Brincidofovir inhibits polyomavirus infection in vivo.

Polyomaviruses are species-specific DNA viruses that can cause disease in immunocompromised individuals. Despite their role as the causative agents for several diseases, there are no currently approved antivirals for treating polyomavirus infection. Brincidofovir (BCV) is an antiviral approved for the treatment of poxvirus infections and has shown activity against other double-stranded DNA viruses. In this study, we tested the efficacy of BCV against polyomavirus infection in vitro and in vivo using mouse polyomavirus (MuPyV). BCV inhibited virus production in primary mouse kidney cells and brain cortical cells. BCV treatment of cells transfected with MuPyV genomic DNA resulted in a reduction in virus levels, indicating that viral inhibition occurs post-entry. Although in vitro BCV treatment had a limited effect on viral DNA and RNA levels, drug treatment was associated with a reduction in viral protein, raising the possibility that BCV acts post-transcriptionally to inhibit MuPyV infection. In mice, BCV treatment was well tolerated, and prophylactic treatment resulted in a reduction in viral DNA levels and a potent suppression of infectious virus production in the kidney and brain. In mice with chronic polyomavirus infection, therapeutic administration of BCV decreased viremia and reduced infection in the kidney. These data demonstrate that BCV exerts antiviral activity against polyomavirus infection in vivo, supporting further investigation into the use of BCV to treat clinical polyomavirus infections. IMPORTANCE: Widespread in the human population and able to persist asymptomatically for the life of an individual, polyomavirus infections cause a significant disease burden in the immunocompromised. Individuals undergoing immune suppression, such as kidney transplant patients or those treated for autoimmune diseases, are particularly at high risk for polyomavirus-associated diseases. Because no antiviral agent exists for treating polyomavirus infections, management of polyomavirus-associated diseases typically involves reducing or discontinuing immunomodulatory therapy. This can be perilous due to the risk of transplant rejection and the potential development of adverse immune reactions. Thus, there is a pressing need for the development of antivirals targeting polyomaviruses. Here, we investigate the effects of brincidofovir, an FDA-approved antiviral, on polyomavirus infection in vivo using mouse polyomavirus. We show that the drug is well-tolerated in mice, reduces infectious viral titers, and limits viral pathology, indicating the potential of brincidofovir as an anti-polyomavirus therapeutic.

A Case of Anti-NXP2 Antibody-positive Juvenile Dermatomyositis with Characteristic Fascial Thickening on Muscle Ultrasound and Improvement with Immunotherapy.

We herein report a 12-year-old boy who presented with a fever, erythematous rash on the cheeks, back pain, and dysphagia. Blood tests revealed increased creatine kinase levels, and muscle ultrasonography (MUS) revealed characteristic fascial thickening in the lumbar paraspinal muscles, where myalgia was prominent. Sarcoplasmic expression of myxovirus-resistant protein A on a muscle biopsy and the presence of anti-nuclear matrix protein 2 (NXP2) antibodies confirmed the diagnosis of dermatomyositis. Prednisolone and intravenous immunoglobulin therapy improved the clinical and laboratory parameters as well as fascial thickening. MUS is useful for evaluating fasciitis associated with anti-NXP2 autoantibodies and monitoring therapeutic efficacy.

Segmental Zoster Paresis Accompanied by Horner's Syndrome.

We herein report a 90-year-old immunocompromised woman who developed right upper limb weakness and right ptosis with a miotic pupil 1 week after oral therapy for zoster on the right T2 dermatome. The right pupil was dilated with instillation of 1% apraclonidine, indicating Horner's syndrome. The patient was treated with intravenous acyclovir and methylprednisolone. Focal weakness related to zoster, generally known as segmental zoster paresis, improved over five months, but Horner's syndrome remained. We suggest that aggressive intravenous treatment should be considered for rare cases of zoster that occur with a combination of these two neurological conditions.

Correlation of muscle ultrasound with clinical and pathological findings in idiopathic inflammatory myopathies.

