Stimulatory Capacity Research Articles

Characterization of Antigen-Presenting Cell Subsets in Human Liver-Draining Lymph Nodes.

T-cell immunity in the liver is tightly regulated to prevent chronic liver inflammation in response to antigens and toxins derived from food and intestinal bacterial flora. Since the main sites of T cell activation in response to foreign components entering solid tissues are the draining lymph nodes (LN), we aimed to study whether Antigen-Presenting Cell (APC) subsets in human liver lymph-draining LN show features that may contribute to the immunologically tolerant liver environment. Healthy liver LN, iliac LN, spleen and liver perfusates were obtained from multi-organ donors, while diseased liver LN were collected from explanted patient livers. Inguinal LN were obtained from kidney transplant recipients. Mononuclear cells were isolated from fresh tissues, and immunophenotypic and functional characteristics of APC subsets were studied using flowcytometry and in ex vivo cultures. Healthy liver-draining LN contained significantly lower relative numbers of CD1c+ conventional dendritic cells (cDC2), plasmacytoid DC (PDC), and CD14+CD163+DC-SIGN+ macrophages (MF) compared to inguinal LN. Compared to spleen, both types of LN contained low relative numbers of CD141hi cDC1. Both cDC subsets in liver LN showed a more activated/mature immunophenotype than those in inguinal LN, iliacal LN, spleen and liver tissue. Despite their more mature status, cDC2 isolated from hepatic LN displayed similar cytokine production capacity (IL-10, IL-12, and IL-6) and allogeneic T cell stimulatory capacity as their counterparts from spleen. Liver LN from patients with inflammatory liver diseases showed a further reduction of cDC1, but had increased relative numbers of PDC and MF. In steady state conditions human liver LN contain relatively low numbers of cDC2, PDC, and macrophages, and relative numbers of cDC1 in liver LN decline during liver inflammation. The paucity of cDC in liver LN may contribute to immune tolerance in the liver environment.

Gluten Free Wheat: Are We There?

Gluten proteins, major determinants of the bread-making quality of wheat, are related to several digestive disorders. Advances in plant genetic breeding have allowed the production of wheat lines with very low gliadin content through the use of RNAi and gene editing technologies. In this review, we carried out a comprehensive study of the application of these cutting-edge technologies towards the development of wheat lines devoid of immunogenic gluten, and their genetic, nutritional and clinical characterization. One line, named E82, showed outstanding nutritional properties, with very low immunogenic gluten and a low stimulation capacity of T-cells from celiac patients. Moreover, a clinical trial with non-celiac wheat sensitivity (NCWS) patients showed that the consumption of bread made with this E82 low gliadin line induced positive changes in the gut microbiota composition.

Immunological modulation effects of an acid Epimedium polysaccharide on immune response in chickens

The purpose of the present study is to investigate the immunological activities of EPS-1 in the non-specific immune response and specific immune response of chickens. In vitro, the results showed that EPS-1 could increase the proliferation and cytokine secretion (IL-2, IL-4, IFN-γ and TNF-α) of spleen lymphocytes, expression of key surface molecules (MHC II, CD11c, CD40 and CD86) and cytokine secretion (TNF-α and IL-10) of matured chBM-DCs, phagocytic rate of matured chBM-DCs, and enhance the maturation and stimulating capacity of chBM-DCs. In vivo, EPS-1 could also prompt the HI antibody titer, boost the peripheral lymphocyte proliferation, enhance the release of cytokine products in blood (IFN-γ, IL-4 and IL-2) and duodenum (IL-17 and sIgA) of chickens. These results indicated that EPS-1 may have the potential as a powerful immune adjuvant in the treatment of chicken diseases.

