Biomedical subjects

S M Michalek

Publications and source records attributed to S M Michalek.

At least 55 records · Page 3Linked to original sources

PubMed1997

Quantitative assessment of inflammatory cytokine gene expression in chronic adult periodontitis.

Adult periodontitis is a chronic destructive disease characterized by an interaction between gram-negative bacteria and the host inflammatory response. Microbial substances such as lipopolysaccharide can activate host cells, e.g., macrophages, fibroblasts and keratinocytes, to secrete proinflammatory cytokines including tumor necrosis factor alpha and interleukin 1 beta (IL-1 beta). This study examined the hypothesis that periodontitis tissue contains increased levels of cytokines that promote osseous and connective tissue destruction. To test this hypothesis, diseased and healthy gingival biopsies were examined for differences in the expression of cytokine mRNA for the pro-inflammatory cytokines tumor necrosis factor alpha and IL-1 beta and the anti-inflammatory cytokine IL-1ra using quantitative reverse transcriptase polymerase chain reaction and in situ hybridization methods. The levels of tumor necrosis factor alpha and IL-1ra mRNA were shown to be significantly higher in diseased than healthy tissues. Additionally, a significantly correlated expression of IL-1 beta and IL-1ra mRNA was seen in all tissue examined. Analysis of tissue sections by immunohistochemical and in situ hybridization techniques revealed a mononuclear cell infiltrate that consisted of a higher average number of cells staining positive for tumor necrosis factor alpha mRNA, CD14, and CD3 in the diseased than healthy tissues. Although both diseased and healthy tissues expressed IL-1 beta and IL-1ra mRNA in the epithelium, the diseased tissue biopsies expressed more IL-1 beta and IL-1ra mRNA in the connective tissue. These results implicate the potential involvement of both the pro- and anti-inflammatory cytokines in the regulation of the chronic inflammatory disease adult periodontitis.

Adult↗

PubMed1997

Mucosal immunogenicity of a recombinant Salmonella typhimurium-cloned heterologous antigen in the absence or presence of coexpressed cholera toxin A2 and B subunits.

An avirulent Salmonella typhimurium vaccine strain expressing a streptococcal protein adhesin and a similar clone which produces the same streptococcal antigen linked to the cholera toxin (CT) A2 and B subunits (CTA2/B) were compared for the ability to induce antibody responses to the expressed heterologous antigen after oral or intranasal immunization of mice. Expression of cloned immunogens in these systems is temperature regulated, being optimal at 37 degrees C, and the two clones under comparison were shown to produce similar levels of the streptococcal antigen. Both clones were found to stimulate high levels of serum immunoglobulin G (IgG) and mucosal IgA antibodies to the cloned immunogen. A consistent trend was observed toward higher mucosal IgA but lower serum IgG responses in the case of the S. typhimurium vector that coexpressed CTA2/B, a potential mucosal adjuvant, regardless of the route of administration. Also noteworthy was the capacity of these antigen delivery systems to induce anamnestic systemic and secretory responses to the cloned immunogen 15 weeks after the primary immunization, despite preexisting immunity to the Salmonella vectors. These antibody responses were sustained for at least 7 months following the booster immunization, at which time the secretory IgA antibody levels were significantly higher in mice given the Salmonella clone that coexpressed CTA2/B. Although the serum IgG response against the Salmonella vector was characterized by a high IgG2a/IgG1 ratio (indicative of the T helper type 1 [Th1]/Th2 profile), a mixed IgG1 and IgG2a pattern was observed for the carried heterologous antigen, which displayed a dominant IgG1 response when administered as a purified immunogen. Our findings indicate that the recombinant streptococcal antigen and CTA2/B are strong immunogens when expressed by the antigen delivery system used in this study and suggest that CTA2/B may have an additional immunoenhancing activity in the mucosal compartment besides its ability to target antigen uptake into the mucosal inductive sites. CTA2/B may thus be useful as an S. typhimurium-cloned adjuvant for coexpressed protein antigens.

