Axonal spheroids in the cochlear nucleus of normal beagle dogs.

In the course of drug evaluation studies, sporadic axonal spheroids were identified in the cochlear nucleus of 8-15-month-old Beagle dogs. These structures were identified by Bielschowski histochemical and anti-neurofilament immunohistochemical stains and by ultrastructural examination. No cellular reaction or significant neuropil alterations were associated with the presence of the spheroids. Their presence was unrelated to treatment and were considered to be an incidental background finding.

Chemopreventive activity of tamoxifen, N-(4-hydroxyphenyl)retinamide, and the vitamin D analogue Ro24-5531 for androgen-promoted carcinomas of the rat seminal vesicle and prostate.

We evaluated the ability of dietary N-(4-hydroxyphenyl)retinamide; 1 alpha,25-dihydroxy-16-ene-23-yne-26,27-hexafluorocholecalcifero l (Ro24-5531); and tamoxifen to inhibit the development of androgen-promoted carcinomas of the accessory sex organs of male Lobund-Wistar rats. Invasive carcinomas of the seminal vesicle (SV) and anterior prostate (AP) were induced in Lobund-Wistar rats with three different combinations of initiator [N-nitroso-N-methylurea (NMU)] and promoter [testosterone propionate (TP)]: (a) high-dose NMU (30 mg/kg) + high-dose TP (20 mg via implant every 2 months); (b) high-dose NMU + low-dose TP (10 mg implanted every 2 months); or (c) low-dose NMU (15 mg/kg) + low-dose TP. During the period of TP administration, rats were fed a diet supplemented with either N-(4-hydroxyphenyl)retinamide (1 or 2 mmol/kg diet), Ro24-5531 (1.25 or 2.5 nmol/kg diet), tamoxifen (0.5 or 5 mg/kg diet), or vehicle alone. After sacrifice at 8.5 or 11 months, the prostate-seminal vesicle complex from each rat was processed in toto and histologically staged as to the extent of tumor involvement. In animals given low-dose TP, all three agents were significantly effective at reducing the incidence of invasive carcinomas of the SV and, to a lesser degree, the AP. Of the three agents, tamoxifen given in high dose (5 mg/kg) had the strongest activity, reducing the occurrence of invasive SV carcinomas from 72-83% in controls to 6% (P = 0.0001) and the occurrence of invasive AP carcinomas from 50-72% to 18-22% (P < 0.05).

Nitric oxide: cytokine-regulation of nitric oxide in host resistance to intracellular pathogens.

To discover how nitric oxide (NO) synthesis is controlled in different tissues as cells within these tissues combat intracellular pathogens, we examined three distinctively different experimental murine models designed for studying parasite-host interactions: macrophage killing of Leishmania major; nonspecific protection against tularemia (Francisella tularensis) by Mycobacterium bovis (BCG); and specific vaccine-induced protection against hepatic malaria with Plasmodium berghei. Each model parasite and host system provides information on the source and role of NO during infection and the factors that induce or inhibit its production. The in vitro assay for macrophage antimicrobial activity against L. major identified cytokines involved in regulating NO-mediated killing of this intracellular protozoan. L. major induced the production of two competing cytokines in infected macrophages: (1) the parasite activated the gene for tumor necrosis factor (TNF), and production of TNF protein was enhanced by the presence of interferon-gamma (IFN-gamma). TNF then acted as a autocrine signal to amplify IFN-gamma-induced production of NO; and (2) the parasite upregulated production of transforming growth factor-beta (TGF-beta), which blocked IFN-gamma-induced production of NO. Whether parasite-induced TNF (parasite destruction) or TGF-beta (parasite survival) prevailed depended upon the presence and quantity of IFN-gamma at the time of infection. The relationship between NO production in vivo and host resistance to infection was demonstrated with M. bovis (BCG).(ABSTRACT TRUNCATED AT 250 WORDS)

Surfactant therapy and high-frequency jet ventilation in the management of a piglet model of the meconium aspiration syndrome.

