Modulation of Lianbizi Injection (Andrographolide ) on Some Immune Functions

Objective: To study the effects of andrographolide on immune functions and the immune mechanism in clinical therapy.Methods: The amounts of IFN-α,IFN-γ, TNF-α, IL-8 in the peripheral blood mononuclear cells (PBMC) culture supernatants dealt with by different concentrations of LianBiZhi (LBZ) injection, the effective component of which is andrographolide, were detected by biological activity test or ELISA in vitro. The effects of LBZ injection on macrophage phagocytotic function and natural killer cells cytotoxicity were examined by means of macrophage to phagocytize cock erythrocyte and measurement of lactate dehydrogenase (LDH) activity released from the damaged cells, respectively.Results: The LBZ injection could not only enhance the phagocytosis activity of peritoneal macrophage from guinea pig to phagocytosis cock erythrocytes, but also augment the cytotoxicity mediated by natural killer cells from PBMCs.Conclusion: Andrographolide is an immunostimulant agent which can modulate both antigen specific and nonspecific immune function by means of its natural killer cells, macrophage and cytokines.

Effect of in vitro interferon-beta administration on hepatitis C virus in peripheral blood mononuclear cells as a predictive marker of clinical response to interferon treatment for chronic hepatitis C

AIM:To test whether in vitro incubation of peripheral blood mononuclear cells (PBMC) with interferon (IFN) could efficiently decrease hepatitis C virus-RNA (HCV-RNA) amount and to analyze whether this effect was associated with clinical response to IFN.METHODS:Twenty-seven patients with histologically proven chronic hepatitis C were given intravenous administration of 6 million units (MU) IFN-β daily for 6 weeks followed by three times weekly for 20 weeks. PBMC collected before IFN therapy were incubated with IFN-β and HCV-RNA in PMBC was semi-quantitatively determined.RESULTS: Twenty-five patients completed IFN therapy.Eight patients (32%) had sustained loss of serum HCV-RNA with normal serum ALT levels after IFN therapy (complete responders).HCV-RNA in PBMC was detected in all patients,whereas it was not detected in PBMC from healthy subjects.In vitro administration of IFN-β decreased the amount of HCV-RNA in PMBC in 18 patients (72%). Eight of these patients obtained complete response. On the other hand,none of the patients whose HCV-RNA in PBMC did not decrease by IFN-β was complete responders. Multiple logistic regression analysis revealed that the decrease of HCV-RNA amount in PBMC by IFN-β was the only independent predictor for complete response (P<0.05).CONCLUSION:The effect of in vitro IFN-β on HCV in PBMC reflects clinical response and would be taken into account as a predictive marker of IFN therapy for chronic hepatitis C.

Intrahepatic natural killer T cell populations are increased in human hepatic steatosis

AIM: To determine if natural killer T cell (NKT) populations are affected in nonalcoholic fatty liver disease (NAFLD). METHODS: Patients undergoing bariatric surgery underwent liver biopsy and blood sampling during surgery. The biopsy was assessed for steatosis and immunocyte infiltration. Intrahepatic lymphocytes (IHLs) were isolated from the remainder of the liver biopsy, and peripheral blood mononuclear cells (PBMCs) were isolated from the blood. Expression of surface proteins on both IHLs and PBMCs were quantified using flow cytometry. RESULTS: Twenty-seven subjects participated in thisstudy. Subjects with moderate or severe steatosis had a higher percentage of intrahepatic CD3+/CD56+ NKT cells (38.6%) than did patients with mild steatosis (24.1%, P = 0.05) or those without steatosis (21.5%, P = 0.03). Patients with moderate to severe steatosis also had a higher percentage of NKT cells in the blood (12.3%) as compared to patients with mild steatosis (2.5% P = 0.02) and those without steatosis (5.1%, P = 0.05). CONCLUSION: NKT cells are significantly increased in the liver and blood of patients with moderate to severe steatosis and support the role of NKT cells in NAFLD.

