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Recommendations (best practices) are provided by the Society of Toxicologic Pathology’s Adversity Working Group for making consistent interpretations of test article–related effects as “adverse” and assigning a “no observed adverse effect level” (NOAEL) in nonclinical toxicity studies. Adverse is a term indicating “harm” to the test animal, while nonadverse indicates lack of harm. Adverse findings in the study reports should be defined in relation to effects on the test species used and within the context of the given study. Test article–related effects should be described on their own merits, and decisions to consider them as adverse or nonadverse should be justified. Related effects may be discussed together; in particular, markers of toxicity that are not in and of themselves adverse ideally should be discussed in conjunction with the causal toxicity to determine adversity. Adverse findings should be identified in subreports (clinical data, pathology data, etc.) if sufficient information is available, and/or in the final study report as individual or grouped findings, but study NOAELs should be established at the level of the overall study report. Interpretations such as “not biologically relevant” or “not toxicologically important” should be avoided unless defined and supported by scientific rationale. Decisions defining adverse findings and the NOAEL in final study reports should combine the expertise of all contributing scientific disciplines. Where possible, use of NOAELs in data tables should be linked to explanatory text that places them in context. Ideally, in nonclinical summary documents, NOAELs from multiple studies are considered together in defining the most important adverse responses in the most sensitive species. These responses are then considered along with an understanding of their likely mechanisms, as well as other information such as variability in species sensitivity, comparative pathology, reversibility and progression, kinetics, and metabolism of the test substance to help assess human risk.

One of the principal challenges facing a toxicologic pathologist is to determine and differentiate a true adverse effect from a nonadverse or an adaptive response. Recent publications from the Society of Toxicologic Pathology (STP) and the European STP provide guidance for determining and communicating adversity in nonclinical toxicology studies. In order to provide a forum to inform and engage in a discussion on this important topic, a continuing education (CE) course was held during the 2016 STP Annual meeting in San Diego, CA. The lectures at this course provided guidance on determining and communicating adversity using case studies involving both clinical pathology and anatomic pathology. In addition, one talk also focused on data quality, study design, and interpretation of artifacts that could hinder the determination of adversity. The CE course ended with a talk on understanding adversity in preclinical studies and engaging the regulatory agencies in the decision-making process. This manuscript is designed to provide brief summaries of all the talks in this well-received CE course.

Peroxisome proliferator-activated receptors (PPARs) represent therapeutic targets for the management of type 2 diabetes mellitus and dyslipidemia. Rodent carcinogenicity studies have revealed a link between γ and dual γ/α PPAR agonist treatment and the increased incidence of subcutaneous (SC) liposarcomas/fibrosarcomas or hemangiosarcomas, but very little has been reported for potent and selective PPARα agonists. We present a mode of action framework for the development of SC mesenchymal tumors in rodents given PPAR agonists. (1) Tumor promotion results from pharmacologically mediated recruitment (proliferation and differentiation), thermogenesis and adipogenesis of stromovascular cells, and subsequent generation of oxidative free radicals. (2) Tumor initiation consists of chemotype-driven mitochondrial dysfunction causing uncontrolled oxidative stress and permanent DNA damage. Promotion is characterized by enhanced adipogenesis in the SC adipose tissue, where the baseline PPARγ expression and responsiveness to PPARγ ligands is the highest, and by thermogenesis through expression of the uncoupling protein 1 (UCP-1) and the PPARγ co-activator 1 α (PGC-1α), two factors more highly expressed in brown versus white adipose tissue. Initiation is supported by the demonstration of mitochondrial uncoupling and OXPHOS Complexes dysfunction (Complexes III, IV and V) by compounds associated with increased incidences of sarcomas (muraglitazar and troglitazone), but not others lacking malignant tumor effects (pioglitazone, rosiglitazone).

CHARLOTTE KEENAN, SUSAN ELMORE, SABINE FRANCKE-CARROLL, RAMON KEMP, ROY KERLIN, SHYAMAL PEDDADA, JOHN PLETCHER, MATTHIAS RINKE, STEPHEN PETER SCHMIDT, IAN TAYLOR, AND DOUGLAS C. WOLF GlaxoSmithKline, King of Prussia, Pennsylvania, USA National Toxicology Program (NTP), National Institute of Environmental Health Sciences (NIEHS), Research Triangle Park, North Carolina, USA Center for Food Safety and Applied Nutrition (CFSAN), U.S. Food and Drug Administration, College Park, Maryland, USA Merck Research Laboratories, Riom, France Pfizer Inc., Groton, Connecticut, USA NIEHS, Research Triangle Park, North Carolina, USA Charles River, Frederick, Maryland, USA Bayer Schering Pharma AG, Wuppertal, Germany Huntingdon Life Sciences, Eye, United Kingdom U.S. Environmental Protection Agency, Research Triangle Park, North Carolina, USA

