lines were tested for cytotoxicity in primary monolayer cultures of human glioblastoma multiforme. The cytotoxicity of the lines was assessed using a cell analyzer that records the cell index based on cell impedance. YT-CISH^(-/-) has been shown to be significantly more effective than wild-type YT in eliminating primary glioblastoma cells in an in vitro cell monolayer experiment. The cytotoxicity of the YT-VAV1^(+)-CISH^(-/-) and YT-VAV1^(+)B2M^(-/-) lines against glioblastoma cells was the highest, but overall, it did not significantly differ from the initially increased cytotoxicity of the YT-VAV1^(+) line. The lines of NK-like cells obtained may serve as a prototype for the creation of "enhanced" allogeneic and autologous NK- and CAR-NK cells for the immunotherapy of glioblastoma multiforme.

Hepatic cytokine-inducible SH2-containing protein (CISH) regulates gluconeogenesis via cAMP-responsive element binding protein (CREB).

Impairment of gluconeogenesis is a key factor responsible for hyperglycemia in patients with type 2 diabetes. As an important member of the suppressors of cytokine signaling (SOCS) protein family, many physiological functions of cytokine-inducible SH2-containing protein (CISH) have been described; however, the role of hepatic CISH in gluconeogenesis is poorly understood. In the present study, we observed that hepatic CISH expression was reduced in fasted wild-type (WT) mice. Overexpression of CISH decreased glucose production in mouse primary hepatocytes, while silencing of CISH had the opposite effects. In addition, adenovirus-mediated hepatic CISH overexpression resulted in improved glucose tolerance and decreased gluconeogenesis in WT and leptin receptor-deficient diabetic (db/db) mice. In contrast, adenovirus-mediated hepatic CISH knockdown impaired glucose tolerance and increased gluconeogenesis in WT mice. We also generated liver-specific CISH knockout (LV-CISH KO) mice and discovered that these mice had a similar phenotype in glucose tolerance and gluconeogenesis as mice injected with adenoviruses that knockdown CISH expression. Mechanistically, we found that CISH overexpression decreased and CISH knockdown increased the mRNA and protein levels of glucose-6-phosphatase (G6Pase) and phosphoenolpyruvate carboxykinase 1 (PEPCK), two key enzymes involved in gluconeogenesis, in vitro, and in vivo. Moreover, we discovered that the phosphorylation of cAMP-responsive element binding protein 1 (CREB), a transcription factor of G6pase and Pepck, was required for regulating gluconeogenesis by CISH. Taken together, this study identifies hepatic CISH as an important regulator of gluconeogenesis. Our results also provide important insights into the metabolic functions of the SOCS protein family and the potential targets for the treatment of diabetes.

Internal checkpoint regulates T cell neoantigen reactivity and susceptibility to PD1 blockade.

BACKGROUND: Adoptive transfer of tumor-infiltrating lymphocytes (TIL) fails to consistently elicit tumor rejection. Manipulation of intrinsic factors that inhibit T cell effector function and neoantigen recognition may therefore improve TIL therapy outcomes. We previously identified the cytokine-induced SH2 protein (CISH) as a key regulator of T cell functional avidity in mice. Here, we investigate the mechanistic role of CISH in regulating human T cell effector function in solid tumors and demonstrate that CRISPR/Cas9 disruption of CISH enhances TIL neoantigen recognition and response to checkpoint blockade. METHODS: Single-cell gene expression profiling was used to identify a negative correlation between high CISH expression and TIL activation in patient-derived TIL. A GMP-compliant CRISPR/Cas9 gene editing process was developed to assess the impact of CISH disruption on the molecular and functional phenotype of human peripheral blood T cells and TIL. Tumor-specific T cells with disrupted Cish function were adoptively transferred into tumor-bearing mice and evaluated for efficacy with or without checkpoint blockade. FINDINGS: CISH expression was associated with T cell dysfunction. CISH deletion using CRISPR/Cas9 resulted in hyper-activation and improved functional avidity against tumor-derived neoantigens without perturbing T cell maturation. Cish knockout resulted in increased susceptibility to checkpoint blockade in vivo. CONCLUSIONS: CISH negatively regulates human T cell effector function, and its genetic disruption offers a novel avenue to improve the therapeutic efficacy of adoptive TIL therapy. FUNDING: This study was funded by Intima Bioscience, U.S. and in part through the Intramural program CCR at the National Cancer Institute.

