Levels of Nitric Oxide Metabolites and Myeloperoxidase in Subjects with Type 2 Diabetes Mellitus on Metformin Therapy .

INTRODUCTION: Endothelial dysfunction is involved in the pathogenesis of insulin resistance, diabetes mellitus type 2, diabetic complications and preceded clinical manifestation of cardiovascular complications. Increased myeloperoxidase activity has been linked to a number of pathologies with compelling evidence in initiation and progression of inflammatory events. The aim of this study was to compare concentrations of metabolite nitric oxide and myeloperoxidase in the plasma of diabetes mellitus type 2 patients on metformin therapy, without clinical signs of cardiovascular disease and healthy subjects, as well as evaluation of concentrations of analytes in association with glycemic control. MATERIALS AND METHODS: Forty four study subjects with diabetes mellitus type 2 and thirty healthy subjects were included in this study. The concentration of myeloperoxidase was determined by enzyme-linked immunosorbent assay, the concentration of nitrate and nitrite with high performance liquid chromatography method. Student's t test, Mann-Whitney U test, Chi-square test and Fisher's exact test were used for statistical analysis. RESULTS: The mean concentration of myeloperoxidase was significantly higher in the diabetic group compared to the control group (16.2±4.9 vs. 3.7±1.8; P<0.001).The nitrite concentration was comparable in both groups while the concentration of nitrate was significantly higher in the diabetic group (41.2 [42.9] vs 31.9 [23]; P=0.017). In this study, plasma myeloperoxidase (Spearman's rho=0.421; P=0.004) and nitrate concentration was significantly positively associated with the HbA1c levels while nitrate concentration (Spearman's rho=- 0.308; P=0.047) were was significantly positively negatively associated with the HbA1c levels. CONCLUSION: Concertation of MPO and nitric oxide were significantly increased in a T2DM subject even when on metformin therapy. However, increased concentration of NO strongly correlates with lower levels of HbA1c showing a postive effect of a gylcemic control on endothelial dysfuction. Increased concentrations of NO3- in T2DM subject compared to control, indicates the variety of NO pathways that should be taken into consideration win relation to endothelial function.

Comparative analyses of biofilm formation among different Cutibacterium acnes isolates.

The Gram-positive anaerobic bacterium Cutibacterium acnes is a commensal of the human skin, but also an opportunistic pathogen that contributes to the pathophysiology of the skin disease acne vulgaris. Moreover, C. acnes, in addition to other skin-colonizing bacteria such as S. epidermidis and S. aureus, is an emerging pathogen of implant-associated infections. Notably, C. acnes isolates exhibit marked heterogeneity and can be divided into at least 6 phylotypes by multilocus sequence typing. It is becoming increasingly evident that biofilm formation is a relevant factor for C. acnes virulence, but information on biofilm formation by diverse C. acnes isolates is limited. In this study we performed a first comparative analysis of 58 diverse skin- or implant-isolates covering all six C. acnes phylotypes to investigate biofilm formation dynamics, biofilm morphology and attachment properties to abiotic surfaces. The results presented herein suggest that biofilm formation correlates with the phylotype, rather than the anatomical isolation site. IA1 isolates, particularly SLST sub-types A1 and A2, showed highest biofilm amounts in the microtiter plate assays, followed by isolates of the IC, IA2 and II phylotypes. Microscopic evaluation revealed well-structured three-dimensional biofilms and relatively high adhesive properties to abiotic surfaces for phylotypes IA1, IA2 and IC. Representatives of phylotype III formed biofilms with comparable biomass, but with less defined structures, whereas IB as well as II isolates showed the least complex three-dimensional morphology. Proteinase K- and DNase I-treatment reduced attachment rates of all phylotypes, therefore, indicating that extracellular DNA and proteins are critical for adhesion to abiotic surfaces. Moreover, proteins seem to be pivotal structural biofilm components as mature biofilms of all phylotypes were proteinase K-sensitive, whereas the sensitivity to DNase I-treatment varied depending on the phylotype.

ABO and RhD Blood Groups in Nasal Polyposis.

OBJECTIVE: The aim of this study was to determine ABO and RhD blood group distribution in nasal polyposis (NP) patients and whether there is a specific ABO or RhD blood phenotype associated with susceptibility to or protection with respect to development of NP. METHODS: The study group comprised 126 consecutive patients with chronic rhinosinusitis and bilateral NP. The control group comprised 126 healthy blood donors. All participants were from the same geographical region. Distribution of ABO and RhD phenotypes in all participants was studied. RESULTS: There were no significant differences between patients and controls in the distribution of the A (p=0.520), B (p=0.306), AB (p=0.673), O (p=0.894), and RhD (p=0.742) phenotypes. CONCLUSION: According to the present results, the ABO and RhD blood group systems are not associated with development of NP.

