NMDA receptors (NMDARs) are ionotropic glutamate receptors that play a key role in excitatory neurotransmission. The number and subtype of surface NMDARs are regulated at several levels, including their externalization, internalization, and lateral diffusion between the synaptic and extrasynaptic regions. Here, we used novel anti-GFP (green fluorescent protein) nanobodies conjugated to either the smallest commercially available quantum dot 525 (QD525) or the several nanometer larger (and thus brighter) QD605 (referred to as nanoGFP-QD525 and nanoGFP-QD605, respectively). Targeting the yellow fluorescent protein-tagged GluN1 subunit in rat hippocampal neurons, we compared these two probes to a previously established larger probe, a rabbit anti-GFP IgG together with a secondary IgG conjugated to QD605 (referred to as antiGFP-QD605). The nanoGFP-based probes allowed faster lateral diffusion of the NMDARs, with several-fold increased median values of the diffusion coefficient (D). Using thresholded tdTomato-Homer1c signals to mark synaptic regions, we found that the nanoprobe-based D values sharply increased at distances over 100 nm from the synaptic edge, while D values for antiGFP-QD605 probe remained unchanged up to a 400 nm distance. Using the nanoGFP-QD605 probe in hippocampal neurons expressing the GFP-GluN2A, GFP-GluN2B, or GFP-GluN3A subunits, we detected subunit-dependent differences in the synaptic localization of NMDARs, D value, synaptic residence time, and synaptic-extrasynaptic exchange rate. Finally, we confirmed the applicability of the nanoGFP-QD605 probe to study differences in the distribution of synaptic NMDARs by comparing to data obtained with nanoGFPs conjugated to organic fluorophores, using universal point accumulation imaging in nanoscale topography and direct stochastic optical reconstruction microscopy.SIGNIFICANCE STATEMENT Our study systematically compared the localization and mobility of surface NMDARs containing GFP-GluN2A, GFP-GluN2B, or GFP-GluN3A subunits expressed in rodent hippocampal neurons, using anti-green fluorescent protein (GFP) nanobodies conjugated to the quantum dot 605 (nanoGFP-QD605), as well as nanoGFP probes conjugated with small organic fluorophores. Our comprehensive analysis showed that the method used to delineate the synaptic region plays an important role in the study of synaptic and extrasynaptic pools of NMDARs. In addition, we showed that the nanoGFP-QD605 probe has optimal parameters for studying the mobility of NMDARs because of its high localization accuracy comparable to direct stochastic optical reconstruction microscopy and longer scan time compared with universal point accumulation imaging in nanoscale topography. The developed approaches are readily applicable to the study of any GFP-labeled membrane receptors expressed in mammalian neurons.

• MeSH
• hipokampus metabolismus   MeSH
• imunoglobulin G metabolismus   MeSH
• jednodoménové protilátky * metabolismus   MeSH
• králíci MeSH
• krysa rodu rattus MeSH
• neurony metabolismus   MeSH
• receptory N-methyl-D-aspartátu * metabolismus   MeSH
• savci MeSH
• synapse fyziologie   MeSH
• zvířata MeSH
• Check Tag
• králíci MeSH
• krysa rodu rattus MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Despite the interest of researchers in IgG4-related disease (IgG4-RD), many questions still remain unanswered regarding the thyroid gland. We aimed to clarify the relationship between IgG4-positive plasma cells and the histopathological pattern in the Hashimoto thyroiditis (HT) in a Finnish series. HT specimens (n = 280) were retrieved from the Department of Pathology, Fimlab Laboratories. After re-evaluation, 82 (29%) cases (72 females and 10 males, 52 ± 17 years) with significant fibrosis were selected. CD38, IgG and IgG4 positivity in plasma cells was evaluated by immunohistochemistry. Adjusted IgG4-positive plasma cells per HPF > 20 and IgG4- to IgG-positive plasma cell ratio > 30% were adopted as threshold criteria and related to other morphological features. IgG4-positive HT group included 13 cases (15% from fibrotic HT, 4.6% from all HT, 50 ± 15 years, 11 females) with adjusted HPF count 30 ± 5 (23-40) IgG4-positive cells. IgG4-positivity significantly correlated with the presence of lobulation, oncocytic metaplasia and certain type of fibrosis, fibrosis spread outside the gland, lymphocytes/plasma cells epithelial penetration, the predominance of microfollicles and follicular atrophy in the present study. Despite the persisting uncertainty whether HT is IgG4-RD, HT with IgG4-positive plasma cells is histopathologically distinct entity with some geographic variability.

