Peripheral arterial disease and diabetes: effects on endothelial progenitor cell differentiation and nitric oxide metabolism

In the recent years it is emerged that peripheral arterial disease (PAD) has become a growing health problem in Western countries. This is a progressive manifestation of atherothrombotic vascular disease, which results into the narrowing of the blood vessels of the lower limbs and, as final consequence, in critical leg ischemia. PAD often occurs along with other cardiovascular risk factors, including diabetes mellitus (DM), low-grade inflammation, hypertension, and lipid disorders. Patients with DM have an increased risk of developing PAD, and that risk increases with the duration of DM. Moreover, there is a growing population of patients identified with insulin resistance (IR), impaired glucose tolerance, and obesity, a pathological condition known as “metabolic syndrome”, which presents increased cardiovascular risk. Atherosclerosis is the earliest symptom of PAD and is a dynamic and progressive disease arising from the combination of endothelial dysfunction and inflammation. Endothelial dysfunction is a broad term that implies diminished production or availability of nitric oxide (NO) and/or an imbalance in the relative contribution of endothelium-derived relaxing factors. The secretion of these agents is considerably reduced in association with the major risks of atherosclerosis, especially hyperglycaemia and diabetes, and a reduced vascular repair has been observed in response to wound healing and to ischemia. Neovascularization does not only rely on the proliferation of local endothelial cells, but also involves bone marrow-derived stem cells, referred to as endothelial progenitor cells (EPCs), since they exhibit endothelial surface markers and properties. They can promote postnatal vasculogenesis by homing to, differentiating into an endothelial phenotype, proliferating and incorporating into new vessels. Consequently, EPCs are critical to endothelium maintenance and repair and their dysfunction contributes to vascular disease. The aim of this study has been the characterization of EPCs from healthy peripheral blood, in terms of proliferation, differentiation and function. Given the importance of NO in neovascularization and homing process, it has been investigated the expression of NO synthase (NOS) isoforms, eNOS, nNOS and iNOS, and the effects of their inhibition on EPC function. Moreover, it has been examined the expression of NADPH oxidase (Nox) isoforms which are the principal source of ROS in the cell. In fact, a number of evidences showed the correlation between ROS and NO metabolism, since oxidative stress causes NOS inactivation via enzyme uncoupling. In particular, it has been studied the expression of Nox2 and Nox4, constitutively expressed in endothelium, and Nox1. The second part of this research was focused on the study of EPCs under pathological conditions. Firstly, EPCs isolated from healthy subject were cultured in a hyperglycaemic medium, in order to evaluate the effects of high glucose concentration on EPCs. Secondly, EPCs were isolated from the peripheral blood of patients affected with PAD, both diabetic or not, and it was assessed their capacity to proliferate, differentiate, and to participate to neovasculogenesis. Furthermore, it was investigated the expression of NOS and Nox in these cells. Mononuclear cells isolated from peripheral blood of healthy patients, if cultured under differentiating conditions, differentiate into EPCs. These cells are not able to form capillary-like structures ex novo, but participate to vasculogenesis by incorporation into the new vessels formed by mature endothelial cells, such as HUVECs. With respect to NOS expression, these cells have high levels of iNOS, the inducible isoform of NOS, 3-4 fold higher than in HUVECs. While the endothelial isoform, eNOS, is poorly expressed in EPCs. The higher iNOS expression could be a form of compensation of lower eNOS levels. Under hyperglycaemic conditions, both iNOS and eNOS expression are enhanced compared to control EPCs, as resulted from experimental studies in animal models. In patients affected with PAD, the EPCs may act in different ways. Non-diabetic patients and diabetic patients with a higher vascular damage, evidenced by a higher number of circulating endothelial cells (CECs), show a reduced proliferation and ability to participate to vasculogenesis. On the other hand, diabetic patients with lower CEC number have proliferative and vasculogenic capacity more similar to healthy EPCs. eNOS levels in both patient types are equivalent to those of control, while iNOS expression is enhanced. Interestingly, nNOS is not detected in diabetic patients, analogously to other cell types in diabetics, which show a reduced or no nNOS expression. Concerning Nox expression, EPCs present higher levels of both Nox1 and Nox2, in comparison with HUVECs, while Nox4 is poorly expressed, probably because of uncompleted differentiation into an endothelial phenotype. Nox1 is more expressed in PAD patients, diabetic or not, than in controls, suggesting an increased ROS production. Nox2, instead, is lower in patients than in controls. Being Nox2 involved in cellular response to VEGF, its reduced expression can be referable to impaired vasculogenic potential of PAD patients.

