Inhaled hexavalent chromium (Cr(VI)) promotes lung injury and pulmonary diseases through poorly defined mechanisms. One hypothesis for this lung pathogenesis is that Cr(VI) silences induction of cytoprotective genes, such as heme oxygenase-1 (HO-1), whose total lung mRNA levels were reduced 21 days after nasal instillation of potassium dichromate in C57BL/6 mice. To investigate the mechanisms for this inhibition, Cr(VI) effects on basal and arsenic (As(III))-induced HO-1 expression were examined in cultured human bronchial epithelial (BEAS-2B) cells. An effect of Cr(VI) on the low basal HO-1 mRNA and protein levels in BEAS-2B cells was not detectible. In contrast, Cr(VI) added to the cells before As(III), but not simultaneously with As(III), attenuated As(III)-induced HO-1 expression. Transient transfection with luciferase reporter gene constructs controlled by the full length ho-1 promoter or deletion mutants demonstrated that this inhibition occurred in the E1 enhancer region containing critical antioxidant response elements (ARE). Cr(VI) pretreatment inhibited As(III)-induced activity of a transiently expressed reporter construct regulated by three ARE tandem repeats. The mechanism for this Cr(VI)-attenuated transactivation appeared to be Cr(VI) reduction of the nuclear levels of the transcription factor Nrf2 and As(III)-stimulated Nrf2 transcriptional complex binding to the ARE cis element. Finally, exposing cells to Cr(VI) prior to co-exposure with As(III) synergized for apoptosis and loss of membrane integrity. These data suggest that Cr(VI) silences induction of ARE-driven genes required for protection from secondary insults. The data also have important implications for understanding the toxic mechanisms of low level, mixed metal exposures in the lung.
The immediate-early protein ICP0 from herpes simplex virus 1 (HSV-1) plays pleiotropic roles in promoting viral lytic replication and reactivation from latency. Most of the known actions of ICP0 occur in the nucleus and are thought to involve the E3 ubiquitin ligase activity of its RING finger domain, which targets proteins for degradation via the proteasome. Although ICP0 translocates to the cytoplasm as the infection progresses, little is known about its activities in this location. Here, we show that cytoplasmic ICP0 has two distinct functions. In primary cell cultures and in an intravaginal mouse model, cytoplasmic ICP0 promotes viral replication in the absence of an intact RING finger domain. Additionally, ICP0 blocks the activation of interferon regulatory factor 3 (IRF3), a key transcription factor of the innate antiviral response, in a mechanism that requires the RING finger domain but not the proteasome. To our knowledge, this is the first observation of a proteasome-independent function of the RING finger domain of ICP0. Collectively, these results underscore the importance of cytoplasm-localized ICP0 and the diverse nature of its activities.
IMPORTANCE Despite ICP0 being a well-studied viral protein, the significance of its cytoplasmic localization has been largely overlooked. This is, in part, because common experimental manipulations result in the restriction of ICP0 to the nucleus. By overcoming this constraint, we both further characterize the ability of cytoplasmic ICP0 to inhibit antiviral signaling and show that ICP0 at this site has unexpected activities in promoting viral replication. This demonstrates the importance of considering location when analyzing protein function and adds a new perspective to our understanding of this multifaceted protein.
Internationally, recruiting the best candidates is central to the success of postgraduate training programs and the quality of the medical workforce. So far there has been little theoretically informed research considering selection systems from the perspective of the candidates. We explored candidates’ perception of the fairness of a National Assessment Centre (NAC) approach for selection into Australian general practice training, where candidates were assessed by a Multiple Mini Interview (MMI) and a written Situational Judgment Test (SJT), for suitability to undertake general practice (GP) training.
In 2013, 1,930 medical practitioners, who were eligible to work in Australia attended one of 14 NACs in each of 5 states and 2 territories. A survey was distributed to each candidate at the conclusion of their assessment, which included open-ended questions aimed at eliciting candidates’ perceived benefits and challenges of the selection process. A framework analysis was informed by the theoretical lens of Social Validity Theory.
Qualitative data was available from 46% (n = 886/1,930) of candidates, who found the NAC experience fair and informative for their training and career goals, but wanted to be provided with more information in preparation. Candidates valued being able to communicate their skills during the MMI, but found some difficulty in interpreting the questions. A significant minority had concerns that a lack of relevant GP experience may inhibit their performance. Candidates also expressed concerns about the time limits within the written paper, particularly if English was not their first language. They also expressed a desire for formative feedback during the interview process.
During any job selection process, not only is the organisation assessing the candidates, but the candidates are also assessing the organisation. However, a focus on the candidate experience throughout an organisation’s selection process may provide benefits to both candidates and the organisation, regardless of whether or not candidates secured the job. Social Validity Theory is a useful addition to the methods for demonstrating the reasonableness of any selection system.