In vitro data have shown a concentration-dependent inhibition of surfactant by meconium, while anecdotal reports demonstrate improved oxygenation after surfactant replacement in babies with meconium aspiration syndrome, particularly in conjunction with high-frequency jet ventilation. We randomized 70 newborn piglets to either conventional or high-frequency jet ventilation, followed by insufflation of 3 mL/kg of a 33% meconium solution. Each group was further randomized to one of five surfactant therapies: 1) control, 2) 4 mL/kg Survanta, 3) 8 mL/kg Survanta, 4) 5 mL/kg Exosurf, or 5) 10 mL/kg Exosurf. We followed arterial blood gases and ventilator requirements over 6 h of ventilation. Aspirates of airway fluids were obtained for surface tension measurements, as well as total protein and phospholipid concentrations. Using a previously established scoring system, a pathologist blinded to treatment evaluated four sections of lung per animal for histologic changes of meconium aspiration syndrome. There were no differences noted between groups in any physiologic parameter measured (mean airway pressure, arterial partial pressure of oxygen/alveolar partial pressure of oxygen ratio, etc.) during the 6 h of ventilation. Airway fluid aspirate total protein concentrations increased significantly after meconium instillation (4- to 5-fold, p < 0.007) and remained elevated in spite of surfactant therapy. There was an initial decline in airway phospholipid concentrations after meconium instillation followed by a rise to levels equal to or greater than premeconium levels. Surface tension measurements increased in all groups after meconium insufflation (p < 0.012) and did not decline thereafter, despite standard and twice-standard surfactant doses of both types.(ABSTRACT TRUNCATED AT 250 WORDS)

Induction of nitric oxide synthase protects against malaria in mice exposed to irradiated Plasmodium berghei infected mosquitoes: involvement of interferon gamma and CD8+ T cells.

Exposure of BALB/c mice to mosquitoes infected with irradiated Plasmodium berghei confers protective immunity against subsequent sporozoite challenge. Immunized mice challenged with viable sporozoites develop parasitemia when treated orally with substrate inhibitors of nitric oxide synthase (NOS). This suggests that the production of nitric oxide (NO) prevents the development of exoerythrocytic stages of malaria in liver. Liver tissue from immunized mice expressed maximal levels of mRNA for inducible NOS (iNOS) between 12 and 24 h after challenge with sporozoites. Intraperitoneal injection of neutralizing monoclonal antibody against interferon gamma (IFN-gamma) or in vivo depletion of CD8+ T cells, but not CD4+ T cells, at the time of challenge blocked expression of iNOS mRNA and ablated protection in immunized mice. These results show that both CD8+ T cells and IFN-gamma are important components in the regulation of iNOS in liver which contributes to the protective response of mice immunized with irradiated malaria sporozoites. IFN-gamma, likely provided by malaria-specific CD8+ T cells, induces liver cells, hepatocytes and/or Kupffer cells, to produce NO for the destruction of infected hepatocytes or the parasite within these cells.

Histogenesis of induced prostate and seminal vesicle carcinoma in Lobund-Wistar rats: a system for histological scoring and grading.

We have developed a grading system for the evaluation of the histogenesis of neoplastic lesions of the prostate and seminal vesicle of the laboratory rat. Prostatic and seminal vesicle carcinomas were induced in Lobund-Wistar rats by initiation with 30 mg/kg N-nitroso-N-methylurea i.v., followed by promotion with 40 mg testosterone propionate implants 1 week later and at 3-month intervals thereafter. Experimental and control groups were sacrificed at various time points between 5 and 11 months after dosing with N-nitroso-N-methylurea in order to visualize progressive stages of carcinogenesis of the dorsolateral prostate, the anterior prostate, and the seminal vesicle. A system of staging was created which allows three different categories (in situ change, invasion, desmoplasia) of tumor development to be ranked progressively in a manner conducive to nonparametric analysis. Each category was then further subdivided to create a total of six stages. This system can be used to evaluate agents which modify tumor induction or suppression. The application of this staging system to the measurement of the effects of the synthetic retinoid, 4-hydroxyphenyl retinamide, on prostatic carcinogenesis in the Lobund-Wistar rat is described.

