Prope tolerance to heart allografts in mice associated with persistence of donor interleukin-10-transduced stem cells.

BACKGROUND: We previously reported that transduction of the human interleukin (IL)-10 gene into the total fetal liver stem cells (hIL-10-TFLs) of mice protects against their rejection in an allogeneic host. In this study, we explored the effects of these cells in two different models of organ transplantation. METHODS: Balb/c mice were sublethally irradiated before receiving skin or vascularized heterotopic heart grafts from C57Bl/6 mice. TFLs from C57Bl/6 mice transduced with hIL-10 or untransduced TFLs were injected on the day of transplantation into recipient mice once or also every 20 days thereafter. RESULTS: Skin allograft survival was prolonged for up to 17.8±0.6 days, vs. 9.0±0.4 days, in mice that received hIL-10-TFLs or untransduced TFLs, respectively. Allogeneic heart transplants survived for 86.25±13.8, 46.3±4.6, 28.1±6.1, or 11.5±0.6 days in mice that received repeated injections of hIL-10-TFLs, a single injection of hIL-10-TFLs, repeated injections of untransduced TFLs, or controls, respectively. Histological analyses of the grafts showed fewer inflammatory foci and CD8+ infiltrating cells in mice injected with hIL-10-TFLs compared with untreated mice. Expressions of H-2b and hIL-10 were found in several organs, including the thymus, liver, and the transplant, in hIL-10-TFL-injected mice. Finally, in hIL-10-TFL-injected mice, FoxP3 T cells were present inside the transplanted heart as late as 140 days after transplantation. CONCLUSIONS: In this study, we showed that repeated injections of hIL-10-TFLs are efficient in mitigating transplant rejection. This "prope" tolerance was associated with survival of donor hematopoietic cells in the host.

Bromohydrin pyrophosphate-stimulated Vgamma9delta2 T cells expanded ex vivo from patients with poor-prognosis neuroblastoma lyse autologous primary tumor cells.

Gamma/delta T cells (Vgamma9delta2) contribute to innate immunity and exert natural cytotoxicity against a variety of tumors. Using a synthetic phosphoantigen (Bromohydrin Pyrophosphate, BrHPP), we amplified Vgamma9delta2 T cells in vitro from neuroblastoma patients. In the presence of BrHPP and low doses of IL-2, robust proliferation of Vgamma9delta2 T cells was obtained from peripheral blood mononuclear cells (PBMC) harvested at diagnosis. Moderate proliferation was observed from PBMC harvested after stem cell transplantation, whereas modest levels of Vgamma9delta2 T cells were obtained from PBMC harvested after induction therapy. Proliferation was observed after a single in vitro stimulation with BrHPP. After 21 days in culture, Vgamma9delta2 T cells represented more than 80% of cultured cells (a 50-fold expansion from baseline). Moreover, BrHPP-amplified Vgamma9delta2 T cells from patients-expressed activation markers and were able to lyse allogeneic and autologous neuroblasts. This cytotoxic activity was gammadelta T-cell receptor-dependent. Clinical trials using BrHPP are warranted in patients with poor-prognosis neuroblastoma, either to expand patient-derived Vgamma9delta2 T cells ex vivo or by direct administration to in vivo to boost the pool of resident Vgamma9delta2 T cells in vivo.

Expression of sphingosine-1 phosphate receptor in rat renal ischemia-reperfusion injury.

Sphingosine-1 phosphate receptor (S1PR) has come to the fore as a mediator of extracellular signaling through its interaction with G-protein-coupled receptors, which results in the induction of peripheral T-cell depletion. The mechanisms involved in renal ischemia-reperfusion (I/R) injury are complex, but appear to involve the early participation of bone marrow-derived cells, such as T lymphocytes. In this study, we investigated the expression of SIPR in a rat model of renal I/R injury. By means of a laparotomy, the right kidney was harvested and the left renal artery and vein were clamped. The kidney was reperfused after 90 min of ischemia, and rats were sacrificed at 0, 3, 6, 12 and 24 h after reperfusion. S1PR expression was analyzed by immunohistochemistry, and was observed only in endothelial cells of the normal kidneys. From 0 to 3 h after reperfusion, S1PR expression gradually became stronger in endothelial cells, reaching its peak intensity at 3 h after reperfusion. Twelve hours after reperfusion, necrosis had extended throughout the ischemic kidney, and nearly all the tubular epithelial cells had been destroyed. From 3 to 12 h after reperfusion, S1PR expression gradually weakened. At 24 h after reperfusion, levels of S1PR expression had almost reached those of the normal kidneys. In conclusion, S1PR was found to be expressed in a rat model of renal I/R injury. Several hours after achieving the maximum level of S1PR expression, the maximum level of renal I/R injury was observed. These results suggest a relationship between S1PR and renal I/R injury.

