Transfer of the full-length dystrophin-coding sequence into muscle cells by a dual high-capacity hybrid viral vector with site-specific integration ability

Duchenne muscular dystrophy (DMD) is caused by mutations in the DMD gene, making it a potential target for gene therapy. There is, however, a scarcity of vectors that can accommodate the 14-kb DMD cDNA and permanently genetically correct muscle tissue in vivo or proliferating myogenic progenitors in vitro for use in autologous transplantation. Here, a dual high-capacity adenovirus-adeno-associated virus (hcAd/AAV) vector with two full-length human dystrophin-coding sequences flanked by AAV integration-enhancing elements is presented. These vectors are generated from input linear monomeric DNA molecules consisting of the Ad origin of replication and packaging signal followed by the recently identified AAV DNA integration efficiency element (p5IEE), the transgene(s) of interest, and the AAV inverted terminal repeat (ITR). After infection of producer cells with a helper Ad vector, the Ad DNA replication machinery, in concert with the AAV ITR-dependent dimerization, leads to the assembly of vector genomes with a tail-to-tail configuration that are efficiently amplified and packaged into Ad capsids. These dual hcAd/AAV hybrid vectors were used to express the dystrophin-coding sequence in rat cardiomyocytes in vitro and to restore dystrophin synthesis in the muscle tissues of mdx mice in vivo. Introduction into human cells of chimeric genomes, which contain a structure reminiscent of AAV proviral DNA, resulted in AAV Rep-dependent targeted DNA integration into the AAVS1 locus on chromosome 19. Dual hcAd/AAV hybrid vectors may thus be particularly useful to develop safe treatment modalities for diseases such as DMD that rely on efficient transfer and stable expression of large genes.     

Transplantation of stem cell-derived astrocytes for the treatment of amyotrophic lateral sclerosis and spinal cord injury

Neglected for years, astrocytes are now recognized to fulfill and support many, if not all, homeostatic functionsof the healthy central nervous system(CNS). During neurodegenerative diseases such as amyotrophic lateral sclerosis(ALS) and spinal cord injury(SCI), astrocytes in the vicinity of degenerating areas undergo both morphological and functional changes that might compromise their intrinsic properties. Evidence from human and animal studies show that deficient astrocyte functions or loss-of-astrocytes largely contribute to increased susceptibility to cell death for neurons, oligodendrocytes and axons during ALS and SCI disease progression. Despite exciting advances in experimental CNS repair, most of current approaches that are translated into clinical trials focus on the replacement or support of spinal neurons through stem cell transplantation, while none focus on the specific replacement of astroglial populations. Knowing the important functions carried out by astrocytes in the CNS, astrocyte replacement-based therapies might be a promising approach to alleviate overall astrocyte dysfunction, deliver neurotrophic support to degenerating spinal tissue and stimulate endogenous CNS repair abilities. Enclosed in this review, we gathered experimental evidence that argue in favor of astrocyte transplantation during ALS and SCI. Based on their intrinsic properties and according to the cell type transplanted, astrocyte precursors or stem cell-derived astrocytes promote axonal growth, support mechanisms and cells involved in myelination, are able to modulate the host immune response, deliver neurotrophic factors and provide protective molecules against oxidative or excitotoxic insults, amongst many possible benefits. Embryonic or adult stem cells can even be genetically engineered in order to deliver missing gene products and therefore maximize the chance of neuroprotection and functional recovery. However, before broad clinical translation, further preclinical data on safety, reliability and therapeutic efficiency should be collected. Although several technical challenges need to be overcome, we discuss the major hurdles that have already been met or solved by targeting the astrocyte populationin experimental ALS and SCI models and we discuss avenues for future directions based on latest molecular findings regarding astrocyte biology.     

Changes in serum parameters associated with iron metabolism in male rat exposed to lead

Due to the severe hazardous influences of lead (Pb²⁺) on iron-related diseases, the effects of Pb²⁺ on serum parameters associated with iron metabolism have been studied in this project. Male Wistar rats weighing 200–250 g were treated with Pb²⁺ for the short and long period of times. The animals received daily intraperitoneal injection of 100 mg Pb²⁺ kg^?1 body weight (BW) for 5 days and 4 mg?kg^?1 BW of Pb²⁺ for 30 and 45 days, respectively. The results show that when animals were treated with both low and high concentrations of Pb²⁺, serum iron concentration decreased markedly, by 23.2, 32.8, and 39.9 %, while the sera TIBC and transferrin concentrations increased significantly (p?<?0.05). Following short- and long-term exposures to Pb²⁺, the percentage of serum transferrin saturation was also decreased in comparison with the untreated control group (p?<?0.05). Concentrations of serum copper and ceruloplasmin following Pb²⁺ treatments also reduced significantly (p?<?0.05). The percentage of hematocrit and hemoglobin levels was reduced (p?<?0.05) in all Pb²⁺-treated animals in comparison with the controls. These results suggest that Pb²⁺ changes the serum parameters related to iron metabolism, which may play an important role in producing iron-related diseases.     

