Percentage Of CD4-positive Cells Research Articles

Resveratrol suppresses lung cancer by targeting cancer stem-like cells and regulating tumor microenvironment

Increasing evidence indicate that cancer stem cells (CSCs) are the key driver of tumor initiation and recurrence. The cellular and soluble components of the tumor microenvironment (TME) impact on cancer initiation and progression, such as cytokines and chemokines. Thus, targeting CSCs and TME is a novel anti-cancer approach. Resveratrol (RES), a bioactive phytochemical extracted from various plants, exhibits tumor-suppressing activities in lung cancer, yet the mechanism remains poorly understood. Our data showed that the expression level of IL-6 was positively correlated with the presence of lung cancer stem-like cells (LCSCs) in human lung cancer tissues. In vitro results showed that IL-6 was highly elevated in lung cancer sphere-forming cells and could enhance the stemness of LCSCs, including tumor sphere formation ability, the percentage of CD133 positive cells, and the expression of LCSC specific markers (CD133, ALDH1A1 and Nanog). Simultaneously, our results confirmed that RES effectively inhibited LCSC properties, downregulated Wnt/β-catenin signaling and reduced IL-6 level in vitro and in vivo. Furthermore, we found RES treatment attenuated the activation of Wnt/β-catenin signaling by LiCl (GSK3β agonist). IL-6-promoted LCSC properties and Wnt/β-catenin signaling was also reversed by RES. Taken together, these data illustrated that RES inhibited lung cancer by targeting LCSCs and IL-6 in TME. The novel findings from this study provided evidence that RES exhibited multi-target effects on suppression of lung cancer and could be a novel potent cancer-preventive compound.

Bone Marrow Osteoblasts Promotes the Proliferation Leukemia Stem Cell by Up-regulating Interleukin-1

To explore the extrinsic regulation mechanism of bone marrow microenvironment in leukemia cells, and investigate the promoting effect of osteoblast niche on the proliferation and self-renewal of leukemia stem cell by up-regulating the expression of interleukin-1 (IL-1) in leukemia cell. The gene expression profiles on leukemia cells derived from AE9a mouse bone marrow endosteum and central bone marrow were determined by RNA sequencing and gene set enrichment analysis (GSEA). Quantitative real-time PCR (qRT-PCR) was used to detect the expression of IL-1 in AE9a mouse leukemia cells co-cultured with or without osteoblasts in vitro. In addition, qRT-PCR was also used to determine the expression of IL-1 in bone marrow mononuclear cell (BMMNC) from 43 patients with acute myeloid leukemia (AML). For leukemia cells co-cultured with osteoblasts or treated with IL-1β, colony forming ability of AE9a leukemia cells was determined by colony formation assay. In AE9a leukemia mouse, RNA-seq data and GSEA showed that the enrichment of the upregulated genes in leukemia cells located in endosteum fell into inflammatory response gene set, among them, IL-1α and IL-1β were significantly higher expressed in AE9a leukemia cells that located osteoblast niche (IL-1α: P<0.001, IL-1β:P<0.001). After AE9a leukemia cells were co-cultured with osteoblasts in vitro, the expression of IL-1α and IL-1β in leukemia cells were increased by 2.5 and 3.5 times respectively. In colony formation assay, the number of colonies was increased significantly after leukemia cells were co-cultured with osteoblasts (P<0.001). In addition, when AE9a leukemia cells were treated with IL-1β, the number of colonies was also increased significantly (P<0.01). In AML patients, BMMNC with high percentage of CD34 positive cells exhibited higher level of IL-1 expression. Osteoblast niche can promote leukemia cell proliferation and self-renewal through up-regulating the expression of IL-1 in leukemia cells. In AML patients, the expression level of IL-1 was correlated to the percentage of CD34 positive cells in BMMNC.

