Sterilizing Activity Research Articles

Nlrp3 Inflammasome Activation in Bone Marrow-Residing Innate Immunity Cells Governs Circadian Changes in the Number of Circulating Hematopoietic Stem/Progenitor Cells in Peripheral Blood

Background. The number of hematopoietic stem/progenitor cells (HSPCs) in peripheral blood (PB) undergoes a circadian oscillation, with the peak occurring in the early morning hours and the nadir at night, and, as nicely demonstrated, this peak has been attributed to the enhanced tonus of the vegetative nervous system in the early morning hours (Nature 2008, 452, 442-447). Moreover, our group has demonstrated that release of HSPCs from bone marrow (BM) into PB is regulated during stress- or pharmacology-induced mobilization by activation of three ancient serum proteolytic cascades, the complement cascade (ComC), the coagulation cascade (CoaC), and the fibrynolytic cascade (FibC) (Stem Cell Rev. 2018; 14:677-685). Since it is known that the ComC, CoaC, and FibC show circadian activation at late night/early morning hours due to deep sleep hypoxia, regulation of the circadian oscillation of HSPC numbers in PB becomes more complex. Moreover, as we recently demonstrated, an important role in egress of HSPCs from BM into PB is played by purinergic signaling involving adenosine triphosphate (ATP) released from cells, which, as signaling mediators in the extracellular space, activate the Nlrp3 inflammasome in hematopoietic cells (Leukemia 2019; 33:815-825). Activation of the Nlrp3 inflammasome induces a state of sterile inflammation in the BM microenvironment and activates the ComC, CoaC, and FibC. Hypothesis. Since Nlrp3 inflammasome activation regulates egress of HSPCs from BM into PB by inducing BM sterile inflammation and activation of the ComC, CoaC, and FibC undergoes circadian activation, we became interested in whether Nlrp3 protein complex orchestrates circadian changes in the number of HSPCs circulating in PB.Materials and Methods. To address this important question, we studied the circadian oscillation in the number of circulating HSPCs in mice. Mice were accustomed to alternating periods of 12 hours light and 12 hours darkness. Light was turned on at 6 AM (ZT0), and the numbers of circulating white blood cells (WBCs), Sca-1+kit+Lin- HSCs, Sca-1+Lin-CD45+ HSCs, clonogenic CFU-GM progenitors, and non-hematopoietic Sca-1+Lin-CD45- cells (VSELs) were measured at 7 AM (ZT1), 11 AM (ZT5), 7 PM (ZT13), and 3 AM (ZT21). At the same time points, we evaluated expression of the Nlrp3 inflammasome at the mRNA level; Nlrp3 activation by measuring Nlrp3 inflammasome activation markers, such as interleukin-1beta, interleukin-18, and Hmgb1, at the mRNA and protein levels; ComC activation (by C5a ELISA); CoaC activation (by thrombin/antithrombin ELISA); and FibC activation (by plasmin/antiplasmin complex ELISA). To confirm the role of the Nlrp3 inflammasome in the circadian oscillation of HSPCs released into PB, we inhibited its activity by employing the specific small-molecule inhibitor MCC950. Results. We observed circadian changes in the expression and activation of the Nlrp3 inflammasome, with a peak in the early morning hours at ZT1 that preceded the peak in the number of circulating HSPCs at ZT5. This increase in activation of the Nlrp3 inflammasome and the number of circulating cells in WT animals was preceded by an increase in C5a concentration in PB at ZT1 as well as activation of the CoaC and FibC at ZT21. As expected, inhibition of the Nlrp3 inflammasome by MCC950 inhibited circadian oscillation of circulating HSPCs in PB. Conclusions. Our study confirms circadian activation of the Nlrp3 inflammsome due to the ComC, CoaC, and FibC in mice at late-night/early-morning hours preceding the release of HSPCs from BM into PB. The fact that we observed significant decrease in circadian changes in the number of circulating cells in PB in mice exposed to an Nlrp3 inflammasome inhibitor confirms its pivotal role in executing circadian release of HSPCs from BM into PB. Moreover, the fact that mice exposed to an Nlrp3 inhibitor show defective activation of the ComC and normal activation of the the CoaC and FibC indicates that, of the ancient proteolytic cascades tested, the ComC is the major player regulating Nlrp3 inflammasome-dependent circadian egress of HSPCs. Disclosures No relevant conflicts of interest to declare.

