Kaposi’s sarcoma-associated herpesvirus (KSHV) is linked to human malignancies. The majority of tumor cells harbor latent virus and a small percentage undergo spontaneous lytic replication. Both latency and lytic replication are important for viral pathogenesis and spread but the cellular players involved in the switch between the two viral lifecycle phases are not clearly understood. We conducted a siRNA screen targeting the cellular kinome and identified Tousled-like kinases (TLKs) as cellular kinases that control KSHV reactivation from latency. Upon treatment of latent KSHV-infected cells with siRNAs targeting TLKs, we saw robust viral reactivation. Knockdown of TLKs in latent KSHV-infected cells induced expression of viral lytic proteins and production of infectious virus. TLKs were also found to play a role in regulating reactivation from latency of another related oncogenic gammaherpesvirus, Epstein-Barr virus (EBV). Our results establish the TLKs as cellular repressors of gammaherpesviral reactivation.
KSHV; EBV; TLK1; TLK2; Latency; Reactivation; siRNA screen
We previously identified a gene signature predicted to regulate the epithelial-mesenchymal transition (EMT) in both epithelial tissue stem cells and breast cancer cells. A phenotypic RNA interference (RNAi) screen identified the genes within this 140-gene signature that promoted the conversion of mesenchymal epithelial cell adhesion molecule-negative (EpCAM−) breast cancer cells to an epithelial EpCAM+/high phenotype. The screen identified 10 of the 140 genes whose individual knockdown was sufficient to promote EpCAM and E-cadherin expression. Among these 10 genes, RNAi silencing of the SWI/SNF chromatin-remodeling factor Smarcd3/Baf60c in EpCAM− breast cancer cells gave the most robust transition from the mesenchymal to epithelial phenotype. Conversely, expression of Smarcd3/Baf60c in immortalized human mammary epithelial cells induced an EMT. The mesenchymal-like phenotype promoted by Smarcd3/Baf60c expression resulted in gene expression changes in human mammary epithelial cells similar to that of claudin-low triple-negative breast cancer cells. These mammary epithelial cells expressing Smarcd3/Baf60c had upregulated Wnt5a expression. Inhibition of Wnt5a by either RNAi knockdown or blocking antibody reversed Smarcd3/Baf60c-induced EMT. Thus, Smarcd3/Baf60c epigenetically regulates EMT by activating WNT signaling pathways.
MEK1/2 inhibitors such as AZD6244 are in clinical trials for the treatment of multiple cancers, including breast cancer. Targeted kinase inhibition can induce compensatory kinome changes, rendering single therapeutic agents ineffective. To identify target proteins to be used in a combinatorial approach to inhibit tumor cell growth, we used a novel strategy that identified microRNAs (miRNAs) that synergized with AZD6244 to inhibit the viability of the claudin-low breast cancer cell line MDA-MB-231. Screening of a miRNA mimic library revealed the ability of miR-9-3p to significantly enhance AZD6244-induced extracellular signal-regulated kinase inhibition and growth arrest, while miR-9-3p had little effect on growth alone. Promoter methylation of mir-9 genes correlated with low expression of miR-9-3p in different breast cancer cell lines. Consistent with miR-9-3p having synthetic enhancer tumor suppressor characteristics, miR-9-3p expression in combination with MEK inhibitor caused a sustained loss of c-MYC expression and growth inhibition. The β1 integrin gene (ITGB1) was identified as a new miR-9-3p target, and the growth inhibition seen with small interfering RNA knockdown or antibody blocking of ITGB1 in combination with MEK inhibitor phenocopied the growth inhibition seen with miR-9-3p plus AZD6244. The miRNA screen led to identification of a druggable protein, ITGB1, whose functional inhibition synergizes with MEK inhibitor.
MAP3K1 is a member of the mitogen-activated protein kinase kinase kinase (MAP3K) family of serine/threonine kinases. MAP3K1 regulates JNK activation and is unique among human kinases in that it also encodes an E3 ligase domain that ubiquitylates c-Jun and ERK1/2. Full length MAP3K1 regulates cell migration and contributes to pro-survival signaling while its caspase 3-mediated cleavage generates a C-terminal kinase domain that promotes apoptosis. The critical function of MAP3K1 in cell fate decisions suggests that it may be a target for deregulation in cancer. Recent large-scale genomic studies have revealed that MAP3K1 copy number loss and somatic missense or nonsense mutations are observed in a significant number of different cancers, being most prominent in luminal breast cancer. The alteration of MAP3K1 in diverse cancer types demonstrates the importance of defining phenotypes for possible therapeutic targeting of tumor cell vulnerabilities created when MAP3K1 function is lost or gained.
