Arterial Blood Supply of Liver Segment IV and Its Possible Surgical Consequences.

The risk of ischemia of segment IV after split liver resection is high. This anatomical study was done to identify the arterial blood supply and the intrahepatic distribution of liver segment IV. The anatomy of segment IV was studied in 29 livers from adult cadavers. To identify the arterial blood supply of segment IV, water and ink were injected into the various branches of the hepatic artery and the outflow through segment IV and discoloration of the liver parenchyma were observed. In 23 of the 29 livers (79.3%), the arterial perfusion of segment IV was separated by a line drawn from the left side of the inferior vena cava at the top of and lateral to the falciform ligament to the medial point of the gallbladder bed. The area lateral to this line was supplied mainly by the right hepatic artery, and the area medial to it was supplied mainly by the left hepatic artery. In addition to the classification system of Couinaud, we describe here a new division of liver segment IV based on arterial blood supply. These anatomical findings may be useful in defining the resection line for split liver to prevent necrosis of segment IV.

Metastatic recurrence after complete resection of colorectal liver metastases: impact of surgery and chemotherapy on survival.

PURPOSE: Surgery is the standard of care for resectable colorectal liver metastases (CRC-LM). Unfortunately, 60% of patients develop secondary metastatic recurrence (SMR) after R0-resection of CRC-LM. We investigated the impact of surgical re-intervention and chemotherapy (Ctx) on survival in a consecutive series of patients with SMR. METHODS: From 01/2001 to 11/2011, 104 out of 178 consecutive patients with R0-resection of CRC-LM developed SMR and were evaluated. The impact of surgical and Ctx re-interventions on recurrence free (RFS) and cancer-specific survival (CSS) was analyzed. Median follow-up was 28.0 (95%CI: 19.4-37.4) months. RESULTS: SMR occurred in 81 patients at a single site (49× liver, 18× lung, 14× other) and in 23 patients at multiple sites. Forty-two patients were scheduled for primary surgery. Fifty-three patients were classified as non-resectable and treated with median 5.0 [IQR, 3.0-10.0] cycles of Ctx, combined with an EGFR/VEGF-antibody in 27 patients. Nine patients received best supportive care only. R0/R1 resection could be achieved in 35 patients primarily and even in 8 patients secondarily after Ctx. Surgical morbidity and mortality were 16 and 0%, respectively. The 5-year RFS rates for patients with R0 versus R1-resection were 22 and 24% (p = 0.948). The 5-year CSS rate for R0/R1-resected patients was 38% versus 10% for those patients treated by Ctx alone (p < 0.001). CONCLUSION: In SMR, surgical re-intervention is feasible and safe in a remarkable number of patients and offers significantly longer CSS compared to patients without resection.

Calcineurin inhibitor sparing with mycophenolate mofetil in liver transplantion: a systematic review of randomized controlled trials.

Liver transplant recipients are at high risk of developing acute and chronic renal failure. Moreover, introduction of the model for end-stage liver disease (MELD) score for primary allocation of liver grafts favors patients with pretransplant kidney dysfunction, which in turn have a higher risk of posttransplant renal failure. Calcineurin inhibitors (CNI) further increase the risk of renal failure and therefore sparing CNI with the use of mycophenolate mofetil (MMF) may improve renal function. MMF may either be used de novo in the immediate posttransplant period in combination with low-dose CNI (scenario 1) or patients that receive immunosuppression based on CNI may be converted to MMF in combination with minimization or elimination of CNI (scenario 2). Although many retrospective cohort studies and nonrandomized trials have implicated efficacy of this approach the evidence from randomized controlled studies has not been summarized. In the current review we report the results of a systematic review and meta-analysis of randomized controlled trials.

Bilobar spreading of colorectal liver metastases does not significantly affect survival after R0 resection in the era of interdisciplinary multimodal treatment.

