Prior Priming Research Articles

Abstract 2462: Live attenuated Salmonella ZH9 as novel microbial immunotherapy for the treatment of bladder cancer

Abstract Microbial immunotherapy with Bacillus Calmette-Guérin (BCG) has been the standard-of-care for non-muscle invasive bladder cancer (NMIBC) for over 30 years. Today, global demand for BCG exceeds supply, leaving many patients untreated. BCG is associated with high disease recurrence and presents high treatment burden for patients. There remains therefore significant unmet need for novel therapeutic approaches for NMIBC and we sought to establish the utility of the live-attenuated Salmonella enterica Typhi ZH9 as a more effective microbial immunotherapy. Efficacy of ZH9 was established in orthotopic, syngeneic, mouse MB49 tumor models in which animals were treated in the bladder via catheterization, with or without prior subcutaneous immune priming. Immune responses were measured by flow cytometry and Nanostring transcriptomic analyses of bladder tissues. An intravesical dose of ZH9 after orthotopic MB49 tumour inoculation demonstrated significant survival benefit compared to both vehicle control and equivalent colony forming unit (CFU) BCG treatments. Repeat, weekly dosing studies showed a single administration of ZH9 was sufficient for maximum efficacy. To explore the durability of protection, surviving intravesical ZH9 treated animals were rechallenged, demonstrating protection from both bladder and subcutaneous MB49 tumors suggestive of lasting local and systemic anti-tumor immunity. Flow cytometry analysis of bladders showed that intravesical ZH9 treatment resulted in cellular immune response characterised by recruitment of NK cells, CD4+ and CD8+ T cells, and dendritic cells with an activated, cross-presenting phenotype, in all cases of greater magnitude than after treatment with an equivalent dose of BCG. Based on observations that pre-existing immunity to BCG has been associated with improved outcomes to intravesical BCG therapy in NMIBC, we explored whether systemic priming with ZH9 benefits local bladder therapy. Systemic prime with ZH9 followed by a single intravesical dose of ZH9 showed a trend for improved survival and will be explored in the clinical setting. This systemic immune priming effect was associated with enhanced local bladder immune cell infiltrates and the upregulation of more than 150 immune response genes in the bladder compared to intravesical therapy alone. These data form the basis of preclinical package accepted by the FDA for a Phase 1/1b study evaluating the safety, pharmacology, and clinical effect of ZH9 treatment in patients with NMIBC, currently recruiting patients. The investigation includes thorough translational and biomarker studies to delve into the mechanism of action of ZH9 and explore patient stratification approaches. By easing the treatment burden on patients, lowering healthcare costs, and featuring a scalable manufacturing process, ZH9 has the potential to revolutionize the treatment paradigm in NMIBC and reshape the patient journey. Citation Format: Nicholas Glanville, Oluwatobiloba Oke, Claudia Prevosto, Alexandra Sevko, Andrew Scott, Sonia Domingos-Pereira, Lenka Polak, Denise Nardelli-Haefliger, Livija Deban. Live attenuated Salmonella ZH9 as novel microbial immunotherapy for the treatment of bladder cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2462.

Cold priming on pathogen susceptibility in the Arabidopsis eds1 mutant background requires a functional stromal Ascorbate Peroxidase

ABSTRACT 24 h cold exposure (4°C) is sufficient to reduce pathogen susceptibility in Arabidopsis thaliana against the virulent Pseudomonas syringae pv. tomato (Pst) strain even when the infection occurs five days later. This priming effect is independent of the immune regulator Enhanced Disease Susceptibility 1 (EDS1) and can be observed in the immune-compromised eds1–2 null mutant. In contrast, cold priming-reduced Pst susceptibility is strongly impaired in knock-out lines of the stromal and thylakoid ascorbate peroxidases (sAPX/tAPX) highlighting their relevance for abiotic stress-related increased immune resilience. Here, we extended our analysis by generating an eds1 sapx double mutant. eds1 sapx showed eds1-like resistance and susceptibility phenotypes against Pst strains containing the effectors avrRPM1 and avrRPS4. In comparison to eds1–2, susceptibility against the wildtype Pst strain was constitutively enhanced in eds1 sapx. Although a prior cold priming exposure resulted in reduced Pst titers in eds1–2, it did not alter Pst resistance in eds1 sapx. This demonstrates that the genetic sAPX requirement for cold priming of basal plant immunity applies also to an eds1 null mutant background.

"No-Primer" Resin Cementation of Lithium Disilicate Ceramic: A Microtensile Bond Strength Evaluation.

