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Biomedical subjects

L M Wetzler

Publications and source records attributed to L M Wetzler.

15 recordsLinked to original sources
PubMed1996

Neisserial porins induce B lymphocytes to express costimulatory B7-2 molecules and to proliferate.

The neisserial porins are the major protein components of the outer membrane of the pathogenic Neisseria (N. meningitidis and N. gonorrhoeae). They have been shown to be able to enhance the immune response to poorly immunogenic substances (e.g., polysaccharides, peptides, glycolipids, etc.). To explore the basis of their potent adjuvant activity, the effect of the neisserial porins on T-B cell interactions and T cell costimulation was examined. Neisserial porins increased the surface expression of the costimulatory ligand B7-2 (CD86) but did not affect the expression of B7-1 (CD80). In addition, incubation with the neisserial porins increased the T lymphocyte costimulatory ability of B lymphocytes, which was inhibited by anti-B7-2 but not anti-B7-1 monoclonal antibodies. Upregulation of B7-2 on the surface of B lymphocytes may be the mechanism behind the immunopotentiating activity of neisserial porins.

Adjuvants, Immunologic↗
PubMed1996

Functional characterization of a sialyltransferase-deficient mutant of Neisseria gonorrhoeae.

Previous studies indicate that sialylation of lipopolysaccharide (LPS) by host CMP-N-acetylneuraminic acid (CMP-NANA) catalyzed by bacterial sialyltransferase rendered gonococci resistant to killing by phagocytes, to entry into epithelial cell lines, to killing by immune serum and complement, and to absorption of complement component C3. These results have been confirmed by comparing a sialyltransferase-deficient mutant (strain JB1) with its parent (strain F62) in appropriate tests. In contrast to F62, JB1 was very susceptible to killing by human polymorphonuclear phagocytes in opsonophagocytosis tests and incubation with CMP-NANA did not decrease the level of killing. The inherent resistance of F62 in these tests was probably due to LPS sialylation by CMP-NANA and lactate present in the phagocytes. A JB1 variant expressing the invasion-associated Opa protein was as able to enter Chang human conjunctiva epithelial cells as an Opa-positive variant of F62, suggesting that the sialyltransferase is not required for Opa-mediated entry. After incubation with CMP-NANA, the number of F62 variant gonococci entering cells but not that of JB1 variant gonococci was drastically reduced. Both JB1 and F62 were killed by incubation with rabbit antibody to gonococcal major outer membrane protein, protein I, and human complement, but only F62 was rendered resistant to the killing by incubation with CMP-NANA. Finally, both JB1 and F62 absorbed similar amounts of complement component C3 and the binding was decreased by incubation with CMP-NANA only for the wild type, F62.

Animals↗
PubMed1995

Vaccines for gonorrhea: where are we on the curve?

Human antibodies that bind the gonococcal outer membrane modulate gonorrheal transmission and disease. The effects of antibody binding can favor either the host or the bacteria, and depend on the antigen involved. An effective gonococcal vaccine is feasible, but only by the careful selection and formulation of gonococcal antigens that elicit only host-protective antibodies.

Bacterial Vaccines↗
PubMed1995

Neisserial porins inhibit human neutrophil actin polymerization, degranulation, opsonin receptor expression, and phagocytosis but prime the neutrophils to increase their oxidative burst.

Porins are trimeric proteins that constitute water-filled pores that allow transmembrane diffusion of small solutes through the outer membrane layer of gram-negative bacteria. The porins are capable of inserting into the membranes of eucaryotic cells, and in the present study we have examined the in vitro effects on neutrophil functions of the following purified porins: meningococcal outer membrane protein classes 1 and 3 and gonococcal outer membrane protein 1B (P1B). The neisserial porins inhibited human neutrophil chemoattractant-induced actin polymerization and degranulation of both primary and secondary granules. The neutrophil expression of immunoglobulin G (IgG) Fc receptors II (Fc gamma RII; CDw32) and III (Fc gamma RIII; CD16), as well as the activation-dependent downregulation of Fc gamma RIII, were reduced by the meningococcal and gonococcal porins. The neisserial porins impaired the upregulation of complement receptors 1 (CD35) and 3 (CD11b) and inhibited the phagocytic capacity of neutrophils, as evaluated by the uptake of meningococci (strain 44/76) in the presence of patient serum containing known amounts of IgG against meningococcal porins. The porins also primed neutrophils to increase their intracellular hydrogen peroxide production in response to FMLP, whereas no such priming was observed if the neutrophil protein kinase C was stimulated directly with phorbol myristate acetate. The neisserial porins influenced neutrophil functions in a time- and concentration-dependent manner. The meningococcal class 1 outer membrane protein and the gonococcal P1B tended to alter neutrophil functions more than the meningococcal class 3 protein. Thus, the neisserial porins inhibited human neutrophil actin polymerization, degranulation, opsonin receptor expression, and phagocytosis but primed the neutrophils to increase their oxidative burst. It remains to be determined whether these in vitro observations reflect mechanisms that may be of importance for the interaction between neutrophils and Neisseria species in vivo.

