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Biomedical subjects

A Kunkl

Publications and source records attributed to A Kunkl.

At least 37 records · Page 2Linked to original sources
PubMed1993

Non-covalent complexes of HIV gp120 with CD4 and/or mAbs enhance activation of gp120-specific T clones and provide intermolecular help for anti-CD4 antibody production.

The 'dangerous liaison' between CD4 and gp120 that offers the first entry opportunity to HIV may also provoke perturbations of the immune control of the host with far-reaching immunopathological consequences. We wondered whether a mechanism of intermolecular help (T help across the gap of a non-covalent bond, in contrast to the intramolecular help of carrier to hapten) could break self-tolerance and be the cause of the frequent anti-CD4 autoantibodies found in AIDS patients. To determine whether this hypothesis deserves further testing, we designed a series of in vitro and in vivo experiments of increasing complexity, focused on the presentation of gp120 to specific T cells by antigen presenting cells (APC) exposed to the envelope protein in the form of non-covalent complexes. Bi-molecular complexes were constructed by allowing gp120 or gp160 to bind specific human mAbs. Tri-molecular complexes were constructed by introducing CD4 as an intermediate ligand between gp120 and mouse mAbs specific for CD4. In all cases the use of complexes did enhance the immunogenic capacity of substimulatory doses of gp120 or gp160 by facilitating uptake by APC via Fc receptor and consequent presentation to specific human T cell clones. Finally, help for the production in vivo of anti-CD4 antibodies was obtained from T lymphocytes specific for gp120 when CD4-primed memory B cells were pulsed with CD4 complexed with gp120, thus demonstrating in the mouse the entire cycle of intermolecular help via non-covalent interaction, and setting the stage for future experiments on self-tolerance breakage in a human molecular context.

Amino Acid Sequence↗
PubMed1992

Kinetic immunodominance: functionally competing antibodies against exposed and cryptic epitopes of Escherichia coli beta-galactosidase are produced in time sequence.

The murine antibody response to Escherichia coli beta-galactosidase (GZ) was analyzed in vivo and in vitro by focusing on two families of antibodies that exert distinct conformational/functional activity on the antigen. Activating antibodies--defined by their capacity to increase the enzymatic activity of defective GZ produced by mutant strains of E. coli--are detected early after secondary challenge. Inhibiting antibodies, which interfere with antibody-mediated enzyme activation, appear later and cause the abrupt fall of activating titer, a scenario suggesting either idiotype/anti-idiotype interaction or opposite pulsions exerted on the antigen molecule. Supporting the latter mechanism, the confrontation of mAbs of the two families produced classical competitive inhibition curves when the readout was enzyme activation, although they recognize two different epitopes of the same molecule: the activating mAb a quaternary conformation-dependent site of wild-type GZ, the inhibiting mAb a sequential determinant exposed only in denatured or in defective enzyme. The different timing of generation of these antibodies during the response may depend on a processing step necessary for unfolding of native antigen and consequent display of certain cryptic epitopes before they can trigger specific B cells. A picture emerges where the response to the various epitopes of a complex antigen is sequentially connected and where the uptake by antigen-presenting cells of antigen complexed with antibodies specific for the exposed epitopes may favor revelation of the cryptic ones.

Animals↗
PubMed1991

Effect of antigen/antibody ratio on macrophage uptake, processing, and presentation to T cells of antigen complexed with polyclonal antibodies.

Activation of a galactosidase-specific murine T hybridoma clone and of a human tetanus toxoid-specific T clone by antigen-presenting cells (APC) was used to evaluate the regulatory function of antibodies complexed with the relevant antigen. Complexed antigen, in fact, is taken up with high efficiency thanks to Fc receptors borne by APC. Antibody/antigen ratio in the complexes proved to be a critical parameter in enhancing antigen presentation. Complexes in moderate antibody excess provided optimal T cell activation independently of the physical state of the complexes (precipitated by a second antibody or solubilized by complement). Complexes in extreme antibody excess, on the contrary, did not yield T cell activation although taken up by APC efficiently. The effect of antibodies at extreme excess was observed with substimulatory dose of antigen (loss of potentiation) and with optimal dose of antigen (loss of stimulation). An excess of specific polyclonal antibodies hampers proteolytic degradation of antigen in vitro, supporting the view that a similar mechanism may operate within the APC that have internalized immune complexes in extreme antibody excess. The possibility that immune complex forming in extreme antibody excess may turn off the T cell response is proposed as a regulatory mechanism.

Animals↗
PubMed1989

In vitro evidence for impaired cellular immune responsiveness in children with chronic renal failure.

We used mixed lymphocyte culture (MLC) as an assay to evaluate in vitro the cellular immune functions of children with chronic renal insufficiency (CRI) or with chronic renal failure (CRF). The proliferative response of lymphocytes from both groups of patients was significantly reduced as compared to the response of control individuals. In addition, the proliferative response of CRF patients treated with hemodialysis was significantly lower than the response of continuous ambulatorial peritoneal dialysis (CAPD) patients. These data confirm that a marked depression of the cellular immune response is detectable in a pediatric population, as predicted from data from the literature on the adult renal failure population.

