Helicobacter pylori (Hp) injects the CagA effector protein into host epithelial cells and induces growth factor-like signaling, perturbs cell-cell junctions, and alters host cell polarity. This enables Hp to grow as microcolonies adhered to the host cell surface even in conditions that do not support growth of free-swimming bacteria. We hypothesized that CagA alters host cell physiology to allow Hp to obtain specific nutrients from or across the epithelial barrier. Using a polarized epithelium model system, we find that isogenic ΔcagA mutants are defective in cell surface microcolony formation, but exogenous addition of iron to the apical medium partially rescues this defect, suggesting that one of CagA's effects on host cells is to facilitate iron acquisition from the host. Hp adhered to the apical epithelial surface increase basolateral uptake of transferrin and induce its transcytosis in a CagA-dependent manner. Both CagA and VacA contribute to the perturbation of transferrin recycling, since VacA is involved in apical mislocalization of the transferrin receptor to sites of bacterial attachment. To determine if the transferrin recycling pathway is involved in Hp colonization of the cell surface, we silenced transferrin receptor expression during infection. This resulted in a reduced ability of Hp to colonize the polarized epithelium. To test whether CagA is important in promoting iron acquisition in vivo, we compared colonization of Hp in iron-replete vs. iron-deficient Mongolian gerbils. While wild type Hp and ΔcagA mutants colonized iron-replete gerbils at similar levels, ΔcagA mutants are markedly impaired in colonizing iron-deficient gerbils. Our study indicates that CagA and VacA act in concert to usurp the polarized process of host cell iron uptake, allowing Hp to use the cell surface as a replicative niche. Helicobacter pylori (Hp) is a bacterium that chronically infects the stomach of humans and can lead to serious illness. To survive in the stomach, the bacteria intimately interact with the epithelial lining. Some inject the virulence protein CagA into the host cells, and we previously showed that CagA helps Hp survive and grow directly on the epithelial cell surface. Iron is one of the limiting factors that infectious bacteria must acquire from their host. Using a model polarized epithelium system, we discovered that CagA is able to alter the internalization, intracellular transport, and polarity of the transferrin/transferrin receptor iron uptake system. This allows the bacteria to shuttle iron across the epithelium and suggests a novel mechanism of iron acquisition from host cells, enabling Hp growth on the cell surface. Another major virulence factor of Hp, VacA, is also involved in this process. To test the role of CagA in iron acquisition in vivo, we infected iron-deficient Mongolian gerbils and found that CagA-deficient bacteria had a decreased ability to colonize the stomach. Our study illustrates how microbes that chronically infect our mucosal surfaces can manipulate the epithelium to acquire micronutrients from host cells and grow on the cell surface. Helicobacter pylori (Hp) is a mucosal colonizer that infects the stomachs of more than half of the world's population Hp infection is a major cause of gastric and duodenal ulcer disease, a risk factor for gastric cancer Hp (∼20%) adhere to the host cell surface via various adhesins Hp can colonize and replicate directly while adhered to the epithelial surface, and can grow in this niche even in conditions where growth of the free-swimming bacteria is not supported Hp virulence factor CagA, which is injected directly into host cells via the bacterium's type IV secretion system, plays an important role in enabling Hp colonization of the epithelium Hp has evolved specialized mechanisms to perturb cell polarity to acquire essential factors directly from the polarized epithelium. However, the nature of the factors transferred from the host cells to the bacteria and the molecular mechanisms involved remain unclear.Hp and several other pathogens Hp is known to possess several iron uptake systems, the sources of iron that Hp utilizes during colonization of the gastric mucosa remain unclear Hp has not been shown to synthesize siderophores Hp is not found in the gastric lumen but rather colonizes the neutral environment of the epithelial cell surface and the overlying mucus layer Hp is unable to compete with partially saturated lactoferrin for iron acquisition Hp cannot compete with partially saturated transferrin for iron Hp is able to utilize iron from fully saturated transferrin Successful colonization of mucosal surfaces by bacteria implies an ability to extract essential micronutrients from their immediate environment, either from epithelial secretions near the cell surface, from the polarized host cells themselves, and/or from the interstitial side, across the epithelial cell layer. Iron is a micronutrient critical for the survival and growth of many mucosal colonizers and its availability controls expression of bacterial virulence factors in Hp colonizing the apical surface are able to acquire iron from host epithelial cells. We also show that the Hp virulence factors CagA and VacA work in concert to shift the basolateral transferrin/transferrin receptor recycling process apically, directing transferrin and its receptor to sites of microcolony formation on the cell surface. Silencing expression of the transferrin receptor interferes with the colonization of the epithelium by Hp. Finally, we show, using a Mongolian gerbil model of Hp infection, that host iron depletion results in a decreased ability of CagA-deficient Hp to colonize the gastric niche.In this study, we utilize a model polarized epithelium to show that Hp are able to colonize the apical cell surface of polarized MDCK monolayers when the apical medium bathing the cells contains only DMEM, a medium that cannot support Hp growth in vitroHp (ΔcagA) are defective in colonizing this niche. This suggested that we could use this system to identify host factors obtained by WT Hp but unavailable to ΔcagA by enriching the apical medium with specific nutrients and testing which would rescue the growth defect of ΔcagA. We found that addition of iron in the form of ferric chloride to the apical chamber partially rescued the growth defect of ΔcagA in a saturable and dose dependent manner  t manner . In contt manner . Imagingithelium . In addiin vitro , suggestcagA growing on the cell surface by iron suggests that CagA affects host epithelial cell function to allow Hp access to micronutrients that are found in the epithelium or across its barrier.The rescue of ΔHp growing on the cell surface gain access to interstitial transferrin and utilize this form of iron for survival. However, in vitro reports have shown that Hp cannot grow on partially saturated transferrin Hp growth on the cell surface was inhibited by the presence of partially saturated transferrin in the apical medium, indicating that Hp is unable to compete with partially saturated transferrin for iron even in the presence of cells  via endocytosis, as its affinity for the transferrin receptor is 2000X greater than iron-free transferrin of ΔcagA . These eHp in iron acquisition from host cells, we asked if Hp colonization of the apical cell surface affects host cell transferrin recycling. To study this, we utilized MDCK cells stably expressing human transferrin receptor Hp colonized the apical cell surface while ΔcagA exhibited a 100X defect in colonization ability, as seen with untransfected MDCK cells  led to restoration of the phenotype of increased transferrin internalization on infection with the bacteria  was less than that observed with WT (774 arbitrary units/bacterium), but this was not statistically significant  may be involved in this phenomenon. Another major virulence factor of Hp, vacuolating cytotoxin (VacA), has been reported to interfere with the endocytic pathway of host cells Hp mutant (ΔvacA), and found that in monolayers infected with ΔvacA, transferrin receptor was absent from the sites of bacterial microcolonies on the apical cell surface  restored the ability of the bacteria to mislocalize transferrin receptor apically to sites of bacterial microcolonies  was still able to mislocalize proteins to the sites of bacterial microcolonies . However, ΔvacA formed significantly smaller microcolonies on the cell surface at day 5 post-infection (vacA (VacA*) led to restoration of the ability of the bacteria to effectively colonize the polarized epithelium, with the formation of large microcolonies . This double mutant resembled the single CagA-deficient mutant, and exogenous addition of iron apically partially rescued its ability to colonize the polarized epithelium  (To directly test if the transferrin receptor pathway is important for pression . Cells tcolonies . More imA (eGFP) .Hp allowed to colonize the apical surface of these cells formed microcolonies similar in size to those formed by Hp colonizing cells transfected with a control siRNA  resulted in restoration of the phenotype of increased transferrin transcytosis , and since CagA-negative strains are common in nature, it is likely that in vivo, Hp utilizes multiple modes of iron acquisition. However, our findings suggest that CagA may be more important for the bacteria when colonizing hosts that are iron-depleted or during conditions of poor dietary iron content. To address this question, we utilized the Mongolian gerbil model of Hp infection to determine if the iron status of the host would affect WT or ΔcagA colonization.Hp strain 7.13, which has been previously shown to reproducibly