CRMP2 tethers kainate receptor activity to cytoskeleton dynamics during neuronal maturation.

The CRMP2 and CRMP4 proteins are strongly expressed in the developing nervous system, mediating neurite outgrowth, neuronal polarity, and axon guidance. In the present study, we demonstrate the interaction of the CRMP2 and CRMP4 proteins with the GluK5 subunit of the kainate (KA) receptor (KAR) and investigated the role of KARs in modulating the development of cultured mouse DRG neurons. We found that KARs modulate neuronal maturation and neurite outgrowth in a bidirectional manner. Accordingly, low concentrations of KA delayed maturation and enhanced neurite outgrowth, whereas maturation was promoted by higher concentrations of KA that attenuated neuritic elongation. The effects of weak KAR activation were prevented by blocking their noncanonical signaling and involved a differential regulation of CRMP2. Whereas the delay in maturation involves PKC-mediated phosphorylation of CRMP2 at T555 leading to a downregulation of membrane Cav2.2, the promotion of neurite outgrowth is achieved by dephosphorylation at T514 at the growth cones, the latter reflecting PKC-driven enhancement of GSK3ß phosphorylation at S9. Together, these findings indicate that noncanonical KAR signaling influences neuronal development by modulating CRMP2 activity.

Comprehensive antigen screening identifies Moraxella catarrhalis proteins that induce protection in a mouse pulmonary clearance model.

Moraxella catarrhalis is one of the three most common causative bacterial pathogens of otitis media, however no effective vaccine against M. catarrhalis has been developed so far. To identify M. catarrhalis vaccine candidate antigens, we used carefully selected sera from children with otitis media and healthy individuals to screen small-fragment genomic libraries that are expressed to display frame-selected peptides on a bacterial cell surface. This ANTIGENome technology led to the identification of 214 antigens, 23 of which were selected by in vitro or in vivo studies for additional characterization. Eight of the 23 candidates were tested in a Moraxella mouse pulmonary clearance model, and 3 of these antigens induced significantly faster bacterial clearance compared to adjuvant or to the previously characterized antigen OmpCD. The most significant protection data were obtained with the antigen MCR_1416 (Msp22), which was further investigated for its biological function by in vitro studies suggesting that Msp22 is a heme binding protein. This study comprises one of the most exhaustive studies to identify potential vaccine candidate antigens against the bacterial pathogen M. catarrhalis.

Panel 6: Vaccines.

OBJECTIVE: To update progress on the effectiveness of vaccine for prevention of acute otitis media (AOM) and identification of promising candidate antigens against Streptococcus pneumoniae, nontypeable Haemophilus influenzae, and Moraxella catarrhalis. REVIEW METHODS: Literature searches were performed in OvidSP and PubMed restricted to articles published between June 2007 and September 2011. Search terms included otitis media, vaccines, vaccine antigens, and each of the otitis pathogens and candidate antigens identified in the ninth conference report. CONCLUSIONS: The current report provides further evidence for the effectiveness of pneumococcal conjugate vaccines (PCVs) in the prevention of otitis media. Observational studies demonstrate a greater decline in AOM episodes than reported in clinical efficacy trials. Unmet challenges include extending protection to additional serotypes and additional pathogens, the need to prevent early episodes, the development of correlates of protection for protein antigens, and the need to define where an otitis media vaccine strategy fits with priorities for child health. IMPLICATIONS FOR PRACTICE: Acute otitis media continues to be a burden on children and families, especially those who suffer from frequent recurrences. The 7-valent PCV (PCV7) has reduced the burden of disease as well as shifted the pneumococcal serotypes and the distribution of otopathogens currently reported in children with AOM. Antibiotic resistance remains an ongoing challenge. Multiple candidate antigens have demonstrated the necessary requirements of conservation, surface exposure, immunogenicity, and protection in animal models. Further research on the role of each antigen in pathogenesis, in the development of correlates of protection in animal models, and in new adjuvants to elicit responses in the youngest infants is likely to be productive and permit more antigens to move into human clinical trials.

Th17/Th1 biased immunity to the pneumococcal proteins PcsB, StkP and PsaA in adults of different age.

