Caenorhabditis elegans displays a spectrum of DNA damage responses similar to humans. In order to identify new DNA damage response genes, we isolated in a forward genetic screen 14 new mutations conferring hypersensitivity to ionizing radiation. We present here our characterization of lem-3, one of the genes identified in this screen. LEM-3 contains a LEM domain and a GIY nuclease domain. We confirm that LEM-3 has DNase activity in vitro. lem-3(lf) mutants are hypersensitive to various types of DNA damage, including ionizing radiation, UV-C light and crosslinking agents. Embryos from irradiated lem-3 hermaphrodites displayed severe defects during cell division, including chromosome mis-segregation and anaphase bridges. The mitotic defects observed in irradiated lem-3 mutant embryos are similar to those found in baf-1 (barrier-to-autointegration factor) mutants. The baf-1 gene codes for an essential and highly conserved protein known to interact with the other two C. elegans LEM domain proteins, LEM-2 and EMR-1. We show that baf-1, lem-2, and emr-1 mutants are also hypersensitive to DNA damage and that loss of lem-3 sensitizes baf-1 mutants even in the absence of DNA damage. Our data suggest that BAF-1, together with the LEM domain proteins, plays an important role following DNA damage – possibly by promoting the reorganization of damaged chromatin.The small nematode  Living organisms are continuously exposed to genotoxic stress, caused both endogenously by cellular metabolism and exogenously by e.g. UV light. To maintain genome integrity, cells possess a number of DNA damage response pathways, which ensure that lesions are removed from the DNA or that cells with irreparable damage are removed by apoptosis. Defects in DNA damage responses can lead to a number of diseases, including predisposition to cancer  domain and a GIY-YIG nuclease domain. Biochemical analysis confirms that LEM-3 acts as nuclease in vitro. Loss of lem-3 function does not impair cell cycle arrest or apoptosis in the germ line but leads to high embryonic lethality following treatment with X-rays, UV-C light and cisplatin. Following DNA damage lem-3 embryos display severe defects during cell division, including chromosome mis-segregation and anaphase bridges. These mitotic defects are similar to those found in baf-1 (barrier-to-autointegration factor) mutants baf-1 gene encodes an essential and highly conserved protein known to interact with the other two C. elegans LEM domain proteins, LEM-2 and EMR-1 baf-1, lem-2, and emr-1 mutants are also hypersensitive to DNA damage and that loss of lem-3 sensitizes baf-1 mutants even in the absence of DNA damage. Our data uncover a novel function for baf-1 following DNA damage, which is dependent on the LEM domain proteins.As the original screen by Hartman and Herman op444 for further analysis because it showed the highest increase in sensitivity to ionizing radiation but had no strong defect in the absence of exogenous DNA damage. Dose-response studies showed that op444 mutants were an order of magnitude more sensitive to ionizing radiation (IR) than wild type  induces cyclobutane pyrimidine dimers and 6-4 photoproducts, which are mainly repaired by the nucleotide excision repair (NER) pathway. To determine whether op444 mutants are also sensitive to UV-C light, we exposed adult hermaphrodites to 60 J/m2 and assessed embryonic lethality of their progeny  mutants, which carry a deletion in the C. elegans homolog of human XPA, a component of the nucleotide excision repair (NER) pathway We next tested whether  progeny . We founop444 animals with cisplatin, a potent DNA damaging agent frequently used in chemotherapy, which generates intra- and interstrand DNA crosslinks. op444 mutants displayed increased embryonic lethality at both concentrations tested  , one of them is shown in We took advantage of fragment length polymorphisms (FLP) and single nucleotide polymorphisms (SNP) between the Hawaii isolate CB4856 and the Bristol wild-type strain N2 42H11.2) , a gene 42H11.2) . We usedrad (radiation hypersensitive) genes. One of these, rad-1(mn155), showed striking phenotypic similarities to lem-3(op444) and mapped within 2 cM of lem-3rad-1(mn155) is allelic to lem-3. Indeed, rad-1(mn155) failed to complement the radiation sensitivity phenotype of lem-3(op444)  had, in a screen similar to ours, isolated and genetically characterized nine 3(op444) . Sequenc3(op444) . This R1lem-3(op444) also fails to complement tm3468, a 330 bp in-frame deletion generated by the Japanese National Bioresource Project  domain and a GIY-YIG domain of the type COG3680 at the C-terminus (C homologue).The LEM-3 protein contains two ankyrin repeats in its N-terminus, followed by a LEM (terminus . The LEMlem-3 and lem-3(op444) and expressed the respective proteins in insect cells (lem-3(op444) greatly diminishes this activity.As LEM-3 contains a GIY-YIG domain, we wanted to test whether LEM-3 also has nuclease activity. We cloned the cDNAs of wild-type ct cells . Incubatct cells . This rect cells . Again, C. elegans germ line (lem-3(op444) mutants showed a normal proliferation arrest and recovery following exposure to ionizing radiation  animals (lem-3(op444) animals. Given that cell cycle arrest and apoptosis following DNA damage are both normal in lem-3(op444) mutants, we speculate that lem-3(op444) mutants are not impaired in DNA damage signalling but rather in another part of the DNA damage response system, most likely DNA repair.During oocyte development , approxi animals . We conclem-3(op444) mutants in the absence of exogenous DNA damage  mutants to repair exogenously generated dsDNA breaks, we analyzed the subcellular localisation of YFP::RAD-54. In the absence of exogenous damage, YFP::RAD-54(opIs257) localized to distinct foci in the meiotic zone of the germ line, but showed a diffuse nuclear staining pattern in the mitotic zone (lem-3(op444) (lem-3(op444). Only a few, enlarged nuclei still showed RAD-54 foci (lem-3(op444).Rad54 has been implicated in several steps during homologous recombination and repair tic zone . This istic zone . Quantit3(op444) . After 1-54 foci . These llem-3 mutants are also hypersensitive to UV-C light, we investigated a possible involvement of lem-3 in the NER pathway. We built and analyzed a double mutant with xpa-1(ok698), a factor common to transcription-coupled repair and global genomic repair, the two branches of the NER pathway. Interestingly, we found that the double mutant showed a significant increase in embryonic lethality compared to both single mutants  mutants, we investigated in more detail the cause of the lethality observed following DNA damage. To get a first insight into the nature of the defect, we stained chromatin in early stage embryos derived from irradiated hermaphrodite worms. Whereas chromosome segregation was mostly normal in irradiated wild type embryos , 77% (n = 31) of irradiated lem-3(op444) embryos showed chromosome mis-segregation starting at the second cell division, as evidenced by the generation of extra nuclear chromatin material and chromatin bridges following mitosis , 61% had a protruding vulva (P-vul)). In contrast, 86% of irradiated wild type L1 larvae developed to phenotypically normal adults  lmn-1 or baf-1 or co-depletion of emr-1 and lem-2 leads to severe defects in mitosis, including abnormal chromosome segregation and anaphase bridges, which ultimately leads to embryonic lethality Three LEM-domain proteins, EMR-1 (Ce-emerin), LEM-2 (Ce-MAN1), and LEM-3 have been identified in baf-1 or co-depletion of emr-1 and lem-2 leads to embryonic lethality lem-3 mutants following IR, we analyzed whether baf-1, lem-2 or emr-1 mutants might be sensitive to DNA damage. We found that emr-1 mutants were weakly sensitive, while lem-2 mutants displayed moderate sensitivity to IR (baf-1(t1639ts) mutant at permissive temperature was also hypersensitive to irradiation treatment ) greatly increased the lethality of the baf-1(t1639) mutation, even in the absence of exogenous DNA damage. By contrast, the double mutant with the point mutation (lem-3(op444)) showed only a minor increase in lethality, likely due to residual activity of the LEM-3 (L659F) protein. This result suggests that when BAF-1 function is limiting, LEM-3 function becomes important even in the absence of DNA damage. Taken together, the phenotypes described in irradiated lem-3 mutants and animals depleted of either lmn-1 or baf-1, or co-depleted of emr-1 and lem-2 suggest a possible common cause of the observed molecular defects.As downregulation of either ty to IR . The temreatment . To testlem-3 as a new gene required for proper DNA damage response in C. elegans. We isolated the lem-3(op444) mutation in a forward genetic screen for animals hyper-sensitive to ionizing radiation. DNA damage response mutants are often defective in DNA lesion-induced cell cycle arrest or apoptosis. As cell cycle arrest  mutant was largely reduced , however, failed to cause DNA damage under the same conditions. These observations suggest that the molecular function of LEM-3/Ankle1 might be conserved through evolution.LEM-3 is evolutionary conserved. A report by Brachner et al. (co-submitted) demonstrates that the human LEM-3 homologue Ankle1 is capable of inducing DNA cleavage when forced to localize to the nucleus. A leucine to phenylalanine mutation corresponding to the mutation in We found that YFP::LEM-3 localizes in a distinct foci-like structure clearly outside of the nucleus . To datelem-3 mutants do not show any defects in the adult germ line following DNA damage, embryos generated from irradiated germ lines show major defects in chromosome segregation starting at the second cell division. This phenotype is reminiscent of embryos depleted of lmn-1, baf-1 or co-depleted of lem-2 and emr-1. We found that a reduction of BAF-1 function in lem-3 mutants causes high embryonic lethality even in the absence of any genotoxic insult. Our data suggest a model in which LEM-3 is required to support BAF-1 function. We postulate that, for a currently unknown reason, requirement for BAF-1 function increases following DNA damage. What exact function BAF-1 plays following DNA damage and how LEM domain proteins contribute to this function remains to be determined. Loss of lem-3 function uncovers this requirement. This model is supported by the fact that also the other C. elegans LEM domain protein mutants lem-2 and emr-1 show a moderate and a weak sensitivity to IR, respectively.Whereas N2 Bristol strain lem-3(op444) (this study), rad-1(mn155)lem-3(tm 3468) (this study), rad-5(mn159) xpa-1(ok698)cku-80(ok861)lem-2(tm1582) (this study), emr-1(gk119)baf-1(t1639)opIs257 [Prad-54::rad-54::YFP::3′UTRrad-54] opIs309 [Pnpp-1::lem-3::3′UTRlem-3] (this study), opIs384 [Pnpp-1::lem-3::3′UTRlem-3] (this study), opIs383 [Pnpp-1::YFP::lem-3::3′UTRlem-3] (this study), ruIs32 [unc-119(+) pie-1::GFP::H2B] Synchronized L4-stage wild-type worms An Isovolt Titan 160 with Isovolt 160 M2/0.4–3.0 (Seifert) and a Stratalinker UV crosslinker, model 1800 (Stratagene) were used to deliver the indicated doses.Animals were irradiated 24 h post L4/adult molt, individualized on plates and allowed to lay eggs for 4 hours. Eggs were quantified; unhatched eggs were counted 24 h later, and the percentage of embryonic lethality was calculated. All assays were performed on 20°C except otherwise stated.Animals were synchronized (freshly hatched L1s) and irradiated. Phenotypes of those animals that had reached adulthood were quantified 3 days later using a dissection microscope. The Uncoordinated (Unc) phenotype was scored on the basis of sluggish movement.For embryonic survival assays synchronized animals (L4/adult molt) were exposed to different doses of cisplatin (Sigma-Aldrich) on agar plates by evenly distributing 1.2 ml of the corresponding concentration of cisplatin on a 10 cm plate. Animals were transferred to fresh plates 24 h post-treatment. Animals were allowed to lay eggs for 12 hr. Adults were then removed and eggs were counted. Unhatched eggs were quantified 24 h later, and the percentage of embryonic lethality was calculated.Staged animals (12 hours post L4/adult molt) were irradiated with 60 Gy. Apoptotic corpses were quantified in the meiotic zone of the germ line at indicated time points, as previously described http://rsb.info.nih.gov/ij).Staged animals (L4/adult molt) were irradiated. Germ lines were dissected at the indicated time points. Images were taken using an ORCA-ER