Polarization of granulocytic myeloid‐derived suppressor cells by hepatitis C core protein is mediated via IL‐10/STAT3 signalling

Myeloid‐derived suppressor cells (MDSCs) have been described as suppressors of T‐cell function in many malignancies. Impaired T‐cell responses have been observed in patients with chronic hepatitis C virus infection (CHC), which is reportedly associated with the establishment of persistent HCV infection. Therefore, we hypothesized that MDSCs also play a role in chronic HCV infection. MDSCs in the peripheral blood of 206 patients with CHC and 20 healthy donors were analyzed by flow cytometry. Peripheral blood mononuclear cells (PBMCs) of healthy donors cultured with hepatitis C virus core protein (HCVc) were stimulated with or without interleukin 10 (IL‐10). Compared to healthy donors and certain CHC patients with sustained viral response (SVR), CHC patients without SVR presented with a dramatic elevation of G‐MDSCs with the HLA‐DR^?/lowCD33⁺CD14^?CD11b⁺ phenotype in peripheral blood. The frequency of G‐MDSCs in CHC patients was positively correlated with serum HCVc, and G‐MDSCs were induced from healthy PBMCs by adding exogenous HCVc. Furthermore, we revealed a potential mechanism by which HCVc mediates G‐MDSC polarization; activation of ERK1/2 resulting in IL‐10 production and IL‐10‐activated STAT3 signalling. Finally, we confirmed that HCVc‐induced G‐MDSCs suppress the proliferation and production of IFN‐γ in autologous T‐cells. We also found that the frequency of G‐MDSCs in serum was associated with CHC prognosis. HCVc maintains immunosuppression by promoting IL‐10/STAT3‐dependent differentiation of G‐MDSCs from PBMCs, resulting in the impaired functioning of T‐cells. G‐MDSCs may thus be a promising biomarker for predicting prognosis of CHC patients.     

Regulatory T Cells and Myeloid-Derived Suppressor Cells in Patients with Peptic Ulcer and Gastric Cancer

Background: Regulatory T Cells (Tregs) and Myeloid-Derived Suppressor Cells (MDSCs) are two main regulatory cells modulating the immune responses in inflammation and cancer. Objective: To investigate and compare Tregs and MDSCs in peptic ulcer and gastric cancer. Methods: Patients with dyspepsia were selected and divided into three groups of non-ulcer dyspepsia (NUD, n=22), peptic ulcer disease (PUD, n=25), and gastric cancer (GC, n=27) according to their endoscopic and histopathological examinations. Helicobacter pylori infection was diagnosed by rapid urease test and histopathology. The number of peripheral blood CD4+CD25+FoxP3+Tregs and CD14+HLA-DR- MDSCs were determined in all patients, by flow cytometry. The number of FoxP3+ regulatory T cells was also determined by immunohistochemistry (IHC). Results: The percentage of peripheral blood Treg cells in both PUD )0.81 ± 0.39, p<0.001) and GC groups )0.98 ± 0.65, p<0.001) were significantly higher than in NUD group (0.46 ± 0.10). These results were also confirmed by IHC. A significantly higher percentage of MDSCs in patients with PUD )0.73 ± 0.19, p<0.001) and GC )0.73 ± 0.16, p<0.001) was also observed when compared to NUD group )0.46 ± 0.16). There was no difference in the percentages of these two cell types between the PUD and GC groups. The percentages of Tregs and MDSCs in patients with PUD and GC were not significantly correlated. Conclusions: Both Tregs and MDSCs showed higher frequencies in PUD and GC. These results suggest that immune-modulation by the Tregs and MDSCs may play a role in the pathogenesis of PUD and GC.     