This study was conducted to characterize spontaneous testicular and epididymal microscopic findings in eighty control beagle dogs from toxicity studies. Hypospermatogenesis, characterized by randomly scattered missing spermatids and/or spermatocytes within seminiferous tubules, was observed in 75% of dogs six to seven months of age and declined to fewer than 10% in dogs over eleven months of age. Atrophy/hypoplasia of seminiferous tubules, characterized by subcapsular triangular clusters of tubules containing no germ cells, was observed in 25 to 40% of dogs under twelve months old, decreasing with age to 14 to 17% in dogs twelve to thirty-six months old. Retained spermatids, multinucleate giant cells, intracytoplasmic vacuoles (presumably in Sertoli cells), and swollen spermatocytes were common findings of minimal severity. Six- and seven-month-old dogs had lower testicular weights, less filling of the epididymal tails with sperm, and a two-fold higher incidence of abnormal epididymal content compared to dogs more than eight months of age. Most male beagles were histologically sexually mature by eight to nine months of age. This study confirms published reports that dogs at least ten months of age at necropsy usually are adequate for routine microscopic evaluation of the testes. If evaluation of spermatogenesis is critical, the incidental findings can be minimized by using males over twelve months of age.

Two young female Beagle dogs in a laboratory colony with clinical signs of loose stools and fecal blood were confirmed to have histiocytic ulcerative colitis by histologic evaluation. This syndrome is well recognized in other dog breeds such as Boxers and related French Bulldogs, Mastiffs, Alaskan malamutes and Doberman Pinschers. Formalin-fixed paraffin sections of large intestine from one dog demonstrated the presence of Escherichia coli strain LF82 by immunohistochemistry and 16S ribosomal RNA gene sequencing. E coli strain LF82 has been implicated in the pathogenesis of Crohn's disease and similar bacteria have been cultured from cases of histiocytic ulcerative colitis in Boxer dogs. Spontaneous histiocytic ulcerative colitis must be differentiated from test article-related findings in nonclinical toxicity studies in Beagle dogs.

Laser scanning cytometry (LSC) is a new technology that combines the properties and advantages of flow cytometry (FC) and immunohistochemistry (IHC), thus providing qualitative and quantitative information on protein expression with the additional perspective provided by cell and tissue localization. Formalin-fixed, paraffin embedded liver sections from rats exposed to a Peroxisome Proliferator Activated Receptor (PPAR) agonist were stained with antibodies against peroxisomal targeting signal-1 (PTS-1) (a highly conserved tripeptide contained within all peroxisomal enzymes), Acyl CoA oxidase (AOX) (the rate limiting enzyme of peroxisomal β oxidation), and catalase (an inducible peroxisomal antioxidant enzyme) to evaluate peroxisomal β oxidation, oxidative stress, and peroxisome proliferation. The LSC showed increased AOX, catalase, and PTS-1 expression in centrilobular hepatocytes that correlated favorably with the microscopic observation of centrilobular hepatocellular hypertrophy and with the palmitoyl CoA biochemical assay for peroxisomal β oxidation, and provided additional morphologic information about peroxisome proliferation and tissue patterns of activation. Therefore, the LSC provides qualitative and quantitative evaluation of peroxisome activity with similar sensitivity but higher throughput than the traditional biochemical methods. The additional benefits of the LSC include the direct correlation between histopathologic observations and peroxisomal alterations and the potential utilization of archived formalin-fixed tissues from a variety of organs and species.

Post-mortem pathology data from nonclinical laboratory studies contain critical information for characterizing a new drug's safety and are pivotal for gaining an appreciation of potential hazards to humans. An understanding of the historical regulatory and scientific perspective on the nature of pathology data is valuable for assuring appropriate and useful audits of pathology material within nonclinical studies. This discussion reviews the specialty of pathology, the process of medical diagnosis, the definitions of ‘raw data’ as they pertain to histopathology data, and the rationale behind the consensus decisions made by industry and the Food and Drug Administration (FDA) in interpreting good laboratory practice (GLP) regulations for this type of data. Copyright © 2004 John Wiley & Sons, Ltd.

