A walk in the PARC: developing and implementing 21st century chemical risk assessment in Europe.

Current approaches for the assessment of environmental and human health risks due to exposure to chemical substances have served their purpose reasonably well. Nevertheless, the systems in place for different uses of chemicals are faced with various challenges, ranging from a growing number of chemicals to changes in the types of chemicals and materials produced. This has triggered global awareness of the need for a paradigm shift, which in turn has led to the publication of new concepts for chemical risk assessment and explorations of how to translate these concepts into pragmatic approaches. As a result, next-generation risk assessment (NGRA) is generally seen as the way forward. However, incorporating new scientific insights and innovative approaches into hazard and exposure assessments in such a way that regulatory needs are adequately met has appeared to be challenging. The European Partnership for the Assessment of Risks from Chemicals (PARC) has been designed to address various challenges associated with innovating chemical risk assessment. Its overall goal is to consolidate and strengthen the European research and innovation capacity for chemical risk assessment to protect human health and the environment. With around 200 participating organisations from all over Europe, including three European agencies, and a total budget of over 400 million euro, PARC is one of the largest projects of its kind. It has a duration of seven years and is coordinated by ANSES, the French Agency for Food, Environmental and Occupational Health & Safety.

Body mass index and body fat distribution and new-onset atrial fibrillation: Substudy of the European Prospective Investigation into Cancer and Nutrition in Norfolk (EPIC-Norfolk) study.

BACKGROUND AND AIM: Obesity is a recognized risk factor for new-onset atrial fibrillation (AF). The association between body fat distribution, which is measured by body mass index (BMI) and waist-hip ratio (WHR), its changes, and new-onset AF is conflicting. METHODS AND RESULTS: Participants of the European Prospective Investigation into Cancer and Nutrition in Norfolk cohort study were included, with exclusion criteria of prevalent AF, rheumatic heart disease, and cancer. AF was confirmed by the International Classification of Diseases-10 hospital discharge code I48. Adjusted sex-specific Cox proportional hazards models were used to quantify the AF risk per 1 standard deviation increase and for quintiles of adiposity indices. A total of 10,885 men and 12,857 women were followed up for a median of 19 years, yielding 451,098 person-years. New-onset AF was diagnosed in 1408 (12.9%) men and 1102 (8.6%) women. Multivariable analyses showed that BMI predicted new-onset AF in all, while WHR predicted only in men. New-onset AF risk gradually increased across the range of adiposity indices: for men in the highest BMI quintile, HR: 1.59 (CI 1.32-1.91, p for trend<0.001), whereas for women in the highest BMI quintile, HR: 1.52 (CI 1.23-1.88, p for trend<0.001). Further, for men in the highest WHR quintile, HR: 1.31 (CI 1.09-1.57, p for trend: 0.01), whereas for women in the highest WHR quintile, HR: 1.12 (CI 0.90-1.41, p for trend: 0.17). The change in BMI and WHR was similar in participants with or without new-onset AF. CONCLUSIONS: An increased body mass, as measured by BMI, is associated with an increased risk of developing new-onset AF. More abdominal fat distribution, as measured by WHR, is associated with an increased risk of developing new-onset AF in men but not in women.

A physiologically based pharmacokinetic model for chickens exposed to feed supplemented with monensin during their lifetime.

We developed a flow-limited physiologically based pharmacokinetic model for residues of monensin in chickens and evaluated its predictive ability by comparing it with an external data set describing concentration decays after the end of treatment. One advantage of this model is that the values for most parameters (34 of 38) were taken directly from the literature or from field data (for growth and feed intake). Our model included growth (changes in body weight) to describe exposure throughout the life of the chicken. We carried out a local sensitivity analysis to evaluate the relative importance of model parameters on model outputs and revealed the predominant influence of 19 parameters (including three estimated ones): seven pharmacokinetic parameters, five physiological parameters and seven animal performance parameters. Our model estimated the relative bioavailability of monensin as feed additive at 3.9%, which is even lower than the absolute bioavailability in solution (29.91%). Our model can be used for extrapolations of farming conditions, such as monensin supplementation or building lighting programme (which may have a significant impact for short half-life molecules such as monensin). This validated PBPK model may also be useful for interspecies extrapolations or withdrawal period calculations for modified dosage regimens.