Streptococcus pneumoniae is a major human pathogen, causing high morbidity and mortality in children, and also in the elderly, who are particularly susceptible to S. pneumoniae infections due to the dysregulated function of the aged immune system. As the current generation of polysaccharide vaccines do not provide sufficient protection for elderly, new vaccination strategies are urgently needed. To learn whether pneumococcal proteins are able to induce adaptive immune responses in adults in different age groups, we determined serum IgG antibody titers and T cell immunity (IFN-γ, IL-17A and IL-5 production) to three pneumococcal antigens, PcsB, StkP and PsaA, that are components of an investigational protein-based pneumococcal vaccine, IC47. Therefore, sera and PBMCs of 108 healthy adults in three different age groups (young, middle-aged and elderly) were analyzed by ELISA and ELISpot, respectively. We found naturally acquired antibodies to all three proteins in all age groups against all three antigens. However, elderly individuals had significantly lower IgG levels to PcsB and PsaA compared to those of younger donors. There was no significant age-related difference in the overall rate of T cell immunity for the three pneumococcal proteins. We found that the Th17 response was dominant in all age groups and was frequently combined with a Th1 or Th2 response in young and middle-aged subjects. However, in elderly persons there was a lower percentage of PBMC samples producing more than one cytokine upon antigenic stimulation. The narrow cytokine secretion pattern was the most striking difference between elderly and younger adult age groups. Our results demonstrate that in the majority of adults there is a naturally acquired humoral and cellular immune response to the three pneumococcal proteins tested. The dominance of the Th17 response is especially interesting in the light of new insights regarding the role of Th17 cells in mucosal protection against this pathogen.

A role for SNAP25 in internalization of kainate receptors and synaptic plasticity.

Regulation of surface insertion and internalization of AMPA and NMDA receptors has emerged as a key mechanism for the control of synaptic strength. Regulatory elements for synaptic kainate receptors (KARs) are, however, largely undetermined. We have found that SNAP25 is critical for the synaptic removal of KARs, acting via GluK5 (i.e., KA2) subunits. SNAP25 coimmunoprecipitates with protein complexes containing PICK1, GRIP1, and GluK5 and colocalizes with GluK5 in both hippocampal neurons and transfected HEK293 cells. In hippocampal slices, purified SNAP25 antibodies and blocking peptides caused a GluK5-dependent run-up of KARs-mediated EPSC (EPSC(KAR)) recorded from CA3 pyramidal neurons when included in the patch pipette and prevented activity-dependent long-term depression of EPSC(KAR). As EPSC(KAR) LTD, SNAP25/PICK1/GluK5 interactions are dynamically regulated by PKC.

Block of kainate receptor desensitization uncovers a key trafficking checkpoint.

A prominent feature of ionotropic glutamate receptors from the AMPA and kainate subtypes is their profound desensitization in response to glutamate-a process thought to protect the neuron from overexcitation. In AMPA receptors, it is well established that desensitization results from rearrangements of the interface formed between agonist-binding domains of adjacent subunits; however, it is unclear how this mechanism applies to kainate receptors. Here we show that stabilization of the binding domain dimer by the generation of intermolecular disulfide bonds apparently blocked desensitization of the kainate receptor GluR6. This result establishes a common desensitization mechanism in both AMPA and kainate receptors. Surprisingly, however, surface expression of these nondesensitizing mutants was drastically reduced and did not depend on channel activity. Therefore, in addition to its role at the synapse, we now propose an intracellular role for desensitization in controlling maturation and trafficking of glutamate receptors.

Identification of GRASP-1 as a novel 97 kDa autoantigen localized to endosomes.

We have identified an autoantigen that is recognized by antibodies from an 18-year-old female with a history of recurrent infections who later in her clinical course developed Raynaud's phenomenon and telangiectasias. By indirect immunofluorescence (IIF), the index serum produced a unique cytoplasmic discrete speckled (CDS) staining pattern that partially colocalized with early endosome antigen 1 (EEA1) but not Golgi complex or other cytoplasmic organelles in HEp-2 cells. When HEp-2 cells were treated with 0.1 N HCl, the cytoplasmic speckled staining of the index serum was markedly decreased, suggesting that the reactive antigen was soluble. Western blot analysis showed a reactive approximately 97 kDa protein in a saline soluble protein preparation from HeLa cells. Mass spectrometric analysis of the excised 97 kDa band that was immunoprecipitated from HeLa cell extracts identified GRASP-1 as a possible target. The index serum and anti-GRASP-1 antibodies colocalized to structures in the cytoplasm of HEp-2 cells. Synthetic peptides representing the full-length GRASP-1 protein were used to identify reactive epitopes. Like many other cytoplasmic autoantigens, GRASP-1 has numerous coiled-coil domains throughout the protein with the exception of short segments at the amino and carboxyl terminus.

