The APC is composed of at least 11 subunits and associates with additional regulatory activators during mitosis and interphase cycles. Despite extensive research on APC and activator functions in the cell cycle, only a few components have been functionally characterized in plants.The orderly progression through mitosis is regulated by the Anaphase-Promoting Complex (APC), a large multiprotein EHere, we describe an in-depth search for APC subunits and activator genes in the Arabidopsis, rice and poplar genomes. Also, searches in other genomes that are not completely sequenced were performed. Phylogenetic analyses indicate that some APC subunits and activator genes have experienced gene duplication events in plants, in contrast to animals. Expression patterns of paralog subunits and activators in rice could indicate that this duplication, rather than complete redundancy, could reflect initial specialization steps. The absence of subunit APC7 from the genome of some green algae species and as well as from early metazoan lineages, could mean that APC7 is not required for APC function in unicellular organisms and it may be a result of duplication of another tetratricopeptide (TPR) subunit. Analyses of TPR evolution suggest that duplications of subunits started from the central domains.The increased complexity of the APC gene structure, tied to the diversification of expression paths, suggests that land plants developed sophisticated mechanisms of APC regulation to cope with the sedentary life style and its associated environmental exposures. Substrates that have a destruction box (D-box), KEN-box or A-box motifs are recognized and ubiquitinated by the APC [Cell proliferation is controlled by an universally conserved molecular machinery in which the key players are cyclin-dependent kinases (CDK) and cyclins (reviewed in ). Eukary cyclins . Degrada cyclins -8. APC i cyclins . The APC cyclins ,11. The  the APC -14.1-S-phase and in post-mitotic differentiated cells [The APC has important functions in mitosis, meiosis, Ged cells ,16. The ed cells ). The pred cells ,19.Although the role of the APC in controlling cell-cycle progression has been extensively investigated, has recent work shown the unexpected presence of APC subunits in differentiated tissues, in particular in the nervous system of vertebrates (reviewed in ). In plaIn this article, we describe thorough searches for the predicted sequences of the APC subunits and the activators CDC20/CCS52 in plant sequence databases. Our results indicate that land plants and green algae orthologs have an ancient evolutionary origin. We present phylogenetic analysis of TPR subunits and activators, and their orthologs from other plants. An evolutionary analysis of Arabidopsis TPR domain suggests that tandem regions could have been created from duplication of internal sequence domains. Overall, our data support the proposal that APC subunits and activators have been conserved in the course of evolution. However, while other eukaryotes like fungi and metazoans have only one copy of each APC subunit and its regulators, gene duplication of different subunits have occurred in Arabidopsis, rice and poplar, and in other plant genomes as well. An attractive proposal is that gene duplication of APC subunits may result in the formation of APC subcomplexes that evolved to assume restricted specialized roles during plant development. Indeed, in Arabidopsis, duplication of the CDC27 subunit led to substantial sequence divergence and specialization . Here weIn order to carry out the cross-species comparison of the APC and activators, we have searched for homologous sequences in the Genomic Research (TIGR) Rice genome database and DOE Join Genome Institute (JGI) Poplar genome database. The sequences of the Arabidopsis APC subunits have been published ,26,27. EAtCDC27a and AtCDC27b [PtCDC27_1 and PtCDC27_2. However, a careful inspection of the PtCDC27_2 deposited sequence revealed a stop codon TAG at nucleotide position 1471-1473. This fragment was amplified and sequenced and the presence of the stop codon was discarded. The expression of both genes in leaves was confirmed by quantitative PCR  and APC11 (VvAPC11_1 and VvAPC11_2) genes in grapevine [PtAPC13 were found in the poplar and grapevine genomes, a feature that has not been reported in any other eukaryotic species so far.Two CDC27 homologs have been identified in Arabidopsis AtCDC27b . In popls genome . Howeversubunits . We examrapevine . Two homOsAPC1 was not found in the TIGR database, but a partial CDS was identified in the Gene Index database  showed major differences from conserved structures of other CDC20; the CDS does not have the C-box sequence element (consensus DR(F/Y)IPxR) that was first identified in the N-terminal region of CDC20, although it is conserved in all known APC/C co-activators  that occupies the C terminus of APC activators and the APC subunit APC10. Comparing with EST databases, all sequences were deduced and the IR-tail was identified  and could represent another layer of complexity in the organization and function of the APC. We selected only a single variant for further analysis.Arabidopsis contains three CCS52 genes, 20 genes . In addi0_6 At5g2945 showeDuplication of cell-cycle machinery components is rare in metazoans but it is a widespread phenomenon in plants -38.It haThe close similarity and presence of putative subunits and paralogs prompted us to investigate the genome distribution of plant APC/activators. APC genes were found in the five Arabidopsis chromosomes, 10 of the 12 rice chromosomes and 10 of the 19 poplar chromosomes (and seven in scaffolds)  and Trebouxiophytes  have apparently lost the APC7 gene, but the Prasinophytes  and land plants retained this TPR subunit. These data also suggest that the primitive ancestor of Chlorophyceae and Trebouxiophyceae classes lost APC7 gene during green algae evolution , while two other clades included the CCS52A and CCS52B sequences (except CmCCS52). The CDC20 clade is divided into two subfamilies: one with S. moellendorffii and P. patens and another with algae and land plants. The number of CDC20 copies varies according to species. Arabidopsis has six copies, rice three, poplar five, grapevine four, sorghum three, S. moellendorffii three and P. patens four. The CCS52A clade contains more duplications than the CCS52B clade. All algae CCS52 genes, two copies of PtCCS52A, AtCCS52A and SmCCS52, and three copies of PpCCS52 genes are grouped into the CCS52A branch.The Arabidopsis genome codes for six CDC20 genes and three CCS52 genes. In addition, CCS52 genes have been divided into two types (A and B) on the basis of functional and sequence analysis . To gainP. patens and S. moellendorffii is interesting because these species are at the base of plant evolution where divergence into A-type and B-type has not occurred yet. Like Arabidopsis, poplar has two copies of CCS52A; however poplar has an additional copy of CCS52B. Analyses of poplar gene evolution must take into account the most significant event in the recent evolution of the genus: a genome-wide duplication event at approximately 65Mya and is still detectable over approximately 92% of the genome [Curiously, only one copy of the CDC20 and CCS52 (possibly a primitive A-type) genes was found in algae genomes. The absence of CCS52B type genes in algae, e genome . Based oProgress in comparative genomics allows assessing the impact of gene and genome evolution on the appearance of novel biological functions and their effect on organismal complexity . The impOsCDC23_2 has 5 exons  [OsAPC11_1 and OsCDC26 mRNA levels increased in darkness. Interestingly, the two OsCDC23 genes are differentially expressed in etiolated roots. While OsCDC23_1 mRNA levels did not change in roots grown in the dark or light, OsCDC23_2 mRNA levels in roots were much lower when rice plants were grown in the dark. Similarly, in etiolated roots OsAPC11_1 mRNA levels increased while OsAPC11_2 levels decreased. These results could indicate that, following gene duplication, the APC11 and CDC23 genes are assuming new functions in rice.Growth response to dark is part of an integrated developmental change throughout all the plant organs . In Arabll cycle . G1-S trpre-RCs) . Almost OsCCS52A and OsCCS52B exhibited high levels of mRNA in darkness and OsCDC20_3 showed a lower level. One hypothesis for the high mRNA levels of CCS52 genes is that the APCCCS52 acts as a negative regulator of CYCA2;3/CDKA;1 complex in Arabidopsis [The activators bidopsis . The CYCbidopsis .OsAPC1 and OsAPC5, which were present in higher amounts in the inflorescence. The duplicated OsCDC23 genes displayed similar expression profiles in the seed tissues examined. However, the OsAPC11 gene seems to be differentially expressed; OsAPC11_2 showed reduced amounts of mRNA in the inflorescence when compared to the pericarp and endosperm. Using the Genevestigator Analysis tool, we compared the expression levels of the APC genes with qRT-PCR results  http://rice.plantbiology.msu.edu, Populus trichocarpa Nisqually-1 - DOE Join Genome Institute http://genome.jgi-psf.org/Poptr1_1/Poptr1_1.home.html, Sorghum bicolor BTx623 - DOE Join Genome Institute http://genome.jgi-psf.org/Sorbi1/Sorbi1.home.html, Vitis Vinifera PN40024 - Genoscope http://www.genoscope.cns.fr/externe/GenomeBrowser/Vitis/, Ostreococcus sp RCC809 - DOE Join Genome Institute http://genome.jgi-psf.org/OstRCC809_1/OstRCC809_1.home.html, Volvox carteri - DOE Join Genome Institute http://genome.jgi-psf.org/Volca1/Volca1.home.html, Chlorella sp. NC64A - DOE Join Genome Institute http://genome.jgi-psf.org/ChlNC64A_1/ChlNC64A_1.home.htmlMicromonas sp NOUM17 (RCC299) - DOE Join Genome Institute http://genome.jgi-psf.org/MicpuN3/MicpuN3.home.html, Physcomitrella patens - DOE Join Genome Institute http://genome.jgi-psf.org/Phypa1_1/Phypa1_1.home.html, Selaginella moellendorffii - DOE Join Genome Institute http://genome.jgi-psf.org/Selmo1/Selmo1.home.html and Cyanidioschyzon merolae http://merolae.biol.s.u-tokyo.ac.jp databases using the BLASTP and TBLASTN programs. In addition, Arabidopsis APC and activator sequences were used to search against Zea mays, Medicago truncatula and Saccharum officinarum EST databases at Gene Index DFCI http://compbio.dfci.harvard.edu/tgi/cgi-bin/tgi/Blast/index.cgi. Sequences with significant similarity to Arabidopsis proteins were downloaded into our database and annotated accordingly. Sequence conservation analysis of APC and activators genes was conducted using the BLAST tool with default curve calculation parameters; amino acids sequence "conservation similarity" of 50%. Chromosomal locations of APC subunits and CDC20/CCS52 genes were obtained using the BLAST server and additional physical localization tools of each genomic browser.Searching of multiple databases was performed to find members of APC, CDC20 and CCS52 in Arabidopsis, rice and polar. The strategy to obtain each gene in a genome was the following; Arabidopsis sequences have been published , but addhttp://smart.embl-heidelberg.de was used to confirm whether each predicted proteins sequence maintains conserved domains and motifs. The TPRpred http://toolkit.tuebingen.mpg.de/tprpred search program was used to confirm the TPR domain in specific sequences [The SMART database equences .Phylogenetic analysis of TPR subunits and activators CDC20 and CCS52 was carried out using the Neighbor-Joining method in the Molecular Evolutionary Genetics Analysis software package - MEGA4 . AlignmeExon-intron information was obtained from TAIR, TIGR and JGI databases. The sequences of the repeating domains were extract and aligned to each other using the Smith-Waterman alignment tool in the EMBOSS package and default parameters ,57. ThisOryza sativa L. cv. Nipponbare) were disinfected with 5% sodium hypochlorite for 20 min and thoroughly washed with water. For darkness treatment, seedlings were grown hydroponically (0.5X Hoagland's solution) in a greenhouse at 25-28°C for 2 weeks. The control group was grown with a photoperiod of 12 h light/12 h dark and the etiolated group was grown in darkness. For auxin treatment, seedlings were grown in Petri dishes (0.5X Hoagland's solution agar) in the greenhouse at 25-28°C for 2 weeks. Seedlings were transferred to new Petri dishes and incubated for 2 and 8 h in either water or 2 μM NAA solution. Harvested seedlings were frozen in liquid nitrogen immediately and store at -70°C until RNA isolation. Leaf poplar RNA was provided by Rodrigo T. Lourenço .Rice seeds  jp/PLACE/. For eacjp/PLACE/. This apThe ratio of nonsynonimous to synonymous nucleotide substitution rates (Ka/Ks) was calculated as described by Siltberg and Liberles Oryza sativa japonica subspecies was used to prepare all generic material. Total RNA was extract from materials according to [® Reverse Transcription Reagent kit (Perkin-Elmer Applied Biosystem) on the GeneAmp 9700 thermocycler (Applied Biosystems) under standard conditions. Transcript levels were determined by qRT-PCR using a 7500 Real-Time PCR System (Applied Biosystems). For poplar gene expression analysis, cDNA synthesis and qRT-PCR were performed as described for rice. The data were first normalized to the level of expression of Os28 S, OsActin and PtActin for each RNA sample. Primers used for real-time RT-PCR were designed in gene specific region using Primer Express V3.0. Gene primer sequences used in the qRT-PCR analysis are listed: Os28 S forward (F) 5' GCGAAGCCAGAGGAAACT 3', Os28 S reverse (R) 5'GACGAACGATTTGCACGTC 3', OsActin F 5' CTTCATAGGAATGGAAGCTGCGGGTA 3', OsActin R 5' CGACCACCTTGATCTTCATGCTGCTA 3', OsAPC1F 5' CTTGAGCTCTGCTTGCATCT 3', OsAPC1 R 5' GCTTACAGCCATCTGCAGTC 3', OsAPC2 F 5' AATGCTGGGGACAATCTTCT 3', OsAPC2 R 5' TAATGGGTCTGCTTCCACAG 3', OsCDC27 F 5' AGCGACTTGCTACCTTCACA 3', OsCDC27 R 5' TTGACAGGACACAAGGCTTC 3', OsAPC4 F 5' CGACAAGGATGGCCTGTTAT 3', OsAPC4 R 5' GAAGCGCTTGAAAATTCCTG 3', OsAPC5 F 5' TTCGTTGGTCTATGCAACCT 3', OsAPC5 R 5' AACGGGAACTTCTCTTCAGC 3', OsCDC16 F 5' ACAGGAGGAGGGTGATCAAG 3', OsCDC16 R 5' GATTTTGCTTGCGTGAAGAA 3', OsAPC7 F 5' AGACTTCAGGGGAGCTCAAG 3', OsAPC7 R 5' TTGAGAGCTTTTGCAGACTGA 3', OsCDC23_1 F 5' CGCTGAAGCTTAATCGAAAGT 3', OsCDC23_2 R 5' GACCAAGACCATACCAAGCA 3', OsCDC23_2 F 5' CAGTTCTGGTGGAATCTGTCA 3', OsCDC23_2 R 5' CAAGATGTGCACTAGCAAGGA 3', OsAPC10 F 5' TGATCCCCGAGAAACATTC 3', OsAPC10 R 5' GAAGTGAAGTGAAATGGCTGAT 3', OsAPC11_1 F 5' CCAGGATGAAACCTGTGGTA 3', OsAPC11_1 R 5' GTTTGAGAATTGACCCACTTGA 3', OsAPC11_2 F 5' AAACATGCGGCATATGCA 3', OsAPC11_2 R 5' AAGTGGCGTAGATGTCTGAGAA 3', OsCDC26 F 5' ATCGGCCTCCCTACCAT 3', OsCDC26 R 5' GAGGAGAGGCTAGGGTTTGG 3', OsAPC13 F 5' TTGTGCTGGTTGGCTTTC 3', OsAPC13 R 5' CGTCCTCATCGACATCGT 3', OsCDC20_1 F 5' ATGATCGGTGCATCAGGTT 3', OsCDC20_1 R 5' GGCAAAGAACACGAGCAGT 3', OsCDC20_2 F 5' CCTGTCCGGAATAAACCTGT 3', OsCDC20_2 R 5' CACTCGATCTCATCGGAGAA 3', OsCDC20_3 F 5' TTCACATTTGGGATGTGTCC 3', OsCDC20_3 R 5' TACCACCACCTCCAGTTGC 3', OsCCS52A F 5' GCCCCAGGAAGATCCCTA 3', OsCCS52A R 5' TGCTGCATGCATTCCATAA 3', OsCCS52B F 5' GTCACCAAGCTCTGCGATT 3', OsCCS52B R 5' TCCTCCCATGTTCCTAATCC 3', PtActin F 5' GGTCAAGGCTGGGTTTGCT 3', PtActin R 5' TCGCCAACATAGGCATCTTTT 3', PtAPC10 F 5' ATGACCCTAGGGAAACATTT 3', PtAPC10 R 5' AAAAGGCTGATGCGGAAAAG 3', PtCDC27_1 F 5' TGGCCTTCCAAACCTGTCAT 3', PtCDC27_1 R 5' GGCATGCTAAGATTGGAACCA 3', PtCDC27_2 F 5' AGCCGGATGTTATTTGCAAAA 3', PtCDC27_2 R 5' ATGCCGCTTCAGCTTCATTT 3', PtCDC20_1 F 5' TTTATTCCAAACCGGTCAGC 3', PtCDC20_2 R 5' TCGGTTCATGTTCAAGGATTC 3', PtCCS52A F 5' TTCCCCCTCGAGACCTATTT 3', PtCCS52A R 5' CATTGGGATTGTCCTCCTTC 3', PtCCS52B F 5' CGTATCATCACCCAGAGCAA 3', PtCCS52B R 5' TCGTTGCCTCCTTCTTTAACA 3'. Data were analyzed using 7500 SDS software V1.4 (Applied Biosystems).rding to . After tMFL carried out the molecular genetic studies, analyzed the data and drafted the manuscript. NBE, CP, RS, CR, TB, LV, AE and ACO carried out experiments and analyzed the data. ASH and PCGF conceived of the study, and participated in its design and coordination and helped to draft the manuscript. All authors read and approved the final manuscript.Text file containing amino acid sequences in FASTA format for APC and activators genes used in these analyses.Click here for filePtCDC27_2Confirmation of functionality of Poplar . A, sequencing of genomic DNA corresponding to PtCDC27_2 locus. Red box shows the actual triplet. B, Real-time PCR detection of PtCDC27_2 mRNA, as well of PtCDC27_1, PtCDC20_1, PtAPC10, PtCCS52A1_1 and PtCCS52B.Click here for fileOsAPC1Gene Index  sequence of . An EST was recovered from the Gene Index Project, showing part of (upper). The coding region was underlined and stop codon was marked in red. This sequence was translated in silico (lower).Click here for filePtAPC5 and fragments from the 3'regions of genes PtAPC4 and PtAPC5 were found in the EST databaseFragments from 5'region of . The additional sequence of OsAPC4 (not present in Arabidopsis nor poplar), product of incorporation of an intron, is boxed in yellow. Differences in 5'region of OsAPC5 are also highlighted in yellow.Click here for fileCDC16 and APC11_2 genes between Arabidopsis, poplar and riceComparison of 5'region of . The additional sequences are boxed in yellow.Click here for fileAtCDC20_6Re-sequencing of 5'region of .Click here for fileCorrection of APC activator sequences. Gray boxes: The conserved APC interacting motifs C-box, CSM, IR-tail, CBM. Differences are boxed in yellow.Click here for fileAPC subunits in plant genomes and red algae. Cr, Chlamydomonas reinhardtii; Vc, Volvox carteri; Csp, Chlorella sp; Msp, Micromonas sp; Osp, Ostreococcus sp; Cm, Cyanidioschyzon merolae; Pp, Physcomitrella patens; Sm, Selaginella moellendorffii; Bd, Brachypodium distachyon; Sb, Sorghum bicolor; Mt, Medicago truncatula; Vv, Vitis vinifera; Cp, Carica papaya; At, Arabidopsis thaliana; Os, Oryza sativa; Pt, Populus trichocarpa; Abbreviations: NI, not identified.Click here for fileChromosomal locations of rice and poplar APC subunits and activators. Chromosome numbers are indicated at the bottom of each chromosome. Paralogs are linked by dashed lines. A, chromosomal positions of genes in rice. B, chromosomal positions of genes in poplar. Seven poplar genes were assigned to scaffolds.Click here for fileNeighbor-joining tree inferred from Poisson-corrected evolutionary distances for genes involved in the cell cycle and multiple sequence alignments of plant TPR subunits proteins. The TPR subunits (A) and activator gene family (B). The abbreviations of species names are as follows: At, Arabidopsis thaliana; Pt, Populus trichocarpa; Os, Oryza sativa; Vv, Vitis vinifera; Sb, Sorghum bicolor; Pp, Physcomitrella patens; Sm, Selaginella moellendorffii; Msp, Micromonas sp; Osp, Ostreococcus sp; Csp, Chlorella sp; Vc, Volvox carteri; Cm, Cyanidioschyzon merolae; Zm, Zea mays; So, Saccharum officinarum; Mt, Medicago truncatula.Click here for filePhylogenetic relationships between Plants and algae.Click here for fileOsCDC23_2 genomic sequence. Exon (yellow boxes), intron and possible exon (gray boxes). Sequencing result - frameshift discarded.Click here for filePattern of internal domain duplications in Arabidopsis TPR subunits. The intensity of shading reflects the alignment score, with a dark shading for higher scores. The numbers on each axis indicate the domains in N-to-C terminal orientation within the repeat.Click here for fileExpression patterns of rice APC genes based on Genevestigator. Expression patterns of OsAPC2, OsAPC7, OsAPC10, OsAPC11_1 and OsAPC13 are shown in different tissues (A) and developmental stages (B).Click here for fileSummary statistics for Ka and Ks. http://services.cbu.uib.no/tools/kaks.Click here for filePromoter analysis. The upstream 1000 bp regions of all rice APC subunits genes were considered to contain the full length promoters. Table 2, motifs found in the promoters and their occurrences. Table 3, motifs found in each duplicated gene .Click here for file


