Temperature is a cue for gene expression in the post-infective L3 of the parasitic nematode Brugia pahangi

The temporal expression pattern of two genes, Bp-cdd and Bp-S3, was studied at defined points throughout the life cycle of Brugia pahangi. Both mRNAs were up-regulated to coincide with the transition of the L3 from the vector to the mammalian host. Bp-cdd was expressed almost exclusively in the post-infective (p.i.) L3 and L4 stages of the life cycle while Bp-S3 was also expressed in adult worms, but at a much lower level than in the larval stages. Immunogold labelling with an antiserum raised to the recombinant Bp-CDD localised the native antigen to the hypodermis in the p.i. L3 and L4. Specific labelling was not detected in the adult worm. The expression of both mRNAs could be triggered by exposure of the vector-derived L3 to a simple mammalian culture system. Analysis of the factors, which induced expression suggested that the temperature shift which accompanies the transition from mosquito to mammal was the most important cue for expression of both genes.     

NK T cells are a source of early interleukin-4 following infection with third-stage larvae of the filarial nematode Brugia pahangi

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(-).     

An evaluation of different methods for labelling the surface of the filarial nematode Brugia pahangi with 125iodine

The specificity of a range of 125I labelling techniques (Chloramine T, Iodogen, Bolton and Hunter reagent, lactoperoxidase and iodosulfanilic acid) to the surface of the filarial nematode Brugia pahangi was evaluated by autoradiography of sections of labelled worms and of dried SDS-polyacrylamide gels following electrophoresis of homogenised worm extracts. It was concluded that Bolton and Hunter reagent was not surface specific but labelled proteins throughout the body of the worm. At the light microscope level autoradiography of worms labelled using Chloramine T, Iodogen, lactoperoxidase and iodosulfanilic acid demonstrated that the 125I labelling was restricted to the worm surface. Electrophoresis and autoradiography showed that each method produced a different pattern of labelled polypeptide. A polypeptide of molecular weight 30 kDa was labelled using each method except Bolton and Hunter reagent, and appears to be a major surface component.     

Proteins secreted by the parasitic nematode Nippostrongylus brasiliensis act as adjuvants for Th2 responses

Infections with parasitic helminths such as Nippostronglyus brasiliensis induce dominant type 2 responses from antigen-specific T helper cells. The potency of the Th2 bias can also drive Th2 responses to bystander antigens introduced at the same time as infection. We now report that the Th2-promoting effect of infection can be reproduced with soluble N. brasiliensis excretory-secretory proteins (NES) released by adult parasites in vitro. Immunization of BALB/c mice with NES results in the production of IL-4 with elevated total serum IgE and specific IgG1 antibodies. NES is also able to stimulate IL-4 and polyclonal IgE production in other mouse strains (C57BL/6, B10.D2, CBA). These features are seen whether NES is administered without adjuvant as soluble protein in phosphate-buffered saline or with complete Freund's adjuvant which normally favors Th1 responses. Thus, NES possesses intrinsic adjuvanticity. Moreover, co-administration of hen egg lysozyme (HEL) with NES in the absence of other adjuvants results in generation of HEL-specific lymphocyte proliferation, IL-4 release and IgG1 antibody responses, documenting that NES can act as an adjuvant for third-party antigens. Proteinase K digestion or heat treatment of NES before immunization abolished the IL-4-stimulating activity, indicating that the factors acting to promote Th2 induction are proteins secreted by the adult parasite.     

Functional and phenotypic characteristics of alternative activation induced in human monocytes by interleukin-4 or the parasitic nematode Brugia malayi

Human monocytes from patients with patent filarial infections are studded with filarial antigen and express markers associated with alternative activation of macrophages (MΦ). To explore the role of filaria-derived parasite antigen in differentiation of human monocytes, cells were exposed to microfilariae (mf) of Brugia malayi, and their phenotypic and functional characteristics were compared with those of monocytes exposed to factors known to generate either alternatively (interleukin-4 [IL-4]) or classically (macrophage colony-stimulating factor [MCSF]) activated MΦ. IL-4 upregulated mRNA expression of CCL13, CCL15, CCL17, CCL18, CCL22, CLEC10A, MRC1, CADH1, CD274, and CD273 associated with alternative activation of MΦ but not arginase 1. IL-4-cultured monocytes had a diminished ability to promote proliferation of both CD4(+) and CD8(+) T cells compared to that of unexposed monocytes. Similar to results with IL-4, exposure of monocytes to live mf induced upregulation of CCL15, CCL17, CCL18, CCL22, CD274, and CD273 and downregulation of Toll-like receptor 3 (TLR3), TLR5, and TLR7. In contrast to results with MCSF-cultured monocytes, exposure of monocytes to mf resulted in significant inhibition of the phagocytic ability of these cells to the same degree as that seen with IL-4. Our data suggest that short exposure of human monocytes to IL-4 induces a phenotypic characteristic of alternative activation and that secreted filarial products skew monocytes similarly.     

