Dipylidiasis cases in Japan-an update by literature survey.

Dipylidium caninum is a cosmopolitan parasite of companion animals such as dogs and cats. Accidental infection in humans occur mostly in children. Although considerable number of cases were reported from Europe and the Americas, case reports of this zoonotic disease are rather scarce from Asian countries. The aim of this study is to report the results of literature survey on dipylidiasis cases in humans in Japan. Conclusively, we have found a total of 17 cases since the first case report in from Aichi Prefecture in 1925.

Differentiation of embryonic stem cells into lung-like cells using lung-derived matrix sheets.

Various extracellular matrix (ECM) in the lungs regulate tissue development and homeostasis, as well as provide support for cell structures. However, few studies regarding the effects of lung cell differentiation using lung-derived ECM (LM) alone have been reported. The present study investigated the capability of lung-derived matrix sheets (LMSs) to induce lung cell differentiation using mouse embryonic stem (ES) cells. Expressions of lung-related cell markers were significantly upregulated in ES-derived embryoid bodies (EBs) cultured on an LMS for two weeks. Moreover, immunohistochemical analysis of EBs grown on LMSs revealed differentiation of various lung-related cells. These results suggest that an LMS can be used to promote differentiation of stem cells into lung cells.

Local administration of shikonin improved the overall survival in orthotopic murine glioblastoma models with temozolomide resistance.

BACKGROUND: Glioblastoma is a type of intracranial malignancy. Shikonin, a Chinese traditional medicine, has been shown to have anti-tumor efficacy toward human glioblastoma cells in vitro. However, shikonin cannot easily cross the blood-brain barrier. To address this issue, we evaluated the anti-tumor effects of direct intracranial infusion of shikonin in in vivo orthotopic syngeneic murine glioblastoma models using C57BL/6 mice. MATERIALS AND METHODS: The cytotoxic effects of shikonin against murine glioblastoma cells, SB28 and CT-2A, were reported resistance to temozolomide, were evaluated using an allophycocyanin-conjugated annexin V and propidium iodide assay with flow cytometry. Impedance-based real-time cell analysis (RTCA) was used to analyze the inhibitory effects of shikonin on growth and proliferation. To evaluate the anti-tumor activity of shikonin in vivo, we used orthotopic syngeneic murine glioblastoma models with SB28 and CT-2A cells. RESULTS: In flow cytometry-based cytotoxic assays, shikonin induced apoptosis. RTCA indicated that shikonin decreased the cell index of murine glioblastoma cells, SB28 and CT-2A, in a dose-dependent manner (p < 0.0001 for both cell lines), while temozolomide did not (p = 0.91 and 0.82, respectively). In murine glioblastoma models, SB28 and CT-2A, direct intracranial infusion of shikonin, as a local chemotherapy, improved the overall survival of mice in a dose-dependent manner compared with control groups (p < 0.0001 and p = 0.02, respectively). While temozolomide did not (p = 0.48 and 0.52, respectively). CONCLUSIONS: The direct intracranial infusion of shikonin has potential as a local therapy for patients with glioblastoma.

Rapid whole genome sequencing methods for RNA viruses.

