Manei Oku

Carbon monoxide reduces pulmonary ischemia–reperfusion injury in miniature swine

OBJECTIVES Carbon monoxide is produced endogenously as a by-product of heme catalysis and has been shown to reduce ischemia-reperfusion injury in a variety of organs in murine models. The aims of this translational research were to establish an in situ porcine lung model of warm ischemia-reperfusion injury and to evaluate the cytoprotective effects of low-dose inhaled carbon monoxide in this model. METHODS Warm ischemia was induced for 90 minutes by clamping the left pulmonary artery and veins in 8 Clawn miniature swine (Japan Farm CLAWN Institute, Kagoshima, Japan). The left main bronchus was also dissected and reanastomosed just before reperfusion. Four animals were treated with inhaled carbon monoxide at a concentration of approximately 250 ppm throughout the procedure. Lung function and structure were serially accessed via lung biopsy, chest x-ray films, and blood gas analysis. RESULTS Carbon monoxide inhalation dramatically decreased the lung injury associated with ischemia and reperfusion. Two hours after reperfusion, the arterial oxygen tension of the carbon monoxide-treated group was 454 +/- 34 mm Hg, almost double the arterial oxygen tension of the control group (227 +/- 57 mm Hg). There were fewer pathologic changes seen on chest x-ray films and in biopsy samples from animals in the carbon monoxide-treated group. Animals in the carbon monoxide-treated group also had fewer inflammatory cell infiltrates and a markedly smaller increase in serum concentrations of the proinflammatory cytokines interleukin 1beta, interleukin 6, and high-mobility group box 1 after ischemia-reperfusion injury. CONCLUSIONS The perioperative administration of low-dose inhaled carbon monoxide decreases warm ischemia-reperfusion injury in lungs in miniature swine. This protective effect is mediated in part by the downregulation of proinflammatory mediators.

Human neutrophil peptide-1 aggravates dextran sulfate sodium-induced colitis.

Background: Human neutrophil peptide (HNP)‐1, HNP‐2, and HNP‐3 (HNP‐1–3) are useful biomarkers for ulcerative colitis (UC). The precise roles of these peptides in UC are poorly understood, however. The aim of this study was to determine whether HNP‐1 affects disease activity in mice with experimental colitis. Methods: Experimental colitis was induced in BALB/c or severe combined immunodeficiency (SCID) mice using dextran sulfate sodium (DSS). Mice were subsequently treated intraperitoneally with HNP‐1 (100 &mgr;g/day) or phosphate‐buffered saline (PBS) from day 4 to day 6. The severity of colitis was evaluated based on a disease activity index, histologic score, and cytokine expression. Results: Body weight and colon length significantly decreased and the disease activity index score, histologic score, and myeloperoxidase activity significantly increased in HNP‐1‐treated BALB/c mice compared with PBS‐treated mice. Interferon‐&ggr; and tumor necrosis factor‐&agr; levels in colon culture supernatants‐derived HNP‐1‐treated mice were also significantly higher, and interleukin (IL)‐1&bgr; levels tended to increase in response to HNP‐1. In addition, treating SCID mice with HNP‐1 aggravated DSS‐induced colitis and IL‐1&bgr; levels in colon culture supernatants from these mice were significantly higher than in cultures obtained from control mice. Furthermore, in both BALB/c and SCID mice increased recruitment of F4/80‐positive macrophages was observed in the inflamed colonic mucosa following HNP‐1 injections. Conclusions: High concentrations of HNP‐1 aggravate DSS‐induced colitis, including upregulated expression of such macrophage‐derived cytokines as IL‐1&bgr;. These results indicate that high concentrations of HNP‐1–3 in patients with UC may exacerbate disease activity via increased cytokine production. (Inflamm Bowel Dis 2011;)

Beneficial effects of perioperative low-dose inhaled carbon monoxide on pulmonary allograft survival in MHC-inbred CLAWN miniature swine.

Background. We have recently reported that perioperative low-dose carbon monoxide (CO) inhalation decreases lung ischemia-reperfusion injury in miniature swine. The aims of this study were to establish a large animal model of pulmonary allograft rejection using polymerase chain reaction-typed major histocompatibility complex (MHC)-inbred CLAWN miniature swine and to examine the effects of CO on allograft survival. Methods. Eleven CLAWN miniature swines received fully MHC-mismatched lungs followed by 12 days of tacrolimus (days 0–11; blood level 35–45 ng/mL). Six recipients received tacrolimus alone (control group). Five recipients were additionally treated with inhaled CO (180 min for donors until graft harvest; 390 min for recipients until 2 hr after reperfusion). Results. All recipients treated with tacrolimus alone uniformly rejected their grafts by postoperative day 63 with development of cytotoxic antidonor antibodies. CO treatment was effective in prolonging allograft survival from a mean of 47±7 to 82±13 days (P=0.017), with one CO-treated animal maintaining function until postoperative day 120. Development of antidonor antibodies and donor-specific responsiveness by cell-mediated lympholysis and mixed lymphocyte reaction assays was delayed in animals that received CO therapy. Furthermore, serum concentrations of proinflammatory cytokines (interleukin-1&bgr; and -6) 1 day after transplant were significantly decreased in the CO-treated group. Conclusions. Fully MHC-mismatched lungs in CLAWN miniature swine were consistently rejected within 63 days, suggesting that this is a robust large animal model ideal for investigating mechanisms and treatment of lung rejection. Perioperative low-dose CO inhalation prolonged graft survival and inhibited antidonor antibody production and was associated with decreased proinflammatory mediators in this model.

