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PubMed2003

Oxidized lipid depresses canine growth, immune function, and bone formation.

Dietary oxidized lipids can increase oxidative stress and potentially contribute to a variety of disease syndromes. This research describes the first use of a canine model to assess the effects of dietary oxidized lipids on growth, antioxidant status, and some immune functions. Three groups of eight, two-month old coon-hound puppies were pair fed diets for 16 weeks. The control diet contained <50 ppm aldehydes, and two additional diets contained thermally oxidized lipids targeted to contain 100 ppm aldehydes (medium-oxidation) and 500 ppm aldehydes (high-oxidation). Dogs fed the high-oxidation diet weighed less than those from the medium-oxidation (P < 0.05) and control groups (P < 0.001) at the end of the study. Oxidized lipids reduced serum vitamin E levels, total body fat content, and bone appositional rate. At different time points of the study, peripheral blood neutrophils and monocytes from dogs fed the HO diet had reduced oxidative burst capacity and produced less superoxide and hydrogen peroxide when stimulated with phorbol esters compared to the control group. Lymphocyte blastogenesis in response to concanavalin A was suppressed by dietary oxidized lipid. This study indicates that dietary oxidized lipids negatively affect the growth, antioxidant status, and some immune functions of dogs. Importantly, some effects are evident at 100 ppm aldehydes in the diet, which is a moderate level of oxidation. The rapid growth and weight gain of the dog during the first 6 months of life may also provide a better model for assessing the risks of dietary oxidized lipid in children and adolescents than previously used rodent models.

Aldehydes↗
PubMed2003

Pyrazolinone-piperidine dipeptide growth hormone secretagogues (GHSs). Discovery of capromorelin.

Novel pyrazolinone-piperidine dipeptide derivatives were synthesized and evaluated as growth hormone secretagogues (GHSs). Two analogues, capromorelin (5, CP-424391-18, hGHS-R1a K(i)=7 nM, rat pituicyte EC(50)=3 nM) and the des-methyl analogue 5c (hGHS-R1a K(i)=17 nM, rat pituicyte EC(50)=3 nM), increased plasma GH levels in an anesthesized rat model, with ED(50) values less than 0.05 mg/kg iv. Capromorelin showed enhanced intestinal absorption in rodent models and exhibited superior pharmacokinetic properties, including high bioavailabilities in two animal species [F(rat)=65%, F(dog)=44%]. This short-duration GHS was orally active in canine models and was selected as a development candidate for the treatment of musculoskeletal frailty in elderly adults.

Animals↗
PubMed2005

Tubular complexes as a source for islet neogenesis in the pancreas of diabetes-prone BB rats.

Tubular complexes (TC) in the pancreas contain duct-like structures with low cuboidal or flattened cells surrounding a large lumen and are thought to be a response to pancreatic injury. TC have been studied in animal models of chemical or surgically induced pancreatic damage but their occurrence has not been reported in rodent models of spontaneous autoimmune type I diabetes. We hypothesized that TC would be increased during the active phase of islet destruction in autoimmune diabetes and could contain islet progenitor cells. We analyzed TC in pancreas of Wistar Furth (WF), control (BBc) and diabetes-prone BioBreeding (BBdp) rats using immunohistochemistry and morphometry. TC were observed in all rat strains during active pancreas remodeling ( approximately 13 days). They increased between 60 and 93 days only in BBdp rats coincident with the increase in diabetes cases. Most TC were infiltrated with CD3(+) T-cells. Duct-like cells in the TC had low expression of the exocrine marker amylase, increased expression of epithelial cell markers, keratin and vimentin, and remarkably high cell proliferation and cell death. TC islets contained cells stained positive for insulin, glucagon, somatostatin, pancreatic polypeptide, as well as PDX-1, chromogranin, and hepatocyte-derived growth factor receptor, c-met. Transitional cells that were keratin(+)/insulin(+) and keratin(+)/amylase(+) cells were present in TC. The stem cell marker, nestin was upregulated in the TC region. Duct-like cells in TC of BBdp rats expressed markers of committed endocrine precursors: PDX-1, neurogenin 3 and protein gene product 9.5. This study demonstrates that TC are upregulated during beta-cell destruction and contain potential endocrine progenitors.

