Tumor inflating lymphocytes. Purification, expanding and cytotoxicity analisys on primary tumor cultures

Background. Tumor Infiltrating Lymphocytes (TILs) is one of the most promising sources of autologous cytotoxic T-cells for adoptive immunotherapy, which has already shown high efficiency in the treatment of metastatic melanoma. However, the isolation of TILs from solid tumors is technically difficult. A suppressive tumor microenvironment, in particular, a high level of expression of check-point inhibitors PD-1 CTLA4, tissue hypoxia and other factors cause that T cells isolated from the tumor do not proliferate well and do not exhibit cytotoxic properties. Aims. In this study, we isolated TILs from surgical material obtained by resection of solid tumors (primary and metastatic adenocarcinomas of various localization, melanoma, glioblastoma), studied their population composition and developed protocols for the purification expanding, and activation of CD4+, CD8+ cytotoxic antitumor lymphocytes. Methods. An urgent task is the activation of TILs, turning off immunosuppressive mechanisms and increasing their antitumor cytotoxic activity. Various approaches are used for this: activation by a cocktail of cytokines and antibodies, editing the lymphocyte genome by knocking out suppressor genes or, conversely, transduction of activating genes, coincubation with feeder cells, etc. Cells were obtained from samples of resected tumors in 16 patients; in each case we obtain an autologous pair: the primary tumor culture and the TILs culture. Results. We could isolate viable lymphocytes in 100% of cases. Isolated TILs were successfully expanded in our specialized medium using various combinations of IL-2, IL-15, IL-21, IL-7, anti-CD3 and anti-CD28. Immunophenotyping showed that the obtained TILs are a heterogeneous mixture of CD4+, CD8+ cells containing populations of CD3+CD8+CD45+(CTL) CD3+CD4+CD45+ (T-helpers), CD4+CD25+CD127- (Т-regulatory cells), CD3-CD56+CD45+ (NK-cells), CD3+CD56+CD45+ (Т-NK-cells). The initial cultures of TILs were also characterized by a high level of PD1 expression, indicating their low antitumor cytotoxicity. Using different protocols of isolation, expansion, and activation, we obtained a cell preparation containing 80% of CD8+ PD-1- activated TILs in an amount sufficient for adoptive therapy (500106 or more). An in vitro study of the cytotoxicity of obtained TILs in primary cultures of homologous tumors using RTCA Icelligence showed high cytotoxicity, providing almost 100% tumor cell death. Conclusion. Our developed protocol for the production and activation of TILs can be recommended for the phase III clinical trials of adoptive immunotherapy of recurrent, highly metastatic solid tumors

A Comparative Cross-Sectional Study Assessing the Psycho-Emotional State of Intensive Care Units’ Physicians and Nurses of COVID-19 Hospitals of a Russian Metropolis

Working in intensive care units (ICUs) is stressful and potentially leads to various psychoemotional disorders. Today, this issue represents a serious concern to the healthcare sector and affects the quality of healthcare provided. This study aimed to assess and compare the psycho-emotional state in COVID-19 and non-COVID-19 hospitals’ ICU healthcare workers (HCWs). From January to July 2021, we conducted an anonymous cross-sectional web survey of ICU physicians and nurses (N = 1259) of various hospitals in a metropolis with a population of over 10 million people. The statistical distributions of non-COVID-19 ICU HCWs showed the following results: emotional exhaustion levels (low 14.6%, average 30.8%, and high 54.6%); depersonalization levels (low 11.6%, average 16.5%, and high 71.9%); and reduced personal accomplishment levels (low 23.5%, average 40.3%, and high 36.2%). The statistical distributions of COVID-19 ICU HCWs showed the following results: emotional exhaustion levels (low 16.5%, average 31.5%, and high 52%); depersonalization levels (low 7.4%, average 9.4%, and high 83.1%); and reduced personal accomplishment levels (low 25.4%, average 45.4%, and high 29.1%). This study found a strong correlation between emotional exhaustion, aggression, and depersonalization in non-COVID-19 ICU HCWs and also found a correlation between their age, aggression, emotional exhaustion, and occupational stress

