The effects of chimeric cells following donor bone marrow infusions as detected by PCR-flow assays in kidney transplant recipients.

40 recipients of first cadaver kidney transplants were given perioperative donor vertebral bone marrow infusions (DBMC), compared with 100 controls who did not receive donor bone marrow. The immunosuppressive regimen included OKT3, Tacrolimus, and steroid maintenance therapy, and, in some patients, newly introduced mycophenolate mofetil. This report describes the 24-mo actuarial follow-up and several immunological monitoring studies including sequential measurements of donor bone marrow lineage subset chimerism by the recently reported PCR-flow assay. This is a sensitive in situ PCR detection system for donor versus recipient histocompatibility genes as well as cell surface CD epitope markers using flow cytometry. The results indicate (a) the stabilization of the donor CD3+ and CD34+ cells in recipient peripheral blood at levels below 1% between 6 mo and 1 yr postoperatively, with a 10-fold higher level of donor cell chimerism of these lineages in recipient iliac crest marrow; (b) significantly lower levels of chimerism in peripheral blood up to 6 mo postoperatively in patients who had early acute (reversible) rejection episodes compared with those who did not; (c) a higher degree of chimerism seen in patients who were class II MHC HLA DR identical with their donors; (d) the identification of a high proportion of the donor bone marrow derived CD3 dimly staining subset of T cells (to which regulatory functions have been ascribed) in recipient peripheral blood and especially in recipient bone marrow; and (e) an unexpectedly increased susceptibility to clinically significant infections (primarily viral), and even death in the DBMC-infused group, compared with controls, but no graft losses because of rejection in the DBMC-infused group. Mixed lymphocyte culture assays showed a trend toward a greater number of nonspecifically low reactors in the DBMC group, as well as a greater number of nonspecifically high reactors in the controls (P = 0.058). The autologous mixed lymphocyte reaction also indicated a trend towards nonspecific immune activation in the DBMC group. Finally, anti-cytomegaloviral IgG antibody reactivity was significantly inhibited in the DBMC group 4-6 mo postoperatively (P = < 0.05). In the controls, there were no donor cell lineages detected by PCR-flow in the peripheral blood. These rather unexpected findings, indicating a more depressed cellular and humoral immune capacity in the DBMC cadaver kidney transplant recipients in this relatively early follow-up period, are discussed relevant to chimerism, MHC restriction, and suppressor activity brought about by specialized DBMC subsets, which still need to be defined.

An association of lower serum citrulline levels within 30 days of acute rejection in patients following small intestine transplantation.

INTRODUCTION: In a prospective protocol we studied whether serum citrulline level within 30 days of an acute rejection was predictive of the episode. METHODS: An acute rejection episode was defined as the date of occurrence of any biopsy-proven rejection in which treatment was initiated until two successive biopsies showed no further rejection. We compared the mean citrulline level based on values determined within 30 days of the start of an acute rejection episode with the mean citrulline level measured on the same patient during a rejection-free period. Serum citrulline measurements were available immediately prior to the occurrence of rejection for 22 patients who experienced 37 episodes. RESULTS: For the 12 episodes of mild rejection, the mean serum citrulline level +/- SE (standard error) was 15.0 + 2.3 micromol/L prior to rejection and 18.8 +/- 2.4 micromol/L during the rejection-free periods. A paired t test of the mean differences was not significant (P = 17). For the 25 episodes of moderate or severe rejection, the mean serum citrulline level was 12.4 +/- 1.1 micromol/L before rejection and 18.8 +/- 2.0 micromol/L during the rejection-free periods. A paired t test of the mean difference was statistically significant (P = .002). CONCLUSIONS: Although further study of citrulline as a marker for the early detection of acute rejection episodes is needed, our hope is that its use will help to prevent some of these early episodes from evolving into full-blown moderate or severe grades of rejection.

Long-term function (6 years) of islet allografts in type 1 diabetes.

