Unstimulated Conditions Research Articles

IRAG2 Interacts with IP3-Receptor Types 1, 2, and 3 and Regulates Intracellular Ca2+ in Murine Pancreatic Acinar Cells.

The inositol 1,4,5-triphosphate receptor-associated 2 (IRAG2) is also known as Jaw1 or lymphoid-restricted membrane protein (LRMP) and shares homology with the inositol 1,4,5-triphosphate receptor-associated cGMP kinase substrate 1 (IRAG1). IRAG1 interacts with inositol trisphosphate receptors (IP3 receptors /IP3R) via its coiled-coil domain and modulates Ca2+ release from intracellular stores. Due to the homology of IRAG1 and IRAG2, especially in its coiled-coil domain, it is possible that IRAG2 has similar interaction partners like IRAG1 and that IRAG2 also modulates intracellular Ca2+ signaling. In our study, we localized IRAG2 in pancreatic acinar cells of the exocrine pancreas, and we investigated the interaction of IRAG2 with IP3 receptors and its impact on intracellular Ca2+ signaling and exocrine pancreatic function, like amylase secretion. We detected the interaction of IRAG2 with different subtypes of IP3R and altered Ca2+ release in pancreatic acinar cells from mice lacking IRAG2. IRAG2 deficiency decreased basal levels of intracellular Ca2+, suggesting that IRAG2 leads to activation of IP3R under unstimulated basal conditions. Moreover, we observed that loss of IRAG2 impacts the secretion of amylase. Our data, therefore, suggest that IRAG2 modulates intracellular Ca2+ signaling, which regulates exocrine pancreatic function.

Metabolic adaptation to the chronic loss of Ca2+ signaling induced by KO of IP3 receptors or the mitochondrial Ca2+ uniporter

Calcium signaling is essential for regulating many biological processes. Endoplasmic reticulum inositol trisphosphate receptors (IP3Rs) and the mitochondrial Ca2+ uniporter (MCU) are key proteins that regulate intracellular Ca2+ concentration. Mitochondrial Ca2+ accumulation activates Ca2+-sensitive dehydrogenases of the tricarboxylic acid (TCA) cycle that maintain the biosynthetic and bioenergetic needs of both normal and cancer cells. However, the interplay between calcium signaling and metabolism is not well understood. In this study, we used human cancer cell lines (HEK293 and HeLa) with stable KOs of all three IP3R isoforms (triple KO [TKO]) or MCU to examine metabolic and bioenergetic responses to the chronic loss of cytosolic and/or mitochondrial Ca2+ signaling. Our results show that TKO cells (exhibiting total loss of Ca2+ signaling) are viable, displaying a lower proliferation and oxygen consumption rate, with no significant changes in ATP levels, even when made to rely solely on the TCA cycle for energy production. MCU KO cells also maintained normal ATP levels but showed increased proliferation, oxygen consumption, and metabolism of both glucose and glutamine. However, MCU KO cells were unable to maintain ATP levels and died when relying solely on the TCA cycle for energy. We conclude that constitutive Ca2+ signaling is dispensable for the bioenergetic needs of both IP3R TKO and MCU KO human cancer cells, likely because of adequate basal glycolytic and TCA cycle flux. However, in MCU KO cells, the higher energy expenditure associated with increased proliferation and oxygen consumption makes these cells more prone to bioenergetic failure under conditions of metabolic stress.

Nutrigenomic Effect of Hydroxytyrosol in Vascular Endothelial Cells: A Transcriptomic Profile Analysis

Hydroxytyrosol (HT), a peculiar olive and olive oil phenolic antioxidant, plays a significant role in the endothelial and cardiovascular protection associated with olive oil consumption. However, studies examining the effects of HT on the whole-genome expression of endothelial cells, which are prominent targets for vasculo-protective effects of olive oil polyphenols, have been lacking. This study aims to comprehensively evaluate the genomic effects exerted by HT, at the transcriptional level, in endothelial cells under resting or proinflammatory conditions. Human umbilical vein endothelial cells (HUVECs) were treated with 10 µmol/L HT for 1 h and then stimulated with 5 ng/mL interleukin (IL)-1β for 3 h. Total RNA was extracted, and gene expression profile assessed with microarray analysis. Functional enrichment analysis and pathway analysis were performed by Ingenuity Pathways Analysis. Microarray data were validated by qRT-PCR. Fixing a significance threshold at 1.5-fold change, HT affected the expression of 708 and 599 genes, respectively, in HUVECs under resting and IL-1β-stimulated conditions; among these, 190 were common to both conditions. Unfolded protein response (UPR) and endoplasmic reticulum stress resulted from the two top canonical pathways common between HT and HT-IL-1β affected genes. IL-17F/A signaling was found in the top canonical pathways of HT modified genes under resting unstimulated conditions, whereas cardiac hypertrophy signaling was identified among the pathways affected by HT-IL-1β. The transcriptomic analysis allowed pinpointing immunological, inflammatory, proliferative, and metabolic-related pathways as the most affected by HT in endothelial cells. It also revealed previously unsuspected genes and related gene pathways affected by HT, thus broadening our knowledge of its biological properties. The unbiased identification of novel genes regulated by HT improves our understanding of mechanisms by which olive oil prevents or attenuates inflammatory diseases and identifies new genes to be enquired as potential contributors to the inter-individual variation in response to functional food consumption.

