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Glutamate dehydrogenase (GLDH) is a liver-specific biomarker of hepatocellular damage currently undergoing qualification as a drug development tool. Since GLDH is located within the mitochondrial matrix, it has been hypothesized that it might also be useful in assessing mitotoxicity as an initiating event during drug-induced liver injury. According to this hypothesis, hepatocyte death that does not involve primary mitochondrial injury would result in release of intact mitochondria into circulation that could be removed by high speed centrifugation and result in lower GLDH activity measured in spun serum vs un-spun serum. A single prior study in mice has provided some support for this hypothesis. We sought to repeat and extend the findings of this study. Accordingly, mice were treated with the known mitochondrial toxicant, acetaminophen (APAP), or with furosemide (FS), a toxicant believed to cause hepatocyte death through mechanisms not involving mitotoxicity as initiating event. We measured GLDH levels in fresh plasma before and after high speed centrifugation to remove intact mitochondria. We found that both APAP and FS treatments caused substantial hepatocellular necrosis that correlated with plasma alanine aminotransferase (ALT) and GLDH elevations. The plasma GLDH activity in both the APAP- and FS- treated mice was not affected by high-speed centrifugation. Interestingly, the ratio of GLDH:ALT was 5-fold lower during FS compared to APAP hepatotoxicity. Electron microscopy confirmed that both APAP- and FS-treatments had resulted in mitochondrial injury. Mitochondria within vesicles were only observed in the FS-treated mice raising the possibility that mitophagy might account for reduced release of GLDH in the FS-treated mice. Although our results show that plasma GLDH is not clinically useful for evaluating mitotoxicity, the GLDH:ALT ratio as a measure of mitophagy needs to be further studied.

Dysregulation of hepatic lipid and cholesterol metabolism is a significant contributor to cardiometabolic health, resulting in excessive liver lipid accumulation and ultimately non-alcoholic steatohepatitis (NASH). Therapeutic activators of the AMP-Activated Protein Kinase (AMPK) have been proposed as a treatment for metabolic diseases; we show that the AMPK β1-biased activator PF-06409577 is capable of lowering hepatic and systemic lipid and cholesterol levels in both rodent and monkey preclinical models. PF-06409577 is able to inhibit de novo lipid and cholesterol synthesis pathways, and causes a reduction in hepatic lipids and mRNA expression of markers of hepatic fibrosis. These effects require AMPK activity in the hepatocytes. Treatment of hyperlipidemic rats or cynomolgus monkeys with PF-06409577 for 6 weeks resulted in a reduction in circulating cholesterol. Together these data suggest that activation of AMPK β1 complexes with PF-06409577 is capable of impacting multiple facets of liver disease and represents a promising strategy for the treatment of NAFLD and NASH in humans., Highlights • PF-06409577 is a potent activator of AMPK β1 containing complexes. • PF-06409577 improves liver function and systemic dyslipidemia in rodents through hepatic AMPK activation. • PF-06409577-mediated reductions in circulating cholesterol was observed in monkeys and diabetic rats. NAFLD and NASH remain poorly treated and are diseases which are growing rapidly in societal cost. Therapeutic mechanisms that impact multiple aspects of the dysregulated metabolic regulation of NAFLD and NASH are needed. Pharmacological AMPK activation has long held promise as a treatment for NAFLD because of its impact on hepatic lipid and cholesterol synthesis, as well as its proposed anti-inflammatory and anti-lipolytic actions. Recent development of clinically viable small molecule AMPK activators, including PF-06409577, has enabled their more thorough characterization in preclinical disease models.

Purpose: Adverse reactions reported in patients treated with antibody–calicheamicin conjugates such as gemtuzumab ozogamicin (Mylotarg) and inotuzumab ozogamicin include thrombocytopenia and sinusoidal obstruction syndrome (SOS). The objective of this experimental work was to investigate the mechanism for thrombocytopenia, characterize the liver injury, and identify potential safety biomarkers. Experimental Design: Cynomolgus monkeys were dosed intravenously at 6 mg/m2/dose once every 3 weeks with a nonbinding antibody–calicheamicin conjugate (PF-0259) containing the same linker-payload as gemtuzumab ozogamicin and inotuzumab ozogamicin. Monkeys were necropsied 48 hours after the first administration (day 3) or 3 weeks after the third administration (day 63). Results: PF-0259 induced acute thrombocytopenia (up to 86% platelet reduction) with nadirs on days 3 to 4. There was no indication of effects on megakaryocytes in bone marrow or activation of platelets in peripheral blood. Microscopic evaluation of liver from animals necropsied on day 3 demonstrated midzonal degeneration and loss of sinusoidal endothelial cells (SECs) associated with marked platelet accumulation in sinusoids. Liver histopathology on day 63 showed variable endothelial recovery and progression to a combination of sinusoidal capillarization and sinusoidal dilation/hepatocellular atrophy, consistent with early SOS. Among biomarkers evaluated, there were early and sustained increases in serum hyaluronic acid (HA) that correlated well with serum aspartate aminotransferase and liver microscopic changes, suggesting that HA may be a sensitive diagnostic marker of the liver microvascular injury. Conclusions: These data support the conclusion that target-independent damage to liver SECs may be responsible for acute thrombocytopenia (through platelet sequestration in liver sinusoids) and development of SOS. Clin Cancer Res; 23(7); 1760–70. ©2016 AACR.

Diabetic nephropathy remains an area of high unmet medical need, with current therapies that slow down, but do not prevent, the progression of disease. A reduced phosphorylation state of adenosine monophosphate-activated protein kinase (AMPK) has been correlated with diminished kidney function in both humans and animal models of renal disease. Here, we describe the identification of novel, potent, small molecule activators of AMPK that selectively activate AMPK heterotrimers containing the β1 subunit. After confirming that human and rodent kidney predominately express AMPK β1, we explore the effects of pharmacological activation of AMPK in the ZSF1 rat model of diabetic nephropathy. Chronic administration of these direct activators elevates the phosphorylation of AMPK in the kidney, without impacting blood glucose levels, and reduces the progression of proteinuria to a greater degree than the current standard of care, angiotensin-converting enzyme inhibitor ramipril. Further analyses of urine biomarkers and kidney tissue gene expression reveal AMPK activation leads to the modulation of multiple pathways implicated in kidney injury, including cellular hypertrophy, fibrosis, and oxidative stress. These results support the need for further investigation into the potential beneficial effects of AMPK activation in kidney disease.

Several chemicals and pharmaceuticals increase the incidence of hemangiosarcomas (HSAs) in mice, but the relevance to humans is uncertain. Recently, canine HSAs were identified as a powerful tool for investigating the pathogenesis of human HSAs. To characterize the cellular phenotype of canine HSAs, we evaluated immunoreactivity and/or messenger RNA (mRNA) expression of markers for hematopoietic stem cells (HSCs), endothelial cells (ECs), a tumor suppressor protein, and a myeloid marker in canine HSAs. Neoplastic canine cells expressed EC markers and a myeloid marker, but expressed HSC markers less consistently. The canine tumor expression results were then compared to previously published immunoreactivity results for these markers in human and mouse HSAs. There are 2 noteworthy differences across species: (1) most human HSAs had HSC marker expression, indicating that they were comprised of tumor cells that were less differentiated than those in canine and mouse tumors; and (2) human and canine HSAs expressed a late-stage EC maturation marker, whereas mouse HSAs were negative, suggesting that human and canine tumors may retain greater differentiation potential than mouse tumors. These results indicate that HSA development is variable across species and that caution is necessary when discussing translation of carcinogenic risk from animal models to humans.