Correlation of serum levels of complement C4a desArg with pathologically estimated severity of glomerular lesions and mesangial hypercellularity scores in patients with IgA nephropathy

The aim of the present study was to explore serum biomarkers for the pathology of IgA nephropathy using serum proteomics. The subjects were 57 patients with IgA nephropathy who were divided into two groups (group 1, n=25; group 2, n=32) and 14 healthy controls. Serum protein profiles were analyzed using the ProteinChip surface-enhanced laser desorption ionization (SELDI) system. Associations between signal intensities of proteins and histological findings in patients with IgA nephropathy were studied in group 1. Serum levels of a candidate biomarker protein (complement component C4a desArg) for IgA nephropathy were determined by enzyme linked-immunosorbent assay (ELISA) in group 2 and the relationships of these levels with histological findings were evaluated. There were significant differences in 93 protein signals between patients in group 1 and controls. Among these signals, 3 proteins at 8592, 8757 and 8806 m/z were significantly correlated with the severity of glomerular lesions. The protein at 8592 m/z was identified as C4a desArg and the signal intensity of 8592 m/z was strongly correlated with serum C4a levels, including C4a desArg, determined by ELISA. In addition, the serum levels of C4a (mainly C4a desArg) were significantly higher in patients in group 2 compared to controls and were correlated with the severity of glomerular lesions and with mesangial hypercellularity scores. In conclusion, the serum levels of complement C4a desArg are significantly higher in patients with IgA nephropathy compared to healthy controls and are significantly correlated with the severity of glomerular lesions and mesangial hypercellularity scores. Thus, serum C4a desArg is a potential biomarker for the severity of histological findings in patients with IgA nephropathy.

Hepatocyte growth factor sustains T regulatory cells and prolongs the survival of kidney allografts in major histocompatibility complex-inbred CLAWN-miniature swine.

Background. Although 12 days of high dose of FK506 permits the induction of tolerance of fully major histocompatibility complex (MHC)-mismatched allogeneic kidneys in MGH-miniature swine, we found that the same dose of FK506 is insufficient to induce such tolerance CLAWN-miniature swine. The CLAWN swine model was therefore chosen to study the potential immunoregulatory effects of human-recombinant hepatocyte growth factor (HGF). Methods. Ten CLAWN miniature swine received fully MHC-mismatched kidneys with 12 days (days 0–11) of FK506. Among these 10 recipients, 4 received 7 or 14 days of human-recombinant HGF starting at day 11. Graft function was assessed by daily serum creatinine and biopsies. Immunologic assays, including CD4/CD25 DP and FoxP3+ cells and development of antidonor antibodies, were performed. Results. Without HGF, all six CLAWN recipients developed severe acute rejection (Cre >9 mg/dL) within 3 weeks of transplantation. In contrast, in the four animals that received HGF for 7 to 14 days, stable renal function was observed for more than 50 days, although all grafts were ultimately rejected by postoperative day 80. Percent FoxP3+ cells in the CD4+CD25+ double positive population (T regulatory cells) in peripheral blood monocyte cells decreased in recipients with FK506 induction monotherapy while no reduction was observed in recipients treated with FK506 and HGF. Conclusion. This study demonstrates that in CLAWN swine treated with a dose of FK506 insufficient to induce tolerance across a fully MHC mismatched barrier, a short course of HGF may inhibit acute rejection while maintaining T regulatory cells. To our knowledge, this study provides the first evidence in a large animal transplantation model of HGFs immunoprotective effects.

Insulin-like growth factor binding protein-1 levels are increased in patients with IgA nephropathy.

The mechanisms underlying the pathogenesis of immunoglobulin A (IgA) nephropathy (IgAN) are not well understood. In this study, we examined gene expression profiles in kidneys obtained from mice with high serum IgA levels (HIGA mice), which exhibit features of human IgAN. Female inbred HIGA, established from the ddY line, were used in these experiments. Serum IgA levels, renal IgA deposition, mesangial proliferation, and glomerulosclerosis were increased in 32-week-old HIGA mice in comparison to ddY animals. By microarray analysis, five genes were observed to be increased by more than 2.5-fold in 32-week-old HIGA in comparison to 16-week-old HIGA; these same five genes were decreased more than 2.5-fold in 32-week-old ddY in comparison to 16-week-old ddY mice. Of these five genes, insulin-like growth factor (IGF) binding protein (IGFBP)-1 exhibited differential expression between these mouse lines, as confirmed by quantitative RT-PCR. In addition, serum IGFBP-1 levels were significantly higher in patients with IgAN than in healthy controls. In patients with IgAN, these levels correlated with measures of renal function, such as estimated glomerular filtration rate (eGFR), but not with sex, age, serum IgA, C3 levels, or IGF-1 levels. Pathologically, serum IGFBP-1 levels were significantly associated with the severity of renal injury, as assessed by mesangial cell proliferation and interstitial fibrosis. These results suggest that increased IGFBP-1 levels are associated with the severity of renal pathology in patients with IgAN.

