Steven E. Weisbrode

Proposal of a 2-Tier Histologic Grading System for Canine Cutaneous Mast Cell Tumors to More Accurately Predict Biological Behavior

Currently, prognostic and therapeutic determinations for canine cutaneous mast cell tumors (MCTs) are primarily based on histologic grade. However, the use of different grading systems by veterinary pathologists and institutional modifications make the prognostic value of histologic grading highly questionable. To evaluate the consistency of microscopic grading among veterinary pathologists and the prognostic significance of the Patnaik grading system, 95 cutaneous MCTs from 95 dogs were graded in a blinded study by 28 veterinary pathologists from 16 institutions. Concordance among veterinary pathologists was 75% for the diagnosis of grade 3 MCTs and less than 64% for the diagnosis of grade 1 and 2 MCTs. To improve concordance among pathologists and to provide better prognostic significance, a 2-tier histologic grading system was devised. The diagnosis of high-grade MCTs is based on the presence of any one of the following criteria: at least 7 mitotic figures in 10 high-power fields (hpf); at least 3 multinucleated (3 or more nuclei) cells in 10 hpf; at least 3 bizarre nuclei in 10 hpf; karyomegaly (ie, nuclear diameters of at least 10% of neoplastic cells vary by at least two-fold). Fields with the highest mitotic activity or with the highest degree of anisokaryosis were selected to assess the different parameters. According to the novel grading system, high-grade MCTs were significantly associated with shorter time to metastasis or new tumor development, and with shorter survival time. The median survival time was less than 4 months for high-grade MCTs but more than 2 years for low-grade MCTs.

Follistatin Gene Delivery Enhances Muscle Growth and Strength in Nonhuman Primates

A vector delivered into muscles of monkeys generates a natural regulatory molecule, which increases muscle size and strength and may be useful therapeutically. Beyond Mighty Mouse: Building Muscle Mass Strength in Monkeys Patients with progressive neuromuscular disorders all experience the foreboding of the severe disability that awaits them and from which there is little to no relief. Although this class of disorders has multiple genetic and physiological origins, a therapy that directly addresses the debilitating muscle weakness that is the hallmark of these maladies would enhance the lives of millions. Now, in an extension of their previous work in dystrophic mice, Kota et al. describe such a therapeutic approach in preclinical studies performed in nonhuman primates. This treatment mode is applicable to several progressive neuromuscular disorders whether or not scientists have defined their precise genetic defects. The authors used a gene therapy approach to introduce a version of the human gene encoding follistatin into the muscles of the femurs of healthy cynomolgus macaques. Follistatin is a potent inhibitor of myostatin, a signaling molecule that regulates skeletal muscle mass. Follistatin blocks myostatin signaling and augments muscle size and strength safely in mice but, until now, has not been tested in primates. Kota et al. injected a follistatin-producing gene therapy vector into the leg muscles of the monkeys and measured increases in muscle mass and strength. Sustained follistatin expression caused no aberrations in the structures or functions of a variety of organs. This promising progress comes with some caveats. Because healthy monkeys served as subjects for this therapeutic protocol, these findings are not predictive of the outcome in a clinical setting with patients suffering from muscle disorders. In certain genetic neuromuscular diseases, the muscles undergo a repeated cycle of degeneration and regeneration. The vector used in this study does not integrate into the muscle cell genome and thus can be lost from the cells during the degeneration-regeneration cycles. However, the authors point out that the enhancement of muscle size and strength observed in similarly treated dystrophic mice persisted for more than a year even though there was appreciable muscle turnover. More study is needed before follistatin enters the clinic, such as a molecular assessment of gene and vector sequences in multiple tissues. Nonetheless, the work of Kota et al. constitutes proof of principle for the use of myostatin inhibitors to build muscle in primates. Antagonists of myostatin, a blood-borne negative regulator of muscle growth produced in muscle cells, have shown considerable promise for enhancing muscle mass and strength in rodent studies and could serve as potential therapeutic agents for human muscle diseases. One of the most potent of these agents, follistatin, is both safe and effective in mice, but similar tests have not been performed in nonhuman primates. To assess this important criterion for clinical translation, we tested an alternatively spliced form of human follistatin that affects skeletal muscle but that has only minimal effects on nonmuscle cells. When injected into the quadriceps of cynomolgus macaque monkeys, a follistatin isoform expressed from an adeno-associated virus serotype 1 vector, AAV1-FS344, induced pronounced and durable increases in muscle size and strength. Long-term expression of the transgene did not produce any abnormal changes in the morphology or function of key organs, indicating the safety of gene delivery by intramuscular injection of an AAV1 vector. Our results, together with the findings in mice, suggest that therapy with AAV1-FS344 may improve muscle mass and function in patients with certain degenerative muscle disorders.