Decreased Frequencies of Gamma/Delta T Cells Expressing Th1/Th17 Cytokine, Cytotoxic, and Immune Markers in Latent Tuberculosis-Diabetes/Pre-Diabetes Comorbidity.

Antigen-specific gamma-delta (γδ) T cells are important in exhibiting anti-mycobacterial immunity, but their role in latent tuberculosis (LTB) with diabetes mellitus (DM) or pre-DM (PDM) and non-DM comorbidities have not been studied. Thus, we have studied the baseline, mycobacterial (PPD, WCL), and positive control antigen-stimulated γδ T cells expressing Th1 (IFNγ, TNFα, IL-2) and Th17 (IL-17A, IL-17F, IL-22) cytokine as well as cytotoxic (perforin [PFN], granzyme [GZE B], granulysin [GNLSN]) and immune (GMCSF, PD-1, CD69) markers in LTB (DM, PDM, NDM) comorbidities by flow cytometry. In the unstimulated (UNS) condition, we did not observe any significant difference in the frequencies of γδ T cells expressing Th1 and Th17 cytokine, cytotoxic, and immune markers. In contrast, upon PPD antigen stimulation, the frequencies of γδ T cells expressing Th1 (IFNγ, TNFα) and Th17 (IL-17F, IL-22) cytokine, cytotoxic (PFN, GZE B, GNLSN), and immune (CD69) markers were significantly diminished in LTB DM and/or PDM individuals compared to LTB NDM individuals. Similarly, upon WCL antigen stimulation, the frequencies of γδ T cells expressing Th1 (TNFα) and Th17 (IL-17A, IL-22) cytokine, cytotoxic (PFN), and immune (PD-1, CD69) markers were significantly diminished in LTB DM and/or PDM individuals compared to LTB NDM individuals. Finally, upon P/I stimulation we did not observe any significant difference in the γδ T cell frequencies expressing cytokine, cytotoxic, and immune markers between the study populations. The culture supernatant levels of IFNγ, TNFα, and IL-17A cytokines were significantly increased in LTB DM and PDM after stimulation with Mtb antigens compared to LTB NDM individuals. Therefore, diminished γδ T cells expressing cytokine, cytotoxic, and other immune markers and elevated levels of cytokines in the supernatants is a characteristic feature of LTB PDM/DM co-morbidities.

Vibration-induced nystagmus in patients with chronic unilateral Meniere's disease

Background and Aim: Meniere's disease (MD) is one of the inner ear disorders associated with fluctuating hearing loss, vertigo, ear fullness, and tinnitus. Vestibular stimulation delas with the int­egrity of the peripheral vestibular system and may cause nystagmus due to the functional asy­mmetry between right and lef peripheral vesti­bular system. This study aimed to assess the vibration-induced nystagmus (VIN) in patients with chronic unilateral MD and investigate the effectiveness of this test in detecting the affected ear in these patients. Methods: This study was conducted on 29 pati­ents with chronic unilateral MD. For this pur­pose, spontaneous nystagmus (SN) and VIN at frequencies of 30 Hz and 100 Hz were recorded by videonystagmography test under five recor­ding conditions. The vibratory stimulation was presented to both healthy and affected ears. Coll­ected were analyzed in SPSS v.22 software. Results: Vibratory stimulation compared to the unstimulated condition, revealed a significant difference in eye movements for both healthy and affected ears. Moreover, the difference between VIN and SN in the affected ear was much greater than in the healthy ear. Conclusion: In patients with chronic unilateral MD, 100 Hz vibratory stimulation of the affected ear induces more reliable nystagmus than 30 Hz stimulation and unstimulated condition. The VIN test can be used for the evaluation of the vesti­bular system function and is a promising techni­que to detect the MD ear. Keywords: Meniere's disease; spontaneous nystagmus; vibration-induced nystagmus; chronic; definite; vestibular vibrator

CAMP triggers Na+ absorption by distal airway surface epithelium in cystic fibrosis swine.

