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1.
Vet Parasitol ; 194(2-4): 101-5, 2013 May 20.
Article in English | MEDLINE | ID: mdl-23465441

ABSTRACT

The aim of this study was to evaluate differences between the small and large intestines (SI and LI) with regard to colonization and immunity during infection with Trichinella spiralis. In orally infected C57BL/6 mice, the gender ratios of worms differed among the SI, cecum, and LI. Mucosal mastocytosis developed in the SI but not in the LI, consistent with reduced IL-9 and IL-13 production by explants from the LI. Despite these differences, worms were cleared at the same rate from both sites. Furthermore, IL-10 production was reduced in the LI, yet it was instrumental in limiting local inflammation. Finally, passive immunization of rat pups with tyvelose-specific antibodies effectively cleared fist-stage larvae from all intestinal regions. We conclude that despite regional differences in immune responsiveness and colonization, immune mechanisms that clear T. spiralis operate effectively throughout the intestinal tract.


Subject(s)
Cytokines/immunology , Intestine, Large/parasitology , Intestine, Small/parasitology , Trichinella spiralis/immunology , Trichinellosis/immunology , Animals , Animals, Newborn , Antibodies, Helminth/immunology , Cytokines/metabolism , Female , Immunization, Passive , Interleukin-10/metabolism , Intestine, Large/immunology , Intestine, Small/immunology , Larva , Male , Mast Cells/immunology , Mastocytosis/immunology , Mastocytosis/parasitology , Mice , Mice, Inbred BALB C , Mice, Inbred C57BL , Rats , Specific Pathogen-Free Organisms , Trichinellosis/parasitology
2.
Parasite Immunol ; 35(1): 21-31, 2013 Jan.
Article in English | MEDLINE | ID: mdl-23094823

ABSTRACT

Intestinal infection with the parasitic nematode, Trichinella spiralis, provides a robust context for the study of mucosal mast cell function. In rats, mucosal mast cells are exposed to parasites during the earliest stage of infection, affording an opportunity for mast cells to contribute to an innate response to infection. During secondary infection, degranulation of rat mucosal mast cells coincides with expulsion of challenge larvae from the intestine. The goal of this study was to evaluate the rat bone marrow-derived mast cells (BMMC) and the rat basophilic leukaemia cell line (RBL-2H3) as models for mucosal mast cells, using parasite glycoproteins and antibody reagents that have been tested extensively in rats in vivo. We found that BMMC displayed a more robust mucosal phenotype. Although T. spiralis glycoproteins bound to mast cell surfaces in the absence of antibodies, they did not stimulate degranulation, nor did they inhibit degranulation triggered by immune complexes. Parasite glycoproteins complexed with specific monoclonal IgGs provoked release of rat mast cell protease II (RMCPII) and ß-hexosaminidase from both cell types in a manner that replicated results observed previously in passively immunized rats. Our results document that RBL-2H3 cells and BMMC model rat mucosal mast cells in the contexts of innate and adaptive responses to T. spiralis.


Subject(s)
Immunity, Mucosal , Mast Cells/immunology , Trichinella spiralis/immunology , Trichinellosis/immunology , Animals , Antigen-Antibody Complex , Bone Marrow Cells/immunology , Cell Degranulation , Cell Line , Cells, Cultured , Glycoproteins/metabolism , Helminth Proteins/metabolism , Larva/immunology , Mast Cells/cytology , Rats , Rats, Inbred Lew , Trichinella spiralis/growth & development
3.
Clin Vaccine Immunol ; 19(7): 1019-26, 2012 Jul.
Article in English | MEDLINE | ID: mdl-22593238

