INFECTIOUS DISEASE AND IMMUNOLOGY PROJECT PROPOSALS 2007 CONVENOR: DR. J E RAMESAR Project Title: CHOLESTEROL: THE PATHOGENS DOORWAY TO PARADISE Supervisor: Prof. Frank Brombacher, Division of Immunology; Tel:406 6424; e-mail: firstname.lastname@example.org Co-supervisor: Dr. Reto Guler, Division of Immunology; 406 6813 / 084 417 16 19; e-mail: email@example.com Keywords: Infection diseases, intracellular pathogen, macrophage, lipid metabolism Background: Listeria monocytogenes is a facultative intracellular bacterium responsible for disseminated infections in immunocompromised people and pregnant women resulting in septicemia, meningitis and abortion. 1 Listeria mainly infects one of the principal cells of the immune system, the macrophage. The bacilli attaches to the cell membrane and through complex interactions enter the cell. Therefore it is important to focalize the host versus pathogen interaction on the level of the cell membrane. The membrane contains many essential lipids such as cholesterol which could be used by the pathogen to enhance the infection.2 In order for the cell to metabolize and store cholesterol in the membrane is has to be taken up by lipid receptors such as the very low density lipoprotein receptor (VLDLR). We obtained mice which are deficient in VLDLR and therefore can investigate the fascinating relationship between lipid metabolism and infection by intracellular pathogens. Objectives: The aim of this honours project is to investigate in detail the immune responses induced by the intracellular pathogen Listeria monocytogenes infection in the absence of VLDLR in mice and to further identify the action of mechanisms involved. Research Plan: VLDL receptor deficient and their corresponding control mice will be infected with Listeria bacilli. At different time points after infection mice will be sacrificed for analyses of bacilli burden, histology and restimulation assays. Furthermore we will investigate their cytokine response by FACS and ELISA. A special focus is directed to the macrophage responses. Bone marrow derived macrophages from VLDLR deficient and their control mice will be infected in vitro with Listeria and their bacilli load, cytokine and effector killing mechanisms will be investigated. Future work will focus on mechanistic studies directly edging the cell membrane compartment which could be integrated into a potential master project, where we will measure regulation of lipid metabolism in the absence of VLDLR and their effect on pathogen survival. Personal formation: This honours proposal aims to attract a highly motivated student which will be integrated into a young dynamic team work. The student will learn different immunological techniques, to analyse and present their scientific data. Excellent supervision will be provided to promote the fun in science. References: 1. Hamon, M., Bierne, H. & Cossart, P. Listeria monocytogenes: a multifaceted model. Nat Rev Microbiol 4, 423-34 (2006). 2. Pucadyil, T.J. & Chattopadhyay, A. Cholesterol: a potential therapeutic target in Leishmania infection? Trends Parasitol 23, 49-53 (2007). Generation of functional full-length Subtype C HIV-1 genomic vectors lacking a functional env gene for pseudovirion assays Supervisor: A/Professor Carolyn Williamson; Co-Supervisors: Dr Zenda Woodman, Melissa-Rose Abrahams Tel: 406 6683; 406 7463 Email: firstname.lastname@example.org; email@example.com Background: The HIV-1 genome comprises 9 kb with a long terminal repeat (LTR) and 9 genes coding for the various viral proteins. Up to now, characterization of HIV-1 replication has focused on subtype B and thus reagents needed for the study of subtype C viral infectivity is limited. One of the methods used to investigate viral replication and compare changes in viral proteins make use of the pseudovirion infection assay which is designed to restrict viral replication to a single round making it safer to use within a laboratory setting and enabling standardization of research protocols. The way this is achieved is by co-transfecting a cloned envelope gene with an HIV-1 genome vector that lacks a functional env gene thus allowing for incorporation of envelope proteins and viral RNA into the viral capsid upon co-expression within mammalian cells while limiting its ability to produce new envelope proteins once it has infected a host cell. This method is used successfully to study the process of viral entry and