INTRODUCTION/AIMS: In idiopathic inflammatory myopathies (IIMs), the change in muscle echogenicity and its histopathological basis are not well understood. We quantitatively measured muscle echogenicity in patients with IIMs and evaluated its correlation with disease activity and histopathological findings. METHODS: This study involved patients with IIMs who underwent both ultrasonography (US) and muscle biopsy, as well as age- and sex-matched rheumatoid arthritis patients as inflammatory disease controls. On US, axial images of the right biceps brachii and vastus medialis were obtained. Standardized histopathological scoring was used to quantitatively measure each pathological domain. RESULTS: Forty-two patients (17 with inclusion body myositis [IBM] and 25 with IIMs other than IBM) and 25 controls were included. The muscle echo intensity (EI) of patients with IIMs was significantly higher than that of controls. Muscle EI showed significant correlations with creatine kinase (r = 0.66, p < .001) and muscle strength (r = -0.73, p < .0001) in patients with non-IBM IIMs. In patients with IBM, moderate correlation was found between muscle EI and quadriceps muscle strength (r = -0.53, p = .028). Histopathologically, the number of infiltrating CD3+ inflammatory cells correlated with muscle EI in the non-IBM group (r = 0.56, p = .017), but not in the IBM group. DISCUSSION: Muscle EI may be useful as a surrogate marker of muscle inflammation in non-IBM IIM. Increased muscle EI may be difficult to interpret in patients with long-standing IBM, which has advanced and complex histopathology.

A phase I/II study of 10-min dosing of bendamustine hydrochloride (rapid infusion formulation) in patients with previously untreated indolent B-cell non-Hodgkin lymphoma, mantle cell lymphoma, or relapsed/refractory diffuse large B-cell lymphoma in Japan.

PURPOSE: This phase I/II clinical study was conducted to examine the safety, tolerability, pharmacokinetics, and efficacy of 10-min dosing of bendamustine in patients with previously untreated indolent B-cell non-Hodgkin lymphoma (iNHL) or mantle cell lymphoma (MCL) (Group 1) and patients with relapsed/refractory diffuse large B-cell lymphoma (rrDLBCL) (Group 2). METHODS: Rituximab 375 mg/m2 was administered intravenously every 28 days to Group 1 patients on day 1 and every 21 days to Group 2 patients on day 1. Bendamustine 90 mg/m2/day was administered to the former on days 1 and 2; bendamustine 120 mg/m2/day was administered to the latter on days 2 and 3. Each regimen was delivered up to six cycles for both groups. The primary endpoints were safety and tolerability in Groups 1 and 2, respectively. RESULTS: Among 37 enrolled patients, safety was assessed in 36. In Group 1 (n = 30), 27 patients (90%) had follicular lymphoma. Adverse events (AEs) were observed in all 30 patients in Group 1. Dose-limiting toxicities were observed in two of six patients in Group 2. Common AEs included lymphocyte count decreased (86.7%, 100%). In Group 1, overall response and complete response rates were 93.1% (95% confidence interval [CI] 77.2-99.2%) and 75.9% (95% CI 56.5-89.7%), respectively. The Cmax and AUC of bendamustine tended to be higher in Group 2 than in Group 1. CONCLUSIONS: This study showed that bendamustine is safe, well-tolerated and effective for patients with previously untreated iNHL, MCL or rrDLBCL. Pharmacokinetic data were equivalent to those obtained outside of Japan. REGISTRATION NUMBERS: Registration NCT03900377; registered April 3, 2019.

Application of ultrasound in a case of eosinophilic fasciitis mimicking stiff-person syndrome.

Eosinophilic fasciitis (EF) is a rare disorder characterized by muscle stiffness mimicking other neuromuscular diseases. The diagnosis of EF is made on the basis of typical skin lesions. We report a case of a 36-year-old male patient with suspected stiff-person syndrome (SPS), who presented with progressive limb muscle stiffness and limited mobility of both wrists without obvious skin changes. Ultrasound revealed fascial thickening of bilateral upper and lower limb muscles and enlargement of hypoechoic tissues around the flexor digitorum tendons of the wrist. Skin and fascia biopsy confirmed the diagnosis of EF. Prednisolone therapy resulted in the improvement of muscle stiffness and tightness. Our findings suggest the need to consider connective tissue diseases such as EF in a patient with atypical features of SPS. Ultrasound is helpful for visualizing the causes of muscle stiffness and joint contractures in EF patients.

Early diagnosis of amyotrophic lateral sclerosis based on fasciculations in muscle ultrasonography: A machine learning approach.

