Severity of effect considerations regarding the use of mutation as a toxicological endpoint for risk assessment: A report from the 8th International Workshop on Genotoxicity Testing (IWGT).

Exposure levels without appreciable human health risk may be determined by dividing a point of departure on a dose-response curve (e.g., benchmark dose) by a composite adjustment factor (AF). An "effect severity" AF (ESAF) is employed in some regulatory contexts. An ESAF of 10 may be incorporated in the derivation of a health-based guidance value (HBGV) when a "severe" toxicological endpoint, such as teratogenicity, irreversible reproductive effects, neurotoxicity, or cancer was observed in the reference study. Although mutation data have been used historically for hazard identification, this endpoint is suitable for quantitative dose-response modeling and risk assessment. As part of the 8th International Workshops on Genotoxicity Testing, a sub-group of the Quantitative Analysis Work Group (WG) explored how the concept of effect severity could be applied to mutation. To approach this question, the WG reviewed the prevailing regulatory guidance on how an ESAF is incorporated into risk assessments, evaluated current knowledge of associations between germline or somatic mutation and severe disease risk, and mined available data on the fraction of human germline mutations expected to cause severe disease. Based on this review and given that mutations are irreversible and some cause severe human disease, in regulatory settings where an ESAF is used, a majority of the WG recommends applying an ESAF value between 2 and 10 when deriving a HBGV from mutation data. This recommendation may need to be revisited in the future if direct measurement of disease-causing mutations by error-corrected next generation sequencing clarifies selection of ESAF values.

N-Nitrosodimethylamine investigations in Muta™Mouse define point-of-departure values and demonstrate less-than-additive somatic mutant frequency accumulations.

The N-nitrosamine, N-nitrosodimethylamine (NDMA), is an environmental mutagen and rodent carcinogen. Small levels of NDMA have been identified as an impurity in some commonly used drugs, resulting in several product recalls. In this study, NDMA was evaluated in an OECD TG-488 compliant Muta™Mouse gene mutation assay (28-day oral dosing across seven daily doses of 0.02-4 mg/kg/day) using an integrated design that assessed mutation at the transgenic lacZ locus in various tissues and at the endogenous Pig-a gene-locus, along with micronucleus frequencies in peripheral blood. Liver pathology was determined together with NDMA exposure in blood and liver. The additivity of mutation induction was assessed by including two acute single-dose treatment groups (i.e. 5 and 10 mg/kg dose on Day 1), which represented the same total dose as two of the repeat dose treatment groups. NDMA did not induce statistically significant increases in mean lacZ mutant frequency (MF) in bone marrow, spleen, bladder, or stomach, nor in peripheral blood (Pig-a mutation or micronucleus induction) when tested up to 4 mg/kg/day. There were dose-dependent increases in mean lacZ MF in the liver, lung, and kidney following 28-day repeat dosing or in the liver and kidney after a single dose (10 mg/kg). No observed genotoxic effect levels (NOGEL) were determined for the positive repeat dose-response relationships. Mutagenicity did not exhibit simple additivity in the liver since there was a reduction in MF following NDMA repeat dosing compared with acute dosing for the same total dose. Benchmark dose modelling was used to estimate point of departure doses for NDMA mutagenicity in Muta™Mouse and rank order target organ tissue sensitivity (liver > kidney or lung). The BMD50 value for liver was 0.32 mg/kg/day following repeat dosing (confidence interval 0.21-0.46 mg/kg/day). In addition, liver toxicity was observed at doses of ≥ 1.1 mg/kg/day NDMA and correlated with systemic and target organ exposure. The integration of these results and their implications for risk assessment are discussed.

Ames test study designs for nitrosamine mutagenicity testing: qualitative and quantitative analysis of key assay parameters.