Molecular approach to Neurodegenerative Disorders: Role of Nociceptin/Orphanin FQ - NOP System in Parkinson and Epigenetic Mechanism in Alzheimer's Disease

With life expectancies increasing around the world, populations are getting age and neurodegenerative diseases have become a global issue. For this reason we have focused our attention on the two most important neurodegenerative diseases: Parkinson’s and Alzheimer’s. Parkinson’s disease is a chronic progressive neurodegenerative movement disorder of multi-factorial origin. Environmental toxins as well as agricultural chemicals have been associated with PD. Has been observed that N/OFQ contributes to both neurotoxicity and symptoms associated with PD and that pronociceptin gene expression is up-regulated in rat SN of 6-OHDA and MPP induced experimental parkinsonism. First, we investigated the role of N/OFQ-NOP system in the pathogenesis of PD in an animal model developed using PQ and/or MB. Then we studied Alzheimer's disease. This disorder is defined as a progressive neurologic disease of the brain leading to the irreversible loss of neurons and the loss of intellectual abilities, including memory and reasoning, which become severe enough to impede social or occupational functioning. Effective biomarker tests could prevent such devastating damage occurring. We utilized the peripheral blood cells of AD discordant monozygotic twin in the search of peripheral markers which could reflect the pathology within the brain, and also support the hypothesis that PBMC might be a useful model of epigenetic gene regulation in the brain. We investigated the mRNA levels in several genes involve in AD pathogenesis, as well DNA methylation by MSP Real-Time PCR. Finally by Western Blotting we assess the immunoreactivity levels for histone modifications. Our results support the idea that epigenetic changes assessed in PBMCs can also be useful in neurodegenerative disorders, like AD and PD, enabling identification of new biomarkers in order to develop early diagnostic programs.

Gene therapy for a novel mouse model of Canavan disease

Canavan disease (CD) is a rare leukodystrophy caused by loss-of-function mutations in the gene encoding aspartoacylase (ASPA), an oligodendrocyte-enriched enzyme. It is characterised by the accumulation of the ASPA substrate N-acetylaspartate (NAA) in brain, blood and urine, leading to a spongiform vacuolisation of the brain, severe motoric and cognitive impairments and premature death. To date, no therapy is available due to the lack of a gene-transfer system allowing transgene expression in oligodendrocytes (OLs) and the restoration of the missing enzyme. Hence, the aim of this study was to establish a novel gene-transfer system and its preclinical evaluation in a CD animal model.rnIn the first part of this thesis, a novel ASPA mouse mutant was generated. A βgeo cassette (including the genes encoding β-galactosidase and neomycin) flanked by frt sites was inserted into intron 1 of the intact aspa gene. Additionally, exon 2 was flanked by loxP sites for optional conditional deletion of the targeted locus. The resulting ASPA-deficient aspalacZ/lacZ-mouse was found to be an accurate model of CD and an important tool to identify novel aspects of its complex pathology. Homozygous mutants showed a CD-like histopathology, neurological impairment, behavioural deficits as well as a reduced body weight. Additionally, MRI data revealed changes in brain metabolite composition. rnRecombinant adeno-associated viral (rAAV) vectors have become a versatile tool for gene transfer to the central nervous system because they are efficient, non-toxic and replication-deficient. Based on the natural neurotropism of AAV vectors, AAV-based gene delivery has entered the clinics for the treatment of neurodegenerative diseases. However, the lack of AAV vectors with oligodendroglial tropism has precluded gene therapy for leukodystrophies. In the second part of this work, it was shown that the transduction profile of established AAV serotypes can be targeted towards OLs in a transcriptional approach, using the oligodendrocyte-specific myelin basic protein (MBP) promoter to drive transgene expression in OLs.rnIn the last part of this work, the therapeutic efficacy of AAV-mediated aspa gene transfer to OLs of juvenile aspalacZ/lacZ mice was evaluated. AAV-aspa injections into multiple sites of the brain parenchyma resulted in transduction of OLs in the grey and white matter throughout the brain. Histological abnormalities in the brain of ASPA-deficient mice were ameliorated and accompanied by a reduction of NAA levels. Furthermore, the treatment resulted in normalisation of body weight, motor function and nest-building behaviour. These data provide a proof-of-concept for a successful gene therapy of Canavan disease. This might pave the way towards translation into clinical application and serve as the basis for the genetic treatment of other leukodystrophies.

