Hideyoshi Toyokawa

Ex Vivo Application of Carbon Monoxide in University of Wisconsin Solution to Prevent Intestinal Cold Ischemia/Reperfusion Injury

Carbon monoxide (CO), a byproduct of heme catalysis, was shown to have potent cytoprotective and anti‐inflammatory effects. In vivo recipient CO inhalation at low concentrations prevented ischemia/reperfusion (I/R) injury associated with small intestinal transplantation (SITx). This study examined whether ex vivo delivery of CO in University of Wisconsin (UW) solution could ameliorate intestinal I/R injury. Orthotopic syngenic SITx was performed in Lewis rats after 6 h cold preservation in control UW or UW that was bubbled with CO gas (0.1–5%) (CO‐UW). Recipient survival with intestinal grafts preserved in 5%, but not 0.1%, CO‐UW improved to 86.7% (13/15) from 53% (9/17) with control UW. At 3 h after SITx, grafts stored in 5% CO‐UW showed improved intestinal barrier function, less mucosal denudation and reduced inflammatory mediator upregulation compared to those in control UW. Preservation in CO‐UW associated with reduced vascular resistance (end preservation), increased graft cyclic guanosine monophosphate levels (1 h), and improved graft blood flow (1 h). Protective effects of CO‐UW were reversed by ODQ, an inhibitor of soluble guanylyl cyclase. In vitro culture experiment also showed better preservation of vascular endothelial cells with CO‐UW. The study suggests that ex vivo CO delivery into UW solution would be a simple and innovative therapeutic strategy to prevent transplant‐induced I/R injury.

Heart allograft protection with low-dose carbon monoxide inhalation: effects on inflammatory mediators and alloreactive T-cell responses.

Background. Carbon monoxide (CO), a byproduct of heme catalysis, has lately received considerable attention as a regulatory molecule in cellular and biological processes. CO has been shown to provide potent protection against a variety of tissue injuries. We hypothesized in this study that low concentration CO would be beneficial for organ allografts, which frequently undergo several types of injury such as ischemia/reperfusion, alloimmune reaction, and inflammation Methods. The efficacy of low-dose CO was examined in a fully allogeneic LEW to BN rat heterotopic heart transplantation (HHTx) model. Recipients were kept in air or exposed to low-dose CO (20 ppm) for 14, 28, or 100 days after HHTx under short-course tacrolimus Results. CO treatment (d0–28, 0–100) was remarkably effective in prolonging heart allograft survival to a median of >100 from 45 days in the air-control group, with significant reductions of arteritis, fibrosis, and cellular infiltration, including macrophages and T cells. CO inhibited intragraft upregulation of Th1 type cytokines (IL-2, IFN&ggr;), proinflammatory mediators (IL-1&bgr;, TNF&agr;, IL-6, COX-2), and adhesion molecule. Shorter CO exposure in early (0–13d) and late (14–28d) posttransplant periods also prolonged graft survival, with a significant inhibition of inflammatory mediators Conclusions. These results show that low dose CO inhalation protects heart allografts and can considerably prolong their survival. CO appears to function via multiple mechanisms, including direct inhibition of Th1 type cytokine production and regulation of inflammatory responses.

Characterization of Transplanted Green Fluorescent Protein+ Bone Marrow Cells into Adipose Tissue

Following transplantation of green fluorescent protein (GFP)‐labeled bone marrow (BM) into irradiated, wild‐type Sprague‐Dawley rats, propagated GFP+ cells migrate to adipose tissue compartments. To determine the relationship between GFP+ BM‐derived cells and tissue‐resident GFP− cells on the stem cell population of adipose tissue, we conducted detailed immunohistochemical analysis of chimeric whole fat compartments and subsequently isolated and characterized adipose‐derived stem cells (ASCs) from GFP+ BM chimeras. In immunohistochemistry, a large fraction of GFP+ cells in adipose tissue were strongly positive for CD45 and smooth muscle actin and were evenly scattered around the adipocytes and blood vessels, whereas all CD45+ cells within the blood vessels were GFP+. A small fraction of GFP+ cells with the mesenchymal marker CD90 also existed in the perivascular area. Flow cytometric and immunocytochemical analyses showed that cultured ASCs were CD45−/CD90+/CD29+. There was a significant difference in both the cell number and phenotype of the GFP+ ASCs in two different adipose compartments, the omental (abdominal) and the inguinal (subcutaneous) fat pads; a significantly higher number of GFP−/CD90+ cells were isolated from the subcutaneous depot as compared with the abdominal depot. The in vitro adipogenic differentiation of the ASCs was achieved; however, all cells that had differentiated were GFP−. Based on phenotypical analysis, GFP+ cells in adipose tissue in this rat model appear to be of both hematopoietic and mesenchymal origin; however, infrequent isolation of GFP+ ASCs and their lack of adipogenic differentiation suggest that the contribution of BM to ASC generation might be minor.

Carbon monoxide induces hypothermia tolerance in Kupffer cells and attenuates liver ischemia/reperfusion injury in rats.

