text
string | predicted_class
string | confidence
float16 |
|---|---|---|
The biological significance of clustered DNA damage is not only based on the difficulty encountered by the different DNA repair proteins that process these closely spaced DNA lesions, but also to the fact that several of these OCDLs can be converted into de novo DSBs during repair . There are still many questions open as to which type of clusters will be more prone to be converted to potentially dangerous DSBs, but some parameters such as the presence of an SSB in one strand that delays the simultaneous process of other base lesions on the other strand, the nucleotide distance between the various lesions and the direction 3′ or 5′ to each other have been found to be critical .For example, some recent in vitro data using plasmid pUC18 DNA exposed to high-LET IR (He2+ or C6+ ions) or low-LET (X-rays) and under varying radical-scavenging conditions, suggest that base lesion clusters appear three or more base pairs apart and are promptly converted to a DSB by a glycosylase, regardless of the order of enzymatic treatment . These and other similar results are in good agreement with Monte Carlo (MC) track structure calculations, suggesting an increase of complexity with LET and specific base to SSB ratio etc. . Additionally, one cannot disregard that the initial repair steps at clustered damage sites is a major parameter that directs towards the conversion of MDS into DSB or not . Unrepaired clustered DNA lesions can lead to chromosomal breaks and significant GI as primarily manifested during the induction of clustered DNA damage by high-LET radiations .
|
study
| 99.94 |
The experimental validation of DNA damage clustering induction, as well as, the repair mechanisms involved have not been an easy task. There are some discrepancies that can still be found between experimental evidence or data and prediction models using Monte Carlo (MC)-based methodologies . Significant advancement in the understanding of expected clustered DNA damage induction mechanisms has been achieved using a fast and cell-level MC code, the Monte Carlo Damage Simulation (MCDS) code, integrated into the general-purpose MC N-particle radiation transport code system (MCNP) . At the same time, a better understanding of the processes and mechanisms involved in the repair of clustered DNA lesions has been provided by the development of analytical biochemical models for DSB and base lesion repair .
|
study
| 99.8 |
Towards the history and advances in the field of experimental detection of clustered DNA lesions, the reader can refer to the above mentioned references. Current research in this field is based on the idea that theory and predictions do not always coincide with experimental evidence. The major challenges towards the detection of clustered DNA damages have been: (1) the accurate measurement of DSBs and OCDLs levels and their types, especially at the cellular level and (2) theirin situ detection and reliable quantitative measurement.
|
review
| 99.9 |
During the last decade and since its initial discovery in 1998, the application of the γH2AX methodology has provided a significant boost towards reliable measurements of DSBs at a cellular or tissue level . On the other hand, for the measurement of non-DSB lesions at least in situ, significant advancements have been made using adaptations of fluorescence microscopy and foci colocalization as reviewed in , but still there is no reliable in situ-technique to detect closely spaced DNA lesions within 1–20 bp apart. The colocalization of two or more antibodies (corresponding to DNA repair proteins presumably working on a clustered damage site), certainly provides valuable information, but this only gives an idea on how many different proteins maybe present in a chromosome region of a few Mbp. In each case, measurement of DNA lesions is being performed indirectly by the use of usually two DNA damage/repair proteins specific primary antibodies (e.g., against γ-H2AX:DSB and OGG1:oxidized purines or NTH1:oxidized pyrimidines etc.) each detected by the appropriate fluorescent labeled secondary antibodies. The simultaneous use of more than three different antibodies requires highly advanced microscopic systems and it is considered to be highly challenging. This microscopy-based methodology however, is very distant from the original definition of clustered DNA damage located in a very small DNA region . Based on the above and in an attempt to make a rough comparison between the originally used adaptations of gel electrophoresis to measure different types of DNA clusters (DSBs and non-DSBs) as introduced by Sutherland and colleagues and afterwards by others one can conclude that: (1) there are two main methodologies to measure complex DNA lesions at the cellular level; one based on DNA fragmentation measurement using gel electrophoresis with repair enzymes as damage probes, and in situ immunofluorescence microscopic approaches using different antibodies to allow foci colocalization centered around the DSB focus (usually γH2AX/53BP1) (Figure 1 and Figure 2) both methodologies are necessary and useful, but they are complementary; when it comes to measurement of damage complexity of DSBs and non-DSBs one should consider applying them both. A short description on the powerful γH2AX methodology follows.
|
review
| 99.9 |
To date, a large volume of studies supports the notion that the γH2AX epigenetic biomarker has been established as the most sensitive and specific epigenetic biomarker for DSB detection and quantification. H2AX is a mammalian variant that belongs to the H2A histone family that has a phosphorylation site at a serine 139. This site becomes rapidly phosphorylated when DSBs are generated into DNA. It has been well documented that this phosphorylation is specific to DSBs . This specific phosphorylation is denoted as “γ-phosphorylation” and the H2AX histone molecules that “carry” this phosphorylation are designated as “γH2AX” accordingly.
|
study
| 95.9 |
One of the most intrinsic features of γH2AX is that γ-phosphorylation extents at megabase-long domains in chromatin. The γ-phosphorylation of H2AX is evident within minutes after the generation of DSBs. Nevertheless, γ-phosphorylation is not restricted to the vicinity of the sites of the DSB, but extends both sides of the damage, and reaches megabase-long domains in chromatin . This feature of γ-phosphorylation is very important; it represents a biological amplification mechanism where one DSB induces the γ-phosphorylation of thousands of H2AX molecules along megabase-long domains of chromatin that are adjusted to the sites of DSBs. The γ-phosphorylated megabase-long chromatin domains that are adjusted to the sites of one DSB are the basis for a very important technological implication. As one DSB is surrounded by thousands of γ-phosphorylated H2AX nucleosomes, specific antibodies enable the microscopy observation of the site of one DSB by immunocytochemistry. When detected with epifluorescence or confocal microscopy, γH2AX foci appear as large, roughly spherical conformations in cells that are in the G0, G1, S, or G2 phase of the cell cycle .
|
study
| 100.0 |
On the contrary, it has been demonstrated that γH2AX foci appear as band-like conformations in deer mitotic cells, resembling perhaps the known bands in human mitotic chromosomes as seen in routine karyotype tests. Though, these conformations have not been detected in human mitotic cells, perhaps due to intrinsic features of human mitotic chromatin. Additionally, the possible detection of only one DSB in the nucleus by γH2AX immunocytochemistry renders this technology as currently the most sensitive assay for the detection of DSBs.
|
study
| 99.94 |
Although the amount of H2AX, as well as the percentage of H2AX in respect to the total H2A of the histone family is not the same between differentiated cell types, the percentage of chromatin that becomes phosphorylated per one DSB has a roughly constant average. That permits the quantification of the γH2AX foci in cell lines, primary cells, and tissues. Assays based on specific antibodies against the characteristic γH2AX epitope (e.g., confocal and epifluorescent microscopy, flow cytometry, ELISA, immunoprecipitation etc.) have been incomparably successful for the detection of DSBs . Among them, immunocytochemical detection of γH2AX has become the primary method of detection, as it is several orders of magnitude more sensitive than other methods and has the potential for quantification . In addition, it has been shown that γH2AX foci are formed preferentially in euchromatin after IR-exposure .
|
study
| 99.94 |
In general, the γH2AX assays share four important technical features: (i) a general acceptance for specificity to DSBs, (ii) sensitivity, (iii) quantification of DSBs, and (iv) repeatability and reproducibility. Regarding the technical supremacy of the specific methodology one can assign the following features: (i) specificity to DSBs: the γH2AX has been shown to detect specifically DSBs rather than other DNA damages . However, it has been reported that γH2AX can be formed at other types of lesions and in high frequencies in S-phase cells undergoing replication , or some other cell types undergoing for example chromatin remodeling , (ii) sensitivity: immunoassays utilizing specific antibodies for γH2AX show the highest score in sensitivity. Even one DSB can be detected by anti-γH2AX immunocytochemistry . The biology of γ-phosphorylation provides the explanation for this remarkable sensitivity; visualization of only one DSB in the whole nucleus is feasible, as γ-phosphorylation spans megabaselong domains in chromatin juxtaposed to the break, (iii) quantification: the presence of γH2AX detected by antibody based techniques can be quantified by various methods, such as confocal and epifluorescence microscopy (measured manually or automatically), flow cytometry, western blot quantification, etc. and (iv) repeatability and reproducibility: to date, the repeatability and reproducibility of the method have been demonstrated by numerous diverse research laboratories all over the world, as demonstrated by the number of scientific publications .
|
review
| 99.8 |
At this point, it must be mentioned that a variety of tumor cells have been found with increased numbers of γH2AX foci suggesting to be related to the overall chromosomal instability of these cells . Last but not least, it has been also indicated by Banath et al. that persistence of DNA damage-induced γH2AX foci can be suggestive of lethal DNA damage so that it may be possible to predict tumor cell killing by different DNA damaging therapeutic agents by measuring the fraction of cells that retain γH2AX signalling .
|
study
| 99.94 |
The study of complex DNA damage in terms of in situ detection involves the concept of DNA repair colocalization (DNA repair centers) as previously introduced for DSBs and non-DSB damage . The term “colocalization” actually refers to the spatiotemporal coexistence of two or more proteins of different type. The detection of complex DNA lesions consisting of a variety of DSBs and OCDLs is made possible through the visualization of proteins participating in a distinct DNA repair mechanism, e.g. one protein participating in the base excision repair (BER) processing base lesions and another one participating in the homologous recombination (HR) or the non-homologous end joining (NHEJ) for the repair of DSBs. As shown in Figure 2, upon the induction of a cluster of DNA lesions, several DNA repair pathways and proteins will be involved. For short-patch BER, a DNA glycosylase will arrive, excise the damaged base and the repair will be completed presumably by the human AP endonuclease 1 (APE1), a polymerase and ligase III to seal the broken ends. In the nearby DSB area (within a few bp apart), the Ku heterodimer (Ku70/80) initiates NHEJ by binding to the free DNA ends and engaging other NHEJ factors such as DNA-dependent protein kinase (DNA-PK), XRCC4, and DNA Ligase IV to the site of the break. DNA-PK becomes activated upon DNA binding, and phosphorylates a number of substrates including p53, Ku, and DNA Ligase IV cofactor XRCC4. Phosphorylation of these factors is believed to further facilitate the processing of the break. Finally, in order for ligation to occur, a partial processing of the ends by nucleases Artemis, MRE11/Rad50/NBS1 complex and FEN-1 is taking place.
|
study
| 99.94 |
Although the in situ immunofluorescence has been extensively utilized for the detection of single/simple DNA damage including one type of lesions , the simultaneous detection of DSBs and non-DSB lesions has been reported only in a few studies . The difficulty in achieving visualization of base lesions, in terms of foci, lies in the fact that only a few molecules of every specific DNA repair protein (e.g., OGG1, NTH1, APE1 etc.) are taking part in the repair of a single lesion, in contrast with DSB repair where hundreds/thousands of molecules of the same DNA repair protein (like γH2AX/53BP1) may contribute to the process, as discussed above. Moreover, unlike γH2AX protein which becomes present mainly upon a DSB formation, most of the non-DSB repair proteins have endogenous concentrations within the cell nucleus, therefore resulting in increased background signal. A pre-extraction step in the experimental procedure, as well as the introduction of the Pclc colocalization parameter in image analysis have helped researchers overcome these obstacles (Figure 1). In Figure 1, the theoretical description of the Pclc-parameter is given in detail (panel A), along with its application for the detection of complex DNA damage (panel B) and an additional application for the derivation of useful data regarding the localization of DNA repair proteins in euchromatin/heterochromatin regions (panel C).
|
study
| 99.94 |
Triage in medical situations refers to the assignment of degrees of urgency to wounds or illnesses to decide the order of treatment of a large number of patients or casualties. Radiation injury for the cell can be considered as a major “wound to its crucial organs” and in many cases a matter of life or death. The delineation of how DDR exerts immune responses still can be considered as a puzzling topic . Based on the above ideas, it is generally accepted that once complex and/or persistent DNA damage is induced and most probably GI, immune signaling is initiated by different components of the DDR/R pathway including DNA damage sensors, transducer kinases, effectors and repair proteins . In general, association between innate immune system response and persistent DNA damage has been shown in various cases as reviewed in . In the same direction, Ermolaeva et al. used the nematode Caenorhabditis elegans eukaryotic system to show that DNA damage in germ cells induces an innate immune response that consequently leads to activation of the ubiquitin-proteasome system (UPS) in somatic tissues, which confers enhanced proteostasis and systemic stress resistance . Rodier et al. have shown that X-ray damaged human HCA2 fibroblasts develop persistent chromatin lesions bearing DSBs detected using γH2AX/53BP1 foci as surrogate markers, which triggers the secretion of inflammatory cytokines such as interleukin-6 (IL-6) . It is important to notice, that this cytokine secretion occurred only after establishment of persistent and heavy DNA damage (10 Gy of X-rays), associated with senescence and not after transient DNA damage responses (X-ray dose of 0.5 Gy). On the other hand, systemic DNA damage responses are part of the organism’s defense system in order to secure removal of damaged and malfunctioning cells and preserve tissue integrity and functionality i.e., tissue homeostasis . For example, it has been shown that in repair deficient Ataxia-telangiectasia (AT) patients, where the repair protein ATM is defective, small DNA fragments generated from the excessive DNA-breaks accumulate in the cytoplasm of these patients' cells. The DNA fragments are consequently recognized by innate immune receptors that normally detect viral DNA. This “false alarm” of viral invasion results in the production of type I interferon which in turn drives the innate immune system into an activated state .
