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Title of Thesis

Shagufta Khaliq
Institute/University/Department Details
University of the Punjab, Lahore
Number of Pages
Keywords (Extracted from title, table of contents and abstract of thesis)
cytogfenetic, molecular genetic studies, symphoid phenotypes, mycobacterial diseases, tb patients, blood lymulated, lectin, human lymphocytes, polymorphism

The study of human populations is of interest both from the clinical and anthropological points of view. This thesis examines Pakistani populations in the context of genetically transmitted susceptibility or resistance to mycobacterial diseases such as tuberculosis and leprosy.

Although it has been a long-held belief that genetic differences between individuals contribute to the variability in response to these infections, it has been difficult to get supporting empirical evidence. However, studies with mice have shown that the macrophage resistance gene Lsh/Bcg/lty has a profound effect on early resistance or susceptibility to mycobacterial infections. This gene is located between Idh-I and In on mouse chromosome 1, which is assumed to be in a region of homology with the long arm of human chromosome 2 (2q).

It has also been suggested that the Lsh gene regulates the ability of macrophages to become primed/activated in response to lipopolysaccharides (LPS) and interferon-γ (IFN-γ). IFN- γ is an effective inducer of antimicrobial mechanisms. This cytokine is released by CD4 + TH1 cells which playa central role both in the expression of acquired cellular resistance (ACR) and delayed type hypersensitivity (DTH). These and other observations are encouraging in the belief that, in human family studies, it may be possible to identify resistant individuals in their enhanced T-cell responsiveness to mycobacterial antigens.

To study T cell responsiveness in the presence of various mycobacterial antigens (purified protein derivative; PPD, Mycobacterium leprea soluble antigen; MLSA), blood samples were collected from multi case (primarily 2 generational) mycobacterial disease families. Chapter 3 compares the proliferation of T cells and the production of IFN- γ in the peripheral blood lymphocytes (PBLs) of Pakistani TB patients and their unaffected contacts in response to mycobacterial antigens. The results show that there is no clear correlation between T cell proliferation and IFN-γ production. However, it was found that the PBLs of patients produced less IFN-γ in response to the mycobacterial antigens PPD and MLSA compared to the unaffected family members (contacts) of these patients. It is speculated that the lower response in patients could be due to the treatment they were receiving with antituberculous drugs. Another explanation could be that since a spectrum of cytokines are produced in response to mycobacterial infection, a molecule like IL-10, which is known to inhibit IFN-γ production, could be responsible for the comparatively reduced production of this cytokine in TB patients. It is also suggested that more meaningful results may be obtained with lymphoid cells obtained from the sites of infection (e.g., pleural fluid from TB patients) in preference to the PBLs.

The study of restriction fragment length polymorphisms (RFLPs) has made it possible to identify specific genetic loci that are uniquely associated with (human) diseases. For this, a continuous supply of large quantities of cells is required. This can be done by €œimmortalizing€ cells with Epstein-Barr virus (EBV). This methodology has been established in this laboratory for the first time in Pakistan.

The work described here shows that the highest success for EBV transformation is obtained if the procedure is initiated within the shortest time possible after the blood is drawn. EBV transformation should be carried out within 24-48 hours after bleeding. If blood is transported on ice from the field to the laboratory, it must be allowed to equilibrate for 1-2 hours at room temperature (22-24oC) before processing. The addition of cyclosporin A to the transformation medium increases the success of transformations to > 95 %. Fluorescence activated cell sorter (FACS) analysis of the cell surface markers of these cells shows that although 95 % of the transformed cells are B-cells, approximately 5 % helper T cells persist with the EBV transformed lymphoblastoid cells. A second method described here for large scale preparation of T -cells, from 5-10ml of blood samples, is by continuous allogeneic stimulation of PBLs. These cells can also be kept frozen and expanded by restimulation with the irradiated stimulator cell line (JY) in the presence of the lectin phytohemagglutinin-P (PHA.P). This method also provides an inexhaustible supply of cells, but is tedious and time consuming (Chapter 4).

T cells playa pivotal role in response to mycobacterial infections. To examine the possibility of allelic segregation of T cell receptor (TCR) genes in (mycobacterial) diseases, using restriction fragment polymorphism (RFLP), the present work describes the detection of polymorphisms in the human TCR α-and β-gene segments in randomly sampled normal Pakistani individuals. In agreement with other laboratories, polymorphic bands were obtained with probes for both the α-and β -chain gene segments. However, the data presented here, with DNA obtained from 17 multi case TB families, show a lack of linkage between the TCR α-and β-gene segment polymorphisms with tuberculous disease.