MAP3K; MEKK; protein kinase; apoptosis
Recent advances in proteomics have facilitated the analysis of the kinome ‘en masse’. What these studies have revealed is a surprisingly dynamic network of kinase responses to highly selective kinase inhibitors, thereby illustrating the complex biological responses to these small molecules. Moreover these studies have identified key transcription factors, such as c-Myc and FOXO (forkhead box O), that play pivotal roles in kinome reprogramming in cancer cells. Since many kinase inhibitors fail despite a high efficacy of blocking their intended targets, elucidating kinome changes at a more global level will be essential to understanding the mechanisms of kinase inhibitor pharmacology. The development of technologies to study the kinome, as well as examples of kinome resilience and reprogramming, will be discussed in the present review.
Cardiac troponin levels help risk-stratify patients presenting with an acute coronary syndrome (ACS). Although they may be elevated in patients presenting with Non-ACS conditions, specific diagnoses and long-term outcomes within that cohort are unclear.
Methods and Results
Using the Veterans Affairs (VA) centralized databases, we identified all hospitalized patients in 2006 who had a troponin assay obtained during their initial reference hospitalization. Based on ICD-9 diagnostic codes, primary diagnoses were categorized as either ACS or Non-ACS conditions. Of a total of 21,668 patients with an elevated troponin level who were discharged from the hospital, 12,400 (57.2%) had a Non-ACS condition. Among that cohort, the most common diagnostic category involved the cardiovascular system and congestive heart failure (N=1661) and chronic coronary artery disease (N=1648) accounted for the major classifications. At one-year following hospital discharge, mortality in patients with a Non-ACS condition was 22.8% and was higher than the ACS cohort (Odds Ratio=1.39; 95%CI: 1.30–1.49). Despite the high prevalence of cardiovascular diseases in patients with a Non-ACS diagnosis, utilization of cardiac imaging within 90 days of hospitalization was low compared with ACS patients (Odds Ratio=0.25; 95%CI: 0.23–0.27).
Hospitalized patients with an elevated troponin level most often have a primary diagnosis that is not an acute coronary syndrome. Their long-term survival is poor and justifies novel diagnostic or therapeutic strategy-based studies to target the highest risk subsets prior to hospital discharge.
outcomes; troponins; non-ACS diagnosis; cardiac imaging; coronary artery disease
Glaucoma is an optic neuropathy and one of the leading causes of blindness. Its hereditary forms are classified into primary closed-angle (PCAG), primary open-angle (POAG) and primary congenital glaucoma (PCG). Although many loci have been mapped in human, only a few genes have been identified that are associated with the development of glaucoma and the genetic basis of the disease remains poorly understood. Glaucoma has also been described in many dog breeds, including Dandie Dinmont Terriers (DDT) in which it is a late-onset (>7 years) disease. We designed clinical and genetic studies to better define the clinical features of glaucoma in the DDT and to identify the genetic cause. Clinical diagnosis was based on ophthalmic examinations of the affected dogs and 18 additionally investigated unaffected DDTs. We collected DNA from over 400 DTTs and a genome wide association study was performed in a cohort of 23 affected and 23 controls, followed by a fine mapping, a replication study and candidate gene sequencing. The clinical study suggested that ocular abnormalities including abnormal iridocorneal angles and pectinate ligament dysplasia are common (50% and 72%, respectively) in the breed and the disease resembles human PCAG. The genetic study identified a novel 9.5 Mb locus on canine chromosome 8 including the 1.6 Mb best associated region (p = 1.63×10−10, OR = 32 for homozygosity). Mutation screening in five candidate genes did not reveal any causative variants. This study indicates that although ocular abnormalities are common in DDTs, the genetic risk for glaucoma is conferred by a novel locus on CFA8. The canine locus shares synteny to a region in human chromosome 14q, which harbors several loci associated with POAG and PCG. Our study reveals a new locus for canine glaucoma and ongoing molecular studies will likely help to understand the genetic etiology of the disease.
Anticancer drug development is inefficient, but genetically engineered murine models (GEMM) and orthotopic, syngeneic transplants (OST) of cancer may offer advantages to in vitro and xenograft systems.
