In vitro anthelmintic effects of cysteine proteinases from plants against intestinal helminths of rodents

Infections with gastrointestinal (GI) nematodes are amongst the most prevalent worldwide, especially in tropical climates. Control of these infections is primarily through treatment with anthelmintic drugs, but the rapid development of resistance to all the currently available classes of anthelmintic means that alternative treatments are urgently required. Cysteine proteinases from plants such as papaya, pineapple and fig are known to be substantially effective against three rodent GI nematodes, Heligmosomoides polygyrus, Trichuris muris and Protospirura muricola, both in vitro and in vivo. Here, based on in vitro motility assays and scanning electron microscopy, we extend these earlier reports, demonstrating the potency of this anthelmintic effect of plant cysteine proteinases against two GI helminths from different taxonomic groups - the canine hookworm, Ancylostoma ceylanicum, and the rodent cestode, Rodentolepis microstoma. In the case of hookworms, a mechanism of action targeting the surface layers of the cuticle indistinguishable from that reported earlier appears to be involved, and in the case of cestodes, the surface of the tegumental layers was also the principal location of damage. Hence, plant cysteine proteinases have a broad spectrum of activity against intestinal helminths (both nematodes and cestodes), a quality that reinforces their suitability for development as a much-needed novel treatment against GI helminths of humans and livestock.     

Gating and inward rectifying properties of the MthK K+ channel with and without the gating ring

In MthK, a Ca2+-gated K+ channel from Methanobacterium thermoautotrophicum, eight cytoplasmic RCK domains form an octameric gating ring that controls the intracellular gate of the ion conduction pore. The binding of Ca2+ ions to the RCK domains alters the conformation of the gating ring, thereby opening the gate. In the present study, we examined the Ca2+- and pH-regulated gating and the rectifying conduction properties of MthK at the single-channel level. The open probability (Po) of MthK exhibits a sigmoidal relationship with intracellular [Ca2+], and a Hill coefficient >1 is required to describe the dependence of Po on [Ca2+], suggesting cooperative Ca2+ activation of the channel. Additionally, intracellular Ca2+ also blocks the MthK pore in a voltage-dependent manner, rendering an apparently inwardly rectifying I-V relation. Intracellular pH has a dual effect on MthK gating. Below pH 7.5, the channel becomes insensitive to Ca2+. This occurs because the gating ring is structurally unstable at this pH and tends to disassemble (Ye, S., Y. Li, L. Chen, and Y. Jiang. 2006. Cell. 126:1161-1173). In contrast, above pH 7.5, a further increase in pH shifts the Po-[Ca2+] relation towards a lower Ca2+ concentration, augments Po at saturating [Ca2+], and activates the channel even in the absence of Ca2+. Channel activity is marked by bursts of rapid openings and closings separated by relatively longer interburst closings. The duration of interburst closing and the burst length are highly Ca2+ and pH dependent, whereas the kinetics of intraburst events is Ca2+ and pH independent. The rapid intraburst openings and closings are also observed with the isolated MthK pore lacking the attached intracellular gating ring. The fast kinetic events, independent of both Ca2+ and pH, therefore appear to be determined by processes occurring within the ion conduction pore, whereas the slow events reflect the gating process controlled by Ca2+ and pH through the gating ring.     

Cutting Edge: IFN-gamma-producing CD4 T lymphocytes mediate spore-induced immunity to capsulated Bacillus anthracis

Virulent strains of Bacillus anthracis produce immunomodulating toxins and an antiphagocytic capsule. The toxin component-protective Ag is a key target of the antianthrax immune response that induces production of toxin-neutralizing Abs. Coimmunization with spores enhances the antitoxin vaccine, and inactivated spores alone confer measurable protection. We aimed to identify the mechanisms of protection induced in inactivated-spore immunized mice that function independently of the toxin/antitoxin vaccine system. This goal was addressed with humoral and CD4 T lymphocyte transfer, in vivo depletion of CD4 T lymphocytes and IFN-gamma, and Ab-deficient (muMT(-/-)) or IFN-gamma-insensitive (IFN-gammaR(-/-)) mice. We found that humoral immunity did not protect from nontoxinogenic capsulated bacteria, whereas a cellular immune response by IFN-gamma-producing CD4 T lymphocytes protected mice. These results are the first evidence of protective cellular immunity against capsulated B. anthracis and suggest that future antianthrax vaccines should strive to augment cellular adaptive immunity.     