Eight type 1 diabetic patients, ages 29-41 years, with mean diabetes duration of 23 years (range 18-29 years) received islet transplants from 1 to 5 donors. Seven patients had stable kidney allografts 1-11 years before the islet transplant, and one patient had a simultaneous islet-kidney allograft. Patients' blood glucose control was poor as reflected by the mean +/- SD HbA1c of 9.1 +/- 1.7% before transplant. Of the first three patients, two (1 and 3) achieved insulin independence for 36 and 38 days, respectively. Two recipients rejected their islet grafts within 1 month (2 and 8) and therefore were excluded from analysis. The HbA1c and insulin requirement of the six remaining patients who had persistent islet function for more than 60 days was significantly reduced from 9.3 +/- 1.9 to 6.4 +/- 1.0% (P = 0.002) and from 0.75 +/- 0.15 to 0.35 +/- 0.12 U x kg(-1) x day(-1) (P < 0.001), respectively. The two patients with the longest graft survival (4 and 6) achieved a normalization or near-normalization of their HbA1c levels during 6 years in the absence of severe episodes of hypoglycemia. As demonstrated by a decline in C-peptide response during Sustacal challenge tests over a 6-year period, there was a diminution of islet allograft function over time, despite persistence of normal or near normal HbA1c. We concluded that transplantation of allogeneic islets with an islet mass comparable with whole or segmental pancreas transplants in type 1 diabetic patients can result in long-term islet allograft function; further, we concluded that, in conjunction with small dosages of exogenous insulin, a functioning islet allograft can result in near-normalization of blood glucose levels and significant improvement in HbA1c. The occurrence of severe hypoglycemic episodes observed for patients in the Diabetes Control and Complications Trial was not observed in recipients with functioning islet transplants, despite the continuous need for exogenous insulin therapy to sustain normal HbA1c over the 6-year follow-up. The significant improvement in metabolic control observed for the patients described in this study, and the potential to significantly decrease or halt the progression of diabetic complications, support the continued application of islet allotransplantation as a treatment modality for type 1 diabetic patients.

Abrogation of the alloreactive responses of cadaveric donor intestinal lymphocytes by intraoperative Campath-1H exposure.

A number of recent reports in clinical and experimental intestinal transplantation have suggested that the donor lymphocytes present in and around the gastrointestinal system are potent mediators of graft-versus-host (GvH) reactivity and that GvH responses may contribute to posttransplant morbidity. Therefore, we have tested the proliferative and cytotoxic capabilities of gut-associated lymphocytes from cadaveric donors obtained prerevascularization (pre-r) and about 6 hours postrevascularization (post-r) in recipients pretreated with Campath-1H antibody (alemtuzumab). Mixed lymphocyte reactions (MLR) were performed with lymphoid cells isolated from intestinal epithelial mucosa, lamina propria, and lymph nodes. The pre-r lymphocytes responded strongly to both the recipient and third-party alloantigenic stimulating cells. However, similar preparations from the post-r samples responded in MLR at significantly lower levels (P < .01). This post-r decrease in responsiveness was not observed in similar lymphocyte samples obtained from donors of recipients not treated with Campath-1H. Both the pre-r and post-r samples had similar flow cytometric profiles, suggesting that there was no receptor loss in these lymphoid tissues by the short-term 6-hour exposure to Campath-1H given to the recipient. Conversely, in preliminary experiments where the donor were treated with Campath-1H, it was observed that very few lymphocytes could be obtained from intestinal tissues (n = 3). These results suggested that Campath-1H treatment of the recipient could bring about a drastic reduction in an otherwise strong GvH reactivity by the donor intestinal immune cells.

Transplantation for the treatment of intra-abdominal fibromatosis.

