Luminex Dotaz Zobrazit nápovědu
- Klíčová slova
- LabMAP, Luminex,
- MeSH
- amplifikace genu genetika MeSH
- biomedicínský výzkum MeSH
- chromozom Y genetika MeSH
- čipová analýza proteinů metody využití MeSH
- finanční podpora výzkumu jako téma MeSH
- jednonukleotidový polymorfismus MeSH
- lidé MeSH
- oligonukleotidové sondy diagnostické užití MeSH
- polystyreny diagnostické užití MeSH
- reagenční diagnostické soupravy trendy využití MeSH
- Check Tag
- lidé MeSH
- Geografické názvy
- Česká republika MeSH
The significance of B-cell crossmatching in kidney transplantation is controversial. Recipients (n = 471) transplanted in a single centre from 1987 to 2005 with complete T- and B-cell crossmatch records were studied. Sera from 83 patients transplanted across a positive B-cell crossmatch, with concomitant negative T-cell crossmatch (T-B+) on either current and/or peak sera were studied using Luminex to determine presence of donor-specific antibodies (DSA). Clinical outcomes of T-B+ patients were compared with 386 T-B- patients. T-B+ predicted vascular (p = 0.01), but not cellular (p = 0.82) or glomerular (p = 0.14) rejection. IgG HLA DSA were found in 33% (n = 27) of the T-B+ patients and were associated with higher risk of any (p = 0.047), vascular (p = 0.01) or glomerular (p < 0.001) rejection at 6 months. Of 27 patients with DSA, 18/21 (86%) were the complement-fixing IgG(1) and/or IgG(3) subclass antibodies. DSA imposed a statistically significant higher risk of graft loss 5 years posttransplant (1.8 [1.0-3.3], p = 0.045). This study showed that only one-third of positive B-cell crossmatch (BXM) was caused by DSA and was associated with late graft loss. Thus, using BXM to preclude kidney transplantation may potentially disadvantage >60% of patients in whom BXM is not indicative of the presence of DSA.
Cytokines, chemokines, and growth factors are key mediators of cell proliferation, migration, and immune response, and in tumor microenvironment, such factors contribute to regulation of tumor growth, immune cell recruitment, angiogenesis, and metastasis. In body fluids, levels of inflammatory mediators reflect the patient immune response to the disease and may predict the effects of targeted therapies. Significant improvements in cytokine detection techniques have been made during last 10 years leading to sensitive quantification of such potent molecules present in low pg/mL levels. Among the techniques, Luminex xMAP® multiplex assays allow for simultaneous quantification of up to 100 analytes with high sensitivity, broad dynamic range of quantification, high throughput, and minimal sample requirements. In this chapter we describe a detailed protocol for the application of xMAP assays using Luminex® 200™ analyzer with xPonent® acquisition software to quantify cytokines, chemokines, and growth factors secreted to blood serum and plasma of cancer patients. We also discuss how sample preparation, instrument settings, and standard curve fitting algorithms can influence validity of obtained results. Special attention is paid to data analysis using open source R statistical environment and we provide an example dataset of cytokine levels measured in serum and corresponding R script for standard curve fitting and concentration estimates.
- Publikační typ
- abstrakt z konference MeSH
BACKGROUND: Anxiety and depressive disorders are highly prevalent mental health conditions, affecting millions worldwide. Advancements in neurobiology have identified the effects of various neuropeptides in modulating mood and stress responses. Some of the well-researched neuropeptides in plasma are oxytocin (OXT), alpha-melanocyte-stimulating hormone (alpha-MSH), beta-endorphin, neurotensin, and substance P. In this study, we used methods of liquid biopsy to acquire saliva samples to analyze the concentrations of neuropeptides associated with depression. METHODS: The study was conducted in Bratislava, Slovakia, from January to June 2022. Participants were 20 subjects treated for depression and anxiety without medication; the control group consisted of 20 healthy individuals with no personal history of depression or anxiety. Salivary samples were collected using buccal swabs to measure the concentrations of the examined neuropeptides. Laboratory analysis was based on detecting fluorescent signals performed on the Luminex MAGPIX® System (Luminex Corporation, Austin, Texas). Means and standard deviations were calculated for individual neuropeptide levels. To determine if there are statistically significant differences in neuropeptide levels between individuals with and without depression, independent t-tests and a one-way ANOVA were conducted. RESULTS: Our findings indicate a significant decrease in all studied neuropeptides in subjects compared to healthy controls. Reductions in mean levels were observed for OXT (7.3), alpha-MSH (3.9), beta-endorphin (2.9), neurotensin (15.1), and a 6.9-fold decrease for substance P. Alpha-MSH and beta-endorphin showed higher variability in measured levels within both groups. CONCLUSION: The results of this study indicate that the levels of the studied salivary neuropeptides, OXT, alpha-MSH, beta-endorphin, neurotensin, and substance P, are statistically significantly reduced in individuals with depression compared to healthy controls.
