Biomedical subjects

D Abraham

Publications and source records attributed to D Abraham.

At least 73 records · Page 4Linked to original sources

PubMed1994

Expression and shedding of intercellular adhesion molecule 1 and lymphocyte function-associated antigen 3 by normal and scleroderma fibroblasts. Effects of interferon-gamma, tumor necrosis factor alpha, and estrogen.

OBJECTIVE: To examine intercellular adhesion molecule 1 (ICAM-1) and lymphocyte function-associated antigen 3 (LFA-3) in cultures of normal and systemic sclerosis (SSc) dermal fibroblasts. METHODS: The surface and soluble forms of ICAM-1 and LFA-3 were measured by flow cytometry and capture enzyme-linked immunosorbent assay, respectively. RESULTS: Surface ICAM-1 was significantly higher on SSc fibroblasts compared with normal controls. Beta-estradiol did not directly enhance ICAM-1 or LFA-3 expression in either normal or SSc cells, but significantly augmented the cytokine-induced increase in ICAM-1. Soluble ICAM-1 (sICAM-1) and sLFA-3 were detected in fibroblast cultures. While no difference was found in the level of sLFA-3, the shedding of sICAM-1 was significantly increased (P < 0.001) in cells from SSc patients. CONCLUSION: SSc fibroblasts express intrinsically elevated levels of surface ICAM-1 and release higher levels of sICAM-1 in vitro. Increased expression of ICAM-1 by interferon-gamma and tumor necrosis factor alpha alone, and the further induction in combination with beta-estradiol may underlie an aspect of fibroblast dysfunction in SSc and the female predisposition to the disease.

Antigens, CD↗

PubMed1994

Regulation of CD2-mediated heterotypic interactions of murine T lymphocytes.

In this study we have investigated the expression and function of the murine CD2 receptor in T cells. The surface level of CD2 increased between 300 and 400% when T cells were activated, which also induced a rapid increase of nearly 40-fold in the steady-state levels of CD2 mRNA. Although the activated lymphocytes were found to internalize and degrade nearly 50% of membrane-bound CD2, in addition to shedding the CD2 receptor from the cell surface, this does not account for the discrepancy between the increase in mRNA and antigen levels during activation. These findings suggest that the expression of CD2 is also regulated by post-transcriptional processes which control the translational efficiency of the CD2 message. Activation of T cells enhanced their interaction with mesenchymal cell targets (fibroblasts) via a CD2-dependent adhesion pathway which was not inhibited by the anti-mouse CD48 (sgp-60) mAb OX78, the only counter-receptor thus far identified on other cells as a ligand for the mouse CD2 receptor. Moreover, since murine fibroblasts were found not to express CD48, our results implicate a novel ligand for CD2, possibly a homologue of the human LFA-3. This new pathway of heterotypic T cell interaction would be facilitated by utilization of the large intracellular pool of CD2 transcripts to up-regulate CD2 expression and, as this receptor is a major signal-transducing molecule, would further enhance T cell activation and increase CD2-mediated adhesion.

Animals↗

PubMed1994

Inhibition of lipid biosynthesis induces the expression of the pspA gene.

Treatment of Escherichia coli with diazaborine strongly induces the synthesis of a 28 kDa protein which is associated with the cytoplasmic membrane. The partial amino acid sequence proved that this protein is identical to the phage shock protein PspA. The kinetics of the expression of the pspA gene were determined in an E. coli strain which carried a pspA-lacZ fusion in the chromosome. PspA synthesis is independent of the growth phase. It is, however, strongly induced when fatty acid biosynthesis is inhibited by diazaborine or cerulenin. Treatment with either compound also causes dose-dependent inhibition of phospholipid biosynthesis whose degree correlates with the induction of PspA. Another cause of induction of PspA synthesis is treatment of E. coli with globomycin, which is an inhibitor of the processing of lipoproteins.

Anti-Bacterial Agents↗

PubMed1993

Multi-level regulation of lysosomal gene expression in lymphocytes.

