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Wen, Dong-Mei, Sheng-Nan Xu, Wei-Jia Wang, Xiu-Ming Zhang, Ming-Huan Suo y De-Cai Zhang. "Evaluation of the Interference of Hemoglobin Variant J-Bangkok on Glycated Hemoglobin (HbA1c) Measurement by Five Different Methods". Experimental and Clinical Endocrinology & Diabetes 125, n.º 10 (20 de septiembre de 2017): 655–60. http://dx.doi.org/10.1055/s-0043-118535.
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Abstract Objective The interference of the hemoglobin variant (Hb J-Bangkok) was evaluated on 4 different glycated hemoglobin assays and compared with a reference immuno assay. Methods An overall test of coincidence of 2 least-squares linear regression lines was performed to determine whether the presence of Hb J-Bangkok caused a statistically significant difference in HbA1c results compared with a reference immuno assay. Statistical analysis was performed on the difference of the estimated average glucose calculated from HbA1c values and fasting plasma glucose in the Hb J-Bangkok variant group using the different detection systems. Deming regression analysis was used to determinate whether Hb J-Bangkok had a significant interference on HbA1c results using an HbA1c±10% relative bias at 6% and 9% HbA1c as evaluation limits. Results Turbidimetric inhibition immunoassay method, and enzymatic methods were not affected by Hb J-Bangkok. However, Hb J-Bangkok showed statistically significant interference to the two ion-exchange high-performance liquid chromatography methods. Conclusion When performing HbA1c tests, clinical laboratory personnel should identify the Hb variant and select the appropriate methods or use alternative indicators.
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Thompson, S. G., F. Duckert, F. Haverkate y J. M. Thomson. "The Measurement of Haemostatic Factors in 16 European Laboratories: Quality Assessment for the Multicentre ECAT Angina Pectoris Study". Thrombosis and Haemostasis 61, n.º 02 (1989): 301–6. http://dx.doi.org/10.1055/s-0038-1646581.
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SummaryAs part of a European multicentre prospective study involving the measurement of a number of haemostatic factors, a quality assessment (QA) scheme was organized. This paper describes the preparation, design and results of the first Qa exercise, involving 16 European laboratories and 10 haemostatic assays. The design allowed the investigation, for each assay, of the variability between duplicates and the variability between days within each centre, and of the agreement between centres. A graphical presentation of each centre’s performance in comparison to that of others was adopted, which preserved the confidentiality of each centre’s results. The factor VIII clotting activity assay (VIII: C) and the rocket immuno-electrophoresis assays of von Willebrand factor related antigen (vWF R:Ag), antithrombin III, protein C and histidine-rich glycoprotein showed the highest betweenduplicate and between-day coefficients of variation (CVs), whereas the clotting assays of activated partial thromboplastin time and fibrinogen had the lowest CVs. CVs for the enzymatic assays using synthetic substrates of antithrombin III, plasminogen and alpha-2-antiplasmin were between these extremes. The between-centre CVs were high for both the VIII:C and vWFR:Ag assays. The QA exercise showed that, in multicentre studies involving the measurement of haemostatic factors, it is feasible to undertake analysis locally at each centre.
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Koua, N’Zi Daniel, Joël Henry, Erwan Corre, Julien Pontin, Benoît Bernay y Jésus Nunez. "Immuno-Enzymatic and Proteomic Approaches for Sexing the African Bonytongue (Heterotis niloticus Cuvier, 1829)". Fishes 7, n.º 3 (6 de mayo de 2022): 106. http://dx.doi.org/10.3390/fishes7030106.
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Heterotis niloticus is an African species of Osteoglossiformes that presents biological peculiarities and zootechnical performances favorable for fish farming. However, the absence of a sexual dimorphism hinders the optimization of its reproduction in captivity and limits the understanding of its reproductive behavior. This study is aimed at developing a minimally invasive and reliable sexing method to detect vitellogenin (Vtg) in female plasma. A commercial sexing kit (Acobium, Montpellier, France) for Arapaima gigas—a phylogenetically sister species of H. niloticus—successfully identified only 20% of mature H. niloticus females. Enzyme-linked immunosorbent assays (ELISA) were carried out using three Vtg antibodies. The A. gigas Vtg1 antibody cross-reacted significantly with plasma dilutions of female H. niloticus ranging from 1:1000 to 1:10,000, but with relatively low intensity. The Vtg antibody from Osteoglossum bicirrhosum, another species of Osteoglossiformes, showed non-specific binding with the Vtg of H. niloticus female plasma. Finally, an antibody for H. niloticus Vtg developed in this study allowed us to differentiate the two sexes with plasma coating dilutions ranging from 1:1000 to 1:10,000. The results of the assay were validated by a proteomic approach showing that Vtg-targeted mass spectrometry analysis of H. niloticus blood protein extracts could be used to accurately determine the presence of Vtg in the plasma of mature females. The final validation of the ELISA technique using the H. niloticus Vtg antibody was confirmed by visual sexing of a significant number of blood-sampled fish gonads; 100% of the fish were correctly sexed by the ELISA method.
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Delatour, Vincent, Noémie Clouet-Foraison, Stéphane Jaisson, Patricia Kaiser y Philippe Gillery. "Trueness assessment of HbA1c routine assays: are processed EQA materials up to the job?" Clinical Chemistry and Laboratory Medicine (CCLM) 57, n.º 10 (25 de septiembre de 2019): 1623–31. http://dx.doi.org/10.1515/cclm-2019-0219.
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Abstract Background With the worldwide increase of diabetes mellitus prevalence, ensuring that HbA1c assays are accurate is essential. External quality assessment (EQA) programs enable laboratories to verify that analytical methods perform according to the manufacturers’ specifications. However, assessing trueness requires commutable materials, a property that is rarely characterized for EQA materials. Methods The difference in bias approach was used to assess commutability of 26 processed quality control materials for 17 of the most frequently used HbA1c assays. Involved assays included immuno-assays, enzymatic assays, affinity, ion-exchange HPLC boronate affinity HPLC and capillary electrophoresis. The measurements were performed at manufacturers or expert laboratories. Assay trueness was additionally assessed against the IFCC reference measurement procedure using fresh clinical specimens that were distributed to 450 medical laboratories. Results Commutability of processed EQA materials was highly heterogeneous and globally insufficient to rigorously assess the trueness of HbA1c assays. Using fresh clinical specimens, mean bias was −0.13 mmol/mol for low HbA1c (34 mmol/mol), between +1.0 and +1.3 mmol/mol for intermediate HbA1c (49 and 58 mmol/mol) and +1.2 mmol/mol for elevated HbA1c (90 mmol/mol). Conclusions This study demonstrates that due to insufficient commutability, most processed EQA materials are unsuitable to assess trueness of HbA1c assays and agreement between the different assays. These materials can only provide information on comparability of individual laboratory results with its peers and on assay precision. Using fresh whole blood samples, this study additionally shows that most HbA1c assays are fairly accurate and meet the total allowable error quality target of 5 mmol/mol.
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Santos, Fred Luciano Neves, Paola Alejandra Fiorani Celedon, Nilson Ivo Tonin Zanchin, Amanda Leitolis, Sandra Crestani, Leonardo Foti, Wayner Vieira de Souza, Yara de Miranda Gomes y Marco Aurélio Krieger. "Performance Assessment of a Trypanosoma cruzi Chimeric Antigen in Multiplex Liquid Microarray Assays". Journal of Clinical Microbiology 55, n.º 10 (19 de julio de 2017): 2934–45. http://dx.doi.org/10.1128/jcm.00851-17.
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ABSTRACT Diagnosing chronic Chagas disease (CD) requires antibody–antigen detection methods, which are traditionally based on enzymatic assay techniques whose performance depend on the type and quality of antigen used. Previously, 4 recombinant chimeric proteins from the Instituto de Biologia Molecular do Paraná (IBMP-8.1 to 8.4) comprising immuno-dominant regions of diverse Trypanosoma cruzi antigens showed excellent diagnostic performance in enzyme-linked immunosorbent assays. Considering that next-generation platforms offer improved CD diagnostic accuracy with different T. cruzi -specific recombinant antigens, we assessed the performance of these chimeras in liquid microarrays (LMAs). The chimeric proteins were expressed in Escherichia coli and purified by chromatography. Sera from 653 chagasic and 680 healthy individuals were used to assess the performance of these chimeras in detecting specific anti- T. cruzi antibodies. Accuracies ranged from 98.1 to 99.3%, and diagnostic odds ratio values were 3,548 for IBMP-8.3, 4,826 for IBMP-8.1, 7,882 for IBMP-8.2, and 25,000 for IBMP-8.4. A separate sera bank (851 samples) was employed to assess cross-reactivity with other tropical diseases. Leishmania , a pathogen with high similarity to T. cruzi , showed cross-reactivity rates ranging from 0 to 2.17%. Inconclusive results were negligible (0 to 0.71%). Bland–Altman and Deming regression analysis based on 200 randomly selected CD-positive and negative samples demonstrated interchangeability with respect to CD diagnostic performance in both singleplex and multiplex assays. Our results suggested that these chimeras can potentially replace antigens currently used in commercially available assay kits. Moreover, the use of multiplex platforms, such as LMA assays employing 2 or more IBMP antigens, would abrogate the need for 2 different testing techniques when diagnosing CD.
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Cavuslu, Saban, William G. Starkey, Barbara Kell, Jennifer M. Best y John Cason. "Detection of human papillomavirus type 16 in microtitre plate based immuno-enzymatic assays: use to determine E5 gene expression in cervical carcinomas". Clinical and Diagnostic Virology 5, n.º 2-3 (mayo de 1996): 215–18. http://dx.doi.org/10.1016/0928-0197(96)00225-5.
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Kuzan, Aleksandra, Emilia Królewicz, Karolina Nowakowska, Kamilla Stach, Krzysztof Kaliszewski, Paweł Domosławski, Łukasz Kotyra, Andrzej Gamian y Irena Kustrzeba-Wójcicka. "Contribution of Glycation and Oxidative Stress to Thyroid Gland Pathology—A Pilot Study". Biomolecules 11, n.º 4 (10 de abril de 2021): 557. http://dx.doi.org/10.3390/biom11040557.
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The patho-mechanism of changes in the thyroid gland, including carcinogenesis, is a complex process, which involves oxidative stress. The goal of our investigation was to verify the extent of stress in the thyroid gland related to glycation. The study samples were comprised of blood sera, thyroid, and adipose tissue sections probed from 37 patients diagnosed with thyroid cancers and goiter. Using immuno-enzymatic and fluorometric assays we analyzed the content of advanced glycation end-products (AGEs), pentosidine, receptors for advanced glycation end-products (RAGE), scavenger receptor class (SR)-A, SR-B, glutathione, malondialdehyde and nitric oxide synthase. In addition to classic AGEs, a recent study detected the melibiose-derived glycation (MAGE) product. We demonstrated the presence of AGEs, MAGE and their receptors of the RAGE and SR-A. In addition, in the control samples of thyroid glands SR-B groups were detected as well as of pathological groups without noticeable tendency to antigen concentration in the area of carcinogenesis. Fluorescent AGEs correlate positively with glutathione, which supports the assumption that glycation stress leads to augmentation of oxidative stress and increase of the intensity of antioxidant mechanisms.
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Ali Khan, Mohd Wajid. "Glycation end-products specific auto-antibodies in Systemic Lupus Erythematosus". Bioinformation 18, n.º 3 (31 de marzo de 2022): 127–33. http://dx.doi.org/10.6026/97320630018127.
