Within the nuclear receptor superfamily, Nur77, Nurr1, and NOR1 constitute the nuclear receptor subfamily 4 group A. Modulation of NOR1 function would be therapeutic potential for diseases related to dysfunction of NOR1, including extraskeletal myxoid chondrosarcoma and autoimmune diseases. By screening arachidonate metabolites for their capacity of transcriptional activation, we have identified prostaglandin (PG) A 2 as a transactivator for NOR1. PGA 2 acted as a potent activator of NOR1-dependent transcription through the GAL4-based reporter system. The putative ligand-binding domain (LBD) of the receptor directly bound PGA 2 , and LBD-deleted receptor showed little transcriptional activation by PGA 2 . Primary cultured spleen cells derived from transgenic mice overexpressing NOR1, showed higher sensitivity to PGA 2 compared to those from wild-type mice. These observations suggest that PGA 2 can serve as a transactivator of NOR1, and thus suggest a possibility of pharmacological modulation of the NOR1 pathways by PGA 2 -related compounds.

SUMMARYInvestigation of differentially expressed genes in eosinophils of patients with allergic diseases such as atopic dermatitis (AD) will provide important information for elucidating possible mechanisms of pathology. To identify novel genes that are expressed in AD, we compared gene expression in samples of peripheral blood eosinophils from AD patients and healthy volunteers. RNA was extracted from peripheral blood eosinophils. The expression of various genes, such as those for cytokine receptors, eosinophil activation marker, platelet activating factor (PAF) receptor, eosinophil-specific granular proteins and apoptosis-related genes, was confirmed using real-time reverse transcription-polymerase chain reaction (RT-PCR). Peripheral blood eosinophils of healthy volunteers were also isolated and stimulated for introduction of various cytokines. RNA was extracted and gene expression was monitored. Several genes, such as those for cytokine receptors (granulocyte-macrophage colony-stimulating factor (GM-CSF) receptor a and b chain and interleukin (IL)-3 receptor a chain), CD44 and PAF receptor were expressed at significantly higher levels in AD patients than in healthy volunteers. In addition, the anti-apoptotic genes, bcl-2 and bcl-xL, were expressed at increased levels in AD patients. No single gene expression correlated with clinical markers, such as eosinophil count or IgE levels. Expression of GM-CSF receptor b chain and IL-3 receptor a chain in isolated blood eosinophils of healthy volunteers was stimulated by IL-5, IL-4, interferon (IFN)-g and GM-CSF. Expression of bcl-2 and bcl-xL was also increased after stimulation with IL-5, IL-4 or IFN-g . The in vitro enhancement of cytokine-stimulated gene expression correlated well with the enhancement observed in clinical samples of eosinophils, suggesting that cytokines may affect gene expression in vivo in eosinophils of patients with AD.

To clarify the relation between the expression of genes such as eosinophil-specific granular proteins and cytokine receptors and the pathogenesis of allergic disease, cord blood-derived CD34+ cells were cultured and differentiated into eosinophils. Gene expression in the cells during the differentiation was determined by real-time reverse transcription PCR (ABI PRISM 7700). CD34+ mononuclear cells cultured with stem cell factor, granulocyte-macrophage colony-stimulating factor (GM-CSF), IL-3, and IL-5 in Iscove’s MEM, and proliferated until the 2nd week, when the cell number reached a plateau. Under these conditions, more than 90% of the cells differentiate into mature eosinophils in 3 weeks. The expression of major basic protein and eosinophil-derived neurotoxin in the treated cells increased until week 2 and decreased between week 2 and 3. However, the expression of membrane receptor genes, such as IL-5 receptor (α chain), IL-3 receptor (α chain), GM-CSF-α receptor, GM-CSF-β receptor, CC chemokine receptor 3, interferon-γ receptor, platelet-activating factor receptor and leukotriene D₄ receptor, increased until the 3rd week of eosinophil maturation. Our study suggests that the in vitro eosinophil differentiation and maturation model is useful for clarifying the relation between eosinophil-specific gene expression during allergic diseases and the progression of the disease.

Hepatitis B virus (HBV) reactivation in hepatitis B surface antigen (HBsAg)-negative patients following treatment with rituximab has been reported increasingly. The aim of this study was to investigate the molecular mechanisms underlying HBV reactivation in an HBsAg-negative patient. HBV was reactivated in a 75-year-old man following chemotherapy with rituximab, without elevation of HBsAg. The patient's full-length HBV genome was cloned and the entire sequence was determined. Transfection studies were performed in vitro using recombinant wild-type HBV (wild-type), the patient's HBV (patient), and two chimeric HBV constructs, in which the preS/S region of the patient and wild-type virus had been exchanged with one another. Secreted HBsAg and intra- and extra-cellular HBV DNA were measured. The number of amino acid substitutions in HBV from this patient was much higher than in previous reports of HBV mutants, such as occult HBV and vaccine escape HBV mutants. Levels of HBsAg and HBV DNA production in vitro were significantly lower in the patient compared to wild-type transfections. From analyses of the chimeric constructs, the altered preS/S region was responsible mainly for this impairment. These results show that highly mutated HBV can reactivate after chemotherapy with rituximab, despite an unusually large number of mutations, resulting in impaired viral replication in vitro. Severe immune suppression, probably caused by rituximab, may permit reactivation of highly mutated HBV. These findings have important clinical implications for the prevention and management of HBV reactivation and may explain partially the mechanism of recent, unusual cases of HBV reactivation.

To identify the genes related to atopic dermatitis (AD), we compared gene expression in eosinophils from AD patients and healthy volunteers. RNA was prepared from peripheral blood eosinophils. Gene expression was monitored by fluorescent differential display (DD) and real-time RT-PCR. Eighteen new sequences, including expressed sequence tags (ESTs), were expressed at higher levels in eosinophils from AD patients than in those from healthy volunteers. The functions of most of these genes are unknown. We found no correlation between the expression of a particular gene and clinical markers such as the number of eosinophils and the amount of IgE. Multivariate analysis of the gene expression data in each sample showed a very high coefficient of correlation among the copy numbers of each gene. The genes under investigation were also expressed in cultured blood eosinophils after IL-4, IL-5 and IFN-γ stimulation. We were able to predict the function of some of the sequences by scanning for homologies within either the human or mouse genome databases. The mouse counterpart of one of these genes, intersectin 2, was expressed dramatically, as measured by ear edema, in 1-fluoro-2,4-dinitrobenzene-induced mouse contact dermatitis and in NC/Nga mouse dermatitis.