The purpose of this paper is to report a patient with otherwise unexplained cerebellar ataxia with serum antibodies against contactin-associated protein-2 (CASPR-2) and provide a detailed description of the composition of cellular infiltrates in the cerebrospinal fluid (CSF) compared to the peripheral blood (PB). CASPR-2 antibodies strongly labeling axons of cerebellar granule neurons have recently been identified in sera from nine patients with otherwise unexplained progressive cerebellar ataxia with mild to severe cerebellar atrophy.This is a report of a single case.The study methods used were neurologic examination, magnetic resonance imaging, fluorodeoxyglucose positron emisson tomography, lumbar puncture and multicolor flow-cytometry.+ plasma cells as well as human leukocyte antigen (HLA)-DR+ CD8+ T cells suggesting that both B cells and CD8+ T cells were preferentially recruited to and activated within the CSF- -) compartment.A 23-year-old Caucasian male presented with a two-year history of a progressive cerebellar and brainstem syndrome. Magnetic resonance imaging (MRI) showed pronounced cerebellar atrophy, especially of the medial parts of the hemispheres and the vermis. Cerebral fluorodeoxyglucose positron emission tomography (FDG-PET) showed pronounced hypometabolism of the whole cerebellum. CASPR-2 antibodies were detected in the serum but not the CSF, and none of the staging and laboratory assessments revealed other causes of progressive cerebellar degeneration. Interestingly, flow-cytometry of the CSF as compared to the PB showed increased fractions of CD138We confirm the association of CASPR-2 serum antibodies with cerebellar ataxia and provide the first evidence for a combined humoral and cellular immune response in this novel antibody-associated inflammatory CNS disease. In thesUsing multicolor flow cytometry, we add excessive cellular CSF and peripheral blood (PB) analysis of another patient with non-paraneoplastic cerebellar ataxia with CASPR-2 antibodies.MRI was performed on 3-tesla scanners. Diffusion weighted imaging (DWI) with calculation of ADC-map, axial and coronar T1-SE before and after application of gadolinium, axial and coronar FLAIR-, axial and saggital T2-FFE- and T2-TSE sequences were performed.Flow cytometry was performed on a NaviosTM Flow Cytometer  and results were analyzed using the Kaluza Software 1.1  as previously described . Referen-3), an intrathecal IgG (35%) and IgM (10%) synthesis and four CSF-specific oligoclonal bands. Glucose and lactate levels were normal.A 23-year-old Caucasian male complained of progressive imbalance of gait, slurred speech, tremor of the upper and lower legs, and double vision two years prior to admission. Severe pancerebellar and brainstem dysfunction was evident in the neurological examination. An initial cerebral magnetic resonance imaging (MRI), performed approximately six months after symptom onset, was unremarkable Figure A, C, butA computed tomography (CT)-scan of the chest and abdomen showed a nodular lesion of the apical thymus Figure F, arrow.+ HLA-DR+ T cells  and CD8+ HLA-DR+ T cells  were only slightly elevated in the PB. Likewise, the fraction of CD19+ B cells  and CD138+ plasma cells  were normal in the PB.Flow cytometry  of the p+ CD56- T cells (95%) with a low CD4/CD8 T cell ratio of 2.2 . This was due to a relatively small CD4+ T cell fraction  together with a relatively large CD8+ T cell fraction . Moreover, numbers of activated CD4+ HLA-DR+ T cells , but especially CD8+ HLA-DR+ T cells , were strongly increased in the CSF. Likewise, the fraction of CD19+ B cells  was elevated in the CSF and accompanied by strongly increased numbers of CD138+ plasma cells . However, considering CD138+ plasma cells as activated B cells only about 26% of all B cells displayed an activated phenotype.In contrast, flow cytometry of the CSF  compartment in CASPR-2 antibody associated cerebellar ataxia as suggested recently [+ T cells, it may in general be subject to a larger margin of experimental variance. Moreover, a majority of about 70% of the cytotoxic CD8+ T cells displayed an activated phenotype , whereas only a minority of about 26% of the B cells displayed an activated phenotype . Thus, we consider the activation of cytotoxic CD8+ T cells within the CSF (and putatively CNS) stronger than that of CD19+ B cells.Hence, although standard CSF analysis showed only very mild inflammatory changes, detailed cellular CSF assessment by multicolor flow cytometry clearly revealed that as compared to controls, cytotoxic CD8recently -9. HowevThe patient received intravenous methylprednisolon pulse-therapy together with plasma-exchange, which had no significant clinical effect. Three months later an immunoadsorption was applied, which, together with thymectomy, led to some deceleration of disease progression. Currently, the patient is undergoing regular immunoadsorption, but will receive rituximab and/or cyclophosphamide in case of further progression.The use of human subjects for this study was approved by the local ethics committee at the University of Münster, Germany.+ channels (VGKC) are predominantly of the IgG4 subclass supposed to reversibly disrupt the respective protein complexes leading to altered synaptic transmission and plasticity as well as neuronal excitability and axonal conduction without overt cytotoxicity [Antibodies to the complex of voltage-gated Ktoxicity ,10.+ T cells  has been observed, recently [+ T cells to neurons was not detected by histological techniques, many of these cells displayed a functionally activated phenotype , and neurons at inflammatory sites expressed major histocampatibility class I (MHC I) molecules enabling them to present autoantigens [+ T cell-mediated neurotoxcity together with antibody-mediated cytotoxicity may contribute to an increasing resistance to systemic immunotherapies at later disease stages as observed in our patient.This concept is consistent with a good response, especially to antibody-depleting therapies, and a rather strong correlation between serum antibody levels and clinical disease course, at least at early disease stages ,10. At lrecently . Althougantigens . Hence, + T cells usually recognize antigens from interior cell compartments following their MHC I-bound presentation on the surface membrane. Whether peptides derived from neuronal surface antigens, such as components of the VGKC complex, are also presented to cytotoxic CD8+ T cells in the context of MHC I molecules enabling them to clonally expand and contribute to neuronal dysfunction needs further investigation [+ T cells specific for intracelluar neuronal antigen might become activated secondary to antibody-mediated neuronal damage in VGKC complex encephalitis.Generally, both effector arms of the adaptive immune response may be activated irrespective of whether the respective neuronal antigen (or its antigenic epitope) is localized on the surface membrane or the interior cellular compartments of the neuron . Howevertigation -13. AlteWe confirm the association of serum antibodies to CASPR-2 with otherwise unexplained cerebellar ataxia of non-paraneoplastic origin. By detailed analysis of the PB and CSF, our case provides first evidence for a combined cellular and humoral immune response in this novel antibody-associated inflammatory CNS disease.Written informed consent was obtained from the patient for publication of this case report and any accompanying images. A copy of the written consent is available for review by the Editor-in-Chief of this journal.CASPR-2, Contactin-associated protein-2; CD, Cluster of differentiation; CNS, Central nervous system; CSF, Cerebrospinal fluid; CT, Computer tomography; DWI, Diffusion weighted imaging; FDG-PET, Fluorodeoxyglucose positron emission tomography; HLA, Human leukocyte antigen; LGI-1, Leucine-rich glioma inactivated 1 protein; MHC I, Major histocampatibility class I; MRI, Magnetic resonance imaging; NMDA, N-Methyl-D-Aspartate; PB, Peripheral blood; VGKC, Voltage-gated K+ channels.All authors report no competing interests. SGM has received honoraria for lecturing, travel expenses for attending meetings, and has received financial research support from Bayer, Biogen Idec, Sanofi-Aventis, Bayer Schering, Merck Serono and Teva. HW has received funding for travel and speaker honoraria from Bayer Schering Pharma, Biogen Idec/Elan Corporation, Sanofi-Aventis, Merck Serono and Teva Pharmaceutical Industries Ltd. HW has served/serves as a consultant for Merck Serono, Medac, Inc., Sanofi-Aventis/Teva Pharmaceutical Industries Ltd., Biogen Idec, Bayer Schering Pharma, Novartis, and Novo Nordisk; and receives research support from Bayer Schering Pharma, Biogen Idec/Elan Corporation, Sanofi-Aventis, Merck Serono and Novo Nordisk.NM and KSG acquired and analyzed clinical data and wrote the manuscript. CCG acquired and analyzed flow cytometry data and performed statistical analysis. SGM and HW designed the study and revised the manuscript. All authors have read and approved the final version of the manuscript.This study received no specific funding.


One of the biggest challenges for HIV vaccine design is identifying conserved HLA Class I- and Class II-restricted epitopes that would impart substantial fitness cost to the virus, thereby controlling or preventing infection. Here, we set out to develop an epitope-driven, DNA-prime/pseudoprotein-boost HIV vaccine (GAIA vaccine) composed of such epitopes.Analysis was performed in 2003 on 10,803 HIV-1 sequences, and again in 2009, on an expanded set of 43,822 sequences. These were searched for conserved 9-10-mer segments with the EpiMatrix suite of immunoinformatic algorithms. From the most highly conserved (>5% isolates) or top 1,000 scoring 9-mers, HLA Class I binding sequences and Class II immunogenic consensus sequences (ICS) were identified for vaccine design. Validation was performed in Thailand, the USA, and Mali via in vitro binding and IFN-γ ELISpot assays, using HIV-infected donor peripheral blood. HLA transgenic mice were immunized in DNA-prime/peptide-boost vaccine studies including these epitopes.Epitopes selected as described are more broadly conserved than those selected for other epitope-based vaccines . Antigenicity of 98% of ICS epitopes and Class I epitopes  was confirmed in HIV-infected subjects. Fifteen ICS peptides and 12 A2 peptides were confirmed in both Mali and the USA. Sixteen A3 peptides were confirmed weighting sequences for conservation and immunogenicity (as determined by EpiMatrix score), in vitro binding, and positive ELISpot responses. Epitope immunogenicity was validated in HLA transgenic mouse immunization studies.The GAIA vaccine approach is an effective means of triaging HIV epitope sequences to identify the most immunogenic and conserved epitope candidates across HLA allotypes. HLA transgenic mouse studies showed prototype DNA vaccines including these epitopes to be immunogenic, suggesting the epitope-rich GAIA vaccine would induce greater immunogenicity than other DNA/viral vector prime-boost vaccines in humans.


