A variety of hematopoietic lineage cells have been produced from embryonic stem (ES) cells, but their differentiation processes have not been elucidated well, especially from the point of view of ...progenitor analysis. In this study, we utilized our coculture system, in which ES-derived Flk-1
+ cells differentiated into TER-119
+ primitive erythroid (EryP) cells on OP9 cells, and looked for progenitors in primitive erythropoiesis.
We studied the kinetics of TER-119
+ erythroblast generation from Flk-1
+ cells by monitoring the expression of TER-119, CD41, VE-cadherin, CD34, and c-kit antigens. Multicolor analysis was performed to detect CD41
+TER-119
+ cells and the stained cells were sorted to examine their morphology and EryP-producing potential in colony formation.
Kinetic studies showed that the CD41
+ population appeared early in the coculture and its expression pattern implied a role as an immediate progenitor of TER-119
+ EryP cells. Multicolor analysis and colony-formation study supported this notion. Other progenitor markers such as VE-cadherin, CD34, and c-kit did not seem to define an immediate progenitor of EryP cells. One interesting observation is the detection of unique populations, CD41
dim and CD41
bright, detectable after 48 hours of the coculture. Majority of the CD41
dim population progressed to the EryP lineage, whereas the CD41
bright population seemingly advanced on a pathway distinct from the CD41
dim population.
CD41 expression was a useful marker to trace hematopoietic progenitors in ES-derived differentiation system. In particular, the CD41
dim but not CD41
bright population could serve as immediate precursors of EryP cells.
We studied the role of reactive oxygen intermediates (ROIs) in experimental liver metastasis induced in mice by the inoculation of COLON 26-M5 murine colon cancer cells, a highly metastatic variant ...of COLON 26 cells, and the effect of ROIs on the invasive capacity of the cells in an in vitro chemo-invasion assay model using reconstituted basement membrane matrigel. We also measured the release of ROIs from cells using electron spin resonance (ESR) spectrometry. Hydroxyl radicals (.OH) were constitutively released from the cells. This release was augmented by pre-treatment with phorbol 12-myristate 13-acetate (PMA). In experimental liver metastasis in CDF1 mice, the administration of recombinant human superoxide dismutase (r-hSOD) significantly increased the number of metastatic nodules, while administration of catalase significantly inhibited metastasis formation. In vitro pre-treatment of cells with PMA significantly increased the number of metastatic nodules. Invasive capacity of the cells was markedly augmented by pre-treatment with PMA. PMA-induced augmentation was significantly inhibited by the simultaneous addition of r-hSOD to the assay. Catalase had no significant effect. Our findings suggest that ROIs play an important role in tumor invasion and metastasis, and that hydrogen peroxide (H2O2) may contribute to the retention or extravasation of circulating tumor cells. Furthermore, the superoxide anion (O2-) released by tumor cells may play an important role in basement membrane degradation.
. Although a number of transcription factors (TFs) have been identified that play a pivotal role in the development of hematopoietic lineages, only little is known about factors that may influence ...development and lineage commitment of natural killer (NK) or NK‐like T (NKT)‐cells. Obviously to fully appreciate the NK‐ and NKT‐cell differentiation process, it is important to identify and characterize the TFs effecting the NK‐ and NKT‐cell lineage. Furthermore, these TFs may play a role in NK‐ or NKT‐cell leukemias, in which the normal differentiation program is presumably disturbed. The present study analyzed the expression of the following 13 TFs: AML1, CEBPA, E2A, ETS1, GATA1, GATA2, GATA3, IKAROS, IRF1, PAX5, PU1, TBET and TCF1 in 7 malignant NK‐cell lines together with 5 malignant NKT‐cell lines, 5 T‐cell acute lymphoblastic leukemia (ALL) cell lines including 3 γ/δ T‐cell receptor (TCR) type and 2 α/β TCR type, and 3 B‐cell precursor (BCP) leukemia cell lines. AML1, E2A, ETS1, IKAROS and IRF1 were found to be positive for all cell lines tested whereas GATA1 turned out to be universally negative. CEBPA, PAX5 and PU1 were negative for all cell lines tested except in the three positive BCP‐cell lines. GATA2 was positive for 3/5 T‐cell lines but negative for the other cell lines. GATA3 was positive for 7/7 NK‐, 4/5 NKT‐, 5/5 T‐and 2/3 BCP‐cell lines. TBET was positive for all NK‐ and NKT‐cell lines and negative for all T‐ and BCP‐cell lines except one BCP‐cell line. In contrast to the expression of TBET, TCF1 was negative for all NK‐ and NKT‐cell lines, being positive for 4/5 T‐ and 1/3 BCP‐cell lines. Expression analysis of TFs revealed that NK‐ and NKT‐cell lines showed identical profiles, clearly distinct from those of the other T‐ALL or BCP‐ALL leukemia‐derived cell lines.
