Prothrombotic Phenotype in COVID-19: Focus on Platelets

COVID-19 infection is associated with a broad spectrum of presentations, but alveolar capillary microthrombi have been described as a common finding in COVID-19 patients, appearing as a consequence of a severe endothelial injury with endothelial cell membrane disruption. These observations clearly point to the identification of a COVID-19-associated coagulopathy, which may contribute to thrombosis, multi-organ damage, and cause of severity and fatality. One significant finding that emerges in prothrombotic abnormalities observed in COVID-19 patients is that the coagulation alterations are mainly mediated by the activation of platelets and intrinsically related to viral-mediated endothelial inflammation. Beyond the well-known role in hemostasis, the ability of platelets to also release various potent cytokines and chemokines has elevated these small cells from simple cell fragments to crucial modulators in the blood, including their inflammatory functions, that have a large influence on the immune response during infectious disease. Indeed, platelets are involved in the pathogenesis of acute lung injury also by promoting NET formation and affecting vascular permeability. Specifically, the deposition by activated platelets of the chemokine platelet factor 4 at sites of inflammation promotes adhesion of neutrophils on endothelial cells and thrombogenesis, and it seems deeply involved in the phenomenon of vaccine-induced thrombocytopenia and thrombosis. Importantly, the hyperactivated platelet phenotype along with evidence of cytokine storm, high levels of P-selectin, D-dimer, and, on the other hand, decreased levels of fibrinogen, von Willebrand factor, and thrombocytopenia may be considered suitable biomarkers that distinguish the late stage of COVID-19 progression in critically ill patients.

Development of a complex anti-radiation protective drug

The article reports results of development of a drug for the prevention and treatment of radiation lesions in animals based on radio modified microorganisms E. coli PL-6 and B. bifidum 1. Aimed at target changing of metabolism, bacteria were exposed to gamma irradiation at doses from 1 to 20 kGy, studying the interaction of microorganisms in the consortium, as well as the safety of produced drugs. Irradiation of E. coli at a dose of 2 kGy led to the appearance of individual cells with polymorphism and having a length exceeding the initial size by 3-7 times. The irradiation of B. bifidum 1 at a dose of 4 kGy contributed to the formation for atypical, polychromic cells, multiple darkening of pigment granules and cell fragments as a result of destruction of microorganisms. The original E. coli PL-6 did not produce the enzymes superoxide dismutaseand catalase, but the metabolites of radiomodified strains of E. coli PL-6 (R10) contained 0.97±0.09 m.c.M/g SOD and 27.38±0.59 mcat/g/ml of catalase activity. In the metabolites of radio-resistant bifidobacteria, as compared with the initial ones, a 1.45-fold excess of peroxidase was recorded.

207 Enhanced antigen capture, antigen-presenting cell (APC)-like function, and cytotoxic responses with chimeric engulfment receptor (CER) T cells

BackgroundActivated T cells have limited antigen presenting capability due to inefficient capture.1 This process can be enhanced through novel chimeric engulfment receptors (CERs) expressing a human Tim-4 phagocyte receptor that recognizes phosphatidylserine (Ptd-Ser)2 fused to T cell and macrophage/dendritic cell-derived signaling domains. CERs can facilitate antigen capture, processing, and presentation, and impart target-dependent cytotoxic function when expressed in T cells. This combined function is hypothesized to improve tumor clearance and durability of response, making CER T cell products ideal clinical candidates.MethodsWe generated Tim-4 receptors fused to toll-like receptor (TLR)-2 or -8, CD28 or CD3 zeta and tested phagocytic, antigen presentation and cytotoxic function in healthy donor T cells. To assess phagocytosis, target cells treated with a small molecule to induce Ptd-Ser externalization were labeled with pH-Rodo followed by co-culture with CER T cells. Activated CER T cells were evaluated by transmission electron microscopy (TEM) or flow cytometry (FC) for lysosomal uptake of cell fragments. Antigen capture and presentation were characterized by FC for the capacity of human papilloma virus 16 (HPV 16) E7 peptide-pulsed CER T cells to activate and induce proliferation of autologous HPV 16 E7-TCR transduced T cells. Cytotoxic function was evaluated in co-culture assays of CER T cells in the presence of subtherapeutic doses of BTKi (ibrutinib)-treated JeKo-1 lymphoma cells.ResultsTEM imaging demonstrated that CER T cells engulfed target cell fragments, illustrated by multi-vesicular bodies containing tumor fragments (some measuring >0.5 uM) and pseudo-pod like formations around apoptotic target cell blebs. RNA analysis revealed upregulation of TLR, myeloid differentiation, and antigen presentation pathways. In the HPV 16 E7 co-culture model, T-cell surface activation markers CD25 and CD69 were upregulated 41% and 23%, respectively, on E7-TCR-T cells relative to controls. In addition, the percentage of dividing E7-TCR-T cells was increased (44% vs 8%) after 6 days in co-culture. Addition of CER T cells to JeKo- 1 target cells in the presence of BTKi at low effector: target ratios enhanced cytotoxicity by over 99%, demonstrating synergy with a targeted small molecule to fully eliminate lymphoma cells.ConclusionsNovel Tim-4/TLR containing CERs can capture tumor cell fragments and present soluble antigen, a function previously demonstrated to be a barrier to effective antigen presentation in T cells. Enhanced T-cell antigen capture and presentation capability alongside inducible and target-specific cytotoxic function in single T cells represents a significant advancement in the potential for chimeric receptor-based therapies.ReferencesLanzavecchia A, Roosnek E, Gregory T, Berman P, Abrignani S. T cells can present antigens such as HIV gp120 targeted to their own surface molecules. Nature 1988 Aug 11;334(6182):530–2.Caronni N, Piperno GM, Simoncello F, Romano O, Vodret S, Yanagihashi Y, et al. TIM4 expression by dendritic cells mediates uptake of tumor-associated antigens and anti-tumor responses. Nat Commun 2021 Apr 14;12(1):2237.