infectivity. However, it has been demonstrated that viral infectivity measurements differ when comparing the ability of a single env gene to infect a host cell within the context of different HIV-1 genome backbone vectors (Luo and Garcia, 1996). Moreover, there is evidence to suggest that subtype C HIV-1 is biologically different compared to other subtypes (Tobiume et al, 2001). This project aims to compare expression of the same env gene in the context of different vector backbones to determine if there is an influence of subtypes on viral entry and infectivity during a pseudovirion assay. Objectives and Research Plan: 1. Clone full-length subtype C genomes into the HIV-1 vector, pSG3.1, by replacing the subtype B backbone sequence but keeping the subtype B LTR intact. 2. Introduce a premature stop codon within the envelope gene by restriction digestion and re-ligation so that the genome is unable to replicate and confirm mutation by sequencing. 3. Co-transfect 293T cells with the Subtype C genome vectors and a functional env clone, Du151a-env, to generate pseudoviral stocks and determine p24 concentration using ELISA 4. Determine whether the cloned HIV-1 subtype C genome is functional by infecting TZM-bl cells with pseudoviral stocks and determine infectivity by measuring the cell-associated luminescence produced by the luciferase gene under the control of the HIV-1 LTR integrated within the host cell genome. 5. Compare the tissue culture infectious dose (TCID50) and p24 concentration of the different pseudovirion stocks that were generated with the same cloned env gene but different HIV-1 subtype-specific backbone vectors. References 1.Luo T, Garcia JV 1996 The association of Nef with a serie/threonine kinase and its enhancement of infectivity are viral isolate dependent 1996, J Viro. 70, 6493-6496 2. Tobiume M, Takahoko M, Tatsumi M, Matsuda M, Establishment of a magi-derived indicator cell line that detects the Nef enhancement of HIV-1 infectivity with high sensitivity. 2001, J Viro Meth. 97 151-158 Role of spatial expression of IL-4R in bleomycin induced pulmonary fibrosis. Supervisor: Prof. Frank Brombacher. Division of Immunology. Email: firstname.lastname@example.org Co-supervisors: Ms Helen Mearns and Dr William Horsnell Keywords: Fibrosis, cytokines, lung, bleomycin Background: Pulmonary fibrosis is an important and often overlooked medical condition suspected to be the result of a number of causes including particulate pollution, genetic prediposition and parasitic nematodes. However the precise causes remain to be definitively identified. Pulmonary fibrosis is defined by elevated levels of collagen deposition and a chronic inflammatory state in the lung resulting in decreased and irreversible lung function. Immunologically this pathology appears to be driven by TH2 associated cytokines especially IL-4 and IL-13 which modulate TGF-beta responses which is recognised as being an essential inducer of the fibrotic pathology . In the project proposed here we will investigate the role of the IL-4R in bleomycin induced experimental pulmonary fibrosis. IL-4Ris a key component for IL-4 and IL-13 dependent signalling and work from our group has been central to describing its role in a number of TH2 pathologies [2, 3]. Objectives: To ascertain the role of IL-4R in bleomycin induced pulmonary fibrosis in our collection of cell specific IL-4Rknock out mice. Research Plan: The project will specifically involve measuring the effects of bleomycin induced pulmonary fibrosis on airway function, collagen deposition, antibody and cytokine responses. This will involve the researcher being fully trained in the use of our plethysomograph, classic histological techniques and ELISAs. Depending on the progress made by the student there will also be scope for training in further techniques such as flow cytometry and Real-Time PCR. References 1. Fichtner-Feigl, S., et al., IL-13 signaling through the IL-13alpha2 receptor is involved in induction of TGF-beta1 production and fibrosis. Nat Med, 2006. 12(1): p. 99-106. 2. Horsnell, W.G., et al., Delayed Goblet Cell Hyperplasia, Acetylcholine Receptor Expression, and Worm Expulsion in SMC-Specific IL-4Ralpha- Deficient Mice. PLoS Pathog, 2007. 3(1): p. e1. 3. Herbert, D.R., et al., Alternative macrophage activation is essential for survival during schistosomiasis and downmodulates T helper 1 responses and immunopathology. Immunity, 2004. 