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Systemic lupus erythematosus (SLE) is an autoimmune disease, which is highly inflammatory. Compared to a healthy control group, SLE patients exhibit a higher concentration of advanced glycation end products (AGEs) and a lower concentration of receptors for AGEs (RAGE) in serum, however, the exact aetiology is still unclear. In the present study, non-enzymatic glycation induced modification of human serum albumin (HSA) has been studied by biophysical techniques. Glycated HSA (G-HSA) was used as an antigen, and serum autoantibody levels were estimated in SLE and normal humans (NH) against it, using direct binding ELISA and competitive inhibition ELISA. Compared to N-HSA, remarkable structural modifications were observed in G-HSA. Modified HSA also showed increased pentosidine fluorescence (213.7 ± 13.4 AU). Glycation of HSA induced a conversion of α-helix and random coil to β-sheet and β-turns. Serum immuno assays results exhibited significantly (p < 0.001) higher binding of G-HSA with serum autoantibodies from SLE patients when compared with native HSA (N-HSA). Furthermore, competitive ELISA results showed significantly (p < 0.001) high percent inhibition of serum IgG from SLE patients with modified antigen. Chronic inflammation with excessive oxidative stress in SLE patients could be a possible reason for structural alterations in blood proteins, generating highly immunogenic unique new-epitopes. These in turn induce the generation of specific autoantibodies against G-HSA that may serve as a potential biomarker for SLE pathogenesis.
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Owen, Valerie M. "Uruguay - immuno-lectin-enzymatic assay". Biosensors and Bioelectronics 11, n.º 8 (enero de 1996): xxii. http://dx.doi.org/10.1016/0956-5663(96)85952-5.
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Cull, Alyssa, Brooke Snetsinger, Iqra Mumal, Flora Shan, David Good y Michael J. Rauh. "Increased Arginase 1 Expression In Human MDS, CMML and Murine Models Points To Dysregulation Of Common Immunosuppressive Signaling Networks". Blood 122, n.º 21 (15 de noviembre de 2013): 1578. http://dx.doi.org/10.1182/blood.v122.21.1578.1578.
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Abstract Introduction Our group and others have reported the murine SHIP1-deficient mouse model of MDS/CMML is characterized by expansion of immunosuppressive, arginase 1 (Arg1)-positive M2-macrophages and myeloid-derived suppressor cells (MDSC) (Rauh et al., Immunity, 2005). Moreover, translational studies confirmed increased Arg1 in bone marrow aspirate cells of subsets of MDS and CMML patients (Rauh et al., Blood Abstract 2010: 1855), although involving impractical Arg1 enzymatic assays and Western blots. Herein, our goals were to 1) confirm this Arg1 immune signature in an independent cohort of patients, using more clinically applicable immunohistochemistry (IHC), 2) determine the associated risk profile with recent prognostic scoring systems, and 3) begin to connect this Arg1 immune signature with recurring MDS/CMML-acquired mutations. Methods With ethics approval, 29 BM biopsies (decalcified, FFPE) and clinical parameters were retrieved from the archives of Kingston General Hospital: 6 controls (4 negative lymphoma staging BM and 2 mild anemia NYD; mean age +/- std = 57 +/- 13 y), 13 MDS (1 RA, 1 RARS, 1 del(5q), 5 RCMD, 3 RAEB-1, 2 RAEB-2; 76 +/- 14 y), and 10 CMML patients (7 CMML-1, 3 CMML-2; 72 +/- 10 y). H&E and anti-human Arg1 IHC (1/2500 dilution, clone HPA003595, Sigma) were conducted under optimized, automated conditions (Ventana). IHC scoring was recorded independently by 2 blinded Hematopathologists. IPSS, IPSS-R (Greenberg), WPSS (Malcovati), CMML PSS (Such) and Mayo CMML (Patnaik) scores were calculated. Floxed TET2 and Vav-Cre mice were obtained from JAX and used according to approved Queen's University Animal Care protocols. TET2 sequences were obtained from genomic DNA using custom AmpliSeq primer pools and the Ion Torrent PGM platform (LifeTech). Linear regression and student's t-tests were conducted with Prism software (GraphPad). Results 1) We demonstrated increased BM biopsy Arg1 IHC expression in CMML (22 +/- 17% Arg1-positive cells; n = 10; p = 0.0093) and low-grade MDS, particularly RCMD (14 +/- 11%; n = 5; p = 0.012) relative to control subjects (0.4 +/- 1%; n = 6) (Figure 1). Significantly increased mean Arg1 expression was not seen in other MDS subtypes (n = 8). These findings were consistent with our previously reported 40-subject (Toronto) BM aspirate Arg1 assay/Western blot cohort (Rauh et al. Blood Abstract 2010:1855), suggesting the reproducibility of these findings and the clinical utility of IHC-based assessment. Parallel Arg1 IHC is underway on the Toronto cohort, to determine correlations with enzymatic assays/Westerns. 2) We previously reported Arg1 over-expression in the Toronto cohort was significantly associated with the lowest IPSS and WPSS MDS clinical risk categories and now extend this to IPSS-R. In our Kingston cohort, only trends to lower MDS risk were observed. In contrast, increased Arg1 expression was not associated with clinical risk (CPSS and Mayo scores) in either CMML cohort. Thus, Arg1 expression associated with neutral risk in CMML and neutral to lower risk in MDS patients. Both the clinical risk profiles and proportions of Arg1 over-expressing MDS/CMML patients were reminiscent of reported TET2 mutation risk profiles/percentages. 3) TET2-deficient mice demonstrated increased monocytes, macrophages and CD11b+Gr1+ splenocytes (immuno-phenotypically consistent with MDSC), phenocopying SHIP1-deficient mice. We are currently determining whether TET2-/- mouse macrophages are similarly M2-skewed. In parallel, were are obtaining TET2 genomic sequences (along with other recurring mutated genes) for our Toronto and Kingston cohorts, in order to determine if the Arg1 immune signature associates with a particular mutation(s). These findings will be discussed. Conclusions 1) Using two independent patient cohorts, we demonstrated significant Arg1 over-expression in CMML and low-grade (RCMD) MDS. Arg1 IHC warrants further investigation as an ancillary diagnostic test. 2) Increased Arg1 expression had neutral prognostic significance in CMML and neutral to low-risk MDS associations. 3) Increased Arg1 expression in MDS/CMML may be driven by mutant TET2, impacting the epigenetics of MDSC and M2-macrophage expansion, controlled by SHIP1 and related signaling networks. Confirmatory studies are in progress. Disclosures: No relevant conflicts of interest to declare.
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Francina, A., H. Cloppet, R. Guinet, M. Rossi, D. Guyotat, O. Gentilhomme y M. Richard. "A rapid and sensitive non-competitive avidin-biotin immuno-enzymatic assay for lysozyme". Journal of Immunological Methods 87, n.º 2 (marzo de 1986): 267–72. http://dx.doi.org/10.1016/0022-1759(86)90541-7.
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Ogawa, Haruko y Uri Galili. "Profiling terminal N-acetyllactosamines of glycans on mammalian cells by an immuno-enzymatic assay". Glycoconjugate Journal 23, n.º 9 (18 de noviembre de 2006): 663–74. http://dx.doi.org/10.1007/s10719-006-9005-0.
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Wang, Yan, Guang He Wu, Wei Sheng, Yan Zhang, Meng Yuan y Shuo Wang. "Rapid Determination of Fumonisin B1 in Food Samples by a Clean-Up Tandem Immunoassay Column". Advanced Materials Research 488-489 (marzo de 2012): 1568–73. http://dx.doi.org/10.4028/www.scientific.net/amr.488-489.1568.
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An immunochemistry-based assay for non-instrumental simultaneous detection of fumonisins in food was developed. The method was based upon the direct competitive immuno-reaction and the horse radish peroxidase enzymatic reaction. The assay was developed to show a visual detection result, according to a yes/no response to the LOD of fumonisins. The limit of detection (LOD) was 40 μg L-1. The assay could be accomplished within 15 min in all and 4 min for chromogenic substrate application. The fumonisin contaminations in different kinds of food were analyzed by the proposed method and the results were confirmed by ELISA. Avoiding time-consuming reaction steps and complicated pre-treatment procedures, this assay was demonstrated as a promising tool for on-site sample detections.
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Israels, Sara J. y Eileen M. McMillan-Ward. "Platelet Tetraspanin Complexes and Their Relation to Lipid Rafts." Blood 108, n.º 11 (16 de noviembre de 2006): 1530. http://dx.doi.org/10.1182/blood.v108.11.1530.1530.
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Abstract CD63 and CD9 are members of the tetraspanin superfamily of integral membrane proteins that function as organizers of multi-molecular signaling complexes involved in cell morphology, motility and proliferation. CD63 is located in the membranes of lysosomes and dense granules in resting platelets. Following platelet activation and granule exocytosis, CD63 is expressed on the platelet plasma membrane and co-localizes with the αIIbβ3-CD9 complex. D545, a monoclonal antibody (MoAb) directed at the second extracellular loop of CD63, inhibits activated platelet spreading on immobilized fibrinogen and FAK phosphorylation in the adherent platelets. To identify CD63-associated signaling enzymes that could be involved in the signaling complex, lipid kinase assays were performed on D545 immunoprecipitates. CD63 co-immunoprecipitated with a lipid kinase with the enzymatic properties of PI4-kinase type II, confirmed by re-precipitation and immunoblotting with 4C5G (MoAb specific for the 55kDa PI4-kinase, PI4K55). The CD63-PI4K55 complex could be co-precipitated from both resting and activated platelets using anti-CD63 MoAb, and co-localized on the filopodia of thrombin-activated platelets using immuno-electron microscopy. Previous studies have demonstrated that tetraspanins associate with cholesterol-enriched membrane domains in a variety of cells including platelets. There is evidence, however, that these tetraspanin-enriched microdomains (TEMs) can be distinguished from prototypic lipid rafts on the basis of detergent solubility and protein composition. To investigate the association of the CD63-PI4K55 complex with lipid rafts in platelets, resting and thrombin-activated platelets were lysed in buffer containing either 1% Brij 35, or Triton X-100, the low- and high-density membrane fractions separated by isopycnic sucrose gradient centrifugation, and the identification of the low-density membrane fractions (LDMF) confirmed by the presence of LAT. CD63, CD9 and PI4K55 were present in the LDMF of platelets lysed in Brij 35 but not in Triton X-100; they were also present in the denser membrane fractions. CD63 and CD9 associated with cholesterol, as demonstrated by recovery of these proteins in the pellet following centrifugation of platelets lysed with 1% digitonin(a cholesterol-precipitating reagent), but not from lysates made with Brij 35/Triton X-100. Incubation of platelets with methyl-β-cyclodextrin(mβCD) to partially deplete cholesterol and disrupt the lipid rafts shifted LAT, CD63, CD9 and PI4K55 to denser fractions within the gradient. Immunoprecipitation of mβCD-treated platelets with anti-PI4K55 MoAb co-precipitated CD63 and CD9, demonstrating that the complexes were not dependent on residence within LDMFs, but remained intact in the denser fractions and pellet. Platelet tetraspanin complexes associate with cholesterol-enriched domains under conditions of mild detergent extraction. The maintenance of the complexes, however, was not dependent on their residence within lipid rafts, as the complexes remained intact following cholesterol depletion. Their presence in LDMF suggests that tetraspanin complexes may associate with platelet lipid rafts under some conditions, which could bring tetraspanin protein partners into proximity with raft residents, and facilitate the assembly and interaction of signaling complexes following platelet activation.
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Mehena, A. A., M. Wishahi, S. Hassan y N. El-Sayed. "MP-04.02: Enzymatic immuno-assay quantification of C-erbB-2 oncogene expression in urinary bladder cancer". Urology 70, n.º 3 (septiembre de 2007): 58. http://dx.doi.org/10.1016/j.urology.2007.06.228.
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Saputra, Elizabeth, Garrett Cornelison, Jennifer Mitchell, Karia Williams, Andrea Mendiola, Rachael Orlandella, John Majercak, Joseph Dekker, Chris Moore y Swati Khanna. "884 Engineered toxin body mediated depletion of TIGIT expressing immune cells for cancer immunotherapy". Journal for ImmunoTherapy of Cancer 9, Suppl 2 (noviembre de 2021): A926. http://dx.doi.org/10.1136/jitc-2021-sitc2021.884.
