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Biomedical subjects

N Zavazava

Publications and source records attributed to N Zavazava.

At least 55 records · Page 3Linked to original sources
PubMed1994

Quantitative and biochemical characterization of soluble HLA class I antigens shed by cultured human cells.

sHLA has been described in human serum and other body fluids. In this study sHLA shed by cultivated human cells and their biochemical nature in solution were studied. EBV-transformed human B-lymphoblastoid cell lines (n = 4), permanent human lymphoblastoid tumor cell lines (n = 4), and PBLs from three donors were cultivated in vitro and sHLA measured in the supernatants. The Daudi cell line was used as a negative control in all experiments. Maximum expression of sHLA was measured after 8 hours, after which the concentrations gradually declined. The allospecificities A2 and B7 were also detectable in the ELISA. sHLA in the supernatants was further characterized by 1D-IEF. All bands representing the allotypes were detected, showing that cell supernatants can be used as antigen sources for biochemical tissue typing. These data show that sHLA expression is a characteristic of viable cells.

Antibodies, Monoclonal↗
PubMed1993

Interaction of papain-digested HLA class I molecules with human alloreactive cytotoxic T lymphocytes (CTL).

Acute immunological rejection events of transplanted allogeneic organs are strongly dependent on T cell reactivity against foreign MHC products. The recognition requirements of alloreactive cytotoxic T cells are of particular interest for finding approaches to modulating alloreactivity. The role of the allogeneic MHC molecule itself and/or an associated peptide in the interaction with the T cell receptor is still, however, unclear. Our studies have focused on the interactions of papain-digested HLA class I molecules with alloreactive CD8+ CTL. These polypeptides, consisting of the polymorphic alpha 1 and alpha 2 and the monomorphic alpha 3 domains, were used in both soluble and immobilized form to study their functional effects on anti-HLA-A2 reactive CTL. Purified polypeptides were of molecular mass 32-34 kD. HLA-A2 polypeptides (0.55 micrograms/ml) in soluble form induced half-maximal reduction of CTL cytotoxicity. These concentrations were quantitatively comparable to the effective doses of intact HLA class I molecules, which contain the hydrophobic transmembrane domain and the intracytoplasmic tail. In addition, specific activation requirements of these CTL were investigated in a serine esterase release assay. Maximal degranulation was observed after 2 h of antigen contact. Purified HLA class I molecules allospecifically activated the anti-HLA-A2 CTL to degranulate serine esterase, when immobilized on plastic microtitre plates. Thus, polypeptides containing the polymorphic alpha 1 and alpha 2 domains of human class I molecules potentially modulate the cytotoxic T cell response. This might have implications for the reduction or prevention of allograft rejection in recipients of foreign organs.

Cytotoxicity, Immunologic↗
PubMed1993

Soluble MHC class I antigens (sHLA) and anti-HLA antibodies in heart and kidney allograft recipients.

Soluble HLA (sHLA) antigens were measured in a panel of 50 renal and 50 cardiac graft recipients by a quantitative ELISA over periods ranging between 2 months and 3 years. 72% of the renal patients and 68% of the cardiac graft patients experienced episodes of acute rejection during the observation period. sHLA were elevated to over 2-5 times the normal levels up to 10 days before histological evidence of rejection. This duration was for each individual patient very variable. Cytomegalovirus (CMV) infections did not appear to elevate sHLA levels. Donor-specific HLA-A2 was measured in only 1 cardiac recipient. 15% of the sera obtained from cardiac patients had cytotoxic anti-HLA antibodies, when tested against a panel of 50 test cells. After thermal inactivation at 56 degrees C, 42.5% of the sera became positive against at least 10% of the test cells. This finding suggests thermal dissociation of immune complexed anti-HLA antibodies. sHLA measurement and careful monitoring of serum for the presence of cytotoxic anti-HLA antibodies in graft patients are useful techniques which could help predict the long-term fate of an allograft.

Antibodies↗
PubMed1993

In vitro cultivation and immunogenicity of human cardiac valve endothelium.