Baicalin inhibits IgG production by regulating Treg/Th17 axis in a mouse model of red blood cell transfusion

This study was conducted to evaluate whether baicalin inhibits red blood cell (RBC) immunization and elucidate the underlying mechanism. We used human RBCs with adjuvant lipopolysaccharide (LPS) and transfused mice to induce antibodies as an experimental system for studying the effect of baicalin on RBC immunization. Mice were divided into a human RBC transfused positive control group administered with human RBC and LPS intravenously once or weekly for 4 weeks, control group administered dexamethasone (DEX) intraperitoneally daily for 4 weeks, and treatment group administered baicalin intraperitoneally daily for 4 weeks. Assessment of human RBC immunization was performed by measuring serum immunoglobulin G (IgG) and immunoglobulin M (IgM) against human RBC weekly. Lymphocyte changes in spleen were monitored by flow cytometry. We found that baicalin treatment significantly decreased serum IgG but not IgM production in a time and does dependent manner, with a concomitant reduction in Th17 cells and increase in CD4 regulatory T cells in the spleen. The percentage of CD4-positive cells in the spleen was not decreased in the baicalin-treated group but was decreased in the dexamethasone-treated group. In conclusion, baicalin inhibited RBC immunization, particularly IgG production by regulating the Treg/Th17 axis without damaging spleen function.

Preparation of 99Tcm-SHNH-AC133 and imaging on colon cancer bearing mice

Objective To prepare 99Tcm-succinimidyl-6-hydrazinonicotinate hydrochloride (SHNH)-AC133 and evaluate its targeting ability on CD133 positive colon cancer by Gamma imaging, and to explore its feasibility as a molecular probe for cancer stem cells (CSCs). Methods CD133 expression was detected by immunofluorescence assay and flow cytometry on Lovo cell lines and tumor xenografts. CD133 specific monoclonal antibody (AC133) was conjugated with SHNH, and then labeled with 99Tcm to prepare 99Tcm-SHNH-AC133. The radiolabeled yield and radiochemical purity were investigated. Colon cancer xenografts were developed and Gamma imaging were performed. Region of interest (ROI) was drawn and the tumor/non-tumor (T/NT) ratio was calculated. For the blocking experiment, animals were pre-injected with excess unlabeled AC133. Two-sample t test was used to analyze the data. Results CD133 was expressed on the surface of Lovo cells. And the percentage of CD133 positive cells in Lovo tumor tissues was (29.5±3.4)%. The radiolabeled yield of 99Tcm-SHNH-AC133 was more than 85% and radiochemistry purity was (97.7±2.4)%. Gamma imaging demonstrated that 99Tcm-SHNH-AC133 could specifically target to Lovo tumors which could be gradually visible after 12 h. The tumor uptake was obvious at 24 h and T/NT ratio was 8.16±0.45. In blocking study, the tumor uptake was significantly reduced by pre-injection of excess unlabeled AC133 (3.52±0.13; t=19.8, P<0.05). Conclusions 99Tcm-SHNH-AC133 has high labeling yield and radiochemistry purity. The excellent targeting properties of 99Tcm-SHNH-AC133 on CD133 positive colon cancer demonstrate that it is a promising imaging agent for CSCs tracking in vivo. Key words: Antibodies, monoclonal; Succinimides; Technetium; Isotope labeling; Colonic neoplasms; Radionuclide imaging; Mice, nude

Expression of Notch signaling transduction pathway in lung cancer stem cells

Objective To explore the expression of Notch transduction signaling pathway in lung cancer stem cells and the effect on the cellular growth in vitro after blocking Notch signaling pathway. Methods Lung cancer stem cells and general tumor cells were isolated from human lung adenocarcinoma cell line A549 by the high speed sorting of flow cytometry based on surface marker of CD133. Expression of Notch transduction signaling pathway in lung cancer stem cells and general tumor cells were detected by real-time fluorescent quantitative polymerase chain reaction(FQ- PCR)and Western blotting.The proliferation of lung cancer stem cells and general tumor cells in vitro was detected by cell counting kit-8 (CCK-8)respectively, and the growth curves of lung cancer stem cells and general tumor cells were described and compared.After blocking the Notch transduction signaling pathway by γ- secretase Inhibitor (DAPT), a γ- secretase inhibitor, cellular growth in lung cancer stem cells and general tumor cells was both observed and analyzed. Results CD133 positive cells(served as lung cancer stem cell)occupied(0.40±0.11)% of all A549 cells before the high speed sorting by flow cytometry, while the percentage of CD133 positive cells reached to(95.00±0.63)% after the high speed sorting of flow cytometry, with significant difference(P 0.05).However,after 48 h of DAPT intervention, variant inhibitions of growth were shown.Compared to general tumor cells[(21.5±3.4)%], growth inhibitory rate of cancer stem cells[(33.7±1.9)%]increased obviously, indicating a statistically significant difference(P< 0.05). Conclusion Conclusion Inhibitory effect on the growth of lung cancer stem cells was more significant. It may be related to overexpression of Notch inhibited by DAPT and its negative feedback effect on lung cancer cells. Key words: Notch pathway; Tumor stem cell; Growth inhibition; CD133