The robust control of genotoxic N-nitrosamine (NA) impurities is an important safety consideration for the pharmaceutical industry, especially considering recent drug product withdrawals. NAs belong to the 'cohort of concern' list of genotoxic impurities (ICH M7) because of the mutagenic and carcinogenic potency of this chemical class. In addition, regulatory concerns exist regarding the capacity of the Ames test to predict the carcinogenic potential of NAs because of historically discordant results. The reasons postulated to explain these discordant data generally point to aspects of Ames test study design. These include vehicle solvent choice, liver S9 species, bacterial strain, compound concentration, and use of pre-incubation versus plate incorporation methods. Many of these concerns have their roots in historical data generated prior to the harmonization of Ames test guidelines. Therefore, we investigated various Ames test assay parameters and used qualitative analysis and quantitative benchmark dose modelling to identify which combinations provided the most sensitive conditions in terms of mutagenic potency. Two alkyl-nitrosamines, N-nitrosodimethylamine (NDMA) and N-nitrosodiethylamine (NDEA) were studied. NDMA and NDEA mutagenicity was readily detected in the Ames test and key assay parameters were identified that contributed to assay sensitivity rankings. The pre-incubation method (30-min incubation), appropriate vehicle (water or methanol), and hamster-induced liver S9, alongside Salmonella typhimurium strains TA100 and TA1535 and Escherichia coli strain WP2uvrA(pKM101) provide the most sensitive combination of assay parameters in terms of NDMA and NDEA mutagenic potency in the Ames test. Using these parameters and further quantitative benchmark dose modelling, we show that N-nitrosomethylethylamine (NMEA) is positive in Ames test and therefore should no longer be considered a historically discordant NA. The results presented herein define a sensitive Ames test design that can be deployed for the assessment of NAs to support robust impurity qualifications.

Error-corrected next generation sequencing - Promises and challenges for genotoxicity and cancer risk assessment.

Error-corrected Next Generation Sequencing (ecNGS) is rapidly emerging as a valuable, highly sensitive and accurate method for detecting and characterizing mutations in any cell type, tissue or organism from which DNA can be isolated. Recent mutagenicity and carcinogenicity studies have used ecNGS to quantify drug-/chemical-induced mutations and mutational spectra associated with cancer risk. ecNGS has potential applications in genotoxicity assessment as a new readout for traditional models, for mutagenesis studies in 3D organotypic cultures, and for detecting off-target effects of gene editing tools. Additionally, early data suggest that ecNGS can measure clonal expansion of mutations as a mechanism-agnostic early marker of carcinogenic potential and can evaluate mutational load directly in human biomonitoring studies. In this review, we discuss promising applications, challenges, limitations, and key data initiatives needed to enable regulatory testing and adoption of ecNGS - including for advancing safety assessment, augmenting weight-of-evidence for mutagenicity and carcinogenicity mechanisms, identifying early biomarkers of cancer risk, and managing human health risk from chemical exposures.

Next Generation Sequencing Workshop at the Royal Society of Medicine (London, May 2022): how genomics is on the path to modernizing genetic toxicology.

The use of error-corrected Next Generation Sequencing (ecNG) to determine mutagenicity has been a subject of growing interest and potentially a disruptive technology that could supplement, and in time, replace current testing paradigms in preclinical safety assessment. Considering this, a Next Generation Sequencing Workshop was held at the Royal Society of Medicine in London in May 2022, supported by the United Kingdom Environmental Mutagen Society (UKEMS) and TwinStrand Biosciences (WA, USA), to discuss progress and future applications of this technology. In this meeting report, the invited speakers provide an overview of the Workshop topics covered and identify future directions for research. In the area of somatic mutagenesis, several speakers reviewed recent progress made with correlating ecNGS to classic in vivo transgenic rodent mutation assays as well as exploring the use of this technology directly in humans and animals, and in complex organoid models. Additionally, ecNGS has been used for detecting off-target effects of gene editing tools and emerging data suggest ecNGS potential to measure clonal expansion of cells carrying mutations in cancer driver genes as an early marker of carcinogenic potential and for direct human biomonitoring. As such, the workshop demonstrated the importance of raising awareness and support for advancing the science of ecNGS for mutagenesis, gene editing, and carcinogenesis research. Furthermore, the potential of this new technology to contribute to advances in drug and product development and improve safety assessment was extensively explored.

