Toxicologic Pathology Forum: Opinion on Obligatory Microscopic Examination of Intermediate-Dose Groups in Toxicity Studies With Biotherapeutics in Cynomolgus Monkeys.

In nonrodent toxicity studies that are usually conducted in cynomolgus monkeys or beagle dogs, the added value of examining all tissues from all dose groups (current practice) versus all tissues in only control and high-dose groups and target tissues in intermediate-dose groups by default, is a subject of debate. A previous retrospective review of 325 nonrodent toxicity studies that included a limited number of biotherapeutics suggested that the evaluation of all tissues from all groups was not justified as a routine practice and recommended the examination of all tissues in control and high-dose groups and only target tissues in intermediate-dose groups. In contrast, the present retrospective review which examined 213 nonrodent studies (212 in cynomolgus monkeys and 1 in dog) from 4 multinational pharmaceutical companies (Bristol-Myers Squibb, Novartis, Pfizer Inc, and Roche) conducted only with biotherapeutics showed that restricting the microscopic examination in intermediate-dose groups to target tissues has the potential to miss findings in 6.6% of studies, possibly impacting the overall study interpretation and conclusion. In conclusion and in the opinion of the authors, all tissues from all dose groups should be examined in toxicity studies with biotherapeutics conducted in nonrodent species.

Spontaneous Findings in the Eyes of Cynomolgus Monkeys ( Macaca fascicularis) of Mauritian Origin.

Spontaneous findings noted in the eyes of Mauritian cynomolgus monkeys are described and descriptions are supplemented with illustrations. Findings observed after extensive histopathologic examinations (20 to 44 sections per eye) from 20 control, 17 treatment-naive stock monkeys, and 2 findings noted in drug-treated monkeys that were considered to be spontaneous are included. Also included are findings from 361 control monkeys of routine toxicity studies performed at our laboratories, for most of which a standard histopathological examination of 1 section per eye was conducted. Common observations in monkeys examined extensively and in historical controls were limited to lymphocytic or mononuclear cell infiltrations of the uvea and/or conjunctiva/sclera and, less commonly observed, melanocytoma of the ciliary body or iris. Findings noted only in monkeys examined extensively consisted of inflammation of the conjunctiva, ora serrata cysts, glial nodules, focal degeneration of the retina, cystoid degeneration of the central retina, ballooning degeneration of the ciliary epithelium, cyst of the ciliary body, and decreased pigmentation of the retinal pigment epithelium. Changes recorded only in historical controls included retinal atrophy and nuclear displacement in the retina. Lesions are discussed and compared with pertinent literature.

Severe Acquired Idiopathic Thrombocytopenia in a Female Cynomolgus Macaque (Macaca fascicularis).

A 4-y-old female cynomolgus macaque presented for veterinary evaluation prior to placement in a preclinical study showed markedly low platelet counts that continued to decrease over time. Physical examination over the next several days showed areas of pale red discoloration in forelimbs, anterior thorax, and inguinal area and multifocal pinpoint areas of erythema or scabs. An area of dark red discoloration approximately 2 cm in diameter on the dorsal surface of the tongue was discovered on day 9. The macaque was euthanized, and histopathologic evaluation showed multifocal, ulcerative or erosive, hemorrhagic, lymphohistiocytic and neutrophilic glossitis and tonsillitis. The lesions on the tongue were associated with opportunistic fungi consistent with Candida albicans. The bone marrow showed megakaryocytic hyperplasia. There was no evidence of increased consumption of platelets, sequestration of platelets, or bone marrow suppression. The monkey was serologically negative for simian retrovirus, SIV, and simian T-lymphotropic virus. In light of cases reported in humans, immune-mediated destruction of platelets due to autoantibodies secondary to Candida albicans infection was considered. However, we were unable to detect antiplatelet antibodies on the platelet surface or in serum to support this etiology; therefore idiopathic thrombocytopenia was diagnosed. To our knowledge, this case represents the second reported observation of acquired thrombocytopenia in a nonhuman primate and the first reported observation in a cynomolgus macaque.

Comparison of physiologic and pharmacologic parameters in Asian and mauritius cynomolgus macaques.

This comparative study was conducted to assess background physiologic and pharmacologic parameters of cynomolgus macaques (Macaca fascicularis) from Cambodia, from a mixed Asian source (Cambodia, Vietnam and Indonesia), and from Mauritius. This evaluation provides a comprehensive assessment of several of these parameters in a single study. Ten male and 10 female captive-bred, age-matched macaques from each source were evaluated. Criteria for evaluation included weight gain, assessment of drug metabolizing enzyme activity, metabolomic analysis, immunologic assessments (lymphocyte subsets, TDAR, and serum Ig isotyping), clinical pathology evaluations, physical (respiratory, neurologic, cardiovascular, and ophthalmologic) examinations, pathogen screening, organ weights, and gross and microscopic pathology analyses. The results of this evaluation indicate that, compared to macaques of Asian origin, macaques from Mauritius had the lowest incidence and/or severity of spontaneous pathologic findings in several organs and tissues (lymphoid organs, stomach, kidney, urothelium, heart, arteries and lung) and better testicular maturity at a given age with minimal variability in organ weights. Although slight differences were observed in other parameters, none were considered detrimental to the use of macaques of Asian or Mauritius origin in pharmaceutical candidate safety studies with the use of a consistent source, concomitant controls, and appropriate background knowledge and screening.

