Antibody-based methods for the measurement of α-synuclein concentration in human cerebrospinal fluid - method comparison and round robin study.

α-Synuclein is the major component of Lewy bodies and a candidate biomarker for neurodegenerative diseases in which Lewy bodies are common, including Parkinson's disease and dementia with Lewy bodies. A large body of literature suggests that these disorders are characterized by reduced concentrations of α-synuclein in cerebrospinal fluid (CSF), with overlapping concentrations compared to healthy controls and variability across studies. Several reasons can account for this variability, including technical ones, such as inter-assay and inter-laboratory variation (reproducibility). We compared four immunochemical methods for the quantification of α-synuclein concentration in 50 unique CSF samples. All methods were designed to capture most of the existing α-synuclein forms in CSF ('total' α-synuclein). Each of the four methods showed high analytical precision, excellent correlation between laboratories (R2 0.83-0.99), and good correlation with each other (R2 0.64-0.93), although the slopes of the regression lines were different between the four immunoassays. The use of common reference CSF samples decreased the differences in α-synuclein concentration between detection methods and technologies. Pilot data on an immunoprecipitation mass spectrometry (IP-MS) method is also presented. Our results suggest that the four immunochemical methods and the IP-MS method measure similar forms of α-synuclein and that a common reference material would allow harmonization of results between immunoassays.

A Comparison of Cathelicidin Levels in the Skin of Leprosy Patients and Their Household Contacts.

OBJECTIVE: This study aimed to compare cathelicidin levels in the skin of leprae patients and leprae contacts. PATIENTS AND METHODS: This research is an analytic observational study with a cross-sectional approach. Fifty-four research subjects participated in this study. They consisted of leprae patients, household contacts, and healthy individuals. Cathelicidin levels were measured using the ELISA method. Data analysis was carried out with the help of SPSS software, and univariate and bivariate analysis was conducted. RESULTS: Cathelicidin levels in the leprae group (256.8±22.9 pg/ml) were higher than in the contact group (25.9±2.7 pg/ml). Likewise, the contact group had higher cathelicidin levels than healthy controls (1.4±0.1 pg/ml). Statistically, there were differences in cathelicidin levels between groups, P<0.050. CONCLUSION: Cathelicidin levels in leprae patients were higher than those in household contacts.

Childhood Bullous Pemphigoid with atypical immunopathology: A case series

@#Bullous pemphigoid (BP) is a rare autoimmune blistering disorder primarily affecting older adults, with limited occurrences in children. BP in children typically manifests as large, tense blisters on the skin, often on flexural areas. It also more often affects the oromucosal areas and the face in children than in adults. Diagnosis involves histopathological examination revealing eosinophilic spongiosis or subepidermal split, immunofluorescence tests highlighting immunoglobulin G (IgG) and C3 depositions, and immunological assays detecting BP180 and BP230 IgG autoantibodies. This report presents two cases of childhood BP (CBP) with atypical immunopathological findings. Clinically, the two cases had generalized plaques and bullae, including the face. The first case exhibited the characteristic linear deposits of IgG and C3 on the basement membrane through direct immunofluorescence (DIF) and revealed negative anti‑BP180 antibodies on enzyme‑linked immunosorbent assay (ELISA). In contrast, the second case showed negative DIF results, despite clinical suspicion, but had positive anti‑BP180 IgG antibodies on ELISA. It is, therefore, crucial to consider the complete clinical presentation of the patient, in conjunction with the histological findings and immunopathologic assessments to diagnose CBP.

A combination of in vitro techniques for efficient discovery of functional monoclonal antibodies against human CXC chemokine receptor-2 (CXCR2).