A controversial hypothesis pertaining to cystic fibrosis (CF) lung disease is that the CF transmembrane conductance regulator (CFTR) channel fails to inhibit the epithelial Na+ channel (ENaC), yielding increased Na+ reabsorption and airway dehydration. We use a non-invasive self-referencing Na+-selective microelectrode technique to measure Na+ transport across individual folds of distal airway surface epithelium preparations from CFTR-/- (CF) and wild-type (WT) swine. We show that, under unstimulated control conditions, WT and CF epithelia exhibit similar, low rates of Na+ transport that are unaffected by the ENaC blocker amiloride. However, in the presence of the cyclic AMP (cAMP)-elevating agents forskolin+IBMX (isobutylmethylxanthine), folds of WT tissues secrete large amounts of Na+, while CFTR-/- tissues absorb small, but potentially important, amounts of Na+. In cAMP-stimulated conditions, amiloride inhibits Na+ absorption in CFTR-/- tissues but does not affect secretion in WT tissues. Our results are consistent with the hypothesis that ENaC-mediated Na+ absorption may contribute to dehydration of CF distal airways.

Rapid Elemental Extraction from Ordered and Disordered Solutes by Acoustically-Stimulated Dissolution

Alkaline industrial wastes (e.g., slags: ordered crystalline solids, and fly ashes: disordered solids) represent abundant reservoirs of elements such as silicon and calcium. Rapid elemental extractions from these wastes, however, have often relied on the use of “stoichiometric additives” (i.e., acids or bases). Herein, we demonstrate that acoustic stimulation enhances the release of network-forming Si species from crystalline blast furnace slags and amorphous fly ashes at reaction temperatures less than 65 °C. These additive-free enhancements are induced by cavitation processes which reduce the apparent activation energy of solute dissolution (Ea, kJ/mol) by up to 40% as compared to unstimulated conditions. Because of the reduction in the apparent activation energy, acoustic stimulation features an energy intensity that is up to 80% lower in promoting dissolution, as compared to other additive-free methods such as enhancing the solute’s surface area, introducing heat, or convectively mixing the solvent. Based on atomic topology analysis, we show that the reduction in apparent dissolution activation energy upon acoustic stimulation scales with the number of weak topological constraints per atom in the atomic network of the dissolving solute, independent of their ordered or disordered nature. This suggests that sonication breaks the weakest constraints in the solute’s atomic network, which, in turn, facilitates dissolution. The results suggest the ability of acoustic stimulation to enhance waste utilization and circularity, by enabling efficient resource extraction from industrial wastes.

Selective induction of rapid cytotoxic effect in glioblastoma cells by oscillating magnetic fields.

The mechanisms underlying anticancer effects of electromagnetic fields are poorly understood. An alternating electric field-generating therapeutic device called Optune™ device has been approved for the treatment of glioblastoma (GBM). We have developed a new device that generates oscillating magnetic fields (OMF) by rapid rotation of strong permanent magnets in specially designed patterns of frequency and timing and have used it to treat an end-stage recurrent GBM patient under an expanded access/compassionate use treatment protocol. Here, we ask whether OMF causes selective cytotoxic effects in GBM and whether it is through generation of reactive oxygen species (ROS). We stimulated patient derived GBM cells, lung cancer cells, normal human cortical neurons, astrocytes, and bronchial epithelial cells using OMF generators (oncoscillators) of our Oncomagnetic Device and compared the results to those obtained under unstimulated or sham-stimulated control conditions. Quantitative fluorescence microscopy was used to assess cell morphology, viability, and ROS production mechanisms. We find that OMF induces highly selective cell death of patient derived GBM cells associated with activation of caspase 3, while leaving normal tissue cells undamaged. The cytotoxic effect of OMF is also seen in pulmonary cancer cells. The underlying mechanism is a marked increase in ROS in the mitochondria, possibly in part through perturbation of the electron flow in the respiratory chain. Rotating magnetic fields produced by a new noninvasive device selectively kill cultured human glioblastoma and non-small cell lung cancer cells by raising intracellular reactive oxygen species, but not normal human tissue cells.