Amylases↗
PubMed1997

Inhibition of poly (ADP-ribose) synthetase attenuates neutrophil recruitment and exerts antiinflammatory effects.

A cytotoxic cycle triggered by DNA single-strand breakage and poly (ADP-ribose) synthetase activation has been shown to contribute to the cellular injury during various forms of oxidant stress in vitro. The aim of this study was to investigate the role of poly (ADP-ribose) synthetase (PARS) in the process of neutrophil recruitment and in development of local and systemic inflammation. In pharmacological studies, PARS was inhibited by 3-aminobenzamide (10-20 mg/kg) in rats and mice. In other sets of studies, inflammatory responses in PARS-/- mice were compared with the responses in corresponding wild-type controls. Inhibition of PARS reduced neutrophil recruitment and reduced the extent of edema in zymosan- and carrageenan-triggered models of local inflammation. Moreover, inhibition of PARS prevented neutrophil recruitment, and reduced organ injury in rodent models of inflammation and multiple organ failure elicited by intraperitoneal injection of zymosan. Inhibition of PARS also reduced the extent of neutrophil emigration across murine mesenteric postcapillary venules. This reduction was due to an increased rate of adherent neutrophil detachment from the endothelium, promoting their reentry into the circulation. Taken together, our results demonstrate that PARS inhibition reduces local and systemic inflammation. Part of the antiinflammatory effects of PARS inhibition is due to reduced neutrophil recruitment, which may be related to maintained endothelial integrity.

Animals↗
PubMed2006

The Drosophila pigmentation gene pink (p) encodes a homologue of human Hermansky-Pudlak syndrome 5 (HPS5).

Lysosome-related organelles comprise a group of specialized intracellular compartments that include melanosomes and platelet dense granules (in mammals) and eye pigment granules (in insects). In humans, the biogenesis of these organelles is defective in genetic disorders collectively known as Hermansky-Pudlak syndrome (HPS). Patients with HPS-2, and two murine HPS models, carry mutations in genes encoding subunits of adaptor protein (AP)-3. Other genes mutated in rodent models include those encoding VPS33A and Rab38. Orthologs of all of these genes in Drosophila melanogaster belong to the 'granule group' of eye pigmentation genes. Other genes associated with HPS encode subunits of three complexes of unknown function, named biogenesis of lysosome-related organelles complex (BLOC)-1, -2 and -3, for which the Drosophila counterparts had not been characterized. Here, we report that the gene encoding the Drosophila ortholog of the HPS5 subunit of BLOC-2 is identical to the granule group gene pink (p), which was first studied in 1910 but had not been identified at the molecular level. The phenotype of pink mutants was exacerbated by mutations in AP-3 subunits or in the orthologs of VPS33A and Rab38. These results validate D. melanogaster as a genetic model to study the function of the BLOCs.

Amino Acid Sequence↗
PubMed2006

Strain-dependent variations in the response of cancellous bone to ovariectomy in mice.

