Early IFN-γ production together with decreased expression of TLR3 and TLR9 characterizes EAE development conditional on the presence of myelin

Experimental autoimmune encephalomyelitis (EAE) is a model for the study of multiple sclerosis, which is an inflammatory and demyelinating disease of the central nervous system (CNS). Despite increased efforts to elucidate the function of toll-like receptors (TLRs) in autoimmune diseases of the CNS, the relative contribution of other factors, including the immunomodulatory properties of TLR signaling, role of the innate response and the presence or absence of myelin peptides remain unclear. The aim was to evaluate TLR expression in the CNS during EAE development by investigating the expression of TLRs in the initial phase of EAE and establishing correlations with the modulation of inflammatory factors. Mice were subcutaneously immunized at the tail base with 100?μg of myelin oligodendrocyte glycoprotein peptide (MOG_35–55), emulsified in complete Freund’s adjuvant (CFA) supplemented with 400?μg of attenuated Mycobacterium tuberculosis H37RA. Pertussis toxin (300?ng per animal) was intraperitoneally injected on the day of immunization and 48?h later. Another group (MOG^?) received an equal emulsion of CFA and M. tuberculosis, without MOG_35–55, and the same protocol of Pertussis toxin. The immunized mice presented signs of disease with increased IFN-γ production and presence of NK cells on Day 2 postimmunization and reduced the expression of TLR-3 and TLR-9. In the spinal cord, CCL5 and CCL20 were higher in EAE. This study establishes a correlation between TLR-3 and TLR-9 expression with the development of EAE. In addition, evidence of a role for the myelin peptide in targeting the innate inflammatory response to the CNS is presented.     

Design Principles of Reptilian Muscles: Calcium Cycling Strategies

The ultrastructure of the sarcoplasmic reticulum (SR) in skeletal muscles was compared among different reptile species (watersnake, boa constrictor, lizard, and turtle) and a mammal (mouse). Morphometric analysis demonstrates a pattern of increasing calsequestrin (CASQ) content in the lumen of SR from turtle to lizard, watersnake, and boa constrictor, and this content is in all cases higher than in mouse. In all reptiles sampled except turtle, CASQ is not confined to the junctional sarcoplasmic reticulum (jSR) cisternae as it is in other species. It instead fills the entire longitudinal (free) SR (fSR) regions, and in the extreme case of snakes, the shape of the SR is modified around the extra CASQ. We suggest that high CASQ content may represent an ATP‐saving adaptation that permits relatively low metabolic rates during prolonged periods of fasting and inactivity, particularly in watersnake and boa constrictor. Anat Rec, 299:352–360, 2016. © 2015 Wiley Periodicals, Inc.     

Pentraxin‐3 is upregulated in the central nervous system during MS and EAE,but does not modulate experimental neurological disease

Pentraxin‐3 (PTX3), an acute‐phase protein released during inflammation, aids phagocytic clearance of pathogens and apoptotic cells, and plays diverse immunoregulatory roles in tissue injury. In neuroinflammatory diseases, like MS, resident microglia could become activated by endogenous agonists for Toll like receptors (TLRs). Previously we showed a strong TLR2‐mediated induction of PTX3 in cultured human microglia and macrophages by HspB5, which accumulates in glia during MS. Given the anti‐inflammatory effects of HspB5, we examined the contribution of PTX3 to these effects in MS and its animal model EAE. Our data indicate that TLR engagement effectively induces PTX3 expression in human microglia, and that such expression is readily detectable in MS lesions. Enhanced PTX3 expression is prominently expressed in microglia in preactive MS lesions, and in microglia/macrophages engaged in myelin phagocytosis in actively demyelinating lesions. Yet, we did not detect PTX3 in cerebrospinal fluid of MS patients. PTX3 expression is also elevated in spinal cords during chronic relapsing EAE in Biozzi ABH mice, but the EAE severity and time course in PTX3‐deficient mice did not differ from WT mice. Moreover, systemic PTX3 administration did not alter the disease onset or severity. Our findings reveal local functions of PTX3 during neuroinflammation in facilitating myelin phagocytosis, but do not point to a role for PTX3 in controlling the development of autoimmune neuroinflammation.     

