Exposure of cells to a wide variety of chemoprotective compounds confers resistance to a broad set of carcinogens. For a subset of the chemoprotective compounds, protection is generated by an increase in the abundance of the protective phase II detoxification enzymes, such as glutathione S-transferase (GST). We have recently developed a cell culture system, using rat liver epithelial RL 34 cells, that potently responds to the phenolic antioxidants resulting in the induction of GST activity (Kawamoto, Y., Nakamura, Y., Naito, Y., Torii, Y., Kumagai, T., Osawa, T., Ohigashi, H., Satoh, K., Imagawa, M., and Uchida, K. (2000) J. Biol. Chem. 275, 11291-11299.) In the present study, we investigated the phase II-inducing potency of an isothiocyanate compound in vitro and in vivo and examined a possible induction mechanism. Based on an extensive screening of vegetable extracts for GST inducer activity in RL34 cells, we found Japanese horseradish, wasabi (Wasabia japonica, syn. Eutrema wasabi), as the richest source and identified 6-methylsulfinylhexyl isothiocyanate (6-HITC), an analogue of sulforaphane (4-methylsulfinylbutyl isothiocyanate) isolated from broccoli, as the major GST inducer in wasabi. 6-HITC potently induced both class ␣ GSTA1 and class GSTP1 isozymes in RL34 cells. In animal experiments, we found that 6-MSHI was rapidly absorbed into the body and induced hepatic phase II detoxification enzymes more potently than sulforaphane. The observations that (i) 6-HITC activated the antioxidant response element (ARE), (ii) 6-HITC induced nuclear localization of the transcription factor Nrf2 that binds to ARE, and (iii) the induction of phase II enzyme genes by 6-HITC was completely abrogated in the nrf2-deficient mice, suggest that 6-HITC is a potential activator of the Nrf2/ARE-dependent detoxification pathway.

B19 parvovirus is pathogenic in humans, causing fifth disease, transient aplastic crisis, some cases of hydrops fetalis, and acquired pure red cell aplasia. Efforts to develop serologic assays and vaccine development have been hampered by the virus's extreme tropism for human bone marrow and the absence of a convenient culture system. We constructed recombinants containing either the major (VP2) or minor (VP1) structural proteins of B19 in a baculovirus-based plasmid, from which the polyhedrin gene had been deleted; these recombinant plasmids were used to generate recombinant infectious baculovirus. Subsequent infection of insect cells in vitro resulted in high-level expression ofeither B19 VP1 or VP2. Parvovirus capsids were obtained by self-assembly in cell cultures coinfected with either VP1-and VP2-containing baculoviruses or, surprisingly, VP2-containing baculoviruses alone. Empty B19 capsids composed of VP1 and VP2 could replace serum virus as a source of antigen in a conventional inmmunoassay for detection ofeither IgG or IgM antiparvovirus antibodies in human serum. Immunization of rabbits with capsids composed of VP1 and VP2 resulted in production of antisera that recognized serum parvovirus on immunoblot and neutralized parvovirus infectivity for human erythroid progenitor cells. Baculovirus-derived parvovirus antigen can substitute for scarce viral antigen in immunoassays and should be suitable as a human vaccine.B19 parvovirus is the only member ofthe family Parvoviridae pathogenic in humans (1, 2). Acute infection results in fifth disease, a common childhood exanthem that in adults more usually manifests as an arthralgia/arthritis syndrome. In persons with underlying hemolysis, acute infection produces transient aplastic crisis, precipitous anemia due to hypoproliferative erythropoiesis. B19 infection can be persistent (3). In the setting of immunodeficiency, due to congenital, acquired, or iatrogenic immunodeficiency, persistent parvovirus results in chronic pure red cell aplasia. In utero infection causes hydrops fetalis, with fetal death due to severe anemia and congestive heart failure (4).Clinical studies of B19 parvovirus infection have been hampered by limited supplies of viral antigen. The virus has extraordinary tropism for human erythroid progenitor cells and has only been propagated in explanted human bone marrow (5-8), fetal liver (9), and (to a lesser degree) erythroleukemia cells (10). We describe here expression of the virus's structural proteins in a baculovirus system. Large quantities of empty viral capsids were produced, which can substitute for serum virus in clinical assays and stimulate the production of neutralizing antibodies in animals. Only the major protein was required for capsid assembly; the minor capsid protein serves an important role in virus function independent of virion assembly. MATERIALS AND METHODSCell Culture and Virus Stocks. Autographa californica nuclear polyhedrosis virus (AcMNPV) and recombinant viruses were grown in monolayers of Sf9 cells. Sf9 c...

