Arnaud J. Van Wettere

Ex vivo effect of gold nanoparticles on porcine synovial membrane

Gold nanoparticles (AuNPs) have great potential as carriers for local drug delivery and as a primary therapeutic for treatment of inflammation. Here we report on the AuNP-synovium interaction in an ex vivo model of intra-articular application for treatment of joint inflammation. Sheets of porcine femoropatellar synovium were obtained post mortem and each side of the tissue samples was maintained in a separate fluid environment. Permeability to AuNPs of different sizes (5−52 nm) and biomarker levels of inflammation were determined to characterize the ex vivo particle interaction with the synovium. Lipopolysaccharide or recombinant human interleukin-1β were added to fluid environments to assess the ex vivo effect of pro-inflammatory factors on permeability and biomarker levels. The synovium showed size selective permeability with only 5 nm AuNPs effectively permeating the entire tissues’ width. This process was further governed by particle stability in the fluid environment. AuNPs reduced matrix metalloproteinase and lactate dehydrogenase activity and hyaluronic acid concentrations but had no effect on prostaglandin E2 levels. Exposure to pro-inflammatory factors did not significantly affect AuNP permeation or biomarker levels in this model. Results with ex vivo tissue modeling of porcine synovium support an anti-inflammatory effect of AuNPs warranting further investigation.

Anchoring Hepatic Gene Expression with Development of Fibrosis and Neoplasia in a Toxicant-induced Fish Model of Liver Injury

Fish have been used as laboratory models to study hepatic development and carcinogenesis but not for pathogenesis of hepatic fibrosis. In this study, a dimethylnitrosamine-induced fish model of hepatic injury was developed in Japanese medaka (Oryzias latipes) and gene expression was anchored with the development of hepatic fibrosis and neoplasia. Exposed livers exhibited mild hepatocellular degenerative changes 2 weeks’ postexposure. Within 6 weeks, hepatic fibrosis/cirrhosis was evident with development of neoplasia by 10 weeks. Stellate cell activation and development of fibrosis was associated with upregulation of transforming growth factor beta 1 (tgfb1), tgfb receptor 2, mothers against decapentaplegic homolog 3 (smad3a), smad3b, beta-catenin (ctnnb1), myc, matrix metalloproteinase (mmp2), mmp14a, mmp14b, tissue inhibitors of metalloproteinase (timp) 2a, timp2b, timp3, collagen type I alpha 1a (col1a1a), and col1a1b and a less pronounced increase in mmp13 and col4a1 expression. Tgfb receptor I expression was unchanged. Immunohistochemistry suggested that biliary epithelial cells and stellate cells were the main producers of TGF-β1. This study identified a group of candidate genes likely to be involved in the development of hepatic fibrosis and demonstrated that the TGF-β pathway likely plays a major role in the pathogenesis. These results support the medaka as a viable fish model of hepatic fibrosis.

Adenovirus Infection in Taita Falcons (Falco fasciinucha) and Hybrid Falcons (Falco rusticolus × Falco peregrinus)

Abstract Three taita falcon (Falco fasciinucha) nestlings and 3 cohoused hybrid falcon (F rusticolus × F peregrinus) nestlings died after showing lethargy and anorexia for 4–5 days. Major lesions in the falcons were necrotizing hepatitis, splenitis, and lymphoplasmacytic nephritis. One hybrid falcon also had a heterophilic nephritis. Intranuclear inclusion bodies were present in the hepatocytes of the taita falcons and in the renal tubular epithelial cells of 1 hybrid falcon. Adenoviral particles were found in the cloacal content of 2 taita falcons. DNA in situ hybridization revealed aviadenoviral nucleic acid within the nuclei of hepatocytes of a taita falcon and a hybrid falcon and in the tubular epithelial cells of a hybrid falcon. Aviadenoviral DNA was amplified by polymerase chain reaction in the liver of taita falcons and hybrid falcons. The death of these birds was attributed to the aviadenovirus infection; however, the source of the virus was not determined. Predisposing factors for the outbreak of the viral infection in these falcons may have included transport stress, lack of quarantine measures, and cohousing of falcon species originating from isolated populations with other falcon species.