digital CCD camera. The diameters of 10 nuclei (in focus) were measured and the average diameter of nuclei per germ line was calculated using ImageJ 1.40 g software .Staged animals . The eggshell was cracked as previously described For time lapse microscopy, an Olympus BX61 was used equipped with a Retiga 2000R camera. Dissected embryos were put on a 2% agarose pad. Images were taken every 20 seconds at 2× binning. Pictures were processed using Openlab software (Improvision).Alignments were ClustalW alignment done with the Jalview software (Version 11.0).Transgenic lines were obtained by microparticle bombardment in a Biolistic PDS- 1000 (Bio-Rad) transformation, as previously described PCR amplification was done with Phusion High-Fidelity DNA Polymerase (Finnzymes). Oligonucleotide primers were synthesized by Microsynth.npp-1 promoterPnpp-1Sbf_fwGATCCCTGCAGGTTATTGGTGTCATTTCGGTGATTATGATTGPnpp-1Asc_revCCATGGCGCGCCTTCGCTGAAAACAAACGATTTTTAAGAGAAATGlem-3 genomic plus 3′UTR3lem3AscIfwGATCGGCGCGCCATGCCTCCAAACGGAGCAATCACCACGA3lem3ApaIrevCTGAGGGCCCAGCCAATTCAACCGACTAATAAGGTAGATT6 cells per ml) were infected following the User's Manual (Gibco BRL). Sf9 cells expressing LEM-3 were harvested 48 h post infection by centrifugation, washed once in PBS and snap frozen. For enrichment, the pellet was resuspended in Column-buffer  containing 1 mM PMSF and a proteinase inhibitor cocktail (Roche), and sonicated. After centrifugation , the supernatant was incubated with Amylose resin (NEB). Subsequently, the column was washed with Column-buffer and MBP-LEM-3 was eluted . Fractions containing MBP-LEM-3 were pooled, snap frozen and stored at −80°C.The cDNA of LEM-3 WT and L→F mutant was cloned into a modified pFastBac1 vector (a kind gift of Petr Cejka) that contains an N-terminal MBP-tag. Baculovirus stocks were generated by a procedure described earlier and Sf9 cells . After incubation at 25°C for 1 h, the reaction was terminated by the addition of 0.1% SDS, 14 mM EDTA and 0.1 mg/ml Proteinase K and incubation at 55°C for 10 min. 10% glycerol was added and the samples were separated on a 0.8% agarose gel for 45 min at 80 V. 80 ng of supercoiled pFastBac1 plasmid were incubated with increasing amounts of LEM-3 wild type or its op444 mutant in endonuclease buffer  for 1 h at 25°C ; The reaction was stopped by addition of 0.1% SDS, 14 mM EDTA and 0.1 mg/ml Proteinase K and incubation at 55°C for 10 min. 10% glycerol was added and the DNA species were separated on a 0.8% agarose gel for 1 h at 70 V. Subsequently, the gel was stained with SYBR Gold and analyzed with a Imager (Typhoon).PhiX174 circular virion ssDNA (NEB) was incubated with LEM-3 wild type and L→F mutant in endonuclease buffer Click here for additional data file.Figure S2YFP::LEM-3 expression pattern. (A) The transgene opIs383 [Pnpp-1::YFP::lem-3::3′UTRlem-3] rescues the hypersensitivity of lem-3(op444) mutants. Data represent the average of three experiments ± S.D. The progeny of 10 worms were analysed for each experiment. (B) Representative DIC and fluorescence images of embryos expressing YFP::LEM-3; in green: YFP::LEM-3, in red: membrane staining with the dye FM4-64.(EPS)Click here for additional data file.Figure S3Complementation tests.lem-3(op444) fails to complement (A) rad-1(mn155) and (B) lem-3(tm3468). F1 embryonic lethality after irradiation with 30 Gy was quantified. Data shown represent the average embryonic lethality of the progeny of 10 hermaphrodites.(EPS)Click here for additional data file.Figure S4Enrichment of Sf9-expressed MBP-LEM-3 fusion protein. The protein, containing an N-terminal MBP-tag, was enriched over an Amylose-column. M: marker; prior inf.: prior infection; post inf.: post infection; sup.: supernatant; WT: LEM-3 wild type; F→L mutant corresponding to lem-3(op444).(EPS)Click here for additional data file.Movie S1Chromatin segregation in wild-type embryos after irradiation. Wild-type hermaphrodites carrying the transgene GFP::H2B were irradiated with 30 Gy. Embryos were dissected 2 h after irradiation treatment. Images were taken every 20 sec (as described in (RAR)Click here for additional data file.Movie S2Chromatin segregation defects in lem-3(op444) embryos after irradiation.lem-3(op444) hermaphrodites carrying the transgene GFP::H2B were irradiated with 30 Gy. Embryos were dissected 2 h after irradiation treatment. Images were taken every 20 sec (as described in (RAR)Click here for additional data file.


The distribution of viable and infarcted myocardium is typically visualized using inversion recovery (IR) late gadolinium enhancement (1) or phase-sensitive inversion recovery (PSIR) sequences (2). Transitioning PSIR from breath-hold 2D to respiratory navigator-gated 3D imaging promises higher SNR and CNR, and whole heart coverage . However, the optimal method for motion compensation with 3D PSIR is undetermined. With standard PSIR, the IR-prepared volume (first heartbeat) is corrected with the phase from the reference volume (second heartbeat), Fig . Current1 values were placed on a pneumatic motor that oscillated 20 mm in the antero-posterior direction every second. Animals: Under ACUC-approved protocol, six swine underwent 2 hr LAD occlusion. Imaging took place 4-25 weeks post-infarction and 10-3 min post double-dose injection of Magnevist . A 3D GRE sequence with 1.25x1.25x4 mm3 resolution and 2.5 min scan time ( assuming 100% gating efficiency) was used.Imaging was performed on a 3T system  using a 32-channel cardiac array coil . Phantoms: agar gel phantoms with varying TFigure INPSIR allows for improved reference images, uncorrupted by respiratory motion with no additional penalties in navigator efficiency. The lung-liver-heart boundaries were all sharper with INPSIR. We expect INPSIR will have a greater effect when using parallel imaging and may help obtain additional useful contrasts from phase reference images (5).


Facing excess numbers of target cells, CNS-invading CD8+ T cells cause neuronal cell death either via confined release of cytotoxic effector molecules towards neurons, or via spillover of cytotoxic effector molecules from 'leaky’ immunological synapses and non-confined release by CD8+ T cells themselves during serial and simultaneous killing of oligodendrocytes or astrocytes.Neural-antigen reactive cytotoxic CD8+ T cells were stimulated in vitro, their activation status was assessed by flow cytometry, and supernatant glutamate levels were determined using an enzymatic assay. Expression regulation of molecules involved in vesicular glutamate release was examined by quantitative real-time PCR, and mechanisms of non-vesicular glutamate release were studied by pharmacological blocking experiments. The impact of CD8+ T cell-mediated glutamate liberation on neuronal viability was studied in acute brain slice preparations.Wild-type and T cell receptor transgenic CD8+ T cells acquire the molecular repertoire for vesicular glutamate release: (i) they upregulate expression of glutaminase required to generate glutamate via deamination of glutamine and (ii) they upregulate expression of vesicular proton-ATPase and vesicular glutamate transporters required for filling of vesicles with glutamate. Subsequently, CD8+ T cells release glutamate in a strictly stimulus-dependent manner. Upon repetitive T cell receptor stimulation, CD25high CD8+ T effector cells exhibit higher estimated single cell glutamate release rates than CD25low CD8+ T memory cells. Moreover, glutamate liberation by oligodendrocyte-reactive CD25high CD8+ T effector cells is capable of eliciting collateral excitotoxic cell death of neurons (despite glutamate re-uptake by glia cells and neurons) in intact CNS gray matter.Following T cell receptor stimulation, CD8+ T cells, contributing to excitotoxicity in CNS inflammation.Glutamate release may represent a crucial effector pathway of neural-antigen reactive CD8 Volumes (V) of the culture medium were determined at the beginning (b) and the end (e) of the individual time intervals. Single cell glutamate secretion rates (∆Glu/∆t/CD8 T cell) during repetitive stimulation were calculated using the following formula:For estimation of single cell glutamate secretion rates, splenocytes from OT-I mice were plated at a density of 1 × 10+ T cells before and after three days of anti-CD3/CD28 bead-stimulation. cDNA was used in quantitative real-time PCR (qRT-PCR) assays with specific TaqMan® primers and probes for murine glutaminase (Mm01257297_m1), V-type proton ATPase (Mm01222963_m1), VGluT1 (Mm00812886_m1), VGluT2 (Mm00499876_m1) and VGluT3  as well as primers for 18sRNA  as endogenous control. qRT-PCR was performed according to the manufacturer’s protocol.For analysis of glutaminase-, vesicular (V-type) proton ATPase-, and vesicular glutamate transporter (VGluT)1-3-mRNA expression regulation, RNA was purified using Trizol reagent  and complementary DNA (cDNA) synthesis was performed using a standard protocol with random hexamer primers  from WT CD82 on poly-D-lysine  coated cover-slips placed in four-well plates . All neuronal cell cultures were maintained at 37°C and 5% CO2 for up to five to seven days prior to experiments. The neuronal cell culture system exhibited a purity of about 80% cyclohepten-5,10-imine maleate (MK801) and 2,3-dihydroxy-6-nitro-7-sulphamoyl-benzo(F)quinoxaline  were dissolved in Ht-test for normally distributed samples or Mann–Whitney test for not-normally distributed datasets cyclohepten-5,10-imine maleate; NBQX: 2,3-dihydroxy-6-nitro-7-sulphamoyl-benzo(F)quinoxaline; NMDA: N-methyl-D-aspartate; ODC: Oligodendrocyte; OT-I: Ovalbumin-reactive CD8The authors declare that they have no competing interests.NM, GH, SB, NB, KG and AMH conducted experiments, NM, HW and SGM designed research, supervised experiments and wrote the manuscript. All authors read and approved the final manuscript.+ T cells were isolated from splenocytes (see Methods) yielding a purity of about 90% of CD8+ cells (A) of which about 90% were CD3+ T cells (B) as revealed by standard flow cytometry analysis. (C) The presence of a variety of glutamate release blockers (see text) did not affect the activation status of CD8+ T cells after 72 hours (d 3) of CD3/28 bead-stimulation as under all conditions the fraction of activated CD8+ CD25+ T cells was > 95%, whereas it was < 1% before CD3/28 bead-stimulation (d 0). (D) The presence of a variety of glutamate release blockers (see text) did not impact CD8+ T cell viability after 72 hours (d 3) of CD3/28 bead-stimulation as the fraction of viable annexin V- PI- cells was always > 90%. WT CD8Click here for file


A randomized trial of Traditional Chinese Medicine (TCM) for temporomandibular joint dysfunction (TMD) showed a linear decline in pain over 16 TCM visits. Here we investigate whether medication increases could account for improvement, or whether pain medications were also reduced.One hundred sixty-eight TMD patients received TCM or enhanced self-care using a stepped-care design where those who failed on self-care were offered TCM. This report includes 121 patients during their first 16 TCM visits. The initial 8 occurred more often than weekly; patients and practitioners determined subsequent schedules. Outcome data were collected via study-administered questionnaires at standard times, and self-report at every treatment visit. Here we report on average pain (VAS 0-10) and pain medications over the previous week, collected at treatment visits. We converted pain medication intake to equivalent weekly doses of aspirin (for NSAIDs and acetaminophen) or morphine (7.5mg for narcotics). Pain was analyzed by linear regression with random effects for within-individual correlations. Medication use was log-transformed and analyzed using quadratic splines.The sample was 85% female and on average 44 (SD=13) years old. Narcotics users’ (n=32) average pain improved -2.56 units over 16 visits (p<0.001). Narcotics use declined until visit 11 , and then increased to week 16 . NSAID use declined linearly (p=0.019), -0.47 and -0.20 doses/wk over visits 1-11 and 11-16, respectively. For the 18 participants in the top quartile of NSAIDS-only users, average pain decreased linearly over 16 visits . Dose of NSAIDs declined between visits 1-7  and increased between visits 7-16 . NSAID use among the rest remained low and stable.Among patients using narcotics and those who had the highest NSAID intakes, we observed a short-term reduction in medication use that was partially sustained as TCM visits became less frequent.