Induction of murine T-helper-cell responses to the filarial nematode Brugia malayi.

The purpose of this study was to examine the murine T-helper-cell (Th) cytokine response to the human filarial parasite Brugia malayi. In the first 14 days following intraperitoneal inoculation of live microfilariae into BALB/c mice, filarial antigen-driven splenic lymphoid cells produced gamma interferon (IFN-gamma) and little or no interleukin-5 (IL-5). After this time, IL-5 production increased (to 10 to 12 ng per 5 x 10(6) cells) coincident with a marked diminution in IFN-gamma generation. A single subcutaneous immunization with soluble microfilarial antigens also induced an IFN-gamma but no IL-5 response, whereas immunization three times elicited a predominant Th2-like reaction characterized by IL-4 and IL-5 production by CD4+ lymph node lymphocytes and a 10-fold increase in serum immunoglobulin E. The importance of IL-10 in establishing the balance between parasite-specific Th1 and Th2 responses was demonstrated by the ability of neutralizing monoclonal antibody to this cytokine to increase IFN-gamma production by splenic and lymph node cells from mice chronically exposed to live microfilariae or immunized multiple times with soluble filarial antigens.     

Identification, synthesis and immunogenicity of cuticular collagens from the filarial nematodes Brugia malayi and Brugia pahangi

The major structural proteins of the cuticle of the filarial nematode parasites Brugia malayi and Brugia pahangi were identified by extrinsic iodination and sensitivity to clostridial collagenase. At least 16 acidic components were identified in adult worms by 2-dimensional electrophoresis, with molecular weights ranging from 35,000 to 160,000. These proteins appear to be cross-linked by disulphide bonds, and localised in the basal and inner cortical layers of the cuticle. The outer cortex, containing the epicuticle, is insoluble in 1% sodium dodecyl sulphate and 5% 2-mercaptoethanol, and can be isolated free of cellular material. Despite their inaccessibility to the immune system in intact worms, antibodies to the cuticular collagens are provoked in humans infected with a variety of filarial parasites. Immunological cross-reactivity was demonstrated between a 35 kDa component and human type IV (basement membrane) collagen. Autoantibodies to type IV collagen were detected in a number of individuals with lymphatic filariasis, although no correlation could be drawn with observed pathology. Synthesis of cuticular collagens is discontinuous, occurs at negligible levels in mature adult male worms, and does not appear to involve the production of small molecular weight precursors, in contrast to Caenorhabditis elegans. Hybridisation with a heterologous cDNA probe coding for the alpha 2 chain of chicken type 1 collagen suggests that they are encoded by a multigene family.     

How to deal with polarized Th2 cells: exploring the Achilles' heel.

The central effector cells in the pathogenesis of atopic allergic diseases are type 2 T helper (Th2) cells, which display an aberrant cytokine profile dominated by type 2 cytokines. Initial reports from mouse studies indicated that established and committed Th2 cells are stable and unsusceptible to modulation. However, there is a growing awareness that in humans, established effector Th2 cells are more flexible and can be reverted to predominant Th1 phenotypes. In fact, the Th1-driving cytokine interleukin (IL)-12 is the crucial factor in this respect. IL-12 is mainly produced by dendritic cells (DC), which can be primed for high or low IL-12 production, depending on inflammatory and/or microbial signals they encounter during their residence in the peripheral tissues. Accordingly, both the regulation of and the priming for IL-12 production in DC form ideal targets for therapeutic intervention. The development of new therapies for atopic allergy now focuses on local IL-12-promoting substances to target both the development of new Th2 cells and the persistent population of established allergen-specific Th2 cells.     

Expression of small heat shock proteins by the third-stage larva of Brugia pahangi.

Changes in proteins synthesised by the infective third-stage larvae (L3) of the filarial nematode Brugia pahangi were examined with respect to the temperature shift encountered by the parasite as it migrates from insect to mammal, and the presence of serum in the culture medium. While the synthesis of a number of polypeptides is regulated by the temperature shift of the L3 from 28 degrees C to 37 degrees C in vitro, there is no evidence that serum has any significant effect on protein synthesis. Two complexes of small acidic polypeptides (22-24 kDa and 18 kDa) are synthesised for a limited period only by L3 transferred to 37 degrees C. One component of each complex appears to be constitutively expressed at 28 degrees C, but its synthesis is up-regulated at 37 degrees C, while the remaining members of each complex are synthesised only at 37 degrees C. Subjection of L3 and post-infective (p.i.) L3 to heat shock (41 degrees C) also induces synthesis of both complexes, indicating that these heat-inducible polypeptides are related to the family of small heat shock proteins. The possible role of the heat shock-related proteins in this important environmental transition is considered.     