colonize the Mongolian gerbil stomach, is able to deliver CagA into host cells, and whose isogenic ΔcagA mutant exhibits a defect in colonization of the polarized epithelium in vitro, similar to the Hp strain G27-MA cagA grow with similar kinetics -like signaling Hp, has previously been reported to affect endosomal trafficking For Hp growing as microcolonies on the apical cell surface increased internalized transferrin. This required signaling via the EPIYA motifs in the C-terminus of the protein, which are important in the activation of RTK-like signaling vacA Hp mutant failed to recruit transferrin receptor apically. We were able to show a direct involvement of the transferrin/transferrin receptor recycling process in the colonization of the polarized epithelium by Hp, since siRNA knockdown of transferrin receptor expression resulted in decreased growth of Hp microcolonies on the cell surface. Our model suggests that Hp colonizing the apical cell surface induce mis-sorting of a subset of the transferrin/transferrin receptor complex apically. In accord with this, we observed significantly increased transcytosis of transferrin from the basal to the apical compartment and its release into the apical media when the epithelium is infected by WT Hp.We found that CagA injected into host cells by Hp. Hp can live as a free-swimming population in vitro without the need for contact with epithelial cells. In vivo, Hp may obtain iron through several mechanisms and from various sources. For example, Hp residing as free-swimming bacteria in the mucus layer may obtain iron conjugated in mucus glycoproteins, or perhaps from the release of dietary iron by the acidic stomach lumen, or from inflammatory exudates, although these potential sources need study. Of note is that in addition to its role in causing peptic ulcers and its association with gastric cancer, Hp infection has recently been associated with iron deficiency anemia unrelated to blood loss Hp in cases of unexplained iron deficiency anemia Hp eradication improved or cured previously unexplained cases of iron deficiency anemia Hp extracts iron from the host epithelium can be fully understood. Several of the known effects of CagA and VacA on host cell physiology may have important roles in altering trafficking of transferrin and other molecules for the benefit of colonizing bacteria. For example, we showed here that signaling through the EPIYA motifs of CagA increase host internalization of transferrin. CagA also has multiple effects mimicking growth factor activity which could influence the way in which gastric epithelial cells uptake and process nutrients that could be used by colonizing HpHp colonization of the apical cell surface vacA-infected polarized cell monolayers (data not shown). The delivery of VacA can be local through contact by adhered bacteria Several important questions remain regarding this model. First, it is clear that iron acquisition from the epithelium is only one of several mechanisms of iron acquisition by Hp colonization of the epithelium. For example, the “East Asian” form of CagA has been reported to bind more strongly to Par1b than the “Western” form of CagA Hp on the cell surface, and perhaps uncover additional iron acquisition methods by Hp.The potential cooperation between CagA and VacA to affect polar transport of micronutrients to colonizing microcolonies is exciting, since CagA and certain VacA alleles have been linked genetically Hp may obtain iron from host epithelial cells. We subsequently found that ΔcagA mutant Hp have a decreased ability to colonize the stomachs of Mongolian gerbils that are iron-deficient whereas WT bacteria do not, indicating that CagA aids Hp in acquiring iron from the host during in vivo bacterial colonization. Our findings also raise the question of how host micronutrient levels impact the pathogenesis of colonizing Hp. For example, might decreased levels of micronutrients in the host lead to increased pathogenicity of the bacteria? We note here that expression of both cagA and vacA have been reported to be upregulated in conditions of iron starvation in vitrovacA expression Hp infection Our use of a simplified experimental system allowed us to focus on the microenvironment of the apical cell surface as a replicative niche, and to uncover one possible mechanism by which Hp appears to be a specific and subtle process, not due to a generalized loss of epithelial polarity, since biotinylated proteins from the basolateral membrane form patches specifically under apical bacterial microcolonies, and not all basolateral proteins are found in these membrane patches. Although we focused on transferrin receptor mislocalization in this study, the technique of basal biotinylation of host membrane proteins in infected monolayers suggests that there are multiple proteins that are mislocalized to the apical sites of bacterial growth. The identity of these proteins may shed other important insights into the way microbes colonize the epithelial surface. CagA and VacA play major roles in this mislocalization, but other bacterial factors do appear to be involved as well. The ability to manage host epithelial polarity is emerging as an important facet of bacterial-host interactions. For example, Listeria monocytogenes has been shown to take advantage of cell polarity changes for invasion Escherichia coli and Pseudomonas aeruginosaP. aeruginosa infection of polarized epithelia, phosphatidylinositol 3, 4, 5-trisphosphate (PIP3), a basolateral membrane lipid important in cellular signaling and polarity, is mislocalized to the sites of bacterial attachment on the apical cell surface Hp microcolonies on the apical cell surface, and whether PIP3 function is involved in Hp's colonization of the polarized epithelium.We suggest that manipulation of host epithelial polarity is akin to utilizing the epithelium as a filter to acquire essential micronutrients while maintaining a barrier that protects the microbes from innate immune defenses present in the interstitial space. Perturbation of host epithelial polarity by Hp is able to obtain from host cells during colonization of the apical cell surface, and illustrate a way in which CagA and VacA work in concert to aid the bacteria in establishing a replicative niche. We hypothesize that growth on the epithelial surface involves more than just iron acquisition from the host. For example, it has previously been shown that Hp can acquire cholesterol directly from host cells via contact Hp has evolved sophisticated mechanisms to manipulate host cell physiology for its own benefit, and that these features have side effects that result in disease in a subset of the infected population. Instead of destroying the epithelium as may occur in some types of acute bacterial infection, mucosal colonizers like Hp use more subtle mechanisms of local epithelial perturbation. We propose that mucosal colonizers may be utilizing the polarized epithelium as a “filter” both to protect themselves from potentially toxic host defense molecules, and to selectively extract micronutrients that are present inside the host in a form that is usable by the bacteria. Future exploration of the nature of host proteins associated with apical bacterial microcolonies, and the possible role of other bacterial factors in perturbing polarity, will give us a better understanding of how Hp and other mucosal colonizers affect the epithelial surface for their benefit.In summary, our results show that iron is one important factor that All animal experiments were performed in accordance to NIH guidelines, the Animal Welfare Act, and US federal law. Such experiments were carried out with the approval of the Institutional Animal Care and Use Committee of Vanderbilt University, which has been accredited by the Association of Assessment and Accreditation of Laboratory Animal Care International (AAALAC). All animals were housed in an AAALAC-accredited research animal facility fully staffed with trained technical, husbandry, and veterinary personnel.2 atmosphere. Caco-2 cells (ATCC) were maintained in DMEM containing 10% FBS, at 37°C in a 5% CO2 atmosphere. Polarized MDCK and Caco-2 monolayers were cultured by seeding cells at confluent density onto 12 mm, 0.4 µm-pore polycarbonate tissue culture inserts . Polarized MDCK monolayers were maintained as previously described Madin-Darby Canine Kidney II (MDCK) cells  Hp strain G27-MA and its isogenic ΔcagA mutant have been previously described cagA has been previously described vacA mutant of strain G27-MA was constructed by deletion of the vacA open reading frame (ORF) beginning from the start codon to 14 base pairs before the end of the ORF, and replacement with the aphA gene (conferring kanamycin resistance), by a PCR based method without recombinant cloning vacA was accomplished by natural transformation with a construct containing the vacA ORF with the cat gene (conferring chloramphenicol resistance) immediately downstream, flanked by the upstream and downstream regions of vacA to allow for homologous recombination. Verification of the ΔvacA mutant and of the complemented strain (VacA*) was performed by immunoblotting bacterial whole cell lysates with a polyclonal rabbit anti-VacA antibody   was constructed by transformation with a previously described allele aphA gene inserted immediately downstream of the mutant CagA sequence for selection of transformants. Hp strain 7.13 and its isogenic ΔcagA mutant have been previously described Hp on Columbia blood agar plates and co-culture of Hp with MDCK cells were as previously described Hp from co-cultures were used for infections. Co-culture media for Hp with MDCK cells consists of DMEM + 5% FBS +10% Brucella broth +10 µg/ml vancomycin.ogicals) . Immunobectively . A G27-M3. 