The presence of abnormal hematologic findings such as lymphopenia, thrombocytopenia, and pancytopenia were diagnosed in severe cases of avian influenza A H5N1. Whether direct viral dissemination to bone marrow (BM) cells causes this phenomenon remains elusive. We explore the susceptibility of the two stem cell types; hematopoietic stem cells (HSCs) and mesenchymal stromal cells (MSCs) isolated from human BM cells or cord blood, to infection with avian H5N1 viruses. For the first time, we demonstrated that the H5N1 virus could productively infect and induce cell death in both human stem cell types. In contrast, these activities were not observed upon human influenza virus infection. We also determined whether infection affects the immunomodulatory function of MSCs. We noted a consequent dysregulation of MSC-mediated immune modulation as observed by high cytokine and chemokine production in H5N1 infected MSCs and monocytes cocultures. These findings provide a better understanding of H5N1 pathogenesis in terms of broad tissue tropism and systemic spread. The highly pathogenic avian influenza A virus of the H5N1 subtype was originally endemic to poultry, but crossed the avian-human species barrier. It has emerged as a highly fatal infectious disease in the human population with a 60% mortality observed in more than ten countries, as reported to the World Health Organization since 2003 + cell is a population that includes hematopoietic stem cells (HSCs), myeloid, erythroid and lymphoid progenitors Bone marrow (BM) is an important source from which progenitor cells are generated. It contains two types of progenitor cells; hematopoietic stem cells and non-hematopoietic stem cells which can differentiate into blood cells and cells of mesenchymal lineages , respectively + HSCs and MSCs. Sialic acid (SA) receptors on the target cells surface were believed to be the major receptor mediating virus binding and entry. H5N1 viral infection induced cell death in both CD34+ HSCs and MSCs. We also observed that pro-inflammatory cytokines and chemokines were enhanced in cocultures of MSCs and primary monocytes indicating that H5N1 infection altered the immunomodulative effect of MSCs.The fact that BM is a rich source of progenitor cells + expressing hematopoietic stem cells (CD34+ HSCs) were isolated from the above two sources, whereas, Mesenchymal Stromal Cells (MSCs) were BM derived. Stem cells were isolated no later than 15 hours (h) after collection of the material. All samples for the present study were obtained with written informed consent from all the donors as per the approval of Institutional Review Board, Faculty of Medicine Ramathibodi Hospital, Mahidol University, Thailand.Umbilical cord blood (CB) was obtained from full term newborns and collected in sterile collection bags containing anti-coagulant citrate-phosphate dextrose. Bone marrow (BM) samples were aspirated from the posterior superior iliac spine of healthy donors. The CD34+ HSCs were isolated from both CBMCs and BMMCs using CD34 MACS Microbeads  following the manufacturer's instructions. The efficiency of CD34+ HSCs purification was determined by staining of isolated cell surface markers with phycoerythrin (PE)-conjugated anti-CD34 monoclonal antibody  for 30 min at 4°C. Cells were washed in PBS and analyzed by Flow Cytometry  on an instrument equipped with an argon laser tuned at 488 nm. The percentage of obtained CD34+ HSCs ranged from 98% to 99% in all of the purified CB and BM samples. Cells expressing >98% CD34 antigen were cultured in Stemline II media  supplemented with specific hematopoietic growth factor cocktail that included 50 ng/ml stem cell factor, 50 ng/L rh-IL-6 and 50 ng/L rh-IL-3 (R&D Systems). CD14+ monocytes used in the cocultures study were isolated as previously described + monocytes were isolated by using CD14 MACs Microbeads. Monocytes were cultured in RPMI 1640 media  supplemented with 10% fetal bovine serum (FBS), 2 mM L-glutamine and 1% penicillin-streptomycin (Pen/Strep).Mononuclear Cells (MCs) were isolated from both CB (CBMCs) and BM (BMMCs) using IsoPrep  density centrifugation. CD346 cells/ml of BMMCs were cultured in DMEM complete medium supplemented with 10% FBS and 1% Pen/Strep at 37°C, 5% CO2. After 72 hours of cultivation, non-adherent cells were discarded and this process was repeated every four days. Upon reaching 90% confluency, MSCs were trypsinized by 0.05% trypsin-EDTA and passaged for the next expansion.BMMCs were separated by density gradient centrifugation with IsoPrep. Briefly, 10 ml of heparinized bone marrow samples were mixed in with an equal volume of Dulbecco's Modified Eagle's Medium (DMEM)  and centrifiuged at 1000 g for 30 min at room temperature. The interface mononuclear cells were collected and washed twice with DMEM. Total cell count and viablility were evaluated by 0.2% Trypan blue. A total of 2×105 each of HSCs and MSCs were incubated with fluorescein isothiocyanate (FITC)-labelled lectins Sambucus nigra agglutinin (SNA) and Peridinin Chlorophyll Protein Complex (PerCP)-labelled Maackia amurensis agglutinin (MAA)  which primarily detects SAα2,6galactose and SAα2,3galactose, respectively To identify sialyoligosaccharides reactive with SAα2,3Gal- or SAα2,6Gal-specific lectins, 1×10+ HSCs, cultured cells were washed with serum free media and allowed to adsorb virus for 1 hour for each type of viruses at a multiplicity of infection (MOI) of 1 and 10. All experiments with H5N1 virus were performed inside a bio-safety level 3 facility by trained researchers. Following adsorption, cells were again washed with serum free medium and cultured in Stemline II media supplemented with specific hematopoietic growth factor cocktails. For H1N1 and H3N2 virus infection, the same media was used but contained 0.02 ug/ml of TPCK trypsin (Sigma-Aldrich) was added into the wells The different subtypes of influenza A virus: HPAI H5N1 (A/open-billed stork/Nakhonsawan/BBD0104F/04) and human influenza A viruses; H1N1 (A/WS/33) and H3N2 (A/Hong Kong/8/68) were propagated and quantified as previously described A fragment of the HA gene covering the multibasic cleavage site had been modified to a low-pathogenic sequence was cloned into pHw2000 plasmid, sequenced, and used for construction of reverse genetic (rg) viruses as described previously 2 overnight. Freshly isolated 3×105 CD14+ monocytes were added to each well (MSC:monocyte ratio 1∶5). Controls included monocultured MSCs and monocytes.MSCs were seeded the day before the experiment in 24-well flat-bottom plates in RPMI 1640 supplemented with 10% FBS and 1% Pen/Strep at 37°C, 5% CO+ monocytes were washed with serum free media and then, infected with H5N1 virus at an MOI of 0.04, and incubated at 37°C for 1 hour. Untreated cells were used as a control. Following infection, the inoculum was removed, cells were washed with serum free media and then 1 ml of RPMI 1640 media supplemented with 10% FBS was placed into the wells. Cytokines produced from supernatants of infected cocultures, MSCs monoculture and monocytes monoculture were determined at 24 hours post infection by a Bio-plex Pro Human Cytokine Assay.Confluent monolayers of cocultures, MSCs and CD144 cells grown on a slide culture. At 24 hours post infection (p.i.), cells were fixed with 4% paraformaldehyde in PBS and permeablised by cytofix/cytoperm solution (BD Biosciences). Viral nucleoprotein (NP) was stained with FITC-conjugated antibody (green) (DAKO). CD34+ cells were counterstained with phycoerythrin (PE)-conjugated anti-CD34 monoclonal antibody (red) (Miltenyi Biotec). The stained cells were analyzed using a lasers canning confocal microscope  at the Division of Medical Molecular Biology, Office for Research and Development, Faculty of Medicine Siriraj Hospital, Mahidol University.An immunofluorescence assay was performed on 2.5×10+ and CD14+ cells using PE and PerCP-conjugated specific monoclonal antibodies, respectively, according to the manufacturer's instructions . For detection of viral antigen, all infected cells were incubated with FITC-conjugated NP antibody (green) . These cells were washed, resuspended in 3.7% formaldehyde prior to analysis by flow cytometry.The surface markers of cells were analyzed to detect CD347 copies) containing the respective cloned gene target. The results were standardized due to the variable quantities of RNA and cDNA and finally expressed as numbers of target genes.Infected MSCs were harvested at various time points, total viral RNA was extracted by use of an RNeasy minikit (Qiagen) following the manufacturer's instructions. Cells were washed for three times with PBS to remove free virions. cDNA was synthesized from viral genomic RNA using oligodT primers and AMV reverse transcriptase (Promega). These cDNA samples were used as template. The M gene of the H5N1 virus was amplified as previously described + HSCs and MSCs at various time points. The viral titers in supernatants were quantified by plaque assay. In brief, confluent monolayers of MDCK cells were inoculated with 10-fold dilutions of H5N1 virus and incubated at 37°C for 1 h. The inoculum was removed, and