Cell Activation Markers Research Articles

Cell cycle parameters and ornithine decarboxylase activity in the red bone marrow of hibernating ground squirrels Urocitellus undulatus

During the hibernation season, the values for the parameters of the cell cycle of red bone marrow cells in the hibernating ground squirrels Urocitellus undulatus, when they return to an active-like state between periods of torpor and interbout arousal, do not differ from those observed in summer-active animals. In animals that enter a state of torpor, the cumulative percentage of cells in the resting phase (G0 phase) and pre-synthesis phase (G1 phase) increased from 71.8 to 76.0%, the percentage of cells in the synthesis phase (S phase) decreased from 19.3 to 15.3% compared to those animals that return to an active-like state between periods of torpor and interbout arousal. The cumulative percentage of cells in the post DNA synthesis phase (G2 phase) and mitosis (M) does not change, but (G2 + M)/S ratio increases. When animals enter a state of torpor, changes in parameter values are observed when the animal’s body temperature drops below 25°C, this effect refers to a system whose thermal relaxation time is a nonmonotonic function of the initial temperature. The activity of the key enzyme of polyamine synthesis ornithine decarboxylase, a marker of cell activation and proliferation during interbout arousal does not significantly differ from that observed in summer-active animals; the enzymatic activity decreases sharply, when animals decrease their body temperature below 25°C and enter a state of torpor, and this activity remains at a low level during hibernation and arousal until body temperature reaches 30°C. The role of changes in the parameter values associated with proliferative activity in adaptation of hematopoietic tissue during hibernation of the Yakutian ground squirrel is discussed.

Corneal neuroepithelial compartmentalized microfluidic chip model for evaluation of toxicity-induced dry eye

Part of the lacrimal functional unit, the cornea protects the ocular surface from numerous environmental aggressions and xenobiotics. Toxicological evaluation of compounds remains a challenge due to complex interactions between corneal nerve endings and epithelial cells. To this day, models do not integrate the physiological specificity of corneal nerve endings and are insufficient for the detection of low toxic effects essential to anticipate Toxicity-Induced Dry Eye (TIDE).Using high-content imaging tool, we here characterize toxicity-induced cellular alterations using primary cultures of mouse trigeminal sensory neurons and corneal epithelial cells in a compartmentalized microfluidic chip.We validate this model through the analysis of benzalkonium chloride (BAC) toxicity, a well-known preservative in eyedrops, after a single (6h) or repeated (twice a day for 15 min over 5 days) topical 5.10−4% BAC applications on the corneal epithelial cells and nerve terminals.In combination with high-content image analysis, this advanced microfluidic protocol reveal specific and tiny changes in the epithelial cells and axonal network as well as in trigeminal cells, not directly exposed to BAC, with ATF3/6 stress markers and phospho-p44/42 cell activation marker. Altogether, this corneal neuroepithelial chip enables the evaluation of toxic effects of ocular xenobiotics, distinguishing the impact on corneal sensory innervation and epithelial cells. The combination of compartmentalized co-culture/high-content imaging/multiparameter analysis opens the way for the systematic analysis of toxicants but also neuroprotective compounds.

Skeletal muscle adaptations in patients with lung cancer: Longitudinal observations from the whole body to cellular level.

