Despite the presence of papillary structures and papillary tumors in humans, the mechanism of papillae formation is unknown. We describe herein a novel role for Niemann-Pick disease type 2C (NPC2) protein, a cholesterol binding protein in the lysosome, in papillae formation.NPC2 shRNA. The xenografts of wild H460 cells and empty shRNA vector cells showed distinct papillae formation, whereas NPC2 protein-depleted H460 cells displayed markedly reduced or no papillae. Since all papillary tissues have open spaces we examined whether NPC2 protein might also contribute to the creation of open spaces. The TUNEL assay in the xenografts of wild and empty shRNA vector H460 cells showed massive cell death, and NPC2 protein-depleted cells displayed minimal cell death. Measurement of caspase 3/7 activities in cultured H460 cells supported NPC2 protein-mediated apoptotic cell death. The presence of excess NPC2 protein, however, did not always produce papillae as seen in the xenografts of CHO cells that were stably transfected with NPC2.We examined NPC2 protein expression in surgical samples of papillary tissues by immunohistochemical stain, and all papillary tissues expressed NPC2 protein in the epithelium. To examine our hypothesis of NPC2 protein-mediated papillae formation, we carried out xenograft experiments using wild H460 cells (large cell lung carcinoma cell line) that constitutively expressed abundant NPC2 protein and NPC2 protein-depleted H460 cells by The NPC2 protein of certain cells forms papillae coupled with apoptosis that creates open space. This protein may have future applications to modulate papillae formation and papillary growth in tumor tissues. Papillary structure consists of a fibrovascular core covered by epithelium creating nipple-like projections, called fronds. One unique feature of all papillary tissues is the presence of open spaces. There has been no report describing the mechanism of how papillae and open spaces are formed. We previously reported unique upregulation of All papillary tissues examined so far showed cytoplasmic NPC2 protein in the epithelium as representative samples of normal papillae, papilloma and papillary cancer are shown in shRNA vector H460 cells showed the same cell morphology; cells grew attaching each other. H460 cells containing NPC2-shRNA grew as separate and individual cells  into nude mice and examined xenografts for papillae formation. The xenografts of wild H460 and empty vector cells showed distinct papillae formation, whereas NPC2-depleted H460 cells failed to show papilla ; both cells were cultured under the identical conditions in the presence of hygromycin B. As shown in NPC2 showed a distinct caspase 3/7 activity expressed as relative luminescent unit (RLU) of 78×104±15×104 (mean ± SD of 3 determinations), whereas wild CHO cells had almost no caspase activity (RLU of 0 to 66). The presence of apoptotic cells was confirmed in surgical samples of human papillary tissues by the TUNEL assay . The xenografts of the mixed cells always showed many papillae as seen in the xenograft of wild H460 cells probably due to overwhelming growth of wild H460 cells over NPC2-shRNA containing cells. Decreased cell proliferation activity by NPC2-shRNA containing cells  and live animal model (born with NPC2 defect) exists. It is speculated that this mouse model may have NPC2 independent mechanisms or might have developed alternative mechanisms to form papillae. In humans, patients with Niemann-Pick Disease type 2C represent human model of NPC2 protein depletion. However, there is no autopsy report examining papillary tissues in this disorder.The question can be raised whether the expression of abundant NPC2 protein alone is sufficient to form papillae regardless of cell types. We examined this question in CHO cells that were stably transfected with  protein . This fiNPC2 in human papillary thyroid cancer NPC2-depleted H460 cells  with protocol number of 02004-05. All animal experiments were carried out under the regulation of IACUC.H460 cells (large cell lung cancer cell line) and CHO cells (Chinese ovarian cells) were obtained from ATCC, Manassas, VA., and cultured in RPMI1640 medium and F-12 medium, respectively, in the presence of 10% fetal bovine serum and antibiotic cocktail.NPC2. For the NPC2 transfection experiment, we obtained the entire coding region of human NPC2 cDNA (NM_006432) from 3 µg total RNA of BHP 2-7 human papillary thyroid cancer cells by two-step RT-PCR using Superscript III , Expand High Fidelity PCR System , and custom primers (Invitrogen), 5′-GATGCGTTTCCTGGCAGCTA-3′ and 5′-CTGCTAACCAAGTGCTGCAT-3′. We introduced the amplicons into the pCR 2.1 vector (Invitrogen), confirmed the correct sequences of the insert, and sub-cloned it into a mammalian expression vector pcDNA3 (Invitrogen) with HindIII and XbaI . We carried out transfection of CHO cells with NPC2 in pcDNA3 using Effectene Transfection Reagents  and selected clones expressing constant and abundant NPC2 protein in the presence of geneticin. Wild CHO cells contained very little NPC2 protein.We used wild H460 cells, since this cell line constitutively expressed abundant NPC2 protein. We also made NPC2 protein-rich CHO cells by stable transfection of CHO cells with NPC2-shRNA using the online program  and ligated the target DNA to the pSilencer3.1-H1 hygro vector (Applied Biosystems/Ambion). After confirming the correct sequences of the insert from the amplified plasmid, we transfected H460 cells with NPC2-shRNA or shRNA vector alone (empty vector) using the T-20 program of the Amaxa Nucleofector II . We selected stably transfected clones by culturing cells in the presence of 500 µg/ml hygromycin B (Invitrogen). The silencing effect of NPC2-shRNA on NPC2 and its protein expression was confirmed by RT-PCR and immunohistochemistry staining, respectively. The most effective NPC2-shRNA insert inhibiting NPC2 protein expression contained the following sequences: 5′GATCCGTGGTATCCAGTGATTCGTTCTCAAGAGAAACGAATCACTGGATACCATTTTTTGGAAA-3′, and 5′AGCTTTTCCAAAAAATGGTATCCAGTGATTCGTTTCTCTTGAGAACGAATCACTGGATACCACG-3′.We designed the target DNA sequences corresponding to 6 cells in 0.1 ml phosphate buffered saline (PBS) subcutaneously to the flank region of mice. After tumors grew to 1–1.5 cm in diameter, we sacrificed animals in a CO2 chamber, removed tumors, and kept sliced tumor samples in formalin for immunostaining.We obtained female nude mice (15 g) from Charles-River Laboratories INC, Wilmington, MA, and all animals were kept in sterile cages. We injected 10Immunohistochemical studies were performed on formaldehyde-fixed, paraffin-embedded tissues. Deparaffinized sliced sections were incubated for 60 min at room temperature with the rabbit polyclonal anti-human NPC2 antiserum (1∶1000). Polyclonal anti-human NPC2 antibody was kindly provided by Dr. Peter Lobel, Rutgers University. Immunostaining was carried out using a Dako autostainer in conjunction with the EnVision™ detection kits . Positive controls  and negative controls (without antibody) were used for NPC2 immunostaining. For TUNEL assay in sliced tissues, we used Apop Tag Peroxidases In Site Apoptosis Detection kit . For image analysis, we used Metamorph™ image analysis software version 7.5 .shRNA vector and NPC2-shRNA. These cells were thawed on the same day and cultured in 6-well plate in the presence of hygromycin B until 80% confluence was achieved. Cells were washed with PBS twice and sonicated in 1 ml PBS. An aliquot of 50 µl sonicate corresponding to 6×104 cells was used immediately for caspase 3/7 assay. Each assay was run with or without 5 units Z-VAD-FMK, caspase inhibitor (Promega), by incubating the reaction mixture for 30 min at 37°C, and relative luminescent unit (RLU) was measured by GloMax 20/20 luminometer (Promega). We defined caspase activity as the RLU that was inhibited in the presence of caspase inhibitor. Similarly, we measured caspase3/7 activities from wild CHO cells and transfected CHO cells with NPC2.Caspase 3/7 activities were measured from cultured cells using Promega caspase-Glo luminescent method . We used H460 cells containing empty NPC2-shRNA cells by plating 1000 cells/well in a 96-well plate; they were cultured under the identical condition in the presence of hygromycin B. Cell proliferation was measured using a quick cell proliferation assay kit  on first, third and fifth days of cell culture. The optical density from each well was measured at 440 nm using a microplate reader .We used empty vector cells and t-test based on difference of variance (F test). The p value less than 0.05 were considered significant.The results of caspase activity and cell proliferation assay were analyzed by Student's


Subtype-C alone accounts for approximately 50% of global and more than 95% human immunodeficiency virus-1 (HIV-1) infection in India. Identification of antigenic epitopes that induce antibodies with cross-clade activity will be crucial to address the HIV-1 viral diversity.80 HIV-1 infected drug naive patients were recruited for this study. The study was approved by the institute ethics committee and informed consent was obtained from all the participants. The relative binding of anti-V3 polyclonal plasma antibodies to 35 mer consensus-B and C V3 peptides was done by ELISA binding assay. Statistical analysis was performed by Graphpad Prism 5.Assessment of the relative binding revealed that 86% (69/80) and 99% (79/80) of the plasma were able to reach an IC50 binding titer with V3B peptide and V3C peptides respectively; with substantially low antibody titers that bind to V3B than V3C  (p<0.0001). The finding suggests that although majority of the antibodies were subtype specific, a good proportion of cross reactive anti-V3 antibodies also exist in these plasma (mean=23% range=0.11-97%). We observed a positive correlation between percent cross reactive anti-V3 antibodies and days from first diagnosis (p=0.008) while no such association was found with other clinical and immunological parameters like plasma viral load (p=0.24), CD4 count (p=0.34) and total plasma IgG levels (p=0.45).This is the first study to demonstrate the presence of cross-clade reactive anti-V3 antibodies and their association with time in the plasma of HIV-1 infected Asian Indians from north India.


