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M R Garovoy

Publications and source records attributed to M R Garovoy.

At least 19 recordsLinked to original sources
PubMed1992

Characterization and minimization of cellular autofluorescence in the study of oligonucleotide uptake using confocal microscopy.

Intrinsic autofluorescent signals can interfere with extrinsic fluorophore signals when living cells are viewed under a confocal laser scanning microscope. The general pattern of this endogenous fluorescence is initially diffuse and cytoplasmic, but it can redistribute and intensify to become punctate and perinuclear as cells age. To reduce the contribution of autofluorescence when tracking the location of an extrinsic fluorophore, such as a fluorescently-labeled oligonucleotide, laser power settings, aperture settings, laser scanning rates, pH buffering environments, and excitatory wavelengths can be modulated. Decreasing laser power settings and aperture sizes, increasing laser scanning rates and excitatory wavelengths, and surrounding cells in a pH buffer all act to delay the signal transformation. In addition, the presence of an exogenous fluorophore can hasten the autofluorescent redistribution and intensification when compared to similar untreated cells.

Cells, Cultured↗
PubMed1992

Cytokine and T cell receptor gene expression at the site of allograft rejection.

Intragraft cytokine and T cell receptor gene expression was analyzed in rejecting renal allografts by polymerase chain reaction (PCR). Message for IL-1 beta, IL-6, and TNF-alpha was detected in nephrectomy tissue with pathological evidence of acute or chronic rejection. Similarly, mRNA for both IL-6 and TNF-alpha was present in renal biopsies from acute rejecting kidneys. IL-2R, IL-4, and IL-5 mRNA was present in both rejecting and rejected kidney allografts, indicating that these cytokines may play a role in ongoing renal allograft rejection. Conversely, IL-2, IL-7, and IFN-gamma message was detected infrequently. In order to address the diversity of T cells in rejecting kidneys, we have analyzed the clonality of the TcR present within the allograft tissue. Rearranged TcR genes were identified in all allografts examined (n = 16) indicating the presence of T cells bearing the alpha/beta TcR. We have determined that there is a heterogeneous infiltration of T cells in the rejected allograft with TcR representing x = 7.47 +/- 2.4 families rearranged in samples obtained from nephrectomies, whereas x = 5.33 +/- 0.58 families were detected in samples obtained from biopsy tissue. These data indicate that (1) cytokines are produced locally which may contribute to graft cell destruction, (2) the heterogeneity of intragraft T cells during kidney allograft rejection may exist because nonspecific lymphocytes have been recruited to the site by locally produced cytokines or because T cells are responding to multiple epitopes or multiple donor antigens. Detection of intragraft cytokines and TcR may prove useful in elucidating the mechanism of rejection and therefore lead to improved immunosuppression.

Adolescent↗
PubMed1991

Are blood transfusions beneficial in the cyclosporine era?

In patients treated with conventional immunosuppression (azathioprine and prednisone) after renal transplantation, there is a beneficial effect of pre-transplant blood transfusions on graft survival; in patients treated with cyclosporine, this effect may be lost. In 66 children who received living-related donor transplants after donor-specific transfusions (DST) and were treated with azathioprine-prednisone in our center, 1- and 5-year graft survival rates were 99% and 77% respectively. These rates were similar to those reported for children who did not receive DST but were treated with cyclosporine in other centers. There were 634 adult and pediatric recipients of cadaver transplants in our center who were treated with cyclosporine and prednisone (non-sequential therapy, n = 89) or antilymphoblast globulin, azathioprine preduisone, and cyclosporin (sequential therapy, n = 545). When all patients were considered, graft survival rates were higher in transfused than in non-transfused patients at 3-5 years, but in the sequential therapy group, there were no differences in graft survival rates between transfused and non-transfused patients. The results suggest that transfusions do not improve cadaver graft survival in patients receiving optimal cyclosporine therapy and that equally good related donor graft survival can be achieved with DST and conventional immunosuppression or no DST and cyclosporine.

Blood Transfusion↗
PubMed1991

An improved method for the detection of soluble interleukin 2 receptors in liver transplant recipients by flow cytometry.

A new flow cytometric method (FCM) for detection of soluble interleukin 2 receptor (sIL-2R) in serum was established. Using this method, results from one to forty samples can be obtained in less than 2 hr as compared with 5 hr in an ELISA. Comparing over 300 sera samples tested by both methods, we found the FCM to be as specific and sensitive as the ELISA, but even more reproducible. Regression analysis of ELISAt on FCMt showed there is a strong relationship between the two assays and they mimic each other over time. We studied the daily serum levels of sIL-2R by FCM in 18 liver allograft recipients: 7 were in the stable group, 6 in the rejection group, and 5 had both rejection and infection. Low or decreasing sIL-2R levels correlated with absence of rejection. In patients with rejection episodes, a typical elevation of sIL-2R was followed by a rapid decrease of sIL-2R after successful response to rejection therapy. Patients with multiple complications displayed elevated sIL-2R levels. In conclusion, because of its rapid turnover time and accuracy, the FCM has the potential to determine the appropriate timing of post-transplant liver biopsies.

