Activation Of Cellular Responses Research Articles

Activation of cellular responses by cyclic dinucleotides and Porphyromonas gingivalis lipopolysaccharide: a proteomic study on gingival fibroblasts

ABSTRACT Background Bacterial cyclic dinucleotides (CDNs), cyclic di-guanosine monophosphate (c-di-GMP), and cyclic di-adenosine monophosphate (c-di-AMP) upregulate interferon signaling proteins of human gingival fibroblasts (HGFs). However, the simultaneous effect of bacterial CDNs and lipopolysaccharides (LPS) on the HGF proteome is unknown. Aim The aim was to apply an unbiased proteomics approach to evaluate how simultaneous exposure to CDNs and Porphyromonas gingivalis (Pg) LPS affect the global proteome of HGFs. Methods The proteomic responses of HGFs were examined under three different treatment conditions (c-di-AMP+Pg LPS, c-di-GMP+Pg LPS, and Pg LPS alone) by label-free quantitative mass spectrometry analysis. Results Simultaneous exposure to CDNs and Pg LPS significantly upregulated innate immunity-related and interferon signaling-related proteins, such as ubiquitin-like protein ISG15 (ISG15), deoxynucleoside triphosphate triphosphohydrolase (SAMHD1), interferon regulatory factor 9 (IRF-9), interferon-induced GTP-binding protein Mx (MX)1, and MX2. Interferon signaling pathway was the most significantly regulated canonical pathway in both CDN treatment groups. Conclusion Simultaneous exposure to CDNs and Pg LPS stimulates the periodontal immune response by activating the anti-microbial cellular responses of HGFs with some notable differences from individual exposures.

Evaluation of the <i>GhMAPK</i> gene expression level under salt stress in cotton cultivars

Background: Abiotic environmental stress factors such as salt stress, drought, oxidative stress adversely affect the development and productivity of plants. To combat adverse environmental conditions, plants have developed a number of protective mechanisms. MAP kinases are protein kinases that regulate cellular activity in response to extracellular stimuli. Given the significant role of MAP kinase mechanisms in universal biological processes, elucidation of its role and mechanisms can be used to create stress-resistant genotypes. The use of stimulators and blockers of MAP kinase mechanisms is promising as a new direction in the management of plant stress resistance. The main goal of the conducted research is the comparative analysis of expression patterns of cotton accessions under salt stress conditions, the study of the relation between an increase in salt concentration and the change in the level of transcripts. Thirteen cultivars from Azerbaijan, nine from Turkey, four from Uzbekistan, four from Greece and one from Kyrgyzstan were used in the research, and changes in the expression level of the GhMAPK gene in cotton accessions was studied with the application of the real-time PCR analysis. In the present study, the evaluation of thirty-one cotton cultivars under different salt stress (NaCl) concentrations revealed significant differences in gene expression levels between cultivars of the same species. The ‘Navai-9’ cultivar from Uzbekistan had the highest expression level at both 100 mM and 200 mM salt concentrations compared to all other cultivars. Among local cultivars, cv. ‘Ganja-110’ (at 100 mM salt concentration), and cv. ‘Zafar’ (at 200 mM) differed from others in the level of changes in transcripts. In addition, there was a wide variation in the expression levels of stress-related genes between groups of accessions identified as resistant and sensitive, and within groups. Thus, both the increase and decrease in the expression level were found within these groups. This diversity in gene expression in sensitive and resistant cultivars in response to the salt stress shows that the mechanisms providing salt tolerance in the studied cultivars are different.

PTP1B phosphatase dampens iPSC-derived neutrophil motility and antimicrobial function.

Neutrophils are rapidly recruited to sites of infection and are critical for pathogen clearance. Therapeutic use of primary neutrophils has been limited, as they have a short lifespan and are not amenable to genetic manipulation. Human induced pluripotent stem cells (iPSCs) can provide a robust source of neutrophils for infusion and are genetically tractable. However, current work has indicated that dampened intracellular signaling limits iPSC-derived neutrophil (iNeutrophil) cellular activation and antimicrobial response. Here, we show that protein tyrosine phosphatase 1B (PTP1B) inhibits intracellular signaling and dampens iNeutrophil effector function. Deletion of the PTP1B phosphatase increased PI3K and ERK signaling and was associated with increased F-actin polymerization, cell migration, and phagocytosis. In contrast, other effector functions like NETosis and reactive oxygen species production were reduced. PTP1B-deficient neutrophils were more responsive to Aspergillus fumigatus and displayed rapid recruitment and control of hyphal growth. Accordingly, depletion of PTP1B increased production of inflammatory factors including the neutrophil chemokine interleukin-8. Taken together, these findings suggest that PTP1B limits iNeutrophil motility and antimicrobial function.

