Detection of hepatitis E virus in slaughtered pigs in Italy

The stools of slaughtered pigs were screened for hepatitis E virus (HEV). HEV RNA was detected in 7.3% of the samples. HEV strains were characterized as genotype 3 subtype c, a cluster previously not described in Italy. These findings provide evidence that slaughterhouse workers may be exposed to HEV infection.     

The effects of a novel histamine-3 receptor inverse agonist on essential tremor in comparison to stable levels of alcohol

Essential tremor (ET) is a common movement disorder. Animal studies show that histaminergic modulation may affect the pathological processes involved in the generation of ET. Histamine-3 receptor inverse agonists (H3RIA) have demonstrated attenuating effects on ET in the harmaline rat model. In this double-blind, three-way cross-over, single-dose, double-dummy study the effects of 25 mg of a novel H3RIA (MK-0249) and a stable alcohol level (0.6 g L(-1)) were compared with placebo, in 18 patients with ET. Tremor was evaluated using laboratory tremorography, portable tremorography and a clinical rating scale. The Leeds Sleep Evaluation Questionnaire (LSEQ) and a choice reaction time (CRT) test were performed to evaluate potential effects on sleep and attention, respectively. A steady state of alcohol significantly diminished tremor as assessed by laboratory tremorography, portable tremorography and clinical ratings compared with placebo. A high single MK-0249 dose was not effective in reducing tremor, but caused significant effects on the LSEQ and the CRT test. These results suggest that treatment with a single dose of MK-0249 does not improve tremor in alcohol-responsive patients with ET, whereas stable levels of alcohol as a positive control reproduced the commonly reported tremor-diminishing effects of alcohol.     

Development and Fit-for-Purpose Validation of a Soluble Human Programmed Death-1 Protein Assay

Programmed death-1 (PD-1) protein is a co-inhibitory receptor which negatively regulates immune cell activation and permits tumors to evade normal immune defense. Anti-PD-1 antibodies have been shown to restore immune cell activation and effector function—an exciting breakthrough in cancer immunotherapy. Recent reports have documented a soluble form of PD-1 (sPD-1) in the circulation of normal and disease state individuals. A clinical assay to quantify sPD-1 would contribute to the understanding of sPD-1-function and facilitate the development of anti-PD-1 drugs. Here, we report the development and validation of a sPD-1 protein assay. The assay validation followed the framework for full validation of a biotherapeutic pharmacokinetic assay. A purified recombinant human PD-1 protein was characterized extensively and was identified as the assay reference material which mimics the endogenous analyte in structure and function. The lower limit of quantitation (LLOQ) was determined to be 100 pg/mL, with a dynamic range spanning three logs to 10,000 pg/mL. The intra- and inter-assay imprecision were ≤15%, and the assay bias (percent deviation) was ≤10%. Potential matrix effects were investigated in sera from both normal healthy volunteers and selected cancer patients. Bulk-prepared frozen standards and pre-coated Streptavidin plates were used in the assay to ensure consistency in assay performance over time. This assay appears to specifically measure total sPD-1 protein since the human anti-PD-1 antibody, nivolumab, and the endogenous ligands of PD-1 protein, PDL-1 and PDL-2, do not interfere with the assay.

Electronic supplementary material

The online version of this article (doi:10.1208/s12248-015-9762-4) contains supplementary material, which is available to authorized users.KEY WORDS: biomarker, nivolumab, reagent characterization, reagent characterizationsoluble PD-1, reference standard     

Analysis of Synchronous and Asynchronous In Vitro Infections with Homologous Murine Norovirus Strains Reveals Time-Dependent Viral Interference Effects

