Human skeletal muscle-derived stem/progenitor cells modified with connexin-43 prevent arrhythmia in rat post-infarction hearts and influence gene expression in the myocardium.

Stem cell therapy in combination with genetic modification (e.g., transfection with the coding sequence for the connexion 43 gene, GJA1) may solve the problems associated with the occurrence of additional (secondary) stimulation in the post-infarcted heart (arrhythmia). Human skeletal muscle-derived stem/progenitor cells (SkMDS/PCs) were transfected with the pCiNeo-GJA1 plasmid at an efficiency of approximately 96%. Gene overexpression was assessed using qPCR, and subsequent analysis revealed that GJA1 expression increased more than 40-fold in SkMDS/PCs transfected with the appropriate coding sequence (SkMDS/PCsCX43) compared to that of the 'native' SkMDS/PCs control (SkMDS/PCsWT). Enhanced (4-fold) protein expression of connexin-43 was also confirmed by Western immunoblotting. Furthermore, using the arrhythmic score, we demonstrated the positive effects of SkMDS/PCsCX43 cell intervention in reducing additional secondary stimulations in rat post-infarcted hearts compared with that of wild-type cell delivery. Selected gene responses (Kcnq1, Cacna1c, Ncx1, Serca2a, and Tgfb1) showed significantly altered expression profiles in the rat myocardium upon intervention with SkMDS/PCsCX43. The genetic modification of human skeletal muscle-derived stem/progenitor cells with connexin-43 prevented the pro-arrhythmic effects of myogenic implanted stem cells on the host myocardium and positively influenced myocardial gene expression profiles in respect to myocardium conductivity.

Sperm mitochondrial dysfunction and oxidative stress as possible reasons for isolated asthenozoospermia.

Reduced sperm motility, defined as asthenozoospermia, is a frequent cause of male infertility, and is mainly connected with the dysfunction of sperm mitochondria. The aim of this study was to identify the proteins, and thereby the metabolic pathways, responsible for asthenozoospermia, using 2-DE and MALDI-TOF MS, and correlate the results obtained with those of two mitochondrial tests: JC-1 and MitoSox Red. The JC-1 test was performed to test sperm mitochondrial activity, and the MitoSox Red test was performed to check whether the observed sperm poor motility is associated with mitochondrial reactive oxygen species (ROS) production. To identify proteins strictly connected with reduced sperm motility, men with isolated asthenozoospermia (n = 4 versus 10 normozoospermic controls) alone were included in the study. The proteomic analyses resulted in the identification of 25 sperm proteins that are differentially expressed in asthenozoospermic individuals. Most of the identified proteins were downregulated and were involved in energy production; however, we have also identified structural sperm proteins and proteins secreted by the epididymis. The latter, together with the results from MitoSox Red assay, may provide insights into the pathophysiological basis of asthenozoospermia.

Influence of hypoxia prevailing in post-infarction heart on proangiogenic gene expression and biological features of human myoblast cells applied as a pro-regenerative therapeutic tool.

Cardiovascular diseases along with MI (myocardial infarction) lead to regional ischaemia and hypoxic conditions, which prevail after infarction. Diminished O2 saturation which is related to elevated level of hypoxia inducible factor 1 (HIF-1) transcription factor, may switch the expression of many genes. To maximize effect of therapies proposed by regenerative medicine, it is essential to verify (within different time points after MI) the expression of proangiogenic genes and their receptors that are regulated, along with the expression of HIF-1α. We demonstrated a connection between the expression of Hif-1α (in murine post infarcted heart model) and the proangiogenic genes Vegf-a; and Plgf and their receptors during myocardial hypoxia. The innovative part of the study required establishment of the most accurate in vitro O2 level corresponding to the hypoxia level prevailing in myocardium after MI. We determined the influence of hypoxia on the biology of human myoblasts in in vitro oxygen conditions (3%), corresponding to those prevailing in the heart after an infarction using a murine model. We also tested myoblasts that were genetically modified with VEGF-A/FGF-4 and PlGF under hypoxic conditions and compared their characteristics with cells cultured under normoxia and hyperoxia (standard in vitro conditions) with respect to myogenic gene expression, cell proliferation, fusion potential and proangiogenic function. The examination of genetically modified myoblasts under optimized in vitro hypoxia conditions led to the conclusion that hypoxia did not negatively influence the biological functions of the myoblasts, such as cell proliferation and/or proangiogenic characteristics. These results support the expected increased proregenerative effects of such genetically modified human myoblasts.

Human myoblast transplantation in mice infarcted heart alters the expression profile of cardiac genes associated with left ventricle remodeling.

BACKGROUND: Myocardial infarction (MI) and left ventricle remodeling (LVR) are two of the most challenging disease entities in developed societies. Since conventional treatment cannot fully restore heart function new approaches were attempted to develop new strategies and technologies that could be used for myocardial regeneration. One of these strategies pursued was a cell therapy--particularly applying skeletal muscle stem cells (SkMCs). METHODS AND RESULTS: Using NOD-SCID murine model of MI and human skeletal myoblast transplantation we were able to show that SkMC administration significantly affected gene expression profile (p<0.05) (NPPB, CTGF, GATA4, SERCA2a, PLB) of the heart ventricular tissue and this change was beneficial for the heart function. We have also shown, that the level of heart biomarker, NT-proBNP, decreased in animals receiving implanted cells and that the NT-proBNP level negatively correlated with left ventricle area fraction change (LVFAC) index which makes NT-proBNP an attractive tool in assessing the efficacy of cell therapy both in the animal model and prospectively in clinical trials. CONCLUSIONS: The results obtained suggest that transplanted SkMCs exerted beneficial effect on heart regeneration and were able to inhibit LVR which was confirmed on the molecular level, giving hope for new ways of monitoring novel cellular therapies for MI.

