FBXO11 governs macrophage cell death and inflammation in response to bacterial toxins.

Staphylococcus aureus causes severe infections such as pneumonia and sepsis depending on the pore-forming toxin Panton-Valentine leukocidin (PVL). PVL kills and induces inflammation in macrophages and other myeloid cells by interacting with the human cell surface receptor, complement 5a receptor 1 (C5aR1). C5aR1 expression is tighly regulated and may thus modulate PVL activity, although the mechanisms involved remain incompletely understood. Here, we used a genome-wide CRISPR/Cas9 screen and identified F-box protein 11 (FBXO11), an E3 ubiquitin ligase complex member, to promote PVL toxicity. Genetic deletion of FBXO11 reduced the expression of C5aR1 at the mRNA level, whereas ectopic expression of C5aR1 in FBXO11-/- macrophages, or priming with LPS, restored C5aR1 expression and thereby PVL toxicity. In addition to promoting PVL-mediated killing, FBXO11 dampens secretion of IL-1ß after NLRP3 activation in response to bacterial toxins by reducing mRNA levels in a BCL-6-dependent and BCL-6-independent manner. Overall, these findings highlight that FBXO11 regulates C5aR1 and IL-1ß expression and controls macrophage cell death and inflammation following PVL exposure.

Armadillo regulates nociceptive sensitivity in the absence of injury.

Abnormal pain has recently been estimated to affect ∼50 million adults each year within the United States. With many treatment options for abnormal pain, such as opioid analgesics, carrying numerous deleterious side effects, research into safer and more effective treatment options is crucial. To help elucidate the mechanisms controlling nociceptive sensitivity, the Drosophila melanogaster larval nociception model has been used to characterize well-conserved pathways through the use of genetic modification and/or injury to alter the sensitivity of experimental animals. Mammalian models have provided evidence of ß-catenin signaling involvement in neuropathic pain development. By capitalizing on the conserved nature of ß-catenin functions in the fruit fly, here we describe a role for Armadillo, the fly homolog to mammalian ß-catenin, in regulating baseline sensitivity in the primary nociceptor of the fly, in the absence of injury, using under- and over-expression of Armadillo in a cell-specific manner. Underexpression of Armadillo resulted in hyposensitivity, while overexpression of wild-type Armadillo or expression of a degradation-resistant Armadillo resulted in hypersensitivity. Neither underexpression nor overexpression of Armadillo resulted in observed dendritic morphological changes that could contribute to behavioral phenotypes observed. These results showed that focused manipulation of Armadillo expression within the nociceptors is sufficient to modulate baseline response in the nociceptors to a noxious stimulus and that these changes are not shown to be associated with a morphogenetic effect.

The brinker repressor system regulates injury-induced nociceptive sensitization in Drosophila melanogaster.

Chronic pain is a debilitating condition affecting millions of people worldwide, and an improved understanding of the pathophysiology of chronic pain is urgently needed. Nociceptors are the sensory neurons that alert the nervous system to potentially harmful stimuli such as mechanical pressure or noxious thermal temperature. When an injury occurs, the nociceptive threshold for pain is reduced and an increased pain signal is produced. This process is called nociceptive sensitization. This sensitization normally subsides after the injury is healed. However, dysregulation can occur which results in sensitization that persists after the injury has healed. This process is thought to perpetuate chronic pain. The Hedgehog (Hh) signaling pathway has been previously implicated in nociceptive sensitization in response to injury in Drosophila melanogaster. Downstream of Hh signaling, the Bone Morphogenetic Protein (BMP) pathway has also been shown to be necessary for this process. Here, we describe a role for nuclear components of BMP's signaling pathway in the formation of injury-induced nociceptive sensitization. Brinker (Brk), and Schnurri (Shn) were suppressed in nociceptors using an RNA-interference (RNAi) "knockdown" approach. Knockdown of Brk resulted in hypersensitivity in the absence of injury, indicating that it normally acts to suppress nociceptive sensitivity. Animals in which transcriptional activator Shn was knocked down in nociceptors failed to develop normal allodynia after ultraviolet irradiation injury, indicating that Shn normally acts to promote hypersensitivity after injury. These results indicate that Brk-related transcription regulators play a crucial role in the formation of nociceptive sensitization and may therefore represent valuable new targets for pain-relieving medications.