Cancer and its treatment can adversely affect skeletal muscle, impacting physical function, treatment response and survival. No studies, however, have comprehensively characterized these muscle adaptations longitudinally in human patients at the cellular level. We examined skeletal muscle size and function from the whole body to the sub-cellular level in 11 patients with non-small cell lung cancer (NSCLC; 6 male/5 female, mean age 58±3years) studied over a 2-month observation period starting during their first cycle of standard of care cancer treatment and in 11 age- and sex-matched healthy controls (HC) without a current or past history of cancer. Biopsies of the vastus lateralis were performed to assess muscle fibre size, contractility and mitochondrial content, along with assessments of physical function, whole muscle size and function, and circulating cytokines. Body weight, composition and thigh muscle area and density were unaltered over time in patients with NSCLC, while muscle density was lower in patients with NSCLC versus HC (P=0.03). Skeletal muscle fibre size decreased by 18% over time in patients (all P=0.02) and was lower than HC (P=0.02). Mitochondrial fractional area and density did not change over time in patients, but fractional area was lower in patients with NSCLC compared with HC (subsarcolemmal, P=0.04; intermyofibrillar, P=0.03). Patients with NSCLC had higher plasma concentrations of IL-6 (HC 1.40±0.50; NSCLC 4.71±4.22; P<0.01), GDF-15 (HC 569±166; NSCLC 2071±1168; P<0.01) and IL-8/CXCL8 (HC 4.9±1.8; NSCLC 10.1±6.0; P=0.02) compared with HC, but there were no changes in inflammatory markers in patients with NSCLC over time. No changes were observed in markers of satellite cell activation or DNA damage in patients and no group differences were noted with HC. Whole-muscle strength was preserved over time in patients with NSCLC coincident with improved single fibre contractility. This study is the first to comprehensively examine longitudinal alterations in skeletal muscle fibre size and function in patients with NSCLC and suggests that muscle fibre atrophy occurs during cancer treatment despite weight stability and no changes in conventional clinical measurements of whole body or thigh muscle size over this period.

Analysis of the Immunostimulatory Effects of Cytokine-Expressing Internal Ribosome Entry Site-Based RNA Adjuvants and Their Applications.

Developing new adjuvants that can effectively induce humoral and cellular immune responses while broadening the immune response is of great value. In this study, we aimed to develop single-stranded RNA adjuvants expressing (1) granulocyte monocyte colony-stimulating factor or (2) interleukin 18 based on the encephalomyocarditis virus internal ribosome entry site; we also tested their efficacy in combination with ovalbumin or inactivated influenza vaccines. Notably, cytokine-expressing RNA adjuvants increased the expression of antigen-presenting cell activation markers in mice. Specifically, when combined with ovalbumin, RNA adjuvants expressing granulocyte monocyte colony-stimulating factor increased CD4+ T-cell responses, while those expressing interleukin 18 increased CD8+ T-cell responses. Cytokine-expressing RNA adjuvants further increased the frequency of polyclonal T cells with the influenza vaccine and reduced the clinical illness scores and weight loss of mice after viral challenge. Collectively, our results suggest that cytokine-expressing RNA adjuvants can be applied to protein-based or inactivated vaccines to increase their efficacy.

Elevated baseline soluble FcεRI may be linked to early response to omalizumab treatment in chronic spontaneous urticaria.

Omalizumab, an anti-IgE monoclonal antibody, is an effective treatment in chronic spontaneous urticaria (CSU). Predictors of fast and good response for omalizumab treatment have not yet been identified and characterized. To evaluate whether soluble FcεRI (sFcεRI), a marker of IgE-mediated mast cell activation, predicts the time of response to omalizumab in CSU. Sera of 67 CSU patients were obtained before omalizumab treatment and analysed for sFcεRI levels by ELISA (2 ng/mL was used as cut-off for elevated sFcɛRI). Treatment response during the first 4 weeks was assessed with the urticaria activity score (UAS7), urticaria control test (UCT) and the rolling UAS7 (rUAS7). Elevated pre-treatment sFcɛRI levels were detected in more than 70% of patients with completely controlled disease (UCT = 16) and well-controlled disease (UCT = 12-15) and were significantly associated with disease control (χ2 = 4.94, p < 0.05). More than half of the patients (14/25) with low levels had poor disease control (UCT < 12). Of the patients who achieved complete and marked UAS7 response, respectively, 75% and 63% had elevated baseline sFcɛRI levels. Post-treatment UAS7 scores were lower in patients with elevated sFcɛRI levels reaching statistical significance at Week 3 (p < 0.05). Patients with elevated baseline sFcɛRI levels achieved rUAS7 ≤ 6 and = 0 earlier than those with lower levels (Days 9 vs. 13 and Days 12 vs. 14, respectively). Elevated sFcεRI serum levels predict early and good response to treatment with omalizumab, which may help to better design treatment options for CSU patients.