Single‐nuclei RNA sequencing provides evidence for glial senescence in Alzheimer’s disease

AbstractBackgroundAgeing is the greatest risk factor for Alzheimer’s disease (AD). While accumulation of senescent cells is one of the major hallmarks of ageing, the effect of the senescence burden is yet to be explored in AD. The aims of this study were to i) quantify senescent microglia, oligodendrocyte and astrocyte in human brain in AD, ii) determine the driver of cell senescence in AD, and iii) AD pathology associated senescence.MethodWe multiplexed Image Mass Cytometry (IMC) to co‐localise cell type markers (IBA1, OLIG2, GFAP), senescence markers (GLB1 and p16) and β‐amyloid (4G8) to detect senescence in tissues from Middle Temporal Gyrus (MTG) of 10 AD and 10 non‐disease control (NDC). We also generated snRNA‐seq from three different brain regions of the same subject namely Entorhinal (EC), MTG (Mid‐temporal Cortex) and Somatosensory Cortex (SSC) of 9 AD and 9 NDC. We performed geneset enrichment analysis using a set of curated senescence associated genesets in snRNA‐seq using AUcell and dream (variancePartition). We further performed trajectory analysis using Monocle3 to characterise β‐amyloid associated senescence signatures in the snRNAseq data.ResultMore than 25% microglia, oligodendrocyte and astrocyte were positive for GLB1 in AD brains while only around 5% glia showed GLB1 expression in NDC. Around 25% peri‐plaque microglia within 10 µm around β‐amyloid plaques in AD showed expression of GLB1 while only 3% of microglia >10 µm had associated senescence markers. Senescence genes were significantly upregulated in AD compared to NDC and were positively correlated with increase amyloid densities in all three glial cells (not in neurons). Trajectories of the glial nuclei described increased expression of pathways related to replicative senescence, cellular activation and stress response in microglia and oxidative stress in oligodendroglia, and astrocyte. Senescence gene signatures were positively associated with both β‐amyloid and pTau.ConclusionOur results highlight the high burden of senescent glia and provide evidence for distinct mechanisms of senescence in microglia (both replicative and stress‐induced) relative to oligodendroglia and astrocyte (stress‐induced) in AD. Both β‐amyloid and pTau associated increase in senescence gene signatures suggest that senescence is an important feature in the late stage of AD.

Immune Activation Profiles Elicited by Distinct, Repeated TLR Agonist Infusions in Rhesus Macaques.

TLR agonists are a promising class of immune system stimulants investigated for immunomodulatory applications in cancer immunotherapy and viral diseases. In this study, we sought to characterize the safety and immune activation achieved by different TLR agonists in rhesus macaques (Macaca mulatta), a useful preclinical model of complex immune interactions. Macaques received one of three TLR agonists, followed by plasma cytokine, immune cell subset representation, and blood cell activation measurements. The TLR4 agonist LPS administered i.v. induced very transient immune activation, including TNF-α expression and monocyte activation. The TLR7/8 agonist 2BXy elicited more persistent cytokine expression, including type I IFN, IL-1RA, and the proinflammatory IL-6, along with T cell and monocyte activation. Delivery of 2BXy i.v. and i.m. achieved comparable immune activation, which increased with escalating dose. Finally, i.v. bacillus Calmette-Guérin (BCG) vaccination (which activates multiple TLRs, especially TLR2/4) elicited the most pronounced and persistent innate and adaptive immune response, including strong induction of IFN-γ, IL-6, and IL-1RA. Strikingly, monocyte, T cell, and NK cell expression of the proliferation marker Ki67 increased dramatically following BCG vaccination. This aligned with a large increase in total and BCG-specific cells measured in the lung. Principal component analysis of the combined cytokine expression and cellular activation responses separated animals by treatment group, indicating distinct immune activation profiles induced by each agent. In sum, we report safe, effective doses and routes of administration for three TLR agonists that exhibit discrete immunomodulatory properties in primates and may be leveraged in future immunotherapeutic strategies.

Evaluation of cellular activity in response to sleep deprivation by a comprehensive analysis of the whole mouse brain.

