Treatment for hypergranulation at gastrostomy sites with sprinkling salt in paediatric patients.

OBJECTIVE: To estimate the efficacy of sprinkling salt treatment (SS) of hypergranulation at gastrostomy sites in paediatric patients. METHOD: A prospective case-series study of paediatric patients with hypergranulation at gastrostomy sites. About one-third of a 5ml-teaspoon of table salt was sprinkled over the tissue once a day, either by the doctors/nurses in the wards or doctors in the outpatient setting. Treatment was continued by the patients or carers in the home, until the hypergranulation was seen to flatten. The patients were followed up by health professionals once a month. RESULTS: Eight paediatric patients (seven females and one male) were included in the study. The hypergranulation decreased in size and became almost flat in all patients within 3 days to 2 months (median 7 days). Five patients had a recurrence of hypergranulation, but were treated successfully by repeated SS. Skin erosion was observed in one patient when SS was continued too long; therefore, the treatment protocol was altered so that the salt was irrigated 10 minutes after SS. A biopsy of the hypergranulation tissue was taken from a patient and salt was sprinkled on the tissue in a dish to simulate SS for histopathological examination. Histopathologically, hypergranulation treated by SS exhibited reduced interstitial oedema. CONCLUSION: The results of this study suggest that hypertonic environment induced by SS effectively reduced the oedematous hypergranulation tissue. SS is advantageous over traditional treatment such as silver nitrate in that SS is inexpensive and can be continued by the parents and repeated if necessary at home.

Hemoadsorption of high-mobility group box chromosomal protein 1 using a column for large animals.

BACKGROUND: High-mobility group box chromosomal protein 1 (HMGB1) has recently been identified as an important mediator of various kinds of acute and chronic inflammation. A method for efficiently removing HMGB1 from the systemic circulation could be a promising therapy for HMGB1-mediated inflammatory diseases. MATERIALS AND METHODS: In this study, we produced a new adsorbent material by chemically treating polystyrene fiber. We first determined whether the adsorbent material efficiently adsorbed HMGB1 in vitro using a bovine HMGB1 solution and a plasma sample from a swine model of acute liver failure. We then constructed a column by embedding fabric sheets of the newly developed fibers into a cartridge and tested the ability of the column to reduce plasma HMGB1 levels during a 4-hour extracorporeal hemoperfusion in a swine model of acute liver failure. RESULTS: The in vitro adsorption test of the new fiber showed high performance for HMGB1 adsorption (96% adsorption in the bovine HMGB1 solution and 94% in the acute liver failure swine plasma, 2 h incubation at 37°C; p < 0.05 vs. incubation with no adsorbent). In the in vivo study, the ratio of the HMGB1 concentration at the outlet versus the inlet of the column was significantly lower in swine hemoperfused with the newly developed column (53 and 61% at the beginning and end of perfusion, respectively) than in those animals hemoperfused with the control column (94 and 93% at the beginning and end of perfusion, respectively; p < 0.05). Moreover, the normalized plasma level of HMGB1 was significantly lower during perfusion with the new column than with the control column (p < 0.05 at 1, 2, and 3 h after initiation of perfusion). CONCLUSION: These data suggest that the newly developed column has the potential to effectively adsorb HMGB1 during hemoperfusion in swine.

Pharmacokinetics of mizoribine in adult living donor liver transplantation.

We investigated the pharmacokinetics of mizoribine in the acute phase after adult living donor liver transplantation (LDLT). Between February 2004 and October 2009, 16 recipients received immunosuppressive therapy that included mizoribine (100 to 200 mg/d) after undergoing LDLT. We determined the serum levels of mizoribine before (C0) and 3 (C3), 4 (C4), and 10 (C10) hours after administration on postoperative days 3, 7, and 21. We assessed area under the concentration time curve (AUC) (hour · µg/mL), normalized serum concentration (NSC) at C0 [concentration (µg/mL)/dose (mg/kg body weight)], and estimated glomerular filtration rate (eGFR). The mizoribine concentration showed increases at C3 and C4 followed by a decrease at C10 on all days. AUC was 4.3, 5.9, and 8.3 in the 200-mg/d dose group on days 3, 7, and 21, respectively. NSC at C0 increased for 3 weeks after LDLT. There was a significant correlation between the NSC at C0 and eGFR on day 21, but not on days 3 and 7. There were no correlations between the NSC at C0 and either aspartate aminotransferase, total bilirubin, albumin, trough cyclosporine, or trough tacrolimus on any day. The pharmacokinetics of mizoribine in the acute phase after LDLT seems to be affected by postoperative day and renal function.

