La población de Cuautlancingo y sus Indicadores Sociales 127.

4.1 Donors

Eight healthy donors (three female, five male), aged between 25 - 36 years contributed blood samples for the experiments described in Chapter 5 after giving informed

consent. By ethnicity, three were European Caucasians, one was south Asian of Indian descent, two were east Asians of Chinese descent, and two were of African descent. By residence, three had resided in the United Kingdom or West Europe from birth, two had moved around in three continents (Africa, America and Asia) over the past 15 years, and the other three had been in the UK for the past 3 - 5 years having previously stayed in east Asia or Africa from birth. Only four were involved in any form of

laboratory or clinical work. All had received BCG vaccination more than 10 years ago, and none had any significant history of chronic illness including tuberculosis and atopic disorders. None had ever been on long-term steroid therapy, or received immunomodulatory drugs. All blood samples were taken at 0930 h (maximum 2 h variation) on each occasion to avoid effects due to diurnal cortisol rhythms. Venous blood was collected in sterile heparinised tubes and processed within 10 min. For each experiment described, cells from at least three donors were studied on different

occasions.

4.2 PBMC cultures

4.2,1 Culture conditions

PBMCs were isolated by density-gradient centrifugation as described previously from freshly-collected whole blood of the donors. Cells were resuspended in RPMI 1640 supplemented with 2 mM L-glutamine, 100 U/ml penicilin, 100 pg/ml streptomycin (all from Gibco-BRL) and 10% (v/v) autologous semm, then seeded into 24-well plates (Nunclon surface, Nalge Nunc International, Denmark) at a density of 5 x 10^

cells/ml, and incubated at 37°C in a humid 5% COj incubator. Only non-adherent cells (NAC) were harvested for further assays.

4.2.2 M onitoring cell growth and viability

Observations on cell growth, density, viability, morphological changes and

contamination checks were routinely performed by microscopy every two days during culture and always before harvesting. Cell membrane integrity may be used as a crude gauge of cell viability; thus the trypan blue exclusion test was used to count viable and non-viable cells. An aliquot of the cell suspension was pelleted and resuspended in 1 ml phosphate-buffered saline (PBS, Appendix A-9). One part of 0.4% trypan blue and

1 part cell suspension were mixed and incubated for 3 min at room temperature. A drop of the mixture was applied to a haemocytometer (Improved Neubauer, Assistent, Germany), and the cells counted under a light microscope. Stained cells were regarded as non-viable. The cells were not used for experiments if more than 40% were non- viable by this criterion.

4.3 Mycobacteria preparations

Crude whole cell sonicates of Mycobacterium tuberculosis H37Rv (a strain known to be virulent in mice) and Mycobacterium vaccae NCTC 11659 (a non-pathogenic environmental strain) were prepared and donated by Dr E. Bayley. The sonicates are henceforth denoted MtbS and MvacS respectively. They were prepared by the method described by Paul et al (Paul et a l, 1975). Briefly, mycobacteria grown on Sautons’ slopes were harvested by centrifigation at 20,000 g for 15 min, washed twice with PBS (pH 6.8) and suspended in 50 ml of the same buffer. The suspensions were sonicated for 15 min in a 100-watt ultrasonic disintegrator with the wave peak distance set at 8-9 pm, then centrifuged at 70,000 g for 30 min to remove cellular debris. The supernatants were then filter-sterilised (0.22 pm-pore size), and the protein

concentration quantified by the Lowry method (Lowry e ta l, 1951). The sonicates were then stored in single-use aliquots at -70°C without adding other diluents or additives. The preparations were not boiled or autoclaved at any time. (The MvacS preparation is not SRL172; the latter is a heat-killed preparation of the same strain used currently in immunotherapy chnical trials.) Live mycobacterial cultures were not used for these experiments because of limitations in containment facilities.

To determine cellular responses to M. tuberculosis antigens, MtbS was added to the PBMC cultures at 50 pg/ml (final concentration) before incubation. It has been shown that lymphocytes cultured in the presence of this concentration of MtbS had increased surface expression of CD30 (Munk et a l, 1997). To assess whether responses were M. tuberculosis-s^Qcific, control wells were set up with the cells from the same donors treated either with 50 pg/ml MvacS or culture medium alone. Culture conditions and durations were otherwise identical. Cytokines and other additional growth factors were not added during the course of culture, unless otherwise specified.

4.4 Antibodies and recombinant proteins

Affinity-purified anti-human antibodies (Ab) and recombinant human proteins (all from R&D Systems, Abingdon, Oxon unless otherwise stated) were added to the cultures in certain experiments. Equivalent concentrations of isotype-matched antibodies (from the same manufacturers) and bovine serum albumin (BSA), fraction V were used

respectively as negative controls in other wells treated otherwise identically.

In some experiments neutralising anti-IL-4 (10 pg/ml) was added to the cultures, this concentration was chosen after titration of its effect on CD30 expression in MtbS- treated cultures (Chapter 5, figure 5-13A). To neutralise TNF-a activity in the

cultures, either 1 pg/ml of anti-human neutralising TNF-a Ab (Insight Biotechnology) or potent TNF-a inhibitors (5 pg/ml recombinant human TNF sRI/Fc chimera or TNF

RII/Fc chimera, both R&D) were used in different experiments (Lesslauer et al,

1991). The anti-TNF-a Ab concentration was determined by titration of its effect on lymphocyte apoptosis in MtbS-treated cultures (Chapter 5, figure 5-8A). To study the effect of neutrahsing Fas-mediated cytotoxicity, recombinant human Fas/Fc chimera was added to cultures to a final concentration of 10 pg/ml (Ju et a l, 1995).

Competitive inhibition of CD28-mediated signalhng was achieved using recombinant human CTLA-4/Fc chimeric protein (R&D) at a final concentration of 100 pg/ml, based on the principle that both bind CD80 (B7-1) and CD86 (B7-2) but CTLA-4 binds with 20-100 fold higher affinity than CD28 (Lenschow et a l, 1996).