PURPOSE: Bilobar colorectal liver metastases (CRLM) are often considered incurable or associated with poor prognosis even after R0 resection. In this single-center study, we evaluate the impact of CRLM spreading on recurrence-free survival (RFS) and cancer-specific overall survival (CSS) after R0 resection of CRLM with respect to multimodal treatment strategies including perioperative chemotherapy and multistep resections. METHODS: Between January 2001 and December 2010, R0 resection could be achieved in 70 patients with bilobar and 100 with unilobar CRLM. Extent of disease, perioperative chemotherapy, surgical procedures, adjuvant treatment, histopathological workup, RFS, and CSS were compared between both cohorts. RESULTS: Forty-six (66 %) patients with bilobar and 26 (26 %) patients with unilobar CRLM received preoperative chemotherapy (p < 0.001). For bilobar CRLM, more extended and multistep resection including portal vein occlusion were performed (29 % versus 3 %; p < 0.001). Morbidity (39 % versus 28 %, p = 0.183) and mortality (1 % versus 3 %, p = 0.644) rates were comparable in both patients' cohorts. Postoperative therapy was applied in adjuvant intent to 42 (60 %) versus 51 (51 %) patients (p = 0.275). The 5-year RFS and CSS rates were 24 % versus 31 % (p = 0.169) and 42 % versus 55 % (p = 0.131), respectively. CONCLUSIONS: To our single-center experience, there is no significant effect of CRLM spreading (bilobar versus unilobar) on RFS and CSS rates. Bilobar CRLM are more likely to require extended multimodal efforts to achieve R0 resection.

Role of the thymus in transplantation tolerance in miniature swine. I. Requirement of the thymus for rapid and stable induction of tolerance to class I-mismatched renal allografts.

The almost uniform failure in transplant patients of tolerance-inducing regimens that have been found to be effective in rodents, has made it necessary to examine large animal models before testing of new approaches clinically. Miniature swine have been shown to share many relevant immunologic parameters with humans, and because of their reproducible genetics, have proved extremely useful in providing such a large animal model. We have previously shown that indefinite systemic tolerance to renal allografts in miniature swine is induced in 100% of cases across a two-haplotype class I plus minor histocompatibility antigen disparity by a 12-d course of Cyclosporine A (CyA), in contrast to irreversible rejection observed uniformly without CyA treatment. In the present study, we have examined the role of the thymus during the induction of tolerance by performing a complete thymectomy 21 d before renal transplantation. This analysis demonstrated a striking difference between thymectomized and nonthymectomized animals. Thymectomized swine developed acute cellular rejection characterized by a T cell (CD25(+)) infiltrate, tubulitis, endothelialitis and glomerulitis, and anti-donor CTL reactivity in vitro. Nonthymectomized and sham thymectomized animals had a mild T cell infiltrate with few CD25(+) cells and no anti-donor CTL response in vitro. These results indicate that the thymus is required for rapid and stable induction of tolerance.

Two-stage hepatectomy (R0) with portal vein ligation--towards curing patients with extended bilobular colorectal liver metastases.

BACKGROUND AND AIMS: Patients with bilobular colorectal liver metastases (CRLM) experience poor prognosis, especially when curative resection cannot be achieved. However, resectability in these patients is often limited by low future remnant liver volume (FRLV). The latter can be enhanced by a two-stage liver resection, using portal vein ligation to induce liver hypertrophy. The aim of this prospective pilot study was to evaluate safety, secondary resectability, and time to recurrence of two-stage hepatectomy with portal vein ligation (PVL) and complete surgical clearance of the FRLV in patients with bilobular CRLM. MATERIALS AND METHODS: Out of 24 patients (63+/-8.26 years) with extended bilobular CRLM (metachronous n=10, synchronous n=14), 18 received preoperative 5-FU-based chemotherapy combined with oxaliplatin or irinotecan. Staging included thoracoabdominal computed tomography and (18)F-fluorodeoxyglucose-positron emission tomography scans. First-stage procedure consisted of PVL, resection of all CRLM in the FRLV, and radiofrequency ablation (RFA) of CRLM situated near the future resection plane. RESULTS: During first-stage procedure, 7x RFA, 4x non-anatomical resections, and 4x bisegmentectomies were performed additionally to PVL. FRLV/body-weight ratio increased from 0.4% to 0.6% within 55 days (median) after PVL. Second-stage hepatectomy was performed in 19 patients without tumor progression. R0 resection was possible in 14 patients. During a median follow-up of 17 months, intrahepatic recurrence occurred in two, and extrahepatic recurrence in nine out of 14 patients. CONCLUSION: Two-stage hepatectomy with PVL and complete surgical clearance of FRLV is safe even after intensified systemic chemotherapy resulting in a curative resection rate of 58.3% (73.7% of re-explored cases).