Sleep deprivation (SD) causes several adverse functional outcomes, and understanding the associated processes can improve quality of life. Although the effects of SD on neuronal activity in several brain regions have been identified, a comprehensive evaluation of the whole brain is still lacking. Hence, we performed SD using two different methods, gentle handling and a dedicated chamber, in targeted recombination in active populations 2 (TRAP2) mice crossed with Rosa-ZsGreen reporter mice and visualized cellular activity in the whole brain. Using the semi-automated post-imaging analysis tool Slice Histology Alignment, Registration, and Cell Quantification (SHARCQ), the number of activated cells was quantified. From the analysis of 14 brain regions, cellular activity was significantly increased in the olfactory areas and decreased in the medulla by the two SD methods. From the analysis of the further subdivided 348 regions, cellular activity was significantly increased in the vascular organ of the lamina terminalis, lateral hypothalamic area, parabigeminal nucleus, ventral tegmental area, and magnocellular reticular nucleus, and decreased in the anterior part of the basolateral amygdalar nucleus, nucleus accumbens, septohippocampal nucleus, reticular nucleus of the thalamus, preoptic part of the periventricular hypothalamic nucleus, ventromedial preoptic nucleus, rostral linear nucleus raphe, facial motor nucleus, vestibular nuclei, and some fiber tracts (oculomotor nerve, genu of corpus callosum, and rubrospinal tract) by the two SD methods. Two subdivided regions of the striatum (caudoputamen and other striatum), epithalamus, vascular organ of the lamina terminalis, anteroventral preoptic nucleus, superior colliculus optic layer, medial terminal nucleus of the accessory optic tract, pontine gray, and fiber tracts (medial lemniscus, columns of the fornix, brachium of the inferior colliculus, and mammillary peduncle) were differentially affected by the two SD methods. Most brain regions detected from these analyses have been reported to be involved in regulating sleep/wake regulatory circuits. Moreover, the results from the connectivity analysis indicated that the connectivity of cellular activity among brain regions was altered by SD. Together, such a comprehensive analysis of the whole brain is useful for understanding the mechanisms by which SD and/or sleep disruption affects brain function.

Analysis of the influences of social isolation on cognition and the therapeutic potential of deep brain stimulation in a mouse model.

Social interaction is a fundamental human need. Social isolation (SI) can have negative effects on both emotional and cognitive function. However, it is currently unclear how age and the duration of SI affect emotion and recognition function. In addition, there is no specific treatment for the effects of SI. The adolescence or adult mice were individually housed in cages for 1, 6 or 12 months and for 2 months to estabolish SI mouse model. We investigated the effects of SI on behavior in mice at different ages and under distinct durations of SI, and we explored the possible underlying mechanisms. Then we performed deep brain stimulation (DBS) to evaluate its influences on SI induced behavioral abnormalities. We found that social recognition was affected in the short term, while social preference was damaged by extremely long periods of SI. In addition to affecting social memory, SI also affects emotion, short-term spatial ability and learning willingness in mice. Myelin was decreased significantly in the medial prefrontal cortex (mPFC) and dorsal hippocampus of socially isolated mice. Cellular activity in response to social stimulation in both areas was impaired by social isolation. By stimulating the mPFC using DBS, we found that DBS alleviated cellular activation disorders in the mPFC after long-term SI and improved social preference in mice. Our results suggest that the therapeutic potential of stimulating the mPFC with DBS in individuals with social preference deficits caused by long-term social isolation, as well as the effects of DBS on the cellular activity and density of OPCs.

Comprehensive Profiling of Secreted Factors in the Cerebrospinal Fluid of Moyamoya Disease Patients.

