The FMCA-GM assays,high throughput non-clonogenic alternatives to CFU-GM in preclinical hematotoxicity testing

One of the most common dose limiting adverse effects in cancer treatment is myelotoxicity. The aim of this study was to develop an in vitro method for measuring potential myelotoxic properties of a drug candidate in a high throughput setting. Human CD34⁺ progenitor cells from umbilical cord blood were plated in 384-well microplates with drugs in liquid culture, supplemented with specific cytokines for the granulocytopoietic-macrophage lineage. After 7 or 14 days of proliferation and differentiation the cells were analyzed using the automated non-clonogenic fluorometric microculture cytotoxicity assay (FMCA). Two types of assays setups were evaluated, the FMCA-GM7 where cells were exposed to drugs directly after thawing and cytotoxicity measured on day 7 in contrast to the FMCA-GM14 where the cells were cultured 7 days prior to plating and drug exposure, with viability analysis on day 14 of differentiation. Drug sensitivity was similar in both assays and method validation was performed using 24 drugs with known myelotoxic profile (acyclovir, bortezomib, busulfan, carboplatin, chloramphenicol, chlorpromazine, cisplatin, cytarabine, clozapine, doxorubicin, erlotinib, etoposide, 5-fluorouracil, fludarabine, gefitinib, gemcitabine, hydroxyurea, imatinib, lomustine, melphalan, sorafenib, sunitinib, taxol and 6-thioguanine). The 50% inhibitory concentrations (IC₅₀) from the FMCA-GM7 and the FMCA-GM14 correlated highly (r = 0.83) and (r = 0.82), respectively, with IC₅₀ from the established clonogenic assay (CFU-GM), obtained from the literature. The current data suggests that the FMCA-GM could offer a simple and robust alternative to the CFU-GM assay in preclinical hematotoxicity studies.     

Valproic acid perturbs hematopoietic homeostasis by inhibition of erythroid differentiation and activation of the myelo-monocytic pathway

As a histone deacetylase inhibitor, valproic acid (VPA) is a candidate for anticancer therapy. Besides, VPA exhibits various mechanisms of action and its effects on the molecular basis of hematopoiesis remain unclear. To study the effects of VPA on the hematopoietic system, we performed microarray analysis using K562 cells treated with 1 mM VPA over a 72 h time course. The association between gene ontology (GO) terms and the lists of differentially expressed genes was tested using the Bioconductor package GOstats. Enrichment analysis for cellular differentiation pathways was performed based on manually curated gene lists. Results from microarray analysis were confirmed by studying cell differentiation features at the molecular and cellular levels using other hematopoietic cell lines as well as hematopoietic stem/progenitor CD34⁺ cells. Microarray analysis revealed 3440 modulated genes in the presence of VPA. Genes involved in the granulo-monocytic differentiation pathway were up-regulated while genes of the erythroid pathway were down-regulated. This was confirmed by analyzing erythrocytic and myeloid membrane markers and lineage-related gene expression in HEL, MEG01, HL60 as well as CD34⁺ cells. Moreover, GATA-1 and its co-factors (FOG1, SP1) were down-regulated, while myelopoiesis activator PU.1 was up-regulated, in agreement with an inhibition of erythropoiesis. Our functional profiling and cell phenotyping approach demonstrates that VPA is able to alter hematopoietic homeostasis by modifying the cell population balance in the myeloid compartment. This may lead to a potential failure of erythropoiesis in patients with cancer or chronic inflammatory diseases having a well-described propensity to anemia.     

Effect of Ultrafilterable Platinum Concentration on Cisplatin and Carboplatin Cytotoxicity in Human Tumor and Bone Marrow Cells in Vitro

