Identification of Antigenic Proteins of Setaria Cervi by Immunoblotting Technique

Identification and characterization of antigenic proteins of Setaria cervi (bovine filarial parasite) adults and microfilariae was done by immunoblotting technique using hyperimmune rabbit sera against S. cervi and Brugia malayi. The antigens recognized by these sera were detected by using 125Iprotein-A followed by autoradiography. Fifteen different antigens were observed to be common between adult and microfilarial stages of the parasite. Some stage specific antigens were also identified. Many antigens of S. cervi adults and microfilariae were also recognized by rabbit anti-B. malayi serum showing the existence of common antigenic determinants between the bovine and human filarial parasites.     

Analysis of B. malayi microfilarial antigens by immunoblotting

BALB/C mice were immunized with heavy or low infection of live B. malayi microfilariae or immunised with different fractions of the microfilarial antigens. Antibody levels and antibody dependent macrophage mediated cytotoxicity to B. malayi microfilariae were determined in the immunized sera. Antigens responsible for induction of antibodies were recognised in B. malayi microfilarial extract by immunoblotting. Appearance of cytotoxic antibodies correlates with recognition of certain common antigens in microfilarial extract such as 45, 54, 62, 66 and 76 KDa mol. wt. proteins.     

Analysis of B. Malayi Microfilarial Antigens by Immunoblotting

BALB/C mice were immunized with heavy or low infection of live B. malayi microfilariae or immunised with different fractions of the microfilarial antigens. Antibody levels and antibody dependent macrophage mediated cytotoxicity to B. malayi microfilariae were determined in the immunized sera. Antigens responsible for induction of antibodies were recognised in B. malayi microfilarial extract by immuno blotting. Appearance of cytotoxic antibodies correlates with recognition of certain common antigens in microfilarial extract such as 45, 54, 62, 66 and 76 KDa mol. wt. proteins.     

IgE-dependent cellular adhesion and cytotoxicity to Litomosoides carinii microfilariae--nature of effector cells.

Albino rats immunized with sonicated microfilarial antigen incorporated in Freund's complete adjuvant, produce antibodies that promote cell-mediated adhesion and killing of Litomosoides carinii microfilariae in vitro. Using highly purified cell populations, it has been shown that macrophages and neutrophils are most active in this phenomenon. Eosinophils, while adhering readily to parasites in the presence of the antibody, did not affect the viability of the parasites when observed after 24 hr incubation.     

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.     

Antibodies to diethylcarbamazine cross-react with microfilariae of Wuchereria bancrofti

Antibodies directed against the microfilarial sheath have been instrumental in the immune elimination of circulating microfilariae in human lymphatic filariasis. We report here that antibodies to diethylcarbamazine (DEC, the most commonly used anti-filarial drug) cross-react with the sheath of Wuchereria bancrofti microfilariae. Antibodies with reactivity to DEC were raised in rabbits by immunization with a conjugate of methylpiperazine carboxylic acid (MPCA, an acid hydrolysis product of DEC) coupled to bovine serum albumin. The reactivity of these antibodies with microfilarial sheath of W. bancrofti was demonstrated by indirect immunofluorescent assay and indirect immunoperoxidase assay. This reactivity could be effectively inhibited by pre-incubation of the antisera with different haptens such as DEC, MPCA or piperazine citrate.     

Kidney Immunopathology and Pathophysiology in Rats Immunized with Proximal Tubule Cell Brush Border or Basolateral Membrane Vesicles

Proximal tubule pathology in Heymann nephritis has been attributed to anti-brush border antibodies, but antibodies with other specificities might also be important. To determine whether injury to the basolateral membranes of proximal tubules could occur independently of brush border injury, LEW rats were immunized either with partially purified basolateral or brush border membrane vesicles. Both immunogens produced glomerular immunopathology and pathophysiology identical in magnitude and time course to that seen in Heymann nephritis. Antibodies eluted from the kidneys of rats immunized with either antigen preparation stained the brush border in vitro. However, circulating anti-brush border antibodies were in significant titers only in rats immunized with brush border vesicles, whereas antibodies that stained the cytoplasm of both proximal and distal tubules predominated in rats immunized with basolateral membranes. With the onset of proteinuria, rats immunized with brush border membranes developed the proximal tubule pathology of Heymann nephritis. In rats immunized with basolateral membranes, the brush border and apical aspect of proximal tubule cells remained essentially normal. However, defects of basolateral membrane transport function were present, indicating that those defects need not necessarily be secondary to brush border damage. The dissociation of brush border damage from glomerular injury suggests that different antibody populations may account for each. Furthermore, anti-brush border antibodies may not account for all aspects of proximal tubule pathology in Heymann nephritis.     