The objective of this study was to evaluate the resin-ceramic adhesion of a long-carbon-chain silane (LCSI)-containing resin cement. Polished lithium disilicate ceramic discs were etched with hydrofluoric acid and randomly assigned into four groups; (PSAP), cemented using a silane-free resin cement with no prior priming; (PSAP-S), primed using a silane-containing primer before cementation using a silane-free resin cement; (PSAU), cemented using a LCSI-containing resin cement with no prior priming; (PSAU-S), primed as for the group (PSAP-S) and cemented using a LCSI-containing resin cement. The cemented blocks were sectioned into microbeams. The resin-ceramic microtensile bond strength (μTBS) was measured at 1 week and after thermocycling. The failure modes of the tested microbeams were evaluated. The μTBS of the LCSI-containing and silane-free resin cements, either with or without a prior priming step, did not significantly differ. The adhesion of the LCSI-containing resin cement to lithium disilicate ceramic, either with or without a prior priming step, did not significantly deteriorate after artificial aging. The long-carbon-chain silane (LCSI) monomer incorporated in the resin cement eliminated the need for a silane priming step of a hydrofluoric acid-etched lithium disilicate ceramic.

How can Cytokine-induced killer cells overcome CAR-T cell limits.

The successful treatment of patients affected by B-cell malignancies with Chimeric Antigen Receptor (CAR)-T cells represented a breakthrough in the field of adoptive cell therapy (ACT). However, CAR-T therapy is not an option for every patient, and several needs remain unmet. In particular, the production of CAR-T cells is expensive, labor-intensive and logistically challenging; additionally, the toxicities deriving from CAR-T cells infusion, such as cytokine release syndrome (CRS) and immune effector cell-associated neurotoxicity syndrome (ICANS), have been documented extensively. Alternative cellular therapy products such as Cytokine-induced killer (CIK) cells have the potential to overcome some of these obstacles. CIK cells are a heterogeneous population of polyclonal CD3+CD56+ T cells with phenotypic and functional properties of NK cells. CIK cell cytotoxicity is exerted in a major histocompatibility complex (MHC)-unrestricted manner through the engagement of natural killer group 2 member D (NKG2D) molecules, against a wide range of hematological and solid tumors without the need for prior antigen exposure or priming. The foremost potential of CIK cells lies in the very limited ability to induce graft-versus-host disease (GvHD) reactions in the allogeneic setting. CIK cells are produced with a simple and extremely efficient expansion protocol, which leads to a massive expansion of effector cells and requires a lower financial commitment compared to CAR-T cells. Indeed, CAR-T manufacturing involves the engineering with expensive GMP-grade viral vectors in centralized manufacturing facilities, whereas CIK cell production is successfully performed in local academic GMP facilities, and CIK cell treatment is now licensed in many countries. Moreover, the toxicities observed for CAR-T cells are not present in CIK cell-treated patients, thus further reducing the costs associated with hospitalization and post-infusion monitoring of patients, and ultimately encouraging the delivery of cell therapies in the outpatient setting. This review aims to give an overview of the limitations of CAR-T cell therapy and outline how the use of CIK cells could overcome such drawbacks thanks to their unique features. We highlight the undeniable advantages of using CIK cells as a therapeutic product, underlying the opportunity for further research on the topic.

A common magma source for plutonic and volcanic rocks of The Geysers geothermal field, California: Volume and intrusive history derived from zircon

