In vivo mammary tumourigenesis in the Sprague-Dawley rat and microdosimetric correlates.

Standard methods for risk assessments resulting from human exposures to mixed radiation fields in Space consisting of different particle types and energies rely upon quality factors. These are generally defined as a function of linear energy transfer (LET) and are assumed to be proportional to the risk. In this approach, it is further assumed that the risks for single exposures from each of the radiation types add linearly. Although risks of cancer from acute exposures to photon radiations have been measured in humans, quality factors for protons and ions of heavier atomic mass are generally inferred from animal and/or cellular data. Because only a small amount of data exists for such particles, this group has been examining tumourigenesis initiated by energetic protons and iron ions. In this study, 741 female Sprague-Dawley rats were irradiated or sham irradiated at approximately 60 days of age with 250 MeV protons, 1 GeV/nucleon iron ions or both protons and iron ions. The results suggest that the risk of mammary tumours in the rats sequentially irradiated with 1 GeV/nucleon 56Fe ions and 250 MeV protons is less than additive. These data in conjunction with earlier results further suggest that risk assessments in terms of only mean LETs of the primary cosmic rays may be insufficient to accurately evaluate the relative risks of each type of particle in a radiation field of mixed radiation qualities.

National Institutes of Health--supported opportunities in Comparative Medicine.

The Comparative Medicine (CM) area of the National Institutes of Health (NIH) is a major source of support for research on laboratory animals, training of laboratory animal specialists, and support of shared, regional animal resources. We present a brief history of CM at NIH and the major mechanisms by which it accomplishes its goals in programs located across the United States.

Cestodiasis with intestinal diverticulosis in a lesser flamingo (Phoeniconaias minor).

An adult female lesser flamingo (Phoeniconaias minor), caught in the African Rift Valley in 1991 and subsequently housed at the Baltimore Zoo, died of severe visceral gout in 1996. Necropsy revealed a white, moderately firm, nodular lesion, 1 cm in diameter, in the serosal wall of the small intestine. Although it was initially thought to be a tumor or focal granuloma, histologic examination revealed multiple cestodes deeply embedded at the base of the crypts between the intestinal villi, with their massive scolices (up to 3.4 mm in diameter) distending these spaces into multiple diverticulae. The mucosal epithelium surrounding the scolices was severely attenuated. Around the diverticulae, in the submucosa and muscularis, was a mild to moderate lymphocytic reaction and mild fibrosis. The proximity of multiple scolices and extensive invasion of host tissue suggested that the infection occupied a preexisting lesion. The cestodes were cyclophyllids but were distinct from any species previously reported from flamingos. Helminths should be included in differential diagnoses for gastrointestinal nodules in flamingos.

Nontraumatic osteonecrosis: MR perfusion imaging evaluation in an experimental model.

RATIONALE AND OBJECTIVES: Because the nature and time course of changes in early, nontraumatic osteonecrosis at perfusion and magnetic resonance (MR) imaging are unknown, the authors evaluated this technique in the assessment of early osteonecrosis with a nontraumatic model. MATERIALS AND METHODS: Five rabbits underwent intravenous injection of lipopolysaccharide endotoxin followed by intramuscular injection of methylprednisolone. MR imaging of the femora was performed before and at weekly intervals after endotoxin injection. Histologic findings from the areas of osteonecrosis were correlated with the findings of MR imaging and MR perfusion studies. RESULTS: Histologic evaluation showed osteonecrosis in six femora of four animals 2-4 weeks after endotoxin injection. Findings on T1-weighted images of the femur were normal in all animals; T2-weighted images of one femur showed equivocal changes. On MR perfusion images, the baseline mean peak percentage of enhancement was 52.7% +/- 12.6. In the six areas without osteonecrosis, the mean percentage of enhancement was similar to the baseline percentage of enhancement at 1 week (62.2% +/- 31.2). In the four areas with diffuse osteonecrosis, there was essentially no contrast enhancement 1-4 weeks after endotoxin injection. CONCLUSION: T1- and T2-weighted MR imaging is insensitive to the presence of early nontraumatic osteonecrosis. MR perfusion imaging might be useful to detect early nontraumatic osteonecrosis.

