Viper bite during pregnancy: case report.

Viper bites in pregnant women have rarely been reported thus far. Moreover, there is no consensus regarding the treatment of such cases. In this paper, the authors report the successful treatment of viper bite during pregnancy without using antivenom.

Generation of large numbers of dendritic cells from mouse bone marrow cultures supplemented with granulocyte/macrophage colony-stimulating factor.

Antigen-presenting, major histocompatibility complex (MHC) class II-rich dendritic cells are known to arise from bone marrow. However, marrow lacks mature dendritic cells, and substantial numbers of proliferating less-mature cells have yet to be identified. The methodology for inducing dendritic cell growth that was recently described for mouse blood now has been modified to MHC class II-negative precursors in marrow. A key step is to remove the majority of nonadherent, newly formed granulocytes by gentle washes during the first 2-4 d of culture. This leaves behind proliferating clusters that are loosely attached to a more firmly adherent "stroma." At days 4-6 the clusters can be dislodged, isolated by 1-g sedimentation, and upon reculture, large numbers of dendritic cells are released. The latter are readily identified on the basis of their distinct cell shape, ultrastructure, and repertoire of antigens, as detected with a panel of monoclonal antibodies. The dendritic cells express high levels of MHC class II products and act as powerful accessory cells for initiating the mixed leukocyte reaction. Neither the clusters nor mature dendritic cells are generated if macrophage colony-stimulating factor rather than granulocyte/macrophage colony-stimulating factor (GM-CSF) is applied. Therefore, GM-CSF generates all three lineages of myeloid cells (granulocytes, macrophages, and dendritic cells). Since > 5 x 10(6) dendritic cells develop in 1 wk from precursors within the large hind limb bones of a single animal, marrow progenitors can act as a major source of dendritic cells. This feature should prove useful for future molecular and clinical studies of this otherwise trace cell type.

A prostaglandin D2 receptor antagonist modifies experimental asthma in sheep.

BACKGROUND: Prostaglandin (PG) D(2) is the major cylooxygenase metabolite released by mast cells upon allergen stimulation, and elicits responses through either the prostanoid DP1 receptor and/or the chemoattractant receptor homologous molecule expressed on T-helper type 2 (Th2) cells (CRTH2/DP2). Experimental evidence suggests that stimulation of one or both these receptors contributes to asthma pathophysiology. OBJECTIVE: The aim of this study was to test the hypothesis that the prostanoid DP1 receptor contributes to asthma pathophysiology by determining the efficacy of an orally active antagonist for this receptor, S-5751, on allergen-induced bronchoconstriction, airway hyperresponsiveness (AHR) and cellular inflammation in the sheep model of asthma. METHODS: PGD(2)-induced cyclic adenosine monophosphate (cAMP) production in platelet-rich plasma was used to establish the in vitro efficacy of S-5751. In vivo, sheep naturally allergic to Ascaris suum were challenged with an aerosolized antigen with and without S-5751 treatment (given 4 days before and for 6 days after the challenge). RESULTS: S-5751 inhibited PGD(2)-induced cAMP production in platelet-rich plasma with an IC(50) value of 0.12 microm. S-5751 at 30 mg/kg, but not at 3 mg/kg, reduced the early bronchoconstriction and inhibited the late bronchoconstriction. AHR and inflammatory cell infiltration in bronchoalveolar lavage fluid at days 1 and 7 were also inhibited with the 30 mg/kg dose. The responses observed with S-5751 at 30 mg/kg were comparable with those with montelukast treatment (0.15 mg/kg, twice a day, intravenous); however, S-5751 did not block inhaled leukotrieneD(4)-induced broncoconstriction. CONCLUSION: Prostanoid DP1 receptor inhibition may represent an alternative target for asthma therapy.

Association study of vascular endothelial growth factor gene polymorphisms in endometrial carcinomas in a Japanese population.