Several classes of drugs have been shown to cause drug-induced vascular injury (DIVI) in preclinical toxicity studies. Measurement of blood flow and vessel diameter in numerous vessels and across various tissues by ultrasound imaging has the potential to be a noninvasive translatable biomarker of DIVI. Our objective was to demonstrate the utility of high-frequency ultrasound imaging for measuring changes in vascular function by evaluating blood flow and vessel diameter in the superior mesenteric arteries (SMA) of rats treated with compounds that are known to cause DIVI and are known vasodilators in rat: fenoldopam, CI-1044, and SK&F 95654. Blood flow, vessel diameter, and other parameters were measured in the SMA at 4, 8, and 24 hr after dosing. Mild to moderate perivascular accumulations of mononuclear cells, neutrophils in tunica adventitia, and superficial tunica media as well as multifocal hemorrhage and necrosis in the tunica media were found in animals 24 hr after treatment with fenoldopam and SK&F 95654. Each compound caused marked increases in blood flow and shear stress as early as 4 hr after dosing. These results suggest that ultrasound imaging may constitute a functional correlate for the microscopic finding of DIVI in the rat.

In a 2-year rat carcinogenicity study, pegvisomant injected subcutaneously on a daily basis at doses of 0, 2, 8, or 20 mg/kg/day produced malignant fibrous histiocytomas (MFHs) at the injection sites of 3 male rats (5%) given 8 mg/kg/day and 5 males (8%) given 20 mg/kg/day. MFH was characterized by unencapsulated dermal and subcutaneous sheets of fusiform and spindle-shaped cells sometimes with areas of round and/or irregular, pleomorphic cells and variable numbers of large multinucleated giant cells. Some regions of MFH had a fibroblastic appearance with streaming cells forming storiform patterns, while other areas consisted primarily of round to plump irregular cells with more giant cells. Pegvisomant did not increase the incidence of MFH in female rats and did not produce any other neoplastic responses in rats. In the dermis and subcutis at the injection sites of many males and females, pegvisomant produced dose-related increased incidences and severity of histiocytic infiltrates consisting of vacuolated macrophages with variable mature or immature fibrous tissue. Neoplasms at injection sites did not result in marketing restrictions or a label warning for human cancer risk, highlighting that injection-site neoplasms in rats have low relevance for human risk assessment.

Diabetic nephropathy remains an area of high unmet medical need, with current therapies that slow down, but do not prevent, the progression of disease. A reduced phosphorylation state of adenosine monophosphate-activated protein kinase (AMPK) has been correlated with diminished kidney function in both humans and animal models of renal disease. Here, we describe the identification of novel, potent, small molecule activators of AMPK that selectively activate AMPK heterotrimers containing the

A series of insertion patterns for chemically modified nucleotides [2'-O-methyl (2'-OMe), 2'-fluoro (2'-F), methoxyethyl (MOE), locked nucleic acid (LNA), and G-Clamp] within antisense gapmers is studied in vitro and in vivo in the context of the glucocorticoid receptor. Correlation between lipid transfection and unassisted (gymnotic--using no transfection agent) in vitro assays is seen to be dependent on the chemical modification, with the in vivo results corresponding to the unassisted assay in vitro. While in vitro mRNA knockdown assays are typically reasonable predictors of in vivo results, G-Clamp modified antisense oligonucleotides have poor in vivo mRNA knockdown as compared to transfected cell based assays. For LNA gapmers, knockdown is seen to be highly sensitive to the length of the antisense and number of LNA insertions, with longer 5LNA-10DNA-5LNA compounds giving less activity than 3LNA-10DNA-3LNA derivatives. Additionally, the degree of hepatoxicity for antisense gapmers with identical sequences was seen to vary widely with only subtle changes in the chemical modification pattern. While the optimization of knockdown and hepatic effects remains a sequence specific exercise, general trends emerge around preferred physical properties and modification patterns.

Summary In order better to evaluate the extent to which degradation of the lamellar basement membrane (LBM) by matrix metalloproteinases (MMP) occurs in equine laminitis, we determined the concentration of type IV collagen and laminin in normal and laminitic horses, using specific immunoassays. Blood samples were obtained from both the jugular and the cephalic veins of horses (n = 10) before and after the induction of acute alimentary laminitis by carbohydrate overload. Jugular and cephalic venous blood samples were also obtained from horses affected with naturally occurring laminitis (n = 16) and nonlaminitic controls (n = 8). The serum collagen IV concentration was not changed following the induction of laminitis in the experimental group. Serum collagen IV concentration was increased in jugular venous blood obtained from cases of naturally occurring laminitis (mean ± s.e. 218.04 ± 18.59 ng/ml) compared with nonlaminitic controls (157.50 ± 10.93 ng/ml) (P

Summary Reasons for performing study: The role of glucocorticoids (GCs) in the pathogenesis of laminitis is incompletely understood. Local tissue activity of GC is regulated by the steroid converting enzyme, 11β-hydroxysteroid dehydrogenase-1 (11β-HSD-1). Changes in integumentary (skin and hoof lamellar) 11β-HSD activity occurring during laminitis could affect the extent to which GCs are involved in its development. Hypothesis: That changes in integumentary 11β-HSD-1 activity associated with the laminitic condition would lead to elevated local tissue levels of GCs, which could subsequently contribute, through paracrine and autocrine mechanisms, to the further development of laminitis; and that similar changes in 11β-HSD-1 activity would be evident in both skin and hoof lamellar tissue. Methods: Activity of 11β-HSD-1 was determined in skin and hoof lamellar tissue specimens obtained from normal and laminitic horses using a radiometric assay. Skin samples were obtained from 10 normal horses and from 10 horses before and after induction of acute laminitis following administration of starch via nasogastric tube. Hoof lamellar samples were obtained from 10 normal horses, 10 horses following induction of acute laminitis and 4 chronically-foundered horses. Bidirectional 11β-HSD-1 activity was measured in both skin and lamellar tissues. Results: 11-ketoreductase activity exceeded 11β-dehydrogenase activity in both skin and lamellar tissues. Cutaneous activity was higher than lamellar 11β-HSD-1 activity in all groups. Both ketoreductase and dehydrogenase activity increased in skin and lamellae following experimental induction of acute laminitis, but the increase in ketoreductase activity was substantially greater than that for dehydrogenase in the lamellae. Induction of acute laminitis was attended by increases of 227 and 220% in cutaneous dehydrogenase and ketoreductase activity, respectively, and 173 and 398% in lamellar dehydrogenase and ketoreductase activity, respectively (P

Reported clinical signs of coccidiosis in South American camelids include anorexia of a few days duration, sudden death, and diarrhea. Antemortem diagnosis of clinical coccidiosis is usually based on clinical signs and supported by detection of coccidial oocysts in feces. This report describes 2 atypical cases of coccidiosis in South American camelids that had no coccidial oocysts detected on antemortem fecal flotation, prolonged weight loss, and normal fecal consistency.

An adult alpaca presented with a large maxillary swelling. Facial trauma or a tooth root abscess was suspected. Radiographically there was a large expansile lesion displacing the right maxillary teeth. An ameloblastoma was diagnosed histologically and palliative radiation therapy was attempted. Because of poor response to therapy and anorexia the animal was euthanized. Necropsy findings confirmed an ameloblastoma of acanthomatous type.

