High-dose immunosuppressive therapy with autologous hematopoietic stem cell transplantation as a treatment option in multiple sclerosis

High-dose immunosuppressive therapy (HDIT) with autologous hematopoietic stem cell transplantation (auto-HSCT) is a new and promising approach to the treatment of multiple sclerosis (MS) patients because currently there are no effective treatment methods for this disease. In this article, we present results of a prospective clinical study of efficacy of HDIT + auto-HSCT in MS patients. The following treatment strategies were employed in the study: "early," "conventional," and "salvage/late" transplantation. Fifty patients with various types of MS were included in this study. No toxic deaths were reported among 50 MS patients; transplantation procedure was well-tolerated by the patients. The efficacy analysis was performed in 45 patients. Twenty-eight patients achieved an objective improvement of neurological symptoms, defined as at least 0.5-point decrease in the Expanded Disability Status Scale (EDSS) score as compared to the baseline and confirmed during 6 months, and 17 patients had disease stabilization (steady EDSS level as compared to the baseline and confirmed during 6 months). The progression-free survival at 6 years after HDIT + auto-HSCT was 72%. Magnetic resonance imaging data were available in 37 patients before transplantation showing disease activity in 43.3%. No active, new, or enlarging lesions were registered in patients without disease progression. In conclusion, HDIT + auto-HSCT suggests positive results in management of patients with different types of MS. Identification of treatment strategies based on the level of disability, namely "early," "conventional," and "salvage/late" transplantation, appears to be feasible to improve treatment outcomes.     

Detection and phasing of single base de novo mutations in biopsies from human in vitro fertilized embryos by advanced whole-genome sequencing

Currently, the methods available for preimplantation genetic diagnosis (PGD) of in vitro fertilized (IVF) embryos do not detect de novo single-nucleotide and short indel mutations, which have been shown to cause a large fraction of genetic diseases. Detection of all these types of mutations requires whole-genome sequencing (WGS). In this study, advanced massively parallel WGS was performed on three 5- to 10-cell biopsies from two blastocyst-stage embryos. Both parents and paternal grandparents were also analyzed to allow for accurate measurements of false-positive and false-negative error rates. Overall, >95% of each genome was called. In the embryos, experimentally derived haplotypes and barcoded read data were used to detect and phase up to 82% of de novo single base mutations with a false-positive rate of about one error per Gb, resulting in fewer than 10 such errors per embryo. This represents a ∼100-fold lower error rate than previously published from 10 cells, and it is the first demonstration that advanced WGS can be used to accurately identify these de novo mutations in spite of the thousands of false-positive errors introduced by the extensive DNA amplification required for deep sequencing. Using haplotype information, we also demonstrate how small de novo deletions could be detected. These results suggest that phased WGS using barcoded DNA could be used in the future as part of the PGD process to maximize comprehensiveness in detecting disease-causing mutations and to reduce the incidence of genetic diseases.Worldwide, more than 5 million babies (Ferraretti et al. 2013) have been born through in vitro fertilization (IVF) since the birth of the first in 1978 (Steptoe and Edwards 1978). Exact numbers are difficult to determine, but it has been estimated that currently 350,000 babies are born yearly through IVF (de Mouzon et al. 2009, 2012; Centers for Disease Control and Prevention 2011; Ferraretti et al. 2013). That number is expected to rise, as advanced maternal age is associated with decreased fertility rates and women in developed countries continue to delay childbirth to later ages. In 95% of IVF procedures, no diagnostic testing of the embryos is performed (https://www.sartcorsonline.com/rptCSR_PublicMultYear.aspx?ClinicPKID=0). Couples with prior difficulties conceiving or those wishing to avoid the transmission of highly penetrant heritable diseases often choose to perform preimplantation genetic diagnosis (PGD). PGD involves the biopsy of one cell from a 3-d embryo or the recently more preferred method, due to improved implantation success rates (Scott et al. 2013b), of up to 10 cells from a 5- to 6-d blastocyst-stage embryo. Following biopsy, genetic analysis is performed on the isolated cell(s). Currently this is an assay for translocations and the correct chromosome copy number (Hodes-Wertz et al. 2012; Munne 2012; Yang et al. 2012; Scott et al. 2013a; Yin et al. 2013), a unique test designed and validated for each specific heritable disease (Gutierrez-Mateo et al. 2009), or a combination of both (Treff et al. 2013). Importantly, none of these approaches can detect de novo mutations.Advanced maternal age has long been associated with an increased risk of producing aneuploid embryos (Munne et al. 1995; Crow 2000; Hassold and Hunt 2009) and giving birth to a child afflicted with Down syndrome or other diseases resulting from chromosomal copy number alterations. Conversely, children of older fathers have been shown to have an increase in single base and short multibase insertion/deletion (indels) de novo mutations (Kong et al. 2012). Many recent large-scale sequencing studies have found that de novo variations spread across many different genes are likely to be the cause of a large fraction of autism cases (Michaelson et al. 2012; O’Roak et al. 2012; Sanders et al. 2012; De Rubeis et al. 2014; Iossifov et al. 2014), severe intellectual disability (Gilissen et al. 2014), epileptic encephalopathies (Epi4K Consortium and Epilepsy Phenome/Genome Project 2013), and many other congenital disorders (de Ligt et al. 2012; Veltman and Brunner 2012; Yang et al. 2013; Al Turki et al. 2014). Additionally rare and de novo variations have been suggested to be prevalent in patients with schizophrenia (Fromer et al. 2014; Purcell et al. 2014), and Michaelson et al. (2012) found that single base de novo mutations affect conserved regions of the genome and essential genes more often than regions of unknown function. Current targeted approaches to PGD would miss many of these important functional changes within the embryonic DNA sequence, and even a whole-genome sequencing (WGS)–based carrier screen of both parents would not enable comprehensive preimplantation or prenatal diagnoses due to de novo mutations. As more parents delay childbirth into their mid-30s and later, these studies suggest we should try to provide better diagnostic tests for improving the health of newborns. In this study, we demonstrate the use of an advanced WGS process that provides an accurate and phased genome sequence from about 10 cells, allowing highly sensitive and specific detection of single base de novo mutations from IVF blastocyst biopsies.     

