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.

All genes encoding enzymes participating in melatonin biosynthesis in the chicken pineal gland are transcribed rhythmically.

Our recent research on the pineal gland of young chickens confirmed that three genes encoding enzymes involved in pineal melatonin biosynthesis, tryptophan hydroxylase 1 (Tph1), arylalkylamine-N-acetyltransferase (Aanat) and acetylserotonin O-methyltransferase (Asmt), are transcribed rhythmically under light:dark (L:D) 12:12 conditions in vivo. Additionally, in the pineal gland of maturing chickens, the dopa decarboxylase (Ddc) gene is transcribed rhythmically at a specific stage of the developmental process. Therefore, the aim of the present study was to verify whether all of these genes are transcribed rhythmically in vivo under constant darkness (D:D) and in pinealocyte cultures under both L:D and D:D. Experiments were performed on chickens maintained under L:D 12:12 conditions. Chickens at 15 days of age were divided into two groups; chickens from the first group remained under the same conditions, whereas those from the second group were kept in darkness. Subsequently, 16-day-old animals were sacrificed every 2 hours over a 24-h period. For the in vitro experiments, 16-day-old chickens were sacrificed at ZT 6, and their pineal glands were isolated. Pineal cultures were maintained for up to two days in L:D conditions. Then, the pinealocyte cultures were divided into two groups: the first remained under L:D conditions, whereas the second was transferred to D:D conditions. Pinealocytes were subsequently collected every 2 hours over a 24-h period. Transcription was evaluated using the RT-qPCR method, and the rhythm percentage was calculated through Cosinor analysis. The mRNA levels of all genes examined were rhythmic under all conditions. Moreover, in silico analysis of the promoters of all of the genes examined revealed the presence of enhancer box sequences in all of the promoters as well as DBP/E4BP4 binding elements in the promoters of Tph1 and Asmt. This suggests that these genes may all be regulated transcriptionally by the molecular clock mechanism and may be considered clock as controlled genes.

Acroangiodermatitis in a leg amputee using a suction-socket prosthesis: clinical, histological as well as HHV-8 and CD34 immunohistochemical study.

Acroangiodermatitis (AAD) is a rare, vascular phenomenon of unclear pathogenesis. Itchy, lichenoid, purple/violaceous/yellowish/brownish papules/nodules, plaques/patches mainly on lower limbs occasionally evolve into verrucous lesions and recurrent painful ulcerations. Elevated vein and capillary pressure due to the sub-atmospheric suspension system seems to be the triggering factor for angioproliferation in the amputation stump. A middle-aged male amputee, a suction-socket prosthesis user, showing combined clinical, histological and immunohistochemical (HHV-8 negative; CD34 and CD31 expressed in endothelial, but not perivascular, cells) features of AAD is presented. Dermatologists, orthopedic surgeons, pathomorphologists, but also prosthesis makers and amputees themselves, should be aware of AAD as suction-socket prostheses become increasingly popular.

Season-dependent postembryonic maturation of the diurnal rhythm of melatonin biosynthesis in the chicken pineal gland.

Previously, we have demonstrated that the timing of the nocturnal peak of activity of the pineal arylalkylamine-N-acetyltransferase - a key enzyme in the melatonin biosynthesis pathway - in 3-wk-old chickens kept from the day of hatch under controlled laboratory conditions (L:D 12:12) varies depending on the season of hatch (summer vs. winter). The present study was undertaken to answer the following questions: (1) are season-related differences seen in the level of transcription of genes encoding enzymes of the melatonin biosynthesis pathway? (2) Does the pineal content of the main precursor (serotonin) and the final product (melatonin) exhibit age- and season-related changes? (3) At which step in postembryonic development are these season-related variations in pineal gland function most pronounced? Male Hy-line chickens hatched in the summer or winter, from eggs laid by hens held on L:D 16:8, were kept from the day of hatch under L:D 12:12 conditions. At the age of 2, 9, or 16 d, chickens were sacrificed every 2 h over a 24-h period and their pineal glands, isolated under dim red light, were processed for the measurement of (i) the level of Aanat and Asmt (acetylserotonin O-methyltransferase) mRNAs encoding the two last enzymes involved in melatonin biosynthesis, (ii) the activity of these enzymes, and (iii) the pineal content of serotonin and melatonin. Circadian rhythmicity of all the measured parameters was evaluated by the cosinor method. The levels of Aanat mRNA, AANAT enzymatic activity, and the pineal melatonin content changed during postembryonic development in a manner that was dependent on the season of hatch. Furthermore, the diurnal profile of Asmt mRNA was elevated during the light phase. In "winter" birds, the pattern and amplitude of the diurnal rhythm of accumulation of this transcript did not change with age, while in "summer" birds it increased in an age-related way. In contrast, the enzymatic activity of hydroxyindole-O-methyltransferase (HIOMT; encoded by the Asmt gene) did not change rhythmically, although it increased with age in a season-related way. In "winter" chickens, the pineal serotonin content was low, regardless of age, and did not change rhythmically, whereas in "summer" individuals the serotonin rhythm was already well established by day 2, with the amplitude increasing with age. These results confirm the existence of a "seasonal memory" operating within the chicken pineal gland, although the mechanism(s) underlying this phenomenon have yet to be characterized.

