Dark Sweet Cherry (Prunus avium) Phenolics Enriched in Anthocyanins Induced Apoptosis in MDA-MB-453 Breast Cancer Cells through MAPK-Dependent Signaling and Reduced Invasion via Akt and PLCγ-1 Downregulation.

Dark sweet cherries (DSCs) are rich source of phenolics known to exert anticancer and anti-invasive activities. This study elucidated the molecular mechanisms underlying the activity of DSC phenolics against MDA-MB-453 breast cancer cells In Vitro. Cells were treated with DSC phenolics in whole extract (WE), and fractions enriched in anthocyanins (ACN) and proanthocyanidins (PCN) at concentrations that inhibited cell growth by 50%. Results showed that DSC phenolics suppressed Akt and PLCγ-1 activation, and inhibited cell motility and invasion, but only ACN reached significance. The extrinsic and intrinsic apoptotic pathways were also activated by DSC phenolics via caspase-8 cleavage and increased Bax/Bcl-2 ratio, with ACN exhibiting significant activation and stronger PARP-1 cleavage. Furthermore, sustained activation of mitogen-activated protein kinases (MAPKs) ERK1/2 and p38 was observed wherein ERK1/2 (U0126) and p38 (SB203580) inhibitors confirmed crosstalk ERK1/2-Akt and MAPK intrinsic mitochondrial pathways. In conclusion, DSC phenolics inhibited MDA-MB-453 breast cancer cells by targeting cell signaling pathways that induce apoptosis and suppress cell invasion, with ACN showing enhanced chemopreventive activities.

Preparation of drip samples from leg of lamb with extended shelf life for nuclear magnetic resonance metabolomics studies.

Metabolomics has been used for the analysis of meat samples for different applications. Using drip as a proxy for meat could offer an easy and non-invasive way of sampling meat, yielding a homogenous liquid sample easy to prepare for metabolomics analysis. There is currently no standard method for the preparation of drip samples for quantitative metabolomics. The aim of this study was to evaluate six different sample preparation methods for quantitative Nuclear Magnetic Resonance (NMR) metabolomics analysis of drip from a lamb leg with extended shelf life: centrifugation, ultrafiltration, and solvent precipitation using four different solvents or solvent mixtures. The six methods were evaluated based on protein removal efficiency, ability to quantify metabolites, metabolite concentrations, reproducibility, speed and relative cost. Three methods (ultrafiltration, solvent precipitation with either acetonitrile/acetone/methanol or chloroform/methanol) resulted in excellent protein removal, high concentrations of metabolites and high reproducibility and are therefore recommended for preparation of extended shelf life lamb leg drip samples for NMR metabolomics.

Membranes replace irradiated blast cells as growth requirement for leukemic blast progenitors in suspension culture.

The blast cells of acute myeloblastic leukemia (AML) may be considered as a renewal population, maintained by blast stem cells capable of both self-renewal and the generation of progeny with reduced or absent proliferative potential. Blast progenitor renewal is manifested in suspension culture by an exponential increase in clonogenic cells. This growth requires that two conditions be met: first, the cultures must contain growth factors in media conditioned either by phytohemagglutinin (PHA)-stimulated mononuclear leukocytes (PHA-LCM), or by cells of the continuous bladder carcinoma line HTB9 (HTB9-CM). Second, the cell density must be maintained at 10(6) blasts/ml; this may be achieved by adding irradiated cells to smaller numbers of intact blasts. We are concerned with the mechanism of the feeding function. We present evidence that (a) cell-cell contact is required. (b) Blasts are heterogeneous in respect to their capacity to support growth. (c) Fractions containing membranes from blast cells will substitute for intact cells in promoting the generation of new blast progenitors in culture. (d) This membrane function may be specific for AML blasts, since membranes from blasts of lymphoblastic leukemia or normal marrow cells were inactive.

Antitumor potential of dark sweet cherry sweet (Prunus avium) phenolics in suppressing xenograft tumor growth of MDA-MB-453 breast cancer cells.