20(5): p. 623-35. Utility of antigen-specific interferon gamma release assays (IGRAs) in the diagnosis of pleural tuberculosis in a high burden setting Supervisor: Dr Keertan Dheda, Pulmonologist and general physician, Division of Pulmonology, Department of Medicine, 021- 4066509; email@example.com Co-supervisor: Miss Surita Marais Keywords: ESAT-6, tuberculosis, human, cytokine, IFN-γ Background: Tuberculosis is a global public health emergency and is out of control in South Africa. The main organ affected by tuberculosis is the lung and involvement of the pleural space (area around the lung) is a frequently encountered clinical problem. The diagnosis is problematic because traditional methods, such as smear and culture of the pleural fluid, have a low yield (< 20%). More recently IFN-γ responses to TB-specific antigens (ESAT-6 and CFP-10) have shown to be sensitive and specific for the diagnosis of TB infection, particularly in low burden settings [1-3]. However, the utility of these responses in the diagnosis of pleural tuberculosis in a clinical high burden setting, compared to traditional and other methods, have not previously been studied. Objectives: To evaluate the performance outcomes of mononuclear cell antigen-specific responses for the diagnosis of TB pleural effusions. Research Plan: (i) measurement, using an immunoassay, of antigen-specific (ESAT-6, CFP- 10 and HBHA) responses in pleural fluid (TB and non-TB) and whole blood, and evaluating performance outcomes (sensitivity, specificity etc) compared to other methods (ADA, pleural biopsy, smear and culture). (ii) comparison of different methods (absolute cell number vs fixed volume) and their impact on outcomes. (iii) cell phenotype producing IFN-γ in pleural fluid vs whole blood. This work in being led by UCT in collaboration with University College London (UK) and University of Sardinia (Italy). It should be noted that this work will involve use of potentially infectious material but appropriate training will be provided. References: 1. Dheda K, Lalvani A, Miller RF, et al. Performance of a T-cell-based diagnostic test for tuberculosis infection in HIV-infected individuals is independent of CD4 cell count. AIDS 2005; 19: 2038-2041. 2. Dheda K, Udwadia ZF, Huggett JF, Johnson MA and Rook GA. Utility of the antigen-specific interferon-gamma assay for the management of tuberculosis. Curr Opin Pulm Med 2005; 11: 195-202. 3. Pai M, Kalantri S and Dheda K. New tools and emerging technologies for the diagnosis of tuberculosis: part I. Latent tuberculosis. Expert Rev Mol Diagn 2006; 6: 413-22. GENETIC ANALYSIS OF PENGUINPOX VIRUS, A POTENTIAL VACCINE VECTOR Supervisor: Prof Anna-Lise Williamson, Division of Medical Virology, (Tel: 0214066124, email: firstname.lastname@example.org) Co-supervisors: Nicola Douglass, Division of Medical Virology, (Tel: 0214066681, email: email@example.com). Dr Dianne Marais, Division of Medical Virology, (Tel: 0214066756, email: firstname.lastname@example.org). Keywords: Penguin pox, non-essential genes, vaccine vector Background: Avipoxviruses are being increasingly investigated and employed as recombinant vaccine vectors. Transgenes inserted into Avipoxviruses are expressed in immunologically favourable ways but infection of mammalian cells is considered abortive. Penguin pox virus is a novel avipoxvirus recently isolated from Jackass penguins1. It was shown that the simian CV-1 cell line was able to support the replication of penguin pox virus DNA but no infectious virus was recovered2. Further characterisation of penguin pox virus has been done3 but its full potential as a vaccine vector needs to be explored. Objectives: The project will aim to assess the potential of penguinpox virus as a vaccine vector. Random sequencing of the genome will give information about the positioning of genes, which can be compared to published poxvirus genome sequences (including fowlpox virus and vaccinia virus). The aim is to identify a nonessential gene which can be used for further vector construction for the purpose of making a recombinant penguinpox virus. Research plan: Growth of penguinpox virus (PPV) on fertilized chick embyo CAMs Isolation of genomic PPV DNA Restriction enzyme digestion and agarose gel electrophoresis of PPV DNA Shotgun cloning of PPV DNA fragments DNA sequencing of cloned fragments Sequence comparison with known poxvirus sequences References: 1. Kow D. (1992) MSc thesis. University of Cape Town 2. Stannard et al (1998) J General Viology 79: 1637-1646. 