Angiogenesis involvement by octacalcium phosphate-gelatin composite-driven bone regeneration in rat calvaria critical-sized defect

Effect of octacalcium phosphate/gelatin composite (OCP/Gel) on angiogenesis was studied by its implantation in rat calvaria critical-sized defect in relation to bone regeneration for 2 and 4 weeks. The implantation of OCP/Gel disks was analyzed by radiomorphometry using a radiopaque material perfusion (Microfil®) method and histomorphometry by hematoxylin and eosin-staining before and after the decalcification. Effect of the OCP dose in the range up to 4 mg per well on the capillary-like tube formation by human umbilical vein endothelial cells (HUVECs) was also examined in a transwell cell culture. The results showed that the blood vessels formation by OCP/Gel group was significantly higher at 2 weeks than other groups but decreased at 4 weeks during the advancement of new bone formation. The capillary-like tube formation was highest in an OCP dose of 1 mg per well while other OCP doses above or below 1 mg did not show such a stimulatory effect. The results established both in vivo and in vitro confirmed that OCP has a positive effect on angiogenesis during bone regeneration in a suitable dose ranges, suggesting that the angiogenesis stimulated by OCP could be involved in the OCP/Gel-enhanced bone regeneration. Statement of significanceWe have reported that octacalcium phosphate (OCP) materials display stimulatory capacities on the bone tissue-related cells. However, the effect of OCP on the angiogenesis and its relation to the OCP-enhanced bone regeneration is unknown. This study confirmed the capacity of OCP on angiogenesis before increasing the new bone mass after the implantation of a composite of OCP and gelatin (OCP/Gel). The blood vessels formation took place associated with the area beginning of the new bone formation, which finally decreased together with development of bone formation. Because OCP was ascertained stimulating the capillary-like tube formation in HUVEC culture with a certain OCP dose, the present study is the first report showing the capacity of OCP on angiogenesis during the OCP/Gel-enhanced bone regeneration.

Bioactivities of Nizamuddinia zanardinii sulfated polysaccharides extracted by enzyme, ultrasound and enzyme-ultrasound methods.

Sulfated polysaccharide (fucoidan) was isolated from Nizamuddinia zanardinii by enzyme (alcalase), ultrasonic and enzyme-ultrasonic methods. The extracted fucoidans were assessed for their chemical compositions, molecular characteristics, anticancer and immunomodulatory activities. Enzyme-ultrasonic isolated fucoidan showed the maximum extraction yield (7.87%) while that obtained by ultrasonic had the minimum value (3.6%). fucoidans were composed of different levels of carbohydrates (52.78-58.65%), proteins (6.98-8.91%), sulfates (21.78-29.6%) and uronic acids (0.42-1.08%). The weight mean average molecular weight of fucoidans varied between 443.7 and 1020.85kDa. The polysaccharide chains were consisted of fucose, galactose, glucose, mannose and xylose. All the recovered fucoidans showed strong growth inhibition against HeLa and Hep-G2 cancer cells. The isolated fucoidans were non-toxic and considerably stimulated the macrophage cells to release nitric oxide. Enzyme extraction produced fucoidan with the most macrophage stimulation capacity (> 42µmol). These results suggested that enzyme treatment preserved more sulfate groups in fucoidan structure influencing its anticancer and immunostimulatory activities.

Parylene-Based Cuff Electrode With Integrated Microfluidics for Peripheral Nerve Recording, Stimulation, and Drug Delivery

