HIV and viral hepatitis coinfection analysis using surveillance data from 15 US states and two cities.

Coinfection with human immunodeficiency virus (HIV) and viral hepatitis is associated with high morbidity and mortality in the absence of clinical management, making identification of these cases crucial. We examined characteristics of HIV and viral hepatitis coinfections by using surveillance data from 15 US states and two cities. Each jurisdiction used an automated deterministic matching method to link surveillance data for persons with reported acute and chronic hepatitis B virus (HBV) or hepatitis C virus (HCV) infections, to persons reported with HIV infection. Of the 504 398 persons living with diagnosed HIV infection at the end of 2014, 2.0% were coinfected with HBV and 6.7% were coinfected with HCV. Of the 269 884 persons ever reported with HBV, 5.2% were reported with HIV. Of the 1 093 050 persons ever reported with HCV, 4.3% were reported with HIV. A greater proportion of persons coinfected with HIV and HBV were males and blacks/African Americans, compared with those with HIV monoinfection. Persons who inject drugs represented a greater proportion of those coinfected with HIV and HCV, compared with those with HIV monoinfection. Matching HIV and viral hepatitis surveillance data highlights epidemiological characteristics of persons coinfected and can be used to routinely monitor health status and guide state and national public health interventions.

Expression cloning of a new member of the ABO blood group glycosyltransferases, iGb3 synthase, that directs the synthesis of isoglobo-glycosphingolipids.

The large array of different glycolipids described in mammalian tissues is a reflection, in part, of diverse glycosyltransferase expression. Herein, we describe the cloning of a UDP-galactose: beta-d-galactosyl-1,4-glucosylceramide alpha-1, 3-galactosyltransferase (iGb(3) synthase) from a rat placental cDNA expression library. iGb(3) synthase acts on lactosylceramide, LacCer (Galbeta1,4Glcbeta1Cer) to form iGb(3) (Galalpha1,3Galbeta1, 4Glcbeta1Cer) initiating the synthesis of the isoglobo-series of glycosphingolipids. The isolated cDNA encoded a predicted protein of 339 amino acids, which shows extensive homology (40-50% identity) to members of the ABO gene family that includes: murine alpha1, 3-galactosyltransferase, Forssman (Gb(5)) synthase, and the ABO glycosyltransferases. In contrast to the murine alpha1, 3-galactosyltransferase, iGb(3) synthase preferentially modifies glycolipids over glycoprotein substrates. Reverse transcriptase-polymerase chain reaction revealed a widespread tissue distribution of iGb(3) synthase RNA expression, with high levels observed in spleen, thymus, and skeletal muscle. As an indirect consequence of the expression cloning strategy used, we have been able to identify several potential glycolipid biosynthetic pathways where iGb(3) functions, including the globo- and isoglobo-series of glycolipids.

Cloning of Gb3 synthase, the key enzyme in globo-series glycosphingolipid synthesis, predicts a family of alpha 1, 4-glycosyltransferases conserved in plants, insects, and mammals.

We have cloned Gb(3) synthase, the key alpha1, 4-galactosyltransferase in globo-series glycosphingolipid (GSL) synthesis, via a phenotypic screen, which previously yielded iGb(3) synthase, the alpha1,3-galactosyltransferase required in isoglobo-series GSL (Keusch, J. J., Manzella, S. M., Nyame, K. A., Cummings, R. D., and Baenziger, J. U. (2000) J. Biol. Chem. 33). Both transferases act on lactosylceramide, Galbeta1,4Glcbeta1Cer (LacCer), to produce Gb(3) (Galalpha1,4LacCer) or iGb(3) (Galalpha1, 3LacCer), respectively. GalNAc can be added sequentially to either Gb(3) or iGb(3) yielding globoside and Forssman from Gb(3), and isogloboside and isoForssman from iGb(3). Gb(3) synthase is not homologous to iGb(3) synthase but shows 43% identity to a human alpha1,4GlcNAc transferase that transfers a UDP-sugar in an alpha1, 4-linkage to a beta-linked Gal found in mucin. Extensive homology (35% identity) is also present between Gb(3) synthase and genes in Drosophila melanogaster and Arabidopsis thaliana, supporting conserved expression of an alpha1,4-glycosyltransferase, possibly Gb(3) synthase, throughout evolution. The isolated Gb(3) synthase cDNA encodes a type II transmembrane glycosyltransferase of 360 amino acids. The highest tissue expression of Gb(3) synthase RNA is found in the kidney, mesenteric lymph node, spleen, and brain. Gb(3) glycolipid, also called P(k) antigen or CD77, is a known receptor for verotoxins. CHO cells that do not express Gb(3) and are resistant to verotoxin become susceptible to the toxin following transfection with Gb(3) synthase cDNA.

