Granulomatous inflammatory response to recombinant filarial proteins of Brugia species.
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The lymphatic inflammatory response in Brugia-infected jirds peaks early during primary infections and then decreases in severity as judged by the numbers of lymph thrombi present within these vessels. Antigen-specific hypersensitivity reactions in these animals was measured by a pulmonary granulomatous inflammatory response (PGRN) induced by somatic adult worm antigen (SAWA)-coated beads, and by cellular proliferative responses of renal lymph node cells. The kinetics of these responses temporally correspond to lymphatic lesion formation. The importance of any single antigen to the induction of this inflammatory response has not been elucidated. In this study, the PGRN was used to measure the cellular immune response to four recombinant filarial proteins during the course of a primary B. pahangi infection. These proteins were BpL4, glycoprotein (glutathione peroxidase) gp29, heat shock protein (hsp) 70, and filarial chitinase. All were fusion proteins of maltose-binding protein (MBP). Control beads included those coated with diethanolamine (DEA), SAWA, or MBP. The measurements of PRGN were made at 14, 28, 56, and > 150 days postinfection (PI) in infected jirds, in jirds sensitized with SAWA, and in uninfected jirds. The secretory homolog of glutathione peroxidase gp29 was the only recombinant protein tested that induced a significantly greater PGRN (P < 0.05) than controls. This was seen at 28 days PI. These observations indicate that gp29 may be part of the worm antigen complex that induces an early inflammatory response, a response similar to that observed with SAWA. These studies indicate that this approach is useful in investigating the functional ability of specific proteins in the induction and down-regulation of immune-mediated inflammatory responses elicited by filarial parasites. Absence of a granulomatous response to the other recombinant proteins used may be related to the nature and sensitivity of the assay used or the character of recombinant proteins tested. (+info)
Interleukin-10 and antigen-presenting cells actively suppress Th1 cells in BALB/c mice infected with the filarial parasite Brugia pahangi.
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Infection with the third-stage larvae (L3) of the filarial nematode Brugia results in a Th2-biased immune response in mice and humans. Previously we have shown that the production of interleukin 4 (IL-4) is critical for down-regulating polyclonal Th1 responses in L3-infected mice. However, the in vitro neutralization of IL-4 did not fully recover the defective polyclonal Th1 responses, nor did it result in the production of any antigen (Ag)-specific Th1 cytokines, suggesting that perhaps infection with L3 does not result in priming of Th1 cells in vivo. In this study, we analyzed the role of IL-10 and Ag-presenting cells (APCs) in the spleen as additional factors controlling the Th2 bias in infected mice. Our data show that IL-10 and APCs also contribute to the suppression of mitogen-driven Th1 responses of spleen cells from infected mice. In addition, the neutralization of IL-10 or the replacement of the resident APC population from spleen cell cultures resulted in the production of Ag-specific Th1 cytokines. Irradiated spleen cells from either L3-infected or uninfected mice were able to restore Ag-specific Th1 responses in vitro. Therefore, it appears that Brugia-reactive Th1 cells are primed following infection with L3, but are actively suppressed in vivo by a mechanism that involves IL-10 and the resident APC population, but not IL-4. These results indicate that a complex interplay of cytokines and cell populations underscores the Th2-polarized response in L3-infected mice. (+info)
Cloning and characterization of two nuclear receptors from the filarial nematode Brugia pahangi.
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Nuclear receptors (NRs) encompass a superfamily of cytoplasmic/nuclear localized receptors that on ligand binding (or by phosphorylation) directly regulate the transcription of target genes. NRs are involved in many developmental processes, including moulting in insects and dauer larva formation in Caenorhabditis elegans. Here we report the isolation of two genes related to NRs from the filarial nematode Brugia pahangi. Bp-nhr-1 is a member of the NGF1-B subfamily of NRs and is expressed at very low levels in post-infective larval stage 3 (L3) after their transmission to the mammalian host. The second gene, Bp-nhr-2, is related to XR78E/F of Drosophila, a gene involved in the ecdysone response, over the region of its DNA-binding domain. cDNA and genomic clones have been isolated that correspond to Bp-nhr-2. The most striking feature of the encoded protein is that, although there is a DNA-binding domain similar to that of other NRs, the ligand-binding domain is absent. To investigate the pattern of transcription of Bp-nhr-2 in the filarial life cycle, semi-quantitative reverse-transcriptase-mediated PCR was performed; this analysis demonstrated that the gene is expressed in early stages after infection and in the adult and microfilariae, and is up-regulated just before the moult between L3 and L4 but is not expressed during the moult between L4 and adult. Antibodies raised against a peptide corresponding to the transactivation domain of Bp-nhr-2 demonstrate that the protein is expressed in microfilariae and adult samples and that another cross-reactive protein is expressed in these life-cycle stages. (+info)
B1 B lymphocytes play a critical role in host protection against lymphatic filarial parasites.