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BackgroundTIGIT (T cell immunoreceptor with Ig and ITIM domains) is an exciting novel target for immuno-oncology which functions as an immune checkpoint on multiple immune cell types including memory CD8+, CD4+ Treg, and memory CD4+ cells. TIGIT upregulation on tumor infiltrating lymphocytes (TILs) has been observed in multiple cancer types and contributes to an immunosuppressive tumor microenvironment (TME). Interestingly, TIGIT is commonly co-expressed with PD-1 on Tregs in the TME, tumor antigen specific CD8+ T cells and CD8+ TILs, leading to weakened anti-tumor immune responses.1–2 To date, TIGIT inhibiting monoclonal antibodies (mAb) have shown little activity as a monotherapy in clinical and preclinical studies. 3–4 Therefore, current clinical trials are now focused on combining TIGIT mAbs with known commercial PD-1 or PD-L1 mAbs. A TIGIT-specific engineered toxin body (ETB) represents a wholly new approach to targeting TIGIT expressing cells including those co-expressing TIGIT and PD-1.MethodsETBs targeting TIGIT were designed to deplete TIGIT-expressing TILs, including Tregs, directly in the TME. ETBs are proteins that consist of an antibody fragment genetically fused to a proprietary de-immunized (DI) form of the Shiga-like toxin A subunit (SLTA). These proteins are specific for a cell surface receptor, and function through triggering rapid internalization upon binding, followed by an enzymatic and irreversible termination of ribosomal protein synthesis resulting in cellular apoptosis. Here we provide proof of concept for ETBs as a novel modality for the depletion of TIGIT-expressing immune cells.ResultsTIGIT-targeting ETBs exhibit potent in vitro cytotoxicity of TIGIT over-expressing cell lines (IC50<1nM). These ETBs also lead to apoptotic depletion of ex vivo TIGIT-expressing regulatory T cells (Tregs) from healthy donors. In mixed culture assays, TIGIT ETBs increase the proliferation of TIGIT negative T cells by depleting TIGIT-expressing T cells.ConclusionsStudies to assess pharmacodynamics and efficacy of TIGIT targeting ETBs using a double knock-in (TIGIT and PD-1) mouse tumor model are ongoing, but these early proof of concept in vitro data support the hypothesis that ETBs can deplete TIGIT positive immune cell populations including those co-expressing PD-1. It is possible that targeted TIGIT inhibition through ETB-induced cell death could tip the balance towards tumor regression by eliminating this novel checkpoint (and TIGIT/PD-1 co-expression) at the level of the TME.ReferencesJinhua X, Ji W, Shouliang C, Liangfeng Z. Expression of immune checkpoints in T cells of esophageal cancer patients. Oncotarget 2016;7(39):1–10.Blessin NC, Simon R, Kluth M, Fischer K, et al. Patterns of TIGIT expression in lymphatic tissue, inflammation and cancer. Dis Markers 2019;2019:1–13.Johnston RJ, Comps-Agrar L, Hackney J, Yu X, et al. The immunoreceptor TIGIT regulates anti-tumor and antiviral CD8(+) T effector function. Cancer Cell 2014;26(6):923–927.Bendell JC, Bedrad P, Bang Y-J, LoRusso P, et al. Phase Ia/Ib dose-escalation study of the anti-TIGIT antibody Tiragolumab as a single agent and in combination with atezolizumab in patients with advanced solid tumors. Proceedings: AACR Annual Meeting 2020; April 27–28, 2020 and June 22–24, 2020; Philadelphia, PA.
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Cairoli, E., M. Varangot, S. Signorelli, C. Batthyany, S. Bianchi, M. Beuzelin, E. Phillips, I. Musé, A. Roseto y E. Osinaga. "1274 Detection of soluble human carcinoma-associated TN-glycoproteins by a new immuno-lectin-enzymatic assay (CA83.4)". European Journal of Cancer 31 (noviembre de 1995): S265. http://dx.doi.org/10.1016/0959-8049(95)96520-n.
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Muratori, Luigi, Gaia Deleonardi, Claudine Lalanne, Erica Barbato, Alessandra Tovoli, Alessia Libra, Marco Lenzi, Fabio Cassani y Paolo Muratori. "Autoantibodies in Autoimmune Hepatitis". Digestive Diseases 33, Suppl. 2 (2015): 65–69. http://dx.doi.org/10.1159/000440748.
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Background: The detection of diagnostic autoantibodies such as antinuclear antibodies (ANA), anti-smooth muscle antibodies (SMA), anti-liver/kidney microsomal type 1 (anti-LKM1), anti-liver cytosol type 1 (anti-LC1) and anti-soluble liver antigen (anti-SLA) is historically associated with the diagnosis of autoimmune hepatitis. Key Messages: When autoimmune hepatitis is suspected, the detection of one or any combination of diagnostic autoantibodies, by indirect immunofluorescence or immuno-enzymatic techniques with recombinant antigens, is a pivotal step to reach a diagnostic score of probable or definite autoimmune hepatitis. Conclusions: Diagnostic autoantibodies (ANA, SMA, anti-LKM1, anti-LC1, anti-SLA) are a cornerstone in the diagnosis of autoimmune hepatitis. Other ancillary autoantibodies, associated with peculiar clinical correlations, appear to be assay-dependent and institution-specific, and validation studies are needed.
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Prévot, J., S. Dubrou y J. Maréchal. "Detection of Human Hepatitis a Virus in Environmental Water by an Antigen-Capture Polymerase Chain Reaction Method". Water Science and Technology 27, n.º 3-4 (1 de febrero de 1993): 227–33. http://dx.doi.org/10.2166/wst.1993.0351.
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To detect hepatitis A virus (HAV) in environmental water samples, sensitive and specific methods are needed. An hemi-nested enzymatic amplification procedure was developed. When it was associated with an immuno-capture (IC) step, specificity and sensitivity were increased. The presence of a specific DNA fragment of 318 bp was detected by the analysis of the electrophoresis of the PGR products and confirmed on the autoradiogram of the Southern blot of the gel, using a labeled oligoprobe PAl selected in a very highly conserved region of the genome coding for the capsid protein VPl. The IC/PCR detected less than one infectious unit of virus in 75 µ1 sample. Eleven Seine River samples and two drinking water specimens were monitored by this assay. The IC/PCR brings a significant contribution for increasing our knowledge of the incidence of HAV on public health.
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RAMIREZ, DARIO C., SANDRA E. GOMEZ-MEJIBA, JEAN T. CORBETT y RONALD P. MASON. "Novel immuno-spin trapping-based assay for the analysis of myeloperoxidase (100.13)". Journal of Immunology 178, n.º 1_Supplement (1 de abril de 2007): S199. http://dx.doi.org/10.4049/jimmunol.178.supp.100.13.
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Abstract Myeloperoxidase (MPO) is an important biomarker of inflammatory diseases. The analyses of MPO in biological samples are based on detection of its peroxidase activity, chloramine formation, and MPO-capture immunoassays. We have developed an immuno-spin trapping (IST)-based approach to determine MPO in biological samples. In this approach, MPO/H2O2/Cl system produces hypochlorous acid (HOCl);HOCl reacts with proteins forming chloramines;chloramines decay forming protein radicals,protein radicals are trapped by the nitrone spin trap 5,5-dimethyl-1-pyrroline N-oxide (DMPO) forming DMPO-protein nitrone adducts, andnitrone adducts are determined using a chemiluminescent direct ELISA with the anti-DMPO antiserum. We used this assay to quantify MPO in plasma, bronchoalveolar fluid and lung parenchyma of rats exposed to lipopolysaccharide, and compared its sensitivity with a capture-based commercial ELISA kit. Hemoglobin does not interfere and the assay can be used in animal and human samples. The assay has three levels of amplification: enzymatic formation of HOCl;many DMPO molecules bound per single protein molecule;a sensitive developing system. The IST-based assay for quantification of MPO will enhance the power of this biomarker for early detection of environmental- and metabolic-induced inflammatory disorders in both experimental and clinical settings. NIH#1K99 ES015414-01
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Seldimirova, O. A. y I. R. Galin. "INFLUENCE OF FLURIDONE ON THE CONTENT OF HORMONES IN CALL OF BARLEY CV. STEPTOE AND TS ABA‑DEFICIENT MUTANT AZ34". ÈKOBIOTEH 4, n.º 1 (2021): 41–49. http://dx.doi.org/10.31163/2618-964x-2021-4-1-41-49.
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The effect of the inhibitor of endogenous ABA synthesis fluridone on the content and distribution of endogenous ABA and IAA in the calli of ABA-deficient mutant AZ34 barley and its parental cultivar Steptoe was studied using the methods of immuno-enzymatic solid-phase assay and immunolocalization of phytohormones. It was found that by the 4th week of in vitro culture, fluridone causes a significant decrease in the ABA level in calli of both genotypes compared to the control, and the inhibitory effect of fluridone in AZ34 is more pronounced than in Steptoe. In the calli of both genotypes, a significant increase in the IAA content was revealed against the background of a decrease in the ABA content upon treatment with fluridone as compared to the control. It was concluded that ABA plays an important role in the process of embryoido-genesis in vitro.
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Arteaga, Maria Francisca, Jan-Henrik Mikesch y Chi Wai Eric So. "Discovery of Critical Functions of Histone Demethylase, PHF8, in Mediating ATRA Response in APL". Blood 118, n.º 21 (18 de noviembre de 2011): 226. http://dx.doi.org/10.1182/blood.v118.21.226.226.
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Abstract Abstract 226 Introduction: Transcription deregulation plays a key role in acute leukemogenesis, which is mostly initiated by chimeric transcription factors such as PML–RARa that accounts for almost 100% of acute promyelocytic leukemia (APL). While APL is uniquely sensitive to all trans retinoic acid (ATRA) treatment and has been the paradigm of differentiation/epigenetic therapies, the underlying mechanisms remain largely unknown and are major interests in the field with a potential of extending the success to other hematological malignancies. Previously, others and we have shown that aberrant recruitment of histone deacetylases and polycomb repressive complexes by oligomeric PML-RARa are key for suppression of its downstream targets and promote cellular transformation. ATRA treatment disrupts their bindings and results in de-repression of target genes. However, the identification of the co-activator complex responsible for gene activation upon ATRA treatment remains a major hurdle that significantly hinders the progress of understanding mechanisms underlying the epigenetic therapy. Results and Discussion: Given the critical functions of JmjC-domains containing histone demethylases in mediating transcriptional regulation, we performed a systematic screening for differential interaction between JmjC-histone demethylases and PML–RARa upon ATRA treatment. We identified a highly specific interaction between PML–RARa and Plant Homeodomain Finger 8 (PHF8). To assess its effect on histone methylation, we detected specific reduction of H3K9me2 and enhancement of H3K4me3 in PHF8 transfected cells, consistent with its function as a transcriptional activator. This was further supported by results from chromatin immuno-precipitation (ChIP) assays in human APL cell line (NB4) showing that PHF8 differentially bound and mediated the same histone modifications in RARb promoter and activated its expression. To investigate the functional significance of this interaction, PHF8 was expressed in human NB4 cells harboring PML-RARa or murine primary bone marrow cells transformed by APL fusion proteins. Induction of PHF8 expression significantly decreased their in vitro transformation capacity in the presence of physiological concentrations of ATRA. Conversely, specific down-modulation of PHF8 expression by shRNAs reduced ATRA sensitivity of these cells, suggesting a critical function of PHF8 in mediating ATRA response. We hypothesized that PHF8 may be able to sensitize ATRA resistant cells to the treatment. Hence, we induced expression of PHF8 in the ATRA resistant variant of NB4 line, NB4-MR2. We were able to demonstrate that PHF8 sensitized NB4-MR2 cells to ATRA treatment in vitro. In contrast, NB4-MR2 cells expressing PHF8-F279S, a catalytically inactive mutant could not be sensitized to ATRA treatment, indicating that the enzymatic activity is critical for mediating the ATRA response. Most importantly, NB4-MR2 cells expressing wild type PHF8 were also sensitive to ATRA treatment in vivo and failed to induce leukemia in NOD/SCID mice, which would otherwise succumb to leukemia in a very brief latency. To gain further insights into the molecular regulation of PHF8 in ATRA response, we characterized the potential functions of CDK1-mediated phosphorylation of PHF8. It is known that in ATRA treated leukemic cells Cyclin A translocates into the nucleus where it interacts with CDK1. Activated CDK1 induces phosphorylation of PHF8 at two serine (S33)/ threonine (T84) phosphorylation sites. Our results showed that PHF8 in its phosphorylated form had a much higher binding affinity to PML-RARa. Consistently, ChIP analyses revealed that the binding of PHF8 to the RARb promoter and the resultant activation were significantly augmented when PHF8 was constitutively phosphorylated. Moreover, inhibition of PHF8 dephosphorylation by Okadaic Acid, sensitized NB4-MR2 cells to ATRA treatment. Conclusions: We discovered a novel function of the histone demethylase PHF8 in mediating therapeutic response in both ATRA sensitive and ATRA resistant cells. This function of PHF8 is critically dependent on two phosphorylation sites as well as its histone modification activity. Thus, therapeutic interventions such as phosphatase inhibitors that enhance the PHF8 activity might become useful tools for development of new epigenetic therapies sensitizing leukemic cells to ATRA treatment. Disclosures: No relevant conflicts of interest to declare.