Differential cytokine and chemokine expression during rejection and infection following intestinal transplantation

Background/objectivesRejection and infectious enteritis in intestinal transplant (ITx) patients present with virtually identical symptoms. Currently, the gold standard for differentiating between these two conditions is endoscopy, which is invasive and costly. Our primary aim was to identify differences in peripheral blood cytokines during episodes of acute cellular rejection (ACR) and infectious enteritis in patients with intestinal transplants. MethodsThis was a prospective, cross-sectional study involving ITx patients transplanted between 2000 and 2016. We studied 63 blood samples collected from 29 ITx patients during periods of normal (n = 24) and abnormal (n = 17) allograft function. PBMCs from whole blood samples were cultured under unstimulated or stimulated conditions with phytohemagglutinin (PHA). The supernatant from these cultures were collected to measure cytokine and chemokine levels using a 38-plex luminex panel. ResultsOur study found that cytokines and chemokines are differentially expressed in normal, ACR, and infectious enteritis samples under unstimulated conditions based on heatmap analysis. Although each cohort displayed distinctive signatures, only MDC (p = 0.037) was found to be significantly different between ACR and infectious enteritis. Upon stimulation of PBMCs, patients with ACR demonstrated increased immune reactivity compared to infectious enteritis; though this did not reach statistical significance. ConclusionsTo our knowledge, this is the first comprehensive study comparing cytokine expression during acute rejection and infectious enteritis in intestinal transplant recipients. Our results suggest that cytokines have the potential to be used as clinical markers for risk stratification and/or diagnosis of ACR and infectious enteritis.

NGPF2 triggers synaptic scaling up through ALK-LIMK-cofilin-mediated mechanisms.

Synaptic scaling is an extensively studied form of homeostatic plasticity critically involved in various brain functions. Although it is accepted that synaptic scaling is expressed through the postsynaptic accumulation of AMPA receptors (AMPARs), the induction mechanism remains elusive. In this study, we show that TTX treatment induces rapid but transient release of the neurite growth-promoting factor 2 (NGPF2), and this release is necessary and sufficient for TTX-induced scaling up. In addition, we show that inhibition of the anaplastic lymphoma kinase (ALK)-LIMK-cofilin signaling pathway blocks TTX- and NGPF2-induced synaptic scaling up. Furthermore, we show that TTX-induced release of NGPF2 is protein synthesis dependent and requires fragile X mental retardation protein 1 (FMRP1). These results indicate that activity blockade induces NGPF2 synthesis and release to trigger synaptic scaling up through LIMK-cofilin-dependent actin reorganization, spine enlargement, and stabilization of AMPARs at the synapse.

Daily apple consumption reduces plasma and peripheral blood mononuclear cell–secreted inflammatory biomarkers in adults with overweight and obesity: a 6-week randomized, controlled, parallel-arm trial

Obesity-associated low-grade inflammation contributes to the development of cardiovascular disease (CVD). Apples are rich in anti-inflammatory bioactives including polyphenols and fiber. We aimed to determine the effects of regular apple consumption on fasting plasma biomarkers of inflammation (primary outcome), endotoxemia, carbohydrate and lipid metabolism (glucose, insulin, triacylglycerol; secondary outcomes), and peripheral blood mononuclear cell (PBMC)-secreted cytokines (secondary outcome) in individuals with overweight and obesity. A randomized, controlled, parallel-arm trial was conducted with n=46 participants. After avoiding foods and beverages rich in polyphenols and fiber for 2 wk, participants consumed 3 whole Gala apples (∼200 g edible parts)/d as part of their habitual diet (n=23) or avoided apples (control, n=23) for 6 wk. All participants limited consumption of polyphenols and fiber during the 6-wk trial. Fasting blood samples were collected before and after 6 wk for analysis of plasma biomarkers and isolation of PBMCs, which were cultured for 24 h unstimulated or stimulated with LPS (10 ng/mL). Forty-four participants completed the trial (30 female, 14 male; mean±SEM age: 45.4±2.2 y; BMI: 33.4±0.9 kg/m2). After ANCOVA and correcting for multiple comparisons, apples decreased fasting plasma C-reactive protein by 17.0% (range: 14.3%-19.6%, P=0.005), IL-6 by 12.4% (range: 6.7%-17.5%, P<0.001), and LPS-binding protein by 20.7% (range: 14.1%-26.4%, P<0.001) compared with control. Apples also decreased PBMC-secreted IL-6 by 28.3% (range: 22.4%-33.5%, P<0.001) and IL-17 by 11.0% (range 5.8-15.6%, P=0.003) in the unstimulated condition compared with control. Exploratory analysis showed apples also increased plasma total antioxidant capacity by 9.6% (range: 1.7-18.9%, P=0.002) compared with control. However, apples had no effect on anthropometric or other CVD risk markers. Six-week daily whole Gala apple consumption may be an effective dietary strategy to mitigate the obesity-associated inflammation that exacerbates CVD risk, without weight loss. This trial was registered at clinicaltrials.gov as NCT03523403.

