Identification of Babesia bovis merozoite antigens separated by continuous-flow electrophoresis that stimulate proliferation of helper T-cell clones derived from B. bovis-immune cattle.

To characterize Babesia bovis merozoite antigens that stimulate anamnestic T helper (Th)-cell responses from B. bovis-immune cattle, B. bovis-specific Th-cell lines and clones, previously assigned to different antigenic groups (W. C. Brown, S. Zhao, A. C. Rice-Ficht, K. S. Logan, and V. M. Woods, Infect. Immun. 60:4364-4372, 1992), were tested in proliferation assays against fractionated merozoite antigens. The antigenic groups were determined by the patterns of response of Th clones to different parasite isolates and soluble or membrane forms of merozoite antigen. Soluble antigen fractionated by anion-exchange chromatography or gel filtration by using fast-performance liquid chromatography resolved two or three antigenic peaks, respectively. To enable fractionation of membrane-associated proteins and to resolve more precisely the proteins present in homogenized merozoites, a novel technique of continuous-flow electrophoresis was employed. Merozoite membranes or whole merozoites were homogenized and solubilized in sodium dodecyl sulfate-sample buffer, electrophoresed under reducing conditions on 15% or 10% acrylamide gels, eluted, and collected as fractions. Individual fractions were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and tested for the ability to stimulate Babesia-specific CD4+ T-cell lines and clones. CD4+ Th-cell lines from two cattle displayed differential patterns of reactivity and detected numerous peaks of antigenic activity, ranging from < 14 to 76 kDa. Th-cell clones previously categorized into different antigenic groups detected antigenic peaks unique for clones representative of a given group. Antigens of 29, 51 to 52, and 85 to 95 kDa (group I), 40 kDa (group III), 20 kDa (group IV), 58 to 60 kDa (group VI), and 38, 45, and 83 kDa (group VII) were identified in the stimulatory fractions. Immunization of rabbits with selected fractions produced a panel of antisera that reacted specifically on Western blots (immunoblots) with merozoite antigens of similar sizes, leading to the tentative identification of candidate antigens of B. bovis merozoites with molecular masses of 20, 40, 44, 51 to 52 or 95, and 58 to 60 kDa that stimulate proliferation of Th clones representative of five different antigenic groups. These antisera may be useful for isolating recombinant proteins that are immunogenic for Th cells of immune cattle and therefore potentially useful for vaccine development.     

Antibody responses to MAP 1B and other Cowdria ruminantium antigens are down regulated in cattle challenged with tick-transmitted heartwater

Serological diagnosis of heartwater or Cowdria ruminantium infection has been hampered by severe cross-reactions with antibody responses to related ehrlichial agents. A MAP 1B indirect enzyme-linked immunosorbent assay that has an improved specificity and sensitivity for detection of immunoglobulin G (IgG) antibodies has been developed to overcome this constraint (A. H. M. van Vliet, B. A. M. Van der Zeijst, E. Camus, S. M. Mahan, D. Martinez, and F. Jongejan, J. Clin. Microbiol. 33:2405-2410, 1995). When sera were tested from cattle in areas of endemic heartwater infection in Zimbabwe, only 33% of the samples tested positive in this assay despite a high infection pressure (S. M. Mahan, S. M. Samu, T. F. Peter, and F. Jongejan, Ann. N.Y. Acad. Sci 849:85-87, 1998). To determine underlying causes for this observation, the kinetics of MAP 1B-specific IgG antibodies in cattle after tick-transmitted C. ruminantium infection and following recovery were investigated. Sera collected weekly over a period of 52 weeks from 37 cattle, which were naturally or experimentally infected with C. ruminantium via Amblyomma hebraeum ticks, were analyzed. MAP 1B-specific IgG antibody responses developed with similar kinetics in both field- and laboratory-infected cattle. IgG levels peaked at 4 to 9 weeks after tick infestation and declined to baseline levels between 14 and 33 weeks, despite repeated exposure to infected ticks and the establishment of a carrier state as demonstrated by PCR and xenodiagnosis. Some of the serum samples from laboratory, and field-infected cattle were also analyzed by immunoblotting and an indirect fluorescent-antibody test (IFAT) to determine whether this observed seroreversion was specific to the MAP 1B antigen. Reciprocal IFAT and immunoblot MAP 1-specific antibody titres peaked at 5 to 9 weeks after tick infestation but also declined between 30 and 45 weeks. This suggests that MAP 1B-specific IgG antibody responses and antibody responses to other C. ruminantium antigens are down regulated in cattle despite repeated exposure to C. ruminantium via ticks. Significantly, serological responses to the MAP 1B antigen may not be a reliable indicator of C. ruminantium exposure in cattle in areas of endemic heartwater infection.     

