Baylisascaris Potosis Larvae in Mice of Different Strains and Infectivity of Tissue Larvae.

Migration of Baylisascaris potosis larvae in different mouse strains were compared, and infectivity of the persisting larvae in mice tissues were investigated. Five strains of mice, BALB/c, C57BL/6, AKR, B10.BR, and ICR were inoculated with 1,000 B. potosis eggs/mouse, and necropsied at week 13 post inoculation (PI). The other uninfected ICR mice (secondary host) were inoculated with 43 larvae/ mouse recovered from mice at week 13 PI with eggs, and necropsied at day 21 PI. Larvae in organs or tissues were counted at necropsy. One AKR mouse showed torticollis and circling at day 56 PI. At necropsy at week 13 PI, larvae were recovered from all mice. A mean total larvae recovered were 124.1 (n=40). Majority of larvae were found in the carcass (mean 113.9) and some in the viscera (mean 9.9). Zero to 1 larva were found in the brain or eyes of some mice. There were no differences among the mouse strains in the number of larvae, except in the viscera; more larvae were seen in BALB/c or ICR than in B10.BR mice. No larvae were found in the secondary host mice. Present study demonstrated that B. potosis larvae migrate well in the carcass of any strains of mice, however, the tissue larvae did not infect the secondary host. Results of our present study suggest that B. potosis larvae is less aggressive for the nervous tissue migration than that of B. procyonis larvae which is commonly known to migrate in central nervous system of mammals and birds.

Experimental Infection with Baylisascaris Potosis in Chickens.

The larvae of the genus Baylisascaris can cause larva migrans in mammals and birds. This study investigated the larval migration of Baylisascaris potosis, the roundworm of kinkajou (Potos flavus), in chickens and the associated clinical manifestations of the host. Thirty-six 3-week-old chickens divided into 6 groups were orally inoculated with 3,000 B. potosis eggs/chick. Each group of chicken was necropsied at days 1, 2, 3, 7, 30 and 90 PI (post inoculation), and the number of larvae in various organs were counted until day 90 PI. No clinical signs were observed in chickens during the study. Larvae were detected from the liver, lungs or breast-muscles of 13/36 (36.1%) chickens. The mean total number of larvae in the liver, lungs and breast-muscles at days 1, 2, 3, 7, 30 and 90 PI were 0.34, 0.17, 1.66, 1.01, 0.17 and 0, respectively. No larvae were found in the brain, eyes, hid-limb muscles, heart, kidneys and spleen. Although infectivity of larvae in egg-inoculated chickens was low, the present study demonstrated that B. potosis larvae can migrate in chickens tissues up to day 30 PI. The result suggests that chickens can serve as a paratenic host for B. potosis and may underline a public health importance of B. potosis infection as a potential foodborne disease in humans.

High IL-1α Production Was Induced in the WBN/Kob-Leprfa Type 2 Diabetes Mellitus Rat Model and Inhibited by Syphacia Muris Infection.

The novel WBN/Kob-Leprfa (fa/fa) congenic rat strain is considered a useful rat model of type 2 diabetes mellitus (T2DM). Accumulating findings suggest that low-grade inflammation is a causative factor in T2DM and that circulating levels of inflammatory cytokines are associated with insulin resistance. However, inflammatory cytokine profiles and their correlations with T2DM development/ progression in fa/fa rats have not been studied. In this study, we found that the fa/fa rats had considerably high plasma levels of interleukin (IL)-1α. Abundant cecal IL-1α mRNA expression and cecal inflammation with infiltrating IL-1α-producing macrophages was observed in fa/fa rats. Bone marrow derived macrophages from fa/fa rats expressed high levels of IL-1α upon lipopolysaccharide stimulation. Furthermore, Syphacia muris infection, which delays the onset of T2DM, reduced both plasma and cecal IL-1α levels in fa/fa rats. These results suggest that macrophage infiltration and IL-1α secretion comprise an important part of T2DM development and that S. muris infection inhibits pro-inflammatory cytokine expression in fa/fa rats.