Viral recombination is a key mechanism in the evolution and diversity of noroviruses. In vivo, synchronous single-cell coinfection by multiple viruses, the ultimate prerequisite to viral recombination, is likely to be a rare event and delayed secondary infections are a more probable occurrence. Here, we determine the effect of a temporal separation of in vitro infections with the two homologous murine norovirus strains MNV-1 WU20 and CW1 on the composition of nascent viral populations. WU20 and CW1 were either synchronously inoculated onto murine macrophage cell monolayers (coinfection) or asynchronously applied (superinfection with varying titres of CW1 at half-hour to 24-h delays). Then, 24 h after initial co-or superinfection, quantification of genomic copy numbers and discriminative screening of plaque picked infectious progeny viruses demonstrated a time-dependent predominance of primary infecting WU20 in the majority of viral progenies. Our results indicate that a time interval from one to two hours onwards between two consecutive norovirus infections allows for the establishment of a barrier that reduces or prevents superinfection.     

Cutaneous manifestations as presenting sign of autoimmune lymphoproliferative syndrome in childhood

Autoimmune lymphoproliferative syndrome is a disorder due to a defect of lymphocyte apoptosis, whose clinical manifestations consist of hyperplasia of lymphoid tissues and autoimmune diseases. We report on a 26-month-old child who presented with frequent eruptions of weals and angioedema without any apparent triggering factor, who subsequently developed an erythematopapular rash with a histological pattern of a lymphoplasmacellular infiltrate. Familial anamnesis revealed a history of lymphoadenomegaly and massive spleen and liver enlargement in her sister. Functional and molecular analysis led to a diagnosis of type 1a autoimmune lymphoproliferative syndrome. Immunophenotyping of the cutaneous lesion revealed the presence of an inflammatory infiltrate with a considerably high number of Langerhans cells. Cutaneous features such as urticaria, angioedema and vasculitis in children with a personal and familial history of hyperplasia of lymphoid tissues may be a presenting sign of a systemic disease, such as autoimmune lymphoproliferative syndrome.     