Cholesterol granulomas in mice deficient in apolipoprotein E.

Three 12- to 16-month-old female B6,129 apolipoprotein E-deficient mice were presented for necropsy because of bilateral masses in the gluteal region. Histopathologic examination revealed cholesterol granulomas extending from the superficial dermis to the underlying gluteal muscles. Microscopic granulomas were found on the face, perivaginal tissues, mesenteric lymph nodes, spleen, joints, kidneys, and choroid plexus. Other lesions included severe atherosclerosis of the aortic valves, aorta, pulmonary artery, and renal artery. Here we show that macroscopic cholesterol granulomas can develop in apolipoprotein E-deficient that receive a normal rodent diet.

Processing postmortem specimens with C18-carboxypropylbetaine and analysis by PCR to develop an antemortem test for Mycobacterium avium infections in ducks.

Mycobacterium avium is the causative agent of the avian mycobacteriosis commonly known as avian tuberculosis (ATB). This infection causes disseminated disease, is difficult to diagnose, and is of serious concern because it causes significant mortality in birds. A new method was developed for processing specimens for an antemortem screening test for ATB. This novel method uses the zwitterionic detergent C18-carboxypropylbetaine (CB-18). Blood, bone marrow, bursa, and fecal specimens from 28 ducks and swabs of 20 lesions were processed with CB-18 for analysis by smear, culture, and polymerase chain reaction (PCR). Postmortem examination confirmed nine of these birds as either positive or highly suspect for disseminated disease. The sensitivities of smear, culture, and PCR, relative to postmortem analysis and independent of specimen type, were 44.4%, 88.9%, and 100%, respectively, and the specificities were 84.2%, 57.9%, and 15.8%, respectively. Reductions in specificity were due primarily to results among fecal specimens. However, these results were clustered among a subset of birds, suggesting that these tests actually identified birds in early stages of the disease. Restriction fragment length polymorphism mapping identified one strain of M. avium (serotype 1) that was isolated from lesions, bursa, bone marrow, blood, and feces of all but three of the culture-positive birds. In birds with confirmed disease, blood had the lowest sensitivity and the highest specificity by all diagnostic methods. Swabs of lesions provided the highest sensitivity by smear and culture (33.3% and 77.8%, respectively), whereas fecal specimens had the highest sensitivity by PCR (77.8%). The results of this study indicate that processing fecal specimens with CB-18, followed by PCR analysis, may provide a valuable first step for monitoring the presence of ATB in birds.

Feline asthma syndrome in African lions (Panthera leo).

Feline asthma syndrome, previously recognized only in domestic cats, was diagnosed in three captive African lions (Panthera leo), one of which died as a result of the condition. Two of the lions displayed progressive signs for 7 yr, including severe bouts of coughing, wheezing, dyspnea, rhonchi, and tachypnea that were most severe during the spring and summer, and the third lion displayed acute signs only once. Scattered to diffuse increased interstitial markings, peribronchial cuffing, and focal atelectasis were visible in radiographs. At necropsy, multiple subpleural bullae, 2-3 cm in diameter, were scattered throughout the lung tissue. There were thick-walled bronchi and bronchioles filled with thick grayish mucus, and alveolar spaces were enlarged with severe, diffuse, banded multifocal areas of alveolar wall fibrosis. The lions had significantly elevated IgE type I immediate hypersensitivity responses to recognized aeroallergens. The captive management of lions should address the design and maintenance of allergen-free air supplies. Ventilation systems should be examined routinely and thoroughly cleaned of any residue. The frequency of examination should increase during the summer. Lions and other large cats should be routinely screened for IgE aeroallergen-specific titers, asthma cases should be treated promptly with prednisolone, and investigations of etiology should be initiated.

Comparison of acute endotoxin-induced lesions in A/J and C57BL/6J mice.

