Similarities and differences in RANTES and (AOP)-RANTES-triggered signals : implications for chemotaxis

11 páginas, 8 figuras.Chemokines are a family of proinflammatory cytokines that attract and activate specific types of leukocytes. Chemokines mediate their effects via interaction with seven transmembrane G proteinÐcoupled receptors (GPCR). Using CCR5-transfected HEK-293 cells, we show that both the CCR5 ligand, RANTES, as well as its derivative, aminooxypentane (AOP)-RANTES, trigger immediate responses such as Ca2+ influx, receptor dimerization, tyrosine phosphorylation,and Gai as well as JAK/STAT association to the receptor. In contrast to RANTES, (AOP)-RANTES is unable to trigger late responses, as measured by the association of focal adhesion kinase (FAK) to the chemokine receptor complex, impaired cell polarization required for migration, or chemotaxis. The results are discussed in the context of the dissociation of the late signals, provoked by the chemokines required for cell migration, from early signals.Peer reviewe

Loewenstein Occupational Therapy Cognitive Assessment to Evaluate People with Addictions

Background. The LOTCA (Loewenstein Occupational Therapy Cognitive Assessment) battery is a cognitive screening test which is widely used in occupational health. However, no work has been found that explores its use in addiction treatment. Objectives of Study. To explore the convergent validity of LOTCA with neuropsychological tests that assess related cerebral functional areas. Methods. The LOTCA, along with a battery of neuropsychological tests, was administered to a sample of 48 subjects who start a treatment by substance or gambling addictions. Findings. A correlational pattern was observed of a considerable magnitude between the effects of the LOTCA scales and those of some neuropsychological tests, but not with others. There is barely any convergence in measures with memory and executive function tests. Relevance to Clinical Practice. There is a lack of research applying test of occupational assessment to populations of patients treated by addictive behaviors. The LOTCA seems to be a reliable and valid test for preliminary screening of function in certain cognitive areas, easy, and quick to use (around 30 minutes). However, it must be supplemented with other tests for a full and ecological assessment of patients. Limitations. An incident, small-size sample. Recommendations for Further Research. New studies are needed to explore the applicability, diagnostic validity, and whole psychometric quality of the test in addiction-related treatment

Deregulated cellular circuits driving immunoglobulins and complement consumption associate with the severity of COVID-19 patients

SARS-CoV-2 infection causes an abrupt response by the host immune system, which is largely responsible for the outcome of COVID-19. We investigated whether the specific immune responses in the peripheral blood of 276 patients were associated with the severity and progression of COVID-19. At admission, dramatic lymphopenia of T, B, and NK cells is associated with severity. Conversely, the proportion of B cells, plasmablasts, circulating follicular helper T cells (cTfh) and CD56–CD16+ NK-cells increased. Regarding humoral immunity, levels of IgM, IgA, and IgG were unaffected, but when degrees of severity were considered, IgG was lower in severe patients. Compared to healthy donors, complement C3 and C4 protein levels were higher in mild and moderate, but not in severe patients, while the activation peptide of C5 (C5a) increased from the admission in every patient, regardless of their severity. Moreover, total IgG, the IgG1 and IgG3 isotypes, and C4 decreased from day 0 to day 10 in patients who were hospitalized for more than two weeks, but not in patients who were discharged earlier. Our study provides important clues to understand the immune response observed in COVID-19 patients, associating severity with an imbalanced humoral response, and identifying new targets for therapeutic interventionThe study was funded by grants SAF2017- 82886-R to FS-M from the Ministerio de Economía y Competitividad, and from “La Caixa Banking Foundation” (HR17-00016) to FS-M. Grant PI018/01163 to CMC and grant PI19/00549 to AA were funded by Fondo de Investigaciones Sanitarias, Ministerio de Sanidad y Consumo, Spain. SAF2017-82886-R, PI018/01163 and PI19/00549 grants were also co-funded by European Regional Development Fund, ERDF/FEDER. This work has been funded by grants Fondo Supera COVID (CRUE-Banco de Santander) to FSM, and “Ayuda Covid 2019” from Comunidad de Madri

Inhibitory Role of Growth Hormone in the Induction and Progression Phases of Collagen-Induced Arthritis