Animals↗

PubMed1997

Depletion of alveolar macrophages exacerbates respiratory mycoplasmosis in mycoplasma-resistant C57BL mice but not mycoplasma-susceptible C3H mice.

Indirect evidence suggests that innate immune mechanisms involving alveolar macrophages (AMs) are of major importance in antimycoplasmal defense. We compared the effects of AM depletion on intrapulmonary killing of Mycoplasma pulmonis during the early phase of infection in mycoplasma-resistant C57BL/6NCr (C57BL) and mycoplasma-susceptible C3H/HeNCr (C3H) mice. More than 80% of AMs were depleted in both strains of mice by intratracheal insufflation of liposome-encapsulated dichloromethylene bisphosphonate (L-Cl2MBP), compared to no significant AM depletion in either strain following insufflation of liposome-encapsulated phosphate-buffered saline (L-PBS), PBS alone, or no treatment. AM-depleted (L-Cl2MBP) and control (L-PBS) mice were infected intranasally with 10(5) CFU of M. pulmonis UAB CT, and their lungs were quantitatively cultured to assess intrapulmonary killing at 0, 8, 12, and 48 h postinfection. AM depletion exacerbated the infection in C57BL mice by reducing killing of the organism to a level comparable to that in C3H mice without AM depletion. In contrast, AM depletion did not alter killing in C3H mice. These results directly identify the AM as the main effector cell in early pulmonary antimycoplasmal defense and suggest that differences in mycoplasmal killing by AMs may explain the resistance of C57BL mice and the susceptibility of C3H mice to mycoplasmal infection.

Animals↗

PubMed1997

Effects of Porphyromonas gingivalis and Escherichia coli lipopolysaccharides on mononuclear phagocytes.

The mononuclear phagocyte plays an important role in the regulation of microbe-induced inflammation, in part through its ability to secrete mediators, particularly cytokines, in response to microorganisms and their products. To evaluate the effects of the microbial flora associated with chronic adult periodontitis on cytokine induction, lipopolysaccharide (LPS) from the periodontopathogen Porphyromonas gingivalis was used to stimulate naive and phorbol ester-primed U937 monocytic cells, as well as elutriated human peripheral blood monocytes. We assessed the effect of this LPS, in comparison to that of LPS from Escherichia coli, on cell proliferation, cytokine induction, and surface expression of the LPS receptor CD14. P. gingivalis LPS stimulated proliferation of U937 cells at concentrations of greater than 1 ng/ml, while E. coli LPS inhibited proliferation. Phorbol myristic acid (PMA)-treated U937 cells and elutriated monocytes responded to E. coli LPS activation by producing tumor necrosis factor alpha (TNF-alpha) mRNA and protein; however, P. gingivalis LPS induced greater numbers of TNF-alpha mRNA-positive cells and higher (P < 0.05) levels of protein than did E. coli LPS. Both cell types expressed interleukin-1 beta (IL-1beta) mRNA and protein in response to either LPS treatment. Compared with E. coli LPS, P. gingivalis LPS induced significantly (P < 0.05) higher numbers of IL-1 mRNA-positive U937 cells and elutriated monocytes, as well as production of significantly more (P < 0.05) IL-1 protein by the monocytes. The PMA-treated U937 cells and the monocytes produced high levels of IL-1 receptor antagonist mRNA and protein which were only marginally affected by the LPS preparations. While E. coli LPS induced expression of CD 14 on the surface of PMA-primed U937 cells and monocytes, P. gingivalis LPS exhibited a significantly (P < 0.05) greater ability to enhance receptor levels. Our results indicate that P. gingivalis LPS can activate the mononuclear phagocyte for proliferation, cytokine production, and CD14 expression, providing evidence for the potential of this bacterial component to act as a critical regulatory factor in the chronic inflammatory response associated with periodontitis.

Cell Line↗

PubMed1997

Passive immunization against dental plaque formation in humans: effect of a mouth rinse containing egg yolk antibodies (IgY) specific to Streptococcus mutans.

