Interleukin-21 is a type I cytokine mainly produced by activated CD4+ T cells that acts as a regulator of immune system. In this work, hIL-21cDNA was amplified from human peripheral blood lymphocytes by RT-PCR, and then inserted into pPIC9K. The recombinant vector pPIC9K-hIL21cDNA was linearized by Sac I, and transformed into Pichia pastoris strain GS115 by electroporation. Transformants were selected by G418 and confirmed by PCR. The recombinant protein was expressed and secreted into the supernatant after inducing by methanol. SDS-PAGE analysis indicated the molecular weight of rhIL-21 was about 16 kD. ELISA results show that the yield of rhIL-21 reach 229.28 mg/L, rhIL-21 was purified from culture supernatants, and it was purified to about 95% purity with ion-exchange chromatography. When co-stimulate with Con A, rhIL-21 can promote the proliferation of human lymphocytes. This is the first expression of bio-active rhIL-21 in Pichia pastoris. It lays a foundation for further research in immunotherapy and cancer therapy.
Electroporation ; Genetic Vectors ; genetics ; Humans ; Interleukins ; biosynthesis ; genetics ; Pichia ; genetics ; metabolism ; Recombinant Proteins ; analysis ; biosynthesis ; genetics

To investigate the effect of fluoxetine, one of selective serotonin reuptake inhibitors (SSRIs), on the immune system, human peripheral blood mononuclear cells (PBMC) were treated with fluoxetine (10 7 M) for 24 h, and immune-related genes were analyzed by cDNA microarray. Expression of the immune- related genes such as CD107b (LAMP-2), CD47 receptor (thrombospondin receptor), CD5 antigen-like (scavenger receptor cysteine rich family), copine III (CPNE3), interleukin (IL) -18 (interferon-gamma- inducing factor), integrin alpha 4 (CD49d), integrin alpha L subunit (CD11a), IL-3 receptor alpha subunit, L apoferritin, and small inducible cytokine subfamily A (Cys-Cys) member 13 (SCYA13) was induced by fluoxetine. This result suggests that fluoxetine may affect the immune system, and provides fundamental data for the involvement of SSRIs on immunoregulation.
Apoferritins ; Cysteine ; DNA, Complementary* ; Fluoxetine ; Humans* ; Immune System ; Interleukins ; Oligonucleotide Array Sequence Analysis* ; Receptors, Interleukin-3 ; Serotonin Uptake Inhibitors

PURPOSE: To elucidate the characteristic gene transcription profiles among various hepatic ischemia conditions, immediately transcribed genes and the degree of ischemic injury were compared among total ischemia (TI), intermittent clamping (IC), and ischemic preconditioning (IPC). METHODS: Sprague-Dawley rats were equally divided into control (C, sham-operated), TI (ischemia for 90 minutes), IC (ischemia for 15 minutes and reperfusion for 5 minutes, repeated six times), and IPC (ischemia for 15 minutes, reperfusion for 5 minutes, and ischemia again for 90 minutes) groups. A cDNA microarray analysis was performed using hepatic tissues obtained by partial hepatectomy after occluding hepatic inflow. RESULTS: The cDNA microarray revealed the following: interleukin (IL)-1beta expression was 2-fold greater in the TI group than in the C group. In the IC group, IL-1alpha/beta expression increased by 2.5-fold, and Na+/K+ ATPase beta1 expression decreased by 2.4-fold. In the IPC group, interferon regulatory factor-1, osteoprotegerin, and retinoblastoma-1 expression increased by approximately 2-fold compared to that in the C group, but the expression of Na+/K+ ATPase beta1 decreased 3-fold. CONCLUSION: The current findings revealed characteristic gene expression profiles under various ischemic conditions. However, additional studies are needed to clarify the mechanism of protection against IPC.
Adenosine Triphosphatases ; Apoptosis ; Constriction ; Hepatectomy ; Interferon Regulatory Factor-1 ; Interleukins ; Ischemia ; Ischemic Preconditioning ; Microarray Analysis ; Necrosis ; Oligonucleotide Array Sequence Analysis ; Osteoprotegerin ; Rats, Sprague-Dawley ; Reperfusion ; Reperfusion Injury ; Transcriptome

Alcoholic liver disease continues to be a significant cause of liver-related morbidity and mortality throughout the world. A number of diagnostic and prognostic models have been developed in the management of this condition, although specific roles for liver biopsy still remain particularly in the setting of alcoholic hepatitis. Despite a large number of recent treatment trials, the ideal pharmacotherapy approach remains undefined. Most essential is the supportive care and focus on abstinence and nutrition. Owing in part to a great deal of attention from governmental funding sources, a number of new treatment approaches are undergoing rigorous evaluation, hopefully providing future treatment options in this very severe condition.
Adrenal Cortex Hormones/therapeutic use ; Biomarkers/analysis ; Humans ; Interleukins/metabolism ; Liver Diseases, Alcoholic/diagnosis/*therapy ; Liver Transplantation ; Pentoxifylline/therapeutic use ; Prognosis

