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					 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:
fbrombac@uctgsh1.uct.ac.za
Co-supervisor:
Dr. Reto Guler, Division of Immunology; 406 6813 / 084 417 16 19; e-mail:
rguler@uctgsh1.uct.ac.za

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: cwilliam@curie.uct.ac.za; zwoodman@curie.uct.ac.za

 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:
fbrombac@mweb.co.za
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 [1].

In the project proposed here we will investigate the role of the IL-4R in bleomycin
induced experimental pulmonary fibrosis. IL-4Ris 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-4Rknock 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; keertandheda@uct.ac.za
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: annalise@curie.uct.ac.za)
Co-supervisors: Nicola Douglass, Division of Medical Virology, (Tel: 0214066681,
email: niki.douglass@uct.ac.za).
Dr Dianne Marais, Division of Medical Virology, (Tel: 0214066756, email:
di.marais@uct.ac.za).

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:
mjacobs@uctgsh1.uct.ac.za)
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: cwilliam@curie.uct.ac.za; dchopera@curie.uct.ac.za
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:
mschulze@uctgsh1.uct.ac.za)
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; evansj@curie.uct.ac.za

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.

				
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