Evidence of immune tolerance to blood group antigens in a case of ABO-incompatible pediatric liver transplantation.

In a 12-year-old patient with blood group O, who had received a partial liver graft 10 years ago from his father with blood group A, the levels of anti-A-specific antibodies (Abs) were persistently undetectable after the transplantation, while the levels of anti-B-specific Abs gradually increased and attained a plateau. Peripheral blood mononuclear cells (PBMCs) from this patient were engrafted into NOD/SCID mouse in order to investigate the immune response to donor-type blood group antigens. Even after sensitization with blood group-A erythrocytes, no anti-A Abs were detected in the serum samples of the mouse that received PBMCs from the blood group-O recipient of group-A liver allograft, however, immunoglobulins specific for antigens other than the A antigens were produced. Thus, we provide a possible evidence of immune tolerance to blood group antigens in this ABO-incompatible pediatric liver transplantation.

Discontinuation of living donor liver transplantation for PSC due to histological abnormalities in intraoperative donor liver biopsy.

Liver transplantation is the only curative treatment known to date for end-stage liver disease occurring as a result of primary sclerosing cholangitis (PSC). Here, we report a case in which living donor liver transplantation (LDLT) for PSC was cancelled because of histological abnormalities in intraoperative biopsy of the donor liver. The donor was the mother of the recipient, and her preoperative evaluation revealed no abnormalities. In the donor operation, the donor liver biopsy revealed expansion of the portal zone with lymphocytic infiltration and dense concentric fibrosis developed around a bile duct. These histological findings were identical to those of early-stage PSC; therefore, the LDLT was called off. The experience in this case suggests that preoperative liver biopsy may be useful to exclude first-degree relative donors with potential PSC prior to LDLT for PSC.

Neuropathy in miniature swine after administration of the mutant diphtheria toxin-based immunotoxin, pCD3-CRM9.

BACKGROUND: Effective in vivo T-cell depletion is a critical component of many transplantation tolerance protocols. We have previously demonstrated T-cell depletion in miniature swine using a CRM9-based CD3-immunotoxin, pCD3-CRM9. CRM9 is a mutant form of diphtheria toxin (DT) that binds less efficiently than wild-type DT to the DT receptor (proHB-EGF) of primates. In this report, we describe and characterize the dose-dependent neurotoxicity associated with CRM9-based immunotoxin administration in swine. METHODS: Miniature swine were treated with varying doses of pCD3-CRM9 followed by daily monitoring for symptoms of neuropathy, including limb weakness, paresis, sluggishness, and/or respiratory distress. Animals demonstrating severe respiratory distress were euthanized and peripheral nerve, spinal cord, and skeletal muscle tissue samples were obtained at autopsy for microscopic examination. Unconjugated CRM9 was administered to one animal to define its toxicity independent of the effects of T-cell depletion. RESULTS: Excellent T-cell depletion was obtained using doses of pCD3-CRM9 greater than 0.1 mg/kg. However, neurotoxicity was observed at these doses, as manifested by transient muscle weakness or paresis, which in some cases progressed to respiratory failure and death. Dorsal root ganglia samples revealed pathological changes typical of diphtheritic polyneuropathy. The animal receiving unconjugated CRM9 exhibited the same neurotoxic side effects as those receiving the pCD3-CRM9 conjugate. CONCLUSIONS: Administration of pCD3-CRM9 immunotoxin provides excellent T-cell depletion in miniature swine but is associated with significant dose-dependent neurotoxicity. A possible reason for CRM9-associated neurotoxicity in swine, but not primates, is suggested on the basis of a known amino acid difference in the exodomain of the DT receptor (proHB-EGF) of swine compared with that of primates.