Prognostic value of the monoethylglycinexylidide (MEGX)-test prior to liver resection.

BACKGROUND/AIMS: The critical issue before major hepatic resection is to evaluate and detect patients with a potentially increased risk of hepatic failure. In this study the prognostic value of the monoethylglycinexylidide (MEGX)- liver function test was evaluated with regards to clinical course and survival after partial liver resection. METHODOLOGY: Between 1995 and 2000 a total of 55 patients (29 male, 26 female) underwent a partial liver resection at the Georg-August University of Göttingen. Forty-two patients were treated for malignant, and 13 for benign, disease. MEGX-testing was performed 15 and 30 minutes after a single-dose of 1mg/kg BW Lidocaine i.v. was applied. RESULTS: MEGX-test results after 30 minutes had significant influence on hospital mortality. Patients who died during the hospital stay showed median MEGX-30 minutes results of 32 microg/L in (4-107 microg/L) in comparison to the surviving patients with a median 68 microg/L (16-176 microg/L) (p = 0.026). Furthermore, patients with MEGX scaled categories of 3 and 4 had a significantly lower surivial at 150 days (p = 0.008) and overall (p = 0.0002). There was an indirect impact of MEGX on hospital stay, costs and mortality reflecting high fluid loss: patients with lower loss of fluid over drainages had a significantly lower mortality at 150 days (p = 0.00046) and overall (p = 0.00008), than did patients with higher fluid loss. Low MEGX-values significantly influenced long hospital stay (p = 0.00001) and high costs (p = 0.00001). Pathologic MEGX in combination with increased age, increased BMI and extensive surgical procedures including resection of over 50% volume of the liver had a significant influence on complications (p = 0.015). CONCLUSION: The preoperative MEGX-test, especially the 30 minutes value, is a useful medium to estimate the liver reserve in non-cirrhotic patients prior to liver resection. In combination with the resection volume it may be very useful to identify patients with a high risk of developing a postoperative liver failure.

Reduction of severe ischemia/reperfusion injury in rat kidney grafts by a soluble P-selectin glycoprotein ligand.

BACKGROUND: Inflammatory leukocyte-endothelium interactions, mediated by selectins, contribute to renal ischemia/reperfusion (I/R) injury. We examined the influence of the soluble P-selectin glycoprotein ligand 1 (sPSGL) on early I/R-induced changes in a rat kidney transplantation model with long cold ischemia. METHODS: After 24 hr of cold storage, syngeneic kidneys were grafted into bilaterally nephrectomized rats. Before transplantation, recipients received either 1 mg/kg of sPSGL or vehicle (n=8 per group). Six hours after reperfusion, grafts were removed for light microscopy and immunohistochemistry. Capillary blood flow was measured under a fluorescence microscope by using the concentric-circles method. RESULTS: A greater proportion, 74.7+/-7.2% (sPSGL) vs. 28+/-7.4% (controls), of all dye-labeled outer medullary capillaries appeared in the 12-microm radius (P<0.01), indicating dense blood flow, whereas 7.6+/-2.9% vs. 43.3+/-9.7%, respectively, appeared in the 60-microm radius (P<0.05), indicating rarefied blood flow. In the sPSGL-treated group, the extent of severe tubular damage within the inner stripe of the outer medulla was lower compared with controls (37.5+/-8.3% vs. 78.4+/-3.5%, P<0.01). Outer medullary heat shock protein 72 expression was 14.5+/-1.6% in the sPSGL-treated group compared with 9.6+/-1.4% in controls (P<0.05). The number of infiltrating polymorphonuclear leukocytes was similar in both groups. Treatment with sPSGL had no influence on the serum creatinine level. CONCLUSIONS: Our data suggest that impairment of outer medullary blood flow is crucial in I/R injury of kidney grafts with prolonged cold storage. Reduction of capillary blood flow perturbations by sPSGL protects tubular cells from severe structural damage. Blocking early selectin-mediated leukocyte adhesion may have therapeutic implications in improving the prognosis of renal transplants with severe I/R injury.