Multivalent human antibody-centyrin fusion protein to prevent and treat Staphylococcus aureus infections.

Treating and preventing infections by antimicrobial-resistant bacterial pathogens is a worldwide problem. Pathogens such as Staphylococcus aureus produce an array of virulence determinants, making it difficult to identify single targets for the development of vaccines or monoclonal therapies. We described a human-derived anti-S. aureus monoclonal antibody (mAb)-centyrin fusion protein ("mAbtyrin") that simultaneously targets multiple bacterial adhesins, resists proteolysis by bacterial protease GluV8, avoids Fc engagement by S. aureus IgG-binding proteins SpA and Sbi, and neutralizes pore-forming leukocidins via fusion with anti-toxin centyrins, while maintaining Fc- and complement-mediated functions. Compared with the parental mAb, mAbtyrin protected human phagocytes and boosted phagocyte-mediated killing. The mAbtyrin also reduced pathology, reduced bacterial burden, and protected from different types of infections in preclinical animal models. Finally, mAbtyrin synergized with vancomycin, enhancing pathogen clearance in an animal model of bacteremia. Altogether, these data establish the potential of multivalent mAbs for treating and preventing S. aureus diseases.

Use of the bacterial reverse mutation assay to predict carcinogenicity of N-nitrosamines.

Under ICH M7, impurities are assessed using the bacterial reverse mutation assay (i.e., Ames test) when predicted positive using in silico methodologies followed by expert review. N-Nitrosamines (NAs) have been of recent concern as impurities in pharmaceuticals, mainly because of their potential to be highly potent mutagenic carcinogens in rodent bioassays. The purpose of this analysis was to determine the sensitivity of the Ames assay to predict the carcinogenic outcome with curated proprietary Vitic (n = 131) and Leadscope (n = 70) databases. NAs were selected if they had corresponding rodent carcinogenicity assays. Overall, the sensitivity/specificity of the Ames assay was 93-97% and 55-86%, respectively. The sensitivity of the Ames assay was not significantly impacted by plate incorporation (84-89%) versus preincubation (82-89%). Sensitivity was not significantly different between use of rat and hamster liver induced S9 (80-93% versus 77-96%). The sensitivity of the Ames is high when using DMSO as a solvent (87-88%). Based on the analysis of these databases, the Ames assay conducted under OECD 471 guidelines is highly sensitive for detecting the carcinogenic hazards of NAs.

Design, Synthesis, and Characterization of TNP-2198, a Dual-Targeted Rifamycin-Nitroimidazole Conjugate with Potent Activity against Microaerophilic and Anaerobic Bacterial Pathogens.

TNP-2198, a stable conjugate of a rifamycin pharmacophore and a nitroimidazole pharmacophore, has been designed, synthesized, and evaluated as a novel dual-targeted antibacterial agent for the treatment of microaerophilic and anaerobic bacterial infections. TNP-2198 exhibits greater activity than a 1:1 molar mixture of the parent drugs and exhibits activity against strains resistant to both rifamycins and nitroimidazoles. A crystal structure of TNP-2198 bound to a Mycobacterium tuberculosis RNA polymerase transcription initiation complex reveals that the rifamycin portion of TNP-2198 binds to the rifamycin binding site on RNAP and the nitroimidazole portion of TNP-2198 interacts directly with the DNA template-strand in the RNAP active-center cleft, forming a hydrogen bond with a base of the DNA template strand. TNP-2198 is currently in Phase 2 clinical development for the treatment of Helicobacter pylori infection, Clostridioides difficile infection, and bacterial vaginosis.

Staphylococcus aureus in Non-Cystic Fibrosis Bronchiectasis: Prevalence and Genomic Basis of High Inoculum ß-Lactam Resistance.