Administration of the selective cyclooxygenase (COX)-2 inhibitor etodolac prolongs cardiac allograft survival in a mouse model.

Etodolac, a selective cyclooxygenase-2 (COX-2) inhibitor, is a non-steroidal anti-inflammatory drug. COX-2 is a key factor in the progression of inflammation. Although inflammation is an essential pathologic feature of cardiac allograft rejection, the role of COX-2 in this process remains unclear. The aim of this study was to investigate the expression of COX and the effects of etodolac in a mouse cardiac allograft model. Balb/c mice (H-2d) were used as recipients and C57BL/6 (H-2b) mice as heart donors. Heart function was evaluated daily after transplantation by regular abdominal palpation of the heart and by laparotomy in cases where the beating became weak. Rejection was defined as total cessation of cardiac muscle contraction. COX-2 expression was analyzed by immunohistochemistry. Cardiac isograft was well tolerated (>150 days, n=5), while non-treated cardiac allograft was rapidly rejected (mean 10.9±2.4, n=7). In the etodolac-treated cardiac allograft (10 mg/kg/day by hypodermic injection), survival was extended to 18.53±2.1 days (n=7). The necrotic area and the grade of COX-2 immunostaining were more significantly reduced in the etodolac-treated cardiac allograft than in the non-treated cardiac allograft at day 14. These results indicate that etodolac contributes to protection against rejection after heart transplantation. Etodolac could therefore be used to suppress graft rejection by means of its anti-inflammatory properties.

Telmisartan inhibits human urological cancer cell growth through early apoptosis.

Angiotensin II receptor blockers (ARBs) are widely used as hypertensive therapeutic agents. In addition, studies have provided evidence that ARBs have the potential to inhibit the growth of several types of cancer cells. It was reported that telmisartan (a type of ARB) has peroxisome proliferator-activated receptor (PPAR)-γ activation activity. We previously reported that the PPAR-γ ligand induces growth arrest in human urological cancer cells through apoptosis. In this study, we evaluated the effects of telmisartan and other ARBs on cell proliferation in renal cell carcinoma (RCC), bladder cancer (BC), prostate cancer (PC) and testicular cancer (TC) cell lines. The inhibitory effects of telmisartan and other ARBs (candesartan, valsartan, irbesartan and losartan) on the growth of the RCC, BC, PC and TC cell lines was investigated using an MTT assay. Flow cytometry and Hoechst staining were used to determine whether the ARBs induced apoptosis. Telmisartan caused marked growth inhibition in the urological cancer cells in a dose- and time-dependent manner. Urological cancer cells treated with 100 µM telmisartan underwent early apoptosis and DNA fragmentation. However, the other ARBs had no effect on cell proliferation in any of the urological cancer cell lines. Telmisartan may mediate potent anti-proliferative effects in urological cancer cells through PPAR-γ. Thus, telmisartan is a potent target for the prevention and treatment of human urological cancer.

Telmisartan as a peroxisome proliferator-activated receptor-γ ligand is a new target in the treatment of human renal cell carcinoma.

Angiotensin II receptor blockers (ARBs) are widely used as hypertensive therapeutic agents. However, it has been reported that Telmisartan (a type of ARB) additionally activates peroxisome proliferator-activated receptor (PPAR)-γ. We previously reported that PPAR-γ ligand induced the growth arrest of renal cell carcinoma (RCC) cells through apoptosis, and that Telmisartan had the potential to inhibit prostate cancer cell growth through apoptosis. In this study, we evaluated the effects of Telmisartan and other ARBs on cell proliferation in an RCC cell line using normal proximal tubular endothelial cells (PRTECs) and the human RCC (Caki-1) cell line. The effects of Telmisartan as well as of other ARBs (Candesartan, Valsartan, Irbesartan and Losartan) on RCC cell growth were examined by MTT assay. Flow cytometry and Hoechst staining were used to determine whether or not the ARBs induced apoptosis. Telmisartan caused marked inhibition in RCC cells in a concentration- and time-dependent manner. Treatment with 100 µM of Telmisartan induced early apoptosis and DNA fragmentation in the RCC cells, but not in the PRTECs. None of the other ARBs had an effect on cell proliferation in the RCC cells or the PRTECs. Telmisartan may mediate potent antiproliferative effects against RCC cells through PPAR-γ. Thus, Telmisartan is a potential target for prevention and treatment in RCC.

