Effects of cytokines on hematopoietic progenitor cells in cord blood,in bone marrow,and in peripheral blood mobilized by chemotherapy and G-CSF

We compared the effects of various combinations of cytokines (stem cell factor [SCF], interleukin [IL] ?3, IL-6, granulocyte-colony stimulating factor [G-CSF], erythropoietin [EPO]) among the growth of human hematopoietic progenitor cells from cord blood (CB), bone marrow (BM), and peripheral blood mononuclear cells (MNC) mobilized by chemotherapy and G-CSF (PB) in a semi-solid medium. Macroscopic colonies, that were visible to the naked eye, were formed from PB-MNC within 1 week even without cytokines. They consisted of blasts containing macrophage-like cells with immature nuclei on Wright stain, and were strongly accelerated by IL-3. Macroscopic colonies were also formed from CB-MNC. However, they appeared after 1–3 weeks and synergistic effects of SCF with other cytokines, especially EPO, were prominent. Macroscopic colonies were not formed from BM-MNC. Granulocyte-colony stimulating factor was effective in increasing colony forming units of granulocyte macrophage from BM-MNC and they appeared between 1 and 2 weeks. These results suggested that the quality of hematopoietic progenitor cells was different among blood sources. This might lead to different bone marrow recovery patterns after transplantation of each blood source. The appropriate cytokines should be added to evaluate their exact potential.     

Ex vivo expansion of umbilical cord blood hematopoietic progenitor cells by combinations of cytokines

Ex vivo expansion of hematopoietic progenitor cells in the umbilical cord blood mononuclear cells (CB-MNC) was investigated in liquid culture system with various combinations of cytokines (stem cell factor [SCF], interleukin [IL]-3, IL-6, granulocyte-colony stimulating factor [G-CSF], erythropoietin [EPO], and interferon [INF]-γ). Non-lineage-committed hematopoietic progenitor cells and lineage committed hematopoietic progenitor cells were represented as CD34⁺CD38^? and CD34⁺CD38⁺ subpopulations, respectively. Although absolute CD34⁺CD38^? cell numbers decreased even in the presence of multicytokines, the combinations of SCF plus IL-6 and SCF plus IL-3 plus IL-6 plus INF-γ were significantly effective in maintaining CD34⁺CD38^? cells than the other combinations (P < 0.05). After 4 weeks of culture, CD34⁺CD38^? cells disappeared in all combinations of cytokines. Absolute CD34⁺CD38⁺ cell numbers increased in the presence of cytokines. Maximal expansion of CD34⁺CD38⁺ cells were observed in the combinations of SCF plus IL-3 plus IL-6 plus EPO (19.8 ± 3.3 -fold) and SCF plus IL-3 plus IL-6 plus G-CSF (18.3 ± 2.6). The combination of SCF plus IL-3 plus IL-6 was also effective to expand CD34⁺CD38⁺ cells (15.8 ± 3.9). However, the expansion was transient and they decreased to zero within 3 weeks. In the combinations of SCF plus IL-6 and SCF plus IL-3 plus IL-6 plus INF-γ, maximal expansion was inferior to the others but CD34⁺CD38⁺ cells were maintained more than 4 weeks. These results suggested that the indication of CBT can be expanded into older children by ex vivo augmentation of CB hematopoietic progenitor cells using multi-cytokines.     

Establishment and characterization of human urothelial cancer xenografts in severe combined immunodeficient mice

AIM: To establish and characterize a murine xenograft model of human urothelial cancer in severe combined immunodeficient (SCID) mice for therapeutic simulation. METHODS: Pieces of 30 freshly resected urothelial tumors (24 obtained from bladder and 6 from ureter or pelvis) were implanted subcutaneously into SCID mice, and xenograft tumors were passed in tumorigenic cases. At each passage, histopathology, TP53 mutational status assessed by yeast p53 functional assay, and the Ki-67 labeling index (LI) were examined to evaluate the preservation of original features. A growth delay assay after single-dose irradiation was performed in four representative xenografts. RESULTS: Tumor growth was observed in 18 mice (60%, 18/30). Histologically, 15 of the 18 were epithelial carcinomas similar to the original tumors, whereas the other 3 were Epstein-Barr virus-associated lymphoproliferative disease, resulting in a 50% (15/30) take rate. No correlation was found between the tumor take rate and the clinicopathologic features, TP53 mutational status, or Ki-67 LI of the patients' tumors. Of these 15 xenografts, 11 xenografts were passed from 3 to 10 generations. TP53 mutational status remained stable during the passages, and the Ki-67 LI of eight xenografts was within a range of 50% of the LI of the original tumors, although the other three xenografts increased by over 50%. Specific growth delay after irradiation, independent of the original tumor growth speed and Ki-67 LI, was observed in four xenografts. CONCLUSIONS: SCID mice are useful recipients for investigations of human urothelial cancer with a wide biological range. This easy-to-handle xenograft system can help to develop a better in vivo preclinical evaluation system for therapeutic agents as well as the investigation of tumor pathophysiology.     