Objective: The MPL gene encodes the thrombopoietin receptor. Recently MPL mutations (MPL W515L or MPL W515K) were described in patients with essential thrombocythemia (ET) and primary (idiopathic) myelofibrosis (PMF). The prevalence and the clinical importance of these mutations are not clear. In the present study, we aimed to investigate the frequency and clinical significance of MPL W515L/K mutations in our patients with ET and PMF. Materials and Methods: A total of 77 patients (66 were diagnosed with ET and 11 with PMF) and 42 healthy controls were included in the study. Using peripheral blood samples, the presence of MPL W515L/K mutations and JAK-2 V617F mutation were analyzed by real-time polymerase chain reaction.Results: In our study, MPL W515L/K or JAK-2 V617F mutations were not observed in healthy controls. JAK-2 V617F mutation was present in 35 patients, of whom 29 had ET  and 6 had PMF . In the patient group, MPL W515L/K mutations were found in only 2 PMF cases, and these cases were negative for JAK-2 V617F mutation. The prevalence of MPL W515L/K mutations in the patient group was 2.6%, and the prevalence of MPL W515L/K mutations among the cases negative for the JAK-2 V617F mutation was found to be 4.8%. The 2 cases with MPL W515L/K mutations had long follow-up times , had no thrombotic or hemorrhagic complications, and had no additional cytogenetic anomalies.Conclusion: MPL W515L/K mutations may be helpful for identifying clonal disease in MPN patients with no established Ph chromosome or JAK-2 V617F mutation.Conflict of interest:None declared. Myeloproliferative neoplasms (MPNs) consist of various clonal hematological diseases that are considered to develop from the hematopoietic stem cell transformation. Overproduction of mature, functional blood cells and a chronic clinical course are the main characteristics of these diseases. Chronic myeloid leukemia (CML) is a MPN that is characterized by the development of the Philadelphia (Ph) chromosome and the BCR-ABL fusion gene. Ph-negative MPNs are polycythemia vera (PV), essential thrombocythemia (ET), and primary (idiopathic) myelofibrosis (PMF). The basic feature of these 3 MPNs is that they show hypercellularity in the bone marrow . Individuals with these conditions have a tendency toward both thrombotic and hemorrhagic complications, and are at risk for leukemic transformation ,2. Janus kinase-2 (JAK-2) is a cytoplasmic tyrosine kinase and has an important role in intracellular signal transduction in hematopoietic cells through the erythropoietin, thrombopoietin (TPO), interleukin-3, granulocyte colony stimulating factor, and granulocyte-macrophage colony stimulating factor receptors. In 2005, an acquired point mutation in the JAK-2 gene (JAK-2 V617F mutation) was discovered in BCR-ABL-negative MPN patients [Sequence analysis of the MPL gene coding TPO receptor led to discovery of a new molecular abnormality in JAK-2 mutation-negative MPN patients. MPL mutations are located in the juxtamembrane region of the receptor. The most common MPL mutations are W515L (tryptophan-to-leucine substitution) and W515K (tryptophan-to-lysine substitution) ,7,8. TheIn the present study, we aimed to investigate the frequency and clinical significance of MPL W515L/K mutations in our patients with ET and PMF. A total of 77 patients (66 with ET and 11 with PMF) and 42 healthy individuals were analyzed for MPL W515L/K mutations. Patients and ControlsThis study was approved by the Ethics Committee of İstanbul University, İstanbul Faculty of Medicine No: 2009-1976) and was carried out in accordance with the principles of the Helsinki Declaration. The patient group was chosen from among cases followed by the Hematology Division. A total of 77 patients (66 with ET and 11 with PMF) who were followed between 1980 and 2010 and were diagnosed with MPN according to the World Health Organization (WHO) criteria [976 and wMethodsPeripheral venous blood was taken from all subjects. The presence of the MPL W515 mutations was screened using melting curve analysis at the molecular hematology laboratory. All samples were analyzed for JAK-2 V617F mutation and MPL W515L/K mutations. Genomic DNA was isolated from peripheral venous blood leukocytes using a spin-colon DNA isolation kit . Genotyping was performed using melting curve analysis with LightCycler 2.0 (Roche Diagnostics). Specific primers and probes were designed using Primer Express 3.0 software. '-ATGGAGTATGTGTCTATGGAGTATGTGTCTGTGG-fluorescein-3') and anchor (5'-LCR640-ACGAGAGTAAGTA AAACTACAGGTC- phosphate-3') probes using the following PCR program: initial denaturation at 95 °C for 10 min and 45 amplification cycles at 55°C for 10 s and 72 °C for 10 s. Melting analysis was performed by denaturing at 95 °C for 30 s and cooling to 35 °C for 1 min, followed by heating at the rate of 0.1 °C/s from 35 °C to 80 °C. Melting peaks generated with the LightCycler displayed a distinct melting temperature for the wild type (60 °C) and the mutant (52 °C). For the JAK-2 V617F LightCycler assay, polymerase chain reaction (PCR) was carried out in capillaries in a total reaction volume of 20 µL containing 25 ng of genomic DNA, 200 µmol/L of dNTPs, 4 mmol/L of MgCl2, 0.1 µmol/L of forward primer (TTCCTTAGTCTTTCTTTGAAGGT), 0.5 µmol/L of reverse primer (GTGATCCTGAAACTGAATTTTCT), and 0.2 µmol/L each of the sensor  of the target DNA, it had a higher melting temperature (62.5 °C). MPL W515 mutation-positive samples melted at a lower temperature (52.5 °C). For the W515 mutations, PCR reactions included 25 ng genomic DNA, 200 µmol/L dNTP, 4 mmol/L MgCl2, 0.2 µmol/L forward primer (TGGGCCGAAGTCTGACCCTTTA), 0.5 µmol/L reverse primer (ACAGAGCGAACCAAGAATGCCTGTG), and 0.2 µmol/L anchor probe (LC640-AGGCCCAGGACGGCGT) and sensor probe (CTGCCACCTCAGCAGCAT-fluorescein-3'). The PCR procedure was performed with 45 amplification cycles of 10 min of denaturation at 95 In evaluating the findings obtained in the study, NCSS  2007 and PASS 2008 Statistical Software  were used for the statistical analyses. In our study, MPL W515L/K and JAK-2 V617F mutations were not found in healthy controls. In the patient group, the JAK-2 V617F mutation was present in a total of 35 patients, 29 of which had ET and 6 of which had PMF. The frequency of JAK-2 V617F mutation in ET patients was 43.9%, and in PMF patients it was 54.5%. MPL W515L/K mutation was found in 2 cases. Both cases were from the PMF group and were negative for JAK-2 V617F mutation. The prevalence of the MPL W515 L/K mutations was found to be 2.6% (2/77) in the patient group, 4.8% (2/42) in patients who were negative for JAK-2 V617F mutation, and 18.2% in the PMF group (2/11). The clinical, laboratory, and molecular characteristics of the PMF patients found to have a MPL W515 mutation are presented in Patient 1, a 62-year-old male patient, complained of abdominal swelling and weakness. Upon physical examination, it was determined that he had hepatomegaly extending beyond the rib curve by 6 cm and splenomegaly extending beyond the rib curve by 10 cm. The patient had known diagnoses of chronic hepatitis B infection and atrial fibrillation with no history of thrombosis. His peripheral smear revealed 3% blast cells, 2% myelocytes, 4% metamyelocytes, tear drop-shaped erythrocytes, and giant platelets . A bone Patient 2, a 58-year-old female patient, presented with complaint of fatigue. Physical examination revealed splenomegaly extending beyond the rib curve by 9 cm. She had no additional disease or history of thrombosis. The peripheral smear revealed erythroblastosis and tear drop-shaped erythrocytes. Hypocellular bone marrow displayed elevated levels of dense reticulin fibers, as shown in The presence of MPL mutations in patients with MPN was first published by Pikman et al. in 2006 . Beer etIn Asian studies, however, the frequencies of MPL W515L/K mutations were not consistent. Lieu et al.  evaluateIn our study, MPL W515L/K mutations were investigated in 77 patients who were diagnosed with ET and PMF according to the WHO criteria. Forty-two patients were negative for JAK-2 V617F mutation. We found that MPL W515L/K mutations were present in only 2 patients with PMF (18.2%), and both of those patients were negative for JAK-2 V617F mutation. These patients had no thrombotic or hemorrhagic complications and had a long disease course (124 and 115 months). In conclusion, MPL W515L/K mutations may be helpful for identifying clonal disease in MPN patients with no established Ph chromosome or JAK-2 V617F mutation. Further studies including larger cohorts of patients will help to understand the phenotypic effects of these mutations. Conflict of Interest StatementThe authors of this paper have no conflicts of interest, including specific financial interests, relationships, and/ or affiliations relevant to the subject matter or materials included.


