Block of postjunctional muscle-type acetylcholine receptors in vivo causes train-of-four fade in mice.

BACKGROUND: Train-of-four (TOF) fade during nerve-mediated muscle contraction is postulated to be attributable to inhibition of prejunctional nicotinic α3ß2 acetylcholine receptors (nAChRs), while decrease of twitch tension is attributable to block of postjunctional muscle nAChRs. The validity of these presumptions was tested using specific prejunctional and postjunctional nAChR antagonists, testing the hypothesis that fade is not always a prejunctional phenomenon. METHODS: Pentobarbital anaesthetized mice had TOF fade measured after administration of: either 0.9% saline; the prejunctional α3ß2 nAChR antagonist, dihydro-ß-erythroidine (DHßE); the postjunctional nAChR antagonists, α-bungarotoxin (α-BTX) or α-conotoxin GI; and a combination of α-BTX and DHßE; or a combination of α-conotoxin GI and DHßE. RESULTS: Saline caused no neuromuscular changes. Administration of muscle nAChR antagonists, α-BTX or α-conotoxin GI caused significant decrease of twitch tension and TOF fade compared with baseline (P<0.01). DHßE alone caused no change of twitch tension or fade even after 90 min, but its coadministration with α-BTX or α-conotoxin GI significantly accelerated the onset of paralysis and degree of fade compared with α-BTX or α-conotoxin GI alone (P<0.01). CONCLUSIONS: Occupation of postjunctional nAChRs alone by α-BTX or α-conotoxin GI causes fade. As the prejunctional effects of DHßE on fade became manifest only when co-administered with α-BTX or α-conotoxin GI, specific inhibition of prejunctional nAChR alone is not necessary and sufficient to cause fade. Fade observed during repetitive nerve stimulation can be because of block of either postjunctional nAChRs alone, or block of prejunctional and postjunctional nAChRs together.

Anti-arthritic properties of FK506 on collagen-induced arthritis in rats.

OBJECTIVE AND DESIGN: To determine the effect of FK506 (tacrolimus) on paw inflammation, TNF-alpha expression in joint, and bone and cartilage destruction in type II collagen-induced arthritis (CIA) model in rats. METHODS: CIA was induced by immunization of female Lewis rats with an emulsion of bovine type II collagen and incomplete Freund's adjuvant. Paw inflammation was assessed by the increase in paw volume. Tumor necrosis factor (TNF) -alpha expression in hind knee joint was assessed by immunohistochemical analysis. Lesions of bone and cartilage were assessed on the basis of histological change in knee joint, radiographic analysis in hind paw, bone mineral density in femora and proteoglycan contents in the cartilage of femoral heads. FK506 at doses of 1, 1.8 and 3.2 mg/kg or its placebo formulation was orally administered to rats for 28 days from the day after immunization (n = 10). Effect of FK506 was compared with that of vehicle (distilled water). RESULTS: FK506 at a dose of 1.8 mg/kg significantly suppressed paw swelling (p < 0.01) and histological change in knee joint (p < 0.05). Tumor necrosis factor (TNF)-alpha was mainly expressed in the region with a marked infiltration of inflammatory cells in the hind knee joint. FK506 (3.2 mg/kg) markedly reduced TNF-alpha expression. FK506 at a dose of 1.8 mg/kg suppressed radiographic changes in hind paw (p < 0.05) and also recovered the decrease in bone mineral density in the femora (p < 0.05). Proteoglycan contents in the cartilage of femoral heads were determined to evaluate the cartilage destruction more quantitatively and found to significantly decrease in CIA rats. FK506 at a dose of 1.8 mg/kg recovered the loss of proteoglycan contents (p < 0.01). CONCLUSION: These results show that FK506 is effective in suppressing inflammation, TNF-alpha expression in joint, and damage to bone and cartilage in rat CIA, and may be useful in the treatment of rheumatoid arthritis.

Atopic dermatitis-like skin lesions induced by topical application of mite antigens in NC/Nga mice.

