Nerve Growth Factor, Antimicrobial Peptides and Chemotherapy: Glioblastoma Combination Therapy to Improve Their Efficacy.

Glioblastoma (GBM) is an aggressive and lethal malignancy of the central nervous system with a median survival rate of 15 months. We investigated the combined anticancer effects of nerve growth factor (NGF), cathelicidin (LL-37), and protegrin-1 (PG-1) with chemotherapy (temozolomide, doxorubicin, carboplatin, cisplatin, and etoposide) in the glioblastoma U251 cell line to overcome the limitations of conventional chemotherapy and to guarantee specific treatments to succeed. The MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay was used to study cell viability and to determine the cytotoxic effects of NGF, LL-37, and PG-1 and their combination with chemotherapy in U251 cells. Synergism or antagonism was determined using the combination index (CI) method. Caspase-3 activity was evaluated spectrophotometrically using a caspase-3 activity assay kit. Apoptosis was analyzed with flow cytometry using propidium iodide (PI) and YO-PRO-1. NGF and the peptides showed a strong cytotoxic effect on U251 glioma cells in the MTT test (IC50 0.0214, 3.1, and 26.1 µM, respectively) compared to chemotherapy. The combination of PG-1 + etoposide had a synergistic effect on apoptosis of U251 glioma cells. It should be noted that the cells were in the early and late stages of apoptosis, respectively, compared with the control cells. The caspase-3 activation analysis revealed that the caspase-3 level was not significantly (p > 0.05) increased in U251 cells following PG-1 with etoposide treatment compared with that in the untreated cells, suggesting that the combination of PG-1 and etoposide may induce caspase-independent apoptosis in U251 cells. NGF, LL-37, and PG-1 represent promising drug candidates as the treatment regimen for GBM. Furthermore, the synergistic efficacy of the combined protocol using PG-1 and etoposide may overcome some of the typical limitations of the conventional therapeutic protocols, thus representing a promising approach for GBM therapy.

Drug Loaded 3D-Printed Poly(ε-Caprolactone) Scaffolds for Local Antibacterial or Anti-Inflammatory Treatment in Bone Regeneration.

Annual bone grafting surgeries due to bone fractures, resections of affected bones, skeletal anomalies, osteoporosis, etc. exceed two million worldwide. In this regard, the creation of new materials for bone tissue repair is one of the urgent tasks of modern medicine. Additive manufacturing, or 3D printing, offers great opportunities for the development of materials with diverse properties and designs. In this study, the one-pot technique for the production of 3D scaffolds based on poly(ε-caprolactone) (PCL) loaded with an antibiotic or anti-inflammatory drug was proposed. In contrast to previously described methods to prepare drug-containing scaffolds, drug-loaded PCL scaffolds were prepared by direct 3D printing from a polymer/drug blend. An investigation of the mechanical properties of 3D-printed scaffolds containing 0.5-5 wt% ciprofloxacin (CIP) or dexamethasone (DEX) showed almost no effect of the drug (compression modulus ~70-90 MPa) compared to unfilled PCL (74 MPa). At the same time, introducing the drug and increasing its content in the PCL matrix contributed to a 1.8-6.8-fold decrease in the specific surface area of the scaffold, depending on composition. The release of CIP and DEX in phosphate buffer solution and in the same buffer containing lipase revealed a faster release in enzyme-containing medium within 45 days. Furthermore, drug release was more intensive from scaffolds with a low drug load. Analysis of the release profiles using a number of mathematical dissolution models led to the conclusion that diffusion dominates over other probable factors. In vitro biological evaluation of the scaffolds containing DEX showed moderate toxicity against osteoblast-like and leukemia monocytic cells. Being 3D-printed together with PCL both drugs retain their biological activity. PCL/CIP and PCL/DEX scaffolds demonstrated antibacterial properties against Pseudomonas aeruginosa (a total inhibition after 48 h) and anti-inflammatory activity in experiments on TNFα-activated monocyte cells (a 4-time reduction in CD-54 expression relative to control), respectively.

Regulated Arginine Metabolism in Immunopathogenesis of a Wide Range of Diseases: Is There a Way to Pass between Scylla and Charybdis?

More than a century has passed since arginine was discovered, but the metabolism of the amino acid never ceases to amaze researchers. Being a conditionally essential amino acid, arginine performs many important homeostatic functions in the body; it is involved in the regulation of the cardiovascular system and regeneration processes. In recent years, more and more facts have been accumulating that demonstrate a close relationship between arginine metabolic pathways and immune responses. This opens new opportunities for the development of original ways to treat diseases associated with suppressed or increased activity of the immune system. In this review, we analyze the literature describing the role of arginine metabolism in the immunopathogenesis of a wide range of diseases, and discuss arginine-dependent processes as a possible target for therapeutic approaches.

