Sixth International Congress of Mucosal Immunology, July 23, 1990. Dental caries vaccines.
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Animals↗
Biomedical subjects
T Lehner
Publications and source records attributed to T Lehner.
The reactivity of naturally sensitized human CD4 cells and IgG antibodies to synthetic peptides derived from the amino terminal sequences of a 3800 MW Streptococcus mutans antigen.
Natural immunity to synthetic peptides (SP) derived from the sequences of a 3800 MW streptococcal antigen (SA) was found in human subjects. Significant serum IgG antibodies were detected both to the native SA and to peptides consisting of residues 3-13, 1-15 and 1-20. Inhibition studies confirmed cross-reactivity between the native SA and SP. A series of short peptides with deletions at the amino and carboxy termini were then tested to determine the sequence of B-cell epitopes. Residues 8-13 and 1-6 bound significant serum IgG antibodies, but residues 8-13 were more effective and consistent in inhibiting human antibodies than residues 1-6. These results suggest that residues 8-13 constitute a major B-cell epitope but that residues 1-6 may represent a minor B-cell epitope. The human CD4 subset of T cells was then examined by stimulating the cells with SA or SP and measuring the uptake of [3H]thymidine [( 3H]TdR). The cells were found to be sensitized in vivo to both the native SA and the SP and cross-reactivity between the SA and SP was shown by enrichment and depletion experiments on antigen-coated monocytes. As with the B-cell epitope, the series of short peptides was used to stimulate CD4 cells, in order to determine the T-cell epitope. Residues 6-15 were the shortest SP which stimulated significant [3H]TdR uptake and this peptide was designated as a T-cell epitope. The results suggest that natural oral immunization with Streptococcus mutans induces serum antibodies and T-cell sensitization to a peptide in which a T-cell epitope (residues 6-15) overlaps with a B-cell epitope (residues 8-13). Furthermore, a comparison between linear and cycled peptides suggests that unlike immunogenicity which is commonly enhanced by the more rigid cyclized peptides, antigenicity is favoured by linear peptides. This was evident not only for antibodies but also for T-cell proliferative responses.
Amino Acid Sequence↗
Genetic mapping of X-linked albinism-deafness syndrome (ADFN) to Xq26.3-q27.I.
X-linked albinism-deafness syndrome (ADFN) was described in one Israeli Jewish family and is characterized by congenital nerve deafness and piebaldness. The ADFN mutation probably affects the migration of neural crest-derived precursors of the melanocytes. As a first step toward identifying the ADFN gene, a linkage study was performed to localize the disease locus on the X chromosome. The family was found to be informative for 11 of 107 RFLPs along the X, and two-point analysis showed four of them--factor 9 (F9), DXS91, DXS37, and DNF1--to have definite or suggestive linkage with ADFN. Multipoint linkage analysis indicated two possible orders within this cluster of loci, neither of which was preferable. In both orders F9 was the most distal, and the best estimate for the location of ADFN was between F9 and the next proximal marker (8.6 cM from F9 [Z = 8.1] or 8.3 cM from F9 [Z = 7.9]). These results suggest that the ADFN is at Xq26.3-q27.1. Disagreement between our data and previous localization of DXS91 at Xq11-q13 was resolved by hybridization of the probe pXG-17, which detects the DXS91 locus, to a panel of somatic cell hybrids containing different portions of the X chromosome. This experiment showed that this locus is definitely at Xq24-q26. Together with the linkage data, our results place DXS91 at Xq26 and underscore the importance of using more than one mapping method for the localization of molecular probes.
Albinism↗
Sequence analysis of the cloned streptococcal cell surface antigen I/II.
The gene spa P (formerly designated as spa P1) encoding the Mr 185,000 surface antigen (I/II) of Streptococcus mutans, serotype c (NG5), has been sequenced. The gene (4683 bp) encodes a protein of 1561 amino acid residues including putative signal peptide (residues 1-38) and transmembrane (residues 1537-1556) sequences. The N-terminal region (60-550) has alanine-rich repeats and is predicted to be alpha-helical. However, the C-terminal region (800-1540) is proline-rich and favours an extended structure. Except for a short central variable region the sequences appear to be highly conserved for S. mutans serotype c. N-Terminal sequencing of separated antigen I and antigen II polypeptides suggests that the former represents the N-terminal and the latter the C-terminal portions of the intact antigen.
