Galectin-4 Controls Intestinal Inflammation by Selective Regulation of Peripheral and Mucosal T Cell Apoptosis and Cell Cycle

Galectin-4 is a carbohydrate-binding protein belonging to the galectin family. Here we provide novel evidence that galectin-4 is selectively expressed and secreted by intestinal epithelial cells and binds potently to activated peripheral and mucosal lamina propria T-cells at the CD3 epitope. The carbohydrate-dependent binding of galectin-4 at the CD3 epitope is fully functional and inhibited T cell activation, cycling and expansion. Galectin-4 induced apoptosis of activated peripheral and mucosal lamina propria T cells via calpain-, but not caspase-dependent, pathways. Providing further evidence for its important role in regulating T cell function, galectin-4 blockade by antisense oligonucleotides reduced TNF-alpha inhibitor induced T cell death. Furthermore, in T cells, galectin-4 reduced pro-inflammatory cytokine secretion including IL-17. In a model of experimental colitis, galectin-4 ameliorated mucosal inflammation, induced apoptosis of mucosal T-cells and decreased the secretion of pro-inflammatory cytokines. Our results show that galectin-4 plays a unique role in the intestine and assign a novel role of this protein in controlling intestinal inflammation by a selective induction of T cell apoptosis and cell cycle restriction. Conclusively, after defining its biological role, we propose Galectin-4 is a novel anti-inflammatory agent that could be therapeutically effective in diseases with a disturbed T cell expansion and apoptosis such as inflammatory bowel disease

Cannabidiol causes activated hepatic stellate cell death through a mechanism of endoplasmic reticulum stress-induced apoptosis

The major cellular event in the development and progression of liver fibrosis is the activation of hepatic stellate cells (HSCs). Activated HSCs proliferate and produce excess collagen, leading to accumulation of scar matrix and fibrotic liver. As such, the induction of activated HSC death has been proposed as a means to achieve resolution of liver fibrosis. Here we demonstrate that cannabidiol (CBD), a major non-psychoactive component of the plant Cannabis sativa, induces apoptosis in activated HSCs through a cannabinoid receptor-independent mechanism. CBD elicits an endoplasmic reticulum (ER) stress response, characterized by changes in ER morphology and the initiation of RNA-dependent protein kinase-like ER kinase-, activating transcription factor-6-, and inositol-requiring ER-to-nucleus signal kinase-1 (IRE1)-mediated signaling cascades. Furthermore, CBD induces downstream activation of the pro-apoptotic IRE1/ASK1/c-Jun N-terminal kinase pathway, leading to HSC death. Importantly, we show that this mechanism of CBD-induced ER stress-mediated apoptosis is specific to activated HSCs, as it occurs in activated human and rat HSC lines, and in primary in vivo-activated mouse HSCs, but not in quiescent HSCs or primary hepatocytes from rat. Finally, we provide evidence that the elevated basal level of ER stress in activated HSCs has a role in their susceptibility to the pro-apoptotic effect of CBD. We propose that CBD, by selectively inducing death of activated HSCs, represents a potential therapeutic agent for the treatment of liver fibrosis

Differential pattern and prognostic significance of CD4+, FOXP3+ and IL-17+ tumor infiltrating lymphocytes in ductal and lobular breast cancers

<p>Abstract</p> <p>Background</p> <p>Clinical relevance of tumor infiltrating lymphocytes (TILs) in breast cancer is controversial. Here, we used a tumor microarray including a large series of ductal and lobular breast cancers with long term follow up data, to analyze clinical impact of TIL expressing specific phenotypes and distribution of TILs within different tumor compartments and in different histological subtypes.</p> <p>Methods</p> <p>A tissue microarray (TMA) including 894 ductal and 164 lobular breast cancers was stained with antibodies recognizing CD4, FOXP3, and IL-17 by standard immunohistochemical techniques. Lymphocyte counts were correlated with clinico-pathological parameters and survival.</p> <p>Results</p> <p>CD4⁺lymphocytes were more prevalent than FOXP3⁺TILs whereas IL-17⁺TILs were rare. Increased numbers of total CD4⁺and FOXP3⁺TIL were observed in ductal, as compared with lobular carcinomas. High grade (G3) and estrogen receptor (ER) negative ductal carcinomas displayed significantly (<it>p </it> < 0.001) higher CD4⁺and FOXP3⁺lymphocyte infiltration while her2/neu over-expression in ductal carcinomas was significantly (<it>p </it> < 0.001) associated with higher FOXP3⁺TIL counts. In contrast, lymphocyte infiltration was not linked to any clinico-pathological parameters in lobular cancers. In univariate but not in multivariate analysis CD4⁺infiltration was associated with significantly shorter survival in patients bearing ductal, but not lobular cancers. However, a FOXP3⁺/CD4⁺ratio > 1 was associated with improved overall survival even in multivariate analysis (<it>p </it>= 0.033).</p> <p>Conclusions</p> <p>Ductal and lobular breast cancers appear to be infiltrated by different lymphocyte subpopulations. In ductal cancers increased CD4⁺and FOXP3⁺TIL numbers are associated with more aggressive tumor features. In survival analysis, absolute numbers of TILs do not represent major prognostic indicators in ductal and lobular breast cancer. Remarkably however, a ratio > 1 of total FOXP3⁺/CD4⁺TILs in ductal carcinoma appears to represent an independent favorable prognostic factor.</p

