Effect of Chinese Herbal Medicinal Ingredients on IL-2 mRNA Levels of T Lymphocytes in Mice Measured Using Semiquantification RT-PCR

In this study, the IL-2 mRNA levels of T lymphocytes in normal mice stimulated by nine Chinese herbal medicinal ingredients （CHMIs） were measured using semiquantification reverse transcription polymerase chain reaction. The results showed that astragalus polysaccharide （APS）, epimedium polysaccharide （EPS）, Chinese angelica polysaccharide （CAPS）, propolis flavone （PF）, and astrogalosides （AS） promoted IL-2 mRNA levels in T lymphocytes in vitro and in vivo to differing degrees, and the level of IL-2 mRNA induced by propolis polysaccharide （PPS） in vitro was higher than that induced by the control, which differed from that of PPS in vivo.

Recombinant Mycobacterium smegmatis expressing Hsp65-hIL-2 fusion protein and its influence on lymphocyte function in mice

Objective:To coastruct a strain of recombinant Mycobacterium smegmatis expressing the heat shock protein 65(Hsp65) and human interleukin 2(IL-2) fusion protein(rMS-Hsp65/IL-2) and to explore the effect of this construct on lymphocyte function in mice.Methods:The fusion gene encoding Hsp65-hIL-2 was cloned into shuttle vector pSMT3.The recombinant plasmid pSMT3- Hsp65-hIL-2 was transferred to Mycobacterium smegmatis by electroporation.Positive clones were selected by hygromycin and identified by PCR.The expression of fusion protein Hsp65- hIL-2 was verified using indirect immunofluorescence staining.Mice were immunized for two times by subcutaneously injection with 1×10~6 CFU rMS-Hsp65/IL-2 at a three-week interval.Two weeks after the second immunization,mice were sacrificed and the serum samples were collected for determination of anli-Hsp65 specific IgG.Splenic lymphocytes were isolated and treated with the rMS-Hsp65/IL-2 to determine lymphocytic proliferation activity by MTT assay.IFN-γand IL-2 in the medium of the treated cells were also determined by ELISA.Results:Successful construction of rMS-Hsp65/IL-2 was verified by PCR and immunofluorescence staining.Compared to the splenic lymphocytes isolated from mice immunized with Bacille Calmette-Guerin or mice immunized with Mycobacterium smegmatis alone,the splenic lymphocytes isolated from mice immunized with rMS-Hsp65/IL-2 showed a marked increase in the proliferation of lymphocytes, together with an increased production of important cytokines such as IFN- 7 and IL-2. Conclusions:rMS-Hsp65/IL-2 markedly enhances lymphocyte function.Therefore,the fusion protein generated by rMS-Hsp65/lL-2 may be of potential value in generating an effective vaccine against tuberculosis.

Analysis of IL-2-like factor in lymphocyte culture supernatant of olive flounder, Paralichthys oliveaceus

To study immune mechanism of fish lymphocyte we performed a proliferation assay and ELISA using monoclonal antibody against human IL-2. The result showed that an interleukin-2 （IL-2）-like factor was detected in the supernatant of plant haemoglutinin （PHA）-stimulated lymphocyte culture from peripheral blood,spleen and head kidney of olive flounder, Paralichthys olivaceus. The quantities of IL-2-1ike factor in the supernatant from different lymphoid tissues were quite different. The IL-2 like factor in the supernatant from cultured head kidney lymphocytes was much higher than those of peripheral blood lymphocytes and spleen lymphocytes （P〈0.01）. The IL-2 activity was found in either mouse thymocyte proliferation assay or flounder head kidney lymphocyte proliferation assay and shown to have obvious enhancing effect on proliferation of the above two types of cell. The recombinant human IL-2 （rhIL-2） was able to stimulate flounder thymocyte proliferation and used to detect the IL-2 receptor （IL-2R） on the surface of flounder lymphocyte. The cross-reaction between the lymphocytes of flounder peripheral blood and CD25（IL-2R） was detected with flow cytometry and shown that the percentage of CD25-positive cell in peripheral blood was 7.74±0.67%.

