Immunomodulation of fetal pig islet-like cell clusters by gamma-irradiation

Pretreatment of tissues to reduce their immunogenicity is an attractive option, and exposure of donor islets to gamma-irradiation has previously been shown to result in their prolonged survival when transplanted into rodents. Fetal pig islet-like cell clusters (ICCs) are currently under trial as a potential xenogeneic tissue for the treatment of type 1 diabetes. The purpose of this study was to examine in vivo and in vitro the immunomodulatory effects of gamma-irradiation on ICCs in a xenogeneic situation. The immunogenicity of gamma-irradiated ICCs was determined in a mixed islet lymphocyte culture (MILC), in which fetal pigs ICCs were able to stimulate human peripheral blood mononuclear cells (PBMCs). Exposure of the ICCs to gamma-irradiation significantly reduced their ability to stimulate PBMCs in a MILC when 10 Gy but not lower doses of irradiation were applied. However, this effect of gamma-irradiation was variable and was present only in those experiments in which the stimulation index was relatively low. Gamma-irradiation was toxic to ICCs in vitro, causing a reduction in the [³H]-thymidine incorporation of 82–94% at 5–20 Gy. This toxic effect of gamma-irradiation was also demonstrated in vivo: the insulin content of ICCs beneath the renal capsule in SCID mice treated with 5–20 Gy significantly was reduced (P < 0.05) 6 weeks after transplantation. Exposure of ICCs to gamma-irradiation (2.5 Gy) alone in vitro or in combination with injection of cyclosporine (12.5 mg/kg per day) did not prevent the rejection of ICCs transplanted beneath the renal capsule of BALB/c mice. We conclude that gamma-irradiation is toxic to fetal pig ICCs at a higher dose and at a lower dose, alone or in combination with cyclosporine, and was unable to prolong discordant islet xenograft survival in mice.     

Delayed type hypersensitivity-associated cytokines in islet xenotransplantation: limited efficacy of interleukin-2- and tumor necrosis factor-alpha-blockade in interferon-gamma receptor-deficient mice

Abstract: To investigate the role of interferon (IFN)- and tumor necrosis factor (TNF)- and their potential to replace each other in the process of fetal porcine islet-like cell cluster (ICC) xenograft rejection, mice with a targeted disruption of the IFN- receptor gene and wild-type controls were transplanted with fetal porcine ICCs under the kidney capsule and given post-transplant treatment with the TNF--inhibiting agent MDL 201,449A. Some of the MDL 201,449A-treated IFN- receptor-deficient mice received additional treatment with cyclosporinee (CsA). Evaluation of the xenografts was performed 7 days after transplantation (all groups), and in IFN- receptor-deficient mice treated with MDL 201 449 A, also 10 and 13 days after transplantation. On day 7 after transplantation, a few CD3⁺ cells were seen accumulated peripherally in the ICC xenograft. Moderate to abundant numbers of F4/80⁺ and Mac-1⁺ cells surrounded a few remaining ICCs present within the xenograft. Histochemical visualization of cyanide-resistant endogenous peroxidase activity for detection of eosinophils demonstrated only small numbers of eosinophils present within the xenograft by day 7 after transplantation. An increased amount of eosinophilic granulocytes was not found until day 10 after transplantation, i.e. at a time when ICC xenograft rejection has already been completed. However, two out of six IFN- receptor-deficient mice given post-transplant treatment with CsA and MDL 201,449A exhibited intact ICC xenografts with ICCs arranged in chords and duct-like structures on day 7 after transplantation. Taken together, findings in this study indicate that, in the pig-to-mouse model, IFN-, TNF-, and interleukin-2 seem to be of importance to fetal porcine ICC xenograft rejection. Nevertheless, in a majority of animals, other cytokines eventually substitute for the lack of IFN-, TNF- and interleukin-2.     