Fascicular Parasystole Associated with Tachycardia-Dependent Right Bundle Branch Block

A 43-year-old female with a chief complaint of palpitation was subjected to clinical electrophysiological studies. Initial standard 12-lead ECG revealed that her palpitation was caused by fascicular parasystole firing at the basic cycle length of 1.25-1.40 seconds, and that both sinus and parasystolic beats were associated with left anterior fascicular block and tachycardia-dependent RBBB. His-bundle electrocardiogram suggested that the parasystolic focus was located in the proximal portion of the anterior fascicle of the left bundle branch and that the site of tachycardia-dependent conduction block was located in the main right bundle branch. These findings suggest that diffuse pathological changes in the intraventricular conducting system were responsible for both the conduction block and automatic impulse formation in the present case.     

The effects of dichloroacetate on liver damage and circulating fuels in rats exposed to carbon tetrachloride

Abstract It has been known that carbon tetrachloride-induced liver damage in starved rats is ameliorated simply by restoration of feeding. An analogue of dichloroacetate has been reported to ameliorate carbon tetrachloride-induced liver damage, and dichloroacetate has been shown to have a variety of effects on fuel metabolism. We investigated simultaneously the effects of dichloroacetate on liver damage and on circulating fuels in rats exposed to carbon tetrachloride. The effects of carbon tetrachloride varied with the rat's condition. In starved rats, the liver damage was more severe, and serum ketone body concentration decreased. In non-starved rats, the liver damage was not as severe and the serum ketone body concentration increased. The administration of dichloroacetate ameliorated liver damage both in starved and in non-starved rats given carbon tetrachloride: the administration of dichloroacetate protected from the liver damage particularly in starved rats. There were associated changes in the concentractions of circulating fuels. When the pyruvate-lowering effect of dichloroacetate was diminished in carbon tetrachloride-injected, starved rats, the alanine aminotransferase-lowering effect of dichloroacetate was also diminished. We propose that dichloroacetate's effect on fuel metabolism may produce a hepato-protective effect.     

Prediction of engraftment after peripheral blood stem cell transplantation by CD34-positive cells in grafts

total of 91 peripheral blood stem cell collections were performed in 26 children with various malignant tumors and peripheral blood stem cell transplantations (PBSCT) were performed in 15 of the children. There was a positive correlation between logarithm of total CD34⁺ cells/kg and logarithm of colony-forming unit-granulocyte macrophage (CFU-GM)/kg (r = 0.86). The time elapsed until the white blood cells (WBC) exceeded 1000/μL was related to both CFU-GM (r = 0.67) and CD34⁺ cell count (r = 0.60). The number of days elapsed until platelet count exceeded 5 times 10⁴/μL was not related to the logarithm of CFU-GM count/10⁵ per kg transfused (r = 0.47), but was related to the logarithm of CD34⁺ cell counts/10⁶ per kg transfused (r = 0.73). The number of days elapsed until the reticulocytes exceeded 5 times 10⁴/μL was not related to the logarithm of CFU-GM count/10⁵ per kg (r = 0.52), but was related to the logarithm of CD34⁺ cell counts/10⁶ per kg transfused (r = 0.91). Although CD34⁺ cell counts correlated with the number of CFU-GM, bone marrow regeneration rates in three lineages were predicted more accurately by the number of CD34⁺ cells transfused than by the number of CFU-GM. These results suggest that measurement of the CD34⁺ cell count may be useful in predicting bone marrow regeneration rate after PBSCT.