PLCD1 and PLCE1, were successfully validated in CRC biopsies (n = 70) and cell lines (n = 19) by quantitative analyses. The repression of both genes was found associated with KRAS mutations , and we observed that microsatellite stable carcinomas with reduced PLCD1 expression more frequently had TP53 mutations (P = 0.002). Promoter methylation analyses of PLCD1 and PLCE1 performed in cell lines and tumor biopsies revealed that methylation of PLCD1 can contribute to reduced expression in 40% of the microsatellite instable carcinomas. In conclusion, we have identified significantly deregulated pathways in CRC, and validated repression of PLCD1 and PLCE1 expression. This illustrates that the GSEA approach may guide discovery of novel biomarkers in cancer.Colorectal cancer (CRC) is one of the most common cancer types in developed countries. To identify molecular networks and biological processes that are deregulated in CRC compared to normal colonic mucosa, we applied Gene Set Enrichment Analysis to two independent transcriptome datasets, including a total of 137 CRC and ten normal colonic mucosa samples. Eighty-two gene sets as described by the Kyoto Encyclopedia of Genes and Genomes database had significantly altered gene expression in both datasets. These included networks associated with cell division, DNA maintenance, and metabolism. Among signaling pathways with known changes in key genes, the “Phosphatidylinositol signaling network”, comprising part of the PI3K pathway, was found deregulated. The downregulated genes in this pathway included several members of the Phospholipase C protein family, and the reduced expression of two of these,  Colorectal cancer  ranks third in cancer incidence world-wide with an estimated 1.2 million new cases per year Messenger RNA (mRNA) expression profiling studies have provided improved understanding of the roles of several genes important in initiation and progression of cancer, including CRC 2) and generate the important second messenger diacylglycerol (DAG) and inositol 1,4,5-triphospate (IP3). Imbalance of phosphoinositide levels (PIP2 and PIP3) has been shown to induce defects in channel activity, trafficking, and normal cell polarity The importance of the canonical Phosphatidylinositol 3-kinase (PI3K) pathway in cancer has been established in numerous publications during the last decade PLCD1 (HGNC:9060) and PLCE1 (HGNC:17175), for detailed studies and compared the findings with clinicopathological data and mutation status of several genes known to be altered in CRC.By exploring gene expression of predefined biological processes and molecular circuits in two CRC and normal colonic mucosa datasets obtained using different microarray platforms, a list of pathways significantly altered by differential gene expression in CRC was identified. We selected the “Phosphatidylinositol signaling network” and two of the deregulated components therein, P≤0.05; P≤0.05) in two independently analyzed datasets, obtained from different microarray platforms, gives a risk of only 1/400 for each pathway to be included as significantly altered merely by chance. Hence, no additional correction for multiple testing is performed  that were closely related isozymes belonging to the Phospholipase C (PLC) family of proteins. PLCD1 and PLCE1, the two PLC genes in the “Phosphatidylinositol signaling system” most significantly downregulated compared with the normal mucosa samples in the HuEx and AB datasets, respectively, were chosen for further validation.The “Phosphatidylinositol signaling system” was one of the molecular pathways found to be downregulated in CRC compared to normal mucosa, ectively , Table 1PLCD1 and PLCE1 were clearly repressed in cancerous tissue samples compared with normal mucosa (P = 3×10−16 and P = 4×10−15), confirming the downregulation found in the microarray expression data (P = 2×10−10 and P = 1×10−8).From quantitative reverse transcription-PCR, ion data . All thePLCD1 and PLCE1 and KRAS mutations in CRC samples  and significantly decreased in stage III tumors (P = 0.005), compared to stage I tumors. However, this was not the case for distant metastatic disease (stage IV). The same trend was also seen for PLCD1, where expression levels were significantly decreased in stage III compared to stage I (P = 0.03). No associations were found between PLCD1 and PLCE1 expression and mutations in BRAF, PIK3CA, or PTEN. For Interestingly, there were strong associations between low expression of both  samples . FurtherPLCD1 and PLCE1 could be targets for promoter methylation since their expression levels were low in CRC compared with normal colonic mucosa samples. Both genes possess CpG islands in their promoters and their expression became upregulated in cell lines treated with epigenetic reactivating agents . Complete or partial methylation of the PLCD1 promoter was found in 79% (15/19) of the colon cancer cell lines, detected by qualitative as well as quantitative methylation-specific polymerase chain reaction . The remaining four cell lines were completely unmethylated. The methylation status of the colon cancer cell lines was confirmed by bisulfite sequencing using two non-overlapping sets of primer pairs covering altogether 69 CpG sites. There was no association between methylation and MSI-status of the cell lines. For primary carcinomas, only nine out of 70 (13%) samples were methylated, as assessed by MSP and qMSP. The majority of the tumors considered positive for methylation displayed low PMR values (median 5.69). Interestingly, eight of the nine methylated tumors were MSI, yielding a methylation frequency of 40% (8/20) in this subgroup. Furthermore, all the methylated MSI tumors had mutations in BRAF, whereas the methylated MSS tumor was wild type. For PLCE1, only one of 19 colon cancer cell lines showed promoter methylation. This was confirmed by bisulfite sequencing covering 47 CpG sites. Due to the low PLCE1 promoter methylation frequency among the cell lines, tissue samples were not subjected to methylation analyses.By exploring gene expression within predefined molecular pathways in two independent CRC transcriptome datasets, we have identified several molecular networks and components therein that are deregulated in CRC and might contribute to carcinogenesis.i.e. “Oxidative phosphorylation” was the most significantly downregulated metabolic network across both datasets, whereas metabolism of purines, pyrimidines, and N-glycans, in addition to the “Pentose phosphate pathway” were upregulated. The purine and pyrimidine metabolism were newly found to be upregulated also in adenomas compared to normal mucosa, indicating that alterations of these networks are early events in tumorigenesis The “Cell cycle” was not surprisingly the most extensively altered gene set in our analyses. Its proper regulation is essential for ensuring correct transmission of genetic information from one generation to the next, thus deregulation of several cell cycle components is defined as cancer hallmarks. Our finding is in line with several comparable studies of CRC where gene sets restricted exclusively to cancer-related pathways PIK3CA and PTEN were expressed in tumor samples, but not at significantly different levels from normal colon tissue in both datasets. We and others have shown that these genes often suffer from alterations on DNA- or protein levels rather than by changes in mRNA expression In addition to more general cancer cell traits, central circuits in CRC like MAPK-, TP53-, and Phosphatidylinositol signaling systems, where usually one or a few prominent players are reported to be frequently altered at the DNA level , gastric and breast cancer PLCD1 were strongly associated with mutations in TP53 and KRAS as well as to tumors of the MSS phenotype; although the biological significance of these associations remains to be determined.In order to reveal additional genes in the “Phosphatidylinositol signaling system” involved in tumorigenesis, we explored the expression level of all its components as annotated in KEGG. Both inositol phosphatases, diacylglycerol kinases, and phospholipases were found to have altered expression. Among the phospholipase enzymes, members of the Phospholipase C family were well represented, indicating an important role for this family in CRC. This was underscored by RT-PCR analyses validating that the expression levels of PLCE1, the second gene investigated in detail, is supported by the results from Sorli et al. who also demonstrated significantly reduced mRNA expression levels in colon and rectum cancer samples and colon cancer cell lines, as well as by Wang and colleagues who reported a frequency of 36% loss of heterozygosity (LOH) of 10q23 where PLCE1 is located and downregulation of PLCE1 in 21/50 colorectal cancer samples compared with matched normal tissue PLCE1 were decreasing with more advanced stages, indicating that the repression of this gene is beneficial for cancer progression. Notably, the level appears to rise when reaching metastatic disease (stage IV), a phenomenon previously described for PLCD1 and PLCD3 in breast cancer PLCD1 and PLCE1 were correlated, and as expected, reduced expression of both genes was associated to more advanced tumor stages (data not shown). However, the individual expression levels and the expression levels of both genes combined were not correlated with patient survival (data not shown). Intriguingly, as for PLCD1, low expression of PLCE1 was strongly associated with mutations in KRAS. Since PLCE1 is found to be both an upstream and downstream effector of Ras family proteins KRAS with the reduced expression levels most likely plays a critical role in CRC tumorigenesis. Further, it was recently reported that overexpression of PLCE1 in a colon cancer cell line inhibited tumor cell proliferation, reduced number of colonies formed, reduced migration, and increased apoptosis The observed repression