Manuel Carreno

A randomized trial of three renal transplant induction antibodies: early comparison of tacrolimus, mycophenolate mofetil, and steroid dosing, and newer immune-monitoring.

Background. New trends in immunosuppression in clinical transplantation include the use of antibody induction agents in protocols that emphasize reduction or avoidance of steroids and calcineurin inhibitors. Methods. In a randomized trial using three different antibody induction agents in 90 first renal transplant recipients from cadaver donors, group A received Thymoglobulin, group B received Alemtuzumab, and group C received Daclizumab. Maintenance immunosuppression included tacrolimus and mycophenolate in all three arms, and methylprednisolone in groups A and C only (standard clinical institutional practice). The targeted trough level of tacrolimus was between 8 and 10 ng/mL for groups A and C, respectively, with a targeted mycophenolate dose of 1 g twice daily. However, in group B, the target tacrolimus trough level was 4 to 7 ng/mL to reduce long-term nephrotoxicity, with 500 mg twice-daily doses of mycophenolate, without steroid maintenance. Results. In this 15-month median postoperative interval report, there were no notable differences in demographics and patient and graft survivals. Acute rejection rates at 1 year were equivalent, that is, 5 of 30 in all three groups (16.6%). In group B, there was slightly lower renal function at 1 month, but no difference at 1 year. There was also significantly more leukopenia, but a greater percentage of T-regulatory cells and number of Fox-P3 mRNA copies by flow cytometry and semiquantitative polymerase chain reaction analysis, respectively, in group B. Conclusions. This preliminary analysis indicates that 80% of the patients in group B remained steroid-free 1 year postoperatively, with lower tacrolimus trough levels and no difference in other adverse events.

De novo membranoproliferative glomerulonephritis in hepatitis C virus-infected renal allograft recipients.

Hepatitis C virus (HCV) is the leading cause of non-A, non-B hepatitis among renal allograft recipients. We sought to identify and describe a proteinuric renal disease occurring in our HCV-infected renal transplant patients. Patients with proteinuria exceeding 1 g/day were identified from a cohort of 98 HCV-infected kidney recipients. Qualitative and quantitative reverse transcriptase polymerase chain reaction (RT-PCR) and restriction fragment-length polymorphism of the amplified RT-PCR product was performed to detect circulating HCV RNA, viral titer, and strain type, respectively. An immune complex nephritis (ICN) of the membranoproliferative pattern (MPGN) was found on five of eight biopsies. Two patients infected with the Hutch strain-type developed nephrotic-range proteinuria within three months posttransplant while the remaining three MPGN patients had been transplanted greater than 5 years prior to the onset of proteinuria. Testing for rheumatoid factors, cryoglobulins, hypocomplementemia, and circulating immune complexes failed to show a consistent pattern. Sucrose density gradient (SDG) equilibrium centrifugation was used to determine the buoyant-density of HCV virions from control (HCV-infected nonproteinuric recipients; n = 5) and nephrotic patients (n = 5). Whereas HCV virions from the control patients had a low buoyant density on sucrose gradients, a substantial percentage of the circulating HCV RNA from the MPGN patients was present in the high-density fractions in association with IgM and IgG. Treatment of the pooled high-density layers with NP40 followed by recentrifugation resulted in a shift of the HCV RNA to the medium-density layers. In conclusion, MPGN developed in five HCV-infected kidney recipients despite pharmacologic immunosuppression. Both the physicochemical properties of the HCV virions on SDG and their association with IgG and IgM in the high-density layers provide indirect evidence for the presence of circulating complexes of anti-HCV antibody and HCV antigen(s).

Six-year clinical effect of donor bone marrow infusions in renal transplant patients.