Rationale: The pathobiology of Staphylococcus aureus in non-cystic fibrosis bronchiectasis (nCFB) is poorly defined. When present at high density or "inoculum," some methicillin-sensitive S. aureus (MSSA) can inefficiently degrade antistaphylococcal ß-lactam antibiotics via BlaZ penicillinases (termed the "inoculum effect" [IE]). Given the high burden of organisms in bronchiectatic airways, this is particularly relevant. Objectives: Drawing from a prospectively collected biobank, we sought to understand the prevalence, natural history, potential for transmission, and antibiotic resistance profiles among nCFB-derived MSSA isolates. Methods: All individuals attending a regional consultancy nCFB clinic with sputum collected between 1981 and 2017 were considered, and those with one or more S. aureus-positive cultures composed the cohort. Each individual's most recent biobank isolate was subjected to whole-genome sequencing (including the blaZ gene), antibacterial susceptibility testing, and comparative ß-lactam testing at standard (5 × 105 colony-forming unit [cfu]/ml) and high (5 × 107 cfu/ml) inocula to assess for the IE and pronounced IE. Results: Seventy-four (35.4%) of 209 individuals had one or more sputum samples with S. aureus (68 MSSA, 6 methicillin-resistant S. aureus). Those with S. aureus infection were more likely to be female. Among 60 of 74 MSSA isolates subjected to whole-genome sequencing, no evidence of transmission was identified, although specific multilocus sequence typing types were prevalent, including ST-1, ST-15, ST-30, and ST-45. Antibiotic resistance was uncommon, except for macrolides (∼20%). Among the 60 MSSA samples, the prevalence of IE and pronounced IE was observed to be drug specific: meropenem (0% and 0%, respectively), cefepime (3% and 5%, respectively), ceftazidime (8% and 0%, respectively), cloxacillin (12% and 0%, respectively), cefazolin (23% and 0%, respectively), and piperacillin-tazobactam (37% and 17%, respectively). The cefazolin IE was associated with blaZ type A (P < 0.01) and ST-30 (P < 0.01), whereas the piperacillin-tazobactam IE was associated with type C blaZ (P < 0.001) and ST-15 (P < 0.05). Conclusions:S. aureus infection was common, although no evidence of transmission was apparent in our nCFB cohort. Although routine susceptibility testing did not identify significant resistance, inoculum-related resistance was found to be relevant for commonly used nCFB antibiotics, including cefazolin and piperacillin-tazobactam. Given previous associations between IEs and negative patient outcomes, further work is warranted to understand how this phenotype impacts nCFB disease progression.

Inter-laboratory automation of the in vitro micronucleus assay using imaging flow cytometry and deep learning.

The in vitro micronucleus assay is a globally significant method for DNA damage quantification used for regulatory compound safety testing in addition to inter-individual monitoring of environmental, lifestyle and occupational factors. However, it relies on time-consuming and user-subjective manual scoring. Here we show that imaging flow cytometry and deep learning image classification represents a capable platform for automated, inter-laboratory operation. Images were captured for the cytokinesis-block micronucleus (CBMN) assay across three laboratories using methyl methanesulphonate (1.25-5.0 µg/mL) and/or carbendazim (0.8-1.6 µg/mL) exposures to TK6 cells. Human-scored image sets were assembled and used to train and test the classification abilities of the "DeepFlow" neural network in both intra- and inter-laboratory contexts. Harnessing image diversity across laboratories yielded a network able to score unseen data from an entirely new laboratory without any user configuration. Image classification accuracies of 98%, 95%, 82% and 85% were achieved for 'mononucleates', 'binucleates', 'mononucleates with MN' and 'binucleates with MN', respectively. Successful classifications of 'trinucleates' (90%) and 'tetranucleates' (88%) in addition to 'other or unscorable' phenotypes (96%) were also achieved. Attempts to classify extremely rare, tri- and tetranucleated cells with micronuclei into their own categories were less successful (≤ 57%). Benchmark dose analyses of human or automatically scored micronucleus frequency data yielded quantitation of the same equipotent concentration regardless of scoring method. We conclude that this automated approach offers significant potential to broaden the practical utility of the CBMN method across industry, research and clinical domains. We share our strategy using openly-accessible frameworks.

