Affinity Chromatography Purification of Recombinant Human Interleukin-6 from Its Fusion Protein with GST

Recombinant E.coli JM109, containing pHZ1818 plasmid which included the fused gene encoding human interleukin 6(IL 6), expressed a fusion protein with glutathion S transferase(GST). The fusion protein existed both in the supernatant and inside the bacterial cell,but the insoluble protein had no biological activity and could not be refolded. The rotative speed of the shaker and the temperature of induction were optimized to maximize the expression of the soluble fusion protein. From the supernatant of the cell sonicates Glutathion Sephrose 4B affinity column chromatography was employed to isolate the fusion protein which could be purified to >80 0 0 in a single step. The yield of soluble GST IL 6 was about 10 mg per liter culture. The GST was site specifically cloven by 6 hours of treatment with thrombin and from the thrombin digest mixture IL 6 was purified by Q high performance ion exchange chromatography. From 1 liter of E.coli culture 2 mg refined IL 6 was obtained. The purified IL 6 had a purity of more than 95 0 0 and a biological activity of 1.02×10 8 IU/mg.

Cloning and Sequence Analysis of Interleukin 10 (IL-10) Full-length cDNA from Cyprinus carpio L.

[Objective] This study aimed to obtain IL-10 (interleukin 10) full-length cDNA of common carp (Cyprinus carpio L.) and conduct the sequence analysis. [Method] The differentially expressed cDNA fragment was obtained by DD-RTPCR (differential display RT-PCR). The cDNA library of peripheral blood leukocytes which were separated from common carp and stimulated by mitogen was screened with a probe labeled with DIG (digoxigenin). The IL-10 full-length cDNA was cloned from 0.8×104 pfu of recombinant phages, and the sequence analysis and homology comparison were carried out. [Result] Sequence analysis indicated that the IL-10 full-length cDNA of common carp was 1 117 bp long, containing a 55 bp 5’-UTR, a 522 bp 3’-UTR, and a 540 bp open reading frame(ORF) encoding 179 amino acids. In addition, there were three mRNA instability motifs (ATTTA) in the 3’-untranslated region. The deduced protein sequence shared typical sequence features of the IL-10 family. Homology comparison indicated that the obtained sequence shared 89.1% homology with the carp IL-10 gene from GenBank. [Conclusion] This study laid foundation for further study of the expression manner, functional characteristic and regulation mechanism of IL-10 in vivo and the interaction mechanism in the inflammatory reaction and immune response.

Clinical evaluation of serum interleukin 10 in patients with acute pancreatitis

OBJECTIVES: To evaluate the behavior of serum interleukin 10 (IL-10) in patients with acute pancreatitis and to explore the relationship between this cytokine and the severity of the disease. METHODS: In 45 patients with acute pancreatitis, the serum concentrations of IL-10 was determined on days 1, 2, 3, 4, 5 after admission. Twelve healthy subjects were also studied as controls. These subjects were tested using a commercial ELISA kit. The severity of pancreatitis was determined according to APACHE Ⅱ score and Balthazar CT criteria. RESULTS: Healthy subjects had no detectable serum levels of IL-10. In acute pancreatitis patients, the serum IL-10 levels were increased on the first day after the onset of the disease and then progressively decreased in the following days. On the first day after the onset of acute pancreatitis, the serum levels of IL-10 in patients with mild acute pancreatitis were significantly higher than in those with severe acute pancreatitis. In the following days, however, no statistically significant difference was observed between the two groups. CONCLUSIONS: Serum IL-10 concentration reflects the severity of acute pancreatitis. IL-10 is a useful variable for early prediction of the prognosis of acute pancreatitis. The low values of serum IL-10 in patients with severe acute pancreatitis suggests that there may be altered down-regulation of immune system response. An enhanced release of IL-10 may be a method for early treatment of acute pancreatitis.

