ABSTRACT
Currently there is minimal concern that islet allograft failure could result from the development of anti-human leukocyte antigen (HLA) antibodies reactive to the allograft. We report here a case of islet allograft failure where the recipient developed immunoglobulin G anti-HLA class I antibodies reactive to HLA antigens present in two of the three islet cell donors. The patient had no detectable anti-HLA antibodies prior to the transplant but these antibodies were detected approximately 4 months posttransplant. Of concern, these antibodies developed despite induction with anti-IL2R antibodies (Zenapex) prior to intraportal islet cell infusion, low-dose tacrolimus (12-hour troughs 3 to 5 ng/mL) and rapammune (target troughs 12 to 15 ng/mL). The patient was not presensitized with blood products or a previous allograft. Her husband, however, shared antigens present in one of the islet donors and the recipient could have been presensitized to her husband during her two pregnancies. This case clearly demonstrates that islet allografts can lead to development of anti-HLA antibodies, which can cause islet allograft failure, as is the case with solid organ transplants, and hence emphasizes the need to monitor for such antibodies pre- and posttransplant. Additionally it appears that currently recommended immunosuppression may not be sufficient to inhibit a humoral response to both alloantigens and autoantigens.
Subject(s)
Antibodies, Anti-Idiotypic/blood , Histocompatibility Antigens Class I/immunology , Islets of Langerhans Transplantation/immunology , Adult , B-Lymphocytes/immunology , Diabetes Mellitus, Type 1/drug therapy , Diabetes Mellitus, Type 1/immunology , Diabetes Mellitus, Type 1/surgery , Female , Histocompatibility Testing , Humans , Insulin/therapeutic use , T-Lymphocytes/immunology , Transplantation, Homologous , Treatment FailureABSTRACT
The purpose of our study is to assess the extent of racial differences in the access to simultaneous pancreas-kidney (SPK) transplantation and evaluate the potential influence of socioeconomic factors on access to transplantation. We performed a retrospective analysis of the US Renal Data System and United Network for Organ Sharing data on all patients with end-stage renal disease (ESRD) due to diabetes mellitus from 1988 to 1996 (n = 562, 814), including all dialysis, wait list, and transplant patients. Racial differences in incidence, prevalence, insurance coverage, employment status, and transplantation rates were calculated. Caucasians had the highest prevalence of ESRD caused by type 1 diabetes (73%), followed by blacks (22%), Hispanics (3%), Native Americans (2%), and others (<1%). Both blacks and Native Americans increased their annual incidence of ESRD caused by insulin-dependent diabetes mellitus by 10% compared with only a 3.5% increase in Caucasians, whereas incidence rates increased annually by almost 8% for both blacks and Native Americans compared with a 3% increase for Caucasians. However, Caucasians received 92% of all SPK transplants, whereas all other racial groups combined received a disproportionate minority of the remaining transplants. Lack of private insurance and unemployment status were associated with annual changes in both incidence of ESRD caused by type 1 diabetes and SPK transplant rates. In conclusion, we observed striking racial disparities for access to SPK transplantation in the United States today, which may be related to employment status, access to private insurance, and subsequent health care. Our preliminary data support current efforts to encourage Medicare and Medicaid coverage for all patients requiring SPK transplantation regardless of racial or financial status.
Subject(s)
Diabetes Mellitus, Type 1/ethnology , Diabetic Nephropathies/ethnology , Diabetic Nephropathies/surgery , Ethnicity/statistics & numerical data , Kidney Failure, Chronic/ethnology , Kidney Failure, Chronic/surgery , Kidney Transplantation/statistics & numerical data , Pancreas Transplantation/statistics & numerical data , Black or African American/statistics & numerical data , Asian/statistics & numerical data , Diabetes Mellitus, Type 1/complications , Diabetes Mellitus, Type 1/epidemiology , Diabetic Nephropathies/epidemiology , Employment/statistics & numerical data , Hispanic or Latino/statistics & numerical data , Humans , Incidence , Indians, North American/statistics & numerical data , Kidney Failure, Chronic/epidemiology , Prevalence , Retrospective Studies , Socioeconomic Factors , United States/epidemiology , United States/ethnology , White People/statistics & numerical dataABSTRACT
The purpose of our study was to evaluate the association of race and ethnicity with outcomes in the living related donor (LRD) renal transplant population, using multivariable adjustment for potential confounding variables. We prospectively analyzed 14,617 patients from the UNOS Renal Transplant Registry who underwent LRD renal transplantations in the United States between January 1, 1988 and December 31, 1996 using the Cox proportional hazards model. This model adjusts for the effects of potential genetic, social, and demographic confounding variables that may be associated with race or ethnicity long-term graft survival. Blacks were 1.8 times as likely as whites (P < 0.01, RR = 1.77) to suffer graft failure during the 9-year study period, which decreased minimally to 1.7 (P < 0.01, RR = 1.65) after controlling for potential confounding variables. Neither genotypic nor phenotypic HLA matching improved outcomes in blacks. Black renal transplant recipients had lower graft survival even after adjustment for matching and rejection, suggesting that non-HLA or socioeconomic mechanisms may contribute to racial differences in transplantation outcomes.
