Treatment of asystole and PEA.

Recent reports consistently point to a substantial decline in the incidence of ventricular fibrillation (VF) as the initial rhythm observed by Emergency Medical Service (EMS) responders and a complementary increase in pulseless electrical activity (PEA) and asystole. Historically, efforts at improving survival have focused primarily on patients found in VF. Consequently, the approach for other patients has included frequent pauses in cardiopulmonary resuscitation (CPR) to check for VF followed by shock when VF is observed. However, the "yield" of survivors comes largely from the non-shocked patients. Therefore, it is critical that we start evaluating treatments specifically for the PEA and asystole groups.

Comparison of neurological outcomes following witnessed out-of-hospital ventricular fibrillation defibrillated with either biphasic or monophasic automated external defibrillators.

BACKGROUND: Biphasic waveform defibrillation results in higher rates of termination of fibrillation than monophasic waveform defibrillation but has not been shown to improve survival outcomes. OBJECTIVE: To compare the effectiveness of a biphasic automated external defibrillator (AED) with a monophasic AED for witnessed out-of-hospital cardiac arrest (OHCA) due to ventricular fibrillation (VF). METHODS: In a prospective population-based cohort study, adults with witnessed VF OHCA were treated with either monophasic or biphasic waveform AED shocks. The primary outcome measure was neurologically favourable 1-month survival, defined as a Cerebral Performance Categories score of 1 or 2. RESULTS: Of 366 adults with witnessed OHCA of presumed cardiac aetiology, 74 (20%) had VF. Termination of VF with the first shock tended to occur more frequently after biphasic AED shocks (36/44 (82%) vs 20/30 (67%), p = 0.14). Return of spontaneous circulation (ROSC) occurred more frequently after biphasic AED shocks (29/44 (66%) vs 8/30 (27%), p = 0.001). Neurologically favourable 1-month survival was also more frequent in the biphasic group (10/44 (23%) vs 1/30 (3%), p = 0.04). The median time interval from the first shock to the second shock was 67 s in the monophasic group and 24 s in the biphasic group (p = 0.001). CONCLUSIONS: Treatment with biphasic AED shocks improved the likelihood of ROSC and neurologically favourable 1-month survival after witnessed VF compared with monophasic AED shocks. In addition to waveform differences, a shorter time interval from the first shock to the second shock could account for the better outcomes with biphasic AED.

Recombination hot spot of hepatitis B virus genome binds to members of the HMG domain protein family and the Y box binding protein family; implication of these proteins in genomic instability.

OBJECTIVE: Previously we hypothesized that the occurrence of hepatocellular carcinoma (HCC) is enhanced by genomic instability induced by the integrated hepatitis B virus (HBV) DNA. Using an in vitro recombination assay, we showed that a subgenomic fragment of HBV DNA designated 15AB (nt1855-1914) is indispensable for in vitro recombination, and also showed the existence of 15AB binding protein. On the assumption that the 15AB binding protein may be a candidate cellular recombinogenic protein which accelerates genomic instability and hepatocarcinogenesis, we tried to isolate it by southwestern screening. RESULTS AND CONCLUSION: We obtained several positive clones including mouse upstream binding factor (UBF) and DNA binding protein A (dbpA). UBF belongs to an HMG domain protein family and dbpA belongs to a Y box binding protein family. 15AB binding seemed to be mediated by the conserved DNA binding domains in these families, because other members in the families such as HMG1 and YB-1 also bound to 15AB. We report them here because several documents have already suggested the possible association of these families and DNA recombination.

Hepatoprotective drugs for the treatment of virus-induced chronic hepatitis: from hypercarcinogenic state to hypocarcinogenic state.

Interferon (IFN)-based therapy is a standard treatment for chronic hepatitis caused by hepatitis C virus (HCV) infection. This treatment is effective in approximately 30-40% of the patients and using ribavirin in combination with IFN increases the rate of sustained virologic clearance. For the remaining patients, glycyrrhizin is often used. Glycyrrhizin is known to prevent the development of hepatocellular carcinoma (HCC), but glycyrrhizin is usually administered intravenously. Drugs that are effective by oral administration are convenient for patients for long-term administration, and development of more effective drugs than glycyrrhizin is preferable. However, studies on drugs for the treatment of hepatitis are not actively conducted, and promotion of the study of drugs in this area is encouraging. For that reason, we show our approach to study drugs for the treatment of hepatitis. We analyzed the effect of glycyrrhizin on hepatitis as a standard chemical using the mouse liver injury model. Based on this, we screened drugs and found that a coumarin derivative seems to be one of model chemicals for the treatment of hepatitis.

Hepatitis C virus core protein activates the MAPK/ERK cascade synergistically with tumor promoter TPA, but not with epidermal growth factor or transforming growth factor alpha.