The importance of the ultrafilterable platinum (fPt) fraction of cisplatin (CDDP) and carboplatin (CBDCA) for cytotoxicity and myelotoxicity was studied in vitro. By incubating CDDP or CBDCA with fetal calf serum (PCS) various fractions of fPt were prepared and determined by atomic absorption spectroscopy. A relation of % fPt fraction and incubation time (h) of 87^e-1123t(r = –0.99) and 101^e-o.oo87t (r = –0.99) were determined for CDDP and CBDCA, respectively. Cytotoxicity in the human small cell lung carcinoma cell line GLC₄ and fPt fraction were closely related for CDDP (r = 0.99) and for CBDCA r = 0.97). However, at a similar fPt fraction the concentrations inhibiting cell survival by 50% (IC50) of CBDCA exceeded that of CDDP by a factor of 10-18 with 4 h exposure and a factor of 5 with continuous exposure. Tested in the range of peak concentrations in plasma of patients and at a clinically relevant fPt fraction of 10%, CDDP was not toxic for human bone marrow cells in the CFU-GM assay, whereas it was toxic at fPt fractions of 50% and 90%. However, CBDCA was myelotoxic at a (clinically relevant) fPt fraction of 50%, and also at 75% and 90%. The use of different fPt fractions, produced by the incubation method described in this study, permits the study of platinum drugs in vitro while approximating in vivo conditions might be used to evaluate myelotoxicity of new platinum drugs prospectively. For CDDP and CBDCA the fraction fPt determines cytotoxicity on tumor cells, and their different fPt fraction in patients account at least partly for their difference in myelotoxicity.     

Phloroglucinol Inhibits the in vitro Differentiation Potential of CD34 Positive Cells into Endothelial Progenitor Cells

Inhibiting the bioactivities of circulating endothelial progenitor cells (EPCs) results in significant inhibition of neovessel formation during tumor angiogenesis. To investigate the potential effect of phloroglucinol as an EPC inhibitor, we performed several in vitro functional assays using CD34⁺ cells isolated from human umbilical cord blood (HUCB). Although a high treatment dose of phloroglucinol did not show any cell toxicity, it specifically induced the cell death of EPCs under serum free conditions through apoptosis. In the EPC colony-forming assay (EPC-CFA), we observed a significant decreased in the small EPC-CFUs for the phloroglucinol group, implying that phloroglucinol inhibited the early stage of EPC commitment. In addition, in the in vitro expansion assay using CD34⁺ cells, treatment with phloroglucinol was shown to inhibit endothelial lineage commitment, as demonstrated by the decrease in endothelial surface markers of EPCs including CD34⁺, CD34⁺/CD133⁺, CD34⁺/CD31⁺ and CD34⁺/CXCR4⁺. This is the first report to demonstrate that phloroglucinol can inhibit the functional bioactivities of EPCs, indicating that phloroglucinol may be used as an EPC inhibitor in the development of biosafe anti-tumor drugs that target tumor angiogenesis.     

Cell screening assay for identifying inhibitors of eosinophil proliferation

The purpose of this study was to develop a cell‐based screening assay for identification of small molecules for the treatment of asthma. Eosinophils are leukocytes that contribute to the pathology of asthma. Lidocaine inhibits interleukin‐5 (IL‐5)‐mediated survival and activation of human eosinophils, and it is able to replace inhaled glucocorticoids for the treatment of asthma; however, lidocaine has many side effects, including anesthesia. Therefore, a collection of commercial and novel, synthesized lidocaine analogues were investigated for inhibitory activity of the IL‐5‐stimulated proliferation of TF‐1 cells, a CD34⁺, cytokine‐dependent, erythroleukemic cell line model for eosinophil growth. Among 74 investigated compounds, 10 were more potent inhibitors of cell proliferation than lidocaine (average IC₅₀ = 223 µM), with IC₅₀ values ranging within 1–119 µM. This cell‐based assay is an effective method for screening chemical compounds and has revealed promising lead compounds for the treatment of asthma. Drug Dev Res 72: 353–360, 2011. © 2011 Wiley‐Liss, Inc.     

Development and characterization of CD22-targeted pegylated-liposomal doxorubicin (IL-PLD)

Non-Hodgkin’s lymphoma (NHL) is the sixth most common cause of cancer deaths in the U.S. Most NHLs initially respond well to chemotherapy, but relapse is common and treatment is often limited due to the toxicity of chemotherapeutic agents. Pegylated-liposomal doxorubicin (PLD, Ben Venue Laboratories, Inc), a produces less myelotoxicity than non-liposomal (NL) doxorubicin. To further enhance efficacy and NHL targeting and to decrease toxicity, we conjugated an anti-CD22 monoclonal antibody (HB22.7) to the surface of PLD, thereby creating CD22-targeted immunoliposomal PLD (IL-PLD). HB22.7 was successfully conjugated to PLD and the resulting IL-PLD exhibits specific binding to CD22-expressing cells as assessed by immunofluorescence staining. IL-PLD exhibits more cytotoxicity than PLD in CD22 positive cell lines but does not increase killing of CD22 negative cells. The IC₅₀ of IL-PLD is 3.1 to 5.4 times lower than that of PLD in CD22+ cell lines while the IC₅₀ of IL-PLD is equal to that of PLD in CD22- cells. Furthermore, IL-PLD remained bound to the CD22+ cells after washing and continued to exert cytotoxic effects, while PLD and NL- doxorubicin could easily be washed from these cells.     