High-throughput RNA sequencing (RNA-seq) is considered a powerful tool for novel gene discovery and fine-tuned transcriptional profiling. The digital nature of RNA-seq is also believed to simplify meta-analysis and to reduce background noise associated with hybridization-based approaches. The development of multiplex sequencing enables efficient and economic parallel analysis of gene expression. In addition, RNA-seq is of particular value when low RNA expression or modest changes between samples are monitored. However, recent data uncovered severe bias in the sequencing of small non-protein coding RNA , such that the expression levels of some RNAs appeared to be artificially enhanced and others diminished or even undetectable. The use of different adapters and barcodes during ligation as well as complex RNA structures and modifications drastically influence cDNA synthesis efficacies and exemplify sources of bias in deep sequencing. In addition, variable specific RNA G/C-content is associated with unequal polymerase chain reaction amplification efficiencies. Given the central importance of RNA-seq to molecular biology and personalized medicine, we review recent findings that challenge small non-protein coding RNA-seq data and suggest approaches and precautions to overcome or minimize bias. Based on the detection of open reading frames (ORFs), entire transcriptomes are differentiated into two main RNA classes. The first comprises all protein coding messenger RNAs (mRNAs), and various subclasses of non-protein coding RNAs constitute the second ,2. AlthoThe high capacity and comparably low cost of modern deep-sequencing analysis suggest that high-throughput, small non-protein coding RNA sequencing  might be a superior tool when applied to personalized medicine. The commonly used microarray-based analysis has to compensate for differences in hybridization efficacies in order to ensure true representation of relative expression levels. Hybridization-based assays must also be sensitive enough to permit isoform-specific miRNA detection. Such requirements are sometimes hard to attain since individual miRNAs often display very different melting temperatures . FurtherHere, we review recent data indicating various sources of bias in sRNA-seq inherent to the common multiple experimental steps involved in library preparation. Experimental strategies and methods to overcome or minimize such distortion in expression levels are suggested wherever possible. Application of sRNA-seq-related methods in mRNA deep sequencing and personalized medicine are also discussed.Supplementary Data and Supplementary Table S1). The template-independent addition of oligo(C) or (A) stretches is catalyzed by Escherichia coli poly(A) polymerase. Alternative strategies of RNA end-modification rely on RNA ligation to introduce adapter oligonucleotides. RNA ligation is also applied to modify RNA 5′-ends.The analysis of full-length non-protein coding RNAs in sequencing projects requires RNA end-modification or equivalent strategies to ensure identification of native RNA termini as a precondition for cDNA construction . IrrespeE. coli poly(A) polymerase is a well-established approach  polymerase accepts only ATP as a substrate; in vitro it is reported to utilize, albeit with unequal affinity, all NTPs as substrates for transfer - and oligo(C)-tailing reactions  or plant miRNAs, is achieved by periodate treatment. The reaction relies on vicinal hydroxyl groups to oxidize sugar moieties and is accompanied by pentose ring opening. The resulting vicinal dialdehyde reacts with water and forms a chemical equilibrium with the corresponding dioxane derivative upon ring closure. The 2′-O-methyl group-containing RNAs are insensitive to oxidation and remain accessible for adapter ligation or tailing . Irrespective of the actual reason, the advice for minimizing this bias is identical and relies on the application of adapter pools to increase the likelihood of productive interactions .The chemical properties of RNA 5′-ends display structural variations. Depending on the starting material and experimental design, it may be necessary to alter the chemistry of RNA 5′-termini prior to cDNA generation. For example, neither 5′-triphosphorylated RNAs in prokaryotes nor primary eukaryotic RNA polymerase III transcripts are accessible for direct 5-adapter ligation. Similarly, RNA polymerase II-specific trimethylguanosine cap structures render RNA 5′-ends inaccessible. Thus, enzymatic pretreatment is usually conducted to avoid bias in the starting material (Supplementary Data and Supplementary Table S1). RNAs that contain monophosphorylated 5′-ends and 3′-hydroxyl groups are directly accessible to cDNA generation procedures. Consequently, unaltered RNA starting material leads to its specific enrichment (Supplementary Table S1). In contrast, triphosphorylated or 5′-capped RNAs require TAP treatment to generate 5′-monophosphorylated RNAs and to permit 5′-end ligation.Although the various chemical properties of RNA 5′-ends establish limitations for unbiased representation, they also permit specific enrichments  . Therefore, different chemistries of RNA 5′-ends not only distort sRNA-seq, but also allow for specific enrichment protocols. Subsequent analysis permits the distinction among RNA subsets, such as primary transcripts, capped RNAs and other products of RNA 5′-end processing  molecules ,74. ReceTo investigate potential influences on 5′-end modification derived from RNA secondary structure, ligation yields were compared among test sets of fully randomized RNAs. A remarkable underrepresentation of double-strand folds at 5′-ligation sites indicated strong preferences for single-stranded RNA substrates in ligation reactions conducted with T4Rnl1 .DNA-based adapter oligonucleotides perform well in RNA 3′-end ligation reactions , whereas RNA-based adapters are preferred for RNA 5′-end modification . Kineticin vitro transcribed non-protein coding RNAs and MID-modified adapters, ligation product levels  ranged from 60–70% to undetectable  experimental goal and (ii) lane capacity. According to Illumina® TruSeq Small RNA Sample Prep Kit guidance, 5 million sRNA-seq reads are considered appropriate for novel miRNA discovery within human-sized genomes. For miRNA profiling usually about 2.5 million reads are recommended. These approximations permit recalculation geared toward specific needs and requirements.et al. . Therefore, techniques to reduce the amount of reads that represent empty adapter dimers are of central importance, as they permit an increase in sequencing depth, reducing costs and ultimately enabling novel small non-protein coding RNA gene discovery.Apart from novel third-generation sequencing technologies that permit direct sequence analysis of unamplified cDNA, all commonly used protocols implement PCR amplification . In addiRecent analysis also emphasized the impact of PCR buffer compositions on PCR amplification bias . Remarkahttp://biz.yahoo.com/e/121115/hlcs8-k.html)) system, avoid PCR amplification steps in cDNA preparation and permit direct sequencing of unamplified samples  . In partThe miRNAs lack structural features such as mRNA poly(A) tails that could serve as class-specific enrichment or priming of reverse transcription . AlternaThus far, two alternative approaches are commonly utilized to reverse transcribe miRNA templates ,106. miRBecause the relative RNA G/C-content dictates primer Tm, qRT-PCRs are also strongly influenced by forward primer design, thereby affecting both qRT-PCR specificity and sensitivity. Altering the length of primer-template complementarity permits adjustment of the actual primer Tm. Conversely, in cases where RNA nucleotide composition would dictate very low melting temperatures, LNA-modified nucleotides might be incorporated into the primer sequence to increase the corresponding primer Tm . HoweverDeep sequencing is not limited to expression profiling of miRNA or other small non-protein coding RNAs. Even entire transcriptomes, including all protein coding and long non-protein coding RNAs, are subjected to high-throughput sequencing methods. Currently, as the length of single sequence runs is limited , longer RNAs require fragmentation prior to cDNA synthesis . MillionBias in general describes systematic errors that reflect method-related distortion from the truth. Bias might be detected easily when data generated by different methods are subjected to analytical comparison. Because it is systematic in nature, bias is not subject to variation in repeated experiments. Here, based on biochemical and deep sequencing data analyses, we have presented various steps in sRNA-seq protocols likely to cause severe distortions in the relative expression levels of individual sRNAs.The efficacy of RNA end-modification to permit cDNA construction depends not only on differences in strategies and reagents but also on changes in, among others, buffer compositions and additives that could increase molecular crowding. Therefore, meta-analysis of data generated with non-identical methods, reagents or even buffers is likely to be error-prone ,39,63,78In addition to the more obvious quantitative aspects of bias, there are also qualitative aspects because RNA-seq methods are also employed for RNA discovery. Individual RNAs might ‘drop out’ from detection because they are not amenable to cDNA construction under chosen conditions. Certainly, to increase the likelihood of productive RNA end-modification and increase the complexity of sRNA-seq library content, the parallel application of different strategies and reagents is required ,39,63,79The source of expression level distortion and the failure to detect certain RNA species at all, appear not to be limited to reactions of RNA/cDNA-end modification, but also apply to cDNA amplification by PCR . DiffereThe sRNA-seq approach is also attractive to medical research. Deep sequencing might permit the screening of multiple patient samples to monitor the progression of disease or treatment. Therefore, it is urgently necessary to establish rigid international standardizations to minimize distortion and to avoid misleading conclusions during RNA-seq data interpretation. In summary, qualitative and quantitative RNomics remain more challenging than anticipated.Supplementary Data are available at NAR Online.National Genome Research Network [NGFNIII 01GS0808 to J.B. and T.S.R.]; the Deutsche Forschungsgemeinschaft [BR 754/4-1 to J.B.]; USM Research University Grant [1001/CIPPT/81305 to T.H.T.]. Funding for open access charge: The licence was funded by Dr. Brosius’s research budget.Conflict of interest statement. None declared.