Oligodeoxynucleotide inhibition of Toll‐like receptors 3, 7, 8, and 9 suppresses cytokine production in a human rheumatoid arthritis model

Toll‐like receptors (TLRs) are innate immune receptors that respond to both exogenous and endogenous stimuli and are suggested to contribute to the perpetuation of chronic inflammation associated with rheumatoid arthritis (RA). In particular, the endosomal TLRs 3, 7, 8, and 9 have more recently been postulated to be of importance in RA pathogenesis. In this study, pan inhibition of the endosomal TLRs by a phosphorothioate‐modified inhibitory oligodeoxynucleotide (ODN) is demonstrated in primary human B cells, macrophages, and RA fibroblasts. Inhibition of TLR8 was of particular interest as TLR8 has been associated with RA pathogenesis in both human and murine arthritis models. ODN1411 competitively inhibited TLR8 signaling and was observed to directly bind to a purified TLR8 ectodomain, suggesting inhibition was through a direct interaction with the receptor. Addition of ODN1411 to human RA synovial membrane cultures significantly inhibited spontaneous cytokine production from these cultures, suggesting a potential role for one or more of the endosomal TLRs in inflammatory cytokine production in RA and the potential for inhibitory ODNs as novel therapies.     

Understanding N‐Acetyl‐L‐Glutamate Synthase Deficiency: Mutational Spectrum,Impact of Clinical Mutations on Enzyme Functionality,and Structural Considerations

N‐acetyl‐L‐glutamate synthase (NAGS) deficiency (NAGSD), the rarest urea cycle defect, is clinically indistinguishable from carbamoyl phosphate synthetase 1 deficiency, rendering the identification of NAGS gene mutations key for differentiation, which is crucial, as only NAGSD has substitutive therapy. Over the last 13 years, we have identified 43 patients from 33 families with NAGS mutations, of which 14 were novel. Overall, 36 NAGS mutations have been found so far in 56 patients from 42 families, of which 76% are homozygous for the mutant allele. 61% of mutations are missense changes. Lack or decrease of NAGS protein is predicted for ～1/3 of mutations. Missense mutations frequency is inhomogeneous along NAGS: null for exon 1, but six in exon 6, which reflects the paramount substrate binding/catalytic role of the C‐terminal domain (GNAT domain). Correspondingly, phenotypes associated with missense mutations mapping in the GNAT domain are more severe than phenotypes of amino acid kinase domain‐mapping missense mutations. Enzyme activity and stability assays with 12 mutations introduced into pure recombinant Pseudomonas aeruginosa NAGS, together with in silico structural analysis, support the pathogenic role of most NAGSD‐associated mutations found. The disease‐causing mechanisms appear to be, from higher to lower frequency, decreased solubility/stability, aberrant kinetics/catalysis, and altered arginine modulation.     

DNA Diagnostics of Hereditary Hearing Loss: A Targeted Resequencing Approach Combined with a Mutation Classification System

Although there are nearly 100 different causative genes identified for nonsyndromic hearing loss (NSHL), Sanger sequencing‐based DNA diagnostics usually only analyses three, namely, GJB2, SLC26A4, and OTOF. As this is seen as inadequate, there is a need for high‐throughput diagnostic methods to detect disease‐causing variations, including single‐nucleotide variations (SNVs), insertions/deletions (Indels), and copy‐number variations (CNVs). In this study, a targeted resequencing panel for hearing loss was developed including 79 genes for NSHL and selected forms of syndromic hearing loss. One‐hundred thirty one presumed autosomal‐recessive NSHL (arNSHL) patients of Western‐European ethnicity were analyzed for SNVs, Indels, and CNVs. In addition, we established a straightforward variant classification system to deal with the large number of variants encountered. We estimate that combining prescreening of GJB2 with our panel leads to a diagnosis in 25%–30% of patients. Our data show that after GJB2, the most commonly mutated genes in a Western‐European population are TMC1, MYO15A, and MYO7A (3.1%). CNV analysis resulted in the identification of causative variants in two patients in OTOA and STRC. One of the major challenges for diagnostic gene panels is assigning pathogenicity for variants. A collaborative database collecting all identified variants from multiple centers could be a valuable resource for hearing loss diagnostics.     