Dilemma of antiepileptic drugs withdrawal in symptomatic epilepsy

A successful treatment of epilepsy depends on numerous factors such as etiology, genetics and environmental impact. An exact diagnosis, treatment and an adequate selection of antiepileptic drugs (AED) are important from the very beginning. The patient with symptomatic epilepsy caused by the brain tumor (low-grade astrocytoma in the left parietal lobe, surgically removed 17 years after the first manifestation of illness) is presented in this study. He has been seizure free for 6 years. The represented case study deals with the risk-benefit analysis of the discontinuation of the prescribed antiepileptic treatment that has lasted for 23 years.     

An analysis of the pollution problem in Slavonski Brod (eastern Croatia)

H2S, PM2.5, O3, NO2, SO2 and meteorological parameters such as temperature, relative humidity, precipitation, wind speed and wind direction were measured simultaneously in an eastern Croatian town called Slavonski Brod during the season winter/spring 2010. Emissions from the nearby cross-border (Bosnia and Herzegovina) oil refinery were identified as sources of temporary elevated concentrations of H2S. The maximum daily averages of PM2.5 concentrations during the winter period were as high as 240 microg m(-3) which is a value 10 times greater than the threshold prescribed by the World Health Organization. It is considered that the heating season, dense traffic, intense industrial activities and temperature inversion during stable weather conditions are prevailing contributors to higher winter concentrations of PM2.5. The results of the principal component analysis technique (PCA) have shown that lower air temperature, lower wind speed and higher relative humidity play a significant role in the winter pollution episodes. From a public health point of view, implementation of measures aimed at reducing the levels of H2S and PM2.5 should be considered.     

Impact of body composition on performance in fitness tests among personnel of the Croatian navy

The purpose of this study was to determine the impact of body weight on fitness tests among the personnel of the Croatian navy. Forty two naval personnel (age 27 +/- 4.1 years; body mass 86.2 +/- 4.9 kg; height 184.6 +/- 7.4 cm; body fat percentage 17.3 +/- 5.2) participated in this study. In order to evaluate the fitness of the naval servicemen, we applied a testing procedure that included measurements of 7 fitness tests and 15 body anthropometric tests. A negative correlation was found between the body fat percentage and all the analyzed sprint tests and three anaerobic power tests (r), SP5 (r = -0.42), SP10 (r = -0.51), SP20 (r = -0.53), SJ (r = -0.45), CM (r = -0.57), SLJ (r = -0.67). Also a negative correlation was found between the body fat percentage and VO2(max) (r = -0.44). A positive correlation was found between the sprint test and the power performance test and thigh and calf girth. Spiriting ability is influenced by the strength of a person. This is one of the reasons why we found a positive correlation between the sprint test (SP5, SP10 and SP20) and thigh and calf girth. In this study we found a negative correlation between body fat percentages and all the sprint tests and three anaerobic power tests and VO2(max). The ectomorph somatotypes have positive correlations with all variables. The mesomorph somatotypes have the greatest positive correlations with all variables. The endomorph somatotypes have negative correlations with all variables. According to the body composition of Croatian naval servicemen we can conclude that they need a sufficient level of strength and endurance for everyday tasks. The effectiveness of a weight-management program is determined by the success of the participants in losing the necessary amount of weight and being able to maintain that weight loss. This requires long-term tracking of these individuals in a naval environment.     

The role of fruit drinks in daily diet of some Osijek inhabitants, Croatia

The aim of the study was to assess the role of fruit drinks in daily diet of Osijek inhabitants. A special questionnaire was administered to 199 patients visiting a family physician's office during December 2003. The concentration of vitamin C (L-ascorbic acid) was determined in 42 commercially available fruit drinks. Only 11% (22/199) of study subjects did not take fruit drinks. The mean concentration of vitamin C in all samples was 150.10 +/- 166.83 mg/L. The questionnaire revealed that 89% (177/199) of study subjects using fruit drinks were taking a mean of 0.4 L of fruit drink per day, yielding a mean of 60.04 mg of vitamin C, i.e. 100% of the recommended daily allowance of 60 mg. Study results indicated fruit drinks to be a significant source of vitamin C in daily diet, however, great variation in vitamin C intake according to socioeconomic status of study subjects and type of fruit drink should be noted.     

