Themis controls T cell activation, effector functions, and metabolism of peripheral CD8+ T cells.

Themis is important in regulating positive selection of thymocytes during T cell development, but its role in peripheral T cells is less understood. Here, we investigated T cell activation and its sequelae using a tamoxifen-mediated, acute Themis deletion mouse model. We find that proliferation, effector functions including anti-tumor killing, and up-regulation of energy metabolism are severely compromised. This study reveals the phenomenon of peripheral adaptation to loss of Themis, by demonstrating direct TCR-induced defects after acute deletion of Themis that were not evident in peripheral T cells chronically deprived of Themis in dLck-Cre deletion model. Peripheral adaptation to long-term loss was compared using chronic versus acute tamoxifen-mediated deletion and with the (chronic) dLck-Cre deletion model. We found that upon chronic tamoxifen-mediated Themis deletion, there was modulation in the gene expression profile for both TCR and cytokine signaling pathways. This profile overlapped with (chronic) dLck-Cre deletion model. Hence, we found that peripheral adaptation induced changes to both TCR and cytokine signaling modules. Our data highlight the importance of Themis in the activation of CD8+ T cells.

Prevalence and risk factors for alcohol use disorders, substance use disorders, and depression anxiety and stress among users of sexual health services in Singapore: a cross-sectional survey study.

Syndemics of poor mental health also drive poorer sexual health outcomes. This study used three scales, the Alcohol Use Disorders Identification Test (AUDIT), the Drug Abuse Screening Test (DAST-10), and the Depression Anxiety Stress Scale (DASS-21) among beneficiaries of sexual health services in Singapore (n =975), respectively. We found that a prevalence of 20.4% and 18.6% of hazardous and moderate-severe alcohol use disorders and substance use risks, respectively. About 13.7%, 18.1% and 10.5% of participants reported severe to extremely severe symptoms of depression, anxiety, and stress, respectively. Further investigation and integrated interventions for mental health in sexual health settings are warranted.

Epstein-Barr virus-induced ectopic CD137 expression helps nasopharyngeal carcinoma to escape immune surveillance and enables targeting by chimeric antigen receptors.

Non-keratinizing nasopharyngeal carcinoma (NPC) is a malignancy with a poor prognosis for relapsing patients and those with metastatic disease. Here, we identify a novel disease mechanism of NPC which may be its Achilles' heel that makes it susceptible to immunotherapy. CD137 is a potent costimulatory receptor on activated T cells, and CD137 agonists strongly enhance anti-tumor immune responses. A negative feedback mechanism prevents overstimulation by transferring CD137 from T cells to CD137 ligand (CD137L)-expressing antigen presenting cells (APC) during cognate interaction, upon which the CD137-CD137L complex is internalized and degraded. We found ectopic expression of CD137 on 42 of 122 (34.4%) NPC cases, and that CD137 is induced by the Epstein-Barr virus latent membrane protein (LMP) 1. CD137 expression enables NPC to hijack the inbuilt negative feedback mechanism to downregulate the costimulatory CD137L on APC, facilitating its escape from immune surveillance. Further, the ectopically expressed CD137 signals into NPC cells via the p38-MAPK pathway, and induces the expression of IL-6, IL-8 and Laminin γ2. As much as ectopic CD137 expression may support the growth and spread of NPC, it may be a target for its immunotherapeutic elimination. Natural killer cells that express a CD137-specific chimeric antigen receptor induce death in CD137+ NPC cells, in vitro, and in vivo in a murine xenograft model. These data identify a novel immune escape mechanism of NPC, and lay the foundation for an urgently needed immunotherapeutic approach for NPC.

Expansion of an Unusual Virtual Memory CD8+ Subpopulation Bearing Vα3.2 TCR in Themis-Deficient Mice.

Deletion of the gene for Themis affects T cell selection in the thymus, which would be expected to affect the TCR repertoire. We found an increased proportion of cells expressing Vα3.2 (TRAV9N-3) in the peripheral CD8+ T cell population in mice with germline Themis deficiency. Analysis of the TCRα repertoire indicated it was generally reduced in diversity in the absence of Themis, whereas the diversity of sequences using the TRAV9N-3 V-region element was increased. In wild type mice, Vα3.2+ cells showed higher CD5, CD6 and CD44 expression than non-Vα3-expressing cells, and this was more marked in cells from Themis-deficient mice. This suggested a virtual memory phenotype, as well as a stronger response to self-pMHC. The Vα3.2+ cells responded more strongly to IL-15, as well as showing bystander effector capability in a Listeria infection. Thus, the unusually large population of Vα3.2+ CD8+ T cells found in the periphery of Themis-deficient mice reflects not only altered thymic selection, but also allowed identification of a subset of bystander-competent cells that are also present in wild-type mice.