Background. To date, several single- and multicenter clinical trials have attempted to induce specific immunological unresponsiveness using donor bone marrow cell infusions to augment solid organ transplantation, but the outcomes have not been definitive. Methods. Between September 1994 and May 1998, 63 cadaver (CAD) renal transplant recipients of either one or two postoperative donor bone marrow cell (DBMC) infusions were prospectively compared with 219 non-infused controls given equivalent immunosuppression. There was at least a 1 HLA DR antigen match present between donors and recipients. The immunosuppressive regimen included a 10-day course of OKT3 induction, and tacrolimus, mycophenolate mofetil, and methylprednisolone maintenance. A total 7.01×108±1.9×108 (SD) DBMC/kg was infused into the CAD recipients on either days 4 and 11 (n=42) or one half of that dose on day 4 (n=21) postoperatively. Clinical follow-up has ranged from 2.9 to 6.3 years (mean, 4.7 years). Studies were also performed of humoral immunity and quantitative cellular chimerism. Results. There is clear-cut equivalence in immunosuppressive dosaging and in the other major demographic variables in both groups. However, only 2/63 DBMC recipients had biopsy-proven chronic rejection, whereas 41/219 showed chronic rejection in the controls (P =<0.01). In both groups, mortality was not associated with rejection. The actuarial graft survival at 6.3 years in the CAD DBMC group was 84.3% compared with 72.2% in the control group (not statistically significant). However, if death with a functioning graft was excluded, graft survival was 94.1% in the DBMC group and 79.8% in the controls (P =0.039). Forty patients in the control group continue to have deteriorating renal function (increasing serum creatinine concentrations to 2 mg/dl and higher), compared with 2 patients in the DBMC group (P =0.04). In the DBMC group, chimerism in iliac crest marrow aspirates has increased 3-fold in yearly sequential measurements between 1 and 4 years postoperatively averaging 1.3±0.36% (SE) most recently. This has not occurred in the controls. Conclusions. There now appears to be more solid long-term evidence, in kidney transplant recipients prospectively receiving DBMC infusions, of an improvement in long-term graft survival, and of the degree of chimerism positively correlating with the absence of graft loss.

The Use of Campath-1h as Induction Therapy in Renal Transplantation: Preliminary Results

Background. In an attempt to reduce both initial and long-term (nephrotoxic) calcineurin inhibitor maintenance dosage and totally eliminate maintenance corticosteroids, alemtuzumab (Campath-1H) was used as induction therapy in first cadaver and non-HLA-identical living donor renal transplantation. Methods. Forty-four de novo renal allograft recipients were treated with Campath-1H (0.3 mg/kg) on days 0 and 4 postoperatively, preceded by methylprednisolone boluses. Maintenance target 12-hr tacrolimus trough levels of 5 to 7 ng/mL were operational from the outset as well as (reduced) mycophenolate mofetil dosage of 500 mg twice daily. No corticosteroids were planned to be given after the first week postoperatively. Results. With a median follow-up of 9 (range, 1–19) months, patient and graft survival rates are each at 100%. Biopsy-proven acute rejection was diagnosed in four patients. Infections requiring hospitalization developed in four patients. Thirty-eight recipients remain without the need for long-term corticosteroid therapy. Conclusions. In an early assessment, the combination of Campath-1H, low dosing of tacrolimus and mycophenolate mofetil, and avoidance of maintenance corticosteroid use seems to be safe and effective for kidney transplant recipients. Long-term outcomes will be reported in the future.

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.

Localization of collagenase in the growth plate of rachitic rats.

