In vivo infectivity of liver extracts after resolution of hepadnaviral infection following therapy associating DNA vaccine and cytokine genes.

DNA-based vaccination appears of promise for chronic hepatitis B immunotherapy, although there is an urgent need to increase its efficacy. In this preclinical study, we evaluated the therapeutic benefit of cytokine (IL-2, IFN-γ) genes co-delivery with DNA vaccine targeting hepadnaviral proteins in the chronic duck hepatitis B virus (DHBV) infection model. Then, we investigated the persistence of replication-competent virus in the livers of apparently resolved animals. DHBV carriers received four injections of plasmids encoding DHBV envelope and core alone or co-delivered with duck IL-2 (DuIL-2) or duck IFN-γ (DuIFN-γ) plasmids. After long-term (8 months) follow-up, viral covalently closed circular (ccc) DNA was analysed in duck necropsy liver samples. Liver homogenates were also tested for in vivo infectivity in neonatal ducklings. Co-delivery of DuIFN-γ resulted in significantly lower mean viremia starting from week 21. Viral cccDNA was undetectable by conventional methods in the livers of 25% and 57% of animals co-immunized with DuIL-2 and DuIFN-γ, respectively. Interestingly, inoculation of liver homogenates from 7 such apparently resolved animals, exhibiting cccDNA undetectable in Southern blotting and DHBV expression undetectable or restricted to few hepatocytes, revealed that three liver homogenates transmitted high-titre viremia (3-5×10(10) vge/mL) to naïve animals. In conclusion, our results indicate that IFN-γ gene co-delivery considerably enhances immunotherapeutic efficacy of DNA vaccine targeting hepadnaviral proteins. Importantly, we also showed that livers exhibiting only minute amounts of hepadnaviral cccDNA could induce extremely high-titre infection, highlighting the caution that should be taken in occult hepatitis B patients to prevent HBV transmission in liver transplantation context.

Analysis of EURADOS neutron intercomparison results according to new ISO standards criteria.

The European Radiation Dosimetry Group has carried out several different types of intercomparison (IC) exercises in the past that qualify as proficiency tests for different dosimetry systems and types of radiation. The first neutron dosemeter IC was held in 2012 (IC2012n) and was followed by a second in 2017/2018 (IC2017n). In sum, 31 Individual Monitoring Services (IMSs) entered 34 dosimetry systems in IC2012n, and 32 IMSs entered 33 dosimetry systems for IC2017n. Such exercises provided a rare opportunity to see how neutron dosemeters perform. For the IC2012n exercise, there were no applicable performance standards for neutron personal dosemeters. ISO/TC85/SC2 updated the ISO Standard 14146 in 2018 (ISO 14146:2018. Radiation protection-Criteria and performance limits for the periodic evaluation of dosimetry services) to include neutron dosimetry. It was thus possible to analyse the IC2017n exercise in accordance with the requirements given by this new standard. It is now of interest to reanalyse the results of IC2012n to quantify any modifications to the conclusions.

Decision-making for extremely preterm infants with severe hemorrhages on head ultrasound: Science, values, and communication skills.

Severe intracranial hemorrhages are not rare in extremely preterm infants. They occur early, generally when babies require life-sustaining interventions. This may lead to ethical discussions and decision-making about levels of care. Prognosis is variable and depends on the extent, location, and laterality of the lesions, and, importantly also on the subsequent occurrence of other clinical complications or progressive ventricular dilatation. Decision-making should depend on prognosis and parental values. This article will review prognosis and the uncertainty of outcomes for different lesions and provide an outline of ways to conduct an ethically appropriate discussion on the decision of whether to continue life sustaining therapy. It is possible to communicate in a compassionate and honest way with parents and engage in decision-making, focussing on personalized information and decisions, and on function, as opposed to diagnosis.

2017 French health authority guidelines for neonates at risk of early-onset sepsis: Adherence and barriers in a tertiary hospital.