• MeSH
• antigeny CD38 metabolismus   MeSH
• dítě MeSH
• dospělí MeSH
• fibróza MeSH
• Hashimotova nemoc imunologie   patologie   MeSH
• imunoglobulin G metabolismus   MeSH
• lidé středního věku MeSH
• lidé MeSH
• membránové glykoproteiny metabolismus   MeSH
• mladiství MeSH
• mladý dospělý MeSH
• plazmatické buňky metabolismus   patologie   MeSH
• počet lymfocytů MeSH
• senioři nad 80 let MeSH
• senioři MeSH
• štítná žláza imunologie   patologie   MeSH
• zánět MeSH
• Check Tag
• dítě MeSH
• dospělí MeSH
• lidé středního věku MeSH
• lidé MeSH
• mladiství MeSH
• mladý dospělý MeSH
• mužské pohlaví MeSH
• senioři nad 80 let MeSH
• senioři MeSH
• ženské pohlaví MeSH
• Publikační typ
• časopisecké články MeSH
• Geografické názvy
• Finsko MeSH

Lactococcus lactis is a food-grade lactic acid bacterium that is used in the dairy industry as a cell factory and as a host for recombinant protein expression. The nisin-controlled inducible expression (NICE) system is frequently applied in L. lactis; however new tools for its genetic modification are highly desirable. In this work NICE was adapted for dual protein expression. Plasmid pNZDual, that contains two nisin promoters and multiple cloning sites (MCSs), and pNZPolycist, that contains a single nisin promoter and two MCSs separated by the ribosome binding site, were constructed. Genes for the infrared fluorescent protein and for the human IgG-binding DARPin were cloned in all possible combinations to assess the protein yield. The dual promoter plasmid pNZDual enabled balanced expression of the two model proteins. It was exploited for the development of a single-plasmid inducible CRISPR-Cas9 system (pNZCRISPR) by using a nisin promoter, first to drive Cas9 expression and, secondly, to drive single guide RNA transcription. sgRNAs against htrA and ermR directed Cas9 against genomic or plasmid DNA and caused changes in bacterial growth and survival. Replacing Cas9 by dCas9 enabled CRISPR interference-mediated silencing of the upp gene. The present study introduces a new series of plasmids for advanced genetic modification of lactic acid bacterium L. lactis.

• MeSH
• antibakteriální látky farmakologie   MeSH
• CRISPR-Cas systémy MeSH
• editace genu metody   MeSH
• fermentace MeSH
• genetické inženýrství metody   MeSH
• genom bakteriální * MeSH
• guide RNA, Kinetoplastida genetika   metabolismus   MeSH
• imunoglobulin G genetika   metabolismus   MeSH
• klonování DNA MeSH
• Lactococcus lactis genetika   metabolismus   MeSH
• lidé MeSH
• methyltransferasy genetika   metabolismus   MeSH
• nisin farmakologie   MeSH
• plazmidy chemie   metabolismus   MeSH
• promotorové oblasti (genetika) účinky léků   MeSH
• proteiny teplotního šoku genetika   metabolismus   MeSH
• regulace genové exprese u bakterií * MeSH
• rekombinantní fúzní proteiny genetika   metabolismus   MeSH
• transgeny * MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

The synthesis and characterization of large-bore silica-based monolithic capillary columns (0.32mm×150mm) are presented. Columns were prepared by acidic hydrolysis of a mixture containing tetramethoxysilane (TMOS) and 1,2-bis(trimethoxysilyl)ethane (BTME) in different molar ratios in the presence of polyethylene glycol and urea. The monoliths were modified by zwitterionic monomer [2-(methacryloyloxy)ethyl]-dimethyl-(3-sulfopropyl)-ammonium hydroxide via 3-(trimethoxysilyl)propyl methacrylate. Prepared stationary phases were evaluated by scanning electron microscopy and chromatographic separation of nucleobases and their derivatives in the HILIC mode. The best chromatographic results were obtained with the column prepared from the reaction mixture containing BTME and TMOS in a 1:4 molar ratio. The permeability of such column reached 1.68×10(-14)m(2) and the efficiency, expressed as a height equivalent of the theoretical plate, did not exceed 10.5μm for the tested compounds. The columns were successfully applied to HILIC separation of native and labeled oligosaccharides and glycans released from bovine ribonuclease B and human immunoglobulin G.