UNLABELLED: The goal of this study was to characterize the skeletal response to ovariectomy in mice (129P3, C57BL/6, and B6129PF2) commonly used in gene manipulation studies to evaluate their potential as preclinical models of postmenopausal osteoporosis. The magnitude of cancellous bone loss and cellular indices of increased bone turnover in response to ovariectomy varied with mouse type and skeletal site, but in general, were less pronounced and less consistent than in Sprague-Dawley rats, the established preclinical model for postmenopausal bone loss. INTRODUCTION: The ovariectomized (OVX) rat is the most widely used preclinical rodent model for postmenopausal osteoporosis. However, the underlying mechanisms of bone disorders, including osteoporosis, have been explored predominantly in the mouse. The purpose of this study was to evaluate mice (129P3 and C57BL/6 inbred strains and their F2 hybrid offspring, B6129PF2), commonly used for gene knockout and overexpression studies, for their potential as preclinical models of postmenopausal bone loss. MATERIALS AND METHODS: The mice were OVX or sham-operated at 4 months of age and killed at 1 or 3 months after surgery. Lumbar vertebrae and distal femora were subjected to histomorphometric assessment. RESULTS: Mice in the two strains and the F2 hybrids (will be referred to as strain for the remainder of the abstract) lost vertebral cancellous bone after OVX; bone volume (BV/TV) was 20% and 27% lower at 1 and 3 months after surgery, respectively. The decreased cancellous BV/TV was associated with an increase in osteoclast surface at 1 month after OVX in the 129P3 strain only. Osteoblast surface was increased by 20% with OVX at both 1 and 3 months after surgery, irrespective of mouse strain. However, bone formation rate was not altered by OVX in any of the mouse strains. In contrast to the lumbar vertebrae, cancellous bone loss in response to OVX differed in the distal femur among the three mouse strains. OVX had no significant effect on distal femur BV/TV in the B6129PF2 mouse strain. In the C57BL/6 strain, cancellous BV/TV was reduced by OVX at 1 month after surgery but not at 3 months after surgery, whereas distal femur BV/TV in 129P3 mice was reduced at 3 months after surgery. Osteoclast surface was not affected by OVX at either time-point in the C57BL/6 strain, but was increased by 116% at 1 month after surgery in the 129P3 strain. Osteoblast surface was increased with OVX at 1 month after surgery, irrespective of strain, whereas bone formation rate was not altered by OVX at either time-point in any of the strains. CONCLUSIONS: The magnitude of cancellous bone loss and cellular indices of increased bone turnover in response to OVX varied with mouse strain and skeletal site, but in general, were less pronounced and less consistent than in the Sprague-Dawley rat. Although mouse models will continue to provide insights into genetic influences on bone mass and turnover, caution should be exercised when using 129P3 and C57BL/6 mice, and their F2 hybrids, as models for postmenopausal bone loss and preclinical testing of potential therapies for osteoporosis.

Animals↗
PubMed2001

Parameters of glucose and lipid metabolism in the male Göttingen minipig: influence of age, body weight, and breeding family.

The pig is useful as a model for human physiology and pathophysiology and could be an important supplement to the many available rodent models of diabetes mellitus. Due to their small size, Göttingen minipigs are especially suitable for long-term studies. The aim of the study reported here was to establish reference values for a range of glucose and lipid homeostasis parameters of interest that could be used to identify possible diabetes-prone male Göttingen minipig individuals, families, or age groups. Plasma samples from nonfed animals were analyzed for glucose, leptin, fructosamine, insulin, C-peptide, triglyceride, free fatty acids, and total cholesterol values. Breeding family had significant effects only on plasma triglyceride concentrations (P < 0.001). Plasma concentrations of glucose (P = 0.012), fructosamine (P < 0.001) and triglycerides (P < 0.001) increased significantly with age, whereas total cholesterol concentration decreased significantly (P = 0.001) with age. Age did not influence other parameters. In conclusion, glycemia and insulinemia increased with age and body weight, possibly indicating a small deterioration in insulin sensitivity with age. It is, therefore, hypothesized that older, compared to younger animals may be more useful in the development of a model of type-2 diabetes mellitus. Furthermore, on the basis of decrease in cholesterol concentration with age, animals fed ad libitum with possibly a high calorie diet might be even more useful in the development of a type-2 diabetes mellitus model.

Aging↗
PubMedDate not supplied

Porcine heterotopic composite tissue allograft transplantation using a large animal model for preclinical studies.