Randomized phase III study of bevacizumab plus FOLFIRI and bevacizumab plus mFOLFOX6 as first-line treatment for patients with metastatic colorectal cancer (WJOG4407G).

BACKGROUND: FOLFIRI and FOLFOX have shown equivalent efficacy for metastatic colorectal cancer (mCRC), but their comparative effectiveness is unknown when combined with bevacizumab. PATIENTS AND METHODS: WJOG4407G was a randomized, open-label, phase III trial conducted in Japan. Patients with previously untreated mCRC were randomized 1:1 to receive either FOLFIRI plus bevacizumab (FOLFIRI + Bev) or mFOLFOX6 plus bevacizumab (mFOLFOX6 + Bev), stratified by institution, adjuvant chemotherapy, and liver-limited disease. The primary end point was non-inferiority of FOLFIRI + Bev to mFOLFOX6 + Bev in progression-free survival (PFS), with an expected hazard ratio (HR) of 0.9 and non-inferiority margin of 1.25 (power 0.85, one-sided α-error 0.025). The secondary end points were response rate (RR), overall survival (OS), safety, and quality of life (QoL) during 18 months. This trial is registered to the University Hospital Medical Information Network, number UMIN000001396. RESULTS: Among 402 patients enrolled from September 2008 to January 2012, 395 patients were eligible for efficacy analysis. The median PFS for FOLFIRI + Bev (n = 197) and mFOLFOX6 + Bev (n = 198) were 12.1 and 10.7 months, respectively [HR, 0.905; 95% confidence interval (CI) 0.723-1.133; P = 0.003 for non-inferiority]. The median OS for FOLFIRI + Bev and mFOLFOX6 + Bev were 31.4 and 30.1 months, respectively (HR, 0.990; 95% CI 0.785-1.249). The best overall RRs were 64% for FOLFIRI + Bev and 62% for mFOLFOX6 + Bev. The common grade 3 or higher adverse events were leukopenia (11% in FOLFIRI + Bev/5% in mFOLFOX6 + Bev), neutropenia (46%/35%), diarrhea (9%/5%), febrile neutropenia (5%/2%), peripheral neuropathy (0%/22%), and venous thromboembolism (6%/2%). The QoL assessed by FACT-C (TOI-PFC) and FACT/GOG-Ntx was favorable for FOLFIRI + Bev during 18 months. CONCLUSION: FOLFIRI plus bevacizumab was non-inferior for PFS, compared with mFOLFOX6 plus bevacizumab, as the first-line systemic treatment for mCRC. CLINICAL TRIALS NUMBER: UMIN000001396.

The first trial of CIM331, a humanized antihuman interleukin-31 receptor A antibody, in healthy volunteers and patients with atopic dermatitis to evaluate safety, tolerability and pharmacokinetics of a single dose in a randomized, double-blind, placebo-controlled study.

BACKGROUND: The cytokine interleukin-31 (IL-31) is considered to be responsible for the development of pruritus in humans. At present, no available evidence has been provided on the safety and efficacy of blocking the IL-31 signal in humans for the amelioration of pruritus in atopic dermatitis (AD). CIM331 is a humanized antihuman IL-31 receptor A (IL-31RA) monoclonal antibody, which binds to IL-31RA to inhibit subsequent IL-31 signalling. OBJECTIVES: To assess the tolerability, safety, pharmacokinetics and preliminary efficacy of CIM331 in healthy Japanese and white volunteers, and Japanese patients with AD. METHODS: In this randomized, double-blind, placebo-controlled phase I/Ib study, CIM331 was administered in a single subcutaneous dose. The primary outcomes were safety and tolerability; the exploratory analysis was efficacy. RESULTS: No deaths, serious adverse events (AEs) or discontinuations due to AEs were reported in any part of the study. No dose-dependent increase in the incidence of AEs occurred in any part of the study. In healthy volunteers, all AEs occurred once in the placebo groups, and increased creatine phosphokinase was more common in the CIM331 groups. In patients with AD, CIM331 reduced pruritus visual analogue scale score to about -50% at week 4 with CIM331 compared with -20% with placebo. CIM331 increased sleep efficiency and decreased the use of hydrocortisone butyrate. CONCLUSIONS: A single subcutaneous administration of CIM331 was well tolerated in healthy volunteers and patients with AD. It decreased pruritus, sleep disturbance and topical use of hydrocortisone. CIM331 may become a novel therapeutic option for AD by inhibiting IL-31.