Dosing of MMF in combination with tacrolimus for steroiD-resistant vascular rejection in pediatric renal allografts

Abstract SteroiD-resistant vascular rejection was treated in seven adolescent renal allograft recipients using the combination of mycopheno-late mofetil (MMF) and tacrolimus (FK506). Since there are no published pediatric dose recommendations for MMF using this combination, trough concentrations and pharmacokinetic profiles were used for therapeutic drug monitoring. In order to keep the mycophenolic acid (MPA) concentrations between 2–5 μg/ml, mean MMF doses were reduced from 600 to 250 mg/m² b. i. d. Apparent clearance of MPA decreased from 5 to 1 ml/min per kg within 2 weeks. Pharmacokinetic monitoring revealed substantial variability among patients of both MMF and FK506. The MPA dose ranged from 178 to 1008 mg/m² per day to achieve an area under the curve (AUC) of 59.9 μg × h/ml ± 10.5 SD (range 49–65 μg). FK506 dose ranged from 1.3 to 8.8 mg/m² per day to achieve an AUC of 116 ng × h/ml ± 27 SD (range 83–145). We recommend adjusting MMF doses using therapeutic drug monitoring.     

Twenty-Year Experience With Heart Transplantation for Infants and Children With Restrictive Cardiomyopathy: 1986–2006

Idiopathic restrictive cardiomyopathy (RCM) is a rare cardiomyopathy in children notable for severe diastolic dysfunction and progressive elevation of pulmonary vascular resistance (PVR). Traditionally, those with pulmonary vascular resistance indices (PVRI) >6 W.U. × m² have been precluded from heart transplantation (HTX). The clinical course of all patients transplanted for RCM between 1986 and 2006 were reviewed. Preoperative, intraoperative and postoperative variables were evaluated. A total of 23 patients underwent HTX for RCM, with a mean age of 8.8 ± 5.6 years and a mean time from listing to HTX of 43 ± 60 days. Preoperative and postoperative (114 ± 40 days) PVRI were 5.9 ± 4.4 and 2.9 ± 1.5 W.U. × m², respectively. At time of most recent follow-up (mean = 5.7 ± 4.6 years), the mean PVRI was 2.0 ± 1.0 W.U. × m². Increasing preoperative mean pulmonary artery pressure (PA) pressure (p = 0.04) and PVRI > 6 W.U. × m² (χ²= 7.4, p < 0.01) were associated with the requirement of ECMO postoperatively. Neither PVRI nor mean PA pressure was associated with posttransplant mortality; 30-day and 1-year actuarial survivals were 96% and 86%, respectively. Five of the seven patients with preoperative PVRI > 6 W.U. × m² survived the first postoperative year. We report excellent survival for patients undergoing HTX for RCM despite the high proportion of high-risk patients.     

Effect of interleukin-10 on human anti-porcine xenogeneic cellular response in vitro