3. Abrahams M. (2002) BSc Hons Thesis. University of Cape Town Cloning, expression and purification of HIV-1 Tat, Nef & Reverse Transcriptase (RT) in E. coli Supervisor: Nyasha Chin’ombe Co-supervisor: Professor Anna-Lise Williamson Introduction: Previous studies have shown that cloning of GFP and HIV-1 Gag fused to lacZ peptide could result in their inducible overexpression in E. coli (and constitutive overexpression in a Salmonella enterica serovar Typhimurium vaccine vector). Using the same approach, HIV-1 Tat, Nef and RT can be overexpressed and purified from the bacteria for possible use as reagents for ELISA experiments in vaccine testing setting. The honours student will clone the three genes for overexpression in E. coli. The student will further purify the antigens and test them for specificity. The constructs developed in the study may be used in future delivery of the antigens by Salmonella vaccine vectors Objectives 1. Cloning of codon-optimized HIV-1 tat, nef & rt 2. Expression of the three antigens in E. coli (and Salmonella) 3. PAGE and Western blotting 4. Purification of the antigens using His-tag 5. Checking specificity of the antigens using ELISA protocol Techniques to be learnt by the student 1. Cloning; mainly gel electrophoresis, restriction digestion, restriction mapping, ligation, bacterial transformation, bacterial growth 2. Protein expression, isolation and blotting 3. Western blotting 4. Protein purification 5. ELISA GENOTYPIC CHARACTERIZATION OF ISONIAZID RESISTANCE IN MULTI-DRUG RESISTANT M. TUBERCULOSIS STRAINS FROM GSH Supervisor: Dr Heidi Segal; x6793; Heidi.Segal@uct.ac.za Co-supervisor: Dr Mark Nicol; x6793; Mark.Nicol@uct.ac.za Key words: Mycobacterium tuberculosis; isoniazid resistance; genotyping. Background: The emergence of multidrug-resistant Mycobacterium tuberculosis (MDR-TB) worldwide poses a serious problem to the treatment of tuberculosis. These MDR strains are at least resistant to two primary chemotherapeutic agents rifampicin and isoniazid (WHO, 1997) and require treatment with more costly, more toxic second-line drugs. Isoniazid (INH) resistance is commonly associated with mutations in the bifunctional catalase-peroxidase enzyme KatG (codon 315) and in the promoter region of an enoyl acyl carrier protein reductase, inhA (Post et al 2004; Lavender et al 2005). However, mutations in genes other than these two major predictors of INH resistance have been described in M. tuberculosis (Ramaswamy et al 2003). The aim of this study is to investigate the genetic basis of INH resistance in MDR-TB strains isolated from patients at Groote Schuur Hospital in 2006. This project will form part of a larger study aiming to fully characterize the genetic basis of multi-drug resistance in M. tuberculosis in patients from GSH. Objectives 1. To determine the prevalence of MDR-TB strains that contain mutations in KatG and the promoter region of inhA in M. tuberculosis from GSH in 2006. 2. To characterize the genetic basis of INH resistance in the remaining MDR-TB strains. 3. To determine whether strains with identical INH resistance mutations are related. Research Plan 1. The MDR-TB strains identified at GSH in 2006 will be included in this study. 2. Information concerning the drug susceptibilities of these strains will be tabulated. 3. PCR assays will be carried out to amplify katG and the promoter region of inhA, using genomic DNA extracted from the MDR-TB strains as template. 4. Following PCR amplification, the products will be sequenced to determine whether mutations are present at codon 315 of KatG and at nucleotide -15 upstream of inhA start codon. 5. Other genetic loci will be investigated for mutations that may account for the INH resistance phenotype observed in the MDR-TB strains. 