In vitro human lymphocyte proliferation assay under different extraction conditions and doses of two types of test samples / 中国组织工程研究

BACKGROUND:In vitro lymphocyte proliferation test is often used to detect the potential immunogenicity of medical devices,but no detailed extraction conditions and dose are given in the relevant standards. OBJECTIVE:To investigate the effects of different extraction conditions of the test product and different doses of the extract on in vitro human lymphocyte proliferation,and to consider the factors that need to be considered when selecting test conditions for in vitro lymphocyte proliferation test. METHODS:In the experiment,the homogenous bone repair material and heparin-modified intraocular lens were divided into the following 12 groups:(1)Experimental group 1:24-hour complete medium(RPMI modified medium containing 10%fetal bovine serum)extract of 200 μL + lymphocyte suspension of 50 μL;(2)negative control group 1:24-hour complete medium 200 μL + lymphocyte suspension 50 μL;(3)experimental group 2:24-hour complete medium extract 100 μL + lymphocyte suspension 100 μL;(4)negative control group 2:24-hour complete medium 100 μL + lymphocyte suspension 100 μL;(5)experimental group 3:72-hour RPMI modified medium extract(addition of 10%fetal bovine serum before experiment)200 μL + lymphocyte suspension 50 μL;(6)negative control group 3:72-hour RPMI modified medium(addition of 10%fetal bovine serum before experiment)200 μL + lymphocyte suspension 50 μL;(7)experimental group 4:72-hour RPMI modified medium extract(addition of 10%fetal bovine serum before experiment)100 μL + lymphocyte suspension 100 μL;(8)negative control group 4:72-hour RPMI modified medium(addition of 10%fetal bovine serum before experiment)100 μL + lymphocyte suspension 100 μL;(9)positive control group 1:complete medium containing 10 μg/mL plant hemagglutinin-M 200 μL + lymphocyte suspension 50 μL;(10)positive control group 2:complete medium containing 10 μg/mL plant hemagglutinin-M 100 μL + lymphocyte suspension 100 μL;(11)blank control group 1:250 μL complete medium;(12)control group 2:200 μL complete medium.After 3 days of culture,the proliferation of lymphocytes was detected by CCK-8 assay. RESULTS AND CONCLUSION:(1)Under different test conditions,the extracts of the allogeneic bone repair material could enhance the activity of human lymphocytes.Under the condition of 72-hour leaching in RPMI modified medium and the volume ratio of leaching solution and lymphocyte suspension was 4:1,the most significant effect was observed.Heparin-modified intraocular lens extract also had obvious inhibitory effect on lymphocyte activity under this condition;its inhibitory effect on lymphocyte activity may be related to the heparin in the extract.However,the activity of lymphocytes was slightly enhanced by heparin-modified intraocular lens extract under the experimental conditions of complete medium extraction for 24 hours and the volume ratio of extract to lymphocyte suspension was 4:1.(2)Under different extraction conditions and doses,the results of in vitro lymphocyte proliferation test may be quite different.The selection of test conditions should be combined with the clinical application of the product,and the inherent characteristics of the product should also be considered.

Immunomodulatory characteristics of human umbilical cord mesenchymal stem cells in vitro / 中国组织工程研究

