CD209, is a receptor capable of binding and internalizing Mycobacterium tuberculosis. Previous studies have reported that the CD209 promoter single nucleotide polymorphism (SNP)-336A/G exerts an effect on CD209 expression and is associated with human susceptibility to dengue, HIV-1 and tuberculosis in humans. The present study investigates the role of the CD209 -336A/G variant in susceptibility to tuberculosis in a large sample of individuals from sub-Saharan Africa.Tuberculosis causes significant morbidity and mortality worldwide, especially in sub-Saharan Africa. DC-SIGN, encoded by CD209 -336A/G SNP (rs4804803). Significant overall protection against pulmonary tuberculosis was observed with the -336G allele when the study groups were combined . In addition, the patients with -336GG were associated with a decreased risk of cavitory tuberculosis, a severe form of tuberculosis disease . This direction of association is opposite to a previously observed result in a smaller study of susceptibility to tuberculosis in a South African Coloured population, but entirely in keeping with the previously observed protective effect of the -336G allele.A total of 2,176 individuals enrolled in tuberculosis case-control studies from four sub-Saharan Africa countries were genotyped for the CD209 -336G variant allele is associated with significant protection against tuberculosis in individuals from sub-Saharan Africa and, furthermore, cases with -336GG were significantly less likely to develop tuberculosis-induced lung cavitation. Previous in vitro work demonstrated that the promoter variant -336G allele causes down-regulation of CD209 mRNA expression. Our present work suggests that decreased levels of the DC-SIGN receptor may therefore be protective against both clinical tuberculosis in general and cavitory tuberculosis disease in particular. This is consistent with evidence that Mycobacteria can utilize DC-SIGN binding to suppress the protective pro-inflammatory immune response.This study finds that the  Mycobacterium tuberculosis infection causes significant morbidity and mortality throughout the world, particularly in resource-poor countries. M. tuberculosis infects one third of the human population and approximately two million persons die of tuberculosis every year M. tuberculosis and more than 25 million will die from the disease, despite the availability of antibiotics CD209 on chromosome 19p13.3, is a C-type lectin that is expressed on subsets of dendritic cells (DCs) and alveolar macrophages M. tuberculosis, M. leprae, Bacillus Calmette-Guérin (BCG), Heliobacter pylori, Streptococcus pneumoniae, Klebsiella pneumoniae, HIV-1, HIV-2, SIV-1, Dengue virus, Ebola Virus, Cytomegalovirus, Hepatitis C virus, Schistosoma mansoni, Leishmania pifanoi and Candida albicansDendritic Cell-Specific ICAM3-Grabbing Non-integrin (DC-SIGN), encoded by M. tuberculosis on immune cell subsets and has been shown to bind mannosylated lipoarabomannan (ManLAM) present on virulent, but not avirulent, strains of Mycobacterium spp.M. tuberculosis and infection of DCs and alveolar macrophages M. tuberculosis-induced immune suppression DC-SIGN is a major receptor for CD209 variation in human susceptibility to infectious diseases including M. tuberculosis and M. leprae, HIV-1, and Dengue M. leprae case-control study found an association of the CD209 -336A/G promoter SNP with infectious disease susceptibility or protection. Martin and colleagues demonstrated that the -336G allele was associated with susceptibility to parenteral but not mucosal HIV-1 infection, although this was not replicated in individuals of recent African descent CD209 -871 SNP was found to be the polymorphism most significantly associated with tuberculosis susceptibility in the South African Cape Coloureds, Barreiro and colleagues found that this SNP is not polymorphic in other African countries. The relatively high prevalence of the -871 SNP in the South African Cape Coloureds cases and controls is due to their inherent admixture with European and Asian populations.There has been considerable recent interest in the role of CD209 -336G allele disrupts a potential SP-1-like transcription factor binding site and that this allele is associated with lower CD209 expression in vitro than the ancestral -336A variant M. tuberculosis binding and signaling events, and in an attempt to replicate the findings of Barreiro and colleagues in a larger population, we genotyped the CD209 -336A/G SNP in five tuberculosis case-control studies from four different sub-Saharan African countries.Sakuntabhai and colleagues also presented evidence that the M. tuberculosis positivity by culture, smear, or histology. HIV-1 testing was routinely performed and the percentage of HIV-1 positive individuals among the tuberculosis index cases and controls for the various studies are as follows: The Gambia (A), cases 7.5%, controls 5.2%; Republic of Guinea, cases 9.0%, controls 5.1%; Guinea Bissau, cases 23.8%, controls 16.2%; Malawi, cases 62.2%, controls 33.5%. The Gambia (B) cohort has 330 confirmed HIV-1 negative tuberculosis cases, with an additional 17 cases which were not tested for HIV-1 status. Controls for the various studies were defined as persons without a previous history of tuberculosis and were frequency matched for age, sex and area of residence. A number of clinical correlates of tuberculosis were recorded for the The Gambia (A), Republic of Guinea and Guinea Bissau studies including haemoptysis, duration of cough, degree of sputum positivity, and the presence of cavitation and number of zones affected on chest radiograph. Cavitory tuberculosis status and number of zones affected was determined by radiological analysis by board-certified radiologists.The tuberculosis case control studies from The Gambia (A), Republic of Guinea and Guinea Bissau Informed written consent was obtained from patients or their parents or guardians. Ethical approval was provided by the joint Gambian Government/MRC Ethical Committee, Ministry of Public Health  and National Ethics Committee, Ministry of Health, Conakry, Republique de Guinee. Human experimentation guidelines of these ministries were followed. Ethical approval for the Malawi study protocol was obtained from the National Health Sciences Research Committee of Malawi and the London School of Hygiene and Tropical Medicine Ethics Committee.CD209 -871A/G (rs735239) and -336A/G (rs4804803) SNP assays were designed using the SPECTRODESIGNER software (Sequenom). The primers for the CD209 -871A/G were ACGTTGGATGCTCTGTCTGGGTCCTTTTAC, ACGTTGGATGACAGCAATGAAAAAGCAAAG, GCAAAGTACTAGTACATTTAATAAC and for CD209 -336A/G were ACGTTGGATGTGTTACACCCCCTCCACTAG, ACGTTGGATGAAAGCAGGAAAGCCAGGAGG, CCCTCCACTAGGGCAAGGGT. The assays were in 384-well plates containing 10 ng of DNA in each well were amplified by a touch-down PCR using Titanium Taq polymerase (Clontech). The touch-down PCR cycling conditions were as follows: 95°C for 15 minutes; 94°C for 20 seconds; 65°C for 30 seconds; 72°C for 30 seconds; steps 2 to 4 repeated for 5 cycles; 94°C for 20 seconds; 58°C for 30 seconds; 72°C for 30 seconds; steps 5 to 7 repeated for 5 cycles; 94°C for 20 seconds; 53°C for 30 seconds; 72°C for 30 seconds; steps 8 to 10 repeated for 38 cycles; final extension at 72°C for 3 minutes.DNA extraction from blood was performed using Nucleon II kits . DNA concentrations were determined using the PicoGreen™ kit . Genotyping was performed utilizing the Sequenom™ system  which uses mass spectrometry (MALDI-TOF) to discriminate products by their absolute mass P value was significant then further analysis was carried out using a stepwise forward binary logistic regression correction to adjust for the potential confounding factors of HIV status, age, ethnicity, and sex. Mantel-Haenszel for the combined analysis was performed using SPSS by weighting cases by frequency and stratifying by population. The Gambian (B) study utilizes the Gambian (A) study controls for this Mantel-Haenszel analysis. The Breslow-Day and Tarone's tests for homogeneity of odds ratios were found to be non-significant indicating that the odds ratio for each study did not significantly deviate from the overall, combined odds ratio. All control genotype distributions were in Hardy-Weinberg equilibrium (P>0.20).Statistical analysis of genotype associations and logistic regression was performed using the program SPSS v14.0. The genotypic and allelic tests of association utilized Pearson's chi-square test with the respective degrees of freedom. All statistics are two-tailed. If the unadjusted CD209 -871A/G and -336A/G SNPs were initially genotyped in 329 tuberculosis cases and 327 control individuals from The Gambia (study group A) CD209 -871 SNP was found to be polymorphic with controls and cases both having a 2.3% allele frequency  consistent with the Barreiro result 2 = 7.79, P = 0.020)  (The = 0.020) Table 1.P = 0.006 (P = 0.32), suggesting that there was a good consistency between the combined odds ratio with the individual odds ratios for each study.Study groups from Republic of Guinea, Guinea-Bissau, and Malawi were then used in an attempt to replicate this finding  = 0.006 Table 2.P = 0.003  and  = 0.003 Table S1CD209 -336A/G SNP was further examined in the cases for association with clinical correlates of tuberculosis, with no significant difference between sex, HIV status, age, haemoptysis, number of zones affected on chest radiograph, duration of cough or degree of sputum positivity. However, a significant association was obtained between CD209 -336GG genotype status and lung cavitation in tuberculosis patients. Patients homozygous for the CD209 -336GG allele were associated with 2.4-fold protection against cavitory tuberculosis disease  mannosylated termini which closely resemble the high mannose N-linked oligosaccharides of newly produced glycoproteins in eukaryotic cells M. tuberculosis modulate the immune response. If so, DC-SIGN is a prime target for this virulence strategy.Maturation of DCs, defined by DC upregulation of co-stimulatory molecules such as CD80/CD86, can occur on exposure to TLR ligands. This process can however be inhibited by a variety of unknown mechanisms. One possible mechanism of inhibition of DC maturation is the binding of pattern recognition receptors to molecular patterns present on host cells M. tuberculosis may use the DC-SIGN receptor to subvert the immune response.Overall, our results suggest that lower levels of DC-SIGN on immune subsets may be protective against tuberculosis in the sub-Saharan Africa setting. The findings implicate the involvement of DC-SIGN in the pathogenesis of clinical tuberculosis in general, and severe, cavitory tuberculosis in particular. This has wider implications since it provides further evidence that Table S1CD209-336A/G SNP Allelic Association in Tuberculosis Case Control Studies Excluding All HIV-1 Positive Cases and Controls. The allelic P value for HIV-1 negative individuals enrolled in tuberculosis case control studies. The Gambia (A), Republic of Guinea and Guinea-Bissau (ref. 33), The Gambia (B) (ref. 34) and Malawi (ref. 35). *The Gambia (B) study utilizes The Gambia (A) controls. Significance levels of P<0.05 are indicated in bold.(0.06 MB DOC)Click here for additional data file.


