Characterization of a non-T, non-B human lymphocyte (L cell) with use of monoclonal antibodies. Its regulatory role in B lymphocyte function.

These studies investigate the role of L lymphocytes in regulating terminal B lymphocyte differentiation. L cells have abundant Fc IgG receptors and comprise 10--15% of human peripheral blood mononuclear cells (PBMC). L cells lack the conventional markers of B and T lymphocytes and in culture, do not develop into B cells, T cells, or macrophages. Additionally, use of monoclonal antibodies failed to detect on L cells, surface antigens specific for B cells, T cells, and macrophages. In these studies, purified L cell subpopulations depleted of macrophages were co-cultured with autologous PBMC in the presence of pokeweed mitogen and at the end of 8 d, development of intracytoplasmic immunoglobulin (Ig) was determined. L cells were depleted of B and T cells by rosetting techniques and, in addition, by cytotoxicity techniques using monoclonal-specific antisera to T cells. In 14 individuals, L cells when co-cultured with PBMC, enhanced Ig synthesis by 83% +/- 62 SD, and also enhanced cell proliferation. Radiated L cells lost enhancing properties. To study the role of their high density Fc IgG receptors, L cells pretreated with IgG antibody-sensitized erythrocytes were used (i.e., after lysis of rosettes). Such L cells significantly inhibited Ig synthesis (by greater than 50%) despite promoting cell proliferation. Antibody-sensitized erythrocyte-rosetted macrophages did not inhibited Ig synthesis. Thus, positive and negative influences can be mediated by the same cell, depending on the state of Fc-receptor stimulation. Such cells may play a more prominent role in "feed-back" regulation of Ig synthesis by virtue of having abundant Fc IgG receptors.

Use of anti-HLA antibodies to mask major histocompatibility complex gene products on tumor cells can enhance susceptibility of these cells to lysis by natural killer cells.

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.

Inhibition of humoral and cell-mediated immune responses in man by distinct suppressor cell systems.

Studies were designed to investigate whether the suppressor cell systems that regulate the humoral and cell-mediated immune responses belong to the same subsets of T cells or different subsets. Mitogen-activated suppressor cells were simultaneously assayed for their ability to inhibit (a) pokeweed mitogen-induced generation of plasma cells, (b) blastogenic response of lymphocytes to allogeneic cells, and (c) generation of killer cells in the cell-mediated lymphocytotoxicity assay. We found that suppressor cells that inhibited the generation of plasma cells were activated by concanavalin A (Con A) and were both radiation and prednisone sensitive. Suppressors that inhibited the blastogenic response in lymphocytes to allogenic cells were also activated by Con A but differed in that they were both radiation and prednisone resistant. In contrast, suppressors that inhibited the generation of the killer cells were activated with phytohemagglutinin and not Con A. These suppressors were prednisone and radiation resistant. These observations cannot be explained by differences at the pro-suppressor or suppressor activator levels as both T cell subsets are radiosensitive. Alternatively, heterogeneity of suppressor cell systems may explain these differences.

Characterizaiton of two populations of human lymphocytes bearing easily detectable surface immunoglobulin.

Two separate lymphocyte populations, each bearing easily detectable surface immunoglobulin, have been detected in human peripheral blood. The first, B cells, has surface determinants that are stable at 37degreeC, but are removed by pronase and regenerate in culture. The cells are nylon adherent and have a receptor for C3, and studies with unit gravity sedimentation indicate they are mostly small lymphocytes. B cells comprise 9.5% of the total lymphocytes, with the normal range from 3-16%. As many or more lymphocytes lack membrane-incorporated Ig determinants but have an Fc receptor that binds IgG1 and IgG3 in normal serum maximally at 4degreeC. This receptor for cytophilic IgG is removed by pronase but not by trypsin. The second population has been named L lymphocytes because of membrane-labile IgG determinants. L cells do not adhere to nylon, do not form rosettes with sheep erythrocytes sensitized with antibody and mouse complement, and are larger than small lymphocytes. These lymphocytes with cold-reactive Fc receptors for serum IgG do not form E-rosettes or respond to phytohemaggutinin. Since L cells do not have surface markers of T and B lymphocytes, it is likely that they comprise a separate population.