CISH Expression Is Associated with Metastasis-Free Interval in Triple-Negative Breast Cancer and Refines the Prognostic Value of PDL1 Expression.

Strategies are being explored to increase the efficiency of immune checkpoint inhibitors (ICIs) targeting PD1/PDL1 in triple-negative breast cancer (TNBC), including combination with therapies inhibiting intracellular immune checkpoints such as CISH (Cytokine-induced SH2 protein). Correlation between CISH expression and TNBC features is unknown. We retrospectively analyzed CISH expression in 1936 clinical TNBC samples and searched for correlations with clinical variables, including metastasis-free interval (MFI). Among TNBCs, 44% were identified as "CISH-up" and 56% "CISH-down". High expression was associated with pathological axillary lymph node involvement, more adjuvant chemotherapy, and Lehmann's immunomodulatory and luminal AR subtypes. The "CISH-up" class showed longer 5-year MFI (72%) than the "CISH-down" class (60%; p = 2.8 × 10-2). CISH upregulation was associated with activation of IFNα and IFNγ pathways, antitumor cytotoxic immune response, and signatures predictive for ICI response. When CISH and PDL1 were upregulated together, the 5-year MFI was 81% versus 52% when not upregulated (p = 6.21 × 10-6). The two-gene model provided more prognostic information than each gene alone and maintained its prognostic value in multivariate analysis. CISH expression is associated with longer MFI in TNBC and refines the prognostic value of PDL1 expression. Such observation might reinforce the therapeutic relevance of combining CISH inhibition with an anti-PD1/PDL1 ICI.

Cytokine-inducible SH2 domain containing protein contributes to regulation of adiposity, food intake, and glucose metabolism.

The cytokine-inducible SH2 domain containing protein (CISH) is the founding member of the suppressor of cytokine signaling (SOCS) family of negative feedback regulators and has been shown to be a physiological regulator of signaling in immune cells. This study sought to investigate novel functions for CISH outside of the immune system. Mice deficient in CISH were generated and analyzed using a range of metabolic and other parameters, including in response to a high fat diet and leptin administration. CISH knockout mice possessed decreased body fat and showed resistance to diet-induced obesity. This was associated with reduced food intake, but unaltered energy expenditure and microbiota composition. CISH ablation resulted in reduced basal expression of the orexigenic Agrp gene in the arcuate nucleus (ARC) region of the brain. Cish was basally expressed in the ARC, with evidence of co-expression with the leptin receptor (Lepr) gene in Agrp-positive neurons. CISH-deficient mice also showed enhanced leptin responsiveness, although Cish expression was not itself modulated by leptin. CISH-deficient mice additionally exhibited improved insulin sensitivity on a high-fat diet, but not glucose tolerance despite reduced body weight. These data identify CISH as an important regulator of homeostasis through impacts on appetite control, mediated at least in part by negative regulation of the anorexigenic effects of leptin, and impacts on glucose metabolism.

A 12-bp indel in the 3'UTR of porcine CISH gene associated with Landrace piglet diarrhea score.

Variations in Cytokine inducible SH2-containing protein (CISH) gene influence human susceptibility to common infectious diseases, but little is known about CISH in swine. The objectives of this study were to 1) determine porcine CISH (pCISH) mRNA expression level in different tissues of piglets, 2) predict putative functional genetic variations within pCISH, 3) investigate the association between a identified variation in the 3'UTR and piglets phenotype traits in Min (n = 226) and Landrace (n = 186) population, and explore the function of this variation. Results of quantitative PCR showed pCISH mRNA expressed in all the collected tissues with higher level in lung and ileum than colon (p < 0.05). In-silico analysis indicated none of the functional ns-SNPs existed in pCISH coding region. Results from the characterizing of 3'UTR presented a novel 12-bp insertion/deletion (indel) mutation. Statistical analysis demonstrated that this 12-bp indel associated with piglets diarrhea score in the Landrace population, and animals with AA genotype (12-bp insertion) presented lower diarrhea score when compared with BB (p < 0.05) or AB (p < 0.01) carriers. The in vitro study indicated that the luciferase activity of reconstruct plasmid psiCHECK-2-CISH-AA or psiCHECK-2-CISH-BB was significantly lower than the negative control (p < 0.05), and luciferase activity of psiCHECK-2-CISH-AA was higher than that of the psiCHECK-2-CISH-BB (p < 0.05). Although results herein suggested the 12-bp indel might affect Landrace piglet susceptibility to diarrhea, further association studies in more populations are needed before this preliminary finding could be used for pig breeding.