Objective—To elucidate tissue inhibitor of metalloproteinase (TIMP)-mediated effects on chondrocytes.Sample Population—Articular cartilage from humeral heads of 6 dogs.Procedure—Chondrocytes from harvested specimens were cultured in 3-dimensional (3-D) agarose at 106cells/mL. We prepared 3-D constructs exposed to only tumor necrosis factor (TNF)-α (50 ng/mL). Recombinant human TIMP-1 (255nM), -2 (285nM), or -3 (250nM) was added to liquid media bathing 3-D constructs cultured with TNF-α. Chondrocytes cultured without TIMP or TNF-α served as control samples. Samples of liquid media were collected on days 6, 9, 15, and 21 of culture for evaluation of glycosaminoglycan (GAG) and nitric oxide concentrations. The 3-D constructs were collected on days 9, 15, and 21 for evaluation of GAG, hydroxyproline (HP), and DNA contents.Results—GAG content in control samples increased significantly during the study, whereas GAG content in 3-D constructs cultured with TNF-α or TNF-α plus TIMP did not increase. On day 9, GAG release from 3-D constructs cultured with TNF-α was significantly higher than that in other constructs. The HP content in control samples increased during the study and was significantly higher than that in all other constructs on day 21. Concentrations of nitric oxide were significantly lower in control samples on day 6, compared with concentrations for all other constructs.Conclusions and Clinical Relevance—Addition of TIMPs did not counteract suppression of GAG and HP accumulation in 3-D constructs exposed to TNF-α. Apparently, adverse effects on chondrocytes exposed to TNF-α cannot be prevented by addition of TIMP alone. (Am J Vet Res2004;65:1611–1615)

In previous studies, novel putative viral pathogens designated that asinine herpesvirus 4 (AsHV4) and asinine herpesvirus 5 (AsHV5) were associated with fatal interstitial pneumonia in donkeys ( Equus asinus). Nucleotide sequence analysis of a portion of the DNA polymerase gene identified these putative pathogens as herpesviruses and possibly as members of the Gammaherpesvirinae subfamily. Although similar to equine herpesvirus 2 (EHV2) and equine herpesvirus 5 (EHV5), sequence diversity was observed among the detected viruses. In this study, novel sequence is reported for a DNA-packaging protein gene of EHV5 plus AsHV4, AsHV5, and a newly described putative pathogen herein designated asinine herpesvirus 6 (AsHV6). Phylogenetic analysis of these sequences suggested that the equine gammaherpesviruses may form a separate clade within the Gammaherpesvirinae subfamily. Based on the sequence of EHV2 and the novel sequences reported in this study, a PCR assay was developed to detect equine gammaherpesviruses. Products of the predicted size were produced after amplification of DNA from EHV2, EHV5, AsHV4, AsHV5, and AsHV6. This nonnested assay was shown to consistently amplify approximately 10 genomic copies of EHV2. Amplification products were not produced from DNA template of other alpha- and gammaherpesviruses. Because the role of gammaherpesviruses has not been well defined in equine disease, it is envisioned that a single, sensitive PCR assay to detect these potential pathogens will facilitate further assessment of their role in disease.

Objective—To evaluate biocompatibility and effects of implantation of 3-dimensional chondrocyte-agarose autografts in tibial defects in rabbits and to compare in vitro and in vivo chondrocyte-agarose constructs with respect to cell viability, differentiation, and matrix production.Animals—24 adult New Zealand White rabbits.Procedure—Three-dimensional constructs with (grafted group) or without (control group) autogenous chondrocytes were implanted into tibial defects of rabbits and cultured in vitro. During an 8-week period, defects were evaluated radiographically, grossly, histologically, biochemically, and immunohistochemically. In vitro constructs were evaluated histologically, biochemically, and immunohistochemically.Results—Tibial defects had significantly higher radiographic densitometry values at 4 and 6 weeks after implantation in grafted group rabbits, compared with control group rabbits. Number of observed centers of endochondral ossification was significantly greater in defects of grafted group rabbits, compared with control group rabbits. On day 14, glycosaminoglycan concentration was significantly higher in tibial defects of grafted group rabbits, compared to defects of control group rabbits or in vitro constructs. At weeks 2, 4, and 8, glycosaminoglycan concentrations were significantly lower in the in vitro control constructs, compared with other groups. Collagen type I was present in bone and bony callous in defects of grafted and control group rabbits. Collagen type II was identified in cartilaginous tissues of grafted and control group rabbits. Collagen type X was associated with hypertrophic chondrocytes. Only type II collagen was found in the in vitro chondrocyte constructs.Conclusion and Clinical Relevance—Chondrocyte-agarose grafts are biocompatible in large tibial defects and appear to provide a cell source for augmenting endochondral ossification. (Am J Vet Res2003;64:12–20)

The administration of exogenously administered GCs and syndromes associated with GC excess are both attended by increased risk for the development of laminitis in adult horses. However, there exists substantial controversy as to whether excess GCs cause laminitis de novo. If true, the pathogenesis of laminitis arising from the effects of GC excess is probably different from that associated with diseases of the gastrointestinal tract and endotoxemia. Although a satisfactory explanation for the development of laminitis as a consequence of GC action is currently lacking, numerous possible and plausible theoretical mechanisms do exist. Veterinarians must exert caution with respect to the use of GCs in adult horses. The extent to which individual horses are predisposed to laminitis as a result of GC effect cannot be predicted based on current information. However, the administration of systemic GCs to horses that have been previously affected by laminitis should be used only with extreme caution, and should be accompanied by careful monitoring for further signs of laminitis. The risk of laminitis appears to be greater during treatment using some GCs (especially dexamethasone and triamcinalone) compared with others (prednisone and prednisolone). Whenever possible, to reduce the risk of laminitis, GCs should be administered locally. For example, the risk of GC-associated laminitis is evidently considerably reduced in horses affected with chronic obstructive pulmonary disease (COPD) if GC treatment is administered via inhalation. We have hypothesized that structural changes in the equine hoof that resemble laminitis may arise as a consequence of excess GC effect. Although these changes are not painful per se, and are not associated with inflammation, they could likely predispose affected horses to the development of bona fide laminitis for other reasons. Moreover, the gross morphological appearance of the chronically GC-affected hoof resembles that of a chronically foundered hoof in some respects. Further investigation into the effect of GC on the hoof lamellar interface is clearly needed.

Pertinent patient data and biopsied tissue from 16 cases of canine synovial sarcoma (SS) were reviewed. Histopathological grade, clinical stage, and tissue immunoreactivity to cytokeratin (broad stain, AE1/AE3 and cytokeratin 7) and vimentin were determined and correlated with survival. Effect of treatment on survival was similarly evaluated. Neither clinical stage nor histopathological grade significantly affected survival patterns. Tissues from all cases stained >30% positively with vimentin, whereas no tissue from any case exhibited cytokeratin immunoreactivity. Dogs receiving surgical tumor excision or amputation had a significantly higher survivability than those receiving no treatment (P

Paranasal meningioma was diagnosed in a 5-year-old Appaloosa gelding. The mass occupied the right maxillary, frontal, and sphenopalatine sinuses but did not invade the calvarium. The diagnosis was based on histologic evaluation, positive immunohistochemical staining for vimentin and cytokeratin, and ultrastructural features including the presence of interdigitating spindle cells with numerous desmosomes.

