Osteopontin Gene Polymorphism and Urinary OPN Excretion in Patients with Immunoglobulin A Nephropathy

Osteopontin (OPN) is a glycoprotein involved in the pathogenesis of multiple autoimmune and inflammatory conditions. However, the association of variants of secreted phosphoprotein 1 gene (SPP1), which encodes OPN, with immunoglobulin A nephropathy (IgAN) has not been examined up to date. Moreover, the role of OPN in disease pathogenesis and clinical manifestations is not fully known. Therefore, the aim of the study was to determine the frequency of four single nucleotide polymorphisms (SNiPs) of SPP1 gene, as well as the urinary OPN excretion in IgAN patients and healthy controls. In total, 58 Caucasian patients with biopsy-proven IgAN and 184 gender-, age-, and ethnically-matched healthy controls were genotyped for rs1126616, rs1126772, rs9138, and rs7687316/rs3841116 polymorphisms by real time polymerase chain reaction (RT-PCR). Urinary OPN concentration was determined by enzyme-linked immunosorbent assay (ELISA) in 58 IgAN patients and 19 controls. SPP1 SNiPs, as well as urinary OPN excretion, were analyzed in relation to their possible associations with the clinicopathological parameters. The frequency of the minor TT/CT genotypes of rs1126616 was significantly higher in IgAN patients compared to controls (P = 0.0217). Similarly, the minor (CC/AC) genotypes and the C allele of rs9138 were more frequent in IgAN patients (P = 0.0425 and P = 0.0112, respectively). Moreover, these two SNiPs were associated with the higher urinary OPN excretion (P < 0.05). These findings suggest that rs1126616, as well as rs9138, may be associated with IgAN development, however future studies in this field are required

Immunomodulatory Properties of Polysaccharides from Lentinula edodes

Lentinula edodes (Berk.) Pegler, also known as shiitake mushroom, is a popular edible macrofungus and a source of numerous bioactive substances with multiple beneficial health effects. L. edodes-derived polysaccharides are the most valuable compounds, with anticancer, antioxidant, antimicrobial, and immunomodulatory properties. It has been demonstrated that their biological activity depends on the extraction method, which affects monosaccharide composition, molecular weight, branching degrees, and helical conformation. In this review, we discuss the immunomodulatory properties of various polysaccharides from L. edodes in animal models and in humans

Differences in Immune Checkpoints Expression (TIM-3 and PD-1) on T Cells in Women with Recurrent Miscarriages—Preliminary Studies

Background: Immune checkpoints are molecules that regulate the function of immune cells and control inflammation processes. An important role in this regard is played by TIM-3/Gal-9 and PD-1/PDL-1 interactions. Previous research performed in a mouse model of pregnancy loss confirmed that blocking TIM-3 could induce fetal loss. Similarly, the PD-1 molecule maintains protective interactions between the mother’s immune cells and the fetus. The purpose of this study was to assess the expression of these molecules on a range of T lymphocyte subpopulations from non-pregnant women with recurrent spontaneous abortion (RSA) versus healthy fertile women. Methods: PBMCs were isolated by gradient centrifugation of blood obtained from 12 healthy women and 24 women with RSA and immediately stained for flow cytometry analysis. Standard immunophenotyping of PBMC was performed with the antibodies against classical lymphocyte markers: CD3, CD4, CD8, and CD56. Immune checkpoints were investigated using antibodies against PD-1(CD279) and TIM-3(CD366). Results: We found that expression of TIM-3 was significantly decreased on CD8+ T lymphocytes in the RSA group, and expression of PD-1 was upregulated on CD4+ T lymphocytes in the RSA group in comparison to the healthy controls. Conclusions: Considering our findings, therapeutic intervention towards immune checkpoints may be a promising treatment option for recurrent spontaneous abortion

