PubMed Health⌕ Search

Biomedical subjects

S M Michalek

Publications and source records attributed to S M Michalek.

At least 73 records · Page 4Linked to original sources
PubMed1995

Oral immunization with recombinant Salmonella typhimurium expressing surface protein antigen A of Streptococcus sobrinus: dose response and induction of protective humoral responses in rats.

An attenuated, recombinant Salmonella typhimurium mutant, chi 4072(pYA2905), expressing the surface protein antigen A (SpaA) of Streptococcus sobrinus was investigated for its effectiveness in inducing protective immune responses against S. sobrinus-induced dental caries in an experimental caries model. Fischer rats were orally immunized with either 10(8) or 10(9) CFU of S. typhimurium chi 4072(pYA2905). Persistence of salmonellae in Peyer's patches and spleens and the induction of immune responses were determined. Maximum numbers of salmonellae were recovered from Peyer's patches of rats within the first week of immunization, with higher numbers recovered from rats given 10(9) CFU than from those given 10(8) CFU. Serum anti-Salmonella and anti-SpaA responses increased more rapidly in rats given 10(9) CFU than in rats given 10(8) CFU. The salivary antibody response to SpaA increased with time, but the response varied in the two groups. In a separate study, rats were orally immunized with the recombinant Salmonella mutant and then challenged with cariogenic S. sobrinus 6715. The levels of serum and salivary antibody and caries activity were assessed at the termination of the experiment. Higher levels of salivary immunoglobulin A antibody to SpaA and Salmonella carrier were detected in rats given 10(9) CFU than in those given 10(8) CFU, and these responses were higher than those in nonimmunized controls. Mandibular molars from immunized rats had lower numbers of recoverable streptococci and less extensive carious lesions than those from nonimmunized, control rats. These data indicate that oral immunization with an attenuated recombinant S. typhimurium expressing SpaA of S. sobrinus induces the production of antigen-specific mucosal antibody and confers protection against dental caries.

Administration, Oral↗
PubMed1994

Oral immunization of humans with dehydrated liposomes containing Streptococcus mutans glucosyltransferase induces salivary immunoglobulin A2 antibody responses.

Seven healthy adult volunteers each ingested an enteric-coated capsule containing 500 micrograms of Streptococcus mutans glucosyltransferase (GTF) in dehydrated liposomes for 3 consecutive days. The immunization regimen was repeated 28 days later. Parotid saliva and plasma were collected prior to and at a weekly interval for 8 weeks following the first immunization for analysis of anti-GTF activity by enzyme-linked immunosorbent assay. The levels of immunoglobulin A1 (IgA1) and IgA2 anti-GTF activities increased in the parotid saliva from 5 of 7 individuals after immunization. Increases in the mean level of IgA1 and IgA2 anti-GTF responses peaked on day 35 (77% and 175% increase over baseline, respectively), although variation was noted in the kinetics and subclass of responses between individuals. No salivary IgG or IgM responses were observed. Low plasma IgM, IgG and IgA anti-GTF responses were seen in immunized subjects. Oral immunization with a dehydrated liposome-protein vaccine was effective in inducing a secretory IgA antibody response, which was primarily of the IgA2 subclass. These results provide the first evidence for the use an oral dehydrated liposome-protein vaccine in humans.

Administration, Oral↗
PubMed1994

Oral immunization with recombinant Salmonella typhimurium expressing surface protein antigen A of Streptococcus sobrinus: persistence and induction of humoral responses in rats.