This paper investigates the role of specific immune factors on the course of infection in genetic knockout (gko) mice infected with 3 different strains of Neospora caninum. Survival time and parasite persistence were examined in interferon-g (IFN- g), tumor necrosis factor receptor-2 (TNFR2), interleukin 10 (IL-10), beta 2 microglobulin ( b2M), and inducible nitric oxide synthase (iNOS2) gko or wild-type (wt) mice following infection with either pathogenic (NC-1 or NC-2) or attenuated (NCts-8) N. caninum strains. Infection with NC-1 was 100% lethal in IFN-g gko mice, as evidenced by mean survival times of 10-13 days, depending on the challenge dose used. TNFR2 and b2M gko mice infected with NC-1 or NC-2 strain demonstrated partial susceptibility to disease, as evidenced by histopathology and survival curves. TNFR2 or b2M gko mice were not susceptible to infection with NCts-8, on the basis of absence of pathology and lack of mortality. Lack of mortality and minimal histopathology scores demonstrated that NC-1, NC-2, and NCts-8 infections were avirulent in IL-10 and iNOS2 gko mice. Adoptive transfer of immune cells from NCts-8 vaccinated normal syngeneic mice into IFN-g gko mice significantly (P , 0.05) prolonged mean survival times at all 3 challenge doses of NC-1 but failed to protect against mortality. Interestingly, there was a notable change in the tissue tropism of tachyzoites from the lung and brain in immunocompetent wt, TNFR2 gko, IL-10 gko, b2M gko, and iNOS2 gko mice to the liver and spleen in IFN-g gko mice when challenged with N. caninum. On the basis of these results in gko mice, IFN-g is a critical cytokine in the host response against acute neosporosis. Neospora caninumis an apicomplexan parasite that causes a variety of symptoms in several mammalian species. The largest economic cost caused by this parasite is because of abortions in dairy and beef cows. If abortions do not occur, infected off- spring can be born weak or lame or both. Asymptomatic, se- ropositive, replacement calves can lead to maintenance of N. caninum infection in these dairy and beef herds. Because of the close taxonomic relationship between Toxo- plasma gondii and N. caninum, it can be postulated that the same immune cells and soluble mediators important for protec- tion in T. gondii infections (Lee et al., 1999) may play a similar role in host protection against N. caninum. For example, T. gondii infections in interferon

Cystic degeneration/spongiosis hepatis in rats has been proposed to be a preneoplastic and/or neoplastic lesion by some authors, because of its proliferative properties and persistent increased cell turnover rate in stop experiments using hepatocarcinogens , and the assumption that it can develop into a sarcoma. The neoplastic potential of cystic degeneration is questioned in this review article. Cystic degeneration, which appears to derive from altered Ito cells, does not have neoplastic histomorphologi c characteristics, although it may be composed of cells with an increased mitotic index. In this regard, persistent proliferation is also seen with other nonneoplastic lesions. Arguments are presented to show that the induced, probably extremely rare sarcoma that was associated with cystic degeneration most likely derives from the very rare induced spherical Ito-cell aggregate with an unusually high cellular turnover rate in rats treated with hepatocarcinogens , and not from cystic degeneration. Also, in none of 12 referenced standard oncogenicity studies with chemically induced cystic degeneration was the lesion associated with mesenchymal (Ito-cell) tumors. Consequently, evidence is lacking that cystic degeneration in rats should be classifi ed as a preneoplastic or neoplastic lesion. The 12 oncogenicity studies in rats with induced cystic degeneration showed a marked sex predilection, with males more likely to develop either spontaneous or chemically induced lesions. In these 12 studies, cystic degeneration was more often associated with hepatocellular hypertrophy or hepatotoxicity, rather than hepatocarcinogenicity. Thus, it is concluded that hepatocarcinogens induce cystic degeneration, not because they are carcinogenic, but because they have other effects on the liver, and that cystic degeneration may be a secondary/reparative change. Cystic degeneration in fi sh parallels the situation in rats in many respects, yet the existence of the lesion in other species, including man, is not as well supported. Based on the data presented in this review, spontaneous and induced cystic degeneration in rats and fi sh is not a preneoplastic or neoplastic lesion and risk assessment for man can be based on no-effect levels and safety margins, as for other nonneoplasti c adverse effects that have no counterpart in man.

The process of drug discovery and development is highly complex, demanding extraordinary scientific acumen as well as an understanding of the regulatory milieu. Veterinary pathologists in industry find themselves at the vanguard of this process, helping companies to bring innovative new therapies to patients in need. In this chapter we focus on the role of the toxicologic pathologist in understanding both the nature of drug efficacy, as well as the pathogenic mechanisms of drug toxicity, and the way in which both perspectives play into decisions to either develop or halt development of a potentially promising drug candidate. We review the role of the toxicologic pathologist in discovering and developing small molecules as well as some of the extraordinary variety of biotherapeutic agents, with their inherent challenges and often unexplored potentialities. Other issues such as reversibility or delayed toxicity, imaging, GLP regulations, and the use of some advanced scientific tools used by toxicologic pathologists developing pharmaceutical agents are discussed. In progressive companies, as well as regulatory agencies, toxicologic pathologists bring their expertise to bear across the entire drug discovery and development pipeline to improve our understanding of the advantages and potential liabilities of new therapies.