Heligmosmoides polygyrus fourth stages induce protection against DSS-induced colitis and change opioid expression in the intestine.

Primary exposure of mice to the nematode Heligmosomoides polygyrus infection reduces inflammation in an experimental model of colitis. The aim of the present investigation was to evaluate whether the reduced inflammation provoked by H. polygyrus L4 larvae in BALB/c mice treated with dextran sulphate sodium is associated with changed expression of opioids in the small intestine and colon. Colitis was induced by 5% Dextran sulphate sodium (DSS) oral administration for 3 days before oral infection with 200 infective larvae (L3) H. polygyrus until the end of the experiment, 6 days post-infection. Clinical disease symptoms were monitored daily. The expressions of proopiomelanocortin POMC1, MOR1 (Oprm1) - opioid receptor and ß-endorphin were determined by RT-PCR, Western blot and immunoassay, respectively, in the colon and small intestine of mice. RT-PCR analysis of colon tissues showed up-regulation of the expression of POMC and MOR1 opioid-dependent genes in mice with DSS-induced colitis. H. polygyrus L4 larvae inhibited DSS-induced colitis symptoms that were correlated with increased IL-1ß, TNF-α, IL-6, myeloperoxidase (MPO) concentration, macrophages infiltration and MOR1, POMC and ß-endorphin increased expression in the small intestine and inhibition of those in the colon.

Pouchitis may increase the risk of dysplasia after restorative proctocolectomy in patients with ulcerative colitis.

AIM: Dysplasia of the pouch mucosa after restorative proctocolectomy is rare. The aim of this study was to establish whether there is a correlation between pouchitis and dysplasia. METHOD: A group of 276 patients treated for ulcerative colitis by restorative proctocolectomy between 1984 and 2009 was analysed. The presence or absence of pouchitis and dysplasia within the pouch was evaluated. RESULTS: Inflammation was diagnosed in 66 (23.9%) patients, low-grade dysplasia in five (1.8%), high-grade dysplasia in three (1.1%), and cancer in one patient (0.4%). The prevalence of low-grade dysplasia was significantly higher in patients with inflammation than in those without (P < 0.04). High-grade dysplasia was significantly more frequent in pouchitis than in non-inflamed pouches (P < 0.01). Logistic regression analysis suggested that the occurrence of mucosal inflammation increased the risk of low grade dysplasia. CONCLUSION: Patients with chronic pouchitis are at risk of dysplasia and require surveillance of the pouch.

Auriculo-vestibular symptoms related to structural and functional disorders of stomatognatic system.

Auriculo-vestibular symptoms are otolaryngological complaints which frequently co-occur with functional disorders of masticatory organ. These symptoms include: earache, plugged ears sensation, sudden hearing impairment, burning pain of the throat, tinnitus, and dizziness. The aim of the study was assessment of co-occurrence of functional disorders of masticatory organ and auriculo-vestibular symptoms, in patients referred for otolaryngological treatment. Forty-two patients aged 24-46 years of both sexes referred for otolaryngological treatment, because of auriculo-vestibular symptoms, were qualified to our study within the framework of research project. After otolaryngological diagnostics (Department of Otolaryngology), these patients were referred for prosthetic consultation to the Department of Dental Prosthetics of Jagiellonian University Medical College. In Otolaryngology Clinic the following specialist examinations were carried out: basic clinical examinations, including otoscopy, tuning fork trials, tonal and verbal audiometry, and tympanometry. These examinations were supplemented with electronystagmometry. On prosthetic consultation, specialist functional investigations of masticatory organ, and electromyographic assessment of the activity of masseter muscles and the anterior part of the temporal muscle, were carried out. Results of the investigation revealed otolarygological causes of the reported symptoms in 30 patients, whereas in 12 patients (out of 42 patients referred for prosthetic consultation), numerous functional disorders of the stomatognathic system were observed. The investigation confirmed the occurrence of functional disorders in patients with auriculo-vestibular symptoms and appropriateness of treatment by a multi-specialist team.