Dehalobacter restrictus strain PER-K23 (DSM 9455) is the type strain of the species Dehalobacter restrictus. D. restrictus strain PER-K23 grows by organohalide respiration, coupling the oxidation of H2 to the reductive dechlorination of tetra- or trichloroethene. Growth has not been observed with any other electron donor or acceptor, nor has fermentative growth been shown. Here we introduce the first full genome of a pure culture within the genus Dehalobacter. The 2,943,336 bp long genome contains 2,826 protein coding and 82 RNA genes, including 5 16S rRNA genes. Interestingly, the genome contains 25 predicted reductive dehalogenase genes, the majority of which appear to be full length. The reductive dehalogenase genes are mainly located in two clusters, suggesting a much larger potential for organohalide respiration than previously anticipated. Dehalobacter restrictus strain PER-K23 (DSM 9455), is the type strain of the species Dehalobacter restrictus . Th. ThD. reb-type cytochrome , two six-subunits membrane-bound energy-conserving Ni/Fe hydrogenases (Dehre_1568-1573 and 1645-1650), and three Fe-only hydrogenases . The Fe-only hydrogenases consist of the catalytic subunit and two to three putative electron transferring subunits.Another interesting feature is the presence of genes predicted to code for eight different hydrogenases. These include three periplasmic membrane-bound Ni/Fe uptake hydrogenases, consisting of three subunits: a catalytic unit, an Fe/S cluster protein and a membrane-bound Desulfitobacterium spp., whereas Dehalococcoides mccartyi strains only have one uptake hydrogenase [Dehalococcoides mccartyi strain 195 [Desulfitobacterium spp [The presence of multiple uptake hydrogenases has also been observed in rogenase ,44,53. Train 195 , as wellrium spp ,44,55.Desulfitobacterium dehalogenans led to loss of the ability to grow using lactate or formate as electron donor and 3-chloro-4-hydroxyphenylacetate as electron acceptor, indicating that hydrogenases may play an important role in the electron transport chain to RD catalytic subunits, even when hydrogen is not used as the initial electron donor [Disrupting either one uptake hydrogenase or the six-subunits energy-conserving hydrogenase in on donor .Dehalococcoides mccartyi strain 195 was cultivated in the presence of varying concentrations of hydrogen [Dehalobacter restrictus strain PER-K23 still needs further studies.The role of the six-subunit hydrogenase complexes are still poorly understood. It has been speculated that they play a role in generating low potential electrons for OHR by reverse electron flow. However, this was considered as unlikely in one study where hydrogen . The exaDesulfitobacterium hafniense strains Y51 and DCB-2 both contain genes predicted to encode a full Wood-Ljungdahl pathway, and strain DCB-2 has been shown to fix CO2 [Dehalococcoides mccartyi strains have been shown to contain partial Wood-Ljungdahl pathways, but its exact role in the metabolism of these organisms remains unclear [The genome also encodes an intact Wood-Ljungdahl pathway (Dehre_0130-0155 and 2348-2351). The presence of a whole or partial Wood-Ljungdahl pathway has been observed in other OHRB. The closely related  fix CO2 ,44. The  unclear ,58.D. restrictus contains 72 genes annotated as encoding transposases or inactive derivatives thereof, whereas it only contains few phage-associated genes despite the lack of a CRISPR phage immunity system.The genome of Dehalobacter restrictus strain PER-K23 are motile [Geobacter, some members of this genus have been shown to be OHRB. Chemotactic behavior towards organohalides has, however, not been described for Geobacter spp [Cells of e motile . The gencter spp -61.Dehalobacter restrictus strain PER-K23 is attracted by organohalides in a chemotactic manner. The complete genome sequence of Dehalobacter restrictus strain PER-K23, the type strain of the genus Dehalobacter, represents a significant leap towards understanding the physiology, ecology and evolution of this specialized organohalide respiring group of bacteria. Current work focuses on obtaining a deeper understanding of the expression and regulation of the RDH genes, and thereby expanding the known organohalide substrate range of this organism. Shot-gun proteome analysis will aid in deciphering the metabolism of D. restrictus strain PER-K23 and allow generation of refined genome scale metabolic models of these dedicated degraders.The presence of an unexpectedly large number of putative RDH encoding genes suggests a far larger potential for use in bioremediation than previously anticipated, especially if


Diffuse large B-cell lymphoma (DLBCL), the most common form of non-Hodgkin Lymphoma, comprises a heterogeneous group of diseases that can be classified into at least three different entities with distinct gene expression signatures. Among these, the activated B cell-like (ABC)-DLBCLs are the most resistant to current standard immune-chemotherapy regimens . DevelopEfforts to identify recurrent somatic mutations in ABC-DLBCLs have revealed common biological themes despite their considerable genetic heterogeneity. Most notably frequent activating somatic mutation in B-cell receptor (BCR) and Toll like receptor (TLR) signaling pathways, leading to constitutive NF-κB activity . Along tMALT1 gene itself is affected by translocations in certain mucosa associated lymphoid tissue lymphomas that may either lead to its deregulated expression or to gain of function activities [The tivities . More sptivities .Its location downstream of multiple lymphomagenic pathways makes MALT1 an excellent therapeutic target. Along these lines, a peptide that can bind and inhibit the MALT1 catalytic pocket (Z-VRPR-FMK) was shown to downregulate NF-κB activity, suppress proliferation and induce apoptosis in ABC-DLBCL cell lines known to contain mutations in TLR and BCR signaling components , 5. Thesin vitro and in vivo [Fortunately, two recent studies identified small molecule inhibitors of the MALT1 protease activity with potent activity against ABC-DLBCL cells both  in vivo , 7. One  in vivo . Irrever in vivo . In cont in vivo  suggestiInhibition of the BCR signaling mediator BTK (Bruton Tyrosine Kinase) has emerged as another promising therapeutic approach for ABC-DLBCLs. The irreversible BTK inhibitor Ibrutinib has potent activity in pre-clinical models and promising activity in early phase clinical trials . However


Postnatal transmission via breastfeeding is a leading cause of infant HIV infection in the developing world. However, only a small minority of breastfed infants born to HIV-infected women become infected. As a genetic bottleneck severely restricts the number of postnatally-transmitted variants, genetic or phenotypic differences in the virus Envelope (Env) may play a role in its ability to breech the mucosal barrier in the infant gastrointestinal tract.We examined the biologic properties of HIV Env pseudoviruses cloned from breast milk of postnatally-transmitting mothers (n=14 viruses), clinically-matched nontansmitting mothers (n=16 viruses), and early viruses from postnatally-infected infants (n = 6).There was no difference in epithelial cell attachment, internalization, or gp120 interaction with the putative HIV epithelial cell receptor, galactosylceramide, between milk HIV Env variants from transmitting and nontransmitting mothers. Similarly, there was no difference in the efficiency of milk Env variants to bind to monocyte-derived dendritic cells (DC). However, there was trend towards more efficient DC-mediated trans-infection of CD4-expressing target cells by milk Env variants of transmitting women compared to those of non-transmitting women (p = 0.06). Moreover, early infant Env variants were more efficiently transferred from DC than milk variants of nontransmitting women (p = 0.0009). The high-efficiency DC trans-infection was not attributable to higher infectivity or fusion efficiency of the postnatally-transmitted viruses. Infant Env variants were more sensitive to neutralization by broadly- neutralizing antibodies  than Env variants from milk of nontransmitting women. In addition, Env variants from transmitting and nontransmitting mothers were equally-sensitive to neutralization by autologous plasma.While resistance to broadly-neutralizing antibodies does not appear to be a defining feature of postnatally-transmitted Env variants, efficient HIV Env co-interaction with DCs and CD4-expressing target cells may be required for postnatal HIV transmission via breastfeeding.


Senile plaques and neurofibrillary tangles (NFTs) are characteristic hallmarks of the neuropathology of Alzheimer’s disease (AD). Numerous drug discovery efforts aimed at reducing the production or enhancing the clearance of amyloid-beta peptides, the principal component of senile plaques, have so far provided mixed results in late stage clinical development. In contrast, drug discovery programs aimed at modulating NFT pathology have been relative sparse although they present a substantial opportunity beyond AD since NFT pathology is also a major cause of tauopathies. NFTs are composed of aggregates of the microtubule associated protein, tau. Tau undergoes a variety of posttranslational modifications that can regulate its aggregation state. One such modification is the addition of O-linked N-acetylglucosamine (O-GlcNAc) moieties to serine and threonine residues by the O-linked N-acetylglucosaminyltransferase (OGT). O-GlcNAcylation of tau is a highly dynamic process driven by the addition of O-GlcNAc moieties by OGT versus the removal by O-linked-N-acetylglucosaminidase (O-GlcNAcase). Recently, it was shown that increasing O-GlcNAcylation of tau in the brain using the O-GlcNAcase inhibitor Thiamet G could decrease tau pathology in transgenic mice and stabilise against aggregation. Although Thiamet G provided the initial conceptual data in tau transgenic mice this compound is a carbohydrate substrate mimetic and as such has poor CNS drug properties. Given these limitations we initiated a drug discovery program to identify structurally distinct O-GlcNAcase inhibitor scaffolds with CNS drug properties.in vivo pharmacodynamic response to chemically optimized inhibitors was assessed using wild-type and JNPL3 tau transgenic mice.To identify novel inhibitors of O-GlcNAcase we performed a high throughput screen against recombinant human enzyme. Subsequent medicinal chemistry was initiated to optimize the potency and pharmacokinetic properties of the hits. The Novel and selective non-carbohydrate inhibitors of O-GlcNAcase were identified and optimized. Focusing on a series with a particularly good CNS profile we synthesized compound A which inhibited recombinant OGA with an IC50 of 155 nM and exhibited > 190-fold selectivity over the related enzyme, Hexosaminidase A. Single oral administration of compound A to wild-type mice resulted in a dose-dependent increase in total protein O-GlcNAcylation in the brain with a minimal effective dose between 3-10 mg/kg. Sub-chronic administration of compound A to JNPL3 tau transgenic mice resulted in an approximate 6-fold increase in the levels of O-GlcNAcylated tau in the brain as detected with our proprietary O-GlcNAc tau antibody.We have identified novel, selective and highly brain penetrant O-GlcNAcase inhibitors. These compounds have a unique non-carbohydrate backbone and show a robust pharmacodynamic response in preclinical animal models with a minimal effective dose between 3-10 mg/kg. Further chemical optimization to yield a molecule suitable for preclinical proof-of-concept in tau transgenic mice and to identify a clinical candidate for human testing is on-going.