Actins↗
PubMed1994

Serum resistance of Neisseria gonorrhoeae. Does it thwart the inflammatory response and facilitate the transmission of infection?

N. gonorrhoeae differentially subvert the effectiveness of complement (C) and alter the inflammatory responses elicited in human infection. Disseminated (DGI) isolates typically resist killing by normal serum (are serum-resistant), inactivate more C3b (to iC3b preferentially bound via amide linkages), generate less C5a, and result in less inflammation at local sites. Pelvic inflammatory disease isolates are serum-sensitive, inactivate less C3b (while maintaining active C3b via stable amide linkages), generate more C5a, and result in more inflammation at local sites. Sialylation of SS gonococci, presumed to occur in vivo, converts them to serum-resistant, but it does not change the patterns of C3b inactivation and therefore may not affect local inflammation. IgG antibody directed against gonococcal reduction modifiable protein (Rmp) blocks C-mediated killing of N. gonorrhoeae. Anti-Rmp blocking antibodies may harbor specificity for OmpA sequences shared with other neisserial species or Enterobacteriaceae or may be directed against unique Rmp upstream cysteine loop specific sequences, or both. Preexisting antibodies directed against Rmp facilitate transmission of gonococcal infection to exposed women; exclusion of highly immunogenic Rmp antigens from vaccine candidates may be important.

Antibodies, Bacterial↗
PubMed1994

Humoral immune response to class 1 outer membrane protein during the course of meningococcal disease.

We have determined the amounts of specific anti-class 1 outer membrane protein antibodies in sera from 25 patients during the course of systemic meningococcal disease, using purified class 1 protein as the sensitizing antigen in an enzyme-linked immunosorbent assay. The class 1 protein was obtained from a variant of strain 44/76 (B:15:P1.7,16) lacking class 3 and class 4 outer membrane proteins. Specific anti-class 1 (serosubtype P1.7,16) outer membrane protein immunoglobulin G (IgG) antibody levels increased significantly in 12 patients (12 of 25; 48%), regardless of the serotype of the infecting strain, indicating that the antibodies reacted in part with epitopes not determined by the monoclonal antibodies used for serotyping. Most patients had low levels of anti-class 1 IgG antibodies during the acute illness. The antibody levels peaked during the second week of disease and returned to near baseline levels in sera collected 6 weeks to 12 months after the onset of the disease. The majority of the specific anti-class 1 IgG antibodies bound to surface-exposed epitopes on whole bacteria and belonged to the IgG1 and IgG3 subclasses. Anti-class 1 IgA and IgM antibodies were not detected in any of the patient sera. Prior to disease, seven patients had been immunized with a meningococcal outer membrane vesicle vaccine developed from strain 44/76 (P1.7,16). None of these patients was infected with meningococcal strains containing class 1 protein homologous or partly homologous to that of the vaccine strain, indicating serosubtype-specific protection. The highest anti-class 1 IgG antibody peak levels were seen in immunized patients infected with strains of heterologous serotype, suggesting an anamnestic response. However, these patients were not protected from meningococcal disease after immunization.

Adolescent↗
PubMed1993

Humoral immune response to the class 3 outer membrane protein during the course of meningococcal disease.

We have determined the amounts of specific anti-class 3 outer membrane protein antibodies of immunoglobulin G (IgG), IgM, and IgA isotypes in patient sera during the course of meningococcal disease by using purified class 3 protein as the sensitizing antigen in an enzyme-linked immunosorbent assay. The class 3 protein was obtained from a variant of strain 44/76 (B:15:P1.7,16) lacking class 1 and class 4 outer membrane proteins. Serum samples from 25 patients with systemic meningococcal disease caused by organisms of various serotypes were collected during the course of disease. Seven of these patients had been immunized with a meningococcal outer membrane vesicle vaccine made from strain 44/76 prior to disease. An increase in specific anti-class 3 (type 15) outer membrane protein IgG antibodies was demonstrated in 22 of 25 patients (88%), regardless of the serotype of the infecting strain. This indicates that the specific anti-class 3 antibodies were reacting in part with epitopes not determined by the monoclonal antibodies used for serotyping. A considerable heterogeneity in antibody levels and IgG subclass response was seen. Most patients had low levels of anti-class 3 antibodies during the acute illness, with antibodies peaking during the second week of disease and returning to near baseline in sera collected 6 to 12 months after the onset of the disease. The majority of the specific anti-class 3 IgG antibodies were shown to bind to surface-exposed epitopes on the whole bacteria and to belong to IgG1 and IgG3. The highest anti-class 3 IgG peak levels were seen in patients infected with strains of the homologous serotype after vaccination with the meningococcal outer membrane vesicle vaccine, suggesting an anamnestic response. However, these patients were not protected from meningococcal disease after immunization.

Adolescent↗
PubMed1992

Gonococcal porin vaccine evaluation: comparison of Por proteosomes, liposomes, and blebs isolated from rmp deletion mutants.