The role of RTX toxins in host specificity of animal pathogenic Pasteurellaceae

RTX toxins are bacterial pore-forming toxins that are particularly abundant among pathogenic species of Pasteurellaceae, in which they play a major role in virulence. RTX toxins of several primary pathogens of the family of Pasteurellaceae are directly involved in causing necrotic lesions in the target organs. Many RTX toxins are known as haemolysins because they lyse erythrocytes in vitro, an effect that is non-specific, but which serves as a useful marker in bacteriological identification and as an easily measurable signal in vitro in experimental studies. More recent studies have shown that the specific targets of most RTX toxins are leukocytes, with RTX toxins binding to the corresponding beta-subunit (CD18) of beta2 integrins and then exerting cytotoxic activity. After uptake by the target cell, at sub-lytic concentrations, some RTX toxins are transported to mitochondria and induce apoptosis. For several RTX toxins the binding to CD18 has been shown to be host specific and this seems to be the basis for the host range specificity of these RTX toxins. Observations on two very closely related species of the Pasteurellaceae family, Actinobacillus suis, a porcine pathogen particularly affecting suckling pigs, and Actinobacillus equuli subsp. haemolytica, which causes pyosepticaemia in new-born foals (sleepy foal disease), have revealed that they express different RTX toxins, named ApxI/II and Aqx, respectively. These RTX toxins are specifically cytotoxic for porcine and equine leukocytes, respectively. Furthermore, the ApxI and Aqx toxins of these species, when expressed in an isogenetic background in Escherichia coli, are specifically cytotoxic for leukocytes of their respective hosts. These data indicate the determinative role of RTX toxins in host specificity of pathogenic species of Pasteurellaceae.

Cloning of farm animals: impact on animal health and welfare and implications in trade

The commercial use of animal cloning for breeding food producing animals has been limited so far by biological and technical constraints such as adverse effects on the health and welfare of animals, especially high perinatal and postnatal disease and mortality of clones. However, the improvement of the technique may overcome those problems in future and contribute to the spread of cloning in agricultural production, which raises concern not only on health and welfare aspects but also on food safety and ethics. This may cause conflict in international trade. The present article reviews these topics on the basis of up-to-date scientific opinions.

Characterization of the porcine transferrin gene (TF) and its association with disease severity following an experimental Actinobacillus pleuropneumoniae infection

Transferrin (TF)-mediated provision of iron is essential for a productive infection by many bacterial pathogens, and iron-depletion of TF is a first line defence against bacterial infections. Therefore, the transferrin (TF) gene can be considered a candidate gene for disease resistance. We obtained the complete DNA sequence of the porcine TF gene, which spans 40 kb and contains 17 exons. We identified polymorphisms on a panel of 10 different pig breeds. Comparative intra- and interbreed sequence analysis revealed 62 polymorphisms in the TF gene including one microsatellite. Ten polymorphisms were located in the coding sequence of the TF gene. Four SNPs (c.902A>T, c.980G>A, c.1417A>G, c.1810A>C) were predicted to cause amino acid exchanges (p.Lys301Ile, p.Arg327Lys, p.Lys473Glu, p.Asn604His). We performed association analyses using six selected TF markers and 116 pigs experimentally infected with Actinobacillus pleuropneumoniae serotype 7. The analysis showed breed-specific TF allele frequencies. In German Landrace, we found evidence for a possible association of the severity of A. pleuropneumoniae infection with TF genotypes.

[Fetal programming of cardiovascular disease: new causes and underlying mechanisms]

There exists an association between pathologic events occurring during early life and the development of cardiovascular disease in adulthood. For example, transient perinatal hypoxemia predisposes to exaggerated hypoxic pulmonary hypertension and preeclampsia predisposes the offspring to pulmonary and systemic endothelial dysfunction later in life. The latter finding offers a scientific basis for observations demonstrating an increased risk for premature cardiovascular morbidity in this population. Very recently, we showed that offspring of assisted reproductive technologies also display generalized vascular dysfunction and early arteriosclerosis. Studies in animal models have provided evidence that oxidative stress and/or epigenetic alterations play an important pathophysiological role in the fetal programming of cardiovascular disease.