BACKGROUND: Atopic dermatitis (AD) is a chronic relapsing inflammation usually observed in patients with an individual or a familial history of atopic diseases, precipitated by environmental factors including mite antigens (Ag). However, the exact etiology of AD is unclear. To further explore the pathogenesis and treatment of AD, a suitable animal model is necessary. In this study, we developed a new animal model of AD induced by mite Ag in NC/Nga mice. METHODS: We injected the extracts of mite Ag intradermally at the ventral side of the ear of SPF NC/Nga mice on days 0, 2, 4, 7, 9, 11, 14 and 16, and measured the clinical symptoms and the ear thickness. On day 18, we collected blood and submandibular lymph nodes (LN) of the immunized ear to perform a histochemical analysis, and to measure the plasma immunoglobulins and cytokines. RESULTS: The NC/Nga mice immunized with mite Ag suffered from AD-like skin lesions including erythema followed by edema, excoriation and scaling. The histological and immunohistochemical examinations of the affected skin showed epidermal hyperplasia with hyperkeratosis, severe infiltration of CD4+ T lymphocytes, eosinophils and macrophages, and degranulation of mast cells. The total plasma IgE level was markedly elevated in mite Ag-treated mice. LN cells of mice immunized with mite Ag synthesized IgE in an Ag-dependent manner and secreted interleukin-4 (IL-4) and IL-5 but not interferon-gamma. CONCLUSIONS: NC/Nga mice treated with mite Ag manifest clinical and immunological aspects similar to patients with AD, suggesting that this model is suitable for exploring the pathogenesis of human AD.

Tacrolimus suppressed the production of cytokines involved in atopic dermatitis by direct stimulation of human PBMC system. (Comparison with steroids).

Tacrolimus (FK506) ointment showed remarkable efficacy against atopic dermatitis in animal models and clinical trials. The suppressive effect of tacrolimus on the production of the cytokines involved in atopic dermatitis (IL-2, IL-3, IL-4, IL-5, IFN-gamma and GM-CSF) from human peripheral blood mononuclear cells (PBMC) was investigated. We constructed a new cytokine production system in which T cells are activated by direct stimulation in vitro with anti-CD3/CD2 or anti-CD3/CD28 antibody combination. Tacrolimus inhibited the production of these cytokines by both stimulations. In a comparative study with steroids (alclometasone dipropionate and betamethason valerate) in anti-CD3/CD2 system, tacrolimus and both steroids inhibited Th1 cytokines (IL-2, IFN-gamma), Th2 cytokines (IL-4, IL-5) and IL-3, GM-CSF (produced by both Th1 and Th2). The suppressive effect of tacrolimus on cytokine production was stronger than that of alclometasone dipropionate and equal to or stronger than that of betamethason valerate. The effective dose of tacrolimus (IC50, 0.02-0.11 ng/ml) is almost the same as for Th1 and Th2 cytokines, and 1 ng/ml of tacrolimus suppressed all cytokines completely. These results suggest that tacrolimus suppresses the allergic cytokines from T cells, and that tacrolimus ointment is effective against atopic dermatitis through the inhibition of cytokine production.

FK506 suppresses neutrophil chemoattractant production by peripheral blood mononuclear cells.

To understand the mechanism of action of FK506 (Tacrolimus) on neutrophil chemotaxis, we examined its effect on human neutrophil chemotaxis and neutrophil chemoattractant production by peripheral blood mononuclear cells. FK506 and cyclosporin A had no direct suppressive effect on neutrophil chemotaxis induced by interleukin-8, leukotriene B(4), complement 5a (C5a), zymosan-activated serum and formyl-Met-Leu-Phe (fMLP). FK506 and cyclosporin A only slightly suppressed the chemotactic activity of platelet-activating factor (PAF). Dexamethasone did not inhibit the chemotactic activity of any chemoattractant. The supernatant of peripheral blood mononuclear cells stimulated with anti-CD3 and CD2 antibodies induced neutrophil chemotaxis. FK506 and cyclosporin A suppressed the chemotactic activity of the supernatant in parallel to the suppression of interleukin-8 production by peripheral blood mononuclear cells. Anti-interleukin-8 antibody completely suppressed the chemotactic activity of the supernatant without drugs. These studies indicate that FK506 may exert a beneficial effect on human inflammatory diseases by suppressing neutrophil chemotaxis secondary to inhibition of chemoattractant (for example, interleukin-8) production by leukocytes.