Implantation of autologous muscle-derived stem cells in treatment of fecal incontinence: results of an experimental pilot study.

BACKGROUND: The aim of this study is to present results of the implantation of autologous myoblasts into the external anal sphincter (EAS) in ten patients with fecal incontinence. METHODS: After anatomical and functional assessment of the patients' EAS, a vastus lateralis muscle open biopsy was performed. Stem cells were extracted from the biopsy specimens and cultured in vitro. Cell suspensions were then administered to the EAS. Patients were scheduled for follow-up visits in 6-week intervals. Total follow-up was 12 months. RESULTS: All biopsy and cell implantation procedures were performed without complications. Nine of the patients completed a full 12-month follow-up. There was subjective improvement in six patients (66.7 %). In manometric examinations 18 weeks after implantation, squeeze anal pressures and high-pressure zone length increased in all patients, with particularly significant sphincter function recovery in five patients (55.6 %). Electromyographic (EMG) examination showed an increase in signal amplitude in all patients, detecting elevated numbers of propagating action potentials. Twelve months after implantation two patients experienced deterioration of continence, which was also reflected in the deterioration of manometric and EMG parameters. The remaining four patients (44.4 %) still described their continence as better than before implantation and retained satisfactory functional examination parameters. CONCLUSIONS: Implantation of autologous myoblasts gives good short-term results not only in a subjective assessment, but also in objective functional tests. It seems that this promising technology can improve the quality of life of patients with fecal incontinence, but further study is required to achieve better and more persistent results.

In vitro and in vivo characteristics of connexin 43-modified human skeletal myoblasts as candidates for prospective stem cell therapy for the failing heart.

BACKGROUND: Previously, connexin 43-modified skeletal myoblasts (MbCx) were shown to reduce the pro-arrhythmic effect during the regeneration of heart tissue in an animal model of infarction. To increase the relevance to clinical implementation, in this study, we introduced connexin 43 into human myoblasts using a highly safe non-viral vector and demonstrated that their transplantation had a positive effect on the function of the injured heart. METHODS AND RESULTS: Myoblasts were efficiently transfected with a pCiNeo-GJA1 plasmid (65.72%). qPCR analysis revealed over 32-fold higher expression of the connexin 43 gene in the MbCx cell population compared to 'native' controls. The susceptibility of the myoblasts to oxidative stress conditions (p<0.001) and the fusion index (p<0.01) were increased in the MbCx cells. Additionally, we observed changes in the MYOG and MYH2 gene expression levels in the GJA1-modified myoblasts. Finally, we observed a significant improvement in the post-infarction echocardiographic parameters after intervention using MbCx cells compared with non-transfected myoblasts (MbWt) and the control (0.9% NaCl), wherein a significant decrease in the left ventricular area change in the short axis (SAX AC%) was observed at the two-month follow-up (p<0.05 and p<0.01, respectively). CONCLUSIONS: We demonstrated the positive effect of connexin 43 overexpression on the biology and function of human skeletal myoblasts in the context of their potential clinical applications. Our preclinical studies using a mouse infarction model indicated the positive effect of MbCx implantation on the function of the injured heart.

PET imaging with a [68Ga]gallium-labelled PSMA ligand for the diagnosis of prostate cancer: biodistribution in humans and first evaluation of tumour lesions.

PURPOSE: Prostate-specific membrane antigen (PSMA) is a cell surface protein with high expression in prostate carcinoma (PC) cells. Recently, procedures have been developed to label PSMA ligands with (68)Ga, (99m)Tc and (123/124/131)I. Our initial experience with Glu-NH-CO-NH-Lys-(Ahx)-[(68)Ga(HBED-CC)]((68)Ga-PSMA) suggests that this novel tracer can detect PC relapses and metastases with high contrast. The aim of this study was to investigate its biodistribution in normal tissues and tumour lesions. METHODS: A total of 37 patients with PC and rising prostate-specific antigen (PSA) levels were subjected to (68)Ga-PSMA positron emission tomography (PET)/CT. Quantitative assessment of tracer uptake was performed 1 and 3 h post-injection (p.i.) by analysis of mean and maximum standardized uptake values (SUVmean/max) of several organs and 65 tumour lesions. Subsequently, tumour to background ratios were calculated. RESULTS: The PET/CT images showed intense tracer uptake in both kidneys and salivary glands. Moderate uptake was seen in lacrimal glands, liver, spleen and in small and large bowel. Quantitative assessment revealed excellent contrast between tumour lesions and most normal tissues. Of 37 patients, 31 (83.8 %) showed at least one lesion suspicious for cancer at a detection rate of 60 % at PSA <2.2 ng/ml and 100 % at PSA >2.2 ng/ml. Median tumour to background ratios were 18.8 (2.4-158.3) in early images and 28.3 (2.9-224.0) in late images. CONCLUSION: The biodistribution of the novel (68)Ga-PSMA tracer and its ability to detect PC lesions was analysed in 37 patients. Within healthy organs, kidneys and salivary glands demonstrated the highest radiotracer uptake. Lesions suspicious for PC presented with excellent contrast as early as 1 h p.i. with high detection rates even at low PSA levels.

Substituiçäo do rádio distal pelo perônio proximal em tumor de células gigantes / Replacement of the distal end of the radius by the proximal fibula in giant cell tumor

Apresenta-se experiência no tratamento do tumor de células gigantes da porçäo distal do rádio mediante ressecçäo em bloco e substituiçäo deste pelo perônuio proximal, mostra-se a técnica utilizada e analisam-se os resultados em 5 pacientes com seguimento mínimo de 7 meses e máximo de 18 anos