Amino Acid Sequence↗
Immunogenicity of synthetic peptides derived from the sequences of a Streptococcus mutans cell surface antigen in nonhuman primates.
The immunogenicity and antigenicity of synthetic peptides (SP) derived from the sequences of a cell surface Ag of Streptococcus mutans were investigated in macaque monkeys. Immunization with the free peptides of 11, 17, and 21 residues failed to elicit serum antibodies or T cell responses. However, immunization with the SP17 and SP21 linked to tetanus toxoid (TT) as a carrier elicited serum antibodies and proliferative responses of lymphocytes, not only to the SP but also to the native streptococcal Ag. In vivo recall of SP-TT immunized monkeys with suboptimal doses of the native streptococcal Ag resulted in a significant increase in antibodies, both to the SP and the streptococcal Ag, confirming that the SP shares antigenic epitopes with the native Ag. B and T cell epitopes were then determined and a B cell epitope was found in residues 8-13, whereas an overlapping T cell epitope was located in residues 7-15. The T cell epitope has an amino-terminal leucine and carboxy-terminal glycine and alanine added to residues 8-13 of the B cell epitope. In spite of the B and T cell epitopes being expressed in SP17 (residues 1-15), the monomer failed to induce serum antibodies without a carrier. However, immunization with a dimer of SP17 elicited both serum antibodies and proliferative responses of lymphocytes without a carrier. The results suggest that the monomeric SP17 is not immunogenic and needs to be dimerised in order to elicit antibodies and T cell responses, both to the SP and to the streptococcal Ag.
Amino Acid Sequence↗
T cell interactions generated by synthetic peptides covalently linked to a carrier.
We have attempted to extend the synthetic peptide-carrier bridge concept of T cell-B cell interaction to T cell-T cell interaction. DNA synthesis of human CD4 cells that were sensitized in vivo to a native streptococcal antigen (SA) was stimulated in vitro with synthetic peptides (SP) derived from the sequence of native SA. The SP were linked to tetanus toxoid (TT) as a carrier which was recognized by primed T cells. The uptake of [3H]thymidine was significantly greater when stimulated with covalently linked SP-TT than that with non-covalently mixed SP and TT. The TT- and SP-sensitized CD4 cells were then enriched and depleted by panning on TT- or SP-treated monocyte layers. When TT-enriched CD4 cells were reconstituted with SP-enriched cells, [3H]thymidine uptake was significantly greater with the linked SP-TT than with the mixed SP and TT. However, reconstitution of the TT-enriched with SP-depleted CD4 cells or the converse failed to increase significantly DNA synthesis by cells stimulated with the linked SP-TT. The production of interleukin 2 (IL 2) and expression of IL 2 receptors were then assayed to examine any difference in stimulation between TT and SP. Both IL2 and IL2 receptors were diminished and delayed when T cells were stimulated with SP as compared with TT. The results suggest that epitope-linked clusters of monocytes, TT-sensitized CD4 and SP-sensitized CD4 cells enable IL2 released by the TT-sensitized CD4 cells to stimulate the SP-sensitized CD4 cells that produce inadequate amounts of IL2. Indeed, addition of recombinant IL2 to T cells stimulated with mixed SP and TT induces an increase in DNA synthesis which becomes similar to that resulting from stimulation with the linked SP-TT.
Antigens, Bacterial↗
Haplotype and multipoint linkage analysis in Finnish choroideremia families.