OBJECTIVE: Although fasciculation on muscle ultrasonography (MUS) is useful in diagnosing amyotrophic lateral sclerosis (ALS), its applicability to early diagnosis remains unclear. We aimed to develop and validate diagnostic models especially beneficial to early-stage ALS via machine learning. METHODS: We investigated 100 patients with ALS, including 50 with early-stage ALS within 9 months from onset, and 100 without ALS. Fifteen muscles were bilaterally observed for 10 s each and the presence of fasciculations was recorded. Hierarchical clustering and nominal logistic regression, neural network, or ensemble learning were applied to the training cohort comprising the early-stage ALS to develop MUS-based diagnostic models, and they were tested in the validation cohort comprising the later-stage ALS. RESULTS: Fasciculations on MUS in the brainstem or thoracic region had high specificity but limited sensitivities and predictive profiles for diagnosis of ALS. A machine learning-based model comprising eight muscles in the four body regions had a high sensitivity (recall), specificity, and positive predictive value (precision) for both early- and later-stage ALS patients. CONCLUSIONS: We developed and validated MUS-fasciculation-based diagnostic models for early- and later-stage ALS. SIGNIFICANCE: Fasciculation detected in relevant muscles on MUS can contribute to the diagnosis of ALS from the early stage.

Bendamustine plus rituximab in Japanese patients with relapsed or refractory diffuse large B-cell lymphoma.

This single-arm phase 3 study was conducted to confirm the results of our phase 2 study of bendamustine (B)-rituximab (R) in patients with relapsed/refractory diffuse large B cell lymphoma (rrDLBCL). The primary endpoint was overall response rate (ORR). Autologous stem cell transplantation-ineligible rrDLBCL patients with ≤ 2 prior chemotherapy regimens received R 375 mg/m2 IV on day 1 and B 120 mg/m2/day IV on days 2 and 3 every 21 days up to 6 cycles. Thirty-eight patients with a median age of 74 years (range, 43-86) received BR. The ORR and complete response rates were 76.3% and 47.4%, respectively. With a median follow-up of 19.5 months including long-term follow-up, median progression-free survival was 11.9 months. Median OS was 29.2 months. Discontinuation of treatment due to Gr3-5 TEAE was observed among 13 of 38 patients (34.2%). One patient with cytomegalovirus enterocolitis died during follow-up. This BR regimen was confirmed to be effective and tolerable in studied patients. ClinicalTrials.gov Identifier: NCT03372837 registered on 14 December 2017, NCT04354402 registered on 21 April, 2020.

Sensitivity of Human Mastadenovirus, the Causal Agent of Pharyngoconjunctival Fever, Epidemic Keratoconjunctivitis, and Hemorrhagic Cystitis in Immunocompromised Individuals, to Brincidofovir.

Human mastadenovirus (HAdV), a linear double-stranded DNA (dsDNA) virus, is the causal agent of several diseases, including pharyngoconjunctival fever, epidemic keratoconjunctivitis, and hemorrhagic cystitis, in immunocompromised individuals. There are more than 100 reported types of adenoviruses, but the pathogenicity of many HAdVs remains unknown. Brincidofovir (BCV) is a hexadecyloxypropyl lipid conjugate of cidofovir (CDV) that is active against dsDNA viruses. Clinical effectiveness of BCV against certain HAdV species has been reported; however, its activity against novel HAdV types remains unknown. We investigated the half-maximal inhibitory concentration (IC50) values of BCV for novel HAdV types and found that the epidemic keratoconjunctivitis-associated HAdV-D54 prevalent in the Asian region was the most susceptible. The mean overall IC50 value of BCV was lower than that of CDV, indicating that BCV is effective against HAdVs, including the novel types. IMPORTANCE We investigated the IC50 values of BCV for novel HAdV types and found that the epidemic keratoconjunctivitis-associated HAdV-D54 prevalent in the Asian region was the most susceptible. In addition, the mean overall IC50 value of BCV was lower than that of CDV, indicating that BCV is effective against HAdVs.

A comparison of overall survival with 40 and 50mg/m2 pegylated liposomal doxorubicin treatment in patients with recurrent epithelial ovarian cancer: Propensity score-matched analysis of real-world data.

