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Biomedical subjects

D Gebhard

Publications and source records attributed to D Gebhard.

16 recordsLinked to original sources
PubMed2000

Infection of a canine macrophage cell line with leishmania infantum: determination of nitric oxide production and anti-leishmanial activity.

We have previously shown that resistance to Leishmania infantum in dogs is associated with a Th1 type of immune response. In this study, we use a canine macrophage cell line (030-D) that can readily be infected with this protozoan parasite. Our aim is to further characterize the effector mechanisms involved in killing of Leishmania parasite in dogs. We observed that activation of 030-D cells by incubation with a supernatant derived from a Leishmania-specific T cell line containing IFN-gamma, TNF-alpha and interleukin-2 (IL-2) resulted in enhanced nitric oxide (NO) production by these cells. In addition, we observed enhanced anti-leishmanial activity of infected 030-cells after activation. Both, NO production and anti-leishmanial activity were abrogated by addition of L-N(G)-nitroargininemethyl ester (L-NAME), an analogue of L-arginine. Thus, NO play an important role in the anti-leishmanial activity of these canine macrophages. We propose the infection of the 030-D cell line as a good in vitro model to further investigate parasite-host cell interactions in dogs, a natural host of Leishmania parasites.

Animals↗
PubMed1997

Auto IgG anti-IgE and IgG x IgE immune complex presence and effects on ELISA-based quantitation of IgE in canine atopic dermatitis, demodectic acariasis and helminthiasis.

Atopic dermatitis is a common allergic disease manifestation in dogs; however, there is no correlation between clinical disease and detectable total serum IgE. Auto antibodies of the IgG subclass against IgE may affect the detection of serum IgE by immunoassay and may be important in the regulation of IgE production by B cells. ELISA were developed to detect serum antibodies specific for IgE using a newly available canine monoclonal IgE of known antigen specificity, generated from a canine x murine heterohybridoma. To test for correlation of auto IgG anti-IgE levels with manifestation of atopic dermatitis, the sera from 101 atopic dogs were compared with sera from non-atopic dogs of various breeds, foxhounds manifesting clinical signs of demodectic acariasis and helminth parasitized random bred dogs for quantities of IgG anti-IgE measured in units/ml compared to a high titer standard serum. To test for serum effects on quantitation of IgE, known amounts of canine monoclonal IgE were added to various sera and measured by capture ELISA with detecting monoclonal antibodies specific for heat labile or heat stabile epitopes. Unheated sera from dogs manifesting clinical atopic dermatitis and helminth parasitized dogs had levels of IgG anti-IgE that were significantly lower than various breeds of dogs not manifesting dermatologic lesions and foxhounds manifesting demodectic acariasis. Heating sera at 56 degrees C for 3 h to denature the high affinity binding site on the IgE heavy chain caused a marked increase over non-heated sera in detectable IgG anti-IgE in almost all dogs. This increase was most profound in helminth-infected dogs and foxhounds manifesting demodectic mange with 7 fold increases each, respectively, and in atopic dogs with a 5 fold increase compared to 3 fold increases for clinically-normal springer spaniels and all soft coated wheaten terriers. The terriers demonstrated an association of lower heated serum values of IgG anti-IgE with manifestation of a familial syndrome of protein-losing enteropathy and protein-losing nephropathy. The ability of mouse anti-canine IgE monoclonal antibodies specific for either heat labile or heat stabile epitopes to detect canine monoclonal IgE added to sera in known amounts varied from serum to serum and at different concentrations of the same serum, but did not correlate with IgG anti-IgE values for these sera. The range of absolute levels of serum IgE in dogs showing little or no inhibition of detection of added IgE was < 0.5 ng/micromilligram to 2 micrograms/micromilligram. It was concluded that the increase in detectable IgG anti-IgE after heating sera indicates that IgG x IgE immune complexes are normally present in most dogs; however, the increase over uncomplexed IgG anti-IgE was most pronounced in dogs manifesting atopic dermatitis and demodectic acariasis. A quantitative comparison of IgG anti-IgE or IgG x IgE to total serum IgE was not made because the ability of monoclonal antibodies specific for either heat labile or heat stable epitopes on the IgE heavy chain to detect IgE added to serum, as well as innate serum IgE, was highly variable in different dilutions of serum from individual to individual.

Animals↗
PubMed1997

Overexpression of bcl-2 alters usage of mutational hot spots in germinal center B cells.