Mobilization of progenitor cells into peripheral blood by gamma-tocotrienol: A promising radiation countermeasure

Gamma-tocotrienol (GT3), a vitamin E isoform, is shown to induce high levels of granulocyte colony stimulating factor (G-CSF) in mice. G-CSF is a key cytokine used for stimulation of hematopoiesis, and mobilization of hematopoietic stem and progenitor cells into peripheral blood. GT3 is also shown to induce vascular endothelial growth factor (VEGF), another important cytokine necessary for vasculogenesis and endothelial progenitor mobilization. Since GT3 induces both these cytokines, we tested whether GT3 mobilizes hematopoietic and endothelial progenitors in mice. GT3 (200 mg/kg) was injected in 10-week-old CD2F1 mice and mobilization of progenitors in peripheral blood was analyzed at 24, 48, and 72 h post-administration. Circulating hematopoietic progenitor cells (HPCs, Lin^?, cKit⁺), endothelial progenitor cells (EPCs, Lin^?, CD34⁺, Flk⁺), and stromal progenitor cells (SPCs, Lin^?, CD29⁺, CD105⁺) in peripheral blood mononuclear cells (PBMCs) were analyzed simultaneously by flow cytometry. Mobilized HPCs, EPCs and SPCs in PBMC were also measured by colony-forming unit (CFU) assay in progenitor-specific media. Three groups of mice received vehicle, GT3 and GT3 plus AMD3100, a receptor antagonist used to enhance mobilization. GT3 induced significant mobilization of all three progenitor cell types compared to vehicle in peripheral blood; AMD3100 enhanced GT3-induced mobilization even further. Mobilization of progenitor cells in peripheral blood by GT3 indicates that GT3 can be used as an alternative to G-CSF and VGEF to mobilize HPCs and EPCs.     

Comparativein vitro myelotoxicity of FCE 24517, a distamycin derivative,to human,canine and murine hematopoietic progenitor cells

Summary FCE 24517, a derivative of distamycin A, exhibits an unusual antitumor profile in experimental models. As part of its preclinical development, we evaluated thein vitro myelotoxicity of FCE 24517 to human, canine and murine hematopoietic cells. Marrow cells were exposed to the agent (2.7×10^–5–2.7 nM) for 4 h and then assayed in capillary (human) or Petri dish (canine, murine) clonal cultures. FCE 24517 inhibited myeloid (CFU-gm), erythroid (BFU-e, CFU-e) and megakaryocytic (CFU-meg) colony formation in a concentration-dependent manner. The progenitor cells were generally similar in their response to FCE 24517 within a species. Comparing the different progenitor cell response to FCE 24517, canine CFU-gm and CFU-e were 26- to 221-fold more sensitive to this drug's toxic effects than their human and murine counterparts. This was demonstrated by extremely low IC₇₀ values for the canine CFU-gm (0.001 nM) and CFU-e (0.007 nM). Murine progenitors displayed 1.3- to 10.9-times higher IC₇₀ values than human CFU-gm, BFU-e and CFU-e following 4 hr exposure to FCE 24517. The data demonstrated that a mouse model may better predict humanin vitro myelotoxicity to FCE 24517 than beagle dogs.     

Cytotoxicity of various chemicals and mycotoxins in fresh primary duck embryonic fibroblasts: a comparison to HepG2 cells