BACKGROUND: Pigs are being used as an alternative source of tissues for humans and we are interested in the xenotransplantation of fetal pig islet-like cell clusters (ICC) into type 1 diabetic patients. Interleukin-(IL) 10 is a Th2 cytokine with immunosuppressive properties that down-regulate the cell-mediated response. In this study, we evaluated the effects of recombinant human IL-10 on human anti-pig xenogeneic cellular response in mixed lymphocyte culture (MLC) and in mixed islet lymphocyte culture (MILC). METHODS: Human peripheral blood mononuclear cells as responder cells were cultured in one-way MLC with pig and human peripheral blood mononuclear cells as stimulant cells in xeno and allo-MLC, respectively, and also with fetal pig ICCs in MILC. IL-10 was added at the time of culture. RESULTS: The addition of IL-10 significantly inhibited the xeno-MLC (human anti-pig) in a dose-dependent manner, the percentage inhibition being 36, 60, and 73% at 1, 10, and 50 ng/ml, respectively. Inhibition in xeno-MLC was significantly lower than that of the allo-MLC (human anti-human) at all concentrations used, the percentage inhibition of the latter being 58, 84, and 92% at 1, 10, and 50 ng/ml, respectively. Further, the addition of IL-10 also significantly inhibited the proliferation of human peripheral blood mononuclear cells when they were cocultured with fetal pig ICCs, the inhibition being 59, 72, and 80% at 1, 10, and 50 ng/ml, respectively. IL-10 was not toxic to ICCs as determined by 3H-thymidine incorporation over 5 days culture. Preincubation of IL-10 with the pig stimulant cells or the human responder cells did not confer additional benefit in the inhibition of xeno-MLC. IL-10 needs to be present at the start or at an early stage (within 4 hr) in the xeno-MLC because if the addition of IL-10 was delayed by 4 hr, the effect was lost. Next, the production of cytokines was examined in MLC and MILC. In xeno-MLC, levels (pg/ml) of tumor necrosis factor-alpha (TNF-alpha) (163+/-17), interferon-gamma (IFN-gamma) (278+/-60), IL-5 (24+/-10), IL-6 (2959+/-923), and IL-10 (17+/-2) were produced in greater amounts than autologous controls (P<0.05). The levels of TNF-alpha, IFN-gamma, IL-6, and IL-10 but not IL-5 were significantly (P<0.05) lower in xeno-MLC than those produced in allo-MLC. All of these cytokines were also produced in MILC when human peripheral blood mononuclear cells (PBMC) were cocultured with ICCs, levels (pg/ml) being TNF-alpha (308+/-47), IFN-gamma (93+/-17), IL-5 (6.2+/-3), IL-6 (5649+/-421), and IL-10 (122+/-18). No detectable levels of IL-2 and IL-4 were produced in the MLC and in MILC. Addition of IL-10 significantly inhibited the production of TNF-alpha, IFN-gamma, IL-5, and IL-6 by 76, 96, 100, and 93%, respectively, in xeno-MLC. Addition of IL-10 also significantly (P<0.05) inhibited the production of TNF-alpha, IFN-gamma, IL-5, and IL-6 by 88, 91, 100, and 96%, respectively, in MILC. Exogenous addition of IL-2 was partially able to reverse the effect of IL-10 although addition of TNF-alpha had no effect on xeno and allo-MLC. Synergism was seen between IL-10 and cyclosporine in the inhibition of xeno and allo-MLC. CONCLUSION: Taken together, the results demonstrated that IL-10 has an immunomodulatory role to play in the inhibition of cellular immune responses associated with the xenotransplantation of fetal pig ICCs.     

Ultraviolet light immunomodulation of canine islets for prolongation of allograft survival

Ultraviolet (UV) light treatment of donor islets has been shown to be effective for the prolongation of islet allograft survival in rodent models. This study evaluated UV as an immunomodulator of canine islets. The effects of UV irradiation on islet secretory function in vitro revealed a trend of increasing basal insulin release with increasing doses of UV and a corresponding significant decrease in glucose-mediated insulin release (expressed as percentage of basal fractional insulin release) beginning at UV light exposures of 200-300 J/m2 (n = 3, P less than 0.05). Proliferative responses to UV-irradiated allogeneic peripheral blood leukocytes and islets were significantly decreased by 53-112% (P less than 0.05) in 27 of 29 mixed-lymphocyte cultures and by 35-74% (P less than 0.05) in 4 of 5 mixed-lymphocyte islet culture experiments, respectively, beginning at 200-600 J/m2. Autotransplantation of nonirradiated (n = 8) and irradiated islets (600 J/m2, n = 6) resulted in a 1-mo graft survival rate of 75% for the control group and 50% for the irradiated group. Allotransplantation of irradiated islets (600 J/m2) into either nonimmunosuppressed recipients (1 donor to 1 recipient, n = 8) or recipients of subimmunosuppressive doses of cyclosporin (2 donors to 1 recipient, n = 4) resulted in 100% rejection by day 10. In contrast, when islets were cultured for 24 h postirradiation and transplanted into cyclosporin-treated pancreatectomized recipients (2 donors to 1 recipient), 3 of 7 grafts were prolonged beyond day 10 to days 16, 26, and greater than 100.(ABSTRACT TRUNCATED AT 250 WORDS)     

Impact of low-dose rituximab on splenic B cells in ABO-incompatible renal transplant recipients

The purpose of this study was to assess the effect of a low-dose rituximab (RIT) at <375 mg/m² on B cells in the spleen and peripheral blood. Five renal transplant recipients received a single dose of RIT at 10, 15, 35, 150, or 300 mg/m² 3–13 days before transplantation. One patient who received the same immunosuppressive regimen except for RIT was also enrolled as a control. Splenectomy was performed at the time of transplantation in all patients. The B-cell count in the peripheral blood was analysed with a fluorescence-activated cell sorter using anti-CD19 antibodies, and the B cells in the spleen were analysed by immunohistochemistry using anti-CD20 and -CD79a antibodies. All but one dosage (10 mg/m²) of RIT completely eliminated B cells from the circulation within 30 days. Immunohistochemical examination of the spleen showed a marked reduction of B cells in the white pulps in all five recipients compared with that in the control patient. The observations in this study indicated that RIT has a potent effect of depleting B cells in the spleen and peripheral blood at low-doses of <375 mg/m².     