Child↗
PubMed1989

Analysis of cellular populations in peritoneal effluents of children on CAPD.

The characterization of cellular populations in peritoneal effluents (PE) of CAPD patients has been the subject of few studies. In order to establish a possible correlation between PE cells and clinical parameters, we studied the immunocompetent cells that are found in uncomplicated patients. In this study we used cytofluorimetric analysis to characterize phenotypically immunocompetent PE cells. We studied 17 children with a mean age of 8.7 +/- 5.3 years, who had been on CAPD for a mean duration of 16 months. The patients never suffered from peritonitis. The effluents were collected after overnight dialysis and centrifuged to concentrate cells. Surface phenotype of PE cells was tested with a panel of monoclonal antibodies. The analysis of 40 PE samples showed that the various cell types were present with different frequencies, suggesting that a dominant phenotype cannot be defined. The ratio between M3 positive cells (monocytic-macrophagic lineage) and T3 positive cells (T lymphocytes) showed a tendency to decrease after initiation of CAPD. T4/T8 ratio in all samples did not differ from that of peripheral lymphocytes. A high frequency of activated T cells (41% +/- 13), defined by the presence of DR antigens on T3+ M3- cells, was seen in PE and confirmed by the high frequency of T cells expressing the IL-2 receptor.

Adolescent↗
PubMed1988

Differential activation of T cell clones stimulated by macrophages exposed to antigen complexed with monoclonal antibodies. A possible influence of paratope specificity on the mode of antigen processing.

We have used the enhanced uptake by FcR-bearing cells observed when Ag is administered as an immune complex to investigate the possible impact of specific antibodies on processing and presentation of antigen by accessory cells. The Ag Escherichia coli beta-galactosidase alone or bound to different mAb was incubated with peritoneal macrophages. These were subsequently exposed to a battery of Ag-specific T hybridoma clones. The resulting production of IL-2 was taken as a measure of effective presentation. The results of 43 mAb-T clone combinations showed a potentiation of presentation of Ag at substimulatory concentration in the majority of the cases, indicating that each mAb is conducive to FcR-mediated uptake by macrophages, and that each T clone can be stimulated by properly presented Ag. In contrast, nine combinations yielded a lower response, two of them falling to baseline values. We attribute these results, which corroborate our previous evidence of directional help in the beta-galactosidase system, to a modulation in enzymatic processing of Ag and its subsequent presentation imposed by the paratope of the mAb binding to the relevant epitope.

Antibodies, Monoclonal↗
PubMed1988

Effect of cyclosporine on the antigen-presenting function of human and murine accessory cells.

Recent reports have challenged the belief that accessory cells are resistant to cyclosporine. Such a tenet was based on the observation that several functions of accessory cells, such as IL-1 production and phagocytosis, are resistant to the drug. On the other hand, when a less primitive, more refined function of accessory cells was examined--i.e., the capacity to take up, process, and present antigen in an MHC-restricted fashion to antigen-specific T lymphocytes, CsA proved to be an effective inhibitor. In contrast to this finding, when antigen was provided in the form of an immune complex prepared with a monoclonal antibody, uptake of antigen--likely mediated by the Fc receptors--and subsequent processing and presentation were not affected by CsA. These results suggest that, depending on whether the antigen is taken up by constitutive or by receptor-mediated endocytosis, accessory cells can be functionally defined as resistant or sensitive to CsA.

Animals↗
PubMed1986

Difference in sensitivity to cyclosporine in vitro of human alloreactive lines and clones.

The effectiveness of cyclosporine (CsA) as immunosuppressive agent in human kidney graft rejection is well established. However, in spite of efforts to maintain optimal plasma levels, a fraction of transplanted patients undergo rejection episodes and/or irreversible chronic rejection. This suggests that immunosuppression by CsA cannot control the alloreactive response if there is a high degree of histoincompatibility for HLA or non-HLA antigens, or it has little effect on the "high responder" patient. Both possibilities are difficult to test in the human system. A third hypothesis, the existence of individual CsA resistance, was tested by evaluating the in vitro inhibitory activity of CsA on alloreactive T cell lines from several individuals. A different degree of in vitro sensitivity to the drug was observed among alloreactive lines generated from different individuals and among clones obtained from the same bulk line. The variability at the individual level and at the clonal level may account for the onset of CsA-resistant rejection assuming that in vivo a positive selection in the presence of the drug occurs and allows for the resistant clones, if present, to dominate the sensitive ones.

Cell Line↗
PubMed1985

Constraints in T-B cooperation related to epitope topology on E. coli beta-galactosidase. I. The fine specificity of T cells dictates the fine specificity of antibodies directed to conformation-dependent determinants.