Antibodies, Monoclonal↗
PubMed1990

Non-inherited maternal HLA antigens and protection against sensitisation.

To test the hypothesis that non-inherited maternal antigens (NIMAs), which reportedly afford protection against sensitisation by random transfusions, can provide protection against repetitive antigenic exposure of donor-specific transfusion (DST), 140 DST patients whose donors were mismatched for NIMAs and 71 whose donors were mismatched for non-inherited paternal antigens (NIPAs) were studied. The rate of sensitisation in the two groups of patients was similar (22.1% vs 15.5%). There was no difference in sensitisation to NIMAs and NIPAs between patients who received azathioprine and those who did not. The formation of donor specific HLA antibody was comparable in the two groups. After kidney transplantation there were no differences in 1-year graft survival or the incidence of rejection episodes. These findings suggest that NIMAs do not provide lifelong protection against subsequent repetitive antigen challenge and sensitisation.

Adult↗
PubMed1990

IL-4 inhibits IL-2 receptor expression and IL-2-dependent proliferation of human T cells.

Recent studies have shown that IL-4 can affect lymphocyte responses to IL-2. To evaluate the effects of IL-4 on T cell responses to physiologically relevant stimuli, we studied normal human T cells cultured with a low concentration of anti-CD3 mAb and IL-2 in the presence and absence of added IL-4. The addition of IL-4 to cultures of T cells stimulated with anti-CD3 mAb and IL-2 reduced the proliferative response by 49 to 59%. The inhibitory effect was observed in 3-, 5-, and 7-day cultures. Inhibition was dose-dependent with maximal inhibition at concentrations greater than or equal to 5 to 10 U/ml IL-4. IL-4-mediated inhibition occurred early during the T cell response, inasmuch as addition of IL-4 after stimulation for 24 h did not result in significant inhibition. Phenotypic analyses of cells cultured in the presence of anti-CD3 mAb, IL-2, and IL-4 suggested that the mechanism of regulation by IL-4 involves the inhibition of IL-2R expression. The proportion of both CD4+ and CD8+ cells that expressed IL-2R in response to IL-2 was diminished in the presence of IL-4, although HLA-DR levels were unaffected. Soluble IL-2R was also reduced in supernatants of cultures stimulated with anti-CD3 mAb, IL-2, and IL-4 as compared to cultures stimulated with anti-CD3 mAb and IL-2. These findings indicate that when normal human T cells are stimulated in vitro in a manner that approximates a physiologic interaction with Ag in vivo, rIL-4 provides a potent inhibitory signal to IL-2 responsive cells that is likely mediated by IL-4-induced inhibition of IL-2R expression.

Antigens, CD↗
PubMed1990

Effect of food on the pharmacokinetics of cyclosporine in healthy subjects following oral and intravenous administration.

The pharmacokinetics of cyclosporine were studied in healthy subjects following administration of cyclosporine both orally (10 mg kg-1) and intravenously (4 mg kg-1) without and with high fat meals. Both blood and plasma samples (separated at 37 degrees C) were analyzed for cyclosporine concentration. Blood and plasma clearances of cyclosporine were calculated to be 0.36 and 0.47 L hr-1 Kg-1, respectively, and volume of distribution at steady state was calculated to be 1.21 L Kg-1, when cyclosporine was administered without a high fat meal. Using plasma analysis, the oral bioavailability of cyclosporine was estimated to be 21 and 79%, when administered without and with a high fat meal, respectively. When cyclosporine was administered intravenously together with a high fat meal, both clearance and volume of distribution increased significantly. Blood and plasma clearances of cyclosporine were 0.44 and 0.70 L hr-1 Kg-1, respectively, when cyclosporine was administered along with a high fat meal. We conclude that food not only enhances the absorption of cyclosporine but also enhances its clearance and volume of distribution. The observed variability in clearance, bioavailability, and volume of distribution values for cyclosporine across various pharmacokinetic studies can be partially accounted by the type of food administered and the sampling matrix used for analysis.

Administration, Oral↗
PubMed1990

Altered monocyte function in uremia.

Uremia appears to suppress immune function predisposing patients to infections. When the defect in cellular immunity was studied by exposing mononuclear cells (MNC) from uremic patients and controls to tetanus toxoid, diptheria toxoid, or Candida albicans antigen in vitro, the uremic cells were far less responsive. Monocytes and T cells, which are both involved in the proliferative response to soluble antigens, were isolated from MNC of uremic patients and HLA class II matched controls and incubated with tetanus toxoid. Tetanus toxoid-pulsed uremic monocytes were unable to stimulate the proliferation of HLA identical control T lymphocytes. Lymphocytes from uremic patients, however, were stimulated by tetanus toxoid-pulsed control monocytes. Therefore, the ability of monocytes to function as accessory cells is severely affected by uremia. The uremic monocytes were FcR+, produced IL-1 beta, and expressed levels of HLA class II antigens comparable to controls. Although the biochemical defect in uremic monocytes remains unknown, the abnormality could explain many of the immunological changes of uremia.