The present and future of withdrawal period calculations for milk in the European Union: focus on heterogeneous, nonmonotonic data.

Harmonization of the method for calculating the withdrawal period for milk dates from the 1990s. European harmonization has led to guidance with three accepted methods for determining the withdrawal period for milk that are currently applicable. These three methods can be used by marketing authorization holders, but, in some cases, their diversity can lead to very different withdrawal periods. This is particularly the case when concentrations in milk are nonmonotonic and heterogeneous, meaning that concentrations strictly increase and then strictly decrease with significant interindividual variability in the time to reach the maximal concentration. Here, we first describe the concepts associated with the different methods used in the harmonized approach currently applicable for the determination of milk withdrawal periods, and then, we propose the application of a modern pharmacometric tool. Finally, with a nonmonotonic heterogeneous dataset, we illustrate the usefulness of this tool in comparison with the three currently applicable methods and discuss the limitations and advantages of each method.

The present and future of withdrawal period calculations for milk in the European Union: dealing with data below the limit of quantification.

The assessment of withdrawal periods for milk is affected by the occurrence of data below the lower analytical quantification limit (BLQ data) and the resulting uncertainty. The current regulatory approach for dealing with BLQ residues is simple and easy: BLQ data (and missing data) are arbitrarily reassigned a value of one-half the LOQ before any calculation on the data with one of the three currently applicable methods. Here, we reconsider the determination of the withdrawal period of milk with data below the limit of quantification. Theoretical background on analytical limits and pharmacometric considerations will be established. Then, we analyze the uncertainty problems caused by the current approach and propose a calculation solution (maximum-likelihood estimation handling left-censored data) included in nonlinear mixed-effects modeling. Finally, we illustrate this issue using a case example.

CD8+ T cells drive autoimmune hematopoietic stem cell dysfunction and bone marrow failure.

Bone marrow (BM) failure syndrome encompasses a group of disorders characterized by BM stem cell dysfunction, resulting in varying degrees of hypoplasia and blood pancytopenia, and in many patients is autoimmune and inflammatory in nature. The important role of T helper 1 (Th1) polarized CD4+ T cells in driving BM failure has been clearly established in several models. However, animal model data demonstrating a functional role for CD8+ T cells in BM dysfunction is largely lacking and our objective was to test the hypothesis that CD8+ T cells play a non-redundant role in driving BM failure. Clinical evidence implicates a detrimental role for CD8+ T cells in BM failure and a beneficial role for Foxp3+ regulatory T cells (Tregs) in maintaining immune tolerance in the BM. We demonstrate that IL-2-deficient mice, which have a deficit in functional Tregs, develop spontaneous BM failure. Furthermore, we demonstrate a critical role for CD8+ T cells in the development of BM failure, which is dependent on the cytokine, IFNγ. CD8+ T cells promote hematopoietic stem cell dysfunction and depletion of myeloid lineage progenitor cells, resulting in anemia. Adoptive transfer experiments demonstrate that CD8+ T cells dramatically expedite disease progression and promote CD4+ T cell accumulation in the BM. Thus, BM dysregulation in IL-2-deficient mice is mediated by a Th1 and IFNγ-producing CD8+ T cell (Tc1) response.

Antimicrobial residues in foods of animal origin in Africa: public health risks.

The authors report on the current status of work on residues of veterinary medicinal products and, in particular, antimicrobial residues in foods of animal origin. This review focuses on residues of veterinary antimicrobials, antimicrobials used in livestock production, the concept of residues, and antimicrobial residues in foods of animal origin. Only one antimicrobial substance has been approved in the West African Economic and Monetary Union, compared with 16 substances in Benin and 56 in the European Union. The issue of antimicrobial residues in foods of animal origin has rarely been a serious concern in developing countries, in contrast to the situation in Europe. However, while the prevalence of veterinary drug residues in foods of animal origin is less than 1% in Europe, in some African countries it can be as high as 94%. Antimicrobial residues in foods of animal origin can cause allergies, cancer, alterations in the intestinal flora, bacterial resistance and the inhibition of fermentation in the dairy industry. The harmonisation of regulations in Africa could reduce the circulation of prohibited antimicrobials and lead to the implementation of a plan for the control and surveillance of residues from veterinary medicinal products in foods of animal origin.