Metformin induces apoptosis of pancreatic cancer cells

AIM：To assess the role and mechanism of metformin in inducing apoptosis of pancreatic cancer cells.METHODS：The human pancreatic cancer cell lines ASPC-1,BxPc-3,PANC-1 and SW1990 were exposed to metformin.The inhibition of cell proliferation and colony formation via apoptosis induction and S phase arrest in pancreatic cancer cell lines of metformin was tested.RESULTS：In each pancreatic cancer cell line tested,metformin inhibited cell proliferation in a dose dependent manner in MTS（3-（4,5-dimethylthiazol-2-yl）-5-（3-carboxymethoxyphenyl）-2-（4-sulfophenyl）-2H-tetrazolium assays）.Flow cytometric analysis showed that metformin reduced the number of cells in G1 and increased the percentage of cells in S phase as well as the apoptotic fraction.Enzymelinked immunosorbent assay（ELISA） showed that metformin induced apoptosis in all pancreatic cancer cell lines.In Western blot studies,metformin induced poly-ADP-ribose polymerase（PARP） cleavage（an indicator of caspase activation） in all pancreatic cancer cell lines.The general caspase inhibitor（VAD-fmk） completely abolished metformin-induced PARP cleavage and apoptosis in ASPC-1 BxPc-3 and PANC-1,the caspase-8 specific inhibitor（IETD-fmk） and the caspase-9 specific inhibitor（LEHD-fmk） only partially abrogated metformin-induced apoptosis and PARP cleavage in BxPc-3 and PANC-1 cells.We also observed that metformin treatment dramatically reduced epidermal growth factor receptor（EGFR） and phosphorylated mitogen activated protein kinase（P-MAPK） in both a time-and dose-dependent manner in all cell lines tested.CONCLUSION：Metformin significantly inhibits cell proliferation and apoptosis in all pancreatic cell lines.And the metformin-induced apoptosis is associated with PARP cleavage,activation of caspase-3,-8,and-9 in a time-and dose-dependent manner.Hence,both caspase-8 and-9-initiated apoptotic signaling pathways contribute to metformin-induced apoptosis in pancreatic cell lines.     

HIV-1 infection increases the expression of human endogenous retroviruses type K (HERV-K) in vitro

Antibodies to HERV-K antigens have been linked to HIV-1 infection and expression of HERV-K proteins generates T-cell cytotoxic responses in many cancers. HERV-K RNA and protein abundance was measured in HIV-1-infected and control cells. In vitro exposure of HIV-1 laboratory-adapted and primary isolates on U87MG cells increased the expression of HERV-K RNA in a dose-dependent manner. HERV-K RNA and protein burdens were significantly increased in HIV-1-producing H9 cell lines compared to H9 cells. The expression of HERV-K was synergistically increased in HIV-1-infected PBMCs after stimulation with PMA/ionomycin. Furthermore, the expression of HERV-K in PBMCs, and particularly in CD4(+) T cells, was higher in HIV-1 patients compared to control subjects. The expression of HERV-K might be related to HIV-1 pathogenesis and AIDS-associated cancers.     

Methotrexate attenuates the Th17/IL-17 levels in peripheral blood mononuclear cells from healthy individuals and RA patients

To investigate whether the inhibition of Th17/interleukin (IL)-17 contributes to the beneficial effects of methotrexate (MTX) in the treatment of rheumatoid arthritis (RA). Peripheral blood mononuclear cells (PBMCs) from healthy donors and RA patients were collected. The cells were stimulated with monoclonal antibodies to CD3 and CD28 in the absence or presence of MTX. After coincubation, IL-17 production was detected at both the mRNA and protein levels, and the percentage of cells positive for both CD4 and IL-17 in PBMCs was analyzed by flow cytometry. PBMCs of healthy donors and RA patients were stimulated with CD3 and CD28 monoclonal antibodies to produce high levels of IL-17. The augmentation of IL-17 at the mRNA and protein levels was significantly inhibited when PBMC cultures were preincubated with MTX. Compared with PBMCs of healthy donors, PBMCs of RA patients produced higher levels of IL-17, and this increase in IL-17 levels was more inhibited by MTX pretreatment. MTX inhibited IL-17 at the mRNA level in a dose-dependent manner, but not at the protein level, in both PBMCs of healthy donors and RA patients. MTX did not affect the percentage of CD4- and IL-17-positive cells in PBMCs. MTX dose dependently suppressed the production of IL-17 at the mRNA level by PBMCs from healthy donors and RA patients. Suppression of IL-17 by MTX may contribute to its potent anti-inflammatory role in RA therapy.     