Over a period of 6 years, antemortem and postmortem examinations were performed on a number of donkeys suffering from respiratory disease. For many cases, initial diagnostic efforts failed to identify an etiology consistent with the pathologic findings. However, retrospective examination of these cases using consensus primer polymerase chain reaction, designed to recognize herpesviruses from all 3 subfamilies of the Herpesviridae, amplified a fragment of the highly conserved herpesvirus DNA polymerase gene from a number of these animals. Two novel herpesviruses, herein designated asinine herpesvirus 4 (AHV4) and asinine herpesvirus 5 (AHV5), were consistently detected in lung tissue from donkeys in which the histopathology was characterized by interstitial pneumonia and marked syncytial cell formation but not in lung tissue from donkeys with evidence of bacterial or verminous pneumonia. Nucleotide sequence and phylogenetic analysis places these new viruses within the Gammaherpesvirinae subfamily and indicates that they are most closely related to the recently identified zebra herpesvirus and wildass herpesvirus as well as equine herpesviruses 2 and 5.

OBJECTIVE To characterize chondrocytes from naturally occurring osteochondrosis (OC) lesions of the humeral head of dogs. SAMPLE POPULATION 15 cartilage specimens from 13 client-owned dogs with humeral head OC and 10 specimens from the humeral head of healthy dogs (controls). PROCEDURE Chondrocytes were isolated and cultured in a 3-dimensional system. On days 7, 10, 15, 20, and 25, glycosaminoglycan and hydroxyproline content and cytologic characteristics were evaluated. Expression of collagen types I, II, and X was assessed by use of immunohistochemistry. RESULTS Chondrocytes from OC lesions were less viable, compared with control chondrocytes. Glycosaminoglycan content in the OC group was significantly less than in the control group on all days except day 20. Hydroxyproline content was also significantly less in the OC group on days 10, 20, and 25. Expression of collagen type II was significantly less in the OC group, compared with the control group on all days, whereas expression of collagen type I was significantly greater in the OC group on days 20 and 25. Expression of collagen type X was significantly less in the OC group on all days except day 25. CONCLUSIONS AND CLINICAL RELEVANCE Chondrocytes from naturally occurring OC lesions of the humeral head of dogs cultured in a 3-dimensional system were less viable and less capable of producing appropriate extracellular matrix molecules than chondrocytes from unaffected dogs. Alterations in the synthetic capabilities of chondrocytes from OC-affected cartilage may be a cause or an effect of the disease process.

Immunohistochemistry, using a monoclonal antibody to Melan A and a polyclonal antibody to S100 protein, was applied to 48 formalin-fixed, paraffin-embedded specimens of feline melanoma. Forty-two cutaneous, three oral, one mucocutaneous, and two metastatic melanomas comprised the tumors. Thirty-two tumors (67%) were positive for Melan A and 42 (87.5%) were positive for S100. All but one of the tumors that were positive for Melan A were also positive for S100. S100 was detected in 11 of 16 tumors that were negative for Melan A. Seventy-five percent (9 of 12) of amelanotic melanomas were negative for Melan A. Normal adrenal cortex, the cerebellum, and the skin had cells that were positive for Melan A. Sebaceous adenoma was the only nonmelanocytic tumor examined that reacted with antibody to Melan A. Although less sensitive than S100 protein, Melan A is more specific for melanoma and is useful in differentiating feline cutaneous melanoma from the more common pigmented basal cell tumor.

Acquired portosystemic shunts (PSS) are a clinical entity distinct from congenital PSS. Their apparent incidence in cats is low, which may reflect the rarity of predisposing hepatic parenchymal disease, such as cirrhosis, in this species. Two cats with acquired PSS associated with primary hepatobiliary disease are described. Relevant findings in acquired PSS are discussed, as are potential reasons for the apparently low incidence in the cat.

SummaryThe objective of this study was to evaluate the effects of [D-Ala2, Nme-Phe4, Gly5-ol] enkephalin (DAMGO), a μ-opioid receptor agonist, and β-funaltrexamine (β-FNA), a μ-opioid receptor antagonist, on the biosynthetic capabilities of canine chondrocytes cultured in the presence of interleukin-1 β (IL-1 β). Articular chondrocytes were harvested from the humeral heads of three adult dogs and cultured in a three-dimensional (3-D) gel medium made from low-melting agarose and cell culture medium. Chondrocytes in 3-D culture were exposed to IL-1 β (0 or 20 ng/ml), DAMGO (0,0.1,1.0, or 10 μM), and β-FNA (0 or 10 μM) by addition to the liquid media in all possible combinations. On days five and 15 of 3-D culture, liquid medium samples were harvested for subsequent analysis of glycosaminogly- can (GAG), prostaglandin E2 (PGE2) and matrix metalloprotease-3 (MMP-3) content. On the same days, gel plugs were also harvested and evaluated for GAG content.Incubation with IL-1 β decreased the amount of GAG in the gel plugs and caused an increase in PGE2 production on days five and 15 of 3-D culture. Treatment with DAMGO or β-FNA did not significantly modulate PGE2 production, MMP-3 production, GAG loss to the medium or GAG content of the gel plugs on either day five or 15 of 3-D culture in the presence or absence of IL-1 β. We concluded that DAMGO and β-FNA had neither protective nor detrimental effects on the biosynthetic capabilities of chondrocytes in the presence or absence of IL-1 β.

Delayed-type hypersensitivity (DTH) is a T-cell-mediated immune response that may be used for immunotoxicity testing in non-clinical species. However, in some cases DTH assays using T-dependent antigens may be confounded by the production of antibodies to the antigen. The authors have previously modified a DTH assay, initially validated in the mouse, for use in juvenile rats to assess the effect of immunosuppressive drugs on the developing rat immune system. The assay measures footpad swelling induced by subcutaneous footpad injection of Candida albicans (C. albicans) derived-chitosan in rats previously sensitized with C. albicans. Antibodies to chitosan are not produced in this model. However, considerable inter-animal variability inherent in the footpad swelling assay can make it difficult to precisely quantify the magnitude of the immune response and inhibition by immunosuppressants, particularly if complete suppression is not observed. This report describes the development of an ex vivo assay to assess DTH in rats using interferon (IFN)-γ production by splenocytes, obtained from rats sensitized with C. albicans, as the quantifiable measure of the DTH response. Adult and neonatal rats administered dexamethasone (DEX), a known immunosuppressant, exhibited immunosuppression as evidenced by a reduction in ex vivo IFNγ production from splenocytes challenged with C. albicans-derived chitosan. Current data indicate that the ex vivo based DTH assay is more sensitive than the conventional footpad swelling assay due to a lower background response and the ability to detect a response as early as post-natal day (PND) 12. The ex vivo based rat DTH assay offers a highly sensitive and quantitative alternative to the footpad swelling assay for the assessment of the immunotoxic potential of drugs. The increased sensitivity of the ex vivo DTH assay may be useful for identifying smaller changes in response to immunotoxic drugs, as well as detecting responses earlier in animal development.

