Ovarian choriocarcinoma in a rhesus monkey associated with elevated serum chorionic gonadotropin levels.

A clinically normal, 3-year-old female rhesus monkey (Macaca mulatta), which was part of a routine toxicology study, had a mass in the right ovary with metastases to the adjacent mesentery and lungs. The histologic features and immunohistochemistry results were consistent with the diagnosis of choriocarcinoma. Neoplastic cell types included cytotrophoblast (positive for cytokeratin), syncytiotrophoblast (positive for human chorionic gonadotropin), and extravillous trophoblast (positive for human placental lactogen). Because the neoplasm was present in the ovary, the uterus was normal, and the animal was not currently pregnant, this was considered a primary ovarian neoplasm of germ cell origin. The monkey had elevated serum levels of chorionic gonadotropin at the beginning of the study, indicating that, as in women, choriocarcinomas in monkeys can be associated with increased gonadotropin levels and that the tumor was preexisting at the start of the toxicology study.

Immune modulator studies in primates: the utility of flow cytometry and immunohistochemistry in the identification and characterization of immunotoxicity.

Exposure to natural environmental products, biopharmaceuticals, or investigational adjuvants has the potential to negatively impact the immune system, resulting in either up- or downregulation of immune function (immunomodulation). Many current protocols for primate toxicologic testing call for the evaluation of changes in immune cell number (peripheral blood or tissue), alterations in the weights of immune system organs (lymph nodes, spleen, thymus), and/or increases in the overall incidence of infections or neoplasms; these data are relied upon to suggest altered immune function. However, these are informative only when clear differences in frequency and/or severity of effects can be distinguished across control and dosed groups. In the absence of such distinct morphologic or clinical pathologic changes, the identification of potential immunomodulatory effects can present a much greater challenge. Additional evaluations may be needed to detect altered immune system integrity; these are based on in vivo assessments in primates of cellular or humoral responsiveness. Immunomodulatory effects can be characterized by in vitro or in vivo immune function tests: these tests require prestudy planning to integrate assessments into ongoing toxicology programs. These methods also involve specialized training and equipment, particularly if the intent is to evaluate parameters in a GLP laboratory setting. In primate toxicology, the added costs required to perform a complete functional analysis of the immune system can be substantial, but may be warranted depending on the clinical development plans. Two analytical methods that are easily incorporated into the standard toxicology profile in primates are flow cytometry and immunohistochemistry. Flow cytometry (FC) is used to assess changes in the relative distribution of immune cell marker expression, and where marker expression is known to fluctuate with the state of cell activation, can also provide information on functional attributes of immune cells. Immunohistochemistry (IHC) provides a means to evaluate similar characteristics of immune cells within tissue sections. Used together, FC and IHC can aid in the identification of changes in immune system that may not be apparent by traditional testing procedures (such as H&E staining), thus aiding in the characterization of immune system alterations. This presentation focused on the utility of flow cytometry and immunohistochemistry in a standard primate toxicology evaluation, with representative examples showing the benefits of these technologies in the diagnosis of potential immunomodulatory effects.

Hypertrophy and prolonged DNA synthesis in smooth muscle cells characterize pulmonary arterial wall thickening after monocrotaline pyrrole administration to rats.

