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This paper develops a second-order explicit predictor–corrector numerical approach for solving a mathematical model on the dynamics of cytokine expressions and human immune cell activation in response to the bacterium staphylococcus aureus (S. aureus). The proposed algorithm is at least zero-stable and second-order accurate. Mathematical modeling works that analyze the human body in response to some antigens have predicted concentrations of a broad range of cells and cytokines. This study deals with a coupled cellular-cytokine model which predicts cytokine expressions in response to gram-positive bacteria S. aureus. Tumor necrosis factor alpha, interleukin 6, interleukin 8 and interleukin 10 are included to assess the relationship between cytokine release from macrophages and the concentration of the S. aureus antigen. Ordinary differential equations are used to model cytokine levels while the cellular responses are modeled by partial differential equations. Interactions between both components provide a more robust and complete systems of immune activation. In the numerical simulations, a low concentration of S. aureus is used to measure cellular activation and cytokine expressions. Numerical experiments indicate how the human immune system responds to infections from different pathogens. Furthermore, numerical examples suggest that the new technique is faster and more efficient than a large class of statistical and numerical schemes discussed in the literature for systems of nonlinear equations and can serve as a robust tool for the integration of general systems of initial-boundary value problems. [ABSTRACT FROM AUTHOR]

Basophils are rare granulocytes in circulation which home to tissues in a process depending on rolling, adhesion and cytokine exposure. However, it is still unclear how these steps affect basophil degranulation. Our aim was to imitate these processes associated with homing by sequential crosslinking of adhesion molecules and cytokine exposure and evaluate the effect on basophil piecemeal (PMD) and anaphylactic degranulation (AND). Blood donors with or without allergic asthma were recruited from an ongoing cohort study. Basophils were subjected to CD62L‐, CD49d‐ or CD11b crosslinking and IL‐3 or IL‐33 stimulation in different orders followed by anti‐IgE and fMLP stimulation. Basophil CD203c and CD63 expression were analysed by flow cytometry to determine PMD and AND, respectively. IL‐3 induced PMD in basophils and combined with CD62L‐ or CD11b crosslinking, IL‐3 potentiated the degranulation regardless of sequential order. IL‐3 priming followed by adhesion molecule crosslinking induced AND and potentiated the effect of anti‐IgE. CD62L‐ and CD11b crosslinking did not further potentiate this effect. CD49d crosslinking followed by IL‐3 increased CD63 expression following anti‐IgE. IL‐3 potentiated the effect of fMLP on AND while adhesion molecule crosslinking did not. IL‐33 had impact on PMD only when followed by adhesion molecule crosslinking but did not potentiate neither IgE‐dependent nor IgE‐independent degranulation. Our data indicate that sequential interactions between basophils, cytokines and adhesion molecule ligands have a decisive effect on basophil degranulation and that these interactions are operational for fine‐tuning the activity of tissue dwelling basophils. These data should be considered when the effect of different pharmaceutical on basophil function is studied. [ABSTRACT FROM AUTHOR]

To investigate the effects of 3-methyladenine (3-MA) on activation and autophagy of rat he⁃ patic stellate cell line HSC-T6 induced by transforming growth faction-β（TGF-β) . Methods HSC-T6 cells in the loga⁃ rithmic growth phase were divided into the blank group, TGF-β+PBS group, and TGF-β+3-MA group. Cells of TGF-β+3- MA group were cultured with 2 ng/mL TGF-β for 72 h and treated with 3-MA (0. 5 mg/mL) for 24 h. Cells of TGF-β+PBS group were cultured with 2 ng/mL TGF-β for 72 h and treated with the same volume of PBS for 24 h. Cells in the blank group were treated with equal volume of PBS, and the mRNA levels of cell activation markers α-SMA and TGF-β and au⁃ tophagy markers LC3, Beclin-1 and Atg5 in each group were detected by real-time fluorescence quantitative PCR. Cell ac⁃ tivation markers α-SMA, TGF-β proteins and autophagy markers LC3, Beclin-1, Atg5 proteins were detected by Western blotting. Results The mRNA and protein levels of cell activation markers α-SMA and TGF-β in the TGF-β+PBS group and TGF-β+3-MA group were higher than those in the blank group, and those were lower in the TGF-β+3-MA group than in the TGF-β+PBS group (all P<0. 05) . The mRNA and protein levels of autophagy markers LC3, Beclin-1 and Atg5 in the TGF-β+PBS group and TGF-β+3-MA group were higher than those in the blank group, and those were lower in the TGF-β+3-MA group than in the TGF-β+PBS group (all P<0. 05) . Conclusion 3-MA can inhibit TGF-β-induced HSCT6 activation and autophagy in rat hepatic astrocytes. [ABSTRACT FROM AUTHOR]

Abstract Background Idiopathic pulmonary fibrosis (IPF) is a progressive interstitial lung disease (ILD) with unknown etiology, characterized by sustained damage repair of epithelial cells and abnormal activation of fibroblasts, the underlying mechanism of the disease remains elusive. Methods To evaluate the role of Tuftelin1 (TUFT1) in IPF and elucidate its molecular mechanism. We investigated the level of TUFT1 in the IPF and bleomycin-induced mouse models and explored the influence of TUFT1 deficiency on pulmonary fibrosis. Additionally, we explored the effect of TUFT1 on the cytoskeleton and illustrated the relationship between stress fiber and pulmonary fibrosis. Results Our results demonstrated a significant upregulation of TUFT1 in IPF and the bleomycin (BLM)-induced fibrosis model. Disruption of TUFT1 exerted inhibitory effects on pulmonary fibrosis in both in vivo and in vitro. TUFT1 facilitated the assembly of microfilaments in A549 and MRC-5 cells, with a pronounced association between TUFT1 and Neuronal Wiskott-Aldrich syndrome protein (N-WASP) observed during microfilament formation. TUFT1 can promote the phosphorylation of tyrosine residue 256 (Y256) of the N-WASP (pY256N-WASP). Furthermore, TUFT1 promoted transforming growth factor-β1 (TGF-β1) induced fibroblast activation by increasing nuclear translocation of pY256N-WASP in fibroblasts, while wiskostatin (Wis), an N-WASP inhibitor, suppressed these processes. Conclusions Our findings suggested that TUFT1 plays a critical role in pulmonary fibrosis via its influence on stress fiber, and blockade of TUFT1 effectively reduces pro-fibrotic phenotypes. Pharmacological targeting of the TUFT1-N-WASP axis may represent a promising therapeutic approach for pulmonary fibrosis.

Background: Ischemia/reperfusion injury (IRI) is generally unavoidable following liver transplantation. Here, we investigated the role of protein phosphatase, Mg2+/Mn2+ dependent 1G (PPM1G) in hepatic IRI. Methods: Hepatic IRI was mimicked by employing a hypoxia/reperfusion (H/R) model in RAW 264.7 cells and a 70% warm ischemia model in C57BL/6 mice, respectively. In vitro, expression changes of tumor necrosis factor-α and interleukin were detected by quantitative real-time polymerase chain reaction (qRT-PCR), western blot analysis, and enzyme-linked immunosorbent assay. The protein expressions of PPM1G and the stimulator of interferon genes (STING) pathway components were analyzed by western blot. Interaction between PPM1G and STING was verified by coimmunoprecipitation (CO-IP). Immunofluorescence was applied for detection of p-IRF3. Flow cytometry, qRT-PCR and western blot were utilized to analyze markers of macrophage polarization. In vivo, histological analyses of mice liver were carried out by TUNEL and H&E staining. Changes in serum aminotransferases were also detected. Results: Following H/R intervention, a steady decline in PPM1G along with an increase in inflammatory cytokines in vitro was observed. Addition of plasmid with PPM1G sequence limited the release of inflammatory cytokines and downregulated phosphorylation of STING. CO-IP validated the interaction between PPM1G and STING. Furthermore, inhibition of PPM1G with lentivirus enhanced phosphorylation of STING and its downstream components; meanwhile, p65, p38, and Jnk were also surged to phosphorylation. Expression of INOS and CD86 was surged, while CD206, Arg-1, and IL-10 were inhibited. In vivo, PPM1G inhibition further promoted liver damage, hepatocyte apoptosis, and transaminases release. Selective inhibition of STING with C-176 partially reversed the activation of STING pathway and inflammatory cytokines in vitro. M1 markers were also suppressed by C-176. In vivo, C-176 rescued liver damage and transaminase release caused by PPM1G inhibition. Conclusion: PPM1G suppresses hepatic IRI and macrophage M1 phenotype by repressing STING-mediated inflammatory pathways. [ABSTRACT FROM AUTHOR]

