T. Karatzas

High-dose donor bone marrow infusions to enhance allograft survival: The effect of timing

BACKGROUND The development of strategies to enhance survival of transplanted organs and to potentially lower or even discontinue immunosuppressive therapy would represent a significant advance in posttransplant patient care. The aim of this clinical trial was to determine the effect of timing and dose of peripheral donor bone marrow cell (DBMC) infusion on graft and patient survival after liver transplantation. METHODS DBMC, obtained from vertebral bodies, were administered in 101 recipients of liver allografts (OLTX). There were 107 patients for whom DBMC could not be obtained; they received OLTX alone (controls). A total of 5 x 10(8)/kg DBMC were infused at day 0 (group 1; n=9); at days 0 and 11 (group 2; n=26); or at days 5 and 11 (group 3; n=26). In group 4 (n=40), patients received up to five infusions of 2 x 10(8)/kg DBMC at days 5, 14, 21, 28, and 90 after OLTX. RESULTS When the results from patients receiving two or more DBMC infusions (groups 2, 3, and 4) are considered, both patient and graft survival were significantly improved compared with the control group (P=0.02 and P=0.01, respectively). In groups 3 and 4, 88.5% and 95% of patients were alive with mean follow-up of 536 and 265 days, respectively, compared with 77.6% of patients in the control group (average follow-up of 452 days) (P=0.02). Graft survival was also significantly improved in groups 3 (88.5%) and 4 (92.5%), compared with the controls (72%) (P=0.007). CONCLUSIONS The results suggest that dose and timing of DBMC infusions may be important variables affecting allograft survival. A randomized prospective trial is now in progress to compare group 3 DBMC infusion protocol with controls receiving OLTX alone.

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 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.

The Immune Reactivity Role of HCV-Induced Liver Infiltrating Lymphocytes in Hepatocellular Damage

Liver infiltrating lymphocytes (LIL) were isolated from HCV-positive (+) and HCV-negative (−) end-stage livers. Phenotypic analysis and functional studies using proliferative and lymphocytotoxic assays were performed with the isolated LIL. Two CD3+ lymphocyte populations were found in LIL using FITC anti-CD3 monoclonal antibodies (mAb). One was a bright fluorescence intensity population (as in PBL), and the other dim. We calculated the number of FITC-anti-CD3 mAbs bound per lymphocyte on PBL and LIL and found 80,040 ± 4628 and 39,615 ± 3932, respectively. Therefore, HCV+ and HCV− patient PBL contained approximately twice the number of CD3 molecules per cell than patient CD3+ LIL. LIL also contained approximately a threefold higher concentration of TCRαβ+, CD4−CD8−, and CD56,16 (NK) cells than the patient PBL. Thus, a major subset of LIL is phenotypically similar to mouse NK1.1+ “intermediate” T cells. LIL freshly isolated from HCV+ livers exhibited weak CTL activity against EBV- or Con A-transformed lymphoblast targets infected with vaccinia–HCV recombinant virus (rHCV) or primary hepatocyte cultured cells. However, after in vitro coculture of LIL with rHCV, these cells developed a strong cytotoxicity for the above targets. In contrast, LIL from HCV− livers were not cytotoxic against the same targets. Histochemical studies (in situ) demonstrated that these hepatocytes express CD95, and stains demonstrated apoptosis. The HCV+ hepatocytes also express class I MHC molecules and ICAM-1. The addition of mAb specific for these adhesion molecules inhibited CML activity. Short-term cultured hepatocytes (targets) from HCV+ and HCV− patients produced low levels of cytokines IL-1β, IL-2, IL-6, TNFα, and IFN-γ but a high level of IL-8. It is speculated that LIL expressing reduced numbers of CD3 molecules may even function as immune regulators as proposed for intermediate T cells in mice.

Human islet allografts in patients with type 2 diabetes undergoing liver transplantation.

BACKGROUND Most patients with cirrhosis have insulin resistance and impaired glucose tolerance, and 20% eventually develop diabetes. Although diabetes in this setting may be reversible after orthotopic liver transplantation (OLTx), immunosuppressive agents administered after transplantation could exacerbate this disease. We report the results of the first pilot trial of islet cell transplantation (ICTx) in patients with diabetes undergoing OLTx. METHODS Five patients with diabetes and liver cirrhosis underwent OLTx and ICTx. Donor bone marrow cells were also infused to enhance the acceptance of the graft. We identified seven patients who received only OLTx and donor bone marrow cells as historical controls. RESULTS Preliminary results suggest that ICTx in conjunction with OLTx may improve glucose metabolism (insulin requirement, hemoglobin A1c) in patients with liver cirrhosis. However, there was virtually no change in pre- and posttransplant basal C-peptide levels in the recipients of OLTx + ICTx. CONCLUSIONS We are planning to further evaluate the effect of OLTx with or without ICTx in a randomized prospective trial, using euglycemic insulin clamp studies.

Transplantation of islets of Langerhans in patients with insulin-requiring diabetes mellitus undergoing orthotopic liver transplantation - the Miami experience

Most patients with cirrhosis of the liver have detectable insulin resistance. In 60–80% of patients with cirrhosis, impaired glucose tolerance can be uncovered; approximately 20% of these patients eventually develop overt diabetes. Theoretically, insulin resistance and glucose intolerance could be improved or reversed by orthotopic liver transplantation alone or in association with a simultaneous transplant of pancreatic islet cells from the same donor. To investigate these possibilities we initiated a pilot study of simultaneous liver and pancreatic islet cell transplantation in seven patients with diabetes and liver cirrhosis. Donor bone marrow cells were also infused to enhance the acceptance of the grafts. Seven patients who received only orthotopic liver transplantation and donor bone marrow cells were used as historical controls. The preliminary results of this pilot trial suggest that islet cell transplantation in conjunction with orthotopic liver transplantation improves glucose metabolism in patients with liver cirrhosis in association with reduced insulin requirements and HbA1c levels. These results were evident in spite of pre- and post-transplant basal C-peptide levels that were unchanged. Further evaluation of the effects of orthotopic liver transplantation with or without islet cell transplantation will require a randomized prospective trial including accurate metabolic evaluation with the euglycemic insulin clamp technique.

