Efficient gene delivery to the cone-enriched pig retina by dual AAV vectors.

Gene therapy with adeno-associated viral (AAV) vectors is limited by AAV cargo capacity that prevents their application to the inherited retinal diseases (IRDs), such as Stargardt disease (STGD) or Usher syndrome type IB (USH1B), which are due to mutations in genes larger than 5 kb. Trans-splicing or hybrid dual AAV vectors have been successfully exploited to reconstitute large gene expression in the mouse retina. Here, we tested them in the large cone-enriched pig retina that closely mimics the human retina. We found that dual AAV trans-splicing and hybrid vectors transduce pig photoreceptors, the major cell targets for treatment of IRDs, to levels that were about two- to threefold lower than those obtained with a single AAV vector of normal size. This efficiency is significantly higher than that in mice, and is potentially due to the high levels of dual AAV co-transduction we observe in pigs. We also show that subretinal delivery in pigs of dual AAV trans-splicing and hybrid vectors successfully reconstitute, albeit at variable levels, the expression of the large genes ABCA4 and MYO7A mutated in STGD and USH1B, respectively. Our data support the potential of dual AAV vectors for large gene reconstitution in the cone-enriched pig retina that is a relevant preclinical model.

AAV-mediated photoreceptor transduction of the pig cone-enriched retina.

Recent success in clinical trials supports the use of adeno-associated viral (AAV) vectors for gene therapy of retinal diseases caused by defects in the retinal pigment epithelium (RPE). In contrast, evidence of the efficacy of AAV-mediated gene transfer to retinal photoreceptors, the major site of inherited retinal diseases, is less robust. In addition, although AAV-mediated RPE transduction appears efficient, independently of the serotype used and species treated, AAV-mediated photoreceptor gene transfer has not been systematically investigated thus so far in large animal models, which also may allow identifying relevant species-specific differences in AAV-mediated retinal transduction. In the present study, we used the porcine retina, which has a high cone/rod ratio. This feature allows to properly evaluate both cone and rod photoreceptors transduction and compare the transduction characteristics of AAV2/5 and 2/8, the two most efficient AAV vector serotypes for photoreceptor targeting. Here we show that AAV2/5 and 2/8 transduces both RPE and photoreceptors. AAV2/8 infects and transduces photoreceptor more efficiently than AAV2/5, similarly to what we have observed in the murine retina. The use of the photoreceptor-specific rhodopsin promoter restricts transgene expression to porcine rods and cones, and results in photoreceptor transduction levels similar to those obtained with the ubiquitous promoters tested. Finally, immunological, toxicological and biodistribution studies support the safety of AAV subretinal administration to the large porcine retina. The data presented here on AAV-mediated transduction of the cone-enriched porcine retina may affect the development of gene-based therapies for rare and common severe photoreceptor diseases.

Time-dependent changes of cytokines mRNA in bronchoalveolar lavage fluid from symptomatic recurrent airway obstruction-affected horses.

During an 18 day test, we measured the cytokine mRNA expression (Interleukin-1beta [IL-1beta], Interleukin-8 [IL-8], Interferon-gamma [IFN-gamma], Tumor Necrosis Factor-alpha [TNF-alpha]) of cells from bronchoalveolar lavage fluid [BALF] in five horses previously diagnosed with RAO, before and during challenge exposure, and after the desensitization phase which involved dexamethasone treatment and environmental modification. Simultaneously, the same cytokine mRNA expression of cells from BALF in four asymptomatic RAO-affected horses maintained outdoors was analyzed. An evident respiratory distress was observed in the challenge group within 3 days, with a significant overexpression of IL-8 and TNF-alpha mRNA on the ninth day. The pharmacological and environmental desensitization provided a down regulation of all the cytokines. No statistical modification characterized the cytokine kinetics of the asymptomatic horses maintained outdoors. A comparison for each time point of the cytokines between the exposed and unexposed horses showed no significant differences. The study suggested that a standardized exposure protocol and sampling time in experimental studies of RAO is mandatory for a correct comparison of the results obtained by different Authors. However, the absence of significant changes between the exposed and unexposed horses could depend on the lack of the sample uniformity since the evolution of the disease represents a continuum from a healthy to a pathological condition.