The receptor tyrosine kinase (RTK) KIT and its ligand stem cell factor (SCF) are essential for human mast cell (huMC) survival and proliferation. HuMCs expressing oncogenic KIT variants secrete large numbers of extracellular vesicles (EVs). The role KIT plays in regulating EV secretion has not been examined. Here, we investigated the effects of stimulation or inhibition of KIT activity on the secretion of small EVs (sEVs). In huMCs expressing constitutively active KIT, the quantity and quality of secreted sEVs positively correlated with the activity status of KIT. SCF‐mediated stimulation of KIT in huMCs or murine MCs, or of transiently expressed KIT in HeLa cells, enhanced the release of sEVs expressing exosome markers. In contrast, ligand‐mediated stimulation of the RTK EGFR in HeLa cells did not affect sEV secretion. The release of sEVs induced by either constitutively active or ligand‐activated KIT was remarkably decreased when cells were treated with KIT inhibitors, concomitant with reduced exosome markers in sEVs. Similarly, inhibition of oncogenic KIT signalling kinases like PI3K, and MAPK significantly reduced the secretion of sEVs. Thus, activation of KIT and its early signalling cascades stimulate the secretion of exosome‐like sEVs in a regulated fashion, which may have implications for KIT‐driven functions. [ABSTRACT FROM AUTHOR]

Background: Idiopathic pulmonary fibrosis (IPF) is a progressive interstitial lung disease (ILD) with unknown etiology, characterized by sustained damage repair of epithelial cells and abnormal activation of fibroblasts, the underlying mechanism of the disease remains elusive. Methods: To evaluate the role of Tuftelin1 (TUFT1) in IPF and elucidate its molecular mechanism. We investigated the level of TUFT1 in the IPF and bleomycin-induced mouse models and explored the influence of TUFT1 deficiency on pulmonary fibrosis. Additionally, we explored the effect of TUFT1 on the cytoskeleton and illustrated the relationship between stress fiber and pulmonary fibrosis. Results: Our results demonstrated a significant upregulation of TUFT1 in IPF and the bleomycin (BLM)-induced fibrosis model. Disruption of TUFT1 exerted inhibitory effects on pulmonary fibrosis in both in vivo and in vitro. TUFT1 facilitated the assembly of microfilaments in A549 and MRC-5 cells, with a pronounced association between TUFT1 and Neuronal Wiskott-Aldrich syndrome protein (N-WASP) observed during microfilament formation. TUFT1 can promote the phosphorylation of tyrosine residue 256 (Y256) of the N-WASP (pY256N-WASP). Furthermore, TUFT1 promoted transforming growth factor-β1 (TGF-β1) induced fibroblast activation by increasing nuclear translocation of pY256N-WASP in fibroblasts, while wiskostatin (Wis), an N-WASP inhibitor, suppressed these processes. Conclusions: Our findings suggested that TUFT1 plays a critical role in pulmonary fibrosis via its influence on stress fiber, and blockade of TUFT1 effectively reduces pro-fibrotic phenotypes. Pharmacological targeting of the TUFT1-N-WASP axis may represent a promising therapeutic approach for pulmonary fibrosis. [ABSTRACT FROM AUTHOR]

Summary: Adoptive T‐cell transfer (ACT) therapies, including of tumor infiltrating lymphocytes (TILs) and T cells gene‐modified to express either a T cell receptor (TCR) or a chimeric antigen receptor (CAR), have demonstrated clinical efficacy for a proportion of patients and cancer‐types. The field of ACT has been driven forward by the clinical success of CD19‐CAR therapy against various advanced B‐cell malignancies, including curative responses for some leukemia patients. However, relapse remains problematic, in particular for lymphoma. Moreover, for a variety of reasons, relative limited efficacy has been demonstrated for ACT of non‐hematological solid tumors. Indeed, in addition to pre‐infusion challenges including lymphocyte collection and manufacturing, ACT failure can be attributed to several biological processes post‐transfer including, (i) inefficient tumor trafficking, infiltration, expansion and retention, (ii) chronic antigen exposure coupled with insufficient costimulation resulting in T‐cell exhaustion, (iii) a range of barriers in the tumor microenvironment (TME) mediated by both tumor cells and suppressive immune infiltrate, (iv) tumor antigen heterogeneity and loss, or down‐regulation of antigen presentation machinery, (v) gain of tumor intrinsic mechanisms of resistance such as to apoptosis, and (vi) various forms of toxicity and other adverse events in patients. Affinity‐optimized TCRs can improve T‐cell function and innovative CAR designs as well as gene‐modification strategies can be used to coengineer specificity, safety, and function into T cells. Coengineering strategies can be designed not only to directly support the transferred T cells, but also to block suppressive barriers in the TME and harness endogenous innate and adaptive immunity. Here, we review a selection of the remarkable T‐cell coengineering strategies, including of tools, receptors, and gene‐cargo, that have been developed in recent years to augment tumor control by ACT, more and more of which are advancing to the clinic. [ABSTRACT FROM AUTHOR]

Mo6S8 in the Chevrel Phase (CP) and 14‐polyanthraquinone (14PAQ) cathode materials‐based coin cells were assembled against Mg‐foil as an anode by using 0.3 M magnesium tetrakis (hexafluoroisopropyloxy) borate Mg[B(hfip)4]2/dimethoxyethane (DME), 0.5 M Mg[B(hfip)4]2/DME and 0.5 M Mg[B(hfip)4]2/tetraglyme (G4) electrolytes. The heat generation of those three variants was compared using a sensitive MS80 Tian‐Calvet calorimeter. The Chevrel Phase Mo6S8 was found to generate less heat than the organic 14PAQ. However, its specific capacity was also comparatively lower than for the organic cathode material. It is equally important for battery kinetics to have a well‐designed electrolyte, therefore different solvents with the same electrolyte salt were utilized. Noticeable differences were observed and in tetraglyme solvent stable cycling and fewer self‐discharge phenomena were detected. However, the activation process needs more cycles to achieve the required capacity in the case of the Chevrel Phase. The generated heat during cycling indicated the high resistances, swelling/contraction in organic cathodes leading to higher heat generation, and poor capacity retention. To overcome self‐discharging in Mg batteries, side reactions/dissolution of cathode materials, electrolyte saturation and the formed interfaces on the anode side must be considered. [ABSTRACT FROM AUTHOR]

The central function of telomerase is maintaining the telomere length. However, several extra-telomeric roles have been identified for this protein complex. In this study, we evaluated the effect of the silencing of the catalytic subunit of telomerase (TERT) on the expression of candidate microRNAs, cell activation markers and glial-related genes in a glioblastoma cell line (T98G). The silencing was performed by a siRNA and the qPCR method was used to analyze the expression of TERT and downstream genes. Flow cytometry was used to quantify the TERT protein, and bioinformatics analysis was carried out to analyze the functions of microRNA target genes. Here, it was observed that after a 50% reduction of the TERT gene, the expression of ARG1 (Arginase 1) was upregulated, whereas NES (Nestin), GLUL (Glutamate-Ammonia Ligase), VIM (Vimentin) and the hsa-miR-29b-3p microRNA were downregulated (P-value <0.05). A bioinformatic analysis showed that target genes of hsa-miR-29b are associated with focal adhesion, the PI3K-Akt signaling pathway, among others. These results are important because they contribute to the knowledge of extratelomeric functions by providing relevant evidence about novel genes modulated by TERT. [ABSTRACT FROM AUTHOR]