New perspectives in occult hepatitis C virus infection

Occult hepatitis C virus (HCV) infection, defined as the presence of HCV RNA in liver and in peripheral blood mononuclear cells (PBMCs) in the absence of detectable viral RNA in serum by standard assays, can be found in anti-HCV positive patients with normal serum levels of liver enzymes and in anti-HCV negative patients with persistently elevated liver enzymes of unknown etiology. Occult HCV infection is distributed worldwide and all HCV genotypes seem to be involved in this infection. Occult hepatitis C has been found not only in anti-HCV positive subjects with normal values of liver enzymes or in chronic hepatitis of unknown origin but also in several groups at risk for HCV infection such as hemodialysis patients or family members of patients with occult HCV. This occult infection has been reported also in healthy populations without evidence of liver disease. Occult HCV infection seems to be less aggressive than chronic hepatitis C although patients affected by occult HCV may develop liver cirrhosis and even hepatocellular carcinoma. Thus, anti-HCV negative patients with occult HCV may benefit from antiviral therapy with pegylated-interferon plus ribavirin. The persistence of very low levels of HCV RNA in serum and in PBMCs, along with the maintenance of specific T-cell responses against HCV-antigens observed during a long-term follow-up of patients with occult hepatitis C, indicate that occult HCV is a persistent infection that is not spontaneously eradicated. This is an updated report on diagnosis, epidemiology and clinical implications of occult HCV with special emphasis on anti-HCV negative cases.

MicroRNA and histopathological characterization of pure mucinous breast carcinoma

Objective: Pure mucinous breast carcinoma (PMBC) is an uncommon histological type of breast cancer characterized by a large amount of mucin production. MicroRNA (miRNA) is a large class of small noncoding RNA of about 22 nt involved in the regulation of various biological processes. This study aims to identify the miRNA expression profile in PMBC. Methods: MiRNA expression profiles in 11 PMBCs were analyzed by miRNA-microarray and real-time polymerase chain reaction (PCR). Thirty-one PMBCs and 27 invasive ductal carcinoma of no special types (IDC-NSTs) were assessed by immunohistochemistry using antibodies against ER, PR-progesterone receptor, HER2, Ki-67, Bcl-2, p53, PCNA, and CK5 and 6. Results: We analyzed the miRNA expression in 11 PMBCs and corresponding normal tissues using miRNA-microarray and real-time PCR, and found that miR-143 and miR-224-5p were significantly downregulated in mucinous carcinoma tissue. Compared with IDC-NSTs, PMBC showed a significantly higher ER positive rate, lower HER-2 positive rate, and lower cell proliferation rates. Conclusions: To our knowledge, this is the first study to demonstrate the miRNA expression profile of PMBC, and our findings may lead to further understanding of this type of breast cancer.

Clinical study of co-treatment with DC-CIK cells for advanced solid carcinomas

Objective： The aim of this study was to observe the therapeutic effect of cytokine induced killer （CIK） cells in combination with dendritic cells （DCs） on advanced solid carcinoma patients. Methods： Isolated peripheral blood mononuclear cells （PBMCs） from 110 advanced solid tumor patients. Added granulocyte-macrophage colony-stimulating factor （GM-CSF）, tumor necrosis factor-a （TNF-a） and interleukin-4 （IL-4） to adherent cells to induce DCs, and sensitized DCs with antigens of autologous tumor cells or extrinsic tumor cell lines. Cultured suspending cells with interferon-y （IFN-y）, interleukin-2 （IL-2） and CD3 monoclonal antibody （CD3 McAb） to prepare CIK cells, then co-cultured with DCs. After analyzing the phenotype and checking tumor markers and immune function, the autologous CIK cells and DCs were transfused into the cancer patients. Results： Forty-two patients with measurable nidus, 2 achieved complete remission （CR）, 9 partial remission （PR） and 15 stable disease （SD）, while 37 patients with immeasurable nidus, 25 had efficient response. The tumor markers and immune function both improved significantly compared with those before treatment. Conclusion： DCs and CIK cells combinational treatment is safe and effective on advanced solid carcinoma and provide a new and efficacious immunity therapeutic methods for the cancer patients.