Recombinant Salmonella typhimurium has been used as an oral vaccine for various microbial pathogens. Here we report immune responses in Fischer rats orally immunized with a recombinant S. typhimurium strain encoding surface protein antigen A (SpaA) of Streptococcus sobrinus. The attenuated S. typhimurium chi 4072 delta cya delta crp delta asd mutant used in this study contains the Asd+ plasmid pYA2905 expressing a fragment of the SpaA protein. Salmonella cells were cleared from spleens by 7 days and from Peyer's patches by 14 days in rats receiving a single oral immunization of 10(9) CFU of chi 4072. In animals receiving multiple (i.e., days 0 and 7 or days 0, 7, and 21) immunizations, Salmonella cells were cleared from the Peyer's patches by 25 days following the initial immunization. Antigen-specific systemic and mucosal antibody responses were greater in rats receiving multiple immunizations than in those receiving a single immunization. Serum anti-Salmonella activity was potentiated following boosting on day 21. Mucosal immunoglobulin A antibody responses were also greater in rats receiving multiple immunizations than in rats receiving a single immunization. Anti-Salmonella and anti-Streptococcus immunoglobulin A activity persisted longer in rats boosted on day 21 than in rats immunized on days 0 and 7. These data indicate that oral immunization of rats with the recombinant S. typhimurium chi 4072(pYA2905) vaccine induces systemic as well as mucosal antibody responses specific to the Salmonella cells and to the cloned SpaA protein. This is the first report of the use of an attenuated mutant of the murine pathogen S. typhimurium as an oral vaccine in rats.

Administration, Oral↗
PubMed1994

Effectiveness of sealed dental prophylaxis angles inoculated with Bacillus stearothermophilus in preventing leakage.

It was the purpose of this in vitro investigation to evaluate the effectiveness of several brands of sealed, reusable prophylaxis angles to keep internal materials within the internal portions of the head of the prophylaxis angle, and not allowing contaminates to leak out. Three brands of sealed, reusable dental prophylaxis angles were autoclaved and then taken apart under a biocontainment flow hood. Testing conditions were designed to prevent a "worst case scenario" by inoculating dental prophylaxis angles with 10(6) of the heat resistant spores of Bacillus stearothermophilus and 20% bovine serum albumin to simulate the presence of human serum. The concentration of Bacillus stearothermophilus spores was verified before testing procedures were initiated. The internal portions of the sterile prophylaxis angles were inoculated with a 1:1 mixture of the Bacillus stearothermophilus spores and bovine serum albumin, at a concentration of 1.15 x 10(6) spores/inoculation. The prophylaxis angles were reassembled under sterile conditions, and a sterile rubber cup was inserted into each of the prophylaxis angles. The prophylaxis angles were attached to a sterile dental handpiece and then submerged in a 50 ml tube containing sterile trypticase soy broth and run at 3000 rpm for 30 seconds. The tube of medium was incubated for 7 days. No growth of Bacillus stearothermophilus spores could be cultured from one of the brands of prophylaxis angles at any time during the incubation period. The other two brands of prophylaxis angles did produce some leakage of the Bacillus stearothermophilus spores.

Cross Infection↗
PubMed1993

Protective salivary immunoglobulin A responses against Streptococcus mutans infection after intranasal immunization with S. mutans antigen I/II coupled to the B subunit of cholera toxin.

The B subunit of cholera toxin (CTB) has been shown to augment mucosal responses to microbial virulence antigens, including those of Streptococcus mutans, which is the principal etiologic agent of dental caries. In the present study, the surface fibrillar protein antigen of S. mutans, antigen I/II (Ag I/II), was chemically coupled to CTB (Ag I/II-CTB), and the conjugate was examined for its effectiveness in inducing salivary immune responses protective against S. mutans infection. Weanling Fischer rats were given Ag I/II-CTB (50 micrograms) by the intranasal route and then orally infected with a virulent strain of S. mutans. Gnotobiotic or conventional rats were given two or three additional immunizations, respectively, at about 2-week intervals. One week after each immunization, individual serum, saliva, and fecal samples were collected and stored frozen until assayed for antibody activity to Ag I/II and cholera toxin (CT) by an enzyme-linked immunosorbent assay. The rats were sacrificed 1 week after the last immunization, when mandibles were also collected from individual rats for assessment of S. mutans levels in plaque and caries activity. Rats immunized only or both immunized and infected showed a salivary immunoglobulin A (IgA) anti-Ag I/II response which reached significantly (P < 0.05) higher levels than those seen in nonimmunized, infected controls. A salivary IgA anti-Ag I/II response was also seen in rats infected only with S. mutans. Essentially no salivary antibody activity to CT was detected. Some serum anti-Ag I/II and anti-CT responses were seen in immunized animals. Serum IgG anti-Ag I/II responses were seen in immunized, infected rats and also in infected-only rats, suggesting that the responses were a result of infection with S. mutans. The immunized and infected rats had significantly (P < 0.05) lower levels of S. mutans in plaque and lower caries activity than nonimmunized, infected rats. These results indicated that intranasal immunization of rats with Ag I/II-CTB induced a protective salivary immune response which was associated with a reduction in S. mutans colonization and S. mutans-induced dental caries.