DNA-mediated Annealing, extension, Selection and Ligation (DASL) assay has become a suitable gene expression profiling system for degraded RNA from paraffin-embedded tissue. We examined assay characteristics and the performance of the DASL 502-gene Cancer Panelv1 (1.5K) and 24,526-gene panel (24K) platforms at differentiating nine human epidermal growth factor receptor 2- positive (HER2+) and 11 HER2-negative (HER2-) paraffin-embedded breast tumors.The ct-statistics tested for differences in expression levels between HER2 + and HER2 - tumors. Regulatory network analysis was performed using Metacore .Bland-Altman plots and Spearman correlations evaluated intra/inter-panel agreement of normalized expression values. Unequal-variance erythroblastic leukemia viral oncogene homolog 2 (ERBB2) was the most differentially expressed gene between the HER2 + and HER2 - tumors and seven additional genes had p-values < 0.05 and log2 -fold changes > |0.5| in expression between HER2 + and HER2 - tumors: topoisomerase II alpha (TOP2A), cyclin a2 (CCNA2), v-fos fbj murine osteosarcoma viral oncogene homolog (FOS), wingless-type mmtv integration site family, member 5a (WNT5A), growth factor receptor-bound protein 7 (GRB7), cell division cycle 2 (CDC2), and baculoviral iap repeat-containing protein 5 (BIRC5). The top 52 discriminating probes from the 24K panel are enriched with genes belonging to the regulatory networks centered around v-myc avian myelocytomatosis viral oncogene homolog (MYC), tumor protein p53 (TP53), and estrogen receptor α (ESR1). Network analysis with a two-step extension also showed that the eight discriminating genes common to the 1.5K and 24K panels are functionally linked together through MYC, TP53, and ESR1.Technical replicate correlations ranged between 0.815-0.956 and 0.986-0.997 for the 1.5K and 24K panels, respectively. Inter-panel correlations of expression values for the common 498 genes across the two panels ranged between 0.485-0.573. Inter-panel correlations of expression values of 17 probes with base-pair sequence matches between the 1.5K and 24K panels ranged between 0.652-0.899. In both panels, The relative RNA abundance obtained from two highly differing density gene panels are correlated with eight common genes differentiating HER2 + and HER2 - breast tumors. Network analyses demonstrated biological consistency between the 1.5K and 24K gene panels. Gene expression profiling is a rapidly advancing field and has become a useful tool in clinical oncology to identify molecular differences and similarities that can be correlated with clinical behavior and drug responsiveness. Numerous genes are controlled by complex regulatory networks and are involved in the development and progression of breast cancer, and these genes are the key factors in determining each characteristic of the tumor ,2. The rFormalin-fixed, paraffin-embedded (FFPE) tumor samples are routinely used for clinical diagnostic purposes and are the most widely available materials for which patient outcomes are known. However, many microarray-based analyses use intact ribonucleic acid (RNA) from fresh frozen tissue, not a commonly available source of tissue. Thus, FFPE tissue is an invaluable resource for cancer research, particularly for phase III adjuvant clinical trials. These large clinical sample sets are critical for validating molecular profiles of tumor classification, treatment response, and clinical outcome prediction. Although RNA isolated from FFPE is usually highly degraded posing several challenges for microarray based gene-expression profiling, a reverse transcriptase/polymerase chain reaction (RT-PCR)-based microarray technology has been developed to allow high-throughput profiling of paraffin block tissue samples -15.DNA-mediated Annealing, extension, Selection and Ligation (DASL®) assay  is a gene expression profiling system suitable for use with degraded RNAs such as those derived from FFPE tumor samples , direct comparisons of the raw intensities will yield seemingly poor cross-platform correlations. However, fold-change correlations of the gene intensities between the two platforms provide a common metric for comparisons.ERBB2 gene expression between HER2+ and HER2- breast tumors, and the HER2 gene was the most differentially expressed gene for both panels. These results indicate that both panels correctly classified the HER2 status of the tumors when comparing gene expression to protein expression determined by IHC (gold standard) and when considering IHC score of 0-1+ as HER2- and IHC scores of 2-3+ as HER2+. The two tumors that had an IHC score of 2+ as defined by the 2007 ASCO/CAP guidelines [ERBB2 and GRB7, are in the 10-gene HER2 cluster observed by Perou and Sorlie [Both panels detected significant differential idelines  were inid Sorlie ,5. We sed Sorlie ,37. Seved Sorlie . Severald Sorlie .MYC and TP53/ESR1 networks. Nine of the 10 genes in the HER2 gene cluster from the Perou/Sorlie dataset [TP53 and ESR1. In addition, four  of the top five HER2 discriminating genes from the 24K panel are genes commonly amplified in the HER2 amplicon (17q12-q21) and were overexpressed in HER2+ tumors. Their gene expressions were also highly correlated . Lastly, network analyses showed that the top eight discriminating genes common to both panels are connected by the shortest path network analysis with a two-step extension. Interconnecting genes include c-Myc (MYC), TP53, and ESR1.As the main purpose of gene expression studies using microarrays is to reveal the underlying biological differences between groups, functional networks were generated using MetaCore. We observed that the top 52 discriminating probes from the 24K panels are enriched with genes functionally linked to MYC, TP53, and ESR1 regulatory networks are important in differentiating between HER2-positive and -negative tumors. HER2 expression has been shown to be influenced by the presence of ESR1 [ESR1 expression (≥ 12), whereas only two of the seven HER2 3+ tumors had high ESR1 expression (Fisher's Exact p = 0.022). In addition, significant correlations between ESR1 gene expression and ER protein expression levels were observed for the 1.5K  and 24K  panels J.E.P. (statistician)S.K.A. (statistician)Y.A.W. (bioinformatician)M.A.Z. (technician)A.L.O. (statistician)A.E.M. (pathologist)A.C.D. (statistician)B.C. (pathologist)C.S.A. (researcher at Illumina)C.S.A. (researcher at Illumina)E.W-G. (researcher at Illumina)R.B.J. J.M.C. (Director of the Mayo Clinic Cancer Center Genotyping Shared Resource)J.J. (Director of the Mayo Clinic Cancer Center Microarray Shared Resource)E.A.P. (co-PI of NIH funding)J-B.F. (researcher and study lead at Illumina)W.L.L. (Director of the Mayo Clinic Cancer Center TACMA Shared Resource)The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1755-8794/3/60/prepubFigures S1-S4 and Tables T1-T3. Additional Figures S1-S3 are additional figures (S1-S2) that explain the associations between 1) block procurement year and RP13a qPCR Cq, 2) block procurement year and scanner p95 readings, and 3) RP13a and qPCR Cq and scanner p95 readings. An additional figure (S3) explains the correlation between gene panels according to number of probes per 24K gene symbol. Additional Figure S4 explains the relationship between ER immunohistochemical staining and ESR1 gene expression intensity. Additional Tables T1-T3 define the gene symbols presented in Figure 5.Click here for file


Gene therapy for ischemic diseases is a prospective strategy. However, excessive expression of therapeutic genes may produce undesired side effects. Recently, multiple copies hypoxia response elements (HRE) were developed to conditionally regulate gene expression under hypoxia. As a nerve growth factor, Neurotrophin-3 (NT-3) possesses neural protect effects either in vitro or in vivo. To explore hypoxia-controlled NT-3 expression, we constructed a recombinant retrovirus vector with 5HRE and NT-3, and generated a gene transferred cell line PC12-5HRE-NT3 to determine effects of conditionally expressed NT-3 on apoptosis induced by hypoxia in PC12 cells.Five copies of HRE from human VEGF gene and simian virus 40 minimal promoter (SV40mp) were employed to construct a cassette and neurotrophin-3 was inserted into its downstream to generate RV-5HRE-NT3. Mediated by retrovirus, 5HRE-NT3 was transferred into PC12 cells and screened with G418, gene transferred cell lines were generated and identified by reporter gene EGFP, Reverse transcription PCR (RT-PCR) and immunofluorescence cell staining. Conditional expression of NT-3 was detected by RT-PCR, ELISA and Western blot. Apoptosis induced by hypoxia was evaluated by TUNEL. Apoptosis related molecules, phosphorylated p38 and active Caspase-3 were assayed by Western blot.We successfully constructed the retroviral vector RV-5HRE-NT3 and generated the gene transferred cell line PC12-5HRE-NT3. Under normoxia, NT-3 expressed at an extremely low level in PC12-5HRE-NT3, whereas it significantly increased under hypoxia (P < 0.05). There was no significant difference in PC12-NT3 between normoxia and hypoxia. Conditionally expressed NT-3 reduced apoptosis induced by hypoxia in gene transferred cell line PC12-5HRE-NT3(P < 0.05) but not PC12-5HRE-EGFP or PC12 cells. Additionally, activation of p38 and Caspase-3 were depressed in PC12-5HRE-NT3 under hypoxia, which indicated that both of them were involved in the protective effect against apoptosis.These results suggest that 5HRE-SV40mp is an ideal cassette to regulate therapeutic gene expression in response to hypoxia, and 5HRE-NT3 can be further developed as an effective therapeutic gene to provide neuroprotection against cerebral ischemia.