Sterile activation of invariant natural killer T cells by ER-stressed antigen-presenting cells

Invariant NKT (iNKT) cells have the unique ability to shape immunity during antitumor immune responses and other forms of sterile and nonsterile inflammation. Recent studies have highlighted a variety of classes of endogenous and pathogen-derived lipid antigens that can trigger iNKT cell activation under sterile and nonsterile conditions. However, the context and mechanisms that drive the presentation of self-lipid antigens in sterile inflammation remain unclear. Here we report that endoplasmic reticulum (ER)-stressed myeloid cells, via signaling events modulated by the protein kinase RNA-like ER kinase (PERK) pathway, increase CD1d-mediated presentation of immunogenic endogenous lipid species, which results in enhanced iNKT cell activation both in vitro and in vivo. In addition, we demonstrate that actin cytoskeletal reorganization during ER stress results in an altered distribution of CD1d on the cell surface, which contributes to enhanced iNKT cell activation. These results define a previously unidentified mechanism that controls iNKT cell activation during sterile inflammation.

Two Cases of Palmoplantar Pustulosis Treated with Oral Rinse Using Ozone Nano-bubble Water in Addition to Conventional Topical Therapy

Background: Palmoplantar pustulosis (PPP) is a unique, chronic inflammatory skin disease characterized by pustules and erythematous lesions on specific sites, such as the palms and soles; however, the etiology and treatment of the condition are not clearly established. Recently, oral bacteria have been proposed as the possible causative factor of PPP. Ozone nano-bubble (ONB) water, treated with nano-sized ozone gas particles, shows strong sterilizing activity and has been attracting attention as a new treatment method not only for periodontal disease but also for PPP. Case history: This report presents the cases of two female patients with PPP who underwent treatment using a mixture of topical vitamin D and steroid ointments for over 6 months. However, the skin lesions did not show much improvement. Owing to the antimicrobial activity of ozone, they voluntarily agreed to rinsing their oral cavities with ozone water, particularly the periodontal areas, for a couple of minutes with 10–20 ml of ONB water, each night, in conjunction with the previous topical treatment.Results: Their skin lesions resolved completely within several months of addition of ONB water oral rinse to the routine topical treatment. Conclusion: The results support the usefulness of ONB water as an effective therapeutic means for the resolution of skin lesions in patients with PPP.

Fabrication of Cu2O-Ag nanocomposites with enhanced durability and bactericidal activity

Hybrid Cuprous oxide-silver (Cu2O-Ag) have attracted tremendous attention due to their various applications in photocatalysis, surface enhanced Raman scattering (SERS), and optical features. Here we expanded the application to exhibit excellent chemical stability and synergistic bactericidal. We prepared Cu2O-Ag heterostructure through thermally decomposing Ag-acetate to deposit Ag nanoparticles (Ag NPs) onto Cu2O surface. Cu2O-Ag heterostructure bears exceptional stability while exposing to oxygen, water, and light, owing to the physical coating of Ag NPs and transferring the electrons and holes inside the Cu2O to the surface through a Schottky barrier to prevent photocorrosion. The deposition of Ag NPs also improved the intensity and time of oxidative stress reaction of Cu2O, proved by reactive oxygen species (ROS) examination. Ag NPs distributed on the surface of Cu2O particles formed a large of ion release channel, resulting in excellent sustained release of copper ions. Density functional theory (DFT) calculations were used to investigate the mechanism of photocatalysis and ROS generation. The constructed Cu2O-Ag heterostructure exhibit highly long-term sterilization activity against Staphylococcus aureus (S. aureus) and Pseudomonas aeruginosa (P. aeruginosa) which were maintained around 70% and 80% and were increased by 40% and 50% compared with free Cu2O after being immersed in phosphate buffer saline (PBS) solutions within 14 days.

