Natural killer cells and innate immunity to protozoan pathogens

Natural killer (NK) cells are lymphoid cells that mediate significant cytotoxic activity and produce high levels of pro-inflammatory cytokines in response to infection. During viral infection, NK cell cytotoxicity and cytokine production is induced principally by monocyte-macrophage- and dendritic cell-derived cytokines but virally encoded ligands for NK cells are also beginning to be described. NK derived interferon-gamma (IFN-gamma) production is also essential for control of several protozoal infections including toxoplasmosis, trypanosomiasis, leishmaniasis and malaria. The activation of NK cells by protozoan pathogens is also believed to be cytokine-mediated although some recent studies suggest that direct recognition of parasites by NK cells also occurs. Both indirect signalling via accessory cell-derived cytokines and direct signalling, presumably through NK receptors, are needed in order for human malaria parasites (Plasmodium falciparum) to optimally stimulate NK activity.     

Triterpenoid saponins from a cytotoxic root extract of Sideroxylonfoetidissimum subsp. gaumeri

Evaluation of the cytotoxicity of an ethanolic root extract of Sideroxylonfoetidissimum subsp. gaumeri (Sapotaceae) revealed activity against the murine macrophage-like cell line RAW 264.7. Systematic bioassay-guided fractionation of this extract gave an active saponin-containing fraction from which four saponins were isolated. Use of 1D (¹H, ¹³C, DEPT135) and 2D (COSY, TOCSY, HSQC, and HMBC) NMR, mass spectrometry and sugar analysis gave their structures as 3-O-(β-d-glucopyranosyl-(1 → 6)-β-d-glucopyranosyl)-28-O-(α-l-rhamnopyranosyl-(1 → 3)[β-d-xylopyranosyl-(1 → 4)]-β-d-xylopyranosyl-(1 → 4)-α-l-rhamnopyranosyl-(1 → 2)-α-l-arabinopyranosyl)-16α-hydroxyprotobassic acid, 3-O-β-d-glucopyranosyl-28-O-(α-l-rhamnopyranosyl-(1 → 3)[β-d-xylopyranosyl-(1 → 4)]-β-d-xylopyranosyl-(1 → 4)-α-l-rhamnopyranosyl-(1 → 2)-α-l-arabinopyranosyl)-16α-hydroxyprotobassic acid, 3-O-(β-d-glucopyranosyl-(1 → 6)-β-d-glucopyranosyl)-28-O-(α-l-rhamnopyranosyl-(1 → 3)-β-d-xylopyranosyl-(1 → 4)[β-d-apiofuranosyl-(1 → 3)]-α-l-rhamnopyranosyl-(1 → 2)-α-l-arabinopyranosyl)-16α-hydroxyprotobassic acid, and the known compound, 3-O-β-d-glucopyranosyl-28-O-(α-l-rhamnopyranosyl-(1 → 3)[β-d-xylopyranosyl-(1 → 4)]-β-d-xylopyranosyl-(1 → 4)-α-l-rhamnopyranosyl-(1 → 2)-α-l-arabinopyranosyl)-protobassic acid. Two further saponins were obtained from the same fraction, but as a 5:4 mixture comprising 3-O-(β-d-glucopyranosyl)-28-O-(α-l-rhamnopyranosyl-(1 → 3)-β-d-xylopyranosyl-(1 → 4)[β-d-apiofuranosyl-(1 → 3)]-α-l-rhamnopyranosyl-(1 → 2)-α-l-arabinopyranosyl)-16α-hydroxyprotobassic acid and 3-O-(β-d-apiofuranosyl-(1 → 3)-β-d-glucopyranosyl)-28-O-(α-l-rhamnopyranosyl-(1 → 3)[β-d-xylopyranosyl-(1 → 4)]-β-d-xylopyranosyl-(1 → 4)-α-l-rhamnopyranosyl-(1 → 2)-α-l-arabinopyranosyl)-16α-hydroxyprotobassic acid, respectively. This showed greater cytotoxicity (IC₅₀ = 11.9 ± 1.5 μg/ml) towards RAW 264.7 cells than the original extract (IC₅₀ = 39.5 ± 4.1 μg/ml), and the saponin-containing fraction derived from it (IC₅₀ = 33.7 ± 6.2 μg/ml).     