|
review
| 99.9 |
Regarding IR exposure as a genuine genotoxic stress, accumulating experimental evidence suggests a diverse range of radiation effects for non-irradiated areas often referred to as non-targeted effects (NTE) or under the general umbrella of systemic effects . The NTE can be separated in two major groups: near (bystander), where non-irradiated cells exhibit a response similar to their neighboring irradiated cells, and distant (e.g., the clinically relevant abscopal effect) while different mechanisms are implicated in each case, as discussed recently in . The NTE usually involve the discharge of various chemical and biological mediators from the irradiated cells and thus promoting the communication of the radiation attack via the so-called damage-associated molecular patterns (DAMPs), which is based on the originally introduced idea of “danger” signals .
|
review
| 99.5 |
Recent work by Redon et al. showed that growing tumors may act as a type of stress in the organism and induce complex DNA damage (DSBs and OCDLs) in distant proliferative tissues in vivo . According to this study, rapidly growing normal tissues, such as colon and skin were found to be particularly susceptible to remotely induced DNA damage and a signaling molecule involved in inflammation, the chemokine CCL2 (monocyte chemoattractant protein-1: MCP-1) appeared to be a major player in promoting this distant effect. Interestingly, later studies by the same groups showed that this systemic DNA damage accumulation under tumor growth can be inhibited by the antioxidant Tempol suggesting the involvement of oxidative stress . The involvement of CCL2 and macrophage activation in tumor-induced distant DNA damage suggests some resemblances with the chronic tissue stress responses usually referred to as para-inflammation , which relies mostly on alternatively activated macrophages (M2) rather than on classically activated macrophages (M1) associated with the acute inflammatory response .
|
study
| 99.94 |
A CCL2-based mechanism has been also suggested for other cases of stresses i.e., exposure to IR. Specifically, it was shown that a single-dose whole-body γ-irradiation (8 Gy) induced DNA damage in mice neuronal retina, which was complemented by a low-grade chronic inflammation, para-inflammation, characterized by upregulated expression of chemokines (CCL2, CXCL12, and CX3CL1) and microglial activation . Recent patient studies also suggest an actual involvement of cytokines in the induction of RT-induced systemic DNA damage in normal tissues distant to the irradiation site . More specifically in this study, sixteen patients with non-small cell lung carcinoma (NSCLC) received 60 Gy in 30 fractions of definitive thoracic RT with or without concurrent chemotherapy (chemoRT) and peripheral blood lymphocytes (PBL) and eyebrow hairs samples were taken prior, during, and after RT. The results showed an elevation of DSBs manifested as γH2AX foci in PBL, representing normal tissues in the irradiated thorax volume, 1 hour after fraction one and γH2AX foci numbers returned to near baseline values in 24 hours after treatment. Most importantly, unirradiated hair follicles, exhibited delayed systemic (abscopal) DDR measured as γH2AX foci which increased at 24 hours post-fraction one, and remained elevated during treatment in a dose-independent manner. This distant radiation effect was related with changes in plasma levels of MDC/CCL22 and MIP-1α/CCL3 cytokines. Interestingly and consistent with the unifying model suggestion introduced by Georgakilas uniting different types of stress i.e., radiations and a growing tumor , MCP-1 blockade by neutralizing antibodies was found to inhibit lung cancer tumor growth by altering macrophage phenotype and activating cytotoxic CD8+ T lymphocytes (CTLs) . Another side of the same coin of cytokines-inflammation is the reverse activity. Earlier studies have shown that ROS/RNS could be generated in vitro by a mixture of inflammatory cytokines (IL-1β, IFN-γ and tumor necrosis factor α) in three human cholangiocarcinoma cell lines by a nitric oxide (NO)-dependent response, as assessed by alkaline (denaturing) comet assay . In addition, a parallel inhibition of global DNA repair activity by 70% was detected. These and later data indicate that activation of iNOS and excess production of NO in response to inflammatory cytokines can cause DNA damage and inhibit DNA repair, at least partially. Recent extensive bioinformatics-based metanalysis studies have verified the interactions between mediators of systemic effects and DDR/R components, as well as interactions between pattern recognition receptors (PRPs) and DNA repair proteins like BRCA1, XRCC1, DNA-PK, Ku70/80 and others . Recently, Nikitaki et al. produced a detailed list of proteins implicated in different categories of radiation-induced systemic effects, including the clinically relevant abscopal phenomenon, using improved text-mining and bioinformatics tools from the literature. Genes belonging to the DDR/R pathway and protein-protein interaction (PPi) networks as well as KEGG pathway analyses have revealed that the main pathways participating in NTE are: apoptosis, TLR-like and NOD-like receptor signaling pathways .
|
study
| 99.9 |
Conclusively, one can wonder how cells triage this scenario of the interaction between complex DNA damage, immune signaling and systemic effects, which is the most important in regulating the overall outcome of this complex crosstalk (Figure 2). It is rather secure to suggest that complex and persistent DNA damage constitutes a major “danger” signal for the cells and this probably alarms the whole cell or tissue about something “peculiar” happening in this area of damage. If this complex form of damage is processed correctly and all problems have been resolved then the alarm goes off, but the “danger” signaling may already have generated an immune response. In this case, the outcome is uncertain. Immune response manifested initially at least as innate and later on as adaptive and inflammation maybe present, especially when specific “danger” signals are produced due to cell death or senescence. As so, a continuing systemic effect of unknown severity and duration will be induced resulting to a chronic state of immune response and a precursor of pathological evolution and disease as presented with red in Figure 2. Recent evidence obtained using mice carrying an ERCC1-XPF DNA repair defect systematically or in adipocytes, suggests that persistent DNA damage-driven autoinflammation plays a causative role in adipose tissue degeneration, with important complications for advanced lipodystrophies and aging . In any case, the knowledge of the exact mechanisms and mediators of systemic responses will be very useful in various applications that involve complex DNA damage formation, such as RT, chemotherapy and tumor growth early detection. As nicely presented in a recent work by Pateras et al. continuous triggering of DDR/R can lead to excessive innate and adaptive immune response which, in turn, can lead to pathological conditions and disease .
|
review
| 99.9 |
As well-known, IR exposure can be considered for humans as a double-edged sword to either hurt or save. On one hand it can induce significant levels of complex and usually unrepairable DNA damage that can lead to enhanced mutation levels, GI and cancer, but on the other hand it can be used as the ultimate weapon against tumors . Treatment options for patients with various kinds of malignancies have expanded with discoveries of druggable targets as well as technological advances. Surgical resection, chemotherapy and RT are the three major available modalities for the treatment of most cancers and are utilized either in combination or separately, as deemed appropriate. In case of chemotherapy and RT, the main aim is to spare normal cells while inducing sufficient, non-repairable DNA damage in tumor cells. Consequently, cancer cells may exit the cell cycle permanently, a phenomenon referred to as senescence, or triggered apoptosis. The mechanism of action of chemotherapeutic agents and the dose and type of RT determines the spectrum of DNA damage induced by treatment. As discussed earlier, complex DNA lesions are the most challenging type of damage for a cell to repair. This section focuses on whether there is evidence linking efficacy of chemotherapeutic drugs or RT to the type of DNA damage they incur. Additionally, a discussion is made on evidence from literature that highlights the drawback of using these agents for therapy, since normal cells affected by these insults to their DNA can also lead to a second primary cancer development.
|
review
| 99.9 |
The therapeutic index is high when molecular targets overexpressed specifically in tumor cells can be targeted by small molecule inhibitors. Multi-kinase inhibitors have dramatically improved patient survival in hematologic malignancies, while drugs targeting cancer specific mutations have improved survival in select patient populations. In the mid-1970s, 5 year survival estimates were at 41% for patients diagnosed with acute lymphocytic leukemia while they are reported at 71% for patients diagnosed between 2006 and 2012. Similar improvement has been witnessed for chronic myeloid leukemia, 22% to 66% in the same time intervals . However, currently available standard chemotherapeutic agents and even the latest technologies in radiation physics fail to qualify as curative options for several cancer types. Commonly used chemotherapy regimens include platinum based DNA alkylating agents, topoisomerase poisons, antimetabolites, microtubule inhibitors, antitumor antibiotics, proteasome inhibitors etc. . Antitumor antibiotics include a class of drugs called anthracyclines that inhibit pathways that generate DNA nucleotides. Non anthracycline drugs in this class include a compound called bleomycin. Bleomycin portrays the strongest evidence for clustered DNA damage being used as the mechanism of action for a chemotherapeutic agent. The mechanism of action of bleomycin and the similarities in base damage produced when compared with IR makes it a “radiomimetic” chemotherapeutic . The drug creates reactive aldehyde groups at the sugar moiety that is capable of reacting with cytosine residues in its proximity and creating clustered DNA damage. Use of bleomycin has been inhibited due to severe pulmonary toxicity and risk of pulmonary fibrosis despite tolerable myelotoxicity. Since the clinical trials establishing the correlation between bleomycin use and pulmonary toxicity in the 1980s there has been years of research that indicates the importance of ROS at the site of action in propagation of the oxidative DNA damage. Even low levels of ROS have been reported to cause GI via NHEJ-mediated DNA repair .
|
review
| 99.9 |
One of the major treatment modalities for several types of cancers is RT, and ROS and clustered DNA damage are thought to be critical to mediate the effect of IR. Many decades of experimental research in cellular and molecular radiation biology provide evidence suggesting that nuclear DNA is the critical target of IR, and both the initial and residual levels of DSBs are linked to the biological effects of radiation, and that DNA damage and repair is relevant to carcinogenesis . Precise delivery of radiation beams to site of solid tumors has improved with advances in medical physics and engineering. Among these, the use of proton beams as an alternative to traditional high energy electrons has at least in theory, improved accuracy of targeting and reduction in surrounding tissue toxicity. Long term follow-up data for significant patient cohort sizes will enable us to compare the potential benefits of proton beam therapy. OCDLs are a hallmark of IR although their endogenous levels are relatively low . Radiation dose and quality dictates the complexity of DNA damage induced by the particle. Increasing dosage and LET (linear energy transfer) correlates with higher accumulation of clustered lesions in cancer cells . The recruitment kinetics of DNA repair proteins is dependent on the level of LET . The fact that DNA repair capability is compromised with increasing complexity of damage underscores the importance of these lesions in therapy . One of the significant and therapeutic advantages of high LET IR is that there are extensive amounts of clustered damage leading to increased relative biological effectiveness (RBE) vs. both photon-based and even proton-based modalities . As recently reviewed by Mohamad et al. , comparison of conventional photon-based external beam radiation to carbon ion radiotherapy reveals that carbon ions result in a better and more targeted-to-the-tumor dose distribution, higher LET and RBE. This improved RBE relates to the unique high-LET radiation-induced complex DNA damage that overpowers the DNA repair system of tumor cells as also showed for example by earlier studies using human monocytes exposed to 56Fe ions (LET=148 keV/µm) . The use of carbon or other high-LET particles maybe a solution in the case of difficult to treat tumors, including those that are hypoxic, radio-resistant, or located deeper in the body . On the history of carbon-ion based RT, one of the pioneers was the National Institute of Radiological Sciences (NIRS) which started treating patients with beams in the Heavy Ion Medical Accelerator (HIMAC) in Chiba, Japan in 1994. Following Japan, Germany in 1997 in the Gesellschaftfür Schwerionenforschung (GSI) in Darmstadt, treated their first patient and later in the Heidelberg Ion Therapy Center (HIT) in 2009. Therefore based on clinical evidence, mostly originating from Japan and Germany, high-LET radiations maybe a promising RT modality with limited radiation toxicity .
|
review
| 99.9 |
The precise contribution to the effects of clustered DNA lesions after proton treatment on cells is a matter of debate that remains to be studied in further detail . The highest LET along the path of a proton beam around the Bragg peak has been reported to correlate with maximum complexity of DNA damage .
|
study
| 100.0 |
The rise in the population of cancer survivors has led to a better understanding of the effects of RT to treat cancer patients . A significant portion of cancer survivors are patients with a history of childhood cancer. According to the American Cancer Society (ACS) the 5-year survival rate for childhood cancer patients is now over 80%. However, exposure to radiation treatment can lead to the occurrence of secondary primary cancers (SPCs) in the future. An analysis of thyroid cancer in childhood cancer survivors showed that the relative risk of thyroid cancer in these patients increased linearly with the dose of radiation for treatment through 10 Gy . The relative risk of thyroid cancer post RT was lowered at high treatment doses. Another study looking at chest RT to treat childhood cancers showed an increased risk of breast cancer in these patients . Especially treatment involving whole-lung irradiation increased this risk. This shows the importance of localized RT to reduce the risk of SPCs. The need for improvement in RT techniques that spare normal tissue is also highlighted by the incidence of metachronous cancers (multiple primary cancers developing at intervals) in adults. The incidence of secondary primary lung tumors increased by a stark 8.5% per Gy in women who had undergone RT for breast cancer . An analysis of prostate cancer patients also told a similar story. Patients with prostate cancer undergoing RT had an increased overall risk of developing hematologic, liver, esophageal, and urinary bladder cancers . Certainly, the induction of DNA damage by RT is an important factor towards the prediction of SPCs. Recent studies for example show that simulated radiation-induced persistent telomere-associated DNA damage foci can be used to predict excess relative risk of developing secondary leukemia after fractionated radiotherapy . In general, the incidence of secondary malignant neoplasms (SMN) depends on several factors like patient’s lifestyle, genetic susceptibility, DNA repair efficiency and radiosensitivity of the patient or specific organ .