Abstract 127: Time Restricted Feeding In Diet Induced Obesity Reduces Kidney Cd8 + T Cells

Diet induced obesity (DIO) is one of the leading risk factors for chronic kidney disease. Previous studies identified a role for pro-inflammatory T cells in mediating DIO-driven renal damage. We found that time restricted feeding (TRF; food available only in 12-hr dark phase) during the final 2 weeks of a 20-week DIO model (45% fat) significantly improves renal damage compared to DIO ad libitum fed male mice. DIO+TRF also significantly reduced total T cells in the kidney adjacent to vasa recta in the outer medulla compared to DIO ad libitum mice assessed at the start of the dark phase or Zeitgeber Time (ZT) 12. We hypothesized that DIO+TRF decreases renal and circulating pro-inflammatory CD4 + and CD8 + T cells compared to DIO ad libitum in a time-of-day dependent manner . Using flow cytometry, we found that DIO significantly increases total renal T cells compared to normal diet (ND; 10% fat) at ZT13-15 (dark phase) only. DIO+TRF significantly decreases total renal T cells compared to DIO in the dark phase (n=7-14; 2-way ANOVA, Interaction: p=0.016, Effect of Time of Day: p=0.010, Effect of Diet: p=0.001). Our data showed no significant changes in renal T cell numbers in the light phase. Compared to ND, DIO increased renal CD8 + T cells at ZT13-15 and DIO+TRF normalized renal CD8 + T cells (n=7-14; 2-way ANOVA, Interaction: p=0.007, Effect of Diet: p=0.004). DIO and DIO+TRF did not alter renal CD4 + T cells at either ZT1-3 or ZT13-15 compared to ND. We further examined the kidney CD8 + T cell phenotype with DIO and TRF. We also found no significant changes in CD8 + T cell activation markers, perforin and granzyme B, CD8 + T cell development marker EOMES, or CD8 + T cell pro-inflammatory cytokines, IFNγ and TNFα. We assessed circulating CD8 + and CD4 + T cells via flow cytometry to analyze systemic effects of TRF. DIO+TRF led to a diurnal rhythm of CD8 + T cells with increased circulating CD8 + T cells in the dark phase compared to the light phase that was not observed in the ND or DIO groups (n=7-17; 2-way ANOVA, Interaction: p=0.012). No significant changes were observed in circulating CD8 + T cell activation markers or cytokines. DIO and TRF had no significant effect on numbers of circulating CD4 + T cells. We conclude that DIO+TRF influences rhythms of circulating and renal CD8 + T cells, but not CD4 + T cells.

CAR-T cells targeting CD38 and LMP1 exhibit robust antitumour activity against NK/T cell lymphoma

BackgroundNatural killer/T cell lymphoma (NKTCL) is an aggressive lymphoma with a poor prognosis. Chimeric antigen receptor-transduced T (CAR-T) cell therapy has become a promising immunotherapeutic strategy against haematologic malignancies.MethodsIn this study, four CAR-T cell lines (CD38-CAR, LMP1-CAR, CD38-LMP1 tandem CAR 1 and CD38-LMP1 tandem CAR 2) were generated. The effect of CAR-T cells against NKTCL cells was evaluated both in vitro and in vivo. Expression of T cell activation markers and cytokines produced by CAR-T cells were detected by flow cytometry.ResultsThe four CAR-T cell lines could effectively eliminate malignant NKTCL cells. They could be activated and produce inflammatory cytokines in a target-dependent manner. In vivo tests showed that the CAR-T cells exhibited significant antitumour effects in a xenotransplanted NKTCL mouse model.ConclusionsIn summary, four CAR-T cell lines exhibited significant cytotoxicity against NKTCL cells both in vitro and in vivo. These results indicated the effective therapeutic promise of CD38 and LMP1 CAR-T cells in NKTCL.

