To investigate if nuclear factor-kappa B (NF-kappa B) p65 antisense oligonucleotides might affect the expression of NF-kappa B p65 and cytokines in lamina propria mononuclear cells(LPMC) from patients with ulcerative colitis (UC). LPMC were isolated from intestinal mucosal biopsy specimens from 3 patients with UC, and cultured with or without NF-kappa B p65 antisense oligonucleotides (5'-GGAACAGTTCGTCCTATGG-3'), missense oligonucleotides (5'-GGAACAGTTCGTCTATGG-3') and dexamethasone. NF-kappa B p65 expression was determined by western blot analysis. The expression of cytokine mRNA was studied by reversal transcription-polymerase chain reaction (RT-PCR). The cytokine levels were measured by enzyme linked immunosorbent assay. The results showed that NF-kappa B p65 antisense oligonucleotides resulted in down-regulation of NF-kappa B p65 expression, blocked the expression of IL-1 beta mRNA and IL-8 mRNA, and strikingly reduced the production of IL-1 beta and IL-8, and these effects were greater than those of dexamethasone in cultured LPMC from patients with UC(P < 0.05). Therefore, the application of NF-kappa B p65 antisense oligonucleotides may serve as a novel molecular approach for the treatment of patients with UC.
Cells, Cultured ; Colitis, Ulcerative ; drug therapy ; pathology ; Cytokines ; biosynthesis ; genetics ; Humans ; Interleukin-1 ; biosynthesis ; genetics ; Interleukin-8 ; biosynthesis ; genetics ; Intestinal Mucosa ; cytology ; Monocytes ; drug effects ; metabolism ; NF-kappa B ; biosynthesis ; genetics ; Oligonucleotides, Antisense ; pharmacology ; RNA, Messenger ; biosynthesis

Interleukin-1 receptor antagonist (IL-1ra), a member of IL-1 family, is a naturally occurring IL-1 inhibitor as "receptor antagonist", which blocks biological responses mediated by IL-1. Recombinant human IL-1ra (rhIL-1ra, Kineret) was introduced in clinical trials involving patients with RA. Between 2001 to approximately 2002, rhIL-1 ra was approved by the US Food and Drug Administration and the European Agency for the Evaluation of Medicine Procedure. Unfortunately, 10,000 to 100,000-fold excess amounts of IL-1ra are needed to relieve disease because minimal IL-1 can induce complete biological responses, and the dosage of 100 to approximately 150mg/day in a RA patient is so big that it greatly influence patients' physical, psychological and economical situation. In this study, IL-1ra mutants were established by site-specific mutagenesis to improve its stability. The sites of mutagenesis included R6 K7-AA,R93 K94-AA and K97 R98-AA. IL-1ra and its mutants were expressed in E. coli BL21 (DE3) using pTIG-Trx expressing system with the induction of IPTG. The recombinant proteins were purified by Ni2+ chelate chromatography and Sephadex G75 gel filtration chromatography. The activity of mutants is as high as IL-1ra. We characterized the pharmacokinetic profile of IL-1ra and its mutants. The third mutant's half life is 2.26 times than wt IL-1ra. The study has provided some approaches and experience for further research to improve the metabolism stability of IL-1ra.
Animals ; Escherichia coli ; genetics ; metabolism ; Female ; Humans ; Interleukin 1 Receptor Antagonist Protein ; biosynthesis ; genetics ; pharmacokinetics ; Mutagenesis, Site-Directed ; methods ; Mutant Proteins ; biosynthesis ; pharmacokinetics ; Recombinant Proteins ; biosynthesis ; genetics ; pharmacokinetics

Animals ; Interleukin-1 ; biosynthesis ; genetics ; Liver ; blood supply ; Lung ; metabolism ; Male ; RNA, Messenger ; biosynthesis ; genetics ; Rats ; Rats, Wistar ; Reperfusion Injury ; metabolism ; Tumor Necrosis Factor-alpha ; biosynthesis ; genetics

OBJECTIVETo investigate the neuro-immune regulatory mechanism of Heart Benefiting recipe (HBR), an effective recipe for treatment of Alzheimer's disease (AD).

METHODSUsing immunohistochemical and RT-PCR methods, the neuro-immunological pathological changes in the AD rat model induced by beta-amyloid protein (A beta1-40) via lateral cerebral ventricle injection, including mainly the glial fibrillary acidic protein expression and inflammatory cytokines IL-1beta, IL-6mRNA and beta-amyloid protein precursor (APPmRNA) gene expression were studied. And the effects of HBR on these parameters were observed.

RESULTSDeposition of A beta in cerebral tissue could induce activation of stellate glial cells and abnormal increased levels of inflammatory cytokines (IL-1beta and IL-6mRNA), also the elevation of APPmRNA level. HBR could effectively control the above-mentioned pathological changes.