Expression of cysteinylLT1 receptor in human testicular cancer and growth reduction by its antagonist through apoptosis.

The metabolism of arachidonic acid by either cyclooxygenase or lipoxygenase is believed to play an important role in carcinogenesis. Leukotriene (LT) D4 is a pro-inflammatory mediator derived from arachidonic acid through various enzymatic steps, and 5-lipoxygenase is an important factor in generating LTD4. We investigated LTD4 receptor (cysteinylLT1 receptor; CysLT1R) expression in testicular cancer (TC), as well as the effects of the CysLT1R antagonist on cell proliferation in a TC cell line. CysLT1R expression in tissue from TC patients and normal testes (NTs) was detected using immunohistochemistry and RT-PCR. The effects of the CysLT1R antagonist on TC cell growth were examined using the MTT assay. Flow cytometry was used to determine whether or not the CysLT1R antagonist induces apoptosis. Immunohistochemistry indicated that CysLT1R expression was strong in all types of TC tissues, but very weak in NT tissues. The TC cell line expressed CysLT1R mRNA as detected by RT-PCR. MTT and flow cytometry revealed that the CysLT1R antagonist caused marked inhibition of TC cells through early apoptosis. In conclusion, CysLT1R was induced in TC. The results suggest that the CysLT1R antagonist may mediate potent anti-proliferative effects against TC cells. Thus, CysLT1R may become a new therapeutic target for the treatment of TC.

Telmisartan is a potent target for prevention and treatment in human prostate cancer.

Angiotensin II receptor blockers (ARBs) are widely used as hypertensive therapeutic agent. Recent studies have reported that ARBs have the potential to inhibit the growth of prostate cancer (PC) cells. Moreover, it was recently reported that Telmisartan (a kind of ARB) has peroxisome proliferator-activated receptor (PPAR)-gamma activation. We previously reported that PPAR-gamma ligand induces growth arrest of PC cells through apoptosis. In this study, we evaluated the effects of the Telmisartan and other ARBs on cell proliferation in several PC cell lines. We used normal prostate stromal cell (NPC), human hormone-refractory PC (PC3), androgen-independent PC (DU-145) and androgen-dependent PC (LNCaP) cell lines. Effects of Telmisartan and other ARBs (Candesartan, Valsartan, Irbesartan and Losartan) on PC cell growth were examined by MTT assay. Flow cytometry and Hoechst staining were used to determine whether or not ARBs induce apoptosis. Telmisartan caused marked inhibition of PC cells in concentration-dependent and time-dependent manner. PC cells with treatment of 100 microM Telmisartan induced early apoptosis and DNA fragmentation. However, NPC with treatment of 100 microM Telmisartan did not induce apoptosis or DNA fragmentation. Furthermore, other ARBs had no effect on cell proliferation in the PC cells and NPC. Telmisartan may mediate potent antiproliferative effects against PC cells through PPAR-gamma. Thus, Telmisartan is a potent target for prevention and treatment in PC.

The effect of neutrophil elastase inhibitor on acute tubular necrosis after renal ischemia-reperfusion injury.