Persistent hepatitis C virus (HCV) infection is associated with the development of human hepatocellular carcinoma (HCC), although the mechanism of HCV-related hepatocarcinogenesis remains unclear. Recently, however, the close relationships between the development of HCC and the mitogen-activated protein kinase (MAPK)/extracellular signal-regulated protein kinase (ERK) cascade have been described. In the present study, we investigated the effects of HCV core protein on this MAPK/ERK cascade. HCV core protein significantly activated the MAPK/ERK cascade, including Elk1. We also examined whether HCV core protein acted synergistically along with hepatocyte mitogen-mediated MAPK/ERK activation. Interestingly, Elk-1 activities were further enhanced by the tumor promoter, 12-O-tetradecanoyl phorbol 13-acetate (TPA), but not by hepatocyte mitogens (epidermal growth factor [EGF] and transforming growth factor alpha [TGF-alpha]) in NIH3T3 cells and HepG2 cells expressing HCV core protein. Moreover, the MAPK/ERK activation by HCV core protein was blocked in the presence of the specific MEK1 inhibitor, PD98059. These results indicate that ERK activation by HCV core protein may be independent of hepatocyte mitogen-mediated signaling but synergistic with TPA, and HCV core protein may function at MEK1 or farther upstream of that component.

A novel gene "Niban" upregulated in renal carcinogenesis: cloning by the cDNA-amplified fragment length polymorphism approach.

A modified AFLP (amplified fragment length polymorphism) method was employed to isolate genes differentially expressed in renal carcinogenesis of Tsc2 gene mutant (Eker) rats. One gene, selected for further investigation, was named "Niban" "second" in Japanese), because it is the second new gene to be found after Erc (expressed in renal carcinoma) in our laboratory. Importantly, "Niban" is well expressed even in small primary rat Eker renal tumors, more than in progressed cell lines, and is also expressed in human renal carcinoma cells, but not in normal human or rat kidneys. Chromosome assignment was to RNO 13 in the rat, and HSA 1. This "Niban" gene is a candidate as a marker for renal tumor, especially early-stage renal carcinogenesis.

VCP, a weak ATPase involved in multiple cellular events, interacts physically with BRCA1 in the nucleus of living cells.

BRCA1, a breast/ovarian cancer susceptibility gene, undergoes mutations in as many as 50% of familial breast tumors. Recent studies indicate that BRCA1 may be involved in DNA damage repair. Here, we demonstrate that the BRCA1 protein physically associates with valosin-containing protein (VCP), a member of the ATPases associated with a variety of cellular activities (AAA) superfamily. In vitro studies revealed that VCP, via its N- terminal region, binds to amino acid residues 303-625 in the BRCA1 protein. Although found predominantly in the cytoplasm and, less abundantly, in the nucleus, VCP can be translocated from the nucleus after stimulation with epidermal growth factor. Collectively, our results suggest that VCP, by binding to BRCA1, participates in the DNA damage-repair function as an ATP transporter, possibly facilitating the transcription-coupled repair.

beta-catenin mutations are absent in hepatocellular carcinomas of SV40 T-antigen transgenic mice.

beta-catenin mutations have been found not only in melanoma and prostatic carcinoma but also in hepatocellular carcinomas in human, c-myc, H-ras genes transgenic mice and chemically-induced models. We investigated beta-catenin mutations in human hepatocellular carcinomas (HCCs), Hep G2 cell line and HCCs in SV40 T-antigen transgenic mice, in order to examine whether beta-catenin mutations are frequently observed in HCC in general. We found a point mutation of beta-catenin in one of nine HCCs in human and a deletion of it in Hep G2 cell line. However, we found no mutation in HCC in SV40 TG mice liver.

[Prevalence of TT virus infection and viral integration in human hepatocellular carcinoma].

In 1997, Nishizawa et al cloned a novel DNA virus designated as TT virus (TTV) from a patient with post-transfusion hepatitis and this virus is being thought to be a new hepatitis virus. Approximately 5 to 10% of hepatocellular carcinomas (HCCs) in Japan occurs in hepatitis B virus-negative and hepatitis C virus-negative (NBNC) patients. In order to study the possible role of TTV in hepatocarcinogenesis, we studied the prevalence of the TTV DNA in liver tissue of HCC patients. As a result, TTV was shown not to be specific for NBNC HCC and TTV integration into host DNA was not detected in any HCC patient by Southern blotting.

Cloning of differentially expressed genes in highly and low metastatic rat osteosarcomas by a modified cDNA-AFLP method.