To screen cost‐effectively the overall toxicity of a sample, particularly in the case of food and feed ingredient quality control, a sensitive bioassay is necessary. With the wide variety of cytotoxicity assays, performance comparison between assays using different cells has become of interest. Fresh primary duck embryonic fibroblasts (DEF) were hypothesized to be a sensitive tool for in vitro cytotoxicity screening; cell viability of DEF in response to various cytotoxins was determined and compared with response of HepG2 cells. The IC₅₀ values by the alamar blue assay in the DEF cells had a high correlation (R² = 0.96) with those obtained in HepG2 cells. Within the same toxin, primary DEF yielded significantly lower IC₅₀ values than that obtained from HepG2 cells using the MTT and alamar blue assay. Additionally, primary DEF responded to all mycotoxins tested using the alamar blue assay, while HepG2 was less sensitive, particularly at short exposure times. The estimated IC₅₀ for aflatoxin B₁, fumonisins B₁ and deoxynivalenol in DEF after 72 h incubation were 3.69, 4.19 and 1.26 μg ml^–1, respectively. Results from the current study suggest that primary DEF are more sensitive to cytotoxins and mycotoxins compared to HepG2, and thus may have great potential as an effective tool for cytotoxicity assessment. The question remains whether in vitro IC₅₀ values can accurately predict in vivo toxicity; however, the current study accentuates the need for further attention to identify sensitive cell models for in vitro cytotoxicity screening and subsequent exploration of species‐specific prediction models for in vivo toxicity. Copyright © 2016 John Wiley & Sons, Ltd.     

Generation of CD2+CD8+ NK Cells from c-kit+ Bone Marrow Cells in Porcine

Natural killer (NK) cells provide one of the initial barriers of cellular host defense against pathogens, in particular intracellular pathogens. Because bone marrow-derived hematopoietic stem cells (HSCs), lymphoid protenitors, can give rise to NK cells, NK ontogeny has been considered to be exclusively lymphoid. Here, we show that porcine c-kit⁺ bone marrow cells (c-kit⁺ BM cells) develop into NK cells in vitro in the presence of various cytokines [interleukin (IL)-2, IL-7, IL-15, IL-21, stem cell factor (SCF), and fms-like tyrosine kinase-3 ligand (FLT3L)]. Adding hydrocortisone (HDC) and stromal cells greatly increases the frequency of c-kit⁺ BM cells that give rise to CD2⁺CD8⁺ NK cells. Also, intracellular levels of perforin, granzyme B, and NKG2D were determined by RT-PCR and western blotting analysis. It was found that of perforin, granzyme B, and NKG2D levels significantly were increased in cytokine-stimulated c-kit⁺ BM cells than those of controls. And, we compared the ability of the cytotoxicity of CD2⁺CD8⁺ NK cells differentiated by cytokines from c-kit⁺ BM cells against K562 target cells for 28 days. Cytokines-induced NK cells as effector cells were incubated with K562 cells as target in a ratio of 100:1 for 4 h once a week. In results, CD2⁺CD8⁺ NK cells induced by cytokines and stromal cells showed a significantly increased cytotoxicity 21 days later. Whereas, our results indicated that c-kit⁺ BM cells not pretreated with cytokines have lower levels of cytotoxicity. Taken together, this study suggests that cytokines-induced NK cells from porcine c-kit⁺ BM cells may be used as adoptive transfer therapy if the known obstacles to xenografting (e.g. immune and non-immune problems) were overcome in the future.     

In vitro myelotoxicity assessment of the emerging mycotoxins Beauvericin, Enniatin b and Moniliformin on human hematopoietic progenitors

The aim of this study was to screen potential myelotoxicity of the emerging mycotoxins Beauvericin, Enniatin b and Moniliformin using human hematopoietic progenitor clonogenic assays.Depending on mycotoxins, inhibitory effects on proliferation of white blood cells progenitors (CFU-GM), platelet progenitors (CFU-MK) and red blood cells progenitors (BFU-E) have been detected at various concentrations. Beauvericin was cytotoxic at 32 μM, 3.2 μM and 6.4 μM, had no effect on proliferation in the presence of 0.032 μM, 0.16 μM and 0.064 μM, and the IC₅₀ was equal to 3.4 μM, 0.7 μM and 3.7 μM for CFU-GM, CFU-MK and BFU-E, respectively. Enniatin b was cytotoxic at 6 μM, 1.8 μM and 5 μM, had no effect on proliferation in the presence of 1 μM, 1.1 μM and 1.2 μM and the IC₅₀ was equal to 4.4 μM, 1.3 μM and 3.3 μM for CFU-GM, CFU-MK and BFU-E, respectively. Moniliformin was not cytotoxic at tested concentrations for CFU-GM and CFU-MK and cytotoxic at 10 μM for BFU-E, had no effect on proliferation in the presence of 5 μM, 0.1 μM and 0.1 μM and the IC₅₀ was equal to 31 μM, 39 μM and 4.1 μM for CFU-GM, CFU-MK and BFU-E, respectively.Inhibition of the BFU-E differentiation has been observed in the presence of Enniatin b or Moniliformin. For the three mycotoxins, variation of distribution of CFU-MK colonies according to their size has been observed. These in vitro effects may be responsible for in vivo hematological troubles in case of consumption of contaminated commodities. In vivo studies have to be performed to test this hypothesis.     