Hepatitis B virus-associated hepatocellular carcinoma (HBV-HCC) and hepatitis C virus (HCV)-HCC are the main indications for liver transplantation. We compared differences in survival outcomes between these two conditions.The China Liver Transplant Registry (CLTR) contains data collated from all transplants performed in 86 liver transplantation centers across China. We analyzed CLTR data from January 1999 to December 2010. In all, 7,658 patients  were included in this study. Clinical characteristics were compared between the HBV-HCC and HCV-HCC groups; Kaplan–Meier analysis was used to calculate the overall, tumor-free and hepatitis-free survival rates. The 1-year, 3-year and 5-year overall survival was significantly higher in HBV-HCC recipients than in HCV-HCC recipients . The corresponding tumor-free survival rates  and hepatitis-free survival rates  were both superior in HBV-HCC recipients (both P<0.001). Multivariate analyses identified hepatitis, preoperative alpha-fetoprotein (AFP) level, size of largest tumor, number of tumor nodules, TNM stage, vascular invasion and preoperative model for end-stage liver disease (MELD) score as independent predictors of overall, tumor-free and hepatitis-free survival.Survival outcomes after liver transplantation were significantly better in HBV-HCC patients than in HCV-HCC patients. This finding may be used to guide donor liver allocation in transplantation programs. Hepatocellular carcinoma (HCC) resulting from chronic hepatitis B virus (HBV) and/or hepatitis C virus (HCV) infection is an important cause of liver disease worldwide Liver transplantation is a life-saving therapy for HBV-infected and HCV-infected patients with HCC. However, recurrence of HCC and hepatitis after liver transplantation has long been recognized in both hepatitis B and hepatitis C patients, which makes these infections the most important risk factors that determine prognosis and outcome after liver transplantation The differences between HBV and HCV infections may result in different outcomes after liver transplantation. There are currently a number of studies that have been published regarding the different clinical characteristics of HBV-associated HCC (HBV-HCC) and HCV-associated HCC (HCV-HCC) We therefore analyzed the China Liver Transplant Registry (CLTR) data to evaluate clinical characteristics and compare survival rates after liver transplantation between recipients with HBV-HCC and those with HCV-HCC.Ethical approval was obtained from Committee of Ethics in Biomedical Research of Zhejiang University. Written informed consent was obtained from all participants.http://www.cltr.org/), which was initiated by the joint effort of the 20 most reputable liver transplant centers across the country in February 2005 and authorized as the only national liver transplant registry in Mainland China by the Ministry of Health in May 2008. Its database warehouse is administered by Center of Study for Liver Disease, Queen Mary Hospital, Department of Surgery, The University of Hong Kong.The research design was hospital-based and retrospective, with all cases being well evaluated. The research was approved by the CLTR  without symptoms, patients with HCC recurrence after primary liver resection, patients who underwent liver transplantation after interventional therapy for HCC, patients with other undefined tumors who underwent radiofrequency ablation (RFA) or transcatheter arterial chemoembolization (TACE) or other interventional therapies for HCC were included.Type of liver transplantation: Both whole cadaveric donor liver transplant recipients and living donor liver transplant recipients were included.Hepatitis B: Recipients diagnosed with “hepatitis B” according to etiology, and/or recipients who tested positive for “hepatitis B surface antigen (HBsAg)” and/or for “HBV-DNA on branched DNA (bDNA)” and/or “polymerase chain reaction (PCR)” assays were included.Hepatitis C: recipients diagnosed with hepatitis C according to etiology were included.HCC recurrence: Post-transplant patients with records for definite mentions of HCC recurrence or the date of first detection of HCC recurrence, and/or who underwent treatments for intra- and/or extrahepatic recurrence  were included.Hepatitis recurrence: The judgment of hepatitis B recurrence was on the basis of the following parameters included in the CLTR database during postoperative follow-up: a) histological change was marked with hepatitis B recurrence during postoperative follow-up; b) postoperative serological status changed from HBsAg or HBV-DNA (bDNA or PCR assay) negative to positive or there was the date of the transition. Post-transplant patients who were diagnosed with hepatitis C recurrence based on post-transplant liver biopsy were also included.Using the above criteria, we excluded 11,202 patients from this study. We analyzed data pertaining to the remaining 7,658 patients who underwent liver transplantation for HBV-HCC or HCV-HCC .The 7,658 patients were divided into two groups according to the type of viral infection: the HBV-HCC group with 7,162 patients and the HCV-HCC group with 496 patients.We compared the following clinical parameters between the two groups: age, gender, vascular invasion, downstaging, number of tumor nodules, size of largest tumor nodule, sum of tumor diameters and preoperative alpha fetoprotein (AFP) level. In addition, we compared the frequency of major postoperative complications between the two groups, including biliary and vascular complications, postoperative infections, renal failure and graft dysfunction.The antiviral therapeutic schedules for hepatitis recurrence were mainly judged by the date of commencement of antiviral therapy with lamivudine, entecavir, adefovir, or telbivudine in the “postoperative data-recipient hepatitis serological status” column in the CLTR database. The above four antiviral drugs are all used to prevent hepatitis B. As for postoperative hepatitis C antiviral prevention programs, CLTR database does not include related drugs in a short time.Patient survival was assessed using the Hangzhou criteria, which are as follows: patients without macrovascular invasion who have one of the two following items: (a) total tumor diameter less than or equal to 8 cm; (b) total tumor diameter more than 8 cm, with histopathologic grade I or II and preoperative AFP level less than or equal to 400 ng/ml, simultaneously. We have previously shown that the Hangzhou criteria are similar to the Milan criteria for defining good prognosis The judgment for hospital mortality of the transplant patient is based on the postoperative information. In the CLTR database, the cases marked with hospital mortality have following features: a) the last follow-up date = the date of hospital discharge = the date of death; and/or b) the last follow-up date = transplant date; and/or c) the last follow-up date is later than the hospital discharge date and transplant date, but only within a few days. The 1-year, 3-year and 5-year overall, tumor-free and hepatitis-free survival rates were also analyzed after excluding cases of hospital mortality.Descriptive statistics were expressed as median (interquartile range). The chi square test or Fisher test was used for univariate comparisons where appropriate. For univariate survival analysis, plots were created and comparisons were made using the Kaplan-Meier method. The multivariate Cox proportional hazards regression analysis was used to identify predictors of recipient survival. Differences were considered statistically significant at P≤0.05. All statistical analyses were performed using SAS software, version 9.2.The patient profiles and overall characteristics are listed in No significant differences existed in the rates of postoperative biliary and vascular complications and infections. However, compared to the HBV-HCC recipients, the HCV-HCC recipients showed a high incidence of renal failure and graft dysfunction , 26.39% of HBV-HCC patients  and 9.07% of HCV-HCC patients (45/496) (P<0.001). In all, 186 recipients developed hepatitis recurrence, including 173 patients with hepatitis B and 13 with hepatitis C (P = 0.774). In the HBV-HCC group, HCC recurrence was significantly more common in patients with recurrent hepatitis B  than in patients without recurrent hepatitis B . However, in the HCV-HCC group, no significant difference was observed in the incidence of HCC recurrence between patients with recurrent hepatitis C  and those without recurrent hepatitis C .The 1-year, 3-year and 5-year overall survival rates  were significantly higher in the HBV-HCC group than in the HCV-HCC group . The corresponding tumor-free survival rates  were also significantly higher in the HBV-HCC group than in the HCV-HCC group . In addition, the corresponding hepatitis-free survival rates were significantly higher in the HBV-HCC group  than in the HCV-HCC group .Pre-transplant status, as judged by number of tumor nodules, size of largest tumor, preoperative AFP level and neoadjuvant treatment, significantly differed between the HBV-HCC and HCV-HCC groups, and may have influenced the survival rates. We therefore repeated the survival analysis for only recipients who satisfied the Hangzhou criteria. HCC recurrence was observed in 16.98%  of all patients who met the Hangzhou criteria, and 17.95%  of HBV-HCC patients and 5.56% (13/234) of HCV-HCC patients who met the Hangzhou criteria (P<0.0001). Moreover, hepatitis recurrence occurred in 80 (2.66%) recipients who met the Hangzhou criteria, including 69 (2.49%) in the HBV-HCC group and 11 (4.70%) in the HCV-HCC group (P = 0.043).Moreover, among the patients who met the Hangzhou criteria, the 1-year, 3-year and 5-year overall survival rates were significantly higher in the HBV-HCC group  than in the HCV-HCC group  in the HBV-HCC group and 6.25% (31/496) in the HCV-HCC group (P = 0.0616). Survival analysis was repeated after excluding cases of hospital mortality, and results similar to those shown above were obtained (data not shown).Although multiple predictors of survival were identified using univariate analysis , the impHBV-HCC and HCV-HCC are the main indications for liver transplantation. The early outcomes of liver transplantation for viral hepatitis-related HCC were unsatisfactory We found that the overall, tumor-free and hepatitis-free survival rates after liver transplantation were significantly worse in HCV-HCC patients than in HBV-HCC patients. In our patients, the pre-transplant status of recipients with HBV-HCC differed considerably from that of recipients with HCV-HCC. The HBV-HCC patients tended to be younger. Comparisons of HBV-HCC and HCV-HCC patients who underwent liver transplantations in the USA, Japan and Italy have revealed similar findings We also found a higher male/female ratio, greater size of largest tumor and higher AFP level in the HBV-HCC group, and a greater number of tumors per patient and downstaging treatments in the HCV-HCC group. These clinicobiological differences, which have also been observed in other studies In our study, postoperative graft dysfunction was more common in the HCV-HCC group; this finding is consistent with the results of Waki et al. In our analysis, the overall, tumor-free and hepatitis-free survival rates were all significantly better in the HBV-HCC group, both when the entire cohort was considered and when only patients meeting the Hangzhou criteria were considered. These findings demonstrated that survival after liver transplantation did differ between recipients with HBV-HCC and those with HCV-HCC, regardless of differences in pre-transplant status. In addition, the multivariate analysis also identified type of viral hepatitis as an important predictor of survival, implying that the difference in virus infection has significantly affected post-transplant survival and recurrence.The differences in survival between the HBV-HCC and HCV-HCC groups may largely attribute to two factors. First, differences in the prophylactic regimens for hepatitis recurrence between HBV and HCV might lead to differences in graft survival between these two groups of recipients, and therefore to different overall survival. Second, differences in the mechanisms of hepatocarcinogenesis between HBV and HCV may lead to different clinical profiles as mentioned above and affect post-transplantation recovery and HCC or hepatitis recurrence.Our findings demonstrate that the differences in survival rates after liver transplantation between the HBV-HCC and HCV-HCC groups were attributable to not only differences in tumor recurrence rates but also differences in hepatitis recurrence rates. Hepatitis may recur because of intra- and extrahepatic viral reservoirs Our study has some limitations. First, as the study was not randomly assigned and included data from transplant centers all around China, the heterogenous nature of the data due to various criteria, definitions, as well as protocols among different centers - including definitions of HBV recurrence, antiviral therapy, post-transplant screening procedures for HCC, etc. - may have confounded our analysis. Second, because this was a clinical research study, which lacks the strong evidence of basic research studies, we could not fully explain the mechanisms underlying hepatitis or tumor recurrence that probably led to differences in survival between the HBV-HCC and HCV-HCC patients. Thirdly, the large number of missing data in the variable‘sum of tumor diameter’ may result in a potential bias in statistical analysis. This may be largely due to the factor that many recipients have diffuse tumor nodules, especially for those exceeding Hangzhou criteria, making it difficult to access the parameter accurately in reality. Fourthly, the relative short follow-up period (median of about1 year) makes it difficult to invest the long-term effects of different viruses on liver transplant recipients. Despite these limitations, our study represents the most comprehensive assessment of survival after liver transplantation in HBV-HCC and HCV-HCC patients to date, because we used data from the CLTR, which contains information on all liver transplants performed in China. Therefore, our results remain important in the development of donor liver allocation in patients with different types of viral hepatitis.In conclusion, HCV-HCC patients have significantly worse overall, tumor-free and hepatitis-free survival after liver transplantation than HBV-HCC patients. Liver transplant recipients with HCV-HCC tended to be older and developed postoperative graft dysfunction and renal failure more frequently than recipients with HBV-HCC. Given the scarcity of donor livers, our findings might be considered to guide the development of future donor liver allocation protocols in transplantation programs, to take recipients with the same secondary diagnoses but with different primary diagnoses/etiologies into different considerations, in order to benefit those candidates who might obtain relatively superior survival rates more and thus improve overall survival in the total transplant population.File S1Univariate survival analysis for patients with hepatocellular carcinoma.(DOC)Click here for additional data file.File S2Multivariate analysis for patients with hepatitis B virus or hepatitis C virus -associated hepatocellular carcinoma.(DOC)Click here for additional data file.


