A highly efficient, stable, and rapid approach for ex vivo human liver gene therapy via a FLAP lentiviral vector

Allogenic hepatocyte transplantation or autologous transplantation of genetically modified hepatocytes has been used successfully to correct congenital or acquired liver diseases and can be considered as an alternative to orthotopic liver transplantation. However, hepatocytes are neither easily maintained in culture nor efficiently genetically modified and are very sensitive to dissociation before their reimplantation into the recipient. These difficulties have greatly limited the use of an ex vivo approach in clinical trials. In the present study, we have shown that primary human and rat hepatocytes can be efficiently transduced with a FLAP lentiviral vector without the need for plating and culture. Efficient transduction of nonadherent primary hepatocytes was achieved with a short period of contact with vector particles, without modifying hepatocyte viability, and using reduced amounts of vector. We also showed that the presence of the DNA FLAP in the vector construct was essential to reach high levels of transduction. Moreover, transplanted into uPA/SCID mouse liver, lentivirally transduced primary human hepatocytes extensively repopulated their liver and maintained a differentiated and functional phenotype as assessed by the stable detection of human albumin and antitrypsin in the serum of the animals for months. In conclusion, the use of FLAP lentiviral vectors allows, in a short period of time, a high transduction efficiency of human functional and reimplantable hepatocytes. This work therefore opens new perspectives for the development of human clinical trials based on liver-directed ex vivo gene therapy.info:eu-repo/semantics/publishedVersio

Fractionated Stereotactic Radiation Therapy for Pituitary Adenomas: An alternative escalating protocol of hypofractionated stereotactic radiotherapy delivering 35Gy in 5 fractions

PURPOSE: Evaluate efficacy and toxicity of hypofractionated stereotactic radiotherapy (HSRT) for patients treated for pituitary adenoma (PA) with an alternative HSRT escalating protocol delivering 35Gy in 5 fractions. MATERIAL AND METHODS: From June 2007 to March 2017, 29 patients with pituitary adenoma were treated in Antoine Lacassagne Cancer Centre with an alternative HSRT protocol. Prescribed dose was 35Gy in 5 fractions of 7Gy. Radiographic responses were assessed by annual MRI. Hormone blood samples were evaluated each year after HSRT. RESULTS: A total of 29 patients aged between 23 and 86 years (median 54 years) were included. Twelve patients received HSRT for recurrent cases and 12 received postoperative adjuvant HSRT, 5 patients did not have surgery. After a median follow-up period of 47 months local control rate was 96%. One patient presented an out-field tumor regrowth 73 months after HSRT. The majority of PA were endocrine-active (18 patients, 62%). After HSRT, 8 patients (44%) presented complete response on initial secretion, 4 patients (23%) presented partial response on initial secretion. Four patients (14%) presented grade 2 or more acute radiation toxicities. One grade 4 visual disorder was observed for one patient. CONCLUSIONS: HSRT delivering 35Gy in 5 fractions represents a feasible treatment and shows promising results to reduce hormonal overproduction and to improve local control in PA

Cerebellar Ataxia With Anti-DNER Antibodies: Outcomes and Immunologic Features

BACKGROUND AND OBJECTIVES: There is no report on the long-term outcomes of ataxia with antibodies against Delta and Notch-like epidermal growth factor-related (DNER). We aimed to describe the clinical-immunologic features and long-term outcomes of patients with anti-DNER antibodies. METHODS: Patients tested positive for anti-DNER antibodies between 2000 and 2020 were identified retrospectively. In those with available samples, immunoglobulin G (IgG) subclass analysis, longitudinal cerebellum volumetry, human leukocyte antigen isotyping, and CSF proteomic analysis were performed. Rodent brain membrane fractionation and organotypic cerebellar slices were used to study DNER cell-surface expression and human IgG binding to the Purkinje cell surface. RESULTS: Twenty-eight patients were included (median age, 52 years, range 19-81): 23 of 28 (82.1%) were male and 23 of 28 (82.1%) had a hematologic malignancy. Most patients (27/28, 96.4%) had cerebellar ataxia; 16 of 28 (57.1%) had noncerebellar symptoms (cognitive impairment, neuropathy, and/or seizures), and 27 of 28 (96.4%) became moderately to severely disabled. Half of the patients (50%) improved, and 32.1% (9/28) had no or slight disability at the last visit (median, 26 months; range, 3-238). Good outcome significantly associated with younger age, milder clinical presentations, and less decrease of cerebellar gray matter volumes at follow-up. No human leukocyte antigen association was identified. Inflammation-related proteins were overexpressed in the patients' CSF. In the rodent brain, DNER was enriched in plasma membrane fractions. Patients' anti-DNER antibodies were predominantly IgG1/3 and bound live Purkinje cells in vitro. DISCUSSION: DNER ataxia is a treatable condition in which nearly a third of patients have a favorable outcome. DNER antibodies bind to the surface of Purkinje cells and are therefore potentially pathogenic, supporting the use of B-cell-targeting treatments

Modelling Hepatic Endoderm Development: Highly Efficient Differentiation of Human Embryonic Stem Cells to Functional Hepatic Endoderm Requires ActivinA and Wnt3a Signalling.

