The role of cysteinyl-LT(1)receptor (CysLT(1)R) in renal ischemia-reperfusion injury.

The pathogenesis of ischemia-reperfusion (I/R) injury is known to involve cytokines and particularly surface adhesion molecules, the expression of which initiates the attachment of inflammatory cells. Renal I/R injury, a clinically important problem, is an invariable consequence of renal transplantation. The problem begins at the onset of acute tubular necrosis (ATN), when the transplantation includes a long ischemic interval or by use of a cardiac arrest donor's kidney. The cysteinyl leukotriene-1 (CysLT(1)), a potent lipid mediator in allergic disease, acts through the CysLT(1)R receptor. We researched the expression of CysLT(1)R in rat renal I/R injury as well as correlations with the degree of ATN. The right kidney was harvested and the left renal artery and vein were clamped at laparotomy. The kidney was reperfused after 90 minutes of ischemia; rats were sacrificed at 0, 3, 5, 12, and 24 hours after reperfusion. CysLT(1)R expression was analyzed by immunohistochemistry. CysLT(1)R expression was observed only in endothelial cells of a normal kidney. CysLT(1)R expression was most intense on endothelial cells at 3 hours after reperfusion, and CysLT(1)R expression on endothelial cells gradually became weaker. Twelve hours after reperfusion, ATN extended throughout the ischemic kidney. Renal I/R injury gradually progressed at time after reperfusion. Several hours after the maximal CysLT(1)R expression, we observed the maximum renal I/R injury.

Study of cysteinyl leukotriene-1 receptor in rat renal ischemia-reperfusion injury.

Renal ischemia-reperfusion (I/R) injury is a major cause of renal transplant dysfunction. Recent studies of I/R injury have focused on the function of neutrophils, the mechanisms of action of inflammatory cytokines, and oxygen free radicals, as well as other mediators. However, few reports address the cysteinyl leukotriene-1 receptor (CysLT1R), an important mediator of bronchial asthma in human beings. We examined the expression of CysLT1R in rat renal I/R injury. At laparotomy, the right kidney was harvested and the left renal artery and vein were clamped. The kidney was reperfused after 90 minutes of ischemia, and the rats were killed after 0, 3, 5, 12, or 24 hours. Expression of CysLT1R analyzed at immunohistochemistry was observed only in endothelial cells in nonischemic kidney. At 0 to 3 hours after reperfusion, CysLT1R expression on endothelial cells gradually became stronger, being most intense at 3 hours after reperfusion. Twelve hours after reperfusion, necrosis extended throughout the ischemic kidney; nearly all of the tubular epithelial cells were destroyed. At 3 to 12 hours after reperfusion, CysLT1R expression gradually became weaker on endothelial cells. At 24 hours after reperfusion, CysLT1R expression was almost at the level of that in nonischemic kidney. Expression of CysLT1R was noted in a rat model of renal I/R injury. Several hours after the maximal CysLT1R expression, we observed the maximum renal I/R injury. These results may suggest a relationship between the CysLT1R and renal I/R injury.

Human interleukin-10 transduced fetal liver stem cells prolong survival of mouse skin and heart allografts.

Interleukin (IL)-10 regulates immune responses, acting as a suppressive cytokine by inhibiting the synthesis of Th1 cytokines, such as IL-2 and interferon (IFN)-gamma. It also strongly down-regulates major histocompatibility complex (MHC) class II determinants on antigen presenting cells (APC). On the other hand, long-term tolerance is well correlated with the persistence of a peripheral microchimerism. In this study, we investigated the synergistic effect of human IL-10 (huIL-10) and hematopoietic microchimerism for the induction of long-term tolerance. Irradiated Balb/c mice (H-2d) were used as recipients (fetal liver stem cells [FLSC], skin and heart) and C57BL/6 (H-2b) mice were used as donors of FLSC, skin and heart. Recipients were simultaneously transplanted with the heart, the skin and with huIL-10 gene-transduced FLSC. Microchimerism was checked using fluorescent flow cytometry, huIL10 production using enzyme-linked immunosorbent assay (ELISA), and graft survival was evaluated by daily observation. Significant level of huIL10 (up to 900 pg/mL) was detected for more than 2 weeks in the serum of mice that underwent transplantation. Four weeks after the FLSC injection, microchimerism was identified in the recipient lymphoid organs (spleen, thymus, and bone marrow) by the presence of donor cells (H-2b). Finally, in the group of mice treated with huIL-10 gene-transduced FLSC, skin allografts survived for 18.9 +/- 1.8 days compared with 9.5 and 9.6 days in the groups of mice treated with nontransduced FLSC or huIL-10 alone, respectively. The same pattern for heart allograft survival was observed. HuIL-10 transduction of donor hematopoietic stem cells resulted in production of huIL-10, cell engraftment, and chimerism. Although full tolerance was not obtained, specific and highly significant (P < .001) prolongation of the survival of donor heart allografts with (more than 2-fold compared with nontreated groups) was observed. The infiltration of the transplanted heart and its late rejection demonstrate that stem cells transduced with huIL-10 gene induce "prope" tolerance in this model.