BACKGROUND: Human umbilical cord mesenchymal stem cells (hUC-MSCs) have been drawing a great attention due to their potential therapeutic effect in a variety of diseases, including immune-mediated diseases. Further characterization of the immunomodulatory properties and action pathways of hUC-MSCs is necessary to ensure their safety and effectiveness in clinical application. OBJECTIVE: To investigate the immunomodulatory properties of hUC-MSCs. METHODS: HUC-MSCs were directly co-cultured with CFSE-labeled peripheral blood mononuclear cells (PBMCs) at the ratio of 1:5, 1:10, and 1:20, or indirectly co-cultured with CFSE-labeled PBMCs at the ratio of 1:5 via the Transwell co-culture system. Phytohemagglutinin- stimulated PBMC proliferation and the percentages of Th1, Th17 and Treg subgroups in the CD4+ T cells were determined by flow cytometry. The levels of tumor necrosis factor α and interferon γ were determined by ELISA. RESULTS AND CONCLUSION: After direct co-culture, hUC-MSCs significantly inhibited the phytohemagglutinin-stimulated PBMCs proliferation in a dose-dependent manner, whereas the inhibitory effect disappeared in the Transwell co-culture system. A significant decrease of Th1, Th17 cells and an increase of Treg cells were detected in the PBMCs co-cultured with hUC-MSCs compared to the PBMCs cultured alone. Furthermore, hUC-MSCs co-culture significantly reduced tumor necrosis factor α and interferon γ levels in the PBMCs. These findings indicate that cell-to-cell contact is essential for hUC-MSCs to inhibit the proliferation, differentiation and inflammatory factor secretion of immune cells.

The effects of different stimulators on the activation of human lymphocytes / 药学学报

The research aims to study the effects of different stimulants on the activation of human lymphocytes. Human peripheral blood mononuclear cells were prepared by density centrifugation. The blood's sample was provided by National Institutes for Food and Drug Control and approved by its Ethics Committee. The expressions of CD69 in CD3+CD4+ and CD3+CD8+ human T cells were detected by flow cytometry after administrated with CD3/CD28 antibody, phytohaemagglutinin (PHA), Staphylococcus auresus enterooxin B (SEB), interleukin (IL27) and PMA plus ionomycin for 24 h. The proliferation of lymphocyte was detected by CellTiter-Glo kit. The secreted IFNγ in supernatant of medium was examined by ELISA kit. The proliferation of lymphocytes had no change after exposed of CD3/CD28 antibody, SEB, IL27 and PMA plus ionomycin for 24 h. However, the CD69 expressions in CD3+CD4+ and CD3+CD8+ T cells and IFNγ productions were significantly increased by CD3/CD28 antibody, SEB, IL27 and PMA plus ionomycin at 24 h, indicating that CD3+CD4+ and CD3+CD8+ T cells were activated under above-mentioned stimulated condition. CD3/CD28 antibody, SEB, IL27 and PMA plus ionomycin were valid stimulants for T cell activation.

Clinical and laboratory characteristics of 11 cases of chronic B lymphocyte proliferation disease without lymphoid proliferation / 肿瘤研究与临床

Objective To explore the clinical and laboratory characteristics of chronic B lymphocyte proliferation disease (B-CLPD) without typical lymphoid proliferation. Methods The clinical records of patients with B-CLPD only characterized by pancytopenia form January 2007 to March 2016 in hematology department of Xinjiang Uygur Autonomous Region People ' s Hospital were collected, and the cell morphology, bone marrow pathology, cytogenetics and molecular characteristics were retrospectively analyzed. Results The median age of 11 patients was 68 years old. The lymphocyte ratio of peripheral blood smears in all patients increased in different level (0.36-0.68), but absolute lymphocyte count was normal or decreased (0.59×109-1.99×109). Lymphocyte-like plasma cell or small numbers of plasma cell can be seen in the bone marrow smears of 4 cases and lymphocytes with irregular burr-like protrusions were observed in 2 cases while there were no characteristic morphological changes in remained 5 cases. Immunophenotypical analysis showed that all patients expressed CD19, CD20, CD22, SmIg, not expressed CD5, CD10 which with scores of 0-2 according to chronic lymphocytic leukemia (CLL) points system;CD103, CD11C, CD25 and FMC7 were highly expressed in 2 cases while there were no characteristic expression in remaining cases. There were no abnormal karyotypes observed from the conventional cytogenetic and fluorescence in situ hybridization (FISH) analysis (both of IgH/CCND1, bcl-2/IgH were negative) in all patients. 8 patients were found IgH gene rearrangement, MYD88L256P and BRAF V600E was positive in 5 cases and 2 cases respectively. 5 cases were diagnosed as Waldenstrom macroglobulinemia, 3 cases were B-CLPD, 2 cases were hairy cell leukemia, 1 case was nodal marginal zone B-cell lymphoma after comprehensive analysis of their clinical and laboratory data. Conclusion Even if there are no increased peripheral blood lymphocytes in pancytopenia patients, it is necessary to perform bone marrow smears, immunophenotyping, IgH gene rearrangement, cytogenetics and other molecular laboratory tests to exclude B-CLPD, and reduce misdiagnosis.

