Dengue virus (DENV) causes a wide range of illnesses in humans: dengue fever (DF), dengue hemorrhagic fever (DHF) and dengue shock syndrome (DSS). Animal models that constantly develop high levels of viraemia are required for the development of protective and preventive measures. Common marmosets (Callithrix jacchus) demonstrated high levels of viraemia after inoculation with clinical isolates of four serotypes of DENV; in particular, over 10 6 genome copies ml "1 after inoculation with DENV-2. Non-structural protein 1 and DENV-specific IgM and IgG antibodies were consistently detected. The DENV-2 genome was detected in lymphoid organs including the lymph nodes, spleen and thymus, and also in non-lymphoid organs. DENV antigen was detected by immunohistochemistry in the liver and spleen from inoculated marmosets. Four marmosets were reinoculated with DENV-2 at 33 weeks after primary inoculation with DENV-2. The DENV-2 genome was not detected in any of these marmosets, indicating protection from a secondary infection. The results indicate that common marmosets are highly sensitive to DENV infection, and suggest that marmosets could be a reliable primate model for the evaluation of candidate vaccines.

The reemergence of dengue virus (DENV) infection has created a requirement for improved laboratory diagnostic procedures. In this study, DENV genome detection in urine was evaluated as a diagnostic method. The DENV genome was detected by realtime reverse transcriptase PCR (RT-PCR) in urine and serum of dengue patients. The detection rate of DENV genome in urine was 25% (2/8) on disease days 0 to 3 and 32% (7/22) on days 4 to 5. The rate was 50% or higher on days 6 to 16, 52% (11/21) on days 6 to 7, 78% (7/9) on days 8 to 9, 80% (4/5) on days 10 to 11, 50% (2/4) on days 12 to 13, and 60% (3/5) on days 14 to 16. The last positive urine sample was on day 16. The detection rates in serum were highest on days 0 to 3 and were greater than 50% on days 0 to 7. Detection rates decreased thereafter, and the last positive detection was on day 11. These results indicate that the time frames for positive detection differ between urine and serum samples, whereby detection rates of 50% or higher are evident between days 6 to 16 for urine samples and days 0 to 7 for serum samples. Nucleotide sequences of PCR products were identical between urine and serum samples. The detection of DENV genome in urine samples by real-time RT-PCR is useful to confirm DENV infection, particularly after viremia disappears. Dengue virus (DENV) infections occur in most of the tropical and subtropical areas of the world. DENV infection with any of four serotypes leads to a broad spectrum of clinical symptoms and severity, including asymptomatic infection, dengue fever (DF), and fatal dengue hemorrhagic fever (DHF). DF/DHF is considered one of the most important reemerging infectious diseases (4). Physicians and pediatricians in countries in which these diseases are not endemic are often unfamiliar with the symptoms and unaware of the potential importation of patients with DF/DHF. As such, DF/DHF often may not be considered part of a differential diagnosis. Furthermore, laboratory diagnosis is hampered in areas where the disease is endemic because of the limited number of facilities with diagnostic capacity, and specimen collection in a proper time frame is not easy in areas were DF/DHF is endemic.Several laboratory diagnostic techniques have been used for the confirmation of dengue virus infection: viral isolation, viral antigen detection, viral genome detection, and antibody (Ab) detection. IgM capture enzyme-linked immunosorbent assay (ELISA) and real-time reverse transcriptase PCR (RT-PCR) are commonly used (6,8,16). NS-1 antigen detection tests have also recently become commercially available (10); however, they cannot determine specific viral types. The antibody/antigen detection of DENV provides less information than the other detection assays, and the virus can be successfully isolated only during limited stages of infection. For detailed analyses, the detection of the DENV genome in serum samples by RT-PCR is widely used. A fluorogenic probe-based assay, which has a number of advantages over conventional RT-PCR, has recently been developed. I...

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In previous work, we engineered functional cell sheets using bone marrow-derived mesenchymal stem cells (BM-MSCs) to promote islet graft survival. In the present study, we hypothesized that a cell sheet using dermal fibroblasts could be an alternative to MSCs, and then we aimed to evaluate the effects of this cell sheet on the functional viability of human islets. Fibroblast sheets were fabricated using temperature-responsive culture dishes. Human islets were seeded onto fibroblast sheets. The efficacy of the fibroblast sheets was evaluated by dividing islets into three groups: the islets-alone group, the coculture with fibroblasts group, and the islet culture on fibroblast sheet group. The ultrastructure of the islets cultured on each fibroblast sheet was examined by electron microscopy. The fibroblast sheet expression of fibronectin (as a component of the extracellular matrix) was quantified by Western blotting. After 3 days of culture, islet viabilities were 70.2 ± 9.8%, 87.4 ± 5.8%, and 88.6 ± 4.5%, and survival rates were 60.3 ± 6.8%, 65.3 ± 3.0%, and 75.8 ± 5.6%, respectively. Insulin secretions in response to high-glucose stimulation were 5.1 ± 1.6, 9.4 ± 3.8, and 23.5 ± 12.4 µIU/islet, and interleukin-6 (IL-6) secretions were 3.0 ± 0.7, 5.1 ± 1.2, and 7.3 ± 1.0 ng/day, respectively. Islets were found to incorporate into the fibroblast sheets while maintaining a three-dimensional structure and well-preserved extracellular matrix. The fibroblast sheets exhibited a higher expression of fibronectin compared to fibroblasts alone. In conclusion, human dermal fibroblast sheets fabricated by tissue-engineering techniques could provide an optimal substrate for human islets, as a source of cytokines and extracellular matrix.