LAMP1 as a novel molecular biomarker to predict the prognosis of the children with autism spectrum disorder using bioinformatics approaches

Autism spectrum disorder (ASD) is a neurodevelopmental disorder that usually manifests in childhood and is thought to be caused by a complex interaction of genetic, environmental, and immune factors. The majority of current ASD diagnostic methods rely on subjective behavioral observation and scale assessment, making early detection difficult. In this study, we confirmed that lysosomal-associated membrane protein 1 (LAMP1), a functional marker of immune cell activation and cytotoxic degranulation, was upregulated in ASD blood, brain cortex, and various genetic animal models or cells using bioinformatics approaches. The prognostic value of LAMP1 was investigated by correlating its expression with clinical ASD rating scales, and the receiver operating characteristic (ROC) curve analysis in ASD also revealed that it has a favorable diagnostic ability in distinguishing ASD from control cohort. According to gene set enrichment analysis (GSEA) results, LAMP1 correlated with genes that were enriched in natural kill and T cell immune function. Taking all of the evidence into account, we discovered that abnormal elevations of LAMP1 mRNA and protein in the blood of ASD children, may influence the development of ASD through its involvement in immune cell activity regulation. This report highlights a novel marker for ASD early detection as well as potential therapeutic targets.

Unraveling the immune and metabolic changes associated with metritis in dairy cows

The objective was to unravel the peripartum immune and metabolic changes associated with metritis in Holstein cows. Holstein cows (n = 128) had blood collected at -14, 0, 3, and 7 d relative to parturition (DRP). Flow cytometry was used to evaluate blood leukocyte counts, proportions, and activation. Total cells, live cells, single cells, monocytes (CD172α+/CD14+), polymorphonuclears (CD172α+/CD14-/SSChigh), B-cells (CD21+/MHCII+), CD4+ T-cells (CD4+), CD8+ T-cells (CD8+), and γδ T-cells (γδTCR+) were evaluated. Both CD62L and CD11b were used as markers of cell activation. Major histocompatibility complex class II was used as a marker of antigen presentation in monocytes. A Milliplex Bovine Cytokine/Chemokine 08-plex kit was used to evaluate plasma concentrations of IFN-γ, IL-1α, IL-1β, IL-4, IL-6, IL-8, IL-10, and tumor necrosis factor-α. The body weight (BW) change prepartum was calculated as the difference between calving BW and prepartum BW divided by the number of days between measurements. Plasma fatty acids (FA) were measured at -14 and 0 DRP using untargeted gas chromatography with time-of-flight mass spectrometry. Data were analyzed by ANOVA for repeated measures. Cows that developed metritis (n = 57) had greater prepartum BW, prepartum BW loss, and greater FA concentrations at calving. Plasma FA at calving was positively correlated with IL-1β. Cows that developed metritis had persistent systemic inflammation, which was demonstrated by greater B-cell activation, greater pro-inflammatory cytokine concentrations, and greater cell damage pre- and postpartum. Postpartum, we observed greater polymorphonuclear cell activation and extravasation but lesser monocytes and CD4+ T-cells activation and extravasation, which suggests postpartum immune tolerance. Greater prepartum adiposity in cows that developed metritis may lead to systemic inflammation pre- and postpartum and immune tolerance postpartum, which may lead to failure to prevent bacterial infection, and development of puerperal metritis.

Universal Near-Infrared Fluorescent Nanoprobes for Detection and Real-Time Imaging of ATP in Real Food Samples, Living Cells, and Bacteria.

Adenosine triphosphate (ATP), an essential metabolite for active microorganisms to maintain life activities, has been widely regarded as a marker of cell activity and an indicator of microbial contamination. Herein, we designed two near-infrared (NIR) fluorescent nanoprobes named CYA@ZIF-90 and CYQ@ZIF-90 by encapsulating the NIR dye CYA/CYQ in ZIF-90 for the rapid detection of ATP. Between them, nanoprobe CYA@ZIF-90 can achieve higher NIR emission (702 nm) and rapid detection (2 min). Based on the superior spatiotemporal resolution imaging of ATP fluctuations in living cells, the applicability of CYA@ZIF-90 for imaging and detection of ATP in living bacteria was explored for the first time. The nanoprobe indirectly realizes the quantitative detection of bacteria, and the detection limit can be as low as 74 CFU mL-1. Therefore, the prepared nanoprobe is expected to become a universal ATP sensing detection tool, which can be further applied to evaluate cell apoptosis, cell proliferation, and food-harmful microbial control.