Evidence indicates an intimate connection between the neuroendocrine and the immune systems. A number of in vitro and in vivo studies have demonstrated growth hormone (GH) involvement in immune regulation. The GH receptor is expressed by several leukocyte subpopulations, and GH modulates immune cell proliferation and activity. Here, we found that sustained GH expression protected against collagen-induced arthritis (CIA); in GH-transgenic C57BL/6 (GHTg) mice, disease onset was delayed, and its overall severity was decreased. The anti-collagen response was impaired in these mice, as were inflammatory cytokine levels. Compared to control arthritic littermates, immunized GHTg mice showed significantly lower RORγt (retinoic acid receptor-related orphan receptor gamma 2), IL-17, GM-CSF, IL-22, and IFNγ mRNA expression in draining lymph nodes, whereas there were no differences in IL-21, IL-6, or IL-2 mRNA levels. Data thus suggest that Th17/Th1 cell plasticity toward a pathological phenotype is reduced in these mice. Exogenous GH administration in arthritic DBA/1J mice reduced the severity of established CIA as well as the inflammatory environment, which also shows a GH effect on arthritis progression. These results indicate that GH prevents inflammatory joint destruction in CIA. Our findings demonstrate a modulatory GH role in immune system function that contributes to alleviating CIA symptoms and underlines the importance of endocrine regulation of the immune response

Use of eltrombopag for patients 65 years old or older with immune thrombocytopenia

Background Eltrombopag is useful for immune thrombocytopenia (ITP). However, results of clinical trials may not accurately mirror clinical practice reality. Here we evaluated eltrombopag for primary and secondary ITP in our ≥65‐year‐old population. Methods A total of 106 primary ITP patients (16 with newly diagnosed ITP, 16 with persistent ITP, and 74 with chronic ITP) and 39 secondary ITP patients (20 with ITP secondary to immune disorders, 7 with ITP secondary to infectious diseases, and 12 with ITP secondary to lymphoproliferative disorders [LPD]) were retrospectively evaluated. Results Median age of our cohort was 76 (interquartile range, IQR, 70‐81) years. 75.9% of patients yielded a platelet response including 66.2% complete responders. Median time to platelet response was 14 (IQR, 8‐21) days. Median time on response was 320 (IQR, 147‐526) days. Sixty‐three adverse events (AEs), mainly grade 1‐2, occurred. The most common were hepatobiliary laboratory abnormalities (HBLAs) and headaches. One transient ischemic attack in a newly diagnosed ITP and two self‐limited pulmonary embolisms in secondary ITP were the only thrombotic events observed. Conclusion Eltrombopag showed efficacy and safety in ITP patients aged ≥65 years with primary and secondary ITP. However, efficacy results in LPD‐ITP were poor. A relatively high number of deaths were observed

Technical Advance: Surface plasmon resonance-based analysis of CXCL12 binding using immobilized lentiviral particles

Use of SPR-based biosensors is an established method for measuring molecular interactions. Their application to the study of GPCRs is nonetheless limited to detergent-solubilized receptors that can then be reconstituted into a lipid environment. Using the chemokine receptor CXCR4 and its specific ligand CXCL12, we outline here a highly reproducible biosensor method based on receptor presentation on the surface of lentiviral particles; the approach is simple and does not require the use of antibodies to achieve correct receptor orientation on the sensorchip surface. We measured the kinetic parameters of CXCR4/CXCL12 binding in a single step and in real time and evaluated the effect of GAG presentation of chemokines on this interaction. The data indicate that at low concentrations, soluble heparin modulates CXCR4/CXCL12 interaction and at high concentrations, abrogates binding. These observations suggest that in addition to their known role in modulating local chemokine availability, GAG affect the receptor/ligand interaction, although their influence on affinity parameters is very limited. The method will also be useful for quantifying these biomarkers in biological fluids and for the development of high-throughput screening for their antagonists.B.V. is supported by a fellowship from the Spanish Ministry of Health FISS. This work was supported in part by grants from the Spanish Ministry of Science and Innovation (SAF 2008- 03388; SAF 2008-00908), the FISS (RD08/0075/0010), and the European Union (Innochem LSHB-CT-2005-518167 and FP7 Integrated Project Masterswitch 223404).Peer Reviewe

Implementation of a SPR immunosensor for the simultaneous detection of the 22K and 20K hGH isoforms in human serum samples

We have implemented a Surface Plasmon Resonance (SPR) immunosensor based on a sandwich assay for the simultaneous detection of the two main hGH isoforms, of 22 kDa (22K) and 20 kDa (20K). An oriented-antibody sensor surface specific for both hormone isoforms was assembled by using the biotin-streptavidin system. The immunosensor functionality was checked for the direct detection of the 22K hGH isoform in buffer, which gave high specificity and reproducibility (intra and inter-assay mean coefficients of variation of 8.23% and 9% respectively). The selective determination of the 22K and 20K hGH isoforms in human serum samples in a single assay was possible by using two specific anti-hGH monoclonal antibodies. The detection limit for both hormone isoforms was 0.9 ng mL -1 and the mean coefficient of variation was below 7.2%. The excellent reproducibility and sensitivity obtained indicate the high performance of this immunosensor for implementing an anti-doping test. © 2013 Elsevier B.V. All rights reserved.This research was carried out with the financial support from the M. Botín Foundation.Peer Reviewe