ABSTRACT

The parasitic nematode Parelaphostrongylus tenuis is an important cause of neurologic disease of camelids in central and eastern North America. The aim of this study was to determine whether alpacas develop resistance to disease caused by P. tenuis in response to a previous infection or a combination of controlled infection and immunization. Alpacas were immunized with a homogenate of third-stage larvae (L3) and simultaneously implanted subcutaneously with diffusion chambers containing 20 live L3. Sham-treated animals received adjuvant alone and empty chambers. The protocol was not effective in inducing resistance to oral challenge with 10 L3, and disease developed between 60 and 71 days following infection. Immediately following the onset of neurologic disease, affected animals were treated with a regimen of anthelmintic and anti-inflammatory drugs, and all recovered. One year later, a subset of alpacas from this experiment was challenged with 20 L3 and the results showed that prior infection induced resistance to disease. Primary and secondary infections induced production of conventional and heavy-chain IgGs that reacted with soluble antigens in L3 homogenates but did not consistently recognize a recombinant form of a parasite-derived aspartyl protease inhibitor. Thus, the latter antigen may not be a good candidate for serology-based diagnostic tests. Antibody responses to parasite antigens occurred in the absence of overt disease, demonstrating that P. tenuis infection can be subclinical in a host that has been considered to be highly susceptible to disease. The potential for immunoprophylaxis to be effective in preventing disease caused by P. tenuis was supported by evidence of resistance to reinfection.


Subject(s)
Antibodies, Helminth/blood , Metastrongyloidea/immunology , Strongylida Infections/immunology , Adjuvants, Immunologic/administration & dosage , Animals , Anthelmintics/administration & dosage , Anti-Inflammatory Agents/administration & dosage , Antigens, Helminth/administration & dosage , Antigens, Helminth/immunology , Camelids, New World , Male , Strongylida Infections/drug therapy , Strongylida Infections/prevention & control , Treatment Outcome , Vaccination/methods
4.
Clin Vaccine Immunol ; 17(12): 2007-15, 2010 Dec.
Article in English | MEDLINE | ID: mdl-20926693

ABSTRACT

In addition to conventional immunoglobulins, camelids produce antibodies that do not incorporate light chains into their structures. These so-called heavy-chain (HC) antibodies have incited great interest in the biomedical community, as they have considerable potential for biotechnological and therapeutic application. Recently, we have begun to elucidate the immunological functions of HC antibodies, yet little is known about their significance in maternal immunity or about the B lymphocytes that produce them. This study describes the application of isotype-specific reagents toward physiological assessments of camelid IgGs and the B cells that produce them. We document the specificities of monoclonal antibodies that distinguish two conventional IgG1 isotypes and two HC IgG3 variants produced by alpacas. Next, we report that the relative concentrations of five isotypes are similar in serum, milk, and colostrum; however, following passive transfer, the concentrations of HC IgG2 and IgG3 declined more rapidly than the concentration of conventional IgG1 in the sera of neonates. Finally, we assessed the distribution of B cells of distinct isotypes within lymphoid tissues during fetal and adult life. We detected IgG1, IgG2, and IgG3 in lymphocytes located in lymph node follicles, suggesting that HC B cells affinity mature and/or class switch. One IgG3 isotype was present in B cells located in ileal Peyer's patches, and one conventional IgG1 isotype was detected in splenic marginal zone B cells. Our findings contribute to the growing body of knowledge pertaining to HC antibodies and are compatible with functional specialization among conventional and HC IgGs in the alpaca.


Subject(s)
Camelids, New World/immunology , Immunoglobulin G/immunology , Immunoglobulin Heavy Chains/immunology , Animals , Animals, Newborn , B-Lymphocytes/immunology , Colostrum/immunology , Female , Male , Milk/immunology , Serum/immunology
5.
Clin Vaccine Immunol ; 17(2): 239-46, 2010 Feb.
Article in English | MEDLINE | ID: mdl-19955323

ABSTRACT

Three classes of IgG have been described for camelids. IgG1 has a conventional four-chain structure, while IgG2 and IgG3 do not incorporate light chains. The structures and antigen-binding affinities of the so-called heavy-chain classes have been studied in detail; however, their regulation and effector functions are largely undefined. The aim of this study was to examine the participation of conventional and heavy-chain IgG antibodies in the camelid immune defense directed against West Nile virus (WNV). We found that natural infection or vaccination with killed WNV induced IgG1 and IgG3. Vaccination also induced IgG1 and IgG3; IgG2 was produced during the anamnestic response to vaccination. When purified IgGs were tested in plaque-reduction neutralization titer (PRNT) tests, IgG3 demonstrated PRNT activities comparable to those of conventional IgG1. In contrast, IgG2 demonstrated only suboptimal activity at the highest concentrations tested. Flow cytometric analysis revealed that macrophages bound IgG1, IgG2, and IgG3. Furthermore, subneutralizing concentrations of all three isotypes enhanced WNV infection of cultured macrophages. Our results document distinctions in regulation and function between camelid heavy-chain isotypes. The reduced size and distinct structure of IgG3 did not negatively impact its capacity to neutralize virus. In contrast, IgG2 appeared to be less efficient in neutralization. This information advances our understanding of these unusual antibodies in ways that can be applied in the development of effective vaccines for camelids.


