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Mycolic acids from Mycobacterium tuberculosis: purification by countercurrent distribution and T-cell stimulation.

Bacterial cell wall lipids are recognized as immunostimulatory molecules which make an important component of vaccines against bacterial diseases. Even mycolic acids, forming the waxy outer layer of the bacilli which cause tuberculosis, have been shown to stimulate human CD4/8 double negative T-cells. The role of these cells in resistance to tuberculosis is currently still debated. In this work, a method is described to purify mycolic acids from bacterial crude extracts in a single step using countercurrent distribution. Mycolic acids obtained in this way approach 100% purity and stimulate both double negative and CD4 positive T-cells in peripheral blood leucocytes obtained from healthy human donors. Stimulation of CD4 cells by mycolic acid antigens has not been reported before, emphasizing the potential importance of mycolic acids in the context of the fight against tuberculosis.

CD4 Antigens↗

Countercurrent distribution of human peripheral blood lymphocytes: isolation of a subpopulation enriched with natural killer and K cells.

Aqueous solutions of dextran and of poly(ethylene glycol) when mixed give rise to two-phase systems suitable for the separation by partitioning of cells based on subtle differences in their membrane surface properties. Human peripheral blood mononuclear cells were obtained by centrifuging blood over Hypaque-Ficoll and removing the cells from the cushion. Clean lymphocytes were then isolated by velocity sedimentation of the mononuclear cells. Lymphocytes were subjected to countercurrent distribution (i.e., a multiple extraction procedure) in a two-polymer aqueous phase system which separates cells primarily on the bases of surface charge-associated properties. It was found that a small subpopulation of cells with highest partition coefficient (presumably highest surface charge-associated properties) contain virtually all of the natural killer (NK) and K cell activities.

Antibody-Dependent Cell Cytotoxicity↗

Prediction of fertility by centrifugal countercurrent distribution (CCCD) analysis: correlation between viability and heterogeneity of ram semen and field fertility.

The prediction of the fertilizing ability of a sire or a given insemination dose is a primary aim in the field of artificial insemination. Centrifugal countercurrent distribution analysis (CCCD) was used to determine the relationship between some sperm parameters and the in vivo fertility rate obtained with the same sample after cervical artificial insemination. A total of 522 ewes from 26 different farms was inseminated with 53 ejaculates obtained from 25 mature Rasa aragonesa rams. Semen was diluted to 1.6 x 10(9) cells ml-1 and doses of 0.25 ml were prepared and kept at 15 degrees C until used for insemination. The same ejaculates were used for analysis of standard semen parameters and CCCD analysis. Sperm motility, concentration and viability were determined before and after CCCD. Post-CCCD parameters were derived from the analysis of the profile obtained after CCCD. The recovered viability showed the highest correlation with fertility, especially in the central chambers (V2), r = 0.415, P < 0.005). The ejaculate heterogeneity also showed a positive correlation with field fertility (r = 0.23), with a tendency towards significance (P < 0.1). The mean fertility value of all ejaculates used in this study was 46.75%, ranging from 12.5% to 75.0%. Ejaculates were classified into two categories according to their fertility: higher and lower than the mean value. Only the viability recovered in the central chambers (V2) was a parameter with a predictive capacity to discriminate between the two groups (P < 0.05). A predictive equation for field fertility with a correlation coefficient r = 0.488 and a very high level of significance (P < 0.005) was deduced by multiple analysis: PF = 6.02 + 0.069V2 + 0.315H (where PF is predictive fertility, V2 is the recovered viability in the CCCD profile central chambers and H is heterogeneity).

Animals↗