We assessed the activity of 16 treatment regimens in a RAS-driven, Ink4a/Arf-deficient melanoma GEMM. In addition, we tested a subset of treatment regimens in three breast cancer models representing distinct breast cancer subtypes: claudin-low (T11 OST), basal-like (C3-TAg GEMM), and luminal B (MMTV-Neu GEMM).
Like human RAS-mutant melanoma, the melanoma GEMM was refractory to chemotherapy and single-agent small molecule therapies. Combined treatment with AZD6244 [mitogen-activated protein–extracellular signal-regulated kinase kinase (MEK) inhibitor] and BEZ235 [dual phosphoinositide-3 kinase (PI3K)/mammalian target of rapamycin (mTOR) inhibitor] was the only treatment regimen to exhibit significant antitumor activity, showed by marked tumor regression and improved survival. Given the surprising activity of the "AZD/BEZ" combination in the melanoma GEMM, we next tested this regimen in the "claudin-low" breast cancer model that shares gene expression features with melanoma. The AZD/BEZ regimen also exhibited significant activity in this model, leading us to testing in even more diverse GEMMs of basal-like and luminal breast cancer. The AZD/BEZ combination was highly active in these distinct breast cancer models, showing equal or greater efficacy compared with any other regimen tested in studies of over 700 tumor-bearing mice. This regimen even exhibited activity in lapatinib-resistant HER2+ tumors.
These results show the use of credentialed murine models for large-scale efficacy testing of diverse anticancer regimens and predict that combinations of PI3K/mTOR and MEK inhibitors will show antitumor activity in a wide range of human malignancies.
Protein kinases play key roles in oncogenic signaling and are a major focus in the development of targeted cancer therapies. Imatinib, a BCR-Abl tyrosine kinase inhibitor, is a successful front-line treatment for chronic myelogenous leukemia (CML). However, resistance to imatinib may be acquired by BCR-Abl mutations or hyperactivation of Src family kinases such as Lyn. We have used multiplexed kinase inhibitor beads (MIBs) and quantitative mass spectrometry (MS) to compare kinase expression and activity in an imatinib-resistant (MYL-R) and -sensitive (MYL) cell model of CML. Using MIB/MS, expression and activity changes of over 150 kinases were quantitatively measured from various protein kinase families. Statistical analysis of experimental replicates assigned significance to 35 of these kinases, referred to as the MYL-R kinome profile. MIB/MS and immunoblotting confirmed the over-expression and activation of Lyn in MYL-R cells and identified additional kinases with increased (MEK, ERK, IKKα, PKCβ, NEK9) or decreased (Abl, Kit, JNK, ATM, Yes) abundance or activity. Inhibiting Lyn with dasatinib or by shRNA-mediated knockdown reduced the phosphorylation of MEK and IKKα. Because MYL-R cells showed elevated NF-κB signaling relative to MYL cells, as demonstrated by increased IκBα and IL-6 mRNA expression, we tested the effects of an IKK inhibitor (BAY 65-1942). MIB/MS and immunoblotting revealed that BAY 65-1942 increased MEK/ERK signaling and that this increase was prevented by co-treatment with a MEK inhibitor (AZD6244). Furthermore, the combined inhibition of MEK and IKKα resulted in reduced IL-6 mRNA expression, synergistic loss of cell viability and increased apoptosis. Thus, MIB/MS analysis identified MEK and IKKα as important downstream targets of Lyn, suggesting that co-targeting these kinases may provide a unique strategy to inhibit Lyn-dependent imatinib-resistant CML. These results demonstrate the utility of MIB/MS as a tool to identify dysregulated kinases and to interrogate kinome dynamics as cells respond to targeted kinase inhibition.