Surface antigens of a filarial nematode: analysis of adult Brugia pahangi surface components and their use in monoclonal antibody production

The surface antigens of adult worms of the filarial nematode Brugia pahangi have been investigated further by surface radioiodination and detergent solubilisation techniques. In addition to yielding new information on the distribution of antigenic components of this stage, detergent-solubilised molecules were used in both radiometric and enzyme-linked assays for human and mouse antibody. These assays were subsequently used in screening for monoclonal antibodies from hybrid cells derived from animals infected with living parasites and boosted with detergent-extracted antigen. Three monoclonal antibody-producing cell lines were isolated, with differing antigenic specificities: Bp-1, which binds a non-iodinatable antigen with high ELISA activity; Bp-2, which reacts with a determinant found on but not unique to the major surface Iodogen-labelled 29 kDa antigen; and Bp-3, which is specific for a minor antigen of 20 kDa revealed by Iodogen labelling.     

Kinetics of cellular responses to intraperitoneal Brugia pahangi infections in normal and immunodeficient mice

Filarial infections evoke exuberant inflammatory responses in the peritoneal cavities of immunocompetent mice. Clearance of infection appears to be dependent on complex interactions between B1 and B2 B lymphocytes, T cells, eosinophils, macrophages, and the products of these cells. In an earlier communication, we described the course of infection in normal immunocompetent mice. In this study, we utilize mice with well-characterized mutations that disable one or more effector components of adaptive immunity in order to determine their roles in host protection. We characterize peritoneal exudate cells by flow cytometry and determine the kinetics of accumulation of each of the different cell types following infection with Brugia pahangi. We find that (i) four-color flow-cytometric analysis of peritoneal exudate cells using anti-CD3, -CD11b, -CD19, and -Gr1 can distinguish up to six different populations of cells; (ii) an initial influx of neutrophils occurs within 24 h of infection, independent of the adaptive immune status of mice, and these cells disappear by day 3; (iii) an early influx of eosinophils is seen at the site of infection in all strains studied, but a larger, second wave occurs only in mice with T cells; (iv) the presence of T cells and eosinophils is important in causing an increase in macrophage size during the course of infection; and (v) most unexpectedly, T-cell recruitment appears to be optimal only if B cells are present, since JHD mice recruit significantly fewer T cells to the site of infection.     

Interleukin-10 and antigen-presenting cells actively suppress Th1 cells in BALB/c mice infected with the filarial parasite Brugia pahangi

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.     

Adjuvant-dependent modulation of Th1 and Th2 responses to immunization with beta-amyloid

The role of adjuvant on the T(h)1 and T(h)2 immune responses to Abeta-immunotherapy (Abeta(42 )peptide) was examined in wild-type mice. Fine epitope analysis with overlapping oligomers of the Abeta(42) sequence identified the 1-15 region as a dominant B cell epitope. The 6-20 peptide was recognized only weakly by antisera from mice administrated with Abeta(42) peptide formulated in complete Freund's adjuvant (CFA), alum or TiterMax Gold (TMG). However, mice immunized with Abeta(42) mixed with QS21 induced a significant antibody response to the 6-20 peptide. The only T cell epitope found was within the 6-28 sequence of Abeta(42). QS21 and CFA induced the strongest humoral response to Abeta, alum was intermediate, and TMG the weakest adjuvant. Analysis of antibody isotypes specific for Abeta indicates that alum induces primarily T(h)2-type immune response, whereas TMG, CFA and QS21 shift the immune responses toward a T(h)1 phenotype. Stimulation of splenocytes from Abeta-immunized mice with Abeta(40) peptide induced strikingly different cytokine expression profiles. QS21 and CFA induced significant IFN-gamma, IL-4 and tumor necrosis factor-alpha expression, whereas alum induced primarily IL-4 production. As T(h)1-type immune responses have been implicated in many autoimmune disorders, whereas T(h)2-type responses have been shown to inhibit autoimmune disease, the choice of adjuvant may be critical for the design of a safe and effective immunotherapy for Alzheimer's disease.     

Effect of cyclophosphamide on the immune responsiveness of jirds infected with Brugia pahangi

The in vitro immune responsiveness of lymphocytes from Brugia pahangi-infected jirds was examined after serial administration of cyclophosphamide (20 mg/kg). Cyclophosphamide had no effect on parasite burdens, anti-B. pahangi antibody titers, or suppressed spleen cell reactivity to B. pahangi antigens. Cyclophosphamide restored cellular responsiveness to the mitogens phytohemagglutinin, concanavalin A, and pokeweed mitogen.     

