Cytologic diagnosis of generalized cutaneous sporotrichosis in a hunting hound

A 1-year-old male Foxhound/Walker Hound mix was presented to the small animal internal medicine service at Louisiana State University School of Veterinary Medicine with a 6-week history of progressive, multifocal, ulcerative and draining, well-circumscribed lesions in a generalized distribution. Prior to referral, a presumptive diagnosis was made of sterile pyogranulomatous disease; immunosuppressive therapy was instituted but resulted in clinical deterioration. At presentation, the dog had marked neutropenia (1100 neutrophils/microL), and a mild toxic left shift (400 bands/microL). Cytologic findings in the exudates from a draining skin lesion included high numbers of markedly degenerate neutrophils (about 95% of nucleated cells) as well as low numbers of macrophages, small mature lymphocytes, and eosinophils. Low numbers of intracellular (within neutrophils and macrophages) and extracellular, pleomorphic, cigar-to-ovoid shaped organisms ( approximately 3x9 microm) consistent with Sporothrix were observed. Histopathologic examination of a skin biopsy showed marked, chronic, active, ulcerative, pyogranulomatous dermatitis and panniculitis, with intralesional yeast consistent with Sporothrix sp. The etiologic agent was confirmed as Sporothrix schenckii by macerated tissue fungal culture. The patient was treated with itraconazole, enrofloxacin, and clindamycin, with clinical resolution occurring over a 3-month period. This case is a rare example of the cytologic diagnosis of Sporothrix schenckii in a canine patient. Diagnosis of canine sporotrichosis is often challenging and usually requires tissue culture, as infected dogs typically harbor very few organisms. The patient\u27s prior immunosuppressive therapy likely contributed to higher numbers of organisms in exudates from the cutaneous lesions, facilitating cytologic diagnosis

Functional characterization of a recombinant adeno-associated virus 5-pseudotyped cystic fibrosis transmembrane conductance regulator vector

Despite extensive experience with recombinant adeno-associated virus (rAAV) 2 vectors in the lung, gene expression has been low in the context of cystic fibrosis (CF) gene therapy, where the large size of the cystic fibrosis transmembrane conductance regulator (CFTR) coding sequence has prompted the use of compact endogenous promoter elements. We evaluated the possibility that gene expression from recombinant adeno-associated virus (rAAV) could be improved by using alternate AAV capsid serotypes that target different cell-surface receptors (i.e., rAAV5) and/or using stronger promoters. The relative activities of the cytomegalovirus (CMV) Rous sarcoma virus (RSV) promoter, the CMV enhancer/beta-actin (CB) promoter combination, and the CMV enhancer/RSV promoter hybrid were assessed in vitro in a CF bronchial cell line. The CB promoter was the most efficient. AAV capsid serotypes, rAAV2 and rAAV5, were also compared, and rAAV5 was found to be significantly more efficient. Based on these studies a rAAV5-CB-promoter-driven CFTR minigene vector was then used to correct the CF chloride transport defect in vitro, as well as the hyperinflammatory lung phenotype in Pseudomonas-agarose bead challenged CF mouse lungs in vivo. These studies provide functional characterization of a new version of rAAV-CFTR vectors

In vitro and in vivo functional characterization of gutless recombinant SV40-derived CFTR vectors

In cystic fibrosis (CF), respiratory failure caused by progressive airway obstruction and tissue damage is primarily a result of the aberrant inflammatory responses to lung infections with Pseudomonas aeruginosa. Despite considerable improvement in patient survival, conventional therapies are mainly supportive. Recent progress toward gene therapy for CF has been encouraging; however, several factors such as immune response and transduced cell turnover remain as potential limitations to CF gene therapy. As alternative gene therapy vectors for CF, we examined the feasibility of using recombinant SV40-derived vectors (rSV40s), which may circumvent some of these obstacles. To accommodate the large cystic fibrosis transmembrane conductance regulator (CFTR) cDNA, we removed not only SV40 Tag genes, but also all capsid genes. We, therefore, tested whether \u27gutless\u27 rSV40s could be packaged and were able to express a functional human CFTR cDNA. The results from our in vitro analysis determined that rSV40-CFTR was able to successfully result in the expression of CFTR protein, which localized to the plasma membrane and restored channel function to CFTR-deficient cells. Similarly, in vivo experiments delivering rSV40-CFTR to the lungs of Cftr-/- mice resulted in a reduction of the pathology associated with intra-tracheal P. aeruginosa challenge. rSV40-CFTR-treated mice had less weight loss when compared with control-treated mice as well as demonstrably reduced lung inflammation as evidence by histology and reduced inflammatory cytokines in the broncho-alveolar lavage. The reduction in inflammatory cytokine levels led to an evident decrease in neutrophil influx to the airways. These results indicate that further study of the application of rSV40-CFTR to CF gene therapy is warranted