Acute otitis media is the most common illness diagnosed during early childhood that can cause significant morbidity (Brook, 1994) and sometimes can cause irreversible sequelae such as a hearing defect and subsequent learning difficulties (Klein, 1994). The aims of the research presented here were to study some aspects of the middle ear defence mechanisms in both immune and non-immune rats following experimental otitis media (OM) with two pathogens nontypeable Haemophilus influenzae (NTHi) and Moraxella catarrhalis (M. catarrhalis). This study also aimed at developing a suitable technique for preparing immunohistochemical staining of middle ear sections (chapter 2). A previous study has shown that a regime where rats received an IPP immunisation combined with an IT boost was effective in enhancing clearance of a middle ear infection with the same strain of NTHi and also in the presence of a concomitant viral infection (Moore et al, 2001). Results of this study have shown that for NTHi infection a distinct cellular influx to the middle ear in the immune rats was accompanied by an enhanced bacterial clearance compared to the non-immunised rats (chapter 3). This cellular influx was responsible for the remarkable reduction in the bacterial number. The sharp decline in PMNs numbers in the NTHi immunised rats that followed complete bacterial clearance at 72h post infection (Table 3.1) indicate a more effectively controlled down regulation of this cell infiltrate than the non-immunised rats. For M. catarrhalis infection, there was no difference in cell infiltrate between immune and non-immune rats, but enhanced clearance of the bacteria were observed for the immune animals. The histopathological changes in the middle ear mucosa of rats with experimentally induced infection were studied to provide a better understanding about the distribution of the inflammatory cells and changes in the mucosa during the first 24h post challenge with NTHi and M. catarrhalis (Chapter 4). These changes have not been previously studied for the two pathogens at 24h post challenge in rats. Induced infections with the two pathogens were found to produce similar histopathological changes but more inflammatory infiltration was observed within the infected mucosa with NTHi than that seen with M. catarrhalis. The infections were characterized by increased thickness of the middle ear mucosa, Eustachian tube mucosa, periosteum and tympanic membrane. There was also an increase in the number and size of small blood vessels at all sites, and these small blood vessels seem to be the source of the inflammatory infiltration into the middle ear mucosa and middle ear cavity during the infection. These findings provided an essential background to the immunohistochemical study. The effect of mucosal immunisation on the distribution of CD4+T cells and CD8+T cells has not been investigated previously. Results of the present study (Chapter 5) show the pattern of distribution of these cells during the first 48h post infection with NTHi in the rat. The number of CD4+and CD8+T cells peaked at 24h post infection in the nonimmunised animal and were highest at 48h post-infection in the immunised rats. The difference in response in the immunised rats may represent regulation of the inflammatory response by the immune system. The inflammatory response regulation is indicated by the difference in cellular influx into the immune rats and the response in the immune rats that corresponds to enhanced bacterial clearance prior to a decrease in numbers of inflammatory cells once the bacteria was no longer detected (Chapter 3). This resolution of the inflammatory mass would reduce the opportunity for continued damage to local tissue. These changes are also supported by the reduction in the thickness of the middle ear mucosa of the immunised rats especially at 24h and 48h post-infection (Chapter 5). This study has shown that there are distinct differences in the rate of bacterial clearance and cellular changes in the middle ear mucosa and tympanic bulla in immunised rats during a middle ear infection. Future studies are still required to gain a better understanding of differences in the inflammatory response for both pathogens, NTHi and M. catarrhalis.

Copy of author's previously published work inserted. / Bibliography: leaves 139-160. / 160, [10] leaves, [41] leaves of plates : ill. (chiefly col.) ; 30 cm. / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / Thesis (Ph.D.)--University of Adelaide, Dept. of Molecular Biosciences, 2001?