Bcl-2 is an anti-apoptotic gene important in B cell development. In order to study how apoptosis regulates somatic hypermutation and selection of B cell clones in the germinal center, we examined the antibody response to phosphorylcholine (PC) in transgenic mice overexpressing bcl-2 in the B cell compartment. The anti-PC antibody response is dominated by the S107V1 variable region heavy chain gene. We, therefore, analyzed S107V1-encoded heavy chains from germinal center cells. The proportion of germinal center sequences that were mutated, and the frequency of mutations did not differ significantly between the two groups of mice. No significant differences were found in the clustering of replacement mutations in the complementarity determining regions (CDRs) and in replacement to silent (R:S) mutation ratios. A significant difference between bcl-2 transgenic mice and controls, however, was found in the targeting of mutations to oligonucleotide motifs presumed to be mutational "hot spots." While non-transgenic mice displayed the expected clustering of mutations in hot spots, mutations from bcl-2 transgenic mice lacked this pattern. This observation suggests that the mechanism for somatic hypermutation includes two distinct functions, a non-specific mutational apparatus and a mechanism to target mutation to hot spots, and that in certain circumstances these functions may be uncoupled.

Amino Acid Sequence↗
PubMed1995

Purification of cell populations from human fetal brain using flow cytometric techniques.

We recently established primary cultures from dissociated second trimester human fetal brains using a novel spin seeding method and characterized cellular populations with distinct phenotypes in these cultures. Here, we report that these neural cultures can be dissociated to single-cell suspensions, sorted by size using flow cytometry and re-seeded to yield cultures selectively enriched for the neuronal and glial cell populations. Sorted neurons were highly homogeneous, viable and extended processes, by one day after re-seeding. These neurons expressed immunoreactivity for neurofilament protein, retained their GABAergic phenotype and were electrically excitable. Re-seeded astrocytes proliferated in culture and expressed glial fibrillary acidic protein. We describe the conditions required for the flow cytometric sorting and tissue culture assays as well as the morphological, immunocytochemical and electrophysiological characteristics of the sorted neuronal population.

Brain↗
PubMed1995

Leishmania infantum-specific T cell lines derived from asymptomatic dogs that lyse infected macrophages in a major histocompatibility complex-restricted manner.

Protective immunity to leishmaniasis has been demonstrated in murine models to be mediated by T cells and the cytokines they produce. We have previously shown that resistance to experimental Leishmania infantum infection in the dog, a natural host and reservoir of the parasite, is associated with the proliferation of peripheral blood mononuclear cells (PBMC) to parasite antigen and to the production of interleukin-2 and tumour necrosis factor. In this study we show that PBMC from asymptomatic experimentally infected dogs produce interferon-gamma upon parasite antigen-specific stimulation, whereas lymphocytes from symptomatic dogs do not. In addition, we report for the first time the lysis of L. infantum-infected macrophages by PBMC from asymptomatic dogs and by parasite-specific T cell lines derived from these animals. These T cell lines were generated by restimulation in vitro with parasite soluble antigen and irradiated autologous PBMC as antigen-presenting cells. We show that lysis of infected macrophages by T cell lines is major histocompatibility complex restricted. Characterization of parasite-specific cytotoxic T cell lines revealed that the responding cells are CD8+. However, for some animals, CD4+ T cells that lyse infected macrophages were also found. In contrast to asymptomatic dogs, lymphocytes from symptomatic dogs failed to proliferate and produce interferon-gamma after Leishmania antigen stimulation in vitro and were not capable of lysing infected macrophages. These results suggest that both the production of interferon-gamma and the destruction of the parasitized host cells by Leishmania-specific T cells play an important role in resistance to visceral leishmaniasis.

Animals↗
PubMed1995

Enhanced expression of novel CD57+CD8+ LAK cells from cats infected with feline immunodeficiency virus.

As a model for lymphokine-activated killer (LAK) function in HIV infection, we studied LAK cells in cats infected with feline immunodeficiency virus (FIV), which causes an acquired immunodeficiency syndrome. Peripheral blood mononuclear cells cultured in concanavalin A and interleukin-2 developed LAK cytotoxicity against chronically FIV-infected CrFK cells and acutely infected CD4+ lymphocytes but not uninfected cells. LAK cells from FIV+ cats were more cytotoxic than LAK cells from uninfected cats. Enhanced FIV+ LAK cytotoxicity against feline leukemia virus-infected cells (FL74) suggested that the cytotoxicity was not antigen specific. Two-color fluorescence-activated cell sorter analysis and antibody depletion studies demonstrated that the majority of LAK cells and their progenitors were positive for both CD8 and CD57. The in vitro induction of dual positive CD8+CD57+ LAK cells was enhanced in FIV+ cats, as reported for HIV+ patients. These CD8+CD57+ LAK cells may play a role in maintaining the long asymptomatic stage of infection in FIV+ cats.