Functionally and phenotypically mature mouse CD8+ T cells develop in porcine thymus grafts in mice

Abstract: Mouse CD4⁺ T cells efficiently develop in fetal pig thymus (FPTHY) grafts and repopulate the periphery of T cell and NK cell-depleted, thymectomized (ATX) mice. However, efficient peripheral repopulation of mouse CD8⁺ T cells does not occur in these mice. We have therefore evaluated the maturation and function of mouse CD8 single positive (SP) thymocytes in fetal pig thymus and liver fragment (FP THY LIV) grafts. Phenotypic maturity, as measured by upregulated expression of TCR, class I MHC, and Qa-2, and downregulated expression of heat stable antigen (HSA) on CD8 SP cells in FP THY grafts, was similar to that in host thymi of euthymic control mice. Cytolytic T lymphocyte (CTL) activity of thymocytes from FP THY grafts was similar to that of thymocytes from host thymi of euthymic mice, indicating that functional maturation of CD8 SP cells had taken place in the grafts. Furthermore, similarly efficient deletion of Vβ5.1/5.2⁺ and Vβ11⁺ CD8 SP cells was observed in FP THY grafts as in host thymi of euthymic control mice. Similar percentages of Vβ6, Vβ7, and Vβ8.1/8.2 expressing cells were also detected among CD8 SP cells in FP THY grafts and host thymi of euthyrnic controls. Together, our results suggest that normal positive and negative selection occurs, and that mouse CD8⁺ cells can undergo normal functional and phenotypic maturation in FPTHY grafts. Thus, other explanations must be sought for the failure of CD8'cells to repopulate the peripheral lymphoid tissues of ATX, T cell-depleted, pig THYLIV-grafted mice.     

The Impact of Obesity on Long-term Outcomes in Liver Transplant Recipients—Results of the NIDDK Liver Transplant Database

The impact of obesity on outcomes following liver transplantation has been difficult to determine, in part due to the confounding effects of ascites on BMI. We evaluated the impact of pretransplant recipient obesity on outcomes following liver transplantation using the NIDDK Liver Transplantation Database. Pretransplant BMI, corrected for ascites, was categorized as underweight (BMI <18 kg/m²), normal weight (BMI 18–25 kg/m²), overweight (BMI 25.1–30 kg/m²), Class I obese (BMI 30.1–35 kg/m²), Class II obese (BMI 35.1–40 kg/m²) and Class III obese (BMI >40 kg/m²). Primary outcomes were patient and graft survival. Secondary outcomes included days in hospital and days in ICU. Data from 704 adult liver transplant recipients from the NIDDK LTD and a further 609 patients from the Mayo Clinic were analyzed. Early and late patient and graft survival was similar across all BMI categories. Correcting for ascites volume resulted in 11–20% of patients moving into a lower BMI classification. The relative risk for mortality increased by 7% for each liter of ascites removed. We conclude that corrected BMI is not independently predictive of patient or graft survival. Obesity, within the ranges observed in this study, should not be considered to be a contraindication to liver transplantation in the absence of other relative contraindications.     