of PLCD1 and PLCE1 could be due to promoter hypermethylation. This is a common way to inactivate tumor suppressor genes, and both phopholipases have been put forward as candidates for promoter hypermethylation PLCD1 within a range of 52–62% methylation in ESCC, gastric and breast cancer PLCE1 as a target for this type of epigenetic silencing, and despite high frequency of methylation in colon cancer cell lines, methylation in the PLCD1 promoter can only explain reduced expression in a subgroup of the carcinomas. Interestingly though, we discovered that all but one of the methylated tumors were MSI, yielding a methylation frequency of nearly forty percent among carcinomas with defect mismatch repair. Additionally, the methylated MSI tumors were localized in the right side of the bowel and had BRAF mutation, in line with the CIMP phenotype We then investigated whether the reduced expression levels of PLCD1 and PLCE1, confirmed reduced levels of these transcripts in CRC compared to normal mucosa samples. Both a tumor suppressive and tumor promoting role of these genes and protein products have been suggested in relation to cancer development, depending on tissue of origin PLCD1 and PLCE1 in CRC, which for PLCD1 possibly can be explained by an epigenetic mechanism in microsatellite unstable carcinomas.By subjecting two CRC transcriptome datasets to GSEA, we have identified a number of statistically significant deregulated gene sets in CRC, and shown that this approach can guide discovery of novel molecular targets and biomarkers for cancer. Verification analyses of selected genes within one of the identified deregulated signaling pathways, Written informed consent was obtained from all subjects included. All samples were retrieved from research biobanks as a part of research projects approved according to national ethical guidelines. The biobank series have been registered according to national legislation and is approved by the Regional Committee for Medical Research Ethics .Two independent colorectal tissue transcriptome profiling datasets previously generated in our laboratory were used for this GSEA study The patient samples included in target verification analyses constituted a subset of the tumors used in the HuEx dataset (n = 70), and 17 normal colonic mucosa samples (including six from the HuEx dataset). All samples were taken from patients undergoing primary surgery for CRC at Oslo University Hospital, Aker, Oslo, between 2005 and 2008. The normal colonic mucosa samples were taken from disease-free areas in the resection margin of the CRC specimens (≥10 cm from the tumor). A clinicopathological description can be found in Nineteen colon cancer cell lines  were included in the study, and are thoroughly described in DNA from fresh-frozen colonic tissue and colon cancer cell lines was extracted by a standard phenol/chloroform protocol. RNA from tumor tissue was extracted using the Qiagen AllPrep DNA/RNA Mini Kit , whereas RNA from normal colonic mucosa samples and colon cancer cell lines was isolated by the Ambion RiboPure™ Kit  and Trizol , respectively. All procedures were performed according to the manufacturers' protocols.versus normal colonic mucosa were independently subjected to GSEA. Data preprocessing was performed as previously described P-values were computed based on the vector of probabilities of the t-statistics across the included genes For GSEA gene sets were defined according to the KEGG pathway collection (Homo sapiens (human) Release 53.0) ACTB (Hs99999903_m1) and GUSB (Hs99999908_m1), as well as cDNA of the genes PLCD1 (Hs00979908_m1) and PLCE1 (Hs00275279_m1) was amplified separately in 384 well-plates as described by the manufacturer (Applied Biosystems). Gene expression was measured real-time using the 7900 HT Sequence Detection System (Applied Biosystems). The samples were analyzed in triplicates and the median value was used for data analysis. A serial dilution of cDNA from the Universal Human Reference RNA  was used to generate the standard curve. The expression levels of PLCD1 and PLCE1 were normalized against the mean value of the endogenous controls.Total RNA from CRC tissue and normal mucosa samples was converted to cDNA using the High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems). The cDNA of two endogenous controls, Prior to qualitative and quantitative MSP and bisulfite sequencing, 1.3 µg DNA from each sample was sodium bisulfite treated using the Epitect Bisulfite Kit (Qiagen) according to the manufacturer's protocol. The DNA cleansing was performed using a QiaCube (Qiagen), and the final elution volume was set to 40 µl.Primers for MSP and bisulfite sequencing were designed using the Methyl Primer Express v.1 software (Applied Biosystems), whereas primers and probe for the qMSPs were designed using the Primer Express v3.0 software (Applied Biosystems). Primers were purchased from Medprobe  and the probe from Applied Biosystems. Primer and probe sequences, product fragment lengths and conditions are listed in PLCD1 (NM_006225.3) and PLCE1 (NM_016341) was analyzed by MSP using the HotStarTaq DNA Polymerase (Qiagen). Positive results were verified by a second round of PCR. Bisulfite modified SssI methyltransferase  treated human placental DNA  and DNA from normal lymphocytes served as positive controls for the methylated and unmethylated reaction, respectively. Water was used as negative control in both reactions.The promoter methylation status of both PLCD1 promoter was also analyzed by quantitative MSP according to a protocol previously described ALUC4 was used as an internal reference to normalize for the amount of input DNA. The level of DNA methylation was calculated as Percent of Methylated Reference (PMR) by the following equation: [(PLCD1/ALU)sample / (PLCD1/ALU)positive control] ×100. Samples with a PMR value exceeding the highest values of normal mucosa samples, i.e. PMR>0, were considered methylated.The PLCD1 and PLCE1 gene promoters were performed in nineteen colon cancer cell lines. Seventy CpG sites in the PLCD1 promoter were analyzed, including the MSP and qMSP primer and probe binding sites  and PTEN (HGNC:9588), as well as of mutation hot spots in KRAS , BRAF , and PIK3CA  have previously been determined . Associations between gene expression levels and clinicopathological features were analyzed by Mann-Whitney-Wilcoxon (MWW) test or Kruskal-Wallis test. The MWW test was also used to examine expression level differences between normal colonic mucosa, CRC samples, and colon cancer cell lines. All reported Figure S1Plots showing gene expression of the most significantly up- and downregulated pathways. Gene expression in CRC versus normal colonic mucosa for the three most significantly up- and downregulated pathways in two gene expression datasets, A–C and D–F, respectively. The plots are based on values from the HuEx dataset, but are representative also for the AB dataset.(TIF)Click here for additional data file.Figure S2Primers and probe location relative to transcription start site. Promoter region of A) PLCD1 (NM_006225.3) and B) PLCE1 (NM_016341) with location of all primers and the probe used in the present study. Vertical bars represent CpG sites, whereas +1 designate transcription start site. Abbreviations: Bisulf.seq, bisulfite sequencing; MSP, qualitative methylation-specific polymerase chain reaction; qMSP, quantitative methylation-specific polymerase chain reaction; ps, present study; s, sense; as, antisense. aPrimers from Hu et.al, Oncogene, 28, 2009.(TIF)Click here for additional data file.Table S1Significantly deregulated KEGG pathways in CRC compared to normal colonic mucosa across two gene expression datasets.*the total number of genes included in each pathway after quality controls § values less than 2.6E-162.(DOC)Click here for additional data file.Table S2Genes significantly deregulated in the most significantly up- and downregulated pathways in CRC. The three most significantly upregulated pathways with the significantly differentially expressed genes in each/both datasets are presented in the upper part of the table. Similarly, the lower part of the table lists the three most significantly downregulated pathways and the differentially expressed genes. Of notice, KEGG pathway 00980: Metabolism of xenobiotics by cytochrome P450 were more downregulated than KEGG pathways 00830 and 00280 displayed in the table, but were due to substantial overlap with KEGG pathway 00982: Drug metabolism – cytochrome P450 replaced with the next pathways on the list.(DOC)Click here for additional data file.Table S3Summary of patient clinical data in the transcriptome datasets.aTumor stage according to Union for International Cancer Control (UICC)/American Joint Committee on Cancer (AJCC) staging system.(DOC)Click here for additional data file.Table S4Clinical, pathological, and molecular data for patient samples included in the validation and methylation analyses.aTumor stage according to UICC/AJCC staging system Abbreviations: M, male; F, female; MMR, mismatch repair; MSI, microsatellite instable; MSS, microsatellite stable; Wt, wild type; Mut, mutated; Unmeth, unmethylated; Meth, methylated.(DOC)Click here for additional data file.Table S5Primers and probe used for qualitative and quantitative methylation-specific polymerase chain reaction and bisulfite sequencing. For PLCD1 (NM_006225.3) the MSP primers were obtained from Hu et al.  whereas the bisulfite sequencing was performed with primers from Hu et al. combined with primers specifically designed for the present study. For PLCE1 (NM_016341), all primers were designed for the present study. Abbreviations: MSP, methylation-specific polymerase chain reaction; M, methylated-specific primers; U, unmethylated-specific primers; qMSP, quantitative methylation-specific polymerase chain reaction; MGB, minor groove binder; BS, bisulfite sequencing.(DOC)Click here for additional data file.