The gonococcal major outer membrane protein, Por (protein I), is a potential vaccine candidate. A large-scale animal trial compared the immunogenicity of Por inserted in liposomes (mimicking its in vivo structure) with previously used vaccines containing Por. Por was purified from Rmp (protein III)-negative gonococcal mutants and made into five different formulations: proteosomes, proteosomes absorbed to alum, liposomes, gonococcal membrane blebs, and gonococcal membrane blebs absorbed to alum. Por liposomes induced the greatest amount of Por antibodies; proteosomes and both preparations of blebs induced minimal amounts of Por antibodies. Proteosomes absorbed to alum induced slightly lower amounts of Por antibodies than did liposomes, especially when protein IA was used. Whole-organism absorption studies revealed that a significantly greater percentage of liposome-induced Por antibodies recognized exposed portions of the protein than did proteosome- or proteosome/alum-induced antibodies.

Adjuvants, Immunologic↗
PubMed1992

Gonococcal lipooligosaccharide sialylation prevents complement-dependent killing by immune sera.

Previous investigators have demonstrated that a sialic acid residue is added to the terminal galactose moiety of gonococcal lipooligosaccharide (LOS) when incubated with 5'-CMP-N-acetylneuraminic acid. When this in vitro sialylation occurs, gonococci become resistant to the bactericidal activity of normal human serum. This is believed to result because the added sialic acid residue blocks the binding of bactericidal anti-LOS antibodies present in normal human serum. We extend these studies by demonstrating that sialylated gonococci also become resistant to the bactericidal effect of immune sera containing antibodies that recognize exposed components of the outer membrane besides LOS. Prevention of antibody binding to the organism was not the cause, since the same percentage of bactericidal antibodies to the major outer membrane protein, Protein I, can be absorbed with sialylated organisms as with wild-type organisms. In addition, gonococcal sialylation prevents opsonophagocytosis by antigonococcal antisera. The negative effect of sialic acid on the complement pathway might be the reason for the findings in this study.

Antigens, Bacterial↗
PubMed1989

The construction and characterization of Neisseria gonorrhoeae lacking protein III in its outer membrane.

Protein III (PIII) is a highly conserved, antigenically stable gonococcal outer membrane protein that is closely associated with the major outer membrane protein, protein I (PI). We have previously reported the cloning of the PIII gene. This gene was inserted into the Eco RI site of the runaway plasmid pMOB45. The beta-lactamase (beta la) Bam HI restriction fragment from the gonococcal plasmid pFA3 was inserted at the Xba I site in the PIII gene. The plasmid construct was Hae III methylated and the PIII/beta la insert was excised with Eco RI and used to transform gonococcal strain F62. One beta la+, ampicillin-resistant transformant was isolated and designated 2D. A Western blot of 2D whole cell lysate was probed with affinity-purified polyclonal PIII antisera. No PIII reactivity was detected. Southern blot analysis was performed on F62 and 2D chromosomal DNA that were cut with Eco RI or Cla I. A PIII DNA probe hybridized with fragments 2.2 kb larger in strain 2D than strain F62. This corresponds to the size of the beta la insert. A beta la-specific probe hybridized with the same 2D restriction fragments as above, but did not react with any F62 fragments, confirming that homologous recombination had occurred. There were minimal phenotypic changes between 2D and its parent strain, F62. Chromosomal DNA from 2D was able to transform gonococcal strains F62, UU1, and Pgh 3-2, rendering these PIII-. 2D and other PIII- transformants can now be used to study the role of PIII in gonococcal physiology, metabolism, membrane structure, and pathogenesis. Moreover, we now have organisms from which we can purify gonococcal proteins without PIII contamination.

Ampicillin Resistance↗
PubMed1988

Characterization and specificity of antibodies to protein I of Neisseria gonorrhoeae produced by injection with various protein I-adjuvant preparations.

A major goal of gonococcal research is the development of a gonorrheal vaccine. A vaccine candidate is the major outer membrane protein (PI) of the gonococcus, which has limited antigenic variability. Two main subtypes, PIA and PIB, and nine main serotypes have been described. To avoid raising anti-protein III (PIII)-blocking antibodies and limit potential lipooligosaccharide toxicity, PI was chromatographically isolated with minimal PIII contamination (less than 1%) from Pgh 3-2 (PIB), a serum-sensitive gonococcal strain and UU1 (PIA), a serum-resistant gonococcal strain. Alum was used as an adjuvant and the antibodies raised in rabbits did not agglutinate the organisms, were not opsonic, and bactericidal titers were not increased. To present PI in a form mimicking its in vivo disposition, it was inserted into liposomes. The resulting antisera did agglutinate the organism and contained opsonic and bactericidal activity greater than the preimmune sera or alum-generated sera. The PIB liposome antisera also had higher ELISA titers to a synthetic peptide equivalent to an exposed portion of PIB and a higher percentage of antibodies absorbed by whole organisms than the PIB alum antisera. We speculate that when PI is presented in liposomes, the antibodies raised are mainly to surface-exposed epitopes of the protein as opposed to when PI is presented absorbed to alum, where the antibodies are produced mainly to buried epitopes.

Adjuvants, Immunologic↗