Social validity; Postgraduate medical training; Assessment centre; Multiple-mini-interview; Situational judgement test; General practice; Selection; Admissions
Oncolytic viruses (OVs) are attractive avenues of cancer therapy due to the absence of toxic side effects often seen with current treatment modalities. Bovine herpesvirus 1 (BHV-1) is a species-specific virus that does not induce cytotoxicity in normal primary human cells but can infect and kill various human immortalized and transformed cell lines. To gain a better understanding of the oncolytic breadth of BHV-1, the NCI panel of established human tumor cell lines was screened for sensitivity to the virus. Overall, 72% of the panel is permissive to BHV-1 infection, with corresponding decreases in cellular viability. This sensitivity is in comparison to a sensitivity of only 32% for a herpes simplex virus 1 (HSV-1)-based oncolytic vector. Strikingly, while 35% of the panel supports minimal or no BHV-1 replication, significant decreases in cellular viability still occur. These data suggest that BHV-1 is an OV with tropism for multiple tumor types and is able to induce cytotoxicity independent of significant virus replication. In contrast to other species-specific OVs, cellular sensitivity to BHV-1 does not correlate with type I interferon (IFN) signaling; however, mutations in KRAS were found to correlate with high levels of virus replication. The knockdown or overexpression of KRAS in human tumor cell lines yields modest changes in viral titers; however, overexpression of KRAS in normal primary cells elicits permissivity to BHV-1 infection. Together, these data suggest that BHV-1 is a broad-spectrum OV with a distinct mechanism of tumor targeting.
IMPORTANCE Cancer remains a significant health issue, and novel treatments are required, particularly for tumors that are refractory to conventional therapies. Oncolytic viruses are a novel platform given their ability to specifically target tumor cells while leaving healthy cells intact. For this strategy to be successful, a fundamental understanding of virus-host interactions is required. We previously identified bovine herpesvirus 1 as a novel oncolytic virus with many unique and clinically relevant features. Here, we show that BHV-1 can target a wide range of human cancer types, most potently lung cancer. In addition, we show that enhanced KRAS activity, a hallmark of many cancers, is one of the factors that increases BHV-1 oncolytic capacity. These findings hold potential for future treatments, particularly in the context of lung cancer, where KRAS mutations are a negative predictor of treatment efficacy.
Scaling up innovative healthcare programs offers a means to improve access, quality, and health equity across multiple health areas. Despite large numbers of promising projects, little is known about successful efforts to scale up. This study examines trans-national scale, whereby a program operates in two or more countries. Trans-national scale is a distinct measure that reflects opportunities to replicate healthcare programs in multiple countries, thereby providing services to broader populations.
Based on the Center for Health Market Innovations (CHMI) database of nearly 1200 health programs, the study contrasts 116 programs that have achieved trans-national scale with 1,068 single-country programs. Data was collected on the programs' health focus, service activity, legal status, and funding sources, as well as the programs' locations (rural v. urban emphasis), and founding year; differences are reported with statistical significance.
This analysis examines 116 programs that have achieved trans-national scale (TNS) across multiple disease areas and activity types. Compared to 1,068 single-country programs, we find that trans-nationally scaled programs are more donor-reliant; more likely to focus on targeted health needs such as HIV/AIDS, TB, malaria, or family planning rather than provide more comprehensive general care; and more likely to engage in activities that support healthcare services rather than provide direct clinical care.
This work, based on a large data set of health programs, reports on trans-national scale with comparison to single-country programs. The work is a step towards understanding when programs are able to replicate their services as they attempt to expand health services for the poor across countries and health areas. A subset of these programs should be the subject of case studies to understand factors that affect the scaling process, particularly seeking to identify mechanisms that lead to improved health outcomes.
Entry into specialty training was determined by a National Assessment Centre (NAC) approach using a combination of a behavioural Multiple-Mini-Interview (MMI) and a written Situational Judgement Test (SJT). We wanted to know if interviewers could make reliable and valid decisions about the non-cognitive characteristics of candidates with the purpose of selecting them into general practice specialty training using the MMI. Second, we explored the concurrent validity of the MMI with the SJT.
A variance components analysis estimated the reliability and sources of measurement error. Further modelling estimated the optimal configurations for future MMI iterations. We calculated the relationship of the MMI with the SJT.