Elevation of CD56brightCD16- Lymphocytes in MDR Pulmonary Tuberculosis

Background: Protective immune responses induced in the majority of people infected with Mycobacterium tuberculosis enable them to control TB infection. Objective: The aim of this study was to investigate CD56 and CD16 positive peripheral blood mononu-clear cells (PBMCs) and leukocyte subsets from multi-drug resistant pulmonary tuber-culosis (MDR-TB), and compare them with nonresistant (NR) TB patients and healthy controls. Methods: 13 MDR-tuberculosis patients, 20 NR-TB individuals and 40 healthy subjects were included. Peripheral blood mononuclear cells were double stained with fluorochrome conjugated antibodies against CD56 and CD16 cell surface markers. The phenotype of positive cells was then analyzed by flow cytometry and the percent-ages of CD56+ CD16+, CD56- CD16+, CD56dimCD16+/-, and CD56brightCD16+/- subsets were calculated. Results: There was a significant decline in the percentage of CD56+CD16+ lymphocytes in both MDR and NR-TB patients compared with healthy controls. We also observed lower proportions of CD56dim/brightCD16+ in addition to higher percentages of CD56dim/brightCD16- subsets in all TB patients (p≤0.05). In MDR-TB, our findings demonstrated a distinct phenotypic feature with increased levels of CD56brightCD16- in comparison with both NR-TB and healthy subjects. Conclusion: Considering the function of CD56/CD16 expressing cells in TB, we suggest that pheno-typic characteristics of PBMCs in MDR-TB may correlate with their status of drug re-sistance and probably with their high mortality rates.     

原发性肝细胞癌患者CD4+CD25+调节性T淋巴细胞的频率、表型及功能

目的探讨CD4^＋CD25^＋调节性T淋巴细胞（CD4^＋CD25^＋Treg）在HCC患者外周血及肿瘤组织中的频率、抑制功能及临床意义。方法收集18例原发性HCC患者的PBMC、肝癌组织及相应的癌旁组织标本,以及10例CHB患者和15名健康对照的外周血标本,以三色与四色流式分析法分析PBMC以及肿瘤浸润的淋巴细胞中CD4^＋CD25^＋Treg的频率及表型,并同时分析其与HBV持续感染、肿瘤TNM分期和其他临床指标的关系,用BrdU掺入法评价CD4^＋CD25^＋Treg的免疫抑制功能。结果与健康对照组相比HCC患者、慢性HBV感染者外周血中CD4^＋CD25^＋Treg频率明显上升（P＜0．01）,且HCC患者肿瘤浸润的淋巴细胞中CD4^＋CD25^＋Treg的频率也明显上调（P＜0．01）;随着肿瘤的进展HCC患者外周血中CD4^＋CD25^＋Treg呈上升趋势（P＜0．05）; HCC患者CD4^＋CD25^＋Treg可非特异性地抑制自身活化的CD4^＋CD25^- T淋巴细胞,并呈剂量依赖性,且抑制能力与健康对照组相比差异无统计学意义。结论HCC患者外周血中及肿瘤组织中CD4^＋CD25^＋Treg的频率升高,增多的CD4^＋CD25^＋Treg可能参与抑制抗HCC的免疫应答,从而使肝癌细胞逃脱机体的“免疫监视”作用。     