A novel management strategy of steroid-free immunosuppression after liver transplantation: efficacy and safety of tacrolimus and mycophenolate mofetil.

BACKGROUND: Corticosteroids have been used traditionally for immunosuppression after solid organ transplantation. The variety of modern immunosuppressive agents offers the chance to replace drugs with an unfavorable risk-benefit ratio. The objective of this prospective pilot study was to investigate a novel steroid-free immunosuppressive regimen after clinical liver transplantation. METHODS: 30 adult liver graft recipients were included in an intent-to-treat analysis. Dual induction immunosuppression consisted of tacrolimus and mycophenolate mofetil. Prophylactic steroids were not given. Efficacy and safety parameters analyzed were patient and graft survival, incidence and severity of rejection, and adverse events in correlation to immunosuppressive drug levels. RESULTS: Patient and graft survival at 2 years was 86.7 and 83.9%, respectively. Acute rejection occurred in 26.2%, and was associated with subtherapeutic tacrolimus blood levels and diarrhea. All rejections were completely reversible by temporary addition of steroids. Acute renal failure was seen in 10/30 patients, and was related to high tacrolimus blood levels together with primary liver graft dysfunction. 43% of all patients never received any steroids, and 73% were on a steroid-free maintenance regimen. CONCLUSIONS: These results confirm that corticosteroids can be completely avoided from the beginning after liver transplantation. Double drug immunosuppression with tacrolimus and mycophenolate mofetil is effective and safe in terms of patient and graft survival as well as incidence and severity of rejection. In order to avoid under- or over-immunosuppression, which may be caused by impaired absorption or metabolism, close drug monitoring is advised.

Selective increase in CD4-positive graft-infiltrating mononuclear cells among the infiltrates in class I disparate kidney grafts undergoing rejection.

Long-term tolerance to kidney allografts across a two-haplotype class I disparity is uniformly induced in miniature swine with a short course of cyclosporine (CsA). In the absence of CsA, all recipients acutely reject kidney allografts within 2 weeks. Previous experiments have shown that graft-infiltrating mononuclear cells (GIC) migrate to the allograft in both CsA-treated and untreated animals. To evaluate the correlation between GIC phenotype and the clinical status, infiltrating cells were examined by flow cytometry, using selective gating to distinguish them from other renal cells. GIC from tolerant and rejector animals were mostly mature T cells, with 84% CD8+ cells, which consisted of 68% CD8+/CD4- and 16% CD8+/ CD4+ cells. This cellular phenotype was, however, markedly different from that of peripheral blood lymphocytes, suggesting a selective migration of cells into the graft. This selective process counterselected the CD3+/CD2- subset of GIC, which was never found in the graft. The distribution of GIC subsets was initially comparable in tolerated and rejected kidneys, but the CD4 single-positive subset then increased specifically in the allograft destined to rejection. The absence of CD4 single-positive cells in tolerated grafts was unlikely to be due to a direct effect of the CsA, because long-term tolerant animals, which received a second kidney without further immunosuppression, also showed no increase in CD4 single-positive cells. The fact that CD4 single-positive cells appeared only within the rejected kidneys, strongly suggests that this cell subset may be important in mediating immune rejection and supports the hypothesis that the development of tolerance in this model depends on a relative deficit of T-cell help.

Mechanism of tolerance to class I-mismatched renal allografts in miniature swine: regulation of interleukin-2 receptor alpha-chain expression on CD8 peripheral blood lymphocytes of tolerant animals.

