A conjugate of gemcitabine with bisphosphonate (Gem/BP) shows potential as a targeted bone-specific therapeutic agent in an animal model of human breast cancer bone metastases.

Bone metastases in advanced breast cancer patients remains a significant treatment challenge. Bisphosphonates are now a routine first line treatment for prevention and treatment of skeletal damage caused by malignancies and, moreover, have shown an ability to transport therapeutic drugs to the bone. Here, we describe the effect of a conjugate between the potent anticancer drug gemcitabine and a bisphosphonate molecule (Gem/BP) in an animal model of breast cancer metastases. We have previously demonstrated the targeting of this compound to bone in normal mice using an analog labeled with the radionuclide 99mTc. Using a bone metastasis model in nude mice produced by intracardiac injection of the human breast cancer cell line MDA-MB-231BO, we examined the effect of Gem/BP and gemcitabine in reducing the frequency and severity of osteolytic bone lesions. High-resolution radiographs and microPET images showed that Gem/ BP reduced the number and size of bone metastases relative to the gemcitabine-treated and the untreated control groups. Histological examination of the humeri and femurs of the control and gemcitabine groups revealed large metastatic cancer lesions in the outer and inner cortices and the medullary cavities. In contrast, Gem/BP-treated mice showed occasional small wedge-shaped metastases under the periosteum of the outer cortex and very occasionally in the medulla. These findings suggest that Gem/BP should be further evaluated for use in the treatment of bone metastases in breast cancer.

Unexpected preferential brain metastases with a human breast tumor cell line MDA-MB-231 in BALB/c nude mice.

Animal models are useful tools to study etiology, progress, and new treatments of disease and are an approximation of human disease for experimental study. Intracardiac injection of the human estrogen-independent breast cancer cell line MDA-MB-231 in nude mice is a well-characterized animal model of bone metastasis mainly used to study new treatments for late-stage breast cancer. According to the published literature, this model should produce radiologically distinguishable bone tumors within 17 days after injection. Mice should develop complications such as cachexia, paraplegia, and morbidity within 28 days and require euthanasia within 35 days after injection. We report a study in which injection of MDA-MB-231 cell line led to brain rather than bone metastasis. Unexpected alterations in biological behavior are an important confounding variable in the use of tumor cell lines, and the occurrence and cause of such variants is poorly documented.

Mechanism and treatment of sulfide-induced coma: a rat model.

Sodium hydrosulfide and dimethylsulfide duplicate the effects of hydrogen sulfide in causing coma in Sprague-Dawley rats and are additive for lethality. Nitrite, pyruvate and dithiothreitol had no significant effect on coma or lethality but bicarbonate with and without glucose reduced duration of coma. This finding suggests an antidotal treatment.

Increased circulating monocyte activation in patients with unstable coronary syndromes.

OBJECTIVES: The primary objective of this research was to assess the activation level of circulating monocytes in patients with unstable angina. BACKGROUND: Markers of systemic inflammatory responses are increased in patients with unstable coronary syndromes, but the activation state and invasive capacity of circulating monocytes have not been directly assessed. METHODS: Peripheral blood mononuclear cell (MC) activation in blood samples isolated from patients with stable and unstable coronary artery disease was measured in two studies. In study 1, a modified Boyden chamber assay was used to assess spontaneous cellular migration rates. In study 2, optical analysis of MC membrane fluidity was correlated with soluble CD14 (sCD14), a cellular activation marker. RESULTS: Increased rates of spontaneous monocyte migration (p < 0.01) were detected in patients with unstable angina (UA) (Canadian Cardiovascular Society [CCS] angina class IV) on comparison to patients with acute myocardial infarction (MI), stable angina (CCS angina classes I to III) or normal donors. No significant increase in lymphocyte migration was detected in any patient category. Baseline MC membrane fluidity measurements and sCD14 levels in patients with CCS class IV angina were significantly increased on comparison with MCs from normal volunteers (p < 0.001). A concomitant reduction in the MC response to activation was detected (p < 0.05). CONCLUSIONS: Using two complementary assays, activated monocytes with increased invasive capacity were detected in the circulation of patients with unstable angina. This is the first demonstration of increased monocyte invasive potential in unstable patients, raising the issue that systemic inflammation may both reflect and potentially drive plaque instability.