Sarcoidosis and Autoimmune Inflammatory Syndrome Induced by Adjuvants.

Currently, sarcoidosis remains one of the diseases with unknown etiology, which significantly complicates its diagnosis and treatment. Various causes of sarcoidosis have been studied for many years. Both organic and inorganic trigger factors, provoking the development of granulomatous inflammation are considered. However, the most promising and evidence-based hypothesis is the development of sarcoidosis as an autoimmune disease, provoked by various adjuvants in genetic predisposed individuals. This concept fits into the structure of the autoimmune/inflammatory syndrome, induced by adjuvants (ASIA) that was proposed in 2011 by Professor Shoenfeld Y. In this paper, the authors reveal the presence of major and minor ASIA criteria for sarcoidosis, propose a new concept of the course of sarcoidosis within the framework of ASIA, and point out the difficulties in creating a model of the disease and the selection of therapy. It is obvious that the data obtained not only bring us closer to understanding the nature of sarcoidosis, but also potentiate new studies confirming this hypothesis by obtaining a model of the disease.

Sarcoidosis-related autoimmune inflammation in COVID-19 convalescent patients.

Currently, there are a large number of reports about the development of autoimmune conditions after COVID-19. Also, there have been cases of sarcoid-like granulomas in convalescents as a part of the post-COVID-19 syndrome. Since one of the etiological theories of sarcoidosis considers it to be an autoimmune disease, we decided to study changes in the adaptive humoral immune response in sarcoidosis and SARS-CoV-2 infection and to find out whether COVID-19 can provoke the development of sarcoidosis. This review discusses histological changes in lymphoid organs in sarcoidosis and COVID-19, changes in B cell subpopulations, T-follicular helper cells (Tfh), and T-follicular regulatory cells (Tfr), and analyzes various autoantibodies detected in these pathologies. Based on the data studied, we concluded that SARS-CoV-2 infection may cause the development of autoimmune pathologies, in particular contributing to the onset of sarcoidosis in convalescents.

Immunopathological Inflammation in the Evolution of Mucositis and Peri-Implantitis.

The purpose of this study was to provide an immuno-mediated substantiation of the etiopathogenesis of mucositis and peri-implantitis based on the results of experimental, laboratory and clinical studies. The biopsy material was studied to identify impregnated nanoscale and microscale particles in the structure of pathological tissues by using X-ray microtomography and X-ray fluorescence analyses. Electron microscopy with energy-dispersive analysis identified the composition of supernatants containing nanoscale metal particles obtained from the surfaces of dental implants. The parameters of the nanoscale particles were determined by dynamic light scattering. Flow cytometry was used to study the effect of nanoscale particles on the ability to induce the activation and apoptosis of immunocompetent cells depending on the particles' concentrations during cultivation with the monocytic cell line THP-1 with the addition of inductors. An analysis of the laboratory results suggested the presence of dose-dependent activation, as well as early and late apoptosis of the immunocompetent cells. Activation and early and late apoptosis of a monocytic cell line when THP-1 was co-cultured with nanoscale metal particles in supernatants were shown for the first time. When human venous blood plasma was added, both activation and early and late apoptosis had a dose-dependent effect and differed from those of the control groups.

Cytokine Storm Signature in Patients with Moderate and Severe COVID-19.

Hypercytokinemia, found in SARS-CoV-2 infection, contributes to multiple organ dysfunctions with acute respiratory distress syndrome, shock etc. The aim of this study was to describe cytokine storm signatures in patients with acute COVID-19 and to investigate their influence on severity of the infection. Plasma levels of 47 cytokines were investigated in 73 patients with moderate and severe COVID-19 (41 and 32, respectively) and 11 healthy donors (HD). The most elevated levels comparing patients and the HD were observed for seven pro-inflammatory cytokines (IL-6, IL-8, IL-15, IL-18, IL-27, IFNγ, TNFα), three chemokines (GROα, IP-10, MIG), two anti-inflammatory cytokines (IL-1RA, IL-10), and two growth factors (G-CSF, M-CSF). The patients with severe disease had significantly higher levels of FGF-2/FGF-basic, IL-1ß, and IL-7 compared to the HD. The two groups of patients differed from each other only based on the levels of EGF, eotaxin, and IL-12 p40. Pneumonia lung injury, characterized by computer tomography, positively correlated with levels of EGF, IP-10, MCP-3 levels and negatively with IL-12 p40. Pro-inflammatory factors including IL-6, TNFα, and IP-10 negatively correlated with the frequency of the circulating T-helper17-like cells (Th17-like) and follicular Th cells that are crucial to develop SARS-CoV-2-specific plasma cells and memory B cells. Obtained data on the cytokine levels illustrate their influence on progression and severity of COVID-19.

