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

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

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

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

OBJECTIVETo study the roles of CD4(+)CD25(+)Foxp3(+) regulatory T cells (Treg) and IL-33 in the pathogenesis of asthma in children.

METHODSFlow cytometry was used to detect peripheral blood CD4(+)CD25(+)Foxp3(+)Treg proportion in CD4(+)T lymphocytes in.45 children with asthma, 50 children with wheezing caused by respiratory syncytial virus infection and 40 healthy children. Serum levels of IFN-γ, IL-4, IL-5 and IL-33 were measured using ELISA.

RESULTSThe level of peripheral blood CD4(+)CD25(+)Foxp3(+)Treg in the asthma group was significantly lower than in the wheezing and control groups (P<0.05). In contrast, serum levels of IL-33 in the asthma group was significantly higher than in the wheezing and control groups (P<0.05). Peripheral blood CD4(+)CD25(+)Foxp3(+)Treg level was negatively correlated with serum IL-33 level in the asthma group(r=-0.156, P<0.01).

CONCLUSIONSCD4(+)CD25(+)Foxp3(+)Treg may interact with IL-33 in the pathogenesis of childhood asthma.

Asthma ; etiology ; immunology ; Child ; Child, Preschool ; Female ; Forkhead Transcription Factors ; analysis ; Humans ; Infant ; Interleukin-2 Receptor alpha Subunit ; immunology ; Interleukin-33 ; Interleukins ; physiology ; Male ; T-Lymphocytes, Regulatory ; physiology

AIMTo investigate if interleukin-6 (IL-6), interleukin-8 (IL-8), interleukin-10 (IL-10), interferon-gamma (IFN-gamma) or tumor necrosis factor-alpha (TNF-alpha) are able to stimulate the level of lipid peroxidation of sperm membranes, either alone or in the presence of leukocytes.

METHODSSemen samples from normozoospermic donors were prepared by density gradient. The sperms were exposed to the indicated cytokines, at physiological and infection-inflammation concentrations, in the absence or presence of leukocytes. Lipid peroxidation of the sperm membranes was determined by measuring malondialdehyde (MDA) and 4-hydroxialkenals (HAE) formation.

RESULTSTNF-alpha, IL-8 and IFN-gamma increased the level of sperm membrane lipid peroxidation when tested at physiological concentrations. At infection-inflammation concentrations, only IL-8 was able to produce a higher effect. When assayed in the presence of leucocytes, IL-8 and TNF-alpha showed a higher effect at infection-inflammation concentrations than at physiological concentrations. Finally, IL-8 showed a higher effect in the presence of leukocytes than in their absence at both physiological and infection-inflammation concentrations. TNF-alpha also showed a higher effect when assayed in the presence of leukocytes than in their absence, but only at infection-inflammation concentrations. There was no effect of IL-6 or IL-10 in any of the tested conditions.

CONCLUSIONSeveral pro-inflammatory cytokines at physiological concentrations increase the level of lipid peroxidation of sperm membranes, which could be important for the sperm fecundation process. However, infection-inflammation concentrations of some cytokines, such as IL-8 and TNF-alpha, either alone or in the presence of leukocytes, could drive the lipid peroxidation of the spermatozoa plasma membrane to levels that can affect the sperm fertility capacity.

Cytokines ; analysis ; Humans ; Inflammation ; physiopathology ; Interferon-gamma ; metabolism ; Interleukins ; metabolism ; Leukocyte Count ; Lipid Peroxidation ; Male ; Reference Values ; Spermatozoa ; physiology ; Tumor Necrosis Factor-alpha ; metabolism