The effect on immunoglobulin glycosylation of altering in vivo production of immunoglobulin G.

The effect on murine immunoglobulin G (IgG) glycosylation of altering IgG production in vivo was assessed in interleukin (IL)-6 transgenic and CD4 knockout mice. C57BL/6 mice carrying the IL-6 transgene showed increased levels of circulating IgG. This was associated with decreased levels of galactose on the IgG oligosaccharides. No decrease in beta4-galactosyltransferase mRNA or in enzyme activity was seen in IL-6 transgenic mice. MRL-lpr/lpr mice normally have elevated levels of circulating IgG, again accompanied by decreased levels of IgG galactose. Disruption of the CD4 gene in MRL-lpr/lpr mice led to a substantial decrease in the concentration of circulating IgG, but IgG galactose levels remained low. Thus, an enforced decrease in IgG levels in the lymphoproliferative MRL-lpr/lpr mice did not alter the percentage of agalactosyl IgG in these mice, suggesting that agalactosyl IgG production is not simply caused by excessive IgG synthesis leading to an insufficient transit time in the trans-Golgi, but rather to a molecular defect in the interaction between galactosyltransferase and the immunoglobulin heavy chain.

The effect on IgG glycosylation of altering beta1, 4-galactosyltransferase-1 activity in B cells.

An absence of galactose on the N-linked oligosaccharides of immunoglobulin G (IgG) has been shown to affect the functional activity of the antibody molecule. In patients with rheumatoid arthritis there is an increased proportion of IgG which lacks galactose and correspondingly lower levels of beta1, 4-galactosyltransferase (beta4Gal-T) activity. The recent demonstration of several expressed beta4Gal-T genes in man raises the possibility that the enzyme responsible for the decreased IgG galactose is not the "classical" beta4Gal-T (beta4Gal-T1). To directly address the question of whether reduced beta4Gal-T1 would lead to reduced IgG galactose, the level of beta4Gal-T1 in a human IgG-secreting B cell line was specifically altered using stable transfection with sense (SpcDNA3-Gal-T1) or antisense (ASpcDNA3-Gal-T1) human beta4Gal-T1 cDNA. SpcDNA3-Gal-T1 B cell transfectants expressed up to a 2.5-fold higher level of beta4Gal-T enzyme activity for the exogenous neoglycoconjugate acceptor GlcNAc-pITC-BSA than did ASpcDNA3-Gal-T1 transfectants. Flow cytometric analysis with Ricinus communis agglutinin I (RCAI) revealed an overall greater number of Galbeta1,4GlcNAc structures in the fixed and permeabilized SpcDNA3-Gal-T1 B cell transfectants compared with the ASpcDNA3-Gal-T1 transfectants. Moreover, there was increased galactosylation of IgG secreted from the SpcDNA3-Gal-T1 transfectants relative to the ASpcDNA3-Gal-T1 B cell transfectants. Alteration of the level of the "classical" beta4Gal-T (beta4Gal-T1) in B cells therefore affects IgG glycosylation.

Antibody recognition of epitopes on wild-type and mutant beta-(1-->4)-galactosyltransferase-1.

The epitopes present on beta-(1-->4)-galactosyltransferase-1 (beta 4Gal-T1) have been explored using a panel of monoclonal antibodies (mAbs) raised against the soluble form of the human enzyme. Reactivity of the antibodies with site-specific and truncated mutants of human beta 4Gal-T1 suggests the presence of a major immunogenic epitope cluster consisting of four epitopes within the stem region and mapping between amino acids 42 and 115. The catalytic activity of the enzyme is increased in the presence of stem region-specific antibody. Two of the epitopes were further localized to a region between amino acids 42 and 77, sequences which are not shared with the recently cloned beta 4Gal-T2 and beta 4Gal-T3 enzymes. An epitope located close to or within the catalytic domain is also identified, and the mAb to this region binds synergistically with antibodies to the stem region.

B lymphocyte galactosyltransferase protein levels in normal individuals and in patients with rheumatoid arthritis.