The expression of the gene coding for the lysosomal enzyme, beta-glucuronidase (Gus), was examined in functionally distinct T, B and plasma cell lines. Each of the different groups of cells had different intracellular levels of active Gus enzyme and numbers of Gus mRNA copies per cell. Analysis of the molecular forms of Gus mRNA and protein by Northern and Western blotting revealed that the different types of cells all produced a single mature 2.7 kb transcript and a 73 kDa polypeptide. However, the utilisation of the Gus mRNA to produce the Gus antigen, and the subsequent posttranslational processing of the polypeptide to generate the mature, enzymically active Gus, were found to be cell type-specific. Control of the functional expression of the Gus gene is thus exerted at both the transcriptional and translational levels, and appears to differ between different types of lymphocyte.

Animals↗

PubMed1993

Lysosomal enzyme transfer from different types of lymphoid cell.

The direct transfer of certain lysosomal enzymes during cell-to-cell contact between normal lymphocytes and enzyme-deficient recipient cells has previously been reported in vitro and may play an important role in the correction of lysosomal storage diseases by bone marrow transplantation in vivo. In the present study we have used a number of different T, B, and plasma cell lines to examine the expression and immunological specificity of the transfer of the lysosomal enzyme, beta-glucuronidase (Gus). Each of these groups of cell had differing intracellular and secreted levels of Gus activity, which were nevertheless similar within each group. Dermal fibroblasts deficient in the Gus enzyme acquired substantial amounts of additional activity when they were cultured together with the T cells, the B cells, or the plasma cells. This occurred by the direct transfer of Gus from all three types of cell. In addition, with plasma cells, which had very high intracellular enzyme activity and also secreted high levels of Gus into their culture medium, the secreted enzyme was readily internalized by the fibroblasts via the mannose 6-phosphate receptor (MPR). It was notable that the purified endogenous enzymes from plasma cells as well as from B cells, but not from T cells, were also endocytosed by the fibroblasts utilizing this receptor-mediated process. Although the Gus activity from all the cell lines examined had the same molecular size, polyacrylamide electrophoresis and isoelectric focusing patterns showed that the immunologically distinct types of lymphoid cell have characteristic, unique pathways of post-translational lysosomal enzyme processing. These results show that the transfer of lysosomal enzymes from lymphoid cells can occur by two distinct mechanisms, both likely to have important roles in enzyme replacement therapy.

B-Lymphocytes↗

PubMed1993

Lysosomal storage diseases: mechanisms of enzyme replacement therapy.

Lysosomal diseases result from deficiency of one of the many enzymes involved in the normal, step-wise breakdown of macromolecules. Studies in vitro have shown that cells from enzyme-deficient patients can be corrected by an exogenous supply of the missing enzyme. This occurs by receptor-mediated endocytosis of normal enzyme added to tissue culture medium and also by direct transfer from normal leukocytes during cell-to-cell contact. Immunohistochemical analysis has revealed that these processes have similar pathways of intracellular transport of the acquired enzymes, which ultimately reach mature lysosomes in the recipient cells. Moreover, recent studies suggest that both mechanisms are important in the therapy of lysosomal storage diseases by bone marrow transplantation. Advances in gene technology are likely to improve the successful treatment of these disorders, by facilitating the large scale production of clinically effective proteins and also by enabling the stable and safe introduction of normal lysosomal genes into cells of affected patients.

Bone Marrow Transplantation↗

PubMed1993

Fewer protective cytotoxic T-cell epitopes than T-helper-cell epitopes on vesicular stomatitis virus.

Cytotoxic T-lymphocyte (CTL) and T-helper-cell responses in various mouse strains were monitored. Protective CTL responsiveness against three proteins of vesicular stomatitis virus was H-2 linked and inducible only in half of the 15 combinations tested (each of five H-2 haplotypes combined with each of three viral proteins), whereas biologically relevant T-helper-cell responses were inducible in all. This suggests that vesicular stomatitis virus exhibits more T-helper-cell than CTL epitopes.

Animals↗

PubMed1993

Induction of protective immunity against larval Onchocerca volvulus in a mouse model.

