Transition of Akabane virus genogroups and its association with changes in the nature of disease in Japan.

Akabane virus (AKAV) is teratogenic to the foetus of domestic ruminants and causes a significant reproduction loss in cattle in Japan. In several past epizootics in cattle, AKAV was also associated with post-natal encephalomyelitis, mainly in calves and young stock. Previously analysed AKAV isolates in East Asia form two major clusters, genogroups I and II, with isolates involved in encephalomyelitis belonging mainly to the former. Between 2007 and 2013, AKAV epizootics were regularly observed in Japan during the summer/autumn season, and abnormal deliveries and post-natal encephalomyelitis caused by the virus in cattle were reported. During this period, 30 AKAV isolates were obtained from diseased and sentinel cattle, a piglet and Culicoides biting midges throughout Japan and were subjected to genetic comparison and phylogenetic analysis with previous isolates. In 2007, 2011 and 2013, AKAV belonging to genogroup I was identified in the central nervous systems of calves showing neurological disorders. Notably, a total of 165 cases of bovine encephalomyelitis were reported in 2011 and the isolated viruses from affected animals shared high genetic identities with a South Korean isolate that was associated with a large outbreak in 2010, suggesting some epidemiological linkage between these epizootics. Epizootics of genogroup II were observed in 2008 and 2010, but bovine post-natal encephalomyelitis cases rarely occurred. Our findings suggest that the frequent incursion of genogroup I isolates has increased the frequency of post-natal encephalomyelitis cases in Japan in recent years. Infection by genogroup I virus was also identified in piglets with neurological disorders or congenital malformations in 2011 and 2013. The aetiological role of AKAV in pigs should be elucidated in the future.

Bovine Arboviruses in Culicoides Biting Midges and Sentinel Cattle in Southern Japan from 2003 to 2013.

Epizootic congenital abnormalities, encephalomyelitis and febrile illnesses in cattle caused by arthropod-borne viruses (arboviruses) are prevalent in Japan. Causative viruses including orthobunyaviruses, orbiviruses and rhabdovirus are thought to be transmitted by Culicoides biting midges. Recently, the incursions of several arboviruses, potentially Culicoides-borne, were newly confirmed in Japan. However, their spread pattern and exact vector species are currently uncertain. Attempts to isolate arboviruses from Culicoides biting midges and sentinel cattle were conducted in Kagoshima, located at the southernmost end of the main islands of Japan, a potentially high-risk area for incursion of arboviral diseases and outbreak of endemic ones. Seventy-eight isolates comprising Akabane, Peaton and Sathuperi viruses of the genus Orthobunyavirus of the family Bunyaviridae, bluetongue virus serotype 16, D'Aguilar virus, Bunyip Creek virus and epizootic haemorrhagic disease virus serotype 1 of the genus Orbivirus of the family Reoviridae, a potentially novel rhabdovirus of the genus Ephemerovirus and unidentified orbivirus-like viruses were obtained from Culicoides biting midges and sentinel cattle between 2003 and 2013. Akabane, Sathuperi, D'Aguilar and Bunyip Creek viruses were selectively isolated from Culicoides oxystoma, suggesting this vector's responsibility for these arbovirus outbreaks. The results of virus isolation also implied that C. tainanus, C. jacobsoni and C. punctatus are competent for the transmission of bluetongue virus serotype 16, Peaton virus and epizootic haemorrhagic disease virus serotype 1, respectively. Our monitoring in Culicoides biting midges and sentinel cattle detected the circulation of Akabane virus just prior to the accumulations of bovine congenital abnormalities and encephalomyelitis by it around study sites in 2003, 2006, 2008 and 2013. Silent circulations of the other arboviruses, including potentially new viruses, were also detected during the study period.

Nationwide surveillance of West Nile virus targeting mosquitoes and dead birds from April 2004 through March 2007 in Japan.