Immunomodulating property of Ocimum sanctum by regulating the IL-2 production and its mRNA expression using rat's splenocytes

Objective:To investigate the regulatory effect of aqueous extract of leaves of Ocimum sanctum on IL-2 cytokine production in vivo and in vitro,and the effect of leaves extract on general blood picture including T& B lymphocytes.Methods:For in vivo studies albino Wistar rats were treated with aqueous crude leaves extract of Ocimum sanctum for 20 consecutive days.Spleen cells were harvested and assayed for IL-2 production by using sandwich enzyme-linked immunosorbent assay(ELISA) and mRNA expression methods.For in vitro study aqueous Ocimum sanctum leaves extract= in different concentrations(25-500μ/mL) was added into culture plates containing ConA stimulated splenocytes.To study the overall effect on blood picture,density gradient purified lymphocytes analysis and conventional methodology for total and differential leukocyte count and hemoglobin level were also done.Results:It indicated that the rats treated with Ocimum sanctum leaves extract had significantly enhanced(P【0.001) ability of spleen cells to secrete IL-2.Investigation in vitro also showed regulation of IL-2 production.Blood study exhibited leucocytosis and augmentation of T& B lymphocytes by 25%approximately.4-5%increase in Hemoglobin value was also noticed.Conclusion:Aqueous Ocimum sanctum leaves extract may have stimulatory effect on T & B lymphocytes particularly on Th 1 subset of lymphocytes as shown by enhancement in IL-2 production.

PREPARATION AND CYTOLYTIC TUMOR ACTIVITY OF OSTEOSARCOMA TUMOR INFILTRATING LYMPHOCYTES IN VITRO

In this study, the isolation, purification and differentiation of tumor-lnflltratlng lymphocytes (TIL) from 6 fresh osteosarcoma specimens were achieved by discontinuous density gradient centrifugation. One specimen of the osteosarcoma TIL were enlarged in IL-2 for long time in vitro, reaching 28 days and their cytolytic activity against different tumor cell lines was Investigated. The experimental results indicated that the preparation of osteosarcoma TIL adopted by the mechanical means was simple, having higher purifity, keeping higher effects on killing NK- sensitive tumor cell lines and NK-insensitive tumor cell lines as well as rapid proliferation in vitro cultured in IL-2.

Construction,Expression and in vitro Biological Effects of Idiotype Ig Fab Fragment of B-Chronic Lymphocytic Leukemia

The purpose of this study was to construct expression vectors of idiotype （Id） SmIg in patients with B-chronic lymphocytic leukemia and to express them in E.coli to obtain recombinant Id, and to investigate the effect of the protein on the proliferation and secretion of IL-2 and IFN-γ of stimulated peripheral blood mononuclear cells （PBMC） in vitro. Light chain gene and Fd fragment of heavy chain gene were inserted into fd-tet-DOG2 vector to construct fd-tet-DOG2-Fab. Fab gene was further cloned into expression vector pHEN2 to construct the soluble expression vector pHEN2-Fab. After induction by IPTG, Fab protein was purified by Ni-NTA-chromatography. MTT was used to determine the effects of purified protein on the proliferation of stimulated PBMC in vitro and the concentrations of IL-2 and IFN-γ in the culture supernatants were detected by ELISA. The results showed that recombinant pHEN2-Fab expression vector was constructed successfully. Fab protein was expressed in positive clone after induced by IPTG and two specific bands at 24-25 kD position were observed by SDS-PAGE electrophoresis. Proliferation of PBMC could be induced by purified Fab and the concentrations of IL-2 and IFN-γ in culture supernatants were increased significantly af- ter induction. It was suggested that the expression vector of SmIg Fab fragment was constructed suc- cessfully, and expressed and secreted from E. coli. The Fab protein could induce proliferation of PBMC and promote secretion of IL-2 and IFN-γ.