Submicroscopic characteristics of the lacrimal fluid in vitreomacular traction syndrome, diabetic retinopathy compared with the norm

Study of the lacrimal fluid (LF) as a constant microenvironment of the anterior part of the eye which is the only atraumatically accessible substrate for the diagnosis and research of ophthalmic diseases, such as vitreomacular traction syndrome (VMTS), diabetic retinopathy (DR), makes it possible to study it using electronic microscopy methods. All studied LF samples contain cells and cell fragments; exosomes which are vesicles (40–100 nm) localized in multivesicular bodies, transmitting signals between cells and carrying markers of many diseases. Analysis of the samples revealed changes in the occurrence of these structures in VMTS and DR in comparison with healthy subjects. In this work, the components of the LF were visualized and their changes were established in DR and VMTS, which proves the value of the LF as a diagnostic substrate and determines the need for further research in order to formulate clear criteria for the diagnosis of these diseases in the early stages. Key words: lacrimal fluid; electronic microscopy; vitreomacular traction syndrome; diabetic retinopathy.

Simple and Efficient Protocol for Subcellular Fractionation of Normal and Apoptotic Cells

Subcellular fractionation approaches remain an indispensable tool among a large number of biochemical methods to facilitate the study of specific intracellular events and characterization of protein functions. During apoptosis, the best-known form of programmed cell death, numerous proteins are translocated into and from the nucleus. Therefore, suitable biochemical techniques for the subcellular fractionation of apoptotic cells are required. However, apoptotic bodies and cell fragments might contaminate the fractions upon using the standard protocols. Here, we compared different nucleus/cytoplasm fractionation methods and selected the best-suited approach for the separation of nuclear and cytoplasmic contents. The described methodology is based on stepwise lysis of cells and washing of the resulting nuclei using non-ionic detergents, such as NP-40. Next, we validated this approach for fractionation of cells treated with various apoptotic stimuli. Finally, we demonstrated that nuclear fraction could be further subdivided into nucleosolic and insoluble subfractions, which is crucial for the isolation and functional studies of various proteins. Altogether, we developed a method for simple and efficient nucleus/cytoplasm fractionation of both normal and apoptotic cells.

Platelets as drivers of ischemia/reperfusion injury after stroke

Ischemic stroke is a leading cause of morbidity and mortality worldwide and, despite reperfusion either via thrombolysis or thrombectomy, stroke patients often suffer from lifelong disabilities. These persistent neurological deficits may be improved by treating the ischemia/reperfusion (I/R) injury that occurs following ischemic stroke. There are currently no approved therapies to treat I/R injury, and thus it is imperative to find new targets to decrease the burden of ischemic stroke and related diseases. Platelets, cell fragments from megakaryocytes, are primarily known for their role in hemostasis. More recently, investigators have studied the nonhemostatic role of platelets in inflammatory pathologies, such as I/R injury after ischemic stroke. In this review, we seek to provide an overview of how I/R can lead to platelet activation and how activated platelets, in turn, can exacerbate I/R injury after stroke. We will also discuss potential mechanisms by which platelets may ameliorate I/R injury.