Resistance to the action of endotoxin varies among inbred strains of mice, indicating that a component of this resistance has a genetic basis. Different responses to endotoxin that are characteristic of individual inbred strains represent phenotypes that can be used to genetically map the response modifier genes. This study compares the acute histologic lesions in 8-week-old male A/J and C57BL/6J (B6) mice injected intraperitoneally with endotoxin of E. coli O265:B6 (15 mg/kg). Animals of both strains exhibited splenitis, splenic lymphoid hyperplasia, splenic lymphoid necrosis, and sequestration of neutrophils in the pulmonary alveoli. The B6 mice showed increased margination of white blood cells to the pulmonary vascular endothelium relative to A/J mice. A large number of degenerating neutrophils was observed in the liver sinusoids of most B6 animals, while this lesion was much less severe in A/J mice. This difference was quantified, demonstrating a highly significant difference in neutrophil infiltration in B6 mice relative to A/J mice. Analysis of this phenotype in F1 mice demonstrates that major genes encoding the trait are not X-linked, imprinted, or maternally inherited and do not show the codominant inheritance expected if Lps(d) were primarily responsible. The distinctive, quantitative nature of these differences provides a useful assay for mapping genes that modify endotoxin responsiveness using the AXB and BXA recombinant inbred (RI) strains derived from A/J and B6 mice.

Gongylonema macrogubernaculum in captive African squirrels (Funisciurus substriatus and Xerus erythropus) and lion-tailed macaques (Macaca silenus).

Necropsies performed between 1989 and 1995 on 15 African rope squirrels (Funisciurus substriatus) and 20 African ground squirrels (Xerus erythropus) from the Baltimore Zoo revealed 13 cases of gongylonemiasis. Nematodes were embedded in the epithelium of the esophagus, pharynx, buccal mucosa, and tongue, resulting in varying degrees of esophagitis, pharyngitis, stomatitis, and glossitis, respectively. Routine fecal examinations were negative, and the nematodes appeared to be unaffected by repeated treatments with ivermectin. Most of the affected animals had shown clinical signs of dyspnea and/or inanition and emaciation. Suppurative rhinitis was also a frequent finding at necropsy and was associated with the presence of the nematodes in eight animals. Dissection of whole nematodes from formalin-fixed specimens revealed morphologic features consistent with Gongylonema macrogubernaculum, a species previously only reported in nonhuman primates. The squirrels were housed in the same building with numerous primate species, and a review of pathology records revealed esophageal gongylonemiasis in three lion-tailed macaques (Macaca silenus), lingual gongylonemiasis in a spotnose monkey (Cercopithecus buettikoferi), and buccal gongylonemiasis in a brown-headed tamarin (Saguinus fuscicollis). Examination of whole nematodes dissected from one of the lion-tailed macaques also demonstrated the unique morphology of G. macrogubernaculum. Nematodes belonging to the species Gongylonema are acquired by ingestion of the intermediate host, the cockroach. This is the first report of G. macrogubernaculum in a nonprimate species and suggests that captive African squirrels can serve as reservoir hosts for this parasite in a zoo environment.

Age-dependent and lobe-specific spontaneous hyperplasia in the brown Norway rat prostate.

We showed previously that exogenously administered testosterone caused age- and lobe-specific overgrowth of the prostate in Brown Norway rats. A common feature observed in testosterone-treated animals was cell hypertrophy in each of the ventral, dorsal, and lateral lobes of both young (6 mo old) and old (24 mo old) rats. By contrast, hyperplasia was seen only in the dorsal and lateral lobes of old rats treated with testosterone. These observations prompted us to examine whether age- and lobe-specific overgrowth might also occur in untreated rats as a consequence of the endogenous hormonal milieu. To this end, blood and prostates were collected from a large number (25-30 rats per group) of 4- to 6-mo-old (young) and 21- to 24-mo-old (old) Brown Norway rats. Both serum testosterone (-45%) and estradiol (-22%) concentrations decreased significantly with age, but the greater magnitude of the decrement in testosterone relative to estradiol led to a reduction in the serum testosterone:estradiol ratio. Paradoxically, although the prostate is androgen dependent, the wet weight, protein, and DNA contents increased significantly with age in the dorsal and lateral lobes of old rats despite the decrease in testosterone level. Histologic examination revealed that the increased weights and DNA contents of the dorsal and lateral lobes in old rats coincided with an increased number of epithelial cells in the distal and intermediate segments of these lobes, indicative of hyperplasia but independent of change in cell size. Taken together, these results show a spontaneous age-related overgrowth of cells in the dorsal and lateral prostatic lobes of old Brown Norway rats despite diminished serum testosterone concentrations. The aging Brown Norway rat, therefore, may be a useful model for studies of some aspects of the pathogenesis underlying spontaneous age-related prostatic hyperplasia.