Prostate cancer is the most common solid malignancy in men, with 32,000 deaths annually. Piperine, a major alkaloid constituent of black pepper, has previously been reported to have anti-cancer activity in variety of cancer cell lines. The effect of piperine against prostate cancer is not currently known. Therefore, in this study, we investigated the anti-tumor mechanisms of piperine on androgen dependent and androgen independent prostate cancer cells. Here, we show that piperine inhibited the proliferation of LNCaP, PC-3, 22RV1 and DU-145 prostate cancer cells in a dose dependent manner. Furthermore, Annexin-V staining demonstrated that piperine treatment induced apoptosis in hormone dependent prostate cancer cells (LNCaP). Using global caspase activation assay, we show that piperine-induced apoptosis resulted in caspase activation in LNCaP and PC-3 cells. Further studies revealed that piperine treatment resulted in the activation of caspase-3 and cleavage of PARP-1 proteins in LNCaP, PC-3 and DU-145 prostate cancer cells. Piperine treatment also disrupted androgen receptor (AR) expression in LNCaP prostate cancer cells. Our evaluations further show that there is a significant reduction of Prostate Specific Antigen (PSA) levels following piperine treatment in LNCaP cells. NF-kB and STAT-3 transcription factors have previously been shown to play a role in angiogenesis and invasion of prostate cancer cells. Interestingly, treatment of LNCaP, PC-3 and DU-145 prostate cancer cells with piperine resulted in reduced expression of phosphorylated STAT-3 and Nuclear factor-κB (NF-kB) transcription factors. These results correlated with the results of Boyden chamber assay, wherein piperine treatment reduced the cell migration of LNCaP and PC-3 cells. Finally, we show that piperine treatment significantly reduced the androgen dependent and androgen independent tumor growth in nude mice model xenotransplanted with prostate cancer cells. Taken together, these results support further investigation of piperine as a potential therapeutic agent in the treatment of prostate cancer.
Cerebral cavernous malformation (CCM) involves the homozygous inactivating mutations of one of three genes, ccm1, -2, or -3 resulting in hyperpermeable blood vessels in the brain. The CCM1, -2, and -3 proteins form a complex to organize the signaling networks controlling endothelial cell physiology including actin dynamics, tube formation, and adherens junctions. The common biochemical defect with the loss of CCM1, -2, or -3 is increased RhoA activity leading to the activation of Rho-associated coiled coil-forming kinase (ROCK). Inhibition of the ROCK rescues CCM endothelial cell dysfunction, suggesting that the inhibition of RhoA-ROCK signaling may be a therapeutic strategy to prevent or arrest the progression of the CCM lesions.
actin dynamics; CCM; cerebral cavernous malformation; RhoA; ROCK
Kinase inhibitors have limited success in cancer treatment because tumors circumvent their action. Using a quantitative proteomics approach, we assessed kinome activity in response to MEK inhibition in triple negative breast cancer (TNBC) cells and genetically engineered mice (GEMMs). MEK inhibition caused acute ERK activity loss, resulting in rapid c-Myc degradation that induced expression and activation of several receptor tyrosine kinases (RTKs). RNAi knockdown of ERK or c-Myc mimicked RTK induction by MEK inhibitors, whereas prevention of proteasomal c-Myc degradation blocked kinome reprogramming. MEK inhibitor-induced RTK stimulation overcame MEK2 but not MEK1 inhibition, reactivating ERK and producing drug resistance. The C3Tag GEMM for TNBC similarly induced RTKs in response to MEK inhibition. The inhibitor-induced RTK profile suggested a kinase inhibitor combination therapy that produced GEMM tumor apoptosis and regression where single agents were ineffective. This approach defines mechanisms of drug resistance, allowing rational design of combination therapies for cancer.
Analysis of patient tumors suggests multiple MAP3kinases (MAP3Ks) are critical for growth and metastasis of cancer cells. MAP3Ks selectively control the activation of ERK1/2, JNK, p38 and ERK5 in response to receptor tyrosine kinases and GTPases. We used MDA-MB-231 cells because of their ability to metastasize from the breast fat pad to distant lymph nodes for an orthotopic xenograft model to screen the function of seven MAP3Ks in controlling tumor growth and metastasis. Stable shRNA knockdown was used to inhibit the expression of each of the seven MAP3Ks, which were selected for their differential regulation of the MAPK network. The screen identified two MAP3Ks, MEKK2 and MLK3, whose shRNA knockdown caused significant inhibition of both tumor growth and metastasis. Neither MEKK2 nor MLK3 have been previously shown to regulate tumor growth and metastasis in vivo. These results demonstrated that MAP3Ks, which differentially activate JNK, p38 and ERK5 are necessary for xenograft tumor growth and metastasis of MDA-MB-231 tumors. The requirement for MAP3Ks signaling through multiple MAPK pathways explains why several members of the MAPK network are activated in cancer. MEKK2 was required for EGF receptor and Her2/Neu activation of ERK5, with ERK5 being required for metastasis. Loss of MLK3 expression increased mitotic infidelity and apoptosis in vitro. Knockdown of MEKK2 and MLK3 resulted in increased apoptosis in orthotopic xenografts relative to control tumors in mice, inhibiting both tumor growth and metastasis; MEKK2 and MLK3 represent untargeted kinases in tumor biology for potential therapeutic development.