BALB/cBYJ mice were immunized against larval Onchocerca volvulus by subcutaneous injection of normal, irradiated, or freeze-thaw-killed Onchocerca sp. larvae. The mice received challenge infections of O. volvulus third-stage larva (L3) contained in diffusion chambers implanted subcutaneously. At two-weeks postinfection, the diffusion chambers were removed and larval survival was assessed. When mice were immunized a single time with 35-krad-irradiated or normal O. volvulus L3, there was a significant reduction in the survival of challenge parasites. However, there was little or no reduction in challenge worm survival when mice were immunized a single time with freeze-thaw-killed O. volvulus L3 or fourth-stage larva (L4), or irradiated O. lienalis L3. When a second dose of freeze-thaw killed O. volvulus L3 or irradiated O. lienalis L3 was administered, there was a significant reduction in parasite survival in immunized mice. Immunization with O. volvulus L4 or a combination of L3 and L4 failed to confer protection. These results demonstrate that mice can be immunized against larval O. volvulus and that diffusion chambers are an efficient method for studying protective immunity to this parasite in a mouse model.

Animals↗

PubMed1993

Survival and development of larval Onchocerca volvulus in diffusion chambers implanted in primate and rodent hosts.

Third-stage larvae (L3) of Onchocerca volvulus were implanted in diffusion chambers in chimpanzees, mangabey monkeys, rhesus monkeys, squirrel monkeys, and inbred strains of mice, jirds, and rats for 3-63 days. At different times during the experimental period, larvae were recovered and assessed for their viability and development. Survival and growth rates were equal regardless of whether the implanted larvae were fresh or cryopreserved. Survival and growth rates of the larvae did not differ among the primate and rodent hosts tested, with the exception of squirrel monkeys and rats, which were resistant to infection. Molting from L3 to fourth-stage larvae began on day 3 and continued through day 14 in the primates and rodents. The primate and rodent models developed in the present study will be useful for the study of the immunology and chemotherapy of onchocerciasis.

Animals↗

PubMed1992

Animal models for the study of immunity in human filariasis.

A major challenge to the development of vaccines against human lymphatic filariasis and onchocerciasis is to direct the immune response toward elimination of the early, prepathogenic larval stages and away from responses that mediate pathology. In this review, James Lok and David Abraham discuss the various animal models that have been used to investigate the pathways leading to immunity, immunological tolerance and chronic pathology in these diseases. Owing to the strict host specificities of the human-dwelling filariae, no single model serves to duplicate exactly all these aspects. Nevertheless, it has been possible to demonstrate a protective immune response invoked by and directed against incoming third-stage larvae of both lymphatic and skin-dwelling filariae. The fact that subsets of the sequelae of human filarial infection can be duplicated in animal systems should also aid in unravelling the mechanisms determining the course of infection and in ensuring that vaccine candidates do not produce an inappropriate immunopathological response. A proposed scheme for using animal models in screening candidates for a vaccine against Onchocerca volvulus is presented.

Journal Article↗

PubMed1992

Identification of surrogate rodent hosts for larval Onchocerca lienalis and induction of protective immunity in a model system.

The objectives of this project were to screen a variety of inbred rodent species and strains to determine their usefulness as surrogate hosts for the study of the early larval development of Onchocerca lienalis and then to use a selected model to study the induction of protective immunity. In the primary screen, 6 strains of mice, 5 strains of rats, jirds, and multimammate rats were tested. Animals were infected with fresh O. lienalis by subcutaneous implantation of third-stage larvae (L3) contained in diffusion chambers covered with 5.0-microns pore-size membranes. After 7 days the chambers were recovered, and larval viability and growth were assessed. Approximately one-half of inoculated larvae were recovered alive regardless of the host tested. Larvae were implanted in CBA/J and DBA/2J mice in chambers covered with membranes that prevented host cells from entering; survival and growth rates of the larvae were not altered by the absence of cells from the chambers. Cryopreserved larvae were implanted in chambers with 5.0-microns pore-size membranes in CBA/J and DBA/2J mice and Wistar Furth rats for 3-28 days. No statistically significant difference was seen in the larval recoveries on days 3-28 in all 3 hosts. Statistically significant increases in length were seen in the 3 strains from day 3 to day 14, after which growth appeared to cease. Molting from L3 to fourth-stage larvae was observed in all 3 hosts beginning on day 3, with most larvae completing the molt by day 7.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1991

Simultaneous measurement of cell surface and intracellular antigens by multiple flow cytometry.