Macroautophagy is an evolutionarily conserved mechanism for bulk intracellular degradation of proteins and organelles. Pathological studies have implicated macroautophagy defects in human neurodegenerative disorders of aging including Alzheimer’s disease and tauopathies. Neuronal deficiency of macroautophagy throughout mouse embryonic development results in neurodevelopmental defects and early postnatal mortality. However, the role of macroautophagy in mature CNS neurons, and the relationship with human disease neuropathology, remains unclear. Here we describe mice deficient in an essential macroautophagy component, Atg7, specifically within postnatal CNS neurons.Postnatal forebrain-specific Atg7 conditional knockout (cKO) mice displayed age-dependent neurodegeneration and ubiquitin- and p62-positive inclusions. Phosphorylated tau was significantly accumulated in Atg7 cKO brains, but neurofibrillary tangles that typify end-stage human tauopathy were not apparent. A major tau kinase, glycogen synthase kinase 3β (GSK3β), was also accumulated in Atg7 cKO brains. Chronic pharmacological inhibition of tau phosphorylation, or genetic deletion of tau, significantly rescued Atg7-deficiency-mediated neurodegeneration, but did not suppress inclusion formation.These data elucidate a role for macroautophagy in the long-term survival and physiological function of adult CNS neurons. Neurodegeneration in the context of macroautophagy deficiency is mediated through a phospho-tau pathway. For secondary detection, Cy3- or FITC-conjugated antibodies were incubated for 1 h (Jackson ImmunoResearch). Photographs were taken using a Zeiss LSM 510 Meta confocal microscope.TM Fluorescent Nissl stain . MAP2-positive neurons were visualized using a Cy3-conjugated secondary antibody (Jackson ImmunoResearch). MAP2 and Nissl double-positive neurons in the CA1 regions were counted manually. In order to count TH-positive neurons, sections covering the entire substantia nigra (25-30 sections / mouse) were stained with sheep anti-TH antibody  at a dilution of 1:250. TH-positive neurons were visualized using the ABC Kit  and DAB . TH-positive neurons in the substantia nigral regions were counted manually under the light microscope.To obtain neuronal cell count, 50 μm coronal brain sections were made using a vibratome. In order to count CA1 neurons, the first 30 sections from the rostral hippocampus were stained with rabbit anti-MAP2 antibody  at a dilution of 1:500, as well as NeuroTraceElectron microscopic analysis was as described. AnesthePreparation of soluble and insoluble fractions was performed as described with some modifications. CorticaCamK-Atg7 cWT and cKO mice littermates (~12 weeks of age) were quickly removed and transverse hippocampal slices (400 μm) were isolated with a Leica VT1200 Vibratome , and placed in ice-cold cutting solution  and maintained at 32°C in ACSF (2 ml/min) containing 125 mM NaCl, 2.5 mM KCl, 1.25 mM NaH2PO4, 25 mM NaHCO3, 25 mM D-glucose, 2 mM CaCl2, and 1 mM MgCl2. All solutions were constantly caboxygenated with 95% O2 + 5% CO2. Slices were allowed to recover for 120 min on the electrophysiology rig prior to experimentation. Bipolar stimulating electrodes (92:8 Pt:Y) were placed at the border of area CA3 and area CA1 along the Schaffer-Collateral pathway. ACSF-filled glass recording electrodes (1–3 MΩ) were placed in stratum radiatum of area CA1. Basal synaptic transmission was assessed for each slice by applying gradually increasing stimuli (0.5–15V), using a stimulus isolator  and determining the input:output relationship. All subsequent stimuli applied to slices was equivalent to the level necessary to evoke a fEPSP that was ~40% of the maximal initial slope that could be evoked. Synaptic efficacy was continuously monitored (0.05 Hz). Sweeps were averaged together every 2 min. fEPSPs were amplified (A-M Systems Model 1800) and digitized  prior to analysis . Stable baseline synaptic transmission was established for 30 min. Slices were given high-frequency stimulation (HFS) to induce long-term potentiation (LTP) using one train of 100 Hz for one second. Stimulus intensity of the HFS was matched to the intensity used in the baseline recordings. fEPSP initial slopes from averaged traces were normalized to those recorded during baseline. Two-way RM-ANOVA were used for electrophysiological data analysis with p < 0.05 as significance criteria.Brains from CamK-Atg7 cWT or CamK-Atg7 cKO mice were used (n = 8 - 10). The mice were placed in a conditioning chamber (Med Associates) for 2 min before the onset of a tone (conditioned stimulus)  and conditioned by a single electrical foot shock (0.45 mA) in the last 2 s. The mice were left in the chamber for another 30 s and placed back into their home cage. Contextual fear learning was measured in the same chamber 24 h after the training by monitoring the freezing for 5 min without electrical shock. Cued fear learning was measured 24 h after the contextual testing. The mice were placed in a novel chamber for 2 min (pre-conditioning). After that, the mice were exposed to the conditioned stimulus for 3 min, and the freezing was monitored. Freezing behavior was scored using FreezeView software (Med Associates Inc.).10-13-mon-old male Dat-Atg7 cWT and Dat-Atg7 cKO mice were treated with Alsterpaullone [Dat-Atg7 cWT mice as controls for Dat-Atg7 cKO mice, to address potential phenotypes due to Cre transgene inserted at the DAT locus[Five-week-old entific). The druAT locus.p value less than 0.05 is considered significant.All comparisons between groups were made using the Mann-Whitney U-test (for two samples) or non-repeated measures ANOVA (for multiple samples). The values are expressed as the means ± S.E. A The authors declare no competing interests.KI, JR, HK, EC, JK, and MK performed the experiments. KI, HK, EK, EC, and AA analyzed the results. KI and AA designed the study and wrote the manuscript. All authors read and approved the final manuscript.CamK-Atg7 cKO mice.Intracellular ubiquitin and p62 positive inclusions in 6-month-old  Ubiquitin-positive inclusions are almost completely overlapped with p62-positive inclusions in the cerebral cortex of CamK-Atg7 cKO mice. Ubiquitin/p62-positive inclusions were already seen at 2-month-old Atg7 cKO mice. Bar, 10 μm.Click here for fileProgressive neurodegeneration in midbrain DA neuron-specific Atg7-deficient (Dat-Atg7 cKO) mice. (a-b) Progressive loss of DA neurons in Dat-Atg7 cKO mice. a, Representative midbrain sections stained with polyclonal antibody specific for TH.Bar, 250 μm. b, Quantification of TH-positive DA neuron number as in (a). White bars, Dat-Atg7 cWT. Black bars, Dat-Atg7 cKO. n = 3 –7 for each group. **, P<0.01. (c-d) Cytoplasmic and dendritic inclusions in Dat-Atg7 cKO mice. Ubiquitin-positive  and p62/SQSTM1-positive  inclusions were present in TH-positive DA neurons (green) of 1-month-old Dat-Atg7 cKO mice, but were never seen in control Dat-Atg7 cWT mice. Bars, 10 μm. (e) Phospho-tau-positive inclusions in TH-positive DA neurons in Dat-Atg7 cKO mice. Phospho-tau specific antibodies (red), AT8, AT100, and TG3, stained inclusions (arrows) in the soma and dendrites of TH-positive DA neurons (green) in Dat-Atg7 cKO mice. AT8, tau phosphorylated at Ser202/Thr205. AT100, tau phosphorylated at Ser212/Thr214. TG3, tau phosphorylated at Thr231/Ser235. Bar, 10 μm. (f) GSK3β-positive inclusions in TH-positive DA neurons in Dat-Atg7 cKO mice. Antibodies recognizing total, activated form (Tyr279/Tyr216), and inactivated form (Ser9) of GSK3β (red), stained the inclusions (arrows) in TH-positive DA neurons (green) in Dat-Atg7 cKO mice. Bar, 10 μm.Click here for fileAPP/Aβ-negative, α-Synuclein-negative, and Thioflavin S-negative inclusions in CamK-Atg7 cKO mice. (a) The ubiquitin-positive inclusions (green) in 1-year-old CamK-Atg7 cKO mice did not contain mouse Aβ (red) (left). 4G8, monoclonal antibody to amino acid residues 17-24 of Aβ, was used. Ten-month-old transgenic mice bearing a mutant form of human APP  were used as positive control for Aβ plaque staining (right). Similar negative results were obtained by 6E10, another antibody to amino acid residues 1-16 of Aβ (data not shown). Bars, 10 μm. (b) The ubiquitin-positive inclusions (green) in 1-year-old CamK-Atg7 cKO mice did not contain mouse α-Synuclein (red). Four different anti-α-synuclein antibodies were used for the double staining. None of four anti-α-synuclein antibodies could detect any positive signals (red) in ubiquitin-positive inclusions (green). Bar, 10 μm. (c) Ubiquitin-positive inclusions (red) in 1-year-old CamK-Atg7 cKO mice were negative for Thioflavin S staining . Thioflavin S stains plaques from β-amyloid and neurofibrillary tangles. Ten-month-old J20 APP transgenic mice were used as positive control for Thioflavin S staining (right). Bars, 20 μm.Click here for fileImmunohistochemical analyses of Atg7-deficient neurons. (a-b) CRMP2-positive inclusions in cortical neurons in CamK-Atg7 cKO mice. a, An antibody recognizing total CRMP2 (red), stained p62-positive inclusions (green) in cortical neurons of CamK-Atg7 cKO mice. Bar, 10 μm. b, An antibody recognizing phosphorylated forms of CRMP2 at Thr514 residues (red), stained p62-positive inclusions (green) in cortical neurons of CamK-Atg7 cKO mice. Bar, 10 μm. (c) β-Catenin-negative inclusions in TH-positive DA neurons in Dat-Atg7 cKO mice. Antibodies recognizing β-Catenin (red) did not stain the inclusions in TH-positive DA neurons (green) in Dat-Atg7 cKO mice. Bar, 10 μm. (d) CDK5-negative inclusions in TH-positive DA neurons in Dat-Atg7 cKO mice. Antibodies recognizing CDK5 (red) did not stain the inclusions in TH-positive DA neurons (green) in Dat-Atg7 cKO mice. Bar, 10 μm.Click here for fileNeuroprotection of Atg7-deficient CNS neurons in vivo. (a) Alsterpaullone can reduce phospho-tau accumulation in the context of macroautophagy inhibition. N2a cells were treated with 1 μM Alsterpaullone in the context of 100 μM chloroquine treatment for 24 h. Cells were lysed in RIPA buffer and subjected to standard Western blotting analysis. Phospho-tau levels were detected by AT8 antibody. (b-c) Ubiquitin-positive inclusion formation was unaffected by systemic injection of Alsterpaullone in the context of Dat-Atg7 cKO mice. Bar, 10 μm. c, Quantification of ubiquitin-positive inclusion number per TH-neuron in Dat-Atg7 cKO mice. No inclusions were observed in Dat-Atg7 cWT mice. n > 60 neurons per genotype. (d-e) Ubiquitin-positive inclusion formation (red) was not changed in TH-positive DA neurons (green) of Dat-Atg7/tau double cKO mice relative to Dat-Atg7 cKO mice. No ubiquitin-positive inclusions were detected in tau KO mice. Bar, 10 μm. e, Quantification of ubiquitin-positive inclusion number per TH-neuron in Dat-Atg7/tau double cKO mice. n > 60 neurons per genotype.Click here for file


In vitro results demonstrated that HSV-sr39tk-transfected cells exhibited enhanced sensitivity to the GCV prodrug, which was not affected by co-expression of the mIL-3 gene. However, in vivo studies showed that co-expression of the mIL-3 gene significantly increased the HSV-sr39tk/GCV-induced tumor growth delay and even cured the tumor. The TRAMP-C1-specific immune response of spleen lymphocytes from mice bearing HSV-sr39tk- and IL-3-expressing TRAMP-C1 tumors was measured by ELISA. Results showed that IL-3-activated IL-4-dominant lymphocytes became IFN-γ- dominant lymphocytes after combined HSV-sr39tk/GCV therapy. The efficacy of combined therapies on tumor regression was reduced when macrophages populations were depleted by carrageenan or NO production was inhibited by administration of the iNOS inhibitor, L-NAME. These results suggest that utilizing a bicistronic vector to express HSV-sr39tk and the IL-3 gene induced an enhanced macrophage- or NO-dependent anti-tumor effect.The herpes simplex virus thymidine kinase/ganciclovir (HSV-sr39tk/GCV) system is a well-established prodrug system used in cancer gene therapy. However, this system is currently not effective enough to eradicate malignant tumors completely. This study aimed to evaluate whether co-expression of interleukin-3 (IL-3) could enhance the anti-tumor activity of HSV-sr39tk/GCV prodrug gene therapy using a murine TRAMP-C1 prostate tumor model.  