The Geysers Plutonic Complex (GPC) in the California Coast Ranges underlies one of the world's premier geothermal resources. The GPC consists of three major intrusive phases, orthopyroxene-biotite microgranite porphyry, orthopyroxene-biotite granite, and hornblende-biotite-orthopyroxene granodiorite (in sequence of emplacement). These are nearly coeval and compositionally equivalent to lavas of the overlying Cobb Mountain Volcanic Center which includes (from older to younger) rhyolite of Alder Creek, dacite of Cobb Mountain, and dacite of Cobb Valley. Zircon from GPC plutonic and associated volcanic rocks were analyzed at high spatial resolution for their trace element abundances along with oxygen and hafnium isotopic compositions. Pronounced negative Eu anomalies and high incompatible trace element (e.g., Y, Hf, and U) abundances in zircon along with low Ti-in-zircon temperatures reveal that the GPC microgranite porphyry magma was more evolved than GPC granite and granodiorite. Isotopically, GPC microgranite porphyry, granite, and granodiorite zircon crystals closely overlap (δ18O = +4.76 to +9.18; εHf = +1.4 to +10.7), but a subpopulation with elevated δ18O (∼8.05) and lower εHf (∼4.4) is only present in GPC granite and granodiorite. Zircon from coeval volcanic units share this dichotomy, with a dominant population at (δ18O = +4.92 to +9.38; εHf = +3.4 to +11.3) and a generally minor population with elevated δ18O and low εHf, which is particularly prominent in dacite of Cobb Valley. Anticorrelated δ18O and εHf values indicate progressive assimilation of Franciscan Complex basement rocks, but fractional crystallization was decoupled from crustal assimilation. Together with published U-Th-Pb geochronology, these data reveal distinct degrees of crust-mantle interaction and constrain the thermochemical conditions during different stages in the evolution of a composite intrusive body that fed the GPC and contemporaneous volcanoes. A thermal model for such a body emplaced at ∼7 km depth quantitatively matches the GPC zircon age distribution when magma accumulation started at low recharge rates (0.1 km3/ka), intermittently peaked during a brief flare-up (4 km3/ka for 50 ka), and then returned to a low recharge flux (0.1 km3/ka). This model also qualitatively explains the initial presence of small-volume, highly evolved melts forming the microgranite porphyry, followed by massive emplacement of less evolved magmas forming the GPC granite-granodiorite complex. During this second stage, crustal assimilation locally intensified due to prior thermal priming of the country rock. The total injected magma volume into the upper-crustal reservoir between c. 2.1 and 1.1 Ma amounts to ∼300 km3, which is about three to four times the known volume from geothermal well penetration into the GPC. The accumulation of large volumes of silicic magma along the western North American continental margin in the wake of the northward migrating Gorda slab edge thus appears limited to a brief (c. 50 ka) pulse of high magma influx from the mantle that was pre- and post-dated by protracted low-flux magmatism.

Prior immunization against an intracellular antigen enhances subsequent red blood cell alloimmunization in mice

AbstractAntibodies against red blood cell (RBC) alloantigens can increase morbidity and mortality among transfusion recipients. However, alloimmunization rates can vary dramatically, as some patients never generate alloantibodies after transfusion, whereas others not only become alloimmunized but may also be prone to generating additional alloantibodies after subsequent transfusion. Previous studies suggested that CD4 T–cell responses that drive alloantibody formation recognize the same alloantigen engaged by B cells. However, because RBCs express numerous antigens, both internally and externally, it is possible that CD4 T–cell responses directed against intracellular antigens may facilitate subsequent alloimmunization against a surface RBC antigen. Here, we show that B cells can acquire intracellular antigens from RBCs. Using a mouse model of donor RBCs expressing 2 distinct alloantigens, we demonstrate that immune priming to an intracellular antigen, which would not be detected by any currently used RBC compatibility assays, can directly influence alloantibody formation after exposure to a subsequent distinct surface RBC alloantigen. These findings suggest a previously underappreciated mechanism whereby transfusion recipient responders may exhibit an increased rate of alloimmunization because of prior immune priming toward intracellular antigens.

Abstract GS113: Inflammasome Primed Neutrophils Regulate Myocardial Infarction Induced Myelopoiesis

Background: Acute myocardial infarction (MI) results in overzealous production and infiltration of neutrophils to the ischemic heart. This is mediated in-part by granulopoiesis induced by the S100A8/A9-NLRP3-IL-1β signaling axis in injury-exposed neutrophils. Despite the transcriptional upregulation of the NLRP3 inflammasome and associated signaling components in neutrophils, the serum levels of IL-1β, the effector molecule in granulopoiesis was not impacted by MI suggesting that IL-1β is not released systemically. We hypothesize that IL-1β is released locally within the bone marrow (BM) by inflammasome-primed and reverse-migrating neutrophils. Methods: Using a combination of time-dependent parabiosis and flow cytometry techniques, we first characterized the migration patterns of different blood cell types across the parabiotic barrier. We next induced MI in parabiotic mice by permanent ligation of the LAD artery, and examined the ability of injury-exposed neutrophils to permeate the parabiotic barrier and induce granulopoiesis in non-infarcted parabionts. Finally, utilizing multiple neutrophil adoptive and BM transplant studies, we studied the molecular mechanisms that govern reverse migration and retention of the primed neutrophils, IL-1β secretion and granulopoiesis. Cardiac function was assessed by echocardiography. Results: MI promoted greater accumulation of the inflammasome-primed neutrophils in the BM. Introducing a time-dependent parabiotic barrier to the free movement of neutrophils inhibited their ability to stimulate granulopoiesis in the non-infarcted parabionts. Prior priming of the NLRP3 inflammasome is not a prerequisite, but the presence of a functional CXCR4 (C-X-C-motif chemokine receptor 4) on the primed neutrophils and elevated serum S100A8/A9 levels are necessary for homing and retention of the reverse-migrating neutrophils. In the BM, the primed neutrophils secrete IL-1β through formation of gasdermin D pores and, promote granulopoiesis. Pharmacological and/ or genetic strategies aimed at inhibition of neutrophil homing or release of IL-1β in the BM markedly suppressed MI-induced granulopoiesis and, improved cardiac function. Conclusions: Our data reveal a new paradigm of how circulatory cells establish a direct communication between organs by delivering signaling molecules (e.g., IL-1β) directly at the sites of action rather through systemic release. We suggest that this pathway may exist to limit the off-target effects of systemic IL-1β release