Endothelial cells were derived from aortic and mitral valves (n = 17) by collagenase digestion and subsequently cultivated in RPMI medium supplemented with 20% fetal calf serum. The cells were stained in an alkaline phosphatase-anti-alkaline phosphatase stain for the expression of MHC Class I and Class II antigens, ICAM-1, ELAM-1, F VIII, and H/Y. The endothelium showed a strong expression of Class I, H/Y, and ICAM-1 molecules, and weak expression of MHC Class II molecules. In contrast to vascular endothelium that is known to express F VIII constitutively, cardiac valve endothelium was found to be negative. F VIII and ELAM-1 were only expressed after stimulation with recombinant interferon-gamma. To analyze the immunogenicity of valve endothelium, cells were used as stimulator cells in a mixed cell culture reaction using lymphocytes as responder cells. Endothelial cells had a 2 to 3 times higher stimulatory effect than peripheral blood lymphocytes. These data allow speculation on whether the observed degeneration of homografts can be reduced if HLA matching is performed prior to valve implantation.

Cell Adhesion Molecules↗
PubMed1993

[HLA typing in high risk keratoplasty].

UNLABELLED: Improved microsurgical techniques and better immunosuppressive treatment have led to an increase in the number of corneal transplantations. However, graft rejection due to a disparity between donor and recipient major histocompatibility antigens remains the major complication. The risk of graft rejection is further increased in patients with vascularized graft beds and in presensitized persons. Well-designed experimental studies and clinical experience have clearly shown the importance of class-I-antigens. However, class-II antigens have not yet been studied to the same extent. METHODS: Multiorgan donors and corneal graft recipients were HLA typed using the lymphocytotoxicity test previously described by Terasaki. The recipients were typed retrospectively. Cadaver corneal graft donors were typed using a novel HLA typing technique on retinal pigment epithelial cells, as previously described by us. Postoperatively, all recipients were routinely examined for rejection over a period of 2 years. RESULTS: Graft rejection was observed in 25% of the 133 high-risk patients. Of the 13 class-I-typed patients who showed a rejection 9 persons had two or more major histocompatibility mismatches. Of the 12 patients without rejection problems only 5 had two or more HLA class-I-mismatches. Of the patients with one or two matches at the HLA-DR locus there were only 4 out of 9 patients with at least one graft rejection; 5 out of 6 patients showed no signs of rejection. CONCLUSIONS: Mismatches at either class-I- or class-II- gene loci lead to a higher frequency in corneal graft rejection. Apart from the major histocompatibility mismatches, other factors also influence graft rejection, such as the distance between the corneal transplant and corneal vessels, presensitization, and the indication for corneal grafting. If we are to define the factors influencing graft rejection better and further increase our knowledge on the role the major histocompatibility antigens play in corneal graft rejection in a larger patient group, then cooperation with other transplantation centers will be required.

Graft Rejection↗
PubMed1992

Clinical relevance of soluble HLA and interaction of papain derived class I molecules with alloreactive CTL.

MHC class I and class II molecules are expressed in soluble form in the serum of both healthy and diseased individuals. Our aim was to investigate whether soluble class I (sHLA) levels in allograft patients correlate with their clinical status. Altogether, 20 renal and 30 cardiac graft recipients were examined. High levels of sHLA were measured at least 5 days preceding acute rejection episodes. Immune complexes between anti-HLA antibodies and sHLA were detected in a patient who died of a severe vascular rejection. In another study the interaction of papain-derived sHLA on alloreactive CTL in vitro was investigated. In a chromium-51-release cytotoxicity assay, 1.25 microg/ml of papain-digested class I molecules reduced CTL cytotoxicity to background levels. On the contrary, immobilized molecules triggered the release of serine esterase allospecifically. These data showed that the MHC molecule alone was a sufficient ligand for the interaction with alloreactive CTL.

Biomarkers↗
PubMed1992

Post-mortem HLA tissue typing of retinal pigment epithelial cells.

Retinal pigment epithelial cells (RPE) were derived from bulbi of cornea donors and maintained in culture. The time-span between donor's death and cell cultivation ranged from 2 to 122 h. The mean numbers of hours was 25.6 (n = 130). After IFN-gamma stimulation, cells were serologically typed for class I and class II antigens. Unclear class I allospecificities were verified by one-dimensional isoelectric focusing. Data from the serological typing of lymphocytes and those of the serological and biochemical typing of RPE from the same donors were compared in 22 cases. There was a discrepancy of less than 5%, whereby either the typing on lymphocytes could not identify some specificities declared as blanks or the RPE typing had failed to clearly define a specificity. Our data show that the strategy adopted here is very successful for tissue typing post mortem, thus increasing the number of available HLA-matched corneas and consequently reducing the number of corneal graft rejections.

Adolescent↗
PubMed1991

Inhibition of anti-HLA-B7 alloreactive CTL by affinity-purified soluble HLA.