Detection of Antibodies against Norovirus Genogroup GIV in Carnivores

Noroviruses (NoVs) resembling human NoV genotype GIV (Alphatron-like) have recently been detected in carnivores. By using an enzyme-linked immunosorbent assay based on baculovirus-expressed capsid protein VP1 of lion strain GGIV.2/Pistoia/387/06/ITA, NoV-specific antibodies were detected in cats (16.11%) and dogs (4.8%), demonstrating that these animals are exposed to infections caused by NoVs.Noroviruses (NoVs) have been identified as the most common cause of viral gastroenteritis in humans. NoV infections affect persons of all age groups (13) and are predominantly transmitted through the fecal-oral route, either indirectly through contaminated food, water, or surfaces or directly from person to person (4). NoVs are small, rounded, nonenveloped, single-stranded RNA viruses belonging to the family Caliciviridae. On the basis of the VP1 capsid gene sequence, NoVs have been classified into five genogroups (genogroups GI to GV) and at least 32 genotypes (11, 16, 18); but only GI, GII, and GIV are known to infect humans, with GII viruses accounting for the majority of NoV infections (15).NoVs genetically and antigenically closely related to human NoVs have been discovered in cows and pigs (14, 16). Recently, NoVs have been detected in the stools of a captive lion cub (strain Pistoia/387/06/ITA) (11) and a dog (strain Bari/170/07/ITA) (10) with enteric signs. Upon sequence analysis, the lion and canine NoVs were found to share 90.1% amino acid (aa) identity in the capsid protein and were closely related to human NoV GIV (Alphatron-like) (69.3% to 70.1% aa identity). According to widely accepted classification criteria (18), the lion and canine NoVs were classified as having a novel genotype, genotype GIV.2, within genogroup GIV (10, 11). However, whether these novel caliciviruses are common in domestic carnivores or whether these were detected serendipitously remains unclear. In the study described here, the prevalence of antibodies specific for NoV GIV in carnivores was assessed by using virus-like particles (VLPs).VLPs were developed with the RNA of the prototype lion NoV GIV strain Pistoia/387/06/ITA (GenBank accession number {"type":"entrez-nucleotide","attrs":{"text":"EF450827","term_id":"148888574","term_text":"EF450827"}}EF450827) (11). Briefly, the full-length VP1 (open reading frame 2) was cloned into the vector pRN16 (kindly provided by Polly Roy), and transfection was performed with triple-cut linearized Autograph californica multiple nucleopolyhedrosis virus DNA. The recombinant baculovirus was selected and plaque purified on Spodoptera frugiperda (Sf21) cells, as described previously (2). For the large-scale production of the VLPs, a 100-ml Sf9 cell suspension culture was infected with the recombinant baculovirus at a multiplicity of infection of 3 PFU/cell. The recombinant protein was isolated from the culture medium at 48 h postinfection. After purification by rate-zonal centrifugation on a discontinuous sucrose gradient (60, 40, 30, 20%), the recombinant VP1 and the assembled VLPs were analyzed by electrophoresis on a 12% SDS-polyacrylamide gel and by electron microscopy. Hyperimmune serum against the purified lion VLPs was raised in two rabbits. The specificity of the serum was tested by Western blotting (WB), with the lion GIV VLPs and dog GIV strain Bari/170/07/ITA being used as positive controls and wild-type baculovirus and vaccine FCV strain F9 being used as negative controls (Fig. ​(Fig.11).Open in a separate windowFIG. 1.Western blotting analysis of lion GIV VLPs using rabbit hyperimmune serum. Lane 1, Precision Plus protein standards (Bio-Rad, Italy); lane 2, mock-infected Sf9 cells; lane 3, wild-type baculovirus Sf9 insect cells; lane 4, FCV strain F9 purified from the supernatant of CrFK cells; lane 5, dog GIV strain Bari/170/07/IT purified from a fecal sample suspension; lane 6, lion GIV VLPs purified from the supernatants of Sf9 insect cells.For the development of the enzyme-linked immunosorbent assay (ELISA), purified VLPs were coated onto 96-well enzyme immunoassay plates (Costar, Italy) at 100 μl per well (final concentration, 8 μg/ml) in carbonate-bicarbonate buffer (0.05 M, pH 9.6), and the plates were incubated at 4°C overnight. After the plates were blocked with 1% bovine serum albumin in phosphate-buffered saline (PBS) buffer at room temperature (RT) for 2 h, the VLP-coated microplates were incubated with 100 μl of dog and cat serum samples diluted to 1:50 in PBS at 37°C for 1 h. The plates were washed three times in PBS with 0.1% Tween 20 (PBST) and were then incubated with goat anti-cat IgG (1:1,000) and anti-dog IgG (1:2,000) conjugated with horseradish peroxidase (Sigma-Aldrich, Italy) for 1 h at 37°C. The plates were washed three times in PBST prior to the addition of 2,2′-azino-di-(3-ethylbenzthiazoline-6-sulfonate) (ABTS) substrate. Each reaction was completed by incubation at room temperature for 20 min, and the absorbance was measured at 405 nm. Wild-type baculovirus Sf9 insect cells were used to obtain a positive/negative ratio (optical density of the GIV VLPs/optical density of the wild-type baculovirus Sf9 insect cells) to evaluate the background binding. In order to establish the cutoff value, 25 cat serum samples negative for the lion GIV VLPs by WB assay and a rabbit negative control serum sample were tested. A mean with a standard deviation (SD) was calculated. The cutoff value was established as the mean value plus 3 SDs. A total of 211 serum samples collected from adult cats (ages, >1 year) from several geographical settings in Italy were tested. Ninety-six serum samples were collected from private veterinary clinics in Teramo, Italy; 44 were from rescue colonies in Reggio Emilia, Italy; 34 were from the clinic of the Faculty of Veterinary Medicine of Bari (Bari, Italy); and 37 were from stray cats living in the Rome, Italy, Biopark. In addition, 103 serum samples from adult dogs (ages, >1 year) collected in Teramo from 2006 to 2008 were tested.The overall prevalence of lion NoV GIV-specific antibodies in cats was 16.1% (34/211), with a higher seroprevalence rate (32.0%) being detected in stray cats living in the Rome Biopark than in the other cats (14.6% to 6.8%). The difference in the estimated prevalence between the two groups was statistically significant (χ² = 8.8393, P > 0.01). Five of 103 (4.8%) serum samples from dogs were also positive for antibodies against the lion NoV GIV.2 (Table ​(Table11).