Dendritic cell function can be modulated through cooperative actions of TLR ligands and invariant NKT cells

The quality of signals received by dendritic cells (DC) in response to pathogens influences the nature of the adaptive response. We show that pathogen-derived signals to DC mediated via TLRs can be modulated by activated invariant NKT (iNKT) cells. DC maturation induced in vivo with any one of a variety of TLR ligands was greatly improved through simultaneous administration of the iNKT cell ligand alpha-galactosylceramide. DC isolated from animals treated simultaneously with TLR and iNKT cell ligands were potent stimulators of naive T cells in vitro compared with DC from animals treated with the ligands individually. Injection of protein Ags with both stimuli resulted in significantly improved T cell and Ab responses to coadministered protein Ags over TLR stimulation alone. Ag-specific CD8(+) T cell responses induced in the presence of the TLR4 ligand monophosphoryl lipid A and alpha-galactosylceramide showed faster proliferation kinetics, and increased effector function, than those induced with either ligand alone. Human DC exposed to TLR ligands and activated iNKT cells in vitro had enhanced expression of maturation markers, suggesting that a cooperative action of TLR ligands and iNKT cells on DC function is a generalizable phenomenon across species. These studies highlight the potential for manipulating the interactions between TLR ligands and iNKT cell activation in the design of effective vaccine adjuvants.     

Poly(I:C) and lipopolysaccharide innate sensing functions of circulating human myeloid dendritic cells are affected in vivo in hepatitis C virus-infected patients

The role of peripheral dendritic cells (DCs) in hepatitis C virus (HCV) infection is unclear. To determine if persistent infection exerts an inhibitory pressure on HCV-specific innate responses, we analyzed DC function in blood through quantification of cell-associated HCV RNA levels in conjunction with multiparametric flow cytometry analysis of pathogen recognition receptor-induced cytokine expression. Independently of the serum viral load, fluorescence-activated cell sorter-purified total DCs had a wide range of cell-associated HCV genomic RNA copy numbers (mean log(10), 5.0 per 10(6) cells; range, 4.3 to 5.8). Here we report that for viremic patients with high viral loads in their total DCs, the myeloid DC (MDC) subset displayed impaired expression of interleukin-12 (IL-12) and tumor necrosis factor alpha (TNF-alpha) but normal IL-6 or chemokine CCL3 expression in response to poly(I:C) and lipopolysaccharide (LPS). IL-6-expressing cells from this subgroup of viremic patients demonstrated a significant increase (sixfold more) in TNF-alpha(-) IL-12(-) cell frequency compared to healthy donors (mean, 38.8% versus 6.5%; P < 0.0001), indicating a functional defect in a subpopulation of cytokine-producing MDCs ( approximately 6% of MDCs). Attenuation of poly(I:C) and LPS innate sensing was HCV RNA density dependent and did not correlate with viremia or deficits in circulating MDC frequencies in HCV-infected patients. Monocytes from these patients were functionally intact, responding normally on a per-cell basis following stimulation, independent of cell-associated HCV RNA levels. Taken together, these data indicate that detection of HCV genomic RNA in DCs and loss of function in the danger signal responsiveness of a small proportion of DCs in vivo are interrelated rather than independent phenomena.     