30 µl of a mixture of 100 mM disodium nitrilotriacetate (Sigma) and 12.5 mM FeCl3 (Sigma) was then added to the solution. After incubation at 37°C for 1 hour, the sample was passed through a HiTrap desalting (Sephadex G-25 Superfine) column , previously equilibrated with a solution of 0.02 M Tris-Cl, pH 7.4, containing 0.15 M NaCl. The ratio of absorbance at 465 nm to 280 nm was measured to provide an estimate of the amount of iron bound by transferrin Transferrin was saturated with iron essentially as previously described To remove unbound iron from the preparations, the samples were subsequently treated with Chelex resin (BioRad) Hp growth assays in media without cells, Hp grown overnight on Columbia blood agar plates were resuspended in DMEM, and aliquots inoculated into the appropriate media in 6-well plates. Where added, ferric chloride  was added at a final concentration of 100 µM, and human transferrin (Sigma) was added at a final concentration of 75 µg/ml. Data are shown as means ± SD. We also tested holotransferrin (prepared as described above) added to DMEM at a final concentration of 75 µg/ml, which does not allow growth of Hp without the presence of cells (For of cells .Hp was carried as previously described Hp (∼108 bacteria/ml) were added to the apical chamber, allowed to adhere for 5 minutes, and cell monolayers washed 5 times with fresh DMEM to remove non-adherent bacteria. Appropriate media was added back to the apical chamber, and the cells incubated at 37°C in a 5% CO2 atmosphere. Basal media was changed daily. After sampling for colony forming unit (CFU) counts from the apical chamber each day, cell monolayers were washed 3 times with fresh DMEM, before appropriate media added back and the cells returned to the incubator. Data are shown as means ± SD. Hp express both CagA and VacA during colonization of the polarized epithelium as evaluated by immunoblotting  (Infection of polarized MDCK monolayers with blotting . We had hnology) .Hp culture, which has been reported to contain 13.9 µM of iron 3 (Sigma) was added to the media in the apical chamber at a final concentration of 100 µM (unless otherwise stated). Holotransferrin (prepared as described above) or transferrin (Sigma) was added to the media in the apical chamber at 75 µg/ml when used. For experiments with transferrin added to the co-culture media in the basal chamber, transferrin was added at a final concentration of 75 µg/ml.DMEM is iron-poor, containing 0.248 µM ferric nitrate (Invitrogen media formulation), in contrast to Brucella broth media often used in Hp antibodies Hp antibodies. We did not use the 488 nm channel in these experiments, and visualized transferrin or transferrin receptor in the 594 nm channel to avoid overlap of the fluorescence spectra and prevent signal bleed-through from one channel into the next. Alexa-fluor 647-conjugated phalloidin (Molecular Probes) was pseudocolored yellow in these experiments. For all other experiments, either Alexa-fluor 594 or 647-conjugated phalloidin were used for visualization of the actin cytoskeleton and pseudocolored red or blue respectively. Nuclei were visualized with DAPI (Invitrogen). Samples were imaged with a BioRad MRC-1024 confocal microscope, or with a Zeiss LSM 700 confocal microscope, and z-stacks reconstructed into 3D using Volocity software (Improvision). Quantification of microcolony sizes was carried out as previously described Samples were processed for confocal immunofluorescence as previously described Hp from the apical side for two days. For assays with polarized Caco-2 cells, Hp were infected from the apical side for 18 hours. Monolayers were washed 3 times apically and 5 times basally with fresh, pre-warmed DMEM. DMEM was added back to the apical chamber, and DMEM + 25 µg/ml human transferrin conjugated to Alexa Fluor 594 (Invitrogen) added to the basal chamber. The samples were then incubated on ice for 30 minutes. After this time, monolayers were washed 5 times with fresh DMEM basally. Samples were then either immediately fixed and processed for confocal immunofluorescence, or co-culture media added back to the basal chamber and the samples incubated for 30 minutes at 37°C in a 5% CO2 atmosphere before fixation and processing.MDCK cells stably expressing human transferrin receptor were seeded on Transwell filters and allowed to polarize before use in assays. Polarized cells were left uninfected or infected with For quantification of transferrin signal, we randomly sampled 300 µm X 300 µm optical fields by confocal microscopy. Stacks containing the full thickness of the monolayers were acquired at 0.5 µm z-steps. The stacks were reconstructed in 3D and the fluorescence sum of the transferrin signal present in the monolayers was measured. The background fluorescence was calculated from voxels imaged below the monolayers in each sample. Statistical differences between the data sets were determined by non-parametric Mann-Whitney test.Lysates were prepared for Western blots as previously described Hp antibodies, and the fluorescence sum of the transferrin receptor signal associated with the microcolonies. For background measurements, regions on the apical cell surface of ∼20 µm3 with no bacteria were also measured for the fluorescence sum of the transferrin receptor signal present. Statistical differences between the data sets were determined by non-parametric Mann-Whitney test.Polarized monolayer samples were fixed and non-permeabilized, apical staining carried out as previously described 2, 10 mM HEPES, pH 7.4). Ringer's buffer containing 200 µg/ml sulfo-NHS-SS-biotin (Pierce) was added to either the basal or apical chambers for selective basal vs. apical membrane protein biotinylation 2 atmosphere before fixation and processing. Non-permeabilized, apical staining with Alexa Fluor 488-conjugated streptavidin (Molecular Probes) was carried out as described previously Polarized monolayers were rinsed 3 times with Ringer's buffer  and a control enhanced green fluorescent protein (eGFP) Silencer siRNA (#AM4626) were also obtained from Ambion. siRNAs were transfected into MDCK cells using a reverse transfection protocol with Lipofectamine RNAiMAX Transfection Reagent (Invitrogen). For each well to be transfected in a 6-well plate, 50 pmoles of RNAi duplex and 7.5 µl of Lipofectamine RNAiMAX Transfection Reagent were gently mixed in 500 µl of OPTI-MEM I Reduced Serum Medium. The mixture was incubated at room temperature for 20 minutes, and 5×105 cells suspended in 2 ml of DMEM + 5% FBS added to the mixture. The cells were then incubated at 37°C in a 5% CO2 atmosphere for 1–3 days. Samples were collected for Western blotting as previously described siRNAs directed against canine transferrin receptor were designed by Ambion, Applied Biosystems Inc., using their Hp infection of polarized cells transfected with siRNA, cells reverse-transfected with siRNA as above were trypsinized after a 24 hour incubation at 37°C in a 5% CO2 atmosphere, and seeded at high confluent density onto Transwell filters. 30 hours after seeding, infection of the polarized epithelial cells was then carried out as described earlier for the Transwell Hp growth assays, with DMEM present in both the apical and basal chambers. Quantification of microcolony sizes was carried out as previously described For Hp from the apical side for 2 days. After this time, the media in the Transwell basal chamber was replaced with co-culture media containing 50 µg/ml biotinylated transferrin (Invitrogen) and 20 µg/ml biotinylated albumin (Sigma). The basal chamber contains 1.5 ml of media, while the apical chamber contains 0.5 ml of media. After a 24 hour incubation at 37°C in a 5% CO2 atmosphere, the media from the apical chamber was collected and diluted 1∶1 in 2X SDS-sample buffer. Samples were separated on a SDS-PAGE gel, and transferred to nitrocellulose membranes for immunoblotting. Detection of biotinylated transferrin and biotinylated albumin was carried out by blotting with streptavidin conjugated to Alexa Fluor 660 (Molecular Probes), and scanning on a LI-COR Odyssey Scanner (LI-COR Biosciences). The detection limit and linear range of measurements of the biotinylated transferrin and biotinylated albumin were determined from standard curves generated by use of a dilution series of the co-culture media containing 50 µg/ml biotinylated transferrin and 20 µg/ml biotinylated albumin, diluted 1∶1 in SDS-sample buffer.MDCK cells stably expressing human transferrin receptor were seeded on Transwell filters and allowed to polarize before use in assays. Co-culture media was added basally, and DMEM apically. Polarized cells were left uninfected or infected with Hp strain 7.13 WT or a 7.13 ΔcagA mutant, and sacrificed 6–8 weeks post-inoculation Mongolian gerbils (Harlan Laboratories) were placed on a regular, iron-replete diet (Modified TestDiet AIN-93M with 250 ppm iron), or an iron-deficient diet (Modified TestDiet AIN-93M with no iron) , for 3 weeks prior to infection. Animals were then inoculated with either Figure S1Hp growth in the absence of host cells. (A) Addition of iron to DMEM is not sufficient to support Hp growth in liquid culture. Plate-grown WT was used to inoculate co-culture media (solid black line), DMEM (red line) or DMEM containing 100 µM ferric chloride , in the absence of host cells. Samples were taken over time and plated for CFU counts. (B) Partially saturated transferrin can inhibit Hp growth in broth. Plate-grown WT was used to inoculate co-culture media (solid black line), DMEM (red line) or