Tlr1 and Ang4 which are involved in the recognition and response to gut bacteria. A higher expression of Pla2g2a was detected in C3H mice. In addition, a number of interferon-inducible genes were higher expressed in C3H than in C57BL/10 mice including Gbp1, Mal, Oasl2, Ifi202b, Rtp4, Ly6g6c, Ifi27l2a, Usp18, Ifit1, Ifi44, and Ly6g indicating that interferons may play an essential role in microbiota regulation. However, genes coding for interferons, their receptors, factors involved in interferon expression regulation or signaling pathways were not differentially expressed between the two mouse strains. Taken together, our study confirms that the host genotype is involved in the establishment of host-specific bacterial communities in the gut. Based on expression differences after colonization with the same bacterial inoculum, we propose that Pla2g2a and interferon-dependent genes may contribute to this phenomenon.The host genotype has been proposed to contribute to individually composed bacterial communities in the gut. To provide deeper insight into interactions between gut bacteria and host, we associated germ-free C3H and C57BL/10 mice with intestinal bacteria from a C57BL/10 donor mouse. Analysis of microbiota similarity between the animals with denaturing gradient gel electrophoresis revealed the development of a mouse strain-specific microbiota. Microarray-based gene expression analysis in the colonic mucosa identified 202 genes whose expression differed significantly by a factor of more than 2. Application of bioinformatics tools demonstrated that functional terms including signaling/secretion, lipid degradation/catabolism, guanine nucleotide/guanylate binding and immune response were significantly enriched in differentially expressed genes. We had a closer look at the 56 genes with expression differences of more than 4 and observed a higher expression in C57BL/10 mice of the genes coding for  The intestine is colonized by a complex community of bacteria. These bacteria convert indigestible food components into absorbable fermentation products and modify non-nutritive plant metabolites and drugs . Deep-seBacteroides fragilis mutant strain that does not produce the capsular polysaccharide A. This strain failed to establish in the gut of germ-free mice. In contrast, the wild-type strain induced tolerance via TLR2 and successfully colonized the murine gut [Many genes that have been proposed to influence microbiota composition are involved in immune functions such as the pattern recognition receptors (PRRs) including Toll-like receptors (TLRs) and the nucleotide-binding oligomerization domain-containing protein 2 (NOD2). For instance, NOD2-deficient and wild-type control mice differ in microbiota composition ,13. Efferine gut . Howeverrine gut . Recognirine gut  and Panerine gut . Interesrine gut .Taken together, there is good indication that host-specific sensing of bacterial antigens by PRRs and the subsequent production of antimicrobial compounds is involved in the establishment of an individual microbiota. However, a large intercross study in mice suggested that complex interactions between polygenic host traits and environmental factors are responsible for microbiota individuality . In ordeThe protocol for the animal experiment was approved by the Animal Welfare Committee of the Ministry of Environment, Health and Consumer Protection of the Federal State of Brandenburg (Germany), State Office of Environment, Health and Consumer Protection . The regulations of the German Animal Welfare Act  were strictly followed.The study was conducted in 12 week-old male C3HHeOuJ (C3H) and C57BL/10ScSn (C57BL/10) mice (12 mice per group). Animals were bred and maintained germ-free in Trexler-type isolators under highly controlled conditions . Animals had free access to irradiated (25 kGy) standard chow  and autoclaved distilled drinking water. Conventional C3H and C57BL/10 (C57BL/10-MRL) mice from our breeding facilities and C57BL/10 (C57BL/10-Harlan) and C57BL/6 (C57BL/6-Harlan) mice purchased from the Harlan Laboratories  were tested for mutations in the Pla2g2a-encoding gene (see below). Conventional animals were housed in individually ventilated cages under standard housing conditions.2HPO4, 0.24 g KH2PO4) and 100 µl of this suspension containing ~107 bacterial cells was intragastrically applied to the experimental animals. During the experimental period, the associated mice were housed in one single isolator in polycarbonate cages on irradiated wood chips (one mouse per cage). After 13 weeks, animals were killed by cervical dislocation and colonic contents were collected for microbiota analysis. Colonic mucosa was carefully scratched, homogenized and immediately frozen in liquid nitrogen and stored at -80 °C until further processing.Germ-free C3H and C57BL/10 mice were associated with the unspecified microbiota of a single C57BL/10 donor mouse. A fresh fecal sample was diluted 1:50 with sterile phosphate buffered saline  and shipped on dry ice to ServiceXS . At ServiceXS, the quality of the RNA was checked with the Agilent Bioanalyzer . Biotin-labelled cDNA was synthesized using the Affymetrix One-Cycle Target Labeling. After testing the cDNA quality , hybridization was performed using 12.5–20 µg of cDNA on a customized Affymetrix nugomm 1a520177 chip . For unkinterferon-induced guanylate-binding protein 1 (Gbp1), Cluster of differentiation 14 (Cd14), angiopoietin-4 (Ang4), and phospholipase A2, group IIA (Pla2g2a). Interferon-induced guanylate-binding protein 1, cluster of differentiation 14, and phospholipase A2, group IIA were selected because differences between C3H and mice with a C57 genetic background in the expression of these genes have already been published [Angiopoietin-4 was selected because it is a known Paneth cell marker [Hypoxanthine-guanine phosphoribosyltransferase (Hprt1) and ribosomal protein L13a (Rpl13a) were selected as the reference genes. For quantitative real-time PCR analysis, RNA from three single mice per group and one pooled sample of the remaining mice per group were used. The RNA (0.7 µg) was transcribed with the QuantiTect Reverse Transcription Kit  and quantitative real-time PCR was performed with the Applied Biosystems 7500 Fast Real-Time PCR System  using the SYBR Green PCR kit (Qiagen). All reactions were carried out in triplicates. The primer sequences for Gbp1 were taken from the literature [Cd14 (5'- CGA ACA AGC CCG TGG AAC CT-3' and 5'-CAA GCA CAC GCT CCA TGG TC-3'), for Ang4 (5'-TGG CCA GCT TTG GAA TCA CTG-3' and 5'-GCT TGG CAT CAT AGT GCT GAC G-3'), and for Pla2g2a (5'-GGC CTT TGG CTC AAT ACA GGT C-3' and 5'-ACA GTG GCA TCC ATA GAA GGC A-3') were designed with the PerlPrimer software [Hprt1 and Rpl13a were 5'-CGT TGG GCT TAC CTC ACT GCT-3', 5'-CAT CAT CGC TAA TCA CGA CGC T-3' and 5'-GTT CGG CTG AAG CCT ACC AG-3', 5'-TTC CGT AAC CTC AAG ATC TGC T-3', respectively.The relative expression of the following genes was determined by quantitative real-time PCR: ublished . We therl marker  and we aterature . The prisoftware . Primer 5’-CTG GCT TTC CTT CCT GTC AGC CTG GCC-3’ and 5’-GGA AAC CAC TGG GAC ACT GAG GTA GTG-3’ [BamHI restriction enzyme  was applied.Genomic DNA was extracted from the tail tip of three mice per group . The exon 3 of the Pla2g2a gene was amplified with the primers A GTG-3’ . The ampDGGE gels were analyzed with the software GelCompar II . Differences in bacterial community structure were evaluated using the Dice similarity coefficient and bottom-up cluster analysis using the Unweighted Pair Group Method with Arithmetic mean (UPGMA). ANOVA followed by the Scheffé test was performed using SPPS 16.0  in order to identify significant differences (p ≤ 0.05) between mice with the same genetic background (intra-strain differences in microbiota composition) and between the C3H and C57BL/10 mice (inter-strain differences in microbiota composition).Mucosal affymetrix microarray gene expression data  was quality checked and evaluated in R (version 2.15.2) with Bioconductor (version 2.11) tools . The micThe qRT-PCR data was analyzed with the 7500 software version 2.0.5 (Applied Biosystems). The relative target gene expression levels were determined with the relative standard curve method after normalization to the reference gene expression. Differences in gene expression were tested for statistical significance (p < 0.05) with the Mann-Whitney test. Prism 5.0  was used for graphical data presentation and re-testing of statistical significances.Thirteen weeks after association of germ-free C3H and C57BL/10 mice with the unspecified intestinal microbiota from a single conventional C57BL/10 mouse, we analyzed the luminal and mucosa-associated microbiota composition in the colon of the recipients using a DGGE approach. The observed band patterns were used as an indicator for differences between C3H and C57BL/10 mice in microbiota composition. C3H and C57BL/10 mice grouped according to their luminal  and mucoWe compared the expression of ~16.000 genes in the colonic mucosa of previously germ-free C3H and C57BL/10 mice 13 weeks after association with intestinal bacteria. The expression of 210 genes significantly (FDR ≤ 0.05) differed by a factor of ≥ 2 . Twenty-phospholipase A2, group IIA, secretory leukocyte peptidase inhibitor, and lactoperoxidase all of which were higher expressed in C3H mice. In contrast, the angiogenin, ribonuclease Afamily, member 4 was higher expressed in the BL/10 mice. The LPS receptor cluster of differentiation 14 was higher expressed in C3H mice and the toll-like receptor 1 was higher expressed in the BL/10 animals. Interferon-inducible genes were higher expressed in C3H than in C57BL/10 mice. These genes included guanylate nucleotide binding protein 1, myelin and lymphocyte protein, 2'-5' oligoadenylate synthetase-like 2, interferon activated gene 202B, receptor transporter protein 4, lymphocyte antigen 6 complex, locus G6C, interferon alpha-inducible protein 27 like 2A, ubiquitin-specific peptidase 18, interferon-induced protein with tetratricopeptide repeats 1, interferon-induced protein 44, and lymphocyte antigen 6 complex, locus G.We had a closer look at the functions of genes with fold changes of more than 4. Many of these genes are involved in the synthesis of antibacterial factors and in immune functions Genes with (possible) antibacterial functions include the phospholipase A2, group IIA (Pla2g2a), cluster of differentiation 14 (CD14) and guanylate nucleotide binding protein 1 (Gbp1) was higher in the colonic mucosa of the C3H than in C57BL/10 mice. In contrast, the expression of Ang4 was significantly higher in C57BL/10 than in C3H mice  strains used are homozygous for the wild-type Pla2g2a genotype. In contrast, C57BL/6 mice and C57BL/10 purchased from Harlan laboratories were homozygous for the defective Pla2g2a gene.Interactions between intestinal bacteria and the host at the mucosal interface influence host health and disease. For instance, excessive host responses towards commensal gut bacteria are associated with the onset and perpetuation of chronic gut inflammation . On the angiogenin 4 . Direct effects of these two genes on colonic microbiota composition have so far not been demonstrated. However, differences in Ang4 expression have been reported between germ-free mice and mice monoassociated with Bacteroidesthetaiotaomicron [Enterococcus faecalis and Listeria monocytogenes. Gram negative bacteria such as Bacteroidesthetaiotaomicron and Escherichia coli are less sensitive. Interestingly, intestinal Ang4 mRNA expression is induced by complex microbiota and by Bthetaiotaomicron.  