A novel Parylene C-based peripheral nerve interface that combines both electrodes and microfluidic channels in an adjustable cuff was designed, fabricated, and characterized. This minimally invasive interface incorporates a drug delivery system for targeted delivery of lysing agents and neurotrophic factors to the nerve surface to locally disrupt the epineurium and allow fascicular selectivity. Multiple platinum (Pt) electrodes were embedded in the microfluidic channels for neural stimulation and recording. The lyse-and-attract cuff electrode (LACE) uses a simple locking mechanism that is adjustable for close contact with nerves of varying diameters. Devices were fabricated using standard Parylene microfabrication techniques resulting in low variability and high device yield. A procedure for the implantation of the LACE was developed and successfully demonstrated in vivo around rat sciatic nerves. The adjustable locking mechanism demonstrated adequate holding strength and fit around the nerves. Benchtop electrochemical characterization of the thin-film Pt electrodes showed that the electrodes possessed high charge storage capacity (>1 mC/cm <sup xmlns:mml="http://www.w3.org/1998/Math/MathML" xmlns:xlink="http://www.w3.org/1999/xlink">2</sup> ) and low impedances ( <inline-formula xmlns:mml="http://www.w3.org/1998/Math/MathML" xmlns:xlink="http://www.w3.org/1999/xlink"> <tex-math notation="LaTeX">$&lt; 2~\text{k}\Omega $ </tex-math></inline-formula> at 1 kHz) suitable for neural stimulation and recording. As expected, embedded electrodes demonstrated higher impedance values. Acute neural recording from the rat sciatic nerve verified the capability of the LACE to record evoked neural activity (compound action potentials). Controlled and localized microfluidic infusions were achieved at low flow rates ( <inline-formula xmlns:mml="http://www.w3.org/1998/Math/MathML" xmlns:xlink="http://www.w3.org/1999/xlink"> <tex-math notation="LaTeX">$&lt; 1~\mu \text{L}$ </tex-math></inline-formula> /min). Finally, infusion experiments in vivo demonstrated targeted drug delivery to the sciatic nerve fascicles. This multifunctional peripheral nerve interface has the potential to enhance implant-tissue integration in vivo and provide reliable chronic performance not available in the existing extraneural or intraneural interfaces. [2018-0192]

Development of a Closed-Loop Stimulator for Laryngeal Reanimation, Part 1: Devices.

The goal of this work was to create implantable stimulator systems that could be used in animal experiments on laryngeal paralysis, including "closed-loop" stimulation of impaired muscles triggered by electromyographic (EMG) potentials from healthy muscles. Iterative device design and testing. A series of microcontroller-based implantable devices were built that incorporated increasingly sophisticated features for stimulation, EMG recording, and communication across the skin. Specific engineering challenges included minimizing power consumption, achieving charge-balanced and relatively high stimulation capacity, implementing noninvasive communication across the skin, providing real-time processing of EMG signals, and mitigating effects of shock artifacts. Bench testing was used to verify performance. Two prototypes are described in detail. Each system is based on an "implant" and an external "communication adapter" that interfaces both with the implant and with external computers for adjustments and monitoring. The first version described is inductively powered and referred to as the "inductive laryngeal stimulator." It uses inductive coupling for both power and communication and performs EMG processing in the communication adapter module. The second version, a "battery-powered laryngeal stimulator," consists of an autonomous battery-powered implant with onboard EMG processing and artifact control; it communicates by infrared light with the external communication adapter for setup and monitoring. The devices met design and performance specifications and have proved useful in the animal experiments that are described in Part 2 of this series. Detailed descriptions of the circuits and their firmware are made available in the Appendix. Level of Evidence: NA.

Phenotype and Function of Activated Natural Killer Cells From Patients With Prostate Cancer: Patient-Dependent Responses to Priming and IL-2 Activation.