Subject(s)
Kidney Transplantation/mortality , Living Donors , Postoperative Complications/mortality , Racial Groups , Adult , Black People , Female , Graft Survival , Histocompatibility Testing , Humans , Male , Multivariate Analysis , Proportional Hazards Models , Prospective Studies , Registries/statistics & numerical data , Risk Factors , Survival Rate , United States , White PeopleABSTRACT
Whole Nigella sativa (N. sativa) proteins were purified on a DEAE Sephadex A50 ion exchange column. Complete fractionation was achieved in four peaks. Analysis of the purified peaks was carried out by sodium dodecyl sulphate polyacrylamide gel electrophoresis. Whole N. sativa showed a number of protein bands ranging from 94-10 kDa molecular mass. In mixed lymphocyte cultures (MLC), whole N. sativa and its purified proteins were found stimulatory as well as suppressive and this effect varied from one donor to another. Maximum stimulation (mean + S.E. of % relative index was 63.73 + 20.78) was observed with fractionated N. sativa proteins (P1) (10 microg/ml) in MLC. In MLC, also N. sativa peaks (P1 and P2) were stimulatory at all concentrations (10 microg/ml, 1 microg/ml or 0.1 microg/ml) used. However, a uniformly suppressive effect of N. sativa and its all four peaks at a concentration of 10 microg/ml was noticed when lymphocytes were activated with pokeweed mitogen (PWM). The effect of N. sativa proteins was further evaluated on the production of cytokines which were measured by using specific enzyme-linked immunosorbent assay. Large quantities of IL-1beta were secreted by whole N. sativa in culture medium with non-activated peripheral blood mononuclear cells (PBMC) (450 pg/ml) and with allogeneic cells (410 pg/ml). Fractionated N. sativa was less effective when compared with whole N. sativa proteins. No effect on IL-4 secretion was seen either by using non-activated, PWM-activated or allogeneic-cells. Whole N. sativa suppressed as well as stimulated the production of IL-8 in non-activated and PWM-activated PBMC respectively. All N. sativa peaks with protein concentration of 2 microg/ml were stimulatory for the induction of IL-8 by PWM-activated cells. However, no effect on IL-8 was seen either with whole N. sativa or its peaks when allogeneic PBMC were used. Stimulatory effect of whole N. sativa and fractionated proteins was also noticed on the production of TNF-alpha either using non-activated or mitogen activated cells.
Subject(s)
Adjuvants, Immunologic/pharmacology , Plant Proteins/pharmacology , Adjuvants, Immunologic/isolation & purification , Chromatography, Ion Exchange/methods , Electrophoresis, Polyacrylamide Gel , Humans , Interleukins/biosynthesis , Leukocytes, Mononuclear/drug effects , Leukocytes, Mononuclear/immunology , Leukocytes, Mononuclear/metabolism , Lymphocyte Activation/drug effects , Lymphocyte Culture Test, Mixed , Plant Extracts/isolation & purification , Plant Extracts/pharmacology , Plant Lectins , Plant Proteins/isolation & purification , Plants, Medicinal/chemistry , Pokeweed Mitogens/pharmacology , Seeds/chemistry , Sodium Dodecyl Sulfate , Tumor Necrosis Factor-alpha/biosynthesisABSTRACT
PURPOSE: To evaluate the utility and potential nephrotoxicity of gadolinium-based contrast angiography when used with carbon dioxide angiography in renal transplant patients with suspected vascular causes of renal insufficiency and/or accelerated hypertension. MATERIALS AND METHODS: Thirteen consecutive renal transplant patients with suspected vascular causes of renal insufficiency and/or accelerated hypertension were evaluated with gadolinium-based contrast and CO2 angiography with use of digital subtraction techniques. Stenotic lesions were treated with angioplasty with/or without stent placement. No iodinated contrast agents were used. Serum creatinine levels were obtained before and at 24 and 48 hours after the procedure. An increase in creatinine levels greater than 0.5 mg/dL (44 micromol/L) was considered significant. RESULTS: Nine patients were studied for renal insufficiency, two for accelerated hypertension, and two for both. All 13 studies were considered diagnostic. Significant stenoses were treated in four patients with angioplasty with or without stent placement. Two patients had progression of their renal insufficiency. One of these patients underwent biopsy and was found to have both acute and chronic rejection. The other patient underwent cardiac catheterization 2 days after a transplant renal artery angioplasty. In the remaining nine patients with renal insufficiency (creatinine range, 1.8-3.9 mg/dL [159-345 micromol/L]; mean, 2.7 mg/dL [239 micromol/L]), renal function improved or did not worsen. CONCLUSION: Based on this limited study, gadolinium-based contrast angiography appears to be a promising supplement to CO2 angiography for the diagnosis and treatment of vascular lesions in patients with renal transplant insufficiency and/or accelerated hypertension. Further study is necessary to determine safety, optimal gadolinium dosage, and imaging parameters.