Subject(s)
Antibodies, Viral/immunology , Camelids, New World/immunology , Immunoglobulin G/immunology , West Nile Fever/immunology , West Nile Virus Vaccines/immunology , West Nile virus/immunology , Animals , Antibodies, Neutralizing/immunology , Antibody-Dependent Enhancement , Female , Macrophages/immunology , Neutralization Tests , New Jersey , Viral Plaque Assay
6.
Vet Parasitol ; 159(3-4): 245-8, 2009 Feb 23.
Article in English | MEDLINE | ID: mdl-19070961

ABSTRACT

Trichinella spiralis larvae establish chronic infections in skeletal muscles of immunocompetent hosts. Muscle infection is crucial to transmission and survival of the parasite in nature. Chronic infections by this highly immunogenic parasite are associated with modulation or escape from potentially destructive immune responses. This review summarizes our current knowledge of immunity to muscle infection with T. spiralis.


Subject(s)
Muscle, Skeletal/immunology , Muscle, Skeletal/parasitology , Trichinella spiralis/immunology , Trichinellosis/immunology , Animals , Mice
7.
Clin Diagn Lab Immunol ; 12(3): 380-6, 2005 Mar.
Article in English | MEDLINE | ID: mdl-15753251

ABSTRACT

Of the three immunoglobulin G (IgG) isotypes described to occur in camelids, IgG2 and IgG3 are distinct in that they do not incorporate light chains. These heavy-chain antibodies (HCAbs) constitute approximately 50% of the IgG in llama serum and as much as 75% of the IgG in camel serum. We have produced isotype-specific mouse monoclonal antibodies (MAbs) in order to investigate the roles of HCAbs in camelid immunity. Seventeen stable hybridomas were cloned, and three MAbs that were specific for epitopes on the gamma chains of llama IgG1, IgG2, or IgG3 were characterized in detail. Affinity chromatography revealed that each MAb bound its isotype in solution in llama serum. The antibodies bound to the corresponding alpaca IgGs, to guanaco IgG1 and IgG2, and to camel IgG1. Interestingly, anti-IgG2 MAbs bound three heavy-chain species in llama serum, confirming the presence of three IgG2 subisotypes. Two IgG2 subisotypes were detected in alpaca and guanaco sera. The MAbs detected llama serum IgGs when they were bound to antigen in enzyme-linked immunosorbent assays and were used to discern among isotypes induced during infection with a parasitic nematode. Diseased animals, infected with Parelaphostrongylus tenuis, did not produce antigen-specific HCAbs; rather, they produced the conventional isotype, IgG1, exclusively. Our data document the utility of these MAbs in functional and physiologic investigations of the immune systems of New World camelids.


Subject(s)
Antibodies, Monoclonal , Camelids, New World/immunology , Immunoglobulin Heavy Chains/immunology , Animals , Antibodies, Monoclonal/biosynthesis , Chromatography, Affinity , Enzyme-Linked Immunosorbent Assay , Epitopes , Immunoglobulin G/immunology , Immunoglobulin Isotypes/immunology , Mice , Mice, Inbred BALB C , Nematode Infections/immunology
8.
Infect Immun ; 70(4): 1853-9, 2002 Apr.
Article in English | MEDLINE | ID: mdl-11895947

ABSTRACT

Trichinella spiralis is an obligate parasite of animals that has an unusual intracellular life cycle. Investigation of parasitism at the cellular and molecular levels has been challenging because of a shortage of tools for in vitro cultivation of T. spiralis. We have found that T. spiralis larvae molt, ecdyse, develop to adulthood, and reproduce when they are inoculated onto cultured intestinal epithelial cells. Initially, larvae invade and migrate through cells in a monolayer (T. ManWarren, L. Gagliardo, J. Geyer, C. McVay, S. Pearce-Kelling, and J. Appleton, Infect. Immun. 65:4806-4812, 1997). During prolonged culture in Caco-2 epithelial cells, L1 larvae molted and ecdysed with efficiencies as high as 50%. Molting and ecdysis in vitro required entry of the parasite into cells; conditions that prevented entry into cells also prevented ecdysis. When larvae were inoculated at a low density and cultured for 5 to 9 days, as many as 50% of the larvae developed to adult stages. Low numbers of mature male worms with copulatory appendages were observed in these cultures. The majority of worms that survived for five or more days were unfertilized females. Low-density cultures supported development of female worms with embryos at rates of 4 to 5%. These results show that the intestinal life cycle of T. spiralis can be supported entirely by host epithelial cells. Our model should allow more detailed investigation of intracellular parasitism by T. spiralis.