Data were available from 1382 candidates, 254 interviewers, six MMI questions, five alternate forms of a 50-item SJT, and 11 assessment centres. For a single MMI question and one assessor, 28% of the variance between scores was due to candidate-to-candidate variation. Interviewer subjectivity, in particular the varying views that interviewer had for particular candidates accounted for 40% of the variance in scores. The generalisability co-efficient for a six question MMI was 0.7; to achieve 0.8 would require ten questions. A disattenuated correlation with the SJT (r = 0.35), and in particular a raw score correlation with the subdomain related to clinical knowledge (r = 0.25) demonstrated evidence for construct and concurrent validity. Less than two per cent of candidates would have failed the MMI.
The MMI is a moderately reliable method of assessment in the context of a National Assessment Centre approach. The largest source of error relates to aspects of interviewer subjectivity, suggesting enhanced interviewer training would be beneficial. MMIs need to be sufficiently long for precise comparison for ranking purposes. In order to justify long term sustainable use of the MMI in a postgraduate assessment centre approach, more theoretical work is required to understand how written and performance based test of non-cognitive attributes can be combined, in a way that achieves acceptable generalizability, and has validity.
Selection; Validity; Postgraduate training; Assessment; Multiple-mini-interview; Situational judgment test; Assessment centre; General practice
Asbestos exposure is related to various diseases including asbestosis and malignant mesothelioma (MM). Among the pathogenic mechanisms proposed by which asbestos can cause diseases involving epithelial and mesothelial cells, the most widely accepted one is the generation of reactive oxygen species and/or depletion of antioxidants like glutathione. It has also been demonstrated that asbestos can induce inflammation, perhaps due to activation of inflammasomes.
The oxidation state of thioredoxin was analyzed by redox Western blot analysis and ROS generation was assessed spectrophotometrically as a read-out of solubilized formazan produced by the reduction of nitrotetrazolium blue (NTB) by superoxide. Quantitative real time PCR was used to assess changes in gene transcription.
Here we demonstrate that crocidolite asbestos fibers oxidize the pool of the antioxidant, Thioredoxin-1 (Trx1), which results in release of Thioredoxin Interacting Protein (TXNIP) and subsequent activation of inflammasomes in human mesothelial cells. Exposure to crocidolite asbestos resulted in the depletion of reduced Trx1 in human peritoneal mesothelial (LP9/hTERT) cells. Pretreatment with the antioxidant dehydroascorbic acid (a reactive oxygen species (ROS) scavenger) reduced the level of crocidolite asbestos-induced Trx1 oxidation as well as the depletion of reduced Trx1. Increasing Trx1 expression levels using a Trx1 over-expression vector, reduced the extent of Trx1 oxidation and generation of ROS by crocidolite asbestos, and increased cell survival. In addition, knockdown of TXNIP expression by siRNA attenuated crocidolite asbestos-induced activation of the inflammasome.
Our novel findings suggest that extensive Trx1 oxidation and TXNIP dissociation may be one of the mechanisms by which crocidolite asbestos activates the inflammasome and helps in development of MM.
Asbestos; Malignant mesothelioma; Thioredoxin; Thioredoxin interacting protein; Inflammasomes
Malignant mesothelioma (MM) is a devastating disease with a need for new treatment strategies. In the present study we demonstrated the importance of ERK5 in MM tumor growth and treatment.
ERK5 as a target for MM therapy was verified using mesothelial and mesothelioma cell lines as well as by xenograft SCID mouse models.
We first showed that crocidolite asbestos activated ERK5 in LP9 cells and mesothelioma cell lines exhibit constitutive activation of ERK5. Addition of doxorubicin resulted in further activation of ERK5 in MM cells. ERK5 silencing increased DOX-induced cell death and DOX retention in MM cells. In addition, shERK5 MM lines exhibited both attenuated colony formation on soft agar and invasion of MM cells in vitro that could be related to modulation of gene expression linked to cell proliferation, apoptosis, migration/invasion and drug resistance as shown by microarray analysis. Most importantly, injection of shERK5 MM cell lines into SCID mice showed significant reduction in tumor growth using both subcutaneous and intraperitoneal models. Assessment of selected human cytokine profiles in peritoneal lavage fluid from IP shERK5 and control tumor-bearing mice showed that ERK5 was critical in regulation of various proinflammatory (RANTES/CCL5, MCP-1) and angiogenesis related (IL-8, VEGF) cytokines. Finally, use of doxorubicin and cisplatin in combination with ERK5 inhibition showed further reduction in tumor weight and volume in the IP model of tumor growth.
; ERK5 inhibition in combination with chemotherapeutic drugs is a beneficial strategy for combination therapy in MM patients.