co-culture media containing 75 µg/ml partially saturated transferrin , in the absence of host cells. Samples were taken over time and plated for CFU counts. (C) Addition of holotransferrin to DMEM is not sufficient to support Hp growth in liquid culture. Plate-grown WT was used to inoculate co-culture media (solid black line), DMEM (red line) or DMEM containing 75 µg/ml holotransferrin , in the absence of host cells. Samples were taken over time and plated for CFU counts.Addition of iron to DMEM is not sufficient to support (TIF)Click here for additional data file.Figure S2cagA. Co-culture media (+) was added basally and DMEM (−) added apically. Samples were taken daily from the apical chamber and plated for CFU counts.Characterization of MDCK cells stably expressing human transferrin receptor. (A) Basolateral localization of transferrin receptor. Fluorescent transferrin (red) was added to the basal chamber of a polarized monolayer of MDCK cells stably expressing human transferrin receptor, and incubated for 30 minutes on ice before fixation. A cross section through the monolayer is shown. Scale bar 10 µm. (B) MDCK cells stably expressing human transferrin receptor were polarized on Transwell filters and infected with WT or Δ(TIF)Click here for additional data file.Figure S3Hp colonization affects Caco-2 cell transferrin internalization. Polarized Caco-2 cells were infected for 18 hours with WT or ΔcagA. Fluorescent transferrin was added to the basal chamber and incubated on ice for 30 minutes, unbound transferrin washed away, then further incubated for 30 minutes at 37°C to allow uptake of bound transferrin. The graph shows quantitative data of the transferrin fluorescence signal at 30 minutes post-uptake, determined from multiple 3D confocal images. p-value was obtained with a Mann-Whitney statistical test. (B and C) Differences in WT vs. ΔcagA effects on host cell transferrin internalization are not due to differences in bacterial numbers. Polarized MDCK cells stably expressing human transferrin receptor in the Transwell system, with 100 µM ferric chloride added to the apical chamber, were infected for 2 days with WT or ΔcagA. Fluorescent transferrin uptake assay was carried out as in (A). The transferrin fluorescence signal was quantified at 30 minutes post-uptake (B). p-value was obtained with a Mann-Whitney statistical test. 3D confocal images of the monolayers at 30 minutes post-uptake of fluorescent transferrin (red) are shown in (C). Bacteria are visualized with anti-Hp antibodies (green). Scale bar 20 µm.CagA-dependent increase of internalized transferrin occurs in multiple polarized epithelial lines. (A) (TIF)Click here for additional data file.Figure S4Hp to host cells does not result in transferrin receptor mislocalization. Polarized MDCK cells in the Transwell system were infected with WT for 5 minutes and fixed immediately. Apical staining with anti-transferrin receptor antibodies was carried out on non-permeabilized samples. Bacteria are visualized with anti-Hp antibodies (blue), transferrin receptor (tfR) is stained red, and phalloidin staining of f-actin is shown in yellow. 3D confocal images are shown. Scale bar 5 µm. (B) Not all basolateral proteins are mislocalized to bacterial microcolonies. Polarized MDCK cells on Transwell filters were infected with WT for 2 days, then fixed and stained with antibodies against E-cadherin (red) either from the apical surface without permeabilization (top panels), or with permeabilization . Bacteria are stained with anti-Hp antibodies (blue), and phalloidin staining of f-actin is shown in yellow. Scale bars 5 µm.Mislocalization of transferrin receptor to sites of bacterial microcolonies is specific. (A) Initial attachment of (TIF)Click here for additional data file.Figure S5cagA, or ΔvacA for 18 hours. Apical staining with anti-transferrin receptor antibodies was carried out on non-permeabilized samples. Bacteria are visualized with anti-Hp antibodies (blue), transferrin receptor (tfR) is stained red, and phalloidin staining of f-actin is shown in yellow. 3D confocal images are shown, and cross-sectional view is also presented for WT (second row). Scale bars 5 µm.Apical mislocalization of transferrin receptor to sites of bacterial microcolonies occurs in multiple cell types. Polarized Caco-2 cells in the Transwell system were infected with WT, Δ(TIF)Click here for additional data file.Figure S6cagA, ΔvacA, or ΔcagAΔvacA for 2 days were selectively biotinylated on ice at the basolateral surface, then incubated for 30 minutes at 37°C before fixation and apical streptavidin staining. Each point represents the total fluorescence intensity of apically-exposed biotin associated with a microcolony, divided by the number of bacteria present in that microcolony. p-values were obtained with a Mann-Whitney statistical test.CagA and VacA both contribute to mislocalization of basolateral proteins to the apical surface at sites of bacterial attachment. Polarized cells infected apically with WT, Δ(TIF)Click here for additional data file.Figure S7cagA, ΔvacA, and ΔcagAΔvacA mutants grow as well as WT in the presence of nutrients. Polarized MDCK cells in the Transwell system were infected with WT, ΔcagA, ΔvacA, or ΔcagAΔvacA. Co-culture media (+) was added both apically and basally. Samples were taken daily from the apical chamber and plated for CFU counts.Δ(TIF)Click here for additional data file.Figure S8Hp microcolony sizes on MDCK cells stably expressing human transferrin receptor, transfected with siRNAs directed against canine transferrin receptor (ctfR) or eGFP as a control. Data from 0 and 2 days post-infection are shown. Microcolony sizes were determined from multiple 3D confocal images. Each point on the graph represents a microcolony. p-values were obtained with a Mann-Whitney statistical test. N.S. indicates no statistical significance.Effect of canine transferrin receptor knockdown is specific. (A) siRNA against canine transferrin receptor is specific. MDCK cells stably expressing human transferrin receptor were mock transfected, or transfected with siRNA against canine or human transferrin receptor (tfR). After polarization, fluorescent human transferrin (red) was added to the basal chamber, and incubated for 30 minutes on ice before fixation. Cross sections through the monolayers are shown. Nuclei are stained with DAPI (blue), phalloidin staining of f-actin is shown in yellow, and cellular tight junctions are visualized with anti-ZO-1 (green). Scale bar 10 µm. (B) Quantification of (TIF)Click here for additional data file.Figure S9Detection limit and linear range of biotin-albumin and biotin-transferrin measurements. Samples of co-culture media containing biotin-albumin (range 0.4 ng to 10 ng) and biotin-transferrin (range 1 ng to 25 ng) were loaded and separated by SDS-PAGE and transferred to a nitrocellulose membrane. The membrane was probed with Alexa-fluor 647-conjugated streptavidin and bands visualized by the LI-COR Odyssey Scanner and quantified. Arbitrary units were used for the integrated intensity graph. The data was plotted as integrated intensity minus the background (bkg). Best-fit linear curves for the data are shown, as are the linear formulas and their fit.(TIF)Click here for additional data file.Figure S10in vitro growth curves of Hp strain 7.13 WT and its isogenic ΔcagA mutant. (A) Dietary iron restriction leads to decreased iron levels. Mongolian gerbils were maintained on a regular, iron-replete diet, or on an iron-deficient diet for 3 weeks prior to WT Hp infection, and throughout the course of the 6-week infection. Liver samples were analyzed by inductively coupled plasma-mass spectrometry . p-values were obtained with a Mann-Whitney statistical test. (B) Hp strain 7.13 WT and its isogenic ΔcagA mutant grow equally well in nutrient-rich broth. Growth of Hp strain 7.13 WT and ΔcagA in Brucella broth + 10% FBS was followed over 30 hours by measurement of optical density at 600 nm (OD600). Similar results were obtained in 3 independent experiments.Iron depletion of Mongolian gerbils and (TIF)Click here for additional data file.Figure S11vacA restores VacA expression. Lysates of Hp grown on Columbia blood agar plates were separated by SDS-PAGE, then either stained with Coomassie Blue or transferred to a nitrocellulose membrane and immunoblotted with polyclonal antibodies against VacA. VacA* is the complemented ΔvacA mutant. (B) CagA and VacA expression is not affected by deletion of vacA and cagA respectively. Lysates of Hp grown on Columbia blood agar plates were separated by SDS-PAGE, then either stained with Coomassie Blue or transferred to a nitrocellulose membrane and immunoblotted with polyclonal antibodies against CagA or VacA.Verification of mutants by immunoblot. (A) Complementation of Δ(TIF)Click here for additional data file.Figure S12Hp during infection in the Transwell system. (A) Hp colonizing the polarized epithelium express CagA and VacA. Polarized MDCK cells in the Transwell system were infected with WT. Free-swimming bacteria were washed away with DMEM and lysates of infected cells were collected at 2 or 5 days post-infection, separated by SDS-PAGE, then either stained with Coomassie Blue or transferred to a nitrocellulose membrane and immunoblotted with antibodies against CagA or VacA. (B) Hp colonizing the polarized epithelium deliver VacA into the host cells. Polarized MDCK cells in the Transwell system were infected with WT for 2 days, then fixed and stained with antibodies against Hp (green) and VacA (red). Phalloidin staining of f-actin is shown in blue. Scale bar 10 µm.CagA and VacA expression by (TIF)Click here for additional data file.


