Malignant lymphoma in macaques: a clinicopathologic study of 45 cases.

Malignant lymphoma was diagnosed in 42 rhesus macaques (Macaca mulatta) and 3 stumptail macaques (M. arctoides) between February 1969 and December 1977. The distribution of tumor masses in the tissues of individual animals varied widely. Solitary tumor masses were present in 14 animals and multiple masses in the remaining 31 animals. Visceral lymph nodes, gastrointestinal tract, heart, and kidneys were most commonly affected. Peripheral lymph nodes were rarely involved. Most malignant lymphomas were of an undifferentiated cell type, although tumors of histiocytic, lymphocytic, poorly differentiated, and mixed lymphocytic and histiocytic cell types were also observed. Concurrent bacterial and/or viral infections were evident in 30 of the 45 macaques with malignant lymphoma. Amyloidosis was present in 9 animals. This high incidence of malignant lymphoma suggested that their immune responses were abnormal. The development of malignant lymphoma in the macaques may have been secondary to or enhanced by immunodeficiency.

Prevention of doxorubicin-induced hematotoxicity in mice by interleukin 1.

Interleukin 1 alpha and interleukin 1 beta induce peripheral neutrophilia with stimulation of granulopoiesis in bone marrow. The continuous administration of interleukin 1 (100 ng/day) to mice for 7 days by s.c.-implanted Alzet osmotic minipumps induced marked stimulation of granulopoiesis in marrow and spleen in normal mice, and protected against the marked depletion of myeloid and erythroid cells in bone marrow of mice treated with single injections of either 20 or 30 mg/kg doxorubicin (DXN). Interleukin 1 beta infusion also protected against DXN-induced atrophy of thymus and secondary lymphoid organs. Single i.p. injection of either interleukin 1 alpha or interleukin 1 beta at doses up to 1000 ng 24 h prior to treatment with DXN did not protect against the hematopoietic and lymphoid toxicities of DXN.

Reduction of alpha-naphthylisothiocyanate-induced hepatotoxicity by recombinant human hepatocyte growth factor.

Hepatocyte growth factor (HGF) is a potent stimulator of DNA synthesis in cultured hepatocytes. To determine whether HGF has any activity in vivo, we have tested HGF in rats in which intrahepatic cholestasis was induced by acute administration of alpha-naphthylisothiocyanate (ANIT). The hepatotoxic effects of a single injection of ANIT were manifested 48 h later as large increases in serum bilirubin, alanine aminotransferase, aspartate aminotransferase, and alkaline phosphatase. These biochemical changes were accompanied by widespread periportal edema, hypertrophy of bile duct epithelium, and randomly scattered areas of liquifaction necrosis in the hepatic parenchyma. The increases in bilirubin, alanine aminotransferase, aspartate aminotransferase and alkaline phosphatase were markedly attenuated when HGF was administered 30 min before ANIT and again at 6, 12, 24, 30, and 36 h after ANIT. In addition, this HGF dosing regimen completely prevented the occurrence of parenchymal lesions, although it had no effect on periportal histopathology. The effect of ANIT was dose dependent; a maximal response was observed at 320 micrograms/kg per injection, with an intermediate response at 105 micrograms/kg. Delaying the administration of HGF until 12 h after ANIT was as effective as when administration was begun 30 min before ANIT. Taken together these results show that HGF can prevent some aspects of ANIT hepatotoxicity.

An evaluation of the functions of the 22-kilodalton (kDa), the 20-kDa, and the N-terminal polypeptide forms of human growth hormone using transgenic mice.

