Immune "tolerance profiles" in donor bone marrow infused kidney transplant patients using multiple ex vivo functional assays.

Ex vivo identification of donor-specific unresponsiveness in organ transplant recipients is important for immunosuppression (IS) minimization. We tested three groups of stable living, related-donor kidney transplant patients up to 11 years postoperatively, i.e.

Evaluation of the tolerogenic effects of donor bone marrow cells using a severe combined immunodeficient mouse-human islet transplant model.

The immunoregulatory role of human donor bone marrow cells (DBMC) has been studied extensively in our laboratory using in vitro and ex vivo assays. However, new experimental systems that can overcome the limitations of tissue culture assays but with more clinical relevance than purely animal experimentation, needed to be generated. Therefore we have developed a new human peripheral blood lymphocyte (PBL) severe combined immunodeficient (SCID) mouse islet transplantation model without the occurrence of graft-versus-host disease (GvHD) and have used it to evaluate the tolerogenic effects of DBMC.

Detection of HLA and MICA antibodies before kidney graft failure.

390 serum samples taken from a series of 28 patients who lost their grafts were retrospectively investigated for antibodies by single antigen HLA Class I&II and MICA Luminex beads. In 20 patients with immunological failure, 10 patients had DSA, 16 had epitope-related NDSA, and 16 had NDSA or MICA antibodies. In 16 which increased antibodies leading up to the graft failure, DSA were found in 7 patients.

Improved metabolic control and quality of life in seven patients with type 1 diabetes following islet after kidney transplantation.

Background: The beneficial effects of glycemic control on both survival and function of transplanted kidneys in patients with type 1 diabetes mellitus (T1DM) and end-stage renal disease (ESRD) have been recognized.

Methods: Herein, we present the clinical outcome of a single-center pilot trial of islet after kidney (IAK) transplantation in seven patients with T1DM. The immunosuppression protocol for the kidney graft was converted to sirolimus+tacrolimus regimen 6 months before islet transplantation to exclude negative effects on kidney graft function.

Campath-1H induction therapy in African American and Hispanic first renal transplant recipients: 3-year actuarial follow-up.

Background: In a retrospective study of the first 75 primary renal transplant patients given alemtuzumab induction at our center, 20 were African American (27%), 32 were Hispanic (43%), and 23 were non-African American, non-Hispanic (31%).

Methods: Alemtuzumab was given intraoperatively and 4 days later (0.3 mg/kg), with planned low-dose maintenance mycophenolate mofetil (500 mg twice daily) and tacrolimus (targeted trough levels of 5 to 7 ng/ml) and no corticosteroid therapy after the first week.

Allosensitization of islet allograft recipients.

Background: The immune monitoring of islet transplant recipients includes the assessment of panel reactive antibodies (PRA). A negative association of PRA+ with allogeneic solid organ graft survival has been recognized, but scattered data is available for islet transplantation.

Methods: We performed a retrospective analysis of PRA status in 66 patients with type 1 diabetes mellitus recipient of islet allografts between 1985 and 2006.

Blood citrulline level is an exclusionary marker for significant acute rejection after intestinal transplantation.

Background: Serum citrulline is a marker for acute cellular rejection (ACR) after intestinal transplantation; however, its clinical utility has not yet been established. The goal of this study was to determine clearcut serum levels beyond which the diagnosis of acute rejection could be supported or refuted, and predictors of citrulline levels posttransplant from which more accurate estimates of sensitivity and specificity could be obtained.

Methods: Since March 2004, we obtained 2135 dried blood spot (DBS) citrulline samples from 57 intestinal transplant recipients at or beyond 3 months posttransplant.

Inhibition of NF-kappa B during human dendritic cell differentiation generates anergy and regulatory T-cell activity for one but not two human leukocyte antigen DR mismatches.

We examined the in vitro inhibition of human monocyte-derived dendritic cells (DC) maturation via NF-kappaB blockade on T-cell allostimulation, cytokine production, and regulatory T-cell generation. DC were generated from CD14+ monocytes isolated from peripheral blood using GM-CSF and IL-4 for differentiation and TNF-alpha, IL-1beta, and PGE2 as maturational stimuli with or without the NF-kappaB inhibitors, BAY 11-7082 (BAY-DC) or Aspirin (ASA-DC). Stimulator and responder cells were one versus two HLA-DR mismatched in direct versus indirect presentation assays.

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.

A novel approach to detect donor/recipent immune responses between HLA-identical pairs.

Renal transplant rejection and graft versus host reactions between HLA genetically-identical sibling (HLAgi) donor/recipient (D/R) pairs are thought to result from minor histocompatibility antigen (mHAg) disparities. We have compared two methods of measuring HLAgi D/R T lymphocyte responses to "matured" dendritic cells: 1.) a modified Cylex assay of CD4(+) ATP levels (MLDC-ATP) versus 2.

Induction of auto-reactive regulatory T cells by stimulation with immature autologous dendritic cells.

We have shown in ex vivo studies in donor bone marrow-infused kidney transplant recipients, that chimeric cells of either donor or recipient origin taken from the recipient's bone marrow down-regulated the recipient's cellular immune responses. In the present study, we have now induced regulatory T cells from peripheral blood mononuclear cells (PBMC) of renal transplant recipients or laboratory volunteers by multi-stimulation with autologous immature dendritic cell (iDC) enriched populations derived from either bone marrow cells (BMC) of the (immunosuppressed) kidney transplant recipients or PBMC of the laboratory volunteers (i.e.

Utilization of dried blood spot citrulline level as a noninvasive method for monitoring graft function following intestinal transplantation.

Background: Citrulline concentrations have been proposed as a marker for intestinal allograft rejection. We instituted dried blood spot (DBS) specimen monitoring of citrulline to simplify sample collection posttransplant. This study demonstrates the correlation between plasma and dried blood spot specimen citrulline concentrations after intestinal transplantation.