Towards integrated modeling of the long-term impacts of oil spills.

Although great progress has been made to advance the scientific understanding of oil spills, tools for integrated assessment modeling of the long-term impacts on ecosystems, socioeconomics and human health are lacking. The objective of this study was to develop a conceptual framework that could be used to answer stakeholder questions about oil spill impacts and to identify knowledge gaps and future integration priorities. The framework was initially separated into four knowledge domains (ocean environment, biological ecosystems, socioeconomics, and human health) whose interactions were explored by gathering stakeholder questions through public engagement, assimilating expert input about existing models, and consolidating information through a system dynamics approach. This synthesis resulted in a causal loop diagram from which the interconnectivity of the system could be visualized. Results of this analysis indicate that the system naturally separates into two tiers, ocean environment and biological ecosystems versus socioeconomics and human health. As a result, ocean environment and ecosystem models could be used to provide input to explore human health and socioeconomic variables in hypothetical scenarios. At decadal-plus time scales, the analysis emphasized that human domains influence the natural domains through changes in oil-spill related laws and regulations. Although data gaps were identified in all four model domains, the socioeconomics and human health domains are the least established. Considerable future work is needed to address research gaps and to create fully coupled quantitative integrative assessment models that can be used in strategic decision-making that will optimize recoveries from future large oil spills.

Framework for a Community Health Observing System for the Gulf of Mexico Region: Preparing for Future Disasters.

The Gulf of Mexico (GoM) region is prone to disasters, including recurrent oil spills, hurricanes, floods, industrial accidents, harmful algal blooms, and the current COVID-19 pandemic. The GoM and other regions of the U.S. lack sufficient baseline health information to identify, attribute, mitigate, and facilitate prevention of major health effects of disasters. Developing capacity to assess adverse human health consequences of future disasters requires establishment of a comprehensive, sustained community health observing system, similar to the extensive and well-established environmental observing systems. We propose a system that combines six levels of health data domains, beginning with three existing, national surveys and studies plus three new nested, longitudinal cohort studies. The latter are the unique and most important parts of the system and are focused on the coastal regions of the five GoM States. A statistically representative sample of participants is proposed for the new cohort studies, stratified to ensure proportional inclusion of urban and rural populations and with additional recruitment as necessary to enroll participants from particularly vulnerable or under-represented groups. Secondary data sources such as syndromic surveillance systems, electronic health records, national community surveys, environmental exposure databases, social media, and remote sensing will inform and augment the collection of primary data. Primary data sources will include participant-provided information via questionnaires, clinical measures of mental and physical health, acquisition of biological specimens, and wearable health monitoring devices. A suite of biomarkers may be derived from biological specimens for use in health assessments, including calculation of allostatic load, a measure of cumulative stress. The framework also addresses data management and sharing, participant retention, and system governance. The observing system is designed to continue indefinitely to ensure that essential pre-, during-, and post-disaster health data are collected and maintained. It could also provide a model/vehicle for effective health observation related to infectious disease pandemics such as COVID-19. To our knowledge, there is no comprehensive, disaster-focused health observing system such as the one proposed here currently in existence or planned elsewhere. Significant strengths of the GoM Community Health Observing System (CHOS) are its longitudinal cohorts and ability to adapt rapidly as needs arise and new technologies develop.

Novel 2D and 3D Assays to Determine the Activity of Anti-Leishmanial Drugs.