TLR5 agonist flagellin is an effective mucosal adjuvant via intranasal administration. Previous studies demonstrated that the mucosal adjuvanticity of flagellin depends on TLR5 signaling of airway epithelial cells. Since dendritic cells play a central role in antigen sensitization and the initiation of primary immune responses, we wondered how dendritic cells were modulated by the intranasally administrated flagellin. In this study, a mouse model of intranasal immunization with ovalbumin, a model antigen, in the presence or absence of flagellin was utilized. We found that nasal administration of flagellin enhanced the coadministered antigen-specific antibody responses and T-cell clonal expansion in a TLR5-dependent manner. However, neither the entering of flagellin to nasal lamina propria nor the uptake of coadministered antigen by nasal resident dendritic cells was associated with TLR5 signaling. In contrast, migration of antigen-loaded dendritic cells from the nasal cavity to the cervical lymph nodes and activation of dendritic cells in the cervical lymph nodes were both enhanced through TLR5 signaling. Furthermore, for the dendritic cells, flagellin enhanced the expression of CCR7, which was pivotal for dendritic cells in the priming site migrating to draining lymph nodes. Interestingly, the migration, activation, and chemokine receptor expression levels of antigen-loaded dendritic cells were all significantly higher than that of bystander dendritic cells. In conclusion, intranasally administrated flagellin enhanced TLR5-dependent antigen-loaded dendritic cells' migration and activation but not antigen uptake. [ABSTRACT FROM AUTHOR]

Aquaporins are a family of ubiquitously expressed transmembrane water channels implicated in a broad range of physiological functions. We have previously reported that aquaporin 4 (AQP4) is expressed on T cells and that treatment with a small molecule AQP4 inhibitor significantly delays T cell mediated heart allograft rejection. Using either genetic deletion or small molecule inhibitor, we show that AQP4 supports T cell receptor mediated activation of both mouse and human T cells. Intact AQP4 is required for optimal T cell receptor (TCR)-related signaling events, including nuclear translocation of transcription factors and phosphorylation of proximal TCR signaling molecules. AQP4 deficiency or inhibition impairs actin cytoskeleton rearrangements following TCR crosslinking, causing inferior TCR polarization and a loss of TCR signaling. Our findings reveal a novel function of AQP4 in T lymphocytes and identify AQP4 as a potential therapeutic target for preventing TCR-mediated T cell activation. [ABSTRACT FROM AUTHOR]

For years, the biochemical processes that are triggered by harmful and non-harmful stimuli at the central nervous system level have been extensively studied by the scientific community through numerous techniques and animal models. For example, one of these techniques is the use of immediate expression genes, which is a useful, accessible, and reliable method for observing and quantifying cell activation. It has been shown that both the c-fos gene and its protein c-Fos have rapid activation after stimulus, with the length of time that they remain active depending on the type of stimulus and the activation time depending on the stimulus and the structure studied. Fos requires the participation of other genes (such as c-jun) for its expression (during hetero-dimer forming). c-Fos dimerizes with c-Jun protein to form factor AP-1, which promotes the transcription of various genes. The production and removal of c-Fos is part of cellular homeostasis, but its overexpression results in increased cell proliferation. Although Fos has been used as a marker of cellular activity since the 1990s, which molecular mechanism participates in the regulation of the expression of this protein is still unknown because the gene and the protein are not specific to neurons or glial cells. For these reasons, this work has the objective of gathering information about this protein and its use in neuroscience.

Objective: In this study, we aimed to investigate the effect of transient postprandial in vivo and prolonged experimental in vitro hyperlipidemia on human peripheral blood monocytes. Methods: Peripheral blood was collected from seven healthy subjects after an overnight fast and three hours after a standardized high-fat meal. Both fasting and postprandial samples were stained for surface markers CD14/CD11b and intracellular lipids using BODIPY493/503. Postprandial samples only were used for isolation of peripheral blood mononuclear cells that were further incubated overnight with postprandial hyperlipidemic autologous plasma, then stained as described above. All samples were analyzed on a FACSAria III flow cytometer.

Single cell technologies, lineage tracing mouse models and advanced imaging techniques unequivocally improved the resolution of the cellular landscape of atherosclerosis. Although the discovery of the heterogeneous nature of the cellular plaque architecture has undoubtedly improved our understanding of the specific cellular states in atherosclerosis progression, it also adds more complexity to current and future research and will change how we approach future drug development. In this review, we will discuss how the revolution of new single cell technologies allowed us to map the cellular networks in the plaque, but we will also address current (technological) limitations that confine us to identify the cellular drivers of the disease and to pinpoint a specific cell state, cell subset or cell surface antigen as new candidate drug target for atherosclerosis. [Display omitted] • Single cell omics unveiled the heterogeneous cellular landscape of atherosclerosis. • Intraplaque heterogeneity adds complexity to current and future (drug) research. • Studying cell activation and cell-cell interactions is the next step forward. • More disease and site-specific therapies for atherosclerosis are needed. [ABSTRACT FROM AUTHOR]

The mechanism of action underlying the intriguing prominent bioactivity of urinary bladder matrix (UBM) for in situ tissue regeneration of soft tissue defects remains to be elucidated. It is speculated that the activity of UBM for cell adhesion, migration, and activation is inherent. The bioactivity of UBM for in situ tissue regeneration and its relation with the structure and intact soluble components of UBM were investigated in comparison to a collagen-based scaffold, PELNAC (PEL). We isolated the soluble component of the two materials with urea buffer, and evaluated the respective effect of these soluble components on the in vitro adhesion and migration of L929 fibroblasts. The spatiotemporal pattern of endogenous-cell ingrowth into the scaffolds and cell activation were investigated using a model of murine subcutaneous implantation. UBM is more capable of promoting the adhesion, migration, and proliferation of fibroblasts than PEL in a serum-independent manner. In vivo, as compared with PEL, UBM exhibits significantly enhanced activity for fast endogenous cell ingrowth and produces a more prominent pro-regenerative and pro-remodeling microenvironment by inducing the expression of TGF-β1, VEGF, MMP-9, and murine type I collagen. Overall, our results suggest the prominent bioactivity of UBM for in situ tissue regeneration is inherent. [ABSTRACT FROM AUTHOR]

Oral anticoagulants are a critical component of stroke prevention, but carry a risk of brain hemorrhage. These hemorrhagic complications tend to occur in elderly individuals, especially those with predisposing conditions such as cerebral amyloid angiopathy (CAA). Clinical evidence suggests that non-vitamin K antagonist oral anticoagulants are safer than traditional oral anticoagulants. We analyzed whether the anticoagulant dabigatran produces cerebral microhemorrhage (the pathological substrate of MRI-demonstrable cerebral microbleeds) or intracerebral hemorrhage in aged mice with and without hemorrhage-predisposing angiopathy. We studied aged (22 months old) Tg2576 (a model of CAA) and wild-type (WT) littermate mice. Mice received either dabigatran etexilate (DE) (Tg N = 7; WT N = 10) or vehicle (Tg N = 9; WT N = 7) by gavage for 4 weeks. Anticoagulation effects of DE were confirmed using thrombin time assay. No mice experienced intracerebral hemorrhage. Cerebral microhemorrhage analysis, performed using Prussian-blue and H&E staining, showed no significant change in either number or size of cerebral microhemorrhage in DE-treated animals. Analysis of biochemical parameters for endothelial activation (ICAM-1), blood-brain barrier disruption (IgG, claudin-5, fibrinogen), microglial activation (Iba-1), or astrocyte activation (GFAP) showed neither exacerbation nor protective effects of DE in either Tg2576 or WT mice. Our study provides histological and biochemical evidence that aged mice, with or without predisposing factors for brain hemorrhage, tolerate anticoagulation with dabigatran. The absence of dabigatran-induced intracerebral hemorrhage or increased frequency of acute microhemorrhage may provide some reassurance for its use in high-risk patient populations.

