Richard R. Dubielzig

Systemic Administration of an Attenuated, Tumor-Targeting Salmonella typhimurium to Dogs with Spontaneous Neoplasia: Phase I Evaluation

Purpose: Genetically modified bacteria are a potentially powerful anticancer therapy due to their tumor targeting capacity, inherent antitumor activity, and ability to serve as efficient vectors for gene delivery. This study sought to characterize the acute and short-term toxicities and tumor colonization rates of a genetically modified Salmonella typhimurium (VNP20009) in dogs with spontaneous tumors, in the context of a phase I dose escalation trial. Experimental Design: Forty-one pet dogs with a variety of malignant tumors received weekly or biweekly i.v. infusions of VNP20009, at doses ranging from 1.5 × 105 to 1 × 108 cfu/kg. Vital signs and clinicopathologic variables were monitored regularly. Incisional biopsies were obtained before and 1 week following the first infusion for histopathology and bacterial culture. Results: The nominal maximum tolerated dose was 3 × 107 cfu/kg, with refractory fever and vomiting being the dose-limiting toxicities. One treatment-related acute death occurred. Bacteria were cultured from tumor tissue in 42% of cases. Thirty-five patients were evaluable for antitumor response. Major antitumor responses were seen in 15% (4 complete response and 2 partial response), and disease stabilization for at least 6 weeks in 10%. Conclusions: Administration of VNP20009 at doses with acceptable toxicity results in detectable bacterial colonization of tumor tissue and significant antitumor activity in tumor-bearing dogs.

Central Nervous System Neosporosis in a Foal

Cutaneous or subcutaneous cysts of all types are considered rare in cats. A literature search yielded no reports of cutaneous or subcutaneous dermoid cysts in cats. The structures reported here were histologically compatible with the description of the dorsal midline structures in dogs but, because they did not communicate with the spinal canal, these cysts posed no danger or potential danger to the animal from central nervous system infection. These structures were present in the flank instead of along the dorsal midline, possibly as a result of faulty embryologic fusion of adjacent dermatomes. The dermoid cyst in cat no. 2, considering its young age, probably was a congenital disorder; however, cat no. 1 was 10 years old at the time of presentation. It was not known how long the cyst in cat 1 had been present or if it had grown in size immediately prior to presentation. Dermoid cysts have been classified according to depth of penetration of the sinus. Class I cysts extend from the skin to the supraspinous ligament, class II cysts do not extend as deeply but are connected to the supraspinous ligament by a fibrous band, and class III cysts are similar to class II cysts but have no connecting band to the ligament. A fourth class has been proposed, in which the cyst extends to the spinal canal and is attached to the dura mater. This class is analogous to the pilonidal sinus of human beings, which usually occurs in the coccygeal region. The term pilonidal cyst, which by definition means any cyst containing a tuft of hair, is usually used synonymously with the term dermoid cyst in veterinary medicine. Perhaps 1 reason for the confusion over the term dermoid cyst is that many deep anomalous structures can have structural components of epithelium and can be loosely termed dermoid cysts by pathologists. A recent textbook has subclassified these cysts in an effort to more clearly define each type from a histologic standpoint. In this classification system, dermoid cysts are considered a type of follicular tumor. More widespread application of the classification system may be helpful to pathologists and clinicians for separating benign lesions from those with potential for serious complications. The surgical treatment of dermoid cysts is straightforward and involves careful dissection of the cyst. Although dermoid cysts are reported to involve only the skin or subcutaneous tissues, the cyst in cat no. 1 was found isolated between muscle layers of the flank and the sinus in cat no. 2 extended to the peritoneum. When excision of the cyst involves creation of an abdominal wall defect, care should be taken to ensure that anatomic closure of the defect is accomplished to avoid subsequent herniation of abdominal contents.

Prevention of bacterial colonization of contact lenses with covalently attached selenium and effects on the rabbit cornea.

Purpose: Although silicone hydrogel materials have produced many corneal health benefits to patients wearing contact lenses, bacteria that cause acute red eye or corneal ulcers are still a concern. A coating that inhibits bacterial colonization while not adversely affecting the cornea should improve the safety of contact lens wear. A covalent selenium (Se) coating on contact lenses was evaluated for safety using rabbits and prevention of bacterial colonization of the contact lenses in vitro. Methods: Contact lenses coated with Se were worn on an extended-wear schedule for up to 2 months by 10 New Zealand White rabbits. Corneal health was evaluated with slit-lamp biomicroscopy, pachymetry, electron microscopy, and histology. Lenses worn by the rabbits were analyzed for protein and lipid deposits. In addition, the ability of Se to block bacterial colonization was tested in vitro by incubating lenses in a Pseudomonas aeruginosa broth followed by scanning electron microscopy of the contact lens surface. Results: The covalent Se coating decreased bacterial colonization in vitro while not adversely affecting the corneal health of rabbits in vivo. The Se coating produced no noticeable negative effects as observed with slit-lamp biomicroscopy, pachymetry, electron microscopy, and histology. The Se coating did not affect protein or lipid deposition on the contact lenses. Conclusion: The data from this pilot study suggest that a Se coating on contact lenses might reduce acute red eye and bacterial ulceration because of an inhibition of bacterial colonization. In addition, our safety tests suggest that this positive effect can be produced without an adverse effect on corneal health.