Peripheral regulation of graft-versus-host alloreactivity in mixed chimeric miniature swine.

BACKGROUND: Despite the presence of circulating donor-derived T cells during the induction of mixed chimerism across MHC barriers in miniature swine, severe graft-versus-host disease was avoided in the majority of animals. In this study, we investigated the possible roles of recipient and donor lymphoid populations in the regulation of donor-anti-recipient alloreactivity. METHODS: Mixed chimerism across a full MHC-mismatch barrier was established in miniature swine using a high-dose allogeneic peripheral blood stem cell protocol. Peripheral blood mononuclear cells from mixed chimeric swine were co-cultured with naïve donor-matched responders and naïve recipient-matched stimulators in mixed lymphocyte reactions. RESULTS: Peripheral blood mononuclear cells from mixed chimeras inhibited donor-anti-recipient proliferation. This suppression was radioresistant to 25 Gy. Suppression of donor-anti-recipient alloreactivity was not observed in mixed lymphocyte co-cultures when donor-derived cells were added in the absence of recipient-derived cells. CONCLUSIONS: These results suggest an association between the presence of an active and relatively radioresistant cell population, demonstrable in vitro, and the regulation of graft-versus-host disease across MHC barriers in mixed chimeric miniature swine.

Skin-specific alloantigens in miniature swine.

BACKGROUND: The acceptance of skin allografts has historically been among the most challenging problems in the field of transplantation, attributed, at least in part, to the existence of antigens expressed by skin but not by other tissues. Many studies have suggested the existence of skin-specific antigens in rodents, but data in large-animal models are more limited. METHODS: We have recently developed protocols for attaining stable mixed hematopoietic chimerism in miniature swine, using MHC-matched donors and recipients. We have now assessed tolerance to donor-derived skin and cardiac allografts in these chimeric animals. RESULTS: Skin-graft rejection was seen in four of six animals receiving skin grafts taken from the respective hematopoietic donors. In the other two animals, donor-derived skin grafts survived indefinitely. No cardiac-allograft rejection was observed in mixed-chimeric animals that received heart transplants from their hematopoietic donors, even in animals that had already rejected skin allografts from the same donors. In all animals assessed, in vitro hyporesponsiveness to donor hematopoietic cells persisted. CONCLUSION: These findings support the concept that skin expresses immunogenic alloantigens that either are not expressed or are not immunogenic in cardiac or hematopoietic tissue.

Posttransplantation lymphoproliferative disease in miniature swine after allogeneic hematopoietic cell transplantation: similarity to human PTLD and association with a porcine gammaherpesvirus.

Posttransplantation lymphoproliferative disease (PTLD) is a major complication of current clinical transplantation regimens. The lack of a reproducible large-animal model of PTLD has limited progress in understanding the pathogenesis of and in developing therapy for this clinically important disease. This study found a high incidence of PTLD in miniature swine undergoing allogeneic hematopoietic stem cell transplantation and characterized this disease in swine. Two days before allogeneic peripheral blood stem cell transplantation, miniature swine were conditioned with thymic irradiation and in vivo T-cell depletion. Animals received cyclosporine daily beginning 1 day before transplantation and continuing for 30 to 60 days. Flow cytometry and histologic examination were performed to determine the cell type involved in lymphoproliferation. Polymerase chain reaction was developed to detect and determine the level of porcine gammaherpesvirus in involved lymph node tissue. PTLD in swine is morphologically and histologically similar to that observed in human allograft recipients. Nine of 21 animals developed a B-cell lymphoproliferation involving peripheral blood (9 of 9), tonsils, and lymph nodes (7 of 9) from 21 to 48 days after transplantation. Six of 9 animals died of PTLD and 3 of 9 recovered after reduction of immunosuppression. A novel porcine gammaherpesvirus was identified in involved tissues. Miniature swine provide a genetically defined large-animal model of PTLD with many characteristics similar to human PTLD. The availability of this reproducible large-animal model of PTLD may facilitate the development and testing of diagnostic and therapeutic approaches for prevention or treatment of PTLD in the clinical setting.

