Tuberculosis (TB), caused by Mycobacterium tuberculosis (Mtb), is the leading cause of death from an infectious disease worldwide. Over the course of its life cycle in vivo, Mtb is exposed to a plethora of environmental stress conditions. Temporal regulation of genes involved in sensing and responding to such conditions is therefore crucial for Mtb to establish an infection. The Rv2745c (clgR) gene encodes a Clp protease gene regulator that is induced in response to a variety of stress conditions and potentially plays a role in Mtb pathogenesis. Our isogenic Mtb:ΔRv2745c mutant is significantly more sensitive to in vitro redox stress generated by diamide, relative to wild-type Mtb, implicating a role for ClgR in the management of intraphagosomal redox stress. Our data indicates that ClgR plays a role in multiple regulatory networks in response to different stress conditions. Thus, redox stress leads to dysregulation of the σH/σE regulon in Mtb:ΔRv2745c. Induction of clgR in Mtb and Mtb:ΔRv2745c (comp) did not lead to Clp protease induction, indicating that clgR has additional functions. Disruption of genes involved in sulfate assimilation also occurred in the knock out, implicating clgR as a possible regulator of downstream signaling cascades that facilitate Mtb survival. On the other hand, the expression of clgR during hypoxia is known to result in Clp protease induction. As such, the isogenic mutant has a significantly different growth profile upon hypoxia and reaeration. Transcriptomics reveal disruption of the dosR regulon, σH/σE regulon, and mycolic acid synthesis genes. Clearly, the Mtb Rv2745c-encoded ClgR performs different functions during stress response and is important for the pathogenicity of Mtb in vivo. Our in vivo findings in a low dose aerosolized model reveal deficiencies of the isogenic mutant when establishing an infection, leading to skewed immune responses throughout the course of infection. Thus, clgR plays a critical role in both establishing an infection that influence the immunogenic outcome. Additional studies investigating the role of clgR in a nonhuman primate model will further elucidate the contributions of clgR to the pathogenesis of Mtb in an animal model that is more representative of human TB disease. / acase@tulane.edu

Digital dermatitis (DD) is a superficial infectious dermatitis of the digital skin of cattle and sheep that can be very painful, causing severe lameness in affected animals. Bovine digital dermatitis (BDD) in dairy cattle has now been reported in most countries they are farmed, and DD in sheep, known as contagious ovine digital dermatitis (CODD) is rapidly emerging as a severe infectious foot disease since first reports from the UK in 1997. Spirochaetes, of the genus Treponema have frequently been found in large numbers in BDD lesions and are now considered the primary causative bacteria of BDD. Three treponeme phylogroups are consistently isolated from dairy cattle BDD in the UK and the USA, which are known as Treponema medium- like, Treponema phagedenis- like spirochaetes and Treponema pedis. Over the past 40 years research has focused on dairy cattle BDD and overlooked whether the disease exists in beef cattle herds in the UK, and whether the same aetiological agents are causal. There is also limited information on the causative bacteriological agents of CODD. Furthermore, no definitive transmission routes or infection reservoirs of DD in either cattle or sheep had thus far been delineated, with only a single study finding a potential reservoir site of DD treponemes in the dairy cattle gastrointestinal (GI) tract. Using molecular bacteriological studies it was found that CODD and beef cattle BDD, as in dairy cattle BDD, show a high association with the three DD treponeme phylogroups. All CODD and beef BDD lesions investigated had at least one of the three DD treponeme phylogroups present in the lesions and these treponemes were also isolated from a high proportion of lesions. No DD treponemes were detected in healthy sheep or beef cattle foot tissue. Upon 16S rRNA gene sequence analysis all isolates showed a high similarity, if not 100% identity, to representatives of each treponeme phylogroup isolated from dairy cattle BDD lesions, indicating a shared aetiology between DD in all three animals. Additionally, the same treponeme bacteria were detected and isolated from a new undefined foot disease in dairy goats in the UK indicating that cross-species transmission of DD may have occurred causing DD infection in a previously unaffected domestic livestock species. To understand potential transmission routes and infection reservoirs of DD, the host GI tract and hoof trimming equipment were investigated. Of the sheep gingival (n= 40) and rectal tissues (n= 40), 1/40 gingival tissues were positive for DD- associated treponemes (T. pedis), and 3/40 rectal tissues (one containing T. medium- like and two tissues containing T. pedis). No DD- associated treponeme DNA was amplified from beef cattle rectal tissues (n= 40), however 4/40 beef gingival tissues were positive for DD- associated treponemes (all containing T. phagedenis- like). A T. phagedenis- like DD treponeme was isolated from the rectal tissue of a CODD symptomatic sheep. Beef cattle (n= 41) and sheep (n= 79) faeces failed to amplify DD- associated Treponema DNA. Twenty two treponemes were isolated from sheep faeces; however, upon phylogenetic analysis these clustered with considered non-pathogenic treponemes, which interestingly exhibited farm specific diversity in their 16S rRNA gene. Trimming equipment was tested after being used to trim cattle and sheep hooves, and subsequently after disinfection of equipment. Of the blades used to trim DD symptomatic animals (n= 26, cattle and sheep combined), 25/26 were found to be positive for at least one of the DD Treponema phylotypes. This figure was reduced to 10/26 (38%) after disinfection of the blades. Following culture of a swab, an isolate belonging to the T. phagedenis- like spirochaetes was isolated from a knife sample after trimming a DD positive cow. Beef cattle sera from DD positive and negative farms were investigated to understand whether beef cattle’s perceived lower prevalence of BDD in the UK is due to a lack of exposure to treponemes, or a protective immune response. Beef cattle from DD positive farms appeared to produce a strong immunological response to treponemes, compared with DD negative farm animal sera. Therefore the perceived lower prevalence of DD in beef cattle does not appear to be due to a protective response in these animals, but more likely due to a lack of exposure to DD treponemes. In conclusion, these studies have produced vital information describing DD in beef cattle and sheep and their respective aetiological agents allowing for more appropriate treatments in the future. Additionally, given the two potential transmission routes delineated from the data, effective actions can be taken to prevent the spread of DD within current hosts and to limit emergence into yet unknown additional host species.