Animals↗
PubMed1995

Functional and phenotypic analysis of in vitro stimulated canine peripheral blood mononuclear cells.

The inter-species cross-reactivity of cytokine bioassays for interleukin-2 (IL2) and interleukin-6 (IL6) was investigated in the canine species. The kinetics of normal canine peripheral blood mononuclear cells (PBMC) stimulated with pokeweed mitogen (PWM), were analyzed in terms of cytokine release and responsiveness to cytokine stimulation, in conjunction with determination of cell proliferation, de novo antibody synthesis and cell surface phenotype. PBMC were stimulated with PWM at the beginning of the culture and human recombinant IL2 (rIL2) was added 3-4 days post stimulation (d.p.s.). Mitogenically stimulated cells proliferated and synthesized antibody in a linear fashion up to 6 d.p.s. Resting PBMC had a mean CD4+/CD8+ ratio of 1.7:1; whereas cells stimulated with PWM were predominantly of CD8 phenotype at 7 d.p.s.. Three days after addition of IL2, stimulated cells were predominantly of the Thy+, sIg-, CD4+, CD8- phenotype, with an increase in the CD4+/CD8+ ratio. The magnitude of de novo antibody synthesis was lower in rIL2-supplemented cultures than in cultures stimulated only with PWM, and suggested a negative relationship between de novo antibody synthesis and proliferative responses of the same cultures. Supernatants from mitogen-stimulated cultures induced proliferation of mouse IL2- and IL6-dependent cell lines. Antibodies reactive with human IL2 or IL6 inhibited these responses. IL2-like activity in PWM-stimulated culture peaked by 2 d.p.s. and decreased thereafter. IL6-like activity peaked later (4-6 d.p.s.).

Animals↗
PubMed1995

Canine IgE monoclonal antibody specific for a filarial antigen: production by a canine x murine heterohybridoma using B cells from a clinically affected lymph node.

Canine popliteal lymph node cells taken at the onset of clinical disease from a rear limb infected with the filarial nematode Brugia pahangi were fused with mouse myeloma cell line P3X63.Ag8.653 cells. Of the several canine immunoglobulin-producing clones from this fusion, one was found to produce canine IgE specific for a filarial nematode antigen. The cell line has undergone limiting dilution cloning six times over the past 3 years and continues to produce monoclonal antibody of the IgE subclass at a rate of greater than 3 mg/l. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) of the cell culture supernatant protein that bound to protein A beads, showed bands at molecular weights (MW) of approximately 75,000 and 25,000 that were characteristic of epsilon and kappa or lambda chains, respectively. A mouse monoclonal antibody specific for canine IgE bound the 75,000 MW band, as demonstrated by Western blot. Western blots of aqueous extracts of adult filarial nematodes demonstrated binding of the canine IgE monoclonal antibody to a single 35,000 MW peptide from B. pahangi but not Dirofilaria immitis; immunochemistry using frozen sections of adult worms, microfilariae and fourth stage larvae revealed focal binding of the monoclonal IgE to worm tissue adjacent to dorsal and ventral cords of only Brugia adults.

Animals↗
PubMed1991

Immune-mediated thrombocytopenia in horses infected with equine infectious anemia virus.

An adult horse infected with a virulent, cell culture-adapted strain of equine infectious anemia virus (EIAV) developed cyclical thrombocytopenia in which the nadir of platelet counts coincided with peak febrile responses. In order to investigate the mechanism of thrombocytopenia during acute febrile episodes, four adult horses were experimentally infected with the wild-type Wyoming strain of EIAV. Platelet counts decreased from baseline as rectal temperature increased. Serum reverse transcriptase activity increased above background levels in all horses, coincident with increase in rectal temperature. All horses developed an EIAV-specific immune response detectable by Western immunoblot by postinfection day 10. Increases in platelet-associated immunoglobulins G and M were detectable by direct fluorescent-antibody test and flow cytometric assay. Viral replication in bone marrow megakaryocytes was not detectable by in situ hybridization. Results suggest an immune-mediated mechanism of thrombocytopenia in horses infected with EIAV. Despite an inability to identify virion particles in association with platelet-bound antibody, the cyclical nature of the thrombocytopenia and the occurrence of a marked cell-free viremia concomitant with fever and thrombocytopenia suggest immune complex deposition on platelets. We propose that clearance of virus and antibody-coated platelets from the peripheral circulation by hepatic Kupffer cells and splenic macrophages may target infectious virus particles, in the form of immune complexes, to host cells most permissive for in vivo viral replication.