CD4+ T cells, without CD8+ or B lymphocytes, can reject xenogeneic skin grafts

Abstract: Previous experiments have shown that rejection of xenogeneic skin grafts by mice is particularly dependent on CD4⁺ T cells. There are two possible explantations for this finding: either 1) "help" provided by CD4⁺ T cells is essential for CD8⁺ T cell-, B cell-, or NK cell-mediated effector mechanisms of rejection, or 2) CD4⁺ cells are themselves responsible for rejection, perhaps by some nonspecific effector mechanism. To examine these two hypotheses, we transplanted pig skin onto SCID mice and then reconstituted the mice with selected subpopulations of lymphocytes. Mice that did not received CD4⁺ T cells were unable to reject their xenografts, whereas those receiving CD4⁺ cells could do so in the absence of CD8⁺ cells or B cells and even when additionally depleted of NK cells by treatment with anti-Asialo GM1 antibody. Additional experiments were performed both in vivo and vitro to confirm the absence in test mice of CD4⁺ or CD8⁺ and B lymphocytes, respectively. These results suggest that CD4⁺ T cells are not only necessary for rejection of xenogeneic skin grafts by mice, but that they can do so without CD8⁺ cells or B cells, and probably without NK cells. Since CD4⁺ cells in mice have been shown to recognize xenogeneic antigens indirectly, this suggests that a nonspecific effector mechanism may be involved in the rejection of xenografts. In these experiments allogeneic skin grafts behave quite differently as they could not be rejected by this mechanism.     

Differentiation of transplanted microencapsulated fetal pancreatic cells

BACKGROUND: Fetal beta cells are a potential form of cell therapy for type 1 diabetes. To protect transplanted cells from cellular immune attack, microencapsulation using barium alginate can be employed. Whether microencapsulated fetal pancreatic cells will differentiate as occurs with nonencapsulated fetal pancreatic cells is presently unknown. It is suggested that such differentiation would occur in encapsulated cells, similar to previous experiments conducted using encapsulated embryonic stem cells. METHODS: Streptozotocin-induced diabetic severe combined immunodeficient mice were transplanted with 5,000 to 38,000 fetal pig islet-like cell clusters (ICCs) within barium alginate microcapsules of diameter 300, 600, or 1000 microm. Viability, insulin secretion, and content of encapsulated cells were measured prior to transplantation. Blood glucose levels (BGL) were measured twice weekly and porcine C-peptide monthly. Encapsulated cells were recovered from mice at 6 months posttransplantation for analysis. RESULTS: Encapsulated cells became glucose responsive and normalized BGL within 13 to 68 days posttransplantation, with 5,000 to 10,000 ICCs required. Microcapsule diameter did not affect the time required to achieve normoglycemia. BGL remained normal for the 6-month duration of the experiments. After removal of grafts at 25 weeks posttransplantation, glucose stimulated insulin secretion of the explants was enhanced 96-fold, insulin content was enhanced 34-fold, and the percentage of insulin and glucagon positive cells increased 10-fold and threefold, respectively, from the time of transplantation. CONCLUSIONS: This study demonstrates that fetal pancreatic cells differentiate and function normally when placed within barium alginate microcapsules and transplanted.     

Does Additional Doxorubicin Chemotherapy Improve Outcome in Patients with Hepatocellular Carcinoma Treated by Liver Transplantation?

The aim of this prospective randomized study was to determine whether additional doxorubicin chemotherapy improves outcome in patients with hepatocellular carcinoma (HCCA) treated by liver transplantation. Stratification parameters were tumor stage (UICC I-IVa), gender, age 50 years, α-fetoprotein 20 ng/mL, cirrhosis and HbsAg status. For pre-operative chemotherapy doxorubicin (15 mg/m²) was given biweekly, intra-operative chemotherapy was a single dose administered before surgical manipulation. Post-operative chemotherapy from day 10 was as given preoperatively for a total dosage of 300 mg/m². Outcome parameters were overall survival (OS) and disease-free survival. Of the 75 consecutive patients who received liver transplantation for treatment of HCCA, 62 patients were enrolled. Thirty-four patients were randomized in the chemotherapy group; 28 patients were in the control group and transplanted only. OS rates at 5 years were 38% in the chemotherapy group and 40% in the control group, disease-free survival rates at 5 years 43% and 53%, respectively. Tumor stage and vascular invasion were identified as independent risk factors for recurrence of disease. Doxorubicin chemotherapy did not improve organ survival and disease-free survival in patients undergoing liver transplantation for HCCA.     

