SUBJECTS

Blood samples were obtained from 29 individuals who had recovered from pulmonary MAC infection, 10 of whom also had HTLV-I (all women, mean (SD) age 70.1 (10.2) years) and 19 who did not have HTLV-I (16 women, mean (SD) age 70.9 (9.5) years).2 They had neither systemic nor local underlying disorders which might predispose to pulmonary MAC infection such as alcoholism and residual pulmonary damage from previous infection, nor any unexplained pulmonary diseases or other pathogens. HTLV-I infection was serologically diagnosed with an Eitest-ATL kit (Eisai Inc, Tokyo, Japan). The HTLV-I positive cases had never had adult T cell leukaemia or HTLV-I associated myelopathy. MAC infection had been diagnosed based on culture of sputum or bronchial washing fluids on Ogawa egg medium on at least two separate occasions. They had all received treatment and were now clinically well.

Fifteen healthy volunteers were also recruited, seven of whom also had HTLV-I (five women, mean (SD) age 68.8 (8.1) years) and eight who did not have HTLV-I (six women, mean (SD) age 69.2 (8.8) years) as healthy controls. None of the volunteers had any disease such as collagen disease or AIDS which might cause immunological abnormalities and pulmonary disorders, and none had ever been infected with MAC. The HTLV-I positive volunteers had never had adult T cell leukaemia or HTLV-I associated myelopathy.

All the cases and volunteers gave written consent to participate in the study.

T CELL SUBSETS IN PERIPHERAL BLOOD LYMPHOCYTES

Total lymphocyte counts and lymphocyte subsets in the peripheral blood of MAC infected HTLV-I carriers, MAC infected non-carriers, and healthy volunteers were measured by flow cytometry on at least two separate occasions.

EXTRACTION AND PURIFICATION OF GPL FROM MAC

Serovar 4 of MAC was identified by thin layer chromatography3 and GPL was extracted as reported previously,5 after which it was dissolved in propanol:methanol (2:1).

PROLIFERATION ASSAY

The GPL solution was added to a 96-well round bottom plate (Iwaki Glass Co Ltd, Tokyo, Japan) and dried under sterile conditions. Peripheral blood lymphocytes were separated from heparinised venous blood by the Ficoll-Hypaque sedimentation method as described previously.8 The lymphocytes were suspended at 5 × 10⁵/ml in RPMI-1640 with 10% heat inactivated fetal bovine serum (Flow Laboratories Inc, McLean, VA, USA). Lymphocytes (5×10⁴) were transferred to the wells in the 96-well plates with or without coated GPL. PHA (Wellcome Research Laboratories, Beckenham, UK) was then added to each well at a concentration of 10 μg/ml. Cells were incubated at 37°C in 5% CO₂ for 72 hours.

During the final 4 hours of incubation Alamar Blue (20 μl/well; Nalge Nunc International, NY, USA) was added to each well and the fluorescence was measured using Fluoroskan Ascent (Labsystems, Helsinki, Finland).9 We measured the absorbance at a wavelength of 590 nm. The assay was carried out in duplicate. The absorbance showed a significant positive correlation with direct cell count by Trypan blue staining (r = 0.95, p<0.0001, Pearson's correlation coefficient, data not shown). The proliferation index (PI) was used to measure the lymphocyte proliferation rate by the following formula:

PI = (count in the presence of PHA − count in medium alone)/(count without PHA − count in medium alone)

To compare the degree of GPL inhibition the relative reduction in PI was calculated using the following formula:

% reduction = (PI with GPL)/(PI without GPL) × 100

STATISTICAL ANALYSIS

The Mann-Whitney U test and one way factorial ANOVA with Banferroni-Dunn test were used to compare the PI and percentage reduction in PI in the four groups. p values below 0.025 were considered significant.