A Single-Dose Intramuscular Nanoparticle Vaccine With or Without Prior Intrauterine Priming Triggers Specific Uterine and Colostral Mucosal Antibodies and Systemic Immunity in Gilts but Not Passive Protection for Suckling Piglets.

An effective single-dose vaccine that protects the dam and her suckling offspring against infectious disease would be widely beneficial to livestock animals. We assessed whether a single-dose intramuscular (i.m.) porcine epidemic diarrhea virus (PEDV) vaccine administered to the gilt 30 days post-breeding could generate mucosal and systemic immunity and sufficient colostral and mature milk antibodies to protect suckling piglets against infectious challenge. The vaccine was comprised of polymeric poly-(lactide-co-glycolide) (PGLA)-nanoparticle (NP) encapsulating recombinant PEDV spike protein 1 (PEDVS1) associated with ARC4 and ARC7 adjuvants, a muramyl dipeptide analog and a monophosphoryl lipid A (MPLA) analog, respectively (NP-PEDVS1). To establish whether prior mucosal exposure could augment the i.m. immune response and/or contribute to mucosal tolerance, gilts were immunized with the NP-PEDVS1 vaccine via the intrauterine route at breeding, followed by the i.m. vaccine 30 days later. Archived colostrum from gilts that were challenged with low-dose PEDV plus alum was used as positive reference samples for neutralizing antibodies and passive protection. On day 100 of gestation (70 days post i.m. immunization), both vaccinated groups showed significant PEDVS1-specific IgG and IgA in the serum, as well as in uterine tissue collected on the day of euthanasia. Anti-PEDVS1 colostral IgG antibody titers collected at farrowing were significantly higher relative to the negative control gilts indicating that the NP vaccine was effective in contributing to the colostral antibodies. The PEDVS1-specific colostral IgA and anti-PEDVS1 IgG and IgA antibodies in the mature milk collected 6 days after farrowing were low for both vaccinated groups. No statistical differences between the vaccinated groups were observed, suggesting that the i.u. priming vaccine did not induce mucosal tolerance. Piglets born to either group of vaccinated gilts did not receive sufficient neutralizing antibodies to protect them against infectious PEDV at 3 days of age. In summary, a single i.m. NP vaccine administered 30 days after breeding and a joint i.u./i.m. vaccine administered at breeding and 30 days post-breeding induced significant anti-PEDVS1 immunity in systemic and mucosal sites but did not provide passive protection in suckling offspring.

CDK Inhibition Primes for Anti-PD-L1 Treatment in Triple-Negative Breast Cancer Models.