Structural and electro-anatomical characterization of the equine pulmonary veins: implications for atrial fibrillation.

INTRODUCTION/OBJECTIVES: Spontaneous pulmonary vein (PV) activity triggers atrial fibrillation (AF) in humans. Although AF frequently occurs in horses, the origin remains unknown. This study investigated the structural and electro-anatomical properties of equine PVs to determine the potential presence of an arrhythmogenic substrate. ANIMALS, MATERIALS AND METHODS: Endocardial three-dimensional electro-anatomical mapping (EnSite Precision) using high-density (HD) catheters was performed in 13 sedated horses in sinus rhythm. Left atrium (LA) access was obtained retrogradely through the carotid artery. Post-mortem, tissue was harvested from the LA, right atrium (RA), and PVs for histological characterization and quantification of ion channel expression using immunohistochemical analysis. RESULTS: Geometry, activation maps, and voltage maps of the PVs were created and a median of four ostia were identified. Areas of reduced conduction were found at the veno-atrial junction. The mean myocardial sleeve length varied from 28 ± 13 to 49 ± 22 mm. The PV voltage was 1.2 ± 1.4 mV and lower than the LA (3.4 ± 0.9 mV, P < 0.001). The fibrosis percentage was higher in PV myocardium (26.1 ± 6.6%) than LA (14.5 ± 5.0%, P = 0.003). L-type calcium channel (CaV1.2) expression was higher in PVs than LA (P = 0.001). T-type calcium channels (CaV3.3), connexin-43, ryanodine receptor-2, and small conductance calcium-activated potassium channel-3 was expressed in PVs. CONCLUSIONS: The veno-atrial junction had lower voltages, increased structural heterogeneity and areas of slower conduction. Myocardial sleeves had variable lengths, and a different ion channel expression compared to the atria. Heterogeneous properties of the PVs interacting with the adjacent LA likely provide the milieu for re-entry and AF initiation.

Risk assessment of coccidostatics during feed cross-contamination: animal and human health aspects.

Coccidiosis, an intestinal plasmodium infection, is a major infectious disease in poultry and rabbits. Eleven different coccidiostats are licensed in the EU for the prevention of coccidiosis in these animal species. According to their chemical nature and main biological activity, these compounds can be grouped as ionophoric (monensin, lasalocid sodium, salinomycin, narasin, maduramicin and semduramicin) or non-ionophoric (robenidine, decoquinate, nicarbazin, diclazuril, and halofuginone) substances. Coccidiostats are used as feed additives, mixed upon request into the compounded feed. During the technical process of commercial feed production, cross-contamination of feed batches can result in the exposure of non-target animals and induce adverse health effects in these animals due to a specific sensitivity of mammalian species as compared to poultry. Residue formation in edible tissues of non-target species may result in unexpected human exposure through the consumption of animal products. This review presents recent risk assessments performed by the Scientific Panel on Contaminants in the Food Chain (CONTAM) of the European Food Safety Authority (EFSA). The health risk to non-target species that would result from the consumption of cross-contaminated feed with coccidostats at levels of 2, 5 or 10% was found to be negligible for most animal species with the exception of salinomycin and monensin in horses because of the particular sensitivity for which toxicity may occur when cross-contamination exceeds 2% and 5% respectively. Kinetic data and tissue analyses showed that residues of coccidiostats may occur in the liver and eggs in some cases. However, the level of residues of each coccidiostat in edible animal tissues remained sufficiently low that the aggregate exposure of consumers would not exceed the established acceptable daily intake (ADI) of each coccidiostat. It could be concluded that technical cross-contamination of animal feeds would not be expected to adversely affect the health of consumers.

