Hepatocytes Traffic and Export Hepatitis B Virus Basolaterally by Polarity-Dependent Mechanisms

Viruses commonly utilize the cellular trafficking machinery of polarized cells to effect viral export. Hepatocytes are polarized in vivo, but most in vitro hepatocyte models are either nonpolarized or have morphology unsuitable for the study of viral export. Here, we investigate the mechanisms of trafficking and export for the hepadnaviruses hepatitis B virus (HBV) and duck hepatitis B virus (DHBV) in polarized hepatocyte-derived cell lines and primary duck hepatocytes. DHBV export, but not replication, was dependent on the development of hepatocyte polarity, with export significantly abrogated over time as primary hepatocytes lost polarity. Using Transwell cultures of polarized N6 cells and adenovirus-based transduction, we observed that export of both HBV and DHBV was vectorially regulated and predominantly basolateral. Monitoring of polarized N6 cells and nonpolarized C11 cells during persistent, long-term DHBV infection demonstrated that newly synthesized sphingolipid and virus displayed significant colocalization and fluorescence resonance energy transfer, implying cotransportation from the Golgi complex to the plasma membrane. Notably, 15% of virus was released apically from polarized cells, corresponding to secretion into the bile duct in vivo, also in association with sphingolipids. We conclude that DHBV and, probably, HBV are reliant upon hepatocyte polarity to be efficiently exported and this export is in association with sphingolipid structures, possibly lipid rafts. This study provides novel insights regarding the mechanisms of hepadnavirus trafficking in hepatocytes, with potential relevance to pathogenesis and immune tolerance. © 2011, American Society for Microbiology

Human papillomavirus E7 oncoprotein expression by keratinocytes alters the cytotoxic mechanisms used by CD8 T cells

Cervical cancer is a malignant transformation of keratinocytes initiated by the E7 oncoprotein of human papillomavirus (HPV). These tumors are characterized by keratinocyte hyperproliferation and are often infiltrated with activated CD8 T cells. HPV infection confers changes to gain immunological advantage to promote chronic infection, and these persist with malignant transformation. We investigated the relative importance of the many redundant mechanisms of cytotoxicity used by CD8 T cells to kill keratinocytes expressing HPV E7 oncoprotein using extendedduration time-lapse microscopy that allows examination of cell-to-cell interactions during killing. E7 expression by keratinocytes increased susceptibility to cell-mediated killing. However, while killing of non-transgenic keratinocytes was traditional, perforin-mediated, and caspase-dependent, E7-expression favored killing by perforinindependent, caspase-independent mechanisms. The roles of perforin, TNFα, IFNγ, Fas/FasL and PD1/PD-L1 were graded according to target cell survival to produce a hierarchy of killing mechanisms utilized in killing E7-expressing cells. TNFα was essential for perforin-mediated killing of E7-expressing cells, but not perforinindependent killing. IFNγ facilitated killing by Fas/FasL interaction, especially in the absence of perforin. Additionally, expression of E7 offered protection from killing by up regulation of PD-L1, Fas and FasL expression on keratinocytes promoting fight-back by target cells, resulting in effector cell death. This study shows that keratinocytes expressing E7 are highly susceptible to killing by CD8 T cells, but utilizing different armamentarium. Down-regulation of CD8 T cell cytotoxicity in HPV-related tumors may be due to suppression by E7-expressing keratinocytes. Immunotherapy for HPVrelated cancers may be improved by suppression of PD-L1, or by suppression of FasL

mRNA Structural Constraints on EBNA1 Synthesis Impact on In Vivo Antigen Presentation and Early Priming of CD8(+) T Cells

Recent studies have shown that virally encoded mRNA sequences of genome maintenance proteins from herpesviruses contain clusters of unusual structural elements, G-quadruplexes, which modulate viral protein synthesis. Destabilization of these G-quadruplexes can override the inhibitory effect on self-synthesis of these proteins. Here we show that the purine-rich repetitive mRNA sequence of Epstein-Barr virus encoded nuclear antigen 1 (EBNA1) comprising G-quadruplex structures, limits both the presentation of MHC class I-restricted CD8(+) T cell epitopes by CD11c(+) dendritic cells in draining lymph nodes and early priming of antigen-specific CD8(+) T-cells. Destabilization of the G-quadruplex structures through codon-modification significantly enhanced in vivo antigen presentation and activation of virus-specific T cells. Ex vivo imaging of draining lymph nodes by confocal microscopy revealed enhanced antigen-specific T-cell trafficking and APC-CD8(+) T-cell interactions in mice primed with viral vectors encoding a codon-modified EBNA1 protein. More importantly, these antigen-specific T cells displayed enhanced expression of the T-box transcription factor and superior polyfunctionality consistent with the qualitative impact of translation efficiency. These results provide an important insight into how viruses exploit mRNA structure to down regulate synthesis of their viral maintenance proteins and delay priming of antigen-specific T cells, thereby establishing a successful latent infection in vivo. Furthermore, targeting EBNA1 mRNA rather than protein by small molecules or antisense oligonucleotides will enhance EBNA1 synthesis and the early priming of effector T cells, to establish a more rapid immune response and prevent persistent infection