MATERIALS AND METHODS: During the last 9 years we treated 14 patients with a diagnosis of intra-abdominal fibromatosis. The 11 patients who received an intestinal allograft included isolated intestine (n = 6), liver-intestine (n = 1), intestine-kidney (n = 1), multivisceral (n = 1), multivisceral-kidney (n = 1), multivisceral-no liver (n = 1). Three patients received an intestinal autograft after partial abdominal evisceration and ex vivo tumor resection. Three patients additionally underwent an abdominal wall allograft. RESULTS: At follow-up until August 2004, all autotransplant patients are alive. Four intestinal transplant patients died within the first postoperative month. There were three graft losses. A patient who lost his graft early postoperatively was retransplanted but died of sepsis shortly there after. Two more patients lost their graft due to severe rejection and were retransplanted successfully. Two patients developed desmoid tumor recurrence in their abdominal or thoracic wall. Ten patients are alive 1 to 9 years posttransplantation. Nine have fully functioning grafts and one patient requires TPN supplementation at night due to dysmotility of her autograft. CONCLUSION: Intestinal allo-, or autotransplantation combined with transplantation of the abdominal wall can be lifesaving for patients suffering from extensive intra-abdominal fibromatosis.

The impact of Campath 1H induction in adult liver allotransplantation.

BACKGROUND: We report our experience with Campath 1H in adult liver allotransplantation. METHODS: Between December 2001 and February 2004, 77 patients underwent liver transplantation using Campath 1H induction and low-dose maintenance tacrolimus immunosuppression. The control group consisted of 50 patients with similar baseline characteristics and the same eligibility criteria, transplanted under our standard Tacrolimus/steroids regimen. Hepatitis C patients were excluded from the study. RESULTS: Patient and graft survival were similar for both groups. The incidence of rejection was significantly lower in the Campath vs the control group (51% vs 65% at 12 months, P = .009). Tacrolimus trough levels and conversion from Tacrolimus or the addition of other immunosuppressive drugs due to nephrotoxicity were also significantly lower in the Campath 1H group. CONCLUSION: Campath 1H induction with low-dose Tacrolimus maintenance immunosuppression is an effective regimen in reducing acute rejection in adult liver transplantation, while maintaining lower tacrolimus levels and less nephrotoxicity than our conventional immunosuppressive regimen.

Cloning and expression of canine interleukin-10.

We describe here the cloning of canine IL-10 cDNA (GenBank accession No. U33843) and the expression of recombinant IL-10 in a dog kidney cell line (DK6247) and Chinese hamster ovary cells (CHO). Canine IL-10 exhibits strong sequence homology to the known sequences of human, mouse, rat, and bovine genes at nucleotide and amino acid levels. The IL-10 gene, when introduced into DK and CHO cell lines, produces recombinant IL-10 that causes an inhibitory effect on allogeneic MHC-driven lymphoproliferative responses.

In vitro generation of human mixed lymphocyte culture suppressor cells. I. Cellular characterization and specificity.

The mixed leukocyte culture (MLC) of peripheral blood lymphocytes (PBLs) from two prospective kidney transplant recipients and their respective donors, produced a primed cell population that functioned as suppressors. The primed cells suppressed the primary and secondary MLC when added as third components, without demonstrable cytotoxic cells. Suppressor cells were derived from the MLCs of related pairs that were genotyped to be: (1) serologically defined (SD) identical and lymphocyte-defined (LD) nonidentical, and (2) HLA (total MHC) identical. Primed cells derived from MLC of unrelated HLA nonidentical subjects also showed similar effects. Two general patterns of inhibition were observed: a marked specific suppression of the autologous responding cells and, to a lesser degree, nonspecific suppression. The T cell fraction derived from the primed suppressor population was suppressive, the cells with B cell characteristics were not suppressive. The suppressive effect of cells generated in MLC was serially monitored after kidney transplantation. The suppressive activity of the primed cells was absent in the early postoperative period (4 weeks), perhaps because of high dose immunosuppression, but later in the course suppression was evident again (2 to 5 months). Manipulation of this T suppressor cell population may prove to be of value in enhancing graft acceptance.

Modulatory effects of human donor bone marrow cells on allogeneic cellular immune responses.