Identification of local and circulating cancer stem cells in human liver cancer

Increasing evidence has revealed the importance of cancer stem cells (CSCs) in carcinogenesis. Although liver CSCs have been identified in hepatocellular carcinoma (HCC) cell lines, no data have shown the presence of these cells in human settings. The present study was designed to delineate CSCs serially from HCC cell lines, human liver cancer specimens to blood samples, using CD90 as a potential marker. The number of CD90(+) cells increased with the tumorigenicity of HCC cell lines. CD45(-)CD90(+) cells were detected in all the tumor specimens, but not in the normal, cirrhotic, and parallel nontumorous livers. In addition, CD45(-)CD90(+) cells were detectable in 90% of blood samples from liver cancer patients, but none in normal subjects or patients with cirrhosis. A significant positive correlation between the number of CD45(-)CD90(+) cells in the tumor tissues and the number of CD45(-)CD90(+) cells in the blood samples was identified. CD90(+) cells sorted from cell lines and CD45(-)CD90(+) cells from the tumor tissues and blood samples of liver cancer patients generated tumor nodules in immunodeficient mice. Serial transplantation of CD90(+) cells from tumor xenografts generated tumor nodules in a second and subsequently third batch of immunodeficient mice. Treatment of CD90(+) CSCs with anti-human CD44 antibody induced cell apoptosis in a dose-dependent manner. CONCLUSION: Identification of CD45(-)CD90(+) CSCs in both tumor tissues and circulation suggests that CD45(-)CD90(+) could be used as a marker for human liver cancer and as a target for the diagnosis and therapy of this malignancy.     

Application of the ELISPOT assay to the characterization of CD8(+) responses to Epstein-Barr virus antigens

CD8(+) cells have an important role in controlling Epstein-Barr virus (EBV) infection. We adapted the interferon-gamma ELISPOT assay to the quantitative analysis of EBV-specific CD8(+) cells. Using peripheral blood mononuclear cells (PBMCs) from healthy donors, we measured both the aggregate response to the virus, using EBV-transformed lymphoblastoid cell lines (LCLs) as stimulators, and the specific responses to 2 A2-restricted peptide epitopes: the subdominant latency membrane protein-2 (LMP2) peptide CLGGLLTMV and the early lytic BMLF1 peptide GLCTLVAML. LCL-responsive CD8(+) cells were detected in all EBV-seropositive donors (range 954 to 37 830 spots/10(6) CD8(+) cells). LMP2 peptide-responsive CD8(+) cells were detected in 10 of 11 healthy seropositive A2 donors (range 11 to 83 spots/10(6) PBMC). BMLF1 peptide-responsive CD8(+) cells were detected in all seropositive A2 donors examined (range 13 to 943 spots/10(6) PBMC). Cytotoxic T-lymphocyte (CTL) lines generated with weekly stimulation of LCLs for therapeutic purposes were also studied. Relative to PBMCs, these CTL lines showed a marked increase in the level of LCL-responsive and LMP2 peptide-responsive CD8(+) cells and a lesser degree of expansion of BMLF1 peptide-responsive CD8(+) cells. Finally, we applied the ELISPOT assay to monitor adoptive infusion of EBV CTL lines. In 2 patients examined, a transient increase in LCL-responsive CD8(+) cells could be detected after infusion. Thus, the ELISPOT assay can be applied to the analysis of CD8(+) responses to EBV antigens in PBMCs, in ex vivo expanded CTL lines, and in PBMCs from patients treated with ex vivo expanded CTL lines. (Blood. 2000;95:241-248)     

Increased central memory T cells in patients with chronic pancreatitis.

BACKGROUND/AIMS: A dysregulated immune response has been suggested to be important for the pathogenesis of chronic pancreatitis (CP). Formation of immunological memory is based on the differentiation of naive T lymphocytes to memory T lymphocytes after exposure to antigens and specific cytokines. The aim of this study was to analyze peripheral blood mononuclear cells (PBMCs) in patients with CP for different T lymphocyte subsets including naive and memory T cells. METHODS: PBMCs from 9 patients who had undergone pancreatic resection due to CP, 9 CP patients who had not been resected and 9 healthy controls were analyzed by flow cytometry. RESULTS: Patients with CP had a skewed distribution of T lymphocytes, with an increased level of CCR7+/CD45RA- central memory T lymphocytes compared to healthy controls. Nonresected CP patients and subjects who had undergone pancreatic resection due to CP had similar levels of central memory T lymphocytes. CONCLUSION: Our results indicate that the dysregulation of the immune system in chronic pancreatitis seems to persist even after removal of large parts of the local inflammatory site. We suggest that the increase of central memory T lymphocytes may be important for maintaining the inflammatory process in chronic pancreatitis.     