This study investigated in vivo the antitumor activity of dark sweet cherry (DSC) whole extracted phenolics (WE) and fractions enriched in anthocyanins (ACN) or proanthocyanidins (PCA) in athymic mice xenografted with MDA-MB-453 breast cancer cells. Mice were gavaged with WE, ACN or PCA extracts (150 mg/kg body weight/day) for 36 days. Results showed that tumor growth was suppressed at similar levels by WE, ACN and PCA compared to control group (C) without signs of toxicity or significant changes in mRNA oncogenic biomarkers in tumors or mRNA invasive biomarker in distant organs. Tumor protein analyses showed that WE, ACN and PCA induced at similar levels the stress-regulated ERK1/2 phosphorylation, known to be linked to apoptosis induction. However, ACN showed enhanced antitumor activity through down-regulation of total oncogenic and stress-related Akt, STAT3, p38, JNK and NF-kB proteins. In addition, immunohistochemistry analysis of Ki-67 revealed inhibition of tumor cell proliferation with potency WE ≥ ACN ≥ PCA. Differential quantitative proteomic high-resolution nano-HPLC tandem mass spectrometry analysis of tumors from ACN and C groups revealed the identity of 66 proteins associated with poor breast cancer prognosis that were expressed only in C group (61 proteins) or differentially up-regulated (P<.05) in C group (5 proteins). These findings revealed ACN-targeted proteins associated to tumor growth and invasion and the potential of DSC ACN for breast cancer treatment. Results lead to a follow-up study with highly immunodeficient mice/invasive cell line subtype and advanced tumor development to validate the anti-invasive activity of DSC anthocyanins.

Non-anthocyanin phenolics in cherry (Prunus avium L.) modulate IL-6, liver lipids and expression of PPARδ and LXRs in obese diabetic (db/db) mice.

Anthocyanin-rich cherries are known for preventing/decreasing risk factors associated with obesity; however, the specific benefits exerted by cherry non-anthocyanin phenolics are not clear. Obese diabetic (db/db) mice fed a diet supplemented with anthocyanin-depleted cherry powder (cherry) were compared to db/db (obese) or lean counterparts (lean) fed a control isocaloric diet for 12 weeks. The reduced plasma interleukin (IL)-6 and improved liver health may be mediated by cherry fibre and non-anthocyanin phenolics. Benefits for liver health included reduction of lipids and protein carbonyls, and modulation of peroxisome proliferator-activated receptor (PPAR)δ mRNA to resemble levels in lean. Lack of plasma antilipidemic, improvement of antioxidant defenses, and PPARα/γ mRNA modulation in liver suggest cherry anthocyanins specific benefits. This is the first study to elucidate in vivo the potential benefits of cherry non-anthocyanin phenolics for diabetes-induced liver disorders and the importance of choosing processing technologies that preserve anthocyanins and health benefits of whole cherries.

Effect of dark sweet cherry powder consumption on the gut microbiota, short-chain fatty acids, and biomarkers of gut health in obese db/db mice.

Cherries are fruits containing fiber and bioactive compounds (e.g., polyphenolics) with the potential of helping patients with diabetes and weight disorders, a phenomenon likely related to changes in the complex host-microbiota milieu. The objective of this study was to investigate the effect of cherry supplementation on the gut bacterial composition, concentrations of caecal short-chain fatty acids (SCFAs) and biomarkers of gut health using an in vivo model of obesity. Obese diabetic (db/db) mice received a supplemented diet with 10% cherry powder (supplemented mice, n = 12) for 12 weeks; obese (n = 10) and lean (n = 10) mice served as controls and received a standard diet without cherry. High-throughput sequencing of the 16S rRNA gene and quantitative real-time PCR (qPCR) were used to analyze the gut microbiota; SCFAs and biomarkers of gut health were also measured using standard techniques. According to 16S sequencing, supplemented mice harbored a distinct colonic microbiota characterized by a higher abundance of mucin-degraders (i.e., Akkermansia) and fiber-degraders (the S24-7 family) as well as lower abundances of Lactobacillus and Enterobacteriaceae. Overall this particular cherry-associated colonic microbiota did not resemble the microbiota in obese or lean controls based on the analysis of weighted and unweighted UniFrac distance metrics. qPCR confirmed some of the results observed in sequencing, thus supporting the notion that cherry supplementation can change the colonic microbiota. Moreover, the SCFAs detected in supplemented mice (caproate, methyl butyrate, propionate, acetate and valerate) exceeded those concentrations detected in obese and lean controls except for butyrate. Despite the changes in microbial composition and SCFAs, most of the assessed biomarkers of inflammation, oxidative stress, and intestinal health in colon tissues and mucosal cells were similar in all obese mice with and without supplementation. This paper shows that dietary supplementation with cherry powder for 12 weeks affects the microbiota and the concentrations of SCFAs in the lower intestinal tract of obese db/db diabetic mice. These effects occurred in absence of differences in most biomarkers of inflammation and other parameters of gut health. Our study prompts more research into the potential clinical implications of cherry consumption as a dietary supplement in diabetic and obese human patients.