We conducted nationwide West Nile virus (WNV) surveillance targeting mosquitoes and dead birds to reveal whether the virus and its potential vectors are present in Japan. A total of 12 766 mosquitoes and 230 dead birds were collected in April 2004-March 2005 (the 2004-2005 period), 10 755 mosquitoes and 267 dead birds in April 2005-March 2006 (the 2005-2006 period), and 8624 mosquitoes and 245 dead birds in April 2006-March 2007 (the 2006-2007 period). The species of most of the mosquitoes collected over the 3 years were Culex tritaeniorhynchus (47.82%) and Anopheles sinensis (28.49%), and other species included Aedes albopictus (6.75%), the Culex pipiens group (Cx. pipiens pallens and Cx. pipiens molestus: 5.37%), Aedes vexans nipponii (2.54%), Armigeres subalbatus (1.08%), and Aedes japonicus (0.95%). As for the dead birds, most were Passeriformes (456 specimens), which included several crow species, and the other orders included Anseriformes, Columbiformes and Ciconiiformes (78, 66 and 36 specimens, respectively). All the specimens tested negative for WNV RNA by reverse-transcriptase polymerase chain reaction (RT-PCR) in the 2004-2005 period and by real-time RT-PCR in the 2005-2006 and the 2006-2007 periods, respectively. Our surveillance provided no evidence for WNV in Japan as of the end of the surveillance period, but on the other hand, it revealed that several species of potential WNV vectors are distributed widely in Japan, which suggests that WNV in principle could be transmitted by the potential vectors if introduced. Thus, it is essential to take continued precautions against WNV introduction.

Identification and characterization of cross-reactive antigens from Neospora caninum and Toxoplasma gondii.

Murine monoclonal antibodies (mAbs) against Neospora caninum tachyzoites were produced to identify the cross-reactive antigens between N. caninum and Toxoplasma gondii. Ten mAbs recognizing cross-reactive antigens of both parasites were obtained and tentatively classified into 6 different groups based on their reactivity patterns in an indirect fluorescent antibody test and Western blot analysis. Three mAbs in group 1 recognized antigens located on the surface of parasites with molecular masses ranging from 28 to 76 kDa; one mAb in group 2 recognized antigens located on interior organelles of parasites with a molecular mass of 50 kDa; one mAb in group 3 recognized antigens located on interior organelles of parasites with molecular masses of 35 kDa and 14 kDa; three mAbs in group 4 recognized antigens located on interior organelles with a molecular mass of 64 kDa; one mAb in group 5 recognized antigens located on the surface of parasites with an unknown molecular mass; one mAb in group 6 recognized antigens located on the apical end of parasites with an unknown molecular mass. The mAbs in groups 1, 2, 3, and 5 showed inhibitory effects on the growth of the two parasites in vitro in a dose-dependent manner. A cDNA expression library prepared from N. caninum tachyzoite mRNA was immunoscreened with the mAb panel. Three kinds of proteins, protein disulfide isomerase (PDI), heat-shock protein 70 (HSP70), and ribosomal protein 1 (RP1), were identified as cross-reactive antigens recognized by mAbs in groups 2, 3, and 4, respectively. Some of the proteins could be useful in developing vaccines or drugs for controlling the diseases caused by the two parasites.

Role of gob-5 in mucus overproduction and airway hyperresponsiveness in asthma.

Airway hyperresponsiveness (AHR), goblet cell metaplasia, and mucus overproduction are important features of bronchial asthma. To elucidate the molecular mechanisms behind these pulmonary pathologies, we examined for genes preferentially expressed in the lungs of a murine model of allergic asthma by using suppression subtractive hybridization (SSH). We identified a gene called gob-5 that had a selective expression pattern in the airway epithelium with AHR. Here, we show that gob-5, a member of the calcium-activated chloride channel family, is a key molecule in the induction of murine asthma. Intratracheal administration of adenovirus-expressing antisense gob-5 RNA into AHR-model mice efficiently suppressed the asthma phenotype, including AHR and mucus overproduction. In contrast, overexpression of gob-5 in airway epithelia by using an adenoviral vector exacerbated the asthma phenotype. Introduction of either gob-5 or hCLCA1, the human counterpart of gob-5, into the human mucoepidermoid cell line NCI-H292 induced mucus production as well as MUC5AC expression. Our results indicated that gob-5 may play a critical role in murine asthma, and its human counterpart hCLCA1 is therefore a potential target for asthma therapy.

