A simple flow cytometric technique for rapid measurement of multilog cytotoxic responses to photosensitization of cellular systems is described. This technique is particularly useful for cell lines with a low colony-forming efficiency, for which a nonclonogenic assay is required. The assay separates cell-sized objects from cellular debris by gating on forward scatter versus side scatter, identifies viable cells by positive calcein AM and negative ethidium homodimer-1 staining and measures cell concentration relative to an internal standard of polystyrene beads. Large numbers of cells can be analyzed rapidly. Two patient-derived small cell lung cancer cell lines, NCI-H209 and SV-E, were used to test the technique. Photordiation survival curves of the response of these cell lines to 5-aminolevulinic acid-induced protoprophyrin IX photosensitization correlated with the extent of photosensitizer accumulation. There was good agreement between the results obtained using the tritiated thymidine incorporation assay and the flow cytometric cytotoxicity assay. The technique can be used to measure cytotoxic responses to photosensitization of cell lines regardless of their plating efficiencies.

Allorecognition is known to involve a large number of lymphocytes carrying diverse T-cell receptor repertoire. Thus, one way to understand allorecognition and rejection mechanisms is via high-throughput sequencing of T-cell receptors. In this study, in order to explore and systematize the properties of the alloreactive T-cell receptor repertoire, we modeled direct and indirect allorecognition pathways using material from inbred mice in vitro and in vivo. Decoding of the obtained T-cell receptor genes using high-throughput sequencing revealed some features of the alloreactive repertoires. Thus, alloreactive T-cell receptor repertoires were characterized by specific V-gene usage patterns, changes in CDR3 loop length, and some amino acid occurrence probabilities in the CDR3 loop. Particularly pronounced changes were observed for directly alloreactive clonotypes. We also revealed a clustering of directly and indirectly alloreactive clonotypes by their ability to bind a single antigen; amino acid patterns of the CDR3 loop of alloreactive clonotypes; and the presence in alloreactive repertoires of clonotypes also associated with infectious, autoimmune, and tumor diseases. The obtained results were determined by the modeling of the simplified allorecognition reaction in inbred mice in which stimulation was performed with a single MHCII molecule. We suppose that the decomposition of the diverse alloreactive TCR repertoire observed in humans with transplants into such simple reactions will help to find alloreactive repertoire features; e.g., a dominant clonotype or V-gene usage pattern, which may be targeted to correct the entire rejection reaction in patients. In this work, we propose several technical ways for such decomposition analysis, including separate modeling of the indirect alloreaction pathway and clustering of alloreactive clonotypes according to their ability to bind a single antigen, among others.

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BackgroundRheumatoid arthritis (RA) is one of the most common autoimmune diseases. Currently, disease-modifying drugs and biological agents are used to treat RA [1]. The available drugs are not perfect: they have serious side effects and do not always cause a stable improvement or remission [2]. The above sets the task of finding new approaches to treatment that will be effective, more specific and safe. In this connection, it is necessary to develop and apply experimental models as close as possible in pathogenesis to rheumatoid arthritis. One such model, rarely used at present, is the combined antigen-collagen-induced arthritis [3].ObjectivesTo show immunological and histological changes similar to RA in the AIA/CIA model and the validity of its application in research activities.MethodsExperimental AIA/CIA was induced according to 2 different protocols in 50 BALB/c mice. Clinical assessment of arthritis was made by measuring the swelling of the paws with a caliper at different times. The assessment of immunological changes included the analysis of the content of antibodies to type II collagen by ELISA, the content of T-regulatory cells by flow cytometry. Also, a histological analysis of the obtained data was carried out.ResultsOn the 10th day, a significant increase in paw thickness was recorded in animals induced both according to the first and second protocols. The intensity of swelling subsided by the 23rd day. A significant increase in the content of antibodies to type II collagen was observed in all experimental groups, but in animals from Protocol No. 1, the amount of antibodies to type II collagen was significantly higher. A high level of T-regulatory cells was registered only in mice induced according to the first protocol on the 10th day. Histological changes in the form of synovial hyperplasia, pannus, usurations were observed to varying degrees in all experimental groups, but the most pronounced changes were in animals from the first protocol.ConclusionIn experimental animals, in all the presented protocols, changes were observed that were closest to RA, when compared with classical models of experimental arthritis induction. Based on the fact that protocol 1 animals showed an increase in the content of T-regulatory cells, the levels of antibodies to type 2 collagen were consistently high, and the histological changes were the most pronounced, it can be assumed that protocol 1 of the combined AIA/CIA model on the line of Balb/c mice, is the most suitable for testing and developing new methods of RA therapy.References[1]Abbasi M, Mousavi MJ, Jamalzehi S, Alimohammadi R, Bezvan MH, Mohammadi H, Aslani S. Strategies toward rheumatoid arthritis therapy; the old and the new. J Cell Physiol. 2019 Jul;234(7):10018-10031. doi: 10.1002/jcp.27860. Epub 2018 Dec 7. PMID: 30536757.[2]Greenberg JD, Reed G, Kremer JM, Tindall E, Kavanaugh A, Zheng C, Bishai W, Hochberg MC; CORRONA Investigators. Association of methotrexate and tumour necrosis factor antagonists with risk of infectious outcomes including opportunistic infections in the CORRONA registry. Ann Rheum Dis. 2010 Feb;69(2):380-6. doi: 10.1136/ard.2008.089276. Epub 2009 Apr 8. PMID: 19359261; PMCID: PMC2861900.[3]Baddack U, Hartmann S, Bang H, Grobe J, Loddenkemper C, Lipp M, Müller G. A chronic model of arthritis supported by a strain-specific periarticular lymph node in BALB/c mice. Nat Commun. 2013;4:1644. doi: 10.1038/ncomms2625. PMID: 23552059; PMCID: PMC3644064Disclosure of InterestsNone declared