Subject(s)
Acute Kidney Injury/diagnostic imaging , Angiography, Digital Subtraction/methods , Carbon Dioxide , Contrast Media , Gadolinium DTPA , Hypertension, Renal/diagnostic imaging , Kidney Transplantation/diagnostic imaging , Acute Disease , Adult , Aged , Angioplasty, Balloon , Biopsy , Cardiac Catheterization , Chronic Disease , Contrast Media/administration & dosage , Creatinine/blood , Disease Progression , Female , Follow-Up Studies , Gadolinium DTPA/administration & dosage , Graft Rejection/diagnosis , Humans , Injections, Intra-Arterial , Male , Middle Aged , Prospective Studies , Renal Artery Obstruction/diagnostic imaging , Renal Artery Obstruction/therapy , StentsABSTRACT
The current studies were aimed at investigating why certain murine anti-CD3 isotypes (e.g. IgG2b) were poor activators of human T cells despite binding to the same epitope as the IgG2a anti-CD3. Most experiments were conducted with a human T cell line (HuT-78) and U937 a human histiocytic cell line with Fc gamma RI and Fc gamma RII. HuT-78 cells lack Fc gamma R. Anti-CD3-TCR of IgG2a isotypes and some antibodies of the IgG1 isotype, but not IgG2b, activated HuT-78 to secrete IL-2 only in the presence of U937. Epitope binding differences could not entirely explain these observations as OKT3E(IgG2a) but not its switch variant (IgG2b) activated HuT-78 in the presence of U937. Of interest, OKT3D(IgG1) in its intact form, but not its F(ab')2, activated HuT-78 to secrete IL-2 in the absence of U937 suggesting therefore that OKT3D activates T cells in the absence of macrophages and provided its Fc domain is intact. All three anti-CD3 with IgG2b isotypes (but none with the IgG2a isotypes) activated HuT-78 to secrete IL-2 only when these antibodies were immobilized onto plastic (in the absence of U937). Of considerable importance, anti-CD3 activation of human T cells with the IgG2b isotype was efficient in the presence of murine macrophages. These observations would suggest that murine anti-CD3 with IgG2b and certain antibodies of IgG1 isotypes, have inherent human T cell activating potential provided the Fc domain is in some manner activated, as for example, by binding to plastic or to Fc gamma R on murine macrophages. Macrophage cytokines are not essential. Therefore, ineffectual anti-CD3-mediated human T cell activation when using murine mAb with the IgG2b isotypes can best be explained on lack of Fc domain activation by human macrophages. This may indeed be the case as IgG2b and certain antibodies of IgG1 isotype, failed to bind to Fc gamma R on macrophages at 37 degrees C. These studies would indicate that murine mAb with the IgG2b isotype may be potentially useful for human use, especially in situations where receptor blockade (without cell activation) is of prime importance.
Subject(s)
Antibodies, Monoclonal , CD3 Complex , Immunoglobulin G , Lymphocyte Activation , T-Lymphocytes/immunology , Animals , Antibodies, Monoclonal/metabolism , Cell Line , Humans , Immunoglobulin G/classification , Immunoglobulin G/metabolism , Interleukin-1/pharmacology , Interleukin-2/metabolism , Interleukin-2/pharmacology , Interleukin-6/pharmacology , Macrophages/immunology , Mice , Receptors, Antigen, T-Cell , Receptors, IgG/metabolismABSTRACT
We present a report of a transplant recipient who lost her renal allograft from hyperacute rejection. This was secondary to a weak IgG anti-HLA class I antibody that was only reactive to donor B lymphocytes. This antibody was not detected in her pretransplant serum by the conventional complement-dependent cytotoxicity assays using donor blood lymphocytes. Pretransplant sera were analyzed retrospectively by two-color flow cytometric crossmatching (FCXM). It was difficult to determine if the recipient's serum contained an IgG antibody specific for HLA on donor B cells since IgG from control AB sera and pretransplant sera bound equally well to CD19 B cells. However, when donor lymphocytes were pretreated with pronase to digest the membrane receptor for Fc domain of IgG (Fc gamma R) on non-T-cells, control IgG in AB serum did not bind to B cells and, hence, it was easy to detect binding of IgG (in pretransplant sera) to HLA on B cells. This case underscores the importance of identifying weak anti-HLA class I antibodies reactive only to B cells. Moreover, it shows that the currently used two-color FCXM lacks the specificity to detect such antibodies.
