RESUMO
BACKGROUND: Immune thrombocytopenia (ITP) is characterized by immune response dysregulations. Cytotoxic T lymphocyte-associated antigen-4 (CTLA-4) plays a central role in immune checkpoint pathways and preventing autoimmune diseases by regulating immune tolerance. We aimed to explore the potential association between CTLA-4 gene polymorphisms and ITP as well as study their impact on the response to therapy. METHODS: We investigated two CTLA-4 single-nucleotide polymorphisms (SNPs; rs: 231775 and rs: 3087243) using real-time PCR as well as the plasma levels of CTLA-4 by ELISA in 88 patients with ITP and 44 healthy participants (HC). RESULTS: CTLA-4 (rs: 3087243) A > G polymorphism analysis showed most HC had the homozygous AA genotype, which was statistically significant compared to patients with ITP. Plasma levels of CTLA4 were statistically lower in patients with acute ITP. There was no correlation between CTLA-4 (rs: 231775 and rs: 3087243) A/G SNPs were not correlated to the response to all lines of therapy assessed (corticosteroids, thrombopoietin receptor agonists, splenectomy, and rituximab). CONCLUSION: CTLA-4 CT 60 A/G may affect the susceptibility of ITP, but both CTLA-4 + 49 A/G and CT60 A/G did not impact the response of patients with ITP to different lines of therapy.
RESUMO
In a precarious world of rapidly growing pandemics, the field of vaccine production has witnessed considerable growth. Bacillus Calmette-Guérin (BCG) is a live-attenuated vaccine and a part of the immunization program in 157 countries. The quality control is based on a potency test through viable cell enumeration. The colony-forming unit (CFU) assay is the official method, however, it often yields fluctuating results, suffers from medium cracking, and requires lengthy analysis (~ 28 days). Flow cytometric analysis was proposed earlier, but it was coupled with a Coulter counter for measuring the entire bacterial population (live/dead). In the present study, thiazole orange/propidium iodide dyes supplemented with fluorogenic reference beads were employed for viable counting, eliminating the need for a Coulter counter. Both the flow cytometry and the colorimetric technique employing tetrazolium salt were validated and compared to the CFU assay. The colorimetric assay displayed high precision, accuracy, and a strong positive correlation with the CFU assay. The flow cytometry assay demonstrated high precision and a notable ability to distinguish different forms of BCG cells (live, injured, and dead). It also exhibited a perfect positive correlation with the CFU assay. Both methods reduced the analysis time by > 26 days and eliminated the need for human intervention by automating the test.