Comparison of porcine corneal decellularization methods and importance of preserving corneal limbus through decellularization

Background The aim of this study is to compare the three previously applied, conventional porcine corneal decellularization methods and to demonstrate the importance of preserving the corneal limbus through decellularization. Methods Fresh, wild-type (with or without) limbus porcine corneas were decellularized using three different methods, including (i) sodium dodecyl sulfate (SDS), (ii) hypertonic saline (HS), and (iii) N2 gas (NG). Post-treatment evaluation was carried out using histological, residual nuclear material, and ultrastructural analyses. Glycerol was used to help reduce the adverse effects of decellularization. The corneas were preserved for two weeks in cornea storage medium. Results All three decellularization methods reduced the number of keratocytes at different rates in the stromal tissue. However, all methods, except SDS, resulted in the retention of large numbers of cells and cell fragments. The SDS method (0.1% SDS, 48h) resulted in almost 100% decellularization in corneas without limbus. Low decellularization capacity of the NG method (<50%) could make it unfavorable. Although HS method had a more balanced damage-decellularization ratio, its decellularization capacity was lower than SDS method. Preservation of the corneoscleral limbus could partially prevent structural damage and edema, but it would reduce the decellularization capacity. Conclusion Our results suggest that SDS is a very powerful decellularization method, but it damages the cornea irreversibly. Preserving the corneoscleral limbus reduces the efficiency of decellularization, but also reduces the damage.

Encephalitozoon cuniculi takes advantage of efferocytosis to evade the immune response

Microsporidia are recognized as opportunistic pathogens in individuals with immunodeficiencies, especially related to T cells. Although the activity of CD8+ T lymphocytes is essential to eliminate these pathogens, earlier studies have shown significant participation of macrophages at the beginning of the infection. Macrophages and other innate immunity cells play a critical role in activating the acquired immunity. After programmed cell death, the cell fragments or apoptotic bodies are cleared by phagocytic cells, a phenomenon known as efferocytosis. This process has been recognized as a way of evading immunity by intracellular pathogens. The present study evaluated the impact of efferocytosis of apoptotic cells either infected or not on macrophages and subsequently challenged with Encephalitozoon cuniculi microsporidia. Macrophages were obtained from the bone marrow monocytes from C57BL mice, pre-incubated with apoptotic Jurkat cells (ACs), and were further challenged with E. cuniculi spores. The same procedures were performed using the previously infected Jurkat cells (IACs) and challenged with E. cuniculi spores before macrophage pre-incubation. The average number of spores internalized by macrophages in phagocytosis was counted. Macrophage expression of CD40, CD206, CD80, CD86, and MHCII, as well as the cytokines released in the culture supernatants, was measured by flow cytometry. The ultrastructural study was performed to analyze the multiplication types of pathogens. Macrophages pre-incubated with ACs and challenged with E. cuniculi showed a higher percentage of phagocytosis and an average number of internalized spores. Moreover, the presence of stages of multiplication of the pathogen inside the macrophages, particularly after efferocytosis of infected apoptotic bodies, was observed. In addition, pre-incubation with ACs or IACs and/or challenge with the pathogen decreased the viability of macrophages, reflected as high percentages of apoptosis. The marked expression of CD206 and the release of large amounts of IL-10 and IL-6 indicated the polarization of macrophages to an M2 profile, compatible with efferocytosis and favorable for pathogen development. We concluded that the pathogen favored efferocytosis and polarized the macrophages to an M2 profile, allowing the survival and multiplication of E. cuniculi inside the macrophages and explaining the possibility of macrophages acting as Trojan horses in microsporidiosis.

Optimization of megakaryocyte trapping for platelet formation in microchannels

Platelets (PLTs) are responsible for stopping the bleeding. They are small cell fragments produced from megakaryocytes (MKs) in the bone marrow. Low platelet count is a significant health problem for a patient. PLTs can usually be stored for up to 5 days prior to transfusion. Instantaneous production of PLTs from isolated and stored MKs is crucial for the patient?s health. Thanks to microfluidic platforms, PLTs can be produced instantaneously from MKs. Herein, we have computationally studied fluid dynamics in the microchannels with slit structures and different inlet geometries. Analysis of the flow dynamics was performed by the commercial analysis software. The effects of flow rates and the angle between the inlet channels on the MKs trapping were investigated. The optimization of the angle between inlet channels and flow rates of main and pressure flows was done with Response Surface Methodology (RSM) by counting the trapped MKs. The optimum conditions lead to the percentage of trapped MKs were 100 with a relative deviation of <1%. We also concluded that flow rates to trapping a higher amount of MKs are as important as the angle between the inlet channels.