pcDNA3.1tdTomato is superior to pDsRed2-N1 for optical fluorescence imaging in the F344/AY-27 rat model of bladder cancer

PURPOSE: Animal models are important for pre-clinical assessment of novel therapies in metastatic bladder cancer. The F344/AY-27 model involves orthotopic colonisation with AY-27 tumour cells which are syngeneic to F344 rats. One disadvantage of the model is the unknown status of colonisation between instillation and sacrifice. Non-invasive optical imaging using red fluorescence reporters could potentially detect tumours in situ and would also reduce the number of animals required for each experiment.

MATERIALS AND METHODS: AY-27 cells were stably transfected with either pDsRed2-N1 or pcDNA3.1tdTomato. The intensity and stability of fluorescence in the resultant AY-27/DsRed2-N1 and AY-27/tdTomato stable cell lines were compared using Xenogen IVIS®200 and Olympus IX51 systems.

RESULTS: AY-27/tdTomato fluorescence intensity was 60-fold brighter than AY-27/DsRed2-N1 and was sustained in AY-27/tdTomato cells following freezing and six subsequent sub-cultures. After sub-cutaneous injection, fluorescence intensity from AY-27/tdTomato cells was threefold stronger than that detected from AY-27/DsRed2-N1 cells. IVIS®200 detected fluorescence from AY-27/tdTomato and AY-27/DsRed2-N1 cells colonising resected and exteriorised bladders, respectively. However, the deep-seated position of the bladder precluded in vivo imaging. Characteristics of AY-27/tdTomato cells in vitro and in tumours colonising F344 rats resembled those of parental AY-27 cells. Tumour transformation was observed in the bladders colonised with AY-27/DsRed2-N1 cells.

CONCLUSIONS: In vivo whole-body imaging of internal red fluorescent animal tumours should use pcDNA3.1tdTomato rather than pDsRed2-N1. Optical imaging of deep-seated organs in larger animals remains a challenge which may require proteins with brighter red or far-red fluorescence and/or alternative approaches.

Na+/H+ exchange inhibitor cariporide attenuates skeletal muscle infarction when administered before ischemia or reperfusion

Administration of Na(+)/H(+) exchange isoform-1 (NHE-1) inhibitors before ischemia has been shown to attenuate myocardial infarction in several animal models of ischemia-reperfusion injury. However, controversy still exists as to the efficacy of NHE-1 inhibitors in protection of myocardial infarction when administered at the onset of reperfusion. Furthermore, the efficacy of NHE-1 inhibition in protection of skeletal muscle from infarction (necrosis) has not been studied. This information has potential clinical applications in prevention or salvage of skeletal muscle from ischemia-reperfusion injury in elective and trauma reconstructive surgery. The objective of this research project is to test our hypothesis that the NHE-1 inhibitor cariporide is effective in protection of skeletal muscle from infarction when administered at the onset of sustained ischemia or reperfusion and to study the mechanism of action of cariporide. In our studies, we observed that intravenous administration of cariporide 10 min before ischemia (1 or 3 mg/kg) or reperfusion (3 mg/kg) significantly reduced infarction in pig latissimus dorsi muscle flaps compared with the control, when these muscle flaps were subjected to 4 h of ischemia and 48 h of reperfusion (P <0.05; n = 5 pigs/group). Both preischemic and postischemic cariporide treatment (3 mg/kg) induced a significant decrease in muscle myeloperoxidase activity and mitochondrial-free Ca(2+) content and a significant increase in muscle ATP content within 2 h of reperfusion (P <0.05; n = 4 pigs/group). Preischemic and postischemic cariporide treatment (3 mg/kg) also significantly inhibited muscle NHE-1 protein expression within 2 h of reperfusion after 4 h of ischemia, compared with the control (P <0.05; n = 3 pigs/group). These observations support our hypothesis that cariporide attenuates skeletal muscle infarction when administered at the onset of ischemia or reperfusion, and the mechanism involves attenuation of neutrophil accumulation and mitochondrial-free Ca(2+) overload and preservation of ATP synthesis in the early stage of reperfusion.