Regulation of interleukin 33/ST2 signaling of human corneal epithelium in allergic diseases

AIM:To identify the function of ST2 and explore the role of IL-33/ST2 signaling in regulating the pro-allergic cytokine production in human corneal epithelial cells (HCECs). METHODS:Human corneal tissues and cultured primary HCECs were treated with IL-33 in different concentrations without or with different inhibitors to evaluate the expression, location and signaling pathways of ST2 in regulating production of pro-allergic cytokine and chemokine. The expression of mRNA was determined by reverse transcription and real time PCR, and protein production was measured by enzyme-linked immunosorbent assay (ELISA), immunohistochemical and immunofluorescent staining. ST2 protein was detected in donor corneal epithelium, and ST2 signal was enhanced by exposure to IL-33. ·RESULTS:IL-33 significantly stimulated production of pro-allergic cytokines thymic stromal lymphopoietin (TSLP) and chemokine (CCL2, CCL20, CCL22) in HCECs at both mRNA and protein levels. These stimulated productions of pro-allergic mediators by IL-33 were blocked by ST2 antibody or soluble ST2 protein(P 【0.05). Interestingly, the IκB-α inhibitor BAY11-7082 or NF-κB activation inhibitor quinazoline blocked NF-κB p65 protein nuclear translocation, and also suppressed the productions of these pro-allergic cytokines and chemokine induced by IL-33. CONCLUSION:These findings demonstrate that IL-33/ ST2 signaling plays an important role in regulating IL-33 induced pro-allergic responses. IL-33 and ST2 could become novel molecular targets for the intervention ofallergic diseases in ocular surface.

INTERLEUKIN 10 INHIBITS THE RAT VSMC PROLIFERATION AND COLLAGEN SECRETION STIMULATED BY ANGIOTENSIN II

Objective. To study the effect of interleukin 10 (IL- 10) on the angiotensin II (AngII) stimulated rat VSMC proliferation and collagen secretion, and furthermore, explore its mechanism. Methods. On cultured VSMC of rat, 3H- thymine (3H- TdR) and 3H- proline incorporations were used to evaluate the DNA and collagen synthesis, respectively. Western blot and immunoprecipitation were applied to assay the expression and activity of focal adhesion kinase (FAK), respectively. Results. IL- 10 (10－ 8～ 10－ 10g/ml) inhibited the increase of 3H- TdR and 3H- proline incorporation as well as FAK activity, which was induced by 10－ 7mol/L AngII (P< 0.05 or P< 0.01). IL- 10 also obviously downregulated the synthesis and secretion of collagen by AngII stimulated VSMC. But there was no difference in the protein expression of FAK among all the groups (P >0.05). Conclusion. IL- 10 antagonizes the VSMC proliferation and collagen synthesis by regulating FAK activity stimulated by AngII.

Clinicopathological significance of human leukocyte antigen F-associated transcript 10 expression in colorectal cancer

BACKGROUND Colorectal cancer(CRC) is a common malignancy of the gastrointestinal tract. The worldwide mortality rate of CRC is about one half of its morbidity. Ubiquitin is a key regulatory factor in the cell cycle and widely exists in eukaryotes. Human leukocyte antigen F-associated transcript 10(FAT10), known as diubiquitin, is an 18 kDa protein with 29% and 36% homology with the N and C termini of ubiquitin. The function of FAT10 has not been fully elucidated, and some studies have shown that it plays an important role in various cell processes.AIM To examine FAT10 expression and to analyze the relationship between FAT10 expression and the clinicopathological parameters of CRC.METHODS FAT10 expression in 61 cases of CRC and para-cancer colorectal tissues was measured by immunohistochemistry and Western blotting. The relationship between FAT10 expression and clinicopathological parameters of CRC was statistically analyzed.RESULTS Immunohistochemical analysis showed that the positive rate of FAT10 expression in CRC(63.93%) was significantly higher than that in tumor-adjacent tissues(9.84%, P < 0.05) and normal colorectal mucosal tissue(1.64%, P < 0.05). Western blotting also indicated that FAT10 expression was significantly higher in CRC than in tumor-adjacent tissue(P < 0.05). FAT10 expression was closely associated with clinical stage and lymphatic spread of CRC. FAT10 expression also positively correlated with p53 expression.CONCLUSION FAT10 expression is highly upregulated in CRC. FAT10 expression is closely associated with clinical stage and lymphatic spread of CRC.

