Biomedical subjects

J M Griffiss

Publications and source records attributed to J M Griffiss.

At least 55 records · Page 3Linked to original sources

PubMed1991

Endogenous sialylation of the lipooligosaccharides of Neisseria meningitidis.

Monoclonal antibodies (MAb) 3F11 and 06B4 recognize epitopes that are conserved on gonococcal lipooligosaccharides (LOS), present on some meningococcal LOS, and conserved on human erythrocytes. LOS of some group B and C prototype meningococcal LOS strains (LOS serotypes L1 to L8) treated with neuraminidase showed increased expression of the 3F11 and 06B4 MAb-defined epitopes. Neuraminidase-treated LOS separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and silver stained showed a shift in migration from a component with a mass of approximately 4.8 kDa to a component with a mass of between 4.5 and 4.6 kDa. The same strains grown in medium with excess CMP-N-acetylneuraminic acid had LOS that shifted in migration to a slightly higher component (mass, approximately 4.8 kDa). Chemical analysis of the neuraminidase-digested products from one LOS indicated it contained approximately 1.5% sialic acid. Covalent linkage between sialic acid and the LOS was confirmed by analysis of de-O-acylated and dephosphorylated LOS by liquid secondary ion mass spectrometry. Three studies show that some meningococci contain sialic acid in their LOS, that the sialic acid is cleaved and lost in conventional acetic acid hydrolysis, and that the sialic acid alters the expression of MAb-defined epitopes.

Antibodies, Monoclonal↗

PubMed1990

In vitro and in vivo modification of Neisseria gonorrhoeae lipooligosaccharide epitope structure by sialylation.

After growth of gonococci in the presence of cytidine monophospho-N-acetyl-neuraminic acid (CMP-NANA), their 4.5-kD lipooligosaccharide (LOS) component was increased by approximately 400 daltons, whereas the LOS of strains lacking the 4.5-kD component were unaffected. Expression of mAb-defined epitopes on the 4.5-kD component was decreased on LOS of strains grown in CMP-NANA, and treatment of the LOS with neuraminidase reversed this affect. Gonococci incubated with human PMNs also had decreased expression of the 4.5-kD+ epitopes. A detergent extract of gonococci incorporated radiolabeled NANA in the LOS, suggesting the presence of a sialyltransferase in gonococci. Exogenous sialyltransferases also could use LOS as an acceptor.

Animals↗

PubMed1990

Structural models for the cell surface lipooligosaccharides of Neisseria gonorrhoeae and Haemophilus influenzae.

A structural model is proposed for the surface glycolipids, or lipooligosaccharides (LOS), of gram-negative pathogenic bacteria that colonize human mucosae, e.g. Neisseria gonorrhoeae and Haemophilus influenzae. The development of this model has involved analysis of a series of pyocin-resistant mutants with altered LOS and other recent immunochemical and structural data. A comprehensive approach to determining the necessary structural data has been constructed that utilizes liquid secondary ion mass spectrometry, tandem mass spectrometry, methylation analysis and nuclear magnetic resonance. To prepare purified oligosaccharides for these analyses, chromatographic and chemical techniques have been developed that include high-pH anion-exchange chromatography of underivatized oligosaccharides and reverse-phase chromatography after derivatization with hydrazino alkyl benzoates. The proposed LOS model has several unique features that distinguish it from models developed for the lipopolysaccharides of enteric bacteria. This information should lead to an understanding of the unique structure/function relationship of LOS and to the development of carbohydrate-based vaccines.

Chromatography, Gel↗

PubMed1990

Modification by sialic acid of Neisseria gonorrhoeae lipooligosaccharide epitope expression in human urethral exudates: an immunoelectron microscopic analysis.