• MeSH
• chromatografie kapalinová přístrojové vybavení   metody   MeSH
• ethan analogy a deriváty   chemie   MeSH
• hmotnostní spektrometrie s elektrosprejovou ionizací MeSH
• hydrofobní a hydrofilní interakce MeSH
• imunoglobulin G metabolismus   MeSH
• lidé MeSH
• methakryláty chemie   MeSH
• mikroskopie elektronová rastrovací MeSH
• oligosacharidy analýza   izolace a purifikace   MeSH
• organické sloučeniny křemíku chemie   MeSH
• oxid křemičitý chemie   MeSH
• ribonukleasy metabolismus   MeSH
• skot MeSH
• trimethylsilylové sloučeniny chemie   MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• skot MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH

BACKGROUND: Epidermolysis bullosa acquisita (EBA) is a rare, potentially devastating autoimmune disease of the skin. IgG autoantibodies directed against type VII collagen (Col7), the major component of anchoring fibrils, induce skin fragility leading to cutaneous and mucocutaneous blister formation, which is mostly of a scarring phenotype. Thus, powerful and reproducible diagnostic assays are critical to establish the diagnosis of EBA early to avoid irreversible sequelae. OBJECTIVES: The present international, retrospective multicentre study included a large cohort of patients with EBA and evaluated the diagnostic power of four different diagnostic assays for the detection of anti-Col7 IgG autoantibodies. METHODS: Overall, 95 EBA sera and 200 control sera consisting of 100 bullous pemphigoid sera, 50 pemphigus vulgaris sera and 50 sera of healthy controls were tested for anti-Col7 IgG autoantibodies using indirect immunofluorescence (IIF), two commercial enzyme-linked immunosorbent assay (ELISA) systems and Western blot (WB) analysis. EBA sera were taken from patients with positive direct immunofluorescence and IgG reactivity in at least one of the immunoserological assays (IIF, ELISA, WB). RESULTS: A Col7-NC1/NC2 ELISA (MBL, Nagoya, Japan) showed the highest sensitivity (97·9%), followed by a Col7-NC1 ELISA (Euroimmun, Lübeck, Germany) (89·5%), WB with Col7-NC1 (85·3%), and IIF on saline-split human skin (74·7%). The specificities of both ELISA systems were comparable (NC1 98·7%, NC1/NC2 99·3%). Furthermore, WB was more sensitive than IIF, which was more specific. CONCLUSIONS: The two commercially available ELISA systems allow for a highly sensitive and specific diagnosis of EBA. The sensitivity of the Col7-NC1/NC2 ELISA is significantly higher compared with the ELISA based on the Col7-NC1 domain only.

• MeSH
• autoprotilátky metabolismus   MeSH
• ELISA MeSH
• epidermolysis bullosa acquisita diagnóza   MeSH
• fluorescenční mikroskopie MeSH
• imunoglobulin G imunologie   metabolismus   MeSH
• kolagen typ VII imunologie   MeSH
• lidé MeSH
• puchýř imunologie   MeSH
• retrospektivní studie MeSH
• studie případů a kontrol MeSH
• western blotting MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• hodnotící studie MeSH
• multicentrická studie MeSH

• MeSH
• autoprotilátky škodlivé účinky   MeSH
• bakteriální proteiny farmakologie   terapeutické užití   MeSH
• glykosidhydrolasy terapeutické užití   MeSH
• HLA antigeny imunologie   MeSH
• imunoglobulin G imunologie   metabolismus   MeSH
• lidé MeSH
• proteolýza MeSH
• rejekce štěpu * etiologie   imunologie   MeSH
• transplantace ledvin MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• klinické zkoušky, fáze II MeSH
• klinické zkoušky, fáze III MeSH