The discovery of novel anti-leishmanial compounds remains essential as current treatments have known limitations and there are insufficient novel compounds in development. We have investigated three complex and physiologically relevant in vitro assays, including: (i) a media perfusion based cell culture model, (ii) two 3D cell culture models, and (iii) iPSC derived macrophages in place of primary macrophages or cell lines, to determine whether they offer improved approaches to anti-leishmanial drug discovery and development. Using a Leishmania major amastigote-macrophage assay the activities of standard drugs were investigated to show the effect of changing parameters in these assays. We determined that drug activity was reduced by media perfusion (EC50 values for amphotericin B shifted from 54 (51-57) nM in the static system to 70 (61-75) nM under media perfusion; EC50 values for miltefosine shifted from 12 (11-15) µM in the static system to 30 (26-34) µM under media perfusion) (mean and 95% confidence intervals), with corresponding reduced drug accumulation by macrophages. In the 3D cell culture model there was a significant difference in the EC50 values of amphotericin B but not miltefosine (EC50 values for amphotericin B were 34.9 (31.4-38.6) nM in the 2D and 52.3 (46.6-58.7) nM in 3D; EC50 values for miltefosine were 5.0 (4.9-5.2) µM in 2D and 5.9 (5.5-6.2) µM in 3D (mean and 95% confidence intervals). Finally, in experiments using iPSC derived macrophages infected with Leishmania, reported here for the first time, we observed a higher level of intracellular infection in iPSC derived macrophages compared to the other macrophage types for four different species of Leishmania studied. For L. major with an initial infection ratio of 0.5 parasites per host cell the percentage infection level of the macrophages after 72 h was 11.3% ± 1.5%, 46.0% ± 1.4%, 66.4% ± 3.5% and 75.1% ± 2.4% (average ± SD) for the four cells types, THP1 a human monocytic cell line, mouse bone marrow macrophages (MBMMs), human bone marrow macrophages (HBMMs) and iPSC derived macrophages respectively. Despite the higher infection levels, drug activity in iPSC derived macrophages was similar to that in other macrophage types, for example, amphotericin B EC50 values were 35.9 (33.4-38.5), 33.5 (31.5-36.5), 33.6 (30.5-not calculated (NC)) and 46.4 (45.8-47.2) nM in iPSC, MBMMs, HBMMs and THP1 cells respectively (mean and 95% confidence intervals). We conclude that increasing the complexity of cellular assays does impact upon anti-leishmanial drug activities but not sufficiently to replace the current model used in HTS/HCS assays in drug discovery programmes. The impact of media perfusion on drug activities and the use of iPSC macrophages do, however, deserve further investigation.

Targeting NLRP3 and Staphylococcal pore-forming toxin receptors in human-induced pluripotent stem cell-derived macrophages.

Staphylococcus aureus causes necrotizing pneumonia by secreting toxins such as leukocidins that target front-line immune cells. The mechanism by which leukocidins kill innate immune cells and trigger inflammation during S. aureus lung infection, however, remains unresolved. Here, we explored human-induced pluripotent stem cell-derived macrophages (hiPSC-dMs) to study the interaction of the leukocidins Panton-Valentine leukocidin (PVL) and LukAB with lung macrophages, which are the initial leukocidin targets during S. aureus lung invasion. hiPSC-dMs were susceptible to the leukocidins PVL and LukAB and both leukocidins triggered NLPR3 inflammasome activation resulting in IL-1ß secretion. hiPSC-dM cell death after LukAB exposure, however, was only temporarily dependent of NLRP3, although NLRP3 triggered marked cell death after PVL treatment. CRISPR/Cas9-mediated deletion of the PVL receptor, C5aR1, protected hiPSC-dMs from PVL cytotoxicity, despite the expression of other leukocidin receptors, such as CD45. PVL-deficient S. aureus had reduced ability to induce lung IL-1ß levels in human C5aR1 knock-in mice. Unexpectedly, inhibiting NLRP3 activity resulted in increased wild-type S. aureus lung burdens. Our findings suggest that NLRP3 induces macrophage death and IL-1ß secretion after PVL exposure and controls S. aureus lung burdens.

Assessment of a student peer-tutoring program focusing on the benefits to the tutors.

CONTEXT: The University of Pittsburgh School of Dental Medicine incorporates a voluntary student peer-tutoring program as 1 resource available to pre-doctoral students. It uses peer-tutoring in didactic and preclinical courses in order to provide additional help to struggling students. OBJECTIVE: The goal of this article is to describe an initial program assessment using data collected between 2015 and 2017. In addition to assessing the program, this report also investigates the benefits of the program to the tutors. DESIGN: Data were collected using surveys from tutors (N = 133) and tutees (N = 115), as well as reflective journals written by the tutors (response rate varies across instruments and questions). Responses to the surveys were analyzed using quantitative analysis, and content analysis was completed for coding the open-ended short responses and reflective journals. RESULTS: Results show tutors' increased preparedness at the end of the term to provide tutoring, an increase in communication and teaching skills due to participation in the program, and other cited benefits to the tutor. CONCLUSIONS: In addition to academic credit, tutors benefit from reviewing the course materials and practicing skills relevant to their future careers. Tutees provided overwhelmingly positive feedback on the tutors' strengths and effectiveness, the program in general, and the extent to which tutoring helped their performance in the class. Based on the initial program assessment, the program provides benefits to both tutors and tutees; the tutors gain a valuable experience impacting them both academically and personally.