We have employed a method for permeabilizing lymphocytes with the detergent saponin in order to detect an intracellular protein simultaneously with surface antigens by flow cytometry (FCM). Using monoclonal antibodies specific for the murine CD2 receptor and for the lysosomal enzyme, beta-glucuronidase (Gus), we found that the expression of both of these antigens increased markedly when T cells were activated. Two sensitive methods were used to show that FCM provided an accurate measure of the actual number of CD2 and Gus molecules present in the lymphocytes. Immunogold electron microscopy revealed the precise ultrastructural localization of these different components and corroborated the specificity of the multiple labelling procedure for the simultaneous detection of surface and intracellular antigens. We also developed a three-colour FCM technique which we used to examine the changes in Gus expression in the CD4 and CD8 T cell sub-sets during activation.

Animals↗

PubMed1991

Expression and function of surface antigens on scleroderma fibroblasts.

Dermal fibroblasts from patients with systemic sclerosis (SSc) bound a much greater number of T lymphocytes than did normal dermal fibroblasts. Monoclonal antibodies (MAb) against classes I and II antigens of the major histocompatibility complex (MHC) and their receptors, CD8 and CD4, had no effect on T cell interaction with SSc and normal cells, while MAb against lymphocyte function-associated antigen type 3 (LFA-3) and CD2 both strongly inhibited lymphocyte attachment. MAb against intercellular adhesion molecule type 1 (ICAM-1) and LFA-1 also prevented binding of T lymphocytes, but had a more marked effect on adhesion to SSc fibroblasts than to normal fibroblasts; they also completely abolished the increased binding to fibroblasts treated with interleukin-1 alpha, tumor necrosis factor alpha, and interferon-gamma. No difference was found in the proportion of normal and SSc fibroblasts that expressed MHC classes I and II and LFA-3, but more SSc cells expressed ICAM-1, and at a higher level, than did normal fibroblasts. These results show that cultured SSc cells have elevated binding to T lymphocytes, which possibly results from expansion of a subset of fibroblasts that produces high levels of ICAM-1.

Adult↗

PubMed1991

Histochemical localization of gene expression in Onchocerca volvulus: in situ DNA histohybridization and immunocytochemistry.

We report here the development of in situ hybridization and immunohistochemistry protocols which permit the histological identification of gene expression of a cloned antigen of Onchocerca volvulus, OI5, in the parasite. Skin nodules containing female adult worms were fixed in a modified Carnoy's fixative and embedded in paraffin. Histological staining of tissue sections revealed uniformly excellent morphology and RNA preservation. To localize mRNA by in situ hybridization, tissue sections were incubated with biotin-labeled pOI5, the plasmid containing the genomic sequence of the antigen, and hybridization signals were histochemically visualized using a streptavidin-enzyme conjugate and chromogenic substrates. The protein antigen was localized immunohistochemically by incubating the sections with specific antibodies prepared against a recombinant fusion protein containing the OI5 sequence (OI3), and visualized via a secondary antibody-biotin-enzyme conjugate procedure. The results reported here showed distinct localization of the OI5 mRNA and OI3 antigen in specific cellular and tissue regions of the adult parasite, and in microfilariae located within the uteri and in the surrounding host tissue. The specificity and high sensitivity of these histological detection methods should be generally applicable for the characterization of gene expression in the filarial parasite, particularly the insect-borne, infective filarial larvae, which are severely limited in quantity.

Animals↗

PubMed1991

Contact-dependent transfer of a lysosomal enzyme from lymphocytes to fibroblasts.

In this study we have examined the mechanism underlying the contact-mediated transfer of a lysosomal enzyme from lymphocytes to fibroblasts in culture. We found that although antibody against the mannose 6-phosphate lysosomal targetting receptor (MPR) completely inhibited fibroblast uptake of the lysosomal enzyme beta-glucuronidase (Gus) from the culture medium, it had no effect on the transfer of the enzyme from normal lymphocytes. In contrast, the presence of antibody that prevented the adhesion of the lymphocytes to the fibroblasts inhibited Gus acquisition but had no effect on endocytosis. Immunogold electron microscopy of the contact site between the two types of cell showed that the transfer of Gus involved uncoated vesicles localized near the cell surface of the fibroblast at sites of contact with the lymphocytes. The acquired lymphocyte enzyme was shown to be transported to the fibroblast lysosomes.

Animals↗

PubMed1991

Passive transfer of protective immunity to larval Dirofilaria immitis from dogs to BALB/c mice.