Blastogenic responses of lymphocytes from mice immunized by sublethal infection with yeast cells of Histoplasma capsulatum.

Blastogenic responses of spleen cells to histoplasmin and ribosomal antigens and to the mitogens concanavalin A. phytohemagglutinin, and lipopolysaccharide were studied in normal and immunized mice (10(5) live yeast cells of Histoplasma capsulatum given by the subcutaneous route). Cells (10(6) per well) were cultured with and without antigens and mitogens in microtiter plates with RPMI 1640-5% heat-inactivated normal mouse serum for 72 h at 37 degrees C. Cells were harvested after a 16- to 18-h pulse with 1 microCi of [3H]thymidine (6.7 Ci/mol), and thymidine incorporation was measured by scintillation counting. The initial blastogenic response to concanavalin A (54 X 10(3) cpm) was suppressed (P less than 0.001) from 4 to 14 days post-immunization and returned to control levels on day 21. The response to phytohemagglutinin was suppressed up to 21 days. Lipopolysaccharide responses, however, were affected to a lesser degree. Blastogenic responses to histoplasmin and H. capsulatum ribosomes were similar on day 0 in normal and immune lymphocytes, but by day 4 cells from immunized mice were more responsive (P less than 0.01). The maximum response to H. capsulatum antigens was detected on day 42 and was 9- to 16-fold higher than in controls. These results demonstrate in vitro responses of primed lymphocytes on exposure to H. capsulatum antigens and suppressed responses to mitogens during early stages of the immune response.     

In vitro proliferation of lymphocytes from cyclophosphamide-pretreated mice immunized with antigen mixed with dimethyl dioctadecyl ammonium bromide

A blastogenesis assay employing lymphocytes from cyclophosphamide-pretreated mice immunized with antigen mixed with the immunopotentiating compound dimethyl dioctadecyl ammonium bromide is described. The model antigen used for determining the assay parameters was inactivated purified measles virus. The optimal time for removal of immunologically primed T cells was 7 days after immunization of mice pretreated 2 days previously with 200 mg of cyclophosphamide/kg. The peak lymphoproliferative response was found to occur after 3-5 days in culture, depending on the concentration of antigen used. Although fetal bovine serum and syngeneic mouse serum each worked well as a medium supplement, significantly higher specific and lower non-specific lymphoproliferation were obtained when the mouse serum was used. Most of the lymphocytes responding to antigen were of the Ly 1.2 phenotype. Specificity of the blastogenic response was shown by a lack of cross-reactivity among measles virus, herpes simplex virus type 1 and vesicular stomatitis virus antigens. This approach to a mouse blastogenesis assay involves an easy way to induce strong T cell priming in mice, while still providing an assay which has an ideal combination of low non-specific and high antigen-specific responses.     

Identification of surface antigens of schistosomula of Schistosoma mansoni recognized by antibodies from mice immunized by chronic infection and by exposure to highly irradiated cercariae.

Surface components of mechanically transformed schistosomula of Schistosoma mansoni were labeled by lactoperoxidase-catalyzed iodination. After solubilization with Triton X-100, antigens were identified by immunoprecipitation. Serum from chronically infected Swiss mice reproducibly precipitated seven major polypeptides with approximate molecular weights (X 10(3] of 94, 68, 45, 40 to 32, 22, and 16. The antigens of molecular weights (X 10(3] of 94, 40 to 32, 22, and 16 were shown to be exposed on the parasite surface by interaction of the antibodies with intact labeled schistosomula. Sera from several strains of infected inbred mice precipitated the same polypeptides. The antibodies produced during chronic infection were found to be stimulated by adult worms since sera from 6-week-infected animals precipitated none of the surface antigens, and the pattern produced by precipitation with antibodies from a mouse infected with male worms only was indistinguishable from the pattern obtained with sera from mice with bisexual infections. Antibodies from mice immunized with highly irradiated cercariae reproducibly precipitated major polypeptides of approximately (X 10(3] 94, 68, 45, 32, 22, 19, and 15 daltons. The antigens of (X 10(3] 94, 43, 32, 22, and 15 daltons were shown to be exposed on the parasite surface by interaction of the antibodies with intact labeled schistosomula. The 15 X 10(3)-dalton surface protein was recognized by sera from vaccinated, but not chronically infected, mice, suggesting that it represents a stage-specific immunogen present on schistosomula but not on adult worms. Sera from two inbred strains of mice which develop different degrees of immunity recognized the same antigens.     

