T cells of colorectal cancer patients' stimulated by neoantigenic and cryptic peptides better recognize autologous tumor cells.

BACKGROUND: Patients with cancers that exhibit extraordinarily high somatic mutation numbers are ideal candidates for immunotherapy and enable identifying tumor-specific peptides through stimulation of tumor-reactive T cells (Tc). METHODS: Colorectal cancers (CRC) HROC113 and HROC285 were selected based on high TMB, microsatellite instability and HLA class I expression. Their HLA ligandome was characterized using mass spectrometry, compared with the HLA ligand atlas and HLA class I-binding affinity was predicted. Cryptic peptides were identified using Peptide-PRISM. Patients' Tc were isolated from either peripheral blood (pTc) or tumor material (tumor-infiltrating Tc, TiTc) and expanded. In addition, B-lymphoblastoid cells (B-LCL) were generated and used as antigen-presenting cells. pTc and TiTc were stimulated twice for 7 days using peptide pool-loaded B-LCL. Subsequently, interferon gamma (IFNγ) release was quantified by ELISpot. Finally, cytotoxicity against autologous tumor cells was assessed in a degranulation assay. RESULTS: 100 tumor-specific candidate peptides-97 cryptic peptides and 3 classically mutated neoantigens-were selected. The neoantigens originated from single nucleotide substitutions in the genes IQGAP1, CTNNB1, and TRIT1. Cryptic and neoantigenic peptides inducing IFNγ secretion of Tc were further investigated. Stimulation of pTc and TiTc with neoantigens and selected cryptic peptides resulted in increased release of cytotoxic granules in the presence of autologous tumor cells, substantiating their improved tumor cell recognition. Tetramer staining showed an enhanced number of pTc and TiTc specific for the IQGAP1 neoantigen. Subpopulation analysis prior to peptide stimulation revealed that pTc mainly consisted of memory Tc, whereas TiTc constituted primarily of effector and effector memory Tc. This allows to infer that TiTc reacting to neoantigens and cryptic peptides must be present within the tumor microenvironment. CONCLUSION: These results prove that the analyzed CRC present both mutated neoantigenic and cryptic peptides on their HLA class I molecules. Moreover, stimulation with these peptides significantly strengthened tumor cell recognition by Tc. Since the overall number of neoantigenic peptides identifiable by HLA ligandome analysis hitherto is small, our data emphasize the relevance of increasing the target scope for cancer vaccines by the cryptic peptide category.

A case of Legionnaires' disease with severe rhabdomyolysis misdiagnosed as COVID-19.

Background: COVID-19 case numbers have begun to rise with the recently reported Omicron variant. In the last two years, COVID-19 is the first diagnosis that comes to mind when a patient is admitted with respiratory symptoms and pulmonary ground-glass opacities. However, other causes should be kept in mind as well. Here we present a case of Legionnaires' disease misdiagnosed as COVID-19. Case presentation: A 48-year-old male was admitted with complaints of dry cough and dyspnea. Chest computed-tomography revealed bilateral ground-glass opacities; therefore, a preliminary diagnosis of COVID-19 was made. However, two consecutive COVID PCR tests were negative and the patient deteriorated rapidly. As severe rhabdomyolysis and acute renal failure were present, Legionnaires' disease was suspected. Urine antigen test for Legionella and Legionella pneumophila PCR turned out to be positive. The patient responded dramatically to intravenous levofloxacin and was discharged successfully. Discussion: Legionnaires' disease and COVID-19 may present with similar signs and symptoms. They also share common risk factors and radiological findings. Conclusions: Shared clinical and radiological features between COVID-19 and other causes of acute respiratory failure pose a challenge in diagnosis. Other causes such as Legionnaires' disease must be kept in mind and appropriate diagnostic tests should be performed accordingly.

Effects of Tooth Brushing and MouthWashing on Leaching Bisphenol A Levels From an Orthodontic Adhesive: An In Vitro Study.

OBJECTIVE: To assess the levels of bisphenol A (BPA) released from an orthodontic adhesive with respect to the effects of tooth brushing and mouth washing. METHODS: Three groups, each containing fifteen adhesive samples were prepared. In Group 1, samples were polymerized according to manufacturer instructions. In Group 2, after the same polymerization protocol, each sample was brushed with a fluoride-containing toothpaste. For Group 3, samples were immersed in a mouth washing solution after polymerization. Later, all samples were placed into glass tubes containing 5 mL distilled water. High-performance liquid chromatography (HPLC) measurements were performed to assess the leaching amount of BPA. Intergroup comparison was performed by one way ANOVA test. RESULTS: Mean amounts of BPA were found to be 0.2674 µg/L, 0.2692 µg/L, and 0.2705 µg/L, respectively. Only a significant difference was found between Group 1 and 3 (P < .01), revealing higher BPA levels with the mouth washing solution. CONCLUSION: Measurable amounts of BPA release were observed in all groups of orthodontic adhesive samples, but the detected amounts were below the toxic levels. From a clinical point of view, alcohol-containing mouth washing solutions might increase the amount of leaching monomer, since alcohol is solvent of BPA.

Trictide, a tricellulin-derived peptide to overcome cellular barriers.

The majority of tight junction (TJ) proteins restrict the paracellular permeation of solutes via their extracellular loops (ECLs). Tricellulin tightens tricellular TJs (tTJs) and regulates bicellular TJ (bTJ) proteins. We demonstrate that the addition of recombinantly produced extracellular loop 2 (ECL2) of tricellulin opens cellular barriers. The peptidomimetic trictide, a synthetic peptide derived from tricellulin ECL2, increases the passage of ions, as well as of small and larger molecules up to 10 kDa, between 16 and 30 h after application to human epithelial colorectal adenocarcinoma cell line 2. Tricellulin and lipolysis-stimulated lipoprotein receptor relocate from tTJs toward bTJs, while the TJ proteins claudin-1 and occludin redistribute from bTJs to the cytosol. Analyzing the opening of the tricellular sealing tube by the peptidomimetic using super-resolution stimulated-emission depletion microscopy revealed a tricellulin-free area at the tricellular region. Cis-interactions (as measured by fluorescence resonance energy transfer) of tricellulin-tricellulin (tTJs), tricellulin-claudin-1, tricellulin-marvelD3, and occludin-occludin (bTJs) were strongly affected by trictide treatment. Circular dichroism spectroscopy and molecular modeling suggest that trictide adopts a ß-sheet structure, resulting in a peculiar interaction surface for its binding to tricellulin. In conclusion, trictide is a novel and promising tool for overcoming cellular barriers at bTJs and tTJs with the potential to transiently improve drug delivery.