New insights into the mechanisms underlying 5-fluorouracil-induced intestinal toxicity based on transcriptomic and metabolomic responses in human intestinal organoids.

5-Fluorouracil (5-FU) is a widely used chemotherapeutical that induces acute toxicity in the small and large intestine of patients. Symptoms can be severe and lead to the interruption of cancer treatments. However, there is limited understanding of the molecular mechanisms underlying 5-FU-induced intestinal toxicity. In this study, well-established 3D organoid models of human colon and small intestine (SI) were used to characterize 5-FU transcriptomic and metabolomic responses. Clinically relevant 5-FU concentrations for in vitro testing in organoids were established using physiologically based pharmacokinetic simulation of dosing regimens recommended for cancer patients, resulting in exposures to 10, 100 and 1000 µM. After treatment, different measurements were performed: cell viability and apoptosis; image analysis of cell morphological changes; RNA sequencing; and metabolome analysis of supernatant from organoids cultures. Based on analysis of the differentially expressed genes, the most prominent molecular pathways affected by 5-FU included cell cycle, p53 signalling, mitochondrial ATP synthesis and apoptosis. Short time-series expression miner demonstrated tissue-specific mechanisms affected by 5-FU, namely biosynthesis and transport of small molecules, and mRNA translation for colon; cell signalling mediated by Rho GTPases and fork-head box transcription factors for SI. Metabolomic analysis showed that in addition to the effects on TCA cycle and oxidative stress in both organoids, tissue-specific metabolic alterations were also induced by 5-FU. Multi-omics integration identified transcription factor E2F1, a regulator of cell cycle and apoptosis, as the best key node across all samples. These results provide new insights into 5-FU toxicity mechanisms and underline the relevance of human organoid models in the safety assessment in drug development.

Permitted daily exposure limits for noteworthy N-nitrosamines.

A genotoxic carcinogen, N-nitrosodimethylamine (NDMA), was detected as a synthesis impurity in some valsartan drugs in 2018, and other N-nitrosamines, such as N-nitrosodiethylamine (NDEA), were later detected in other sartan products. N-nitrosamines are pro-mutagens that can react with DNA following metabolism to produce DNA adducts, such as O6 -alkyl-guanine. The adducts can result in DNA replication miscoding errors leading to GC>AT mutations and increased risk of genomic instability and carcinogenesis. Both NDMA and NDEA are known rodent carcinogens in male and female rats. The DNA repair enzyme, methylguanine DNA-methyltransferase can restore DNA integrity via the removal of alkyl groups from guanine in an error-free fashion and this can result in nonlinear dose responses and a point of departure or "practical threshold" for mutation at low doses of exposure. Following International recommendations (ICHM7; ICHQ3C and ICHQ3D), we calculated permissible daily exposures (PDE) for NDMA and NDEA using published rodent cancer bioassay and in vivo mutagenicity data to determine benchmark dose values and define points of departure and adjusted with appropriate uncertainty factors (UFs). PDEs for NDMA were 6.2 and 0.6 µg/person/day for cancer and mutation, respectively, and for NDEA, 2.2 and 0.04 µg/person/day. Both PDEs are higher than the acceptable daily intake values (96 ng for NDMA and 26.5 ng for NDEA) calculated by regulatory authorities using simple linear extrapolation from carcinogenicity data. These PDE calculations using a bench-mark approach provide a more robust assessment of exposure limits compared with simple linear extrapolations and can better inform risk to patients exposed to the contaminated sartans.

Synergistic Activity of Nitroimidazole-Oxazolidinone Conjugates against Anaerobic Bacteria.

The introductions of the bicyclic 4-nitroimidazole and the oxazolidinone classes of antimicrobial agents represented the most significant advancements in the infectious disease area during the past two decades. Pretomanid, a bicyclic 4-nitroimidazole, and linezolid, an oxazolidinone, are also part of a combination regimen approved recently by the US Food and Drug Administration for the treatment of pulmonary, extensively drug resistant (XDR), treatment-intolerant or nonresponsive multidrug-resistant (MDR) Mycobacterium tuberculosis (TB). To identify new antimicrobial agents with reduced propensity for the development of resistance, a series of dual-acting nitroimidazole-oxazolidinone conjugates were designed, synthesized and evaluated for their antimicrobial activity. Compounds in this conjugate series have shown synergistic activity against a panel of anaerobic bacteria, including those responsible for serious bacterial infections.