Myeloid suppressor cells induced by hepatitis C virus suppress T-cell responses through the production of reactive oxygen species

Impaired T-cell responses in chronic hepatitis C virus (HCV) patients have been reported to be associated with the establishment of HCV persistent infection. However, the mechanism for HCV-mediated T-cell dysfunction is yet to be defined. Myeloid-derived suppressor cells (MDSCs) play a pivotal role in suppressing T-cell responses. In this study we examined the accumulation of MDSCs in human peripheral blood mononuclear cells (PBMCs) following HCV infection. We found that CD33(+) mononuclear cells cocultured with HCV-infected hepatocytes, or with HCV core protein, suppress autologous T-cell responses. HCV core-treated CD33(+) cells exhibit a CD14(+) CD11b(+/low) HLADR(-/low) phenotype with up-regulated expression of p47(phox) , a component of the NOX2 complex critical for reactive oxygen species (ROS) production. In contrast, immunosuppressive factors, arginase-1 and inducible nitric oxide synthase (iNOS), were not up-regulated. Importantly, treatment with an inactivator of ROS reversed the T-cell suppressive function of HCV-induced MDSCs. Lastly, PBMCs of chronic HCV patients mirror CD33(+) cells following treatment with HCV core where CD33(+) cells are CD14(+) CD11b(+) HLADR(-/low) , and up-regulate the expression of p47(phox). CONCLUSION: These results suggest that HCV promotes the accumulation of CD33(+) MDSC, resulting in ROS-mediated suppression of T-cell responsiveness. Thus, the accumulation of MDSCs during HCV infection may facilitate and maintain HCV persistent infection.     

Effect of rhG-CSF on the mobilization of CD38 and HLA-DR subfractions of CD34+ peripheral blood progenitor cells

Several studies have demonstrated that both CD34+/CD38– and CD34+/HLA-DR- human hematopoietic progenitor cells have properties associated with hematopoietic stem cells. However, the kinetics of these two cell populations in human peripheral blood (PB) after priming with granulocyte colony-stimulating factor (rhG-CSF) has not been investigated. By using flow-cytometric analysis we have shown that administration of rhG-CSF to 14 patients eligible for peripheral blood progenitor cell (PBPC) transplantation led to an increment of CD34+/CD38+ and CD34+/HLA-DR+ cells in the PB that paralleled the increase of total CD34+ cells, indicating that such subpopulations are responsible for the major release of CD34+ cells. Furthermore, rhG-CSF priming led to a significant mobilization of fractions of more immature CD34+/CD38– and CD34+/HLA-DR- cells to the PB. In the leukapheresis preparations, the average frequency of CD34+ cells lacking the CD38 or HLA-DR antigens was low (5% and 30%, respectively), with little overlap between the CD38- and HLA-DR- subpopulations. In addition, the yield of each subset of CD34+ cells (CD34+/CD38± and CD34+/HLA-DR±) in the PB correlated with the numbers in the collected material. The results of the present study indicate that administration of rhG-CSF causes a significant increase of CD34+/CD38± and CD34+/HLA-DR± cells in PB, and that such cells can be then safely harvested by leukapheresis procedures.     

Reconstitution of herpes simplex virus-specific T cell immunity in HIV-infected patients receiving highly active antiretroviral therapy

Production of herpes simplex virus (HSV)-specific interferon- gamma by peripheral-blood mononuclear cells (PBMCs) of HSV-seropositive healthy donors and human immunodeficiency virus-infected persons was determined by use of ELISPOT. The mean +/- SD number of spot-forming cells/10(6) PBMCs was 314 +/- 74 in 11 healthy donors, 360 +/- 69 in 3 long-term nonprogressors (LTNPs), 186 +/- 52 in 9 newly diagnosed patients, and 181 +/- 59 in 33 patients who were receiving highly active antiretroviral therapy (HAART) for a median period of 30 months (range, 1-109 months). In 9 patients monitored prospectively while receiving virologically and immunologically successful first-line HAART, the number of spot-forming cells increased by 5.6/month (95% confidence interval, 1.2-9.9 [P=.01]) and 21.3/100 CD4 cells/mm(3) gained (95% confidence interval, 13.8-28.7 [P<.0001]). Responses were correlated with LTNP status and CD4 cell count.     