Trends in the use of implantable cardioverter defibrillators in Australia: a 10-year nationwide study from 2000-2009.

BACKGROUND: Implantable cardioverter defibrillators (ICD) have been demonstrated to reduce mortality in survivors of life-threatening arrhythmias (secondary prevention) and in patients at increased risk of sudden cardiac death (primary prevention). Other nations have reported significant increases in ICD use in recent years. AIM: To investigate Australian nationwide trends of ICD procedures over a 10-year period (2000-2009). METHODS: A retrospective analysis of the Australian Institute of Health and Welfare's National Hospital Morbidity Database was performed to determine the annual number of ICD implantation and replacement procedures between 2000 and 2009. Rates were calculated using Australian Bureau of Statistics data on the annual estimated population. Time trends in the yearly procedure number and rate were analysed using negative binomial regression models with comparisons made by age and sex. RESULTS: The number of new ICD implantations increased from 708 to 3198 procedures between 2000 and 2009. Replacement procedures increased from 290 to 1378. The implantation rate (per million) increased from 37.0 to 145.6 and the replacement rate from 15.1 to 62.7. When rates were adjusted for age and sex, the implantation rate increased annually by 15.8% and the replacement rate by 16.6% (P < 0.0001). Procedures occurred most commonly in men (implantations: 80.1%; replacements: 78.0%) between ages 70-79. CONCLUSIONS: ICD procedures increased significantly in Australia between 2000-2009. Despite these increases, other studies have suggested ICD devices are currently under-utilised. During the study period, males accounted for the majority of ICD procedures. While there are numerous reasons for this, it is not known if device under-use is more common in females.

The Bayesian microbial subtyping attribution model: robustness to prior information and a proposition.

Attributing foodborne illnesses to food sources is essential to conceive, prioritize, and assess the impact of public health policy measures. The Bayesian microbial subtyping attribution model by Hald et al. is one of the most advanced approaches to attribute sporadic cases; it namely allows taking into account the level of exposure to the sources and the differences between bacterial types and between sources. This step forward requires introducing type and source-dependent parameters, and generates overparameterization, which was addressed in Hald's paper by setting some parameters to constant values. We question the impact of the choices made for the parameterization (parameters set and values used) on model robustness and propose an alternative parameterization for the Hald model. We illustrate this analysis with the 2005 French data set of non-typhi Salmonella. Mullner's modified Hald model and a simple deterministic model were used to compare the results and assess the accuracy of the estimates. Setting the parameters for bacterial types specific to a unique source instead of the most frequent one and using data-based values instead of arbitrary values enhanced the convergence and adequacy of the estimates and led to attribution estimates consistent with the other models' results. The type and source parameters estimates were also coherent with Mullner's model estimates. The model appeared to be highly sensitive to parameterization. The proposed solution based on specific types and data-based values improved the robustness of estimates and enabled the use of this highly valuable tool successfully with the French data set.

Comparison of the oral bioavailability and tissue disposition of monensin and salinomycin in chickens and turkeys.

The current study describes the pharmacokinetic parameters of two carboxylic polyether ionophores: monensin in turkeys and salinomycin in chickens. These data can be used to understand and predict the occurrence of undesirable residues of coccidiostats in edible tissues of these animal species. Special attention is paid to the distribution of residues between the different edible tissues during and at the end of the treatment period. For the bioavailability studies, monensin was administered to turkeys intravenously, in the left wing vein, at a dose of 0.4 mg /kg and orally at a dose of 20 mg /kg. Salinomycin was administered to chickens intravenously, in the left wing vein, at a dose of 0.25 mg /kg and orally at a dose of 2.5 mg /kg. Residue studies were carried out with supplemented feed at the rate of 100 mg /kg of feed for monensin in turkeys and 70 mg /kg for salinomycin in chickens, respectively. Coccidiostats had a low bioavailability in poultry (around 30% for monensin in chickens, around 1% for monensin in turkeys and around 15% for salinomycin in chickens). Monensin in chickens had a longer terminal half-life (between 3.07 and 5.55 h) than both monensin in turkeys (between 1.36 and 1.55 h) and salinomycin in chickens (between 1.33 and 1.79 h). The tissue /plasma partition coefficients showed a higher affinity of both monensin and salinomycin for fat, followed by liver and muscle tissue. The depletion data showed a fairly rapid elimination of coccidiostats in all the tissues after cessation of treatment. According to the results of depletion studies, a withdrawal period of 1 day seems sufficient to avoid undesirable exposure of consumers.

