C-type lectin-like receptors (CTLRs) play critical roles in immunity and homeostasis by recognising a variety of microbial or endogenous ligands. Clecsf8 is a member of the Dectin-2 family of CTLRs. Clecsf8 shares important similarities with its relatives Mincle and Dectin-2, such as the lack of an integral signalling motif and a single, calcium dependent ligand binding domain. They were shown to associate with the FcRγ adaptor, which is essential for receptor surface expression and downstream signalling. Recent publications revealed an important role for Clecsf8 in anti-mycobacterial immunity. It was reported to recognise the mycobacterial cord factor (TDM), similar to the related CTLR Mincle, as well as a possible role in candidiasis. In this study, we characterised the underlying mechanism of Clecsf8 expression in a context of mycobacterial disease. The generation of novel anti-Clecsf8 monoclonal antibodies allowed us to characterise the expression of Clecsf8 in detail in homeostasis and inflammation in murine models in vivo and culture systems in vitro. We found Clecsf8 to be predominantly expressed on monocytes/macrophages and neutrophils within e. g. the peritoneal cavity, blood and bone marrow. Notably, Clecsf8 was expressed only weakly in the lung, but strongly upregulated in a pulmonary mycobacterial infection model. In vitro, Clecsf8 expression on elicited macrophages was strongly induced upon treatment with microbial stimuli in a Myd88- and Mincle dependent manner. Interestingly, surface expression of Clecsf8 in a murine fibroblast cell line was greatly enhanced by co-transfection of Mincle, but not another related CTLR, Dectin-2. Notably, we confirmed mycobacteria as a ligand of CLECSF8, but found no role for the receptor in Candida immunity. In conclusion, Clecsf8 is a myeloid expressed, mycobacterial receptor, showing significant interdependence with Mincle and is regulated through the Myd88 pathway.

Little is known about the effect of different modes of expression of an antigen in rBCG on immune response. An appropriate wing of the immune system, with different degrees, is activated upon encounter with a foreign antigen. Knowledge of these responses is vital to the development of future recombinant vaccine. Various E. coli-mycobacterial species shuttle vector constructs were made using a combination of mycobacterial promoters and signal sequences. Thus enabling foreign antigens to be expressed cytoplasmically or secreted outside rBCG as native proteins or membrane-associated lipoproteins. A pivotal study using an E. coli beta-lactamase as a reporter gene is described for the evaluation of the strength of promoter and signal sequence constructs both in vitro and most importantly in vivo using the mouse macrophage cell line J-774. Expression of the diphtheria toxin fragment B as a foreign antigen was detected in vitro with all constructed plasmid vectors in rBCG using a western blot as a means of detection. It was observed that all hsp60 promoter-based constructs exhibited a high frequency with variable degree of plasmid DNA deletions Using three different rBCG substrains, the BCG Tokyo was found to be more stable (P < 0.01) and exhibited less degree of deletion (P < 0.001) compared to either BCG Moreau or BCG Pasteur. Sequence analysis of deleted plasmid DNA revealed a specific region common with nearly all plasmid deletions. Such a region of the DNA was found to correspond to the first transcriptional starting site of the hsp60 promoter. Furthermore no differences were observed in the level of expression among the three-rBCG substrains, retaining plasmid DNA, when detected by immunoblotting.