PRNP) and many of these generate infectious prions as evidenced by their experimental transmissibility by inoculation to laboratory animals. However, some, and in particular an extensively studied type of Gerstmann-Sträussler-Scheinker syndrome (GSS) caused by a PRNP A117V mutation, are thought not to generate infectious prions and instead constitute prion proteinopathies with a quite distinct pathogenetic mechanism. Multiple attempts to transmit A117V GSS have been unsuccessful and typical protease-resistant PrP (PrPSc), pathognomonic of prion disease, is not detected in brain. Pathogenesis is instead attributed to production of an aberrant topological form of PrP, C-terminal transmembrane PrP (CtmPrP). Barriers to transmission of prion strains from one species to another appear to relate to structural compatibility of PrP in host and inoculum and we have therefore produced transgenic mice expressing human 117V PrP. We found that brain tissue from GSS A117V patients did transmit disease to these mice and both the neuropathological features of prion disease and presence of PrPSc was demonstrated in the brains of recipient transgenic mice. This PrPSc rapidly degraded during laboratory analysis, suggesting that the difficulty in its detection in patients with GSS A117V could relate to post-mortem proteolysis. We conclude that GSS A117V is indeed a prion disease although the relative contributions of CtmPrP and prion propagation in neurodegeneration and their pathogenetic interaction remains to be established.Prions are infectious agents causing fatal neurodegenerative diseases of humans and animals. In humans, these have sporadic, acquired and inherited aetiologies. The inherited prion diseases are caused by one of over 30 coding mutations in the human prion protein (PrP) gene  associated with the substitution of valine for alanine at amino acid position 117 (GSS A117V) of the human prion protein gene have not. This has led to the suggestion that such syndromes are not true prion diseases and are better designated non-transmissible proteinopathies. Since prions may transmit more efficiently when the host's normal prion protein amino acid sequence matches that of the infecting prion, we generated transgenic mice expressing human prion protein with the same amino acid sequence found in A117V GSS. We found that brain tissue from GSS A117V patients could transmit disease to these mice, producing the typical brain lesions associated with GSS A117V. We therefore conclude that GSS A117V is an authentic prion disease. Sc) of host-encoded cellular prion protein (PrPC) is the principal, and possibly the sole, constituent of the transmissible agent or prion C onto pre-existing seeds or fibrils (for review see PRNP) According to the widely accepted “protein-only” hypothesis Sc, with pathogenesis mediated by aberrant production of CtmPrP, a transmembrane form of PrP Sc accumulation in other forms of prion disease may cause pathology by inducing the synthesis of CtmPrP de novoBy definition, prion diseases are transmissible, and while all the sporadic and acquired human prion diseases have been transmitted to laboratory animals, not all of the inherited forms have. It has been suggested therefore that some of these inherited neurodegenerative syndromes are prion proteinopathies with a distinct pathogenesis that may not involve production of infectious prions. One inherited prion disease (IPD) in particular, associated with an alanine to valine substitution at residue 117 of PrP (A117V), has been proposed to cause neurodegeneration in the absence of PrPPRNP sequencing A117V is one of the IPD mutations associated phenotypically with the Gerstmann-Sträussler-Scheinker syndrome (GSS) which usually presents clinically as a progressive cerebellar ataxia with dementia occurring later in a clinical course usually far more protracted than that of Creutzfeldt-Jakob disease (CJD) Sc, referred to as molecular strain typing Sc types have been reported associated with the same PRNP pathogenic mutation and this may in part explain phenotypic heterogeneity , designated Tg-31 (hereafter referred to as 117VV Tg31), with human PrP expression levels three times that of pooled normal human brain (data not shown). We studied an ageing cohort of 20 mice for evidence of spontaneous neurodegeneration, however all of these uninoculated mice died of intercurrent illnesses or old age between 460 and 904 days without developing neurological disease. In addition, three out of a further control group of five mice mock-inoculated with PBS buffer lived to between 344 and 735 days post-inoculation without developing neurological signs. One mouse was scored clinically sick at 303 days post inoculation  caused clinical disease in 2/6 mice with relatively short incubation periods of 263 and 303 days post-inoculation  and found that five brain samples analysed showed variable PK resistance  conditions of 100 µg/ml at 37°C for 1 hour . Of note, immunoblotting of the same samples in the absence of PK digestion showed that PrPC remained relatively stable in these samples (data not shown). These results strongly suggest that 117V PrPSc is significantly more labile than that seen in CJD and other human prion diseases. The remarkable difference in migration patterns between classical CJD-challenged -30 (designated 117VV Tg30), with the same three IPD A117V prion isolates in addition to one case of classical CJD and the same case of vCJD . We are currently investigating whether this pathology is transmissible on sub-passage.PRNP 129VV genotype We also investigated the pattern of neuropathology produced in vCJD-inoculated 117VV Tg30 mice. One of 3 positive samples showed abundant plaques in the cerebral cortex, hippocampus, thalamus and cerebellum . AlthougSc molecular signature to type 5 PrPScSc fragment size in vCJD-inoculated 117VV Tg30 mouse brain (Sc propagated in the vCJD-inoculated 129MM Tg45 control brain (lane 1) clearly shows that the 117V mutation on the valine-129 allele does not influence the previously established strain shift phenomenon. Interestingly, a truncated PrP peptide of about 8 kDa that is associated with GSS A117V mutation was also seen on longer exposure in PK-digested , 6/7 117VV Tg30 mice challenged with the same inoculum survived in the range of 627–811 days post-inoculation. These data suggest that very prolonged replication periods may be required for pathological PrP to become detectable in vCJD-challenged 117V transgenic mice by either IHC or immunoblotting. Subpassage of apparently negative brains could be used to explore this, however this is not a central part of this study.Sc in our study suggests that A117V-associated PrPSc is labile and readily susceptible to proteases. This results in progressive reduction of PrPSc to undetectable, yet still potentially infectious levels. In this regard, failure to detect low levels of PrPSc in the past from patient brain samples could be due to technical limitations of currently available biochemical techniques, rather than its absence.Our results may have wider implications for other inherited prion diseases that have not been shown to be transmissible as yet. Firstly, it is possible that demonstration of transmissibility of such inherited prion diseases would require specific transgenic models with over-expression of the relevant mutant human PrP, rather than endogenous levels of mutant PrP expression, if transmissibility is to be demonstrated within the lifespan of a mouse. The transient detection of PrPStorage and biochemical analysis of human tissue samples and transmission studies to mice were performed with written informed consent from patients or relatives under approval from the Local Research Ethics Committee of UCL Institute of Neurology/National Hospital for Neurology and Neurosurgery and the code of practice specified in the Human Tissue Authority licence held by UCL Institute of Neurology. Work with mice was performed under licence granted by the UK Home Office  Act 1986 ; Project Licence number 70/6454) and conformed to University College London institutional and ARRIVE guidelines.GTCGACCAGTCATTATGGCGAACCTT-3′5′- and reverse primer CTCGAGAAGACCTTCCTCATCCCACT-3′5′-. Restriction sites Sal I and XhoI (underlined) were introduced in the forward and reverse primers respectively for cloning. The sequence was confirmed and ligated into the cosmid vector CosSHaTet Prnp null mice 117V,129V+/+o/oPrnp)-30 (designated human PrP 117VV Tg30) and Tg-31 (human PrP 117VV Tg31) with mutant transgene expression levels of 2 and 3 times respectively, compared to pooled normal human brain levels.The 759 bp human PrP ORF was amplified by PCR with pfu polymerase from genomic DNA prepared from the brain of a patient with the inherited prion disease A117V mutation, using forward primer Sc) Strict bio-safety protocols were followed. Inocula were prepared, using disposable equipment for each inoculum, in a microbiological containment level 3 laboratory and