FK506 potently inhibits T cell activation induced TNF-alpha and IL-1beta production in vitro by human peripheral blood mononuclear cells.

The aim of this study was to elucidate the in vitro inhibitory potency of FK506 on production of the inflammatory cytokines, tumour necrosis factor (TNF)-alpha and interleukin (IL)-1beta, with a view to assessing this immunosuppressive agent as a potential anti-rheumatic drug. We employed an in vitro model which produces TNF-alpha and IL-1beta through T cell activation. Human peripheral blood mononuclear cells (PBMC) were cultured with immobilized anti-CD3/CD28 monoclonal antibody in this model. FK506 inhibited anti-CD3/CD28 induced TNF-alpha and IL-1beta production at concentrations less than 1 ng ml(-1). Flow cytometric analysis of intracellular TNF-alpha and IL-1beta positive cells showed that FK506 potently suppresses inflammatory cytokine production from CD14+ monocytes as well as from T cells. Cyclosporin A (CsA) and dexamethasone (DEX) also inhibited the anti-CD3/CD28 induced cytokine production, but were less potent than FK506. FK506 and CsA, but not DEX, specifically inhibited anti-CD3/CD28 induced inflammatory cytokine production without affecting the lipopolysaccaride (LPS) induced effect. Methotrexate (MTX) was completely inactive for suppressing cytokine production under either condition. Anti-CD3/CD28 stimulated PBMC culture supernatants were found to enhance the expression of adhesion molecules in human vascular endothelial cells. FK506, CsA and DEX led to the suppression of adhesion molecule expression probably by inhibiting cytokine production from PBMC. The inhibitory potency of agents on TNF-alpha and IL-1beta production was compared with cytotoxicity and FK506 was not cytotoxic at concentrations several orders of magnitude greater than those required for cytokine inhibition. These results strongly suggest that FK506 may be most effective to specifically prevent T cell activation mediated inflammatory cytokine production in a clinical setting.

Cloning and sequencing of cDNA encoding NG,NG-dimethylarginine dimethylaminohydrolase from rat kidney.

Three cDNA clones encoding NG,NG-dimethylarginine dimethylamino-hydrolase (EC 3.5.3.18) have been isolated from a rat kidney lambda gt11 library using a probe prepared by PCR on the basis of partially determined amino-acid sequences of the enzyme. The sequence analyses of the cDNAs established that the 3008 bp cDNA contains 855 bp open reading frame encoding 285 amino acids (M(r) 31414), 3'-flanking region (1722 bp) and 5'-flanking region (431 bp). The amino-acid sequences of all the peptides isolated from the purified enzyme were shown to be included in the deduced amino-acid sequence. Northern blotting analyses showed that the mRNA (4 kb) is ubiquitously expressed in various rat tissues.

Study of cytokines in ulcerative colitis.

We investigated the lymphocyte-activation antigens and the expression of cytokine genes in the mucosa of ulcerative colitis (UC). Fresh colonic mucosal biopsy specimens from patients with UC and controls were fixed for the immunohistochemical study of CD4, HLA-DR, and CD25, and other specimens were prepared for the RNA analysis of cytokines. Gene expression was evaluated by the reverse transcription-polymerase chain reaction, and the radioactivity of dot-blotted amplified cDNA was standardized by co-amplified beta-actin cDNA. The inflamed mucosa of active UC showed increased CD4+DR+ and CD25+ cells in comparison with control subjects. Active UC showed significantly increased mRNA expression of IL-1 beta, IL-2R alpha, IL-6, IL-8, and TNF alpha compared with the controls. We found no significant difference in the mRNA expression for IL-2, IL-4, IL-10, and IFN-gamma between active UC and controls. Increased CD4+DR+ and CD25+ cells in active UC mucosa indicate mucosal CD4(+) T cell activation in the lamina propria, but we did not clarify Th1 or Th2 specific T cell activation from our study of cytokine mRNA expression. The increased mRNA expression for IL-1 beta, IL-6, and TNF alpha in the mucosal lesions of UC indicates that these inflammatory cytokines may play important roles in the pathogenesis of UC.

Production of functional chick liver HMG 2a protein in Escherichia coli.