Inhibition of snake venom induced sterile inflammation and PLA2 activity by Titanium dioxide Nanoparticles in experimental animals

Sterile inflammation (SI) is an essential process in response to snakebite and injury. The venom induced pathophysiological response to sterile inflammation results into many harmful and deleterious effects that ultimately leads to death. The available treatment for snakebite is antiserum which does not provide enough protection against venom-induced pathophysiological changes like haemorrhage, necrosis, nephrotoxicity and often develop hypersensitive reactions. In order to overcome these hindrances, scientists around the globe are searching for an alternative therapy to provide better treatment to the snake envenomation patients. In the present study TiO2 (Titanium dioxide)-NPs (Nanoparticles) has been assessed for antisnake venom activity and its potential to be used as an antidote. In this study, the synthesis of TiO2-NPs arrays has been demonstrated on p-type Silicon Si < 100 > substrate (∼30 ohm-cm) and the surface topography has been detected by Field-emission scanning electron microscopy (FESEM). The TiO2-NPs successfully neutralized the Daboia russelii venom (DRV) and Naja kaouthia venom (NKV)-induced lethal activity. Viper venom induced haemorrhagic, coagulant and anticoagulant activities were effectively neutralized both in in-vitro and in vivo studies. The cobra and viper venoms-induced sterile inflammatory molecules (IL-6, HMGB1, HSP70, HSP90, S100B and vWF) were effectively neutralised by the TiO2-NPs in experimental animals.

Optimization of alkaline protease production by rational deletion of sporulation related genes in Bacillus licheniformis

BackgroundOur laboratory has constructed a Bacillus licheniformis strain that secretes alkaline protease (AprE) with excellent enzymatic properties. B. licheniformis is generally regarded as safe and has a high industrial exoenzyme secretion capacity, but the host retains some undomesticated characteristic that increase its competitiveness and survival, such as spore-formation, which increases the requirements and difficulties in industrial operations (e.g. sterilization and enzyme activity control). Furthermore, the influence of sporulation on alkaline protease production in B. licheniformis has not been elucidated in detail.ResultA series of asporogenic variants of the parent strain were constructed by individually knocking out the master regulator genes (spo0A, sigF and sigE) involved in sporulation. Most of the variants formed abortively disporic cells characterized by asymmetric septa at the poles and unable to survive incubation at 75 °C for 10 min. Two of them (ΔsigF and ΔsigE) exhibited superior characteristics in protease production, especially improving the expression of the aprE gene. Under the currently used fermentation conditions, the vegetative production phase of ΔsigF can be prolonged to 72 h, and the highest protease production of ΔsigF reached 29,494 ± 1053 U/mL, which was about 19.7% higher than that of the wild-type strain.ConclusionWe first constructed three key sporulation-deficient strain to investigate the effect of sporulation on alkaline protease synthesis. The sigF mutant retained important industrial properties such as facilitating the sterilization process, a prolonged stable phase of enzyme production and slower decreasing trend, which will be superior in energy conservation, simpler operations and target product controlling effect. In summary, the work provides a useful industrial host with preferable characteristics and a novel strategy to enhance the production of protease.

In vivo imaging of sterile microglial activation in rat brain after disrupting the blood-brain barrier with pulsed focused ultrasound: [18F]DPA-714 PET study

BackgroundMagnetic resonance imaging (MRI)-guided pulsed focused ultrasound combined with the infusion of microbubbles (pFUS+MB) induces transient blood-brain barrier opening (BBBO) in targeted regions. pFUS+MB, through the facilitation of neurotherapeutics’ delivery, has been advocated as an adjuvant treatment for neurodegenerative diseases and malignancies. Sterile neuroinflammation has been recently described following pFUS+MB BBBO. In this study, we used PET imaging with [18F]-DPA714, a biomarker of translocator protein (TSPO), to assess for neuroinflammatory changes following single and multiple pFUS+MB sessions.MethodsThree groups of Sprague-Dawley female rats received MRI-guided pFUS+MB (Optison™; 5–8 × 107 MB/rat) treatments to the left frontal cortex and right hippocampus. Group A rats were sonicated once. Group B rats were sonicated twice and group C rats were sonicated six times on weekly basis. Passive cavitation detection feedback (PCD) controlled the peak negative pressure during sonication. We performed T1-weighted scans immediately after sonication to assess efficiency of BBBO and T2*-weighted scans to evaluate for hypointense voxels. [18F]DPA-714 PET/CT scans were acquired after the BBB had closed, 24 h after sonication in group A and within an average of 10 days from the last sonication in groups B and C. Ratios of T1 enhancement, T2* values, and [18F]DPA-714 percent injected dose/cc (%ID/cc) values in the targeted areas to the contralateral brain were calculated. Histological assessment for microglial activation/astrocytosis was performed.ResultsIn all groups, [18F]DPA-714 binding was increased at the sonicated compared to non-sonicated brain (%ID/cc ratios > 1). Immunohistopathology showed increased staining for microglial and astrocytic markers in the sonicated frontal cortex compared to contralateral brain and to a lesser extent in the sonicated hippocampus. Using MRI, we documented BBB disruption immediately after sonication with resolution of BBBO 24 h later. We found more T2* hypointense voxels with increasing number of sonications. In a longitudinal group of animals imaged after two and after six sonications, there was no cumulative increase of neuroinflammation on PET.ConclusionUsing [18F]DPA-714 PET, we documented in vivo neuroinflammatory changes in association with pFUS+MB. Our protocol (utilizing PCD feedback to minimize damage) resulted in neuroinflammation visualized 24 h post one sonication. Our findings were supported by immunohistochemistry showing microglial activation and astrocytosis. Experimental sonication parameters intended for BBB disruption should be evaluated for neuroinflammatory sequelae prior to implementation in clinical trials.