Ph.D. / Background: AIDS is characterized by a number of opportunistic infections and the immune depletion caused by HIV infection is the strongest risk factor for both reactivation of tuberculosis (TB) and progression of Mycobacterium tuberculosis (Mtb) infection to disease. Numerous studies have shown that concurrent infection of the same host cell by HIV,and M.tb stimulates replication of both pathogens. The interaction between the two is lethal. A synergistic relationship exists between Mtb and HIV. While HIV spurs the spread of TB, mycobacterial infection results in acceleration of HIV disease progression. The requirement for iron as a crucial factor for cellular processes has long been demonstrated. Excess iron leads to infections with harmful consequences such as cell death and function impairment. During infection, iron is required by both the host cell and the pathogens. Iron chelation is believed to modulate some of these effects. Objectives: Mtb, HIV and Fe-overload are common in sub-Saharan Africa and iron plays a major role in determining the outcome of several infections. In view of this, we wanted to (1) investigate the effect of excess iron on host cell defences during co-infection with the mentioned microorganisms, (2) evaluate the differences in both host and pathogen responses during acute and chronic infection in the presence of iron overload and (3) Determine the efficacy of iron chelation (with DFO) as a means of counteracting conditions associated with iron overload. Hypotheses: The combination of Fe-overload and co-infection of host cells with HIV and Mtb in an in vitro model should stimulate replication of the pathogens, which would ultimately result in host cell stress manifesting as lower viability or cell death and impaired immune defence functions. Also the detrimental effects of excess iron on host cell viability could be counteracted through the use of iron chelators. Methods: We analyzed the in vitro effect of Mtb in bothchronically and acutely HI V-infected cells (PBMC's and monocytes), exposed to 500 uM FeSO 4 and/or DFO for 4 days. Host cell viability, survival and death were assessed through viability assays (MIT and Alamar Blue) and flow cytometric analyses of apoptosis/necrosis (using Annexin V and propidium iodide). Secretion of IL- 6 and TNF-a and production of total nitrate were monitored as host immune/defence responses using specialized ELISAs. HIV replication was investigated by looking at core protein (p24) contents and reverse transcriptase (RT) activity. Mtb replication and growth was monitored using the microplate Alamar Blue assay (MABA) and quantitative culturing.Results: Co-infection caused a reduction of host cell viability (± 20% and 45% inhibition during chronic and acute infection respectively;, as measured by MTT), increases in the numbers of viral particles (2.3 times and 20% increases for chronic and acute infections respectively) and stimulation of both bacterial viability (36%) and host defence responses (30% increase in TNF-ct secretion). Excess iron further decreased viability with a marked increase in necrosis of cells and was found to enhance pathogen replication and growth (26% for HIV and 47% for Mtb). Chelation of iron with DFO abrogated the enhanced replication of the pathogens with a marginal restoration of host viability. Conclusion: The results obtained demonstrate the deleterious effect of excess iron during concurrent infection with both pathogens as well as its stimulating/enhancing properties on pathogens. On the other hand, DFO inhibited pathogen replication and host viability.

No description available.

In this study the effects of repeated antigen injections on total complement (C') and C'4 of guinea pig serum were investigated to determine if constant antigenic stimulation would show changes in the C' and C'4 titers. Attempts were also made to correlate any changes with variations in antibody titers during the repeated antigen injections.

In order to further elucidate the interaction between CD23 and β2 integrins (CD11b/CD18) the following objectives were established: Expression and purification of CD11b I-domain as a GST-fusion protein using Escherichia coli; Cloning, synthesis and expression of CD18 I-Like domain.CD11b I-domain has previously been expressed as a GST-fusion protein (Daniels, 2010) and consequently led to comparable expression of CD18 I-like domain as a GST-fusion protein; Preparation of two site-directed mutants of CD18 I-Like domain in order to study the function of the serine residue involved in the S116P mutation. The serine was mutated to proline, as in LAD patients, as well as alanine, a non-polar alternative, in order to contrast and compare binding characteristics.  Expression, refolding and purification of sCD23, and a double mutatedsCD23 (RKΔAA) from E. coli; This was performed according to the method described by Daniels et al. (2005); Investigation of the CD23-CD11b I-like domain interaction through surface plasmon resonance spectroscopy.

The effect of continual antigenic stimulation on the immune system of mice was studied using two different experimental approaches. A GVHR was induced in Fi mice by the injection of parental spleen cells at weekly intervals. Several weeks later the spleen cells of mice undergoing a GVHR were shown to be immunosuppressed as their in vitro responses to the mitogens Con A and LPS were substancially lower than control animals. The serum from these treated mice was also immunosuppressive to normal spleen cells. The proliferative response to Con A and allogeneic cells of normal: syngeneic, allogeneic, and parental spleen cells was 90% suppressed when GVH serum was added in comparison to the addition of normal serum. Similarly, the in vitro antibody response to a T dependant antigen was impaired; however, the antibody response to a T independant antigen was not impaired. These results indicate that T cell functions are more sensitive than are B cell function to immunosuppressive factors in the serum of mice undergoing a GVHR. The serum was fractionated by gel filtration on a Bio-Gel P-200 column. The inhibitory material in GVH serum eluted in the immunoglobulin fraction of serum which indicate that it has a molecular weight of 150,000 or greater. The second approach studied involved continual allogeneic stimulation. Parental type mice were injected at five day intervals with Fi spleen cells in order to induce a HVG reaction. After several injections the spleen cells from these mice were tested in vitro. The spleen cells from HVG mice responded the same as normal spleen cells to the mitogens Con A and LPS. The spleen cells from HVG mice showed an enhanced in vitro antibody response as compared to normal spleen cells. This enhancement was attributed to the allogeneic effect. This series of experiments have shown that the induction of a GVHR in mice can later lead to immunosuppression and production of immunosuppressive factors in the serum of these mice. The induction of a short term HVG reaction has no adverse effects on the immune system except for enhancing an antibody response. It is possible that a more prolonged HVG reaction would parallel the immunosuppression observed in mice undergoing a GVHR. / Science, Faculty of / Microbiology and Immunology, Department of / Graduate

