Preliminary Study on Antiradiation Effect of Kuei-Pi-Tang

In order to evaluate the potential action of Kuei-Pi-Tang as an antiradiation agent, colony forming units of bone marrow cells in the spleen (CFUs) were used. Different sequences of X-ray irradiation with or without Kuei-Pi-Tang administration in the groups of ICR strain mice were intraperitoneally injected 10mg/20g or 20mg/20g, once a day, for consecutive seven days before or after 4Gy X-ray irradiation. After the different treatments, whole blood was collected from the tail endings to observe the fluctuation of leukocytes, erythrocytes and thrombocytes. The administration of 20mg/20g was more effective than that of 10mg/20g. Lower radiosensitivity was observed with the treatment of 20mg/20g of Kui-Pi-Tang than that of 10mg/20g. The injection of Kuei-Pi-Tang accelerated the recovery of blood cell counts of leukocytes, erythrocytes and thrombocytes in mice irradiated with 4Gy, especially for leukocytes under the treatments with 20mg/20g of Kuei-Pi-Tang administered after irradiation.

Download Full-text

Preliminary Study on the Anti-radiation Effect of Jen-Sheng-Yang-Yung-Tang

The American Journal of Chinese Medicine ◽

10.1142/s0192415x93000224 ◽

1993 ◽

Vol 21 (02) ◽

pp. 187-195 ◽

Cited By ~ 7

Author(s):

Hsue-yin Hsu ◽

Yau-hui Ho ◽

Shi-Iong Lian ◽

Chun-ching Lin

Keyword(s):

Bone Marrow ◽

Radiation Effect ◽

Bone Marrow Cells ◽

X Rays ◽

Male Mice ◽

X Ray ◽

Colony Forming Units ◽

Before And After ◽

Preliminary Study ◽

Different Doses

Six to seven week old male mice of ICR strain were exposed to different doses of x-rays to determine if Jen-Sheng-Yang-Yung-Tang could be a modifier in the elimination of radiation damage. Colony forming units of bone marrow cells in the spleen (CFUs) were measured before and after x-ray irradiation with intraperitoneal injection of 10 mg/20 g or 20 mg/20 g body weight of Jen-Sheng-Yang-Yung-Tang, once a day for seven consecutive days. The recovery of CFUs and hemocytes counts by 4 Gy irradiation with Jen-Sheng-Yang-Yung-Tang administration was faster for a concentration of 20 mg/20 g than 10 mg/20 g. The measurement of 10-day CFUs showed an increase of radiotolerance in the treatment of 20 mg/20 g administration before x-ray irradiation. The injection of Jen-Sheng-Yang-Yung-Tang accelerated the recovery of hemocyte counts in mice irradiated with 4 Gy x-ray; the effect was especially profound for leukocytes with 20 mg/20 g Jen-Sheng-Yang-Yung-Tang administration after irradiation.

Download Full-text

Preferential Inhibition of An Activated Form of Janus Kinase 2 (JAK2) by a Novel JAK2 Inhibitor, NS-018

Blood ◽

10.1182/blood.v116.21.4107.4107 ◽

2010 ◽

Vol 116 (21) ◽

pp. 4107-4107 ◽

Cited By ~ 2

Author(s):

Yohei Nakaya ◽

Haruna Naito ◽

Junko Homan ◽

Seishi Sugahara ◽

Tatsuya Horio ◽

...

Keyword(s):