The objective of this study was to elucidate the interaction of naturally occurring soluble MHC class I molecules with alloreactive CTL and to discuss its possible relevance to graft acceptance. An anti-HLA-B7 specific CTL-line, BV.B7, was generated in vitro. On phenotyping the cells after 6 weeks, 80% were found to be CD8+, 14% CD4+ and 6% CD8+CD4+. CD4+ CTL were depleted using immunomagnetic beads precoated with an anti-CD4 antibody. Of the recovered CTL greater than 96% were CD8+. A total of 12 HLA-B7 target cell lines and PHA blasts tested were specifically lysed in a 51Cr-release assay. Soluble HLA class I molecules were isolated on affinity chromatography columns using the anti-HLA-B7 ME 1 and the anti-heavy chain W6/32 monoclonal antibodies. Antigen purity was confirmed by analysis on SDS-PAGE gels. CTL were preincubated with 0.1-1.8 micrograms/ml soluble HLA for 30 min at 37 degrees C and subsequently tested for cytotoxicity in the 51Cr-release assay; 1.1 micrograms/ml HLA-B7 molecules reduced CTL cytotoxicity by 50% whereas non-B7 HLA had no effect. Further, CTL cytotoxicity was reduced by preincubation with anti-CD8, anti-TcR, and anti-CD3 antibodies. We anticipate a possible down-regulatory role of soluble HLA on CTL in allogeneic transplantation.

Antibodies↗
PubMed1991

Expression of MHC class I and II molecules by cadaver retinal pigment epithelium cells: optimization of post-mortem HLA typing.

The objective of this study was to investigate the expression of MHC antigens by retinal pigment epithelium cells (RPE) after stimulation with interferon-gamma (IFN-gamma) and to improve the currently practised technique of cadaver HLA typing. A concentration of 100 U/ml IFN-gamma induced expression of class I molecules up to greater than 90% 3 days after stimulation, whereas 50 U/ml were required for the expression of HLA-DR to greater than 90%. A concentration of 750 U/ml induced 35-45% expression of HLA-DP and less than 25% HLA-DQ after 3 days. Cells were serologically typed using the standard lymphocytotoxicity assay 3 days after stimulation with 250 U/ml IFN-gamma. Typing of class I specificities was complemented by one-dimensional isoelectric focusing (1D-IEF). We observed high concordance between the results of the RPE typing and the lymphocytotoxicity test on the same donors. Our results show complete typing of class I and II antigens post-mortem, which, in particular, enables graft matching and improvement of graft survival in recipients of organs removed many hours after death such as the cornea.

Adolescent↗
PubMed1991

[Postmortem serologic and biochemical HLA typing with cultivated retinal pigment epithelium cells].

A quick and reliable method for HLA typing of human cadavers has been established. Retinal pigment epithelial cells (RPE) were obtained from cadaver bulbi 8-36 h post mortem (n = 24). After 24 hours, cell growth correlated inversely with the time lapse between the donor's death and bulbus explantation. Cultivated human RPE were stimulated by different Y-interferon (IFN) concentrations and examined by FACS analysis for the expression of MHC class I and II molecules. A concentration of 100 units/ml human Y-IFN was sufficient to induce maximal class I antigen expression by about 90% of cells after 3 days. At 50 units/ml Y-IFN, the cells showed maximal class II antigen expression for HLA-DR, whereas 100 units/ml I-IFN was required for maximal HLA-DQ expression. Higher concentrations of Y-IFN induced a further HLA-DP expression. However, serological HLA typing was performed after cell stimulation with 250 units/ml Y-IFN for 3 days. A concentration of 500 units/ml Y-IFN yielded equivalent results, but 750 units/ml Y-IFN led to poor identification of class I MHC specificities. Unclear serological results on class I specificities were clarified by 1D-isoelectric focusing (1D-IEF), thus allowing full HLA typing of all donor bulbi examined. In summary, successful HLA typing of cultivated RPE is dependent on both the Y-IFN concentration and the time lapse between stimulation and typing of the RPE. The method provides a much improved basis for cadaver cornea grafting.

Adolescent↗
PubMed1990

Measurement of soluble MHC class I molecules in renal graft patients: a noninvasive allograft monitor.

