Cytokinetic parameters of erythropoiesis under normal health conditions and during the development of acute lymphoblastic and acute myeloblastic leukemia

The article presents the application of the method for determining the quantitative parameters of erythropoiesis to patients with acute leukemia. The objective of the work is an investigation of erythropoiesis cytokinetic parameters under the normal health conditions and during the development of the acute lymphoblastic leukemia and the acute myeloblastic leukemia. It was found that the distribution of reticulocytes shifts towards an increase of immature reticulocyte fraction while the ratio between maturing and immature cells remains unchanged. The method presented can be used in clinical diagnostic and scientific research of bone marrow hematopoietic activity.

TET2 Inhibitory Effects of Eltrombopag Contribute Its Hematopoietic Activity

Severe aplastic anemia (sAA) is bone marrow (BM) failure syndrome characterized by immune mediated BM hypoplasia and pancytopenia usually treated with immunosuppressive therapy (IST) or allogeneic BM transplant. Despite the successes of IST in treating acquired, idiopathic sAA, most of the available treatment options only convert severe into non-severe forms of disease. Recently, eltrombopag (Epag), a small molecule thrombopoietin receptor (TPOR) agonist has been introduced into the routine treatment of de-novo and refractory AA with robust clinical response. Epag treatment showed remarkable tri-lineage responses in AA, suggesting an effect on hematopoietic stem and progenitor cells (HSPCs) amplification. The use of hematopoietic growth factors in AA that increases HSPCs has been associated with justified fears of facilitating AA evolution to MDS. Similarly, Epag has parallel effects on the amplification of HSPCs and there has been recent signals that long term treatment of Epag may increase the MDS risk. Using an in-house developed highly stable cell free high throughput screening of bioactive small molecule chemical library, we identified Epag as one of the most potent inhibitor of TET2. TET2 is the most abundant DNA dioxygenase in HSPCs. The loss-of-function mutations of TET2 frequently occurs in myeloid neoplasia. Here using in vitro and in vivo model system we report that effect of Epag, in part, is mediated by its ability to inhibit TET-dioxygenase activity. We demonstrate that Epag directly binds to TET2 in the presence of Fe2+ and inhibits its dioxygenase function in cell free system. In cell culture model we observed that nearly 40% of Epag partitioned into to nucleus suggesting its potential TET2 inhibitory role in cells. To test if TPOR signaling activation by Epag has any impact on TET activity we engineered murine cell lines BaF3 and 32D by introducing human TPOR. Irrespective of the status of TPOR-JAK-STAT pathway activation, as reflected in STAT5 phosphorylation, Epag demonstrated a robust TET-inhibition activity. However, in similar experiments peptide agonist of TPOR or another small molecule TPOR agonist Avotrombopag did not have any TET inhibitory effects. Consistent with these observations, Epag but not TPO or Avotrombopag inhibited TET activity in murine BM. Thus, Epag mediated TET2 inhibition in these cells are specific and independent of TPOR signaling. Consistent with previous reports, we observed that Epag treatment can significantly expand murine HSPCs even in the absence of TPOR signaling activation. The hematopoietic activity of Epag in murine system is dependent on its ability to inhibit TET2 as confirmed by the lack of Epag effects on Tet2-/- HSPCs. We further confirmed that TET-inhibition by Epag is central for its ability to expand HSPCs in murine model under in vivo settings using BM transplant experiments. We transplanted a mixture of cells derived from Tet2+/+ Pep Boy (CD45.1, 95%) and Tet2-/- C57BL/6 (CD45.2, 5%) into lethally irradiated recipient PepBoy mice. Epag treatment of mice reconstituted with the mixture of BM cells increases the fraction of Tet2+/+ BM cells only. Interestingly, Epag mimic loss of Tet2 in vivo as observed in the expansion of myeloid compartments of Tet2+/+ fractions reflected in CD11b-CD11c+, CD11b+CD11c-, CD11b+CD11c-Ly6C+Ly6G- (monocytes) and CD11b+CD11c-Ly6ClowLy6G+ (neutrophils). We did not observe any significant change in B220+, CD4+ or CD8+ lymphoid populations. On the contrary, we observed a growth restrictive effect of Epag treatment on Tet2-/- myeloid cells. Consistent with our hypothesis that Epag inhibits TET-dioxygenase activity, we observed hypermethylation in the mononuclear cells derived AA patients (n= 16) after Epag treatment. This was further confirmed in ex vivo Epag treatment of BM cells derived from healthy donor. Epag treatment significant increase clonogenic expansion of normal BM cells but not TET2 mutant myeloid cells. In summary, here we demonstrate that Epag is a potent inhibitor of TET-dioxygenase and its biological consequences are independent of TPOR-JAK-STAT activation. Epag treatment mimics TET2 loss of function in AA cells, however it restricts clonal expansion of TET2 mutant myeloid cells. Disclosures Patel: Alexion: Other: educational speaker. Nazha:Jazz: Research Funding; Incyte: Speakers Bureau; MEI: Other: Data monitoring Committee; Novartis: Speakers Bureau. Saunthararajah:EpiDestiny: Consultancy, Current equity holder in private company, Patents & Royalties: University of Illinois at Chicago. Sekeres:Pfizer: Consultancy; BMS: Consultancy; Takeda/Millenium: Consultancy. Maciejewski:Alexion, BMS: Speakers Bureau; Novartis, Roche: Consultancy, Honoraria.