Multiple peptide hormones can be derived from the single human GH gene. In addition to the full-length 191-amino acid 22-kilodalton (kDa) form, a 20-kDa variant can be produced by alternative splicing, and a 5-kDa variant can be produced by posttranslational cleavage. To more fully appreciate the physiological roles of these proteins, we have made a comparison of transgenic mice that constitutively overexpress one or another of these variants. We have found that both the 22-kDa and the 20-kDa forms of human GH stimulate linear growth and liver hypertrophy. The increase in linear growth in 22-kDa transgenic mice does not, however, correlate with an increase in circulating IGF-I; rather, the increase in IGF-I that does finally occur correlates with marked liver pathology. Both groups of mice also develop glomerulosclerosis and suffer from hyperinsulinemia. Although there are histologically obvious lesions in the livers of both the 22-kDa and the 20-kDa transgenic mice, only the former exhibit hyperalbuminemia and hypercholesterolemia. Both forms of GH lead to anemia, which is normocytic in the 20-kDa transgenic mice and macrocytic in the 22-kDa transgenic mice. Despite the presence of high levels of the 5-kDa N-terminal form of human GH, the transgenic mice that express this protein are indistinguishable from their nontransgenic littermates.

Utilization of homologous proteins to evaluate the safety of recombinant human proteins--case study: recombinant human interferon-gamma (rhIFN-gamma).

Interferon-gamma is an immunomodulatory cytokine that has an extremely restricted host range of activities. RhIFN-gamma was one of the first species-specific recombinant proteins to be assessed in conventional safety models typically utilized for xenobiotics. Acute, subchronic and Segment I and II reproductive studies in rats revealed no evidence of toxicity at any of the doses tested; these results were not predictive of clinical toxicity, which is not unexpected since rodents are known to be pharmacologically nonresponsive to rhIFN-gamma. In contrast, 4- and 13-week multidose toxicity studies in cynomolgus monkeys with rhIFN- were predictive of many of the dose-limiting clinical toxicities. RhIFN- is active on non-human primate cells, though not at the same level as on human cells. In addition, qualitative similarities were observed between toxicity studies employing rhIFN-gamma in the cynomolgus monkey and recombinant murine interferon-gamma (rmuIFN-gamma) in the mouse. These results suggest that in situations where a high degree of species specificity is encountered, studies employing a recombinant protein in a homologous species may provide a useful test system for preclinical safety assessment. This information should be evaluated in conjunction with data from studies conducted with the human protein in pharmacologically responsive animal models when possible.

Immunologic surface markers on nonhuman primate lymphocytes.

Peripheral blood lymphocytes from 37 healthy rhesus macaques (Macaca mulatta) and thymocytes from 10 fetal and neonatal rhesus macaques were studied for membrane characteristics. Spontaneous rosette formation with sheep erythrocytes, a characteristic of human T lymphocytes, was evaluated. The presence of membrane-bound immunoglobulin and surface receptors for fixed complement was measured, using fluorescent antibody techniques and erythrocyte-antibody-complement rosettes, respectively. The mean percentages +/- 1 standard error of the lymphocyte markers in the peripheral blood lymphocytes from the macaques were: spontaneous rosettes, 63 +/- 1.0; erythrocyte-antibody-complement rosettes, 14.9 +/- 1.2; and membrane immunoglobulin-positive cells, 21.9 +/- 2.2. These values are very similar to values reported for human beings.

Lymphocyte subpopulations in spontaneous disease of rhesus macaques.

The immunologic status of rhesus macaques (Macaca mulatta) with naturally occurring disease was evaluated by determining the percentages of B and T lymphocytes and mitogen responsiveness of lymphocytes in peripheral blood and lymph nodes. The B lymphocytes were identified by the presence of cell surface immunoglobulin and receptors for complement. The T lymphocytes were identified by their ability to form spontaneous rosettes with sheep red blood cells. Rhesus macaques with idiopathic or primary amyloidosis had normal lymphocyte characteristics. Peripheral blood lymphocytes from rhesus macaques with atypical tuberculosis had decreased percentages of spontaneous rosette-forming cells and depressed responses to concanavalin A, and those with chronic diarrhea or chronic arthritis were also found to have abnormal peripheral blood lymphocyte characteristics. The percentage of B and T lymphocytes in normal lymph nodes was variable, making simultaneous histologic examination necessary for evaluation of diseased animals.

Cellular immunologic studies of malignant lymphoma in rhesus macaques.