BACKGROUND: Donor-specific tolerance to renal allografts in miniature swine is uniformly induced across a two-haplotype class I plus minor histocompatibility antigen disparity by a 12-day course of cyclosporine. Recent studies have demonstrated that the thymus is essential for rapid and stable tolerance induction, because either prior thymectomy or a series of thymic biopsies induce a spontaneously reversible rejection crisis after the 12-day course of cyclosporine. The present study examined the peripheral cellular mechanisms of tolerance by analyzing cytotoxic effector pathways in peripheral blood lymphocytes (PBL) of tolerant animals. METHODS: The phenotype and cytotoxic T lymphocyte response of alloantigen-activated PBL cultures using cells from a series of tolerant animals with stable renal function (no thymic manipulation), or during a rejection crisis (induced by thymic biopsies), were studied. The in vitro findings were correlated with the in vivo clinical course of experimental animals. RESULTS: The data demonstrated that in vivo and in vitro tolerance was associated with a specific deficiency of interleukin-2 receptor (IL-2R) alpha-chain up-regulation on CD8 single-positive (SP) T cells expressing high levels of CD8 (CD8high) when PBL from tolerant animals are stimulated with donor class I alloantigen. Stimulation by third party class I alloantigen, or by donor antigen during a rejection crisis, produced efficient cytotoxic T lymphocyte responses and expression of IL-2Ralpha on CD8high SP cells. CONCLUSION: Antigen-specific regulation of the IL-2Ralpha expression on CD8high SP PBL is a principal event associated with and potentially involved in the mechanism of tolerance in this preclinical large animal model.

Tolerance to class I-disparate renal allografts in miniature swine. Maintenance of tolerance despite induction of specific antidonor CTL responses.

Miniature swine that become tolerant to renal allografts across an MHC class I barrier following a short course of cyclosporine are unresponsive to donor class I antigens in cell-mediated lymphocytotoxicity. However, skin grafts bearing donor class I plus third-party class II antigens are promptly rejected, and the animals then develop marked cell-mediated lymphocytoxic reactivity to donor class I antigens in vitro, but do not reject the kidney transplants. We show here that CTL generation is directed toward the same donor class I antigens as are expressed by the kidney donor, and is not the result of recognition in vitro of the tolerated class I antigen plus peptides of minor antigens shared between the skin graft donor and the stimulator/target cells. We also show that detection by CTLs of peptides expressed by skin but not by kidney is also not a sufficient explantation of the results, since the survival of skin grafts from the kidney donor is also prolonged, even after precursor CTL can be detected in vitro. The data are most consistent with suppression in vivo in tolerant animals of the helper pathways necessary for activation of precursor CTLs. Differences in patterns of cytokine expression by graft infiltrating cells may provide a mechanism for local suppression of help in this model. Finally, we have examined antibody production after sensitizing by skin grafts in long-term tolerant animals and have found that anti-donor class I antibodies are not produced, even though the same animals produce both anti-class II and anti-third-party class I antibodies.

Role of the thymus in transplantation tolerance in miniature swine: II. Effect of steroids and age on the induction of tolerance to class I mismatched renal allografts.

BACKGROUND: Recent studies in young (5-7 months) miniature swine have demonstrated that the thymus is involved in the rapid induction of stable tolerance to class I mismatched renal allografts after a 12-day course of Cyclosporine (CyA). Because both steroids and age are known to influence the structure and function of the thymus, we have now studied the effects of these two parameters on tolerance induction in this model. MATERIALS AND METHODS: In young swine, the administration of methylprednisolone (MP) during the standard tolerance-inducing regimen (a 12-day course of CyA) produced severe renal dysfunction and acute cellular rejection histologically. However, the renal allografts recovered and were accepted for >100 days with histological evidence of chronic rejection. To test the effect of age, two relatively old swine (55 and 71 months) received transplants of class I mismatched renal allografts and the standard 12-day course of CyA. One animal rejected the allograft acutely on postoperative day 22, and the second also rejected, but more slowly, with manifestations of chronic rejection. CONCLUSION: These findings suggest that both MP and old age interfere with the induction of stable tolerance in a fashion similar to the previously described effect of thymectomy. These results may have important implications for the mechanism of thymic-dependent tolerance, for the use of steroids in clinical protocols for the induction of allograft tolerance, and for the application of such protocols to adult patients.