Protective effectiveness of the blood sera and immune system cells from mice immunized with live or inactivated influenza virus

The results of the study of comparative protective role of humoral and cell-mediated immunity factors obtained from inbred donor mice immunized with live or inactivated influenza virus are presented. The superiority of the live virus over the inactivated preparation as the inducer of not only humoral but especially cell-mediated immune response was demonstrated by the effectiveness of passive intranasal protection of the infected mice, by the degree of inhibition of virus reproduction in the lungs of the protected mice, and by the capacity for interferon production by the cells of the immune system of donor mice.     

Bacterial antigens stimulate the production of histamine releasing factor (HRF) by lymphocytes from intrinsic asthmatic patients.

Lymphocyte from 12 intrinsic asthmatic patients and 10 healthy controls were studied for their capability to produce histamine releasing factor (HRF) in vitro. Spontaneous HRF production was measured by culturing the lymphocytes alone for 20 h. In another set of experiments lymphocytes were first preincubated separately with phytohaemagglutinin, antigens of Haemophilus influenzae, Streptococcus viridans, Staphylococcus sp. and Neisseria catarrhalis for 4 h then carefully washed three times and cultured alone for an additional 16 h. Cell-free supernatant was assayed for histamine releasing activity using basophils from healthy donors. It was observed that lymphocytes from intrinsic asthmatic patients spontaneously produced HRF. The production of this lymphokine was enhanced following preincubation of lymphocytes with phytohaemagglutinin or bacterial antigens. Results of skin test with bacterial antigens did not correlate with the magnitude of the production of HRF by lymphocytes. At gel chromatography over Sephadex G-75 bacterial antigen-stimulated lymphocyte supernatant revealed two peaks of HRF activity in the molecular weight ranges 35,000-50,000 and 3,000-7,000.     

Development and evaluation of PCR assay for detection of low levels of Cowdria ruminantium infection in Amblyomma ticks not detected by DNA probe.

The sensitivities of a PCR assay and a DNA probe assay were compared for the detection of Cowdria ruminantium in Amblyomma ticks that were fed on C. ruminantium-infected, clinically reacting, and recovered carrier animals. The PCR assay and DNA probe detected infection in 86.0 and 37.0%, respectively, of 100 ticks fed on a febrile animal. In 75 ticks fed on carrier animals, PCR and the DNA probe detected infection in 28.0 and 1.33% of ticks, respectively. This demonstrates that the DNA probe has poor sensitivity for the detection of low levels of infection in ticks and that PCR is necessary for this purpose. The PCR assay had a detection limit of between 1 and 10 C. ruminantium organisms and did not amplify DNA from Ehrlichia canis, which is phylogenetically closely related to C. ruminantium, Theileria parva, or uninfected Amblyomma hebraeum or A. variegatum. PCR detected infection in A. hebraeum and A. variegatum adult ticks infected with one of six geographically different C. ruminantium strains. Amplification was also possible from desiccated ticks and ticks fixed in 70% ethanol, 10% buffered formalin, or 2% glutaraldehyde. The PCR assay supersedes the DNA probe and older detection methods for the detection of C. ruminantium in ticks, particularly those fed on carrier animals, and is suitable for both prospective and retrospective studies which require accurate detection of C. ruminantium in individual ticks. Application of the PCR assay should significantly improve the understanding of heartwater epidemiology, particularly through the determination of field tick infection rates.     

Inhibition of the proliferative response of peripheral blood lymphocytes to mycobacterial or fungal antigens by co-stimulation with antigens from various mycobacterial species.

