Hexamethylene bisacetamide protects peritoneal mesothelial cells from glucose

Hexamethylene bisacetamide protects peritoneal mesothelial cells from glucose.BackgroundPeritoneal dialysis causes damage to peritoneal mesothelial cells primarily because dialysis fluids have a high glucose concentration. This study examined the abnormalities of gap junctional intercellular communication (GJIC) in human peritoneal mesothelial cells (HPMCs) exposed to relatively high levels of glucose. Also, ability of hexamethylene bisacetamide (HMBA) to up-regulate GJIC in HPMCs exposed to high levels of glucose was measured.MethodsAn assay that monitors the recovery of fluorescence after photobleaching was used to measure GJIC in primary cultured HPMCs. The cells were exposed to a low (10 mmol/L) or high (50 or 90 mmol/L) glucose level for a total of six days, and some cells were also incubated with or without HMBA (1 or 6 mmol/L) from day 4. The effects of incubation in these various environments on expression of the connexin 43 (Cx43) gene were investigated by the reverse transcription-polymerase chain reaction (to detect Cx43 mRNA) or by immunofluorescence and Western blotting (to detect Cx43 protein). To evaluate the influence of protein kinase C (PKC) or mitogen-activated protein kinase (MAPK) on GJIC, specific inhibitors were added to cultures in a high glucose medium.ResultsGap junctional intercellular communication was inhibited in a concentration- and time-dependent manner when cells were exposed to high glucose. The addition of 6 mmol/L HMBA to cultures significantly enhanced GJIC despite the presence of a high glucose concentration. High glucose also down-regulated Cx43 mRNA and protein expression, with the dose-dependent decrease of Cx43 protein at gap junctions paralleled by a decrease in the phosphorylation of this protein. As expected, treatment of cells with 6 mmol/L HMBA increased both Cx43 mRNA and protein levels despite exposure to high glucose. The addition of PKC or MAPK inhibitors to high glucose cultures did not restore GJIC, and there was no significant change of Cx43 phosphorylation in the presence of these inhibitors.ConclusionsHigh glucose down-regulates GJIC in human peritoneal mesothelial cells. It also decreases the levels of both Cx43 mRNA and Cx43 protein, with the latter becoming hypophosphorylated. HMBA appears to reverse all of these changes. These results are consistent with our hypothesis that HMBA protects HPMCs from the adverse effects of high glucose by reversing various processes that would otherwise lead to harmful loss of GJIC

A Novel Method for Screening Monoclonal Antibodies Reacting with Antigenic Determinants on Soluble Antigens; A Reversed Indirect-Enzyme Linked Immunosorbent Assay(RI-ELISA)

A novel screening method was established to select new monoclonal antibodies which react with unknown antigenic determinants on molecules bearing antigen determinants reactive with established monoclonal antibodies. This new method is a sandwich assay termed "reversed indirect-enzyme linked immunosorbent assay" (RI-ELISA). Goat antimouse immunoglobulin antibodies are used as the primary immobilized antibody in this assay. They allow the non-purified monoclonal antibodies contained in hybridoma culture supernatants to bind to the microtest plate for enzyme immunoassay (EIA plate) much more efficiently than in the usual sandwich assay where the non-purified monoclonal antibodies are adsorbed directly to the polystyrene surface. The antigen solution is then reacted with the monoclonal antibodies and thereafter enzyme labeled monoclonal antibody with known specificity is added. Therefore, if the hybridoma culture supernatant contains monoclonal antibodies which were bound to the EIA plate and react with antigenic determinants on the soluble molecules which have antigen determinants recognized by the enzyme labeled antibody, the enzyme labeled antibodies will bind to induce an enzymatic reaction. The most important technical consideration in the RI-ELISA is the inhibition of direct binding of the enzyme labeled monoclonal antibodies to free sites remaining in the immobilized goat anti-mouse immunoglobulins antibodies. This problem could be effectively overcome by using normal mouse serum as blocking substance. These studies indicate that the RI-ELISA may be a useful screening method for selecting new monoclonal antibodies which react with unknown antigenic determinants on soluble molecules

Reactivity of the Serum from A-Bomb Survivors with the Tissues of Stomach, Liver and Kidney of Normal Rats

In order to evaluate delayed effects of radiation on pathological immune response an attempt was made to detect antibodies in the serum of atomic bomb survivors against kidney, liver, and parietal cells from rats. The following results were observed. Analysis of changes in antibody detection frequencies by age and exposure dose without considering sex showed that the rates for those exposed to 100 + rad showed a trend to increase with age for all three organs (P<0.01). However, in the 0 rad group, a significant trend to increase with age was noted for anti-kidney and antiliver antibodies only (P<0.01 for both). Analysis of changes in antibody detection frequencies by sex, age, and exposure dose showed that the detection frequencies increased significantly with age for all three organs in males exposed to 100 + rad (P < 0.05), but only the anti-liver antibody frequency increased significantly with age in males in the O rad exposure group. Females failed to shown any statistical changes in any exposure group

CD43-independent augmentation of mouse T-cell function by glycoprotein cleaving enzymes

Previous work has shown that the function of mouse CD4 + T cells can be augmented by an enzyme, O -sialoglycoprotein endopeptidase (OSGE), which cleaves surface CD43, suggesting the idea that the high levels of glycosylated CD43 found on T cells from aged mice may contribute to immune senescence. New results now show that OSGE improves T-cell function even in mice lacking CD43, showing that other glycoproteins must contribute to the OSGE effect on function. Evaluation of other enzymes found two whose ability to stimulate CD4 activation was higher in aged than in young T cells. One of these, PNGase F, is a glycosidase specific for N-linked glycans, and the other, ST-Siase(2,3) from Salmonella typhimurium , is specific for α2,3-linked terminal sialic acid residues. Parallel lectin-binding experiments showed that removal of α2,3-linked sialic acid residues vulnerable to PNGase F and ST-Siase(2,3) was also greater in old than in young T cells. The preferential ability of PNGase F and ST-Siase(2,3) to improve the function of T cells from aged mice may involve cleavage of glycoproteins containing α2,3-linked sialic acid residues on N-linked or O-linked glycans or both.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/75621/1/j.1365-2567.2006.02419.x.pd

