Nitric oxide synthase immunoreactivity in the nematode Trichinella britovi. Evidence for nitric oxide production by the parasite

Nitric oxide has been extensively studied as an effector molecule of the host immune response against both protozoa and helminths, but parasites can also produce this molecule, through the action of nitric oxide (NO) synthases or NO synthases-like enzymes. The aim of this study was to verify the possible production of NO by Trichinella britovi L(1) larvae and the enzymes involved in this process. The NO synthase immunoreactivity and putative nitric oxide synthase-activity was analysed using antibodies to mammalian NO synthase III and to nitrotyrosine with immunohistochemistry, gold immunocytochemistry and immunoblot analysis and NADPH-diaphorase histochemistry. Our results show that T. britovi L(1) larvae possess an enzymatic activity capable of producing NO. The localisation of this activity, according to the NADPH-diaphorase histochemistry, is both at the cuticular and the internal level. This localisation is confirmed by nitrotyrosine immunohistochemistry both under optical and electron microscopy. Using the NO synthase III antibody, a similar pattern of labelling was found: in particular, electron microscopy showed a localisation of this immunoreactivity in the cuticle and in the stichocytes, where only the alpha2 granules contained gold particles, mainly concentrated at their periphery. Four polypeptides reacting to the NO synthase III antibody are revealed by Western blotting. Their molecular weight ranged from 38 to 50 kDa. A significant reaction of the anti-nitrotyrosine antibody to polypeptides 95, 60, 48 and 39 kDa from the same sample suggested the presence of different nitrosylated proteins.     

Stimulation by parathyroid hormone of a NHERF-1-assembled complex consisting of the parathyroid hormone I receptor, phospholipase Cbeta, and actin increases intracellular calcium in opossum kidney cells

Parathyroid hormone (PTH) binds its cognate G-protein-coupled receptor (PTH1R) and signals through both adenylyl cyclase and phospholipase C (PLC). C-terminal determinants of the PTH1R interact with the Na+/H+ exchanger regulatory factor 1 (NHERF-1) by binding the first of two PDZ (psd95, discs-large, ZO-1) domains. Compared with wild-type opossum kidney (OK) cells, OKH cells, a sub-clone, do not display PTH-mediated increases of [Ca2+]i and express NHERF-1 at markedly lower levels. Stable expression of NHERF-1 in the OKH parent (OKH-N1) restores the PTH-mediated increase of [Ca2+]i that arises from an influx of extracellular calcium and is both PLC-dependent and pertussis toxin-sensitive. From a morphological perspective, NHERF-1 and the PTH1R co-localize to apical patches of OKH-N1 cells, an expression pattern that is absent in OKH cells and depends on a direct NHERF-1-PTH1R interaction in OKH-N1 cells. Actin and PLCbeta1 and -beta3 co-localize with NHERF-1 and the PTH1R in OKH-N1 cell apical patches. Actin is also an integral component of the NHERF-1-assembled complex because cytochalasin D disrupts apical localization of both NHERF-1 and the PTH1R and inhibits the PTH-mediated increase of [Ca2+]i. Expression of the first PDZ domain of NHERF-1 acts as a dominant-negative interactor by blocking apical localization of the PTH1R and inhibiting PTH-elicited increases of [Ca2+]i. Thus, NHERF-1 assembles a signaling complex in the apical domains of OK cells that contains the PTH1R, PLCbeta, and the actin cytoskeleton. Disruption of this complex blocks the PTH mediated increases of intracellular calcium.     

The Australian White Ibis (Threskiornis molucca) as a Reservoir of Zoonotic and Livestock Pathogens

Over the last 20 years, Australian white ibis populations (Threskiornis molucca) have expanded into urban areas, leading to increased contact between ibis, domestic animals, and humans. This has led to concern that ibis may transmit pathogens that threaten public health or food production. Here we report results from a study of ibis viral serology and bacterial culture that indicate that ibis are hosts of zoonotic and livestock pathogens such as Salmonella spp., Newcastle disease virus, avian influenza virus, and flaviviruses in Australia. We also performed a behavioral study to measure contact rates among ibis, people, and livestock that determine the potential for disease transmission.     