Efficacy and mechanism of hypoxic postconditioning in salvage of ex vivo human rectus abdominis muscle from hypoxia/reoxygenation injury

In reconstructive surgery, skeletal muscle may endure protracted ischemia before reperfusion, which can lead to significant ischemia/reperfusion injury. Ischemic postconditioning induced by brief cycles of reperfusion/reocclusion at the end of ischemia has been shown to salvage skeletal muscle from ischemia/reperfusion injury in several animal models. However, ischemic postconditioning has not been confirmed in human skeletal muscle. Using an established in vitro human skeletal muscle hypoxic conditioning model, we tested our hypothesis that hypoxic postconditioning salvages ex vivo human skeletal muscle from hypoxia/reoxygenation injury and the mechanism involves inhibition of opening of the mitochondrial permeability transition pore (mPTP) and preservation of ATP synthesis. Muscle strips (~0.5×0.5×15mm) from human rectus abdominis muscle biopsies were cultured in Krebs-Henseleit-HEPES buffer, bubbled with 95%N(2)/5%CO(2) (hypoxia) or 95%O(2)/5%CO(2) (reoxygenation). Samples were subjected to 3h hypoxia/2h reoxygenation. Hypoxic postconditioning was induced by one or two cycles of 5min reoxygenation/5min hypoxia after 3h hypoxia. Muscle injury, viability and ATP synthesis after 2h of reoxygenation were assessed by measuring lactate dehydrogenase (LDH) release, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) reduction and ATP content, respectively. Hypoxic postconditioning or treatment with the mPTP-opening inhibitors Cyclosporine A (CsA, 5×10(-6)M) or N-Methyl-4-isoleucine Cyclosporine (NIM811, 5×10(-6)M) 10min before reoxygenation decreased LDH release, increased MTT reduction and increased muscle ATP content (n=7 patients; P

Development of an in vitro model for study of the efficacy of ischemic preconditioning in human skeletal muscle against ischemia-reperfusion injury

Ischemia-reperfusion (I/R) injury causes skeletal muscle infarction and ischemic preconditioning (IPC) augments ischemic tolerance in animal models. To date, this has not been demonstrated in human skeletal muscle. This study aimed to develop an in vitro model to investigate the efficacy of simulated IPC in human skeletal muscle. Human skeletal muscle strips were equilibrated in oxygenated Krebs-Henseleit-HEPES buffer (37 degrees C). Aerobic and reperfusion phases were simulated by normoxic incubation and reoxygenation, respectively. Ischemia was simulated by hypoxic incubation. Energy store, cell viability, and cellular injury were assessed using ATP, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT), and lactate dehydrogenase (LDH) assays, respectively. Morphological integrity was assessed using electron microscopy. Studies were designed to test stability of the preparation (n = 5-11) under normoxic incubation over 24 h; the effect of 1, 2, 3, 4, or 6 h hypoxia followed by 2 h of reoxygenation; and the protective effect of hypoxic preconditioning (HPC; 5 min of hypoxia/5 min of reoxygenation) before 3 h of hypoxia/2 h of reoxygenation. Over 24 h of normoxic incubation, muscle strips remained physiologically intact as assessed by MTT, ATP, and LDH assays. After 3 h of hypoxia/2 h of reoxygenation, MTT reduction levels declined to 50.1 +/- 5.5% (P <0.05). MTT reduction levels in HPC (82.3 +/- 10.8%) and normoxic control (81.3 +/- 10.2%) groups were similar and higher (P <0.05) than the 3 h of hypoxia/2 h of reoxygenation group (45.2 +/- 5.8%). Ultrastructural morphology was preserved in normoxic and HPC groups but not in the hypoxia/reoxygenation group. This is the first study to characterize a stable in vitro model of human skeletal muscle and to demonstrate a protective effect of HPC in human skeletal muscle against hypoxia/reoxygenation-induced injury.

Conjugated linoleic acid and atherosclerosis: no effect on molecular markers of cholesterol homeostasis in THP-1 macrophages

Macrophage cholesterol homeostasis is a key process involved in the initiation and progression of atherosclerosis. Peroxisome proliferator-activated receptors (PPARs) regulate the transcription of the genes involved in cholesterol homeostasis and thus represent an important therapeutic target in terms of reducing atherosclerosis. Conjugated linoleic acid (CLA) is a potent anti-atherogenic dietary fatty acid in animal models of atherosclerosis and is capable of activating PPARs in vitro and in vivo. Therefore, this study examined whether the anti-atherogenic effects of CLA in vivo could be ascribed to altered cholesterol homeostasis in macrophages and macrophage derived foam cells. Of several genes that regulate cholesterol homeostasis investigated, CLA had most effect on the class B scavenger receptor CD36. The cis-9,trans-11 CLA (c9,t11-CLA) and trans-10,cis-12 CLA (t10,c12-CLA) isomers augmented CD36 mRNA expression (P