The monoclonal antibody A103 to the melanocytic differentiation antigen Melan A stains human steroid-producing cells and their tumors. A total of 200 formalin-fixed, paraffin-embedded canine normal tissues and hyperplastic and neoplastic lesions of the adrenal gland, testis, and ovary were immunohistochemically tested for Melan A with antibody A103. Leydig cell tumors (23/23, 100%), Sertoli cell tumors (14/15, 93%), and adrenocortical adenomas (12/13, 92%) were consistently positive. Adrenocortical carcinomas (23/35, 65%) and granulosa cell tumors (10/17, 59%) were less frequently positive. All pheochromocytomas, seminomas, and dysgerminomas were negative. The pattern of staining was cytoplasmic, but nuclear staining was also frequently seen in normal Leydig cells and their tumors. As in human tumors, immunohistochemistry for Melan A stains many canine steroid-producing tumors and can be used to distinguish these tumors from those of nonstereidogenic cells.

Objective—To determine glycosaminoglycan (GAG) concentration and immunohistochemical staining characteristics of type-I, -II, and -X collagen from cartilage affected by osteochondritis dissecans (OCD) in dogs. Animals—31 dogs with OCD and 11 clinically normal purpose-bred dogs. Procedure—Cartilage samples were evaluated microscopically, and GAG content was determined. Immunohistochemical staining was performed for type-I, -II, and -X collagen. Sections were subjectively evaluated for location and intensity of staining. Results—Cartilage affected by OCD had a variety of pathologic changes and significantly lower GAG concentrations than did normal cartilage. Normal cartilage had no detectable type-I collagen. For dogs < 9 months of age, cartilage affected by OCD had significantly more type-I collagen but significantly less type- X collagen than did control cartilage. For dogs > 12 months of age, cartilage affected by OCD contained significantly more type-I collagen than did control cartilage. There was a significant negative correlation between immunoreactivity of type-I collagen and that of type-II and -X collagen. A significant positive correlation was found between immunoreactivity of type-II and -X collagen. Conclusions and Clinical Relevance—Cartilage affected by OCD contains less GAG, more type-I collagen, and less type-X collagen, compared with normal cartilage. A direct correlation between these changes and the etiopathogenesis of OCD was not established. (Am J Vet Res 2001;62:876–881)

Porcine small intestinal submucosa (SIS) was used to replace large, avascular defects in the medial menisci of dogs. Twelve dogs received SIS grafts and 3 dogs were left untreated as controls. Dogs were evaluated at 4, 8, and 12 weeks by means of lameness scoring and ultrasonography. Dogs were sacrificed at 1, 6, or 12 weeks after implantation, and the tissue at the site of meniscal resection was evaluated for gross and histologic appearance, cross-sectional and surface area, and collagen types I and II. The femoral and tibial condyles were assessed for articular cartilage damage. Control dogs were significantly more lame than grafted dogs 8 and 12 weeks after instrumentation. Grafted dogs' replacement tissue appeared meniscal-like when evaluated grossly and ultrasonographically 12 weeks after instrumentation. The amount of replacement tissue was significantly greater in both cross-sectional and surface area for grafted dogs than for controls at all time points. Histologically, the SIS biomaterial could be identified in all grafted dogs at 1 week post-implantation, but in none at 6 weeks post-implantation. Subjectively, grafted dogs' replacement tissue was histologically superior to that of controls with respect to tissue type, organization, and architecture. Collagen types I and II immunoreactivity in grafted menisci were similar to that of normal menisci. Control dogs had significantly more articular cartilage damage than grafted dogs. SIS appears to induce regeneration of meniscal-like tissue in large, avascular meniscal defects in dogs, resulting in superior clinical function and articular cartilage protection compared to ungrafted controls.

Objective—To evaluate the clinical and pathologic characteristics of mammary duct ectasia in dogs.Design—Retrospective study.Animals—51 dogs with mammary duct ectasia.Procedure—Information regarding body condition, history, number and location of affected mammary glands, appearance of lesions, surgical treatment, nonsurgical treatment, and evidence of recurrence or development of mammary neoplasia was obtained from surveys sent to referring veterinarians. Results of information from examination of histologic sections and referring veterinarians were evaluated for all mammary duct ectasia biopsies performed between 1992 and 1999.Results—Duct ectasia was the primary diagnosis in 51 of 1,825 (2.8%) mammary biopsy specimens and comprised 48% of nonneoplastic mammary diseases. Affected dogs were evenly distributed over a range of 1 to 13 years of age, with a mean age at the time of diagnosis of 6.1 ± 3.1 years. All dogs were female (31 sexually intact, 20 spayed); 10 of 26 had whelped. Duct ectasia was described as nodular (26 dogs), cystic (13), and multiglandular (11) and located in caudal (31) more often than cranial (14) or middle glands (10). Ectasia recurred in 3 dogs. One dog had a history of previously excised mammary adenocarcinoma; another subsequently developed mammary carcinoma.Conclusions and Clinical Relevance—Duct ectasia affected mature, sexually intact and spayed female dogs over a wide age range. Certain breeds were affected more commonly than expected. Increased risk for mammary neoplasia was not evident. Duct ectasia should be considered as a cause for mammary enlargement, especially in young dogs or when its cystic nature is evident. Mastectomy is usually curative, and neoplasia should be ruled out in dogs with ectasia. (J Am Vet Med Assoc2001;218:1303–1307)

SummaryThe purpose of this study was to determine collagen type II and X immunohistochemical staining characteristics of naturally-occurring fragmented medial coronoid processes (FMCP) in order to help delineate potential pathophysiological events associated with FMCP.34 surgically excised FMCP from 21 client-owned dogs and 16 intact medial coronoid processes from 8 mongrel dogs were examined. The specimens were categorized by the dog's age: 24 months.The excised FMCP and normal medial coronoid processes were sectioned and stained with haematoxylin and eosin, safranin-O fast green, and toluidine blue. Sections were subjectively evaluated for tissue morphology, cell and matrix content, and proteoglycan staining. Immunohistochemical staining was performed for collagen types II and X. Sections were then subjectively evaluated for the location and intensity of staining.FMCP tissue demonstrated a wide variety of histological and immunohistochemical characteristics compared to normal medial coronoid processes. Significantly (P = 0.016) more normal dogs stained positive for collagen type X than dogs with FMCP in the Excised fragmented medial coronoid processes were examined to determine the collagen immunohistochemical staining characteristics when compared to intact processes.

Objective— To determine if there is a difference in in vitro growth of fibroblasts isolated from the trunk and distal aspect of the limb of horses and ponies. To determine the effects of a corticosteroid and monokine on in vitro growth of fibroblasts isolated from the trunk and distal aspect of the limb of horses and ponies. Study Design— Growth of fibroblasts from tissues harvested from the trunk and limb were compared from horse and pony samples grown in control media and control media with triamcinolone or monokine added. Animals or Sample Population— Dermal and subcutaneous tissue from 22 horses and 17 ponies of various ages and breeds. Methods— Fibroblast growth was assessed by tritiated thymidine uptake using standard cell culture techniques. The effect of a monokine or triamcinolone plus control media were compared with control media for fibroblast growth. Results— Fibroblast growth from tissues isolated from the horse limb was significantly less than growth from the horse trunk and the limb and trunk of ponies. Monokine was more effective than triamcinolone in suppressing fibroblast growth from tissues isolated from the trunk and limb in both horses and ponies. Conclusions— There are growth differences in fibroblasts isolated from the limb of horses compared with those isolated from the trunk and from the limb and trunk of ponies. Clinical Relevance— The difference in fibroblast growth from tissues isolated from the trunk and limb of horses and ponies may provide evidence for the difference reported in the healing characteristics of limb wounds in horses and ponies. Influencing fibroblast growth may provide a key to controlling the development of exuberant granulation tissue in horses and ponies.

