Аналіз вибіркових даних при оцінюванні наукового потенціалу і характер статистичних властивостей вербальних моделей

OBJECTIVE: To determine sensitivity and specificity of a standardized recombinant cell-based indirect immunofluorescence assay (RC-IFA) for anti-Tr antibodies in comparison to a reference procedure. METHODS: Delta/Notch-like epidermal growth factor-related receptor (DNER) was expressed in HEK293 and used as a substrate for RC-IFA. HEK293 control cells expressing CDR2/Yo and CDR2L as well as mock-transfected HEK293 cells were used as controls. Serum samples from 38 patients with anti-Tr antibodies (33 with paraneoplastic cerebellar degeneration [PCD] and Hodgkin lymphoma), 66 patients with anti-Tr-negative PCD, 53 patients with Hodgkin lymphoma without neurologic symptoms, 40 patients with rheumatic diseases, and 42 healthy blood donors were tested for anti-DNER reactivity in the RC-IFA. In addition, RC-IFA results were compared to those from a commercial tissue-based IFA using monkey cerebellum. RESULTS: Using the RC-IFA, anti-DNER was detected in all anti-Tr-positive patients but in none of the controls (sensitivity 100%, 95% confidence interval [CI] 92.8%-100%; specificity 100%, 95% CI 98.7%-100%). In comparison, anti-Tr was not detected in 4 samples with low-titer autoantibodies using the commercial tissue-based assay. Preadsorption of sera with either recombinant full-length DNER or its extracellular domain selectively abolished anti-Tr reactivity. CONCLUSION: Anti-Tr antibodies bind to the extracellular domain of DNER and can be detected by RC-IFA using HEK293 cells expressing the recombinant receptor. The new method performs better than a frequently used commercial tissue-based indirect immunofluorescence assay (IFA) in samples with low-titer antibodies. CLASSIFICATION OF EVIDENCE: This study provides Class II evidence that RC-IFA accurately detects anti-Tr as compared to conventional IFA

Solubilization of human cells by the styrene-maleic acid copolymer : Insights from fluorescence microscopy

Extracting membrane proteins from biological membranes by styrene-maleic acid copolymers (SMAs) in the form of nanodiscs has developed into a powerful tool in membrane research. However, the mode of action of membrane (protein) solubilization in a cellular context is still poorly understood and potential specificity for cellular compartments has not been investigated. Here, we use fluorescence microscopy to visualize the process of SMA solubilization of human cells, exemplified by the immortalized human HeLa cell line. Using fluorescent protein fusion constructs that mark distinct subcellular compartments, we found that SMA solubilizes membranes in a concentration-dependent multi-stage process. While all major intracellular compartments were affected without a strong preference, plasma membrane solubilization was found to be generally slower than the solubilization of organelle membranes. Interestingly, some plasma membrane-localized proteins were more resistant against solubilization than others, which might be explained by their presence in specific membrane domains with differing properties. Our results support the general applicability of SMA for the isolation of membrane proteins from different types of (sub)cellular membranes

Molecular and celllar mechanisms underlying anti-neuronal antibody mediated disorders of the central nervous system

Over the last decade multiple autoantigens located on the plasma membrane of neurons have been identified. Neuronal surface antigens include molecules directly involved in neurotransmission and excitability. Binding of the antibody to the antigen may directly alter the target protein's function, resulting in neurological disorders. The often striking reversibility of symptoms following early aggressive immunotherapy supports a pathogenic role for autoantibodies to neuronal surface antigens. In order to better understand and treat these neurologic disorders it is important to gain insight in the underlying mechanisms of antibody pathogenicity. In this review we discuss the clinical, circumstantial, in vitro and in vivo evidence for neuronal surface antibody pathogenicity and the possible underlying cellular and molecular mechanisms. This review shows that antibodies to neuronal surface antigens are often directed at conformational epitopes located in the extracellular domain of the antigen. The conformation of the epitope can be affected by specific posttranslational modifications. This may explain the distinct clinical phenotypes that are seen in patients with antibodies to antigens that are expressed throughout the brain. Furthermore, it is likely that there is a heterogeneous antibody population, consisting of different IgG subtypes and directed at multiple epitopes located in an immunogenic region. Binding of these antibodies may result in different pathophysiological mechanisms occurring in the same patient, together contributing to the clinical syndrome. Unraveling the predominant mechanism in each distinct antigen could provide clues for therapeutic interventions

Solubilization of human cells by the styrene-maleic acid copolymer : Insights from fluorescence microscopy

Extracting membrane proteins from biological membranes by styrene-maleic acid copolymers (SMAs) in the form of nanodiscs has developed into a powerful tool in membrane research. However, the mode of action of membrane (protein) solubilization in a cellular context is still poorly understood and potential specificity for cellular compartments has not been investigated. Here, we use fluorescence microscopy to visualize the process of SMA solubilization of human cells, exemplified by the immortalized human HeLa cell line. Using fluorescent protein fusion constructs that mark distinct subcellular compartments, we found that SMA solubilizes membranes in a concentration-dependent multi-stage process. While all major intracellular compartments were affected without a strong preference, plasma membrane solubilization was found to be generally slower than the solubilization of organelle membranes. Interestingly, some plasma membrane-localized proteins were more resistant against solubilization than others, which might be explained by their presence in specific membrane domains with differing properties. Our results support the general applicability of SMA for the isolation of membrane proteins from different types of (sub)cellular membranes