淋巴细胞亚群变化与糖皮质激素治疗早期乙肝肝衰竭患者的相关性研究

目的 探讨糖皮质激素治疗早期乙肝肝衰竭患者的病情转归及与淋巴细胞亚群的相关性。方法 纳入2011年12月—2014年12月收治的乙型病毒性肝炎慢加急(亚急性)早期肝衰竭患者60例,按知情同意分为2组,糖皮质激素治疗组32例,对照组28例。流式细胞仪检测治疗前后淋巴细胞亚群绝对计数的变化,观察2组的病情转归。结果 2组治疗前淋巴细胞亚群差异无统计学意义。治疗后,与对照组相比,治疗组CD4⁺/CD8⁺比例、CD3^-CD16⁺CD56⁺细胞增加,差异有统计学意义。与治疗前相比,治疗后治疗组CD3⁺CD4⁺T细胞绝对计数、对照组CD3⁺CD4⁺T细胞、CD3⁺CD8⁺T细胞绝对计数增多,差异有统计学意义。2组救治成功率比较,治疗组75.00%,对照组64.29%,差异无统计学意义(Z=0.816,P=0.366)。但10 d病情稳定率治疗组71.88%,高于对照组46.43%,差异有统计学意义(Z=4.029,P=0.044)。救治成功患者住院天数比较,治疗组35.00 d(29.00,51.00),低于对照组50.50 d(38.25,58.00),差异有统计学意义(Z=-2.241,P=0.025)。结论 在早期乙肝肝衰竭患者中应用糖皮质激素可较快稳定病情,减少住院时间;治疗后CD3⁺CD4⁺T淋巴细胞增高,CD3⁺CD8⁺水平稳定,CD4⁺/CD8⁺比例升高,CD3^-CD16⁺CD56⁺细胞增高可能预示患者预后良好。     

骨髓造血干细胞分离 保存的研究

目的探讨骨髓造血干细胞分离及保存的方法。方法应用羟乙基淀粉(HES)或percoll液分离骨髓造血干细胞;联合应用二甲基亚砜(DMSO)和HES对造血干细胞进行液氮保存。应用血细胞计数法、锥虫蓝拒染实验、粒-巨噬细胞集落生成单位(CFU-GM)的体外培养等方法对造血干细胞冷冻前后的有核细胞(NC)数、存活率、体外分化能力进行检测;应用流式分析法计数CD34+细胞数。结果利用HES沉降法分离的单个核细胞数、CD34+细胞数、CFU-GM集落数均比percoll液离心法明显增多;骨髓造血干细胞冷冻保存1年后的有核细胞数、CD34+细胞数、锥虫蓝活率、CFU-GM集落计数与保存前差异无统计学意义。结论HES法分离骨髓造血干细胞方法安全、有效;通过程序降温,联合使用DMSO及HES的低温冻存方法对骨髓造血干细胞的长期保存是适合的。     

UM171 promotes expansion of autologous peripheral blood hematopoietic stem cells from poorly mobilizing lymphoma patients

BackgroundAutologous hematopoietic stem cell transplantation is an effective therapeutic strategy for lymphoma patients. However, some patients have to give up receiving transplantation because of failing to obtain sufficient CD34⁺ cells yields. Therefore, we ex vivo expanded HSCs of lymphoma patients using UM171 to solve the problem of HSCs deficiency.MethodsMobilized peripheral blood-derived CD34⁺ cells from lymphoma patients were cultured for 10 days with or without UM171. The fold of cell expansion and the immunophenotype of expanded cells were assessed by flow cytometry. RNA-seq experiment was performed to identify the mechanism by which UM171 promoted HSCs expansion.ResultsUM171 treatment increased the proportion of CD34⁺ (68.97 ± 6.91%), CD34⁺ CD38⁻ cells (44.10 ± 9.20%) and CD34⁺CD38⁻CD45RA⁻CD90⁺ LT-HSCs (3.05 ± 2.08%) compared to vehicle treatment (36.08 ± 11.14%, 18.30 ± 9.49% and 0.56 ± 0.45%, respectively). UM171 treatment led to an 85.08-fold increase in LT-HSC numbers relative to initial cells. Importantly, UM171 promoted expansion of LT-HSCs achieved 138.57-fold in patients with poor mobilization. RNA-seq data showed that UM171 upregulated expression of HSC-, mast cell-specific genes and non-canonical Wnt signaling related genes, and inhibited genes expression of erythroid, megakaryocyte and inflammatory mediated chemokine.ConclusionsOur study shows that UM171 can efficiently promote ex vivo expansion of HSCs from lymphoma patients, especially for poorly mobilizing patients. In terms of mechanism, UM171 upregulate HSC-specific genes expression and suppress erythroid and megakaryocytic differentiation, as well as activate non-classical Wnt signaling.     

