Purification and partial characterization of a 32-kilodalton sialic acid-specific lectin from Aspergillus fumigatus

Adherence of the opportunistic fungus Aspergillus fumigatus to the extracellular matrix components is considered a crucial step in the establishment of the infection. Given the high carbohydrate content of these glycoproteins and the role of carbohydrate-protein interactions in numerous adherence processes, the presence of a lectin in A. fumigatus was investigated. Different fungal extracts obtained by sonication or grinding in liquid nitrogen from resting or swollen conidia, as well as from germ tubes and mycelium, were tested by hemagglutination assays using rabbit erythrocytes. A lectin activity was recovered in all the extracts tested. However, sonication of resting conidia resulted in the highest specific activity. Purification of the lectin was achieved by gel filtration followed by ion-exchange and hydrophobic-interaction chromatographies. Analysis of the purified lectin by sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed an apparent molecular mass of 32 kDa, which is similar to that of the alkaline protease already identified from different strains of A. fumigatus. However, as evidenced by the use of an alkaline protease-deficient mutant, the two activities were supported by distinct proteins. In addition, hemagglutination inhibition experiments using different saccharides and glycoproteins demonstrated the specificity of the lectin for sialic acid residues. Together these results suggest that this lectin may contribute to the attachment of conidia to the extracellular matrix components through the recognition of the numerous terminal sialic acid residues of their carbohydrate chains.     

Inhibition of Migration of Mouse Macrophages by Tuberculin-Sensitive Mouse Lymphocytes and by Mouse Migration Inhibitory Factor

The guinea pig migration inhibition technique, an accepted in vitro correlate of delayed hypersensitivity, has been adapted to a murine system. Peritoneal exudate cells from CF-1 mice vaccinated with viable cells of the H37Ra strain of Mycobacterium tuberculosis were inhibited in vitro by purified protein derivative (PPD) or whole H37Ra microorganisms. Peritoneal exudate cells from the inbred C57Bl/6 mice immunized with H37Ra cells also were inhibited in vitro by PPD or whole H37Ra microorganisms. Migration inhibitory factor (MIF) was produced by splenic lymphocytes from the H37Ra-immunized C57Bl/6 mice when incubated with either antigen. Intravenous injection of PPD or viable H37Ra organisms into H37Ra mice resulted in MIF production in vitro by splenic lymphocytes without further antigenic stimulation. Peritoneal exudate cells from nonimmunized C57Bl/6 mice and supernatant fluids from cultures of lymphocytes from nonimmunized C57Bl/6 mice were not inhibited in the presence of antigen. The production of MIF by splenic lymphocytes from immunized C57Bl/6 mice depended upon the conditions under which the lymphocytes were cultured, the time of exposure to antigen (3 days), the use of a higher concentration of PPD for stimulation of lymphocytes than that required for guinea pig cells, and also the use of cells from a highly inbred mouse strain.     

Modification of collagen matrices for enhancing angiogenesis

The vascularization of engineered tissues in many cases does not keep up with the ingrowth of cells. Nutrient and oxygen supply are not sufficient, which ultimately leads to the death of the invading cells. The enhancement of the angiogenic capabilities of engineered tissues therefore represents a major challenge in the field of tissue engineering. The immobilization of angiogenic growth factors may be useful for enhancing angiogenesis. The most potent angiogenic growth factor specific to endothelial cells, vascular endothelial growth factor (VEGF), occurs in several splice variants. The variant with 165 amino acids both has a high angiogenic activity and a high affinity for heparin. We therefore incorporated heparin molecules into collagen matrices by covalently cross-linking them to amino functions on the collagen. Physical binding of VEGF to the heparin may then prevent a rapid clearance from the implant, while the release rate may become coupled to the degradation of the collagen matrix. The modified matrices were characterized by determination of the extent of the heparin immobilization, the in vitro degradation rate by collagenase. For testing the angiogenic properties, non-modified and heparinized collagen specimens were--either loaded with VEGF or non-loaded--subcutaneously implanted on the back of rats. Specimens were explanted after varying periods of implantation, the dry weights and the hemoglobin contents, as well as immunostained histological sections were evaluated: heparinized collagen matrices loaded with VEGF are vascularized to a substantially higher extent as compared to non-modified matrices.     

Babesia bovis merozoite surface antigen 1 and rhoptry-associated protein 1 are expressed in sporozoites, and specific antibodies inhibit sporozoite attachment to erythrocytes

We examined Babesia bovis sporozoites for the expression of two molecules, merozoite surface antigen 1 (MSA-1) and rhoptry-associated protein 1 (RAP-1), that are postulated to be involved in the invasion of host erythrocytes. Both MSA-1 and RAP-1 were transcribed and expressed in infectious sporozoites. Importantly, monospecific MSA-1 and RAP-1 antisera each inhibited sporozoite invasion of erythrocytes in vitro. This is the first identification of antigens expressed in Babesia sp. sporozoites and establishes that, at least in part, sporozoites and merozoites share common targets of antibody mediated inhibition of erythrocyte invasion.     