Background: Although immunotherapy has emerged as the “next generation” of cancer treatments, it has not yet been shown to be successful in the treatment of patients with prostate cancer, for whom therapeutic options remain limited to radiotherapy and androgen (hormone) deprivation therapy. Previous studies have shown that priming natural killer (NK) cells isolated from healthy individuals via co-incubation with CTV-1 cells derived from an acute lymphoblastic leukemia (ALL) enhances their cytotoxicity against human DU145 (metastatic) prostate cancer cells, but it remains unknown to what extent NK cells from patients with prostate cancer can be triggered to kill. Herein, we explore the phenotype of peripheral blood NK cells in patients with prostate cancer and compare the capacity of CTV-1 cell-mediated priming and IL-2 stimulation to trigger NK cell-mediated killing of the human PC3 (metastatic) prostate cancer cell line.Methods: The phenotype of resting, primed (co-incubation with CTV-1 cells for 17 h) and IL-2 activated (100 IU/ml IL-2 for 17 h) NK cells isolated from frozen-thawed peripheral blood mononuclear cell (PBMC) preparations from patients with benign disease (n = 6) and prostate cancer (n = 18) and their cytotoxicity against PC3 and K562 cells was determined by flow cytometry. Relationship(s) between NK cell phenotypic features and cytotoxic potential were interrogated using Spearman Rank correlation matrices.Results and Conclusions: NK cell priming and IL-2 activation of patient-derived NK cells resulted in similar levels of cytotoxicity, but distinct NK cell phenotypes. Importantly, the capacity of priming and IL-2 stimulation to trigger cytotoxicity was patient-dependent and mutually exclusive, in that NK cells from ~50% of patients preferentially responded to priming whereas NK cells from the remaining patients preferentially responded to cytokine stimulation. In addition to providing more insight into the biology of primed and cytokine-stimulated NK cells, this study supports the use of autologous NK cell-based immunotherapies for the treatment of prostate cancer. However, our findings also indicate that patients will need to be stratified according to their potential responsiveness to individual therapeutic approaches.

Regulation of Dendritic Cell Immune Function and Metabolism by Cellular Nutrient Sensor Mammalian Target of Rapamycin (mTOR).

Dendritic cell (DC) activation is characterized by an acute increase in glucose metabolic flux that is required to fuel the high anabolic rates associated with DC activation. Inhibition of glycolysis significantly attenuates most aspects of DC immune effector function including antigen presentation, inflammatory cytokine production, and T cell stimulatory capacity. The cellular nutrient sensor mammalian/mechanistic Target of Rapamycin (mTOR) is an important upstream regulator of glycolytic metabolism and plays a central role in coordinating DC metabolic changes and immune responses. Because mTOR signaling can be activated by a variety of immunological stimuli, including signaling through the Toll-like Receptor (TLR) family of receptors, mTOR is involved in orchestrating many aspects of the DC metabolic response to microbial stimuli. It has become increasingly clear that mTOR's role in promoting or attenuating inflammatory processes in DCs is highly context-dependent and varies according to specific cellular subsets and the immunological conditions being studied. This review will address key aspects of the complex role of mTOR in regulating DC metabolism and effector function.

Analysis of 24-Hour Heart Rate Variability among Panic Disorder Patients without Previous Drug Treatment and Comorbid Disorders

One of the METHODS used to assess autonomic nervous system dysfunction in the etiology of panic disorder (PD) is heart rate variability (HRV). HRV is controlled by the sympathetic and parasympathetic (vagal) branches of the autonomic nervous system and reflects the capacity of autonomic stimulation by the parasympathetic system. The aim of this study was to evaluate heart rate variability (HRV) time domain parameters based on twenty four hour holter ECG analysis among drug-naive patients with panic disorder (PD) without any other medical and psychiatric comorbidity. The study group consisted of 41 patients with PD and 46 healthy controls. Participants were evaluated with SCID-1 for psychiatric diagnoses. Then Hamilton Depression Rating Scale (HDRS), Hamilton Anxiety Rating Scale (HARS), Panic Disorder Severity Scale (PDSS) and Clinical Global Impression Scale (CGI-S) were applied to participants. Twenty four hour Holter ECG outcomes were analyzed on a computer program and time domain parameters were evaluated. Among the parameters analyzed from HRV, SDANN was significantly higher (p <0.001); duration of RMSSD, NN50 and pNN50 were lower in PD group than the control group (p = 0.003, p = 0.005, p = 0.047, respectively). In the correlation analysis, there was a moderate negative correlation between CGI-S and NN50 and pNN50. In logistic regression analysis, the increase in SDNN was found to increase the probability of PD by 1.11 (95% CI, 1.010-1.209); the increase in SDANN was found to decrease the probability of PD by 0.892 (95% CI, 0.818-0.973), and the increase in pNN50 was found to decrease the probability of PD by 0.523 (95% CI, 0.342-0.801). The data obtained in our study confirm that there is a decrease in some HRV parameters like RMSSD, NN50 and pNN50 reflecting parasympathetic activity among patients with PD.