Calcium-restricted ovariectomized sinclair s-1 minipigs: An animal model of osteopenia and trabecular plate perforation

The ovariectomized rat model has now been generally accepted as a useful model for screening different therapeutic agents, but there is a major requirement to identify reliable large animal models for osteoporosis research. In this study, the calcium restricted, ovariectomized minipig has been thoroughly investigated in order to define a large animal model with trabecular and cortical bone remodeling which would be reliable for further testing of agents that had shown promise of efficacy during the screening procedure. Twenty six female, 4-month old minipigs were randomized into four groups and fed either normal diet (0.90% calcium (Ca.)) or diet with restricted calcium content (0.75%, 0.50%). At the age of ten months, 3 groups were ovariectomized (OVX) while one group on normal diet was shamoperated. The groups were followed for six months after the operation. At death, bone mass was determined by densitometry and by ashing. Additionally, biomechanical competence was assessed in trabecular bone cores from the vertebral bodies. Finally, histomorphometry (static and dynamic parameters) and structural analyses (star volume) were performed on the vertebral bodies. The study revealed an OVX-related decline of 6% in vertebral bone mineral density (BMD) and a decline of 15% in trabecular bone volume (BV/TV). In contrast, a 15% increase in mean trabecular plate separation (Tb.Sp.) and a small increase in marrow space star volume (Ma. Star V.) were detected. The structural changes became more pronounced when OVX was combined with mild Ca. restriction (0.75% Ca.) with an increase in Ma. Star V. to 164%.(ABSTRACT TRUNCATED AT 250 WORDS)

Osteogenic gene regulation and relative acceleration of healing by adenoviral‐mediated transfer of human BMP‐2 or ‐6 in equine osteotomy and ostectomy models

This study evaluated healing of equine metatarsal osteotomies and ostectomies in response to percutaneous injection of adenoviral (Ad) bone morphogenetic protein (BMP)‐2, Ad‐BMP‐6, or beta‐galactosidase protein vector control (Ad‐LacZ) administered 14 days after surgery. Radiographic and quantitative computed tomographic assessment of bone formation indicated greater and earlier mineralized callus in both the osteotomies and ostectomies of the metatarsi injected with Ad‐BMP‐2 or Ad‐BMP‐6. Peak torque to failure and torsional stiffness were greater in osteotomies treated with Ad‐BMP‐2 than Ad‐BMP‐6, and both Ad‐BMP‐2‐ and Ad‐BMP‐6‐treated osteotomies were greater than Ad‐LacZ or untreated osteotomies. Gene expression of ostectomy mineralized callus 8 weeks after surgery indicated upregulation of genes related to osteogenesis compared to intact metatarsal bone. Expression of transforming growth factor beta‐1, cathepsin H, and gelsolin‐like capping protein were greater in Ad‐BMP‐2‐ and Ad‐BMP‐6‐treated callus compared to Ad‐LacZ‐treated or untreated callus. Evidence of tissue biodistribution of adenovirus in distant organs was not identified by quantitative PCR, despite increased serum antiadenoviral vector antibody. This study demonstrated a greater relative potency of Ad‐BMP‐2 over Ad‐BMP‐6 in accelerating osteotomy healing when administered in this regimen, although both genes were effective at increasing bone at both osteotomy and ostectomy sites.