Myeloid dendritic cells (mDC) are lost from blood in individuals with human immunodeficiency virus (HIV) infection but the mechanism for this loss and its relationship to disease progression are not known. We studied the mDC response in blood and lymph nodes of simian immunodeficiency virus (SIV)-infected rhesus macaques with different disease outcomes. Early changes in blood mDC number were inversely correlated with virus load and reflective of eventual disease outcome, as animals with stable infection that remained disease-free for more than one year had average increases in blood mDC of 200% over preinfection levels at virus set-point, whereas animals that progressed rapidly to AIDS had significant loss of mDC at this time. Short term antiretroviral therapy (ART) transiently reversed mDC loss in progressor animals, whereas discontinuation of ART resulted in a 3.5-fold increase in mDC over preinfection levels only in stable animals, approaching 10-fold in some cases. Progressive SIV infection was associated with increased CCR7 expression on blood mDC and an 8-fold increase in expression of CCL19 mRNA in lymph nodes, consistent with increased mDC recruitment. Paradoxically, lymph node mDC did not accumulate in progressive infection but rather died from caspase-8-dependent apoptosis that was reduced by ART, indicating that increased recruitment is offset by increased death. Lymph node mDC from both stable and progressor animals remained responsive to exogenous stimulation with a TLR7/8 agonist. These data suggest that mDC are mobilized in SIV infection but that an increase in the CCR7-CCL19 chemokine axis associated with high virus burden in progressive infection promotes exodus of activated mDC from blood into lymph nodes where they die from apoptosis. We suggest that inflamed lymph nodes serve as a sink for mDC through recruitment, activation and death that contributes to AIDS pathogenesis. Myeloid dendritic cells (mDC) are essential innate immune system cells that are lost from blood in human immunodeficiency virus infection through an ill-defined mechanism. We studied the kinetics of the mDC response in blood and lymph nodes of rhesus macaques infected with the closely related simian immunodeficiency virus. We found that differences in the number of blood mDC correlated with eventual disease outcome, as at virus set-point mDC were increased in blood in animals remaining disease free but lost from blood in animals that progressed rapidly to AIDS. mDC loss was linked to an increase in the chemokine axis responsible for mDC recruitment to lymph nodes; however, mDC did not accumulate in tissues but rather died from apoptosis. Lymph node mDC remained responsive to stimulation with a TLR7/8 agonist during infection. Importantly, mDC dysregulation was partially reversed by antiretroviral therapy. These data indicate that chronic mDC recruitment, activation and death within lymph nodes precede development of disease in SIV infected monkeys and may play a role in AIDS pathogenesis. Myeloid dendritic cells (mDC) are professional antigen-presenting cells that are critical for the induction of acquired immune responses to pathogens The impact of antiretroviral therapy (ART) on mDC loss and recovery in HIV infection is also unclear, as several studies indicate that ART is not effective at increasing blood mDC In the present study we followed the mDC response in blood and lymph nodes over time in two cohorts of SIV-infected animals that received ART and adenovirus (Ad)-based immunotherapy with different disease outcomes. We find that loss of blood mDC at virus set-point is predictive of disease progression, whereas an increase in blood mDC is predictive of long-term absence of disease, and that even relatively short periods of ART are beneficial to mDC homeostasis. In animals that progress to AIDS the early loss of mDC from blood is associated with evidence of increased CCR7-CCL19-mediated recruitment to lymph nodes and increased apoptosis within these tissues.Animals in this study were enrolled in an immunotherapy protocol using Ad-based vectors the majority of which has been previously described 7 RNA copies/ml plasma; however virus loads began to diverge by week 4 and at virus set point virus loads differed by ∼1 log between groups , although when each group was analyzed separately this decrease was not significant. However, in the post-acute period the mDC response diverged, as mDC in progressor animals continued to decline to week 12 when they were significantly reduced in number relative to preinfection time points. In contrast, mDC in stable animals significantly increased from weeks 2 to 12 (P = 0.3). Initiation of the second round of ART again reduced mDC number in the majority of stable animals, concurrent with the reduction in virus load, after which the number of mDC remained relatively constant  as CD45c+ cells  and were 2 to 12 . The rel 2 to 12 . This ch 2 to 12 . Exposur 2 to 12 . However 2 to 12 . The magconstant . The divnfection . These d+ mDC in progressor animals also expressed CD86 with a smaller proportion expressing CD80, consistent with activation . Surgeries were performed under anesthesia induced and maintained with ketamine hydrochloride and medetomidine hydrochloride, and all efforts were made to minimize suffering.Macaca mulatta) used in this study were housed at the University of Pittsburgh Primate Facility for Infectious Disease Research. All animals were infected by intravenous inoculation with 1,000 TCID50 of uncloned, pathogenic SIVmac251 . Virus load in plasma was determined as described previously gag, env and nef with and without rhesus IL-15.FLAG or empty Ad-ψ5 were given by intramuscular injection at week 16  and week 22 , and boosting injections of the same quantity of Ad35-based vectors expressing the same transgenes were given at week 36 and 42. All Ad vectors were E1/E3-deleted with the exception of Ad35 containing the env gene which was E3 deleted. Lymph nodes were taken from the axillary or inguinal regions prior to infection and at weeks 12, 24 and 32 post infection and single cell suspensions were generated by disruption and digestion with collagenase D, as described Twenty one Indian-origin rhesus macaques  that contained a known number of fluorescent beads to provide internal calibration, as previously reported + T cells in PBMCs at the same time point Identification of mDC was performed as previously described with some modifications Intracellular cytokine production by lymph node mDC was measured as described previously for plasmacytoid DC with minor modifications −ΔCT method using β-glucuronidase as the endogenous control Total lymph node RNA was extracted and purified from cell suspensions generated from biopsies taken prior to or 12 weeks after infection using the RNAeasy kit (Qiagen) after treatment with DNAse I (Invitrogen). cDNA was synthesized using random primers and Superscript II reverse transcriptase (Invitrogen). Primers and probes from Taqman human gene expression arrays  were utilized for real time PCR analysis of CCL19, CCL21 and β-glucuronidase expression as previously described P values are two-sided with significance considered to be P<0.05.Comparisons between two groups were carried out using the Mann-Whitney U test. Comparison of DC numbers across different time points was carried out using the Wilcoxon signed-rank test. Correlations were determined using the non-parametric Spearman rank test. Graphpad Prism 5 (Graphpad Software) was used for statistical analysis. All The identification of genes analyzed in this paper as defined by Entrez-Gene are 574386 (CCL19), 574183 (CCL21) and 677692 (β-glucuronidase).


Further, single nucleotide polymorphisms (SNPs) in its sequence have been linked with risk of developing severe influenza virus infections in humans. The number of viruses restricted by this host protein has continued to grow since it was first demonstrated as playing an antiviral role; all of which enter cells via the endosomal pathway. We therefore sought to test the limits of antimicrobial restriction by Ifitm3 using a knockout mouse model. We showed that Ifitm3 does not impact on the restriction or pathogenesis of bacterial  or protozoan (Plasmodium berghei) pathogens, despite in vitro evidence. However, Ifitm3 is capable of restricting respiratory syncytial virus (RSV) in vivo either through directly restricting RSV cell infection, or by exerting a previously uncharacterised function controlling disease pathogenesis. This represents the first demonstration of a virus that enters directly through the plasma membrane, without the need for the endosomal pathway, being restricted by the IFITM family; therefore further defining the role of these antiviral proteins.The interferon-inducible transmembrane (IFITM) family of proteins has been shown to restrict a broad range of viruses  Intrinsic cellular defense molecules are able to detect and restrict invading pathogens at the level of the infected cell and constitute an initial repertoire of proteins that prevent infection. Such intrinsic defenses have the ability to detect the pathogen, and either directly block a component of the pathogen’s life cycles and / or signal to the innate and adaptive immune system to further control the infection. In certain cases, these intrinsic restriction factors recognise non-self pathogen-assocated molecular patterns, such as lipids, proteins and nucleic acids, from a broad range of pathogens through pathogen recognition receptors. This ability allows the host to detect bacterial, viral, fungal and protozoan pathogens . For exaWe and others have found that the ISG interferon-inducible transmembrane 3 (IFITM3), initially defined as playing a developmental role in germ cell homing , has a pRecently, the role of IFITM3 has been expanded by the discovery of nonenveloped reoviruses’ restriction . This haIFITM3 in vivo shows it is crucial restriction factor for preventing the onset of severe influenza viral infections in a knockout mouse model [IFITM3 gene in two cohorts of patients hospitalised with influenza virus during the 2009 H1N1 pandemic shows that the rs12252 CC genotype confers an 4-5 fold increased risk developing a severe influenza virus infection [The role of se model . Furthernfection ,19.-/-) mice to bacteria , a parasite (Plasmodium berghei) and a virus  to determine the specificity of this crucial antimicrobial protein. We show that Ifitm3 is specifically an antiviral protein; yielding no significant phenotype in mice when challenged with bacteria and protozoa, despite studies implicating the IFITM family in restriction of these pathogens [Here we sought therefore to expand and define the role of Ifitm3 in pathogen restriction by assessing the susceptibility of Ifitm3-deficient  Act 1986 Amendment Regulations 2012.All animal experiments were conducted under Prof. Gordon Dougan’s project licence No. 80/2596, entitled “The characterization of microbial and murine genes required for infection”. This was brought before the Wellcome Trust Sanger Institute’s Animal Welfare and Ethical Review Body  on 30-/- (Wellcome Trust Sanger Institute [-/- mice (Jackson Laboratories), all of which were >95% C57BL/6, were maintained in accordance with UK Home Office regulations, UK Animals Scientific Procedures Act 1986 under the project license PPL 80/2596. Animals were supplied with food and water ad libitum and were monitored daily for signs of illness. Ifitm3-/- mice were phenotyped through pipelines at the Wellcome Trust Sanger Institute as described previously [Background-matched 8-10 week old wild type, Ifitm3nstitute ) and Ifneviously ,24. 