Clostridium difficile infection aggravates colitis in interleukin 10-deficient mice

AIM: To investigate the effect of Clostridium difficile (C. difficile) infection in an interleukin 10-deficient (IL-10-/-) mouse model of inflammatory bowel disease.

Study of recombinant human interleukin-12 for treatment of complications after radiotherapy for tumor patients

AIM To evaluate the treatment effects of recombinant human interleukin-12(rh IL-12) on radiotherapy complications, such as severe myelosuppression or pancytopenia, the decline or imbalance of immune function, etc.METHODS The patients received high-dose and short-course precise radiotherapy, such as Cyber knife and image-guided radiotherapy(IGRT), which can cause myelosuppression or pancytopenia and immune function decline within a short time. One-hundred subjects were enrolled in the study, and 50 were randomized to a treatment group which used rh IL-12 and 50 were randomized to a control group which used symptomatic and supportive therapy after radiotherapy. The 50 subjects in the treatment group were further divided into five subgroups and intervenedwith rh IL-12 at a dose of 50, 100, 150, 200 or 250 ng/kg respectively. The dose-effect relationship was observed. RESULTS Rh IL-12 significantly attenuated the decrease of peripheral blood cells in the treatment group, and immune function was improved after treatment. Due to the different radiation doses, there was a fluctuation within 12 h after treatment but mostly showing an increasing trend. As to the clinical manifestations, 2 patients in the 250 ng/kg subgroup showed low fever after administration, 1 patient in the 200 ng/kg subgroup and 2 patients in the 250 ng/kg subgroup showed mild impairment of liver function during the observation period.CONCLUSION Rh IL-12 has effective therapeutic and protective effects on complications following radiotherapy, such as the decline of blood cells, myelosuppression and the decline or imbalance of immune function, which indicated good prospects for development and application.

Autosomal recessive 333 base pair interleukin 10 receptor alpha subunit deletion in very early-onset inflammatory bowel disease

BACKGROUND Interleukin 10 receptor alpha subunit(IL10RA)dysfunction is the main cause of very early-onset inflammatory bowel disease(VEO-IBD)in East Asians.AIM To identify disease-causing gene mutations in four patients with VEO-IBD and verify functional changes related to the disease-causing mutations.METHODS From May 2016 to September 2020,four young patients with clinically diagnosed VEO-IBD were recruited.Before hospitalization,using targeted gene panel sequencing and trio-whole-exome sequencing(WES),three patients were found to harbor a IL10RA mutation(c.301C>T,p.R101W in one patient;c.537G>A,p.T179T in two patients),but WES results of the fourth patient were not conclusive.We performed whole-genome sequencing(WGS)on patients A and B and reanalyzed the data from patients C and D.Peripheral blood mononuclear cells(PBMCs)from patient D were isolated and stimulated with lipopolysaccharide(LPS),interleukin 10(IL-10),and LPS+IL-10.Serum IL-10 levels in four patients and tumor necrosis factor-α(TNF-α)in the cell supernatant were determined by enzyme-linked immunosorbent assay.Phosphorylation of signal transducer and activator of transcription 3(STAT3)at Tyr705 and Ser727 in PBMCs was determined by western blot analysis.RESULTS The four children in our study consisted of two males and two females.The age at disease onset ranged from 18 d to 9 mo.After hospitalization,a novel 333-bp deletion encompassing exon 1 of IL10RA was found in patients A and B using WGS and was found in patients C and D after reanalysis of their WES data.Patient D was homozygous for the 333 bp deletion.All four patients had elevated serum IL-10 levels.In vitro,IL-10-stimulated PBMCs from patient D failed to induce STAT3 phosphorylation at Tyr705 and only minimally suppressed TNF-αproduction induced by LPS.Phosphorylation at Ser727 in PBMCs was not affected by LPS or LPS+IL-10 in both healthy subjects and in patient D.CONCLUSION WGS revealed a novel 333-bp deletion of IL10RA in four patients with VEO-IBD,whereas the WES results were inconclusive.