Effects of dexmedetomidine on cellular immune function of immunosuppression juvenile rats / 临床麻醉学杂志

Objective To explore the effects of dexmedetomidine on peripheral blood T lymphocyte proliferation and T lymphocyte subsets of juvenile rats with splenectomy.Methods Twenty-four healthy male Sprague-Dawley rats,weighing 130-150 g,aged six weeks were enrolled in this study.Half of the rats received splenectomy to make an immunosuppressive model,then they were randomly divided into 2 groups (n=6 each): splenectomy+normal saline group (group SN) and splenectomy+dexmedetomidine group(group SD).The another half of the rats without splenectomy were randomly divided into 2 groups: normal saline group(group S) and dexmedetomidine group(group D).After one week of normal feeding,normal saline 10 ml/kg was injected intraperitoneally (ip) in groups S and SN,dexmedetomidine 50 μg/kg was injected ip in groups D and SD respectively.Two hours after the injection,blood samples were collected.MTT was utilized to examine the peripheral blood T lymphocyte proliferative capability.T lymphocyte subsets CD4+,CD8+ were determined by flow cytometry.CD4+/CD8+ was calculated.Results Compared with group S,T lymphocyte proliferative capability,the percentages CD4+,CD8+ and CD4+/CD8+ ratio were significantly decreased in group SN (P<0.05);T lymphocyte proliferative capability in group D was decreased (P<0.05),but no significant changes was found in the percentages CD4+,CD8+ and CD4+/CD8+ ratio.Compared with the group D,T lymphocyte proliferative capability,the percentages CD4+,CD8+ and CD4+/CD8+ ratio in group SD were significantly decreased (P<0.05).Compared with the group SN,T lymphocyte proliferative capability in group SD was significantly decreased (P<0.05).Conclusion Cellular immune function of juvenile rats with or without splenectomy is suppressed by dexmedetomidine,and the suppressive function is more severe in splenectomy rats than that in normal juvenile rats.

Impact of compound 48/80 activated mast cell on dendritic cell homing to lymph nodes / 中国免疫学杂志

Objective:To investigate the effect of compound 48/80(C48/80) activated mast cell on dendritic cell homing to draining lymph nodes(DLN) in order to explore the potential mechanism of C48/80 promoting adaptive immune response.Methods: C57BL/6 mice bone marrow derived dendritic cells(BM-DC) and MC/9 mast cell line were propagated in vitro.Chemotaxis of BM-DC supplemented with supernatant of C48/80 treated MC/9 to CCL21 was measured by transwell chemotaxis assay.C48/80 was injected into murine scapular site,local skin mast cell degranulation was detected by toluidine blue staining method.The number of CD11c+ DC in draining lymph nodes(DLN) was detected by Flow cytometry.Exogenous green microbeads labeled BM-DC homing to DLN was detected by fluromicroscope.The mice pretreated with or without C48/80 were vaccinated by ovalbumin pulsed DC(OVA-DC),DLN lymphocyte proliferation was detected by WST-8 reagent.Results: A large amount of typical BM-DC were harvested from media supplemented with murine recombinant IL-4 and GM-CSF.Product of activated MC/9 enhanced chemotaxis of BM-DC to CCL21(P<0.01).Intradermal injection of C48/80 induced local mast cell obvious degranulation and local inflammation.Mice pretreated with C48/80 demonstrated high number of total cells and DC cells in DLN.The number of fluorescent positive BM-DC in DLN also increased in C48/80 pretreated mice.Antigen specific lymphocyte proliferation enhanced in OVA-DC inoculated C48/80 pretreated mice.Conclusion: Mast cell activation induced by C48/80 can enhance DC homing to DLN and increase specific lymphocyte proliferation,which may be one of a mechanism of C48/80 in promoting adaptive immune response.