Prospects for NK Cell Therapy of Sarcoma

Natural killer (NK) cells are innate lymphoid cells with potent antitumor activity. One of the most NK cell cytotoxicity-sensitive tumor types is sarcoma, an aggressive mesenchyme-derived neoplasm. While a combination of radical surgery and radio- and chemotherapy can successfully control local disease, patients with advanced sarcomas remain refractory to current treatment regimens, calling for novel therapeutic strategies. There is accumulating evidence for NK cell-mediated immunosurveillance of sarcoma cells during all stages of the disease, highlighting the potential of using NK cells as a therapeutic tool. However, sarcomas display multiple immunoevasion mechanisms that can suppress NK cell function leading to an uncontrolled tumor outgrowth. Here, we review the current evidence for NK cells’ role in immune surveillance of sarcoma during disease initiation, promotion, progression, and metastasis, as well as the molecular mechanisms behind sarcoma-mediated NK cell suppression. Further, we apply this basic understanding of NK–sarcoma crosstalk in order to identify and summarize the most promising candidates for NK cell-based sarcoma immunotherapy

Perspectives for 3D-Bioprinting in Modeling of Tumor Immune Evasion

In a living organism, cancer cells function in a specific microenvironment, where they exchange numerous physical and biochemical cues with other cells and the surrounding extracellular matrix (ECM). Immune evasion is a clinically relevant phenomenon, in which cancer cells are able to direct this interchange of signals against the immune effector cells and to generate an immunosuppressive environment favoring their own survival. A proper understanding of this phenomenon is substantial for generating more successful anticancer therapies. However, classical cell culture systems are unable to sufficiently recapture the dynamic nature and complexity of the tumor microenvironment (TME) to be of satisfactory use for comprehensive studies on mechanisms of tumor immune evasion. In turn, 3D-bioprinting is a rapidly evolving manufacture technique, in which it is possible to generate finely detailed structures comprised of multiple cell types and biomaterials serving as ECM-analogues. In this review, we focus on currently used 3D-bioprinting techniques, their applications in the TME research, and potential uses of 3D-bioprinting in modeling of tumor immune evasion and response to immunotherapies

Sildenafil Citrate Downregulates PDE5A mRNA Expression in Women with Recurrent Pregnancy Loss without Altering Angiogenic Factors—A Preliminary Study

In our previous study, we showed that sildenafil citrate (SC), a selective PDE5A blocker, modulated NK cell activity in patients with recurrent pregnancy loss, which correlated with positive pregnancy outcomes. It was found that NK cells had a pivotal role in decidualization, angiogenesis, spiral artery remodeling, and the regulation of trophoblast invasion. Thus, in the current study, we determined the effects of SC on angiogenic factor expression and production, as well as idNK cell activity in the presence of nitric synthase blocker L-NMMA. Methods: NK cells (CD56+) were isolated from the peripheral blood of 15 patients and 15 fertile women on MACS columns and cultured in transformation media containing IL-15, TGF-β, and AZA—a methylation agent—for 7 days in hypoxia (94% N2, 1% O2, 5% CO2). Cultures were set up in four variants: (1) with SC, (2) without SC, (3) with NO, a synthase blocker, and (4) with SC and NO synthase blocker. NK cell activity was determined after 7 days of culturing as CD107a expression after an additional 4h of stimulation with K562 erythroleukemia cells. The expression of the PDE5A, VEGF-A, PIGF, IL-8, and RENBP genes was determined with quantitative real-time PCR (qRT-PCR) using TaqMan probes and ELISA was used to measure the concentrations of VEGF-A, PLGF, IL-8, Ang-I, Ang-II, IFN–γ proteins in culture supernatants after SC supplementation. Results: SC downregulated PDE5A expression and had no effect on other studied angiogenic factors. VEGF-A expression was increased in RPL patients compared with fertile women. Similarly, VEGF production was enhanced in RPL patients’ supernatants and SC increased the concentration of PIGF in culture supernatants. SC did not affect the expression or concentration of other studied factors, nor idNK cell activity, regardless of NO synthase blockade

Selective Biological Effects of Selenium-Enriched Polysaccharide (Se-Le-30) Isolated from <i>Lentinula edodes</i> Mycelium on Human Immune Cells