Network Pharmacology-Based Investigation of the System-Level Molecular Mechanisms of the Hematopoietic Activity of Samul-Tang, a Traditional Korean Herbal Formula

Hematopoiesis is a dynamic process of the continuous production of diverse blood cell types to meet the body’s physiological demands and involves complex regulation of multiple cellular mechanisms in hematopoietic stem cells, including proliferation, self-renewal, differentiation, and apoptosis. Disruption of the hematopoietic system is known to cause various hematological disorders such as myelosuppression. There is growing evidence on the beneficial effects of herbal medicines on hematopoiesis; however, their mechanism of action remains unclear. In this study, we conducted a network pharmacological-based investigation of the system-level mechanisms underlying the hematopoietic activity of Samul-tang, which is an herbal formula consisting of four herbal medicines, including Angelicae Gigantis Radix, Rehmanniae Radix Preparata, Paeoniae Radix Alba, and Cnidii Rhizoma. In silico analysis of the absorption-distribution-metabolism-excretion model identified 16 active phytochemical compounds contained in Samul-tang that may target 158 genes/proteins associated with myelosuppression to exert pharmacological effects. Functional enrichment analysis suggested that the targets of Samul-tang were significantly enriched in multiple pathways closely related to the hematopoiesis and myelosuppression development, including the PI3K-Akt, MAPK, IL-17, TNF, FoxO, HIF-1, NF-kappa B, and p53 signaling pathways. Our study provides novel evidence regarding the system-level mechanisms underlying the hematopoiesis-promoting effect of herbal medicines for hematological disorder treatment.

Effect of Sinorhizobium meliloti lipopolysaccharide on blood cell composition in experiment