Impact of the TCR Signal on Regulatory T Cell Homeostasis, Function, and Trafficking

Signaling through the T cell antigen receptor (TCR) is important for the homeostasis of naïve and memory CD4+ T cells. The significance of TCR signaling in regulatory T (Treg) cells has not been systematically addressed. Using an Ox40-cre allele that is prominently expressed in Treg cells, and a conditional null allele of the gene encoding p56Lck, we have examined the importance of TCR signaling in Treg cells. Inactivation of p56Lck resulted in abnormal Treg homeostasis characterized by impaired turnover, preferential redistribution to the lymph nodes, loss of suppressive function, and striking changes in gene expression. Abnormal Treg cell homeostasis and function did not reflect the involvement of p56Lck in CD4 function because these effects were not observed when CD4 expression was inactivated by Ox40-cre.The results make clear multiple aspects of Treg cell homeostasis and phenotype that are dependent on a sustained capacity to signal through the TCR

ABC Transporter Pdr10 Regulates the Membrane Microenvironment of Pdr12 in Saccharomyces cerevisiae

The eukaryotic plasma membrane exhibits both asymmetric distribution of lipids between the inner and the outer leaflet and lateral segregation of membrane components within the plane of the bilayer. In budding yeast (Saccharomyces cerevisiae), maintenance of leaflet asymmetry requires P-type ATPases, which are proposed to act as inward-directed lipid translocases (Dnf1, Dnf2, and the associated protein Lem3), and ATP-binding cassette (ABC) transporters, which are proposed to act as outward-directed lipid translocases (Pdr5 and Yor1). The S. cerevisiae genome encodes two other Pdr5-related ABC transporters: Pdr10 (67% identity) and Pdr15 (75% identity). We report the first analysis of Pdr10 localization and function. A Pdr10-GFP chimera was located in discrete puncta in the plasma membrane and was found in the detergent-resistant membrane fraction. Compared to control cells, a pdr10∆ mutant was resistant to sorbate but hypersensitive to the chitin-binding agent Calcofluor White. Calcofluor sensitivity was attributable to a partial defect in endocytosis of the chitin synthase Chs3, while sorbate resistance was attributable to accumulation of a higher than normal level of the sorbate exporter Pdr12. Epistasis analysis indicated that Pdr10 function requires Pdr5, Pdr12, Lem3, and mature sphingolipids. Strikingly, Pdr12 was shifted to the detergent-resistant membrane fraction in pdr10∆ cells. Pdr10 therefore acts as a negative regulator for incorporation of Pdr12 into detergent-resistant membranes, a novel role for members of the ABC transporter superfamily

Practical guidelines for rigor and reproducibility in preclinical and clinical studies on cardioprotection

The potential for ischemic preconditioning to reduce infarct size was first recognized more than 30 years ago. Despite extension of the concept to ischemic postconditioning and remote ischemic conditioning and literally thousands of experimental studies in various species and models which identified a multitude of signaling steps, so far there is only a single and very recent study, which has unequivocally translated cardioprotection to improved clinical outcome as the primary endpoint in patients. Many potential reasons for this disappointing lack of clinical translation of cardioprotection have been proposed, including lack of rigor and reproducibility in preclinical studies, and poor design and conduct of clinical trials. There is, however, universal agreement that robust preclinical data are a mandatory prerequisite to initiate a meaningful clinical trial. In this context, it is disconcerting that the CAESAR consortium (Consortium for preclinicAl assESsment of cARdioprotective therapies) in a highly standardized multi-center approach of preclinical studies identified only ischemic preconditioning, but not nitrite or sildenafil, when given as adjunct to reperfusion, to reduce infarct size. However, ischemic preconditioning—due to its very nature—can only be used in elective interventions, and not in acute myocardial infarction. Therefore, better strategies to identify robust and reproducible strategies of cardioprotection, which can subsequently be tested in clinical trials must be developed. We refer to the recent guidelines for experimental models of myocardial ischemia and infarction, and aim to provide now practical guidelines to ensure rigor and reproducibility in preclinical and clinical studies on cardioprotection. In line with the above guideline, we define rigor as standardized state-of-the-art design, conduct and reporting of a study, which is then a prerequisite for reproducibility, i.e. replication of results by another laboratory when performing exactly the same experiment

Three-dimensional analysis of mitochondrial crista ultrastructure in a patient with Leigh syndrome by in situ cryoelectron tomography

Mitochondrial diseases produce profound neurological dysfunction via mutations affecting mitochondrial energy production, including the relatively common Leigh syndrome (LS). We recently described an LS case caused by a pathogenic mutation in USMG5, encoding a small supernumerary subunit of mitochondrial ATP synthase. This protein is integral for ATP synthase dimerization, and patient fibroblasts revealed an almost total loss of ATP synthase dimers. Here, we utilize in situ cryoelectron tomography (cryo-ET) in a clinical case-control study of mitochondrial disease to directly study mitochondria within cultured fibroblasts from a patient with LS and a healthy human control subject. Through tomographic analysis of patient and control mitochondria, we find that loss of ATP synthase dimerization due to the pathogenic mutation causes profound disturbances of mitochondrial crista ultrastructure. Overall, this work supports the crucial role of ATP synthase in regulating crista architecture in the context of human disease