mRNA Expression of Chemokine Receptors on Peripheral Blood Mononuclear Cells and Correlation with Clinical Features in Systemic Lupus Erythematosus Patients

Objective To investigate the expressions of chemokine receptors and interleukin （IL） receptors on the peripheral blood mononuclear cells （PBMCs） from systemic lupus erythematosus （SLE） patients and their correlations with clinical features as well as SLE disease activity index （SLEDAI）. Methods The mRNA expressions of chemokine receptors and IL receptors on PBMCs of 93 SLE patients and 30 healthy controls were detected by reverse transcription-polymerase chain reaction, including CCR2, CCR3, CCR4, CCR5, CCR6, CCR8, CXCR3, CXCR5, CX3CR1, XCR1, IL-4R, and IL-10R. The clinical features of SLE patients were recorded. The correlations of chemokine receptors and IL receptors mRNA expressions with clinical features as well as SLEDAI were assayed using linear regression analysis. Results The level of CCR5 mRNA in SLE patients （including active and inactive SLE） was signifi- cantly higher than that in healthy controls （P〈0.05）, and there was no significant difference between active and inactive patients in this respect （P〉0.05）. CX3CR1 mRNA expression significantly increased from healthy control to inactive SLE to active SLE in sequence. The others （except for CCR8, CXCR3, and IL-1 OR） in active SLE patients weresignificantly higher than those in both inactive SLE patients and healthy controls （all P〈0.05）. There were positive correlations between SLEDAI and CCR2 （r=0.424, t=4.313, P〈0.001）, CCR3 （r=0.518, t=5.410, P〈0.001）, CCR4 （r=0.376, t=3.851, P〈0.001）, CCR6 （r=0.457, t=4.513,P〈0.001）, CXCR5 （r=0.455, t=4.629, P〈0.001）, CX3CR1 （r=0.44-5, t=4.523, P〈0.001）, as well as XCRI （r=0.540, t=5.445, P〈0.001）. And CCR5 mRNA expression level was positively correlated with IL-4R mRNA （r=0.313, t=2.353, P〈0.05）. The patients with myositis and cutaneous vasculitis simultaneously showed lower levels of CCR5 and CX3CRI, and CCR5 expression was negatively correlated with the scores of SLEDAI in SLE cases accompanied by photosensitivity （r=0.426, t=- 2.155, P〈0.05）. Conclusion Increased expressions of CCR5 and CX3CRI on PBMCs may be indicators in clinical survey for SLE.

Effect of Intracoronary Infusion of Bone Marrow Mononuclear Cells or Peripheral Endothelial Progenitor Cells on Myocardial Ischemia-reperfusion Injury in Mini-swine

Objective To simulate and assess the clinical effect of intracoronary infusion of bone marrow mononuclear cells or peripheral endothelial progenitor cells on myocardial reperfusion injury in mini-swine model. Methods Twenty-three mini-swine with myocardial reperfusion injury were used as designed in the study protocol. About （3.54±0.90）×10^7 bone marrow mononuclear cells （MNC group, n=9） or （1.16± 1.07）× 10^7 endothelial progenitor cells （EPC group, n=7） was infused into the affected coronary segment of the swine. The other mini-swine were infused with phosphate buffered saline as control （n=7）. Echocardio- graphy and hemodynamic studies were performed before and 4 weeks after cell infusion. Myocardium infarc- tion size was calculated. Stem cell differentiation was analyzed under a transmission electromicroscope. Results Left ventricular ejection fraction dropped by 0% in EPC group, 2% in MNC group, and 10% in the control group 4 weeks after cell infusion, respectively （P〈0.05）. The systolic parameters increased in MNC and EPC groups but decreased in the control group. However, the diastolic parameters demonstrated no significant change in the three groups （P〉0.05）. EPC decreased total infarction size more than MNC did （1.60±0.26 cm2 vs. 3.71±1.38 cm2, P〈0.05）. Undermature endothelial cells and myocytes were found under transmission electromlcroscope. Conclusions Transplantation of either MNC or EPC may be beneficial to cardiac systolic function, but might not has obvious effect on diastolic function. Intracoronary infusion of EPC might be better than MNC in controlling infarction size. Both MNC and EPC may stimulate angiogenesis, inhibit flbrogenesis, and differentiate into myocardial cells.