The potential of local immunomodulation and tolerance induction in porcine islet xenotransplantation

Transplantation of pancreas or isolated islet cells is currently the only option to cure type 1 diabetes. The success of islet transplantation is still limited by the requirement of large numbers of high quality islets and the shortage of organ donors. Porcine islets are a promising cell source, but the intensive immunosuppressive regimen required to suppress rejection prevents the translation into clinical practice. We aimed to develop a novel method to inhibit the human‐anti‐pig immune reaction by the expression of immunomodulatory molecules in porcine beta cells. Thus, a transgenic pig was generated expressing LEA29Y – a second generation human CTLA4‐Ig fusion protein, which inhibits activation of T cells by CD80/CD86‐CD28 costimulation – under the control of the porcine insulin promotor. Islet‐like clusters (ICC) from neonatal pigs were isolated and transplanted under the kidney capsule of diabetic NOD‐scid‐IL2γnull (NSG) mice. After an in vivo maturation period mice transplanted with wildtype (wt) as well as with LEA29Y transgenic (tg) ICCs developed normal glucose homeostasis. Within 30 days after the transfer of human PBMCs 80% of NSG mice transplanted with wt‐ICCs developed diabetes indicating xenograft rejection. By contrast, LEA‐tg ICCs were completely protected from rejection in all animals (1). Immunohistochemistry revealed a massive intra‐islet T cell infiltration, which was absent in the LEA‐tg ICCs. This proof of principle study suggests that specific expression of immunomodulatory molecules in beta cells does not disturb beta cell function and may have the potential to modulate immune response locally at the transplantation site without systemic immunosuppression. To overcome the strong xenogeneic barrier of the human and cellular immune system a combination of LEA29Y with additional immunomodulatory factors may be required. Recently, Yi and coworkers demonstrated that the treatment with in vitro expanded regulatory T cells (Treg) prevents porcine islet rejection in humanized NSG mice by the suppression of the T cell‐mediated graft destruction (2). Other potential candidates to induce a state of tolerance against porcine islets currently under investigation are molecules targeting innate immunity and factors that prevent the reoccurrence of autoimmunity. Recent advances in xenotransplantation suggest that it may be possible to start with clinical trials using porcine neonatal or adult islets within the near future. References: 1. KLYMIUK N, VAN BÜRCK L, BÄHR Aet al. Xenografted islet‐cell‐clusters from INSLEA29Y transgenic pigs rescue diabetes and prevent immune rejection in humanized mice. Diabetes 2012; 61:1527–1532. 2. YI S, JI M, WU J et al. Adoptive transfer with in vitro expanded human regulatory T cells protects against porcine islet xenograft rejection via interleukin‐10 in humanized mice. Diabetes 2012; 61:1180–1191.     

Acute cell-mediated rejection in orthotopic pig-to-mouse corneal xenotransplantation

Abstract: Background:  To investigate the role of T cells and natural killer (NK) cells in mediating corneal xenograft rejection in a pig-to-mouse model.
Methods:  Pig corneas were orthotopically transplanted into BALB/c, C57BL/6, nude, severe combined immunodeficiency (SCID), and NOD/SCID/γc^null (NOG) mice. Graft survival was clinically assessed by slit-lamp biomicroscopy and median survival times (MST) were calculated. The rejected grafts were histologically evaluated using antibodies against CD4, CD8, NK1.1, and F4/80.
Results:  The pig corneal xenografts were acutely rejected by BALB/c and C57BL/6 mice (MST 9.0 days), while nude, SCID and NOG mice rejected pig corneas in a more delayed fashion (MST 16.0, 16.4, and 16.9 days, respectively). The majority of infiltrating cells found in rejected grafts in C57BL/6 mice were macrophages and CD4⁺ T cells, while CD8⁺ T cells and NK cells were rarely found. The grafts in nude mice had markedly decreased inflammatory infiltration with small numbers of macrophages and CD4⁺ T cells. Infiltration was even more modest in grafts in SCID and NOG mice.
Conclusions:  T cells play an important role in acute rejection of pig corneal xenografts in mice, although acute rejection is not solely the result of T-cell-mediated immunity. NK cells are less likely to be involved in the rejection process.     

Does tacrolimus cause more severe anemia than cyclosporine A in children after renal transplantation?