Protein and Antigenic Composition of Adult and Microfilarial Stages of Setaria Cervi

The protein and antigenic pattern of adult (female/male) and microfilarial stages of Setaria cervi, a bovine filarial parasite, was studied using certain immunochemical techniques. SDS-polyacrylamide gel electrophoretic analysis showed the presence of 35-40 protein bands in adults and 25-29 protein bands in microfilariae in molecular weight range of 10,000-200,000. Immunoelectrophoresis revealed the presence of 9-10 precipitin lines in adult and only 4 precipitin lines in microfilarial antigenic preparations. Crossed immuno-electrophoresis resolved these antigenic preparations further, and revealed the presence of 22-24 antigens in adults and 12-14 in microfilariae. These results demonstrate complex nature of somatic extracts of adult stage as compared to microfilariae and also reveal some qualitative and quantitative differences between these stages.     

Antigenic analysis of Brugia timori, a filarial nematode of man: initial characterization by surface radioiodination and evaluation of diagnostic potential.

The antigenic composition of Brugia timori has been investigated with surface labelling techniques and defined sets of parasite molecules have been identified on infective larvae, adult worms and microfilariae. Iodinated preparations from all three stages were assessed for immunodiagnostic potential with a small number of serum samples from human filariasis patients. In these tests, reaction with infective larval antigen was the clearest indicator of infection. Reactivity to microfilarial antigens however, correlated poorly with incidence of infection. These experiments show that levels of anti-parasite antibody appear to increase as filarial disease becomes more severe. In contrast to some reports, antibody to microfilarial surface antigens is present in sera from several patients with circulating microfilariae. The immunodiagnostic potential of these tests is indicated by the detection of a few individuals who have high levels of antibody but no outward signs of infection.     

Protein and Antigenic Composition of Adult and Microfilarial Stages of Setaria Cervi

The protein and antigenic pattern of adult (female/male) and microfilarial stages of Setaria cervi, a bovine filarial parasite, was studied using certain immunochemical techniques. SDS-polyacrylamide gel electrophoretic analysis showed the presence of 35–40 protein bands in adults and 25–29 protein bands in microfilariae in molecular weight range of 10,000–200,000. Immunoelectrophoresis revealed the presence of 9–10 precipitin lines in adult and only 4 precipitin lines in microfilarial antigenic preparations. Crossed immuno-electrophoresis resolved these antigenic preparations further, and revealed the presence of 22–24 antigens in adults and 12–14 in microfilariae. These results demonstrate complex nature of somatic extracts of adult stage as compared to microfilariae and also reveal some qualitative and quantitative differences between these stages.     

Immunopotentiation and Anabolism Induced by Sodium Diethyldithiocarbmate

Sodium diethyldithiocarbamate, DTC, enhances over a large range of doses macrophage listericidal capacity and T cell activities in terms of increased IgG-antibody forming spleen cells and delayed hypersensitivity levels. Such immunopotentiation is not associated with splenomegalia or increase in lymphocyte counts. Imunopotentiation requires a preexisting link between carbon disulfide and diethylamine, since both moieties were inactive if administered alone or on separate body sites. DTC demonstrates also an anabolic effect on mice emaciated by administering a B. melitensis cell-wall fraction. The role of DTC on hormonal production is discussed in relation to hormone-mediated action on T cell induction.     

Development of a recombinant antigen vaccine against infection with the filarial worm Onchocerca volvulus

Efforts to control Onchocerca volvulus, the etiologic agent of river blindness, have been limited to vector control and drug treatment to eliminate microfilariae, with no means available to prevent infection. The goal of this study was to develop a vaccine against this infection using recombinant antigens that are expressed in the early larval stages of the parasite. Five recombinant antigens, Ov7, Ov64, OvB8, Ov9M, and Ov73k, were identified by screening adult and larval cDNA libraries with antibodies from immune humans, chimpanzees, or rabbits. When mice were immunized with the five individual recombinant antigens, statistically significant reductions in parasite survival were induced in mice immunized with Ov7, OvB8, or Ov64, when administered in alum but not when injected in Freund's complete adjuvant (FCA). Live larvae recovered from control and immunized mice were analyzed to determine their developmental stages. A decrease in the percentage of larvae molting from the third stage to the fourth stage was observed with mice immunized with Ov7, Ov64, or OvB8 in alum but not with mice immunized with Ov9 and Ov73k or with mice immunized with any of the five antigens in FCA. Mice immunized with a cocktail of the three protective antigens developed protective immunity equal to that seen with mice immunized with individual antigens. This study has identified, for the first time, three recombinant antigens capable of inducing protective immunity to O. volvulus. Furthermore, since the antigens functioned with alum as the adjuvant, this vaccine could potentially be used clinically to prevent river blindness in humans.     