MiR- 16 Regulates Crosstalk in NF-κB Inflammatory Signaling between Myeloma Cells and Bone Marrow Macrophages

Introduction: Multiple myeloma plasma cells (MM-PCs) home to and dynamically interact with the bone marrow (BM) microenvironment (BM-ME). Of the different factors secreted by the BM-ME, NF-κB-induced cytokines including IL-6 play a central role in MM-PC survival and resistance to treatments. Previously published data have shown that in the BM of MM patients the presence of CD163+ tumor associated macrophages (TAM) correlates with IL-6 production. The canonical NF-κB pathway is induced by most physiological stimuli to produce low levels of inflammatory cytokines, through the activation of the IκB kinase complex (IKKβ, IKKα, and IKKγ). This pathway seems constitutively activated in TAM, where instead the activation of the non-canonical pathway seems to play a critical role in the anti-cancer phenotype of BM macrophages (BM-Mϕ).Extracellular vesicles (EV) are membrane-covered cell fragments released by all cell types, and recently they have been shown to play a pivotal role in creating a tumor supportive BM-ME. Our group was the first to show that MM cells produce EV (Harshman et.al. 2013). Previous studies have shown that miR- 16 encapsulated inEV(EV-miR- 16) down-modulates IL-6 production in the BM-ME, and we showed that higher levels of circulating miR- 16 is a prognostic factor that correlates with better survival in newly diagnosed MM patients treated with proteasome inhibitor (PI) based therapy (Rocci et al. 2013). Here, we investigated whether MM derived EV can play a pivotal role in supporting monocyte to TAM differentiation and defined the molecular mechanisms of IL-6 modulation by miR- 16 .Methods: Monocyte to TAM differentiation was evaluated by flow cytometry analysis, light microscopy, q-RT-PCR and phagocytosis assays. EV were isolated via a serial centrifugation method. EV RNA content was analyzed by NanoString. EV isolated from producing cells (0.5x106cells/ml) were used to differentiate 1 monocyte (4:1 effector:target ratio). Cytokine arrays in the supernatant of BM-Mϕ and in the BM-Mϕ depleted fractions were performed. Cloning, Western blot and RT-PCR were used to confirm IKK complex targeting by miR- 16 . Single cell flow and NF-kB activation assays were conducted.Results: Our in vitro data show that EV isolated from the supernatant of MM cell lines (MM-EV) can promotemonocyte differentiation to CD163+ TAM. Mϕ differentiation was also observed when peripheral blood monocytes (PB-M) obtained from both MM and healthy donors were treated with EV isolated from the BM acellular fraction of MM patients (n=4). Limited differentiation was instead observed when the acellular fraction was EV-depleted (Fig. 1). Our data show that PB-M differentiation was partially reversed by the addition of an EV-miR- 16 mimic. We found that miR- 16 is dramatically down-regulated when PB-M is induced by macrophage colony stimulating factor to differentiate to TAM, and it is also down-regulated in primary BM-Mϕ as compared to PB-M of the same patients and to the BM-Mϕ of 3 age matched healthy donors. We learned that BM-Mϕ isolated from MM patients are the main source of NF - κB-induced cytokines including IL-6 and IL-10 under unstimulated conditions as well as IL-6, IL-8, IL-10, TNF-α and VEGF under Toll-like receptor stimulated conditions, as the Mϕ-depleted BM cellular fraction had significantly low levels. We also found that miR -16 ectopic expression in MM cells and in a human monocytic cell line downregulates both IKKβ/IKKα protein and mRNA levels, with subsequent downregulation of NF-κB activation signaling and decreased expression of inflammatory cytokines. Further, cell growth and apoptotic assays show that the attenuation of PI-based anti-MM activity by TAM is partially reversed by ectopic miR- 16 in both TAM and MM-PCs.Conclusions: MM-EV support monocyte differentiation to TAM, causing miR- 16 downregulation and an increase in NF-κB inflammatory signaling. We also show that this effect can be impaired by an ectopic miR- 16 mimic. Since constitutive activation of NF-κB decreases MM cells dependency on extrinsic growth factors, at the onset of the disease miR- 16 downregulation in MM-PCs can support NF-kB intrinsic activation and increased fitness of MM cells. Because NF-kB activation in MM-PCs counterbalances the anti-MM activity of PI, it is not surprising that miR- 16 expression can potentiate the killing effect of PI. We are exploring these hypotheses through use of miR -16 knock out mouse models. [Display omitted] DisclosuresHofmeister:Adaptive Biotechnologies: Honoraria, Membership on an entity's Board of Directors or advisory committees; Janssen: Research Funding; Karyopharm: Research Funding; Bristol-Myers Squibb: Research Funding; Celgene: Research Funding; Roche: Research Funding; Thrassos: Honoraria, Membership on an entity's Board of Directors or advisory committees; Takeda: Research Funding. Krishnan:Takeda: Speakers Bureau; Onyx: Speakers Bureau; Sutro: Consultancy; Janssen: Consultancy, Speakers Bureau; Celgene: Consultancy, Equity Ownership, Speakers Bureau.