IL-6, TNF-alpha, IL-1-beta arterial wall expression is independent of serum concentration in patients sustaining primary or secondary open vascular reconstructions.

AIM: The aim of this study was to assess morphological and immunohistochemical differences in arterial wall inflammation during primary and secondary vascular reconstructions. METHODS: Forty patients with lower extremities ischemia underwent vascular reconstruction: Group I included patients undergoing primary vascular reconstruction with 25 minor arterial reconstructive procedures; Group II included patients undergoing secondary reconstruction due to limb ischemia after at least 12 months since first operation (15). Immunohistochemical expression of IL-1-BETA,IL-6,and TNF-alpha in arterial wall was correlated with serum concentration. RESULTS: No differences were observed in IL-1-beta, IL-6, TNF-alpha expression among groups (P=0.06, P=0.13, P=0.9). Cytokines expression was not accompanied by the growth of its serum concentrations (P=0.8, P=0.9). IL-6 serum concentration depended on the degree of limb ischemia and was very high in case of critical limb ischemia (P=0.006). Arterial morphology was comparable among the groups. CONCLUSION: IL-6, IL-1-BETA, TNF-alpha expression in the arterial wall is comparable in primary and secondary lesions. Serum concentrations of IL-6, IL-1-BETA, TNF-alpha are independent of its arterial wall expression.

Campylobacter protein oxidation influences epithelial cell invasion or intracellular survival as well as intestinal tract colonization in chickens.

The Dsb family of redox proteins catalyzes disulfide bond formation and isomerization. Since mutations in dsb genes change the conformation and stability of many extracytoplasmic proteins, and since many virulence factors of pathogenic bacteria are extracytoplasmic, inactivation of dsb genes often results in pathogen attenuation. This study investigated the role of 2 membrane-bound oxidoreductases, DsbB and DsbI, in the Campylobacter jejuni oxidative Dsb pathway. Campylobacter mutants, lacking DsbB or DsbI or both, were constructed by allelic replacement and used in the human intestinal epithelial T84 cell line for the gentamicin protection assay (invasion assay) and chicken colonization experiments. In C. coli strain 23/1, the inactivation of the dsbB or dsbI gene separately did not significantly affect the colonization process. However, simultaneous disruption of both membrane-bound oxidoreductase genes significantly decreased the strain's ability to colonize chicken intestines. Moreover, C. jejuni strain 81-176 with mutated dsbB or dsbI genes showed reduced invasion/intracellular survival abilities. No cells of the double mutants (dsbB⁻ dsbI⁻) of C. jejuni 81-176 were recovered from human cells after 3 h of invasion.

The influence of elastic components of the venous wall on the biomechanical properties of different veins used for arterial reconstruction.

OBJECTIVE: To evaluate the relationship between the biomechanical properties and the structure of elastic components in different veins used for vascular reconstruction. DESIGN: In vitro experimental study. MATERIAL AND METHODS: Groups of 30 samples of incompetent saphenous veins (rSV), competent saphenous veins (cSV) and femoral veins (FVs) were compared following immunohistochemical staining for the presence of collagen types I, III and IV and elastin. The percentage area of transverse section of veins occupied by each type of collagen and elastin was measured using a computer-image-analysis system connected to a microscope. For all three groups of veins, the storage modulus, E', and the loss modulus, E'', were measured with a mechanical analyser, DMA-242, and changes in the function of temperature and frequency, and duration of exposure to the applied force were determined. RESULTS: The rSV showed the highest percentage share of collagen I and the lowest percentage share of collagen IV. These samples also showed the greatest expression of elastin and the highest elastin to collagen ratio. The rSV were also found to have the highest E' and E'', and during the long-term exposure achieved maximum stiffness in the least time as compared to cSV and FV. CONCLUSION: The histological structure directly influences the biomechanical properties of venous wall with rSV showing least compliance and cSV the greatest compliance.