An arabinogalactan isolated from Boswellia carterii: Purification, structural elucidation and macrophage stimulation via NF-κB and MAPK pathways

Polysaccharides from Boswellia carterii were isolated using hot water and fractionated on a DEAE Sepharose Fast Flow column to evaluate their structural characteristics and macrophage stimulating capacities. Crude and fractionated polysaccharides (BCF1 and BCF2) were mainly consisted of different amounts of neutral sugars (63.7–83.3%) and uronic acids (6.2–22.3%). The weight average molecular weights (Mw) of polysaccharides greatly varied between 295.5 and 504.7 × 103 g/mol. The BCF2 was a highly active polysaccharide on RAW264.7 murine macrophage cells inducing considerable release of nitric oxide and proinflammatory cytokines including TNF-α, IL-1β and IL-6. The presence of p-NF-κB, p-JNK, p-ERK and p-p38 proteins in the cytoplasm of the stimulated macrophages indicated the activation of cells via NF-κB and MAPKs signaling pathways which was followed after CBF2-macrophage interaction through TLR4 and TLR2 receptors. The most potent macrophage activating polysaccharide, BCF2, contained a backbone of (1 → 6)-β-galactopyranosyl units branched at C-3.

Characterization of the innate stimulatory capacity of plant-derived virus-like particles bearing influenza hemagglutinin

Cell-mediated immunity is an important component of immediate and long-term anti-viral protection. Dendritic cells (DCs) are essential for the induction of cell-mediated immunity by instructing the activation and differentiation of antigen-specific T cell responses. Activated DCs that express co-stimulatory molecules and pro-inflammatory cytokines are necessary to promote the development of type 1 immune responses required for viral control. Here we report that plant-derived virus-like particles (VLPs) bearing influenza hemagglutinins (HA) directly stimulate mouse and human DCs. DCs exposed to H1- and, to a lesser extent, H5-VLPs in vitro rapidly express co-stimulatory molecules and produce pro-inflammatory cytokines including IL-12, IL-6 and TNFα. Furthermore, these VLPs support the activation and differentiation of antigen-specific T cell responses. Mechanistically, H1-VLPs stimulate the activation of kinases typically activated downstream of pattern recognition receptors including AKT, p38, and p42/44 ERK. In vivo, immunization with plant-derived VLPs induce the accumulation of both cDC1s and cDC2 in the draining lymph node and a corresponding increase in T and B cells. VLPs devoid of HA protein activate DCs, suggesting they are intrinsically immunostimulatory. Together, the results demonstrate that these candidate plant-derived VLP vaccines have an inherent and direct stimulatory effect on DCs and can enhance the ability of DCs to promote Type 1 immune responses.

Staphylococcus aureus PSM Peptides Modulate Human Monocyte-Derived Dendritic Cells to Prime Regulatory T Cells.