Reversible myocardial dysfunction in a dog after resuscitation from cardiopulmonary arrest.

A 4-year-old Dachshund was referred for management of a mandibular fracture. The dog underwent cardiopulmonary arrest after sedation for skull radiography. Cardiopulmonary resuscitation was started immediately, and return of spontaneous circulation was rapidly obtained. However, after resuscitation, the dog was hemodynamically unstable. Additionally, global left ventricular systolic dysfunction and a focal hyperechoic myocardial lesion were found echocardiographically, and serum cardiac troponin I was severely elevated (82.80 ng/mL, upper hospital limit <0.7 ng/mL). The dog remained hospitalized in the intensive care unit and received supportive medical therapies, including intravenous inotropes (pimobendan, dobutamine). Over the following days, progressive hemodynamic and echocardiographic improvement was achieved in response to treatment. Moreover, after 72 h, a significant reduction of serum cardiac troponin I (9.80 ng/mL) was documented. On recheck, 3 weeks after discharge, the dog was clinically stable, and both left ventricular systolic function and serum cardiac troponin I (0.10 ng/mL) were normal. Based on clinical and instrumental findings after cardiopulmonary resuscitation, a probable diagnosis of reversible postresuscitation myocardial dysfunction associated with transient myocardial injury was made.

Survey on antimicrobial prescribing patterns in small animal veterinary practice in Emilia Romagna, Italy.

This investigation provides for the first time a general view of the prescribing patterns of antimicrobials in small animal practice in Emilia Romagna, Italy. In the context of a project on antimicrobial resistance managed by the Regional Veterinary Service, veterinary clinicians were invited to voluntarily complete an online questionnaire. This was designed to gather information on antimicrobial prescribing practices and biosecurity measures and to understand the perception of the issue specific to this region of Italy. In total, 266 questionnaires correctly completed were collected. Although clinicians seemed to follow different approaches when using antimicrobials, the data analysis revealed a general awareness on resistance. Penicillins were the most commonly prescribed class, followed by (fluoro)quinolones and cephalosporins. Among those who use laboratory testing more or less frequently (microbiological analysis and susceptibility testing) to support their prescribing habits, only 7 per cent make a habit of always waiting for the results before starting the treatment. Seventy-eight per cent of the respondents declared the use of antimicrobials licensed for human beings. Biosecurity measures were carefully taken into account by the majority of the veterinarians. The results identified the antimicrobial classes that are commonly prescribed and highlighted that perioperative hygiene measures and the use of laboratory diagnosis are critical aspects that need to be emphasised in drawing up guidelines on the prudent use of these drugs in pets.

Evaluation of the delta neutrophil index from an automated blood cell analyser in septic dogs.

Immature granulocytes (IG) are a marker of severe inflammatory states in human beings and animals, and have been linked to a diagnosis of sepsis and poor prognosis. The delta neutrophil index (DNI), automatically calculated by a haematological analyser, provides an estimate of circulating IG. In particular, an increased DNI value has been associated with the severity of sepsis, and mortality, in critically ill human beings. The aims of this study were to determine the DNI reference interval (RI) in healthy dogs, and to evaluate its diagnostic and prognostic significance in dogs with sepsis. A total of 118 dogs with sepsis undergoing a complete blood cell count (CBC) at the time of hospital admission were included retrospectively. Dogs with sepsis were compared to 20 dogs with primary immune-mediated haemolytic anaemia (IMHA) and 99 healthy controls. The DNI RI was set from 0 to 9.2%. The DNI was significantly higher in dogs with sepsis compared to dogs with IMHA and healthy dogs (P<0.001), and significantly higher in dogs with septic shock compared to septic dogs without circulatory failure (P<0.03). No differences were detected between survivors (78/118) and non-survivors (40/118). Septic dogs with a DNI above the RI had significantly higher frequencies of IG and toxic neutrophil changes on manual blood smear evaluation (P=0.03 and P<0.001, respectively). The DNI had a fair performance in identifying dogs with sepsis in this population and predicted septic shock. Larger prospective studies are needed to validate DNI measurement in dogs and to test its clinical utility.

Hodgkin's lymphoma: the pathologist's viewpoint.