Circulating monocytes have been demonstrated to relocate to the intestinal mucosa during intestinal inflammation, but the underlying mechanisms remain poorly understood. Here, we have investigated a subpopulation of blood monocytes expressing high levels of HLA-DR, CCR9 and CCR7 in patients with inflammatory bowel disease (IBD).+HLA-DRhi monocytes was monitored weekly in peripheral blood using flow cytometry. The surface phenotype and cytokine profile of these monocytes were established using flow cytometry and real-time PCR.51 patients with mild-to-severe ulcerative colitis (UC) or Crohn’s disease (CD) were included together with 14 controls. The frequency of CD14+HLA-DRhi monocytes was significantly higher in IBD patients with moderate-to-severe disease compared to healthy controls. Furthermore, these monocytes correlated to disease activity in patients with UC and CD. CD14+HLA-DRhi monocytes are defined by their high production of pro-inflammatory cytokines and their surface expression of CCR7 and CCR9.The frequency of CD14+HLA-DRhi blood monocytes were increased in patients with active inflammatory bowel disease. These monocytes exhibit a pro-inflammatory gut-homing phenotype with regards to their production of inflammatory mediators and expression of CCR9. Our results suggest that these monocytes are important in mediating intestinal inflammation, and provide potential therapeutic targets in IBD.CD14


NK cells present in the peripheral blood (PB) respond rapidly to pathogens or pathogen-infected cells by various means including cytotoxicity and release of cytokines and chemokines. In addition they modulate adaptive immunity via the interaction with dendritic cells. Decidual NK cells (dNK) are poorly cytotoxic in healthy pregnancy, both in humans and rodents, when compared to their PB counterparts. We will discuss recent findings that may contribute to answer the following questions: (i) Do dNK possess functional killing machinery in normal healthy pregnancy? (ii) If so, what are the regulatory mechanisms that negatively control this effector function? (iii) Have dNK from early pregnant uterus the intrinsic ability to kill pathogen-infected autologous maternal uterine cells and/or produce soluble factors that stimulate the anti-pathogen adaptive immune response? (iv) Do dNK undergo a receptor repertoire profile shift when they are in contact with pathogen-infected uterine cells? (v) Which pathogen-mediated signal(s) and molecular interactions subvert the inhibition of dNK cytolytic activity? More activity . dNK celactivity . This KIactivity . PB-NK mactivity . In healactivity –15. Untidecidua basalis by interacting with the CXCR3 and CXCR1 receptors expressed by the trophoblast cells  and placental growth factor (PlGF) , 17–19 ast cells . Such prst cells –28. Actist cells . Other rst cells , 30. Durst cells .Human PB-NK are important innate immune effector cells that rapidly respond to and destroy malignant or virally infected cells . PB-NK kdecidua basalis  , human ilciparum , bacterilciparum , 47 can us space , 48. We us space . Areas ous space , dNK couus space .A recent report from our lab has demonstrated that dNK play a role in the control of decidual cell hCMV infection . When exin vitro, the frequency of early in utero mother-to-child transmission of HIV-1 is low, suggesting efficient inhibitory regulatory mechanisms (Co-culture of dNK from early gestation with hCMV-infected autologous fibroblast induced a significant increased secretion of VEGF-A, sICAM, CXCL-1, IL-6, Granzyme-B, and MCP-1 by comparison with dNK in contact with uninfected similar target cells . IL-6 ischanisms . dNK secchanisms . The kilchanisms  as dNK wchanisms .dim dNK, associated with appearance of CD16 (dim CD16+ dNK subpopulation as early as 12 h after in vitro infection with Toxoplasma when compared to uninfected dNK (hCMV infection modulates dNK cell receptor repertoire ability to kill virally infected decidual cells. Such a killing requires interaction between dNK cell activating receptors and specific ligands expressed at the cell surface of autologous decidual infected cells. Co-culture of dNK for 48 h with hCMV-infected decidual fibroblasts and without addition of IL-2 or IL-15, significantly increased the number of CD56cted dNK . Furthercted dNK . It is octed dNK , highlighigh dNK subset and decrease of KIR2DL1, KIR2DL4, and LILRB1/ILT2 expression were also observed on dNK in contact with hCMV-infected decidual fibroblasts (Toxoplasma-infected dNK which correlated with enhanced cytolytic activity against human extravillous cytotrophoblast target cells (Disappearance of the NKp46roblasts . Thus, eet cells . Based oUsing Fc-chimeric proteins, we found that hCMV infection of decidual fibroblast modulated the expression of several NKR ligands: NKp30L and NKG2DL were significantly increased whereas NKp44L and NKp46L decreased . BlockinAll of these data, including those dealing with hCMV infection and summarized in Table in situ, dNK are likely to contribute to local pathogen innate immune control and clearance during the initial phase of decidua infection.Instead of being detrimental, dNK cells have positive effects in healthy human pregnancy as they promote placental development through the crucial release of angiogenic factors and attraction of extravillous trophoblast. After contact with hCMV-infected autologous decidual fibroblasts, dNK acquire cytotoxic capacity by subverting their killing inhibitory regulatory mechanisms occurring in normal pregnancy. Effector functions of dNK in pathogen-infected pregnancy require engagement of shifted repertoire of activating and/or inhibiting dNK cell receptors by specific ligands up- or down-regulated in pathogen-infected decidual cells, including HLA-E and NKG2DL. These results, if confirmed by further studies dealing with different viruses or pathogens, strongly suggest that, The author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Several reports have documented similar efficacies and tolerable toxicities of radioimmunotherapy (RIT) consolidation and rituximab maintenance after initial R-chemotherapy of follicular lymphoma. The relative merits of these two interventions are currently under discussion. We now raise the question whether both RIT consolidation and rituximab maintenance should be used together aiming to augment the results achievable with R-chemotherapy. The recent review of T.M. Illidge on radioimmunotherapy (RIT) of lymphoma highlighted the inherent potential of this particular treatment . While c®, GlaxoSmithKline, Brentford, UK), we experienced several long-lasting complete remissions with six of 12 patients (50%) reaching 10-years progression free survival without any further treatment [131I-tositumomab in relapse have been reported by another group, though at a somewhat lower rate [131I-tositumomab [131I-tositumomab [131I-tositumomab alone in the initial treatment of follicular lymphoma [We would like to elaborate further on the biological agents that have shown efficacy in treatment of follicular lymphoma. In a small series of relapsed indolent lymphoma patients treated in Switzerland with 131I-tositumomab  is the only RIT currently approved in Europe, and its successful use in consolidation treatment following chemotherapy has been well documented [®, Rituxan®, Roche Ltd., Genentech, Basel, Switzerland) maintenance treatment after R-chemotherapy was recently shown to improve the relapse-free survival in a large phase III study [cumented . On the 131I-tositumomab and 90Y-ibritumomab are targeted radiation therapies administered in combination with two injections of moderate amounts of unlabeled antibody. The latter approach is supported by long-standing evidence showing that radiotherapy may occasionally be curative when used as initial treatment for localized follicular lymphoma [Several reports have documented similar efficacies of RIT consolidation and rituximab maintenance, though these approaches have not been formally tested in a randomized trial. Both complementary treatments have moderate and/or transient side effects. The mode of action of these two added therapies, however, are different. Rituximab maintenance is an immunotherapy while lymphoma . We envilymphoma ,3,5.131I-tositumomab either alone or combined with chemotherapy, upfront or at relapse of follicular lymphoma, appears to be an important milestone. It is anticipated that this approach might afford a low recurrence rate in subsequent years, analogous to that observed after external beam radiotherapy [131I are also prone to eradicate microscopic disease, as has been shown by others and us in RIT of small-sized lung or liver metastatic disease, respectively [The 10-year progression-free survival as observed after ectively ,12.Further improved biological efficacy in NHL might be achieved by combining anti-CD20 rituximab treatment with other antibodies directed against other antigens, such as anti-CD22 or anti-CD40, utilizing humanized antibodies, or novel anti-CD20 antibodies with modified Fc domains providing increased affinity for Fc receptors and improved effector functions as discussed previously ,14.In current practice, rituximab has appropriately assumed a dominant position in treatment of lymphoma both in combination with chemotherapy and as maintenance . We now 131I-tositumomab consolidation and 4 years of rituximab maintenance. This study will assess the potential impact of administering rituximab anti-CD20 antibody with CHOP prior to anti-CD20 RIT [We acknowledge that there is currently little published data demonstrating the efficacy of anti-CD20 RIT following rituximab-containing induction chemotherapy regimens. This information gap will be partially remedied by a phase II study investigating this combined approach that has recently been piloted by SWOG in the NCT00770224 trial, which is assessing the toxicity and efficacy of R-CHOP induction chemotherapy followed by CD20 RIT , though RIT: radioimmunotherapy; R-chemotherapy :rituximab and chemotherapy; R-CHOP: combined rituximab, cyclophosphamide, doxorubicin, vincristine, and prednisone; SWOG (US): Southwest Oncology Group.OWP declares a compensated consultant and advisory role and having received honoraria from Hoffmann-LaRoche/Genentech and Spectrum Pharmaceuticals as well as having received research funding from Hoffmann-LaRoche/Genentech. FB has no competing interest to declare.Both authors express in this letter their opinion, edited and corrected this letter and approved its final version.