Subject(s)
Antibodies/immunology , B-Lymphocytes/immunology , Graft Rejection/immunology , Histocompatibility Antigens Class I/immunology , Immunoglobulin G/analysis , Kidney Transplantation/immunology , Adult , Antibodies/analysis , Female , Flow Cytometry/methods , Humans , PronaseABSTRACT
We have previously shown that mutant B lymphoblastoid cell lines, totally deficient in expression of human leucocyte antigen (HLA)-class II molecules, but with normal HLA-class I expression, develop enhanced susceptibility to natural killer (NK) and lymphokine-activated killer (LAK) cell lysis. The current investigations were aimed at examining the role of HLA-DR and native peptides occupying the antigen-presenting grooves of HLA-class II molecules in protecting mutants of the same B-lymphoid lines against LAK-mediated lysis. No augmentation in LAK lysis was observed despite using two mutant B-cell lines (9.22.3 and 3.1.0) that lacked HLA-DR. Both these lines expressed HLA-DP and HLA-DQ. However, when using other B-cell lines with point mutations in certain regions of the HLA-DR alpha-chain (78, 80 and 96) significantly increased their susceptibility to LAK lysis despite normal expression of HLA-DR and the other class I and II molecules. Of particular interest was the finding that absence of native peptides in antigen-presenting grooves of all the HLA-class II molecules did not render the mutant B cell (9.5.3) susceptible to LAK lysis. These observations support the concept that there are different NK or LAK clones. Certain LAK clones recognize 'self' major histocompatibility complex (MHC) antigens (but not the native peptides in their antigen-presenting grooves). Presence of 'self' MHC antigens inhibits such clones. Conversely, other NK or LAK clones recognize 'non-self' in the context of MHC antigens. Hence, point mutations at certain specific sites on the MHC molecules or foreign peptides in the antigen-presenting grooves enhances the susceptibility of these cells to LAK clones recognizing 'non-self'.
Subject(s)
Cytotoxicity, Immunologic/immunology , HLA-D Antigens/immunology , Killer Cells, Lymphokine-Activated/immunology , Killer Cells, Natural/immunology , Lymphoma, B-Cell/immunology , Antigen Presentation , HLA-DR Antigens/immunology , Humans , Peptides/immunology , Tumor Cells, CulturedABSTRACT
Currently there is a paucity of data regarding the influence of high serum triglyceride levels on cyclosporin A (CyA) levels and dosing. We therefore undertook a retrospective study to determine the relationship of serum lipid levels to CyA levels and CyA dosages. Renal transplant patients at a 0.5-to-3-year post-transplant stage, with a stable CyA dosage, who were not on medications that affect CyA metabolism or renal function, were entered into the study. The CyA dosage was adjusted by clinicians to maintain whole blood. 12-h CyA trough levels between 200 and 250 ng/ml (monoclonal TDX method, which measures the parent compound). Forty-four patients qualified for the study. The data clearly indicated that high cholesterol levels (> 300 mg/dl and with normal triglyceride levels) did not influence the CyA levels or the dosages. Conversely, high triglyceride levels ( > 500 mg/dl) significantly reduced the amount of CyA required. A decreased clearance of CyA in the presence of hypertriglyceridemia led to high CyA levels in some patients. Reducing the CyA dosage to achieve levels between 200 and 250 ng/ml improved renal allograft function and decreased other side effects attributed to CyA toxicity. These studies indicate that high triglyceride levels, but not high cholesterol levels, increase CyA levels, which can lead to CyA toxicity.