Multipoint linkage analysis of choroideremia (TCD) and seven X chromosomal restriction fragment length polymorphisms (RFLPs) was carried out in 18 Finnish TCD families. The data place TCD distal to PGK and DXS72, very close to DXYS1 and DXYS5 (Zmax = 24 at theta = 0) and proximal to DXYS4 and DXYS12. This agrees with the data obtained from other linkage studies and from physical mapping. All the TCD males and carrier females studied have the same DXYS1 allele in coupling with TCD. In Northeastern Finland, 66/69 chromosomes carrying TCD had the same haplotype at loci DXS72, DXYS1, DXYS4, and DXYS12. The same haplotype is seen in only 15/99 chromosomes not carrying TCD. Moreover, in 71/104 non-TCD chromosomes, the haplotype at six marker loci is different from those seen in any of the 76 TCD chromosomes. This supports the previously described hypothesis that the large Northern Finnish choroideremia pedigrees, comprising a total of over 80 living patients representing more than a fifth of all TCD patients described worldwide, carry the same mutation. These linkage and haplotype data provide improved opportunities for prenatal diagnosis based on RFLP studies.
Choroideremia↗
Monoclonal antibodies against micro-organisms.
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Animals↗
Physical mapping, linkage analysis of a putative schizophrenia locus on chromosome 5q.
Two recent studies have suggested that a schizophrenia susceptibility locus may lie on the proximal long arm of chromosome 5. Partial trisomy of a 20-30 centimorgan region of chromosome 5 (5q11.2-13.3) was found to cosegregate with schizophrenia in a Canadian family of Chinese descent. Moreover, DNA markers from proximal 5q (D5S39, D5S76) were found to be linked to schizophrenia and related disorders in seven British and Icelandic families. We now report an initial physical map of DNA markers relative to the partial trisomy chromosome 5, as well as preliminary evidence against linkage of this region to schizophrenia in four American families.
Chromosome Mapping↗
Characterisation of monoclonal antibodies to common protein epitopes on the cell surface of Streptococcus mutans and Streptococcus sobrinus.
Three monoclonal antibodies (MAb) were prepared against a cell surface antigen which cross-react between Streptococcus mutans (serotypes c, e and f) and Streptococcus sobrinus (serotypes d and g). Two of the MAb also recognise a determinant on the surface of Streptococcus cricetus (serotype a). The common antigen shared between S. mutans and S. sobrinus was demonstrated by Western blotting to be about 200 kD in size. This antigen is shared not only by the cell surfaces of serotypes a, c, d, e, f and g, but also by the major cell surface antigen of S. mutans of 185 kD and another of 150 kD. These MAb identify all but one mutans type of streptococci and can be utilised as analytical reagents.
Antibodies, Monoclonal↗
Immunohistology of oral lesions from patients with recurrent oral ulcers and Behçet's syndrome.
A qualitative and quantitative immunohistological investigation was performed on biopsies of oral ulcers from patients with Behcet's syndrome (BS) and those with recurrent oral ulcers (ROU). The results were compared with control oral biopsies from patients with other diseases and normal oral mucosa. The expression of HLA-DR on the cell membrane of keratinocytes was found in 13 out of 15 lesions from patients with BS and ROU, as compared with only one out of 15 controls. The relative density of HLA-DR was investigated quantitatively by microdensitometry and this confirmed that DR expression in the epithelial cells of patients with BS and ROU was significantly greater than in diseased and normal control oral tissues. A prominent mononuclear cell infiltration consisted predominantly of T lymphocytes and mature macrophages. Analysis of the CD4 and CD8 subsets of T cells failed to show significant differences between BS, ROU and control diseased tissues. Increased numbers of Langerhans cells were found in the epithelium by morphometric analysis with the CD1 monoclonal antibody in BS and ROU but an increased number was also found in lichen planus. The results suggest that the immunohistological changes in oral lesions of BS and ROU manifest an enhanced immune response in the epithelium, keratinocytes express HLA-class II antigen and increased number of Langerhans cells as well as in the lamina propria with a prominent infiltration of CD4, CD8 and macrophage-like cells. The characteristic pattern of exacerbations and remissions of oral ulceration can be interpreted by the hypothesis that an initiating microbial agent may induce a mononuclear cell infiltration, with the release of cytokines, expression of class II antigen in keratinocytes and causing ulceration, followed by down-regulation of immunity by tolerant T cells induced by the class II positive keratinocytes, leading to a remission.