Considerations for using sucrose to reduce procedural pain in preterm infants

Preterm and critically ill newborns admitted to a NICU undergo repeated skin-breaking procedures that are necessary for their survival. Sucrose is rapidly becoming the accepted clinical standard nonpharmacologic intervention for managing acute procedural pain for these infants. Although shown to be safe in single doses, only 4 studies have evaluated the effects of repeated doses of sucrose over relatively short periods of time. None has examined the use of sucrose throughout the NICU stay, and only 1 study evaluated the neurodevelopmental outcomes after repeated doses of sucrose. In that study, infants born at 10 doses per day in the first week of life were more likely to show poorer attention and motor development in the early months after discharge from the NICU. Results of studies in animal models have suggested that the mechanism of action of sucrose is through opioid pathways; however, in human infants, little has been done to examine the physiologic mechanisms involved, and the findings reported thus far have been ambiguous. Drawing from the growing animal literature of research that has examined the effects of chronic sugar exposure, we describe alternative amine and hormone pathways that are common to the processing of sucrose, attention, and motor development. In addition, a review of the latest research to examine the effects of repeated sucrose on pain processing is presented. These 2 literatures each can inform the other and can provide an impetus to initiate research to examine not only the mechanisms involved in the calming mechanisms of sucrose but also in the long-term neurodevelopmental effects of repeated sucrose in those infants born extremely preterm or critically ill.

Neonatal procedural pain exposure predicts lower cortisol and behavioral reactivity in preterm infants in the NICU

Data from animal models indicate that neonatal stress or pain can permanently alter subsequent behavioral and/or physiological reactivity to stressors. However, cumulative effects of pain related to acute procedures in the neonatal intensive care unit (NICU) on later stress and/or pain reactivity has received limited attention. The objective of this study is to examine relationships between prior neonatal pain exposure (number of skin breaking procedures), and subsequent stress and pain reactivity in preterm infants in the NICU. Eighty-seven preterm infants were studied at 32 (+/-1 week) postconceptional age (PCA). Infants who received analgesia or sedation in the 72 h prior to each study, or any postnatal dexamethasone, were excluded. Outcomes were infant responses to two different stressors studied on separate days in a repeated measures randomized crossover design: (1) plasma cortisol to stress of a fixed series of nursing procedures; (2) behavioral (Neonatal Facial Coding System; NFCS) and cardiac reactivity to pain of blood collection. Among infants born

Prenatal exposure to maternal depression, neonatal methylation of human glucocorticoid receptor gene (NR3C1) and infant cortisol stress responses

In animal models, variations in early maternal care are associated with differences in hypothalamic-pituitary-adrenal(HPA) stress response in the offspring, mediated via changes in the epigenetic regulation of glucocorticoid receptor (GR) gene (Nr3c1) expression.

Epigenetics in Vascular Disease - Therapeutic Potential of New Agents

Vascular diseases, including atherosclerosis, angioplasty-induced restenosis, vessel graft arteriosclerosis and hypertension-related stenosis, remain the most prevalent cause of death in the developed world. The aetiology of vascular diseases is multifactorial with both genetic and environmental factors. Recently, some of the most promising research identifies the epigenetic modification of the genome to play a major role in the disease development, linking the environmental insults with gene regulation. In this process, modification of DNA by methylation, and histone modification by acetylation, methylation, phosphorylation and/or SUMOylation are reported. Importantly, recent studies demonstrated that histone deacetylase (HDAC) enzymes are crucial in endothelial integrity, smooth muscle proliferation and in the formation of arteriosclerosis in animal models. The study of HDACs has shown remarkable specificity of HDAC family members in vascular cell growth/death that influences the disease process. Interestingly, the effects of HDACs on arteriosclerosis development in animal models have been observed after HDAC inhibition using specific inhibitors. This provides a new approach for the treatment of vascular disease using the agents that influence the epigenetic process in vascular cells. This review updates the rapid advances in epigenetics of vascular diseases focusing on the role of HDAC family in atherosclerosis. It will also discuss the underlying mechanisms of histone acetylation in vascular cells and highlight the therapeutic potential of such agents.