Constitutive TNF-α signaling in neonates is essential for the development of tissue-resident leukocyte profiles at barrier sites.

Newborn infants have a high disposition to develop systemic inflammatory response syndromes (SIRSs) upon inflammatory or infectious challenges. Moreover, there is a considerable trafficking of hematopoietic cells to tissues already under noninflammatory conditions. These age-specific characteristics suggest a hitherto unappreciated crucial role of the vascular endothelium during the neonatal period. Here, we demonstrate that healthy neonates showed already strong endothelial baseline activation, which was mediated by a constitutively increased production of TNF-α. In mice, pharmacological inhibition of TNF-α directly after birth prevented subsequent fatal SIRS but completely abrogated the recruitment of leukocytes to sites of infection. Importantly, in healthy neonates, blocking TNF-α at birth disrupted the physiologic leukocyte trafficking, which resulted in persistently altered leukocyte profiles at barrier sites. Collectively, these data suggest that constitutive TNF-α-mediated sterile endothelial activation in newborn infants contributes to the increased risk of developing SIRS but is needed to ensure the postnatal recruitment of leukocytes to organs and interfaces.-Bickes, M. S., Pirr, S., Heinemann, A. S., Fehlhaber, B., Halle, S., Völlger, L., Willers, M., Richter, M., Böhne, C., Albrecht, M., Langer, M., Pfeifer, S., Jonigk, D., Vieten, G., Ure, B., von Kaisenberg, C., Förster, R., von Köckritz-Blickwede, M., Hansen, G., Viemann, D. Constitutive TNF-α signaling in neonates is essential for the development of tissue-resident leukocyte profiles at barrier sites.

Evolution of rifampicin treatment for tuberculosis

Rifampicin was discovered in 1965 and remains one of the most important drugs in tuberculosis treatment that is valued for its sterilizing activity and ability to shorten treatment. Antimicrobial activity of rifampicin was initially proved in vitro; subsequently numerous in vivo studies showed the bactericidal properties and dose-dependent effect of rifampicin. Rifampicin was first during the late 1960s to treat patients suffering from chronic drug-resistant pulmonary TB. Decades later, rifampicin continues to be studied with particular emphasis on whether higher doses could shorten the duration of treatment without increasing relapse or having adverse effects. Lesion-specific drug penetration and pharmacokinetics of rifampicin are improving our understanding of effective concentration while potentially refining drug regimen designs. Another prospective aspect of high-dose rifampicin is its potential use in treating discrepant mutation thereby eliminating the need for MDR treatment. To date, several clinical trials have shown the safety, efficacy, and tolerability of high-dose rifampicin. Currently, high-dose rifampicin has been used successfully in a routine clinical setting for the treatment of high-risk patients. However, the WHO and other relevant policy makers have not committed to implementing a controlled rollout thereof. This review describes the course that rifampicin has travelled to the present-day exploration of high-dose rifampicin treatment.

Guideline Implementation: Sterilization.

Ensuring that reusable surgical instruments and medical devices have been sterilized is an important factor in preventing surgical site infections. The sterilization method for a particular device is based on the device design, material, packaging, compatibility with the sterilant, load limitations, and safety requirements. Perioperative team members must review the manufacturer's instructions for cleaning, packaging, and sterilizing the device to determine the correct sterilization process. The AORN "Guideline for sterilization" provides guidance for processing reusable medical devices for use in perioperative and procedural settings. This article elaborates on key takeaways from the guideline, including processing a device based on the intended use of the item, protecting sterile items during transport, reprocessing by immediate-use steam sterilization when certain conditions can be met, and educating personnel who perform sterile processing activities. Perioperative RNs should review the complete guideline for additional information and for guidance when writing and updating policies and procedures.