Immunoprophylactic potential of cloned Shiga toxin 2 B subunit.

The Shiga toxins Stx1 and Stx2 contribute to the development of enterohemorrhagic O157:H7 Escherichia coli-mediated colitis and hemolytic-uremic syndrome in humans. The Stx2 B subunit, which binds to globotriaosylceramide (GB3) receptors on target cells, was cloned. This involved replacing the Stx2 B subunit leader peptide nucleotide sequences with those from the Stx1 B subunit. The construct was expressed in the TOPP3 E. coli strain. The Stx2 B subunits from this strain assembled into a pentamer and bound to a GB3 receptor analogue. The cloned Stx2 B subunit was not cytotoxic to Vero cells or apoptogenic in Burkitt's lymphoma cells. Although their immune response to the Stx2 B subunit was variable, rabbits that developed Stx2 B subunit-specific antibodies, as determined by immunoblot and in vitro cytotoxicity neutralization assays, survived a challenge with Stx2 holotoxin. This is thought to be the first demonstration of the immunoprophylactic potential of the Stx2 B subunit.

Inhibition of transplant vasculopathy in a rat aortic allograft model after infusion of anti-inflammatory viral serpin.

BACKGROUND: Transplant vasculopathy remains a difficult therapeutic problem, resulting in the majority of late cardiac graft losses. This chronic vascular disease is thought to be triggered by alloantigen-dependent and alloantigen-independent inflammatory factors. Despite improved 1-year survival, the incidence of transplant vasculopathy has not improved with current immunosuppressive protocols. Highly effective strategies have evolved in the large DNA viruses that shield infecting viruses from host inflammatory responses. Serp-1 is a secreted myxoma virus anti-inflammatory serine proteinase inhibitor. Serp-1 inhibits plasminogen activators in a manner similar to plasminogen activator inhibitor (PAI-1), a vascular protein that plays a pivotal regulatory role in vascular wound healing. In this study, we tested the ability of purified Serp-1 protein to ameliorate posttransplant vasculopathy after rat aortic allograft surgery. METHODS AND RESULTS: Serp-1 protein or controls were infused into 98 rats immediately after segmental aortic allograft transplantation. After either late (28 days, 64 rats) or early (12 to 48 hours, 24 rats) follow-up, transplanted aortic segments were harvested for morphological and immunohistochemical analysis. Significant reductions in intimal plaque growth (P<0.002) and mononuclear cell invasion (P<0.033) were detected after Serp-1 infusion at nanogram doses. Serp-1 reduced early macrophage (P<0.0016) and nonspecific lymphocyte (P<0.0179) invasion into medial and adventitial layers and inhibited associated depletion of medial smooth muscle cells (P<0.0006). CONCLUSIONS: Infusion of a viral anti-inflammatory serpin, Serp-1, significantly reduces early inflammatory responses and later luminal occlusion in a rat aortic allograft model.

Detection of transplant vasculopathy in a rat aortic allograft model by fluorescence spectroscopic optical analysis.

BACKGROUND AND OBJECTIVE: Transplant vasculopathy is a leading cause of late cardiac graft loss. We have examined laser-induced fluorescence (LIF) spectroscopy as an optical diagnostic tool for detection of intimal plaque development and inflammatory cellular invasion in a rat model of aortic allograft transplant. STUDY DESIGN/MATERIALS AND METHODS: Infrarenal aortic segments were transplanted from Lewis to Sprague Dawley rats. A range of vasculopathy development was produced by treatment with a viral anti-inflammatory protein. LIF spectra were recorded from the intima of aortic implants at 28 days. Fluorescence intensity was analyzed for correlation with vasculopathy development. RESULTS: Significant differences in LIF intensity at 400-450 nm (P < or = 0.05 by ANOVA) were detected. LIF emission was correlated with plaque growth (R2 = 0.980), vessel narrowing (R2 = 0.964), and cellular invasion (R2 = 0.971) by regression analysis. CONCLUSION: LIF optical analysis provides a nontraumatic diagnostic approach for detection of atherosclerosis prior to cardiac transplant or during development of vasculopathy after transplant.

