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Biomedical subjects

D Abraham

Publications and source records attributed to D Abraham.

At least 91 records · Page 5Linked to original sources
PubMed1990

Dirofilaria immitis: molting process of third-stage larvae.

The objective of this study was to determine the molting process of Dirofilaria immitis third-stage larvae (L-3) to fourth-stage larvae (L-4), as it occurred in vitro. After 48 hr in vitro, the L-4 epicuticle was completely formed, and by 72 hr there was a clear separation between the L-3 and L-4 cuticles. The thickness of the newly formed L-4 cuticle was significantly less than that which has been described for larvae recovered from dogs after a similar incubation time period. If culture conditions were lacking in bovine albumin or proper temperature, larvae successfully developed the L-4 epicuticle but did not complete ecdysis. The molting process of D. immitis L-3 was thus shown to be multistepped with different factors required to induce the various developmental phases.

Animals↗
PubMed1990

Genetic control of murine immune responses to larval Dirofilaria immitis.

Previous studies have demonstrated that BALB/c mice, immunized against infection with Dirofilaria immitis, were capable of killing a significant percentage of challenge larvae found within diffusion chambers. The percentage of larvae killed by immunized mice was, however, less than in immunized dogs and unlike immunized dogs, mice were unable to retard the development of the surviving larvae. The objective of the present study was to test 3 inbred strains of mice to determine whether a higher level of protective immunity would develop in these hosts and if larval growth retardation would occur. DBA/2J and C57BL/6J mice and their F1 hybrids B6D2F1/J were used in these studies; it was determined that there were differences in susceptibility among the 3 strains but no difference in ability to eliminate larvae from challenge infections. Growth retardation was seen in larvae recovered from immunized DBA/2J and C57BL/6J mice but not in B6D2F1/J. No difference was noted between immune and control mice in the cell types found in the diffusion chambers. The predominant cell types seen were mononuclear macrophages, multinucleate syncytial cells, and neutrophils. Antibody responses to soluble third- and fourth-stage larval antigens and larval excretory/secretory antigens were measured. Although antibodies to all 3 antigen groups were found in higher concentrations in immunized mice than in their respective controls, only antibody responses to soluble L-3 antigens provided a clear correlation with protective immunity.

Animals↗
PubMed1989

Adhesion of lymphoid cells to fibroblasts in tissue culture.

In this study we have examined the cellular and molecular specificity of lymphocyte interaction with fibroblasts. Using mitogen-activated T-cells, we found that attachment to fibroblasts was highly sensitive to protease treatment, and to an antibody raised against the purified lymphocyte plasma membrane, but it was not mediated by the MEL-14 surface antigen or phosphomannosyl receptors. Lymphocyte interaction with fibroblasts was also unaffected by monoclonal antibodies against the LFA-1, Mac-1, and Class II MHC antigen complexes. In contrast, adhesion of both T- and B-lymphocytes was strongly inhibited by fucoidan, a polymer of sulphated fucose, whereas fucose, mannan, and mannose 6-phosphate had no effect. Both B- and T-lymphoid cell lines were able to recognise and adhere to fibroblasts, although the marked differences between the attachment of the different types of cell did not appear to be related to their immunological function. The attachment of most of the cell lines was prevented by the presence of fucoidan, whereas the inhibition of binding of each of the lymphoid lines in the presence of the anti-T-lymphocyte plasma membrane antibody varied widely. These findings suggest that lymphocyte attachment to fibroblasts involves multiple cell surface receptors, and that these are expressed at different levels on specific T- and B-cells.

Animals↗
PubMed1989

Human T-cell stimulation, molecular characterization and in situ mRNA localization of a Brugia malayi recombinant antigen.

Cellular immune responses play a major role in lymphatic filarial infections. To further our understanding of the host-parasite interaction, we investigated T-cell stimulation by purified filarial recombinant antigens in peripheral blood mononuclear cells derived from filarial-infected individuals. One of a subset of cloned Brugia malayi antigens involved in the humoral immune response to filarial infection was found to be a T-cell-stimulating antigen. The fusion protein encoded by clone lambda Bm19 induced proliferation of human T cells in a parasite-specific, antigen dose-dependent manner. The deduced amino acid sequence from this cloned region revealed 4 predicted T-cell recognition sites. The lambda Bm19 DNA sequence hybridizes to a 3-kb transcript, and in situ mRNA hybridization analyses of the adult female worm demonstrated that this gene is expressed in developing uterine microfilariae. The native parasite protein is present in several developmental stages since clone lambda Bm19 was initially identified with antiserum directed against the infective larval stage; this protein is therefore a potential target for the host's immune system.