Simple SummaryThe cyclin E/CDK2 complex may present a promising target axis for the treatment of triple-negative breast cancers (TNBC); however, therapeutically relevant doses of CDK2 inhibitors have been associated with toxicities. Here, we report that the suboptimal dosing of the CDK 2, 7 and 9 inhibitor SNS-032 reduced the viability of TNBC cells and upregulated the checkpoint ligand PD-L1 expression in surviving cancer cells in vitro and in human orthotopic MDA-MB-231 TNBC xenografts grown in immunodeficient mice. Moreover, in immunodeficient, TNBC xenograft-bearing mice engrafted with human immune cells, SNS-032 treatment was associated with the infiltration of CD45+ human immune cells in tumors. In these orthotopic MDA-MB-231 TNBC-bearing mice, suboptimal SNS-032 doses given sequentially ahead of dosing with the anti-PD-L1 antibody avelumab significantly restricted tumor growth compared with monotherapy. These findings suggest that surviving cancer cells following suboptimal CDK inhibitor treatment may be responsive to checkpoint immunotherapy.Triple-negative breast cancers (TNBC) expressing PD-L1 qualify for checkpoint inhibitor immunotherapy. Cyclin E/CDK2 is a potential target axis in TNBC; however, small-molecule drugs at efficacious doses may be associated with toxicity, and treatment alongside immunotherapy requires investigation. We evaluated CDK inhibition at suboptimal levels and its anti-tumor and immunomodulatory effects. Transcriptomic analyses of primary breast cancers confirmed higher cyclin E/CDK2 expression in TNBC compared with non-TNBC. Out of the three CDK2-targeting inhibitors tested, the CDK 2, 7 and 9 inhibitor SNS-032 was the most potent in reducing TNBC cell viability and exerted cytotoxicity against all eight TNBC cell lines evaluated in vitro. Suboptimal SNS-032 dosing elevated cell surface PD-L1 expression in surviving TNBC cells. In mice engrafted with human immune cells and challenged with human MDA-MB-231 TNBC xenografts in mammary fat pads, suboptimal SNS-032 dosing partially restricted tumor growth, enhanced the tumor infiltration of human CD45+ immune cells and elevated cell surface PD-L1 expression in surviving cancer cells. In tumor-bearing mice engrafted with human immune cells, the anti-PD-L1 antibody avelumab, given sequentially following suboptimal SNS-032 dosing, reduced tumor growth compared with SNS-032 alone or with avelumab without prior SNS-032 priming. CDK inhibition at suboptimal doses promotes immune cell recruitment to tumors, PD-L1 expression by surviving TNBC cells and may complement immunotherapy.

Assessment of Rational Antibiotic Use in Pediatric Ward in A Teaching Hospital

Introduction: Antibiotics are a class of natural and synthetic compounds that inhibit the growth of or kill other microorganisms. These are among the most frequently prescribed medications in modern medicine. The use of antibiotics has contributed to the dramatic fall in morbidity from communicable and infectious diseases over the last 50 years globally. Overuse of antibiotic is one of the most important factors for the development and spread of resistance in the hospital, as well as in the community. Methodology: The present study was conducted in pediatric ward of a tertiary care institute. The study population included 250 children. The group consisted of 250 consecutive children admitted in one unit of the pediatric ward and they were analyzed for antibiotic use without any prior priming of the members of the unit regarding rationality of antibiotic use. Results: Age and sex wise distribution of 250 children included in the study. Of these 166(57%) were males and 84 (43%) were females. 60 received antibiotics for respiratory tract infections, this being the commonest disease for which antibiotics were received. 55 (44%) children received antibiotics in rational way. Conclusion: From this study it is clear that there is lack of proper rational antibiotic usage by unit doctors. To conclude it can be stated that inappropriate use of antibiotics is increasing and is responsible for rapidly developing antimicrobial resistance. To increase rational use of antibiotics the members of the unit (treating physicians) should be motivated regarding correct uses of antibiotics and hazards of their inappropriate use. A proper workshop should be arranged for this.

Autoimmune response and myocarditis associated with COVID-19 vaccination

Recently, there have been numerous reports of suspected myocarditis cases following vaccination against Corona Virus Disease 2019 (COVID-19), although the exact mechanism behind it is still unclear [1,2]. Shaw KE and colleagues reported four cases of acute myocarditis that were temporally related to the receiving of COVID-19 vaccine based on their Cardiac Magnetic Resonance (CMR) imaging findings [3]. Among them, two patients with previous COVID-19 infection developed symptoms after the first dose, while the other two patients with no previous COVID-19 infection developed symptoms after the second dose. Therefore, the authors argued that there might be a potential immune-boosting mechanism flowing prior immune exposure or priming.

Monosodium urate crystals regulate a unique JNK-dependent macrophage metabolic and inflammatory response.

Monosodium urate crystals (MSUc) induce inflammation invivo without prior priming, raising the possibility of an initial cell-autonomous phase. Here, using genome-wide transcriptomic analysis and biochemical assays, we demonstrate that MSUc alone induce a metabolic-inflammatory transcriptional program in non-primed human and murine macrophages that is markedly distinct to that induced by LPS. Genes uniquely upregulated in response to MSUc belong to lipid and amino acid metabolism, glycolysis, and SLC transporters. This upregulation leads to a metabolic rewiring in sera from individuals and mice with acute gouty arthritis. Mechanistically, the initiating inflammatory-metabolic changes in acute gout flares are regulated through a persistent expression and increased binding of JUN to the promoter of target genes through JNK signaling-but not P38-in a process that is different than after LPS stimulation and independent of inflammasome activation. Finally, pharmacological JNK inhibition limits MSUc-induced inflammation in animal models of acute gouty inflammation.

A Novel Hyperactive Nud1 Mitotic Exit Network Scaffold Causes Spindle Position Checkpoint Bypass in Budding Yeast.