Effects of human interleukin 10 gene transfer on the expression of Bcl-2 Bax and apoptosis of hepatocyte in rats with acute hemorrhagic necrotizing pancreatitis

Acute necrotising pancreatitis is characterized by inflammatory and necrotic events, which followthe initial intra-acinar injury involving enzyme activation, and disruption of the acinar cytoskeleton. At present, apoptosis has become a hot topic in many kinds of disease. Bax and Bcl-2 are the important control gene during cell apoptosis. 2 Previous study has shown that interleukin 10 （IL-10） is a kind of important antiinflammatory cytokine and plays a role of selfdefense mechanism,

Air Quality Indices, Sources and Impact on Human Health of PM₁₀and PM_2.5in Alexandria Governorate, Egypt

In this study, PM10 and PM2.5 were measured in seven sites representing different activities (the same sites of EEAA monitoring stations) in addition to eighth site that used as a background. All results were higher than AQLs of EEAA, US/EPA, and EC although PM10 and PM2.5 are considered to be a direct cause of cardiovascular diseases as well as lead to death and it may be a reason for a number of chest diseases in short-term as well as long-term. Results were compared to the Air Quality Forecast system which developed by EEAA and AQI which created by US/EPA was calculated for some PM10 and PM2.5. Probable potential anthropogenic sources for such high concentrations of PM included unpaved roads, indiscriminate demolition and construction work, industrial activities, and solid wastes. This study resulted in a number of suggestions and recommendations include: 1) Implementation of integrated ISO 26000 and ISO 14001, 2) EIMP/EEAA monitoring stations need restructuring plan to cover all areas in Alexandria, 3) EIMP/EEAA must be supported with PM2.5 monitors, 4) PM control systems must be used in all industrial activities to reduce PM pollution from the source, 5) AQL of PM2.5 in the ambient environment must be reduced and it must be included in the working environment parameters, 6) Environmental law must be applied strictly, and 7) Multidisciplinary co-operation especially between environment and public health specialists must be increased.

Neuronal changes in the retinal ganglion cell layer following recombinant human interleukin-2 intravitreal injection in a rat model of chronically elevated intraocular pressure

Intraperitoneal injection of recombinant human interleukin-2（rhIL-2）inhibits neuronal apoptosis in the chronic ocular hypertension retinal ganglion cell layer.Intravitreous injection was performed on retinal ganglion cells in a Wistar rat model of chronically elevated intraocular pressure to observe the effects of LY294002 and AG490 on retinal ganglion cell survival,macrophage activation,and PI3K/Akt and JAK/STAT activation.The number of retinal ganglion cells in the rhIL-2 treatment group was much greater than in the normal control and phosphate-buffered saline groups.Western blot analysis revealed low Akt and STAT3 protein expression in the retina after 3-hour intravitreous injections of rhIL-2.However,protein expression was increased at 12 hours,but decreased again at 24 hours,with very low expression at 96 hours.LY294002 and AG490,which are inhibitors of the PI3K/Akt and JAK/STAT3 signal pathways,prevented upregulation of Akt and STAT3 protein expression in the retina,respectively.Intravitreous injection of rhIL-2 exhibited neuroprotective effects by decreasing retinal ganglion cell layer damage in a rat model of chronic glaucoma.These results suggest that intravitreal injection of rhIL-2 could induce the PI3K/Akt and JAK/STAT3 signaling pathways to protect retinal ganglion cells in chronically elevated intraocular pressure models.

Upregulation of stromal cell-derived factor-1 alpha/CXCR4 axis-induced migration of human neural progenitors by tumor necrosis factor-alpha and interleukin-8