Experiments to test the relationship between the epitopes on a protein antigen recognized by T and B cells in their collaboration to produce antibody cannot rely solely on hapten-carrier models. In the present work we used E. coli beta-galactosidase, a molecule whose tertiary and quaternary epitopes have been well characterized, as the model antigen. T helper cells were raised by stimulating mice with the intact or the denatured molecule or with any of several beta-galactosidase cyanogen bromide peptides. In a series of in vitro helper T cell assays we confronted the various T populations with B cells preimmunized with the native antigen, and we tested their capacity to help production of (a) binding antibodies and (b) antibodies directed to single conformational epitopes, characterized by their capacity to protect the enzyme from heat denaturation or to activate defective beta-galactosidase. According to our results, (a) equivalent T cell help can be provided by T helper cells primed with native or denatured antigen, even for the production of "conformational" antibodies; (b) one of the peptides (CB-18) is most efficient in raising help for binding antibodies; and (c) two peptides (CB-20 and CB-21) rank highest in priming T helper cells for the eventual production of protecting and activating antibodies, respectively. Thus, not every beta-galactosidase-specific T helper cell is useful in providing help to B cells specific for any particular epitope on the molecule, but rather preferential pairings exist, possibly governed by a proximity rule.

Animals↗
PubMed1985

Inhibition of the accessory function of murine macrophages in vitro by cyclosporine.

The accessory function of macrophages, which is strictly related to the induction of T cell activation, has been studied to determine whether it is affected by cyclosporine (CsA). Irradiated spleen cells, used as a source of macrophages, were pulsed overnight with beta-galactosidase (GZ) in the presence of CsA. After washing of the pulsed macrophages, cells from a GZ-specific T cell line were added to cultures and 3H-thymidine incorporation was measured 72 hr later. We found that 500 ng/ml CsA present during macrophage pulsing with GZ reduced T cell proliferation to 5%. On the other hand, 100 ng/ml CsA almost completely abrogated the proliferative response when present for the duration of the culture. Similar results were also obtained using antigen-pulsed peritoneal-adherent macrophages to stimulate the T cell line to proliferate, or a T hybridoma clone to produce interleukin-2 (IL-2). The possibility that CsA actually affects interleukin-1 (IL-1) production by macrophages by inhibiting uninvolved T cells could be ruled out. We conclude that CsA-induced inhibition of T cell functions (proliferation and IL-2 production) is partially due to the effect of the drug on the accessory function of macrophages. This immunosuppressive mechanism of action of CsA on macrophages has not been previously described.

Animals↗
PubMed1984

A simple procedure for freezing and storing lymphocyte panels in trays.

Lymphocytes from different donors may be frozen in Terasaki trays by a simple procedure. This method provides the tissue typing laboratory with a readily available panel that can be routinely used to test for lymphocytotoxic antibodies sera from patients waiting for kidney transplant, transplanted patients and polytransfused individuals. The advantage of this procedure is that expensive programmed freezing apparatus is not required.

Blood Preservation↗
PubMed1983

Effects of cyclosporine on the immune system of the mouse. II. Cyclosporine inhibits the effector function of primary T helper cells, but not helper cell priming.

This paper describes various effects of cyclosporine on T helper (TH) cells for antibody production in the mouse. Immunosuppressive doses of cyclosporine abolish the help provided by virgin (primary) TH cells, but allow the priming of normal (and under certain conditions supranormal) numbers of TH cells as assayed by adoptive transfer experiments. The helper function of primed (secondary) TH cells is normally resistant to cyclosporine. However, if T cells are exposed to cyclosporine during priming their helper function remains totally cyclosporine-sensitive as long as the drug therapy is continued. The mechanisms involved in these selective effects are unknown, but the results indicate that in vivo cyclosporine can dissociate effector function (primary help) from TH cell proliferation (priming).

Animals↗
PubMed1983

Recognition of donor fibroblast antigens by lymphocytes homing in the human grafted kidney.

A human transplanted kidney, surgically removed because of untreatable chronic rejection, was used as the source of lymphocytes (K-L) of recipient origin that were expanded with interleukin-2 (IL-2), and of kidney fibroblasts (K-F) of donor origin that were maintained as an established line. Cytotoxicity assays were performed using K-L and peripheral blood lymphocytes (PBL) as effectors, and K-F and donor PBL as targets. From the results the following conclusions can be drawn: (1) cytotoxic lymphocytes, presumably involved in the process of chronic graft rejection, home in the kidney (from which they can be recovered) but are not detected in the circulation; (2) cytotoxic lymphocytes can be generated from peripheral lymphocytes by mixed lymphocyte culture (MLC) and further expansion in vitro with IL-2 (MLC-L); and (3) although both K-L and MLC-L are cytotoxic toward K-F, the former are not cytotoxic toward donor PBL. This suggests that although MLC-L recognize antigens shared by K-F and PBL, K-L recognize antigens specific for K-F only. These results, if confirmed, indicate that antigens not present on PBL, and possibly tissue-restricted are important in graft rejection. Thus, while monitoring transplanted patients, a lack of cytotoxicity in the recipient PBL may be misleading because the relevant cytotoxic effector cells may have disappeared from the periphery and the appropriate antigenic target may be absent on donor PBL.

Adolescent↗