Staphylococcus aureus (Sa), as one of the major human pathogens, has very effective strategies to subvert the human immune system. Virulence of the emerging community-associated methicillin-resistant Sa (CA-MRSA) depends on the secretion of phenol-soluble modulin (PSM) peptide toxins e.g., by binding to and modulation of innate immune cells. Previously, by using mouse bone marrow-derived dendritic cells we demonstrated that PSMs in combination with various Toll-like receptor (TLR) ligands induce a tolerogenic DC phenotype (tDC) characterized by the production of IL-10 and impaired secretion of pro-inflammatory cytokines. Consequently, PSM-induced tDCs favored priming of CD4+CD25+FoxP3+ Tregs with suppressor function while impairing the Th1 response. However, the relevance of these findings for the human system remained elusive. Here, we analyzed the impact of PSMα3 on the maturation, cytokine production, antigen uptake, and T cell stimulatory capacity of human monocyte-derived DCs (moDCs) treated simultaneously with either LPS (TLR4 ligand) or Sa cell lysate (TLR2 ligand). Herein, we demonstrate that PSMs indeed modulate human moDCs upon treatment with TLR2/4 ligands via multiple mechanisms, such as transient pore formation, impaired DC maturation, inhibited pro- and anti-inflammatory cytokine secretion, as well as reduced antigen uptake. As a result, the adaptive immune response was altered shown by an increased differentiation of naïve and even CD4+ T cells from patients with Th1/Th17-induced diseases (spondyloarthritis and rheumatoid arthritis) into CD4+CD127−CD25hiCD45RA−FoxP3hi regulatory T cells (Tregs) with suppressor function. This Treg induction was mediated most predominantly by direct DC-T-cell interaction. Thus, PSMs from highly virulent Sa strains affect DC functions not only in the mouse, but also in the human system, thereby modulating the adaptive immune response and probably increasing the tolerance toward the bacteria. Moreover, PSMα3 might be a novel peptide for tolerogenic DC induction that may be used for DC vaccination strategies.

Legionella feeleii: pneumonia or Pontiac fever? Bacterial virulence traits and host immune response.

Gram-negative bacterium Legionella is able to proliferate intracellularly in mammalian host cells and amoeba, which became known in 1976 since they caused a large outbreak of pneumonia. It had been reported that different strains of Legionella pneumophila, Legionella micdadei, Legionella longbeachae, and Legionella feeleii caused human respiratory diseases, which were known as Pontiac fever or Legionnaires' disease. However, the differences of the virulence traits among the strains of the single species and the pathogenesis of the two diseases that were due to the bacterial virulence factors had not been well elucidated. L. feeleii is an important pathogenic organism in Legionellae, which attracted attention due to cause an outbreak of Pontiac fever in 1981 in Canada. In published researches, it has been found that L. feeleii serogroup 2 (ATCC 35849, LfLD) possess mono-polar flagellum, and L. feeleii serogroup 1 (ATCC 35072, WRLf) could secrete some exopolysaccharide (EPS) materials to the surrounding. Although the virulence of the L. feeleii strain was evidenced that could be promoted, the EPS might be dispensable for the bacteria that caused Pontiac fever. Based on the current knowledge, we focused on bacterial infection in human and murine host cells, intracellular growth, cytopathogenicity, stimulatory capacity of cytokines secretion, and pathogenic effects of the EPS of L. feeleii in this review.

Purification, structural analysis and mechanism of murine macrophage cell activation by sulfated polysaccharides from Cystoseira indica

Sulfated polysaccharides were isolated and purified from the water extract of Cystoseira indica using DEAE Sepharose Fast Flow column to evaluate their structure and macrophage stimulating capacity. Crude and fractionated polysaccharides, CIF1 and CIF2, were mostly composed of neutral sugars (73.1%–78.6%) with relatively lower amounts of acidic sugars (1.3%–9.0%) and sulfate esters (6.9%–9.7%). The polymer chains of polysaccharides were mainly built of different levels of glucose (2.1%–30.8%), fucose (17.2%–24.4%), mannose (17.8%–20.6%) and galactose (16.7%–17.3%). The weight average molecular weight (Mw) of polysaccharides varied between 573.1 × 103 g/mol to 1146.6 × 103 g/mol. The CIF2 polysaccharide, as the most immunostimulating polysaccharide, remarkably induced the release of nitric oxide and inflammatory cytokines including TNF-α, IL-1β, IL-6 and IL-10 from RAW264.7 murine macrophage cells through NF-κB and PAMKs transduction signaling pathways via cell surface TLR4. The interconnections of sugars in CIF2 polysaccharide were complex with (1→3)-fucopyranose, (1→2,3,4)-glucopyranose, (→1)-galactopyranose, (→1)-xylopyranose, (1→2)-rhamnopyranose and (1→2,3)-mannopyranose units being the most predominant residues.