TABLE 1.

Results of serological investigation by the ELISA with feline and canine sera

Factors associated with clinicians’ dispositions in an out-patient psychiatric department

AIM: This cross-sectional study attempted to identify factors associated with clinicians' dispositions of patients after the first visit in an out-patient psychiatric department. METHODS: Over a 33-month period, all new episodes of care with the department were included in the study. For each patient, socio-demographic, clinical information and contact characteristics were prospectively collected in relation to the first visit, as was information on case disposition. Factors associated with clinicians' disposition were analysed. RESULTS: Of the 1,138 patients who met the study criteria, 848 (75%) were followed up by the department, 150 (13%) were referred to other services and 140 (12%) were discharged. Suffering from a major psychiatric disorder, being younger and not living in an institution influenced clinicians' disposition to follow-up patients. Older age increased the chances of being referred to other services rather than discharged. CONCLUSIONS: Examining decision-making behaviour in out-patient psychiatric departments is a worthwhile endeavour because this setting represents the main entry point of modern and accessible community-based systems of care. The findings confirmed the importance of psychiatric determinants in the dispositional process and contribute to make clinicians more aware of other factors related to their decision-making.     

Hepatitis E virus in sheep in Italy

Hepatitis E virus (HEV) is the leading cause of human enterically transmitted viral hepatitis occurring around the world both as outbreaks and as sporadic cases. The accumulating literature indicates that domestic pigs and wild boars are the main reservoirs of genotype 3 and genotype 4 for human infections in industrialized countries. However, the recent identification of HEV from various animal species poses additional potential concerns for HEV zoonotic infection. In this study, the role of sheep as potential host of hepatitis E virus (HEV) was investigated. By screening 192 sheep from seven farms located in Abruzzo Region (Southern Italy), HEV‐specific antibodies were detected in the sera of 41 animals (21.3%) whilst the RNA of HEV, genotype 3, was detected in 20 faecal (10.4%) and three serum samples (1.6%). Upon sequence analyses of a partial ORF2 gene region of eight HEV positive samples, the sheep sequences all grouped together within HEV genotype 3 subtype c, being most closely related to HEV strains identified in goat and wild boar from Abruzzo. This is the first study that demonstrates, serologically and molecularly, the presence of HEV in sheep population in a European country.     

Rapid and simple determination of inulin in biological fluids by high-performance liquid chromatography with light-scattering detection

We report a new high-performance liquid chromatography method developed for measuring inulin in plasma and urine using ion moderated partition chromatography and evaporative light-scattering detection. Samples are deproteinized with a zinc acetate and phosphotungstic acid solution and added with melezitose as an internal standard. The chromatographic separation is carried out in 16 min at a flow-rate of 0.6 ml/min using deionized water as the mobile phase. Within-run precision, measured at four different concentrations (0.050 mg/ml, 0.150 mg/ml, 0.300 mg/ml and 1.200 mg/ml), ranges from 1.7 to 3.4% in plasma and from 1.5 to 3.5% in urine. Similarly, between-run precision is in plasma from 2.0 to 4.3% and in urine from 2.0 to 4.4%. Analytical recovery ranges from 97.9 to 100.1% in plasma and from 99.1 to 99.7% in urine, respectively. Detection limit (signal-to-noise ratio=3) is 5 microg/ml both in plasma and urine. The method is simple, sensitive, without interference due to hexoses or drugs commonly taken by patients with renal diseases, and offers the advantage of measuring inulin without previous hydrolysis of the molecule.