Biochemical analysis of CTLA-4 immunoreactive material from human blood

<p>Abstract</p> <p>Background</p> <p>CTLA-4 was initially described as a membrane-bound molecule that inhibited lymphocyte activation by interacting with B7.1 and B7.2 molecules on antigen presenting cells. Alternative splicing of mRNA encoding the CTLA-4 receptor leads to the production of a molecule (sCTLA-4) that lacks a membrane anchor and is therefore secreted into the extracellular space. Despite studies finding that people with autoimmune disease more frequently express high levels of sCTLA-4 in their blood than apparently healthy people, the significance of these findings is unclear.</p> <p>Methods</p> <p>Molecules isolated from blood using CTLA-4 specific antibodies were analyzed with ligand binding assays, mass spectroscopy, and biochemical fractionation in an effort to increase our understanding of CTLA-4 immunoreactive material.</p> <p>Results</p> <p>Mass spectroscopy analysis of the molecules recognized by multiple CTLA-4-specific antibodies failed to identify any CTLA-4 protein. Even though these molecules bind to the CTLA-4 receptors B7.1 and B7.2, they also exhibit properties common to immunoglobulins.</p> <p>Conclusion</p> <p>We have identified molecules in blood that are recognized by CTLA-4 specific antibodies but also exhibit properties of immunoglobulins. Our data indicates that what has been called sCTLA-4 is not a direct product of the CTLA-4 gene, and that the CTLA-4 protein is not part of this molecule. These results may explain why the relationship of sCTLA-4 to immune system activity has been difficult to elucidate.</p

Traceability methods for cell line authentication and mycoplasma detection

Many laboratories struggle with mycoplasma contamination and cell line misidentification when growing cells in culture. These well-documented issues affect the scientific research community and have detrimental downstream effects. Research published with suspect cultures can produce misleading results. There is increasing pressure to verify the integrity of experimental and established cell lines before publishing. Therefore, laboratories need to define how and when to perform these critical tests, analyze the results, and determine action plans if disparities exist. Our laboratory is committed to producing cell lines of the highest quality for use in experiments; thus, we created a surveillance strategy for these potential problems. We developed processes for both testing and tracing cell line authentication and mycoplasma detection data. Using these methods, we can protect the integrity of our patient and commercial cell lines, maintaining reliable cultures for our research

Creating an affordable, user-friendly electronic inventory system for lab samples

Sharing physical laboratory space optimizes resource use among research departments, improves collaboration, and reduces costs. Universal inventory management is essential to successful space sharing, item tracking and availability, and data mining; therefore, our lab was interested in a tool for these purposes. Different research groups have differing needs, priorities, and considerations. We investigated prebuilt inventory management programs, and we found a custom Microsoft (MS) Access reference database to be optimal. We designed a custom MS Access abstract inventory data model around previously established needs with information gathered in staff interviews and meetings. We performed a physical inventory of all cryopreserved samples to allow uniform data entry when migrating to the database. We created manuals and protocols, and the staff was trained on entering data. Using a universal inventory system allows for quick report generation of physical inventory, which in turn preserves sample quality by reducing redundancy as well as time locating vials. Users can run queries to report a wide range of sample characteristics. Storage information can be easily analyzed for statistically significant trends, samples have reliable traceability, and data are auditable for accuracy

A mechanistic study of immune system activation by fusion of antigens with the ligand-binding domain of CTLA4

Fusion proteins consisting of the ligand-binding domain of CTLA4 covalently attached to an antigen (Ag) are potent immunogens. This fusion strategy effectively induces Ag-specific immunity both when introduced as a DNA-based vaccine and as a recombinant protein. CTLA4 is a ligand for B7 molecules expressed on the surface of antigen-presenting cells (APCs), and this interaction is critical for the fusion protein to stimulate Ag-specific immunity. We show that interaction of the fusion protein with either B7-1 or B7-2 is sufficient to stimulate immune activity, and that T cells are essential for the development of IgG responses. In addition, we demonstrate that human dendritic cells (DCs) pulsed with CTLA4-Ag fusion proteins can efficiently present Ag to T cells and induce an Ag-specific immune response in vitro. These studies provide further mechanistic understanding of the process by which CTLA4-Ag fusion proteins stimulate the immune system, and represent an efficient means of generating Ag-specific T cells for immunotherapy

Association between the 10-Year ASCVD risk score and COVID-19 complications among healthy adults (analysis from the National Cohort COVID Collaborative)