PLASMACHEMICAL GENERATION OF NITRIC OXIDES IN AIR PLASMAS FOR MEDICAL APPLICATIONS

Nitric oxide is well known as a poly functional regulator of different physiological processes in human body and therefore could be used for therapeutic purposes in different medical applications. In this review mechanism of nitric oxide generation in gas plasma and biological mechanisms of nitric oxide containing plasma gas treatment of tissues are described. In addition to nitric oxide the other biologically active species like hydrogen peroxide and nitrogen dioxide are formed in air plasma at atmospheric pressure. Synergetic action of molecules NO/H2O2 и NO/O2, generated in plasma gas results to manifold increase of sterilization activity of plasma mixture to bacteria and at the same time the toxicity of these species to living cells is low. Plasma gas exhibits therapeutic action on human tissues. On the one hand plasma gas contains molecules Н2О2, NO and NO2 acting as a antimicrobial agents and promoting sterilization, on the other hand the presence of significant concentration of NO leads to effective regeneration of damaged tissue. These processes are complementary and finally result to effective healing of diabetic trophic ulcer and other diseases in the oncology, ophthalmology, dentistry, purulent surgery, battlefield surgery and so on. Peculiarities of different discharges which could be used for generation of nitric oxide, like arc discharge, gliding arc discharge, microwave discharge, radiofrequency and pulsed discharges are discussed. The production of nitric oxide depending on type of discharge and plasma parameters like discharge power, gas flow rate and electrode configuration are analyzed. The efficacy of nitric oxide generation in different discharges is compared.

Death Receptor Interactions With the Mitochondrial Cell Death Pathway During Immune Cell-, Drug- and Toxin-Induced Liver Damage.

Due to its extensive vascularization and physiological function as a filter and storage organ, the liver is constantly exposed to infectious and tumorigenic threat, as well as damaging actions of xenobiotics. Detoxification reactions are essential for the excretion of harmful substances, but harbor also the risk of “side effects” leading to dangerous metabolites of otherwise harmless substances, a well known effect during paracetamol overdose. These drugs can have detrimental effects, which often involves the induction of sterile inflammation and activation of the immune system. Therefore, the role of certain immune cells and their effector molecules in the regulation of drug-induced liver damage are of special interest. Hepatocytes are type II cells, and death receptor (DR)-induced cell death (CD) requires amplification via the mitochondrial pathway. However, this important role of the mitochondria and associated CD-regulating signaling complexes appears to be not restricted to DR signaling, but to extend to drug-induced activation of mitochondrial CD pathways. We here discuss the role of members of the TNF family, with a focus on TRAIL, and their interactions with the Bcl-2 family in the crosstalk between the extrinsic and intrinsic CD pathway during xenobiotic-induced liver damage.

Monocyte-Derived Interleukin-1β As the Driver of S100A12-Induced Sterile Inflammatory Activation of Human Coronary Artery Endothelial Cells: Implications for the Pathogenesis of Kawasaki Disease.

Kawasaki disease (KD) is an acute vasculitis of childhood, predominantly affecting the coronary arteries. S100A12, a granulocyte-derived agonist of both the receptor for advanced glycation end products (RAGE) and Toll-like receptor 4 (TLR-4), is strongly up-regulated in KD. This study was undertaken to investigate the potential contributions of S100A12 to the pathogenesis of KD. Serum samples from patients with KD (n = 30) at different stages pre- and post-intravenous immunoglobulin (IVIG) treatment were analyzed for the expression of S100A12, cytokines, chemokines, and soluble markers of endothelial cell activation. Primary human coronary artery endothelial cells (HCAECs) were analyzed for responsiveness to direct stimulation with S100A12 or lipopolysaccharide (LPS), as assessed by real-time quantitative reverse transcription-polymerase chain reaction analysis of cytokine and endothelial cell adhesion molecule messenger RNA expression. Alternatively, HCAECs were cultured in conditioned medium obtained from primary human monocytes that were stimulated with LPS or S100A12 in the absence or presence of IVIG or cytokine antagonists. In the serum of patients with KD, pretreatment S100A12 levels were associated with soluble vascular cell adhesion molecule 1 titers in the course of IVIG therapy (rs = -0.6, P = 0.0003). Yet, HCAECs were not responsive to direct S100A12 stimulation, despite the presence of appropriate receptors (RAGE, TLR-4). HCAECs did, however, respond to supernatants obtained from S100A12-stimulated primary human monocytes, as evidenced by the gene expression of inflammatory cytokines and adhesion molecules. This response was strictly dependent on interleukin-1β (IL-1β) signaling (P < 0.001). In its role as a highly expressed mediator of sterile inflammation in KD, S100A12 appears to activate HCAECs in an IL-1β-dependent manner. These data provide new mechanistic insights into the contributions of S100A12 and IL-1β to disease pathogenesis, and may therefore support current IL-1-targeting studies in the treatment of patients with KD.