Differentially Tolerized Mouse Antigen Presenting Cells Share a Common miRNA Signature Including Enhanced mmu-miR-223-3p Expression Which Is Sufficient to Imprint a Protolerogenic State.

Dendritic cells (DCs) are pivotal for the induction and maintenance of antigen-specific tolerance and immunity. miRNAs mediate post-transcriptional gene regulation and control in part the differentiation and stimulation-induced immunogenic function of DCs. However, the relevance of miRNAs for the induction and maintenance of a tolerogenic state of DCs has scarcely been highlighted yet. We differentiated mouse bone marrow cells to conventional/myeloid DCs or to tolerogenic antigen presenting cells (APCs) by using a glucocorticoid (dexamethasone) or interleukin-10, and assessed the miRNA expression patterns of unstimulated and LPS-stimulated cell populations by array analysis and QPCR. Differentially tolerized mouse APCs convergingly down-regulated a set of miRNA species at either state of activation as compared with the corresponding control DC population (mmu-miR-9-5p, mmu-miR-9-3p, mmu-miR-155-5p). These miRNAs were also upregulated in control DCs in response to stimulation. In contrast, miRNAs that were convergingly upregulated in both tolerized APC groups at stimulated state (mmu-miR-223-3p, mmu-miR-1224-5p) were downregulated in control DCs in response to stimulation. Overexpression of mmu-miR-223-3p in DCs was sufficient to prevent stimulation-associated acquisition of potent T cell stimulatory capacity. Overexpression of mmu-miR-223-3p in a DC line resulted in attenuated expression of known (Cflar, Rasa1, Ras) mRNA targets of this miRNA species shown to affect pathways that control DC activation. Taken together, we identified sets of miRNAs convergingly regulated in differentially tolerized APCs, which may contribute to imprint stimulation-resistant tolerogenic function as demonstrated for mmu-miR-223-3p. Knowledge of miRNAs with protolerogenic function enables immunotherapeutic approaches aimed to modulate immune responses by regulating miRNA expression.

Tuning of human MAIT cell activation by commensal bacteria species and MR1-dependent T-cell presentation.

Human mucosal-associated invariant T (MAIT) cell receptors (TCRs) recognize bacterial riboflavin pathway metabolites through the MHC class 1-related molecule MR1. However, it is unclear whether MAIT cells discriminate between many species of the human microbiota. To address this, we developed an in vitro functional assay through human T cells engineered for MAIT-TCRs (eMAIT-TCRs) stimulated by MR1-expressing antigen presenting cells (APC). We then screened 47 microbiota-associated bacterial species from different phyla for their eMAIT- TCR stimulatory capacities. Only bacteria species that encoded the riboflavin pathway were stimulatory for MAIT-TCRs. Most species that were high-stimulators belonged to Bacteroidetes and Proteobacteria phyla, whereas low/non-stimulator species were primarily Actinobacteria or Firmicutes. Activation of MAIT cells by high- vs low-stimulating bacteria also correlated with the level of riboflavin they secreted or after bacterial infection of macrophages. Remarkably, we found that human T cell subsets can also present riboflavin metabolites to MAIT cells in MR1- restricted fashion. This T-T cell mediated signaling also induced IFN𝛄, TNF and GranzymeB from MAIT cells, albeit at lower level than professional APC. These findings suggest that MAIT cells can discriminate and categorize complex human microbiota through computation of TCR signals depending on antigen load and presenting cells, and fine-tune their functional responses.