Change in the localization of heat shock protein 27 (HSP 27) in BG-1 human ovarian cancer cells following treatment by the ether lipid ET-18-OCH3.

We have demonstrated a higher nuclear protein content in the hypodiploid fraction of BG-1 human ovarian cancer cells following treatment with one of the ether lipids, ET-18-OCH3. In this study, we have attempted to identify the overexpressed nuclear protein induced in those dying or dead cells in the hypodiploid fraction and its localization before and after ET-18-OCH3 treatment. The pattern of nuclear proteins was analyzed by two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) before and after ET-18-OCH3 treatment. The partial amino acid sequence of the most dominantly and consistently up-regulated protein spot after ET-18-OCH3 treatment was determined and it was found to be heat shock protein 27 (HSP27). Immunofluorescence staining disclosed that HSP27 localizes in the cytoplasm of the BG-1 cells before ET-18-OCH3 treatment. Condensation of HSP27 around the nuclei was observed following treatment by ET-18-OCH3. Ultimately, the nuclei of the cells in the hypodiploid fraction were stained by immunofluorescent HSP27. These results indicate that change of the localization of HSP27 may play an important role as a component of the signal transduction pathways affected by ether lipids.

Cloning and expression of a novel lysophospholipase which structurally resembles lecithin cholesterol acyltransferase.

Lecithin cholesterol acyltransferase (LCAT) is the key enzyme in the esterification of plasma cholesterol and in the reverse cholesterol transport on high-density lipoprotein (HDL). We have found a novel LCAT-related gene among differentially expressed cDNA fragments between two types of foam cells derived from THP-1 cells, which are different in cholesterol efflux ability, using a subtractive PCR technique. The deduced 412-amino-acid sequence has 49% amino acid sequence similarity with human LCAT. In contrast to the liver-specific expression of LCAT, mRNA expression of the gene was observed mainly in peripheral tissues including kidney, placenta, pancreas, testis, spleen, heart, and skeletal muscle. The protein exists in human plasma and is probably associated with HDL. Moreover, we discovered that the recombinant protein hydrolyzed lysophosphatidylcholine (lysoPC), a proatherogenic lipid, to glycerophosphorylcholine and a free fatty acid. We have therefore named this novel enzyme LCAT-like lysophospholipase (LLPL), through which a new catabolic pathway for lysoPC on lipoproteins could be elucidated.

Cytokinetic and morphologic differences in ovarian cancer cells treated with ET-18-OCH3 and the DNA-interacting agent, etoposide.

New antineoplastic agents with different cytotoxic mechanisms are of interest for their ability to overcome resistance to conventional DNA-interacting agents. Ether lipids are known to be active against ovarian carcinoma both in vitro and in vivo, and the cell membrane is believed to be the target of their antitumor activity. In this study we have investigated the different cytokinetic and morphologic responses of human ovarian carcinoma cells (BG-1) to one of the ether lipids (ET-18-OCH3) and to etoposide. Etoposide induced a significantly greater G2/M block. However, the proportion of the cycling cell fraction decreased significantly in cells treated by ET-18-OCH3 and induction of the hypodiploid traction was strongly correlated with reduction of the cycling cell fraction. On the other hand, the hyperdiploid fraction was found to correlate with reduction of the cycling cell fraction in etoposide treated cells. Despite the significant appearance of the hypodiploid fraction, apoptosis was not observed by DNA-gel assay. Microscopic study showed that the hyperdiploid fraction represented cells with multiple nuclei. These observations support the unique lethal effect of ET-18-OCH3 on ovarian carcinoma cells, distinguishing it from the action of a typical DNA-interacting agent. The membrane-targeted ether lipids deserve consideration for the future chemotherapy of ovarian carcinoma, perhaps in combination with the appropriate DNA-interacting agent. New antineoplastic agents with different cytotoxic mechanisms are of interest not only for their unique inhibitory properties but also for their potential of overcoming resistance to conventional DNA-interacting agents. Ether lipids are known to be active against ovarian carcinoma both in vitro (1, 2, 3) and in vivo (4, 5), and the cell membrane is believed to be the target of their antitumor activity. Etoposide, a DNA-interacting agent, is also active against human ovarian cancer cells in vitro (6) or in clinical trials either as a single agent (7) or in combination with cisplatin (8). We have reported that a cytotoxic dose of one of the ether lipids, ET-18-OCH3, induces a G2/M block in BG-1 human ovarian cancer cells, and also a hypodiploid fraction as shown on DNA analysis by flow cytometry (FCM) (9). The G2/M block was also observed in BG-1 cells following etoposide treatment (6). In the present study, we have investigated the differences in the cytokinetic and morphologic responses of BG-1 cells to ET-18-OCH3 and to etoposide.

