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Systematic approaches for accurate repurposing of targeted therapies are needed. We developed and aimed to biologically validate our therapy predicting tool (TPT) for the repurposing of targeted therapies for specific tumor types by testing the role of Bromodomain and Extra-Terminal motif inhibitors (BETi) in inhibiting BRD4 function and downregulating Notch3 signaling in ovarian cancer.Utilizing established ovarian cancer preclinical models, we carried out in vitro and in vivo studies with clinically relevant BETis to determine their therapeutic effect and impact on Notch3 signaling.Treatment with BETis or siRNA-mediated BRD4 knockdown resulted in decreased cell viability, reduced cell proliferation, and increased cell apoptosis in vitro. In vivo studies with orthotopic mouse models demonstrated that treatment with BETi decreased tumor growth. In addition, knockdown of BRD4 with doxycycline-inducible shRNA increased survival up to 50% (P < 0.001). Treatment with either BETis or BRD4 siRNA decreased Notch3 expression both in vitro and in vivo. BRD4 inhibition also decreased the expression of NOTCH3 targets, including HES1. Chromatin immunoprecipitation revealed that BRD4 was present at the NOTCH3 promoter.Our findings provide biological validation for the TPT by demonstrating that BETis can be an effective therapeutic agent for ovarian cancer by downregulating Notch3 expression.The TPT could rapidly identify candidate drugs for ovarian or other cancers along with novel companion biomarkers.

Raw RPPA data for OVCAR 5.

File contains synthesis of CN210, sequences for siRNAs, supplementary tables 1 to 3, supplementary figures 1 through 8 and figure legends.

Raw RPPA data for OVCAR 4.

Inhibitors of bromodomain and extra-terminal motif (BET) proteins are emerging epigenetic therapeutics that suppress gene expressions that drive cancer and inflammation. The present study examined anti-inflammatory effects of a quinazoline-based BET inhibitor, CN210, in a murine ileitis model. CN210 was given orally 30 min before and 24 h after a subcutaneous administration of indomethacin. Macroscopic and histological evidences of ileitis, mucosal myeloperoxidase (MPO) activity and cytokine expressions were evaluated 48 h after the indomethacin administration. To further characterize the anti-inflammatory pathways modulated by CN210, its effects on RAW264 cells treated with lipopolysaccharide (LPS) were investigated. Competitive ligand binding and docking studies of CN210 to CREB-binding protein (CBP) and p300 were also performed. Oral administration of CN210 significantly reduced the severity of ileitis, normalized both proinflammatory MPO activity and concomitant cytokine expressions induced by indomethacin administration. Furthermore, CN210 attenuated the expression of cytokines and reversed the activation of nuclear factor κB (NF-κB) and mitogen-activated protein kinases (MAPK) induced by LPS. Competitive ligand binding assays showed that CN210 bound to the bromodomains of two paralogous histone acetyltransferases, CBP and p300, in addition to the bromodomains of BET proteins. Docking studies of CN210 to the bromodomains of CBP and p300 showed a similarity to the binding mode of SGC-CBP30, a specific CBP/p300 inhibitor. CN210 ameliorates indomethacin-induced ileitis by inhibiting the expression of inflammatory cytokines through the attenuation of NF-κB and MAPK pathways. CN210 thus represents a new mode of therapy for non-steroidal anti-inflammatory drug-induced ileitis and inflammatory bowel disease.

Acute lymphoblastic leukemia with chromosomal rearrangements involving the mixed-lineage leukemia (MLL) gene (MLL-r ALL) remains an incurable disease. Thus, development of a safe and effective therapeutic agent to treat this disease is crucial to address this unmet medical need. BRD4, a member of the bromodomain and extra-terminal domain (BET) protein family, and cyclic AMP response element binding protein binding protein (CBP) and p300, two paralogous histone acetyltransferases, are all considered cancer drug targets and simultaneous targeting of these proteins may have therapeutic advantages. Here, we demonstrate that a BET/CBP/p300 multi-bromodomain inhibitor, CN470, has anti-tumor activity against MLL-r ALL in vitro and in vivo. CN470, potently inhibited ligand binding to the bromodomains of BRD4, CBP, and p300 and suppressed the growth of MLL-r ALL cell lines and patient-derived cells with MLL rearrangements. CN470 suppressed mRNA and protein expression of MYC and induced apoptosis in MLL-r ALL cells, following a cell cycle arrest in the G1 phase. Moreover, CN470 reduced BRD4 binding to acetylated histone H3. The in vivo effects of CN470 were investigated using SEM(Luc/GFP) cells expressing luminescent markers in an orthotopic mouse model. Mice administered CN470 daily had prolonged survival compared to the vehicle group. Further, CN470 also showed anti-tumor effects against an MLL-r ALL patient-derived xenograft model. These findings suggest that inhibition of BET/CBP/p300 by the multi-bromodomain inhibitor, CN470, represents a promising therapeutic approach against MLL-r ALL.

Adult T-cell leukemia/lymphoma (ATL) is an aggressive T-cell lymphoproliferative malignancy caused by human T-cell leukemia virus type 1 (HTLV-1). ATL is an orphan disease with no curative drug treatment regimens urgently needing new combination therapy. HTLV-1-infected cells rely on viral proteins, Tax and HBZ (HTLV-1-b-ZIP factor), to activate the transcription of various host genes that are critical for promoting leukemic transformation. Inhibition of bromodomain and extraterminal motif (BET) protein was previously shown to collapse the transcriptional network directed by BATF3 super-enhancer and thereby induced ATL cell apoptosis. In the current work, by using xenograft, ex vivo, and in vitro models, we demonstrated that I-BET762 (BETi) synergized with copanlisib (PI3Ki) and bardoxolone methyl (NF-κBi) to dramatically decrease the growth of ATL cells. Mechanistically, the triple combination exhibited synergistic activity by down-regulating the expression of c-MYC while upregulating the level of the glucocorticoid-induced leucine zipper (GILZ). The triple combination also enhanced apoptosis induction by elevating the expression of active caspase-3 and cleaved PARP. Importantly, the triple combination prolonged the survival of ATL-bearing xenograft mice and inhibited the proliferation of ATL cells from peripheral blood mononuclear cells (PBMCs) of both acute and smoldering/chronic ATL patients. Therefore, our data provide the rationale for a clinical trial exploring the multiagent combination of BET, PI3K/AKT, and NF-κB inhibitors for ATL patients and expands the potential treatments for this recalcitrant malignancy.