Abstract Initial reports indicated the possibility of severe anemia associated with tacrolimus (TC) therapy. We investigated the degree of anemia under TC treatment in comparison to cyclosporine A (CsA) treatment in children after renal transplantation. A cross-sectional analysis of 95 children successfully transplanted for at least 3 months was performed. Eighty-five children received CsA and 10 TC. TC-treat-ed patients were compared with CsA-treated patients who were matched according to age, gender, creatinine clearance, and time after transplantation. No patient received additional therapy with mycophenolate mofetil or azathioprine. The creatinine clearance of the whole group of transplanted children was 58 ml/min per 1.73 m². The patients within the matcheD-pair analysis had a lower creatinine clearance (TC 46 and CsA 48 ml/min per 1.73 m²). The hemoglobin was 10.3 g/dl for the TC-treated children and 10.4 g/dl among the CsA-treated patients. Numerically, EPO was higher and iron lower in the TC group than in the CsA group. Among children with functioning renal grafts, a correlation exists between Hb and creatinine clearance. A significant difference in the degree of anemia between TC- and CsA-treated children could not be found.     

Comparison of fetal porcine aggregates of purified beta-cells versus islet-like cell clusters as a treatment of diabetes

Fetal pig islet-like cell clusters (ICCs) have the potential to reverse diabetes 1-5 months after transplantation. In a fetal ICC, however, beta-cells constitute only 6-8% of the cells, in contrast to 65% in an adult pig islet. Attempts to purify fetal beta-cells from cell clusters and compare their function to that of ICCs have not been shown previously. Beta-cells were purified from ICCs isolated from the fetal pig pancreas. These were then aggregated and maintained in culture for 3 days. ICCs were isolated from fetal pig pancreas and allowed to round up in culture for 3 days. Transplantation of aggregates and ICCs (10,000 and 12,600, respectively) into diabetic immunoincompetent mice resulted in normoglycemia at 18 +/- 2 and 8 +/- 1 weeks, respectively (p = 0.0006). Removal of grafts after normalization of blood glucose levels resulted in rapid return of hyperglycemia in both groups. In conclusion, a purified population of immature beta-cells can be produced from the fetal pig pancreas. The reason these cells take longer than ICCs to reverse diabetes when transplanted is postulated to be because of the relative lack of precursor cells from which beta-cells differentiate. This finding may have implications for stem cell therapy, as other cell types, other than purified beta-cells, may be necessary for appropriate function in vivo.     

Hyperlipidemia Is Associated With Accelerated Chronic Kidney Disease Progression After Lung Transplantation

Hyperlipidemia is associated with faster progression of chronic kidney disease (CKD) in the general public. We sought to investigate this association after lung transplantation. Data was retrospectively collected on 230 lung recipients transplanted between January 1997 and December 2003. Estimated glomerular filtration rates (eGFR) and lipid levels were recorded at regular intervals posttransplant. Independent associations between lipid levels early posttransplant and pertinent renal endpoints were investigated. Baseline LDL was 110 ± 35 mg/dL and remained unchanged at 6 months. A faster decline in eGFR was seen in those with 6 month LDLs > versus < the mean level of 110 mg/dL (p = 0.05). By 6 months posttransplant eGFRs were lower in the 6 month LDL > versus < 110 mg/dL group (53 ± 23 vs. 62 ± 29 mL/min/1.73 m², p = 0.01), a difference that persisted at 60 months (39 ± 24 vs. 73 ± 57 mL/min/1.73 m², p = 0.05). On univariate analysis, a 6 month LDL in the highest quartile, i.e. >140 mg/dL, predicted faster progression to CKD, defined as declining to an eGFR < 30 mL/min/1.73 m² (HR 1.5, p = 0.01). This finding persisted in the multivariate Cox-proportional model (HR 1.4, p = 0.02). Hyperlipidemia predicts faster decline in renal function after lung transplant. Prospective trials are needed to confirm this finding.     