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.

Expression of Peptidylarginine Deiminase 4 and Protein Tyrosine Phosphatase Nonreceptor Type 22 in the Synovium of Collagen-Induced Arthritis Rats

Objective To study the expression level of peptidylarginine deiminase 4(PADI4) and protein tyrosine phosphatase nonreceptor type 22(PTPN22) in the synovium of rat model of collagen-induced arthritis, and to explore their possible therapeutic role in rheumatoid arthritis. Methods Thirty-two female Wistar rats weighing 100±20 g were randomly assigned into 3-week collagen-induced arthritis(CIA) model group(n=8), 4-week CIA model group(n=8), 6-week CIA model group(n=8), and the control group(n=8). The body weight changes of each group were recorded. The expression levels of PADI4 and PTPN22 were detected and compared by the methods of immunohistochemical staining and Western blot. Results Arthritis of rat began to form 14 days after sensitization and the joint swelling reached peak at 28 days. The weights of the rats slowly grew both in CIA model groups and the control group. Immunohistochemical staining results showed that the positive expression of PADI4 and PTPN22 was mainly located in cartilage peripheral mononuclear cells, the cytoplasm of infiltrated cells, and bone marrow cavity. There were significant differences in the optical density of PADI4 and PTPN22 among CIA model groups and the control group(PADI4, 0.2898±0.012, 0.2982±0.022, 0.2974±0.031, 0.2530±0.013 in 3-week CIA model, 4-week CIA model, 6-week CIA model and control groups; PTPN22, 0.2723±0.004, 0.2781±0.010, 0.2767±0.008, 0.2422±0.019; all P <0.05). The expression bands of PADI4 were observed in Western blot 3 weeks after initial immunization, the thickest in the 4th week, and decreased in the 6th week. The expression bands of PTPN2 were observed at all the time points, with no obvious time-dependent trend. Conclusions PADI4 and PTPN22 are obviously correlated with CIA in rat model. PADI4 is expressed at early stage of the disease, while the expression of PTPN22 sustains throughout the course.

Cilostazol inhibits plasmacytoid dendritic cell activation and antigen presentation

Background Cilostazol, an anti-platelet drug for treating coronary heart disease, has been reported to modulate immune cell functions Plasmacytoid dendritic cells （pDCs） have been found to participate in the progression of atherosclerosis mainly through interferon ct （IFN-ct） production. Whether cilostazol influences pDCs activation is still not clear. In this study, we aimed to investigate the effects of cilostazol on cell activation and antigen presentation ofpDCs in vitro in this study. Methods Peripheral blood mononuclear cells isolated by Ficoll cen- trifugation and pDCs sorted by flow cytometry were used in this study. After pretreated with cilostazol for 2 h, cells were stimulated with CpG-A, R848 or virus for 6 h or 20 h, or stimulated with CpG-B for 48 h and then co-cultured with naive T cell for five days. Cytokines in supernatant and intracellular cytokines were analyzed by ELISA or flow cytometry respectively. Results Our data indicated that cilostazol could inhibit IFN-α and tumor necrosis factor α （TNF-α） production from pDCs in a dose-dependent manner. In addition, the ability of priming na ve T cells of pDCs was also impaired by cilostazol. The inhibitory effect was not due to cell killing since the viability of pDCs did not change upon cilostazol treatment. Conclusion Cilostazol inhibits pDCs cell activation and antigen presentation in vitro, which may explain how cilostazol protects against atherosclerosis.