Passive immunization involving the delivery of antibodies specific to pathogens of infectious diseases to the host has been an attractive approach to establish protective immunity against a variety of microbial pathogens, including Streptococcus mutans, which is the principal etiologic agent of dental caries in humans. The overall purpose of the present study was to determine the effectiveness of a mouth rinse containing antibodies to S. mutans in preventing the establishment of this bacterium in dental plaque of humans. The antibodies were derived from egg yolks obtained from hens immunized with whole cells of S. mutans grown in sucrose-containing medium. The immunoglobulin derived from the yolks (IgY) of immunized hens was characterized in vitro and in vivo in human volunteers. Cross-reactivity tests showed that immune IgY reacted with every serotype, except serotype b, which had lost its GTase activity, when the bacteria were cultured in sucrose-containing medium. Immune IgY inhibited S. mutans adherence to saliva-coated hydroxyapatite discs by 59.2%, while control IgY caused an inhibition of only 8.2%. In the short-term (4-hour) test using a mouth rinse containing 10% sucrose, immune IgY decreased the ratio of the percentage of S. mutans per total streptococci in saliva. In the long-term (7-day) test using a mouth rinse without sucrose, the ratio in saliva was not significantly reduced in the volunteers using the immune IgY due to the large standard deviation. However, comparing the ratios of the percentage of S. mutans per total streptococci in plaque of individual subjects, there was a tendency for a reduction of the ratios in the volunteers receiving the mouth rinse containing immune IgY. These results support the effectiveness of IgY with specificity to S. mutans grown in the presence of sucrose as an efficient method to control the colonization of mutans streptococci in the oral cavity of humans.

Antibodies, Bacterial↗

PubMed1997

Profile of cytokine mRNA expression in chronic adult periodontitis.

Chronic inflammation induced by bacteria often leads to host-mediated destruction of tissues adjacent to the sites of microbial insult. The chronic inflammatory process of adult periodontitis results in the destruction of supporting osseous and connective tissues of the teeth. We hypothesized that virulence factors of periodontal pathogens such as lipopolysaccharide stimulate inflammatory cytokine expression by mononuclear cells of the host which contribute to disease development. In this study, to elucidate the role of these cytokines in chronic adult periodontitis, we tested whether the prevalence of mRNA for inflammatory cytokines generally associated with mononuclear phagocytes was higher in diseased than in healthy gingival tissue. Gingival mononuclear cells or whole gingival biopsies from 32 adult periodontitis patients and five healthy individuals used as controls were evaluated for inflammatory cytokine mRNA expression by reverse-transcription polymerase chain-reaction (RT-PCR) procedures. The cytokines assessed included IL-1 alpha, IL-1 beta, IL-1ra, IL-6, IL-8, IL-12, IL-13, TNF-alpha, TGF-beta, and IFN-gamma. The monocyte/macrophage lipopolysaccharide (LPS) receptor CD14 was also assessed. Results showed that TNF-alpha mRNA was present significantly more frequently in diseased than in healthy biopsies, whereas IL-1 alpha, IL-1 beta, and IL-1ra mRNA were found in most (from 80 to 100%) healthy tissues. Message for CD14 was present in both healthy and diseased tissue samples (100%). This study provides evidence for a major role of TNF-alpha in chronic adult periodontitis. Moreover, our results suggest that the mononuclear cells derived from periodontal tissues have the capacity to respond to components of periodontal pathogens and express both pro- and anti-inflammatory cytokines in these tissues.

Adult↗

PubMed1997

Routes of immunization and antigen delivery systems for optimal mucosal immune responses in humans.