5-µm sections of paraffin-embedded tissue were incubated with anti-Ifitm3 antibody (Abcam), which was subsequently bound to a secondary horse radish peroxidase-conjugated anti-rabbit antibody (Dako). Sections were counterstained with hematoxylin (Sigma-Aldrich) and were assessed for expression by microscopy.-/- and 8 C57BL/6J mice were challenged intravenously with 5 x 105 colony forming units (cfu) Salmonella Typhimurium M525 containing TetC, , and followed for 28 days. On day 14 post-infection (pi), four from each group of mice are culled and organs  removed. A small piece of the spleen and liver was fixed in 4% formalin and then later processed to paraffin blocks as a biobank of infected tissue for histological study if interesting. The rest of the organs were weighed then homogenized, serially diluted and plated to determine viable bacterial load. At day 28pi, the remaining four mice are culled following a terminal bleed and the organs removed and processed as above. The blood was allowed to clot then centrifuged when the serum was removed and used to detect TetC antigen specific antibodies by enzyme-linked immuosorbent assay (ELISA). Mice were weighed and monitored daily for signs of clinical illness.Groups of 8 Ifitm3-/- and 8 C57BL6j mice were orally infected by gavage with 1 x 109 cfu of and followed for 28 days. Every 2-3 days faeces from infected mice are collected. These were then weighed and homogenized in 1 ml per 100 mg of faeces. This was then serially diluted and plated to determine viable bacterial load. On day 14pi four mice per group were culled and organs  removed. A small piece of the distal colon was fixed in 4% formalin and processed to paraffin blocks as a biobank of infected tissue for histological analysis. The rest of the organs were weighed then homogenized, serially diluted and plated to determine viable bacterial load. On day 28pi the remaining four mice were culled and the above is repeated. Mice were weighed and monitored daily for signs of clinical illness.Groups of 8 Ifitm3M. tuberculosis using a Glas-Col  aerosol generator calibrated to deliver approximately 100 bacteria into the lungs. Bacterial counts in the lungs at each time point of the study were determined by plating serial dilutions of individual lung homogenates on duplicate plates of Middlebrook 7H11 agar containing OADC enrichment. Colony-forming units were counted after 3–4 weeks incubation at 37 °C. Mice were infected by low-dose aerosol exposure with H37Rv Plasmodium berghei ANKA reporter line, PbGFP-LUCCON (RMgm-28), that constitutively expresses a fusion protein of GFP and Firefly Luciferase [5 infected red blood cells was injected intraperitoneally. From day six pi mice were monitored twice daily and scored for clinical signs of neurologic disease using a ten parameter murine coma and behaviour scale adapted from that of Carroll et al. [A transgenic ciferase , was mail et al. . Mice clP. berghei infected mice were analysed for cytokines using a Cytometric Bead Array Inflammation kit (BD Biosciences) according to manufacturer’s instructions.  Samples were acquired on a BD FACS Aria II, and data analysed using BD FCAP array software. Plasma samples from 5 plaque forming units (PFU) under isoflurane anesthesia. Weight was measured daily to monitor disease severity. To collect bronchoalveolar lavage (BAL) fluid, the lungs of each mouse were inflated five times with 1 ml of PBS and BAL fluid kept on ice; 100 μl was centrifuged onto glass slides and stained with hematoxylin and eosin for cell differentiation. The remainder was centrifuged, the supernatant retained at −80°C, and the pellet resuspended in RPMI medium with 10% fetal calf serum. Lungs were removed, the smaller lobe was snap frozen in liquid nitrogen for RNA extraction and the remainder was homogenized by passage through 100-μm cell strainers . Red blood cells in the lung sample were lysed in ammonium chloride buffer, and the remaining cells resuspended in RPMI medium with 10% fetal calf serum. Viable cell numbers were determined by trypan blue exclusion and lung cells types were differentiated by flow cytometry on a BD FACS Aria II using antibodies from BD and eBioscience. RSV viral load was measured by quantitative RT-PCR for the RSV L gene using primers and probes previously described [RSV strain A2  was grown in HEp-2 cells and viral titer determined by plaque assay. Mice were infected intranasally (i.n.) with 5 x 10escribed , with L t-tests for the comparison of two groups. Non-normally distributed data were assessed by Mann-Whitney U test. Results regarding mouse survival were analysed by a Log-Rank (Mantel-Cox) test. All data regarding mouse phenotyping, including how individual traits were statistically analysed can be found at the Wellcome Trust Sanger Institute’s Mouse Resources Portal  All calculations were performed using GraphPad Prism 5.0 software, and results were considered significant at p < 0.05.All results are expressed as mean +/- S.D.; statistical significance was calculated by analysis of variance (ANOVA) followed by Bonferroni's Multiple Comparison Test when there were more than two groups and Student’s -/- mice have no discernable phenotype [http://www.sanger.ac.uk/mouseportal/). We assessed the tissue distribution of Ifitm3 by immunohistochemistry of wild type compared to Ifitm3-/- mice, including lymph node, lung, spleen, liver and intestine. The expression of Ifitm3 was absent in all Ifitm3-/- mouse organs, as expected, but was highly expressed in all wild type organs  and colon showed no significant differences in bacterial colonisation and clearance , that constitutively expresses a fusion protein of GFP and Firefly Luciferase [-/-) mice were included to act as control, as these mice do not succumb to lethal episodes of ECM. The experimental challenge revealed there to be no significant difference in phenotype seen in Ifitm3-/- mice compared with wild type littermate controls, with both showing susceptibility to ECM  and were monitored daily for weight loss for seven days pi. Cohorts of mice were sacrificed on days four and seven pi to quantify viral burden and immunological changes over the course of the challenge.Wild type and Ifitm3-/- mice showed highly significant weight loss on days six and seven pi compared to wild type littermates (p < 0.01) , which remained higher than wild type mice at seven days pi (Ifitm3 < 0.01) . Further days pi .-/- mice  in the lungs  was performed at days four and seven pi and lungs harvested at day seven for cell and cytokine measurements. Cellular infiltrate was quantified over the course of infection, which showed a significant increase in total cells resident in the lungs on day seven pi in Ifitm3he lungs  and granhe lungs . Analysihe lungs , with siin vivo in the absence of infection is not well understood. Here we show Ifitm3 is expressed in many different murine tissues and cell types and does affect the response to RSV infection in mice. Ifitm3 does not contribute to the infection phenotype of Citrobacter, Salmonella or Mycobacterium bacterial infections. The role of IFITM3 in restricting virus infections, where the virus enters the cell through the acidic endosomal pathway, is well established ,15,29. HSalmonella enterica serovar Typhimurium (S. Typhimurium) is an intracellular bacteria that enters cells through phagocytosis or by a bacterial-triggered entry mechanism and replicates within endosomal-like structures known as Salmonella-containing vacuoles [Citrobacter rodentium (C. rodentium) is a non-invasive, Gram-negative bacterium used to model enteropathogenic and enterohaemorrhagic  E. coli infections of the gut [Mycobacterium tuberculosis (M. tuberculosis) is an intracellular respiratory bacterium that replicates primarily within macrophages and dendritic cells, before forming latent granulomas in the infected organs [M. tuberculosis bacterial growth in murine lungs, despite the fact that the pathogen triggers a type I IFN response [IFITM3 with susceptibility to TB [M. tuberculosis infection. However, it should be noted that we only assayed for initial infection and colonisation of the lungs and that the long term dormancy characteristic of human TB is not observed in the mouse model. Furthermore, these differences may have arisen due to our challenge strain differing from that used in the human study; therefore inducing different gene expression signatures.These bacterial species encompass a range of physiological and pathogenic niches. vacuoles . In cont the gut ,32, and d organs , and is response , which sty to TB , whereinPlasmodium berghei ANKA infection, although it is well established that malarial parasites elicit strong type I and type II IFN responses in their hosts, which have been shown to impact on the severity of infection [IFNAR1, have been associated with the development of cerebral malaria in children; a finding that is corroborated in Ifnar-/- mice, which also do not develop cerebral malaria [IFITM3, along with several other ISGs, are significantly up-regulated in patients that have become infected with P. falciparum [Tbk1 and the double knockout of Irf3 and Irf7 prevented mice from developing lethal ECM [IFITM3 is not an ISG involved in the pathogenesis of experimental cerebral malaria.Further, we found that Ifitm3 does not impact on the development of ECM in nfection ,36, with malaria . IFITM3,lciparum . It was thal ECM ,36. Our -/- mice, as assessed by weight loss, viral load and a dysregulated immune response. Although these trends were seen with influenza virus infection of Ifitm3-/- mice, the phenotype seen in the RSV challenge is not as striking [However, we saw that Ifitm3 was important in the control of RSV infection, leading to more severe disease in Ifitm3striking ,38. Thisstriking  and othestriking  have shoRSV is one of the commonest respiratory pathogens in children that necessitates hospitalisation ; accountin vivo, or exerts a hitherto uncharacterised function controlling virus infection in vivo is novel and supports associations seen in the mouse model [Our discovery that the lack of Ifitm3 can alter the pathogenesis of RSV infection suggests IFITM3 either directly restricts RSV cell infection se model ,44, in ase model  and in bse model . Strikinse model ,48. RSV -/- mice [Ifitm1 has been shown to be up-regulated during RSV infection [Recently, Li and colleagues  suggeste-/- mice , may pro-/- mice ,8. Impor-/- mice , possiblnfection . With the recent recognition of IFITM3-mediated restriction of nonenveloped reoviruses, the pleotropic effect of IFITM3 on diverse virus infections, or the co-operative role of all antiviral IFITM proteins as a layered defense to different virus infections, remains to be determined.