Study on Chinese Medicine Shouwu Capsules on Immune Function in Athletes

To observe the effect of Chinese medicine health care products Shouwu capsules on immune function in athletes during training, and to explore its possible mechanism. Methods: the test group received can strengthen the immune function of Chinese medicine health Shouwu capsules, the control group did not take. CD4+CD8+ CD4+, CD8+, T lymphocyte subsets ( + ) and CD4+/CD8+ ratio, serum immunoglobulin ( Ig ) IgA, IgG, IgM were observed before and after the experimental period, 30 days of observation, changes of immune function in athletes. Results : after the experiment, the test group CD4+ significantly increased the proportion of IgG ratio, numerical experiment, compared with before the experiment with statistical difference, P ≤ 0.05, CD4+CD8+ group ( + ) ratio decreased significantly, P ≤ 0.05. Conclusion: take Shouwu capsules can improve athletes the percentage of CD4 positive cells, can enhance the immune function of T lymphocytes, immune response in the balance of IgG, can regulate humoral immune function. Can effectively improve the athletes sleep, appetite, physical strength, improve the athlete ‘s body function, more conducive to the improvement of sports performance.

Abstract 3483: Inhibition of Monocarboxylate Transporter 4 (MCT4) targets stem-like cells in glioblastoma

Abstract Glioblastomas (GBM) are the most common adult malignant brain tumors and contain a hypoxic/necrotic core surrounded by proliferative cells. To unmask genes important in hypoxia, we exposed 2 GBM neurosphere lines, HSR-GBM1 and JHH-GBM10, to 1% and 21% oxygen levels for 24 hours and compared gene expression using Agilent oligonucleotide microarrays. We identified SLC16A3 (Monocarboxylate transporter-4, MCT4) as one of the most upregulated genes in response to hypoxia. To investigate the clinical importance of MCT4 in GBM, we examined the Kaplan-Meier survival curves of glioma patients using public databases. We found that patients with upregulation of MCT4 (≥2.0X) have a significantly shorter survival (p&amp;lt;0.0001) than patients with intermediate expression. Consistent with this, MCT4 upregulation correlated with the aggressive mesenchymal subset of GBM (p&amp;lt;0.0001). We next examined MCT4 protein levels using immunohistochemical analysis of tissue microarrays, confirming that MCT4 protein levels were increased in high-grade as compared to lower grade astrocytomas (p&amp;lt;0.0001). These data clearly demonstrated that MCT4 is a clinically relevant target. We next tested the requirement of MCT4 in vitro. We found that when neurospheres were transduced with viruses encoding short hairpin RNA (shRNA) against MCT4, cell growth was inhibited by 30-69% in hypoxia. Interestingly, similar results were observed in normoxia, suggesting that MCT4 may be critical to tumor growth and survival independent of oxygen levels. We next explored the role that MCT4 may play in the maintenance of the stem-like population in our neurosphere lines. We found that MCT4 is overexpressed in sorted CD133-positive cells compared to CD133-negative cells in both HSR-GBM1 and JHH-GBM10. Furthermore, flow cytometric analysis of shMCT4-expressing neurospheres showed a decrease in the percentage of CD133-positive cells as compared to controls. This suggested to us that MCT4 silencing might inhibit stem-like cell proliferation/survival, offering a potential explanation for growth inhibition following MCT4 knockdown in normoxia. To test this hypothesis, we examined the effect of MCT4 inhibition functionally using a clonogenic assay. Cells were exposed to normoxia or hypoxia for 48 hours followed by recovery in normoxia. We found that MCT4 silencing resulted in significant reduction in sphere number and size in both normoxic and hypoxic cells, consistent with a loss of clonogenicity. In agreement with what we found in vitro, MCT4 silencing also slowed GBM intracranial xenograft growth in vivo (p=0.009). Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3483. doi:1538-7445.AM2012-3483

Intrinsic gemcitabine resistance in a novel pancreatic cancer cell line is associated with cancer stem cell-like phenotype