Recent work has shown that in vitro, the cytotoxic immune response to cell surface antigens is enhanced if the antigen to which the immune response is directed, is on cells bearing major histocompatibility antigens identical to those of the responding cells. This 'H-2 restriction' has been demonstrated in the mouse using virally infected cells, haptenated cells, cells bearing the male Y antigen, and cells differing at the minor histocompatibility loci. Other investigations have shown that antigenic determinants coupled to tolerated antigens or isologous serum proteins, elicit a humoral response which is weaker than that to the same determinant coupled to a heterologous carrier. This and other evidence suggest an inverse relationship between humoral and cell mediated immunity. The purpose of this investigation was to explore the humoral response to antigens on cells which are syngeneic or allogeneic to the recipient, in order to determine the influence of a tolerated as opposed to allogeneic background. The approach used in this study was as follows: Mice were immunized with antigen (rabbit Fab' fragments) attached to syngeneic, allogeneic, or F₁ (semi syngeneic), irradiated spleen cells. Specific anti-rabbit Fab' plaque forming cell numbers were determined five days after the third, weekly injection of Fab' coated spleen cells. Some of the spleen cells taken from the responding animals, on the day of sacrifice, were incubated in vitro with soluble antigen (rabbit Fab' fragments not specific for mouse cells) for four days. The results showed that the humoral response to antigens attached to cells bearing 'self histocompatibility antigens (i.e. syngeneic or F₁ semi syngeneic cells) was significantly weaker than the humoral response to the same antigen on allogeneic cells. The effect of in vitro incubation of responder spleen cells for four days with soluble antigen was to reverse this difference. Those spleen cells exhibiting lowered plaque forming cell numbers initially (i.e. those cells from mice immunized with antigen on syngeneic or F₁ cell surfaces) showed, after incubation, a response equal to or greater than those cells which intially (before in vitro incubation) demonstrated a larger response (i.e. cells from those mice immunized with antigen on allogeneic cell surfaces). / Science, Faculty of / Microbiology and Immunology, Department of / Graduate

Although it is known that cell-mediated immunity (CMI) plays a key role in protection against brucellosis, the exact immune mechanisms leading to protection are still not fully understood. Better understanding of the mechanisms would help in the development of a human Brucella vaccine and help in improving animal vaccines. In this research, B. abortus strain RB51 and a closely-related, nonpathogenic Ochrobactrum anthropi (strain 49237) bacterium were used to study the immune response against brucellosis in mice. Both O. anthropi strain 49237 and recombinant strain 49237 expressing Brucella protective antigen copper-zinc superoxide dismutase (Cu/Zn SOD) induced a mix of Th1 and Th2 type immune responses but failed to provide protection against virulent Brucella challenge. After changing the immune response to a predominantly Th1 type of response using CpG oligonucleotides as an adjuvant, both strains provided protection with the recombinant strain inducing significantly higher protection. It was also demonstrated that vaccination with strain RB51 induced Th1 immune responses characterized by high interferon-gamma (IFN-g) production with no interleukin-4 (IL-4) secretion as well as high IgG2a and minimal IgG1 production. A colorimetric cytotoxic T lymphocytes (CTL) assay was developed to demonstrate that strain RB51 induced an antigen-specific CTL reaction that probably plays an important role in protection. The results suggest that optimal protection against brucellosis requires IFN-g-secreting T cells and antigen-specific CTLs. Recombinant strain RB51 overexpressing Brucella Cu/Zn SOD and simultaneously expressing mycobacterial 85A antigen induced higher IFN-g production and CTL activity than the parent RB51 strain. The combined results suggest that the recombinant O. anthropi strain could be used as a human vaccine against brucellosis and that the recombinant RB51 strain could be used as an effective vaccine against both brucellosis and tuberculosis in animals. / Ph. D.

Sepsis, a major clinical problem with high morbidity and mortality, is caused by overwhelming systemic host-inflammatory response. Toll-like receptors (TLRs) play a fundamental role in induction of hyperinflammation and tissue damage in sepsis. In this study, we demonstrate a protective role of TLR9 inhibition against the dysregulated inflammatory response and tissue injury in sepsis. TLR9 deficiency decreased the mortality of mice following cecal ligation and puncture (CLP)-induced sepsis. TLR9 knockout mice showed dampened p38 activation and augmented Akt phosphorylation in the spleen, lung and liver. In addition, TLR9 deficiency decreased the levels of inflammatory cytokines and attenuated splenic apoptosis after CLP. These results indicate that TLR9 inhibition might offer a novel therapeutic strategy for the management of sepsis.