Aberrant B-cell activation and B-cell subpopulations in rheumatoid arthritis: analysis by clinical activity, autoantibody seropositivity, and treatment.

Few studies analyze the role of B-cell subpopulations in rheumatoid arthritis (RA) pathophysiology. Therefore, this study aimed to analyze the differences in B-cell subpopulations and B-cell activation according to disease activity, RA subtype, and absence of disease-modifying antirheumatic drugs (DMARDs) therapy. These subgroups were compared with control subjects (CS). One hundred and thirty-nine subjects were included, of which 114 were RA patients, and 25 were controls. Patients were divided into 99 with seropositive RA, 6 with seronegative RA, and 9 without DMARDs. The patients with seropositive RA were subclassified based on the DAS28 index. A seven-color multicolor flow cytometry panel was used to identify B-cell immunophenotypes and cell activation markers. There were no changes in total B-cell frequencies between RA patients and controls. However, a lower frequency of memory B cells and pre-plasmablasts was observed in seropositive RA compared to controls (P < 0.0001; P = 0.0043, respectively). In contrast, a higher frequency of mature B cells was observed in RA than in controls (P = 0.0002). Among patients with RA, those with moderate activity had a higher percentage of B cells (P = 0.0021). The CD69+ marker was increased (P < 0.0001) in RA compared to controls, while the CD40+ frequency was decreased in patients (P < 0.0001). Transitional, naïve, and double-negative B-cell subpopulations were higher in seronegative RA than in seropositive (P < 0.01). In conclusion, in seropositive and seronegative RA patients, there are alterations in B-cell activation and B-cell subpopulations, independently of clinical activity and DMARDs therapy.

Co-expression of HLA-E and HLA-G on genetically modified porcine endothelial cells attenuates human NK cell-mediated degranulation.

Natural killer (NK) cells play an important role in immune rejection in solid organ transplantation. To mitigate human NK cell activation in xenotransplantation, introducing inhibitory ligands on xenografts via genetic engineering of pigs may protect the graft from human NK cell-mediated cytotoxicity and ultimately improve xenograft survival. In this study, non-classical HLA class I molecules HLA-E and HLA-G were introduced in an immortalized porcine liver endothelial cell line with disruption of five genes (GGTA1, CMAH, β4galNT2, SLA-I α chain, and β-2 microglobulin) encoding three major carbohydrate xenoantigens (αGal, Neu5Gc, and Sda) and swine leukocyte antigen class I (SLA-I) molecules. Expression of HLA-E and/or HLA-G on pig cells were confirmed by flow cytometry. Endogenous HLA-G molecules as well as exogenous HLA-G VL9 peptide could dramatically enhance HLA-E expression on transfected pig cells. We found that co-expression of HLA-E and HLA-G on porcine cells led to a significant reduction in human NK cell activation compared to the cells expressing HLA-E or HLA-G alone and the parental cell line. NK cell activation was assessed by analysis of CD107a expression in CD3-CD56+ population gated from human peripheral blood mononuclear cells. CD107a is a sensitive marker of NK cell activation and correlates with NK cell degranulation and cytotoxicity. HLA-E and/or HLA-G on pig cells did not show reactivity to human sera IgG and IgM antibodies. This in vitro study demonstrated that co-expression of HLA-E and HLA-G on genetically modified porcine endothelial cells provided a superior inhibition in human xenoreactive NK cells, which may guide further genetic engineering of pigs to prevent human NK cell mediated rejection.

Natriuretic Peptides and Cardiac Troponins: Markers of Disease Progression and Risk in Light Chain Cardiac Amyloidosis.