Antiproliferative and differentiative effect of granulocyte-macrophage colony-stimulating factor on a variant human small cell lung cancer cell line.

A variant clone was adopted during passages of a small cell lung cancer cell line, GKT3-1.3. The variant clone exhibited distinct characteristics with alterations in morphology, positive staining with nonspecific esterase stain, and an increase in surface specific markers OKM5, HLA-DR, Mo1, and My7, usually found on monocytes or their precursors. However, it exerted a very rapid proliferation just like immature cells. This new clone, GKT3-1.3V, was shown to have specific binding capacity to granulocyte-macrophage colony-stimulating factor (GM-CSF), with a number of binding sites comparable to that of myelomonocytes or monocytic cell lines. Thus its proliferation was inhibited by GM-CSF in clonogenic assay and suspension culture. Increase in the percentage of cells with surface marker Mo1 by the addition of GM-CSF suggested its differentiative effect. Cell cycle analysis showed that the antiproliferative effect of GM-CSF was due to a block in G0 or G1. The antiproliferative effect of GM-CSF was abolished by the addition of anti-GM-CSF antibody.

Effects on leukemic clonogenic cells in murine myeloid leukemia of 1-beta-D-arabinofuranosylcytosine and the anthracyclines adriamycin, daunomycin, aclacinomycin A, and 4'-epidoxorubicin.

M-3 murine myeloid leukemic cells undergo terminal divisions making colonies in methylcellulose culture and also renew themselves in methylcellulose and suspension; leukemic clonogenic cells are characteristic as stem cells. The effects of 1-beta-D-arabinofuranosylcytosine and four anthracyclines (Adriamycin, daunomycin, aclacinomycin A, and 4'-epidoxorubicin) on M-3 leukemic clonogenic cells were studied. 1-beta-D-Arabinofuranosylcytosine was effective in reducing primary and secondary colonies in methylcellulose and the growth of clonogenic cells in suspension. In contrast, the anthracyclines were not so effective in reducing secondary colonies in methylcellulose or clonogenic cells in suspension as to suppress primary colonies in methylcellulose. The results suggest that 1-beta-D-arabinofuranosylcytosine but not the anthracyclines is effective for not only terminal divisions but also self-renewal of leukemic clonogenic cells. The study will be used as a practical screening test to examine the effects of antitumor agents on leukemic blast progenitors.

Cytotoxicity of 4-hydroperoxycyclophosphamide for the blast progenitors of acute myeloblastic leukemia.

The effects of 4-hydroperoxycyclophosphamide (4-HC), an analogue of cyclophosphamide, on the blast progenitors from eight acute myeloblastic leukemia patients were studied in methylcellulose and suspension cultures. Leukemic blast progenitors undergo terminal divisions in methylcellulose culture, making blast colonies. Cells in primary colonies can make secondary colonies after replating in fresh methylcellulose medium. Leukemic blast progenitors grow exponentially in suspension culture for periods of weeks. The ability to form secondary colonies and the exponential growth in suspension culture are considered to reflect the self-renewal of blast progenitors. 4-HC suppressed primary blast colonies in a dose-responsive manner. Secondary colonies were not significantly affected by 4-HC. Although the clonogenic cell recovery was suppressed by 4-HC, leukemic blast progenitors were less sensitive to 4-HC in suspension culture than in methylcellulose culture. The results suggest that 4-HC is effective in suppressing the terminal divisions of blast progenitors but not as effective against the self-renewal of blast progenitors.

Production of growth potentiating factor(s) for autologous blast cells by acute myeloblastic leukemia cells.