alone in previously germ-free mice [To identify genotype-specific responses towards bacterial colonization of the intestine, we analyzed with a microarray approach the mucosal gene expression in our experimental animals. This technique has been previously applied by others to describe differences in intestinal gene expression between germ-free and conventional C3H mice revealing that more than 50% of the differentially regulated genes were higher expressed in the conventional mice. The majority of these genes grouped in Gene Ontology biological processes involved in water transport across the gut wall and immune responses. Since mainly immunoglobulin-associated genes were identified in the “defense/immunity protein activity molecular function cluster”, it can be assumed that presence of microbiota influenced adaptive immune responses in this model . The catomicron . The antree mice .Ang4, phospholipase A2, group IIA (Pla2g2a), guanylate nucleotide binding protein 1 (Gbp1), and cluster of differentiation 14 (Cd14) were higher expressed in C3H than in C57BL/10 mice. The same observation has been reported in C3H and C57BL/6 mice and was associated with a strain-specific susceptibility to microbiota-driven colitis: interleukin 10-deficient mice with a C3H background are susceptible whereas the C57BL/6 background renders the mice colitis resistant [Cd14, which is involved in the detection of lipopolysaccharides by TLR4, it was later demonstrated that a higher expression by gut epithelial cells is responsible for a lower colitis susceptibility in IL-10-/- mice with a C3H than with a C57BL/6 background [In contrast to esistant . The latckground . WhetherPla2g2a gene in C57BL/6 mice which results in a defective gene product [Pla2g2a gene expression is equal in germ-free and conventional mice [The high genetic homogeneity that can be assumed for C57BL/6 and C57BL/10 mice suggests that our experimental mice were affected by a known frameshift mutation in exon 3 of the  product . This wo product . In conjnal mice , these fnal mice .Gbp1, these genes included Mal, Oasl2, Ifi202b, Rtp4, Ly6g6c, Ifi27l2a, Usp18, Ifit1, Ifi44, and Ly6g. Interestingly, the interferon regulatory factor 9-encoding gene (IRF9) was 2-fold higher expressed in C3H mice. It may therefore well be that IFN-dependent epithelial responses towards gut bacteria differ in a host-specific manner. However, whether individual IFN-dependent immune responses are the cause or the consequence of differences in intestinal microbiota composition remains elusive. On the one hand mouse strain specific Gbp1 expression has been demonstrated. Among the 46 inbred strains tested, mice with a C3H but not with a C57BL background displayed IFN-inducible GBP-1 synthesis [Bacteroidesthetaiotaomicron and Bifidobacteriumlongum have been observed in mice. Based on gene interaction network analysis it has been concluded that host responses to Bthetaiotaomicron.  are centered on tumor necrosis factor alpha triggered gene expression but that the gene expression network associated with Bifidobacteriumlongum is centered on INF-gamma responsive genes [The expression in human bronchioepithelial and nasal epithelial cells of type IIA phospholipase A2 is up-regulated by IFN-gamma  indicatiynthesis . The impynthesis . On the ve genes . Taken tSLPI encoding the secretory leucocyte protease inhibitor (28-fold higher expressed in C3H) and Mep1a coding for meprin 1 α (8-fold higher expressed in C57BL/10). SLPI expression is up-regulated by bacterial antigens and under inflammatory conditions. The protein protects the tissue from immune cell-derived proteases and is implicated in the priming of innate immunity and tissue repair. It also acts as an antibacterial protein and is active against both Gram positive and Gram negative bacteria [E. coli to bind to host receptors and to induce the expression of pro-inflammatory IL-8 [Differentially expressed genes with functions in the immune system included bacteria . Meprinsory IL-8 . These fO-glycosylated mucus protein MUC2. The glycans may serve as substrates and, in addition as adhesion sites for gut bacteria. Therefore, different mucin glycosylation pattern may be involved in the selection of a host specific microbiota [β-1,4-N-acetylgalactosaminyltransferase 2  which may contribute to mucin decoration with N‑acetylgalactosamine influences gut microbiota in mice [B4galnt2 expression. Since closely related bacterial species appeared to replace each other in B4galnt2-/- and B4galnt2+/+ mice, respectively, it is difficult to interpret whether B4galnt2-induced changes influence microbiota function. Interestingly, Helicobacter spp. were rarely detected in the intestine of B4galnt2-/- mice indicating that adhesion of the pathogen and possibly by other adherent bacteria to N-acetogalactosamine residues in the intestine is impaired in these animals.The intestinal mucus layer in the colon consists of a dense inner bacteria-free and a loose outer layer that is colonized by commensal bacteria. The mucins that form this layer are mainly composed of the densely crobiota . Interes in mice . Using aLin7c, Plscr2, AMN) and cell adhesion and cell signaling  were also differentially regulated in the experimental mice but their role in intestinal host-microbe interactions was not obvious from literature research.Genes with functions in plasma membrane structure or functions  and at the clonic mucosa (B).(PDF)Click here for additional data file.Table S2Genes differentially expressed (fold change >2) between C57BL/10 and C3H mice.(PDF)Click here for additional data file.Table S3DAVID functional gene list: signaling/secretion.(PDF)Click here for additional data file.Table S4DAVID functional gene list: peptidase inhibition.(PDF)Click here for additional data file.Table S5DAVID functional gene list: guanine binding.(PDF)Click here for additional data file.Table S6DAVID functional gene list: response to bacteria/defense.(PDF)Click here for additional data file.Table S7DAVID functional gene list: hormone activity.(PDF)Click here for additional data file.Table S8DAVID functional gene list: lipoprotein-associated.(PDF)Click here for additional data file.


We have previously studied the histone acetylation in primary human leukemia cells. However, histone H3 methylation in these cells has not been characterized.This study examined the methylation status at histone H3 lysine 4 (H3K4) and histone H3 lysine 9 (H3K9) in primary acute leukemia cells obtained from patients and compared with those in the non-leukemia and healthy cells. We further characterized the effect of phenylhexyl isothiocyanate (PHI), Trichostatin A (TSA), and 5-aza-2’-deoxycytidine (5-Aza) on the cells.We found that methylation of histone H3K4 was virtually undetectable, while methylation at H3K9 was significantly higher in primary human leukemia cells. The histone H3K9 hypermethylation and histone H3K4 hypomethylation were observed in both myeloid and lymphoid leukemia cells. PHI was found to be able to normalize the methylation level in the primary leukemia cells. We further showed that PHI was able to enhance the methyltransferase activity of H3K4 and decrease the activity of H3K9 methyltransferase. 5-Aza had similar effect on H3K4, but minimal effect on H3K9, whereas TSA had no effect on H3K4 and H3K9 methyltransferases.This study revealed opposite methylation level of H3K4 and H3K9 in primary human leukemia cells and demonstrated for the first time that PHI has different effects on the methyltransferases for H3K4 and H3K9. The N-terminal tails of histones go through a plethora of posttranslational modifications, including phosphorylation, ubiquitination, acetylation, and methylation. Each modification can affect chromatin architecture, and the sum of these modifications ultimately determines the chromatin state, which governs gene transcription ,2. HistoBrassica. These include the common vegetables cabbages, broccoli, Boi choi (Chinese cabbage), etc. ITCs have been successfully synthesized. More than a dozen of ITC derivatives distinguish from the natural forms by the length in the aliphatic carbon chains. Phenylhexyl isothiocyanate (PHI) is one of the most potent ITC derivatives. Our group has shown that PHI suppressed growth of the leukemic cell line HL-60 by inducing apoptosis both in vitro and in vivo in immunodeficient mice. We further demonstrated that PHI has preferential activity in leukemia cells and spares normal cells and tissues [Natural isothiocyanates (ITCs) are found in the cruciferous vegetables, genus  tissues ,18. PHI  tissues . PHI was tissues . We rece tissues ,21.In this report, we have identified the presence of aberrant histone methylation at H3K4 and H3K9 in fresh primary acute leukemia cells from patients. We further demonstrated that PHI is capable of correcting the aberrant methylation in the human leukemia cells. We demonstrated for the first time that PHI has differential effects on methyltransferases for H3K4 and H3K9.The laboratory analysis of bone marrow and peripheral blood specimens after diagnosis were approved by the human ethics committee of Zhangzhou Affiliated Hospital of Fujian Medical University, China. Informed consent was obtained. This study analyzed samples from twenty seven patients with primary untreated acute leukemias. These patients were enrolled from September 2007 to December 2008, including patients with ALL (acute lymphoblastic leukemia), AML (acute myeloid leukemia), or BAL (biphenotypic acute leukemia) with their age ranged from 6–78.Histone methylation was examined in the mononuclear cells isolated from the bone marrow of nineteen patients by Ficoll-Hypaque density gradient centrifugation. These patients included eleven males and eight females, with a median age of 38 (6–78). They included one case of M1, two cases of M2a, three cases of M3a, one case of M5a, two cases of M5b, three cases of L1, five cases of L2 and two cases of BAL. To study the effect of PHI on leukemia cells, bone marrow mononuclear cells were isolated from an additional group of eight patients. The cells were cultured and treated with PHI, tricostatin (TSA) and 5-Aza-2-dexoy-citidine (5-Aza). These patients included five males and three females, with a median age of 34 (12–78). They included two cases of M2a, one case of M3a, one case of M5b, one case of L1, two cases of L2 and one case of BAL. Individuals without leukemia, including two patients with iron-deficiency anemia (IDA), one with infection disease, two with idiopathic thrombocytopenic purpura (ITP), as well as three healthy volunteers were enrolled in the study and served as controls.5 cells/ml of RPMI-1640 medium with 15% heat-inactivated fetal calf serum, 100 IU/ml of penicillin and streptomycin, and maintained at 37 °C in a 5% CO2 atmosphere. Cultures were exposed to various concentrations of PHI , TSA at 10 μmol/L, or 5-Aza at 10 μmol/L. Cultures supplemented with the methanol medium were used as a vehicle control. After PHI treatment, bone marrow cell preparations were evaluated for histone methylation at H3K4 and H3K9 by Western blotting.PHI, greater than 98% pure, was purchased from LKT Lab . PHI was dissolved in a stock solution of 75% methanol. TSA (Sigma Inc.) was dissolved in dimethyl sulfoxide (DMSO). 5-Aza (Sigma Inc) was prepared in phosphate buffer (PBS). Bone marrow mononuclear cells, isolated from eight acute leukemia (AL) patients  specific for H3K4 and H3K9 modifications were used to quantify the H3 methyltransferase activity. The procedure was done according to the manufacturer’s instructions (Epigentek). For methylation assays, a blank lane was always included as a control to determine the spontaneous background.P≤0.05 was considered to be statistically significant.Statistical analysis was performed with the software SPSS 11.5. P = 0.005). Figure P = 0.001). These results clearly demonstrated that histone H3 was hypomethylated at H3K4, and hypermethylated at H3K9 in leukemia patients.The methylation status of histone H3 at lysines 4 and 9 was compared in mononuclear cells from patients with acute leukemia, and from individuals without leukemia and health volunteers Figure . The levWe have demonstrated that PHI enhanced acetylation of histones H3 and H4 ,20,21. ITo further study the mechanism, we examined the methytransferase activity in HL-60 cell line. Figure This study demonstrated for the first time that PHI has different effects on the methyltransferases of H3K4 and H3K9 in the leukemia cells. The methylation at H3K4 is generally associated with transcriptional activation, while methylation of H3K9 signifies heterochromatinization and transcriptional inhibition of euchromatin ,23. Our Methylation and acetylation of histone H3 are interconnected post-translational modifications. The methylation of histone H3K4 appears to correlate with its acetylation. PHI is known to augment acetylation at H3K4 via augmenting the activity of P300 ,20, whicH3K4, Histone H3 lysine4; H3K9, Histone H3 lysine9; K, Lysine; PHI, Phenyhexyl isothiocyanate; TSA, Trichostatin A; 5-Aza, 5-aza-2’-deoxycytidine; IDA, Iron deficiency anemia; ITP, Idiopathic thrombocytopenic purpura; PBS, Phosphate buffer; AML, Acute myeloid leukemia; ALL, Acute lymphoblastic leukemia; AL, Acute leukemia; BAL, Biphenotypic acute leukemia; HDACi, Histone deacetylase inhibitor; ITC, Isothiocyanate; DMSO, Dimethyl sulfoxide.The authors declare that they have no competing interests.XM is responsible for study design, data analysis, and manuscript preparation. YZ and YH carried out laboratory study and data collection. LH is responsible for collecting data and analyzing the data. JwC is involved in manuscript preparation. All authors have read and approved the final manuscript.


