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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., Competing Interests: Authors SJS, GGB, JMK, and JA were employees of Pfizer Inc. at the time this study was completed. This does not alter our adherence to PLOS ONE policies on sharing data and materials.

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.

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 6weeks 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., (Copyright © 2018 The Authors. Published by Elsevier B.V. All rights reserved.)

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 β 1 subunit. After confirming that human and rodent kidney predominately express AMPK β 1, 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., (Copyright © 2017 by The American Society for Pharmacology and Experimental Therapeutics.)

The mononuclear phagocyte system (MPS) which provides protection against infection is made up of phagocytic cells that engulf and digest bacteria or other foreign substances. Suppression of the MPS may lead to decreased clearance of pathogenic microbes. Drug delivery systems and immunomodulatory therapeutics that target phagocytes have a potential to inhibit MPS function. Available methods to measure inhibition of MPS function use uptake of radioactively-labeled cells or labor-intensive semi-quantitative histologic techniques. The objective of this work was to develop a non-radioactive quantitative method to measure MPS function in vivo by administering heat-killed E. coli conjugated to a pH-sensitive fluorescent dye (Bioparticles(®)). Fluorescence of the Bioparticles(®) is increased at low pH when they are in phagocytic lysosomes. The amount of Bioparticles(®) phagocytosed by MPS organs in rats was determined by measuring fluorescence intensity in livers and spleens ex vivo using an IVIS(®) Spectrum Pre-clinical In Vivo Imaging System. Phagocytosis of the particles by peripheral blood neutrophils was measured by flow cytometry. To assess method sensitivity, compounds likely to suppress the MPS [clodronate-containing liposomes, carboxylate-modified latex particles, maleic vinyl ether (MVE) polymer] were administered to rats prior to injection of the Bioparticles(®). The E. coli particles consistently co-localized with macrophage markers in the liver but not in the spleen. All of the compounds tested decreased phagocytosis in the liver, but had no consistent effects on phagocytic activity in the spleen. In addition, administration of clodronate liposomes and MVE polymer increased the percentage of peripheral blood neutrophils that phagocytosed the Bioparticles(®). In conclusion, an in vivo rat model was developed that measures phagocytosis of E. coli particles in the liver and may be used to assess the impact of test compounds on MPS function. Still, the detection of inhibition of splenic macrophage function will require further assay development.

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., (© 2014 by The Author(s).)

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., (© 2014 by The Author(s).)

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.

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.

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<.001). GAG or collagen content had a significant positive correlation with stiffness values in 6 of 13 sites (P<.05, r>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.

Objective: To address possible roles of matrix metalloproteinases (MMPs) and mechanical stress in the pathogenesis of osteochondrosis (OC)., Methods: Naturally-occurring canine OC lesions (n=50) were immunohistochemically analyzed for MMP-1, -3, and -13, and normal canine articular cartilage explants (n=6) cultured under 0-, 2-, or 4-MPa compressive loads (0.1 Hz, 20 min every 8 h up to 12 days) were compared to OC samples (n=4) biochemically and molecularly., Results: MMP-1 and -3 immunoreactivities were readily detected in both OC samples and control tissues obtained from age-matched dogs (n=11) whereas MMP-13 was only detectable in OC samples. MMP-13 gene expression as determined by real-time reverse transcription polymerase chain reaction was elevated in OC samples and cartilage explants cultured without mechanical stimuli (0 MPa groups) compared to normal cartilage (day 0 controls). Glycosaminoglycan content (per weight) in cartilage explants cultured under no load was significantly (P<0.05) lower on day 12 than in the day 0 controls. Gene expression levels of aggrecan and type II collagen in OC samples were lower than those in the day 0 controls. High levels of aggrecan and collagen II expression were seen in the 2 MPa groups., Conclusions: These findings imply that impaired biochemical characteristics in OC-affected cartilage may be attributable to decreased extracellular matrix production that may stem from disruption of normal weight bearing forces.

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.

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 10(6) cells/mL. We prepared 3-D constructs exposed to only tumor necrosis factor (TNF)-alpha (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-alpha. Chondrocytes cultured without TIMP or TNF-alpha 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-alpha or TNF-alpha plus TIMP did not increase. On day 9, GAG release from 3-D constructs cultured with TNF-alpha 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-alpha. Apparently, adverse effects on chondrocytes exposed to TNF-alpha cannot be prevented by addition of TIMP alone.

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.