Subject(s)
Intestinal Mucosa/parasitology , Trichinella/physiology , Animals , Caco-2 Cells , Dogs , Female , Humans , Molting , Reproduction , Transforming Growth Factor beta/pharmacology
9.
Vet Parasitol ; 101(3-4): 249-60, 2001 Nov 22.
Article in English | MEDLINE | ID: mdl-11707300

ABSTRACT

The nematode Trichinella spiralis demonstrates a simple but novel parasitic life-cycle, completing all of its development in intracellular habitats. This feature of the life-cycle has challenged investigators aiming to elucidate mechanisms of parasitism. Investigations of immunity showed a dominant influence of N-glycans in the responses to larval T. spiralis. It has become evident that novel glycans are positioned to play important roles in parasitism, as well as immunity, in infection with this nematode.


Subject(s)
Muscle, Skeletal/parasitology , Polysaccharides/physiology , Trichinella spiralis/immunology , Trichinellosis/veterinary , Animals , Antibodies, Helminth/biosynthesis , Histocytochemistry , Host-Parasite Interactions/physiology , Humans , Immunity, Cellular , Immunodominant Epitopes/immunology , Intestines/immunology , Intestines/parasitology , Larva , Life Cycle Stages , Muscle, Skeletal/chemistry , Polysaccharides/immunology , Trichinella spiralis/physiology , Trichinellosis/immunology , Trichinellosis/parasitology
10.
Parasite ; 8(2 Suppl): S48-50, 2001 Jun.
Article in English | MEDLINE | ID: mdl-11484381

ABSTRACT

It has been known for many years that Trichinella spiralis initiates infection by penetrating the columnar epithelium of the small intestine, however, the mechanisms used by the parasite in the establishment of its intramulticellular niche in the intestine are unknown. The recent demonstration that invasion also occurs in vitro when infective larvae of T. spiralis are inoculated onto cultures of epithelial cells provides a model that allows the direct observation of the process by which the parasite recognizes, invades and migrates within the epithelium. The finding that penetration of the cell membrane or induction of plasma membrane wounds by larvae do not always result in invasion argue in favor of some kind of host-parasite communication in successful invasion. In this sense, the in vitro model of invasion provides a readily manipulated and controlled system to investigate both parasite, and host cell requirements for invasion.


Subject(s)
Epithelial Cells/parasitology , Trichinella spiralis/physiology , Trichinella spiralis/pathogenicity , Animals , Cells, Cultured , Humans , Larva
11.
Glycobiology ; 10(9): 941-50, 2000 Sep.
Article in English | MEDLINE | ID: mdl-10988255

ABSTRACT

Although the presence of phosphorylcholine (PC) in Trichinella spiralis is well established, the precise structure of the PC-bearing molecules is not known. In this paper, we report structural studies of N-glycans released from T.spiralis affinity-purified antigens by peptide N-glycosidase F. Three classes of N-glycan structures were observed: high mannose type structures; those which had been fully trimmed to the trimannosyl core and were sub-stoichiometrically fucosylated; and those with a trimannosyl core, with and without core fucosylation, carrying between one and eight N-acetylhexosamine residues. Of the three classes of glycans, only the last was found to be substituted with detectable levels of phosphorylcholine.