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Iacomelli, Iacopo, Giuseppina Barberio, Piero Pucci, Vittoria Monaco, Massimo Maffei, Massimo Mogni, Cristina Curcio et al. "Hemoglobin Yamagata [β132(H10)Lys→Asn; (HBB: c.399A>T)]: a mosaic to be put together". Clinical Chemistry and Laboratory Medicine (CCLM) 59, n.º 10 (23 de abril de 2021): 1670–79. http://dx.doi.org/10.1515/cclm-2021-0376.
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Abstract Objectives Artifactually altered glycated hemoglobin (HbA1c) concentrations are frequently linked to hemoglobin (Hb) variants. Their expression and detection require in-depth analysis. Methods Cation exchange high performance liquid chromatography (HPLC) (Bio-Rad Variant™ II; Trinity Biotech Premier Hb9210 Resolution), capillary electrophoresis (CE) (Sebia Capillarys 2 Flex Piercing) and mass spectrometry (MS) (Waters) were used for variant detection; Sanger sequencing, multiplex ligation-dependent probe amplification (MLPA) and next generation sequencing (NGS) were used for DNA analysis; HbA1c was measured with cation exchange HPLC (Bio-Rad Variant™ II; Arkray Adams HA-8180V; Tosoh HLC-723 G7), CE (Sebia Capillarys 2 Flex Piercing), boronate affinity HPLC (Trinity Biotech Hb9210 Premier), immunoassay (Cobas c501 Tina-quant HbA1c Gen. 3; Nihon Kohden CHM-4100 Celltac chemi HbA1c HA-411V) and enzymatic assay (Abbott Architect c 8000 HbA1c). Results Hb Yamagata [β132(H10)Lys→Asn; (HBB: c.399A>T)] was identified in the proband by MS after the observation of an abnormal peak in HPLC and CE. A mosaic expression of this variant was detected by NGS (mutant: 8%; wild type: 92%), after negative results in Sanger sequencing. Hb Yamagata interfered with HbA1c measurements by cation exchange HPLC and CE whereas immuno and enzymatic assay values showed good agreement with boronate affinity HPLC measurement. Conclusions A mosaicism of Hb Yamagata was found in a patient with altered HbA1c values. This rare gene variant was detected only by advanced technologies as MS and NGS. The variant interfered with common HbA1c determination methods.
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Kalifa, Sonia Ben Hadj, Ferid Limam, M. Issam Smaali, Thierry Maugard y M. Nejib Marzouki. "β-glucosidase from Sclerotinia sclerotiorum: a new and efficient purification procedure and use as a suitable marker in immuno-enzymatic assay". World Journal of Microbiology and Biotechnology 23, n.º 10 (3 de abril de 2007): 1363–70. http://dx.doi.org/10.1007/s11274-007-9374-y.
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Liu, Shuwen, Louise Boyer-Chatenet, Hong Lu y Shibo Jiang. "Rapid and Automated Fluorescence-Linked Immunosorbent Assay for High-Throughput Screening of Hiv-1 Fusion Inhibitors Targeting gp41". Journal of Biomolecular Screening 8, n.º 6 (diciembre de 2003): 685–93. http://dx.doi.org/10.1177/1087057103259155.
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The human immuno deficiency virus type 1 (HIV-1) envelope glycoprotein gp41 plays animportant role in the virus entry. During the process of fusion between the viral and target cell membranes, the N-and C-terminal heptad repeat (HR) regions of the gp41 extracellular domain associate to form a 6-helical bundle, corresponding to the fusion-active gp41 core. Any compound that blocks the gp41 6-helix bundle formation between the N- and C-peptides, which are derived from the N- and C-terminal HR regions, respectively, may inhibit HIV-1 mediated membrane fusion. Based on this principle, we previously established a sandwich enzyme-linked immunosorbent assay (ELISA) for drug screening by using the N-peptide N36 and the C-peptide C34 and a monoclonal antibody (NC-1) which specifically recognizes the gp41 6-helix bundle. In the present study, a fluorescence-linked immunosorbent assay (FLISA) was developed by using fluorescein isothiocyanate (FITC)-conjugated C34 to replace C34 and by directly detecting fluorescence intensity instead of more complicated enzymatic reaction. Compared with the sandwich ELISA, this FLISA has similar sensitivity and specificity, but it is much more rapid, economic and convenient. Using an Integrated Robotic Sample Processing System, this assay has been applied for high-throughput screening of organic compounds on a large scale for HIV-1 fusion inhibitors targeting gp41.
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CIMINO, R. O., M. MONJE RUMI, P. RAGONE, J. LAUTHIER, A. ALBERTI D'AMATO, I. R. LÓPEZ QUIROGA, J. F. GIL et al. "Immuno-enzymatic evaluation of the recombinant TSSA-II protein ofTrypanosoma cruziin dogs and human sera: a tool for epidemiological studies". Parasitology 138, n.º 8 (26 de abril de 2011): 995–1002. http://dx.doi.org/10.1017/s0031182011000540.
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SUMMARYThe rTSSA-II (recombinant Trypomastigote Small Surface II) antigen was evaluated by ELISA to detect anti-Trypanosoma cruziantibodies in sera from naturally infected dogs and humans. For this evaluation ELISA-rTSSA-II was standardized and groups were classified according to the results obtained through xenodiagnosis, ELISA and PCR. Sensitivity (Se), Specificity (Sp), Kappa index (KI) and area under curve (AUC) were determined. The Se was determined by using 14 sera from dogs infected withT. cruziVI (TcVI) whereas Sp was determined by using 95 non-chagasic sera by xenodiagnosis, ELISA-Homogenate and PCR. The performance of ELISA-rTSSA-II in dog sera was high (AUC=0·93 and KI=0·91). The Se was 92·85% (1 false negative) and Sp was 100%. Two sera from dogs infected with TcI and 1 with TcIII were negative. For patients infected withT. cruzi, reactivity was 87·8% (36/41), there was only 1 indeterminate, and Sp was 100%. Fifty-four sera from non-chagasic and 68 sera from patients with cutaneous leishmaniasis did not react with rTSS-II. ELISA-rTSSA-II showed a high performance when studying sera from naturally infected dogs and it also presented 100% Sp. This assay could be an important tool to carry out sero-epidemiological surveys on the prevalence ofT. cruzicirculating lineages in the region.
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Guillory, Georgia y Marianne Bronner-Fraser. "An in vitro assay for neural crest cell migration through the somites". Development 98, n.º 1 (1 de noviembre de 1986): 85–97. http://dx.doi.org/10.1242/dev.98.1.85.
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Neural crest cells in the trunk of the avian embryo come into contact with the somites and neural tube during the course of their migration. However, the relationship between the somites and the early migratory routes followed by these cells is not yet completely understood. Here, we use a tissue culture assay to examine if avian neural crest cells migrate through the somites. Cultures of quail somites were prepared from four adjacent regions along the neural axis in the trunk. Each region had four pairs of consecutive somites with region I being most anterior and region IV containing the last four segments. Within each region, the somites were separated from other tissues by enzymatic digestion and plated onto collagen-coated dishes. Immuno-cytochemical techniques were used to confirm that no neural crest cells, recognized by the HNK-1 antibody, were present on the surface of the somites at the time of explantation. After several days in culture, the explanted somites were screened to identify pigment cells. Because neural crest cells give rise to all of the melanocytes in the trunk, the presence of pigment cells indicated that neural crest precursors were contained within the initial explant. After 5–11 days in vitro, the percentage of somite cultures containing pigment cells in regions I through IV, respectively, was 36%, 51%, 31% and 1%. These results suggest that neural crest cells migrate through the somitic mesenchyme and first enter the somites between 5 to 9 segments rostral to the most recently formed somite.
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HIRAGA, KAZUAKI. "Correlation of blood morphine concentration by enzymatic immuno assay ( enzyme immunoassay ) and high performance liquid chromatograhy, and significance of blood morphine concentration measurements." Rinsho yakuri/Japanese Journal of Clinical Pharmacology and Therapeutics 28, n.º 1 (1997): 303–4. http://dx.doi.org/10.3999/jscpt.28.303.
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B. Jagan Mohan Reddy y S.P. Girish. "Assessment of Usefulness of CRP, PMN Elastase, PCT and Il- 6 as Prognostic Factors in Patients with Acute Pancreatitis". Academia Journal of Surgery 3, n.º 2 (27 de diciembre de 2020): 1–4. http://dx.doi.org/10.47008/ajs/2020.3.2.1.
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Background: Acute pancreatitis is an inflammatory disease of the exocrine pancreas with rapid onset. The present study was conducted to assess the usefulness of CRP, PMN elastase, PCT and IL- 6 as prognostic factors in patients with acute pancreatitis. Subjects and Methods: The present study comprised 53 patients who presented with a diagnosis of Acute Pancreatitis. CRP was estimated by turbidimetric immunoassay using CRP/U2A-000 kit. PMN-Elastase was estimated by solid-phase enzyme immunoassay. Procalcitonin was estimated by the immuno- chromatographic test. IL-6 was estimated by Immuno-enzymatic assay. Results: There were 47 males and 6 females in the present study. The mean SD CRP in patients with mild pancreatitis was 44.35 53.04 and in severe pancreatitis was 174.80 14.55, PCT was seen in 4 in mild pancreatitis patients and 12 in severe pancreatitis patients, PMN- elastase level was 3.89 1087 in mild pancreatitis and 3.99 2.75 inn severe pancreatitis patients, IL-6 level was 129.63 319.08 in mild pancreatitis and 1166.76 818.06 in severe pancreatitis patients. The difference was significant (P< 0.05). CRP had higher (100) specificity as compared to PCT (81), PMN- E (10) and IL- 6 (90), Specificity found to be 88, 81, 97 and 94 respectively, PPV was 84, 74, 67 and 90 respectively, NPV was 100, 87, 62 and 94 respectively, accuracy was 92, 81, 62 and 92 respectively, AUC was 0.97, 0.81, 0.43 and 0.95 respectively. Conclusion: Authors found that CRP is the single best predictor of the severity of acute pancreatitis. IL-6 and PCT also are reliable predictors. PMN-Elastase needs to be assessed in patients with acute pancreatitis presenting early in the course of the illness.
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MacKinnon, Andrew, Deepthi Bhupathi, Jason Chen, Tony Huang, Weiqun Li, Yong Ma, Natalija Sotirovska, Susanne Steggerda, Winter Zhang y Francesco Parlati. "705 Anti-tumor activity of CB-668, a potent, selective and orally bioavailable small-molecule inhibitor of the immuno-suppressive enzyme Interleukin 4 (IL-4)-Induced Gene 1 (IL4I1)". Journal for ImmunoTherapy of Cancer 8, Suppl 3 (noviembre de 2020): A747. http://dx.doi.org/10.1136/jitc-2020-sitc2020.0705.
