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Funder: Kennedy Trust Rheumatology Research Prize Studentship, Funder: DFG Cluster of Excellence ���Precision Medicine in Chronic In-flammation��� (PMI; ID: EXC2167), Funder: EC | EC Seventh Framework Programm | FP7 Ideas: European Research Council (FP7-IDEAS-ERC - Specific Programme: ���Ideas��� Implementing the Seventh Framework Programme of the European Community for Research, Technological Development and Demonstration Activities (2007 to 2013)); doi: https://doi.org/10.13039/100011199; Grant(s): 715772, Funder: NWO-VIDI grant 016.178.056, the Netherlands Heart Foundation CVON grant 2018-27, and NWO Gravitation grant ExposomeNL, Funder: Li Ka Shing Foundation (Li Ka Shing Foundation Limited); doi: https://doi.org/10.13039/100007421, Irritable bowel syndrome (IBS) results from disordered brain���gut interactions. Identifying susceptibility genes could highlight the underlying pathophysiological mechanisms. We designed a digestive health questionnaire for UK Biobank and combined identified cases with IBS with independent cohorts. We conducted a genome-wide association study with 53,400 cases and 433,201 controls and replicated significant associations in a 23andMe panel (205,252 cases and 1,384,055 controls). Our study identified and confirmed six genetic susceptibility loci for IBS. Implicated genes included NCAM1, CADM2, PHF2/FAM120A, DOCK9, CKAP2/TPTE2P3 and BAG6. The first four are associated with mood and anxiety disorders, expressed in the nervous system, or both. Mirroring this, we also found strong genome-wide correlation between the risk of IBS and anxiety, neuroticism and depression (rg > 0.5). Additional analyses suggested this arises due to shared pathogenic pathways rather than, for example, anxiety causing abdominal symptoms. Implicated mechanisms require further exploration to help understand the altered brain���gut interactions underlying IBS.

Background TH2 cells were thought to be a pivotal factor for initiation of the autoimmune blistering disease pemphigus. However, the role of other T-cell subsets in pemphigus pathogenesis remained unclear. Objective We aimed to characterize the exact phenotype of T cells responsible for the development of pemphigus. Methods Whole transcriptome shotgun sequencing was performed to determine differential gene expression in pemphigus lesions and skin of healthy individuals. The cutaneous cytokine signature was further evaluated by real-time quantitative PCR. In peripheral blood, the distribution of TH cell and folliclular helper (TFH) cell subsets was analyzed by flow cytometry. Finally, the capacity of TH and TFH cell subsets to induce desmoglein (Dsg)-specific autoantibodies by memory B cells was evaluated in coculture experiments. Results Transcriptome analysis of skin samples identified an IL-17A–dominated immune signature in patients with pemphigus, and Kyoto Encyclopedia of Genes and Genomes pathway analysis confirmed the dominance of the IL-17A signaling pathway. Increased expression of IL17A and associated cytokines was also detected by real-time quantitative PCR comparing lesional with perilesional or healthy skin. Interestingly, utilization of flow cytometry showed that patients with active pemphigus had elevated levels of circulating IL-17+, TH17, TFH17, and TFH17.1 cells. Notably, levels of TH17 and TFH17 cells correlated with levels of Dsg-specific CD19+CD27+ memory B cells, and patients with acute pemphigus showed higher levels of Dsg3-autoreactive TFH17 cells. Coculture experiments revealed TFH17 cells as primarily responsible for inducing Dsg-specific autoantibody production by B cells. Conclusion Our findings show that TFH17 cells are critically involved in the pathogenesis of pemphigus and offer novel targets for therapeutic intervention.

Hair is a defining feature of mammals and has critical functions, including protection, production of sebum, apocrine sweat and pheromones, social and sexual interactions, thermoregulation, and provision of stem cells for skin homeostasis, regeneration, and repair. The hair follicle (HF) is considered a “mini-organ,” consisting of intricate and well-organized structures which originate from HF stem and progenitor cells. Dermal papilla cells are the main components of the mesenchymal compartments in the hair bulb and are instrumental in generating signals to regulate the behavior of neighboring epithelial cells during the hair cycle. Mesenchymal-epithelial interactions within the dermal papilla niche drive HF embryonic development as well as the postnatal hair growth and regeneration cycle. This review summarizes the current understanding of HF development, repair, and regeneration, with special focus on cell signaling pathways governing these processes. In particular, we discuss emerging paradigms of molecular signaling governing the dermal papilla-epithelial cellular interactions during hair growth and maintenance and the recent progress made towards tissue engineering of human hair follicles.

Clinical and translational allergy : CTA 11(8), e12058 (2021). doi:10.1002/clt2.12058, Published by BioMed Central, London

Frontiers in microbiology 12, 728989 (2021). doi:10.3389/fmicb.2021.728989, Published by Frontiers Media, Lausanne

The epidermis is the primary area of contact between the body and the environment, and it distinguishes between harmful exposures and those that should be tolerated. Discrimination between insults, and in particular the recognition of danger signals such as UVB, is mediated by innate immune receptors. Inflammasomes are one major innate mechanism that activate inflammatory caspases. In human keratinocytes, the importance of inflammasomes and the sensing of UVB and other danger signals are a matter of debate. Fenini et al. now provide evidence that the NLRP1 (rather than the NLRP3) inflammasome plays a key role in UVB sensing and subsequent IL-1β and -18 secretion by human keratinocytes.

European journal of immunology eji.202048879 (2020). doi:10.1002/eji.202048879, Published by Wiley-VCH, Weinheim

Prediction of spatial heterogeneity in disease incidence based on measurable spatial factors is a major goal of spatial epidemiology. There are a number of applied goals of these predictions, including appropriately targeting resources for surveillance and intervention and accurately quantifying disease burden. Although spatial heterogeneity is evident in the epidemiology of many diseases, several aspects of the biology of vector-borne diseases amplify this form of heterogeneity. Here, we review several aspects of this biology, highlighting seven distinct ways in which the biology of vector-borne diseases impacts understanding spatial heterogeneity in disease incidence. Whereas traditional methods place emphasis on spatial regression and other forms of statistical analysis of empirical data, the goal here is to offer a perspective on potential pitfalls of analyses that take data at face value and do not acknowledge the complex, nonlinear, and dynamic relationships between spatial patterns of disease incidence and spatial heterogeneity in transmission.

International journal of molecular sciences 21(22), 8859 (2020). doi:10.3390/ijms21228859 special issue: "Special Issue "Cell Death in Biology and Diseases" / Special Issue Editors: Dr. Peter Richter, Guest Editor; Dr. Marcus Krüger, Co-Guest Editor", Published by Molecular Diversity Preservation International, Basel