BACKGROUND: In clinical practice, 40mg/m2 of pegylated liposomal doxorubicin (PLD40) has been used as an initial dosage for treating recurrent epithelial ovarian cancer (OC) instead of the recommended dose of 50mg/m2 (PLD50). However, no robust evidence is available to support the use of PLD40. This post-hoc study aimed to compare the efficacy and safety of initial PLD dosages in propensity score (P-score)-matched dataset. METHODS: The data source was a PLD postmarketing surveillance dataset (n=2189) conducted in Japan. Eligibility criteria for the present study were as follows: recurrent OC, history of chemotherapy, and treatment with PLD monotherapy at a dosage between 35.5 and 54.4mg/m2. Overall survival (OS) was compared between PLD50- and PLD40-treated groups using the log-rank test. Incidences of palmar-plantar erythrodysesthesia (PPE) and stomatitis were also compared between the groups. RESULTS: Overall, 503 matched pairs were generated using P-score analysis. The median survival time with PLD50 and PLD40 was 383 and 350days, respectively, with a hazard ratio of 1.10 (95% confidence interval, 0.98-1.26; p=0.211), although the difference was not statistically significant in the P-score-matched dataset. However, the incidence and severity of PPE and stomatitis were significantly lower with PLD40. CONCLUSIONS: Our study showed that the efficacy of PLD did not differ based on initial dosages, but the risk of adverse events was reduced with PLD40. Considering the balance between patient benefits and risks, our results support the use of PLD40 in clinical practice.

Predictive factors for 24 weeks sustained virologic response (SVR24) and viral relapse in patients treated with simeprevir plus peginterferon and ribavirin.

BACKGROUND: Simeprevir with peginterferon and ribavirin has been used for the treatment of chronic hepatitis caused by genotype 1 hepatitis C virus (HCV). We explored the predictive factors for sustained virological response (SVR) and viral relapse using datasets from four Japanese phase 3 studies (CONCERTO). METHODS: We used a multiple logistic regression model. First, an integrated dataset comprising 357 patients was analyzed. Subsequently, prior treatment-naïve and relapser (223 patients) and nonresponder (134 patients) of interferon-based treatment subsets were analyzed to identify predictors of SVR. A subset of nonresponders (106 patients) who were treated ≥24 weeks was also analyzed to identify predictors for viral relapse. RESULTS: In the integrated dataset, prior treatment response was significantly associated with SVR. In subset analyses, interleukin-28B (IL28B) TT genotype and undetectable plasma HCV RNA level at week 4 were associated in treatment-naïve patients and relapsers [odds ratio (OR); 4.106 and 3.701, respectively]. In the nonresponders, the IL28B TT genotype population was very small, and inosine triphosphatase (ITPA) and undetectable plasma HCV RNA at week 4 were associated (OR; 2.506 and 3.333, respectively). Furthermore, ribavirin dose intensity (RBV-DI) and detectable plasma HCV RNA at week 4 were significantly associated with viral relapse (OR; 0.327 and 2.922, respectively). CONCLUSION: IL28B and plasma HCV RNA level at week 4 were clinically relevant predictive factors for SVR in treatment-naïve patients and relapsers. Moreover, RBV-DI and plasma HCV level at week 4 were identified as relevant predictive factors for viral relapse in nonresponders.

Regulation of hepatitis C virus secretion by the Hrs-dependent exosomal pathway.

The molecular mechanisms of assembly and budding of hepatitis C virus (HCV) remain poorly understood. The budding of several enveloped viruses requires an endosomal sorting complex required for transport (ESCRT), which is part of the cellular machinery used to form multivesicular bodies (MVBs). Here, we demonstrated that Hrs, an ESCRT-0 component, is critical for the budding of HCV through the exosomal secretion pathway. Hrs depletion caused reduced exosome production, which paralleled with the decrease of HCV replication in the host cell, and that in the culture supernatant. Sucrose-density gradient separation of the culture supernatant of HCV-infected cells revealed the co-existence of HCV core proteins and the exosome marker. Furthermore, both the core protein and an envelope protein of HCV were detected in the intraluminal vesicles of MVBs. These results suggested that HCV secretion from host cells requires Hrs-dependent exosomal pathway in which the viral assembly is also involved.

Decadal electrocardiographic changes between age 40 and 50 in military pilots.

