Identification of a putative pathway for the muscle homing of stem cells in a muscular dystrophy model

Attempts to repair muscle damage in Duchenne muscular dystrophy (DMD) by transplanting skeletal myoblasts directly into muscles are faced with the problem of the limited migration of these cells in the muscles. The delivery of myogenic stem cells to the sites of muscle lesions via the systemic circulation is a potential alternative approach to treat this disease. Muscle-derived stem cells (MDSCs) were obtained by a MACS(R) multisort method. Clones of MDSCs, which were Sca-1+/CD34-/L-selectin+, were found to adhere firmly to the endothelium of mdx dystrophic muscles after i.v. or i.m. injections. The subpopulation of Sca-1+/CD34- MDSCs expressing L-selectin was called homing MDSCs (HMDSCs). Treatment of HMDSCs with antibodies against L-selectin prevented adhesion to the muscle endothelium. Importantly, we found that vascular endothelium from striate muscle of young mdx mice expresses mucosal addressin cell adhesion molecule-1 (MAdCAM-1), a ligand for L-selectin. Our results showed for the first time that the expression of the adhesion molecule L-selectin is important for muscle homing of MDSCs. This discovery will aid in the improvement of a potential therapy for muscular dystrophy based on the systemic delivery of MDSCs.     

Both apoptosis and necrosis occur early after intracerebral grafting of ventral mesencephalic tissue: a role for protease activation

Neural transplantation is an experimental treatment for Parkinson's disease. Widespread clinical application of the grafting technique is hampered by a relatively poor survival (around 10%) of implanted embryonic dopamine neurones. Earlier animal studies have indicated that a large proportion of the grafted cells die during graft tissue preparation and within the first few days after intracerebral implantation. The present study was designed to reveal the prevalence of cell death in rat intrastriatal grafts at 90 min, 1, 3, 6 and 42 days after implantation. We examined apoptotic cell death using semi-thin and paraffin sections stained with methylene blue and an antibody against activated caspase 3, respectively. We identified abundant apoptotic cell death up to 3 days after transplantation. In addition, we studied calpain activation using an antibody specific for calpain-cleaved fodrin. We report a peak in calpain activity 90 min after grafting. Surprisingly, we did not observe any significant difference in the number of dopaminergic neurones over time. The present results imply that grafted cells may be victims of either an early necrotic or a later apoptotic cell death and that there is substantial cell death as early as 90 min after implantation.     

Mitochondrial dysfunction in liver disease and organ transplantation

The mitochondria play a crucial role in maintaining hepatocyte integrity and functions. Mitochondrial defects are either inherited or acquired. Mitochondria dysfunction occurs when the hepatocyte experience excessive physiologic stress. Its clinical presentation depends on the severity of the stress. It varies from mild abnormalities in liver biochemical tests to manifestations of acute or chronic liver failure. Mitochondria dysfunction is implicated in most liver disease and in early graft dysfunction after liver transplantation. This review will address the role of mitochondria in liver disease.     

The cagA status of Helicobacter pylori isolates from dyspeptic children in Turkey

There are inconsistent reports regarding cytotoxin-associated gene A (cagA) status of Helicobacter pylori isolates and the severity of the mucosal lesions in children. The aim of this study was to determine the prevalence of cagA(+) strains and to evaluate its correlation with clinic and endoscopic findings. We examined 45 H. pylori strains that were grown on brain-heart infusion agar supplemented with 7% horse blood. Following 72 h of incubation colonies were harvested and bacterial DNA was extracted. Polymerase chain reaction primers F1 and B1 were used to amplify a 348-bp internal fragment of cagA. The prevalence of cagA in Turkish pediatric patients was 55.6%. No association was found between cagA status and the severity of gastro-duodenal lesions.     

Coral reef rehabilitation through transplantation of staghorn corals: effects of artificial stabilization and mechanical damages

In order to develop and test a low-cost method of coral reef rehabilitation, the staghorn corals Acropora muricata and A. vaughani were transplanted to a shallow site with unstable substrate. To avoid abrasion, dislodgement and transport due to water movement, the transplanted corals were tied to string sections, which were connected at the seabed to form a grid. This created stability and improved the survival of the corals. The average increase in weight of live coral over 1 year was 56%, eight times more than the control treatment with unattached coral branches. This difference was mainly due to a reduced partial mortality among smaller coral fragments in the stabilized treatment. Survival was positively related to initial size among the loosely placed coral branches, whereas the attached treatment showed a negative relation between size and relative increase in weight of the surviving parts of the coral branches. Coral fragments were not significantly affected by severe physical damage simulating the effects of handling.     