|
review
| 99.9 |
The advent of proton beam therapy (PBT) brings new promise of reduced radiation treatment related morbidity by minimizing the dose to critical normal tissues . Proton therapy has shown great therapeutic potential in treating various adult malignancies including the central nervous system and gastrointestinal tract, but with uncertain benefits for example for lung cancers. At the same time, it has been estimated that excess fatal SPCs may be further reduced with proton therapy by two-thirds compared to conventional photon therapy . However, evidence that PBT reduces occurrence of metachronous cancers is limited. A study looking at PBT for advanced cholangiocarcinomas showed gastrointestinal toxicities and early metastatic progression still remains a treatment obstacle . Another study looking at cardiac events post RT in patients with thymic malignancies, showed that the lower dose to organs with PBT reduced the occurrence of major cardiac events post treatment . A long-term follow-up of patients with pediatric tumors showed fewer late adverse events and a reduced risk of metachronous malignancies with PBT . Similar studies seem to indicate that the reduced dose to normal structures with PBT as opposed to intensity-modulated RT is better tolerated by the patient population . But, it may be too soon to draw a conclusion on the benefits of PBT over traditional photon RT. A study comparing RT usage trends in men with localized prostate cancers pointed differences in demographic and prognostic factors between patients treated with proton and photon RT . Thus, although the physical theory of it may indicate to clear benefits, there is a need for more long-term assessments and more studies in general looking at the potential profits of PBT over traditional RT.
|
review
| 99.9 |
In this mini-review, we present the idea of complex (clustered DNA damage), the signature of IR, by a different perspective that of its clinical implications and its involvement in the route to carcinogenesis. As recently discussed in Pateras et al. , enthralling evidence supports the idea that DNA damage response and repair (DDR/R) and immune response signaling networks work together towards the proper function of organisms and homeostasis. We believe that there is a strong linkage between the induction of complex DNA damage, deficient or incomplete DNA repair, constant DDR/R triggering and the continuous activation of the immune system. This vicious relationship which is usually accompanied by GI can be considered without any doubt as the major pathway leading to carcinogenesis . Chronic inflammation which is synonymous to the activation of innate immune system can lead to the downregulation of DNA repair pathways and cell cycle checkpoints due to the release of inflammatory mediators and ROS which can lead to GI . Towards this direction, Colotta et al. suggested a few years ago, that cancer-related inflammation can promote GI by the various inflammatory mediators, leading to accumulation of random genetic modification in cancer or healthy cells. According to the authors, this cancer-relating inflammation represents the seventh hallmark of cancer in addition to the six hallmarks suggested initially by Hanahan and Weinberg . The understanding of the mechanisms that repair-resistant DNA damage is processed by the cells will benefit significantly therapeutic applications maximizing tumor killing and minimizing radiation toxicity for the cancer patient under RT.
|
review
| 99.9 |
Therefore, one can easily understand the importance of detecting correctly not only DSBs but also all other forms of non-DSB clustered lesions (OCDLs) and especially in the context of chromatin. A special effort must be made by the scientific community to optimize the specificity and accuracy of all current methodologies for the detection of complex DNA damage in situ and even better under live-cell imaging conditions.
|
other
| 99.44 |
The integrity of the blood brain barrier (BBB) is one of the major obstacles to effective chemotherapy for malignant brain tumors. Previous research has focused on how to circumvent the BBB with direct delivery of chemotherapy to the tumor or by mechanically opening the BBB using focused ultrasound or intra-arterial mannitol [1–6]. These direct methods are often associated with comorbidities, hospitalization or added expenses. Very few systemic pharmacologic agents have been evaluated for effectiveness of transient BBB disruption [7–9]. Yet, there is a significant need to identify agents that can transiently disrupt the BBB to improve chemotherapy delivery for patients with such CNS malignancies.
|
review
| 99.9 |
Previous studies have demonstrated the limited permeability of an intact blood–brain barrier [10–12]. However with the presence of tumor cells the BBB becomes heterogeneously disrupted and has been noted as the blood-tumor barrier (BTB) . The BTB and BBB provide a physical barrier with collaborative cells that inhibit entry of toxins, including chemotherapy. Specifically, the BTB amongst malignant gliomas is unique with a high proliferative index of microvasculature and evident alterations in astrocytic endfeet and transcytotic mechanisms; making the BTB more leaky in certain areas of the tumor but peritumoral brain less permeable with a normal BBB [10, 13–15]. These factors collectively play a role in restricting drug entry and have guided extensive research on how best to enhance transport to the CNS.
|
review
| 99.5 |
Adenosine appears to play an important role in the integrity of the BBB [16–21]. The function of adenosine is controlled by four G-protein coupled receptors: A1, A2A, A2B and A3. A1 and A3 receptors inhibit and A2A and A2B stimulate downstream activation of adenylate cyclase resulting in calcium influx and vasodilation [17, 22]. Inhibitory receptor A1 and stimulating receptor A2A exhibit high expression and functionality within the heart and brain; specifically impacting local vasodilation [18, 21, 23]. Regadenoson is an FDA-approved A2A receptor agonist which is routinely used for pharmacologic stress testing in patients with suspected cardiac disease and an inability to perform an exercise stress test. Single-photon emission computed tomography (SPECT) is often performed with a radiotracer to measure myocardial perfusion both at rest and then at the time of stress induced by regadenoson administration. Pre-clinical models have demonstrated the effectiveness of A1 and/or A2A receptor agonism to increase BBB permeability to a 70 kD dextran molecule in both mice and rat brains . The large dextran was detected in the brain for up to 180 min following a single injection in both mice and rats. In additional studies that evaluated CNS barrier permeability with regadenoson, there was a 60% increase in temozolomide brain concentrations in non-tumor bearing rats, without changing the systemic pharmacology of temozolomide . These findings prompted clinical studies of regadenoson followed by brain SPECT and CT imaging to evaluate CNS permeability differences, but there was no detectable change in permeability of the BBB in patients . However, no previous study has directly investigated whether regadenoson is capable of increasing temozolomide concentrations in the human brain.
|
study
| 99.7 |
Temozolomide is an FDA approved oral alkylating agent used in newly diagnosed and recurrent high grade gliomas. While temozolomide with radiotherapy has modestly improved overall survival rates in high grade gliomas, previous studies have proven that levels of temozolomide in the brain are only 20% of systemic drug levels [26, 27]. The peak concentration of temozolomide in the brain occurs approximately 1–2 h after ingestion. Once ingested, temozolomide undergoes degradation from its prodrug form to the highly reactive alkylating agent, methyl-triazenyl imidazole carboxamide (MTIC). Previous studies have utilized CSF sampling and intracerebral microdialysis catheters (MDC) to measure temozolomide brain extracellular concentrations in primary or metastatic brain tumors. Use of an indwelling MDC for long term tissue monitoring in the cerebrum is not new, and this technique has been utilized mainly in the traumatic brain injury setting. Prolonged catheter placement allows for continued fluid collections in alert and mobile patients [28–30]. These catheters are often placed in the operating room with verification of placement determined by brain CT. The presence of a gold filament at the catheter tip allows for easy visibility on non-contrast CT brain imaging. The semi-permeable catheter performs similarly to a capillary when perfusion fluid is pumped continuously through it. The presence of the microvial at the end of the catheter allows for regular interval sampling of the dialysate fluid. Then, drug recovery is assessed in each dialysate sample as an indirect measurement of free drug concentration.
|
study
| 96.44 |
Limited clinical studies have been performed in brain tumor patients evaluating drug delivery to the tumor bed using intracerebral microdialysis measurements [26, 31–34]. To date, the only chemotherapeutic agents evaluated have been methotrexate, temozolomide, bafetinib and 5-flucytosine [26, 32, 33, 35]. Portnow and colleagues studied serum and brain extracellular concentrations of temozolomide via MDC collected at 30 min time intervals post oral drug administration over 24 h. This study evaluated temozolomide drug delivery to the peritumoral non-contrast enhancing area in both primary and metastatic patients (n = 10). Collectively, they found that oral administration of temozolomide yielded an average brain:plasma AUC ratio of 17.8 ± 13.3%, with a peak drug concentration of approximately 2–3 h after administration and undetectable concentrations by 18 h . We designed this study to determine if FDA-approved doses of regadenoson would increase the temozolomide concentration in human brains with malignant glioma as measured by serial brain interstitial fluid assessments.
|
study
| 99.94 |
In this pilot feasibility study, we utilized MDC to determine the neuropharmacokinetics of temozolomide co-administered with regadenoson to assess temozolomide drug entry. We hypothesized that regadenoson would transiently impact the permeability of temozolomide as it did in rodents resulting in increased brain interstitium (BI) and brain:plasma AUC ratio. The primary aim of this trial was to measure brain interstitial temozolomide concentrations pre and post regadenoson using MDC in patients with recurrent high grade glioma. The secondary endpoint was to evaluate tolerability of temozolomide with a single dose of regadenoson in the post-operative setting.
|
study
| 97.44 |
This study was approved by Institutional Review Boards at Johns Hopkins and the National Institutes of Health, and all patients provided informed consent. Eligible patients were ≥ 18 years old with a diagnosis of recurrent high grade glioma suspected by MRI findings. All patients had a clinically indicated need for surgical intervention. Patients were required to have: Karnofsky performance status (KPS) of ≥ 60%; normal liver and kidney function; absolute neutrophil count ≥ 1500 cells/mm3; and a platelet count ≥ 100,000 cells/mm3. Patients were excluded if they were currently receiving chemotherapy or radiation therapy, allergic to temozolomide, pregnant or breast-feeding, had a serious medical or psychiatric illness or social situation that could interfere with catheter placement/monitoring. Patients with a prior use of VEGF or VEGFR-targeted therapy, use of investigational agents within the past 4 weeks, NCI CTC grade 3 or greater baseline neurologic symptoms, history of cardiac, bronchospastic lung disease, or a contraindication to adenosine were all excluded from study participation. Additionally, patients were asked to refrain for caffeine use at least 24 h prior to regadenoson administration, secondary to its ability to blunt the effect of regadenoson.
|
other
| 96.9 |
Once intra-operative pathology was confirmed, one to two MDialysis 70 Microdialysis Brain Catheters (membrane length 10 mm; shaft length 60 mm; ref. no. P00049) were placed into contrast-enhancing and/or non-contrast peritumoral tissue (within 5 mm from the resection cavity). Post-operative non-contrast CT imaging confirmed catheter placement with identification of an enhancing gold tip. After transfer to the intensive critical care unit, the inlet tubing of the catheter was connected to a portable syringe pump (MDialysis 107 Microdialysis Pump, ref no. P000127), containing artificial CSF (Perfusion Fluid CNS, ref no. P000151) at a rate of 1 µl/min. A microvial was connected at the end of the outlet tubing to continuously collect dialysate samples. To account for the correction factor, fractional recovery of temozolomide by ICMD was calculated based on previous in vitro sampling using CMA 70 Microdialysis catheter . All microdialysis supplies were purchased from MDialysis, (Stockholm, Sweden).
|
clinical case
| 99.9 |
Once the patient was clinically stable, at least 24 h after the completion of surgery on post-operative day 1, and tolerating oral intake, they were administered temozolomide 150 mg/m2 orally once. On post-operative day 2, each patient was again given temozolomide 150 mg/m2 orally, and approximately 60 min later, each patient was administered intravenous regadenoson 0.4 mg once over 10 s. Previous studies have demonstrated temozolomide peak brain concentrations occur 90–120 min after administration [26, 27]. Additionally, preclinical studies with regadenoson have demonstrated the peak effect on barrier permeability occurs 30–60 min after administration [19, 24, 36]. Thus, we opted to administer regadenoson 60 min after temozolomide to ensure peak concentration of temozolomide in the brain and optimal mechanism of regadenoson action on brain vasculature simultaneously. Regadenoson was given with continuous ECG monitoring for a total of 10 min post injection, in the presence of a staff cardiologist (standard cardiac dosing regimen). Pre-temozolomide blood samples were collected 15 min prior to drug administration and then 1, 2, 3, 4, 8, and 18 h after the dose of temozolomide (5 ml per collection). The samples of blood were collected in heparinized syringes, promptly mixed by inversion, and then placed on wet ice until centrifugation at 1300×g for 10 min at 4 °C (within 1 h). The samples were processed to plasma within 30 min from centrifugation, and the pH of each sample was adjusted to < 4 with the use of 8.5% phosphoric acid. Plasma was then stored frozen at − 70 °C or below until subsequent batch analysis via liquid chromatography-tandem mass spectrometry (LC–MS/MS).
|
clinical case
| 99.9 |
Dialysate samples were continuously collected with microvial changes every 3 h after portable syringe pump connection on post-operative day 0. At least 24 h after surgery, the microvial was changed pre-temozolomide ingestion and then 1, 2, 3, 4, 6, 8, 10, 12, 14, 16, and 18 h after temozolomide intake on post-operative day 1 and 2. For temozolomide stabilization each microvial was prefilled with 6–12 µl of acetic acid. Microvials containing dialysate samples were stored on dry ice until all microdialysis samples were collected from the patient. Thereafter, the dialysate samples were stored at or below − 70 °C until LC–MS/MS analysis.