Animals↗
PubMed1989

Increased density of ecto 5' nucleotidase antigen on leukemic T cells from patients with cutaneous T-cell lymphoma and adult T-cell leukemia/lymphoma.

Malignant CD4+ T cells in adult T-cell leukemia/lymphoma (ATL) and cutaneous T-cell lymphoma (CTCL) express a number of cell surface molecules that are upregulated on normal T cells activated by foreign antigen. In this report we describe an interesting exception to the parallel phenotypic features of activated T cells and malignant CD4+ T cells. A monoclonal antibody (MoAb; termed 27.2) that was raised to HTLV-1+, CD4+25+ leukemic T cells stained weakly 25% of peripheral T cells, including approximately 50% of CD8+ T cells and 20% of CD4+ T cells. Flow cytometry analysis indicated that the surface density of the 27.2 antigen was unchanged or diminished when normal T cells were activated by antigen. However, 3/4 Sezary cases and 4/8 cases of ATL had relatively high densities of the 27.2 antigen. Immunoprecipitation and sodium dodecylsulfate polyacrylamide gel electrophoresis of the NP-40-solubilized membranes of surface-iodinated ATL cells indicated that MoAb 27.2 reacted with a 75 Kd molecule. The size and distribution of the 27.2 antigen on T cell subsets suggested that it might be the enzyme ecto-5' nucleotidase (NT), a phosphatidylinositol-linked enzyme that catalyzes dephosphorylation of monophosphate nucleotides to their respective nucleosides. This was confirmed by demonstrating that lymphocyte ecto-5'NT activity was blocked partially and inhibited completely by preincubating cells with MoAb 27.2 for 1 hour at 4 degrees C and 24 hours at 37 degrees C, respectively. When used with a second MoAb (27.1) to a novel T cell activation antigen found on all CTCL and ATL leukemias examined, 27.2 was found to discriminate between normal and leukemic T cells in two patients with ATL. These studies suggest that ecto-5'NT has diagnostic value in T cell malignancies and may be aberrantly expressed in some cases of ATL and CTCL.

5'-Nucleotidase↗
PubMed1986

Surface immunoglobulin light chain expression in pre-B cell leukemias.

Cytofluorographic analysis of surface immunoglobulin (sIg) light chain clonal excess (CE), defined as (%kappa+ - %lambda+)/(%kappa+ + %lambda+) cells per discrete level of fluorescence intensity, was carried out on mononuclear cells of 32 leukemic patients. Eight demonstrated sIg light chain CE, including four blastic chronic myeloid leukemias (BL-CML), three "null" acute lymphoblastic leukemias (ALL), and one leukemic lymphoblastic lymphoma. Six of the leukemias demonstrated a kappa CE and two had a lambda CE. Sorted kappa+ PB cells from a BL-CML patient were shown to have a diploid DNA stem line and to bear the "common" ALL antigen. To provide further support for our finding of the expression of sIg light chains in ALL, we studied the REH cell line, derived from a "common" ALL patient and found cytoplasmic mu heavy chain and surface Ig lambda CE. Nucleic acid blotting experiments on REH revealed that both kappa genes had been deleted and that lambda genes had been rearranged, as expected in B cells expressing lambda light chains. Moreover, REH cells contained mu and lambda RNA. When REH cells were treated with TPA the amount of mu chain RNA increased by approximately fivefold and the amount of lambda chain RNA increased by approximately twofold. The finding of sIg light chain in pre-B cell leukemias and in the REH cell line, suggests that these leukemic cells are further differentiated along the B-cell lineage than was previously believed.

Antigens, Neoplasm↗
PubMed1985

Analysis of cell surface light chain immunoglobulin expression by flow cytometry in normal controls: a new mathematical approach.

It has been observed in flow cytometric studies that in normal individuals there are proportionally more kappa+ than lambda+ bearing light chain B-cells. Overt predominance of one type of light chain bearing cell over the other is a characteristic of B-cell neoplasias, a phenomenon called clonal excess (CE). A mathematical model using the Weibull distribution is proposed for studying such an excess. The new approach is desirable for two reasons: First, it is parametric and hence offers a more sensitive and versatile analysis than its nonparametric counterparts. Second, it utilizes only the relevant information from the upper tails of the distributions of the fluorescence intensity of the kappa+ and lambda+ cells. Two measures of CE based on the Weibull model are proposed, and a normal range of variability was determined for each measure using a random sample of 48 normal controls. Such normal ranges are particularly useful in detecting cancer patients with minimal B-cell neoplasias. A comparative study of the new measures, Ault's maximum difference measure, and a measure based on Ligler's method showed that the parametric approach provides much more sensitivity than both the nonparametric ones.