Salmonella spp. pose significant health risks to humans and animals. S. Typhi, the causative agent of typhoid fever, is responsible for 21 million new cases of enteric fever each year and an estimated 200,000 deaths worldwide. Approximately 5-8% of infected individuals will become lifelong bacterial carriers. It is currently unknown why bacteria persist within the host in the face of robust anti-bacterial immune responses. We hypothesize a stalemate between bacterial persistence and the host immune response is determined by anatomical location, and that this dictates CD4 T cell function and infection outcome. Using a mouse model of persistent S. Typhimurium infection, we show lymphoid Salmonella-specific Th1 cells are potent producers of IFN-γ and protect mice from challenge when adoptively transferred into naïve animals. Conversely, Salmonella-specific CD4 T cells from chronically infected livers exhibit a Tr1-like phenotype, produce large amounts of IL- 10, and increase mouse susceptibility to bacterial challenge. These differences in CD4 T cell phenotypes may inhibit macrophage ability to control intracellular bacterial replication; liver Tr1-like cells fail to activate bacterial killing, possibly through the production of IL-10, which reduces the expression of iNOS and nitric oxide production by macrophages. Additionally, we demonstrate liver macrophages from chronically infected mice exhibit an immunosuppressive, M2-like phenotype and are not classically primed to kill intracellular bacteria, unlike macrophages from lymphoid sites. Furthermore, we show that the liver may be responsible for inducing these Tr1-like cells, as liver macrophages are capable of activating and expanding Salmonella-specific CD4 T cells during infection. Thus, we believe the immunosuppressive environment in the liver affords a permissive niche for Salmonella persistence in vivo. However, we believe vaccination can protect mice from Salmonella infection, if potent Th1 cells are induced, as seen in lymphoid tissue during infection. Therefore, we developed a CD4 T cell peptide vaccine against a known Salmonella secreted epitope. We show vaccinated mice generate potent Th1 responses against Salmonella and mice are significantly protected from challenge. These studies provide new insight into the immunological mechanisms regulating bacterial persistence, as well as the role tissue microenvironments play in modulating pathogen-specific immune responses. / acase@tulane.edu

No description available.

