Biomedical subjects

R M Hamadeh

Publications and source records attributed to R M Hamadeh.

7 recordsLinked to original sources

PubMed1996

Bacterial enzymes can add galactose alpha 1,3 to human erythrocytes and creates a senescence-associated epitope.

Humans have abundant circulating anti-alpha (1,3-di)-galactosyl (alpha Gal) antibodies (anti-Gal). Anti-Gal has been implicated in the clearance of senescent human erythrocytes (RBCs). The nature of the anti-Gal-binding RBC epitope has defied explanation, given that humans repress expression of the alpha 1,3 galactosyltransferase (alpha 1,3 GT) enzyme. This study explored whether alpha Gal epitopes on human RBCs might be synthesized by alpha 1,3 GTs of bacterial origin that are translocated into the circulation during commensal colonization of the gut by gram-negative bacteria. We found that an acellular Klebsiella pneumoniae sonicate could add 3H-UDP-Gal to human RBCs in the alpha configuration at 37 degrees C in the presence of 6 mM MnCl2 (pH 7.6). Gradient anion-exchange chromatography of the Klebsiella sonicate yielded four fractions that could catalyze the addition of 3H-Gal to human RBCs. Size-exclusion chromatography of these anion-exchange fractions yielded peaks of high GT activity for each, but only those derived from the first, third, and last anion-exchange fractions incorporated Gal such that the RBCs bound anti-Gal by fluorescence-activated cell sorter, suggesting that these three GTs are alpha 1,3 GTs. Thus, Klebsiella spp. make at least four GTs that can add an alpha Gal to human cell surface acceptor structures. Three of these GTs can form alpha 1,3 Gal structures on human RBCs that bind anti-Gal, thereby creating "autoimmune" senescence-associated RBC epitopes.

Bacteria↗

PubMed1995

Anti-alpha-galactosyl immunoglobulin A (IgA), IgG, and IgM in human secretions.

Anti-alpha-galactosyl (anti-Gal) is a natural human serum antibody that binds to the carbohydrate Gal alpha 1,3Gal beta 1,4GlcNAc-R (alpha-galactosyl epitope) and is synthesized by 1% of circulating B lymphocytes in response to immune stimulation by enteric bacteria. We were able to purify secretory anti-Gal from human colostrum and bile by affinity chromatography on silica-linked Gal alpha 1,3Gal beta 1,4GlcNAc. We found similar secretory anti-Gal antibodies in human milk, saliva, and vaginal washings. Secretory anti-Gal from milk and saliva was exclusively immunoglobulin A (IgA); that from colostrum and bile also contained IgG and IgM isotypes. Serum was also found to contain anti-Gal IgM and IgA in addition to the previously reported IgG. Anti-Gal IgA purified from colostrum and bile had both IgA1 and IgA2. Secretory anti-Gal from saliva, milk, colostrum, and bile agglutinated rabbit erythrocytes (RRBC) and bound to bovine thyroglobulin, both of which have abundant alpha-galactosyl epitopes. The RRBC-hemagglutinating capacity of human saliva, milk, bile, and serum was specifically adsorbed by immobilized Gal alpha 1,3Gal beta 1,4GlcNAc but not by Gal alpha 1,4Gal beta 1,4GlcNAc, Gal beta 1,3GalNAc, Gal beta 1,4GlcNAc, Gal beta 1,4GlcNAc alpha 1,2Man, or Fuc alpha 1,2Gal beta 1,4GlcNAc. No RRBC-hemagglutinating activity could be detected in rat milk, rat bile, cow milk, or rabbit bile, suggesting a restricted species distribution for secretory anti-Gal similar to that found for serum anti-Gal. Colostral anti-GaI IgA bound strongly to a sample of gram-negative bacteria isolated from the throats and stools of well children as well as to an Escherichia coli K-1 blood isolate. Colostral anti-GaI IgA inhibited the binding of a Neisseria meningitidis strain to human buccal epithelial cells, suggesting that this antibody may play a protective role at the mucosal surface.