Mitotic exit is a critical cell cycle transition that requires the careful coordination of nuclear positioning and cyclin B destruction in budding yeast for the maintenance of genome integrity. The mitotic exit network (MEN) is a Ras-like signal transduction pathway that promotes this process during anaphase. A crucial step in MEN activation occurs when the Dbf2-Mob1 protein kinase complex associates with the Nud1 scaffold protein at the yeast spindle pole bodies (SPBs; centrosome equivalents) and thereby becomes activated. This requires prior priming phosphorylation of Nud1 by Cdc15 at SPBs. Cdc15 activation, in turn, requires both the Tem1 GTPase and the Polo kinase Cdc5, but how Cdc15 associates with SPBs is not well understood. We have identified a hyperactive allele of NUD1, nud1-A308T, that recruits Cdc15 to SPBs in all stages of the cell cycle in a CDC5-independent manner. This allele leads to early recruitment of Dbf2-Mob1 during metaphase and requires known Cdc15 phospho-sites on Nud1. The presence of nud1-A308T leads to loss of coupling between nuclear position and mitotic exit in cells with mispositioned spindles. Our findings highlight the importance of scaffold regulation in signaling pathways to prevent improper activation.

Advancement of chimeric antigen receptor-natural killer cells targeting hepatocellular carcinoma.

With the advance of genome engineering technology, chimeric antigen receptors (CARs)-based immunotherapy has become an emerging therapeutic strategy for tumors. Although initially designed for T cells in tumor immunotherapy, CARs have been exploited to modify the function of natural killer (NK) cells against a variety of tumors, including hepatocellular carcinoma (HCC). CAR-NK cells have the potential to sufficiently kill tumor antigen-expressing HCC cells, independent of major histocompatibility complex matching or prior priming. In this review, we summarize the recent advances in genetic engineering of CAR-NK cells against HCC and discuss the current challenges and prospects of CAR-NK cells as a revolutionary cellular immunotherapy against HCC.

Impact of Educational Intervention on Rational Antibiotic Use In Pediatric Ward

Introduction: Antibiotics are a class of natural and synthetic compounds that inhibit the growth of or kill other microorganisms. Overuse of antibiotic is one of the most important factors for the development and spread of resistance in the hospital, as well as in the community. Present study was designed to describe antibiotic use in children and to assess the impact of an educational intervention on antibiotic prescription. Methods: The present study was conducted in pediatric ward of a tertiary care institute. The study population included two groups of children – each having 250 children. The first group consisted of 250 consecutive children admitted in one unit of the pediatric ward and they were analyzed for antibiotic use without any prior priming of the members of the unit regarding rationality of antibiotic use. This was followed by an intervention in the form of a 3hour workshop for all members of the unit. The second group included another 250 children consecutively admitted in the wards after this intervention. Results: In Pre intervention group 55 (44%) children received antibiotics in rational way, while in Post intervention group 90(60.40%) children received antibiotic in rational way. One hundred and thirty-six children (49.4%) received antibiotics for respiratory tract infections, this being the commonest disease for which antibiotics were received. As seen in the table, unindicated use of antibiotic was significantly reduced in Post intervention group (p = 0.03). Conclusion: The present study included 500 children. There were 285 (57%) males and 215 (43%) females. Amongst these 500 patients, 178 (35.6%) were below 1 year of age. This group had the highest number of patients. In the Pre intervention group, 55 (44%) children received antibiotics rationally, while in Post intervention group 90(60.40%) children received antibiotic rationally.

Trained Immunity in Anopheles gambiae: Antibacterial Immunity Is Enhanced by Priming via Sugar Meal Supplemented With a Single Gut Symbiotic Bacterial Strain.