Plasmodium falciparum is responsible for most malaria-related morbidity and mortality, mostly affecting young children, non-immune adults and pregnant women. A characteristic feature of the pathogenesis of infection caused by P. falciparum is the cytoadherence of infected erythrocytes to the endothelial cells lining the microvessels of host organs. This phenomenon, termed ''sequestration'', mainly results from the adhesive interactions between P. falciparum erythrocyte membrane protein-1 (PfEMP1) proteins on the surface of infected erythrocytes and various host endothelial receptors such intercellular adhesion molecule 1 (ICAM-1), which is hypothesised to have a role in cerebral malaria. PfEMP1 molecules consist of several Duffy binding-like (DBL) and cysteine rich interdomain region (CIDR) domains that have different cytoadhesive functions. The second class of DBL domains, DBLβ, has been associated with adhesion to ICAM-1 receptors. In the present study, we selected four recombinant PfEMP1ICAM-1-DBLβ domains for mouse immunisations. Thirteen monoclonal (mAbs) and polyclonal antibodies (pAbs) were raised to three recombinant domains (DBL13, DBL31 and DBL41). All mouse mAbs and pAbs comprised IgM antibodies that recognised homologous and heterologous DBLβ domains. Most mAbs and pAbs labelled the surface of erythrocytes infected by P. falciparum isolates, with an IgM labelling capacity ranging from 10.1% to 67.6% of total IEs. Mouse antibodies showed similar patterns of reactivity with ICAM-1-binding and non-binding isolates, and reacted with a parasite isolate from a different genome (3D7). Surprisingly, we detected a remarkable reduction in IE population after incubation with mouse mAbs and pAbs, and this was mainly observed with antibodies that strongly labelled the surface of IEs. We demonstrated that this haemolysis was resulted from an immunological interaction between mouse IgMs and a parasite-derived component on the surface of live IEs. Antibodies raised to DBL41 were the most effective in all assays. Of these, three antibodies (pAb B5, mAb B4, mAb G6) and an anti-DBL31 mAb (E7) significantly blocked IE adhesion to purified proteins (ICAM-1 and CD36) under static and flow conditions. These antibodies also blocked parasite adhesion to HUVEC under conditions of blood flow. In a separate work, we characterised the immune response of eight semi-immune serum samples obtained from female adults living in Kilifi County, Kenya. Our results indicated that semi-immune sera specifically recognised five recombinant DBLβICAM-1 domains and a VAR2CSA DBL domain, and recognised the surface of erythrocytes infected by diverse parasite isolates with variable levels of reactivity. Some sera, particularly JA225 and JA235, significantly inhibited IE adhesion to ICAM-1 under both static and flow conditions. To our knowledge, this is the first study to examine the use of PfEMP1ICAM-1-DBLβ domains for the development of mouse mAbs and pAbs that recognise homologous and heterologous parasite isolates and block IE adhesion. However, further work is required to identify the surface ligand(s) involved in interaction with mouse IgM and to investigate the mechanisms of IgM-mediated IE lysis.

Immunotherapy for renal cell carcinoma (RCC) has yielded some clinical responses. However this approach frequently fails, possibly due to inefficient migration of T-cells to tumour tissue or immunosuppressive mechanisms within the tumour environment. To aid development of T-cell therapy for RCC I investigated how T-cells are recruited to this tumour, which T-cell subsets infiltrate, and how they function. Analysis of the expression of all 19 chemokine receptors on matched TIL and PBMC demonstrated that CCR5, CXCR3 and CXCR6 were expressed at significantly higher levels on tumour-infiltrating T-cells than memory T-cells in PBMC, suggesting a role for these receptors in recruitment to RCC. Immunohistochemistry showed the corresponding ligands were present in RCC, and transwell assays confirmed the ligands induce migration of TIL. I demonstrated Foxp3\(^+\)CD25\(^{hi}\)CD127\(^{low}\) Tregs were enriched within the tumour, and also expressed high levels of CCR5, CXCR3 and CXCR6, as well as CCR6. They lacked expression of IL-2 and IFN-\(\gamma\) post-stimulation, consistent with a regulatory phenotype. Functional characterisation of Foxp3\(^-\) TIL demonstrated they can function ex vivo, however their high expression of the inhibitory molecule PD-1 may indicate exhaustion in vivo. Double positive CD4\(^+\)CD8\(^+\) T-cells were also enriched in TIL and had a similar functional profile to CD8 T-cells.