Cells from malignant lymphoma in 10 rhesus macaques were examined for lymphocyte surface markers. Three had features of T cells, 5 had features of B cells, and 2 lacked evidence of either B- or T-cell differentiation. Correlation between the histologic classification of cell type and the B- or T-cell nature of the neoplasms was not evident. Evaluation of serum electrophoresis, mitogen responses tests, and previous histologic studies suggest that the development of the neoplastic lymphocyte proliferation occurred following or during an abnormal immunologic response.

Polyclonal gammopathies associated with chronic diseases in nonhuman primates.

Serum samples collected from clinically healthy rhesus macaques (Macaca mulatta) were compared with serums from animals diagnosed as having malignant lymphoma, atypical tuberculosis, progressive multifocal leukoencephalopathy, or more than 1 of these. Electrophoretic analyses of serum proteins (in grams per deciliter) indicated that mean values in a clinically normal group were as follows--total protein, 8.30; albumin, 4.07; alpha-globulin, 0.85; beta-globulin, 2.23; and gamma-globulin, 1.15. In macaques with malignant lymphoma, mean values were--total protein, 7.60; albumin, 2.72; alpha-globulin, 0.91; beta-globulin, 1.71; and gamma-globulin, 2.23. In macaques with atypical tuberculosis, these values were 8.47, 2.92, 0.84, 2.18, and 2.52, respectively, and those with progressive multifocal leukoencephalopathy, 9.06, 4.01, 6.71, 2.42, and 1.93. Where multiple serum samples were available from 1 macaque for analysis, gamma-globulin values were increased before clinical disease was apparent. Evaluation of serums from macaques may be used as a means of detecting preclinical lymphoproliferative disease.

Fibrous osteodystrophy in an opossum.

A free living opossum (Didelphis marsupialis) was found to have severe fibrous osteodystrophy of the maxilla and mandibles. No significant lesions were found in the kidneys, ruling out an etiology of renal secondary hyperparathyroidism. An etiology of primary phperthyroidism or nutritional secondary hyperparathyroidism is suggested.

Idiopathic hypoparathyroidism in a dog.

Idiopathic hypoparathyroidism in a 3-year-old dog was manifested clinically as periodic episodes of depression, vomiting, and tetanic convulsions. Hypocalcemia and hyperphosphatemia were detected late in the course of the illness, but the dog died before corrective measures could be instituted. The pathologic features were similar to those reported in dogs in which hypoparathyroidism was induced by injection of homologous parathyroid tissue, thus suggesting an auto-immune pathogenesis.

Paramyxo-like virus infection in a rock rattlesnake.

A rock rattlesnake (Crotalus lepidus) with a history of progressive central nervous disease was submitted for necropsy. The histopathologic findings included evidence of interstitial pneumonia, multifocal areas of gliosis in the brain, and ballooning degeneration and demyelination of brainstem and upper spinal cord axons. By electron microscopy, brainstem tissue was found to contain numerous virus particles in the extracellular spaces. A paramyxo-like virus, isolated in viper heart cells from lung tissue, was observed by electron microscopy to be similar in size and shape to the particles seen in nerve tissue.

Obstructive jaundice associated with chronic active hepatitis in a dog.

A cholestatic syndrome was the primary manifestation of chronic active hepatitis in a dog. Enzyme determinations did not differentiate between extrahepatic and intrahepatic cholestasis as the cause of the conjugated hyperbilirubinemia, thus necessitating percutaneous liver biopsy. The dog was treated with prednisolone but developed gastrointestinal hemorrhage and terminal hepatic encephalopathy. Morphologic variations in liver biopsy specimens obtained simultaneously and in the specimen obtained at necropsy demonstrated that the disease activity varied by locales in the liver.

Ontogeny of the immune system.

Cellular and humoral aspects of the immune response develop sequentially in the fetus. Although there is evidence of immunologic activity to some antigens, many of the nonimmune effector systems are neither developed nor fully functional in the fetus. The lack of fully functional systems make the fetus a highly susceptible candidate for invading bacteria and viruses. Often, infection prior to immune competence may lead to abortion, malformation, and in some instances, viral persistence and immune tolerance. On the other hand, immune responses tend to elevate immunoglobulin values and cause specific antibody that may be used for diagnostic purposes. Little work has been done to appreciate fully the immunologic and nonimmune effector systems' role in normal development or during congenital infections.