Several hundred thousand Zika cases have been reported across the Americas since 2015. Incidence of infection was likely much higher, however, due to a high frequency of asymptomatic infection and other challenges that surveillance systems faced. Using a hierarchical Bayesian model with empirically-informed priors, we leveraged multiple types of Zika case data from 15 countries to estimate subnational reporting probabilities and infection attack rates (IARs). Zika IAR estimates ranged from 0.084 (95% CrI: 0.067–0.096) in Peru to 0.361 (95% CrI: 0.214–0.514) in Ecuador, with significant subnational variability in every country. Totaling infection estimates across these and 33 other countries and territories, our results suggest that 132.3 million (95% CrI: 111.3-170.2 million) people in the Americas had been infected by the end of 2018. These estimates represent the most extensive attempt to determine the size of the Zika epidemic in the Americas, offering a baseline for assessing the risk of future Zika epidemics in this region., Author summary During the recent Zika epidemic in the Americas millions of people were likely infected, but the true size of the epidemic is unknown because of gaps in the surveillance system. The infection attack rate (IAR)—defined as the proportion of the population that was infected over the course of the epidemic—has important implications for the longer-term epidemiology of Zika in the region, such as the timing, location, and likelihood of future outbreaks. To estimate the IAR and the total number of people infected, we leveraged multiple types of Zika case data from 15 countries and territories where subnational data were publicly available. Datasets included confirmed and suspected Zika cases in pregnant women and in the total population, Zika-associated Guillan-Barré syndrome cases, and cases of congenital Zika syndrome. We used a hierarchical Bayesian model with empirically-informed priors that leveraged the different case report types to simultaneously estimate national and subnational reporting probabilities, the fraction of symptomatic infections, and subnational IARs. In these 15 countries and territories, estimates of Zika IAR ranged from 0.084 (95% CrI: 0.067–0.096) in Peru to 0.361 (95% CrI: 0.214–0.514) in Ecuador. Totaling these infection estimates across these and 33 other countries and territories in the region, our results suggest that 132.3 million (95% CrI: 111.3-170.2 million) people in the Americas were infected with ZIKV by the end of 2018.

BACKGROUND: HVTN 098, a randomized, double-blind, placebo-controlled trial, evaluated the safety, tolerability, and immunogenicity of PENNVAX-GP HIV DNA vaccine, administered with or without plasmid IL-12 (pIL-12), via intradermal (ID) or intramuscular (IM) electroporation (EP) in healthy, HIV-uninfected adults. The study tested whether PENNVAX-GP delivered via ID/EP at one-fifth the dose could elicit equivalent immune responses to delivery via IM/EP and whether inclusion of pIL-12 provided additional benefit. METHODS: Participants received DNA encoding HIV-1 env/gag/pol in 3 groups: 1.6 mg ID (ID no IL-12 group, n = 20), 1.6 mg ID + 0.4 mg pIL-12 (ID + IL-12 group, n = 30), 8 mg IM + 1 mg pIL-12 (IM + IL-12 group, n = 30), or placebo (n = 9) via EP at 0, 1, 3, and 6 months. Results of cellular and humoral immunogenicity assessments are reported. RESULTS: Following vaccination, the frequency of responders (response rate) to any HIV protein based on CD4(+) T cells expressing IFN-γ or IL-2 was 96% for both the ID + IL-12 and IM + IL-12 groups; CD8(+) T cell response rates were 64% and 44%, respectively. For ID delivery, the inclusion of pIL-12 increased CD4(+) T cell response rate from 56% to 96%. The frequency of responders was similar (≥90%) for IgG binding antibody to gp140 consensus Env across all groups, but the magnitude was higher in the ID + IL-12 group compared with the IM + IL-12 group. CONCLUSION: PENNVAX-GP DNA induced robust cellular and humoral immune responses, demonstrating that immunogenicity of DNA vaccines can be enhanced by EP route and inclusion of pIL-12. ID/EP was dose sparing, inducing equivalent, or in some aspects superior, immune responses compared with IM/EP. TRIAL REGISTRATION: ClinicalTrials.gov NCT02431767. FUNDING: This work was supported by National Institute of Allergy and Infectious Diseases (NIAID), U.S. Public Health Service grants, an HIV Vaccine Design and Development Team contract, Integrated Preclinical/Clinical AIDS Vaccine Development Program, and an NIH award.

Despite the routine development and distribution of seasonal influenza vaccines, influenza remains an important pathogen contributing to significant human morbidity as well as mortality each year. The seasonal variability of influenza creates a significant issue for vaccine development of seasonal strains that can afford protection from infection or disease based on serotype matching. It is appreciated that the globular head of the HA antigen contained in the vaccines generates antibodies that result in HAI activity that are a major correlates of the protection against a particular strain. Due to seasonal genetic changes in the HA protein, however, new vaccine strains are needed to be developed continually to match the new HA antigen of that seasons virus. A distinct advantage in seasonal vaccine development would be if a small group of antigens could be developed that could span many seasons without needed to be replaced due to this genetic drift. Here we report on a synthetic microconsensus approach that relies on a small collection of 4 synthetic H1HA DNA antigens which together induce broad protective HAI immunity spanning decades of H1 influenza viruses in mice, guinea pigs and non-human primates. The protective HAI titers induced by microconsensus immunogens are fully functional in vivo as immunized ferrets were completely protected from A/Mexico/InDRE4487/2009 virus infection and morbidity associated with lethal challenge. These results are encouraging that a limited easy-to-formulate collection of invariant antigens can be developed which can span seasonal vaccine changes allowing for continued immune protection.

This study has been conducted at the city of Diwaniah located 180 km south of Baghdad to examine the effect of potassium and copper floral application on the growth and yield of potato plants (var.Burren) during the 2015 spring season. Factorial experiment has been carried out in Random Complete Block Design (RCBD) with three replications and Nine treatments have been used which have three potassium concentrations at 0, 3000, and 6000 mg KL-1 designated respectively and three copper concentrations at 0, 2.5, and 5 mg CuL-1 designated respectively in addition to their interactions. The treatments are applied at three different timeframes which are 45 days after sowing, 15 days after the first spraying, and 15 days after the second spraying. Vegetative growth have been collected at the maturation stage then dried to obtain dry weight and N, P, K, and Cu have been estimated in plant leaves. Tubers are harvested to measure number and yield/plant, tubers average weight, total marketing yield and yield. The obtained data are statistically analyzed and averages are compared according to the least significant differences (LSD) at 5% level of significance and the results were as follows: The yield components which include tubers number as compared with the control treatment. Copper foliar application significantly increase vegetative traits of the yield. The K2 treatment has given the highest significant increase of main stem vegetative growthof the components which include tubers number as compared with the control treatment. The interaction treatments have given a significant result in terms of the both the vegetative and yield traits where K2* Cu2 give the most significant increase in term of vegetative growth while K2* Cu1 significantly increase the plant height and K1* Cu2 significantly affect the number of leaves. As for the yield component, K2*Cu2 show to have the most significant result in all traits under investigation. Finally.

Although Zika virus (ZIKV) infection is typically self-limiting, other associated complications such as congenital birth defects and the Guillain-Barré syndrome are well described. There are no approved vaccines against ZIKV infection.In this phase 1, open-label clinical trial, we evaluated the safety and immunogenicity of a synthetic, consensus DNA vaccine (GLS-5700) encoding the ZIKV premembrane and envelope proteins in two groups of 20 participants each. The participants received either 1 mg or 2 mg of vaccine intradermally, with each injection followed by electroporation (the use of a pulsed electric field to introduce the DNA sequence into cells) at baseline, 4 weeks, and 12 weeks.The median age of the participants was 38 years, and 60% were women; 78% were White and 22% Black; in addition, 30% were Hispanic. At the interim analysis at 14 weeks (i.e., after the third dose of vaccine), no serious adverse events were reported. Local reactions at the vaccination site (e.g., injection-site pain, redness, swelling, and itching) occurred in approximately 50% of the participants. After the third dose of vaccine, binding antibodies (as measured on enzyme-linked immunosorbent assay) were detected in all the participants, with geometric mean titers of 1642 and 2871 in recipients of 1 mg and 2 mg of vaccine, respectively. Neutralizing antibodies developed in 62% of the samples on Vero-cell assay. On neuronal-cell assay, there was 90% inhibition of ZIKV infection in 70% of the serum samples and 50% inhibition in 95% of the samples. The intraperitoneal injection of postvaccination serum protected 103 of 112 IFNAR knockout mice (bred with deletion of genes encoding interferon-α and interferon-β receptors) (92%) that were challenged with a lethal dose of ZIKV-PR209 strain; none of the mice receiving baseline serum survived the challenge. Survival was independent of the neutralization titer.In this phase 1, open-label clinical trial, a DNA vaccine elicited anti-ZIKV immune responses. Further studies are needed to better evaluate the safety and efficacy of the vaccine. (Funded by GeneOne Life Science and others; ZIKA-001 ClinicalTrials.gov number, NCT02809443.).

