A sensitive enzyme immunoassay (EIA) specific for hepatitis B virus core antigen (HBcAg) and hepatitis B e antigen (HBeAg) was developed. We designated the precore/core gene products as hepatitis B virus (HBV) core-related antigens (HBcrAg). In order to detect HBcrAg even in anti-HBc/e antibody-positive specimens, the specimens were pretreated in detergents. The antibodies are inactivated by this pretreatment and, simultaneously, the antigens are released and the epitopes are exposed. The assay demonstrated 71 to 112% recovery using HBcrAg-positive sera. We observed no interference from the tested anticoagulants or blood components. When the cutoff value was tentatively set at 10 3 U/ml, all healthy control (HBsAg/HBV-DNA negative; n ‫؍‬ 108) and anti-HCV antibody-positive (n ‫؍‬ 59) sera were identified as negative. The assay showed a detection limit of 4 ؋ 10 2 U/ml using recombinant antigen. Detection limits were compared in four serially diluted HBV high-titer sera. The HBcrAg assay demonstrated higher sensitivity than HBV-DNA transcription-mediated amplification ( Many hepatitis B virus (HBV) markers are used for diagnosing and monitoring hepatitis B patients. HBV-DNA tests, such as the branched-chain DNA (b-DNA) signal amplification assay (7, 31), and transcription-mediated amplification (TMA)-based (11) or PCR-based (12,14,20) assays are used to diagnose and monitor the efficacy of treatment. However, these methods require cumbersome procedures and expensive equipment, thus requiring considerable skill and high costs. These gene amplification assays also present some limitations (22,23,35). The b-DNA assay provides quantitative results but requires a long incubation time and lacks adequate sensitivity. Amplification assays have adequate sensitivity but are less quantitative.Immunoassays are generally easy and inexpensive. There have been a few reports of serum HBcAg assays with specimen pretreatment (4, 32). The concentration of HBcAg in these assays correlated with levels of HBV-associated DNA polymerase (4). Thus, HBcAg could be a marker for virus load. However, the use of these assays is limited because of relatively low sensitivity and complex procedures.Serum HBeAg concentration reflects virus replication and hepatitis activity and is closely correlated with virus load in anti-HBe antibody-negative patients (8). Seroconversion of HBeAg to anti-HBe antibody reveals the inactive phase of infection (17,25). However, after seroconversion, many patients may exhibit reactivation and high viral load (3,10,18). In these cases, HBeAg is usually negative due to masking by anti-HBe antibody (24), although the HBeAg/anti-HBe immune complex can be indirectly detected according to the levels of alanine aminotransferase (ALT) and HBV-DNA (6). Therefore, HBcAg and HBeAg could be expected to be efficient markers of virus load if antibodies were inactivated and the antigens released.In the present study, for the purpose of developing a simple, sensitive, and inexpensive assay for determining HBV virus load, we targete...

Endometriosis, a common disease among women of reproductive age, is characterized by the presence of endometrial-like tissue outside the uterus. We previously reported that TNFalpha promoted proliferation of endometriotic stromal cells by inducing IL-8 gene and protein expression. We hypothesize that TNFalpha may induce IL-8 production in endometriotic cells through nuclear factor-kappa B (NF-kappa B) activation. Western blot analyses and electrophoretic mobility shift assays revealed that incubation with TNF alpha induced the expression of phosphorylated inhibitor kappa B (p-I kappa B) and activation of NF-kappa B in endometriotic stromal cells. The NF-kappa B inhibitor, N-tosyl-L-phenylalanine chloromethyl ketone, reduced TNFalpha-induced IL-8 gene and protein expression. The medical treatment of endometriosis with GnRH agonist (GnRHa) has been shown to induce hypoestrogenemia and reduce the observable number of endometriotic implants. We compare the expression of IL-8 gene and protein in endometriotic stromal cells of patients treated with GnRHa and those of patients without treatment before laparoscopic cystectomy for endometrioma. The addition of TNFalpha (0.1 ng/ml) significantly increased protein and gene expression of IL-8 in the cells of patients without GnRHa treatment, but this expression was not observed in the cells of patients with GnRHa. The addition of estradiol (E2; 10(-7) M) enhanced the expression of IL-8. However, in the cells of patients who received GnRHa treatment, TNFalpha and E2 did not show any significant effect. In endometriotic stromal cells without GnRHa treatment, TNFalpha and E2 increased the expression of p-I kappa B. In contrast, TNFalpha and E2 had no significant effect on the expression of p-I kappa B in cells that received GnRHa treatment. These findings demonstrate that NF-kappa B activation is critical for TNFalpha-induced IL-8 expression in endometriotic stromal cells. The current study showed for the first time that GnRHa treatment attenuated the expression of IL-8 by reducing TNFalpha-induced NF-kappa B activation.

