Hyperplastic and neoplastic lesions of the mammary gland in macaques.

Macaques provide an important animal model for the study of hormonal agents and their effects on risk biomarkers for breast cancer. A common criticism of this model is that spontaneous breast cancer has rarely been described in these animals. In this report, we characterize 35 mammary gland lesions ranging from ductal hyperplasia to carcinoma in situ and invasive ductal carcinoma in cynomolgus and rhesus macaques. Based on a retrospective analysis, we estimated the lifetime incidence of mammary gland neoplasia in aged female macaques to be about 6%. Hyperplastic lesions (n = 19) occurred segmentally along ducts and included such features as columnar alteration, micropapillary atypia, and fibroadenomatous change. In situ carcinomas (n = 8) included solid, comedo, cribriform, and micropapillary elements, encompassing 4 of the major architectural patterns seen in human lesions. Invasive ductal carcinomas (n = 8) were generally solid, with prominent central necrosis and mineralization, often on a background of micropapillary ductal hyperplasia and in situ carcinoma. Cytologic changes of invasive lesions included increased mitoses, nuclear pleomorphism, extensive microinvasion, and stromal desmoplasia. Axillary lymph-node metastases were confirmed in 5 of the 8 invasive carcinomas. On immunohistochemistry, intraductal and invasive carcinomas had increased Ki67/MIB1 and HER2 expression and selective loss of estrogen and progesterone receptors. These findings suggest that breast cancer is an underreported lesion in macaques and highlight unique morphologic and molecular similarities in breast cancer between human and macaque species.

Colonic spirochetosis of colony-raised rhesus macaques associated with Brachyspira and Helicobacter.

Colonic spirochetosis is an inflammatory bowel disease that affects a broad range of hosts, including human and non-human primates. The disease in humans and non-human primates is characterized by intimate attachment of the anaerobic spirochetes Brachyspira aalborgi and B. pilosicoli, and some unclassified flagellated microbes along the apical membrane of colonic enterocytes. Although the presence of spiral-shaped bacteria with single polar flagella and blunted ends in colonic spirochetosis is well established, the identities of many of these organisms is still unknown. Recently, Helicobacter species with a morphology similar to the flagellated bacteria present in colonic spirochetosis have been cultured from intestinal specimens obtained from rhesus macaques, some with idiopathic colitis. The purpose of the present study was to determine whether or not the flagellated bacteria seen in the colons of rhesus macaques with colonic spirochetosis are Helicobacter. The presence of flagellated bacteria alone (n=2) or together with spirochetes (n=1) in formalin-fixed and paraffin-embedded colons of three rhesus macaques with the naturally occurring disease was demonstrated by immunohistochemical staining and ultrastructural examination. Total DNA extracted from affected and control intestinal specimens was amplified by polymerase chain reaction (PCR) using Helicobacter 16S rRNA gene-specific primers. Comparative nucleotide sequence analysis of PCR products cloned from positive reactions indicated that two distinct Helicobacter genomospecies were present either alone or in combination with Brachyspira in the colons of rhesus macaques with microscopic lesions indicative of colonic spirochetosis.

Two cases of digital defects in Macaca mulatta infants and a survey of the literature.

Although congenital digital defects, particularly polydactyly, have been reported frequently in humans, their occurrence in rhesus macaques is relatively rare. We observed two cases of spontaneous digital defects in male rhesus monkey infants recently born at the California Regional Primate Research Center. One infant exhibited bilateral postaxial polydactyly and the other infant had bilateral oligodactyly with unilateral phalangeal duplication. In this report, we present the clinical/pathological details of these cases as well as discuss the embryology of normal and abnormal limb development. We will also summarize a variety of spontaneous and experimentally induced digital defects that have been reported in several nonhuman primate species.

Spontaneous rat bite fever in non-human primates: a review of two cases.

Rat bite fever is a worldwide zoonotic, non-reportable disease. This entity encompasses similar, yet distinct, disease syndromes caused by Streptobacillus moniliformis or Spirillum minus. Naturally occurring rat bite fever has not been previously described in non-human primates. This report describes two cases of non-human primate rat bite fever caused by S. moniliformis; a rhesus macaque (Macaca mullata) with valvular endocarditis, and a titi monkey (Callicebus sp.) with septic arthritis. Potential sources of infection included direct contact, and ingestion of surface water or feed contaminated with rodent feces.

Effect of dioxin on ovarian function in the cynomolgus macaque (M. fascicularis).