Inducible nitric oxide synthetase is expressed in adult but not fetal pig pancreatic islets

Abstract: Cytokine-induced expression of inducible nitric oxide synthetase (iNOS) and production of nitric oxide (NO) by pancreatic islet cells has been suggested as one potential mechanism for beta cell destruction. In this study, we investigated the role of iNOS and NO in islet primary non-function. Islets were assessed for their function, viability and expression of iNOS. Adult rat and pig islets isolated by collagenase digestion and fetal pig pancreas (FPP) grafts isolated by collagenase digestion or high oxygen culture were transplanted into C57BL6 mice and nude mice. iNOS protein was detected by immunohistochemistry. iNOS protein was found in normal rat and pig pancreas and adult rat and pig islets that were isolated by collagenase digestion and transplanted into either C57BL6 mice or nude mice. iNOS was not detected in fetal pig islet grafts, regardless of whether collagenase was used in the isolation process. In adult pig islet grafts, the presence of iNOS protein correlated with high levels of islet cell apoptosis and primary non-function. Despite the persistent presence of iNOS in rat islets, there was no evidence that it had a deleterious effect on rat islet viability, or function. Therefore, in isolated adult pig islets, there was a correlation between iNOS expression and apoptosis, suggesting that iNOS activation may be deleterious to the adult pig islets. However, other factors such as the fragility of the islet capsule may be equally important. By contrast, fetal pig islets did not express iNOS and this may be an important reason for their enhanced viability when compared with adult islet tissue.     

A comparison of the sensitivity of pig and human peripheral blood mononuclear cells to the antiproliferative effects of traditional and newer immunosuppressive agents

Difficulty in preventing rejection of fetal pig islet-like cell clusters (ICCs) transplanted into pigs using traditional forms of immunotherapy has been reported. An in vitro study of the efficacy of seven different immunosuppressive agents to inhibit proliferation of pig peripheral blood mononuclear cells (PBMC) was performed, and a comparison was made between the human and pig to determine if the efficacy of these agents differed between species. The efficacy of cyclosporine (CsA), azathioprine (Aza), methylprednisolone (MP), FK506, rapamycin (RAP), mycophenolate mofetil (MMF) and deoxymethylspergualin (MeDSG) to inhibit pig and human PBMC proliferation in mitogenic experiments using phytohaemagglutinin (PHA) as a stimulus was performed. Further, allogeneic pig mixed lymphocyte reactions (MLR) were used to determine the activity of these agents in a model more comparable to the allograft rejection process. It was found that pig PBMC stimulated with PHA or in a MLR were inhibited by the agents tested, with the exception of MeDSG that was ineffective in mitogenic experiments. The inhibitory effects of these agents differed between PHA and MLR, the respective (50% inhibitory concentration) IC50 values for pig PBMC being 1.7 and 0.08 microg/ml for CsA, 1.4 and 4.4 microg/ml for Aza, 0.11 and 0.002 microg/ml for MP, 3.0 and 2.8 ng/ml for FK506, 2.1 and 0.3 ng/ml for RAP and 10.8 and 454 ng/ml for MME Pig PBMC were less sensitive than human PBMC to the antiproliferative effects of CsA, Aza, FK506, RAP and MMF, but not MP on PHA stimulation, the ratio of the pig to human IC50 values being 19, 11, 13, 2.3, 1.4, and 0.4, respectively. These data suggest that the doses of most immunosuppressive agents administered to prevent rejection in pigs need to be higher than those used to achieve therapeutic benefit in humans.     

Central venous oxygen saturation for the diagnosis of low cardiac output in septic shock patients

Background: Simple diagnostic tests are needed to screen septic patients for low cardiac output because intervention is recommended in these patients. We assessed the diagnostic value of central venous oxygen saturation in the superior vena cava (ScvO₂) for detecting low cardiac output in patients with septic shock.
Methods: We conducted a prospective observational study in three general intensive care units (ICUs) of adult patients with septic shock, who were to have a catheter inserted for thermodilution measurement of cardiac index (CI_TD). Paired measurements of CI_TD and central venous oximetry values were obtained when the clinician first measured CI_TD.
Results: We included 56 patients with septic shock and a mean sequential organ failure assessment score of 12 (range 3–20). Baseline CI_TD was 3.5 l/min/m² (1.0–6.2) and ScvO₂ of 70% (33–87). The best cut-off of ScvO₂ for CI_TD>2.5 l/min/m² ( n =42) was a value ≥64% with positive and negative predictive values of 91% (95% confidence interval 79–98) and 91% (59–100), respectively. The diagnostic values were not improved by using instead central venous O₂ tension or the difference between arterial and central venous O₂ saturation.
Conclusions: This prospective, observational study found that a ScvO₂ measurement of ≥64% indicated CI_TD>2.5 l/min/m² in ICU patients with septic shock.