Innate IFNs and plasmacytoid dendritic cells constrain Th2 cytokine responses to rhinovirus: a regulatory mechanism with relevance to asthma

Human rhinoviruses (RV) cause only minor illness in healthy individuals, but can have deleterious consequences in people with asthma. This study sought to examine normal homeostatic mechanisms regulating adaptive immunity to RV in healthy humans, focusing on effects of IFN-αβ and plasmacytoid dendritic cells (pDC) on Th2 immune responses. PBMC were isolated from 27 healthy individuals and cultured with RV16 for up to 5 d. In some experiments, IFN-αβ was neutralized using a decoy receptor that blocks IFN signaling, whereas specific dendritic cell subsets were depleted from cultures with immune-magnetic beads. RV16 induced robust expression of IFN-α, IFN-β, multiple IFN-stimulated genes, and T cell-polarizing factors within the first 24 h. At 5 d, the production of memory T cell-derived IFN-γ, IL-10, and IL-13, but not IL-17A, was significantly elevated. Neutralizing the effects of type-I IFN with the decoy receptor B18R led to a significant increase in IL-13 synthesis, but had no effect on IFN-γ synthesis. Depletion of pDC from RV-stimulated cultures markedly inhibited IFN-α secretion, and led to a significant increase in expression and production of the Th2 cytokines IL-5 (p = 0.02), IL-9 (p < 0.01), and IL-13 (p < 0.01), but had no effect on IFN-γ synthesis. Depletion of CD1c(+) dendritic cells did not alter cytokine synthesis. In healthy humans, pDC and the IFN-αβ they secrete selectively constrain Th2 cytokine synthesis following RV exposure in vitro. This important regulatory mechanism may be lost in asthma; deficient IFN-αβ synthesis and/or pDC dysfunction have the potential to contribute to asthma exacerbations during RV infections.     

Synthesis, structure-activity relationships and molecular modeling studies of new indole inhibitors of monoamine oxidases A and B

New monoamine oxidase inhibitors were synthesized as indole analogues of a previously reported pyrrole series. Several compounds were potent MAO-A (12, 17, 19-22, 31, 36, and 37) or MAO-B (14, 20, 24, 38, 44, and 46) inhibitors, and had K(i) values in the nanomolar concentration range. In particular, 22 (K(i)=0.00092 microM, and SI=68,478) was exceptionally potent and selective as MAO-A inhibitor. In molecular modeling studies, compounds 22, 24, 44, and 46 positioned the indole ring into an aromatic cavity of MAO-A, and established pi-pi stacking interactions with Tyr407, Tyr444, and FAD cofactor. However, only compound 22 was able to form hydrogen bonds with FAD, a finding which was in accordance with its potent anti-MAO-A activity. Conversely, 22/MAOB complex was highly unstable during the MD simulation.     

Macrophage-tropic simian/human immunodeficiency virus chimeras use CXCR4, not CCR5, for infections of rhesus macaque peripheral blood mononuclear cells and alveolar macrophages

After the nearly complete and irreversible depletion of CD4(+) T lymphocytes induced by highly pathogenic simian/human immunodeficiency virus chimeric viruses (SHIVs) during infections of rhesus monkeys, tissue macrophages are able to sustain high levels (>10(6) viral RNA copies/ml) of plasma viremia for several months. We recently reported that the virus present in the plasma during the late macrophage phase of infection had acquired changes that specifically targeted the V2 region of gp120 (H. Imamichi et al., Proc. Natl. Acad. Sci. USA 99:13813-13818, 2002); some of these SHIV variants were macrophage-tropic (M-tropic). Those findings have been extended by examining the tropic properties, coreceptor usage, and gp120 structure of five independent SHIVs recovered directly from lymph nodes of late-stage animals. All of these tissue-derived SHIV isolates were able to infect alveolar macrophages. These M-tropic SHIVs used CXCR4, not CCR5, for infections of rhesus monkey PBMC and primary alveolar macrophages. Because the starting highly pathogenic T-tropic SHIV inoculum also utilized CXCR4, these results indicate that the acquisition of M-tropism in the SHIV-macaque system is not accompanied by a change in coreceptor usage. Compared to the initial T-tropic SHIV inoculum, tissue-derived M-tropic SHIVs from individual infected animals carry gp120s containing similar changes (specific amino acid deletions, substitutions, and loss of N-linked glycosylation sites), primarily within the V1 and/or V2 regions of gp120.