Pharmacodynamic modeling of in vitro activity of marbofloxacin against Escherichia coli strains.

A mathematical pharmacodynamic model was developed to describe the bactericidal activity of marbofloxacin against Escherichia coli strains with reduced susceptibility levels (determined using MICs) under optimal and intestinal growth conditions. Model parameters were estimated using nonlinear least-square curve-fitting procedures for each E. coli strain. Parameters related to bactericidal activity were subsequently analyzed using a maximum-effect (E(max)) model adapted to account for a direct and a delayed effect. While net growth rates did not vary significantly with strain susceptibility, culture medium had a major effect. The bactericidal activity of marbofloxacin was closely associated with the concentration and the duration of exposure of the bacteria to the antimicrobial agent. The value of the concentration inducing a half-maximum effect (C(50)) was highly correlated with MIC values (R(2) = 0.87 and R(2) = 0.94 under intestinal and optimal conditions, respectively). Our model reproduced the time-kill kinetics with good accuracy (R(2) of >0.90) and helped explain observed regrowth.

Patient preferences for next generation neural prostheses to restore bladder function.

STUDY DESIGN: A survey administered to 66 individuals with spinal cord injury (SCI) implementing a choice-based conjoint (CBC) analysis. Six attributes with three levels each were defined and used to generate choice sets with treatment scenarios. Patients were asked to choose the scenario that they preferred most. OBJECTIVES: To determine the utility weights for treatment characteristics as well as the overall preference for the three types of neural prostheses (NP), that is Brindley, rhizotomy-free Brindley, and pudendal nerve stimulation. Earlier studies have revealed the importance of restoration of bladder function, but no studies have been performed to determine the importance of NP features. SETTING: Two academic affiliated medical systems' SCI outpatient and inpatient rehabilitation programs, Cleveland, OH. METHODS: CBC analysis followed by multinomial logit modeling. Individual part-worth utilities were estimated using hierarchical Bayes. RESULTS: Side effects had the greatest significant impact on subject choices, followed by the effectiveness on continence and voiding. NPs with rhizotomy-free sacral root stimulation were preferred (45% first choice) over pudendal afferent nerve stimulation (39% second choice) and sacral root stimulation with rhizotomy (53% third choice). Almost 20% did not want to have an NP at all times. CONCLUSION: CBC has shown to be a valuable tool to support design choices. The data showed that persons would prefer a bladder NP with minimally invasive electrodes, which would give them complete bladder function, with no side effects and that can be operated by pushing a button and they do not have to recharge themselves.

Reflection paper on MRSA in food-producing and companion animals: epidemiology and control options for human and animal health.

The scope of this reflection paper was to review the latest research on the risk of MRSA infection and colonization in animals. Attention focused on occurrence, risk factors for colonization and infection, and human contact hazard for livestock, horses, and companion animals. Whereas the clonal relationship between MRSA strains of CC398 is straightforward in livestock this is less obvious in horses. Small companion animals typically share MRSA strains that seem to exchange with a human reservoir. Management and therapeutic options have been suggested for livestock, horses, companion animals, as well as instructions on safety measures for persons in contact with animals. Conclusions were drawn with emphasis on future research activities, especially to confirm the apparent evolution of the organism and to demonstrate efficiency of control strategies.

Practical applications of studies on the TSH receptor and TSH receptor autoantibodies.