Expression of lipooligosaccharide (LOS) antigenic determinants during human gonococcal infection was studied in secretions from seven men with gonococcal urethritis. Five monoclonal antibodies with distinct gonococcal LOS specificities and an H.8 lipoprotein monoclonal antibody were used in combination with immunogold electron microscopic analysis. The LOS epitope defined by antibody 6B7 was present on all seven strains in secretions and after in vitro growth. Gonococci from six of seven patients, when grown in vitro, expressed the 6B4 LOS epitope. The 6B4 epitope is a Gal beta 1-4-GlcNAc residue, which is immunochemically similar to the precursor of the human erythrocyte i antigen. This epitope was found unmodified on gonococcal LOS in urethral secretions from two patients. The unmodified epitope could not be demonstrated on organisms in five secretions. Neuraminidase digestion exposed the 6B4 epitope on organisms in these secretions and increased the 6B4 epitope density in the two secretions, which contained the unmodified epitope. These studies indicate that in vivo modification by sialylation of gonococcal LOS Gal beta 1-4-GlcNAc residue occurs during human infection.

Antibodies, Monoclonal↗

PubMed1990

Localization of host immunoglobulin G to the surface of Pneumocystis carinii.

In immunoblotting studies of Pneumocystis carinii surface proteins, we found that a secondary antibody, anti-human immunoglobulin G (IgG), recognized a 52-kilodalton (kDa) band in homogenates of P. carinii purified from human autopsy lungs and bronchoalveolar lavage fluids, even when serum as a source of primary antibody was omitted. The electrophoretic mobility of the 52-kDa band is identical to that of IgG heavy chains. In addition to affinity-purified, anti-human IgG, monoclonal antibodies specific for the Fab and Fc regions of human IgG recognized the 52-kDa band. To determine whether the 52-kDa band represents IgG bound to the surface of P. carinii, we treated intact organisms with Triton X-100 and acid in order to elute immunoglobulin from the surface of P. carinii. After purification over a protein G column, the eluate comigrated with human IgG, was recognized by anti-IgG, and bound to discrete bands with molecular sizes of 65 to 70, 60, 50, and 35 kDa in purified, rat-derived P. carinii. To confirm the presence of human IgG on the surface of P. carinii, we performed immunocytochemical and immunoelectron microscopic studies. Staining of intact P. carinii aggregates by anti-human IgG was pronounced and was abolished by acid treatment. IgA was also present. Ultrastructural studies showed the presence of IgG on the cyst wall and on fine membranous structures and vesicles adjacent to cysts. We conclude that the surface of P. carinii is coated with human IgG. The close association of human IgG with P. carinii may have implications for the pathogenesis of P. carinii pneumonia in acquired immunodeficiency syndrome.

Acquired Immunodeficiency Syndrome↗

PubMed1990

Measurement of the human immune response to meningococcal lipooligosaccharide antigens by using serum to inhibit monoclonal antibody binding to purified lipooligosaccharide.

We developed a human inhibition monoclonal enzyme-linked immunosorbent assay (HIMELISA) to investigate the human immune response to the lipooligosaccharides (LOS) of Neisseria meningitidis. Monoclonal antibodies (MAb) were used to define seven epitopes on four LOS molecules of a meningococcal strain (126E) previously shown to express immunogenic LOS epitopes. The assay could distinguish epitope-specific antibody within whole sera. Neither the specificity nor the amount of the antibody measured by HIMELISA in sera of vaccinates changed during the immune response to meningococcal capsular polysaccharides, a chemically unrelated antigen. By using the HIMELISA, it was determined that sera from adults convalescing from meningococcal disease strongly inhibited MAb binding to two of the seven defined epitopes. The 3.6-kilodalton LOS of strain 126E expressed both of these epitopes. In addition, one of the inhibited epitopes was also expressed on the 4.0-kilodalton LOS of strain 126E. The convalescent-phase sera inhibited MAb binding to these two epitopes when they were expressed on LOS of diverse meningococcal strains. An acute-phase serum blocked MAb to the two epitopes to a lesser degree than did a convalescent-phase serum from the same patient. Immunoblotting the sodium dodecyl sulfate-polyacrylamide gel electrophoresis-separated LOS with convalescent-phase sera confirmed the specificity of the human anti-LOS antibody identified by HIMELISA.