Ras-associated autoimmune leukoproliferative disorder: a report of 2 cases and literature review / 中华儿科杂志

Objective@#To investigate the clinical features and genetic characteristics of cases with Ras-associated autoimmune leukoproliferative disorder(RALD).@*Method@#Characteristics of clinical data and gene mutation of the first two cases in China with RALD were retrospectively analyzed. The related literature was searched by using search terms "NRAS" , "KRAS" or "RALD" .@*Result@#Case1, a seven-year-seven-month old girl, was admitted due to "thrombocytopenia and splenomegaly for three years" . Palpation showed enlargement of submandibular lymph nodes and hepatosplenomegaly.The platelet count fluctuated between 15×109/L and 60×109/L. Hemoglobin was as 57 g/L and Coomb's test was positive.Lung computed tomography revealed interstitial lung disease, bilateral pleural effusion, pericardial effusion, myocardial injury and ascites. Case2, a seven-year-five-month old girl, was admitted due to "recurrent thrombocytopenia for seven years, intermittent eyelid and abdominal swelling for three years" . Palpation showed enlargement of cervical and right inguinal lymph nodes, and hepatosplenomegaly.The number of platelet and monocyte were 9×109/L and 5.46×109/L, respectively. Bone marrow smear revealed an increase in the proportion of primitive immature cells (0.09 to 0.11). Lung computed tomography revealed interstitial lung disease, pericardial effusion, cardiac enlargement and pulmonary hypertension. The gene sequencing results showed KRAS gene c.38G> A somatic mutation in case1, and p.G12D and NRAS gene c.38G> A, p.G13D somatic mutation in case2. A total of 8 reports were retrieved including 23 cases caused by NRAS(10 cases) or KRAS(13 cases) gene somatic mutation. All the 23 cases showed hypergammaglobulinemia, splenomegaly, B cells hyperplasia or mononucleosis.@*Conclusion@#RALD often manifests as hepatosplenomegaly,lymphoproliferation, autoimmune hematocytopenia, B cells hyperplasia or mononucleosis, hypergammaglobulinemia. Gene sequencing analysis can help diagnose the disease.

Inhibitory effect of the Pseudobrickellia brasiliensis (Spreng) R.M. King & H. Rob. aqueous extract on human lymphocyte proliferation and IFN-γ and TNF-α production in vitro

Pseudobrickellia brasiliensis (Asteraceae) is a plant commonly known as arnica-do-campo and belongs to the native flora of the Brazilian Cerrado. The alcoholic extract of the plant has been used as an anti-inflammatory agent in folk medicine, but the biological mechanism of action has not been elucidated. The present study evaluated the composition of P. brasiliensis aqueous extract and its effects on pro-inflammatory cytokine production and lymphocyte proliferation. The extracts were prepared by sequential maceration of P. brasiliensis leaves in ethanol, ethyl acetate, and water. Extract cytotoxicity was evaluated by trypan blue exclusion assay, and apoptosis and necrosis were measured by staining with annexin V-FITC and propidium iodide. The ethanolic (ETA) and acetate (ACE) extracts showed cytotoxic effects. The aqueous extract (AQU) was not cytotoxic. Peripheral blood mononuclear cells stimulated with phorbol myristate acetate and ionomycin and treated with AQU (100 μg/mL) showed reduced interferon (IFN)-γ and tumor necrosis factor (TNF)-α expression. AQU also inhibited lymphocyte proliferative response after nonspecific stimulation with phytohemagglutinin. The aqueous extract was analyzed by liquid chromatography coupled with photodiode array detection and mass spectrometry. Quinic acid and its derivatives 5-caffeoylquinic acid and 3,5-dicaffeoylquinic acid, as well as the flavonoids luteolin and luteolin dihexoside, were detected. All these compounds are known to exhibit anti-inflammatory activity. Taken together, these findings demonstrate that P. brasiliensis aqueous extract can inhibit the pro-inflammatory cytokine production and proliferative response of lymphocytes. These effects may be related to the presence of chemical substances with anti-inflammatory actions previously reported in scientific literature.