Subject(s)
Cholesterol/blood , Cyclosporine/administration & dosage , Graft Rejection/prevention & control , Hypertriglyceridemia/blood , Immunosuppressive Agents/administration & dosage , Kidney Transplantation/immunology , Adolescent , Creatinine/blood , Dose-Response Relationship, Drug , Follow-Up Studies , Graft Rejection/blood , Humans , Hypertriglyceridemia/drug therapy , Middle Aged , Multivariate Analysis , Retrospective StudiesABSTRACT
Since endogenous 1:25 vitamin D (1:25VD) is principally involved with involution of secondary hyperparathyroidism post-renal transplant we correlated 1:25VD levels with intact PTH in 82 random patients with a serum creatinine of < 2 mg/dl and with normal hepatic function. All patients studied were normocalcemic with normal phosphorus and received azathioprine, cyclosporin A and prednisone. Of considerable interest, of the 42 patients studied after 2 years post-transplant, there were 8 (19%) patients with intact PTH of more than twice the upper limit of normal (normal 10-65 pg/ml) and other 15 (36%) with PTH levels above normal. Secondly, in no patient did we see 1:25VD above normal (normal 15-60 pg/ml) despite levels of PTH of > 200 pg/ml. Of concern, 20% of 73 patients had 1:25VD deficiency (< 15 pg/ml). This may not have been previously appreciated because of the number of patients studied. Like previous investigators, we failed to understand why 1:25VD levels were relatively low. There was no correlation between 1:25VD and serum creatinine. Of 25 patients with a serum creatinine of 1.4 or less, there were 10 patients (40%) with 1:25VD of less than 20 pg/ml. Since persistently high PTH can contribute to bone demineralization, which is not uncommon post-transplant, we treated 8 patients with small doses of oral 1:25VD (rocaltrol). In less than 6 months PTH levels returned to normal in 7 of the 8 patients. The current studies clearly indicate that asymptomatic hyperparathyroidism is common even after 2 years post-renal transplant. Monitoring for PTH and 1:25VD will help prevent bone disease post-transplant now that rocaltrol is available.
Subject(s)
Calcitriol/blood , Calcitriol/therapeutic use , Calcium/blood , Hyperparathyroidism, Secondary/blood , Hyperparathyroidism, Secondary/drug therapy , Kidney Transplantation , Adolescent , Adult , Aged , Azathioprine/therapeutic use , Calcitriol/deficiency , Child , Child, Preschool , Chronic Kidney Disease-Mineral and Bone Disorder/prevention & control , Creatinine/blood , Cyclosporine/therapeutic use , Female , Follow-Up Studies , Glucocorticoids/therapeutic use , Humans , Immunosuppressive Agents/therapeutic use , Kidney Transplantation/adverse effects , Liver/physiology , Male , Middle Aged , Parathyroid Hormone/blood , Phosphorus/blood , Prednisone/therapeutic useABSTRACT
The effects of Nigella sativa (N. sativa) seeds and their soluble fractions were studied in vitro on lymphocyte response to different mitogens and on polymorphonuclear leukocyte phagocytic activity. No stimulatory effect of N. sativa was detected on lymphocyte response to phytohemagglutinin, concanavalin-A or pokeweed mitogen. A stimulatory effect of N. sativa was noticed on the lymphocyte response to pooled allogeneic cells. This effect was more pronounced when the low molecular weight (< 10 kDa) fraction was used and varied from one normal individual to another (25% to 825%). N. sativa enhanced the production of interleukin-3 by human lymphocytes when cultured with pooled allogeneic cells or without any added stimulator. N. sativa did not, however, enhance or suppress interleukin-2 secretion by mitogen activated peripheral blood mononuclear cells. Interestingly, N. sativa increased interleukin-1 beta, suggesting therefore, that it has an effect on macrophages. It also suppressed the leukocyte chemiluminescence activity using phorbol myristate acetate and Zymosan as stimulants. No effect of N. sativa or its fractions was, however, noticed on bacterial phagocytosis or killing when Staphylococcus aureus was used, indicating that the decrease in chemiluminescence activity in the presence of N. sativa is not relevant to the bactericidal activity.
Subject(s)
Lymphocytes/immunology , Neutrophils/immunology , Phagocytosis , Plants, Medicinal , Humans , In Vitro Techniques , Interleukin-2/biosynthesis , Interleukin-3/biosynthesis , Luminescent Measurements , Lymphocyte Activation/drug effects , Lymphocyte Culture Test, Mixed , Lymphocytes/drug effects , Mitogens/pharmacology , Neutrophils/drug effects , Phagocytosis/drug effects , Plant Extracts/pharmacology , Staphylococcus aureus/immunologyABSTRACT
The current prospective investigation was conducted to determine whether development of IgG donor-specific lymphocytotoxins detected at the onset of acute rejections was predictive of a poor-prognosis acute rejection. Between January 1990 and August 1993, 206 kidney transplants were performed. Cadaver kidney recipients were managed with antilymphocyte globulin as induction therapy and all recipients (i.e., cadaver and living related donor kidneys) received triple immunosuppressive therapy, i.e., CsA, AZA, and prednisone. Rejections were treated with intravenous Solu-Medrol and OKT3. Presence of donor-specific IgG lymphocytotoxin was detected by using dithiothreitol-pretreated sera (obtained at onset of rejection) and frozen donor cells. In addition, percentage of panel reactive antibody was determined on this dithiothreitol-pretreated sera. Of the 82 patients with biopsy-proven acute rejections, 19 were found to have developed donor-specific IgG lymphocytotoxin and a marked increase in panel reactive antibody. One-year graft survival in this group was dismal (16%), despite OKT3 therapy. Over 90% of these patients lost their graft within 2 months of rejection diagnosis. In 63 recipients who had acute rejections without development of IgG anti-HLA antibody, 1-year graft survival was 72%. The majority of these patients lost their grafts from chronic rejection. No anti-HLA activity was found in patients who did not have rejection episodes. Based on this study, evidence indicates that assaying for IgG donor-specific antibody at time of rejection is a valuable tool for selecting a subset of patients with poor-prognosis acute rejections. Identifying this subset will become important as we enter an era of new immunosuppressive agents.