Limits on the spin-dependent WIMP-nucleon cross sections from the first science run of the ZEPLIN-III experiment.

We present new experimental constraints on the WIMP-nucleon spin-dependent elastic cross sections using data from the first science run of ZEPLIN-III, a two-phase xenon experiment searching for galactic dark matter weakly interacting massive particles based at the Boulby mine. Analysis of approximately 450 kg x days fiducial exposure allow us to place a 90%-confidence upper limit on the pure WIMP-neutron cross section of sigma(n)=1.9x10(-2) pb at 55 GeV/c(2) WIMP mass. Recent calculations of the nuclear spin structure based on the Bonn charge-dependent nucleon-nucleon potential were used for the odd-neutron isotopes 129Xe and 131Xe. These indicate that the sensitivity of xenon targets to the spin-dependent WIMP-proton interaction could be much lower than implied by previous calculations, whereas the WIMP-neutron sensitivity is impaired only by a factor of approximately 2.

Single brain metastasis from a minute, well differentiated, but invading beyond the tunica muscularis mucosa rectal carcinoid.

We report the case of a 48-year-old woman with small-sized (< 10 mm), highly differentiated, aggressive rectal carcinoid, who developed a solitary distant metastasis to the brain. The primary lesion, initially removed by conventional polypectomy, invaded the mucosa/tunica muscularis mucosa and had positive resection margins. Afterwards, an assessment of 5-hydroxyindoloacetic acid (5-HIAA) 24 h urine excretion revealed a significantly increased level. Thus, a partial rectal resection was performed. Because of constantly elevated carcinoid markers: serum chromogranin A (CGA) and 5-HIAA, a somatostatin receptor scintigraphy was performed, which disclosed a focus of pathological marker accumulation in the left frontal area. The pathological finding after neurosurgical excision was meningioma. An unexpected normalization of the biochemical markers prompted us to verify this diagnosis. The final histopathological report was a well-differentiated neuroendocrine brain metastasis. Our case shows that in well differentiated, of diameter < 10 mm rectal carcinoids, an invasion even beyond the mucosa/tunica muscularis mucosa seems to be an independent factor predicting a malignant metastatic potential of these tumors. Hence, in such cases, behind the endoscopic submucosal resection with ligation device a more radical surgery should be considered. Additionally, a systematic CGA and 5-HIAA follow-up assessment and whole body somatostatin receptor scintigraphy, if necessary, are required.

Measurement of occlusal forces in the therapy of functional disorders with the use of botulinum toxin type A.

Functional disorders of the stomatognathic system include dysfunctions leading to pathological increase of the occlusal forces generated by mandibular adductors. High values of these forces are the cause of numerous disorders within the masticatory organ such as pathological abrasion of the teeth, tension headaches and pain in the region of the temporomandibular joints. The aim of this study was assessment of occlusal forces in the course of the therapy of painful types of functional disorders with the use of botulinum toxin type A - the drug causing muscle relaxation. The material for the study comprised adult patients aged 24-42 years who presented with a painful type of functional disorders in the University Hospital, Institute of Dentistry Jagiellonian University Medical College in Cracow. The patients were qualified to the study on the basis of the results of specialist functional examination of the masticatory organ in which additionally the VAS (Visual Analogue Scale) was used for pain assessment of the masseters and temporomandibular joints. The measurement of maximal occlusal forces in the examined patients was carried out in all patients in the region of the central incisors and first molars on the both side with the use of a special measuring instrument for dental examination, specially constructed (patent number P 334933). The examinations were performed before the beginning of the treatment, 10 days and 16 weeks after intramuscular administration of botulinum toxin type A at a dosage of 21 mouse units - U for one masseter. The results of the examination of maximal occlusal forces obtained in the first examination, that is, before the beginning of the treatment, markedly exceeded the physiological values. In the control examinations, significant, persistent decrease of the studied forces has been noted.

Experimental research on the possibilities of maintaining thermal conditions within the limits of the physiological conditions during intraoral preparation of dental implants.