Systematic approaches for accurate repurposing of targeted therapies are needed. We developed and aimed to biologically validate our therapy predicting tool (TPT) for the repurposing of targeted therapies for specific tumor types by testing the role of Bromodomain and Extra-Terminal motif inhibitors (BETi) in inhibiting BRD4 function and downregulating Notch3 signaling in ovarian cancer. Utilizing established ovarian cancer preclinical models, we carried out in vitro and in vivo studies with clinically relevant BETis to determine their therapeutic effect and impact on Notch3 signaling. Treatment with BETis or siRNA-mediated BRD4 knockdown resulted in decreased cell viability, reduced cell proliferation, and increased cell apoptosis in vitro. In vivo studies with orthotopic mouse models demonstrated that treatment with BETi decreased tumor growth. In addition, knockdown of BRD4 with doxycycline-inducible shRNA increased survival up to 50% (P < 0.001). Treatment with either BETis or BRD4 siRNA decreased Notch3 expression both in vitro and in vivo. BRD4 inhibition also decreased the expression of NOTCH3 targets, including HES1. Chromatin immunoprecipitation revealed that BRD4 was present at the NOTCH3 promoter. Our findings provide biological validation for the TPT by demonstrating that BETis can be an effective therapeutic agent for ovarian cancer by downregulating Notch3 expression. The TPT could rapidly identify candidate drugs for ovarian or other cancers along with novel companion biomarkers.

A new series of quinazoline-based analogs as potent bromodomain-containing protein 4 (BRD4) inhibitors is described. The structure-activity relationships on 2- and 4-position of quinazoline ring, and the substitution at 6-position that mimic the acetylated lysine are discussed. A co-crystallized structure of 48 (CN750) with BRD4 (BD1) including key inhibitor-protein interactions is also highlighted. Together with preliminary rodent pharmacokinetic results, a new lead (65, CN427) is identified which is suitable for further lead optimization.

Pulmonary fibrosis is a progressive disease with poor prognosis and limited therapeutic options. In this study, we evaluated the potential therapeutic effects of CG223, a novel inhibitor of bromodomain and extra-terminal motif (BET) proteins, on pulmonary fibrosis by focusing on the transforming growth factor-β1 (TGF-β1) pathway. In a murine model of bleomycin-induced pulmonary fibrosis, CG223 attenuated fibrosis while reducing the infiltration of inflammatory cells into the lungs. Fibroblasts expressing BRD4, a member of the BET protein family, were enriched in the tissue regions corresponding to bleomycin-induced fibrotic lesions. Additionally, pulmonary fibroblasts isolated from bleomycin-instilled mice showed a significantly increased association of BRD4 with the promoters of two pro-fibrotic genes linked to the entry into the TGF-β1 autocrine/paracrine loop, thrombospondin 1 (Thbs1) and integrin β3 (Itgb3), as well as with the promoter of a myofibroblast marker gene, actin alpha 2 (Acta2). Subsequent in vitro studies with murine primary lung fibroblasts showed that the mRNA induction of Thbs1, Itgb3, and Acta2 by TGF-β1 can be inhibited by CG223 in a dose-dependent manner. Taken together, CG223-induced BRD4 inhibition suppressed lung fibrogenesis by affecting multiple genes, including those involved in the triggering of the TGF-β1 autocrine/paracrine loop.

Multiple myeloma (MM) is characterized by the clonal proliferation of neoplastic plasma cells. Despite a stream of new molecular targets based on better understanding of the disease, MM remains incurable. Epigenomic abnormalities contribute to the pathogenesis of MM. bromodomain 4 (BRD4), a member of the bromodomain and extraterminal (BET) family, binds to acetylated histones during M/G1 transition in the cell cycle promoting progression to S phase. In this study, we investigated the effects of a novel BET inhibitor CG13250 on MM cells. CG13250 inhibited ligand binding to BRD4 in a dose-dependent manner and with an IC50 value of 1.1 μM. It inhibited MM proliferation in a dose-dependent manner and arrested cells in G1, resulting in the induction of apoptosis through caspase activation. CG13250 inhibited the binding of BRD4 to c-MYC promoter regions suppressing the transcription of the c-MYC gene. Administered in vivo, CG13250 significantly prolonged survival of an orthotopic MM-bearing mice. In conclusion, CG13250 is a novel bromodomain inhibitor that is a promising molecular targeting agent against MM.

The consultation rate for uterine cervical cancer screening in Japan is markedly low in comparison with other developed countries. The purpose of this study is to investigate the trends in uterine cervical cancer screening during regular company checkups and to identify potential problems. Questionnaires were sent to occupational health physicians through Sansuiken (Alumni Association of the University of Occupational and Environmental Health, Japan). Overall, 127 valid responses showed that Papanicolaou (Pap) tests are conducted in 100 companies (79%). The detailed information from 50 of the 100 responses was analyzed. Mandatory cervical cancer screenings are performed at just 6 companies (12%). Pap test are started at 30 years of age at 9 of 49 companies, and only 18 of 49 companies (37%) start Pap tests for employees at 20 years old. Of the 86,695 women, 31,294 (36%) received cervical cancer screening. Abnormal Pap test results were detected in 3.0%. Although cervical cancer screening rates have slightly increased compared to our previous studies (17% in 2004, 23% in 2008), it remains at a low level. Complete examinations with colposcopy and punch biopsy were carried out in 70% (61 of 87 women) of those with an abnormal Pap test. Twelve of 26 companies had no information about detailed examination results. It is important to note that cervical cancer incidence and mortality are increasing among young women in Japan. Occupational physicians and health nurses should manage female health education and care at the workplace, by including uterine cervical cancer screening in the growing female working population.