Mortalities Associated with Sepsis, Parasitism, and Disseminated Round Cell Neoplasia in Yellow-Lipped Sea Kraits (Laticauda colubrina)

Abstract This case series describes multiple mortalities associated with sepsis, neoplasia, and endoparasitism in yellow-lipped sea kraits (Laticauda colubrina) at an exhibit aquarium. Over a 2-yr period, the facility kept 42 L. colubrina, of which 38 died and 19 were suitable for necropsy and histopathology. The common clinical syndrome seen in these animals consisted of partial to compete anorexia, increased time spent “hauled-out” on land, intermittent regurgitation, chronic lethargy, and weight loss. Few animals died without premonitory signs. Nutritional support and treatment for presumptive parasitism and sepsis were unsuccessful. The mortality seen in this collection of sea kraits could be placed into three groups; one group of animals (n = 9) died of sepsis secondary to necrotizing enteritis or pneumonia; one group (n = 6) remained apparently healthy for over 1 yr and then died with multifocal granulomas and sepsis; and the last group (n = 3) died as a result of multicentric lymphoid neoplasia with secondary sepsis. The unifying factor in the majority of these cases is the presence of septicemia as the proximate cause of death. Based on the clinical picture, it is presumed that an immunosuppressive event, such as transport, captivity stress, or possible concurrent viral infection, resulted in a septic event and death.

Mechanical Evaluation of Various External Skeletal Fixator-Intramedullary Pin Tie-in Configurations Using a Tubular Plastic Bone Model

Abstract Use of external skeletal fixator–intramedullary pin tie-in (ESF-IM pin tie-in) fixators is an adjustable and effective method of fracture fixation in birds. The objective of this study was to evaluate the elements of the ESF-IM tie-in configuration used in birds. Ten variations of constructs were applied to a plastic bone model with a standard gap. Variants included non-tied and tie-in configurations, use of a 6- or 10-mm acrylic bar or a thermoplastic connecting bar, variation in the placement of the proximal fixation pin, use of 1.1-mm (0.045-in) or 1.6-mm (0.062-in) fixation pins, and configurations of 2, 3, or 4 fixation pins. The various constructs were loaded in bending, torque, and compression, and response variables were determined from resulting load-displacement curves (stiffness, load at 1-mm displacement). Results showed that, by using the tie-in configuration, increasing the diameter of the acrylic connecting bar, and increasing the diameter or number of fixation pins, each significantly increased the stiffness in all assessments. Placing the fixation pin distally in the proximal bone model segment increased the stiffness in bending, and adding a fixation pin to the distal bone model segment increased the stiffness in torque and bending. These results quantified the relative importance of specific parameters that effect stiffness and safe load of the ESF-IM tie-in construct as applied to a plastic bone fracture model.

Mechanical Evaluation of External Skeletal Fixator- Intramedullary Pin Tie-in Configurations Applied to Cadaveral Humeri From Red-tailed Hawks (Buteo jamaicensis)

Abstract Use of external skeletal fixator-intramedullary pin (ESF-IM) tie-in fixators is an adjustable and effective method of fracture fixation in birds. The objective of this study was to determine the contribution of each of the following parameters to the compressive and torsional rigidity of an ESF-IM pin tie-in applied to avian bones with an osteotomy gap: 1) varying the fixation pin position in the proximal bone segment and 2) increasing the number of fixation pins in one or both bone segments. ESF-IM pin tie-in constructs were applied to humeri harvested from red-tailed hawks (Buteo jamaicensis) (n u200a=u200a 24) that had been euthanatized for clinical reasons. Constructs with a variation in the placement of the proximal fixation pin and with 2, 3, or 4 fixation pins applied to avian bone with an osteotomy gap were loaded to a defined displacement in torque and axial compression. Response variables were determined from resulting load-displacement curves (construct stiffness, load at 1-mm displacement). Increasing the number of fixation pins from 1 to 2 per bone segment significantly increased the stiffness in torque (110%) and compression (60%), and the safe load in torque (107%) and compression (50%). Adding a fixation pin to the distal bone segment to form a 3-pin fixator significantly increased the stiffness (27%) and safe load (20%) in torque but not in axial compression. In the configuration with 2 fixation pins, placing the proximal pin distally in the proximal bone segment significantly increased the stiffness in torque (28%), and the safe load in torque (23%) and in axial compression (32%). Results quantified the relative importance of specific parameters affecting the rigidity of ESF-IM pin tie-in constructs as applied to unstable bone fracture models in birds.

Canine cutaneous epitheliotropic T-cell lymphoma with vesiculobullous lesions resembling human bullous mycosis fungoides.