Bone Marrow ◽

Bone Marrow Cells ◽

Jak2 V617f ◽

Janus Kinase ◽

Wild Type ◽

X Ray ◽

Jak2 Inhibitor ◽

Colony Forming Units ◽

Ic50 Value ◽

Marrow Cells

Abstract Abstract 4107 A somatic point mutation of Janus Kinase 2 (JAK2) tyrosine kinase (JAK2 V617F) has been shown to occur at a high frequency in myeloproliferative neoplasm (MPN) patients. JAK2 V617F is a constitutively activated kinase that activates the JAK/STAT signaling pathway and dysregulates cell growth and function. These findings suggest that the inhibition of aberrant JAK2 activation has a therapeutic benefit. Our novel JAK2 inhibitor, NS-018, is highly active against JAK2 with an IC50 value of less than 1 nM, and it has 30–50-fold selectivities for JAK2 over other JAK-family kinases such as JAK1, JAK3 and Tyk2. We determined the X-ray structure of JAK2 in complex with NS-018. An Asp-Phe-Gly (DFG) motif is located at the N-terminus of the activation loop and regulates ATP binding. The resolved X-ray structure showed that NS-018 bound to JAK2 in the “DFG-in” active conformation. A molecular modeling study indicated that NS-018 would hardly bind to JAK2 in the “DFG-out” inactive conformation. In accordance with the structural analysis, NS-018 preferentially suppressed the growth of bone-marrow cells expressing activated JAK2. Thus, NS-018 reduced in a dose-dependent manner the number of erythroid colony-forming units (CFU-E) derived from bone-marrow cells taken from JAK2 V617F transgenic mice, but had only a limited effect on the number of colonies from wild-type mice (Figure A). NS-018 had no effect on the number of granulocyte-macrophage colony-forming units (CFU-GM) from either mouse strain. Furthermore, NS-018 showed potent antiproliferative activity against Ba/F3 cells expressing JAK2 V617F with an IC50 value of <100 nM but showed only minimal cytotoxicity against most other hematopoietic and non-hematopoietic cell lines (IC50 >3 μ M). In a mouse Ba/F3-JAK2 V617F leukemia model, NS-018 significantly prolonged survival during repeated oral administrations at 6.25 mg/kg bid and reduced splenomegaly at doses as low as 1.5 mg/kg bid. NS-018 was well tolerated at dosages of more than 100 mg/kg bid. In conclusion, NS-018 is a potent JAK2 inhibitor which preferentially inhibits an activated form of JAK2 and has potent in vitro and in vivo efficiency in preclinical studies. NS-018 is expected to be suitable for the treatment of MPN caused by aberrant JAK2 activation and its effectiveness will be verified by early-phase clinical investigations in the near future. JAK2 V617F preferential inhibition of erythrocyte colony growth Bone-marrow cells were collected from femurs of JAK2 V617F transgenic mice and same-strain BDF1 wild-type mice. (a) To detect CFU-E colonies, cells were treated with NS-018 in semisolid methylcellulose containing erythropoietin (EPO) and cell clusters were counted after incubation for two days. (b) To detect CFU-GM colonies, cells were treated with NS-018 in semisolid methylcellulose containing EPO, interleukin-3 (IL-3), IL-6 and stem cell factor and colonies were counted on day 7. Disclosures: Nakaya: Nippon Shinyaku Co., Ltd: Employment. Naito:Nippon Shinyaku Co., Ltd: Employment. Homan:Nippon Shinyaku Co., Ltd: Employment. Sugahara:Nippon Shinyaku Co., Ltd: Employment. Horio:Nippon Shinyaku Co., Ltd: Employment. Niwa:Nippon Shinyaku Co., Ltd: Employment. Shimoda:Nippon Shinyaku Co., Ltd: Research Funding.

Download Full-text

PKCδ Negatively Regulates Primary Megakaryocyte Proliferation and Differentiation

Blood ◽

10.1182/blood.v120.21.853.853 ◽

2012 ◽

Vol 120 (21) ◽

pp. 853-853

Author(s):

Satya P. Kunapuli ◽

John C Kostyak

Keyword(s):

Bone Marrow ◽

Blood Cell ◽

Dna Content ◽

Bone Marrow Cells ◽

Conflicts Of Interest ◽

Platelet Counts ◽

Cell Counts ◽

Apoptotic Protein ◽

Primary Mouse ◽

Marrow Cells

Abstract Abstract 853 Megakaryocytes are large, polyploid cells that give rise to platelets in the bone marrow and spleen. Megakaryocytes achieve their size and DNA content through a process known as endomitosis via signaling from the cytokine thrombopoietin (Tpo). We have previously determined that the novel Protein Kinase C isoforms theta (PKCθ) and delta (PKCδ) regulate a number of platelet functions including aggregation and secretion. However, the function of these two PKC isoforms in primary megakaryopoiesis has not yet been elucidated. Therefore we chose to utilize primary mouse megakaryocytes from WT, PKCδ−/− and PKCθ−/− mice to characterize the roles of PKCδ and PKCq in megakaryopoiesis. We were first able to determine via western blotting that megakaryocytes express more PKCd than either mononuclear bone marrow cells (p < 0.05) or progenitor cells isolated from bone marrow (p < 0.05). Deletion of PKCδ in mice caused an increase in white blood cell and platelet counts compared to WT mice (p < 005). However, deletion of PKCθ had no effect on murine blood cell counts. Observed increases in platelet counts in PKCδ−/− mice are due to increased platelet production as PKCδ−/− mice contain more thiazole orange positive platelets than WT mice (p < 0.05). Furthermore, we determined via flow cytometry that PKCδ−/− mice had more bone marrow megakaryocytes than WT mice (p < 0.05), although megakaryocyte DNA content was unaltered. Conversely, there was no alteration in megakaryocyte number or DNA content with PKCθ deletion. Interestingly, the increase in bone marrow-derived megakaryocyte count observed in PKCδ−/− mice was heightened following culture of bone marrow cells in 50ng/mL exogenous Tpo (Figure 1). Furthermore, in similar experiments, megakaryocyte DNA content was also enhanced in PKCδ−/− mice compared to WT mice (p < 0.05). PKCδ is an important pro-apoptotic protein, and heightened megakaryocyte number following culture could be due to reduced apoptosis in PKCδ−/− megakaryocytes. However, neither apoptosis nor necrosis was altered with PKCδ deletion as the number of AnnexinV+/7AAD- cells, and the number of AnnexinV+/7AAD+ cells, were not different from WT. Therefore, the observed increases in megakaryocyte number and DNA content could be due to elevated Tpo-induced signaling as ERK1/2 phosphorylation was heightened in PKCδ−/− megakaryocytes compared to WT, in response to exogenous Tpo (Figure 2). These data suggest that PKCδ is an important megakaryopoietic protein, which negatively regulates signaling induced by Tpo in megakaryocytes, while PKCθ is dispensable for primary mouse megakaryopoiesis. Disclosures: No relevant conflicts of interest to declare.