A Jew for Roman Tastes: The Parting of the Ways in Justin Martyr's Dialogue with Trypho from a Post-Colonial Perspective

This article offers a new and distinctly Roman perspective on Trypho the Jew in Justin's Dialogue. While generations of scholars have debated whether Trypho was a historical Jew, who engaged in a real dialogue with Justin, or rather a literary construct invented by the author for Christian readers, I apply post-colonial theory and argue that Justin constructed Trypho for a Roman audience, whether "pagan" or Christian. Trypho highlights by his Otherness the Roman character of Christianity and thus helps to position the new religion firmly at the center of the empire. The article offers a close reading of Trypho's prominent features, namely 1) his circumcision, which is analyzed in the context of Antonius Pius's new legislation on this Jewish rite following the Bar Kokhba Revolt, 2) Trypho's insistence on ethnicity and exclusivism, which is evaluated in view of Roman stereotypes concerning Jewish xenophobia, and 3) his recourse to detailed Bible exegesis, which is interpreted in view of Roman philosophers' disdain for exegesis as a spiritually sterile activity. On all these accounts Trypho the Jew emerges as deviant and rather more Greek than Roman. He and Marcion the "Gnostic" are relegated to the eastern provinces of the empire and serve as mirror images of what Justin considers as authentic, authoritative, and distinctly Roman Christianity.

Inflammasome-Mediated Regulation of Hematopoiesis in the Vertebrate Embryo

Hematopoietic stem and progenitor cells (HSPCs) arise in the embryonic dorsal aorta (DA) through a Runx1-dependent process of endothelial-to-hematopoietic transition (EHT). Subsequently, HSPCs colonize secondary niches to proliferate, differentiate and maintain lifelong hematopoiesis. We previously reported that elevated glucose metabolism and sterile inflammatory signaling each stimulate zebrafish HSPC production from hemogenic endothelium. Consistent with the hypothesis that transient metabolic activation induces an inflammatory response to influence HSPC formation, we found that glucose stimulation from 12-36 hours post fertilization (hpf) increased expression of pro-inflammatory cytokines and receptors by quantitative PCR. In particular, morpholino-mediated knockdown of il1b blunted the inductive effects of glucose metabolism on runx1 expression in the DA and numbers of CD41+ HSPCs in the caudal hematopoietic tissue (CHT). In contrast, overexpression of mature il1b increased HSPC numbers as assessed by flow cytometry and runx1/cmyb in situ hybridization. As IL-1β is cleaved and activated by the inflammasome, which is a well-known sensor of metabolic stimuli, we treated embryos with validated inflammasome activators, such as nigericin, and found that they also increased runx1/cmyb expression and Flk1+cMyb+ HSPCs. Inflammasome stimulation required NF-κB activity, and upregulated canonical IL-1β targets, including IL-6 and IL-8, which have been previously shown by our laboratory to play significant roles in embryonic HSPC formation. Importantly, loss of the inflammasome adapter pycard or effector caspa reduced HSPC numbers, and prevented expansion of the HSPC pool in response to glucose stimulation. In further support of a role for sterile inflammatory activity in regulating HSPC number downstream of metabolic activation, we found that inflammasome-mediated IL-1β action bypassed the suppressive effect of antioxidant treatment on runx1/cmyb expression in the DA. Conversely, inflammasome inhibition partially blocked effects of metabolism-associated HIF1α activation on runx1/cmyb expression, indicating that the inflammasome acts downstream of reactive-oxygen-species (ROS) generation and HIF1α activation to promote HSPC production and/or expansion from hemogenic endothelium. Inflammasome components are highly expressed in myeloid cells; targeted knockdown and cell ablation studies indicate that macrophages are necessary to mediate the effects of inflammasome stimulation on HSPC numbers in the CHT at 72hpf. Interestingly, prolonged inflammasome stimulation also expands myeloid and lymphoid progenitors, as cmyb, rag1 and mpo expression were each elevated at 120hpf; increased numbers of Rag2+ and Mpo+ cells were confirmed by flow cytometry. To determine whether the effects of inflammasome activation were conserved in higher vertebrates, we induced inflammasome activation during differentiation of human induced pluripotent stem cells (iPSC) into hematopoietic progenitors. In this system, nigericin treatment of iPSC-derived hemogenic endothelial cells yielded an increased frequency of functional colony-forming units by day 7 of EHT culture. Taken together, inflammasome-mediated IL-1β action appears to serve as an integrator of metabolic activity, downstream of ROS/ HIF1α, to promote HSPC formation and development of myeloid and lymphoid lineages in vivo and in vitro. These studies identify the inflammasome as a promising target to promote human HSPC production from iPSCs for therapeutic purposes. DisclosuresNo relevant conflicts of interest to declare.

