Eberhard Buse

Reproductive/developmental toxicity and immunotoxicity assessment in the nonhuman primate model.

Nonhuman primates are being used increasingly as a non-rodent animal model during preclinical toxicology and safety assessment on the basis of proven similarity and comparability between nonhuman primates and humans. The validity of the nonhuman primate models applies to many aspects of toxicological testing and holds particularly true for the evaluation of reproductive toxicology and developmental toxicology. More recently, the advent of humanized antibodies and vaccines imposed further demand on nonhuman primate models since many immunotherapeutics do not interact with rodent receptors but frequently only cross-react with primate tissue. In this paper we discuss the suitability of primate models for reproductive, developmental and immunotoxicology testing, and present our initial data on the development of lymphatic organs and immune system in a nonhuman primate model.

Postnatal development in cynomolgus monkeys following prenatal exposure to natalizumab, an α4 integrin inhibitor

BACKGROUND Natalizumab is a humanized monoclonal IgG4 antibody to human alpha4 integrin that blocks the interaction of alpha4beta1 and alpha4beta7 integrins with their ligands, including fibronectin, vascular cell adhesion molecule-1, and mucosal addressin cellular adhesion molecule-1. Because alpha4 integrins and their ligands are widely involved in mammalian development, lymphopoeisis, and hematopoiesis, natalizumab may interfere with these processes. METHODS The effects of prenatal exposure to natalizumab on postnatal development were assessed in cynomolgus monkeys at doses of 0 and 30 mg/kg administered intravenously every other day from gestational day (GD) 20 to 70 or GD 20 to term. Infants were delivered by natural birth and evaluated for general health, survival, development, and immunological structure and function at 12 or 18 months. RESULTS An increase in abortions was seen in the first cohort of natalizumab-treated dams (39.3 vs. 7.1% in the controls) but not in the second cohort (33.3, 37.5%). Infants in the term treatment group had elevated lymphocyte ( approximately 150%) and nucleated red blood cell counts ( approximately 400%), consistent with the pharmacological effect of natalizumab, and reductions in platelet counts ( approximately 28%), which were reversible following clearance of natalizumab. No anemia was observed. Infants in the term treatment group had significantly increased spleen weights at 12 months but not at 18 months. All other experimental observations in infants from natalizumab-treated dams were comparable with those of controls. CONCLUSION Natalizumab had no adverse effects on the general health, survival, development, or immunological structure and function of infants born to dams treated with natalizumab during pregnancy.

Summary Comparison of Female Reproductive System in Human and the Cynomolgus Monkey (Macaca fascicularis)

Competing Interests: This article was sponsored by Covance Inc. and Schering-Plough. Gerhard F. Weinbauer and Eberhard Buse are employed by Covance Inc. Eveline P. C. T. de Rijk and Eric Van Esch are employed by Schering-Plough. No other competing interests were declared.

Selected Background Findings and Interpretation of Common Lesions in the Female Reproductive System in Macaques.

The authors describe a selection of normal findings and common naturally occurring lesions in the reproductive system of female macaques, including changes in the ovaries, uterus, cervix, vagina, and mammary glands. Normal features of immature ovaries, uteri, and mammary glands are described. Common non-neoplastic lesions in the ovaries include cortical mineralization, polyovular follicles, cysts, ovarian surface epithelial hyperplasia, and ectopic ovarian tissue. Ovarian neoplasms include granulosa cell tumors, teratomas, and ovarian surface epithelial tumors. Common non-neoplastic uterine findings include loss of features of normal cyclicity, abnormal bleeding, adenomyosis, endometriosis, epithelial plaques, and pregnancy-associated vascular remodeling. Hyperplastic and neoplastic lesions of the uterus include endometrial polyps, leiomyomas, and rarely endometrial hyperplasia and endometrial adenocarcinoma. Vaginitis is common. Cervical lesions include endocervical squamous metaplasia, polyps, and papillomavirus-associated lesions. Lesions in the mammary gland are most often proliferative and range from ductal hyperplasia to invasive carcinoma. Challenges to interpretation include the normal or pathologic absence of menstrual cyclicity and the potential misinterpretation of sporadic lesions, such as epithelial plaques or papillomavirus-associated lesions. Interpretation of normal and pathologic findings is best accomplished with knowledge of the life stage, reproductive history, and hormonal status of the animal. Competing Interests: This article was sponsored by Covance Inc. and Schering-Plough. Gerhard F. Weinbauer and Eberhard Buse are employed by Covance Inc. Eveline P. C. T. de Rijk and Eric Van Esch are employed by Schering-Plough. No other competing interests were declared.

Development of the immune system in the cynomolgus monkey: the appropriate model in human targeted toxicology?

Toxicological research for humans requires model animals that are physiologically and/or developmentally closely related to one another. The development of the immune system (IS) in the cynomolgus monkey (Macaca fascicularis) was assessed and compared with selected data of humans and mice with the aim of contributing arguments to the discussion of the most appropriate animal model in toxicological research. Details of the developing IS in the cynomolgus monkey from embryonic day 35 to birth are investigated utilizing cluster development (CD) antibodies. Early immunoreactivity with CD 68, CD 117, and HLA-DR antibody is apparent from days 40 and 45 onward. All principal cell lines—T-, B-, and NK-cells—are present on day 100 in both thymus and peripheral organs. We discuss investigations of the cynomolgus monkey IS development with the reported development in humans and mice and stress 4 topics of significant interspecies differences to be considered in the decision for the appropriate animal model in a toxicological study.