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, 11beta-hydroxysteroid dehydrogenase-1 (11beta-HSD-1). Changes in integumentary (skin and hoof lamellar) 11beta-HSD activity occurring during laminitis could affect the extent to which GCs are involved in its development., Hypothesis: That changes in integumentary 11beta-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 11beta-HSD-1 activity would be evident in both skin and hoof lamellar tissue., Methods: Activity of 11beta-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 11beta-HSD-1 activity was measured in both skin and lamellar tissues., Results: 11-ketoreductase activity exceeded 11beta-dehydrogenase activity in both skin and lamellar tissues. Cutaneous activity was higher than lamellar 11beta-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<0.05)., Conclusions: The 11-ketoreductase moiety of 11beta-HSD-1 plays a role in equine skin and hoof lamellae regarding the regulation of local glucocorticoid activity. Increased 11-ketoreductase activity will lead to increased local tissue GC activity by virtue of conversion of cortisone to cortisol., Potential Relevance: The laminitic condition is attended by integumentary biochemical changes that enhance the local concentration of cortisol, especially in the hoof lamellar interface. Through multiple and diverse actions, increased local GC activity contributes to the pathogenesis and morbidity associated with laminitis. Pharmacological manipulation of 11beta-HSD-1 deserves further investigation regarding the prevention and treatment of laminitis.

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 ovariohysterectomized 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-alpha (ER alpha). Adenosarcoma stromal cells were positive for vimentin and ER alpha 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 ER alpha. 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 investigate the effects of tissue inhibitor of metalloproteinase (TIMP)-1 and -2 on chondrocytes cultured with or without interleukin (IL)-1 beta., Design: Canine articular chondrocytes were cultured in three-dimensional (3-D) agarose constructs. Cells were distributed into each of the two groups, those without IL-1 beta and those with IL-1 beta added to the liquid media. Each group was subdivided into three groups, based on the presence of TIMP-1 or -2. IL-1 beta and TIMPs were added to liquid media bathing the 3-D constructs beginning on day 3. The liquid media and the 3-D constructs were collected on days 9, 15, and 24, and analyzed histologically, biochemically, and immunohistochemically., Results: Addition of TIMP-1 or -2 resulted in decreases in matrix metalloproteinase (MMP)-3 concentrations of 37 and 41%, and MMP-1 immunoreactivity of 32 and 36%, respectively, compared with the IL-1 beta group, on day 9. Chondrocytes in groups without IL-1 beta maintained viability and produced abundant extracellular matrix (ECM). Chondrocytes in IL-1 beta groups appeared less viable and produced less ECM compared with those without IL-1 beta. Glycosaminoglycan (GAG) concentrations in 3-D constructs (GAG/weight) were significantly (P<0.001) higher in groups without IL-1 beta than in those with IL-1 beta, on days 15 and 24., Conclusions: The addition of TIMP was not detrimental to chondrocytes, as used in this study. Despite evidence of decreased MMP levels, TIMPs did not prevent IL-1 beta-associated changes in cellular or ECM characteristics. Further study is necessary before clinically relevant conclusions can be drawn regarding the use of TIMPs in the treatment of osteoarthritis.

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., Conclusions and Clinical Relevance: Chondrocyte-agarose grafts are biocompatible in large tibial defects and appear to provide a cell source for augmenting endochondral ossification.

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]-1beta, AG plus carprofen [4 microg/mL], AG plus dexamethasone [1 mg/mL], AG plus IL-1beta [20 ng/mL] plus carprofen [4 microg/mL], and AG plus IL-1beta (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-1beta 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-1beta., Conclusions and Clinical Relevance: Human recombinant IL-1beta 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.

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., Conclusions 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.

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.

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.

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<0.02). Treatment aggressiveness may be more appropriate than clinical staging or tumor grading in predicting survival. Reliability of diagnosing and prognosticating canine SS with current immunohistochemistry protocols should be questioned.

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.