Epigenetic Clock and Circadian Rhythms in Stem Cell Aging and Rejuvenation

This review summarizes the current understanding of the interaction between circadian rhythms of gene expression and epigenetic clocks characterized by the specific profile of DNA methylation in CpG-islands which mirror the senescence of all somatic cells and stem cells in particular. Basic mechanisms of regulation for circadian genes CLOCK-BMAL1 as well as downstream clock-controlled genes (ССG) are also discussed here. It has been shown that circadian rhythms operate by the finely tuned regulation of transcription and rely on various epigenetic mechanisms including the activation of enhancers/suppressors, acetylation/deacetylation of histones and other proteins as well as DNA methylation. Overall, up to 20% of all genes expressed by the cell are subject to expression oscillations associated with circadian rhythms. Additionally included in the review is a brief list of genes involved in the regulation of circadian rhythms, along with genes important for cell aging, and oncogenesis. Eliminating some of them (for example, Sirt1) accelerates the aging process, while the overexpression of Sirt1, on the contrary, protects against age-related changes. Circadian regulators control a number of genes that activate the cell cycle (Wee1, c-Myc, p20, p21, and Cyclin D1) and regulate histone modification and DNA methylation. Approaches for determining the epigenetic age from methylation profiles across CpG islands in individual cells are described. DNA methylation, which characterizes the function of the epigenetic clock, appears to link together such key biological processes as regeneration and functioning of stem cells, aging and malignant transformation. Finally, the main features of adult stem cell aging in stem cell niches and current possibilities for modulating the epigenetic clock and stem cells rejuvenation as part of antiaging therapy are discussed

Oncolytic therapy with recombinant vaccinia viruses targeting the interleukin-15 pathway elicits a synergistic response

We developed recombinant variants of oncolytic vaccinia virus LIVP strain expressing interleukin-15 (IL-15) or its receptor subunit alpha (IL-15Rα) to stimulate IL-15-dependent immune cells. We evaluated their oncolytic activity either alone or in combination with each other in vitro and in vivo using the murine CT26 colon carcinoma and 4T1 breast carcinoma models. We demonstrated that the admixture of these recombinant variants could promote the generation of the IL-15/IL-15Rα complex. In vitro studies indicated that 4T1 breast cancer cells were more susceptible to the developed recombinant viruses. In vivo studies showed significant survival benefits and tumor regression in 4T1 breast cancer syngeneic mice that received a combination of LIVP-IL15-RFP with LIVP-IL15Ra-RFP. Histological analysis showed recruited lymphocytes at the tumor region, while no harmful effects to the liver or spleen of the animals were detected. Evaluating tumor-infiltrated lymphocytes represented profound activation of cytotoxic T cells and macrophages in mice receiving combination therapy. Thus, our experiments showed superior oncolytic effectiveness of simultaneous injection of LIVP-IL15-RFP and LIVP-IL15Ra-RFP in breast cancer-bearing mice. The combined therapy by these recombinant variants represents a potent and versatile approach for developing new immunotherapies for breast cancer

cultural exclusion and frontier zones

The functioning of cultural memory is always accompanied by the emergence of suppression zones covering the experiences and phenomena which were excluded, for some reason or another, from conventional cultural practices. The scope of excluded phenomena is as broad as possible and reaches from inconvenient events, works of art, historical or cultural figures to certain forms of expression, gestures, emotions, material objects, attitudes, discourse frameworks and narration patterns. Being repressed as they are, they, however, still influence the drawing of cultural borderlines and the processes of identity construction. Such dialectics of repression and (re-) actualization can be best characterized through the notion of “cultural exclusion zones” (similar to Chernobyl exclusion zone). In the current issue of “Rivista di Estetica” we thus try to discover and to describe those exclusion zones, the mechanisms of their formation and their multifold impact upon the contents of culture in different social, historical, epistemological and cultural contexts