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BackgroundTumors evade destruction by the immune system through multiple mechanisms including altering metabolism in the tumor microenvironment. Metabolic control of immune responses occurs through depletion of essential nutrients or accumulation of toxic metabolites that impair immune cell function and promote tumor growth. The secreted enzyme interleukin 4 (IL-4)-induced gene 1 (IL4I1) is an L-phenylalanine oxidase that catabolizes phenylalanine and produces phenyl-pyruvate and hydrogen peroxide. IL4I1 regulates several aspects of adaptive immunity in mice, including inhibition of cytotoxic T cells through its production of hydrogen peroxide (reviewed in1). In human tumors, IL4I1 expression is significantly elevated relative to normal tissues and is notably high in ovarian tumors and B cell lymphomas. Motivated by the hypothesis that IL4I1 is an immuno-metabolic enzyme that suppresses anti-tumor immunity, we discovered CB-668, the first known small-molecule inhibitor of IL4I1.MethodsIL4I1 enzymatic activity was measured using an HRP-coupled enzyme assay. RNA in-situ hybridization was carried out on the RNAScope platform. Syngeneic mouse tumor models were used to evaluate the anti-tumor activity of CB-668. The level of phenyl-pyruvate in tumor homogenates was measured by LC/MS.ResultsOur clinical candidate, CB-668 is a potent and selective non-competitive inhibitor of IL4I1 (IC50 = 15 nM). CB-668 has favorable in vitro ADME properties and showed low clearance and high oral bioavailability in rodents. Twice-daily oral administration of CB-668 was well-tolerated in mice and resulted in single-agent anti-tumor activity in the syngeneic mouse tumor models B16-F10, A20, and EG7. Oral CB-668 administration reduced the levels of phenyl-pyruvate in the tumor, consistent with inhibition of IL4I1 enzymatic activity. Anti-tumor activity of CB-668 was immune cell-mediated since efficacy was abrogated in CD8-depleted mice, and CB-668 treatment caused increased expression of pro-inflammatory immune genes in the tumor. Moreover, CB-668 had no direct anti-proliferative activity on tumor cells grown in vitro (IC50 > 50 µM). CB-668 also favorably combined with anti-PD-L1 therapy to reduce tumor growth in the B16-F10 tumor model.ConclusionsThese data support an immune-mediated anti-tumor effect of IL4I1 inhibition by CB-668, and suggest inhibition of IL4I1 represents a novel strategy for cancer immuno-therapy.ReferencesMolinier-Frenkel V, Prévost-Blondel A, and Castellano F. The IL4I1 Enzyme: A New Player in the Immunosuppressive Tumor Microenvironment. Cells 2019;8:1–9.
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Kamal, Achmad Fauzi, Deded Yudha Pranatha, Waluyo Sugito, Faisal Rahman, Eka Susanto, Silmi Mariya y Wei Ming Chen. "Isolation, Culture and Characterization of Cancer Stem Cells from Primary Osteosarcoma". Open Stem Cell Journal 5, n.º 1 (28 de septiembre de 2018): 1–13. http://dx.doi.org/10.2174/1876893801805010001.
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Background:Osteosarcoma cancer stem cells (CSCs) are defined as a subpopulation of osteosarcoma cells, which have the ability of self-renewal, proliferation and differentiation. This study aimed to identify CSCs from human osteosarcomain vitro.Methods:Osteosarcoma CSCs were isolated and cultured with sphere-forming assay technique on an ultra-low well attachment surface plate. After sarcosphere colonies were formed, we conducted reverse transcriptase-polymerase chain reaction (RT-PCR) to detect the expression of genes of embryonic stem cells such asNANOG, Oct3/4, STAT3 and gene of MSC CD133. Immunofluorescence analysis (IFA) of alkaline phosphatase (ALP), osteocalcin, and CD 133 was also performed to see the expression of osteosarcoma CSC surface protein with immuno-enzymatic staining principle. We also performed alizarin red staining to evaluate calcification in osteosarcoma CSCs.Results:The culture sphere-of the osteosarcoma cells showed three dimension round shaped colonies (sarcospheres) in slightly hypoxicand serum free condition which was not attached to the substrate with tight density. RT-PCR demonstrated that sarcospheres expressed genes which encodeNANOG, Oct3/4 STAT 3, but not for CD 133. IFA showed positive protein expression of ALP, osteocalcin and CD 133 which was moderate, strong, and weak positive respectively. Sarcospheres also had a positive reaction toward alizarin red staining.Conclusion:Osteosarcoma CSCs could be isolated from human osteosarcoma by sphere-forming assay technique and characterized by the expression of genes of embryonic stem cells,such asNANOG, Oct3/4, STAT3 and IFA of ALP, osteocalcin, and CD 133.
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Li, Shijie, Wenjun Wen, Jianping Guo, Shuo Wang y Junping Wang. "Development of non-enzymatic and photothermal immuno-sensing assay for detecting the enrofloxacin in animal derived food by utilizing black phosphorus-platinum two-dimensional nanomaterials". Food Chemistry 357 (septiembre de 2021): 129766. http://dx.doi.org/10.1016/j.foodchem.2021.129766.
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Terpos, Evangelos, Ioannis Ntanasis-Stathopoulos, Gerasimos-Petros Papassotiriou, Efstathios Kastritis, Alexandra Margeli, Nikolaos Kanellias, Evangelos Eleutherakis-Papaiakovou et al. "Circulating Soluble Urokinase-Type Plasminogen Activator Receptor Levels Reflect Renal Function in Newly Diagnosed Patients with Multiple Myeloma Treated with Bortezomib-Based Induction". Journal of Clinical Medicine 9, n.º 10 (3 de octubre de 2020): 3201. http://dx.doi.org/10.3390/jcm9103201.
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(1) Background: Soluble urokinase-type plasminogen activator receptor (suPAR) has been implicated in the pathogenesis of kidney disease in different disease settings. The aim of this study was to investigate a possible link between suPAR circulating levels and renal impairment (RI) in newly diagnosed patients with symptomatic multiple myeloma (NDMM) before and after frontline therapy with bortezomib-based regimens. (2) Methods: We studied 47 NDMM patients (57% males, median age 69.5 years) before the administration of anti-myeloma treatment and at best response to bortezomib-based therapy. suPAR was measured in the serum of all patients and of 24 healthy matched controls, using an immuno-enzymatic assay (ViroGates, Denmark). (3) Results: suPAR levels were elevated in NDMM patients at diagnosis compared to healthy individuals (p < 0.001). suPAR levels strongly correlated with disease stage (p-ANOVA < 0.001). suPAR levels both at diagnosis and at best response negatively correlated with estimated glomerular filtration rate (eGFR) values (p < 0.001). Interestingly, no significance changes in suPAR levels were observed at best response compared to baseline values (p = 0.31) among 18 responding patients with baseline eGFR < 50 mL/min/1.73 m2. (4) Conclusions: SuPAR levels reflect renal function in NDMM patients treated with bortezomib-based induction. Responders may have elevated circulating suPAR levels, possibly reflecting persistent kidney damage, despite their renal response.
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Borella, F., R. Cantoni, I. Volpato, M. Magara, F. Aredia y V. Rizza. "Quantitative Determination of the Semi-Synthetic Immunomodulator 7 BV Derived from strain ATCC 53966 of Corynebacterium Granulosum Using an Immuno-Enzymatic Assay and its Validation by Gas Chromatography". Analytical Letters 26, n.º 2 (febrero de 1993): 233–45. http://dx.doi.org/10.1080/00032719308017381.
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Lu, Hongtao, Dawei Sun, Jun Sun, Yanan Geng, Jinhui Zhang, Rui Gao, Lei Li, Zhihao Wu, Lily Tang y Yangsheng Qiu. "792 Creating an immune-favorable tumor microenvironment by a novel anti-CD39/TGFβ-Trap bispecific antibody". Journal for ImmunoTherapy of Cancer 9, Suppl 2 (noviembre de 2021): A827. http://dx.doi.org/10.1136/jitc-2021-sitc2021.792.
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BackgroundAdenosine and TGFβ are two key immune suppressors in tumor microenvironment (“TME”) that cause broad immune suppression resulting in resistance to current CPI immunotherapies. Cancers frequently express transforming growth factor-β (TGFβ), which drives immune dysfunction in the tumor microenvironment by inducing regulatory T cells (Tregs), inhibiting CD8+ activation and infiltration into TME, and promoting epithelial–mesenchymal transition (EMT). We observed that TGFβ induces the expression of CD39, a critical enzyme that regulates adenosine generation. CD39 is highly expressed in Tregs within TME, it drives the production of adenosine, an immunoinhibitory molecule that partly mediates Treg inhibitory function. To inhibit CD39-Adenosine and TGFβ simultaneously to create an immune favorable tumor microenvironment, we designed a bi-specific antibody targeting both CD39 and TGFβ (ES014), which aims to inhibit the generation of adenosine and iTreg in TME. The immuno-stimulating effect of ES014 was demonstrated in a PD-1-unresponsive mouse model where tumor growth was significantly inhibited after the treatment of the bi-specific antibody.MethodsThe bifunctional antibody–ligand trap ES014 was created by fusing the TGFβ receptor II ectodomain to an antibody targeting CD39. ES014 molecule could simultaneously inhibit CD39 enzymatic function to prevent extracellular ATP from degradation and neutralize autocrine/paracrine TGFβ in the target cell microenvironment. The immunological function of ES014 was studied in an in vitro Elpiscience proprietary ImmunoShine platform which includes T cell activation and apoptosis assay, iTreg differentiation and suppression assay, NK cell activation assay and DC maturation activity. The in vivo efficacy of ES014 was investigated in a human PBMC engrafted cancer model.ResultsWe demonstrated that ES014 bispecific antibody can inhibit CD39 enzymatic activity and neutralizes TGFβ-induced effect, resulting in greater T cell activation and suppression of Treg differentiation. Interestingly, we found ES014 molecule demonstrated a unique mechanism by significantly protecting effector T cell from anti-Fas induced apoptosis or activation induced cell death (AICD) that is not observed in monotherapy or combo treatment. The ES014 molecule is more effective in inhibiting tumor growth as compared with anti-CD39 antibody or TGFβ-trap in a human PBMC engrafted in vivo model.ConclusionsWe find that by simultaneously targeting CD39 and TGFβ by a novel bi-specific molecule ES014, a more immune-favorable TME and synergistic anti-tumor effects can be achieved. Our pre-clinical data demonstrate that ES014 counteracts TGFβ-mediated inhibitory effect and adenosine induced immune tolerance and has a unique ability to protect T cell from apoptosis. ES014 demonstrated strong efficacy in in vivo tumor growth inhibition.
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Jayakumar, Thanasekaran, Ming-Ping Wu, Joen-Rong Sheu, Chih-Wei Hsia, Periyakali Saravana Bhavan, Manjunath Manubolu, Chi-Li Chung y Chih-Hsuan Hsia. "Involvement of Antioxidant Defenses and NF-κB/ERK Signaling in Anti-Inflammatory Effects of Pterostilbene, a Natural Analogue of Resveratrol". Applied Sciences 11, n.º 10 (19 de mayo de 2021): 4666. http://dx.doi.org/10.3390/app11104666.
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Pterostilbene (PTE), a natural stilbenoid occurring in grapes and berries, is recognized as a dimethylated analogue of resveratrol. This compound shows numerous notable pharmacological activities, including antiaging, anticancer, antidiabetes, antioxidant, and neuroprotection. This study investigates the anti-inflammatory properties of PTE in macrophage cells (RAW 264.7) against the lipoteichoic acid (LTA) stimulation. The expression of inflammatory tumor necrosis factor (TNF-α), interleukin-1β (IL-1 β), and inducible nitric oxide synthase (iNOS) and the content of nitric oxide (NO) were detected in LTA-induced cells. In addition, a Western blot assay was used to detect mitogen-activated protein kinases: extracellular signal-regulated kinase (ERK)1/2, p38 MAPK, and c-Jun N-terminal kinase (JNK). The phosphorylation of IκB and p65 and translocation of nuclear factor kappa B (NF-κB) were assessed by Western blot and immuno-fluorescence staining. The results showed that PTE significantly attenuated NO production and TNF-α, IL-1 β, and iNOS expression in LTA stimulated cells. Among the activation of ERK, JNK, and p38 in cells treated with LTA, PTE at higher concentration had only inhibited ERK activation. However, PTE blocked IκB phosphorylation, phosphorylation and nuclear translocation of p65NF-κB. Fascinatingly, PTE enhanced antioxidant defense molecules as verified by the enhanced heme oxygenase-1 (HO-1) expression, catalase (CAT) antioxidant enzyme, and non-enzymatic antioxidant, and reduced glutathione (GSH) in LTA-induced RAW 264.7 cells. These results suggest that PTE exerts an anti-inflammatory property via attenuating NF-κB/ERK signaling pathways as well as enriching antioxidant defense mechanisms.