Objectives: To develop an in vivo perineal hernia model, to develop a technique for using small intestinal submucosa (SIS) in perineal hernia repair, to further elucidate the biological behavior of SIS, and to compare SIS herniorrhaphy with the internal obturator muscle transposition (IOT) technique., Study Design: Prospective evaluation comparing SIS herniorrhaphy with IOT., Animals: Twelve adult castrated male, large-breed dogs., Methods: All dogs had bilateral pelvic diaphragm defects created by complete excision of the levator ani muscle. Each dog had one side repaired using SIS and the other by IOT. Pain and inflammation were subjectively scored. Dogs were killed 2 weeks (n = 4), 12 weeks (n = 4), or 16 weeks (n = 4) after surgery. Each pelvic diaphragm was biomechanically tested to failure. The pelvic diaphragms from 2 normal dogs (n = 4 sides) were also biomechanically tested. Failure site, maximum pressure, displacement at failure, and initial linear stiffness values were determined. Histologic assessment was performed. Statistical analysis was performed with significance set at P <.05, Results: No significant postoperative complications were noted. There were no significant differences in maximum pressure to failure, displacement, or stiffness when comparing normal, SIS, and IOT at any time point. The SIS group had significantly less displacement (P =.004) at 2 weeks than at weeks 12 or 16. For all herniorrhaphy techniques, the failure site was central (n = 22) or at the suture line (n = 2). At 2 weeks, histologic evaluation of tissues from the IOT group showed inflammation, mineralization, and necrosis, which were not present in tissues from the SIS group. Histologic examination at 12 and 16 weeks showed no microscopic differences in cell population or tissue characteristics between the IOT and SIS groups., Conclusions: SIS herniorrhaphy was successfully performed in this in vivo model of perineal hernia in the dog., Clinical Relevance: This study suggests that SIS can be used as a primary means of repair, as augmentation when the internal obturator muscle is thin and friable, or as a salvage procedure in cases of recurrence in dogs with perineal hernia., (Copyright 2002 by The American College of Veterinary Surgeons)

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.

African trypansosomes are tsetse-transmitted parasites of chief importance in causing disease in livestock in regions of sub-Saharan Africa. Previous studies have demonstrated that certain breeds of cattle are relatively resistant to infection with trypanosomes, and others are more susceptible. Because of its extracellular location, the humoral branch of the immune system dominates the response against Trypanosoma congolense. In the following study, we describe the humoral immune response generated against T. congolense in SCID mice reconstituted with a bovine immune system (SCID-bo). SCID-bo mice infected with T. congolense were treated with an agonistic anti-CD40 antibody and monitored for the development of parasitemia and survival. Anti-CD40 antibody administration resulted in enhanced survival compared with mice receiving the isotype control. In addition, we demonstrate that the majority of bovine IgM+ B cells in SCID-bo mice expresses CD5, consistent with a neonatal phenotype. It is interesting that the percentage of bovine CD5+ B cells in the peripheral blood of infected SCID-bo mice was increased following anti-CD40 treatment. Immunohistochemical staining also indicated increased numbers of Ig+ cells in the spleens of anti-CD40-treated mice. Consistent with previous studies demonstrating high IL-10 production during high parasitemia levels in mice and cattle, abundant IL-10 mRNA message was detected in the spleens and peripheral blood of T. congolense-infected SCID-bo mice during periods of high parasitemia. In addition, although detected in plasma when parasites were absent or low in number, bovine antibody was undetectable during high parasitemia. However, Berenil treatment allowed for the detection of VSG-specific IgG 14 days postinfection in T. congolense-infected SCID-bo mice. Overall, the data indicate that survival of trypanosome-infected SCID-bo mice is prolonged when an agonistic antibody against bovine CD40 (ILA156) is administered. Thus, stimulation of B cells and/or other cell types through CD40 afforded SCID-bo mice a slight degree of protection during T. congolense infection.

The objective of this study was to determine the effects of ascorbate and two different culture media on cell morphology and extracellular matrix formation of canine chondrocytes grown in a three-dimensional (3-D) culture system. Articular cartilage harvested from the humeral head of three adult canine cadavers was used to obtain chondrocytes for primary culture. Subcultured chondrocytes were seeded in a 3-D medium of RPMI-1640 (R), RPMI-1640 with 50 microg/mL ascorbate (RA), Ham's F-12 (F), or Ham's F-12 with 50 microg/mL ascorbate (FA) in agarose. Samples were harvested at 5, 10, 15, and 20 days of 3-D culture and analyzed for histologic appearance and proteoglycan staining, electron microscopic appearance, dimethylmethylene blue assay for glycosaminoglycan (GAG) content, and immunohistochemical staining for collagen type II production. Chondrocytes in all four groups maintained appropriate morphology and produced matrix over the entire study period. Chondrocytes from groups R and RA produced more GAG and collagen type II than did those from groups F and FA on days 10 (P = .00791) and 15 (P = .0173). Chondrocytes from group RA produced more GAG on days 5 (P = .0154) and 20 (P = .0044) than did those in groups R, F, and FA. With respect to matrix production, RPMI-1640 is superior to Ham's F-12 for 3-D culture of canine chondrocytes. The addition of ascorbate at 50 microg/mL to RPMI-1640 did have a positive effect on the production of GAG but had minimal effect on type II collagen production. Determining the most ideal in vitro microenvironment for canine chondrocytes grown in a 3-D culture system has important implications to the in vivo application of this technique.

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.

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 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.