Adult↗
PubMed1989

Immunity to larval Brugia malayi in BALB/c mice: protective immunity and inhibition of larval development.

The objective of this study was to analyze the immune response of mice to the larval stages of Brugia malayi. Male BALB/c mice were inoculated with 3 doses of irradiated third-stage larvae (L-3) of B. malayi and were subsequently challenged with L-3 implanted ip within diffusion chambers. After 3 weeks, larvae were recovered to determine their viability, length, and stage of development. A significant reduction in parasite survival was observed in immunized mice. Furthermore, larvae recovered from immunized mice were significantly shorter than larvae recovered from control mice. All larvae recovered from immunized mice were L-3, whereas 96% of larvae recovered from controls were fourth-stage larvae (L-4). Sera collected from control and immunized mice were tested for the presence of antibodies reactive with L-3 and L-4 antigens using an indirect fluorescent antibody assay employing frozen larval cross-sections as antigen. Sera recovered after challenge of control mice reacted with internal, but not surface, antigens of L-3 and L-4. Alternatively, sera from immunized mice reacted with both internal and external antigens of both L-3 and L-4.

Animals↗
PubMed1989

Fibroblast matrix and surface components that mediate cell-to-cell interaction with lymphocytes.

The interaction between lymphocytes and fibroblasts in vitro has been examined using a quantitative ELISA assay to measure the binding of T and B cells to monolayer cultures of human dermal fibroblasts. This was carried out on microtiter culture plates, using an anti-Thy-1 monoclonal antibody, to determine the attachment of murine T lymphocytes and an affinity-purified polyclonal anti-IgM antibody to measure B cell binding. Both types of lymphocyte were found to adhere strongly to intact human fibroblasts, and also had high levels of attachment to purified fibroblast plasma membranes and extracts of the fibroblast extracellular matrix. Attachment, particularly of B lymphocytes, also took place onto plastic surfaces coated with fibronectin, but not to collagens or to intact fibroblasts that had been fixed with a low concentration of paraformaldehyde. Lymphocyte binding to fibroblasts was partially prevented by a monoclonal antibody against fibroblast MHC class II antigens, but not against the class I membrane complex, or by polyclonal antiserum to the cell surface mannose 6-phosphate receptor. In addition, although both lymphocyte types were able to adhere to fibro-nectin, the presence of antibody against fibronectin or the synthetic peptide Arg-Gly-Asp-Ser, had no effect on their attachment to fibroblasts. Thus, lymphocyte adhesion may occur by fibronectin, but other types of interactions with fibroblasts also appear to take place.

Animals↗
PubMed1988

Cell contact induces the synthesis of a lysosomal enzyme precursor in lymphocytes and its direct transfer to fibroblasts.

The activity of a lysosomal enzyme, alpha-D-mannosidase (EC 3.2.1.24), increased markedly in normal lymphocytes when they were cultured together with fibroblasts from a patient with an inherited deficiency of this enzyme. Cell-to-cell contact was obligatory for this increase in activity, which also required new protein synthesis. The enzyme induced in the co-cultured lymphocytes was a high molecular weight form of alpha-D-mannosidase that was not detected in lymphocytes cultured alone, which had only the low molecular weight mature enzyme. It was this precursor form alone that was directly transferred to the mannosidosis fibroblasts, where it was present initially in organelles of low density. When the culture period was extended the lymphocyte precursor enzyme was transported to the heavy lysosomes in the recipient cells, and correctly processed to the functionally effective mature enzyme.

Animals↗
PubMed1988

Mycetoma of the forearm due to Actinomadura madurae.

A case of mycetoma, with abscess-like lesions which appeared on the right forearm of a 43-year-old male, is briefly reported. A few whitish granules were detected in the oily-like discharge, the same as in the histologic examination. No bone involvement was discovered in X-ray examination. Actinomadura was identified in the culture. A daily dose of 4 g bactrin brought significant improvement to the patients continuing the treatment.

Adult↗
PubMed1988

Lymphocytes transfer only the lysosomal form of alpha-D-mannosidase during cell-to-cell contact.