Subject(s)
Antilymphocyte Serum/immunology , Graft Rejection/immunology , HLA Antigens/immunology , Immunoglobulin G/blood , Kidney Transplantation , Tissue Donors , Adolescent , Adult , Aged , Child , Child, Preschool , Female , Graft Rejection/prevention & control , Humans , Lymphotoxin-alpha/immunology , Male , Methylprednisolone Hemisuccinate/administration & dosage , Middle Aged , Muromonab-CD3/administration & dosage , Prospective StudiesABSTRACT
Two-color fluorescence cytometry (FCXM) has recently been introduced to improve the detection of anti-HLA antibodies that react to donor cells, especially in recipients receiving kidney allografts. Although this assay system is highly sensitive, it lacks specificity. Between 70% and 90% of potential kidney recipients with a positive FCXM would have been denied transplant if such an assay had been used alone to detect antidonor antibodies. Lack of specificity is principally due to normal or irrelevant IgG in aggregates or immune complexes binding to Fc gamma R receptors on lymphocytes including B cells and a significant subset of T cells. To circumvent this problem, we digested Fc gamma R receptors on lymphocytes with pronase. We present data demonstrating that pronase digestion of lymphocytes does not alter HLA antigenicity. In addition, pronased lymphocytes allow one to use either single- or two-color FCXM. With single-color FCXM, one can quantitate antibody reactivity to lymphocytes via a cursor (on the fluorescence histogram) that separates lymphocytes that do not bind to antibodies. We present data demonstrating that this modification renders FCXM highly sensitive and specific. In addition, one can discriminate between IgG and IgM antibodies that react to lymphocytes.
Subject(s)
Flow Cytometry/methods , HLA Antigens/immunology , Lymphocytes/immunology , Pronase , Fluorescent Antibody Technique, Indirect , Humans , Immunoglobulin M/immunology , Sensitivity and SpecificityABSTRACT
There are several lines of evidence clearly demonstrating that major histocompatibility complex (MHC) class I antigens are important in protecting haemopoietic tumour cells from natural killer (NK)-mediated cell lysis. In the present studies we examined the role of MHC class II antigens in affording such protection to haemopoietic tumour cell lines. NK and lymphokine-activated killer (LAK) lysis were performed on two human B lymphoma cell lines and their mutants lacking HLA class II expression, i.e. DR, DP and DQ. Raji and T5-1 were compared to their mutants RM3 and 6.1.6, respectively. Significantly more lysis was observed with the mutants compared to the parent cell line. Effectors used included (1) peripheral blood NK effectors, (2) a human NK cell line (NK 3.3), and (3) peripheral blood LAK effectors. The increased lysis with the mutants could not be explained on the basis of (1) increased conjugate formation, (2) increased cell fragility or (3) ineffectual expression of HLA class I and other non-HLA antigens. These findings suggest that HLA class II molecules may have a novel role. They may be relevant not only in antigen presentation but may also protect tumour cells (and possibly normal activated lymphoid cells) against lysis mediated by NK and LAK cells.
Subject(s)
Cytotoxicity, Immunologic/immunology , HLA-D Antigens/immunology , Killer Cells, Lymphokine-Activated/immunology , Killer Cells, Natural/immunology , Lymphoma, B-Cell/immunology , Antigens, Neoplasm/immunology , HLA-D Antigens/analysis , Histocompatibility Antigens Class I/analysis , Humans , Mutation/immunology , Osmotic Fragility/immunology , Tumor Cells, CulturedABSTRACT
Recent reports indicate a higher incidence of both acute and chronic liver allograft rejection when, at the time of transplantation, the recipients serum contains donor-specific anti-HLA antibodies. From 9/89 to 5/91, 133 liver allografts were performed at our institution. Thirteen liver recipients had donor-specific IgG anti-HLA antibodies (complement-fixing) at the time of transplantation. In eleven patients, antibodies reacted to donor class I antigens while in 1 patient the donor-specific antibody had class II reactivity. Twelve patients have been followed for a minimum of 12 months (median 18 months, range 28-12 months). No hyperacute rejection was seen in any of the cases and four patients had acute rejections. Thus far only one of the twelve patients has biopsy evidence suggestive of chronic liver injury. The remaining have normal liver enzymes and bilirubin. Three of these twelve patients died (one from a myocardial infarction and the others from sepsis) accounting for a one-year graft survival of 75%. There was no significant statistical difference in the one-year graft survival in those recipients without donor-specific antibodies (i.e., 80.5%). In eight of the twelve patients, pretransplant preformed antibody level (PRA) was > 50%. In six of the thirteen patients donor-specific antibody was present at dilutions greater than 1:64. As previously reported, the donor-specific antibody disappeared from the serum posttransplant within hours and did not reappear. In vitro studies demonstrated no factor in portal or hepatic artery blood that could inhibit rabbit complement mediated lysis of anti-HLA antibodies. We conclude that it is not a contraindication to do liver transplants in the presence of donor-specific anti-HLA antibodies.