Mixed hematopoietic chimerism induces long-term tolerance to cardiac allografts in miniature swine.

BACKGROUND: Tolerance to cardiac allografts has not been achieved in large animals using methods that are readily applicable to human recipients. We investigated the effects of mixed hematopoietic chimerism on cardiac allograft survival and chronic rejection in miniature swine METHODS: Recipients were T-cell depleted using a porcine CD3 immunotoxin, and each received either of two nonmyeloablative preparative regimens previously demonstrated to permit the establishment of stable mixed hematopoietic chimerism across MHC-matched, minor antigen-mismatched histocompatibility barriers. Five to 12 months after the chimerism was induced, hearts from the original cell donors were heterotopically transplanted into the stable mixed chimeras. RESULTS: Cardiac allografts transplanted into untreated recipients across similar minor antigen barriers were rejected within 44 days (within 21, 28, 35, 39, 44 days among individual study subjects). In contrast, hearts transplanted into the mixed chimeras were all accepted long term ( > 153, > 225, > 286, > 362 days) without immunosuppressive drugs and developed minimal vasculopathy. CONCLUSIONS: Mixed hematopoietic chimerism, established in miniature swine using clinically relevant, non-myeloablative conditioning regimens, permits long-term cardiac allograft survival without chronic immunosuppressive therapy, significant vasculopathy, or graft-versus-host disease.

Mixed chimerism and tolerance without whole body irradiation in a large animal model.

Mixed hematopoietic chimerism may provide a treatment for patients with nonmalignant hematologic diseases, and may tolerize patients to organ allografts without requiring chronic immunosuppression. However, the toxicity of the usual conditioning regimens has limited the clinical applicability of this approach. These regimens generally include some level of whole body irradiation (WBI), which is thought to facilitate engraftment either by making room for donor hematopoietic stem cells or by providing sufficient host immunosuppression to enable donor cells to engraft. Here, we have established mixed chimerism across both minor and major histocompatibility barriers in swine, by using high doses of peripheral blood stem cells in the absence of WBI. After mixed chimerism was established, swine leukocyte antigen-matched (SLA-matched) donor skin grafts were tolerated and maintained for a prolonged period, whereas third-party SLA-matched skin was rejected promptly. Donor-matched kidney allografts were also accepted without additional immunosuppression. Because of its low toxicity, this approach has potential for a wide range of clinical applications. Our data may indicate that niches for engrafting stem cells are filled by mass action and that WBI, which serves to empty some of these niches, can be omitted if the donor inoculum is sufficiently large and if adequate host T-cell depletion is achieved before transplant.

Cytokine-mobilized peripheral blood progenitor cells for allogeneic reconstitution of miniature swine.

BACKGROUND: Because of the relative ease of acquisition, increased yield, and improved engraftment characteristics, mobilized peripheral blood progenitor (stem) cells (PBSCs) have recently become the preferred source for hematopoietic stem cell transplantation. In our laboratory, procurement of a megadose of PBSCs is necessary for on-going studies evaluating non-myelosuppressive transplant regimens for the induction of mixed chimerism and allograft tolerance. To exploit hematopoietic growth factor synergy, we have sought to combine growth factors with proven utility to improve PBSC mobilization and maximize our PBSC procurement through an automated collection procedure. METHODS: Mobilization characteristics of PBSCs were determined in 2-5-month-old miniature swine. Animals received either swine recombinant stem cell factor (pSCF, 100 microg/kg) and swine recombinant interleukin 3 (pIL-3, 100 microg/kg), administered intramuscularly for 8 days, or pSCF, pIL-3, and human recombinant granulocyte-colony stimulating factor (hG-CSF), at 10 microg/kg. Leukapheresis was performed beginning on day 5 of cytokine treatment and continued daily for 3 days. RESULTS: Collection of PBSCs from cytokine-mobilized animals via an automated leukapheresis procedure demonstrated a 10-fold increase in the number of total nucleated cells (TNC) (20-30 x 10(10) TNC) compared to bone marrow harvesting (2-3 x 10(10) total TNC). A more rapid rise in white blood cells (WBCs) was seen after administration of all three cytokines compared to pSCF and pIL-3 alone. An increase in colony-forming unit granulocyte-macrophage frequency measured daily from peripheral blood during cytokine treatment, was seen with the addition of hG-CSF to pSCF/pIL-3 correlating well with the rise in WBCs. Similarly, the addition of hG-CSF demonstrated a notable increase in the median progenitor cell yield from the 3-day leukapheresis procedure. Cytokine-mobilized PBSCs were capable of hematopoietic reconstitution. PBSCs mobilized with pSCF/pIL-3 were infused into an SLA-matched recipient conditioned with cyclophosphamide (50 mg/kg) and total body irradiation 1150 cGy. Neutrophil and platelet engraftment occurred on days 5 and 7, respectively, with minimal evidence of graft-versus-host disease. Complete donor chimerism has been demonstrated 331 days after transplant. CONCLUSIONS: Our preliminary results show that in this well-defined miniature swine model, recombinant swine cytokine combinations (pSCF, pIL-3 with or without hG-CSF) successfully mobilize a high yield of progenitor cells for allogeneic transplantation. Furthermore, these cytokine-mobilized PBSCs demonstrate the potential to reconstitute hematopoiesis and provide long-term engraftment in miniature swine.