BACKGROUND: In order to evaluate whether immunoregulatory mechanisms are brought about by human donor bone marrow cell infusions accompanying organ transplantation, we established in vitro culture systems analogous to the transplant model. METHODS: Cell-mediated lympholysis (CML) and mixed lymphocyte culture (MLC) responses of peripheral blood lymphocytes or spleen cells stimulated with irradiated cadaver donor spleen cells in the presence of specific donor vertebral-body bone marrow cell (DBMC) modulators were tested. RESULTS: When compared with spleen cells as modulator controls, DBMC inhibited both the proliferative and cytotoxic responses in a dose-dependent manner. Use of unirradiated and T cell-depleted DBMC was required for detection of the inhibitory activity. Furthermore, DBMC had to be added within the first 2 days after the initiation of the cultures for the down-regulation of CML (MLC) to occur. The down-regulation of MLC responses could not be shown to be antigen (donor) specific. Physical separation of DBMC from the responder-stimulator cells using the transwell system abrogated modulation of the CML (and MLC) reactions, suggesting the requirement of cell-cell contact for modulatory effect. The inhibitory activity by DBMC could not be overcome by addition of up to 200 U/ml of exogenous recombinant interleukin 2 to the cultures. However, it could be abrogated by restimulation with donor spleen cells, indicating that donor reactive cells were not deleted by DBMC in short-term cultures. CONCLUSIONS: These results showed a regulatory role for DBMC in cellular immune responses against donor cell alloantigens, supporting the rationale for DBMC for facilitating clinical allograft acceptance.

Immunochemical and biochemical characterization of purified canine interferon-gamma. Production of a monoclonal antibody, affinity purification, and its effect on mixed lymphocyte culture and mixed lymphocyte kidney culture reactions.

We have advanced the hypothesis that the primary autolymphoproliferative response of dog T cells in mixed lymphocyte kidney cultures (MLKC) results from their recognition of tissue-specific (kidney-associated) antigen(s) presented in conjunction with class II MHC antigens. Lymphocyte culture-derived supernatants had been found previously to upregulate class II antigen expression on kidney cells and enhance T cell activation. In the present study we have isolated and characterized dog IFN-gamma, a class II-inducing substance that is secreted in the culture supernatant of activated T lymphocytes. Dog IFN-gamma was induced with A-23187 and PMA and purified stepwise using controlled-pore glass, Mono Q anion exchange chromatography, and Superose 6-gel filtration on FPLC. The purification resulted in two molecules of 42 Kd and 31 Kd molecular weights. An IgG1 monoclonal antibody was engendered to these molecules. With this mAb reagent, in immunochemical experiments, we have developed a sensitive ELISA and a method for purifying dog IFN-gamma by affinity chromatography. Species specificity studies indicated that purified dog IFN-gamma reacted with a polyclonal rabbit antihuman IFN-gamma, but not with a mAb to human IFN-gamma. However, the antidog IFN-gamma mAb that was generated also reacted with recombinant human IFN-gamma. In in vitro biological studies, the purified IFN-gamma (two mol. wt. species) upregulated the expression of canine class II MHC molecules on dog tubular epithelial cells and the dog kidney epithelial cell line (MDCK). The antidog IFN-gamma mAb blocked T cell proliferative response to kidney cell and, by inference, the interaction between endogenously released IFN-gamma in vitro with its cell surface receptor, thus inhibiting the induced upregulation of class II. Interestingly, although antidog IFN-gamma markedly blocked the MLKC (10 micrograms mAb/well), there was no effect on the allogeneic MLC. This observation indicates that the cytokine IFN-gamma may be a uniquely key substance amplifying the immune response of T cells to tissue-associated antigens on surrogate antigen-presenting cells that require induced upregulation of class II MHC antigen expression (MLKC), in contrast to reactions in which these antigens are already constitutively expressed on the antigen-presenting cells (mixed lymphocyte culture).

Detection of antibody to hepatitis C virus in renal transplant recipients.