No evidence for the presence of neuronal surface autoantibodies in plasma of patients with schizophrenia

The immune system has been implicated in the etiology of schizophrenia. Autoimmunity by antibodies against neuronal cell surface antigens has been proposed as one of the pathological mechanisms. We examined plasma samples of 104 patients diagnosed with schizophrenia for the presence of autoantibodies against neuronal cell surface antigens using cultured hippocampal neurons and transfected HeLa cells. None of the samples tested positive for the presence of these autoantibodies. Based on our results it seems unlikely that autoantibodies against neuronal cell surface antigens play a role in the pathogenesis of schizophrenia, although further studies using cerebrospinal fluid are needed

No evidence for the presence of neuronal surface autoantibodies in plasma of patients with schizophrenia

The immune system has been implicated in the etiology of schizophrenia. Autoimmunity by antibodies against neuronal cell surface antigens has been proposed as one of the pathological mechanisms. We examined plasma samples of 104 patients diagnosed with schizophrenia for the presence of autoantibodies against neuronal cell surface antigens using cultured hippocampal neurons and transfected HeLa cells. None of the samples tested positive for the presence of these autoantibodies. Based on our results it seems unlikely that autoantibodies against neuronal cell surface antigens play a role in the pathogenesis of schizophrenia, although further studies using cerebrospinal fluid are needed.publisher: Elsevier articletitle: No evidence for the presence of neuronal surface autoantibodies in plasma of patients with schizophrenia journaltitle: European Neuropsychopharmacology articlelink: http://dx.doi.org/10.1016/j.euroneuro.2015.09.017 content_type: article copyright: Copyright © 2015 Elsevier B.V. and ECNP. All rights reserved.status: publishe

Plasticity-related gene 5 : A novel surface autoantigen in paraneoplastic cerebellar degeneration

Paraneoplastic cerebellar degeneration (PCD) is one of the most frequent paraneoplastic syndromes affecting the CNS. It is associated with antibodies targeting intracellular neuronal antigens (Hu, Yo, Ri, CV2/CRMP5), which are not thought to be directly pathogenic, and surface antigens (DNER, mGluR1, VGCC), which are potentially pathogenic.1,2 However, in many patients the immunologic target remains unidentified, resulting in diagnostic and therapeutic challenges. We report a patient with PCD and a squamous cell lung carcinoma with antibodies to a novel neuronal surface antigen, plasticity-related gene 5 (PRG5)

Plasticity-related gene 5: A novel surface autoantigen in paraneoplastic cerebellar degeneration

Paraneoplastic cerebellar degeneration (PCD) is one of the most frequent paraneoplastic syndromes affecting the CNS. It is associated with antibodies targeting intracellular neuronal antigens (Hu, Yo, Ri, CV2/CRMP5), which are not thought to be directly pathogenic, and surface antigens (DNER, mGluR1, VGCC), which are potentially pathogenic.1,2 However, in many patients the immunologic target remains unidentified, resulting in diagnostic and therapeutic challenges. We report a patient with PCD and a squamous cell lung carcinoma with antibodies to a novel neuronal surface antigen, plasticity-related gene 5 (PRG5)

Conservation of the Use1 5′UTR between species.

<p>(<b>A</b>) Top: a cartoon representing consensus splice sites. Bottom: Alignment of the 5′UTR of <i>Mus musculus Use1</i> with <i>Rattus norvegicus</i> and <i>Homo sapiens</i>. Splice donor and –acceptor sites, and the branching point (A) are indicated by capitals and underlined. (<b>B</b>) Top: alignment of the G-rich sequence preceding the AUG start codon of <i>Use1</i>. The stopcodon in the human sequence is underlined, asterixes indicate identical nucleotides. Bottom: G-quadruplex structures predicted in the sequences shown in (A) by the QGSRS mapper, including the score and length. (<b>C</b>) CD spectroscopy experiments were performed using a 40 nt nucleotide probe of the <i>Use1</i> 5′UTR wt sequence (5′-GUA-GCA-GGG-CGG-ACC-UCG-GAG-GGG-AAG-GAC-CUC-ACU-CAG-G-3′), in the presence of 100 mM of KCl (black line), 100 mM NaCl (dark grey line) or in the absence of salts (light grey line). Data were accumulated from ten scans. Presented data are representative for 3 independent experiments. (<b>D</b>) The human 5′UTR of <i>USE1</i> was cloned in front of the luciferase reporter, and transfected to HEK-293T cells with increasing amounts of Grsf1 expression vector. Luciferase activity is corrected for β-gal expression and given as fold-change (fc) compared to WT reporter in absence of Grsf1 overexpression (n = 3, error bars indicate SD, *p<0.01, **p<0.005).</p

Identification of the Use1 5′UTR.

<p>(<b>A</b>) NM025917.4 represents the <i>Use1</i> cDNA sequence in Genbank. The <i>Use1</i> ORF is 831 nt long, the 3′UTR is only 43 nt long. Nested primers (GSP1-3) located downstream of the AUG start codon were used for 5′RACE which revealed a 387 nt 5′UTR with a 156 nt alternatively spliced intron just upstream of the AUG start codon (see also <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0104631#pone-0104631-g005" target="_blank">figure 5A</a>). (<b>B</b>) The GSP2 primer in the 5′RACE yielded two products of 260 and 420 nt (RT-: cDNA reaction without reverse transcriptase). Fragments size is indicated in nucleotides.</p