Weak peroxisome proliferator–activated receptor (PPAR) α agonists (fibrates) are used to treat dyslipidemia. This study compared the effects of the potent and selective PPARα agonist CP-778875 on peroxisomal β-oxidation and cardiac and/or skeletal muscle injury with those of the weak PPARα agonist fenofibrate. We hypothesized that these muscle effects are mediated through the PPARα receptor, leading to increased β-oxidation and consequent oxidative stress. CP-778875 (5 or 500 mg/kg) and fenofibrate (600 or 2,000→1,200 mg/kg, dose lowered because of intolerance) were administered to rats for six weeks. Standard end points, serum troponin I, heart and skeletal muscle β-oxidation of palmitoyl-CoA, and acyl co-oxidase (AOX) mRNA were assessed. Both compounds dose-dependently increased the incidence and/or severity of cardiomyocyte degeneration and necrosis, heart weight, troponin I, and skeletal muscle degeneration. Mean heart β-oxidation (3.4- to 5.1-fold control) and AOX mRNA (2.4- to 3.2-fold control) were increased with CP-778875 500 mg/kg and both doses of fenofibrate. β-Oxidation of skeletal muscle was not affected by either compound; however, a significant increase in AOX mRNA (1.6- to 2.1-fold control) was observed with CP-778875 500 mg/kg and both doses of fenofibrate. Taken together, these findings were consistent with PPARα agonism and support the link between increased cardiac and skeletal muscle β-oxidation and resultant muscle injury in the rat.

Summary Secretory/excretory products (sec/ex) of parasitic larvae of the sheep blowfly Lucilia cuprina potently inhibited proliferation of peripheral blood leucocytes stimulated by mitogens in vitro. Suppression of proliferation was not due to irreversible damage because cells cultured for 24 h in high concentrations of sec/ex appeared viable (assessed by Trypan blue exclusion) and did not show impaired proliferation after washing. Furthermore, suppression induced by sec/ex could be overcome by increasing concentrations of mitogen. The inhibitory activity could be demonstrated in cultures where sec/ex was added at different times during the culture period. Inhibitory activities in sec/ex were heat-labile and sensitive to treatment with trypsin. In addition to effects in vitro, sec/ex was strongly immunosuppressive in vivo. Sheep given combined injections of myoglo-bin and sec/ex had markedly lower anti-myoglobin antibody levels in sera than sheep that received injections of myoglobin alone. There was no significant antibody response to sec/ex itself. Immunosuppressive moieties in sec/ex produced by blowfly larvae may promote parasite survival by inhibiting the immune response of host sheep.

CHARLOTTE KEENAN (CHAIR), SUSAN ELMORE, SABINE FRANCKE-CARROLL, ROY KERLIN, SHYAMAL PEDDADA, JOHN PLETCHER, MATTHIAS RINKE, STEPHEN PETER SCHMIDT, IAN TAYLOR, AND DOUGLAS C. WOLF GlaxoSmithKline, King of Prussia, Pennsylvania 19406, USA National Toxicology Program, National Institute of Environmental Health Sciences, Research Triangle Park, North Carolina 27709, USA Center for Food Safety and Applied Nutrition, U.S. Food and Drug Administration, College Park, Maryland 20740, USA Pfizer Inc., Groton, Connecticut 06340, USA National Institute of Environmental Health Sciences, Research Triangle Park, North Carolina 27709, USA Charles River, Frederick, Maryland 21701, USA Bayer Schering Pharma AG, Wuppertal, Germany Huntingdon Life Sciences, Eye, Suffolk IP23 7PX, UK U.S. Environmental Protection Agency, Research Triangle Park, North Carolina 27711, USA

Zoniporide, an inhibitor of the Na+-H+ exchanger-1, was administered by continuous intravenous infusion to rats and dogs for up to 1 month. In 1-month studies, histological and functional changes were observed in select portions of the peripheral nervous system; however, these findings were not detected in 2-week studies at similar or higher doses. In the 1-month rat study, there was dose-dependent, minimal, focal, or multifocal nerve fiber (axonal) degeneration in the spinal cord and/or sciatic nerve. In a follow-up rat study, findings included slowing of caudal nerve conduction velocity and axonal degeneration in the spinal cord (dorsal funiculus), dorsal roots, dorsal root ganglia (DRG), radial, sciatic, and tibial nerves. In the 1-month dog study, there was impairment of the patellar reflex and associated postural reaction changes, minimal to marked proximal nerve fiber degeneration in the DRG, and minimal nerve fiber degeneration in the dorsal roots and funiculi of the spinal cord. Minimal nerve fiber degeneration of equivocal significance was noted in various peripheral nerves. Taken together, these findings were consistent with a specific effect on peripheral sensory nerve fibers. These studies demonstrated that zoniporide produces clinical, electrophysiologic, and microscopic evidence of peripheral sensory axonopathy and establishes the importance of careful preclinical evaluation of neurological function.