Ivermectin and the reduction of the rate of infection of simuliids in a forest focus of human onchocerciasis

Eight patients from a forest onchocercian area "Grandes rivières", in Ivory Coast, were treated with a single oral dose of ivermectin (12 mg). The density of dermic microfilariae was estimated at days 0, 7 and 180; the mean numbers of ingested microfilariae and of developing larvae in the vectors S. soubrense-S. sanctipauli, engorged on the treated patients were recorded. Comparisons were made with non treated patients, having a similar density of microfilariae than the 8 treated patients at day 7. Results confirm the reduction induced by ivermectin of the dermic microfilarial density and the resulting reduction of the infection of the simuliids. Furthermore 7 days after treatment, a new phenomenon is demonstrated: for a similar dermic microfilarial density, simuliids take up a number of microfilariae 100 times lower from treated than from untreated patients. To explain this phenomenon, it is suggested that ivermectin induces a change in the microfilarial distribution in the layers of the dermis. Six months after treatment, this low uptake of microfilariae by the vectors had disappeared, and the infection rate of the engorged similiids was much higher than at day 7 although the dermic microfilarial density was similar.     

Mycoplasma Neurolyticum Membranes: A T-Independent Antigen in the Rat

Our understanding of the mechanisms involved in B cell activation, proliferation and differentiation to immunoglobulin secreting cells has been facilitated by the use of T-independent and T-dependent antigens. The majority of these studies have used the murine system and only recently, the rat. Because membranes isolated from Mycoplasma neurolyticum are potent B cell mitogens in the rat and some T-independent antigens also activate DNA synthesis in B cells, the in vitro and in vivo antibody responses induced by M. neurolyticum membranes in T-deficient rat systems were examined. The three groups of rats used, i.e., nude; anti-thymocyte serum-treated, neonatally-thymectomized (ATS-Tx); and normal Fischer 344 produced a non-polyclonal antibody response against the membranes. Spleen cell cultures that were T cell deficient and B cell enriched produced plaque-forming cells against the Mycoplasma membranes. Antibody production was depleted upon removal of Sephadex G-10 adherent cells. The antibody response is comprised of both antigen-specific and polyclonal responses. Lipoglycan, found in the aqueous phenol extract of the membranes, is the mitogenic fraction of the membranes, and this study suggests that it may also be the T-independent antigenic component of the M. neurolyticum membranes.     

Cytotoxic Antibodies to Cultured Human Lymphoid Cells in Polytransfused Thrombocytopenic Patients: Association with Reduced Survival of Allogeneic Transfused Platelets

Four out of 8 thrombocytopenic patients without detectable cytotoxic antibodies to human peripheral lymphocytes contained cytotoxic antibodies to cultured human lymphoid cells. The presence of such antibodies was associated with reduced survival of infused allogeneic platelets in these patients. The antibodies reacted in a distinct fashion with a panel of cultured human lymphoid cells and are directed to B cell antigens. Cytotoxic antibodies to cultured human lymphoid cells did not react in vitro with platelets suggesting that the antibodies play no significant role in the accelerated destruction of infused allogeneic platelets, although their presence predicts it. Therefore, screening of sera with cultured human lymphoid cells appears to be a useful test in addition to those now used to select patients for transfusion of allogeneic platelets. Sera from polytransfused patients without cytotoxic antibodies to peripheral lymphocytes may be a useful source of antibodies to B cell antigens.     

Immunochemical characterization and biosynthesis of major antigens of iodo-bead surface-labeled Brugia malayi microfilariae

The objectives of the present study were to identify and characterize biochemically the major antigens of Brugia malayi microfilariae, a filarial parasite that infects humans. IgG antibodies in sera of mice which had cleared parasites from the bloodstream reacted with 30, 55, 94 and 150 kDa molecules of living microfilariae radioiodinated by the Iodo-bead method. Sera of humans infected with the related filariae Wuchereria bancrofti, Loa loa or Onchocerca volvulus immunoprecipitated molecules of similar size as well as two additional proteins of 22 and 43 kDa. Sera of uninfected North Americans or mice infected with Trichinella spiralis or Schistosoma mansoni did not recognize these radioiodinated antigens. Experiments to examine the possible surface localization and metabolism of these antigens showed that they were removed from intact parasites exposed to chymotrypsin or trypsin and that immunogenic molecules of 30, 55, and 150 kDa were released into excretory-secretory products by viable microfilariae. [35S]Methionine biosynthetically labeled polypeptide antigens of 22, 30, 35 and 150 kDa were detected by antibody reacted with intact microfilariae and/or their excretion products. Antigens of 30, 55, and 150 kDa appear to be glycoproteins as they bound wheat germ agglutinin and were biosynthetically labeled with [14C]N-acetyl-D-glucosamine. These data suggest that the surface of B. malayi microfilariae is a dynamic structure which synthesizes and sheds antigens.     