Proposed by Moolten in 1986, the activation of a suicide gene encoding an enzyme protein that is nontoxic to genetically modified cells has become an extensively adopted approach for prodrug gene therapies designed to treat cancer in vitro and in vivo models. These studies have demonstrated that cells transfected with either of the mutant enzymes were more sensitive to the cytotoxic effects of GCV and acyclovir (ACV) when compared to cells transfected with wild-type HSV-tk. In addition to improving GCV activation, combining the HSV-tk/GCV system with other strategies, such as cytokine therapy, has been demonstrated in several tumor systems to be more effective than using a single treatment Although the bystander effect promotes tumor cell death, inefficient activation of GCV by HSV-tk and prodrug-associated negative side effects limit the clinical efficacy of this system. Using HSV-tk mutants is one approach to improving the activity and specificity for GCV + cells Interleukin-3 (IL-3) is well characterized as a multicolony-stimulating factor that affects the growth of most hemopoietic lineages and is produced by cytotoxic and helper T lymphocytes in mice in vivo. The results demonstrated that bicistronic expression of HSV-sr39tk and the mIL-3 gene followed by GCV administration induced a more effective anti-tumor response compared to that achieved with either HSV-sr39tk/GCV or mIL-3 therapy alone. In addition, we reveal that the anti-tumor mechanism of the combined HSV-sr39tk/GCV and mIL-3 therapy was associated with macrophages or nitric oxide (NO)-dependent T helper 1 (Th1) immune response.In the current study, we investigated whether intratumoral expression of murine IL-3 (mIL-3) enhances the anti-tumor effect of the HSV-tk/GCV system in a murine TRAMP-C1 prostate cancer model Six- to eight-week-old C57BL/6J male mice were purchased from the National Laboratory Animal Center, Taiwan. C.B17/Icr-scid mice were purchased from the Laboratory Animal Center of National Taiwan University College of Medicine, Taiwan. The recommendations of the approved guide for the care and use of laboratory animals by the Institutional Animal Care and Use Committee (IACUC) of National Tsing Hua University, Taiwan (approved number: IACUC:10012), were followed at all times. All surgery was performed under ketamine/xylazine anesthesia, and all efforts were made to minimize suffering.−8 M dehydrotestosterone . RAW264.7 cells were maintained in Dulbecco’s modified Eagle’s medium with 10% fetal bovine serum, 1% penicillin-streptomycin. Cell lines were incubated at 37°C in humidified 5% CO2/air atmosphere.TRAMP-C1 mouse prostate cancer cell line was purchased from American Type Culture Collection (CRL-2730) and RAW264.7 mouse macrophage cell line was purchased from Bioresource Collection and Research Center (BCRC-60001), Taiwan. TRAMP-C1 cells were maintained in Dulbecco’s modified Eagle’s medium  with 10% fetal bovine serum , 1% penicillin-streptomycin (Gibco), 5 µg/ml insulin , and 105′-ATGCTAGCATGGCTTCGTACCCCGGCCAT-3′ and 3′-primer with XhoI site: 5′-ATCTCGAGTCAGTTAGCCTCCCCCATCTC-3′). After restriction enzyme digestion, sr39tk DNA fragments were cloned into IRES or IRES-mIL3 vector to generate IRES-sr39tk or IRES-mIL3-sr39tk plasmid. The three constructed plasmids, IRES-sr39tk, IRES-mIL3 and IRES-mIL3-sr39tk, were transfected into TRAMP-C1 cell line by Effectene Transfection Reagent . After 48 h of transfection, cells were selected in a culture medium containing 1.5 mg/ml G418 for two weeks. The transfected cells were called TRAMP-C1/sr39tk, TRAMP-C1/mIL3, or TRAMP-C1/mIL3-sr39tk.The bicistronic expression vector, IRES , was used to express full length of HSV-sr39tk or/and mIL-3 genes. HSV-sr39tk DNA fragments were amplified from SP72-sr39tk plasmid by PCR with specific primers  and , respectively.Total RNA was isolated from cells by TRIzol reagent according to manufacturer’s protocol . Two µg of total RNA was reversely transcripted into cDNA by Omniscript reverse transcriptase kit (Qiagen). The primer pairs for sr39tk and mIL-3 were:  and cultured overnight. After 24 h, medium were exchanged for fresh medium containing serial diluted GCV (Sigma-Aldrich) concentration from 0 to 200 µg/mL. After incubation for three days, viability of cells were quantified by MTT  assay, and O.D. values were measured at 570 nm wavelength. The percentage of surviving cells was calculated as a percentage of absorbance found in the GCV-treated cells divided by that found in the cells without GCV treatment.5 macrophages were co-cultured with 2×105 tumor cell lines in 1.0 mL of culture media in 24-well tissue culture plates, and treated with GCV for 3 days at 37°C. The concentration of IFN-γ in the media was quantified using IFN-γ immunoassay kit .Mice were intraperitoneally (i.p.) injected with 2 mL each of 3% fluid thioglycolate medium (Sigma-Aldrich) and sacrificed four days later, followed by peritoneal lavage with 10 mL of cold PBS twice. About 90% of collected peritoneal cells were CD11b positive as determined by staining with PE-conjugated anti-Mac-I antibody. 2×106), TRAMP-C1/sr39tk (1.5×106), TRAMP-C1/mIL3 (3×106) and TRAMP-C1/mIL3-sr39tk (3×106) tumor cells were injected subcutaneously into the right flanks of C57BL/6J mice to establish tumor model. Tumor volumes were measured every two days with a caliper until the experiment was completed. When tumor diameter reached 5 mm, each group of mice was randomly divided into two subgroups: PBS and GCV treatment. GCV (33 mg/kg) were dissolved in 100 µl PBS and administered intraperitoneally twice per day for five consecutive days.TRAMP-C1 . Splenocytes were re-suspended in RPMI 1640 with 10% FBS and 1% penicillin-streptomycin and re-stimulated by adding irradiated (70 Gy) TRAMP-C1 cell at 10∶1 ratio for 48 h. Cell supernatants were harvested for IFN-γ and IL-4 immunoassay kits (BD Pharmingen).The phenotype of tumor-infiltrating cells was evaluated by specific antibodies. First, tumors were digested into single cell suspension by dispase (BD Pharmingen) and filtered through 70 µm cell strainer. To avoid non-specific binding, cells were blocked by anti-mouse CD16/CD32 antibody (BD Pharmingen) and serum before antibody staining and incubated on ice for 30 min. After washed twice, cell suspensions were stained by PE-CD11b, FITC-Gr-1 (BD Pharmingen), FITC-F4/80, FITC-CD68, isotype control antibodies (Serotec) on ice for 1 h, and then washed twice. For intracellular staining, cell surface staining were performed first and then fixed by Fixation buffer (BioLegend) for 20 min on ice. Cells were washed by Permeabilization buffer (BioLegend) twice and resuspended in Permeabilization buffer for intracellular staining. FACS analysis was performed on FACSCanto II flow cytometer (Becton Dickinson) and data were analyzed by FACSDiva software.Tumor tissue samples were embedded in OCT compound , snap-frozen in liquid nitrogen and stored in −80°C. Cryostat sections of frozen tissues were fixed in cold methanol for 5 min, followed by two rinses in PBS. To avoid non-specific staining, tumor tissue slides were blocked and permeabilized with blocking buffer  containing 0.01% Tween-20 and 0.1%Triton for 30 min at room temperature. The slides were subsequently incubated with rat anti-mouse CD11b, CD68, and rabbit anti-mouse iNOS (BD Pharmingen) antibodies. After 24 h, slides were washed and incubated with secondary antibodies conjugated with Alexa Fluor 488 or 594 (Invitrogen) for 1 h. After wash, slides were mounted with DAPI (Invitrogen) to visualize nuclei.P-values <0.05 and are derived from the Student’s t test using a two-tailed distribution or one-way ANOVA. The P-values were calculated using the GraphPad Prism software version 5 package .Where indicated, the results of tests of significance are reported as 50∶ 0.558 µM) and TRAMP-C1/mIL3-sr39tk (IC50∶ 0.575 µM) cells were more sensitive to the cytotoxic action of the GCV prodrug, compared to parental TRAMP-C1 (IC50∶ 218.358 µM) and TRAMP-C1/mIL3 cells (IC50∶ 179.4 µM)  . This inin vivoin vivo experiments. After tumor reached 5 mm in diameter, mice were injected intraperitoneally (i.p.) with GCV. The growth of subcutaneous tumors was markedly suppressed in the TRAMP-C1/sr39tk and TRAMP-C1/mIL3-sr39tk groups following GCV treatment , indicating that long-term immunity had developed.To determine the anti-tumor effect of combined HSV-sr39tk/GCV and IL-3 therapy, transfected cells were inoculated subcutaneously (s.c.) into the right flanks of mice. Since IL-3-transfected cells do not readily form tumor reatment . In addiin vitro. Lymphocytes isolated from mice bearing TRAMP-C1 or TRAMP-C1/sr39tk tumors did not develop a specific immune response against challenge with irradiated-TRAMP-C1 cells. In contrast, lymphocytes from mice bearing mIL-3-transfected tumors (both TRAMP-C1/mIL3 and TRAMP-C1/mIL3-sr39tk) developed a specific immune response to TRAMP-C1 cells by producing higher levels of IFN-γ and IL-4 . These findings are consistent with those from our previous study using a fibrosarcoma tumor model, which demonstrated that IL-3 could act as a macrophage attractant Several studies have demonstrated that macrophages are the prime cells responsible for the IL-3-associated anti-tumor T cell response in vitro IFN-γ cytokine production by peritoneal macrophages was measured after co-cultured with tumor cell lines with or without GCV treatment for three days. Neither HSV-sr39tk/GCV-treated nor mIL-3-expressing cells could enhance IFN-γ production by macrophages; however, GCV treatment of TRAMP-C1/mIL3-sr39tk cells increased IFN-γ production by peritoneal macrophages  staining. The majority of iNOS-positive cells in TRAMP-C1/sr39tk tumors co-expressed CD11b and CD68  produced both IL-4 and IFN-γ in response to challenge with irradiated-TRAMP-C1 cells. When mice bearing TRAMP-C1/mIL3-sr39tk tumors were treated with GCV, increased IFN-γ production and decreased IL-4 production were found in the splenocytes in response to challenge with irradiated-TRAMP-C1. This may be associated with the development of long-term immunity in these mice and indicates that the function of macrophages within TRAMP-C1/mIL3-sr39tk tumors was altered following GCV administration. The interaction between IL-3-activated macrophages and HSV-sr39tk/GCV cytotoxicity was further illustrated by co-culture assay + and CD8+ T cells was observed after HSV-tk/GCV therapy + and CD8+ T cells was observed in the spleen following HSV-sr39tk/GCV therapy. However, unlike that found in other tumor models 1 immune response or long-term immunity in our TRAMP-C1 prostate tumor model despite that GCV-treated mice having TRAMP-C1/sr39tk re-grew earlier in T cell deficient SCID mice than in immune competent C57BL/6J mice. This discrepancy may be the result of a lack of suitable stimulating factors in the TRAMP-C1 tumor microenvironment, which was overcome when IL-3 was co-expressed.Several studies have demonstrated the occurrence of anti-tumor immunity when tumors were reduced or ablated by HSV-tk/GCV suicide gene therapy. Vile and colleagues reported that the induction of a pronounced intratumoral infiltration by macrophages, CD42 immune response to a Th1 immune response 2 immune response to a Th1 immune response. The unique ability of IL-3 to convert the Th2-dominant microenvironment into a Th1-dominant microenvironment when combined with cytotoxic therapy makes it an ideal immune adjuvant cytokine to cytotoxic therapies such as radiation therapy or chemotherapy. However, it should be noted that it is difficult to form tumors using IL-3-expressing cells alone in vivoOur early studies using fibrosarcoma tumor models + TAMs, but in tumors carrying IL-3 gene (TRAMP-C1/mIL3 tumor), the iNOS-positive cells were mainly CD11b− non-TAMs. Since TRAMP-C1/mIL3-sr39tk tumors express both IL-3 and HSV-sr39tk genes, iNOS-positive cells were found in both TAMs and non-TAM cells. It is unclear how iNOS were induced in these gene-transfected tumors. Nevertheless, we observed that administration of the iNOS inhibitor, L-NAME, diminished the combined therapeutic effects of HSV-sr39tk/GCV and IL-3, but had no effect on HSV-sr39tk/GCV prodrug therapy or IL-3 immune therapy individually. This suggests that NO may play an important role in the interaction between the cytotoxic effect of the HSV-sr39tk/GCV system and the IL-3-mediated immune reaction. However, it may be just an issue of the level of NO production, since TRAMP-C1/mIL3-sr39tk tumors produced more NO than TRAMP-C1/sr39tk or TRAMP-C1/mIL-3 tumors.Another interesting finding of this study was that NO appeared to play different roles in IL-3 immune therapy and HSV-sr39tk/GCV cytotoxicity. The critical role of NO in tumor progression has been well studied. Within the tumor microenvironment, NO can be generated by macrophages, neutrophils, endothelial cells, fibroblasts, and, in certain cases, by tumor cells themselves. NO has been hypothesized to play a dual role within the tumor microenvironment. Despite its established role in anti-tumor responses 2 response into a Th1 immune response. Furthermore, we showed that the interaction between IL-3-activated macrophages and HSV-sr39tk/GCV therapy is associated with NO production by TAMs. The current study suggests a feasible strategy by which immune gene therapy in conjunction with a suicide gene system can significantly improve the efficacy of prostate cancer treatment. Importantly, prodrug-based suicide gene therapy possesses the added benefits of reduced side effects on normal tissue and increased specificity for tumor cells.In summary, we have demonstrated that co-expression of IL-3 can enhance the cytotoxic effects of HSV-sr39tk/GCV prodrug therapy, and this could convert the IL-3-directed ThFigure S1+CD25+ cells in the spleen after GCV administration as determined by flow cytometry.Percentage of CD4(TIF)Click here for additional data file.