|
study
| 99.94 |
Temozolomide concentrations were quantified in acidified sodium heparin plasma and acidified brain interstitial dialysate. For plasma, 15 µl of 8.5% phosphoric acid was added per 0.5 ml of plasma. For microdialysis fluid, 1 µl of glacial acetic acid was added for every 10 µl of the dialysate. Perfusion fluid CNS (artificial CSF) was used as a surrogate matrix for dialysate standards and QCs. Temozolomide was extracted from samples (50 µl of acidified plasma or 20 µl of acidified microdialysis fluid) by adding 600 µl of ethyl acetate containing IS (20 ng/ml of caffeine-13C3) (Sigma–Aldrich, St. Louis, MO). Samples were vortex-mixed and centrifuged at 2000 rpm for 10 min. The top layer was transferred to a clean glass tube and dried under a nitrogen air stream. Samples were reconstituted with 200 µl of 0.5% formic acid in water and stored in the autosampler at 5 °C for LC–MS/MS analysis.
|
study
| 100.0 |
Liquid chromatography-tandem mass spectrometry analysis was performed on an AB Sciex 5500 QTrap mass spectrometer (Sciex, Foster City, CA) coupled with an Acquity UPLC system (Waters, Milford MA). The LC separation was achieved using a Zorbax XDB C18 column (4.6 × 50 mm, 5 µm) (Agilent, Santa Clara, CA) at room temperature. The mobile phase solvent A was water containing 0.1% formic acid and mobile phase solvent B was methanol containing 0.1% formic acid. The mobile phase was delivered at a flow rate of 0.3 ml/min. The initial mobile phase composition was 60% solvent A and 40% solvent B. From 0.5 to 4.0 min, solvent B was increased to 100% and conditions held until 5.0 min. At 5.1 min, the mobile phase composition was then returned to 40% solvent B until 6.0 min. The total runtime was 6 min.
|
study
| 99.94 |
The column eluent was monitored using a Sciex 5500 QTrap mass spectrometer using electrospray ionization operating in positive mode. The mass spectrometer was programmed to monitor the following multiple reaction monitoring (MRM) m/z transitions: 195.15 → 138.10 and 198.00 → 140.00 for temozolomide and IS, respectively. Calibration curves for temozolomide were computed using the area ratio peak of the analysis to the internal standard by using a quadratic regression with 1/x2 weighting for plasma and 1/x weighting for artificial CSF, both with a calibration range of 0.005–1.0 µg/ml, and dilutions up to 1:10 (v/v) were accurately quantitated.
|
study
| 100.0 |
Using non-compartmental methods, the pharmacokinetic parameters within plasma and dialysate temozolomide were determined by concentrations vs. time. While the maximum concentration (Cmax) and time maximum concentration (Tmax) were determined directly from the measured data points, half-lives (t1/2) were calculated from the elimination rate constant derived from the terminal slope. The AUC0–18 h for each day was estimated by standard non-compartmental analysis performed by Phoenix® WinNonlin version 6.3 (Pharsight Corporation, Mountain View, CA, USA). The pharmacokinetics variables were tabulated, using descriptive statistics calculated pre and post regadenoson. The differences of the AUCs were summarized by mean and standard deviation. Means and standard deviations were presented for peak drug concentration (Cmax), time of peak drug concentration (Tmax), drug half-life (t1/2) and area under the curve from time 0–18 h after temozolomide administration (AUC0–18 h).
|
study
| 100.0 |
Six patients were enrolled on the study from May 2015 to April 2017. Five of the patients were deemed as evaluable. One patient was deemed unevaluable due to microdialysis catheter displacement, which occurred approximately 24 h after insertion. This displacement was attributed to manipulation of the patient’s surgical head dressing post-operatively. This patient was removed from study before any study medications were administered. Table 1 summarizes patient demographics. All patients underwent surgical debulking for their recurrent high grade glioma. Each catheter was placed in peritumoral tissue that was deemed to be non-contrast enhancing tissue by pre-operative MRI. In two patients, catheters were also placed in contrast enhancing areas approximately 5 mm from the surgical bed due to a subtotal resection. Catheter tip locations were accurately determined by superimposed CT and MRI imaging (Fig. 1). All evaluable patients tolerated microdialysis catheter insertion without associated bleeding, infection, pain or other incidents attributable to foreign material placement.Table 1Clinical demographicsPatientAgeGenderDiagnosisCatheter(s) tip placement area149MGlioblastomaNon-contrast and contrast enhancing268MGlioblastomaNon-contrast enhancing332MGlioblastomaNon-contrast and contrast enhancing460MGlioblastomaNon-contrast enhancing551MGlioblastomaNon-contrast enhancing670FGlioblastomaNon-contrast enhancingAll patients diagnosed with recurrent glioblastoma and catheter tips were placed in non-contrast enhancing tumor. In patients 1 and 3, tips were also placed in contrast enhancing tissue Fig. 1Catheter placement imaging. Brain CT and MRI superimposed delineating catheter placement and tumor margins. Patient 1 had one catheter tip placed in non-contrast enhancing area and the second in contrast enhancing tissue (a). Patient 2 had one catheter placed in non-contrast enhancing tissue (b). Superimposed images denote contrast enhancement in green. The white triangle indicates placement of catheter tips
|
clinical case
| 98.6 |
Catheter placement imaging. Brain CT and MRI superimposed delineating catheter placement and tumor margins. Patient 1 had one catheter tip placed in non-contrast enhancing area and the second in contrast enhancing tissue (a). Patient 2 had one catheter placed in non-contrast enhancing tissue (b). Superimposed images denote contrast enhancement in green. The white triangle indicates placement of catheter tips
|
clinical case
| 99.9 |
Post-operatively, each patient was transferred and cared for in the intensive care unit during the entire duration of the microdialysis sampling period. Dialysate samples were obtained from 5 patients, with sampling obtained for approximately 72 h from catheter insertion. Drug administration and monitoring while in the ICU included regadenoson administration by the trial-associated cardiologist. Each patient received temozolomide 150 mg/m2 daily for 2 days and on day 2 was administered regadenoson by the cardiologist; who noted expected regadenoson side effects of transient tachycardia, elevated blood pressure and flushing. Only two patients experienced grade 1 headaches, (patient 2 and 4) which resolved within 30 min from regadenoson administration. No subjects required aminophylline as a reversal agent to regadenoson. No unanticipated adverse events were noted from temozolomide, regadenoson drug administrations, or microdialysis catheter sample collections.
|
clinical case
| 99.4 |
Dexamethasone was administered to each patient as part of their standard post-operative care; in an effort to minimize post-operative vasogenic brain edema. For each patient on study, dexamethasone was given via intravenous administration every 8 h. Specifically, on the days of study, all patients except patient 4 received dexamethasone approximately 2 h after temozolomide administration (1 h after regadenoson administration). For patient 4, dexamethasone was given at the same time as temozolomide (1 h prior to regadenoson administration).
|
other
| 78.9 |
Additional file 1: Table S1 summarizes the plasma and brain interstitial pharmacokinetic data for each patient, accounting for in vitro fractional recovery . We opted to only assess plasma and brain dialysate samples for temozolomide because the active temozolomide metabolite, MTIC, was associated with poor acid stabilization and recovery . Comparing plasma concentrations of temozolomide alone vs. temozolomide with regadenoson, neither the Cmax nor AUCs were impacted by regadenoson (Fig. 2, Additional file 2: Fig S1). Peak temozolomide plasma concentrations with temozolomide alone or combined with regadenoson were: 3.5 ± 1.6 µg/ml vs. 4.8 ± 1.2 µg/ml, respectively. Non-contrast enhancing brain concentrations for temozolomide alone or combined with regadenoson were: 0.55 ± 0.26 µg/ml vs. 0.57 ± 0.32 µg/ml, respectively. The non-contrast enhancing brain:plasma AUC ratio was 19.1 ± 10.7% when temozolomide was administered alone and 18.0 ± 7.8% when combined with regadenonson Three patients (patients 2, 3 and 5) demonstrated a rise in non-contrast enhancing mean brain AUC with regadenoson by approximately 53%. But this increase can be mostly attributed to patient 2 who demonstrated a significant rise in temozolomide brain AUC concentration with regadenoson; doubling AUC from 0.6 to 1.2 µg/ml h. Overall, evaluation of non-enhancing brain interstitium mean concentrations for all 5 patients demonstrated no significant difference in Cmax or AUCs between treatment groups, but individual variations existed.Fig. 2Mean temozolomide concentrations. Plasma temozolomide systemic concentrations unchanged with regadenoson treatment (a). Non-contrast enhancing brain interstitium (BI) temozolomide concentration over time failed to increase with regadenoson (b). Contrast enhancing BI temozolomide concentration demonstrated a slight increase in temozolomide concentration over time (c). Error bars represent standard error of mean values
|
study
| 100.0 |
Mean temozolomide concentrations. Plasma temozolomide systemic concentrations unchanged with regadenoson treatment (a). Non-contrast enhancing brain interstitium (BI) temozolomide concentration over time failed to increase with regadenoson (b). Contrast enhancing BI temozolomide concentration demonstrated a slight increase in temozolomide concentration over time (c). Error bars represent standard error of mean values
|
study
| 99.94 |
Two patients (patients 1 and 3) had catheters placed in contrast enhancing brain. The dialysate AUCs increased slightly by 10.0 and 19.1% when temozolomide was administered with regadenoson. For patient 1, brain AUC increased from 4.4 to 5.4 µg/ml h with temozolomide alone to combination with regadenoson, respectively. And for patient 3, brain AUC increased from 3.2 to 4.2 µg/ml h with temozolomide alone to combination with regadenoson, respectively. Generally, treatment with regadenoson exhibited a quicker rise to peak concentration but failed to demonstrate a prolonged increase of brain interstitial temozolomide concentrations (Fig. 2). The variations in peak time, Tmax and AUC can be seen in individual patient neuropharmacokinetics with temozolomide alone vs. temozolomide + regadenoson (Fig. 3, Additional file 1: Table S1).Fig. 3Maximum temozolomide concentration (Cmax) and area under the curve (AUC). Cmax of contrast enhancing and non-contrast enhancing tissue (a). Brain AUC of contrast enhancing and non-contrast enhancing tissue (b). Brain:Plasma AUC of contrast enhancing and non-contrast enhancing tissue (c). Black circles represent non-contrast enhancing tissue, white circles represent contrast-enhancing tissue
|
study
| 99.5 |
Maximum temozolomide concentration (Cmax) and area under the curve (AUC). Cmax of contrast enhancing and non-contrast enhancing tissue (a). Brain AUC of contrast enhancing and non-contrast enhancing tissue (b). Brain:Plasma AUC of contrast enhancing and non-contrast enhancing tissue (c). Black circles represent non-contrast enhancing tissue, white circles represent contrast-enhancing tissue
|
other
| 99.9 |
Despite numerous clinical studies using chemotherapeutic agents, novel biologics and immunotherapeutic agents, the overall survival of patients with high grade gliomas has not changed drastically over the last decade . The clinical impact of many cytotoxic agents has likely been limited in patients with malignant gliomas by their inability to cross the BBB. This poses an issue not only for primary brain tumors but also for metastatic brain disease. Unfortunately, while systemic therapy options have improved over the years for solid tumors, metastatic tumor cells are able to invade the CNS and proliferate with shelter from an impermeable BBB. Thus, with a lack of effective drug entry of varied chemotherapy agents, there has been no improvements in the overall survival of both malignant glioma and metastatic brain tumors. With this small clinical study, we evaluated regadenoson as a tool to facilitate CNS entry of a mildly permeable agent, temozolomide, from the proposed mechanism of enabling transient BBB disruption.
|
study
| 99.94 |
Overall, our study failed to demonstrate that brain interstitial temozolomide concentrations were increased by use of standard dose regadenoson; which we pre-specified as an increase in temozolomide brain concentration by ≥ 50%. Importantly, regadenoson did not alter temozolomide plasma concentrations which could result in changes in temozolomide related efficacy or toxicity. Although these results are consistent with our previous negative imaging study , they are at odds with the preclinical data that demonstrated increased drug delivery with one small dose of regadenoson [16, 19, 24, 36]. This difference in effect has raised further questions regarding BBB differences between mice and humans relating to expression and function of CNS adenosine A2A receptors. Alternatively, activation of A2A receptors and subsequent BBB disruption in the brains of glioblastoma patients may differ from the activation of A2A receptors in the normal brain vasculature. Previous preclinical studies demonstrated regadenoson’s ability to decrease cell–cell adhesion integrity while potentially modifying efflux transporter expression within 0.5–2 h after administration [19, 36, 38]. While we anticipated that regadenoson might increase drug entry across the BBB, drug exit from the CNS could also be facilitated resulting in decreased temozolomide concentrations in brain interstitium. The effect on transport is further compounded by the studies by that demonstrated temozolomide’s ability to bind to the multi-drug resistance protein, P-glycoprotein; which likely plays a significant role in glioblastoma temozolomide resistance . Interestingly, regadenoson has been shown to downregulate P-glycoprotein expression in brain endothelial cells thus increasing CNS drug delivery in in vitro BBB models and non-tumor bearing rodents . Thus, these combined findings add to the plausibility of temozolomide efflux by P-glycoprotein along with inadequate P-glycoprotein inhibition within brain/brain tumor parenchyma by regadenoson, thereby not causing a significant increase in brain interstitial temozolomide concentrations.
