Proteome of Rhipicephalus sanguineus tick saliva induced by the secretagogues pilocarpine and dopamine.

One dimensional gel electrophoresis was used to separate proteins from the saliva of Rhipicephalus sanguineus female ticks fed on rabbits. Gel slices were subjected to tryptic digestion and analyzed by reversed-phase HPLC followed by MS/MS analysis. The data were compared to a database of salivary proteins of the same tick and to the predicted proteins of the host. Saliva was obtained by either pilocarpine or dopamine stimulation of partially fed ticks. Electrophoretic separations of both yielded products that were identified by mass spectrometry, although the pilocarpine-derived sample was of much better quality. The majority of identified proteins were of rabbit origin, indicating the recycling of the host proteins in the tick saliva, including hemoglobin, albumin, haptoglobin, transferring, and a plasma serpin. The few proteins found that were previously associated with parasitism and blood feeding include 2 glycine-rich, cement-like proteins, 2 lipocalins, and a thyropin protease inhibitor. Among other of the 19 tick proteins identified, albeit with undefined roles, were SPARC and cyclophilin A. This catalog provides a resource that can be mined for secreted molecules that play a role in tick-host interactions.

The D7 family of salivary proteins in blood sucking diptera.

The D7 subfamily of salivary proteins is widespread in blood sucking Diptera and belongs to the superfamily of pheromone/odourant binding proteins. Although D7 proteins are among the most abundant salivary proteins in adult female mosquitoes and sand flies, their role in blood feeding remains elusive. In the present work we report the sequence of seventeen novel D7 proteins, and propose an evolutionary scenario for the appearance of the several forms of this protein, based on a total of twenty-one sequences from Culex quinquefasciatus, Aedes aegypti, Anopheles gambiae, An. arabiensis, An. stephensi, An. darlingi mosquitoes and Lutzomyia longipalpis and Phlebotomus papatasi sand flies.

Mannan-binding lectin enhances susceptibility to visceral leishmaniasis.

Levels of the serum opsonin mannan-binding lectin (MBL) were directly correlated with the probability of developing visceral leishmaniasis. Monocytes infected with MBL-opsonized Leishmania chagasi promastigotes secreted higher levels of tumor necrosis factor alpha and interleukin-6 than cells infected with nonopsonized parasites. Our findings indicate that MBL can modulate the clinical outcome of infection with L. chagasi and the function of infected macrophages.

Tumor necrosis factor-alpha production and disease severity after immunization with enriched major core protein (p26) and/or infection with equine infectious anemia virus.

Cardinal features of equine infectious anemia (EIA) include fever, hemolytic anemia and thrombocytopenia during the acute phase of the disease, and cachexia and anemia seen during the chronic phase. These signs are thought to result from the release of inflammatory cytokines such as TNF-alpha. In order to determine if TNF-alpha has a role in the pathogenesis of acute EIA and vaccine-induced disease enhancement, we measured plasma concentrations of TNF-alpha in ponies immunized with virus enriched major core protein-p26 and/or experimentally infected with EIAV. Naturally infected inapparent EIAV carriers were also studied. TNF-alpha levels were determined by means of a WEHI 164, clone 13 cytotoxicity assay. We show a significant positive temporal correlation between TNF-alpha levels, severity of symptoms (fever and thrombocytopenia) and viremia. Furthermore, TNF-alpha levels also correlate with strain virulence and the disease enhancement seen in vaccinated ponies. Of this group of animals, those challenged with a heterologous virulent strain presented the most unfavorable outcome as well as the highest levels of TNF-alpha and viremia. The TNF-alpha activity observed in the bioassay was completely abrogated by a polyclonal rabbit anti-human TNF-alpha antiserum, thus confirming the specificity of the plasma cytotoxicity. Our observations indicate that TNF-alpha production correlates with the outcome of infection with EIAV.

Immune evasion by Trypanosomatidae: normal aggregated immunoglobulin protects against lysis by the alternative complement pathway.

Immunoglobulin Fc receptors (FcRs), present in Trypanosomatidae pathogenic for mammals, may be a mechanism by which these parasites escape the host immune response. We studied the possible role of these receptors in evasion by the alternative complement pathway. Promastigotes of Leishmania amazonensis and trypsinized trypomastigotes of Trypanosoma cruzi treated with heat-aggregated normal gamma globulin and then incubated with fresh normal guinea pig serum were shown to be more resistant to lysis. When compared to log phase Leishmania promastigotes, this resistance was at least 4.5-fold greater in parasites harvested in the stationary growth phase. EDTA and EGTA plus MgCl2 inhibited the cytotoxic effect of serum, suggesting the participation of the alternative complement pathway. The distribution of FcRs among genera of Trypanosomatidae that are pathogenic, infective or noninfective for mammals and their affinity for mammalian and fowl immunoglobulin were also examined. These receptors are present only in species infective or pathogenic for mammals, a finding that suggests that this structure is essential for the establishment of infection but is not necessarily a virulence factor. Furthermore, the ligand specificity is limited to the immunoglobulin of mammalian but not of fowl origin.