BACKGROUND： Studies of several animal models of central nervous system diseases have shown that neural progenitor cells （NPCs） can migrate to injured tissues. Stromal cell-derived factor 1 alpha （SDF-la）, and its primary physiological receptor CXCR4, have been shown to contribute to this process. OBJECTIVE： To investigate migration efficacy of human NPCs toward a SDF-1α gradient, and the regulatory roles of tumor necrosis factor-α （TNF-α） and interleukin-8 （IL-8） in SDF-1α/CXCR4 axis-induced migration of NPCs. DESIGN, TIME AND SETTING： An in vitro, randomized, controlled, cellular and molecular biology study was performed at the Laboratory of Department of Cell Biology, Medical College of Soochow University between October 2005 and November 2007. MATERIALS： SDF-1α and mouse anti-human CXCR4 fusion antibody were purchased from R＆D Systems, USA. TNF-αwas purchased from Biomyx Technology, USA and IL-8 was kindly provided by the Biotechnology Research Institute of Soochow University. METHODS： NPCs isolated from forebrain tissue of 9 to 10-week-old human fetuses were cultured in vitro. The cells were incubated with 0, 20, and 40 ng/mL TNF-α, or 0, 20, and 40 ng/mL IL-8, for 48 hours prior to migration assay. For antibody-blocking experiments, cells were further pretreated with 0, 20, and 40 μg/mL mouse anti-human CXCR4 fusion antibody for 2 hours. Subsequently, the transwell assay and CXCR4 blockade experiments were performed to evaluate migration of human NPCs toward a SDF-1α gradient. Serum-free culture medium without SDF-1α served as the negative control. MAIN OUTCOME MEASURES： The transwell assay was performed to evaluate migration of human NPCs toward a SDF-1α gradient, which was blocked by fusion antibody against CXCR4. In addition, CXCR4 expression in human NPCs stimulated by TNF-α and IL-8 was measured by flow cytometry. RESULTS： Results from the transwell assay demonstrated that SDF-1α was a strong chemoattractant for human NPCs （P 〈 0.01）, and 20 ng/mL produced the highest levels of migration. Anti-human CXCR4 fusion antibody significantly blocked the chemotactic effect （P 〈 0.05）. Flow cytometry results showed that treatment with TNF-α and IL-8 resulted in increased CXCR4 expression and greater chemotaxis efficiency of NPCs towards SDF-1α（P 〈 0.01）. CONCLUSION： These results demonstrated that SDF-la significantly attracted NPCs in vitro, and neutralizing anti-CXCR4 antibody could block part of this chemotactic function. TNF-α and IL-8 increased chemotaxis efficiency of NPCs towards the SDF-1αgradient by upregulating CXCR4 expression in NPCs.

The effect of interleukin-6 gene transfer on human cord blood megaka ryopoiesis

Objective:To investigate the effect of IL-6 gene transfe r into human cord blood hematopoietic stem cells on the production of megakaryocy t ic progenitors.Methods:IL-6 gene was transfected into human c o rd blood CD34+ cells using a retrovirus vector with the aid of recombinant fibro nectin fragments in the presence of a cocktail of cytokines (SCF,IL-6,sIL-6R,F L,and TPO).Colony-forming units-megakaryocyte (CFU-MK) assays were performed as IL-6 gene transduced CD34+ cells were incubated alone or in combination with I L -3 or sIL-6R, controlled with neoR gene transduced CD34+ cells.Result s :IL-6 alone or sIL-6R alone stimulated few CFU-MK colonies,the addit ion of sIL-6R to IL-6 gene transduced CD34+ cells significantly enhanced the prod uction of CFU-MK colonies.IL-6 gene transduced CD34+ cells showed a modest syn er gistic effect with IL-3.Conclusion:These results suggest that IL-6 gene transfer may protect patients from chemotherapy-induced thrombocytop en ia.

Differential IFN-Gamma (IFN-γ), Interleukin 10 (IL-10) and Cardiac Troponin I (cTnI) Responses in Natural Bovine Trypanosomosis in Nigeria