Twenty of 25 horses in a well-managed Missouri boarding stable were diagnosed with gingivitis/stomatitis. Gross examination of the affected horses revealed varying degrees of gingivitis ranging from mild periodontal swelling to marked swelling and erythema with ulceration and hemorrhage. Fine hair-like material was embedded within the intensely affected areas. Gingival biopsies from 4 affected horses contained pyogranulomatous inflammation with, in some cases, numerous eosinophils and several grass awns in cross and longitudinal section. Numerous foxtail seed heads were identified in hay samples. Examination of the records revealed that all of the affected horses had been fed the suspect hay, with the exception of 1 horse. Although not deliberately fed the suspect hay, this horse did have access to the hay when turned out into the exercise paddock. The lesions resolved following a change in hay source.

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.

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<0.05). Serum collagen IV concentration was also increased in jugular venous blood obtained from severely laminitic horses (219.50 +/- 18.18 ng/ml) compared with nonlaminitic controls (157.50 +/- 10.93 ng/ml) (P<0.05). A difference in serum concentration of collagen IV was not identified based on chronicity of naturally occurring laminitis. Serum laminin concentration did not differ between laminitic and nonlaminitic horses. Differences in serum laminin concentration were not identified based on sampling location (jugular or cephalic vein), severity of laminitic pain, or chronicity of spontaneous laminitis. In conclusion, the circulating concentration of collagen IV was increased in horses affected with naturally occurring laminitis. The potential role for serum collagen IV assay for characterisation of equine laminitis warrants further investigation.

Objective: To determine the effects of interleukin (IL)-1beta on matrix synthesis and degradation by chondrocytes cultured in a 3-dimensional (3-D) gel medium., Sample Population: Chondrocytes from 7 dogs., Procedure: Articular chondrocytes were harvested and cultured in 3-D gel medium alone or with 10 or 20 ng IL-1beta/ml that was added beginning on day 0, 3, 6, or 9. On days 3, 6, 12, and 20 of 3-D culture, samples of the liquid medium were evaluated for glycosaminoglycan (GAG), prostaglandin E2 (PGE2), and matrix metalloprotease (MMP)-3 content. The 3-D plug in each well was evaluated for histologic characteristics of viability, cell morphology, and proteoglycan staining, immunohistochemically stained for collagen type II, and spectrophotometrically analyzed for GAG content., Results: Significant differences for all variables were detected between controls and each IL-1beta group, among groups with different IL-1beta concentrations, and among groups with IL-1beta added at various time points. Chondrocytes exposed to IL-1beta had loss of GAG, increased PGE2 and MMP-3 concentrations, and lack of collagen type-II synthesis. These IL-1beta effects appeared to be time and concentration dependent., Conclusions: Addition of IL-1beta to chondrocytes in 3-D gel medium results in time- and concentration-dependent effects on matrix synthesis and degradation and provides an appropriate in vitro model for many of the pathophysiologic events associated with osteoarthritis.

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.

Objective: To determine effects of glucosamine and acetylsalicylate on canine chondrocytes in 3-dimensional culture., Sample Population: Chondrocytes isolated from articular cartilage of 2 adult female dogs recently euthanatized for reasons unrelated to orthopedic abnormalities., Procedure: Chondrocytes were cultured in a 3-dimensional agarose-based medium alone (control), with glucosamine (100 microg/ml; GL), or with acetylsalicylate (18 microg/ml; AS). Supernatant and agarose plugs from 4 wells/group/d were collected on days 3, 6, and 12 of culture. Agarose plugs were evaluated for percentage of viable cells, percentage of cells producing pericellular or territorial matrix, glycosaminoglycan (GAG) concentration, and type-II collagen production. Prostaglandin E2 concentration in supernatants was determined., Results: Chondrocytes in all groups had characteristics indicative of viability and differentiation; however, on day 12, a lower percentage of viable cells was detected in the AS group, compared with the other 2 groups. On day 6, GAG concentration in the AS group was significantly greater than concentrations in the other 2 groups. On day 12, GAG concentrations in the GL and AS groups were significantly less than in the control group. Within the GL and AS groups, cell viability was significantly less on day 12, compared with day 3. Significant differences in PGE2 concentration among or within groups and evidence of type II collagen production were not detected., Conclusions: 3-dimensional culture of canine chondrocytes allows for production of hyaline cartilage matrix constituents and growth of cells with morphologic characteristics similar to those of articular cartilage. Acetylsalicylate and glucosamine, at the single concentration evaluated, had detrimental effects on chondrocyte viability, GAG production, or both.

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.