inoculations performed within a class 1 microbiological safety cabinet. Ten mice per group of 117VV Tg31 transgenic mice were inoculated with prion isolates comprising human brain homogenates from: three separate IPD A117V cases; two sporadic CJD cases; three iatrogenic CJD cases; one case of vCJD and one mouse brain isolate from vCJD passaged once in Tg152 mice expressing wild-type human PrP V129 . All mice were thereafter examined daily for clinical signs of prion disease. Mice were killed if they exhibited any signs of distress or once a diagnosis of prion disease was established.The genotype of each mouse was confirmed by PCR of tail DNA prior to inclusion and all mice were uniquely identified by sub-cutaneous transponders. Disposable cages were used and all cage lids and water bottles were also uniquely identified by transponder and remained with each cage of mice throughout the incubation period. Mice were anaesthetised with a mixture of halothane and O2 asphyxiation. Brain was fixed in 10% buffered formol saline and then immersed in 98% formic acid for 1 hour and paraffin wax embedded. Serial sections of 4 µm thickness were pre-treated by boiling for 10 min in a low ionic strength buffer  before exposure to 98% formic acid for 5 min. Abnormal PrP accumulation was examined using anti-PrP monoclonal antibody ICSM 35  on a Ventana automated immunohistochemical staining machine  using proprietary secondary detection reagents  before development with 3′3 diaminobenzedine tetrachloride as the chromogen. Harris haematoxylin and eosin staining was done by conventional methods. Appropriate controls were used throughout.Mice were culled by COPreparation of brain homogenates , proteinase K digestion (titration up to 100 µg/ml for 1 h at 37°C), and subsequent western blotting was performed as described previously Figure S1H&E staining showing semi-quantitative scale used in scoring variable neuronal loss in the hippocampus of A117V-inoculated mice and PrP plaques in brain of a PBS-inoculated 117VV Tg31 mouse. (A) Coronal section of the anterior commissure on level Bregma +2 mm showing PrP deposits in a PBS-inoculated Tg31 mouse. a, location of PrP deposits within and surrounding the anterior commissure (ac). Other structures seen on this level are the piriform cortex (Pir), the anterior olfactory nucleus, medial part (AOM) and the forceps minor of the corpus callosum (fmi). b, high power magnification of the anterior commissure shows multiple small plaques within the white matter and immediately adjacent to it. Note the spongiform changes in the anterior commissure. Scale bar = 1200 µm (A) and 250 µm (B). (B) Upper row  shows progressive thinning of the neuronal layer of the hippocampus. Lower row  shows close up of the neuronal layer in each corresponding figure above, with blue arrows highlighting drop outs of neurones. Definition of values for neuronal loss: 0: No neuronal loss; +: Drop out of single neurones either focally or within the Ammon's horn (AH), leaving the AH continuity intact; ++: Focal or regional drop out, interrupting the continuity of the AH and creating a small-medium gap (up to 1/3 of the length of the AH); +++: Neuronal drop out leaving gaps of more than 1/3 of the AH's length. Scale bar = 500 µm for top panel and 125 µm for the bottom panel.(TIF)Click here for additional data file.Figure S2Immunoblot analyses of the brains of 117VV Tg31 mice challenged with GSS A117V prions showing time-course of degradation due to freeze-thawing. (A) Samples initially digested at 100 µg/ml PK at 37°C for 1 hour (lanes 3–8) showed variable digestion but the 8 kDa fragment was already visible (lanes 3 and 4). (B) Repeat immunoblotting performed on the same samples after 1 freeze-thaw, using the same PK digestion conditions, confirmed the 8 kDa fragment as the main detectable PrP fragment in brains of A117V-challenged Tg31 mice (lanes 3 and 4). All other fragments are almost completely degraded (lanes 3–8). The positive control used in lanes 1 and 2 of both panels A and B was from a transgenic mouse expressing wild type HuPrP-129MV challenged with sporadic CJD inoculum.(TIF)Click here for additional data file.


Identification of an inflammatory asthma phenotype currently requires sputum induction. This technique is invasive, has a high variability, is time-consuming and a burden for patients. Therefore, there is a strong need for a routine blood test to establish the inflammatory phenotype of asthma. We designed a clinical cohort study  in 115 asthma patients visiting the outpatient clinic to compare the results of such a blood test to sputum analysis. This abstracts focuses at the preliminary results in 21 patients.To assess whether expression of active FcgammaRII (CD32) and MAC-1( CD11b) on neutrophils and eosinophils in peripheral blood enables the diagnosis of an inflammatory phenotype of asthma.21 asthma patients were recruited at the outpatient clinic of the University Medical Centre Utrecht. Clinical parameters were gathered and FENO , sputum induction and blood tests were performed. Eosinophils and neutrophils in whole blood were stained with a FITC labeled antibody against active FcgammaRII receptor (clone A17) and a PE-labeled antibody against CD11b in the absence and presence of the activator fMLP (1 µM). Subsequently, fluorescence intensity was measured by flowcytometry.Expression of Mac-1 (CD11b) and activeFcgammaRII (CD32) on eosinophils at basal level and after stimulation (fMLP) showed refractory cells (low responsiveness for fMLP) in case of granulocytic asthma . FENO values were overall lower in paucigranulocytic asthma compared to granulocytic asthma .‘activation prone’ eosinophils migrated to the lung.Refractory blood eosinophils determined by a simple blood test were found in those patients that were more likely to suffer from granulocytic asthma . Therefore, paucigranulocytic asthma can be distinguished from other phenotypes purely based on blood analysis and FENO measurement. A low expression of activation epitopes on eosinophils in peripheral blood might indicate that


FH, the gene that encodes for the tricarboxylic acid TCA (TCA) cycle enzyme fumarate hydratase, are associated with an inherited form of cancer referred to as Hereditary Leiomyomatosis and Renal Cell Cancer (HLRCC). Individuals with HLRCC are predisposed to the development of highly malignant and lethal renal cell carcinoma (RCC). The mechanisms of tumorigenesis proposed have largely focused on the biochemical consequences of loss of FH enzymatic activity. While loss of the tumor suppressor gene von Hippel Lindau (VHL) is thought to be an initiating event for the majority of RCCs, a role for FH in sporadic renal cancer has not been explored. Here we report that FH mRNA and protein expression are reduced in clear cell renal cancer, the most common histologic variant of kidney cancer. Moreover, we demonstrate that reduced FH leads to the accumulation of hypoxia inducible factor- 2α (HIF-2α), a transcription factor known to promote renal carcinogenesis. Finally, we demonstrate that overexpression of FH in renal cancer cells inhibits cellular migration and invasion. These data provide novel insights into the tumor suppressor functions of FH in sporadic kidney cancer.Germline mutations of  Individuals with HLRCC are predisposed to the development of leiomyomas of the skin and uterus in addition to highly malignant and lethal RCC. The mechanisms of tumorigenesis proposed have largely focused on the biochemical consequences of loss of FH enzymatic activity. It has been proposed that loss of FH leads to fumarate accumulation and promotes a pseudohypoxic state in which hypoxia response pathways are aberrantly activated despite normoxic conditions VEGF and other growth factors that may be pro-tumorigenic when dysregulated. Pseudohypoxia has also been implicated in the most common variant of RCC, clear cell carcinoma (ccRCC), in which loss of VHL is a common genetic event Germline mutations of VHL loss is clearly critical to HIF-2α stabilization, alternate mechanisms, besides the prevention of degradation, may play a role in the maintenance of HIF-2α in renal cancer. Previous work by Block et al. established a role for reactive oxygen species (ROS) generated by NADPH oxidases in maintaining HIF-2α protein expression through an AKT-dependent mRNA translational mechanism in VHL-deficient cells VHL null renal carcinoma cells While FH mutations have primarily been linked to papillary type II renal cancer, a histologic variant that accounts for less than 10% of all renal cancers FH mutations have not been identified in sporadic clear cell renal cancer. However, a recent report has linked FH to the development of clear cell renal cancer in a patient with a germline mutation of FHFH in ccRCC has not examined. Therefore, we investigated the role of FH in sporadic clear cell renal cancer.FH in tumor tissue as compared to patient-matched normal renal tissue in specimens from the Cooperative Human Tissue