Observations on animal and human health during the outbreak of Mycobacterium bovis in game farm wapiti in Alberta.

This report describes and discusses the history, clinical, pathologic, epidemiologic, and human health aspects of an outbreak of Mycobacterium bovis infection in domestic wapiti in Alberta between 1990 and 1993, shortly after legislative changes allowing game farming. The extent and seriousness of the outbreak of M. bovis in wapiti in Alberta was not fully known at its onset. The clinical findings in the first recognized infected wapiti are presented and the postmortem records for the herd in which the animal resided are summarized. Epidemiologic findings from the subsequent field investigation are reviewed, the results of recognition and investigation of human exposure are updated, and recommendations for reduction of human exposure are presented.

Atherosclerosis in Marek's disease virus infected hypercholesterolemic roosters is reduced by HMGCoA reductase and ACE inhibitor therapy.

OBJECTIVES: Accelerated atherosclerosis is associated with herpesviral infection both in transplant patients and after balloon angioplasty. Marek's disease virus (MDV) is a herpesvirus that induces accelerated atherosclerosis associated with the development of an invasive lymphoma in hyperlipemic roosters. We have examined the effects of pravastatin, a 3-hydroxy-3-methylglutaryl-coenzyme A (HMGCoA) reductase inhibitor and quinapril, an angiotensin converting enzyme (ACE) inhibitor, on atherosclerosis development in MDV infected, cholesterol fed rooster chicks. METHODS: The effects of these drugs on plaque growth after MDV infection were examined in two studies. In Study 1, MDV infected White Leghorn rooster chicks were divided into 4 groups assigned to normal or high cholesterol diet, and treated at three months of age with either pravastatin or saline. In Study 2, cholesterol fed rooster chicks infected with MDV were divided into 3 groups for treatment with either pravastatin, quinapril, or saline control. RESULTS: A significant decrease in plaque area was detected after 60 days of treatment with both pravastatin and quinapril in cholesterol fed chicks (P < 0.001). Lymphocyte infiltration into the arterial wall or target organs was not inhibited by treatment with either drug. CONCLUSIONS: (1) HMGCoA reductase inhibitor and ACE inhibitor therapy reduce atherosclerosis induced by virus infection and cholesterol diet, but this decrease in plaque growth is not due to a reduction in lymphocyte invasion. (2) MDV infection in cholesterol fed roosters provides a model for virus-induced arterial injury in atherogenesis.

Treatment of experimentally induced pneumonic pasteurellosis of young calves with tilmicosin.

Twenty four (24) healthy male Holstein calves (< 70 kg) were each experimentally infected by intrabronchial inoculation of 4.0 x 10(9) viable cells of Pasteurella haemolytica-AI (B122) at Time = 0 h. At 1 h following inoculation animals received either: 1) Sham treatment with sterile 0.85% saline SC (n = 12); or 2) a single injection of 10 mg tilmicosin per kg body weight (n = 12). Calves that were non-infected and tilmicosin-treated were also included for determining tilmicosin concentrations in serum and lung tissue at 1, 2, 4, 6, 8, 24, 48, and 72 h (n = 3-per time). In the infected calves, response to therapy was monitored clinically. Serum samples were collected for determination of tilmicosin concentrations using HPLC. Any animal becoming seriously ill was humanely killed. Complete necropsy examinations were performed on all animals and included gross pathologic changes, bacteriologic analysis, histopathology, and determination of pulmonary concentrations of tilmicosin. Tilmicosin treated animals responded significantly better to therapy than saline-treated control calves. Clinical assessment of calves during the study indicated that tilmicosin-treated calves had significantly improved by T = 8 h compared to satine-treated animals (P < 0.05). At necropsy tilmicosin-treated calves had significantly less severe gross and histological lesions (P < 0.05) of the pulmonary tissue. Of the 12 saline-treated calves, 92% (11/12) had Pasteurella haemolytica-A1 in lung tissue, while of the tilmicosin-treated calves 0% (0/12) cultured positive for P. haemolytica. Mean (+/- standard error) serum tilmicosin concentrations in infected calves peaked at 1 h post-injection (1.10 +/- 0.06 micrograms/mL) and rapidly decreased to 0.20 +/- 0.03 microgram/mL, well below the MIC of 0.50 microgram/mL for P. haemolytica-A1 (B122), by 12 h. These serum concentrations were very similar to serum concentrations of tilmicosin in non-infected tilmicosin-treated calves. Lung tissue concentrations of the antibiotic were comparatively high, even at 72 h post-infection (6.50 +/- 0.75 ppm). Lung tissue concentrations at 72 h were significantly higher in experimentally infected calves than in non-infected tilmicosin-treated animals (P < 0.05). These data demonstrate that tilmicosin was effective in treating experimentally-induced pneumonic pasteurellosis as determined by alleviation of clinical signs, pathological findings at post mortem, and presence of viable bacteria from the lung. Concentrations substantially above MIC for P. haemolytica were present in lung tissue even at 72 h following a single subcutaneous injection of 10 mg tilmicosin per kg body weight.