Background. We report the first-in-human safety and immunogenicity evaluation of PENNVAX-G DNA/modified vaccinia Ankara-Chiang Mai double recombinant (MVA-CMDR) prime-boost human immuonodeficiency virus (HIV) vaccine, with intramuscular DNA delivery by either Biojector 2000 needle-free injection system (Biojector) or CELLECTRA electroporation device. Methods. Healthy, HIV-uninfected adults were randomized to receive 4 mg of PENNVAX-G DNA delivered intramuscularly by Biojector or electroporation at baseline and week 4 followed by intramuscular injection of 10(8) plaque forming units of MVA-CMDR at weeks 12 and 24. The open-label part A was conducted in the United States, followed by a double-blind, placebo-controlled part B in East Africa. Solicited and unsolicited adverse events were recorded, and immune responses were measured. Results. Eighty-eight of 100 enrolled participants completed all study injections, which were generally safe and well tolerated, with more immediate, but transient, pain in the electroporation group. Cellular responses were observed in 57% of vaccine recipients tested and were CD4 predominant. High rates of binding antibody responses to CRF01_AE antigens, including gp70 V1 V2 scaffold, were observed. Neutralizing antibodies were detected in a peripheral blood mononuclear cell assay, and moderate antibody-dependent, cell-mediated cytotoxicity activity was demonstrated. Discussion. The PVG/MVA-CMDR HIV-1 vaccine regimen is safe and immunogenic. Substantial differences in safety or immunogenicity between modes of DNA delivery were not observed.

Background Chronic inflammatory skin diseases are characterized by controlled proliferation of keratinocytes. Here, activating transcription factor 3 (ATF3) might play a fundamental role. In these inflammatory diseases, proliferation is controlled and only rarely leads to cancer development which can be supported by an inflammatory microenvironment. ATF3 is a dual function protein as it suppresses pro-inflammatory IL-6 and IL-8, but also acts as a pro-oncogenic factor by the suppression of p53. We therefore analyzed ATF3 expression comparing myeloid cells with keratinocytes. Objective To dissect the bi-modal role of ATF3 we pharmacologically induced ATF3 and analyzed its influence on cytokine expression and secretion in a cell type specific manner. Methods Since inflammatory skin diseases can be treated systemically with Cyclosporin A or Dimethylfumarate we stimulated myeloid cells and primary human keratinocytes with these drugs and analyzed gene expression by quantitative real-time PCR. Cytokine secretion was measured by ELISA. Results In the present study, we could show that ATF3 is induced in PBMCs by DMF and weakly by Ebselen, while CsA is the most prominent inducer of ATF3 in keratinocytes without enhancing HO-1 transcription. Further we could show that induction of stress by LPS treatment elevates IL-1β and IL-6 and weakly ATF3 transcription in PBMCs. While transcription of both cytokines is elevated, LPS treatment mediates IL-6 secretion with only little IL-1β secretion. Treatment with DMF dampens LPS-induced transcription. Conclusions Taken together, our results shed light into the different carcinogenic potential of CsA and DMF, which both target ATF3. Collectively our data demonstrate that CsA strongly induces pro-carcinogenic ATF3 in keratinocytes, whereas ATF3 induction by DMF in myeloid cells acts anti-inflammatory.

Tumor-associated antigens have emerged as important immunotherapeutic targets in the fight against cancer. Germline tumor antigens, such as WT1, Wilms’ tumor gene 1, are overexpressed in many human malignancies but have low expression in somatic tissues. Recent vaccination approaches to target WT1 have been hampered by poor in vivo immune potency, likely due to the conserved self-antigen nature of WT1. In this study, we use a novel synthetic micro-consensus SynCon DNA vaccine approach with the goal of breaking tolerance and increasing vaccine immune potency. This approach induced new, neo-antigen-like responses that were superior to those induced by native WT1 DNA immunogens for driving T cell immunity and breaking tolerance. Non-human primates (NHPs) vaccinated with SynCon WT1 antigens elicited immune responses against native rhesus WT1 peptides. When delivered by electroporation (EP) in mice, SynCon-based WT1 constructs elicited strong CD4 and CD8 T cell responses (including IFN-γ, CD107a, and TNF-α) to both native and consensus peptides. In addition, SynCon WT1 vaccine-induced antibodies recognized native WT1 in vitro. Vaccination with the SynCon WT1 immunogens was capable of slowing tumor growth in therapeutic models in vivo. These data support the further study of synthetic consensus DNA vaccines for breaking tolerance to important germline antigens.

Background and objectives Intralesional injection of anti-CD20 antibody (rituximab) has been described as effective therapeutic option for patients with indolent primary cutaneous B-cell lymphoma (PCBL). To date, no parameters that reproducibly predict favorable clinical outcome of this treatment have been identified. The study aims to evaluate the clinical response and adverse effects as well as patients' self-perception of intralesional injection of anti-CD20 antibody for treatment of indolent PCBL compared to other treatment modalities. Patients and methods Eleven patients with PCBL, namely primary cutaneous follicle center lymphoma (n = 9) and primary cutaneous marginal zone lymphoma (n = 2), treated with intralesional anti-CD20 antibody were retrospectively evaluated for response rate and adverse events as well as their self-perception of anti-CD20 antibody therapy and other therapies of PCBL. Results Patients treated with intralesional anti-CD20 antibody for PCBL showed complete response or partial response in 45 % or 27 % of patients, respectively. Particularly, patients with marked flu-like symptoms after intralesional injection of rituximab responded very well to rituximab. The majority of patients considered rituximab as best therapy compared to other therapies such as excision or radiotherapy. Conclusions Intralesional rituximab is an effective therapy with high patient satisfaction. Strong therapy induced side effects of fever, chills and headache after administration of rituximab might be used as indicator for favorable response.

Since their discovery, the matrix metalloproteinase (MMP) family proteases have been considered as therapeutic targets in numerous diseases and disorders. Unfortunately, clinical trials with MMP inhibitors have failed to yield any clinical benefits of these inhibitors. These failures were largely due to a lack of MMP-selective agents; accordingly, it has become important to identify a platform with which high selectivity can be achieved. To this end, we propose using MMP-targeting antibodies that can achieve high specificity in interactions with their targets. Using a scaffold of single-domain antibodies, here we raised a panel of MMP10-selective antibodies through immunization of llamas, a member of the camelid family, whose members generate conventional heavy/light-chain antibodies and also smaller antibodies lacking light-chain and CH1 domains. We report the generation of a highly selective and tightly binding MMP10 inhibitor (K(i) < 2 nm). Using bio-layer interferometry–based binding assays, we found that this antibody interacts with the MMP10 active site. Activity assays demonstrated that the antibody selectively inhibits MMP10 over its closest relative, MMP3. The ability of a single-domain antibody to discriminate between the most conserved MMP pair via an active site–directed mechanism of inhibition reported here supports the potential of this antibody as a broadly applicable scaffold for the development of selective, tightly binding MMP inhibitors.