Since γ/δ T cells are preferentially expanded upon infection of PBLs with M. tuberculosis, these studies need also to be extended with gene probes for segments of the γ/δ TCR genes (Chapter 5).

The mycobacterial diseases tuberculosis (TB) and leprosy and leishmaniasis are characterized by a wide spectrum of disease phenotypes, and by the fact that the majority of individuals exposed to the causative organisms Mycobacterium tuberculosis, M. leprae, and Leishmania sp. become infected but do not present with clinical disease. In order to determine whether a human homologue to the murine macrophage resistance Lsh/Ity/Bcg influences susceptibility to human disease, multi case families for all three diseases have been collected, and linkage analysis performed using a panel of markers in the region of human chromosome 2q33-q37, known to be conserved with the Lsh/Ity/Bcg-containing region of murine chromosome 1. Because of the paucity, of available polymorphic markers, linkage information for 2q33-q37, data from 35 multi case TB, leprosy and visceral leishmaniasis families (310 individuals) were first pooled to produce a detailed . RFLP map of the region. Peak LOD scores well in excess of 3 were observed for linkage between adjacent pairs of a more proximal (2q33-q35) set of markers CRYGPl, MAP2, FNI, VILI and DES, and between adjacent pairs 'of a more distal (2q35-q37) set COL6A3, D2S55 and D2S3. These peak LOD score and the corresponding values for θ were used in the MAP92 programme to generate a multiple two-point map with gene order/map intervals (cM) of: CRYGPI-4.65-MAP2-3.45-FNl-5.95-TNPI-3.41-YILl 3.01-DES-20.14-COL6A3-1O.91-D2S55-3.67-D2S3. Although local support for the placement of loci in this order was weak (LOD < 2, except for DES-COL6A3 where LOD = 6.02), the map is consistent with the gene order for those loci (Cryg, Fn-l, Tp-1, Vil, Des, Co16a3) previously mapped in the mouse. Data from 17 multi case leprosy families (149 individuals) were further analysed for linkage between a putative disease susceptibility locus (DSL) controlling susceptibility to leprosy per se and each of the loci. Assuming 100% penetrance for the susceptibility allele no positive LaD score was obtained for linkage between DSL and any of the marker genes. Instead, the data provide convincing evidence (LaD scores <-2) that a DSL does not fall within 10-20cM of CRYGPI, MAP2, TNPI, YILI, DES or D2S55 or within 5-10cM of FN1, COL6A3 or D2S3. This effectively excludes a putative DSL controlling susceptibility to leprosy per se from the entire region 2q33-q37. Even with reduced penetrance (80% and 60%) for the susceptibility allele the data argue against a putative DSL within the region TNP1-DES where the murine Lsh/Ity/Bcg gene is located. Analysis of the data for these loci using affected pedigree-member linkage analysis also failed to provide evidence for co-segregation of these markers with susceptibility to disease per se. Nor could any evidence be found for a gene in this region controlling susceptibility to the tuberculoid form of disease, or to T-cell responder phenotypes for proliferative responses to the mycobacterial antigens purified protein derivative (PPD) or M. leprae soluble antigen (MLSA). The RFLP map generated is now the most detailed genetic map of the region 2q33-q37, mostly comprising genes encoding molecules of known function. This provides an anchor map and a set of typed families around which new highly polymorphic microsatellite markers can be ordered.

Download Full Thesis
1749.34 KB
S. No. Chapter Title of the Chapters Page Size (KB)
1 0 contents
82.98 KB
2 1 General introduction 1
189.14 KB
3 2 Literature Survey 22
280.32 KB
4 3 Cellular immune response of peripheral blood lymulated from TB patients 48
186.61 KB
  3.1 Introduction 48
  3.2 Materials and Methods 50
  3.3 Results and Discussion 55
  3.4 Fingers and Tables 61
  3.5 References 69
5 4 Analusis of the cell surface receptors of lectin stimulated and EBV transformed human lymphocytes 72
175.14 KB
  4.1 Introduction 72
  4.2 Materials and Methods 75
  4.3 Results and Discussion 80
  4.4 Figures and Tables 85
  4.5 References 91
6 5 Restriction fragment length polymorphism of the human T cell receptor genes 93
431.01 KB
  5.1 Introduction 93
  5.2 Materials and Methods 96
  5.3 Results and Discution 108
  5.4 Figures and Tables 112
  5.5 References 124
7 6 Evidence for human homologue of Lsh/Ity/Bcg disease susceptibility gene 127
381.02 KB
  6.1 Introduction 127
  6.2 Materials and Methods 131
  6.3 Results 137
  6.4 Discussion 143
  6.5 Figures and Tables 150
  6.6 References 166
  6.7 Work in Support of ph.d Candidature.
  6.8 Publication 174