Expression of sulfated glycoprotein 2 is associated with carcinogenesis induced by N-nitroso-N-methylurea in rat prostate and seminal vesicle.

To understand the molecular mechanism of carcinogenesis in androgen-dependent tumors, we have searched for new markers which are associated with this process. In normal rat prostate and seminal vesicle, sulfated glycoprotein 2 (SGP-2) messenger RNA is barely detectable. However, we have found high levels of SGP-2 expression in the epithelial component of carcinomas of the prostate and seminal vesicle after initiation with N-nitroso-N-methylurea and promotion with testosterone propionate. We have also observed induction of SGP-2 expression in epithelial cells at early stages in carcinogenesis when cytologically malignant cells first begin to appear. SGP-2 has been reported previously to be associated with a variety of models of programmed cell death (apoptosis), including the prostate following castration. Our present findings provide a novel marker for carcinogenesis in the rat prostate and seminal vesicle.

Management of a piglet model of the meconium aspiration syndrome with high-frequency or conventional ventilation.

OBJECTIVE: To assess the usefulness of three methods of high-frequency ventilation in the early management of a piglet model of the meconium aspiration syndrome. DESIGN: Randomized, block design. SETTING: Animal research facility. SUBJECTS: Fifty-six mixed-breed newborn piglets aged 1 to 4 days and weighing 1.8 to 2.7 kg. INTERVENTIONS: After instillation of a 2.2-mL/kg solution of 25% meconium, 56 piglets were randomized to receive treatment with (1) a conventional positive-pressure infant ventilator, (2) the Bunnell Life Pulse high-frequency jet ventilator, (3) the Bird model VDR high-frequency flow interruptor, or (4) the Infant Star high-frequency flow interruptor. We adjusted ventilator settings to maintain partial pressures of arterial oxygen (PaO2) of 80 to 120 cm H2O and partial pressures of arterial carbon dioxide (PaCO2) of 30 to 50 cm H2O during the 6 hours of ventilation. MEASUREMENTS AND MAIN RESULTS: We compared pulmonary histologic alterations and four physiologic parameters: (1) mean airway pressure, (2) PaCO2, (3) ratio of PaO2 to partial alveolar oxygen pressure (PAO2), and (4) oxygenation index ([(fraction of inspired oxygen)(mean airway pressure)(100)]/PaO2). The two measures of oxygenation were similar for all four devices except at 4 hours, when the PaO2/PAO2 ratio on positive-pressure ventilation was significantly higher than that on high-frequency jet ventilation (P = .008). The histologic changes on positive-pressure ventilation (atelectasis, inflammation, presence of meconium, and exudative debris) were significantly worse than those on high-frequency jet ventilation or flow interruption (P < .0001). CONCLUSIONS: The finding of less severe pathologic alterations with various types of high-frequency ventilators justifies further investigations into the management of the meconium aspiration syndrome with these devices.

Live vaccine strain of Francisella tularensis: infection and immunity in mice.