Animals↗
PubMed1993

Immune response of the female rat genital tract after oral and local immunization with keyhole limpet hemocyanin conjugated to the cholera toxin B subunit.

The immune response of the female rat genital tract was evaluated with Lewis rats given primary and secondary immunizations with keyhole limpet hemocyanin (KLH) alone or coupled to the cholera toxin (CT) B subunit (CTB) by the oral or intravaginal-uterine route or a combination of routes. CT (2 to 5 micrograms) was administered as an adjuvant with the KLH-CTB conjugate. While a significant mucosal immunoglobulin A (IgA) response was induced by KLH, there were no significant differences among the immunized groups in the levels of IgA antibodies in salivary gland, gut, vaginal, and uterine secretions, with the exception that rats immunized only orally with the KLH-CTB conjugate lacked a detectable vaginal response. Levels of IgA antibodies to CT, however, were significantly increased in genital tract secretions of rats immunized locally versus orally with the KLH-CTB conjugate. Antibody activity of the IgG isotype against both KLH and CT was significantly elevated in genital tract secretions of rats immunized with KLH-CTB by the oral or intravaginal-uterine route and given genital tract boosters, in comparison with the results for the other groups. IgM antibody titers were generally negligible in the different secretions. An enzyme-linked spot-forming assay revealed IgA and IgG antibody-secreting cells in salivary gland and uterine tissues. A highly significant correlation between the numbers of antibody-secreting cells and antibody titers existed for uterine IgG but not IgA responses to KLH among the different groups of rats. In conclusion, a vigorous local immune response was induced after immunization of the female rat reproductive tract alone or in combination with peroral challenge with the KLH-CTB conjugate.

Adjuvants, Immunologic↗
PubMed1993

Role of M protein in pharyngeal colonization by group A streptococci in rats.

As the initial step in infection, group A streptococci (GAS) colonize either the nasopharyngeal mucosa or the skin of humans. A number of virulence factors have been implicated in the colonization phase of pathogenesis based upon their in vitro activities, but the in vivo data supporting their role in colonization of the host tissues is lacking. In this investigation, the potential requirement for M protein in pharyngeal colonization by GAS was explored by using near-isogenic strains in experimental animals studies. Fischer rats were infected by intranasal and oral inoculation with both M-positive and M-negative Streptococcus pyogenes strains. Colonization of the pharyngeal area by the streptococci was monitored at various time intervals. Both M-positive and M-negative strains colonized during the first week following infection, indicating that M protein was not necessary for this initial colonization. Two M-positive strains of S. pyogenes were recovered from the rats up to 23 weeks following inoculation, while the colonization levels for M-negative strains decreased rapidly in the second and third weeks, becoming negligible by the fourth week. This indicates a potential role for M protein in the persistence of colonization at this mucosal surface. Colonization of rats with either M-positive strain of S. pyogenes also resulted in the appearance of salivary and serum antibody responses. This in vivo model should allow further investigation into factors required for GAS disease, including the examination of the potential role of the host immune response both in modulation of the pharyngeal surface and in modulation of antigenic changes in M protein or other surface factors.