Acute lymphoblastic leukemia (ALL) is the most common form of childhood leukemia. While the prognosis of patients with ALL has improved significantly, ALL with chromosomal rearrangements involving mixed-lineage leukemia (MLL) gene remains an incurable disease. Thus, development of a safe and efficacious therapeutic agent based on the biology of MLL rearrangement is of high importance to address this unmet medical need. Bromodomains are protein motifs that bind to acetylated histones and are recognized as new epigenetic drug targets in recent years. Bromodomain and extra-terminal motif proteins (“BET proteins”) are epigenetic ‘reader' proteins that are characterized by containing two of such bromodomains. BRD4, a member of BET protein family, is a key component of multi-protein complex that induce oncogene transcription at Super-Enhancers. Currently, small-molecule BET inhibitors are being investigated in the clinic as potential therapeutics for hematological cancers. CBP and p300, two paralogous histone acetyltransferases, also contain bromodomains and are considered as drug targets for cancer. Because BET and CBP/p300 proteins are key components of the etiology of MLL-rearranged (MLL-r) leukemia, an agent that simultaneously target both classes of proteins may have therapeutic advantages. We previously demonstrated that a novel BET inhibitor, CG13250, suppressed multiple myeloma cell proliferation in vitro and in vivo (Biochem Biophys Res Commun, 484:262-268, 2017) and subsequently synthesized additional BET inhibitors (Bioorg Med Chem Lett, 29:1220-1226, 2019). In the present study, we demonstrate that some of our BET inhibitors can also inhibit bromodomains of CBP/p300 proteins. One such compound, CN470, potently suppresses BRD4, CBP, and p300 with BROMOscan KD values of 33, 32, and 20 nM, respectively, and suppressed mRNA and protein expression of c-MYC as expected. CN470 potently inhibited the growth of MLL-r ALL cell lines in a dose-dependent manner and flow cytometric analyses showed that CN470 caused apoptosis in MLL-r ALL cells following a cell cycle arrest at the G1 phase. Moreover, co-immunoprecipitation assay demonstrated that CN470 reduced the binding of BRD4 to acetylated H3 (H3K27), an effect absent with a reference BET inhibitor, OTX-015. Taken together, CN470 may represent an effective strategy against MLL-r ALL cells. As a confirmatory study, we investigated the in vivo effects of CN470 by using SEMLuc/GFP cells in an orthotopic mouse model of MLL-AF4 leukemia. Mice were orally administered with CN470 (10 mg/kg) once daily resulting in a prolonged survival compared to vehicle control group (mean 37 days vs. 28 days; p=0.0246). In conclusion, simultaneous inhibition of BET/CBP/p300 with a multi-bromodomain inhibitor represents a promising novel therapeutic approach against MLL-r ALL. Citation Format: Natsuki Imayoshi, Makoto Yoshioka, Shyh-Ming Yang, Koshi Akahane, Yuki Toda, Shigekuni Hosogi, Takeshi Inukai, Seiji Okada, David J. Maloney, Jefferey W. Strovel, Eishi Ashihara. Application of BET/CBP/p300 multi-bromodomain inhibitors as a novel therapeutic strategy for MLL-rearranged acute lymphoblastic leukemia [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1745.

Extensive optimization of quinazoline-based lead 8 is described. The structure-activity relationship studies indicate the S-configuration is preferred for the phenylmorpholine substitution. Together with incorporation of a (2-hydroxyl-2-methylpropyl)pyrazole moiety at the 2-position leads to analogs with comparable potency and marked improvement in the pharmacokinetic profile over our previously reported lead compounds. Further in vivo efficacy studies in Kasumi-1 xenograft mouse model demonstrates that the selected inhibitors are well tolerated and highly efficacious in the inhibition of tumor growth. Additionally, the representative analog 19 also demonstrated significant improvement of arthritis severity in a collagen-induced arthritis (CIA) mouse model. These results indicate potential use of these quinazoline-based BET inhibitors for treatment of cancer and inflammatory diseases. A brief discussion of the co-crystallized structure of 19 with BRD4 (BD1) is also highlighted.

In an attempt to simulate both large scale and high-intensity turbulence within a limited entry length, an innovative wind tunnel of “multi-fan type” is developed. The airflow is driven by an array of fans (9 columns×11 rows), each of which is independently controlled by a computer. Details of the wind tunnel were introduced, and two typical methods (“uniformly-active” and “quasi-grid” methods) were applied to drive the wind tunnel. Comparison with the quasi-grid method revealed the spatial structure of the turbulent flow generated by the uniformly-active method.

症例は30歳男性.発熱,咳嗽,呼吸困難を主訴に,入院精査した.心室中隔欠損(VSD, subarterial type)と大動脈弁の感染性心内膜炎(IE)を伴う今野分類I型のValsalva洞動脈瘤破裂と診断された.手術所見として,大動脈弁と肺動脈弁の感染性心内膜炎が認められたため,右Valsalva洞動脈瘤切除,大動脈弁,肺動脈弁切除とVSD周囲の右室壁の炎症性脆弱組織の切除術を施行した結果,VSDと右Valsalva洞切除部が連続した欠損孔を生じ,ウマ心膜パッチを用いて閉鎖を行った.大動脈弁置換術は機械弁(ATS18AP)の弁輪の一部をパッチに縫着し,肺動脈弁置換術は機械弁(ATS23A)を用い肺動脈弁位で施行した.術後13ヵ月を経過し,厳重なワーファリン管理下,感染や心不全徴候なく経過良好である.本症例はVSDから大動脈閉鎖不全症をきたし,Valsalva洞動脈瘤の形成と破裂,さらにIEが大動脈弁と肺動脈弁にまで波及し,心不全がより重症化したことが考えられた.重篤な心不全やIEが併発する前の早期にこのような適切な手術が不可避であることが示唆された症例と思われた.