Comparison of 5-HT4 and 5-HT7 receptor expression and function in the circular muscle of the human colon

Serotonin receptors are potential targets for treating functional bowel disorders. This study investigated the functional roles and expression of the 5-HT4 and the 5-HT7 receptor, which coexist in human colon circular smooth muscle. 5-HT3 receptor expression was also investigated. Part of the relaxant response to 5-HT was due to activation of 5-HT4 receptors as the apparent pKB value of the selective 5-HT4 antagonist, GR 113808, was 9.36. 5-HT4 mRNA levels were low in five tissues and undetectable in four others, but all responded to 5-HT with an EC50 value of 102.54+/-19.32 nM. The contribution of 5-HT7 receptors to the response was not readily demonstrated using the selective 5-HT7 antagonist, SB-269970, as its apparent pKB value of 7.19 (5-HT4 block with 1 microM GR 113808) was lower than the value obtained using the 5-HT7 guinea pig ileum assay (8.62). Nevertheless, the 5-HT7 receptor was expressed more consistently than the 5-HT4, but at similar levels. The 5-HT(3Ashort) and 5-HT(3B) subunits were co-expressed at similar levels, but the 5-HT(3Along) subunit was detected in only five of the nine samples tested. The findings show that 5-HT4-induced relaxation occurs at low to undetectable levels of tissue mRNA, as measured by qPCR. Although 5-HT7 receptor mRNA is detected at low, but consistent levels, the functional activity of this receptor is not readily identified given the currently available drugs.     

Multiplexed immunoassays for proteins using magnetic luminescent nanoparticles for internal calibration

Suspension arrays present a promising tool for multiplexed assays in large-scale screening applications. A simple and robust platform for quantitative multiprotein immunoanalysis has been developed with the use of magnetic Co:Nd:Fe(2)O(3)/luminescent Eu:Gd(2)O(3) core/shell nanoparticles (MLNPs) as a carrier. The magnetic properties of the MLNPs allow their manipulation by an external magnetic field in the separation and washing steps in the immunoassay. Their optical properties enable the internal calibration of the detection system. The multiplexed sandwich immunoassay involves dual binding events on the surface of the MLNPs functionalized with the capture antibodies. Secondary antibodies labeled with conventional organic dyes (Alexa Fluor) are used as reporters. The amount of the bound secondary antibody is directly proportional to the concentration of the analyte in the sample. In our approach, the fluorescence intensity of the reporter dye is related to the luminescence signal of the MLNPs. In this way, the intrinsic luminescence of the MLNPs serves as an internal standard in the quantitative immunoassay. The concept is demonstrated for a simultaneous immunoassay for three model proteins (human, rabbit, and mouse IgGs). The method uses a standard bench plate reader. It can be applied to disease diagnostics and to the detection of biological threats.     

A rapid assay for dihydropteroate synthase activity suitable for identification of inhibitors

The enzymes 6-hydroxymethylpterin pyrophosphokinase (HPPK) and dihydropteroate synthase (DHPS) catalyze sequential steps in folate biosynthesis. They are present in microorganisms but absent in mammals and therefore are especially suitable targets for antimicrobials. Sulfa drugs (sulfonamides and sulfones) currently are used as antimicrobials targeting DHPS, although resistance to these drugs is increasing. The most widely used assay that measures activity of these enzymes, to assess new inhibitors in vitro, is not amenable to automation. This article describes a simple, coupled, enzymatic spectrophotometric assay where the product of the DHPS reaction, dihydropteroate, is reduced to tetrahydropteroate by excess dihydrofolate reductase (DHFR) using the cofactor NADPH. The oxidation of NADPH is monitored at 340 nm. The activity of both HPPK and DHPS can be measured in this assay, and it has been used to measure kinetic parameters of wild-type and sulfa drug-resistant DHPS enzymes to demonstrate the utility of the assay. It is a sensitive and reproducible assay that can be readily automated and used in multiwell plates. This NADPH-coupled microplate photometric assay could be used for rapid screening of chemical libraries for novel inhibitors of folate biosynthesis as the first step in developing new antimicrobial drugs targeting the folate biosynthetic pathway.