Glycans on the human immunodeficiency virus (HIV) envelope glycoprotein (Env) play an important role in viral infection and evasion of neutralization by antibodies. In this study, all 25 potential N-linked glycosylation sites (PNGS) on the HIV-1 CRF07_BC Env, FE, were mutated individually to study the effect of their removal on viral infectivity, virion production, and antibody-mediated neutralization.Removal of specific N-glycosylation sites has a significant effect on viral infectivity and antibody-mediated neutralization phenotype. Six of these glycosylation mutants located on the V1/V2 and C1/C2 domains lost infectivity. PNGS mutations located on V4/C4/V5 (except N392 on V4), were shown to increase viral infectivity. Furthermore, FE is much more dependent on specific glycans than clade B Env YU-2. On neutralization effect, PNGS mutations at N197 (C2), N301 (V3), N442 (C4) and N625 (gp41) rendered the virus more susceptible to neutralization by the monoclonal antibodies (MAbs) that recognize the CD4 binding site or gp41. Generally, mutations on V4/V5 loops, C2/C3/C4 regions and gp41 reduced the neutralization sensitivity to PG16. However, mutation of N289 (C2) made the virus more sensitive to both PG9 and PG16. Furthermore, we showed that mutations at N142 (V1), N355 (C3) and N463 (V5) conferred resistance to neutralization by anti-gp41 MAbs. We used the available structural information of HIV Env and homology modeling to provide a structural basis for the observed biological effects of these mutations.This report provides the first systematic experimental account of the biological role of the entire PNGS on an HIV-1 Env, which should provide valuable insights for understanding the function of Env in HIV infection cycle and for developing future anti-HIV strategies. The human immunodeficiency virus type 1 (HIV-1) Env consists of a trimer of heterodimers of the gp120 surface protein and the gp41 transmembrane protein . HIV-1 gThis cascade of conformational changes leads to the exposure of new epitopes on gp120 and gp41 for antibodies to recognize. Classes of broadly neutralizing monoclonal antibodies (MAbs) have been shown to neutralize HIV-1 by binding different regions of Env, including the gp120 CD4 binding site , the membrane proximal region of the gp41 ectodomain , and clusters of glycans on the surfaces of gp120  [HIV-1 gp120 is heavily glycosylated by the infected host with glycan moiety comprising about 50% of its total mass . These genvelope. The CRF07_BC recombinant strain has been one of the most predominantly circulated HIV-1 strains in China. The total potential N-linked glycosylation sites (PNGS) on CRF07_BC Env range between 24–35 (mean=30), with a mean of 25.8 in gp120 (range 20–30) and 4.2 in gp41 (range 2–6) [The circulating recombinant forms (CRFs) of HIV-1, CRF07_BC and CRF08_BC, are descendants of the parental subtypes B from Thailand and C from India, and are comprised of mostly subtype C in In the present study, the effect of PNGS on viral infectivity and antibody-mediated neutralization was evaluated through systematic mutations of each PNGS of Env and using pseudoviruses expressing the mutated Env to assess the infectivity and sensitivity to the MAb-mediated neutralization. In addition, we have utilized structural data of gp120 and gp41 and molecular modeling to evaluate the structural/functional relationship of the PNGS based on what we have observed in our systematic mutational study.We aligned the FE Env sequence with other HIV-1 isolates (12 isolates of each clade): clade B ; clade We compared the infectivity of FE with that of YU-2 (clade B), SF162.LS(clade B), 92BR025.9 (clade C) and sc19-15 (clade 07_BC), and found that the infectivity of FE strain was similar to those of the other HIV-1 strains from the different clades . Except for N181, the location of all gp120 PNGS can be visualized on the FE gp120 monomer model as shown in Figure In order to determine whether Env mutants with PNGS mutations could be incorporated into viral particles and support viral infection, we generated pseudoviruses with the PNGS mutants of Env and tested the capacity of these pseudoviruses to infect TZM-bl cells (CD4p<0.05). Six of these ten mutants  were non-infectious. These nine PNGS were mainly located in the C1, V1/V2, C2 regions and one in gp41 (N616Q). Another group of seven mutants  showed slight reduction of viral infectivity (p<0.05). There are five mutants (N408Q (V4), N411Q (V4), N442Q (C4), N448Q (C4) and N463Q (V5)) that had a significant increase in viral infectivity (p<0.05), and three mutants  showed no difference in viral infectivity compared to the wt.Among these 25 mutants with N->Q mutations individually, nine  had significant reduction in viral infectivity . Additionally, N88D showed a ~9-fold increase in infectivity compared to N88Q mutant and N234D showed a ~2-fold increase. Others showed no difference in viral infectivity compared with the N to Q mutants.To determine the effects of substitution other than Gln (Q) at PNGS, we mutated to Asp (D) (N->D) for the PNGS at positions 88, 156, 160, 181, 197, 234, 241, 262, 289 and 616. Interestingly, compared with the completely inactive N197Q and N289Q mutants, N197D and N289D recovered some level of viral infectivity  with N->Q mutation that resulted in complete loss of the infectivity, we also made the S/T to A mutation to abrogate the glycosylation motif . We found that the S/T->A mutants showed no difference in viral infectivity compared to the N->Q mutants, and that no activity was detected in both N->Q and S/T->A mutants (data not shown). These data suggest that the loss of infectivity of N->Q mutation in these six mutants probably is due to the loss of glycosylation.The effect of PNGS mutations on viral infectivity in different HIV-1 isolates was also investigated by generating PNGS mutations in other isolates that belongs to different clades. We made mutations in HIV isolates Sc19-15 (clade BC) and YU-2 (clade B) on those PNGS sites that significantly influenced the FE infectivity  by performing both N->Q and N->K/T/S mutations. Additionally, we also made N->K/T/S mutations on these sites in FE isolates. The result showed that N->K/T/S/Q mutation of N156, N160, N197, N262 and N289 resulted in complete loss of the infectivity for FE, most of these mutations (except for N156 and N289) also lost infectivity for the Sc19-15 isolate  or FE Env-expressing plasmid alone (expressing gp120). As expected, after ultra-centrifugation, no gp120 was present, but a similar level of p24 was detected from the single pSG3ΔEnv transfected pellet, and neither p24 nor gp120 were detected from the FE Env-expressing plasmid transfected pellet.To establish whether the loss of infectivity is caused by solely the mutations of PNGS or by a defect in the mutant gp120 incorporation into virions, we measured virus-associated gp120 glycoprotein for each PNGS mutant of FE Env. The mutant viruses were pelleted through a 25% sucrose cushion, and then subjected to p24 and gp120 ELISAs. The ratios of gp120/p24 were calculated for each virus to measure gp120 incorporation into virions, and are shown in Figure p<0.05) . Nine mutants  had slightly reduced gp120 incorporation than wt (around 83% to 97% of wt), mutants N289D and N637Q showed a 60%-66% of wt level. Much lower levels of virus-associated gp120 glycoprotein (2% - 47% of wt) was observed for mutants N133Q, N142Q, N156Q/D/S, N160Q/D, N181Q/D, N234Q/D, N241Q, N262Q/D, N289Q, N301Q, N355Q, N392Q, N611Q, N616Q/D (p<0.05).Among all Env mutants, N88Q and N442Q in-creased ~30% of gp120 incorporation compared to wt (p<0.05). The gp120 incorporation for N241Q was 12% of wt, but for N241D was 85% of wt, even though both mutants essentially lost all infectivity . However, even though wt FE also was resistant to mAb 3869, all of the three V1/V2 mutants of FE became more sensitive to mAb 3869, with the N142Q mutant being 19 fold more sensitive  were tested with four different monoclonal antibodies targeting areas of the V3 domain: 447-52D, 2191, 2442 and 3869. All four strains were resistant to mAbs 447-52D, 2191 and 2442 at highest ICWe made a complete set of systematically mutated N-glycosylation Env mutants and conducted comprehensive evaluation and analysis on their effects on viral infectivity and neutralization by different classes of neutralizing MAbs.We showed that all Env mutations on or near the V1/V2 domain had no infectivity or a reduced viral infectivity  are detrimental for infectivity for isolate YU-2 and that only N262 is also detrimental in another clade B isolate JRFL . In our  and N386For those glycosylation mutants that showed viral infectivity, we examined their sensitivity to seven broadly neutralizing MAbs ,44. The Unlike our results seen from the CD4bs MAbs, almost all of the PNGS mutants tested displayed a 2–10 fold increase in susceptibility to neutralization by the anti-gp41 MAbs, 2F5 and 4E10 (Table 50) than PG9  (data not shown). FE lost the N295, N332 and N386 PNGS that were suggested to be involved in 2G12 binding. We re-introduced these three PNGS into FE Env together, which remained resistant to 2G12 in our experiments (data not shown). 2G12 broadly neutralizes clade B isolates and is less effective against other clades, particularly clade C viruses, which have a somewhat different oligomannose glycan arrangement than clade B viruses. The continued insensitivity to 2G12 for the re-introduced mutant suggests the role of other residues in forming 2G12 epitope. A previous study showed that 447-52D can neutralize most subtype B viruses which contain the peptide GPGR at the tip of the V3 tip, but infrequently neutralized other clades . The wt In summary, through systematic mutations of all the PNGS of the Env gp120 and gp41 proteins, we have charted for the first time the relative significance of each of the PNGS of the FE Env for their biological functions in virion formation, viral infectivity, and MAb sensitivity. Using molecular modeling based on the available rich structural information in the literature, we provide the structural and functional correlation for the biological effects of most of the PNGS observed in our study, which should be valuable for our understanding of the roles of the respective glycans in HIV infection and host immunity. Furthermore, the results of the increased or decreased sensitivity to neutralizing antibodies after PNGS mutations will be valuable information for future design of anti-HIV/AIDS strategy.