Липолисахариды (ЛПС, эндотоксины) грамотрицательных бактерий обладают выраженной биологической активностью, в том числе терапевтической, однако для S. meliloti таких данных нет. Цель работы - экспериментальное изучение гемопоэтической активности 4 фракций липополисахаридов, выделенных из S. meliloti, при индуцированном иммунодефиците у мышей. Методика. Сформировано 7 групп лабораторных мышей (по 10 особей в каждой): 1-я группа - интактные (контроль 1), 2-я - 7-я группа - мыши с иммунодефицитным состоянием, индуцированным однократным внутрибрюшинным введением циклофосфамида. Через 1 сут после моделирования иммунодефицита в течение 21 сут ежедневно мышам 3-й группы вводили препарат сравнения Ликопид® (химическое название: [4-O-(2-ацетиламино-2-дезокси-β-D-глюкопиранозил)-N-ацетилмурамил]-L-аланил-D-α-глутамиламид - синтетический аналог бактериальных гликопептидов из группы иммуностимулирующих средств). Мышам 4-7-й групп - вводили исследуемые фракции липолисахаридов - ЛПС-1, ЛПС-2, ЛПС-3 и ЛПС-4 соответственно. Для ликопида разовая доза составляла 0,1 мл (0,05 мг/мл), для исследуемых фракций ЛПС S. meliloti - 0,2 мл (10 пг/мл). Иммунодефицитным мышам 2-й группы фракции липополисахаридов и препарат сравнения Ликопид® не вводили Через 21 сут мышей выводили из эксперимента. Изучали весовые характеристики органов подопытных животных и лейкоцитарную формулу. Результаты. Введение мышам на фоне вторичного экспериментального иммунодефицита ликопида сопровождалось снижением количества палочкоядерных нейтрофилов и моноцитопенией; при введении фракции ЛПС-1 возрастало количество сегментоядерных нейтрофилов; ЛПС-2 - имели место снижение содержания палочкоядерных нейтрофилов и лимфоцитоз; ЛПС-3 - наблюдали снижение содержания палочкоядерных нейтрофилов и лимфоцитоз на фоне значимого увеличения количества сегментоядерных нейтрофилов; ЛПС-4 - констатировалось увеличение числа базофилов, снижение содержания палочкоядерных нейтрофилов и лимфоцитоз на фоне значимого увеличения количества сегментоядерных нейтрофилов. Заключение. Фракции ЛПС Sinorhizobium meliloti проявляют модулирующие эффекты, схожие с механизмами «экстренного миелопоэза» при физиологичном варианте течения бактериальных инфекций. Lipolysaccharides (LPS, endotoxins) of gram-negative bacteria have a pronounced biological activity, including therapeutic activity; however, there is no such data for S. meliloti. Aim. To conduct an experimental study of hematopoietic activity of four lipopolysaccharide fractions isolated from S. meliloti under induced immunodeficiency in mice. Methods. 7 groups of 10 laboratory mice each were formed: group 1, intact mice (control 1); groups 2-7, mice with immunodeficiency induced by a single intraperitoneal injection of cyclophosphamide. Mice of group 3 were daily injected with a comparison agent, Licopid® (Chemical name: [4-O- (2-acetylamino-2-deoxy-β-D-glucopyranosyl) -N-acetylmuramyl] -L-alanyl-D-α-glutamyl amide; single dose, 0.1 ml (0.05 mg/ml)) for 21 days starting one day after the induction of immunodeficiency. Mice of groups 3-7 were injected with the studied S. meliloti LPS fractions, LPS-1, LPS-2, LPS-3, and LPS-4, respectively (single dose, 0.2 ml (10 pg/ml)). Immunodeficient mice of group 2 received neither the comparison agent, Licopid® nor LPS fractions. The mice were euthanized at 21 days. Weight characteristics of animal organs and white blood count were studied. Results. Administration of Licopid® to mice with secondary experimental immunodeficiency was associated with decreased count of stab neutrophils and monocytopenia; LPS-1 fraction increased the count of segmented neutrophils; LPS-2 decreased the count of stab neutrophils and induced lymphocytosis; LPS-3 decreased the count of stab neutrophils and induced lymphocytosis associated with a significant increase in the count of segmented neutrophils; LPS-4 induced basophilia, decreased count of stab neutrophils, and lymphocytosis associated with a significant increase in the count of segmented neutrophils. Conclusion. Sinorhizobium meliloti LPS fractions exerted modulating effects similar to the mechanisms of “emergency myelopoiesis” in the physiological course of bacterial infections.

Comparative analysis of hematopoietic activity of total RNA from bone marrow cells and splenocytes of rats with chronic benzene-induced anemia