Immune checkpoint blockade has revolutionized cancer treatment, Patients developing immune mediated adverse events, such as colitis, appear to particularly benefit from immune checkpoint inhibition. Yet, the contributing mechanisms are largely unknown. We identified a systemic LP5 signature in melanoma patients with colitis following anti-cytotoxic T lymphocyte-associated antigen 4 (anti-CTLA-4) checkpoint inhibitor treatment and hypothesized that intestinal microbiota-derived LPS contributes to therapeutic efficacy. Because activation of immune cells within the tumor microenvironment is considered most promising to effectively control cancer, we analyzed human and murine melanoma for known sentinels of LPS. We identified mast cells (MCs) accumulating in and around melanomas and showed that effective melanoma immune control was dependent on LP5-activated MCs recruiting tumor-infiltrating effector T cells by secretion of CXCL10. Importantly, CXCL10 was also upregulated in human melanomas with immune regression and in patients with colitis induced by anti-CTLA-4 antibody. Furthermore, we demonstrate that CXCL10 upregulation and an MC signature at the site of melanomas are biomarkers for better patient survival, These findings provide conclusive evidence for a "Trojan horse treatment strategy" in which the plasticity of cancer-resident immune cells, such as MCs, is used as a target to boost tumor immune defense.

Since the first Zika virus (ZIKV) infection was confirmed in Brazil in May 2015, several hundred thousand cases have been reported across the Americas. This figure gives an incomplete picture of the epidemic, however, given that asymptomatic infections, imperfect surveillance, and variability in reporting rates imply that the incidence of infection was likely much higher. The infection attack rate (IAR)—defined as the proportion of the population that was infected over the course of the epidemic—has important implications for the longer-term epidemiology of Zika in the region, such as the timing, location, and likelihood of future outbreaks. To estimate the IAR and the total number of people infected, we leveraged multiple types of Zika case data from 15 countries and territories where subnational data were publicly available. Datasets included confirmed and suspected Zika cases in pregnant women and in the total population, Zika-associated Guillan-Barré syndrome cases, and cases of congenital Zika syndrome. We used a hierarchical Bayesian model with empirically-informed priors that leveraged the different case report types to simultaneously estimate national and subnational reporting rates, the fraction of symptomatic infections, and subnational IARs. In these 15 countries and territories, estimates of Zika IAR ranged from 0.084 (95% CrI: 0.067 − 0.096) in Peru to 0.361 (95% CrI: 0.214 − 0.514) in Ecuador, with significant subnational variability in IAR for every country. Totaling these infection estimates across these and 33 other countries and territories in the region, our results suggest that 132.3 million (95% CrI: 111.3-170.2 million) people in the Americas have been infected by ZIKV since 2015. These estimates represent the most extensive attempt to date to determine the size of the Zika epidemic in the Americas, and they offer an important baseline for assessing the risk of future Zika epidemics in this region.

Bacterial infections resulting from nonsurgical traumatic wounds can be life threatening, especially those caused by multidrug-resistant (MDR) bacteria with limited therapeutic options. The antimicrobial activity of polymyxin B (1) and curcumin (2) alone and in combination was determined versus MDR bacterial isolates associated with traumatic wound infections. Cytotoxicity assays for 1 and 2 were undertaken in keratinocyte cell lines. Minimum inhibitory concentrations of 1 were significantly reduced in the presence of 2 (3- to 10-fold reduction), with synergy observed. Time-kill assays showed the combinations produced bactericidal activity. Cytotoxicity assays indicate the toxicity of 2 was reduced in the presence of 1.

Background Vaccination and passive antibody therapies are critical for controlling infectious diseases. Passive antibody administration has limitations, including the necessity for purification and multiple injections for efficacy. Vaccination is associated with a lag phase before generation of immunity. Novel approaches reported here utilize the benefits of both methods for the rapid generation of effective immunity. Methods A novel antibody-based prophylaxis/therapy entailing the electroporation-mediated delivery of synthetic DNA plasmids encoding biologically active anti-chikungunya virus (CHIKV) envelope monoclonal antibody (dMAb) was designed and evaluated for antiviral efficacy, as well as for the ability to overcome shortcomings inherent with conventional active vaccination and passive immunotherapy. Results One intramuscular injection of dMAb produced antibodies in vivo more rapidly than active vaccination with an anti-CHIKV DNA vaccine. This dMAb neutralized diverse CHIKV clinical isolates and protected mice from viral challenge. Combination of dMAb and the CHIKV DNA vaccine afforded rapid and long-lived protection. Conclusions A DNA-based dMAb strategy induced rapid protection against an emerging viral infection. This method can be combined with DNA vaccination as a novel strategy to provide both short- and long-term protection against this emerging infectious disease. These studies have implications for pathogen treatment and control strategies.

There have been encouraging results for the development of an effective HIV vaccine. However, many questions remain regarding the quality of immune responses and the role of mucosal antibodies. We addressed some of these issues by using a simian immunodeficiency virus (SIV) DNA vaccine adjuvanted with plasmid-expressed mucosal chemokines combined with an intravaginal SIV challenge in rhesus macaque (RhM) model. We previously reported on the ability of CCR9 and CCR10 ligand (L) adjuvants to enhance mucosal and systemic IgA and IgG responses in small animals. In this study, RhMs were intramuscularly immunized five times with either DNA or DNA plus chemokine adjuvant delivered by electroporation followed by challenge with SIVsmE660. Sixty-eight percent of all vaccinated animals (P

Despite a long history of mosquito-borne virus epidemics in the Americas, the impact of the Zika virus (ZIKV) epidemic of 2015–2016 was unexpected. The need for scientifically informed decision-making is driving research to understand the emergence and spread of ZIKV. To support that research, we assembled a data set of key covariates for modeling ZIKV transmission dynamics in Colombia, where ZIKV transmission was widespread and the government made incidence data publically available. On a weekly basis between January 1, 2014 and October 1, 2016 at three administrative levels, we collated spatiotemporal Zika incidence data, nine environmental variables, and demographic data into a single downloadable database. These new datasets and those we identified, processed, and assembled at comparable spatial and temporal resolutions will save future researchers considerable time and effort in performing these data processing steps, enabling them to focus instead on extracting epidemiological insights from this important data set. Similar approaches could prove useful for filling data gaps to enable epidemiological analyses of future disease emergence events.

Background: Temporal incidence patterns provide a crucial window into the dynamics of emerging infectious diseases, yet their utility is limited by the spatially aggregated form in which they are often presented. Weekly incidence data from the 2015-2016 Zika epidemic were available only at the national level for most countries in the Americas. One exception was Colombia, where data at departmental and municipal scales were made publicly available in real time, providing an opportunity to assess the degree to which national-level data are reflective of temporal patterns at local levels. Methods: To characterize differences in epidemic trajectories, our analysis centered on classifying proportional cumulative incidence curves according to six features at three levels of spatial aggregation. This analysis used the partitioning around medoids algorithm to assign departments and municipalities to groups based on these six characteristics. Examination of the features that differentiated these groups and exploration of their temporal and spatial patterns were performed. Simulations from a stochastic transmission model provided data that were used to assess the extent to which groups identified by the classification algorithm could be associated with differences in underlying drivers of transmission. Results: The timing of departmental-level epidemic peaks varied by three months, and departmental-level estimates of the time-varying reproduction number, R(t), showed patterns that were distinct from a national-level estimate. The classification algorithm identified moderate support for two to three clusters at the departmental level and somewhat stronger support for this at the municipal level. Variability in epidemic duration, the length of the tail of the epidemic, and the consistency of cumulative incidence data with a cumulative normal distribution function made the greatest contributions to distinctions across these groups. Applying the classification algorithm to simulated data showed that municipalities with basic reproduction number, R0, greater than 1 were consistently associated with a particular group. Municipalities with R0 < 1 displayed more diverse patterns, although in this case that may be due to simplifications of how the model represented spatial interaction among municipalities. Conclusions: The diversity of temporal incidence patterns at local scales uncovered by this analysis underscores the value of spatially disaggregated data and the importance of locally tailored strategies for responding to emerging infectious diseases.