BACKGROUND:Cerebral microhemorrhages (CMH) are commonly found in the aging brain. CMH are also the neuropathological substrate of cerebral microbleeds (CMB), demonstrated on brain MRI. Recent studies demonstrate the importance of systemic inflammation in CMH development, but the relationships among inflammation, aging, and CMH development are not well-defined. In the current study, we hypothesized that the pathogenesis of inflammation-induced CMH in mice differs by age. METHODS:We studied young (3 months, n = 20) and old (18 months, n = 25) C57BL/6 mice injected with low-dose lipopolysaccharide (LPS; 1 mg/kg, i.p.) or saline at 0, 6, and 24 h. Seven days after the first LPS/saline injection, brains were harvested, sectioned, and stained with hematoxylin and eosin (H&E) and Prussian blue (PB) to estimate acute/fresh and sub-acute CMH development, respectively. The relationships between microglial/macrophage activation (ionized calcium-binding adapter molecule-1), astrocyte activation (glial fibrillary acidic protein), blood-brain barrier (BBB) disruption (brain immunoglobulin G), aging, and CMH development were examined using immunohistochemistry. RESULTS:Aging alone did not increase spontaneous H&E-positive CMH development but significantly increased the number, size, and total area of LPS-induced H&E-positive CMH in mice. LPS- and saline-treated aged mice had significantly larger PB-positive CMH compared with young mice, but the total area of PB-positive CMH was increased only in LPS-treated aged mice. Aged mice had significantly increased microglial/macrophage activation, which correlated with H&E- and PB-positive CMH development. Aged mice treated with LPS had significantly increased astrocyte activation and BBB disruption compared with young LPS-treated mice. CONCLUSIONS:Aging makes the brain more susceptible to inflammation-induced CMH in mice, and this increase in CMH with aging is associated with microglial/macrophage activation.

The deformability of leukocytes is relevant to a wide array of physiological and pathophysiological behaviors. The goal of this study is to provide a detailed, quantitative characterization of the mechanical properties of T cells and how those properties change with activation. We tested T cells and CD8+ cells isolated from peripheral blood samples of healthy donors either immediately (naïve population) or after 7 days of activation in vitro. Single-cell micropipette aspiration was used to test the mechanical properties. T cells exhibit the general characteristics of a highly viscous liquid drop with a cortical “surface” tension, Tcort. The time course of each cell entry into the micropipette was measured at two different aspiration pressures to test for shear thinning behavior. The data were analyzed in the framework of an approximate mechanical model of the cell deformation to determine the cortical tension, the cell volume, the magnitude of the initial cell entry, the characteristic viscosity μo, and the shear thinning coefficient, b. Activation generally caused increases in cellular resistance to deformation and a broadening of the distribution of cell properties. The cell volume increased substantially upon cell activation from ∼200 μm3 to ∼650 μm3. Naive and activated T cells had similar mean cortical tension (∼150 pN/μm). However, compared to naïve CD8+ cells, the cortical tension of activated CD8+ cells increased significantly to ∼250 pN/μm. Dynamic resistance of naive CD8+ T cells, as reflected in their characteristic viscosity, was ∼870 Pa and significantly increased to 1,180 Pa after in vitro activation. The magnitude of the instantaneous projection length as the cell enters the pipette (Linit) was more than doubled for activated vs. naive cells. All cell types exhibited shear thinning behavior with coefficients b in the range 0.5–0.65. Increased cell size, cortical tension, and characteristic viscosity all point to increased resistance of activated T cells to passage through the microvasculature, likely contributing to cell trapping. The increased initial elastic response of cells after activation was unexpected and could point to instability in the cell that might contribute to spontaneous cell motility.

The human akna gene encodes an AT-hook transcription factor, the expression of which is involved in various cellular processes. The goal of this study was to identify potential AKNA binding sites in genes that participate in T-cell activation and validate selected genes. Here we analyzed ChIP-seq and microarray assays to determine AKNA-binding motifs and the cellular process altered by AKNA in T-cell lymphocytes. In addition, we performed a validation analysis by RT-qPCR to assess AKNA's role in promoting IL-2 and CD80 expression. We found five AT-rich motifs that are potential candidates as AKNA response elements. We identified these AT-rich motifs in promoter regions of more than a thousand genes in activated T-cells, and demonstrated that AKNA induces the expression of genes involved in helper T-cell activation, such as IL-2. The genomic enrichment and prediction of AT-rich motif analyses demonstrated that AKNA is a transcription factor that can potentially modulate gene expression by recognizing AT-rich motifs in a plethora of genes that are involved in different molecular pathways and processes. Among the cellular processes activated by AT-rich genes, we found inflammatory pathways potentially regulated by AKNA, suggesting AKNA is acting as a master regulator during T-cell activation. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Peripheral endothelial cells are capable of erythrophagocytosis, but data on brain endothelial erythrophagocytosis are limited. We studied the relationship between brain endothelial erythrophagocytosis and cerebral microhemorrhage, the pathological substrate of MRI-demonstrable cerebral microbleeds. To demonstrate the erythrophagocytic capability of the brain endothelium, we studied the interactions between brain endothelial cells and red blood cells exposed to oxidative stress in vitro, and developed a new in vitro cerebral microbleeds model to study the subsequent passage of hemoglobin across the brain endothelial monolayer. Using multiple approaches, our results show marked brain endothelial erythrophagocytosis of red blood cells exposed to oxidative stress compared with control red blood cells in vitro. This brain endothelial erythrophagocytosis was accompanied by passage of hemoglobin across the brain endothelial monolayer with unaltered monolayer integrity. In vivo and confocal fluorescence microscopy studies confirmed the extravasation of RBC exposed to oxidative stress across brain endothelium. These findings, demonstrating erythrophagocytosis mediated by the brain endothelial monolayer and the subsequent passage of iron-rich hemoglobin in vitro and RBC in vivo, may have implications for elucidating mechanisms involved in the development of cerebral microbleeds that are not dependent on disruption of the microvasculature.

The T-cell synapse is essentially a "circular" lamellipodia, and T-cell synapse formation and T-cell migration rely on many of the same cell-cell interactions (e.g. integrin ligation) and signaling mediators. Keywords: T lymphocytes; signaling cascade; T-cell receptors/receptor complex; cell activation; T-cell responses EN T lymphocytes signaling cascade T-cell receptors/receptor complex cell activation T-cell responses 541 543 3 07/20/23 20230601 NES 230601 Aquaporins (AQPs) are water channels that control the transport of water molecules across plasma membranes. [Extracted from the article]

Uncontrolled inflammation of the airways in chronic obstructive lung diseases leads to exacerbation, accelerated lung dysfunction and respiratory insufficiency. Among these diseases, asthma affects 358 million people worldwide. Human bronchial epithelium cells (HBEC) express both anti‐inflammatory and activating molecules, and their deregulated expression contribute to immune cell recruitment and activation, especially platelets (PLT) particularly involved in lung tissue inflammation in asthma context. Previous results supported that HLA‐G dysregulation in lung tissue is associated with immune cell activation. We investigated here HLA‐F expression, reported to be mobilised on immune cell surface upon activation and displaying its highest affinity for the KIR3DS1‐activating NK receptor. We explored HLA‐F transcriptional expression in HBEC; HLA‐F total expression in PBMC and HBEC collected from healthy individuals at rest and upon chemical activation and HLA‐F membrane expression in PBMC, HBEC and PLT collected from healthy individuals at rest and upon chemical activation. We compared HLA‐F transcriptional expression in HBEC from healthy individuals and asthmatic patients and its surface expression in HBEC and PLT from healthy individuals and asthmatic patients. Our results support that HLA‐F is expressed by HBEC and PLT under healthy physiological conditions and is retained in cytoplasm, barely expressed on the surface, as previously reported in immune cells. In both cell types, HLA‐F reaches the surface in the inflammatory asthma context whereas no effect is observed at the transcriptional level. Our study suggests that HLA‐F surface expression is a ubiquitous post‐transcriptional process in activated cells. It may be of therapeutic interest in controlling lung inflammation. [ABSTRACT FROM AUTHOR]