Primary endpoint was survival. 233 eligible patients were randomised. Chemotherapy could be significantly accelerated in arm B with increased absolute dose-intensity. Best response rates, in the population of evaluable patients, were, respectively for arm A, B and C, 59%, 76% and 70%. The response rate was significantly higher in arm B in comparison to arm A (P = 0.04). There was, however, no survival difference with respective median duration and 2-year rate of 286 days and 5% for arm A, 264 days and 6% for arm B and 264 days and 6% for arm C. Severe thrombopenia occurred more frequently in arm B but without an increased rate of bleeding. Non-severe infections were more frequent in arm B and severe infections were less frequent in arm C. Our trial failed to demonstrate, in ED-SCLC, a survival benefit of chemotherapy acceleration by using GM-CSF support.   http://www.bjcancer.comThe European Lung Cancer Working Party (ELCWP) designed a 3-arm phase III randomised trial to determine the role of accelerated chemotherapy in extensive-disease (ED) small-cell lung cancer (SCLC). Eligible patients were randomised between the 3 following arms: (A) Standard chemotherapy with 6 courses of EVI (epirubicin 60 mg m


Rana pipiens oocytes indicate that progesterone binds to a plasma membrane site within the external loop between the M1 and M2 helices of the α-subunit of Na/K-ATPase, triggering a cascade of lipid second messengers and the release of the block at meiotic prophase. We have characterized this site, using a low affinity ouabain binding isoform of the α1-subunit.Progesterone binding to the surface of the amphibian oocyte initiates the meiotic divisions. Our previous studies with Rana oocytes demonstrate that physiological levels of progesterone (or the non-metabolizable progestin R5020) successively activate phosphatidylethanolamine-N-methyltransferase (PE-NMT) and sphingomyelin synthase within seconds. Inhibition of PE-NMT blocks the progesterone induction of meiosis in intact oocytes, whereas its initial product, phosphatidylmonomethylethanolamine (PME), can itself initiate meiosis in the presence of the inhibitor. Published X-ray crystallographic data on Na/K-ATPase, computer-generated 3D projections, heptad repeat analysis and hydrophobic cluster analysis of the transmembrane helices predict that hydrophobic residues L, V, V, I, F and Y of helix M2 of the α1-subunit interact with F, L, G, L, L and F, respectively, of helix M3 of PE-NMT.Preparations of isolated plasma membranes from We propose that progesterone binding to the first external loop of the α1-subunit facilitates specific helix-helix interactions between integral membrane proteins to up-regulate PE-NMT, and, that successive interactions between two or more integral plasma membrane proteins induce the signaling cascades which result in completion of the meiotic divisions. Rana pipiens oocyte surface appears to be localized within the N-terminal external loop of the α1-subunit of Na/K-ATPase PME in plasma-vitelline membranes during the first 2 min after addition of 800 nM progesterone to the medium. Isolated membranes were preincubated in Ringer's solution containing [3H]S-adenosyl methionine (SAM) for 5 min at 20°C. Progesterone was then added and groups of 10 membranes were collected, rinsed, and extracted at the times indicated (see Methods). Incorporation was expressed as fmols per 10 plasma-vitelline membranes. Following preincubation, control plasma-vitelline membranes contained 2.2 ± 0.04 fmols of [3H]PME/10 membranes . Progesterone induced a 20-fold increase in plasma membrane PME within the first 15 sec, increasing 100-fold within 1-2 min. The response was dose-dependent, with maximal PME synthesis at 600-800 nM progesterone. In contrast, only a small increase in N-methylated phospholipids (~30%) was found in untreated (control) membranes over the 2 min period (data not shown). The phospholipid products of N-methylation were analyzed by paper chromatography of the water-soluble bases released after acid hydrolysis, as described by Percy et al. [Figure y et al. . More th3H]Sphingomyelin (SM) synthesis also increased 10 - 15 seconds after the initial rise in [3H]PME. [3H]SM continued to rise as PME synthesis fell, typical of a precursor-product relationship  and blue (lower) dotted lines. The β (blue) and γ (green) subunits span the plasma membrane at approximately 45°angles. As seen in Figure The architecture of the α1-Na/K-ATPase-β γ complex isolated from porcine kidney (Primary Accession #P05024) is illustrated in Figure Figure Comparison of the 3D projections of all 10 helices of the α1-subunit Figure  using ChIt is useful to visualize the helices both by Chem 3D projections and by computer graphics approaches such as hydrophobic cluster analysis  and heptThe 3D projection of the M2 helix . Table The helical wheel is an additional useful device for visualizing amphipatic helices . Figure The most favorable way for two such helices to arrange themselves in an aqueous environment is to wrap the hydrophobic strands against each other, forming a coiled-coil. As shown for PE-NMT M3 Table . M3 of P1/2 of about 15 min glycerol-labeled PME rises within 1-2 min after exposure to progesterone and is derived from the conversion of about 50% of the plasma membrane PE to PME . Our prSince the progesterone-induced reactions alter the lipid composition of the oocyte plasma membrane , it is iTransmembrane helix-helix interactions may have at least two major functions: 1) intramolecular helix-helix interactions would serve to stabilize the tertiary structure of the protein in the membrane bilayer, and 2) helix-helix interactions between two or more integral membrane proteins appear to regulate ligand-initiated response systems. X-Ray crystallography of the α1-subunit of Na/K-ATPase Figure  indicateIn our model of progesterone binding to the external M1-M2 loop , the polSince a translayer movement of newly formed phosphatidylcholine (PC) from the inner to the outer bilayer occurs in the plasma membrane , SM woul2 and pooled, as described previously [3H]methionine (80 Ci/mmol) and R5020 were obtained from Amersham Corp., Arlington Heights, IL. 2-Methyl(amino)ethane (2-MAE) was obtained from Sigma Chemical Company . Lipids were obtained from Avanti Polar Lipids .Fully grown Rana pipiens oocytes, arrested in first meiotic prophase, were stripped of their follicle envelopes and freed from all thecal cells . These oeviously . Nuclear2, 20 mM Mg2+ (acetate), 0.24 M sucrose] containing 2.5 μCi S-Adenosyl-L-[methyl-3H]methionine at 20°C (isotope was used without addition of carrier). After preincubation, 20 μl of steroid were added, and the samples collected and frozen in liquid nitrogen at the times indicated. Isotope was also added to membranes at ice bath temperatures, which were immediately frozen in liquid nitrogen as a reagent control. All experiments were carried out in during the normal breeding period (April-May) for R. Pipiens and were repeated on oocytes from 3 females.Oocyte plasma-vitelline membranes were obtained from oocytes in prophase arrest, and the solutions prepared immediately before use. Progesterone  was dissolved in 95% ethanol; with 1.0 μl added per ml of Ringer's solution where indicated. Five-six isolated membranes per sample were preincubated for 5 min in 200 μl aliquots of medium .The UniprotKB/Swiss-Prot/EMBL database http://cti.itc.Virginia.EDU/~cmg/Demo/wheel/wheelApp.html.A Residue-based Diagram editor (RbDe) Web site maintained by the Department of Physiology and Biophysics, Weill Medical College of Cornell University, New York, NY, was employed to illustrate the topologies of the α1-subunit of the Na/K-ATPase, PE NMT and SM synthase. Chem 3D Ultra v. 11  was used to visualize the 3D structure of the transmembrane helices. Hydrophobic cluster analysis  of the ahttp://www.pymol.org and the King Display Software, Kinemage v. 2.1 (Biochem.duke.edu/software/king/php). The RasMol image of a phosphatidylcholine bilayer by E. Martz was used as a spatial reference to the 3D projection of transmembrane helices http://www.umass.edu/microbio/rasmol/cutctw.gif.The X-ray crystallographic projections of the Na/K-ATPase (3B8E) were processed with the PyMOL Molecular Graphics System, Delano Scientific LLC, Palo Alto, CA GAM was responsible for computer modeling and drafted the manuscript. ABK and GAM carried out the biochemical studies and collaborated on the manuscript. AA jointly conceived of the problem with GAM and participated in design and coordination of the study. All authors read and approved of the final manuscript.