Stimulation of T-helper cell gamma interferon and immunoglobulin G responses specific for Babesia bovis rhoptry-associated protein 1 (RAP-1) or a RAP-1 protein lacking the carboxy-terminal repeat region is insufficient to provide protective immunity against virulent B. bovis challenge

Rhoptry-associated protein 1 (RAP-1) is a targeted vaccine antigen for Babesia bovis and Babesia bigemina infections of cattle. The 60-kDa B. bovis RAP-1 is recognized by antibodies and T lymphocytes from cattle that recovered from infection and were immune to subsequent challenge. Immunization with native or recombinant protein was reported to reduce parasitemias in challenged animals. We recently reported that the NT domain of B. bovis RAP-1 contained immunodominant T-cell epitopes, whereas the repeat-rich CT domain was less immunostimulatory for T lymphocytes from cattle immune to B. bovis. The present study was therefore designed to test the hypothesis that the NT region of RAP-1, used as a vaccine with interleukin-12 and RIBI (catalog no. R-730; RIBI Immunochem Research, Inc., Hamilton, Mont. [now Corixa, Seattle, Wash.]) adjuvant to induce a type 1 response, would prime calves for antibody and T-helper cell responses comparable to or greater than those induced by full-length RAP-1 containing the C-terminal repeats. Furthermore, a type 1 immune response to RAP-1 was hypothesized to induce protection against challenge. Following four inoculations of either recombinant full-length RAP-1 or RAP-1 NT protein, RAP-1-specific immunoglobulin G (IgG) titers, T-lymphocyte proliferation, and gamma interferon production were similar. Similar numbers of NT region peptides were recognized. However, in spite of the presence of strong RAP-1-specific IgG and CD4(+)-T-lymphocyte responses that were recalled upon challenge, neither antigen stimulated a protective immune response. We conclude that successful priming of calves with recombinant RAP-1 and adjuvants that elicit strong Th1 cell and IgG responses is insufficient to protect calves against virulent B. bovis challenge.     

Altered chromatin landscape in circulating T follicular helper and regulatory cells following grass pollen subcutaneous and sublingual immunotherapy

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Interleukin-6 regulates the cytotoxic effect of tumour necrosis factor on U937 cells.

In this study, we demonstrate that low but not high concentrations of interleukin-6 (IL-6) potentiate the cytotoxic effect of tumour necrosis factor-alpha (TNF-alpha) on U937 cells, in a dose-dependent manner. Killing of U937 cells by 100 U/ml of TNF-alpha, was maximally potentiated by 50 U/ml of IL-6. No potentiation of cell killing was observed when the concentration of IL-6 was increased to 4000 U/ml. At a concentration of 50 U/ml, IL-6 up-regulated TNF receptor expression but no change in TNF receptor number was observed when the concentration of IL-6 was increased to 4000 U/ml. Low concentrations of IL-6 can also induce sub-cytotoxic doses of TNF-alpha (0.1 and 0.33 U/ml) to kill U937 cells. Up-regulation of TNF receptors by IL-6 is dependent on de novo protein synthesis since receptor induction is abolished in the presence of cycloheximide. Taken together the data suggest that the potentiation of cell killing observed by a combination of these lymphokines is mediated in part by IL-6-induced changes in TNF receptor expression.     

Expression of Anaplasma marginale major surface protein 2 operon-associated proteins during mammalian and arthropod infection

The antigenically variant major surface protein 2 (MSP2) of Anaplasma marginale is expressed from a 3.5-kb operon that contains, in a 5'-to-3' direction, four open reading frames, opag3, opag2, opag1, and msp2. This operon structure was shown to be conserved among genotypically and phenotypically distinct A. marginale, A. ovis, and A. centrale strains. The individual OpAG amino acid sequences are highly conserved among A. marginale strains, with identities ranging from 95 to 99%. OpAG2 and OpAG3 were expressed by all examined A. marginale strains during the acute rickettsemia in the mammalian host and, like MSP2, localize to the bacterial surface. OpAG2 and OpAG3 were also expressed in an infected Ixodes scapularis tick cell line. In contrast, the same A. marginale strains expressed only OpAG2 in two different Dermacentor spp. during transmission feeding. OpAG1 expression was not detected in the infected mammalian host, the infected tick cell line, or within infected Dermacentor ticks. The differential expression of outer membrane proteins from within an operon is a novel finding in tick-transmitted bacteria, and the regulation of expression may be broadly applicable to understanding how the pathogen adapts to the mammalian host-tick vector transition.