COVID-19 complications have been linked to worse outcomes among patients with established atherosclerotic cardiovascular disease (ASCVD). Less is known about the cumulative consequences of multiple ASCVD risk factors on COVID-19 outcomes. We evaluated the dose-response associations between 10-year ASCVD risk scores and COVID-19 complications. The National COVID-19 Cohort Collaborative collects electronic health record data from over 70 US health systems. Our analysis was limited to patients with positive COVID-19 tests without documented ASCVD events at the time of the first positive test. We evaluated the dose-response associations between 10-year ASCVD risk scores, categorized into categorized as low (20.0%), and COVID-19 complications, including hospitalizations and mortality. We reported the outcomes using multivariable-adjusted hazard ratios and 95% confidence intervals (CIs). Our cohort included 120,335 patients with documented positive COVID-19 test results who were free of ASCVD events. The mean age was 51.9 ± 16.1 years, 59.4% were women, 15.3% were Black, and 13.7% were Hispanic/Latino. Overall, 15,363 patients (12.8%) were hospitalized and 2,058 (1.7%) died. Patients at intermediate risk of developing ASCVD were had a 1.49 (95% CI 1.41 to 1.56) increased risk of hospitalization and 1.77 (95% CI 1.76 to 1.79) increased risk of mortality compared with patients at low risk. Patients at high risk had a 2.23 (95% CI 2.10 to 2.38) increased risk of hospitalization and a 5.98 (95% CI 5.93 to 6.03) increased risk of mortality. In conclusion, patients in this nationwide cohort at high risk of developing ASCVD are at substantially greater risk of COVID-19 complications. COVID-19 mitigation efforts should focus on these patient populations

Concordant genomic vulnerabilities of patient-derived cell line and matched xenograft-derived cell line through whole exome sequencing in breast-brain metastatic cancer

Background: Brain metastasis (mets) is often associated with the worst prognosis among all disseminated disease, and one of the two main sources of brain mets is the breast, with reported 1-year survival rate of 20% once developed. Breast-to-brain mets (BBM) usually happens in a late stage of breast cancer, and options of treatment are very limited, which becomes a major limitation of life expectancy. Therefore, developing a cost-efficient, robust model that could precisely recapitulate the features of tumor origin would be beneficial to discovery of novel therapeutic strategies to further improve outcomes. Purpose: Patient-derived xenografts (PDXs) often accurately recapitulate the tumor of origin in terms of genomic landscape, histopathology, and therapeutic response; however, restrictions 362 JPCRR • Volume 7, Issue 4 • Fall 2020 Supplement such as cost, high maintenance, and limited amenability for large-scale screening for potential therapies remain a challenge. To overcome these issues, we established a platform to derive cell lines from both a patient’s BBM tumor and the matched PDX to further investigate the similarity of their pathogenic genomic variants. Methods: DNAs from the first passage of cells derived from patient tumor along with 2 different passages of cells derived from PDXs were extracted, followed by whole exome sequencing analysis. Raw FASTQ files were quality controlled using FASTQC and then aligned to hg38 using BWA-MEM without trimming. The aligned BAM files were processed using GATK4, following best practice. Mutations were called using mutect2 without normal control. Classification of variants was finally referred to ClinVar. Results: Cells derived from PDXs (PDXL) has a significantly shorter doubling time than cells derived from patient tumor (PTL). PDXL was able to recapitulate the entire spectrum of PTL’s pathogenic variants. Exome sequencing analyses delineated several pathogenic variants of some common oncogenes and tumor suppressors, which potentially contributed to progression of this tumor. PDXL and PTL have shown consistent response to drugs that target the certain pathogenic variants. Conclusion: Our patient tumor-PDX cell line platform represents a preclinical tool for functional gene validation and proof-of-concept studies to identify novel druggable vulnerabilities in BBM, which could be further applied to other types of brain mets

Cardiac Troponin T and i Release after a 30-km Run

Prolonged endurance-type exercise is associated with elevated cardiac troponin (cTn) levels in asymptomatic recreational athletes. It is unclear whether exercise-induced cTn release mirrors a physiological or pathological underlying process. The aim of this study was to provide a direct comparison of the release kinetics of high-sensitivity cTnI (hs-cTnI) and T (hs-cTnT) after endurance-type exercise. In addition, the effect of remote ischemic preconditioning (RIPC), a cardioprotective strategy that limits ischemia-reperfusion injury, was investigated in a randomized controlled crossover manner. Twenty-five healthy volunteers completed an outdoor 30-km running trial preceded by RIPC (4 × 5 min 220 mm Hg unilateral occlusion) or control intervention. hs-cTnT, hs-cTnI, and sensitive cTnI (s-cTnI) concentrations were examined before, immediately after, 2 and 5 hours after the trial. The completion of a 30-km run resulted in a significant increase in circulating cTn (time: all p <0.001), with maximum hs-cTnT, hs-cTnI, and s-cTnI levels of 47 ± 27, 69 ± 62, and 82 ± 64 ng/L (mean ± SD), respectively. Maximum hs-cTnT concentrations were measured in 60% of the participants at 2 hours after exercise, compared with maximum hs-cTnI and s-cTnI concentrations at 5 hours in 84% and 80% of the participants. Application of an RIPC stimulus did not reduce exercise-induced cTn release (time × trial: all p >0.5). In conclusion, in contrast to acute myocardial infarction, maximum hs-cTnT levels after exercise precede maximum hs-cTnI levels. Distinct release kinetics of hs-cTnT and hs-cTnI and the absence of an effect of RIPC favors the concept that exercise-induced cTn release may be mechanistically distinct from cTn release in acute myocardial infarction