PURPOSE: This study was performed to investigate whether therapeutic hypercapnia could attenuate systemic inflammatory responses in hemorrhagic shock in rats. METHODS: Male Sprague-Dawley rats were mechanically ventilated and underwent pressure-controlled (mean arterial pressure: 38+/-1 mmHg) hemorrhagic shock. At 10 minutes after the induction of hemorrhagic shock, the rats were divided into the normocapnia (PaCO2=35-45 mmHg, n=10) and the hypercapnia (PaCO2=60-70 mmHg) groups. The PaCO2 concentration was adjusted by using the concentration of inhaled CO2 gas. After 90 minutes of hemorrhagic shock, rats were resuscitated with shed blood for 10 minutes and were observed for 2 hours. The mean arterial pressure (MAP) and the heart rate were monitored continuously, and the results of arterial blood gas analyses, as well as the plasma concentrations of interleukin (IL)-6, IL-10, and nitrite/nitrate were compared between the normocapnia and the hypercapnia groups. RESULTS: The MAP and the heart rate were not different between the two groups. The plasma concentration of IL-6 was significantly lower in the hypercapnia group than in the normocapnia group (p<0.05). The IL-10 concentration was not different and the IL-6 to IL-10 ratio was significantly lower in the hypercapnia group compared to the normocapnia group. The plasma nitrite/nitrate concentration of the hypercapnia group was lower than that of the normocapnia group. CONCLUSION: Therapeutic hypercapnia attenuates systemic inflammatory responses in hemorrhagic shock.
Animals ; Arterial Pressure ; Blood Gas Analysis ; Cytokines ; Heart Rate ; Humans ; Hypercapnia ; Inflammation ; Interleukin-10 ; Interleukin-6 ; Interleukins ; Male ; Nitric Oxide ; Plasma ; Rats ; Rats, Sprague-Dawley ; Shock, Hemorrhagic

BACKGROUND: Macrophages generated in vitro using macrophage-colony stimulating factor (M-CSF) and interleukin (IL)-6 from bone marrow cells (BM-Mp) are defective in antigen presenting cell (APC) function as shown by their ability to induce the proliferation of anti-CD3 mAb-primed syngeneic T cells. However, they do express major histocompatibility (MHC) class I and II molecules, accessory molecules and intracellular adhesion molecules. Here we demonstrate that the defective APC function of macrophages is mainly due to production of TGF-beta1 by BM-Mp. METHODS: Microarray analysis showed that TGF-beta1 was highly expressed in BM-Mp, compared to a macrophage cell line, B6D, which exerted efficient APC function. Production of TGF-beta1 by BM-Mp was confirmed by neutralization experiments of TGF-beta1 as well as by real time-polymerase chain reaction (PCR). RESULTS: Addition of anti-TGF-beta1 monoclonal antibody to cultures of BM-Mp and anti-CD3 mAb-primed syngeneic T cells efficiently induced the proliferation of syngeneic T cells. Conversely, the APC function of B6D cells was almost completely suppressed by addition of TGF-beta1. Quantitative real time-PCR analysis also confirmed the enhanced expression of TGF-beta1 in BM-Mp. CONCLUSION: The defective APC function of macrophages generated in vitro with M-CSF and IL-6 was mainly due to the production of TGF-beta1 by macrophages.
Antigen-Presenting Cells ; Bone Marrow Cells ; Cell Line ; Histocompatibility ; Interleukin-6 ; Interleukins ; Macrophage Colony-Stimulating Factor ; Macrophages ; Microarray Analysis ; T-Lymphocytes ; Transforming Growth Factor beta1

BACKGROUND: Macrophages generated in vitro using macrophage-colony stimulating factor (M-CSF) and interleukin (IL)-6 from bone marrow cells (BM-Mp) are defective in antigen presenting cell (APC) function as shown by their ability to induce the proliferation of anti-CD3 mAb-primed syngeneic T cells. However, they do express major histocompatibility (MHC) class I and II molecules, accessory molecules and intracellular adhesion molecules. Here we demonstrate that the defective APC function of macrophages is mainly due to production of TGF-beta1 by BM-Mp. METHODS: Microarray analysis showed that TGF-beta1 was highly expressed in BM-Mp, compared to a macrophage cell line, B6D, which exerted efficient APC function. Production of TGF-beta1 by BM-Mp was confirmed by neutralization experiments of TGF-beta1 as well as by real time-polymerase chain reaction (PCR). RESULTS: Addition of anti-TGF-beta1 monoclonal antibody to cultures of BM-Mp and anti-CD3 mAb-primed syngeneic T cells efficiently induced the proliferation of syngeneic T cells. Conversely, the APC function of B6D cells was almost completely suppressed by addition of TGF-beta1. Quantitative real time-PCR analysis also confirmed the enhanced expression of TGF-beta1 in BM-Mp. CONCLUSION: The defective APC function of macrophages generated in vitro with M-CSF and IL-6 was mainly due to the production of TGF-beta1 by macrophages.
Antigen-Presenting Cells ; Bone Marrow Cells ; Cell Line ; Histocompatibility ; Interleukin-6 ; Interleukins ; Macrophage Colony-Stimulating Factor ; Macrophages ; Microarray Analysis ; T-Lymphocytes ; Transforming Growth Factor beta1