6. The genetic relatedness of the strains will be determined using spoligotyping. References Post et al 2004 Journal of Infectious Diseases 190:99-106. Lavender et al 2005 Antimicrobial Agents and Chemotherapy 49:4068-4074. Ramaswamy et al 2003 Antimicrobial Agents and Chemotherapy 47:1241-1250. CD4+ T-cell derived TNF in M. bovis BCG infection Supervisor: Dr. Muazzam Jacobs, Division of Immunology, (Tel.: 406 6078, e-mail: email@example.com) Co-supervisor: Nasiema Allie Keywords: Tumour necrosis factor, M. bovis BCG, tuberculosis, granuloma Background: Tumour Necrosis Factor alpha (TNFα) and their corresponding receptors, TNFRp55 and TNFRp75, are members of supergene families (Locksley et al., 2001). TNFα mediated signaling has a significant influence on the outcome of several infectious diseases and contributes to protective immunity against fungal, viral, protozoan and bacterial pathogens (Schluter and Deckert, 2000). TNFα is synthesized as a membrane bound protein which is cleaved to a soluble form by TNFα converting enzyme (TACE) (Perez et al., 1990). Both forms of the ligand are bioactive. TNF is synthesised by several different cell-types of which macrophages and lymphocytes are thought to be important for protective immunity against infections. Objectives: To investigate the role of CD4+ T cell derived TNF in immunity against M. bovis BCG infection. Research Plan: The project makes use of a novel genetically modified mouse strain in which TNF is deleted only in CD4+ T cells (CD4-TNF KO mouse). Mice will be challenged with M. bovis BCG and analysed for their ability to generate a protective immune response against pathogen insult. Parameters of investigation will include (1) determination of bacilli burden (2) organ histology (3) cytokine and chemokine analysis by ELISA (4) Analysis of cellular recruitment by flowcytometry. Mice will be subjected to PCR analysis to confirm strain genotypes. References: Jacobs M, Marino MW, Brown N, Abel B, Bekker LG, Quesniaux VJ, Fick L, Ryffel B. Correction of defective host response to Mycobacterium bovis BCG infection in TNF-deficient mice by bone marrow transplantation. Lab Invest. 2000 Jun;80(6):901-14. Grivennikov SI, Tumanov AV, Liepinsh DJ, Kruglov AA, Marakusha BI, Shakhov AN, Murakami T, Drutskaya LN, Forster I, Clausen BE, Tessarollo L, Ryffel B, Kuprash DV, Nedospasov SA. Distinct and nonredundant in vivo functions of TNF produced by t cells and macrophages/neutrophils: protective and deleterious effects. Immunity. 2005 Jan;22(1):93-104. Saunders BM, Briscoe H, Britton WJ. T cell-derived tumour necrosis factor is essential, but not sufficient, for protection against Mycobacterium tuberculosis infection. Clin Exp Immunol. 2004 Aug;137(2):279-87. INVESTIGATE WHETHER A CTL ESCAPE MUTATION WITHIN A NEF EPITOPE AFFECTS DISEASE PROGRESSION Supervisor: A/Professor Carolyn Williamson; Co-Supervisors: Denis Chopera; Dr Zenda Woodman; Desiree Bowers. Tel: 406 6683; 406 7463 ; 406 6017 Email: firstname.lastname@example.org; email@example.com Key-words: HIV-1, CTL, Nef, FACS analysis Background: A striking feature of HIV-1 infection is the extraordinary viral diversity within individuals. During the course of infection, HIV-1 is under intense evolutionary selective pressure to escape from both humoral and cellular immune responses. In fact HIV-1 can readily mutate essential residues within CTL epitopes in order to escape immune detection and clearance. In some cases these mutations occur within functional domains of the viral proteins so that the HIV strain evades immune detection but compromises its ability to replicate. Individuals infected with attenuated viral strains tend to have low viral loads and high CD4 counts and can survive for years without progressing to AIDS (Long-term non-progressors- LTNP). One of the proteins targeted by the cell-mediated immune responses is the accessory gene product, Nef, a 27-34 kDa protein which has been shown to be inactive within LTNP (Deacon et al, 1995). Nef functions to enhance viral infectivity and also down- regulates CD4 and MHC class I molecules at the surface of infected cells which affects immune escape and fitness of viral progeny. The individual functions are largely independent of one another and functional residues and domains have been mapped (Arold and Bauer, 2001). As part of a larger longitudinal study, a participant has been identified with a putative escape mutation within a CTL epitope of Nef and the appearance of this mutation corresponds to a loss of immune response and a concomitant decrease in viral load. The overall aim of this project is therefore to determine whether the identified escape mutation affects Nef function which will provide evidence that the patient is infected with an attenuated virus. Identification of functionally constrained CTL epitopes is important for vaccine design. Objectives and Research Plan: 1. Cloning Nef wt and mutants: Amplification of the nef gene and cloning into the mammalian expression vector pcDNA 3.1D/V5-His-TOPO 5514. 