ConverGene has developed a first-in-class dual-active small molecule inhibitor that i) inhibits BET family of bromodomain-containing proteins, and ii) antagonizes dopamine receptor D2 (DRD2). BET protein family includes BRD4, an epigenetic reader protein that mediates expression of MYC oncogene. Thus, BRD4 is considered as a cancer therapeutic target to indirectly suppress MYC expression. In addition to being a therapeutic target for psychiatric diseases, DRD2 is emerging as a potential therapeutic target in neuroendocrine tumors, subsets of pancreatic ductal adenocarcinoma and small cell lung cancer. Our lead compound showed high activity in a binding test against BRD4 (Ki = 34 nM); exhibited high bioavailability upon oral administration; profoundly suppressed MYC expression both in vitro and in vivo; inhibited growth of AML and solid tumor cells in xenograft models; potently inhibited both isoforms of DRD2 (IC50 0.1 µM); and interfered with DRD2/β-arrestin/Akt pathway in vitro. Therefore, our BRD4/DRD2 dual-active compounds may hold promise as a novel class of therapeutics that interferes with both cancer growth and maintenance by simultaneously interfering with MYC and DRD2 pathways. We currently are investigating these dual-active compounds in multiple in vitro and in vivo models and expect to report the outcomes at the AACR Annual Meeting in 2017. Citation Format: Makoto Yoshioka, Jay Chauhan, Steven Fletcher, Jeffrey W. Strovel. Development of a first in class inhibitor of BET bromodomains and dopamine receptor 2 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5062. doi:10.1158/1538-7445.AM2017-5062

Coumestrol, a phytoestrogen found in high levels in alfalfa and red clover, is of concern since endocrine disorders have been observed in farm animals exposed to high levels of phytoestrogens. Previous studies found that coumestrol was an effective inducer of DNA strand breaks, micronuclei, and mutations in the Hypoxanthine phosphoribosyl transferase ( HPRT ) gene of Chinese hamster ovary cells. In the experiments presented here, we extended the previous studies to examine the effect of coumestrol exposure on AHH-1 TK +/− human lymphoblastoid cells. Micronuclei were induced with the highest frequency occurring at day 2 after exposure. Flow cytometric analysis of annexin V-FITC–7-aminoactinomycin D stained cells indicated that the primary pathway of cell death was by apoptosis. Mutations were induced in the Thymidine Kinase ( TK ) gene and were due primarily to the induction of clones with the slow-growth phenotype. Subsequent molecular analysis revealed the loss of exon 4 in the coumestrol-induced clones, indicative of loss-of heterozygosity and consistent with a proposed inhibition of topoisomerase-II activity as a mechanism of action for coumestrol. Taken together, these results suggest that coumestrol exhibits both mutagenic and clastogenic properties in cultured human lymphoblastoid cells.

We previously demonstrated that a novel BRD4 inhibitor, CA2, suppressed MM cell proliferation in vitro in the 57th ASH Annual Meeting. In the present study, we investigated the inhibitory mechanisms of CA2 on myeloma cell proliferation in vitro and in vivo. CA2 has a unique quinolinone core and inhibited the proliferation of MM cell lines, MM.1s, AMO-1, NCI-H929, OPM-2, and IM-9. As expected, CA2 suppressed the expression of c-MYC mRNA and protein in a dose- and time-dependent manner. In addition, CA2 decreased BRD4 binding to c-MYC promoters and c-MYC enhancers in a ChIP assay using IM-9 cells and an anti-BRD4 antibody (Figure 1). Flow cytometric Annexin-V/propodium iodide staining analyses demonstrated that CA2 induced apoptosis in MM cells. Taken together, CA2 may induce apoptosis in MM cells by inhibiting the binding of BRD4 to promoters and enhancers of c-MYC gene thereby suppressing c-MYC expression. We next performed a pharmacokinetic study of CA2 in mice. Half-lives (t1/2) of CA2 following oral administration (50 mg/kg) and subcutaneous administration (10 mg/kg) were approximately 61 min and 139.8 min, respectively. The values of area under the curve for oral and subcutaneous administrations were 179,000 hr·ng/mL and 31,000 hr·ng/mL, respectively. Lastly, we assessed the in vivo effects of CA2 using orthotopic MM mouse model, where IM-9Luc cells were inoculated intravenously through the tail veins of SCID mice that had received 2 Gy of irradiation. The mice in the vehicle-control arm died of MM by approximately 30 days after transplantation; IM-9Luc cells engrafted in many organs including bone marrow of spine and femurs, kidneys, ovaries, and stomach. CA2 treatment consisting of 20 cycles of 100 mg/kg/day b.i.d. oral dosing resulted in reduced luminescence intensity of IM-9Luc cells and prolonged the survival of mice (mean 33.7 days) compared to the vehicle-control arm (mean 27.1 days; p=0.029, Figure 2). In conclusion, CA2 is a BRD4 inhibitor with a novel structure and could be a promising molecular targeting-agent against MM. Disclosures Yoshioka: ConverGene LLC: Employment. Kado:Japan Community Health Care Organization Kyoto Kuramaguchi Medical Center: Employment. Strovel:ConverGene LLC: Employment.

To elucidate the influence of osteoporosis on the fracture healing, we produced a rat osteoporosis model by ovariectomy and by maintaining a low calcium diet; and monitored the healing process radiographically, histologically, and biomechanically for 12 weeks. Radiologic, histologic and biomechanical findings of the fracture areas 6 weeks after making the fractures were almost identical in both the osteoporosis group and the control group. However, 12 weeks after making the fractures, newly generated bones in the osteoporosis group showed histological osteoporotic changes and their bone mineral density on the fracture site decreased. These findings show that estrogen-deficient and low calcium conditions greatly affect the bone in the later period of the healing process, but do not affect remarkably the early healing period. This is clinically important when we consider fracture treatments for patients with osteoporosis due to menopause.

Limited information is available regarding the frequency, spectrum, and clinical relevance of somatic mutations in the developing fetus. The goal of this study was to determine somatic mutant frequencies (Mfs) at the hypoxanthine phosphoribosyltransferase (HPRT) reporter gene in cord blood T lymphocytes from preterm infants to gain insight intoin uteromutational events. Mf determinations were made by using theHPRTT cell cloning assay on cord blood samples from 52 preterm infants. Natural logarithm Mfs (lnMfs) from preterm infants were compared with results from our database for full-term infants. Our analysis revealed higher lnMfs in cord blood T lymphocytes from preterm compared with full-term infants (P= 0.008). In addition, preterm females had significantly higher lnMfs compared with full-term females (P< 0.001), whereas preterm males were found to have significantly lower lnMfs than preterm females (P= 0.005). Regression analyses also demonstrate a significant relationship between lnMf and gestational age for preterm females that does not exist for preterm males. These results demonstrate the gender-specific association between Mf and age in humans.