CONCLUSIONHBR could effectively control the inflammation and the A beta immune cascade reaction in brain of AD patients, it is one of the important therapeutic mechanisms of the recipe.

Alzheimer Disease ; chemically induced ; metabolism ; Amyloid beta-Peptides ; Amyloid beta-Protein Precursor ; biosynthesis ; genetics ; Animals ; Brain ; metabolism ; Drugs, Chinese Herbal ; pharmacology ; Interleukin-1 ; biosynthesis ; genetics ; Interleukin-6 ; biosynthesis ; genetics ; Male ; Neuroprotective Agents ; pharmacology ; RNA, Messenger ; biosynthesis ; genetics ; Random Allocation ; Rats

OBJECTIVEThis study aimed at investigating effects of exogenous interleukine-10 (IL-10) on IL-6 and intercellular adhesion molecular (ICAM-1) expression in inflamed gingival tissue.

METHODSThe expression of IL-6 and ICAM-1 was examined using immunohistochemical techniques. Inflammatory gingival tissue treated with IL-10 was taken as the experimental group and the same patient's inflammatory gingival tissue without treatment of IL-10 was included into the control group.

RESULTSIL-6 expression was found mainly in monocytes, macrophages, lymphocytes, endotheliocytes and fibroblasts. The expression of ICAM-1 was found mainly in epithelial cells, monocot-macrophages, lymphocytes, endotheliocytes and fibroblasts. The immunohistochemical optical density (IOD) of the expression of IL-6 and ICAM-1 was detected by using Image-Proplus software, and the results showed that the expression in the experimental group differed significantly from that in the control group.

CONCLUSIONThe exogenous IL-10 may down-regulate IL-6 and ICAM-1 expression in inflammatory gingival tissue.

Adult ; Down-Regulation ; Female ; Gingiva ; metabolism ; Gingivitis ; metabolism ; Humans ; Intercellular Adhesion Molecule-1 ; biosynthesis ; genetics ; Interleukin-10 ; pharmacology ; Interleukin-6 ; biosynthesis ; genetics ; Male ; Middle Aged

OBJECTIVETo investigate the influence of baicalin on the IL-1beta induced pro-MMP-1 in HGF and the effects of baicalin on MMP-3 expression in periodontal ligament cells (PDLCs).

METHODSThe amount of secreted pro-MMP-1 and MMP-3 expression was detected by ELISA and cell immunochemistry.

RESULTS(1) The amount of secreted pro-MMP-1 (3.333 +/- 0.123) microg/L increased significantly following 1 microg/L of IL-1beta, compared with control group (1.960 +/- 0.180) microg/L. Addition of baicalin to cell culture medium for 1 hour following IL-1beta decreased pro-MMP-1 secretion in a dose-dependent manner in the range of 10 approximately 1,000 microg/L. (2) 1 microg/L IL-1beta could significantly stimulate the synthesis and secretion of MMP-3 in PDLCs. (3) The baicalin could not interfere the synthesis of MMP-3, but could inhibit the release of MMP-3 from PDLCs.

CONCLUSIONSBaicalin could inhibit the secretion of pro-MMP-1 and MMP-3 expression in IL-1beta induced HGF and PDLCs, which suggests that baicalin may play an important role in preventing and treating periodontal disease.

Collagenases ; biosynthesis ; genetics ; Enzyme Precursors ; biosynthesis ; genetics ; Fibroblasts ; enzymology ; pathology ; Flavonoids ; pharmacology ; Gingiva ; enzymology ; pathology ; Humans ; Interleukin-1 ; pharmacology ; Interleukin-1beta ; Matrix Metalloproteinase 1 ; Metalloendopeptidases ; biosynthesis ; genetics ; Peptide Fragments ; pharmacology ; Periodontal Ligament ; enzymology ; pathology ; Periodontitis ; enzymology ; pathology ; Scutellaria ; chemistry

