No description available.

No description available.

Lactobacilli are intestinal bacteria with known immunomodulatory competence. Numerous strains of this genus have been implicated in both the prevention and treatment of intestinal inflammation as well as in maintenance of immunological homeostasis. The frequent inclusion of lactobacilli in probiotic products attests to this ability. These lactic acid bacteria colonise the murine forestomach and burgeon in other environments similarly rich in carbohydrate-containing substrates. Accordingly, lactobacilli may utilise fermentable carbohydrates to synthesise exopolysaccharides (EPS). These polymers are secreted into the cellular milieu and, while the ecological function of EPS is yet to be defined, evidence points towards a protective role. This function may include bacterial protection from immunological attack, via EPS recognition by immune cells, resulting in modulation of immunological activity. Dendritic cells (DC) are potent antigen presenting cells, providing an essential link between the innate and adaptive arms of the intestinal immune system. DC efficiently sample intestinal antigens and present peptides to cognate naive CD4⁺ T cells in secondary lymphatic tissue. Under the influence of secreted cytokines, DC direct the differentiation of naive CD4⁺ T cells and therefore, instruct the resultant immune response. Anti-inflammatory Th2 and regulatory T cells can down-regulate the destructive Th1 pro-inflammatory effects associated with inflammatory bowel disease (IBD). As such, bioactives with the aptitude to direct DC activity and T cell differentiation have the potential to prevent or reduce intestinal inflammation. Therefore, this study aimed to determine whether heat-killed EPS-producing strains of lactobacilli, and their secreted EPS, exert an immunomodulatory effect on the murine gut which may down-regulate the immune reactions associated with IBD. Lactobacilli were screened for their ability to produce EPS when grown in the presence of glucose, sucrose or lactose. Heat-killed EPS-producing strains were then used to stimulate bone marrow-derived DC (BMDC) and the resultant cytokine profile was analysed. Nine Lactobacillus strains were found to produce EPS when grown in the presence of sucrose. Of these, L. reuteri 100-23 and L. johnsonii 100-33 exhibited potential anti-inflammatory effects. Therefore, these strains, as well as L. johnsonii 100-5 and L. johnsonii #21, with relatively weak BMDC stimulatory effect, were selected for further investigation. EPS of the potentially anti-inflammatory strain L. reuteri 100-23 was analysed. This sample contained approximately 85% carbohydrate and was composed of a (2[to]6)-β-fructofuranan (levan) and a mannan. The fructan, with an estimated molecular weight of 7 kDa, comprised at least 50% of the EPS, while the mannan made up at least 22%. The mannan component was likely linked to a protein and may have originated from the culture medium. The immunostimulatory capacity of heat-killed Lactobacillus bacterial cells and their EPS was determined in vitro. Firstly, the effect of lactobacilli and EPS on BMDC cytokine secretion, particularly levels of anti-inflammatory IL-10 and pro-inflammatory IL-12, as well as the expression of cell surface activation markers, was determined. L. reuteri 100-23 stimulated relatively high IL-10 secretion but low IL-12, while L. johnsonii 100-33-stimulated BMDC produced elevated levels of both IL-10 and IL-12. All bacterial cells up-regulated co-stimulatory molecules CD40 and CD80 on BMDC. The effect of these stimulated BMDC on T cell proliferation and cytokine production was then assessed, employing the ovalbuminDO11.10 T cell model. L. reuteri 100-23-stimulated BMDC down-regulated T cell production of the proliferation-stimulating cytokine, IL-2, up-regulated regulatory TGF-β secretion, but did not affect pro-inflammatory IFN-γ levels. The EPS of all strains did not stimulate significant BMDC cytokine production and failed to alter BMDC activation marker expression. However, BMDC stimulated with L. reuteri 100-23 and L. johnsonii 100-33 EPS significantly enhanced T cell IL-2 secretion, but did not alter TGF-β or IFN-γ levels. The effect of in vivo L. reuteri 100-23 and EPS intestinal stimulation on the reactivity of immune cells was subsequently investigated. Mesenteric lymph node (MLN) cells and splenic T cells from reconstituted Lactobacillus-free mice fed stimulant or PBS on two occasions were co-cultured with stimulated or unstimulated donor CD11c⁺ splenic DC ex vivo. Cellular proliferation as well as TGF-β and IFN-γ secretion was analysed, and IL-10 neutralisation assays were carried out to ascertain the involvement of this cytokine. Primary exposure of MLN cells to L. reuteri 100-23 resulted in suppressed cell proliferation in the presence of enhanced TGF-β levels, which may have also involved IL-10. Primed splenic T cells exhibited increased proliferation in the presence of elevated TGF-β levels following re-exposure to L. reuteri 100-23, and IL-10 may be involved in limiting this proliferation. L. reuteri 100-23 EPS did not alter MLN cell proliferation, possibly due to the suppressive activity of IL-10, but did enhance that of naive and primed splenic T cells. The effect of ingested L. reuteri 100-23 and EPS on intestinal sIgA concentration was assessed by quantifying IgA levels in the faecal supernatant of RLF mice previously ingesting L. reuteri 100-23 and EPS. L. reuteri 100-23 EPS-fed female mice exhibited significantly elevated levels of sIgA, while heat-killed bacteria did not affect antibody levels. The present study demonstrated that oral administration of heat-killed L. reuteri 100-23 and EPS exerts immunomodulatory effects on the murine intestine. These bioactives may promote a suppressive environment by conditioning DC to secrete a cytokine profile conducive to regulatory T cell induction and memory generation. Additionally, mucosal protection may be favoured by the stimulation of elevated sIgA levels. Therefore, a therapeutic composite is possibly obtained to preserve the intestinal barrier by defending against pathogen-induced injury and buffering inflammatory events. In these ways, L. reuteri 100-23 and EPS may confer long-lasting protection against, and down-regulate the immune reactions associated with, IBD.