OBJECTIVE: Vascular endothelial growth factor (VEGF) is one of the most potent endothelial cell mitogens and plays a critical role in angiogenesis of endometrial carcinomas. Several studies have demonstrated positive associations between VEGF gene polymorphisms and several carcinomas. In this study we investigated whether VEGF gene polymorphisms are associated with endometrial carcinomas in a Japanese population. METHODS: The allele frequencies and genotype distributions of VEGF -460 C/T, +405 G/C, and +936 C/T polymorphisms were examined in 105 endometrial carcinomas and 179 controls using PCR-RFLP analysis. An association of these polymorphisms with three-year disease-free survival was evaluated using the Kaplan-Meier method. RESULTS: No significant differences in the allele frequencies and genotype distributions of VEGF -460 C/T (p = 0.54, 0.90), +405 G/C (p = 0.31, 0.17), and +936 C/T polymorphisms (p = 0.46, 0.24) were observed between endometrial carcinoma patients and controls. There were no significant differences in the frequencies of haplotype -460 T/+405 C between patients and controls. Futhermore, VEGF -460 C/T, +405 G/C, and +936 C/T polymorphisms were not associated with three-year disease-free survival of endometrial carcinoma patients. CONCLUSIONS: Although limited by sample size, our study did not demonstrated any evidence that VEGF -460 C/T, +405 G/C, and +936 C/T polymorphisms are associated with an increased risk of endometrial carcinomas in Japanese women.

Association study between catechol-O-methyltransferase polymorphisms and uterine leiomyomas in a Japanese population.

PURPOSE: To investigate a possible association between uterine leiomyomas and catechol-O-methyltransferase (COMT) polymorphisms in a Japanese population. METHODS: We compared the allele frequencies and genotype distributions of the exon 4 NlaIII restriction site polymorphism (RSP), the P2 promoter HindIII RSP at -1217, and the exon 6 BglI RSP in the COMT gene in 250 leiomyoma cases and 182 controls using polymerase chain reaction-restriction fragment-length polymorphism analysis. RESULTS: No significant differences in allele frequencies and genotype distributions of the exon 4 NlaIII RSP, the P2 promoter HindIII RSP at -1217, and the exon 6 BglI RSP were found between uterine leiomyoma cases and controls. Moreover, no associations were noted between these three polymorphisms in COMT genes and leiomyoma size or a family history of uterine leiomyomas. CONCLUSION: COMT gene polymorphisms are unlikely to be associated with an increased risk of uterine leiomyomas in a Japanese population.

Mechanism of killing of Giardia lamblia trophozoites by complement.

Only antibodies of the IgM class support the lytic effect of complement on Giardia lamblia (GL). We sensitized GL trophozoites (SGL) at 4 degrees C with serum containing anti-GL antibodies or IgM purified from this serum, and either normal human serum (NHS), complement 2-deficient human serum (C2d-HS), or C4-deficient guinea pig serum was used as source of complement. SGL were killed by NHS (86%) and by the deficient sera (50 and 40%, respectively), suggesting activation of the alternative pathway. However, the reaction was inhibited by Mg-EGTA. These observations led to studies of the role of C1. The lytic effect of NHS and C2d-HS on SGL was abolished by immunochemically depleting C1 from these sera, and reconstituted by adding purified C1q plus C1r and C1s. Factor B-depleted C2d-HS also lost its capacity to mediate killing, but reconstitution with factor B led to a dose-dependent increase in the killing of SGL. We next investigated the participation of the membrane attack complex in this system. SGL carrying C5b to C7 were lysed when incubated with C8 alone (56%); the addition of C9 further increased killing (98%), while C9 in the absence of C8 had no effect. We concluded that although activation of the classical pathway produces lysis of SGL, lysis may also proceed through a unique pathway of complement activation that requires C1 and factor B, but is independent of C4 and C2. Lysis of SGL can be accomplished by C5b to C8 in the absence of C9.

The effects of exercise in forest and urban environments on sympathetic nervous activity of normal young adults.