ΔEnv) were obtained from the US National Institutes of Health (NIH) AIDS Research and Reference Reagent Program (ARRRP), as contributed by John C. Kappes, Xiaoyun Wu, and Tranzyme . The 293FT cells were obtained from Invitrogen .TZM-bl (JC53-bl) cells, Env-deficient HIV-1 backbone ; MAbs 2F5 and 4E10 were contributed by Hermann Katinger  and b12 was contributed by Dennis Burton and Carlos Barbas ; MAbs VRC01, VRC03, PG9, PG16 and anti-V3 MAbs  were obtained from the ARRRP (NIH).2+ plus) , 4 μL dNTP mixture (2.5 mM) and 0.5 μL PrimeSTAR®HS DNA polymerase (2.5 U/μL) (TaKaRa), then add ddH2O to a total volume of 50 μL. Standard PCR and cloning procedure were used to obtain the mutant clones. All 25 PNGS of wt FE Env were mutated individually to have N to Q/D mutations at the positions shown in Table The motif for an N-linked glycosylation site is Asn-X-Thr/Ser, where X can be any amino acid except proline . Elimina2 rectangular canted neck cell culture flask, Corning, USA) with 5.3 μg pSG3ΔEnv plasmid and 2.7 μg Env-expressing plasmids using the Lipofectamine 2000 reagent (Invitrogen). Supernatants were harvested 48 hr after transfection, filtered (0.45-μm pore size), and stored at −80°C. The concentration of HIV-1 Gag p24 antigen in viral supernatants was measured by enzyme-linked immunosorbent assay (ELISA) .Pseudoviruses were produced by co-transfection of 293FT cells (>90% confluency in a 25 cm10 (RLU of mutant) by Log10 (RLU of wt).A fixed amount of pseudovirus  was added to TZM-bl cells at 70−80% confluency in a 96-well plate in the presence of 15 μg/mL DEAE-dextran, in a total volume of 200 μL. 48 hr after infection, the luciferase activity in infected cells was measured using the Bright-Glo™ luciferase assay system . Relative infectivity was calculated by dividing the Log50) of a single infectious pseudovirus batch was determined in TZM-bl cells, as described previously [The 50% tissue culture infectious dose  48 hr post transfection, then were pelleted through a 25% sucrose cushion by ultracentrifugation at 100,000 × g for 2.5 hr. The layers of supernatant and sucrose were removed, and the resulting viral pellets were resuspended in 250 μl PBS. The viral pellets were subjected to p24 ELISA, and gp120 ELISA  [ΔEnv or FE Env-expressing plasmid were set as control, which were performed as the viral supernatant above.293FT cells were co-transfected with plasmid pSG3rp, USA) -62 to deThe core FE gp120 model was created using the previously solved structure of truncated gp120 from a clade B HIV-1 isolate, JR-FL (PDB: 2B4C), as a template. Based on an alignment using Multalin, deletions were made to the FE gp120 sequence to correspond with the truncations made in the JR-FL sequence of the crystallized construct. The comparative homology modeling program SWISS-MODEL was used to generate the FE gp120 model. The FE V1/V2 domain model was generated similarly using the recently solved structure of the V1/V2 domain from a clade C isolate, CAP4 (PDB: 3U4E) as a template. The FE gp120 model including the V1/V2 domain was created in PyMOL by manually grafting the V1/V2 loop model onto the FE gp120 monomer model based on sequence and structural alignments of the limited overlapping sequences between the two molecules. The model of the FE gp41 was generated using the structure of the gp41 six-helical core bundle of the HXB2 group M subtype B isolate (PDB: 2X7R) as a template. For the “closed” trimer model we first generated an FE monomer model using the unliganded SIV gp120 structure as a template (PDB: 2BF1). The model was generated with Multalin and SWISS MODEL as described above. Our FE unliganded monomer model was then superimposed onto each of the monomers in the “closed” trimer model that was generated from cryo-ET (“closed” state PDB: 3DNN). The FE gp120 trimer model in the “open” CD4 bound state was generated by superimposing our FE gp120 monomer with V1/V2 domain model onto each of the monomers in the CD4 bound trimer model generated from cryo-ET (“open” CD4 bound state PDB: 3DNO).p value <0.05 was considered significant and is depicted by an asterisk in Figure Error bars indicate the standard deviation of the means calculated by Prism software 5.0. One-way analysis of variance with Tukey’s multiple comparison was used to test whether the mean infectivity and gp120 incorporation is significantly different between wild type and mutants by SPSS 10.0. A 50: The 50% tissue culture infectious dose; MAb: Monoclonal antibody; MPER: Membrane proximal external region.Env: Envelope glycoprotein; PNGS: Potential N-linked glycosylation site; CRFs: The circulating recombinant forms; TCIDThe authors declare that they have no competing interests.WW performed the experiments and analyzed data; WWB, XS. C and YC conceived and designed the experiments; JN and SW constructed the wildtype Env expressing plasmid FE; CP, CT and XS. C made the structure modeling; WW, XS. C and YW wrote the paper; all authors read and approved the final document.List of primers used for site-directed mutagenesis to eliminate potential N-linked glycosylation sites in the FE envelope.Click here for file


Avian influenza (AI) infection in poultry can result in high morbidity and mortality, and negatively affect international trade. Because most AI vaccines used for poultry are inactivated, our knowledge of immunity against AI is based largely on humoral immune responses. In fact, little is known about cellular immunity following a primary AI infection in poultry, especially regarding cytotoxic T lymphocytes (CTL’s).2/B2) chickens were infected with low pathogenic AI (LPAI) H9N2 and clinical signs of disease were monitored over a two weeks period. Splenic lymphocytes from infected and naïve birds were examined for cross reactivity against homologous and heterologous (H7N2) LPAI by ex vivo stimulation. Cellular immunity was determined by cytotoxic lysis of B2/B2 infected lung target cells and proliferation of T cells following exposure to LPAI.In these studies, major histocompatibility complex (MHC)-defined  and heterologous (H7N2) infected target cells, whereas lymphocytes obtained from sham-infected birds did not. T cell proliferation was determined to be highest when exposed to the homologous virus.Taken together these data extend the findings that cellular immunity, including CTL’s, is cross reactive against heterologous isolates of AI and contribute to protection following infection. Cell-mediated immunity (CMI) is antigen specific immunity mediated by T lymphocytes and has been suggested to be an important factor to the development of protection in chickens vaccinated against viral diseases . The sub2/B2 defined MHC allele (Line 15.6-2). Birds were placed in Horsfall units in BSL3E facilities  and provided feed and water ad libitum.Two-day-of age chickens were received from the USDA-ARS-Avian Disease Oncology Laboratory  containing the BLow pathogenic AI viruses of H9N2 (A/Chicken/NJ/12220/97) and H7N2 (A/Turkey/Virginia/4259/02) subtypes were propagated in the allantoic cavities of 9-11 day of embryonation SPF chicken eggs. Viral titers were determined as previously described [7 EID50 of H9N2 AI virus or sham inoculum in 0.2 ml volume (1/2 each nare) at 2 weeks of age. Chicken were monitored daily for clinical signs of disease and weight loss monitored at 2, 4 and 7 days post inoculation (p.i.) following H9N2 primary infection. Mean weight was compared by ANOVA using the Tukey Test (SigmaStat).Two-week-old chickens (6 per group) were inoculated via the intranasal route with 102/B2 infected and control chickens, three each, at 10 days post- infection as previously described [Splenic lymphocytes were harvested from Bescribed . MHC-matescribed . Lung ceLymphocyte proliferation as a measure of cellular memory was performed with almarBlue™ (Invitrogen) as previously described, using BPL-inactivated H9N2 (5 µg/ml) or H7N2 (5 µg/ml) . The T cInfection with A\Chicken\NJ\97 (H9N2) following a natural route of exposure  significantly decreased weight gains at day 2, 4 and 7 post-inoculation compared to control (uninfected) chickens Fig . In gene2/B2 lung target cells were lysed, while 43 % of H7N2-infected target cells were lysed. At lower E:T ratios, approximately 18 % (20:1) and 10 % (10:1) of infected target cells were lysed in either H9N2 or H7N2-infected lung cells. The splenic T cells from H9N2 infected birds did not lyse uninfected target cells, although spontaneous lysis was approximately 7 % (data not shown). In addition, lymphocytes obtained from sham-infected birds did not lyse infected or control lung cell cultures (data not shown).Splenic T cells from inbred chickens infected with H9N2 AI lysed target cells infected with either homologous or H7N2 AI at all effector:target (E:T) ratios tested , we found CTL’s isolated from H9N2 infected birds were active against homologous and heterologous isolates of AI based on lysis of MHC-matched target cells and T cell proliferation. In addition, memory T cells demonstrated proliferation in the presence of either isolate tested. These data suggest that antigenic peptides found in both isolates are presented by MHC-class I and II molecules that lead to production of memory CTL’s and stimulated proliferation of T cells, respectively. The proteins containing these peptides remain to be determined, although at least one report has identified a T cell epitope on the HA protein which could activate both chicken CD4+ and CD8+ cells [The main objective of the present research was to examine CMI following infection with low pathogenic AI, and to identify cross reactive T cell immunity in chickens. Using birds with a genetically defined MHC haplotype (B+ lymphocytes from H9N2-infected chickens and demonstrate protection from H5N1 lethal challenge [For this study birds infected with a LPAI H9N2 isolate were the source of lymphocytes and examining cross reactivity against a heterologous H7N2 isolate. We provide evidence that the H9N2 AI virus can induce cross reactive CMI against the H7N2 AI isolate. Because the majority of vaccines used across the world to protect poultry from AI are inactivated, they must be matched to the field subtype. While these vaccines induce protective antibodies against homologous subtypes based on HA, little to no protective cross reactivity is afforded against heterologous isolates . In contTaken together these data extend the findings that CMI, including production of CTL’s, is cross reactive against heterologous isolates of AI infection, and contribute to protection following infection.The authors declare that they have no competing interests.DRK designed the experiments, carried out the analysis, and drafted the manuscript. KL and DP performed the animal experiments in BSL3E, and together with HJ helped perform the ex vivo experiments. HH and GK developed the model and provided expertise with the MHC birds. All authors approved the final manuscript.