Numerous experiments performed in humans and animals have revealed that stimulation of mucosal lymphoid inductive sites such as intestinal Peyer's patches results in parallel immune responses manifested by the appearance of S-IgA antibodies in the external secretions of remote glands. However, recent experiments suggest that inductive sites associated with the upper respiratory tract, rectum, and perhaps genital tract may also function as sources of lymphoid cells that populate, with some selectivity, certain remote mucosal effector sites. Furthermore, antigen-specific IgA antibodies can be induced in certain secretions (e.g., female genital tract) not only by immunization in the vicinity of corresponding mucosal tissues (e.g., vagina and rectum) but also by oral and especially intranasal immunization. The ineffectiveness of simple delivery of soluble antigens to mucosal membranes for immunization has stimulated extensive studies of strategies for effective delivery systems that would (a) increase the antigen absorption, (b) prevent its degradation, and (c) skew the outcome of immunization to a desired goal (protective response to infectious diseases vs. tolerance; B vs. T cell responses; mucosal vs. systemic). The induction of immune responses at a desired mucosal site can be accentuated with the use of a suitable antigen-delivery system including relevant bacterial or viral vectors, edible transgenic plants expressing microbial antigens, incorporation of antigens in biodegradable microspheres or liposomes, and linkage or coadministration of antigens with cholera toxin B subunit. However, only a few antigen-delivery systems extensively used in animal experimentation have been evaluated for their efficacy in humans. The combination of various immunization routes and the use of suitable antigen-delivery systems may accomplish an important task-the induction of mucosal immune responses at a location relevant to the site of entry of a given pathogen.

Animals↗

PubMed1996

Effect of in vitro passage of healthy human gingival fibroblasts on cellular morphology and cytokine expression.

Cytokines have been implicated in the regulation of antibody and inflammatory responses, but their role in periodontal diseases has not been elucidated. In the present study, cytokine production by human gingival fibroblasts (HGF) following in vitro passage was assessed in order to determine the basal levels of cytokine message and protein and to determine if the cellular morphology and the profile of cytokines produced differed with passage. The HGF cell line F-CL was established by explantation from non-inflamed gingival tissue, and cells from passages 1-10 were studied. The number of cells was determined in confluent cultures and cell morphology was examined by light microscopy. Fibroblasts from confluent cultures were examined for cytokine mRNA by reverse transcription-polymerase chain reaction and culture supernatants were assessed for cytokines by enzyme-linked immunosorbent assay. The morphology of F-CL fibroblasts in passages 1-4 was normal, while fibroblasts in passages 5-10 were larger. In general, the number of cells decreased from early to late passage. Fibroblasts from passages 1-10 contained message for interleukin-1 beta, -6 and -8, but not for interleukin-1 alpha or tumour necrosis factor-alpha. Interleukin-6 was detected in culture supernatants of F-CL fibroblasts by the enzyme immunoassay and its level decreased with increasing passage.

Cell Culture Techniques↗

PubMed1996

Oral immunization with recombinant Salmonella typhimurium expressing surface protein antigen A (SpaA) of Streptococcus sobrinus: effects of the Salmonella virulence plasmid on the induction of protective and sustained humoral responses in rats.

Recombinant strains of Salmonella typhimurium have been studied as antigen delivery systems to determine their effectiveness as multivalent vaccines. Here we compare the efficacy of two strains of S. typhimurium. chi 4072 (pYA2905) and chi 3987 (pYA2905), expressing SpaA of Streptococcus sobrinus 6715 as oral vaccines for dental caries. Both strains are attenuated delta cya delta crp delta asd mutants with their Asd phenotypes complemented by the Asd+ plasmid pYA2905, which also encodes a peptide fragment of SpaA. S. typhimurium chi 3987 (pYA2905), unlike S. typhimurium chi 4072 (pYA2905), contains the 100 kb S. typhimurium virulence plasmid. Fischer rats were orally immunized with approximately 10(9) S. typhimurium chi 3987 (pYA2905) or chi 4072 (pYA2905) and then challenged with cariogenic S. sobrinus 6715. Rats orally immunized with either strain of recombinant Salmonella developed salivary IgA anti-SpaA responses and had lower levels of S. sobrinus-induced dental caries than nonimmunized, infected animals. In a second series of experiments, the kinetics and isotype of the serum and salivary antibody responses were determined in rats orally immunized with S. typhimurium chi 3987 (pYA2905) or chi 4072 (pYA2905) on weeks 0 and 8. IgG and IgM serum antibody responses to SpaA and S. typhimurium were detected after the primary and secondary immunizations, and the secondary immunization boosted serum IgG anti-Salmonella activity. In general, animals immunized with chi 3987 (pYA2905) had higher serum anti-SpaA, as well as serum and salivary anti-Salmonella, responses than animals immunized with chi 4072 (pYA2905). This study demonstrates the effective use of two recombinant S. typhimurium strains as oral vaccines for inducing protective and sustained immune responses against a mucosal pathogen and suggests that the recombinant Salmonella vaccine strain carrying the virulence plasmid induced similar or higher protective immune responses than the strain lacking the virulence plasmid.