Ischemia/reperfusion (I/R) injury in liver grafts, which is initiated by cold preservation and is augmented by reperfusion, is a major problem that complicates graft quality, posttransplant patient care, and outcomes of liver transplantation (LT). Kupffer cells (KCs) play important roles in I/R injury; however, little is known about their changes during cold preservation. We examined whether a pretreatment with carbon monoxide (CO), a cytoprotective product of heme degradation, could influence KC activity during cold storage and protect liver grafts against LT‐induced I/R injury. In vitro, primary rat KCs were stimulated for 24 hours under hypothermic conditions (4°C, 20% O2), with lipopolysaccharide, or under hypoxic conditions (37°C, 5% O2) with or without a CO pretreatment. When rat KCs were exposed to hypothermic conditions, they produced reactive oxygen species (ROS), but they did not produce tumor necrosis factor α (TNF‐α) or nitric oxide. The preincubation of KCs with CO up‐regulated heat shock protein 70 (HSP70) and inhibited ROS generation. When liver grafts from donor rats exposed to CO (250 ppm) for 24 hours were transplanted after 18 hours of cold preservation in University of Wisconsin solution, HSP70 expression increased in these grafts versus control grafts, and serum aspartate aminotransferase and alanine aminotransferase levels as well as necrotic areas and inflammatory infiltrates were significantly reduced after LT. CO‐pretreated liver grafts showed less up‐regulation of TNF‐α and inducible nitric oxide synthase messenger RNA (mRNA) and reduced expression of proapoptotic B cell lymphoma 2–associated X protein mRNA, cleaved caspase‐3, and poly(adenosine diphosphate ribose) polymerase. In conclusion, the pretreatment of donors with CO ameliorates LT‐associated I/R injury with increased hepatic HSP70 expression, particularly in the KC population. Liver Transpl, 2011.

Relative contribution of direct and indirect allorecognition in developing tolerance after liver transplantation.

The interaction of donor passenger leukocytes and host leukocytes in recipient secondary lymphoid tissues during the early posttransplantation period is crucial in directing host immune reactions toward allograft rejection or acceptance. Responsible T cell clones could be activated through the direct and indirect pathways of allorecognition. We examined the role of the indirect pathway in liver transplantation (LT) tolerance by depleting host antigen‐presenting cells (APC) with phagocytic activity [e.g., cluster domain (CD)68+/CD163+ macrophages, CD11c+ dendritic cells (DC)] using liposome‐encapsulating clodronate (LP‐CL). After Lewis rat cell or liver graft transplantation, Brown Norway (BN) rat recipients pretreated with LP‐CL showed a significantly reduced type 1 helper T cell cytokine up‐regulation than control‐LP‐treated recipients. In the LT model, LP‐CL treatment and host APC depletion abrogated hepatic tolerance; Lewis liver grafts in LP‐CL‐treated‐BN recipients developed mild allograft rejection, failed to maintain donor major histocompatibility complex (MHC) class II+ leukocytes, and developed chronic rejection in challenged donor heart allografts, while control‐LP‐treated BN recipients maintained tolerance status and donor MHC class II+ hepatic leukocytes. Furthermore, in the BN to Lewis LT model, LP‐CL recipient treatment abrogated spontaneous hepatic allograft acceptance, and graft survival rate was reduced to 43% from 100% in the control‐LP group. In conclusion, the study suggests that host cells with phagocytic activity could play significant roles in developing LT tolerance. Liver Transpl 14:346–357, 2008.

3D-confocal structural analysis of bone marrow-derived renal tubular cells during renal ischemia/reperfusion injury

Bone marrow cells (BMC) have been shown to migrate into injured sites for parenchymal repair. However, the extent of BMC involvement is controversial. To determine whether and to what extent BMC contribute to renal parenchymal repair, we employed three-dimensional (3D) fluorescent confocal microscopy/video in renal warm and cold ischemia/reperfusion (I/R) injury using enhanced green fluorescent protein transgenic rats and their radiation chimeras. After induction of renal warm I/R injury in chimeras, BM-derived renal tubular cells were found in 2D microscopy as isolated single cells or clusters of 2–3 cells. Likewise, cold I/R injury resulted in host-derived tubular cells with frequencies ∼0.2%. However, stringent confocal microscopic analysis and 3D image construction revealed that BM-derived tubules identified in 2D images were frequently artifacts of overlapping cells separately stained with different markers. The actual frequency in 3D analysis was approximately one-fourth of that seen in 2D analysis. 3D confocal imaging precisely detected BM-derived tubular epithelial cells and could be useful to study BMC contribution to tissue repair.

The Difficulty of Eliminating Donor Leukocyte Microchimerism in Rat Recipients Bearing Established Organ Allografts

Background. Unequivocal eradication of donor leukocyte microchimerism from recipients of long-surviving organ transplants has never been reported. Here we describe a drastic attempt to accomplish this objective. Methods. In control experiments, a rank order of microchimerism and of associated donor specific nonreactivity was produced in Brown-Norway (BN) rats by transplantation of Lewis (LEW) liver, bone marrow cell (BMC) and heart allografts under a brief course of tacrolimus. The degree of microchimerism at 60 and 110 days was estimated with semiquanitative immunocytochemical and PCR techniques. Tolerance at 110 days was assessed in the different control groups by challenge transplantation of naïve LEW hearts. In parallel experimental groups, an attempt was made to eliminate microchimerism from the BN recipients. The animals were submitted at 60 days to 9.5-Gy total body irradiation (TBI), reconstituted immediately with naïve BN BMC, and tested for donor specific nonreactivity by LEW heart transplantation at 110 days. Results. After the TBI-reconstitution at 60 days, microchimerism was undetectable in BMC recipients at 110 days, significantly reduced in heart recipients, and least affected in liver recipients. Except in liver recipients, abrogation of LEW-specific nonreactivity was demonstrated by rejection of the priming grafts, or by rejection of the challenge heart grafts, and by in vitro immune assay. Conclusions. It is difficult to eliminate microchimerism in organ recipients once the donor cells have settled into tissue niches.