Animals↗

PubMed1995

Anti-Gal binds to pili of Neisseria meningitidis: the immunoglobulin A isotype blocks complement-mediated killing.

alpha 1,3-Galactosyl antibodies (anti-Gal) are ubiquitous natural human serum and secretory polyclonal antibodies that bind to terminal galactose-alpha 1,3-galactose (alpha-galactosyl) residues. Serum immunoglobulin G (IgG) anti-Gal can block alternative complement pathway-mediated lysis of representative gram-negative enteric bacteria that bind it to lipopolysaccharide alpha-galactosyl structures, thereby promoting survival of such bacteria in the nonimmune host. We wanted to know whether anti-Gal also could bind to the lipooligosaccharides (LOS) of Neisseria meningitidis. To our surprise, we found that serum and secretory anti-Gal bound to pili but not to LOS of certain strains. This suggested the presence of an immunogenic pilus carbohydrate epitope. Mild periodate oxidation of sodium dodecyl sulfate-polyacrylamide gel electrophoresis-separated outer membrane preparations from strains that bound anti-Gal followed by labeling of the neoaldehyde groups resulted in the labeling of bands that corresponded to pilin and LOS, confirming that pilin contains carbohydrate structures. A Bandeiraea simplicifolia lectin that also binds terminal alpha 1,3-galactosyl residues also bound to pilin. Serum IgG, IgA, and IgM anti-Gal as well as colostral secretory IgA anti-Gal bound to pilin, as judged by immunoblotting, and to the pili of intact piliated organisms, as judged by immunoelectron microscopy. Total serum anti-Gal (IgG, IgA, and IgM) and purified serum IgA1 anti-Gal, but not its purified IgG isotype, blocked complement-mediated lysis of a piliated meningococcal strain that bound anti-Gal to its pili. Colostral anti-Gal secretory IgA blocked killing of the same strain. Thus, anti-Gal IgA may promote disease when it binds to the pili of N. meningitidis strains.

Animals↗

PubMed1994

Differences in outer membrane characteristics between gallstone-associated bacteria and normal bacterial flora.

Previous studies with scanning electron microscopy (SEM) have suggested that pigment gallstones contain bacteria. We set out to culture these bacteria and to study their membrane characteristics. We studied gallstones from 54 patients (36 men, 18 women; mean age 55.4 years) admitted consecutively to two hospitals for cholecystectomy. SEM detected bacteria in all of 14 brown pigment stones, 2 of 14 black pigment stones, and in the pigmented centres of 9 of 19 mixed cholesterol stones; no bacteria were detected in 14 pure cholesterol stones or within the cholesterol portions of mixed stones. We were able to culture bacteria from all gallstones with bacteria seen on SEM and for which sufficient material was available (n = 16). 20 bacterial species were recovered from these stones. Gallstones containing bacteria were associated with clinical sepsis and cholangitis. All bacteria obtained from gallstones agglutinated human O P1 erythrocytes, which reflects the presence of P1-specific fimbriae. 5 strains were positive for Forssman-antigen-specific fimbriae. None showed evidence of mannose-specific fimbriae. All of the organisms bound anti-Gal, a ubiquitous naturally occurring IgG specific for alpha-galactosyl residues. The presence of P1 fimbriae and alpha-galactosyl residues and the absence of mannose-specific fimbriae distinguish these organisms from gut flora. We postulate that possession of these unusual properties may enhance the ability of bacteria to colonise the biliary tree and initiate pigment gallstone formation.

Adolescent↗

PubMed1992

Human natural anti-Gal IgG regulates alternative complement pathway activation on bacterial surfaces.

One percent of circulating IgG in humans recognizes galactose alpha 1,3 galactose residues (anti-Gal) and is synthesized in response to stimulation by enteric bacteria. In this study, we found that the prevalence of binding of anti-Gal to blood isolates is significantly higher than its binding to normal stool isolates. When anti-Gal bound onto the lipopolysaccharide of a representative blood isolate, Serratia marcescens #21, it blocked its alternative complement pathway (ACP) lysis and made the organism serum resistant. In contrast, when anti-Gal bound to the capsular polysaccharide of a serum sensitive Serratia, #7, it increased ACP killing of this strain. The mechanism of blockade of ACP lysis by anti-Gal did not involve a decrease in the number of C3 molecules deposited onto Serratia #21 or an inhibition of the binding of C3b to its LPS, nor did it change the iC3b and C3d degradation products of bound C3b or prevent membrane attack complex formation on this organism. Our findings suggest that the effect of anti-Gal on immune lysis is dependent on the bacterial outer membrane structure to which it binds. We postulate that anti-Gal may play a role in the survival of selected Enterobacteriacae in Gram-negative sepsis by blocking ACP-mediated lysis of such bacteria by the nonimmune host, and that this effect depends on where anti-Gal finds its epitope on the bacterial outer membrane.

