Intestinal myofibroblasts: targets for stem cell therapy

The subepithelial intestinal myofibroblast is an important cell orchestrating many diverse functions in the intestine and is involved in growth and repair, tumorigenesis, inflammation, and fibrosis. The myofibroblast is but one of several α-smooth muscle actin-positive (α-SMA(+)) mesenchymal cells present within the intestinal lamina propria, including vascular pericytes, bone marrow-derived stem cells (mesenchymal stem cells or hematopoietic stem cells), muscularis mucosae, and the lymphatic pericytes (colon) and organized smooth muscle (small intestine) associated with the lymphatic lacteals. These other mesenchymal cells perform many of the functions previously attributed to subepithelial myofibroblasts. This review discusses the definition of a myofibroblast and reconsiders whether the α-SMA(+) subepithelial cells in the intestine are myofibroblasts or other types of mesenchymal cells, i.e., pericytes. Current information about specific, or not so specific, molecular markers of lamina propria mesenchymal cells is reviewed, as well as the origins of intestinal myofibroblasts and pericytes in the intestinal lamina propria and their replenishment after injury. Current concepts and research on stem cell therapy for intestinal inflammation are summarized. Information about the stem cell origin of intestinal stromal cells may inform future stem cell therapies to treat human inflammatory bowel disease (IBD).     

Structure based virtual screening of ligands to identify cysteinyl leukotriene receptor 1 antagonist

Montelukast and Zafirlukast are known leukotriene receptor antagonists prescribed in asthma treatment. However, these fall short as mono therapy and are frequently used in combination with inhaled glucocorticosteroids with or without long acting beta 2 agonists. Therefore, it is of interest to apply ligand and structure based virtual screening strategies to identify compounds akin to lead compounds Montelukast and Zafirlukast. Hence, compounds with structures having 95% similarity to these compounds were retrieved from NCBI׳s PubChem database. Compounds similar to lead were grouped and docked at the antagonist binding site of cysteinyl leukotriene receptor 1. This exercise identified compounds UNII 70RV86E50Q (Pub Cid 71587778) and Sure CN 9587085 (Pub Cid 19793614) with higher predicted binding compared to Montelukast and Zafirlukast. It is shown that the compound Sure CN 9587085 showed appreciable ligand receptor interaction compared to UNII 70RV86E50Q. Thus, the compound Sure CN 9587085 is selected as a potent antagonist to cysteinyl leukotriene receptor 1 for further consideration in vitro and in vivo validation.     

The influence of cultivation methods on Shewanella oneidensis physiology and proteome expression

High-throughput analyses that are central to microbial systems biology and ecophysiology research benefit from highly homogeneous and physiologically well-defined cell cultures. While attention has focused on the technical variation associated with high-throughput technologies, biological variation introduced as a function of cell cultivation methods has been largely overlooked. This study evaluated the impact of cultivation methods, controlled batch or continuous culture in bioreactors versus shake flasks, on the reproducibility of global proteome measurements in Shewanella oneidensis MR-1. Variability in dissolved oxygen concentration and consumption rate, metabolite profiles, and proteome was greater in shake flask than controlled batch or chemostat cultures. Proteins indicative of suboxic and anaerobic growth (e.g., fumarate reductase and decaheme c-type cytochromes) were more abundant in cells from shake flasks compared to bioreactor cultures, a finding consistent with data demonstrating that “aerobic” flask cultures were O₂ deficient due to poor mass transfer kinetics. The work described herein establishes the necessity of controlled cultivation for ensuring highly reproducible and homogenous microbial cultures. By decreasing cell to cell variability, higher quality samples will allow for the interpretive accuracy necessary for drawing conclusions relevant to microbial systems biology research.     

Edwardsiella ictaluri evpP is required for colonisation of channel catfish ovary cells and necrosis in anterior kidney macrophages

Edwardsiella ictaluri is a Gram‐negative facultative anaerobe that can survive inside channel catfish phagocytes. E. ictaluri can orchestrate Type VI Secretion System (T6SS) for survival in catfish macrophages. evpP encodes one of the T6SS translocated effector proteins. However, the role of evpP in E. ictaluri is still unexplored. In this work, we constructed an E. ictaluri evpP mutant (EiΔevpP) and assessed its survival under complement and oxidative stress. Persistence of EiΔevpP in catfish as well as attachment and invasion in catfish macrophage and ovary cells were determined. Further, virulence of EiΔevpP in catfish and apoptosis it caused in macrophages were explored. EiΔevpP behaved same as wild type (EiWT) under complement and oxidative stress in complex media, whereas oxidative stress affected mutant's survival significantly in minimal media (p < .05). Persistence of EiΔevpP in live catfish and uptake and survival inside peritoneal macrophages were similar. The attachment and invasion capabilities of EiΔevpP in catfish ovary cells were significantly less than that of EiWT (p < .05). Although EiΔevpP showed reduced attenuation in catfish, causing decreased catfish mortality compared with EiWT (44.73% vs. 67.53%), this difference was not significant. The apoptosis assay using anterior kidney macrophages indicated that the number of live macrophages exposed to EiΔevpP was significantly higher compared with EiWT exposed macrophages at 24‐hr post‐treatment (p < .05). However, there were no significant differences in the early and late apoptosis. Remarkably, necrosis in EiΔevpP exposed macrophages was significantly less than that of EiWT exposed macrophages at 24 hr (p < .05). Our results demonstrated that evpP is required for colonisation of catfish ovary cells and increased apoptosis and necrosis in anterior kidney macrophages.     