Mosquitoes have evolved an effective innate immune system. The mosquito gut accommodates various microbes, which play a crucial role in shaping the mosquito immune system during evolution. The resident bacteria in the gut microbiota play an essential role in priming basal immunity. In this study, we show that antibacterial immunity in Anopheles gambiae can be enhanced by priming via a sugar meal supplemented with bacteria. Serratia fonticola S1 and Enterobacter sp. Ag1 are gut bacteria in mosquitoes. The intrathoracic injection of the two bacteria can result in an acute hemocoelic infection in the naïve mosquitoes with mortality of ∼40% at 24 h post-infection. However, the Enterobacter orSerratia primed mosquitoes showed a better 24 h survival upon the bacterial challenge. The priming confers the protection with a certain degree of specificity, the Enterobacter primed mosquitoes had a better survival upon the Enterobacter but not Serratia challenge, and the Serratia primed mosquitoes had a better survival upon the Serratia but not Enterobacter challenge. To understand the priming-mediated immune enhancement, the transcriptomes were characterized in the mosquitoes of priming as well as priming plus challenges. The RNA-seq was conducted to profile 10 transcriptomes including three samples of priming conditions (native microbiota, Serratia priming, and Enterobacter priming), six samples of priming plus challenges with the two bacteria, and one sample of injury control. The three priming regimes resulted in distinctive transcriptomic profiles with about 60% of genes affected by both bacteria. Upon challenges, different primed mosquitoes displayed different transcriptomic patterns in response to different bacteria. When a primed cohort was challenged with a heterogenous bacterium, more responsive genes were observed than when challenged with a homogenous bacterium. As expected, many canonical immune genes were responsive to the priming and challenge, but much more non-immune genes with various functions were also responsive in the contexts, which implies that the prior priming triggers a delicately coordinated systemic regulation that results in an enhanced immunity against the subsequent challenge. Besides the participation of typical immune pathways, the transcriptome data suggest the involvement of lysosome and metabolism in the context. Overall, this study demonstrated a trained immunity via priming with bacteria in diet.

Assessing the Association of Mitochondrial Function and Inflammasome Activation in Murine Macrophages Exposed to Select Mitotoxic Tri-Organotin Compounds.

Background:Mitochondrial function is implicated as a target of environmental toxicants and found in disease or injury models, contributing to acute and chronic inflammation. One mechanism by which mitochondrial damage can propagate inflammation is via activation of the nucleotide-binding oligomerization domain (NOD)-like receptor (NLR) family, pyrin domain-containing receptor (NLRP)3 inflammasome, a protein complex that processes mature interleukin . plays an important role in the innate immune response and dysregulation is associated with autoinflammatory disorders.Objective:The objective was to evaluate whether mitochondrial toxicants recruit inflammasome activation and processing.Method:Murine macrophages (RAW 264.7) exposed to tri-organotins (triethyltin bromide (TETBr), trimethyltin hydroxide (TMTOH), triphenyltin hydroxide (TPTOH), bis(tributyltin)oxide) [Bis(TBT)Ox] were examined for pro-inflammatory cytokine induction. TMTOH and TETBr were examined in RAW 264.7 and bone marrow-derived macrophages for mitochondrial bioenergetics, reactive oxygen species (ROS) production, and inflammasome activation via visualization of aggregate formation, caspase-1 flow cytometry, enzyme-linked immunosorbent assay and Western blots, and microRNA (miRNA) and mRNA arrays.Results:TETBr and TMTOH induced inflammasome aggregate formation and release in lipopolysaccharide (LPS)-primed macrophages. Mitochondrial bioenergetics and mitochondrial ROS were suppressed. Il1a and Il1b induction with LPS or challenge was diminished. Differential miRNA and mRNA profiles were observed. Lower miR-151-3p targeted cyclic adenosine monophosphate (cAMP)-mediated and AMP-activated protein kinase signaling pathways; higher miR-6909-5p, miR-7044-5p, and miR-7686-5p targeted Wnt beta-catenin signaling, retinoic acid receptor activation, apoptosis, signal transducer and activator of transcription 3, IL-22, IL-12, and IL-10 signaling. Functional enrichment analysis identified apoptosis and cell survival canonical pathways.Conclusion:Select mitotoxic tri-organotins disrupted murine macrophage transcriptional response to LPS, yet triggered inflammasome activation. The differential response pattern suggested unique functional changes in the inflammatory response that may translate to suppressed host defense or prolong inflammation. We posit a framework to examine immune cell effects of environmental mitotoxic compounds for adverse health outcomes. https://doi.org/10.1289/EHP8314

Cytokines Orchestrating the Natural Killer-Myeloid Cell Crosstalk in the Tumor Microenvironment: Implications for Natural Killer Cell-Based Cancer Immunotherapy.