We investigated the biochemistry of naturally occurring major histocompatibility complex (MHC) class I molecules in human serum and established a quantitative enzyme-linked immunoassay (ELISA) to determine soluble human leukocyte antigen (HLA) concentration. Peptides of 46, 40, 37, 35, and 12 kDa were isolated on affinity chromatography columns using two monoclonal antibodies (MoAbs), the anti-heavy chain W6/32 and the anti-beta 2-microglobulin BBM. 1. These peptides were confirmed on Western blots by the HC-10 MoAb, which binds a monomorphic epitope on denatured heavy chains. In detergent-binding experiments, only the 46-kDa peptide could be isolated from solubilized cell membranes. No quantitative differences between serum and plasma HLAs of the same individual were measured. Soluble HLA expression in 12 renal graft recipients was measured over 1-3 mo posttransplantation. A highly significant increase of 50%-100% was noted during rejection episodes. Clinical signs of rejection were accompanied by poor renal function, including elevated creatinine values. After the crises had been managed, class I levels normalized to 0.3-1.5 micrograms/ml, which is the range in healthy persons. Patients who showed no rejection crises maintained constant levels within the period of study. We anticipate the application of soluble HLA measurement in clinical practice as a noninvasive graft monitor.

Enzyme-Linked Immunosorbent Assay↗
PubMed1990

Characterization of soluble HLA molecules in sweat and quantitative HLA differences in serum of healthy individuals.

Soluble class I molecules were immunoprecipitated from human sweat and serum using the BB7.7 monoclonal antibody (mAb) coupled to immunomagnetic beads. Molecules were analysed biochemically on SDS-PAGE gels and finally by ID-isoelectric-focusing (IEF). Serum- and sweat-HLA IEF-band patterns of the same individual were fully identical, showing that HLA excreted in sweat possess polymorphic structures like those in serum. Quantitatively, we used a highly sensitive competitive enzyme-linked immunosorbent (ELISA) assay to determine soluble class I concentrations. The first group was that of non-HLA-A9 and -Bw62 sera, which were found to contain HLA levels with a mean concentration of 0.82 +/- 0.63 microgram/ml (n = 44). However, sera that were HLA-A23 or -24 (splits of HLA-A9) contained higher levels, with a mean of 3.2 +/- 0.94 microgram/ml (n = 20). Similarly, HLA-Bw62 individuals had a higher mean of 2.05 +/- 0.65 micrograms/ml (n = 10). The difference of the HLA-A9 group to the first group was statistically highly significant, P less than 0.0001, and that of the HLA-Bw62 to the first was also significant, P less than 0.004. Individuals who were both HLA-A9 and -Bw62 (n = 5) did not express significantly higher levels than those who only had one of these specificities. Sweat HLA levels had a mean of 0.42 +/- 0.4 microgram/ml (n = 10). These results show for the first time that soluble class I peptides are excreted in relatively high concentrations in sweat and possess polymorphic structures identical to those of serum HLA and that serum HLA levels are allotype dependent.

Female↗
PubMed1988

Diversity of the human monocyte/macrophage system as detected by monoclonal antibodies.

Four monoclonal antibodies against the human monocyte/macrophage system, termed Ki-M1, Ki-M6, Ki-M7, and Ki-M8, are described with regard to their immunohistochemical tissue distribution pattern and their subcellular reactive sites. The differences found applying these analyses are also reflected by the various molecular weights of the recognized antigens. Based on these data it is proposed that the monocyte/macrophage system can be divided into the phagocytosing compartment on one hand and the immune accessory compartment on the other hand; the latter constitutes the interdigitating reticulum cells, the indeterminate dendritic cells, and the Langerhans cells, as well as the follicular dendritic cells (dendritic reticulum cells) as the accessory cells for T- and B-cell immune response, respectively.

Antibodies, Monoclonal↗
PubMedDate not supplied

Molecular forms of soluble HLA in body fluids: potential determinants of body odor cues.

The major histocompatibility complex (MHC) has been linked to encoding for individual olfactory identity. Experiments in mice and rats proved that behavior and mating were, at least in part, determined by genes within the MHC. This study was aimed at investigating whether sHLA are excreted in human urine, saliva and sweat. In particular examination of the molecular forms in these fluids would give clues to whether break down forms of soluble MHC molecules might participate in shaping behavior. Major bands of 45, 40, and 23 kD were detectable. Increased levels of sHLA were measured using a quantitative ELISA in urine shortly before ovulation decreasing to normal levels thereafter. In animal models strain specific MHC-linked odor cues have been detected in urine. Thus, excretion of sHLA in urine might indicate a similar role for these molecules in humans.

Body Fluids↗