Pancreatic ductal adenocarcinoma (PDA) remains one of the most lethal malignancies in the world, often diagnosed at an advanced stage, resistant to conventional chemotherapy and having high invasive and metastatic potential. The mechanism of drug resistance of PDA is still not clear. In the present study, we established two novel pancreatic cancer cell lines PAXC-002 and PAXC-003 from human primary xenograft models. The cell lines were characterized by morphology, karyotype, pancreatic cancer marker and short tandem repeat (STR) analysis, and growth kinetics and tumorigenicity. The in vitro anti-proliferation test revealed that PAXC-002 cell was intrinsically resistant to the standard of care chemotherapy-gemcitabine, compared with that of PAXC-003 and other widely used pancreatic cancer cell lines. Interestingly, the gemcitabine resistant PAXC-002 cell line was more potent in forming colonies in 3-Dimensional matrigel culture conditions and had a higher percentage of CD133 positive cells, which is recognized as a cancer stem cell marker, compared to the gemcitabine-sensitive PAXC-003 cell line. In this study, we present two novel pancreatic cancer cell lines which could be used for gemcitabine resistance investigation, mechanism identification of pancreatic cancer and anticancer drug screening. The preliminary data indicate that the drug resistance of pancreatic carcinoma cells is associated with a cancer stem cell-like phenotype.

Hypocellularity and insufficient expression of angiogenic factors in implanted autologous bone marrow in patients with chronic critical limb ischemia

The aim of this study was to identify the clinical parameters of absolutely poor-prognosis patients with chronic critical limb ischemia (AP-CLI). Sixteen no-option CLI patients with arteriosclerosis obliterans: ASO (nine) and non-ASO patients (seven) treated with bone marrow-mononuclear cell implantation (BMI) were analyzed. There were three AP-CLI patients (all ASO). The mRNA expression of several angiogenic factors in the implanted cells was analyzed in comparison with normal donor bone marrow. To observe the response of bone marrow components to hypoxia, normal bone marrow cells were cultured for 24 h in 2.5% O(2), and mRNA expression of angiogenic factors were measured. AP-CLI patients exhibited extraordinary low bone marrow cellularity as well as the percentage of CD34-positive cells. Among angiogenic factors, only VEGF expression was maintained in response to HIF-1, while other factors such as HGF, Ang-1, PLGF, and SDF-1 decreased in the implanted bone marrow cells of the patients with CLI compared to normal bone marrow cells. HIF-1 and all of the five angiogenic factors increased in vitro in response to hypoxia. Thus it is highly likely that angiogenic factors except VEGF do not respond to chronic ischemia in bone marrow in vivo. An organ-protection system against tissue ischemia may be applied for acute hypoxia, but it may be insufficient for chronic ischemia.

Effects of Health Food Containing Cistanche Deserticola Extract on QOL and Safety in Elderly: An Open Pilot Study of 12-week Oral Treatment

Objective: The purpose of the present study was to elucidate the effects and safety of Cistanche deserticola Y. C. Ma extract (CDX), which was given to elderly persons as a 12-week oral administration via CDX containing health food.Methods: A non-controlled open study was conducted with 25 elderly persons, without severe illness. Participants were given test product containing 100 mg of CDX as well as vitamin E, vitamin B6, ubiquinone, zinc and fukoidan, by oral administration for 12 weeks, with examination conducted before and after the study.Results: Subjective symptoms of “tired eyes” and “feeling tired all the time” were significantly improved after 12 weeks. With regard to anthropometric measurements, pulse rate was significantly reduced (−8.0%, p = 0.002). Biochemical examinations showed significant changes in white blood cell count (−14.1%, p ‹ 0.001), platelet count (−4.3%, p = 0.040), albumin (−3.7%, p ‹ 0.001), albumin/globulin ratio (−3.7%, p = 0.016), and fasting blood sugar (−10.2%, p = .039). Immune function was significantly improved with elevation of percentage of CD4 positive cells (6.1%, p = 0.003), CD4/CD8 ratio (20.2%, p = 0.002) and natural killer (NK) cell activity (11.7%, p = 0.024) accompanied by a decrease in percentage of CD8 positive cells (−7.7%, p = 0.022). Arteriosclerosis examinations by fingertip acceleration pulse wave analysis showed significant improvement in vascular age (−4.2%, p = 0.016) and significant decrease in cardio ankle vascular index (CAVI) (right: −10.9%, p ‹ 0.001, left: −12.6%, p ‹0.001).Conclusion: The test product appears to promote quality of life by decreasing fatigue, activating immune function and favorably affecting the vascular system in the elderly. No severe adverse effects were observed during the study.