RNA viruses are the etiological agents of many infectious diseases. Since RNA viruses are error-prone during genome replication, rapid, accurate and economical whole RNA viral genome sequence determination is highly demanded. Next-generation sequencing (NGS) techniques perform whole viral genome sequencing due to their high-throughput sequencing capacity. However, the NGS techniques involve a significant burden for sample preparation. Since to generate complete viral genome coverage, genomic nucleic acid enrichment is required by reverse transcription PCR using virus-specific primers or by viral particle concentration. Furthermore, conventional NGS techniques cannot determine the 5' and 3' terminal sequences of the RNA viral genome. Therefore, the terminal sequences are determined one by one using rapid amplification of cDNA ends (RACE). However, since some RNA viruses have segmented genomes, the burden of the determination using RACE is proportional to the number of segments. To date, there is only one study attempting whole genome sequencing of multiple RNA viruses without using above mentioned methods, but the generated sequences' accuracy compared to the reference sequences was up to 97% and did not reach 100% due to the low read depth. Hence, we established novel methods, named PCR-NGS and RCA-NGS, that were optimized for an NGS machine, MinION. These methods do not require nucleic acid amplification with virus-specific PCR primers, physical viral particle enrichment, and RACE. These methods enable whole RNA viral genome sequencing by combining the following techniques: (1) removal of unwanted DNA and RNA other than the RNA viral genome by nuclease treatment; (2) the terminal of viral genome sequence determination by barcoded linkers ligation; (3) amplification of the viral genomic cDNA using ligated linker sequences-specific PCR or an isothermal DNA amplification technique, such as rolling circle amplification (RCA). The established method was evaluated using isolated RNA viruses with single-stranded, double-stranded, positive-stranded, negative-stranded, non-segmented or multi-segmented genomes. As a result, all the viral genome sequences could be determined with 100% accuracy, and these mean read depths were greater than 2,500×, at least using either of the methods. This method should allow for easy and economical determination of accurate RNA viral genomes.

Expression and distribution of generated neurons and endogenous precursors in rat cerebral cortical venous ischemia.

Neurogenesis in the subventricular zone (SVZ), subgranular zone (SGZ), and cerebral cortex is now a familiar event to confirm by cerebral arterial ischemia in rat models. However, it remains unclear whether cerebral venous ischemia (CVI) alone causes neurogenesis, and where that neurogenesis occurs. After creating CVI rat models via a two-vein occlusion (2-VO) method, neurogenesis was immunohistochemically evaluated by double-labeling 5-bromo-2'-deoxyuridine (BrdU)-positive cells with neuronal nuclei (NeuN) or doublecortin (DCX) antibody. Fifty Wistar rats were divided into two major groups (BrdU-NeuN and BrdU-DCX) and then separated into two subgroups (2-VO or sham). The total number of double-positive cells expressed inside a predefined region of interest (ROI) covering the ischemic area was compared between the two subgroups. Then, we divided the ROI into six sections to evaluate and compare the distribution of double-positive cells generated in each section between the two subgroups. The 2-VO subgroup presented more double-positive cells than the sham group in both BrdU-NeuN and BrdU-DCX groups, while the BrdU-DCX+2-VO group showed a characteristic distribution of double-positive cells in ROI 2 and ROI 3, suggesting areas of the ischemic core and penumbra, with a significant difference compared to the BrdU-DCX+sham group. This study demonstrates that CVI has the potential to induce endogenous neurogenesis, with significant numbers of both newly generated neurons and precursors observed in the ischemic area. The distribution of these cells suggests that the cortex could be the main origin of neurogenesis after cortical CVI.

Culture of organoids with vestibular cell-derived factors promotes differentiation of embryonic stem cells into inner ear vestibular hair cells.

Vestibular hair cells (V-HCs) residing in the inner ear have important roles related to balance. Although differentiation of pluripotent stem cells into HCs has been shown, an effective method has yet to be established. We previously reported that use of vestibular cell-derived conditioned medium (V-CM) was helpful to induce embryonic stem (ES) cells to differentiate into V-HC-like cells in two-dimensional (2D) cultures of ES-derived embryoid bodies (EBs). In the present report, V-CM was used with three-dimensional (3D) cultures of EBs, which resulted in augmented expression of V-HC-related markers (Math1, Myosin6, Brn3c, Dnah5), but not of the cochlear HC-related marker Lmod3. Gene expression analyses of both 2D and 3D EBs cultured for two weeks revealed a greater level of augmented induction of HC-related markers in the 3D-cultured EBs. These results indicate that a 3D culture in combination with use of V-CM is an effective method for producing V-HCs.

Schistosoma mansoni larvae in vitro cultures using Biomphalaria glabrata extracts.

