C´ alculo, reconstrucci´ on y an´ alisis de dosis

The mean proliferation response of skin-test positive healthy subjects was significantly higher than their skin-test negative counterparts when the whole blood proliferation (WBP) method was used (Fig. 3.1). This suggested that the WB cells reflected the in vivo immune balance better than the PBMCs. Mean values of WBP(SI) to vaccin were higher in healthy people compared to tuberculosis patients (Fig. 3.2) whereas their WBP (SI) to tuberculin were similar (Fig. 3.3). TB patients showed virtually no proliferation response to low doses of vaccin whereas healthy people showed a small response. Low doses of tuberculin gave no proliferation in TB patients nor in healthy people. At higher doses of antigens, both healthy people and TB patients gave a higher response to tuberculin than vaccin.

Owing to wide variations between different individuals there was no significant difference given by any concentration of vaccin o r tuberculin. When the maximal WBP(SI) of each individual was analysed irrespective of the antigen concentration, healthy people showed a significantly higher response to vaccin and chelonin but not to tuberculin (Fig. 3.4). This suggests that the antigens of M.vaccae and M.chelonae

might be recognised differently from antigens of M .tuberculosis in healthy people compared to TB patients.

The size of proliferation response appears to depend on the antigen used. Healthy people showed higher response to vaccin than TB patients (Fig. 3.2). Healthy people are likely to respond to common Ags shared

Figure 3.1 Comparison of proliferation responses between skin-test +ve and skin-test -ve healthy subjects using whole blood proliferation(WBP) and peripheral blood mononuclear cell proliferation(PBMCP) methods (±SD)

5 0 £ 3 £ ‘i 4 0 - 3 0 - 2 0- 1 0- n S T + v e to vaccin (n = 5 ) I I S T -ve to vaccin (n=7) p<0.002 n.s T W B P P B M C P T Y PE S OF CELL C U L T U R E S

Lymphocyte proliferation (LP) responses of twenty one healthy subjects to 10 p,g/ml vaccin were carried out using their ficoll-separated peripheral blood mononuclear cells (PBMCs) and their whole blood (WB) cells. The results were expressed as maximum stimulation indices (SI) or maximum count per minute / back ground count per minute. The p value was calculated using the Mann-Whitney test.

Figure 3.2 Proliferation response to vaccin (mean ± SEM) Healthy (n=23) TB (n=14) 1 5 - 1 0- 8 d o 00 o s <ri (N O cone ( \)g/ml)

Figure 3.3 Proliferation response to tuberculin (mean ±SEM)

52 Pu Healthy (n=23) 1 5 - _{TB (n=14)} l Æ 1 0- o o 8 d GO o d § <n Cone (ug/ml)

Figs 3.2-3.3. TB patients showed particularly lower proliferation responses to low doses of vaccin (< 5 pg/ml) compared to healthy people. However, no statitistical difference was obtained using the Mann-whitney test. Both groups of people showed lower proliferation response to vaccin compared to equivalent concentrations of tuberculin. Healthy people showed their maximal WBP (SI) to 5 |ig/ml of vaccin and 10 |ig/ml tuberculin while TB patients required 10|ig/ml of vaccin and 5|ig/ml of tuberculin.

Figure 3.4 Proliferation responses of TB patients and healthy controls to sonicated mycobacterial antigens (±SD)

I I H ealthy (n = 2 3 ) □ T B (n = 1 4 ) 3 0 - -■ p<0.03 2 0- 1 0- ch elon in tuberculin va ccin Sonicated antigens

The maximum stimulation indices (Sis) shown above were given by different concentrations of sonicated antigens in different individuals. Antigen

concentrations ranging from 1.25 - 10 pg/ml produced maximum proliferation responses.

Using the Mann-Whitney test, statistical differences were found between healthy people and TB patients in their maximum WBP responses to vaccin (p<0.01) and chelonin (p<0.03) but not to tuberculin. When a particular concentration of antigen (eg. 10 pg/ml) was chosen for each individual, no statistical difference could be found between the TB patients and the healthy controls in their proliferation responses to vaccin or chelonin. This shows that individuals respond preferentially to different doses of mycobacterial antigens

by different species of mycobacteria better than TB patients. Reactivity to common antigens might become suppressed in TB patients as they become increasingly sensitised to M.tuberculosis-s^tcifio, antigens. Common antigens may also be described as epitopes that confer similar types of T cell responses rather than epitopes that are structurally similar. Therefore, different subsets of lymphocytes might be selected by common and species-specific antigens.

There is evidence that sensitisation by particular species may be favourable, as in Malawi and Uganda (Brown et al, 1968; Stanford et al,

1976), or unfavourable as in Burma (Bechelli et al, 1974; Lwin et al,

1985). Skin-test positivity to scrofulin in Burmese children is linked with a negative skin-test response to M. tuberculosis and M.leprae and increased susceptibility to respective diseases. High sensitisation with

M.scrofulaceum and M.intacellulare has also been shown to induce unfavourable responses in BCG vaccinees in South Asia.

Although the mean proliferation responses of healthy people and TB patients to tuberculin were similar (Fig. 3.3), healthy people and TB patients could be reacting to different antigenic components of tuberculin. Healthy people could be responding prim arily to the common antigens in tuberculin whereas the TB patients could be responding to M.tuberculosis-^ptcific antigens. This shows that the size of proliferation response alone is not a sufficient indicator of a protective or non-protective response.

It is debatable whether a high proliferation response necessarily correlates with a protective response. Lymphocytes from tuberculoid skin lesions usually show a larger proliferative response to whole o r sonicated M. leprae than lymphocytes from lepromatous lesions (Bjune

et al, 1976). A higher proliferation response in these conditions might be indicative of protective CMI. However, PBMCs from patients with tuberculoid leprosy give a higher proliferation response to sonicated

M. leprae than healthy people. PBMCs from ‘Inflamed’ skin lesions were found to give larger proliferation than ‘silent’ lesion in border-line tuberculoid or lepromatous lesions. High proliferation is therefore also found in hypersensitive or inflammatory conditions.

Proliferation in response to the soluble extracts of M .tuberculosis was found to be decreased slightly in peripheral blood mononuclear cells (PBMCs) from tuberculosis patients compared to healthy controls (Surcel et al, 1995). Lymphocytes isolated from the pleural fluid of tubercular pleurisy show accelerated kinetics and larger proliferation in response to mycobacterial antigens, compared to lymphocytes from their peripheral circulation, and also lymphocytes from non-tubercular pleurisy (Lorgat et al, 1992). The immune response in patients with tubercular pleurisy is thought to be protective because this is a self- limiting condition which can sometimes resolve without chemotherapy.

3.3.2 PROLIFERATION AND SKIN-TEST RESPONSES OF