Yoshito Naiki

Possible Effects of Hepatocyte Growth Factor for the Prevention of Peritoneal Fibrosis

Objective: Some patients who had carried out long-term continuous ambulatory peritoneal dialysis discontinued the treatment because of progressive peritoneal fibrosis. It has been previously reported that transforming growth factor-β1 (TGF-β1) is one of the factors that induces peritoneal fibrosis. Also, hepatocyte growth factor (HGF) plays a role in the prevention of fibrosis and in inhibiting TGF-β1 production. In this study, we examined the effects of HGF on peritoneal fibrosis by TGF-β1 induced by high concentrations of D-glucose. Design: We transfected a full-length human HGF cDNA in an expression vector into human peritoneal mesothelial cells (HPMCs) using the calcium phosphate method. Transfected HPMCs were cultured with high concentrations of D-glucose solution and co-cultured with fibroblasts using a transwell system. Cell proliferation was determined using the Tetra Color One method. TGF-β1 and HGF protein were measured by enzyme-linked immunosorbent assay. Results: In addition to recombinant HGF, the growth inhibition of HPMCs by high concentration D-glucose or TGF-β1 was significant. By transfecting HGF cDNA into HPMCs, growth inhibition by high concentration D-glucose was completely restored. Furthermore, the production of TGF-β1 was also significantly decreased. Conclusion: These results suggested that exogenous HGF could possibly prevent peritoneal fibrosis.

Possible role of hepatocyte growth factor in regeneration of human peritoneal mesothelial cells.

Human peritoneal mesothelial cells (HPMCs) play an important role in peritoneal functions. During long term peritoneal dialysis, it has been reported that HPMCs are damaged by high glucose solution via the signal of transforming growth factor (TGF)- ß1 produced by HPMCs. In this study, we focused on the effect of hepatocyte growth factor (HGF), known as an anti-fibrotic and anti-TGF-ß1 agent, on HPMCs damaged by high glucose solution. HPMCs were isolated from specimens of the omentum from nonuremic patients after informed consent had been obtained. After confirming adhesion for 6 hours, 100 μL of DMEM with 0.5%FCS were added at different concentrations (D-glucose; 6, 30mM) with or without HGF (10, 30, 100 ng/mL) for 48 hours. We examined the effects of a high concentration of glucose and then focused on following four critical points: 1) the production of HGF from HPMCs exposed to a high concentration of glucose, 2) the expression of c-Met on HPMCs, 3) the viability of those cells, and 4) matrix metalloproteinase-2 (MMP-2) and tissue inhibitors of metalloproteinase-2 (TIMP-2). The following significant changes are described herein: high glucose solution and TGF-ß1 i) decreased HGF production from HPMCs and ii) up-regulated expression of c-Met on HPMCs, and addition of HGF iii) restored viability of HPMCs damaged by glucose, iv) suppressed TGF-ß1 production by HGF, and v) induced up-regulation of MMP-2 and decreased TIMP-2 production by HPMCs. Levels of HGF decreased by high concentrations of glucose in the peritoneal cavity may induce the loss of HPMCs and thereby result in peritoneal fibrosis. These results suggest that HGF is an effective agent in the regeneration of peritoneal membrane damaged by high glucose solution.

Possible involvement of soluble erythropoietin receptor in resistance to erythropoietin in patients with renal anemia.

Accessible online at: www.karger.com/journals/ajn Dear Sir, Recombinant human erythropoietin (rHuEpo) is effective in treating anemia associated with chronic renal failure (CRF). However, patients with CRF have sometimes developed a resistance to rEpo. This resistance can be due to iron or folate deficiency, aluminum toxicity, hyperparathyroidism, or autoantibodies for rHuEpo [1]. In this study, we focused on soluble erythropoietin receptor (sEpoR) [2], which can bind to rEpo. To prove the possibility that the sweeping of rEpo by sEpoR results in resistance to rHuEpo, we performed a bioassay using Epo-dependent cell line UT-7/Epo [3]. The growth of UT-7/Epo is not known to be inhibited by any cytokine except sEpoR. Recombinant sEpoR can reduce the proliferation of UT-7/Epo induced by rHu/Epo in a dose-dependent manner. Furthermore, the binding of 125I-labeled rEpo on UT-7/Epo has been seen to be significantly inhibited by recombinant sEpoR. We considered that this cell line could be a useful tool in a bioassay to detect the inhibitory factor(s) against Epo. We selected sera from the two groups of patients with renal anemia associated with CRF receiving hemodialysis 3 times a week, one is patient group with resistance to rHuEpo and the other patient group sensitive to rEpo. The definition of the rHuEpo resistance was described as hemoglobin rise of 1 g/dl/month despite a rHuEpo dose of 1200 U/ kg/week. Furthermore, concerning the clinical characteristics of the rHuPo-sensitive and -resistant groups regarding factors (infection, hyperparathyroid bone disease, aluminum accumulation, vitamin B12 and/ or folate deficiency, hemolysis, bone marrow dysfunction malnutrition and inadequate hemodialysis) known to create Epo resistance, no significant differences were observed between the two groups. Serum was collected the day before regular hemodialysis. Interestingly, the proliferation of UT-7/ Epo determined with [3H]thymidine incorporation was reduced significantly by the addition of sera from Epo-resistant patients, but not by the addition of sera from the Eposensitive group. These results indicate that sEpoR in serum plays an important role in the signal transduction via EpoR on erythroid progenitor in CRF patients. References