Light chain (AL) amyloidosis can cause an infiltrative cardiomyopathy that can result in symptomatic heart failure. The vague, nonspecific onset of signs and symptoms may lead to a delay in diagnosis and treatment leading to poor outcomes. Cardiac biomarkers, such as troponins and natriuretic peptides, play a pivotal role in diagnosis, determining prognosis, and assessing treatment response in patients with AL amyloidosis. Because of the evolving landscape for both diagnosis and treatment of AL cardiac amyloidosis, we review the critical role these and other biomarkers play in the clinical management of this disease. A number of conventional cardiac and noncardiac serum biomarkers are commonly used in AL cardiac amyloidosis and may be surrogates for cardiac involvement and inform prognosis. These include typical heart failure biomarkers such as levels of circulating natriuretic peptides as well as cardiac troponins. Other noncardiac biomarkers frequently measured in AL cardiac amyloidosis included difference between the involved and uninvolved free light chains (dFLC) and markers of endothelial cell activation and damage such as von Willebrand factor antigen and matrix metalloproteinases. AL amyloidosis can lead to cardiac involvement which has been associated with poor outcomes, especially if not identified and treated early. Natriuretic peptides and cardiac troponins are cornerstones for the diagnosis and management of AL cardiac amyloidosis. Their levels may represent cardiac stress, injury, and possibly degree of cardiac involvement, and they play a key role in AL amyloidosis disease staging.

Ginsenoside Rg1 improves anti-tumor efficacy of adoptive cell therapy by enhancing T cell effector functions.

Adoptive cell therapy (ACT) has emerged with remarkable efficacies for tumor immunotherapy. Chimeric antigen receptor (CAR) T cell therapy, as one of most promising ACTs, has achieved prominent effects in treating malignant hematological tumors. However, the insufficient killing activity and limited persistence of T cells in the immunosuppressive tumor microenvironment limit the further application of ACTs for cancer patients. Many studies have focused on improving cytotoxicity and persistence of T cells to achieve improved therapeutic effects. In this study, we explored the potential function in ACT of ginsenoside Rg1, the main pharmacologically active component of ginseng. We introduced Rg1 during the in vitro activation and expansion phase of T cells, and found that Rg1 treatment upregulated two T cell activation markers, CD69 and CD25, while promoting T cell differentiation towards a mature state. Transcriptome sequencing revealed that Rg1 influenced T cell metabolic reprogramming by strengthening mitochondrial biosynthesis. When co-cultured with tumor cells, Rg1-treated T cells showed stronger cytotoxicity than untreated cells. Moreover, adding Rg1 to the culture endowed CAR-T cells with enhanced anti-tumor efficacy. This study suggests that ginsenoside Rg1 provides a potential approach for improving the anti-tumor efficacy of ACT by enhancing T cell effector functions.

Chimeric HLA antibody receptor T cells to target HLA-specific B cells in solid organ transplantation.

HLA-sensitized patients on the transplant waiting list harbor antibodies and memory B cells directed against allogeneic HLA molecules, which decreases the chance to receive a compatible donor organ. Current desensitization strategies non-specifically target circulating antibodies and B cells, warranting the development of therapies that specifically affect HLA-directed humoral immune responses. We developed Chimeric HLA Antibody Receptor (CHAR) constructs comprising the extracellular part of HLA-A2 or HLA-A3 coupled to CD28-CD3ζ domains. CHAR-transduced cells expressing reporter constructs encoding T-cell activation markers, and CHAR-transduced CD8+ T cells from healthy donors were stimulated with HLA-specific monoclonal antibody-coated microbeads, and HLA-specific B cell hybridomas. CHAR T cell activation was measured by upregulation of T cell activation markers and IFNγ secretion, whereas CHAR T cell killing of B cell hybridomas was assessed in chromium release assays and by IgG ELISpot. HLA-A2- and HLA-A3-CHAR expressing cells were specifically activated by HLA-A2- and HLA-A3-specific monoclonal antibodies, either soluble or coated on microbeads, as shown by CHAR-induced transcription factors. HLA-A2 and HLA-A3 CHAR T cells efficiently produced IFNγ with exquisite specificity and were capable of specifically lysing hybridoma cells expressing HLA-A2- or HLA-A3-specific B-cell receptors, respectively. Finally, we mutated the α3 domain of the CHAR molecules to minimize any alloreactive T-cell reactivity against CHAR T cells, while retaining CHAR activity. These data show proof of principle for CHAR T cells to serve as precision immunotherapy to specifically desensitize (highly) sensitized solid organ transplant candidates and to treat antibody-mediated rejection after solid organ transplantation.