Peroxisomal proliferator-activated receptor (PPAR)-alpha is a ligand-activated transcriptional factor that regulates genes involved in lipid metabolism and energy homeostasis. PPAR-alpha activators, including fibrates, have been used to treat dyslipidemia for several decades. In contrast to their known effects on lipids, the pharmacological consequences of PPAR-alpha activation on cardiac metabolism and function are not well understood. Therefore, we evaluated the role that PPAR-alpha receptors play in the heart. Our studies demonstrate that activation of PPAR-alpha receptors using a selective PPAR-alpha ligand results in cardiomyocyte necrosis in mice. Studies in PPAR-alpha-deficient mice demonstrated that cardiomyocyte necrosis is a consequence of the activation of PPAR-alpha receptors. Cardiac fatty acyl-CoA oxidase mRNA levels increased at doses in which cardiac damage was observed and temporally preceded cardiomyocyte degeneration, suggesting that peroxisomal beta-oxidation correlates with the appearance of microscopic injury and cardiac injury biomarkers. Increased myocardial oxidative stress was evident in mice treated with the PPAR-alpha agonists coinciding with increased peroxisomal biomarkers of fatty acid oxidation. These findings suggest that activation of PPAR-alpha leads to increased cardiac fatty acid oxidation and subsequent accumulation of oxidative stress intermediates resulting in cardiomyocyte necrosis.

Cystic degeneration/spongiosis hepatis in rats has been proposed to be a preneoplastic and/or neoplastic lesion by some authors, because of its proliferative properties and persistent increased cell turnover rate in stop experiments using hepatocarcinogens, and the assumption that it can develop into a sarcoma. The neoplastic potential of cystic degeneration is questioned in this review article. Cystic degeneration, which appears to derive from altered Ito cells, does not have neoplastic histomorphologic characteristics, although it may be composed of cells with an increased mitotic index. In this regard, persistent proliferation is also seen with other nonneoplastic lesions. Arguments are presented to show that the induced, probably extremely rare sarcoma that was associated with cystic degeneration most likely derives from the very rare induced spherical Ito-cell aggregate with an unusually high cellular turnover rate in rats treated with hepatocarcinogens, and not from cystic degeneration. Also, in none of 12 referenced standard oncogenicity studies with chemically induced cystic degeneration was the lesion associated with mesenchymal (Ito-cell) tumors. Consequently, evidence is lacking that cystic degeneration in rats should be classified as a preneoplastic or neoplastic lesion. The 12 oncogenicity studies in rats with induced cystic degeneration showed a marked sex predilection, with males more likely to develop either spontaneous or chemically induced lesions. In these 12 studies, cystic degeneration was more often associated with hepatocellular hypertrophy or hepatotoxicity. rather than hepatocarcinogenicity. Thus, it is concluded that hepatocarcinogens induce cystic degeneration, not because they are carcinogenic. but because they have other effects on the liver, and that cystic degeneration may be a secondary/reparative change. Cystic degeneration in fish parallels the situation in rats in many respects, yet the existence of the lesion in other species, including man, is not as well supported. Based on the data presented in this review, spontaneous and induced cystic degeneration in rats and fish is not a preneoplastic or neoplastic lesion and risk assessment for man can be based on no-effect levels and safety margins, as for other nonneoplastic adverse effects that have no counterpart in man.

The in vivo persistence, immunogenicity and pathogenicity of a recently described temperature-sensitive (ts) strain from Neospora caninum, NCts-8, was investigated in normal and immunodeficient mice. Groups of BALB/c and SCID/Bg mice were infected s.c. with 5 x 10(6) wild-type NC-1, control NCts-8 (pass 0) or NCts-8 tachyzoites prepared at four in vitro passage levels (pass 7, 13, 21 and 28). For persistence and immunogenicity studies, BALB/c mice were bled and sacrificed at 4, 6 or 8 weeks p.i. Sera were analysed by IFAT and brain tissues examined for lesions by histology and tested for parasite presence by PCR. For pathogenicity studies, SCID/Bg mice were monitored by clinical signs and survival time. Results from parasite persistence experiments demonstrated microscopic lesions and PCR positive brain tissues in NC-1 infected mice. In contrast, brain tissues from NCts8-infected groups were consistently negative by histology and PCR. Based on IFAT titres, all parasite strains were immunogenic, although parasite-specific IgG levels were lower in the NCts-8 infected groups. Results from pathogenicity studies in SCID/Bg mice demonstrated a significantly (P < 0.0001) longer mean survival time in NCts-8 vs NC-1 infected groups. In addition, there was no significant difference in mean survival time between control NCts-8 and experimental passage NCts-8 infected mice. Collectively, these studies demonstrate that the NCts-8 strain maintains a stable phenotype following multiple passages in vitro, and possesses an attenuated, shorter persistence phenotype in vivo compared with the parental wild-type NC-1.