Chemically induced aneuploidy in germ cells. Part II of the report of the 2017 IWGT workgroup on assessing the risk of aneugens for carcinogenesis and hereditary diseases.

As part of the 7th International Workshops on Genotoxicity Testing held in Tokyo, Japan in November 2017, a workgroup of experts reviewed and assessed the risk of aneugens for human health. The present manuscript is one of three manuscripts from the workgroup and reports on the unanimous consensus reached on the evidence for aneugens affecting germ cells, their mechanisms of action and role in hereditary diseases. There are 24 chemicals with strong or sufficient evidence for germ cell aneugenicity providing robust support for the ability of chemicals to induce germ cell aneuploidy. Interference with microtubule dynamics or inhibition of topoisomerase II function are clear characteristics of germ cell aneugens. Although there are mechanisms of chromosome segregation that are unique to germ cells, there is currently no evidence for germ cell-specific aneugens. However, the available data are heavily skewed toward chemicals that are aneugenic in somatic cells. Development of high-throughput screening assays in suitable animal models for exploring additional targets for aneuploidy induction, such as meiosis-specific proteins, and to prioritize chemicals for the potential to be germ cell aneugens is encouraged. Evidence in animal models support that: oocytes are more sensitive than spermatocytes and somatic cells to aneugens; exposure to aneugens leads to aneuploid conceptuses; and, the frequencies of aneuploidy are similar in germ cells and zygotes. Although aneuploidy in germ cells is a significant cause of infertility and pregnancy loss in humans, there is currently limited evidence that aneugens induce hereditary diseases in human populations because the great majority of aneuploid conceptuses die in utero. Overall, the present work underscores the importance of protecting the human population from exposure to chemicals that can induce aneuploidy in germ cells that, in contrast to carcinogenicity, is directly linked to an adverse outcome.

Targets and mechanisms of chemically induced aneuploidy. Part 1 of the report of the 2017 IWGT workgroup on assessing the risk of aneugens for carcinogenesis and hereditary diseases.

An aneuploidy workgroup was established as part of the 7th International Workshops on Genotoxicity Testing. The workgroup conducted a review of the scientific literature on the biological mechanisms of aneuploidy in mammalian cells and methods used to detect chemical aneugens. In addition, the current regulatory framework was discussed, with the objective to arrive at consensus statements on the ramifications of exposure to chemical aneugens for human health risk assessment. As part of these efforts, the workgroup explored the use of adverse outcome pathways (AOPs) to document mechanisms of chemically induced aneuploidy in mammalian somatic cells. The group worked on two molecular initiating events (MIEs), tubulin binding and binding to the catalytic domain of aurora kinase B, which result in several adverse outcomes, including aneuploidy. The workgroup agreed that the AOP framework provides a useful approach to link evidence for MIEs with aneuploidy on a cellular level. The evidence linking chemically induced aneuploidy with carcinogenicity and hereditary disease was also reviewed and is presented in two companion papers. In addition, the group came to the consensus that the current regulatory test batteries, while not ideal, are sufficient for the identification of aneugens and human risk assessment. While it is obvious that there are many different MIEs that could lead to the induction of aneuploidy, the most commonly observed mechanisms involving chemical aneugens are related to tubulin binding and, to a lesser extent, inhibition of mitotic kinases. The comprehensive review presented here should help with the identification and risk management of aneugenic agents.

Role of aneuploidy in the carcinogenic process: Part 3 of the report of the 2017 IWGT workgroup on assessing the risk of aneugens for carcinogenesis and hereditary diseases.