Survey of Japanese encephalitis virus in pigs and wild boars on Ishigaki and Iriomote Islands in Okinawa, Japan.

SUMMARY We previously revealed that Japanese encephalitis virus (JEV) seroprevalence was 4.5% in pigs on Ishigaki Island from 2005 to 2007. However, a partial E gene sequence (151 bp) of the JEV genome (JEV/sw/Ishigaki/1/2005) was detected in one pig. Phylogenetic analysis showed that JEV/sw/Ishigaki/1/2005 belonged to genotype III and to the same lineages isolated in Taiwan from 2006 to 2008. Serum samples were collected from 128 pigs on Ishigaki from 2009 to 2010, 24 wild boars on Ishigaki from 2008 to 2010, and 117 wild boars on Iriomote Island from 2008 to 2010. Four (3.1%) pigs on Ishigaki were positive for JEV antibody, but all wild boars on the island were negative. Fifty-two (44.4%) wild boars on Iriomote were positive for JEV antibody, in contrast to a seroprevalence of 3.7% in 2000 and 2004. JEV on Iriomote and/or in Taiwan might be related to transmission on Ishigaki.

Tissue rinse liquid-based cytology: a feasible tool for the intraoperative pathological evaluation of sentinel lymph nodes in breast cancer patients.

OBJECTIVES: A unique diagnostic method was designed for the intraoperative pathological evaluation of sentinel lymph nodes (SLNs) in breast cancer patients, and the results were verified with 2 years of experience. METHODS: Excised lymph nodes were cut into 2-mm-thick slices and rinsed thoroughly in CytoRich Red(®). The sliced tissues were embedded in a paraffin block. Three cytological glass slides of the cells exfoliated in CytoRich Red(®) were prepared by the SurePath(®) liquid-based cytology (LBC) technique. Two slides were stained by the Papanicolaou method, and the remaining slide was immunostained with an anti-keratin antibody. This process is called tissue rinse liquid-based cytology (TRLBC). The results of TRLBC were compared with those of the final pathological diagnoses, including immunostaining with an anti-keratin antibody on paraffin blocks (PB). RESULTS: This study analysed 444 SLNs from 247 consecutive breast cancer patients. It required 35 minutes to complete the intraoperative diagnosis on a single node, and it took an additional 5 minutes per node if more than one node was submitted. When the results of PB were assumed to be the gold standard, the sensitivity and specificity of TRLBC were 81.9% and 96.1%, respectively. TRLBC detected all nodes with macrometastasis and 23 of 24 nodes with micrometastasis. Fifteen false-negative TRLBC results were 'isolated tumour cell clusters' on PB, but there was one with micrometastasis histologically. Four of 14 false-positive TRLBC results were proven to be true positive by supplementary examination using step sectioning of the paraffin blocks of the nodes. CONCLUSION: TRLBC is a feasible and promising intraoperative cytopathological tool showing a comparable efficacy to PB while still allowing the conventional postoperative histological examination.

Cytodiagnosis through use of a z-axis video by volunteer observers: a promising tool for external quality assessment.

OBJECTIVE: This study examined whether cytological diagnosis through the use of a video, which shows the changing depth of focus in the microscopic field, described as a z-axis video, is useful compared with a still image. METHODS: From 17 cytology preparations of fine needle aspiration of the breast, we made six z-axis videos per case. A frame exhibiting the characteristic features was then extracted from each video and saved as a representative still image. One hundred and twenty-eight volunteer cytotechnologists were randomly divided into two groups of video observers and still image observers. The participants were asked to make a diagnosis of benign, indeterminate, suspicious or malignant without having any clinical information other than the age of the patient. Diagnoses were categorized as 'recommended' or 'unacceptable' according to degree of correlation with histology. RESULTS: The number of definitive diagnoses of 'benign' or 'malignant' were increased in video observers, and indeterminate or suspicious categories were decreased (P = 0.013). The distribution of diagnostic categories in three of the 17 cases was significantly different; the distribution in the remaining cases was similar between the two groups. The z-axis video observers may have selected the definite diagnoses with confidence because they observed valuable microscopic findings by 'focusing through observation'. The average number of 'recommended' diagnoses by individual observers was significantly higher in the video observer group than in the still image observer group (P = 0.016). In contrast, the average number of 'unacceptable' diagnoses was significantly lower (P = 0.019). CONCLUSIONS: A z-axis video is easy to obtain and is therefore expected to become a powerful diagnostic modality for the external quality assessment of clinical cytology and even in the field of primary cytodiagnosis.