Adhesins, Bacterial↗
PubMed1992

Liposomes and conjugate vaccines for antigen delivery and induction of mucosal immune responses.

In this brief review, emphasis was placed on the effectiveness of liposomes as carriers/vehicles of soluble antigens and as adjuvants for use in oral vaccine development. Evidence was provided that oral administration of antigen in liposomes resulted in a mucosal response which was higher than that obtained when the oral vaccine consisted of antigen alone. Specific mucosal responses were enhanced by incorporating lipophilic MDP into the antigen/liposome vaccines. Antigens shown to be effective in inducing a protective mucosal response when given in an oral liposome vaccine were anti-idiotypic antibodies, purified S. mutans GTF, CHO and Ag I/II. Evidence is also provided that CTB may be an effective oral adjuvant when coupled to proteins or peptides by either chemical or genetic methods. Further studies, however, will be required to characterize the effectiveness and safety of CTB in conjugate vaccines for inducing specific mucosal responses and to develop practical means to prepare oral liposome vaccines for use in humans.

Animals↗
PubMed1992

Cloning of a locus involved in Streptococcus mutans intracellular polysaccharide accumulation and virulence testing of an intracellular polysaccharide-deficient mutant.

The streptococcal transposon Tn916 (Tcr) was used to isolate mutants of Streptococcus mutans altered in glycogen accumulation to investigate whether glycogenlike intracellular polysaccharides (IPS) play an important role in S. mutans-induced caries formation. S. mutans UA130 (serotype c) was transformed with the Escherichia coli plasmid pAM620 (Tn916), and the resultant transposon libraries were screened for glycogen content by iodine staining. A transposon mutant, designated SMS201, demonstrated a glycogen-deficient phenotype on glucose-enriched medium. Quantitative electron microscopy confirmed that IPS concentrations were significantly reduced in SMS201 relative to the wild-type progenitor strain, UA130. Importantly, reversion to wild type correlated at all times with loss of the transposon. Transposon excisants were used to facilitate cloning of the streptococcal gene(s) involved in glycogen biosynthesis and storage. A 2.1-kb chromosomal determinant (glgR) which encodes a putative regulator of S. mutans glycogen accumulation was isolated. A stable deletion mutation (delta glgR) was subsequently generated in E. coli and introduced into S. mutans by allelic exchange. The resultant glycogen synthesis-deficient mutant, SMS203, demonstrated a significantly reduced cariogenic potential (P less than 0.01) on the buccal, sulcal, and proximal surfaces of teeth in germfree rats, relative to animals challenged with the glycogen synthesis-proficient progenitor strain, UA130. These observations confirm previous reports (J. M. Tanzer, M. L. Freedman, F. N. Woodiel, R. L. Eifert, and L. A. Rinehimer, p. 597-616, in H. M. Stiles, W. J. Loesche, and T. L. O'Brien, ed., Proceedings in Microbiology. Aspects of Dental Caries. Special Supplement to Microbiology Abstracts, vol. 3, 1976) which implicate IPS as significant contributors to the S. mutans cariogenic process.

Animals↗
PubMed1992

Humoral response to Porphyromonas (Bacteroides) gingivalis in rats: time course and T-cell dependence.

