Interleukin 33 is induced by tumor necrosis factor alpha and interferon gamma in keratinocytes and contributes to allergic contact dermatitis.

BACKGROUND: Interleukin (IL) 33, a novel member of the IL-1 family, is produced mainly by epithelial cells and endothelial cells in response to various types of stress, including necrosis. The effects of IL-33 on the immune cells involved in allergic contact dermatitis have recently been revealed in vitro. However, in vivo, the induction mechanism and function of IL-33 are not fully understood. OBJECTIVES: Our objectives were to investigate induction of IL-33 in keratinocytes and to evaluate the functions of IL-33 and its inducers in a murine model of allergic contact dermatitis. MATERIAL AND METHODS: KERTr cells, a human keratinocyte cell line, were cultured with various cytokines, including tumor necrosis factor (TNF) alpha and interferon (IFN) gamma. IL-33 expression was detected using quantitative reverse transcriptase polymerase chain reaction, immunocytochemistry, and Western blotting. The functions of IL-33, TNF-a, and IFN-y in allergic contact dermatitis were evaluated using a murine model. RESULTS: TNF-alpha and IFN-gamma induced expression of IL-33 mRNA and protein in KERTr cells. Blockade of IL-33 attenuated swelling in the ears of the experimental mice. Similar effects were noted for blockade of TNF-alpha and IFN-gamma in these mice. CONCLUSIONS: TNF-alpha and IFN-gamma induce expression of IL-33, and IL-33 produced by keratinocytes contributes to allergic contact dermatitis. Blockade of IL-33, TNF-alpha, and IFN-gamma could represent novel and potent strategies to treat allergic contact dermatitis.

Age and sex dependent regulation of the factor IX gene in mice.

Plasma factor IX and liver factor IX mRNA levels in two normal mouse strains (B6D2F1 and BALB/CJNIA) were determined in relation to aging and sex of the animals. With male B6D2F1 mice, mean plasma factor IX activity levels for the 14 and 21 approximately 22 month-old animals were found to be 124% and 226%, respectively, of the 5 month-old group. Similarly, liver factor IX mRNA levels for the same age animal groups were 145% and 227%, respectively, of the reference group. Mean plasma factor IX levels for the same age female animals were 132% and 175%, respectively, and were accompanied by similarly elevated liver factor IX mRNA levels, 119 and 175%, respectively, of the 5 month-old female group. Factor IX activity and mRNA levels for the 5, 14 and 21 approximately 22 month-old female animal groups were lower than those of the corresponding male age groups by 25, 20 and 37%, and 20, 36 and 38%, respectively. With BALB/CJNIA mice, similar correlation was observed between the advancing age and substantial elevations in the factor IX mRNA level as well as on the unequal factor IX mRNA levels in females and males. These results indicate that the plasma factor IX level in both male and female mice is greatly elevated with aging, in general agreement with a similar phenomenon observed for human populations, and that this increase is due to a similar elevation in the factor IX mRNA level in the liver. In mice, both factor IX activity and mRNA levels are significantly higher in males than in females, which has not been described for humans.

Isolation and characterization of canine factor IX.

Canine plasma factor IX was purified to homogeneity by a combination of barium citrate precipitation and three-step column chromatographies of DEAE sepharose, heparin agarose and a monoclonal antifactor IX antibody-linked agarose. Canine factor IX has an apparent molecular size of 61 kDa, which is slightly smaller than that of human factor IX, as determined by denatured polyacrylamide gel electrophoresis. Its amino acid composition, amino-terminal and carboxyterminal amino acid sequences agreed well with those predicted from the reported cDNA. Unlike purified human factor IX, canine factor IX preparation often showed a discrete smaller molecular species (approximately 50 kDa) which was generated by a specific proteolytic cleavage between Arg310 and Val311. When purified canine factor IX was utilized as a standard for enzyme linked immunosorbent assay, the concentration of canine factor IX in the pooled normal dog plasma was determined to be 5.3 micrograms/ml with 11.2% carbohydrate content (or 4.7 micrograms/ml for its polypeptide chain moiety). Concentration of plasma factor IX antigen was measured in six severely affected, unrelated hemophilia B dogs. Four had factor IX antigen of less than 1% of the normal, and two had undetectable levels. The latter two had gross molecular abnormalities in their factor IX genes. Three obligate carrier females had variable but proportionately reduced factor IX antigen and factor IX coagulant activity levels. These results provide a quantitative method for measuring canine factor IX antigen which is a prerequisite for studying hemostasis and development of gene transfer approaches in the canine model of hemophilia B.

Identification of bilirubin UDP-GTs in the human alimentary tract in accordance with the gut as a putative metabolic organ.