Trypanosomosis is major drawback to profitable livestock production in sub-Sahara African, including Nigeria. Knowledge of the cytokines production in the phase of natural infection may help to better diagnose, treat and prevent bovine trypanosomosis. The purpose of the this study was to determine the levels of interferon-gamma (IFN-γ), interleukin-10 (IL-10) and cardiac troponin–I (cTnI) in the sera of cattle naturally infected with T. brucei, T. congolense and T. vivax and correlate these levels with parasitaemia and PCV of the infected animals. Five milliliter of blood samples were collected via the jugular vein from 411 randomly selected cattle into EDTA and non-citrated bottle. PCV was determined manually using HCT. Trypansomes were detected and characterized by microscopy and PCR, respectively. Serum levels of IFN-γ, IL-10 and cTnI were determined using commercial ELISA kit. Data were summarized using descriptive statistic and significance of differences determined by ANOVA. Of the 62 samples positive for trypanosomes by microscopy, 50 samples were confirmed to species level by PCR. The sera levels of IFN-γ, IL-10 and cTnI of infected cattle were higher than non-infected cattle. The differences were not significant (p γ, IL-10 and cTnI in cattle with natural trypanosomosis. Further investigation is required to understand the specific effect of trypanosomes on myocardiac integrity and interaction between the two cytokines in natural trypanosomosis in cattle.

Protective effect of recombinant human IL-1Ra on CCl_4-induced acute liver injury in mice

AIM: To evaluate the effects of positive regulation of recombinant human interleukin 1 receptor antagonist (rhIL-1Ra) on hepatic tissue recovery in acute liver injury in mice induced by carbon tetrachloride (CCl 4 ). METHODS: Acute liver damage was induced by injecting 8-wk-old mice with CCl 4 1 mL/kg (1:3 dilution in corn oil) intraperitoneally (ip). Survival after liver failure was assessed by injecting 8-wk-old mice with a lethal dose of CCl 4 2.6 mL/kg (1:1 dilution in corn oil) ip. Mice were subcutaneously injected with 1 mg/kg recombinant human IL-1Ra twice a day after CCl 4 treatment for 5 d. Serum alanine amino transferase (ALT) and aspartate aminotransferase (AST) levels were determined with a commercial assay kit. Serum IL-1β, IL-1Ra levels were measured by enzyme-linked immunosorbent assay kit. Quantitative real-time polymerase chain reaction was used to determine liver IL-1β, IL-1Ra and IL-6 expression during CCl 4-induced acute liver injury. Liver sections were stained with hematoxylin-eosin. A histology-injury grading system was used to evaluate the degree of necrosis after acute liver injury. Proliferating cell nuclear antigen (PCNA) staining was used to evaluate the role of rhIL-1Ra in promoting hepatocyte proliferation. RESULTS: Quantitative analysis showed a higher level of IL-6 mRNA expression and reduced serum AST and ALT levels in the livers of the rhIL-1Ra-treated group at the early phase of CCl 4-induced acute liver injury. Histological examination indicated a decrease in centrilobular necrotic areas in mice treated with rhIL-1Ra, and a novel role of rhIL-1Ra in promoting hepatocyte proliferation was also supported by an increase of PCNA staining. All these results, accompanied by a strong survival benefit in rhIL-1Ra-treated vs PBS-treated groups, demonstrated that rhIL-1Ra administration ameliorated the histological damage and accelerated the regeneration and recovery process of the liver. CONCLUSION: rhIL-1Ra could be further developed as a novel therapeutic agent for the treatment of acute liver injury because of its ability to reduce hepatocellular damage and facilitate liver regeneration.

<i>In Vitro</i>Evaluation System of Pharmacokinetics and Irradiation Effect in Boron Neutron Capture Therapy (BNCT) Using Three-Dimensional Artificial Human Tumor Tissue Model

Boron neutron capture therapy (BNCT) is based on the incorporation of boron-containing drugs to cancer cells and the nuclear reaction of 10B atoms by thermal neutron irradiation results in tumor degeneration. For the development of this therapy, currently, long time and high cost consuming experiments using many animals are required. In this study, we constructed a new in vitro evaluation system for BNCT by combination of an artificial tumor tissue model, comprised of normal human dermal-derived fibroblast (NHDF) and human pancreatic cancer cell line BxPC3, and the optical plastic material CR-39 as a solid state nuclear track detector. Administration of boronophenylalanine (10BPA) as a boron-containing drug and neutron irradiation up to 2.52 × 1012 n/cm2 to the control tissue constructed by NHDF (NHDF3D) and BxPC3 cell loaded tissue (NHDF3D/BxPC3) resulted in detection of 1.6 times higher number of α-ray/recoiled Li particle tracks in NHDF3D/BxPC3 in comparison to NHDF3D, demonstrating that putative irradiation damage to cancer cells can be evaluated by this system. On a cellular level, the hit number of α-ray/recoiled Li particle tracks per single BxPC3 cells and NHDF was evaluated as 5.46 and 1.71, respectively. The tumor and normal tissue ratio (T/N ratio) was 3.19, which was corresponded with those of BPA as 2 - 4 that reported in the previous studies. This new in vitro evaluation system may provide a useful tool for a low cost, labor-saving, and non-animal method for the development of new boron-containing drugs or improvement of BNCT conditions.