A unique biomaterial, porcine small intestinal submucosa, was used to construct grafts for implantation into surgically created medial meniscal defects in dogs. Five dogs received grafts and two were left untreated as controls. All dogs were evaluated at 4, 8, and 12 weeks by means of lameness scoring, force plate analysis, and ultrasonography. Twelve weeks after implantation the dogs were sacrificed and the replacement tissue was evaluated for gross and histologic appearance, amount, glycosaminoglycan content, and type II collagen immunoreactivity. Four weeks after instrumentation, both groups had lameness scores that were significantly higher than preoperative scores, but at the 8- and 12-week evaluations, scores for the grafted dogs were not different from preoperative values. The ultrasonographic appearance of replacement tissue in grafted defects resembled normal meniscus. In the untreated defects, only unorganized tissue was present. In control dogs, replacement tissue resembled fibrous tissue and cartilage erosions were visible on the medial femoral condyles. In four of the five grafted dogs, replacement tissue was grossly indistinguishable from normal meniscus. The amount of tissue in the defect was significantly greater for the grafted dogs. Histologically, replacement tissue in control dogs was composed of vascularized connective tissue with no evidence of chondroid differentiation. Replacement tissue in grafted dogs closely resembled normal meniscal tissue with respect to chondroid differentiation, collagen content, and zonal architecture. Porcine small intestinal submucosa appeared to have beneficial effects on meniscal regeneration.

Summary We retrospectively evaluated the medical records and obtained follow-up information for nine horses which had been treated for cecocolic intussusception (CCI) between January 1982 and April 1998. During the 16-year study period, CCI was diagnosed in nine of 748 horses in which exploratory celiotomy was undertaken for abdominal pain, representing an incidence of 1.2%. Most affected horses (78%) were less than four years of age (median age was 12 months, age range was five months to 15 years). Cecocolic intussusception affected male horses (78%) more commonly than female horses. The most common clinical presentation was abdominal pain of a severe, acute nature or milder but recurrent signs of abdominal pain persisting in spite of conservative treatment for several days. Correction of CCI by either simple reduction or reduction followed by partial typhlectomy was successful if compromise of the intestine by devitalization and adhesion formation was not found at surgery. Definitive diagnosis of CCI necessitates exploratory celiotomy, although an ultrasonographic examination of the abdomen may confirm the diagnosis in some cases. When recognized early during the course of disease, surgical correction of CCI is associated with a favorable outcome; of the eight horses which underwent surgery in our series, five horses (63%) survived surgical correction of CCI. Handling of compromised gut during reduction of CCI necessitates extreme caution because the risk of intestinal tearing is quite high.

PURPOSE:Mycobacterium paratuberculosis has been proposed as a causative agent in patients with Crohn's disease. The purpose of this study was to determine whetherM. paratuberculosis was present in tissue from patients with Crohn's disease in a defined geographic area. METHODS: We prospectively evaluated, using polymerase chain reaction and culture, whetherM. paratuberculosis was present in 44 specimens (37 from intestinal mucosal biopsies and 7 from surgical resections) from patients with Crohn's disease, ulcerative colitis, or normal colonic mucosa. RESULTS: Of the 25 specimens tested from the 21 Crohn's patients, only 1 positive specimen was noted, whereas the 8 specimens from the 5 ulcerative colitis patients and the 11 specimens from the 11 control patients failed to demonstrate a positive result with polymerase chain reaction. Cultures of all specimens revealed no growth ofM. paratuberculosis. CONCLUSION:M. paratuberculosis was only rarely detected in biopsy or surgical specimens from patients with Crohn's disease. These results do not support a common causative role ofM. paratuberculosis in Crohn's disease.

SummaryThree-dimensional (3-D) culture of chondrocytes has been shown to promote cell proliferation, differentiation, and production of matrix while providing for cell morphology and matrix characteristics that resemble normal articular cartilage (1-3). In a recently published study, enhanced glycosaminoglycan (GAG) production was demonstrated in canine chondrocytes in 3-D culture on cancellous bone substrate (3). The effects of growth factors known to be present in cryopreserved bone were suggested to be responsible. Various growth factors including epidermal growth factor (EGF), fibroblast growth factor (FGF), and plateletderived growth factor (PDGF) have been shown to enhance chondrocyte DNA production, mitotic activity, and matrix production (5-10). The objective of the study reported here was to evaluate the effects of one concentration of EGF, FGF, and PDGF on canine chondrocyte proliferation and production of normal matrix constituents in 3-D culture.Canine articular chondrocytes were cultured in three-dimensional medium for 25 days. EGF, FGF, and PDGF were added to the culture medium. Chondrocytes in 3-D culture maintained viability and differentiation. Cell morphology and matrix production resembled that of intact hyaline cartilage. Significant differences with respect to cell counts, glycosaminoglycan concentration, or collagen type II immuno-reactivity as the result of added growth factors were not found.

In vivo carcinogenicity assessment of chemicals is a long and expensive process, typically including lifetime (2-year) studies in two rodent species. The relevance of these animal data to human cancer risk assessment has been questioned, and there are numerous examples of chemically-induced rodent neoplasms that are not considered applicable to human risk. This chapter summarizes the design, planning, and execution of 2-year rodent carcinogenicity studies as well as the same features in newer alternative (6-month) studies that use mouse models with genetically engineered predispositions to develop xenobiotic-induced cancers (e.g., the rasH2 mouse and p53+/− mouse). Special considerations required when undertaking carcinogenicity assessment of proteins, small nucleic acids, and stem cell-based therapies are also reviewed.

Gemtuzumab ozogamicin (Mylotarg, GO) and inotuzumab ozogamicin (IO) are antibody-drug conjugates (ADCs) comprised of different humanized monoclonal antibodies (against CD33 and CD22 antigen, respectively) and of the same acid-labile linker and calicheamicin payload. GO and IO are developed for the treatment of acute myeloid leukemia and acute lymphoblastic leukemia, respectively. Adverse events reported with these 2 drugs in patients include thrombocytopenia and liver toxicity, which is characterized by increases in serum aminotransferases and bilirubin along with occasional cases of sinusoidal obstruction syndrome (SOS). The platelet and liver effects were seen in patients with conjugates targeting unrelated antigens and are likely target-independent. Since the specific mechanisms of these toxicities remain elusive, an investigative study was performed in cynomolgus monkeys with a non-binding ADC containing the same linker and payload as GO and IO, PF-0259, with the objectives to investigate the mechanism for thrombocytopenia, characterize the liver injury and identify potential safety biomarkers. Cynomolgus monkeys were dosed intravenously with PF-0259 at 6 mg/m2/dose once every 3 weeks for up to 3 doses and were necropsied 48 hours after the 1st administration (Day 3) or 3 weeks after the 3rd administration (Day 63). PF-0259 induced acute thrombocytopenia (up to 86% reduction in platelet count) in monkeys with nadirs on Days 3-4. There was no indication of effects on megakaryocytes in bone marrow or activation of platelets in peripheral blood. Microscopic evaluation of liver samples from animals necropsied on Day 3 demonstrated midzonal degeneration and loss of sinusoidal endothelial cells (SECs) associated with marked platelet accumulation in sinusoids. Liver histopathology on Day 63 showed variable endothelial cell recovery and progression to a combination of sinusoidal capillarization and sinusoidal dilation/hepatocellular atrophy, consistent with early SOS. Among biomarkers evaluated, there were early and sustained increases in serum hyaluronic acid (HA) that correlated well with AST levels and liver microscopic changes, suggesting that HA could be a sensitive diagnostic marker of the liver microvascular injury. In conclusion, this work has demonstrated that target-independent damage to liver SECs was responsible for acute thrombocytopenia (through platelet sequestration in the liver) and development of early SOS in monkeys. The translation of these observations to humans has not been evaluated. We further hypothesize that this toxicity mechanism may operate for other types of non-calicheamicin based ADCs in patients where adverse events of thrombocytopenia, increased liver enzymes and liver microvascular disorders (including nodular regenerative hyperplasia) have been observed. Citation Format: Magali Guffroy, Hadi Falahatpisheh, Kathleen Biddle, John Kreeger, Leslie Obert, Karen Walters, Richard Goldstein, Germaine Boucher, Timothy Coskran, William Reagan, Danielle Sullivan, Sharon Sokolowski, Richard Giovanelli, Hans-Peter Gerber, Martin Finkelstein, Nasir Khan. Liver microvascular injury and thrombocytopenia of antibody-calicheamicin conjugates: mechanism and monitoring. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr LB-202.