Ovarian function was evaluated in mature female cynomolgus macaques 443 to 625 days following a single oral exposure (1, 2, or 4 microg/kg BW) to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). Urinary estrone conjugates (E1C), pregnanediol-3-glucuronide (PdG), and follicle stimulating hormone (FSH) were measured. Three of four animals in the high dose group had no evidence of menstrual cycles while animals in the low and medium dose groups plus one from the high dose group had cycles that were similar to those of control animals. The noncycling animals had baseline E(1)C concentrations without ovulatory midcycle peaks and monotonic PdG profiles. Mean FSH concentrations during the midfollicular phase of the medium dose group and during the entire cycle of the high dose group were elevated compared to those of the control group and the endometria of the noncycling animals were inactive. These data demonstrate that a single exposure of 4 microg/kg BW TCDD leads to long-term adverse effects on ovarian function in primates.

Exposure to the dioxin 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) induces squamous metaplasia in the endocervix of cynomolgus macaques.

Female cynomolgus macaques (n = 11) were treated orally with graded doses of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). Cervical tissue was recovered at necropsy 1.2-2.7 years later and examined using routine histopathology. Results were compared histologically with cervical tissue from untreated, age- and parity-matched controls. Significant squamous epithelial metaplasia was observed in the endocervix of 9 of 11 TCDD-treated animals, and the degree of severity was dose dependent. In contrast, minimal or no pathological changes were observed in eight of nine control animals and one animal had only mild squamous metaplasia. These results suggest that TCDD exposure induces epithelial transdifferentiation in the primate cervix. Consequently, the TCDD-treated macaque may serve as a predictable animal model for the study of cervical epithelial transdifferentiation and for examining the relationship between squamous metaplasia and cervical oncogenesis both at the cellular and at the molecular level.

Spontaneous intestinal adenocarcinoma in geriatric macaques (Macaca sp.).

BACKGROUND AND PURPOSE: Intestinal adenocarcinoma appears to be the most common malignant neoplasm in macaques, and is a substantial cause of morbidity and mortality in the elderly. METHODS: A retrospective review of 32 cases was done. RESULTS: Thirty-two cases were reviewed. Clinical examination had revealed severe weight loss, anorexia, and palpable abdominal mass. Microcytic hypochromic anemia, intermittent fecal occult blood positive test results, hypoproteinemia, and hypoalbuminemia were the predominant clinical laboratory findings. Carcinoembryogenic antigen serologic testing and single-strand conformational polymorphism analysis were performed in selected cases. The most common sites of the intestinal adenocarcinoma were ileocecal junction, colon, ileum, jejunum, and cecum. Metastases were evident in 34% of the cases and involved peripheral nodes, liver, lungs, pancreas, and adrenal gland. Overall survival of 12 macaques that underwent surgical excision was 83% at 6 months, 58% at 1 year, 50% at 1.5 years, 33% at 2 years, and 8% at 4 years. The overall mean survival rate (MSR) was > 483 postoperative days. CONCLUSION: Intestinal adenocarcinomas should be amenable to surgical resection. Early detection of localized, non-invasive neoplasms will increase surgical cure rate. Survivability could be potentially improved by use of adjuvant therapies.

Isolation and characterization of an antigenically distinct 68-kd protein from nonviral intracytoplasmic inclusions in Boa constrictors chronically infected with the inclusion body disease virus (IBDV: Retroviridae).

The relationship between a retroviral infection and the development of nonviral intracytoplasmic inclusion bodies was studied in a Boa constrictor model. Twelve juvenile age- and size-matched inclusion body disease (IBD)-negative boas were randomly divided into three groups. Each group was inoculated intraperitoneally with 1 ml of an IBD virus (IBDV)-infected liver homogenate or 1 ml of normal boa liver homogenate (sham-inoculated control) or was left untreated. All boas were monitored for development of IBD by daily examination and serial liver biopsy over 1 year. The 4 IBDV-inoculated boas became IBDV and inclusion positive by 10 weeks postinoculation. The average size and density of inclusion bodies increased with the duration of infection. Ultrastructurally, inclusion bodies <2 microm in diameter consisted of intracytoplasmic aggregates of granular electron-dense material that were not membrane limited. Larger inclusions (3-6 microm in diameter) were characterized as membrane-bound aggregates of amorphous to granular electron-dense material admixed with membranelike fragments. The sham-inoculated and untreated control snakes did not become inclusion or IBDV positive. Direct comparison of the protein electrophoretograms of IBDV-infected and normal boa tissues demonstrated a prominent 68-kd protein band unique to infected inclusion-positive tissues. Monoclonal antibodies directed against the 68-kd protein band specifically labeled inclusion bodies. The results of this study demonstrate that IBD inclusions represent an intracytoplasmic accumulation of an antigenically distinct IBDV-associated protein.