The live vaccine strain (LVS) of Francisella tularensis caused lethal disease in several mouse strains. Lethality depended upon the dose and route of inoculation. The lethal dose for 50% of the mice (LD50) in four of six mouse strains (A/J, BALB/cHSD, C3H/HeNHSD, and SWR/J) given an intraperitoneal (i.p.) inoculation was less than 10 CFU. For the other two strains tested, C3H/HeJ and C57BL/6J, the i.p. log LD50 was 1.5 and 2.7, respectively. Similar susceptibility was observed in mice inoculated by intravenous (i.v.) and intranasal (i.n.) routes: in all cases the LD50 was less than 1,000 CFU. Regardless of the inoculation route (i.p., i.v., or i.n.), bacteria were isolated from spleen, liver, and lungs within 3 days of introduction of bacteria; numbers of bacteria increased in these infected organs over 5 days. In contrast to the other routes of inoculation, mice injected with LVS intradermally (i.d.) survived infection: the LD50 of LVS by this route was much greater than 10(5) CFU. This difference in susceptibility was not due solely to local effects at the dermal site of inoculation, since bacteria were isolated from the spleen, liver, and lungs within 3 days by this route as well. The i.d.-infected mice were immune to an otherwise lethal i.p. challenge with as many as 10(4) CFU, and immunity could be transferred with either serum, whole spleen cells, or nonadherent spleen cells (but not Ig+ cells). A variety of infectious agents induce different disease syndromes depending on the route of entry. Francisella LVS infection in mice provides a model system for analysis of locally induced protective effector mechanisms.

Rat maze performance after resuscitation with cross-linked hemoglobin solution.

Unmodified stroma-free hemoglobin has been found to produce neurotoxicity and behavioral impairment in rats. In contrast, a recent assessment of a modified (diaspirin alpha-alpha cross-linked) hemoglobin (HbXL) solution found normal memory, learning, and brain histology after infusion of a clinically relevant dose of a 14% HbXL solution. The current study examined this potential resuscitation fluid for evidence of neurobehavioral toxicity under clinical conditions. Rats were trained to complete a water alley maze, had 50% of their total blood volume (30 ml/kg) withdrawn, were resuscitated with 14% HbXL solution (45 ml/kg), Ringer's lactate (60 ml/kg), or autologous shed blood, and were subsequently retested in the water maze. Rats resuscitated with HbXL or autologous shed blood survived resuscitation, while 20% of those resuscitated with Ringer's lactate died during treatment. No significant performance degradation was observed in the HbXL rats following resuscitation, and no brain pathology was observed at necropsy 10 days after treatment. Ischemic brain lesions were observed in three (25%) of the surviving rats resuscitated with Ringer's lactate solution. Renal tubule regeneration indicative of an earlier insult was observed in animals from all three groups. A significant correlation between the total pathology in the five organs examined and maze errors was observed (p less than 0.001). The survival, maze performance, and histology results suggest that resuscitation with 14% HbXL solution does not cause neurotoxicity, as assessed in this lethal hemorrhage model.

Pathogenesis of a phleboviral infection (Punta Toro virus) in golden Syrian hamsters.

The hamster, Mesocricetus auratus, was examined as a possible model for investigating the poorly defined pathogenesis of the family Bunyaviridae, genus Phlebovirus. Punta Toro virus (PTV) isolates from Eastern Panama were highly virulent for two outbred and five inbred hamster strains, while isolates from western Panama were of low virulence. The Adames strain (eastern Panama) of PTV (LD50 approximately 1 PFU, sc) caused an acute fatal disease (average survival time, 3.8 days) in 10-week-old Lak: LVG (SYR) hamsters. Severe necrosis of the liver, spleen, and small intestine was associated with extensive expression of viral antigen in these organs. The Balliet strain (western Panama) of PTV (LD50 greater than 6 log10 PFU, subcutaneously) caused a mild hepatocellular infection with peak viral liver titers of 3-4 log10 PFU/g compared to 8-9 log10 PFU/g for the Adames strain. We observed histological lesions in the red pulp of the spleen or the lamina propria of the small intestine with the Adames strain. Lesions in the hamsters had characteristics of disseminated intravascular coagulation (DIC). The PTV-hamster model shares similarities to Rift Valley fever (phleboviral disease), which causes fatal disease in man and domesticated ruminants.

Administration of monoclonal anti-IFN-gamma antibodies in vivo abrogates natural resistance of C3H/HeN mice to infection with Leishmania major.