Mutant Type Glutathione S-transferase Theta 1 Gene Homologue to mTOR in Myelodysplastic Syndrome: Possible Clinical Application of Rapamycin

In this study, we observed the expression of the GSTT-1 gene in patients with myelodysplastic syndrome (MDS) at the messenger RNA level. Reverse transcription-polymerase chain reaction (RT-PCR) for GSTT-1 was performed with a pair of primers complementary to the 5′ coding section and the 3′ coding section of the GSTT-1 cDNA for amplifying the 623-bp band. Among 20 patients with MDS, 8 patients showed the expected 623-bp band on RT-PCR, and 12 patients showed a 500-bp band on RT-PCR, indicating that a 123-bp sequence was deleted as a mutant of the GSTT-1 gene. Furthermore, a BLAST DNA search showed that the deletion of a 123 bp sequence creates a sequence that is 63% homologous to human FKBP-rapamycin associated protein (FRAP); this protein has been termed a mammalian target of rapamycin (mTOR). We respectively transfected the wild type and the mutant type GSTT-1 gene in an expression vector to two cell lines (K562 and HL-60). The stable transformants for the wild type and the mutant type GSTT-1 genes were made by G418 selection. Interestingly, rapamycin could induce significant growth inhibition of the stable transformants for mutant type GSTT-1, which was indicative of apoptosis, but not that of those for wild type GSTT-1. These results suggest that rapamycin could be included in the therapeutic modality for the patients with MDS who have the mTOR sequences in GSTT-1 gene.

Establishment of a Myeloid Cell Line, YM711, Characterized by Retinoid Resistance

A myeloid cell line (YM711) was established by transfecting exogenous PML/RARα cDNA into peripheral blood stem cells. The cells were positive for CD33, CD34, CD38, CD13, CD14, and CD11b, Cytochemical examination revealed YM711 cells to be positive for peroxidase, α-naphtyl butyrate esterase, and acid phosphatase as well. Karyotypic analysis showed them to be nearly tetraploid (92 XXYY). Reverse-transcription polymerase chain reaction showed that, although PML/RARα mRNA was detected in YM711, these cells could not be differentiated by all-trans retinoic acid (ATRA). We therefore designated the YM711 cell line as being ATRA resistant. Because YM711 expressed multi drug resistance 1 (MDR-1) mRNA and p-glycoprotein cell surface protein, we assessed whether verapamil and ATRA would induce the differentiation of YM711 cells; they did not. An increased expression of cellular retinoic acid-binding protein (CRABP)-II was also detected on YM711 cells compared with that of HL-60. These results suggest that high level of expression of CRABP-II may contribute to be the mechanism of ATRA resistance. This cell line may be useful in evaluating the mechanism of resistance to retinoid.

Transient eosinophilia by HIV infection.

Abstract We describe a case of early human immunodeficiency virus infection characterized by transient eosinophilia without an elevated immunoglobulin E concentration, allergic symptoms, or atopic dermatitis. Possible mechanisms of the eosinophilia are discussed.

Exogenous PML/RARα Fusion Gene Responds to All-trans Retinoic Acid Results in Differentiation of the Human B Cell Line.

The interaction of an exogenous PML/RARα fusion gene, associated with acute promyelocytic leukemia, with all-trans retinoic acid (ATRA) was examined in B-lymphoid cell lines. RPMI8866 cells were transfected with PML/RARα cDNA in the expression vector pGD and two stable transformants (RPMI8866Y-4 and RPMI8866Y-17) were established by selection with G418. ATRA inhibited the growth of those stable transformants, as assessed by [3H]-thymidine incorporation, but had no effect on the growth of control cells stably transformed with neomycin resistant gene alone. ATRA also increased expression of CD38 and immunoglobulin production in RPMI8866Y-4 cells but not in control cells. When these results are taken together, it can be observed that the exogenous PML/RARα fusion gene responds to ATRA, which results in cell differentiation of the human B cell line.