The aim of this study was to examine the relationship between menses-associated health problems of women, such as premenstrual symptoms, menstrual pain and irregular menstrual cycles, and psychosocial stress. A cross-sectional study was conducted among Japanese college students, measuring psychosocial stress levels by means of IMPS (The Inventory to Measure Psychosocial Stress). A total of 264 female students (mean age 19.4 years), who were invited to participate in the study in October 2007, completed the questionnaire, which dealt with anthropometric data, lifestyle, menstrual history, and menstrual health status. Forty-three students were excluded due to missing data, and the remaining 221 were analyzed. The proportions of students who reported premenstrual symptoms, menstrual pain, and the experience of irregular menstrual cycles were 79%, 79%, and 63%, respectively. Students who reported premenstrual symptoms, menstrual pain, and the experience of irregular menstrual cycles had higher stress scores than those who did not. Multiple logistic regression analyses were used to identify independent factors associated with having premenstrual symptoms, menstrual pain, and the experience of irregular menstrual cycles. Stress score, heavy menstrual flow, and menstrual pain were significant predictors for premenstrual symptoms, while age at menarche and having premenstrual symptoms were significant predictors for menstrual pain. Both stress score and body mass index were found to be significant predictors for having experienced irregular menstrual cycles. The results suggest that psychosocial stress is independently associated with premenstrual symptoms and the experience of irregular menstrual cycles among college students, implying that changes in the functional potentiality of women as a result of stress are related with changes in their menstrual function.

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Inducible nitric oxide synthase (iNOS) is involved in pathological processes related to overproduction of nitric oxide (NO), and is expressed in response to pro-inflammatory agents such as interleukin (IL)-1b, tumor necrosis factor (TNF)-a and lipopolysaccharide (LPS) in various cell types including macrophages, endothelial cells, and smooth muscle cells.1) Nuclear factor (NF)-kB is a major transcription factor involved in iNOS, TNF-a, IL-1b, and IL-8 genes expression. NF-kB activation involves dissociation of an inhibitory subunit, IkB, which keeps NF-kB in the cytoplasm, thereby preventing activation of the target gene in the nucleus. Cellular signals lead to phosphorylation of IkB following elimination of IkB from NF-kB by proteolytic degradation. Then, the activated-NF-kB is released and translocated into the nucleus to activate transcription of its target genes.2) Inhibition of iNOS enzyme activity or iNOS induction and inhibition of NF-kB activation may be of therapeutic benefit in various types of inflammation. 10) In the course of our studies on constituents with NO production inhibitory activity from natural medicines, 11) the methanolic extract from the dried bark of M. obovata was found to inhibit nitrite (NO 2 Ϫ , a product of NO) accumulation in LPS-activated mouse macrophages (IC 50 ϭ25 mg/ml).Previous reports demonstrated that two neolignans [magnolol (1), honokiol (2)] showed inhibitory effects on NO production from LPS-activated RAW 264.7 cells.12) However, effects of other constituents on NO production from LPS-activated mouse macrophages and their cytotoxicities for macrophages have not been examined. This report describes the effects of the constituents from the bark of M. obovata on NO production in LPS-stimulated macrophages. In addition, we describe the effects of principal active neolignan constituents [magnolol (1), honokiol (2), obovatol (3)] on iNOS enzyme activity, induction of iNOS, and activation of NF-kB to clarify their action mechanisms. Results and DiscussionIsolation of Chemical Constituents from the Dried Bark of M. obovata The bark of Japanese M. obovata was extracted with methanol under reflux. The methanolic extract was subjected to ordinary-and reversed-phase silica gel column chromatography and finally HPLC to furnish five neolignans, magnolol (1, 13) 2.1% from the natural medicine), honokiol (2, 14) 0.43%), obovatol (3, 15) 0.26%), 4-O-methylhonokiol (4, 16) 0.0031%), and 6Ј-O-methylhonokiol (5, 10,16) 0.0031%), seven sesquiterpene-neolignans, eudesmagnolol (6, 17) 0.096%), clovanemagnolol (7, 18) 0.0061%), caryolanemagnolol (8, 10) 0.0044%), eudeshonokiols A (9, 10) 0.0056%) and B (10, 10) 0.0054%), eudesobovatols A (11, 19) 0.043%) and B (12,19) 0.024%), a trineolignan, magnolianin (13,20) 0.27%), a phenylpropanoid glycoside, syringin (14, 21) 0.39%), two lignan glycosides, liriodendrin (15,22) 0.15%) and (ϩ)-syringaresinol 4Ј-O-b-D-glucopyranoside (16,23) 0.029%), and four sesquiterpenes, caryophyllene oxide (17, 24) 0.0049%), a-eudesmol (18, 11g,24) 0.096%), b...