Biosynthesis of endothelin-1 (ET-1), the most potent endogenous vasoconstrictor yet identified, is increased following myocardial infarction (MI) in man. Pathological events which occur in the connective tissues of the equine hoof during laminitis are similar in some respects, to changes occurring in the myocardial connective tissues following MI in man. The objective of this study was to determine whether ET-1 expression in connective tissues obtained from the hoof of laminitic horses is increased compared with tissues obtained from healthy horses. Expression of ET-1 in connective tissues of the equine hoof was measured following tissue extraction from 3 groups of horses: horses in which acute laminitis had been induced by the administration of starch; chronically foundered horses; nonlaminitic horses. The concentration of ET-1 in laminar connective tissues obtained from all laminitic horses (1573.0 +/- 392.8 pg/g of tissue; n = 10) was increased when compared with tissues obtained from nonlaminitic horses (392.5 +/- 117.4 pg/g of tissue; n = 5) (P<0.05). The concentration of ET-1 in laminar connective tissues obtained from the experimentally induced, acute laminitic horses (1043.6 +/- 254.4 pg/g of tissue; n = 7) and from the spontaneously affected, chronic laminitic horses (2808.3 +/- 878.6 pg/g of tissue; n = 3) was increased compared with the control group (P<0.05, P<0.01, respectively). The concentration of ET-1 in laminar connective tissues obtained from the chronic laminitic horses was greater than that of the experimentally induced, acute laminitic group (P<0.05). It is suggested that the data provide a strong argument that increased ET-1 expression in the connective tissues of the equine hoof represent a potentially important and hitherto unrecognised component of the pathophysiology of equine laminitis. Further studies are needed to determine whether inhibitors of ET-1 converting enzyme or antagonists of ET-1 receptors might be useful in the treatment and prevention of laminitis in horses.

The role of various effector T cell populations in the bovine immune response to Mycobacterium bovis infection is poorly understood. This is largely due to the difficulties associated with performing in vivo challenge studies in the natural host species. In this report, we utilized a fetal bovine-severe combined immunodeficient (SCID-bo) xenochimeric mouse model to study the protective role of two putative effector cell types, CD8+ T cells and a subpopulation of gamma/delta T cells that express WC-1, a member of the cysteine-rich scavenger receptor superfamily (CRSR). We demonstrate that CD8+ T cells play a key role in protection and contribute substantially to bovine IFN-gamma mRNA levels at 30 days post-infection. The role of WC-1 bearing cells to protection was less definitive but our results suggest that this population may play a pivotal role early in infection. Granuloma architecture was altered in anti-WC-1 (ILA29) but not anti-CD8 (ILA51) -treated animals, suggesting that this population may be involved in recruitment of various cell types to sites of infection.

Samples of connective tissue obtained from the hoof of six laminitic and eight non-laminitic adult horses were analysed zymographically to investigate whether connective tissue matrix metalloproteinases are activated or induced during laminitis. The activity or matrix metalloproteinases was substantially greater in the tissues from the laminitic horses than in the tissues from the non-laminitic horses. A comparison of the collagenolytic activity in the laminitic and control tissues showed that collagenolytic activities corresponding to the 92 kDa (P < 0.001), 72 kDa (P < 0.01) and 66 kDa (P < 0.01) bands were induced in the laminitic tissues.

Purpose: Mycobacterium paratuberculosis has been proposed as a causative agent in patients with Crohn's disease. The purpose of this study was to determine whether M. 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, whether M. 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 of M. 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 of M. paratuberculosis in Crohn's disease.

Elbow dysplasia is osteoarthrosis/degenerative joint disease due to abnormal development of the elbow joint. The abnormal development is the result of specific inherited etiologies alone or in combination. This paper attempts to clarify the diagnosis of elbow dysplasia based on the presence of degenerative joint disease by correlating radiographic, necropsy, and histopathologic results using elbows from 8 German Shepherd dogs. All elbows had radiographic changes consistent with osteoarthrosis/degenerative joint disease which were identified best on the flexed medial-lateral projection. Radiographically, a specific diagnosis was made in seven elbows; ununited anconeal process (6) and osteochondrosis (1). At necropsy these lesions were confirmed plus 14 elbows were identified that had fragmented medial coronoid process (6), abnormally shaped medial coronoid processes or fissures in the articular cartilage of the medial coronoid process (8). Additionally, histopathologically there was proliferative synovitis at the radial notch of the ulna and degenerative changes on the proximal, nonarticular surface of the anconeal process at the site of insertion of the olecranon ligament and joint capsule. Therefore, for screening the elbow joint to identify elbow dysplasia, the recognition of osteoarthrosis/degenerative joint disease on an extreme flexed mediolateral radiograph appears to be sufficient.