Subject(s)
Antigens, Helminth/chemistry , Phosphorylcholine/analysis , Polysaccharides/chemistry , Trichinella spiralis/chemistry , Acetylation , Amidohydrolases/metabolism , Animals , Antigens, Helminth/metabolism , Carbohydrate Conformation , Carbohydrate Sequence , Gas Chromatography-Mass Spectrometry , Hydrofluoric Acid/metabolism , Methylation , Molecular Sequence Data , Monosaccharides/analysis , Oligosaccharides/chemistry , Oligosaccharides/classification , Oligosaccharides/metabolism , Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase , Polysaccharides/classification , Polysaccharides/metabolism , Spectrometry, Mass, Fast Atom Bombardment , Trypsin/metabolism
12.
Mol Biochem Parasitol ; 107(2): 207-18, 2000 Apr 15.
Article in English | MEDLINE | ID: mdl-10779597

ABSTRACT

Trichinella spiralis L1 larvae infect susceptible hosts by invading epithelial cells that line the small intestine. Invasion also occurs in vitro when larvae are inoculated into cultures of epithelial cells from several different animal species. To further investigate the mechanism of invasion, we studied the interaction of larvae with the rat epithelial cell line IEC-6. Larvae did not invade IEC-6 cells, but did cause the cells to take up parasite glycoproteins. Glycoprotein bearing cells remained viable and were detectable in monolayers for as long as 24 h, suggesting that the glycoproteins were not toxic for cells. Immunofluorescence revealed that parasite glycoproteins localized in the nuclei, mitochondria and cytoplasm and we found evidence for selection of certain molecules between nuclear and cytoplasmic compartments. Using fluorescent dextrans as fluid phase markers we found 17-38% of the cells in inoculated monolayers were engorged with dextran and that dextran was free in the cytoplasm. Increased dextran uptake was not lethal, required the presence of activated larvae, and was often associated with uptake of parasite glycoproteins. These observations suggest that larvae caused plasma membrane wounds. Our results showed that neither delivery of glycoproteins nor mechanical wounding is sufficient to allow entry of the parasite into resistant epithelial cells. Because both invasion-resistant and susceptible epithelial cells undergo non-lethal wounding, we propose that larvae-induced injury to epithelial cells may result in release of cell-specific mediators that signal larva to invade a particular cell line or, alternatively, to ignore it.


Subject(s)
Cell Membrane/parasitology , Epithelial Cells/parasitology , Glycoproteins/metabolism , Trichinella spiralis/pathogenicity , Animals , Cell Line , Cell Membrane/ultrastructure , Dextrans/metabolism , Endocytosis , Flow Cytometry , Fluorescence , Helminth Proteins/metabolism , Hexoses/metabolism , Larva/metabolism , Larva/pathogenicity , Microscopy, Confocal , Rats , Subcellular Fractions , Trichinella spiralis/growth & development
13.
Infect Immun ; 67(9): 4661-7, 1999 Sep.
Article in English | MEDLINE | ID: mdl-10456913

ABSTRACT

The antibody response to the L1 stage of Trichinella spiralis has been described as biphasic. Worms resident in the intestine during the first week of infection stimulate an antibody response against a subset of larval proteins. L1 larvae in the muscle at the end stage of infection stimulate a second antibody response against tyvelose-bearing glycoproteins. Antityvelose antibodies protect rats against challenge infection with larvae. The aim of this study was to characterize the rat B-cell response against larval antigens during the intestinal phase of T. spiralis infection and to test the antiparasitic effects of such antibodies. Strain PVG rats were infected orally with 500 larvae. Antibodies specific for phosphorylcholine-bearing proteins of L1 larvae first appeared in serum 9 days postinfection. Absorption experiments showed that the majority of antilarval antibodies produced in rats 16 days after infection with T. spiralis were specific for phosphorylcholine-bearing proteins. A fraction of these antibodies bound to free phosphorylcholine. Immunoglobulin G2c (IgG2c) producing cells in the mesenteric lymph node dominated this early antibody response. IgG2c is associated with T-independent immune responses in the rat; however, a comparison of athymic rats with euthymic controls suggested that only a small fraction of the phosphorylcholine-related antibody response against T. spiralis was T independent. Phosphorylcholine is a common epitope in antigens of bacteria and nematode parasites and has been shown to be a target of protective immunity in certain bacteria. A monoclonal IgG2c antibody was prepared from infected rats and shown to be specific for phosphorylcholine. Monoclonal phosphorylcholine-specific IgG2c failed to protect rats against intestinal infection with T. spiralis. Therefore, our findings do not support a role for phosphorylcholine-bearing antigens in immune defense against T. spiralis; however, the potency of the immune response induced suggests an immunomodulatory role for the lymphocytes involved.