Urothelial Cancer Risk in Relation to Genotypes of Glutathione S-Transferase (GST) M1, T1, P1 and N-Acetyltransferase 2 (NAT2), and Tobacco Smoking: Takahiko KAT OH , et al. Department of Health Information Science, School of Health Sciences—Both genetic and environmental factors are involved in the development of cancer. Molecular epidemiological studies were performed in order to elucidate the relationship between four polymorphic metabolic enzymes and susceptibility to urothelial cancer (bladder, renal pelvis and ureter) and cigarette-smoking. The polymorphisms of glutathione S-transferase (GST) M1, T1 and P1, and N- acetyltransferase (NAT) 2 were analyzed by PCR, and a history of cigarette smoking was obtained through an interview with 102 cancer cases and with 100 controls. GSTM1 null genotype was a risk factor (odds ratio (OR), 2.12; 95% confidence interval (CI), 1.17- 3.90), especially in smokers (OR, 3.70; 95%CI, 1.16- 12.79). GSTT1 null genotype and the polymorphism of GSTP1 at nucleotide 313 were not associated with the cancer risk. Slow acetylation genotype of NAT2 was a significant risk factor (OR, 4.75; 95%CI, 1.64- 15.90) in overall samples and the greatest risk for smokers (OR, 10.05; 95% CI, 1.08-240.6). To investigate the relationship between the cancer and the amount of smoking, we performed regression analysis for the relative risk of cancer as a function of the amount of smoking. The regression analysis shows that the susceptibility of people who have the GSTM1 null genotype or NAT2 intermediate acetylator genotype is easily raised by a little smoking compared to the GSTM1 positive genotype or NAT2 rapid acetylation genotype, respectively. These results imply that polymorphisms in the GSTM1 and NAT2 gene are a genetic determinant of urothelial cancer among Japanese, and the interaction between genes and cigarette smoking may be important in the development of urothelial cancer. (J Occup Health 1999; 41: 12-18)

A case-control study was carried out to examine the relation between genetic polymorphisms of five genes, cigarette smoking and colorectal cancer risk. We collected blood samples from 106 colorectal cancer patients and 100 healthy persons, then analyzed them to identify genotypes for glutathione S-transferase (GST) M1, T1, P1, N-acetyltransferase (NAT) 1 and 2 by the PCR method. We also collected smoking history data from all participants by questionnaire. From statistical evaluation on various combinations of genotypes, we observed that the cancer risk of those who have both GSTM1 present genotype and GSTP1 Adenine/Adenine homozygous genotype was significantly less than those who have other combinations of genotypes for two genes. For other combinations of genes, there was no significant association between genotype and cancer risk. There was also no significant association between amount of cigarette smoking and the cancer risk. These findings suggest that it is valuable to study cancer risk when examining genotypes of more than two genes at the same time. For further study, we need to collect more samples to increase statistical reliability, and besides cigarette smoking, include the nutrition data as an environmental factor.

Objective: To clarify the relationship between type or direction of cartilage injury and its repair process, we investigated defects produced in rat knees histologically, immunohistochemically, and histomorphometrically. Methods: A full-thickness cartilage injury (1 mm wide and 5 mm long) was produced on the patellar groove of one knee (L-injury) and transversely on the other knee (T-injury) in 42 male Wistar rats. Six rats each were sacrificed at 1, 2, 3, 4, 6 and 8 weeks after surgery, and cartilage tissues were obtained, prepared into 4 μm-thick histologic specimens, and stained with hematoxylin and eosin. Cartilage thickness, cartilage area, and surface roughness were measured using a computer system. Localization of S-100 proteins was evaluated with immunohistochemistry. Results: Grossly, there were no differences in repair process between L- and T-injuries. However, histological and histomorphometric differences became apparent after the third week: cartilage thickness, repair area, and surface roughness showed better recovery in L-injury than in T-injury. Appearance of S-100 positive protein preceded the appearance of chondrocytes, and L-injury presented S-100 in the entire defect while S-100 in T-injury appeared mainly on the margins of the defect. Conclusions: Repair mechanism of cartilage injury differs according to injury direction. Better repair can be obtained in the injury which is parallel to the direction of joint motion.

成人Ebstein奇形に合併する三尖弁逆流に対し,Hetzer法による手術症例を経験した.症例は51歳,男性.主訴は労作時呼吸困難で,診断はNYHA III度の成人Ebstein奇形である.高度三尖弁逆流と心房化右室を認め,Carpentierの形態分類のType Bであり,さらに前尖には裂隙を認めた.三尖弁形成術はHetzer法にて,プレジェット付き3-0モノフィラメント糸で前尖・後尖の交連部付近の弁輪から中隔尖中央部の弁輪へU字縫合を行い,弁輪部を接合させ,縫縮偏位した部分の弁輪部を計5針のU字縫合で縫縮した.術後三尖弁逆流は認めず,胸部レントゲン写真上,心胸郭比は術前74%より術後60%へ著明に縮小し,またNYHAもIII度よりI度に改善した.Hetzer法は,心房化右室の縫縮を行わず,前尖または後尖,あるいは両者の弁機能を用い,本来の三尖弁輪レベルでの弁接合を得ることである.術式は比較的容易で再現性があり,また,術後結果もきわめて良好で満足いくものであった.

Osteoblast stimulating factor-1 (OSF-1) stimulates in vitro proliferation and differentiation of osteoblastic cells, and its gene is expressed in the bone and brain tissues in mammals and amphibians. To evaluate the in vivo function of OSF-1 in bone metabolism, transgenic mice overexpressing the human osf-1 gene driven by the osteocalcin promoter were generated. Femoral bone mineral content was increased in transgenic mice relative to wild-type controls as estimated by ash assay, depending on the transgene copy number per cell. In ovariectomized mice, bone mass loss due to estrogen deficiency was observed in both transgenic and control mice but bone mass was still higher in transgenic mice than in controls. Bone mass in ovariectomized transgenic mice was comparable to that in wild-type mice without ovariectomy. These observations indicate that OSF-1 may direct in vivo appositional bone formation by increasing osteoblast activity rather than decreasing osteoclast activity, suggesting a new way to treat osteoporosis with OSF-1.