The effects of media conditioned by leukemic cells from 11 acute myeloblastic leukemia patients on the growth of autologous blast progenitors were studied. First, it was shown that T-cell-depleted leukemic cells from some patients release high levels of colony-stimulating activity into the culture medium, whereas following further depletion of phagocytic cells, the levels of colony-stimulating activity become undetectable. Second, media conditioned by purified blast cell fraction depleted of both T-cells and phagocytic cells potentiated autologous blast progenitor growth both in methylcellulose and suspension cultures stimulated by optimal concentration of media conditioned by human bladder carcinoma line 5637. Third, media conditioned by these purified blast cells generally did not contain measurable colony-stimulating activity or interleukin 1, whereas substantial levels of granulocyte colony-stimulating factor, granulocyte-macrophage colony-stimulating factor, and interleukin 1 were observed in media conditioned by human bladder carcinoma line 5637 using bioassays and specific immunological assays. Therefore, purified blast cell fraction from acute myeloblastic leukemia patients appears to produce factor(s) other than granulocyte colony-stimulating factor, granulocyte-macrophage colony-stimulating factor, or interleukin 1, which potentiate the growth of autologous blast progenitors both in methylcellulose and suspension cultures.

Effects of N4-behenoyl-1-beta-D-arabinofuranosylcytosine on blast progenitors of acute myeloblastic leukemia.

To determine the antileukemic effect of N4-behenoyl-1-beta-D-arabinofuranosylcytosine (BH-AC), a synthetic masked compound of 1-beta-D-arabinofuranosylcytosine, the pharmacokinetics and suppressive effect on leukemic blast progenitors of BH-AC were studied. When BH-AC was added to the suspension culture of leukemic cells, BH-AC gradually decreased in concentration in the culture media and was rapidly taken into the cellular fraction. The conversion from BH-AC to 1-beta-D-arabinofuranosylcytosine was noted in both the culture media and the cellular fraction. The concentration of 1-beta-D-arabinofuranosylcytosine converted from BH-AC in the culture medium gradually increased during 7 days of culture, although the rate of conversion was variable among the samples. BH-AC suppressed primary and secondary blast colony formation in a dose responsive manner. BH-AC also suppressed the recovery of clonogenic cells in suspension culture. The suppression by BH-AC was more prominent in secondary blast colony formation and the recovery of clonogenic cells in suspension culture than in primary blast colony formation. Secondary blast colony formation and the recovery of clonogenic cells in suspension are considered to reflect the self-renewal of blast progenitors, while primary blast colony formation is considered to reflect the terminal divisions of blast progenitors. The results obtained in the present study suggest that BH-AC is more effective to suppress the self-renewal of blast progenitors than the terminal divisions. The findings offer a theoretical basis in the utility of BH-AC in the therapy of acute myeloblastic leukemia.

Expression of interleukin 1 beta receptors on blast cells in acute myeloblastic leukemia: comparison with interleukin 1 beta proliferative activity.

We describe here the presence of a single class of interleukin 1 beta (IL-1 beta) receptors on the surface of blast cells freshly obtained from eight acute myeloblastic leukemia patients and one chronic myelocytic leukemia patient in blast crisis. Blast cells possessed a low number of high-affinity receptors (range, less than 10-173 receptors/cell) with a Kd of 1.8-12.8 x 10(-10) M. At the same time, we have investigated the effects of IL-1 on the growth of leukemic blast progenitors, and a significant heterogeneity of responsiveness was observed. IL-1 beta (1 ng/ml) enhanced blast colony formation in six patients. No significant effect was observed by addition of up to 100 ng/ml of IL-1 beta in the remaining three patients. No significant correlation was observed between the receptor number, receptor affinity, and the cellular responsiveness to IL-1; in some acute myeloblastic leukemia cases with apparent IL-1 receptors, no proliferation response to added IL-1 was observed. Our data show that IL-1 alone can enhance blast colony formation and that lack of responsiveness to IL-1 in some acute myeloblastic leukemia patients is not related to the absence of IL-1 receptors on blast cells.

Epidemiological aspects and risk factors for infection by Leishmania infantum chagasi in dogs from municipality of Petrolina, Northeastern Brazil.