BACKGROUND: Development of functional monoclonal antibodies against intractable GPCR targets. RESULTS: Identification of structured peptides mimicking the ligand binding site, their use in panning to enrich for a population of binders, and the subsequent challenge of this population with receptor overexpressing cells leads to functional monoclonal antibodies. CONCLUSION: The combination of techniques provides a successful strategic approach for the development of functional monoclonal antibodies against CXCR2 in a relatively small campaign. SIGNIFICANCE: The presented combination of techniques might be applicable for other, notoriously difficult, GPCR targets. SUMMARY: The CXC chemokine receptor-2 (CXCR2) is a member of the large 'family A' of G-protein-coupled-receptors and is overexpressed in various types of cancer cells. CXCR2 is activated by binding of a number of ligands, including interleukin 8 (IL-8) and growth-related protein α (Gro-α). Monoclonal antibodies capable of blocking the ligand-receptor interaction are therefore of therapeutic interest; however, the development of biological active antibodies against highly structured GPCR proteins is challenging. Here we present a combination of techniques that improve the discovery of functional monoclonal antibodies against the native CXCR2 receptor. The IL-8 binding site of CXCR2 was identified by screening peptide libraries with the IL-8 ligand, and then reconstructed as soluble synthetic peptides. These peptides were used as antigens to probe an antibody fragment phage display library to obtain subpopulations binding to the IL-8 binding site of CXCR2. Further enrichment of the phage population was achieved by an additional selection round with CXCR2 overexpressing cells as a different antigen source. The scFvs from the CXCR2 specific phage clones were sequenced and converted into monoclonal antibodies. The obtained antibodies bound specifically to CXCR2 expressing cells and inhibited the IL-8 and Gro-α induced ß-arrestin recruitment with IC50 values of 0.3 and 0.2 nM, respectively, and were significantly more potent than the murine monoclonal antibodies (18 and 19 nM, respectively) obtained by the classical hybridoma technique, elicited with the same peptide antigen. According to epitope mapping studies, the antibody efficacy is largely defined by N-terminal epitopes comprising the IL-8 and Gro-α binding sites. The presented strategic combination of in vitro techniques, including the use of different antigen sources, is a powerful alternative for the development of functional monoclonal antibodies by the classical hybridoma technique, and might be applicable to other GPCR targets.

Problems with measuring peripheral oxytocin: can the data on oxytocin and human behavior be trusted?

Research on the neurobiological and behavioral effects of oxytocin (OT), as well as on its possible therapeutic applications, has intensified in the past decade. Accurate determination of peripheral OT levels is essential to reach meaningful conclusions and to motivate, support and inform clinical interventions. Different, but concordant, methods for measuring plasma OT have been developed over the past four decades, but since 2004 several commercially available methods have been favored in research with humans. Evaluation of these methods reveals that they lack reliability when used on unextracted samples of human fluids, and that they tag molecules in addition to OT, yielding estimates that are wildly discrepant with an extensive body of earlier findings that were obtained using methods that are well validated, but more laborious. An accurate, specific, and readily available method for measuring OT that can be adopted as the standard in the field is urgently needed for advances in our understanding of OT's roles in cognition and behavior.

D-aspartic acid supplementation combined with 28 days of heavy resistance training has no effect on body composition, muscle strength, and serum hormones associated with the hypothalamo-pituitary-gonadal axis in resistance-trained men.

It was hypothesized that D-aspartic acid (D-ASP) supplementation would not increase endogenous testosterone levels or improve muscular performance associated with resistance training. Therefore, body composition, muscle strength, and serum hormone levels associated with the hypothalamo-pituitary-gonadal axis were studied after 28 days of resistance training and D-ASP supplementation. Resistance-trained men resistance trained 4 times/wk for 28 days while orally ingesting either 3 g of placebo or 3 g of D-ASP. Data were analyzed with 2 × 2 analysis of variance (P < .05). Before and after resistance training and supplementation, body composition and muscle strength, serum gonadal hormones, and serum D-ASP and d-aspartate oxidase (DDO) were determined. Body composition and muscle strength were significantly increased in both groups in response to resistance training (P < .05) but not different from one another (P > .05). Total and free testosterone, luteinizing hormone, gonadotropin-releasing hormone, and estradiol were unchanged with resistance training and D-ASP supplementation (P > .05). For serum D-ASP and DDO, D-ASP resulted in a slight increase compared with baseline levels (P > .05). For the D-ASP group, the levels of serum DDO were significantly increased compared with placebo (P < .05). The gonadal hormones were unaffected by 28 days of D-ASP supplementation and not associated with the observed increases in muscle strength and mass. Therefore, at the dose provided, D-ASP supplementation is ineffective in up-regulating the activity of the hypothalamo-pituitary-gonadal axis and has no anabolic or ergogenic effects in skeletal muscle.

Structural and functional characterization of the single-chain Fv fragment from a unique HCV E1E2-specific monoclonal antibody.