Effects of polysaccharide from Ophiopogon japonicus on immune response to Newcastle disease vaccine in chicken

In order to investigate the immune enhancement effects of Ophiopogon japonicus polysaccharide Ophiopogon japonicus (OJPS) on Newcastle disease (ND) live vaccine, chickens vaccinated against ND live vaccine was orally administered with the OJPS at high, medium and low concentrations respectively. In negative control group, chickens were given orally equal volume of physiological saline. On day 14, 21 and 28, the serum antibody titer, erythrocyte-C3b receptor rosette rate (E-C3bRR), erythrocyte-C3b immune complex rosette rate (E-ICRR) and peripheral lymphocyte proliferation were measured. The results showed that at most time points, the antibody titer, peripheral lymphocyte proliferation, E-C3bRR and elimination rate of immune complex of three OJPS administrating groups were significantly higher (P<0.05) than those in negative control group. It indicated that OJPS could significantly improve the immune efficacy of Newcastle disease live vaccine, Ophiopogon japonicus polysaccharide possessed synergistical immunoenhancement.(AU)

Effects of exercise on spleen T lymphocyte proliferation and T-lymphocyte subsets of SD rats / 中国免疫学杂志

Objective:To explore the effects of exercise on the spleen T lymphocyte proliferation and T lymphocyte subsets of SD rats.Methods:24 SD rats were divided into 3 groups randomly:control group,30 min exercise group,60 min exercise group;MTT and flow cytometry ( FCM ) were utilized to examine the spleen T lymphocyte proliferative capability and T lymphocyte subsets, respectively.Results:Compared with the control group,the T lymphocyte proliferation,levels of CD4+and CD4+/CD8+T lymphocytes in 30 min exercise group rats were significantly increased(P<0.05),levels of CD8+T lymphocytes in 30 min exercise group rats had no statistically significant change;the T lymphocyte proliferation,levels of CD4+,CD8+and CD4+/CD8+T lymphocytes in 60 min exercise group rats had no statistically significant changes.Conclusion: Suitable loaded exercise could enhance the cellular immune function, this is probably related with the mechanisms of improving the T lymphocyte proliferative capability and regulating the CD4+/CD8+T lymphocyte subsets.

Prediction and identification of T-cell epitopes in major group 3 allergen de-rived from Dermatophagoides farina / 中国血吸虫病防治杂志

Objective To predict and identify T cell epitopes of major group 3 allergen derived from Dermatophagoides fari-na(Der f 3). Methods The T cell epitopes of Der f 3 were analyzed through the sequence analysis by using the bioinformatics online tools. The five predicted peptides of T-cell epitopes were artificially synthesized. The spleen lymphocytes were co-cultured with the five T cell epitopes by using the modified MTT method and the levels of IL-2,IFN-γ,IL-4 and IL-5 in the supernatant of the cultures were detected by ELISA. Results Five T cell epitopes of Der f 3 were predicted and three of which could pro-mote the proliferation of the mouse spleen lymphocytes. The secretions of IL-2 and IFN-γwere significantly induced and the se-cretions of IL-4 and IL-5 were significantly decreased by three of five prediction epitopes of Der f 3:37GDCPYQISLQSSSHFC-GG54,98IYQHENYDSMTIDNDVALIKLKTPMT123 and 164SELQRVDIDVVSREQCDQLYS184. Conclusion Three T cell epitopes of Der f 3 have been initially identified,which lays the foundation of the diagnosis and treatment of asthma.

Evaluation and characterisation of A and B fragments of Corynebacterium diphtheriae toxin towards recombinant diphtheria vaccine.