Development of anti-human leukocyte antigen class 1 antibodies following allogeneic islet cell transplantation.

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.

Double immunofluorescence staining of pokeweed mitogen differentiated plasma cells--a sensitive assay to ascertain purity of anti-human Ig reagents as each cell produces only one isotype.

A simple, but highly sensitive assay is described for the determination of the specificity and purity of heterologous antibodies to human immunoglobulin isotypes. In this assay, cytocentrifuge cell preparations of normal pokeweed mitogen differentiated plasma cells are stained initially with an FITC conjugate of an antibody to 1 isotype and then restained with a TRITC conjugate of an antibody to another isotope. No double staining of intracytoplasmic immunoglobulin in plasma cells was observed when highly purified and specific reagents were used. However, some reagents from commercial laboratories, which used immunodiffusion assays and enzyme immunoassay techniques (ELISA) to determine specificity of antisera, were found to lack specificity by the double immunofluorescence technique. This technique of obtaining cultured plasma cells with a wide variety of isotypes is reproducible and utilizes reagents that are easily available. Hence, one need not depend on panels of myeloma plasma cells or spleen cells which can be difficult to obtain.

Human blood B lymphocytes with receptors for sheep erythrocytes--their relevance in techniques to obtain T cells depleted of mature B cells.

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.

In vitro assays to study role of T cell-derived factors on human B lymphocytes should take into consideration inhibiting effect of large granular lymphocyte subset (CD5-,CD16+) that can contaminate B cell preparations.

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.

An apparent paradoxical effect of pretransplant blood transfusions. Its association with decreased anti-HLA antibody formation following unsuccessful renal transplantation.

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.

Kidney transplantation across positive B and T cell crossmatches.

Cadaveric renal transplants were performed despite a positive conventional crossmatch (usually intermediate positive) resulting from donor-specific B cell lymphocytotoxins (both IgG and IgM) or IgM cold-reactive T cell lymphocytotoxins. Graft survival at 2 months was 72% in the 14 patients with B cell-specific antibodies and 71% in the 7 recipients with T cell antibodies. No correlation was observed between graft rejection and warm (mainly IgG) or cold (IgM) B cell-specific antibodies. These results indicate that not all positive crossmatches are a contraindication to transplantation. Attempts should be made to study the nature of the lymphocytotoxins before withholding the allograft from the recipient.

Cold-reactive alloantibodies and allograft malfunction occurring immediately posttransplant.

Certain kidney allografts function promptly, whereas others subjected to similarly optimal procurement and preservation methods do not. Previous reports have indicated that such unexplained allograft malfunction (AM) could be due to the presence of cold-reactive IgM alloantibodies (i.e., lymphocytotoxins and agglutinins) present in renal transplant recipients. These investigations were undertaken to determine whether the presence of such alloantibodies was associated with any histological abnormalities. Pretransplant and 1-hr posttransplant biopsies were analyzed from 49 cadaveric renal allografts that came from ideal donors and were subjected to "optimal" preservation. First, no correlation could be made between AM and the severity of renal tubular cell disruption. However, glomerular lesions in the posttransplant biopsy correlated significantly with the development of AM. Segmental glomerular intracapillary red blood cell aggregates and fibrin deposition were present in 71% of biopsies in the 21 allografts with AM, whereas such lesions were present in 29% of biopsies in the 28 allografts with immediate function (P less than 0.005). Development of glomerular lesions correlated significantly with the presence of cold-reactive lymphocytotoxins (CRL) in the recipient (60% vs. 9%). Sera containing CRL were found to also have IgM antiendothelial cell antibody. These observations suggest that another possible mechanism for lack of prompt allograft function is a self-limiting vascular injury, that occurs in the cold and is immune-mediated.

Utility of protease-digested human peripheral blood lymphocytes for the detection of lymphocyte-reactive alloantibodies by indirect immunofluorescence.

Human peripheral blood lymphocytes were digested briefly with protease prior to application of indirect immunofluorescence techniques for detecting alloantibodies in sera of patients with chronic renal failure on maintenance hemodialysis. Background staining of intrinsic surface IgM and cytophilic IgG bound to Fc receptors was eliminated or greatly reduced, enabling detection of B cell specific antibodies, including cold-reactive types not demonstrable by conventional immunofluorescence or complement-dependent lymphocytotoxicity. The antigenicity of HLA and other surface membrane determinants was not decreased by protease, although reactivity with certain sera was enhanced. In experiments comparing indirect immunofluorescence using protease-treated cells with complement-dependent lymphocytotoxicity and antibody-dependent, lymphocyte-mediated cytotoxicity assays, indirect immunofluorescence was more sensitive and comprehensive, but not less specific, in defining alloantibodies of a variety of types.