Optimal conditions for heart cell cryopreservation for transplantation

Cultured myocyte transplantation into an infarcted myocardium has been shown to improve contractile function. Cryopreservation of cultured muscle cells or heart tissue will be important for the technology to be practical. This study, using fetal cardiomyocytes, evaluated the optimal conditions for muscle cell cryopreservation. Study 1: Fetal rat cardiomyocytes were isolated and cultured. The freshly isolated and passage 1, 2, 3 and 4 cells were cryopreserved in a solution containing 70% IMDM, 20% FBS and 10% DMSO and stored in –196°C for 1, 2, 4, 8, 12 and 24 weeks. The cells were thawed and cultured. Cell number and contractility were evaluated at 0, 2, 4, 6, 8 and 10 days of culture. Study 2: Rat myocardium was cryopreserved in sizes of 0.2, 2 and 6 mm³ for 1 week. The tissue was thawed and cells were isolated. Cell growth and contractility were evaluated. (1) Cardiomyocytes grew and contracted after cryopreservation. Storage time did not affect cell survival rate, beating cell numbers and beating rates. Increasing cell passage prior to cryopreservation decreased the percentage of beating cells. (2) Cells isolated from cryopreserved tissue grew in vitro and contracted normally. Cell yield decreased with increased cryopreserved tissue size. Fetal rat cardiomyocytes survived and functioned after in vitro cryopreservation. Viable cells can be isolated from cryopreserved myocardium and cultured. Cryopreservation of small pieces of myocardium is preferred for maximal cell yields.     

Mossy fiber pathfinding in multilayer organotypic cultures of rat hippocampal slices

1. Using a novel technique of organotypic cultures, in which two hippocampal slices were cocultured in a bilayer style, we found that the mossy fibers arising from the dentate gyrus grafted onto another dentate tissue grew along the CA3 stratum lucidum of the host hippocampal slice. The same transplantation of a CA1 microslice failed to form a network with the host hippocampus.2. Thus, the type of grafted neurons is important to determine whether they can form an appropriate network after transplantation.     

Age of ovary determines remaining life expectancy in old ovariectomized mice

We investigated the capacity of young ovaries, transplanted into old ovariectomized CBA mice, to improve remaining life expectancy of the hosts. Donor females were sexually mature 2-month-olds; recipients were prepubertally ovariectomized at 3 weeks and received transplants at 5, 8 or 11 months of age. Relative to ovariectomized control females, life expectancy at 11 months was increased by 60% in 11-month recipient females and by 40% relative to intact control females. Only 20% of the 11-month transplant females died in the 300-day period following ovarian transplantation, whereas nearly 65% of the ovariectomized control females died during this same period. The 11-month-old recipient females resumed oestrus and continued to cycle up to several months beyond the age of control female reproductive senescence. Across the three recipient age groups, transplantation of young ovaries increased life expectancy in proportion to the relative youth of the ovary. Our results relate to recent findings on the gonadal input upon aging in Caenorhabditis elegans and may suggest how the mammalian gonad, including that of humans, could regulate aging and determine longevity.     

Monitoring method for pre- and post-liver transplantation in patients with primary hyperoxaluria type I

Differential diagnosis of primary hyperoxaluria type I (PH1) may become difficult once end-stage renal failure and anuria have occurred. Here we describe a rapid and sensitive method to simultaneously quantify glycolate and oxalate in plasma using a stable isotope dilution and gas chromatography-mass spectrometry. The results are provided within 2 h. The linearity of the method was validated up to 200 micromol/l of these compounds and the inter-assay precision for glycolate and oxalate was 2.4 and 2.6%, respectively (n=5), when the control plasma was spiked with 50 micromol/l of glycolate and oxalate. For healthy subjects, 1.0 ml of plasma was required and 0.1 ml for the PH1 patients. Using this method, plasma levels in non-PH1 patients under hemodialysis and in healthy subjects were determined. This method proved to be useful when used for differential diagnosis of PH1 and for monitoring the plasma levels of glycolate and oxalate in two PH1 patients before and after dialysis and liver transplantation. Plasma glycolate in these patients was dramatically decreased after liver transplantation, but plasma oxalate decreased more slowly due to remobilization of the calcium oxalate stores deposited throughout the body.     

Asynchronous cytoplast and karyoplast transplantation reveals that the cytoplasm determines the developmental fate of the nucleus in mouse oocytes

The relationship between nucleus and cytoplasm can be well revealed by nuclear transplantation. Here, we have investigated the behavior changes of the reconstructed oocytes after transferring the karyoplasts from mouse GV, MI, and MII oocytes into the cytoplasts at the different developmental stages. When the GV cytoplast was used as recipient and MI or MII karyoplast was used as donor (MI-GV pair and MII-GV pair), the reconstructed pairs extruded a polar body after electrofusion and culture. Both the cytoplasm and the polar body had a metaphase spindle in the MI-GV pair, while only a clutch of condensed chromatin was observed in the cytoplasm and polar body of the MII-GV pair. When the MI cytoplast was used as recipient and GV or MII karyoplast was used as donor (GV-MI pair and MII-MI pair), the reconstructed pairs also extruded a polar body. Each had one spindle and a group of metaphase chromosomes in the cytoplasm and polar body, respectively. When the MII cytoplast was used as recipient and GV or MI karyoplast was used as donor (GV-MII pair and MI-MII pair), the reconstructed pairs were activated, became parthenogenetic embryos and even developed to hatching blastocysts after electrofusion. The result from immunoblotting showed that MAP kinase activity was high in the MI and MII cytoplasts, while not detected in GV cytoplast. The results demonstrate that the cytoplasmic environment determines the behavior of asynchronous donors.