Subject(s)
Antibodies, Helminth/immunology , Antigens, Helminth/immunology , Immunoglobulin G/immunology , Phosphorylcholine/immunology , Trichinella spiralis/immunology , Trichinellosis/immunology , Animals , Antibodies, Helminth/blood , Antibodies, Monoclonal/immunology , Antibody Specificity , Immunoglobulin G/blood , Immunoglobulin G/classification , Immunoglobulin Isotypes , Male , Mice , Rats , T-Lymphocytes/immunology , Time Factors , Trichinellosis/blood , Trichinellosis/prevention & control
14.
Glycobiology ; 7(3): 383-90, 1997 Apr.
Article in English | MEDLINE | ID: mdl-9147047

ABSTRACT

Indirect evidence that the immunodominant N-glycans of the parasite, Trichinella spiralis are capped by novel beta-linked 3,6-dideoxy-D-arabinohexopyranosyl residues (tyvelose, Tyv) was obtained from immunochemical assays employing monoclonal antibodies and synthetic oligosaccharides. Three of four previously characterized monoclonal antibodies generated from the lymphocytes of T.spiralis infected rats bind BSA glycoconjugates bearing the synthetic epitope beta-D-Tyvp(1-->3)-beta-D-GalNAcp but not to the corresponding alpha-D-Tyvp(1-->3)-beta-D-GalNAcp-BSA glycoconjugate. Monosaccharide and disaccharide glycoside inhibition data mirrors the results of the direct binding experiments. The branched tetrasaccharide beta-D-Tyv(1-->3)-beta-D-GalNAcp(1-->4)[alpha-L-Fucp(1 -->3)] beta-D-GalNAcp is the most active synthetic oligosaccharide inhibitor for all four monoclonal antibodies studied, while the corresponding alpha-D-Tyv containing tetrasaccharide and the core trisaccharide beta-D-GalNAcp(1-->4)[alpha-L-Fucp(1-->3)]beta-D-GlcNAcp+ ++ are inactive. The exceptional inhibitory activity of the disaccharide beta-D-Tyvp(1-->3)-beta-D-GalNAcp with one mAb (18H) compared to that of the branched tetrasaccharide beta-D-Tyvp(1-->3)-beta-D-GalNAcp(1-->4)[alpha-L-Fucp( 1-->3)]-beta-D-GlcNAcp is indicative of the presence of linear, nonfucosylated glycan epitopes (beta-D-Tyvp(1-->3)-beta-D-GalNAcp(1-->4) beta-D-GlcNAcp) that lack a fucose residue in one arm of the antigenic, tetra-antennary N-glycan. This observation supports earlier FAB-mass spectrometry evidence for the existence of tetra-antennary, core fucosylated glycans that lack a fucose residue on one of their antennae.


Subject(s)
Antigens, Helminth/chemistry , Epitopes/chemistry , Hexoses/chemistry , Hexoses/immunology , Polysaccharides/immunology , Trichinella spiralis/immunology , Animals , Antibodies, Monoclonal/immunology , Carbohydrate Conformation , Carbohydrate Sequence , Fucose/analysis , Molecular Sequence Data , Molecular Structure , Rats , Serum Albumin, Bovine/immunology , Spectrometry, Mass, Fast Atom Bombardment
15.
Parasite Immunol ; 18(6): 273-84, 1996 Jun.
Article in English | MEDLINE | ID: mdl-9229380

ABSTRACT

In order to characterize immunodominant components of T. spiralis a workshop was organized. In this the reactivity of monoclonal and polyclonal antibodies, provided by different research groups, towards total extracts from adult, new born larvae and muscle larvae as well as to excretory/secretory components of muscle larvae were tested by ELISA, Western blot and immunoprecipitation assays. As a result of this workshop T. spiralis ML antigens have been classified into eight groups (TSL-1-TSL-8) according to their recognition by monoclonal and polyclonal antibodies. Among them, TSL-1 antigens have been the most extensively characterized both biochemically and immunologically. These antigens are stage specific, originate in the muscle stichosome and are abundant in both E/S and on the larval cuticular surface. The TSL-1 antigens share an immunodominant carbohydrate epitope (tyvelose), which is unique for Trichinella and is not associated with phosphorylcholine. The data collected in this workshop has allowed both the unification of the nomenclature for T. spiralis antigens and their biochemical characterization. It also has provided a platform for further studies on the characterization of other T. spiralis antigens and indeed for other Trichinella species.