Despite its well known histological and clinical features, Hodgkin's lymphoma (HL) has recently been the object of intense research activity, leading to a better understanding of its phenotype, molecular characteristics, histogenesis, and possible mechanisms of lymphomagenesis. There is complete consensus on the B cell derivation of the tumour in most cases, and on the relevance of Epstein-Barr virus infection and defective cytokinesis in at least a proportion of patients. The REAL/WHO classification recognises a basic distinction between lymphocyte predominance HL (LP-HL) and classic HL (CHL), reflecting the differences in clinical presentation and behaviour, morphology, phenotype, and molecular features. CHL has been classified into four subtypes: lymphocyte rich, nodular sclerosing, with mixed cellularity, and lymphocyte depleted. The borders between CHL and anaplastic large cell lymphoma have become sharper, whereas those between LP-HL and T cell rich B cell lymphoma remain ill defined. Treatments adjusted to the pathobiological characteristics of the tumour in at risk patients have been proposed and are on the way to being applied.

[Propafenone and verapamil poisoning. Report of 2 clinical cases]. / Intossicazione da propafenone e verapamil. Descrizione di due casi clinici.

We described the self-poisoning of two young adolescents who took improper doses of two major cardiovascular drugs: propafenone and verapamil. The young girls developed markedly different clinical patterns: ECG abnormalities without clinical consequences were found in one case progressively ingravescent ECG abnormalities leading to cardiac arrest in the other. These differences are probably due to varying doses taken and metabolic states. Conventional detoxication and resuscitation techniques proved successful in both cases.

Investigation of the presence of canine adenovirus (CAdV) in owned dogs in Northern Italy.

The use of a modified live canine adenovirus (CAdV) vaccine has greatly reduced the incidence of infectious canine hepatitis (ICH) in dogs. Nevertheless, cases of CAdV type 1 and 2 (CAdV-1 and CAdV-2) infection have been recently reported posing questions about the epidemiological situation of CAdV in dogs. In order to assess the presence of CAdV, samples from 51 dogs presented at a Veterinary Teaching Hospital in Bologna, Italy, for reasons unrelated with CAdV infection, were tested with a polymerase chain reaction (PCR) assay for CAdV. Thirty dogs (58.8%) were PCR positive for CAdV-2 infection and four of them (7.8%) were positive for CAdV-1. Sequence analysis performed on the obtained PCR products suggests that a genetically stable CAdV-1 strain and different CAdV-2 strains circulate in the canine population examined and that coinfections are relatively frequent.

Reversible pulmonary hypertension in a cat.

A 13-year-old, neutered female domestic shorthair cat was presented for sudden respiratory distress following palliative radiotherapy and the combined administration of a single dose of carboplatin for the treatment of recurrent fibrosarcoma. Clinical and radiographic findings were suggestive of pleural effusion. Echocardiography revealed marked right-sided cardiac enlargement associated with tricuspid regurgitation and Doppler evidence of pulmonary hypertension. After 25 days of treatment for congestive heart failure and suspected pulmonary thromboembolism, clinical signs and echocardiographic and Doppler evidence of right-sided cardiac enlargement and pulmonary hypertension had completely resolved. To the best of the authors' knowledge, this is the first report of reversible pulmonary hypertension, likely secondary to pulmonary thromboembolism, in a cat.

Pulmonary kinetic expression of the endothelin system in a swine model of endotoxic shock.

Endothelin (ET)-1 is a potent vasoconstrictor peptide involved in the derangement of respiratory mechanics during endotoxic shock. We measured the kinetics of pulmonary mRNA expression of the key components of the ET system [i.e., ET-1, ET-converting enzyme (ECE), and ETA and ETB receptors] by quantitative real-time reverse transcriptase-polymerase chain reaction in a swine model of endotoxic shock (0, 1, 2, 3, and 4 h of continuous LPS infusion at 40 microg/kg/hour; sham group, 4 hour saline infusion). A significant increase in mRNA expression levels was observed for ET-1 in LPS-treated piglets; the increase began as early as 1 hour. In contrast, no significant variations were observed for the ECE, ETA, or ETB genes. Small gene expression differences observed with respect to our previous results suggest a possible effect of the anesthesia or surgical protocol on ET system regulation.