Small interfering RNA (siRNA)–based therapies allow targeted correction of molecular defects in distinct cell populations. Although efficient in multiple cell populations, dendritic cells (DCs) seem to resist siRNA delivery. Using fluorescence labeling and radiolabeling, we show that cholesterol modification enables siRNA uptake by DCs in vitro and in vivo. Delivery of cholesterol-modified p40 siRNA selectively abolished p40 transcription and suppressed TLR-triggered p40 production by DCs. During immunization with peptide in CFA, cholesterol-modified p40 siRNA generated p40-deficient, IL-10–producing DCs that prevented IL-17/Th17 and IFN-γ/Th1 responses. Only cholesterol-modified p40-siRNA established protective immunity against experimental autoimmune encephalomyelitis and suppressed IFN-γ and IL-17 expression by CNS-infiltrating mononuclear cells without inducing regulatory T cells. Because cholesterol-modified siRNA can thus modify selected DC functions in vivo, it is intriguing for targeted immune therapy of allergic, autoimmune, or neoplastic diseases.

Functional disorders are common conditions with a substantial impact on a patients' wellbeing, and can be diagnostically elusive. There are bidirectional associations between functional disorders and mitochondrial dysfunction. In this study, provided clinical information and the exon sequence of the TRAP1 mitochondrial chaperone were retrospectively reviewed with a focus on the functional categories of chronic pain, fatigue and gastrointestinal dysmotility. Very-highly conserved TRAP1 variants were identified in 73 of 930 unrelated patients. Functional symptomatology is strongly associated with specific variants in the ATPase binding pocket. In particular, the combined presence of all three functional categories is strongly associated with p.Ile253Val (OR 7.5, P = 0.0001) and with two other interacting variants (OR 18, P = 0.0005). Considering a 1-2% combined variant prevalence and high odds ratios, these variants may be an important factor in the etiology of functional symptomatology.

Numerous Zika virus vaccines are being developed. However, identifying sites to evaluate the efficacy of a Zika virus vaccine is challenging due to the general decrease in Zika virus activity. We compare results from three different modeling approaches to estimate areas that may have increased relative risk of Zika virus transmission during 2017. The analysis focused on eight priority countries (i.e., Brazil, Colombia, Costa Rica, Dominican Republic, Ecuador, Mexico, Panama, and Peru). The models projected low incidence rates during 2017 for all locations in the priority countries but identified several subnational areas that may have increased relative risk of Zika virus transmission in 2017. Given the projected low incidence of disease, the total number of participants, number of study sites, or duration of study follow-up may need to be increased to meet the efficacy study endpoints.

The impact of broad-spectrum antibiotics on antimicrobial resistance and disruption of the beneficial microbiome compels the urgent investigation of bacteria-specific approaches such as antibody-based strategies. Among these, DNA-delivered monoclonal antibodies (DMAbs), produced by muscle cells in vivo, potentially allow the prevention or treatment of bacterial infections circumventing some of the hurdles of protein IgG delivery. Here, we optimize DNA-delivered monoclonal antibodies consisting of two potent human IgG clones, including a non-natural bispecific IgG1 candidate, targeting Pseudomonas aeruginosa. The DNA-delivered monoclonal antibodies exhibit indistinguishable potency compared to bioprocessed IgG and protect against lethal pneumonia in mice. The DNA-delivered monoclonal antibodies decrease bacterial colonization of organs and exhibit enhanced adjunctive activity in combination with antibiotics. These studies support DNA-delivered monoclonal antibodies delivery as a potential strategy to augment the host immune response to prevent serious bacterial infections, and represent a significant advancement toward broader practical delivery of monoclonal antibody immunotherapeutics for additional infectious pathogens., DNA-delivered monoclonal antibodies (DMAbs) can be produced by muscle cells in vivo, potentially allowing prevention or treatment of infectious diseases. Here, the authors show that two DMAbs targeting Pseudomonas aeruginosa proteins confer protection against lethal pneumonia in mice.

Epidemic growth rate, r, provides a more complete description of the potential for epidemics than the more commonly studied basic reproduction number, R0, yet the former has never been described as a function of temperature for dengue virus or other pathogens with temperature-sensitive transmission. The need to understand the drivers of epidemics of these pathogens is acute, with arthropod-borne virus epidemics becoming increasingly problematic. We addressed this need by developing temperature-dependent descriptions of the two components of r—R0 and the generation interval—to obtain a temperature-dependent description of r. Our results show that the generation interval is highly sensitive to temperature, decreasing twofold between 25 and 35°C and suggesting that dengue virus epidemics may accelerate as temperatures increase, not only because of more infections per generation but also because of faster generations. Under the empirical temperature relationships that we considered, we found that r peaked at a temperature threshold that was robust to uncertainty in model parameters that do not depend on temperature. Although the precise value of this temperature threshold could be refined following future studies of empirical temperature relationships, the framework we present for identifying such temperature thresholds offers a new way to classify regions in which dengue virus epidemic intensity could either increase or decrease under future climate change., Author summary Recurrent, rapidly intensifying epidemics of dengue–the world’s most prevalent mosquito-borne viral disease–pose a challenge to healthcare systems throughout the tropical and subtropical world. An acute disease that tends to respond well to proper treatment, the sometimes intense nature of dengue epidemics has been known to overwhelm healthcare systems and elevate the morbidity and mortality of patients left without adequate medical treatment under peak epidemic conditions. Here, we quantify the temperature dependence of dengue epidemic intensity by quantifying two distinct determinants of epidemic growth rate: the average number of secondary infections arising from each primary infection and the average time between successive infections in humans. Our results show that the time between successive infections in humans decreases steadily with increasing temperatures, whereas the average number of secondary infections peaks at intermediate temperatures. Altogether, this suggests a peak temperature for dengue epidemic intensity. Applying this result to global temperature projections under future climate change scenarios suggests that dengue epidemics in many regions of the world could become more intense under future temperature increases.

Background Atopic dermatitis (AD) is a common inflammatory skin disease with dysfunction of the skin barrier, an abnormal immune response and frequent allergies to environmental antigens like food antigens. Clinical observations suggest that certain diets can influence the course of AD. Objective Here we compared the phenotype of food allergen-specific T cells activated through skin or gut allergen exposure to transfer skin inflammation into naive recipients upon epicutaenous allergen challenge. Methods Ovalbumin (OVA) TCR-transgenic mice were treated epicutaneously with OVA or were fed OVA. CD4+ T cells from skin lymph nodes or mesenteric lymph nodes were transferred into naive BALB/c mice, which were challenged with OVA epicutaneously. Skin inflammation was determined by histological parameters. In addition, we analyzed the phenotype of the immune response in lymphoid tissues and in skin tissue. Results TCR-transgenic T cells activated through epicutaneous or oral OVA exposure both migrate to skin lymph nodes after adoptive transfer and epicutaenous OVA exposure. AD-like skin inflammation could only be induced by the transfer of epicutaneously primed OVA T cells. Analysis of the immune phenotype demonstrated an IL-22/IL-17A-dominated immune phenotype of skin-pathogenic T cells. Conclusion IL-22 seems to be the critical cytokine for the development of AD and is induced in this model by epicutaneous sensitization with OVA.