A mitochondrial superoxide theory for oxidative stress diseases and aging

Fridovich identified CuZnSOD in 1969 and manganese superoxide dismutase (MnSOD) in 1973, and proposed ”the Superoxide Theory,” which postulates that superoxide (O₂^•−) is the origin of most reactive oxygen species (ROS) and that it undergoes a chain reaction in a cell, playing a central role in the ROS producing system. Increased oxidative stress on an organism causes damage to cells, the smallest constituent unit of an organism, which can lead to the onset of a variety of chronic diseases, such as Alzheimer’s, Parkinson’s, amyotrophic lateral sclerosis and other neurological diseases caused by abnormalities in biological defenses or increased intracellular reactive oxygen levels. Oxidative stress also plays a role in aging. Antioxidant systems, including non-enzyme low-molecular-weight antioxidants (such as, vitamins A, C and E, polyphenols, glutathione, and coenzyme Q₁₀) and antioxidant enzymes, fight against oxidants in cells. Superoxide is considered to be a major factor in oxidant toxicity, and mitochondrial MnSOD enzymes constitute an essential defense against superoxide. Mitochondria are the major source of superoxide. The reaction of superoxide generated from mitochondria with nitric oxide is faster than SOD catalyzed reaction, and produces peroxynitrite. Thus, based on research conducted after Fridovich’s seminal studies, we now propose a modified superoxide theory; i.e., superoxide is the origin of reactive oxygen and nitrogen species (RONS) and, as such, causes various redox related diseases and aging.     

Vibrotactile discriminative capacity is impacted in a digit-specific manner with concurrent unattended hand stimulation

A number of perceptual and neurophysiological studies have investigated the effects of delivering unilateral versus bilateral tactile sensory stimulation. While a number of studies indicate that perceptual discrimination degrades with opposite-hand stimulation, there have been no reports that examined the digit specificity of cross-hemispheric interactions to discriminative capabilities. The purpose of this study was to determine whether unattended hand (UH) stimulation significantly degraded or improved amplitude discriminative capacity on the attended hand (AH) in a digit-specific manner. The methods are based on a sensory perceptual task (vibrotactile amplitude discriminative capacity on the tips of the fingers D2 and D3 of the left hand) in the absence and presence of conditioning stimuli delivered to D2 and D3 of the right hand. Non-specific equal-amplitude stimulation to D2 and D3 of the UH significantly worsened amplitude discrimination (AD) performance, while delivering unequal-amplitude stimuli to D2 and D3 of the UH worsened task performance only under the condition in which the unattended stimuli failed to appropriately match the stimulus parameters on the AH. Additionally, delivering single-site stimuli to D2 or D3 of the UH resulted in degraded performance on the AD task when the stimulus amplitude did not match the amplitude of the stimulus applied to homologous digits of the AH. The findings demonstrate that there is a reduction in performance under conditions where UH stimulation least matched stimulation applied to the AH, while there was little or no change in performance when stimulus conditions on the homologous digit(s) of the contralateral sites were similar. Results suggest that bilateral interactions influence perception in a context-dependent manner that is digit specific.     