Malignant fibrous histiocytoma was diagnosed in seven cats from biopsy specimens received at the University of Missouri Veterinary Medical Diagnostic Laboratory during a 4-year period from 1987–1991. Tissue blocks from formalin-fixed specimens were resectioned and stained for type I (AE1) and type II (AE3) cytokeratins, desmin, S100 protein, vimentin, and alpha1-antitrypsin by the avidin biotin peroxidase complex method with diaminobenzidine (DAB) chromogen. None of the tumors stained positively for alpha1-antitrypsin. Four of seven of the tumors had similar immunohistochemical staining results, with positive staining for type I and type II cytokeratins, desmin, S100 protein, and vimentin. Of the remaining three, one stained positively only for S100 protein and vimentin; one stained positively for vimentin only; and one was negative for all six antigens. Based only on immunohistochemical staining results, three of the tumors could possibly be reclassified: one as a melanoma, one as a probable fibrosarcoma, and one as unknown. These results also indicate that feline malignant fibrous histiocytomas show a diversity of intermediate filament expression, as do human tumors. Our results also do not support the theory that malignant fibrous histiocytomas are of histiocytic origin.

Genetic ablation of the voltage-gated potassium channel Kv1.3 improves insulin sensitivity and increases metabolic rate in mice. Inhibition of Kv1.3 in mouse adipose and skeletal muscle is reported to increase glucose uptake through increased GLUT4 translocation. Since Kv1.3 represents a novel target for the treatment of diabetes, the present study investigated whether Kv1.3 is functionally expressed in human adipose and skeletal muscle and whether specific pharmacological inhibition of the channel is capable of modulating insulin sensitivity in diabetic mouse models. Voltage-gated K+channel currents in human skeletal muscle cells (SkMC) were insensitive to block by the specific Kv1.3 blockers 5-(4-phenoxybutoxy)psoralen (PAP-1) and margatoxin (MgTX). Glucose uptake into SkMC and mouse 3T3-L1 adipocytes was also unaffected by treatment with PAP-1 or MgTX. Kv1.3 protein expression was not observed in human adipose or skeletal muscle from normal and type 2 diabetic donors. To investigate the effect of specific Kv1.3 inhibition on insulin sensitivity in vivo, PAP-1 was administered to hyperglycemic mice either acutely or for 5 days prior to an insulin tolerance test. No effect on insulin sensitivity was observed at free plasma PAP-1 concentrations that are specific for inhibition of Kv1.3. Insulin sensitivity was increased only when plasma concentrations of PAP-1 were sufficient to inhibit other Kv1 channels. Surprisingly, acute inhibition of Kv1.3 in the brain was found to decrease insulin sensitivity in ob/ob mice. Overall, these findings are not supportive of a role for Kv1.3 in the modulation of peripheral insulin sensitivity.

The purpose of this study was to document the levels of tumor necrosis factor-alpha (TNF) in serum of calves experimentally infected intratracheally with Pasteurella haemolytica A1 and to determine if elevated TNF levels correlate with development of pneumonic pasteurellosis in the bovine. Serum samples were collected at sequential time periods from 0 h to 72 h post inoculation with P. haemolytica. TNF levels in those sera were measured by a cytotoxicity assay utilizing the TNF-sensitive WEHI 164 mouse fibrosarcoma cell line. Serum TNF levels in infected cattle began to rise at 2 h post inoculation, peaked at approximately 8 h, and decreased to near control levels by 72 h. There was extreme variability in serum TNF among the inoculated animals with levels varying from 120 pg ml-1 to 5000 pg ml-1 at 8 h post inoculation. These levels did not correspond with the degree of lung involvement. All inoculated calves developed lesions of pneumonic pasteurellosis characterized by fibrinous pleuritis with necrotizing, hemorrhagic pneumonia. These results suggest that TNF is probably a significant inflammatory mediator involved in the pathogenesis of bovine pneumonic pasteurellosis.

Objective— To determine normal cartilage stiffness values in different weight-bearing and non-weight-bearing areas of 3 different equine joints, and to evaluate the relationship between cartilage stiffness and glycosaminoglycan (GAG) and collagen content. Study Design— Compressive stiffness of the articular cartilage was measured in 8 horse cadaver femoropatellar (FP), tarsocrural (TC), and metatarsophalangeal (MT) joints. Gross evaluation, collagen content, GAG content, and histologic appearance were assessed for each measurement location. Animals— Eight equine cadavers (4 intact females, 4 castrated males; 7 Quarter Horse or Quarter Horse type, 1 Arabian; aged 4–12 years, weighing 400–550 kg) Methods— The articular surfaces of 8 equine cadaver FP, TC, and MT joints were grossly evaluated for signs of articular cartilage pathology. Stiffness at preselected sites (FP joint—6 sites; TC joint—3 sites; MT joint—4 sites) was determined using an arthroscopic indentation instrument. Biochemical composition (collagen, GAG content) and histologic evaluation (modified Mankin score) were assessed for each measurement site. Results— All cartilage from all sites evaluated was determined to be normal based on macroscopic and histologic assessments. No significant correlation between Mankin scores and cartilage stiffness values was observed. Site differences in cartilage stiffness were measured in all 3 joints (P 0.622, r2>0.387). Conclusion— Relative cartilage stiffness values measured in healthy equine joints are site dependent and can be measured using an indentation device intended for arthroscopic application. Clinical Relevance— An indentation instrument provided an objective means of determining relative compressive stiffness of articular cartilage. Further research needs to be performed to confirm the site and joint differences observed in this study in clinically normal horses and to determine if the tester can be used clinically to predict articular cartilage pathology.