Human embryonic stem cells (hESCs) are a valuable source of pluripotential primary cells. However, their homogeneous cellular differentiation to specific cell types _in vitro_ has proven difficult thus far. Wnt signalling has been shown to play important roles in coordinating development and we demonstrate that Wnt3a is differentially expressed at critical stages of human liver development _in vivo_. The essential role of Wnt3a in hepatocyte differentiation from hESCs is paralleled by our _in vitro_ model, demonstrating the importance of a physiological approach to cellular differentiation. Our studies provide compelling evidence that Wnt3a signaling is important for coordinated hepato-cellular function _in vitro_ and _in vivo_. In addition, we demonstrate Wnt3a facilitates clonal plating of hESCs capable of hepatic endoderm differentiation. These studies represent an important step forward toward the use of hESC-derived hepatocytes in biomedical applications and has opened the door to high through-put metabolic analysis of human liver function

Targeting Membrane-Bound Viral RNA Synthesis Reveals Potent Inhibition of Diverse Coronaviruses Including the Middle East Respiratory Syndrome Virus

Coronaviruses raise serious concerns as emerging zoonotic viruses without specific antiviral drugs available. Here we screened a collection of 16671 diverse compounds for anti-human coronavirus 229E activity and identified an inhibitor, designated K22, that specifically targets membrane-bound coronaviral RNA synthesis. K22 exerts most potent antiviral activity after virus entry during an early step of the viral life cycle. Specifically, the formation of double membrane vesicles (DMVs), a hallmark of coronavirus replication, was greatly impaired upon K22 treatment accompanied by near-complete inhibition of viral RNA synthesis. K22-resistant viruses contained substitutions in non-structural protein 6 (nsp6), a membrane-spanning integral component of the viral replication complex implicated in DMV formation, corroborating that K22 targets membrane bound viral RNA synthesis. Besides K22 resistance, the nsp6 mutants induced a reduced number of DMVs, displayed decreased specific infectivity, while RNA synthesis was not affected. Importantly, K22 inhibits a broad range of coronaviruses, including Middle East respiratory syndrome coronavirus (MERS–CoV), and efficient inhibition was achieved in primary human epithelia cultures representing the entry port of human coronavirus infection. Collectively, this study proposes an evolutionary conserved step in the life cycle of positive-stranded RNA viruses, the recruitment of cellular membranes for viral replication, as vulnerable and, most importantly, druggable target for antiviral intervention. We expect this mode of action to serve as a paradigm for the development of potent antiviral drugs to combat many animal and human virus infections

High Burden of Non-Influenza Viruses in Influenza-Like Illness in the Early Weeks of H1N1v Epidemic in France

BACKGROUND: Influenza-like illness (ILI) may be caused by a variety of pathogens. Clinical observations are of little help to recognise myxovirus infection and implement appropriate prevention measures. The limited use of molecular tools underestimates the role of other common pathogens. OBJECTIVES: During the early weeks of the 2009-2010 flu pandemic, a clinical and virological survey was conducted in adult and paediatric patients with ILI referred to two French University hospitals in Paris and Tours. Aims were to investigate the different pathogens involved in ILI and describe the associated symptoms. METHODS: H1N1v pandemic influenza diagnosis was performed with real time RT-PCR assay. Other viral aetiologies were investigated by the molecular multiplex assay RespiFinder19®. Clinical data were collected prospectively by physicians using a standard questionnaire. RESULTS: From week 35 to 44, endonasal swabs were collected in 413 patients. Overall, 68 samples (16.5%) were positive for H1N1v. In 13 of them, other respiratory pathogens were also detected. Among H1N1v negative samples, 213 (61.9%) were positive for various respiratory agents, 190 in single infections and 23 in mixed infections. The most prevalent viruses in H1N1v negative single infections were rhinovirus (62.6%), followed by parainfluenza viruses (24.2%) and adenovirus (5.3%). 70.6% of H1N1v cases were identified in patients under 40 years and none after 65 years. There was no difference between clinical symptoms observed in patients infected with H1N1v or with other pathogens. CONCLUSION: Our results highlight the high frequency of non-influenza viruses involved in ILI during the pre-epidemic period of a flu alert and the lack of specific clinical signs associated with influenza infections. Rapid diagnostic screening of a large panel of respiratory pathogens may be critical to define and survey the epidemic situation and to provide critical information for patient management

Vascular clamping in liver surgery: physiology, indications and techniques

This article reviews the historical evolution of hepatic vascular clamping and their indications. The anatomic basis for partial and complete vascular clamping will be discussed, as will the rationales of continuous and intermittent vascular clamping