Since the expression levels of wild-type p53 in nonmalignant tissue are usually much lower in contrast, the p53 protein is an attractive target for cancer immunotherapy. We tested p53 encoded HLA-A24 binding peptides for their capacity to elicit anti-tumour cytotoxic T lymphocytes (CTL) in vitro. These peptides were in murine p53-derived cytotoxic peptides, which were being presented to CTL by H-2K d and H-2K b molecules, because the HLA-A24 peptide binding motifs were similar to the H-2K d and H-2K b. For CTL induction, we used CD8+T lymphocytes from the peripheral blood mononuclear cells (PBMC) of healthy donors and the peptides from pulsed dendritic cells as antigen-presenting cells. We identified the peptide, p53-161 (AIYKQSQHM), which was capable of eliciting CTL lines that lysed tumour cells expressing HLA-A24 and p53. The effectors lysed C1RA24 cells , but not their parental cell lines C1R . These results strongly indicate that the CTL exerts cytotoxic activity in HLA-A24's restricted manner. The identification of this novel p53 epitope for CTL offers the possibility to design and develop specific immunotherapeutic approaches for treating tumours with p53 mutation in HLA-A24-positive patients. © 2001 Cancer Research Campaign http://www.bjcancer.comMutations in the


Bifidobacteria are frequently proposed to be associated with good intestinal health primarily because of their overriding dominance in the feces of breast fed infants. However, clinical feeding studies with exogenous bifidobacteria show they don't remain in the intestine, suggesting they may lose competitive fitness when grown outside the gut.Bifidobacterium longum DJO10A that was minimally cultured in the laboratory, and compared it to that of a culture collection strain, B. longum NCC2705. This comparison revealed colinear genomes that exhibited high sequence identity, except for the presence of 17 unique DNA regions in strain DJO10A and six in strain NCC2705. While the majority of these unique regions encoded proteins of diverse function, eight from the DJO10A genome and one from NCC2705, encoded gene clusters predicted to be involved in diverse traits pertinent to the human intestinal environment, specifically oligosaccharide and polyol utilization, arsenic resistance and lantibiotic production. Seven of these unique regions were suggested by a base deviation index analysis to have been precisely deleted from strain NCC2705 and this is substantiated by a DNA remnant from within one of the regions still remaining in the genome of NCC2705 at the same locus. This targeted loss of genomic regions was experimentally validated when growth of the intestinal B. longum in the laboratory for 1,000 generations resulted in two large deletions, one in a lantibiotic encoding region, analogous to a predicted deletion event for NCC2705. A simulated fecal growth study showed a significant reduced competitive ability of this deletion strain against Clostridium difficile and E. coli. The deleted region was between two IS30 elements which were experimentally demonstrated to be hyperactive within the genome. The other deleted region bordered a novel class of mobile elements, termed mobile integrase cassettes (MIC) substantiating the likely role of these elements in genome deletion events.To further the understanding of genetic attenuation that may be occurring in bifidobacteria cultures, we obtained the complete genome sequence of an intestinal isolate, Deletion of genomic regions, often facilitated by mobile elements, allows bifidobacteria to adapt to fermentation environments in a very rapid manner  and the concomitant loss of possible competitive abilities in the gut. E. coli [Recent molecular studies into the microbial diversity of the human intestine reveal a much greater diversity than previously recognized and very little is currently known of the contribution of individual groups to the human organism . One numE. coli -6. This E. coli . HoweverE. coli -9. This Bifidobacterium longum, that was grown for less than 20 generations in laboratory media, was deciphered. This was compared to an available genome sequence of a culture collection strain, B. longum NCC2705 [To further the understanding of genetic attenuation that may occur in bifidobacteria, the complete genomic sequence of a numerically dominant human intestinal isolate of  NCC2705  and analB. longum NCC2705 [B. longum strains were characterized and strain DJO10A was selected based on its prominent ability to bacteriostatically inhibit other bacteria through the production of siderophores [The power of comparative genomics can provide insights into features that are important for a species to survive and compete in its habitat. The genome sequence of the culture collection strain,  NCC2705 , is avairophores , a chararophores .B. longum DJO10A contained 2,375,792 bp, with 60.15% G+C content and 1,990 encoded genes containing four rRNA operons, 58 tRNAs, 6 insertion sequence (IS) families as well as one prophage  consisting of three different contiguous integrases flanked by an inverted repeat and a palindrome structure sandwiched by two IS3-type IS elements , as well as other intestinal bacteria, Bacteroides (AE015928), Lactobacillus (CP000033), and E. coli (U00096), did not reveal MIC elements, suggesting these structures may be unique to a subset of closely related bifidobacteria.The chromosome of oriC and terC were found in identical locations in the genome of strain DJO10A and the updated genome sequence of strain NCC2705  that is 100% conserved between both strains . The low identity (40%) between the HsdS proteins in the two strains likely reflects the independent evolution of this type I R-M system in these strains following their evolutionary divergence, as these systems evolve by changing their specificity components (HsdS) to enable it to recognize different sequences. This is substantiated by the existence of an hsdS gene that was inactivated by an IS256 insertion event and both parts of this disrupted gene exhibit much higher conservation, suggesting the insertion event occurred before their evolutionary divergence , that is not present in NCC2705. The conserved type II R-M systems in both strains are isoschizomers of Sau3AI and EcoRII which restrict DNA at very frequently occurring sites  peak Fig. , as this3 element in the NCC2705 genome at the precise location of the extra oligosaccharide utilization genes in strain DJO10A  and 100% greater than E. coli K12  involved in the lantibiotic production was found to be missing from 40% of the isolates  remained stable . Clostridium difficile DJOcd1 was isolated from fresh feces by plating on Clostridium difficile Selective Agar (BD Diagnostics) and speciated using a sequence analysis of its 16S rRNA gene. E. coli DJOec1 was obtained from fresh feces by plating on MacConkey agar (Difco) and speciated using a sequence analysis of its 16S rRNA gene. E. coli K12 was obtained from the American Type Culture Collection (ATCC). Bifidobacteria were cultivated at 37°C in MRS (Difco) supplemented with 0.05% L-cysteine·HCl (Sigma), Bifidobacteria Low-Iron Medium (BLIM) [2HPO4, 0.15% MgSO4·7H2O, 0.5% D-glucose) under anaerobic conditions using either the BBL Anaerobic system (BBL) or the Bactron II Anaerobic/Environmental Chamber (Sheldon Manufacturing).'s feces  and B. am (BLIM)  or Bifid30 elements covering A5, A6 and A7 loci were identified and assembled separately. The presence and location of long repeated sequences in genomic DNA samples were verified by direct genomic sequencing of the unique/repeat junctions. The resolution of the most complex high GC-rich repeats was achieved by sequencing of PCR products amplified with a hybrid TopoTaq DNA polymerase with increased strand displacement capacity.Whole-genome shotgun sequencing was carried out at the US Department of Energy Joint Genome Institute (JGI). Sequences were assembled into 227 contigs using the Phred/Phrep/Consed software and the sequence coverage was 9.2-fold. Gap closure and genome sequence finishing was carried out at Fidelity Systems using ThermoFidelase-Fimer direct genome sequencing technology . ShotgunB. longum genomes was conducted using MUMmer3, ACT4 and ClustalX. The origin of replication and terminus were predicted using OriLoc [2 analysis of Artemis8. To predict gene functions, the two conserved protein domain databases of GAMOLA and InterProScan were used. COG functional categories were used for functional classification of all genes in both B. longum genome sequences.Annotation of all open reading frames (ORFs) was carried out using Glimmer, GeneMark, JGI annotation tools and GAMOLA , before g OriLoc . Codon ug OriLoc . The basxl Auto sequencer (Applied Biosystems). All PCR primers are listed in Additional file lanM gene was obtained using PCR with LANT-F and LANT-R primers. Probes for IS elements were also PCR amplified. Probes were DIG-labeled and hybridized with digested genomic DNA according to the manufacturer's instructions (Roche). Pulsed field gel electrophoresis of XbaI-digested B. longum genomes was performed using a CHEF-DR III Variable Angle Pulsed Field Electrophoresis System according to manufacturer's instructions (Bio-Rad).General sequencing was conducted using a Big-Dye terminator and ABI Prism 3730Comparative nucleotide substitution analysis by Nei and Gojobori's algorithm  was used2-, 1 to 100 mM) and sodium arsenate . Freshly grown cultures were sub-inoculated into the arsenite/arsenate media and incubated anaerobically at 37°C for 48 h.To determine the minimal inhibitory concentration of arsenic, BLIM was supplemented with different concentrations of sodium arsenite  and R3  were used to amplify a ~1.8 kb region spanning the deletion and sequencing located the precise borders  and MIC-R1  were used to amplify the upstream region of MIC III and primers MIC-F2  and MIC-R2  were used to amplify the downstream region.To find the precise location of the deletion of the lantibiotic operon in the B. longum DJO10A was inoculated into the center of an MRS agar plate and incubated anaerobically at 37°C for 2 days. After incubation, molten 0.5% top agar of the same medium containing 1% of an indicator strain was overlaid on the plates prior to incubation.B. longum DJO10A compared to its in vitro adapted derivative strain DJO10A-JH1, a simulated in vitro fecal system was developed. Triplicate experiments for each strain were used. Each experiment was conducted in 10 g sterilized feces in an anaerobic chamber, to which 0.38 g Reinforced Clostridial Medium (RCM) and 0.02 g mucin (Porcine gastric type III) was added. The two competitor bacteria were added to all tubes at calculated concentrations of 1.2 × 107 cfu/g for E. coli DJOec1 and 5.1 × 107 for Clostridium difficile DJOcd1. B. longum DJO10A was added to three tubes at a calculated concentration of 4.0 × 107 cfu/g and strain DJO10A-JH1 to the other three tubes at 4.4 cfu/g. Standard viable plate counts were used to calculate all bacterial concentrations. After thorough mixing in