In Japan, forest-air bathing and walking (shinrin-yoku) has been proposed as a health-facilitating activity in which people spend a short period of time in a forest environment. Initially, we examined the usefulness of salivary amylase activity as an indicator of an individual's stress levels in a forest environment. The circadian rhythm of salivary amylase activity was measured in healthy young male subjects under stress-free conditions. The salivary amylase activity remained relatively constant throughout the day. Salivary amylase activity was then measured before and after walking in both urban and forest environments using a hand-held monitor. Our results indicated that (i) the circadian rhythm fluctuations in salivary amylase activity were much smaller than the stressor-induced variations; (ii) salivary amylase activity was an excellent indicator of the changes in sympathetic nervous activity; and (iii) the forest was a good environment in which people could experience much less environment-derived stress.

Non-verbal communication method based on a biochemical marker for people with severe motor and intellectual disabilities.

This study evaluated a novel non-verbal communication method for people with severe motor and intellectual disabilities (SMID) based on a biochemical marker, salivary amylase. The physical and psychological status of 10 people with SMID was quantitatively evaluated using a hand-held salivary amylase activity monitor. Each patient needed daily gastric and/or bronchial tube exchanges and these medical procedures were thought to cause severe distress and pain. Salivary amylase activity and heart rate were simultaneously measured during 32 medical procedures. The medical procedures resulted in a significant mean increase for individuals of 70% in salivary amylase activity. The increase in salivary amylase activity was more than four-fold that observed for heart rate. The structural equation modelling analysis also demonstrated a significant correlation between pain and salivary amylase activity. Our data indicate that salivary amylase activity might be used as a non-verbal method of assessing pain in people with SMID.

The biological properties of a novel ethyl methacrylate resin.

A novel ethyl methacrylate (EMA) resin was developed to overcome the tissue, organ and systemic damage associated with the residual monomer of conventional methyl methacrylate (MMA) resin bone cement. EMA resin is a chemical/ photopolymerizable material and is easy to handle during clinical procedures. The biocompatibility of EMA was evaluated in accordance with ISO10993-6. No inflammatory response was observed 1 and 9 weeks after implantation in the dorsal subcutaneous tissue of ddY mice. EMA resin also demonstrated better biocompatibility when compared with conventional bone cements. Poly-L-lactic acid (PLLA) was used as a carrier for bone morphogenetic protein (BMP) and added to the EMA slurry. The EMA-PLLA composite membrane was sticky and BMP readily adhered to its surface. The EMA-PLLA-BMP composite membrane induced new bone formation, the new bone growing in the shape of the EMA in the thigh muscle pouch of ddY mice. This novel EMA resin has many potential clinical applications.

Anemia-inducing substance from plasma of patients with advanced malignant neoplasms.

Patients with advanced malignant neoplasms develop anemia and immunosuppression. During an attempt to identify the causes, we have found that plasma from such patients makes RBCs more fragile in hypotonic buffer, according to results obtained with a coil planet centrifuge. Plasma from these patients suppresses mitogen-stimulated lymphocyte proliferation. In this study, we identified the substance with these effects as a protein. During two-dimensional gel electrophoresis, two isomers with M(r) 50,000 and slightly different isoelectric points near 6.0 were found. Cell fractionation showed that these proteins were in both the cytosol and the nuclear fraction of cells in neoplasms. Another protein with the same antigenicity and a M(r) 100,000 found in the nuclear fraction of cells in neoplasms.

Alterations of beta- and gamma-catenin in N-butyl-N-(-4-hydroxybutyl)nitrosamine-induced murine bladder cancer.