MAP3K; Metastasis; MAPK network; MEKK2; MLK3
Coronary computed tomographic angiography (CCTA) is associated with high radiation dose to the female breasts. Bismuth breast shielding offers the potential to significantly reduce dose to the breasts and nearby organs, but the magnitude of this reduction and its impact on image quality and radiation dose have not been evaluated.
Radiation doses from CCTA to critical organs were determined using metal-oxide-semiconductor field-effect transistors positioned in a customized anthropomorphic whole-body dosimetry verification phantom. Image noise and signal were measured in regions of interest (ROIs) including the coronary arteries.
With bismuth shielding, breast radiation dose was reduced 46–57% depending on breast size and scanning technique, with more moderate dose reduction to the heart, lungs, and esophagus. However, shielding significantly decreased image signal (by 14.6 HU) and contrast (by 28.4 HU), modestly but significantly increased image noise in ROIs in locations of coronary arteries, and decreased contrast-to-noise ratio by 20.9%..
While bismuth breast shielding can significantly decrease radiation dose to critical organs, it is associated with an increase in image noise, decrease in contrast-to-noise, and changes tissue attenuation characteristics in the location of the coronary arteries.
coronary CT angiography; radiation dose; bismuth shielding; breast shielding
A soft x-ray microbeam using proton-induced x-ray emission (PIXE) of characteristic titanium (Kα 4.5 keV) as the x-ray source has been developed at the Radiological Research Accelerator Facility (RARAF) at Columbia University. The proton beam is focused to a 120 μm × 50 μm spot on the titanium target using an electrostatic quadrupole quadruplet previously used for the charged particle microbeam studies at RARAF. The proton induced x-rays from this spot project a 50 μm round x-ray generation spot into the vertical direction. The x-rays are focused to a spot size of 5 μm in diameter using a Fresnel zone plate. The x-rays have an attenuation length of (1/e length of ~145 μm) allowing more consistent dose delivery across the depth of a single cell layer and penetration into tissue samples than previous ultra soft x-ray systems. The irradiation end station is based on our previous design to allow quick comparison to charged particle experiments and for mixed irradiation experiments.
soft x-ray; microbeam; PIXE
Epithelial-mesenchymal transition (EMT) is an essential developmental program that becomes reactivated in adult tissues to promote the progression of cancer. EMT has been largely studied by examining the beginning epithelial state or the ending mesenchymal state without studying the intermediate stages. Recent studies using trophoblast stem (TS) cells paused in EMT have defined the molecular and epigenetic mechanisms responsible for modulating the intermediate “metastable” stages of EMT. Targeted inactivation of MAP3K4, knockdown of CBP or overexpression of SNAI1 in TS cells induced similar metastable phenotypes. These TS cells exhibited epigenetic changes in the histone acetylation landscape that cause loss of epithelial maintenance while preserving self-renewal and multipotency. A similar phenotype was found in claudin-low breast cancer cells with properties of EMT and stemness. This intersection between EMT and stemness in TS cells and claudin-low metastatic breast cancer demonstrates the usefulness of developmental EMT systems to understand EMT in cancer.
EMT; metastable EMT; TS cells; claudin-low breast cancer; EMT and stemness; epigenetics; MAP3K4; CBP; histone acetylation
L-2-hydroxyglutaric aciduria is a metabolic repair deficiency characterized by elevated levels of L-2-hydroxyglutaric acid in urine, blood and cerebrospinal fluid. Neurological signs associated with the disease in humans and dogs include seizures, ataxia and dementia.
Here we describe an 8 month old Yorkshire terrier that presented with episodes of hyperactivity and aggressive behavior. Between episodes, the dog’s behavior and neurologic examinations were normal. A T2 weighted MRI of the brain showed diffuse grey matter hyperintensity and a urine metabolite screen showed elevated 2-hydroxyglutaric acid. We sequenced all 10 exons and intron-exon borders of L2HGDH from the affected dog and identified a homozygous A to G transition in the initiator methionine codon. The first inframe methionine is at p.M183 which is past the mitochondrial targeting domain of the protein. Initiation of translation at p.M183 would encode an N-terminal truncated protein unlikely to be functional.