Immune evasion by Trypanosomatidae: normal aggregated immunoglobulin protects against lysis by the alternative complement pathway

Immunoglobulin Fc receptors (FcRs), present in Trypanosomatidae pathogenic for mammals, may be a mechanism by which these parasites escape the host immune response. We studied the possible role of these receptors in evasion by the alternative complement pathway. Promastigotes of Leishmania amazonensis and trypsinized trypomastigotes of Trypanossoma cruzi treated with heat-aggregated normal gamma globulin and then incubated with fresh normal guinea pig serum were shown to be more resistant to lysis. When compared to log phase Leishmania promastigotes, this resistance was at least 4.5-fold greater in parasites harvested in the stationary growth phase EDTA and egta PLUS MgCl2 inhibited the cytotoxic effect of serum, suggesting the participation of the alternative complement pathway. The distribution of FcRs among genera of Trypanosomatidae that arepathogenic, infective or noninfective for mammals and their affinity for mammalian and fowl immunoglobulin were also examined. These receptors ara presented only in species infective or pathogenic for mammals, a finding that suggests that this structure is essential for the establishment of infection but in not necessarily a virulence factor. Further more, the ligand specificity is limited to the immunoglobulin of mammalian but not of fowl origin

A crude extract of Artocarpus integrifolia contains two lectins with distinct biological activities.

The crude extract derived from seeds of Artocarpus integrifolia (jack fruit) contains two fractions with different biological activities for lymphocytes. One fraction is the D-galactose-binding lectin, jacalin, obtained by affinity purification on a D-galactose agarose column. The other, which is a component of the flow-through fraction (FT), is responsible for the mitogenic activity observed with human PBMC and murine spleen cells. In contrast, jacalin inhibits FT- and ConA-induced proliferative activity of human PMBC and murine spleen cells. This inhibition is not due to toxicity, because: (1) jacalin induces significant levels of IL-3/GM-CSF but not of IL-2 and/or IL-4 in murine spleen cells; (2) jacalin does not affect the capacity of these cells to secrete IL-2 or IL-4 as supernatants obtained from spleen cells sequentially stimulated with jacalin and ConA contain IL-2 and/or IL-4 as well as IL-3/GM-CSF. The ligand for the mitogen contained in the FT fraction is D-mannose as determined by sugar inhibition studies.

Activation of T and B cells by a crude extract of Artocarpus integrifolia is mediated by a lectin distinct from jacalin.

The biological activities previously described for a crude extract derived from seeds of Artocarpus integrifolia (jack fruit) are shown in the present work to be assigned to two distinct fractions present in this extract. One fraction is the D-galactose binding lectin, jacalin, obtained by affinity purification on a D-galactose Sepharose column. The other fraction is a D-mannose-binding protein which we propose to call 'Artocarpin'. As is well documented, jacalin binds to human IgA1 and is a useful tool for the purification of this immunoglobulin. We show here that the remaining biological activities consisting of the proliferative response of mouse spleen cells and human peripheral blood mononuclear cells and polyclonal activation of human and mouse B cells for the secretion of immunoglobulin are mediated by artocarpin. Artocarpin is unique in its capacity to induce polyclonal activation of B cells in the absence of proliferation. BALB/c nu/nu spleen cells failed to proliferate which indicates that this lectin is a T cell-dependent B cell polyclonal activator.

Lectins discriminate between pathogenic and nonpathogenic South American trypanosomes.

Cell surface carbohydrates of Trypanosoma cruzi, Trypanosoma rangeli, and Trypanosoma conorhini were analyzed by a micro-agglutination assay employing 27 highly purified lectins and by binding assays using various 125I-labeled lectins. The following seven lectins discriminated between the trypanosomes: 1) tomato lectin (an N-acetyl-D-glucosamine-binding protein), both in purified form and as crude tomato juice; 2) Bauhinea purpurea and Sophora japonica lectins (both N-acetyl-D-galactosamine-binding proteins), which selectively agglutinated T. cruzi; 3) Vicia villosa (an N-acetyl-D-galactosamine-binding protein) which was specific for T. rangeli; 4) peanut lectin (a D-galactose-binding protein) both in purified form and as crude saline extract; and 5) Ulex europaeus and Lotus tetragonolobus (both L-fucose-binding proteins) lectins which reacted only with T. conorhini. Binding studies with 125I-labeled lectins were performed to find whether unagglutinated cells of the three different species of trypanosomes might have receptors for these lectins, in which case absence of agglutination could be due to a peculiar arrangement of the receptors. These assays essentially confirmed the agglutination experiments.

Receptor for immunoglobulin Fc on pathogenic but not on nonpathogenic protozoa of the Trypanosomatidae.

Members of the Trypanosomatidae were studied for their ability to acquire host IgG through a possible Fc receptor. A simple rosette test was devised in which the different species and forms of protozoa were mixed with SRBC sensitized with subagglutinating does of IgG, IgM, and F (ab') 2 anti-SRBC, and the pelleted mixture was observed for the number of clumps (rosettes) formed between the parasites and SRBC. Rosettes were formed between parasites and SRBC sensitized with IgG but not with IgM or F(ab')2, indicating the presence of a receptor for IgG Fc. The specificity of this receptor for Fc was confirmed by inhibition experiments with normal rabbit aggregated gammaglobulins or with purified normal rabbit Fc. The receptor is sensitive to treatment with trypsin but regenerates after a short period of incubation (1 h), which indicates that it is synthesized by the parasite itself. Interesting was the observation that only pathogenic members of the Trypanosomatidae formed rosettes with sensitized SRBC. In none of the nonpathogenic forms studied could we demonstrate the Fc receptor. Also important was the finding that freshly isolated blood stream forms of Trypanosoma cruzi from infected mice did not form rosettes. However, after trypsinization, these forms clearly displayed the ability to do so, possibly indicating a previous acquisition of the host IgG by the parasites in the mouse blood stream. These findings point to a possible and important means of parasite evasion of the host immune response by masking their surface with host IgG.