Our previous study presented evidence that the inflammation-related S100A9 gene is significantly upregulated in the brains of Alzheimer's disease (AD) animal models and human AD patients. In addition, experiments have shown that knockdown of S100A9 expression improves cognition function in AD model mice (Tg2576), and these animals exhibit reduced amyloid plaque burden. In this study, we established a new transgenic animal model of AD by crossbreeding the Tg2576 mouse with the S100A9 knockout (KO) mouse. We observed that S100A9KO/Tg2576 (KO/Tg) mice displayed an increased spatial reference memory in the Morris water maze task and Y-maze task as well as decreased amyloid beta peptide (Aβ) neuropathology because of reduced levels of Aβ, C-terminal fragments of amyloid precursor protein (APP-CT) and phosphorylated tau and increased expression of anti-inflammatory IL-10 and also decreased expression of inflammatory IL-6 and tumor neurosis factor (TNF)-α when compared with age-matched S100A9WT/Tg2576 (WT/Tg) mice. Overall, these results suggest that S100A9 is responsible for the neurodegeneration and cognitive deficits in Tg2576 mice. The mechanism of S100A9 is able to coincide with the inflammatory process. These findings indicate that knockout of S100A9 is a potential target for the pharmacological therapy of AD. S100A9, a the member of the calcium binding S100 protein family that is also known as MRP14 or Calgranulin B, is an inflammation-associated protein that is constitutively expressed in neutrophils and inducible in numerous inflammatory cells, including macrophages, epithelial cells, and keratinocytes Neuronal degeneration, which involves synaptic and neuronal loss, and formations of intracellular neurofibrillary tangles and extracellular neuritic plaques containing amyloid beta (Aβ) peptide plays a central role in the pathogenesis of neurodegenerative diseases, particularly in AD It is well known that Tg2576 mice (Tg) harboring the human APP transgene with the familial AD Swedish mutation develop AD-like cerebral amyloidosis There have been many recent studies that have examined S100A9 deficiencies. For example, in one study, S100A9 deficient mice were used to confirm the expression of Interleukin-8-induced CD11b To assess whether S100A9 knockout rescued the cognitive deficit and neuropathology in AD animal mice, S100A9 KO mice were crossbred with Tg2576 mice. These mice allowed for the comparison of four groups of mice; wild type/wild type (WT/WT), S100A9knock out/wild type (KO/WT), wild type/Tg2576 (WT/Tg), and S100A9knock out/Tg2576 (KO/Tg). At 13 months of age, we found that KO/Tg mice showed rescued cognitive impairments compared to WT/Tg mice. We also confirmed differences in pathogenesis, particularly abundant amyloid neuritic plaques containing Aβ and phosphorylated tau proteins and inflammatory process related cytokines in 4 groups of 14-month-old S100A9 KO/Tg crossbred mice.All animal procedures were performed following the National Institutes of Health Guidelines for the Humane Treatment of Animals, with approval from the Institutional Animal Care and Use Committee of Seoul National University (IACUC No. SNU-100611-1). Animals of only male were used in this study.S100A9 KO mice in a C57BL/6 background were kindly provided by Wolfgang Nacken  To analyze the offspring, genomic DNA samples isolated from mice tails was genotyped based on the previously described method To obtain tissues for experiments, the animals were anaesthetized and immediately cardiac-perfused with PBS containing heparin. For morphological analyses, one hemisphere of the brain was fixed in a 4% paraformaldehyde solution for 24 h and embedded in paraffin. For biochemical analyses, including western blotting, enzymatic activity assays and enzyme-linked immunosorbent assays, the other half of the brain was quickly frozen on dry ice and stored at −70°C. Tissues were lysed in RIPA buffer with protease inhibitors cocktail (Roche).Sections were deparaffinized in xylene and dehydrated using graded alcohols to water. Sections were retrieved by 0.01M citric acid (pH 6.0) and blocked with 0.5% triton X-100 and 2% normal serum in TBS. Appropriate primary antibodies were incubated overnight (O/N) and were visualized using an appropriate secondary antibody. For labeling, immunohistochemistry was performed using a Vectastain avidin biotin complex (ABC) elite kit. The reaction product was detected using 3.3-diaminobenzidinetetrahydrochloride (DAB).Tissues were washed with phosphate-buffered saline (PBS) and lysed in RIPA buffer with a cocktail of protease inhibitors (Roche). Proteins were separated using SDS-PAGE and transferred to a PVDF membrane. The PVDF membrane was blocked with 5% nonfat dry milk in Tris-buffered saline containing 0.05% Tween 20 (TBS-T). After 1 h of blocking, the protein blot was confirmed using appropriate antibodies at 4°C O/N and detected using a horseradish peroxidase-conjugated secondary antibody (Amersham Pharmacia). Westen blotting was detected by Gel doc system (Bio-rad) and data was analyzed using quantity one program (Bio-rad).The following primary antibodies were used: anti-Aβ mouse monoclonal antibody 6E10 , anti-mouse S100A9 and S100A8 , S100B , GAPDH (Abfrontier), anti-Amyloid Oligomer, Aβ, ,p-Tau (Ser404) , Phospho-PHF-tau  , Anti-PhosphoTau  , Tau (C-17) , Calnexin (H-70) , and BACE (M-83) .1-42: IBL, mouse IL-10: KMC0102, Invitrogen, mouse IL-6: DY406, R&D systems, TNF-α: DY410, R&D systems) for the quantitative determination of human Aβ1-42 IL-10, IL-6, and TNF-α in brains. All assays were performed according to manufacturer's specific instructions. Levels of these proteins were calculated from a standard curve developed with specific OD versus serial dilutions of known concentration. Each standard and experimental sample was run in duplicate, and the results were averaged.ELISAs were performed using colorimetric sandwich ELISAs kits . Vertical metal poles located at the outer perimeter of the maze provided spatial cues. A mouse was placed at the end of one arm and allowed to move freely through the maze for 8 min without reinforcements, such as, food and water. The total numbers of entries into the arms, including returns to the same arms, was recorded. Alternation was defined as entry into each of the three arms consecutively. The maximum number of alternations was calculated by subtracting two from the total number of arms entered. Percent alternation was calculated by expressing actual alternations as a percentage of maximum alternations As described previously Brain sections (4 µm) were deparaffinized and hydrated using a descending ethanol series. After washing in a freshly prepared alkaline alcoholic saturated sodium chloride reagent  for 20 min at room temperature, the sections were incubated in 0.4% Congo red  in an alkaline alcoholic saturated sodium chloride reagent (freshly prepared and filtered prior to use) for 30 min at room temperature. Sections were washed in distilled water and counterstained with hematoxylin for 1 min. Sections were rinsed using ascending grades of ethanol with a final three changes of 100% reagent-grade ethanol, cleared in xylene and cover slipped with permount (Fisher Scientific) The fluorometric assay of secretase was conducted using β- and γ-secretase activity kits  in accordance with the protocol supplied by the manufacturer. As an enzyme source, total cortical protein lysates were tested. Quantification of substrate cleavage was assessed using a fluorometric reader .p≤0.05, **, p≤0.01, and ***, p≤0.001.Data were expressed as the mean ±SEM value or as fraction of the control value ± SEM. These results were analyzed by ANOVA followed by the Tukey HSD or the LSD test (SPSS version 18). The difference was considered statistically significant for *, −/− plays a key role in AD progression, we crossed F1 male S100A9+/−/Tg2576 (HT/Tg) mice with female S100A9+/−/WT (HT/WT) mice to generate F2 littermates with the following genotypes: S100A9+/+/WT (WT/WT), HT/WT, S100A9−/−/WT (KO/WT), S100A9+/+/Tg2576 (WT/Tg), HT/Tg and S100A9−/−/Tg2576 (KO/Tg) mice. The experimental groups include WT/WT, KO/WT, WT/Tg and KO/Tg. In To elucidate whether S100A9We investigated the levels of other specific calcium-binding proteins, including Calnexin, S100A8 and S100B in the brains of S100A9 KO/Tg crossbred mice by western blot analysis. Our data indicate that there is no change in their levels among all groups . We alsoth day of the learning sessions, analysis of the escape latency of each group showed significant differences between the KO/Tg and WT/Tg groups  . HoweverP = 0.009, F = 54.8%) but not in the KO/Tg group . In our analysis of these behavioral test findings, the S100A9 KO/Tg crossbred mice showed an improvement in cognitive performance.To further examine the learning and memory function in S100A9 KO/Tg crossbred mice, we performed the passive avoidance test. As