BACKGROUND: Composite tissue allograft (CTA) transplantation is currently limited by the risks of side effects resulting from long-term high-dose immunosuppression. Therefore, preclinical animal models are essential to help CTA transplantation advance into clinical reality. Evidence has shown that small-animal model (rodents) immunotherapy protocols cannot be directly applied to humans. This study investigated whether a miniature porcine model is reproducible for preclinical studies. METHODS: Based on the concept of vascularized skeletal tissue allograft transplantation, limb heterotopic allograft tissue from a mismatched donor miniature pig consisting of the distal femur, knee joint, tibia, fibula, and surrounding muscle with a vascularized skin paddle model supplied by the superficial femoral vessels was transplanted into recipient pigs. Swine viability and rejection signs of the allograft were monitored postoperatively. Histopathological changes in the allograft tissues were examined using hematoxylin and eosin staining if the allo-skin flap was rejected. RESULTS: The recipient pigs were ambulatory immediately following surgery. The flaps showed no visible signs of rejection over the first 4 days of observation. The skin flaps appeared bluish-purple and edematous on postoperative days 5 approximately 7, and progressed to tissue necrosis and rejection on postoperative days 8 approximately 13. Histological examination revealed marked mononuclear cell infiltration and necrotic changes in the all rejected tissues, especial in the allograft skin tissues (skin > muscle > bone > cartilage). CONCLUSIONS: The results showed this the porcine CTA model is reproducible and suitable for preclinical training for human CTA transplantation. Monitoring of the allo-skin flap is a useful strategy to evaluate composite tissue allograft rejection.

Animals↗
PubMed1994

Animal model for the study of methanol toxicity: comparison of folate-reduced rat responses with published monkey data.

We attempted to develop a rodent model that exhibits characteristics of human methanol toxicities such as acidosis and visual dysfunction, which are correlated with an accumulation of formate, a toxic metabolite of methanol. Initially three groups of Long-Evans rats with different levels of liver folate were prepared and examined for formate accumulation after methanol administration (3.5 g/kg). The folate-reduced (FR) rats prepared by feeding a folate-deficient diet with 1% succinylsulfathiazole yielded blood formate levels equivalent to those found in methanol-intoxicated humans and developed signs of the visual system toxicity (a manuscript on the latter aspect is in preparation). Responses of FR rats to a variety of methanol exposure scenarios were then investigated, and the results were compared with those reported in the literature for monkeys. Formate accumulation and/or lethality were used as toxic parameters for this comparative evaluation. In FR rats dosed orally with 3 g/kg, the blood formate concentration was 9.2 mmol/L at 24 h postadministration and increased to 15.6 mmol/L at 48 h. The same dose given to monkeys yielded a plateau of 7.4 mmol/L at 12 h after methanol administration, and stayed at this level for an additional 12 h. The area under the concentration vs. time curve for blood formate in FR rats was 2.5-fold greater than that in monkeys when 2.0 g/kg methanol was administered. After a 6-h exposure to 1200 ppm and 2000 ppm methanol, the blood formate concentrations in FR rats were increased by 370% and 636% above the endogenous level, respectively. However, blood formate did not accumulate above the endogenous level when monkeys were exposed to methanol up to 2000 ppm for 6 h. Under acute inhalation exposure conditions, FR rats exposed to 3000 ppm methanol, 20 h/d, could not survive more than 4 d. On the other hand, monkeys exposed to 3000 ppm, 21 h/d, out-lived 20 d. Moreover, monkeys survived for more than 4 d even after an exposure to 10,000 ppm. Thus, these results indicate that FR rats are more sensitive to methanol challenges than monkeys, and suggest that the FR rat could be a congruous animal model for evaluating the health effects of methanol in humans.

Administration, Inhalation↗
PubMed1996

A new experimental model of contusion in the rat. Histopathological analysis and temporal patterns of cerebral blood flow disturbances.