Impact of MTT based tumor chemosensitivity assay in vitro

Objective： The aim of the study was to evaluate the impact of the chemosensitivity assay in vitro and establish a standard process of measuring the anti-cancer drug sensitivity with MTT assay. Methods： Some influencing factors of MTT assay in studying the sensitivity of human peripheral blood mononuclear cells （PBMC） to anti-cancer drugs were observed, including red blood cells, platelets, three different kinds of DMSO and different concentrations of MTT. Meanwhile the stability of tumor drug-coated plate was monitored. Results： The red blood cells and platelets may affect the results at a certain range of concentration. Analytical pure DMSO, both imported and domestic reagents showed the same color with MI3-, and the A values of the reaction were dependent on MTT dose. The stability of the freeze-drying drug-coated plates was superior to non freeze-drying ones. Conclusion： To make clear and definite all kinds of influencing factors might contribute to a kind of standard MTT assay for drug sensitivity test in vitro.

Study on neoplasia of hydatidiform mole by detecting telomerase reverse transcriptase mRNA expression in peripheral blood mononuclear cells

By setting up a real-time fluorescent quantitative RT-PCR assay to detect human telomerase reverse transcriptase （hTERT） mRNA in hydatidiform mole in peripheral blood mononuclear cells, to analyze the correlation between the expression level of hTERT mRNA and the prognosis of hydatidiform mole, and to evaluate the clinic value of quantitative determination of hTERT mRNA in the diagnosis of hydatidiform mole. Methods： A real-time fluorescent quantitative RT-PCR （FQ RT-PCR） assay based on TaqMan fluorescence methodology and the Light-Cycler system was used to quantify the full range of hTERT mRNA copy numbers in 30 samples of hydatidiform mole and the neoplasia of hydatidiform mole. The normalized hTERT （NhTERT） was standardized by quantifying the number of GAPDH transcripts as internal control and expressed as 100x （hTERT/GAPDH） ratio. Based on the prognosis of the hydatidiform mole, the patients were divided into two groups： the experimental group and the control group, to compare the telomerase reverse transcriptase mRNA expression in peripheral blood mononuclear cells. Results： hTERT mRNA was both expressed in the peripheral blood mononuclear cells and pathological tissues in the mole experimental group and the control group. In the mole experimental group, the values were 6.31±0.32 and 6.24±0.44, respectively, and there was no significant difference between them （P〉0.05）. But in the control group the values were 1.21±0.65 and 1.40±0.61, respectively, and there was no significant difference between them （P〉0.05） The values in experimental group was significantly higher than those in the control group （P〈0.01）. Conclusion： Quantitative determination of hTERT mRNA by FQ RT-PCR is a rapid and sensitive method, hTERT in peripheral blood mononuclear cells may have potential use as a biomarker for the early detection of the prognosis of the hydatidiform mole.

Vigorous,but differential mononuclear cell response of cirrhotic patients to bacterial ligands