<p>The humoral immune response is dependent on the formation of antibodies. Antibodies are produced by terminally differentiated B cells, plasma cells. Plasma cells are generated either directly from antigen challenged B cells, memory cells or from cells that have undergone the germinal center (GC) reaction. The GC is the main site for class switch, somatic hypermutation and generation of memory cells.</p><p>Different factors, both internal and external, shape the outcome of the immune response. In this thesis, we have studied a few factors that influence the maturation of the humoral response. We have studied how age affects the response, and we show that responses against thymus dependent antigens (TD) are more affected than responses to thymus independent (TI) antigens, in concordance with the view that the T cell compartment is more affected by age than the B cell compartment. Furthermore, we demonstrate that priming early in life have a big influence on the immune response in the aged individual. Priming with a TI form of the carbohydrate dextran B512 (Dx) induces a reduction of IgG levels in later TD responses against Dx. We have evaluated possible mechanisms for this reduction. The reduction does not seem to be caused by clonal exhaustion or antibody mediated mechanisms. We also showed that the reduced TD response after TI priming can be induced against another molecule than Dx. With the hypothesis that TI antigens induce a plasma cell biased maturation of the responding B cells, we examined the presence of Blimp-1, a master regulator of plasma cell differentiation, in GCs induced by TD and TI antigen. Blimp-1 was found earlier in GCs induced by TI antigen and the staining intensity in these GCs was stronger than in TD antigen induced GCs, indicating that plasma cells might be continuously recruited from these GCs.</p><p>B cells undergoing the GC reaction are thought to be under a strict selection pressure that removes cells with low affinity for the antigen and also cells that have acquired self-reactivity. We investigated the effect of apoptotic deficiencies on the accumulation of somatic mutations in GC B cells. In mice lacking the death receptor Fas, <i>lpr</i> mice, the frequency of mutations was increased but the pattern of the mutations did not differ from wild type mice. In contrast, mice over-expressing the anti-apoptotic protein Bcl-2, had a lowered frequency of mutations and the mutations introduced had other characteristics.</p>

Three experiments were conducted to study the influence of dietary fatty acids on the production performance and immune response of chickens. In experiment I, forty day-old broiler chicks were fed diets containing 5% of either animal fat + conjugated linoleic acid (CLA) (Diet I), sunflower oil (Diet II), flax oil (Diet III) or fish oil (Diet IV). No significant differences (P>0.05) were observed between the live weight of birds. The liver tissue total fat content was lower (P<0.05) in treatment I and II. The fatty acid composition of breast and thigh muscle, liver, heart, pericardial fat, plasma, splenocytes and gut associated lymphoid tissue differed (P<0.05) between treatments. The thiobarbituric acid reactive substances (TBARS) of breast and thigh muscle, liver and heart tissue were lower (P<0.05) in Diet I fed birds. Serum antibody activity was decreased (P<0.05) in Diet II fed birds. In experiment II, 120 day-old broiler chicks were fed diets containing 3.5% of either animal fat + conjugated linoleic acid (CLA) (Diet I), sunflower oil (Diet II), linseed oil (Diet III) or fish oil (Diet IV). Body weight gain was higher (P<0.05) in Diets III and IV compared to Diets I and II fed birds. Feed intake was increased (P<0.05) in Diet IV fed birds. Birds fed Diets III and IV had higher (P<0.05) n-3 fatty acids in all tissues studied. A preferential incorporation of CLA was observed in spleen mononuclear cells. TBARS were higher (P<0.05) in the breast and thigh muscle of Diet IV fed birds. Serum anti-BSA antibody content was higher (P<0.05) in birds fed Diets III and IV. Delayed type hypersensitivity (DTH) response was increased (P<0.05) in Diets IV and III fed birds. Lymphocyte and spleen mononuclear cell CD4⁺, CD8⁺ and IgM⁺ cell population did not differ (P>0.05) among treatments. In experiment III, 120 layer birds were fed diets containing 3% of CLA+animal fat (Diet I), sunflower oil (Diet II), canola+flax oil (Diet III) or fish oil (Diet IV). Egg production, feed consumption and feed efficiency did not differ (P>0.05) among treatments. Birds fed Diets III and IV had higher content of n-3 fatty acids in eggs. Eggs from hens fed Diet I incorporated higher (P<0.05) CLA and saturated fatty acids with a concomitant reduction in (P<0.05) monounsaturated fatty acid content. A preferential incorporation of CLA was observed in eggs over other tissues. TBARS were higher (P<0.05) in breast and thigh muscle of Diet IV fed birds. Egg TBARS content did not differ (P>0.05) among treatments. Serum and yolk anti-BSA antibody contents were higher (P<0.05) in birds fed Diets III and IV. DTH response was increased (P<0.05) in Diets IV and III fed birds. Lymphocyte and spleen mononuclear cell CD4⁺, CD8⁺ and IgM⁺ cell population did not differ (P>0.05) among treatments. Feeding n-3 fatty acids increased antibody mediated immune response while n-6 fatty acids and CLA increased cell mediated immune response. / Graduation date: 2003