Clostridium difficile -associated disease (CDAD) constitutes a large majority of nosocomial diarrhea cases in industrialized nations and is mediated by the effects of two secreted toxins, toxin A (TcdA) and toxin B (TcdB). Patients who develop strong antitoxin antibody responses can clear C. difficile infection and remain disease free. Key toxin-neutralizing epitopes have been found within the carboxy-terminal receptor binding domains (RBDs) of TcdA and TcdB, which has generated interest in developing the RBD as a viable vaccine target. While numerous platforms have been studied, very little data describes the potential of DNA vaccination against CDAD. Therefore, we created highly optimized plasmids encoding the RBDs from TcdA and TcdB in which any putative N -linked glycosylation sites were altered. Mice and nonhuman primates were immunized intramuscularly, followed by in vivo electroporation, and in these animal models, vaccination induced significant levels of both anti-RBD antibodies (blood and stool) and RBD-specific antibody-secreting cells. Further characterization revealed that sera from immunized mice and nonhuman primates could detect RBD protein from transfected cells, as well as neutralize purified toxins in an in vitro cytotoxicity assay. Mice that were immunized with plasmids or given nonhuman-primate sera were protected from a lethal challenge with purified TcdA and/or TcdB. Moreover, immunized mice were significantly protected when challenged with C. difficile spores from homologous (VPI 10463) and heterologous, epidemic (UK1) strains. These data demonstrate the robust immunogenicity and efficacy of a TcdA/B RBD-based DNA vaccine in preclinical models of acute toxin-associated and intragastric, spore-induced colonic disease.

Chronic HCV is a surreptitious disease currently affecting approximately 3% of the world's population that can lead to liver failure and cancer decades following initial infection. However, there are currently no vaccines available for the prevention of chronic HCV. From patients who acutely resolve HCV infection, it is apparent that a strong and broad cytotoxic T lymphocyte (CTL) response is important in HCV clearance. DNA vaccines are naked plasmid DNA molecules that encode pathogen antigens to induce a pathogen-specific immune response. They are inexpensive to produce and have an excellent safety profile in animals and humans. Additionally, DNA vaccines are able to induce strong CTL responses, making them well-suited for an HCV vaccine. We aimed to maximize vaccine recipients' opportunity to induce a broad T cell response with a novel antigenic sequence, multi-antigen vaccine strategy. We have generated DNA plasmids encoding consensus sequences of HCV genotypes 1a and 1b non-structural proteins NS3/4a, NS4b, NS5a, and NS5b. Rhesus macaques were used to study the immunogenicity of these constructs. Four animals were immunized 3 times, 6 weeks apart, at a dose of 1.0mg per antigen construct, as an intramuscular injection followed by in vivo electroporation, which greatly increases DNA uptake by local cells. Immune responses were measured 2 weeks post-immunization regimen (PIR) in immunized rhesus macaques and showed a broad response to multiple HCV nonstructural antigens, with up to 4680 spot-forming units per million peripheral blood mononuclear cells (PBMCs) as measured by Interferon-γ ELISpot. In addition, multiparametric flow cytometry detected HCV-specific CD4+ and CD8+ T cell responses by intracellular cytokine staining and detected HCV-specific CD107a+/GrzB+ CD8+ T cells indicating an antigen specific cytolytic response 2 weeks PIR compared with baseline measurements. At the final study time point, 6 weeks PIR, HCV-specific CD45RA- memory-like T cells remained detectable in peripheral blood. Data presented in this manuscript support the notion that vaccine immunogenicity studies using a macaque model can be used to depict key anti-HCV nonstructural antigenic cellular immune responses and support the development of DNA-based prophylactic HCV vaccines.

Cells sense their mechanical and physical environment through diverse mechanisms, and these interactions specify a wide range of responses including growth, survival, migration and differentiation. Although much work has focused on dissecting the adhesive and structural components of the cell responsible for transducing external mechanical forces into biochemical signalling cascades, only recently have studies begun to examine how mechanical signals are transmitted to the nucleus and activate specific gene expression programmes. One necessary step in these processes is the transport of signalling molecules from the cytoplasm to the nucleus. The SRF (serum-response factor) and YAP (Yes-associated protein)/TAZ (transcriptional co-activator with PDZ-binding motif) pathways are known mediators of this process in multiple cell types, including mesenchymal stem cells, keratinocytes, mammary epithelial cells and smooth muscle cells. In addition, recent evidence suggests a potential role for β-catenin and Smad signalling in mechanotransduction, but further mechanistic studies are needed to prove this hypothesis. As a model system, the epidermis of the skin is one tissue in which nucleocytoplasmic shuttling mediates cellular mechanosensing and is essential for tissue development, homoeostasis and repair. We propose that nuclear translocation is a common element of mechanotransduction conserved across multiple cell types and tissues.

We describe a new species of the basal mammaliaform Docodon, D. apoxys, sp. nov., represented by three nearly complete dentaries from the Upper Jurassic Morrison Formation of central Colorado. This species differs from other known species of Docodon in the relative heights of the principal molar cusps and in the size of the distal molars. In addition, we attribute a partial rostrum preserving much of the maxilla and most of the premaxilla, bearing a complete incisor and premolar count, to Docodon sp. These materials supply previously unknown morphology for this classic North American Jurassic taxon, and help establish a common morphology for Docodonta as a whole. While Docodon differs from the Portuguese docodont Haldanodon in the presence of only five upper incisors (none of which is entirely within the maxilla), an internarial bar is present in both taxa. The new specimens also unambiguously preserve a complete and undistorted angular process that differs conspicuously from the classical interpretation of the holotype of Docodon victor; this structure in docodonts closely resembles the angular process in australosphenidans and trechnotheres. We abandon the concept of a “pseudangular” process and we consider the angular process to be a homologous feature (where present) across Mammaliaformes.

The study included the detection of the effect of the Mycotoxins of the fungal isolates of Alternaria alternata, which was diagnosed both genetically and chemically by PCR. The analysis showed that the ability of A. alternata to produce toxins, including AltratoxinII, was found in one of the isolates 7.5499μg / ml. This concentration is used in the experiment to determine its effect on the tissues and organs of white mice. In the study, the fungus Pleurotus ostreatus was used to reduce the toxicity of the fungus A. alternata. The fungus P.ostreatus gave high tolerance to A. alternata and evaluated the effect of sequential chemical treatments CaCO3 on reducing the auditory effect on laboratory mice. The toxic isolation of pathogenic fungi is extract into the body of the organism. The effect on the levels of liver enzymes (GPT, GOT, ALP) has reached its level (IU / L) (84, 88, 264) respectively compared to control group which reached (22, 35, 147) IU / L respectively as well, And resulted in the effectiveness of the decomposition of liver cells containing enzymes and led to the liberation of the serum, as well as the cause of changes in the tissue pathological clear in the cross section of the liver tissue, including the occurrence of hepatocytes vaculosis and the presence of bubbles in the cells walls and the expansion of liver sinuses causing Hydropic degeneration. The results of the laboratory examination of the kidney function showed significant differences in the increase in the concentration of urea and creatinine in the serum of laboratory mice treated with urea level (59 mg / dl) and creatinine (1.73 mg / dl) compared to control group (29 mg / dl) And (0.7 mg / dl) respectively, and found a clear effect within the kidney tissues leading to atrophy in the glomeruli, infiltration of the lymphocytes and hemorrhage, in addition to the degradation of urinary tubule cells affecting kidney function leading to renal failure.