AIM： To study the role of gram-positive and gram-negative bacteria in the pathogenesis of liver injury, specifically the activation of inflammatory mediators. METHODS： Peripheral blood mononuclear cells of 20 out-patients were studied, 10 of them with cirrhosis,Peripheral blood mononuclear cells were isolated and exposed to lipopolysaccharide or lipoteichoic acid. CD14, Toll-like receptor 2 and 4 expression was determined by flow cytometry, and tumor necrosis factor （TNF） α, interleukin （1L）-1β, IL-6, IL-12 and IL-10 secretion in su- pernatants was determined by ELISA. RESULTS： Higher CD14, Toll-like receptor 2 and 4 expression was observed in peripheral blood mononudear cells from cirrhotic patients, （P 〈 0.01, P 〈 0.006, P 〈 0.111） respectively. Lipopolysaccharide and lipoteichoic acid induced a further increase in CD14 expression （P 〈 0.111 lipopolysaccharide, P 〈 0.013 lipoteichoic acid）, and a decrease in Toll-like receptor 2 （P 〈 0.008 lipopolysaccharide, P 〈 0.008 lipoteichoic acid） and Toll-like receptor 4 （P 〈 0.008 lipopolysaccharide, P 〈 0.028 li- poteichoic acid） expression. With the exception of TNFα, absolute cytokine secretion of peripheral blood mononuclear cells was lower in cirrhotic patients under nonexposure conditions （P 〈 0.070 IL-6, P 〈 0.009 IL-1]5, P 〈 0.022 IL-12）. Once exposed to lipopolysaccharide or lipoteichoic acid, absolute cytokine secretion of peripheral blood mononuclear cells was similar in cirrhotic and non-cirrhotic patients, determining a more vigorous response in the former （P 〈 0.005 TNFα, IL-1β, IL-6, IL-2 and IL-10 lipopolysaccharide; P 〈 0.037 TNFα; P 〈 0.006 IL-113; P 〈 0.005 IL-6; P 〈 0.007 IL-12; P 〈 0.014 IL-10 lipoteichoic acid）. Response of peripheral blood mononuclear cells was more intense after lipopolysaccharide than after lipoteichoic acid exposure. CONCLUSION： Peripheral blood mononuclear cells of cirrhotic patients are able to respond to a sudden bacterial ligand exposure, particularly lipopolysaccharide, suggesting that immune regulation mechanisms are still present.

Seven amino acid peptide mimic from HVR1 of HCV influences cytokine profile of immune cells

Objective:To investigate the actions of cytokine profile in the immune cells by a seven amino acid peptide mimic from HVR1 of HCV (GQTYTSG,named 7P).Methods:The peripheral blood mononuclear cells (PBMCs) from 17 healthy blood donors were stimulated with 7P,and the cytokine levels in CD4+CD8-,CD4-CD8+ T cells,NK,NKT cells were analyzed by the intracellular cytokine staining.Results:The frequency of cells which secreted interferon-γ (IFN-γ) was found to be significantly increased in all cells,interleukin-10(IL-10) was significantly increased in CD4+CD8-,CD4-CD8+ T cells but decreased in NK,NKT cells,and tumor necrosis factor-α (TNF-α) was decreased in CD4+CD8-,CD4-CD8+ but increased in NK cells.Conclusion:7P could induce a cytokine profile in different immune cells in vitro and there was some difference between the CD4+CD8-,CD4-CD8+ T cells which represented the adaptive immunity cells and NK,NKT cells which represented the innate immunity cells.This kind of variation of cytokine profile might contribute to anti-virus and anti-inflammatory immune reaction.

Stimulation of staphylococcal enterotoxin A combined with PML-RARα peptide on the specifical T-cells against NB4 cell line

Objective： To investigate the effects of staphyococcal enterotoxin A （SEA） on the cytotoxicity of T cells stimulated by PML-RARa peptide in vitro. Methods： Peripheral blood mononuclear cells （MNC） from healthy donor were obtained by density gradient centrifugation on Ficoll-Hypaque, MNC were cultured with PML-RARa peptide and SEA for 20 days. After induction, the cytotoxicity of T cells induced against NB4 and K562 cell lines were examined by Cell Counting Kit-8 （CCK-8）. The CD4 and CD8 surface markers on the harvested CD3^＋ T cells were detected by flow cytometry （FCM）. Results： The cytotoxicity of T cells induced by PML-RARa peptide with SEA was higher than that of T cells induced only by PML-RARa peptide against NB4 cells. The FCM assay showed that the ratio of CD4^＋/CD8^＋ T cells were gradually decreased in both groups of PML-RARα peptide whether with SEA or not at the intervals of day 5,10 and 20 day after induction, but the most significantly decreased by PML-RARe peptide with SEA. Conclusion： The specific cytotoxicity of T cells induced by PML-RARa peptide against NB4 cells could be enhanced with superantigen SEA.