A46R gene in a NYVAC vector expressing HIV-1 Env, Gag, Pol and Nef antigens (NYVAC-C) improves immune responses against HIV-1 antigens. This question was examined in human macrophages and in mice infected with a single A46R deletion mutant of the vaccine candidate NYVAC-C (NYVAC-C-ΔA46R). The viral gene A46R is not required for virus replication in primary chicken embryo fibroblast (CEF) cells and its deletion in NYVAC-C markedly increases TNF, IL-6 and IL-8 secretion by human macrophages. Analysis of the immune responses elicited in BALB/c mice after DNA prime/NYVAC boost immunization shows that deletion of A46R improves the magnitude of the HIV-1-specific CD4 and CD8 T cell immune responses during adaptive and memory phases, maintains the functional profile observed with the parental NYVAC-C and enhances anti-gp120 humoral response during the memory phase. These findings establish the immunological role of VACV A46R on innate immune responses of macrophages in vitro and antigen-specific T and B cell immune responses in vivo and suggest that deletion of viral inhibitors of TLR signalling is a useful approach for the improvement of poxvirus-based vaccine candidates.Viruses have developed strategies to counteract signalling through Toll-like receptors (TLRs) that are involved in the detection of viruses and induction of proinflammatory cytokines and IFNs. Vaccinia virus (VACV) encodes A46 protein which disrupts TLR signalling by interfering with TLR: adaptor interactions. Since the innate immune response to viruses is critical to induce protective immunity, we studied whether deletion of  The search for a safe and effective HIV vaccine able to elicit long-lasting protective immunity has encouraged the development of recombinant live vaccine candidates with good safety and immunogenicity profiles. The Thai phase III clinical trial RV144) using the recombinant poxvirus vector ALVAC and the protein gp120 in a prime-boost strategy and showing a 31.2% protection against HIV infection  immunomodulatory genes [B19R and B8R encoding type I and type II IFN binding proteins, respectively, increased the immune responses to HIV antigens in the mouse model [A number of MVA deletion mutants in viral immune modulators have been generated to date and tested in mice ,56,57,58C6L ), apopto12L [UDG ), enhanc41L/B16R ; or C6L/olog MVA1R; A46R, olog MVA1R; A46R, se model .A46R gene in the NYVAC-C genome has no effect on the replication capacity of the virus in CEF cells but triggers expression of immunoregulatory genes in infected macrophages. NYVAC-C also enhances, though to a lesser extent, the production of these proinflammatory cytokines and chemokines compared to NYVAC-WT indicating that the expression of HIV-1 antigens has an effect on innate immune cells. The impact on antigen-presenting cells of the expression of HIV antigens from an attenuated poxvirus vector has been previously reported by using microarray technology in human dendritic cells infected with an MVA-based recombinant virus expressing gp120 and GPN from clade B [In an effort to uncover the role of VACV genes as immune modulators and search for potential applications of these vectors in the development of optimized vaccines, in this investigation we showed that deletion of the viral TLR inhibitor  clade B .p<0.001), maintaining the polyfunctional profile observed with the parental NYVAC-C. In the memory phase, deletion of A46R gene from NYVAC-C genome improved again the magnitude of the HIV-1-specific memory CD8 T cell immune response, while both the polyfunctional profile and memory differentiation pattern observed were similar as those obtained with the parental NYVAC-C. The main phenotype of the memory response was TEM, which is of immunological relevance as this phenotype has been correlated with protection in the macaque-SIV model [Significantly, in mice immunized following a DNA prime/NYVAC boost protocol, the deletion mutant NYVAC-C-ΔA46R enhanced HIV-specific T cell immune responses. Both CD4 and CD8 T cells specific for HIV antigens were activated. In the adaptive phase, the magnitudes of the HIV-1-specific CD4 or CD8 T cell responses in the group immunized with NYVAC-C-ΔA46R were significantly higher than those obtained in the group DNA-C/NYVAC-C . In thi reduced . Since t reduced ,16,17, T reduced  and A46Rand TRAM , our finThe animal studies were approved by the Ethical Committee of Animal Experimentation (CEEA-CNB) of Centro Nacional de Biotecnologia  in accordance with national and international guidelines and with the Royal Decree (RD 1201/2005) (Permit numbers: 152/07 and 080030).2 atmosphere at 37°C. The poxvirus strains used in this work included the genetically attenuated vaccinia virus-based vector NYVAC-WT and the recombinant NYVAC-C expressing gp120 as a cell-released product and Gag-Pol-Nef as an intracellular polyprotein from the clade C CN54 HIV-1 isolate [A46R deletion mutant. Virus infections were performed with 2% NCS or FCS. All viruses were grown in primary CEF cells, similarly purified through two 36% (w/v) sucrose cushions and the virus titers were determined by immunostaining plaque assay in BSC-40 cells as previously described [African green monkey kidney cells  and primary chicken embryo fibroblast cells  were grown in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 100 IU/ml of penicillin, 100 µg/ml of streptomycin and 10% newborn calf serum (NCS) for BSC-40 cells or 10% fetal calf serum (FCS) for CEF cells. The human monocytic THP-1 cells  were cultured in RPMI 1640 medium containing 2 mM L-glutamine, 50 µM 2-mercaptoethanol, 100 IU/ml of penicillin, 100 µg/ml of streptomycin and 10% FCS. Cells were maintained in a humidified air 5% CO isolate , used asescribed . The titA46R ORF deleted, was obtained by the sequential cloning of A46R recombination flanking sequences into the plasmid pGem-Red-GFP wm, containing dsRed2 and rsGFP genes as fluorescent markers, and previously described [A46R gene (432 bp) with oligonucleotides LFA46R-Aat (GACGTCAGAGGAGTTAT-3´5´-CACGAT) (AatII site underlined) and LFA46R-Xba (TCTAGATTATTTTGCTGAG-3´5´-CGTATG) (XbaI site underlined). This left flank was digested with AatII and XbaI and cloned into plasmid pGem-Red-GFP wm previously digested with the same restriction enzymes to generate pGem-RG-LFsA46R wm (4939 bp). The repeated left flank of A46R gene (432 bp) was amplified by PCR from NYVAC genome with oligonucleotides LFA46R’-Eco (GAATTCAGAGGAGTTAT-3´5´-CACGAT) (EcoRI site underlined) and LFA46R’-Cla (ATCGATT TATTTTGCTGAG-3´5´-CGTATG) (Cla I site underlined), digested with EcoRI and Cla I and inserted into the EcoRI / Cla I-digested pGem-RG-LFsA46R wm to generate pGem-RG-LFdA46R wm (5330 bp). The right flank of A46R gene (360 bp) was amplified by PCR from NYVAC genome with oligonucleotides RFA46R-Cla (ATCGATAGGATGAATTTG-3´5´-CTGAGA) (Cla I site underlined) and RFA46R-Bam (GGATCCAGAACGGCAAC-3´5´-ATTTAA) (BamHI site underlined), digested with Cla I and BamHI and inserted into the Cla I / BamHI-digested pGem-RG-LFdA46R wm. The resulting plasmid pGem-RG-A46R wm  according to the manufacturer’s recommendations. Forty-eight hours post-infection, the cells were harvested, lysed by freeze-thaw cycling, sonicated and used for recombinant virus screening. Deletion mutant was selected from progeny virus by consecutive rounds of plaque purification in BSC-40 cells during which plaques were screened for Red2/GFP fluorescence. In the first three passages, viruses from selected plaques expressed both fluorescent proteins, while in the next two passages viral progeny from selected plaques expressed only one fluorescent marker (Red2). In the last two passages , viruses from selected plaques do not express any marker due to the loss of the fluorescent marker by homologous recombination within the repeated flanking DNA sequences. The resulting NYVAC-C-ΔA46R virus was expanded in BSC-40 cells and the crude preparation obtained was used for the propagation of the virus in large cultures of primary chicken fibroblasts (CEF) followed by virus purification through two 36% (w/v) sucrose cushions and titrated by immunoplaque assay in BSC-40 cells.The deletion mutant NYVAC-C-ΔA46R was constructed using dsRed2 and rsGFP as fluorescent markers. 3 x 10A46R flanking regions were used for PCR analysis of A46R locus. The amplification reactions were carried out with Platinum Taq DNA polymerase (Invitrogen) according to the manufacturer’s recommendations. The correct sequence of deleted A46R locus was confirmed by DNA sequence analysis.To test the identity and purity of the recombinant virus NYVAC-C-ΔA46R, viral DNA was extracted from BSC-40 cells infected at 5 PFU/cell with NYVAC-WT or NYVAC-C-ΔA46R. Cell membranes were disrupted using sodium dodecyl sulphate (SDS) followed by proteinase K treatment  and phenol extraction of viral DNA. Primers LFA46R-Aat and RFA46R-Bam spanning A46R deletion mutant, monolayers of BSC-40 cells were mock-infected or infected at 5 PFU/cell with NYVAC-WT, NYVAC-C or NYVAC-C-ΔA46R. At 24 hours post-infection, cells were lysed in Laemmli buffer, cells extracts fractionated by 8% SDS-PAGE and analyzed by Western-blot using the polyclonal anti-gp120 antibody  or the polyclonal anti-gag p24 serum  to evaluate the expression of gp120 and GPN proteins, respectively. The anti-rabbit-HRPO  was used as secondary antibody. The immunocomplexes were detected by enhanced chemiluminescence .To test the correct expression of HIV-1 antigens by the 2 atmosphere. At different times post-infection , cells were harvested by scraping (lysates at 5 x 105 cells/ml), freeze-thawed three times and briefly sonicated. Virus titers in cell lysates were determined by immunostaining plaque assay in BSC-40 cells using rabbit polyclonal anti-vaccinia virus strain WR , followed by anti-rabbit-HRPO .To determine virus growth profiles, monolayers of CEF cells grown in 12-well plates were infected in duplicate at 0.01 PFU/cell with NYCAC-C or NYVAC-C-ΔA46R deletion mutant. Following virus adsorption for 60 min at 37°C, the inoculum was removed. The infected cells were washed once with DMEM without serum and incubated with fresh DMEM containing 2% FCS at 37°C in a 5% COTHP-1 cells were differentiated into macrophages by treatment with 0.5 mM phorbol 12-myristate 13-acetate  for 24 h. The medium was changed and cells were either mock-infected or infected with 1 or 5 PFU/cell of NYVAC-WT, NYVAC-C or NYVAC-C-Δ46R. Cell-free supernatants were collected after 6 hours to quantify the concentrations of TNF, IL-6 and IL-8. The concentrations of human IL-8 (BD Biosciences) in cell-culture supernatants were measured by ELISA as previously described . TNF andCN54gp120 (pcDNA- CN54gp120) and HIV-1 CN54Gag-Pol-Nef (GPN) polyprotein (pcDNA- CN54GPN) have been previously reported [The two DNA constructs expressing the HIV-1 reported . Plasmid140-148 peptide , previously described as immunodominant epitope in BALB/c mice [The HIV-1 peptide pools Gag-1, Gag-2, Env-1, Env-2, GPN-1, GPN-2, GPN-3 and NEF were provided by the EuroVacc Foundation and were previously described . They spB/c mice , was useCN54gp120 + 50 µg of pcDNA- CN54GPN) or 100 µg of DNA-ϕ (100 µg of pcDNA) by intramuscular route (i.m.). Two weeks later, animals were immunized with 1 x 107 PFU of NYVAC-WT, NYVAC-C or NYVAC-C-ΔA46R by intraperitoneal route (i.p.). Mice immunized with sham DNA (DNA-ϕ) followed by NYVAC-WT boost were used as control group. At 10 and 53 days after the last immunization, 4 mice in each group were sacrificed and spleens processed for Intracellular Cytokine Staining (ICS) assay to measure the adaptive and memory cellular immune responses against HIV-1 antigens, respectively. Two independent experiments have been performed for the different groups.BALB/c mice (6-8 weeks old) were purchased from Harlan. For the heterologous DNA prime/NYVAC boost immunization protocol performed to assay the immunogenicity of NYVAC-C-ΔA46R deletion mutant, groups of animals (n=8) received 100 µg of DNA-C (50 µg of pcDNA- 6 splenocytes (depleted of red blood cells) were seeded on 96-well plates and stimulated during 6 hours in complete RPMI 1640 media supplemented with 10% FCS containing 1 µl/ml GolgiPlug (BD Biosciences), anti-CD107a-Alexa 488 (BD Biosciences) and 5 µg/ml of the different HIV peptide pools. At the end of the stimulation period, cells were washed, stained for the surface markers, fixed and permeabilized  and stained intracellularly using the appropriate fluorochromes. Dead cells were excluded using the violet LIVE/DEAD stain kit (Invitrogen). For functional analyses the following fluorochrome-conjugated antibodies were used: CD3-PE-CF594, CD4-APC-Cy7, CD8-V500, IFN-γ-PE-Cy7, IL-2-APC and TNF-α-PE . In addition, for phenotypic analyses the following antibodies were used: CD62L-Alexa 700 (BD Biosciences) and CD127-PerCP-Cy5.5 (eBioscience). Cells were acquired using a GALLIOS flow cytometer (Beckman Coulter). Analyses of the data were performed using the FlowJo software version 8.5.3 . The number of lymphocyte-gated events ranged between 1 x 105 and 1 x 106. After gating, Boolean combinations of single functional gates were then created using FlowJo software to determine the frequency of each response based on all possible combinations of cytokine expression or all possible combinations of differentiation marker expression. For each population, background responses detected in the non-stimulated control samples were subtracted from those detected in stimulated samples for every specific functional combination and the percentages of cells producing cytokines obtained in the DNA-ϕ/NYVAC-WT control populations were also subtracted in all the groups in order to remove the non-specific responses detected as background. Only positive responses are represented.The magnitude, polyfunctionality and phenotype of the HIV-specific T cell responses were analyzed by ICS. After an overnight rest, 4 x 10Binding antibodies to Env protein in serum were determined by enzyme-linked immunosorbent assay (ELISA) as previously described . Serum sp values of hypothesis tests [http://exon.niaid.nih.gov [For the statistical analysis of ICS data, we used a novel approach that corrects measurements for the medium response (RPMI) and allows the calculation of confidence intervals and is tests ,70. Only.nih.gov . Compari.nih.gov . All valFigure S1A46R deletion mutant in the spleen of BALB/c mice after prime/boost immunization.Profile of memory HIV-specific T cell immune responses elicited by Flow cytometry profiles of vaccine-induced CD8 T cell responses against Env pool in splenocytes from immunized animals.(TIF)Click here for additional data file.Figure S2Scheme of construction of the plasmid transfer vector pGem-RG-A46R wm.The plasmid transfer vector pGem-RG-A46R wm was obtained by the sequential cloning of A46R recombination flanking sequences into the plasmid pGem-Red-GFP wm, containing dsRed2 and rsGFP genes as fluorescent markers. NYVAC genome was used as the template to amplify the left flank of A46R gene by PCR. This left flank was digested with AatII and XbaI and cloned into plasmid pGem-Red-GFP wm previously digested with the same restriction enzymes to generate pGem-RG-LFsA46R wm (4939 bp). The repeated left flank of A46R gene was amplified by PCR from NYVAC genome, digested with EcoRI and Cla I and inserted into the EcoRI / Cla I-digested pGem-RG-LFsA46R wm to generate pGem-RG-LFdA46R wm (5330 bp). The right flank of A46R gene was amplified by PCR from NYVAC genome, digested with Cla I and BamHI and inserted into the Cla I / BamHI-digested pGem-RG-LFdA46R wm. The resulting plasmid pGem-RG-A46R wm (5660 bp) directs the deletion of A46R gene from NYVAC-C genome.(TIF)Click here for additional data file.


Here, we investigate the potential of PET-MRI tracking of 19F MRI-visible microencapsulated human mesenchymal stem cells (hMSCs) labeled with triple-fusion (TF) reporter gene in non-immunosuppressed rabbits.Exogenous stem cell therapy has shown benefits for treating peripheral arterial disease patients, who are not amenable for conventional revascularization therapy. Previously, we have demonstrated the ability of imaging-visible cell microencapsulation to overcome the challenges of poor cell retention/survival and difficulties with monitoring cell delivery success. However, in vitro cell viability. Rabbits received either intramuscular injection of PFOB-encapsulated TF-hMSCs in the medial thigh followed by intravenous administration of [18F] 9-[4-fluoro-3-(hydroxymethy) butyl] guanine ([18F]-FHBG) , or PFOB-encapsulated TF-hMSCs that were pre-incubated with [18F]-FHBG . Dynamic PET imaging (Siemens HRRT CPS Innovation) was acquired immediately or 60 min after transplantation for 30-90 min. Proton  and 19F MRIs  were obtained 1-2 days after delivery. PET images were fused with 1H/19F MR to identify the location of transplanted cells. Follow-up PET imaging was repeated within 1-14 days with intravenous or ultrasound-guided intramuscular (0.7±0.3 mCi/thigh) of administration of [18F]-FHBG. Follow-up 19F MRIs were acquired 1-2 days after PET imaging. BLI was performed 2 weeks after delivery.Bone marrow-derived hMSCs were stably transfected with a lentiviral vector encoding firefly luciferase, red fluorescence protein, and thymidine kinase. Alginate cell microencapsulation was performed using a modification to incorporate perfluorooctylbromide (PFOB). Bioluminescence imaging  was acquired before and after cell encapsulation to assess In vitro hMSC viability and transgene expression were not affected by encapsulation or [18F]-FHBG incubation as determined by BLI and live/dead cell staining (91±6%). All PFOB cap injections were identified on 19F MRI in vivo (Figure 19F MRI over 2 weeks. BLI demonstrated viable xenogeneic TF-hMSCs in PFOB microcapsules 2 week post delivery (Figure o Figure . Using Po Figure  and showo Figure . No signy Figure .We demonstrate xenogeneic MSC delivery in non-immunosuppressed large animals using novel MRI-visible microencapsulation and reporter gene labeling. PFOB microencapsulation of TF-hMSCs enables cell tracking and viability assessment using clinical PET-MRI.Funding support was provided by NIH R21/R33-HL89029 & MD-SCRFII-039


Varicella-zoster virus (VZV) causes varicella, establishes a life-long latent infection of ganglia and reactivates to cause herpes zoster. The cell types that transport VZV from the respiratory tract to skin and ganglia during primary infection are unknown. Clinical, pathological, virological and immunological features of simian varicella virus (SVV) infection of non-human primates parallel those of primary VZV infection in humans. To identify the host cell types involved in virus dissemination and pathology, we infected African green monkeys intratracheally with recombinant SVV expressing enhanced green fluorescent protein (SVV-EGFP) and with wild-type SVV (SVV-wt) as a control. The SVV-infected cell types and virus kinetics were determined by flow cytometry and immunohistochemistry, and virus culture and SVV-specific real-time PCR, respectively. All monkeys developed fever and skin rash. Except for pneumonitis, pathology produced by SVV-EGFP was less compared to SVV-wt. In lungs, SVV infected alveolar myeloid cells and T-cells. During viremia the virus preferentially infected memory T-cells, initially central memory T-cells and subsequently effector memory T-cells. In early non-vesicular stages of varicella, SVV was seen mainly in perivascular skin infiltrates composed of macrophages, dendritic cells, dendrocytes and memory T-cells, implicating hematogenous spread. In ganglia, SVV was found primarily in neurons and occasionally in memory T-cells adjacent to neurons. In conclusion, the data suggest the role of memory T-cells in disseminating SVV to its target organs during primary infection of its natural and immunocompetent host. Varicella-zoster virus (VZV) causes varicella, establishes life-long latent infection in ganglia and reactivates later in life to cause zoster. VZV is acquired via the respiratory route, with skin rash occurring up to 3 weeks after exposure. The cell types that transport VZV to skin and ganglia during primary infection are unknown. Simian varicella virus (SVV) infection of non-human primates mimics clinical, pathological and immunological features of human VZV infection. African green monkeys were infected with recombinant SVV expressing enhanced green fluorescent protein (SVV-EGFP) or wild-type SVV (SVV-wt) as a control. By visualizing SVV-EGFP−infected cells in the living animal and in tissue samples, we identified the virus-infected cell types in blood, lungs, skin and ganglia during primary infection. Our data demonstrate that during viremia, SVV predominantly infects peripheral blood memory T-cells. Detection of SVV-infected memory T-cells in lungs, in early varicella skin lesions and also, albeit to a lesser extent, in ganglia suggests a role for memory T-cells in transporting virus to these organs. Our study provides novel insights into the cell types involved in virus dissemination and the overall pathology of varicella in a non-human primate model. Varicella-zoster virus (VZV) is a ubiquitous human neurotropic alphaherpesvirus that causes varicella (chickenpox) as a primary infection and herpes zoster (shingles) upon reactivation of latent virus Most of the current understanding