INTRODUCTION: Cardiovascular diseases can lead to sudden in-flight incapacitation and long-term disability in aircraft pilots. Electrocardiogram (ECG) has been widely used to screen for these diseases in routine aeromedical examinations. Several ECG changes such as complete left bundle-branch block (CLBBB) and left ventricular hypertrophy (LVH) have been associated with increased likelihood of underlying structural cardiac diseases in addition to the emergence of newly recognized cardiovascular diseases such as Brugada syndrome. Therefore, the purpose of this study was to analyze decadal ECG changes in aircraft pilots between 40 and 50 yr in order to make an appropriate evaluation of these ECG changes. METHODS: We analyzed the ECGs from the annual aeromedical examination of age 50 compared to those 40 yr of age in a total of 176 Japan Air Self-Defense Force pilots. RESULTS: With regard to decadal changes, we detected 34 new ECG changes (1 of sinus tachycardia, 8 sinus bradycardia, 1 atrial fibrillation, 2 premature atrial contraction, 1 premature ventricular contraction, 2 left axis deviation, 6 first-degree atrioventricular block, 1 CLBBB, 3 complete right bundle-branch block, 2 incomplete right bundle-branch block, 1 right ventricular conduction delay, and 6 LVH). Although the majority of them were concluded to be normal variants, the results of echocardiography in two hypertensive pilots without good control demonstrated abnormalities: one had mild hypertrophic nonobstructive cardiomyopathy and another had heart enlargement. CONCLUSION: Thus, this study recommends additional cardiovascular examinations, including echocardiography for hypertensive pilots with ECG changes.

Enhanced replication of hepatitis B virus with frameshift in the precore region found in fulminant hepatitis patients.

BACKGROUND: The genotype B of hepatitis B virus (HBV) was reported to associate with fulminant hepatitis (FH). We aimed to clarify the characteristics of HBV obtained from FH patients in an area of Japan where genotype B HBV is prevalent. METHODS: Using serum samples of 16 HBV-associated FH patients, partial HBV sequences were determined. The effects of HBV mutation/insertion/deletion were evaluated using an in vitro HBV replication system. RESULTS: Of the 16 HBV isolates, 31% belonged to subgenotype B1/Bj, 38% were subgenotype B2/Ba, and 31% were subgenotype C2/Ce. Notably, the single nucleotide insertion/deletion that resulted in a frameshift of the precore protein was found exclusively in 60% of B1/Bj strains. An in vitro study showed that all of the frameshift mutants had significantly higher amounts of HBV DNA than did the wild type. One of the isolates had a novel insertion of A between nucleotides 1900 and 1901, which resulted in a 3-nucleotide change within the Kozak sequence of the core protein and enhanced the core protein expression in vitro. CONCLUSIONS: The frameshift insertion/deletion in the precore region enhanced HBV replication and might be associated with the development of FH by the subgenotype B1/Bj HBV.

Plasma L-cystine/L-glutamate imbalance increases tumor necrosis factor-alpha from CD14+ circulating monocytes in patients with advanced cirrhosis.

BACKGROUND AND AIMS: The innate immune cells can not normally respond to the pathogen in patients with decompensated cirrhosis. Previous studies reported that antigen-presenting cells take up L-Cystine (L-Cys) and secrete substantial amounts of L-Glutamate (L-Glu) via the transport system Xc- (4F2hc+xCT), and that this exchange influences the immune responses. The aim of this study is to investigate the influence of the plasma L-Cys/L-Glu imbalance observed in patients with advanced cirrhosis on the function of circulating monocytes. METHODS: We used a serum-free culture medium consistent with the average concentrations of plasma amino acids from patients with advanced cirrhosis (ACM), and examined the function of CD14+ monocytes or THP-1 under ACM that contained 0-300 nmol/mL L-Cys with LPS. In patients with advanced cirrhosis, we actually determined the TNF-alpha and xCT mRNA of monocytes, and evaluated the correlation between the plasma L-Cys/L-Glu ratio and TNF-alpha. RESULTS: The addition of L-Cys significantly increased the production of TNF alpha from monocytes under ACM. Monocytes with LPS and THP-1 expressed xCT and a high level of extracellular L-Cys enhanced L-Cys/L-Glu antiport, and the intracellular GSH/GSSG ratio was decreased. The L-Cys transport was inhibited by excess L-Glu. In patients with advanced cirrhosis (n = 19), the TNF-alpha and xCT mRNA of monocytes were increased according to the Child-Pugh grade. The TNF-alpha mRNA of monocytes was significantly higher in the high L-Cys/L-Glu ratio group than in the low ratio group, and the plasma TNF-alpha was significantly correlated with the L-Cys/L-Glu ratio. CONCLUSIONS: A plasma L-Cys/L-Glu imbalance, which appears in patients with advanced cirrhosis, increased the TNF-alpha from circulating monocytes via increasing the intracellular oxidative stress. These results may reflect the immune abnormality that appears in patients with decompensated cirrhosis.