Cutting edge: IL-4-induced protection of CD4+CD25- Th cells from CD4+CD25+ regulatory T cell-mediated suppression

CD4(+)CD25(+) T regulatory (Treg) cells are a CD4(+) T cell subset involved in the control of the immune response. In vitro, murine CD4(+)CD25(+) Treg cells inhibit CD4(+)CD25(-) Th cell proliferation induced by anti-CD3 mAb in the presence of APCs. The addition of IL-4 to cocultured cells inhibits CD4(+)CD25(+) Treg cell-mediated suppression. Since all cell types used in the coculture express the IL-4Ralpha chain, we used different combinations of CD4(+)CD25(-) Th cells, CD4(+)CD25(+) Treg cells, and APCs from wild-type IL-4Ralpha(+/+) or knockout IL-4Ralpha(-/-) mice. Results show that the engagement of the IL-4Ralpha chain on CD4(+)CD25(-) Th cells renders these cells resistant to suppression. Moreover, the addition of IL-4 promotes proliferation of IL-4Ralpha(+/+)CD4(+)CD25(+) Treg cells, which preserve full suppressive competence. These findings support an essential role of IL-4 signaling for CD4(+)CD25(-) Th cell activation and indicate that IL-4-induced proliferation of CD4(+)CD25(+) Treg cells is compatible with their suppressive activity.     

The role of CXCR2 activity in the contact hypersensitivity response in mice

The recruitment of polymorphonuclear neutrophil leukocytes (PMN) into a challenge site, and their subsequent activation, are thought to play a role in the elicitation of the contact hypersensitivity (CHS) response. The present study investigated the role played by CXCR2 activity in tissue PMN infiltration and subsequent triggering of CHS. Our results show that the cutaneous infiltration by PMN, induced by hapten challenge was dramatically inhibited in sensitized, CXCR2-deficient (CXCR2(-/-)) mice. Inhibition of PMN recruitment into the hapten-challenged ears of CXCR2(-/-) mice was associated with a consistent reduction of the CHS response (ear swelling) in CXCR2(-/-) mice as compared with that observed in neutropenic, wild-type (CXCR2(+/+)) mice. Prevention of skin PMN infiltration and the ear swelling response by the absence of functional CXCR2 was observed regardless of the hapten used. These data clearly suggest that CXCR2 activity plays an essential role in mediating cutaneous recruitment and activation of PMN, and thus indirectly regulates recruitment of hapten-primed T cells into challenge sites, with the subsequent elicitation of the CHS response. The role played by CXCR2 activity in the CHS response provides the rationale for testing CXCR2 inhibitors as a new therapeutic approach to skin diseases.     

Paleoparasitological remains revealed by seven historic contexts from "Place d'Armes", Namur, Belgium

Human occupation for several centuries was recorded in the archaeological layers of "Place d'Armes", Namur, Belgium. Preventive archaeological excavations were carried out between 1996/1997 and seven historical strata were observed, from Gallo-Roman period up to Modern Times. Soil samples from cesspools, latrines, and structures-like were studied and revealed intestinal parasite eggs in the different archaeological contexts. Ascaris lumbricoides, A. suum, Trichuris trichiura, T. suis. Taenia sp., Fasciola hepatica, Diphyllobothrium sp., Capillaria sp. and Oxyuris equi eggs were found. Paleoparasitology confirmed the use of structures as latrines or cesspit as firstly supposed by the archaeologists. Medieval latrines were not only used for rejection of human excrements. The finding of Ascaris sp. and Trichuris sp. eggs may point to human's or wild swine's feces. Gallo-Roman people used to eat wild boar. Therefore, both A. suum and T. suis, or A. lumbricoides and T. trichuris, may be present, considering a swine carcass recovered into a cesspit. Careful sediment analysis may reveal its origin, although parasites of domestic animals can be found together with those of human's. Taenia sp. eggs identified in latrine samples indicate ingestion of uncooked beef with cysticercoid larvae. F. hepatica eggs suggest the ingestion of raw contaminated vegetables and Diphyllobothrium sp. eggs indicate contaminated fresh-water fish consumption. Ascaris sp. and Trichuris sp. eggs indicate fecal-oral infection by human and/or animal excrements.     