Non-A, non-B hepatitis is a significant cause of liver disease among renal allograft recipients. In order to assess the impact and prevalence of hepatitis C in a series of renal allograft recipients, we retrospectively screened 621 consecutive patients transplanted between 1979 and 1989 and 484 cadaver organ donors retrieved in the same interval for serologic evidence of hepatitis C viral (HCV) infection using the enzyme-linked assay for anti-HCV antibody. Of 596 HBsAg negative patients, 180 (30%) were anti-HCV positive at the time of transplant. One-year posttransplant, 117 (22%) had detectable levels of anti-HCV antibody. Chemically significant hepatitis developed in 52/234 (22%) anti-HCV positive patients, and 26 of these followed a clinical course consistent with chronic hepatitis. Significantly more males and patients with antibody to HCV detectable at 1 year posttransplant were in the group experiencing an increase in liver enzymes. Ten-year patient and graft survival was 78% and 50%, respectively, for the anti-HCV positive patients who had an elevation of alanine aminotransferase, and 76% and 57% for the cohort maintaining normal liver function (P = NS). There were also no differences in patient and graft survival among the anti-HCV positive group and the consistently sero-negative patients. Of 484 cadaver organ donors with serum available for analysis (out of 1200 retrieved), 67 (14%) were anti-HCV positive at the time of organ donation. Among 23 anti-HCV negative kidney recipients who received a kidney from an HCV antibody positive donor, only one had seroconverted at 1 year posttransplant. Antibody to HCV appears to be widespread among renal transplant recipients and cadaver organ donors. We were unable to demonstrate any evidence of long-term adverse effects on patient and graft survival among anti-HCV positive patients employing the first generation anti-HCV assay.

The biologic significance of the mixed lymphocyte kidney culture in humans.

The mixed lymphocyte kidney culture (MLKC) in humans has been studied in normal and abnormal clinical conditions. Human renal cortical cells were extracted by collagenase treatment from the kidneys of "normal" heart-beating cadaver organ donors (n = 13), patients with end-stage renal disease (ESRD) at pretransplant bilateral nephrectomy and splenectomy (n = 13), and from irreversibly rejected renal allografts at the time of graft nephrectomy (n = 5). Proliferation of peripheral blood T lymphocytes of 2-DR-mismatched volunteers occurred in response to kidney cortical cells extracted from each of the 3 donor categories in a reaction termed the allogeneic mixed lymphocyte kidney culture. Additionally, splenic T cells from cadavers and patients with ESRD were seen to react to their autologous kidney cells. The renal cortical cells extracted from ESRD kidneys were more stimulatory in the allogeneic and autologous MLKC responses than those extracted from "normal" cadaver kidneys even when the ESRD kidneys were 99% depleted of passenger T and B lymphocytes by treatment with monoclonal antibodies T11 and B1. In order to help define the antigens operative in the MLKC, we pretreated stimulating lymphocytes and renal cortical cells with anti-class II monoclonal antibodies. The allogeneic mixed lymphocyte reaction and MLKC were inhibited ca. 80% and 30%, respectively. The autologous MLKC was unaffected by this treatment. To further support that tissue-specific immune mechanisms were operative in the reaction, experiments were performed with infiltrating lymphocytes isolated from the ESRD kidneys, which were seen to generate a proliferative response when stimulated with autologous cortical cells. However, the response of these same infiltrating lymphocytes when stimulated with allogeneic lymphocytes (MLR), was markedly weaker than the response of the patients' autologous spleen cells. In addition, two kidneys were obtained at rejection from recipients that had received grafts from HLA-MLR-identical sibling donors. A lymphoproliferative reaction of recipient peripheral blood T lymphocytes occurred in response to (donor) renal cortical cells, but not to donor peripheral blood lymphocytes. In contrast, infiltrating (recipient) kidney lymphocytes responded to the kidney cortical cells and to donor peripheral blood lymphocytes. Moreover, peripheral blood T lymphocytes of the HLA-identical donor responded to his own kidney cortical cells, which were isolated from the rejected recipient kidney, and did not respond to recipient peripheral blood lymphocytes. Finally, a "normal" cadaveric kidney was fortuitously available at the same time that a rejected transplant (cadaver)

In vivo and in vitro induction of class II molecules on canine renal cells and their effect on the mixed lymphocyte kidney cell culture.