Growth factor requirements for survival in G0 and entry into the cell cycle of primitive human hemopoietic progenitors.

In this study we have isolated populations of dormant human hemopoietic progenitors by two different approaches. First CD34+ cells isolated by panning were further separated on the basis of absence of HLA-DR expression by using fluorescence-activated cell sorting. Second, CD34+ HLA-DR- cells were isolated by nonadherence to soybean agglutinin, negative immunomagnetic bead selection with lineage-specific antibodies, and two-color cell sorting. Progenitors in either cell population were unable to form colonies in the presence of interleukin (IL)-3 alone but yielded a substantial number of colonies, including multilineage colonies, in the presence of combinations of IL-3 and IL-6. Similarly, IL-3 plus any one of the other synergistic factors, including granulocyte colony-stimulating factor, IL-11, leukemia inhibitory factor, and steel factor, effectively supported colony formation from CD34+ HLA-DR- progenitors. Sequential observation of colony formation from single CD34+ HLA-DR- cells provided definitive evidence that the synergistic factors trigger cell divisions of dormant cells. Studies with delayed addition of factors to the cultures provided evidence that this population of cells also requires IL-3 or granulocyte/macrophage colony-stimulating factor (GM-CSF) to survive even while dormant. In contrast, none of the synergistic factors were able to replace IL-3 or GM-CSF in this function. These findings confirm and extend the model that multiple factors with overlapping functions operate both independently and in combination to regulate early stages of hemopoiesis.     

Detection of tumor stem cell markers in pancreatic carcinoma cell lines

IntroductionT he cell population of most tumors is hetero- geneous with regard to its proliferation capacity, apoptosis-resistance mechanisms, and ability to reconstitute the tumor upon xeno-transplantation. This phenomenon arises as the result of accumulation of multiple genetic and epigenetic changes. Current evidence suggests that only a few cells within the tumor, the cancer stem cells, possess unlimited proliferative capacity and give rise to tumors that phenotypically resemble their init…     

Ex vivo expansion of enriched peripheral blood CD34+ progenitor cells by stem cell factor, interleukin-1 beta (IL-1 beta), IL-6, IL-3, interferon-gamma, and erythropoietin

To provide sufficient numbers of peripheral blood progenitor cells (PBPCs) for repetitive use after high-dose chemotherapy, we investigated the ability of hematopoietic growth factor combinations to expand the number of clonogenic PBPCs ex vivo. Chemotherapy plus granulocyte colony-stimulating factor (G-CSF) mobilized CD34+ cells from 18 patients with metastatic solid tumors or refractory lymphomas were cultured for up to 28 days in a liquid culture system. The effects of interleukin-1 beta (IL-1), IL-3, IL-6, granulocyte-macrophage-CSF (GM-CSF), G-CSF, macrophage-CSF (M-CSF), stem cell factor (SCF), erythropoietin (EPO), leukemia inhibitory factor (LIF), and interferon- gamma, as well as 36 combinations of these factors were tested. A combination of five hematopoietic growth factors, including SCF, EPO, IL-1, IL-3, and IL-6, was identified as the optimal combination of growth factors for both the expansion of total nucleated cells as well as the expansion of clonogenic progenitor cells. Proliferation peaked at days 12 to 14, with a median 190-fold increase (range, 46- to 930- fold) of total clonogenic progenitor cells. Expanded progenitor cells generated myeloid (colony-forming unit-granulocyte-macrophage), erythroid (burst-forming unit-erythroid), as well as multilineage (colony-forming unit-granulocyte, erythrocyte, monocyte, megakaryocyte) colony-forming units. The number of multilineage colonies increased 250- fold (range, 33- to 589-fold) as compared with pre-expansion values. Moreover, the absolute number of early hematopoietic progenitor cells (CD34+/HLA-DR-; CD34+/CD38-), as well as the number of 4-HC-resistant progenitors within expanded cells increased significantly. Interferon- gamma was shown to synergize with the 5-factor combination, whereas the addition of GM-CSF significantly decreased the number of total clonogenic progenitor cells. Large-scale expansion of PB CD34+ cells (starting cell number, 1.5 x 10(6) CD34+ cells) in autologous plasma supplemented with the same 5-factor combination resulted in an equivalent expansion of progenitor cells as compared with the microculture system. In summary, our data indicate that chemotherapy plus G-CSF-mobilized PBPCs from cancer patients can be effectively expanded ex vivo. Moreover, our data suggest the feasibility of large- scale expansion of PBPCs, starting from small numbers of PB CD34+ cells. The number of cells expanded ex vivo might be sufficient for repetitive use after high-dose chemotherapy and might be candidate cells for therapeutic gene transfer.     