Moyamoya disease (MMD) is characterized by progressive occlusion of the intracranial internal carotid arteries, leading to ischemic and hemorrhagic events. Significant clinical differences exist between ischemic and hemorrhagic MMD. To understand the molecular profiles in the cerebrospinal fluid (CSF) of MMD patients, we investigated 62 secreted factors in both MMD subtypes (ischemic and hemorrhagic) and examined their relationship with preoperative perfusion status, the extent of postoperative angiographic revascularization, and functional outcomes. Intraoperative CSF was collected from 32 control and 71 MMD patients (37 ischemic and 34 hemorrhagic). Multiplex Luminex assay analysis showed that 41 molecules were significantly elevated in both MMD subtypes when compared to controls, including platelet-derived growth factor-BB (PDGF-BB), plasminogen activator inhibitor 1 (PAI-1), and intercellular adhesion molecule 1 (ICAM1) (p < 0.001). Many of these secreted proteins have not been previously reported in MMD, including interleukins (IL-2, IL-4, IL-5, IL-7, IL-8, IL-9, IL-17, IL-18, IL-22, and IL-23) and C-X-C motif chemokines (CXCL1 and CXCL9). Pathway analysis indicated that both MMD subtypes exhibited similar cellular/molecular functions and pathways, including cellular activation, migration, and inflammatory response. While neuroinflammation and dendritic cell pathways were activated in MMD patients, lipid signaling pathways involving nuclear receptors, peroxisome proliferator-activated receptor (PPAR), and liver X receptors (LXR)/retinoid X receptors (RXR) signaling were inhibited. IL-13 and IL-2 were negatively correlated with preoperative cerebral perfusion status, while 7 factors were positively correlated with the extent of postoperative revascularization. These elevated cytokines, chemokines, and growth factors in CSF may contribute to the pathogenesis of MMD and represent potential future therapeutic targets.

Robust engraftment of fetal nonhuman primate CD34-positive cells in immune-deficient mice.

Nonhuman primates (NHPs) represent one of the most important models for preclinical studies of novel biomedical interventions. In contrast with small animal models, however, widespread utilization of NHPs is restricted by cost, logistics, and availability. Therefore, we sought to develop a translational primatized mouse model, akin to a humanized mouse, to allow for high-throughput in vivo experimentation leveraged to inform large animal immunology-based studies. We found that adult rhesus macaque mobilized blood (AMb) CD34+-enriched hematopoietic stem and progenitor cells (HSPCs) engrafted at low but persistent levels in immune-deficient mice harboring transgenes for human (NHP cross-reactive) GM-CSF and IL3, but did not in mice with wild-type murine cytokines lacking NHP cross-reactivity. To enhance engraftment, fetal liver-derived HSPCs were selected as the infusion product based on an increased CD34hi fraction compared with AMb and bone marrow. Coupled with cotransplantation of rhesus fetal thymic fragments beneath the mouse kidney capsule, fetal liver-derived HSPC infusion in cytokine-transgenic mice yielded robust multilineage lymphohematopoietic engraftment. The emergent immune system recapitulated that of the fetal monkey, with similar relative frequencies of lymphocyte, granulocyte, and monocyte subsets within the thymic, secondary lymphoid, and peripheral compartments. Importantly, while exhibiting a predominantly naïve phenotype, in vitro functional assays demonstrated robust cellular activation in response to nonspecific and allogenic stimuli. This primatized mouse represents a viable and translatable model for the study of hematopoietic stem cell physiology, immune development, and functional immunology in NHPs. Summary Sentence: Engraftment of rhesus macaque hematopoietic tissues in immune-deficient mice yields a robust BLT/NeoThy-type primatized mouse model for studying nonhuman primate hematopoiesis and immune function in vivo.

Effects of Two Protocols of Low-Level Laser Therapy on the Proliferation and Differentiation of Human Dental Pulp Stem Cells on Sandblasted Titanium Discs: An In Vitro Study.

Introduction: Stem cell activities have different effects on tissue response and its outcomes. Low-level laser therapy (LLLT) can be considered a trigger to modify stem cell activities. The objective of the present experimental investigation was to study the effects of two protocols of LLLT on the proliferation and differentiation of human dental pulp stem cells (hDPSCs) cultured on sandblasted titanium discs. Methods: Cells obtained from human dental pulp were seeded/cultured on titanium discs and were set in 2 main groups: (i) Radiated cells using the gallium-aluminium-arsenide (GaAlAs) diode laser at a continuous wavelength of 808 nm at 3 J/cm2 for 12 sec or 5 J/cm2 for 20 seconds, and (ii) Non-irradiated cells serving as control groups. The impact of LLLTs on hDPSC-proliferation and viability was investigated using the MTT assay after 24, 72 and 96 hours. The alkaline phosphatase activity was studied with p-nitrophenylphosphate after 14 and 28 days. The ability of hDPSCs to express osteocalcin was investigated using real-time polymerase chain reaction after 28 days, while their attachment was observed under a scanning electron microscope (SEM) after 14 and 28 days. Results: Our study showed that LLLTs caused maximum cell proliferation in 96 hours (P<0.001) with 3 J/cm2 resulting in a higher proliferation rate. The highest activity of alkaline phosphatase and osteocalcin expression was observed in the laser radiation groups after 28 days. Conclusion: The outcomes of the current study showed that cultured hDPSCs on sandblasted titanium discs had a tendency towards increased cellular activity in response to LLLTs. Thus, LLLTs could regulate the activities of hDPSCs on bone repair surrounding the sandblasted titanium discs.