Canine renal cortical cells were obtained by collagenase extraction from allogeneic haploidentical, donor-recipient beagle littermate pairs and from unrelated mongrels. Peripheral blood lymphocytes (PBL) of the mongrels, as well as of one member of the beagle pair that exhibited high mixed lymphocyte culture (MLC) reactivity against the other were also stimulated by renal cortical cells derived from both normal and rejected transplanted kidneys in mixed lymphocyte kidney cell culture (MLKC). A moderate autologous MLKC reactivity occurred in response to normal renal cortical cells. However, rejected kidney cortical cells were markedly more stimulatory than normal renal cortical cells in both allogeneic and autologous MLKC reactions. Lymphocytes from donor animals responded more strongly to autologous cortical cells isolated during rejection of the transplant than to cortical cells from normal allogeneic kidneys. Recipient infiltrating lymphocytes and propagated T cell lines extracted from the rejected kidney also responded more strongly than PBL to cortical cells from this kidney. Gradient purification of the stimulating cortical cells resulted in one virtually pure preparation of distal tubular epithelial cells, as demonstrated by immunohistochemical stains and electron microscopy, which caused enhanced stimulation in MLKC. Class II marker analysis of the canine renal cells from rejected kidneys revealed the presence of these molecules on tubular cells that were absent on normal kidney cells. A 16-hr coculture of normal renal cortical cells not exhibiting class II surface markers in the presence of allogeneic or autologous lymphocytes induced the expression of these molecules, associated with an increased stimulatory capacity. This also occurred to a lesser extent with MLC (and MLKC) cell culture media supernatants. However, the low level of class II expression by all the various gradient-purified fractions in the absence of rejection or coculture, and the increased but equivalent expression on all fractions after coculture did not correlate with the preferential stimulatory capacity of the purified distal tubular cell layer. We conclude that two signals are necessary for the MLKC reaction, one involving tissue (kidney)-associated epitopes (the nominal antigen demonstrated in this study to be present in normal distal tubular cells), the other involving class II molecules as costimulatory (amplification) moieties.

Posttransplant hyperglycemia. Increased incidence in cyclosporine-treated renal allograft recipients.

The incidence of posttransplant diabetes mellitus (PTDM) was compared in two groups of renal allograft recipients. These were all nondiabetic patients who had been transplanted between 1979 and 1987 and received either azathioprine-methylprednisolone (group 1) or cyclosporine-methylprednisolone (group 2) therapy as maintenance immunosuppression. The incidence of PTDM in group 1 was 9.1% vs. 18.6% in group 2 (P less than .05). The mean daily dose of methylprednisolone during the initial 2 months posttransplant was not greater among the PTDM patients of groups 1 or 2. Cyclosporine levels and mean daily CsA doses during the initial 2 posttransplant months were also not different among the CsA-PTDM and euglycemic CsA patients. Posttransplant diabetes mellitus occurred rapidly (less than 2 months) and required insulin therapy in the majority of cases. Increased age (greater than 40 years) was associated with a higher risk for PTDM, however, the greater incidence accompanying increased body weight only approached significance. Patient gender and donor source were not associated with significant risk for PTDM. The development of PTDM was accompanied by a significant decrease in graft survival at 3 years in the entire PTDM population and at 4 years in the CsA-PTDM subgroup. Actuarial patient survival was not adversely affected. The current study suggests that CsA may be diabetogenic when administered with methylprednisolone to renal allograft recipients. The adverse effect on allograft survival requires further investigation. These results may also have important implications for pancreatic and islet cell transplantation.

Primary immunosuppression with tacrolimus and mycophenolate mofetil for renal allograft recipients.