High levels of human telomerase reverse transcriptase (hTERT) are detected in more than 85% of human cancers. Immunologic analysis supports that hTERT is a widely applicable target recognized by T cells and can be potentially studied as a broad cancer immunotherapeutic, or a unique line of defense against tumor recurrence. There remains an urgent need to develop more potent hTERT vaccines. Here, a synthetic highly optimized full-length hTERT DNA vaccine (phTERT) was designed and the induced immunity was examined in mice and non-human primates (NHP). When delivered by electroporation, phTERT elicited strong, broad hTERT-specific CD8 T-cell responses including induction of T cells expressing CD107a, IFN-γ, and TNF-α in mice. The ability of phTERT to overcome tolerance was evaluated in an NHP model, whose TERT is 96% homologous to that of hTERT. Immunized monkeys exhibited robust [average 1,834 spot forming unit (SFU)/106 peripheral blood mononuclear cells (PBMC)], diverse (multiple immunodominant epitopes) IFN-γ responses and antigen-specific perforin release (average 332 SFU/106 PBMCs), suggesting that phTERT breaks tolerance and induces potent cytotoxic responses in this human-relevant model. Moreover, in an HPV16-associated tumor model, vaccination of phTERT slows tumor growth and improves survival rate in both prophylactic and therapeutic studies. Finally, in vivo cytotoxicity assay confirmed that phTERT-induced CD8 T cells exhibited specific cytotoxic T lymphocyte (CTL) activity, capable of eliminating hTERT-pulsed target cells. These findings support that this synthetic electroporation-delivered DNA phTERT may have a role as a broad therapeutic cancer vaccine candidate. Cancer Immunol Res; 1(3); 179–89. ©2013 AACR.

The crucial role of the proinflammatory cytokine interleukin 1β (IL-1β) in driving inflammatory disorders, such as Muckle-Wells syndrome and gout, has been extensively characterised. Owing to its high potency to induce inflammation the activation and secretion of IL-1β is tightly regulated. The sensing of various host 'dangers', including infections and metabolic deregulation, results in the formation of large protein complexes, termed inflammasomes. Formation of the inflammasomes leads to the cleavage and activation of caspase-1, which in turn proteolytically processes its substrates, including pro-IL-1β. Biologically active IL-1β is subsequently secreted by the cell. In contrast to IL-1β, little is known about mechanisms underlying the activation and secretion of its close homologue IL-1α. Moreover, the physiological role of IL-1α is still not well defined. Several studies hypothesise that IL-1α serves as a danger signal, which is passively released from dying cells. However, recent studies suggest a more complex function of this cytokine. Indeed, NLRP3 inflammasome agonists such as uric acid crystal or nigericin induce IL-1α cleavage and secretion, leading to the cosecretion of both IL-1β and IL-1α. Depending on the type of NLRP3 agonist, release of IL-1α is NLRP3-inflammasome/caspase-1 dependent or independent, but in both cases IL-1α processing depends on calpain protease activity. Taken together, these results suggest that the promotion and progression of inflammatory diseases is not solely due to IL-1β but also to its close relative IL-1α. This should be considered when IL-1 blockade is applied as a therapeutic strategy for diseases such as cryopyrin-associated periodic syndromes or gout.

Significant concerns have been raised owing to the rapid global spread of infection and disease caused by the mosquito-borne Zika virus (ZIKV). Recent studies suggest that ZIKV can also be transmitted sexually, further increasing the exposure risk for this virus. Associated with this spread is a dramatic increase in cases of microcephaly and additional congenital abnormalities in infants of ZIKV-infected mothers, as well as a rise in the occurrence of Guillain Barre’ syndrome in infected adults. Importantly, there are no licensed therapies or vaccines against ZIKV infection. In this study, we generate and evaluate the in vivo efficacy of a novel, synthetic, DNA vaccine targeting the pre-membrane+envelope proteins (prME) of ZIKV. Following initial in vitro development and evaluation studies of the plasmid construct, mice and non-human primates were immunised with this prME DNA-based immunogen through electroporation-mediated enhanced DNA delivery. Vaccinated animals were found to generate antigen-specific cellular and humoral immunity and neutralisation activity. In mice lacking receptors for interferon (IFN)-α/β (designated IFNAR−/−) immunisation with this DNA vaccine induced, following in vivo viral challenge, 100% protection against infection-associated weight loss or death in addition to preventing viral pathology in brain tissue. In addition, passive transfer of non-human primate anti-ZIKV immune serum protected IFNAR−/− mice against subsequent viral challenge. This study in NHP and in a pathogenic mouse model supports the importance of immune responses targeting prME in ZIKV infection and suggests that additional research on this vaccine approach may have relevance for ZIKV control and disease prevention in humans. Zika virus: A DNA vaccine candidate. A Zika virus vaccine candidate has been developed that elicits an immune response and confers protection in animal models. An international team of researchers headed by David Weiner of The Wistar Institute, Philadelphia, USA, generated a DNA-based vaccine candidate using genes encoding for Zika virus surface proteins—with the intent that these genes, when transferred to a host, would produce characteristic proteins allowing the host to recognise and protect against Zika virus infection. Weiner’s team found that their vaccine candidate generated a specific immune cell response in healthy mice, and offered protection to mice genetically engineered to be vulnerable to the virus. Importantly, their vaccine also elicited functional responses in non-human primates, highlighting that further research into this approach is warranted for the immunisation of humans against Zika virus.

Background The Nod-like receptor NACHT, LRR, and PYD domain–containing protein 3 (NLRP3) and Bruton tyrosine kinase (BTK) are protagonists in innate and adaptive immunity, respectively. NLRP3 senses exogenous and endogenous insults, leading to inflammasome activation, which occurs spontaneously in patients with Muckle-Wells syndrome; BTK mutations cause the genetic immunodeficiency X-linked agammaglobulinemia (XLA). However, to date, few proteins that regulate NLRP3 inflammasome activity in human primary immune cells have been identified, and clinically promising pharmacologic targeting strategies remain elusive. Objective We sought to identify novel regulators of the NLRP3 inflammasome in human cells with a view to exploring interference with inflammasome activity at the level of such regulators. Methods After proteome-wide phosphoproteomics, the identified novel regulator BTK was studied in human and murine cells by using pharmacologic and genetic BTK ablation. Results Here we show that BTK is a critical regulator of NLRP3 inflammasome activation: pharmacologic (using the US Food and Drug Administration–approved inhibitor ibrutinib) and genetic (in patients with XLA and Btk knockout mice) BTK ablation in primary immune cells led to reduced IL-1β processing and secretion in response to nigericin and the Staphylococcus aureus toxin leukocidin AB (LukAB). BTK affected apoptosis-associated speck-like protein containing a CARD (ASC) speck formation and caspase-1 cleavage and interacted with NLRP3 and ASC. S aureus infection control in vivo and IL-1β release from cells of patients with Muckle-Wells syndrome were impaired by ibrutinib. Notably, IL-1β processing and release from immune cells isolated from patients with cancer receiving ibrutinib therapy were reduced. Conclusion Our data suggest that XLA might result in part from genetic inflammasome deficiency and that NLRP3 inflammasome–linked inflammation could potentially be targeted pharmacologically through BTK.