Myofibroblastic sarcoma originating at the site of rabies vaccination in a cat

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Feline Intraocular Tumors May Arise from Transformation of Lens Epithelium

Feline ocular sarcomas are malignant intraocular neoplasms that are frequently associated with a history of ocular trauma. They usually present as fibrosarcomas, but some have both epithelial and mesenchymal features. The purpose of this study was to determine the cell of origin of a subset of feline intraocular sarcomas that display a mixed epithelial-mesenchymal phenotype, with elaboration of basement membrane—type matrix. We examined the morphology and histochemical and immunohistochemical phenotypes of nine feline intraocular sarcomas. Immunohistochemistry and in situ hybridization were performed to detect expression of crystallin alpha A. In addition, tumors were examined for expression of vimentin, cytokeratin, smooth muscle actin, desmin, melan A, neural cell adhesion molecule, S-100, glial fibrillary acidic protein, nerve growth factor receptor, and collagen type IV. Animals ranged from 7 to 17 years of age—no breed or sex predilection for tumor occurrence was present. Tumors were characterized by mixed epithelial and mesenchymal phenotypes, both of which elaborated basement membrane-type material and expressed vimentin highly. On the basis of collagen type IV and crystallin alpha A immunopositivity, we established that three of nine tumors were of lens epithelial origin. Expression of desmin and smooth muscle actin identified one tumor as a leiomyosarcoma. The remainder were undifferentiated sarcomas of myofibroblastic origin. This is the first report of lens epithelial neoplasia in clinical material from any species. The history and morphologic features of feline ocular sarcomas are reminiscent of feline vaccine-induced sarcomas. These tumors may share pathophysiologic similarities unique to this species.

Viral Replication Rate Regulates Clinical Outcome and CD8 T Cell Responses during Highly Pathogenic H5N1 Influenza Virus Infection in Mice

Since the first recorded infection of humans with H5N1 viruses of avian origin in 1997, sporadic human infections continue to occur with a staggering mortality rate of >60%. Although sustained human-to-human transmission has not occurred yet, there is a growing concern that these H5N1 viruses might acquire this trait and raise the specter of a pandemic. Despite progress in deciphering viral determinants of pathogenicity, we still lack crucial information on virus/immune system interactions pertaining to severe disease and high mortality associated with human H5N1 influenza virus infections. Using two human isolates of H5N1 viruses that differ in their pathogenicity in mice, we have defined mechanistic links among the rate of viral replication, mortality, CD8 T cell responses, and immunopathology. The extreme pathogenicity of H5N1 viruses was directly linked to the ability of the virus to replicate rapidly, and swiftly attain high steady-state titers in the lungs within 48 hours after infection. The remarkably high replication rate of the highly pathogenic H5N1 virus did not prevent the induction of IFN-β or activation of CD8 T cells, but the CD8 T cell response was ineffective in controlling viral replication in the lungs and CD8 T cell deficiency did not affect viral titers or mortality. Additionally, BIM deficiency ameliorated lung pathology and inhibited T cell apoptosis without affecting survival of mice. Therefore, rapidly replicating, highly lethal H5N1 viruses could simply outpace and overwhelm the adaptive immune responses, and kill the host by direct cytopathic effects. However, therapeutic suppression of early viral replication and the associated enhancement of CD8 T cell responses improved the survival of mice following a lethal H5N1 infection. These findings suggest that suppression of early H5N1 virus replication is key to the programming of an effective host response, which has implications in treatment of this infection in humans.

Spontaneous Pneumothorax Caused by Pulmonary Blebs and Bullae in 12 Dogs

Spontaneous pneumothorax caused by pulmonary blebs and bullae was diagnosed in 12 dogs based on history, clinical examination, thoracic radiographs, surgical findings, and histopathological examination of resected pulmonary lesions. Radiographic evidence of blebs or bullae was seen in only one dog. None of the dogs responded to conservative treatment with thoracocentesis or thoracostomy tube drainage. A median sternotomy approach was used to explore the thorax in all dogs. Pulmonary blebs and bullae were resected with partial or complete lung lobectomy. Ten of the dogs had more than one lesion, and seven of the dogs had bilateral lesions. The cranial lung lobes were most commonly affected. Histopathology results of the blebs and bullae were consistent in all dogs and resembled lesions found in humans with primary spontaneous pneumothorax. None of the dogs developed recurrence of pneumothorax. Median follow-up time was 19 months. The outcome following resection of the pulmonary blebs and bullae was excellent.