Tunable heat shock protein-mediated NK cell responses are orchestrated by STAT1 in Antigen Presenting Cells

The release of Heat Shock Proteins (HSPs) from aberrant cells can initiate immune responses following engagement of the HSPs with antigen presenting cells (APCs). This is an important mechanism for cancer immunosurveillance and can also be modeled by vaccination with HSPs through various routes, targeting specific APCs expressing the HSP receptor CD91. Immunological outcomes can be varied as a result of the broad expression of CD91 in different dendritic cell and macrophage populations. We investigated the cellular response of different APCs to the prototypical immunogenic HSP, gp96, in the context of Th1 immunity. Although APCs generally express similar levels of the HSP receptor CD91, we uncovered APC-distinct, downstream signaling pathways activating STAT1, and differential STAT1 induced genes. As a result of this differential and unique signaling we determined that gp96-activated macrophages, but not DCs are capable of activating NK cells to produce IFN-gamma. These data demonstrate that different APC subsets elicit unique intracellular signaling responses to HSPs which result in different patterns of downstream cellular activation and immune responses. Collectively this provides a novel tunable and autochthonous immune response to extracellular HSPs which has important implications on the development of immunity to cancer and infectious disease, as well as homeostasis.

Actin Dynamics at the T Cell Synapse as Revealed by Immune-Related Actinopathies

The actin cytoskeleton is composed of dynamic filament networks that build adaptable local architectures to sustain nearly all cellular activities in response to a myriad of stimuli. Although the function of numerous players that tune actin remodeling is known, the coordinated molecular orchestration of the actin cytoskeleton to guide cellular decisions is still ill defined. T lymphocytes provide a prototypical example of how a complex program of actin cytoskeleton remodeling sustains the spatio-temporal control of key cellular activities, namely antigen scanning and sensing, as well as polarized delivery of effector molecules, via the immunological synapse. We here review the unique knowledge on actin dynamics at the T lymphocyte synapse gained through the study of primary immunodeficiences caused by mutations in genes encoding actin regulatory proteins. Beyond the specific roles of individual actin remodelers, we further develop the view that these operate in a coordinated manner and are an integral part of multiple signaling pathways in T lymphocytes.

Immunogenicity of Ad26.COV2.S vaccine against SARS-CoV-2 variants in humans

The Ad26.COV2.S vaccine1–3 has demonstrated clinical efficacy against symptomatic COVID-19, including against the B.1.351 variant that is partially resistant to neutralizing antibodies1. However, the immunogenicity of this vaccine in humans against SARS-CoV-2 variants of concern remains unclear. Here we report humoral and cellular immune responses from 20 Ad26.COV2.S vaccinated individuals from the COV1001 phase I–IIa clinical trial2 against the original SARS-CoV-2 strain WA1/2020 as well as against the B.1.1.7, CAL.20C, P.1 and B.1.351 variants of concern. Ad26.COV2.S induced median pseudovirus neutralizing antibody titres that were 5.0-fold and 3.3-fold lower against the B.1.351 and P.1 variants, respectively, as compared with WA1/2020 on day 71 after vaccination. Median binding antibody titres were 2.9-fold and 2.7-fold lower against the B.1.351 and P.1 variants, respectively, as compared with WA1/2020. Antibody-dependent cellular phagocytosis, complement deposition and natural killer cell activation responses were largely preserved against the B.1.351 variant. CD8 and CD4 T cell responses, including central and effector memory responses, were comparable among the WA1/2020, B.1.1.7, B.1.351, P.1 and CAL.20C variants. These data show that neutralizing antibody responses induced by Ad26.COV2.S were reduced against the B.1.351 and P.1 variants, but functional non-neutralizing antibody responses and T cell responses were largely preserved against SARS-CoV-2 variants. These findings have implications for vaccine protection against SARS-CoV-2 variants of concern.

Regulation of microRNA-221, -222, -21 and -27 in articular cartilage subjected to abnormal compressive forces.