cells were washed and overlaid with plaque medium containing 2% agarose. After two days, cells were stained with 1.25% crystal violet, and the plaque numbers were evaluated.Culture supernatants were collected from infected CD345 cells/well on a 24-well plate at an MOI of 1 or 10, CD34+ HSCs and MSCs were cultured in a specific media as previously described for 16 h. Parallel samples were treated with DNaseI as a positive control. Apoptotic cells were characterized by positive terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling (TUNEL) staining according to the protocol provided by the manufacturer. . Briefly, 16 h after infection with H5N1, H1N1, or H3N2, cells were fixed with 4% paraformaldehyde and permeabilised by cytofix/cytoperm solution (BD Biosciences). After that, cells were then incubated in the labeling reaction mixture containing terminal deoxynucleotidyl transferase enzyme, TMR red-conjugated nucleotide at 37°C for 1 hour and cells were washed with PBS several times. To detect viral antigen after TUNEL staining, cells were incubated at 4°C for 30 min with FITC-conjugated anti-NP antibody as mentioned earlier. Both CD34+ HSCs and MSCs were counterstained with DAPI, and then imaged with a laser scanning confocal microscope .Following viral infection of 1×10Cytokine protein levels were measured (at 24 hours p.i.) by a Bio-Plex Pro Human Cytokine Assay  following the manufacturers' instructions. Cytokine levels were measured in triplicate and compared against a standard curve.p<0.05The Statistical analysis was performed on SPSS Statistics 17.0 using one way analysis of variance (ANOVA) to compare susceptibilities of stem cells to H5N1 infection and to H1N1 or H3N2 infections with statistical significance set at + HSCs and MSCs with MAA and SNA specific for SAα2,3-Gal and SAα2,6-Gal, respectively. We demonstrated that CD34+ HSCs and MSCs expressed receptors of both avian  and human  influenza viruses  on the cell surface is widely recognized as an influenza virus receptor, which can mediate influenza virus binding and fusion. SA linked to galactose  was distributed in the avian gastrointestinal tract, and human lower respiratory tract, whereas, SAα2,6-Gal was expressed in the human upper respiratory tract  viruses . MDCK an+ HSCs from BM and CB were isolated, purified, and then infected with H5N1 virus as described in the + HSCs when infected with live H5N1 virus but not heat-killed virus (+ HSCs were completely resistant to human influenza viruses (H1N1 and H3N2) as shown in immunofluorescent staining in P value <0.05 . MSCs were infected with H5N1, H1N1 and H3N2 viruses. Surprisingly, MSCs were highly susceptible to H5N1 virus which live virus was required . The susue <0.05 . These f+ HSCs after infection. Apoptotic cells were characterized by positive terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling (TUNEL) staining . TUNEL assay confirmed that live H5N1 virus could induce apoptosis in both CD34+ HSCs and MSCs, whereas, human influenza viruses and viral proteins of heat-killed H5N1 could not . We suspected that this was related to apoptosis of these cells. With this thought, we investigated the fate of MSCs and CD34ould not . Virus-iould not . The perpoptosis . This da+ monocytes  onocytes . The cheonocytes . IL-6, Monocytes . In contonocytes . These f+ HSCs and MSCs by other persistent viruses, such as Measles virus (MV) Details regarding the pathogenesis of H5N1 are limited. H5N1 infection is not restricted to respiratory organs, and this systemic spread of the virus makes H5N1 unique from other human influenza viruses. Detection of viral RNA in BM upon autopsy and the presence of hematologic abnormalities in severe H5N1 cases increased our interests in H5N1-mediated BM suppression. It is therefore important to investigate viral tropism in BM cells. Previous studies of direct infection of CD34+ HSCs and MSCs from the BM and CB of healthy donors. Using different techniques including flow cytometry, quantitative RT-PCR, plaque assay, immunofluorescence and confocal microscopy, we demonstrated that CD34+ HSCs and MSCs supported a productive infection of H5N1 virus, although to a lesser degree in CD34+ HSCs. Both stem cells were resistant to human influenza virus (H1N1 and H3N2) infection, even though these two cells expressed both α-2,3 SA and α-2,6 SA, receptors for avian and human influenza viruses, respectively. Our findings suggested that H5N1 virus had BM tropism and CD34+ HSCs and MSCs could serve as key amplifiers of virus within the BM compartment. The mobilizing property of these two cells could support a spread of virus throughout the body et al. et al. In the present study, we first demonstrated that H5N1 virus could directly infect primary human BM progenitor cells: HSCs and MSCs. Viral infection induced cell death and affected the immune modulation activity of MSCs. Our obtained data could better explain viral pathogenesis in extra-pulmonary organs. We started with isolation of CD34+ HSCs to H5N1 virus. Previous studies have demonstrated that H5N1 could infect various targets in particular cells of the immune system in vivo, and the receptor switching of avian to human receptors enabling H5N1 to evade the virus-neutralizing effects of mucins containing α-2,3 SA The susceptibility of each cell type to H5N1 infection is partly dependent on SA receptors since some α-2,3 SA and 2,6-SA expressing cells are unable to support efficient infection and replication of virus in vitro+ HSCs. Apoptosis observed in a majority of infected MSCs was likely due to direct infection, whereas, apoptosis in CD34+ HSCs might be induced by paracrine factors as apoptotic signals were observed in uninfected CD34+ HSCs population  which IL-6 is partially involved in this action + monocytes cocultures were up-regulated, whereas this effect was not seen in MSCs or monocytes monoculture. Elevated IL-6 may enhance inflammation as shown in previous studies The pathogenesis of the H5N1 virus was characterized by broad tissue tropism, systemic replication and hypercytokinemia et al., previously documented that both hypocellular conditions and RHS in BM were the most prominent pathologic features observed in fatal H5N1 cases Hyperactivation of cytokines, including TNF-α, soluble IL-2 receptor, IL-1 and IL-6 may play a role in the pathogenesis of reactive hemophagocytosis syndrome (RHS) Besides BM tissue, MSCs have been identified in many other organs with similar functions including the lung + HSCs and MSCs compared to human influenza viruses in vitro. Apoptosis was a direct result of infection in MSCs, whereas, it might be induced by paracrine factors in CD34+ HSCs. In addition, H5N1 infection could interfere with MSC-mediated immune modulation by shifting immune modulator production towards enhancers, which, in turn, amplifies the cytokine response by the influx of circulating monocytes and macrophages. Our explorations provide an insight into H5N1 pathogenesis in terms of aggressive systemic infection and hematologic abnormalities in patients with severe H5N1 infection.In summary, we demonstrated that H5N1 virus more efficiently infected and replicated in CD34Figure S1+ cells to different H5N1 strains. Infection is determined by using a specific marker (Nucleoprotein), and detected by flow cytometry. Cells were infected by different strains of avian and human influenza viruses at an MOI of 10 for 24 h. The results were obtained individually from three different experiments and are presented as means plus standard errors. *P<0.05 indicates statistically significant differences between H5N1 infection and human influenza viruses infection (H1N1 and H3N2).Susceptibility of CB-derived CD34(TIF)Click here for additional data file.Figure S2Viral production of H5N1-infected MSCs at various doses and incubation times. Avian influenza H5N1 could infect and replicate in MSC. Data are plaque-forming units in the supernatant of MSCs infected with H5N1 at various MOI for 1 h, washed, and incubated for indicated time points.(TIF)Click here for additional data file.Figure S3P<0.05 indicate statistically significant differences of rgPR8-H5 compared with rgPR8-H5monobasic, and rg-PR8 viruses.High susceptibility of MSCs was restricted only to HPAI H5N1. MSCs were infected with rgPR8-H5, rgPR8-H5monobasic, and rg-PR8 which represent HPAI H5N1, LPAI H5N1, and H1N1 viruses, respectively at MOI 1 for 24 hours. LPAI H5N1 is the reverse genetics (rg) virus bearing monobasic amino acids at HA cleavage site as described in (TIF)Click here for additional data file.Figure S4+ HSCs were infected with H5N1 virus and (B) MSCs were infected with both avian and human influenza viruses at an MOI of 10. After 18 h, cells were fixed, permeabilized and multiple stained with TUNEL, NP and DAPI and determined by confocal microscopy as described in +. **P<0.05 indicates the significance of the number of H5N1-induced apoptosis compared with human influenza viruses in MSCs.Percentage of apoptotic cells induced by H5N1 infection. (A) CD34(TIF)Click here for additional data file.Figure S5P values of <0.05 and double asterisks indicate statistically significant differences between cocultures and monoculture groups with P values of <0.05. n.s. means no significance.H5N1 did not induce IL-1β, IL-8, and GM-CSF in cocultures. (A) IL-1β, (B) IL-8, and (C) GM-CSF levels were measured using a Bio-plex Cytokine assay. The two different cell types in this experiment were derived from different donors. Data shown are mean±SD of two independent experiments. Single asterisk indicates statistically significant differences between mock and infected cells with (TIF)Click here for additional data file.