Malignant mesothelioma; asbestos; Mitogen activated protein kinases; Extracellular signal regulated kinase 5; Gene expression
Streptococcus pneumoniae is a common human pathogen that accounts for over a million deaths every year. Colonization of the nasopharynx by S. pneumoniae precedes pulmonary and other invasive diseases, and is therefore a promising target for intervention. Since the receptors Scavenger Receptor A (SRA), Macrophage Receptor with Collagenous Structure (MARCO), and Mannose Receptor (MR) have previously been identified as non-opsonic receptors for S. pneumoniae in the lung, we utilized scavenger receptor knock out mice to study the roles of these receptors in the clearance of S. pneumoniae from the nasopharynx. MARCO−/−, but not SRA−/− or MR−/−, mice had significantly impaired clearance of S. pneumoniae from the nasopharynx. In addition to impairment in bacterial clearance, MARCO−/− mice had abrogated cytokine production and cellular recruitment to the nasopharynx following colonization. Furthermore, macrophages from MARCO−/− mice were deficient in cytokine and chemokine production, including type I interferons, in response to S. pneumoniae. MARCO was required for maximal TLR2- and NOD2-dependent NF-κB activation and signaling that ultimately resulted in clearance. Thus, MARCO is an important component of anti-S. pneumoniae responses in the murine nasopharynx during colonization.
Innate Immunity; Macrophage; Scavenger Receptor; MARCO; Streptococcus pneumoniae; Colonization; Nasopharynx
Oncolytic viruses are novel immunotherapeutic agents that appear to mediate potent antineoplastic effects in both preclinical and clinical settings. Recent studies demonstrate that manipulating the mechanisms whereby cancer cells die in the course of oncolytic virotherapy has potential to boost anticancer immune responses.
chemotherapy; danger-associated molecular patterns; immunogenic cell death; mitoxantrone; oncolytic virus
Malignant mesotheliomas (MMs) are chemoresistant tumors related to exposure to asbestos fibers. The long latency period of MM (30-40 yrs) and heterogeneity of tumor presentation make MM difficult to diagnose and treat at early stages. Currently approved second-line treatments following surgical resection of MMs include a combination of cisplatin or carboplatin (delivered systemically) and pemetrexed, a folate inhibitor, with or without subsequent radiation. The systemic toxicities of these treatments emphasize the need for more effective, localized treatment regimens.
Acid-prepared mesoporous silica (APMS) microparticles were loaded with doxorubicin (DOX) and modified externally with a mesothelin (MB) specific antibody before repeated intraperitoneal (IP) injections into a mouse xenograft model of human peritoneal MM. The health/weight of mice, tumor volume/weight, tumor necrosis and cell proliferation were evaluated in tumor-bearing mice receiving saline, DOX high (0.2 mg/kg), DOX low (0.05 mg/kg), APMS-MB, or APMS-MB-DOX (0.05 mg/kg) in saline.
Targeted therapy (APMS-MB-DOX at 0.05 mg/kg) was more effective than DOX low (0.05 mg/kg) and less toxic than treatment with DOX high (0.2 mg/kg). It also resulted in the reduction of tumor volume without loss of animal health and weight, and significantly decreased tumor cell proliferation. High pressure liquid chromatography (HPLC) of tumor tissue confirmed that APMS-MB-DOX particles delivered DOX to target tissue.
Data suggest that targeted therapy results in greater chemotherapeutic efficacy with fewer adverse side effects than administration of DOX alone. Targeted microparticles are an attractive option for localized drug delivery.
Targeted therapy; Mesoporous silica; Peritoneum; Chemotherapy; Microparticles
Pleural fibrosis and malignant mesotheliomas (MM) occur after exposures to pathogenic fibers, yet the mechanisms initiating these diseases are unclear.
We document priming and activation of the NLRP3 inflammasome in human mesothelial cells by asbestos and erionite that is causally related to release of IL-1β, IL-6, IL-8, and Vascular Endothelial Growth Factor (VEGF). Transcription and release of these proteins are inhibited in vitro using Anakinra, an IL-1 receptor antagonist that reduces these cytokines in a human peritoneal MM mouse xenograft model.
These novel data show that asbestos-induced priming and activation of the NLRP3 inflammasome triggers an autocrine feedback loop modulated via the IL-1 receptor in mesothelial cell type targeted in pleural infection, fibrosis, and carcinogenesis.
Asbestos; Mesothelioma; Mesothelium; Inflammasomes; NLRP3
Presently there is limited research to suggest efficacious interventions for infants at-risk for autism. Pivotal response treatment (PRT) has empirical support for use with preschool children with autism, but there are no reports in the literature utilizing this approach with infants. In the current study, a developmental adaptation of PRT was piloted via a brief parent training model with three infants at-risk for autism. Utilizing a multiple baseline design, the data suggest that the introduction of PRT resulted in increases in the infants’ frequency of functional communication and parents’ fidelity of implementation of PRT procedures. Results provide preliminary support for the feasibility and utility of PRT for very young children at-risk for autism.