The humoral immune response is dependent on the formation of antibodies. Antibodies are produced by terminally differentiated B cells, plasma cells. Plasma cells are generated either directly from antigen challenged B cells, memory cells or from cells that have undergone the germinal center (GC) reaction. The GC is the main site for class switch, somatic hypermutation and generation of memory cells. Different factors, both internal and external, shape the outcome of the immune response. In this thesis, we have studied a few factors that influence the maturation of the humoral response. We have studied how age affects the response, and we show that responses against thymus dependent antigens (TD) are more affected than responses to thymus independent (TI) antigens, in concordance with the view that the T cell compartment is more affected by age than the B cell compartment. Furthermore, we demonstrate that priming early in life have a big influence on the immune response in the aged individual. Priming with a TI form of the carbohydrate dextran B512 (Dx) induces a reduction of IgG levels in later TD responses against Dx. We have evaluated possible mechanisms for this reduction. The reduction does not seem to be caused by clonal exhaustion or antibody mediated mechanisms. We also showed that the reduced TD response after TI priming can be induced against another molecule than Dx. With the hypothesis that TI antigens induce a plasma cell biased maturation of the responding B cells, we examined the presence of Blimp-1, a master regulator of plasma cell differentiation, in GCs induced by TD and TI antigen. Blimp-1 was found earlier in GCs induced by TI antigen and the staining intensity in these GCs was stronger than in TD antigen induced GCs, indicating that plasma cells might be continuously recruited from these GCs. B cells undergoing the GC reaction are thought to be under a strict selection pressure that removes cells with low affinity for the antigen and also cells that have acquired self-reactivity. We investigated the effect of apoptotic deficiencies on the accumulation of somatic mutations in GC B cells. In mice lacking the death receptor Fas, lpr mice, the frequency of mutations was increased but the pattern of the mutations did not differ from wild type mice. In contrast, mice over-expressing the anti-apoptotic protein Bcl-2, had a lowered frequency of mutations and the mutations introduced had other characteristics.

Groupers are an important mariculture fish in Taiwan and Southeast Asian countries. The rapidly growing orange spotted grouper (Epinephelus coioides) has experienced relatively severe bacterial disease problems. The proliferation of pathogens in fish can be suppressed by commensal microbiota. In this context, probiotic seem to offer an attractive alternative. Bacillus subtilisis a probiotic bacteriumthat is administered in diet to suppress proliferation of pathogens. In the present study, E.coioideswere fed for 6 months with diets containing B.subtilis at 0 (control), 0.1 % and 1 %. Percent weight gain and feed efficiency of the 0.1 and 1 % groups were significantlybetter than the control group. The innate cellular response, respiratory burst of the fish fed the 1 % and 0.1 % diet was significantly higher compared to the control group on 10 or 20 days after feeding, and even moresignificanton 30 days.ProbioticBacillus subtilis increased the fish¡¦s intestinal microbial diversity as measured by visible band number and Shannon diversity indexin DGGE analysis. Probiotic Bacillus subtilis also stimulated the population of bacterial species likePaenibacillussp,Lactobacillus oenistrain 59 b, and Methilacidophiluminfernorumstrain V4 that beneficial for Epinephelus coioides. The best dose of probiotic Bacillus subtilis based on growth performances, innate cellular responses and profile of microbiota in fish intestines is 0.1 %, which showed equal efficacy as the 1% diet.

Abstract -1 The immune response occurred in the mice infected with Angiostrongylus cantonensis was mainly humoral immune response. This study was designed to compare the systemic and localized humoral immune responses occurred after primary and secondary infections in C57BL/6J mice. Eight weeks after the primary infection with 20 third-stage larvae, each mouse received a second inoculation of the same dosage. Specific serum IgM, IgG and IgE were found in the second week after primary infection. However, the titers of IgG1 and IgG2b increased at the fourth week after primary infection. Antibodies of these mentioned increased continuously as the progress of infection. On the other hand, the IgM and IgG1 titers increased in brain tissue infusion since the forth week after primary infection, while the titer of IgG start to elevate at the sixth week. Nevertheless, the increase of IgG2B was only noticed at the sixth week and no significant change was observed for IgG2a and IgE. After the secondary infection, serum IgM titers increased while the titer of IgG1 in the brain tissue infusion decreased. Results of Western blot showed that IgG1and IgE in the brain tissue infusion lost the ability to recognize a 42 kDa molecule of the somatic and excreting-secreting antigens of fifth-stage larvae. These variations could be used in the diagnosis of the early stage of mice that re-infected with Angiostrongylus cantonensis. Abstract -2 The radiographic lateral views of the skulls of the mice infected with Angiostrongylus cantonensis were taken. Thus, the parietofrontal index ( PI ) was obtained by measuring and calculating the distances among specific positions on their skulls. Compared with the controls, a significant elevation over the top of the crania of the cases was observed sixty days post-infection. In addition, the phenomenon emerged apparently during the second to the fourth week post-infection. These findings are able to be applied as the external diagnostic references for the infection course of mice infected with Angiostrongylus cantonensis.

Thesis (Ph. D.)--University of Chicago, Committy on immunology, June 2001. / Includes bibliographical references. Also available on the Internet.

Thesis (Ph. D.)--University of Chicago, Dept. of Neurobiology, Pharmacology and Physiology, August 2001. / Includes bibliographical references. Also available on the Internet.

Not available