Atherosclerosis is a generalized disorder that progresses very slowly. Early detection of atherosclerosis is very important to prevent cardiovascular disease such as myocardial infarction, stroke and sudden cardiac death. Various surrogate markers have recently been proposed for the early detection of atherosclerosis in asymptomatic patients who have one or more risk factors. Among them, biomarkers such as CRP, Interleukin, myeloperoxidase, fibrinogen, homocystein and lipoprotein (a) are established as predictors of atherothrobotic events in apparently healthy individuals. Although these novel biomarkers provide important information into the pathophysiology of atherosclerosis, no clear evidence exist that lowering the plasma level of these markers reduces the vascular risk. Imaging markers such as the carotid intima-media thickness and brachial arterial flow mediated vasodilation as assessed by ultrasound, coronary calcification as assessed by CT, and the pulse wave velocity and augmentation index as assessed by tonometry can visualize the arterial wall and directly measure the arterial function. These imaging markers are very useful clinical tools for detecting the early changes of vascular structure and also for predicting cardiovascular events, in addition to being more precise biomarkers in asymptomatic subjects.
Atherosclerosis* ; Biomarkers ; Cardiovascular Diseases ; Carotid Intima-Media Thickness ; Death, Sudden, Cardiac ; Fibrinogen ; Humans ; Interleukins ; Lipoprotein(a) ; Manometry ; Myocardial Infarction ; Peroxidase ; Plasma ; Pulse Wave Analysis ; Risk Factors ; Stroke ; Ultrasonography ; Vasodilation

PURPOSE: The pattern of radioprotection by the combined use of low dose amifostine plus IL-1beta was investigated in mice exposed to an acute whole-body radiation dose of 10 Gy. MATERIALS AND METHODS: Male ICR mice were divided into the control group, the irradiation-only group, the high dose amifostine (400 mg/kg i.p. 30 min before irradiation) group, and the low dose amifostine (200 mg/kg i.p. 30 min before irradiation) plus IL-1beta (5 microgram/kg i.p. 20 h before irradiation) group. The radioprotective effects were evaluated using TUNEL assay and microcolony survival assay at jejunal crypt, bone marrow cell count and CBC in peripheral blood, and survival analysis up to 30 days following irradiation. RESULTS: The apoptotic index (p=0.987), surviving crypt number (p=0.484), and the number of WBCs (p=0.226), RBCs (p=0.544), and platelets (p=0.157) were not significantly different between the high dose amifostine group and the low dose amifostine plus IL-1beta group, although the bone marrow cell count was higher in the combination group. The irradiation-only group was dead within 15 days. However, the survival rate at 30 days in the high dose amifostine and the low dose amifostine plus IL-1beta pretreatments were 61% and 66%, respectively. Moreover, the differences between the two groups were insignificant for both 10 days (p=0.9461) and 30 days (p=0.8030). CONCLUSION: These results indicate that the low dose amifostine plus IL-1beta may be applied as a non-toxic radioprotector, while the high dose amifostine, known as the strongest radioprotector, however, had toxic side effects.
Amifostine* ; Animals ; Bone Marrow Cells ; Humans ; In Situ Nick-End Labeling ; Interleukin-1* ; Interleukin-1beta* ; Interleukins* ; Male ; Mice ; Mice, Inbred ICR ; Radiation Protection ; Survival Analysis ; Whole-Body Irradiation

PURPOSE: To examine the expression pattern of inflammatory cytokines/receptors in the subacromial bursa of patients with rotator cuff disease using a cDNA(Complement DNA) Array technique. MATERIALS AND METHODS: Twenty two human subacromial bursal specimens were obtained intraoperatively from patients during shoulder surgery (18 bursitis, 4 normal bursa). The RNA was isolated from the bursal tissues and the presence of gene expression was analyzed using a cDNA Array technique. The statistical differences between bursitis and the normal bursa specimens were determined using a Mann Whitney U test and Student's t-test. RESULTS: cDNA Array analysis revealed a significant increase in the expression of several cytokine genes and their receptors in patients with subacromial bursitis compared with the controls (p<0.05). These cytokines included the interleukins (IL-1, 6, 12, 13, 15, 16, 17) and their receptors, lymphotoxin, small inducible cytokines, chemokine receptor (CCR 4, 6, 7) and stromal cell derived factor-1 (SDF-1). CONCLUSION: This study demonstrated a significant increase in many inflammatory cytokines in the subacromial bursa of patients with rotator cuff disease. This suggests that there is an active inflammatory reaction at the subacromial bursa in rotator cuff disease.
Bursitis ; Cytokines ; DNA, Complementary ; Gene Expression ; Humans ; Interleukins ; Lymphotoxin-alpha ; Oligonucleotide Array Sequence Analysis ; RNA ; Rotator Cuff ; Shoulder ; Stromal Cells ; Transcriptome