Transient isolated brainstem symptoms  are not consistently classified as transient ischaemic attacks (TIAs) and data for prognosis are limited. If some of these transient neurological attacks (TNAs) are due to vertebrobasilar ischaemia, then they should be common during the days and weeks preceding posterior circulation strokes. We aimed to assess the frequency of TNAs before vertebrobasilar ischaemic stroke.We studied all potential ischaemic events during the 90 days preceding an ischaemic stroke in patients ascertained within a prospective, population-based incidence study in Oxfordshire, UK  and compared rates of TNA preceding vertebrobasilar stroke versus carotid stroke. We classified the brainstem symptoms isolated vertigo, vertigo with non-focal symptoms, isolated double vision, transient generalised weakness, and binocular visual disturbance as TNAs in the vertebrobasilar territory; atypical amaurosis fugax and limb-shaking as TNAs in the carotid territory; and isolated slurred speech, migraine variants, transient confusion, and hemisensory tingling symptoms as TNAs in uncertain territory.vs two of 751 before a carotid stroke, OR 35·8, 8·4–153·5, p<0·0001). Of all 59 TNAs preceding  vertebrobasilar stroke, only five (8%) fulfilled the National Institute of Neurological Disorders and Stroke (NINDS) criteria for TIA. The other 54 cases were isolated vertigo (n=23), non-NINDS binocular visual disturbance (n=9), vertigo with other non-focal symptoms (n=10), isolated slurred speech, hemisensory tingling, or diplopia (n=8), and non-focal events (n=4). Only 10 (22%) of the 45 patients with isolated brainstem TNAs sought medical attention before the stroke and a vascular cause was suspected by their physician in only one of these cases.Of the 1141 patients with ischaemic stroke, vascular territory was categorisable in 1034 (91%) cases, with 275 vertebrobasilar strokes and 759 carotid strokes. Isolated brainstem TNAs were more frequent before a vertebrobasilar stroke (45 of 275 events) than before a carotid stroke , particularly during the preceding 2 days , Thames Valley Primary Care Research Partnership, and the NIHR Biomedical Research Centre, Oxford. The diagnosis of transient ischaemic attack (TIA) is often based purely on the description of symptoms, particularly in patients with vertebrobasilar events in whom positive diffusion weighted imaging is less common16The inconsistency in clinical diagnosis of vertebrobasilar TIA is due mainly to uncertainty about the nature of several transient isolated brainstem symptoms. Most clinical guidelines do not specify which of these symptoms should be deemed as TIA,The Oxford Vascular Study (OXVASC) is a prospective, population-based study of all stroke and TIA in 91 105 individuals of all ages registered with 63 primary care physicians in Oxfordshire, UK. We restricted analysis to patients with a first stroke presenting to medical attention from April 1, 2002, to March 31, 2010.The study methods have been described elsewhere.(1) An open-access TIA service available 7 days per week to which participating general practitioners and the local accident and emergency department send all individuals with suspected TIA or minor stroke whom they would not usually admit to hospital, with emergency provision at weekends supplementing a weekday clinic; (2) daily searches of admissions to the stroke unit, general medical, neurology, and other relevant wards; (3) daily searches of the local accident and emergency department and eye hospital attendance register; (4) monthly computerised searches of general practitioner diagnostic coding and hospital discharge codes; (5) monthly searches of all cranial and carotid imaging studies done in local hospitals; and (6) monthly reviews of all death certificates and coroners' reports.OXVASC was approved by our local ethics committee (Oxfordshire Ethics Committee A: 05/Q1604/70). All patients provided written informed consent and were seen by study physicians as soon as possible after their initial presentation. Relatives provided written assent and clinical details for those unable to provide written consent.The study senior neurologist (PMR) reviewed all cases and classified them as stroke or other disorder using standard definitions.A detailed history about preceding events was obtained from every patient with a standardised questionnaire.2 test for categorical variables and t test for age. Where significant differences were noted, appropriately stratified analyses were done. We used the χ2 test to compare the frequency and type of TNAs reported during the 90 days preceding a vertebrobasilar stroke versus a carotid stroke and to compare the frequency of events preceding stroke with or without symptomatic arterial stenosis. Given that some patients experience multiple TNAs before stroke, to avoid double counting, analysis was based on the first TNA or TIA during the 90 days preceding stroke, rather than the most recent TNA or TIA. We assumed an arbitrary level of 5% significance.We compared baseline characteristics of patients with stroke in the vertebrobasilar territory (vertebrobasilar stroke) versus those with stroke in the carotid territory (carotid stroke) using the χThe funding sources had no role in study design, collection, analysis, and interpretation of data, writing of the report, or the decision to submit for publication. The corresponding author had full access to all of the data in the study and had final responsibility for the decision to submit for publication.Of 1141 consecutive patients with definite stroke, vascular territory could be identified in 1034 (91%). Of these, 742 (72%) patients had a minor stroke and 292 (28%) had a major stroke. 759 (73%) patients had a stroke in the carotid territory and 275 (27%) in the vertebrobasilar territory . Patientvs 65 of 759 patients before a carotid stroke; OR 2·92, 95% CI 1·99–4·28, p<0·0001). Of 275 patients with vertebrobasilar stroke, 59  had at least one event within the preceding 90 days  of which only five (8%) had events that fulfilled the NINDS criteria for TIA (Events (TIAs and other TNAs) were more common in the 90 days preceding a vertebrobasilar stroke than before carotid stroke . This relative rate was similar for isolated brainstem TNAs preceding vertebrobasilar stroke .The temporal relation between isolated vertebrobasilar TNA and vertebrobasilar stroke was very similar to that of definite carotid TIA and carotid stroke , both bevs eight of 558 events before carotid stroke, OR 15·8, 95% CI 17·0–37·3, p<0·0001), but strikingly more so for events within the 2 days immediately preceding the stroke . Of the 759 patients with definite carotid territory stroke, 65 (9%) had at least one TNA or TIA within the preceding 90 days  of which 32 (49%) fulfilled the NINDS criteria for TIA , table 2Only ten (22%) of the 45 patients with isolated brainstem TNAs sought medical attention before the stroke and a vascular cause was suspected by their physician in one of these cases. In the remainder of cases, the initial diagnosis was documented as postural hypotension (three cases), peripheral vestibular disturbance (two cases), sepsis (two cases), migraine (one case), and cranial nerve palsy (one case). Vascular risk factors were more common in those with isolated vertigo, compared with other vertebrobasilar TNAs .Table 3VIn patients with stroke in whom 50% or higher symptomatic stenosis was documented, TNAs were also more frequent preceding vertebrobasilar stroke than preceding carotid stroke . This exPanelSystematic reviewWe searched Medline (1950–2011) and Embase (1988–2011) and also hand-searched relevant journals and the reference lists of included papers using the search terms “transient ischaemic attack”, “transient neurological attack (TNA)”, “stroke”, “vertebrobasilar”, and “stroke symptoms”. Searches were restricted to human studies. All types of study with at least three patients were included. Several studiesInterpretationWe studied all potential ischaemic events during the 90 days preceding an ischaemic stroke in patients ascertained within a prospective, population-based incidence study in Oxfordshire, UK (the Oxford Vascular Study). Isolated brainstem TNAs were more frequent before a vertebrobasilar stroke than before a carotid stroke, particularly during the preceding 2 days. Our study shows a close temporal relation between vertebrobasilar stroke and preceding isolated brainstem transient neurological attacks that do not satisfy the NINDS definition of TIA. More studies of the prognosis of the different transient isolated brainstem symptoms are needed.Our study shows that there is a temporal relation between transient isolated brainstem symptoms and vertebrobasilar stroke . Such evResults from large cohort studies have shown that 12–18% of people without a previous diagnosis of stroke reported TIA-like symptoms.Isolated vertigo was the most common transient symptom preceding vertebrobasilar stroke in our study. Results from the Atherosclerosis Risk in Communities Study (ARIC) studyOur findings have implications for public education to improve recognition and awareness of the significance of symptoms of TIA. In our study, of those patients who did present to medical attention after the initial event, most were not managed as having TIAs, suggesting that broader diagnostic criteria are needed. Although we do not know the rate of TNAs in the population, in patients with isolated vertigo plus vascular risk factors, referral for specialist opinion would appear to be reasonable.The main strengths of our study were the population-based design, the rigorous case-ascertainment, and the prospective definition and classification of the various types of TNA. However, our study has limitations. First, the prevalence of TNAs before stroke events might be inaccurate if patients failed to report them during direct questioning or if recall bias increased recollection and reporting of TNAs. However, the very rapid fall in rate of preceding TNAs over the few days before the stroke is not what would be expected on the basis of recall bias—people would recall events for the preceding few weeks and not just the preceding few hours. Most importantly, in previous studies of definite TIA before stroke, this same time course of preceding TIA was notedIn conclusion, our study shows a close temporal relation between vertebrobasilar stroke and preceding isolated brainstem TNAs which do not satisfy the NINDS definition of TIA. A high index of suspicion is particularly important in view of the short-time window available for prevention of stroke. However, because of the adverse effects of a false-positive diagnosis of TIA, more studies of the prognosis of the different transient isolated brainstem syndromes are needed.