Antigens, Viral↗
Proteolysis of the 185,000 MW streptococcal cell wall antigen generating 4000 and 6000 MW peptides with distinct antigenic determinants.
A 185,000 MW glycoprotein antigen derived from Streptococcus mutans was digested with subtilisin. Purification by reversed phase high-powered liquid chromatography (HPLC) resulted in a homogeneous 20,000 MW protein which possesses the streptococcal antigen (SA) I and II determinants. This protein was immunogenic in mice both for the 20,000 MW and the native 185,000 MW SA. Further proteolysis with subtilisin generated four peptides of 18,000, 10,000, 6000 and 4000 MW. Whereas the 20,000, 18,000 and 10,000 MW peptides contained both SAI and SAII determinants, the 4000 MW peptide possessed only the SAI and the 6000 MW peptide the SAII determinant. The 4000-SAI peptide is of special significance, as the smallest SAI material separated in the past was 150,000 MW. This was difficult to purify and proved to be protective against dental caries on immunization of rhesus monkeys.
Antigens, Bacterial↗
Specificity of monoclonal antibodies in local passive immunization against Streptococcus mutans.
Local oral passive immunization in human subjects with a monoclonal antibody (MoAb) raised against the 185-kD antigen I/II from S. mutans significantly reduced or prevented oral colonization of an exogenous strain of the organism. In subjects sham-immunized with either saline or an unrelated MoAb, however, significantly greater proportions of S. mutans persisted for a longer duration than in those immunized with the specific anti-streptococcal MoAb. Recolonization of indigenous S. mutans after this organism was reduced to undetectable levels by an antimicrobial agent has also been completely prevented with specific MoAb. Indeed, S. mutans was not detected for a period of over 1 year, as compared with recolonization within 10-82 days in the control subjects. The specificity of MoAb in preventing colonization of the streptococci was studied with four MoAb. This revealed that: (1) the sub-class of antibody is not an essential factor, as both MoAb Guy's 1 and 13 prevented colonization, although Guy's 1 is an IgG2a and Guy's 13 is an IgG1 class of antibody; (2) serotype specificity is important, as MoAb Guy's 9, which only recognizes S. sobrinus (serotypes d and g), does not prevent colonisation by S. mutans (serotype c); (3) neither protein nor carbohydrate nature of the putative adhesin was a determining factor, because MoAb Guy's 1 recognizes a carbohydrate and Guy's 13 a protein determinant and both MoAb prevented adherence of S. mutans; and (4) epitope specificity appears to be the most important factor in preventing adherence of S. mutans, as MoAb Guy's 11 and 13 share the same serotype specificity and both recognize a protein determinant, yet only Guy's 13 prevents colonisation. The long duration of protection from re-colonization by indigenous S. mutans, lasting about 1 year after application of the specific MoAb was stopped, cannot be accounted for by functional MoAb remaining on the teeth. We suggest that initially the MoAb prevents colonization by S. mutans and that the ecological niche vacated by this streptococcus is filled by other organisms from the oral flora, thereby discouraging re-colonization by S. mutans.
Adult↗
Linkage analysis for a disease with a complex mode of inheritance in a simulated data set.
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Computer Simulation↗
Local oral immunization with synthetic peptides induces a dual mucosal IgG and salivary IgA antibody response and prevents colonization of Streptococcus mutans.