Adherence and activation of human mesenchymal stromal cells on brushite coated porous titanium

Tri-calcium-phosphate Ca3(PO4)2 is considered a promising material to improve implant and bone tissue linking. Titanium (Ti), a highly porous material, was coated with crystalline brushite using an electrochemical process so that it could be used as an implant material. The aim of this research is to get a faster ingrowth of bone-tissue inside the porous structure. The crystalline coating was characterized by the standard XRD and SEM techniques. Additionally, the phase evolution of Ca3(PO4)2-crystals with different kinds of sterilization techniques was observed. In-vitro tests on the biocompatibility of the coating were done with human mesenchymal stem cells (HMSCs). The HMSCs are the most promising cell type for regenerative medicine and tissue engineering owing to their ability to differentiate into several tissues, such as, bone, cartilage, tendon, and muscle. For the treatment of local bone defects expanded HMSCs could be loaded on Ca3(PO4)2 coated metallic matrices and delivered to the target site. A possible application is brushite-coated porous titanium as a carrier matrix. To determine whether cell functions of HMSCs are influenced by the brushite surface, HMSCs were cultured on brushite-coated and uncoated porous titanium specimens. The viability and proliferation of HMSCs were successfully analyzed. Additionally, the release of cytokines (IL-6, IL-8, IL-11) were quantified as a marker of cell activation. The viable cells could be detected on specimens after 48 h initial seeding. After 8 days cell number on both specimens increased, although the proliferation on brushite-coated titanium was decreased compared to the uncoated surfaces. The release of both, IL-8 and IL-11 increased significantly due to the cultivation of HMSCs on the coated specimens for 8 days.

An In Vitro Alveolar Model Allows for the Rapid Assessment of Particles for Respiratory Sensitization Potential.

Dust, both industrial and household, contains particulates that can reach the most distal aspects of the lung. Silica and nickel compounds are two such particulates and have known profiles of poor health outcomes. While silica is well-characterized, nickel compounds still need to be fully understood for their potential to cause long-term immune responses in the lungs. To assess these hazards and decrease animal numbers used in testing, investigations that lead to verifiable in vitro methods are needed. To understand the implications of these two compounds reaching the distal aspect of the lungs, the alveoli, an architecturally relevant alveolar model consisting of epithelial cells, macrophages, and dendritic cells in a maintained submerged system, was utilized for high throughput testing. Exposures include crystalline silica (SiO2) and nickel oxide (NiO). The endpoints measured included mitochondrial reactive oxygen species and cytostructural changes assessed via confocal laser scanning microscopy; cell morphology evaluated via scanning electron microscopy; biochemical reactions assessed via protein arrays; transcriptome assessed via gene arrays, and cell surface activation markers evaluated via flow cytometry. The results showed that, compared to untreated cultures, NiO increased markers for dendritic cell activation, trafficking, and antigen presentation; oxidative stress and cytoskeletal changes, and gene and cytokine expression of neutrophil and other leukocyte chemoattractants. The chemokines and cytokines CCL3, CCL7, CXCL5, IL-6, and IL-8 were identified as potential biomarkers of respiratory sensitization.

Aberrant expression of T cell activation markers and upregulation of Tregs in bone marrow and peripheral blood in acute myeloid leukemia patients

ABSTRACTBackground:T cells’ function and activation and the immunosuppressive effect of regulatory T cells (Tregs) play a pivotal role in the occurrence and progression of acute myeloid leukemia (AML). In this study, we investigate the expression of T cell activation markers and quantity of Tregs in bone marrow (BM) and peripheral blood (PB) from AML patients and further characterized their correlation with BM leukemic blasts.Methods:Expression of CD25, CD38, CD69, and HLA-DR on the surfaces of CD4+ and CD8+ T cells and the quantity of Tregs in BM and PB from new diagnosed (ND), relapsed-refractory (RR), complete remission (CR) AML patients were measured via flow cytometry.Results:Compared to normal controls (NC), we found higher proportion of CD4+ CD69+ T cells, CD8+ CD69+ T cells and Tregs in PB. CD8+ CD38+ T cells and CD8+ HLA-DR+ T cells in RR were significantly higher than ND, CR and NC). Tregs were normalized when AML patients achieved CR. Moreover, there was a minor positive correlation between AML blasts and CD8+ CD25+ T cells or Tregs, while AML blasts had a minor negative correlation with CD4+ CD69+ T cells.Conclusion:Abnormal activation markers of T cells and Tregs may be involved in the pathological mechanism of ND and RR AML. Our results indicated that CD8+ CD38+ T cells and CD8+ HLA-DR+ T cells might be RR markers of AML patients. Furthermore, Tregs could be used as clinical indicators to evaluate prognosis for AML patients.