As synapses form and mature the synaptic partners produce organizing molecules that regulate each other’s differentiation and ensure precise apposition of pre- and post-synaptic specializations. At the skeletal neuromuscular junction (NMJ), these molecules include agrin, a nerve-derived organizer of postsynaptic differentiation, and synaptic laminins, muscle-derived organizers of presynaptic differentiation. Both become concentrated in the synaptic cleft as the NMJ develops and are retained in adulthood. Here, we used mutant mice to ask whether these organizers are also required for synaptic maintenance. Deletion of agrin from a subset of adult motor neurons resulted in the loss of acetylcholine receptors and other components of the postsynaptic apparatus and synaptic cleft. Nerve terminals also atrophied and eventually withdrew from muscle fibers. On the other hand, mice lacking the presynaptic organizer laminin-α4 retained most of the synaptic cleft components but exhibited synaptic alterations reminiscent of those observed in aged animals. Although we detected no marked decrease in laminin or agrin levels at aged NMJs, we observed alterations in the distribution and organization of these synaptic cleft components suggesting that such changes could contribute to age-related synaptic disassembly. Together, these results demonstrate that pre- and post-synaptic organizers actively function to maintain the structure and function of adult NMJs. At the mammalian skeletal neuromuscular junction (NMJ), pre- and post-synaptic structures are highly differentiated and precisely aligned. This synapse forms in embryos, matures during an early postnatal period, and is remarkably stable in young adult mice In seeking such factors, we considered molecules called synaptic organizers, which are already known to regulate the formation and maturation of the NMJ Agrin is a nerve-derived proteoglycan that is secreted by motor axons and promotes aggregation of acetylcholine receptors (AChRs) in the postsynaptic membrane. Muscle cells also express agrin, but only motor neurons produce an alternatively spliced isoform called “z-agrin” that activates a postsynaptic receptor complex consisting of LRP4 and the receptor tyrosine kinase MuSK −/−lama4) are viable but lack precise apposition of presynaptic neurotransmitter release sites (active zones) to postsynaptic depressions called junctional folds In contrast to nerve-derived agrin, laminins are synthesized by muscles and promote presynaptic differentiation. Laminins are present in all basal laminas (BLs) and are heterotrimers of α, β, and γ subunits drawn from a total of 5α, 3β, and 3γ isoforms floxagrn, ref lama4 mutants, which show subtle developmental effects but are otherwise normal and fertile floxagrn and lama4 mutants were distinct from each other, but resembled in several respects those seen in NMJs of aged wild-type mice. Moreover, the synaptic distribution of agrin and synaptic laminins changed with age. Together, these results suggest that maintenance of the NMJ requires the continuous presence of synaptic organizers and that alterations in the integrity of the synaptic basal lamina may contribute to age-related synaptic disassembly.Here, we asked whether agrin and synaptic laminins are required only for synaptic development and maturation, or whether their continuous presence is also required for synaptic maintenance in adult NMJs. To bypass the neonatal lethality of agrin mutants, we used a conditional allele (floxagrn) in which the genomic sequence from exon 6 to intron 33 is flanked by loxp sites Thy1 gene −/floxagrn).Mice lacking all agrin isoforms or only z-agrin die soon after birth due to severe neuromuscular defects In initial studies, we assessed levels of agrin at the NMJ by immunohistochemistry 1 to 3 months post-tamoxifen injection. As expected, agrin was concentrated at NMJs in control muscle  AChRs remained normally clustered 3 months after tamoxifen administration, and terminal branches of individual axons exactly opposed postsynaptic sites , which binds tightly and specifically to AChRs. In control heterozygous animals . Some YFP+ nerve terminals in mutants were apposed to postsynaptic sites containing normal levels of AChRs . Together, these results suggest that postsynaptic alterations precede presynaptic alterations.agrn had not been deleted.To determine the sources of variability among affected nerve terminals, we performed three additional studies. First, we determined the fraction of YPF+ terminals with structural alterations at monthly intervals following tamoxifen administration. The percent of YFP+ terminals exhibiting one or more of the alterations described above increased from ∼12% at 1 month post deletion when agrin levels are fairly normal, to ∼75% at 3 months when agrin is largely depleted Fig. 3D.Second, we examined several muscles that differ in position and fiber type composition. Changes in AChR clustering and nerve terminal architecture similar to those documented in Third, we examined the variability in structural alterations among nerve terminals arising from a single motor axon. Because multiple YFP+ axons were present in most muscles, it was not feasible to reconstruct entire motor units, so we examined pairs of terminals in sparsely labeled regions that we knew arose from a single axon because we could trace them back to a common branch point. In nearly all cases the terminal structure of each member of a pair was similarly affected Fig. 4A Finally, we extended our analysis to 5–6 months after tamoxifen injection to assess the fate of motor axons lacking agrin. By this time, pretzel shaped presynaptic nerve terminals had nearly all disappeared Fig. 5A.−/−lama4 mice are viable and fertile but have small NMJs in which active zones are misaligned with postsynaptic junctional folds −/−lama4 mice with anti-neurofilaments to label axons, anti-synaptotagmin-2 to visualize nerve terminals, f-BTX to label AChRs, and anti-laminin-α4 to confirm deletion. At 40 days of age , −/−lama4 NMJs were smaller than those controls, but appeared largely normal by light microscopic criteria  and synaptic disassembly following deletion of agrin in adults . Alterations observed in the two mutants are related but distinct, and both resemble some of those seen during normal aging and in diseases of the motor unit A key feature of the adult NMJ is that it is remarkably stable under ordinary circumstances floxagrn), we could allow synapses to develop normally and then assess the effect of agrin loss in adulthood. A role for agrin in synaptic maintenance was not unexpected given its critical role in development By using a conditional agrin mutant  AChR aggregates were lost from the postsynaptic membrane; (2) other components of the postsynaptic membrane (β-dystroglycan), subsynaptic cytoskeleton (rapsyn), and synaptic cleft (laminins-α4 and -α5) were depleted; and (3) nerve terminals remodeled, became dystrophic and eventually withdrew from the end-plate. Why does loss of agrin lead to so many structural alterations at the NMJ? The best-characterized role of agrin is to induce and stabilize AChR clusters. Other alterations could either reflect direct roles for agrin in many aspects of synaptic maintenance, or secondary effects of AChR loss. In favor of the latter possibility is the observation that postsynaptic defects appear to precede presynaptic ones: presynaptic alterations are found only at defective postsynaptic structures whereas postsynaptic alterations often occur at synapses with structurally normal nerve terminals. An attractive model is that dispersion of AChRs leads to rapid loss of other components, including presynaptic organizers such as ß2 laminins and LRP4 agrn deletion presumably reflects the slow loss of z-agrin from the synaptic cleft. Although we have no data on the rate at which agrn mRNA and protein levels decline within motoneurons following gene inactivation, our results are consistent with the idea that agrin turns over slowly, as has been shown for other basal lamina components in many tissues, and for recombinant agrin injected into muscles agrn deletion.The slow pace of change following −/− mice are viable and fertile. We were therefore able to ask whether laminin-α4 is required for long-term maintenance of the NMJ as well as its earlier maturation.To assess roles of synaptic laminins in maintenance of the NMJ, we examined mice lacking laminin-α4. Five laminin subunits are present in the basal lamina of the synaptic cleft -α2, α4, α5 and β2 and γ1- of which α4, α5 and β2 are present at several-fold higher levels synaptically than extrasynaptically. The rationale for analyzing α4 was as follows: lack of either broadly distributed subunit, α2 or γ1, has devastating effects on muscle integrity. Lack of β2 severely perturbs presynaptic development, but the null mutant is lethal −/− mice, the normal precise apposition of active zones to junction folds is lost In contrast to the complete synaptic disassembly that occurred following loss of neural agrin in adults, effects of deleting laminin-α4 were largely confined to presynaptic structures. Moreover, loss of laminin-α4 had no obvious effect on the molecular architecture of the synaptic cleft or postsynaptic membrane, either in young adults NMJs react differently to the loss of agrin and laminin-α4. After agrin is deleted, specializations of the postsynaptic membrane and synaptic cleft are lost; presynaptic alterations occur later, perhaps, as discussed above, as an indirect consequence of the initial disruptions. In the absence of laminin-α4, in contrast, nerve terminals show gradual structural changes, but the synaptic cleft and postsynaptic membrane are spared. Thus, these two synaptic cleft proteins play distinct roles in synaptic