The lack of long-term detrimental effects on liver allografts caused by donor-specific anti-HLA antibodies.

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.

Evidence demonstrating poor kidney graft survival when acute rejections are associated with IgG donor-specific lymphocytotoxin.

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.

Critical appraisal of complement dependent microlymphocytotoxicity assay for detecting donor-specific alloantibody pretransplant--importance of indirect immunofluorescence as a superior alternative.

The ability of complement-dependent microlymphocytotoxicity assay (CdL) to detect and discriminate between the various types of donor-specific alloantibodies was reevaluated. Data obtained with the CdL assay on purified B and T lymphocytes at warm and cold temperatures was compared to other modes of antibody-detection, i.e., indirect immunofluorescence (IF) and the noncomplement-dependent antibody-dependent cellular cytotoxicity (ADCC). Additionally, the significance of antibodies as detected by CdL and IF was ascertained by correlating with kidney transplant outcome. It became apparent that the CdL assay identified weakly reactive HLA-ABC alloantibodies as being B cell specific. Such weakly reactive HLA-ABC antibodies were also not appreciated in the presence of the cold reactive IgM antibody. Accelerated rejections were the rule in the presence of weakly reactive HLA-ABC alloantibodies indicating that their detection was highly important. The IF assay could discriminate between the antibody class, could detect weakly reactive HLA-ABC alloantibodies, and could detect noncomplement fixing antibodies (ADCC). Further, use of IF prevented us from unnecessarily denying transplants to certain recipients when a positive CdL assay resulted from an IgM antibody or poor cell viability.

Racial disparities in access to simultaneous pancreas-kidney transplantation in the United States.

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.

Racial disparities in renal transplant outcomes.

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.

Wound infections in renal transplant recipients--a complication of urinary tract infections during allograft malfunction.

Both wound and urinary tract infections are common in renal transplant recipients. Certain recipients, however, develop detrimental complications following such infections, and our aim was to analyze factors that predisposed recipients to such complications. Analysis of 174 consecutive transplants performed over a 7-year period ending December 1980 demonstrated that a urinary infection developing during acute tubular malfunction (ATM) led to serious septic complications. The complication rate was 70% in the 30 recipients in whom urinary tract infection occurred during ATM but only 10% in the 20 recipients with infection in the absence of ATM (P less than 0.001). Similarly, analysis of 14 deep wound infections showed that the source of the organisms was the urinary tract (12 cases), especially when the urinary tract infection occurred in the setting of ATM. Deep injections led to high rates of morbidity and mortality. Conversely, superficial wound infections (10 cases) contained staphylococci and healed without complications. We suggest that urinary tract organisms, which are difficult to eradicate with antibiotics because of low urinary concentration of antibiotics during ATM, lead to infection of the perirenal tissues.

Use of hemodialysis in meprobamate overdosage.

A case of meprobamate overdosage successfully treated with hemodialysis is described. The patient was admitted 4 hours after an overdosage of meprobamate (30-40 g) deeply unconscious, hypotensive, in respiratory failure and with a serum meprobamate level of 50 mg/100 ml. Hemodialysis was instituted using a Gambro parallel flow dialyzer and a portable re-circulating dialyzate delivery system (Redy, CCi Life Systems). Meprobamate removal with hemodialysis was 672+/-167 mg/hr with a corresponding clearance of 61.97+/-9.9 ml/min. Drug removal with forced diuresis was 177+/-23.4 mg/hr. Metabolic degradation of the drug was approximately 482 mg/hr with a plasma disappearance rate of 5.2%/hr. No drug could be detected in the dialyzate fluid after its passage through the Redy re-circulating dialyzate system. Because of the rapidity of metabolic degradation of meprobamate, we feel that hemodialysis should be reserved for severe clinical intoxication and either compromised normal excretory routes or progressive clinical deterioration.