The objective of the study was to evaluate the degree of temperature change in the bone, directly adjacent to the implant site during the abutment part of one-piece implants, during procedures involving different cutting techniques. Three different one-piece implants: Osteoplant, Nobel Direct, Q-implant were cut with air-turbine burs after insertion in fresh pig ribs. Tests were performed with a variety of cooling techniques including air and air-liquid coolant. Implants were cut on the occlusal and axial surfaces of the abutment part. The temperature changes were evaluated using thermocouple type K during and after cutting procedures. It was found that regardless of the cutting technique, the temperature of the bone always increased during the cutting procedure. The critical temperature threshold Delta T=10 degrees C (47 degrees C) was always exceeded, when only the air coolant was used, although when the air-water spray coolant was utilized during the cutting procedure, it prevented the temperature to exceed this critical level. The extent of thermal changes in the bone depends on the cutting technique. Constant air-water cooling during the cutting procedure is a recommended technique in order to avoid overheating the implant-bone interface. The dry cutting technique, using only an air coolant, should be avoided as it can induce sufficiently high thermal damage in the bone adjacent to the implant, which leads to compromise of the osteointegration process.

Synthesis, surface modifications, and size-sorting of mixed nickel-zinc ferrite colloidal magnetic nanoparticles.

We report on the spontaneous covalent growth of monomolecular adlayers on mixed nickel-zinc nanoferrite colloidal suspensions (ferrofluids). Synthesized nanoparticles were subjected to surface modification by means of acid chloride chemistry, leading to the formation of covalent bonds between the hydroxy groups at the nanoparticle surface and the acid chloride molecules. This procedure can be easily tailored to allow for the formation of adlayers containing both hydrophobic and hydrophilic regions stacked at predetermined distances from the magnetic core, and also providing the nanoferrites with functional carboxy groups capable of further modifications with, for example, drug molecules. Here, fluorophore aminopyrene molecules were bound to such modified nanoferrites through amide bonds. We also used the same chemistry to modify the surface with covalently bound long-chain palmitoyl moieties, and for comparison we also modified the nanoferrite surface by simple adsorption of oleic acid. Both procedures made the surface highly hydrophobic. These hydrophobic colloids were subsequently spread on an aqueous surface to form Langmuir monolayers with different characteristics. Moreover, since uniformity of size is crucial in a number of applications, we propose an efficient way of sorting the magnetic nanoparticles by size in their colloidal suspension. The suspension is centrifuged at increasing rotational speed and the fractions are collected after each run. The mean size of nanoferrite in each fraction was measured by the powder X-ray diffraction (PXRD) technique.

Ghrelin ovarian cell expression in patients with polycystic ovary syndrome: an immunohistochemical evaluation.

INTRODUCTION: The influence of ghrelin on different organs has been studied recently, e.g. in the regulation of pituitary hormone release, regulation of energy homeostasis, glucose metabolism and insulin secretion, cell proliferation, and reproductive function. However, the etiology of polycystic ovary syndrome has not been fully explained. The aim of our study was to estimate the presence of ghrelin in polycystic ovaries cells and evaluation of the relationship between ghrelin occurrence and cells proliferation. METHODS: In the present work we have compared ten polycystic ovaries with ovaries without pathology as the control group. We used immunohistochemical method to detect ghrelin. The cells proliferation was evaluated by Ki 67 proliferation index. RESULTS: Ghrelin immunostaining was demonstrated in cytoplasm of ovarian secondary interstitial cells and in atretic corpus luteum. The cell nuclei were ghrelin positive in granulosa, theca layers of follicular cyst in both groups as well as in luteal cells of young corpus luteum in healthy ovaries. Ki 67 immunostaining was observed in granulosa and theca layers of follicular cyst in polycystic and healthy ovaries. CONCLUSIONS: It is possible that local ghrelin expression plays an important role in the direct control of ovarian development and function and ghrelin may participate in patomechanism of PCOS.

Simultaneous measurement of ionization and scintillation from nuclear recoils in liquid xenon for a dark matter experiment.

We report the first measurements of the absolute ionization yield of nuclear recoils in liquid xenon, as a function of energy and electric field. Independent experiments were carried out with two dual-phase time-projection chamber prototypes, developed for the XENON dark matter project. We find that the charge yield increases with decreasing recoil energy, and exhibits only a weak field dependence. These results are the first unambiguous demonstration of the capability of dual-phase xenon detectors to discriminate between electron and nuclear recoils down to 20 keV, a key requirement for a sensitive dark matter search.