The broad spectrum of clinical signs in canine cutaneous epitheliotropic T-cell lymphoma mimics many inflammatory skin diseases and is a diagnostic challenge. A 13-year-old-male castrated golden retriever crossbred dog presented with multifocal flaccid bullae evolving into deep erosions. A shearing force applied to the skin at the periphery of the erosions caused the epidermis to further slide off the dermis suggesting intraepidermal or subepidermal separation. Systemic signs consisted of profound weight loss and marked respiratory distress. Histologically, the superficial and deep dermis were infiltrated by large, CD3-positive neoplastic lymphocytes and mild epitheliotropism involved the deep epidermis, hair follicle walls and epitrichial sweat glands. There was partial loss of the stratum basale. Bullous lesions consisted of large dermoepidermal and intraepidermal clefts that contained loose accumulations of neutrophils mixed with fewer neoplastic cells in proteinaceous fluid. The lifted epidermis was often devitalized and bordered by hydropic degeneration and partial epidermal collapse. Similar neoplastic lymphocytes formed small masses in the lungs associated with broncho-invasion. Clonal rearrangement analysis of antigen receptor genes in samples from skin and lung lesions using primers specific for canine T-cell receptor gamma (TCRgamma) produced a single-sized amplicon of identical sequence, indicating that both lesions resulted from the expansion of the same neoplastic T-cell population. Macroscopic vesiculobullous lesions with devitalization of the lesional epidermis should be included in the broad spectrum of clinical signs presented by canine cutaneous epitheliotropic T-cell lymphoma.

A Comparison of Heavy Metal Concentrations and Health Assessment in Asian Clams Corbicula fluminea from Florida and North Carolina

The Asian clam Corbicula fluminea was introduced into the United States in 1938 and has since become established in much of the country. This invasive species can compete with native bivalves and compromise industrial water supply systems and power plants. Numerous studies have examined bivalves as bioindicators. The purpose of this study was to compare the heavy metal concentrations of the hard and soft tissues of specimens from Florida and North Carolina and to assess the clams health by microscopic examination of their soft tissues. Although the sample size was small, this study suggests that the Asian clams from the watersheds examined are healthy and that they accumulate lower levels of heavy metals than have been reported for clams from other, more polluted aquatic environments.

Secondary Amyloidosis and Renal Failure in a Captive California Sea Lion (Zalophus californianus)

Abstract A 16-yr-old, captive-born, female California sea lion (Zalophus californianus) was evaluated for intermittent lethargy, partial anorexia, and polydipsia of 2 wk duration. The animal was immobilized for physical examination. It was in thin body condition, with multifocal mucosal ulcerations over the caudal and ventral tongue. Blood was collected for hematology, serum chemistry, and leptospirosis serology. Serum chemistry revealed severe azotemia, mild hyperglycemia, and severe hyperphosphatemia. The animal went into cardiac arrest during recovery from anesthesia and died. On histopathology, abundant amorphous, finely fibrillar, eosinophilic material was deposited in the kidneys, and smaller amounts of the same material were found in the splenic and pancreatic vessels; these findings are consistent with systemic secondary amyloidosis. The animal also had chronic nephritis, which, coupled with renal amyloidosis, resulted in renal failure and death. Systemic amyloidosis should be considered as an additional differential diagnosis for renal failure in California sea lions.

Phenotypic characterization of transgenic Japanese medaka (Oryzias latipes) that express a red fluorescent protein in hepatocytes.

Transgenic organisms that express fluorescent proteins are used frequently for in vivo visualization of proteins and cells. The phenotype of a transgenic medaka (Oryzias latipes) strain that expresses a red fluorescent protein (RFP) in hepatocytes was characterized using light and fluorescence microscopy, immunohistochemistry, and transmission electron microscopy (TEM). Expression of RFP was first detected by confocal fluorescence microscopy in the location of the liver bud of live medaka embryos at 60 hr postfertilization (developmental stage 27). Subsequently, RFP signal was observed exclusively in hepatocytes throughout life using fluorescence microscopy in live fish and immunohistochemistry in formalin-fixed, paraffin-embedded liver sections. As the fish aged, prominent intracytoplasmic eosinophilic inclusions immunoreactive for RFP were observed by light microscopy and were correlated with membrane-bound electron dense inclusions on TEM. These results define the onset and location of RFP expression in the Tg(zf.L-fabp:DsRed) medaka and characterize a histologic phenotype that results from RFP expression in hepatocytes.