The objective of this study was to evaluate the adherence to the French Health Authority's 2017 guidelines concerning early-onset sepsis (EOS) in neonates. This 6-month prospective observational study was conducted in 2020 in a tertiary hospital maternity unit. Neonates were classified according to their risk of EOS and the level of appropriate surveillance was analyzed. The risk of EOS was determined for 69.9% of the 1024 included neonates. Of the 123 neonates with a moderate or high risk of EOS, the optimal clinical surveillance was 88%. Even in a tertiary hospital, with training of caregivers, adherence to guidelines was incomplete. Some corrective measures are suggested and should be applied to enhance the adherence.

Proficiency of transient elastography compared to liver biopsy for the assessment of fibrosis in HIV/HBV-coinfected patients.

Transient elastography (TE) is a noninvasive technique to evaluate liver fibrosis. We compared the performance of TE with liver biopsy (LB) in patients with human immunodeficiency virus (HIV) and hepatitis B virus (HBV) coinfection. Patients prospectively underwent TE and LB. The diagnosis accuracy of TE was calculated using receiver operating characteristic (ROC) curves for different stages of fibrosis, and optimal cut-off values were defined. A sequential algorithm combining TE with biochemical score (Fibrotest) is proposed. Fifty-seven patients had both TE and LB (median time: 3 days) and two with proven cirrhosis, only TE. Forty-six (78%) were under antiretroviral therapy with anti-HBV drugs in 98%, and 19 (32%) had elevated alanine aminotransferase (ALT). A significant correlation was observed between liver stiffness measurement (LSM) and METAVIR fibrosis stages (P < 0.0001). Patients with elevated ALT tended to have higher LSM than those with normal ALT. The areas under the ROC curves were 0.85 for significant fibrosis (≥ F2), 0.92 for advanced fibrosis (≥ F3) and 0.96 for cirrhosis. Using a cut-off of 5.9 kPa for F ≥ 2 and 7.6 kPa for F ≥ 3, the diagnosis accuracy was 83% and 86%, respectively. With an algorithm combining TE and Fibrotest, 97% of patients were well classified for significant fibrosis. Using this algorithm, the need for LB can be reduced by 67%. In HIV/HBV-coinfected patients, most of them with normal ALT under antiretroviral treatment including HBV active drugs, TE was proficient in discriminating moderate to severe fibrosis from minimal liver disease.

Neurodevelopmental impairment at 2 years of age in children born before 29 weeks' gestation with bronchopulmonary dysplasia.

INTRODUCTION: Very preterm children are at a high risk for neurological impairment, especially those with bronchopulmonary dysplasia (BPD). The main goal of this study was to describe the neurodevelopmental impairment (NDI) at 2 years of corrected age in children born before 29 weeks' gestation between 2010 and 2015 and affected by BPD at 28 days of life. We also searched for risk factors associated with NDI, especially postnatal steroid (PNS) administration. MATERIAL AND METHODS: This was a retrospective study comprising a cohort of children hospitalized at the university hospital in Grenoble, born before 29 weeks' gestation between 2010 and 2015, and included in the monitoring network "Naitre et Devenir" (RND). Infants at 2 years of corrected age were classified as having NDI if they had at least one of the following outcomes: a global developmental quotient (DQ) on the revised Brunet-Lézine scale of<85, blindness, deafness, or cerebral palsy (CP) graded as level 3 or more according to the Gross Motor Function Classification System. RESULTS: A total of 129 children were included, of whom 99 were monitored at the age of 2 years: 31.3% of the population had NDI and 4% had CP. The median DQ test result was 90 (interquartile 82-97). Factors associated with NDI in univariate analysis were low gestational age, low birth weight, a cord pH<7.2, chorioamnionitis, treatment for persistent ductus arteriosus, longer oxygen therapy, and outborn status, which almost reached statistical significance. In multivariate analysis, low gestational age and outborn status remained statistically significant, while chorioamnionitis was found to have some association with NDI. While 13.1% of the followed-up population was treated with PNS, this risk factor was not associated with NDI. CONCLUSION: In a population of very preterm children, one third had NDI at 2 years of corrected age. Low gestational age, outborn status, and perinatal inflammation are associated with this unfavorable outcome. The frequency of sequelae confirms the importance of following up these children.