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Host defense against multicellular, extracellular pathogens such as nematode parasites is believed to be mediated largely, if not exclusively, by T lymphocytes. During our investigations into the course of Brugia malayi and Brugia pahangi infections in immunodeficient mouse models, we found that mice lacking B lymphocytes were permissive for Brugian infections, whereas immunocompetent mice were uniformly resistant. Mice bearing the Btk(xid) mutation were as permissive as those lacking all B cells, suggesting that the B1 subset may be responsible for host protection. Reconstitution of immunodeficient recombination activating gene (Rag)-1(-/)- mice with B1 B cells conferred resistance, even in the absence of conventional B2 lymphocytes and most T cells. These results suggest that B1 B cells are necessary to mediate host resistance to Brugian infection. Our data are consistent with a model wherein early resistance to B. malayi is mediated by humoral immune response, with a significant attrition of the incoming infectious larval load. Sterile clearance of the remaining parasite burden appears to require cell-mediated immunity. These data raise the possibility that the identification of molecule(s) recognized by humoral immune mechanisms might help generate prophylactic vaccines. (+info)
Pharmacokinetics of UMF-078, a candidate antifilarial drug, in infected dogs.
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The pharmacokinetics of the filaricidal benzimidazole compounds UMF-078 and UMF-289 were evaluated in beagle dogs experimentally infected with Brugia pahangi. Twenty-four infected microfilaremic beagles were selected and randomly allocated into 4 treatment groups of 6 dogs each: oral (PO) UMF-078, PO UMF-289 (the HCl salt form of UMF-078), intramuscular (IM) UMF-078, and untreated controls. Equivalent doses of 50 mg/kg of the free base were given twice a day for 3 days to the 3 groups of treated dogs. Oral absorption is rapid compared with IM dosing; the absorption half-life (K01-HL) for the IM treatment is approximately 14 hr compared with 1 and 2 hr for the PO regimen of salt and free base forms, respectively. The elimination half-lives (K10-HL) for the PO regimens are 13 and 15 hr for the salt and free base forms, respectively. Because of sustained absorption following IM dosing, the K10-HL is prolonged. In contrast to oral administration, IM dosing of UMF-078 provides sustained, relatively low plasma drug levels, with good tolerance and efficacy. (+info)
Heat shock and developmental expression of hsp83 in the filarial nematode Brugia pahangi.
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hsp83 was cloned from the filarial nematode Brugia pahangi. The mRNA was constitutively expressed at 37 degrees C in life cycle stages that live in the mammalian host (microfilariae and adult worms). Heat shock resulted in only a minimal increase in levels of transcription. A genomic copy of hsp83 was isolated and was shown to contain 11 introns while sequencing of the 5' upstream region revealed several heat shock elements. Using a chloramphenicol acetyltransferase (CAT) reporter gene construct the expression of hsp83 from B. pahangi (Bp-hsp83) was studied by transfection of COS-7 cells. Similar to the expression pattern in the parasite, CAT activity was detected at 37 degrees C and was not influenced by heat shock. When the free-living nematode Caenorhabditis elegans was transfected with the same construct, CAT activity was not observed at normal growth temperatures (21 degrees C) or under moderate heat shock conditions (28 degrees C). However exposure to more severe heat shock (35 degrees C) resulted in an increase in CAT activity. These results suggest that Bp-hsp83 has a temperature threshold > or = 35 degrees C for expression. (+info)
NK T cells are a source of early interleukin-4 following infection with third-stage larvae of the filarial nematode Brugia pahangi.
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Infection of C57BL/6 mice with the third-stage larvae of Brugia pahangi results in a rapid expansion of NK1.1(+) T cells in the spleen and draining lymph nodes. NK T cells produced interleukin-4 in the spleen within 24 h of infection, and these cells were CD4(-). (+info)
Cloning and expression analysis of two mucin-like genes encoding microfilarial sheath surface proteins of the parasitic nematodes Brugia and Litomosoides.
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In several filarial genera the first stage larvae (microfilariae) are enclosed by an eggshell-derived sheath that provides a major interface between the parasite and the host immune system. Analysis of the polypeptide constituents of the microfilarial sheath from the cotton rat filaria Litomosoides sigmodontis identified two abundant surface glycoproteins: Shp3a and Shp3. The corresponding genes and the orthologues of the human parasite Brugia malayi and the rodent filaria Brugia pahangi were cloned and sequenced. They encode secreted, mucin-like proteins with N-terminal Ser/Thr-rich repeats and a C-terminal anchor domain rich in aromatic amino acids. About 75% of the protein molecular masses result from post-translational modifications. The Ser/Thr-rich motifs are supposed to serve as targets for dimethylaminoethanol-phosphate substitutions. These modifications were detected only on the sheaths of the late developmental stage of stretched microfilariae, corresponding with the expression of the proteins in the epithelium of the distal part of the uterus and the specific transcription of shp3 and shp3a in the anterior female worm segment. Genomic analysis of all three species demonstrated a conserved linkage of the two genes. Their transcripts undergo cis- and trans-splicing. The transcription start sites of the primary transcripts were determined for the L. sigmodontis genes. The core promoter regions are remarkably conserved between the paralogue genes Ls-shp3a and Ls-shp3 and their orthologues in Brugia, implicating conserved regulatory elements. (+info)