The nutritional index 'CONUT' is useful for predicting long-term prognosis of patients with end-stage liver diseases.

Organ allocation in Japan remains difficult due to the shortage of deceased-donor livers. The screening tool for controlling nutritional status (CONUT) has been considered to be an established assessment model for evaluating nutritional aspects in surgical patients. However, the application of this CONUT for evaluating the prognosis of patients with end-stage liver diseases has not been evaluated. We assessed the predictability of the prognoses of 58 patients with end-stage liver disease using various prognostic models. The patients registered at the transplantation center of Tohoku University Hospital for the waiting list of Japan Organ Transplant Network for liver transplantation were retrospectively analyzed. The prognoses of the patients were evaluated using the following 5 models: CONUT, the model for ELD with incorporation of sodium (MELD-Na), Child-Turcotte-Pugh score (CTP), prognostic nutritional indices (Onodera: PNI-O), and the Japan Medical Urgency criteria of the liver (JMU). Cox's proportional hazard model, log-rank test and concordance(c)-static were used for the statistics. The indices were 17.74 ± 5.80 for MELD-Na, 9.21 ± 2.19 for CTP, 33.92 ± 11.16 for PNI-O, and 7.57 ± 3.09 for CONUT. Univariate analysis revealed the significance of CONUT (p = 0.017, Odds: 1.325) but not MELD-Na, CTP, JMU or PNI-O for prediction. The cumulative survival rate was clearly discriminated at CONUT point 7. The c-static was 0.081 for the 6-month (M) survival rate, 0.172 for 12M, 0.517 for 36M, 0.821 for 48M, and 0.938 for 60M for CONUT. In conclusion, CONUT shows best predictability for the distant prognoses of patients with ELD.

Mobile and accurate detection system for infection by the 2009 pandemic influenza A (H1N1) virus with a pocket-warmer reverse-transcriptase loop-mediated isothermal amplification.

The 2009 pandemic H1N1 influenza A virus spread quickly worldwide in 2009. Since most of the fatal cases were reported in developing countries, rapid and accurate diagnosis methods that are usable in poorly equipped laboratories are necessary. In this study, a mobile detection system for the 2009 H1N1 influenza A virus was developed using a reverse-transcriptase loop-mediated isothermal amplification (RT-LAMP) kit with a disposable pocket-warmer as a heating device (designated as pwRT-LAMP). The pwRT-LAMP can detect as few as 100 copies of the virus--which is nearly as sensitive as real-time reverse-transcription polymerase chain reaction (RT-PCR)--and does not cross-react with RNA of seasonal influenza viruses. To evaluate the usefulness of the pwRT-LAMP system, nasal swab samples were collected from 56 patients with flu-like symptoms and were tested. Real-time RT-PCR confirmed that the 2009 H1N1 influenza A virus was present in 27 of the 56 samples. Of these 27 positive samples, QuickVue Influenza A+B immunochromatography detected the virus in only 11 samples (11/27; 40.7%), whereas the pwRT-LAMP system detected the virus in 26 of the 56 samples (26/27 of the positive samples; 96.3%). These findings indicate that the mobile pwRT-LAMP system is an accurate diagnostic system for the 2009 H1N1 influenza A virus, and has great potential utility in diagnosing future influenza pandemics.

Lymphotropic HCV strain can infect human primary naïve CD4+ cells and affect their proliferation and IFN-γ secretion activity.