The initial identification of traditionally hepatic enzymes expressed in the gut has led to the hypothesis that the gut may function as a metabolic organ. The UDP glucuronosyltransferases (UDP-GTs) play an important role as phase II metabolizing enzymes. Previously members of this family have been identified in the gut by non-isoform specific immunoreactivity, and a small amount of bilirubin glucuronosyltransferase activity was detected in the colon. Recent reports of gut transplantation to reverse the metabolic defect in Gunn rats raised further interest in the expression and distribution of human bilirubin (UDP-GTs (HUG Br 1 and HUG Br 2) in the human alimentary tract. The availability of molecular genetic probes for HUG Br 1 and HUG Br 2 permits the screening of the alimentary tract for the presence of isoform specific message. RNA samples extracted from pinch biopsy specimens of buccal mucosa, esophagus, stomach body, antrum, duodenum, and colon were analyzed for expression of HUG Br 1 and HUG Br 2. HUG Br 1 hybridization was detected in duodenum > colon, whereas HUG Br2 hybridization was detected in duodenum > esophagus > colon. Immunoreactivity data confirmed the presence of HUG Br 1 protein at low levels in the duodenum, whereas the less abundant HUG Br 2 protein was below the limits of detection of isoform specific anti-peptide antibodies. Bilirubin specific reactivity was demonstrated in duodenal samples but not antrum samples, consistent with the molecular genetic data. The presence of functional bilirubin UDP-GT isoforms in human alimentary tract supports the notion that the gut may function as a metabolic organ and may have diagnostic and therapeutic implications for disorders of bilirubin metabolism.

Age-related changes in the metabolism of neurotransmitters and the effect of scavengers: an in vivo microdialysis study.

Brain microdialysis is a method to study the in vivo release and metabolism of neurotransmitters using freely-moving animals. We studied the changes in neurotransmitter metabolism from very young to the old rats (Sprague-Dawley). For brain microdialysis, a guide cannula, was implanted in the striatum (A +1.0, L +2.5, V -4.0). Extracellular content of neurotransmitters and their metabolites were detected with an electrochemical detector (ECD-100, EICOM Co.). The content was affected by KCl (100 mM), calcium-free Ringer and tetrodotoxin in a perfusion medium. Dopamine and its metabolites such as dihydroxyphenylacetic acid and homovanillic acid were significantly changed with age. The content of these metabolites were maximal around 2-3 months of age. In the case of serotonin metabolism, statisticaly no significant change was observed in the extracellular content of 5-hydroxyindoleacetic acid after sexual maturation (1.5 months of age). Potassium-induced dopamine release which is newly synthesized one, was maximum around 1.5 years of age and the content was about 50% lower in the old. The basal level of dopamine significantly decreased with age. Moreover, the inhibitory effect of pargyline, a monoamine oxidase inhibitor, was also decreased with age. On the basis of these results, it is suggested that the synaptic function shows postnatal development and reaches maximum at sexual maturation. Microdialysis could be a useful procedure for studying the in vivo metabolism of neurotransmitters.

Suppression of Plasma Cholesterol Elevation by Okara Tempe in Rats.

Okara Tempe (OT), an Indonesian fermented traditional food, which is the fermented okara (insoluble residues of homogenized soybean, OC) by Rhizopus oligosporus, and which is interested in as a new high fiber and low energy soybean foodstuff. In this study, the effects of protein and dietary fiber (DF) of OT on sterol metabolism in rats were investigated. When rats were fed by OT or casein as protein source, the cholesterol and bile acid levels in plasma was significantly lower in OT fed group than in casein fed group. When rats were fed by OT or OC as DF source, in OT fed group, the cholesterol and bile acid levels in plasma was similar to them in OC fed group, but the cholesterol level in liver was lower than it in OC fed group, and the excretion of cholesterol and bile acid in feces was larger than in OC fed group. These results suggest that the cholesterol lowering action of OT in plasma would arise from the complementary action of protein and water soluble DF in OT.

Retrovirus-mediated expression of HUG Br1 in Crigler-Najjar syndrome type I human fibroblasts and correction of the genetic defect in Gunn rat hepatocytes.

Crigler-Najjar syndrome type I (CN-I) is a congenital hepatic metabolic deficiency in bilirubin UDP-glucuronosyltransferase activity which leads to profound jaundice and death from kernicterus. UGT1, the gene locus coding for multiple glucuronosyltransferase isoforms, has been well characterized and the cDNA for the most active form, HUG Br1, has been cloned. Recent advances in liver directed gene transfer suggest that this disease could be treated through gene therapy. As an initial step to correct the genetic defect in Crigler-Najjar type I, recombinant retroviruses were used to transduce an HUG Br1 gene into hepatocytes of a rat model of CN-I and CN-I fibroblasts. The retroviral vector gagCMVBA HUG Br1 was constructed and helper-free amphotrophic virus was isolated and used to transfer bilirubin UDP-glucuronosyltransferase activity to genetically deficient cells. The efficiency of transduction as measured by Southern blot analysis of integrated proviral sequences in DNA of recipient cells ranged from 5 to 100%. HUG Br1 gene expression was documented by blot hybridization analysis of total cellular RNA, immunotransblot analysis using a rabbit polyclonal antipeptide HUG Br1 antibody, and lysate enzymatic assay of bilirubin UDP-glucuronosyltransferase activity. HUG Br1 gene transfer was definitively demonstrated by four independent modalities following HUG Br1 retroviral transduction.