Induction of tolerance to renal allografts across single-haplotype MHC disparities in miniature swine.

We have previously demonstrated that a 12-day course of cyclosporine A (CsA) leads to the induction of tolerance to renal allografts in 100% of recipients selectively mismatched at class I for both haplotypes, and in 71% of recipients selectively mismatched at class II for both haplotypes, but in 0% of recipients mismatched for two haplotypes at both class I and class II. We have postulated that the mechanism by which tolerance is induced may therefore require matching for either class I or class II antigens. One might predict from this hypothesis that tolerance would also be induced in donor-recipient combinations sharing one full haplotype (e.g., AC-->AD), which mimics the clinically relevant transplant combination of parent to offspring. We have therefore investigated the effects of the CsA regimen on renal transplants in this combination. Without immunosuppression, such kidney allografts were uniformly rejected (n = 12; 10.6 +/- 2.4 days). In contrast, a course of CsA (10-13 mg/kg/day) during the first 12 postoperative days induced long-term acceptance of the allograft in 67% (4/6) of recipients. Some acceptor animals also showed specific unresponsiveness to donor antigens as measured by in vitro assays and by failure to develop anti-donor antibodies. Tolerance was confirmed in four of these animals by failure to reject a second transplant SLA-matched to the first kidney donor without additional immunosuppression. These results suggest the feasibility of inducing specific tolerance across a single-haplotype mismatch in the majority of the cases, which could have clinical implications for living-related transplants.

Long-term acceptance of primarily vascularized renal allografts in miniature swine. Systemic tolerance versus graft adaptation.

We have previously demonstrated that tolerance to two-haplotype class I-mismatched renal allografts can be induced uniformly by a short course of cyclosporine. We report here that following transplant nephrectomy, 8 such long-term acceptor animals all accepted a second renal transplant MHC matched to the original donor without additional immunosuppression. These results indicate that the mechanism of tolerance to primarily vascularized renal allografts involves modification of the host's immune system by the first transplant. To assess the possibility that "graft adaptation" is also involved in the maintenance of tolerance, we retransplanted class I-disparate kidneys from tolerant animals into naive recipients MHC matched to the original recipient. Three of 4 such transplants were rejected acutely, while one animal demonstrated a markedly prolonged survival, but also eventually rejected. These results, therefore, demonstrate that: (1) graft adaptation is not required in order to maintain tolerance; (2) graft acceptance involves induction of systemic tolerance; and (3) graft adaptation may participate in kidney graft prolongation but is not sufficient to transfer tolerance to a secondary host.

Removal of anti-porcine natural antibodies from human and nonhuman primate plasma in vitro and in vivo by a Galalpha1-3Galbeta1-4betaGlc-X immunoaffinity column.

BACKGROUND: Natural antibodies (NAbs) against a terminal alpha1-3 galactosyl (alphaGal) epitope have been identified as the major human anti-pig NAbs. METHODS AND RESULTS: We used two synthetic alphaGal trisaccharides--type 6 (alphaGal6) and type 2(alphaGal2)--linked to an inert matrix to remove NAbs from human plasma in vitro. Flow cytometry indicated that an average of 85% of the NAb binding activity was depleted by adsorption with alphaGal6. By measuring the binding of NAbs to pig peripheral blood mononuclear cells and bone marrow cells, we demonstrated that alphaGal6 was more effective than alphaGal2 in removing NAbs, and the combination of alphaGal6 + alphaGal2 did not further increase removal of NAbs. The specificity of the removal of NAbs (IgM and IgG) reactive with the alphaGal epitope by alphaGal6 matrix was shown by enzyme-linked immunosorbent assay. In vivo studies in nonhuman primates compared plasma perfusion through a alphaGal6 immunoaffinity column with hemoperfusion through a pig liver for changes in blood pressure, hematocrit, platelets, and NAb adsorption. CONCLUSIONS: Both methods reduced the level of anti-pig IgM and IgG xenoreactive antibodies to nearly background, but column perfusion caused less hypotension and reduction in platelets than liver perfusion. Four pig kidneys transplanted into monkeys after column perfusion did not undergo hyperacute rejection, remaining functional for 2-10 days, with a mean functional period of 7 days, demonstrating that a pig kidney can support renal function in a primate.