The effect of unilateral transection of the anterior cruciate ligament on the confined compression and swelling properties of the distal femoral articular cartilage of skeletally mature rabbits at 9 weeks after surgery was determined. Gross morphological grading of the transected and contralateral control distal femora stained with India ink confirmed that cartilage degeneration had been induced by ligament transection. Osteochondral cores, 1.8 mm in diameter, were harvested from the medial femoral condyles. The modulus, permeability, and electrokinetic (streaming potential) coefficient of the articular cartilage of the osteochondral cores were assessed by confined compression creep experiments. The properties (mean +/- SD) of control cartilage were: confined compression modulus, 0.75 +/- 0.28 MPa; hydraulic permeability, 0.63 +/- 0.28 x 10(-15) m2/Pa*sec; and electrokinetic coefficient, 0.16 +/- 0.31 x 10(-9) V/Pa. In transected knees, the modulus was reduced by 18% (p = 0.04), while the permeability and electrokinetic coefficient were not detectably altered. The change in modulus was accompanied by a trend (p = 0.07) toward a decrease (-11%) in the glycosaminoglycan density within the tissue, a significant increase (p < 0.001) in the water content of the cartilage after equilibration in 1 x phosphate buffered saline from 70.3 +/- 4.1% in control knees to 75.2 +/- 4.0% in transected knees, and little further swelling after tissue equilibration in hypotonic saline. The compressive modulus of the cartilage from both control and transected knees was positively correlated with the density of tissue glycosaminoglycan. The alterations in the physical properties of the articular cartilage after transection of the anterior cruciate ligament in the rabbit show trends similar to those observed in human and other animal models of osteoarthritis and provide further support for the use of this model in the study of cartilage degeneration.

Adult cases of intussusception often have organic diseases including tumors and inflammation of the intestine. We rarely see adult intussusception probably due to an ileus tube in the literature. This paper describes our experience with three cases of such rare intussusception. In one case intussusception occurred during indwelling of an ileus tube for aspiration therapy of intestinal obstruction. In the remaining two cases, intussusception developed after drawing out an ileus tube which was inserted preoperatively as splinting tube to prevent postoperative intestinal obstruction and was left untouched. All invaginated sites were found out in the jejunum. One case demanded excision of the intestine, but the remaining two cases were successfully reduced by Hutchinson's procedure. No organic diseases were noted in the lead of the intussusception, and hence it was thought that the indwelt ileus tube might cause intussusception in these cases. One of these cases was treated 3.5 months after the onset of the deseases. The ileus tube should be used cautiously by observing symptomatic changes even after removal of the tube in terms of possible association of intussusception.

The effects of portal venous inoculation with donor splenocytes on lung allograft survival were evaluated in dogs. Within the dose range examined, portal venous inoculation with donor splenocytes alone did not affect the graft survival time. Although the mean graft survival time was prolonged by FK 506 treatment for 14 days, the transplanted lungs were rejected within 12 days after termination of FK 506 administration. However, when the recipients were given portal venous inoculation with donor splenocytes at the time of transplantation, the graft survival time after termination of FK 506 administration was significantly prolongated. In the recipients that received combined therapy of portal venous inoculation with donor splenocytes and FK 506 treatment, arterial oxygen tension and histologic architecture of the transplanted lungs remained within the normal range for a longer period than these measures did in control animals that received FK 506 treatment alone. Dose-response experiments revealed that 1 × 10 8 cells/recipient was most effective in causing prolonged graft survival after termination of FK 506 treatment. On the basis of these results, perioperative portal venous inoculation with donor splenocytes can enhance the immunosuppressive effect of FK 506 in the canine lung transplantation model. (J T horac C ardiovasc S urg 1996;112:300-5)

Efforts to expand treatment options for articular cartilage repair have increasingly focused on the implantation of cell-polymer constructs. The purpose of this study is to determine the suitability of porous D,D-L,L-polylactic acid as a carrier for delivering repair cells obtained from rib perichondrium into full-thickness articular cartilage defects. In vitro characterization of perichondrocyte-polylactic acid composite grafts was combined with in vivo assessment of the early articular cartilage repair in a clinically relevant model. Using a fluorescent double-stain protocol to visualize live and dead cells in situ, primary cells cultured from perichondrium were found to be capable of attaching to and surviving within a porous D,D-L,L-polylactic acid matrix. These perichondrocyte-polylactic acid composite grafts were then implanted within osteochondral defects drilled into the left medial femoral condyles of 16 adult New Zealand white rabbits. Experimental animals were sacrificed 6 weeks after implantation and the repair tissue was evaluated grossly, histologically, and biochemically. Grossly, 96% (15/16) of the experimental animals demonstrated repairs consisting of a smooth, firm neocartilage which appeared similar in color and texture to the surrounding articular surface. Matrix staining for cartilaginous protein was seen surrounding chondrocyte-like cells in the cartilage regions of the repair. Cellular alignment was found to be related to scaffold architecture. These results suggest that scaffolds composed of porous D,D-L,L-polylactic acid support the growth of cartilaginous repair tissue and are compatible with both in vitro and in vivo survival of chondrogenic cells.

New molecular targeting agents against multiple myeloma (MM) have been developed and the prognosis of patients with MM has been improved. However, MM still remains an incurable disease. It is important to continue to investigate new therapeutic agents based on the biology of MM cells. Bromodomain (BRD4) inhibitors such as JQ-1 and I-BET151 have been shown as candidates for a molecular targeting agent against MM. In this study, we searched for chemical compounds inhibiting BRD4 binding ability and we investigated their effects on MM cell proliferation. We identified novel compounds CA1 and CA2 as a BRD4 inhibitor using an alpha screening assay. The IC50 values of the binding ability of recombinant human BRD4-BD1-BD2 of CA1 and CA2 are 0.44 and 1.1 mM, respectively (Figure 1). We next investigated the effects of CA1 and CA2 on MM cell lines (MM.1s, AMO-1, NCIH929, and OPM-2 cells). Analyses with MTT assay showed that these compounds inhibited the proliferation of MM cells in a dose-dependent manner. c-myc and cyclinD1 mRNA transcripts were decreased by the treatment of CA1 and CA2, while, p21 mRNA transcripts were increased (Figure 2). In flow cytometric analysis, cell cycle arrest occurred in a G1 phase and apoptosis was induced. Western blotting analysis showed the decrease of c-myc protein levels as well as the cleavage of caspase-3. In conclusion, novel BRD4 inhibitors CA1 and CA2 inhibited binding of BRD4 to its ligands, resulting in the suppression of MM cell proliferation and the induction of apoptosis via the caspase activation. Figure 1. Figure 1. Figure 2. Figure 2. Disclosures Yoshioka: ConverGene LLC: Employment. Strovel:ConverGene LLC: Employment.