IRF5 Promotes Influenza Virus-Induced Inflammatory Responses in Human Induced Pluripotent Stem Cell-Derived Myeloid Cells and Murine Models.

Recognition of influenza A virus (IAV) by the innate immune system triggers pathways that restrict viral replication, activate innate immune cells, and regulate adaptive immunity. However, excessive innate immune activation can exaggerate disease. The pathways promoting excessive activation are incompletely understood, with limited experimental models to investigate the mechanisms driving influenza virus-induced inflammation in humans. Interferon regulatory factor 5 (IRF5) is a transcription factor that plays important roles in the induction of cytokines after viral sensing. In an in vivo model of IAV infection, IRF5 deficiency reduced IAV-driven immune pathology and associated inflammatory cytokine production, specifically reducing cytokine-producing myeloid cell populations in Irf5-/- mice but not impacting type 1 interferon (IFN) production or virus replication. Using cytometry by time of flight (CyTOF), we identified that human lung IRF5 expression was highest in cells of the myeloid lineage. To investigate the role of IRF5 in mediating human inflammatory responses by myeloid cells to IAV, we employed human-induced pluripotent stem cells (hIPSCs) with biallelic mutations in IRF5, demonstrating for the first time that induced pluripotent stem cell-derived dendritic cells (iPS-DCs) with biallelic mutations can be used to investigate the regulation of human virus-induced immune responses. Using this technology, we reveal that IRF5 deficiency in human DCs, or macrophages, corresponded with reduced virus-induced inflammatory cytokine production, with IRF5 acting downstream of Toll-like receptor 7 (TLR7) and, possibly, retinoic acid-inducible gene I (RIG-I) after viral sensing. Thus, IRF5 acts as a regulator of myeloid cell inflammatory cytokine production during IAV infection in mice and humans and drives immune-mediated viral pathogenesis independently of type 1 IFN and virus replication.IMPORTANCE The inflammatory response to influenza A virus (IAV) participates in infection control but contributes to disease severity. After viral detection, intracellular pathways are activated, initiating cytokine production, but these pathways are incompletely understood. We show that interferon regulatory factor 5 (IRF5) mediates IAV-induced inflammation and, in mice, drives pathology. This was independent of antiviral type 1 IFN and virus replication, implying that IRF5 could be specifically targeted to treat influenza virus-induced inflammation. We show for the first time that human iPSC technology can be exploited in genetic studies of virus-induced immune responses. Using this technology, we deleted IRF5 in human myeloid cells. These IRF5-deficient cells exhibited impaired influenza virus-induced cytokine production and revealed that IRF5 acts downstream of Toll-like receptor 7 and possibly retinoic acid-inducible gene I. Our data demonstrate the importance of IRF5 in influenza virus-induced inflammation, suggesting that genetic variation in the IRF5 gene may influence host susceptibility to viral diseases.

Loss of IL-10 signaling in macrophages limits bacterial killing driven by prostaglandin E2.

Loss of IL-10 signaling in macrophages (Mφs) leads to inflammatory bowel disease (IBD). Induced pluripotent stem cells (iPSCs) were generated from an infantile-onset IBD patient lacking a functional IL10RB gene. Mφs differentiated from IL-10RB-/- iPSCs lacked IL-10RB mRNA expression, were unable to phosphorylate STAT3, and failed to reduce LPS induced inflammatory cytokines in the presence of exogenous IL-10. IL-10RB-/- Mφs exhibited a striking defect in their ability to kill Salmonella enterica serovar Typhimurium, which was rescuable after experimentally introducing functional copies of the IL10RB gene. Genes involved in synthesis and receptor pathways for eicosanoid prostaglandin E2 (PGE2) were more highly induced in IL-10RB-/- Mφs, and these Mφs produced higher amounts of PGE2 after LPS stimulation compared with controls. Furthermore, pharmacological inhibition of PGE2 synthesis and PGE2 receptor blockade enhanced bacterial killing in Mφs. These results identify a regulatory interaction between IL-10 and PGE2, dysregulation of which may drive aberrant Mφ activation and impaired host defense contributing to IBD pathogenesis.