Antibodies, Bacterial↗

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↗

PubMed1989

Human IgA1 initiates complement-mediated killing of Neisseria meningitidis.

We studied the effect of human IgA1, the predominant IgA subclass in serum, on C-mediated killing of Neisseria meningitidis. We purified monomeric IgA1 from normal human serum and tetravalent meningococcal polysaccharide vaccinate serum by using the following successive chromatographic steps: jacalin lectin affinity, Superose 12 FPLC gel filtration, Mono Q FPLC anion exchange, and anti-IgG affinity. SDS-PAGE, ELISA, and Western immunoblot analyses of the IgA1 detected no trace of contaminating IgG or IgM. IgA1 initiated partial or complete lysis (62 to 100%) of nine group C strains by using either normal, hypogammaglobulinemic, factor B-depleted, or properdin-deficient human serum as a C source, but IgA1 was unable to effect killing in serum chelated with 10 mM MgCl2 and 10 mM EGTA. Lytic activity was dependent on the group C strain and the source of the IgA1; neither IgA1 preparation was bactericidal for all nine strains. Removal of the Fc portion of IgA1 with pepsin completely abolished bactericidal activity. We purified and radiolabeled C component C3, and found that IgA1 did not increase C3 deposition. With the use of a group C polysaccharide ELISA, we found that the vaccinate IgA1 had a high titer of group C polysaccharide antibody, whereas the IgA1 purified from normal human serum had no detectable group C polysaccharide specificity. Absorption of the vaccinate IgA1 with alum-bound group C polysaccharide did not affect the killing of a sensitive strain, but it did potentiate the killing of a previously resistant strain. Western immunoblots of whole cell lysates, outer membrane complex, and purified lipooligosaccharide showed that the bactericidal IgA1 was specific for several outer membrane proteins. Four of the proteins recognized by both IgA1 preparations had apparent Mr of 29, 42, 66, and 74 kDa. We conclude that IgA1, when bound to specific outer membrane proteins, can initiate lysis of group C meningococci via the classical C pathway, and that initiation of lysis is an Fc-dependent event which occurs without an increase in C3 deposition.

Adult↗

PubMed1989

Structure and heterogeneity of the oligosaccharides from the lipopolysaccharides of a pyocin-resistant Neisseria gonorrhoeae.

The compositions and partial structures of the oligosaccharides from the lipopolysaccharides (LPS) of a pyocin-resistant Neisseria gonorrhoeae (strain JW31R) have been determined by liquid secondary ion mass spectrometry (LSIMS), tandem mass spectrometry, and methylation analysis. Four major structures were identified with Mr 2123, 2000, 1961, and 1838, as well as seven species of lower abundance of Mr 1758-1272. The largest of the major oligosaccharides (Mr, 2122) consists of 3-deoxymanno-2-ketooctulosonic acid (KDO)-Hep2GalNAcGlcNAcGal4Glc2 (Hep, heptose) and phosphoethanolamine (PEA). The smaller oligosaccharides are truncated versions of this larger oligosaccharide. The oligosaccharides consist of a common triantennary structure containing KDO at the reducing terminus attached to a heptose disaccharide. A hexose (Hex)2-3 branch is attached to the heptose linked directly to KDO and a GalNAc-Hex3, GlcNAc, and PEA are separately attached to the second heptose. These oligosaccharides are the first structures to be determined for a gonococcal LPS and should further our understanding of the structural and antigenic diversity of these glycolipids.

Bacteriocins↗

PubMed1989

Neisseria lactamica and Neisseria meningitidis share lipooligosaccharide epitopes but lack common capsular and class 1, 2, and 3 protein epitopes.