Soluble antigen preparations from Mycobacterium leprae are reported to inhibit the response to other stimuli, of peripheral blood mononuclear cells from lepromatous leprosy cases (BL/LL) and also from tuberculoid cases (TT/BL) and normal donors. We confirm these findings and in addition, report that a similar suppressive effect is exerted by antigen from Mycobacterium vaccae, Mycobacterium nonchromogenicum and, to a lesser extent, Mycobacterium tuberculosis and Mycobactyerium kansasii. Moreover, suppression is seen using peripheral blood mononuclear cells from individuals unlikely to have encountered the organism used. The suppression is not due to toxicity of the antigen preparation, and is not indomethacin sensitive. It involves a cell found in the E-rosetting population, which loses its ability to suppress or be suppressed, after 48 hr in culture. Possible explanations include a pharmacological effect of cell wall peptidoglycolipids, or the triggering of suppressor cells specific for common mycobacterial antigens.     

Garlic extracts stimulate proliferation of rat lymphocytes in vitro by increasing IL-2 and IL-4 production

Garlic components are known to modulate certain immune functions. However, mechanisms of their action are not sufficiently elucidated. This study was, therefore, undertaken to examine the effects of aqueous and ethanolic extracts prepared from a garlic powder sample on proliferation of rat spleen lymphocytes in culture. Cells were stimulated with the combination of phorbol myristate acetate (PMA) and a Ca ionophore (A23187) or R73 monoclonal antibody (mAb) directed to the alphabeta chain of T cell receptor. It has been shown that both extracts significantly stimulated proliferation of lymphocytes. The effect correlated with upregulation of the Interleukin 2 receptor alpha (IL-2R alpha) expression and the increase in IL-2 production. Stimulation of IL-2 production by the extracts was higher in cultures with PMA/Ca ionophore than in cultures with R73 mAb. In contrast, both extracts stimulated production of IL-4 by splenocytes triggered by R73 mAb. The complete dependence of lymphocyte proliferation in cultures with R73 mAb and garlic extracts on IL-2 and IL-4 was demonstrated using neutralising mAbs to IL-2R alpha and IL-4. These results suggest that the potentiating effect of garlic extracts on lymphocyte proliferation in vitro differs depending on specific stimulators of cell proliferation and probably on the type of responding cells.     

Identification of immunodominant antigens by probing a whole Chlamydia trachomatis open reading frame proteome microarray using sera from immunized mice

Chlamydia trachomatis infections can lead to severe chronic complications, including trachoma, ectopic pregnancy, and infertility. The only effective approach to disease control is vaccination. The goal of this work was to identify new potential vaccine candidates through a proteomics approach. We constructed a protein chip array (Antigen Discovery, Inc.) by expressing the open reading frames (ORFs) from C. trachomatis mouse pneumonitis (MoPn) genomic and plasmid DNA and tested it with serum samples from MoPn-immunized mice. Two groups of BALB/c female mice were immunized either intranasally or intravaginally with live elementary bodies (EB). Another two groups were immunized by a combination of the intramuscular and subcutaneous routes with UV-treated EB (UV-EB), using either CpG and Montanide as adjuvants to favor a Th1 response or alum to elicit a Th2 response. Serum samples collected at regular intervals postimmunization were tested in the proteome array. The microarray included the expression products of 909 proteins from a total of 921 ORFs of the Chlamydia MoPn genome and plasmid. A total of 185 immunodominant proteins elicited an early and sustained antibody response in the mice immunized with live EB, and of these, 71 were also recognized by the sera from mice immunized with UV-EB. The reactive antigens included some proteins that were previously described as immunogenic, such as the major outer membrane protein, OmpB, Hsp60, and IncA and proteins from the type III secretion system. In addition, we identified in mice several new immunogens, including 75 hypothetical proteins. In summary, we have identified a new group of immunodominant chlamydial proteins that can be tested for their ability to induce protection.     

PCR detection of Ehrlichia ruminantium and Babesia bigemina in cattle from Kwara State,Nigeria: unexpected absence of infection

Parasitology Research - Ticks and tick-borne diseases (TBDs) continue to pose an insidious and ever-present threat to livestock and livelihoods across the globe. Two of the most significant TBDs of...     

Sodium dodecyl sulfate- and salt-extracted antigens from various Brucella species induce proliferation of bovine lymphocytes.