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Castro, Juliana Prazeres Gonçalves de, Telma Regina da Silva Aguiar, Gilson Coutinho Tristão, Gutemberg Gomes Alves, Marina Prado Fernandes Pinheiro, Valquiria Quinelato, Priscila Ladeira Casado y George E. Romanos. "Peri-implant health after supportive mucositis therapy is associated with increased levels of FGF-2". Brazilian Dental Journal 32, n.º 5 (septiembre de 2021): 55–66. http://dx.doi.org/10.1590/0103-6440202104027.
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Abstract This study aimed to analyze Fibroblast Growth Factor-2 (FGF-2) levels in the peri-implant crevicular fluid throughout supportive mucositis therapy. Twenty-six participants with Branemark protocol prosthesis were divided into two groups: the control group, characterized by healthy peri-implants, and the mucositis group, presenting a diagnosis of peri-implant mucositis. All participants underwent clinical examination, radiographic analysis, prosthesis removal, and non-invasive peri-implant therapy (mechanical debridement associated with chlorhexidine 0.12%) during a period of 36 days divided into three intervals. Peri-implant crevicular fluid samples were collected at each interval in order to analyze FGF-2 levels by immuno-enzymatic assay. The control and mucositis groups showed difference in keratinized mucosa. The smaller the range of keratinized mucosa the higher susceptibility of peri-implant mucositis. Throughout the treatment intervals, participants were diagnosed in different groups indicating whether or not the non-invasive therapy was able to treat peri-implant mucositis. There was a significant difference of FGF-2 levels between groups, with the higher FGF-2 levels in the control group (p=0.01). After supportive therapy, the mucositis group showed significantly increased FGF-2 levels (p<0.01) compared to initial levels. After 36 days of supportive therapy, there was a reduction of peri-implant mucositis from 70% to 23%. Clinical and laboratory outcomes showed a clear correlation since FGF-2 levels increased after 36 days. It was concluded that the therapy protocol was effective and promoted a regenerative reaction and FGF-2 can be considered a future target for peri-implant mucositis understanding.
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Mazumdar, Reaz Mohammad, Mohammad Razuanul Hoque, Suman Mohajan, Imran Khan, Md Zahid Hassan, Md Omar Faruque, Liaquat Ali y Md Golam Kabir. "THE RELATIONSHIP BETWEEN APOB AND APOA1 WITH INSULINEMIC STATUS IN PREDIABETIC SUBJECTS". Bioresearch Communications 9, n.º 1 (29 de diciembre de 2022): 1170–76. http://dx.doi.org/10.3329/brc.v9i1.63596.
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The present study was mainly aimed at exploring the causal association of the atherogenic (ApoB) and antiatherogenic apolipoproteins (ApoA1) and their ratio in the basic defects of pancreatic β cell dysfunction and insulin resistance in type 2 Diabetes Mellitus (T2DM). An intermediate stage towards diabetes, the prediabetic stage, was chosen to explore the association. Following a standardized selection process, 131 subjects were purposefully recruited for the study, including 18 with impaired fasting glucose (IFG), 56 with impaired glucose tolerance (IGT), and 57 with type 2 diabetes (T2DM). Fifty-nine healthy subjects served as controls. Glucose, lipid and insulin were estimated by glucose-oxidase, enzymatic colorimetric assay and enzyme linked immunosorbent assay (ELISA) respectively. Serum ApoB and ApoA1 were estimated by immuno-nephelometric method. Appropriate statistical tools were used to calculate statistical differences using Statistical Package for Social studies (SPSS) for Windows V12. Absolute insulin (mIU) levels were significantly higher in the IGT and T2DM groups compared to controls (p 0.001 and p = 0.001, respectively). HOMA%B (meanSD) was significantly lower in T2DM groups (p=<0.001) and higher in IGT compared to the controls although it is significantly lower in IFG compared to the controls but mean value is about 90%. HOMA%S was significantly lower in IGT and T2DM group (p=0.001 and 0.002 respectively). ApoA1 levels were significantly higher only in the T2DM group (p = 0.027), whereas ApoB levels were higher in both the IGT and T2DM groups (p = 0.026-0.001).. Neither ApoB nor ApoA1 showed any significant difference in the IFG group as compared to control. ApoB-ApoA1 ratio did not show significant difference among the groups. ApoB showed significant positive correlation with both fasting and postprandial glucose (p=0.006 and 0.040 respectively). In IGT group ApoB was positively correlated with absolute insulin (p=0.025) and HOMA%B (p=0.049) and negatively with HOMA%S (p=0.026). ApoB, but not ApoA1 or the ApoB and ApoA1 ratio, seem to have a causal association with insulin resistance, and elevation of ApoB is also modulated by obesity and atherogenic lipids. Bioresearch Commu. 9(1): 1170-1176, 2023 (January)
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Churashova, Irina A., Alexey V. Sokolov, Valeria A. Kostevich, Nikolay P. Gorbunov, Olga L. Runova, Elvira M. Firova y Vadim B. Vasilyev. "Myeloperoxidase/high-density lipoprotein cholesterol ratio in patients with arterial hypertension and chronic coronary heart disease". Medical academic journal 21, n.º 2 (24 de septiembre de 2021): 75–86. http://dx.doi.org/10.17816/maj71486.
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BACKGROUND: Myeloperoxidase (MPO), the enzyme of leukocytes, catalyzes the production of reactive halogen species, which can modify the structure of lipoproteins. Chlorination and nitration of tyrosine residues in apolipoprotein A-1 lead to the formation of dysfunctional high-density lipoproteins (HDL-p), thus blocking the reverse cholesterol transport. Low level of high-density lipoprotein cholesterol (HDL-C) is associated with exacerbation of coronary heart disease, but the prognostic value of this index is not fully assessed.
AIM: The aim of this study was to examine a possible contribution of MPO to the atherosclerotic plaque development (the stable growth or the erosion and rupture) via the modification of HDL-p. That is to say we investigated the diagnostic values of measuring the total MPO (MPO-T), the active MPO (MPO-A) and the MPO/HDL-С relation in patients with hypertension and various forms of chronic coronary heart disease.
MATERIALS AND METHODS: The cohort under study included 44 patients with arterial hypertension and chronic coronary heart disease. All patients were divided into three groups according to the diagnosis: arterial hypertension without coronary heart disease (Group I, n = 20); arterial hypertension and the initially stable chronic coronary heart disease without acute complications in the anamnesis (Group II, n = 14); arterial hypertension and myocardial infarction (acute coronary syndrome) in the anamnesis (Group III, n = 10). The enzyme-linked immunosorbent assay (ELISA) for MPO-T and specific immuno-extraction followed by enzymatic detection (SIEFED) by fluorogenic substrate for MPO-A were applied. After that the ratio MPO-T/HDL-C or MPO-A/HDL-C was calculated.
RESULTS: The MPO-A and MPO-A/HDL-C ratio were significantly increased in the group III of patients with old myocardial infarction as compared with the patients of group II who had the initially stable coronary heart disease (p = 0.009 and p = 0.003, respectively). Besides, the level of HDL-C in the group III was significantly reduced (p = 0.013). Our measurements revealed the negative correlation between MPO-A and HDL-C concentrations (r = 0.31; p 0.05), which is in line with the presumption of the study accomplished. Surprisingly, the correlation between MPO-T/HDL-C ratio and that MPO-A/HDL-C was stronger (r = 0.72; p 0.05), than between MPO-T and MPO-A (r = 0.36; p 0.05).
CONCLUSIONS: Our study demonstrates the importance of assessing MPO-T and MPO-A plasma concentrations and of calculating the ratio MPO/HDL-C as promising biomarkers in the complicated cases of chronic coronary heart disease. MPO-A and MPO-A/HDL-C values were elevated in the patients with old myocardial infarction, while the concentration of HDL-C remained decreased upon the transition from the acute to chronic phase of the disease.
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Huang, Zhaoliang, Xinghua Pang, Tingting Zhong, Chunshan Jin, Na Chen, Xinrong He, Dennis Xia et al. "750 AK119, a CD73 targeting antibody with dual mechanism of action". Journal for ImmunoTherapy of Cancer 9, Suppl 2 (noviembre de 2021): A783—A784. http://dx.doi.org/10.1136/jitc-2021-sitc2021.750.
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BackgroundAK119 is an Fc-engineered humanized IgG1 monoclonal antibody targeting human CD73. CD73-extracellular adenosine pathway regulates conversion of pro-inflammatory and immuno-stimulatory extracellular adenosine ATP into immunosuppressive adenosine. CD73 expresses on cancer cells, endothelial cells, fibroblasts, lymphocytes and myeloid cells. CD73 upregulated can be a result of tissue hypoxia,1 epithelial-to-mesenchymal transition,2 inflammation3 and/or cytotoxic stress.4 Also, increasing immune response may lead to faster viral clearance, shorter recovery time, less complications, longer immunity and protection from re-infection. Inhibiting CD73 was reported to evoke B cells activation and shows anti-fibrotic effects. The ability of enhancing immune response provides a potential opportunity to treat COVID-19. Thus, we investigated pharmacological activity of AK119 as an agent treating cancers, COVID-19 and fibrosis.MethodsAK119 inhibition of CD73 enzymatic activity was tested in human PBMCs based assay. The ability of AK119 to enhance B cells immune response was detected by cell-based assay. PBMCs were incubated overnight with APCP (inhibitor of CD73 enzyme acitvity) or AK119, CPI006 or MEDI9447. Flow cytometry analysis was performed with gating on B cells (CD19+CD3-) and MFI and positive percent were reported for antibody staining of CD69 or CD83, as well as HLA-DR and IgM. Enhancement of anti-SARS-CoV-2 antibody production was studied using human CD73 transgenic mouse immunized with SARS-CoV-2 spike protein. The in-vivo activity of AK119 was further studied in bleomycin-induced pulmonary fibrosis model in human CD73 transgenic mouse.ResultsAK119 shows a more potent antigen binding (figure 1) and completely CD73 enzyme inhibition activity (figure 2). AK119 promotes B cell proliferation, and upregulating CD69, CD83, HLA-DR and IgM that are markers of B cell activation (figure 3). B cell activation induced by AK119 is independent of adenosine. AK119 show significantly higher bioactivity to induce B cells activation in comparison with MEDI9447 or CPI006 (figure 4). In human CD73 transgenic mice, AK119 increased secretion of anti-S protein IgG (figure 5). In pulmonary fibrosis mouse model, number of inflammatory cell in broncholveolr lavage fluid of AK119 was significantly decreased, and decreased HYP representing collagen content in lung tissue homogenate of mice was found in both AK119 50 mg/kg and 10 mg/kg group (figure 6).ConclusionsAK119 selectively binds to and inhibits the ectonucleotidase activity of CD73 thus reducing adenosine accumulation. Results from non-clinical pharmacology studies reveal potent bioactivities as well as favorable safety properties of AK119. AK119 is intended for advanced solid tumors, pulmonary fibrosis and therapy of COVID-19.ReferencesBullen JW, Tchernyshyov I, Holewinski RJ, DeVine L, Wu F, Venkatraman V, Kass DL, Cole RN, Van Eyk J, Semenza GL, Protein kinase A-dependent phosphorylation stimulates the transcriptional activity of hypoxia-inducible factor 1. Sci Signal 2016;9(430):ra56.Lupia M, Angiolini F, Bertalot G, Freddi S, Sachsenmeier KF, Chisci E, Kutryb-Zajac B, Confalonieri S, Smolenski RT, Giovannoni R, Colombo N, Bianchi F, Cavallaro U. CD73 regulates stemness and epithelial-Mesenchymal transition in ovarian cancer-initiating cells, Stem Cell Rep 2018;10(4):1412–1425.Reinhardt J, Landsberg J, Schmid-Burgk JL, Ramis BB, Bald T, Glodde N, Lopez-Ramos D, Young A, Ngiow SF, Nettersheim D, Schorle H, Quast T, Kolanus W, Schadendorf D, Long GV, Madore J, Scolyer RA, Ribas A, Smyth MJ, Tumeh PC, Tuting T, Holzel M. MAPK signaling and inflammation link melanoma phenotype switching to induction of CD73 during immunotherapy. Cancer Res 2017;77(17):4697–4709.Samanta D, Park Y, Ni X, Li H, Zahnow CA, Gabrielson E, Pan F, Semenza GL. Chemotherapy induces enrichment of CD47(+)/CD73(+)/ PDL1(+) immune evasive triple-negative breast cancer cells, Proc Natl Acad Sci USA. 2018;115(6):E1239–E1248.Abstract 750 Figure 1Binding activity of AK119 to human PBMCs. Binding Curve of AK119 to CD73 expressed on (A) CD8+ T cells and (B) CD19+ B cells in human PBMCsAbstract 750 Figure 2Inhibition activity of CD73 on human PBMCs. AK119 Inhibits Enzymatic Activity of CD73 Expressed on human PBMCsAbstract 750 Figure 3Effect of upregulating B cell markers by AK119. AK119 Upregulates (A) CD69, (B) CD83, (C) HLA-DR and (D) IgM Expression on B cellsAbstract 750 Figure 4Stimulation of B cell Proliferation by AK119Abstract 750 Figure 5Therapeutic activity in the COVID-19 mouse model. Serum Concentration of S protein-specific IgG in Mouse Model of COVID-19Abstract 750 Figure 6Therapeutic activity in the asthma mouse model. (A) AK119 relieves the increased airway resistance and restore the lung function. (B) Reduction of the inflammatory cells in BALF by AK119
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Wang, Seungho, Yi Na Yoon, Mi kwon Son, Soo Jung Kim, Bo Ram Lee, Eun hee Yang, Byeongwook Jeon et al. "600 BR101801 stimulates anti-tumor immunity and enhances the efficacy of radiation in a syngeneic model". Journal for ImmunoTherapy of Cancer 9, Suppl 2 (noviembre de 2021): A630. http://dx.doi.org/10.1136/jitc-2021-sitc2021.600.