An efficient Escherichia coli system for the production of a variant form of high-mobility group-2a protein (HMG 2a), having the additional 5 amino acid residues (Ala-Pro-Thr-Leu-Glu) at the NH2-terminal, has been constructed. cDNA encoding HMG 2a was ligated with the Omp A signal peptide sequence and was inserted into an inducible bacterial expression vector pSH-L. After the plasmid introduced into E. coli was expressed by temperature shift, the recombinant product was purified by trichloacetic acid precipitation followed by Bio-Rex 70 column chromatography. The purified product showed the expected NH2-terminal sequence and the superhelical activity of circular DNA similar to the authentic HMG 2a isolated from chick liver.

Activated CD4+ and CD8+ cells in the colonic mucosa of ulcerative colitis patients: their relationship to HLA-DR antigen expression on the colonic epithelium and serum soluble CD25 levels.

This study was performed to clarify the relationship between activated (HLA-DR-expressing) CD4+ and CD8+ cells in the colonic lamina propria of ulcerative colitis and other immunological factors, i.e., epithelial DR expression, serum soluble CD25 levels, and colonic mucosal CD25+ cells. The frequency of epithelial DR expression was positively correlated with the numbers of CD4+ and CD8+ cells. The percentages activated CD4+/CD4+ cells were higher in mucosae with DR- epithelium than in mucosae with DR+ epithelium. The serum soluble CD25 levels were increased in ulcerative colitis, and there was an inverse correlation between these levels and the relative number of activated CD4+ cells in untreated active disease. These results suggest that interactions among mucosal CD4+ cells, colonic epithelium, and serum soluble CD25 might play an important role in the pathogenesis of ulcerative colitis.

Activated immunocompetent cells in rat colitis mucosa induced by dextran sulfate sodium and not complete but partial suppression of colitis by FK506.

Administration of dextran sulfate sodium (DSS) solutions to rats induced colitis which resembled mucosal lesions of human ulcerative colitis. Recent reports have shown that some cytokines are related to the pathogenesis of ulcerative colitis. In the present report, we describe the production of two cytokines in colitis mucosa in this DSS model. Using a cytotoxicity assay and a radioimmunoassay, we observed significant increases in levels of tumor necrosis factor-alpha (TNF-alpha) in the colitis mucosa and detected interleukin-1 alpha in the mucosa of 3 of 5 DSS rats and an increase in TNF-alpha had a tendency to be inhibited by treatment with FK506. Immunohistochemical investigation of DSS mucosa showed that the number of activated T cells increased at the earlier phase of inflammation. Luminol-dependent chemiluminescence values and myeloperoxidase activities were increased in the late phase of colitis and were suppressed by the FK506 treatment. These findings may support the role of TNF-alpha and T-cell activation in the pathogenesis of DSS colitis.

Developmental changes in the expression of HMG 2a protein.

The levels of HMG 2a chromosomal protein and its mRNA change during the post-hatched development of chicks were investigated. The contents of both HMG 2a and 2b proteins of liver, heart, brain, muscle and gizzard were abundant in the newly hatched chicks but their contents decreased significantly in those tissues of the 70-day-old chicks. The HMG 2a mRNA levels of liver, heart and brain in 70-day-old chick decreased to about 40% of those mRNA in the newly hatched chicks while the HMG 2a mRNA levels of muscle and gizzard in the 70-day-old chicks increased 5- and 3-fold, respectively. These results suggest that the decrease in the HMG 2a protein contents of the muscle and gizzard in the 70-day-old chicks may be largely due to the stimulation of HMG 2a protein degradation or the reduction of HMG 2a mRNA translation.

Molecular cloning of chick liver HMG 2a cDNA and developmental expression of HMG 2a mRNA.

The cDNA clone encoding HMG 2a of chick liver was isolated from a lambda gt11 expression library using polyclonal antibodies. DNA sequence analysis revealed an open reading frame of 201 amino acids. Comparison of the nucleotide sequences of cDNA coding for chick liver HMG 2a with pig thymus HMG 2 and human monocytic leukemia cell HMG 2 showed 70% homology. 2.0 kb and 1.2 kb mRNAs were found in newly hatched chick liver and decreased during postnatal development of chicks.