The biochemical and immunochemical characterisation of the 30 kilodalton surface antigen of Brugia pahangi

The major surface antigen (30 kDa) of Brugia pahangi has been characterised by a number of biochemical and immunochemical means. The 30 kDa polypeptide is a glycoprotein which can be extracted from the worm surface by homogenization in the absence of detergents. The 30 kDa polypeptide can be metabolically labelled with [35S]methionine in adult male and female parasites. In addition small amounts of the 35S-labelled 30 kDa antigen can be detected in the medium of worms cultured in vitro. 125I labelling of the excretory-secretory (ES) products of adult male and female parasites followed by immunoprecipitation and peptide mapping has confirmed the relationship between the surface located 30 kDa polypeptide and that released in vitro.     

Three beta-tubulin cDNAs from the parasitic nematode Haemonchus contortus.

Experimental evidence indicates that tubulin is the site of action of the anthelmintic benzimidazoles. Furthermore, certain residues of β-tubulin seem to be critical for this mechanism. Although the benzimidazoles selectively affect nematode vs. mammalian β-tubulin, the molecular basis for this differential action is not known. To enhance our understanding of this phenomenon, and to provide the basis for investigating benzimidazole resistance in parasitic nematodes, we undertook the cloning of β-tubulin cDNAs from the ruminant parasite, Haemonchus contortus. We have cloned and sequenced three β-tubulin cDNAs from this organism, β12–16, β12–164, and β8–9. The first 2 differ at only 23 nucleotides, which give rise to 4 amino acid changes, β8–9 represents a different isotype class from the other two, since it differs extensively in the carboxyterminus. By comparing the sequences of these and other nematode β-tubulins with mammalian β-tubulins, several regions of consistent difference can be recognized; the functional significance of these regional differences has not been defined. Sequences very similar or identical to β8–9 and β12–16 are present in both benzimidazole-sensitive and benzimidazole-resistant populations of H. contortus. However, it appears that drug-resistant organisms may differ in the presence of a gene product which is closely related to β8–9.     

Activation of jird (Meriones unguiculatus) macrophages by the filarial parasite Brugia pahangi

Peritoneal macrophages from Mongolian jirds (Meriones unguiculatus) with either lymphatic or intraperitoneal infections of Brugia pahangi were studied to determine the effects of infection on macrophage function and morphology. Macrophages were collected at 40, 90, 140, and 200 days after inoculation of infective third-stage larvae and assayed for phagocytic and bactericidal activity by the acridine orange method and for morphological changes by light and electron microscopy. Significant increases in phagocytic and microbicidal activity (P less than or equal to 0.01) were observed in peritoneal macrophages collected from jirds with intraperitoneal infections when compared with peritoneal macrophages from jirds with lymphatic infections and resident peritoneal macrophages from normal, noninfected jirds. Morphological changes in peritoneal macrophages from jirds with intraperitoneal infections were similar to those found in thioglycolate-elicited macrophage populations. Granuloma formation was also observed in the peritoneal cavities of intraperitoneally infected jirds. The peritoneal cavity may serve as a model to study cell-worm interactions in filarial nematode infections.     

Th1 and Th2 cell involvement in immune response to Salmonella typhimurium porins.

In understanding the regulation of the specific immune response to Salmonella typhimurium, the role of a surface major component (porins) was studied. In this study we demonstrate that purified porins are able to induce a different response to that induced by the porins present on the S. typhimurium cell surface. Porin-treated or orally infected mice show anti-porin antibodies with bactericidal activity. The complete adoptive transfer of resistance to S. typhimurium is achieved only using splenic T cells from survivor mice after experimental infection. After stimulation with specific antigen in vitro CD4+ cells from porin-immunized mice released large amounts of interleukin-4 (IL-4), at a time when CD4+ cells from S. typhimurium-infected mice predominantly secreted interferon-gamma (IFN-gamma). Limiting dilution analysis showed that infection resulted in a higher precursor frequency of IFN-gamma-producing CD4+ T cells and a lower precursor frequency of IL-4-producing CD4+ T cells, while immunization with porins resulted in a higher precursor frequency of IL-4-producing cells and a low frequency of IFN-gamma-producing cells. Analysis of polymerase chain reaction-amplified cDNA from the spleens of infected mice revealed that IFN-gamma, IL-2 and IL-12 p40 mRNA were found 5 days after in vitro challenge and increased after 15 days; IL-10 expression was barely present after both 5 and 15 days, while IL-4 mRNA expression was not detected. In immunized mice, the IL-4 mRNA expression increased after 15 days, IFN-gamma mRNA expression disappeared entirely after 15 days, while IL-2, IL-10 and IL-12 mRNA remained relatively unchanged.