Clinical intestinal transplantation: Experience in Miami

Abstract Intestinal transplantation can be a life-saving procedure for patients with intestinal failure and life-threatening complications of the underlying disease or total parenteral nutrition (TPN). The transplantation techniques at the University of Miami were based on our Pittsburgh experience and the organs were separated as needed following Starzls cluster principle. According to the latter all intra-abdominal organs are like a grape cluster. They are all hanging from a central stem consisting of the superior mesenteric artery (SMA), superior mesenteric vein (SMV), celiac axis, and portal vein (PV). Individual clumps can be removed without jeopardizing the integrity of the remaining organs. The three main variants of this concept are the isolated intestinal, the combined liver-intestinal, and multivisceral grafts. Common goals in all variants during the implantation procedure are to prevent injury of the organs to be preserved and resect damaged ones without contamination of the peritoneal cavity. In the isolated intestinal transplant the arterialization of the graft is from the infrarenal aorta by end-to-side anastomosis to the donor SMA. The graft portal or SMV can be anastomosed either to the inferior vena cava (IVC) or to the portal system. In the absence of IVC thrombosis, anastomosis to the IVC is technically easier. Anastomosis to the portal system provides hepatopetal flow and is more physiologic but technically more demanding if there have been previous surgeries involving the mesentery. In combined liver-intestine transplantation the native hepatectomy is usually performed with preservation of the native IVC. The native PV is anastomosed end-to-side to the native IVC in order to provide decompression of the retained viscera. This anastomosis can be taken down after the graft is implanted. The native PV can later be anastomosed end-to-side onto the donor portal or end-to-end to the donor splenic vein. More commonly the portocaval shunt is left in place permanently. A short interposition arterial graft, usually a segment of the thoracic aorta of the donor, is placed end-to-side onto the infrarenal aorta to facilitate the arterialization of the graft. Arterialization of the graft is achieved by anastomosis of the donor celiac and SMA with a common Carrel patch to the distal end of the arterial interposition graft. The outflow of the graft is usually provided by anastomosis of the suprahepatic IVC of the graft to the joined ostia of the native suprahepatic veins (piggyback). In multivisceral transplantation the first objective of the resection is to disconnect the visceral blood supply early in the dissection in order to avoid unnecessary bleeding. Various approaches can be utilized. When the lower abdomen is frozen, transection of the esophagus at the esophagogastric junction facilitates exposure of the celiac axis and SMA and early disconnection. The retroperitoneal structures (kidneys, ureters, bladder, the aorta, IVC, and iliac vessels) are carefully preserved as well as the distal esophageal and colonic stumps. Arterial inflow to the graft is with an interposition graft to the suprarenal or more commonly infrarenal aorta. The venous outflow is to the joined ostia of the native suprahepatic veins. A Mikulicz pyloroplasty is routinely performed. The donor esophagogastric junction is oversewn. The esophago(native)gastro(graft)-stomy is performed in two layers to the dome of the stomach. No biliary reconstruction is necessary.

Transplant-associated autoimmune mechanisms in human hepatitis C virus infection.

In order to define factors which are important for the development of hepatitis C virus (HCV) infection and disease in transplant patients, we examined the role of class II MHC antigen restriction in viral antigen presentation to support a hypothesis of the association of this disease with an autoimmune pathogenesis. A greater degree of histocompatibility match between these donors and their HCV-negative recipients was associated with a greater predisposition to recipient HCV liver disease (ALT elevation) posttransplant. The HCV carrier state could be identified with significant amplification of autologous mixed lymphocyte reactivity (AMLR) in both long-term hemodialysis and long-term renal transplant patients, but the AMLR was absent in end-stage liver disease patients with HCV-associated cirrhosis and was insignificantly elevated in these patients with persistent infection in the first 2 years after a new liver was transplanted. There was also a moderate reduction in autologous reactivity as well as serum HCV titers among renal transplant patients who displayed biochemical evidence of chronic liver disease as opposed to those who did not. This appeared later in the course of the disease. HCV RNA could be detected in peripheral blood mononuclear cells (PBMC) of only a portion of HCV-infected renal transplant patients and these showed significantly higher autologous reactivity. In contrast, despite the fact that observations were earlier after de novo liver transplantation, HCV RNA (i.e., earlier in the course of a new or recurrent disease process) was found in PBMC of all liver transplant recipients tested. The AMLR of noninfected laboratory volunteers could be amplified by preincubating their stimulating cells (APCs) with enriched HCV possibly in immune complex (pHCV-IC). This amplification appeared only with specific combinations of HCV strains with HLA DR serotypes. In addition, HCV-primed T cells could be generated to the virus which displayed accelerated activation kinetics. Liver infiltrating lymphocytes extracted from HCV-positive end-stage diseased livers had significantly higher proliferative and cytotoxic reactivity to autologous (HCV-infected) hepatocytes than the extracted lymphocytes responding to autologous hepatocytes from HCV-negative livers. These findings offer evidence of dynamic autoimmune mechanisms in the spectrum of progression of HCV disease and may help to predict the effect of intervention at various intervals in this progression in organ transplant recipients.