Neisseria lactamica, a common human pharyngeal commensal, contributes to acquired immunity to Neisseria meningitidis. To define the surface antigens shared between these two species, we used monoclonal antibodies (MAbs) to study 35 N. lactamica strains isolated in various parts of the world for cross-reactivity with meningococcal capsules, outer membrane proteins, and lipooligosaccharides (LOS). No N. lactamica strain reacted significantly with MAbs specific for capsular group A, B, C, Y, or W, and we were unable to extract capsular polysaccharide from them. Only 2 of 33 strains reacted weakly with MAbs against class 2 serotype proteins P2b and P2c. None reacted with MAbs specific for meningococcal class 1 protein P1.2 or P1.16 or class 2/3 serotype protein P2a or P15. Most N. lactamica strains (30 of 35) bound one or more of seven LOS-specific MAbs. Two LOS epitopes, defined by MAbs O6B4 and 3F11, that are commonly found on pathogenic Neisseria species were found on 25 of 35 N. lactamica. Analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotting showed that the LOS of N. lactamica are composed of multiple components that are physically and antigenically similar to the LOS of pathogenic Neisseria species. Among four other commensal neisserial species, only Neisseria cinerea shared LOS epitopes defined by MAbs O6B4 and 3F11. Previous studies have shown that pharyngeal colonization with N. lactamica induces bactericidal antibodies against the meningococcus. We postulate that shared N. lactamica and meningococcal LOS epitopes may play an important role in the development of natural immunity to the meningococcus.

Antibodies, Monoclonal↗

PubMed1989

In vitro differentiation of human-derived Pneumocystis carinii.

Two major impediments to the development of an in vitro cultivation system for Pneumocystis carinii are lack of an accurate means of quantitation and difficulty in determination of viability over time. Human-derived P. carinii exists as aggregates of cysts and trophozoites in bronchoalveolar lavage fluid. These aggregates persisted over time in an in vitro system consisting of a monolayer of radiated or nonradiated A549 cells, RPMI 1640, and 10% fetal calf serum. Parallel measurements at specified times after introduction into the in vitro system of the number of aggregates, total aggregate area, and total number of cysts varied and appeared to be a function of the number of aggregates initially added into the system. However, cyst density, the number of cysts per unit aggregate area, was independent of the total number of aggregates added into the system. Cyst density was determined by staining an aggregate with a cyst-specific stain, such as toluidine blue, and counterstaining with Diff-Quik, allowing simultaneous visualization of cysts and aggregate area. Preliminary experiments suggested an increase in cyst density over time. Cyst density may be a means of accurate in vitro quantitation of P. carinii.

Acquired Immunodeficiency Syndrome↗

PubMed1988

Lipooligosaccharides (LOS) of Neisseria gonorrhoeae and Neisseria meningitidis have components that are immunochemically similar to precursors of human blood group antigens. Carbohydrate sequence specificity of the mouse monoclonal antibodies that recognize crossreacting antigens on LOS and human erythrocytes.

We have used mouse mAbs, 3F11 and 06B4, that are specific for highly conserved epitopes of Neisseria gonorrhoeae lipooligosaccharides (LOS) to identify immunochemically similar structures on human erythrocytes. mAb 3F11 agglutinated erythrocytes from all randomly selected adult humans, while mAb 06B4 agglutinated only 80% of the same specimens. The antibodies had an activity with erythrocytes similar to human cold agglutinins in that agglutination occurred at 4 degrees C and decreased with increasing incubation temperature. Human infant erythrocytes were agglutinated less well, but enzymatic treatment of either infant or adult cells resulted in an increase in expression of the 3F11- and 06B4-defined epitopes. Both antibodies bound to a series of neutral glycosphingolipids from human erythrocytes and neutrophils that have a type 2 (Gal beta 1----4GlcNAc) or N-acetyllactosamine structure. Neither antibody bound to glycosphingolipids from human meconium, which have a type 1 (Gal beta 1----3GlcNAc) structure. The antibodies were unable to bind to N-acetyl-lactosamine glycosphingolipids with a nonreducing terminal sialic acid or a Gala1----3Gal disaccharide. Antibody binding also was blocked by the presence of fucose linked to the penultimate glucosamine residue of N-acetyllactosamine glycosphingolipids. Although both antibodies bound to linear and branched-chain N-acetyllactosamine glycosphingolipids, 3F11 had a higher affinity for branched structures than did 06B4. The activity of 3F11 with human adult and infant treated and untreated erythrocytes with N-acetyllactosamine glycosphingolipids, and with LOS was very similar, if not identical, in specificity to 1B2, an mAb prepared from mice inoculated with a linear N-acetyllactosamine glycosphingolipid.