Neuronal nitric oxide synthase in the canine prostate: aging, sex steroid, and pathology correlations.

Nitric oxide synthase (NOS) is expressed in the prostate of various species, including humans. NOS catalyzes the production of nitric oxide (NO), which may function in prostatic smooth-muscle relaxation. To investigate further the role of NO in the prostate, we examined neuronal NOS expression in the aging canine prostate, after hormonal perturbation, and correlated these results with histopathologic findings. The study comprised the following treatment groups: intact dogs (treatment group 1, n = 6); dogs who were castrated at 7 days of age and received testosterone and estrogen replacement at 2 years of age (treatment group 2, n = 10); and dogs who were castrated at 2 years of age and received testosterone and estrogen replacement at 2 years of age (treatment group 3, n = 9). Studies were done on prostates removed from dogs after euthanasia at 6 years of age. In treatment group 1, complex benign prostatic hyperplasia (BPH) was observed in all specimens. In treatment group 2, atrophy was observed in 70%, normal prostate with small areas of hyperplasia in 20%, and BPH in 10% of specimens. In treatment group 3, atrophy was observed in 78%, normal histology with small areas of hyperplasia in 11%, and BPH in 11% of specimens. Neuronal NOS localizations were confirmed by western blot analysis and by immunohistochemistry in 0% and 17%, respectively, of specimens in treatment group 1, in 60% and 70%, respectively, of specimens in treatment group 2, and in 67% and 71%, respectively, of specimens in treatment group 3. Neuronal NOS immunoreactivity was localized in histologically normal prostates of four intact, young-adult control dogs (2 years of age). For all treatment groups, neuronal NOS immunoreactivity was confirmed by western blot in 86% of atrophic prostates but in no prostates with BPH (P < 0.001), and it was confirmed by immunohistochemistry in 75% of atrophic prostates but in only 13% of prostates with BPH (P < 0.02). These data suggest that, in the canine prostate, NO release relates to growth and pathology. Low levels of neuronal NOS expression in BPH tissue, compared with higher levels in atrophic tissue, suggest that neuronal NOS expression is down-regulated in the prostate with benign cellular proliferation whereas it is maintained or possibly up-regulated in the prostate with prostatic involution. Whether altered neuronal NOS expression contributes to the pathogeneses of BPH and prostatic involution or whether it occurs as a consequence of these processes requires further investigation.

Endothelial cells from diverse tissues exhibit differences in growth and morphology.

An increased recognition of the role of endothelial cells in disease and the development of methods for endothelial cell culture has led to an upsurge in in vitro studies of endothelial cell function. However, the cells most often used for these studies do not reflect the in vivo heterogeneity of endothelial cells. To assess intrinsic differences between large and small vessel endothelial cells from different tissues, primary cultures of endothelial cells from capillaries (brain, lung, and adipose tissue) and a large vessel (aorta) of sheep were isolated, purified by fluorescence-activated cell sorting of acetylated low density lipoprotein (DiI-Ac-LDL) labeled cells, and characterized by phase contrast and ultrastructural morphology, expression of von Willebrand factor, and lack of expression of cytokeratin, smooth muscle actin, and glial fibrillary acidic protein (GFAP). Although all endothelial cells were cultured in the same media, only the brain microvascular endothelial cells demonstrated tight junctions by electron microscopy. Only the large vessel (aortic) endothelial cells contained Weibel-Palade bodies. Expression of von Willebrand factor decreased with passage of cells, but uptake of DiI-Ac-LDL was consistently positive regardless of culture conditions or passage number. These studies demonstrate that the unique ultrastructural characteristics of microvascular and macrovascular endothelial cells are intrinsic to the cells themselves and are not determined by differential culture conditions. This system allows the study of pathologic processes that affect endothelial cells of certain target organs selectively and should more accurately represent the response of tissue-specific endothelial cells in inflammatory processes.