an anaerobic environment, the tubes were left at 37°C for 3 days, whereby the entire fecal samples were homogenized in 90 ml peptone water to conduct an accurate serial plate count analysis.To access the competitive 'fitness' of the wild-type CP000605.The Sequence and annotation data have been deposited in GenBank under the accession number B. longum DJO10A into a single contig; SAK assembled the genome sequence of B. longum DJO10A into a single contig, observed the IS30 hyperactivity and critiqued the manuscript; DM facilitated the shotgun sequencing of B. longum DJO10A and critiqued the manuscript; NVP assembled the genome sequence of B. longum DJO10A into a single contig; NNP, assembled the genome sequence of B. longum DJO10A into a single contig; PMR carried out the shotgun sequencing of B. longum DJO10A; VVS assembled the genome sequence of B. longum DJO10A into a single contig; AIS assembled the genome sequence of B. longum DJO10A into a single contig; BW facilitated the shotgun sequencing of B. longum DJO10A; and DJO'S designed the study, analyzed the results and co-wrote the manuscript.J-HL carried out the comparative and functional genomic analysis, and co-wrote the manuscript; VNK assembled the genome sequence of Comparison of serine codon usage between chromosomal and prophage genes in strain DJO10A.Click here for fileB. longum DJO10A. (A) and NCC2705, (B). Orfs 1, 2 and 3 refer to three contiguous, but different xerC integrase genes. P, a conserved 20 bp palindrome (TTAAACCGACATCGGTTTAA), which has a 11 bp extension in MIC III. IR, 96 bp inverted repeat (IR) (GATTAAGCCGGGTTTGTTGTTAAGCCGGGGAACGGTTCGGGGTCTTGGTGGCTGGCCGTGTCCCATGTGGTTTCCCGGCTTAACGTTCCGGGTTAT), that has a 3 bp extension in MIC I and II, a 5 bp extension in MIC III and a 1 bp extension in MIC 1, 2 and 3. IS, insertion sequence.Organization of mobile integrase cassettes (MIC) in Click here for fileB. longum strains DJO10A and NCC2705. (A) Mummer3 plot of both genomes. (B) ACT4 plot showing the relative locations of mobile elements. Red lines indicate the relative location of elements that are orientated in the same direction. Blue lines indicate elements orientated in opposite directions.Comparison of the two genomes of Click here for fileoriC region. This consists of three clusters, in the two B. longum genomes. The DnaA boxes consist of 7 types, designated A to G as follows: Type A (TTATCCACA), Type B (TTGTCCACA), Type C (TTTTCCACA), Type D (TTACCCACA), Type E (TTATCCACC), Type F (TTATTCACA), Type G (TTATGCACA).Conserved structure of the Click here for fileB. longum genomes. (A) Alignment of the genomic locations encoding a type I R-M system between B. longum DJO10 and NCC2705. (B) Comparison of a Sau3AI-type II R-M system  with analogous R-M systems in other bacteria and (C) comparison of a EcoRII-type II R-M system  with analogous R-M systems in other bacteria. Percentage protein sequence identities compared to B. longum DJO10A are indicated in red.Type I and II restriction modification (R-M) systems encoded by the Click here for fileB. longum genomes.COG categories for all genes in both Click here for filemalE; F, malF; G,malG; R, lacI-type repressor; K, ATPase of ABC transporter; αGal, α-galactosidase; βXyl, β-xylosidase; Est, esterase; LCFACS, long-chain fatty acid acetyl CoA synthetase; f, fragmented gene; XylT, D-xylose proton symporter; βGal, β-galactosidase; Arab-βGal, arabinogalactan endo-1,4-β-galactosidase; O157, ORF with homolog only in E. coli O157; αMan, α-mannosidase; GlycH, glycosyl hydrolase; NAc-Glc, N-acetyl glucosaminidase; UhpB, histidine kinase; RfbA, dTDP-glucose pyrophosphorylase; RfbB, dTDP-D-glucose 4,6-dehydratase; RfbC, dTDP-4-dehydrorhamnose 3,5-epimerase; RgpF, lipopolysaccharide biosynthesis protein; TagG, ABC-type polysaccharide/polyol phosphate export systems, permease component; TagH, ABC-type polysaccharide/polyol phosphate transport system, ATPase component; MdoB, phosphoglycerol transferase; ProP, permease; Acyl-Est, acyl esterase. It should be noted that the glycosyl hydrolase gene in cluster 7 was annotated as isomaltase in the NCC2705 genome annotation.Organization of the 11 different types of oligosaccharide utilization gene clusters (11 in DJO10A and 7 in NCC2705). Red-colored arrows indicate strain DJO10A unique genes. IS, insertion sequence; Hyp, hypothetical protein; Arab, arabinosidase; E, Click here for fileB. longum DJO10A and NCC2705, according to the dN:dS ratio.Nucleotide substitution analysis of all gene homologs between Click here for fileSubstitution ratios of the 52 genes in the positive selection category.Click here for fileB. longum DJO10A. The numbers below the signal peptide boxes indicate the location of signal peptides. The size of the respective proteins is indicated in amino acids.Organization of four predicted LPXTG-type, cell surface anchor proteins in Click here for fileB. longum DJO10A-JH1. (A) Detection of DJO10A specific gene clusters in B. longum DJO10A and its fermentation adapted isolate DJO10A-JH1 by PCR. M, 1 kb DNA ladder (Invitrogen); lane 1, unique region 15; lane 2, unique region 6; lane 3, unique region 9; lane 4, unique region 11; lane 5, unique region 5; lane 6, unique region 7; lane 7, unique region 12; lane 8, 16S rRNA partial gene. The red arrow indicates the lantibiotic encoded unique region 12 that is missing from strain DJO10A-JH1. (B) Southern blot analysis using a lanM probe and the EcoRI-digested genomes of B. longum strains DJO10A and DJO10A-JH1. The 1.7 kb EcoRI band containing lanM is indicated with an arrow.Loss of the lantibiotic gene cluster from Click here for fileE. coli DJOec1; green triangles, Clostridium difficile DJOcd1; purple circles, B. longum DJO10A-JH1; and blue diamonds, B. longum DJO10A.Growth curves in RCM medium of the four bacteria used in the fecal competitive growth experiments. All bacteria were inoculated at 1% from freshly grown cultures. Black squares, Click here for filePrimers used in this study.Click here for file


Salmonella enterica serovar Typhi (S. Typhi), the etiologic agent of typhoid fever, has aggravated an already important public health problem and added new urgency to the development of more effective typhoid vaccines. To this end it is critical to better understand the induction of immunity to S. Typhi. CD8+ T cells are likely to play an important role in host defense against S. Typhi by several effector mechanisms, including killing of infected cells and IFN-γ secretion. However, how S. Typhi regulates the development of specific CD8+ responses in humans remains unclear. Recent studies in mice have shown that dendritic cells (DC) can either directly  or indirectly (by bystander mechanisms) elicit Salmonella-specific CD8+ T cells.The emergence of antibiotic-resistant strains of S. Typhi, human DC produced high levels of pro-inflammatory cytokines IL-6, IL-8 and TNF-α, but low levels of IL-12 p70 and IFN-γ. In contrast, DC co-cultured with S. Typhi-infected cells, through suicide cross-presentation, uptake S. Typhi-infected human cells and release high levels of IFN-γ and IL-12p70, leading to the subsequent presentation of bacterial antigens and triggering the induction of memory T cells, mostly CD3+CD8+CD45RA−CD62L− effector/memory T cells.We report here that upon infection with live S. Typhi on human DC maturation and on their ability to prime CD8+ cells and highlights the significance of these phenomena in eliciting adaptive immunity to S. Typhi.This study is the first to demonstrate the effect of  Salmonella enterica serovar Typhi (S. Typhi), the etiologic agent of typhoid fever, has aggravated an already important public-health problem S. Typhi, is a facultative intracellular bacterial pathogen with the capacity to survive and replicate in phagocitic and non-phagocytic cells S. Typhi. We have shown in volunteers immunized orally with attenuated strains of S. Typhi, including Ty21a, as well as with the novel attenuated typhoid vaccine candidate strains CVD 908, CVD 908-htrA and CVD 909, the induction of CD8+ cell-mediated immunity (CMI) mechanisms that involve the secretion of IFN-γ and the killing of S. Typhi-infected cells by cytotoxic T lymphocytes + cells requires the presentation of antigens by specialized cells of the immune system named dendritic cells (DC) S. Typhimurium mouse model have showed that DC can either directly  or indirectly  present Salmonella antigens Typhoid fever remains an important public health priority, particularly in developing countries, with an estimated 16 million new cases annually and 600,000 deaths S. Typhimurium is considered a model for the pathogenesis of human typhoid fever S. Typhi infection is restricted to humans S. Typhimurium and S. Typhi infection are identical S. Typhi regulates the development of CD8+ CMI in humans, we examined the effects of S. Typhi on the maturation of DC and on their ability to prime naive CD8+ T cell responses. We report here that DC, through suicide cross-presentation, uptake apoptotic S. Typhi-infected human cells and release IFN-γ and IL-12p70, leading to the subsequent presentation of bacterial antigens and triggering the induction of mostly CD3+CD8+CD45RA−CD62L− memory T cells. This study is, to our knowledge, the first demonstration of the effects of S. Typhi on human DC maturation and on their ability to prime CD8+ cells and highlights its significance in eliciting adaptive immunity to S. Typhi.Infection of susceptible mice with S. Typhi drives DC maturation, we evaluated the DC surface expression of CD80 and CD83 costimulatory molecules after different stimulatory conditions. Unstimulated DC (media) were used as negative controls. We observed that DC pulsed with live S. Typhi increased their surface expression of CD80 and CD83 in a dose-dependent manner   . Caspased blasts . Both mestaining . IncreasS. Typhi . ApoptosS. Typhi . TogetheS. Typhi-infected blasts, we incubated them with infected blasts labeled with a mAb to CD45, a surface marker present in all human leukocytes. After a 2 hour incubation, an average of 20% of the DC that were incubated with S. Typhi-infected blasts were positive for CD45, indicating capture of blast-derived material  staining (S. Typhi (data not shown) had equal or higher viability than DC exposed to S. Typhi-infected blasts  that the cross-presentation by DC might be related to S. Typhi-antigen itself rather than by the apoptotic nature of the S. Typhi-infected blasts. Thus, the apoptosis elicited by S. Typhi antigens can be the result of at least two pathways leading to cross-presentation: (a) through a bystander effect on neighboring DC and (b) through phagocytosis of S. Typhi–infected blasts which leads to the dead of these DC via irreversible apoptosis . In this way, having more S. Typhi-infected blasts engulfed by DC might result in more Salmonella antigens available for cross-presentation resulting from more DC undergoing apoptosis.Despite the fact that at the beginning of the cultures the numbers of the DC loaded with either d blasts  and 5A. staining . DC celld