Schistosomiasis is one of the most prevalent waterborne parasitic diseases affecting humans. In natural conditions, snails are necessary for maintenance of its lifecycle and also required as intermediate hosts to maintain the lifecycle in laboratory settings. In the present study, the location of S. mansoni larvae in Biomphalaria glabrata snails after infection (inoculation of miracidia) was investigated. Larvae were found located in the head-foot (HF) area of B. glabrata snails at 10 days post-infection (DPI), then their location was predominantly changed to the hepatopancreas and ovotestis (HPOT) area by 56 DPI. Next, the effects of extracts from various organs of B. glabrata snails including HF and HPOT for in vitro culturing of S. mansoni larvae were investigated. The HF extract enabled prolonged culturing of S. mansoni larvae. Furthermore, sequential use of that followed by the HPOT extract supported larval development or reproduction of daughter sporocysts. These results may provide important information for identifying essential factors and molecules for culturing Schistosoma larvae in vitro.

Impaired differentiation potential of CD34-positive cells derived from mouse hair follicles after long-term culture.

Hair follicle epithelial stem cells (HFSCs), which exist in the bulge region, have important functions for homeostasis of skin as well as hair follicle morphogenesis. Although several methods for isolation of HFSCs using a variety of stem cell markers have been reported, few investigations regarding culture methods or techniques to yield long-term maintenance of HFSCs in vitro have been conducted. In the present study, we screened different types of commercially available culture medium for culturing HFSCs. Among those tested, one type was shown capable of supporting the expression of stem cell markers in cultured HFSCs. However, both the differentiation potential and in vivo hair follicle-inducing ability of HFSCs serially passaged using that optimal medium were found to be impaired, probably because of altered responsiveness to Wnt signaling. The changes noted in HFSCs subjected to a long-term culture suggested that the Wnt signaling-related environment must be finely controlled for maintenance of the cells.

Dioctophyme renale (Goeze, 1782) (Nematoda, Dioctophymidae) parasitic in mammals other than humans: A comprehensive review.

A comprehensive review of the infection of mammals with the nematode Dioctophyme renale (Goeze, 1782) (Nematoda, Dioctophymidae) is presented. Mammals, including man, are the definitive hosts for this parasite. Several aspects of the infection with the parasite in mammals other than humans are critically evaluated: geographical distribution, host species recorded so far and the relative importance of the different hosts, location of parasites within the host, prevalence and intensity of the infection, diagnostic methods, pathology induced by the parasites, epidemiology and the methods of control and treatment. The authors provide an updated review about the infection, based on a extensive bibliographic search worldwide, and point out the most relevant aspects of the biology of the parasite as well as several research topics which need to be explored for a better understanding of the biology of this interesting and important parasitic nematode.

Clinical characteristics and molecular epidemiology of invasive Streptococcus agalactiae infections between 2007 and 2016 in Nara, Japan.

Invasive Streptococcus agalactiae (GBS) infections are increasingly common among neonates and the elderly. Therefore, GBS surveillance for better antibiotic treatment and prophylaxis strategies are needed. We retrospectively evaluated the clinical aspects of invasive infections and the phenotypic and genetic diversity of infectious isolates from Nara, Japan, collected between 2007 and 2016, by using information from hospital records. GBS strains collected from the blood and cerebrospinal fluid cultures were evaluated for capsular types, multi-locus sequence typing (MLST), antibiotic susceptibility, antibiotics resistance gene, and pulsed-field gel electrophoresis. Forty GBS isolates (10 from children and 30 from adults) were analyzed, and the distribution of molecular serotype and allelic profiles varied between children and adults. We found the rates of early-onset disease in neonates with birth complications to be higher than that of previous reports, indicating that there could be relevance between complications at birth and early-onset disease. Standard antibiotic prophylaxis strategies may need to be reconsidered in patients with birth complications. In adults, the mean age of the patients was 68 years (male: 63%). Primary bacteremia was the most common source of infection. In the neonates, six had early-onset diseases and four had late-onset diseases. The most frequently identified strains were molecular serotype Ia ST23 (40%) and molecular serotype Ib ST10 (20%) in children and molecular serotype Ib ST10 (17%), molecular serotype VI ST1 (13%), and molecular serotype V ST1 (13%) in adults. Levofloxacin-resistant molecular serotype Ib strains and molecular serotypes V and VI ST1 were common causes of GBS infection in adults but were rarely found in children. Furthermore, pulsed-field gel electrophoresis in our study showed that specific clone isolates, that tend to have antibiotics resistance were widespread horizontally for a decade. Continuous surveillance and molecular investigation are warranted to identify the transmission route and improve antibiotic treatment strategies.