121. Improving Predictive Value of Phase 2a TB Drug Development Models Through PET/CT Imaging (NexGen EBA)

BackgroundCurrent Phase 2a methods in tuberculosis (TB) drug development are severely limited in capturing a drug candidate’s sterilizing activity (ability to prevent TB relapse). We hypothesize that sputum-based measurements of current methods fail to capture drug activity in deep TB lesion compartments, where recalcitrant TB populations are sequestered. In our recent marmoset study, we found complementary 14-day PET/CT delta signatures across different TB lesion compartments that distinguish drugs with long-term sterilizing activity (e.g., rifampin and pyrazinamide) versus drugs with primarily early bactericidal activity (e.g., isoniazid) (Figure 1). We proceeded to evaluate this novel PET/CT Phase 2a approach among TB patients, benchmarked by drugs that are well-characterized clinically and pharmacokinetically.MethodsHIV-negative patients with smear-positive pulmonary tuberculosis from Cape Town, South Africa were enrolled and randomized to receive a single drug [isoniazid (H), rifampin (R), pyrazinamide (Z), moxifloxacin (M)], a 2-drug regimen (HZ, RZ), or a 4-drug regimen (HRZE, MRZE) for 14 days in an inpatient facility. Primary data points were PET/CT scans evaluated at pretreatment baseline and treatment day 14 (and day 28 in the HRZE arm), and daily overnight sputum for early bactericidal activity measurements.ResultsFrom December 2015 to September 2017, 160 participants completed the study, from whom 340 PET/CT scans and 2,238 overnight sputum samples were collected. PET/CT scans from participants demonstrate diversity of lesion architecture and inter-individual variation in PET/CT changes over 14 days of treatment (Figure 2). A preliminary computational algorithm and first- and second-order PET/CT statistics have been developed for initial segmentation and TB lesion categorization of all study scans. Derivation of radiologic signatures by analysis of 14-day PET/CT changes across each lesion type is ongoing; blinded prediction of each participant’s treatment arm based on these signatures is expected by third-quarter 2018.ConclusionIf successful, these signatures may be applied within a 14-day Phase 2a framework to evaluate new TB drug candidates and construct TB drug regimens with complementary lesion pharmacokinetics. Disclosures All authors: No reported disclosures.

Surgical case scheduling with sterilising activity constraints

In this paper, we study a real scheduling problem which consists in scheduling a set of elective surgical cases requiring surgical instruments and tools in several operating rooms. The objectives are to minimise the overtime of the surgical unit staff, the number of operating rooms used and the number of instruments processed in emergency in the sterilising unit while respecting the current level of service represented by the total number of patients operated per month at the orthopaedic surgery unit. This research was performed in collaboration with the University Hospital of Angers in France (CHU Angers), which has also provided historical data for the experiments. We propose a mixed integer linear programming model for the problem which is solved in a lexicographic fashion. We also propose a robust formulation to deal with the uncertain surgery durations. Both the deterministic and robust formulations are then compared over a set of instances provided by the CHU. The solutions obtained are competitive in terms of number of operating rooms and significantly improve those implemented operationally at the CHU in terms of overtime and emergencies at the sterilising unit.