The placenta in toxicology. Part II : Systemic and local immune adaptations in pregnancy

During pregnancy, the maternal immune system is challenged by the semiallogeneic fetus, which must be tolerated without compromising fetal or maternal health. This review updates the systemic and local immune changes taking place during human pregnancy, including some examples in rodents. Systemic changes are induced by contact of maternal blood with placental factors and include enhanced innate immunity with increased activation of granulocytes and nonclassical monocytes. Although a bias toward T helper (Th2) and regulatory T cell (Treg) immunity has been associated with healthy pregnancy, the relationship between different circulating Th cell subsets is not straightforward. Instead, these adaptations appear most evidently at the fetal–maternal interface, where for instance Tregs are enriched and promote fetal tolerance. Also innate immune cells, that is, natural killer cells and macrophages, are enriched, constituting the majority of decidual leukocytes. These cells not only contribute to immune regulation but also aid in establishing the placenta by promoting trophoblast recruitment and angiogenesis. Thus, proper interaction between leukocytes and placental trophoblasts is necessary for normal placentation and immune adaptation. Consequently, spontaneous maladaptation or interference of the immune system with toxic substances may be important contributing factors for the development of pregnancy complications such as preeclampsia, preterm labor, and recurrent miscarriages.

The Macaque Endometrium, with Special Reference to the Cynomolgus Monkey (Macaca fascicularis)

The macaque endometrium undergoes dramatic morphologic and functional changes during the menstrual cycle that are nearly identical to those of the human endometrium. The sequential events that take place in the endometrium are mainly driven by the ovarian steroids and their respective receptors. To be able to interpret the changes and effects induced by mammalian or synthetic hormones and other compounds that could have influence on the hormonal status of the animal, a thorough knowledge of the anatomy, physiology, and histology of the cyclic hormone-mediated processes within the endometrium is indispensable. In this paper we give an overview of uterine growth and development, anatomy, basic histology, aging, spontaneous pathology, and the techniques to study the endometrium in-life. In addtion, a comprehensive description of the receptor-mediated, hormone-driven morphological changes during the menstrual cycle in the cynomolgus monkey (Macaca fascicularis) is given. Where possible, differences between the macaque and human endometria are discussed. Competing Interests: This article was sponsored by Covance Inc. and Schering-Plough. Gerhard F. Weinbauer and Eberhard Buse are employed by Covance Inc. Eric Van Esch is employed by Schering-Plough. No other competing interests were declared.

The Macaque Ovary, with Special Reference to the Cynomolgus Macaque (Macaca fascicularis)

Concerning functional and morphological aspects, the ovary of the cynomolgus macaque is representative for the conditions in higher primates like humans and is therefore of major relevance in toxicological research. Against this background, a comprehensive overview about the cynomolgus macaque ovary is given from its embryonic appearance, throughout the adolescent and adult development until old age. The overview includes morphologic characteristics, a description of the different cell types, comparisons between the expression of selected receptors, and some details on hormonal effects if considered necessary for understanding the unit of ovarian morphology and function. The close correlation of hormones and morphological characteristics of the ovary and of the other reproductive organs is emphasized by several schematic drawings and images. Special emphasis is also laid on the comparison to the human organism indicating the similarity of both species and hence underlining the importance of the cynomolgus macaque as a model in toxicological research. Competing Interests: This article was sponsored by Covance Inc. and Schering-Plough. Martina Zöller and Eberhard Buse are employed by Covance Inc. Eric Van Esch is employed by Schering-Plough. No other competing interests were declared.

The placenta in toxicology. Part IV: Battery of toxicological test systems based on human placenta

This review summarizes the potential and also some limitations of using human placentas, or placental cells and structures for toxicology testing. The placenta contains a wide spectrum of cell types and tissues, such as trophoblast cells, immune cells, fibroblasts, stem cells, endothelial cells, vessels, glands, membranes, and many others. It may be expected that in many cases the relevance of results obtained from human placenta will be higher than those from animal models due to species specificity of metabolism and placental structure. For practical and economical reasons, we propose to apply a battery of sequential experiments for analysis of potential toxicants. This should start with using cell lines, followed by testing placenta tissue explants and isolated placenta cells, and finally by application of single and dual side ex vivo placenta perfusion. With each of these steps, the relative workload increases while the number of feasible repeats decreases. Simultaneously, the predictive power enhances by increasing similarity with in vivo human conditions. Toxic effects may be detected by performing proliferation, vitality and cell death assays, analysis of protein and hormone expression, immunohistochemistry or testing functionality of signaling pathways, gene expression, transport mechanisms, and so on. When toxic effects appear at any step, the subsequent assays may be cancelled. Such a system may be useful to reduce costs and increase specificity in testing questionable toxicants. Nonetheless, it requires further standardization and end point definitions for better comparability of results from different toxicants and to estimate the respective in vivo translatability and predictive value.

The placenta in toxicology. Part I: Animal models in toxicology: placental morphology and tolerance molecules in the cynomolgus monkey (Macaca fascicularis)

The immune system represents a key defense mechanism against potential pathogens and adverse non-self materials. During pregnancy, the placenta is the point of contact between the maternal organism and non-self proteins of the fetal allograft and hence undoubtedly fulfils immune functions. In the placenta bacteria, foreign (non-self) proteins and proteins that might be introduced in toxicological studies or by medication are barred from reaching the progeny, and the maternal immune system is primed for acceptance of non-maternal fetal protein. Both immunologic protection of the fetus and acceptance of the fetus by the mother require effective mechanisms to prevent an immunologic fetomaternal conflict and to keep both organisms in balance. This is why the placenta requires toxicological consideration in view of its immune organ function. The following articles deal with placenta immune-, control-, and tolerance mechanisms in view of both fetal and maternal aspects. Furthermore, models for experimental access to placental immune function are addressed and the pathological evaluation is elucidated. “The Placenta as an Immune Organ and Its Relevance in Toxicological Studies” was subject of a continuing education course at the 2012 Society of Toxicologic Pathology meeting held in Boston, MA.