Adolescent↗
PubMed1990

Human spleen and peripheral blood lymphocytes activated by interleukin-2 have similar phenotypic and functional characteristics.

Phenotypic markers and cytotoxic function were monitored in cultures of normal human mononuclear cells obtained from peripheral blood or spleen and stimulated by recombinant interleukin-2 (IL-2; 1,500 U/ml). Fresh spleen cells contained less than 5% natural killer (NK) cells (CD3-NKH1+), which increased about threefold after activation with IL-2. In both spleen and peripheral blood cultures, T cells with the NKH1 marker showed the highest relative increment among all cell types studied. Lymphokine-activated killer (LAK) cells from peripheral blood and spleen displayed very similar cytotoxic activity against K562, Daudi, and COLO carcinoma cell lines. Killing of the three targets peaked at 7 days of culture. Antibody-dependent cell cytotoxicity against a B-cell line was mediated by both circulating and splenic LAK cells from 2 to 14 days of culture. Cell sorting experiments showed that K562 targets were killed by both CD5+NKH1+ and CD5-NKH1+ cells whereas Daudi targets are only killed by CD5-NKH1+ activated NK cells from both spleen and peripheral blood. In summary, human spleen LAK cells have similar phenotypic and functional properties to circulating LAK cells, and they may be used for adoptive immunotherapy of human cancer.

Antigens, Differentiation↗
PubMed1989

A flow cytometric method for the detection of the development of antibody to Orthoclone OKT3.

Orthoclone OKT3 is a monoclonal antibody used in the treatment of transplant rejection. The development of anti-OKT3 antibodies after therapy with this drug is a side effect which must be monitored. The current ELISA test to detect these antibodies is difficult to standardize between laboratories and does not lend itself to frequent monitoring of small numbers of samples. This prompted us to develop a new method employing flow cytometry to detect the development of anti-murine antibodies. Comparing samples tested by both techniques, we found the flow cytometric method to be more sensitive and as the ELISA methodology. This technique is adaptable to numerous serologic assays and could greatly expand the use of flow cytometry in the clinical laboratory.

Antibodies, Anti-Idiotypic↗
PubMed1989

"Anti-idiotypic" antibodies to HLA in transiently sensitized DST patients.

To test the hypothesis whether "anti-idiotypic" antibodies (Ab2) were involved in the loss of sensitization following donor-specific blood transfusions (DST), we investigated nine potential kidney graft recipients who became transiently sensitized after DST. Inhibiting "anti-idiotypic" activity of post-DST sera was determined using a complement-dependent cytotoxicity inhibition assay. The follow-up after DST ranged from 9 to 66 months. The nine patients developed 12 different anti-HLA antibodies (Ab1). Inhibiting post-DST serum activity was found in relation to four of them, while enhancement of cell killing caused by post-DST sera was observed in relation to six anti-HLA antibodies. In two patients presenting with more than one anti-HLA antibody inhibition was found with only one of the antibodies, indicating that the blocking of Ab1 was specific for the respective antibody. Enhancement was associated with a significantly prolonged sensitization of the patients. On the average, 5.7 +/- 5.0 months were necessary before "anti-idiotypic" activity was developed after the loss of sensitization, whereas enhancement was found immediately thereafter. Testing by flow cytometry indicated that enhancing sera still contained subthreshold levels of Ab1. Our results indicate that DST can induce the development of "anti-idiotypic" antibodies following a short period of sensitization. This finding provides further evidence that anti-idiotypic antibodies might be relevant factors in the eventual disappearance of sensitization to HLA antigens.

Adult↗
PubMed1989

Reduction in HLA-DR, HLA-DQ and HLA-DP expression by Leu-M3+ cells from the peripheral blood of patients with thermal injury.

Monocytes that bear HLA Class II antigens, such as HLA-DR, HLA-DQ, or HLA-DP, are obligatory for many cell-mediated immunological processes. Patients with thermal injury suffer from hypoimmunity and are at risk for developing life-threatening septic episodes. To determine whether an alteration in expression of HLA Class II antigens is involved in the defect, monocytes from the peripheral blood of burn patients and controls were double-stained with anti-Leu-M3 and either anti-HLA-DR, HLA-DQ, or HLA-DP monoclonal antibodies. As analysed by flow cytometry the percentage of Leu-M3+ monocytes from the peripheral blood from patients and controls was the same. The percentage of Leu-M3+ monocytes bearing the HLA Class II antigens and the density of antigen on the monocytes, however, was significantly reduced post-burn compared with controls. In nearly all cases these changes were detected as early as 24 h post-burn before any drug therapy was implemented. In-vivo re-expression of normal levels of HLA Class II coincided with patient recovery. In-vitro exposure of post-burn Leu-M3+ cells to IFN-gamma for 72 h restored HLA Class II expression to control levels. It is possible that the reductions in HLA Class II expression may be involved in the general immunosuppression that follows thermal injury.

Adult↗