Hepatitis C virus down-regulates insulin receptor substrates 1 and 2 through up-regulation of suppressor of cytokine signaling 3

The pathogenesis of hepatitis C virus (HCV)-associated insulin resistance remains unclear. Therefore, we investigated mechanisms for HCV-associated insulin resistance. Homeostasis model assessment for insulin resistance was increased in patients with HCV infection. An increase in fasting insulin levels was associated with the presence of serum HCV core, the severity of hepatic fibrosis and a decrease in expression of insulin receptor substrate (IRS) 1 and IRS2, central molecules of the insulin-signaling cascade, in patients with HCV infection. Down-regulation of IRS1 and IRS2 was also seen in HCV core-transgenic mice livers and HCV core-transfected human hepatoma cells. Carbobenzoxy-l-leucyl-l-leucyl-l-leucinal, a potent proteosomal proteolysis inhibitor, blocked down-regulation of IRS1 and IRS2 in HCV core-transfected hepatoma cells. In human hepatoma cells, HCV core up-regulated suppressor of cytokine signaling (SOCS) 3 and caused ubiquitination of IRS1 and IRS2. HCV core-induced down-regulation of IRS1 and IRS2 was not seen in SOCS3(-/-) mouse embryonic fibroblast cells. Furthermore, HCV core suppressed insulin-induced phosphorylation of p85 subunit of phosphatidylinositol 3-kinase and Akt, activation of 6-phosphofructo-2-kinase, and glucose uptake. In conclusion, HCV infection changes a subset of hepatic molecules regulating glucose metabolism. A possible mechanism is that HCV core-induced SOCS3 promotes proteosomal degradation of IRS1 and IRS2 through ubiquitination.     

Development of a 5' fluorogenic nuclease-based real-time PCR assay for quantitative detection of Actinobacillus actinomycetemcomitans and Porphyromonas gingivalis

A 5' nuclease TaqMan PCR was developed for the quantitative detection of the periodontopathic bacteria Actinobacillus actinomycetemcomitans and Porphyromonas gingivalis. The relative numbers of bacteria were measured by the comparative threshold cycle method. This simplified method is a way of obtaining the relative quantities of these organisms from specimens and of monitoring the effect of therapy.     

Changes in T,B, and NK Lymphocyte Subsets During and After Normal Pregnancy

PROBLEM: Pregnancy affects the maternal immune system and the clinical course of maternal diseases. Here we report the changes in the detailed lymphocyte subsets of helper T cells, suppressor T cells, CD5⁺ B cells, T cell receptor (TCR) αβ-positive T cells (Tαβ cells), TCRαβ-negative T cell (Tγδ cells), and others during and after pregnancy through to one year postpartum, and discuss the significance of the changes. METHOD: The absolute numbers of helper T cells, suppressor T cells, cytotoxic T cells, TCRαβ-negative T cells (Tγδ cells), CD5^— B cells, CD5⁺ B cells, and NK cell subsets were examined by two-color flow cytometry in peripheral blood from 51 healthy non-pregnant women, 106 healthy pregnant women, and 148 healthy postpartum women. RESULTS: In early pregnancy, the numbers of suppressor T cells and NK cells with strong cytotoxicity (NK⁺⁺⁺ cells) increased, and the number of cytotoxic T cells decreased. In late pregnancy, the helper T cell and NK⁺⁺⁺ cell numbers decreased. Tαβ, CD5^— B and CD5⁺ B cells decreased during pregnancy. After delivery, helper T cells and cytotoxic T cells increased from 1 to 4 months postpartum, and suppressor T cells increased at 7 months postpartum. TCRαβ-negative T cells increased at 4 to 10 months postpartum. Both CD5^— and CD5⁺ B cells decreased further at 1 month postpartum, but CD5⁺ B cells increased markedly at 7 to 10 months postpartum. CONCLUSIONS: These data indicate that 1) early increases of suppressor T cells and NK⁺⁺⁺ cells during pregnancy may be related to the mechanism to accept or reject the fetus in early pregnancy, respectively; 2) late decreases of helper T cells and NK⁺⁺⁺ cells may be related to the maintenance of pregnancy: 3) postpartum increases of helper T cells, cytotoxic T cells, TCRαβ-negative T cells (Tγδ cells), and CD5⁺ B cells may be related to the postpartum aggravation of autoimmune diseases; and 4) the immunological effects of pregnancy remains until about 1 year after delivery.     