Synthesis and D(2)-like binding affinity of new derivatives of N-(1-ethyl-2-pyrrolidinylmethyl)-4,5-dihydro-1H-benzo[g]indole-3-carboxamide and related 4H-[1]benzothiopyrano[4,3-b]pyrrole and 5,6-dihydro-4H-benzo[6,7]cyclohepta[b]pyrrole-3-carboxamide analogues

Various new derivatives and structural analogues of N-(1-ethyl-2-pyrrolidinylmethyl)-4,5-dihydro-1H-benzo[g]indole-3-carboxamide (2a), a representative term of a series of 2-aminomethylpyrrolidinyl derived 4,5-dihydrobenzo[g]indolcarboxamides with good D(2)-like affinity, were synthesized and evaluated for their ability to bind to dopamine D(2)-like receptors in vitro. The structural contribution to D(2)-like receptor binding of the 4,5-dihydrobenzo[g]indole portion of the molecule was examined. From these studies, compound 2k, 2-chloro-N-(1-ethyl-2-pyrrolidinylmethyl)-5,6-dihydro-4H-benzo[6,7]cyclohepta[b]pyrrole-3-carboxamide, was found to possess a potent affinity for D(2)-like receptors. Behavioural tests in rats have shown that this compound reduces the hyperactivity induced by amphetamine, a property shared by all antipsychotic drugs, at a dose which failed to induce catalepsy, an effect which is predictive of extrapyramidal side effects in humans. The other compounds demonstrated moderate (2c, 2h, and 2j) or no affinity for D(2)-like receptors.     

Scintillometric determination of DNA repair in human cell lines: A critical appraisal

The ability of a variety of chemical and physical agents to stimulate DNA repair synthesis in human cell cultures was tested by a simplified scintillometric procedure, with the use of hydroxyurea (HU) to suppress DNA replicative synthesis. After incubation with [³H]thymidine, the radioactivity incorporated in to DNA was determined in controls (C) and treated (T) cultures and in the corresponding HU series (C_HU, T_HU). The ratios T_HU/C_HU and T_HU/T:C_HU/C, indicating absolute and relative increases of DNA radioactivity, were calculated. When both ratios were significantly higher than 1, they were taken as indices of DNA repair stimulation, whereas, no stimulation in inferred when both of them are ?1. The scintillometric estimate of DNA repair was always in agreement with the autoradiographic observations, so that the procedure adopted can be used as a rapid test for screening investigations.Agents giving a relative but no an absolute increase of DNA radioactivity are generally not inducers of repair synthesis as estimated by autoradiography. However, the same scintillometric results are also occasionally observed with DNA repair inducers, such as methyl methanesulphonate (MMS) and ethyl methanesulphonate (EMS), owing to alterations of thymidine pool radioactivity. These chemicals, besides affecting the levels of labelled precursors in the intracellular pool in the T series, differently modified the increase of pool radioactivity which is a regular effect of HU. With such chemicals, DNA repair synthesis can be detected only after normalization of th DNA radioactivity on the basis of pool alterations.The quantitative value of the autoradiographic estimate of DNA repair is also affected by the changes in the radioactivity of the thymidine pool although autoradiography retains its qualitative value.Dimethylnitrosamine, mitomycin C and potassium dichromate, described by other authors as inducers of DNA repair, also gave negative results by the scintillometric procedure after normalization of DNA radioactivities. However, in our hands, these agents were unable to stimulated repair synthesis, according to the results of autoradiography and isopynic centrifugation.The proposed scintillometric procedure is effective in indicating false negative inducers of DNA repair, not giving rise to false positives.     

Distribution of Serotonin Receptor of Type 6 (5-HT6) in Human Brain Post-mortem. A Pharmacology, Autoradiography and Immunohistochemistry Study

The aim of this study was to investigate the distribution of serotonin (5-HT) receptors of type 6 (5-HT(6)) in postmortem human prefrontal cortex, striatum and hippocampus. The brain samples were obtained from 6 subjects who had died for causes not involving primarily or secondarily the CNS. The 5-HT(6) receptor distribution was explored by the [(125)I]SB-258585 binding to brain membranes followed by the pharmacological characterization, where possible, and by autoradiographic, immunohistochemical and immunofluorescence evaluations. A specific and saturable [(125)I]SB-258585 binding was detected in striatum only, with a pharmacological characterization consistent with that of a 5-HT(6) receptor. The autoradiography showed the presence of a specific [(125)I]SB-258585 binding distributed homogeneously in caudate, putamen and accumbens. The immunohistochemistry, carried out in the striatum only, coupled with the immunofluorescence with glial fibrillary acidic protein (GFAP) and parvalbumin (PV) showed the co-localization of 5-HT(6) receptor with PV, while indicating that this receptor subtype was expressed in neurons and not in astrocytes. Taken together, the present findings showed the presence of a higher density of 5-HT(6) receptors, as labeled by [(125)I]SB-258585, in striatum than in hippocampus and prefrontal cortex, and specifically within the neuronal body. In addition, they would suggest that striatum is one of the major potential CNS targets linked to 5-HT(6) receptor modulation.