Network (NCI). Quantitative real time RT-PCR demonstrated reduced FH mRNA levels in tumor tissue as compared with normal tissue  demonstrated a greater than twofold reduction in FH mRNA levels in tumor samples relative to normal control. Overall, the average reduction in FH mRNA levels was 2.9 fold. This difference was determined to be statistically significant. These results demonstrate reduced expression of FH at the mRNA and protein levels in ccRCC.FH expression in ccRCC has yet to be explored. Therefore, we first examined the protein expression of FH in a panel of human clear cell renal tumors and patient-matched normal renal parenchyma. Immunoblot analysis of tissue lysates demonstrated a marked reduction of FH protein levels in the tumors as compared to normal adjacent tissue . To confl tissue . AnalysiVHL null cell lines. However, we found that siRNA-mediated knockdown of FH resulted in a further increase of HIF-2α protein levels in two ccRCC lines which are VHL null (786-O and A498)  . In suppnd A498) . TogetheTo further elucidate the mechanism by which FH regulate HIF-2α protein expression, we created stable cells lines overexpressing FH-FLAG in VHL-deficient 786-O cells. We identified 3 clones that stably expressed the FH-FLAG construct. All 3 clones demonstrated reduced HIF-2α levels as compared with control vector transfected cells . We initVHL null cells through a translational mechanism FH in both 786-O and A498 cells results in increased AKT phosphorylation on serine 473 of AKT  in gene regulation where reports suggests that genes involved in oxidative phosphorylation may be subject to regulation by miRNAs In this report, we demonstrate for the first time reduced FH expression at the mRNA and protein levels in clear cell renal carcinoma, the most common histological variant that accounts for the majority of kidney cancers. These findings are significant as previous studies did not identify FH into a FH deficient tumor cell line results in a marked reduction in nuclear HIF-1α levels et al. demonstrated that elevated cellular reactive oxygen species, mediated by p22-phox based Nox oxidases, maintain HIF-2α protein levels in RCC cells through an AKT/4E-BP1 mRNA translational dependent mechanism et al. found that AKT activation, via signaling through the mTOR signaling complex 2 (mTORC2), was required to maintain HIF-2α in VHL null cells We have previously demonstrated that reintroduction of wild type et al. indicates that loss of extramitochondrial FH may contribute to HIF stabilization in vitro and in vivo efficacy of a dual PI3K/mTOR inhibitor in RCC The mechanism by which AKT signaling is activated by FH loss is under current investigation. We have previously demonstrated that loss of FH in renal epithelial cells results in elevated cellular oxidative stress SDHB, SDHC, and SDHD  SDH subunit mutations have been linked to pheochromocytoma and paraganglioma and more recently to gastrointestinal stromal tumor (GIST) et al. demonstrated reduced expression of succinate dehydrogenase subunit B (SDHB) in a subset of pheochromocytomas SDHB or other genes commonly mutated in GISTs including KIT and PDGFRAet al. proposed that oxygen consumption via mitochondrial metabolism may regulate tumor growth by limiting the availability of oxygen for non-mitochondrial activities that are contributory to tumor growth Interestingly, there is precedent for alterations of TCA cycle enzyme in cancer. Multiple other genes encoding enzymes of the tricarboxylic (TCA) cycle are considered tumor suppressor genes including et al. that demonstrated that fh knockdown in immortalized mouse embryonic fibroblasts (iMEFS) resulted in increased motility as compared with untransduced iMEFS et al. demonstrated that HIF-2α knockdown in 786-O cells reduced the expression of multiple integrins that may mediate cell motility and invasion et al. examined the role of VHL loss in cell invasion VHL into VHL-deficient 786-O cells reduced HIF-2α levels and cell invasion. Conversely, re-expression of HIF-2α in VHL reconstituted 786-O cells restored invasive potential. These data indicate a role for HIF-2α in RCC migration and invasion. Additionally, HIF-2α overexpression was found to enhance the growth of RCC xenografts whereas overexpression of HIF-1α was found to inhibit xenograft growth VHL null tumor xenografts Of significant interest is our finding that FH overexpression results in reduced migration and invasion of RCC cells. Our data are in concordance with a recent report by Costa Despite the recent approval of multiple agents for advanced renal cancer, most patients with advanced renal cancer will eventually succumb to their disease. Hence, the identification of novel signaling pathways will be critical to the development of effective therapeutics. There is now mounting evidence that renal cancer is among the tumors that are representative of the emerging paradigm in cancer biology of metabolic links to malignancy. Our findings with FH suggest a metabolic reprogramming in clear cell renal cancer that promotes expression of tumorigenic factors including HIF-2α via multiple mechanisms. Unraveling the mechanisms by which tumor metabolism is altered and the downstream cellular consequences should provide deep insight in renal cancer biology as well as novel therapeutic strategies.2 atmosphere. RCC4 cells were kindly provided by P. Ratcliffe (Oxford).A498, 786-O, and ACHN cells were obtained from the American Type Culture Collection and maintained in DMEM supplemented with 10% heat-inactivated fetal bovine serum at 37°C in a humidified 5% COLY294002 was purchased from Sigma.The FH-FLAG construct has been previously described All immunoblot analyses were performed as previously described Ct method with normalization to 18S rRNA levels. As a statistical test we used the Mann-Whitney paired non-parametric test to compare FH expression in tumor versus normal adjacent tissue .Biospecimens for RNA analysis were obtained from the Cooperative Human Tissue Network of the NCI/NIH. Total RNA was reverse transcribed using the High-Capacity cDNA Archive kit (Applied Biosystems). cDNA was then used as template with Applied Biosystems' assays-on-demand 20× assay mix of primers and Taqman probes. Forty amplification cycles were done on the Applied Biosystems Prism 7900 sequence detector. Fold change values between tumor and normal samples were calculated using the ΔTumor samples and normal corresponding tissue from patients with RCC were obtained from the Department of Urology at the University of Texas Health Science Center at San Antonio. The tumors for this study were histologically classified as clear cell renal carcinoma by a genitourinary pathologist. The collection and handling of human samples was performed according to a protocol approved by the Institutional Review Board of the University of Texas Health Science Center at San Antonio. Based on the protocol, samples were obtained in a deidentified fashion from patients undergoing surgical resection for renal cancer. As samples were obtained and analyzed in an anonymous fashion, patient consent was not required.Cells were allowed to grow to near confluence in 60 mm dishes. A uniform scratch was then made down the center of the plate using a 200 microliter micropipette tip, followed by washing twice with PBS. The same marked field of the scratch wound was photographed using an Olympus light microscope (4× objective) at the indicated time points. The width of the scratch wound was measured at three different areas with Q-Capture pro software. Quantified data represent the mean +/− S.D. from at least two independent experiments.4) were seeded on an 8 µM pore size Thin-cert for 24 well plates (Greiner Bio-One) in serum free media. Seven hundred fifty microliters of 10% FBS medium was added to the bottom chamber as chemotractant. After 48 hrs, cells on the top of the membrane were removed with a cotton swab. The migrated cells at the bottom side were washed with PBS, fixed with 70% ethanol and stained using 0.1% Crystal violet to visualize the migrated cells. Migrated cells attached to the lower side of the membrane were enumerated using a light microscope at 10× magnification. Counts represent the average cell number of ten microscopic fields.786-O subclones cells (1×10Cell invasion was determined by invasion assay (membrane coated with a layer of Matrigel extracellular matrix proteins) according to the manufacturer's instructions. Cells were seeded in serum-free medium into the upper chamber and invaded toward the bottom chamber containing a 10% FBS medium as the chemotractant. Membranes were processed in a similar fashion as the migration assay.For FH and HIF-2α knockdown, cells were transfected with pooled siRNA reagent (Thermo Fisher) with the Amaxa Nucleofector system according to the manufacturer's protocol. Cells were harvested at 48–72 hours following transfection. A non-targeting scramble siRNA pool was used as a negative control (Thermo Fisher).