We have examined the changes in the activities of the different types of alpha-D-mannosidase when fibroblasts from patients deficient in the lysosomal form of the enzyme are cultured together with normal lymphocytes. Our results show that whereas the mannosidosis cells acquired high levels of this enzyme, the activities of both the Golgi and the endoplasmic reticulum forms of alpha-D-mannosidase remained the same as in the fibroblasts cultured alone in the absence of lymphocytes. The increase in the activity of the lysosomal enzyme in the cocultured fibroblasts was not affected by the presence of mannose 6-phosphate or alpha-methyl mannoside, inhibitors of receptor- and lectin-mediated uptake of lysosomal enzymes, respectively, but it did require cell-to-cell contact. Ion-exchange HPLC and electrophoresis in polyacrylamide gradient gels showed that the acquired enzyme had the same elution profile and molecular size as the lysosomal form of the enzyme present in the lymphocytes. Immunoprecipitation studies using antibody specific for the lymphocyte type of lysosomal alpha-D-mannosidase confirmed that the increased activity in the cocultured mannosidosis cells resulted from the acquisition of the lymphocyte enzyme. Cytochemical examination revealed, however, that the transferred lymphocyte enzyme was localized in cytoplasmic organelles in the peripheral regions of the recipient fibroblasts. These results show that lymphocytes transfer only the lysosomal form of alpha-D-mannosidase during cell-to-cell contact with mannosidosis cells.

Animals↗
PubMed1988

Dirofilaria immitis: surface properties of third- and fourth-stage larvae.

The objective of this study was to analyze surface properties of larval Dirofilaria immitis with potential relevance to protective immunity. Comparisons were made between third (L3)- and fourth-stage larvae (L4) based on their net surface charge, surface carbohydrate and antigen composition, ability to nonspecifically absorb host proteins, complement activation, and nonspecific cellular adherence. It was determined that L3 had a net negative surface charge, whereas L4 had either a neutral or weakly positive surface charge. The lectin Con A, but not any of the other lectins tested, bound only to the surface of L4, and not to that of L3. Monoclonal antibodies were prepared which reacted with the surface of L3 or with the surface of L4, but never both. L4 were found to nonspecifically adsorb host protein to their surfaces, whereas L3 did not. Both L3 and L4 were found to activate complement through the alternate pathway. Finally, nonspecific cellular adherence was found on L3 both in vitro and in vivo but not on L4. The surfaces of L3 and L4 were thus shown to be significantly different and, potentially, in ways which would have great impact in the generation and effectiveness of a protective immune response.

Animals↗
PubMed1988

Forms and intracellular distribution of alpha-D-mannosidases in murine liver and spleen.

1. The intracellular distribution of alpha-D-mannosidase in homogenates of murine liver and spleen was investigated by differential and gradient density centrifugation. 2. In both tissues an enzyme with a neutral pH optimum was found in the cytosol together with an alpha-D-mannosidase with optimal activity between pH 5.5 and 6.0 which was also partially membrane-bound. 3. In liver the acidic alpha-D-mannosidase was obtained almost entirely in a particulate form distributed equally between a heterogeneous low density region and heavy density lysosomes. 4. The lysosomal form of the liver enzyme was purified to electrophoretic homogeneity and shown to be a glycoprotein composed of four identical subunits of molecular weight 65 kDa. 5. Antibody raised against the purified liver alpha-D-mannosidase immunoprecipitated a polypeptide from spleen which had the same molecular size. This acidic enzyme was the predominant type of alpha-D-mannosidase in spleen, but in contrast to liver, it was obtained mainly in a cytosoluble form, the remaining activity being present in the heterogeneous light density compartment. 6. Although both tissues contain the same molecular form of the acidic alpha-D-mannosidase, in murine spleen this enzyme does not appear to be associated with stable heavy density lysosomes.

Animals↗
PubMed1988

Solubilization of epicuticular antigen from Dirofilaria immitis third-stage larvae.

The solubilization of epicuticle from third-stage (L3) Dirofilaria immitis larval cuticles was investigated. Cuticles collected after L3 had molted were incubated in 1.5% sodium dodecyl sulfate (SDS) at 37 degrees C with vigorous shaking. Solubilization of epicuticular layers was accomplished as demonstrated by electron microscopy. Diminished binding of an epicuticular specific monoclonal antibody (DIM-229) was seen when SDS-treated cuticles were compared to untreated cuticles in an indirect fluorescence antibody assay. Cuticles which were extracted further by boiling in 1.5% dithiothreitol (DTT) produced less protein than cuticles solubilized in SDS. Both extracts reacted with DIM-229 in an indirect enzyme-linked immunosorbent assay, indicating retention of antigenic reactivity of the solubilized epitope. SDS-polyacrylamide gel electrophoresis of SDS-derived antigens revealed, after silver staining, proteins from 12 to 77 kDa and only 1 band at 15 kDa for SDS-treated cuticles boiled in DTT. Western blot analyses of the extracts with DIM-229 were inconclusive.