|
study
| 100.0 |
The early rapid rise of temozolomide seen with regadenoson administration can be attributed to the fast acting modulation that results from adenosine receptor activation [16, 24, 36]. Preclinical studies demonstrated the effect of regadenoson on brain vasculature with 0.05 mg/kg dosage per mouse (human equivalent dosing of 0.004 mg/kg); which is less than the standard cardiac stress dosing of 0.006 mg/kg per patient. Yet, despite these preclinical studies utilizing lower than standard regadenoson dosing, increased CNS penetration of 70 kD dextran and temozolomide was observed [16, 19, 24]. Interestingly, these studies in rodents demonstrated a bell shaped dose/effect curve, suggesting that regadenoson doses too high or too low result in minimal changes in BBB disruption. With this clinical study, we opted to use the standard clinical dosing of regadenoson (0.4 mg). This FDA approved agent is used daily in the clinical setting of patients with suspected heart disease to induce vasodilation. Clinically, patients receive one dose with associated cardiac imaging. We hypothesized that because approximately 26% of patients with suspected cardiac disease experience brief headaches after regadenoson administration, it is possible that headaches may be a direct correlate/biomarker for the presence or degree of BBB disruption. We opted to start with the clinical dosing of regadenoson as a means to increase temozolomide CNS entry. While optimal dosing has been determined for cardiac stress testing, optimal dosing and schedule of administration has yet to be determined with a focus on BBB permeability. Thus, it is plausible that increased or decreased standard regadenoson dosing could optimally augment CNS temozolomide entry. These studies of varied regadenoson dosing impacting the BBB permeability have not been performed to date in humans.
|
study
| 99.94 |
Given the importance of transiently opening the BBB to facilitate drug entry, further research in this area is desperately needed to improve the outcome of patients with CNS malignancies. For both primary and metastatic tumors, treatment options are very limited and/or exhibit poor sustainability for growth inhibition, and invasion. Several agents have been investigated in the past as a means to transiently “open” the BBB, but very few studies or laboratory investigations are being conducted to identify optimal genes, signaling pathways, or receptors so as to design drugs to influence CNS permeability. Regadenoson may be a potential agent, but more studies are needed to define the optimal dose and dosing schedule with the desired effect on CNS vasculature. These questions, along with the proper dosing and schedule of regadenoson, remain to be further studied, in order to explain our negative findings and improve chances of future success in enhancing transient BBB permeability.
|
review
| 99.9 |
Additional file 1. Pharmacokinetics of plasma and brain dialysate sampling post temozolomide and regadenoson. Additional file 2. Individual temozolomide concentrations within non-contrast enhancing brain interstitium on a log based scale (A). Contrast-enhancing brain interstitium values in patients 1 and 3 (B). Solid line demonstrates treatment with temozolomide alone. Dashed line demonstrates combined treatment with regadenoson.
|
study
| 99.9 |
Additional file 2. Individual temozolomide concentrations within non-contrast enhancing brain interstitium on a log based scale (A). Contrast-enhancing brain interstitium values in patients 1 and 3 (B). Solid line demonstrates treatment with temozolomide alone. Dashed line demonstrates combined treatment with regadenoson.
|
study
| 99.75 |
Toll-like receptor (TLR) 4 is a pattern-recognition receptor (PRR) that recognizes lipopolysaccharide (LPS) from Gram-negative bacteria as a pathogen-associated molecular pattern (PAMP). TLR4 signaling activates the MyD88 (myeloid differentiation primary-response protein 88)- and TRIF (TIR domain-containing adaptor protein-inducing interferon-β)-dependent pathways to induce the production of proinflammatory cytokines and interferons (IFNs), respectively1–4. Since the discovery of TLR4 as a receptor for LPS, several non-LPS TLR4 ligands, which are mostly derived from microbes, have been identified and their roles in TLR4-mediated innate immune responses have been proposed5–8. During the onset of intestinal inflammation in response to PAMPs from colon and possibly non-LPS TLR4 ligands from diet, TLR4 signaling is likely to play an important role in intestinal epithelial cells (IECs) and immune cells, including, macrophages and dendritic cells. IFN and/or inflammatory cytokines produced in the intestine upon stimulation by these PAMPs would systemically act on virus-infected cells locally and/or far away from sites where IFN signaling was initially induced. However, TLR4 signaling in the small intestine is not well characterized9.
|
review
| 99.06 |
Noroviruses are positive-sense, single-stranded RNA viruses belonging to the Caliciviridae family10. Human norovirus (HuNoV) is transmitted via the fecal-oral route and propagates in the gastrointestinal (GI) tract11. It is the most common cause of non-bacterial acute outbreaks of gastroenteritis worldwide across all age groups12. Norovirus has become a global health burden due to its high-sustained viability in environment, high risk of infection with fewer than 100 particles, possibility of causing chronic infection in immune deficient hosts as well as the elderly and infants, and emergence of novel norovirus strains13–16. Despite the breadth of potential antiviral drugs tested in vitro using a HuNoV replicon culture system17, limited studies have validated the efficacy of these drugs in vivo18–20. There are no reports on safe, prophylactic antiviral drugs that could prevent the onset of acute gastroenteritis in immunocompromised hosts. In the absence of approved vaccines and specific antiviral therapies for HuNoV18,21,22, development of prophylactic or therapeutic measures against HuNoV has become the need of the hour.
|
review
| 99.9 |
Poly-γ-glutamic acid (γ-PGA) is an anionic polypeptide synthesized and secreted by Bacillus species. In γ-PGA, d- and/or l-glutamate is polymerized via γ-amide linkages formed between the α-amino and γ-carboxylic acid functional groups23. We had previously reported the activation of TLR4 signaling pathway by γ-PGA, leading to the production of type I interferon (IFN) (α and β)6. In vitro, γ-PGA displays antiviral activity against severe acute respiratory syndrome coronavirus and hepatitis C virus6. In norovirus-infected cells, IFN-α/β production is initiated by the sensing of viral RNA genome via the cytoplasmic PRR MDA5 (melanoma differentiation-associated gene 5) and TLR3, followed by phosphorylation of IFN regulatory factor 3 (IRF-3) through two independent pathways24. This type I IFN signaling plays a major role in clearing mouse norovirus24–26 and HuNoV27,28.
|
study
| 100.0 |
γ-PGA is a major ingredient of “natto”, a Japanese traditional fermented food, and “Chungkookjang” a Korean fermented seasoning, both made from soybeans6 and used for dietary consumption. In the present study, we investigated whether γ-PGA, a non-LPS TLR4 ligand, could activate TLR4 signaling when administered orally. Our results demonstrate the induction of IFN-β by peroral administration of γ-PGA without the production of proinflammatory cytokines, thereby establishing an antiviral state against mouse norovirus. We provide evidence of in vivo efficacy of γ-PGA in limiting norovirus infection, highlighting the potential of γ-PGA as a prophylactic antiviral agent.
|
study
| 100.0 |
HuNoV infection causes epithelial apoptosis and downregulates the level of tight junction proteins involved in sealing, leading to diarrhea29. Comparably, murine norovirus (MNV) infection also induces apoptosis in RAW264.7 cells30. In the absence of a robust cell culture system for HuNoV31, we used murine norovirus 1 (MNV-1), as a surrogate model for HuNoV, to test for the inhibition of virus-induced cell death by γ-PGA. We first determined the range of multiplicity of infection (MOI) of MNV in which viral infection-induced apoptosis is triggered during the course of viral infection. As shown in Fig. 1a, infection of RAW264.7 cells with MNV at an MOI of ≥0.005 produced ≥1 × 1011 viral RNA copies/ml of media within 30 h. With an MOI ≤0.0005, there was a >10-fold reduction in viral RNA levels, delaying the progress of cell death within 2 days. Accordingly, we used an MOI of 0.005 for infecting cells to assess the impact of γ-PGA. As shown in Fig. 1b, treatment with 2,000-kDa γ-PGA significantly inhibited MNV infection-induced cell death as visualized by live cell staining, with a 4.4-fold increase in IFN-β mRNA expression than in non-treated control cells. γ-PGA dose-dependently prevented virus-induced cell death resulting in increased cell viability (from approximately 40% to 80% at 200 nM; Fig. 1c), demonstrating the potent antiviral activity of this non-canonical TLR4 ligand against MNV.Figure 1Inhibition of norovirus infection-induced apoptosis by the TLR4 ligand γ-PGA. (a) Molecular structure of γ-PGA with γ-linkage (top). RT-qPCR quantification of viral genome titers in the culture media from RAW264.7 cells infected with increasing MOI of murine norovirus 1 (MNV-1). Approximately 60–70% cells were dead when the viral genome titer reached to a titer of 1 × 1011 copies/ml media denoted by the dotted line. (b) RT-qPCR quantification of the expression of IFN-β mRNA in RAW264.7 cells treated with 2,000-kDa γ-PGA (100 nM) for 18 h. Shown is the GAPDH-normalized IFN-β mRNA level relative to that in mock-treated cells. Presented below each bar are representative images of RAW264.7 cells stained with neutral red, 30 h post-infection with MNV-1 at an MOI of 0.005. (c) RAW264.7 cells infected with MNV-1 (MOI of 0.005) were treated with the indicated doses of 2,000-kDa γ-PGA. Cell viability was quantified by the MTS assay at 36 h post-infection. (d) MNV-1 (MOI of 0.005)-infected RAW264.7 cells were treated with the indicated concentrations of 2,000-kDa γ-PGA with or without 500-kDa γ-PGA. Viral genome and VP1 levels were analyzed 30 h later by RT-qPCR and immunoblotting (IB), respectively. In (c,d), data are mean ± s.d.
|
study
| 100.0 |
Inhibition of norovirus infection-induced apoptosis by the TLR4 ligand γ-PGA. (a) Molecular structure of γ-PGA with γ-linkage (top). RT-qPCR quantification of viral genome titers in the culture media from RAW264.7 cells infected with increasing MOI of murine norovirus 1 (MNV-1). Approximately 60–70% cells were dead when the viral genome titer reached to a titer of 1 × 1011 copies/ml media denoted by the dotted line. (b) RT-qPCR quantification of the expression of IFN-β mRNA in RAW264.7 cells treated with 2,000-kDa γ-PGA (100 nM) for 18 h. Shown is the GAPDH-normalized IFN-β mRNA level relative to that in mock-treated cells. Presented below each bar are representative images of RAW264.7 cells stained with neutral red, 30 h post-infection with MNV-1 at an MOI of 0.005. (c) RAW264.7 cells infected with MNV-1 (MOI of 0.005) were treated with the indicated doses of 2,000-kDa γ-PGA. Cell viability was quantified by the MTS assay at 36 h post-infection. (d) MNV-1 (MOI of 0.005)-infected RAW264.7 cells were treated with the indicated concentrations of 2,000-kDa γ-PGA with or without 500-kDa γ-PGA. Viral genome and VP1 levels were analyzed 30 h later by RT-qPCR and immunoblotting (IB), respectively. In (c,d), data are mean ± s.d.
|
study
| 100.0 |
Furthermore, treatment with 100 nM γ-PGA resulted in a 98% decrease in plaque titer and significantly reduced the level of VP1, a viral structural protein (Fig. 1d). The antiviral potency of 2,000-kDa γ-PGA was comparable to that of 17-DMAG (Supplementary Fig. 1, mean 50% effective concentration of 17-DMAG: 35 nM), an Hsp90 inhibitor that has been recently shown to be effective in controlling MNV infection32. The antiviral activity of 2,000-kDa γ-PGA was not attenuated by co-treatment with 500-kDa γ-PGA at 100 nM or 500 nM, suggesting the importance of molecular mass or a specific structure formed by 2,000-kDa γ-PGA for the activation of TLR4 signaling.