Early intervention; Pivotal response treatment; Parent education; Infant siblings
In myeloid cells the inflammasome plays a crucial role in innate immune defenses against pathogen- and danger-associated patterns such as crystalline silica. Respirable mineral particles impinge upon the lung epithelium causing irreversible damage, sustained inflammation and silicosis. In this study we investigated lung epithelial cells as a target for silica-induced inflammasome activation.
A human bronchial epithelial cell line (BEAS-2B) and primary normal human bronchial epithelial cells (NHBE) were exposed to toxic but nonlethal doses of crystalline silica over time to perform functional characterization of NLRP3, caspase-1, IL-1β, bFGF and HMGB1. Quantitative RT-PCR, caspase-1 enzyme activity assay, Western blot techniques, cytokine-specific ELISA and fibroblast (MRC-5 cells) proliferation assays were performed.
We were able to show transcriptional and translational upregulation of the components of the NLRP3 intracellular platform, as well as activation of caspase-1. NLRP3 activation led to maturation of pro-IL-1β to secreted IL-1β, and a significant increase in the unconventional release of the alarmins bFGF and HMGB1. Moreover, release of bFGF and HMGB1 was shown to be dependent on particle uptake. Small interfering RNA experiments using siNLRP3 revealed the pivotal role of the inflammasome in diminished release of pro-inflammatory cytokines, danger molecules and growth factors, and fibroblast proliferation.
Our novel data indicate the presence and functional activation of the NLRP3 inflammasome by crystalline silica in human lung epithelial cells, which prolongs an inflammatory signal and affects fibroblast proliferation, mediating a cadre of lung diseases.
Silica; NLRP3 inflammasome; Caspase-1; IL-1β; HMGB1; bFGF
The innate immune response plays a critical role in the host defense against invading pathogens, and TLR2, a member of the Toll-like receptor (TLR) family, has been implicated in the immune response and initiation of inflammatory cytokine secretion against several human viruses. Previous studies have demonstrated that infectious and ultraviolet-inactivated herpes simplex virus 1 (HSV-1) virions lead to the activation of nuclear factor kappa B (NF-κB) and secretion of proinflammatory cytokines via TLR2. However, except for the envelope glycoprotein gH and gL, whether there are other determinants of HSV-1 responsible for TLR2 mediated biological effects is not known yet. Here, we demonstrated that the HSV-1-encoded envelope glycoprotein gB displays as molecular target recognized by TLR2. gB coimmunoprecipitated with TLR2, TLR1 and TLR6 in transfected and infected human embryonic kidney (HEK) 293T cells. Treatment of TLR2-transfected HEK293T (HEK293T-TLR2) cells with purified gB results in the activation of NF-κB reporter, and this activation requires the recruitment of the adaptor molecules myeloid differentiation primary-response protein 88 (MyD88) and tumor necrosis factor receptor-associated factor 6 (TRAF6) but not CD14. Furthermore, activation of NF-κB was abrogated by anti-gB and anti-TLR2 blocking antibodies. In addition, the expression of interleukin-8 induced by gB was abrogated by the treatment of the human monocytic cell line THP-1 with anti-TLR2 blocking antibody or by the incubation of gB with anti-gB antibody. Taken together, these results indicate the importance and potency of HSV-1 gB as one of pathogen-associated molecular patterns (PAMPs) molecule recognized by TLR2 with immediate kinetics.
Recent studies investigating possible causes of male subfertility have largely focused on how lifestyle or environmental factors impact on the process of spermatogenesis. Markedly, fewer studies have investigated those risk factors that result in reduced sperm quality, such as poor sperm motility. The speed at which sperm swim is a major predictor of fertility and is extremely variable in human populations. It has been hypothesized that offspring sex may be adaptively manipulated to maximize the offspring's reproductive fitness (e.g., parents with genes for good male fertility traits, such as high sperm speed, would produce primarily sons and fewer daughters because the offspring will inherit advantageous male fertility genes). Conversely, parents with poor male fertility genes would produce primarily daughters. We tested whether there was an association between how fast a man's sperm swam and the sex bias of his siblings in a sample of men attending clinic for fertility investigations with their partner and with a wide range of semen characteristics, including sperm speed. We found that the sex bias of a man's siblings is associated with his sperm speed; men with female-biased siblings had significantly slower sperm (judged using computer-assisted sperm analysis (CASA)) than men from male-biased sibships. This observation suggests family composition is an important factor that needs to be considered in future epidemiological and clinical studies of human fertility.
