Induction of granulysin and perforin cytolytic mediator expression in 10-week-old infants vaccinated with BCG at birth.

BACKGROUND: While vaccination at birth with Mycobacterium bovis Bacilli Calmette-Guérin (BCG) protects against severe childhood tuberculosis, there is no consensus as to which components of the BCG-induced immune response mediate this protection. However, granulysin and perforin, found in the granules of cytotoxic T lymphocytes and Natural Killer (NK) cells, can kill intracellular mycobacteria and are implicated in protection against Mycobacterium tuberculosis. METHODS: We compared the cellular expression of granulysin and perforin cytolytic molecules in cord blood and peripheral blood from 10-week-old infants vaccinated at birth with either Japanese or Danish BCG, administered either intradermally or percutaneously. RESULTS: In cord blood, only CD56+ NK cells expressed granulysin and perforin constitutively. These cytolytic mediators were upregulated in CD4+ and CD8+ cord blood cells by ex vivo stimulation with BCG but not with PPD. Following BCG vaccination of neonates, both BCG and PPD induced increased expression of granulysin and perforin by CD4+ and CD8+ T cells. There was no difference in expression of cytolytic molecules according to vaccination route or strain. CONCLUSIONS: Constitutive expression of perforin and granulysin by cord blood NK-cells likely provides innate immunity, while BCG vaccination-induced expression of these cytolytic mediators may contribute towards protection of the neonate against tuberculosis.

Rifampin levels, interferon-gamma release and outcome in complicated pulmonary tuberculosis.

Factors that relate to medium-term outcome in patients with pulmonary tuberculosis (PTB) who have completed the 2-month intensive phase of treatment are incompletely understood. The relationship between in vitro production of interferon-gamma (IFN-gamma), interleukins (ILs)-5 and -10 and drug levels determined after 2 months of drug therapy, to outcome at 24 months was studied prospectively. Cytokine concentrations were determined from culture supernatants after stimulation of whole blood with purified protein derivative (PPD) of Mycobacterium tuberculosis. Plasma concentrations of rifampin, isoniazid, pyrazinamide and ethambutol were determined by high-performance liquid chromatography. The treatment failure and relapse free survival probability was 0.54 (95% CI: 0.40-0.67) at 24 months. In multivariate analysis of parameters at 2 months the strongest positive associations with disease free survival were IFN-gamma response to PPD (p=0.002) and serum creatinine (p=0.001). Drug concentrations were not associated with outcome although rifampin exposure correlated with IFN-gamma response to PPD (p=0.0132). These data suggest that the ability to mount a recall immune response to M. tuberculosis may influence treatment outcome. The data support the idea to identify persons at risk of a poor treatment outcome by monitoring of the in vitro response to tuberculosis antigens.

Novel application of a whole blood intracellular cytokine detection assay to quantitate specific T-cell frequency in field studies.

We optimized a whole blood intracellular cytokine assay to quantitate the frequency of specific CD4+ and CD8+ T cells in small volumes of whole blood from infants from developing countries. The assay is performed in two steps. First, whole blood is stimulated in the presence of specific antigens for 6-18 h, ending with cryopreservation of fixed white cells. These stimulation steps were specifically adapted to be practical and reliable in a rural, developing country field setting. Later, in a more resourceful setting, interferon-gamma producing CD4+ or CD8+ T cells are detected by flow cytometry. The assay proved sensitive and specific for detecting mycobacteria-specific immunity 10 weeks after Bacillus Calmette-Guerin (BCG) vaccination of newborns from a rural field site.

Neonatal mycobacterial specific cytotoxic T-lymphocyte and cytokine profiles in response to distinct BCG vaccination strategies.

This study evaluated whether different bacillus Calmette-Guérin (BCG) strains, routes of administration, vaccination age and percutaneous tools influenced immune responses to BCG vaccination in infants. Proliferative responses, cytokine production and cell-mediated cytotoxicity obtained in post-vaccinated children were compared to baseline cord bloods and unvaccinated 10-week-old infants. BCG vaccination generally induced strong lymphoproliferative and T helper type 1 (Th1)-type cytokine responses. There was a trend for greater responsiveness following the intradermal route of vaccination, with Japanese-172 strain and with delaying vaccination until 10 weeks. Cord mononuclear cells differentially stimulated the Th2-type cytokines interleukin-5 (IL-5) and IL-10 selectively in response to BCG, as compared to H37Rv or purified protein derivative stimulation. We document for the first time the generation of mycobacterium-specific cytotoxic T lymphocytes in neonates, following BCG vaccination. Cytotoxic activity correlated with the ratio of interferon-gamma to IL-5, aside from a single instance where use of the Biovac tool resulted in a striking dissociation selectively against H37Rv targets. These data have implications for correlates of protective immunity in design of vaccine studies.