maintenance. We draw two main conclusions from these data. First, synaptic organizing molecules, which are critical for embryonic and early postnatal formation of the NMJ, are also required for its long-term maintenance. Second, maintenance of the adult NMJ relies on combined effects of several distinct organizers.Perhaps our most striking observation is that abnormalities observed in agrin or laminin-α4 mutants resemble those in aged wild-type mice. At present, we have no direct evidence on whether agrin or laminin are involved in the pathogenic mechanisms that underlie synaptic aging. However, the similarity between the mutant phenotypes and normal aging invites the speculation that alterations in these molecules could contribute to age-related synaptic disassembly. Three types of alterations could occur. First, levels of these components could decline with age. We see no dramatic decline, but our methods would not have allowed us to detect a modest decline. Second, the distribution of the proteins could be altered with age, decreasing their ability to mediate highly localized specializations of appositions. In fact, we have observed such alterations in distribution . Third, −/−agrn) flox/floxagrn) −/−lama4) mice +/−agrn mice were mated to SLICKA animals to generate heterozygtes and the +/−;SlickA+agrn offspring were crossed to flox/floxagrn to generate transheterozygots carrying CreER  as well as heterozygote controls . Tamoxifen dissolved in corn oil was administered over a two-week period by oral gavage once a day for 4 consecutive days during each week .Generation and characterization of agrin null  and aged (24 to 28 months) C57BL/6 mice were obtained from the National Institute on Aging or The Jackson Laboratory  or bred in our vivarium. All experiments conformed to NIH guidelines and were carried out in accordance with protocols approved by the Harvard University Standing Committee on the Use of Animals in Research and Teaching.GACCACTGAGTGCATGGTTG; α4 = TGTTTGTTGGAGGTGTTCCA and ATGCTCACAGCACCACTGAC; α5 = TACAACCTGTGACCCGACAA and GGATGACAAGAGCCAGCTTC.For agrin and laminin transcripts analysis, total RNA was prepared from the spinal cords or skeletal muscles of young (3 to 4 months) and old (24 to 28 months) C57BL/6J mice using the RNEasy kit as per the manufacturer’s instructions . cDNA was generated using the QuantiTect Reverse Transcription system (Qiagen) and transcript levels were determined on a Stratagene MX3000p machine using SYBR green. Primers for the various z agrin splicing isoforms  have been described and validated previously Mice were anesthetized with sodium pentobarbital and then transcardially perfused with phosphate buffered saline (PBS) followed by 4% paraformaldehyde (PFA) in PBS. Muscles were postfixed in PFA for 10 min and used for whole mount analysis.For agrin staining, muscles were first exposed to ice cold methanol (100%) for 10 s and then washed in PBS. Tissue was then incubated successively with blocking solution  overnight at 4°C, primary antibodies diluted in blocking solution for 12–14 hours at 4°C, and fluorophore-labeled secondary antibodies or fBTX  for 2 to 3 hours at room temperature.The primary antibodies used in this study were directed against the following molecules: agrin ; laminin-α4 and laminin-ß2 ; laminin-α5 1∶1000, ; rapsyn −/−lama4 mice, sprouting was defined as an axonal profile that extended beyond ≥2 µms of the endplate; partial or complete denervation was assayed by the apposition of the axon terminals and their AChRs; multiple innervation was identified by counting the number of axons impinging onto each endplate. For agrin conditional animals, altered NMJs were quantified as those that displayed any one of the several dystrophies outlined above, including axon swelling, nerve sprouting, atrophy, fragmentation, and thinning. For axon terminal pair analyses, whole triangularis muscles from conditional agrin mice were imaged and reconstructed. Images were scanned to identify those in which two terminals arose from the same axon and their level of dystrophy was then evaluated relative to control animals.Maximum intensity projections of confocal stacks were generated using Image J or Metamorph 6.03 software . For structural analysis in


To date, detailed analyses of walking patterns using accelerometers during the 6-min walk test (6MWT) have not been performed in patients with chronic obstructive pulmonary disease (COPD). Therefore, it remains unclear whether and to what extent COPD patients have an altered walking pattern during the 6MWT compared to healthy elderly subjects.79 COPD patients and 24 healthy elderly subjects performed the 6MWT wearing an accelerometer attached to the trunk. The accelerometer features  and subject characteristics were assessed and compared between groups. Moreover, associations were sought with 6-min walk distance (6MWD) using multiple ordinary least squares (OLS) regression models. COPD patients walked with a significantly lower walking intensity, lower cadence and increased walking variability compared to healthy subjects. Walking intensity and height were the only two significant determinants of 6MWD in healthy subjects, explaining 85% of the variance in 6MWD. In COPD patients also age, cadence, walking variability measures and their interactions were included were significant determinants of 6MWD .COPD patients have an altered walking pattern during 6MWT compared to healthy subjects. These differences in walking pattern partially explain the lower 6MWD in patients with COPD. The 6-minute walk test (6MWT) is commonly used to assess functional exercise performance in patients with chronic obstructive pulmonary disease (COPD) The 6MWD cannot be confidently predicted from conventional descriptors of COPD, such as the Global Initiative for Chronic Obstructive Lung Disease (GOLD) stage or the Medical Research Council (MRC) scale Walking patterns are generally influenced by the trade-off between the requirements to minimize energetic costs and to maintain stability Detailed analyses of walking patterns during the 6MWT have not yet been performed in patients with COPD. Yentes et al. recently reported gross walking abnormalities in patients with COPD, such as the presence of a limp or shuffle A priori, the authors hypothesized that patients with COPD have a different walking pattern during 6MWT compared to healthy elderly subjects, which is related to the reduced 6MWD in COPD independent of the degree of airflow limitation.Features derived from tri-axial accelerometers attached to the lower back can be used to measure walking variability. Accelerometers may also allow monitoring walking abnormalities in patients with COPD, as was done before in patients with chronic heart failure Patients were recruited prospectively during a three-day pre-rehabilitation assessment period at CIRO+, a centre of expertise for chronic organ failure in Horn, the Netherlands In total, 93 patients enrolled, of which 14 (15%) stopped during both 6MWT. Therefore, 79 COPD patients were included in the analyses . None of the remaining patients received long-term oxygen therapy (LTOT). All measurements were part of routine baseline assessment for pulmonary rehabilitation Participants performed two 6MWTs on consecutive days 2). Patients also underwent physical examination and medical history Post-bronchodilator forced expiratory volume in the first second (FEV1) and forced vital capacity (FVC) were determined using spirometry and reference values were from Quanjer et al. For data analyses of the accelerometer signals 5 seconds at the beginning and end of the test were excluded to be sure that possible group differences in walking pattern were not due to start and/or stop of the 6MWT. Dedicated software written in Matlab(c) was used to analyse the remaining 350 seconds of raw acceleration data. The software included algorithms to calculate the walking intensity, spatio-temporal aspects of gait and medio-lateral stability. Walking intensity was calculated from the integral of the modulus accelerometer output Sample size and power sample size calculations are based on outcomes of Moe-Nilsson et al. using the interstride trunk acceleration variability of fit and frail older adults versus anterior-posterior and vertical directions between frail or fatigued persons and fit persons a posteriori walking variability measures were compared in a subset of healthy subjects and COPD patients who had on average a comparable walking distance (range 6MWD: 560 m–640 m). Differences in walking patterns between the best and worst 6MWT in patients with COPD are described in the A priori, results were considered statistically significant when p-value was ≤0.05.Statistical analysis was done using SPSS software . Data are reported as mean ± standard deviation (SD) or percentages, as appropriate. GOLD stages 1 and 2, and GOLD stages 3 and 4 were combined for further analyses due to the small number of GOLD stage 1 (n = 8) and GOLD stage 4 patients (n = 7). The comparisons were conducted with 1-way analysis of variance or chi-square tests as appropriate. Accelerometer features , subject characteristics  and perceived dyspnea and fatigue (before and after the best 6MWT) were tested in their association with the 6MWD via multiple ordinary least squares (OLS) regression models per group. Previously, different patterns were found for the variability in medio-lateral direction Healthy subjects and COPD patients had a similar gender distribution, age and BMI . As expeAccelerometer features showed that COPD patients walked at a significantly lower intensity and a lower cadence . DiffereHealthy subjects (n = 8) and COPD patients (n = 14) with a 6MWD range between 560 m and 640 m did not differ significantly in 6MWD , walking intensity  