blasts  and 5B. nchanged  and 5. TSalmonella on DC maturation and cytokine production described above, as well as their capacity to induce apoptosis in the infected cells, prompted us to investigate the ability of Salmonella antigen-loaded DC to prime naïve human T cells. Previous studies have shown that by exposing DC to antigen and then to naïve T cells in vitro, it is possible to mimic the process of antigen presentation in vivoSalmonella) or indirectly  present Salmonella antigens to naïve T cells. This was studied by co-culturing ex vivo PBMC from individuals non-exposed to S. Typhi (naïve subjects) with DC that had been pulsed with live or heat-killed S. Typhi at 10∶1 MOI (priming), or pulsed with uninfected or S. Typhi-infected blasts at a 1∶1 blast to DC ratio (cross-priming). After 30 hours of incubation, the S. Typhi specificity of primed PBMC was evaluated by their ability to secrete IL-2, IFN-γ and TNF-α detected as measured by intracellular staining. To avoid infection of DC by live bacteria from dying infected-blasts, blasts were harvested, washed 3 times with RMPI containing gentamicin (100 µg/ml) and incubated for 1 hour at 37°C to kill and remove extracellular bacteria before being added to PBMC. Moreover, to assess the pre-existing background from cross-reactive effector T cells, a control consisting of PBMC co-cultured with the S. Typhi-infected blasts in the absence of DC was also included. Although PBMC in the presence of S. Typhi-infected blasts alone (in the absence of DC) produced moderate levels of cytokines, these levels were considerably lower than those observed in PBMC co-cultured with DC pre-mixed with S. Typhi-infected blasts (S. Typhi infection. Results indicate different levels of cytokine production in the various volunteers. Whilst in 2 of 4 volunteers higher induction of cytokine production was observed following DC co-cultured with S. Typhi-infected blasts (S. Typhi or with S. Typhi-infected blasts (data not shown), and in the 4th volunteer the cytokine production was higher in cultures of PBMC with DC infected with live S. Typhi (data not shown). Therefore it is likely that both mechanisms, cross- and direct-priming by DC, might be involved in stimulating cytokine production from PBMC.The effect of d blasts  and 7. Td blasts  and 7, aex vivo PBMC cultures. As expected, CCD markedly inhibited the DC's ability to stimulate cytokine production by naïve PBMC in all 4 volunteers evaluated . A substantial decrease in cytokine production by PBMC was observed when DC were pre-treated with ZVA-D before exposure to live S. Typhi (4 of 4 volunteers) , effector memory T cells  and naïve T cells  + T cells elicited were composed of classical TEM (EMRA. No significant increases were observed in TCM and T naïve subsets (data not shown).We next investigated which CD8 cell populations within PBMC preferentially produced IFN-γ and TNF-α cytokines under the different stimulatory conditions described above. We evaluated the induction of cytokine production in the various memory CD8ical TEM  and 9. MS. Typhi-specificity of PBMC was evaluated by their capacity to secrete IFN-γ by ELISPOT. Significant increases in net frequencies of IFN-γ-SFC were observed between PBMC previously stimulated with DC pulsed with S. Typhi-infected cells and all other groups  or by active phagocytosis of S. Typhi–infected cells (a suicide pathway) and/or bystander effect in neighboring DC are very efficient in priming T cells. These results resemble previous studies using the S. Typhimurium mouse model that demonstrated that DC can either directly  or indirectly (by bystander mechanisms) present Salmonella antigens S. Typhi-antigen cross-presentation by DC rather than by the apoptotic nature of S. Typhi-infected blasts. This is consistent with a previous model showing that necrotic cells or even living cells were also cross-presented by human DC to CD8+ T cells + T cells. Below this level, peripheral antigens were unable to stimulate by cross-presentation and therefore ignored by naive CD8+ T cells Here, we also demonstrate that human DC directly , recruited from the Baltimore-Washington area and University of Maryland at Baltimore campus, participated in this study. The PBMC were isolated from the blood of volunteers by density gradient centrifugation and cryopreserved in liquid NCells were surface stained with mAbs to CD1a, CD8, CD14, CD56, CD62L, TCRαβ, TCRγδ, HLA-DR and DC-Sign , CD4, CD27, CD45, CD45RA, CD80, CD83 , CD3, CD4, CD8, CD19, CD45, CD45RA and CD62L .S. Typhi strain ISP1820 , at different multiplicity of infection (MOI), (c) uninfected or (d) S. Typhi-infected autologous blasts at different blast to DC ratios, or (e) LPS from Salmonella Typhi  for 30–48 hours. DC cultured in media alone were used as negative control.DC were generated by standard procedures from peripheral blood monocytes. The cells were used before or after further treatment with (a) live or (b) 60°C heated-inactivated wild-type S. Typhi at a MOI of 10∶1 To infect DC, cells were incubated for 3 hours at 37°C in RPMI (without antibiotics) in the presence of −CD14−CD19− cells (“dump” channel) DC-Sign+ HLA-DR+ were analyzed for the expression of CD80 and CD83 maturation makers.The phenotype of DC was examined by multichromatic flow cytometry using mAbs to CD14, CD3, CD14, CD19, CD80 and CD83 cell surface antigens. DC gated on CD35 DC at 5∶1 blast to DC ratio. After 2 hours of incubation at 37°C, mixed cells were washed twice with cold PBS to stop endocytosis, and stained with an amine reactive viability dye (ViViD)(Molecular Probe) as a dead cell exclusion marker, followed by surface staining with mAbs to HLA-DR and DC-Sign. Cells were then washed and fixed in cold 1% formalin. The quantitative uptake of CD45-labeled blasts by DC was determined by flow cytometry. DC that either bound together or very close to blast cells without internalizing them were identified by doublet discrimination and excluded from analysis . In some experiments, DC were treated with cytochalasin D (CCD)  or Z-Val-Ala-Asp-FMK (ZVA-D)  for 1 hour before and/or during co-culture with blasts. All surface staining was carried out at 4°C.DC endocytosis was measured by uptake of CD45 ECD-labeled blasts by flow cytometry. Fluorescent CD45-labeled blasts were cocultured with approximately 10S. Typhi, cells were stained with anti-Salmonella common structural antigens (CSA-1)-FITC  and analyzed by flow cytometry Autologous blasts were generated following standard procedures Cytokine levels of IFN-γ, IL-1β, IL-2, IL-5, IL-6, IL-8, IL-10, IL-12p70 and TNF-α by DC was measured by flow cytometry-based BD Cytometric bead array (CBA). Briefly, DC were cultured in the absence (media) or presence of the various culture conditions discussed above for 48 hours. The supernatants were then harvested and kept at -70°C until assayed. CBA assays were carried-out following the manufacturer's instructions. The levels of sensitivity for the various cytokines ranged from 2.5–20 pg/ml.Cell apoptosis was monitored by two assays: (A) by measuring membrane changes by staining cells with annexin-V and propidium iodide (PI)  and (B) by measuring cytosol changes following staining with a rabbit polyclonal antibody to caspase-3 PE (BD-Pharmingen). Annexin V and PI staining were performed following the manufacturer's recommendations.S. Typhi-unvaccinated adult volunteers were co-cultured with autologous DC at PBMC to DC ratios of 15–20∶1. The S. Typhi PBMC specificity or priming was evaluated by their ability to secrete cytokines ex vivo or in vitro detected by intracellular staining or ELISPOT, respectively. DC were stimulated as described above for 30-48 hours before exposure to the PBMC.Priming assays were performed as previously described S. Typhi antigens, brefeldin-A (BFA) , a protein transport blocker, was added to the PBMC. After 16–18 hours, PBMC were surface and intracellularly for IFN-γ, TNF-α, IL-2 (B-D Pharmingen) and CD69 (Beckman-Coulter) to perform up to 13-color stainings. Cells were then resuspended in fixation buffer  and analyzed as soon as possible by flow cytometry on an LSR-II instrument (BD Biosciences).After 1–2 hours of stimulation with in vitro expanded effector cells was established as the frequency of SFC/106 PBMC in co-cultures of effectors with not-infected targets + 2 SE.IFN-γ ELISPOT assays were performed as previously described P values<0.05 were considered significant.All tests were performed using SigmaStat software . Comparisons between groups were performed by the One-way ANOVA test. Specifically, in flow cytometric experiments, a response was considered specific if the differential in the number of positive events between experimental and negative control (media only) cultures was significantly increased by Chi-square tests.


Primary HIV-infected patients display severe and irreversible damage to different blood B-cell subsets which is not restored by highly efficient anti-retroviral therapy (HAART). Because longitudinal investigations of primary HIV-infection is limited by the availability of lymphoid organs, we studied the tissue-specific B-cell dysfunctions in acutely simian immunodeficiency virus (SIV) mac251-infected Cynomolgus macaques.−CD27+) B-cells in spleen and lymph nodes during the first 4 weeks of infection, concomitant to selective homing/sequestration of B-cells to the small intestine and spleen. SIV non-specific Ig production was transiently increased before D14p.i., whereas SIV-specific Ig production was only detectable after D14p.i., coinciding with the presence of CD8+ T-cells and IgG-expressing plasma cells within germinal centres. Transient B-cell apoptosis on D14p.i. and commitment to terminal differentiation contributed to memory B-cell loss. HAART abrogated B-cell apoptosis, homing to the small intestine and SIV-specific Ig production but had minimal effect on early Ig production, increased B-cell proportions in spleen and loss of memory B-cells. Therefore, virus–B-cell interactions and SIV-induced inflammatory cytokines may differently contribute to early B-cell dysfunction and impaired SIV/HIV-specific antibody response.Experiments were performed on three groups of macaques infected for 14, 21 or 28 days and on three groups of animals treated with HAART for two-weeks either initiated at 4 h, 7 or 14 days post-infection (p.i.). We have simultaneously compared changes in B-cell phenotypes and functions and tissue organization of B-cell areas in various lymphoid organs. We showed that SIV induced a steady decline in SIgG-expressing memory (SIgDThese data establish tissue-specific impairments in B-cell trafficking and functions and a generalized and steady memory B-cell loss in secondary lymphoid organs. Characterization of underlying mechanisms would be helpful in designing new therapeutic strategies to dampen B-cell activation and increases HIV/SIV specific antibody response. B-cell dysfunction represents a central feature of HIV infection and an important pathogenic mechanism mac251 strain for 14, 21 or 28 