shown in Insoluble deposits of Aβ plaques are strong candidates for initiating the inflammatory response 1–17 of Aβ.In this study, we examined amyloid plaque load and protein levels of Aβ and CT in the brains of 14-month-old WT/WT, KO/WT, WT/Tg and KO/Tg mice using Congo red staining and western blot analysis with the 6E10 antibody, which specifically recognizes amino acidsP = 0.025; Student's t-test; Fibrilar plaques were observed by Congo red and Thio-S staining in the cortex and hippocampus of KO/Tg and WT/Tg mice . HoweverP = 0.007; APP-CT, from1 to 0.613, P = 0.020; Based on the Congo red staining data , we exam1-42 levels in the cortex and hippocampus of all groups. Similar to the Congo red staining and western blot results, Aβ1-42 in the brain of the KO/Tg group was significantly decreased by 51.17% in the cortex , detected near Aβ plaques was reduced in the hippocampus and cortex of KO/Tg mice brains . And som2+ levels S100 proteins, including S100A8, S100A9 and S100A12, are known to contribute to chronic inflammation P = 0.04) compared to WT/Tg mice  compared to WT/Tg mice brain (P = 0.035) compared to WT/Tg mice  In AD animal mice, such as the Tg2576 mice, a rapid increase of Aβ begins at from 6 months, amyloid plaques are formed after 9–12 months, and memory deficits begin after 12 months We crossbred Tg2576 and S100A9KO mice and investigated the behavioral and pathological characteristics of S100A9KO/Tg2576 crossbred mice. Previous results have shown that S100A9 deficiency results in attenuated spatial learning and memory behavior in tests, including the Morris water maze, passive avoidance test, and Y-maze tasks, in 14-month-old mice. In our study, we found that S100A9 is related with learning and memory impairment in the AD mice model.1-42 was greatly decreased in KO/Tg mice compared with WT/Tg mice. These results raise the question that S100A9 may be involved in the formation of plaques and may contribute to Aβ aggregation. We previously showed that knockdown using short hairpin RNA reduced the amount of Aβ and CT by decreasing BACE activity in Tg2576 mice. In S100A9 KO/Tg mice, we did not detect significant changes in BACE activity and expression of BACE  For genotyping, DNA levels of S100A9 and Swedish APP were measured in each group by PCR analysis with each primer . The absence of S100A9was shown in KO/WT and KO/Tg mice and the DNA band of Swedish APP was detected in WT/Tg and KO/Tg mice. Actin was used as a loading control. (B) At the age of 14-months, S100A9 expression was observed in the brain by immunohistochemisty using the anti-S100A9 antibody. In the hippocampus and cortex of mice brain, S100A9 expression was significantly reduced in KO/Tg mice compared with WT/Tg mice. Significant differences were observed in the Frontal Cortex (FC) and Parietal Cortex (PC). Sections are 4 µm thick. Scale bar; 200 µm.(TIF)Click here for additional data file.Figure S2Expression of calcium binding proteins in the brains of S100A9 crossbred mice. (A) At the age of 14-months, western-blot analysis was performed with total lysates from the cortical region of the brains in each group using anti-Calnexin, anti-S100A8 and anti-S100B antibodies. The membrane was stripped and reprobed with GAPDH to confirm equal loading. There were no noticeable differences among all groups. This is a representative blot from at least five independent experiments. (B) Immunoreactivities of S100A8 were examined in the cortex and hippocampus of 14-month-old S100A9 crossbred mice brains. There were no noticeable differences among all groups. ((a)-(l) Scale bar; 50 µm).(TIF)Click here for additional data file.Figure S3Congo-red staining in the brains of S100A9 crossbred mice. Congo-red staining was performed in the cortex of S100A9 crossbred mice brains.(TIF)Click here for additional data file.Figure S4Expression of P-tau was decreased in KO/Tg mice brain. (A) P-tau expression such as AT8  and PHF-13 (S396) were decreased in KO/Tg mice brain compare with WT/Tg mice brain.(TIF)Click here for additional data file.Figure S5Enzymatic activity of the β-secretase in the brains of S100A9 crossbred mice. (A) 60 min after adding the substrate, enzymatic activity of the β-secretase from the mice brain lysates was assessed using fluorometric reaction. β-secretase activity was assessed as time passed. In S100A9 KO/Tg mice, we did not detect significant changes in BACE activity. (B) Expression of BACE showed no significant difference.(TIF)Click here for additional data file.


A swine influenza outbreak occurred on a commercial pig farm in Thailand. Outbreak investigation indicated that pigs were co-infected with pandemic (H1N1) 2009 virus and seasonal influenza (H1N1) viruses. No evidence of gene reassortment or pig-to-human transmission of pandemic (H1N1) 2009 virus was found during the outbreak. Pigs showed mild respiratory signs; only 1 pandemic (H1N1) 2009 virus was isolated from 80 nasal swab specimens.In April 2009, a novel swine origin influenza A (H1N1) virus, now referred to as pandemic (H1N1) 2009 virus, emerged in humans in Mexico and the United States and spread worldwide  was reported in Thailand in 1981 (We report an outbreak of infection with pandemic (H1N1) 2009 virus during November 2009–March 2010 on a commercial pig farm in Thailand. The outbreak presumably resulted from human-to-pig transmission because 1 of the workers on this farm had influenza-like clinical signs at the beginning of the outbreak. Infection in this worker was not confirmed because he quit his job on the farm after the start of the outbreak and could not be located.In early November 2009, a small commercial pig farm in central Thailand reported respiratory problems in pigs  in nursery pigs. The farm contained 3,235 pigs . It has a conventional open-house production system in which both sides of the unit have natural air flow ventilation. The farm also has continuous nursery herd flow in which new pigs are continuously added when they are old enough. This process results in pigs of different ages being in the same unit. Sick pigs had clinical signs  of infection.–Nasal swabs from 20 nursery pigs (4–9 weeks of age) were submitted to Chulalongkorn University Veterinary Diagnostic Laboratory. All samples were positive for porcine circovirus type 2 and porcine reproductive and respiratory syndrome virus (these viruses are major causes of swine respiratory disease), and 2 samples were positive for influenza A virus by reverse transcription–PCR (RT-PCR) with primers for each specific pathogen , and sows (10 per group) with clinical signs were submitted to the diagnostic laboratory by the end of December 2009. Two samples from nursery pigs were positive for influenza virus A (H1N1) by multiplex RT-PCR . Genome www.fao.org) sample collection recommendations, we obtained 20 nasal swabs from pigs with SIV-like illness. In addition, nasal swab specimens (n = 10 per group) were collected from gilts, sows, and finishing pigs to test for pandemic (H1N1) 2009 virus, although no clinical signs were observed in any pigs from these age groups. All SIV-positive samples were subjected to virus isolation (Pandemic (H1N1) 2009 investigations on the farm included clinical surveillance and sample collection from sick and contact pigs and close monitoring of swine workers and farm pets for influenza-like illness. Nasal swab specimens were obtained from pigs on January 17, 2010, January 30, 2010, and March 9, 2010. Because initial laboratory findings indicated that the outbreak involved the nursery herd, weaned pigs were moved to a separate site on the farm to control disease in the nursery pigs. Following Food and Agriculture Organization (www.megasoftware.net/) and the neighbor-joining method with 1,000 bootstrap replicates. Whole genome analysis showed that contemporary SIV (H1N1) and pandemic (H1N1) 2009 virus were concurrently circulating in the nursery herd . Phylogenetic trees were constructed by using MEGA4  2009 virus interspecies transmission, we obtained serum samples on January 17, 2010, from 40 pigs in 8 age groups (5/group), 15 workers, and 4 farm pets (3 dogs and 1 cat). Samples were subjected to hemagglutination-inhibition (HI) testing with SIV (H1N1) and pandemic (H1N1) 2009 virus antigens (Control rabbit antibodies against SIV (H1N1) viruses did not cross-react with pandemic (H1N1) 2009 virus. Serologic results showed that only 2 (9.5%) of 21 test samples from the nursery group had positive HI titers for pandemic (H1N1) 2009 virus and 8 (38%) of 21 had positive HI titers for SIV (H1N1) virus. For pigs in other age groups, 11 (55%) of 20 had antibodies against pandemic (H1N1) 2009 virus and 14 (70%) of 20 had antibodies against SIV (H1N1) by HI test. No human cases of co-infection were observed. We found no evidence of pandemic (H1N1) 2009 virus interspecies transmission from pigs to humans or to farm pets.,Consistent with findings of previous reports (