In the transition from proliferation to hypertrophic cell zones in the growth plate, there is an increase in chondrocyte volume and a corresponding decrease in collagen content to accommodate the enlarging cells. It is postulated that collagenase accounts for this collagen loss. To test this hypothesis, tibial growth plates were obtained from normal rats, rachitic rats deficient in vitamin D and phosphate, and rats after 48 and 72 h of healing from rickets. Collagenase was quantitated by a pellet assay based on the release of solubilized collagen from the endogenous insoluble collagen in the tissue homogenates. A fourfold greater collagen release and a concomitant sixfold greater hypertrophic cell volume were measured in rachitic growth plates compared with normal age-matched controls. During healing of rickets, collagenase activity and hypertrophic cell volume returned almost to control levels. Rachitic growth plates were dissected into the juxtaepiphyseal 1/3 and the juxtametaphyseal 2/3. The latter portion contained greater than 95% of the hypertrophic cells and 86% of the collagenase. The collagen-degrading activity was extracted from this region and was shown to be a true collagenase by its production of typical A fragments of tropocollagen produced by collagenase action. The enzyme was activated by aminophenylmercuric acetate and trypsin and was inhibited by EDTA, 1,10-phenanthroline, and a tissue inhibitor of metalloproteinases from human articular cartilage. Inhibitors of aspartic, cysteine, and serine proteases had no effect. Micropuncture fluids aspirated from rachitic cartilage contained latent collagenase activity, indicating an extracellular localization. Negative tests for hemoglobin in the rachitic cartilage samples indicated that there was no contamination by capillaries and that this was not a source of collagenase. It is concluded that extracellular collagenase accounts for the loss of cartilage matrix in the hypertrophic zone, and that this process may be distinct from that of capillary invasion.

Heterotopic Uterus Transplantation in a Swine Model

Background. The aim of our study was to examine the feasibility of allogeneic uterine transplantation in a large animal model. Methods. We performed heterotopic uterine transplants in genetically defined mini-pigs. Immunosuppression was tacrolimus administered intravenously for the first 12 days posttransplantation followed by oral cyclosporine maintenance immunosuppression. The graft was transplanted heterotopically in the lower abdominal cavity of the recipient. The vaginal vault was exteriorized as a stoma in the lower right abdominal wall. The uterine grafts were followed with endoscopies and biopsies. Results. Ten transplants were performed. Follow-up was until July 2008. At the end of the follow-up period, 5 animals were alive and healthy, 0.5 to 12 months posttransplantation. There were 5 deaths due to pneumonia (n=1), intussusception of the graft (n=1), cardiorespiratory arrest during anesthesia (n=1), and complications of the stoma (n=2). Acute rejections of the graft presented during the 2nd and 3rd month posttransplantation were treated successfully with increase of the maintenance immunosuppression and steroids. Other complications included prolapse and infections of the graft stoma. Pathological changes seen in the endometrial biopsies included acute rejection and acute endometritis. Conclusion. These findings demonstrate that successful uterus transplantation in a large animal model (miniature swine) is feasible using this heterotopic model, and it can be useful for the study of these transplants.

An assessment of the effects of cadaver donor bone marrow on kidney allograft recipient blood cell chimerism by a novel technique combining PCR and flow cytometry

A new technique, the PCR-flow assay is described that has allowed for the serial identification and quantitation of discrete mononuclear cell subsets of donor (or recipient) bone marrow derived cells in cadaver kidney transplant recipients infused postoperatively with donor vertebral body bone marrow cells. With fixed permeabilized cells in flow cytometry the amplification power of the polymerase chain reaction (PCR), using fluorescent-labeled primers to identify single copy HLA class II DRbeta1 genes of either donor or recipient origin, is combined with multi-color fluorochrome-labeled CD epitope-specific monoclonal antibodies. The details of the methodology are described; these support the utility of the assay. Initial observations were made on the chimeric makeup of the peripheral blood as well as iliac crest bone marrow between six months and one year posttransplantation in recipients serially followed weekly and then monthly, concomitantly compared with a control group of stable kidney transplant recipients using similar therapeutic protocols, who did not receive cadaver bone marrow. Several findings are of note. In 14 recipients of two bone marrow infusions totalling a mean of 6.29+/-2.18x10(10) cells, donor CD34 positive (+) (immature) cells were fourteen times as numerous in peripheral blood six months postoperatively as in six recipients given half as many bone marrow cells in one infusion (averaging 3.02+/-0.5x10(10)). These donor CD34+ cells unexpectedly averaged 36+/-7% of the total (donor plus recipient) CD34+ subset counted. Moreover, iliac crest bone marrow aspirates contained an average of thirteen times this number of CD34+ cells than in the peripheral blood, supporting the notion of engraftment. Of additional interest, between six months and one year posttransplant although no donor cells could be detected in peripheral blood of the controls there was an identifiable presence of donor CD34+ cells in their iliac crest bone marrow, albeit 10-fold less than the marrow-infused patients. In the clinical follow-up, although there were three unrelated mortalities, there were no additional kidney losses with current serum creatinine concentrations averaging 1.3+/-0.06 mg/dl. In conclusion, the PCR-flow assay presents the possibility of identifying discrete subsets of donor or recipient cells that may have an immunoregulatory function.