Expression of the stem cell marker CD133 in recurrent glioblastoma and its value for prognosis

Experimental data suggest that glioblastoma cells expressing the stem cell marker CD133 play a major role in radiochemoresistance and tumor aggressiveness. To date, however, there is no clinical evidence that the fraction of CD133-positive cells in glioblastoma that recurs after radiochemotherapy may be relevant for prognosis. The authors used immunohistochemistry to assess CD133 expression in 37 paired glioblastoma samples, including 1 primary tumor sample and 1 recurrent tumor sample, after patients received adjuvant radiochemotherapy. To assess the actual composition of the CD133-positive glioblastoma cell population, fluorescence-associated cell sorting (FACS) analysis was used to sort CD133-positive/CD45-negative cells that were assayed for tumor-specific chromosomal aberrations using interphase fluorescence in situ hybridization. To rule out endothelial precursor cells, CD133-positive fractions also were assayed with anti-CD34 by FACS. In recurrent glioblastomas, the percentage of CD133-positive cells was increased by 4.6-fold compared with the percentage in primary glioblastomas, although, in some tumors, it increased up to 10-fold and 20-fold. Unexpectedly, the increase in CD133 expression was associated significantly with longer survival after tumor recurrence. An analysis of tumor-specific chromosomal aberrations and in vivo studies revealed that the CD133-positive cell compartment of recurrent glioblastoma was composed of both cancer stem cells and nontumor neural stem cells. The latter cells represented from 20% to 60% of the CD133-positive cell population, and their relative percentage favorably affected the survival of patients with recurrent glioblastoma. Endothelial CD133-positive/CD34-positive precursors did not contribute to the CD133-positive cell population. The authors hypothesized that, similar to the phenomenon described in glioblastoma models, neural stem/progenitor cells that are recruited by the tumor from surrounding brain may exert an antitumorigenic effect.

Correlation of stem cell marker nestin expression on circulating melanoma cells with extension of disease and survival.

8551 Background: Within circulating melanoma cells (CMCs) there may be a subset of cells with stem cell characteristics able to develop distant metastasis. We evaluated the presence of melanoma cells bearing stem cell phenotype in the bloodstream of patients (pts) with metastatic melanoma. Stem cell marker assessment included nestin (NES) and CD133. Methods: Twenty-five ml blood were collected after informed consent from 32 consecutive pts affected by metastatic uveal (n = 14) or cutaneous (n = 18) melanoma and from (n = 6) healthy volunteers according to ethically approved protocols. Blood was enriched for tumour cells by CD45 depletion of the non-melanoma cell fraction using a magnetic bead separation technique. Potential melanoma cells were identified using flow cytometry with the markers MELAN-A and HMB45. Multiparameter cytometry was carried out to co-stain with the combinations of CD133 and NES. Five tissue samples from metastatic lesions of five different pts were stained with the same antibodies by immunohistochemistry. Results: No cells positive for the melanocyte markers were detected in blood from volunteers. In pts MELANA and/or HMB45 positive cells were detected in 28 samples (87.5%). The absolute number of melanoma cells ranged from 0 to 1233 (median 53) per 10 mL of blood. NES expressing cells were more represented compared to CD133 expressing cells (median 18.0% [0.3%-85.1%] vs. median 0.1% [0%-23.4%]). Percentage of NES-positive cells correlated significantly with tumor burden (p = 0.01) and number of metastatic sites (p = 0.03), whereas percentage of CD133-positive cells and absolute total number of cells did not. Cox regression analysis revealed levels of LDH (HR: 12.8 [1.35-121.5]; p = 0.02), number of metastatic sites (HR 3.87 [1.66-9.03]; p = 0.02), tumor burden (HR 5.72 [1.57- 20.9]; p = 0.01) and high level of NES expression (HR 3.5 [0.92-13.6]; p = 0.04) to be factors related to shorter overall survival. CD133 and NES expression profiles on CMCs and in matched metastatic tissue were similar. Conclusions: Melanoma cells in peripheral blood expressed stem cell associated markers NES and CD133. Higher expression of NES on CMCs might represent an index of poor prognosis. No significant financial relationships to disclose.