Key points microRNAs (miRs) are small non‐coding molecules that regulate post‐transcriptional target gene expression.miRs are involved in regulating cellular activities in response to mechanical loading in all physiological systems, although it is largely unknown whether this response differs with increasing magnitudes of load.miR‐221, miR‐222, miR‐21‐5p and miR‐27a‐5p were significantly increased in ex vivo cartilage explants subjected to increasing load magnitude and in in vivo joint cartilage exposed to abnormal loading. TIMP3 and CPEB3 are putative miR targets in chondrocytesIdentification of mechanically regulated miRs that have potential to impact on tissue homeostasis provides a mechanism by which load‐induced tissue behaviour is regulated, in both health and pathology, in all physiological systems. MicroRNAs (miRs) are small non‐coding molecules that regulate post‐transcriptional target gene expression and are involved in mechano‐regulation of cellular activities in all physiological systems. It is unknown whether such epigenetic mechanisms are regulated in response to increasing magnitudes of load. The present study investigated mechano‐regulation of miRs in articular cartilage subjected to ‘physiological’ and ‘non‐physiological’ compressive loads in vitro as a model system and validated findings in an in vivo model of abnormal joint loading. Bovine full‐depth articular cartilage explants were loaded to 2.5 MPa (physiological) or 7 MPa (non‐physiological) (1 Hz, 15 min) and mechanically‐regulated miRs identified using next generation sequencing and verified using a quantitative PCR. Downstream targets were verified using miR‐specific mimics or inhibitors in conjunction with 3′‐UTR luciferase activity assays. A subset of miRs were mechanically‐regulated in ex vivo cartilage explants and in vivo joint cartilage. miR‐221, miR‐222, miR‐21‐5p and miR‐27a‐5p were increased and miR‐483 levels decreased with increasing load magnitude. Tissue inhibitor of metalloproteinase 3 (TIMP3) and cytoplasmic polyadenylation element binding protein 3 (CPEB3) were identified as putative downstream targets. Our data confirm miR‐221 and ‐222 mechano‐regulation and demonstrates novel mechano‐regulation of miR‐21‐5p and miR‐27a‐5p in ex vivo and in vivo cartilage loading models. TIMP3 and CPEB3 are putative miR targets in chondrocytes. Identification of specific miRs that are regulated by increasing load magnitude, as well as their potential to impact on tissue homeostasis, has direct relevance to other mechano‐sensitive physiological systems and provides a mechanism by which load‐induced tissue behaviour is regulated, in both health and pathology.

A Cytoplasmic Receptor-like Kinase Contributes to Salinity Tolerance.

Receptor-like cytoplasmic kinases (RLCKs) are receptor kinases that lack extracellular ligand-binding domains and have emerged as a major class of signaling proteins that regulate plant cellular activities in response to biotic/abiotic stresses and endogenous extracellular signaling molecules. We have identified a rice RLCK (OsRLCK311) that was significantly higher in transgenic pSARK-IPT rice (Oryza sativa) that exhibited enhanced growth under saline conditions. Overexpression of OsRLCK311 full-length protein (RLCK311FL) and the C-terminus of OsRLCK311 (ΔN) in Arabidopsis confirmed its role in salinity tolerance, both in seedlings and mature plants. Protein interaction assays indicated that OsRLCK311 and ΔN interacted in-vivo with the plasma membrane AQP AtPIP2;1. The RLCK311-PIP2;1 binding led to alterations in the stomata response to ABA, which was characterized by more open stomata of transgenic plants. Moreover, OsRLCK311-ΔN effect in mediating enhanced plant growth under saline conditions was also observed in the perennial grass Brachypodium sylvaticum, confirming its role in both dicots and monocots species. Lastly, OsRLCK311 interacted with the rice OsPIP2;1. We suggest that the rice OsRLCK311 play a role in regulating the plant growth response under saline conditions via the regulation of the stomata response to stress. This role seems to be independent of the RLCK311 kinase activity, since the overexpression of the RLCK311 C-terminus (ΔN), which lacks the kinase full domain, has a similar phenotype to RLCK311FL.