Visceral leishmaniasis (VL) is a disease of great concern for public health because of its high incidence and lethality. Here, we performed a serologic study of domestic dogs in the municipality of Petrolina in northeastern Brazil to evaluate the possible risk factors associated with canine seropositivity for Leishmania infantum chagasi. Blood samples from 1245 dogs in urban and rural areas were collected and examined by indirect immunofluorescent antibody test (IFAT) and an indirect enzyme-linked immunosorbent assay (ELISA). The dogs were subjected to physical examination and classified according to their clinical manifestations. A questionnaire was administered to the owners to detect potential risk factors for infection with Leishmania spp. using logistic regression models. Of the 1245 dogs evaluated, 11.2% (140/1245) were seropositive in both tests (CI 95%: 9.5% to 13.1%). Approximately 60.7% of the reactive dogs were clinically suspect, with lymphadenomegaly, cutaneous ulcerations, onychogryphosis, pale mucous membranes and alopecia being the most obvious symptoms of infection. The seroprevalences in urban and rural areas were 5.4% (CI 95%: 4% to 7.1%) and 23.6% (CI 95%: 19.5% to 28.1%), respectively. The possible risk factors for the presence of anti L. infantum chagasi antibodies were the presence of a green area close to the home of the animal (OR=3.63; p<0.001), a mongrel breed (OR=2.11; p=0.025) and male gender (OR=1.51, p=0.034). The seroprevalence of L. infantum chagasi in the canine population is distributed in a heterogeneous manner, with a higher prevalence in rural areas.

Diversity and similarity in signaling pathways of hematopoietic growth factors in human leukemia cell lines.

Diversity and similarity in signaling pathways of granulocyte colony-stimulating factor (G-CSF), granulocyte-macrophage colony-stimulating factor (GM-CSF), interleukin-3 (IL-3), and stem cell factor (SCF) in five human factor-responsive leukemia cell lines were investigated by immunoblotting to detect tyrosine phosphorylation of intracellular proteins. G-CSF induced tyrosine phosphorylation of a set of proteins with few different components according to the cell lines. IL-3 also induced phosphorylation of several proteins. In a lymphoid cell line, phosphorylation patterns induced by IL-3 were somewhat different from that in myeloid cell lines. Phosphorylation patterns by G-CSF and those by IL-3 were similar in myeloid cell lines. In a cell line which responded to both IL-3 and SCF, almost similar sets of proteins were phosphorylated by each, although phosphorylation of a 92-KDa protein was specific to IL-3 and that of a 140-200-KDa protein was specific to SCF. Taken together, proliferative growth factors induced tyrosine phosphorylation of similar sets of proteins with little difference according to each growth factor and each target cell line.

Effects of recombinant human tumor necrosis factor on the self-renewal capacity of leukemia blast progenitors in acute myeloblastic leukemia.

The effects of human recombinant tumor necrosis factor type alpha (rTNF alpha) on the blast progenitors from 14 acute myeloblastic leukemia (AML) patients and 1 chronic myelogenous leukemia patient in blastic crisis were studied in methylcellulose and suspension cultures. Blast progenitors renew themselves and/or undergo terminal divisions. Plating efficiency of primary colony formation (PE1) in methylcellulose, which is considered to reflect the terminal divisions of blast progenitors, was suppressed by rTFN alpha in a dose-dependent manner in all cases. Plating efficiency of secondary colony formation (PE2) and the recovery of clonogenic cells in suspension culture, which are considered to reflect the self-renewal capacity of blast progenitors, were also suppressed by rTNF alpha in a dose-dependent manner in almost all cases. rTNF alpha also inhibited both PE2 and clonogenic cells in suspension culture, even in relapsed AML patients who were very refractory to intensive chemotherapies. The results demonstrate that rTNF alpha inhibits not only terminal divisions but also the self-renewal capacity of leukemic blast progenitors. The finding that rTNF alpha suppressed the self-renewal capacity of leukemic blast progenitors proposes the utility of rTNF alpha to AML therapy.

Effect of protein kinase inhibitors on the proliferation of leukemic cells stimulated by granulocyte colony-stimulating factor, granulocyte-macrophage colony-stimulating factor or interleukin-3.

The effects of the inhibitor for protein kinase A or C, or tyrosine kinase (H-8, staurosporine, or genistein, respectively) on the proliferation of leukemic and normal bone marrow cells stimulated by granulocyte colony-stimulating factor (G-CSF), granulocyte-macrophage colony-stimulating factor (GM-CSF), or interleukin-3 (IL-3) were studied using the MTT assay. These inhibitors suppressed the proliferation of leukemic and normal bone marrow cells in a dose-dependent manner. Although the suppressive effect of each inhibitor on cell proliferation was varied in each instance, the effects were almost similar whichever CSF was added. A significant difference was not recognized between leukemic and normal bone marrow cells in terms of sensitivity to these inhibitors. The data indicate that protein kinase inhibitors have an inhibitory effect on leukemic and normal hematopoietic cell proliferation and that further studies are required to determine if this effect is due to the inhibition of protein kinases acting as the second messenger of CSFs.