Evaluation of pharyngeal swallowing pressure using high-resolution manometry during transoral surgery for oropharyngeal cancer.

BACKGROUND: Transoral robotic surgery is frequently described, driven by the desire to offer a less morbid alternative to chemoradiation. However, the objective evaluation of post-operative function has rarely been reported. Therefore, high-resolution manometry was used in this study to evaluate the impact of changes in peri-operative swallowing function on pharyngeal pressure events. METHODS: Ten patients with various stages of oropharyngeal cancer underwent transoral surgery. High-resolution manometry and videofluoroscopic swallow studies were performed before surgery and two months afterwards. The following parameters were obtained: velopharyngeal and mesopharyngeal post-deglutitive upper oesophageal sphincter pressures, velo-meso-hypopharyngeal contractile integral, upper oesophageal sphincter relaxation pressure, and pharyngeal velocity. RESULTS: There was no significant difference in pharyngeal pressure or contractile integral pre- versus post-operatively. However, pharyngeal velocity was significantly higher post-operatively than pre-operatively. CONCLUSION: High-resolution manometry showed that transoral surgery in patients without pre-operative dysphagia preserved pharyngeal constriction. However, transoral surgery might produce scar formation in the pharynx, which could lead to narrowing of the pharynx.

Performance enhancement of semiconductor devices by control of discrete dopant distribution.

As semiconductor devices are scaled down to the nanometre level, random dopant fluctuation in the conducting channel caused by the small number of dopant atoms will significantly affect device performance. We fabricated semiconductor devices with random discrete dopant distribution in the drain side and then evaluated how well we could control the drain current of the devices. The results showed that the drain current in devices with the dopant distribution in the drain side was several per cent higher than that in devices with the dopant distribution in the source side. We believe that this increase in current is caused by the suppression of injection velocity degradation in the source side. The capability to control the location of individual dopant atoms enhances drain current and, therefore, the performance of nanodevices. Accurately controlling both the amount and the positioning of dopant atoms is critical for the advancement of true nanoelectronics.

Efficacy of fluoroscopy-guided endoscopic cricopharyngeal myotomy.

BACKGROUND: In endoscopic cricopharyngeal myotomy, surgeons sometimes have concerns about performing an adequate incision with only a narrow intra-cavital view from one direction. In order to overcome these issues, fluoroscopic radiography was used during endoscopic cricopharyngeal myotomy. METHODS: Peri-operative fluoroscopic radiography was utilised to check the position of the diverticuloscope, and to confirm the extent of the incision during surgery. A balloon catheter was used to determine whether the cricopharyngeal muscle was sufficiently resected. Blood loss, peri-operative complications, and functional oral swallowing scale and penetration aspiration scale scores were evaluated. RESULTS: In 12 out of 15 patients, intra-operative fluoroscopic radiography showed the diverticuloscope positioned in the post-cricoid area, and the cricopharyngeal muscle was raised and the surgery completed without adverse effect. Swallowing functions improved following surgery. CONCLUSION: Intra-operative fluoroscopy might improve endoscopic cricopharyngeal myotomy by allowing surgeons to confirm the extent of resection, and by reducing peri-operative morbidity and complication rates.

Formation of a catalytically active dimer by tRNA(Val)-driven short ribozymes.

