Compartmentalized bronchoalveolar IFN-gamma and IL-12 response in human pulmonary tuberculosis.

Human tuberculosis (TB) principally involves the lungs, where local immunity impacts on the load of Mycobacterium tuberculosis (M.tb). Because concomitants of local Th1 immunity are still under-explored in humans, we characterized immune responses in bronchoalveolar cells (BACs) and systemically in peripheral blood mononuclear cells (PBMCs) in persons with active pulmonary TB and in healthy community controls. PPD- and live M.tb-induced IFN-gamma-production were observed in CD4(+), CD8(+), gammadeltaTCR(+), and CD56(+) alveolar T cell subpopulations and NK cells (CD3(-)CD56(+)). IFN-gamma-producing CD4(+) T cells (mostly CD45RO(+)) were more abundant (p<0.05). M.tb-induced IL-12p70, but interestingly also IL-4, was increased (p<0.05) in BACs from TB patients. Constitutive expression of IL-12Rbeta1 and IL-12Rbeta2 mRNA in BACs and PBMCs and IFN-gammaR1 in BACs was similar in both study groups. Data were normalized to account for differences in proportions of alveolar T cells and macrophages in the study groups. IFN-gamma-production and its induction by IL-12R engagement occur virtually unimpaired in the bronchoalveolar spaces of patients with pulmonary TB. The reasons for the apparent failure to control M. tuberculosis growth during active pulmonary TB disease is unknown but could be the expression of locally acting immunosuppressive mechanisms that subvert the antimycobacterial effects of IFN-gamma.

Human {beta}-defensin 2 is expressed and associated with Mycobacterium tuberculosis during infection of human alveolar epithelial cells.

To determine the role of human beta-defensin 2 (HBD-2) in human tuberculosis, we studied the in vitro induction of HBD-2 gene expression by Mycobacterium tuberculosis H37Rv infection in the human lung epithelial cell line A549, in alveolar macrophages (AM), and in blood monocytes (MN) by reverse transcription-PCR. We also studied the induction of HBD-2 gene expression by mannose lipoarabinomannan (manLAM) from M. tuberculosis. Intracellular production of HBD-2 peptide was detected by immunocytochemistry and electron microscopy. Our results demonstrated that there was induction of HBD-2 mRNA in A549 cells after infection with M. tuberculosis at various multiplicities of infection (MOI) and that there was stimulation with manLAM. AM expressed the HBD-2 gene only at a high MOI with M. tuberculosis. MN did not express HBD-2 at any of the experimental M. tuberculosis MOI. Immunostaining revealed the presence of intracellular HBD-2 peptide in A549 cells following infection with M. tuberculosis, and the staining was more intense in areas where there were M. tuberculosis clusters. By using electron microscopy we also demonstrated production of HBD-2 after M. tuberculosis infection and adherence of HBD-2 to the membranes of M. tuberculosis. Alveolar epithelial cells are among the first cells to encounter M. tuberculosis following aerogenic infection. As HBD-2 has been shown to control growth of M. tuberculosis and has chemotactic activity, our results suggest that HBD-2 induction by M. tuberculosis may have a role in the pathogenesis of human tuberculosis.

Founder mutation R245H of Sanfilippo syndrome type A in the Cayman Islands.

Sanfilippo A syndrome is an autosomal recessive lysosomal storage disease. This disease was reported in the Cayman Islands population with carrier frequency of 1/7 to 1/10 in the West Bay district of Grand Cayman. The carrier testing of Sanfilippo A disease for families at risk was carried out using the thermal characteristics of sulfamidase activity. In the present study, a search for mutations in the sulfamidase gene in an index family was performed. In addition, 77 individuals, relatives of children with Sanfilippo A syndrome, were also studied by single-strand conformation polymorphism (SSCP), restriction fragment-length polymorphism (RFLP) analyses, and sequencing. A single mutation, G746A (R245H), was found in the family, with the patient being homozygous and both parents and 1 of the 3 siblings being carriers. Among the 77 family members of the patient with Sanfilippo syndrome, the same mutation was found among carriers of the disease. The finding of a single mutation supports the idea of a founder effect, which facilitates accurate carrier identification of Sanfilippo A syndrome in the population of Cayman Islands.

Improved mucosal protection against Venezuelan equine encephalitis virus is induced by the molecularly defined, live-attenuated V3526 vaccine candidate.

The genetically engineered, live-attenuated Venezuelan equine encephalitis (VEE) virus vaccine candidate, V3526, was evaluated as a replacement for the TC-83 virus vaccine. Protection from lethal subcutaneous or aerosol challenge was evaluated in vaccinated mice clinically and immunohistochemically. Subcutaneous administration of V3526 induced systemic and mucosal protection more efficiently than did the TC-83 vaccine. The bronchial IgA responses induced in mice by subcutaneous administration of vaccines significantly corresponded to the ability to survive aerosol challenge with virulent virus. Furthermore, V3526 delivered by aerosol induced more complete mucosal protection than either vaccine administered subcutaneously. The ability of V3526 to induce protection in mice warrants its consideration for further testing as a potential vaccine candidate for human use.

Comparative neurovirulence and tissue tropism of wild-type and attenuated strains of Venezuelan equine encephalitis virus administered by aerosol in C3H/HeN and BALB/c mice.

To assess the potential for aerosol administration of vaccines for Venezuelan equine encephalitis virus (VEE), we compared the neurovirulence and tissue tropism of the wild-type Trinidad donkey (TrD) strain to those of the attenuated TC83 and V3526 strains of VEE in mice. Six to 8-week-old female C3H/HeN and BALB/c mice were aerosol exposed to one of the three VEE strains. Three mice of each strain were euthanatized at different times and their tissues were processed and stained using hematoxylin and eosin, immunohistochemistry, and in situ hybridization. All three viral strains infected the brains of mice and induced encephalitis. TrD spread caudally from the olfactory bulbs to all regions of the brain, caused widespread necrotizing panencephalitis by day 5, and resulted in 100% mortality (geometric mean = 7 days) in both mouse strains. By comparison, TC83 relatively spared the caudal regions of the brain but still caused 100% mortality in the C3H/HeN mice (geometric mean = 12 days), yet it did not kill any BALB/c mice. V3526 infectivity of the brain was the most limited, mainly affecting the neocortex and diencephalon. This virus was not lethal in either mouse strain. The TrD strain also infected the olfactory neuroepithelium, local lymphoid tissues, teeth, and vomeronasal organs, whereas the affinity of TC83 and V3526 outside the brain was essentially limited to the olfactory neuroepithelium. Attenuated VEE strains administered to mice by aerosol have restricted tissue tropism as compared with wild-type virus; however, even attenuated strains can infect the brain and induce encephalitis.