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BackgroundBR101801 is an inhibitor of PI3K γ/δ and DNA-PK. It has received clinical approval from the U.S. FDA as an anticancer drug candidate, and phase 1a/1b is ongoing in the U.S. and South Korea. According to the prior studies PI3K γ/δ inhibition exhibits anticancer immune effects by changing the tumor microenvironment [1]. In addition, ionizing radiation (IR) activates the immune response by causing the destroyed cells to act as antigens [2]. Therefore, the combination of BR101801 and IR can induce cancer cell death and amplify anticancer immune effects. This study aims to demonstrate efficacy of the BR101801 as a potent cancer immunotherapy.MethodsThe enzymatic potency of PI3K isotype and DNA-PK was analyzed by Eurofins. The effects of BR101801 on cell viability were evaluated in 4T1 (breast cancer) and CT-26 (colon cancer) cells for 72 h using WST-8 assay. For in vivo studies, the tumor (4T1 or CT-26)-bearing syngeneic mice were treated with BR101801. To evaluate the synergistic effect, CT-26 tumor-bearing syngeneic mice were treated with vehicle, BR101801, IR (2 Gy or 7.5 Gy), and BR101801 + IR. Immune cells from the spleen or tumor were quantified by flow cytometry.ResultsIn vitro selectivity and target potency of BR101801 on different PI3K isotypes and DNA-PK were studied in a cell-free system. The biochemical IC50 values of BR101801 for PI3K -γ, -δ, and DNA-PK were 15 nM, 2 nM, and 6 nM, respectively. In vitro 50% of maximal inhibition of cell proliferation (GI50) in 4T1 and CT26 cell lines were both above 10 μM. In 4T1 and CT-26 syngeneic models, BR101801 showed the highest tumor inhibitor efficacy (Figure 1). In particular, regulatory T cells (Tregs) & Myeloid derived suppressor cells (MDSC) were decreased and CD8+ T cells were increased in the spleens isolated from the tumor-bearing mice. Compared with other PI3K inhibitors, BR101801 had the highest efficacy, confirming that it changes the immune microenvironment. Moreover, BR101801 was synergistic in combination with 2 Gy or 7.5 Gy of IR in the syngeneic model. Notably, Tregs & Macrophage2 were decreased and CD8+ T cells were increased in the tumor tissue, confirming that the anticancer efficacy.Abstract 600 Figure 1Synergistic effect with ionizing radiation In VivoThe combination of BR101801 and ionising radiation showed synergistic effects in the CT-26 Syngeneic model. BR101801 increases anti-cancer immune cells, CD8 + T cells, and decreases immune suppressor cells Tregs and macrophages through a combination of radiation, resulting in immuno-cancer effects.ConclusionsBR101801 demonstrated an anticancer immune effect by changing the tumor microenvironment and showed synergistic effects with radiation combination therapy. We will confirm the anticancer immunity effect in ongoing clinical trials.ReferencesOkkenhaug K, Graupera M, Vanhaesebroeck B. Targeting PI3K in Cancer: Impact on Tumor Cells, Their Protective Stroma, Angiogenesis, and Immunotherapy. Cancer Discov. 2016; 10: 1090–1105.McKelvey K, Hudson A, Back M, Eade T, Diakos C. Radiation, inflammation and the immune response in cancer. Mammalian Genome. 2018;9:843–865Ethics ApprovalThe protocol and any amendment(s) or procedures involving the care and use of animals in this study were reviewed and approved by the Institutional animal Car and Use Committee (IACUC) of BoRyung Pharm. prior to conduct.[Approval number:BR18130]
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Rawley, Orla, Jamie O'Sullivan, Gudmundur Bergsson, Alain Chan, Rachel Therese McGrath, Maartje van den Biggelaar, Jan Voorberg, Vince Jenkins y James S. O'Donnell. "Specific N- and O-Linked Carbohydrate Structures Mediate Von Willebrand Factor Interaction with Galectins -1 and -3". Blood 118, n.º 21 (18 de noviembre de 2011): 2234. http://dx.doi.org/10.1182/blood.v118.21.2234.2234.
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Abstract Abstract 2234 Von Willebrand Factor (VWF) is extensively glycosylated with both N- and O-linked carbohydrates. Moreover, these complex glycan structures influence VWF functional properties, including susceptibility to ADAMTS13 proteolysis, and plasma clearance. The molecular mechanisms through which VWF glycosylation (including ABO blood group antigens) act to influence VWF physiology remains unexplained. However, recent data suggest that VWF circulates in normal plasma bound to various carbohydrate-binding proteins, including specific members of the galectin family. In addition, galectin-3 binding has been reported to influence VWF cleavage by ADAMTS13. In this context, we sought to elucidate the role of specific VWF glycan determinants in modulating galectin interaction. VWF was purified from human plasma (pdVWF) by cryoprecipitation and gel filtration. VWF glycosylation was then modified using exoglycosidases and quantified by specific lectin ELISAs. Blood group specific VWF was also purified from pooled group AB, O, or Bombay plasmas. Galectins-1 and -3 were transiently expressed in competent E-coli cells with an N-terminal histidine tag, and purified by nickel chromatography. Finally, binding interactions were characterized via modified immunosorbant assay. In keeping with the previous report of Lenting et al, human pdVWF bound to both galectin-1 and galectin-3 in a dose-dependent manner. Enzymatic desialylation of pdVWF with α2-3,6,8,9 neuraminidase (Neu-VWF) markedly enhanced binding to galectin-1 (231±6%, p<0.0001). Similarly, removal of terminal sialic acid also increased binding to galectin-3, albeit to a lesser extent (136±6%, p<0.05). To further define the role of VWF glycans in regulating galectin binding, pdVWF was exposed to sequential neuraminidase and galactosidase digestions to remove terminal sialic acid and sub-terminal galactose residues (NeuGal-VWF). In contrast to the enhanced binding of Neu-VWF, binding of NeuGal-VWF to both galectin -1 and -3 was significantly reduced (51±5% and 52±6% compared to pdVWF; p<0.005). Cumulatively these findings suggest that loss of capping sialic acid and exposure of sub-terminal galactose critically regulates VWF-galectin binding. Treatment with PNGase F to completely remove N-linked carbohydrate structures (PNG-VWF) markedly decreased binding to galectin -1 and -3 (13±1% and 57±2%, p<0.001). Moreover, combined PNGase F and O-glycosidase digestions further attenuated galectin-3 binding (21±1%, p<0.001), suggesting that both the N- and O-linked glycans are involved in mediating the VWF-galectin interaction. ABO(H) blood group antigens are expressed on both the N-linked and O-linked glycans of human VWF. Moreover, ABO(H) determinants influence VWF susceptibility to ADAMTS13 proteolysis and plasma VWF half-life, through unknown mechanisms. Purified VWF from normal group AB individuals bound to both galectin-1 and galectin-3 significantly better than group O VWF (146±8% and 483±19%; p<0.01). Conversely, no significant difference in binding was observed between Group O and Bombay VWF. Consequently, although terminal A (GalNAc) and B (Gal) sugar moieties promote galectin binding, expression of terminal α1–2 fucose residues is not important. The glycosylation profile of platelet-VWF differs from that of pdVWF. In particular, platelet-VWF expresses reduced levels of both capping sialic acid and sub-terminal galactose residues (∼50%), and lacks AB blood group antigens. To characterize the effects of this differential sugar expression on galectin binding, platelet-derived VWF was isolated and purified (platelet freeze-thawing followed by immuno-affinity chromatography with monoclonal CLB-Rag20). In keeping with the reduction in Gal and AB blood group antigen expression, platelet VWF bound less well to galectin-1 and galectin-3 (72±6% and 67±7% versus pdVWF; p<0.05). These novel data demonstrate that both the N- and O-linked oligosaccharide structures of VWF are involved in mediating galectin binding. In particular, expression of terminal AB blood group antigens, and expression of sub-terminal galactose moieties following loss of capping sialic acid, both markedly enhance galectin binding affinity. Further studies will be required to define how galectin binding is involved in mediating the functional consequences of variation in VWF glycans. Disclosures: No relevant conflicts of interest to declare.
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Oğuz, OSMAN, Huriye Serin y Fatma Hocaoğlu Emre. "Alkaline phosphatase interference in immuno-enzymatic assays." Journal of Medical Biochemistry, 16 de diciembre de 2021. http://dx.doi.org/10.5937/jomb0-33981.
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Background: Alkaline phosphatase (ALP) enzymes are widely used as signal amplifiers in immunoenzymatic methods. Conditions that cause ALP elevations, such as bone or liver diseases can cause interference in immunoenzymatic methods. Objective: We aimed to examine ALP's effect on immunoenzymatic assay by adding isolated pure ALP to the prepared serum pool. Material and Methods: We prepared a serum pool and divided into 4 groups. By adding isolated pure ALP at different concentrations to each group, we obtained sample groups containing ALP enzyme at concentrations of 85 U/L, 340 U/L, 870 U/L and 1570 U/L. In each group, 20-repetition of βhCG, Ferritin, FT4, TSH, Troponin I and Vit B12 tests were performed. Coefficient of variation, bias, and total error were calculated. All groups were compared by using Friedman test for paired samples. Result: After ALP addition, the calculated total error values of FT4, βhCG and troponin I tests were found to be above the acceptable error limits. There were statistically significant differences in βhCG ,FT4, troponin I and Vit B12 tests when compared to the baseline ALP level (P<0,0125).Conclusion: Isolated ALP elevations can be a source of interference for immunoenzymatic methods.KeywordsAlkaline phosphatase, ALP, bias, immunoenzymatic, total error
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Osinaga, E., A. Babino, J. Grosclaude, E. Cairoli, C. Batthyany, S. Bianchi, S. Signorelli, M. Varangot, I. Muse y A. Roseto. "Development of an immuno-lectin-enzymatic assay for the detection of serum cancer-associated glycoproteins bearing Tn determinant". International Journal of Oncology, 1 de febrero de 1996. http://dx.doi.org/10.3892/ijo.8.2.401.