Subject(s)
Antibodies/pharmacology , HLA Antigens/immunology , Liver Transplantation/immunology , Adolescent , Adult , Aged , Antibody Specificity , Child , Female , Graft Rejection/etiology , Graft Survival/immunology , Humans , Liver Transplantation/physiology , Male , Middle Aged , Time FactorsABSTRACT
The role of major histocompatibility gene products (i.e., HLA molecules) in rendering tumor cells resistant to natural killer (NK) cell-mediated lysis was investigated by using mouse monoclonal antibodies to bind and mask HLA or non-HLA gene products on the cell membrane of human allogeneic tumor targets. Enhanced lysis of resistant lymphoid and certain other solid tumor cell lines was observed only when monoclonals used reacted to class I and II HLA molecules but not non-HLA molecules on tumor targets. Enhanced lysis was not due to antibody dependent cellular cytotoxicity or due to an effect of antibody on NK effectors. Of importance, normal autologous and allogeneic human lymphocytes could not be lysed by NK cells despite blast transformation with mitogens or masking of HLA membrane determinants on blasts with monoclonal antibodies. Enhanced lysis, in the presence of antibody to HLA antigens, was not due to increased NK cell binding to tumor targets, but a consequence of enhanced postbinding lysis. Studies using granules obtained from NK cells indicated that masking of HLA antigens did not enhance the susceptibility of tumor targets to cytolysins. Such observations would suggest that HLA antigens on tumor targets inhibit the triggering of effector cells (and release of cytolysins) after recognition and binding of NK cells to target cells.
Subject(s)
Genes, MHC Class II , Histocompatibility Antigens/immunology , Killer Cells, Natural/immunology , Animals , Antibodies, Monoclonal , Antibody-Dependent Cell Cytotoxicity , Cell Line , Epitopes/immunology , Histocompatibility Antigens Class I/immunology , MiceABSTRACT
In these studies, the inhibitory role of a large granular lymphocyte (LGL) subset (CD5-,CD16+) on pokeweed mitogen (PWM)-induced B lymphocyte differentiation was examined. CD5-,CD16+ LGL cells are the predominant subset of LGL cells and are possibly distinct from other LGL subsets in that they lack B and T cell markers. CD5-,CD16+ LGL possess abundant FcIgG receptors and previous studies have clearly demonstrated that in the presence of insoluble immune complexes, this LGL subset will inhibit B lymphocyte differentiation in the presence of T cells. In the present studies, we analyzed the inhibiting role of CD5-,CD16+ LGL cells that had not been activated by immune complexes. B + L preparations obtained by removal of E rosette-forming T cells were further depleted of T lymphocytes by complement-dependent lysis of T cells using a monoclonal antibody reactive to total T cells (Leu-1, CD5 antigen, Becton-Dickinson). B lymphocytes in such B + L preparations failed to differentiate into plasma cells containing intracytoplasmic immunoglobulin (Ig), in the presence of PWM, T cell-derived helper supernatants (THS), and interleukin-2 (IL-2). However, B cells differentiated under these conditions, when B + L preparations were further depleted of CD5-,CD16+ LGL cells by complement-dependent lysis using a monoclonal antibody (Leu-11) reactive to CD16 antigen of FcIgG receptors present on LGL cells. These studies indicated that CD5-,CD16+ cells unlike the CD8-positive T suppressor cell, will directly inhibit B lymphocyte differentiation into plasmacytoid cells containing intracytoplasmic Ig when T lymphocytes are not present. However, addition of a few T lymphocytes (less than 10%) to purified B + L preparations abrogated the CD5-,CD16+ LGL cell inhibition of B cell differentiation.