Purification and immunochemical studies of type b carbohydrate antigen of oral Streptococcus milleri.

The type-specific antigen of serotype b Streptococcus milleri was extracted with trichloroacetic acid from a purified cell wall preparation of the strain NCTC 10708 and then purified on a DEAE-Sephadex A-25 column, followed by a Sephadex G-100 column. The antigen was composed of rhamnose and glucose in a molar ratio of 1.7:1.0, with a trace of galactosamine (0.1). The quantitative precipitin inhibition test with various haptenic sugars showed that rhamnose gave the greatest inhibition, whereas glucose and its related carbohydrates were less effective. The major carbohydrate components of the Rantz-Randall extracts from cells of all four serotype b strains tested were also rhamnose and glucose. These results suggest that rhamnose is structurally involved in the immunodeterminant of the serotype b-specific antigen of oral S. milleri.     

Increase of Thy-1 antigen on the thymocytes accompanied with their augmented adhesion capacity to thymic epithelial cells in the mice infected with Listeria monocytogenes.

It becomes increasingly clear that adhesion systems such as CD2/LFA-3 (lymphocyte function-associated antigen-3), LFA-1/ICAM-1 (intercellular adhesion molecule-1) and Thy-1/putative Thy-1 ligand participate in the association between murine thymocytes and thymic epithelial cells. In the present study, thymocytes showed an increase in surface Thy-1 levels in mice infected with Listeria monocytogenes, but no significant changes in the levels of CD2 or LFA-1. No alteration was found either in the ratio of CD3high/CD3low/CD3- or in that of CD4/CD8 subsets in these thymocytes compared with uninfected control thymocytes which excluded the possibility of enrichment of 'cortical thymocytes' with Thy-1high/CD3low/CD4+ CD8+ in the thymocyte population of infected mice. Moreover such Thy-1high thymocytes exhibited a highly augmented ability of adhesion to a thymic epithelial cell line due to the increase of surface Thy-1 antigens as an adhesion molecule. At such intervals after infection, the total number of thymocytes was found to be reduced. These results suggest that the expression level of surface Thy-1 on thymocytes is regulated in response to in vivo stimulation and may play a role in the intrathymic development of thymocytes by affecting the adhesion of thymocytes with thymic stromal cells. The implication of the enhanced ability of adhesion in the decrease in the number of thymocytes is discussed.     

Cellular Adherence, Glucosyltransferase Adsorption, and Glucan Synthesis of Streptococcus mutans AHT Mutants

Streptococcus mutans AHT mutants M1, M2, and M13 failed to adhere to a glass surface, whereas mutants M9 and M35 exhibited decreased and increased adherence, respectively, as compared with the parent strain, when grown in sucrose broth. Extracellular glucosyltransferase prepared from glucose-grown cultures of the adherent strains (wild type, M9, and M35) induced adherence of heat-killed cells of the homologous and heterologous streptococcal strains as well as of Escherichia coli K-12 and uncoated resin particles. The glucosyltransferase was adsorbed on all the streptococcal cells and glucan-coated resins, but not on E. coli cells and the uncoated resins. Glucosyltransferase from the nonadhering mutants (M1, M2, M13) neither was significantly adsorbed on nor induced adherence of any of the cells and resins. Cell-free enzymes from the glucose-grown adherent strains produced water-soluble and water-insoluble glucans, whereas those from the nonadhering mutants produced only water-soluble glucans. Small amounts of alkali-soluble, cell-associated glucan were recovered from the sucrose-grown nonadhering mutants. Thus, the relative proportions of glucosyltransferase isozymes elaborated by the S. mutans mutants, insofar as they affect the physico-chemical properties of the glucans produced, seem to determine the adherence abilities of the cells. The adsorption of glucosyltransferase on glucan molecules on the cell surface is not required for the adherence of S. mutans, but de novo glucan synthesis is important in the adherence process.     

Immunochemical and structural characterization of a serotype-specific polysaccharide antigen from Actinobacillus actinomycetemcomitans Y4 (serotype b).