Prophylactic and therapeutic benefits of short-term 9-[2-(R)-(phosphonomethoxy)propyl]adenine (PMPA) administration to newborn macaques following oral inoculation with simian immunodeficiency virus with reduced susceptibility to PMPA.

Simian immunodeficiency virus (SIV) infection of newborn macaques is a useful animal model of human pediatric AIDS to study pathogenesis and to develop intervention strategies aimed at preventing infection or delaying disease progression. In previous studies, we demonstrated that 9-¿2-(R)-(phosphonomethoxy)propyladenine (PMPA; tenofovir) was highly effective in protecting newborn macaques against infection with virulent wild-type (i.e., drug-susceptible) SIVmac251. In the present study, we determined how reduced drug susceptibility of the virus inoculum affects the chemoprophylactic success. SIVmac055 is a virulent isolate that has a fivefold-reduced in vitro susceptibility to PMPA, associated with a K65R mutation and additional amino acid changes (N69T, R82K, A158S, S211N) in reverse transcriptase (RT). Eight newborn macaques were inoculated orally with SIVmac055. The three untreated control animals became SIVmac055 infected; these animals had persistently high viremia and developed fatal immunodeficiency within 3 months. Five animals were treated once daily with PMPA (at 30 mg/kg of body weight) for 4 weeks, starting 24 h prior to oral SIVmac055 inoculation. Two of the five PMPA-treated animals had no evidence of infection. The other three PMPA-treated infant macaques became infected but had a delayed viremia, enhanced antiviral antibody responses, and a slower disease course (AIDS in 5 to 15 months). No reversion to wild-type susceptibility or loss of the K65R mutation was detected in virus isolates from any of the PMPA-treated or untreated SIVmac055-infected animals. Several additional amino acid changes developed in RT, but they were not exclusively associated with PMPA therapy. The results of this study suggest that prophylactic administration of PMPA to human newborns and to adults following exposure to human immunodeficiency virus will still be beneficial even in the presence of viral variants with reduced susceptibility to PMPA.

9-[2-(Phosphonomethoxy)propyl]adenine (PMPA) therapy prolongs survival of infant macaques inoculated with simian immunodeficiency virus with reduced susceptibility to PMPA.

Simian immunodeficiency virus (SIV) infection of newborn rhesus macaques is a useful animal model of human immunodeficiency virus infection for the study of the emergence and clinical implications of drug-resistant viral mutants. We previously demonstrated that SIV-infected infant macaques receiving prolonged treatment with 9-[2-(phosphonomethoxy)propyl]adenine (PMPA) developed viral mutants with fivefold reduced susceptibility to PMPA in vitro and that the development of these mutants was associated with the development of a K65R mutation and additional compensatory mutations in reverse transcriptase (RT). To study directly the virulence and clinical implications of these SIV mutants, two uncloned SIVmac isolates with similar fivefold reduced in vitro susceptibilities to PMPA but distinct RT genotypes, SIVmac055 (K65R, N69T, R82K A158S,S211N) and SIVmac385 (K65R, N69S, I118V), were each inoculated intravenously into six newborn rhesus macaques; 3 weeks later, three animals of each group were started on PMPA treatment. All six untreated animals developed persistently high levels of viremia and fatal immunodeficiency within 4 months. In contrast, the six PMPA-treated animals, despite having persistently high virus levels, survived significantly longer: 5 to 9 months for the three SIVmac055-infected infants and > or = 21 months for the three SIVmac385-infected infants. Virus from only one untreated animal demonstrated reversion to wild-type susceptibility and loss of the K65R mutation. In several other animals, additional RT mutations, including K64R and Y115F, were detected, but the biological role of these mutations is unclear since they did not affect the in vitro susceptibility of the virus to PMPA. In conclusion, this study demonstrates that although SIVmac mutants with the PMPA-selected K65R mutation in RT were highly virulent, PMPA treatment still offered strong therapeutic benefits. These results suggest that the potential emergence of HIV mutants with reduced susceptibility to PMPA in patients during prolonged PMPA therapy may not eliminate its therapeutic benefits.

Early short-term 9-[2-(R)-(phosphonomethoxy)propyl]adenine treatment favorably alters the subsequent disease course in simian immunodeficiency virus-infected newborn Rhesus macaques.