computer-assisted sperm analysis (CASA); fertility; intralocus sexual conflict; male; offspring; sex ratio; sexual antagonism
We hypothesized that normal human mesothelial cells acquire resistance to asbestos-induced toxicity via induction of one or more epidermal growth factor receptor (EGFR)–linked survival pathways (phosphoinositol-3-kinase/AKT/mammalian target of rapamycin and extracellular signal–regulated kinase [ERK] 1/2) during simian virus 40 (SV40) transformation and carcinogenesis. Both isolated HKNM-2 mesothelial cells and a telomerase-immortalized mesothelial line (LP9/TERT-1) were more sensitive to crocidolite asbestos toxicity than an SV40 Tag-immortalized mesothelial line (MET5A) and malignant mesothelioma cell lines (HMESO and PPM Mill). Whereas increases in phosphorylation of AKT (pAKT) were observed in MET5A cells in response to asbestos, LP9/TERT-1 cells exhibited dose-related decreases in pAKT levels. Pretreatment with an EGFR phosphorylation or mitogen-activated protein kinase kinase 1/2 inhibitor abrogated asbestos-induced phosphorylated ERK (pERK) 1/2 levels in both LP9/TERT-1 and MET5A cells as well as increases in pAKT levels in MET5A cells. Transient transfection of small interfering RNAs targeting ERK1, ERK2, or AKT revealed that ERK1/2 pathways were involved in cell death by asbestos in both cell lines. Asbestos-resistant HMESO or PPM Mill cells with high endogenous levels of ERKs or AKT did not show dose-responsive increases in pERK1/ERK1, pERK2/ERK2, or pAKT/AKT levels by asbestos. However, small hairpin ERK2 stable cell lines created from both malignant mesothelioma lines were more sensitive to asbestos toxicity than shERK1 and shControl lines, and exhibited unique, tumor-specific changes in endogenous cell death–related gene expression. Our results suggest that EGFR phosphorylation is causally linked to pERK and pAKT activation by asbestos in normal and SV40 Tag–immortalized human mesothelial cells. They also indicate that ERK2 plays a role in modulating asbestos toxicity by regulating genes critical to cell injury and survival that are differentially expressed in human mesotheliomas.
mesothelioma; asbestos; toxicity; epidermal growth factor receptor; protein kinase B/AKT
The interferon (IFN)-mediated antiviral response is a major defense of the host immune system. In order to complete their life cycle, viruses must modulate host IFN-mediated immune responses. Herpes simplex virus 1 (HSV-1) is a large DNA virus containing more than 80 genes, many of which encode proteins that are involved in virus-host interactions and show immune modulatory capabilities. In this study, we demonstrate that the US11 protein, an RNA binding tegument protein of HSV-1, is a novel antagonist of the beta IFN (IFN-β) pathway. US11 significantly inhibited Sendai virus (SeV)-induced IFN-β production, and its double-stranded RNA (dsRNA) binding domain was indispensable for this inhibition activity. Additionally, wild-type HSV-1 coinfection showed stronger inhibition than US11 mutant HSV-1 in SeV-induced IFN-β production. Coimmunoprecipitation analysis demonstrated that the US11 protein in HSV-1-infected cells interacts with endogenous RIG-I and MDA-5 through its C-terminal RNA-binding domain, which was RNA independent. Expression of US11 in both transfected and HSV-1-infected cells interferes with the interaction between MAVS and RIG-I or MDA-5. Finally, US11 dampens SeV-mediated IRF3 activation. Taken together, the combined data indicate that HSV-1 US11 binds to RIG-I and MDA-5 and inhibits their downstream signaling pathway, preventing the production of IFN-β, which may contribute to the pathogenesis of HSV-1 infection.
New and effective treatment strategies are desperately needed for malignant mesothelioma (MM), an aggressive cancer with a poor prognosis. We have shown previously that acid-prepared mesoporous microspheres (APMS) are nontoxic after intrapleural or intraperitoneal (IP) administration to rodents. The purpose here was to evaluate the utility of APMS in delivering chemotherapeutic drugs to human MM cells in vitro and in two mouse xenograft models of MM. Uptake and release of doxorubicin (DOX) alone or loaded in APMS (APMS-DOX) were evaluated in MM cells. MM cell death and gene expression linked to DNA damage/repair were also measured in vitro. In two SCID mouse xenograft models, mice received saline, APMS, DOX, or APMS-DOX injected directly into subcutaneous (SC) MM tumors or injected IP after development of human MMs peritoneally. Other mice received DOX intravenously (IV) via tail vein injections. In comparison to DOX alone, APMS-DOX enhanced intracellular uptake of DOX, MM death, and expression of GADD34 and TP73. In the SC MM model, 3X weekly SC injections of APMS-DOX or DOX alone significantly inhibited tumor volumes, and systemic DOX administration was lethal. In mice developing IP MMs, significant (p<0.05) inhibition of mesenteric tumor numbers, weight, and volume was achieved using IP administration of APMS-DOX at one-third the DOX concentration required after IP injections of DOX alone. These results suggest APMS are efficacious for the localized delivery of lower effective DOX concentrations in MM, and represent a novel means of treating intracavitary tumors.