BACKGROUND AND AIMS: Advances in the diagnosis and treatment of multiple myeloma (MM), place increasing demands on accurate stratification of patients as the starting point for optimal individualized therapy. The present study focused on assessing the association between HLC levels and the HLC-r to parameters of MM activity, prognosis and tumor mass volume.The objective was to assess the correlation of immunoglobulin (Ig), heavy/light chain (HLC) pairs (IgG-κ and-λ, IgA-κ and -λ HLC) and the ratio of monoclonal involved-HLC (i-HLC) to polyclonal uninvolved (u-HLC) Ig concentrations assessed by the Hevylite(TM) method with the free light chain κ/λ ratio (FLC-r), selected prognostic laboratory parameters i.e. Hb, platelets, albumin, β2-microglobulin (β2-M), Ca, lactate dehydrogenase (LDH), creatinine and the Durie-Salmon (D-S) and International Staging System (ISS), stages (1-3) for MM. METHODS: Hevylite assays were done on the sera of 132 MM patients at the time of diagnosis (IgG 94, IgA 38). HLC-r was calculated in the case of i-HLC-κ from the i-HLC-κ/u-HLC-λ ratio and for i-HLC-λ from the i-HLC-λ/u-HLC-κ ratio. D-S and ISS stages were evenly distributed. RESULTS: Md IgG-κ HLC-r was 64.8 (2.7-2222) and of IgG-λ HLC-r 49.6 (0.7-465.1), in the case of IgA-κ, Md HLC-r was 408.9 (3.4-3966) and for IgA-λ HLC-r the Md was 180.0 (0.1-3110). Normal levels of HLC pairs and HLC-r did not always rule out the diagnosis of MM. HLC-r correlated with FLC-r in IgG (r = 0.244, P = 0.018), but not in the IgA type. For IgG, HLC-r values were significantly different in patients with abnormal vs normal levels of Hb (P < 0.0001), albumin (P < 0.043), β2-M (P < 0.0001) and creatinine (P = 0.034) but not thrombocyte count, Ca or LDH. For the IgA isotype, we found a significant difference in HLC-r values only for thrombocyte count (P = 0.026) and β2-M (P = 0.016) but not for Hb, albumin, Ca, LDH or creatinine. For the IgG isotype there was a significant relationship of HLC-r index to stages 1-3 (P = 0.038) and substage A vs B (P = 0.048) according to D-S, and with high significance to stages 1-3 according to ISS (P = 0.005) and between stages 1 vs 3 (P = 0.001). For the IgA isotype, we found significant differences in HLC-r only between stages 1-3 (P = 0.025) according to D-S but not in the case of ISS. There were no significant correlations between i-HLC Ig levels and D-S or ISS stages in both IgG-κ and λ and IgA-κ and λ. Exceptions were significant differences for stages 1 vs 3 (P = 0.012) and 2 vs 3 (P = 0.017) for the IgG-λ isotype. There were no correlations of the HLC-r and u-HLC levels for either D-S or ISS stratifications in all HLC isotypes. CONCLUSION: We found a significant positive contribution of HLC-r using the i-HLC/u-HLC ratio even in the case of i-HLC-λ i.e. i-HLC-λ/u-HLC-κ. Variable results for the relationship of important laboratory parameters and D-S and ISS stratifications (stage 1-3) to HLC-r values in IgG and IgA isotypes make separate interpretation of the Hevylite method results necessary in clinical practice.

• MeSH
• dospělí MeSH
• imunoglobulin A metabolismus   MeSH
• imunoglobulin G metabolismus   MeSH
• lidé středního věku MeSH
• lidé MeSH
• mnohočetný myelom krev   klasifikace   diagnóza   MeSH
• prognóza MeSH
• prospektivní studie MeSH
• senioři nad 80 let MeSH
• senioři MeSH
• těžké řetězce imunoglobulinů metabolismus   MeSH
• Check Tag
• dospělí MeSH
• lidé středního věku MeSH
• lidé MeSH
• mužské pohlaví MeSH
• senioři nad 80 let MeSH
• senioři MeSH
• ženské pohlaví MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Investigation of protein glycosylation is an important area in biomarker discovery and biopharmaceutical research. Alterations in protein N-glycosylation can be an indication of changes in pathological conditions in the medical field or production parameters of biotherapeutics. Rapid development of these disciplines calls for fast, high-throughput, and reproducible methods to analyze protein N-glycosylation. Currently used methods require either long deglycosylation times or large excess of enzymes. In this paper, we report on the use of PNGase F immobilization onto the surface of magnetic microparticles and their use in rapid and efficient removal of N-glycans from glycoproteins. The use of immobilized PNGase F also allowed reusability of the enzyme-coated beads as the magnetic microparticles can be readily partitioned from the sample by a magnet after each deglycosylation reaction. The efficiency and activity of the PNGase F coated magnetic beads was compared with in-solution enzyme reactions using standard glycoproteins possessing the major N-glycan types of neutral, high mannose, and highly sialylated carbohydrates. The PNGase F coated magnetic beads offered comparable deglycosylation level to the conventional in-solution based method in 10-min reaction times for the model glycoproteins of immunoglobulin G (mostly neutral carbohydrates), ribonuclease B (high mannose type sugars), and fetuin (highly sialylated oligosaccharides) with the special features of easy removal of the enzyme from the reaction mixture and reusability.