Subject(s)
B-Lymphocytes/immunology , Cell Differentiation , Cytotoxicity, Immunologic , Interleukin-2/physiology , Killer Cells, Natural/immunology , Adult , Animals , B-Lymphocytes/cytology , Binding, Competitive , Cell Communication , Cell Separation , Cell-Free System , Humans , Killer Cells, Natural/classification , Lymphocyte Activation , Mice , Phenotype , Pokeweed MitogensABSTRACT
After B lymphocyte depletion, blood lymphocytes (PBMC) from certain individuals will generate substantial quantities of immunoglobulin (Ig) when cultured with pokeweed mitogen (PWM). The present investigations were aimed at analysing the cell subset in these T cell preparations, that was responsible for Ig production especially since the quantity of Ig generated was disproportionate to numbers of contaminating sIg-bearing B cells. Ficoll-Hypaque PBMC from 17 individuals were subjected to overnight sheep erythrocyte rosetting. Rosetted cells were subjected to very slow (100 X g) density gradient centrifugation to isolate T rosettes (and deplete PBMC of B cells). In 6 of these 17 individuals, such enriched T cells repeatedly generated substantial quantities of plasmacytoid cells after an 8-day culture in the presence of PWM and helper factors. Mean values for plasmacytoid cells per 1000 cells recovered were as follows: T cells 168.16 +/- 96.7 SD, B cells 226.67 +/- 161.1, PBMC 225.67 +/- 78.9. In further experiments, contaminating surface immunoglobulin (sIg)-positive B cells (less than 2% sIg-positive) were removed from the T cell preparations by the "panning" method, i.e. layering T cells on plates precoated with antisera specific for human Ig (polyvalent), and lysis of B cells with a monoclonal antibody BA-1. In these 6 individuals, removal of B cells by both these techniques completely abolished generation of plasmacytoid cells, thus confirming that it is a contaminating B cell subset which is responsible for Ig production in these T cell preparations. These data indicate that in certain individuals there are B cells that separate out with T cells during the E-rosette isolation procedure. With double immunofluorescence techniques, it became apparent that 4.8 +/- 1.3% of TRITC-labeled sIgM-bearing B cells were also labeled with FITC-conjugated monoclonal antibody to the sheep erythrocyte receptor. Kuritani and Cooper have previously demonstrated that PWM-responsive B cell precursors of IgM, IgG, or IgA plasmacytoid cells lack sIgD and, hence, comprise about 10-15% of the total B cells in PBMC. Our data would indicate that in certain individuals, about half this subset forms E-rosettes, which may explain why both E-rosette separated T cells and enriched B cells make similar quantities of plasmacytoid cells.
Subject(s)
B-Lymphocytes/cytology , Receptors, Immunologic/immunology , T-Lymphocytes/cytology , Animals , B-Lymphocytes/immunology , Cell Separation/methods , Complement System Proteins/immunology , Cytotoxicity, Immunologic , Erythrocytes/immunology , Fluorescent Antibody Technique , Humans , Lymphocyte Activation , Rosette Formation , Sheep , T-Lymphocytes/immunologyABSTRACT
Many potential renal transplant recipients develop multispecific anti-HLA antibodies after a previous unsuccessful transplant. Therefore, it became important to analyze factors that could predispose to multispecific anti-HLA antibodies in the hope of preventing their occurrence because their presence hinders early retransplantation. In these studies, we elected to retrospectively analyze the impact of previous transfusions and the degree of HLA-A,B antigen mismatch on the development of these antibodies. Patients transplanted within the South Eastern Organ Procurement Foundation ( SEOPF ) after January 1977 were analyzed. All patients in these studies were immunosuppressed with prednisone and azothioprine . Antibodies to HLA antigens were determined in a complement-dependent microcytotoxicity assay utilizing recipient's sera and lymphocyte cell panels from random donors. Multispecificity of antibody was quantitated and expressed as the percentage of reactivity to lymphocyte panel (PRL). Only patients who lost their first cadaveric kidney allograft, and who, in addition, had a peak of less than 15% pretransplant and no prior pregnancies were analyzed. Peak posttransplant PRL had to be determined within 3 months after allograft failure. In the 146 patients with accurate transfusion data, it became evident that patients who received more than 5 pretransplant transfusions seldom developed greater than or equal to 50% PRL posttransplantation (P less than 0.001). Only 12% of patients receiving more than 5 pretransplant transfusions developed greater than or equal to 50% PRL, whereas 50% of patients with minimal pretransplant transfusions developed greater than or equal to 50% PRL. This protective effect of pretransplant transfusions was seen even in recipients receiving 3 and 4 HLA-A,B mismatched kidneys (P less than 0.01). The reason for this apparent protective effect is not clear from our data; it could be explained either on the basis of a selection process (i.e., exclusion of high responders pretransplant) or by suppression of the immune system. Nonetheless, these observations warrant attention, because the protective effect may be useful in preventing development of high PRL posttransplant in the event of a rejection.