A Genome-Wide Knockout Screen in Human Macrophages Identified Host Factors Modulating Salmonella Infection.

A genome-scale CRISPR knockout library screen of THP-1 human macrophages was performed to identify loss-of-function mutations conferring resistance to Salmonella uptake. The screen identified 183 candidate genes, from which 14 representative genes involved in actin dynamics (ACTR3, ARPC4, CAPZB, TOR3A, CYFIP2, CTTN, and NHLRC2), glycosaminoglycan metabolism (B3GNT1), receptor signaling (PDGFB and CD27), lipid raft formation (CLTCL1), calcium transport (ATP2A2 and ITPR3), and cholesterol metabolism (HMGCR) were analyzed further. For some of these pathways, known chemical inhibitors could replicate the Salmonella resistance phenotype, indicating their potential as targets for host-directed therapy. The screen indicated a role for the relatively uncharacterized gene NHLRC2 in both Salmonella invasion and macrophage differentiation. Upon differentiation, NHLRC2 mutant macrophages were hyperinflammatory and did not exhibit characteristics typical of macrophages, including atypical morphology and inability to interact and phagocytose bacteria/particles. Immunoprecipitation confirmed an interaction of NHLRC2 with FRYL, EIF2AK2, and KLHL13.IMPORTANCESalmonella exploits macrophages to gain access to the lymphatic system and bloodstream to lead to local and potentially systemic infections. With an increasing number of antibiotic-resistant isolates identified in humans, Salmonella infections have become major threats to public health. Therefore, there is an urgent need to identify alternative approaches to anti-infective therapy, including host-directed therapies. In this study, we used a simple genome-wide screen to identify 183 candidate host factors in macrophages that can confer resistance to Salmonella infection. These factors may be potential therapeutic targets against Salmonella infections.

Using a Systems Biology Approach To Study Host-Pathogen Interactions.

The rapid development of genomics and other "-omics" approaches has significantly impacted how we have investigated host-pathogen interactions since the turn of the millennium. Technologies such as next-generation sequencing, stem cell biology, and high-throughput proteomics have transformed the scale and sensitivity with which we interrogate biological samples. These approaches are impacting experimental design in the laboratory and transforming clinical management in health care systems. Here, we review this area from the perspective of research on bacterial pathogens.

Infection by Brucella melitensis or Brucella papionis modifies essential physiological functions of human trophoblasts.

Brucellosis is a zoonosis caused by bacteria of the Brucella genus. In ruminants, brucellosis causes abortion, followed by chronic infection and secretion of bacteria in milk. In humans, it usually presents as flu-like symptoms, with serious complications if untreated. Epidemiological studies have only recently established that brucellosis can also cause pregnancy complications in women, but the pathogenic mechanisms are unknown. Pioneering studies in ruminants showed that Brucella infect trophoblasts and then colonise the placenta where they grow to high density. A recent study showed that the main zoonotic Brucella species can infect human cytotrophoblasts (CTB) and extravillous trophoblasts (EVT). In this work, we show that Brucella papionis (associated with stillbirth in primates) also infects human trophoblasts. However, it replicates actively in CTB, whereas its replication is very restricted within EVT. We also observed alteration of several trophoblastic functions upon infection by B. papionis or Brucella melitensis (the most prevalent species in human brucellosis). Infection altered the production of hormones, the ability of CTB to form syncytiotrophoblasts, and the invasion capacity of EVT. We also found that infection can spread between different types of trophoblasts. These findings constitute a new step in understanding how Brucella infection causes adverse pregnancy outcomes.

International Consortium for Health Outcome Measurement Set of Outcomes That Matter to People Living With Inflammatory Arthritis: Consensus From an International Working Group.