- Publikační typ
- časopisecké články MeSH
- Publikační typ
- abstrakt z konference MeSH
- Publikační typ
- abstrakt z konference MeSH
Tato práce popisuje zavedení a optimalizaci metody umožňující analýzu produkce prozánětlivých cytokinů buňkami vrozené imunity. Diagnos-tika autoinflamatorních onemocnění, jako jsou familiární středozemní horečka (Familial Mediterranean Fever, FMF), syndrom Schnitzlerové či hyper IgD syndrom (HIDS), imunodysregulačních a autoimunitních onemocnění s autoinflamatorní složkou, je často složitá pro nespecifický klinický obraz. Přesná diagnóza je možná až genetickým vyšetřením, které však není u všech pacientů průkazné a u některých onemocnění ani není genetický původ znám. Nejasnosti v diagnostice onemocnění a rozlišení patogenetických pochodů u konkrétních pacientů ztěžují či zcela znemožňují nastavení cílené terapie, která v současné době zahrnuje i možnosti specifické blokády jednotlivých prozánětlivých cytokinů. Z tohoto důvodu jsme se zaměřili na zavedení, nastavení a optimalizaci metody, která by umožnila rychle a ekonomicky v malém množství lehce dostupného biomateriálu (periferní krve) odhalit aberantní produkci vybraných prozánětlivých cytokinů, a přispěla by tak ke správné diagnostice a výběru vhodné terapie. Produkce základních prozánětlivých cytokinů (IL-1β, IL-6, TNFα) tvořených zejména monocyty po stimulaci lipopo-lysacharidem (LPS) byla v práci testována pomocí dvou rozdílných metod – průtokovou cytometrií detekující intracelulární produkci cytokinů a metodou ALBIA (Addressable Laser Bead Immuno Assay) – Luminex, která umožňuje stanovení množství cytokinů uvolněných do superna-tantu. Na zdravých dárcích byla optimalizována délka stimulace, koncentrace LPS a čas zastavení uvolňování cytokinů z buněk Brefeldinem A.
This work aims at the introduction and optimization of methods for innate immune parameters analysis with a detailed focus on proinflammatory cytokines. Autoinflammatory diseases like Familial Mediterranean Fever (FMF), Schnitzler syndrome and Hyper-IgD syndrome (HIDS), as well as autoimmune diseases that involve the inflammatory component, are usually characterized by a non-specific clinical manifestation. Therefore, obtaining a proper diagnosis of these disorders remains complicated. Genetic examination serves as an accurate diagnostic method for the esta-blishment of monogenic autoinflammatory disease diagnosis. However, genetic profiling cannot be provided to all patients. Moreover, some of the mutations responsible for the development of the disease haven’t been defined yet. Therapy of inflammatory diseases is currently based on molecules that are capable of blocking the function of inflammatory cytokines. In inflammatory diseases, the proper understanding of disease pathogenesis is still far from satisfactory and therefore, selecting the right therapeutic treatment modality keeps causing difficulties. For this reason, we focused on the introduction and optimization of a novel method which has a potential to detect activation of inflammatory cytokines and may, therefore, contribute to the correct diagnosis and direct therapeutic strategy. Production of main proinflammatory cytokines (IL-1β, IL-6, TNFα), which are produced by monocytes after lipopolysaccharide stimulation, was tested with two different methods – flow cytometry, detecting the intracellular production of cytokines, and ALBIA (Addressable Laser Bead Immuno Assay) - Luminex method, which can detect extracellular cytokine release into supernatant. Using flow cytometry we optimized the setting by detecting the optimal time of stimulation, concentration of LPS and the optimal time to stop cytokine exocytosis after adding Brefeldin A.
- Klíčová slova
- metoda Luminex,
- MeSH
- analýza dat MeSH
- časové faktory MeSH
- cytokiny * imunologie krev MeSH
- horečka neznámého původu * diagnóza genetika imunologie MeSH
- lidé MeSH
- průtoková cytometrie MeSH
- Check Tag
- lidé MeSH