Download Full-text

Effect and Kinetics of Depleting ACK-2 Anti C-Kit Monoclonal Antibody on Hematopoeisis and Hematopoetic Progenitors and Ability To Condition for Bone Marrow Transplantation.

Blood ◽

10.1182/blood.v104.11.4963.4963 ◽

2004 ◽

Vol 104 (11) ◽

pp. 4963-4963

Author(s):

Daniel L. Kraft ◽

Irving L. Weissman

Keyword(s):

Monoclonal Antibody ◽

Bone Marrow ◽

Stem Cell ◽

Blood Cell ◽

Peripheral Blood ◽

Bone Marrow Cells ◽

Peripheral Blood Cell ◽

Post Transplant ◽

Cell Counts ◽

Blood Cell Counts

Abstract c-kit is expressed on hematopoetic stem and progenitor cell populations. ACK-2 is an anti-mouse c-Kit monoclonal antibody which has been shown (by Nishikawa et al) to antagonize the function of c-kit and deplete the bone marrow of treated mice. We wished to characterize the effect of the anti c-kit mAb ACK-2 on the peripheral blood cell counts and marrow hematopoetic stem cell and progenitor populations. We also tested the hypothesis that treatment with ACK-2 may serve as a novel means of non-myeloablative conditioning for hematopoeitic stem cell transplantation. METHODS: Adult C57 black mice were injected either intravenouslyl or intraperitonealy with 1mg of ACK2 mAb every other day on Day 0, Day 2 and Day 4. Peripheral blood cell counts, including white cell differentials were followed over time in recipient mice. Peripheral blood and bone marrow was analyzed at Day 7, and marrow was analyzed for fraction and proportions of hematopoetic stem cells (HSC), common myeloid progenitors (CMP), granulocyte macrophage progenitors (GMP) and macrophage erythrocyte progenitors (MEP). A subset of Ly5.1 mice which had received ACK2 mAb on Days 0,2 and 4 received a bone marrow transplant on Day 7 with 1 Million bone marrow cells from a Ly5.2 donor. Peripheral blood from transplanted recipient mice was analyzed three weeks post transplant for presence of Ly5.2 donor derived cell engraftment. RESULTS: Both intravenous and intraperitoneal administration of ACK-2 resulted in rapid development of anemia, neutropenia and thrombocytopenia. In mice treated with intravenous ACK-2 on Days 0,2,4, the WBC dropped from a mean (3 mice) of 12.12 at Day 0 in untreated controls, to 6.6 at Day 2 to 4.2 at Day 4, and to 2.56 at day 7, recovering to a WBC of 9.5 by Day 11. The mean neutrophil dropped rapidly from a control mean of 796 prior to treatment, to 180 at Day 2, to 32 on Day 4, 26 on Day 7, recovering to a mean of 320 by Day 11. Hemoglobin dropped from a pretreatment mean of 15.1 to 14.5, 13.2, 6.2 and 5.3 on Days 2,4,7,11 respectively. Platelet counts fell from 1107 pre ACK-2 to 763 at Day 7 and 220 and Day 11, recovering to 1226 by Day 16. Analysis of Day 7 peripheral blood revealed an decrease in Mac-1+ cells from 12.2 of circulating white cells in untreated controls to 3.1%, while the proportion of B220+ B cells increased from 49.7 to 72.7%. The fraction of circulating T cells decreased from 24.5% to 11.1% at Day 7. The bone marrow fraction of KTLS HSC decreased from.07% in controls to.003% in Day 7 treated mice. Marrow fraction CMP decreased from 12% in untreated to 4.7% in treated, wheras the GMP population dropped from 66 to 53%. In ACK-2 treated mice which received 1 Million Ly5.2 syngeneic bone marrow cells analyzed at Day 21 post transplant, there was no evidence of Ly5.2+ donor derived peripheral blood white cells, as compared to 80% Ly5.2 donor derived cells in control mice which had received 9 Gray of radiation for conditioning. CONCLUSIONS: The anti-kit Monocolonal antibody rapidly induces anemia, neutropenia and thrombocytopenia with decreases in the marrow HSC and other progenitor populations. ACK-2 does not appear to facilitate non-myelablative conditioning for a syngeneic graft when given alone.

Download Full-text