Microparticles; Mesoporous silica; Mesothelioma; Doxorubicin; Intracavitary tumors
Thiostrepton (TS) is a thiazole antibiotic that inhibits expression of FOXM1, an oncogenic transcription factor required for cell cycle progression and resistance to oncogene-induced oxidative stress. The mechanism of action of TS is unclear and strategies that enhance TS activity will improve its therapeutic potential. Analysis of human tumor specimens showed FOXM1 is broadly expressed in malignant mesothelioma (MM), an intractable tumor associated with asbestos exposure. The mechanism of action of TS was investigated in a cell culture model of human MM. As for other tumor cell types, TS inhibited expression of FOXM1 in MM cells in a dose-dependent manner. Suppression of FOXM1 expression and coincidental activation of ERK1/2 by TS were abrogated by pre-incubation of cells with the antioxidant N-acetyl-L-cysteine (NAC), indicating its mechanism of action in MM cells is redox-dependent. Examination of the mitochondrial thioredoxin reductase 2 (TR2)-thioredoxin 2 (TRX2)-peroxiredoxin 3 (PRX3) antioxidant network revealed that TS modifies the electrophoretic mobility of PRX3. Incubation of recombinant human PRX3 with TS in vitro also resulted in PRX3 with altered electrophoretic mobility. The cellular and recombinant species of modified PRX3 were resistant to dithiothreitol and SDS and suppressed by NAC, indicating that TS covalently adducts cysteine residues in PRX3. Reduction of endogenous mitochondrial TRX2 levels by the cationic triphenylmethane gentian violet (GV) promoted modification of PRX3 by TS and significantly enhanced its cytotoxic activity. Our results indicate TS covalently adducts PRX3, thereby disabling a major mitochondrial antioxidant network that counters chronic mitochondrial oxidative stress. Redox-active compounds like GV that modify the TR2/TRX2 network may significantly enhance the efficacy of TS, thereby providing a combinatorial approach for exploiting redox-dependent perturbations in mitochondrial function as a therapeutic approach in mesothelioma.
The syndrome of cerebellar ataxia with bilateral vestibulopathy was delineated in 2004. Sensory neuropathy was mentioned in 3 of the 4 patients described. We aimed to characterize and estimate the frequency of neuropathy in this condition, and determine its typical MRI features.
Retrospective review of 18 subjects (including 4 from the original description) who met the criteria for bilateral vestibulopathy with cerebellar ataxia.
The reported age at onset range was 39–71 years, and symptom duration was 3–38 years. The syndrome was identified in one sibling pair, suggesting that this may be a late-onset recessive disorder, although the other 16 cases were apparently sporadic. All 18 had sensory neuropathy with absent sensory nerve action potentials, although this was not apparent clinically in 2, and the presence of neuropathy was not a selection criterion. In 5, the loss of pinprick sensation was virtually global, mimicking a neuronopathy. However, findings in the other 11 with clinically manifest neuropathy suggested a length-dependent neuropathy. MRI scans showed cerebellar atrophy in 16, involving anterior and dorsal vermis, and hemispheric crus I, while 2 were normal. The inferior vermis and brainstem were spared.
Sensory neuropathy is an integral component of this syndrome. It may result in severe sensory loss, which contributes significantly to the disability. The MRI changes are nonspecific, but, coupled with loss of sensory nerve action potentials, may aid diagnosis. We propose a new name for the condition: cerebellar ataxia with neuropathy and bilateral vestibular areflexia syndrome (CANVAS). Neurology® 2011;76:1903–1910
Varicella-zoster virus (VZV) infection of differentiated cells within the host and establishment of latency likely requires evasion of innate immunity and limits secretion of antiviral cytokines. Here we report that its immediate-early protein ORF61 antagonizes the beta interferon (IFN-β) pathway. VZV infection down-modulated the Sendai virus (SeV)-activated IFN-β pathway, including mRNA of IFN-β and its downstream interferon-stimulated genes (ISGs), ISG54 and ISG56. Through a primary screening of VZV genes, we found that ORF61 inhibited SeV-mediated activation of IFN-β and ISRE (IFN-stimulated response element) promoter activities but only slightly affected NF-κB promoter activity, implying that the IFN-β pathway may be blocked in the IRF3 branch. An indirect immunofluorescence assay demonstrated that ectopic expression of ORF61 abrogated the detection of IRF3 in SeV-infected cells; however, it did not affect endogenous dormant IRF3 in noninfected cells. Additionally, ORF61 was shown to be partially colocalized with activated IRF3 in the nucleus upon treatment with MG132, an inhibitor of proteasomes, and the direct interaction between ORF61 and activated IRF3 was confirmed by a coimmunoprecipitation assay. Furthermore, Western blot analysis demonstrated that activated IRF3 was ubiquitinated in the presence of ORF61, suggesting that ORF61 degraded phosphorylated IRF3 via a ubiquitin-proteasome pathway. Semiquantitative reverse transcription-PCR (RT-PCR) analysis demonstrated that the level of ISG54 and ISG56 mRNAs was also downregulated by ORF61. Taken together, our results convincingly demonstrate that ORF61 down-modulates the IRF3-mediated IFN-β pathway by degradation of activated IRF3 via direct interaction, which may contribute to the pathogenesis of VZV infection.