P059 GB004 drives protective effects on immune cells and epithelial cells using human ex vivo monolayer and co-culture systems

Abstract Background GB004 is a small molecule that stabilizes hypoxia inducible factor (HIF-1α), a key transcription factor involved in the cellular responses at the intersection of hypoxia and inflammation. GB004 induction of HIF-1a target genes improves barrier integrity and reduces barrier permeability in rodent models of colitis. Here we investigated gene expression and secretion of soluble factors elicited by GB004 treatment on immune cells and epithelial cells, human monolayer assays and immune co-culture studies to further explore its protective mechanism. Methods In the BioMAP human co-culture assay, cells were treated with GB004 one hour prior to stimulation and incubated for 24–168 hours. Biomarkers, cell viability and proliferation were assessed. Repligut human stem-cell-derived monolayer platforms were used to assess GB004 effects under unstimulated or TNFa stimulation conditions. Barrier integrity was assessed by Transepithelial Endothelial Electric Resistance (TEER), HIF-1a target genes were assessed in cell lysates, and tight junction formation and epithelial monolayer viability were investigated by immunofluorescence staining. Results GB004 demonstrated activity in 11 of 12 systems in the BioMAP human co-culture assay, which covers a range of disease-relevant immune and non-immune mechanisms under stimulation conditions. Collectively, GB004 treatment led to changes in biomarkers associated with inflammation (sTNFa, IL-8, MCP-1, CXCL11), immunomodulation (sIL-17F, sIL-17A), and tissue remodeling (collagen I, collagen IV). GB004 treatment also reduced the proliferative activity in the in vitro lymphocyte assay systems. In the human Repligut intestinal epithelium assay, GB004 significantly reduced cell death and improved barrier integrity in response to pro-inflammatory cytokines. The protective role of GB004 on barrier integrity was further supported by immunostaining experiments demonstrating that GB004 treatment protected epithelial cells from TNFa-induced cell apoptosis, improving both epithelial cell number and tight junction protein ZO-1 staining. Conclusion GB004 modulates key anti-inflammatory and immunomodulatory activities in human co-culture systems. Furthermore, GB004 demonstrates protective effects on human derived monolayer cultures by reducing cell death, promoting tight junction formation, and improving barrier integrity. Targeting both barrier function and local colonic inflammation represents a multi-faceted approach to treatment of inflammatory bowel disease. A phase 2 clinical study of GB004 is ongoing in patients with ulcerative colitis (NCT04556383). Sponsored by GB004, Inc., a wholly owned subsidiary of Gossamer Bio, Inc.

Distinct phenotypic expression levels of macrophages in neonatal lungs.

Alveolar macrophages are the front-line defense against environmental pathogens. However, to the best of our knowledge, differences in function and phenotypic expression levels of macrophages between neonatal and adult lungs have not previously been determined. The present study investigated lung tissues and analyzed blood samples to find cell markers of M1 and M2 macrophages in neonatal and adult rats. Pulmonary sepsis was induced by intrapleural instillation of lipopolysaccharide (LPS; 20 mg/kg) and survival time after administration of LPS was measured. In certain neonates, a selective inducible nitric oxide synthase (iNOS) inhibitor, 1400w, was administered prior to induction of pulmonary sepsis. Compared with adults, fetal and neonatal lung tissues had significantly higher levels of iNOS and CD86 (M1 markers), whereas the expression levels of CD206 and arginase-1 (M2 markers) were lower in the neonatal lung. The circulating cells that co-expressed CD68 (monocytes and macrophages) and CD86 in the blood were also significantly higher in neonates than in adults (25.9±6.6 vs. 11.6±2.2%; P=0.007. At basal unstimulated conditions, lung tissue concentrations of nitrite and nitrate (NOx) were significantly lower in the neonates than in adults (112.1±55.9 vs. 340.9±124.9 µM/g; P<0.001). However, NOx was increased following administration of LPS. Administration of 1400w suppressed lung tissue levels of NOx and improved the survival time in neonatal rats treated with LPS. The present study demonstrated that M1 is the primary macrophage phenotype in the neonatal lung and that higher iNOS expression levels do not have a protective effect against pulmonary endotoxins in neonates. Overproduction of NO by iNOS in neonatal alveolar macrophages may result in detrimental effects during pulmonary inflammation.

