Objective To investigate the expression of Toll-like receptor 4 (TLR4) of peripheral blood mononuclear cell (PBMC) in the different prognosis of patients with severe sepsis and its relation with endotoxin-tolerant.Methods Thirty-five patients with severe sepsis (severe sepsis group) and 15 healthy volunteers (control group),respectively stayed in ICU 24 h and physical examination in the day measured TLR4 expression of PBMC by flow cytometry and measured the serum of tumor necrosis factor (TNF)-α and interleukin (IL)-10 by enzyme-linked immunosorbentassay.Simultaneous separation of peripheral blood of PBMC,addition of 40 μ g/ml lipopolysaccharide (LPS) cultured for 24 h,and TLR4 expression of PBMC was measured in the same manner and the culture supernatant of TNF-α and IL-10.Results Thirty-five patients with severe sepsis survivor 25 cases (survivor group) and death 10 cases (death group),28 d death rate was 28.6％ (10/35).The TLR4 expression of PBMC in severe sepsis group was significantly lower than that in control group [(11.09 ± 8.90) MFI vs.(33.72 ± 12.59) MFI,P ＜ 0.01],the serum TNF-α and IL-10 in severe sepsis group were significantly higher than those in control group [(96.66 ± 45.33) ng/L vs.(2.53 ± 1.21) ng/L,(149.79 ± 67.15) ng/L vs.(34.56 ± 19.08) ng/L,P ＜ 0.01].There was no significant difference in TLR4 expression of PBMC between survivor group and death group (P ＞ 0.05).The TLR4 expression of PBMC in control group after LPS stimulation was significantly higher than that before LPS stimulation [(50.22 ± 19.23) MFI vs.(33.72 ± 12.59) MFI,P ＜ 0.05],survivor group and death group after LPS stimulation were significantly lower than those before LPS stimulation [(4.55 ± 2.30) MFI vs.(11.21 ±7.92) M FI and (5.46 ± 3.44) M FI vs.(10.15 ± 9.70) MFI,P ＜ 0.01].The culture supernatant of TNF-α,IL-10 in survivor group and death group were significantly lower than those in control group [(22.34 ± 8.27)and (19.49 ±5.35) ng/L vs.(88.70 ±34.21) ng/L,(54.29 ±32.89) and (98.04 ±40.26) ng/L vs.(146.56 ± 52.18) ng/L,P ＜ 0.01],but the culture supernatant of IL-10 in death group was significantly higher than that in survivor group (P ＜ 0.05).Conclusion Different prognosis of patients with severe sepsis have endotoxin tolerance and different inflammatory cytokine secretion abifity.

Objective To explore the differences of the proliferation ability and the antineoplastic activity of CIK cells against origenal hepatocellullar cancer cells between healthy adults and tumor patients in vitro. Methods Peripheral blood mononuclear cells (PBMC) from healthy donors and tumor patients were incubated to induce CIK cells in the presence of interferon gamroa(IFN-γ) ,IL-2 and anti-CD3 monoclonal antibody (mAb). The changes in the proliferation activity and phenotypes of the CIK cells were identified by flow cytometric analysis. Single cell suspension was prepared bom the fresh hepatocellular carcinoma tissue by using mechanical trituration method. MTT assays were used to determine the cytotoxicity of CIK cells against origenal hepatocellullar cancer cells. Results The CIK cells from both healthy donors and tumor patients were significantly increased with the extension of time. The expression rate of CD3+/CD56+ cells from healthy donors rose from 1.053% ±0.22% on 1st day ,25.36% ±2.19% on the 7th day to 55.12% ±1.99% on the 14th day ,which was significantly higher than that of tumor patients ( P ＜ 0.05). MTT assays showed that the cytotoxicity of CIK cells from both enhanced obviously with the addition of Effect/Target rate and extension of time(P ＜0.05). After the origenal hepatocellullar cancer cells were treated by CIK cells 24 hours, the cytotoxicity of CIK cells from healthy donors at the effector:target ratio of 10 ∶ 1,20 ∶1 and 40 ∶1 was significantly higher than that of tumor patients(P＜0.05) ,respectively. After treated 48 hours, compared with tumor patients, the cytotoxicity of healthy donors? CIK at the three effector:target ratio was alsosignificantly higher(P ＜0.05), respectively. Conclusion CIK cells from both have amplification ability and cytotoxic activity in vitro,and the proliferation ability and killing activity of healthy adults CIK is stronger than that of tumor patients,which provides an experimental basis for CIK to clinical application as an adoptive immunotherapy.