BackgroundWe describe a novel scoring system, namely the inflammatory response biomarker (IRB) score. The aim of this study is to evaluate the clinical value of IRB score in patients undergoing curative resection for esophageal squamous cell carcinoma (SCC).MethodsWe retrospectively reviewed patients who underwent curative esophagectomy. We evaluated IRB score in both non-elderly (<70 years) and elderly (≥70 years) SCC patients. The IRB score was determined as follows: a high lymphocyte-to-monocyte ratio (LMR) (>4), a high neutrophil-to-lymphocyte ratio (NLR) (>1.6), and a low platelet-to-lymphocyte ratio (PLR) (<147) were each scored as 1, and the remaining values were scored as 0; the individual scores were then summed to produce the IRB score (range 0−3).ResultsUnivariate analyses demonstrated that the TNM pStage (p < 0.0001), tumor size (p = 0.002), LMR (p = 0.0057), PLR (p = 0.0328) and IRB score (p = 0.0003) were significant risk factors for a worse prognosis. On multivariate analysis, the TNM pStage (p < 0.0001) and IRB score (p = 0.0227) were independently associated with worse prognosis in overall patients. Among non-elderly patients, multivariate analyses demonstrated that the pStage (p = 0.0015) and IRB score (p = 0.0356) were independent risk factors for a worse prognosis. Among elderly patients, multivariate analysis demonstrated that the pStage (p = 0.0016), and IRB score (p = 0.0102) were independent risk factors for a worse prognosis.ConclusionThe present study provides evidence that the preoperative IRB score can be considered a promising independent prognostic factor of cancer-specific survival in patients undergoing curative resection for SCC, and that its predictive ability is useful in both non-elderly and elderly patients.

Highlights 8.1% of COVID-19 patients were mechanically ventilated in the first outbreak in Osaka. The 30-day mortality rate of mechanically ventilated COVID-19 patients was 24.0%. Among these, 97% of patients were intubated within 14 days from clinical onset. Age ≥65 years and men were associated with a higher 30-day mortality rate. Among these, the median duration of viral RNA shedding from onset was 35 days.

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TO THE EDITORS:A 53-year-old male patient underwent living donor liver transplantation (LDLT) for hepatitis C virusinfected liver cirrhosis complicated with hepatocellular carcinoma. Preoperative 3-dimensional (3D) images were obtained with a 3D image analysis system (Synapse Vincent; Fujifilm Medical, Tokyo, Japan) so that we could evaluate the graft volume and possible congested volume after implantation in LDLT. This revealed that a large middle hepatic vein (MHV) drained a vast area in the right lobe (Fig. 1A,B). The estimated volume of the donor's whole liver was 1048 mL. The extended left graft was considered to be small for the size of the recipient and corresponded to 30% of the recipient's standard liver volume; also, it had an estimated congested area of 407 mL, which was equivalent to 39% of the donor's liver volume in the remnant right lobe (Fig. 1C). As a result, if a left lobe graft could be procured, the functional remnant liver volume was estimated to become 20% of the donor's liver volume. Hence, the left lobe was considered inappropriate not only because of the small-for-size graft for the recipient but also because of safety concerns for the donor.After ensuring sufficient drainage of the left lobe by medial segmental vein (V4) and the left hepatic vein, we decided to use a right lobe graft with the MHV because the volume was considered sufficient and was equivalent to 47.5% of the standard liver volume of the recipient. A preoperative contrast-enhanced computed tomography scan revealed a distance of 2 cm between the donor's right hepatic vein (RHV) and MHV at the estimated Cantlie line. Because of the location and alignment, we planned to use an autologous portal vein (PV) Y-graft interposition for the hepatic vein anastomosis. The image of the Y-graft from the recipient's PV was also made with the Synapse Vincent program (Fig. 2).An extended right lobe graft was transplanted from the patient's wife, and the actual graft weight was 493 g, which corresponded to 42.3% of the recipient's standard liver volume. A hilar PV was harvested from the recipient, and ex vivo hepatic vein reconstruction was performed through the connection of a right portal branch to the MHV and a left portal branch to the RHV with continuous 5-0 polypropylene monofilament sutures. An end-to-end anastomosis was performed between the explanted main PV graft and the inferior vena cava with continuous 5-0 polypropylene monofilament sutures. In addition, the right inferior hepatic vein (RIHV) was directly anastomosed to the inferior vena cava. After reperfusion, intraoperative Doppler ultrasound showed a stable and sufficient hepatic inflow and outflow from the 3 anastomosed hepatic veins. The length of the recipient operation was 13 hours 15 minutes, and the blood loss was 11,700 g. At the time of this writing, 8 months after LDLT, the patient was doing well with good liver function. The fact that both the MHV and the RHV were patent in the early postoperative period is considered to have contributed to the patient's recov...