Necessity to screen and treat latent tuberculosis before ruxolitinib treatment-Ruxolitinib-associated disseminated tuberculosis: A case report and literature review.

Ruxolitinib, a Janus kinase inhibitor, considerably improves symptoms of patients with polycythemia vera and primary or secondary myelofibrosis. However, its association with the development of infectious complications is a concern. Herein, we report the case of an 80-year-old man with primary myelofibrosis who developed disseminated tuberculosis during treatment with ruxolitinib at 15 mg twice daily and prednisone at 5 mg. We also reviewed the literature on patients who developed tuberculosis during treatment with ruxolitinib. There are 13 case reports of patients who developed tuberculosis during treatment with ruxolitinib, including our case. Disseminated tuberculosis manifestations were observed in 84.6 % of the patients and 50 % of them died. Although the interferon-gamma release assay was performed for seven of the patients with six positive results at the time of tuberculosis diagnosis, none were tested before the commencement of ruxolitinib. We suggest taking a history of tuberculosis and screening for and treating latent tuberculosis before administering ruxolitinib, especially in areas where the risk of tuberculosis is high.

Spinal epidural abscess caused by non-typhoidal Salmonella: A case report and literature review.

Non-typhoidal Salmonellae are Gram negative bacilli commonly causing self-limiting gastroenteritis, representing a public health issue particularly in tropical countries. Further, the epidemiology of invasive infection by non-typhoidal Salmonella species is poorly understood. Herein, we presented a case of an unusual Salmonella enterica subsp. enterica serovar Altona epidural abscess that cause osteomyelitis and psoas abscess in a 52-year-old Japanese man. To ensure adequate antibiotics penetration into the epidural space, the patient was treated with antibiotics in doses similar to those administered for meningitis. We also reviewed the literature on patients who developed non-typhoidal Salmonella epidural abscesses, and we found 10 other previously reported cases. Salmonella Enteritidis was the pathogen most commonly identified, similar to gastroenteritis. More surveillance of non-typhoidal Salmonella serovars, especially in cases of severe infection, and investigation of antibiotic penetration rate into the epidural space are warranted to decide the best treatment course.

Isolation and culture of pleural mesothelial cells.

Purpose: Although the isolation of rat and mouse mesothelial cells has previously been reported, most mesothelial cells used for experimental studies are obtained from peritoneal cells. Here, we describe an optimized method for the isolation and in vitro propagation of rodent pleural mesothelial cells without the requirement for specialized surgical techniques. Materials and Methods: To harvest pleural mesothelial cells, the pleural space of 8-9-week-old rats or older mice was filled with 0.25% trypsin in ethylenediaminetetraacetic acid (EDTA) buffer for 20 min at 37 °C. Cells were then harvested, and incubated at 37 °C in a humidified atmosphere with 5% CO2. Immunofluorescence analysis of plated pleural mesothelial cells was performed using Alexa 546 (calretinin). To investigate optimal proliferation conditions, medium enriched with various concentrations of fetal calf serum (FCS) was used for pleural mesothelial cell proliferation. Results: By day 10, confluent cell cultures were established, and the cells displayed an obvious cobblestone morphology. Immunofluorescence analysis of the cells demonstrated that all stained positive for Alexa 546 (calretinin) expression. Mesothelial cells grew better in medium containing 20% FCS than with 10% FCS. Conclusions: This is a simple procedure for the efficient collection of primary pleural mesothelial cells, which were obtained in defined culture conditions from the euthanized rodent thoracic cavity using trypsin-EDTA treatment. The ability to easily culture and maintain identifiable pleural mesothelial cells from rodents will be helpful for future experiments using these cells.

Differentiation of embryonic stem cells into inner ear vestibular hair cells using vestibular cell derived-conditioned medium.