Mutation of Plekha7 attenuates salt-sensitive hypertension in the rat

PLEKHA7 (pleckstrin homology domain containing family A member 7) has been found in multiple studies as a candidate gene for human hypertension, yet functional data supporting this association are lacking. We investigated the contribution of this gene to the pathogenesis of salt-sensitive hypertension by mutating Plekha7 in the Dahl salt-sensitive (SS/JrHsdMcwi) rat using zinc-finger nuclease technology. After four weeks on an 8% NaCl diet, homozygous mutant rats had lower mean arterial (149 ± 9 mmHg vs. 178 ± 7 mmHg; P < 0.05) and systolic (180 ± 7 mmHg vs. 213 ± 8 mmHg; P < 0.05) blood pressure compared with WT littermates. Albumin and protein excretion rates were also significantly lower in mutant rats, demonstrating a renoprotective effect of the mutation. Total peripheral resistance and perivascular fibrosis in the heart and kidney were significantly reduced in Plekha7 mutant animals, suggesting a potential role of the vasculature in the attenuation of hypertension. Indeed, both flow-mediated dilation and endothelium-dependent vasodilation in response to acetylcholine were improved in isolated mesenteric resistance arteries of Plekha7 mutant rats compared with WT. These vascular improvements were correlated with changes in intracellular calcium handling, resulting in increased nitric oxide bioavailability in mutant vessels. Collectively, these data provide the first functional evidence that Plekha7 may contribute to blood pressure regulation and cardiovascular function through its effects on the vasculature.Hypertension is a complex disease that is characterized by increased blood pressure, renal damage, and vascular dysfunction which collectively increase risk of atherosclerosis, stroke, heart disease, and renal failure in one-quarter of all adults worldwide (1–3). Because there is strong evidence of heritability in hypertension (2, 4, 5), considerable effort has been put toward identifying novel candidate genes and their molecular mechanisms. Genome-wide association studies (GWAS) have identified many potential hypertension loci, which shed light on the genetic complexity of this disease (5–8) but have provided little mechanistic insight. As such, validation and elucidation of the functional roles and disease mechanisms for these gene candidates are the next important challenges (4).Because hypertension is a complex disease (i.e., multiple variants of small effect sizes contributing to disease risk), we hypothesized candidate gene targeting on a genetically sensitized background would reveal functional role(s) of genetic disease modifiers. The Dahl salt-sensitive (SS) rat is an inbred genetic model of salt-sensitive hypertension that displays hypertension-induced renal damage, cardiac hypertrophy and vascular dysfunction (9–11). These phenotypes are induced by exposing SS rats to a high-salt diet, which results in rapid induction of hypertensive phenotypes that closely resemble salt-induced hypertension seen in humans (12–15). Knockout of specific genes in this disease model using zinc-finger nuclease (ZFN) technology have revealed the importance of key mechanisms contributing to hypertension risk, such as the protection from salt-induced hypertension and renal injury by selective ablation of adaptive immune cells in the SS-Rag1^em1Mcwi and SS-Cd247^em1Mcwi knockout rats (16, 17) and reduced hypertension and renal injury in the SS-Ncf2^em1Mcwi (p67phox) null model exhibiting reduced medullary oxidative stress (18). Additionally, we have recently demonstrated multiple genes at a single hypertension GWAS-nominated locus (Agtrap-Plod1 locus) can have additive or subtractive effects on blood pressure and renal function when mutated in the SS rat (19). These previous studies highlight the utility of this model system for testing the roles of GWAS candidate human disease genes by disrupting their specific rat orthologs using ZFN technology (20).A single-nucleotide polymorphism (SNP) (rs381815, minor allele frequency 0.26) in intron 1 of the pleckstrin homology domain containing family A member 7 (PLEKHA7) gene, was identified by five independent GWAS to be associated with elevated systolic blood pressure and hypertension in multiple populations (5, 6, 8, 21, 22). The associated locus contains only the PLEKHA7 gene (5); however, the genetic mechanism(s) underlying this locus have not yet been functionally characterized. PLEKHA7 is highly expressed in the kidney and heart, where it may be involved in formation and maintenance of the apical junction complex of epithelial cells (23). However, limited data on PLEKHA7 function are available to extrapolate its potential role(s) in the pathogenesis of hypertension. Here we used ZFN mutagenesis to obtain the first evidence to our knowledge in any model system that Plekha7 has a functional role in several hypertension-associated phenotypes in the rat. We found that mutation of Plekha7 in the SS rat attenuated salt-induced hypertension, reduced renal damage, and improved cardiac function. We also show that Plekha7 modulates calcium handling and nitric oxide (NO) bioavailability, both of which are required for normal vascular health. Collectively, these studies provide significant mechanistic insight to the role of Plekha7 in salt-sensitive hypertension.     