The authors have devised a simple reproducible rodent model of focal cortical injury that uses a mechanical suction force applied through intact dura. The time course and pattern of changes in neurons, glia, and microvasculature were investigated using this model. Early traumatic disruption of the blood-brain barrier and hemorrhage do not occur in this model; however, many of the features of human contusion seen with light and electron microscopy are closely reproduced. At the site of injury, early swelling and lucency of neural dendritic processes have been shown to precede an astrocyte response. In the absence of perivascular hemorrhage, delayed perivascular protein leakage and polymorphonuclear infiltration of the damaged cortex occurs, which is suggestive of an acute inflammatory response. Cerebral blood flow (CBF) has been measured using 14C-iodoantipyrine autoradiography at 30 minutes, 4 hours, and 24 hours after induction of negative-pressure injury in rats anesthetized with halothane and in time-matched sham-operated controls. A significant reduction in blood flow in the sensorimotor cortex at the site of the injury was present at 30 minutes, 4 hours, and 24 hours after induction of the lesion, compared to the contralateral cortex (superficial lamina, ipsilateral 50 +/- 7 ml/100 g/minute, contralateral 112 +/- 26 ml/100 g/minute). The CBF was significantly reduced at the ipsilateral entorhinal cortex at 30 minutes postinjury but no significant reduction was demonstrated at later time points. Although marked alterations in CBF occurred in this cortical injury model, the magnitude and duration of the reduction in CBF are not consistent with those necessary for production of ischemic cell damage. These data indicate that this model of cortical injury can be used to examine biomechanical aspects of contusion without domination by ischemic pathophysiology.

Animals↗
PubMed2002

A tumor-expressed inhibitor of the early but not late complement lytic pathway enhances tumor growth in a rat model of human breast cancer.

Membrane-bound complement inhibitors protect host cells from inadvertent complement attack, and complement inhibitors are often up-regulated on tumors, possibly representing a selective adaptation by tumors to escape elimination by a host antitumor immune response. Relevant in vivo studies using rodent models of human cancer have been hampered by the fact that human complement inhibitors are not effective against rodent complement. Using nude rats and MCF7 cells expressing different rat complement inhibitors, a model of human breast cancer was established to investigate the role of complement and complement inhibitors in tumor progression. Expression of rat CD59, an inhibitor of the terminal cytolytic membrane attack complex of complement, had no effect on the incidence or growth rate of MCF7 tumors. In contrast, expression of rat Crry, an inhibitor of complement activation, dramatically enhanced the tumorigenicity of MCF7 cells. The expression of rat Crry on MCF7 inhibited the in vivo deposition of complement C3 fragments that serve as opsonins for receptors on phagocytes and natural killer cells. These data provide direct in vivo evidence that an inhibitor of complement activation can facilitate tumor growth by modulating C3 deposition. These data indicate an important role for complement opsonization in promoting cell-mediated antitumor immune function, a conclusion further supported by the demonstration that expression of rat Crry, but not rat CD59, on MCF7 cells inhibits rat cell-mediated cytotoxicity in vitro. Rat complement activation on MCF7 tumors was mediated by tumor-reactive antibodies present in the serum of naïve nude rats, but there was also an IgM response to MCF7 tumors, a situation with similarities to some human cancers. These data support a hypothesis that blocking complement inhibitor function on tumor cells will not only enhance monoclonal antibody-mediated immunotherapy but may also be effective at enhancing a normally ineffective humoral immune response in the absence of administered antitumor antibody.

Animals↗
PubMed2004

Systemic neutrophil priming by lipid mediators in post-shock mesenteric lymph exists across species.