BackgroundCerebral microbleeds (CMB) are MRI-demonstrable cerebral microhemorrhages (CMH) which commonly coexist with ischemic stroke. This creates a challenging therapeutic milieu, and a strategy that simultaneously protects the vessel wall and provides anti-thrombotic activity is an attractive potential approach. Phosphodiesterase 3A (PDE3A) inhibition is known to provide cerebral vessel wall protection combined with anti-thrombotic effects. As an initial step in the development of a therapy that simultaneously treats CMB and ischemic stroke, we hypothesized that inhibition of the PDE3A pathway is protective against CMH development.MethodsThe effect of PDE3A pathway inhibition was studied in the inflammation-induced and cerebral amyloid angiopathy (CAA)-associated mouse models of CMH. The PDE3A pathway was modulated using two approaches: genetic deletion of PDE3A and pharmacological inhibition of PDE3A by cilostazol. The effects of PDE3A pathway modulation on H&E- and Prussian blue (PB)-positive CMH development, BBB function (IgG, claudin-5, and fibrinogen), and neuroinflammation (ICAM-1, Iba-1, and GFAP) were investigated.ResultsRobust development of CMH in the inflammation-induced and CAA-associated spontaneous mouse models was observed. Inflammation-induced CMH were associated with markers of BBB dysfunction and inflammation, and CAA-associated spontaneous CMH were associated primarily with markers of neuroinflammation. Genetic deletion of the PDE3A gene did not alter BBB function, microglial activation, or CMH development, but significantly reduced endothelial and astrocyte activation in the inflammation-induced CMH mouse model. In the CAA-associated CMH mouse model, PDE3A modulation via pharmacological inhibition by cilostazol did not alter BBB function, neuroinflammation, or CMH development.ConclusionsModulation of the PDE3A pathway, either by genetic deletion or pharmacological inhibition, does not alter CMH development in an inflammation-induced or in a CAA-associated mouse model of CMH. The role of microglial activation and BBB injury in CMH development warrants further investigation.

Myeloperoxidase (MPO) is one of the most abundantly expressed proteins in neutrophils. It serves as a critical component of the antimicrobial defense system, facilitating microbial killing via generation of reactive oxygen species (ROS). Interestingly, emerging evidence indicates that in addition to the well-recognized canonical antimicrobial function of MPO, it can directly or indirectly impact immune cells and tissue responses in homeostatic and disease states. Here, we highlight the emerging non-canonical functions of MPO, including its impact on neutrophil longevity, activation and trafficking in inflammation, its interactions with other immune cells, and how these interactions shape disease outcomes. We further discuss MPO interactions with barrier forming endothelial and epithelial cells, specialized cells of the central nervous system (CNS) and its involvement in cancer progression. Such diverse function and the MPO association with numerous inflammatory disorders make it an attractive target for therapies aimed at resolving inflammation and limiting inflammation-associated tissue damage. However, while considering MPO inhibition as a potential therapy, one must account for the diverse impact of MPO activity on various cellular compartments both in health and disease. [ABSTRACT FROM AUTHOR]

Blood-based preparations are used in clinical practice for the treatment of several eye disorders. The aim of this study is to analyze the effect of freeze-drying blood-based preparations on the levels of growth factors and wound healing behaviors in an in vitro model. Platelet-rich plasma (PRP) and serum (S) preparations from the same Cord Blood (CB) sample, prepared in both fresh frozen (FF) and freeze-dried (FD) forms (and then reconstituted), were analyzed for EGF and BDNF content (ELISA Quantikine kit). The human MIO-M1 glial cell line (Moorfield/Institute of Ophthalmology, London, UK) was incubated with FF and FD products and evaluated for cell migration with scratch-induced wounding (IncuCyte S3 Essen BioScience), proliferation with cyclin A2 and D1 gene expression, and activation with vimentin and GFAP gene expression. The FF and FD forms showed similar concentrations of EGF and BDNF in both the S and PRP preparations. The wound healing assay showed no significant difference between the FF and FD forms for both S and PRP. Additionally, cell migration, proliferation, and activation did not appear to change in the FD forms compared to the FF ones. Our study showed that reconstituted FD products maintained the growth factor concentrations and biological properties of FF products and could be used as a functional treatment option. [ABSTRACT FROM AUTHOR]

Our ability to understand the cellular complexity of tissues has been revolutionized in recent years with significant advances in proteogenomic technologies including those enabling spatial analyses. This has led to numerous consortium efforts, such as the human cell atlas initiative which aims to profile all cells in the human body in healthy and diseased contexts. The availability of such information will subsequently lead to the identification of novel biomarkers of disease and of course therapeutic avenues. However, before such an atlas of any given healthy or diseased tissue can be generated, several factors should be considered including which specific techniques are optimal for the biological question at hand. In this review, we aim to highlight some of the considerations we believe to be important in the experimental design and analysis process, with the goal of helping to navigate the rapidly changing landscape of technologies available., (© The Author(s) 2024. Published by Oxford University Press on behalf of the British Society for Immunology.)

Astrocytes are an abundant cell type in central nervous system (CNS) and play a critical role in CNS homeostasis. Abnormal astrocytes have been consistently observed in diverse CNS diseases, which lead to enlarged inflammatory reactions and glial scars. As a naturally extracted monomer with anti-inflammatory properties, berberine shows a potential therapeutic effect on abnormal astrocytes. However, the mechanism of berberine in the regulation of abnormal astrocytes is still unclear. Here, we have shown that berberine restored lipopolysaccharide (LPS)-induced mitochondrial abnormalities by upregulating NDUFC2, a subunit of ubiquinone oxidoreductase, expression resulting in astrocyte rehabilitation. Pretreatment of 10 µg/ml berberine inhibited LPS-induced astrocyte activation and migration. The knockdown of NDUFC2 repressed the inhibitory action of berberine on LPS-induced astrocyte activation and migration; and, as a result, mitochondria injury was increased. These findings prove a new mechanism in astrocyte reaction and provide a potential curative target of berberine for inflammatory diseases. [ABSTRACT FROM AUTHOR]

Objective To investigate the effect of sodium arsenite (NaAsO2) on activation of human hepatic stellate cells（LX-2 cells) and its mechanism. Methods LX-2 cells were cultured in vitro. The LX-2 cells in the logarithmic growth phase were cultured with NaAsO2 medium at the concentrations of 0，5，15，20，30，40，50 µmol/L. The cell viability was detected by MTT method and the experimental concentration of NaAsO2 was determined. LX-2 cells in the logarithmic growth phase were randomly divided into the control group, low-，medium- and high-concentration NaAsO2 groups， which were treated with 0，5，10，and 15 µmol/L NaAsO2 for 48 h, respectively. The autophagy of the cells was detected by transmission electron microscope, and the expression levels of the cell activated protein (ɑ-SMA) and autophagy protein（AKT，Hes1，and mTOR) were detected by Western blotting. Results With the increase of NaAsO2 concentration， the inhibition rate of cell proliferation increased gradually. Five，10，and 15 µmol/L were selected as the experimental concentrations of NaAsO2 according to the half-maximal inhibitory concentration and the cell viability. Autophagosome and autophagolysosome were observed in the control group. In the low-，medium- and high-concentration NaAsO2 groups， the cell gap widened graduallym and "False Dee gap" was observed. The number of autophagosomes increased, and the distribution of lipid droplets was more dense. Some mitochondria were swollen and vacuolated, and a large number of mitochondrial cristae were broken, and the above changes were the most obvious in the high-concentration NaAsO2 group. Compared with the control group, the relative expression of ɑ-SMA protein gradually increased, the protein expression of AKT and mTOR gradually decreased, and while the protein expression of Hes1 gradually increased in the low-, medium and high-concentration NaAsO2 groups（all P<0. 05）. Conclusion NaAsO2 may induce the activation of LX-2 cells by promoting autophagy. [ABSTRACT FROM AUTHOR]