Real-time monitoring of the Bragg-peak position in ion therapy by means of single photon detection.

For real-time monitoring of the longitudinal position of the Bragg-peak during an ion therapy treatment, a novel non-invasive technique has been recently proposed that exploits the detection of prompt gamma-rays issued from nuclear fragmentation. Two series of experiments have been performed at the GANIL and GSI facilities with 95 and 305 MeV/u (12)C(6+) ion beams stopped in PMMA and water phantoms. In both experiments, a clear correlation was obtained between the carbon ion range and the prompt photon profile. Additionally, an extensive study has been performed to investigate whether a prompt neutron component may be correlated with the carbon ion range. No such correlation was found. The present paper demonstrates that a collimated set-up can be used to detect single photons by means of time-of-flight measurements, at those high energies typical for ion therapy. Moreover, the applicability of the technique both at cyclotron and at synchrotron facilities is shown. It is concluded that the detected photon count rates provide sufficiently high statistics to allow real-time control of the longitudinal position of the Bragg-peak under clinical conditions.

[Protein-losing enteropathy of non neoplastic origin in the dog: a retrospective study of 34 cases]. / Les entéropathies exsudatives d'origine non néoplastique du chien: étude rétrospective de 34 cas.

This study reports the results in 34 dogs with protein-losing enteropathy (PLE). Rottweilers and Yorkshire Terriers were overrepresented. Most obvious clinical signs and laboratory findings were diarrhea (91 %), weight loss (74 %), anorexia (56 %), lethargy (51 %), ascites (18 %), ascites without gastrointestinal signs (9 %), hypoalbuminemia (<12 g/l, 65 %) and hypocholesterolemia (51 %). Ultrasonographic findings included intestinal wall thickening in 71 %, abnormal echogenicity in 68 %, and no abnormalities in 29 %. The most important endoscopic findings were diffuse and profound dilation of villi in 35 %, a more granular appearing mucous membrane in 32 % and a "rice-grain" appearing villus dilation in 12 %. In 21 % of the dogs the mucous membrane was considered unremarkable. Histologically, 62 % had lymphangiectasia, in 86 % of these associated with moderate to severe inflammatory infiltrates, and in 71 % associated with dilatation of intestinal crypts. In 68 % of the dogs lesions in intestinal crypts were found, 35 % of these without lymphangiectasia. In 12 % of the dogs the histological findings failed to explain intestinal protein loss. This study shows the important prevalence of crypts' lesions in protein-losing enteropathy.

CALCULATION OF CONVERSION COEFFICIENTS FOR VOXELIZED PHANTOMS FOR CRITICALITY ACCIDENT DOSIMETRY.

In the event of a criticality accident, not only the maximal doses received by the victims must be determined but it is also crucial to evaluate the doses to the different organs. With a neutron component, morphology is a key parameter in the organ dose calculation. As the simulation tools can be time consuming to proceed, especially if morphology is taken into account, for all the victims, it may be very useful to have a database of conversion coefficients that allow to obtain the organ doses from the dose measured in the dosemeter for different kinds of morphology. In this paper, we present a study performed to evaluate such conversion coefficients using voxelized anthropomorphic phantoms. These coefficients take into account two crucial parameters having an impact on the dose at the organs: the orientation of the victim in the radiation field and the morphology, that is to say the body mass index of the different victims.

Discordance between biochemical markers of liver activity and fibrosis (Actitest-Fibrotest) and liver biopsy in patients with chronic hepatitis C.