We previously found that enveloped virus binding and penetration are necessary to initiate an interferon-independent, IRF3-mediated antiviral response. To investigate whether membrane perturbations that accompany membrane fusion-dependent enveloped-virus entry are necessary and sufficient for antiviral-state induction, we utilized a reovirus fusion-associated small transmembrane (FAST) protein. Membrane disturbances during FAST protein-mediated fusion, in the absence of additional innate immune response triggers, are sufficient to elicit interferon-stimulated gene induction and establishment of an antiviral state. Using sensors of membrane disruption to activate an IRF3-dependent, interferon-independent antiviral state may provide cells with a rapid, broad-spectrum innate immune response to enveloped-virus infections.
Members of the extracellular signal-regulated kinase (ERK) family may have distinct roles in the development of cell injury and repair, differentiation and carcinogenesis. Here we show, using a synthetic small molecule MEK1/2 inhibitor (U0126) and RNA silencing of ERK1 and 2, comparatively, that ERK2 is critical to transformation and homeostasis of human epithelioid malignant mesotheliomas (MMs), asbestos-induced tumors with a poor prognosis. Whereas MM cell (HMESO) lines stably transfected with shERK1 or shERK2 both exhibited significant decreases in cell proliferation in vitro, injection of shERK2 cells, and not shERK1 cells, into immunocompromised SCID mice showed significant attenuated tumor growth in comparison to shControl cells. Inhibition of migration, invasion, and colony formation occurred in shERK2 MM cells in vitro, suggesting multiple roles of ERK2 in neoplasia. Microarray and qRT-PCR analyses revealed gene expression that was significantly increased (CASP1, TRAF1, FAS) or decreased (SEMA3E, RPS6KA2, EGF, BCL2L1) in shERK2-transfected MM cells in contrast to shControl-transfected MM cells. Most striking decreases were observed in mRNA levels of Semaphorin 3 (SEMA3E), a candidate tumor suppressor gene linked to inhibition of angiogenesis. These studies demonstrate a key role of ERK2 in novel gene expression critical to the development of epithelioid MMs. After injection of sarcomatoid human MM (PPMMill) cells into SCID mice, both shERK1 and shERK2 lines showed significant decreased tumor growth, suggesting heterogeneous effects of ERKs in individual MMs.
Asbestos; mesothelioma; extracellular signal regulated kinase (ERK1/2); Mitogen activated protein kinases; gene expression
The innate host response to virus infection is largely dominated by the production of type I interferon and interferon stimulated genes. In particular, fibroblasts respond robustly to viral infection and to recognition of viral signatures such as dsRNA with the rapid production of type I interferon; subsequently, fibroblasts are a key cell type in antiviral protection. We recently found, however, that primary fibroblasts deficient for the production of interferon, interferon stimulated genes, and other cytokines and chemokines mount a robust antiviral response against both DNA and RNA viruses following stimulation with dsRNA. Nitric oxide is a chemical compound with pleiotropic functions; its production by phagocytes in response to interferon-γ is associated with antimicrobial activity. Here we show that in response to dsRNA, nitric oxide is rapidly produced in primary fibroblasts. In the presence of an intact interferon system, nitric oxide plays a minor but significant role in antiviral protection. However, in the absence of an interferon system, nitric oxide is critical for the protection against DNA viruses. In primary fibroblasts, NF-κB and interferon regulatory factor 1 participate in the induction of inducible nitric oxide synthase expression, which subsequently produces nitric oxide. As large DNA viruses encode multiple and diverse immune modulators to disable the interferon system, it appears that the nitric oxide pathway serves as a secondary strategy to protect the host against viral infection in key cell types, such as fibroblasts, that largely rely on the type I interferon system for antiviral protection.