days (placebo-treated groups) and on three groups of animals treated with HAART for two-weeks either initiated at 4 h, 7 or 14 days post-infection (p.i.) and sacrificed at D14, 21 and D28 p.i. (HAART-treated groups). After having characterized naive, memory and MGZ B-cell subsets in various lymphoid organs from non-infected animals, we have compared the changes in these B-cell subsets in blood, lymph nodes (LN) and spleen of placebo- and HAART-treated macaques and examined whether correlations could be established with immunological or virological parameters. Changes in B-cell functions in vitro  production) as well as in tissue organization of areas populated by B-cells within lymphoid tissues  were concomitantly examined in placebo- or HAART-treated animals.The study of primary HIV-1 infection in humans is limited by the availability of lymphoid organs and the difficulties associated with performing longitudinal investigations using tissues other than blood or tonsils. We thus used a model of experimental pathogenic infection in cynomolgus macaques—a well-known, suitable model that reproduces long-lasting HIV-1 disease Our data show that SIV induced a transient increase in B-cell apoptosis and SIV non-specific Ig production by D14p.i., a steady loss of memory B-cells in spleen and peripheral LN but promoted preferential B-cell trafficking to the small intestine and spleen. HAART initiated 4 hp.i. strongly decreased B-cell apoptosis and B-cell seeding of gut mucosa, but not memory B-cell loss. The production of SIV non-specific IgG was similar in placebo- and HAART-treated animals whereas that of SIV-specific antibodies, only detectable after D14p.i., was abrogated by HAART initiated on D7p.i.. These data establish tissue-specific impairments in B-cell trafficking and functions but a generalized and steady memory B-cell loss in secondary lymphoid organs. Our results provide a clearer understanding of the effects of SIV/HIV-1 on the B-cell compartment during the acute/primary phase of infection.We compared the proportions of lymphocyte subsets in blood, spleen and peripheral  LN from various groups of animals infected for 14, 21 or 28 days. Baseline values for blood samples were obtained from each animal before infection, whereas those for spleen and LN were obtained from non-infected animals (controls).+ and CD8+ T-cell and NK-cell counts, detectable by D7 p.i., but reaching their nadir on D14 p.i., with reductions of 53% for CD4+, 49% for CD8+ and 85% for NK cells . Plasma VL was inversely correlated with CD4+  and CD8+  T-cell counts  and CD4+ cell number  and inversely correlated with pVL  in SIV-infected animals  B-cells and higher proportions of CD20+CD27+ B-cells are present in blood and LN of cynomolgus macaques than those in humans data not shown).Consistent with previous data obtained in humans data not shown). In spleen, memory B-cells were decreased by a factor of 2.3 by D14, 5.6 by D21 and 3.9 by D28 p.i.. The percentages observed for MGZ B-cells between D14 and D28 p.i. were between 1.2 and 1.5 times lower than baseline. In contrast, the percentage of naive B-cells steadily increased from D14p.i. by a factor of 2.3. (p<0.05) at each time point (p<0.05). These results reveal a substantial depletion of memory and MGZ B-cells in all organs during the first four weeks of SIV infection. This depletion was transient in blood but longer lasting in lymphoid organs.SIV infection induced an early but transient loss of peripheral memory B-cells, with a two- and 1.4-fold decrease observed in the percentage of cells at D7p.i. and D28p.i., respectively. A 4.3- and three-fold increase in peripheral naive B-cells was observed on D7 and D28p.i., respectively. Changes in MGZ B-cells were only detected on D21 and D28 p.i.   co-expressed CD80 and CD86 in non-infected animals . A significant correlation was observed between the percentages of SIgD−CD27+ memory B-cells and those of SIgD−CD86+  or SIgD−CD80+  B-cells. In LN, most memory B-cells were either CD80+ or CD86+ in non-infected animals (data not shown). By D14 p.i., the percentage of CD80+ cells had decreased by a factor of 1.5, CD86+ had decreased by a factor of 1.2 and total memory B-cells by a factor of 1.3. At D21 and D28 p.i., the percentage of CD80+ had increased, approaching the baseline value, whereas the percentages of both CD86+ and total memory B-cells continued to decrease by similar amounts. The percentage of total SIgD−CD27+ memory B-cells was correlated with that of SIgD−CD86+  but not SIgD−CD80+ B-cells. In spleen, the percentage of CD80+ was lower than that of CD86+ B-cells within the memory B-cell subset, about 10% of memory B-cells were CD80+CD86+ (data not shown). After infection, the percentages of CD80 and CD86 memory B-cells had decreased by factors of 2.2 and 2 by D14 p.i., by factors of 1.6 and 2.3 by D21 p.i. and by factors of 3 and 3.5, respectively, by D28 p.i.. At the same time, the proportion of total memory B-cells decreased by factors of 2.3, 5.5 and 3.9 by D14, D21 and D28 p.i.. The percentage of total (SIgD−CD27+) memory B-cells was correlated with that of SIgD−CD86+  but not SIgD−CD80+ B-cells.As in humans, macaque memory B-cells can express CD80 and CD86 co-stimulatory molecules: most peripheral memory B-cells  and LN  during acute SIV-infection.Consistent with the expression of CD95 by most memory B-cells in all organs, the percentage of total and CD95-expressing memory B-cells decreased concomitantly in SIV-infected animals Table 2.+CD86+CD95+ memory B-cells without modification of the ratio between SIgM- and SIgG-expressing cells. In contrast, a preferential and sustained depletion of SIgG-, CD86-, CD95-expressing memory B-cells was observed in LN and spleen.Our data thus show a transient depletion of blood CD80To assess whether the decrease in memory B-cells results from apoptosis, anergy or terminal differentiation, we investigated the effect of SIV infection on spleen B-cell function. The proliferation rate of SIV-infected spleen B-cells to B-cell receptor (BCR) stimulation was three (IL2+ SAC) and four (IL4+ anti-Ig H+L Ab) times lower than in non-infected animals at D14 p.i., but three and 1.4 times higher thereafter. Similarly, the response to CD40 stimulation was four times lower at D14p.i. and six and 8.4 times greater at D21 and D28 p.i., respectively  and 10-fold (H28) decreases in pVL .To improve our understanding of the kinectics of B-cell dysfunctions during the acute phase of SIV infection, we compared data obtained in placebo- and HAART-treated macaques. Animals were subjected to HAART for two-weeks initiated at 4 h (beginning of the infection), 7 or 14 (peak of pVL) days p.i. and sacrificed at the end of HAART (i.e.: on D14 (H14), 21 (H21) and 28 (H28) p.i.) . However, percentages of memory and MGZ blood B-cells were partially restored at D21 and D28p.i.. In H28 animals, percentages of naive, MGZ and memory B-cells almost reached baseline values ( right panel). Only the difference between percentages of MGZ B-cells between P28 and H28 was statistically significant (p<0.05). The SIV-mediated increase in total spleen B-cells  was less marked in animals receiving HAART than in placebo-treated animals but loss of memory and MGZ B-cells was still prominent at D21 and D28 p.i. . The differences between percentages of total B-cells in placebo- and HAART-treated groups were not statistically significant but those between percentages of naive and memory B-cells on D21/28p.i. and of MGZ B-cells on D14p.i. were statistically significant (p<0.05).The percentage of total blood B-cells was 20% and 10% higher in H14 and H21/28 animals respectively, than in placebo-treated animals . These differences were not statistically significant (in vitro (data not shown). HAART potently reduced SIV-specific Ab and total IgM production when initiated on D7 p.i. but was less efficient when initiated on D14p.i.  mAb. Measurement of the width of MGZ did not reveal any significant increase in size.To extend our data obtained from cell suspensions, we investigated the tissue organization in lymphoid organs of placebo- or HAART-treated SIV-infected animals. Spleen and mesenteric LN (MLN) taken on D14, 21 and 28 p.i. were analyzed by immunohistochemistry (IHC) with a large panel of primary antibodies  and MLN on D28 p.i.  (data not shown) suggesting a depletion in follicular helper T cells.According to the loss in CD4D28 p.i. Figure 5+ cells in the EFZ could be detected from D14p.i., infiltrating CD8+ cells were only detectable in GC from D21p.i. (+ cells were four (D21p.i.) and twenty (D28p.i.) times higher in the spleen of infected animals and five (D21p.i.) and 11 (D28p.i.) times higher in MLN  but remained similar in MLN  . CD23 staining was distributed throughout the GC of control animals, but was frequently patchy, with preferential staining at one side of GC, probably in the light zone (LZ) in placebo-treated animals. These observations are indicative of an impaired expansion of the FDC network and LZ during early SIV infection. HAART significantly reduced these alterations  and GC (staining with Bcl6) B cell-populated areas were already increased on D14p.i. and even more at D28p.i. in spleen Figure 6data not shown) whereas they were present in similar proportions in the extrafollicular zones of the spleen (data not shown). HAART significantly reduced these alterations  with a nadir at D14p.i., coinciding with the plasma viremia peak and the nadir in NK and CD4+ or CD8+ T-cells. Similar panleukopenia is observed during the acute phase of most viral infections in primates and mice.Impaired production of neutralizing HIV-specific antibodies during natural exposure to virus or during vaccination protocols is one major hallmark of HIV infection data not shown). During the same time, the percentage of total B-cells did not vary in peripheral LN  whereas a consistent influx of B-cells into the small intestine occurs from D14p.i. with the number and size of follicles progressively increased between D14 and D28p.i.. Altogether these data suggest that blood B-cells traffick to lymphoid organs but preferentially accumulate in the spleen and the small intestine. A drop in blood B-cells associated with a selective tissue homing has previously been reported in mice acutely infected by various pathogens In contrast to blood, the percentage of total B-cells increased in spleen from D14p.i. despite few changes in the percentage of T-cells in spleen before D21p.i. . Given that HAART initiated 4 h p.i. fully prevented apoptosis but partially reduced in vitro IgG production, independent control mechanisms may be involved. Previous data obtained in SIV- or HIV-infected individuals suggest that apoptosis is induced by various virus-induced signals Memory B-cells in spleen or LN steady declined until D28p.i., correlating with a preferential depletion in SIgG-expressing memory B-cells. This cell loss peaked at D14p.i. in spleen but was more progressive in LN. This loss may have been initiated by SIV-induced B-cell apoptosis by