Comparative pathology of recombinant murine interferon-gamma in mice and recombinant human interferon-gamma in cynomolgus monkeys.

Interferon-gamma is a highly species-specific cytokine and has the most restricted host range of activity of the interferons. Recombinant human IFN-gamma was one of the first species-specific recombinant proteins to be thoroughly assessed in conventional safety models used for xenobiotics. Acute single-dose intravenous toxicity studies with rHuIFN-gamma were performed in rats, marmosets, and squirrel monkeys with no indications of toxicity. A complete series of subchronic toxicity studies and segment I and II reproductive studies in the rat revealed no evidence of toxicity at any of the doses tested. These results suggested that studies conducted in pharmacologically nonresponsive species may not be predictive of clinical toxicity. Human IFN-gamma is active on nonhuman primate cells, though not at the same level as on human cells. Multidose studies in cynomolgus monkeys with rHuIFN-gamma for 28 or 90 days were predictive of many of the dose-limiting clinical toxicities. Qualitative similarity was observed between toxicity studies employing rHuIFN-gamma in the cynomolgus monkey and rMuIFN-gamma in the mouse. The adverse effects seen in toxicity studies with cytokines and growth factors are often exaggerated pharmacological effects of the molecules, and therefore can only be studied in a responsive species. In situations in which a high degree of species specificity is encountered, studies employing a recombinant protein in a homologous species may provide a useful test system for preclinical safety assessment.

Issues with biotechnology products in toxicologic pathology.

The emergence of the biotechnology industry and introduction of drugs derived from recombinant DNA technology has generated many new issues in approaches to preclinical safety evaluation and extrapolation of results to risk assessment in humans. Products or therapeutic approaches for consideration include hormones, growth factors, cytokines, monoclonal antibodies, vaccines, blood products, antisense, and gene therapy. In many instances the application of standard safety tests conventionally used for small molecules are of limited value or are inappropriate. Studies should be designed to answer specific scientific questions rather than simply to fulfill regulatory requirements. Special consideration must be given to study design and species selection in terms of biological activity and species specificity, implications of immunological responses in the animal studies, and effects of systemic administration of molecules at clinically relevant doses. A full understanding of the clinical relevance of toxicological and pathologic findings associated with administration of these molecules to laboratory animals requires definition of the pathogenic mechanism of lesion induction.

Uveitis induction in the rabbit by muramyl dipeptides.

Intraocular inflammation (uveitis) was produced in rabbits by intravenous or subcutaneous treatment with N-acetylmuramyl-L-alanyl-D-isoglutamine and several of its synthetic analogs at doses of greater than or equal to 0.2 mg/kg in saline. A dose-dependent increase in permeability of the ocular blood-aqueous barrier as measured by leakage of protein or fluoresceinated dextran from the serum into the eye was observed from 2 to 14 h after glycopeptide treatment. Peak response occurred at approximately 3 h postdose. The lowest dose found to produce maximal vascular leakage for the most active glycopeptide analogs was 1 mg/kg. The adjuvant-inactive L-L stereoisomer of N-acetylmuramyl-L-alanyl-D-isoglutamine was inactive, even at doses as high as 10 mg/kg. Analogs of N-acetylmuramyl-L-alanyl-D-isoglutamine which were homologous in the lactyl side chain were found to cause less uveitis. Chronic biweekly intravenous treatment of rabbits for 1 month with either N-acetyl-L-alpha-aminobutyryl-D-isoglutamine or its lipophilic 6-O-stearoyl derivative at 1 mg/kg, but not with murabutide, resulted in leukocytic inflammatory lesions unique to the uveal tract of the eye. The uveitis was potentially reversible and occurred with decreased severity as long as 2 months after cessation of chronic treatment. Vascular leakage but not cellular infiltrate in the choroid could be modulated by pharmacologic means. Pyrogenicity but not adjuvanticity correlated with ability of glycopeptides to induce vascular leakage. Several adjuvant-active muramyl dipeptide analogs with minimal ability to cause acute vascular leakage or chronic inflammation in the rabbit eye have been identified.