Fatal cryptosporidiosis in a juvenile captive African hedgehog (Ateletrix albiventris).

Fatal intestinal cryptosporidiosis of unknown source and unexplained epizootiology is reported in a neonatal captive African hedgehog (Ateletrinx albiventris) and for the first time in a hedgehog species. The infection, confined to ileum, jejunum, and colon, was extremely severe in the lower jejunum where over 75% of the epithelial cells harbored the pathogen. The ileum and the jejunum displayed moderate and severe villus atrophy and mucosal hyperplasia. Lamina propria and mucosa were infiltrated by eosinophils, lymphocytes, and macrophages. Developmental stages of Cryptosporidium sp. produced a positive reaction with immunofluorescent antibody for detection of the human pathogen, Cryptosporidium parvum. Personnel of captive centers with hedgehogs should be alerted and undertake appropriate precautions to prevent zoonotic transmission. Commercially offered hedgehog-pets may pose a risk for Cryptosporidium infection for human immunodeficiency virus-infected people.

Intestinal Cryptosporidium sp. infection in the Egyptian tortoise, Testudo kleinmanni.

An adult Egyptian tortoise (Testudo kleinmanni) presented with clinical signs of enteritis and died 5 weeks after initiation of antibiotic therapy. Histological examination of the small intestine revealed heavy infection with Cryptosporidium sp.; over 80% of epithelial cells harboured the pathogen. No Cryptosporidium developmental stages were present in the stomach or the lungs. The intestinal lamina propria and mucosa were infiltrated by heterophils, lymphocytes and macrophages. The present study constitutes the first report of Cryptosporidium sp. infection in T. kleinmanni, and the first histological documentation of intestinal cryptosporidiosis in Chelonia.

Differential replication of ovine lentivirus in endothelial cells cultured from different tissues.

Blood-brain barrier dysfunction has been postulated to be important in the pathogenesis of HIV dementia. This study used an in vitro model of the blood-brain barrier to determine the effects of ovine lentivirus (OvLV) infection on endothelial cells. The replication of two American OvLV isolates and two lcelandic OvLV isolates in pure cultures of endothelial cells isolated from brain was compared to replication in endothelial cells from adipose, lung, and aorta. Inoculation with the two American isolates resulted in 100 times greater reverse transcriptase (RT) activity in supernatant of the microvascular endothelial cells (brain, lung, and adipose) than in the macrovascular endothelial cells (aorta). Conversely, inoculation with the two lcelandic isolates resulted in 100 times higher RT activity in aortic, lung, and adipose endothelial cells than in the brain endothelial cells. Transmission electron microscopy of the brain capillary endothelial cells infected with the American isolates revealed polarized viral budding from the lateral cell membrane and a loss of tight junctions. Replication of OvLV in brain capillary endothelial cells could play a role in the pathogenesis of lentiviral encephalitis by altering blood-brain barrier integrity.

Perinatal lethality and multiple craniofacial malformations in MSX2 transgenic mice.

MSX2 is a homeodomain transcription factor that has been implicated in craniofacial morphogenesis on the basis of its expression pattern during mouse development and the finding of a missense mutation (P148H) in humans affected with Boston-type craniosynostosis. We have generated transgenic mice carrying a 34 kb DNA fragment encompassing a human MSX2 gene encoding either wild-type or mutant (P148H) MSX2. Inheritance of either transgene resulted in perinatal lethality and multiple craniofacial malformations of varying severity, including mandibular hypoplasia, cleft secondary palate, exencephaly, and median facial cleft, which are among the severe craniofacial malformations observed in humans. Transgenic mice also manifested aplasia of the interparietal bone and decreased ossification of the hyoid. Transgene-induced malformations involved cranial neural-crest derivatives, were characterized by a deficiency of tissue, and were similar to malformations associated with embryonic exposure to ethanol or retinoic acid, teratogens that cause increased cell death. Together with previous observations implicating MSX2 expression in developmentally-programmed cell death, these results suggest that wild-type levels of MSX2 activity may establish a balance between survival and apoptosis of neural crest-derived cells required for proper craniofacial morphogenesis.