Monocrotaline pyrrole (MCTP) is a highly reactive pneumotoxic metabolite of the pyrrolizidine alkaloid plant toxin monocrotaline. When administered to rats, it causes a delayed and progressive lung injury, vascular remodeling, and pulmonary hypertension. Structural remodeling consists of endothelial cell swelling followed by increased thickness of the vascular media in small pulmonary arteries and muscularization of normally nonmuscular arteries. Experiments were performed to characterize DNA synthesis and cell proliferation in vascular smooth muscle cells (VSMCs) after MCTP and to determine their relationship to changes in the thickness of the arterial medial layer of pulmonary resistance vessels. Male Sprague-Dawley rats were treated with MCTP (3.5 mg/kg, intravenously) or its vehicle (dimethylformamide). To label cells actively synthesizing DNA, rats were given the thymidine analog, bromodeoxyuridine (BrdU), 3 times by intraperitoneal injection during the 24 hr preceding euthanasia. Using immunohistochemistry, BrdU incorporation was quantified as a ratio of labeled nuclei to total nuclei. Within 5 days after MCTP administration, the thickness of the medial smooth muscle layer in arteries 60-250 microm in diameter was increased, prior to evidence of right heart hypertrophy. BrdU incorporation by VSMCs in pulmonary arteries was not different in vehicle- and MCTP-treated rats for the first 48 hr after treatment. However, MCTP caused a significant increase in DNA synthesis in VSMC on days 3-8 in arteries up to 250 microm in diameter. Although increased DNA synthesis precedes cell proliferation, the relative number of medial VSMCs did not increase over 8 days, suggesting that hypertrophy alone was responsible for the increased thickness of the arterial media. These results demonstrate that MCTP causes thickening of the media of pulmonary vessels through VSMC hypertrophy and that the prolonged DNA synthesis that accompanies VSMC hypertrophy is not followed by proliferation.

Vitamin A or zymosan pretreatment attenuates alpha-naphthylisothiocyanate-induced liver injury.

alpha-Naphthylisothiocyanate (ANIT) is a cholangiolitic hepatotoxicant that causes periportal hepatic injury in the rat that is neutrophil- and platelet-dependent. Since macrophages have recently been implicated as participants in some chemically induced hepatotoxicities, we evaluated the role of these cells in ANIT-induced hepatic injury. Rats were treated with gadolinium chloride (GdCl3), an agent which decreases hepatic macrophage numbers and activity, zymosan, an agent which increases hepatic macrophage numbers, or vitamin A, which increases hepatic macrophage activity. GdCl3 did not ameliorate ANIT-induced hepatotoxicity, as demonstrated by a lack of attenuation of any of the markers of hepatic insult evaluated. In contrast, pretreatment with either zymosan or vitamin A decreased ANIT hepatotoxicity. Zymosan administration reduced blood neutrophil numbers and influx of neutrophils into the peritoneum after intraperitoneal glycogen administration but did not affect hepatic neutrophil accumulation in ANIT-treated rats. To determine if macrophages were important in the protection by vitamin A, rats were cotreated with GdCl3 and vitamin A. GdCl3 did not alter the protection from ANIT hepatotoxicity afforded by vitamin A. Vitamin A treatment decreased ANIT and glutathione concentrations in bile at 1 and 4 hr after ANIT administration but had a minimal effect on plasma ANIT concentration. In summary, pretreatment of rats with zymosan or vitamin A but not GdCl3 attenuated ANIT-induced liver injury. The protection afforded by zymosan may derive from its effects on neutrophils or platelets. The protection by vitamin A appears to result from its effect on the transport of ANIT into bile. The results suggest that hepatic macrophages are not required for the manifestation of ANIT hepatotoxicity.

Disseminated trichosporonosis in a neutropenic murine model.

Life-threatening disseminated infection with Trichosporon beigelii (trichosporonosis) is a rare mycosis most commonly seen in patients with hematologic malignancies made neutropenic by cytotoxic therapy. This infection is usually resistant to conventional antifungal therapies. Poor correlation between therapeutic outcome of trichosporonosis and in vitro susceptibility of clinical isolates of T. beigelii to antifungal agents is often reported. To obtain a better understanding of its pathogenesis, and to aid in the future study of the therapy of this disease, a murine model of trichosporonosis was developed. The in vitro growth of clinical isolates of T. beigelii was first studied. Subsequently, mice made neutropenic with cyclophosphamide were inoculated intravenously with the fungus to produce the disease model. Inoculum size which produced 100% mortality, yet allowed an apparent therapeutic window (6 x 10(6)) was determined. Tissue distribution and burden of organism during the course of infection was examined by viability and histopathologic studies. T. beigelii disseminated rapidly in this model, involving numerous organs including the heart, brain, kidneys, lungs, and liver. The heart and kidneys of the infected animals showed evidence of infection as early as 6 hours following inoculation. Further understanding of the pathogenesis of trichosporonosis in the neutropenic host was imparted by this study. This will aid in the future study of antibiotic treatment of this disease and its untreated progression.