A murine model of pulmonary damage induced by lipopolysaccharide via intranasal instillation.

This study examines the intranasal instillation of lipopolysaccharide (LPS) into BALB/c mice causing acute pulmonary damage, due to neutrophil infiltration and sepsis. A dose response with LPS showed that an intranasal instillation of 167 microg/ml (10 microg/mouse) caused acute lung injury within 2-4 h and reached maximal damage at 24-48 h. We found the method of LPS administration for induction of acute pulmonary damage to be crucial. After 24 h post-LPS injection, a comparison showed a substantial increase in pulmonary damage with intranasal instillation of LPS. As for intravenous injection, it showed a baseline effect. This study indicates that LPS administered intranasally causes acute pulmonary damage, whereas with intravenous and intraperitoneal endotoxin administration a tissue-specific or similar degree of pulmonary injury may not develop.

Evaluation in vitro of epidermal cell keratinization.

Rhodamine B has been used as a histopathological stain for keratinization and cornification. Its ability as an in vitro indicator of the degree of epidermal keratinization was investigated in these preliminary studies. An immortalized human keratinocyte cell line, SVK-14, was evaluated as an alternative to primary human keratinocytes. The influence of extracellular calcium levels was evaluated alongside the effects of exposure to 1,25 (OH)(2) vitamin D(3) in serum-free and serum-containing media. Alamar blue (AB) conversion was used to measure changes in cellular reductive potential, and the amount of bound Rhodamine B relative to total protein per well was taken as an indicator of keratinization. Exposure to 1,25 (OH)(2) vitamin D(3) for 7 or 10 days did not increase Rhodamine B binding to confluent SVK-14 cultures, regardless of calcium concentration. Variation in Rhodamine B dye-binding to cells made it difficult to interpret the data. In addition, concern regarding the ability of SVK-14 cells to differentiate suggests that further studies need to be performed using normal human keratinocytes to validate this in vitro endpoint, with epidermal growth factor, insulin and hydrocortisone removed from the media to enhance epidermal differentiation.

Virus-encoded serine proteinase inhibitor SERP-1 inhibits atherosclerotic plaque development after balloon angioplasty.

BACKGROUND: Recurrent atherosclerotic plaque growth, restenosis, is a significant clinical problem after interventional procedures. Initiation of restenosis involves activation of inflammatory and thrombotic cascades, which are regulated by serine proteinase enzymes and inhibitors. We have investigated the use of a viral serine proteinase inhibitor, SERP-1, to reduce plaque development after primary balloon angioplasty. This is the first experimental report of the use of a viral anti-inflammatory protein for the prevention of atherosclerosis. METHODS AND RESULTS: Seventy-four cholesterol-fed rabbits were treated with either local or systemic infusions of SERP-1 protein (or control solutions) after balloon-mediated injury. Sites of SERP-1 infusion in rabbits had dramatically reduced plaque compared with control infusions at the 4-week follow-up. At low-dose infusions (30 to 300 pg), only the primary infusion site had a demonstrable decrease in plaque, whereas at higher-dose infusions (> 3000 pg), a generalized reduction in plaque development was detected. An associated decrease in mononuclear cell infiltration of the arterial wall was detected after SERP-1 infusion within the first 24 hours. Infusion of an active-site mutant of SERP-1 (P1-P1', ala-ala) lacking serine proteinase inhibitory activity failed to prevent plaque growth. CONCLUSIONS: Purified SERP-1, a virus-encoded secreted glycoprotein, reduces plaque growth after primary balloon-mediated injury. Plaque development is decreased by inhibition of serine proteinase activity and is associated with a focal reduction in macrophage infiltration immediately after injury. Investigation of serine proteinase inhibitors may provide new insight into the regulation of arterial responses to injury.