Helminth infection may modulate the expression of Toll like receptors (TLR) in dendritic cells (DCs) and modify the responsiveness of DCs to TLR ligands. This may regulate aberrant intestinal inflammation in humans with helminthes and may thus help alleviate inflammation associated with human inflammatory bowel disease (IBD). Epidemiological and experimental data provide further evidence that reducing helminth infections increases the incidence rate of such autoimmune diseases. Fine control of inflammation in the TLR pathway is highly desirable for effective host defense. Thus, the use of antagonists of TLR-signaling and agonists of their negative regulators from helminths or helminth products should be considered for the treatment of IBD. Crohn' s disease (CD) and ulcerative colitis (UC) are two forms of inflammatory bowel disease (IBD) that are autoimmune-like disorders characterized by chronic, idiopathic inflammation of the intestinal mucosal tissue, which causes a range of symptoms including abdominal pain, severe diarrhoea, rectal bleeding and wasting ,2. PatieCD and UC are distinguished by the tissues affected: CD can affect any region of the gastrointestinal tract in a discontinuous and transmural manner, whereas pathology in UC is restricted to the surface mucosa of the colon, in particular the rectum . CurrentGenetic studies have provided new evidence to suggest that derangements in innate and adaptive immunity result in human IBD . In 1989et al supported these results by infecting mice with Trichinella spiralis and showed that mice were protected from colitis induced by an intrarectal challenge using dinitrobenzene sulfate (DNBS) [et al. evidenced that mice infected with the tapeworm Hymenolepis diminuta, ameliorated dextran sodium sulfate (DSS)-induced colitis [et al. also shows promise in that natural exposure to helminths, such as T. suis, affords protection from immunological diseases like CD [Khan e (DNBS) . Reardon colitis . Helmint colitis ,18, but  like CD ,20. Epid like CD .An important role for TLR signaling in the pathogenesis of IBD has been established through many studies over the last decade -24. In tIt is now clear that the innate immune system comprised of TLRs and related molecules, plays a key role in the regulation of intestinal inflammation and in the recognition of invading pathogens . TLRs coTLR molecules and their downstream signaling pathways play a crucial role in selected cell types in adaptive immunity and in activating innate immune cells of the immune system ,41. GiveIn the intestine, the end result of TLR signaling is the activation of nuclear factor kappa-B (NF-κB), triggering off the induction of pro-inflammatory cytokines or interferon (IFN) response factors (IRFs), depending on the induction of type I interferons Figure . TLR-depet al. [Schistosoma japonicum eggs could alleviate TNBS-induced colitis in mice. The mechanism for this action was assumed to be due to the regulation of T-helper cell 1/2 balance and TLR4 expression. In brief, these reports make a significant contribution in that helminths will execute positive therapy in IBD by targeting the TLR signaling pathway.Negative regulation of TLRs reduces pro-inflammatory cytokine production, protecting the host from autoimmune pathogenesis . Helmintet al.  reportedThe critical roles of TLRs are to sustain the integrity of the epithelial barrier and to accelerate maturation of the mucosal immune system. Mice deficient in TLRs can develop intestinal inflammation . IECs exToxoplasma gondii on day 8 post-infection [Recent studies have demonstrated that TLR signals can influence intestinal homeostasis . One stunfection . Mucosalnfection . Hence, nfection .Gymnophalloides seoi antigen can induce mucin-related 2 (MUC2) expression by the activation of the TLR pathways in human IECs [Trichinella spiralis and Nippostrongylus brasiliensis [Intestinal parasitic infections also activate mucin hypersecretion, which is a key response of the innate immune system for intestinal homeostasis . One stuman IECs . The expiliensis . These riliensis ,65. ConsThus, the fine control of inflammation by helminths in the TLR pathway is highly feasible for effective host defense via TLR-dependent pro-inflammatory cascades triggered by parasitic infections, which must be tightly regulated to avoid severe pathology or even mortality in IBD patients .TLRs trigger an intracellular signaling cascade through the toll/IL-1 receptor (TIR)  and throSchistosoma derived lysophosphatidyl-serine contains a helminth-specific acyl chain that, through influence on TLR2, promotes the differentiation of DCs that induce regulatory T cells, which secrete the anti-inflammatory cytokine interleukin-10 (IL-10) [et al. [Schistosoma mansoni eggs may alter TLR ligand-induced activation of DCs. The broad effect of excretory-secretory products (ESP) of Fasciola hepatica on different TLR signaling regulation could be an immediate action of these antigens (Ags) on TLR expression. Falcón demonstrated that ESP was also able to affect the MyD88-dependent signaling pathway [A recent report indicates that helminth infection may alter TLR4 expression in mucosal T cells . Schisto (IL-10) . Studies [et al.  suggest  pathway . These r pathway ,75. LogiThe intestinal tract is the largest and most complex immune environment in the human body. Successful therapy for these tissues will require accurate timing and targeting the optimal location. The number of therapeutics being developed for IBD has increased dramatically over the last 2 decades because of rapid gains in our understanding of the mechanisms of inflammation .et al. [Schistosoma infection. Donnelly et al. [et al [G. seoi was employed in inducing the expression of TLR4, TLR2 and the MUC2 gene in a human IEC. MUC2 has been used in alleviating ulcerative colitis of the IBD model mouse [Disturbing TLR signaling by helminths or helminth products is expected to be a promising strategy in IBD treatment because TLR signaling can inhibit inflammatory responses in innate immune cells . van Stiet al.  demonstry et al.  showed ty et al.  is anoth. [et al , the intel mouse .F. hepatica and S. mansoni specifically disturb the MyD88-independent, TRIF-dependent signaling pathways of TLR4 and TLR3 for the modulation of the innate immune responses of their hosts [Cysteine proteases, excretory-secretory (ES) production and antigens from helminths with potential TLR ligands that may obtain more effective agonists or antagonists of a targeted function of TLRs signaling need to be considered in IBD treatment. One study showed that the major cysteine proteases secreted from ir hosts . These rSchistosoma mansoni exerts potent regulatory effects on both DCs and B cells through TLR2 regulation in patients with the autoimmune disease, multiple sclerosis. Summers et al. [Trichuris suis, to patients with CD and UC. The study suggests that it is possible to down-regulate aberrant intestinal inflammation in humans with helminthes. Local treatment using antagonists of TLR-signaling and agonists of their negative regulators from helminthes or helminth products ought to prompt consideration for treatment of IBD instead of infection with live parasites.Epidemiological, experimental and clinical data support the idea that helminths could provide protection against IBD. Correale and Farez  evidences et al.  demonstrIBD: inflammatory bowel disease; DCs: dendritic cells; Treg: regulatory T cell; TLR: Toll-like receptor; CD: Crohn's disease; UC: ulcerative colitis; DNBS: dinitrobenzene sulfate; DSS: dextran sodium sulfate; PRRs: pattern recognition receptors; GI: gastrointestinal; IECs: intestinal epithelial cells; MyD88: myeloid differentiation factor-88; IRAK: IL-I receptor-associated kinase; NF-κB: nuclear factor kappa-B; IFN: interferon; IRFs: interferon response factors; TRIF: toll/IL-1 receptor domain-containing adaptor protein inducing interferon; TIR: toll/IL-1 receptor; NOD: nucleotide-binding oligomerization domain; NLRs: nucleotide-binding oligomerization domain-like receptors; NALPs: neutrophilic alkaline phosphatases; RIP: regulated intramembrane proteolysis; MAP: mitogen-activated protein; AP1: activator protein 1; MUC2: mucin-related 2; TICAM-1: TIR domain-containing adapter molecule-1; TIRAP: TIR domain-containing adaptor protein; TRAM: TRIF-related adaptor molecule; JNK: c-Jun-N-terminal kinase; MAPK: mitogen-activated protein kinases; ERK: extracellular signal-regulated kinase; PAMPs: pathogen-associated molecular patterns; IL-1RI: type I interleukin-1 receptor; IRAKs: IL-1 receptor-associated kinases; TRAF6: tumor necrosis factor receptor-associated factor 6;. IKK: I kappa B kinase; ESP: excretory-secretory products; APCs: antigen-presenting cells; ES: excretory-secretory; SEA: soluble egg Ag.The authors declare that they have no competing interests.SMS, ZLW and MYL drafted the manuscript. XLW, XPW, YZ, FW, XLL and YXS collected material or generalized useful information from the collected material in the paper. All authors approved the final version of the manuscript.


Drosophila infected with Zygomycetes. One way the host can transiently reduce iron bioavailability is by ferritin overexpression. To study the effects of neuronal-specific ferritin overexpression on survival and neurodegeneration we generated flies simultaneously over-expressing transgenes for both ferritin subunits in all neurons. We used two independent recombinant chromosomes bearing UAS-Fer1HCH, UAS-Fer2LCH transgenes and obtained qualitatively different levels of late-onset behavioral and lifespan declines. We subsequently discovered that one parental strain had been infected with a virulent form of the bacterial endosymbiont Wolbachia, causing widespread neuronal apoptosis and premature death. This phenotype was exacerbated by ferritin overexpression and was curable by antibiotic treatment. Neuronal ferritin overexpression in uninfected flies did not cause evident neurodegeneration but resulted in a late-onset behavioral decline, as previously reported for ferritin overexpression in glia. The results suggest that ferritin overexpression in the central nervous system of flies is tolerated well in young individuals with adverse manifestations appearing only late in life or under unrelated pathophysiological conditions.Iron is required for organismal growth. Therefore, limiting iron availability may be a key part of the host’s innate immune response to various pathogens, for example, in  Drosophila melanogaster is used very widely to study basic biological questions and as a model of human disease  reflects the appearance of the bacteria visualized in situ with electron microscopy (EM) and is thought to result from its increased proliferation. The severity of wMelPop-mediated phenotypes has been shown to vary with ambient temperature and the genetic background of the host . The construction of transgenic stocks UAS-Fer1HCH and UAS-Fer2LCH have been described in detail elsewhere  used to recover the UAS-Fer1HCH, UAS-Fer2LCH recombinant on the X-chromosome. Wolbachia-infected D. simulans flies were a kind gift from Kostas Bourtzis.The X-chromosome insertion of the pan-neuronal driver lsewhere . The wMeAdult brains were prepared, cut, and stained as described . UltrathFemale adult flies collected 0-2 days after eclosion were kept throughout their lifetime at 25°C in groups of 20 individuals per vial. For elicited-escape response tests flies were adapted for at least 20 min in an environment of 25°C and 70-80% humidity illuminated by red light. They were then placed in 14 mL polystyrene Falcon tubes individually and each fly was vortexed at the highest speed for 2-3 s and was tested twice in negative geotaxis and horizontal escape assays . All fliWolbachia was determined by PCR using 16s rDNA Wolbachia-specific primers  was unknown to us when experiments were originally planned and performed. In Figure 1 our controls were derived from uninfected white (w) mutant mothers. We also tested the effect of neuronal overexpression of single subunits, as they have been previously shown to rescue