An Analysis of the Association between Serum Citrulline and Acute Rejection among 26 Recipients of Intestinal Transplant

Small preliminary studies suggest that serum citrulline levels may act as a marker for acute cellular rejection in small intestinal transplant recipients. The results comparing serum citrulline concentrations with biopsy‐based grades of rejection are summarized here for an expanded group of 26 isolated intestinal and multivisceral transplant recipients. Other factors considered included patient and donor age and sex, ischemia time, serum creatinine, and type of transplant. Straight‐line fits reasonably described how each patients citrulline levels changed over time. Among 21 patients who demonstrated increasing citrulline levels over time, the estimated median time‐to‐achieve normal citrulline (≥30 μmol/L) was 79 days post‐transplant. Using stepwise linear regression, two characteristics were associated with a significantly higher maximum grade of rejection after 14 d post‐transplant: longer time‐to‐achieve normal citrulline (using ranks, p < 0.00001) and the patient not receiving a multivisceral transplant (p = 0.0005). Only the latter characteristic was significantly associated with maximum grade of rejection during the first 14 d post‐transplant (p = 0.01). Clearly, time‐to‐normalization of citrulline was delayed by the incidence of rejection, and in some cases with moderate‐to‐severe rejection, normalization of citrulline levels never occurred. We plan to further examine the use of citrulline as a marker for rejection in larger prospective studies.

Donor bone marrow-derived chimeric cells present in renal transplant recipients infused with donor marrow. I. Potent regulators of recipient antidonor immune responses.

Background. Even though a number of transplant centers have adopted donor-specific bone marrow cell (DBMC) infusions to enhance donor cell chimerism, to date there has been no direct evidence linking chimerism with tolerance induction in human organ transplant recipients. Methods. Cells of donor phenotype were isolated 1 year postoperatively from the peripheral blood lymphocytes and iliac crest bone marrow of 11 living-related-donor (LRD) renal transplant recipients, who had received perioperative donor bone marrow cell infusions. These recipient-derived donor (RdD) cells were characterized phenotypically by flow cytometric analysis and functionally as modulators in mixed lymphocyte reaction (MLR) and cell-mediated lympholysis (CML) assays. Results. The yield of RdD cells ranged from 0.1 to 0.9% of the starting material with the majority being TcR&agr;&bgr;, CD3 positive T cells, a substantial percentage of which coexpressed CD28. At 1 year posttransplant almost 50% of the LRD-kidney/DBMC recipients tested so far exhibited donor-specific unresponsiveness in MLR (7/17) and CML (6/13) reactions and this trend was further enhanced at 2–3 years. In the recipients with residual positive antidonor immune responses, the RdD cells inhibited recipient antidonor MLR and CML responses significantly more strongly than freshly isolated and similarly treated iliac crest bone marrow cells from the donor. RdD cells also inhibited the MLR of the recipient to third party allogeneic stimulator cells; however, this nonspecific effect was significantly weaker than specific inhibition. We also established long-term bone marrow cultures stimulated every 2 weeks with irradiated allogeneic feeder cells, that had similar functional properties thus possibly providing us with an in vitro correlate the RdD cells. Conclusions. These results clearly support the notion that the infused donor cells play a positive role in the induction and/or maintenance of transplant tolerance.