Stable mixed chimerism and tolerance using a nonmyeloablative preparative regimen in a large-animal model.

Bone marrow transplantation (BMT) has considerable potential for the treatment of malignancies, hemoglobinopathies, and autoimmune diseases, as well as the induction of transplantation allograft tolerance. Toxicities associated with standard preparative regimens for bone marrow transplantation, however, make this approach unacceptable for all but the most severe of these clinical situations. Here, we demonstrate that stable mixed hematopoietic cell chimerism and donor-specific tolerance can be established in miniature swine, using a relatively mild, non-myeloablative preparative regimen. We conditioned recipient swine with whole-body and thymic irradiation, and we depleted their T-cells by CD3 immunotoxin-treatment. Infusion of either bone marrow cells or cytokine-mobilized peripheral blood stem cells from leukocyte antigen-matched animals resulted in stable mixed chimerism, as detected by flow cytometry in the peripheral blood, thymus, and bone marrow, without any clinical evidence of graft-versus-host disease (GvHD). Long-term acceptance of donor skin and consistent rejection of third-party skin indicated that the recipients had developed donor-specific tolerance.

An allelic non-histocompatibility antigen with wide tissue distribution as a marker for chimerism in pigs.

It is frequently useful in studies of transplantation to have available an antibody to a cell surface antigen, which is not itself responsible for transplant rejection. In this paper, we identify and describe such an antibody/antigen system in miniature swine. The monoclonal antibody, 1038H-10-9, was found to react to a pig allelic antigen (called PAA), found on a variety of pig cells and tissues, including peripheral blood mononuclear cells (PBMC), thymocytes, lymph node, bone marrow, and skin. Analysis for recipient sensitization against PAA was performed by in vitro cell-mediated lympholysis (CML) assay, mixed lymphocyte reaction (MLR) assay, antibody binding studies, and skin graft rejection patterns were examined. No evidence was found to indicate detection of PAA by any of these assays of alloreactivity. We therefore conclude that PAA is an allelic swine cell surface antigen, with wide tissue distribution, and that it is not a histocompatibility antigen. It should provide a powerful tool for studies of transplantation biology in miniature swine, such as identification and quantification of chimerism following organ transplantation.

Relationship between chimerism and tolerance in a kidney transplantation model.