Abnormal degradation of beta-catenin caused by alteration of the glycogen synthase kinase-3beta (GSK-3beta) consensus motif is an important step for carcinogenesis. We hypothesize that beta- and gamma-catenin may play an important role in the pathogenesis of bladder cancer. We tested this hypothesis through analysis of beta- and gamma-catenin in both murine and human bladder cancers. A murine bladder cancer model was prepared by use of N-butyl-N-(-4-hydroxybutyl)nitrosamine (BBN) in 6-week-old male B6D2F1 mice. After 4, 8, 12, 16, 20, 24, and 28 weeks of BBN treatment, bladder specimens were harvested and analyzed for both protein and gene expression for beta- and gamma-catenin. Mutational analysis of the NH(2)-terminal regulatory domains of beta- and gamma-catenin was performed in each specimen by PCR-single-strand conformational polymorphism (SSCP) analysis. Mutations were further confirmed by direct DNA sequencing with a dye terminator method. Human bladder cancer specimens with normal tissues, dysplasia, carcinoma in situ, and carcinoma of grades, 1, 2, and 3 were also analyzed for beta- and gamma-catenin expression. beta- and gamma-catenin were analyzed for mutations by SSCP and direct DNA sequencing. Intracellular accumulation of beta- and gamma-catenin was observed in 6 of 20 invasive carcinoma specimens. There was no intracellular accumulation of beta- and gamma-catenin in mucosal dysplasia, papillary or nodular dysplasia, and carcinoma in situ specimens. On an SSCP analysis for beta-catenin, abnormal bandshifts were detected in two invasive carcinomas with intracellular beta-catenin accumulation. Further sequencing revealed two mutations [AGT(S) to ATT(I) and TCT(S) to CCT(P)] within the consensus motif for GSK-3beta phosphorylation. On the other hand, SSCP analysis for gamma-catenin followed by sequencing revealed three mutations in two invasive carcinomas with intracellular accumulation of gamma-catenin. These three alterations affected the 3' downstream region outside the GSK-3beta phosphorylation site [ACC(T) to GCC(A), CTC(L) to ATC(I), and CTC(L) to ATG(M)]. In human bladder cancer, beta- and gamma-catenin expression was significantly weaker than in normal bladder. On SSCP analysis one abnormal bandshift was observed in high-grade human bladder cancer with intracellular beta-catenin accumulation. DNA sequencing revealed mutation TCT(S) to TGT(C). In summary, alterations in beta- and gamma-catenin are late events favoring tumor progression in mouse BBN-induced bladder cancer. Changes affecting the GSK-3beta phosphorylation site appear to be associated with activation of beta-catenin, but not with activation of gamma-catenin. In human blabber cancer, beta- and gamma-catenin expression is similar to the expression in the mouse model. The present study demonstrates that beta- and gamma-catenin may play an important role in bladder cancer progression.

Isolation and characterization of mammalian homologues of Caenorhabditis elegans lin-7: localization at cell-cell junctions.

In Caenorhabditis elegans, the vulval induction is mediated by the let-23 receptor tyrosine kinase (RTK)/ Ras signaling pathway. The precise localization of the let-23 RTK at the epithelial junctions is essential for the vulval induction, and requires three genes including lin-2, -7, and -10. The mammalian homologue of lin-2 has been identified as a protein interacting with a neuronal adhesion molecule, neurexin, and named CASK. CASK has recently been reported to interact with syndecans and an actin-binding protein, band 4.1, at epithelial and synaptic junctions, and to play central roles in the formation of cell-cell junctions. The product of C. elegans lin-7 directly interacts with let-23 RTK and localize it at epithelial junctions. Here, we report three rat homologues of lin-7 ubiquitously expressed in various tissues. These homologues are accumulated at the junctional complex region in cultured Madin-Darby canine kidney cells, and are also localized at the synaptic junctions in neurons. The mammalian homologues of lin-7 may be implicated in the formation of cell-cell junctions.

Localization of membrane-associated guanylate kinase (MAGI)-1/BAI-associated protein (BAP) 1 at tight junctions of epithelial cells.