Enhanced T helper 1 (Th1)-type immunity was observed in patients with hepatitis C virus (HCV) infection during investigations on the efficiency of interferon (IFN) therapy. The mechanism for the ...shift to Th1-type immunity is, however, obscure in HCV infection. In this study, we examined the in vitro effect of IFN-α subtypes (IFN-α1, -α2, -α5, -α8 and -α10) on the Th1/Th2 balance in the peripheral blood mononuclear cells (PBMC) obtained from healthy control volunteers and HCV-infected patients. A two-day incubation without IFN stimulation did raise the Th1-type cell percentage but not the Th2-type cell percentage in the PBMC of the control group. The Th1-type cell percentage and Th1/Th2 ratio were significantly larger in the PBMC of patients when compared to controls both before and after treatment with the IFNs. IFN-α5 treatment induced an increase of the Th2-type cell percentage in both control and patient PBMC but did not show any significant changes in the Th1/Th2 ratio. Furthermore, IFN-α8 treatment slightly promoted an increase in the Th1/Th2 ratio only in patient PBMC. Statistical analysis revealed that effects of the IFN-α subtypes on the Th1/Th2 balance differed between two patient groups with severe liver damage (alanine aminotransferase; ALT: ≥ 80 IU/ml) and mild liver damage (ALT: 〈 80 IU/ml). IFN-α5 treatment lowered the Th1/Th2 ratio in patients with mild liver damage, whereas IFN- α 8 treatment raised the Th1/Th2 ratio in patients with severe liver damage without IFN-α5-induced decrease in the ratio. These findings imply that HCV infection and its disease status modify the effects of IFN-α subtypes on Th1 and Th2 immune balance in patients. Our findings should help to elucidate the mechanisms underlying IFN therapy for HCV infection.
The de novo methylation activity is essential for embryonic development as well as embryonic stem (ES) cell differentiation, where the intensive and extensive DNA methylation was detected. In this ...study, we investigated the effects of a demethylating agent, 5-azacytidine (5-AzaC), on differentiated ES cells in order to study the possibility of reversing the differentiation process. We first induced differentiation of ES cells by forming embryoid bodies, and then the cells were treated with 5-AzaC. The cells showed some undifferentiated features such as stem cell-like morphology with unclear cell-to-cell boundary and proliferative responsiveness to LIF. Moreover, 5-AzaC increased the expressions of ES specific markers, SSEA-1, and alkaline phosphatase activity as well as ES specific genes, Oct4, Nanog, and Sox2. We also found that 5-AzaC demethylated the promoter region of H19 gene, a typical methylated gene during embryonic differentiation. These results indicate that 5-AzaC reverses differentiation state of ES cells through its DNA demethylating activity to differentiation related genes.
Background. The preoperative intratumoral injection with OK‐432 (Picibanil, Chugai Pharmaceutical Co., Tokyo, Japan), an immunomodulatory agent prepared from an attenuated strain of streptococcus ...pyogenes, activates the regional immune system and causes degeneration of cancer tissue in carcinoma of the stomach.