OBJECTIVE: The implementation of value-based health care in inflammatory arthritis requires a standardized set of modifiable outcomes and risk-adjustment variables that is feasible to implement worldwide. METHODS: The International Consortium for Health Outcomes Measurement (ICHOM) assembled a multidisciplinary working group that consisted of 24 experts from 6 continents, including 6 patient representatives, to develop a standard set of outcomes for inflammatory arthritis. The process followed a structured approach, using a modified Delphi process to reach consensus on the following decision areas: conditions covered by the set, outcome domains, outcome measures, and risk-adjustment variables. Consensus in areas 2 to 4 were supported by systematic literature reviews and consultation of experts. RESULTS: The ICHOM Inflammatory Arthritis Standard Set covers patients with rheumatoid arthritis (RA), axial spondyloarthritis, psoriatic arthritis, and juvenile idiopathic arthritis (JIA). We recommend that outcomes regarding pain, fatigue, activity limitations, overall physical and mental health impact, work/school/housework ability and productivity, disease activity, and serious adverse events be collected at least annually. Validated measures for patient-reported outcomes were endorsed and linked to common reporting metrics. Age, sex at birth, education level, smoking status, comorbidities, time since diagnosis, and rheumatoid factor and anti-citrullinated protein antibody lab testing for RA and JIA should be collected as risk-adjustment variables. CONCLUSION: We present the ICHOM inflammatory arthritis Standard Set of outcomes, which enables health care providers to implement the value-based health care framework and compare outcomes that are important to patients with inflammatory arthritis.

A Simple Multistep Protocol for Differentiating Human Induced Pluripotent Stem Cells into Functional Macrophages.

Macrophages differentiated from human induced pluripotent stem cells (hiPSCs) provide an alternative new tool overcoming some of the limitations of existing models for human macrophages, such as human macrophage-like cell lines and primary monocyte-derived macrophages. A combination of different cytokines and growth factors can differentiate hiPSCs toward myeloid lineage. Here we describe a simple multistep protocol for differentiating hiPSCs into functional macrophages. This includes derivation of three germ-line containing embryoid bodies (EBs) from iPSCs, generation of myeloid precursors from EBs, and finally maturation of myeloid precursors into functional macrophages. Technical procedure and specific culture conditions associated with each of these steps are discussed in detail.

Inter- and intraspecific variation in leaf economic traits in wheat and maize.

Leaf Economics Spectrum (LES) trait variation underpins multiple agroecological processes and many prominent crop yield models. While there are numerous independent studies assessing trait variation in crops, to date there have been no comprehensive assessments of intraspecific trait variation (ITV) in LES traits for wheat and maize: the world's most widespread crops. Using trait databases and peer-reviewed literature, we compiled over 700 records of specific leaf area (SLA), maximum photosynthetic rates (Amax) and leaf nitrogen (N) concentrations, for wheat and maize. We evaluated intraspecific LES trait variation, and intraspecific trait-environment relationships. While wheat and maize occupy the upper 90th percentile of LES trait values observed across a global species pool, ITV ranged widely across the LES in wheat and maize. Fertilization treatments had strong impacts on leaf N, while plant developmental stage (here standardized as the number of days since planting) had strong impacts on Amax; days since planting, N fertilization and irrigation all influenced SLA. When controlling for these factors, intraspecific responses to temperature and precipitation explained 39.4 and 43.7 % of the variation in Amax and SLA, respectively, but only 5.4 % of the variation in leaf N. Despite a long history of domestication in these species, ITV in wheat and maize among and within cultivars remains large. Intraspecific trait variation is a critical consideration to refine regional to global models of agroecosystem structure, function and food security. Considerable opportunities and benefits exist for consolidating a crop trait database for a wider range of domesticated plant species.

Shared genetic effects on chromatin and gene expression indicate a role for enhancer priming in immune response.

Regulatory variants are often context specific, modulating gene expression in a subset of possible cellular states. Although these genetic effects can play important roles in disease, the molecular mechanisms underlying context specificity are poorly understood. Here, we identified shared quantitative trait loci (QTLs) for chromatin accessibility and gene expression in human macrophages exposed to IFNγ, Salmonella and IFNγ plus Salmonella. We observed that ~60% of stimulus-specific expression QTLs with a detectable effect on chromatin altered the chromatin accessibility in naive cells, thus suggesting that they perturb enhancer priming. Such variants probably influence binding of cell-type-specific transcription factors, such as PU.1, which can then indirectly alter the binding of stimulus-specific transcription factors, such as NF-κB or STAT2. Thus, although chromatin accessibility assays are powerful for fine-mapping causal regulatory variants, detecting their downstream effects on gene expression will be challenging, requiring profiling of large numbers of stimulated cellular states and time points.

Two Phase 1, Open-Label, Mass Balance Studies to Determine the Pharmacokinetics of 14 C-Labeled Isavuconazonium Sulfate in Healthy Male Volunteers.