FcγRIIB1 molecules serve as negative feedback regulator for B cell Ag receptor-elicited activation of B cells; thus, any impaired FcγRIIB1 function may possibly be related to aberrant B cell activation. We earlier found deletion polymorphism in the Fcgr2b promoter region among mouse strains in which systemic autoimmune disease-prone NZB, BXSB, MRL, and autoimmune diabetes-prone nonobese diabetic, but not NZW, BALB/c, and C57BL/6 mice have two identical deletion sites, consisting of 13 and 3 nucleotides. In this study, we established congenic C57BL/6 mice for NZB-type Fcgr2b allele and found that NZB-type allele down-regulates FcγRIIB1 expression levels in germinal center B cells and up-regulates IgG Ab responses. We did luciferase reporter assays to determine whether NZB-type deletion polymorphism affects transcriptional regulation of Fcgr2b gene. Although NZW- and BALB/c-derived segments from position −302 to +585 of Fcgr2b upstream region produced significant levels of luciferase activities, only a limited activity was detected in the NZB-derived sequence. EMSA and Southwestern analysis revealed that defect in transcription activity in the NZB-derived segment is likely due to absence of transactivation by AP-4, which binds to the polymorphic 13 nucleotide deletion site. Our data imply that because of the deficient AP-4 binding, the NZB-type Fcgr2b allele polymorphism results in up-regulation of IgG Ab responses through down-regulation of FcγRIIB1 expression levels in germinal center B cells, and that such polymorphism may possibly form the basis of autoimmune susceptibility in combination with other background contributing genes.

No abstract

INTRODUCTION: The bleeding source of hematochezia is unknown without performing colonoscopy. We sought to identify whether colonoscopy is a risk-stratifying tool to identify etiology and predict outcomes and whether presenting symptoms can differentiate the etiologies in patients with hematochezia. METHODS: This multicenter retrospective cohort study conducted at 49 hospitals across Japan analyzed 10,342 patients admitted for outpatient-onset acute hematochezia. RESULTS: Patients were mostly elderly population, and 29.5% had hemodynamic instability. Computed tomography was performed in 69.1% and colonoscopy in 87.7%. Diagnostic yield of colonoscopy reached 94.9%, most frequently diverticular bleeding. Thirty-day rebleeding rates were significantly higher with diverticulosis and small bowel bleeding than with other etiologies. In-hospital mortality was significantly higher with angioectasia, malignancy, rectal ulcer, and upper gastrointestinal bleeding. Colonoscopic treatment rates were significantly higher with diverticulosis, radiation colitis, angioectasia, rectal ulcer, and postendoscopy bleeding. More interventional radiology procedures were needed for diverticulosis and small bowel bleeding. Etiologies with favorable outcomes and low procedure rates were ischemic colitis and infectious colitis. Higher rates of painless hematochezia at presentation were significantly associated with multiple diseases, such as rectal ulcer, hemorrhoids, angioectasia, radiation colitis, and diverticulosis. The same was true in cases of hematochezia with diarrhea, fever, and hemodynamic instability. DISCUSSION: This nationwide data set of acute hematochezia highlights the importance of colonoscopy in accurately detecting bleeding etiologies that stratify patients at high or low risk of adverse outcomes and those who will likely require more procedures. Predicting different bleeding etiologies based on initial presentation would be challenging.

Left heart disease (LHD) frequently causes lung vascular remodelling and pulmonary hypertension (PH). Yet pharmacological treatment for PH in LHD is lacking and its pathophysiological basis remains obscure. We aimed to identify candidate mechanisms of PH in LHD and to test their relevance and therapeutic potential.In rats, LHD was induced by supracoronary aortic banding. Whole genome microarray analyses were performed, candidate genes were confirmed by RT-PCR and Western blots and functional relevance was tested in vivo by genetic and pharmacological strategies.In lungs of LHD rats, mast cell activation was the most prominently upregulated gene ontology cluster. Mast cell gene upregulation was confirmed at RNA and protein levels and remodelled vessels showed perivascular mast cell accumulations. In LHD rats treated with the mast cell stabiliser ketotifen, or in mast cell deficient Ws/Ws rats, PH and vascular remodelling were largely attenuated. Both strategies also reduced PH and vascular remodelling in monocrotaline-induced pulmonary arterial hypertension, suggesting that the role of mast cells extends to noncardiogenic PH.In PH of different aetiologies, mast cells accumulate around pulmonary blood vessels and contribute to vascular remodelling and PH. Mast cells and mast cell-derived mediators may present promising targets for the treatment of PH.

No abstract

We have established a recombinant HIV gene transfer system based on transient expression of the HIV packaging functions and a recombinant vector genome in monkey kidney Cos cells. The recombinant HIV retroviral vector introduced the neoR gene into CD4+ cells with high efficiency, comparable to that achieved with the highest titer amphotropic murine recombinant retrovirus. Vector preparations were devoid of replication competent, infectious HIV. Gene transfer was dependent on CD4 expression, as shown by expression of the CD4 gene in HeLa cells, and could be inhibited by soluble CD4. This specific and efficient gene transfer system may be useful for development of gene therapy for which T cells are the desired targets. (J. Clin. Invest. 1991. 88:1043-1047