D14p.i. and early commitment to terminal differentiation. Most of the IgG molecules produced +SIgM+SIgD+ MGZ and CD27+ SIgD−SIgG+ memory B-cells poised to differentiate rapidly into plasma cells in response to T-independent signals, including BAFF, APRIL and IL21 +SIgD+) MGZ B-cells decreased transiently in LN between D14 and D21p.i. and steadily in spleen and blood from D14p.i. which could be due to their commitment to T-independent terminal differentiation. Similarly, a loss in IgM-only memory B-cells, associated withan impaired response to T-independent antigens has been reported in primary and chronically HIV-infected patients per se but is correlated with SIV-induced inflammatory signals, including BAFF as also suggested by the work of He et al Plasma IgG and IgM levels were increased by D14p.i., whereas SIV-specific antibodies were detectable only from D21p.i. on. Therefore, we suggest that an early T-independent response takes place before D14p.i.. In humans, unlike in mice, MGZ contains both CD27et al. in chronically infected patients subjected to short-term interruption of treatment IgG-expressing plasma cells were only detectable in GC from D21p.i.. The SIV-specific response was therefore likely to have been initiated between D14 and D21p.i.. Initiation of the specific response is likely to be delayed until viral antigens are locally available in sufficient amounts. Consistent with a recent study by Cinamom, showing that MGZ B-cells transport immune complexes into the GC in situ or prevent their homing to GC. In contrast, infiltrated GrB+ cells and CD8+ cells were present in GC from D14p.i. and D21p.i. respectively. Given previous findings in chronically infected macaques GC hyperplasia was detected in spleen and MLN of SIV-infected animals from D14 and D21p.i., respectively. The less intense staining observed in spleen and MLN suggests that fewer CD4+ T-cells may be present in EFZ of infected animals than in non-infected ones. However, the distribution of CD4+ cells in GC of infected animals was similar to those observed in non-infected animals before D28p.i.. This contrasts with the impaired polarization of GC observed in chronically infected patients In conclusion, we have combined IHC studies of lymphoid organs with phenotypic and functional analyses of B-cells to further elucidate the mechanisms underlying B-cell dysfunction during acute SIV infection. Consistent with unresponsiveness to early HAART, we suggest that T-independent signals, possibly triggered by direct contact with virions and relayed by inflammatory cytokines, are important in early B-cell dysfunction—a major feature of the SIV/HIV-specific B-cell response strongly favoring non-specific Ig production. A better understanding of the inflammatory signals acting on B-cell subsets should help to design of new therapeutic strategies, potentially delaying the initiation of HAART which strongly decreases the HIV/SIV humoral response.Macaca fascicularis) were inoculated intravenously (i.v) with 50 AID50 of the pathogenic SIVmac251 strain, as previously described Thirty adult male cynomolgus macaques  were obtained by analysis of 15 µl of blood, collected on EDTA, in Micro Diff II . B-cells (CD20), monocytes (CD14), total T (CD3), CD4, CD8 cells and NK (CD3−CD8+) cells were quantified by flow cytometry with 50 µl of titrated combinations of fluorochrome–conjugated mAbs added to 100 µl of whole blood, as previously described supplemental Spleen, axillary and inguinal LN were collected from SIV-infected animals and four non-infected ones as controls. Splenocytes and LN mononuclear cells were obtained by mechanical disruption, passed through nylon mesh cell strainers with 40-µm pores and further purified by Ficoll gradient centrifugation . Freshly isolated cells were subjected to phenotypic analysis or kept at −80°C until use. Blood, spleen and LN B-cell subsets were identified by three-colour flow cytometry detecting the combined expression of CD20 with CD27, CD80, CD86, CD95, surface IgD (SIgD), SIgM or SIgG. Briefly, cells were stained with 50 µl of titrated combinations of fluorochrome–conjugated mAbs listed in Spleen B-cells were purified using PE-conjugated CD20 mAb  and PE-conjugated magnetic beads . The cell suspension was incubated with CD32 mAb  before CD20-PE addition according to the manufacturer's instructions. Only B-cell fractions that contained ≥80% CD20+ cells were used for functional studies. Splenic B-cells were cultured in RPMI 1640-glutamax medium supplemented with 1 mM sodium pyruvate, 100 µg/ml streptomycin, 100 U/ml penicillin, 10 mM HEPES buffer, 2 mM non-essential amino acids  and 2% (proliferation) or 10% (apoptosis and IgG production) heat-inactivated FCS .2 rabbit anti-human Ig(H+L) Ab  or medium (spontaneous apoptosis) for 24 h. After several washes, cells were fixed and permeabilized in 70% ethanol overnight, at 4°C. Cells were then treated for 30 min at 37°C with 100 µg/ml RNAse A  and labelled with DAPI  for 15 min at 20°C and analyzed by flow cytometry. Apoptotic cells were defined as hypodiploid cells.Splenocytes were cultured in triplicate with 25 µg/ml F(ab')2 rabbit anti-human Ig(H+L) Ab (Jackson ImmunoResearch Laboratories) and 20 ng/ml IL4 , Staphylococcus Aureus cowan I  and 20 ng/ml IL2 or 10 µg/ml CD40 mAb (G28.5) plus 20 ng/ml IL2 and 50 ng/ml IL10 (both cytokines from R&D systems) for 72 h. Proliferative responses were measured by pulsing cultures with 1 µCi per well [methyl-3H] thymidine  for the last 12 h of the culture. Results are cpm means±SEM of triplicate measurements. Stimulation index (SI) was calculated as the ratio between the cpm means for cultures with and without stimuli.Splenocytes were cultured in triplicate with 25 µg/ml F(ab')Splenocytes were cultured for 10 days at 37°C with medium, 20 ng/ml IL2 plus 50 ng/ml IL10, with or without 10 µg/ml CD40 mAb. Ig concentrations were determined in cell-free culture supernatants or in plasma by specific ELISA. Rabbit anti-human IgG+M+A, HRP-conjugated goat anti-rhesus IgM (μ chain-specific)  and rhesus IgM  were used for IgM quantification. Goat anti-monkey IgG, HRP-conjugated goat anti-rhesus IgG  and rhesus IgG  were used for IgG quantification. Results are expressed as mean concentration  of duplicate measurements.450 nm readings.Specific anti-SIV Ab were detected in plasma by ELISA using Genscreen HIV1/2 kit, version2 . Results are expressed in OD obtained from Asupplemental IHC were performed on deparaffinized spleen, MLN or small intestine sections, with primary mAb , captured by a Microfire microscope camera system  and analyzed with Mercator 4.42 software .p value≤0.05 was considered as significant.Data are expressed as mean±SEM, unless otherwise indicated. Non-parametric analysis (Wilcoxon signed-rank test and Mann-Whitney test) and correlations (Spearman's rank coefficient) were performed using StatView software . Table S1Antibodies used in FCM(0.28 MB DOC)Click here for additional data file.Table S2Antibodies used in IHC(0.23 MB DOC)Click here for additional data file.Figure S1Correlation between plasma viral load and blood T- or B-cell counts during SIV infection. Correlation between plasma viral load  and blood CD4 (A), CD8 (B) or CD20 (C) cell count (cells/µl) in SIV-infected animals is shown. Correlation between blood B-cell count and percentage (D) or CD4 cell count (E) in SIV-infected animals is shown. Statistical significance was assessed by Spearman's rank correlation test; Rho and p values are indicated.(0.38 MB TIF)Click here for additional data file.Figure S2Phenotypic change within MLN B-cell areas from placebo- and HAART-treated SIV-infected animals. MLN sections from two non-infected (Ctl), P28 and H28 animals were stained for CD20 (A), CD23 (B), Ki67 (C), Vs38c (D) and IgG (E) expression. Staining from one representative animal of each group is shown. Brown indicates positive staining; cell nuclei were counterstained in blue by hematoxylin. Original magnification ×100 for A to E and ×200 for C.(8.77 MB TIF)Click here for additional data file.Figure S3Phenotype change within mantle zone of follicles from placebo and HAART-treated SIV-infected animals. Spleen sections from two non-infected (Ctl), P28 and H28 animals were stained for CD3 (A) and CD68 (B) expression. Staining from one representative animal of each group is shown. Brown indicates positive staining; cell nuclei were counterstained in blue by hematoxylin. Original magnification 100× and 400× for both markers.(4.49 MB TIF)Click here for additional data file.


The phosphatase PTEN governs the phosphoinositide 3-kinase (PI3K)/Akt signaling pathway which is arguably the most important pro-survival pathway in neurons. Recently, PTEN has also been implicated in multiple important CNS functions such as neuronal differentiation, plasticity, injury and drug addiction. It has been reported that loss of PTEN protein, accompanied by Akt activation, occurs under excitotoxic conditions (stroke) as well as in Alzheimer's (AD) brains. However the molecular signals and mechanism underlying PTEN loss are unknown.2O2-mediated oxidation. We found that S-nitrosylation of PTEN was markedly elevated in brains in the early stages of AD (MCI). Surprisingly, there was no increase in the H2O2-mediated oxidation of PTEN, a modification common in cancer cell types, in the MCI/AD brains as compared to normal aged control. Using several cultured neuronal models, we further demonstrate that S-nitrosylation, in conjunction with NO-mediated enhanced ubiquitination, regulates both the lipid phosphatase activity and protein stability of PTEN. S-nitrosylation and oxidation occur on overlapping and distinct Cys residues of PTEN. The NO signal induces PTEN protein degradation via the ubiquitin-proteasome system (UPS) through NEDD4-1-mediated ubiquitination.In this study, we investigated redox regulation of PTEN, namely S-nitrosylation, a covalent modification of cysteine residues by nitric oxide (NO), and H2O2-mediated PTEN oxidation, known to only inactivate the enzyme. This novel regulatory mechanism likely accounts for the PTEN loss observed in neurodegeneration such as in AD, in which NO plays a critical pathophysiological role.This study demonstrates for the first time that NO-mediated redox regulation is the mechanism of PTEN protein degradation, which is distinguished from the H PTEN, the phosphatase and tensin homologue deleted on chromosome 10, is one of the most frequently mutated tumor suppressors in human cancers. The major, and best characterized, function of PTEN is its lipid phosphatase activity which dephosphorylates PIP3 to generate PIP2, and thus antagonizes the PI3K activity in the activation of Akt , neuronaStudies conducted in experimental models for cancer and diabetes have shown that PTEN regulation is rather complex. Multiple mechanisms might be involved in a decrease or loss of PTEN function, in addition to gene mutation and deletion. These mechanisms may include transcription and post-translational modifications (PTMs) which include