PEDOT: PSS coating on gold microelectrodes with excellent stability and high charge injection capacity for chronic neural interfaces

PEDOT: PSS has become one of the best choices as coating material of neural interfaces electrodes for its excellent biocompatibility, chemical steadiness and extremely inhomogeneous topography that facilitate reversible faradaic charge injection. However, chronic application of this material is still a matter of challenge for its poor adhesion to metallic electrodes. This study presents two new methods that increases stability and stimulation capacity of PEDOT: PSS (poly(3,4-ethylenedioxythiophene): poly(styrene sulfonate)) coated neural interfaces electrodes. These improvements in the characteristics make PEDOT: PSS as a promising coating material for the long-term stable bidirectional neural application. The first process ensures long-term stability of PEDOT: PSS coating. The stability improvement is done by creating a rough and porous surface with selective iodine etching of Au electrodes. The pores act as anchors and increases mechanical bonding between PEDOT: PSS coating and electrode surface, hence the stability of the coating increases. PEDOT: PSS coating on iodine etched gold electrode showed 100% stability under strong ultrasonic stability test compared to non-etched electrodes. Long-term in vitro continuous stimulation for 7 days was performed to evaluate the stability of the coating. 1 μm thick PEDOT:PSS coating on iodine etched Au electrode completely survived under 604 million continuous current pulses that was evaluated by SEM image and electrochemical impedance spectroscopy (EIS). The second process improves the characteristics of PEDOT: PSS coated electrodes in terms of stimulation capacity. This is done by electrochemical modification of PEDOT: PSS coating surface by applying a cyclic voltammetry method. A perfect and effective method is developed by comparative analysis and optimizing the parameters of cyclic voltammetry. The PEDOT: PSS coating presented here (1 μm thick) reduces the impedance of bare Au electrode by around 99%. This electrochemical modification method further reduces the impedance of this PEDOT: PSS coated electrode by 33%. Moreover, this method reduces electrode polarization under stimulation by 30.5%. Reduced electrode impedance and polarization under stimulation are beneficial for increasing safe charge injection limit of the electrode for stimulation. Both processes facilitate the use of PEDOT: PSS coating as a true long-term stable and promising material on gold microelectrodes of neural interfaces.

3-Day monocyte-derived dendritic cells stimulated with a combination of OK432, TLR7/8 ligand, and prostaglandin E2 are a promising alternative for cancer immunotherapy.

Numerous trials using dendritic cell (DC)-based vaccinations for the treatment of cancer are being carried out. However, an improvement of the quality of DC used is highly warranted. We here generated human monocyte-derived dendritic cells using a 3day protocol and stimulated the cells using a combination of OK432 (Picibanil), TLR7/8 ligand CL097, and reduced amounts of prostaglandin (PG)E2. We analyzed phenotype, migratory, and T-cell stimulatory capacity compared to a cytokine cocktail consisting of IL-1β, IL-6, TNF, and PGE2. The OK432 cocktail stimulated cells had a similar mature phenotype with upregulated co-stimulatory molecules, HLA-DR and CCR7 as the cytokine cocktail-matured cells and a similar cytokine profile except increased amounts of IL-12p70. Chemotaxis towards CCL19 was reduced compared to the cytokine cocktail, but increased compared to OK432 alone. The T-cell stimulatory capacity was similar to the cytokine cocktail stimulated cells. In conclusion, the OK432 cocktail has the advantage of inducing IL-12p70 production without impairing phenotype or T-cell stimulatory capacity of the cells and might, therefore, be an advantageous alternative to be used in DC-based immunotherapy.

Вопросы регулирования трансфертного ценообразования в системе налогового контроля России

In article the questions of tax control of the prices of transactions between vertically integrated structures of multinational corporations, holdings and other business combinations are considered. An assessment of modern model of tax administration, from the point of view of the preferences and privileges provided to corporations is given. Features of assessment of criteria of controllability of transactions and instruments of control of transfer pricing are revealed. Constructive proposals on realization of the stimulating capacity of tax system taking into account the interests of development of small and medium business are given.