Colorectal polypectomy and endoscopic mucosal resection: European Society of Gastrointestinal Endoscopy Cascade Guideline

No abstract availabl

Leveraging human capital to reduce maternal mortality in India: enhanced public health system or public-private partnership?

Developing countries are currently struggling to achieve the Millennium Development Goal Five of reducing maternal mortality by three quarters between 1990 and 2015. Many health systems are facing acute shortages of health workers needed to provide improved prenatal care, skilled birth attendance and emergency obstetric services – interventions crucial to reducing maternal death. The World Health Organization estimates a current deficit of almost 2.4 million doctors, nurses and midwives. Complicating matters further, health workforces are typically concentrated in large cities, while maternal mortality is generally higher in rural areas. Additionally, health care systems are faced with shortages of specialists such as anaesthesiologists, surgeons and obstetricians; a maldistribution of health care infrastructure; and imbalances between the public and private health care sectors. Increasingly, policy-makers have been turning to human resource strategies to cope with staff shortages. These include enhancement of existing work roles; substitution of one type of worker for another; delegation of functions up or down the traditional role ladder; innovation in designing new jobs;transfer or relocation of particular roles or services from one health care sector to another. Innovations have been funded through state investment, public-private partnerships and collaborations with nongovernmental organizations and quasi-governmental organizations such as the World Bank. This paper focuses on how two large health systems in India – Gujarat and Tamil Nadu – have successfully applied human resources strategies in uniquely different contexts to the challenges of achieving Millennium Development Goal Five

Global Retinoblastoma Presentation and Analysis by National Income Level.

Importance: Early diagnosis of retinoblastoma, the most common intraocular cancer, can save both a child's life and vision. However, anecdotal evidence suggests that many children across the world are diagnosed late. To our knowledge, the clinical presentation of retinoblastoma has never been assessed on a global scale. Objectives: To report the retinoblastoma stage at diagnosis in patients across the world during a single year, to investigate associations between clinical variables and national income level, and to investigate risk factors for advanced disease at diagnosis. Design, Setting, and Participants: A total of 278 retinoblastoma treatment centers were recruited from June 2017 through December 2018 to participate in a cross-sectional analysis of treatment-naive patients with retinoblastoma who were diagnosed in 2017. Main Outcomes and Measures: Age at presentation, proportion of familial history of retinoblastoma, and tumor stage and metastasis. Results: The cohort included 4351 new patients from 153 countries; the median age at diagnosis was 30.5 (interquartile range, 18.3-45.9) months, and 1976 patients (45.4%) were female. Most patients (n = 3685 [84.7%]) were from low- and middle-income countries (LMICs). Globally, the most common indication for referral was leukocoria (n = 2638 [62.8%]), followed by strabismus (n = 429 [10.2%]) and proptosis (n = 309 [7.4%]). Patients from high-income countries (HICs) were diagnosed at a median age of 14.1 months, with 656 of 666 (98.5%) patients having intraocular retinoblastoma and 2 (0.3%) having metastasis. Patients from low-income countries were diagnosed at a median age of 30.5 months, with 256 of 521 (49.1%) having extraocular retinoblastoma and 94 of 498 (18.9%) having metastasis. Lower national income level was associated with older presentation age, higher proportion of locally advanced disease and distant metastasis, and smaller proportion of familial history of retinoblastoma. Advanced disease at diagnosis was more common in LMICs even after adjusting for age (odds ratio for low-income countries vs upper-middle-income countries and HICs, 17.92 [95% CI, 12.94-24.80], and for lower-middle-income countries vs upper-middle-income countries and HICs, 5.74 [95% CI, 4.30-7.68]). Conclusions and Relevance: This study is estimated to have included more than half of all new retinoblastoma cases worldwide in 2017. Children from LMICs, where the main global retinoblastoma burden lies, presented at an older age with more advanced disease and demonstrated a smaller proportion of familial history of retinoblastoma, likely because many do not reach a childbearing age. Given that retinoblastoma is curable, these data are concerning and mandate intervention at national and international levels. Further studies are needed to investigate factors, other than age at presentation, that may be associated with advanced disease in LMICs