C3H/HeN mice that are naturally resistant to cutaneous disease and systemic infections with the protozoan parasite, Leishmania major, were treated at the time of infection, and weekly thereafter, with mouse anti-rat IFN-gamma mAb or an irrelevant antibody of similar isotype. Anti-IFN-gamma-treated mice developed cutaneous lesions; parasites spread to the regional lymph nodes and then metastasized to spleens and livers. The course of disease in these animals was similar to that of genetically susceptible BALB/c mice. Two exceptions in the pathology of L. major infections were noted between BALB/c and anti-IFN-gamma-treated C3H/HeN mice: 1) BALB/c mice died of systemic complications, whereas C3H/HeN mice did not, and 2) multinucleated giant cells were observed in lymph nodes and spleens of infected BALB/c mice, whereas these cells were not observed in infected C3H/HeN mice. Control mice, those treated with either saline or irrelevant antibody of the same isotype as the anti-IFN-gamma monoclonal, showed no evidence of cutaneous disease (development of footpad lesions) or systemic infection (by histopathology). Total abrogation of the natural resistance of C3H/HeN mice could be achieved by treatment with as little as 0.5 mg/mouse/wk of anti-IFN-gamma antibody, or by a single dose of 1 mg/mouse anti-IFN-gamma antibody administered at the time of parasite inoculation. If antibody treatment was delayed for as little as 1 wk after parasite inoculation, the infections in treated animals resembled that of untreated or control antibody-treated mice: no cutaneous lesions (by footpad swelling or viable counts of leishmania in footpad tissue) or systemic disease (by microscopic analysis of touch preparations of internal organs, and histopathology of same). The production of IFN-gamma during the initial interaction of the parasite and host cells appears to be a major component of genetic control of natural resistance to infection with L. major in C3H/HeN mice.

Hemostatic derangement produced by Rift Valley fever virus in rhesus monkeys.

Rift Valley fever (RVF) is an important cause of disease in animals and humans in sub-Saharan Africa. In a small percentage of human cases, the disease is complicated by hemorrhage, which often is associated with a fatal outcome. Inoculation of rhesus monkeys with the Zagazig Hospital strain of RVF virus produced a clinical picture similar to illness in humans. Ten of 17 monkeys developed clinical evidence of hemostatic impairment. When coagulation tests were performed, this group of monkeys had significant abnormalities, including evidence for disseminated intravascular coagulation. These abnormalities were much less pronounced in the remaining seven monkeys-whose only sign of illness was transient fever-and, in general, they paralleled the level of viremia and the degree of elevation in levels of serum hepatic enzymes. Autopsy of the three monkeys with severe disease revealed hepatic necrosis.

Nasal cavity carcinosarcoma in a bonnet macaque (Macaca radiata).

An example of a rare, highly aggressive malignant intranasal neoplasm in a male Bonnet macaque is presented. The tumor invaded the bony orbit, distorted the position of the globe, and metastasized to regional lymph nodes and lungs. Histologically the tumor was composed of both mesenchymal and epithelial components and probably was of minor salivary gland origin.

Naturally occurring melioidosis in a colonized rhesus monkey (Macaca mulatta).

An aged wild-caught male rhesus monkey (Macaca mulatta), maintained in a research facility for 10 years, developed bilateral pelvic limb paralysis without other signs of disease. Unresponsive to therapy, the monkey was killed and necropsied. Chronic inflammation with osteolysis of thoracic vertebrae 10-13 was observed. Pseudomonas pseudomallei was cultured and identified from cerebrospinal fluid obtained at the site of the thoracic lesion. This Gram-negative bacterium can cause infection in animals and man and may remain latent for years before the appearance of clinical signs.

Feline malignant lymphoma: log-linear multiway frequency analysis of a population involving the factors of sex and age of animal and tumor cell type and location.

A method appropriate for the analysis of multiway frequency tables is described. This multivariate approach was used to evaluate the relationship between age, sex, cell type, and tumor location based on information available from 1,733 cases of feline malignant lymphoma. The analysis identified 6 bivariate relationships to be statistically significant, P less than 0.001; there were no significant higher order interactions observed. Emphasis was placed on 3 interactions: age to tumor location, age to sex, and tumor location to cell types.