Histopathologic appearance of arterial occlusions with hydrogel and polyvinyl alcohol embolic material in domestic swine.

PURPOSE: This study observes the histologic changes resulting from a hydrogel embolic agent (polyacrylonitrile [PAN]) compared with polyvinyl alcohol particles (PVA) of similar size. MATERIALS AND METHODS: Hepatic and renal embolizations were performed in 13 domestic swine by selecting small (1-mm) branches utilizing a coaxial 3-F microcatheter. The hydrogel embolic agent (tantalum-loaded and plain) and PVA were delivered through microcatheters. The longest follow-up period was 8 weeks. Postmortem examination of the embolized tissues included gross examination and histologic analysis. RESULTS: Tantalum-loaded PAN particles were radiopaque and seen in groups fluoroscopically and individually with specimen radiography. Histologic studies showed similar luminal and cellular response to PVA and the hydrogel embolic agents. The arterial lesion induced by the hydrogel embolic agents led to an absence of the arterial wall locally in the area of deployment. Hydrogel embolic particles became surrounded in fibrous connective tissue with no arterial wall. PVA and porous hydrogel capsules produced an inflammatory response, resulting in less wall reorganization, and surrounding fibrous connective tissue at 8 weeks than the solid PAN particles. CONCLUSION: These hydrogel embolic create a permanent arterial occlusion by transmural arterial damage. Mechanical effects and, to a lesser degree, inflammatory changes are responsible.

Characterization of prostatic basal cell hyperplasia and neoplasia in aged macaques: comparative pathology in human and nonhuman primates.

There are very few reports of proliferative prostatic lesions occurring spontaneously in nonhuman primates. We found that 15 of 19 glands in aged macaques contained one or more epithelial lesions in the cranial lobe. These originated in the basal cell compartment and were characterized as hyperplasia and benign neoplasia. The adenomas contained variable gland formation, with morphologic and immunohistochemical evidence of secretory, mucigenous, neuroendocrine, transitional, and squamous cell differentiation. These cell types are resident in the normal prostate or appear in metaplastic lesions, and their presence in the macaque tumors is consistent with differentiation of a stem cell along multiple phenotypic pathways. The macaque growths are similar to human prostatic basal cell lesions and could provide insights into their pathogenesis as well as cellular ontogeny and general mechanisms of carcinogenesis in this organ.

High-intensity focused ultrasound ablation of rabbit kidney tumors.

High-intensity focused ultrasound (HIFU) is a noninvasive surgical technique in which ultrasound energy is delivered transcutaneously to a discrete area within the body. This energy can result in a well-defined zone of cellular death within the targeted tissue. We used HIFU in an effort to ablate rabbit VX-2 kidney tumors. A tumor cell suspension was injected into a renal segmental artery (Phase 1, nine rabbits) or directly into the lower pole parenchyma (Phase 2, nine rabbits). After a 2-week incubation period, open direct contact (Phase 1) or transcutaneous ablation (Phase 2) was performed. In Phase 1, after sonablation, there was pathologic evidence of tissue destruction in nine animals, and seven had both gross and histologic evidence of tumor ablation. There was sharp demarcation between viable and ablated tissue. In Phase 2, pathologic evidence of kidney ablation was seen in seven of nine animals. However, only two rabbits showed the well-demarcated effects of ablation in the tumor. High-intensity focused ultrasound can be effective at causing cell death in renal tumors and surrounding renal tissue. However, with the present ultrasound technology, imaging of renal lesions in the rabbit model is not adequate to consistently localize and completely ablate tumor.