of VZV pathogenesis is based on experimental infection of human fetal tissue transplanted in severe combined immunodeficient mice (SCID-hu model) Simian varicella virus (SVV) produces a naturally occurring disease in non-human primates with clinical, pathological and immunological features that parallel human VZV infection The aim of the present study was to characterize the kinetics of virus infection and the cell types involved in the dissemination of SVV during primary infection. We have previously shown that infection of macaques with recombinant measles virus expressing EGFP (rMV-EGFP) facilitated the identification of the cell types involved in MV pathogenesis with unprecedented sensitivity Five SVV-seronegative adult AGMs were infected intratracheally with SVV-wt (n = 2) or SVV-EGFP (n = 3). A transient increase in body temperature was seen between 6 and 11 dpi . SVV-wt−in situ, lung tissue sections were analyzed by dual-IF staining with SVV-specific antiserum and anti-keratin, -CD3, -CD68 and -CD11c mouse monoclonal antibodies (mAbs). SVV-infected cells were readily detected in lungs at 9 dpi, but not at later times (data not shown). At 9 dpi, abundant SVVposkeratinpos lung epithelial cells were observed (posCD3pos T-cells (posCD11cpos dendritic cell (DC)-like cells displaying multiple branched projections were observed adjacent to bronchi  cells were obtained at 5 dpi, 9 dpi and at necropsy . SVV DNA load and infectious virus titers in BAL cells peaked at 5 dpi and declined rapidly thereafter . Infectipos  and CD45neg cells, most likely bronchial and alveolar epithelial cells, were detected . Most EGFPposCD45pos BAL cells were alveolar myeloid cells and T-cells  isolated at multiple dpi from SVV-infected monkeys were analyzed. SVV DNA was detected in PBMC from 2 dpi until necropsy . Viral Dat 7 dpi . Togethepos cells were detected at similar frequencies in all major PBMC subsets   . Howeverc cells) , T-cellsc cells) . Moreove T-cells  [28]. Wh T-cells . Importa T-cells . The appIn vitro infection studies on human tonsil-derived lymphocytes showed that VZV preferentially infects T-cells expressing the activation marker CD69 and skin-homing markers CCR4 and CLA pos T-cells obtained at 5 and 7 dpi were analyzed for expression of both CCR4 and the T-cell activation marker CD137, the latter marker is selectively expressed by T-cells early after recognition of their cognate antigen in vivo   . Thus, S  , peakingey test) , as mighntiserum . Dual-IF neurons . Occasio neurons . SGC for neurons . Notably neurons .Our findings in ganglia contrast with the pronounced VZV-induced histopathology of both SGCs and neurons found in VZV-infected human fetal ganglia xenografts in the SCID-hu mouse model The current study is the first to present experimental evidence  66 and ORF67 in vivo, and identical PBMC types in vitro. The recent cloning of the SVV-wt full-length genome in a bacterial artificial chromosome facilitates the generation of a potentially less attenuated recombinant EGFP-expressing SVV by inserting the EGFP gene adjacent to SVV genes dispensable for growth in vitroLike VZV, SVV is considered to spread to naive monkeys via aerosols and therefore most likely targets mucosal epithelial cells of the upper respiratory tract, although – depending on the size of the aerosols – some virus may also directly reach the lower respiratory tract Future studies on juvenile African green monkeys, infected with less-attenuated SVV-EGFP strains and via alternative inoculation routes , are warranted. Particularly, analysis of tissues obtained from infected animals euthanized shortly after primary infection are needed to unequivocally determine the early target cell types of SVV, their role in virus dissemination to the target organs affected during primary infection and the route of SVV entry into sensory ganglia ad libitum. Cages also contained sources of “environmental enrichment” such as hiding places and hanging ropes, tires and other toys. During infection, study animals were housed in HEPA-filtered, negatively pressurized BSL-3 isolator cages. Animal welfare was monitored daily and all animal handling was performed under light anesthesia (ketamine) or deep anesthesia (ketamine and medetomidine) to minimize animal discomfort. After deep anesthesia, atipamezole was administered to antagonize the effect of medetomidine. Animals were euthanized by sedation with ketamine and medetomidine followed by exsanguination.This study was performed in strict accordance with European guidelines  and Dutch legislation . The protocol was approved by the independent animal experimentation ethical review committee DCC in Driebergen, the Netherlands (Erasmus MC permit number EMC2374). Animals were housed in groups, received standard primate feed and fresh fruit daily, and had access to water Mycoplasma-free. SVV-EGFP was generated by insertion of the EGFP gene downstream from a Rous sarcoma virus promoter between SVV ORF66 and ORF67 Low-passage clinical isolates of the Delta herpesvirus strain of SVV-wt and SVV-EGFP were obtained from PBMC of acutely infected AGM and propagated less than 5 times in AGM- kidney epithelial cell line BSC-1 (American Tissue Type Culture no. CCL-26) to generate virus stocks as described 5) with SVV-EGFP–infected Vero cells (0.5–1×105), showing 70% virus-induced cytopathic effect (CPE), in 0.5 ml DMEM supplemented with antibiotics and 10% heat-inactivated fetal bovine serum (FBS) for 24 hr in 24-well plates at 37°C in a CO2-incubator. Mock-infected PBMC were similarly generated by co-cultivating PBMC with uninfected Vero cells. SVV-EGFP−infected PBMC were stained and analyzed by flow cytometry or spotted on microscope slides, fixed and stained by immunofluorescence for SVV as described below.PBMC from SVV-naive AGM were infected by co-cultivating PBMC (5×10Cercopithecus aethiops) with intraperitoneal implanted temperature transponders were inoculated intratracheally with ∼106 plaque-forming units (pfu) of SVV-EGFP  or SVV-wt  diluted in 5 ml of phosphate-buffered saline Five adult (10- to 12-year-old) SVV-seronegative AGMs . qPCR was performed in triplicate on a ABI Prism 7500 using Taqman 2× PCR Universal Master Mix (Applied Biosystems) with primers and probes specific for SVV open reading frame 21 (ORF21) and the pan-primate single-copy gene oncostatin-M (OSM) as described APC-Cy7 (clone SP34-2), CD4AmCyan (L200), CD8PerCp (SK1), CD14PE (M5E2), CD16AF647 (3G8), CD20PE-Cy7 (L27) and HLA-DRPacificBlue (L243)  to delineate SVV-infected PBMC subsets. To identify SVV-infected T-cell subtypes, PBMC from infected AGMs were stained with mAbs specific for CD3APC-Cy7 (SP34-2), CD4PacificBlue (L200), CD8MCyan (SK1), CD28APC (28.2), CD95PerCp (DX2), CCR4PE-Cy7 (1G1)  and CD137PE . T-cells were categorized into naive, central memory (CM) and effector memory (EM) T-cells based on differential expression of CD28 and CD95 (pos T-cell populations based on expression of CD4 and CD8α: CD4posCD8αneg (CD4pos), CD4neg CD8αdim (CD8dim) and CD4negCD8αbright (CD8bright)  instead of anti-CD16 mAb. Fluorescence was detected on a FACS Canto II and analyzed using FACS Diva software (BD Biosciences). At least 106 viable cells were measured to accurately identify EGFPpos cells.PBMC were either directly used for flow cytometry to detect EGFP+ cells or stained for indicated markers using fluorochrome-conjugated mAbs: CD3and CD95  [28]. In8bright) [28]. BALImmunohistochemical and immunofluorescence staining was performed using predefined optimal dilutions of primary mAbs directed against: CD3 , CD11c , CD20 , CD68 , NCAM , GFAP , keratin , α-smooth muscle actin  and rabbit polyclonal antibodies directed against GFP  and SVV nucleocapsid proteins For immunofluorescence staining, sections were incubated with secondary Alexa Fluor 488 (AF488)- or AF594-conjugated goat-anti-mouse and/or goat-anti-rabbit antibodies and mounted in Prolong Gold Antifade reagent with 4′,6-diamidino-2-phenylindole (Invitrogen) Figure S1Gating strategy for flow cytometric differentiation of bronchoalveolar lavage (BAL) cells of African green monkeys. BAL cells were gated on viable cells based on forward scatter (FSC) and sideward scatter (SSC) properties and defined as CD45neg cells or CD45pos leukocytes. CD45pos BAL leukocyte subsets were defined as follows: CD3negCD20negMHC-IIposCD14pos/dim = alveolar macrophages (AM) or dendritic cells (DC); CD20posMHC-IIpos = B-cells; CD3posT-cells; CD4negCD8αhigh = CD8bright T-cells, CD4negCD8αdim = CD8dim T-cells, and CD4posCD8αneg = CD4pos T-cells.(TIF)Click here for additional data file.Figure S2Gating strategy for flow cytometric differentiation of PBMC subsets from African green monkeys. (A) Viable lymphocytes were selected based on forward scatter (FSC) and sideward scatter (SSC) properties and PBMC subsets were defined as follows: CD3posCD16neg = T-cells; CD3negCD16pos = natural killer (NK) cells; CD3negCD14posMHC-IIpos = monocytes; CD20posMHC-IIpos = B-cells; CD3negCD20negCD14negCD16negMHC-IIpos = dendritic cells (DC). (B) AGM-specific T-cell subsets were categorized based on the expression of CD8α and CD4: CD4negCD8αhigh = CD8bright T-cells, CD4negCD8αdim = CD8dim T-cells, and CD4posCD8αneg = CD4pos T-cells. (C) Based on the differential expression of CD28 and CD95, T-cells were categorized as naive (CD28posCD95neg), central memory  and effector memory  T-cells.(TIF)Click here for additional data file.Figure S3pos and CD137pos T-cells were not preferentially infected in African green monkeys.Peripheral blood CCR4 Flow cytometric detection of EGFP expression in central memory (CM) and effector memory (EM) T-cells at 5 dpi (A) and 7 dpi (B). Gating strategy was according to (TIF)Click here for additional data file.Figure S4Memory T-cells were preferentially infected in vitro. (A) SVV-naive African green monkey peripheral blood mononuclear cells (PBMC) were infected with SVV-EGFP in vitro and stained 24 hr later for SVV proteins to show that EGFP fluorescence (green) co-localized with SVV proteins (red). Nuclei were counterstained with DAPI (blue). Magnification: 400×. (B) African green monkey PBMC were infected with SVV-EGFP in vitro and analyzed 24 hr later by flow cytometry for EGFP expression in the indicated lymphocyte subsets. Data are plotted as the frequency of EGFPpos cells within individual PBMC subsets (within subset) or as the percentage of EGFPpos cells within each lymphocyte subset relative to the total number of PBMC (absolute).  Percentage of EGFPpos cells in the indicated T-cell subsets as assessed by flow cytometry. The lymphocyte subsets were defined as described in p<0.05 by Mann-Whitney test.(TIF)Click here for additional data file.