Complement C3↗

PubMed1990

Immunophysical characterization of human isolates of Serratia marcescens.

The immunophysical characteristics of 29 Serratia marcescens strains isolated from hospitalized patients in three different cities were studied. Their outer membrane antigens were compared by solid-phase radioimmunoassay inhibition, and their proteinase K-treated, whole-cell lysates were compared by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblot analysis. The strains had a limited number of unique outer membrane lipopolysaccharide (LPS) and capsular polysaccharide (K) antigens. By solid-phase radioimmunoassay inhibition, these strains could be divided into four distinct LPS and five K antigenic groups. By SDS-PAGE, the LPS groups could be further divided into three distinct SDS-PAGE core polysaccharide profiles and five distinct O-side-chain polysaccharide profiles. Immunoblot analysis with rabbit antiserum confirmed the limited heterogeneity of these isolates. Of the strains tested, no PAGE profile was unique to blood or nonblood isolates or to organisms collected from a given hospital. Variability of O and core PAGE profiles was not a function of organism growth cycle. Five representative Serratia strains were tested by SDS-PAGE and immunoblot analysis and in a bactericidal assay with normal human serum. We found that (i) the normal human serum had antibodies to the LPS of each of the strains, (ii) the anti-LPS antibody measured by immunoblot did not correlate with the level of bactericidal activity in the normal human serum, (iii) three of four sepsis isolates were serum sensitive, (iv) two Serratia strains serum sensitive in log-phase growth became serum resistant in late stationary-phase growth and under limiting nutrient conditions, and (v) no LPS PAGE profile distinguished serum-sensitive from serum-resistant strains.

Antigens, Bacterial↗

PubMed1988

Interaction between human natural anti-alpha-galactosyl immunoglobulin G and bacteria of the human flora.

Anti-alpha-galactosyl immunoglobulin G (anti-Gal) is a natural antibody present in unusually high amounts in human sera. It constitutes as much as 1% of circulating immunoglobulin G in humans and displays a distinct specificity for the carbohydrate epitope galactosyl alpha(1----3) galactosyl (Gal alpha 1----3Gal). Recently, it has been suggested by various investigators that anti-Gal may be related to some autoimmune phenomena, since marked elevation of its titer was found in sera of patients with autoimmune thyroid disorders, rheumatoid arthritis, glomerulonephritis, and Chagas' disease. In view of the ubiquitous presence of anti-Gal in high titers in humans, throughout life, we hypothesized that, analogous with synthesis of anti-blood group antibodies against bacterial antigens, bacteria within normal intestinal flora may provide constant antigenic stimulation for the synthesis of anti-Gal. This hypothesis would imply that anti-Gal may bind to a variety of bacterial strains of human flora. In the present study, the interaction between affinity chromatography-purified anti-Gal and various bacterial strains was studied. By the use of a direct immunostaining assay and an enzyme-linked immunosorbent assay, anti-Gal was found to interact with a variety of Escherichia coli, Klebsiella, and Salmonella strains, some of which were isolates from normal stool. Furthermore, the anti-Gal-binding sites in some strains were found to be present on the carbohydrate portion of bacterial lipopolysaccharides. It is thus suggested that Gal alpha 1----3Gal epitopes in the outer membranes of normal flora enterobacteria may provide a continuous source for antigenic stimulation. Since there is no immune tolerance to the Gal alpha 1----3Gal carbohydrate structure in humans, anti-Gal seems to be constantly produced in response to these enterobacteria. In addition, bacteria which express Gal alpha----3Gal epitopes and which may adhere to various cells mediated binding of anti-Gal to human cell lines. These findings raise the possibility that anti-Gal may damage normal human tissues via inflammatory processes facilitated by bacterial Gal alpha 1----3Gal epitopes.

Antigen-Antibody Reactions↗