Immunohistochemical study of matrix metalloproteinases-2 and -9, macrophage inflammatory protein-2 and tissue inhibitors of matrix metalloproteinases-1 and -2 in normal, purulonecrotic and fungal infected equine corneas.

OBJECTIVE: Determine the effects of matrix metalloproteinases (MMPs)-2, -9, macrophage inflammatory protein-2 (MIP-2), tissue inhibitors of matrix metalloproteinase (TIMP)-1 and -2 by immunohistochemical expression in fungal affected and purulonecrotic corneas. PROCEDURE: Paraffin-embedded equine corneal samples; normal (n = 9), fungal affected (FA; n = 26), and purulonecrotic without fungi (PN; n = 41) were evaluated immunohistochemically for MMP-2, -9, MIP-2, TIMP-1 and -2. The number of immunoreactive inflammatory cells was counted and statistics analyzed. Western blot was performed to detect MMP-2, MMP-9, TIMP-1 and TIMP-2 proteins. RESULTS: Matrix metalloproteinases-2, -9, MIP-2, TIMP-1 and -2 immunoreactivity was identified in corneal epithelium of normal corneas, and in corneal epithelium, inflammatory cells, keratocytes, and vascular endothelial cells of both FA and PN samples. Inflammatory cell immunoreactivity was significantly higher in FA and PN samples than in the normal corneas. There was positive correlation between MMP-2 and MIP-2, MMP-9 and MIP-2, and MMP-9 and TIMP-1 in inflammatory cell immunoreactivity in FA samples. There was positive correlation between MMP-9 and MIP-2, MMP-9 and TIMP-2, MIP-2 and TIMP-1, and MIP-2 and TIMP-2 in inflammatory cell immunoreactivity in PN samples. Western blot confirmed the presence of all four proteins in equine corneal samples. CONCLUSION: Increased immunoreactivity of MMP-2 and -9 in FA and PN samples is indirectly related to MIP-2 through its role in neutrophil chemo-attraction. Tissue inhibitors of matrix metalloproteinase-1 and TIMP-2 are up-regulated in equine purulonecrotic and fungal keratitis secondary to MMP-2 and MMP-9 expression. The correlation between MMPs -2 and -9, MIP-2, TIMPs -1 and -2 suggests that these proteins play a specific role in the pathogenesis of equine fungal keratitis.

In vitro and in vivo characterization of alkyl hydroperoxide reductase mutant strains of Helicobacter hepaticus.

Mutant strains in the tsaA gene encoding alkyl hydroperoxide reductase were more sensitive to O(2) and to oxidizing agents (paraquat, cumene hydroperoxide and t-butylhydroperoxide) than the wild type, but were markedly more resistant to hydrogen peroxide. The mutant strains resistance phenotype could be attributed to a 4-fold and 3-fold increase in the catalase protein amount and activity, respectively compared to the parent strain. The wild type did not show an increase in catalase expression in response to sequential increases in O(2) exposure or to oxidative stress reagents, so an adaptive compensatory mutation has probably occurred in the mutants. In support of this, chromosomal complementation of tsaA mutants restored alkyl hydroperoxide reductase, but catalase was still up-expressed in all complemented strains. The katA promoter sequence was the same in all mutant strains and the wild type. Like its Helicobacter pylori counterpart strain, a H. hepaticus tsaA mutant contained more lipid hydroperoxides than the wild type strain. Hepatic tissue from mice inoculated with a tsaA mutant had lesions similar to those inoculated with the wild type, and included coagulative necrosis of hepatocytes. The liver and cecum colonizing abilities of the wild type and tsaA mutant were comparable. Up-expression of catalase in the tsaA mutants likely permits the bacterium to compensate (in colonization and virulence attributes) for the loss of an otherwise important oxidative stress-combating enzyme, alkyl hydroperoxide reductase. The use of erythromycin resistance insertion as a facile way to screen for gene-targeted mutants, and the chromosomal complementation of those mutants are new genetic procedures for studying H. hepaticus.

Helicobacter hepaticus hydrogenase mutants are deficient in hydrogen-supported amino acid uptake and in causing liver lesions in A/J mice.

Helicobacter hepaticus, a causative agent of chronic hepatitis and hepatocellular carcinoma in mice, expresses a nickel-containing hydrogen-oxidizing hydrogenase enzyme. Growth of a hyaB gene-targeted mutant was unaffected by the presence of hydrogen, unlike the wild-type strain, which showed an enhanced growth rate when supplied with H(2). Hydrogenase activities in H. hepaticus were constitutive and not dependent on the inclusion of H(2) during growth. Addition of nickel during growth significantly stimulated both urease (for wild-type and hyaB) and hydrogenase (for wild-type) activities. In a 5-h period, the extent of (14)C-labeled amino acid uptake by the wild type was markedly enhanced in the presence of hydrogen and was >5-fold greater than that of the hyaB mutant strain. In the presence of H(2), the short-term whole-cell amino acid uptake V(max) of the parent strain was about 2.2-fold greater than for the mutant, but the half-saturation affinity for amino acid transport was the same for the parent and mutant strain. The liver- and cecum-colonizing abilities of the strains was estimated by real-time PCR quantitation of the H. hepaticus-specific cytolethal distending toxin gene and showed similar animal colonization for the hyaB mutant and the wild type. However, at 21 weeks postinoculation, the livers from mice inoculated with wild type exhibited moderate lobular lymphoplasmacytic hepatitis with hepatocytic coagulative necrosis, but the hydrogenase mutants exhibited no histological evidence of lobular inflammation or necrosis.