|
study
| 100.0 |
Inhibition of MNV by 2,000-kDa γ-PGA was further verified by measuring intracellular viral genome titers. γ-PGA reduced viral genome titer by up to 92% in cells infected with a low MOI (0.000005). Its inhibitory effect decreased slightly (86% inhibition) at an MOI of 0.05 (Fig. 2a). Additionally, it led to a 30% reduction in the level of Norwalk virus sub-genomic replicon (derived from a GI strain of HuNoV) at 100 nM. In HG23 cell line, 500-kDa γ-PGA had little impact on the replication of sub-genomic replicon (Fig. 2b). The HG23 replicon cell line derived from the human hepatocellular carcinoma cell line Huh7 normally fails to respond to TLR4 ligands because of the low-level expression of TLR4 expression together with the lack of the expression of its accessory proteins, CD14 and MD26. Thus, the insufficient, transient expression of TLR4 and the associated accessory proteins might have contributed to the relatively lower inhibitory effect. The critical role of these accessory proteins in γ-PGA sensing and TLR4-mediated IFN-β expression was further investigated in human embryonic kidney 293 T cells (HEK293T), which express no endogenous TLR4, MD2, and CD1433,34. By confocal microscopy, we found that γ-PGA sensing and intracellular entry occurred upon ectopic expression of both TLR4 and its two known accessory factors (Supplementary Fig. 2a–c). Substantial induction of IFN-β expression and IRF-3 activation were observed only when TLR4, MD2, and CD14 were expressed (Supplementary Fig. 2d). This TLR4-mediated IFN-β induction capability of γ-PGA was also verified in RAW264.7 cells in which RNAi-mediated depletion of TLR4 abrogated IFN-β expression and IRF-3 activation (Supplementary Fig. 2e).Figure 2Analysis of the antiviral activity of γ-PGA against mouse and human noroviruses. (a) RAW264.7 cells infected with the indicated MOI of MNV-1 were treated with 100 nM 2,000-kDa γ-PGA. Viral genome copy number and infectious virus titer in the culture media were determined by RT-qPCR at 40 h post-infection. (b) The HG23 cell line harboring a Norwalk virus sub-genomic replicon was transfected with plasmids expressing TLR4, MD2, and CD14 individually. After incubation for 6 h, transfected cells were treated with γ-PGA (100 nM) for 2 days, prior to RT-qPCR quantification of replicon RNA titer two days later. Neo, neomycin phosphotransferase-coding gene conferring resistance to G418. (c) RAW264.7 cells were pre-treated for 6 h before MNV-1 infection or treated after the viral infection with increasing concentrations of 2,000-kDa γ-PGA. Viral genome titer was determined at 30 h post-infection. (d,e) Inhibition of MNV-1 entry by γ-PGA. Q-dot (QD)-conjugated MNV-1, which was pre-incubated with 2,000-kDa γ-PGA (100 nM) for 30 min, was applied to RAW264.7 cells. Two hours after adsorption, viral entry was quantified by fluorescence microscopy (d), and the fluorescence intensity signals were quantified by image analysis using ImageJ software (e). BF, bright field image. Scale bar, 50 μm. Shown as an inset in (d) is the transmission electron micrograph of purified MNV particles negatively stained with uranyl acetate (Scale bar, 100 nM). (f) Expression of TLR1-9 in RAW264.7 cells infected with MNV-1 (MOI of 0.005). Shown are the GAPDH-normalized TLR expression levels relative to those in mock-treated cells, analyzed by RT-qPCR at 24 h post-infection. Results are from two independent experiments, each analyzed by triplicate assay. (g) Immunoblot assay representing the activation of IRF-3 by γ-PGA (100 nM) and MNV-1 infection (MOI of 0.005) without affecting TLR2, TLR3, and TLR4 expression levels. pIRF-3, phosphorylated IRF-3. In all panels, data are mean ± s.d. Statistical significance of differences between groups was determined via two-tailed unpaired Student’s t-test. *P < 0.05; **P < 0.01; n.s., not significant.
|
study
| 100.0 |
Analysis of the antiviral activity of γ-PGA against mouse and human noroviruses. (a) RAW264.7 cells infected with the indicated MOI of MNV-1 were treated with 100 nM 2,000-kDa γ-PGA. Viral genome copy number and infectious virus titer in the culture media were determined by RT-qPCR at 40 h post-infection. (b) The HG23 cell line harboring a Norwalk virus sub-genomic replicon was transfected with plasmids expressing TLR4, MD2, and CD14 individually. After incubation for 6 h, transfected cells were treated with γ-PGA (100 nM) for 2 days, prior to RT-qPCR quantification of replicon RNA titer two days later. Neo, neomycin phosphotransferase-coding gene conferring resistance to G418. (c) RAW264.7 cells were pre-treated for 6 h before MNV-1 infection or treated after the viral infection with increasing concentrations of 2,000-kDa γ-PGA. Viral genome titer was determined at 30 h post-infection. (d,e) Inhibition of MNV-1 entry by γ-PGA. Q-dot (QD)-conjugated MNV-1, which was pre-incubated with 2,000-kDa γ-PGA (100 nM) for 30 min, was applied to RAW264.7 cells. Two hours after adsorption, viral entry was quantified by fluorescence microscopy (d), and the fluorescence intensity signals were quantified by image analysis using ImageJ software (e). BF, bright field image. Scale bar, 50 μm. Shown as an inset in (d) is the transmission electron micrograph of purified MNV particles negatively stained with uranyl acetate (Scale bar, 100 nM). (f) Expression of TLR1-9 in RAW264.7 cells infected with MNV-1 (MOI of 0.005). Shown are the GAPDH-normalized TLR expression levels relative to those in mock-treated cells, analyzed by RT-qPCR at 24 h post-infection. Results are from two independent experiments, each analyzed by triplicate assay. (g) Immunoblot assay representing the activation of IRF-3 by γ-PGA (100 nM) and MNV-1 infection (MOI of 0.005) without affecting TLR2, TLR3, and TLR4 expression levels. pIRF-3, phosphorylated IRF-3. In all panels, data are mean ± s.d. Statistical significance of differences between groups was determined via two-tailed unpaired Student’s t-test. *P < 0.05; **P < 0.01; n.s., not significant.
|
study
| 100.0 |
Pre-treatment of cells (6 h before infection) with 2,000-kDa γ-PGA significantly increased its antiviral activity compared to post-infection treatment (1 h after infection) (Fig. 2c). This finding suggests that treatment with γ-PGA predisposes cells to acquire resistance to viral infection that is effective in suppressing MNV propagation. Additionally, the observation raised the possibility of γ-PGA interfering with the entry of MNV. Accordingly, a virus entry assay using Q-dot-conjugated norovirus was conducted. The result indicated the capability of γ-PGA to block the entry of norovirus into RAW264.7 cells (Fig. 2d,e) and explains a relatively lower antiviral activity in HG23 replicon cells than in MNV-infected macrophage cells.
|
study
| 100.0 |
Upon virus infection, upregulation of TLR4 or crosstalk between TLR signaling pathways35 might alter the sensitivity of TLR4 to γ-PGA. Alterations in the expression profile of TLRs including TLR4 following norovirus infection was further investigated. TLR1-9 mRNA expression levels were not altered by more than 2-fold in MNV-1-infected RAW264.7 cells (Fig. 2f). Neither norovirus infection nor γ-PGA treatment enhanced TLR4 expression. Immunoblotting analyses detected pIRF-3, indicating that both these stimuli activated TLR4 signaling as evidenced by the activation of IRF-3 (Fig. 2g). Similarly, neither TLR3, a sensor for norovirus dsRNA24 nor TLR2, which could be a potential innate immune sensor for not-yet-identified norovirus-derived or induced PAMPs, was upregulated by norovirus infection or γ-PGA. These results suggest that the antiviral activity of γ-PGA is not dependent on the upregulation of TLRs.
|
study
| 100.0 |
Norovirus consumed with contaminated food and/or water penetrates through intestinal microfold (M) cells and infects its target cells including macrophages and dendritic cells (for MNV)25 or B cells (for HuNoV)36. After infection, norovirus propagates in these sentinel cells that are highly enriched in Peyer’s patches (PPs) in the ileum, before spreading to lymph nodes37,38.
|
study
| 99.94 |
We thus sought to address the question of whether γ-PGA can trigger TLR4 signaling-mediated IFN production in the ileum (composed of epithelial cells plus diverse immune cells in the laminar propria and the PPs) to exert its antiviral activity against norovirus. To this end, distal part of the ileum with or without PPs was retrieved from BALB/c mice. These were treated with γ-PGA in culture dishes for 18 h ex vivo. After treatment, the villi released into the culture media and the remaining basal body of the ileum (ileal submucosa and lamina propria) were collected separately and the IFN-β mRNA levels were quantified in these ileal compartments by reverse-transcription quantitative PCR (RT-qPCR) (Fig. 3a). Interestingly, we found induction of IFN-β production by γ-PGA only in the villi (compartments II and IV) and not in PPs and basal body of the ileum (compartments III and I) (Fig. 3b). This induction was more substantial in the villi derived from the ileum with PPs (compartment II) than from the ileum without PPs (compartment IV). TLR4-stained cells were more abundant in compartment II than in compartment IV (Fig. 3c). These results suggest that to activate TLR4 signaling, γ-PGA transverses the epithelial cell layer of the villi to reach the laminar propria bearing TLR4-positive macrophages and dendritic cells. Despite the abundance of TLR4-positive cells in PPs, γ-PGA treatment did not significantly induce IFN-β mRNA expression, suggesting non-responsiveness of PPs to γ-PGA by an as-yet-unknown mechanism.Figure 3Induction of IFN-β expression by γ-PGA and MNV in mouse ileum. (a) Experimental schemes for ex vivo stimulation of the ileum with γ-PGA. (b) Mouse (n = 4) ileum with or without PPs was treated with γ-PGA ex vivo. After 18 h incubation, IFN-β mRNA levels in the ileal compartments depicted in (a) were determined by RT-qPCR. *P < 0.05; **P < 0.01; ***P < 0.001; n.s., not significant; by two-tailed unpaired Student’s t-test. (c) Microscopic analysis of TLR4-expressing cells in the ileum with or without PPs. Intestinal microvilli stained with a villin-specific antibody. (d) Microscopic analysis of the entry of MNV. Mouse ileum with or without PPs was infected with Qdot fluorophore-tagged MNV ex vivo in serum-free DMEM. At 2 h post-infection, cryosection images were taken after extensively washing the infected tissues. Scale bar, 50 μm.
|
study
| 100.0 |
Induction of IFN-β expression by γ-PGA and MNV in mouse ileum. (a) Experimental schemes for ex vivo stimulation of the ileum with γ-PGA. (b) Mouse (n = 4) ileum with or without PPs was treated with γ-PGA ex vivo. After 18 h incubation, IFN-β mRNA levels in the ileal compartments depicted in (a) were determined by RT-qPCR. *P < 0.05; **P < 0.01; ***P < 0.001; n.s., not significant; by two-tailed unpaired Student’s t-test. (c) Microscopic analysis of TLR4-expressing cells in the ileum with or without PPs. Intestinal microvilli stained with a villin-specific antibody. (d) Microscopic analysis of the entry of MNV. Mouse ileum with or without PPs was infected with Qdot fluorophore-tagged MNV ex vivo in serum-free DMEM. At 2 h post-infection, cryosection images were taken after extensively washing the infected tissues. Scale bar, 50 μm.
|
study
| 100.0 |
In a parallel experiment, the route of entry of MNV in the ileum was monitored. Fluorescence microscopic analysis of Q-dot-conjugated viruses detected MNV particles in compartment II. No prominent sign of virion entry was detected through the villi at the ileum without PPs (Fig. 3d). These results demonstrate that MNV primarily enters into the ileum bearing PPs through intestinal villi, possibly via M cells or transepithelial dendritic cells. To confirm the entry of MNV through this part of the ileum, viral genome was monitored upon delivering Q-dot-conjugated MNV directly to the small intestine of mouse ex vivo. High levels of viral genome was detected in the ileum with PPs [in both compartment I representing PPs and compartment II] 8 h after virus delivery (Supplementary Fig. 3a), in agreement with our results showing MNV particles within villi. Consequently, enhanced IFN-β mRNA levels were detected in both ileal compartments while greater induction was observed in ileal PPs (iPPs) early during virus infection (Supplementary Fig. 3b,c), suggesting that MNV entering through villi reached to the PPs to infect ileal sentinel cells. These results are consistent with the previous findings indicating entry of norovirus through the M cells within the follicle-associated epithelium overlying PPs or in villi to infect macrophages and dendritic cells37,39,40.
|
study
| 100.0 |
IFN-β-inducible activity of γ-PGA was further investigated in mice to ascertain the prophylactic antiviral efficacy of orally delivered γ-PGA. Initial experiments revealed production of both IFN-α and IFN-β at levels below the detection limits of ELISA at 2, 4, 8, 12, and 24 h post-administration of γ-PGA (50 mg/kg body weight per day) to BALB/c mice (Supplementary Fig. 4a,b). These results suggest weak IFN-inducing activity or inefficient delivery of the 2,000-kDa γ-PGA in an intact conformation to the ileum (Fig. 3b,c). When γ-PGA (50 mg/kg body weight) was administered to mice, once a day for five days, serum IFN-β level increased starting from day 3 (Fig. 4a). Induction of IFN-α was negligible (Fig. 4b and Supplementary Fig. 4b). Production of IFN-β was induced by 2,000-kDa γ-PGA but not by 500-kDa γ-PGA (Supplementary Fig. 4c,d), suggesting the importance of its structural entity in activating TLR4 signaling. Furthermore, IFN-β levels (mean values) neither decreased nor increased from day 3 to day 5, indicating that multiple administrations of γ-PGA do not induce TLR4 tolerance to blunt the responsiveness to repetitive challenges with γ-PGA.Figure 4Analysis of cytokines induced by the oral administration of γ-PGA. BALB/c mice (6 weeks old, n = 10 per group) were given 2,000-kDa γ-PGA (50 mg/kg of body weight) in 100 µl saline once daily for five days. Blood samples were collected before (day 0) or 4 h after administering γ-PGA on day 1, 3, or 5 to measure titers of IFN-β (a), IFN-α (b), and other cytokines (c) by ELISA. In all panels, data are mean ± s.d. **P < 0.01; ***P < 0.001; n.s., not significant; by two-tailed unpaired Student’s t-test. LOD, limit of detection.