Increased frequency of CD3/8/56-positive umbilical cord blood T lymphocytes after allo-priming in vitro.

Umbilical cord blood (UCB) or adult peripheral blood mononuclear cells (PBMC) were repeatedly stimulated by HLA-mismatched allogeneic Epstein-Barr virus (EBV)-transformed cell lines, and the bulk responders or single-cloned cells were immunophenotypically analyzed by flow cytometry. One month after the allo-stimulation, not in PBMC but in UCB, the proportion of CD3/8/56 triple positive T lymphocytes significantly increased. Furthermore, UCB clones exhibited those unique CD3/8/56 markers at an extremely higher frequency than PB clones. They showed as much strong killing activity against allo-stimulators as conventional PB CD56-negative, CD8 T-cell clones, whereas they did not kill the other target, Raji cells. UCB CD3/8/56 T cell clones produced a smaller amount of interferon-gamma compared with PB CD4 or PB CD8 T cell clones. We concluded that CD8 T cells coexpressed with CD56 marker expanded after allo-priming in vitro and would become one of the graft-versus-host (GVH) effectors after UCB transplantation.

Expression of cyclooxygenase-2 in patients with bladder carcinoma.

PURPOSE: Cyclooxygenase-2 is considered to have an important role in the development of metastasis in cancer due to angiogenesis function. The expression of cyclooxygenase-2 was found to be up-regulated in colorectal carcinoma and other cancers. We investigated cyclooxygenase-1 and 2 expressions in patients with bladder cancer, chronic cystitis and normal bladder. MATERIALS AND METHODS: A total of 118 specimens were obtained from patients treated at Osaka City University Hospital for bladder cancer, including 10 with chronic cystitis and 8 with normal bladder tissue. Immunohistochemistry, with affinity purified antibodies against human cyclooxygenase-1 and 2 that did not have cross-reactivity with each other, and reverse transcriptase polymerase chain reaction to study the messenger RNA expression were performed. RESULTS: Although no marked expression of cyclooxygenase-2 was observed in the normal bladder, it was slightly seen in infiltrative inflammatory cells of chronic cystitis, and a higher expression was found in cancer cells. The extent and intensity of immunoreactive cyclooxygenase-2 polypeptides in cancer cells was statistically much greater than those in cells from normal bladder tissue. Moreover, correlation between cyclooxygenase-2 expression and tissue type or progression of bladder cancer was observed. Cyclooxygenase-2 expression was higher in grade 3 bladder cancer than in grade 1, and was higher in advanced than in early stage cancer. CONCLUSIONS: These results demonstrate that generated cyclooxygenase-2 in the cells of patients with bladder cancer might be significant in the proliferation of bladder malignant cells and development of invasions.

The efficient generation of CD83 positive immunocompetent dendritic cells from CD14 positive acute myelomonocytic or monocytic leukemia cells in vitro.

The ability of leukemic cells to differentiate to mature dendritic cells (DCs) was investigated in six acute myelomonocytic or monocytic leukemia cases. It was found that CD14 positive cells were more efficiently changed to CD83 positive mature typed DCs with granulocyte-macrophage colony-stimulating factor (GM-CSF)/interleukin-4 (IL-4) and tumor necrosis factor alpha (TNF-alpha) compared with CD14 negative cells. Such leukemia derived DCs expressed a sufficient level of costimulatory molecules (CD80 and CD86), and were shown to be monoclonal based on an the X-inactivation analysis. They also stimulated not only allo- but auto-T lymphocytes, which thereafter became cytotoxic T lymphocytes (CTLs).

Expression of cyclooxygenase-2 in prostate carcinoma.