Background: Diphtheria is a highly communicable disease caused by toxin-producing strains of Corynebacterium diphtheriae. Objectives: To evaluate the efficacy of A and B subunits of diphtheria toxin (DT-A, DT-B) as potential vaccines against C. diphtheriae. A culture of C. diphtheriae (strain PW 8) was grown on Loeffler plates while Lingood medium was used for production of diphtheria toxin (DT). Materials and Methods: DT was purified and digested to obtain pure DT-A and DT-B and detoxified to obtain diphtheria toxin. Four groups of mice were immunised with different antigens (Ag) of C. diphtheriae. Results: The antibody (Ab) titres were significantly increased with immunised groups subsequent to three injections. On the other hand, Ab titres were estimated after the three immunisations and the levels of different Ab isotypes were comparatively measured. The levels of various isotypes immune responses showed variation between immunised groups where the IgG subclasses were significantly increased mainly with DPT immunised group. The IgM and IgA were significantly increased with DT-A more than others. Additionally, the evaluation of the cellular immune responses demonstrated that spleen cells from DPT and DT-A groups gave highly significant proliferative response with production of high levels of IL-2 and IFN-γ (Th1/Th2). Separation and purification of DT gene were performed using polymerase chain reaction (PCR) and sub-cloned in pGEM-T vector, for further studying of recombinant vaccine. Conclusion: Our results showed the possibility to prepare a potent recombinant vaccine containing whole DT gene or DT-A against C. diphtheriae or could be used in treatment of cancer as it give high levels of IL-2 and IFN-γ.

Proliferação de linfócitos e apoptose de células CD5+ de bovinos infectados pelo vírus da leucose enzoótica bovina / Apoptosis of CD5+ cells and lymphocyte proliferation in bovine leukemia virus-infected dairy cows

Avaliaram-se a proliferação de linfócitos e a apoptose de células CD5+ de bovinos naturalmente infectados pelo vírus da leucose enzoótica bovina. Para tal, 100 vacas da raça Holandesa, em lactação, foram triadas quanto ao sorodiagnóstico para a leucose enzoótica bovina e o perfil hematológico, e 15 foram escolhidos e distribuídos uniformemente entre os três grupos, a saber: animais negativos, animais positivos alinfocitóticos e animais positivos e que manifestaram linfocitose persistente (LP). Para a avaliação da proliferação de linfócitos, procedeu-se ao isolamento das células mononucleares por gradiente de centrifugação, em que 2x10(6) linfócitos por mL foram plaqueados por poço e analisados por citometria de fluxo utilizando-se o fluorocromo CFSE-DA. A apoptose do sangue periférico deu-se utilizando a anexina V-FITC, e para a identificação das células CD5+, utilizaram-se anticorpos monoclonais. Ocorreu menor proliferação de linfócitos nos animais infectados e que manifestavam LP, e menor apoptose de células CD5+ do sangue periférico. Pode-se sugerir que o desenvolvimento da LP, resultante do aumento de linfócitos B, deve-se à redução do processo apoptótico das células CD5+, principal população infectada, e que a maior proliferação linfocitária pode se restringir apenas ao estádio inicial do desenvolvimento da LP.

The purpose of the present trail was to evaluate the lymphocyte proliferation and the apoptosis rates of CD5+ cells in dairy cows infected with bovine leukemia virus (BLV) with distinct lymphocyte profiles in infected animals known as alymphocytotic (AL) and persistent lymphocytosis (PL). A total of 100 Holstein cows were sera tested for bovine leukemia virus through agar gel immunodiffusion (AGID) and enzyme-linked immunosorbent-assay (ELISA). From these animals, 15 cows were selected and divided uniformly in 3 groups (negative, AL, LP). The lymphocyte proliferation was performed using flow cytometric measurement of CFSE-DA dye, where 2x10(6)/mL lymphocytes were plated per well. The apoptosis of CD5+ cells from peripheral blood was performed using the annexin V-FITC to measure the apoptosis rates and the identification of CD5+ was accessed using monoclonal antibodies. Animals from the LP group showed lower lymphocyte proliferation and also lower apoptosis rates of CD5+ cells compared with negative and AL animals. The development of PL which resulted from an increase in B cell count, is due to the decrease in the apoptosis rates of CD5+ cells, and the higher lymphocyte proliferation appears to be limited only in the initial stages of development of LP.