Studies on the TSH receptor (TSHR) have numerous practical applications in vitro and in vivo. For example human monoclonal autoantibodies (MAbs) to the TSHR are useful reagents for in vitro diagnostics. Measurement of TSHR autoantibodies (TRAbs) is helpful in diagnosis and management of autoimmune thyroid disease. Currently available highly sensitive and specific assays to measure TRAbs use the human TSHR MAb M22 instead of the TSH. Furthermore, preparations of the human TSHR MAb M22 are useful as the World Health Organisation International Standard for thyroid stimulating antibody and for calibration of the assays for measuring TRAbs. Preparations of thermostabilised TSHR extracellular domain have recently become available and this is likely to have an impact on improvements in specificity testing for TRAb assays. In addition the stable TSHR preparations have practical application for specific immunoadsorption of patient serum TRAbs. Human TSHR MAbs also have promising prospects as new therapeutics. Autoantibodies with TSHR antagonistic activities are "natural" inhibitors of TSHR stimulation and are expected to be helpful in controlling TSHR activity in patients with Graves' disease, Graves' ophthalmopathy and thyroid cancer.

B Cells Improve Overall Survival in HPV-Associated Squamous Cell Carcinomas and Are Activated by Radiation and PD-1 Blockade.

PURPOSE: To characterize the role of B cells on human papilloma virus (HPV)-associated cancer patient outcomes and determine the effects of radiation and PD-1 blockade on B-cell populations. EXPERIMENTAL DESIGN: Tumor RNA-sequencing data from over 800 patients with head and neck squamous cell carcinoma (HNSCC) and cervical cancer, including a prospective validation cohort, was analyzed to study the impact of B-cell gene expression on overall survival (OS). A novel murine model of HPV+ HNSCC was used to study the effects of PD-1 blockade and radiotherapy on B-cell activation, differentiation, and clonality including analysis by single-cell RNA-sequencing and B-cell receptor (BCR)-sequencing. Human protein microarray was then used to quantify B-cell-mediated IgG and IgM antibodies to over 16,000 proteins in the serum of patients treated on a clinical trial with PD-1 blockade. RESULTS: RNA-sequencing identified CD19 and IGJ as novel B-cell prognostic biomarkers for 3-year OS (HR, 0.545; P < 0.001). PD-1 blockade and radiotherapy enhance development of memory B cells, plasma cells, and antigen-specific B cells. BCR-sequencing found that radiotherapy enhances B-cell clonality, decreases CDR3 length, and induces B-cell somatic hypermutation. Single-cell RNA-sequencing identified dramatic increases in B-cell germinal center formation after PD-1 blockade and radiotherapy. Human proteome array revealed enhanced IgG and IgM antibody responses in patients who derived clinical benefit but not those with progressive disease after treatment with PD-1 blockade. CONCLUSIONS: These findings establish a key role for B cells in patient outcomes and responses to PD-1 blockade in HPV-associated squamous cell carcinomas and demonstrate the need for additional diagnostics and therapeutics targeting B cells.

Long-Term Clinical Outcomes of Intercalary Allograft Reconstruction for Lower-Extremity Bone Tumors.

BACKGROUND: Improved survival rates for patients with primary bone tumors of the extremities have increased the demand for reliable and durable reconstruction techniques. Some authors have stated that, after successful ingrowth, allografts are a durable long-term solution. This hypothesis is largely based on small studies with short-to-midterm follow-up. In order to determine the durability of intercalary allograft reconstructions in the lower extremities, we evaluated the long-term clinical outcomes at a minimum of 10 years. METHODS: All patients who received an intercalary allograft reconstruction in a lower extremity between 1980 and 2006 were included in this retrospective multicenter cohort study. One hundred and thirty-one patients with a median age of 19 years were included. Eighty-nine (68%) had a femoral reconstruction, and 42 (32%) had a tibial reconstruction. The most prevalent diagnoses were osteosarcoma (55%), Ewing sarcoma (17%), and chondrosarcoma (12%). The median follow-up was 14 years. A competing risk model was employed to estimate the cumulative incidences of mechanical failure and infection. Patient mortality or progression of the disease was used as a competing event. RESULTS: Nonunion occurred in 21 reconstructions (16%), after a median of 16 months, and was associated with intramedullary nail-only fixation (p < 0.01) and fixation with nonbridging plate(s) (p = 0.03). Allograft fracture occurred in 25 reconstructions (19%) after a median of 42 months (range, 4 days to 21.9 years). Thirteen (52%) of the allograft fractures occurred within 5 years; 8 (32%), between 5 and 10 years; and 4 (16%), at >10 years. With failure for mechanical reasons as the end point, the cumulative incidences of reconstruction failure at 5, 10, and 15 years were 9%, 14%, and 21%, respectively. CONCLUSIONS: Intercalary allograft reconstruction is an acceptable reconstructive option, mainly because of the absence of superior alternatives with a known track record. However, a considerable and continuing risk of mechanical complications should be taken into account. LEVEL OF EVIDENCE: Therapeutic Level IV. See Instructions for Authors for a complete description of levels of evidence.