Thesis (Ph.D.)--University of Melbourne, Dept. of Medical Biology, 2003. / Typescript (photocopy). Includes bibliographical references (leaves 161-194).

Thesis (Ph. D.)--University of Hong Kong, 2009. / Includes bibliographical references (leaves 196-222). Also available in print.

published_or_final_version / Biological Sciences / Doctoral / Doctor of Philosophy

No description available.

There is an abundance of information available on the molecular mechanisms of antigenic variation in Plasmodium falciparum. The variant antigen PfEMP1, which mediates antigenic variation as well as cytoadherence and rosetting, has been extensively characterised. Genes coding for the antigen belong to the gene family var, and several var genes have been cloned and characterised. The rodent malaria parasite P. chabaudi is a widely studied in vivo model for P. falciparum. The P. c. chabaudi AS parasite strain has been shown to exhibit antigenic variation and the variant antigen has been detected by surface fluorescence. As with P. falciparum, there is a link between antigenic variation and cytoadherence, however genes coding for the variant antigen in P. chabaudi have not been cloned to date. Therefore, potentially useful in vivo experiments on antigenic variation are restricted. In this thesis it is shown for the first time that the P. c. adami DS parasite strain also exhibits antigenic variation. / Chapter 3 describes efforts to locate genes coding for variant antigens in P. c. adami DS. The main strategy involved a genome survey, by sequencing and analysing randomly selected clones from a P. c. adami DS genomic library. DNA sequences were compared to Plasmodium spp. sequence databases to look for similarity to var genes or other genes encoding variant antigens. Of the 297 clones analysed none had significant sequence similarity to genes coding for variant antigens. However, in a small proportion of sequences some similarity to var genes was noted. Several genes of potential interest were identified, most importantly the gene coding for the vaccine candidate rhoptry associated protein 1 (RAP1), which was subsequently cloned and characterised. Further attempts to locate var gene homologues in P. c. adami involved amplification of P. c. adami genomic DNA using degenerate oligonucleotide primers corresponding to conserved regions of var genes. This strategy proved to be unsuccessful, most likely due to lack of sequence similarity between P. falciparum and P. c. adami genes. In several vaccination studies with the apical membrane antigen 1 (AMA1) of P. c. adami DS, mice were significantly protected against homologous parasite challenge. However, some mice developed late, low-level breakthrough parasitaemias. In Chapter 4, the characterisation of two such breakthrough parasitaemias is described. The ama1 genes of the breakthrough parasites were found to be identical to the ama1 gene of the parental parasites. Similarly, no alteration in AMA1 expression was observed. However, the breakthrough parasites were found to be more resistant than the parental parasites to the effects of passive immunisation with rabbit antisera to AMA1, RAP1 and possibly also MSP119. P. chabaudi infections in mice have been previously shown to consist of a primary parasitaemia followed by a short period of subpatency, and a recrudescent parasitaemia. In surface immunofluorescence studies with P. c. chabaudi, parasites of the recrudescence were shown to be distinct from parasites of the primary parasitaemia, with respect to antigens expressed on the surface of late trophozoite- and schizont-infected erythrocytes. / Chapter 4 describes similar surface immunofluorescence assays carried out with P. c. adami infected erythrocytes, and quantitation of fluorescence by flow cytometry. As with P. c. chabaudi, the recrudescent parasites were found to be antigenically distinct from the primary parasitaemia, indicating that antigenic variation had taken place. Because breakthrough parasites from the AMA1 vaccination trial were similar to recrudescences in peak and duration, we hypothesised that breakthrough parasitaemias, like recrudescent parasitaemias, occur as a result of antigenic variation. In Chapter 4 it was shown by surface immunofluorescence and flow cytometry using hyperimmune sera raised against different parasite populations, that breakthrough parasites express antigens on the surface of late trophozoite- and schizont infected erythrocytes that differ from those expressed by the parental and recrudescent parasites. These results support the hypothesis that switching of the variant antigen on the infected erythrocyte surface enables parasites to evade protective antibody responses directed against merozoite antigens. / Chapter 5 describes the cloning and characterisation of P. c. adami RAP1 which was identified in the process of the genomic survey described in Chapter 3, as well as P. berghei RAP1. Both rodent parasite orthologues of RAP1 were found to have 30% sequence similarity to P. falciparum RAP1, and 6 of 8 cysteines were conserved in the rodent parasite orthologues. However the three polypeptides vary significantly in size. P. c. adami RAP1 and P. berghei RAP1 consist of 691 aa and 604 aa respectively, whereas P. falciparum RAP1 consists of 783 aa residues. These size differences reflect very different N-terminal sequences prior to the first cysteine, whereas the cysteine-rich C-terminal regions are more conserved. Both P. falciparum RAP1 and P. c. adami RAP1 contain N-terminal repeats, however they bear no sequence similarity to each other. P. berghei RAP1 lacks N-terminal sequence repeats that are characteristic of P. falciparum and P. c. adami RAP1. The large cysteine-rich C-terminal region P. c. adami RAP1 (PcRAP1 C3) was expressed in E. coli as a hexa-his fusion protein. Rabbit antiserum to recombinant PcRAP1 C3 was used to characterise the expression and sub-cellular localisation of the RAP1 antigen. P. c. adami RAP1 was found to have a Mr of approximately 80,000 and was shown by immunofluorescence to localise to the merozoite rhoptries. Passive immunisation of mice with rabbit anti-RAP1 serum was shown to protect against fulminant parasitaemia and mortality. In a mouse vaccination trial using the recombinant PcRAP1 C3 polypeptide partial protection was conferred against homologous parasite challenge.