To the Editor: We report a human infection with avian-like swine A(H1N1) influenza virus first identified through a surveillance system for influenza like illness (ILI) in mainland China. An influenza virus, isolated from a patient with ILI, was originally subtyped as influenza A(H1N1)pdm09 virus with a hemagglutination inhibition (HI) test, but it was identified as a Eurasian avian-like influenza A(H1N1) virus (EA-H1N1) by full genome sequencing on January 30, 2013. The virus was named A/Hebei-Yuhua/SWL1250/2012 (H1N1v) (HB/1250/12), according to the definition of the World Health Organization  within 2 weeks before the onset of symptoms, nor a history of travel. He lived with his sister and parents; all other family members did not develop influenza-like symptoms during the period of the investigation.Sporadic human infections with swine influenza virus had been reported previously (HI test with ferret anti-serum against A(H1N1)pdm09 (CA09), seasonal H3N2, classical swine subtype H1N1(NJ76), and the seasonal influenza subtype H1N1 viruses  showed that the HB/1250/12 virus is antigenically indistinguishable from NJ76 and CA09, but different from subtype H3N2 viruses  and seasonal subtype H1N1 viruses . These f>40. Before vaccination, 28% of children (3–5 years) and 6.7% of adults (18–59 years) had HI titers >40, but elderly adults (>60 years) did not. Samples from 56% of children, 56.7% of adults, and 26.7% of elderly adults had HI titers >40 after TIV vaccination; however, a 4-fold antibody rise developed in <30% in any age group  could provide cross-protection, we collected serum samples from children, adults, and elderly adults, before and after 2012–2013 TIV vaccination, and the antibody against HB/1250/12 virus was tested by HI assay. The seroprotection antibody was defined as HI titers ge group . These rAntisera from hyperimmune sheep are usually used for influenza virus typing and subtyping, the CA09 sheep antisera reacted well with the HB/1250/12 virus . This isThis is the first human case of EA-H1N1 infection identified through the national ILI surveillance network in China, indicating that the influenza surveillance network not only plays a critical role in monitoring the seasonal influenza circulation and the vaccine virus selection, but also is useful for early detection of novel influenza viruses with pandemic potential. This study also highlighted the value of, and urgent demand for, a cost-effective sequencing platform on routine influenza surveillance for pandemic preparedness.Table showing cross-reactive antibody response against avian-like influenza A(H1N1) virus in pediatric and adult recipients of seasonal trivalent inactivated influenza vaccines, China, 2013


We observed the invasive behavior only when cells were plated on a stiff matrix (Matrigel/Collagen-1) and not when plated on a soft matrix . Cells lacking two polarity proteins upregulated expression of EGFR and activated Akt. Inhibition of Akt activity blocked the invasive behavior identifying a mechanism by which loss of polarity promotes invasion of epithelial cells. Thus, we demonstrate that loss of polarity proteins confers phenotypic plasticity to epithelial cells such that they display normal behavior under normal culture conditions but display aggressive behavior in response to activation of oncogenes or exposure to cytokines.Changes in expression and localization of proteins that regulate cell and tissue polarity are frequently observed in carcinoma. However, the mechanisms by which changes in cell polarity proteins regulate carcinoma progression are not well understood. Here, we report that loss of polarity protein expression in epithelial cells primes them for cooperation with oncogenes or changes in tissue microenvironment to promote invasive behavior. Activation of ErbB2 in cells lacking the polarity regulators Scribble, Dlg1 or AF-6, induced invasive properties. This cooperation required the ability of ErbB2 to regulate the Par6/aPKC polarity complex. Inhibition of the ErbB2-Par6 pathway was sufficient to block ErbB2-induced invasion suggesting that two polarity hits may be needed for ErbB2 to promote invasion. Interestingly, in the absence of ErbB2 activation, either a combined loss of two polarity proteins, or exposure of cells lacking one polarity protein to cytokines IL-6 or TNF Asymmetric distribution of proteins within cells play critical roles during several biological processes such as secretion, absorption, directional cell migration and asymmetric cell division. This asymmetry is, in part, generated by the action of cell polarity proteins in vivo  RNAi  bed made up of a 1∶1 mixture of Matrigel/collagen-I (M/Col-I). The latter condition was chosen because, recent reports have demonstrated a role for matrix stiffness in promoting invasive behavior of epithelial cells and that increasing collagen concentration to 2.0 or 4.0 mg/ml can lead to an 5–10 fold increase in rigidity compared to stiffness observed in Matrigel We have previously demonstrated that ErbB2 disrupts the Par6 protein complex, and the interaction with Par6/aPKC complex is required for the ability of ErbB2 to disrupt apical-basal polarity To assess whether loss of polarity protein-induced invasion is relevant in contexts other than ErbB2 activation in MCF-10A cells, we used a non-invasive tumor derived human cell line, MCF-7, and xenograft-selected MCF10AT displaying comedo-type Ductal-Carcinoma-In-Situ phenotype (DCIS.COM) cells. In addition, we also analyzed a non-invasive mouse tumor cell line 393P, derived from the lung adenocarcinomas induced by activation of K-RasG12D in p53R72H heterozygous background To test if loss of other polarity genes also cooperated with a non-invasive transformed line, we tested AF-6 and Dlg1 knockdowns in DCIS.COM cells and compared them with Luc control cells . Both coin vivo models, parental 393P and 393P.Scrib.RNAi cells were injected into the tail vein of non-transgenic mice. The parental 393P cells did not result in colonization and growth of cells in the lung. However, 393P.Scrib.RNAi cells were very effective in colonizing the lung demonstrating that loss of Scribble significantly increases the ability of Ras transformed cells to undergo metastasis to the lung  to gain invasive properties even in the absence of expression of transforming oncogenes. In oncogene expressing cells, loss of expression of cell polarity genes cooperates with transformation to promote invasion in culture and metastasis in vivo.In this report we demonstrate that normal epithelial cells with disruption of polarity genes cooperate with changes in the microenvironment  were obtained from Santa Cruz Biotechnology . Mouse monoclonal anti-Laminin and anti-ErbB3 antibodies were obtained from Millipore . Mouse monoclonal antibodies to Dlg and AF6 were obtained from BD Biosciences . Mouse monoclonal anti-Flag, anti-tubulin and anti-actin antibodies were obtained from Sigma . Anti-HA tag monoclonal antibody was obtained from Covance . Rabbit monoclonal antibodies to EGFR, Phospho-Akt (Ser-473), Pan-Akt, Phospho-Erk1/2, and pan-Erk were obtained from Cell Signaling Technologies . Secondary HRP-conjugated antibodies against mouse and rabbit were purchased from GE Healthcare . Secondary HRP-conjugated antibody against goat was obtained from BioRad Laboratories . Pierce Fast Western kit was obtained from Thermo Scientific . CpG Oligonucleotide (ODN 1826) was purchased from TCAG facility .http://cancan.cshl.edu/cgi-bin/Codex/Codex.cgi) in PSM2 vector backbone. A 97-nucleotide oligonucleotide was synthesized containing a 5′ miR30 flanking sequence, a sense strand Dlg1 or AF6 target sequence, a common miR30 loop sequence, an antisense strand targeting Dlg1 or AF6 or PatJ and a common 3′ miR30 flanking sequence . The sequences were amplified using PCR primers that recognize the miR30 flanking sequence and had Xho1 and EcoR1 restriction enzyme sites. The PCR product was subcloned into the MSCV–LTR–PURO–IRESGFP RNAi AF-6 sequence:TGCTGTTGACAGTGAGCGCGGTGGAACATTGAGAATTTATTAGTGAAGCCACAGATGTAATAAATTCTCAATGTTCCACCTTGCCTACTGCCTCGGA; RNAi PatJ sequence: MCF10A cells were maintained in DMEM/F12 with supplements, referred to as Growth medium, as previously described R172Hp53g/+ΔLA1/+ K-ras mouse (129Sv background) has been previously described 2 at 37°C in RPMI 1640 with 5% fetal bovine serum (FBS).The 393P lung adenocarcinoma cell line from a 5 on 8 µm-pore transwell plates in Assay medium without EGF P-values were calculated using two-tailed unpaired t-test.10A.B2 cells were plated at a density of 5×104 onto Transwell plates coated with 0.1% gelatin with RPMI 1640 containing 5% FBS placed in the lower well as the chemoattractant. Each condition was performed in triplicate or quadruplicate. After 16–18 h incubation, the medium was removed and the cells fixed with 90% ethanol. The migrated cells were stained with 0.1% crystal violet, washed with ddH2O, and five microscopic fields counted per filter.393P cells were seeded at 5×103 cells per well. In experiments with 10A.B2 lines, ErbB2 was activated or kept inactive by adding AP1510 or solvent control respectively to 8-day old acinar structures. Acini were monitored for invasive protrusion from day 2 to day 16 after addition of AP1510 with changes to the top medium with fresh AP1510 after every 4 days. Phase images were collected on Zeiss Axiovert 200 M using AxioVision 4.4 as described earlier P-values were calculated using two-tailed unpaired t-test.MCF10A cells were grown as three-dimensional acinar structures in Matrigel:Collagen-I (M/Col-I) matrix as previously described 393P cells were seeded onto GFR-Matrigel (BD Biosciences)-coated transwell filters and invaded cells fixed and stained as in Migration assay.Parental or polarity gene knockdown MCF10A cells were grown for four days on plastic dish in Growth medium, washed twice with PBS and fresh DMEM:F12 with 2% horse serum without growth supplements was added overnight. Next day, cells were brought out from starvation by adding Assay medium with 5 ng/ml EGF for indicated times. At the end of each time interval, cells were washed with ice cold PBS and lysed with RIPA buffer containing protease inhibitors. DNA was sheared using syringe, lysate cleared of debris by spinning and protein estimation performed according to BCA protocol. The sample buffer-treated lysate was run according to routine protein gel separation procedures.For transfer of proteins from gel to PVDF membrane, dry I-blot transfer  at 20 Volts for 7 min was used, the membrane treated according to manufacturer's instructions and blocked with 5% milk in TBST. Phospho-specific antibodies or total protein antibodies were incubated overnight at 4°C, washed three-times with 5% milk in TBST, incubated with HRP-conjugated secondary antibody at room temperature for 1 hour, washed thrice and developed using standard ECL kit. Stripping of membrane was performed by incubation with 0.5 N NaOH in de-ionized water for 10 min at RT on a shaker.2) for 30–45 min. The acini were labeled with primary antibody to laminin overnight at room temperature (RT) and next day with secondary Alexa-568 for 1 hour at RT. DAPI was used for for nuclear staining.M/Col-Igrown MCF10A acini were fixed as described earlier Fluorescence images were obtained with Zeiss Axiovert 200 M using AxioVison 4.4 and ApoTome imaging system or Olympus confocal system.TGATGTCGTCCTTGCGGAGG) and EGFR was (FORWARD: AAGGAGCTGCCCATGAGAAA/REVERSE: TGGCTTCGTCTCGGAATTTG).Total RNA from samples grown in monolayer culture was purified using Trizol  according to the manufacturer's instructions and subjected to Two-Step RT-PCR analyses using SYBR® Green RT-PCR Reagents Kit on a 7500 Fast Real-Time PCR System , using published primers. Housekeeping gene GAPDH gene was used as an internal control for normalization of the data. Data were analyzed with the SDS 2.1 software . One set of primer sequences (out of three tested) for PatJ was , so the entire experiment was stopped, all the animals sacrificed and necropsy performed. The WT cells showed no lesions in the lungs by gross inspection or after H&E staining of sections and the non-targeting vector showed 3 lesions <1 mm in 2 animals and otherwise no disease. The animals receiving the scribble KD cells were all sick  and had large, bilateral, consolidated tumors in the lungs. These were not discrete, quantifiable tumor masses, but as you see in the picture, gross overwhelming disease.All animal experiments were reviewed and approved by the Institutional Animal Care and Use Committee at The University of Texas M. D. Anderson Cancer Center. Wildtype 129Sv mice from our colony  of at least 8 weeks of age were used for the syngeneic tumor experiments. Animals received injections of one million cells in single-cell suspension in a volume of 100 μL of PBS or complete media by lateral tail vein. Animals were monitored regularly. There were three groups , 5 animals per group. 1×10Figure S1Transwell migration and 3D Matrigel invasion upon loss of polarity in 10A.B2 cells and laminin breakdown in 10A.B2.Dlg1 cells. (A) Transwell migration assay showing DAPI-stained nuclei of migrated cells for indicated knockdowns under ErbB2 inactive (ErbB2−) or activated (ErbB2+) conditions. Scale bar, 50 µm. (B) Phase morphology of 10A.B2 acini with indicated polarity gene knockdowns grown in Matrigel alone with (left panels) or without (right panels) ErbB2 activator. Note the multiacinar structures but lack of invasion in ErbB2 activated cultures. Scale bars, 100 µm. Arrows indicate region of the image magnified in the inset.(C) Immunofluorescence images showing disruption of laminin in ErbB2 activated Dlg1 knockdown 10A.B2 cells grown in M/Col-I. Scale bars, 50 µm. Examples of invasion-positive (D) and non-invasive (E) acini. See Materials and (TIF)Click here for additional data file.Figure S2PatJ knockdown measured by quantitative RT-PCR and effect of single or combined polarity gene knockdowns grown in different culture substrates. (A) Knockdown of PatJ was confirmed using two sets of primers Click here for additional data file.Figure S3ELISA quantification of cytokines TNF-α and IL-6 in dendritic cell supernatant and acinar invasion in polarity knockdown 10As treated with indicated cytokines. (A) ELISA analysis of TNF-α and IL-6 in supernatant medium of CpG-stimulated dendritic cells. (B–C) Phase images of M/Col-I grown acini of parental MCF10A cells or cells expressing sh-RNAi for indicated polarity genes showing induction of invasion in the presence of recombinant human TNF-α (B) or IL-6 (C). Scale bar 100 µm. See Materials and (TIF)Click here for additional data file.Figure S4Lack of Erk1/2 activation in combined polarity-gene knockdowns compared to parental or single polarity gene knockdowns. Parental or polarity-gene knockdowns cells were grown overnight in low-serum medium without growth factor supplements and next day replenished with Assay medium with 5 ng/ml EGF for indicated times Click here for additional data file.Figure S5Loss of AF6 or DLG1 leads to increased invasion in response to pro-tumorgenic cytokines. A) Immunoblot for AF6 and DLG using twenty-five and 50 μg total lysates from cells transfected with AF6 or DLG1 siRNA smart pool (Dharmacon) knockdowns, four days after nucleofection with indicated amounts of siRNA in MCF10A cells grown in 2D. B) Phase contrast images of MCF10A cells grown on Matrigel:collagen five days after nucleofection with 2.0 μM siRNA pools compared to control cells. Day 4 acini have been treated with 2.0 ng/ml recombinant TNFα or 25 ng/ml recombinant IL-6 for three days.(TIF)Click here for additional data file.Figure S6Effect of Scribble knockdown on Dlg expression. Immunoblots from independent experiments showing lack of any consistent effect of Scribble knockdown on Dlg expression in MCF10A.ErbB2 (10A.B2.Scrib) cells compared to control Luc cells (10A.B2.Luc).(TIF)Click here for additional data file.Figure S7Effect of polarity gene knockdowns on expression of Par3. Lysates obtained from indicated polarity-gene knockdown cells were immunoblotted with Par3 antibody. Note the absence of any effect of knockdowns on Par3 expression.(TIF)Click here for additional data file.Figure S8Effect of Perifosine treatment on AKTactivation (ser-473) in MCF10A cells. Lysates from MCF10A wild-type (10A) or AF6 and PatJ knockdown cells (10A.AF6.PJ) were obtained as described for phospho-AKT assay (refer to refer to  except o(TIF)Click here for additional data file.