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Pasciu, Valeria, Francesca Daniela Sotgiu, Maria Nieddu, Cristian Porcu y Fiammetta Berlinguer. "Measurement of fecal T3 metabolite levels in sheep: Analytical and biological validation of the method". Frontiers in Veterinary Science 9 (25 de noviembre de 2022). http://dx.doi.org/10.3389/fvets.2022.1011651.
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IntroductionBiological sample collection from wild and farms animals is often associated with difficulties related to the handling and restraint procedures, and most of the time it could induce stress, altering the welfare and physiological homeostasis. The analysis of fecal T3 metabolites (FTMs) allows to test samples collected in a non-invasive manner, providing several information about the animal's physiological conditions and the effects related to environmental and nutritional variations. This procedure has found wide application in wild species, but less in domestic ones.MethodsThe aim of this work was to validate the use of an immuno-enzymatic competitive ELISA kit, designed for T3 quantification in human blood serum samples, for the assessment of FTMs in the sheep. For the analytical validation, precision, recovery and parallelism were evaluated; for biological validation the variations of FTMs in relation to age, sex and the physiological status of the animal were determined.ResultsAfter a verification of the precision (RSD % < 15%), mean recovery (75%) and parallelism (CV% < 10%), the kit was used to measure FTMs in cyclic, pregnant, and early lactating ewes as well as in rams and ewe lambs. The results showed that FTMs concentrations in pregnant ewes were significantly lower (p < 0.05) than in cyclic and early lactation ones. Furthermore, there were no significant differences in FTMs levels between ewes and rams, while in lambs FTMs levels were higher than in adults (p < 0.001).ConclusionIn conclusion the present study demonstrates that FTMs can be reliably and accurately determined in sheep feces, using an ELISA kit formulated for human serum T3 assay. The application of this method in the livestock sector could allow to improve our knowledge about the response of animals to different physiological and environmental conditions, and thus assess their welfare.
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Benjamaa, Rania, Abdelkarim Moujanni, Anass Terrab, Rabiaa Eddoha, Maryam Benbachir, Abderrahman Moujahid, Boubker Nasser et al. "Relationship Among Antibiotic Residues And Antibacterial Activity Of The Endemic Spurge Honey (Euphorbia Resinifera O. Berg) From Morocco". Emirates Journal of Food and Agriculture, 8 de noviembre de 2020, 795. http://dx.doi.org/10.9755/ejfa.2020.v32.i11.2190.
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Antibiotic-resistant bacteria continue to be of major health concern worldwide. In recent years, several reports and scientific articles claim the contamination of honey by antibiotics, detectable concentrations of antibiotic residues in honey are illegal. They, may cause hypersensitivity or resistance to drug therapy in humans, and are perceived by consumers as undesirable. In this sense, the purpose of this work was to examine the antibacterial activity of the Euphorbia resinifera (E. resinifera) honey against Escherichia coli and Staphylococcus aureus in vitro using the well-agar diffusion assay followed by dilution range to obtain more precise minimum inhibitory concentration values. The second aim is to evaluate the presence of antibiotics in honey using a screening test: Evidence InvestigatorTM, an immuno-enzymatic method for detection of 27 antibiotic residues followed by a liquid chromatography-tandem mass spectrometry (LC-MS/MS) for confirmation of suspect samples; in order to assess the relationship between the presence of antibiotic residues and the antibacterial activity of honey. In this study, a total of 37 E. resinifera honey samples were analyzed. The results show that all samples of honey inhibited the growth of bacteria at the dilutions at 50% (v/v); the highest inhibition zone (25.98 ± 0.11 mm) was recorded from sample 5 for Staphylococcus aureus and (13.84 ± 1.10 mm) in sample 17 for Escherichia coli and that 50% (v/v) dilutions showed significant antibacterial effect compared to other dilutions (6.25, 12.5, 25% (v/v)). In all samples, there were no antibiotic residues detected except for one showing the detection of Trimethoprim at 6.48 µg kg-1. Our research is one of the first studies that relate the he relationship between the presence of antibiotic residues and the antibacterial activity of Euphorbia resinifera honey and showed that the antibacterial activity of honey might be due to the high osmotic nature, a low pH, its content of phenolic compounds and hydrogen peroxide and also to its content of methylglyoxal.
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Kryukova, A. V., E. Yu Markov, V. B. Nikolaev, Yu O. Popova, V. T. Klimov, S. V. Igumnova, N. M. Andreevskaya, A. V. Ulanskaya, T. Yu Zagoskina y M. V. Chesnokova. "PHYSICOCHEMICAL AND ANTIGENIC PROPERTIES OF THE UREA-EXTRACTED SURFACE STRUCTURES OF YERSINIA PSEUDOTUBERCULOSIS O:1B". Russian Journal of Infection and Immunity, 12 de abril de 2022. http://dx.doi.org/10.15789/2220-7619-paa-1602.
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Abstract. Immuno-serological diagnostic tools particularly identifying pathogen antigens are the most important methods of pseudotuberculosis studies . The main immunodominant and species-specific antigens located in the surface structures of the bacterial cell are of practical interest. Thereby the aim of the work was to isolate and characterize biologically active surface structures of the pseudotuberculosis microbe. Here, the living cells of Y. pseudotuberculosis 3704 (O:1b) were lysed by using 9 M urea solution to extract antigens localized in the microbial surface structures. The subcellular fractions obtained such as outer membranes (OM), urea extract (UE) and isolated protein-lipopolysaccharide complex (PLPSC) are characterized by physical and chemical parameters. The protein content in the preparations ranged from 42% to 53%. The polypeptide band of the OM preparation, UE polypeptide and PLPSC for pseudotuberculosis microbe was presented by 14, 16 and 9 major polypeptides with molecular weight ranging from 13,9 kDa to 131,5 kDa, 13,5 kDa to 101,6 kDa, and 20,7 kDa to 66,6 kDa, respectively. Proteolytically active proteins and polypeptides were detected in isolated subcellular fractions (OM and UE) by using the radial enzyme diffusion test and substrate-gel electrophoresis found to be presented by 4 and 7 polypeptides with molecular weight ranging from 28,0 kDa to 118,0 kDa and 29,2 kDa to 97,7 kDa in the OM and UE preparation, respectively. The subcellular fractions obtained are capable to exhibit immunogenic activity after inoculation to experimental animals and antigenic activity while interacting with specific antibodies in the radial immunodiffusion (RID) assay and antibodies labeled with colloidal silver nanoparticles in dot immunoassay (DIA). OM and PLPSC preparations in DIA with immunoglobulins isolated from experimental antisera and labeled with colloidal silver nanoparticles were detected at a concentration of ≥ 0.12 μg / ml (dry weight), cells of strain Y. pseudotuberculosis 3704 at a concentration of ≥ 3, 9 × 106 m.c. / ml, which is similar to the results of DIA with immunoglobulins isolated from commercial pseudotuberculosis antiserum (St. Petersburg) and labeled with nanoparticles of colloidal silver. Thus, the subcellular fractions of pseudotuberculosis microbe isolated by using urea as a lysing and decontaminating agent retain their antigenic and immunogenic properties and enzymatic activity suggesting about their potential benefits for use to improve early diagnostics of pseudotuberculosis.
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Gros, Luis, Alicia Roldán, Almudena Cabero-Martínez, Nerea Domínguez-Pinilla, Adolfo de la Fuente, Eva González-Barca, María Tasso et al. "Incidence and management of patients with methotrexate delayed elimination in the clinical practice: A Delphi study". Journal of Oncology Pharmacy Practice, 11 de febrero de 2022, 107815522210795. http://dx.doi.org/10.1177/10781552221079568.
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Introduction High-dose methotrexate (HDMTX) is administered for the treatment of some cancers. HDMTX is usually safe but may crystallize in renal tubules causing acute kidney injury (AKI). Consequently, MTX elimination is delayed, resulting in a severe and life-threatening condition. No studies have been published about the impact of MTX toxicity in Spain. This study aims to estimate the incidence and management of MTX delayed elimination and toxicity. Methods A two-round Delphi study was performed to reach consensus between 10 medical experts on haemato-oncology and paediatric oncology with experience in the management of HDMTX treated patients from leading Spanish hospitals. An online questionnaire was developed based on national and international guidelines and previous evidence regarding HDMTX-related toxicity. Consensus was established at 80% agreement. Median and interquartile ranges were calculated, and incidence data were extrapolated to the Spanish general population. Results Out of 1.475 patients estimated to receive HDMTX treatment annually in Spain, 27.5% present MTX delayed elimination and 11.6% develop HDMTX-induced AKI (35.4% with severe systemic toxicities (>grade 3) and 18.8% develop chronic renal disease). Mortality is estimated in 4.2%. Immuno-enzymatic assay is used in most of the hospitals (90%) for MTX serum level monitoring. All experts use increased supportive care and high leucovorin as first-line treatment. Available treatments in experts’ hospitals in case toxicity persists are haemodialysis (90% of hospitals), glucarpidase (60%) and hemofiltration (50%). Most prevalent non-renal systemic toxicities are haematologic and mucositis (21–40% of patients). Patients with HDMTX-induced AKI require from intensive care (5% of patients), more than 3 sessions and 4 days of dialysis, and about 8.5 days of hospitalization (non-ICU patients) and 12 days in case of patients requiring ICU. Conclusions These results are the first evidence regarding HDMTX-induced AKI in Spain. Incidence and mortality results are in line with previous studies. Clinical management is based on preventive measures and the treatment depend on the availability in the hospital. The need for effective, safe and rapid treatment for the reduction of MTX toxic levels and the improvement of monitoring methods were noted by experts as urgent needs. Further observational studies to validate these results would be needed.
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Kapepula, P. Mutwale, H. Luzayana Wamba, D. Mukundi Lukusa, T. Franck, P. Lokole Bahati, T. Mbemba Fundu, P. Kalenda Dibungi et al. "Congolese edible caterpillars, valuable sources of bioactive compounds with human health benefits". Journal of Insects as Food and Feed, 3 de noviembre de 2022, 1–12. http://dx.doi.org/10.3920/jiff2022.0072.
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Insects are part of the regular diet of more than two billion people around the world and are not only delicacies. Insects provide great opportunities to replace meals but can have important additional benefits as well. In the Democratic Republic of the Congo (DRC), caterpillars are the most consumed insects, and they are consumed by more than 70% of the population throughout the year. The aim of this research was to report the microscopic features, mineral micronutrients, chromatographic fingerprints, antioxidant activities and peroxidase inhibition of edible Congolese caterpillars of the genus Cinabra, Imbrasia and Gonimbrasia from DRC. Microscopic analysis showed the presence of characteristic features, specific to each host plant of caterpillars, such as palisade cells, stomata, trichomes, sclereids, fibres, vessels, pollen and starch grains. Phytochemical screening by chromatographic techniques revealed the presence of phenolic acids, flavonoids and terpenes as major secondary metabolites. Elemental analysis on dry matter showed that studied caterpillars are insects containing significant amounts of micronutrients such as copper, magnesium, manganese, selenium and zinc. Gonimbrasia belina had the highest selenium, magnesium and zinc content (0.12 g/100 g, 0.17 g/100 g and 0.011 g/100 g, respectively) than Cirina forda, Cinabra hyperbius, Imbrasia truncata and Imbrasia sp., C. forda and Imbrasia sp. had the highest copper content (0.003 g/100 g). C. forda had the highest manganese content (0.006 g/100 g). All aqueous extracts displayed high radical-scavenging activities with IC50 values ranging from 10 to 80 μg/ml. Extracts showed the best cellular antioxidant activities on reactive oxygen species-induced chemiluminescence using L012 on human leucocytes 60 monocytes related to their IC50 values less than 0.5 μg/ml. In specific immuno-extraction followed by enzymatic detection of myeloperoxidase assay, all extracts of caterpillars exhibited a dose-dependent inhibitory effect on myeloperoxidase activity in the range concentrations of 1 to 20 μg/ml excepted extracts of Imbrasia epimethea, Imbrasia sp. and I. truncata. Our results showed that insects are not only valuable source of lipids, proteins and micronutrients such as selenium but also are sources of phytochemicals with therapeutic benefits.