The persistence of donor leukocytes in recipients of organ allografts has been associated with long-term graft acceptance. However, it remains unclear whether this peripheral donor cell microchimerism plays an active role in graft acceptance or is simply a consequence of the maintenance of sufficient immunosuppression to avoid rejection. A model of kidney transplantation between swine leukocyte Ag (SLA)-matched miniature swine, in which tolerance can be established with or without immunosuppressive treatment, has been used to study the correlation between donor leukocyte chimerism and kidney graft acceptance. SLA-identical kidney transplants were performed from animals positive for an allelic pig leukocyte Ag to animals negative for this marker. SLA-identical kidney transplant recipients given a 12-day course of cyclosporine (CyA) (n = 3) became tolerant, showing stable serum creatinine levels (1-2 mg/dl) after cessation of CyA treatment. Donor cell chimerism (0.2-0.7%) was present by FACS in all three animals with peak levels detected at 3 wk. Two control animals receiving SLA-identical kidney grafts without CyA also showed stable serum creatinine levels and became tolerant. However, in neither of these animals could donor leukocytes be detected in the peripheral blood beyond 1 wk following transplantation. In one additional control animal, ureteral obstruction occurred at day 10, and was associated with additional peripheral chimerism, presumably related to inflammation rather than to immune status. These results indicate that the persistence of donor cell chimerism is not a requirement for the maintenance of tolerance to organ allografts in this model.

Porcine kidney and heart transplantation in baboons undergoing a tolerance induction regimen and antibody adsorption.

BACKGROUND: Xenotransplantation would provide a solution to the current shortage of organs for transplantation. Our group has been successful in inducing tolerance in mice and monkey models of allogeneic transplantation. The present study attempts to extend the same tolerance-inducing regimen to a pig-to-baboon organ transplantation model. METHODS: Nine baboons underwent a conditioning regimen (consisting of nonmyeloablative or myeloablative whole body and thymic irradiation, splenectomy, antithymocyte globulin, pharmacologic immunosuppression and porcine bone marrow transplantation [BMTx]), which has previously been demonstrated to induce donor-specific allograft tolerance in monkeys. In addition, immunoadsorption of anti-alphaGal antibody (Ab) was performed. Four of the nine baboons received pig kidney transplants (KTx), and one also underwent repeat transplantation with an SLA-matched kidney. Two received heterotopic pig heart transplants (HTx). Three baboons underwent conditioning without organ transplantation for long-term studies of natural Ab kinetics. RESULTS: In the three baboons that received the conditioning regimen without an organ transplant, immunoadsorption reduced Ab by approximately 90%, but recovery of Ab to pretreatment level or higher occurred within 7 days. In contrast, the level of Ab remained low after organ transplant. No Ab to pig antigens other than alphaGal was detected in any baboon before or after BMTx, KTx, or HTx. No graft succumbed to hyperacute rejection. KTx function began to deteriorate within 3-6 days, with oliguria and hematuria progressing to anuria, and the kidneys were excised after 3, 6, 9, 11, and 14 days, respectively. One HTx ceased functioning at 8 days; the second baboon died with a contracting HTx at 15 days. Features of coagulopathy and thrombocytopenia developed in all six transplanted baboons (high D-dimer, prolonged prothrombin time and partial thromboplastin time, and falling fibrinogen) resulting in serious bleeding complications in two baboons, one of which died on day 9. Donor organs showed progressive acute humoral rejection with deposits of IgM, IgG, and complement; a focal mononuclear cellular infiltrate was also observed. The ureter was the earliest structure of the KTx affected by rejection, with progression to necrosis. CONCLUSIONS: This conditioning regimen prevented hyperacute rejection but was ineffective in preventing the return of Ab, which was associated with the development of acute humoral rejection with features of coagulopathy. No baboon developed anti-pig Ab other than alphaGal Ab. Further modifications of the protocol directed toward suppression of production of Ab are required to successfully induce tolerance to pig organs in baboons.

The role of surgery in advanced neuroblastoma.

Recently we performed multi-modal therapy including chemotherapy with bone marrow transplantation (BMT), surgery, and local irradiation in the treatment of advanced neuroblastoma. As a result, all Stage III patients survived, and 4 out of 8 Stage IV patients who underwent our original systemic gross complete resection achieved long-term survival, 4y8m, 5y8m, 5y6m and 5y5m, respectively. Long-term survival patients were treated by local irradiation, brain surgery and intensive induction chemotherapy before primary site operation, and when resolution of distant metastases was obtained, the primary site was resected completely, followed by postoperative supralethal chemotherapy with BMT. The purpose of this report is to elucidate the role of surgery in advanced neuroblastoma based on our clinical results.