Building a nonclinical pathology laboratory of the future for pharmaceutical research excellence.

We describe a roadmap for a fully digital artificial intelligence (AI)-augmented nonclinical pathology laboratory across three continents. Underpinning the design are Good Laboratory Practice (GLP)-validated laboratory information management systems (LIMS), whole slide-scanners (WSS), image management systems (IMS), and a digital microscope intended for use by the nonclinical pathologist. Digital diagnostics are supported by tools that include AI-based virtual staining and deep learning-based decision support. Implemented during the COVID-19 pandemic, the initial digitized workflow largely mitigated disruption of pivotal nonclinical studies required to support pharmaceutical clinical testing. We believe that this digital transformation of our nonclinical pathology laboratories will promote efficiency and innovation in the future and enhance the quality and speed of drug development decision making.

Necrotizing enterotyphlocolitis in dogs treated with a potent antimuscarinic.

To assess the safety of a potent muscarinic receptor antagonist (antimuscarinic) for human clinical testing, repeat-dose oral toxicity studies were conducted in the Beagle dog. Treatment resulted in unexpected gastrointestinal system effects that were likely mediated by the exaggerated pharmacologic effects of the antimuscarinic on intestinal motility. Dogs developed profound anorexia followed by severe bloody diarrhea, and at necropsy, the intestinal tract was distended, filled with red-tinged fluid, and the mucosa was reddened. Histologically, intestinal lesions consisted of severe epithelial necrosis and mucosal atrophy localized predominantly to the ileum and large intestine. Feces from dogs with diarrhea were culture-positive for Clostridium perfringens (CP), suggesting that CP might be the etiologic agent; however, the intestinal lesions were not consistent with histologic findings reported in dogs with hemorrhagic canine enteritis.

Modulation of allergic inflammation in mice deficient in TNF receptors.

Tumor necrosis factor-alpha (TNF) is implicated as an important proinflammatory cytokine in asthma. We evaluated mice deficient in TNF receptor 1 (TNFR1) and TNFR2 [TNFR(-/-) mice] in a murine model of allergic inflammation and found that TNFR(-/-) mice had comparable or accentuated responses compared with wild-type [TNFR(+/+)] mice. The responses were consistent among multiple end points. Airway responsiveness after methacholine challenge and bronchoalveolar lavage (BAL) fluid leukocyte and eosinophil numbers in TNFR(-/-) mice were equivalent or greater than those observed in TNFR(+/+) mice. Likewise, serum and BAL fluid IgE; lung interleukin (IL)-2, IL-4, and IL-5 levels; and lung histological lesion scores were comparable or greater in TNFR(-/-) mice compared with those in TNFR(+/+) mice. TNFR(+/+) mice chronically treated with anti-murine TNF antibody had BAL fluid leukocyte numbers and lung lesion scores comparable to control antibody-treated mice. These results suggest that, by itself, TNF does not have a critical proinflammatory role in the development of allergic inflammation in this mouse model and that the production of other cytokines associated with allergic disease may compensate for the loss of TNF bioactivity in the TNFR(-/-) mouse.

Utilization of genetically altered animals in the pharmaceutical industry.

The study of transgenic and gene-deleted (knockout) mice provides important insights into the in vivo function and interaction of specific gene products. Within the pharmaceutical industry, genetically altered mice are used predominantly in discovery research to characterize the diverse functions of one or multiple gene products or to establish animal models of human disease for proof-of-concept studies. We recently used genetically altered animals in drug discovery to examine the NF-kappaB family of transcriptional regulatory genes and to elucidate their essential role in the early onset of immune and inflammatory responses. Transgenic and knockout mice are also useful in drug development, because questions regarding risk assessment and carcinogenesis, xenobiotic metabolism, receptor- and ligand-mediated toxicity, and immunotoxicity can be evaluated using these genetically altered mice. For example, the p53 knockout mouse is one of several genetically altered mice whose use may increase the sensitivity and decrease the time and cost of rodent carcinogenicity bioassays. As with any experimental model system, data obtained from genetically altered mice must be interpreted carefully. The complete inactivation of a gene may result in altered expression of related genes or physiologic compensation for the loss of the gene product. Consideration must also be given to the genetic background of the mouse strain and the impact of strain variability on disease or toxicity models. Despite these potential limitations, knockout mice provide a powerful tool for the advancement of drugs in the pharmaceutical industry.

Susceptibility to Pneumocystis carinii in mice is dependent on simultaneous deletion of IFN-gamma and type 1 and 2 TNF receptor genes.