Novel, potent, and orally active substance P antagonists: synthesis and antagonist activity of N-benzylcarboxamide derivatives of pyrido[3,4-b]pyridine.

A series of 4-phenylisoquinolone derivatives were synthesized and evaluated for NK1 (substance P) antagonist activity. Highly potent antagonists, 4-phenyl-3-isoquinolone-N-benzylcarboxamides (11), were discovered from the structure-activity relationship studies on the isoquinolone-urea lead 1a. Optimization of the activity in this series resulted in the development of 5-phenyl-6-pyrido[3,4-b]pyridine-N-benzylcarboxamides (30) which are highly potent orally active NK1 antagonists. Among the compounds synthesized, N-[3,5-bis(trifluoromethyl)benzyl]-7,8-dihydro-N,7-dimethyl-8-oxo-5- (substituted phenyl)-6-pyrido[3,4-b]pyridinecarboxamides (30a,f,g) showed excellent antagonist activities with IC50 values (in vitro inhibition of [125I]-BH-SP binding in human IM-9 cells) of 0.21-0.34 nM and ED50 values (in vivo inhibition of capsaicin-induced plasma extravasation in guinea-pig trachea, iv) of 0.017-0.030 mg/kg. These compounds exhibited significantly potent activity upon oral administration with ED50 values of 0.068-0.17 mg/kg. Conformational studies on 30g indicated that the two stable conformers of 30g are quite similar to those of CP-99,994.

[A study on ceftriaxone in the perinatal period].

Ceftriaxone (CTRX), a new cephem antibiotic with high activity against Gram-positive and Gram-negative bacteria, was investigated pharmacokinetically in 30 mothers in the perinatal period. The obtained results are summarized below. 1. The maximum CTRX level in the maternal serum was 135 micrograms/ml between 20 and 25 minutes after an intravenous administration of 1 g of CTRX. 2. The transfer of CTRX into the umbilical cord serum and the amniotic fluid was very good. CTRX levels in these fluids were about 20% and 10% of the maternal serum level, respectively. 3. No side effect was observed in mothers or neonates. 4. CTRX is a useful antibiotic for perinatal infections.

[Study on cefotiam in the perinatal period].

Cefotiam (CTM), a new cephem antibiotic with high activity against Gram-positive and Gram-negative bacteria, has been investigated for use in 60 mothers in perinatal period, and following results were obtained. The concentration of CTM in maternal serum was 38 micrograms/ml at 0.5 hour after an intravenous administration of 1 g. A good transport of CTM into umbilical cord serum and amniotic fluid was observed after the intravenous administration into the mother. No adverse effect appeared in the neonates. The CTM is highly useful antibiotic in perinatal infections, and the safe dose of CTM to the mother in perinatal period is considered to be 1-2 g per day.

[A study on ceftazidime in the perinatal period].

Ceftazidime (CAZ), a new cephem antibiotic with high activity against Gram-negative bacteria, has been investigated for use in mothers in perinatal period, and the results obtained are summarized below. Into maternal serum, umbilical cord serum and amniotic fluid, CAZ showed good transfer after intravenous administration of 1 g or 2 g into mothers, and no adverse effect appeared in their neonates. The CAZ is a very useful antibiotic for the prophylaxis and the treatment of perinatal infections at a dose level of 1-2 g per day.

[Fundamental and clinical study on cefpiramide in obstetrics and gynecology. Obstetrics and Gynecology Study Group for Cefpiramide].