degeneration associated with soluble β-amyloid overexpression . The control flies used in this experiment showed a modest two-fold increase (p < 0.05) in the time required to perform this test during the sixth week of testing. Flies overexpressing either ferritin subunit alone behaved similar to controls through the fifth week, but presented significant performance impairment  when the aging-related decline initiated at week six. The flies overexpressing both ferritin subunits from the transgenes on the third chromosome showed a modest compromise at weeks 5 and 6 . The flies overexpressing both ferritin subunits from the transgenes on the X chromosome showed a robust mobility impediment one week earlier than controls , with a dramatic tenfold increase in the time required to climb 10 cm by week six . Similar findings were found when testing horizontal escape responses, which presumably require less physical effort from individual flies . During their sixth week post-eclosion, elav-Gal4/UAS-Fer1HCH, UAS-Fer2LCH; +/+; +/+ flies could hardly walk.We used the pan-neuronal driver pression . Once weelav-Gal4/+; +/+; +/+ controls , flies overexpressing the single ferritin subunits  and those overexpressing ferritin from the third chromosomal recombinant . In stark contrast, animals overexpressing ferritin from the X-chromosome showed severe neuronal degeneration, evident throughout the brain, including the optic lobes . Furthermore, lifespan determinations, clearly distinguished between ferritin overexpressing flies derived from the third and X-chromosomal recombinants, with the latter presenting precipitous mortality during the sixth week of aging, 2 weeks earlier than controls . Therefore, in addition to the significant difference in the onset of the mobility impediment between the two genotypes of flies overexpressing both ferritin subunits, brain pathology, and reduced lifespan were also associated only with the ferritin overexpression induced from the X-chromosomal transgenes.We then prepared plastic-embedded sections of adult fly heads to monitor the neuroanatomical integrity of the brains of 40-day old flies, at the transition between weeks 5 and 6. No apparent degeneration was evident in UAS-Fer1HCH, UAS-Fer2LCH to male elav-Gal4 or to w control flies and vice-versa. UAS-Fer1HCH, UAS-Fer2LCH/elav-Gal4 flies were therefore derived from UAS-Fer1HCH, UAS-Fer2LCH or elav-Gal4 mothers, whereas UAS-Fer1HCH, UAS-Fer2LCH/+ control flies were derived from UAS-Fer1HCH, UAS-Fer2LCH, or w mothers. The results clearly show that lifespan shortening was observed only in progeny of UAS-Fer1HCH, UAS-Fer2LCH mothers . In agreement with this notion the decrease was also seen in control UAS-Fer1HCH, UAS-Fer2LCH/+ animals that do not overexpress ferritin since they do not carry the elav-Gal4 driver, but their transgene carrying chromosome is maternal in origin. Therefore, the behavioral, neurodegenerative, and reduced life span phenotypes are not associated with over-expression of the ferritin subunits, but rather with the maternal origin of the transgene-bearing chromosome.To investigate the potential cause of the differential shortened lifespan, neurodegeneration and early onset behavioral decline of the X-chromosome driven ferritin overexpression, we tested whether there was a maternal effect associated with these phenotypes. We crossed homozygous female UAS-Fer1HCH, UAS-Fer2LCH/elav-Gal4 flies revealed the presence of bacterial infections, with the characteristic morphology attributed to wMelPop . We verified the presence of Wolbachia in mothers and progeny derived from the stock bearing the UAS-Fer1HCH, UAS-Fer2LCH recombinant on the X-chromosome. We also assessed negative geotaxis on UAS-Fer1HCH, UAS-Fer2LCH/elav-Gal4 flies and in agreement with the life span experiment, we only observed a large time delay in infected individuals at 5 weeks of age . Although the severe neurodegeneration that follows wMelPop infection has been previously documented, it has been assumed that the bacterial infection leads to neuronal necrosis , consistent with the characteristic nuclear changes that accompany programmed cell death. Treatment of the UAS-Fer1HCH, UAS-Fer2LCH stock with the antibiotic tetracycline for four consecutive generations eliminated the infection . UAS-Fer1HCH, UAS-Fer2LCH/elav-Gal4 flies derived from the tetracycline-treated UAS-Fer1HCH, UAS-Fer2LCH mothers retained their brain integrity at 40 days of age  and, by qualitative observations, showed no signs of premature mobility decline and mortality.Transmission electron microscopy images from affected necrosis . Our TEMWolbachia infects arthropods and nematodes . Our experiments did not discriminate between the possibility that ferritin overexpression benefited the pathogen, encouraging its propagation, or, alternatively, that ferritin overexpression on top of pathogen infection further deprived bioavailable brain iron. Our results point to a different outcome between wMelPop and wMel infections when responding to iron deficiency, with the former killing their host more effectively when the latter appear to provide an advantage to their host  that iron limitation stops an infection  thnfection . ClearlyDrosophila glia (D. melanogaster.We previously described the effect of ferritin overexpression in all ila glia  and in tila glia . Ferritiila glia . One parila glia . No suchila glia . Our resila glia , should Wolbachia and the antibiotic treatments. Jose A. Botella and Stephan Schneuwly performed the electron microscopy analysis. Tracey A. Rouault and Efthimios M. C. Skoulakis had continuous input during each stage of the project, including during the writing of the manuscript, with Fanis Missirlis in a coordinating role.All authors designed the original set of experiments aiming to address the consequences of ferritin overexpression in neurons of wild type flies and participated in the data analysis. Fanis Missirlis generated the transgenic flies and measured the life spans. Stylianos Kosmidis performed the behavioral tests, the molecular biology to detect The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


C. albicans cell densities because substrate concentration and solubility are limiting factors in the reaction. Because mature biofilms are composed of high cell density populations we sought to develop a quantitative real-time RT-PCR assay (qRT-PCR) that could accurately assess mature biofilm changes in response to a wide variety of anti-fungal agents, including host immune cells.Fungal biofilms are more resistant to anti-fungal drugs than organisms in planktonic form. Traditionally, susceptibility of biofilms to anti-fungal agents has been measured using the 2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxyanilide (XTT) assay, which measures the ability of metabolically active cells to convert tetrazolium dyes into colored formazan derivatives. However, this assay has limitations when applied to high The XTT and qRT-PCR assays were in good agreement when biofilm changes were measured in planktonic cultures or in early biofilms which contain lower cell densities. However, the real-time qRT-PCR assay could also accurately quantify small-medium size changes in mature biofilms caused by mechanical biomass reduction, antifungal drugs or immune effector cells, that were not accurately quantifiable with the XTT assay.We conclude that the qRT-PCR assay is more accurate than the XTT assay when measuring small-medium size effects of anti-fungal agents against mature biofilms. This assay is also more appropriate when mature biofilm susceptibility to anti-fungal agents is tested on complex biological surfaces, such as organotypic cultures. C. albicans to form biofilms on prosthetic devices and mucosal surfaces is believed to be intimately associated with its ability to trigger systemic or mucosal infection , environmental  or immune cell stressors and viability was measured by the XTT or qRT-PCR assays. To quantify susceptibility to immune cell-inflicted damage we used a neutrophil-like cell line , as previously described . Brieflypofungin ,41) or HThe authors declare that they have no competing interests.ZX participated in the design of the study, performed the experimental procedures, carried out the data analysis, and drafted the manuscript. AT and HK helped in certain experimental procedures. ADB conceived the study and helped to draft the manuscript. All authors read and approved the final manuscript.


We sought to engineer a novel mouse model of polyomavirus middle T antigen (PyV mT)-mediated mammary tumourigenesis in which inducible expression of this well-characterized viral oncoprotein is coupled to Cre recombinase (TetO-PyV mT-IRES-Cre recombinase or MIC).Effective MIC mice were crossed to the mouse mammary tumour virus (MMTV)-reverse tetracycline transactivator (rtTA) strain to generate cohorts of virgin females carrying one or both transgenes. Experimental (rtTA/MIC) and control (rtTA or MIC) animals were administered 2 mg/mL doxycycline beginning as early as eight weeks of age and monitored for mammary tumour formation, in parallel with un-induced controls of the same genotypes.50 of seven days post-induction, while induced or un-induced controls remained tumour-free after one year of induction. Histological analyses of rtTA/MIC mammary glands and tumour revealed that lesions followed the canonical stepwise progression of PyV mT tumourigenesis, from hyperplasia to mammary intraepithelial neoplasia/adenoma, carcinoma, and invasive carcinoma that metastasizes to the lung; at each of these stages expression of PyV mT and Cre recombinase transgenes was confirmed. Withdrawal of doxycycline from rtTA/MIC mice with end-stage mammary tumours led to rapid regression, yet animals eventually developed PyV mT-expressing and -non-expressing recurrent masses with varied tumour histopathologies.Of the rtTA/MIC virgin females studied, 90% developed mammary tumour with complete penetrance to all glands in response to doxycycline and a TWe have successfully created a temporally regulated mouse model of PyV mT-mediated mammary tumourigenesis that can be used to study Cre recombinase-mediated genetic changes simultaneously. While maintaining all of the hallmark features of the well-established constitutive MMTV-PyV mT model, the utility of this strain derives from the linking of PyV mT and Cre recombinase transgenes; mammary epithelial cells are thereby forced to couple PyV mT expression with conditional ablation of a given gene. This transgenic mouse model will be an important research tool for identifying synthetic viable genetic events that enable PyV mT tumours to evolve in the absence of a key signaling pathway. Phosphorylation of the membrane-anchored PyV mT protein by Src family tyrosine kinases leads to the recruitment and activation of specific proto-oncogenes such as Shc and phosphatidyl inositol 3’ kinase. In recent decades, the signalling pathways initiated by these effectors have been found to be activated in several human tumours and, consequently, PyV mT model systems continue to be employed in cancer research  have been essential in understanding cellular signalling and tumourigenesis. PyV mT can readily transform cells in vivo.Transgenic expression of PyV mT in the mouse can lead to tumour formation in many epithelial tissues . PerhapsInitial attempts to study the mammary-specific role of a particular gene in the MMTV-PyV mT model involved crossing in LOXP-flanked alleles of this gene as well as an MMTV-driven Cre recombinase transgene. However, the MMTV promoter is only active in approximately 70% of the mammary epithelium, resulting in stochastic Cre recombinase expression in PyV mT tumour tissue; a selection for PyV mT-positive/Cre recombinase-negative cells arises in the developing tumour, precluding excision of the conditional allele of interest. This phenomenon has occurred in several MMTV-PyV mT/MMTV-Cre recombinase backgrounds as demonstrated by our own laboratory: conditional ablation of β1-integrin, focal adhesion kinase, c-Src or Stat3 was prevented, indicating