The global retinoblastoma outcome study : a prospective, cluster-based analysis of 4064 patients from 149 countries

DATA SHARING : The study data will become available online once all analyses are complete.BACKGROUND : Retinoblastoma is the most common intraocular cancer worldwide. There is some evidence to suggest that major differences exist in treatment outcomes for children with retinoblastoma from different regions, but these differences have not been assessed on a global scale. We aimed to report 3-year outcomes for children with retinoblastoma globally and to investigate factors associated with survival. METHODS : We did a prospective cluster-based analysis of treatment-naive patients with retinoblastoma who were diagnosed between Jan 1, 2017, and Dec 31, 2017, then treated and followed up for 3 years. Patients were recruited from 260 specialised treatment centres worldwide. Data were obtained from participating centres on primary and additional treatments, duration of follow-up, metastasis, eye globe salvage, and survival outcome. We analysed time to death and time to enucleation with Cox regression models. FINDINGS : The cohort included 4064 children from 149 countries. The median age at diagnosis was 23·2 months (IQR 11·0–36·5). Extraocular tumour spread (cT4 of the cTNMH classification) at diagnosis was reported in five (0·8%) of 636 children from high-income countries, 55 (5·4%) of 1027 children from upper-middle-income countries, 342 (19·7%) of 1738 children from lower-middle-income countries, and 196 (42·9%) of 457 children from low-income countries. Enucleation surgery was available for all children and intravenous chemotherapy was available for 4014 (98·8%) of 4064 children. The 3-year survival rate was 99·5% (95% CI 98·8–100·0) for children from high-income countries, 91·2% (89·5–93·0) for children from upper-middle-income countries, 80·3% (78·3–82·3) for children from lower-middle-income countries, and 57·3% (52·1-63·0) for children from low-income countries. On analysis, independent factors for worse survival were residence in low-income countries compared to high-income countries (hazard ratio 16·67; 95% CI 4·76–50·00), cT4 advanced tumour compared to cT1 (8·98; 4·44–18·18), and older age at diagnosis in children up to 3 years (1·38 per year; 1·23–1·56). For children aged 3–7 years, the mortality risk decreased slightly (p=0·0104 for the change in slope). INTERPRETATION : This study, estimated to include approximately half of all new retinoblastoma cases worldwide in 2017, shows profound inequity in survival of children depending on the national income level of their country of residence. In high-income countries, death from retinoblastoma is rare, whereas in low-income countries estimated 3-year survival is just over 50%. Although essential treatments are available in nearly all countries, early diagnosis and treatment in low-income countries are key to improving survival outcomes.The Queen Elizabeth Diamond Jubilee Trust and the Wellcome Trust.https://www.thelancet.com/journals/langlo/homeam2023Paediatrics and Child Healt

Role of endoscopy in primary sclerosing cholangitis

Primary sclerosing cholangitis (PSC) is a progressive disease of the bile ducts that usually results in chronic liver disease often requiring liver transplantation. Endoscopy remains crucial to the care of these patients, although magnetic resonance cholangiopancreatography has replaced endoscopic retrograde cholangiopancreatography (ERCP) as the primary imaging modality for diagnosis. For detection of dysplasia or cholangiocarcinoma, ERCP with intraductal sampling remains compulsory. Moreover, dominant strictures play an important part in the disease development, and management by balloon dilatation or stenting could contribute to long-term prognosis. In addition, endoscopy offers management for adverse events such as bile leaks and anastomotic strictures after liver transplantation. Finally, the special phenotype of inflammatory bowel disease associated with PSC as well as the frequent occurrence of portal hypertension mandates close follow-up with colonoscopy and upper endoscopy. With the emergence of novel techniques, the endoscopist remains a key member of the multidisciplinary team caring for PSC patients

How to access the common bile duct

Biliary access is a prerequisite to all endoscopic interventions in the biliary tract. Successful cannulation of the papilla of Vater is the predominant challenge for the majority of endoscopists training in endoscopic retrograde cholangiopancreaticography (ERCP), and the skills required for success differ substantially from those of regular luminal endoscopy. This paper reviews some of the key elements to successful biliary cannulation, a range of options for problem-solving when cannulation is difficult, and some tips and tricks in select special situations as well. The techniques are described, and available evidence is reviewed