BACKGROUND: Post-hemorrhagic shock mesenteric lymph (PHSML) has been linked with neutrophil (PMN) priming, endothelial cell (EC) activation, and acute lung injury (ALI) in rodent models. We have previously identified the lipid fraction of PHSML as containing the causative agent(s). Due to the lesson learned from the rodent gut bacterial translocation experience, we sought to confirm this phenomenon using a large animal model; hypothesizing that lymph collected from the porcine gut following ischemia/reperfusion (I/R) would cause PMN priming. METHODS: Mesenteric lymph was collected from adult pigs before, during, and for 2 hours after non-lethal hemorrhagic shock (mean arterial pressure = 30 mm Hg x 45 minutes). Whole lymph and the extracted lipid fractions of the lymph were then added to isolated human and porcine PMNs and superoxide production was measured by cytochrome C reduction. RESULTS: Hemorrhagic shock profoundly affected mesenteric lymph flow from baseline (pre-shock) flow rates of 75.63 +/- 8.86 mL/hr to 49.38 +/- 5.76 mL/hr during shock and increasing to 253.38 +/- 27.62 mL/hr after 2 hours of resuscitation. Human PMNs exposed to both whole lymph (PHSML) and its extracted lipids (PHSML Lipid) collected 2 hours after shock exhibited more than a two-fold increase in superoxide release upon activation compared with pre-shock samples: PHSML- 6.27 +/- 0.83 versus 2.56 +/- 0.60 nmolO2(-)/ 3.75 cells/mL/min, respectively (p = 0.007), PHSML Lipid- 4.93 +/- 0.34 versus 2.49 +/- 0.11 nmolO2(-)/ 3.75 cells/mL/min (p < 0.001). Similarly, porcine PMNs exhibited close to a two-fold activation when exposed to the lymph and lipid fraction: PHSML- 4.51 +/- 0.42 versus 1.06 +/- 0.28 nmolO2(-)/ 3.75 cells/mL/min (p = 0.008), PHSML Lipid-4.80 +/- 0.81 versus 1.55 +/- 0.23 nmolO2(-)/ 3.75 cells/mL/min (p = 0.002). CONCLUSION: Mesenteric lymphatics serve as the conduit for inflammatory mediators elaborated by the post-ischemic gut in both small and large animal models. Further, the causal agent(s) exist in the lipid fraction of the lymph and are active on both human and animal PMNs.

Animals↗
PubMed1999

Motor effects and mapping of cerebral alterations in animal models of Parkinson's and Huntington's diseases.

Changes in stimulant-induced behavioral effects and subcortical c-Fos expression were compared between rodent models of Parkinson's disease (PD) and Huntington's disease (HD). Rats received either a unilateral 6-hydroxydopamine (6-OHDA)-induced lesion of the nigrostriatal dopamine pathway (PD model) or a unilateral infusion of antisense oligodeoxynucleotides targeting c-fos into the striatum (HD model). Dopamine-lesioned animals received intraperitoneal injections of either d-amphetamine (6-OHDAamp group) or apomorphine (6-OHDAapo group), whereas all animals that received antisense infusions received d-amphetamine (ASF group). All groups exhibited robust circling behavior upon stimulant challenge. Changes in subcortical activation, as assessed by the induction of Fos-like immunoreactivity (Fos-LI), were examined in several brain regions. The 6-OHDAamp and ASF groups exhibited robust, ipsiversive circling behavior, with similar changes in Fos-LI in the striatum, entopeduncular nucleus, superior colliculus, and ventromedial thalamus. The 6-OHDAapo group exhibited contraversive rotation and had reciprocal patterns of Fos-LI in these regions. Despite exhibiting the same direction of rotation, the 6-OHDAamp and ASF groups had markedly different patterns of Fos-LI in the globus pallidus and the pontine reticular formation. These results suggest that the globus pallidus may undergo distinct alterations in PD and HD and that the pontine reticular formation is particularly susceptible to changes in mesencephalic dopamine sources.

Animals↗
PubMed2006

Cardioprotective effect of des-Aspartate-angiotensin-I (DAA-I) on cytokine gene expression profile in ligation model of myocardial infarction.

We investigate the influence of des-Aspartate-angiotensin-I (DAA-I) on the cytokine expression profile in a rodent model of myocardial infarction. Myocardial infarction model was created in female Wistar rats by coronary artery ligation. Animals were randomized to receive intravenously either a daily dose of 1.2 mug DAA-I/kg body weight (group 1; n = 60) or saline (group 2; n = 60) for 14 days after infarction. Heart function was assessed by echocardiography. Animals were euthanized at 1, 3, 7, 14 and 31 days. Morphometric analysis using tetrazolium chloride staining revealed that infarct size was reduced by 32.2% (p < 0.05) in group 1 after 14 days of DAA-I treatment. Left ventricular ejection fraction in group 1 improved significantly (73.4%) as compared to group 2 (47.7%; p < 0.001). Immunostaining for immune cells at the infarct site showed that CD8+ lymphocytes infiltration at the infarct site declined in group 1 (15 +/- 5 cells) as compared to group 2 (50 +/- 6 cells; p < 0.001). Infiltration of monocytes and macrophages remained high at day 14 in group 2 (126 +/- 40 cells) as compared to group 1 (49 +/- 11 cells; p = 0.006). Multiplex PCR was done for differential gene expression of various pro-inflammatory cytokines. IL-6, TNF-alpha, TGF-beta and GM-CSF expression were significantly down-regulated in the infarct, peri-infarct and contra-lateral zones of the left ventricle in group 1 as compared to group 2. IL-6, TGF-beta and GM-CSF expression started to decline from day 1 of DAA-I treatment while TNF-alpha expression only reduced after 7 days of DAA-I treatment. We conclude that DAA-I prevented infarct expansion through suppression of inflammatory cytokines and immune cell infiltration in the infarct region.