INTRODUCTION: The purpose of this clinical trial was to determine in routine practice and in comparison with liver biopsy the limitations of two blood tests, Actitest and Fibrotest, for the evaluation of hepatic activity and fibrosis in patients with chronic hepatitis C. METHODS: Routine blood tests, Actitest and Fibrotest, and liver biopsy were performed in 96 patients with chronic hepatitis C attending routine outpatient clinics. Receiver operating characteristics (ROC) curves were used to assess the diagnostic value of the biochemical tests in comparison with the METAVIR classification. RESULTS: The study population was predominantly male (63.5%) with a mean age of 48 years; 83.3% of the patients had genotype 1 hepatitis C virus infection. Treatment status was naive (62.5%), nonresponders (17.7%), relapsers (7.3%), or unknown (12.5%). The comparison of F0-F2 versus F3-F4 estimated the negative predictive value at 92% and the positive predictive value at 52% for a cut-off of 0.455. Discrepancies in activity score were more frequently due to a higher score of the biochemical test compared to biopsy (18 cases out of 19). Discrepancies for fibrosis were observed in 18 patients with a higher score for biochemical test in eight and a higher score for liver biopsy in 10 cases. A significant increase of gamma-glutamyl-transferase (GGT) (p=0.0001) and alanine aminotransferase (ALT) (p<0.0001) was observed in case of biochemical test overestimation of activity, and a significant increase of alpha2-macroglobulin (p=0.006) and GGT (p=0.018) in case of biochemical test overestimation of fibrosis. CONCLUSION: This prospective study confirms the good diagnostic value of biochemical tests for necrotico-inflammatory activity and fibrosis as compared with the histological analysis of liver biopsy. Clinicians must interpret Actitest and Fibrotest results with caution in patients with a significant elevation of ALT, and/or GGT and/or alpha2-macroglobulin which could overestimate hepatic injury.

Neonatal fever: A puzzling case.

Toxoplasmosis is a potentially serious fetal infection associated with maternal seroconversion of toxoplasmosis during pregnancy. Follow-up and treatment vary between different countries. We present a case of congenital toxoplasmosis with unusual physiopathology and symptomatology. The mother was immunized before the beginning of pregnancy but immunosuppressive treatments for Crohn disease maintained during the pregnancy could explain toxoplasmosis reactivation in the mother and congenital toxoplasmosis. The baby presented reversible B lymphopenia and hypogammaglobulinemia.

Cloning and transcriptional control of a eucaryotic permease gene.

The uracil permease gene of the yeast Saccharomyces cerevisiae was cloned on a hybrid plasmid which replicates autonomously in both yeast and Escherichia coli. Cloning was carried out by complementation in yeast. The smallest DNA fragment found to complement the uracil permease deficiency in recipient yeast cells measured approximately 2.3 kilobases. In strains transformed by the plasmid with the uracil permease gene inserted, initial rates of uracil uptake increased up to 25 times more than the rates found in the wild type. Using DNA probes carrying several regions of the cloned gene, I showed that a strain carrying the dhul-I mutation, which is not linked to the permease structural gene and is responsible for enhanced uptake velocity of uracil, had enhanced transcription of the permease gene. By using DNA probes recloned in phage M13 mp7, the direction of transcription of the permease gene relative to the restriction map was deduced. A half-life of 2 min was found for the permease mRNA in labeling kinetics experiments.

Membrane insertion of uracil permease, a polytopic yeast plasma membrane protein.

Uracil permease is a multispanning protein of the Saccharomyces cerevisiae plasma membrane which is encoded by the FUR4 gene and produced in limited amounts. It has a long N-terminal hydrophilic segment, which is followed by 10 to 12 putative transmembrane segments, and a hydrophilic C terminus. The protein carries seven potential N-linked glycosylation sites, three of which are in its N-terminal segment. Overexpression of this permease and specific antibodies were used to show that uracil permease undergoes neither N-linked glycosylation nor proteolytic processing. Uracil permease N-terminal segments of increasing lengths were fused to a reporter glycoprotein, acid phosphatase. The in vitro and in vivo fates of the resulting hybrid proteins were analyzed to identify the first signal anchor sequence of the permease and demonstrate the cytosolic orientation of its N-terminal hydrophilic sequence. In vivo insertion of the hybrid protein bearing the first signal anchor sequence of uracil permease into the endoplasmic reticulum membrane was severely blocked in sec61 and sec62 translocation mutants.