Animals↗
PubMed1988

Effect of exposure time on the depth of polymerization of a visible light-cured composite resin.

The effect of exposure time of a visible light source on the depth of polymerization and degree of hardness of a sample of Occlusin posterior composite resin was investigated. The border between cured and non-cured composite resin was identified by a change in colour and by applying pressure with a scalpel. Knoop hardness tests were performed perpendicular to the long axis of illumination. The composite resin nearer to the light source underwent more complete polymerization. Increased exposure time resulted in greater depth of cure. The rate of polymerization was greatest in the first 10 s. Maximum hardness measured up to a depth of 1 mm obtained after 80 s of exposure time. At greater depth, a decrease in Knoop hardness was observed. At exposures under 80 s, maximum hardness was not achieved even at a depth of only 1 mm.

Chemical Phenomena↗
PubMed1988

Induction of protective immunity in dogs to infection with Dirofilaria immitis using chemically-abbreviated infections.

Four dogs were immunized against Dirofilaria immitis infection by a series of 3 larval infections which were each subsequently terminated by ivermectin treatment. Two control dogs received ivermectin treatment alone. Following the final ivermectin treatment, dogs were challenged with infective larvae by subcutaneous inoculation, both free and contained within diffusion chambers. Three weeks after larval challenge the chambers were removed and live larvae were enumerated. Seven months after challenge dogs were killed and necropsied to collect and count adult D. immitis. Chambers recovered from immunized dogs had 63% fewer larvae than chambers from control dogs. At necropsy, control dogs had a mean of 28.5 adult worms whereas the immunized animals had an average of 0.5 worms (range 0-2). Sera collected from immune dogs throughout the study had elevated antibody levels to third- and fourth-larval stage antigens. Significant levels of immune protection were achieved with this immunization regimen. The data suggest that a multiple-stage parasite killing occurs in immune animals. It was not possible to associate immune protection with any of the 5 antigen subsets.

Animals↗
PubMed1988

Ultrastructural studies of a lysosomal enzyme during lymphocyte activation.

A post-embedding immunogold technique has been used for the ultrastructural localization of a lysosomal enzyme, beta-glucuronidase, in resting and activated T- and B-lymphocytes. The results presented here show that mitogen-induced stimulation of T- and B-cells was associated with an increase in the amount of enzyme in the Golgi complex and rough endoplasmic reticulum, organelles which were rarely present in the resting lymphocytes.

Animals↗
PubMed1988

Adhesion of T and B lymphocytes to fibroblasts in tissue culture.

We have examined the direct interaction of lymphocytes with human fibroblasts in vitro. The results of our study, using radiolabelled lymphocytes, show that activated T cells rapidly adhered to the fibroblasts when the two types of cell were cultured together at 37 degrees, and that the residency time of lymphocyte-fibroblast contact was approximately 30 min. Lymphoblast adhesion did not require the presence of divalent cations, but it was temperature-dependent. Although the binding of the lymphocytes was not prevented by the presence of monosaccharides or by treatment with exoglycosidases, it was highly sensitive to low concentrations of trypsin. Attachment was also inhibited partially in the presence of the sulphated fucose polysaccharide, fucoidan. These results suggest that at least two distinct receptors are involved in lymphocyte adhesion to fibroblasts. The binding of B lymphoblasts to fibroblasts was much greater than that of the activated T cells. Resting, non-stimulated, B and T lymphocytes both bound only very poorly, showing that only mature lymphocytes express high levels of functionally effective adhesion proteins. Moreover, the results of competition experiments suggest that different cell surface components mediate the interaction of T and B cells with fibroblasts.

B-Lymphocytes↗
PubMed1988

Active and passive immunization of mice against larval Dirofilaria immitis.

The objective of this study was to determine if Dirofilaria immitis larvae would survive in diffusion chambers implanted in dogs and mice and secondly to determine if mice could be immunized against infection with D. immitis. Dirofilaria immitis third-stage larvae (L3) survived and grew in diffusion chambers implanted in dogs and mice for at least 3 wk. BALB/c mice, which were repeatedly infected with live L3, showed resistance to challenge infections. Dead L3, with or without adjuvants elicited no protective immunity. A correlation was found between the degree of immune protection seen in mice and antibody levels to soluble larval antigen but not to antibody levels to surface antigens. A monoclonal antibody was prepared that reacted with the surface of D. immitis and Onchocerca lienalis L3, but not to the surfaces of other stages and species of various filarial worms. When this antibody was administered to mice prior to challenge no significant reduction in larval survival was observed.

Animals↗