Vestibular hair cells (V-HCs) in the inner ear have important roles and various functions. When V-HCs are damaged, crippling symptoms, such as vertigo, visual field oscillation, and imbalance, are often seen. Recently, several studies have reported differentiation of embryonic stem (ES) cells, as pluripotent stem cells, to HCs, though a method for producing V-HCs has yet to be established. In the present study, we used vestibular cell conditioned medium (V-CM) and effectively induced ES cells to differentiate into V-HCs. Expressions of V-HC-related markers (Math1, Myosin6, Brn3c, Dnah5) were significantly increased in ES cells cultured in V-CM for 2 weeks, while those were not observed in ES cells cultured without V-CM. On the other hand, the cochlear HC-related marker Lmod3 was either not detected or detected only faintly in those cells when cultured in V-CM. Our results demonstrate that V-CM has an ability to specifically induce differentiation of ES cells into V-HCs.

Effect of covering with cross-linked gelatin glue on tissue regeneration in a rat lung injury model.

OBJECTIVES: Thoracic reintervention is a common treatment; however, preventing adhesion of the lung to the thoracic cavity wall remains a problem. This study aimed to investigate the effect on pleural adhesion of covering the postoperative pleural injury site with cross-linked gelatin glue (gelatin plus glutaraldehyde, hereafter 'gelatin glue') and to evaluate the proliferation of healing cells on gelatin glue. METHODS: We created a rat incisional lung-wound model and compared the effects of sealing the wound with gelatin glue (group A, n = 5), fibrin glue (group B, n = 5) or fibrin glue with a polyglycolic acid sheet (group C, n = 5). Adhesions were assessed 28 days postoperatively and compared among the groups using the Karacam's scoring method. Lung-wound healing was studied histologically at day 7 postoperatively. Mesothelial cell proliferation was investigated on gelatin and fibrin glues in vitro. RESULTS: There were no or few adhesions of the chest wall in group A. The adhesion scores (mean ± standard deviation) were 1.2 ± 0.4, 2.6 ± 1.4 and 3.2 ± 1.2 in groups A, B and C, respectively (A vs C, P = 0.0496). During the healing process, the gelatin glue surface was covered by mesothelial-like cells. Proliferation of cultured mesothelial cells was promoted on the gelatin glue compared with the fibrin glue. CONCLUSIONS: Covering lung wounds with the gelatin glue reduced adhesions and promoted the growth of healing cells compared with the fibrin glue. These findings suggest that the gelatin glue may help prevent adhesions and thus be a therapeutically effective biomaterial in lung surgery.

Induction of lung-like cells from mouse embryonic stem cells by decellularized lung matrix.

Decellularization of tissues is a recently developed technique mostly used to provide a 3-dimensional matrix structure of the original organ, including decellularized lung tissues for lung transplantation. Based on the results of the present study, we propose new utilization of decellularized tissues as inducers of stem cell differentiation. Decellularized lung matrix (L-Mat) samples were prepared from mouse lungs by SDS treatment, then the effects of L-Mat on differentiation of ES cells into lung cells were investigated. ES cell derived-embryoid bodies (EBs) were transplanted into L-Mat samples and cultured for 2 weeks. At the end of the culture, expressions of lung cell-related markers, such as TTF-1 and SP-C (alveolar type II cells), AQP5 (alveolar type I cells), and CC10 (club cells), were detected in EB outgrowths in L-Mat, while those were not found in EB outgrowths attached to the dish. Our results demonstrated that L-Mat has an ability to induce differentiation of ES cells into lung-like cells.

Ancylostoma ceylanicum, novel etiological agent for traveler's diarrhea-report of four Japanese patients who returned from Southeast Asia and Papua New Guinea.