Synergistic effects of monensin in combination with permethrin or neomycin on neuronal activity.

Drug combinations have the potential to produce novel and unpredictable responses on nervous tissue. This study was designed to test the hypothesis that the effects of combinations of monensin (an ionophore antibiotic) and either neomycin (an aminoglycoside antibiotic) or permethrin (synthetic pyrethroid) are synergistic. Effects of the drug combinations upon the electrical properties and membrane activities of an in vitro sensory neuron preparation were found to be greater than expected from addition of the effects of the same drugs acting individually, indicating synergism and thus supporting the hypothesis. It was concluded that drugs acting at different neuronal membrane sites and applied in combination produce unpredictable responses. Such drug combinations behave as if they were novel drugs.

Hepatic disease in Alberta horses: A retrospective study of 'alsike clover poisoning' (1973-1988).

Over sixteen years, 49 horses were diagnosed by Alberta Agriculture Animal Health laboratories as having "alsike clover poisoning". There was a distinct northwestern distribution of cases, the majority coming from the Peace River district. This distribution is opposite to that of the Alberta horse population, but coincides with areas of alsike clover cultivation. Cases could be divided into chronic or nervous clinical presentations, as described by Schofield. Tissues from 45 animals were retrieved and examined microscopically. Significant histological lesions were confined to the liver and consisted of biliary fibrosis and epithelial proliferation.I conclude that alsike clover poisoning is a specific disease entity, likely due to exposure to an exogenous toxin. The evidence is not strong enough to incriminate alsike clover as the etiology.

Intestinal protozoa and epithelial cell kinetics, structure and function.

Intestinal protozoa are not only common enteric pathogens in the tropics but also the high incidence of infection among immunocompromised patients in northern countries has evoked an increased interest in these parasites. Although enteric protozoa are a major cause of diarrhea and malabsorption in humans and other animals, the pathophysiology of gut disturbances caused by them remains poorly understood. Clinical signs related to enteric protozoan disease commonly involve malabsorption, diarrhea, weight loss or retarded weight gain and anorexua. Since these infections are most prevalent and most severe in the young, this may translate into considerable illness among children and significant loss to the agricultural economy where domestic animals are prone to infection. In this review we describe the effects of intestinal protozoan diseases on the structure, kinetics and function of absorptive intestinal cells and other epithelial cells, and correlate morphological injury with physiological alterations in the parasitized gut. Some of the interactions between immune responses and pathophysiology will be discussed, but in-depth discussion of intestinal immunity has recently been undertaken by other authors.

Terminal ileitis in lambs.

Terminal ileitis was diagnosed in three flocks of lambs in different areas of Alberta. Salient clinical features in affected lambs were progressive emaciation with diarrhea, and in some lambs, frequent abdominal stretching. Postmortem findings included thickening of the ileal, and in some animals, the jejunal, cecal and colonic mucosa as a result of mucosal infiltrates of many lymphocytes and fewer plasma cells, eosinophils, globular leukocytes, and neutrophils. Hyperplasia of intestinal lymphoid tissue was prominent in most affected lambs. The cause of the condition is unknown.

Maggots, mutilations and myth: Patterns of postmortem scavenging of the bovine carcass.

Based upon what is known about the habits of common carrion eaters in Alberta, we review the patterns of postmortem scavenging of carcasses of cattle. We then compare with these patterns those reported in the lay press and by veterinarians investigating cattle mutilations in Alberta. We conclude that the so-called "mutilation" of cattle in Alberta was due to scavenging of carcasses and further conclude that claims of human involvement in such incidents require, as a first condition, that postmortem scavenging of the carcass be excluded.