Advances in our understanding of diabetic retinopathy

Diabetic retinopathy remains the most common complication of diabetes mellitus and is a leading cause of visual loss in industrialized nations. The clinicopathology of the diabetic retina has been extensively studied, although the precise pathogenesis and cellular and molecular defects that lead to retinal vascular, neural and glial cell dysfunction remain somewhat elusive. This lack of understanding has seriously limited the therapeutic options available for the ophthalmologist and there is a need to identify the definitive pathways that initiate retinal cell damage and drive progression to overt retinopathy. The present review begins by outlining the natural history of diabetic retinopathy, the clinical features and risk factors. Reviewing the histopathological data from clinical specimens and animal models, the recent paradigm that neuroretinal dysfunction may play an important role in the early development of the disease is discussed. The review then focuses on the molecular pathogenesis of diabetic retinopathy with perspective provided on new advances that have furthered our understanding of the key mechanisms underlying early changes in the diabetic retina. Studies have also emerged in the past year suggesting that defective repair of injured retinal vessels by endothelial progenitor cells may contribute to the pathogenesis of diabetic retinopathy. We assess these findings and discuss how they could eventually lead to new therapeutic options for diabetic retinopathy.

Abnormal changes in voltage-gated sodium channels NaV1.1, NaV1.2, NaV1.3, NaV1.6 and in Calmodulin/calmodulin-dependent protein kinase II, within the brains of spontaneously epileptic rats and tremor rats

Voltage-gated sodium channels (VGSCs) play a crucial role in epilepsy. The expressions of different VGSCs subtypes are varied in diverse animal models of epilepsy that may reflect their multiple phenotypes or the complexity of the mechanisms of epilepsy. In a previous study, we reported that NaV1.1 and NaV1.3 were up-regulated in the hippocampus of the spontaneously epileptic rat (SER). In this study, we further analyzed both the expression and distribution of the typical VGSC subtypes NaV1.1, NaV1.2, NaV1.3 and NaV1.6 in the hippocampus and in the cortex of the temporal lobe of two genetic epileptic animal models: the SER and the tremor rat (TRM). The expressions of calmodulin (CaM) and calmodulin-dependent protein kinase II (CaMKII) were also analyzed with the purpose of assessing the effect of the CaM/CaMKII pathway in these two models of epilepsy. Increased expression of the four VGSC subtypes and CaM, accompanied by a decrease in CaMKII was observed in the hippocampus of both the SERs and the TRM rats. However, the changes observed in the expression of VGSC subtypes and CaM were decreased with an elevated CaMKII in the cortex of their temporal lobes. Double-labeled immunofluorescence data suggested that in SERs and TRM rats, the four subtypes of the VGSC proteins were present throughout the CA1, CA3 and dentate gyrus regions of the hippocampus and temporal lobe cortex and these were co-localized in neurons with CaM. These data represent the first evidence of abnormal changes in expression of four VGSC subtypes (NaV1.1, NaV1.2, NaV1.3 and NaV1.6) and CaM/CaMKII in the hippocampus and temporal lobe cortex of SERs and TRM rats. These changes may be involved in the generation of epileptiform activity and underlie the observed seizure phenotype in these rat models of genetic epilepsy.

Clinical trials in cystic fibrosis

In patients with cystic fibrosis (CF), clinical trials are of paramount importance. Here, the current status of drug development in CF is discussed and future directions highlighted. Methods for pre-clinical testing of drugs with potential activity in CF patients including relevant animal models are described. Study design options for phase II and phase III studies involving CF patients are provided, including required patient numbers, safety issues and surrogate end point parameters for drugs, tested for different disease manifestations. Finally, regulatory issues for licensing new therapies for CF patients are discussed, including new directives of the European Union and the structure of a European clinical trial network for clinical studies involving CF patients is proposed.

Antigen-specific IgA and IgG responses in calves inoculated intranasally with ovalbumin encapsulated in poly(DL-lactide-co-glycolide) microspheres

The immunogenicity of proteins encapsulated in poly(DL-lactide-co-glycolide) (PLG) microspheres has not been investigated to any extent in large animal models. In this study, IgG and IgA responses to ovalbumin (OVA), encapsulated in microspheres was investigated following intranasal inoculation into calves. Scanning electron microscopy and flow cytometric analysis demonstrated a uniform microsphere population with a diameter of <2.5 micrometers. Ovalbumin was released steadily from particles stored in PBS almost in a linear fashion, and after 4 weeks many particles showed cracks and fissures in their surface structure. Following intranasal inoculation of calves with different doses of encapsulated antigen, mean levels of ovalbumin-specific IgA were observed to increase steadily but significant differences in IgA levels (from the pre-inoculation level) were only observed following a second intranasal inoculation. With 0.5 and 1.0mg doses of antigen, ovalbumin-specific IgG was also detected in serum. Ovalbumin-specific IgA persisted in nasal secretions for a considerable period of time and were still detectable in four out of seven animals, 6 months after inoculation.