Simian immunodeficiency virus (SIV) infection of newborn macaques is a useful animal model of human pediatric AIDS to study disease pathogenesis and to develop intervention strategies aimed at delaying disease. In the present study, we demonstrate that very early events of infection greatly determine the ultimate disease course, as short-term antiviral drug administration during the initial viremia stage significantly delayed the onset of AIDS. Fourteen newborn macaques were inoculated orally with uncloned, highly virulent SIVmac251. The four untreated control animals showed persistently high virus levels and poor antiviral immune responses; they developed fatal immunodeficiency within 15 weeks. In contrast, SIV-infected newborn macaques which were started on 9-[2-(R)-(phosphonomethoxy)propyl]adenine (PMPA) treatment at 5 days of age and continued for either 14 or 60 days showed reduced virus levels and enhanced antiviral immune responses. This short-term PMPA treatment did not induce detectable emergence of SIV mutants with reduced in vitro susceptibility to PMPA. Although viremia increased in most animals after PMPA treatment was withdrawn, all animals remained disease-free for at least 6 months. Our data suggest that short-term treatment with a potent antiviral drug regimen during the initial viremia will significantly prolong AIDS-free survival for HIV-infected infants and adults.

Morphometric studies of myelination in the spinal cord of mice exposed developmentally to aluminum.

Swiss-Webster mice were exposed to diets containing 7 or 1000 microg aluminum (Al)/g as Al lactate from conception through maturity (45 days of age). This exposure has previously been shown to cause changes in CNS myelin composition and peroxidizability; in this study myelin sheath widths were measured. Initially, samples of epon embedded, toluidine blue stained cervical spinal cord sectioned at 0.5 mm were examined light microscopically. Qualitatively, Al-treated mice appeared to have a diffuse paleness in nerve tracts. No indication of myelin structural damage (splitting, degeneration) was noted. Quantitative microscopy was performed using images captured with Scion Image Dage 1.59 at 1000x with oil. Axon perimeters and sheaths were measured with NIH image using a standardized sampling pattern in the right medial dorsal and ventral regions of the cervical spinal cord in 6 mice (3 male, 3 female) per group. Mean myelin sheath widths were 16% smaller in the Al-treated group compared to controls (p=.03). There was no effect of sex or region (dorsal/ventral). Axon perimeters were also smaller on the average in the Al treated group but this difference was not significant (p=.16). The relationship between sheath width and axon diameter was similar in the two groups. The density of myelinated axons was greater in some areas for the Al-treated group. The data indicate that dietary aluminum exposure can interfere with myelination in the spinal cord.

Spontaneous leprosy in a wild-caught cynomolgus macaque.

Naturally occurring Mycobacterium leprae has been previously documented in only two species of nonhuman primates from West Africa--the chimpanzee and the sooty mangabey. We report here the first known case of spontaneous leprosy in an Asian macaque. A wild-caught cynomolgus macaque imported from The Philippines developed a reaction to a tuberculin skin test after 3 years at the California Regional Primate Research Center (CRPRC), University of California-Davis, Davis, California, U.S.A. Biopsies of concurrent skin lesions suggested a cutaneous mycobacterial infection. Diagnosis of the infection was obtained by a polymerase chain reaction (PCR) assay specific for M. leprae. Clinical presentation, histopathological findings, and ELISA serology for M. leprae-specific PGL-I and to the LAM mycobacterial antigens were consistent with those of human borderline (BB) leprosy. Longitudinal serologic data suggest that the cynomolgus macaque had subclinical leprosy at the time of arrival in the CRPRC quarantine. Intradermal tuberculin testing is the traditional method for screening nonhuman primate populations for mycobacterial infections. Exposure to nontuberculous mycobacteria, such as M. leprae, amy sensitize some individual primates to nonspecific mycobacterial antigens, resulting in false-positive tuberculin reactions. Susceptibility of the cynomolgus macaque and other nonhuman primates to M. leprae should be re-evaluated. Cynomolgus macaques and, possibly, other nonhuman primates may serve as valuable experimental models of leprosy in humans.

Passive immunization of newborn rhesus macaques prevents oral simian immunodeficiency virus infection.

To determine if passively acquired antiviral antibodies modulate virus transmission and disease progression in human pediatric AIDS, the potential of pre- and postexposure passive immunization with hyperimmune serum to prevent oral simian immunodeficiency virus (SIV) infection or disease progression in newborn rhesus macaques was tested. Untreated neonates became infected after oral SIV inoculation and had high viremia, and most animals developed fatal AIDS within 3 months. In contrast, SIV hyperimmune serum given subcutaneously prior to oral SIV inoculation protected 6 newborns against infection. When this SIV hyperimmune serum was given to 3 newborns 3 weeks after oral SIV inoculation, viremia was not reduced, and all 3 infants died within 3 months of age due to AIDS and immune-complex disease. These results suggest that passively acquired antihuman immunodeficiency virus (HIV) IgG may decrease perinatal HIV transmission. However, anti-HIV IgG may not impart therapeutic benefit to infants with established HIV infection.