Correlation between circulating myeloid-derived suppressor cells and Th17 cells in esophageal cancer

AIM: To perform a comprehensive investigation into the potential correlation between circulating myeloidderived suppressor cells (MDSCs) and Th17 cells in esophageal cancer (ECA). METHODS: A total of 31 patients newly diagnosed with ECA and 26 healthy subjects were included in the current study. The frequencies of MDSCs and Th17 cells in peripheral blood were determined by flow cytometry. The mRNA expression of cytokines, arginase 1 (Arg1) and inducible NO synthase (iNOS) in peripheral blood mononuclear cells (PBMCs) and plasma Arg1 were assessed by real-time polymerase chain reaction and enzyme-linked immunosorbent assay, respectively. RESULTS: There was an increased prevalence of MDSCs in the peripheral blood from ECA patients (15.21% ± 2.25%) when compared with healthy control (HC) (1.10% ± 0.12%, P 0.0001). The plasma levels of Arg1 in ECA patients were significantly higher than those in HC (28.28 ± 4.10 ng/mL vs 9.57 ± 1.51 ng/ mL, P=0.0003). iNOS mRNA levels in the peripheral blood of ECA patients also showed a threefold increase compared with HC (P=0.0162). The frequencies of Th17 cells (CD4 + IL-17A + ) were significantly elevated in ECA patients versus HC (3.50% ± 0.33% vs 1.82% ± 0.19%, P=0.0001). Increased mRNA expression of IL-17 and ROR-γt was also observed in ECA patients compared with HC (P=0.0041 and P=0.0004, respectively), while the mRNA expression of IL-6 and tumor necrosis factor-α (TNF-α) showed significant decreases (P=0.0049 and P 0.0001, respectively). No obvious correlations were found between the frequencies of MDSCs and Th17 cells in the peripheral blood from ECA patients(r=-0.1725, P=0.3534). Arg1 mRNA levels were positively correlated with levels of IL-6 (r=0.6404, P=0.0031) and TNF-α (r=0.7646, P=0.0001). Similarly, iNOS mRNA levels were also positively correlated with levels of IL-6 (r=0.6782, P=0.0007) and TNF-α (r=0.7633, P 0.0001). CONCLUSION: This study reveals the relationship between circulating MDSCs and Th17 cells, which may lead to new immunotherapy approaches for ECA based on the associated metabolites and cytokines.     

Fucosylation is a common glycosylation type in pancreatic cancer stem cell-like phenotypes

AIM: To evaluate/isolate cancer stem cells(CSCs) from tissue or cell lines according to various definitions and cell surface markers. METHODS: Lectin microarray analysis was conducted on CSC-like fractions of the human pancreatic cancer cell line Panc1 by establishing anti-cancer drug-resistant cells. Changes in glycan structure of CSC-like cells were also investigated in sphere-forming cells as well as in CSC fractions obtained from overexpression of CD24 and CD44. RESULTS: Several types of fucosylation were increased under these conditions, and the expression of fucosylation regulatory genes such as fucosyltransferases, GDP-fucose synthetic enzymes, and GDP-fucose transporters were dramatically enhanced in CSC-like cells. These changes were significant in gemcitabineresistant cells and sphere cells of a human pancreatic cancer cell line, Panc1. However, downregulation of cellular fucosylation by knockdown of the GDP-fucose transporter did not alter gemcitabine resistance, indicating that increased cellular fucosylation is a result of CSC-like transformation. CONCLUSION: Fucosylation might be a biomarker of CSC-like cells in pancreatic cancer.     