BACKGROUND: Nonsteroidal antiinflammatory drugs inhibiting cyclooxygenase (COX) enzyme activity in both its constitutive (COX-1) and inducible (COX-2) isoforms were shown also to inhibit the development of colon carcinoma in animal models. COX-2 is an inducer of angiogenesis of new blood vessels. The expression of COX-1 and COX-2 in prostate tissues from patients with prostate carcinoma was investigated using reverse transcriptase-polymerase chain reaction (RT-PCR) and immunohistochemistry. METHODS: Tumor specimens were obtained from 28 prostate carcinoma (PC) patients, 8 benign prostatic hyperplasia (BPH) patients, 1 prostatic intraepithelial neoplasia (PIN) patient, and 8 specimens of normal prostate tissue (NP). Affinity-purified COX-1 and COX-2 antibodies were used in immunochemistry. RESULTS: Very weak expression of COX-1 and marked expression of immunoreactive COX-2 in tumor cells was obtained. In contrast, expression of both isoforms was very weak in all cases of BPH and in the NP tissues. Immunoreactive COX-1 also was very weak in all cases of benign tissues. The extent and intensity of immunoreactive COX-2 polypeptides in tumor cells was statistically much greater than those of cells from BPH. Immunostaining with normal rabbit immunoglobulin G was completely negative. By RT-PCR analysis, enhanced expression of COX-2, but not COX-1, was observed in PC tissue. BPH displayed faint expression of COX-2. CONCLUSIONS: The results of the current study demonstrated that human prostate carcinoma cells generated COX-2, and that COX-2 might play an important role in the proliferation of prostate carcinoma cells. These findings suggest that inhibition of COX-2 development may lead not only to inhibition of the proliferation and metastasis of prostate carcinoma but also to the inhibition of prostate carcinogenesis.

Extensive generation of human cord blood CD34(+) stem cells from Lin(-)CD34(-) cells in a long-term in vitro system.

Human CD34(-) hematopoietic stem cells (HSCs) have been identified as potential precursors of CD34(+) HSCs by using xenogeneic transplantation systems. However, the properties of CD34(+) cells generated from CD34(-) cells have not been precisely analyzed due to the lack of an in vitro system in which CD34(+) cells are continuously produced from CD34(-) cells. We conducted this study to determine whether CD34(+) cells generated in vitro from CD34(-) cells have long-term multilineage reconstitution abilities. Lin(-)CD34(-) population isolated from human cord blood was cultured in the presence of murine bone marrow stroma cell line, HESS-5, and human cytokines, thrombopoietin, Flk2/Flt3 ligand, stem cell factor, granulocyte colony-stimulating factor, interleukin 3 (IL-3), and IL-6. They were analyzed weekly for their surface markers expressions, colony-forming cells, long-term culture initiating cells (LTC-IC), and SCID repopulating cells (SRC) abilities up to 30 days of culture. In this culture system, more than 10(7) CD34(+) cells can be continuously generated from 10(4) CD34(-) cells over 30 days. These CD34(+) cells produce colony-forming units, LTC-IC, and SRC with multi-lineage differentiation, all of which are characteristic features of hematopoietic stem/progenitor cells. These findings suggest that CD34(-) HSCs have extensive potential for the generation of CD34(+) HSCs in vitro. This system provides a novel and potentially useful procedure to generate CD34(+) cells for clinical transplantation and gene therapy.

Induction of hyperacute rejection of skin allografts by CD8+ lymphocytes.