Gene directed enzyme prodrug therapy using E.coli the enzyme nitroreductase (NR) to activate the prodrug CB1954, is being developed as an attractive targeted chemotherapy for eradication of localized tumours. In addition to direct killing of NR-expressing tumour cells and potentially also their immediate neighbours via local spread of the activated prodrug, the consequent release of tumour antigens from dying tumour cells has the potential to induce antitumour immune responses. The present study investigates the capacity of NR/CB1954-mediated tumour cell death to activate CD8\(^+\) T cell responses using ovalbumin (OVA), as a model tumour antigen. The transgenic adenocarcinoma mouse prostate tumour cell line (Tramp-C1) was modified to stably express the therapeutic NR gene together OVA. These modified tumour cells were used to seed tumours in mice and OVA-specific T cell responses to gene therapy were investigated. Treatment of mice bearing NR-expressing tumours with CB1954 enhanced expansion of endogenous OVA-specific CD8\(^+\) T cells and marginally enhanced OVA-specific cytotoxic T lymphocyte (CTL) activity, however long-term CD8\(^+\) T cell dependent immunity was insignificant. The possibility of enhancing NR/CB1954-mediated long-term antitumour immune responses by combining with other immunogene therapies namely, 4-1BB costimulatory ligand (4-1BBL) or granulocyte macrophage colony stimulation factor (GM-CSF) was further explored. These combined therapies notably increased the frequency of memory OVA-specific CD8\(^+\) T cell and CTL response in some lymphoid tissue relative to NR/CB1954 monotherapy. One of the obstacles to cancer immunotherapy is the development of T cell anergy early in the course of tumour progression, therefore it was of interest to investigate the potential of NR/CB1954 and 4-1BBL combined tumour therapy to reverse CD8\(^+\) T cells anergy in vivo. This study describes preliminary results showing the effect of this combined therapy on the proliferative and functional responsiveness of anergic CD8\(^+\) T cells. In conclusion, these findings indicate that NR/CB1954-mediated tumour cell death is a weakly immunogenic process that facilitates short-term antitumour CD8\(^+\) T cell responses. Combining NR/CB1954 with intratumoural GM-CSF or 4-1BBL immunotherapy can enhance the frequency and effector function of memory tumour antigen-specific CD8\(^+\) T cells; and thus has the potential to provide long-term antitumour immunity.

Myeloid and lymphoid malignancies are potentially curable through a graft versus leukaemia (GvL) effect following allogeneic haematopoietic stem cell transplantation. Whilst donor T cell are thought to be the main mediators of GvL, the effect of donor NK cells within HLA matched T cell depleted transplant setting is more unclear. Patient blood samples were analysed during the first month post-transplant, with higher reconstitution of NK cells at two weeks conferring a relapse protection association. Donor stem cell graft samples, from which NK cells within the patient at two weeks are thought to be derived, similarly displayed a strong association between high NK cell dose and protection from disease relapse. CD56dimDNAM+ NK cells were found to be the population with the most significant association. The ability of NK cells to kill AML blasts in a DNAM dependent manner was shown indicating that direct killing of residual tumour cells may be a valid mechanism of GvL. These findings suggest that optimising the number of NK cells within stem cell grafts should be considered as a means to prevent disease relapse.

Dr James A. Thomson reached a milestone discovery in 1998, as he managed to isolate and in vitro expand embryonic stem cells originating from human blastocysts. Since then, human embryonic stem (hES) cells have served as excellent tools for the understanding of a plethora of events that take place during embryogenesis. A full and comprehensive analysis of the molecular mechanisms that regulate both pluripotency and differentiation procedures will ultimately allow these cells to be utilised for therapeutic purposes. The first part of the present thesis is dedicated to investigating the implication of ADP-Ribosylation Factor 6 (ARF6) in TGFβ signalling. ARF6 is a low molecular weight GTPase involved in various cellular functions. Our preliminary data indicate that ARF6 interacts with SMAD4. Building on that, we uncover novel interactions of ARF6 with proteins SMAD2/3 and the interconnection between nucleotide status and downstream signalling events. The connection between ARF6 and TGFβ signalling led us to hypothesize a role for the GTPase in hES cells. In that system, we characterise the effects of ARF6 activation or knockout on both Activin A and BMP4 signalling. In addition, we uncover a novel role for the GTPase during mesendoderm specification. In the last part of the thesis, we utilise a broad transcriptomic approach to reveal novel candidates that are implicated in early differentiation of hES cells to mesendoderm. The assay has been carried out using a novel culture system, based on the ability of Activin A to preserve pluripotency and BMP4 to initiate differentiation.

Nontyphoidal Salmonella (NTS) are a major cause of fatal bacteremia in Africa. We investigated the role of bactericidal antibody in complement-mediated killing of NTS. Immunised mice serum lacked such activity due to weak complement activity. Mouse anti-Salmonella antibodies were able to effect killing when given a source of human complement. Human serum bactericidal assays showed that the serum-susceptibility of an African clinical isolate varied based on growth conditions. In vitro kinetics of serum-killing, phagocytosis and antibody and complement deposition indicated that a proportion of Salmonellae are phagocytised before serum-killing occurs and this may explain how the protective effects of anti-Salmonella antibodies are undermined in IFN\(\gamma\) deficiency. We studied targets of bactericidal antibodies using an optimised serum-adsorption procedure and a range of different NTS strains and serovars as well as LPS mutants. Antibodies against the immuno-dominant O-antigen (OAg) were a major target of bactericidal antibodies against NTS in human serum. These data support development of an OAg based vaccine against NTS. Finally, using electron microscopy, we showed the physical effects of serum-killing on Salmonellae and also demonstrated that a major difference between inhibitory and bactericidal serum was the quantity of complement deposited on Salmonellae.