An 18-year-old Spanish Mustang mare was referred for evaluation of progressive weight loss and persistent hyperglycemia. Clinicopathologic abnormalities included marked hyperglycemia and glycosuria. Serum cortisol concentration was appropriately decreased following administration of dexamethasone, indicating that the horse did not have pituitary pars intermedia dysfunction. Serum insulin and plasma C-peptide concentrations were low, suggesting that hyperglycemia was a result of decreased secretion of insulin by pancreatic beta cells. In addition, glucose concentration did not return to the baseline concentration until 5 hours after i.v. administration of a glucose bolus, suggesting that insulin secretion, insulin effect, or both were reduced. However, i.v. administration of insulin caused only a slight decrease in the plasma glucose concentration, giving the impression that the action of insulin was impaired. Within 5 hours after administration of a combination of glyburide and metformin, which is used to treat diabetes mellitus in humans, the glucose concentration was within reference limits. The horse was euthanized, and a postmortem examination was done. Immunohistochemical staining of sections of the pancreas revealed attenuation of the pancreatic islet beta-cell population, with beta cells that remained generally limited to the periphery of the islets. These findings indicate that, albeit rare, pancreatic beta-cell failure may contribute to the development of diabetes mellitus in horses.

Naturally occurring biomaterials, such as small intestine submucosa (SIS), are attractive as potential scaffolds for engineering various tissue types. The aim of this study was to determine whether acellular SIS scaffolds can support cell attachment and ingrowth in a diarthroadial joint without significant intraarticular hemorrhage. Disks of porcine SIS were arthoscopically implanted freely within a randomized knee joint of 21 dogs and harvested 1, 2, 3, and 6 weeks postoperatively. Harvested disks were assessed for gross and histologic appearance, cellular infiltration, and immunoreactivity of collagenase and collagen types I and II. Knee synovium and synovial fluid were also evaluated. All disks were thickened and opacified at harvest. Eleven disks (52%) had adhered to intraarticular tissues and cellular infiltration into the disks was positively correlated with tissue adherence. Further, tissue adherence was positively correlated with duration of intraarticular implantation. Five disks (24%) contained focal areas of homogeneous extracellular matrix. A trend toward more collagenase immunoreactivity was noted in the 3-week disks. Collagen type I was present in remaining SIS and extracellular matrix associated with infiltrated cells. Placed freely within a joint, acellular SIS disks underwent cellular and extracellular matrix modification resulting in fibrocartilage-like tissue. Utilization of SIS as a scaffold for intraarticular tissue-engineering applications is supported as cytoconductivity, appropriate residence time, and absence of untoward effects of implantation are desirable criteria for a tissue-engineering biomaterial.

Thirteen uterine tumors were diagnosed in 13 cats and accounted for 0.29% of all feline neoplasms received during a 9.6-year period. Age at diagnosis ranged from 3 to 16 years; median 9 years. Six were Domestic Shorthair cats, and 7 were purebred cats of 5 different breeds. Eight adenocarcinomas and 1 mixed Müllerian tumor (adenosarcoma) comprised the endometrial tumors. Myometrial tumors included 3 leiomyomas and 1 leiomyosarcoma. One of the adenocarcinomas developed in the uterine stump of an ovario-hysterectomized cat; the other cats were sexually intact. Concurrent mammary adenocarcinoma was diagnosed in 1 cat with uterine adenocarcinoma and in another with uterine leiomyoma. Tumors were discovered during elective ovariohysterectomy in 2 cats, but at least 3 others had experienced reproductive problems (infertility or pyometra). Five cats presented for abdominal or pelvic masses. Endometrial adenocarcinomas were positive immunohistochemically for cytokeratins and negative for smooth muscle actin (SMA); 1 of 6 cats was positive for vimentin and 4 of 8 were positive for estrogen receptor—α (ERα). Adenosarcoma stromal cells were positive for vimentin and ERα but negative for cytokeratins and SMA. Smooth muscle tumors were positive for vimentin and SMA and negative for cytokeratins. Leiomyomas, but not the leiomyosarcomas, were positive for ERα. Adenocarcinomas in 4 cats had metastasized by the time of ovariohysterectomy. Two other cats were euthanized 5 months after ovariohysterectomy; at least one of these cats had developed an abdominal mass that was not examined histologically. Only 2 cats with endometrial adenocarcinoma had disease-free intervals longer than 5 months after surgery. Metastasis was not detected in any mesenchymal tumor; however, these cats were either euthanized on discovery of the tumor or the tumor was first detected at necropsy.

Objective—To determine effects of carprofen and dexamethasone on chondrocytes in a culture model of osteoarthritis (OA). Sample Population—Chondrocytes isolated from articular cartilage of the humeral head of 5 adult dogs. Procedure—Chondrocytes were harvested, cultured and subcultured in monolayer, and then cultured in a 3-dimensional (3-D) medium. Cells from each dog were distributed into 6 groups with differing content of liquid medium for each 3-D construct (agarose [AG], AG plus interleukin [IL]-1β, AG plus carprofen [4 μg/mL], AG plus dexamethasone [1 mg/mL], AG plus IL-1β [20 ng/mL] plus carprofen [4 μg/mL], and AG plus IL-1β (20 ng/mL) plus dexamethasone (1 mg/mL). On days 3, 6, 12, and 20 of culture, samples from all groups were collected. Liquid media were assayed for glycosaminoglycan, prostaglandin (PG)E2, matrix metalloprotease (MMP)-3, and MMP- 13 concentrations. All 3-D constructs were evaluated for viability, cell morphology, proteoglycan staining, and collagen type-II concentration. Total glycosaminoglycan content in each 3-D construct was quantitated by spectrophotometric assay. Results—Addition of IL-1β caused a significant loss of cell viability and matrix production. Addition of carprofen or dexamethasone caused significant decreases in PGE2 in the liquid media, and each was minimally effective in protecting chondrocytes against negative effects of IL-1β. Conclusions and Clinical Relevance—Human recombinant IL-1β resulted in loss of cell viability, alterations in extracellular matrix components, and production of PG and MMP. Carprofen and dexamethasone had little effect on cell and matrix variables but did decrease PGE2 concentrations and primarily affected the inflammatory pathway of osteoarthritis. (Am J Vet Res 2002;63:1363–1369)

Objective—To determine immunoreactivity of matrix metalloproteinase (MMP)-1, -3, and -13 in cartilaginous tumors of dogs, correlate expression of MMP with histologic grade of tumors and clinical outcome of dogs, and compare MMP immunoreactivity between chondrosarcomas and chondromas. Sample Population—Formalin-fixed, paraffin-embedded tissues obtained from samples of naturally occurring chondrosarcomas (n = 31) and chondromas (8) of dogs that were submitted to our veterinary medical diagnostic laboratory. Procedure—Histologic sections from each sample were stained with H&E and monoclonal antibody to MMP-1, -3, and -13 by use of an avidin-peroxidase immunohistochemical technique. For each section, histologic grade (I, II, or III) and immunohistochemical expression (0, 1, 2, or 3) were evaluated. Clinical outcome was obtained from medical records or interviews with referring veterinarians and scored as a good outcome, moderate outcome, or poor outcome. Correlations among variables and differences between chondrosarcomas and chondromas were analyzed. Results—Samples from chondrosarcomas had significantly higher immunoreactivity of MMP-1 and -13, compared with immunoreactivity in samples from chondromas. In chondrosarcomas, a significant positive correlation (r, 0.386) was found between MMP-1 and -13 immunoreactivities, and a significant negative correlation (r, –0.390) was detected between MMP-3 and -13 immunoreactivities. Conclusion and Clinical Relevance—A significant increase in expression of collagenases (MMP-1 and - 13) in chondrosarcomas, compared with expression in chondromas, suggests that collagenases may play an important role in tumor progression, and possibly metastasis, in chondrosarcomas of dogs. (Am J Vet Res 2002;63:1285–1291)