Abstract 4266: Potential role of reactive oxygen species in affecting tumorigenicity of glioblastoma stem cells

Abstract Glioblastoma multiforme (GBM) is a common and highly aggressive primary brain tumor with poor prognosis due in part to drug resistance and high incidence of tumor recurrence. The drug resistant and cancer recurrence phenotype may be ascribed to the presence of cancer stem cells, which seem to reside in special stem-cell niches in vivo and require special culture conditions including certain growth factors and serum-free medium to maintain their stemness in vitro. In this study, we used two lines of stem-like cancer cells (GSC11 and GSC23) derived from the tumor tissues of two GBM patients by classical neurosphere culture system, and showed that addition of serum (FBS) to the medium induced an increase of ROS, leading to aberrant differentiation and eventual cell death. Although both GBM stem-like cell lines showed high percentage of CD133-positive cells (20% in GSC11 and 70% in GSC23), exposure to serum only caused a moderate decrease of CD133 expression at later time points. Several methods were used to further test the role of ROS in affecting the stemness of the GSC cells and their self-renewal capacity. Addition of 5% FBS or removal of the antioxidant supplements (B27) from the culture medium caused the spheroid GSC cells to attach to the culture surface, exhibit differentiation morphology, decrease expression of certain stem-cell markers, and eventually die within two weeks. Serum exposure also resulted in a decrease of the GSC ability to form colonies in soft agar assay and to grow tumors in a mouse xenograft model. Although an increase of general cellular ROS could be detected when GSC cells were exposed to FBS, the mitochondrial superoxide appeared to be prominent and likely to be a primary event, which led to a compensatory increase of the mitochondrial SOD2 expression and upregulation of glutathione synthesis. Interestingly, addition of the antioxidant N-Acetylcysteine to the FBS-containing medium partially blocked the FBS-induced aberrant differentiation and improved cell viability and self-renewal capacity, suggesting that ROS may play an important role in affecting stemness and differentiation status of the cancer stem cells. We postulate that during tumor development, only the progeny cells that acquire the ability to counteract the impact of ROS increase induced by serum may eventually proliferate and grow as the tumor bulk, and that proper redox-modulation may be a potential novel strategy to block tumor formation and possibly kill the stem-like cancer cells. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4266.

The percentage of CD133+ cells in human colorectal cancer cell lines is influenced by Mycoplasma hyorhinis infection

BackgroundMollicutes contamination is recognized to be a critical issue for the cultivation of continuous cell lines. In this work we characterized the effect of Mycoplasma hyorhinis contamination on CD133 expression in human colon cancer cell lines.MethodsMycoAlert® and mycoplasma agar culture were used to detect mycoplasma contamination on GEO, SW480 and HT-29 cell lines. Restriction fragment length polymorphism assay was used to determine mycoplasma species. All cellular models were decontaminated by the use of a specific antibiotic panel (Enrofloxacin, Ciprofloxacin, BM Cyclin 1 and 2, Mycoplasma Removal Agent and MycoZap®). The percentage of CD133 positive cells was analyzed by flow cytometry on GEO, SW480 and HT-29 cell lines, before and after Mycoplasma hyorhinis eradication.ResultsMycoplasma hyorhinis infected colon cancer cell lines showed an increased percentage of CD133+ cells as compared to the same cell lines rendered mycoplasma-free by effective exposure to antibiotic treatment. The percentage of CD133 positive cells increased again when mycoplasma negative cells were re-infected by Mycoplasma hyorhinis.ConclusionsMycoplasma hyorhinis infection has an important role on the quality of cultured human colon cancer cell lines giving a false positive increase of cancer stem cells fraction characterized by CD133 expression. Possible explanations are (i) the direct involvement of Mycoplasma on CD133 expression or (ii) the selective pressure on a subpopulation of cells characterized by constitutive CD133 expression.In keeping with United Kingdom Coordinating Committee on Cancer Research (UKCCCR) guidelines, the present data indicate the mandatory prerequisite, for investigators involved in human colon cancer research area, of employing mycoplasma-free cell lines in order to avoid the production of non-reproducible or even false data.