In renal transplantation, ischemia-reperfusion (I/R) injury is a major cause of renal dysfunction. Activated neutrophils are reported to be closely involved in I/R injury after renal transplantation. Neutrophil elastase, a protease released from activated neutrophils, damages tubular endothelial cells. We investigated the beneficial effect of neutrophil elastase inhibitor (ONO-5046.Na) on renal I/R injury in rats. The study was conducted using 10 male Lewis rats (270-320 g) that were intravenously administered ONO-5046.Na (30 mg/kg before ischemia and after reperfusion) (group A) and control rats (group B) in a 90-min renal warm I/R injury model. Neutrophil elastase expression was analyzed using immunohistochemical staining, and the degree of renal dysfunction was evaluated using H&E staining and blood biochemistry. Neutrophil elastase was detected in tubular endothelial cells. The necrotic area extended to and encompassed nearly all the ischemic kidney within 12 h after reperfusion. The necrotic area and the grade of neutrophil elastase staining were significantly reduced in group A compared to group B. Significant differences in blood urea nitrogen and serum creatinine levels were observed. Survival rates over a 14-day period were examined. No rats survived for more than 4 days in group B. However, 2 of the 10 rats (20%) in group A survived for a 14-day period. To conclude, ONO-5046.Na inhibits neutrophil elastase and reduces acute tubular necrosis. Thus, it is a potent therapeutic agent for the control of renal I/R injury in renal transplantation.

The role of arachidonic acid in a rat renal ischemia-reperfusion injury model.

The metabolism of arachidonic acid by either the cyclooxygenase (COX) or the lipoxygenase (LOX) pathway generates eicosanoids, which have been implicated in the pathogenesis of a variety of human diseases, including ischemia-reperfusion (I/R) injury. Several reports have demonstrated that COX-2 and LOX inhibitors can reduce the damage caused by I/R injury. However, few reports have investigated the effects of COX and LOX expression on renal I/R injury, thus this study aimed to do so in a rat renal I/R injury model. The right kidney was harvested and the left renal artery and vein were clamped under laparotomy. The kidney was reperfused after 90 min of ischemia, and rats were sacrificed at 0, 1.5, 3, 5, 12 and 24 h after reperfusion. COX and LOX expression was analyzed by immunohistochemistry. COX-2 and 5- and 12-LOX expression was most intense in the endothelial cells at 3 and 5 h after reperfusion. The expression of COX-2 was stronger than that of 5- and 12-LOX. However, in the hours following reperfusion there were no significant variations in COX-1 expression. Our results demonstrate that COX-2 and LOX can be induced in a rat renal I/R injury model, and that the arachidonic acid pathways play a very important role in renal I/R injury.

Relationship between peroxisome proliferator-activated receptor-γ and renal ischemia-reperfusion injury.

The pathogenesis of ischemia-reperfusion (I/R) injury is known to involve cytokines and, in particular, surface adhesion molecules, the expression of which initiates inflammatory cell attachment. It has been suggested that peroxisome proliferator-activated receptor (PPAR)-γ is an important immunomodulatory factor as well as a regulator of fatty acid. In this study, we investigated the expression of PPAR-γ in a renal I/R injury rat model. The right kidney was harvested and the left renal artery and vein were clamped by means of a laparotomy. The kidney was reperfused following 90 min of ischemia. Rats were sacrificed at 0, 1.5, 3, 5, 12 and 24 h after reperfusion. PPAR-γ expression was analyzed by immunohistochemical staining using monoclonal antibody. PPAR-γ staining was weak in the endothelial cells, interstitial cells and collecting ducts in the normal kidney. From 1.5 to 5 h after reperfusion, PPAR-γ staining was strong. Twelve hours after reperfusion, necrosis had extended throughout the kidney, and nearly all the tubular epithelial cells were destroyed. However, 12 h after reperfusion, PPAR-γ staining was weak in the endothelial cells and its expression was moderate in the interstitial cells and collecting ducts. PPAR-γ was induced in the endothelial cells, including the mesangial cells, interstitial cells and collecting ducts in a rat model of renal I/R injury.

Prolonged survival of mouse skin allografts after transplantation of fetal liver cells transduced with hIL-10 gene.