Long-term mineralocorticoid receptor blockade reduces fibrosis and improves cardiac performance and coronary hemodynamics in elderly SHR

Aldosterone has been implicated as one of the mediators of cardiovascular injury in various diseases. This study examines whether mineralocorticoid antagonism ameliorates or prevents the adverse cardiac effects of hypertension and aging. Male 22-wk-old spontaneously hypertensive rats (SHR) were divided into two groups, 15 rats in each. One group received no treatment; the other was given eplerenone ( approximately 100 mg.kg(-1).day(-1)). At the age of 54 wk, indexes of cardiovascular mass, systemic and regional hemodynamics, including coronary, left ventricular function, and myocardial collagen content, were determined in all rats. Hemodynamic studies were done in conscious rats. Arterial pressure was lowered only slightly in eplerenone-treated rats, and cardiac output and total peripheral resistance did not differ from control rats. Left and right ventricular and aortic mass indexes were unaffected by eplerenone; however, concentration of hydroxyproline in the right and left ventricle was decreased significantly (P < 0.05) by eplerenone. This was accompanied by an improvement in left ventricular diastolic function and coronary hemodynamics. In conclusion, long-term therapy with the mineralocorticoid receptor antagonist eplerenone ameliorated adverse cardiac effects of both hypertension and aging in SHR. Thus reduction in myocardial fibrosis, paralleled by improvements in left ventricular function and coronary hemodynamics, was observed in eplerenone-treated SHR.     

CYFRA 21-1 in non-small cell lung cancer--standardisation and application during diagnosis

There is no ideal tumour marker at present. The clinical application of CYFRA 21-1 is possible once a thorough standardisation process is carried out. Standardisation is achieved by determining the reference range in asymptomatic population, benign and malignant lung diseases, and benign and malignant diseases of other organs. Furthermore, it depends on knowledge of research population characteristics, patient medical histories and individual diagnostic procedure results, the size of research target samples and the clinically defined control groups. The cut-off level of CYFRA 21-1 for non-small cell lung cancer (NSCLC) is 1.72 ng/mL in the Croatian population. It is based on the clinically applicable sensitivity of 78% and specificity of 95% in benign lung diseases. The cut-off value is verified by clinical findings. For clinicians the level of CYFRA 21-1 is an early sign of NSCLC in relation to all the benign lung diseases and all the benign diseases of other organs, of which it was confirmed that they can influence the above level, provided that NSCLC is verified using standard diagnostic methods. The level of CYFRA 21-1 is also influenced by the time of sampling in relation to other diagnostic invasive procedures. The marker is clinically applicable if clinical findings verify it; otherwise, it is useless. This research has involved 343 healthy persons, 474 patients with a benign disease and 4440 patients with a malignant disease, 2453 of whom suffer from NSCLC. The sensitivity of CYFRA 21-1 in NSCLC is 78%, in squamous cell lung cancer (SQC) 84.6%, in adenocarcinomas (AD) 74.3% and in large cell lung cancer (LCC) 75.3%. The level of CYFRA 21-1 differs significantly between healthy persons, benign and malignant diseases (p<10(-3)). There are differences between the three histological types of NSCLC (p<10(-6)) and according to T and N (p<10(-3)). The level of CYFRA 21-1 prompts clinicians to repeat the clinical procedure during diagnosis, and helps to detect the disease earlier and implement treatment in NSCLC. We have achieved high concordance between marker findings and clinical diagnostic.     

Cardiac structural and functional responses to salt loading in SHR

Increased dietary salt intake induces cardiac fibrosis in the spontaneously hypertensive rat (SHR), yet little information details its effects on left ventricular (LV) function. Additionally, young normotensive rats are more sensitive to the trophic effect of dietary sodium than older rats. Thus cardiac responses to salt loading were evaluated at two ages in the SHR; LV collagen content was also examined. SHR (8 or 20 wk of age) were given an 8% salt diet; their age-matched controls received standard chow. Echocardiographic indexes, arterial pressure, and LV hydroxyproline concentration were measured at 16 and 52 wk in the younger and older SHR groups, respectively. In most SHR, salt excess increased arterial pressure, LV mass, and hydroxyproline concentration and impaired LV relaxation manifested by prolonged isovolumic relaxation time, decreased early and atrial filling velocity ratio (V(E)/V(A)), and slower propagation velocity of E wave (V(P)). LV systolic function remained normal. However, one-quarter of the young salt-loaded SHR developed cardiac failure with systolic and diastolic dysfunction associated with greater LV mass and ventricular fibrosis. They also had lower arterial pressure, decreased fractional shortening, and a restrictive pattern of mitral flow. Moreover, the shorter deceleration time of the E wave and increased V(E)/V(P), an index of LV filling pressure, indicated increased LV stiffness in these rats. These findings demonstrated that sodium sensitivity in SHR is manifested not only by further pressure elevation but also by significant LV functional impairment that most likely is related to enhanced ventricular fibrosis. Moreover, the SHR are more susceptible to cardiac damage when high dietary salt is introduced earlier in life.