Inhibition of neuronal M(2) muscarinic receptor function in the lungs by extracellular nitric oxide.

1. These experiments were carried out to test whether neuronal M(2) muscarinic receptor function in the lungs is affected by nitric oxide (NO) and whether the source of the NO is epithelial or neuronal. 2. In pathogen free, anaesthetized guinea-pigs, the muscarinic agonist pilocarpine inhibited vagally induced bronchoconstriction demonstrating functional neuronal M(2) muscarinic receptors. In the presence of the NO donor, 3-morpholino-sydnonimine (SIN-1), pilocarpine no longer inhibited vagally induced bronchoconstriction. In contrast, inhibiting endogenous NO with N(G)-monomethyl-L-arginine methyl ester (L-NMMA) did not affect the ability of pilocarpine to decrease vagally induced bronchoconstriction. 3. In isolated tracheas, pilocarpine inhibited contractions induced by electrical field stimulation demonstrating that neuronal M(2) muscarinic receptors function in vitro. As in the anaesthetized guinea-pigs, SIN-1 shifted the pilocarpine dose response curve to the right, demonstrating decreased neuronal M(2) receptor function. However, in vitro, L-NMMA shifted the pilocarpine dose response curve to the left, demonstrating that endogenous NO was inhibiting the ability of the M(2) receptors to decrease acetylcholine (ACh) release. 4. Both haemoglobin (Hb), which scavenges NO, and epithelial removal also shifted the pilocarpine dose response curve to the left, demonstrating that the NO inhibiting neuronal M(2) receptor function was extracellular and probably of epithelial origin. 5. In conclusion, extracellular NO appears to inhibit the ability of the M(2) receptors to decrease ACh release from the parasympathetic nerves in the lungs in vivo and in vitro in pathogen free guinea-pigs. However, while the neuronal M(2) receptors will respond to NO (from SIN-1) in vivo, there does not appear to be an endogenous source of NO since L-NMMA had no effect in vivo.

IL-12 regulates T helper type 1 cytokine responses in human infectious disease.

We investigated the role of IL-12 in regulating T cell and cytokine responses in human infectious disease by using the spectrum of leprosy as a model. Tuberculoid patients mount strong T cell responses to Mycobacterium leprae, with production of the type 1 cytokines IL-2 and IFN-gamma in lesions; whereas lepromatous patients manifest weak T cell responses to M. leprae, with production of the type 2 cytokines IL-4 and IL-10 in lesions. We found expression of IL-12 p40 mRNA, as measured by PCR amplification, and IL-12 p70, as measured by immunohistochemistry, to be 10-fold greater in tuberculoid lesions than in lepromatous lesions. The ability of M. leprae to stimulate release of IL-12 from monocytes was inhibited by rIL-4 and rIL-10. M. leprae-induced T cell proliferation in tuberculoid patients was blocked by the addition of neutralizing Abs to IL-12. Furthermore, rIL-12 stimulated proliferation of CD4+ type 1 T cell clones from tuberculoid lesions, but not CD8+ type 2 T cell clones from lepromatous lesions; however, both responded to rIL-2, rIL-12 augmented M. leprae-specific T cell proliferation in lepromatous patients, thereby causing the selective expansion of CD4+ T cells and increasing T cell IFN-gamma production. These data indicate that IL-12 is an important mediator in the generation of the type 1 cytokine response in human infectious disease.

T cells bearing V beta 6 T cell receptors in the cell-mediated immune response to Mycobacterium leprae.

The skin lesions of leprosy provide a window into the immunoregulatory events involved in the human immune response to infection. T cells are thought to play a vital role in the pathogenesis of different forms of the disease. To identify predominant specific T cell subpopulations in leprosy lesions, the TCR-beta chain repertoire was simultaneously studied in skin biopsy specimens and PBMC from both immunologically resistant tuberculoid leprosy and susceptible lepromatous leprosy patients. This was accomplished by obtaining RNA from lesions and PBMC, synthesizing cDNA, and performing the polymerase chain reaction. We found that TCR gene subfamilies V beta 6.1 through V beta 6.4 (V beta 6.1-4) were strikingly overrepresented in lesions vs PBMC of seven of nine tuberculoid patients but only one of nine lepromatous patients. Similarly, V beta 6.5/6.8/6.9 subfamilies were predominant in four of nine tuberculoid patients, but none of the nine lepromatous patients. To explore the influence of the complementarity-determining region 3 (CDR3) in selection of T cells expressing V beta 6 TCR, we sequenced the V beta 6.1-4-C beta polymerase chain reaction products derived from the lesions and PBMC of two tuberculoid patients. From the analysis of deduced amino acid sequences, we found conserved amino acid residues and amino acid motifs in the CDR3 region of the lesion-derived sequences from each patient. Our data suggest that the nominal Ag select T cells bearing V beta 6 TCR in the cell-mediated immune response to Mycobacterium leprae.

Variation of larval susceptibility to Lagenidium giganteum in three mosquito species.

A significantly greater number of Lagenidium giganteum zoospores were found encysting on the dorsal thoracic surface of Anopheles gambiae larvae than on the larvae of Aedes aegypti and Culex pipiens. However, germ tube penetration in the cuticle of A. gambiae provoked an intense and diffuse melanization which encapsulated the fungus and protected 56% from death. Although a small number of zoospores attach to and penetrate the cuticular surface of A. aegypti and C. pipiens approximately 99% of both species succumb to fungal infection. Melanization in A. aegypti is slower, weaker, more localized, and generally ineffective against L. giganteum infection compared to A. gambiae. Upward migration of L. giganteum zoospores to the water surface favored encounters with mosquito larvae and was speculated to be due to negative geotaxis rather than positive aerotaxis and phototaxis. Otherwise, initial contact between larva and zoospore was random.