BACKGROUND: Second-set rejection is generally regarded as a phenomenon mainly mediated by humoral cytotoxic antibodies, although a few discordant data have been presented. In the reported experiments, we have taken advantage of the absence of production of specific cytotoxic alloantibodies contrasting with the normal development of transplantation cellular immunity, in two murine models: chimeric mice and RAG mice. METHODS: Chimeras (BALB/c-->CBA) were obtained by transplantation of 2x10(7) fetal liver cells from BALB/c (H-2d) mice to lethally irradiated CBA (H-2k) mice. After hyperimmunization with third-party C57/ BL6 (B6) (H-2b) skin transplants and with injections of 2x10(7) B6 spleen cells, antibody production, and skin graft survival were analyzed. To identify further the factors or cells responsible for accelerated rejection of B6 skin transplants in hyperimmunized chimeras, transfer experiments were carried out involving the injection of serum, whole spleen cells, spleen T cells, spleen CD8+ T cells or spleen CD4+ T cells from chimeras into BALB/c mice that had received 6 Gy irradiation. The recipient mice were then grafted with B6 skin. Similarly, the immunodeficient RAG mice were used to construct a model of recipient animals with anti-H-2d hyperimmunized B6 T cells in the total absence of antibody. RESULTS: In chimeras, anti-B6 cytotoxic antibodies were not detectable in any of hyperimmunized chimeric mice, yet accelerated rejection of B6 skin transplant occurred: a graft survival of 8.6+/-0.5 days (d), comparable to 8.9+/-0.8 d survival in CBA control mice subjected to the same hyperimmunization procedure, and significantly shorter than that in nonhyperimmunized (BALB/c-->CBA) chimeras (11.6+/-0.5 d) or in non-hyperimmunized CBA control mice (12.1+/-0.6 d). High titers of anti-B6 cytotoxic antibodies were present in the serum of hyperimmunized CBA control mice. In transfer experiments, the graft survival was over 14 d in mice treated with irradiation alone, with irradiation + serum or with irradiation + CD4+ T cells. It was significantly shorter in mice treated with irradiation + whole spleen cells, with irradiation + T cells or with irradiation + CD8+ T cells (8.9+/-0.8 d). Similarly, in immunodeficient RAG mice, reconstitution of the T cell compartment with T cells from hyperimmunized B6 mice led to accelerated rejection of BALB/c skin allografts (11.4+/-1.1 d vs. 18.8+/-0.8 d when T cells were provided by nonimmunized mice). In a second transfer of cells from these reconstituted RAG mice into naive RAG mice, CD8+ T cells were shown to induce accelerated rejection of skin allografts (12.0+/-0.6 d) whereas CD4+ T cells were much less efficient (16.5+/-0.1 d). CONCLUSION: These data indicate that T cells, and especially the CD8+ subset, can be responsible for second-set rejection in the absence of anti-donor antibodies in chimeric and RAG mouse models. These sensitized CD8+ T cells are also likely to play an important role in normal mice, in addition to that of cytotoxic antibodies.

Inhibition of Epstein-Barr virus (EBV)-specific CD8+ cytotoxic T lymphocyte (CTL) activity by soluble HLA class I in vitro.

In the present study, the effects of soluble HLA (sHLA) class I molecules against EBV-specific CTL were examined. Two different sources of sHLA class I, either bioengineered spliced form of HLA-B7 (sB7) or natural production from EBV-transformed B cells (natural sHLA), were added during the induction of CTL or incubated with MHC-restricted CD8+ CTL, which were selected by immunobeads just before testing for their cytotoxic activity. Both sB7 and natural sHLA class I blocked the generation of CD8+ CTL and also inhibited the cytotoxic activity of established CTL in a dose-dependent manner. In both ways, natural sHLA class I was effective in 10-fold lower concentrations compared with sB7. The inhibitory effect did not require a sharing of the HLA allotypes between sHLA and the CTL. CTL, after being treated with sHLA, underwent apoptosis, which was considered here as the main mechanism.

Ex vivo generation of CD34(+) cells from CD34(-) hematopoietic cells.

The human Lin(-)CD34(-) cell population contains a newly defined class of hematopoietic stem cells that reconstitute hematopoiesis in xenogeneic transplantation systems. We therefore developed a culture condition in which these cells were maintained and then acquired CD34 expression and the ability to produce colony-forming cells (CFC) and SCID-repopulating cells (SRCs). A murine bone marrow stromal cell line, HESS-5, supports the survival and proliferation of Lin(-)CD34(-) cells in the presence of fetal calf serum and human cytokines thrombopoietin, Flk-2/Flt-3 ligand, stem cell factor, granulocyte colony-stimulating factor, interleukin-3, and interleukin-6. Although Lin(-)CD34(-) cells do not initially form any hematopoietic colonies in methylcellulose, they do acquire the colony-forming ability during 7 days of culture, which coincides with their conversion to a CD34(+) phenotype. From 2.2% to 12.1% of the cells became positive for CD34 after culture. The long-term multilineage repopulating ability of these cultured cells was also confirmed by transplantation into irradiated NOD/SCID mice. These results represent the first in vitro demonstration of the precursor of CD34(+) cells in the human CD34(-) cell population. Furthermore, the in vitro system we reported here is expected to open the way to the precise characterization and ex vivo manipulation of Lin(-)CD34(-) hematopoietic stem cells.