Themis regulates metabolic signaling and effector functions in CD4+ T cells by controlling NFAT nuclear translocation.

Themis is a T cell lineage-specific molecule that is involved in TCR signal transduction. The effects of germline Themis deletion on peripheral CD4+ T cell function have not been described before. In this study, we found that Themis-deficient CD4+ T cells had poor proliferative responses, reduced cytokine production in vitro and weaker inflammatory potential, as measured by their ability to cause colitis in vivo. Resting T cells are quiescent, whereas activated T cells have high metabolic demands. Fulfillment of these metabolic demands depends upon nutrient availability and upregulation of nutrient intake channels after efficient TCR signal transduction, which leads to metabolic reprogramming in T cells. We tested whether defects in effector functions were caused by impaired metabolic shifts in Themis-deficient CD4+ T cells due to inefficient TCR signal transduction, in turn caused by the lack of Themis. We found that upon TCR stimulation, Themis-deficient CD4+ T cells were unable to upregulate the expression of insulin receptor (IR), glucose transporter (GLUT1), the neutral amino acid transporter CD98 and the mTOR pathway, as measured by c-Myc and pS6 expression. Mitochondrial analysis of activated Themis-deficient CD4+ T cells showed more oxidative phosphorylation (OXPHOS) than aerobic glycolysis, indicating defective metabolic reprogramming. Furthermore, we found reduced NFAT translocation in Themis-deficient CD4+ T cells upon TCR stimulation. Using previously reported ChIP-seq and RNA-seq data, we found that NFAT nuclear translocation controls IR gene expression. Together, our results describe an internal circuit between TCR signal transduction, NFAT nuclear translocation, and metabolic signaling in CD4+ T cells.

Autoimmune responses and inflammation in type 2 diabetes.

Obesity-induced insulin resistance is one of the largest noncommunicable disease epidemics that we are facing at the moment. Changes in lifestyle and greater availability of low nutritional value, high caloric food has led to the highest rates of obesity in history. Obesity impacts the immune system and obesity-associated inflammation contributes to metabolic diseases, such as type 2 diabetes. Both the adaptive and the innate immune system play a role in the regulation of glycemic control, and there is a need to understand how metabolic imbalances drive disease pathogenesis. This review discusses the cell types, mediators, and pathways that contribute to immunologic-metabolic crosstalk and explores how the immune system might be targeted as a strategy to treat metabolic disease.

Expression of α-smooth muscle actin in benign and malignant salivary gland tumors: An immunohistochemical study.

BACKGROUND AND OBJECTIVES: Myoepithelial cells (ME) are known to contribute in the patterning of salivary gland neoplasms (SGN) and possess cytoplasmic smooth muscle actin (SMA) revealed by alpha SMA (α-SMA). The present study aimed to assess the expression of α-SMA in selected benign and malignant SGN (pleomorphic adenoma [PA], mucoepidermoid carcinoma (MEC), adenoid cystic carcinoma (ACC), and polymorphous low-grade adenocarcinoma (PLGA). MATERIALS AND METHODS: The intensity and pattern of expression of α-SMA were studied in 25 cases of SGN's ACC (n = 7), MEC (n = 8), PA (n = 8), and PLGA (n = 2), and correlated with the histological patterns. RESULTS: Maximum expression of α-SMA in the epithelial compartment was seen in ACC, followed by PA, whereas MEC and PLGA showed completely negative staining. The connective tissue expression was mild in ACC and MEC. The myxoid stroma of PA with "melting" pattern was weakly positive for α-SMA. The stroma in PLGA showed complete negativity. In ACC, α-SMA-positive cells were lining the cribriform spaces, small islands, and dispersed within large islands. Small nests showed complete positivity for α-SMA. INTERPRETATION AND CONCLUSION: In ACC, α-SMA expression supports the involvement of ME in epithelial organization explaining the histological patterns seen. In PA, the expression correlates with the predominantly secretory nature of ME. The absence of epithelial positivity in MEC and PLGA suggest that ME has less role to play in their histogenesis. The weak stromal positivity observed in MEC and ACC may be attributed to the positive immunoreactivity of myofibroblasts playing a role in modulating the course of SGN's.