Membrane-associated guanylate kinase (MAGI)-1/BAI-associated protein (BAP) 1 and Synapse-associated protein (SAP) 97/human Discs-large tumor suppressor gene (hDLG) are ubiquitous isoforms of synaptic scaffolding molecule (S-SCAM) and Postsynaptic density (PSD)-95/SAP90, both of which are implicated in the structures of synapses, respectively. SAP97/hDLG is localized at epithelial junctions and may function as a scaffolding protein, but the subcellular localization or the function of MAGI-1/BAP1 has not been clarified. In intestinal epithelial cells, MAGI-1/BAP1 was localized at tight junctions, whereas SAP97/hDLG was localized diffusely at cell - cell junctions. In Madine Darby canine kidney (MDCK) cells, MAGI-1/BAP1 was colocalized with ZO-1, whereas SAP97/hDLG was colocalized with E-cadherin. In MDCK cells, dominant active and negative mutants of Rac1 small G protein changed the amounts of SAP97/hDLG at cell - cell junctions, but not that of MAGI-1/BAP1. When MDCK cells were switched to a low Ca2+ medium, E-cadherin disappeared from the plasma membrane, and cells were dissociated. The phorbol 12-myristate 13-acetate-treatment after the low Ca2+ switch induced a tight junction-like structure. MAGI-1/BAP1 was recruited with ZO-1 to this structure, but SAP97/hDLG or E-cadherin was not. These findings suggest that MAGI-1/BAP1 is a component of tight junctions of epithelial cells, and that its role is different from that of SAP97/hDLG.

Involvement of the C-terminal region of yeast proteinase B inhibitor 2 in its inhibitory action.

Yeast proteinase B inhibitor 2 (YIB2), which is composed of 74 amino acid residues, is an unusual serine protease inhibitor, since it lacks disulfide bonds. To identify its reactive site for proteases, we constructed an expression system for a synthetic YIB2 gene and then attempted to change the inhibitory properties of YIB2 by amino acid replacements. The purified wild-type YIB2 inhibited the activity of subtilisin BPN', a protein homologous to yeast proteinase B, although its binding ability was not strong, and a time-dependent decrease in its inhibitory activity was observed, demonstrating that wild-type YIB2 behaves as a temporary inhibitor when subtilisin BPN' is the target protease. Since YIB2 exhibits sequence homology to the propeptide of subtilisin, which inhibits a cognate protease using its C-terminal region, we replaced the six C-termi nal residues of YIB2 with those of the propeptide of subtilisin BPN' to make the mutant YIB2m1. This mutant exhibited markedly increased inhibitory activity toward subtilisin BPN' without a time-dependent decrease in its inhibitory activity. Replacement of only the C-terminal Asn of YIB2 by Tyr, or deletion of the C-terminal Tyr of YIB2m1, inhibited subtilisin, but the ability of these mutants to bind subtilisin and their resistance to proteolytic attack were weaker than those of YIB2m1, indicating that the C-terminal residue contributes to the interaction with the protease to a greater extent than the preceding five residues and that the resistance of YIB2 to proteolyic attack is closely related to its ability to bind a protease. These results demonstrate that YIB2 is a unique protease inhibitor that involves its C-terminal region in the interaction with the protease.

Elimination of anemia-inducing substance by cyclic plasma perfusion of tumor-bearing rabbits.

We carried out a fundamental study to search for a therapeutic modality that would remove the anemia-inducing substance (AIS) from the plasma of cancer patients because it is thought to be one of the substances responsible for anemia and immunodeficiency in advanced cancer patients. Using AIS isolated from the plasma of patients with advanced ovarian carcinoma, we confirmed that adsorption of AIS to noncoated charcoal was nonspecific and high. Moreover, it was verified that VX2 carcinoma-bearing rabbits are an optimal experimental model for plasma perfusion. The data obtained on day 40 after transplantation (hemoglobin, 9.1+/-2.1 g/dl; osmotic pressure inducing RBC lysis, 137+/-11 mosmol/kg; lymphocyte stimulation index, 8.8+/-8.6; and RBC fragility-inducing activity, 40+/-9 mosmol/kg) proved similar to the hematological findings in patients with cancer cachexia. A 1-h plasma perfusion (3 ml/min) through noncoated charcoal was performed in tumor-bearing rabbits, and it resulted in the restoration of RBC fragility-inducing activity and suppression of lymphocyte blast formation to pretransplantation values. When plasma perfusion was performed every 3 days, RBC fragility-inducing activity, which increased again 3 days after perfusion, was diminished, and RBC osmotic resistance was within the normal range from the fourth perfusion onward. These results showed that cyclic plasma perfusion is effective in sustained removal of RBC fragility-inducing factor from plasma, suggesting that it might have the potential for clinical application.

