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.     

A novel IVS2 -2A>T splicing mutation in the GH-1 gene in familial isolated growth hormone deficiency type II in the spectrum of other splicing mutations in the Russian population

Isolated GH deficiency (IGHD) is characterized by genetic heterogeneity, both in familial and sporadic cases. To determine if this statement can be applied to the Russian population, we performed screening for mutations in the GH-1 gene in children living in Russia with IGHD. Twenty-eight children from 26 families with total IGHD were studied. DNA fragments, covering each of four (2-5) exons of GH-1 were amplified using PCR. Single-strand conformation polymorphism analysis followed by direct DNA sequencing identified five heterozygous mutations of splicing in intron 2, intron 3, and exon 4 of GH-1; three of them were not previously reported. We concentrated here on dominant-negative mutations causing IGHD type II, which were as follows: 1) A>T transversion of the second base of the 3'-acceptor splice site of intron 2 (IVS2 -2A>T); 2) T>C transition of the second base of the 5'-donor splice site of intron 3 (IVS3 +2T>C); 3) G>A transition of the first base of the 5'-donor splice site of intron 3 (IVS3 +1G>A). Our data indicate allelic heterogeneity of IGHD type II (IGHD II). However, all mutations in Russian IGHD II patients affect splicing, a striking difference from the mutation spectrum of other IGHD forms. The IVS2 -2A>T mutation is the first identified mutation in intron 2 of GH-1. The 5'-donor splice site of intron 3 of GH-1 is a mutational hot spot, and the IVS3 +1G>A mutation can be considered to be a common molecular defect in IGHD II in Russian patients.     

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.     

The length‐dependent activation of contraction is equally impaired in impuberal male and female rats in monocrotaline‐induced right ventricular failure

The length‐dependent activation of contraction is attenuated in the failing myocardium of adult male rats. This pathological change is not seen in adult female rats, possibly because of a protective effect of sex hormones. The present study evaluated length‐dependent changes in isometric twitch, Ca²⁺ transient (CaT) and action potential (AP) in the right ventricular myocardium of impuberal healthy male and female rats (control) and in rats treated with a single injection of 50 mg/kg monocrotaline (MCT). Compared with sex‐matched control rats, MCT‐treated male and female rats exhibited increased right ventricular weight (134% and 142% of control, respectively), decreased left ventricular weight (72% and 79%), twitch attenuation (48.8 ± 2.7% and 57.5 ± 1.2%) and prolongation (125 ± 3% and 127 ± 2%), CaT attenuation (37.8 ± 0.4% and 39.1 ± 1.1%) and prolongation (114 ± 1% and 116 ± 1%) and AP prolongation at 90% repolarization (195 ± 2% and 203 ± 1%). The MCT‐treated male rats exhibited a 50% lower integral magnitude and an approximately 25% larger time‐to‐peak ‘bump’ compared with control male rats. These parameters in MCT‐treated female rats tended to show similar changes to those seen in the control female rats, with no significant difference between the two groups. In all groups, integral magnitude and time‐to‐peak ‘bump’ increased with length. In conclusion, the length‐dependent activation of contraction was equally blunted in the failing right ventricular myocardium of impuberal male and female rats. This was related to changes in CaT and AP, which were similar between male and female rats. Therefore, puberty is necessary for manifestation of the protective effects of sex hormones on this remodelling.     

Adenosine receptors in regulation of dendritic cell differentiation and function

Differentiation of functional dendritic cells (DCs) critically depends on the microenvironment. DCs differentiate in hypoxic tumor sites and inflamed or damaged tissue. Because local concentrations of adenosine reach high physiologically relevant levels in these conditions, we assessed the expression of adenosine receptors and the effect of their activation on differentiation of human monocytes and mouse peritoneal macrophages and hematopoietic progenitor cells (HPCs) into myeloid DCs. Stimulation of adenosine receptors skews DC differentiation toward a distinct cell population characterized by expression of both DC and monocyte/macrophage cell surface markers. Pharmacologic analysis and experiments with cells from A(2B) adenosine receptor knockout mice identified A(2B) receptor as the mediator of adenosine effects on DCs. Unlike normal myeloid DCs, adenosine-differentiated DCs have impaired allostimulatory activity and express high levels of angiogenic, pro-inflammatory, immune suppressor, and tolerogenic factors, including VEGF, IL-8, IL-6, IL-10, COX-2, TGF-beta, and IDO. They promoted tumor growth if injected into tumors implanted in mice. Using adenosine desaminase knockout animals, we showed that DCs with proangiogenic phenotype are highly abundant under conditions associated with elevated levels of extracellular adenosine in vivo. Adenosine signaling through A(2B) receptor is an important factor of aberrant DC differentiation and generation of tolerogenic, angiogenic, and proinflammatory cells.     

Morita-Baylis-Hillman Adduct 2-(3-Hydroxy-2-oxoindolin-3-yl)acrylonitrile (ISACN) Modulates Inflammatory Process In vitro and In vivo

Inflammation - Morita-Baylis-Hillman adducts (MBHA) are synthetic molecules with several biological actions already described in the literature. It has been previously described that adduct...     

Graphene oxide quantum dots immobilized on mesoporous silica: preparation,characterization and electroanalytical application

Because of its high surface area and combination of various functional groups, graphene oxide (GO) is currently one of the most actively studied materials for electroanalytical applications. It is not practical to utilize self-supported GO on its own and thus it is commonly integrated with different supporting carriers. Having a large lateral size, GO can only wrap the particles of the support and thus can significantly reduce the surface area of porous materials. To achieve synergy from the high surface area and polyfunctional nature of GO, and the rigid structure of a porous support, the lateral size of GO must essentially be decreased. Recently reported graphene oxide quantum dots (GOQDs) can fulfil this task. Here we report the successful preparation of an SiO₂-GOQDs hybrid, where GOQDs have been incorporated into the mesoporous network of silica. The SiO₂-GOQDs emit a strong luminescence with a band maximum at 404 nm. The Raman spectrum of SiO₂-GOQDs shows two distinct peaks at 1585 cm⁻¹ (G-peak) and 1372 cm⁻¹ (D-peak), indicating the presence of a graphene ordered basal plane with aromatic sp²-domains and a disordered oxygen-containing structure. Covalent immobilization of GOQDs onto aminosilica via such randomly structured oxygen fragments was proven with the help of Fourier transform infrared spectroscopy, solid-state cross-polarization magic angle spinning ¹³C nuclear magnetic resonance, and X-ray photoelectron spectroscopy. SiO₂-GOQDs were used as a modifier of a carbon paste electrode for differential pulse voltammetry determination of two antibiotics (sulfamethoxazole and trimethoprim) and two endocrine disruptors (diethylstilbestrol (DES) and estriol (EST)). The modified electrodes demonstrated a significant signal enhancement for EST (370%) and DES (760%), which was explained by a π–π stacking interaction between GOQDs and the aromatic system of the analytes.

Graphene oxide quantum dots incorporated into a mesoporous silica network have been used as a modifier of a carbon paste electrode for the determination of antibiotics and hormones.     

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.