Simian retrovirus (SRV) type D is a common cause of simian acquired immunodeficiency syndrome (SAIDS), a usually fatal immunosuppressive disease of macaques. Associated gross and histologic lesions have been well described for the rhesus macaque (Macaca mulatta) in experimental and natural infections. However, morphologic changes induced by this virus at the gross and light-microscopic level have not been documented in the cynomolgus macaque (Macaca fascicularis). In 1996, sporadic cases of anemia, weight loss, and diarrhea were noted in a colony of cynomolgus macaques in our research facility. Out of 28 animals, 24 tested positive for SRV by serology or virus isolation. Animals could mainly be classified into 1 of 2 categories: 1) positive for virus isolation but negative for SRV antibody and 2) negative for virus isolation but antibody positive. During the process of eliminating the virus from the colony, a complete postmortem examination was performed on the 24 infected animals that had to be culled. Twelve SRVnegative animals were available as controls. Minimal to mild follicular lymphoid infiltrates were seen in various organ systems in 75% of the negative animals, compared with moderate to marked infiltrates in 83% of infected animals. Lymphoid infiltrates were more common in the brain, bone marrow, and salivary gland of viremic animals and were rare to nonexistent in seropositive or negative animals. Lymphoid hyperplasia was present in 38% of the infected animals, whereas lymphoid depletion was seen in 47% of the infected animals. Overall, lesions were of greater severity in viremic animals than in virus-negative or seropositive animals. Overall, infected animals had lower, statistically significant hematocrit and lymphocyte values. Viremic animals had significantly lower hematocrit, white blood cell, lymphocyte, and neutrophil values than did controls. Only 1 out of 24 infected animals had clinical signs that were consistent with the definition of SAIDS, and none had evidence of opportunistic infections. Lesions were similar to those already reported in other species of macaques, but the absence of severe illness that was consistent with SAIDS in most viremic animals suggests that there may be a different manifestation of disease in the cynomolgus.

Most nonclinical safety studies performed to satisfy regulatory requirements include numerous anatomic and clinical pathology endpoints. Consequently, the traditional predominant role of the pathologist in the pharmaceutical industry has focused upon the interpretation of histologic changes in tissues and of clinicopathologic changes in blood from animals that had been exposed to new chemical entities. In addition to this conventional role, pathologists are increasingly being recognized for their expanded contributions to drug discovery and development. The medical training, specialty or residency training, and comparative medicine perspective place the industrial pathologist in a unique position to contribute broadly to the objectives of drug discovery and development programs. In addition, the technologies in the pathologist’s repertoire have broad application and utility to many areas of biomedical research in the pharmaceutical industry. Such objectives and areas of research include characterization of ! disease states; identification of modified disease states as a response to administration of potential therapies; characterization and quantification of toxic responses to drug administration ; delineation of mechanisms of toxicities and potential relevance to target species; and facilitation of multidisciplinary efforts to monitor for the clinical occurrence, progression, and reversibility of adverse events. The business and science of pharmaceutical research and development is increasingly complex, and the pressures associated with them create competitive advantages for companies that are able to deploy resources in novel and beneficial ways. Use of pathologists to support drug discovery represents one such approach.

An 8-year-old female spayed Cocker Spaniel mix breed dog was presented with generalized erythroderma, scaling and alopecia. Radiographs of the thorax demonstrated a discrete lung mass which was aspirated using ultrasound guidance and cytological analysis revealed large abnormal lymphocytes. Similar cells were observed in the peripheral blood and in skin biopsies. The cells in the skin biopsies were epidermotropic, indicative of an uncommon cutaneous lymphoma termed cutaneous T cell lymphoma (CTCL), sometimes also called mycosis fungoides. Immunohistochemical staining of a skin biopsy was positive for the CD3 antigen demonstrating that the lymphocyte infiltrate was of a T-cell lineage. The presence of neoplastic lymphocytes in the epidermis and peripheral blood indicate that this is a rare variant of Cutaneous Epidermotropic Lymphoma (CEL) called Sezary syndrome based on nomenclature used in the human literature. An unusual feature of this dog, not seen in previous cases, was the presence of a discrete neoplastic lung mass.

Sea star factor (SSF), a protein of 39 kDa purified from macrophage-like coelomocytes of the echinoderm Asterias forbesi , has potent immunosuppressive effects on T-dependent but not T-independent antibody responses in vivo . SSF at a concentration of 0.5 μg/ml markedly inhibits T-dependent antibody production in vitro by fluorescein (Flu)-specific B cells responding in clonal microculture to antigenic stimulation with Flu-conalbumin via the conalbumin-specific T cells D10.G4.1 (D10). At this concentration of SSF, Ig secretion induced by a T cell-independent stimulus, lipopolysaccharide (LPS), is not affected. Inhibition of antibody production in T-dependent microcultures by SSF can be completely overcome in a dose-dependent fashion by addition of lymphokine-rich supernatants from stimulated cultures of D10 cells. The possibility that SSF suppresses production of requisite cytokine growth factors from T cells was substantiated by the finding that SSF diminishes concentrations of stimulatory cytokines detectable in supernatants from antigen-stimulated cultures. Nevertheless, levels of intracytoplasmic mRNA for IL-4 and IL-5 are not detectably altered by concentrations of SSF that suppress antibody production. Furthermore, when cultures of D10 cells stimulated in the presence of SSF are subjected to freezing and thawing to release intracytoplasmic lymphokines, total levels of stimulatory cytokines are not lower than those in cultures without SSF. These results suggest that SSF inhibits antibody responses by limiting the availability of lymphokines produced by helper T cells. The mechanism for this inhibition may involve either direct effects of SSF on T cells or a block in effective T cell-B cell interaction.