A minizyme is a hammerhead ribozyme with a short oligonucleotide linker instead of stem/loop II. Minizymes with low activity as monomers form active dimeric structures with a common stem. We explored the use of dimeric minizymes as gene-inactivating agents by placing minizymes under the control of a tRNA(Val) promoter. The tRNA(Val) portion of the transcript did not hinder dimerization as the tRNA-embedded minizyme formed an active dimeric structure. The cleavage activity of this minizyme that had been expressed either in vitro or in HeLa cells was almost one order of magnitude higher than that of the tRNA(Val)-embedded conventional hammerhead ribozyme. The tRNA(Val)-driven minizyme inhibited reporter gene activity (95%) whereas the tRNA(Val)-driven hammerhead ribozyme resulted in approximately 55% inhibition.

Small interfering RNA expression vector targeting hypoxia-inducible factor 1 alpha inhibits tumor growth in hepatobiliary and pancreatic cancers.

Hepatobiliary and pancreatic carcinomas are hypovascular tumors that can proliferate under hypoxic conditions. Recent reports have demonstrated that hypoxia-inducible factor 1 alpha (HIF1alpha) plays an important role in the survival of these cancers. Given these findings, the inhibition of the HIF1alpha pathway might prove to be a powerful tool in the treatment of these cancers. To inhibit HIF1alpha expression, we used small interference RNA (siRNA) expression vectors in this study. The transient transfection of siRNA expression vectors significantly reduced both HIF1alpha mRNA levels (13% of control) and protein levels (41% of control) and significantly inhibited the growth of cancer cell lines (P<0.05). VEGF, Glut1, and aldorase A expressions were also significantly reduced by transfection with these vectors (P<0.05), and we found that these vectors induced apoptosis but not cell cycle arrest. In a subcutaneous tumor model using nude mice, transfected MIA PaCa-2 cells, stably expressing siRNAs, barely formed tumors compared to control (P<0.05). This study thus demonstrates the usefulness of siRNA expression vector in targeting HIF1alpha and points to a potential clinical role in the treatment of pancreatic and hepatobiliary carcinomas.

Screening and determination of gene function using randomized ribozyme and siRNA libraries.

Rapid progress in the sequencing of the genomes of model organisms, such as the mouse, rat, nematode, fly, and Arabidopsis, as well as the human genome, has provided abundant sequence information, but functions of long stretches of these genomes remain to be determined. RNA-based technologies hold promise as tools that allow us to identify the specific functions of portions of these genomes. In particular, catalytic RNAs, known also as ribozymes, can be engineered for optimization of their activities in the intracellular environment. The introduction of a library of active ribozymes into cells, with subsequent screening for phenotypic changes, can be used for the rapid identification ofa gene function. Ribozyme technology complements another RNA-based tool for the determination of gene function, which is based on libraries of small interfering RNAs (siRNAs).

A further investigation and reappraisal of the thio effect in the cleavage reaction catalyzed by a hammerhead ribozyme.

We synthesized three types of 11mer substrate, namely the natural substrate S11O and the thio-substituted substrates S11 S pS and S11 R pS, in which the respective pro-S p and pro-R p oxygen atoms were replaced by sulfur, and subjected them to detailed kinetic analysis in the cleavage reaction catalyzed by a hammerhead ribozyme. In agreement with previous findings, in the presence of Mg(2+)or Ca(2+)ions the rate of ribozyme-catalyzed cleavage of S11 S pS was as high as that of S11O, whereas the corresponding rate for S11 R pS was nearly four orders of magnitude lower than that for either S11O or S11 S pS. However, the rate of the ribozyme-catalyzed reaction with each of the three substrates was enhanced by Cd(2+)ions. Such results have generally been taken as evidence that supports the direct interaction of the sulfur atom at the R p position of the cleavage site with the added Cd(2+)ion. However, our present analysis demonstrates that (i) the added Cd(2+)ion binds at the P9 site; (ii) the bound Cd(2+)ion at the P9 site replaces two Mg(2+)or two Ca(2+)ions, an observation that suggests a different mode of interaction with the added Cd(2+)ion; and, most importantly and in contrast to the conclusion reached by other investigators, (iii) the Cd(2+)ion does not interact with the sulfur atom at the R p position of the scissile phosphate either in the ground state or in the transition state.

