Development of a High-Throughput Calcium Mobilization Assay for CCR6 Receptor Coupled to Hydrolase Activity Readout.

CCR6 is a chemokine receptor highly implicated in inflammatory diseases and could be a potential therapeutic target; however, no therapeutic agents targeting CCR6 have progressed into clinical evaluation. Development of a high-throughput screening assay for CCR6 should facilitate the identification of novel compounds against CCR6. To develop a cell-based assay, RBL-2H3 cells were transfected with plasmids encoding ß-hexosaminidase and CCR6. Intracellular calcium mobilization of transfected cells was measured with a fluorescent substrate using the activity of released hexosaminidase as readout of the assay. This stable, transfected cell showed a specific signal to the background ratio of 19.1 with low variability of the signal along the time. The assay was validated and optimized for high-throughput screening. The cell-based calcium mobilization assay responded to the specific CCR6 ligand, CCL20, in a dose-dependent manner with an EC50 value of 10.72 nM. Furthermore, the assay was deemed robust and reproducible with a Z' factor of 0.63 and a signal window of 7.75. We have established a cell-based high-throughput calcium mobilization assay for CCR6 receptor. This assay monitors calcium mobilization, due to CCR6h activation by CCL20, using hexosaminidase activity as readout. This assay was proved to be robust, easy to automate and could be used as method for screening of CCR6 modulators.

Covalent Immobilization of Antibodies through Tetrazine-TCO Reaction to Improve Sensitivity of ELISA Technique.

Enzyme-linked immunosorbent assay (ELISA) is routinely used to detect biomolecules related to several diseases facilitating diagnosis and monitoring of these, as well as the possibility of decreasing their mortality rate. Several methods have been carried out to improve the ELISA sensitivity through antibodies immobilization on the microtiter plates. Here, we have developed a strategy of antibodies immobilization to improve the ELISA sensitivity increasing the antibody density surface through the tetrazine (Tz)-trans-cyclooctene (TCO) reaction. For this, we prepared surfaces with tetrazine groups while the captured antibody was conjugated with TCO. The tetrazine surfaces were prepared in two different ways: (1) from aminated plates and (2) from Tz-BSA-coated plates. The surfaces were evaluated using two sandwich ELISA models, one of them using the low-affinity antibody anti-c-myc as a capture antibody to detect the c-myc-GST-IL8h recombinant protein, and the other one to detect the carcinoembryonic human protein (CEA). The sensitivity increased in both surfaces treated with tetrazine in comparison with the standard unmodified surface. The c-myc-GST-IL8h detection was around 10-fold more sensible on both tetrazine surfaces, while CEA ELISA detection increased 12-fold on surfaces coated with Tz-BSA. In conclusion, we show that it is possible to improve the ELISA sensitivity using this immobilization system, where capture antibodies bond covalently to surfaces.

Amino terminal recognition by a CCR6 chemokine receptor antibody blocks CCL20 signaling and IL-17 expression via ß-arrestin.

BACKGROUND: CCR6 chemokine receptor is an important target in inflammatory diseases. Th17 cells express CCR6 and a number of inflammatory cytokines, including IL-17 and IL-22, which are involved in the propagation of inflammatory immune responses. CCR6 antagonist would be a potential treatment for inflammatory diseases such as psoriasis or rheumatoid arthritis. The aim of this study is to develop an antagonistic monoclonal antibody (mAb) against human CCR6 receptor (hCCR6). RESULTS: We generate monoclonal antibodies against hCCR6 immunizing Balb/c mice with hCCR6 overexpressing cells. The antibodies were tested by flow cytometry for specific binding to hCCR6, cloned by limiting dilution and resulted in the isolation and purification monoclonal antibody 1C6. By ELISA and flow cytometry, was determined that the antibody obtained binds to hCCR6 N-terminal domain. The ability of 1C6 to neutralize hCCR6 signaling was tested and we determined that 1C6 antibody were able to block response in ß-arrestin recruitment assay with IC50 10.23 nM, but did not inhibit calcium mobilization. In addition, we found in a chemotaxis assay that 1C6 reduces the migration of hCCR6 cells to their ligand CCL20. Finally, we determined by RT-qPCR that the expression of IL-17A in Th17 cells treated with 1C6 was inhibited. CONCLUSIONS: In the present study, we applied whole cell immunization for successfully obtain an antibody that is capable to neutralize hCCR6 signaling and to reduce hCCR6 cells migration and IL-17 expression. These results provide an efficient approach to obtain therapeutic potential antibodies in the treatment of CCR6-mediated inflammatory diseases.

Clinical and Neurochemical Effects of Transcranial Magnetic Stimulation (TMS) in Multiple Sclerosis: A Study Protocol for a Randomized Clinical Trial.