No description available.

Dendritic cells are a key population in the immune system. There are two main subsets: conventional DC (sometimes called classical DC, cDCs), which survey tissues for pathogens and activate naive T cells, and plasmacytoid dendritic cells (pDCs), which produce high amounts of IFNα in response to viral products. Both subsets begin development in the bone marrow from common dendritic progenitor (CDP), and driven by Flt3 signaling induced by the growth factor Flt3L. The CDP gives rise to a cDC committed preDC which exits the bone marrow and seeds peripheral organs to give rise to cDCs, while pDCs finish development in the bone marrow and migrate into the periphery as mature cells. pDC development is directed by E2-2, a member of the E protein family of basic helix-loop-helix transcription factors that are obligate dimers and bind an E-box sequence. E proteins are antagonized by Id proteins, and Id family member Id2 is required for cDC development. The apparently cell intrinsic choice of pDC vs. cDC fate is determined by the net activity of E proteins. Loss of E2-2 affects only pDC development, but its expression is not as specific, as E2-2 is also expressed in cDCs, macrophages and B cells. E2-2 expression in cDCs was particularly puzzling, considering the dependence of cDCs on Id2, the E2-2 antagonist. We strived to address the discrepancy between the very specific activity of E2-2 in pDC development and its broader expression. This work shows that the E2-2 locus encodes two independently regulated isoforms. E2-2Long, the more active isoform is expressed only in pDCs, and E2-2Short, the less transcriptionally active isoform, is expressed more broadly. Moreover, E2-2Long is required for optimal pDC development. Homozygous loss of just E2-2Long leads to lower pDC frequency in the spleen and phenotypically affected pDCs in the periphery. Although E2-2 is essential for pDC development, no signal has yet been identified that induces E2-2 expression to influence pDC over cDC fate. Given the essential nature of E2-2 in pDC development, we aimed to understand better how E2-2 is regulated during pDC fate specification. We examined an E2-2Long reporter and found that E2-2Long expression precedes pDC commitment in early progenitors. E2-2 is only upregulated in committed pDCs, and actively shut down in cDCs through the expression of Id2. Analysis of E2-2 in an in vitro time controlled model of dendritic cell development showed that all dendritic cells (both cDCs and pDC) go through an E2-2 expressing stage only to resolve into E2-2- cDCs and E2-2+ pDCs. Early E2-2 expression and E2-2 upregulation upon pDC commitment hinted at the presence of an E2-2 controlled cis-regulatory module. ChIP-Seq data showed only one peak of E2-2 binding located 150 kb downstream of the TCF4 gene. Heterozygous deletion of this region in the in vitro DC development model led to impaired pDC development and a fail to undergo the E2-2+ stage described above. The regulatory element is essential for E2-2 upregulation during DC development. This work describes two new mechanisms of E protein regulation during dendritic cell development: cell specific differential isoform usage and a distal cis regulatory element responsible for enforcing and upregulating E2-2 expression. These new mechanisms can lead to better understanding of how this family of broadly expressed and pleiotropic transcription factors is regulated, with implications in overall development, not only dendritic cell fate decision.