Pseudomonas aeruginosa colonization in cystic fibrosis: Impact on neutrophil functions and cytokine secretion capacity.

Chronic colonization with Pseudomonas (P.) aeruginosa worsens the prognosis of cystic fibrosis (CF) patients. This study aims to analyze the functional properties of neutrophils in CF patients with P. aeruginosa colonization. Patients with CF (n = 16) were grouped by positivity of P. aeruginosa in sputum culture, as positive (P.+) or negative (P.-), then compared with age and sex matched healthy controls (n = 8). Adhesion molecules, apoptotic index, intracellular CAP-18, interleukin 8 (IL-8), and tumor necrosis factor α (TNF-α) levels of neutrophils, following P. aeruginosa and lipopolysaccharides (LPS) stimulation, were analyzed by flow cytometry. IL-1β, IL-6, TNF-α, and IL-17 plasma levels were determined by Luminex. Patients with CF had increased phagocytosis of Escherichia coli and P. aeruginosa, upregulated oxidative burst and chemotaxis. Increased neutrophil apoptosis was noted in CF patients. In unstimulated conditions, higher levels of CD16+ TNF-α+ and CD16+ IL-8+ neutrophils were determined, whereas bacteria and LPS stimulation significantly decreased secretion of CAP-18 from CD16+ neutrophils of CF patients. Plasma levels of IL-1β, TNF-α and IL-17 in P.+ patients were higher than in P.- group. Our findings confirm inadequate neutrophil defense towards pathogens in CF. A significant difference in migration, phagocytosis, oxidative burst, percentage of IL-8 producing neutrophils, IL-1β, TNF-α, and IL-17 secretions were noted among CF patients according to their colonization status, which might induce a further destructive effect on airways, resulting in an unfavorable prognosis for children with CF who also have colonization.

Ratiometric imaging of extracellular pH in Streptococcus mutans biofilms exposed to different flow velocities and saliva film thicknesses

ABSTRACT Introduction: Fluid flow has a prominent influence on the metabolism of surface-attached biofilms. Dental biofilms are covered by a thin saliva film that flows at different rates in different locations under stimulated and unstimulated conditions. Methods:The present study employed pH ratiometry to study the impact of different flow velocities, saliva film thicknesses and saliva concentrations on microscale pH developments in Streptococcus mutans biofilms of different age. Results:While saliva flow at a velocity of 0.8 mm/min (unstimulated flow) had little impact on biofilm pH, stimulated flow (8 mm/min; 80 mm/min) affected vertical pH gradients in the biofilms and raised the average pH in 48-h biofilms, but not in 72-h and 168-h biofilms. The saliva film thickness had a strong impact on biofilm pH under both static and dynamic conditions. pH drops were significantly higher in biofilms exposed to a thin saliva film (≤ 50 µm) than a thick saliva film (> 50 µm). pH drops in the biofilms were also strongly dependent on the saliva concentration and thus the buffer capacity of the salivary medium. For 48-h and 72-h biofilms, but not for 168-h biofilms, pH drops in distinct microenvironments were more pronounced when the local biofilm thickness was high.