Aneuploidy is regarded as a hallmark of cancer, however, its role is complex with both pro- and anti-carcinogenic effects evident. In this IWGT review, we consider the role of aneuploidy in cancer biology; cancer risk associated with constitutive aneuploidy; rodent carcinogenesis with known chemical aneugens; and chemotherapy-related malignant neoplasms. Aneuploidy is seen at various stages in carcinogenesis. However, the relationship between induced aneuploidy occurring after exposure and clonal aneuploidy present in tumours is not clear. Recent evidence indicates that the induction of chromosomal instability (CIN), may be more important than aneuploidy per se, in the carcinogenic process. Down Syndrome, trisomy 21, is associated with altered hematopoiesis in utero which, in combination with subsequent mutations, results in an increased risk for acute megakaryoblastic and lymphoblastic leukemias. In contrast, there is reduced cancer risk for most solid tumours in Down Syndrome. Mouse models with high levels of aneuploidy are also associated with increased cancer risk for particular tumours with long latencies, but paradoxically other types of tumour often show decreased incidence. The aneugens reviewed that induce cancer in humans and animals all possess other carcinogenic properties, such as mutagenicity, clastogenicity, cytotoxicity, organ toxicities, hormonal and epigenetic changes which likely account for, or interact with aneuploidy, to cause carcinogenesis. Although the role that aneuploidy plays in carcinogenesis has not been fully established, in many cases, it may not play a primary causative role. Tubulin-disrupting aneugens that do not possess other properties linked to carcinogenesis, were not carcinogenic in rodents. Similarly, in humans, for the tubulin-disrupting aneugens colchicine and albendazole, there is no reported association with increased cancer risk. There is a need for further mechanistic studies on agents that induce aneuploidy, particularly by mechanisms other than tubulin disruption and to determine the role of aneuploidy in pre-neoplastic events and in early and late stage neoplasia.

Staphylococcus aureus Leukocidins Target Endothelial DARC to Cause Lethality in Mice.

The pathogenesis of Staphylococcus aureus is thought to depend on the production of pore-forming leukocidins that kill leukocytes and lyse erythrocytes. Two leukocidins, Leukocidin ED (LukED) and γ-Hemolysin AB (HlgAB), are necessary and sufficient to kill mice upon infection and toxin challenge. We demonstrate that LukED and HlgAB cause vascular congestion and derangements in vascular fluid distribution that rapidly cause death in mice. The Duffy antigen receptor for chemokines (DARC) on endothelial cells, rather than leukocytes or erythrocytes, is the critical target for lethality. Consistent with this, LukED and HlgAB injure primary human endothelial cells in a DARC-dependent manner, and mice with DARC-deficient endothelial cells are resistant to toxin-mediated lethality. During bloodstream infection in mice, DARC targeting by S. aureus causes increased tissue damage, organ dysfunction, and host death. The potential for S. aureus leukocidins to manipulate vascular integrity highlights the importance of these virulence factors.

Identification of biologic agents to neutralize the bicomponent leukocidins of Staphylococcus aureus.

A key aspect underlying the severity of infections caused by Staphylococcus aureus is the abundance of virulence factors that the pathogen uses to thwart critical components of the human immune response. One such mechanism involves the destruction of host immune cells by cytolytic toxins secreted by S. aureus, including five bicomponent leukocidins: PVL, HlgAB, HlgCB, LukED, and LukAB. Purified leukocidins can lyse immune cells ex vivo, and systemic injections of purified LukED or HlgAB can acutely kill mice. Here, we describe the generation and characterization of centyrins that bind S. aureus leukocidins with high affinity and protect primary human immune cells from toxin-mediated cytolysis. Centyrins are small protein scaffolds derived from the fibronectin type III-binding domain of the human protein tenascin-C. Although centyrins are potent in tissue culture assays, their short serum half-lives limit their efficacies in vivo. By extending the serum half-lives of centyrins through their fusion to an albumin-binding consensus domain, we demonstrate the in vivo efficacy of these biologics in a murine intoxication model and in models of both prophylactic and therapeutic treatment of live S. aureus systemic infections. These biologics that target S. aureus virulence factors have potential for treating and preventing serious staphylococcal infections.