To identify differentially expressed genes between highly and low metastatic rat transplantable osteosarcomas, we applied a modified AFLP (amplified fragment length polymorphisms) method for cDNA subtraction. The specific point of our modification is selective amplification using suppression PCR technique after restriction enzyme cutting. Our cDNA-AFLP gave high reproducibility (about 95%) in mRNA patterns and enabled us to clone four dominantly expressed genes in a highly metastatic tumor line. Three showed homology with known genes, encoding Ki-67, a proliferation-associated effective marker of malignancy, type IV collagen alpha-3, a major component of basement membrane, and KIAA77 for which the function is unknown. Although one fragment showed no database homology, we revealed a derivation from the rat homologue of the Drosophila melanogaster diaphanous gene (Dia) by cloning of longer cDNA. Dia genes, known to affect actin filament formation, are downstream effectors of Rho small GTPase. The results suggest that alterations in the expression of cytoskeletal protein, basement membrane elements, and proliferative markers may be important for metastasis of osteosarcomas.

Unique features of dendritic cells in IFN-gamma transgenic mice: relevance to cancer development and therapeutic implications.

Although induction of interferon gamma (IFN-gamma) and activation of antigen presenting dendritic cells (DCs) are two vital events during an immune response, the impact of endogenous IFN-gamma on DC function has yet to be clarified. The phenotype and function of DCs isolated from mice with high (IFN-gamma-transgenic mouse [Tg]) and undetectable levels of circulating IFN-gamma (normal mice [NM]) were therefore compared. The capacity to stimulate allogenic (p < 0.05) and antigen-specific T lymphocytes (p < 0.05), as well as the ability to produce IL-12 (p < 0.05) and to process soluble protein antigens (p < 0.05) was found to be significantly higher in DCs from the Tg mice compared to the NM case. The presence of activated DCs in a microenvironment of endogenous IFN-gamma suggests that the IFN-gamma-Tg mouse is a suitable animal model to study cancer immunotherapy in vivo.

Determination of the clonal origin of multiple human hepatocellular carcinomas by cloning and polymerase chain reaction of the integrated hepatitis B virus DNA.

The poor prognosis of hepatocellular carcinoma (HCC) is partly the result of the high rate of recurrence that is caused either by intrahepatic metastasis (IM) or independent multicentric occurrence (MO). For convenience, discrimination of IM and MO is based on pathological findings, but reliable parameters are not sufficiently established. In the case of hepatitis B virus (HBV)-associated HCC, molecular discrimination of IM from MO can be achieved by comparison of integrated HBV DNAs. However, Southern blotting cannot be used for this purpose when one tumor is saved in frozen form and the other is in paraffin-embedded form. To solve this problem, we employed polymerase chain reaction (PCR) assays to confirm the clonality of primary and recurrent tumors. From the frozen tissue, we determined the junction between the integrated HBV and flanking genomic DNA by molecular cloning, and checked the existence of an identical junction in the DNA of paraffin-embedded tissue by PCR. Using this method, as well as Southern blotting, we proved in 6 of 8 patients that two nodular HCC lesions resected metachronously or simultaneously were caused by MO, while the remaining 2 cases were caused by IM. In 1 IM case, band patterns between two HCCs detected by Southern blotting were not identical.

BRCA1 binds c-Myc and inhibits its transcriptional and transforming activity in cells.

c-Myc, a proto-oncogene that is implicated in tumorigenesis, embryonic development and apoptosis, can physically associate with BRCA1. We have found that BRCA1 interacts with c-Myc in yeast, in in vitro assays and in mammalian cells. Endogenous interactions between BRCA1 and c-Myc were also observed. Efficient BRCA1-Myc association requires the intact helix-loop-helix region of c-Myc, a motif involved in Myc-Max dimerization. BRCA1 does not however bind to Max. Our studies revealed that BRCA1 represses Myc-mediated transcription while having no effect on some other transcriptional activities. Furthermore, BRCA1 reverses the phenotype of embryonic fibroblasts transformed by the activation of Myc and Ras, but only minimally affects the transformed phenotype induced by SV40 virus. These data indicate that BRCA1 may function as a tumor suppressor by regulating the behavior of c-Myc and provide a molecular explanation for some of the effects of the BRCA1 gene product.

Hepatocellular carcinomas infected with the novel TT DNA virus lack viral integration.

A novel DNA virus designated TT virus (TTV) was cloned from a patient with posttransfusion hepatitis and is thought to be a new hepatitis virus. At present, hepatitis B virus (HBV) and hepatitis C virus (HCV) are known to induce hepatocellular carcinoma (HCC). But, actually, in Japan approximately 5 to 10% of HCCs are in HBV-negative and HCV-negative (NBNC) patients. In order to study the possible role of TTV in hepatocarcinogenesis, we investigated the frequency of the TTV genome in liver tissue of 20 HCC patients. As a result, 3 of 8 NBNC HCC patients and 5 of 12 HBV- or HCV-associated HCC patients were TTV positive, and TTV was shown not to be specific for NBNC HCC. For all TTV-positive patients, we also confirmed that the TTV genome was not integrated into host hepatocyte DNA.