Methods. A multi‐institutional randomized trial of OK‐432 to determine its clinical usefulness was conducted. Three hundred and ninety‐five patients with gastric cancer were assigned randomly either to receive or not to receive a preoperative intratumoral injection of OK‐432. Among them, 277 patients with advanced cancer were treated by common postoperative chemoimmunotherapy consisting of mitomycin C, tegafur, and OK‐432. All patients were followed for at least 5 years.
Results. The adverse effects of OK‐432 injected intratumorally predominantly were mild fever, anorexia, and abdominal pain, however, no treatment was required for these symptoms. Overall, there were no differences in outcome between the OK‐432 and control groups. However, analysis based on stage showed that a preoperative intratumoral injection of OK‐432 significantly improved the 5‐year survival rate of patients with Stage III cancer (P = 0.0229), at 47.7% for the OK‐432 group and 27.5% for the control group. In subset analysis, when the 5‐year survival of patients with and without tumor infiltrating lymphocytes (TIL) was compared, OK‐432 injected intratumorally had a significant positive effect on the group showing a moderate to marked number of TIL (P = 0.0438).
Conclusion. These results showed that the intratumoral injection of OK‐432 may improve survival of patients with Stage III gastric cancer.
IL-18 is a new type of inflammatory cytokine similar to but distinct from IL-12 and IL-1beta. One intriguing property of IL-18 is synergism with IL-12 in many respects. In this study we examined the ...in vivo synergistic effects of IL-18/IL-12 in mice and found lethal toxicity accompanying an elevated IFN-gamma level in the serum. Since treatment with IL-18 alone did not have any apparent toxicity, and treatment with IL-12 alone showed only limited toxicity in our system, the synergy between the two cytokines was all the more remarkable. The major symptoms of the toxicity were weight loss, diarrhea, hemorrhagic colitis, splenomegaly, fatty liver, and atrophic thymus, most of which are similarly found in endotoxin-induced septic shock. However, in contrast to septic shock, TNF-alpha was not induced. The involvement of IFN-gamma in the toxicity was further studied in detail. Treatment of athymic nude mice with anti-asialo-GM1 did not reduce the toxicity, whereas anti-IFN-gamma treatment of wild-type mice alleviated it. When IFN-gamma-deficient mice were treated with IL-18/IL-12, the majority of them showed mortality and toxicity with severe pulmonary edema. These results indicate that IL-18/IL-12 treatment induces severe adverse effects through not only IFN-gamma-dependent mechanisms but also IFN-gamma-independent processes.
The deleted-in-colorectal-cancer (DCC) gene, located on chromosome 18q 21.3, is considered to be a tumor suppressor gene related to cellular adhesion receptors. A loss of heterozygosity (LOH) on ...chromosome 18q is frequently observed in adenomatous polyposis coli, as well as in sporadic colon carcinoma and its liver metastatic loci. In this study, we investigated the expression of DCC mRNA in the resected specimens of 38 gastric cancers and 28 colorectal cancers by a reverse transcription-polymerase chain reaction method. In the gastric cancer patients, the mean expression level of DCC mRNA in the tumors was significantly lower than that in normal tissues (p = 0.009), but no difference was observed in the colorectal cancer patients. DCC mRNA expression was decreased in 15 gastric cancers (40%) and 10 colorectal cancers (36%), and there was a significant correlation between the decreased expression of DCC mRNA and nodal metastasis in colorectal cancer (chi 2 = 7.049, DF = 1, P = 0.0079). Two of four gastric cancer patients and none of seven colorectal cancer patients whose cancers were confined to the muscularis propria without metastasis showed decreased expression of DCC mRNA. These findings demonstrate that decreased expression of DCC mRNA may occur at an early stage in gastric cancer and at a late stage in colorectal cancer and that this decreased expression correlates with the potential to develop nodal metastasis.
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