Autism is a behavioral syndrome characterized by a severe impairment of reciprocal social relations, and of verbal and nonverbal communications. Many different etiologic factors such as viral infection and genetic predisposition have been proposed to explain the development of this disorder. Immune abnormalities, such as a decreased lymphoblastic response to T-cell mitogen, defective antibody responses to rubella vaccine, and decreased numbers of T lymphocytes, also have been identified in a subpopulation of patients with autism, which implies that the development of autism in some cases may be due to autoimmune mechanisms. Recent evidence suggests that immune response to the heat-shock proteins 60 and 70 is associated with several autoimmune diseases, including juvenile arthritis, type 1 diabetes, and multiple sclerosis. Therefore, in this study, the plasmas of patients with autism were examined by enzyme linked immunosorbent assay (ELISA) for antibodies to the heat-shock proteins 60 and 70. The autistic subjects were found to have increased levels of antibodies against heat-shock protein 70 as compared to that of age-matched controls (p=O. 0148). However, levels of antibodies to heat shock protein 60 in the autistic subjects showed considerable individual variation and no significant difference was found. Abnormal immune reactions to myelin basic protein have also been found in autistic subjects. Since epitopes on myelin basic protein have been shown to crossreact with determinants on heat-shock protein 60, the similarity between anti-myelin basic protein monoclonal antibodies and antiheat- shock protein 60 antibodies in the autistic subjects was also studied. The results showed no crossreactivity between these two antibodies. In conclusion, the data from the study of antibodies against heat-shock protein 70 suggest an elevated immune response to heat-shock protein 70 in autistic subjects. This result implies that autism could be an autoimmune disease.

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Previous studies by our group showed that vaccinia virus recombinants expressing Brucella abortus (BA) antigens heat shock protein GroEL, 18 kDa protein and Cu/Zn SOD, were unable to induce protective immune responses against Brucella challenge. This dissertation analyzes the possible reasons for this phenomenon, by using other genes/proteins from BA and Listeria monocytogenes (LM), various shuttle plasmids (pSC65, pSC11) and immune response modulators (CpG, IL-12, B7-1). As the first objective, a vaccinia virus recombinant (WRL7/L12), expressing the BA L7/L12 gene was generated. L7/L12 ribosomal protein was used as a T-cell reactive antigen, with protective potential to Brucella challenge. The WRL7/L12 was able to express the gene of interest and induce IgG2A type antibody response, but not a protective immune response against Brucella challenge. As a control, an antigen from LM proven to induce CTL and protective immune responses, was used to test the efficacy of vaccinia virus to induce protection. A portion of hly gene, encoding partial listeriolysin (pLLO), was inserted into the same vaccinia virus stain. This recombinant (WRpLLO) was able to induce protection against a Listeria challenge. Next another vaccinia virus recombinant expressing Brucella abortus Cu/Zn SOD was analyzed. Although a variety of approaches, including the enhancement of the protein expression by the pMCO2 synthetic promoter, booster immunization, addition of the oligomer CpG adjuvant (WRSODCpG) to enhance Th1 type response, were used, the SOD recombinant failed to protect mice against Brucella challenge. Lastly, vaccinia virus produces a family of proteins that bind cytokines, chemokines and interferons to evade the host defensive systems. Therefore, a vaccinia virus strain co-expressing murine IL-12, and cofactor B7-1, were used to generate the recombinant WRIL12L7/L12. In order to further boost the induction of Th 1 type response, the adjuvant CpG was used. A similar recombinant, WRIL12pLLO, was generated with partial hly gene to serve as a positive control for protection. Mice immune responses to these recombinants, with and without adjuvant CpG, were analyzed, and compared with the recombinants generated with vaccinia strain WR. Co-expression of IL12 and B7 abrogated the protective efficacy of the vaccinia/ pLLO recombinant. / Ph. D.

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This study was undertaken to examine the specificity of the cell-mediated immune response to vesicular stomatitis virus in mice, using the recombinant adenovirus vector AdG12. AdG12 contains the coding region for VSV glycoprotein (G) within the genome of adenovirus type 5. Ultimately, these studies attempted to provide a model for the use of adenovirus vectors to elicit specific CTL responses in mice against an inserted foreign protein. Cell-mediated immunity was examined using a standard ⁵¹Cr release assay. Splenocyte effectors from VSV or AdG12 primed mice were tested for their ability to lyse labelled infected target cells. A number of target cell lines were analyzed for productive infection by AdG12 and expression of VSV-G. Of the lines tested, B10.D2 (H-2ᵈ) and PAK (H-2ᵇ) lines were shown to be infectible with AdG12 and expressed VSV-G 36 hours post infection. Cell lines P815 (H-2ᵈ) and EL-4 (H-2ᵇ) did not appear to be AdG12 infectible. Responses were measured in mice intravenously infected with AdG12. Results demonstrated that peak cytotoxic activity from AdG12 primed mice occurred six days post-infection against syngeneic target cells infected with AdG12, Ad5 wt or VSV. However, these effectors also significantly lysed allotargets infected with VSV, implying that VSV infected targets were lysed in a non-MHC restricted manner. In subsequent experiments, it was discovered that VSV infected B10.D2 and PAK targets were markedly lysed by effectors from immunized and non-immunized Balbic, C57B1/6 and CBA/J mice. Thus, it appeared that these mouse strains contained an inherent or natural cytotoxic activity against VSV infected targets that was unlike classical CTL killing. Depletion experiments showed that this activity was not due to adherent or Thy1 bearing cells within spleen cell populations. To further characterize this activity, splenocyte effectors were tested for their ability to lyse NK-sensitive YAC-1 targets, but no significant lysis was demonstrated. However, despite these results, it appeared that this activity was that of an NK-like effector. The presence of NK-like cytotoxicity against VSV infected targets precluded efforts to define specific anti-VSV responses in these mice. / Thesis / Master of Science (MS)

Alginate is a well-known naturally-derived biomaterial that has been widely used in preparing microparticles for drug delivery and in preparing scaffolds for tissue engineering. Despite desirable properties, alginate has been shown to activate inflammatory cells in vivo. The mechanisms are still unclear. In this thesis, the mechanism by which alginate caused innate immune responses was investigated in vitro by using RAW264.7 cells, a macrophage-like cell line. The NF-(kappa)B pathway, an important signaling pathway in macrophages, has been tracked to identify cellular responses. The secretion of cytokines IL-1(beta), IL-6, IL-12(p40) and TNF-(alpha) was quantified to determine the activation outcomes. Also the interaction between alginate and serum was studied. Experimental results indicated that alginate induced the activation of RAW264.7 cells with a time and dose dependent behavior. Like lipopolysaccharide, a bacterial product and known activator of innate immunity, alginate induced macrophage activation through the NF-(kappa)B pathway and eventually led to detectable IL-1(beta), IL-6 and TNF-(alpha) cytokine secretion. Serum influenced alginate recognition by macrophages in an unknown mechanism. Also, alginate promoted cell survival in a nutrition starvation condition. These results revealed in vitro alginate stimulation, and provided much information for further research. / Thesis / Master of Applied Science (MASc)