Natural killer (NK) cells are endowed with germline-encoded receptors that enable them to detect and kill malignant cells without prior priming. Over the years, overwhelming evidence has identified an essential role for NK cells in tumor immune surveillance. More recently, clinical trials have also highlighted their potential in therapeutic settings. Yet, data show that NK cells can be dysregulated within the tumor microenvironment (TME), rendering them ineffective in eradicating the cancer cells. This has been attributed to immune suppressive factors, including the tumor cells per se, stromal cells, regulatory T cells, and soluble factors such as reactive oxygen species and cytokines. However, the TME also hosts myeloid cells such as dendritic cells, macrophages, neutrophils, and myeloid-derived suppressor cells that influence NK cell function. Although the NK-myeloid cell crosstalk can promote anti-tumor responses, myeloid cells in the TME often dysregulate NK cells via direct cell-to-cell interactions down-regulating key NK cell receptors, depletion of nutrients and growth factors required for NK cell growth, and secretion of metabolites, chemokines and cytokines that ultimately alter NK cell trafficking, survival, and cytotoxicity. Here, we review the complex functions of myeloid-derived cytokines in both supporting and suppressing NK cells in the TME and how NK cell-derived cytokines can influence myeloid subsets. We discuss challenges related to these interactions in unleashing the full potential of endogenous and adoptively infused NK cells. Finally, we present strategies aiming at improving NK cell-based cancer immunotherapies via pathways that are involved in the NK-myeloid cell crosstalk in the TME.

The different root apex zones contribute to drought priming induced tolerance to a reoccurring drought stress in wheat

Drought priming is a promising approach to improve tolerance to further drought in wheat. The root apex plays important roles in drought however, its contribution to drought priming remains unknown. To provide mechanistic insights into this process, the transcriptomes and proteomes at three different zones along the root axis under drought stress were analyzed. Physiological assessment of root growth indicated that priming augmented roots growth in response to drought and also the levels of protective proline and glycine betaine. Scanning across the proximal to the distal zones of the root apex indicated increases the transcription of genes involved in primary and secondary metabolism. Conversely, genes related to translation, transcription, folding, sorting and degradation, replication and repair were increased in the apex compared to the proximal zone. A single drought episode suppressed their expression but prior drought priming served to maintain expression with recurrent drought stress. The differentially primed responses genes were mainly involved in the pathways related to plant hormone signaling, stress defense and cell wall modification. The prediction of regulatory hubs using Cytoscape implicated signaling components such as the ABA receptor PYL4 as influencing antioxidant status and the cell cycle. Based our integrative transcriptomic-proteomic assessments we present a model for drought priming protected plant hormone signaling transduction pathways to drive the cell cycle and cell wall loosening to confer beneficial effects on roots to counter the effects of drought. This model provides a theoretical basis for improvement of drought tolerance in wheat, via an increased understanding of drought priming induced drought tolerance.

Infrapatellar fat pad-derived mesenchymal stem cell-based spheroids enhance their therapeutic efficacy to reverse synovitis and fat pad fibrosis

BackgroundTo investigate the in vitro and in vivo anti-inflammatory/anti-fibrotic capacity of IFP-MSC manufactured as 3D spheroids. Our hypothesis is that IFP-MSC do not require prior cell priming to acquire a robust immunomodulatory phenotype in vitro in order to efficiently reverse synovitis and IFP fibrosis, and secondarily delay articular cartilage damage in vivo.MethodsHuman IFP-MSC immunophenotype, tripotentiality, and transcriptional profiles were assessed in 3D settings. Multiplex secretomes were assessed in IFP-MSC spheroids [Crude (non-immunoselected), CD146+ or CD146− immunoselected cells] and compared with 2D cultures with and without prior inflammatory/fibrotic cell priming. Functionally, IFP-MSC spheroids were assessed for their immunopotency on human PBMC proliferation and their effect on stimulated synoviocytes with inflammation and fibrotic cues. The anti-inflammatory and anti-fibrotic spheroid properties were further evaluated in vivo in a rat model of acute synovitis/fat pad fibrosis.ResultsSpheroids enhanced IFP-MSC phenotypic, transcriptional, and secretory immunomodulatory profiles compared to 2D cultures. Further, CD146+ IFP-MSC spheroids showed enhanced secretory and transcriptional profiles; however, these attributes were not reflected in a superior capacity to suppress activated PBMC. This suggests that 3D culturing settings are sufficient to induce an enhanced immunomodulatory phenotype in both Crude and CD146-immunoselected IFP-MSC. Crude IFP-MSC spheroids modulated the molecular response of synoviocytes previously exposed to inflammatory cues. Therapeutically, IFP-MSC spheroids retained substance P degradation potential in vivo, while effectively inducing resolution of inflammation/fibrosis of the synovium and fat pad. Furthermore, their presence resulted in arrest of articular cartilage degradation in a rat model of progressive synovitis and fat pad fibrosis.Conclusions3D spheroids confer IFP-MSC a reproducible and enhanced immunomodulatory effect in vitro and in vivo, circumventing the requirement of non-compliant cell priming or selection before administration and thereby streamlining cell products manufacturing protocols.