The relative binding affinities of Mnt protein are determined for each possible base-pair at position 15 of the operator sequence, and for all combinations of G.C base-pairs at positions 15 and 17. The partitioning of each operator sequence is determined quantitatively with restriction enzymes. At position 15, the wild-type G.C base-pair provides the highest binding affinity but, unlike position 17, the primary distinction is between purine and pyrimidine bases on the top strand. The information content at position 15 is only about 0.16 bit. In comparison with previous measurements at position 17, it is determined that the interactions of the Mnt protein with positions 15 and 17 are independent, i.e. the specific binding energies for the two positions are additive. The relative binding affinities at position 17 are also determined in the background of a G to T mutation at position 5, the position equivalent to 17 on the other half of the symmetric operator. The relative affinities at position 17 are independent of whether position 5 is wild-type or mutant.

The relative binding affinities of Mnt protein from bacteriophage P22 are determined for each possible base pair at position 17 of the operator. These are determined from the partitioning of randomized operators into bound and unbound fractions; quantitation is provided by restriction enzyme analysis. Mnt protein is found to have an unusual specificity at this position: a C.G base pair (the wild-type operator) has the highest affinity, a G.C base pair has the lowest affinity, and both orientations of A.T base pairs are intermediate and nearly equivalent. A specific binding constant and specific binding free energy are defined and shown to be directly related to the information content of the operator sequences bound to the protein, taking into account the quantitative differences in binding affinities.

In the clinical application of hyperthermia, determining the thermotolerance that influences the anti-tumor effect is an important problem. The purpose of this study was to investigate the methods of induction and disappearance of thermotolerance in vivo. After transplanting MH134 cancer cells into the paws of C3H mice, local hyperthermia with warm water was administered, and the movement of thermotolerance in vivo studied in terms of the heating intervals and tumor growth times. When the first heating was applied on the 8th day after transplantation, thermotolerance appeared within 1 hour, increasing gradually to reach a maximum at 18 hours, after which it decreased gradually and disappeared after 48 hours. When the first heating was applied on the 13th day after transplantation, which fell during the rapid tumor proliferation period, the movement of thermotolerance presented a similar pattern of appearance and disappearance. The results of this study made it clear that there was no difference in the movement of thermotolerance between the two periods even though each had a different rate of tumor proliferation.

Jumping metastasis to the mediastinal lymph nodes in non small cell stage III A lung cancer was evaluated. The study population included 76 patients with adenocarcinoma and 74 with squamous cell carcinoma.1) Hilar lymph node involvement was seen in 23 of the 76 cases (30%) with adenocarcinoma and 14 of the 74 cases (19%) with squamous cell carcinoma. Mediastinal lymph node involvement was seen in 29 of the 76 patients with adenocarcinoma (43%) and 14 of the 74 patients with squamous cell carcinoma (19%).2) The frequency of jumping metastases were seen in 12 of the 76 patients (16%) with adenocarcinoma and in 9 of the 74 patients (12%) with squamous cell carcinoma.3) The frequency of lymphatic metastasis was not related to the location and histological type of the primary cancer.4) The 5-year survival rate of the stage MA group with jumping metastasis tended to be longer than that of the stage III A group without jumping metastasis (p

Preparatory education has been provided for both nursing students and teachers to understand the electronic patient record (EPR) since 2004 when EPR was introduced to the hospitals where students are allocated to undertake their work experience. First, the training and management board contacted our medical information department for an appointment and sent us a working group. They taught the nursing training staff how to use EPR and how to assign students to the proper patient record in the EPR system. Second, as preparatory education for the students, they explained the procedure for the use of EPR and the protection of personal information. Students practiced with training in the EPR system, focusing on the functions which are used frequently in practical tasks. As a result of this preparatory education, students understood the protection of personal information very well, although their understanding of the operation and management of the equipment was relatively poor and adversely affected their practice. We need to review our education contents more often. We also need to examine the present state of understanding of EPR and the problems of teaching in practical nursing training.

Two cases of intraaortic balloon entrapment were presented. In patient 1, urokinase was injected into the gas driver lumen of the intraaortic balloon catheter, then it could be removed. In patient 2, the intraaortic balloon was removed retrogradely through the left axillary artery. If the entrapped intraaortic balloon is encountered, it is effective to try a clot-lysis method initially, followed by a surgical approach through a left axillary artery.

Arteriosclerosis in the ascending aorta is widely accepted as a strong risk factor for the occurrence of stroke after coronary artery bypass grafting (CABG). The aortic no-touch technique, with a variety of modifications, has been used to reduce the risk of post-CABG stroke. Saphenous vein grafts (SVGs) have been used for axillocoronary bypass grafting, a modification of the aortic no-touch technique. However, kinking or compression often occurs with SVGs. We report here the successful application of an 8-mm expanded polytetrafluoroethylene graft, of the external bead support type, that was used to cover an SVG during axillocoronary bypass grafting.

When teaching students the hospital information system, it is impossible to use an actual hospital information system because of security reasons. To overcome this problem, a simulator of the hospital information system for student education has been developed. The purpose of this system is to help students understanding a hospital information system from actual experience. The characteristics of this system are as follows: 1) Students can easily learn a hospital information system on the Web pages in the computer training room. 2) The present system is not as complete as a hospital information system, however, helpful explanations regarding the data processing have been inserted. 3) The fictional patient data have been prepared for the pages relating to the electronic medical chart. Consequently, students can understand what kind of data has been saved in this system. Through a questionnaire, students evaluated this system in terms of understandability. The result from four years between 1999 and 2002 showed that 70% of them evaluated it as good system. We therefore consider this system to be effective over a short period of time, and useful for medical education.