Lymphocyte subset characterization associated with persistent hepatitis C virus infection and subsequent progression of liver fibrosis

This study aims to deepen understanding of lymphocyte phenotypes related to the course of hepatitis C virus (HCV) infection and progression of liver fibrosis, in a cohort of atomic-bomb survivors. The study subjects comprise three groups: 162 HCV persistently infected, 145 spontaneously cleared, and 3511 uninfected individuals. We found increased percentages of peripheral blood TH1 and total CD8 T cells and decreased percentages of NK cells in the HCV persistence group, compared with the other two groups, after adjustment for age, gender, and radiation exposure dose. Subsequently, we found that increased TH1 cell percentages in the HCV persistence group were significantly associated with an accelerated time-course reduction in platelet counts―accelerated progression of liver fibrosis―while TC1 and NK cell percentages were inversely associated with the progression. This study suggests that TH1 immunity is enhanced by persistent HCV infection, and that percentages of peripheral TH1, TC1, and NK cells may help predict progression of liver fibrosis.This research was based on RERF Research Protocols 3-09, 4-02, 2-00, 9-92, and was supported in part by the U.S. National Institute of Allergy and Infectious Diseases (NIAID Contract HHSN272200900059C)

Zebrafish homologs of 16p11.2, a genomic region associated with brain disorders, are active during brain development, and include two deletion dosage sensor genes

Deletion or duplication of one copy of the human 16p11.2 interval is tightly associated with impaired brain function, including autism spectrum disorders (ASDs), intellectual disability disorder (IDD) and other phenotypes, indicating the importance of gene dosage in this copy number variant region (CNV). The core of this CNV includes 25 genes; however, the number of genes that contribute to these phenotypes is not known. Furthermore, genes whose functional levels change with deletion or duplication (termed 'dosage sensors'), which can associate the CNV with pathologies, have not been identified in this region. Using the zebrafish as a tool, a set of 16p11.2 homologs was identified, primarily on chromosomes 3 and 12. Use of 11 phenotypic assays, spanning the first 5 days of development, demonstrated that this set of genes is highly active, such that 21 out of the 22 homologs tested showed loss-of-function phenotypes. Most genes in this region were required for nervous system development - impacting brain morphology, eye development, axonal density or organization, and motor response. In general, human genes were able to substitute for the fish homolog, demonstrating orthology and suggesting conserved molecular pathways. In a screen for 16p11.2 genes whose function is sensitive to hemizygosity, the aldolase a (aldoaa) and kinesin family member 22 (kif22) genes were identified as giving clear phenotypes when RNA levels were reduced by ~50%, suggesting that these genes are deletion dosage sensors. This study leads to two major findings. The first is that the 16p11.2 region comprises a highly active set of genes, which could present a large genetic target and might explain why multiple brain function, and other, phenotypes are associated with this interval. The second major finding is that there are (at least) two genes with deletion dosage sensor properties among the 16p11.2 set, and these could link this CNV to brain disorders such as ASD and IDD.Simons Foundation (Grant Number 95091

Heavy Ion Carcinogenesis and Human Space Exploration

Prior to the human exploration of Mars or long duration stays on the Earth s moon, the risk of cancer and other diseases from space radiation must be accurately estimated and mitigated. Space radiation, comprised of energetic protons and heavy nuclei, has been show to produce distinct biological damage compared to radiation on Earth, leading to large uncertainties in the projection of cancer and other health risks, while obscuring evaluation of the effectiveness of possible countermeasures. Here, we describe how research in cancer radiobiology can support human missions to Mars and other planets

The Changing Landscape of Naive T Cell Receptor Repertoire With Human Aging

Human aging is associated with a profound loss of thymus productivity, yet naïve T lymphocytes still maintain their numbers by division in the periphery for many years. The extent of such proliferation may depend on the cytokine environment, including IL-7 and T-cell receptor (TCR) “tonic” signaling mediated by self pMHCs recognition. Additionally, intrinsic properties of distinct subpopulations of naïve T cells could influence the overall dynamics of aging-related changes within the naïve T cell compartment. Here, we investigated the differences in the architecture of TCR beta repertoires for naïve CD4, naïve CD8, naïve CD4+CD25−CD31+ (enriched with recent thymic emigrants, RTE), and mature naïve CD4+CD25−CD31− peripheral blood subsets between young and middle-age/old healthy individuals. In addition to observing the accumulation of clonal expansions (as was shown previously), we reveal several notable changes in the characteristics of T cell repertoire. We observed significant decrease of CDR3 length, NDN insert, and number of non-template added N nucleotides within TCR beta CDR3 with aging, together with a prominent change of physicochemical properties of the central part of CDR3 loop. These changes were similar across CD4, CD8, RTE-enriched, and mature CD4 subsets of naïve T cells, with minimal or no difference observed between the latter two subsets for individuals of the same age group. We also observed an increase in “publicity” (fraction of shared clonotypes) of CD4, but not CD8 naïve T cell repertoires. We propose several explanations for these phenomena built upon previous studies of naïve T-cell homeostasis, and call for further studies of the mechanisms causing the observed changes and of consequences of these changes in respect of the possible holes formed in the landscape of naïve T cell TCR repertoire