Expression of Laminin in Oral Squamous Cell Carcinomas

Background and objectives: Laminin is a significant basement membrane (BM) glycoprotein, the expression of which reflects BM integrity more precisely than do other ECM proteins. The present study aimed to evaluate laminin expression in oral squamous cell carcinomas OSCC and to determine any associations with clinico-pathological parameters (surgical margin status, lymph node involvement, survival and recurrence). Methods: Laminin expression was evaluated in 31 cases of biopsy-proven OSCC by immunohistochemical staining and its association with prognosticators and the Brynes grading system was determined by appropriate statistical analysis. Results: We observed a significant increase in linear staining pattern (p<0.001) at the tumour-host interface in well-differentiated OSCC cases, in contrast to poorly differentiated lesions which exhibited intense cytoplasmic expression within tumour cells. Higher cytoplasmic laminin expression was seen in 33.3% of cases with involved surgical margins and 69.2% of cases with lymph node metastasis (along with weak/absent staining of laminin around the tumour-host interface ­ Basement membrane around tumour islands). Similarly, in 60% of the cases who died and in 81.8% of cases with tumour recurrence, moderately intense cytoplasmic laminin expression was seen within tumour cells. On comparing variables of the Brynes grading system, significant cytoplasmic expression of laminin was linked with mild inflammation (p<0.0016) and increased mitotic activity (p<0.008). Conclusion: Based on these observations, immunohistochemical expression of laminin might be useful to evaluate histological differentiation and aggressiveness of OSCCs.

Tumour-Associated Tissue Eosinophilia in Oral Squamous Cell Carcinoma- A Boon or a Bane?

INTRODUCTION: The infiltration of tumour stroma by eosinophils, Tumour-Associated Tissue Eosinophilia (TATE) is known to modulate the evolution of Oral Squamous Cell Carcinoma (OSCC). Identification of eosinophils in the inflammatory stroma has been proven to be an important factor in prognostication of malignant tumours including cancers of mouth, oesophagus, larynx, pharynx, breast, lung, intestine and genitourinary tract. AIM: Our study aimed to assess the role of TATE as a prognosticator in OSCC as visualized by Haematoxylin and Eosin (H&E) and congo red staining. MATERIALS AND METHODS: Thirty histologically-proven cases of OSCC were retrieved from the archives of Department of Oral Pathology, Manipal College of Dental Sciences, Mangalore, Manipal University, Karnataka, India. Two serial sections of 4µm thickness were made and subjected to routine staining with H&E and modified congo red staining, where eosinophil granules stained red and nuclei stained blue. In 40x magnification, 10 HPF at invasive tumour front were assessed for counting eosinophils by placing a 49 square grid (measuring 0.0289 sq mm). STATISTICAL ANALYSIS: The TATE was compared with the prognosticators using Mann-Whitney U-test. The grades of carcinoma were correlated with TATE using Kruskal-Wallis test followed by Post-hoc Bonferronis correction. Agreement of the number of eosinophils counted in the two staining techniques (H&E and Congo red) in OSCC was achieved using interclass correlation coefficient, and Friedman's test. A value of p< 0.05 was considered statistically significant. RESULTS: Our results showed that tissue eosinophil counts were higher in well-differentiated cases of OSCC, cases with lymph node involvement, decreased survival, without margin involvement and in cases that did not recur. H&E stain showed significantly better visualization of eosinophils resulting in higher eosinophil counts than when seen with Congo red (p=0.008). CONCLUSION: Thus, TATE can be used as a surrogate marker in prediction of survival and recurrence in OSCC. H&E proved to be a better stain for evaluation of eosinophils.

Evaluation of DNA damage in Type 2 diabetes mellitus patients with and without peripheral neuropathy: A study in South Indian population.

BACKGROUND: The increasing incidence of Type 2 diabetes mellitus globally has collaterally increased the incidence of diabetes-associated complications such as neuropathy. Oxidative stress induced DNA damage is one of the mechanisms implicated in the pathogenesis of diabetic complications. Here we aimed to evaluate the extent of DNA damage in diabetes patients with and without clinical neuropathy using the Cytokinesis Block Micronucleus Cytome assay, in a group of South Indian population. MATERIALS AND METHODS: The Cytokinesis Block Micronucleus Cytome assay was performed in lymphocyte cultures of 42 type 2 diabetes patients (22 with neuropathy and 20 without neuropathy) and 42 age and sex matched controls. Nuclear aberrations like Nuclear Buds, Nucleoplasmic Bridges and Micronuclei were analyzed. RESULTS: The frequency of nuclear aberrations in diabetes patients with neuropathy was higher than compared to diabetes patients without neuropathy. The mean frequencies of nuclear aberrations per cell in diabetes patients with neuropathy and without neuropathy were 0.02 ± 0.02 and 0.01 ± 0.01, respectively. This was significantly higher than in the controls (0.002 ± 0.002) (P < 0.0001). An increasing trend of nuclear aberrations in correlation with the duration of diabetes was observed. CONCLUSION: This study highlights the use of the Cytokinesis Block Micronucleus Cytome assay as a potent tool for the identification of DNA damage, which may prove to be useful biomarker to assess the severity diabetes-associated complications such as neuropathy. Implementation of this technique at the clinical level would potentially enhance the quality of management of patients with diabetes and its complications like neuropathy.