Adult↗

PubMed1988

Epitope expression of gonococcal lipooligosaccharide (LOS). Importance of the lipoidal moiety for expression of an epitope that exists in the oligosaccharide moiety of LOS.

Antigenic expression of lipooligosaccharide (LOS) of strain F62 of Neisseria gonorrhoeae, was investigated with mouse monoclonal IgM antibody 3F11. F62 LOS was modified in various ways in order to understand structural requirements for expression of the 3F11-defined epitope. When the LOS was partially deacylated by treating it with 50 mM NaOH at 80 degrees C for 20 min or with anhydrous hydrazine at 80 degrees C for 20 min, the binding of 3F11 to those deacylated LOS samples decreased significantly. Removal of phosphate groups by treatment of the LOS with HF (4 days at 4 degrees C) did not affect the antigenicity at all. Neither did reduction of carboxyl groups in the LOS molecule (by activation of carboxyl groups with a carbodiimide followed by treatment with NaBH4) alter epitope expression. On oxidation with NaIO4, the LOS lost its antigenicity completely. The presence of Mg2+ did not change the circular dichroism (CD) behavior of F62 LOS. However, the partially deacylated LOS samples showed significantly different CD patterns in the 190-200 nm region compared with F62 LOS, which suggests conformational changes of F62 LOS due to the loss of fatty acids in the lipoidal moiety. Oligosaccharide (OS) and lipoidal components obtained after hydrolysis of F62 LOS with 1% acetic acid, were not recognized by the antibody. The antigenicity of OS was not retained by non-stereospecific acylation of OS with decanoyl chloride. We conclude the following: (1) 3F11-defined epitope exists in the OS moiety of F62 LOS; however, for it to be expressed, the carbohydrate moiety must be in a certain conformation that is defined by an overall structure of the LOS molecule. This structure is significantly influenced by some of the fatty acids in the lipoidal moiety of the LOS molecule; (2) the presence of phosphate or 3-deoxy-manno-2-octulosonic acid (dOclA) is not essential for expression of the 3F11-defined epitope; (3) the presence of divalent cations does not affect epitope expression.

Antibodies, Monoclonal↗

PubMed1988

Electromorphic characterization and description of conserved epitopes of the lipooligosaccharides of group A Neisseria meningitidis.

We studied the lipooligosaccharides (LOS) of 28 group A Neisseria meningitidis of epidemiologically diverse origins to investigate whether each of the LOS serotypes found in serogroup A could be identified physically as well as antigenically. Using a dot blot assay with LOS-specific monoclonal antibodies (MAbs), we identified four epitopes that were serotype specific. The LOS from strains of each serotype were electromorphically and antigenically distinct when analyzed by silver-stained sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotting. The LOS of L8 strains contained a 3,600-Mr component that bound the L8 MAb. The LOS of L9 strains contained two major components of 4,500 and 4,200 Mr. They bound the L9 MAb to the larger component. The LOS of L10 strains had a single major component of 4,000 Mr that bound the L10 MAb. The LOS of L11 strains contained a major 3,600-Mr component that could not be distinguished from the 3,600-Mr LOS of L8 strains by SDS-PAGE but that bound the L11 MAb. LOS of group A strains contained a highly conserved epitope in addition to a serotype-specific epitope. This was identified by a MAb that bound to all the strains on dot-blots and to multiple LOS components of various Mrs on immunoblots. We conclude that the LOS which bear the L9, L10, and L11 determinants are physically distinct and can be identified by SDS-PAGE or MAb binding or both. L8 and L11 are both borne on a 3.6-kilodalton LOS and can only be distinguished serologically.

