Rapid diagnosis of tuberculosis and multidrug resistance with the microscopic observation drug susceptibility assay in Ecuador.

A collaborative project was established between the Alli Causai Foundation in Ambato, Ecuador, and the University of Genoa, Italy, to introduce the microscopic observation drug susceptibility (MODS) assay for the rapid identification of Mycobacterium tuberculosis in Ecuador. A total of 507 samples were evaluated during a 10-month period, and DNA was extracted from each isolate and sent to Genoa for confirmatory molecular analysis. M. tuberculosis was identified in 45 samples by MODS, and drug resistance was observed in approximately 21% of the isolates, with four multidrug-resistant strains detected in two patients.

Matrix metalloproteinase 7 and perlecan in oral epithelial dysplasia and carcinoma in situ: an aid for histopathologic recognition of their cell proliferation centers.

BACKGROUND: As one of the valuable tools for differential diagnoses of oral epithelial dysplasia, carcinoma in situ (CIS) and squamous cell carcinoma (SCC), we have proposed the immunohistochemistry for perlecan, a heparan sulfate proteoglycan (HSPG). As HSPGs have been shown to be extracellular docking molecules for matrix metalloproteinase (MMP) 7, our aim was to determine the expression mode of MMP-7 in these lesions for its possible diagnostic aid for oral borderline malignancies. METHODS: Twenty cases each of moderate dysplasia, CIS, SCC, and normal/hyperplastic/mild dysplastic epithelia of the tongue and buccal mucosa were immunohistochemically examined for MMP-1, -2 and -7 in reference to their perlecan immunolocalization. RESULTS: The expression of all three MMPs in the normal mucosal epithelium was restricted mainly to the parabasal layers. The most striking finding was strong expression of MMP-7 in epithelial dysplasia with a two-phase appearance: a clear demarcation of MMP-7-immunopositive (+) lower dysplastic/basaloid cells from non-positive upper keratinized cells. MMP-7+ cells were spread over the whole epithelial layer of CIS. In SCC, MMP-7 positivity was reduced from carcinoma cells but instead appeared in stromal cells. These expression profiles of MMP-7 resembled those of perlecan. MMP-1 and MMP-2 exhibited a similar but much weaker staining than MMP-7. CONCLUSION: These results suggest that the enhanced metabolism of perlecan associated with MMP-7 plays an important role in the cell proliferation of oral epithelia in their malignant transformation process, and that MMP-7 immunohistochemistry may be a valuable aid for identification of the cell proliferation center in oral CIS and dysplasia.

Variation in the level of xenoantigen expression in porcine organs.

Hyperacute rejection of vascularized porcine to primate xenografts is initiated by the binding of xenoreactive natural antibodies to donor endothelium. We tested the hypothesis that the level of xenoantigen expression varies in the population of potential porcine donors and may determine the amount of binding of xenoreactive natural antibodies to a porcine organ perfused by xenogeneic blood. Two hundred ninety pigs were studied using an inhibition ELISA that quantitated the xenoantigen level on porcine platelets. Based on this assay, the levels of xenoantigen expression in the population adhered to a normal distribution. Kidneys from pigs found to express high antigen levels and kidneys from pigs found to express low antigen levels were perfused with baboon blood using an extracorporeal circuit. In multiple experiments, a significant difference was observed in the amount of xenoreactive natural antibody adsorbed by high antigen versus low antigen organs. Normalizing for the weight of the perfused organs and for levels of natural antibody in individual baboons, high antigen organs adsorbed 3.6 +/- 1.3 U of xenoreactive natural antibody/g and low antigen organs adsorbed -0.8 +/- 1.0 U of xenoreactive natural antibody/g (P < 0.002). Immunopathology of tissues from the perfused organs demonstrated more deposition of IgM and C4 in high than in low xenoantigen organs. The quantitative relationship between binding of xenoreactive natural antibodies to platelets and to whole organs suggests that platelets are a valid representation of endothelial cell antigen expression in vivo. Despite the probable importance of Gal alpha(1-3)Gal as an epitope recognized by xenoreactive natural antibodies, differences in the binding to platelets or to organs of the GS-I-B4 lectin that recognizes that sugar had no correlation with the differences in binding of IgM to these tissues. Variation in expression of xenoantigen may be exploited to selectively breed donors for xenotransplantation that are less susceptible to attack by xenoreactive natural antibodies.

Human complement regulatory proteins protect swine-to-primate cardiac xenografts from humoral injury.

The susceptibility of xenografts to hyperacute rejection is postulated to reflect in part failure of complement regulatory proteins (CRPs) to control activation of heterologous complement on graft endothelium. To test this concept, transgenic swine expressing the human CRP decay accelerating factor and CD59 were developed using a novel expression system involving transfer of the proteins from erythrocytes to endothelial cells. Hearts from transgenic swine transplanted into baboons had markedly less vascular injury and functioned for prolonged periods compared to hearts from nontransgenic swine. These results indicate that expression of human CRPs in xenogeneic organs may contribute to successful xenografting and suggest that intercellular protein transfer might be a useful approach for expression of heterologous proteins in endothelial cells.

Cardiac xenografts between primate species provide evidence for the importance of the alpha-galactosyl determinant in hyperacute rejection.

Transplants performed between phylogenetically disparate species are subject to hyperacute rejection initiated by binding of xenoreactive natural Abs to endothelium in the donor organ. Binding of these Abs activates complement, leading to tissue injury and destruction of the graft. Human xenoreactive natural Abs recognize Gal alpha 1-3Gal beta 1-4GlcNAc (galactose alpha 1-3galactose beta 1-4-N-acetylglucosame); however, the relative importance of this Ag in graft rejection has not been proved. The present study was conducted to test the potential importance of alpha-galactosyl (alpha-Gal) determinants in the pathogenesis of hyperacute rejection. To this end, hearts (n = 3) from New World monkeys (Saimiri scureus, squirrel monkey), which can synthesize Gal alpha 1-3Gal, were transplanted heterotopically into Old World monkeys (Papio species, baboon), which do not synthesize Gal alpha 1-3Gal determinants and which have circulating anti-alpha Gal Abs. The xenografts were rejected in 51 to 56 min (mean +/- SD = 53.3 +/- 2.5), results similar to those observed in porcine grafts transplanted into baboons. Histologic analysis of the hearts revealed thrombosis and intraparenchymal hemorrhage and immune deposits consisting of IgM, Clq, C3, C4, C5b, and the membrane attack complex, but not properdin or factor B of the recipient deposited on graft endothelium. Sera obtained from baboons after perfusion of squirrel monkey kidneys revealed depletion of alpha-Gal-specific Abs and anti-pig endothelial cell Abs. These findings provide strong evidence that the Abs that accumulate in New World monkey organs during perfusion with baboon blood are the same Abs that would accumulate in a porcine organ transplanted into a primate and suggest that hyperacute rejection is not necessarily a reflection of phylogenetic distance but that the expression of terminal alpha-Gal residues provides an adequate target to initiate that process.