The acromioclavicular ligament shows an early and dynamic healing response following acute traumatic rupture

PurposeSymptomatic horizontal instability is clinically relevant following acute acromioclavicular joint dislocations. However, the intrinsic healing response is poorly understood. The present study sought to investigate time-dependent healing responses of the human acromioclavicular ligament following acute traumatic rupture.MethodsBiopsies of the acromioclavicular ligament were obtained from patients undergoing surgical treatment for acute acromioclavicular joint dislocations. Specimens were stratified by time between trauma and surgery: group 1, 0–7 days (n = 5); group 2, 8–14 days (n = 6); and group 3, 15–21 days (n = 4). Time-dependent changes in cellularity, collagen (type 1 and 3) concentration, and histomorphological appearance were evaluated for the rupture and intact zone of the acromioclavicular ligament.ResultsGroup 1 was characterized by cellular activation and early inflammatory response. The rupture zone exhibited a significantly higher count of CD68-positive cells than the intact zone (15.2 vs 7.4; P ≤ 0.05). Consistently, synovialization of the rupture end was observed. Within the second week, the rupture zone was subject to proliferation showing more fibroblast-like cells than the intact zone (66.8 vs 43.8; P ≤ 0.05) and a peak of collagen type 3 expression (group 1: 2.2 ± 0.38, group 2: 3.2 ± 0.18, group 3: 2.8 ± 0.57; P ≤ 0.05). Signs of consolidation and early remodeling were seen in the third week.ConclusionsThe acromioclavicular ligament exhibits early and dynamic healing responses following acute traumatic rupture. Our histological findings suggest that surgical treatment of acute ACJ dislocations should be performed as early as possible within a timeframe of 1 week after trauma to exploit the utmost biological healing potential. Prospective clinical studies are warranted to investigate whether early surgical treatment of ACJ dislocations translates into clinical benefits.

Multifunctional Flexible Biointerfaces for Simultaneous Colocalized Optophysiology and Electrophysiology

AbstractRecent developments in optophysiology techniques such as optogenetics have revolutionized the ability to actuate cell activity. Further combining optophysiology and electrophysiology will integrate the advantages from both optical and electrical modalities and yield enabling technologies that allow simultaneous monitoring of cellular activity in response to modulation, which are crucial for biomedical applications. However, multifunctional devices that can deliver optical stimuli to regions beneath the electrodes and perform simultaneous sensing remain largely unexplored. Existing transparent microelectrode technologies depend on external bulk optical instruments for optical interventions. Here, innovative monolithic integrated multifunctional microsystems are demonstrated by applying transparent nanogrid electrodes onto microscale light sources to permit simultaneous electrophysiology and optical modulation at the same anatomical site. The nanogrid electrodes have transmittances &gt; 70% with a low normalized impedance of 5.9 Ω cm2. Additional features of the devices include superior mechanical flexibility, minimized light‐induced electrical artifacts, and excellent biocompatibility. Ex vivo experiments demonstrate that the multifunctional devices can record abnormal heart rhythm in transgenic mouse hearts and simultaneously restore the sinus rhythm via optogenetic pacing. This work provides a versatile approach for constructing multifunctional colocalized biointerfaces containing crosstalk‐free optical and electrical modalities with expanded opportunities in both fundamental and applied biomedical research.

The endoplasmic reticulum stress-autophagy pathway controls hypothalamic development and energy balance regulation in leptin-deficient neonates

Obesity is associated with the activation of cellular responses, such as endoplasmic reticulum (ER) stress. Here, we show that leptin-deficient ob/ob mice display elevated hypothalamic ER stress as early as postnatal day 10, i.e., prior to the development of obesity in this mouse model. Neonatal treatment of ob/ob mice with the ER stress-relieving drug tauroursodeoxycholic acid (TUDCA) causes long-term amelioration of body weight, food intake, glucose homeostasis, and pro-opiomelanocortin (POMC) projections. Cells exposed to ER stress often activate autophagy. Accordingly, we report that in vitro induction of ER stress and neonatal leptin deficiency in vivo activate hypothalamic autophagy-related genes. Furthermore, genetic deletion of autophagy in pro-opiomelanocortin neurons of ob/ob mice worsens their glucose homeostasis, adiposity, hyperphagia, and POMC neuronal projections, all of which are ameliorated with neonatal TUDCA treatment. Together, our data highlight the importance of early life ER stress-autophagy pathway in influencing hypothalamic circuits and metabolic regulation.

Monocyte and CD4+ T-cell antiviral and innate responses associated with HIV-1 inflammation and cognitive impairment.