We have identified a mutation in the initiation codon of L2HGDH that is likely to result in a non-functional gene. The Yorkshire terrier could serve as an animal model to understand the pathogenesis of L-2-hydroxyglutaric aciduria and to evaluate potential therapies.
L-2-hydroxyglutaric aciduria; L2HGDH; Yorkshire terrier; Initiator methionine codon
Ectopic Mer expression promotes pro-survival signaling and contributes to leukemogenesis and chemoresistance in childhood acute lymphoblastic leukemia (ALL). Consequently, Mer kinase inhibitors may promote leukemic cell death and further act as chemosensitizers increasing efficacy and reducing toxicities of current ALL regimens. We have applied a structure-based design approach to discover novel small molecule Mer kinase inhibitors. Several pyrazolopyrimidine derivatives effectively inhibit Mer kinase activity at sub-nanomolar concentrations. Furthermore, the lead compound shows a promising selectivity profile against a panel of 72 kinases and has excellent pharmacokinetic properties. We also describe the crystal structure of the complex between the lead compound and Mer, opening new opportunities for further optimization and new template design.
Mer inhibitors; acute lymphoblastic leukemia; pyrazolopyrimidines; chemosensitizer
Epithelial stem cells self-renew while maintaining multipotency, but the dependence of stem cell properties on maintenance of the epithelial phenotype is unclear. We previously showed that trophoblast stem (TS) cells lacking the protein kinase MAP3K4 maintain properties of both stemness and epithelial-mesenchymal transition (EMT). Here, we show that MAP3K4 controls the activity of the histone acetyltransferase CBP, and that acetylation of histones H2A and H2B by CBP is required to maintain the epithelial phenotype. Combined loss of MAP3K4/CBP activity represses expression of epithelial genes and causes TS cells to undergo EMT while maintaining their self-renewal and multipotency properties. The expression profile of MAP3K4 deficient TS cells defines an H2B acetylation regulated gene signature that closely overlaps with that of human breast cancer cells. Taken together, our data define an epigenetic switch that maintains the epithelial phenotype in TS cells and reveal previously unrecognized genes potentially contributing to breast cancer.
Preliminary results are presented for a personal radiation dosimeter in the form of a clothing button to provide gamma-ray dose estimation for clinically significant external radiation exposures to the general public due to a radiological incident, such as a Radiological Dispersal Device. Rods of thermoluminescent material (LiF:Mg,Ti and LiF:Mg,Cu,P) were encapsulated in plastic “buttons”, attached to shirts, and subjected to three cycles of home or commercial laundering or dry cleaning, including ironing or pressing. The buttons were subsequently exposed to doses of 137Cs gamma rays ranging from 0.75 to 8.2 Gy. The rods were removed from the buttons and their light output compared to their responses when bare or to the responses of a set of calibration rods of the same type and from the same manufacturer. In all three of the comparisons for LiF:Mg,Ti rods the relative responses of the rods in buttons changed by 2-6% relative to the same rods before cleaning. In both comparisons for LiF:Mg,Cu,P rods, the response of laundered rods was 1-3% lower than for the same rods before cleaning. Both these materials are potential candidates for button dosimeters.
detector; thermoluminescent; dosimetry; personnel; emergencies; radiological; screening measurements
Major depressive disorder has been associated with activation of inflammatory processes as well as with reductions in innate, adaptive and non-specific immune responses. The objective of this study was to evaluate the association between major depression and a disease-relevant immunologic response, namely varicella-zoster virus (VZV)-specific immunity, in elderly adults. A cross-sectional cohort study was conducted in 104 elderly community dwelling adults ≥ 60 years of age who were enrolled in the Depression Substudy of the Shingles Prevention Study, a double blind, placebo-controlled vaccine efficacy trial. Fifty-two subjects had a current major depressive disorder, and 52 age- and sex-matched controls had no history of depression or any mental illness. VZV-specific cell-mediated immunity (VZV-CMI) was measured by VZV responder cell frequency (VZV-RCF) and interferon-γ enzyme-linked immunospot (ELISPOT) assays, and antibody to VZV was measured by an enzyme-linked immunosorbent assay against affinity-purified VZV glycoproteins (gpELISA). VZV-CMI, measured by VZV-RCF, was significantly lower in the depressed group than in the controls (p<0.001), and VZV-RCF was inversely correlated with the severity of depressive symptoms in the depressed patients. In addition, an age-related reduction in VZV-RCF was observed in the depressed patients, but not in the controls. Furthermore, there was a trend for depressive symptom severity to be associated with lower ELISPOT counts. Finally, VZV-RCF was higher in depressed patients treated with antidepressant medications as compared to untreated depressed patients. Since lower levels of VZV-RCF appear to explain the increased risk and severity of herpes zoster observed in older adults, these findings suggest that, in addition to increasing age, depression may increase the risk and severity of herpes zoster.