Cytotoxicity Assessment in Cultures of Differentiating Rodent Embryonic Cells

Several techniques for assessing the cytotoxicity of chemical agents in the micromass cell culture systems for rat embryo midbrain (CNS) and limb bud (LB) cells were compared. Cultures were treated with the antitubulin agent alben-dazole (ABZ) and its sulfoxide derivative (SOABZ) and cultured in Primaria 35 mm dishes, Primaria 96-well plates, or collagen-coated 96-well plates. Three assays for cytotoxicity were employed: staining with the vital dye neutral red (NR), mitochondrial reduction of MTT, and total culture protein levels. Comparison of the cytotoxicity IC₅₀ values was also made to the IC₅₀ value for differentiation. The IC₅₀ values for cytotoxicity and differentiation in CNS cultures were statistically equivalent regardless of the procedure employed. However, in LB cultures, the IC₅₀'s for cytotoxicity generated by NR staining of fixed cells in Primaria 96-well plates were significantly different from those for differentiation and total culture protein content. With a single exception (NR staining of unfixed ABZ-treated LBs in collagen-coated 96-well plates), no other cytotoxicity procedures yielded IC₅₀ values that differed significantly from those for differentiation. We conclude that the NR cytotoxicity procedure should be applied to micromass culture with caution due to the sensitivity of this assay to cell density, cell type, and the surface on which the cells are plated. Assaying for total culture protein content yields important supplementary information.     

Stachydrine is effective and selective against blast phase chronic myeloid leukaemia through inhibition of multiple receptor tyrosine kinases

ContextResistance to BCR-ABL tyrosine kinase inhibitor (TKI) is the cause of treatment failure in blast phase chronic myeloid leukaemia (BP-CML). Agents that act synergistically with BCR-ABL TKI are required to improve response.ObjectiveThis work investigated the effects of stachydrine in CML.Materials and methodsCML cells were treated with control or stachydrine at 20, 40 and 80 µM. Proliferation and apoptosis were examined after 72 h treatment. Combination studies were performed in four groups: control, TKI, stachydrine and the combination of stachydrine and TKI. Immunoblotting analysis was performed in CML cells after 24 h treatment.ResultsStachydrine inhibited K562 (IC₅₀ 61 µM), KCL22 (IC₅₀ 141 µM), LAMA84 (IC₅₀ 86 µM), Ba/F3 T315I (IC₅₀ 26 µM), Ba/F3 WT (IC₅₀ 22 µM) and KU812 (IC₅₀ 35 µM) proliferation, and induced apoptosis in these CML cell lines. Stachydrine significantly induced apoptosis, inhibited colony formation and self-renewal in BP-CML CD34⁺ cells. The combination index of stachydrine and TKI combination was <1. Compared to TKI alone, the combination of stachydrine and TKI significantly induced more apoptosis and decreased colony formation in BP-CML CD34⁺ cells. Stachydrine decreased phosphorylation levels of multiple receptor tyrosine kinases in CML cells.Discussion and conclusionsOur study is the first to demonstrate (1) the anticancer activity of stachydrine on primary patient cancer cells; (2) the inhibitory effects of stachydrine on cancer stem cells; (3) the synergism between stachydrine and other anticancer drugs.     