that in each case the conditional gene was most likely essential to PyV mT-driven tumourigenesis -7  to a chemically-inducible approach has been made possible by the advent of the MMTV-reverse tetracycline transactivator (rtTA) strain used in combination with the well-established TetON system . The tetIn order to link expression of the PyV mT oncogene with that of Cre recombinase in an inducible manner, we generated a TetO-PyV mT-IRES-Cre recombinase (MIC) transgenic mouse that, when crossed to the MMTV-rtTA strain and treated with doxycycline, expresses both PyV mT and Cre recombinase from the same bi-cistronic transcript in the mammary epithelium. In the majority of experimental mice, mammary tumours develop within two weeks of induction, progress through the typical PyV mT histological stages, and metastasize to the lung. These tumours were susceptible to regression upon doxycycline withdrawal; however, recurrent tumours ultimately arose in de-induced animals. This research article details the characterization of this novel inducible model and reflects on its potential use in future studies of PyV mT mammary tumourigenesis.The MIC construct was created using the pTE-mElf5-IRES-eGFP vector (a generous gift from Dr. C. Ormandy). Briefly, after removal of the mElf5 and eGFP transgenes, PyV mT cDNA was sub-cloned between the Tet-operator (TetO) and internal ribosome entry sequence (IRES), followed by sub-cloning of Cre recombinase cDNA downstream of the IRES   in light-blocking bottles each week. Mammary tumours were detected by physical palpation and animals were sacrificed at a total tumour burden of six cubic centimetres unless otherwise specified. Material from necropsied mice was flash frozen in liquid nitrogen  or fixed in 10% neutral buffered formalin  and embedded in paraffin wax. Fixed and embedded mammary tissues were sectioned at 4 μm and either stained by haematoxylin and eosin (H&E) or processed further as indicated. H&E-stained lung sections (4 μm) were scanned with a Scanscope XT Digital Slide Scanner and for each sample the area occupied by metastases relative to the total lung area was quantified using Imagescope software . The number and type of lesions in mammary gland sections were counted similarly by analysing 10 fields within a scanned image of each sample. Inguinal mammary glands were whole mounted on glass slides and processed for H&E-staining as described . For resFlash frozen mammary tissues were lysed as in  and 20 μSections of paraffin-embedded samples were immunostained as described previously . AntigenOCT-embedded sections of mammary tumours were processed and stained with X-gal as described previously .Total RNA was extracted from flash frozen mammary tumours and lung lesions using a Qiagen AllPrep DNA/RNA Mini Kit . cDNA was prepared by reverse transcribing the isolated RNA using M-Mulv Reverse Transcriptase (#M0253S), Oligo-dT(23VN) (#S1327S) and a murine RNase inhibitor (#M0314S) . Real-time quantitative PCR was performed on the cDNA using LightCycler 480 SYBR Green I Master  and run on a Roche LightCycler 480 instrument. Primers used for PyV mT were as follows: forward - 5’CCCGATGACAGCATATCCCC-3’; reverse - 5’CTTGTTCCCCCGGTAGGATC-3’. PyV mT transcript levels were normalized to GAPDH transcript levels.Hras, Kras1, Nras and Trp53:Total DNA was extracted from flash frozen mammary tumours and lung lesions using a Qiagen AllPrep DNA/RNA Mini Kit (#80204). Genomic DNA was analysed by Sanger sequencing  using the following primers to detect SNPs in known mutational "hotspots" of Hras exon2/exon3Forward - CCTTGGGTCAGGCATCTATTReverse - AAAGACATAAAGCCTCAGTGTGCKras exon 2Forward- GAAGATGAAAGTACTGGTTTCCAReverse- TGCACCTATGGTTCCCTAACAKras exon 3Forward - TCACCTTGTAAAAGATGCACTGReverse - AAAACAGGAATTCTGCATACTTGANras exon 2Forward - AGTGGAAGGCCACGTGTATCReverse - GGAAATCCTCAGTAAGCACGANras exon 3Forward - TGCATGCGTGTGATTATGTATGReverse - AAAAGTTGTATGTTTCCTAAGTCCATrp53 exon 2Forward - ACGTGGTTGGTTACCTCTGCReverse - GATACAGGTATGGCGGGATGTrp53 exons 3/4Forward - CCAGCCTGGGATAAGTGAGAReverse - GCTAAAAAGGTTCAGGGCAAATrp53 exons 5/6Forward - TGGTGCTTGGACAATGTGTTReverse - CCCTTCTCCCAGAGACTGCTTrp53 exons 7/8/9Forward - GTAGGGAGCGACTTCACCTGReverse - AAGACCTGGCAACCTGCTAATrp53 exon 10Forward - GTTGGGAACCAACTTTCAGAReverse - TGTCCCTCATACCCCTTAACATrp53 exon 11Forward - CAGAAGTATTCCAGTGTGTTCTGTGReverse - CTACTCAGAGAGGGGGCTGAThe Proteome Profiler Mouse Phospho-receptor tyrosine kinase (RTK) Array Kit from R&D Systems/Cedarlane Corp., Burlington, ON, Canada (#ARY014) was used according to the manufacturer’s instructions. Flash frozen mammary tissues were lysed as in  and 200 50 of 40 days of age [Polyomavirus middle T antigen (PyV mT) and Cre recombinase cDNAs were sub-cloned into a pTE vector containing an internal ribosome entry sequence (IRES) to produce a TetO-PyV MIC transgene  remained tumour-free after one year post-induction.Mammary gland masses were detected in rtTA/MIC mice as early as four days post-induction, with 74.4% (29/39) of the cohort developing multifocal tumours within 16 days of induction . Histological analysis of mammary glands and tumours from these animals revealed the presence of all previously characterized stages of PyV mT tumourigenesis, ranging from hyperplasia, to MIN/adenoma, and finally to early and late carcinoma . These lysates were positive for PyV mT expression; Cre recombinase was also detected in rtTA/MIC tumours, although it was lowly expressed in mammary gland lysates which likely have relatively less epithelial content as indicated by E-cadherin levels Figure B. InducelacZ gene is downstream of a LOXP-flanked STOP cassette [lacZ transgene, which can cleave the compound X-gal into an insoluble blue-coloured product. The mammary epithelium of rtTA/MIC/GTRosa tumour sections turned blue upon staining with X-gal, indicating that Cre recombinase had been expressed and active in these cells  in which the cassette . The pres Figure . This ouOne of the most useful features of the original constitutive PyV mT model is the ability of the mammary tumours to effectively metastasize to the lungs, an organ that is a common site of distal lesions in the human disease. To determine if rtTA/MIC mammary tumours were capable of forming pulmonary metastases, we examined sections of the lung lobes from animals that had reached similar end-stage tumour burdens. All tumour-bearing mice presented with lung metastases, albeit to varying degrees, with some lungs harbouring only a few small lesions while others were made up almost entirely of secondary tumour tissue Figure A, B. TheAnother important feature of inducible systems is the capacity to “turn off” the oncogene by withdrawal of the inducing agent; one can then evaluate whether tumours regress and if they have the potential to recur in the absence of transgene expression. To test this in our model, doxycycline treatment was discontinued for a cohort of rtTA/MIC mice bearing end-stage mammary tumours. Upon de-induction of PyV mT expression the tumours began to shrink rapidly Figure A. By 10 The morphology of the recurrent masses exhibited striking intra- and intertumoural heterogeneity, with individual tumours differing among animals and among tumours from the same mouse . This does not impact on overexpression of PyV mT protein in the inducible system, as evidenced by the strong levels detected by immunoblot , ErbB2 and platelet-derived growth factor receptor (PDGFR) β  and of Trp53 that are orthologous to those frequently mutated in human cancers. Notably, mutations in these genes have been previously identified as potential driving events in the recurrence of other doxycycline-driven transgenic mouse tumour models [Trp53 (R245C) in one recurrent mammary tumour . The affected residue corresponds to R248 of human TP53, which is frequently mutated in human cancer. This result suggests that mutations in known tumour suppressor genes can occur in recurrent rtTA/MIC mammary tumours. However, at least in the case of Trp53, they may be relatively infrequent (1/10 samples examined). A more comprehensive mutational analysis  of doxycycline-dependent and recurrent rtTA/MIC mammary tumours could be undertaken in the future to provide additional information on cooperating genetic events during tumour recurrence.To further analyze the potential occurrence of cooperating oncogenic events during the process of doxycycline-independent recurrence of rtTA/MIC mammary tumours, we sequenced regions of the three Ras genes  and treatment (de-induction). The TetO-PyV mT-IRES-Cre recombinase (MIC) strain generated in our laboratory not only utilizes inducible expression of the PyV mT oncoprotein, but incorporates Cre recombinase-mediated genetic changes as well, due to the bi-cistronic linking of these transgenes. In this study, we have chosen a mammary epithelial-specific rtTA (MMTV-rtTA) to characterize a new model of mammary tumourigenesis driven by the MIC transgene.The development of inducible transgene systems for in vitro and hypothetically essential for tumourigenesis in vivo - is achievable in the rtTA/MIC model, leading to abrogation of tumour formation in animals induced for at least 200 days [Induction of rtTA/MIC mice with doxycycline led to the rapid onset of invasive mammary tumours in the majority of animals. MIC-expressing lesions developed in a stepwise fashion that resembled the progression observed in the constitutive MMTV-driven model of PyV mT tumourigenesis (MMTV-PyV mT). Notably, earlier lesions displayed more uniform staining of PyV mT and Cre recombinase protein and at a higher intensity than carcinoma stages, suggesting a potential dampening of transgene expression once the tumour has progressed Figure A. There 200 days  .Click here for fileFigure showing multiple histopathologies from recurrent tumours and expression of PyV mT transcript.Click here for fileFigure showing RTK signalling in doxycycline-independent masses in rtTA/MIC mice.Click here for file


In patients with STEMI, CMR parameters such as infarct size, MO and the MSI add incremental prognostic value above traditional outcome markers.Infarct size, microvascular obstruction (MO) and the myocardial salvage index (MSI) assessed by cardiac magnetic resonance imaging (CMR) have been shown to be associated with adverse clinical outcome. However, in daily clinical routine prognosis after STEMI is evaluated using simple assessable parameters such as ST-segment resolution, enzymatic infarct size, pre- and post-PCI TIMI-flow, left ventricular ejection fraction (LV EF) and TIMI-risk score. Up to date it remains unclear if the assessment of infarct size, MO and MSI by CMR adds incremental prognostic value in comparison to the traditional outcome markers.STEMI patients reperfused by primary angioplasty (n=392) within 12 hours after symptom onset underwent CMR 3 days after the index event (interquartile range [IQR] 2-4). Infarct size and MO were measured 15 min after gadolinium injection. T2-weighted and contrast-enhanced CMR was then used to calculate MSI. In all patients ST-resolution, pre- and post-PCI TIMI-flow, as well as the enzymatic infarct size using creatine kinase-MB were recorded. All patients underwent transthoracic echocardiography for the assessment of LV EF. Finally the TIMI-risk score was calculated. Clinical follow-up was conducted after 19 months (IQR 10-27). The primary endpoint was defined as a composite of death, myocardial reinfarction and congestive heart failure. C-statistics were computed to assess the prognostic value of different models regarding the occurrence of the primary endpoint.Comparison of a model including only traditional outcome markers  to a model including only CMR parameters  revealed an increase of the corresponding c-statistics . Comparison of model 1 to a model including the CMR parameters on top of traditional outcome markers (model 3) demonstrated an incremental prognostic value of infarct size, MO and MSI .In patients with STEMI, CMR parameters such as infarct size, MO and the MSI add incremental prognostic value above traditional outcome markers.None.