In this study, we describe the time course and T-cell dependence of the serum antibody response to the periodontopathogen Porphyromonas (Bacteroides) gingivalis in an experimental rat model. Normal Fischer rats were challenged by a local injection of P. gingivalis (2 x 10(8) bacteria) into gingival tissue or by the administration of a similar number of bacteria by the intravenous (i.v.) route on days 0, 2, and 4. Serum antibody activity was detected within 1 week and peaked at 8 weeks after gingival challenge. A similar but lower response was seen for rats challenged by the i.v. route. The response in both groups of rats was mainly of the immunoglobulin G (IgG) isotype; some IgM but no IgA antibody activity was detected. Analysis of the IgG subclass revealed mainly IgG2c in animals challenged locally in the gingiva with P. gingivalis, whereas IgG2b predominated in rats challenged by the i.v. route. The importance of T cells in the response was established by demonstrating the absence of serum IgG antibodies in nude rats after a local challenge of gingival tissue with P. gingivalis. Nude rats given purified splenic T cells from normal rats immunized systemically with P. gingivalis prior to a local gingival challenge showed a rapid appearance of serum antibody activity that peaked between 4 and 6 weeks. This initial peak occurred 2 to 4 weeks earlier than that seen in normal animals. Fluorescence-activated cell sorter analysis of splenic lymphoid cells from these nude rats revealed a helper T-cell population. The levels of serum IgG antibodies in nude rats given nonimmune T cells rose slowly, and the antibodies were mainly of the IgG2a and IgG2b subclasses. Nude rats given immune T cells showed a rapid increase primarily in IgG2b antibody levels following a local gingival challenge. These findings suggest that the immune helper T-cells contributed to the rapid development of the response to P. gingivalis. Furthermore, it is likely that the IgG subclass response to P. gingivalis in these nude rats was related to the splenic origin of the T cells used for adoptive transfer.

Animals↗
PubMed1991

A method for generating antigen-specific rat T helper cell clones.

T lymphocytes play an important role in host responses to microbial antigens, and therefore, in order to facilitate studies to determine the mechanisms involved in the regulation of these responses, a method for the generation of T cell clones with specificity to microbial whole cell (WC) antigens has been developed. T cells were purified from the spleen of rats immunized with WC antigen of either the gram-positive bacterium Streptococcus mutans or the gram-negative bacterium Bacteroides gingivalis and cultured with irradiated Sephadex G-10-passed spleen (feeder) cells and the homologous WC antigen for the generation of T cell clones. Clonal growth of the T cells was maintained for up to 6 months by co-culturing similar numbers of T cells, feeder cells and WC antigen; however, the addition of exogenous interleukin-2 (IL-2) was not required. Phenotypic characterization of the cloned T cells showed that they were CD4+ T helper (Th) cells which did not express the leukocyte-common antigen, but did express receptors for 1L-2. These T cells required the presence of homologous WC antigen for growth and their antigen specificity was further confirmed by the ability of the T cells to proliferate in response to the homologous WC, but not to heterologous microbial WC, antigen. These T cells were further characterized for their ability to produce cytokines, specifically 1L-2 and interferon-gamma, and to provide help for B cell responses to microbial WC antigen. This study describes a reproducible method for the generation of rat Th cell clones with specificity to microbial WC antigens which will be valuable for defining the mechanisms involved in T cell regulation of responses to either gram-positive or gram-negative bacteria.

Animals↗
PubMed1991

Effect of immune bovine milk on Streptococcus mutans in human dental plaque.

The use of a mouthrinse of bovine milk containing antibodies to Streptococcus mutans resulted in an initial reduction in the numbers of recoverable Strep. mutans in a group of 9 individuals. Ten volunteers who used control bovine milk that contained no antibody activity to Strep. mutans had variable levels of plaque Strep. mutans. In addition, after culture on Mitis Salivarius and Gold's agar, the plaque Strep. mutans from subjects who used the immune bovine milk rinse formed smaller colonies than those from pre-treatment plaque and from all plaque samples of subjects who used the control rinse.

Adult↗
PubMed1991

Repeated DNA sequence involved in mutations affecting transport of sucrose into Streptococcus mutans V403 via the phosphoenolpyruvate phosphotransferase system.