One- and two-dimensional gel electrophoresis and cellular immunoblotting were used to compare the protein profiles and immunogenic capabilities of salt- and sodium dodecyl sulfate-extracted components isolated from different Brucella species. Cellular immunoblotting demonstrated that freshly isolated bovine peripheral blood mononuclear cells proliferated to similar molecular mass components from the various Brucella extractions. One- and two-dimensional gel electrophoresis showed similarities in protein profiles among the different Brucella species that correlated with lymphocyte reactivity. The results presented in this study provide preliminary evidence that common proteins that induce lymphocyte proliferation are present among different Brucella species, suggesting that a genuswide subunit vaccine may be feasible.     

Cultured human alveolar macrophages from smokers with lung cancer: resolution of factors that stimulate fibroblast proliferation, production of collagenase, or prostaglandin E2

Marked connective tissue remodelling involves both destruction and repair in inflammatory lung diseases. Throughout the remodelling event, it was reasoned that alveolar macrophages may release substances similar to those produced by blood monocyte-macrophages that affect fibroblast functions, ie, the interleukin 1 family of monokines (or cytokines). We have examined human alveolar macrophage cultures obtained after bronchoalveolar lavage of freshly excised lungs from heavy smokers with bronchial carcinoma. Crude culture media contained fibroblast proliferative activity and collagenase- and PGE2- production-stimulating activity. The main peak of these biological activities was located around approximately 18 kilodaltons (kD) on gel filtration chromatography. Resolution of this peak by high performance liquid chromatography showed the presence of three distinct peaks, with quantitative and qualitative differences in biological activities. This suggests the presence of heterogeneous factors.     

Identification and molecular weight characterization of antigens from Candida albicans that are recognized by human sera.

Antigenic components in the cytoplasmic extract of Candida albicans were examined after fractionation by concanavalin A-Sepharose and DEAE-Sephacel ion-exchange chromatography. Fractions from the DEAE column were tested by fused rocket immunoelectrophoresis for their reactivity with antibodies in the sera of 20 patients with disseminated candidiasis. Three groups of fractions (regions A, B, and C) from the DEAE column were defined by their reactivity with these sera. Immunoblot analysis with 20 human sera identified 18 antigenic components in regions A, B, and C. Region A contained nine antigens, region B contained four antigens, and region C contained five antigens. Region A contained an antigen with an apparent molecular weight of 48,000 that was recognized by 7 of 10 sera from patients with disseminated candidiasis. Immunoprecipitation experiments with labeled proteins from region A and 51 human sera also demonstrated the presence of a major antigen whose apparent molecular weight is 48,000 to 52,000. The 48- to 52-kilodalton protein is an abundant protein in region A and is the most frequently recognized protein by antibodies in the sera of patients with disseminated candidiasis. Patients with disseminated candidiasis had significantly higher levels of antibody (immunoglobulin G) (P less than 0.001) directed against the 48- to 52-kilodalton protein than did patients with noninvasive forms of candidiasis, patients with other fungal infections, or normal, healthy persons.     

In vitro cytotoxicity by human lymphocytes from individuals immunized against histocompatibility antigens. II. Relation to HL-A incompatibility between effector and target cells

Lymphocytes from humans immunized by allogeneic skin grafts destroyed fibroblast monolayer cultures derived from the skin donor. Cytotoxicity also developed on several allogeneic fibroblast monolayers from unrelated persons. HL-A typing showed that all of these allogeneic fibroblasts shared one or more HL-A antigens with the skin donor. The intensity of the cytotoxic reaction increased with the number of these antigens present on the fibroblast targets, whereas no reaction occurred on allogeneic targets lacking these antigens or on the autochthonous fibroblasts. It is suggested, therefore, that the cytotoxic reaction reflects immunization against antigens within the HL-A system. An analogous correlation between the response of immunized lymphocytes and the number of immunizing HL-A antigens present was demonstrated in mixed lymphocyte cultures.

Lymphocytes from kidney grafted patients were not cytotoxic to any fibroblasts tested, including those from the kidney donor, not even during periods of clinical rejection. Humoral antibodies directed against the donor cells were demonstrated in one patient, but still no cytotoxicity occurred on the donor fibroblasts. Similarly negative results were obtained with lymphocytes from bone grafted patients.