The pharmacologic assessment of a novel lymphocyte function-associated antigen-1 antagonist (SAR 1118) for the treatment of keratoconjunctivitis sicca in dogs.

PURPOSE Keratoconjunctivitis sicca (KCS) is characterized by inflammation and decreased production of tears containing increased levels of cytokines. The release occurs in the setting of conjunctival and lacrimal gland inflammation, potentially mediated by the interaction between lymphocyte function-associated antigen (LFA)-1, a cell surface protein found on lymphocytes, and its cognate ligand intercellular adhesion molecule (ICAM)-1. SAR 1118 is a novel LFA-1 antagonist and may be an effective therapeutic agent for the treatment of KCS. The following studies were performed to assess the in vitro activity of SAR 1118 and to evaluate the clinical efficacy of topical SAR 1118 for the treatment of idiopathic canine KCS. METHOD Pharmacodynamics were assessed by measuring the ability of SAR 1118 to inhibit Jurkat T-cell binding with recombinant human ICAM-1 and to inhibit cytokine release from human peripheral blood mononuclear cells (PBMCs) stimulated by staphylococcal enterotoxin B. For the assessment of clinical efficacy, 10 dogs diagnosed with idiopathic KCS were treated with SAR 1118 1% topical ophthalmic solution three times daily for 12 weeks. Schirmers tear test (STT) was used to measure tear production. RESULTS SAR 1118 demonstrated concentration-dependent inhibition of Jurkat T-cell attachment, inhibition of lymphocyte activation, and release of inflammatory cytokines, particularly the Th1, Th2, and Th17 T-cell cytokines IFN-γ, IL-2, and IL-17F, respectively. Mean STT values increased from 3.4 mm during week 1 to 5.8 mm at week 12 (P < 0.025). No SAR 1118-related adverse events were observed. CONCLUSIONS SAR 1118 appears to be an effective anti-inflammatory treatment for KCS. Additional studies are warranted to establish the efficacy of SAR 1118 for the treatment of KCS in humans.

Prevention of Experimental Choroidal Neovascularization and Resolution of Active Lesions by VEGF Trap in Nonhuman Primates

OBJECTIVE To evaluate the efficacy of systemic and intravitreous administration of VEGF Trap (aflibercept) in a nonhuman primate model of choroidal neovascularization (CNV). METHODS VEGF Trap treatment on laser-induced CNV was evaluated in 48 adult cynomolgus monkeys. In the prevention arms of the study, VEGF Trap was administered by intravenous injection (3 or 10 mg/kg weekly) or intravitreous injection (50, 250, or 500 μg/eye every 2 weeks) beginning before laser injury. In the treatment arm, a single intravitreous injection (500 μg) was given 2 weeks following laser injury. Laser-induced lesions were scored from grade 1 (no hyperfluorescence) to grade 4 (clinically relevant leakage). Representative lesions were evaluated histologically. RESULTS Grade 4 leakage developed at 32.4% and 45.4% of the laser sites in animals receiving intravitreous or intravenous administration of placebo at 2 weeks following laser injury, respectively. In contrast, the development of grade 4 lesions was completely or nearly completely prevented in all groups receiving intravenous or intravitreous injections of VEGF Trap. A single intravitreous injection of VEGF Trap (500 μg) administered following the development of CNV reduced the frequency of grade 4 lesions from 44.4% to 0% within 14 days of treatment. Intravitreous VEGF Trap was well tolerated with either no or only mild ocular inflammation. Histological evaluation showed decreased scores for morphologic features of tissue proliferation in the VEGF Trap prevention groups. CONCLUSIONS VEGF Trap prevented the development of clinically relevant CNV leakage when administered at the lowest doses tested. Moreover, a single intravitreous injection induced inhibition of active CNV leakage. CLINICAL RELEVANCE The animal model used in this study has an established track record as a predictor of pharmacologic efficacy of antineovascular drugs in humans having the neovascular, or wet, form of age-related macular degeneration.

Caries and odontoclastic resorptive lesions in a chinchilla (Chinchilla lanigera)

represented an underestimate. This reflects the fact that each follicle was analysed in only one histological section and that in some follicles only a minority of follicle cells were deemed infected. A comparison of BvDv antigen and RNA expression in infected oocytes (Table 2) suggested that in situ hybridisation was a much more more sensitive technique for the detection of virus since a four-fold greater percentage of infected oocytes was recorded especially in animal 2 where the comparison was between contralateral ovaries. An alternative explanation for this difference may be the absent co-expression of antigen in virus infected tissues. The data also established that viral antigen was present in both primary and secondary follicles (Table 2) indicating that infected follicles had some developmental competence (Figs 1 and 2). Antigen was observed in oocytes residing in follicles possessing from one to more than 10 layers of follicle