Mechanisms underlying immune activation and HIV-associated neurocognitive disorders (HAND) in untreated chronic infection remain unclear. The objective of this study was to identify phenotypic and transcriptional changes in blood monocytes and CD4 T cells in HIV-1-infected and uninfected individuals and elucidate processes associated with neurocognitive impairment. A group of chronically HIV-1-infected Thai individuals (n = 19) were selected for comparison with healthy donor controls (n = 10). Infected participants were further classified as cognitively normal (n = 10) or with HAND (n = 9). Peripheral monocytes and CD4 T cells were phenotyped by flow cytometry and simultaneously isolated for multiplex qPCR-targeted gene expression profiling directly ex vivo. The frequency of HIV-1 RNA-positive cells was estimated by limiting dilution cell sorting. Expression of genes and proteins involved in cellular activation and proinflammatory immune responses was increased in monocytes and CD4 T cells from HIV-1-infected relative to uninfected individuals. Gene expression profiles of both CD4 T cells and monocytes correlated with soluble markers of inflammation in the periphery (P < 0.05). By contrast, only modest differences in gene programs were observed between cognitively normal and HAND cases. These included increased monocyte surface CD169 protein expression relative to cognitively normal (P = 0.10), decreased surface CD163 expression relative to uninfected (P = 0.02) and cognitively normal (P = 0.06), and downregulation of EMR2 (P = 0.04) and STAT1 (P = 0.02) relative to cognitively normal. Our data support a model of highly activated monocytes and CD4 T cells associated with inflammation in chronic HIV-1 infection, but impaired monocyte anti-inflammatory responses in HAND compared with cognitively normal.

Abstract PR17: Targeted blockade of FcγRIIB on dendritic cells and macrophages enhances cellular activation and Fc-dependent immune responses

Abstract FcγRIIB (CD32b) is the sole inhibitory member of the Fcγ family of IgG-binding receptors, suppressing cellular activation in response to Fc-dependent stimuli. Human FcγRIIB is expressed by both dendritic cells (DCs) and macrophages, and the ratio of activating to inhibitory signals from their cell surface Fcγ receptors sets the threshold for their activation. Anti-tumor antibodies with an Fc-dependent mode of action (MoA) can bind to FcγRIIB on these myeloid cells, limiting effector responses such as antibody-dependent cell-mediated cytotoxicity (ADCC) and DC-mediated priming of T cells. Agents that lower the immunologic threshold for Fc-dependent cellular activation therefore represent an ideal means of enhancing antitumor antibody therapeutic efficacy. We hypothesized that anti-FcγRIIB antibodies that can block the Fc-binding domain of human FcγRIIB can enhance the activation and effector functions of human DCs and macrophages, thereby bolstering innate and adaptive antitumor immunity. We found that anti-FcγRIIB treatment specifically blocked the binding of immune complexes (ICs) to human FcγRIIB in vitro, demonstrating a functional consequence of blocking the Fc binding pocket of FcγRIIB. Treatment of human monocyte-derived DCs (moDCs) with anti-FcγRIIB antibodies induced DC maturation as measured by CD86 upregulation and CD14 downregulation, and this enhancement of DC maturation was dependent upon the expression of activating FcγRIIa. Treatment of DC-like cells derived from the FcγRIIB-expressing monocytic human THP-1 cell line with anti-FcγRIIB led to their improved performance when combined with primary human T cells in a mixed lymphocyte reaction, consistent with a functional enhancement of DC maturation. Treatment of human monocyte-derived macrophages with an Fc-enhanced anti-FcγRIIB was also sufficient to induce TNFα secretion and downregulation of cell surface CD163, consistent with robust FcγR-dependent macrophage activation. Ongoing studies to test the cell type-specific roles of FcγRIIB in the context of an in vivo model that can read out Fc-dependent antitumor antibody activity will further clarify the mechanism by which disrupting FcγRIIB signaling can enhance antitumor mAb therapeutic efficacy. In conclusion, our work highlights the ability of a novel antibody directed against the Fc-binding domain of human FcγRIIB to both directly enhance DC and macrophage activation and further bolster the activation and effector functions of these cells in response to Fc-dependent stimuli, offering a viable avenue for enhancing the efficacy of mAbs with an Fc-dependent MoA. This abstract is also being presented as Poster A77. Citation Format: Ryan D. Molony, Sunyoung Jang, Adwait Oka, Sinam Isim, Fangmin Xu, Barbara Platzer, Karrie Wong, Matthew Meyer, Haihui Lu. Targeted blockade of FcγRIIB on dendritic cells and macrophages enhances cellular activation and Fc-dependent immune responses [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2018 Nov 27-30; Miami Beach, FL. Philadelphia (PA): AACR; Cancer Immunol Res 2020;8(4 Suppl):Abstract nr PR17.