BACKGROUND: Studies using tacrolimus and corticosteroids or the combination of cyclosporine, mycophenolate mofetil, and corticosteroids have been shown to reduce the incidence of biopsy-proven acute rejection episodes in cadaveric kidney recipients compared with cyclosporine-based immunosuppression. The current study is a retrospective analysis of our experience with tacrolimus combined with mycophenolate mofetil and steroids as primary immunosuppression for kidney transplant recipients. METHODS: In a retrospective analysis, 72 patients who received primary therapy with tacrolimus, mycophenolate mofetil, and corticosteroids (triple therapy) were compared with a control group of 98 kidney recipients who received tacrolimus and corticosteroids (double therapy). RESULTS: There was a significant reduction in the incidence of biopsy-confirmed acute rejection in the triple therapy group (8.2%) compared with the double therapy group (21%; P=0.003). One-year patient and graft survival did not differ between groups. The incidence of posttransplant diabetes mellitus was 18% and 21% in the triple and double therapy groups, respectively. Leukopenia and gastrointestinal side effects were the most common cause for discontinuation of mycophenolate mofetil. CONCLUSIONS: The combination of tacrolimus with mycophenolate mofetil and corticosteroids is more effective at preventing early acute rejection than tacrolimus and corticosteroids alone. The use of mycophenolate mofetil was associated with a higher incidence of leukopenia and diarrhea, often leading to discontinuation of the drug.

Effects of in vitro and in vivo anticanine T lymphocyte and anticanine class II-specific monoclonal antibodies in the beagle.

Two murine anticanine lymphocyte monoclonal antibodies, designated T83 and B1F6, were assessed for (1) in vitro antiepitope activity to circulating lymphocyte subsets, their functional effects on lymphoproliferative assays and binding specificities to diverse cell suspensions and tissue sections; (2) in vivo effects after administration on cells expressing the target epitopes, lymphoproliferation, and allograft survival; and (3) the host immune response to the injected murine immunoglobulins. The monoclonal antibody T83 (IgG3) appeared to be specific for a T cell subset with an up-regulating function on lymphoproliferation. It caused a profound depletion of cells with the appropriate epitope after intravenous administration but it bound to lymphocyte membrane epitopes on some cells in peripheral blood that did not become depleted and putatively caused other modulating effects on lymphocyte function. The monoclonal antibody B1F6 (IgG2a, previously described) was immunologically specific in vitro for cells expressing class II major histocompatibility complex antigens. In the dog, this also consisted of 50% of T lymphocytes. After intravenous administration, there were functional effects similar to those of T83. A modest prolongation of survival of renal allografts was observed when both mAbs were used as the sole immunosuppressive agent. These studies also demonstrated the occurrence of natural canine antimurine IgG antibodies. Administration of either monoclonal antibody was followed by a rapid increase in the concentration of the antimouse antibody(s). We postulate that the presence of canine natural antimouse IgG markedly influenced the biologic effects of in vivo administered monoclonals.

Involvement of multiple subpopulations of human bone marrow cells in the regulation of allogeneic cellular immune responses.

BACKGROUND: The identity of the cells in the human bone marrow that function as effective regulators of in vitro and possibly in vivo cellular immune responses is not well established. METHODS: Cell subpopulations were isolated from cadaver donor vertebral-body bone marrow cells (DBMC) by using immuno-magnetic microbeads and were tested as inhibitors (modulators) in cell-mediated lympholysis (CML) and mixed lymphocyte reaction (MLR) responses of normal peripheral blood lymphocytes stimulated with irradiated cadaver donor spleen cells. RESULTS: Compared with spleen cells as controls, un-irradiated T-cell depleted DBMC inhibited both the MLR and CML responses of allogeneic responder cells in a dose dependent manner (as in our previous reports). The inhibition was also mediated by a number of purified subpopulations including pluripotent CD34+ stem cells, and their CD34 negative early progeny of both lymphoid and myeloid lineages. These included DBMC enriched for non-T-cell lymphoid precursors (NT-LP/DBMC; i.e., DBMC depleted of CD3, CD15, and glycophorin-A positive cells) and DBMC positively selected for CD38+, CD2+, CD5+, and CD1+ lymphoid cells (all were depleted of CD3+ cells) as well as CD33+ (but CD15 negative) myeloid precursors. However, positively selected CD19+ B-cells and CD15+ myeloid cells did not inhibit the MLR and CML responses. The NT-LP/DBMC that had been repeatedly stimulated with irradiated allogeneic peripheral blood lymphocytes caused the strongest inhibition of the MLR and CML responses of the same allogeneic cells with 200 times fewer modulator cells needed than uncultured DBMC (P<0.001). Flow cytometric analysis revealed that majority of cells in these cell lines had become CD3+ TcR-alphabeta+ CD4+ and CD28+ cells. CONCLUSION: A variety of less differentiated cells of various lineages residing in the human bone marrow are immunoregulatory in vitro. Among them, there is at least one subset that can undergo differentiation in vitro into regulatory T cells that can be maintained in long-term cultures.