and cadence . Nevertheless, the COPD patients maintained to have a significantly lower autocorrelation coefficient in the medio-lateral direction compared to the healthy control subjects .The intensity parameter walking intensity correlated most strongly with the 6MWD in healthy subjects  and COPD patients  . The corThe results of the OLS regression models used to test associations between subject characteristics, accelerometer features and 6MWD for healthy subjects and COPD patients  are summarised in The present study provides the first comprehensive evaluation of qualitative and quantitative measures of the walking pattern during 6MWT in patients with moderate to very severe COPD. It extends previous work on the 6MWT by providing detailed information on walking variability. On average, COPD patients walk with a lower intensity, a lower cadence and show a higher medio-lateral variability during 6MWT in comparison with healthy elderly subjects. The difference in medio-lateral variability remained even if the walking speed was similar. Moreover, walking variability was associated with functional exercise capacity in COPD patients, but not in healthy controls. These results indicate an altered walking pattern in COPD patients.Increased variability in the medio-lateral direction  is an active control strategy to compensate for balance disturbances in order to maintain stability in the anterior-posterior direction (the direction of propulsion). In the present study, walking variability was higher in the medio-lateral direction in COPD patients compared to the control group , suggestA high positive association was found between the intensity parameter walking intensity and 6MWD in both COPD patients and healthy controls . PreviouPreviously, the cadence and the walking intensity have been studied in patients' home-environments to evaluate daily performance in COPD patients or to study the effects of pulmonary rehabilitation The design of this study was cross-sectional. Future investigations using the current methodology should include repeated measures to investigate the effect of a comprehensive pulmonary rehabilitation program on walking pattern in patients with COPD. Indeed, faster walking may be more stable than slow walking Participants who stopped during both 6MWT were excluded from this study, which was necessary to obtain reliable measures of walking variability In conclusion, patients with COPD have a different walking pattern during 6MWT compared to healthy elderly subjects, as objectified by using accelerometer signals. In addition to walking intensity, cadence and walking variability are important variables associated with 6MWD in patients with COPD. These differences in walking pattern partially explain the reduction in 6MWD in patients with COPD.Text S1Intra-individual differences between best and worst 6-min walk tests.(DOCX)Click here for additional data file.Table S1Patients' characteristics for all COPD patients and a subgroup of COPD patients with a 2nd test.(DOCX)Click here for additional data file.Table S2Accelerometer features.(DOCX)Click here for additional data file.


Methanol poisoning is a relatively rare but potentially serious medical emergency. Toxicity results when methanol is successively oxidized to the active metabolites formaldehyde and formic acid. We report a case of a 23-year-old male, a high daily alcohol consumer, who attended the local primary health care centre complaining of sudden visual loss. A presumed diagnosis of methanol intoxication was suggested based on the patient’s visual impairment and the history of alcohol ingestion. Specific therapy was initiated before a definitive diagnosis. Gas chromatographic determination of methanol levels confirmed the initial diagnostic suspicion. In this case, prompt recognition of methanol intoxication and treatment conditioned a favorable clinical outcome. Given that timely diagnosis and antidote administration are crucial issues in terms of prognosis, we underline the necessity for physicians to be alert for entities provoked by rare environmental factors. It is commonly used in a variety of industrial solvents, such as antifreeze, paint remover and in the preparation of adulterated beverages [1] leading to ‘endemics’ of poisoning [2]. Methanol intoxication represents a rare clinical presentation [1] although the total number of cases is probably underreported [3]. It constitutes a severe form of poisoning [1] that, if left untreated, is associated with significant mortality and visual deficits among survivors [3]. Ingestion of only 15 ml of 40% methanol may be fatal [2]. We report a case of visual impairment from methanol poisoning in a 24-year-old Greek male.Methanol or wood alcohol is a colorless liquid with a slightly sweeter odor than ethanol  was 23 mEq/l  [4]. The measured serum osmolality (by freezing point depression) was of 316 mOsm/Kg. The calculated serum osmolarity by the equation 2[Na+ (meq/L)]+[BUN(mg/dL)]/2,8+ [Glucose (mg/dL)]/18 [4] was of 278,66 mOsm/Kg, yielding an osmolar gap of 38,66 mOsm/Kg. Magnetic resonance imaging and computed tomography scan of the brain did not reveal any abnormalities. On admission, the ophthalmologic examination revealed an almost total decline in visual acuity and central scotoma in both eyes. His pupils were normal with a moderate reaction to light. Ocular fundus was bilaterally normal. Since toxicological assays for toxic alcohols were not available in the hospital, blood samples were obtained and transferred to a specialized clinical centre. Thirty minutes after the arrival, the initial laboratory evaluation revealed a complete normal blood count. Serum chemistries disclosed serum sodium of 133 mEq/L, creatinine of 101.6 μmol/l, potassium of 5 mEq/L, chloride of 101 mEq/L, urea of 32mg/dl and glucose of 7.3mmol/l. Liver functions tests were normal. The patient’s arterial blood gas results when he was breathing ambient air were: pH 7.25; PO2 of 118mmHg, PCO2 of 16 mmHg and bicarbonate level of 9 mmol/L. Serum anion gap calculated by the formula: [sodium (Na+)-[chloride (Cl-)+bicarbonate (HCO3-)]. Alcoholics and prisoners are considered high-risk groups that often substitute ethanol with methanol-contained substances [6]. Pure methanol is not toxic, until it is metabolized by alcohol dehydrogenase to formaldehyde and subsequently to formic acid [6]. Accumulation of the later has been reported to be the main cause of the adverse effects typically observed on methanol intoxication such as metabolic acidosis, neurological impairment and ocular injury [6]. Remarkably, the formic acid’s serum concentration level is considered a poor prognostic parameter correlated with high toxicity [7].Methanol intoxication occurs through unintentional or suicidal ingestion as well as after attempted inebriation [1]. In case of concomitant ethanol ingestion, the onset is usually delayed by 24 hours [6]. Clinical manifestations of methanol poisoning are non-specific [5] making the diagnosis challenging [8]. Early signs include abdominal discomfort, nausea, vomiting, and a mild central nervous system depression [1]. Late onset signs include anion gap acidosis, neurological dysfunction and ophthalmological disturbances [5]. Visual symptoms are the most specific clinical features [8] and range from photophobia and a decline of visual acuity to complete blindness [1].Toxical symptoms usually occur after an asymptomatic latent period of 8 to 24 hours following methanol ingestion [5]. Laboratory evidence of metabolic acidosis along with the elevation of anion and osmolar gap are features that suggest a diagnosis of methanol intoxication [8]. However, an isolated normal value of anion or osmolar gap is of limited diagnostic accuracy [6]. This happens because these markers follow a chronological variation. Early after methanol ingestion, a maximum elevation of the osmolar gap usually occurs. Later in the course of poisoning, as metabolic degradation of methanol proceeds, osmolar gap decreases and may be normal while anion gap increases [6].Anion and osmolar gap  are used routinely in the evaluation of patients with a suspicion of toxic alcohol poisoning [6]. The appropriate management relies on the prompt inhibition of enzymatic oxidation of methanol to formic acid through antidote provision (fomepizole or ethanol) [3], which also represents an important prognostic factor associated with recovery from visual impairment [9]. Noteworthy, even though ocular complications are the most specific clinical signs in methanol poisoning, they are underestimated and progress over time even after hospital discharge [10]. Regarding the therapy, although there is a lack of clinical evidence to confirm the superiority of fomepizole over ethanol, the former has been reported to be the antidote of choice in methanol poisoning [6]. Adjunctive hemodialysis and intravenous administration of folinic acid may be useful in order to enhance the elimination and increase the metabolism of formic acid respectively [6].Definitive diagnosis requires measurement of the serum concentration of methanol by the gold standard test which is gas chromatography and confirmation of an elevated methanol level (> 6mmol/L or 20mg/dL) [In this case, the hospital where the patient was admitted was not equipped to perform toxicological assays and a definitive diagnosis would require a waiting for confirmation of blood results. However, the decision to initiate a specific treatment was made only based on the patient’s high daily alcohol consumption history, ocular symptoms and anion and osmolar gap elevation. This case highlights the necessity for physicians to always cultivate a ‘detective judgment’, especially for rare conditions and when patients’ clinical presentation cannot support a likely diagnosis.