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.

FoxP3 mRNA transcripts and regulatory cells in renal transplant recipients 10 years after donor marrow infusion.

BACKGROUND: We update more favorable 10-year deceased donor kidney transplant survival in 63 recipients infused perioperatively with donor vertebral body bone marrow (DBMC-i) vs. 219 noninfused controls having equivalent immunosuppression and demographics. We questioned if this was associated with putatively regulatory FoxP3 mRNA and cell phenotypes (CD4+CD25+high percentages and high DC2:DC1 ratios) in DBMC-i vs. noninfused controls. METHODS: Baseline studies were performed on peripheral blood lymphocytes (PBLs) vs. marrow in normal laboratory volunteers of CD4+CD25+high percentages and DC2:DC1 by flow cytometry, and FoxP3 mRNA in CD3+ cells by real-time polymerase chain reaction. Similar studies were performed on PBL of the majority of the 10-year patients remaining with graft function: 21 (of the remaining 37) DBMC-i vs. 55 (of the remaining 105) controls. RESULTS: In normal subjects, all parameters were significantly higher in marrow than in PBL, supporting our previous reports of ex vivo DBMC immunoregulation. At 9.8+/-.02 years posttransplant in DBMC-i vs. controls, death-censored percent graft failure was 17.5% vs. 32.9% (P=0.02) with 247.6+/-24 vs. 79.9+/-3.1 (mean+/-SE) FoxP3 copies/5,000 CD3+ cells (P=0.0001). PBL CD4+CD25+high percentages were lower, but DC2:DC1 values higher in both recipient groups than in end-stage renal disease patients who had lower FoxP3 levels (40.8+/-5.9, P<0.0001), consistent with non-CD4+CD25+high T regulatory cells generated long-term posttransplant. Individual higher FoxP3 values correlated with higher iliac crest chimerism in DBMC-i, but not in controls (with 50-fold lower chimerism). In chronically rejecting controls, FoxP3 was further decreased. CONCLUSIONS: Peritransplant DBMC-i has higher 10-year renal transplant acceptance, chimerism, and FoxP3 mRNA in thus-far unclarified regulatory cell phenotypes.

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.

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.

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 patient's citrulline levels changed over time. Among 21 patients who demonstrated increasing citrulline levels over time, the estimated median time-to-achieve normal citrulline (>or=30 micromol/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.

Human donor bone marrow cells induce in vitro "suppressor T cells" that functionally suppress autologous B cells.

We have reported a beneficial effect of donor vertebral body bone marrow cells (DBMC) infusions in cadaver renal allograft recipients in a 6-year follow-up, but with a transient increase in early (6 month) postoperative CMV infections and concomitant suppressed immunoglobulins (Ig) production. We also found that although there was no difference between the DBMC-infused and non-infused (control) groups in the development of donor-specific antibody, we now describe an additional difference seen in the percent reactive antibody (PRA) reactivity against a panel of HLA antigens that developed postoperatively. We hypothesize that (allogeneic) antigen presenting cells in the DBMC, systemically infused, caused the generation of recipient T suppressor (T4-suppressor) cells, thereby "inducing" a negative influence on B cell Ig production. We tested this notion in vitro by incubating PBL from CMV IgG positive laboratory volunteers with either (allogeneic) T-cell depleted DBMC or donor spleen cells (DSPC) from (the same) cadaver donors. After 7 days, the (responding) T cells were collected using magnetic beads and placed in culture with purified B cells freshly obtained from the same (autologous) CMV positive volunteer. To these cultures were added either media or 40 ng of CMV antigen. After 3, 5, 7, and 9 days, the expression of surface anti-CMV Ig was measured by flow cytometry using a panel of fluorescent markers double-labeled for activated B cells (CD20, CD19, and HLA DRw) and CMV-FITC. We also determined the phenotype of the cultured T cells using anti-CD3, CD4, and CD62L specific monoclonal antibodies. B cells that had been in contact with autologous T cells derived from DBMC cultures (TBM) were less likely to express anti-CMV surface Ig than those cultured with DSPC (TSP). The flow cytometry analysis revealed an increase in the number of T4 suppressor cells (CD3+, CD4+, CD62L+) in the TBM group, whereas the T4 helper phenotype (CD3+, CD4+, CD62L-) predominated in the TSP group. These in vitro findings support the notion that (allogeneic) DBMC infusions can induce a T4 suppressor (regulatory) influence and thereby indirectly affect B-cell function.