Effect of recombinant human M-CSF on the proliferation of leukemic blast progenitors in AML patients.

We studied the effects of recombinant human macrophage colony-stimulating factor (M-CSF) on the leukemic blast progenitors from 10 acute myeloblastic leukemia patients. Recombinant human (rh)M-CSF stimulated leukemic blast progenitors in methylcellulose in four patients, but the colonies by rhM-CSF were smaller in size and number than those by rh-granulocyte-CSF or human bladder carcinoma cell line 5637 conditioned medium. rhM-CSF did not increase the number of clonogenic cells in long-term suspension culture. The blast colony formation in methylcellulose and the exponential growth of clonogenic cells in long-term suspension culture are considered to reflect the terminal divisions and the self-renewal of blast progenitors, respectively. The results show that M-CSF stimulates terminal divisions weakly but does not stimulate self-renewal of leukemic blast progenitors. M-CSF did not induce differentiation of blasts either in methylcellulose or in suspension culture.

Inhibition of the in vitro growth of blast progenitors from acute myeloblastic leukemia patients by transforming growth factor-beta (TGF-beta).

The effects of transforming growth factor-beta (TGF-beta) on the blast progenitors from nine acute myeloblastic leukemia patients were studied in methylcellulose and suspension cultures. Leukemic blast progenitors undergo terminal divisions with a limited differentiation in methylcellulose culture, making blast colonies. Blast progenitors can renew themselves. The self-renewal can be reflected by secondary colony formation after replating primary blast colonies in fresh methylcellulose media and by the exponential growth of clonogenic cells in suspension culture. TGF-beta suppressed primary and secondary colony-formation in methylcellulose culture. Furthermore, TGF-beta suppressed the recovery of clonogenic cells in suspension. The results indicate that TGF-beta is effective in inhibiting not only terminal divisions but also self-renewal of leukemic blast progenitors.

Mixed colony formation composed of erythroblasts and myeloblasts by leukemic blast progenitors in patients with erythroleukemia.

To determine the clonal origin and growth requirement of leukemic blast progenitors in erythroleukemia, acute myelogenous leukemia (AML) M6, T cell-depleted mononuclear cells obtained from 5 erythroleukemia patients were cultured in methylcellulose media. Although plating efficiency did not significantly differ from those in the other AML subtypes, the morphology of colonies in erythroleukemia was distinct. Two types of colonies were formed; one was composed of myeloblasts, and the other consisted of erythroblasts and myeloblasts. The "mixed" colony formation was not stimulated by interleukin-3, granulocyte-macrophage colony-stimulating factor, granulocyte colony-stimulating factor or erythropoietin. Both types of colonies were formed spontaneously in the absence of supplemented growth-stimulatory factor when cultured at high density. The results suggest that leukemic blast progenitors in erythroleukemia originated at the stage of bipotent hematopoietic precursor capable of differentiating into erythroid and myeloid lineages. The formation of mixed colonies composed of erythroblasts and myeloblasts in the absence of erythropoietin or colony-stimulating factor may indicate the deranged hematopoiesis in erythroleukemia.

Effects of chloramphenicol on hematopoietic inductive microenvironment.

Effects of chloramphenicol (CP) on hematopoietic inductive microenvironment (HIM) were studied using in vitro and in vivo assay systems. HIM was represented as fibroblast colonies (CFUF) in in vitro culture. CP suppressed the growth of not only granuloid committed progenitor cells (CFUC), but also CFUF in in vitro culture at the concentration of 10, 50 and 100 micrograms/ml. To analyze the function of HIM in vivo, the subcutaneous bone implantation method was used. The recovery of hematopoietic stem cells in subcutaneously implanted femora of mice, which were treated with a 500 mg/kg dose of CP daily for 6 days, was significantly decreased compared to the sham treated group. Suppressive effect of CP on HIM was shown. The important role of the derangement of HIM on the pathogenesis of CP-induced aplastic anemia was discussed.