BackgroundCerebral microhemorrhages (CMH) are tiny deposits of blood degradation products in the brain and are pathological substrates of cerebral microbleeds. The existing CMH animal models are β-amyloid-, hypoxic brain injury-, or hypertension-induced. Recent evidence shows that CMH develop independently of hypoxic brain injury, hypertension, or amyloid deposition and CMH are associated with normal aging, sepsis, and neurodegenerative conditions. One common factor among the above pathologies is inflammation, and recent clinical studies show a link between systemic inflammation and CMH. Hence, we hypothesize that inflammation induces CMH development and thus, lipopolysaccharide (LPS)-induced CMH may be an appropriate model to study cerebral microbleeds.MethodsAdult C57BL/6 mice were injected with LPS (3 or 1 mg/kg, i.p.) or saline at 0, 6, and 24 h. At 2 or 7 days after the first injection, brains were harvested. Hematoxylin and eosin (H&E) and Prussian blue (PB) were used to stain fresh (acute) hemorrhages and hemosiderin (sub-acute) hemorrhages, respectively. Brain tissue ICAM-1, IgG, Iba1, and GFAP immunohistochemistry were used to examine endothelium activation, blood-brain barrier (BBB) disruption, and neuroinflammation. MRI and fluorescence microscopy were used to further confirm CMH development in this model.ResultsLPS-treated mice developed H&E-positive (at 2 days) and PB-positive (at 7 days) CMH. No surface and negligible H&E-positive CMH were observed in saline-treated mice (n = 12). LPS (3 mg/kg; n = 10) produced significantly higher number, size, and area of H&E-positive CMH at 2 days. LPS (1 mg/kg; n = 9) produced robust development of PB-positive CMH at 7 days, with significantly higher number and area compared with saline (n = 9)-treated mice. CMH showed the highest distribution in the cerebellum followed by the sub-cortex and cortex. LPS-induced CMH were predominantly adjacent to cerebral capillaries, and CMH load was associated with indices of brain endothelium activation, BBB disruption, and neuroinflammation. Fluorescence microscopy confirmed the extravasation of red blood cells into the brain parenchyma, and MRI demonstrated the presence of cerebral microbleeds.ConclusionsLPS produced rapid and robust development of H&E-positive (at 2 days) and PB-positive (at 7 days) CMH. The ease of development of both H&E- and PB-positive CMH makes the LPS-induced mouse model suitable to study inflammation-induced CMH.

MCL, Mincle and Dectin‐2 are C‐type lectin receptors expressed by subsets of myeloid cells, and their genes cluster together in the APLEC/Dectin‐2 gene complex. We have previously shown that MCL and Mincle form a heterodimer in the rat, and others have shown that MCL and Dectin‐2 form a heterodimer in the mouse. In the rat, Dectin‐2 is a pseudogene, but here, we examine the association of the three receptors in human. In co‐transfection experiments analyzed with flow cytometry and immunoprecipitation, we here show that human MCL and Mincle form a disulphide‐linked heterodimer that associates with the signalling adaptor molecule FcεRIγ, in accordance with our previous findings in the rat. In contrast to previous findings in the rat, data in this paper indicate a direct association of MCL with FcεRIγ, as previously shown for mouse MCL. We were unable to demonstrate the formation of a heterodimer between human MCL and Dectin‐2. Thus, despite similarities, there may be important differences in the conformation of these receptors between rat, mouse and human, and this may have functional consequences. [ABSTRACT FROM AUTHOR]

The incidence of liver fibrosis is increasing worldwide, and if left untreated, it will later develop into cirrhosis with a high mortality rate. In this paper, the activation pathway and related mechanism of stellate cells in liver fibrosis are introduced, and some current therapeutic methods are summarized. These results suggest that stellate cells play an important role in liver fibrosis, and targeted therapy for the purpose of inhibiting the activation of stellate cells and inducing their apoptosis is expected to be an effective regimen to reverse liver fibrosis. However, there are some problems such as insufficient in-depth study of related mechanisms and imperfect experiments. In future animal experiments and clinical trials, more studies can be carried out to provide high-quality protocols for the treatment of liver fibrosis. [ABSTRACT FROM AUTHOR]

Drug hypersensitivity can be an important problem during pharmacological management of various diseases. Patients diagnosed as having a drug allergy usually need to avoid the offending drug, either temporarily or for life. Another way of overcoming a drug allergy is to establish desensitization using the allergen drug itself. We previously investigated in vitro desensitization of human basophils using a subthreshold dose of an IgE-crosslinking reagent. We found that basophil desensitization occurred in a dose-dependent manner over a period of one to several hours. We think that inducible basophil desensitization occurring without histamine release may explain, at least in part, the clinical features of drug desensitization in type 1 drug allergy.

• Organic acids are acid in nature. • Dietary organic acids have no risk factors. • Organic acids have antimicrobial activity. • They attenuate Alzheimer disease via antioxidant activity to affect the amyloid aggregation pathway. Organic acids are specific groups of chemicals that contain COOH in their structures. They have properties of weakly acidic nature and dissolves completely into protons when present in aqueous solutions. In biological systems, organic acids are involved in various activities such as important intermediates in various metabolic cycles, antimicrobial compounds, and prediction of maturity indices. Due to these properties, organic acids are important phytochemicals that have a wide range of commercial applications like animal feeds utilization, improve plant quality, bactericidal and bacteriostatic potential, as well as in the pharmaceutical industry for numerous preparations. In pharmacokinetics, organic acids are primarily involved in the absorption of pH-dependent drugs. Different techniques have been employed for the extraction, purification, and quantification of organic acids. Among them, microwave-assisted extractions (MAE) and ultrasound-assisted extractions (UAE) are the chiefly used techniques where MAE is one of the most valuable methods for isolating different compounds from plant materials for industrial and analytical purposes. Moreover, bioactive chemicals such as natural organic acid from plants and other sources are important and referred as "lifespan essential"since they havea beneficial effect on healthy aging and Alzheimer's disease (AD). Therefore, dietary organic acids and avoiding risk factors, will reduce the risk of neurodegenerative diseases such as AD and increase a healthy lifespan. Thus, the present study aimed to understand the phytochemistry, biological activities, industrial applications, possible interaction with other drugs/foods supplements,techniques of extraction, purification and fractionation, techniques of identification and quantification, the effect of food processing,and molecular mechanisms of organic acid to attenuate AD. [ABSTRACT FROM AUTHOR]

Abstract Background Human mesenchymal stem/stromal cells (hMSCs) represent a promising therapeutic strategy for ventilator-induced lung injury (VILI) and acute respiratory distress syndrome. Translational challenges include restoring hMSC efficacy following cryopreservation, developing effective xenogeneic-free (XF) hMSCs and establishing true therapeutic potential at a clinically relevant time point of administration. We wished to determine whether cytokine pre-activation of cryopreserved, bone marrow-derived XF-hMSCs would enhance their capacity to facilitate injury resolution following VILI and elucidate mechanisms of action. Methods Initially, in vitro studies examined the potential for the secretome from cytokine pre-activated XF-hMSCs to attenuate pulmonary epithelial injury induced by cyclic mechanical stretch. Later, anaesthetised rats underwent VILI and, 6 h following injury, were randomized to receive 1 × 107 XF-hMSC/kg that were (i) naive fresh, (ii) naive cryopreserved, (iii) cytokine pre-activated fresh or (iv) cytokine pre-activated cryopreserved, while control animals received (v) vehicle. The extent of injury resolution was measured at 24 h after injury. Finally, the role of keratinocyte growth factor (KGF) in mediating the effect of pre-activated XF-hMSCs was determined in a pulmonary epithelial wound repair model. Results Pre-activation enhanced the capacity of the XF-hMSC secretome to decrease stretch-induced pulmonary epithelial inflammation and injury. Both pre-activated fresh and cryopreserved XF-hMSCs enhanced resolution of injury following VILI, restoring oxygenation, improving lung compliance, reducing lung leak and improving resolution of lung structural injury. Finally, the secretome of pre-activated XF-hMSCs enhanced epithelial wound repair, in part via a KGF-dependent mechanism. Conclusions Cytokine pre-activation enhanced the capacity of cryopreserved, XF-hMSCs to promote injury resolution following VILI, potentially via a KGF-dependent mechanism.