Results of excision of thymoma in cats and dogs: 20 cases (1984–2005)

OBJECTIVE To provide long-term follow-up information for a series of dogs and cats with invasive and noninvasive thymomas treated by excision alone. DESIGN Retrospective case series. ANIMALS 9 cats and 11 dogs with thymoma. PROCEDURES Medical records were reviewed. The following factors were analyzed for their effect on prognosis: age of dog or cat, invasiveness of the tumor, percentage of lymphocytes in the mass (percentage lymphocyte composition) on histologic evaluation, and mitotic index of the mass. RESULTS All patients were treated with excision of the tumor alone. Median overall survival time for the cats was 1,825 days, with a 1-year survival rate of 89% and a 3-year survival rate of 74%. Median overall survival time for the dogs was 790 days, with a 1-year survival rate of 64% and a 3-year survival rate of 42%. Recurrence of thymoma was observed in 2 cats and 1 dog, and a second surgery was performed in each, with subsequent survival times of 5, 3, and 4 years following the first surgery. Percentage lymphocyte composition of the mass was the only factor that was significantly correlated with survival time; animals with a high percentage of lymphocytes lived longer. CONCLUSIONS AND CLINICAL RELEVANCE Results of this study indicated that most cats and dogs with thymomas did well after excision. Even cats and dogs with invasive masses that survived the surgery and the few cats and dogs with recurrent thymomas or paraneoplastic syndromes had a good long-term outcome. Excision should be considered an effective treatment option for dogs and cats with thymomas.

Spontaneous gastroduodenal perforation in 16 dogs and seven cats (1982-1999).

The records of 23 dogs and cats diagnosed with spontaneous gastroduodenal perforation (GDP) were retrospectively reviewed. Survival was 63% in dogs and 14% in cats. Rottweilers <5 years of age were overrepresented. Clinical evidence of gastrointestinal bleeding was common in dogs but not in cats. Shock was an uncommon presenting condition in dogs and was not closely linked to outcome. In fact, progression of an ulcerating lesion to GDP was not associated with marked changes in symptoms exhibited by many patients in this study. Most GDPs were associated with histopathological evidence of subacute or chronic peritoneal reaction at the time of diagnosis. This suggests that diagnostic methods employed lacked sensitivity in identifying early perforating lesions, and that dramatic signs of acute abdomen following gastroduodenal perforation may not be as common as was previously thought.

Antibiotic‐Responsive Histiocytic Ulcerative Colitis in 9 Dogs

Canine histiocytic ulcerative colitis (HUC) is characterized by colonic inflammation with predominantly periodic acid-Schiff (PAS)-positive macrophages. The inflammation results in colonic thickening, ulcerations, and distortion of normal glandular architecture. Resultant clinical signs consist of chronic large bowel diarrhea, tenesmus, and marked weight loss, and the disease frequently results in euthanasia. Conventional therapy consists of some combination of prednisone, azathioprine, sulfasalazine, and metronidazole. Nine dogs (8 Boxers and 1 English Bulldog) with histologic confirmation of HUC were treated with antibiotic therapy (either with enrofloxacin alone or in combination with metronidazole and amoxicillin). Clinical signs, physical examination findings, laboratory abnormalities, and the histologic severity of the disease were evaluated. Four of the 9 dogs had been treated previously with conventional therapy and had failed to respond favorably; then, these dogs were placed on antibiotic therapy (enrofloxacin, n = 1; enrofloxacin, metronidazole, and amoxicillin, n = 3) and had resolution of clinical signs within 3-12 days. Five dogs were treated solely with antibiotic therapy (enrofloxacin, n = 1; enrofloxacin and metronidazole, n = 1; enrofloxacin, metronidazole, and amoxicillin, n = 3), and clinical signs resolved in 2-7 days. Repeated biopsy specimens were obtained from 5 dogs after treatment, and all showed marked histologic improvement. The increase in body weight after treatment was statistically significant (P = .01). Three dogs currently are not on any treatment and have had resolution of clinical signs for up to 14 months. These observations suggest that an infectious agent responsive to antibiotics plays an integral role in the clinical manifestation of canine HUC, and they support the use of antibiotics in its treatment.