Molecular and cellular events implicated in local tolerance to kidney allografts in miniature swine.

Long-term tolerance to class I-mismatched renal allografts can be induced in miniature swine by treatment with a short course of cyclosporine (CsA). Kidney recipients treated with CsA and untreated control kidney recipients both demonstrated infiltration of the transplanted kidney by mononuclear cells, which reached a maximum between postoperative days 8 and 11. Recipients that did not receive the tolerizing regimen rejected their grafts between postoperative days 8 and 12 in this model. The kinetics of cytokine gene expression, including interleukin (IL)-1alpha, IL-1beta, IL-2, IL-6, IL-10, tumor necrosis factor, and interferon-gamma (IFN-gamma), within the grafted kidney of rejector and acceptor animals, were determined using Northern blot hybridization. A strong correlation between rejection and up-regulation of the IFN-gamma gene was observed, whereas animals with long-term tolerance showed low levels of IFN-gamma, but high levels of IL-10 gene transcription. None of the other cytokine genes demonstrated a reproducible pattern of expression that correlated with acceptance/rejection of allografts. Analysis of transcription patterns of cytokine genes in mononuclear cells purified from renal grafts confirmed the initial observations made on biopsies. The phenotype of graft-infiltrating cells (GIC) showed a dominance of CD8+ cells, with an average of 66% single-positive cells and 19% CD4/CD8 double-positive cells, compared with 30% and 14%, respectively, for peripheral cells. Predominance of CD8+ GIC was dictated neither by the MHC antigen disparity nor the rejector/acceptor status. These results, therefore, suggest that GIC represent a regulated combination of mononuclear cells producing local immune mediators that, in part, control the fate of allografts in this large animal model.

Long-term discordant xenogeneic (porcine-to-primate) bone marrow engraftment in a monkey treated with porcine-specific growth factors.

BACKGROUND: Mixed allogeneic hematopoietic chimerism has previously been reliably achieved and shown to induce tolerance to fully MHC-mismatched allografts in mice and monkeys. However, the establishment of hematopoietic chimerism has been difficult to achieve in the discordant pig-to-primate xenogeneic model. METHODS: To address this issue, two cynomolgus monkeys were conditioned by whole body irradiation (total dose 300 cGy) 6 and 5 days before the infusion of pig bone marrow (BM). Monkey anti-pig natural antibodies were immunoadsorbed by extracorporeal perfusion of monkey blood through a pig liver, immediately before the intravenous infusion of porcine BM (day 0). Cyclosporine was administered for 4 weeks and 15-deoxyspergualin for 2 weeks. One monkey received recombinant pig cytokines (stem cell factor and interleukin 3) for 2 weeks, whereas the other received only saline as a control. RESULTS: Both monkeys recovered from pancytopenia within 4 weeks of whole body irradiation. Anti-pig IgM and IgG antibodies were successfully depleted by the liver perfusion but returned to pretreatment levels within 12-14 days. Methylcellulose colony assays at days 180 and 300 revealed that about 2% of the myeloid progenitors in the BM of the cytokine-treated recipient were of pig origin, whereas no chimerism was detected in the BM of the untreated control monkey at similar times. The chimeric animal was less responsive by mixed lymphocyte reaction to pig-specific stimulators than the control monkey and significantly hyporesponsive when compared with a monkey that had rejected a porcine kidney transplant. CONCLUSION: To our knowledge, this is the first report of long-term survival of discordant xenogeneic BM in a primate recipient.

Expression of an allogeneic MHC DRB transgene, through retroviral transduction of bone marrow, induces specific reduction of alloreactivity.