The human akna gene encodes an AT-hook transcription factor, the expression of which is involved in various cellular processes. The goal of this study was to identify potential AKNA binding sites in genes that participate in T-cell activation and validate selected genes. Here we analyzed ChIP-seq and microarray assays to determine AKNA-binding motifs and the cellular process altered by AKNA in T-cell lymphocytes. In addition, we performed a validation analysis by RT-qPCR to assess AKNA’s role in promoting IL-2 and CD80 expression. We found five AT-rich motifs that are potential candidates as AKNA response elements. We identified these AT-rich motifs in promoter regions of more than a thousand genes in activated T-cells, and demonstrated that AKNA induces the expression of genes involved in helper T-cell activation, such as IL-2. The genomic enrichment and prediction of AT-rich motif analyses demonstrated that AKNA is a transcription factor that can potentially modulate gene expression by recognizing AT-rich motifs in a plethora of genes that are involved in different molecular pathways and processes. Among the cellular processes activated by AT-rich genes, we found inflammatory pathways potentially regulated by AKNA, suggesting AKNA is acting as a master regulator during T-cell activation.

NK cells express activating receptors that signal through ITAM-bearing adapter proteins. The phosphorylation of each ITAM creates binding sites for SYK and ZAP70 protein tyrosine kinases to propagate downstream signaling including the induction of Ca 2 + influx. While all immature and mature human NK cells co-express SYK and ZAP70, clonally driven memory or adaptive NK cells can methylate SYK genes and signaling is mediated exclusively using ZAP70. Here, we examined the role of SYK and ZAP70 in a clonal human NK cell line KHYG1 by CRISPR-based deletion using a combination of experiments and mechanistic computational modeling. Elimination of SYK resulted in more robust Ca + + influx after cross-linking of the CD16 and NKp30 receptors and enhanced phosphorylation of downstream proteins, whereas ZAP70 deletion diminished these responses. By contrast, ZAP70 depletion increased proliferation of the NK cells. As immature T cells express both SYK and ZAP70 but mature T cells often express only ZAP70, we transduced the human Jurkat cell line with SYK and found that expression of SYK increased proliferation but diminished TCR-induced Ca 2 + flux and activation. We performed transcriptional analysis of the matched sets of variant Jurkat and KHYG1 cells and observed profound alterations caused by SYK expression. As depletion of SYK in NK cells increased their activation, primary human NK cells were transduced with a CD19-targeting CAR and were CRISPR edited to ablate SYK or ZAP70 . Deletion of SYK resulted in more robust cytotoxic activity and cytokine production, providing a new therapeutic strategy of NK cell engineering for cancer immunotherapy.

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.

Trypanosoma cruzi is a protozoan parasite that affects millions of people in Latin America. Infection occurs by vectorial transmission or by transfusion or transplacental route. Immune events occurring immediately after the parasite entrance are poorly explored. Dendritic cells (DCs) are target for the parasite immune evasion mechanisms. Recently, we have demonstrated that two different populations of DCs display variable activation after interaction with the two infective forms of the parasite: metacyclic or blood trypomastigotes (mTp or bTp) in vitro. The skin constitutes a complex network with several populations of antigen-presenting cells. Previously, we have demonstrated T. cruzi conditioning the repertoire of cells recruited into the site of infection. In the present work, we observed that mTp and bTp inoculation displayed differences in cell recruitment to the site of infection and in the activation status of APCs in draining lymph nodes and spleen during acute infection. Animals inoculated with mTp exhibited 100% of survival with no detectable parasitemia, in contrast with those injected with bTp that displayed high mortality and high parasite load. Animals infected with mTp and challenged with a lethal dose of bTp 15 days after primary infection showed no mortality and incremented DC activation in secondary lymphoid organs compared with controls injected only with bTp or non-infected mice. These animals also displayed a smaller number of amastigote nests in cardiac tissue and more CD8 T cells than mice infected with bTp. All the results suggest that both Tp infective stages induce an unequal immune response since the beginning of the infection. [ABSTRACT FROM AUTHOR]

Cell-based mathematical models have previously been developed to simulate the immune system in response to pathogens. Mathematical modeling papers which study the human immune response to pathogens have predicted concentrations of a variety of cells, including activated and resting macrophages, plasma cells, and antibodies. This study aims to create a comprehensive mathematical model that can predict cytokine levels in response to a gram-positive bacterium, S. aureus by coupling previous models. To accomplish this, the cytokines Tumor Necrosis Factor Alpha (TNF- α), Interleukin 6 (IL-6), Interleukin 8 (IL-8), and Interleukin 10 (IL-10) are included to quantify the relationship between cytokine release from macrophages and the concentration of the pathogen, S. aureus, ex vivo. Partial differential equations (PDEs) are used to model cellular response and ordinary differential equations (ODEs) are used to model cytokine response, and interactions between both components produce a more robust and more complete systems-level understanding of immune activation. In the coupled cellular and cytokine model outlined in this paper, a low concentration of S. aureus is used to stimulate the measured cellular response and cytokine expression. Results show that our cellular activation and cytokine expression model characterizing septic conditions can predict ex vivo mechanisms in response to gram-negative and gram-positive bacteria. Our simulations provide new insights into how the human immune system responds to infections from different pathogens. Novel applications of these insights help in the development of more powerful tools and protocols in infection biology. [ABSTRACT FROM AUTHOR]

Trypanosoma cruzi is a protozoan parasite that affects millions of people in Latin America. Infection occurs by vectorial transmission or by transfusion or transplacental route. Immune events occurring immediately after the parasite entrance are poorly explored. Dendritic cells (DCs) are target for the parasite immune evasion mechanisms. Recently, we have demonstrated that two different populations of DCs display variable activation after interaction with the two infective forms of the parasite: metacyclic or blood trypomastigotes (mTp or bTp) in vitro. The skin constitutes a complex network with several populations of antigen-presenting cells. Previously, we have demonstrated T. cruzi conditioning the repertoire of cells recruited into the site of infection. In the present work, we observed that mTp and bTp inoculation displayed differences in cell recruitment to the site of infection and in the activation status of APCs in draining lymph nodes and spleen during acute infection. Animals inoculated with mTp exhibited 100% of survival with no detectable parasitemia, in contrast with those injected with bTp that displayed high mortality and high parasite load. Animals infected with mTp and challenged with a lethal dose of bTp 15 days after primary infection showed no mortality and incremented DC activation in secondary lymphoid organs compared with controls injected only with bTp or non-infected mice. These animals also displayed a smaller number of amastigote nests in cardiac tissue and more CD8 T cells than mice infected with bTp. All the results suggest that both Tp infective stages induce an unequal immune response since the beginning of the infection.

MicroRNAs (miRNAs), small non-coding molecules targeting messenger RNAs and inhibiting protein translation, modulate key biological processes, including cell growth and development, energy utilization, and homeostasis. In particular, miRNAs control the differentiation, survival, and activation of CD4+ T conventional (Tconv) cells, key players of the adaptive immunity, and regulate the physiological response to infections and the pathological loss of immune homeostasis in autoimmunity. Upon T-cell receptor (TCR) stimulation, the described global miRNA quantitative decrease occurring in T cells is believed to promote the acquisition of effector functions by relaxing the post-transcriptional repression of genes associated with proliferation and cell activity. MiRNAs were initially thought to get downregulated uniquely by intracellular degradation; on the other hand, miRNA secretion via extracellular vesicles (EVs) represents an additional mechanism of rapid downregulation. By focusing on molecular interactions by means of graph theory, we have found that miRNAs released by TCR-stimulated Tconv cells are significantly enriched for targeting transcripts upregulated upon stimulation, including those encoding for crucial proteins associated with Tconv cell activation and function. Based on this computational approach, we present our perspective based on the following hypothesis: a stimulated Tconv cell will release miRNAs targeting genes associated with the effector function in the extracellular space in association with EVs, which will thus possess a suppressive potential toward other Tconv cells in the paracrine environment. We also propose possible future directions of investigation aimed at taking advantage of these phenomena to control Tconv cell effector function in health and autoimmunity.