A small cell surface antigen of Streptococcus mutans was partially sequenced and the amino terminal peptides of 11, 15 and 20 amino acid residues and a dimer of the 15 and 20 residues peptides were synthesized. The synthetic peptides (SP) were used in topical oral immunization of the gingivomucosal epithelium of macaque monkeys. Sequential examination for antibodies over a period of up to 30 weeks revealed that six applications of the linear or cyclized SP11 and a random SP11 induced negligible or very low antibody levels. In contrast, the SP17 (SP15 with added cysteine at each terminus), SP21 (SP20 with one cysteine) and the dimer (SP35) induced significant anti-SP as well as anti-native streptococcal antibodies in the gingival fluid and in saliva. The functional significance of this immune response was examined by studying its effect on oral colonization of S. mutans following feeding of a carbohydrate-rich diet. Whereas control animals, sham-immunized with a random SP of 11 residues, showed increased colonization of the teeth by S. mutans, there was no colonization or a significant reduction in colonization of animals immunized with the cyclized SP17, linear SP21 or dimerized SP35. These experiments suggest that local immunization with SP derived from the sequences of a streptococcal cell surface antigen induce a dual local immune response of gingival IgG and salivary IgA antibodies against the SP and native SA. These antibodies may be involved in preventing colonization of S. mutans, which is the principal agent in the development of dental caries.
Animals↗
A comparative investigation of three methods of separation of CD4 and CD8 cells from human peripheral blood cells.
A comparison was made of the phenotypic purities and functional efficiencies of CD4 and CD8 cell subsets, separated by three methods. CD4 and CD8 monoclonal antibodies were used for positive and negative selection of T cells by (a) panning, (b) red cell rosetting, and (c) complement mediated killing techniques. The viability of the CD4 and CD8 cell subsets, separated by any of the three methods and assessed by the dye exclusion method, was greater than 95%. An inverse relationship was found between the yields and purities of the CD4 and CD8 cell subset. The most efficient separation of the T cells was achieved by positive selection using the rosetting method; 95.3 +/- 1.8% of the CD4 subset reacted with antibodies to CD4 and 1.4 +/- 0.5% with antibodies to CD8 antigens. The separated CD8 subset showed 90.7 +/- 0.4% reacting with CD8 and 3.5 +/- 1.3% with CD4 antibodies. This separation was slightly better than that achieved by the panning method. However, the positive methods of selection gave lower yields than the negative selection methods. The five CD4 and five CD8 cell subsets were assessed functionally by stimulating with a mitogen (phytohaemagglutinin), a bacterial antigen (purified protein derivative) and a viral antigen (herpes simplex virus). The results suggest that the uptake of [3H]thymidine is generally most efficient with the positively selected rosetting CD4 and CD8 cell subset.
Antibodies, Monoclonal↗
The effect of immunization with a 14-kDa streptococcal antigen on primate T cell and B cell responses.
A streptococcal antigen (SA) of 185 kDa was isolated from Streptococcus mutans and this antigen induced in vitro helper, suppressor and contrasuppressor activities with primate peripheral blood lymphocytes. The 185-kDa SA was then treated by sodium dodecyl sulfate and yielded a 4-kDa SA which was capable of eliciting only helper activity. We have now cleaved the 185-kDa SA with cyanogen bromide, in an attempt to identify suppressor and contrasuppressor determinants. A 14-kDa SA was separated from the cyanogen bromide digest and its ability to elicit T cell and B cell functional activities was tested in rhesus monkeys. Whereas the 185-kDa SA (and 4-kDa SA) elicited high serum anti-SA antibodies and the CD4 cells showed an increase in DNA synthesis, this was not demonstrable with the 14-kDa SA. However, the 14-kDa SA, unlike the 185-kDa SA, activated a significant proportion of CD4 and CD8 cells to bind the Vicia villosa lectin (VV) and this is a characteristic feature of contrasuppressor cells. We then studied the effect of sequential immunization of monkeys with the 14-kDa SA, followed by the 185-kDa SA. The results of this showed suppression of the CD4 proliferative response, in the presence of a normal antibody production. We suggest that the split tolerance between the T cell proliferative and B cell differentiating functions might be interpreted on the basis of suppressor CD8 cells inhibiting the CD4 proliferative phase and the VV-adherent CD8 cells contrasuppressing B cell antibody formation.
Animals↗
Phenotypic and functional characterization of human contrasuppressor cell interactions.
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Antibody-Producing Cells↗