In neuroinflammatory diseases, macrophages can play a dual role in the process of tissue damage, depending on their activation status (M1 / M2). M1 macrophages are considered to exert damaging effects to neurons, whereas M2 macrophages are reported to aid regeneration and repair of neurons. Their migration within the central nervous system may be of critical importance in the final outcome of neurodegeneration in neuroinflammatory diseases e.g. multiple sclerosis (MS). To provide insight into this process, we examined the migratory capacity of human monocyte-derived M1 and M2 polarised macrophages towards chemoattractants, relevant for neuroinflammatory diseases like MS.Primary cultures of human monocyte-derived macrophages were exposed to interferon gamma and lipopolysaccharide (LPS) to evoke proinflammatory (M1) activation or IL-4 to evoke anti-inflammatory (M2) activation. In a TAXIScan assay, migration of M0, M1 and M2 towards chemoattractants was measured and quantified. Furthermore the adhesion capacity and the expression levels of integrins as well as chemokine receptors of M0, M1 and M2 were assessed. Alterations in cell morphology were analysed using fluorescent labelling of the cytoskeleton.Significant differences were observed between M1 and M2 macrophages in the migration towards chemoattractants. We show that M2 macrophages migrated over longer distances towards CCL2, CCL5, CXCL10, CXCL12 and C1q compared to non-activated (M0) and M1 macrophages. No differences were observed in the adhesion of M0, M1 and M2 macrophages to multiple matrix components, nor in the expression of integrins and chemokine receptors. Significant changes were observed in the cytoskeleton organization upon stimulation with CCL2, M0, M1 and M2 macrophages adopt a spherical morphology and the cytoskeleton is rapidly rearranged. M0 and M2 macrophages are able to form filopodia, whereas M1 macrophages only adapt a spherical morphology.Together our results indicate that the alternative activation status of macrophages promotes their migratory properties to chemoattractants relevant for neuroinflammatory diseases like MS. Conversely, classically activated, proinflammatory macrophages have reduced migratory properties. Based on our results, we postulate that the activation status of the macrophage influences the capacity of the macrophages to rearrange their cytoskeleton. This is the first step in understanding how modulation of macrophage activation affects macrophage migration in neuroinflammatory diseases like MS. Infiltration of circulating monocytes is a pathological hallmark of injury to the central nervous system (CNS). Once in the CNS, blood-derived macrophages are thought to contribute to tissue damage and repair in yet unidentified ways -3. In thMigration within the CNS towards sites of injury or inflammation is directed by chemokines. In the current study, we focus on chemokines, which are upregulated in neuroinflammatory diseases, such as multiple sclerosis (MS) -12. In tin vitro[Cell migration is closely regulated by cell polarisation due to dynamic changes of the cytoskeleton. Cytoskeletal changes of differently activated macrophages has been described in terms of morphology of the cells in vitro. Howeverin vitro,15,23-25Peripheral blood mononuclear cells (PBMCs) were isolated from buffy coats  obtained from healthy donors who gave informed consent for use of their blood for research purposes. Research was performed with the approval of the Medical Ethical Committee of the VU University Medical Center. PBMCs were isolated using Ficoll density gradient . Monocytes were isolated from PBMCs using anti-CD14 magnetic beads  with a MACS® MultiStand and LS Column by passing 3 mL of MACS buffer (2 mM ethylenediaminetetraacetic acid (EDTA) and 0.1% FCS in PBS) according to the manufacturer's protocol.6 cells/mL in Petridishes in macrophage medium , supplemented with 5% (v/v) normal human serum , and 1% (v/v) penicillin-streptomycin-glutamine (Invitrogen), at 37°C, 5% CO2. Monocytes matured into macrophages (M0 macrophages) in the course of 5 days of culturing. After 5 days the supernatant was removed and cells were harvested by adding 4% lidocaine (v/v) in PBS to the Petridish, incubated for 10 minutes, scraped to aid detachment, and counted. Macrophages were washed and reseeded in 6-well plates in a concentration of 106 cells/mL in macrophage medium and cultured for an additional 2 days.For maturation into macrophages, monocytes were washed and cultured at a concentration of 2 × 10Escheria coli-derived lipopolysaccharide (LPS)  was added to the macrophage medium for a further 24 h. To generate M2 macrophages, 10 ng/mL recombinant human IL-4  was added to the culture medium for 48 h. As a control (M0) macrophages were cultured for 7 days without additional stimuli.Generation of pro-inflammatory M1 macrophages and anti-inflammatory M2 macrophages was performed as previously described ,26. In bThe production of pro- and anti-inflammatory mediators was assessed by ELISA in cell-free macrophage-conditioned medium sampled 48 h after activation using commercial kits for human IL-4, IL-10, IL-6, and TNF-α (Sanquin blood bank). These cytokines were chosen as they are able to characterize M1  and M2  activation status ,28. In aDirected migration experiments were performed using a TAXIScan  , consist5 cells per well and cultured for 2 h at 37°C and 5% CO2. After 2 h, non-adherent cells were removed. The remaining cells were lysed with 0.1 N NaOH and fluorescence was measured in a Fluostar24 . The percentage of adherent cells was determined by comparing fluorescence intensity to a calibration line ranking from 0 cells to 1.106 cells/mL.The adhesion of M0, M1 and M2 macrophages to extracellular matrix molecules was determined using plates coated with either collagen type 1  or fibronectin isolated from human plasma . M0, M1 and M2 macrophages were harvested (as described above) and washed with PBS, labelled for 15 minutes at 37°C with 1 μL  5 (and6) carboxyfluoresce) (BCECF-AM) (Invitrogen). Cells were washed and seeded onto a 96-well plate in a density of 106 cells. Viable cells were gated (approximately 70%) as described before [Macrophages were harvested 48 h after activation using PBS containing lidocaine 4%  and the supernatants collected for ELISA. Cells were washed twice with PBS and fixed with PBS containing 4% formalin. Subsequently the macrophages were labelled for 1 h at room temperature with primary antibody Table  diluted To analyse the cytoskeleton of the macrophages, the actin cytoskeleton was visualized. Macrophages were harvested after 5 days culture, reseeded on glass coverslips and then activated with IFNγ/LPS or IL-4 to induce M1 or M2 subsets. M0 macrophages were cultured in medium without stimuli. After 48 h, cells were left untreated or activated with CCL2 10 μM  for 10 minutes fixed with 4% paraformaldehyde in PBS for 30 minutes at 4°C, rinsed with PBS and permeabilized with 0.2% Triton X-100 in PBS for 5 minutes. The cells were rinsed with 10 mM Glycine, rinsed with PBS, and incubated with Cdc42  for 1 h. The cells were rinsed with PBS prior to staining with the second antibody goat anti rabbit ALEXA 488  and rhodamine phalloidin  a high affinity, fluorescent filamentous actin probe for 1 h . Cells wP was <0.05.Statistical analysis was performed using Graphpad Prism version 4.03 for Windows . The FACS analysis, morphology, adhesion and migration experiments were analysed using one-way analysis of variance (ANOVA) with the Bonferroni correction. The results were considered statistically significant when To investigate the migration capacities of differently activated macrophages, we examined the rate of migration towards a chemoattractant, their ability to adhere to different extracellular matrices, the differential expression of chemokine receptors and changes in the cytoskeleton.First the effect of M1 and M2 induction was assessed by determining their cytokine profile. The cytokines measured in the supernatants for M1 polarised cells showed a significant increase of IL-6 and TNF-α compared to M0  showed a mixture of round and spindle-shaped cells in which actin was diffusely distributed throughout the cell and partially concentrated in the cell cortex. Upon treatment with IFNy and LPS (M1), macrophages became spindle-shaped, in which the actin fibres were randomly spread throughout the cell, with clusters at the cell cortex. After stimulation with IL-4, M2 macrophages were mostly spherical and displayed actin condensed at protrusions. Cdc42, relevant for making filopodia, was randomly distributed through M1 and in the nucleus, whereas in M2 macrophages it was only present at the nucleus Figure A.To further investigate the role of the cytoskeleton in the capability of migration of the macrophages, M0, M1 and M2 macrophages were activated with CCL2, upon which M0, M1 and M2 macrophages adopted a spherical morphology and the cytoskeleton was rapidly rearranged. M0 macrophages showed no co-localization of actin and Cdc42, and formed filopodia. In M2 macrophages, the actin formed a dense network and was co-localizing with Cdc42 at the part where filopodia are formed. M1 macrophages showed a diffuse distribution of actin which showed almost complete co-localization with Cdc42 Figure B.In neuroinflammatory diseases like MS, macrophages play a dual role. Depending on their activation status, they are considered to play either a detrimental (neuron) damaging, pro-inflammatory M1) or neuroprotective, anti-inflammatory (M2) role, as revealed in experimental autoimmune encephalomyelitis  and a spinal cord injury model  or neuro,31. The M2 macrophages migrated over longer distances towards chemoattractants CCL5, CXCL10, CXCL12 and C1q, whereas M1 macrophages do not respond and remain sessile. The un-activated macrophages (M0) show an intermediate migration capability. The migration of M0 exceeds M1 and M2 towards chemokine CCL2, one of the key factors for monocyte recruitment into MS lesions ,32. Our In our study C1q is the most potent attractant for M2 macrophages; interestingly, it was recently shown that macrophages show an anti-inflammatory phenotype upon activation with C1q . The samTo understand the altered migration between the different subsets of macrophages, the adhesion to different extracellular matrix components was studied as well as the expression of integrins and adhesion molecules -38. In cet al. who described that IL-4 activated human macrophages adopted a spherical morphology and activation with IFNγ without the additional treatment of LPS stimulated elongated morphology [However, we were able to show a clear difference in morphology and cytoskeletal arrangement between M1 and M2 macrophages, which may underlie