Adenofibromasarcoma originating from a mural nodule of ovarian serous cystadenoma.

A 83-year-old woman received bilateral salpingo-oophorectomy and hysterectomy due to a provisional diagnosis of ovarian cystic tumor. The tumor had a unilocular cystic cavity demonstrating serous cystadenoma and a solid mural nodule representing a biphasic pattern with mesenchymal and glandular components. The glandular elements were composed of benign serous cells, whereas the mesenchymal components consisted of an admixture of fibromatous stromal cells without atypia and sarcomatous overgrowth. The area of transition from a fibromatous component to sarcomatous overgrowth was identified. After a 2-year follow-up, there were no signs of tumor recurrence or systemic disease. To the authors' knowledge, this is the first reported case of adenofibrosarcoma originating from a mural nodule of ovarian serous cystadenoma.

Pregnancy complicated with pulmonary lymphangioleiomyomatosis: case report.

A case of a 30-year-old primiparous woman with pulmonary lymphangioleiomyomatosis is described. The patient had experienced six episodes of spontaneous pneumothorax at the age of 27 years and had been diagnosed as having pulmonary lymphangioleiomyomatosis based on histological findings of specimens obtained by transbronchial biopsy. She had undertaken open lung surgery and thoracoscopy. Thereafter, she became pregnant spontaneously. Her antenatal course was uneventful with no exacerbation of respiratory status. At 38 weeks of gestation, she underwent a selective cesarean section and myomectomy under combined spinal and epidural anesthesia. Her postoperative course was uneventful. No remarkable changes in computed tomographic findings of the lung were noted on the 20th day of postoperation compared with those before pregnancy. She has been followed-up in the pulmonary outpatient clinic with no deterioration of the disease.

Cultured murine dermal cells can function like thymic nurse cells.

We have established a dermal fibroblast-like stromal cell line, DFB-1, and a clone, 12E2, from epidermal sheets prepared from the skin of BALB/c mouse ears by trypsin digestion. They were suggested to be fibroblasts or myofibroblasts, as 1) they were polygonal or spindle-shaped under the phase-contrast microscope, 2) they did not possess any tonofilaments or desmosomes, and 3) they did not express any marker for bone marrow-derived cells or macrophages. Interestingly, these cells showed a unique phenomenon of "pseudo-emperiporesis," which was first recognized in the interaction between thymic nurse cells and thymocytes. Namely, two T-cell clones and one T-cell hybridoma migrated beneath the cytoplasmic projections of the fibroblast-like cutaneous stromal cells in culture. Furthermore, secretion of interleukin 7 by these cells was confirmed by bioassay using an IL-7-dependent cell line and by inhibition with anti-interleukin 7 antibody, and the expression of interleukin 7 mRNA was also demonstrated in these cells by a combination of reverse-transcriptase polymerase chain reaction and Southern blot analysis. These data strongly suggest the presence of unique stromal cells even in the skin, probably at the upper dermis, which can function like the nurse cells in the thymus. These stromal cells may play a crucial role in cutaneous immunophysiology.

Exogenous and endogenous type I interferons inhibit interferon-gamma-induced nitric oxide production and nitric oxide synthase expression in murine peritoneal macrophages.

We investigated the effect of type I IFNs (IFN-alpha and IFN-beta) on IFN-gamma-induced nitric oxide (NO) production by murine peritoneal macrophages. It was found that exogenous and also endogenous type I IFNs suppressed IFN-gamma-induced NO production, cytosolic inducible NO synthase (iNOS) activity, and iNOS mRNA accumulation in macrophages. Furthermore, we show here that type I IFNs prevent the NO-mediated deterioration of mitochondrial respiratory activity in macrophages. These results seem to indicate a possible protective role of type I IFNs against the NO-mediated immunosuppressive and/or cytotoxic effect of macrophages.