Porcine CFSE mixed lymphocyte reaction and PKH-26 cell-mediated lympholysis assays

UNLABELLED Mixed lymphocyte reaction (MLR) and cell-mediated lympholysis (CML) are widely used to assess T cell responses. A major limitation of the traditional MLR and CML assays is that they require radioisotope labeling with (3)H for MLR and (51)Cr for CML, thereby limiting their use to laboratories with the capabilities to deal safely with these materials. Recently, flow cytometry with CFSE labeling has been used to detect cell division in rodent and human assays, and flow cytometry with PKH-26 labeling has been used to study cytotoxicity in murine models. Partially inbred miniature swine provide a unique large animal preclinical model for experimental transplantation, helping to bridge the gap between rodent and clinical studies. In this study, we modified the reported CFSE and PKH-26 labeling procedures for use with porcine cells, and established that these radioactive-free MLR and CML assays are comparable to traditional radioactive CML and MLR assays for assessing immunologic responses in miniature swine. To our knowledge, this is the first report that has directly compared the traditional CML/MLR with radiation-free CML/MLR in MHC-defined swine models. OBJECTIVE The aim of this study is to establish non-radiolabeled CSFE and PKH-26 labeling procedures for flow cytometry based CML/MLR assays that are comparable to radioactive CML/MLR assays in preclinical large animals.

ANTI-HIGH-MOBILITY GROUP BOX-1 ANTIBODIES REDUCE PULMONARY ISCHEMIA-REPERFUSION INJURY IN MINIATURE SWINE: 3191

H. Sahara1, M. Oku1, K. Setoyama1, M. Tasaki1, H. Nishimura1, O. Wunimenghe2, M. Okumi3, A. Shimizu4, K. Yamada5 1Xenotransplantation Surgery Unit, Frontier Science Research Center, Kagoshima University, Kagoshima/JAPAN, 2Frontier Science Research Center, Kagoshima University, Kagoshima/JAPAN, 3Urology, Osaka University Graduate School of Medicine, Suita/ JAPAN, 4, Nippon medical school, Tokyo/JAPAN, 5Transplantation Biology Research Center, Harvard Medical School, Boston/MA/ UNITED STATES OF AMERICA

S1697 Human Neutrophil Peptide-1 Has Cytotoxic Effects On Colon Cancer Cells and Aggravates Dextran Sulfate Sodium-Induced Colitis

IFN-γ plays an important role in the generation and perpetuation of mucosal inflammation in IBD. TL1A, a TNF superfamily member, synergizes with IL-12 and IL-18 to augment IFN-γ production in human T cells. Enhanced TL1A expression is detected in IBD with a strong TL1A SNP association reported in Crohns disease. We have previously shown TL1A expression is induced in peripheral monocytes and in-vitro derived dendritic cells (DC). The molecular mechanisms regulating TL1A expression during DC differentiation however, remain poorly defined. The human monocytic cell line, KG-1, provides a valuable model for dendritic cell differentiation. The round non-adherent KG1 cells develop a DC-like phenotype following activation by PMA/ionomycin. These cells become loosely adherent with long cytoplasmic projections. A concomitant induction of membrane TL1A and mRNA is observed with mRNA expression being detectable within 2 hours and peaking at 8 hours following stimulation. A strikingly similar kinetics of TL1A expression was likewise noted following PMA/ionomycin stimulation of peripheral monocytes. Further DC maturation of KG-1 cells following treatment with LPS results in additional enhancement of TL1A expression. Transient transfection of KG-1 with TL1A promoter-constructs up to 1.5 kb in length reveals up to 90-fold enhanced expression compared to cells transfected with the promoterless vector. EMSA analysis demonstrates PMA/ionomycin mediated upregulation of nucleo-protein binding to a putative TL1A NFκB binding site. Binding is competed by excess unlabeled wt TL1A NFκB, but not mutant oligonucleotide and is likewise attenuated following proteasomal inhibition by MG132. Our data suggests in the human monocytic KG-1 cell line, differentiation of immature cells towards a mature DC phenotype is associated with functional upregulation of surface TL1A expression and binding of NFκB to the TL1A promoter. These studies provide a molecular basis for future studies which may help elucidate the mechanism modulating TL1A expression during DC maturation.