|
study
| 100.0 |
Analysis of cytokines induced by the oral administration of γ-PGA. BALB/c mice (6 weeks old, n = 10 per group) were given 2,000-kDa γ-PGA (50 mg/kg of body weight) in 100 µl saline once daily for five days. Blood samples were collected before (day 0) or 4 h after administering γ-PGA on day 1, 3, or 5 to measure titers of IFN-β (a), IFN-α (b), and other cytokines (c) by ELISA. In all panels, data are mean ± s.d. **P < 0.01; ***P < 0.001; n.s., not significant; by two-tailed unpaired Student’s t-test. LOD, limit of detection.
|
study
| 100.0 |
Among the other cytokines analyzed, no significant change was detected in the levels of IL-2, IL-6, IL-10, and TNF-α (Fig. 4c). However, significant decrease was seen in the level of IL-1α and IL-1β at day 3 and 5, respectively. Level of IL-1α remained downregulated until day 5 indicating inverse correlation of its expression with IFN-β. These results are consistent with recent studies suggesting a negative regulatory role of IFN-β in the maturation of IL-1α/β41,42. Type II interferon (IFN-γ) produced primarily by activated T cells was not detected in all cases. Taken together, our results show that unlike LPS, peroral administration of 2,000-kDa γ-PGA selectively induces IFN-β but not inflammatory cytokines.
|
study
| 100.0 |
To test the antiviral activity of γ-PGA in vivo, MNV-1 (1 × 107 PFU per mouse) was orally inoculated into BALB/c mice that received γ-PGA (50 mg/kg body weight) prior to infection once daily for five days to induce IFN-β expression. Two days after MNV inoculation, viral load in the ileum with PPs and mesenteric lymph nodes (MLNs) were quantified using RT-qPCR and plaque-forming assay. γ-PGA administration resulted in 47% and 53% reduction of MNV-1 genome titer in the ileum with PPs and MLNs, respectively (Fig. 5a). Plaque-formation assay revealed 36% and 33% reduction of MNV titer in the ileum with PPs and MLNs, respectively (Fig. 5b). These results indicate that peroral administration of 2,000-kDa γ-PGA effectively suppresses MNV propagation in mouse ileum and MLNs, indicating its prophylactic antiviral efficacy in vivo.Figure 5γ-PGA inhibits MNV-1 in mice. BALB/c mice (6 weeks old, n = 7–8 per group) were administered with 2,000-kDa γ-PGA as described in Fig. 4 for 5 days. Twelve hours after the last γ-PGA administration, mice were inoculated with MNV-1 (1 × 107 PFU in 100 µl saline) perorally and given γ-PGA once daily for another two days. Viral genome and infectious virus titers in the ileum with PPs and the MLNs were determined by RT-qPCR (a) and plaque-forming assay (b), respectively. In all panels, data are mean ± s.d. *P < 0.05 by two-tailed unpaired Student’s t-test.
|
study
| 100.0 |
γ-PGA inhibits MNV-1 in mice. BALB/c mice (6 weeks old, n = 7–8 per group) were administered with 2,000-kDa γ-PGA as described in Fig. 4 for 5 days. Twelve hours after the last γ-PGA administration, mice were inoculated with MNV-1 (1 × 107 PFU in 100 µl saline) perorally and given γ-PGA once daily for another two days. Viral genome and infectious virus titers in the ileum with PPs and the MLNs were determined by RT-qPCR (a) and plaque-forming assay (b), respectively. In all panels, data are mean ± s.d. *P < 0.05 by two-tailed unpaired Student’s t-test.
|
study
| 100.0 |
Given the possibility of crosstalk between TLRs, if expression of TLRs in the ileum was altered in response to norovirus infection, TLR4 response to γ-PGA would also be affected. The ileum with PPs of norovirus-infected mice showed no significant increase in TLR4 expression. Expression levels of TLR2, TLR3, TLR6, TLR8, and TLR9 were slightly increased. Though the increase was less than 2-fold, it was statistically significant (Fig. 6a). The expression of TLR1, which recognizes peptidoglycan and lipoproteins43,44, and TLR5, which recognizes bacterial flagellin45 was upregulated by ~5-fold and 2.5-fold, respectively, suggesting the possibility of crosstalk between TLR signaling through some of these upregulated TLRs. This might have contributed to the amplification of γ-PGA-mediated TLR4 signaling in vivo as several TLR downstream signaling pathways share transcription factors such as NF-κB and IRF-335, used for type I IFN production. In MLNs, expression levels of TLR2, TLR4, TLR6, TLR7, and TLR8 were slightly decreased (less than 2-fold) (Fig. 6b). These results suggest differentially altered TLR expression profile for norovirus infection in the ileum with PPs and MLNs in vivo. Interestingly, this expression profile of TLRs was different from that seen in virus-infected RAW264.7 cells (Fig. 2F), suggesting the involvement of commensal microbiota in the regulation of TLR expression in the ileum, in the context of norovirus infection in mice.Figure 6Impact of norovirus infection on TLR expression in mice. The ileum with PPs (a) and the MLNs (b) from BALB/c mice (6 weeks old, n = 9–10 per group) infected with MNV-1 as in Fig. 5 were retrieved 2 days after viral infection and expression of TLR mRNA was quantified as described in Fig. 2f. Data are represented as mean ± SD. *P < 0.05; **P < 0.01; ***P < 0.001; n.s., not significant; by two-tailed unpaired Student’s t-test.
|
study
| 100.0 |
Impact of norovirus infection on TLR expression in mice. The ileum with PPs (a) and the MLNs (b) from BALB/c mice (6 weeks old, n = 9–10 per group) infected with MNV-1 as in Fig. 5 were retrieved 2 days after viral infection and expression of TLR mRNA was quantified as described in Fig. 2f. Data are represented as mean ± SD. *P < 0.05; **P < 0.01; ***P < 0.001; n.s., not significant; by two-tailed unpaired Student’s t-test.
|
study
| 100.0 |
Thus, in the ileum with PPs, where villi responded to γ-PGA and induced IFN-β expression, level of TLR4 remained unchanged although expression of other TLRs was differentially affected by norovirus infection. It remains to be determined whether γ-PGA’s antiviral activity against norovirus was potentiated by the amplification of TLR4 signaling through sensitization of other upregulated TLRs in the ileum upon norovirus infection.
|
study
| 100.0 |
Type I IFN is the primary cytokine that is induced rapidly in response to viral infections. It amplifies immune responses through the IFN-signaling cascade24,25,46. However, its antiviral effect in the intestinal tract is less clear. In the present study, we investigated the ability of orally administered γ-PGA to induce an innate immune response and to mount an antiviral state in the ileum. Our results indicate induction of IFN-β production without elevating the level of proinflammatory cytokine by γ-PGA, a non-LPS TLR4 ligand, thereby predisposing the ileum to develop a robust antiviral innate immunity against norovirus.
|
study
| 100.0 |
Our results demonstrate induction of type I IFN production primarily by the activation of TRIF-dependent pathway in mice following multiple peroral administration of γ-PGA. Previous studies have reported the primary activation of MyD88-dependent pathway by LPS in eliciting the secretion of numerous proinflammatory cytokines1–4,8. As a TLR4 ligand, γ-PGA is distinct from LPS in selectively increasing the serum IFN-β titers, but not of other inflammatory cytokines, namely IL-1α/β, IL-6, and TNF-α. This difference in the cytokine profile induced by γ-PGA and LPS suggests that a regulatory network different from that of LPS should control γ-PGA-mediated TLR4 activation. It remains unclear whether other non-LPS TLR4 ligands including endogenous TLR4 agonists such as heat shock proteins and cholesteryl ester hydroperoxides8 upon being sensed by the same receptor also behave differently than LPS in terms of the cytokine profile. Another unexpected finding was that the elevated serum IFN-β levels in mice after multiple oral administrations of γ-PGA (once daily for three days) neither increased nor decreased following two additional oral administrations. These results suggest the absence of tolerance phenomena with γ-PGA, which is normally seen with endotoxin, or LPS, wherein, innate immune cells pre-exposed to LPS become refractory to subsequent challenges47. Based on the results of our present study, γ-PGA seems to be a new class of TLR4 agonists that selectively activates the TRIF-dependent pathway.
|
study
| 100.0 |
IECs are normally non-responsive to TLR4 ligands such as LPS because of low-level expression of TLR4 receptors to prevent undesirable inflammation in response to gut microbiomes or pathogens in the GI tract. Minimal TLR4 expression was seen in normal IECs and the lamina propria rich in scattered inflammatory cells in human biopsies48. Although not thoroughly characterized, a previous study had indicated the expression of TLR4 as well as other TLRs (TLR1, TLR2, TLR3, TLR5, and TLR9) in the enterocytes of small intestine49. By immunohistochemistry, we observed no prominent TLR4-staining patterns on apical or basolateral surfaces of IECs; TLR4-positive cells were instead localized in the lamina propria of villi and the PPs in the ileum. Thus, our results together with previous findings provide evidence of TLR4 expression in the ileum. However, its fine localization are yet to be well defined. Low but detectable level of TLR4 expression explains its role at the distal ileum that is continuously exposed to microbes and their products transferred from colon50. This renders the ileum or IECs hyposensitive to PAMPs including LPS and other TLR agonists, thereby preventing aberrant production of proinflammatory cytokines. In this regard, induction of IFN-β in ex vivo cultures of mouse ileum and in mice receiving γ-PGA perorally was surprising, particularly in the absence of upregulation of TLR4 expression by γ-PGA in RAW264.7 cells and in mouse ileum with PPs.
|
study
| 100.0 |
TRIF-dependent pathway of TLR4 signaling is activated in endosomes upon internalization of TLR4-ligand complexes1–4. Accordingly, in the present study, γ-PGA delivered orally seemed to be internalized into IECs or transverses the tight junctions between enterocytes to be internalized into the sentinel cells in the lamina propria to induce IFN-β production. Thus, we further investigated as to how γ-PGA reached a site where functional interaction with TLR4 occurred in the ileum. The first barrier for the entry of γ-PGA was the mucus layer, where access of γ-PGA to the small intestine is more likely to occur as it is covered by a thinner mucus layer compared to the colon51. For induction of IFN production, γ-PGA needs to be sensed by TLR4 in and/or on villi. Immunostaining revealed lack of overlapping between TLR4 staining and villin, a cytoskeletal protein specific to villi in the ileum, indicating a barely detectable level of TLR4 at the apical site of IECs. We therefore hypothesized a model in which γ-PGA transverses the tight junctions between enterocytes to be sensed by sentinel cells such as dendritic cells within villi or is directly uptaken by transepithelial dendrites (Supplementary Fig. 5). Expression of IFN-β mRNA was significantly enhanced solely in the villi with lack of noticeable IFN-β induction in PPs. Absence of response to γ-PGA in PPs resulting in lack of inflammation was consistent with the previous findings suggesting anergy of intestinal macrophages. Anergy was mediated by their inability to activate NF-κB, and low-level expression of TLR4 and its accessory factors CD14 and MD252.
|
study
| 100.0 |
We previously showed that activation of TLR4 signaling by γ-PGA leading to induction of IFN was dependent on TLR4-associated accessary factors CD14 and MD26. In the present study, results from Q-dot-conjugated γ-PGA confirm the necessity of CD14 and MD2 for sensing γ-PGA. Dependence on CD14 in MyD88-dependent pathway-mediated induction of TNF-α was marginal in LPS-stimulated macrophages53,54. However, our results indicate the absolute requirement of CD14 and its prominent role over MD2 in sensing γ-PGA. Our findings suggesting a substantial stimulatory effect of γ-PGA in the lamina propria raises interest on the possibility of differential expression of MD2, CD14, and TLR4 in macrophages and dendritic cells in the lamina propria and PPs. Currently, requirement of these accessory factors in sensing various non-LPS TLR4 ligands remains largely unknown. Irrespective of the mechanisms involved in γ-PGA entry and sensing by TLR4-positive cells in the ileum, both, results from ex vivo culture indicating stimulation of the ileum with γ-PGA and the serum cytokine profile of mice receiving peroral γ-PGA, provide clues as to how γ-PGA activates the TRIF-dependent TLR4 signaling pathway in the ileum.
|
study
| 100.0 |
We further investigated the impact of norovirus infection on TLR4-mediated induction of IFN-β by γ-PGA. Our results suggest lack of significant alteration of TLR4 expression in norovirus-infected mice. Thus, responsiveness of TLR4 to γ-PGA was unlikely to be elevated upon norovirus infection. However, norovirus infection led to a ~2-fold increase in the expression of most TLRs, except TLR4 and TLR6 (Fig. 6a). TLR1 level was increased ~5-fold. Upregulated TLR expression could be in part due to the infiltration of leukocytes to the infected ileum. We envision that TLR4 signaling activation by γ-PGA might be potentiated through crosstalks with other upregulated TLRs. Among the upregulated TLRs, the endosomal TLRs including TLR3, TLR7, TLR8, and TLR9 may influence the sensitivity of TLR4 to γ-PGA early during norovirus infection. Currently, their involvement (except TLR3) in innate immune response to norovirus infection remains unknown24,55. Although norovirus infection did not affect the expression of TLR4 in the ileum, TLR responses to colon-resident microbiota and/or PAMPs derived from them might influence the downstream cascade of TLR4 signaling. Thus, induction of IFN-β by γ-PGA is likely to be modulated by commensal microbiome in the large intestine adjacent to the ileum.