The nucleotide sequence of the unique neutralizing monoclonal antibody D32.10 raised against a conserved conformational epitope shared between E1 and E2 on the serum-derived hepatitis C virus (HCV) envelope was determined. Subsequently, the recombinant single-chain Fv fragment (scFv) was cloned and expressed in Escherichia coli, and its molecular characterization was assessed using multi-angle laser light scattering. The scFv mimicked the antibody in binding to the native serum-derived HCV particles from patients, as well as to envelope E1E2 complexes and E1, E2 glycoproteins carrying the viral epitope. The scFv D32.10 competed with the parental IgG for binding to antigen, and therefore could be a promising candidate for therapeutics and diagnostics.

Dynamic evaluation of antibodies against serogroup A, C, Y and W135 strains in healthy adults im-munized with one dose of quadrivalent meningococcal polysaccharide vaccine / 中华微生物学和免疫学杂志

Objective To dynamically analyze the antibodies with regard to in vitro serum bacteri-cidal activity and the quantity of IgG in healthy adults received one dose of immunization with ACYW 135 me-ningococcal polysaccharide vaccine .To investigate the term of protection with polysaccharide vaccine and the correlation between bactericidal titer and IgG concentration .Methods Twenty healthy adults were given one dose of immunization with quadrivalent meningococcal polysaccharide vaccine .Serum samples were col-lected before and after vaccination at specific time points .Test for serum bactericidal activity ( SBA ) and quantitative ELISA were performed to detect bactericidal titers and IgG concentrations in serum samples .Re-sults Certain levels of antibodies against capsular polysaccharides of serogroup A , C, Y and W135 strains had already existed prior to immunization .Moreover, bactericidal titers against serogroup A , Y and W135 strains except serogroup C strain were relatively high .Specific IgG concentrations and bactericidal titers for all serogroup stains were significantly increased on day 15 after vaccination (P0.05).However, a close correla-tion was demonstrated between GMTs and GMCs of serum samples (r>0.7, P<0.05).Conclusion The geometric mean titers ( GMTs) and geometric mean concentrations ( GMCs) of serum samples collected be-fore and after vaccination at different time points were reliable and consistent parameters for the evaluation of vaccine .The term of protection of quadivalent meningococcal polysaccharide vaccine was about 3 years upon a single dose of immunization .

Clinical Diagnostic Value of CT Scan and Serum Tumor Markers Determination for SCLC / 实用放射学杂志

Objective To investigate the clinic diagnosis value of CT scan and the determination of serum NSE, Cyfra21-1 for SCLC. Methods ① The CT findings and pathologic basis of 220 cases with SCLC were analyzed retrospectively.② Between February 2000 and December 2001, 120 consecutive patients with pulmonary lesions by CT scan, which suspiciously suffered lung cancer, were studied prospectively. Serum samples were obtained by venous puncture from all patients 1 week prior to surgery or puncture biopsy. The serum concentration of NSE, Cyfra21-1 were determinated using ELISA. According to the pathologic result, the diagnostic value of CT scan, NSE and Cyfra21-1 were evaluated.Results ① Most CT findings of SCLC were central and spindly or eggplant-shape mass, hilar and/or mediastinal lymph nodes enlargement.The obstructive pulmonary lesions were found rarely.② Using CT scan, NSE and Cyfra21-1 alone, the sensitivity were 75%,72.5% and 32.5%,the specificity were 73.75%, 83.75% and 48.75%,the accuracy were 74.16%, 81.67% and 43.33% respectively. With combination of CT scan and NSE, the diagnostic accuracy was 78.3%, and the sensitivity was 87.5%. However, CT scan combined with Cyfra21-1 can not be improve the diagnostic value. Conclusion SCLC has specific CT findings. The sensitivity of CT scan was 75%, combinated with NSE, its accuracy was higher(78.3%), and the sensitivity was 87.5%.

Change of serum soluble tumor necrosis factor receptor in rats with multiple organ dysfunction syndrome(MODS) and its clinical implication / 第二军医大学学报

Objective: To investigate the changes of sTNFR in rats with MODS and its clinical implication. Methods: Rat delayed two phase models of MODS were replicated, then serum sTNFR were measured respectively pre and post MODS by ELISA method. Results: Serum sTNFRⅠ pre and post MODS was (112.15?24.95) pg/ml and (438.06? 67.06) pg/ml ( P