A mosaic of functional kainate receptors in hippocampal interneurons.

Although some physiological functions of kainate receptors (KARs) still remain unclear, recent advances have highlighted a role in synaptic physiology. In hippocampal slices, kainate depresses GABA-mediated synaptic inhibition and increases the firing rate of interneurons. However, the sensitivity to agonists of these responses differs, suggesting that the presynaptic and somatic KARs have a distinct molecular composition. Hippocampal interneurons express several distinct KAR subunits that can assemble into heteromeric receptors with a variety of pharmacological properties and that, in principle, could fulfill different roles. To address which receptor types mediate each of the effects of kainate in interneurons, we used new compounds and mice deficient for specific KAR subunits. In a recombinant assay, 5-carboxyl-2,4-di-benzamido-benzoic acid (NS3763) acted exclusively on homomeric glutamate receptor subunit 5 (GluR5), whereas 3S,4aR,6S,8aR-6-((4-carboxyphenyl)methyl) 1,2,3,4,4a,5,6,7,8,8a-decahydroisoquinoline-3-carboxylic acid (LY382884) antagonized homomeric GluR5 and any heteromeric combination containing GluR5 subunits. In hippocampal slices, LY382884, but not NS3763, was able to prevent kainate-induced depression of evoked IPSC. In contrast, neither prevented the concomitant increase in spontaneous IPSC frequency. The selectivity of these compounds was seen additionally in knock-out mice, such that they were inactive in GluR5-/- mice but completely effective in GluR6-/- mice. Our data indicate that in wild-type mice, CA1 interneurons express heteromeric GluR6 -KA2 receptors in their somatic compartments and GluR5-GluR6 or GluR5-KA2 at presynaptic terminals. However, functional compensation appears to take place in the null mutants, a new pharmacological profile emerging more compatible with the activity of homomeric receptors in both compartments: GluR5 in GluR6-/- mice and GluR6 in GluR5-/- mice.

Characterization of early endosome antigen 1 in neural tissues.

The finding that patients and mice bearing autoantibodies directed against early endosome antigen 1 (EEA1) develop neurological signs and deficits prompted an investigation of EEA1 distribution, localization, and interaction with synaptic proteins found in neural tissues. We detected EEA1 in a variety of neural tissues and in cells of neural origin where it co-localized with SNAP-25. The interaction between EEA1 and SNAP-25 was dependent on the leucine zipper and a newly identified methyl-accepting domain of EEA1. The C-terminal zinc-binding FYVE finger motif (EEA1(1271-1411)) of EEA1 also interacted with native SNAP-25 but only in the presence of 100microM Ca(2+). In contrast, EEA1 did not bind to cysteine string protein or synapsin in these binding assays. These results suggest that EEA1 is involved in neuronal synaptic vesicle function and axonal transport and growth. EEA1 may undergo calcium-dependent conformational changes that are required for binding to SNAP-25.

What do young people from Mostar, Bosnia and Herzegovina know about contraception and sexual health?

AIM: To determine the knowledge of adolescents living in Mostar, Bosnia and Herzegovina, about contraception and sexual health. METHODS: We used an anonymous questionnaire to survey a random sample of 120 high school students, 60 from two general high schools and 60 from a vocational school. There were 30 male and 30 female students aged 15-17 years from each type of school. The questionnaire consisted of 24 questions: 17 tested the students' knowledge on the menstrual cycle, contraception, emergency contraception, and sexually transmitted diseases (STDs), and six inquired how they obtained the information on these issues. Student t-test was used to test for score differences between male and female students, and between general high school and vocational school students in their general and specific knowledge. Pearson's r coefficient was used to test the correlation between average grades and knowledge. RESULTS: Female students had greater general knowledge (t=3.69, df=118, p<0.001), knowledge on contraception (t=3.66, df=118, p<0.001), and knowledge on STDs (t=2.71, df=118, p=0.008) than their male peers. General high school students also had greater general knowledge (t=2.44, df=118, p=0.016), and knowledge on contraception (t=2.18, df=118, p=0.031) or STDs (t=2.36, df=118, p=0.020) than their vocational school peers. Major sources of information were magazines (69%), TV/radio (50%), school (37%), and friends (36%). The most common reason that kept our examinees from obtaining information on these issues was shame (52%). The two most common contraception methods known to them were a condom (82%) and contraception pill (77%), whereas 17% of all students were unfamiliar with any contraception method. When asked what could be done to improve their knowledge on sexuality, contraception, and STDs, most students opted for the inclusion of sexual education into the school curricula. CONCLUSION: Female students knew more about contraception and sexual health than their male peers. Young people should be provided with more information on sexual health.