Consequences of unlocking the cardiac myosin molecule in human myocarditis and cardiomyopathies

Myocarditis, often initiated by viral infection, may progress to autoimmune inflammatory heart disease, dilated cardiomyopathy and heart failure. Although cardiac myosin is a dominant autoantigen in animal models of myocarditis and is released from the heart during viral myocarditis, the characterization, role and significance of anti-cardiac myosin autoantibodies is poorly defined. In our study, we define the human cardiac myosin epitopes in human myocarditis and cardiomyopathies and establish a mechanism to explain how anti-cardiac myosin autoantibodies may contribute to heart disease. We show that autoantibodies to cardiac myosin in sera from myocarditis and dilated cardiomyopathies in humans targeted primarily epitopes in the S2 hinge region of cardiac myosin. In addition, anti-cardiac myosin antibodies in sera or purified IgG from myocarditis and cardiomyopathy targeted the beta-adrenergic receptor and induced antibody-mediated cAMP-dependent protein kinase A (PKA) cell signaling activity in heart cells. Antibody-mediated PKA activity in sera was abrogated by absorption with anti-human IgG. Antibody-mediated cell signaling of PKA was blocked by antigen-specific inhibition by human cardiac myosin or the beta-adrenergic receptor but not the alpha adrenergic receptor or bovine serum albumin. Propranolol, a beta blocker and inhibitor of the beta-adrenergic receptor pathway also blocked the antibody-mediated signaling of the beta-adrenergic receptor and PKA. The data suggest that IgG antibody against human cardiac myosin reacts with the beta-adrenergic receptor and triggers PKA signaling in heart cells. In summary, we have identified a new class of crossreactive autoantibodies against human cardiac myosin and the beta-adrenergic receptor in the heart. In addition, we have defined disease specific peptide epitopes in the human cardiac myosin rod S2 region in human myocarditis and cardiomyopathy as well as a mechanistic role of autoantibody in the pathogenesis of disease.

Lactobacillus strains isolated from infant faeces possess potent inhibitory activity against intestinal alpha- and beta-glucosidases suggesting anti-diabetic potential

Purpose: Inhibitors of intestinal alpha-glucosidases are used therapeutically to treat type 2 diabetes mellitus. Bacteria such as Actinoplanes sp. naturally produce potent alpha-glucosidase inhibitor compounds, including the most widely available drug acarbose. It is not known whether lactic acid bacteria (LAB) colonising the human gut possess inhibitory potential against glucosidases. Hence, the study was undertaken to screen LABs having inherent alpha- and beta-glucosidase inhibitory potential. Methods: This study isolated, screened, identified and extracted Lactobacillus strains (Lb1–15) from human infant faecal samples determining their inhibitory activity against intestinal maltase, sucrase, lactase and amylase. Lactobacillus reference strains (Ref1–7), a Gram positive control (Ctrl1) and two Gram negative controls (Ctrl2–3), were also analysed to compare activity. Results: Faecal isolates were identified by DNA sequencing, with the majority identified as unique strains of Lactobacillus plantarum. Some strains (L. plantarum, L. fermentum, L. casei and L. rhamnosus) had potent and broad spectrum inhibitory activities (up to 89 %; p < 0.001; 500 mg/ml wet weight) comparable to acarbose (up to 88 %; p < 0.001; 30 mg/ml). Inhibitory activity was concentration-dependent and was freely available in the supernatant, and was not present in other bacterial genera (Bifidobacterium bifidum and Escherichia coli or Salmonella typhimurium). Interestingly, the potency and spectrum of inhibitory activity across strains of a single species (L. plantarum) differed substantially. Some Lactobacillus extracts had broader spectrum activities than acarbose, effectively inhibiting beta-glucosidase activity (lactase) as well as alpha-glucosidase activities (maltase, sucrase and amylase). Anti-diabetic potential was indicated by the fact that oral gavage with a L. rhamnosus extract (1 g/kg) was able to reduce glucose excursions (Area under curve; 22 %; p < 0.05) in rats during a carbohydrate challenge (starch; 2 g/kg). Conclusion: These results definitively demonstrate that Lactobacillus strains present in the human gut have alpha- and beta-glucosidase inhibitory activities and can reduce blood glucose responses in vivo. Although the potential use of LAB such as Lactobacillus as a dietary supplement, medicinal food or biotherapeutic for diabetes is uncertain, such an approach might offer advantages over drug therapies in terms of broader spectrum activities and fewer unpleasant side effects. Further characterisation of this bioactivity is warranted, and chronic studies should be undertaken in appropriate animal models or diabetic subjects.