Fundamental and clinical studies on a new cephalosporin antibiotic, cefpiramide (CPM), was carried out under a joint study program, in order to evaluate the usefulness of the drug in treating infection of the female genital organs. The results obtained were as follows: CPM was readily transported to female genital organ tissues, and the concentrations of the drug exceeded 35 micrograms/g in various organ tissues in about 1 hour, following intravenous injection of 1 g. A level of more than 2 micrograms/g was maintained even 14 hours after the injection. The transport of CPM to various tissues was also studied following intravenous drip infusion of 1 g for 1 hour. The concentrations in tissues were slightly low but similar to those following intravenous injection. The peak concentration of the drug in the dead space exudate was 3.1-20.4 micrograms/ml, following intravenous injection and intravenous drip infusion of 1 g. The MIC80 of CPM were 3.13-12.5 micrograms/ml against S. aureus, Klebsiella sp., P. mirabilis and P. aeruginosa. Clinical effects of CPM were analyzed in 158 patients, including 56 cases with intrauterine infection, 37 cases with intrapelvic infection, 22 cases with external genital infection, 31 cases with adnexitis, 6 cases with postoperative wound infection and 6 cases with other infections. Excellent response was seen in 28 cases (17.7%), good response in 120 (75.9%) poor response in 10 (6.3%). The rate of response was calculated as 93.7%. Safety of the drug was analyzed in 258 patients, and side effects occurred in 4 (1.6%). Of these 4 patients, rash was in 1 patient, heat sensation in 1 patient, nausea in 1 patient and rash accompanying edema in 1 patient. Abnormal values in clinical laboratory findings were seen in 7 patients. Elevations of transaminase were seen in 5 patients and decrease of platelet was seen in a patient, and then elevations of transaminase with decrease of platelet was seen in a patient, and no other changes of particular note appeared.

[Fundamental studies on aspoxicillin in the field of gynecology. Penetration of the drug into the genital organs and the dead space fluid. Antibacterial activity of the drug against various strains of clinical isolates].

A new semisynthetic penicillin aspoxicillin (ASPC, TA-058) has been evaluated in regard to distribution into the genital organs and the dead space fluid and also in regard to antibiotic activity against various strains clinically isolated in the field of gynecology. The pharmacokinetic studies were made by using two-compartment model on the data of concentration in ovary, oviduct, endometrium, myometrium, cervix uteri and portio vaginalis of the genital organs and in dead space fluid. The following results were obtained. After one shot intravenous administration of 2.0 g ASPC, no marked difference was observed in concentration pattern between peripheral vein and uterine artery. Half-lives were 1.8 hours and 3.5 hours, respectively. The concentrations in various tissues of genital organs reached to the maximum at 36.2-73.6 micrograms/g during 0.07-0.44 hour with half-life varied from 0.53 to 1.3 hours. Area under the curves (AUC) of various tissues were reached to 30-65% of that uterine artery. After 2.0 g administration of ASPC drip intravenously (d.i.v.; for 30 or 60 minutes), there are no remarkable difference on simulation curve of concentration between peripheral vein and uterine artery. The peak level of various tissues attained to 22.5-40.3 micrograms/g during 0.53-0.56 hours and AUC of their concentration achieved about 20-40% of that of uterine artery after intravenous drip infusion for 30 minutes. After intravenous drip infusion for 60 minutes, tissue level reached to the maximum during 1.01-1.82 hours with half-life of 0.65-1.4 hours and AUC of tissue concentration achieved about 20-57% of that of uterine artery. The concentration of the drug in dead space fluid was reached to the maximum at 3.1 hours after the administration and its half-life was 5.3 hours. The in vitro activities of ASPC against clinical isolates were examined. The values of MIC70 and MIC80 were ranged 0.35-5.1 micrograms/ml against S. aureus, S. epidermidis, E. faecalis and P. mirabilis, respectively. Against E. coli, the value of MIC70 was attained 2.9 micrograms/ml, however, the value of MIC80 was more than 100 micrograms/ml due to the two-peaked activity of this drug against the bacteria. AUC and duration time of various tissue concentration were scrutinized regarding to the value of MIC80 of ASPC against S. aureus, S. epidermidis, E. faecalis and P. mirabilis.(ABSTRACT TRUNCATED AT 400 WORDS)

[Fundamental and clinical studies on latamoxef in the perinatal period].