Autoantibodies to protein transport and messenger RNA processing pathways: endosomes, lysosomes, Golgi complex, proteasomes, assemblyosomes, exosomes, and GW bodies.

Over 50 years ago the lupus erythematosus (LE) cell phenomenon was described and this was quickly followed by the introduction of the LE cell test and indirect immunofluorescence (IIF) to detect antinuclear antibodies (ANA) in clinical laboratories. Recently, attention has turned to the identification of the autoantigens that bind to cytoplasmic organelles such as the Golgi complex, endosomes and other "cytoplasmic somes". Three endosome autoantigens include early endosome antigen 1 (EEA1, 160 kDa), cytoplasmic linker protein-170 (CLIP-170, 170 kDa), and lysobisphosphatidic acid (LBPA). Antibodies to EEA1 were seen in a variety of conditions but approximately 40% of the patients had a neurological disease. Despite the prominence of lysosomes in cells and tissues, reports of autoantibodies are limited to the lysosomal antigen h-LAMP-2 and the cytoplasmic antineutrophil antibodies (cANCA). Autoantigens in the Golgi complex include giantin/macrogolgin, golgin-245, golgin 160, golgin-97, golgin 95/gm130, and golgin-67. More recently, there has been an interest in autoantibodies that bind components of the "SMN complex" or the "assemblyosome". Arginine/glycine (RG)-rich domains in components of the SMN complex interact with Sm, like-Sm (LSm), fibrillarin, RNA helicase A (Gu), and coilin proteins, all of which are antigen targets in a variety of diseases. More recently, components of a novel cytoplasmic structure named GW bodies (GWBs) have been identified as targets of human autoantibodies. Components of GWBs include GW182, a unique mRNA-binding protein, like Sm proteins (LSms), and decapping (hDcp1) and exonuclease (Xrn) enzymes. Current evidence suggests that GWBs are involved in the cytoplasmic processing of mRNAs. Autoantibodies to the "cytoplasmic somes" are relatively uncommon and serological tests to detect most of them are not widely available.

Altered neurological function in mice immunized with early endosome antigen 1.

BACKGROUND: Autoantibodies directed against the 160 kDa endosome protein early endosome antigen 1 (EEA1) are seen in patients with neurological diseases. To determine if antibodies to EEA1 have a neuropathological effect, mice from three major histocompatibility haplotype backgrounds (H2q, H2b and H2d) were immunized with EEA1 (amino acids 82-1411) that was previously shown to contain the target EEA1 epitopes. The mice were then subjected to five neuro-behavioural tests: grid walking, forelimb strength, open field, reaching and rotarod. RESULTS: The immunized SWR/J mice with sustained anti-EEA1 antibodies had significantly reduced forelimb strength than the control non-immune mice of the same strain, and BALB/CJ immune mice demonstrated significantly more forelimb errors on the grid walk test than the control group. CONCLUSIONS: Antibodies to recombinant EEA1 in mice may mediate neurological deficits that are consistent with clinical features of some humans that spontaneously develop anti-EEA1 autoantibodies.

Identification of the B-cell epitopes of the early endosome antigen 1 (EEA1).

Early endosome antigen 1 (EEA1) is a target autoantigen in patients diagnosed with neurological and other autoimmune conditions. Eighteen of 65 sera (28%) that displayed a vesicular cytoplasmic staining pattern also immunoprecipitated the recombinant EEA1. These 18 sera were selected for further clinical, serological and epitope mapping studies. Thirty-six percent of the 18 patients had neurological diseases. Seventeen sera (94%) reacted with the partial length EEA1 constructs that included the C-terminal zinc finger (+FYVE) and the methyl accepting domain (LeuMA: amino acids 82-1411) in an addressable laser bead assay suggesting that the assay may be used for rapid laboratory detection of anti-EEA1 antibodies. Three of seven sera selected for epitope mapping studies bound to EEA1 peptides represented by amino acids 1096-1125, and two reacted with peptides represented by amino acids 1296-1320. One serum reacted only with the C-terminal peptide 1096-1125. The remaining serum reacted with other EEA1 epitopes. This data was supported by the observations that all the sera immunoprecipitated the C-terminal +FYVE (EEA1 1064-1411) construct, a peptide that also contained the linear epitopes 1096-1140. The limited epitope mapping studies suggest that the sera from patients with non-neurological diseases recognized epitopes in the central and C-terminal EEA1 domains, whereas the patients with neurological disease recognized a more restricted set of epitopes in the C-terminal.