Renal tissue oxygenation in essential hypertension and chronic kidney disease

Animal studies suggest that renal tissue hypoxia plays an important role in the development of renal damage in hypertension and renal diseases, yet human data were scarce due to the lack of noninvasive methods. Over the last decade, blood oxygenation level-dependent magnetic resonance imaging (BOLD-MRI), detecting deoxyhemoglobin in hypoxic renal tissue, has become a powerful tool to assess kidney oxygenation noninvasively in humans. This paper provides an overview of BOLD-MRI studies performed in patients suffering from essential hypertension or chronic kidney disease (CKD). In line with animal studies, acute changes in cortical and medullary oxygenation have been observed after the administration of medication (furosemide, blockers of the renin-angiotensin system) or alterations in sodium intake in these patient groups, underlining the important role of renal sodium handling in kidney oxygenation. In contrast, no BOLD-MRI studies have convincingly demonstrated that renal oxygenation is chronically reduced in essential hypertension or in CKD or chronically altered after long-term medication intake. More studies are required to clarify this discrepancy and to further unravel the role of renal oxygenation in the development and progression of essential hypertension and CKD in humans.

The role of genetic polymorphisms in alcoholic liver disease

Chronic alcohol consumption is a major cause of liver cirrhosis which, however, develops in only a minority of heavy drinkers. Evidence from twin studies indicates that genetic factors account for at least 50% of individual susceptibility. The contribution of genetic factors to the development of diseases may be investigated either by means of animal experiments, through linkage studies in families of affected patients, or population based case-control studies. With regard to the latter, single nucleotide polymorphisms of genes involved in the degradation of alcohol, antioxidant defense, necroinflammation, and formation and degradation of extracellular matrix are attractive candidates for studying genotype-phenotype associations. However, many associations in early studies were found to be spurious and could not be confirmed in stringently designed investigations. Therefore, future genotype-phenotype studies in alcoholic liver disease should meet certain requirements in order to avoid pure chance observations due to a lack of power, false functional interpretation, and insufficient statistical evaluation.

[Hyena disease in a Brown Swiss heifer]

A heifer with developmental skeletal disorder was presented to the ruminant clinic of the University of Berne. Abnormal long bone growth and a wobbling gait were the main clinical signs. All long bones were examined radiologically, several parameters of body size were measured and the results were compared to the measurements of a healthy control animal. Haematology and blood chemistry were normal. Based on the poor prognosis the animal was slaughtered. The final diagnosis of hyena disease was based on the characteristic growth disturbances in the caudal parts of the body, giving the animal a hyena-like appearance. For the first time a case of hyena disease is reported in Switzerland.

Clinical efficacy of pimobendan versus benazepril for the treatment of acquired atrioventricular valvular disease in dogs

Seventy-six dogs with clinical acquired atrioventricular valvular disease were evaluated to determine the efficacy of pimobendan (n=41) versus benazepril hydrochloride (n=35) in a randomized, positive-controlled, multicenter study. The study was divided into 56-day and long-term evaluation periods. In a subgroup of dogs with concurrent furosemide treatment (pimobendan [n=31], benazepril [n=25]), the Heart Insufficiency Score improved in favor of pimobendan (P=0.0011), equating to a superior overall efficacy rating (P<0.0001) at day 56. Long-term median survival (i.e., death or treatment failure) for dogs receiving pimobendan was 415 days versus 128 days for dogs not on pimobendan (P=0.0022).