Natural indicators provide intrinsic probes of metabolism, biogenesis and oxidative protection. Nicotinamide adenine dinucleotide metabolites (NAD(P)) are one class of indicators that have roles as co-factors in oxidative phosphorylation, glycolysis, and anti-oxidant protection, as well as signaling in the mitochondrial biogenesis pathway. These many roles are made possible by the distinct redox states (NAD(P)(+) and NAD(P)H), which are compartmentalized between cytosol and mitochondria. Here we provide evidence for detection of NAD(P)(+) and NAD(P)H in separate mitochondrial and cytosol pools in vivo in human tissue by phosphorus magnetic resonance spectroscopy ((31)P MRS). These NAD(P) pools are identified by chemical standards (NAD(+), NADP(+), and NADH) and by physiological tests. A unique resonance reflecting mitochondrial NAD(P)H is revealed by the changes elicited by elevation of mitochondrial oxidation. The decline of NAD(P)H with oxidation is matched by a stoichiometric rise in the NAD(P)(+) peak. This unique resonance also provides a measure of the improvement in mitochondrial oxidation that parallels the greater phosphorylation found after exercise training in these elderly subjects. The implication is that the dynamics of the mitochondrial NAD(P)H peak provides an intrinsic probe of the reversal of mitochondrial dysfunction in elderly muscle. Thus, non-invasive detection of NAD(P)(+) and NAD(P)H in cytosol vs. mitochondria yields natural indicators of redox compartmentalization and sensitive intrinsic probes of the improvement of mitochondrial function with an intervention in human tissues in vivo. These natural indicators hold the promise of providing mechanistic insight into metabolism and mitochondrial function in vivo in a range of tissues in health, disease and with treatment.

Zika virus is a mosquito-borne pathogen that is rapidly spreading across the Americas. Due to associations between Zika virus infection and a range of fetal maladies1,2, the epidemic trajectory of this viral infection poses a significant concern for the nearly 15 million children born in the Americas each year. Ascertaining the portion of this population that is truly at risk is an important priority. One recent estimate3suggested that 5.42 million childbearing women live in areas of the Americas that are suitable for Zika occurrence. To improve on that estimate, which did not take into account the protective effects of herd immunity, we developed a new approach that combines classic results from epidemiological theory with seroprevalence data and highly spatially resolved data about drivers of transmission to make location-specific projections of epidemic attack rates. Our results suggest that 1.65 (1.45–2.06) million childbearing women and 93.4 (81.6–117.1) million people in total could become infected before the first wave of the epidemic concludes. Based on current estimates of rates of adverse fetal outcomes among infected women2,4,5, these results suggest that tens of thousands of pregnancies could be negatively impacted by the first wave of the epidemic. These projections constitute a revised upper limit of populations at risk in the current Zika epidemic, and our approach offers a new way to make rapid assessments of the threat posed by emerging infectious diseases more generally.

Apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC) is an important adaptor protein for inflammasome activation, mediating the secretion of protumorigenic innate cytokines. However, ASC is also known to trigger apoptosis in tumor cells, acting as a tumor-suppressor gene, which is lost in several human cancers. The aim of this study was to evaluate the clinical significance of ASC in human cutaneous squamous cell carcinoma (SCC). Initially, ASC expression was immunohistochemically evaluated in non-metastic and metastatic SCC. While ASC expression does not correlate with metastatic potential, it correlates with the degree of dedifferentiation. Using methylation specific PCR we were able to demonstrate ASC silencing by promotor specific methylation and impaired inflammasome function in methylated cell lines, linking epigenetic modifications to innate immune activation in keratinocytes. Interestingly, upon ASC restoration by treatment with demethylating agents, we were able to restore AIM2 and NLRP3 activation. In summary, loss of ASC driven tumor development is counterbalanced in the identical cell by the inhibition of pro-tumorigenic inflammation in the tumor cell itself.

We have previously demonstrated the immunogenicity of VGX-3100, a multicomponent DNA immunotherapy for the treatment of Human Papillomavirus (HPV)16/18-positive CIN2/3 in a phase 1 clinical trial. Here, we report on the ability to boost immune responses with an additional dose of VGX-3100. Patients completing our initial phase 1 trial were offered enrollment into a follow on trial consisting of a single boost dose of VGX-3100. Data show both cellular and humoral immune responses could be augmented above pre-boost levels, including the induction of interferon (IFN)γ production, tumor necrosis factor (TNF)α production, CD8+ T cell activation and the synthesis of lytic proteins. Moreover, observation of antigen-specific regulation of immune-related gene transcripts suggests the induction of a proinflammatory response following the boost. Analysis of T cell receptor (TCR) sequencing suggests the localization of putative HPV-specific T cell clones to the cervical mucosa, which underscores the putative mechanism of action of lesion regression and HPV16/18 elimination noted in our double-blind placebo-controlled phase 2B trial. Taken together, these data indicate that VGX-3100 drives the induction of robust cellular and humoral immune responses that can be augmented by a fourth "booster" dose. These data could be important in the scope of increasing the clinical efficacy rate of VGX-3100.

The interleukin-1 (IL-1) family consists of several pro- or anti-inflammatory proteins, with pro-inflammatory IL-1β being its best characterized member. IL-1β is one of the most prominent mediators of inflammation resulting in fever and immune activation via binding to IL-1 receptor 1. Due to its potency, its secretion is tightly regulated. First the transcription of the biologically inactive proform is induced by TLR activation, TNF, or IL-1 receptor activation by mature IL-1α or IL-1β. For the secretion of IL-1β, inflammasome activation as second stimulus is needed. Inflammasomes are cytosolic protein complexes whose activation results in the maturation of inflammatory caspases such as caspase-1. Caspase-1 then cleaves the inactive pro-IL-1β into its mature form which is then being secreted. While IL-1α and IL-1β are considered pro-inflammatory, IL-1Ra as a naturally occurring receptor antagonist acts as an inhibitor on IL-1 receptor signaling. Further members of the IL-1 family, such as IL-18, IL-33, or IL-36, are even involved in T-helper-cell differentiation and will also be discussed in this chapter.

A chronic inflammatory microenvironment favors tumor progression through molecular mechanisms that are still incompletely defined. In inflammation-induced skin cancers, IL-1 receptor- or caspase-1–deficient mice, or mice specifically deficient for the inflammasome adaptor protein ASC (apoptosis-associated speck-like protein containing a CARD) in myeloid cells, had reduced tumor incidence, pointing to a role for IL-1 signaling and inflammasome activation in tumor development. However, mice fully deficient for ASC were not protected, and mice specifically deficient for ASC in keratinocytes developed more tumors than controls, suggesting that, in contrast to its proinflammatory role in myeloid cells, ASC acts as a tumor-suppressor in keratinocytes. Accordingly, ASC protein expression was lost in human cutaneous squamous cell carcinoma, but not in psoriatic skin lesions. Stimulation of primary mouse keratinocytes or the human keratinocyte cell line HaCaT with UVB induced an ASC-dependent phosphorylation of p53 and expression of p53 target genes. In HaCaT cells, ASC interacted with p53 at the endogenous level upon UVB irradiation. Thus, ASC in different tissues may influence tumor growth in opposite directions: it has a proinflammatory role in infiltrating cells that favors tumor development, but it also limits keratinocyte proliferation in response to noxious stimuli, possibly through p53 activation, which helps suppressing tumors.