Isavuconazonium sulfate is the water-soluble prodrug of the active triazole isavuconazole. Two phase 1 studies were conducted to identify the metabolic profile and mass balance of isavuconazole and BAL8728 (inactive cleavage product). Seven subjects in study 1 (isavuconazole mass balance) received a single oral dose of [cyano-14 C]isavuconazonium sulfate corresponding to 200 mg isavuconazole. Six subjects in study 2 (BAL8728 mass balance) received a single intravenous dose of [pyridinylmethyl-14 C]isavuconazonium sulfate corresponding to 75 mg BAL8728. Pharmacokinetic parameters of radioactivity in whole blood and plasma and of isavuconazole and BAL8728 in plasma were assessed. Radioactivity ratio of blood/plasma, percentage of dose, and cumulative percentage of radioactive dose recovered in urine and feces for isavuconazole and BAL8728 were assessed. Metabolic profiling was carried out by high-performance liquid chromatography and mass spectrometry. Mean plasma isavuconazole pharmacokinetic parameters included apparent clearance (2.3 ± 0.7 L/h), apparent volume of distribution (301.8 ± 105.7 L), and terminal elimination half-life (99.9 ± 44.6 hours). In study 1, isavuconazole-derived radioactivity was recovered approximately equally in urine and feces (46.1% and 45.5%, respectively). In study 2, BAL8728-derived radioactivity was predominantly recovered in urine (96.0%). Isavuconazole (study 1) and M4 (cleavage metabolite of BAL8728; study 2) were the predominant circulating components of radioactivity in plasma.

Exploiting induced pluripotent stem cell-derived macrophages to unravel host factors influencing Chlamydia trachomatis pathogenesis.

Chlamydia trachomatis remains a leading cause of bacterial sexually transmitted infections and preventable blindness worldwide. There are, however, limited in vitro models to study the role of host genetics in the response of macrophages to this obligate human pathogen. Here, we describe an approach using macrophages derived from human induced pluripotent stem cells (iPSdMs) to study macrophage-Chlamydia interactions in vitro. We show that iPSdMs support the full infectious life cycle of C. trachomatis in a manner that mimics the infection of human blood-derived macrophages. Transcriptomic and proteomic profiling of the macrophage response to chlamydial infection highlighted the role of the type I interferon and interleukin 10-mediated responses. Using CRISPR/Cas9 technology, we generated biallelic knockout mutations in host genes encoding IRF5 and IL-10RA in iPSCs, and confirmed their roles in limiting chlamydial infection in macrophages. This model can potentially be extended to other pathogens and tissue systems to advance our understanding of host-pathogen interactions and the role of human genetics in influencing the outcome of infections.

Pharmacokinetic Evaluation of CYP3A4-Mediated Drug-Drug Interactions of Isavuconazole With Rifampin, Ketoconazole, Midazolam, and Ethinyl Estradiol/Norethindrone in Healthy Adults.

This report describes the phase 1 trials that evaluated the metabolism of the novel triazole antifungal isavuconazole by cytochrome P450 3A4 (CYP3A4) and isavuconazole's effects on CYP3A4-mediated metabolism in healthy adults. Coadministration of oral isavuconazole (100 mg once daily) with oral rifampin (600 mg once daily; CYP3A4 inducer) decreased isavuconazole area under the concentration-time curve (AUCτ ) during a dosing interval by 90% and maximum concentration (Cmax ) by 75%. Conversely, coadministration of isavuconazole (200 mg single dose) with oral ketoconazole (200 mg twice daily; CYP3A4 inhibitor) increased isavuconazole AUC from time 0 to infinity (AUC0-∞ ) and Cmax by 422% and 9%, respectively. Isavuconazole was coadministered (200 mg 3 times daily for 2 days, then 200 mg once daily) with single doses of oral midazolam (3 mg; CYP3A4 substrate) or ethinyl estradiol/norethindrone (35 µg/1 mg; CYP3A4 substrate). Following coadministration, AUC0-∞ increased 103% for midazolam, 8% for ethinyl estradiol, and 16% for norethindrone; Cmax increased by 72%, 14%, and 6%, respectively. Most adverse events were mild to moderate in intensity; there were no deaths, and serious adverse events and adverse events leading to study discontinuation were rare. These results indicate that isavuconazole is a sensitive substrate and moderate inhibitor of CYP3A4.