Capacity for differentiation and normalization of tumor cell populations in transplantation into the anterior chamber of the eye. III. Rhabdomyosarcoma A-7 clones

Four clone lines of transplantable cell polymorphic rhabdomyosarcoma A-7 were investigated during transplantation to the subcutaneous connective tissue (SCT) and into eye anterior chamber (EAC). Cell morphology of transplants was studied by light and electron microscopy, the activity of their LDH M- and H-subunits was examined cytochemically, and the quantity of their nuclear DNA--cytophotometrically. In the case of A-7/1, A-7/2 and A-7/3 cell lines of EAC transplants we noticed a decrease in cell element kataplasia levels, differences in LDH M- and H-form ratio, reduction in the karyotype variability. Transplants of A-7/4 clone line were similar in SCT and EAC for all the signs studied. The results obtained show that the transplantable cell polymorphic rhabdomyosarcoma A-7 is heterogeneous for its differentiation and normalizing capacities during EAC proliferation. The data reported elsewhere concerning capability of four lines of murine rhabdomyosarcomas to normalize in EAC are discussed, and some possible mechanisms of this effect are regarded.     

2‐Hexadecenal inhibits growth of C6 glioma cells

2‐Hexadecenal (2HD) formation in the organism occurs via irreversible enzymatic degradation of sphingosine‐1‐phosphate or nonenzymatic γ‐, UV‐, or HOCl‐induced destruction of a number of sphingolipids including S1P. The current research focuses on the study of 2HD effects on C6 glioma cells growth. The results obtained show that 2HD causes a dose‐dependent decrease in proliferative and mitotic indices. The change in the mitotic index is due to the redistribution of cells in the different phases of mitosis. These processes are accompanied by cytoskeleton rearrangement and changes in cell morphology, which are expressed in F‐actin redistribution, change in the number and type of filopodia and fibrils, leading to cell shape changes, decrease in intercellular contacts and monolayer rarefaction. Cells treatment with 2HD leads to apoptosis induction and signalling pathways modification, including activation of JNK, p38, and ERK1/2 MAPK but not PI3K. The effects observed are not related to the cytotoxicity of 2HD. Significance of the study: 2HD—an unsaturated aldehyde, which level can rise under conditions of oxidative stress as a result of nonenzymatic sphingolipids' destruction. The mechanisms of 2HD action on various cell types have not been sufficiently studied. Therefore, the study on functional role of this aldehyde in different cell types that may be its target is relevant. This study demonstrated that 2HD inhibits growth of C6 glioma cells due to modification of intracellular processes of signal transduction, cytoskeleton rearrangement, change in the mitotic regimen and apoptosis induction.     

Copper-induced LDL peroxidation: interrelated dependencies of the kinetics on the concentrations of copper, hydroperoxides and tocopherol

Excessive uptake of oxidized low density lipoprotein plays a role in the onset of atherosclerosis. Lipid-associated antioxidants, the most abundant of which is tocopherol (vitamin E), are therefore believed to have anti-atherogenic properties. By contrast, hydroperoxides enhance the peroxidation of low density lipoprotein. We demonstrate that none of these compounds markedly affect the maximal rate of oxidation of low density lipoprotein, whereas the lag preceding rapid oxidation is prolonged by tocopherol but shortened by hydroperoxides. The corresponding 'prolongation' and 'shortening' can be compensated by each other in low density lipoprotein preparations enriched with both these compounds. The dependence of the balance between the effects of tocopherol and hydroperoxides on the copper concentration indicates that the antioxidative effect of vitamin E increases with the oxidative stress.     

Changes in hepatocyte ultrastructure under the influence of chronic furacillin administration to rats]

The ultrastructure of rat liver cells was studied during a 6 months' furacilline (5-nitro-2-furtfurilsemicarbazon) feeding in dose of 40 mg per day, and also during the next 8 months after the treatment cessation. An irregular swelling of membranous structure in addition to disorganization and partial reduction of the granular endoplasmic reticulum were found in the hepatocyte cytoplasm after the prolonged furacilline feeding as well as glycogen depletion and the tendency of the agranular endoplasmic reticulum to enlargement. These changes rarely reached the essential intensity with the transition to necrobiosis. They disappeared already a month after the cessation of furacilline treatment. In all the terms of experiment, the nucleoli were hypertrophied and retained their loose nucleoloneme structure. No sings of furacilline carcinogenic activity were found in rat liver during the 14 months of investigation.     

Subepithelial myofibroblasts are novel nonprofessional APCs in the human colonic mucosa

The human gastrointestinal mucosa is exposed to a diverse normal microflora and dietary Ags and is a common site of entry for pathogens. The mucosal immune system must respond to these diverse signals with either the initiation of immunity or tolerance. APCs are important accessory cells that modulate T cell responses which initiate and maintain adaptive immunity. The ability of APCs to communicate with CD4+ T cells is largely dependent on the expression of class II MHC molecules by the APCs. Using immunohistochemistry, confocal microscopy, and flow cytometry, we demonstrate that alpha-smooth muscle actin(+), CD90+ subepithelial myofibroblasts (stromal cells) constitutively express class II MHC molecules in normal colonic mucosa and that they are distinct from professional APCs such as macrophages and dendritic cells. Primary isolates of human colonic myofibroblasts (CMFs) cultured in vitro were able to stimulate allogeneic CD4+ T cell proliferation. This process was dependent on class II MHC and CD80/86 costimulatory molecule expression by the myofibroblasts. We also demonstrate that CMFs, engineered to express a specific DR4 allele, can process and present human serum albumin to a human serum albumin-specific and DR4 allele-restricted T cell hybridoma. These studies characterize a novel cell phenotype which, due to its strategic location and class II MHC expression, may be involved in capture of Ags that cross the epithelial barrier and present them to lamina propria CD4+ T cells. Thus, human CMFs may be important in regulating local immunity in the colon.