HPV16 E5 Mediates Resistance to PD-L1 Blockade and Can Be Targeted with Rimantadine in Head and Neck Cancer.

There is a critical need to understand mechanisms of resistance and to develop combinatorial strategies to improve responses to checkpoint blockade immunotherapy (CBI). Here, we uncover a novel mechanism by which the human papillomavirus (HPV) inhibits the activity of CBI in head and neck squamous cell carcinoma (HNSCC). Using orthotopic HNSCC models, we show that radiation combined with anti-PD-L1 immunotherapy significantly enhanced local control, CD8+ memory T cells, and induced preferential T-cell homing via modulation of vascular endothelial cells. However, the HPV E5 oncoprotein suppressed immune responses by downregulating expression of major histocompatibility complex and interfering with antigen presentation in murine models and patient tumors. Furthermore, tumors expressing HPV E5 were rendered entirely resistant to anti-PD-L1 immunotherapy, and patients with high expression of HPV16 E5 had worse survival. The antiviral E5 inhibitor rimantadine demonstrated remarkable single-agent antitumor activity. This is the first report that describes HPV E5 as a mediator of resistance to anti-PD-1/PD-L1 immunotherapy and demonstrates the antitumor activity of rimantadine. These results have broad clinical relevance beyond HNSCC to other HPV-associated malignancies and reveal a powerful mechanism of HPV-mediated immunosuppression, which can be exploited to improve response rates to checkpoint blockade. SIGNIFICANCE: This study identifies a novel mechanism of resistance to anti-PD-1/PD-L1 immunotherapy mediated by HPV E5, which can be exploited using the HPV E5 inhibitor rimantadine to improve outcomes for head and neck cancer patients. GRAPHICAL ABSTRACT: http://cancerres.aacrjournals.org/content/canres/80/4/732/F1.large.jpg.

Macrophage Syk-PI3Kγ Inhibits Antitumor Immunity: SRX3207, a Novel Dual Syk-PI3K Inhibitory Chemotype Relieves Tumor Immunosuppression.

Macrophages (MΦ) play a critical role in tumor growth, immunosuppression, and inhibition of adaptive immune responses in cancer. Hence, targeting signaling pathways in MΦs that promote tumor immunosuppression will provide therapeutic benefit. PI3Kγ has been recently established by our group and others as a novel immuno-oncology target. Herein, we report that an MΦ Syk-PI3K axis drives polarization of immunosuppressive MΦs that establish an immunosuppressive tumor microenvironment in in vivo syngeneic tumor models. Genetic or pharmacologic inhibition of Syk and/or PI3Kγ in MΦs promotes a proinflammatory MΦ phenotype, restores CD8+ T-cell activity, destabilizes HIF under hypoxia, and stimulates an antitumor immune response. Assay for transposase-accessible Chromatin using Sequencing (ATAC-seq) analyses on the bone marrow-derived macrophages (BMDM) show that inhibition of Syk kinase promotes activation and binding of NF-κB motif in SykMC-KO BMDMs, thus stimulating immunostimulatory transcriptional programming in MΦs to suppress tumor growth. Finally, we have developed in silico the "first-in-class" dual Syk/PI3K inhibitor, SRX3207, for the combinatorial inhibition of Syk and PI3K in one small molecule. This chemotype demonstrates efficacy in multiple tumor models and represents a novel combinatorial approach to activate antitumor immunity.