TREC/KREC Levels and T and B Lymphocyte Subpopulations in COVID-19 Patients at Different Stages of the Disease.

BACKGROUND: T and B cell-mediated immunity can be assessed using T cell receptor excision circle (TREC) and Kappa-deleting recombination excision circle (KREC) analysis, respectively, and successful implementation of this method requires evaluation of the correlation between the TREC frequencies and T cell subsets as well as KREC levels and B lymphocyte subsets. The aim of the present study was to evaluate the correlation between the TREC/KREC concentrations and T/B lymphocyte subsets at different stages of COVID-19. METHODS: We examined 33 patients in the acute stage of COVID-19 (including 8 patients with poor outcomes) and 33 COVID-19 survivors. TREC/KREC concentrations were measured using quantitative real-time PCR. T/B lymphocyte subsets were determined using flow cytometry. RESULTS: Blood TREC and KREC levels were found to be significantly lower in the acute stage of COVID-19 compared to control values. Moreover, a zero blood TREC level was a predictor of a poor disease outcome. Reductions in CD3+CD4+CD45RO-CD62L- and CD3+CD8+CD45RO-CD62L- T cell counts (as well as in the main fractions of B1 and B2 B cells) indicated a favorable outcome in COVID-19 patients in the acute stage of the disease. Decreased CD3+CD4+CD45RO-CD62L+ and CD3+CD8+CD45RO-CD62L+ T cell frequencies and increased CD3+CD8+CD45RO-CD62L- cell counts were found to indicate a poor outcome in patients with acute COVID-19. These patients were also found to have increased B1 cell counts while demonstrating no changes in B2 cell counts. The levels of effector T cell subsets an naïve B cells were normal in COVID-19 survivors. The most pronounced correlations between TREC/KREC levels and T/B cell subsets counts were observed in COVID-19 survivors: there were positive correlations with naïve T and B lymphocytes and negative correlations with central and effector memory T cell subsets. CONCLUSIONS: The assessment of correlations between TREC and T cell subsets as well as KREC levels and B cell subset counts in patients with acute COVID-19 and COVID-19 survivors has shown that blood concentrations of TREC and KREC are sensitive indicators of the stage of antigen-independent differentiation of adaptive immunity cells. The results of the TREC and KREC analysis correlated with the stages of COVID-19 and differed depending on the outcome of COVID-19.

Imbalanced Immune Response of T-Cell and B-Cell Subsets in Patients with Moderate and Severe COVID-19.

BACKGROUND: The immunological changes associated with COVID-19 are largely unknown. METHODS: Patients with COVID-19 showing moderate (n = 18; SpO2 > 93%, respiratory rate > 22 per minute, CRP > 10 mg/L) and severe (n = 23; SpO2 < 93%, respiratory rate >30 per minute, PaO2/FiO2 ≤ 300 mmHg, permanent oxygen therapy, qSOFA > 2) infection, and 37 healthy donors (HD) were enrolled. Circulating T- and B-cell subsets were analyzed by flow cytometry. RESULTS: CD4+Th cells were skewed toward Th2-like phenotypes within CD45RA+CD62L- (CM) and CD45RA-CD62L- (EM) cells in patients with severe COVID-19, while CM CCR6+ Th17-like cells were decreased if compared with HD. Within CM Th17-like cells "classical" Th17-like cells were increased and Th17.1-like cells were decreased in severe COVID-19 cases. Circulating CM follicular Th-like (Tfh) cells were decreased in all COVID-19 patients, and Tfh17-like cells represented the most predominant subset in severe COVID-19 cases. Both groups of patients showed increased levels of IgD-CD38++ B cells, while the levels of IgD+CD38- and IgD-CD38- were decreased. The frequency of IgD+CD27+ and IgD-CD27+ B cells was significantly reduced in severe COVID-19 cases. CONCLUSIONS: We showed an imbalance within almost all circulating memory Th subsets during acute COVID-19 and showed that altered Tfh polarization led to a dysregulated humoral immune response.