Selective cysteines oxidation in soluble guanylyl cyclase catalytic domain is involved in NO activation

Nitric oxide (NO) binds to soluble guanylyl cyclase (GC1) and stimulates its catalytic activity to produce cGMP. Despite the key role of the NO-cGMP signaling in cardiovascular physiology, the mechanisms of GC1 activation remain ill-defined. It is believed that conserved cysteines (Cys) in GC1 modulate the enzyme's activity through thiol-redox modifications. We previously showed that GC1 activity is modulated via mixed-disulfide bond by protein disulfide isomerase and thioredoxin 1. Herein we investigated the novel concept that NO-stimulated GC1 activity is mediated by thiol/disulfide switches and aimed to map the specific Cys that are involved. First, we showed that the dithiol reducing agent Tris (2-carboxyethyl)-phosphine reduces GC1 response to NO, indicating the significance of Cys oxidation in NO activation. Second, using dibromobimane, which fluoresces when crosslinking two vicinal Cys thiols, we demonstrated decreased fluorescence in NO-stimulated GC1 compared to unstimulated conditions. This suggested that NO-stimulated GC1 contained more bound Cys, potentially disulfide bonds. Third, to identify NO-regulated Cys oxidation using mass spectrometry, we compared the redox status of all Cys identified in tryptic peptides, among which, ten were oxidized and two were reduced in NO-stimulated GC1. Fourth, we resorted to computational modeling to narrow down the Cys candidates potentially involved in disulfide bond and identified Cys489 and Cys571. Fifth, our mutational studies showed that Cys489 and Cys571 were involved in GC1′response to NO, potentially as a thiol/disulfide switch. These findings imply that specific GC1 Cys sensitivity to redox environment is critical for NO signaling in cardiovascular physiology.

Calcium dynamics and chromatin remodelling underlie heterogeneity in prolactin transcription.

Pituitary cells have been reported to show spontaneous calcium oscillations and dynamic transcription cycles. To study both processes in the same living cell in real time, we used rat pituitary GH3 cells stably expressing human prolactin-luciferase or prolactin-EGFP reporter gene constructs loaded with a fluorescent calcium indicator and measured activity using single-cell time-lapse microscopy. We observed heterogeneity between clonal cells in the calcium activity and prolactin transcription in unstimulated conditions. There was a significant correlation between cells displaying spontaneous calcium spikes and cells showing spontaneous bursts in prolactin expression. Notably, cells showing no basal calcium activity showed low prolactin expression but elicited a significantly greater transcriptional response to BayK8644 compared to cells showing basal calcium activity. This suggested the presence of two subsets of cells within the population at any one time. Fluorescence-activated cell sorting was used to sort cells into two populations based on the expression level of prolactin-EGFP however, the bimodal pattern of expression was restored within 26 h. Chromatin immunoprecipitation showed that these sorted populations were distinct due to the extent of histone acetylation. We suggest that maintenance of a heterogeneous bimodal population is a fundamental characteristic of this cell type and that calcium activation and histone acetylation, at least in part, drive prolactin transcriptional competence.

Betamethasone induces potent immunosuppression and reduces HIV infection in a PBMC in vitro model

Genital inflammation is an established risk factor for increased HIV acquisition risk. Certain HIV-exposed seronegative populations, who are naturally resistant to HIV infection, have an immune quiescent phenotype defined by...

PRMT5-mediated methylation of YBX1 regulates NF-\u03baB activity in colorectal cancer

The multifunctional protein Y-box binding protein 1 (YBX1), is a critical regulator of transcription and translation, and is widely recognized as an oncogenic driver in several solid tumors, including colorectal cancer (CRC). However, very little is known about the upstream or downstream factors that underlie YBX1′s regulation and involvement in CRC. Previously, we demonstrated that YBX1 overexpression correlated with potent activation of nuclear factor κB (NF-κB), a well-known transcription factor believed to be crucial in CRC progression. Here, we report a novel interaction between NF-κB, YBX1 and protein arginine methyltransferase 5 (PRMT5). Our findings reveal for the first time that PRMT5 catalyzes methylation of YBX1 at arginine 205 (YBX1-R205me2), an event that is critical for YBX1-mediated NF-κB activation and its downstream target gene expression. Importantly, when WT-YBX1 is overexpressed, this methylation exists under basal (unstimulated) conditions and is further augmented upon interleukin-1β (IL-1β) stimulation. Mechanistically, co-immunoprecipitation studies reveal that the R205 to alanine (A) mutant of YBX1 (YBX1-R205A) interacted less well with the p65 subunit of NF-κB and attenuated the DNA binding ability of p65. Importantly, overexpression of YBX1-R205A significantly reduced cell growth, migration and anchorage-independent growth of CRC cells. Collectively, our findings shed important light on the regulation of a novel PRMT5/YBX1/NF-κB axis through PRMT5-mediated YBX1-R205 methylation. Given the fact that PRMT5, YBX1 and NF-κB are all among top crucial factors in cancer progression, pharmacological disruption of this pivotal axis could serve as the basis for new therapeutics for CRC and other PRMT5/YBX1/NF-κB-associated cancers.