Recombinant bacille Calmette-Guérin expressing the measles virus nucleoprotein protects infant rhesus macaques from measles virus pneumonia.

Measles virus infection continues to be a major cause of infant mortality. There is a need for a measles vaccine that can be administered at birth in the presence of maternal neutralizing antibody. Infant rhesus monkeys were immunized with recombinant bacille Calmette-Guérin expressing the full-length measles virus nucleoprotein (BCG-N) and subsequently challenged with measles virus. Nucleoprotein-specific lymphocyte proliferative responses were detected in the absence of anti-N antibody after vaccination. Vaccination with BCG-N did not prevent systemic measles virus infection; however, there was a significant reduction of lung inflammation after challenge. Virus titers in lymph nodes were significantly lower, and the duration of nasopharyngeal viral shedding was shorter in some vaccinated monkeys after challenge. These results suggest that measles virus-specific T cells were primed by BCG-N vaccination and that they prevented virus-induced lung pathology.

A zidovudine-resistant simian immunodeficiency virus mutant with a Q151M mutation in reverse transcriptase causes AIDS in newborn macaques.

The simian immunodeficiency virus (SIV)-newborn rhesus macaque model of AIDS can be used to study directly the virulence of viral mutants which are resistant to antiviral drugs. A viral mutant called SIVmac79A6.1, isolated from an SIV-infected macaque after prolonged zidovudine treatment, was found to have a double-base-pair change at codon 151 of reverse transcriptase, resulting in a glutamine to methionine substitution (Q151M). This mutation was associated with more than 100-fold increased resistance to zidovudine and low-level cross-resistance to other dideoxynucleoside analogs. To determine whether this Q151M mutation affects viral virulence, four newborn macaques were inoculated intravenously with a biological clone of this drug-resistant SIVmac79A6.1 mutant; two of these animals were also treated orally with zidovudine. All four animals showed persistent viremia, and two of the four animals developed fatal immunodeficiency at 3 and 8 months of age, respectively. The remaining two animals had CD4+ T-cell depletion and clinical symptoms of AIDS at 22 months. No phenotypic or genotypic reversion of virus to the wild type could be detected in any of the four animals. These results demonstrate that the Q151M mutation in SIV reverse transcriptase does not reduce viral virulence.

Frequency of spontaneous congenital defects in rhesus and cynomolgus macaques.

This paper summarizes the spontaneous incidence of congenital defects in the rhesus and cynomolgus macaque colonies (Macaca mulatta and M. fascicularis) at the California Regional Primate Research Center. The computerized database used in this analysis included fetuses, term infants, juveniles, and adults that underwent a necropsy procedure over a 14-year period (1983-1996). The calculated malformation rates were 0.9% (40/4,390) and 0.3% (3/965) for the rhesus and cynomolgus monkey, respectively. Most of the observed malformations in both species affected the musculoskeletal and the cardiovascular systems, while a smaller number of defects were observed in the gastrointestinal, urogenital, endocrine, and central nervous systems. Inbreeding did not contribute to the spontaneous malformation incidence and there was no predilection for sex (male vs. female) or housing (indoors vs. outdoors) among the malformed cases. This spontaneous malformation database in our macaque colony aids in the interpretation of defects that occur in an experimental study as well as in the ongoing assessment of a healthy nonhuman primate breeding colony.

9-[2-(Phosphonomethoxy)propyl]adenine therapy of established simian immunodeficiency virus infection in infant rhesus macaques.

The long-term therapeutic and toxic effects of 9-[2-(phosphonomethoxy)propyl]adenine (PMPA) were evaluated in simian immunodeficiency virus (SIV)-infected newborn rhesus macaques. Four untreated SIV-infected newborn macaques developed persistently high levels of viremia, and three of the four animals had rapidly fatal disease within 3 months. In contrast, long-term PMPA treatment of four newborn macaques starting 3 weeks after virus inoculation resulted in a rapid, pronounced, and persistent reduction of viremia in three of the four animals. Emergence of virus with fivefold-decreased susceptibility to PMPA occurred in all four PMPA-treated animals and was associated with the development of a lysine-to-arginine substitution at amino acid 65 (K65R mutation) and additional mutations in the reverse transcriptase; however, the clinical implications of this low-level drug resistance are nuclear. No toxic side effects have been seen, and all PMPA-treated animals have remained disease-free for more than 13 months. Our data suggest that PMPA holds much promise for the treatment of human immunodeficiency virus-infected human infants and adults.