Aberrant expression of a cytokeratin in a subset of hepatocytes during chronic WHV infection.

Chronic infection of woodchucks with woodchuck hepatitis virus (WHV) invariably leads, within 2-4 years, to the appearance of hepatocellular carcinoma (HCC). HCC is preceded by an extended period of chronic liver damage, probably resulting from the immune response to viral antigens. It may be that infection itself also induces changes in the hepatocyte population. To begin to identify some of the changes in the liver prior to the appearance of HCC, monoclonal antibodies (MAbs) were generated from mice immunized with hepatocytes from a woodchuck chronically infected with WHV or with a tumor lysate. Immunofluorescence microscopy was used to select MAbs that reacted with host markers whose patterns of expression would distinguish chronically infected from uninfected liver or from liver tumors. One of these MAbs (2F2) reacted strongly with a subset of hepatocytes in chronically infected liver; a similar staining pattern was not detected in uninfected or transiently infected liver. Evidence is presented that this strong staining reaction reflects the overexpression or accumulation of the hepatocyte-specific intermediate filament protein, cytokeratin K18, a protein previously implicated in cryptogenic cirrhosis of the liver in humans (Ku, N. O. , Wright, T. L., Terrault, N. A., Gish, R., and Omary, M. B. J. Clin. Invest. 99: 19-23, 1997). Double immunofluorescent staining with antibodies to K18 and M-envelope protein of WHV suggested that strong reactivity to K18 was limited to cells expressing high levels of one or both of the large viral-envelope proteins, M and L; however, high expression of these viral proteins was not always associated with a strong K18 staining reaction.

Fas- and perforin-independent mechanism of cytotoxic T lymphocyte.

Cytotoxic T lymphocytes (CTLs) play an important role in elimination of virus-infected cells (1). Recent studies revealed at least two distinct mechanisms that CTLs utilize to destroy their target cells. Both mechanisms induce target cell apoptosis specifically and directionally, but these processes are totally different. One is pore formation on target cell membrane by perforin secreted from CTLs (perforin-granzyme pathway), and the other is ligation of Fas, which is expressed on the surface of target cells and Fas ligand, on the surface of CTLs (Fas-FasL pathway) (2). Here we review our work and describe CTL clones that have novel lytic mechanisms derived from CD4-CD8- lymph node cells of gld mice.

Induction of endometriosis and adenomyosis by transvaginal pituitary transplantation in mice with and without natural killer cell activity.

PROBLEM: The aims of this study were to establish a mouse model of endometriosis and adenomyosis and to elucidate the necessity of reduced natural killer (NK)-cell and T-cell activities in the establishment of endometriosis and adenomyosis. METHOD OF STUDY: Pituitary glands, submandibular glands, a hypothalami were transvaginally inoculated into the uteri of syngeneic female mice. Twenty weeks later, the recipient mice were sacrificed and examined. RESULTS: Cysts, adhesion of the uteri to surrounding tissues, and adenomyosis had formed in the uteri of 7 (29.2%), 14 (58.3%), and 22 (91.7%) mice, respectively, out of 24 BALB/c mice after the transplantation of pituitary glands. Similar findings were obtained by experiments with C3H/He and C57BL/6 mice. In NK-cell-deficient C57BL/6-bgJ and T-cell-deficient BALB/c nu/nu mice, an increase in the formation of cysts, adhesion, and adenomyosis was not observed. CONCLUSIONS: These findings indicate that transvaginal pituitary transplantation specifically induces cysts, adhesion, and adenomyosis. Reduced NK-cell activities may not be necessary in the primary development of endometriosis and adenomyosis.

Structure-based design and characterization of exocyclic peptidomimetics that inhibit TNF alpha binding to its receptor.

Exocyclic small peptidomimetics corresponding to three critical binding sites of tumor necrosis factor (TNF)-receptor(I) have been designed based on atomic features deduced from the crystal structures of TNF alpha and the TNF beta/TNF-receptor(I) complex and a model of an anti-TNF alpha monoclonal antibody. TNF alpha antagonistic activities were evaluated by binding assays using soluble receptor or intact receptor on cells as well as an apoptosis/cytotoxicity assay. The most critical interaction site for rational design of peptidomimetics was localized to the loop1/domain3 of the TNF-receptor. The best antagonist showed 5 microM inhibition in the binding assay. Biologically, the mimetics inhibited TNF alpha-mediated apoptosis.