Background: Transcranial Magnetic Stimulation (TMS) is a technique based on the principles of electromagnetic induction. It applies pulses of magnetic radiation that penetrate the brain tissue, and it is a non-invasive, painless, and practically innocuous procedure. Previous studies advocate the therapeutic capacity of TMS in several neurodegenerative and psychiatric processes, both in animal models and in human studies. Its uses in Parkinson's disease, Alzheimer's disease and in Huntington's chorea have shown improvement in the symptomatology and in the molecular profile, and even in the cellular density of the brain. Consequently, the extrapolation of these TMS results in the aforementioned neurodegenerative disease to other entities with etiopathogenic and clinical analogy would raise the relevance and feasibility of its use in multiple sclerosis (MS). The overall objective will be to demonstrate the effectiveness of the TMS in terms of safety and clinical improvement, as well as to observe the molecular changes in relation to the treatment. Methods and Design: Phase II clinical trial, unicentric, controlled, randomized, single blind. A total of 90 patients diagnosed with relapsing-remitting multiple sclerosis (RRMS) who meet all the inclusion criteria and do not present any of the exclusion criteria that are established and from which clinically evaluable results can be obtained. The patients included will be assigned under the 1:1:1 randomization formula, constituting three groups for the present study: 30 patients treated with natalizumab + white (placebo) + 30 patients treated with natalizumab + TMS (1 Hz) + 30 patients treated with natalizumab + TMS (5 Hz). Discussion: Results of this study will inform on the efficiency of the TMS for the treatment of MS. The expected results are that TMS is a useful therapeutic resource to improve clinical status (main parameters) and neurochemical profile (surrogate parameters); both types of parameters will be checked. Ethics and Dissemination: The study is approved by the Local Ethics Committee and registered in https://clinicaltrials.gov (NCT04062331). Dissemination will include submission to a peer-reviewed journal, patients, associations of sick people and family members, healthcare magazines and congress presentations. Trial Registration: ClinicalTrials.gov ID: NCT04062331 (registration date: 19th/ August/2019). Version Identifier: EMTr-EMRR, ver-3, 21/11/2017.

Highly enhanced ELISA sensitivity using acetylated chitosan surfaces.

BACKGROUND: The enzyme-linked immunosorbent assay (ELISA), is the most widely used and reliable clinical routine method for the detection of important protein markers in healthcare. Improving ELISAs is crucial for detecting biomolecules relates to health disorders and facilitating diagnosis at the early diseases stages. Several methods have been developed to improve the ELISA sensitivity through immobilization of antibodies on the microtiter plates. We have developed a highly sensitive ELISA strategy based on the preparation of acetylated chitosan surfaces in order to improve the antibodies orientation. RESULTS: Chitin surfaces were obtained by mixing small quantities of chitosan and acetic anhydride in each well of a microtiter plate. Anti-c-myc 9E10 low affinity antibody fused to ChBD was cloned and expressed in CHO cells obtaining the anti-c-myc-ChBD antibody. We found that anti c-myc-ChBD binds specifically to the chitin surfaces in comparison with anti-c-myc 9E10, which did not. Chitin surface was used to develop a sandwich ELISA to detect the chimeric human protein c-myc-GST-IL8 cloned and expressed in Escherichia coli. The ELISA assays developed on chitin surfaces were 6-fold more sensitive than those performed on standard surface with significant differences (p<0,0001). CONCLUSIONS: As shown here, acetylated chitosan surfaces improve the antibody orientation on the substrate and constitute a suitable method to replace the standard surfaces given the stability over time and the low cost of its preparation.

The apo A-I gene promoter region polymorphism determines the severity of hyperlipidemia after heart transplantation.

BACKGROUND: To study whether the Apolipoprotein A-I (apo A-I) promoter region gene polymorphism produces changes in the lipid profile of heart transplant recipients. METHODS: One hundred and three heart transplant recipients (93 men and 10 women, with a mean age of 47 +/- 13 yr) receiving triple immunosuppressive therapy were submitted to a genetic study of the apo A-I gene promoter region. Anthropometric and analytical data, including lipid profile, arterial blood pressure, were collected prior to transplantation and 3, 6, 12, and 24 months after transplantation. RESULTS: Sixty-three subjects had the GG genotype and 40 the GA genotype. Carriers of the GA genotype had higher triglyceride levels at 6 months and 2 yr (2.50 +/- 1.20 versus 1.93 +/- 0.98 mmol/L and 2.46 +/- 1.58 versus 1.60 +/- 0.68 mmol/L, respectively, p < 0.001), and a greater rise in LDL-cholesterol at 1 yr than the GG subjects (4.57 +/- 1.16 versus 4.16 +/- 1.18 mmol/L, p < 0.05). Multiple regression analyses showed that genetic variants at the apo A-I promoter region are responsible for 11% of the variability in triglyceride levels at 6 months (p = 0.005). CONCLUSIONS: The GA genotype of the apo A-I promoter region produces a greater rise in plasma triglyceride and LDL-cholesterol levels in heart transplant patients.