Single- and multi-analyte determination of gonadotropic hormones in urine by Surface Plasmon Resonance immunoassay

8 páginas, 3 figuras, 4 tablas.Single- and multi-analyte detection of two gonadotropic hormones (follicle stimulating hormone (hFSH) and luteinizing hormone (hLH)) was achieved by a Surface Plasmon Resonance (SPR) immunoassay on untreated human urine samples. Multi-analyte detection was accomplished using two alternative formats which are based in the individual or simultaneous immobilization of the hormones on the sensor surface. The lowest detection limit for both hormones in urine was found to be 1 ng mL−1, which in international units (IU) in terms of the World Health Organization (WHO) standards represents 8 mIU mL−1 of hLH and 14 mIU mL−1 of hFSH, respectively. The reliability of the assay was demonstrated by intra- and inter-assay variabilities <6%, chip-to-chip variabilities <5%, recoveries in the range of 80–120% and stability of the sensor response through more than 100 measurements. The sensitivity of this biosensing methodology renders it in a useful technique for the diagnosis of reproductive disorders, as well as for fertility monitoring.The authors gratefully acknowledge the financial support from Fundació La Marató de TV3 and Fundación M. Botín. The Department of Immunology and Oncology was founded and is supported by the Spanish National Research Council (CSIC) and by Pfizer.Peer reviewe

Surface plasmon resonance immunoassay analysis of pituitary hormones in urine and serum samples

7 páginas, 5 figuras, 1 tabla.Background: Direct determination of four pituitary peptide hormones: human thyroid stimulating hormone (hTSH), growth hormone (hGH), follicle stimulating hormone (hFSH), and luteinizing hormone (hLH) has been carried out using a portable surface plasmon resonance (SPR) immunosensor. Methods: A commercial SPR biosensor was employed. The immobilization of the hormones was optimized and monoclonal antibodies were selected in order to obtain the best sensor performance. Assay parameters as running buffer and regeneration solution composition or antibody concentration were adjusted to achieve a sensitive analyte detection. Results: The performance of the assays was assessed in buffer solution, serum and urine, showing sensitivity in the range from 1 to 6 ng/mL. The covalent attachment of the hormones ensured the stability of the SPR signal through repeated use in up to 100 consecutive assay cycles. Mean intra- and inter-day coefficients of variation were all < 7%, while batch-assay variability using different sensor surfaces was < 5%. Conclusions: Taking account both the excellent reutilization performance and the outstanding reproducibility, this SPR immunoassay method turns on a highly reliable tool for endocrine monitoring in laboratory and point-of-care (POC) settings.The authors gratefully acknowledge the financial support from Fundació La Marató de TV3 and Fundación M. Botín. The Department of Immunology and Oncology was funded and is supported by the Spanish National Research Council (CSIC) and by Pfizer.Peer reviewe

Determination of human growth hormone in human serum samples by surface plasmon resonance immunoassay

6 páginas, 4 figuras, 1 tabla.A surface plasmon resonance immunoassay has been developed to determine human growth hormone (hGH) directly and without pre-treatment in human serum samples. A binding inhibition immunoassay was employed. Antibody concentration, assay buffer and regeneration solution have been optimized in order to reach the best performance and the lower non-specific binding of the matrix components to the sensor surface. The lowest detection limit was 6 ng/mL, with a working range covering the physiological range. Reproducibility of the assay was excellent with both intra-assay and inter-assay relative standard deviations <5%, while a variation of 2.19% was obtained employing different sensor chips. Reutilization of the sensor surface allows its continuous use over 50 measurements with a signal drop <20%. The SPR immunoassay results were validated using enzyme-linked immunosorbent assay (ELISA) showing an excellent correlation (R2 = 0.985). A portable and fully automated system (Sensia SL) was employed in this work. This is the first SPR biosensor assay capable of detecting relevant concentrations of a clinical analyte in serum. This study shows the potentials of this device as a diagnostic tool for the detection of multiple clinical analytes.The authors gratefully acknowledge the financial support from Fundació La Marató de TV3 and Fundación M. Botín. The Department of Immunology and Oncology was found and is supported by the Spanish National Research Council (CSIC) and by Pfizer.Peer reviewe