the altered migration capacity of the different subsets. M1 macrophages are elongated, whereas M2 macrophages have a more circular appearance. M1 macrophages have a dense static actin-network along the cortex, which could explain the limited migration, whereas the M2 macrophages have more randomly distributed actin. Differences in morphology and migration is known to be dependent on changes in the cytoskeleton and the actin reorganization, a process regulated by small guanine triphosphate (GTP)-binding proteins  ,39-43. Lrphology . Our datrphology  or Ca2+ rphology .In summary, our studies reveal substantial differences in the migratory capabilities of M1 versus M2 human macrophages, and that M2 macrophages are more motile and migrate over longer distances towards chemoattractants involved in CNS inflammation. In contrast, macrophages in a pro-inflammatory state, are sessile and do not migrate towards these chemoattractants. M2 macrophages are known to be growth-promoting and are able to secrete neurotrophic factors. Further elucidation of macrophage migration across a variety of tissues holds great potential for understanding the role of M1 and M2 macrophages in neuroinflammatory diseases such as MS.AA/M2: Alternatively activated; BCECF: 2′ 7′′-bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein; BSA: Bovine serum albumin; CA/M1: Classically activated; CCL: C-C motif chemokine ligand; CCR: Receptor for CCL; CNS: Central nervous system; COM: Centre of mass; CXCL: C-X-C motif chemokine ligand; CXCR: Receptor for CXCL; DMEM: Dulbecco’s modified Eagle's medium; EAE: Experimental autoimmune encephalomyelitis; EDTA: Ethylenediaminetetraacetic acid; ELISA: Enzyme-linked immunosorbent assay; FACS: Fluorescence-activated cell scan; FCS: Fetal calf serum; FMI: Forward migration index; IFN-γ: Interferon-γ; IL: Interleukin; LPS: Lipopolysaccharide; MFI: Mean fluorescence intensity; MS: Multiple sclerosis; PBMC: Peripheral blood mononuclear cells; PBS: Phosphate-buffered saline; PECAM/CD31: Platelet endothelial cell adhesion molecule; TNF: Tumour necrosis factor; VLA-4: Very late antigen-4.The authors declare that they have no competing interests.DV was involved in acquisition of data, data analysis and writing of the manuscript. PH, MB performed both data acquisition and analysis. AT and HV were involved in data acquisition and interpretation. SA and CD participated in study design, conceptualization, data analysis and helped to draft the manuscript. All authors read and approved the final manuscript.Quantification of velocity and directness of the macrophage subsets. The velocity of the macrophages in the migration assay was calculated with ImageJ. M2 macrophages have a significantly higher velocity towards CCL5, CXCL10, CXCL12 and C1q compared to M1. M0 have a higher velocity towards CXCL10 and CXCL12 than M1. Towards CCL2 M0 macrophages migrate faster than M2. CCL, Chemokine C-C motif ligand; CXCL, Chemokine C-X-C motif ligand.Click here for fileQuantification of forward migration index. The quantification of the forward migration index of the macrophages in the migration assay was calculated with ImageJ. M2 macrophages have a significantly higher forward migration index towards CXCL10, CXCL12 and C1q compared to M1. Towards CCL2 M0 macrophages exceed M2 macrophages. CCL, Chemokine C-C motif ligand; CXCL, Chemokine C-X-C motif ligand.Click here for fileM0 macrophages migrating towards CXCL12. A few of the M0 macrophages lined up under the horizontal line are migrating towards the CXCL12. CXCL, Chemokine C-X-C motif ligand.Click here for fileM1 macrophages migrating towards CXCL12. None of the M1 macrophages lined up under the horizontal line are migrating towards the CXCL12. The cells are able to transform their cell body, however fail to migrate towards the CXCL12. CXCL, Chemokine C-X-C motif ligand.Click here for fileM2 macrophages migrating towards CXCL12. The majority of M2 macrophages lined up under the horizontal line are migrating towards the CXCL12. These cells show high capacity to transform their cell body migrate towards the CXCL12. CXCL, Chemokine C-X-C motif ligand.Click here for file


P < 0.001 and P < 0.05, resp.). Hospital admission was higher in influenza A group (P = 0.01). In contrast, ICU admission was higher in patients with influenza A(H1N1) pdm09 than influenza A patients (P < 0.05). There were higher numbers of bacterial samples taken and culture positivity in patients with influenza A than patients with influenza A(H1N1) pdm09 . In both groups, the majority of the patients with positive bacterial cultures had underlying diseases. The present study shows that the patient characteristics and the frequency of secondary bacterial infections were different in patients with seasonal influenza A and in patients with influenza A(H1N1) pdm09. The aim of the present study is to analyse the secondary bacterial infections in a large group of patients with seasonal influenza A and influenza A(H1N1) pdm09. Patients diagnosed with seasonal influenza A and influenza A(H1N1) pdm09 between 2005 and 2009 were enrolled in the study. Data was retrieved from medical records and laboratory information systems (LIS). In total, 1094 patients with laboratory confirmed influenza were studied. There were 352 patients with seasonal influenza A and 742 patients with influenza A(H1N1) pdm09. The patients with influenza A were older and had higher comorbidity than patients with influenza A(H1N1) pdm09 ( The interaction between human influenza viruses with different subtypes of human and animal influenza viruses has been shown to give rise to new variants of the virus with pandemic potential –3. All tStreptococcus pneumoniae, Haemophilus influenzae, Staphylococcus aureus, and Streptococcus pyogenes have been important contributors to morbidity and mortality [During previous influenza pandemics, secondary bacterial infections caused by ortality , 7, 8. TPrevious studies have suggested that influenza A(H1N1) pdm09 differs from the previous influenza pandemics, and secondary bacterial infections seem to play a limited role in influenza deaths during the current pandemic . HoweverThe aim of the present study is to analyse the secondary bacterial infections in a large group of patients with seasonal influenza A and influenza A(H1N1) pdm09. A retrospective study analysing the presence of secondary bacterial infections in patients with either seasonal influenza A (between 2005 and 2008) or influenza A(H1N1) pdm09 (2009) was designed. All patients diagnosed with seasonal influenza A and influenza A(H1N1) pdm09 between 2005 and 2009 were enrolled in the study. Patients with influenza between 2005 and 2008 were grouped as “influenza A”. It is important to note that patients in the “influenza A” group were not homogeneous since several different influenza viruses including H1N1 and H3N2 dominated during different periods of time between 2005 and 2008. It was not possible to subgroup these patients according to the type of influenza virus since the molecular typing of respective virus for each patient was not performed. The patients that had positive bacteriological findings in both groups were studied further. Reviewers used a standardized form. All data was abstracted from medical records including age, sex, presence of comorbid conditions, and clinical presentation and course including autopsy reports, length of hospital stay (including intensive care unit stay), and use of antibiotic/antiviral treatment. A positive bacteriological finding was defined as any growth in blood cultures or growth of relevant pathogenic airway bacteria in airway samples. The microbiology results were collected from medical records and laboratory information systems (LIS). Flocked swabs or nasopharyngeal aspirates had been used to collect respiratory epithelial cells from the posterior nasopharynx. μL was applied to glass slides, by cytocentrifugation  at 1200 rpm for 10 min. The slides were air-dried and then fixed in cold acetone for 10 min. The cell spots were stained with 25 μL of conjugated monoclonal antibodies from a commercial DFA kit from PathoDx Respiratory Virus Panel  for 15 minutes at 37°C. Evan's Blue was used as a counterstain. After repeated washing in phosphate-buffered saline, the slides were mounted in 70% glycerol and examined under microscope. A positive result was indicated by the presence of at least one intact cell showing specific fluorescence using a fluorescence microscope . The presence of ≥50 epithelial cells per glass slide was required for the specimen to be considered adequate for DFA testing. Positive and negative controls from PathoDx as well as controls from cultivated influenza A strains were used.The laboratory diagnosis of seasonal influenza A was made by detection of influenza A-antigen with immunofluorescence (IF). Samples were centrifuged at 1000 rpm for 10 min to pellet the cells for direct immunofluorescence (DFA). The cell pellets were then resuspended in a small amount of phosphate-buffered saline, and 100 μL was extracted using the MagAttract Virus Mini M48 Kit  according to the manufacturer's instructions with the Qiagen M48 BioRobot. The RNA was eluted to a final volume of 100 μL. Water was used as a negative control and an influenza virus isolate, A/Stockholm2/2009 H1N1, was used as a positive control for each extraction. The PCR method used was a one-step rRT-PCR provided by the Swedish Institute for Communicable Disease Control. The primers used were 5′ GGC TGC TTT GAA TTT TAC CAC AA 3′ and 5′ TTT GGG TAG TCA TAA GTC CCA TTT T 3′, amplifying the haemagglutinin gene. The probe used was 5′-FAM-TGC GAT AAC ACG TGC ATG GAA AGT GTC-TAMRA-3′. For the PCR, the SuperScript III Platinum One-Step Quantitative RT-PCR system was used . The PCR program used was reverse transcription for 15 minutes at 50°C followed by 2 minutes at 95°C, 45 cycles of 95°C for 5 seconds, 60°C for 60 seconds, and 40°C for 30 seconds using LightCycler 480 . A threshold cycle (CT) of ≤40 together with a sigmoid fluorescence curve was needed for the result to be considered positive.For diagnosing pandemic influenza H1N1, real-time-reverse transcriptase-polymerase chain reaction (rRT-PCR) with subtype-specific primers for influenza A(H1N1) pdm09 was used. A sample volume of 400 All patients with positive seasonal influenza A or influenza A(H1N1) pdm09 were cross-examined in the clinical microbiology laboratory database of Karolinska University Hospital, Huddinge, for the presence of positive bacteria cultures from airways or blood. Blood samples were cultured in the BacT/ALERT 3D  automated blood culture system. Detection of bacteria from clinical samples was made by standard methods. t-test were used in categorical comparison and in comparing