B-Lymphocytes↗
PubMed1984

Analysis of T-cell subsets in B-cell chronic lymphocytic leukemia: a correlation with the stage of disease.

T-cell subsets were determined by the Leu monoclonal antibodies in the peripheral blood and/or bone marrow of 52 patients with B-cell chronic lymphocytic leukemia (B-CLL) not on therapy at the time of study. The diagnosis of B-CLL required that the leukemic cells expressed surface receptors for "la-like" antigen, Fc fragment of IgG, mouse red blood cells (MRBC), C3-coated red cells (EAC), and low density of monoclonal surface immunoglobulin. The Leu-3a+/2a+ ratio was applied to define the balance between the helper/suppressor subsets in the residual T-lymphocytes. Most patients showed a decrease in the Leu-3a+/2a+ ratios at all stages of disease. The decrease in ratio was mainly related to a decrease in the Leu-3a+ T-cell subset. The more advanced stages of B-CLL were associated with lower Leu-3a+/2a+ ratio, higher total white cell and percent lymphocyte counts. There was no correlation between the proportion of EAC or MRBC rosetting cells and stages of B-CLL. This analysis further suggests that B-CLL is an immunosuppressed state that becomes more pronounced in the advanced stages and is characterized by a progressive decrease in the Leu-3a+ (helper) T-cell subset.

Antibodies, Monoclonal↗
PubMed1982

Characterization of B-cell type chronic lymphocytic leukemia cells by surface markers and a monoclonal antibody.

This study was carried out to determine the reactivity of the Leu-1 mouse monoclonal antibody with B-chronic lymphocytic leukemia and other B-cell leukemias. This antibody has been previously reported to recognize a surface antigen expressed by almost all human thymocytes and peripheral T cells. It was also detected on surface immunoglobulin-bearing cells of most patients with chronic lymphocytic leukemia but was not detectable in normal B-cells and B-cell lines. In the present series, the neoplastic lymphocytes in 33 cases of B-chronic lymphocytic leukemia expressed surface immunoglobulin, Ia antigen, and receptors for Fc, C3, and mouse erythrocytes. All but one case expressed the Leu-1 antigen as detected by indirect immunofluorescence using flow cytometry. In three other cases, the leukemic cells in the peripheral blood reacted with anti-Leu-1, whereas the bone marrow lymphocytes did not. Moreover, quantitative differences in the surface density of Leu-1 were apparent by flow cytometry. The peripheral blood and bone marrow lymphocytes in other B-cell leukemias, including 15 cases of leukemic B-cell lymphomas and three cases of hairy-cell leukemia, failed to stain positively with the anti-Leu-1 antibody. The recognition of the Leu-1 antigen adds to the phenotypic characterization of b-chronic lymphocytic leukemia and may contribute to a better understanding of the pathogenesis of the disease and its expression in different tissues.

Antibodies, Monoclonal↗
PubMed1982

Analysis of T-cell differentiation antigens in acute lymphatic leukemia using monoclonal antibodies.

Leukemic cells in 134 patients with ALL were analyzed by a panel of mouse monoclonal antibodies. Two antibodies are reactive with all peripheral blood T cells but define different surface antigens (Leu-1 and Leu-4). Two other antibodies react with antigens that are restricted to suppressor/cytotoxic T cells (Leu-2) and to helper T cells (Leu-3). We also used antibodies to the receptor for sheep red blood cells (SRBC) (Leu-5) and to a human "TL-like" antigen that is found on most thymocytes but not in peripheral T cells (Leu-6). An antibody to the human p29.34 "Ia-like" molecule was also tested. Of the 134 ALL patients, 17 had a predominance of SRBC-rosetting (Leu-5+) lymphoblasts ("T" ALL), expressing different surface phenotypes defined by this panel of monoclonal antibodies. These phenotypes were not readily classifiable according to a scheme of sequential stages of normal differentiation proposed. Moreover, the lymphoblasts in 8 of 113 patients not expressing conventional B- to T-cell markers ("null" ALL) reacted with the monoclonal anti-T-cell antibodies. This study suggests that the classification of lymphoblasts in ALL based on the reactivities observed with this panel of mouse monoclonal antibodies is not easily reconciled with current models of normal T-cell differentiation. However, it should be emphasized that the precise sequence of antigenic expression by cells undergoing thymic differentiation is still not fully known, and further phenotypic analysis of ALL cells might contribute to an improved understanding of this malignancy.

Adolescent↗