2. Functional analysis- CD4 deregulation: The cloned nef genes will be transfected into the 293T cells expressing CD4 and the down-regulation of cell-surface CD4 will be determined by FACS analysis. 3. Functional analysis- Infectivity: The genes will be co-transfected into 293T cells with the HIV-1 backbone vector lacking functional env and nef genes, pNL4.3.luc and a cloned functional env gene to generate pseudovirion stocks. The p24 concentration of the pseudovirion stocks will be determined by ELISA and standardised concentrations of pseudoviral stocks will be used to infect U87 CD4+CCR5+ cells. Infectivity will be determined by luminescence produced by the reporter luciferase gene cloned into the HIV-1 backbone vector. References 1. Deacon NJ., Tsykin A.,Solomon A., Smith K et al Genomic structure of an attenuated quasispecies of HIV-1 from blood transfusion donor and recipients Science1995 Feb 21;363(9409):619-22. 2. Arold ST., Baur AS. Dynamic Nef and Nef dynamics: how structure could explain the complex activities of this small HIV proein. Trends Biochem Sci.2001.26 (6)356-363. The role of in vitro generated donor CD4+CD25+FoxP3+ regulatory T-cells(Treg) in allogeneic immune responses as measurements for graft versus host (GVHD) and graft versus tumor effect (GVT). Supervisor: PD Dr. Maren Schulze, Division of Hematology, (Tel.: 406 6260, e-mail: firstname.lastname@example.org) Co-supervisor: Prof. N. Novitzky, Head of Division of Hematology Keywords: graft versus host reaction, bone marrow transplant, regulatory T-cells, graft versus tumor reaction. Background: The therapeutic potential of allogeneic bone marrow transplantation (BMT) relies on the graft-versus-tumor (GVT) effect, whereby residual malignant cells of the host are eradicated by the donor T cells. Unfortunately, the clinical use of allogeneic BMT for cancer treatment is seriously hampered by the concomitant occurrence of graft-versus-host disease (GVHD), particularly since the beneficial GVT effects are closely associated with the severity of the GVHD. Any treatment that could reduce the GVHD response, without affecting GVT, could provide for a substantial improvement in allogeneic BMT therapy. GVHD is an immune-mediated disease in which donor T cells recognize and attack the genetically disparate cells of the recipient. It has a complex pathophysiology, ultimately involving multiple organs. Objectives: The aim of the study evaluate the immune response by flow cytometry and mixed lymphocyte culture and to specifically monitor the population of donor regulatory T-cells (Treg) that tolerates normal healthy host cells and thus reducing graft-versus-host disease while preserving the desired graft-versus-tumor effect. Research Plan: This study is designed to generate Treg in vitro by exposing donor T- cells to host antigen presenting cells or malignant host cells during a 4 day coculture. The effect on immune response towards normal host cells and host leukemia cells as a model for GVHD or GVT effect in vitro will be evaluated on a cellular. Methods used will include flow cytometry, cell culture and the principals of mixed lymphocyte culture. References: 1. Fändrich F, Dresske B, Bader M, Schulze M (2002) Embryonic stem cells share immune-privileged features relevant for tolerance induction. J Mol Med-JMM 80: 343-350. 2. Fändrich F, Lin X, Chai GX, Schulze M, Ganten D, Bader M, Holle J, Huang DS, Parwaresch R, Zavazava N, Binas B (2002) Preimplantation-stage stem cells induce long-term allogeneic graft acceptance without supplementary host conditioning. Nat Med 8: 171-178. 3. Fändrich F, Schulze M, Zehle G, Ungefroren H (2004) Stem cell-mediated tolerance inducing strategies in organ transplantation. Kidney Int 65: 1548-1550. INVESTIGATION OF P. AERUGINOSA CONVERSION TO HYPERMUTATOR IN CF LUNG Supervisor: Dr Heidi Segal; x6793; Heidi.Segal@uct.ac.za Co-supervisor: Ms Joanna Evans; x6798; email@example.com Key words: Pseudomonas aeruginosa; cystic fibrosis; hypermutability; genotyping; antibiotic resistance. Background: One of the most common causes of death in patients with cystic fibrosis (CF) is collapse of the lungs due to persistent infection with Pseudomonas aeruginosa.1 Certain P. aeruginosa strains seemingly have an ability to disseminate more rapidly than others, have an increased resistance to antibiotic therapy, and are usually more aggressive, leading to worse prognosis than patients not colonized with these strains.2 Adaptation of these P. aeruginosa strains to the CF lung includes conversion to a more resilient mucoid state, development of a protective biofilm, and conversion to a more readily adaptable hypermutator phenotype. The latter affords an ability to rapidly acquire antibiotic resistance mutations. Genes involved in DNA repair mechanisms, as well as transposable insertion sequences (IS), have been implicated in conversion to hypermutability. Objectives 1. To determine whether P. aeruginosa isolated from CF patients at Red Cross Children’s Hospital are hypermutable. 