Alterations of exhaled nitric oxide in pre-term infants with chronic lung disease

Animal models suggest that reduced nitric oxide (NO) synthase activity results in lower values of exhaled NO (eNO) present at birth in those individuals who are going to develop chronic lung disease of infancy (CLDI). Online tidal eNO was measured in 39 unsedated pre-term infants with CLDI (mean gestational age (GA) 27.3 weeks) in comparison with 23 healthy pre-term (31.6 weeks) and 127 term infants (39.9 weeks) at 44 weeks post-conceptional age, thus after the main inflammatory response. NO output (NO output (V'(NO)) = eNO x flow) was calculated to account for tidal- flow-related changes. Sex, maternal atopic disease and environmental factors (smoking, caffeine) were controlled for. The mean eNO was not different (14.9 ppb in all groups) but V'(NO) was lower in CLDI compared with healthy term infants (0.52 versus 0.63 nL x s(-1)). Values for healthy pre-term infants were between these two groups (0.58 nL x s(-1)). Within all pre-term infants (n = 62), V'(NO) was reduced in infants with low GA, high clinical risk index for babies scores and longer duration of oxygen therapy but not associated with post-natal factors, such as ventilation or corticosteroid treatment. After accounting for flow, the lower nitric oxide output in premature infants with chronic lung disease of infancy is consistent with the hypothesis of nitric oxide metabolism being involved in chronic lung disease of infancy.

Lifespan and disease predispositions in the Irish Wolfhound: a review

Several disease predispositions of Irish Wolfhounds are mentioned in the veterinary literature, but these lists vary greatly between different publications. This article reviews findings on lifespan as well as disease predispositions that have been reported in the literature. Hereditary mechanisms found so far are discussed, including their implications for breeding healthier dogs, the ethical necessity of which is stressed under the aspect of animal welfare. An open health registry, combined with the estimation of breeding values, seems to be the most promising approach. Furthermore, routine male castration is discouraged as being associated with an increased osteosarcoma risk. Mean lifespan estimates in Irish Wolfhounds vary between 4.95 and 8.75 years, but bias due to right censored data is common. The diseases reported to occur most frequently are dilated cardiomyopathy, osteogenic sarcoma, gastric dilation and volvulus and diseases of the osteochondrosis spectrum. Furthermore, intrahepatic portosystemic shunt plays an important role. Several other diseases have been reported in the literature, including rhinitis, epilepsy, progressive retinal atrophy, von Willebrand's Disease, and juvenile fibrocartilaginous embolism.

Experimental models of CNS infections. Contributions to concepts of disease and treatment

Lessons learned from studies of experimental meningitis and brain abscess in animal models of infection represent major, highly significant contributions to our understanding of the pathogenesis and antimicrobial chemotherapy of these infections. For example, studies of experimental meningitis in rabbits demonstrated that the subarachnoid space is deficient in local host defenses, a finding that explains why only bactericidal antibiotic regimens are effective in treating this disease; studies of the efficacy of corticosteroids as adjunctive therapy for meningitis yielded data indicating that both beneficial and detrimental effects on the host are imparted by these compounds. These and a number of other key investigations of experimental meningitis and brain abscess, the results of these investigations, and the clinical significance of these results are presented in this article.

A case-control study of sporadic Creutzfeldt-Jakob disease in Switzerland: analysis of potential risk factors with regard to an increased CJD incidence in the years 2001-2004

BACKGROUND: In 2001, the observed annual mortality from Creutzfeldt-Jakob disease (CJD) in Switzerland increased from less than 1.5 to 2.6 per million inhabitants. An underlying cause could not be identified. METHODS: To analyse potential risk factors for sCJD in Switzerland, close relatives of 69 sCJD-patients and 224 frequency age-matched controls were interviewed in a case-control study using a standardised questionnaire. 135 potential risk factors including socio-demographics, medical history, occupation and diet were analysed by logistic regression adjusting for age, sex and education. RESULTS: sCJD patients were more likely to have travelled abroad, worked at an animal laboratory, undergone invasive dental treatment, orthopaedic surgery, ophthalmologic surgery after 1980, regular GP visits, taken medication regularly, and consumed kidney. No differences between patients and controls were found for residency, family history, and exposure to environmental and other dietary factors. CONCLUSION: Although some factors were significantly more frequent among sCJD-cases, this study did not reveal specific explanations for the increased incidence of deaths due to sporadic CJD observed in Switzerland since 2001. Results have to be interpreted with caution due to multiple testing and possible recall bias in association with a long incubation period. The most plausible reason for the increase in Swiss sCJD cases after 2000 is an improved case ascertainment. Therefore, underreporting of cases might well have occurred before the year 2001, and the "real" yearly incidence of sCJD might not be lower than, but rather above 2 per million inhabitants.