Pneumocystis carinii pneumonia is an important cause of morbidity and mortality in immunosuppressed patients, particularly HIV-infected individuals. An improved understanding of pulmonary host response, including the cytokines required for defense, could suggest novel immunotherapeutic approaches to this infection. The cytokines IFN-gamma and TNF have contributory roles in host defense against P. carinii, but their combined and interactive importance is unclear. To test the roles of these cytokines in defense against P. carinii directly, organisms were inoculated intratracheally into wild-type mice and into three groups of gene-deleted mice: those lacking genes for IFN-gamma (IFN-gamma(-/-)), for TNF receptors 1 and 2 (TNFR(-/-)), and for both IFN-gamma and TNFR (TNFR-IFN-gamma(-/-)). Four weeks after P. carinii inoculation, lungs of the wild-type, IFN-gamma(-/-), and TNFR(-/-) mice demonstrated clearance of P. carinii and only mild inflammation. However, TNFR-IFN-gamma(-/-) mice demonstrated severe P. carinii infection and lung inflammation. Our findings demonstrate conclusively that deletion of either IFN-gamma or TNF activity alone does not block clearance of P. carinii. However, simultaneous deletion of IFN-gamma and TNF receptor genes results in susceptibility to P. carinii. Rather than focusing exclusively on individual cytokines, our data suggest that immunotherapy targeted at maximizing both the IFN-gamma and TNF responses to P. carinii may be required to augment host defense against this important opportunistic pathogen.

Pulmonary and mediastinal metastases of a vaccination-site sarcoma in a cat.

Sarcomas at vaccination sites in cats were first reported in 1992. Recent retrospective studies have confirmed an association between these vaccination-site sarcomas (VSS) and feline leukemia virus (FeLV) and/ or rabies vaccines. In most cases, VSS are locally invasive fibrosarcomas that tend to recur but rarely metastasize. We report the mediastinal and pulmonary metastases of a VSS in a FeLV-and feline immunodeficiency virus-negative, 8-year-old, domestic short-haired cat. The primary sarcoma was removed from an interscapular vaccination site and diagnosed as a VSS 3 months prior to radiographic lesions suggestive of pulmonary and mediastinal metastases. At necropsy, there were multiple pulmonary and mediastinal nodules that histologically and ultrastructurally were fibrosarcomas, cytomorphologically similar to the VSS. In addition, immunohistochemical staining patterns of the VSS and metastatic sites were consistent with that described for VSS. Recent reports of pulmonary and mediastinal metastases of interscapular VSS emphasize the importance of early diagnosis and treatment of these tumors.

Baylisascaris procyonis larva migrans in a puppy: a case report and update for the veterinarian.

Baylisascaris larva migrans (LM) has been recognized as a cause of central nervous system (CNS) disease in puppies. A presumptive antemortem diagnosis is based on a history of raccoon exposure, clinical signs, cerebrospinal fluid eosinophilic pleocytosis, and peripheral blood eosinophilia. Early diagnosis is critical for treatment or prevention of disease in other dogs, animals, or humans exposed to the suspected contaminated area. In the present case, an antemortem diagnosis was not made, emphasizing the importance of postmortem examination in cases of CNS disease in puppies.

Evaluation of risk factors for blastomycosis in dogs: 857 cases (1980-1990)

An epidemiologic study was conducted by use of the Veterinary Medical Data Base to investigate risk factors for blastomycosis in dogs. From January 1980 through June 1990, 971 cases of blastomycosis in dogs from 22 North American veterinary teaching hospitals were identified. Of these cases, 114 (11.7%) were excluded from the study because of incomplete information regarding age, body weight, sex, and neuter status. A control group of 417,079 dogs was selected that included all other dogs with medical conditions unrelated to blastomycosis for which records were submitted to the data base during the same period. The prevalence of blastomycosis in dogs was 205/100,000 admissions during the study period. When veterinary teaching hospitals were grouped on the basis of their general geographic location, dogs in the East South central, East North central, West South central, and South Atlantic regions had a significantly (P < 0.05) increased risk of acquiring blastomycosis, compared with that of dogs in the Mountain/Pacific region. When teaching hospitals from all geographic regions were considered, dogs had a significantly (P < 0.05) increased risk of acquiring blastomycosis in autumn, compared with that in spring. Sporting dogs and hounds, as defined by the American Kennel Club, were at increased risk for blastomycosis. At highest risk were Bluetick Coonhounds, Treeing-walker Coonhounds, Pointers, and Weimaraners, compared with mixed-breed dogs. Ages of dogs with blastomycosis tended to be normally distributed. Generally, the highest-risk group was composed of sexually intact male dogs, 2 to 4 years old, weighing 22.7 to 34.1 kg. This same pattern was observed for sporting dogs and hounds.(ABSTRACT TRUNCATED AT 250 WORDS)

Ultrastructural study of Helicobacter pylori adherence properties in gnotobiotic piglets.

Ultrastructural examination of gastric mucosa from Helicobacter pylori-infected gnotobiotic piglets identified four general adherence patterns comparable to those observed in human patients. Intimate associations between the bacterial and mucosal cell membranes, including cuplike invaginations and adherence pedestals, were present and were accompanied by alterations to microvilli and cell membrane morphology.