Exposure to particulate matter 2.5 (PM2.5) induced macrophage-dependent inflammation,characterized by increased Th1/Th17 cytokine secretion and cytotoxicity

Particulate matter PM2.5 is a class of airborne particles and droplets with sustained high levels in many developing countries. Epidemiological studies have shown the association between sustained high level of PM2.5 and the risk of many diseases in the respiratory system, including lung cancer. However, the precise mechanisms through which PM2.5 induces respiratory diseases are still unclear. In this study, we demonstrated that CD4⁺ and CD8⁺ T cells following PM2.5 treatment demonstrated significantly elevated mRNA and protein levels of interferon (IFN)-γ, interleukin (IL)-10, IL-17, and IL-21 production. This increase in cytokines required the presence of macrophages, such that CD4⁺ and CD8⁺ T cells treated with PM2.5 in the absence of macrophages did not present higher IFN-γ, IL-10, or IL-21 expression. In contrast, PM2.5-treated macrophages could significantly upregulate T cell cytokine secretion, even when excess PM2.5 was removed from cell culture. We also observed a macrophage-dependent upregulation of granzyme A and granzyme B expression by CD4⁺ and CD8⁺ T cells following PM2.5 treatment. These PM2.5-stimulated CD4⁺ and CD8⁺ T cells potently induced the death of human bronchial epithelial (HBE) cells. Interestingly, the CD4⁺ and CD8⁺ T cells presented synergistic effects at inducing HBE cytotoxicity, such that CD4⁺ T cells and CD8⁺ T cells combined resulted in higher HBE cell death than the sum of the separate effects of CD4⁺ T cells and CD8⁺ T cells. While blocking cytotoxic molecule release significantly compromised the T cell-mediated cytotoxicity against HBE cells, blocking IFN-γ, but not IL-10, could also slightly but significantly reduce T cell-mediated cytotoxicity. Together, these data demonstrated that PM2.5 could promote the inflammation of cytotoxicity of T cells in a macrophage-dependent manner. In addition, PM2.5-treated macrophages presented long-lasting proinflammatory effects on T cells.     

Cell detachment and cloning efficiency as parameters for cytotoxicity

Cell detachment and cloning efficiency of Baby Hamster Kidney cells (BHK-21 C13) were used as parameters to quantify cytotoxicity in vitro of 3 endogenous chemicals (glutathione, L-methionine, L-cysteine HCl) and 4 organotin compounds (tributyltinoxide, tributylchloride, tetrabutyltin, tetraphenyltin). IC₅₀ values (inhibitory concentration at which the cloning efficiency was reduced to 50%) were estimated to be larger than 10^?3 M for all 3 endogenous substances, which served as a calibration of the cell culture system for non-toxic chemicals. For the 2 tributyltin salts the IC₅₀ values were estimated to be near 10^?6 M and for the 2 tetraalkyltin compounds near 10^?5 M.The estimated CD₅₀ values (concentration at whicb 50% of the cells detach) were at least twice as large as the corresponding IC₅₀ values for all 7 chemicals tested. For the toxic tributyltin salts the cell detachment assay was 30–60 times less sensitive than the cloning efficiency assay. However, both assays rank all compounds tested in the same sequence of toxicity as that known from in vivo studies.     

Circulating endothelial progenitor cells are reduced in hemodialysis patients

SUMMARY

Objective: The risk of cardiovascular disease in hemodialysis patients is far greater than in the general population. Endothelial progenitor cells (EPCs) circulating in the peripheral blood contribute to neovascularization in the ischemic tissue. EPCs are considered to be included in CD34 positive (CD34⁺) or AC133 positive (AC133⁺) mononuclear cells (MNCs). This study's aim was to determine the number and functional activity of EPCs in hemodialysis patients and age-matched control subjects.

Methods: The numbers of CD34⁺ MNCs and AC133⁺ MNCs in the peripheral blood were quantified by flow cytometry. The peripheral blood EPCs were also examined by an in vitro culture assay. The levels of serum vascular endothelial growth factor (VEGF) were measured by sandwich enzyme immunoassay.

Results: The numbers of CD34⁺ MNCs and AC133⁺ MNCs were significantly reduced by 56% and 49%, respectively, in hemodialysis patients (n?=?50) compared with control subjects (n?=?36). The number of EPCs determined by the culture assay was also significantly reduced by 41% in hemodialysis patients compared with control i subjects. Multivariate analysis revealed that none of the atherosclerotic risk factors were independent predictors of reduced CD34⁺ MNC counts. The serum VEGF levels in hemodialysis patients were not different from those in control subjects and did not correlate with CD34⁺ MNC counts.

Conclusion: Circulating EPCs are significantly reduced in hemodialysis patients, which might be related to impaired neovascularization and cardiovascular disease in these patients.