重度冻伤后早期机体免疫功能变化的研究

对兔足重度冻伤后早期ＮＫ 细胞的杀伤活性、Ｔ 和Ｂ 淋巴细胞转化及Ｔ 淋巴细胞ＩＬ—２产生能力进行动态观察。结果表明，重度冻伤后早期免疫功能低下，１２ｈ 免疫功能明显低于对照，２４ ～４８ｈ 处于较低水平，７２ｈ 有所回升。冻伤对ＮＫ 细胞、Ｔ 淋巴细胞和Ｂ 淋巴细胞功能均有影响，但ＮＫ 细胞、Ｔ 淋巴细胞功能受抑程度强于Ｂ 淋巴细胞受抑程度。

补肾方对慢性乙型肝炎T细胞亚群及其功能的影响

目的观察补肾方对慢性乙型肝炎患者免疫功能状态的调节作用。方法收集HBeAg阳性ALT异常者30例,给予补肾方治疗,用药6个月,于用药前、用药3个月、6个月时分别检测下列指标。采用罗氏荧光定量PCR法检测HBVDNA ,外周血单个核细胞(peripheralbloodmononuclearcell,PBMC)加入HBeAg、HBcAg与植物血凝素(Phytohemagglutinin ,PHA)培养48h ,检测培养上清液中IL 10、IFN -γ,血液中CD4+、CD8+、CD8+CD2 8+、CD8+CD2 8-和CD2 8+T细胞的比例及加入HBeAg、HBcAg与PHA培养48h后培养细胞中CD8+CD2 8+的比例。结果补肾方治疗有效组在治疗3个月后,PBMC培养中IFN- γ较治疗前明显上升,IL- 10明显下降(P <0 . 0 5,P <0. 0 1) ;CD8+CD2 8+T、CD2 8+T、培养后CD8+CD2 8+T细胞较治疗前明显上升,CD8+CD2 8-T细胞明显下降(P <0 .0 5或P <0 .0 1)。结论补肾方能明显增强Th1型细胞因子的表达,降低Th2型细胞因子的表达,促进CD8+(CytotoxicTlymphocyte ,CTL)的表达,这可能是使HBVDNA复制得到抑制的机制之一。

The Immunity Induced by Recombinant Spike Proteins of SARS Coronavirus in Balb/c Mice

The immune effect of two recombinant protein fragments of spike protein in severe acute respiratory syndrome coronavirus (SARS CoV) was investigated in Balb/c mice. Two partial spike gene fragments S1 (322-1464 bp) and S2 (2170-2814 bp) of SARS coronavirus were amplified by RT-PCR, and cloned into pET-23a prokaryotic expression vector, then transformed into competent Escherichia E.coli BL21 (DE3)(pLysS) respectively. Recombinant proteins were expressed and puri- fied by Ni2+ immobilized metal ion affinity chromatography. The purified proteins mixed with com- plete Freund adjuvant were injected into Balb/c mice three times at a two-week interval. High titer antibody was detected in the serum of immunized Balb/c mice, and mice immunized with S1 protein produced high titer IgG1, IgG2a, IgG2b and IgG3, while those immunized with S2 protein produced high titer IgG1, IgG2a, but lower titer IgG2b and IgG3. Serum IFN-γ concentration was increased significantly but the concentrations of IL-2, IL-4 and IL-10 had no significant change. And a marked increase was observed in the number of spleen CD8+ T cells. The results showed that recombinant proteins of SARS coronavirus spike protein induced hormonal and cellular immune response in Balb/c mice.

Atomization Inhalation of Terbutaline and Budesonide Efficiently Improved Immunity and Lung Function of AECOPD Patients

Chronic obstructive pulmonary disease （COPD） is a syndrome of chronic progressive airflow limitation as a result of chronic inflammation of the airways and lung parenchyma. COPD patients always have airway hyperreactivity （AHR）, so how to reduce AHR becomes the key purpose of clinical treatment. It is hypothesized that combined inhalation of corticosteroids and β2-agonists can reduce the AHR in COPD. In this study, atomization inhalation of budesonide and terbutaline plus conventional therapies was applied to treat AECOPD （acute exacerbation of chronic obstructive pulmonary disease） patients for two weeks. The results showed that additional inhalation of budesonide and terbutaline could upregulate serum IL-2 levels, the percentages of CD3^＋ T and CD4^＋ T cells, and CD4/CD8 ratio, and decrease eosinophils and serum CRP level more efficiently than conventional treatment in patients with AECOPD. And the lung function of the atomization inhalation group was improved more obviously after the treatment compared with the conventional treatment group. Thus, atomization inhalation of terbutaline and budesonide can control AECOPD effectively, and has wide clinical perspective in controlling and preventing the exacerbation of COPD. Cellular ＆ Molecular Immunology. 2008;5（4）：287-291.