Administration, Oral↗

PubMed1996

Construction and oral immunogenicity of a Salmonella typhimurium strain expressing a streptococcal adhesin linked to the A2/B subunits of cholera toxin.

A major adhesin from the oral pathogen Streptococcus mutans has been shown to be mucosally immunogenic upon genetic fusion with the cholera toxin A2/B subunits. To take advantage of the ability of Salmonella typhimurium to deliver cloned antigens to the mucosal inductive sites that would obviate the need for antigen purification, we expressed this chimeric construct in an attenuated S. typhimurium strain under the control of bacteriophage T7 transcription. Residual expression of the temperature-regulated T7 RNA polymerase at 30 degrees C allowed production of the chimeric protein at 2-3% of the total soluble protein, but it was increased five to six times following induction at 37 degrees C. Oral administration of a single dose of 10(9) recombinant Salmonella to mice resulted in serum IgG and salivary IgA antibody responses to Salmonella, cholera toxin, and the streptococcal adhesin, which were generally enhanced after a booster immunization.

Adhesins, Bacterial↗

PubMed1996

Host responses induced by co-infection with Porphyromonas gingivalis and Actinobacillus actinomycetemcomitans in a murine model.

In this study, evidence is presented that mixed infection with the periodontal pathogens Porphyromonas gingivalis and Actinobacillus actinomycetemcomitans results in a synergistic effect in their pathogenicity and in their ability to induce humoral and cellular host responses. BALB/c mice were injected subcutaneously on the back with P. gingivalis ATCC 53977, A. actinomycetemocomitans 75 or a mixture of both bacteria. Samples of blood and fluid from abscesses formed at the site of injection (first degree) or distant from the injection site were collected for microbiologic analysis. Serum and spleens were obtained for evaluation of humoral and cellular responses to P. gingivalis and A actinomycetemocomitans. Mice injected with A. actinomycetemcomitans had first-degree lesions only, whereas mice injected with P. gingivalis and A. actinomycetemcomitans had lesions at first- and second-degree sites from which both bacterial species were isolated. A serum anti-P. gingivalis response was induced in P. gingivalis-injected mice, which was higher in mice injected with P. gingivalis and A. actinomycetemcomitans. This pattern was not seen in the anti-A, actinomycetemcomitans response. Lymphoproliferative responses to phytohemagglutinin, Escherichia coli lipopolysaccharide and P. gingivalis of spleen cells from infected mice were decreased, especially following co-infection. Furthermore, co-infection of mice resulted in the greatest decrease in the number of CD5+, especially CD4+ lymphocytes.

Aggregatibacter actinomycetemcomitans↗

PubMed1996

Effect of host responses on the pathogenicity of strains of Porphyromonas gingivalis.