BACKGROUND: Countries in the Southeast Asia region have a high prevalence of soil-transmitted helminth, such as roundworm, whipworm, and hookworms [Ancylostoma duodenale, Necator americanus, Ancylostoma ceylanicum]. Recent molecular-based surveys have revealed that A. ceylanicum, a zoonotic hookworm, is likely the second most prevalent hookworm species infecting humans in that part of the world, while others have noted that this infection is an emerging public health risk not only for indigenous people but also for visitors from other countries. CASE PRESENTATION: We recently encountered four cases of A. ceylanicum infection in Japanese individuals who returned from Southeast Asia and Papua New Guinea. Case 1 was a 25-year-old male who stayed in a rainforest in Malaysia for 4 weeks, where he developed abdominal pain and diarrhea in the third week. Eleven adult worms (five males, six females) were expelled after treatment with pyrantel pamoate and identified as A. ceylanicum based on morphological characteristics and DNA sequences of the mitochondrial cytochrome c oxidase subunit 1 (cox1) gene. Case 2 was a 26-year-old male who spent 2 years as an overseas cooperation volunteer for agriculture in Papua New Guinea. He did not note any symptoms at that time, though eggs were detected in feces samples at a medical check-up examination after returning. Although collection of adult worms was unsuccessful, DNA analysis of the eggs for cox1 and the ribosomal internal transcribed spacer (ITS)-1 and ITS-2 genes demonstrated that they were A. ceylanicum. Case 3 was a 47-year-old male who spent 1 month in a rural village in Lao People's Democratic Republic and began suffering from watery diarrhea from the third week. A total of nine adult worms (three males, six females) were collected by endoscopic procedures and following treatment with pyrantel pamoate. Morphological examination and molecular analyses of the cox1 gene showed that they were A. ceylanicum. Case 4 was a 27-year-old male who participated in group travel to India for 5 days. Three weeks after returning, he developed abdominal pain and diarrhea. Hookworm eggs were found in feces samples and developed into larvae in culture, which were identified as A. ceylanicum based on molecular analysis of the cox1 gene. Eosinophilia was observed in all of the cases prior to treatment. CONCLUSIONS: A. ceylanicum should be recognized as an important etiologic pathogen of hookworm diseases in travelers to countries in the Southeast Asia and West Pacific Ocean regions.

Molecular diagnosis and characterization of a culture-negative mycotic aneurysm due to ST54 Haemophilus influenzae type b with PBP 3 alterations.

Mycotic aneurysm is a rare but life-threatening disease that warrants an integrated therapeutic approach involving surgical intervention and prolonged antibiotic use. However, the causative organisms are often unidentified because antibiotics started empirically render blood and tissue cultures negative. Molecular diagnosis has been reported to be useful in such culture-negative cases. We report a case of a culture-negative mycotic aortic aneurysm due to Haemophilus influenzae, diagnosed by direct 16S rRNA polymerase chain reaction (PCR) and sequencing of the resected aneurysm tissue. PCR for serotype revealed type b, and PCR and sequencing of the ftsI gene revealed alterations in penicillin-binding protein 3, suggesting resistance to ampicillin. Multilocus sequence typing demonstrated that the isolate belonged to sequence type 54.

Infective Endocarditis Complicated by Intraventricular Abscesses, Pericarditis, and Mycotic Aneurysm Due to an Emerging Strain of Serotype VI Streptococcus agalactiae.

An increasing number of invasive infections due to Streptococcus agalactiae in non-pregnant adults have been reported. We report a case of infective endocarditis complicated by intraventricular abscesses, pericarditis, and mycotic aneurysm due to S. agalactiae belonging to ST681 with a capsular serotype VI in a woman with diabetes. The patient also had a myocardial infarction and was treated with percutaneous coronary intervention, pericardiocentesis, and 6 weeks of antibiotic treatment. Invasive infections due to serotype VI S. agalactiae are common in Asian countries such as Taiwan and Japan, so continuous monitoring of invasive S. agalactiae strains is warranted.

Ocular Gnathostomiasis-Update of Earlier Survey.

Ocular gnathostomiasis is rather a rare food-borne zoonosis caused by infection with the larvae of several species of genus Gnathostoma and is a representative ocular larva migrans syndrome. In our previous literature survey, we found 73 cases of ocular gnathostomiasis reported up to and including 2009, though additional sporadic cases have been reported in Asia and the Americas since that report. Here, we review 10 additional cases reported since 2010, and also update current findings regarding epidemiological and clinical features in affected patients.