Effects of purified zearalenone on selected immunological and histopathologic measurements of spleen in post-weanling gilts

The present study was aimed at investigating the adverse effects of dietary zearalenone(ZEA) on the lymphocyte proliferation rate(LPR), interleukin-2(IL-2), mRNA expressions of pro-inflammatory cytokines, and histopathologic changes of spleen in post-weanling gilts. A total of 20 crossbred piglets(Yorkshire × Landrace × Duroc) with an initial BW of 10.36 ± 1.21 kg(21 d of age) were used in the study.Piglets were fed a basal diet with an addition of 0.1.1,2.0, or 3.2 mg/kg purified ZEA for 18 d ad libitum.The results showed that LPR and IL-2 production of spleen decreased linearly(P < 0.05) as dietary ZEA increased. Splenic mRNA expressions of interleukin-1β(IL-1β) and interleukin-6(IL-6) were linearly upregulated(P < 0.05) as dietary ZEA increased. On the contrary, linear down-regulation(P<0.05) of mRNA expression of interferon-γ(IFN-γ) was observed as dietary ZEA increased. Swelling splenocyte in1.1 mg/kg ZEA treatments, atrophy of white pulp and swelling of red pulp in 2.0 and 3.2 mg/kg ZEA treatments were observed. The cytoplasmic edema in 1.1 mg/kg ZEA treatments, significant chromatin deformation in 2.0 mg/kg ZEA treatment and phagocytosis in 3.2 mg/kg ZEA treatment were observed.Results suggested that dietary ZEA at 1.1 to 3.2 mg/kg can induce splenic damages and negatively affect immune function of spleen in post-weanling gilts.

Interim results from ongoing PhaseⅢ placebo-controlled,randomized trial of hepcortespenlisimut-L for advanced hepatocellular carcinoma indication

Aim:We aimed to further investigate the role of hepcortespenlisimut-L(Hepko-V5 or V5),a new oral immunotherapy developed by us,for hepatocellular carcinoma(HCC)indication.Methods:The interim data from ongoing PhaseⅢplacebo-controlled,randomized trial were evaluated on the initial group of patients in advanced stage of HCC with emphasis on liver function and tumor marker alpha-fetoprotein levels.Additionally,an in vitro study was undertaken to elucidate the mechanism of action of V5 by measuring with flow cytometry the expression of cytokines such as IL-2,INF-γ,and TNF-αand cell activation markers CD69 and Ki67 on CD4-and CD8-positive lymphocytes isolated from peripheral blood of healthy volunteers.Results:As early as one month after treatment initiation,there was a clear improvement in alanine transaminase,aspartate transaminase,alkaline phosphatase,and bilirubin levels among HCC patients who received daily dose of V5,but not in the placebo group.Additionally,alpha-fetoprotein(AFP)levels among V5 recipients decreased,while in the ;placebo group they rose.Clinical results are in line with in vitro observations indicating immune activation,as evidenced by many-fold enhancement of CD69,Ki67,and INF-γexpression and at the same time marked anti-inflammatory effect resulting in 10-fold decrease in TNF-αoutput and lack of influence on IL-2 production.Conclusion:Hepcortespenlisimut-L,a tableted oral formulation derived from heat-inactivated pooled blood of patients with HCC and viral hepatitis shows beneficial clinical effect,as demonstrated by improvement in liver function and reduction of tumor marker AFP levels.These correlate with in vitro observations showing potent activation of the immune response and pronounced oral tolerance effect.