phosphorylation, acetylation, oxidation and ubiquitination . PTEN is2O2), superoxide, peroxynitrite and nitrosothiol, modify PTEN on the critical cysteine residue (C124) and inactivate its lipid phosphatase activity in multiple cancer cell lines [2O2 and NO-mediated S-nitrosylation; the later, a process of reversible addition of NO to Cys-sulfur in proteins, has emerged as a major regulatory mechanism in fine-tuning many critical molecules in the neuronal death pathway and neurodegeneration [PTEN can be acutely regulated by oxidative stress and by endogenously produced reactive oxygen species (ROS) . Oxidatill lines -24. To iInitially, we sought to investigate whether S-nitrosylated PTEN (SNO-PTEN) is produced in neurodegenerative disorders associated with high levels of nitrosative stress such as stroke, AD and Parkinson's disease (PD). We included in our tests those specimens taken from autopsy patients diagnosed at an early stage of AD, called mild cognitive impairment (MCI), and compared them to aged matched control brain specimens . The patient cohort and information are summarized in the table [Additional file From semi-quantitative profiling of PTEN/Akt in 27 human brains, we found that SNO-PTEN was markedly induced in the entorhinal cortices, the most vulnerable region in AD brains, as early as the MCI stage but was nearly undetectable in age-matched normal brains (NC) Figure . Althoug25-35) also induced robust SNO-PTEN within minutes  also induced SNO-PTEN (data not shown); this is consistent with SNO-PTEN in PD brains and suggests that SNO-PTEN may play a common role in chronic degenerative diseases such as AD and PD. In parallel, we found that after treatment of SNOC/glutamate/Aβ, the majority of PTEN in neuronal cells (>85%) remained unmodified by H2O2-type oxidation, indicating that H2O2 may not be the dominant oxidizing species induced by these treatments  in a dose-dependent manner with detectable nitrosylation achieved by as little as 10 μM SNOC and a plateau with 300 μM Figure . This was Figure  and lasts Figure . This iss Figure .25-35 peptides in a time-dependent manner , or okadaic acid (OA). The strong correlation between SNO-PTEN formation and the reduced PTEN protein level suggests that the PTEN decrease may be a direct consequence of PTEN S-nitrosylation or may be caused otherwise by NO signaling. Indeed, pretreatment with a NOS inhibitor (l-NMMA) or the proteasome inhibitor MG132 for 5 h prior to glutamate exposure rescued the decrease in PTEN by over 70%and 50%, respectively  steady-state levels in neurons 2-4 h after exposure to SNOC, glutamate or Aβr Figure  and 3B. y Figure , suggestWe also found that SNOC/glutamate/Aβ all induce enhanced PTEN ubiquitination with the maximum effect seen 2 h after treatments Figure . ConsistWe next examined the effect of PTEN S-nitrosylation on the lipid phosphatase activity of PTEN. By conventional Malachite Green assay using PIP3 as substrate , SNOC trIn cultured primary neurons, exposure to Aβ or NMDA induced a rapid decrease in PTEN and increase in P-Akt levels. Downregulation of endogenous PTEN via specific siRNA produces neuroprotection, as evidenced by preserved neuronal structures Figure . Cortica2O2, induces PTEN protein degradation. Loss of PTEN protein is reportedly associated with myocardial and brain ischemia [PI3K/Akt is arguably the most important cell-survival signaling pathway for neurons. As the key negative regulator of the PI3K/Akt pathway, PTEN is an important target of study for neuroprotection during neurodegeneration. In this work, we are the first to demonstrate NO-mediated redox regulation as the mechanism of PTEN protein degradation. We also demonstrate that NO rapidly induces S-nitrosylation of PTEN, thereby inactivating it. Moreover, NO, but not Hischemia -29, pres2O2-induced modification and S-nitrosylation represent the two dominant oxidative events through targeted modifications of critical Cys residues in proteins. H2O2-induced PTEN oxidation was reported to cause the formation of an intra-chain disulfide bond between C71-124 [It is widely accepted that oxidative stress is one of the earliest changes that occurs in the pathogenesis of AD, arising from the imbalance between increased production of reactive oxygen and nitrogen species and impaired antioxidant defenses, as reflected in the accumulation of oxidative damage to macromolecules detected in MCI, the clinical precursor of AD, and AD brains ,31. H2O2Although our mutagenesis studies Figure  cannot dOur results demonstrate NO-mediated PTEN protein degradation via UPS, as evidenced by enhanced ubiquitination. There are several precedents showing that protein S-nitrosylation can be functionally coupled to its ubiquitination and modulate protein degradation -38. It iAlthough we made our initial observation in MCI/AD brain samples, the loss of PTEN is also reported to occur in both myocardial  and cereIn summary, our studies have demonstrated that S-nitrosylation of PTEN plays a major role in PTEN regulation in neuronal systems and that NO signaling induces S-nitrosylation and ubiquitination to modulate both PTEN protein degradation and enzymatic activity, which may represent the underlying regulatory mechanism of the PI3K/Akt signaling pathway in both acute and chronic neurodegeneration. A fuller understanding of the molecular mechanisms underlying the cascade of events leading to PTEN inhibition in both acute and chronic settings will advance our knowledge of PTEN regulation in the CNS. It may also be instrumental in future therapeutic design of novel target-based intervention in treating neurodegenerative diseases and perhaps other age-related conditions such as cancers, diabetes, cardiovascular ischemia and stroke.l-cysteine were added to 950 μl H2O. Then, 50 μl of 10 N HCl is added to adjust pH to be 7.4. For glutamate (Sigma-Aldrich), Aβ25-35 peptides , and staurosporine/STS (Sigma-Aldrich), neurons were treated for 4 hours before cell lysates were prepared. For suppression of NOS activity, l-NMMA  was applied to neurons before exposure to neurotoxic reagents.Primary cultured cortical neurons (PRCN) and neuroblastoma N2a cells were prepared as described . For theTransient transfections were performed with plasmid constructs for pEF-PTEN-WT  and its site-directed mutagenized constructs (Cysteine to Alanine substitution).Human brain samples were provided by UCSD, San Diego, CA and were analyzed with institutional permission under California and National Institutes of Health guidelines. Informed consent was obtained following the procedures of the Institutional Review Boards of the Sanford-Burnham Institute for Medical Research.25-35 for 4 h.A biotin switch assay for detection of SNO-PTEN was performed as previously described with minor modification . PRCN cuS-nitrosylation of PTEN was measured as previously described .Cells were lysed in buffer containing 2% SDS and 40 mM N-ethylmaleimide and 20 μg of protein was subjected to 10% SDS-PAGE under non-reducing condition as described ,23.Recombinant PTEN, which was expressed in baculovirus, was assessed for the phosphatase activity as previously described . EnzymatIP-Western experiments were performed as described . The folPRCN cells were incubated with 50 μg/ml cycloheximide  for the indicated times in the presence or absence of glutamate (200 μM). Cells were simultaneously treated with glutamate and cycloheximide. To examine whether the UPS is involved in nitrosative stress induced PTEN reduction, cells were co-treated with glutamate and 25 μM MG132. Cells lysates were then prepared for Western blot analysis.Three different sets of siRNAs were designed by Ambion for rat PTEN . The annealed oligonucleotides encoding siRNAs were cloned into the pIRES-enhanced RFP (Invitrogen) using EcoRI/BamHI sites and the resulting siRNA-IRES-RFP fragment was further cloned into pSin-Rep5 (Invitrogen). The efficiency and specificity of PTEN-siRNAs were then assessed by RT-PCR and western blot analysis. Virus particles were generated and infection performed in primary neurons according to the manufacturer's protocol as described previously .® II Site-Directed Mutagenesis Kits, Stratagene, La Jolla USA) at C71, C83, C105, C124, C136, C211, C218, C250, C296, C304, and its double/triple mutants with various combinations. The primers for C71A forward 5'-TTTAAAGCATAAAAACCATTACAAGATATACAA-3' and reverse 5'-GTCATAATGTCTAGCAGCAAGATTGTATAT-3'; C83A forward 5'-GACACCGCCAAATTTAATGCCAGAGTTGCACAA-3' and reverse 5'-GATATTGTGCAACTCTGGCATTAATGGCGG-3'; C105A forward 5'-GAACTTATCAAACCCTTTGCTGAAGATCTTGAC-3' and reverse 5'-CCATTGGTCAAGATCTTCAGCAAAGGGTTTGAT-3'; C124A forward 5'-CATGTTGCAATTCACGCTAAAGCTGGAAAG-3' and reverse 5'-GTCCCTTTCCAGCTTTGCGTGAATTGCTGGAATTGCTGCAA-3'; C136A forward 5'-GACGAACTGGTGTAATGATAGCCGCATATTTAT-3' and reverse 5'-CCCGATGATATAAATATGCGGCTATCATTACAC-3'; C211A forward 5'-GTTCAGTGGCGGAACTGCCAATCCTCAGTTTG-3' and reverse 5'-CACAAACTGAGGATTGGCAGTTCCGCCACTGAA-3'; C218A forward 5'-CCTCAGTTTGTGGTCGCCCAGCTAAAGGTGAA-3' and reverse 5'-CTTCACCTTTAGCTGGGCGACCACAAACTGAGC-3'; C250A forward 5'-CAGCCGTTACCTGTGGCTGGTGATATCAAAG-3' and reverse 5'-CTTTGATATCACCAGCCACAGGTAACGGCTG-3'; C296A forward 5'-TCAGAAAAAGTAGAAAATGGAAGTCTAGCTGAT-3' and reverse 5'-CAAATGCTATGGATTTCTTGATCAGCTAGACTT-3'; C304A forward 5'- CAAGAAATCAGCATTGCCAGTATAGAGCGT-3' and reverse 5'-CTGCACGCTCTATACTGGCAATGCTATCGATTT-3' were utilized for PCR amplification, respectively. PCR amplification was performed according to the company's protocol. Once single mutants were constructed, we used these mutants as a template for further construction of double/triple mutants.PTEN mutants were created by site-directed mutagenesis (QuikChangeIHC on PTEN (1:500) and MAP-2/NeuN (1:500/each) and apoptotic staining were performed as described  on two wp < 0.05 were considered to be significant.All quantitative data were presented as means ± SDV. Comparison between groups were analyzed with unpaired ANOVA using Graphpad PRIZM software  and values of The authors declare that they have no competing interests.Author contributions: FFL designed research; Y-DK, TM, S-YD, XZ, Y-MC, and JS performed experiments; SAL, EM, HX, analyzed data. FFL and Y-DK wrote the paper. All authors have read and approved the final manuscript.The table of the patient brain information.Click here for fileAdditional data on the Cys mutants. SNO-PTEN levels were determined by biotin-switch assays and data were analyzed by densitometry of the SNO-PTEN/total PTEN ratio, indicating that the majority of C211-304 located in the C2 domain do not seem to be the direct sites of S-nitrosylation.Click here for fileEffect of double and triple Cys mutants on PTEN protein stability upon SNOC treatment. Various mutant plasmids were transiently introduced to N2a cells along with WT control and cells were treated with SNOC (200 μM) 48 h after transfection. The steady-state levels of PTEN were determined at various time points by Western blot analysis using anti-HA antibody to probe on the exogenously expressed PTEN.Click here for file