Histamine receptors on bovine peripheral blood lymphocytes (PBL) were detected by three different methods: a rosetting technique, binding to histamine-bearing Sepharose beads and immunofluorescence staining. The rosetting technique used histamine-rabbit serum albumin (H-RSA) conjugated to bovine red blood cells to detect histamine receptors and this showed that 10.8% of bovine PBL were positive. A method using H-RSA conjugate coupled Sepharose beads also detected histamine receptor bearing PBL but was not quantitative. The indirect immunofluorescence method, by which the subpopulation of histamine receptor bearing lymphocytes can be easily double stained to concurrently identify the B cell marker, revealed that PBL, the B cell and T cell fraction of bovine PBL contained 18.4, 52.8 and 9.3% histamine receptor bearing cells, respectively. This method was found to be more stable and more easily quantifiable than the other two methods. Blocking tests using the histamine H1 receptor antagonist diphenhydramine and the histamine H2 receptor antagonist cimetidine suggested that bovine PBL have both H1 and H2 receptors on their surfaces. Addition of histamine into cultures of PBL at the concentration range 10(-6) to 10(-3) M suppressed the response of PBL to the mitogen phytohemagglutinin. The histamine induced suppression of mitogenesis could be reduced partially by the H2 receptor antagonist cimetidine, but not by the H1 antagonist diphenhydramine. It is possible that histamine induced suppression of PBL mitogenesis was mediated by H2 receptors on T cells.

For many infectious diseases of ruminants vaccination is a feasible and desirable alternative to therapy. Vaccines that result in elevated herd immunity and disease prevention have important advantages in terms of cost effectiveness, animal welfare, and reduction of the environmental contamination that occurs when animals are treated with drugs and chemicals. Currently on the market are many efficacious and successful vaccines for ruminant diseases, but there are very few vaccines available for diseases of the mammary gland and skin. Undoubtedly the diseases that are easiest to control by vaccination have already been conquered; it is equally true that at the time such vaccines were developed the process was not easy! In the 1990s we have at our disposal new technologies such as molecular engineering and peptide synthesis. These technologies have dramatically improved our ability to synthesize usable quantities of antigens, yet many economically important diseases of the skin and mammary gland remain intractable. In this chapter we review progress that has been made with vaccines against such diseases and try to identify areas that may be profitable for future research endeavours.

Many important diseases of domestic animals have been rendered impotent by the development of safe and effective vaccines to prevent or ameliorate infection. Up to now, veterinary vaccines consisted mainly of killed or attenuated whole organisms. Vaccines of this type that are registered for use in the state of Queensland, Australia are listed in Table 1.1 (63,161). While not including many of the vaccines found in other countries, the list is indicative of current vaccine technologies. Vaccines such as these, which mostly contain whole pathogens, are usually highly immunogenic and utilize either no adjuvant or a simple adjuvant, such as alum or oil, to prolong the effect of the vaccine. The term adjuvant was first defined by Ramon (117) in 1925 as a substance that, when used in combination with antigen, enhanced levels of immunity beyond those developed with the antigen alone. A synopsis of the adjuvants currently available for veterinary vaccines was recently published by Vanselow (152).

The number and frequency of spontaneous and cytokine-inducible 'natural' immunoglobulin-secreting cells (ISC) were determined in bone marrow (BM), spleen and Peyer's patch (PP), in vitro. Cells were cultured at limiting dilution in the presence or absence of exogenous recombinant cytokines and supernatants then assayed for total immunoglobulin (Ig) and Ig isotype using an ELISA. Most spontaneous ISC were found in the spleen and BM, with fewer in PP. The addition of recombinant interleukin 5 (rIL-5) promoted a marked increase in both the ISC frequency and the amount of Ig secreted/ISC whereas recombinant IL-6 (rIL-6) promoted only a marginal increase. Recombinant IL-4 (rIL-4) promoted a marginal increase in ISC frequency only. The isotype profile of ISC was in the order IgM greater than IgG2 greater than IgA greater than IgG3 greater than IgG1. The exposure of cells to 1200 rad of gamma-radiation resulted in decreased numbers of spontaneous ISC in all tissues, but the addition of rIL-5 or rIL-6 to the irradiated cells increased both the ISC frequency and Ig secreted. The Ig isotype profile was similar to that of non-irradiated ISC with a few minor exceptions. This large population of potential cytokine-inducible ISC could contribute to 'natural' Ig secretion in vivo.