A common edible mushroom Lentinula edodes, is an important source of numerous biologically active substances, including polysaccharides, with immunomodulatory and antitumor properties. In the present work, the biological activity of the crude, homogenous (Se)-enriched fraction (named Se-Le-30), which has been isolated from L. edodes mycelium by a modified Chihara method towards human peripheral blood mononuclear cells (PBMCs) and peripheral granulocytes, was investigated. The Se-Le-30 fraction, an analog of lentinan, significantly inhibited the proliferation of human PBMCs stimulated with anti-CD3 antibodies or allostimulated, and down-regulated the production of tumor necrosis factor (TNF)-α by CD3+ T cells. Moreover, it was found that Se-Le-30 significantly reduced the cytotoxic activity of human natural killer (NK) cells. The results suggested the selective immunosuppressive activity of this fraction, which is non-typical for mushroom derived polysaccharides

Selenium-Containing Exopolysaccharides Isolated from the Culture Medium of <i>Lentinula edodes</i>: Structure and Biological Activity

In continuation of our research on the influence of selenium incorporation on the biosynthesis, structure, and immunomodulatory and antioxidant activities of polysaccharides of fungal origin, we have isolated from a post-culture medium of Lentinula edodes a selenium (Se)-containing exopolysaccharide fraction composed mainly of a highly branched 1-6-α-mannoprotein of molecular weight 4.5 × 106 Da, with 15% protein component. The structure of this fraction resembled mannoproteins isolated from yeast and other mushroom cultures, but it was characterized by a significantly higher molecular weight. X-ray absorption fine structure spectral analysis in the near edge region (XANES) suggested that selenium in the Se-exopolysaccharide structure was present mainly at the IV oxidation state. The simulation analysis in the EXAFS region suggested the presence of two oxygen atoms in the region surrounding the selenium. On the grounds of our previous studies, we hypothesized that selenium-enriched exopolysaccharides would possess higher biological activity than the non-Se-enriched reference fraction. To perform structure–activity studies, we conducted the same tests of biological activity as for previously obtained mycelial Se-polyglucans. The Se-enriched exopolysaccharide fraction significantly enhanced cell viability when incubated with normal (human umbilical vein endothelial cells (HUVEC)) cells (but this effect was absent for malignant human cervical HeLa cells) and this fraction also protected the cells from oxidative stress conditions. The results of tests on the proliferation of human peripheral blood mononuclear cells suggested a selective immunosuppressive activity, like previously tested Se-polyglucans isolated from L. edodes mycelium. The Se-exopolysaccharide fraction, in concentrations of 10–100 µg/mL, inhibited human T lymphocyte proliferation induced by mitogens, without significant effects on B lymphocytes. As with previously obtained Se-polyglucans, in the currently tested Se-polymannans, the selenium content increased the biological activity. However, the activity of selenium exopolysaccharides in all tests was significantly lower than that of previously tested mycelial isolates, most likely due to a different mode of selenium binding and its higher degree of oxidation

Optimization of Se- and Zn-Enriched Mycelium of <i>Lentinula edodes</i> (Berk.) Pegler as a Dietary Supplement with Immunostimulatory Activity

Mycelial cultures of Lentinula edodes, an edible and medicinal mushroom, have been used in our previous research to obtain selenium-containing immunomodulatory preparations. Our current attempts to obtain a new preparation containing both selenium and zinc, two micronutrients necessary for the functioning of the immune system, extended our interest in the simultaneous accumulation of these elements by mycelia growing in media enriched with selenite and zinc(II) ions. Subsequently, we have studied the effects of new L. edodes mycelium water extracts with different concentrations of selenium and zinc on the activation of T cell fraction in human peripheral blood mononuclear cells (PBMCs). Flow cytometry analysis was used to measure the expression of activation markers on human CD4+ and CD8+ T cells stimulated by anti-CD3 and anti-CD3/CD28 antibodies (Abs). It was demonstrated that statistically significant changes were observed for PD-1 and CD25 antigens on CD8+ T cells. The selenium and zinc content in the examined preparations modified the immunomodulatory activity of mycelial polysaccharides; however, the mechanisms of action of various active ingredients in the mycelial extracts seem to be different