BACKGROUND: Transfer of MHC class II genes, through allogeneic bone marrow (BM) transplantation, induced long-lasting acceptance of renal allografts in miniature swine. To adapt this approach to the clinic, we have now examined whether somatic transfer of allogeneic class II DR genes, into otherwise autologous bone marrow cells (BMC), can provide the matching required for inducing immune tolerance. METHODS: Autologous BMC were transduced ex vivo with recombinant retroviruses for allogeneic DRB followed by BM transplantation. The recipients were then challenged with kidney allografts solely matched to the DRB transgene. RESULTS: Five miniature swine received autologous BMC conditioned with growth factors and transduced with recombinant retrovirus vectors containing allogeneic (n=4) or syngeneic (n=1) class II DRB genes and a drug-resistance marker. Expression of retrovirus-derived products in BM-derived cells was demonstrated by the detection of drug-resistant colony-forming progenitors and the presence of DRB retrovirus transcripts in peripheral cells. Analysis of selective mixed lymphocyte reaction responses to DR or DQ antigens indicated decreased reactivity toward the transduced DR gene product. Among all of the animals receiving fully mismatched kidney allografts, but with DRB matched to the transduced DRB, the one with the highest gene transduction rate showed stable allograft function and essentially normal renal histology for 2.5 years. A control animal, which received a syngeneic DRB gene, rejected its kidney allograft in 120 days after an earlier rejection crisis. CONCLUSIONS: These studies demonstrate that allogeneic MHC gene transfer into BM provides a new strategy for inducing tolerance across MHC barriers.

Pig to monkey bone marrow and kidney xenotransplantation.

BACKGROUND: The intensity of discordant xenograft cellular rejection makes it unlikely that safe doses of immunosuppressive drugs will alone be sufficient to permit long-term survival. We have therefore concentrated our efforts on establishing tolerance to xenogeneic organs through lymphohematopoietic chimerism and the elimination of preformed natural antibodies (nAbs). METHODS: Here we report the most recent series of 11 technically successful porcine to nonhuman primate transplantation procedures. In eight experimental animals induction therapy consisted of (1) 3 x 100 cGy nonlethal whole body irradiation (day -6 and day -5) to all animals, (2) horse anti-human thymocyte globulin (day -2, day -1, and day 0) to seven of the animals, (3) 700 cGy thymic irradiation (day -1) to five of the animals, and (4) pig bone marrow infused on day 0 (2-9 x 10(8)/cells/kg). On day 0, just before the renal xenograft, the recipient was splenectomized, and antipig nAbs were removed by means of perfusion of the monkey's blood through either a pig liver (n = 6) or a Gal-alpha (1,3)-Gal adsorption column (n = 5). There control animals did not receive this pretransplantation induction therapy but did undergo hemoperfusion and posttransplantation immunosuppression identical to the experimental animals. All 11 recipients were treated after transplantation with cyclosporin A and 15-deoxyspergualin. Recombinant pig-specific growth factors (interleukin-3 and stem cell factor) were given to six experimental animals from day 0 until the termination of the experiment. RESULTS: Analysis of recipients' sera by means of flow cytometry indicated the effective removal of immunoglobulin M and immunoglobulin G nAbs by either liver perfusion or column adsorption. In the eight experimental animals, nAb titers remained low until death (up to 15 days), but in the three control animals nAb titers increased substantially with time. The longest surviving recipient maintained excellent kidney function with creatinine levels at 0.8 to 1.3 mg/dl throughout its course. Death occurred at day 15 from complications caused by a urinary leak and pancytopenia. Histologic examination of the xenograft revealed only focal tubular necrosis and cytoplasmic vacuolization, with trace amounts of fibrin and C3 in peritubular capillaries. In this animal a fraction of the peripheral blood cells (3%) at day 7 were of pig origin as detected by pig-specific monoclonal antibodies. In addition, colony-forming assays performed on a bone marrow biopsy specimen taken at day 14 indicated that approximately 30% of the relatively few myeloid progenitors detected were of swine origin. CONCLUSIONS: We have demonstrated that our protocol is effective in the prevention of hyperacute rejection and in the maintenance of excellent function of the renal xenograft for up to 15 days. These results also indicate that at least short-term engraftment of the xenogeneic donor bone marrow cells is possible to achieve in this discordant large animal combination. Longer survivals will be required to assess the possible effect of this engraftment on induction of tolerance.