Expression of mouse PD-1/PD-L1 recombinant protein in prokaryotic cells / 第二军医大学学报

Objective: To establish a molecule binding model of mouse PD-1/PD-L1 protein in vitro, so as to lay a foundation for studying the biological activities of the recombinant protein and to establish a high throughput drug screening model. Methods: Prokaryotic expression plasmid pET28 a (+) /mPDL-1 and pGEX-4T-1/mPD-1 were transformed to E. coli BL21 (DE3), which was then induced with IPTG. The expression products were purified by fast affinity chromatography with FPLC Protein Purification Instrument or gel separation. ELISA and GST pull-down were used to detect the interaction between mPD-1 and mPD-L1. In addition, Alamar blue was used to test the role of protein mixtures in the mixed lymphocyte proliferation. Results: SDS-PAGE and Western blotting analysis showed that the recombinant proteins were successfully expressed. Then purified mPD-1 and mPD-L1 protein was obtained by affinity chromatography, Gel seperation and refolding, etc. ELISA and GST pull-down showed that mPD-1 and mPD-L1 protein had a specific binding activity in vitro. The mPD-1/ mPD-L1 protein had a significantly decreased influence on the proliferation of lymphocytes(P<0. 05). Conclusion: Mouse PD-1/PD-L1 recombinant protein model has been successfully established using purified mPD-1 and mPD-L1 protein expressed prokaryotically, which lays a foundation for high-throughput drug screening.

Physicochemical properties and biological activity of a recombinant inducible co-stimulator-Ig fusion protein / 第二军医大学学报

Objective: To investigate the physicochemical properties and biological activity of self-prepared fusion protein inducible co-stimulator-Ig. Methods: Acid hydrolysis, edman degradation and peptide mass finger printing were used to determine the amino acid composition, N-terminal 15 amino acid sequences, and peptide mapping. In vivo mixed lymphocyte reaction assay was used for identification of its biological activity. Results: The result of amino acids composition analysis was consistent with the theoretical value of ICOS-Ig. N-terminal 15 amino acid sequences of the product were EINGSANYEMFIFHN, consistent with the theoretical value of ICOS-Ig. Peptide match assay identified six peptides of the product which could match the theoretic maps of ICOS-Ig. ICOS-Ig and CsA noticeably inhibited the proliferation of allo-reactive T cells in vivo. Conclusion: The prepared ICOS-Ig fusion protein has a correct structure and can inhibit the proliferation of allogeneic T cells in vivo, which lays a foundation for quality control of ICOS-Ig fusion protein.

Eukaryotic expression of human ICOS-Ig fusion protein and analysis of its biological activity / 第二军医大学学报

Objective: To express human ICOS extracellular region and IgG Fc fusion protein using euckaryotic vector and to analyze its biological activity. Methods: Human ICOS extracellular region and IgG Fc fragment were cloned by RT-PCR and inserted into eukaryotic expression vector pSecTag2. The constructed plasmid pSecTag2-ICOS-Ig was stably transfected into CHO cells. Soluble ICOS-Ig fusion protein was collected from serum-free medium and purified with protein A affinity chromatography. The purified product was analyzed by SDS-PAGE, Western blotting assay, and ELISA. Fluorescence-activated cell sorting (FACS) and mixed lymphocyte reaction (MLR) were used to study the activity of the fusion protein. Results: ICOS extracellular region and IgG Fc fragment were successfully cloned into expression vector; ICOS-Ig fusion protein was expressed and purified in mammal cells. The purified fusion protein specifically bound to ICOSL and inhibited mixed lymphocyte reaction. Conclusion: A ICOS-Ig fusion protein expression system has been successfully constructed, and bioactive ICOS-Ig fusion protein has been obtained.