Angiotensin I↗
PubMed2006

Hepatic oval cell response to the choline-deficient, ethionine supplemented model of murine liver injury is attenuated by the administration of a cyclo-oxygenase 2 inhibitor.

Oval cell proliferation precedes neoplasia in many rodent models of hepatocellular carcinoma and prevention of this proliferative response can reduce the risk of subsequent carcinoma. This study aimed to determine whether a selective cyclo-oxygenase-2 (COX-2) inhibitor, SC-236, affects (i) the oval cell response to liver injury in a mouse model of hepatocarcinogenesis and (ii) an oval cell line. Four-week-old mice were fed either normal chow or a choline deficient, ethionine supplemented (CDE) diet in the presence or absence of SC-236. Liver histology and oval cell numbers were determined after 2, 4, 12 and 52 weeks of treatment. Oval cells were scored using morphological criteria and positive immuno-staining for the M(2)-isozyme of pyruvate kinase (M2PK) or A6. An immortalized oval cell line (PIL-2) was used to study the in vitro effects of SC-236 on oval cell proliferation, apoptosis and Akt phosphorylation. The percentage of M2PK-positive oval cells and COX-2-positive cells was reduced by 80% and 45%, respectively, in CDE-fed mice receiving SC-236 compared with CDE-fed animals not receiving SC-236. Some M2PK-positive oval cells were also COX-2 positive. The percentage of A6-positive cells was not affected by SC-236 administration to CDE-fed mice. Administration of SC-236 increased apoptosis as evidenced by a 73% increase in the number of TUNEL-positive cells at 2 weeks in CDE-fed mice. Primary oval cells and PIL-2 cells expressed COX-2. In vitro treatment of PIL-2 cells with SC-236 resulted in a dose-dependent preferential death of A6-negative cells. Administration of 25 and 50 microM Prostaglandin E(2) partially attenuated SC-236 induced cell death by 25%. In vitro oval cell death was associated with apoptosis and a 70% reduction in Akt phosphorylation. These results suggest that the SC-236 induced reduction of M2PK-positive oval cell numbers may be due to COX-2 dependent inhibition of Akt phosphorylation and induction of apoptosis.

Animals↗
PubMed2006

Heat shock protein 70 neutralization exerts potent antitumor effects in animal models of colon cancer and melanoma.

When overexpressed, the stress protein heat shock protein 70 (HSP70) increases the oncogenic potential of cancer cells in rodent models. HSP70 also prevents apoptosis, thereby increasing the survival of cells exposed to a wide range of otherwise lethal stimuli. These protective functions of HSP70 involve its interaction with and neutralization of the adaptor molecule apoptotic protease activation factor-1, implicated in caspase activation, and the flavoprotein apoptosis-inducing factor (AIF), involved in caspase-independent cell death. We have shown previously that a peptide containing the AIF sequence involved in its interaction with HSP70 (ADD70, amino acids 150-228) binds to and neutralizes HSP70 in the cytosol, thereby sensitizing cancer cells to apoptosis induced by a variety of death stimuli. Here, we show that expression of ADD70 in tumor cells decreases their tumorigenicity in syngeneic animals without affecting their growth in immunodeficient animals. ADD70 antitumorigenic effects are associated with an increase in tumor-infiltrating cytotoxic CD8+ T cells. In addition, ADD70 sensitizes rat colon cancer cells (PROb) and mouse melanoma cells (B16F10) to the chemotherapeutic agent cisplatin. ADD70 also shows an additive effect with HSP90 inhibition by 17-allylamino-17-demethoxygeldanamycin in vitro. Altogether, these data indicate the potential interest of targeting the HSP70 interaction with AIF for cancer therapy.