Summary Objective: Traditionally, histological analysis of cartilage and cartilage repair has been performed qualitatively. While new techniques and grading systems have attempted to improve the quantitative nature of histological assessment, the advent of computer-based analysis systems have enabled development of more quantitative methodologies for cartilage repair. The objective of this study was to develop such a methodology for a more quantitative assessment of cartilage repair using a color-based image analysis system. Design: Repair parameters were defined to describe the degree of cartilage restoration: repair dimensions, degree of attachment, surface roughness and repair location. This technique was experimentally applied to a cartilage repair study using cultured perichondrial cells implanted in a polylactic acid matrix in the rabbit femoral condyle. Specimens were examined and compared with the contralateral normal knee. Results: Results showed increases in cartilage height, repair area, and surface roughness over controls for both 6 and 12 weeks. Surface elevation was significantly decreased at 6 weeks over 12 weeks. The percentage of repair improved between 6 and 12 weeks. Results were compared with a traditional grading system and demonstrated close correlation. Intraobserver and interobserver precision analysis were performed and demonstrated the reproducibility of the quantitative results by and between individuals. Conclusions: The methodology was deemed successful for a more objective analysis of cartilage with the added advantage of providing measured parameters that can assist in making comparisons between different studies using the same methodology.

Articular cartilage repair remains a clinical and scientific challenge with increasing interest focused on the transplantation of chondrogenic cells. This study evaluated the repair response during a 1-year period after implantation of allogenic perichondrium cell polylactic acid composite grafts into 3.7 x 5 mm osteochondral defects drilled into the medial femoral condyles of 82 adult New Zealand White rabbits. The repair tissue was evaluated grossly, histologically, histomorphometrically, biochemically, and biomechanically at 6 weeks, 12 weeks, 6 months, and 1 year after implantation. After gross evaluation, cartilaginous material appeared to fill the defect in 70 experimental knees, for an overall repair frequency of 85%. The histomorphometric results and the histologic appearances were variable. None of the specimens were completely normal at 1 year. Only specimens with subchondral bone reformation displayed a definable cartilage appearing surface with chondrocytes surrounded by dense matrix. Subchondral bone reformation was inconsistent, reaching 50% at 1 year. Biochemically, the repair tissue matured during a 1-year period into a hyaline Type II collagen dominant tissue, whereas glycosaminoglycan content remained low at all time periods. The measured compressive properties of the repair tissue at 1 year were not significantly different from those of the contralateral knee that was not surgically treated. The treatment of osteochondral defects in the rabbit knee with allogenic perichondrium cell polylactic acid composite grafts yielded a high percentage of grossly successful repairs that showed inconsistent subchondral bone reformation. These results suggest that healthy subchondral bone is important to articular cartilage repair. They also highlight that a cartilaginous appearing tissue at gross inspection may not represent structurally normal articular cartilage. Continued multidisciplinary studies on the arthroplastic potential of rib perichondrial cells are needed before human studies, which rarely can extend beyond gross assessment of repair tissue appearance can be undertaken.

SummaryNinety-nine mature New Zealand White (NZW) rabbits underwent unilateral anterior cruciate ligament transection (ACLT) and were divided into three groups. The contralateral non-operated knees served as controls. The first group (SA) received intra-articular injections of 0.3 ml hyaluronan (HA: MW; 8 × 105) beginning 4 weeks after ACLT, once a week for 5 weeks. The second group (SV) was injected with vehicle (carrier of HA) in the same fashion as the SA group. The third group (SN) served as a nontreatment group post ACLT. All animals were killed 9 weeks post-surgery and were assessed by gross morphology, histomorphometry and biochemical analysis. Gross morphologic changes on the femoral cartilage in the SA group were less severe than those in the SV and SN groups. Cartilage thickness, cartilage area, and thickness of synovial lining cell layer histomorphometric parameters were measured, showing a positive effect of HA on the preservation of articular cartilage and synovial tissue. Similarly, the cartilage and synovial tissues from knees injected with HA did not demonstrate significant alterations from contralateral controls as measured by biochemical analysis [i.e., water content, pyridinoline concentration, glycosaminoglycan (GAG) content for the cartilage, and DNA concentration for the synovial tissue].

Summary A new computerized method of histomorphometry was used to assess the development of osteoarthritis (OA) in a rabbit model. Three groups of 10 New Zealand White rabbits with closed epiphyses underwent unilateral anterior cruciate ligament transection (ACLT) and contralateral arthrotomy (sham). Groups were killed at 4, 8 and 12 weeks. At the time of death the femoral condyles were assessed grossly following the application of India ink using the following grading scale. Grade 1: intact surface; grade 2: minimal fibrillation; grade 3: overt fibrillation; grade 4: erosion. All histological sections were assessed using a color image analysis system. The mean thickness and area were measured for a defined cartilage region. The root mean square surface roughness (based on deviations from an idealized smooth surface) was calculated to assess the surface profile of the articular cartilage. The results were as follows. After ACLT, no full-thickness ulceration was noted at 4 weeks. Four of the medial femoral condyles at 8 weeks and six at 12 weeks showed full-thickness ulceration of the articular cartilage. The per cent cartilage area and cartilage thickness (ACLT divided by sham) in almost all regions showed decreases with time, indicating progressive erosion. The surface of the ACLT knees was much rougher than that of sham of the knees. These results demonstrate the usefulness of a quantitative methodology using a computerized video analysis system to assess the articular cartilage following ACLT in a rabbit model for the development of OA.

This paper describes the importance of 3.5" magneto-optical media as the secure hyper storage technology in an information centric global network.

We report herein the rare case of an 11-year-old boy in whom mucoepidermoid carcinoma of the right upper lobe bronchus was successfully treated by bronchoplasty. The patient underwent bronchoscopy to investigate the cause of relapsing respiratory infections over the past 2 years, which revealed a tumor at the orifice of the right upper lobe bronchus. Thus, a right upper sleeve lobectomy was effectively carried out, preserving right pulmonary function. The tumor was observed to partially invade the bronchial wall, but not the lung parenchyma. Histological examination confirmed a diagnosis of mucoepidermoid carcinoma, classified as grade 2 by Conlan's classification. The patient has been well and free of recurrence for 3 years postoperatively.