|
study
| 100.0 |
Type I IFN plays a major role in clearing mouse norovirus24–26. It is effective in limiting norovirus replication in human norovirus sub-genomic replicon culture system17. In spite of the well-established antiviral potency of type I IFN against noroviruses, IFN therapy is not translated into clinical practice because subcutaneous or intramuscular injection of IFN-α/β is associated with various side effects56,57. Our results, demonstrating the prophylactic antiviral activity of γ-PGA in mice using MNV-1 as a model for enteric viruses infecting the small intestine, accentuates its potential use as a safe and prophylactic candidate for the treatment of acute norovirus infection. Although norovirus infection is typically limited to an acute infection that lasts for 2–3 days in most healthy subjects58, it is associated with significant clinical outcomes with ~200,000 deaths related with gastroenteritis in children under 5 years of age59. In young children, the elderly, and immunocompromised patients for whom IFN might not be the best treatment option, γ-PGA can be used as an alternative prophylactic antiviral agent for HuNoV. It is important to note that unlike the MNV replicating mainly in macrophages and dendritic cells25, HuNoV, which was shown to replicate in B cells36, also targets IECs60–62. Therefore, the IFN produced by the sentinel cells in the lamina propria of villi upon sending the γ-PGA might restrict HuNoV infection more effectively at the early stage of infection by acting on nearby IECs through paracrine signaling.
|
study
| 99.94 |
In summary, our results demonstrate that oral administration of γ-PGA induces IFN-β production in the ileum without upregulating the expression of TLR4 and without eliciting the production of inflammatory cytokines. The specific IFN-β induction feature of γ-PGA highlights its potential application as a prophylactic antiviral agent for noroviruses and possibly other enteric viruses.
|
study
| 100.0 |
The murine macrophage cell line RAW264.7 and human embryonic kidney 293 T (HEK293T) cell line were purchased from American Type Culture Collection (ATCC; Rockville, MD, USA) and cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 100 U/ml penicillin, and 100 µg/mL streptomycin. The HG23 cell line stably harboring a HuNoV (Norwalk virus) replicon17 was cultured in DMEM supplemented with 10% FBS, 2 mM l-glutamine, 100 U/ml penicillin, 100 µg/ml streptomycin, and 0.1 mM nonessential amino acids in the presence of 1 mg/ml neomycin (G418 sulfate). Cells were maintained at 37 °C in a 5% CO2 humidified incubator.
|
study
| 99.94 |
Endotoxin-free γ-PGAs with different average molecular masses (500- and 2,000-kDa), which were prepared as described previously60, were provided by BioLeaders (Daejeon, Korea). The plasmids expressing human TLR4, CD14, and MD2 were described61,62. In transient expression experiments, empty vector was added to normalize the total amounts of transfected DNA. Plasmids were transfected into cells using Fugene HD transfection reagent (Roche Diagnostics, Indianapolis, IN, USA). Chemically synthesized siRNAs were purchased from Bioneer (Daejeon, Korea). The siRNA used are as follows: siTLR4 [siRNA targeting mouse TLR4, 5′-GAAUUGUAUCGCCUUCUUAdTdT-3′ (passenger stand) and 5′-UAAGAAGGCGAUACAAUUCdTdT-3′ (guide strand)] and siCtrl [control siRNA targeting GFP, 5′-CUCGCCGGACACGCUGAACUU-3′ (passenger stand) and 5′-GUUCAGCGUGUCCGGCGAGUU-3′ (guide strand)]. siRNAs were transfected into RAW264.7 cells using the ND98 lipidoid as a transfection reagent63. Mouse polyclonal anti-IRF-3 (FL-425), rabbit monoclonal anti-phospho-IRF-3(Ser396), and rabbit polyclonal anti-β-actin antibody were purchased from Cell Signaling Technology (Beverly, MA, USA). Anti-norovirus capsid protein VP1 antibody was purchased from Abcam (Cambridge, UK). Anti-TLR2 (H-175), anti-TLR3 (N-14), anti-TLR4 (25), and anti-villin antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Anti-α-tubulin antibody was purchased from Calbiochem (La Jolla, CA, USA).
|
study
| 99.94 |
MNV-1.CW164 was kindly provided by Herbert W. Virgin (Washington University School of Medicine, St. Louis, MO, USA) and propagated in RAW264.7 cells to generate a viral stock. Briefly, RAW264.7 cells were infected with MNV at a multiplicity of infection (MOI) of 0.5 in a serum-free medium and incubated for 1 h. Following washing, infected cells were further cultivated in fresh complete media for 2 days. Viral stock was prepared by centrifuging harvested media at 450 × g for 10 min. Intracellular virus particles recovered by three freeze/thaw cycles were harvested by centrifugation at 3,000 × g for 10 min. Virus samples were then concentrated using an Amicon Ultra centrifugal filter unit with a molecular weight cutoff of 50 kDa (Merck Millipore, Billerica, MA, USA) and stored at −80 °C until further use as crude MNV preparation. Purification of MNV was performed as previously described with modifications65. Briefly, crude MNV was pelleted by ultracentrifugation at 150,000 × g for 1 h at 4 °C. The pellet was gently suspended in a suspension buffer (50 mM Tris-HCl, pH 7.5, 100 mM NaCl) at 4 °C and pelleted again by ultracentrifugation. The resultant pellet was dissolved in suspension buffer and was subjected to sucrose density gradient (10–60%) centrifugation at 100,000 × g for 150 min at 4 °C to collect the MNV-containing fractions that were identified by SDS- polyacrylamide gel electrophoresis (PAGE) followed by immunoblotting for the VP1 capsid protein. The fractions were subjected to ultracentrifugation as described above to obtain purified virus particles. These purified virus particles were suspended in PBS and used for virus entry experiments.
|
study
| 100.0 |
Plaque-forming assay was performed as described previously66. Briefly, RAW264.7 cells seeded in 6-well plates (2 × 106 cells/well) were cultured overnight and infected with norovirus in serum-free DMEM. After 1 h of infection, cells were washed to remove the inoculum and were overlaid by SeaPlaque Agarose (1% w/v; Lonza, Rockland, ME, USA) in complete DMEM with high glucose. Following 2 or 3 days of culture, plaques were visualized by staining the monolayer cells with neutral red.
|
study
| 100.0 |
RAW264.7 cells seeded in 96-well plates (2 × 104 cells/well) were grown overnight and treated with various concentrations of γ-PGA for 48 h. Cell viability was assessed by an MTS [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assay as described previously67.
|
study
| 100.0 |
For total RNA extraction from mouse ileum, three pieces (1-cm-long segment) of the ileum with Peyer’s patches (~3-mm each in length) were retrieved from the distal ileum (distal 1/3 part of the ileum), opened, cleaned by washing with PBS to remove fecal matter, and homogenized in 1-ml PBS. Similarly, total RNA was recovered from MLNs. Total RNA from cells or culture media was extracted with TRIzol or TRIzol LS reagent (Invitrogen, Carlsbad, CA, USA), respectively. Norwalk virus and MNV-1 RNA copy number was determined by RT-qPCR as described previously68,69. Quantification of mRNA was carried out by RT-qPCR using SYBR Premix ExTaq (Takara, Japan). The specific primer sets used are listed in Supplementary Table 1. Specific primers for human IFN-β and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) were as described previously70. The expression levels of all the target genes normalized with the expression level of GAPDH mRNA were quantified using the ΔΔCt method71.
|
study
| 100.0 |
Cells were lysed in lysis buffer (50 mM Tris-HCl, pH 8.0, 150 mM NaCl, 1 mM EDTA, 1% Nonidet P-40) supplemented with EDTA-free protease inhibitor cocktail (Roche Diagnostics) by incubating on ice for 20 min. Aliquots of cleared cell lysates from the same samples were resolved by SDS-PAGE, transferred onto nitrocellulose membranes, and processed in parallel for immunoblotting analyses for multiple proteins. Membranes were immunoblotted with specific antibodies and the bound antibodies were detected with an enhanced chemiluminescence kit (GE Healthcare Life Sciences, Piscataway, NJ, USA).
|
study
| 99.94 |
Purified MNV particles absorbed onto carbon-coated grids were negatively stained with uranyl acetate. The stained samples were examined under a Tecnai G2 Spirit TWIN transmission electron microscope (FEI Company, OR, USA) at the Division of Electron Microscopic Research, Korea Basic Science Institute (KBSI; Daejeon, Korea).
|
study
| 99.9 |
For microscopic analysis, both, γ-PGA and purified MNV were conjugated with biotin on the exposed amine group using Sulfo-NHS-LC-Biotin (EZ-Link; Pierce, Rockford, IL, USA) according to the manufacturer’s protocol. Biotinylated γ-PGA and MNV-1 were detected using Qdot 625 streptavidin conjugate (Invitrogen).
|
study
| 99.94 |
Intestinal tissues of mice were embedded in a frozen section compound (Surgipath FSC22, Leica Microsystems, Wetzlar, Germany). Thin sections (10 µm) were fixed in 4% paraformaldehyde, blocked in 1% BSA in PBS, and incubated for 2 h at room temperature with an anti-TLR4 mouse monoclonal antibody or an anti-villin rabbit polyclonal antibody with gentle rocking. After washing 3 times with PBS, appropriate fluorescent-conjugated secondary antibodies were used for visualizing the antigen. Nuclei were visualized by staining with 1 μM 4′,6′-diaamidino-2-phenylindole (DAPI) in PBS for 10 min. Confocal images were acquired on a ZEISS LSM 880 confocal laser scanning microscope (Carl Zeiss, Oberkochen, Germany). For z-stack analysis, images were obtained along the z-axis at 0.3-μm intervals and subjected to three-dimensional analysis using ZEN software (Blue edition, 2.3, Carl Zeiss).
|
study
| 99.94 |
The serum levels of IFN-α and β were measured using the VeriKine mouse IFN-α and β ELISA kit (PBL Interferon Source, Piscataway, NJ, USA). The detection limit of mouse IFN-α and β ELISAs were 12.5 and 15.6 pg/ml, respectively. Serum levels of other cytokines (IL-1α, IL-1β, IL-2, IL-6, IL-10, TNF-α, and IFN-γ) were quantified using Luminex multiplex detection assay using antibody-coated magnetic beads (R&D Systems, Minneapolis, MN, USA).
|
study
| 100.0 |
For ex vivo culture of small intestinal tissues of mouse, ileum was isolated and cultured as described previously72 with slight modifications. Briefly, distal ileum segments (1 cm in length) with or without Peyer’s patches were collected, gently opened with a sterilized operating scissors, washed five times with PBS supplemented with 10% penicillin/streptomycin, and transferred to 12-well plates for further incubation in complete DMEM.
|
study
| 99.94 |
BALB/c mice (6 weeks old, n = 10 per group) were orally administered with γ-PGA in 100-µl saline once a day for 1, 3, or 5 days at a dose of 50 mg/kg body weight. Four hours after the last γ-PGA administration, total blood was collected from the abdominal vein of mice for cytokine analyses using ELISA. To test the antiviral efficacy of γ-PGA, BALB/c mice (6 weeks old, n = 7 per group) were orally administered with γ-PGA (50 mg/kg body weight) in 100 µl saline once a day for 5 days. This was followed by peroral inoculation with MNV (1 × 107 PFU/100 µL saline) 12 h after the last γ-PGA administration at day 5. γ-PGA was given once daily for another two days. Two days after inoculation, the distal ileum and MLNs were retrieved from mice for analyses of intracellular viral RNA and infectious virus titers. Homogenized tissues were subjected to centrifugation at 3,000 × g for 10 min to recover supernatants, which were then passed through a 0.20-µm pore-size membrane filter. The filtrates were used for quantification of virus titers using a plaque-forming assay.
|
study
| 100.0 |
All animal experiments were performed in accordance with the Korean Food and Drug Administration guidelines. Protocols were reviewed and approved by the Institutional Animal Care and Use Committee of the Yonsei Laboratory Animal Research Center (Permit No: IACUC-A-201409-280-02; continued on 201502-153-01). For collection of serum and tissues, mice were anesthetized with intraperitoneal injection of tribromoethanol (Avertin). At the termination of the experiment, all mice were euthanized by CO2 inhalation.
|
other
| 99.94 |
Statistical analysis was performed using GraphPad Prism 6 (Graphpad Prism Software Inc., La Jolla, CA, USA). Results are presented as the mean ± standard deviation (s.d) from at least three independent experiments, unless otherwise stated. Specific tests used to determine statistical difference are noted in each figure legend. Differences between groups were considered statistically significant if *P < 0.05.
|
other
| 53.84 |
Over one billion people globally have limited access to health services, with many living in rural areas in low- and middle-income countries (LMICs) (1). One important way to reduce this growing burden is to increase training of frontline health workers worldwide. In particular, community health nurses (CHNs) are vital health-care workers who can extend healthcare in rural areas, especially in LMICs. These nurses are trained health workers based in central health centers who meet the needs of local people by visiting and providing care at individual homes and community group visits. The importance of community health workers and CHNs has been widely recognized on an international scale. In 2008, the World Health Organization and the Global Workforce Alliance challenged the international community “to make sure that everyone has access to a suitable, trained and motivated health worker as part of a functioning health system” (1). In order to ensure the ongoing effectiveness of CHNs, organizations must provide continuing education to health workers.
|
other
| 99.75 |
Subsets and Splits
No community queries yet
The top public SQL queries from the community will appear here once available.