Fundamental and clinical studies on latamoxef (LMOX) in the perinatal period were carried out, and following results were obtained. Concentration of LMOX was showed high peak levels in maternal serum, umbilical serum and amniotic fluid. LMOX seemed to be a very transferable compound to human tissues. LMOX was administered to 28 cases of various perinatal infections. Clinical responses were excellent in 13 cases, good in 15 cases and poor in none. And 140 cases of prophylactic use in the field of perinatal period were evaluated in good. No side effect was seen and an abnormal laboratory finding, the increase of GPT, was observed in only 1 case. LMOX was a highly useful antibiotic in perinatal infections, the safe dose range of LMOX into the perinatal mothers was estimated to be 2 g/day, with the maximum safe dose being 4 g/day.

[Clinical evaluation of latamoxef in the perinatal period].

Latamoxef (LMOX), a new oxacephem antibiotic with high activity against Gram-negative bacteria has been investigated for use in No. of 58 mothers in perinatal period, and obtained following results. Concentration of LMOX in maternal serum was 43.4 micrograms/ml at the 1 hour after intravenous administration of 1 g. In umbilical cord serum and amniotic fluid, LMOX showed good translation after intravenous administration of 1 g into the mother, but no adverse effect appeared in the neonate. LMOX is highly useful antibiotic in perinatal infections, and the safe dose of LMOX to the mother in perinatal period is 1--2 g per day considerably.

[Transfer of fosfomycin sodium into the pelvic organs].

To confirm efficacy and safety of fosfomysin sodium (FOM-Na) in the field of obstetrics and gynecology, hemodynamics and pelvic transference were analyzed using so-called three-compartment model in adult women treated with the drug at a dose of 2 g either as an one shot injection or an intravenous infusion over 1 hour. Following results were obtained. 1. Hemodynamics in these patients were similar to those obtained in healthy adult males, although there was a tendency that levels reached were lower and elimination was more rapid in cases of genital cancer. 2. As to drug concentration of dead cavity fluid, Tmax appeared at 1.75 hours after completion of administration irrespective of administration methods, with Cmax being 67.8 micrograms/ml and 50.5 micrograms/ml of intravenous injection and intravenous infusion, respectively. Thereafter, there was no difference between two administration methods. 3. Uterine tissue concentration reached to its Tmax at 15 to 30 minutes after intravenous injection and within 10 minutes after completion of intravenous infusion, Cmax being higher than 90 micrograms/g. Even at 5 hours, 15 to 25 micrograms/g of the drug was detected. From these results and from susceptibilities of clinical isolates to the drug, it was considered that FOM-Na at a dose level of 2 g is highly effective in the treatment of various infections in the field of obstetrics and gynecology including intrapelvic infections, uterine infections and adnexitis.

[Laboratory and clinical studies on latamoxef in the field of obstetrics and gynecology].

Latamoxef (LMOX) is a new antibiotic synthesized by Shionogi Research Laboratory. Chemically LMOX is especially unique with a sulfur atom replacing the oxygen atom in the 1 position of the conventional cephalosporin nucleus, and in addition, this antibiotic has a cephamycin-like structure. The antibacterial activity of LMOX shows high potency against Gram-negative bacteria, but tends to be weak against Gram-positive bacteria. The tissue levels of LMOX in humans after intravenous injection of 1 g were examined. The levels in uterine and adnexa uteri tissue at 1 hour after administration were 25.4 and 27.4 micrograms/g respectively. LMOX was administered to 147 cases in infections of obstetric and gynecological field. The clinical effect according to disease was 94.6% for intrauterine infections, 95.0% for adnexitis, 87.0% intrapelvic infections, and 100% for external genital organ infections, making a total of 92.5%. The rate of occurrence of side effects or abnormal laboratory findings was similar to or slightly less than that seen with other beta-lactam antibiotics.