the two groups, respectively. Values of P < 0.05 were considered as statistically significant. The Fisher exact test and Students The study was approved by the Regional Ethical Review Board of Stockholm, Sweden (Dnr 2010/266-31). P < 0.0001). In total, 1094 patients with laboratory confirmed influenza were studied. In the study group, there were 352 patients with seasonal influenza A and 742 patients with influenza A(H1N1) pdm09. The numbers of bacteriological sampling were higher in patients with seasonal influenza A than patients with influenza A(H1N1) pdm09, 240/352 (68.18%) versus 99/742 (13.34%), respectively  pdm09 and influenza A patients. Among the influenza A patients, 33/352 (9.38%) had positive bacteriological samples. The numbers of influenza A positive patients who had positive cultures from upper airways, lower airways, and blood were 20/352 (5.68%), 13/352 (3.69%), and 8/352 (2.27%), respectively .Staphylococcus aureus (MRSA) in the studied material.In 38/742 (5.12%) of the influenza A(H1N1) pdm09 positive patients, the cultures were positive for bacterial growth. The numbers of influenza A(H1N1) pdm09 positive patients who had positive cultures from upper airways, lower airways, and blood were 15/742 (2.02%), 12/742 (1.62%), and 12/742 (1.62%), respectively. The different bacterial species identified in cultures are depicted in P = 0.01). Numbers of positive upper- and lower-airway cultures were elevated in patients with influenza A than patients with influenza A(H1N1) pdm09 . No difference was observed in the numbers of positive blood cultures between the two groups  pdm09 . Women were the majority in both patient groups, 57.58% in seasonal influenza A patients and 65.79% among influenza A(H1N1) pdm09 patients.There were differences in baseline characteristics between patients with seasonal influenza A and patients with influenza A(H1N1) pdm09. The median age of patients with seasonal influenza A group was significantly higher than influenza A(H1N1) pdm09 group, 57.5 years versus 30.5 years, respectively (P < 0.05). When comparing prestudy morbidity in the groups, the patients with seasonal influenza A had significantly more cardiovascular disease than the influenza A(H1N1) pdm09 patients 7/33 (21.21%) versus 1/38 (2.63%), respectively (P < 0.05). Two out of 33 (6%) seasonal influenza A patients had diabetes mellitus, compared to none among the influenza A(H1N1) pdm09 patients. Conditions leading to immunosuppression were equally common in the two groups and included lymphoproliferative disease, acute myeloid leukemia (AML) or chronic lymphocytic leukemia (CLL), stem cell transplantation, and secondary immunodeficiency (hypogammaglobulinemia or HIV) (data not shown). The presence of chronic lung disease and chronic renal disease was similar in both groups. Total numbers of patients with comorbidity were significantly higher in the seasonal influenza A group (P = 0.01). Despite high hospital admission rates among the influenza A patients, the duration of the hospital stay was shorter in the seasonal influenza A patients compared to influenza A(H1N1) pdm09 patients, 8.5 versus 13.5 days, respectively, although this difference was not statistically significant (P = 0.06). Interestingly, ICU admittance was lower in seasonal influenza A patients compared to influenza A(H1N1) pdm09 patients, 2/33 (6.06%) versus 11/38 (28.95%), respectively (P < 0.05). Median time between onset of symptoms and time to seeking medical care was similar in both study groups. In contrast, there were considerable differences in length of hospital stay and intensive care unit (ICU) admission. Influenza A patients were more often admitted to hospital than influenza A(H1N1) pdm09 patients 30/33 (90.90%) versus 24/38 (63.16%), respectively  was used in 10/33 (30.30%) patients with seasonal influenza A and in 18/38 (47.37%) patients with influenza A(H1N1) pdm09. Treatment time was 5 days for all seasonal influenza A patients and most of the influenza A(H1N1) pdm09 patients. Extended antiviral treatment was given to 6 patients with influenza A(H1N1) pdm09 that had a long ICU stay (>7 days). Length of antiviral treatment in these patients ranged between 6 and 35 days (mean = 22 days). All of these patients suffered from preexisting hematological disease  or severe chronic lung disease. Several of these patients remained PCR positive for influenza A(H1N1) pdm09 throughout their ICU stay, despite antiviral treatment.Numbers of seasonal influenza A patients and influenza A(H1N1) pdm09 patients who received antibacterial treatment were 26/33 (78.79%) and 25/38 (65.79%), respectively . There wWhen influenza A(H1N1) pdm09 patients were treated with PO antibiotics, amoxicillin or amoxicillin-clavulanic acid was preferred as empiric therapy. When IV therapy was chosen, penicillin G or a second/third-generation cephalosporin was most commonly used. Patients admitted to the ICU always received a second/third-generation cephalosporin. In cases with severe inflammatory response and/or severe respiratory failure, a quinolone (moxifloxacin) was added as empiric therapy. In cases of septic shock/ARDS, cephalosporin therapy was often combined with aminoglycosides. Piperacillin/tazobactam and carbapenems were rarely used in the ICU patients unless they were treated in the ICU for a long time (>7 days) (data not shown). S. pneumoniae and S. aureus have contributed to excess mortality in these patient groups [Secondary bacterial infections have been important contributors to morbidity and mortality during the previous influenza pandemics , 7, 11. t groups . There iHere, we analysed the secondary bacterial infections in a large group of patients with seasonal influenza A and influenza A(H1N1) pdm09 influenza, diagnosed at Karolinska University Hospital, during 2005–2009. The numbers of influenza A(H1N1) pdm09 positive patients in one year were significantly higher than the numbers of patients with seasonal influenza A in 4 years, 742 versus 352 patients, respectively. During 2009, the diagnostic tests in order to identify influenza A(H1N1) pdm09 were used extensively. The clinicians were instructed by the Swedish National Board of Health and Welfare to test every suspected case of influenza A(H1N1) pdm09 influenza to keep track of the extension of the pandemic and to identify the patients in the risk groups that should receive antiviral treatment. The high numbers of influenza A(H1N1) pdm09 positive patients in the study might probably depend on high numbers of sampling for this virus. In the study, the diagnostic methods used for seasonal influenza A and influenza A(H1N1) pdm09 were also different. The PCR method which is used for influenza A(H1N1) pdm09 has previously shown to be more sensitive than IF, which was used for seasonal influenza A between 2005 and 2008 . The difP = 0.01). In contrast, ICU admission was higher in patients with influenza A(H1N1) pdm09 than influenza A patients (P < 0.05). The influenza A(H1N1) pdm09 patients, as a group being younger and healthier, were probably more likely to be discharged from the emergency room, but the individuals that were hospitalized often had a more severe course of infection, especially in the subgroup of individuals suffering from immunosuppression.Previous studies have shown that influenza A(H1N1) pdm09 influenza mostly infects young patients , 15. TheP < 0.0001 and P = 0.01, resp.). However, the two patient groups in the study differed in size and partly in selection since bacterial cultures were performed in a larger proportion in the seasonal influenza A patients. In both groups, a majority of the patients with positive bacterial cultures had underlying diseases. In blood cultures, S. pneumoniae  and coagulase-negative Staphylococcus were the most common isolates in both groups. The three most common bacterial species isolated from lower airway samples in influenza A patients were S. pneumoniae, S. aureus, and H. influenzae. In influenza A(H1N1) pdm09 patients, lower airway cultures were instead dominated by S. aureus, Streptococcus dysgalactiae, and coagulase-negative staphylococci. The reason for this difference is not known.There were higher numbers of bacterial samples taken and culture positivity in patients with influenza A than patients with influenza A(H1N1) pdm09  pdm09 patients received antiviral treatment. During the pandemic, the Swedish National Board of Health and Welfare instructed physicians to initiate antiviral treatment within 2-3 days after the beginning of influenza symptoms. It has been shown that patients with influenza benefit from antiviral treatment if it is initiated within the first 4 days of illness . SeveralS. pneumoniae and S. aureus. In the Swedish setting there is, in general, no need for empiric coverage of MRSA. Despite a low frequency of secondary bacterial infections in the present and previous studies, a majority of the patients with either seasonal influenza A or influenza A(H1N1) pdm09 received antibacterial treatment. Our results concerning bacterial etiology in these infections suggest that empiric antibiotic therapy in influenza A and influenza A(H1N1) pdm09 patients should be directed primarily against In retrospect, the course of the influenza A(H1N1) pdm09 in Sweden was not as severe as first suspected. The lack of a great number of secondary bacterial infections, as presented here, might be one of the underlying reasons for the benign course of the disease, especially when it is compared with the 1918 pandemic . However


At ISICEM 2011, we reported that AN69ST showed the highest capacity to adsorb high mobility group box 1 protein (HMGB1) when compared with polymethylmethacrylate, polysulfone and high cut-off membrane [The test solution contained 100 g HMGB1 and 35 g albumin in 1,000 ml substitution fluid. We executed three different experimental hemofiltrations with solution flow of 100 ml/minute and: ultrafiltrate flow 1,000 ml/hour using AN69ST primed with a heparinized saline, F(+) and H(+); ultrafiltrate flow 1,000 ml/hour using AN69ST with no heparinized saline, F(-) and H(+); and ultrafiltrate flow of 0 ml/hour using AN69ST with no heparinized saline, F(-) and H(-). In addition, AN69ST membrane was immunostained using an antibody that confirmed dying on human kidney tissue.The concentration decreases of HMGB1 at 0, 60 and 360 minutes indicated no significant differences among the three different hemofiltration experiments Figure .Surface heparin or filtration might not contribute to HMGB1 adsorption on the AN69ST membrane. Remarkable adsorption on AN69ST is likely to be influenced by material characteristics, hydrogel structure with moisture content, or negative electric charge and may occur not in bulk layers but on large surfaces of membranes.