We have quantified the level of beta4-galactosyltransferase protein in human B lymphocytes using an ELISA-based assay. Between 1-10ng of beta4-galactosyltransferase was detected per mg total cellular protein, indicating that this enzyme constitutes <0.001% of B lymphocyte cellular protein. Akin to previous studies, individuals with rheumatoid arthritis exhibited reduced lymphocytic galactosyltransferase enzyme activity compared with normal controls when using ovalbumin as the acceptor substrate. The levels of enzyme protein present in B lymphocytes from patients with rheumatoid arthritis was, however, not reduced suggesting that the B lymphocyte galactosyltransferase catalytic activity may be regulated post-translationally.

The stability of lymphocytic beta 1,4-galactosyltransferase expression during pregnancy and lactation.

The authors used murine pregnancy as a model to investigate the regulation of IgG glycosylation. Pregnancy is associated with decreased levels of circulating IgG. The oligosaccharides on this IgG from late (day 15), but not early (day 8), pregnant Balb/c mice exhibited increased levels of terminal galactose. The levels remained elevated 8 days post-partum in lactating mice. Nonetheless, splenic beta 1, 4-GalTase mRNA and enzyme activity remained relatively constant throughout pregnancy and into lactation. This was in contrast to a pregnancy-associated increase in mammary gland beta 1,4-GalTase mRNA. Thus the increased IgG galactose levels seen in pregnancy are regulated by mechanisms which are independent of transcriptional control of beta 1,4-GalTase expression.

Analysis of different glycosylation states in IgG subclasses.

Altered IgG glycosylation affects certain immunological activities of human IgG. Enzyme-linked lectin assays (ELLA) were developed for detecting the glycosylation on IgG and its individual subclasses in sera from healthy controls. Biotinylated Sambucus nigra, Ricinus communis agglutinin I and Bandeiraea simplicifolia II were used to detect the terminal sialic acid (SA), galactose (Gal) and N-acetylglucosamine (GlcNAc) sugar residues, respectively on the captured IgG. A mild oxidation step of the anti-IgG-coated plates obviated background reaction with the lectins. Terminal glycosylation varied significantly with age. The old age group (> 65 years) had less SA in IgG1, and IgG2, when compared to young (0-19 years) and adult (20-39 years) groups, respectively. Also the old age group had less Gal in IgG2, IgG3 and IgG4 subclasses compared to the adult group, and to the young group in the case of IgG3. This ELLA system may be a valuable tool in the detection of glycosylation disorders in patients' sera.

beta 1,4-Galactosyltransferase activity in B cells detected using a simple ELISA-based assay.

Lymphocytic beta 1,4-galactosyltransferase (beta 1,4-GalTase, EC 2.4.1.38) activity was measured in B cells using a neoglyco-protein, N-acetylglucosamine-phenylisothiocyanate-bovine serum albumin (GlcNAc-pITC-BSA), as an acceptor substrate in a novel enzyme-linked immunosorbent assay (ELISA)-based method. This assay proved to be much simpler to use than the lengthy and expensive radiochemical assays commonly used, and has the additional advantage that it specifically detects the enzyme mediating transfer via the Gal beta 1,4GlcNAc linkage. A F(ab')2 antibody against GalTase was able to specifically inhibit the reaction. Greater sensitivity for beta 1,4-GalTase activity was obtained using GlcNAc-pITC-BSA as an acceptor substrate rather than ovalbumin. Low levels of beta-galactosidase activity were detectable in lymphocyte cell lysates at acidic pH, although such activity was not detectable at the neutral pH used in the beta 1,4-GalTase activity assay. Using this assay with the GlcNAc-pITC-BSA acceptor, similar beta 1,4-GalTase activities were observed in CD19+ B cells from patients with rheumatoid arthritis (RA) to those seen in normal control individuals.

Tiopronine-induced reduction of rheumatoid factor functional affinity and asialylated IgG in patients with rheumatoid arthritis.

Serum and synovial fluid (SF) from 16 rheumatoid arthritis patients were evaluated before and after a 2-month treatment with tiopronine (TP). The levels of rheumatoid factor (RF) declined, as did the functional affinity of the remaining RF (p less than 0.01 in serum and p less than 0.05 in SF). Concomitant restoration of the sialylation of IgG was observed (p less than 0.05 in serum and SF). Following an initial increase, a significant reduction was observed in the level of soluble interleukin-2 receptors in serum (p less than 0.05) and SF (p less than 0.05) after treatment with TP.