Safety evaluation of an extract from Salacia oblonga

Plant extracts from the Salacia genus have been found to have intestinal alpha-glucosidase inhibitor activity, which may have application to the development of medical foods for people with diabetes. We evaluated the safety of a hot water extract of S. oblonga (salacinol extract) supplemented to or processed into a medical food. Thirty male Sprague-Dawley rats were assigned among one of three treatments: (1) EN-0178 (control, liquid diet), (2) EN-0178+salacinol (as 1 plus 500 mg of salacinol extract per 253 g diet, which was added to product immediately prior to feeding), (3) EN-0195 (as 1 plus 500 mg of salacinol extract per 253 g diet, which was added during product manufacture). After 14 days of free access to dietary treatments, rats were sacrificed, blood collected and organs weighed. Rats consuming salacinol extract had reduced (P <0.05) weight gain and feed intake. The relative (% of body weight) testicular weight was higher (P<0.05) for rats consuming salacinol extract, whereas, the relative liver and spleen weight was lower (P<0.05) for rats consuming salacinol extract. Of the serum chemistries analyzed, blood urea nitrogen and alkaline phosphatase was lower (P<0.05) for rats consuming salacinol extract. No differences in blood hematology were found. We conclude that salacinol extract, in a medical food consumed for 2 weeks in amounts estimated at 10-fold greater than proposed for human intake, did not result in clinical chemistry or histopathologic indications of toxic effects in male Sprague-Dawley rats.

Dermal fibroblast‐mediated BMP2 therapy to accelerate bone healing in an equine osteotomy model

This study evaluated healing of equine metacarpal/metatarsal osteotomies in response to percutaneous injection of autologous dermal fibroblasts (DFbs) genetically engineered to secrete bone morphogenetic protein‐2 (BMP2) or demonstrate green fluorescent protein (GFP) gene expression administered 14 days after surgery. Radiographic assessment of bone formation indicated greater and earlier healing of bone defects treated with DFb with BMP2 gene augmentation. Quantitative computed tomography and biomechanical testing revealed greater mineralized callus and torsional strength of DFb‐BMP2‐treated bone defects. On the histologic evaluation, the bone defects with DFb‐BMP2 implantation had greater formation of mature cartilage and bone nodules within the osteotomy gap and greater mineralization activity on osteotomy edges. Autologous DFbs were successfully isolated in high numbers by a skin biopsy, rapidly expanded without fastidious culture techniques, permissive to adenoviral vectors, and efficient at in vitro BMP2 protein production and BMP2‐induced osteogenic differentiation. This study demonstrated an efficacy and feasibility of DFb‐mediated BMP2 therapy to accelerate the healing of osteotomies. Skin cell‐mediated BMP2 therapy may be considered as a potential treatment for various types of fractures and bone defects.

The Effects of Radiofrequency Energy Treatment on Chondrocytes and Matrix of Fibrillated Articular Cartilage

Background There is no consensus regarding the safety of radiofrequency energy treatment for chondroplasty. Hypothesis Use of a radiofrequency thermal probe will produce a dose-dependent detrimental effect on chondrocytes in a setting mimicking the arthroscopic procedure. Study Design Controlled laboratory study. Methods Paired patellae from 11 horses were fibrillated; one served as the control while the contralateral patella was treated with 20, 40, and 60 watts of radiofrequency energy for 4 minutes. Proteoglycan synthesis, proteoglycan degradation, and cell viability were measured. Explant histologic sections were scored for cellular characteristics, metachromatic matrix staining intensity, and tissue architecture and were quantitatively analyzed for cell death. Results Mean peak surface articular cartilage temperatures were 35.47°C (20 watts), 40.76°C (40 watts), and 44.81°C (60 watts). Treatment at all three settings significantly decreased proteoglycan synthesis of the chondrocytes. Proteoglycan degradation increased with increased power setting. Treatment at 40 and 60 watts significantly decreased cell viability to 81% and 73%, respectively. Significant histologic changes occurred in the superficial cartilage zone with 60-watt application. Conclusion Use of radiofrequency energy on mechanically fibrillated articular cartilage induced a dose-dependent detrimental effect on chondrocytes and matrix metabolism. Clinical Relevance Cautious use of radiofrequency energy for treatment of articular cartilage is recommended until long-term effects are evaluated.