Frequency of delta F508 mutation in Venezuelan patients with cystic fibrosis

Cystic Fibrosis (CF) is the most common and severe autosomal recessive disease in Caucasian populations, with an incidence of 1 in 2500 live births. It is characterized by a generalized disturbance in exocrine glands and it is caused by over one thousand mutations at the cystic fibrosis conductance regulator gene (CFTR) mapped at 7q31. AF508 is the most frequent mutation worldwide and it consists in a deletion of the codon that encodes fenilalanine at the 508 protein's position. The aim of this study was to determine the frequency of the delta F508 mutation in Venezuelan patients with CF using the Polymerase Chain Reaction (PCR). We studied thirty patients of twenty eight families who were diagnosed with CF based on their clinical features and sweat chloride level > 60 mEq/l in two determinations. Detection of the mutation was performed from the amplification of a 98 pair of bases (pb) CF gene segment which contains the codon that encodes fenilalanine in the 508 position by PCR. This PCR product is absent in those who have the mutation. The delta F508 allelic frequency was 26.79%, distributed in six homozygous and seven compound heterozygote delta F508/X. The reminder mutations (no delta F508) represent 73.21%. The delta F508 frequency in our sample is less than the reported in European countries. On the other hand, a delta F508 frequency highly heterogeneous has been observed in Latin-American countries. This variation results from mixed populations with a different genetic background influenced by external migration and CF molecular alterations, which exists in the analyzed populations. In this study, the delta F508 mutation comes mainly from grandparents (79.41%) who were born in Mediterranean countries and Colombia, while the no delta F508 mutations come from grandparents who were born in Venezuela (79.27%) and Colombia (17.07%).     

Mannose Receptor-Mediated Gene Delivery into Antigen Presenting Dendritic Cells

Dendritic cells are professional antigen presenting cells and are unique in their ability to prime naíve T cells. Gene modification of dendritic cells is of particular interest for immunotherapy of diseases where the immune system has failed or is aberrantly regulated, such as in cancer or autoimmune disease, respectively. Dendritic cells abundantly express mannose receptor and mannose receptor-related receptors, and receptor-mediated gene transfer via mannose receptor offers a versatile tool for targeted gene delivery into these cells. Accordingly, mannose polyethylenimine DNA transfer complexes were generated and used for gene delivery into dendritic cells. Mannose receptor belongs to the group of scavenger receptors that allow dendritic cells to take up pathogenic material, which is directed for degradation and MHC class II presentation. Therefore, a limiting step of transgene expression by mannose receptor-mediated gene delivery is endosomal degradation of DNA. Several strategies have been explored to overcome this limitation including the addition of endosomolytic components to DNA transfer complexes like adenovirus particles and influenza peptides. Here, we review the current understanding of mannose receptor-mediated gene delivery into dendritic cells and discuss strategies to identify appropriate endosomolytic agents to improve DNA transfer efficacy.     

Making Quality Health Websites a National Public Health Priority: Toward Quality Standards

BackgroundMost US adults have limited health literacy skills. They struggle to understand complex health information and services and to make informed health decisions. The Internet has quickly become one of the most popular places for people to search for information about their health, thereby making access to quality information on the Web a priority. However, there are no standardized criteria for evaluating Web-based health information. Every 10 years, the US Department of Health and Human Services'' Office of Disease Prevention and Health Promotion (ODPHP) develops a set of measurable objectives for improving the health of the nation over the coming decade, known as Healthy People. There are two objectives in Healthy People 2020 related to website quality. The first is objective Health Communication and Health Information Technology (HC/HIT) 8.1: increase the proportion of health-related websites that meet 3 or more evaluation criteria for disclosing information that can be used to assess information reliability. The second is objective HC/HIT-8.2: increase the proportion of health-related websites that follow established usability principles.ObjectiveThe ODPHP conducted a nationwide assessment of the quality of Web-based health information using the Healthy People 2020 objectives. The ODPHP aimed to establish (1) a standardized approach to defining and measuring the quality of health websites; (2) benchmarks for measurement; (3) baseline data points to capture the current status of website quality; and (4) targets to drive improvement.MethodsThe ODPHP developed the National Quality Health Website Survey instrument to assess the quality of health-related websites. The ODPHP used this survey to review 100 top-ranked health-related websites in order to set baseline data points for these two objectives. The ODPHP then set targets to drive improvement by 2020.ResultsThis study reviewed 100 health-related websites. For objective HC/HIT-8.1, a total of 58 out of 100 (58.0%) websites met 3 or more out of 6 reliability criteria. For objective HC/HIT-8.2, a total of 42 out of 100 (42.0%) websites followed 10 or more out of 19 established usability principles. On the basis of these baseline data points, ODPHP set targets for the year 2020 that meet the minimal statistical significance—increasing objective HC/HIT-8.1 data point to 70.5% and objective HC/HIT-8.2 data point to 55.7%.ConclusionsThis research is a critical first step in evaluating the quality of Web-based health information. The criteria proposed by ODPHP provide methods to assess website quality for professionals designing, developing, and managing health-related websites. The criteria, baseline data, and targets are valuable tools for driving quality improvement.