BACKGROUND: Lymphotropic hepatitis C virus (HCV) infection of B and T cells might play an important role in the pathogenesis of hepatitis C. Recently, we showed that a lymphotropic HCV (SB strain) could infect established T-cell lines and B-cell lines. However, whether HCV replication interferes with cell proliferation and function in primary T lymphocytes is still unclear. AIM: The aim of this study was to analyze whether HCV replication in primary T lymphocytes affected their development, proliferation, and Th1 commitment. METHODS: SB strain cell culture supernatant (2 × 10(4) copies/ml HCV) was used to infect several kinds of primary lymphocyte subsets. Mock, UV-irradiated SB-HCV, JFH-1 strain, and JFH-1 NS5B mutant, which could not replicate in T cells, were included as negative controls. Carboxyfluorescein succinimidyl ester (CFSE) and CD45RA double staining was used to evaluate the proliferative activity of CD4(+)CD45RA(+)CD45RO(-) naïve CD4(+) cells. Interferon (IFN)-γ and interleukin (IL)-10 secretion assays magnetic cell sorting (MACS) were carried out. RESULTS: Negative strand HCV RNA was detected in CD4(+), CD14(+), and CD19(+) cells. Among CD4(+) cells, CD4(+)CD45RA(+)RO(-) cells (naïve CD4(+) cells) were most susceptible to replication of the SB strain. The levels of CFSE and CD45RA expression gradually declined during cell division in uninfected cells, while HCV-infected naïve CD4(+) cells expressed higher levels of CFSE and CD45RA than Mock or UV-SB infected naïve CD4(+) cells. Moreover, the production of IFN-γ was significantly suppressed in SB-infected naïve CD4(+) cells. CONCLUSIONS: Lymphotropic HCV replication suppressed proliferation and development, including that towards Th1 commitment, in human primary naïve CD4(+) cells.

Exosome secretion of dendritic cells is regulated by Hrs, an ESCRT-0 protein.

Exosomes are nanovesicles derived from multivesicular bodies (MVBs) in antigen-presenting cells. The components of the ESCRT (endosomal sorting complex required for transport) pathway are critical for the formation of MVBs, however the relationship between the ESCRT pathway and the secretion of exosomes remains unclear. We here demonstrate that Hrs, an ESCRT-0 protein, is required for fascilitating the secretion of exosomes in dendritic cells (DCs). Ultrastructural analyses showed typical saucer-shaped exosomes in the culture supernatant from both the control and Hrs-depleted DCs. However, the amount of exosome secretion was significantly decreased in Hrs-depleted DCs following stimulations with ovalbumin (OVA) as well as calcium ionophore. Antigen-presentation activity was also suppressed in exsosomes purified from Hrs-depleted DCs, while no alteration in OVA degradation was seen in Hrs-depleted DCs. These data indicated that Hrs is involved in the regulation of antigen-presentation activity through the exosome secretion.

Characterization of the epithelial cell adhesion molecule (EpCAM)+ cell population in hepatocellular carcinoma cell lines.

Accumulating evidence suggests that cancer stem cells (CSC) play an important role in tumorigenicity. Epithelial cell adhesion molecule (EpCAM) is one of the markers that identifies tumor cells with high tumorigenicity. The expression of EpCAM in liver progenitor cells prompted us to investigate whether CSC could be identified in hepatocellular carcinoma (HCC) cell lines. The sorted EpCAM(+) subpopulation from HCC cell lines showed a greater colony formation rate than the sorted EpCAM(-) subpopulation from the same cell lines, although cell proliferation was comparable between the two subpopulations. The in vivo evaluation of tumorigenicity, using supra-immunodeficient NOD/scid/γc(null) (NOG) mice, revealed that a smaller number of EpCAM(+) cells (minimum 100) than EpCAM(-) cells was necessary for tumor formation. The bifurcated differentiation of EpCAM(+) cell clones into both EpCAM(+) and EpCAM(-) cells was obvious both in vitro and in vivo, but EpCAM(-) clones sustained their phenotype. These clonal analyses suggested that EpCAM(+) cells may contain a multipotent cell population. Interestingly, the introduction of exogenous EpCAM into EpCAM(+) clones, but not into EpCAM(-) clones, markedly enhanced their tumor-forming ability, even though both transfectants expressed a similar level of EpCAM. Therefore, the difference in the tumor-forming ability between EpCAM(+) and EpCAM(-) cells is probably due to the intrinsic biological differences between them. Collectively, our results suggest that the EpCAM(+) population is biologically quite different from the EpCAM(-) population in HCC cell lines, and preferentially contains a highly tumorigenic cell population with the characteristics of CSC.