Microcultures of helper T (Th) cells and a few appropriately primed murine B cells can be used to detect cognate T-B interactions which lead to clonal production of IgM, IgG1, and IgE. However, IgG2, IgG3, and IgA are very rarely expressed. We have found that the addition of dendritic cells to such cultures creates an extremely supportive environment for clones expressing IgA with other isotypes, as well as clones expressing only detectable IgA. Typically, 400 dendritic cells were added to 3000 conalbumin-specific Th cells (D10.G4.1) and 30 hapten-specific Peyer's patch (PP) B cells with antigen in 15 microliters. The response was antigen dependent and clonal. Almost half of the clones expressed only non-IgM isotypes, 43% expressed some IgA, and 14% expressed some IgG3; isotype diversity increased over time. Dendritic cells from PP and spleen were found to be equally supportive, and allowed the number of T cells required in microculture to be decreased from 3000 to 400. However, T cell proliferation was not required for the supportive effect of dendritic cells. Surface IgD-bearing cells were also found to switch to IgA production in microculture as judged by their generating clones expressing IgM along with IgA and other isotypes. Again, IgA was usually expressed only in the presence of dendritic cells. The mechanism may involve dendritic cell-induced T cell activation and/or dendritic cell factors, and is under investigation.

Immunoglobulin (Ig) isotype switching and commitment, generation of memory cells, and the functional potential of B cell subsets have been analyzed in two different clonal microcultures. One is antigen (Ag)-dependent, haplotype-restricted, and requires linked recognition of hapten and carrier. The other is Ag-independent and relies on the stimulation of single I-Ab cells by alloreactive D10 helper T cells. Immunising mice increased the frequency of clonal precursors among hapten-gelatin enriched cells as well as the proportion of clones secreting non-IgM isotypes. IgA was produced when either purified dendritic cells (DC) or IL-5/IL-6 were added to the T-B cultures. sIgD+ cells could be induced to produce a wide range of isotypes including IgA whereas a majority of sIgA+ cells produced IgA exclusively. Germinal centre (GC) cells could be stimulated in both systems to divide and produce antibody and this subset appears to contain a radiation-resistant population that secretes mainly IgA upon allo -recognition by D10 cells in the presence of DC.

We have developed a number of in vivo and in vitro systems for testing the functional potential of gut T- and B- cells primed in vivo via acute or chronic stimulation of the intestinal mucosa with reovirus or Morganella morganii (M.m.) respectively. Reovirus type 3 given orally to neonatal, normal mice or adult severe, combined immunodeficient (scid) mice results in a disseminated fatal infection due to meningoencephalitis at 10–12 d in the pups and to focal hepatitis and liver failure at 4–6 wk. in the scid mice. IELs (106) from adult, immune mice given to pups challenged one d later can prevent development of brain lesions. The protective effect of IELs is decreased after treatment with anti-Thy1 + C’ and eliminated after treatment with anti-CD8 + C’. Transferred Peyer’s patch (PP) cells from immune, congenic normal donors can likewise prevent dissemination of viral infection in scid mice challenged 2 d later. The most effective subset of PP cells is Thy1+, CD8+, and GCT+.

The cellular response within lesions and in draining lymph was examined in sheep following a primary intracutaneous injection of live or killed S. aureus. Microscopic examination of sections from live S. aureus lesions (12, 24, 48, and 96 h following vaccination) revealed a high ratio of neutrophils to macrophages at all times. This ratio was initially high following inoculation of killed S. aureus but decreased steadily at successive sampling times. Representative sections from lesions were subjected to indirect immunofluorescent staining to identify IgM-, IgG1-, and IgG2-containing cells. The ratio of IgG2- to IgG1-containing cells in lesions produced following live S. aureus vaccination was significantly greater than the ratio in lesions produced by killed staphylococci. Lesions induced by live S. aureus recruited significantly greater numbers of 51Cr-labeled allogeneic neutrophils from blood than did lesions induced by killed S. aureus. During the first 6 h this difference was approx. 20-fold. The volume of lymph and the number of leukocytes draining live S. aureus lesions was considerably greater than from lesions produced by killed staphylococci. The proportion of neutrophils in lymph draining both types of lesions increased markedly during the first two days of the response but was observed to be greater and remained higher for a longer period of time in lymph draining vaccine lesions produced following injection of live staphylococci. The increase in proportion of neutrophils in lymph was accompanied by a concomitant decrease in proportion of lymphocytes and macrophages. No immunoglobulin-containing cells or anti-staphylococcal antibody production was detected in lymph draining either type of lesion. These differences in inflammatory responses may contribute to the documented differences in immune responses to live and killed staphylococcal vaccines.