Animals↗
PubMed2000

A modified transorbital baboon model of reperfused stroke.

BACKGROUND AND PURPOSE: Although pathophysiological studies of focal cerebral ischemia in nonhuman primates can provide important information not obtainable in rodent models, primate experimentation is limited by considerations of cost, availability, effort, and ethics. A reproducible and quantitative model that minimizes the number of animals necessary to detect differences between treatment groups is therefore crucial. METHODS: Eight male baboons (weight, 22+/-2 kg) underwent left transorbital craniectomy followed by 1 hour of temporary ipsilateral internal carotid artery occlusion at the level of the anterior choroidal artery together with bilateral temporary occlusion of both anterior cerebral arteries (A1) proximal to the anterior communicating artery. A tightly controlled nitrous oxide-narcotic anesthetic allowed for intraoperative motor evoked potential confirmation of middle cerebral artery (MCA) territory ischemia. Animals survived to 72 hours or 10 days if successfully self-caring. Outcomes were assessed with a 100-point neurological grading system, and infarct volume was quantified by planimetric analysis of both MRI and triphenyltetrazolium chloride-stained sections. RESULTS: Infarction volumes (on T2-weighted images) were 32+/-7% (mean+/-SEM) of the ipsilateral hemisphere, and neurological scores averaged 29+/-9. All animals demonstrated evidence of hemispheric infarction, with damage evident in both cortical and subcortical regions in the MCA vascular territory. Histologically determined infarction volumes differed by <3% and correlated with absolute neurological scores (r=0.9, P:=0.003). CONCLUSIONS: Transorbital temporary occlusion of the entire anterior cerebral circulation with strict control of physiological parameters can reliably produce reperfused MCA territory infarction. The magnitude of the resultant infarct with little interanimal variability diminishes the potential number of animals required to distinguish between 2 treatment regimens. The anatomic distribution of the infarct and associated functional deficits offer comparability to human hemispheric strokes.

Animals↗
PubMed1995

Pharmacological activity of the second generation leukotriene B4 receptor antagonist, SC-53228: effects on acute colonic inflammation and hepatic function in rodents.

Inflammatory bowel disease is a chronic inflammatory disorder of the gastrointestinal tract that includes ulcerative colitis and Crohn's disease. Leukotriene B4 is thought to be a prominent proinflammatory mediator in these diseases, in that leukotriene B4 levels are increased in the colonic mucosa of inflammatory bowel disease patients and there is increased polymorphonuclear leukocyte infiltration of these tissues. SC-53228 [(+)-(S)-7-[3-[2(-cyclopropylmethyl)-3-methoxy-4- [(methylamino)carbonyl]phenoxy]propoxy]-3,4-dihydro-8-propyl-2H-1- benzopyran-2-propanoic acid], a second generation LTB4 receptor antagonist, was evaluated for therapeutic efficacy in a rodent model of acute colonic inflammation induced by short chain organic acids, as well as for effects on rodent liver. When given intracolonically to mice, SC-53228 inhibited neutrophil infiltration, assessed by myeloperoxidase (MPO) levels, with an ED50 value of 9 +/- 1.2 mg/kg. When given by gavage, SC-53228 inhibited neutrophil influx in colitic mice with an ED50 value of 30 mg/kg. These results were also confirmed histologically. Furthermore, high dose oral SC-53228 treatment had no effect on liver cytochrome P-450 content, fatty acyl CoA oxidase or liver weight in rats and mice. Together, these data suggest that SC-53228 may be efficacious orally and locally, as well as safe for use in trials for the medical management of IBD.

Acyl-CoA Oxidase↗