Since the 1960s, the Radiological Research Accelerator Facility (RARAF) has been providing researchers in biology, chemistry and physics with advanced irradiation techniques, using charged particles, photons and neutrons.
We are currently developing a unique facility at RARAF, to simulate neutron spectra from an improvised nuclear device (IND), based on calculations of the neutron spectrum at 1.5 km from the epicenter of the Hiroshima atom bomb. This is significantly different from a standard fission spectrum, because the spectrum changes as the neutrons are transported through air, and is dominated by neutron energies between 0.05 and 8 MeV. This facility will be based on a mixed proton/deuteron beam impinging on a thick beryllium target.
A second, novel facility under development is our new neutron microbeam. The neutron microbeam will, for the first time, provide a kinematically collimated neutron beam, 10–20 micron in diameter. This facility is based on a Proton Microbeam, impinging on a thin lithium target near the threshold of the 7Li(p,n)7Be reaction. This novel neutron microbeam will enable studies of neutron damage to small targets, such as single cells, individual organs within small animals or microelectronic components.
Accelerator applications; Beam optics; Neutron sources; Radiation damage evaluation methods
Activation of caspases is a hallmark of apoptosis. Several methods, therefore, were developed to identify and count the frequency of apoptotic cells based on the detection of caspases activation. The method described in this chapter is based on the use of fluorochrome-labeled inhibitors of caspases (FLICA) applicable to fluorescence microscopy, and flow- and image-cytometry. Cell-permeant FLICA reagents tagged with carboxyfluorescein or sulforhodamine when applied to live cells in vitro or in vivo, exclusively label cells that are undergoing apoptosis. The FLICA labeling methodology is simple, rapid, robust, and can be combined with other markers of cell death for multiplexed analysis. Examples are presented on FLICA use in combination with a vital stain (propidium iodide), detection of the loss of mitochondrial electrochemical potential, and exposure of phosphatidylserine on the outer surface of plasma cell membrane using Annexin V fluorochrome conjugates. Following cell fixation and stoichiometric staining of cellular DNA, FLICA binding can be correlated with DNA ploidy, cell cycle phase, DNA fragmentation, and other apoptotic events whose detection requires cell permeabilization. The “time window” for the detection of apoptosis with FLICA is wider compared to that with the Annexin V binding, making FLICA a preferable marker for the detection of early phase apoptosis and more accurate for quantification of apoptotic cells.
FLICA; Caspases; Apoptosis; Flow cytometry; Laser scanning cytometry; Cell death; Mitochondrial potential; Annexin V binding
Despite the increasing concern about the effect of doses below 0.5 Gy and non-targeted exposures of ionising radiation on living organisms, the majority of radiobiological studies are conducted using in vitro cell lines. In order to be able to extrapolate the in vitro results to in vivo models with confidence, it would be of great benefit to develop a reproducible tissue system suitable for critical radiobiological assays. This manuscript describes the development of a reliable protocol to harvest cells from tissue samples and investigate the radiation damage induced on a single cell basis.
Materials and Methods
To validate this approach as a potential tool for bystander experiments, the method focuses on analysing radiation damage in individual cells as a function of their relative position in the tissue. The experiments reported describe the micronucleus formation following partial irradiation with 3.5 MeV protons (0.1, 0.5 and 1 Gy) in an artificial human skin construct.
The reproducible and low background frequency of micronuclei measured in this system allows detection of small increases following radiation exposures. The effect was statistically significant at doses as low as 0.1 Gy in the directly irradiated as well as in the bystander cells.
The data presented provide evidence of a spatially dependent bystander effect whose magnitude decrease as a function of the distance from the directly exposed area.
bystander; 3D skin tissue; micronuclei