Points to consider regarding the use and implementation of virtual controls in nonclinical general toxicology studies.

The replacement of a proportion of concurrent controls by virtual controls in nonclinical safety studies has gained traction over the last few years. This is supported by foundational work, encouraged by regulators, and aligned with societal expectations regarding the use of animals in research. This paper provides an overview of the points to consider for any institution on the verge of implementing this concept, with emphasis given on database creation, risks, and discipline-specific perspectives.

Glutamate dehydrogenase as a biomarker for mitotoxicity; insights from furosemide hepatotoxicity in the mouse.

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.

Expression of Hematopoietic Stem and Endothelial Cell Markers in Canine Hemangiosarcoma.

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.

Activation of Liver AMPK with PF-06409577 Corrects NAFLD and Lowers Cholesterol in Rodent and Primate Preclinical Models.

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 ß1-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 ß1 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.

Selective Activation of AMPK ß1-Containing Isoforms Improves Kidney Function in a Rat Model of Diabetic Nephropathy.

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 ß1 subunit. After confirming that human and rodent kidney predominately express AMPK ß1, 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.

Liver Microvascular Injury and Thrombocytopenia of Antibody-Calicheamicin Conjugates in Cynomolgus Monkeys-Mechanism and Monitoring.

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.

Evaluation of a novel delayed-type hypersensitivity assay to Candida albicans in adult and neonatal rats.

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.

Development of a fluorescence-based in vivo phagocytosis assay to measure mononuclear phagocyte system function in the rat.

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.

Hemodynamic correlates of drug-induced vascular injury in the rat using high-frequency ultrasound imaging.

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.

Injection-site malignant fibrous histiocytomas in a pegvisomant carcinogenicity study in SD rats.

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.

Study of CRTC2 pharmacology using antisense oligonuceotides.

The cAMP response element binding protein (CREB)-regulated transcriptional coactivator 2 (CRTC2) is a key component of the transcription complex regulating glucagon driven hepatic glucose production and previous evidence suggests that "inhibition" of CRTC2 improves glucose homeostasis in multiple rodent models of type 2 diabetes. Here we describe a process of identifying potential therapeutic antisense oligonucleotides (ASOs) directed against CRTC2. These ASOs were designed as locked nucleic acid (LNA) gapmers and a panel of approximately 400 sequences were first screened in vitro within both human and mouse liver cell lines. A group of active and selective compounds were then profiled in acute studies in mice to determine the level of CRTC2 mRNA reduction in liver as well as to obtain a preliminary indication of safety and tolerability. The compounds with the best activity and safety profiles were then evaluated in subchronic efficacy studies using the diet induced obese (DIO) mouse model of type 2 diabetes and primary human hepatocytes. Efficacy findings broadly confirmed the beneficial effect of reducing CRTC2 mRNA levels towards improving glucose control and other markers of metabolic function. Additionally, for the first time, translation to human cells has been established with demonstration of a reduction in glucagon-mediated glucose production in primary human hepatocytes and a potential clinical biomarker source identified to assess modulation of CRTC2 mRNA following ASO treatment. While the compounds identified herein did not demonstrate a therapeutic index sufficient for further development, this study should facilitate more efficient prosecution of compounds within an in vivo setting.

Immunopathologic characterization of naturally acquired Trypanosoma cruzi infection and cardiac sequalae in cynomolgus macaques (Macaca fascicularis).

Trypanosoma cruzi, the causative agent of Chagas disease, is endemic in south Texas due to the abundant vector and wild small mammalian reservoir populations. This situation predisposes nonhuman primate colonies exposed to outdoor housing to infection from ingestion or bite of triatomid insects. Using a T. cruzi-specific real-time PCR and Trypanosome spp.-specific ELISA, we revealed a prevalence rate of 8.5% in a colony of outdoor-housed cynomolgus macaques. By using a discriminating kinetoplastid minicircle PCR, we eliminated the possibility of mixed prevalence with nonpathogenic trypanosomes and showed the ELISA results were specific for T. cruzi. In this study, we found an inverse relationship between antibody titers and circulating parasite load. Also, 23% of T. cruzi IgG ELISA-positive macaques were negative by real-time PCR. Furthermore, in a subset of infected macaques, cardiac tissue was infiltrated by inflammatory mononuclear cells and contained T. cruzi genomic and kinetoplast DNA despite lacking microscopic evidence of discrete parasite stages. In addition, 19% of the infected macaques had titers for cardiac troponin I autoantibody, which could contribute to autoimmune myocarditis or interfere with circulating troponin I measurements. These findings indicate the possibility of T. cruzi to interfere with the assessment of cardiac safety signals in preclinical toxicology and safety pharmacology studies and the necessity for prestudy screening for T. cruzi in outdoor-housed nonhuman primates from endemic areas.

Chemical modification study of antisense gapmers.

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.

Pharmacological inhibition of Kv1.3 fails to modulate insulin sensitivity in diabetic mice or human insulin-sensitive tissues.

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.

Atypical coccidiosis in South American camelids.

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.

Measurement of articular cartilage stiffness of the femoropatellar, tarsocrural, and metatarsophalangeal joints in horses and comparison with biochemical data.

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.

Diabetes mellitus in a domesticated Spanish mustang.

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.

Acanthomatous ameloblastoma of the maxilla of an adult alpaca.

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.

Fibrochondrogenesis of free intraarticular small intestinal submucosa scaffolds.

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.

Detection and nucleotide sequencing of a DNA-packaging protein gene of equine gammaherpesviruses.

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.