Double potentialities of tumoricide and hematopoiesis of human bone marrow cells activated by IL-2 and IL-3

The biomodulative and hematopoietic potentialities of IL-2 and IL-3 activatedbone marrow(ABM)cells from patients with lung adenocarcinoma were studied in vitro.Human bone marrow(BM)cells could be activated by IL-2 in culture for 7d.TheseIL-2 ABM cells had higher cytolytic activities against cells of H 7402 cell line and freshautologous adenocarcinoma cells and maintained the cytotoxicities longer than IL-2 acti-vated peripheral blood lymphocytes(APBLs),a point of possible importance in adoptiveimmunotherapy for cancer patients.The IL-2 ABM cells also had similar number ofBFU-E and CFU-GM to that had fresh BM cells if 1L-3 was added 48h alter IL-2 inculture.The IL-2 and IL-3 ABM cells might be used to eliminate tumor cells and tosupply reconstitutive elements of BM for autologous bone marrow transplantation.

Ameliorative effects of human adipose tissue-derived mesenchymal stem cells on myelin basic protein-induced experimental autoimmune encephalomyelitis in Lewis rats

Mesenchymal stem cells have been previously shown to exert an immunomodulatory function. The present study sought to investigate the effects of multipotential human adipose tissue-derived mesenchymal stem cells （hAdMSCs） on disease progression and cytokine expression in Lewis rats with experimental autoimmune encephalomyelitis （EAE） induced by myelin basic protein. The duration of EAE paralysis in the group treated on day 7 posfimmunization with 5 × 10^6 hAdMSCs was significantly reduced compared with the vehicle-treated controls and the 1 x 106 hAdMSC- treated group. The duration of EAE paralysis in the groups treated with 5 × 10^6 hAdMSCs on both day 1 and day 7 postimmunization was significantly reduced compared with the vehicle-treated controls and the groups treated with 5 × 10^6 hAdMSCs on both day 7 and day 10 postimmunization. The mRNA expression of interleukin-10 and indoleamine 2, 3-dioxygenase was significantly decreased in the hAdMSC-treated group compared with the vehicle-treated group. These findings suggest that the ameliorative effects of hAdMSCs on EAE symptoms operate in a dose- and time-dependent manner and can be mediated in part by the ample production of anti-inflammatory cytokines.

CONSTRUCTION OF HUMAN INTERLUEKIN-18 DNA VACCINE AND IT'S EXPRESSION IN MAMMALIAN CELLS

Objective To construct the human interleukin 18 DNA plasmids vaccine and to express the eukaryotic plasmids vaccine in mammalian cell lines Cos 7 and D5.Methods Gene recombinant technique was used to construct hIL 18 eukaryotic expression vectors.Calcium phosphate method was performed to transect recombinant hIL 18 eukaryotic expression vectors into Cos 7 and D5 cells. In situ hybridization and Western Blot were implemented to verify the transient expression of recombinant hIL 18 in Cos 7 and D5.Results The eukaryotic expression plasmid pVAX1 IL 18 was constructed successfully.hIL 18 was transiently expressed in Cos 7 and D5.Conclusion The eukaryotic expression plasmid pVAX1 IL 18 was constructed. In situ hybridization and Western Blot results proved the successful transient expression of pVAX1 IL 18 in Cos 7 and D5.Therefore,the work has settled the foundation for further biological research on hIL 18,including immunogene therapy through hIL 18.