Dendritic cells in patients with type I Gaucher disease are decreased in number but functionally normal

Gaucher disease is a lysosomal storage disorder, in which undigested glucosylceramide is deposited in the cytoplasm of mature macrophages, which accumulate in the bone marrow and the reticuloendothelial system. Dendritic cells are bone marrow-derived cells, specialized for the uptake, processing, transport and presentation of antigens to T-lymphocytes. We investigated peripheral blood dendritic cell-precursors, as well as the potential of peripheral blood monocytes and bone marrow-derived progenitor cells, to differentiate into mature dendritic cells in 12 patients with type I Gaucher disease. Results of the 10 adult patients were compared with those of 10 healthy volunteers, matched for age and sex. Six patients were anemic and 9 were thrombocytopenic, but none had severe bone disease. Both myeloid and plasmacytoid dendritic cells of patients with Gaucher disease, as well as the yield of the monocyte-derived dendritic cells, obtained after GM-CSF and IL-4 stimulation, were found significantly decreased, when compared to controls (myeloid dendritic cells: 0.19 +/- 0.07% vs. 0.34 +/- 0.10%, P = 0.009, plasmacytoid dendritic cells: 0.17 +/- 0.12% vs. 0.39 +/- 0.13%, P = 0.004, monocyte-derived dendritic cells: 4.8 +/- 3.5% vs. 8.3 +/- 3.2%, P = 0.036). However, the immunophenotypic profile of dendritic cells, estimated by CD1a, CD40, CD54, CD80, CD83 and HLA-DR expression, the endocytic and allo-stimulatory capacity of the immature, as well as of the TNF-alpha- or lipopolysaccharite-stimulated mature monocyte-derived dendritic cells, was similar to those obtained by healthy controls. In addition, bone marrow-derived CD34+ cells differentiated in the presence of GM-CSF, SCF, TNF-alpha and IL-4 into mature dendritic cells that did not differ in number, phenotype and allo-stimulatory activity from those of controls. Our findings suggest that patients with Gaucher disease exhibit mainly quantitative defects of their dendritic cells' system, demonstrated by decreased circulating dendritic cell precursors of both myeloid and plasmacytoid type. This finding may contribute to the poor immune response against infectious agents and an impaired immune surveillance, associated with an increased risk of developing a neoplastic disease.     

猪带绦虫抗原刺激囊虫病患者PBMC Th1/Th2细胞因子的表达及其免疫调节

目的检测猪带绦虫不同虫期抗原刺激囊虫病患者外周血单个核细胞(PBMC)Th1/Th2细胞因子的表达水平,并分析其在囊虫病免疫调控中的作用。方法用流式细胞仪检测囊虫病患者新鲜血T淋巴细胞亚群,并以猪带绦虫六钩蚴抗原或囊尾蚴抗原刺激囊虫病患者PBMC,分别在刺激的第0d、5d、10d和15d时检测Th1/Th2细胞因子(IFN-γ及IL-4),对分泌IFN-γ或IL-4的不同细胞群体进行数据分析。结果囊虫病患者新鲜血T淋巴细胞亚群CD3+与CD4+细胞较正常人升高,CD4+/CD8+较正常人升高,分泌IFN-γ及IL-4的CD3+细胞百分率较正常人升高;猪囊尾蚴抗原刺激囊虫病患者PB-MC第0d、5d、10d和15d时,分泌IFN-γ的CD3+细胞百分率分别为25.42%±5.53%,30.46%±4.94%,36.52%±4.73%,38.69%±5.58%;分泌IL-4的CD3+细胞百分率分别为2.52%±0.52%,3.00%±0.57%,3.81%±0.70%,5.03%±0.73%。六钩蚴抗原刺激囊虫病患者PBMC分泌IFN-γ及IL-4的CD3+细胞百分率亦呈现相同的变化趋势。结论囊虫病患者与正常人相比PBMC中存在T淋巴细胞的极化水平异常,CD3+与CD4+细胞百分率均显著升高,T细胞亚群比例失调,免疫功能紊乱;随着囊尾蚴抗原刺激外周血PBMC时间延长,分泌IFN-γ和IL-4的CD3+细胞百分率逐渐升高。