Mutants of Streptococcus mutans V403 defective in the intracellular sucrose-6-phosphate hydrolase (product of the scrB gene) are sensitive to sucrose because of the intracellular accumulation of the phosphorylated sugar. Using a scrB mutant prepared by allelic exchange, we have isolated and characterized a number of sucrose-resistant revertants. One such mutant was found to lack the ability to transport sucrose into the cell via the phosphoenolpyruvate-dependent sucrose phosphotransferase system (PTS). Genetic analysis of this strain revealed this lesion to be linked to the scrB gene. This was corroborated by the physical demonstration of an insertion mutation very near scrB. Taken together with DNA sequence information (Y. Sato, F. Poy, G. R. Jacobson, and H. K. Kuramitsu, J. Bacteriol. 171:263-271, 1989), our results indicated that all of the mutations characterized were located in the adjoining scrA gene which encodes the membrane-associated, sugar-specific enzyme II (EIIsucrose) component of the sucrose PTS in S. mutans. Biochemically, such a genetic lesion disables the sucrose PTS and prevents sucrose from entering the cell by this system. In this paper, we detail the nature of two independent insertion mutations and conclude them to be the result of duplicative transposition events into the scrA gene. This region of the chromosome was amplified and purified in large quantities by using the polymerase chain reaction. Examination of the amplified DNA revealed that the two independent insertion mutations were composed of sequences that were indistinguishable by size and by restriction site endonuclease maps. Their insertion points in the scrA gene were approximately 200 bp apart. The amplified DNA fragment was also used as a probe to demonstrate the presence of five copies of this element on the S. mutans V403 chromosome. A second strain, S. mutans V310, also was found to carry similarly arranged, multiple copies of this sequence on its chromosome, suggesting a clonal origin of V403 and V310. The small size of this sequence, its presence in multiple copies on the V403 chromosome, and its ability to duplicate itself semiconservatively into remote sites argue compellingly that it is an insertion sequence element. One such insertion mutant, with a defective sucrose PTS, was tested for virulence in rats and was found to cause caries at levels similar to those of the wild-type strain.

Animals↗
PubMed1991

Cariogenicity of Streptococcus mutans V403 glucosyltransferase and fructosyltransferase mutants constructed by allelic exchange.

Streptococcus mutans produces several enzymes which metabolize sucrose. Three glucosyltransferase genes (gtfB, gtfC, and gtfD) and a single fructosyltransferase gene (ftf) encode enzymes which are important in formation of exopolysaccharides. Mutants of S. mutans V403 carrying single and multiple mutations of the gtfB, gtfC, gtfD, and ftf genes recently have been constructed by allelic exchange in our laboratory. Using selected strains from this panel of mutants, we examined the importance of water-insoluble glucan, water-soluble glucan, and fructan production in cariogenicity while controlling for the effects of strain and species variability. Genetic and biochemical characterization of mutants and assays of glucosyltransferase and fructosyltransferase activities were performed to ensure that the phenotypes of strains coincided with deficiencies predicted by genotype. The young gnotobiotic rat model of cariogenicity was used to assess virulence of the wild-type strain and isogenic mutants. Mutant strains were less virulent than the wild type in almost every location examined for caries on tooth surfaces and level of involvement of lesions (depth and severity). Inactivation of either gtfB and gtfC or ftf dramatically reduced virulence; the subsequent inactivation of gtfD did not enhance the effect of reduced virulence.

Alleles↗
PubMed1990

Low-cariogenicity of the tetrasaccharide nystose.

1. The effect of nystose on cariogenic factors of mutans streptococci was investigated. 2. Nystose was not utilized as a substrate for the growth, lactic acid production, plaque formation and cellular aggregation of serotype a-g strains of mutans streptococci. 3. Furthermore, nystose did not served as a substrate for water-insoluble glucan formation by glucosyltransferase (GTase) from strains used. 4. In the cellular adherence experiment using nystose, the amount of serotype d cells that adhered to glass surface was almost zero. 5. In the presence of sucrose, nystose inhibited GTase activity and adherence of cells. 6. Finally, significantly lower caries score was observed in rats infected with Strep. sobrinus and fed with diet containing nystose as compared with sucrose.

Animals↗