Porphyromonas gingivalis is implicated in the etiology of periodontitis. Strains of P. gingivalis have been classified as invasive or noninvasive based on their ability to form abscesses in a mouse model. The purpose of this study was to investigate the ability of P. gingivalis strains to cause abscesses and periodontal bone loss in an experimental rat model and the effect of serum and salivary responses on the pathogenicity of these strains. Subcutaneous injection of animals with P. gingivalis 33277, A7A1-28, W50 or 381 resulted in abscesses in a higher percentage of mice than rats. P. gingivalis 33277 caused lesions at the site of injection, whereas strains A7A1-28 and W50 induced abscesses at distant sites in both mice and rats. Local lesions were seen in rats injected with strain 381, whereas lesions formed distant from the site of injection in mice. When periodontal bone loss was assessed in the experimental rat model, animals challenged with 33277 had the highest amount of horizontal and vertical bone loss. Rats challenged with strain A7A1-28, W50 or 381 had some or no periodontal bone loss compared with controls. Assessment of antibody responses to P. gingivalis in these animals revealed that rats challenged with 33277 had lower levels of serum immunoglobulin G-(IgG) and especially salivary IgA antibody activity than A7A1-28-challenged rats. Serum IgG and in particular salivary IgA anti-P. gingivalis responses were seen in W50- and 381-challenged rats. These results indicate that the ability of P. gingivalis strains to cause abscesses does not relate directly to their periodontal pathogenicity as assessed by periodontal bone loss in the same animal model. The results further suggest the importance of salivary IgA antibody responses in protection against experimental periodontal bone loss after challenge with P. gingivalis.

Abscess↗

PubMed1996

Properties of practical oral liposome-Streptococcus mutans glucosyltransferase vaccines for effective induction of caries protection.

Here we report the effectiveness of various liposome vaccines containing Streptococcus mutans glucosyltransferase (GTF) in protecting against dental caries after oral immunization. Rats were immunized by gastric intubation of the appropriate liposome vaccine at weaning and boosted 3 times. Rats were infected with S. mutans following initial immunization and fed cariogenic diet (Diet 305). Saliva and serum were collected during the study and assessed for antibody activity by enzyme-linked immunosorbent assay. Mandibles were removed on day 47 and assessed for S. mutans levels and then for caries. Animals immunized with sonicated, filtered and microemulsified GTF liposome preparations had decreased levels of dental caries compared with control animals given empty liposomes. Rats given dehydrated/rehydrated or purified liposomal GTF also had significantly less caries than control group (GTF alone). Because of economy, ease of preparation and efficiency in amount of antigen used, filtered, dehydrated/ rehydrated and purified liposomal GTF preparations are most practical for use in further assessing the efficacy of liposomal GTF in oral immunization.

Analysis of Variance↗

PubMed1996

Persistence of serum and salivary antibody responses after oral immunization with a bacterial protein antigen genetically linked to the A2/B subunits of cholera toxin.

Primary oral immunization of mice with a bacterial protein antigen genetically coupled to the A2 and B subunits of cholera toxin induced specific secretory immunoglobulin A and serum immunoglobulin G antibodies that persisted at substantial levels for at least 11 months. A subsequent single booster immunization did not further enhance the antibody responses. Long-term antibody persistence may be especially important in infections caused by common pathogens for which continuous immunity would be advantageous.

Adhesins, Bacterial↗

PubMed1995

Conjugation of cholera toxin or its B subunit to liposomes for targeted delivery of antigens.

Several immunoadjuvant systems have been proposed to enhance mucosal immune responses of orally administered purified antigens. Cholera toxin (CT) or its B subunit (CTB) have been found to promote immune responses to antigens when they are co-administered via mucosal routes. Oral administration of antigens incorporated into liposomes has also been shown to result in enhanced mucosal immune responses. Here, we describe the covalent coupling of CT and CTB to small unilamellar liposomes for targeting these vesicles to Peyer's patch M cells, following their oral administration. Conjugation was done by means of a thioether bond using succinimidyl(4-N-maleimidomethyl)cyclohexane-1-carboxylate to modify the dipalmitoylphosphatidyl-ethanolamine constituent of liposomes and N-succinimidyl-3-(2-pyridyldithio)propionate to thiolate CT or CTB. The biological activity of CT or CTB bound to liposomes was confirmed by a hemagglutination assay using GM1-enriched human erythrocytes. Furthermore, oral administration of CT-conjugated liposomes to rats resulted in the induction of serum IgG and salivary IgA anti-CT responses. CT-conjugated liposomes may prove to be a useful system for targeted delivery and immunoenhancement of weakly immunogenic antigens.