Pharmacogenomic Analyses Implicate B Cell Developmental Status and MKL1 as Determinants of Sensitivity toward Anti-CD20 Monoclonal Antibody Therapy.

Monoclonal antibody (mAb) therapy directed against CD20 is an important tool in the treatment of B cell disorders. However, variable patient response and acquired resistance remain important clinical challenges. To identify genetic factors that may influence sensitivity to treatment, the cytotoxic activity of three CD20 mAbs: rituximab; ofatumumab; and obinutuzumab, were screened in high-throughput assays using 680 ethnically diverse lymphoblastoid cell lines (LCLs) followed by a pharmacogenomic assessment. GWAS analysis identified several novel gene candidates. The most significant SNP, rs58600101, in the gene MKL1 displayed ethnic stratification, with the variant being significantly more prevalent in the African cohort and resulting in reduced transcript levels as measured by qPCR. Functional validation of MKL1 by shRNA-mediated knockdown of MKL1 resulted in a more resistant phenotype. Gene expression analysis identified the developmentally associated TGFB1I1 as the most significant gene associated with sensitivity. qPCR among a panel of sensitive and resistant LCLs revealed immunoglobulin class-switching as well as differences in the expression of B cell activation markers. Flow cytometry showed heterogeneity within some cell lines relative to surface Ig isotype with a shift to more IgG+ cells among the resistant lines. Pretreatment with prednisolone could partly reverse the resistant phenotype. Results suggest that the efficacy of anti-CD20 mAb therapy may be influenced by B cell developmental status as well as polymorphism in the MKL1 gene. A clinical benefit may be achieved by pretreatment with corticosteroids such as prednisolone followed by mAb therapy.

Procoagulant effect of extracellular vesicles in patients after transcatheter aortic valve replacement or transcatheter aortic valve replacement with percutaneous coronary intervention.

Patients with severe aortic stenosis (AS) after replacement of the transcatheter aortic valve (TAVR) are more likely to develop thrombotic complications such as cerebral embolism and artificial valve thrombosis. However, the mechanism is not yet well defined. We aimed to explore the plasma extracellular vesicles (EVs) levels and their role in the induction of procoagulant activity (PCA) in patients receiving TAVR alone or TAVR with percutaneous coronary intervention (PCI). EVs were analyzed with flow cytometer. Markers of platelet and endothelial cell activation were quantified using selective enzyme-linked immunosorbent assay (ELISA) kits. Procoagulant activity (PCA) was assessed by clotting time, purified clotting complex assays, and fibrin production assays. Our results confirmed that EVs with positive phosphatedylserin (PS+EV), platelet EVs (PEVs) and positive tissue factor EVs (TF+EVs) were higher in patients following TAVR than before TAVR, particularly in TAVR with PCI. Furthermore, endothelial-derived EVs (EEVs) were also higher in patients after TAVR with PCI than pre-TAVR, however, the EEVs levels in TAVR alone patients were gradually reduce than pre-TAVR. In addition, we further proved that total EVs contributed to dramatically shortened coagulation time, increased intrinsic/extrinsic factor Xa and thrombin generation in patients after TAVR, especially in TAVR with PCI. The PCA was markedly attenuated by approximately 80% with lactucin. Our study reveals a previously unrecognized link between plasma EV levels and hypercoagulability in patients after TAVR, especially TAVR with PCI. Blockade of PS+EVs may improve the hypercoagulable state and prognosis of patients.