• MeSH
• elektroforéza MeSH
• enzymy imobilizované metabolismus   MeSH
• glykopeptidasa metabolismus   MeSH
• glykoproteiny chemie   metabolismus   MeSH
• glykosylace MeSH
• imunoglobulin G chemie   metabolismus   MeSH
• mikrosféry * MeSH
• oligosacharidy metabolismus   MeSH
• polysacharidy metabolismus   MeSH
• ribonukleasy chemie   metabolismus   MeSH
• Publikační typ
• časopisecké články MeSH

The aim of the study was to test the hypothesis that oral plasma cell granuloma may represent a mucosal manifestation of immunoglobulin (Ig)G4-related disease (IgG4-RD) in the oral cavity. The study sample comprised two males and four females, aged 54-79 years (median 62 years). The lesions were localized on gingival/alveolar mucosa (four cases), hard palate, and floor of the mouth, measuring 17-40 mm (median 31 mm). The duration of the lesions ranged from 3 months to several years. Information on IgG4 serum levels was available for two patients, and these were increased to 1.85 and 1.65 g/L, respectively. The follow-up period ranged 11-30 months (median 13 months). None of the lesions recurred, and none of the patients developed any manifestation of IgG4-RD. Microscopically, all cases presented as nodular lesions composed of numerous polyclonal plasma cells admixed with lymphocytes, histiocytes, mast cells, and eosinophils, set within collagenized stroma in variable proportions. Obliterative phlebitis was observed in two cases. The number of IgG4-positive plasma cells ranged between 51 and 142 per HPF (median 114), while the IgG4/IgG ratio values ranged between 0.16 and 0.72 (median 0.44) and were above 0.40 in three cases. Based on international criteria, two cases were diagnosed as definite and one as probable IgG4-RD. Oral plasma cell granuloma is a heterogeneous group of lesions, and a subset may represent a mucosal manifestation of IgG4-RD in the oral cavity.

• MeSH
• časové faktory MeSH
• diferenciální diagnóza MeSH
• eozinofily patologie   MeSH
• gingivitida diagnóza   imunologie   patologie   MeSH
• histiocyty patologie   MeSH
• imunoglobulin G metabolismus   MeSH
• lidé středního věku MeSH
• lidé MeSH
• mastocyty patologie   MeSH
• nemoci imunitního systému diagnóza   imunologie   patologie   MeSH
• nemoci úst diagnóza   imunologie   patologie   MeSH
• plazmatické buňky patologie   MeSH
• plazmocelulární granulom diagnóza   imunologie   patologie   MeSH
• senioři MeSH
• ústní sliznice imunologie   patologie   MeSH
• Check Tag
• lidé středního věku MeSH
• lidé MeSH
• mužské pohlaví MeSH
• senioři MeSH
• ženské pohlaví MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• srovnávací studie MeSH

BACKGROUND: Polycystic ovary syndrome (PCOS) is commonly associated with endocrine, metabolic, cardiovascular and other morbidities. However its association with autoimmune diseases is still controversial. AIM: The aim of this study was to assess the prevalence of non organ-specific and antithyroid, antibodies in PCOS women compared to healthy controls. METHODS: The study included 152 women with PCOS and 76 healthy controls for the evaluation of non organ-specific autoimmunity and 64 PCOS and 68 controls for the study of organ-specific autoimmunity. All sera were tested for autoantibodies.using the ELISA method. RESULTS: There were no significant differences in the prevalence of ANA, SSA, SSB, anti-dsDNA, anti-RNP, ANCA/MPO or ANCA/PR3 between PCOS and controls. The prevalence of ACLA IgG was higher in controls than PCOS (5.4% v.s. 0%, P=0.011). Patients had a higher prevalence of anti-TPO antibodies (18.75% v.s. 7.35%, P=0.045) and slightly but not significantly higher prevalence of autoimmune thyroiditis (18.75% v.s. 10.29%) than controls. CONCLUSION: The prevalence of non organ-specific autoantibodies in PCOS women is low and similar to controls. On the other hand, we found a slightly higher prevalence of thyroid autoimmunity in PCOS women.

• MeSH
• autoimunita imunologie   MeSH
• autoimunitní tyreoiditida imunologie   MeSH
• autoprotilátky metabolismus   MeSH
• dospělí MeSH
• ELISA MeSH
• imunoglobulin G metabolismus   MeSH
• lidé středního věku MeSH
• lidé MeSH
• mladiství MeSH
• mladý dospělý MeSH
• studie případů a kontrol MeSH
• syndrom polycystických ovarií imunologie   MeSH
• Check Tag
• dospělí MeSH
• lidé středního věku MeSH
• lidé MeSH
• mladiství MeSH
• mladý dospělý MeSH
• ženské pohlaví MeSH
• Publikační typ
• časopisecké články MeSH