Lack of biological significance of in vitro Brugia malayi microfilarial cytotoxicity mediated by Propionibacterium acnes ("Corynebacterium parvum")-and Mycobacterium bovis BCG-activated macrophages

The effect of nonspecific activation of host macrophages by Propionibacterium acnes ("Corynebacterium parvum") or Mycobacterium bovis BCG on Brugia malayi microfilariae was determined by in vitro and in vivo studies. Intraperitoneal injection of C. parvum or BCG stimulated peritoneal exudate cells, which were toxic to microfilariae. Microfilariae were equally susceptible to damage by C57BL/6J or BALB/cJ peritoneal exudate cells. Furthermore, inhibitors of oxidative metabolism and arginine supplementation did not prevent this toxicity, suggesting that the mechanism of microfilarial damage differs from that seen with another multicellular helminth, Schistosoma mansoni. In vivo studies with both BCG and C parvum, however, did not confirm the importance of nonspecific immunity in resistance to B. malayi microfilaremia. Despite the lack of biologic relevance of this phenomenon, in vitro studies may yield important knowledge about the mechanisms of microfilarial damage.     

Eliciting Antibodies in Chickens to Human Dimeric IgA. Removal of Factors from Human Colostrum Depressing Anti IgA Antibody Production

Contrary to expectation chickens did not readily elicit antibodies to IgA dimers when untreated human colostrum was used as antigen. When colostrum was fractionated by means of a column of 8% granulated agarose equilibrated with 10mM phosphate buffer, pH 7.2, a major and a minor fraction were obtained. The major or “1st fraction” consisted of two components with sedimentation coefficients of 10.9 S and 14.1 S, respectively. The minor or “2nd fraction” consisted of components of S values ranging from 2 to 6 and small amounts of 10.9 and 14.1 S material. When chickens were immunized with the “1st fraction” antibodies to dimeric IgA were produced. When the “1st and 2nd fractions” of the column were remixed and used for immunization of chickens, the immune response was as poor as when the chickens were injected with untreated colostrum. An immuno-depressing agent in colostrum was indicated. When rabbits were immunized with clarified human colostrum, antibodies against five antigens were elicited, one of the antigens being dimeric IgA. The immuno-depressing agent is therefore not universal. The purified agent suppressed antibody formation in chickens against the haemocyanin of Jasus lalandii. The “activity” is therefore not specific for IgA and the remaining four antigens in human colostrum.

The purified component is a glyco-protein with a hexose content in excess of 10%. The derivatized sugars prepared from it were shown by gas liquid chromatography to be an equimolar mixture of galactose, mannose and fucose.

The molecular weight (Mr) of the purified component was found to be 72 000 by sedimentation and diffusion and 80 000 by SDS page using Mr reference standards.

The properties of the immuno-suppressor stronly suggest that it is the secretory piece of dimeric IgA.     

Biochemical and immunochemical characterization of surface and excretory-secretory antigens of Loa loa microfilariae

Detergent solubilized extracts of 125Iodogen surface-labelled Loa loa microfilariae revealed a relatively simple profile of two strongly labelled molecules of 23 and 67 kDa for blood microfilariae and several strongly labelled molecules of 23, 40, 42-67 kDa for in vitro born microfilariae. In addition, there were other weakly labelled molecules which were resolved after prolonged autoradiographic exposure. Surface molecules of 28, 29, and 33 kDa were unique to blood microfilariae, a 14.4 kDa molecule was unique to in vitro born microfilariae and molecules of 23, 40, and 75-84 kDa were common to both forms of microfilariae. The profile of excretory-secretory products consisted of molecules of 14.4-198 kDa. Human albumin was a predominant component of surface molecules and excretory-secretory products from blood microfilariae. Immunoprecipitation with occult and microfilaremic loaiasis sera demonstrated that the 23 kDa surface molecule and excretory-secretory products of 14.4 and 33 kDa were only recognized by occult loaiasis sera whereas surface molecules of 40 and 75-84 kDa and excretory-secretory products of 28 and 67 kDa were recognised by both sera. Studies with heterologous sera demonstrated that with the exception of the 75-84 kDa antigens, all the L. loa microfilarial surface antigens contained epitopes which were restricted to filarial parasites. Further studies revealed that the 23 kDa antigen was a protein which contained neither asparagine-N-linked oligosaccharides nor interchain disulfide-linkages.