Antibodies, Bacterial↗

PubMed1988

Use of transformation to construct Neisseria gonorrhoeae strains with altered lipooligosaccharides.

DNA isolated from a nalidixic acid- and rifampin-resistant derivative of Neisseria gonorrhoeae serum-resistant strain 302 (MUG116), a strain that reacts with monoclonal antibody (MAb) 2-1-L8, was used to transform N. gonorrhoeae DOV, a serum-sensitive strain, to antibiotic resistance and/or reactivity with the MAb. MAb 2-1-L8 binds to a 3.6-kilodalton lipooligosaccharide (LOS). Reactivity with MAb 2-1-L8 transformed as a single marker and was unlinked to either of the antibiotic resistance markers. Immunoblot analysis of LOSs separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed that new LOSs were synthesized in the transformed cells and that these LOSs corresponded to those of the DNA donor. Although multiple LOS components were made by the transformants, the MAb recognized only one. All transformants that were selected for on the basis of strong reactivity with MAb 2-1-L8 were serum resistant; however, the level of resistance correlated with the apparent loss of recipient LOS components. MAb 2-1-L8-reactive transformants that still produced DOV LOS components remained serum sensitive.

Blood Bactericidal Activity↗

PubMed1988

Instability of expression of lipooligosaccharides and their epitopes in Neisseria gonorrhoeae.

We assessed variation in the expression of lipooligosaccharide (LOS) components and their epitopes within populations of a strain of Neisseria gonorrhoeae by using the monoclonal antibodies (MAbs) O6B4 and 3F11 and immunoenzymatic, immuno-colloidal gold electron microscopic, and sodium dodecyl sulfate-polyacrylamide gel electrophoretic procedures. Wild-type organisms varied in binding of both MAbs. We used the intensity of immunoenzymatic colony blot color to distinguish four binding variants for each MAb: red (R), pink (P), and colorless (nonreactive [N]) and an N back to R (N-R) revertant. R to P to R and R to N to R variation occurred at frequencies of 0.2% and 0.02%, respectively. The electrophoretic LOS profiles and MAb immunoblot patterns of the R, P, and N-R variants were the same as those of the wild type. LOSs of the N variants, in contrast, were of lower Mr, bound neither 3F11 nor O6B4 MAb, and contained as their major component the 3.6-kilodalton LOS that bears the L8LOS epitope of N. meningitidis. Results of immunoelectron microscopic studies were consistent with LOS binding patterns. Large number of colloidal gold particles were deposited about both R and P variants, distally from R organisms, but proximally from P organisms. N variant organisms, like their LOS, bound neither of the MAbs. N-R variant organisms were like the wild type in that they showed much variation in the amounts of MAb they bound.

Antibodies, Monoclonal↗

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↗

PubMed1988

Pneumocystis carinii in sputum. Comparable efficacy of screening stains and determination of cyst density.

To determine whether accurate quantification of Pneumocystis carinii cysts in sputum is possible using currently available means and to determine the optimal stain for screening sputum for P carinii, we undertook a comparative evaluation of readily available stains: (1) Diff-Quik, a stain that provides tinctorial detail comparable with Giemsa, (2) toluidine blue, and (3) silver methenamine on simultaneously thawed sputum samples containing P carinii. We also studied the Diff-Quik stain in combination with (4) toluidine blue and (5) silver methenamine. The three stains were comparable for screening purposes. Each disclosed similar numbers of P carinii aggregates on similarly prepared slides. Cysts were difficult to recognize on Diff-Quik due to the negatively staining character of the cyst wall with this stain. The cyst-wall stains, toluidine blue and silver methenamine, clearly stained the cyst wall. The combination of a cyst-wall stain with Diff-Quik allowed visualization of cysts, highlighted by the cyst-wall stain, and aggregate area, delineated by the Diff-Quik, allowing expression of number of cysts per aggregate area, a measure termed cyst density.

Animals↗