2. To determine whether hypermutable strains are associated with increased antibiotic resistance levels. 3. To investigate the role of IS elements in conversion to hypermutability and development of antibiotic resistance. 4. To investigate the epidemiology of P. aeruginosa strains in RCCH. Research Plan 1. Collect P. aeruginosa strains isolated from CF patients at RCCH. 2. Mutator assays will be carried out to determine whether strains are hypermutable. 3. PCR assays will be used to screen for the presence of IS elements, including IS6100 and ISPa25. 4. Clones obtained following shotgun cloning of genomic DNA from strains harboring IS6100 and ISPa25 will be sequenced to determine the genetic environment of these elements and their role in hypermutability and antibiotic resistance. 5. The genetic relatedness of the strains will be determined using PFGE and RAPD. References 1) Koch C, Hoiby N. Pathogenesis of cystic fibrosis. Lancet 1993; 341: 1065-1069 2) Al-Aloul M, Crawley J, Winstanley C, Hart CA, Ledson MJ, Walshaw MJ. Increased morbidity associated with chronic infection by an epidemic Pseudomonas aeruginosa strain in CF patients. Thorax. 2004; 59: 334-6. INNATE IMMUNITY TO MYCOBACTERIUM TUBERCULOSIS IS STRAIN-SPECIFIC Supervisor: Dr Mark Nicol: x6793; Mark.Nicol@uct.ac.za Key words: Mycobacterium tuberculosis; innate immunity; cell culture; flow cytometry Background Inhalation of Mycobacterium tuberculosis (M.tb) may result in a range of clinical outcomes including elimination of infection, latent infection and active tuberculosis. The factors influencing these diverse outcomes remain largely unknown, but epidemiological studies point to influence of both host (human) and bacillary (M.tb) factors(1). There are several lines of evidence relating differences in the virulence of individual strains of M.tb to their ability to interact with the innate immune response. Work we have conducted suggests that successful strains of M.tb from a variety of genetic backgrounds may employ a common strategy of subverting, or suppressing the normal innate immune response(2;3). Objectives 1. To determine whether lineages (or families) of M.tb induce consistently different patterns of innate immune responses. 2. To determine whether the most successful lineages tend to induce similar immune- suppressive phenotypes. Research plan 1. Selection of strains representative of the most successful lineages as well as comparator lineages. We will select M.tb strains representative of the two most successful lineages in the Western Cape, East Asian (including Beijing) and Euro-American (including LAM3/F11) lineages 2. Evaluation of cytokine and gene expression profiles induced following infection of primary monocyte-derived macrophages (MDM) with representative strains. (a) isolate macrophages from blood donor buffy coats using Ficoll density centrifugation and adherence (b) infect them with different strains of M.tb to assess a. the strain-specific induction of innate immune responses. b. the rates of growth of representative strains within macrophages (c) perform cytometric bead array analysis of multiple cytokines in supernatants from macrophage cultures References (1) Reed MB, Domenech P, Manca C, Su H, Barczak AK, Kreiswirth BN et al. A glycolipid of hypervirulent tuberculosis strains that inhibits the innate immune response. Nature 2004; 431(7004):84-87. (2) Gagneux S, DeRiemer K, Van T, Kato-Maeda M, de Jong BC, Narayanan S et al. Variable host- pathogen compatibility in Mycobacterium tuberculosis. Proc Natl Acad Sci U S A 2006; 103(8):2869-2873. (3) Nicol MP, Sola C, February B, Rastogi N, Steyn L, Wilkinson RJ. Distribution of strain families of Mycobacterium tuberculosis causing pulmonary and extrapulmonary disease in hospitalized children in Cape Town, South Africa. J Clin Microbiol 2005; 43(11):5779-5781.