INTRODUCTION: Interleukin-10 (IL-10) is a cytokine with a moleculary weight of 18 kDa, that was first identified as being produced by Th2 cells. It appears to have anti-inflammatory action by diminishing the production of pro-inflammatory cytokines produced by Th1 cells. IL-10 also regulates the differentiation and proliferation of several immune cells such as T cells, B cells, natural killer cells, antigen-presenting cells, mast cells and granulocytes. Recent data suggest, however, that IL-10 also has immunostimulatory properties with important consequences on the prognosis of disease. In this study, we demonstrate the importance of injection of hematopoietic fetal liver cells transduced with the human IL-10 (hIL-10) gene into an allogenic recipient subsequently transplanted with allogenic skin grafts. The immaturity of stem cells and precursor cells from fetal liver and their transient survival in the host, due to the production of hIL-10, may afford 'prope' tolerance. It also explains the lack of graft-vs.-host reaction (GvHR) and the delay in rejection of the specific donor skin grafts after virtual disappearance of donor hematopoietic cells. OBJECTIVES: Transduction of CBA hematopoietic fetal cells with the human IL-10 gene was used with the aim of inducing tolerance to donor antigen in recipient BALB/c mice. The observed effects were prolonged IL-10 production, donor cell chimerism in the host and delayed rejection of skin grafts from the specific donor strain. MATERIALS AND METHODS: To prevent or delay rejection of highly incompatible skin allografts, we used IL-10 gene transfer to establish chimerism with donor hematopoietic cells. Fetal liver cells from CBA mice were transduced with the human IL-10 gene and injected into BALB/c mice. RESULTS: Human IL-10, which is active in mice but does not cross-react with murine IL-10 in ELISA, was produced in vivo for 3 weeks. Donor cells were identified in the recipients during the same time period, on the basis of presence of the H-2 k gene and human IL-10 intracellular protein. Skin allografts from CBA or C57BL/6 mice survived for a mean of 9.5 days in recipient mice injected with non-transduced cells. In contrast, survival of CBA allograft was extended to 18.9+/-1.8 days in recipients injected with hIL-10-transduced fetal liver cells from CBA mice. Human IL-10 alone, without donor hematopoietic cell engraftment, did not prolong graft survival (9.6+/-1.2 days). CONCLUSIONS: IL-10 transduction of donor hematopoietic stem cells resulted in production of IL-10, cell engraftment and chimerism. Although full tolerance was not obtained at this level of donor cell development in the host, a specific and highly significant (P<0.001) prolongation of the survival of donor skin allografts was observed.

Expression of nonmuscle myosin heavy chain B (SMemb) in rat allogeneic kidney transplantation.

BACKGROUND/AIM: Investigators have reported that the nonmuscle myosin heavy chain B (SMemb) expression is enhanced in various types of glomerular diseases which develop into nephrosclerosis. In renal transplantation, transplant glomerulitis is often recognized during acute rejection. Therefore, we hypothesized that SMemb plays important roles in acute kidney rejection. To evaluate the role of SMemb in the development of kidney rejection, we examined its expression in rat kidney transplantation models. METHODS: We used Lewis rats as recipients and Wistar rats as donors. Group I: controls; group II: isograft model; group III: allograft model; group IV: as group III +10 mg/kg/day of ciclosporin A (CsA), and group V: as group III + CsA administration for 5 days postoperatively. Histopathological and SMemb immunohistochemical studies were completed. RESULTS: Clear enhancement of SMemb expression was found on day 3 in group III. In groups I, II, IV, and V, SMemb was faintly expressed in the glomerular cells. However, after termination of CsA treatment, the SMemb expression increased. The expression of SMemb was higher in the allograft model than in either isograft or CsA-treated models. CONCLUSIONS: Immunohistological investigations show that the SMemb expression was significant from an early stage at which histopathological reactions were hardly identifiable. This, therefore, could be useful for an earlier diagnosis of acute rejection.

Hepatocyte growth factor (HGF) as a rapid diagnostic marker and its potential in the prevention of acute renal rejection.

Several investigators have reported that hepatocyte growth factor (HGF) may be related to the protection or reconstruction of the kidney during acute renal rejection. To address this question, we examined the relationship between HGF and acute rejection in the following two studies with rat renal transplantation models. In study 1, the relationship between serum HGF levels and acute renal rejection in iso-, allo- and allograft with cyclosporine (CsA)-treated models was examined. In study 2, the focus was whether or not the injection of recombinant HGF can prevent acute renal rejection. Our results demonstrated that HGF levels were rapidly increased during acute rejection and that recombinant HGF effectively protected the kidney from acute rejection. These results suggest that HGF may be induced as a counter-response to the renal injury and that it can be used as a reliable indicator for the diagnosis of acute rejection. We suggest that recombinant HGF suppresses the onset of the pathological changes of acute rejection.