Shorter-course treatment for Mycobacterium ulcerans disease with high-dose rifamycins and clofazimine in a mouse model of Buruli ulcer.

Starting in 2004, the standard regimen for treatment of Buruli ulcer (BU) recommended by the World Health Organization has been daily treatment for eight weeks with rifampin (RIF) and streptomycin. Based on recent clinical trials, treatment with an all-oral regimen of RIF and clarithromycin (CLR) may be an effective alternative. With the achievement of an all-oral regimen, a new goal is to find a regimen that can shorten the duration of treatment without compromising efficacy. We recently observed that increasing the dose of RIF from the standard 10 mg/kg dose to 20 or 40 mg/kg, or replacing RIF with the more potent long-acting rifamycin, rifapentine (RPT) at 10 mg/kg or 20 mg/kg increased the bactericidal activity of the RIF+CLR regimen in a mouse model of BU. We also recently showed that replacing CLR with clofazimine(CFZ) at 25 mg/kg may have greater sterilizing activity than the RIF+CLR regimen. Here, we demonstrate that combining high-dose rifamycins with CFZ at a lower dose of 12.5 mg/kg results in similar reductions in swelling, bacterial burden and mycolactone concentrations in mouse footpads compared to the standard regimens and more rapid sterilization of footpads as determined by the proportions of footpads harboring viable bacteria three months after completion of treatment. The potential of these high-dose rifamycin and CFZ combinations to shorten BU treatment to four weeks warrants evaluation in a clinical trial.Trial registrationClinicalTrials.gov NCT03474198, NCT01659437

Externalisation de l’activité de stérilisation : impact sur l’état des instruments chirurgicaux

Subcontracting our institution's sterilization activity induced the implementation of an automated cleaning facility. Following this development, some of the resterilizable stainless steel needle holders started to show abnormal corrosion. Our study goal was to investigate the causes of this corrosion in order to optimize the sterilization circuit. A full sterilization process mapping and Ishikawa diagram enabled us to identify potential causes of corrosion. The needle holders' intrinsic characteristics, like steel quality and manufacturing, were analyzed as well as extrinsic factors such as the influence of preprocessing soaking conditions, steel passivation, water quality and the impact of corrosion inhibitors. Each potential factor of corrosion was tested in real conditions on needle holders' kits. The needle holders steel grade complies with medical standards and the tests showed that passivation and pre-processing conditions were not involved in the occurrence of corrosion, contrary to soaking length and use of softened rinsing water, containing more chloride than reverse osmosis water, and, thus conducive to rust formation. Moreover, corrosion inhibitors were deemed ineffective or incompatible. Due to this analysis, the incidence of corrosion was reduced by switching softened water to osmosis water and by introducing dynamic drying in the automated cleaning process. In addition, this work stresses the importance of minimizing waiting times and auditing the sterilization circuit before any subcontracting. Management Guidelines related to sterilization's outsourcing would probably have helped to limit this episode.

Activities of anti-Toxoplasma drugs and compounds against tissue cysts in the last three decades (1987 to 2017), a systematic review.

Currently, there is no approved therapy that can eradicate Toxoplasma gondii tissue cysts, which are responsible for chronic infection. This systematic review was performed to assess drugs or compounds that can be used as anti-T. gondii tissue cysts in vitro and in vivo. English electronic databases (i.e., PubMed, Science Direct, Scopus, Google Scholar, and Web of Science) were systematically searched for articles published up to 2017. A total of 55 papers published from 1987 to 2017 were eligible for inclusion in this systematic review. Among the drugs, atovaquone and azithromycin were found effective after long-term inoculation into mice; however, clinical cases of resistance to these drugs have been reported. Also, FR235222, QUI-11, tanshinone IIA, and hydroxyzine were shown to be effective against Toxoplasma cysts, but their effectiveness in vivo remains unknown. Additionally, compound 32, endochin-like quinolones, miltefosine, and guanabenz can be used as effective antiparasitic with the unique ability to reduce brain tissue cysts in chronically infected mice. Importantly, these antimicrobial agents are significant criteria for drug candidates. Future studies should focus on the biology and drug susceptibility of the cyst form of T. gondii in chronic toxoplasmosis patients to find more effective strategies that have sterilizing activity for eliminating T. gondii tissue cysts from the host, preventing disease relapse and potentially shortening the required duration of drug administration. Graphical abstract.