Burnet envisaged the early presence of self‐antigens in development, or 'the history' of an animal, ablates the animal's ability to immunologically respond against them. Lederberg added the idea that the continuous presence of self‐antigens is required to maintain tolerance throughout life. We refer to Lederberg's proposal as 'The Historical Postulate'. The mechanism of central tolerance, as now understood, is consistent with The Historical Postulate. Some observations, reflecting peripheral tolerance, appear inconsistent with this postulate. For example, some foreign peripheral tissues, grafted onto an animal before the immune system develops, can be rejected as the immune system matures. The original two‐signal model of lymphocyte activation was proposed in part because it accounted for peripheral tolerance in a manner consistent with The Historical Postulate. We proposed that lymphocyte activation required antigen‐mediated lymphocyte cooperation, whereas antigen would inactivate lymphocytes when insufficient in number to achieve activation. We argue here that the exceptions to The Historical Postulate can be explained by the two‐signal model of lymphocyte activation: they reflect the existence of greater numbers of lymphocytes specific for these antigens than for natural peripheral antigens, and so are outside the physiological limits important in selecting through evolution this mechanism of peripheral tolerance. We argue that a consideration of whether The Historical Postulate is valid is important, even if only valid within certain understandable limits. The currently popular DAMP model of CD4 T cell activation is, strictly speaking and in a manner we discuss, in violation of this postulate. [ABSTRACT FROM AUTHOR]

Parachartergus fraternus wasp induces inflammation with a predominance of mononuclear cells, that can acquire macrophage functions at the sting site, amplifying the response. These cells can be activated by venomous animals and are involved in destruction of injurious agents and release of inflammatory mediators. The objective of this work was to evaluate the activity of P. fraternus venom (Pfv) on isolated murine macrophage function. The cells were obtained from peritoneal cavity of Swiss male mice and incubated with Pfv (2.5, 5 and 10 μg/mL). Cytotoxicity was determined using MTT assay. Adhesion and detachment were evaluated using violet crystal dye. Spreading was evaluated based on morphological parameters. Phagocytosis was performed with opsonized zymosan. Production of hydrogen peroxide (H 2 O 2) and nitric oxide (NO) were quantified using the phenol red and Griess assays, respectively. Pfv at concentrations evaluated was not cytotoxic in MTT assay and did not cause macrophage detachment in cell culture plates. However, it increased adhesion of macrophage, spreading and phagocytosis of opsonized zymosan, as well as induced production of H 2 O 2 and NO. Therefore, Pfv induces macrophage activation in vitro and the response of these cells can be correlated with the previously reported inflammatory process triggered by this wasp. • Parachartergus fraternus venom is not cytotoxic up to 10 μg/mL and it is a pro-inflammatory activator of macrophages. • P. fraternus venom increases adhesion and spreading of macrophages. • P. fraternus venom induces phagocytosis in vitro. • P. fraternus venom induces nitric oxide and hydrogen peroxide production by macrophages. [ABSTRACT FROM AUTHOR]

Objectives: Interleukin (IL)-24 has been considered as an inflammatory cytokine in autoimmune diseases. However, conflicting data exist and its biological function remains controversial. Additionally, little is known about its functional impact on natural killer (NK) cells. The aim of this study was to investigate the role of IL-24 in NK cell activation and its clinical implication in systemic lupus erythematosus (SLE). Methods: Serum cohort consisting of 299 SLE patients, 214 RA patients, and 159 healthy controls (HCs) and plasma cohort consisting of 70 SLE patients, 82 RA patients, and 123 HCs were included in evaluating IL-24 concentrations. Impact of IL-24 on NK cell activation was assessed in two NK cell subsets, i.e., CD56dimCD16+ and CD56brightCD16− NK cells. Human NK-92 cell line was applied to evaluate functional potential of IL-24 on NK cell migration and invasion. Results: Serum and plasma levels of IL-24 were comparable between patients with SLE or RA and HCs. While recombinant human (rh) IL-2 consistently induced an increased expression of CD69 on both CD56dimCD16+ and CD56brightCD16− cells derived from both healthy subjects and patients with SLE, IL-24 alone was insufficient to activate the CD56dim and CD56bright NK cells. Similarly, while the migratory NK-92 cell numbers were significantly increased with rhIL-2 stimulation, IL-24 alone was unable to enhance NK-92 cell migratory and invasive capacity. Conclusion: Our data indicate that there were no significant differences in serum and plasma concentrations of IL-24 between SLE patients and healthy controls. Recombinant IL-24 has no effect on NK cell activation and migration. Key points • This is the first study to investigate functional potential of IL-24 on NK cell activation. • Recombinant IL-24 lacks functional capacity on NK cell activation in either CD56dimCD16+or CD56brightCD16-NK cell subsets derived from both healthy subjects and patients with SLE. • No significant differences in serum and plasma levels of IL-24 between SLE patients and healthy controls. [ABSTRACT FROM AUTHOR]

With the objective of reducing energy cost, we study the stochastic optimization of traffic off-loading via dual-connectivity by joint cell activation and user association. Particularly, explicitly considering the dynamic effects of time-varying and random traffic arrivals, we formulate a stochastic problem that quantifies the tradeoff between energy cost and queuing delay. Employing two-time-scale Lyapunov optimization, the formulated problem is transformed as a user association problem at each time slot and a small-cell activation problem at each frame. However, the user association problem turns out to be a mixed-integer nonlinear programming problem and is difficult to be transformed into a convex problem, and traditional game-theoretic solutions, such as Nash equilibrium, cannot be used to solve it. Then, we introduce a two-sided many-to-one matching game and propose a distributed user association algorithm that converges to a local optimum. On the other hand, the cell activation problem belongs to the class of maximum facility location problems that are generally NP-hard. Additionally, its objective function is not a submodular one. Thus, we present an iterative and heuristic algorithm that finds the best set of active small-cell BSs by repeatedly solving the user association problem with different sets of active small cells in each iteration. Furthermore, an online two-time-scale joint cell activation and user association algorithm are developed. Finally, numerical results show the effectiveness of the matching-based user association algorithm on traffic off-loading improvement, the heuristic cell activation algorithm on trading off between energy cost reduction and network throughput enhancement, and the online joint algorithm on balancing between energy cost reduction and queuing delay performance.

Monocytes are the third most frequent type of leukocytes in humans, linking innate and adaptive immunity and are critical drivers in many inflammatory diseases. Based on the differential expression of surface antigens, three monocytic subpopulations have been suggested in humans and two in rats with varying inflammatory and phenotype characteristics. Potential intervention strategies that aim to manipulate these cells require an in-depth understanding of monocyte behavior under different conditions. However, monocytes are highly sensitive to their specific activation state and expression of surface markers, which can change during cell isolation and purification. Thus, there is an urgent need for an unbiased functional analysis of activation in monocyte subtypes, which is not affected by the isolation procedure. Here, we present a flow cytometry-based protocol for evaluating subset-specific activation and cytokine expression of circulating blood monocytes both in humans and rats using small whole blood samples (50 - 100 μL). In contrast to previously described monocyte isolation and flow cytometry visualization methods, the presented approach virtually leaves monocyte subsets in a resting state or fixes them in their current state and allows for an unbiased functional endpoint analysis without prior cell isolation. This protocol is a comprehensive tool for studying differential monocyte regulation in the inflammatory and allogeneic immune response in vitro and vivo.

Introduction: Aging is a natural process involving dysfunction of multiple organs and is characterized by increased susceptibility to infections, cancer and autoimmune diseases. The functionality of the immune system depends on the capacity of lymphocytes to proliferate in response to antigenic challenges, and telomere length has an important role regulating the number of cell divisions. The aim of this study was to determine the possible relationship between telomere length, interleukin 2 (IL-2) production, CD25 expression and proliferation of peripheral blood mononuclear cells (PBMCs) in aged men.Material and Methods: Telomere length was measured by RT-PCR in PBMCs from young and aged men. IL-2 production and CD25 expression were determined by ELISA and flow cytometry, respectively. Cell proliferation was measured by CFSE dilution assays upon in vitro stimulation with concanavalin A (Con A).Results: PBMCs from aged men showed a shorter telomere length and a reduced capacity to proliferate in vitro, compared to young men. In contrast, no significant differences in the level of CD25 expression on T lymphocytes, and in vitro production of IL-2 were detected in both groups. In addition, no significant correlation was detected between levels of CD25 expression, IL-2 production, cell proliferation, and telomere length in aged men.Conclusions: In aged men the telomere length shortening and the reduced T cell proliferation are not related to the capacity of IL-2 production and CD25 expression on T lymphocytes. [ABSTRACT FROM AUTHOR]