Subject(s)
Antigens, Helminth/isolation & purification , Trichinella spiralis/immunology , Animals , Antibodies, Helminth , Antibodies, Monoclonal , Antigens, Helminth/chemistry , Antigens, Helminth/classification , Blotting, Western , Carbohydrates/immunology , Enzyme-Linked Immunosorbent Assay , Immunodominant Epitopes/chemistry , Immunodominant Epitopes/isolation & purification , Microscopy, Immunoelectron , Molecular Weight , Phosphorylcholine/chemistry , Precipitin Tests , Trichinella spiralis/growth & development , Trichinella spiralis/ultrastructure
17.
Vet Microbiol ; 45(1): 81-92, 1995 Jun.
Article in English | MEDLINE | ID: mdl-7653031

ABSTRACT

Antibodies specific for equine influenza viruses are usually quantified using single radial hemolysis (SRH), hemagglutination inhibition (HI) or virus neutralization (VN). Neutralizing antibodies are thought to provide optimum protection to challenged animals. The purpose of this study was to determine the extent to which SRH and HI assays detect antibodies which neutralize equine influenza viruses. Acute and convalescent sera from 41 horses were analyzed using VN, SRH, and HI assays. These horses were present in a population of Thoroughbred racehorses during an epidemic of upper respiratory tract disease associated with influenza A/equine/Saskatoon/1/91 (H3N8), infections. Concentrations of antibodies binding to influenza A/equine/Kentucky/1/81 (H3N8), A/equine/Miami/1/63 (H3N8), and A/equine/Prague/1/56 (H7N7) were determined. Results of the VN assay were compared with results from the SRH and HI assays for acute antibody levels, changes in antibody concentrations between acute and convalescent sampling, and the occurrence of seroconversion. The correlation between assays for pre-exposure antibody levels ranged from 88% to 96%. The correlation between assays for change in antibody concentration ranged from 83% to 90% for the H3N8 viruses. This study shows that antibody concentrations specific for equine influenza virus, measured using SRH and HI assays, are highly correlated with concentrations detected using a VN assay.


Subject(s)
Antibodies, Viral/blood , Horse Diseases/immunology , Horses , Influenza A virus/immunology , Orthomyxoviridae Infections/veterinary , Animals , Hemagglutination Inhibition Tests/veterinary , Hemolytic Plaque Technique/veterinary , Horse Diseases/blood , Horse Diseases/virology , Neutralization Tests/veterinary , Orthomyxoviridae Infections/blood , Orthomyxoviridae Infections/immunology , Orthomyxoviridae Infections/virology
18.
Glycobiology ; 4(5): 593-603, 1994 Oct.
Article in English | MEDLINE | ID: mdl-7881173

ABSTRACT

The larval stage of the intestinal nematode, Trichinella spiralis, secretes and displays on its cuticle a number of antigenically cross-reactive glycoproteins. These so-called TSL-1 antigens induce a powerful antibody response in parasitized animals. In rats, anti-TSL-1 antibodies mediate a protective immunity that expels invading larvae from the intestine. The vast majority of anti-TSL-1 antibodies are specific for glycans. Although the biological functions of TSL-1 antigens are not known, the powerful effect of glycan-specific antibodies on the intestinal survival of T. spiralis suggests that they play an important role in parasite establishment. Little is known about the structures of the glycans present on the TSL-1 glycoproteins. Recent studies have suggested, however, that the antigens contain very unusual glycans (Wisnewski, N., McNeil, M., Grieve, R.B. and Wassom, D.L., Mol. Biochem. Parasitol., 61, 25-36, 1993). Sugar and linkage analysis of the combined secreted products unexpectedly showed that a major terminal sugar is tyvelose (3,6-dideoxy-D-arabino-hexose; Tyv) which has previously been found only in certain gram-negative bacterial lipopolysaccharides. In this paper, we report the first rigorous structural study of oligosaccharides released from TSL-1 antigens by peptide N-glycosidase F digestion. Using strategies based on fast atom bombardment mass spectrometry (FAB-MS), we have discovered a novel family of tri- and tetra-antennary N-glycans whose antennae are comprised of the tyvelose-capped structure: Tyv1,3GalNAc beta 1,4(Fuc alpha 1,3)GlcNAc beta 1-. Thus a major population of TSL-1 glycans contains clusters of hydrophobic terminal structures which are likely to be highly immunogenic.