Protective immunity to larval Dirofilaria immitis has been demonstrated in both the natural host, the dog, and in an experimental host, the mouse. In the present study, sera were collected and pooled from dogs that had been shown to have protective immunity to larval D. immitis. The pooled serum was inoculated into normal BALB/cByJ mice that then were challenged with third-stage larvae (L3) implanted in diffusion chambers. Two weeks postchallenge no significant difference was seen in either parasite survival or growth. Three weeks postchallenge, there was a significant decrease in parasite survival in mice receiving serum from immune dogs. Living larvae recovered at 3 wk postchallenge were significantly shorter than cohorts recovered from control mice. Antibody responses to L3 and forth-stage larvae (L4) surface antigens, to L3 and L4 aqueous soluble antigens, and to an excretory-secretory antigen fraction were measured. Only antibody responses to L3 surface antigens were elevated in the immune serum as compared to controls, thus suggesting a possible role for antibodies with specificity for surface antigens in protective immunity.

Animals↗

PubMed1990

The activation of resting lymphocytes is accompanied by the biogenesis of lysosomal organelles.

The degradative activity of lymphocytes plays by important role in a number of essential immune functions. In the present study we have examined how the activation of resting lymphocytes, by the mitogen concanavalin A (Con A), affects three major components of the lysosomal compartment: the lysosomal enzyme beta-glucuronidase (Gus); an integral lysosomal membrane protein (LAMP-1); and the mannose 6-phosphate receptor (MPR) which directs lymphocyte enzyme transport. Resting T cells were found to contain only very low levels of these proteins, but they were actively synthesized by, and far more abundant in, stimulated lymphoblasts. Although the lysosomal antigens did not have a distinct cytoplasmic localization in the resting lymphocytes, in the activated T lymphoblasts they were present in several highly developed intracellular structures, including the rough endoplasmic reticulum and the Golgi complex. Furthermore, in these latter cells Gus was also found to be accumulated within the lumen of large vesicles which we characterized as lysosomes by the presence of LAMP-1 at the periphery and by the absence of MPR. Subcellular fractionation confirmed that these organelles were present in the activated lymphocytes only, and not in the resting T cells. Our results demonstrate that lymphocyte activation is accompanied by the synthesis of the enzymic and structural components of the lysosomal compartment which are sorted and assembled into distinct organelles in the activated cell.

Animals↗

PubMed1990

Interactions between lymphocytes and dermal fibroblasts: an in vitro model of cutaneous lymphocyte trafficking.

Cultures of dermal fibroblasts were established from skin biopsies of CBA mice and used to study the interactions with murine T-lymphocytes. Electron microscopy showed that zones of contact developed between the fibroblasts and the T-cells, particularly after mitogenic activation. The adhesion of the lymphocytes was temperature-dependent, and many more lymphoblasts than resting cells attached to the fibroblast monolayers. Flow cytometry analysis of the adherent population showed that the most prominent type of resting lymphocyte was of the CD4 phenotype, which was also observed using a T-helper lymphoid cell line. However, neither the CD4 nor the CD8 (T-cytotoxic) antigens were involved in the binding process, and while the fibroblasts expressed Class I MHC molecules (but not Class II), these also had no role in mediating lymphocyte adhesion. Although the fibroblasts did not express the ligand Mala-2, the murine homologue of human ICAM-1, a monoclonal antibody against LFA-1, its cognate receptor on the lymphocytes, nevertheless effectively inhibited binding. T-cell attachment was also partially prevented by antibody against the lymphocyte CD2 antigen and by RGDS, a protein epitope known to mediate a number of receptor-integrin interactions. Moreover, this peptide also rapidly and preferentially detached T-lymphocytes which had previously adhered to the fibroblast monolayers. Lymphocyte binding was substantially elevated following treatment of the fibroblasts with cytokines such as tumor necrosis factor-alpha and interferon-gamma, but not interleukin-1 alpha. This increase in adhesiveness was, however, almost completely abolished by monoclonal antibodies specific for LFA-1 or for Mala-2. The results of this study show that while lymphocytes recognize fibroblasts normally via a number of constitutively expressed receptor-integrin interactions, their adhesion can also be modulated by cytokine-induced changes in the expression of other surface ligands.

Animals↗