Adjuvants, Immunologic↗

PubMed1995

Sucrose-derived exopolymers have site-dependent roles in Streptococcus mutans-promoted dental decay.

We have constructed a panel of mutants of S. mutans V403 which are defective in one or more of the glucosyltransferase genes (gtfB, C or D) or the fructosyltransferase gene (ftf). These strains have been tested for virulence in a gnotobiotic rat caries model with reference to both buccal (smooth surface) and sulcal (pit and fissure) carious lesions. Our data suggest differing roles for degradable and non-degradable polymers at buccal and sulcal sites. Non-degradable polymers (made by products of the gtfB and C genes) contributed significantly to the severity of smooth surface lesions. However, our studies suggested their role in pit and fissure lesions was not as important as the role of degradable polymers. Specifically, a mutant deficient in the major insoluble glucan synthesizing activity (product of the gtfB gene) was 25% more cariogenic on sulcal surfaces than was the wild-type V403 strain. We propose that extracellular glucosyltransferases and fructosyltransferase compete for sucrose and that this competition influences pathogenicity at differing tooth sites.

Animals↗

PubMed1995

Enhancement of protective immune responses to Venezuelan equine encephalitis (VEE) virus with microencapsulated vaccine.

Venezuelan equine encephalomyelitis (VEE) virus is a mosquito-borne arbovirus of major human health significance in the New World. Currently two forms of VEE virus are used for immunization of humans and horses, i.e. a live attenuated and a formalin-inactivated vaccine. Clinical evidence suggests that these vaccines are not fully efficacious and may produce certain undesirable side-effects. In the present study, microspheres composed of biocompatible and biodegradable poly (DL-lactide-co-glycolide) (DL-PLG) were evaluated for their effectiveness as a delivery system of whole, inactivated VEE virus vaccine for the induction of protective immune responses. Mice receiving 50 micrograms VEE virus in microspheres composed of an equimolar ratio of DL-lactide and glycolide (50:50 DL-PLG) exhibited a primary circulating IgG antibody response which was approximately 32-times higher than the response induced with the same dose of unencapsulated (free) virus. A similar difference in responses was seen with antigen doses ranging from 3.1 to 50 micrograms. A rapid increase in antibody activity was seen after the secondary immunization (day 50). Formalin fixation of inactivated VEE virus was important for immunogenicity since the circulating anti-VEE virus antibody response induced with microencapsulated nonformalin-fixed virus vaccine was lower than that induced with microencapsulated formalin-fixed virus vaccine. Furthermore, at low antigen concentrations, DL-PLG microsphere vaccines prepared with the solvent methylene chloride induced higher antibody responses than those prepared using ethyl acetate as the solvent. Microencapsulated vaccine also induced higher VEE virus neutralization titers than did free virus vaccine. Finally, the microencapsulated virus was more effective than the free virus in inducing immune responses protective against systemic challenge with virulent VEE virus. These results demonstrate that DL-PLG microspheres containing formalin-fixed, inactivated VEE virus were effective in augmenting circulating IgG antibody levels and neutralization titers to the VEE virus following systemic immunization and in affording enhanced protection against systemic challenge with virulent VEE virus. The effects of antigen form and the microsphere processing solvent on the immunogenicity of the vaccine are discussed.

Adjuvants, Immunologic↗

PubMed1995

Generation of monoclonal antibodies to Macaca mulatta (rhesus) IgA.

One IgG1 and five IgM murine monoclonal antibodies (mAb) specific for rhesus (Rh) IgA were generated. These mAbs bound to Rh IgA but not IgG or IgM when tested by enzyme-linked immunosorbent assay. Immunoblotting revealed that the mAbs reacted with the alpha heavy chain of Rh but not human IgA. The IgG1 anti-Rh IgA mAb detected IgA-producing cells in sections of monkey gut examined by immunofluorescent staining. These mAbs should be useful for characterizing IgA responses in the Rh monkey.

Animals↗