Multicenter verification of the Sysmex XN-Series.

INTRODUCTION: Verification of hemocytometry equipment deviates significantly from that of clinical chemistry equipment due to the absence of appropriate control material and the need for fresh material. In practice, verification is limited to comparison with the previously used equipment and determination of reproducibilities. Particularly in multicenter settings, harmonization of results is necessary. If the same equipment is used in several laboratory departments, calibration and uniformity are important issues. METHODS: In this study, seven Sysmex XN hematology modules distributed over three laboratories were evaluated with the same set of samples (n=160). RESULTS: Results of each Sysmex XN hematology module were compared with the results of the Sysmex XE-2100 hematology analyzer using linear regression. Although excellent correlation coefficients were obtained, in many cases the criteria for slope and/or intercept were not met. Therefore, the same data were analyzed with Bland-Altman difference plots with three times the specified CV% of the parameter as limits of agreement. At least 90% of the determinations per parameter and per module must comply with those limits of agreement. Almost all parameters on each module fulfilled these criteria, and only RBC and Ht from respectively two and four XN modules had to be recalibrated. Reproducibility of each parameter was determined 10 times in patient samples with low, normal, and high levels. Reproducibility of all parameters was within the specifications of the manufacturer and the biological variability. CONCLUSION: With this straightforward method, all seven Sysmex XN hematology modules demonstrated uniform results, which were identical to those of the previously used Sysmex XE-2100 hematology analyzer, the performance of which was well known.

Risk of eye lens radiation exposure for members of the public.

In 2011, the International Commission on Radiological Protection (ICRP) reviewed its recommendation concerning the equivalent dose limit for the eye lens, lowering it to 20 mSv in a year, for occupational exposure in planned exposure situations. The ICRP's statement does not contain any explicit recommendations regarding the organ dose limit for the eye lens for public exposure. For the moment, no change is proposed. But, to be coherent in the overall approach, the current equivalent limit for the public might be lowered. A similar yardstick than in the former recommendation may be used, that is to say a reduction of 10 times lower than that for occupational exposure. In this context, additional data on potential scenarios for public exposure of the eye lens are necessary. This paper, mainly based on a literature study, aims to provide, as far as possible, an exhaustive list of the situations in which members of the public can be exposed at the level of the eye lens. Once these situations have been defined, some calculations, made to assess the associated doses to the eye lens, are presented. This literature study did not reveal any current situations where members of the public would receive significant radiation doses to the eye lens. Indeed, the situations in which the dose to the eye lens might reach around 1 mSv per year for the public are extremely rare.

Flagging performance of the Sysmex XN2000 haematology analyser.

INTRODUCTION: With the introduction of the Sysmex XN haematology analyser, the white blood cell differentiation channel (WDF) and abnormal cell detection channel (WPC) have been added with algorithms for flagging blasts and abnormal or atypical lymphocytes. METHODS: In this study, 2011 samples were evaluated on a Sysmex XN2000 analyser and microscopically reviewed using a CellaVision DM96 digital microscope. RESULTS: A reference group of apparently healthy blood donors (n = 262) demonstrated in only three samples a positive suspect flag, which could not be confirmed microscopically. Positive WBC suspect flags were demonstrated in 3% of the 2011 samples. From the 55 samples with positive WBC suspect flags, an automatic reflex test was performed within the WPC. The WPC reflex test resulted in 10× Blast?, 15× Abnormal lymph? and 15× Atypical lymph? flags, which could be confirmed microscopically in 33% of these cases. A negative flagging was demonstrated in 15 cases. Microscopic evaluation demonstrated no abnormalities in these 15 cases. However, laboratory technicians also reported the presence of abnormal lymphocytes in 158 samples without an Abnormal lymph? flag. CONCLUSION: In conclusion, the combined use of WDF and WPC resulted in a reduction of approximately 25% of the number blood smears. As a result of the various techniques for light microscopy and haemocytometry, the adequacy of the XN flagging for abnormal and atypical lymphocytes cannot be established with certainty. To improve the quality of the reported results, it is recommended that laboratory technicians incorporate the haemocytometry results and the flagging information in the microscopic slide review.