Subject(s)
Antigens, Helminth/immunology , Hexoses/chemistry , Immunodominant Epitopes/immunology , Polysaccharides/immunology , Trichinella spiralis/immunology , Animals , Carbohydrate Conformation , Carbohydrate Sequence , Glycoside Hydrolases , Hydrolysis , Immunodominant Epitopes/chemistry , Molecular Sequence Data , Polysaccharides/analysis , Spectrometry, Mass, Fast Atom Bombardment , Stereoisomerism
19.
Glycobiology ; 4(5): 585-92, 1994 Oct.
Article in English | MEDLINE | ID: mdl-7881172

ABSTRACT

We have investigated the role of glycans on Trichinella spiralis antigens in recognition by rat monoclonal antibodies (mAbs) which protect rat pups against challenge with the parasite. In pups born to infected dams or pups passively immunized with mAbs, antibodies eliminate a challenge dose from the intestine within hours ('rapid expulsion'). Because such dramatic protection can be afforded by mAbs, we have sought to characterize the parasite antigens they target. In this report we show that protective antibodies were unable to bind excretory/secretory (ES) antigens deglycosylated with trifluoromethanesulphonic acid (TFMS). In addition, oligosaccharides isolated from glycoproteins by alkaline hydrolysis or peptide: N glycosidase F (PNGase F) digestion were bound by protective, but not non-protective, mAbs. Glycans affinity purified with protective mAb 9D bound to all but one protective mAb. These antibodies have been shown previously to bind to the surfaces of intact larvae, indicating that the glycan is exposed on the parasite surface. Candidate glycans that may be involved in binding protective mAbs have unusual tri- and tetra-antennary structures with terminal tyvelose moieties (Reason et al., Glycobiology, 4, 000-000, 1994). Coating of the larval surface with such glycans may serve to protect the parasite and its secreted products from enzymatic attack as the parasite travels to and resides in its epithelial niche.


Subject(s)
Antibodies, Monoclonal/immunology , Polysaccharides/immunology , Trichinella spiralis/immunology , Trichinellosis/prevention & control , Animals , Chromatography, Affinity , Enzyme-Linked Immunosorbent Assay , Glycosylation , Immunization, Passive , Immunohistochemistry , Mesylates , Oxidation-Reduction , Periodic Acid/chemistry , Rats
20.
Mol Biochem Parasitol ; 65(2): 201-11, 1994 Jun.
Article in English | MEDLINE | ID: mdl-7526209

ABSTRACT

Rapid expulsion is a protective immune mechanism which eliminates as much as 99% of a challenge infection of Trichinella spiralis muscle larvae from the gastrointestinal tract of suckling rats. Protective monoclonal antibodies (mAbs) generated against larval excretory-secretory antigens (ESA) specifically recognize a 45-kDa glycoprotein, gp45, in addition to a distinct profile of other cross-reactive antigens that are also recognized by non-protective mAbs. Recent data indicate that protective mAbs recognize carbohydrate epitopes. To complement biochemical studies on the target(s) of rapid expulsion, we describe here the cloning and characterization of the cDNA, TspE1, which belongs to a multigene family and encodes several larval proteins in the 40-50-kDa range. A second cDNA, TspM6 encodes a 45-kDa antigen and is homologous to the published sequence of gp45. Anti-TspE1 antibodies detected antigens within beta- and gamma-stichocytes while anti-TspM6 antibodies detected antigens within alpha-stichocytes of the secretory organs of muscle larvae. Sequence analysis has provided no functional information on the encoded gene products. Neither recombinant antigen is recognized by the mAbs but native parasite molecules with peptide homology to both the TspE1 and TspM6 recombinant antigens bear the glycan recognized by the protective mAbs. These molecules are candidate targets in rapid expulsion.


Subject(s)
Antibodies, Helminth/immunology , Antigens, Helminth/immunology , Trichinella spiralis/immunology , Trichinellosis/immunology , Amino Acid Sequence , Animals , Antibodies, Monoclonal/immunology , Antigens, Helminth/genetics , Base Sequence , Cloning, Molecular , DNA, Protozoan/analysis , Epitopes/immunology , Female , Male , Molecular Sequence Data , Protozoan Proteins/genetics , Protozoan Proteins/immunology , Rats , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/immunology , Trichinellosis/prevention & control
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