A serotype-specific polysaccharide antigen of Actinobacillus actinomycetemcomitans Y4 (serotype b) was extracted from whole cells by autoclaving. The extract was purified by chromatography on DEAE-Sephadex A-25 and Sephacryl S-300 columns. The purified polysaccharide antigen formed a single precipitin line with anti-type b serum but not with anti-type a serum and anti-type c serum. The antigen was composed of 43.9% L-rhamnose, 49.1% D-fucose, and a trace amount of fatty acid. Methylation analysis, Smith degradation, and optical rotation data showed that the antigen was a polymer consisting of a disaccharide repeating unit, ----3)-alpha-D-fucopyranosyl-(1----2)-beta-L-rhamnopyranosyl-(1----. In quantitative precipitin inhibition tests, D-fucose and L-rhamnose showed very low inhibition, but the partial hydrolysate of the purified antigen was an effective inhibitor, suggesting that the serotype b specific antiserum recognizes the larger oligosaccharide units.     

Alterations with time of secretion and ultrastructure of the exocrine pancreas in rats with an obstructed biliary system.

The effects of biliary obstruction on the secretory activity and ultrastructure of the exocrine pancreas were studied in rats. Increased volume, protein and amylase secretion were observed in the early periods, under basal conditions and in response to cerulein-secretin stimulation. Bicarbonate concentrations in the pancreatic juice were not significantly altered during the experiments, under either basal or stimulated conditions. The wet weight of the pancreas, and the tissue protein and amylase content of the pancreas increased progressively after bile duct ligation, under basal conditions. Morphological changes were marked in the acinar cells, the most characteristic being an increase in the size and number of zymogen granules and dilatation of the Golgi cisternae. These findings demonstrate a hyperfunctional state of the acinar cells following progressive suppression of granule discharge. It is suggested that the inhibition of granule discharge from acinar cells in rats with prolonged bile duct ligation may be due to the cytotoxic effects of hyperbilirubinemia and inhibition of normal mitochondrial function.     

Lung adenocarcinoma with bronchioloalveolar carcinoma component is frequently associated with foci of high-grade atypical adenomatous hyperplasia

We assessed the occurrence of atypical adenomatous hyperplasia (AAH) in whole lung lobes with primary cancer lesions. Following surgical resection, tissue specimens were sliced to a thickness of 4 mm (3,641 specimens from 61 cases; mean = 59.7 specimens per case). A total of 119 AAH foci were found and an association was evident in 25 (57%) of 44 adenocarcinomas, 3 (30%) of 10 squamous cell carcinomas, and 2 (29%) of 7 other lung cancers. Histologic evaluation showed that 108 AAH foci were categorized as low-grade and the other 11 as high-grade AAH. These 11 foci of high-grade AAH were present in 7 patients with adenocarcinoma, and in 1 patient there was a synchronous double primary lung adenocarcinoma. High-grade AAH was closely associated with bronchioloalveolar carcinoma (BAC) type adenocarcinoma, and low-grade AAH with non-BAC adenocarcinoma. The mean +/- SD Ki-67 labeling index in high-grade AAH (3.5%+/-2.9%) was significantly higher than for the low-grade index (1.4%+/-1.6%). We propose that foci of high- but not low-grade AAH may be potential precursor lesions of lung adenocarcinoma, especially with the BAC component.     

Bone formation using novel interconnected porous calcium hydroxyapatite ceramic hybridized with cultured marrow stromal stem cells derived from Green rat

The clinical use of cultured marrow stromal stem cells (MSCs) has recently attracted attention in the field of tissue engineering. For the clinical use of the MSCs, a prominent scaffold is needed. A scaffold hybridized with MSCs is transformed into a "bioactive bone substitute," and this provides good osteoconduction. In this study, a novel calcium hydroxyapatite ceramic with an interconnected porous structure (IP-CHA) was used as a scaffold. MSCs were harvested from Green rats containing Green Fluorescent Protein (GFP), and then these hybrids were implanted into the tibias of Sprague-Dawley rats. The purposes of this study were to examine the osteogenic ability of these hybrids without coculture, and to evaluate whether the resulting bone formation originated from the grafted MSCs or the recipient's cells. The hybridized group showed excellent bone formation compared with the IP-CHA-only implant group. Observation of the implanted MSCs revealed that they survived 8 weeks after surgery, and differentiated into osteoblast-like cells, thus providing bone formation. This implantation of the MSCs/IP-CHA composite provides excellent osteoconduction, and is expected to have extensive clinical applications.