Objective To detect by flow cytometry (FCM) acute myeloid leukemia. Methods Fourcolor staining was used for analysis of immunphonotypes of 52 cases with acute myeloid leukemia and 7 cases of mixed leukaemia. Results 52 cases with series of patients with AML expression, with the main expression CD13 (94.2 %), CD117 (90.4 %), cMPO (90.4 %) CD33 (86.5 %), CD34 (57.7 %), HLA-DR (53.8 %). In 7 cases of mixed cell leukemia 3 cases of myeloid/B (M/B), are expressing CD13, CD34, CD117, CD10, CD19, cMPO, cCD79a.Myeloid/T (M/T) 2 cases with expression of CD13, CD117, CD33, CD34, CD5,CD7, cMPO; B department/T (B/T) 2cases, with expression of CD5, CD7, CD10, CD19, CD20, CD34. And with the morphological and organizations with high accuracy of chemical diagnosis. Conclusion FCM can improve the diagnose rate for AML and mixed leukemia. And has played an important clinical significance.

Objective To detect double immuno-phenotype of acute leukemia by the application of flow cytometry (FCM). Methods Four-color FCM was used to analyse 23 cases of acute leukemia of double immune phenotype. Results Among 23 cases, cCD3 was expressed in 10 cases (43.4 %), cCD79a in 16 cases (69.6 %), cMPO in 20 cases (87.0 %), TdT in 14 cases (60.9 %), CD34 in 19 cases (82.6 %) and CD117 in 20 cases (87.0 %). 13 cases (56.5 %) expressed both myeloid and B lymphocyte antigens, in which cCD79a and cMPO were positively expressed, 7 cases (30.4 %) were found to express both myeloid and T lymphocyte antigens of cCD3 and cMPO 3 cases (13.04 %) were positive for both cCD3 and cCD79a. Conclusion cCD3,cCD79a, and cMPO were lineage specific antigen markers, which could be used for diagnosis and differential double phenotype leukemias. FCM is a useful and reliable method for diagnosis of acute leukemia, and guidance of treatment and prognosis of leukemia in clinical.

Objective To evaluate the clinical efficacy and safety of treatment of non-small cell lung cancer (NSCLC) with autologous and half consistency allograft cytokines induced killer cells combined with chemotherapy. Methods We selected 42 patients with NSCLC patients as the research object. According to the group matching principle, the cases were divided into three groups: autologous CIK cells combination chemotherapy group, half consistency allograft CIK cells combination chemotherapy group, pure chemotherapy group. The autologous and allograft CIK cellular immune therapy of security, flow cytometric analysis technique (FCM) comparisons between before and after the treatment group infusion in vivo T lymphocyte subsets changes, and three treatment group clinical short-term curative effect were used in the comparison.Results FCM detection results show that CIK cell infusion after, CD+3, CD+4 / CD+8 ratio, NK cells (CD+3 CD+56)and CIK cells (CD+3 CD+56) ratio obviously higher than before treatment, autologous infusion before treatment,respectively (47.2±10.1) %, 1.0±0.1, (15.1±2.7) %, (0.7±0.2) %. After treatment respectively (58.8±12.3) %,1.3±0.2, (24.6±7.1) %, (3.8±2.2) %; Allograft infusion before treatment for (49.4±11.4) %, 0.9±0.2, (14.8±3.2) %, (0.9±0.3) % for after treatment (57.3±9.2) %, 1.4±0.3, (25.4±6.7) %, (4.3 ± 2.6) % (t = 22, 20, 19,P ＜ 0.05), and the pure chemotherapy group before and after the treatment T lymphocyte subsets level has not seen the obvious change. Clinical short-term curative effect comparison, autologous and allograft CIK cell therapy group objective efficient and disease control rates are slightly higher than the pure chemotherapy group, but the difference was not statistically significant. Respectively 21.4 %, 57.1%, and 35.7 %, 28.6 %,64.3 %, 71.4 % (x2=38.85, x2=41.24, P ＞ 0.05). Conclusion Autologous or half consistency allograft CIK cellular immune therapy is good safety and low toxicity, have certain short-term curative effect, which can effectively slowed tumor recurrence, is a worthy of popularizing clinically tumor adjuvant treatment mode.