Analysis of the sensitivity of glioma stem cells to temozolomide

Objective To investigate the sensitivity of glioma stem cells (GSC) to temozolomide (TMZ).MethodsFive GSC lines were successfully established from fresh glioma tissues.Immunofluoresence was employed to detect the expression of CD133 in undifferentiated GSC and glial fibriUary acid protein (GFAP) in differentiated GSC.The sensitivity of GSC was determined by using MTS.The percentage of CD133-positive cells was measured with flow cytometry.The methylation of MGMT promoter region in GSC was determined by using fluorescent methylation specific PCR (F-MSP).Western blotting was used to determine the protein level of PTEN in GSC.Results (1) The 5 established GSC lines demonstrated characteristics of cancer stem cells.(2) GSC lines were generally resistant to TMZ.The IC50 of TMZ was 22.3 μmol/L for T509,286.3 μmol/L for T411,and more than 1000 μmol/L for T402,T405 and T511,respectively.(3) GSC lines with more than 10% CD133-positive cells were more resistant to TMZ.(4) GSC lines with unmethylated MGMT promoter region were resistant or intermediately sensitive to TMZ.(5) The protein level of PTEN was different in the 5 GSC lines.No relationship between PTEN level and TMZ sensitivity was found.Conclusion A majority of GSC is resistant to TMZ,which is associated with the methylation status of MGMT promoter and CD133-positive cell population. Key words: Glioma; Glioma stem cells; Temozolomide; MGMT; CD133

Prognostic Significance of FLT3 Internal Tandem Duplication in Acute Myeloid Leukemia with Normal Karyotype

Background: Activating mutations of the fms-like tyrosine kinase 3 gene (FLT3) by internal tandem duplication (ITD) in the juxtamembrane region are found in 20-30% of the adults with acute myeloid leukemia (AML). The allelic ratio of FLT3/ITD (ITD-AR), as assessed by Genescan analysis, has been reported to carry prognostic significance in AML patients who have normal karyotype. Methods: FLT3/ITD was studied by PCR in 113 adults with AML including 55 patients with normal karyotype. Genescan analysis was performed for the PCR products to determine the allelic distribution. The results were correlated with the prognostic factors. Results: FLT3/ITD was found in 23% of the total AML patients and in 32.7% of those patients with normal karyotype. The mutation was related to a high WBC count, a high serum LD level and a low percentage of CD34 positive cells. The ITD-AR ranged from 0.05 to 8.27 (median, 0.61). The patients with a high ITD-AR (0.7) had significantly higher WBC count and LD level than those without FLT3/ITD. On multivariate analysis, a high ITD-AR as well as FLT3/ITD were confirmed to be independent adverse prognostic factors for AML patients with normal karyotype. Conclusion: This study demonstrated that a high ITD-AR and FLT3/ITD had major adverse impacts on the prognostic relevance for AML patients with normal karyotype.

Flow cytometric differential of leukocyte populations in normal bone marrow: Influence of peripheral blood contamination1

Availability of immunophenotypic reference values for the various leukocyte populations distributed in bone marrow may be helpful to recognize abnormal bone marrow development and, therefore, useful as first screening of individuals with suspected hematological malignancies or other hematopoietic disorders. A single tube four-color staining panel (CD66abce/CD14/CD45/CD34) together with a predefined gating strategy was utilized to immunologically differentiate the distribution of the major leukocyte populations in bone marrow aspirates of healthy donors. The sample-blood erythrocyte ratio was applied to assess the amount of blood contamination of marrow and account for this in the marrow value estimates. The frequency of the major leukocyte populations in bone marrow of 134 normal donors were for granulocytes: mean, 69.4%; SD, 10.3%; monocytes: mean, 4.7%; SD, 2.3%; lymphocytes: mean, 18.3%; SD, 8.7%. The frequency of the immature cell population that included precursor cells of each of the cell lineages among other cell types were mean 5.0%; SD 2.2%. The mean percentage of CD34 positive cells was 1.5%; SD 0.7%. Our results showed further that the frequency of cell populations, of which the presence is restricted to the bone marrow (e.g., CD34+ progenitor cells), is influenced by the degree of peripheral blood admixture. Between the total immature cells and purity of the bone marrow, there was a significant positive correlation demonstrated, whereas a negative correlation was found between the percentages of both lymphocytes as monocytes and the purity of the bone marrow. With a single tube-staining panel, we obtained reference values for flow cytometric assessment of all relevant leukocyte populations present in bone marrow that can be used as a frame of reference for better recognition of individuals with abnormal hematopoiesis. In addition, we have demonstrated the influence of the degree of peripheral blood admixture in the bone marrow aspirates on those reference values.