Recent advances in the elucidation of the mechanisms of action of ribozymes.

The cleavage of RNA can be accelerated by a number of factors. These factors include an acidic group (Lewis acid) or a basic group that aids in the deprotonation of the attacking nucleophile, in effect enhancing the nucleophilicity of the nucleophile; an acidic group that can neutralize and stabilize the leaving group; and any environment that can stabilize the pentavalent species that is either a transition state or a short-lived intermediate. The catalytic properties of ribozymes are due to factors that are derived from the complicated and specific structure of the ribozyme-substrate complex. It was postulated initially that nature had adopted a rather narrowly defined mechanism for the cleavage of RNA. However, recent findings have clearly demonstrated the diversity of the mechanisms of ribozyme-catalyzed reactions. Such mechanisms include the metal-independent cleavage that occurs in reactions catalyzed by hairpin ribozymes and the general double-metal-ion mechanism of catalysis in reactions catalyzed by the Tetrahymena group I ribozyme. Furthermore, the architecture of the complex between the substrate and the hepatitis delta virus ribozyme allows perturbation of the pK(a) of ring nitrogens of cytosine and adenine. The resultant perturbed ring nitrogens appear to be directly involved in acid/base catalysis. Moreover, while high concentrations of monovalent metal ions or polyamines can facilitate cleavage by hammerhead ribozymes, divalent metal ions are the most effective acid/base catalysts under physiological conditions.

Significantly higher activity of a cytoplasmic hammerhead ribozyme than a corresponding nuclear counterpart: engineered tRNAs with an extended 3' end can be exported efficiently and specifically to the cytoplasm in mammalian cells.

Hammerhead ribozymes were expressed under the control of similar tRNA promoters, localizing transcripts either in the cytoplasm or the nucleus. The tRNA(Val)-driven ribozyme (tRNA-Rz; tRNA with extra sequences at the 3' end) that has been used in our ribozyme studies was exported efficiently into the cytoplasm and ribozyme activity was detected only in the cytoplasmic fraction. Both ends of the transported tRNA-Rz were characterized comprehensively and the results confirmed that tRNA-Rz had unprocessed 5' and 3' ends. Furthermore, it was also demonstrated that the activity of the exported ribozyme was significantly higher than that of the ribozyme which remained in the nucleus. We suggest that it is possible to engineer tRNA-Rz, which can be exported to the cytoplasm based on an understanding of secondary structures, and then tRNA-driven ribozymes may be co-localized with their target mRNAs in the cytoplasm of mammalian cells.

Working at the cutting edge: the creation of allosteric ribozymes.

The appropriate folding of catalytic RNA is a prerequisite for effective catalysis. A novel ribozyme, the maxizyme, has been generated and its activity can be controlled allosterically. The maxizymes work both in vitro and in vivo indicating the potential utility of this novel class of ribozyme as a gene-inactivating agent with a biosensor function.

Prevalence of Baylisascaris Roundworm in Captive Kinkajous in Japan.

Baylisascaris potosis causes larva migrans in animals. The present study evaluated the prevalence of B. potosis in captive kinkajous ( Potos flavus ) and the ability of milbemycin to treat natural infections of B. potosis in 2 female wild-caught kinkajous. In 2012, fecal samples were collected from 16 kinkajous in 6 zoological gardens and 29 imported captive kinkajous from 4 pet traders in Japan. Although all samples from zoological gardens were negative, 8 kinkajous from traders were positive for Baylisascaris eggs, at least 4 of which were wild caught in the Republic of Guyana. No associated human illness was reported from any of the facilities. The 2 infected kinkajous received a single oral administration of Milbemycin® A Tablets, which delivers 0.69-0.89 mg/kg milbemycin oxime. Fecal examinations on days 14 and 30 were negative for Baylisascaris eggs. These results demonstrated that milbemycin oxime has possible anthelmintic efficacy against Baylisascaris roundworms in captive kinkajous. We conclude that Baylisascaris infections are highly prevalent in wild-caught kinkajous in Japan and that most of the infected kinkajous were imported from the Republic of Guyana.