Effects of Nitrobenzene on Immunity Function of Mice / 环境与健康杂志

Objective To study the effects of nitrobenzene on macrophage function and lymphocyte proliferation in mice. Methods ICR mice were divided into groups and treated with nitrobenzene by gavage,once a day,at doses of 2,20 and 200 mg/kg respectively,for 21 consecutive days.The mice were killed after 21 days of treatment and then the effects of nitrobenzene on the organs index,the maerophage function and the lymphocyte proliferation were determined.Results The maerophage function and the lymphocyte proliferation decreased as the increase of the dose of nitrobenzene.Conclusion The results of the present paper show that nitrobenzene may inhibit the immunity function of mice.

Effect of mucilage from yam on activation of lymphocytic immune cells

The immunostimulating activities of mucilage fraction from yam were investigated. The proliferation of BSA-primed lymph node cells was enhanced between 4.1- to 10.9-fold compare to control, when cultured with 1 to 25 microgram/mL of yam-mucilage fraction. It showed strong immunopotentiating activity than ginseng extract and as remarkable as Bifidobacterium adolescentis M101-4 known as a positive immunostimulator. Mitogenicity to lymph node cells was fully induced by concanavalin A and lipopolysaccharide. The proliferation of splenocytes and Peyer's patch cells was enhanced between 5.0- to 14.1-fold and 2.4- to 6.4-fold, respectively, when cultured with 1 to 25 microgram/mL of yam-mucilage fraction. It enhanced the production of cytokines such as tumor necrosis factor-alpha and IL-6 in the culture of RAW 264.7 macrophage cells. In the culture of lipopolysaccharide-stimulated RAW 264.7 cells, production of cytokines was as similar as compared to controls. In unstimulated RAW 264.7 cells, both tumor necrosis factor-alpha and IL-6 production were enhanced between 15.6- to 60.1-fold and 2.3- to 9.1-fold, respectively. Mucilage fraction from yam is expected to be a safe immunopotentiator to maintain the host immunity and develop a physiologically functional food.

Experimental Study of Pharmacology of Chinese Medicine to the Proliferation of Leukocyte in Anemia Mice / 中国康复理论与实践

@# Objective To observe the effect of “Shengbai Tablets” on increasing the numbers of leucocyte, enhancing lymphocyte proliferation and reducing the micronucleus rate of cells.Methods The leukopenia model of mouse was established with cyclophosphamide, and the changes of the leukocyte count, lymphocyte proliferation and micronucleus rate before and after Shengbai Tablets treatment were observed.Results After treatment, the leukocyte count, lymphocyte proliferation and micronucleus rate of the Shengbai Tablets group were ( 7.470 ±1.852)×109/L, (14768.23±8316.86) and (6.02±1.72)‰ respectively, all were significantly better than that of the group without Shengbai Tablets treatment ( P<0.05～0.01).Conclusion Shengbai Tablets is able to increase the numbers of leucocyte, enhance the lymphocyte proliferation and reduce the micronucleus rate.

Protective effect of Poly gala tenui folia willd on genetic damage and enhancement of lymphocyte function induced by cyclophosphamide in mice / 吉林大学学报(医学版)

Objective To study the effect of anti-mutation of Chinese medicinal herb Poly gala tenui folia willd on T lymphocyte proliferation in spleen. Methods The micronucleus test of mouse bone marrow cell (MNT): thirty mice were divided into six groups (n = 5) , negative control (NS) , cyclophosphamide group (CP 3. 0 mg ? kg-1) , Poly gala tenui folia willd antimutagenesis groups (Polygala tenui folia willd with dosage of 0.5, 1.0. 2.0, 4.0 g ? kg-1 + CP 30 mg ? kg-1 ). The improved method was used to detect the micro-nuclei frequency. Lymphocyte transformation test: twenty-four mice were divided into four groups (n = 6), saline control , CP control (3.0 mg ? kg-1), Polygala tenui folia willd (2.0 g ? kg-1), Polygala tenui folia willd + CP (2. 0 g ? kg-1 Polygala tenuifolia willd + CP 30 mg ? kg- ) group. MTT assay was used to calculate the stimulation index (SI). Results The micro-nuclei frequency was significant difference between Poly gala tenui folia willd antimutagenesis groups and CP groups (P