Fresh-stored osteochondral allografts for the treatment of femoral head defects: surgical technique and preliminary results

Purpose

The purpose of this study was to present the preliminary clinical and radiographic outcomes of the treatment of femoral head osteochondral defects in eight consecutive symptomatic patients with fresh-stored osteochondral allografts via a trochanteric osteotomy.

Methods

This study included all consecutive patients treated in our department between 2008 and 2010 for worsening pain and mechanical symptoms of femoral head osteochondral defects. Each patient had preoperative routine hip radiographs and a preoperative magnetic resonance imaging study that determined and recorded the defect size and femoral head diameters. Allograft donors were identified through the Multiple Organ Retrieval and Exchange program (Ontario, Canada).

Results

The osteochondral defects were secondary to osteochondritis dissecans in four patients, avascular necrosis in three and femoral head fracture without dislocation in one. The patients’ average age at surgery was 23.7 (range 17–42), and the average follow-up was 41 months (range 24–54). Follow-up included clinical and radiographic examinations at standard intervals. The average Harris hip scores improved from 57.7 (range 50–65) points preoperatively to 83.9 (range 72–94) points at latest follow-up. Five patients had good-to-excellent clinical outcomes, and one had a fair outcome. One patient was converted to a total hip arthroplasty due to progression of arthritis. Another patient’s graft subsided and he underwent a successful repeat transplantation. An additional patient required the removal of the screws transfixing her trochanter due to persistent irritation.

Conclusions

These findings indicate that fresh-stored osteochondral allograft transplantation using a trochanteric slide and surgical dislocation is a viable treatment option for femoral head defects in young patients.     

A representative of arylcyanomethylenequinone oximes effectively inhibits growth and formation of hyphae in Candida albicans and influences the activity of protein kinases in vitro

In this study, we applied various assays to reveal new activities of phenylcyanomethylenequinone oxime-4-(hydroxyimino) cyclohexa-2,5-dien-1-ylidene](phenyl)ethanenitrile (4-AN) for potential anti-microbial applications. These assays demonstrated (a) the antimicrobial effect on bacterial and fungal cultures, (b) the effect on the in vitro activity of the kinase CK2, (c) toxicity towards human erythrocytes, the Caco-2 cancer cell line, and embryonic development of Zebrafish. We demonstrated the activity of 4-AN against selected bacteria and Candida spp. The MIC ranging from 4?µg/ml to 125?µg/ml proved effective in inhibition of formation of hyphae and cell aggregation in Candida, which was demonstrated at the cytological level. Noteworthy, 4-AN was found to inhibit the CK2 kinase with moderate potency. Moreover, at low concentrations, it did not exert any evident toxic effects on human erythrocytes, Caco-2 cells, or Zebrafish embryos. 4-AN can be a potential candidate as a novel drug against Candida infections.     

Sphenoid wing meningiomas: peritumoral brain edema as a prognostic factor in surgical outcome

Neurosurgical Review - Sphenoidal meningiomas constitute 18% of intracranial masses, and still present a difficult surgical challenge. PTBE has been associated with several complications and future...