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).

Serial ten-year follow-up of HLA and MICA antibody production prior to kidney graft failure.

The role of HLA antibodies in chronic allograft rejection was examined utilizing a unique resource of sera collected annually and stored over a 12-year period from patients with rejected or retained grafts. In patients selected for not having preformed HLA antibodies, 679 postoperative serial serum samples from 39 patients who rejected their grafts and 26 with functioning grafts were tested for HLA Class I and Class II antibodies by flow cytometry and for MICA antibodies by cytotoxicity on recombinant cell lines. HLA antibodies were found in 72% of patients who rejected grafts, compared to 46% with functioning transplants (p<0.

Islet transplantation in type 1 diabetes mellitus using cultured islets and steroid-free immunosuppression: Miami experience.

Following the success obtained with transplantation of fresh human islets under steroid-free immunosuppression, this trial evaluated the transplantation of islets that had undergone a period of in vitro culture and the potential of tumor necrosis factor (TNF-alpha) blockade to improve islet engraftment. Subjects included 16 patients with type 1 diabetes mellitus (T1DM); half were randomly assigned to receive Infliximab immediately preceding initial infusion. Immunosuppression consisted of daclizumab induction and sirolimus/tacrolimus maintenance.

Campath-1H does not alter bone marrow cell regulatory function.

We have previously reported in laboratory volunteers (in vitro) and renal transplant recipients (ex vivo) that bone marrow cells (BMC) are potent downregulators of the immune response. Also, the use of alemtuzumab (Campath-1H, C1H) for immunodepletion is associated with the most potent lasting effects yet seen on T-cell immunity in renal transplantation. We questioned whether the administration of C1H to kidney allograft recipients of donor bone marrow cell (DBMC) infusions would lead to stronger or weaker immunoregulatory effects.

The role of donor bone marrow infusions in withdrawal of immunosuppression in adult liver allotransplantation.

We investigated the role of donor bone marrow cell (DBMC) infusions in immunosuppression withdrawal in adult liver transplantation. Patients enrolled were at least 3 years post-transplantation, with stable graft function. Forty-five (study group: G1) received DBMC, and 59 (control group: G2) did not.

Donor bone marrow infusion in deceased and living donor renal transplantation.

The infusion and persistence in a transplant recipient of donor-derived bone marrow cells (DBMC) of multi-lineage can lead to a state of permanent chimerism. In solid vascular organ transplantation, the donor bone marrow lineage cells can even be derived from the transplant organ, and these cells can be detected in very small numbers in the recipient. This has been called microchimerism.

TNF-alpha, TGF-beta, IL-10, IL-6, and INF-gamma alleles among African Americans and Cuban Americans. Report of the ASHI Minority Workshops: Part IV.

Point mutations or single nucleotide substitutions in the regulatory regions of cytokine genes may affect levels of cytokine expression and have been associated with acute and chronic rejection in organ transplantation, severity of graft-versus-host disease in hematopoietic stem cell transplants, and predisposition to autoimmune disorders. Because these cytokine variants have been studied primarily among Caucasians, we defined the alleles and frequencies of five cytokines among 691 unrelated, adult African Americans and 296 Cuban Americans in the American Society for Histocompatibility/National Institutes of Health Minority HLA Workshops. The genotypes of all cytokines, except for transforming growth factor (TGF)-beta among African Americans, were found to be in Hardy-Weinberg's equilibrium.

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.

Preliminary experience with alemtuzumab (Campath-1H) and low-dose tacrolimus immunosuppression in adult liver transplantation.

Background: The administration of alemtuzumab (Campath-1H [C1H]; Berlex Laboratories, Montville, NJ) at transplantation prevents a vigorous immune response and is believed to allow a gradual engagement of the host immune system. We report our preliminary experience with C1H and tacrolimus (Tac) immunosuppression in adult liver transplantation.

Methods: We administered C1H and low-dose Tac to 40 adult recipients of cadaveric liver allografts between December 2001 and April 2003.

Toward tolerance to human organ transplants: a few additional corollaries and questions.

Several component mechanisms of "central" (thymic) and "peripheral" tolerance in human organ transplant recipients are briefly discussed, in opposition to a more confined view limited to clonal depletion and exhaustion as proposed by Starzl (Transplantation 2004; 77(6): 926). Attention is directed to more than 40 years of experimental work in adult animal species dealing with immunoregulation. This work is in contradistinction to the simpler depletion/exhaustion explanation of Starzl (and Zinkernagel) regarding the potential of human organ transplant recipients to be significantly (or totally) withdrawn from continuous immunosuppression.

Immune responses and their regulation by donor bone marrow cells in clinical organ transplantation.

Infusions of donor bone marrow derived cells (DBMC) continue to be tested in clinical protocols intended to induce specific immunologic tolerance of solid organ transplants based on the observations that donor-specific tolerance is induced this way in animal models. We studied the immunological effects of human DBMC infusions in renal transplantation using modifications in lymphoproliferation (MLR) and cytotoxicity (CML) assays. The salient observations and tentative conclusions are summarized in this review.

Alemtuzumab (Campath-1H) combined with tacrolimus in intestinal and multivisceral transplantation.

Background: We combined alemtuzumab (Campath-1H, Berlex Laboratories, Montville, NJ) and tacrolimus (Tac) immunosuppression for intestinal and multivisceral transplantation.

Materials And Methods: A total of 21 adult patients received 24 grafts: 14 intestinal, nine multivisceral, and one liver-intestinal graft. Alemtuzumab was administered perioperatively in four doses with low-dose Tac (levels 10-15 ng/dL) and no maintenance steroids.

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.