The aims of this study were to investigate the relationships between the production of interleukin-1 (IL-1), and IL-6 system by whole blood cells, and bone mineral density (BMD), and polymorphisms in IL-1 system and IL-6 gene in postmenopausal Korean women. The production of IL-1alpha, IL-1beta, IL-1 receptor antagonist (IL-1ra), IL-6, and soluble IL-6 receptor (sIL-6r) by lipopolysaccharide-stimulated whole blood cells was measured by ELISA in 110 subjects. Serum osteocalcin, C-telopeptide of type I collagen, and BMD at lumbar spine and proximal femur were measured. IL-1alphaC(-889)T polymorphism, IL-1beta C(-511)T polymorphism, 86-base pair variable number tandem repeat polymorphism in the IL-1ra gene, and IL-6 C(-634)G polymorphism were analyzed. The production of IL-1beta correlated positively with BMD at femoral neck, whereas the production of other ILs did not correlate with BMD at the skeletal sites examined. No significant differences in the production of ILs were observed among normal, osteopenic and osteoporotic postmenopausal women, and among the different IL system polymorphisms groups studied. No correlation between bone turnover markers and the production of ILs was noted. In conclusion IL-1beta may regulate bone metabolism at femoral neck, and the IL system polymorphism do not affect the production of ILs by whole blood cells.
Aged ; Blood Cells/drug effects/immunology ; Bone Density/*genetics/*immunology ; Bone Diseases, Metabolic/blood/genetics/immunology ; Cytokines/*biosynthesis/blood ; Female ; Humans ; In Vitro ; Interleukin-1/biosynthesis/blood/genetics ; Interleukin-6/biosynthesis/blood/genetics ; Interleukins/*genetics ; Lipopolysaccharides/pharmacology ; Middle Aged ; Osteoporosis, Postmenopausal/blood/genetics/immunology ; *Polymorphism, Genetic ; Receptors, Interleukin-6/biosynthesis/blood/genetics ; Research Support, Non-U.S. Gov't ; Sialoglycoproteins/biosynthesis/blood/genetics

Recombinant human interleukin-1 receptor antagonist was expressed in E. coli as an insoluble inclusion body. The inclusion body was dissolved in the 8 M urea and then the solution was diluted untill the concentration of urea became 2 M. By ion exchange chromatography the protein in the solution of 2 M urea was refolded and purified. At last the purity of product is more than 95% and its bioactivity is more than 1 x 10(5) IU/mg while it has little endotoxin. Western-Blotting also indicates that recombinant protein can react with antibodies against anti-hIL-1ra.
Escherichia coli ; genetics ; metabolism ; Humans ; Inclusion Bodies ; metabolism ; Interleukin 1 Receptor Antagonist Protein ; biosynthesis ; genetics ; Protein Folding ; Recombinant Proteins ; biosynthesis ; genetics ; isolation & purification

OBJECTIVETo observe the role of Kupffer cells in the postburn production of TNFalpha, IL-1beta and IL-6 in severely scalded rats.

METHODS(1) The production of TNFalpha, IL-1beta and IL-6 from rat Kupffer cells stimulated by burn serum was observed. (2) The postburn change in the expression of cytokine mRNA from rat Kupffer cells was monitored. (3) The change in the plasma cytokine contents in scalded rats was determined after the application of gadolinium chloride, a specific inhibitor of Kupffer cells.

RESULTSKupffer cells could be stimulated by burn serum to release cytokines TNFalpha, IL-1beta and IL-6. The mRNA expression of TNFalpha, IL-1beta and IL-6 from rat Kupffer cells increased significantly after injury. But the postburn plasma levels of TNFalpha, IL-1beta and IL-6 decreased obviously to 34.71%, 36.99% and 33.7% of those in scalding group, respectively, after the Kupffer cell activity was inhibited.

CONCLUSIONThe plasma cytokines, i.e. TNFalpha, IL-1beta and IL-6, were primarily produced from Kupffer cells after injury in scalded rats, initiated by TNFalpha, IL-1beta and IL-6 mRNA transcription.

Animals ; Burns ; immunology ; metabolism ; Gadolinium ; pharmacology ; Interleukin-1 ; biosynthesis ; genetics ; Interleukin-6 ; biosynthesis ; genetics ; Kupffer Cells ; physiology ; RNA, Messenger ; analysis ; Rats ; Rats, Sprague-Dawley ; Tumor Necrosis Factor-alpha ; biosynthesis ; genetics

OBJECTIVETo investigate the causes and influencing factors of heterogeneity of HSA/IL1ra fusion protein expression in Pichia pastoris.

METHODSThe heterogeneity of HSA/IL1ra fusion protein expressed in Pichia pastoris was studied by removing glycosylation and inhibiting glycosylation, as well as by different ways of fusion, different clones, and different expression host.

RESULTGlycosylation caused expression heterogeneity of fusion protein, but in SMD1168 and some GS115 clones there was no this phenomenon.

CONCLUSIONThe expression heterogeneity of HSA/IL1ra fusion protein in Pichia pastoris is due to the glycosylation, and different ways of fusion, different clones, different expression host also have some impact.

Escherichia coli ; genetics ; metabolism ; Genetic Heterogeneity ; Genetic Vectors ; genetics ; Humans ; Interleukin 1 Receptor Antagonist Protein ; biosynthesis ; genetics ; Pichia ; genetics ; metabolism ; Recombinant Fusion Proteins ; biosynthesis ; genetics ; Serum Albumin ; biosynthesis ; genetics