EURADOS IC2012N: FURTHER INFORMATION DERIVED FROM AN EURADOS INTERNATIONAL COMPARISON OF NEUTRON PERSONAL DOSEMETERS.

In 2012, the European Radiation Dosimetry Group (EURADOS) performed an intercomparison for neutron dosemeters that are intended to measure personal dose equivalent, Hp(10). A total of 31 participants registered with 34 dosimetry systems. The irradiation tests were chosen to provide the participants with useful information on their dosimetry systems, i.e. linearity, reproducibility, responses for different energies and angles and to simulated workplace fields. This paper gives details of the extensive information derived from the exercise.

Focal intestinal lipogranulomatous lymphangitis in 10 dogs.

OBJECTIVES: To describe the clinical and pathological features of canine focal lipogranulomatous lymphangitis, to evaluate its underlying infectious cause and to compare it with human Crohn's disease. METHODS: Retrospective review of case records with a histopathological diagnosis of focal lipogranulomatous lymphangitis. Bacterial and fungal colonisation was evaluated using fluorescence in situ hybridisation and histochemical staining, respectively. A comparison with Crohn's disease was performed by a human pathologist. RESULTS: Ten dogs were evaluated. The historical complaints were predominantly chronic diarrhoea (10/10) and vomiting (5/10). The biochemical abnormalities included hypoalbuminaemia (6/10) and hypocobalaminaemia (4/6). Abdominal sonography revealed a thickened distal ileum±ileocolic junction. Colonoscopy showed a swollen caecal ostium and oedematous caecum in 7/10 dogs. A stenotic ileo-colic opening prevented endoscopic intubation in all dogs. Histology from the resected lesions revealed granulomatous inflammation involving the muscularis and serosa. Fluorescence in situ hybridization demonstrated invasive bacteria in 2/10 dogs. Post-resection, all dogs received metronidazole and tapering immunosuppressive doses of prednisolone. Remission (median 17 months) was achieved in 8/10 dogs. CLINICAL SIGNIFICANCE: Focal lipogranulomatous lymphangitis is a rare and severe form of canine inflammatory bowel disease with preferential localisation to the ileum and the ileocolic junction. An underlying infectious aetiology was not identified.

Studies on deoxyribonucleases from Haemophilus influenzae on DNA agarose affinity chromatography. Two-step purification of ATP-dependent deoxyribonuclease.

In a first part of this report, purification and characterization of several nucleased from lysates of Haemophilus influenzae are described. The enzymes bind to DNA with agarose columns and are removed by elution with phosphate buffer. Among the considered enzymes, the exonucleases 1 and 3, and endonuclease, a DNA polymerase and a restriction enzyme were recovered mixed by raising the phosphate concentration from 0.1 to 0.3 M, while the ATP-dependent DNAase recovered well purified, by raising the phosphate concentration to 0.45 M. After a rechromatography, on a second DNA with agarose column, of the peak of the ATP-dependent DNAase, the specific activity tested with 3H-labeled DNA was 125 units/mg of protein, representing a 300-fold purification of the original crude extract. In a second part, we have investigated the inactivation, at various pH, of transforming DNA of Haemophilus influenzae wild strain Rd with the different eluted fractions of the column, in order to determine the importance of contamination with other enzymatic activities, and also in order to confirm the nature of theisolated enzymes with a biological method. Finally, with enzymatic extracts of mutant strain Rd com minus 56, a strain which integrates shorter than normal pieces of DNA and which is suspected to possess and "activated specific endonuclease" able to recognize even small conformational modifications in paired structures, we tried to detect this activity on artificially constructed heteroduplex regions in DNA.