Analysis of gene expression and chemoresistance of CD133+ cancer stem cells in glioblastoma

BackgroundRecently, a small population of cancer stem cells in adult and pediatric brain tumors has been identified. Some evidence has suggested that CD133 is a marker for a subset of leukemia and glioblastoma cancer stem cells. Especially, CD133 positive cells isolated from human glioblastoma may initiate tumors and represent novel targets for therapeutics. The gene expression and the drug resistance property of CD133 positive cancer stem cells, however, are still unknown.ResultsIn this study, by FACS analysis we determined the percentage of CD133 positive cells in three primary cultured cell lines established from glioblastoma patients 10.2%, 69.7% and 27.5%, respectively. We also determined the average mRNA levels of markers associated with neural precursors. For example, CD90, CD44, CXCR4, Nestin, Msi1 and MELK mRNA on CD133 positive cells increased to 15.6, 5.7, 337.8, 21.4, 84 and 1351 times, respectively, compared to autologous CD133 negative cells derived from cell line No. 66. Additionally, CD133 positive cells express higher levels of BCRP1 and MGMT mRNA, as well as higher mRNA levels of genes that inhibit apoptosis. Furthermore, CD133 positive cells were significantly resistant to chemotherapeutic agents including temozolomide, carboplatin, paclitaxel (Taxol) and etoposide (VP16) compared to autologous CD133 negative cells. Finally, CD133 expression was significantly higher in recurrent GBM tissue obtained from five patients as compared to their respective newly diagnosed tumors.ConclusionOur study for the first time provided evidence that CD133 positive cancer stem cells display strong capability on tumor's resistance to chemotherapy. This resistance is probably contributed by the CD133 positive cell with higher expression of on BCRP1 and MGMT, as well as the anti-apoptosis protein and inhibitors of apoptosis protein families. Future treatment should target this small population of CD133 positive cancer stem cells in tumors to improve the survival of brain tumor patients.

Predicting AIDS-related events using CD4 percentage or CD4 absolute counts.

BackgroundThe extent of immunosuppression and the probability of developing an AIDS-related complication in HIV-infected people is usually measured by the absolute number of CD4 positive T-cells. The percentage of CD4 positive cells is a more easily measured and less variable number. We analyzed sequential CD4 and CD8 numbers, percentages and ratios in 218 of our HIV infected patients to determine the most reliable predictor of an AIDS-related event.ResultsThe CD4 percentage was an unsurpassed predictor of the occurrence of AIDS-related events when all subsets of patients are considered. The CD4 absolute count was the next most reliable, followed by the ratio of CD4/CD8 percentages. The value of CD4 percentage over the CD4 absolute count was seen even after the introduction of highly effective HIV therapy.ConclusionThe CD4 percentage is unsurpassed as a parameter for predicting the onset of HIV-related diseases. The extra time and expense of measuring the CD4 absolute count may be unnecessary.

Elevated levels of serum soluble E-selectin in patients with chronic hepatitis B: Correlation with T lymphocyte subsets, NK cells and liver inflammation

It has been reported that serum soluble E-selectin (sE-selectin) may be increased in some inflammatory liver diseases. The purpose of our study was to investigate changes of sE-selectin, T lymphocyte subsets, natural killer (NK) cells, and HBV load in patients with chronic hepatitis B (CHB), analyze the relationship between them, and discuss their significances. Fifty-four patients with chronic hepatitis B and fourteen controls were studied. Serum sE-selectin, T lymphocyte subsets, NK cells, and hepatitis B virus (HBV) load were detected by enzyme-linked immunosorbent assay (ELISA), flow cytometry (FCM), and fluorescence quantitative polymerase chain reaction (FQ-PCR), respectively. sE-selectin level in patients with CHB increased significantly than that in controls (p<0.01). The percentages of CD4 positive cells and NK cells decreased while the percentage of CD8 positive cells increased significantly in CHB patients than in controls (p<0.01, respectively). sE-selectin level significantly related to levels of T lymphocyte subsets, NK cells, serum alanine aminotransferase (sALT) and aspartate aminotransferase (sAST) (p<0.01, respectively), but had no relationship with HBV level. The sE-selectin levels in patients with chronic hepatitis B increase significantly and correlate to liver inflammation, suggesting that sE-selectin can be considered as an additional useful marker of CHB inflammatory activity, and E-selectin may play part of role in pathogenesis of chronic hepatitis B.