Цель - оценка лечебных свойств суммарной РНК на модели хронической гипопластической анемии, выявление особенностей гемопоэтического действия суммарных РНК, выделенных из костного мозга и лимфоидных клеток селезенки. Методика. Работа выполнена на 36 белых беспородных крысах-самках массой 130-250 г. Хроническую токсическую анемию моделировали подкожным введением смеси химически чистого бензола (0,05 мл/100 г) и стерильного растительного масла. Смесь вводили трижды с интервалом в 7 сут. Контрольным животным (группа 1 в каждой серии) в эти же сроки внутривенно вводили равное по объему количество 0,9% раствора NaCl. Выполнено 2 серии экспериментов. В 1-й серии оценивали гемопоэтическую активность суммарных РНК лимфоидных клеток селезенки, во 2-й - суммарных РНК костного мозга. Препараты суммарной РНК получали из костного мозга и лимфоидных клеток селезенки интактных крыс, а также из клеток тех же органов крыс, подвергнутых кровопусканию (2% от массы тела) за 17 ч до выделения из них РНК. Суммарную РНК выделяли методом гуанидин тиоцианат-фенол-хлороформной экстракции. Для выяснения влияния суммарных РНК на состояние эритроидного ростка костного мозга в препаратах, окрашенных по Паппенгейму, оценивали количественный и качественный состав эритроидных островков. Результаты. Сравнение гемопоэтической активности суммарных РНК, выделенных из костного мозга и лимфоидных клеток селезенки здоровых животных, при их введении крысам с экспериментальной хронической бензольной анемией, показало, что различия клеточного состава лимфоидных органов-источников РНК отражается на их гемопоэтических свойствах. Суммарная РНК из лимфоидных клеток селезенки интактных животных в первую очередь способствует восстановлению количества лейкоцитов, в то время как суммарная РНК лимфоидных клеток костного мозга в начальный период регенерации не оказывает заметного влияния на лейкопоэз. Показано также, что суммарная РНК, выделенная из костного мозга, в целом обеспечивает более интенсивный процесс гемопоэза. Кроме того, обнаружена более высокая эритропоэтическая активность суммарных РНК, выделенных из клеток обоих лимфоидных органов крыс-доноров, стимулированных кровопотерей, по сравнению с таковой суммарных РНК, полученных из органов интактных крыс. Заключение. Подтверждена гемопоэтическая активность суммарной РНК костного мозга и лимфоидных клеток селезенки при гипопластической анемии. Выявлена более высокая эритропоэтическая активность суммарных РНК обоих лимфоидных органов животных после кровопускания по сравнению с их лейкопоэтической активностью. Aim. To evaluate therapeutic properties of total RNA on a model of chronic hypoplastic anemia and to elucidate features of the hemopoietic effect of total RNA isolated from bone marrow splenic lymphoid cells. Methods. Experiments were performed on 36 white mongrel rats weighing 230-250 g. Chronic toxic anemia was induced by subcutaneous injections of a mixture of chemically pure benzene (0.05 ml/100 g) and sterile vegetable oil. The mixture was injected three times with an interval of 7 days. Control animals (group 1 in each series) received intravenous injections of an equal volume of saline at the same time intervals. Two experimental series were performed. In series 1 and 2, hemopoietic activity of total RNAs from splenic lymphoid cells and from bone marrow, respectively, was evaluated. Total RNA was obtained from splenic lymphoid cells and from bone marrow of intact rats and rats subjected to hemorrhage (2% of body weight) 17 h prior to the RNA isolation. Total RNA was isolated by guanidine-thiocyanate-phenol-chloroform extraction. The effect of total RNA on the erythroid lineage in the bone marrow was evaluated by the quantitative and qualitative composition of erythroid islets in Pappenheim-stained preparations. Results. Comparing the hematopoietic activity of total RNA isolated from bone marrow and splenic lymphoid cells of healthy animals and administered to rats with experimental chronic benzene anemia showed that the difference in cell composition of the lymphoid organs, sources of the RNAs, impacted on the RNA hemopoietic properties. The total RNA from splenic lymphoid cells of intact animals contributed primarily to the recovery of leukocyte count while the RNA from the bone marrow of intact rats did not significantly influence leukopoiesis during the early regeneration period. It was also shown that the total RNA extracted from the bone marrow generally provided a more intensive hemopoiesis. In addition, the erythropoietic activity of total RNA isolated from the cells of both lymphoid organs of the donor rats stimulated by blood loss was higher than that of the total RNA isolated from intact rats. Conclusion. The total RNA isolated from bone marrow and lymphoid cells spleen exerted a hemopoietic effect in hypoplastic anemia. The erythropoietic activity of total RNA from both lymphoid organs of animals after hemorrhage was higher than the RNA leukopoietic activity.