Polysaccharide, Adenosine and Crude Triterpenoid Contents of Pleurotus Eryngfi and Its Immunostimulant Capacity in vitro

Purpose of the study was to demonstrate the ability of Pleurotus eryngii （PE） extracts to stimulate the production of nitric oxide （NO） and TNF-α as well as to evaluate the cell viability by the use of chicken peripheral blood mononuclear cells （PBMCs） and RAW 264.7 macrophages in vitro. The PE contained 341.0 ± 14.5 mg/g DW, 78,2 ± 4.1 Bg/g DW and 38.4 ＋ 3.3 mg/g DW of crude polysaccharide, adenosine and crude triterpenoid contents, respectively. The results of cell viability assay showed that 95,0% of chicken PBMCs and RAW 264.7 macrophages were survived under the treatment of 9.6 mg/mL of PE extracts by microculture tetrazolium assays （MTAs）. Moreover, the cell inhibitory activity did not show the 50% cyto-toxicity effect （ICs0）. In addition, no production by stimulated chicken PBMCs and RAW 264.7 macrophages exhibited individually the linear relations with the concentration of PE extracts, and they were approximately 65 μM and 45 p.M in 1600 μg/mL, respectively. The tendency with the release of TNF-α by RAW 264.7 maerophages also corresponded with concentration of PE extracts. It is concluded, PE extracts act as an immunostimulant for both tested cells but have no serious effect on inhibiting chicken PBMCs viability, and therefore it may be suggested as immune supplements for broilers.

The Influence of IFN-α on Blood Plasmacytoid Dendritic Cell in Chronic Myeloid Leukaemia

OBJECTIVE To study the mechanism of IFN on CML.METHODS Samples of 15 CML patients and 10 healthy controlswere studied. The flow cytometry was performed to identifycirculating pDCs. The concentration of IFN-α in serum and that inthe supernatant of peripheral blood mononuclear cells (PBMCs)cultured after stimulation with CpG ODN2216 were examinedboth in CML patients and in the healthy controlsRESULTS There was significant reduction in the numberof circulating pDCs, serum concentration of IFN-α and thecapacity of IFN-α producing PBMCs in CML patients comparedwith those in healthy control individuals (P < 0.001). After theactive treatment with IFN-α and hydroxyurea, the quantity andfunction of pDCs were increased in stabilized patients, especiallythe function of pDCs in 2 patients achieving major cytogeneticresponse (MCR). The proportion and function of pDCs and theserum levels of IFN were inversely correlated with both WBC andage of the patients with CML, and positively correlated with thestate of the illness.CONCLUSION CML patients had a reduced number anddysfunction of circulating pDCs. The active treatment with IFN inCML patients may be related to the restoration of pDCs.

Mechanism of peripheral blood mononuclear cell invasion by HBV on artificial immunization in newborns

OBJECTIVE: To study the effect and mechanism of the peripheral blood mononuclear cell (PBMC) invasion by HBV on artificial immunization in newborns. METHODS: Fifty-two newborns of HBsAg positive mothers were immunized with HBIG (hepatitis B immunoglobulin) and HBVac (hepatitis B vaccine) and were followed up for 7 months. The newborns' HBV-DNA in serum and in the PBMCs was detected with nested-PCR; anti-HBs was tested with solid phase radioimmunoassay (SP-RIA). PBMCs isolated from newborn peripheral blood were incubated in the presence of PHA or purified HBsAg. Interleukin-2 (IL-2) level in culture supernatants of activated cells was detected by ELISA. RESULTS: The failure rate of immunization was higher in infants with positive HBV-DNA in PBMCs than those with negative HBV-DNA (P