Acute graft loss secondary to necrotizing vasculitis. Evidence for cytokine-mediated Shwartzman reaction in clinical kidney transplantation.

A small number of kidney transplant recipients abruptly lose function secondary to acute renal artery or vein thrombosis or more rarely a form of necrotizing vasculitis. We report a group of four kidney transplant recipients who lost renal function and share the following features: (1) diabetes (type I, insulin-dependent diabetes mellitus, type II or steroid-induced); (2) abrupt change/loss of renal function; (3) a concomitant clinical event (fever, viral symptoms, menometrorrhagia, viremia, bacteremia); (4) severe necrotizing vasculitis with hemorrhagic necrosis on histopathology; (5) patent renal artery and vein at time of transplant nephrectomy (i.e., no vascular thrombosis); and (6) high levels of peripheral serum gamma-IFN 1-5 days before transplant nephrectomy (467 +/- 175 pg/ml) compared with that of patients experiencing severe rejection (8.4 +/- 3.7 pg/ml) (P < 0.002). These data support the concept of a cytokine (IFN-gamma)-mediated accelerated inflammatory response resulting in graft loss from necrotizing vasculitis--the clinical equivalent of an organ-specific Shwartzman reaction.

A prospective study of hepatitis C virus infection in renal allograft recipients.

Hepatitis C virus (HCV) is the predominant cause of posttransplant non-A, non-B hepatitis among renal allograft recipients. Prior studies evaluating the impact of HCV in kidney transplantation have been retrospective in design and based largely on changes in serum transaminases. We studied a group of HCV-infected end-stage renal disease patients prospectively with pretransplant liver biopsies and close virologic and biochemical follow-up posttransplant. Fourteen patients have been followed a mean of 11.6 +/- 5.6 months posttransplant (range, 5-21 months). Six had changes of chronic hepatitis on pretransplant liver biopsy while 8 showed only mild histologic abnormalities. Circulating viral titers increased several-fold over baseline levels during posttransplant follow-up. Viral replication was particularly enhanced immediately following a course of antilymphocyte therapy. Although all patients showed a 2-3 fold increase in alanine aminotransferase (ALT) following transplantation, there were no association noted between pretransplant liver histology, the use of FK506 and/or cyclosporine-based immunosuppression, and the magnitude of ALT change posttransplant. The only clinical outcome found to differ significantly was a higher incidence of cytomegalovirus infection among patients with chronic hepatitis. All patients are alive with functioning grafts. There have been no episodes of fulmiinant or subfulminant liver failure. We conclude that HCV-infected patients can be safely transplanted with excellent short-term follow-up. Continued monitoring with sequential liver biopsies will be needed to define the long-term course of HCV infection in an immuno-suppressed population.

Association of interleukin-10 with rejection-sparing effect in septic kidney transplant recipients.

Certain cytokines, particularly gamma-interferon (IFN) and interleukin (IL)-2 associated with TH1 cell function, have been shown to play a role in allograft rejection. One paradigm for long-term allograft acceptance involves TH2 cytokine predominance (IL-4 and IL-10). We describe two renal allograft recipients for whom immunosuppression was discontinued due to serious sepsis and who maintained stable renal function over 2-6 months without immunosuppression. During this time, there were higher levels of both IFN-gamma and IL-10 in the peripheral blood than in stable control kidney transplant recipients on immunosuppression. In one of the patients, levels of IL-10 fell, while those of IFN-gamma remained persistently elevated. This was associated with biopsy-proven rejection. Although peripheral blood cytokine levels may not reflect intragraft events, these data are consistent with an allograft protective role for IL-10 offsetting that of IFN-gamma in both patients off immunosuppression.