Bemisia tabaci species complex have the ability to transmit a large number of plant viruses and are some of the most detrimental pests in agriculture. Although whiteflies are known to transmit both DNA and RNA viruses, most of the diversity has been recorded for the former, specifically for the Begomovirus genus. This study investigated the total diversity of DNA and RNA viruses found in whiteflies collected from a single site in Florida to evaluate if there are additional, previously undetected viral types within the B. tabaci vector. Metagenomic analysis of viral DNA extracted from the whiteflies only resulted in the detection of begomoviruses. In contrast, whiteflies contained sequences similar to RNA viruses from divergent groups, with a diversity that extends beyond currently described viruses. The metagenomic analysis of whiteflies also led to the first report of a whitefly-transmitted RNA virus similar to Cowpea mild mottle virus (CpMMV Florida) (genus Carlavirus) in North America. Further investigation resulted in the detection of CpMMV Florida in native and cultivated plants growing near the original field site of whitefly collection and determination of its experimental host range. Analysis of complete CpMMV Florida genomes recovered from whiteflies and plants suggests that the current classification criteria for carlaviruses need to be reevaluated. Overall, metagenomic analysis supports that DNA plant viruses carried by B. tabaci are dominated by begomoviruses, whereas significantly less is known about RNA viruses present in this damaging insect vector.Whiteflies from the  Aleyrodidae), in particular the Bemisia tabaci species complex, are among the most detrimental insect vectors causing considerable economic losses to multiple agricultural industries The majority of vectored plant viruses are transmitted by hemipteran insects, whose piercing-sucking mouthparts allow efficient transmission Begomovirus (Geminiviridae) and a small diversity of RNA viruses have been associated with whiteflies. Whiteflies are known to transmit more than 280 begomovirus species Begomovirus as the group of plant viruses with the largest number of recognized species Crinivirus , Carlavirus , Ipomovirus , and Torradovirus  Among the large diversity of viral types known to infect plants, only DNA viruses belonging to the genus B. tabaci may instead reflect methodological limitations that are only capable of detecting close relatives of known vector-transmitted viruses . In addition, agricultural surveillance efforts typically focus on viral species that negatively affect economically important crops. Therefore it is likely that viruses present in native vegetation that do not show any impact on agricultural crops in the area will be overlooked. Since viruses found in native vegetation may emerge as serious pathogens for crops and asymptomatic hosts may facilitate virus spread by serving as reservoirs a priori knowledge of the plant pathogens present in a given area It is possible that the diversity of whitefly-transmitted viruses reported to date does not accurately represent the total complement of viruses in this insect vector. Viral types known to be carried by B. tabaci specimens collected from an experimental field site in Florida. Begomoviruses were the only DNA plant viruses detected, whereas known and novel RNA viruses from different families were found in whiteflies from this single field site. Furthermore, sequencing efforts resulted in the detection and first report of a whitefly-transmitted carlavirus most similar to Cowpea mild mottle virus (CpMMV) in North America. Although the CpMMV Florida isolate was originally detected in whiteflies, it was subsequently identified in wild and cultivated plants from the same area and its host range was experimentally determined. Analysis of the CpMMV Florida genome suggests that the current classification criteria for carlaviruses need to be reevaluated.In an effort to shed light on the total diversity of viruses carried by whiteflies, this study incorporated high-throughput sequencing into the VEM approach to detect DNA and RNA viruses in B. tabaci specimens used for viral metagenomics were collected in an experimental field in Citra, Florida (29°24′N 82°06′W) in August 2007 as previously described The 4, 0.1 M NaCl, pH 7.5) using a bead-beater (BioSpec) with 1.0 mm glass beads  for 1 min. Cells were removed from homogenates by centrifuging at 10,000 xg for 10 min and filtering the supernatant through a 0.22 µm Sterivex filter (Millipore). Virus particles present in the filtrate were treated with 0.2 volumes of chloroform, followed by DNase I (2.5 U/µl) and RNase A (0.25 U/µl) treatment at 37°C for 3 hrs to remove non-encapsidated nucleic acids.Virus particles were partially purified from the whiteflies before nucleic acid extraction and sequencing. For this purpose, approximately 250 whiteflies were homogenized in SM Buffer  following manufacturer's instructions and sequenced individually. For the RNA fraction, the ‘on-column DNase digestion’ step was used to minimize DNA carryover. The extracted DNA fraction was amplified using the GenomiPhi V2 DNA Amplification Kit  followed by further amplification and fragmentation using the GenomePlex Whole Genome Amplification (WGA) Kit (Sigma-Aldrich). The RNA fraction was amplified using the TransPlex Whole Transcriptome Amplification (WTA) Kit (Sigma-Aldrich). WGA- and WTA-amplified nucleic acids were used for next-generation sequencing using a single lane of a Genome Analyzer IIx System (Illumina) by multiplexing.http://edwards.sdsu.edu/cgi-bin/tagcleaner/tc.cgi) http://metavir-meb.univ-bpclermont.fr/) under the project ‘Whiteflies_Citra_2007’. Sequences (1.4 million from the DNA library and 2.1 million from the RNA library) were then assembled with a minimum identity of 95% over 25 bp using the Geneious software package (Biomatters). Contigs over 80 bp in length were compared against the GenBank non-redundant database using either BLASTn (DNA library) or BLASTx (RNA library) with an e-value cut-off of E<0.001 in June 2011 WGA and WTA adapter sequences as well as multiplexing barcodes were removed from the DNA and RNA sequence libraries, respectively, using the TagCleaner server , 72°C for 1.5 min, followed by a final extension at 72°C for 8 min. The 5′ end of the genome was completed using gene-specific primers with the 5′ RACE System Kit (Invitrogen) according to manufacturer's instructions . Primer pairs were designed to bridge the gaps between contigs and primer pairs that spanned the entire genome were used to complete the genome . cDNA foructions . All PCRFabaceae family which are known hosts of CpMMV, namely peanuts , hairy indigo , and dixie ticktrefoil (Desmodium tortuosum (Sw.) DC.; n = 5). All plant tissues were tested for the presence of CpMMV using a CpMMV-specific ELISA Reagent Set (Neogen Europe Ltd) in accordance with manufacturer's protocols. Samples were considered positive when their absorbance values were greater than the mean of the negative controls plus three standard deviations. Positive samples were verified through a degenerate carlavirus RT-PCR assay targeting part of the capsid protein (CP) and 3′end poly-A tail of these RNA genomes 5′GGBYTNGGBGTNCCNACNGA3′) and oligo-dT (21) primers under the following conditions: 0.5 ul cDNA, Taq DNA Polymerase (New England Biolabs), 1× standard Taq (Mg-free) buffer, 3.0 mM MgCl2, and 1 µM spermidine. Amplification was performed with an initial denaturation at 94°C for 5 min, followed by 35 cycles of 94°C for 1 min, 50°C for 1 min, and 72°C for 1 min ending with a final extension at 72°C for 5 min.To investigate the presence of the carlavirus CpMMV in the vegetation, 90 plants were surveyed in the summer of 2011 from an area around the field site in Citra, FL where whiteflies were originally collected. Leaves exhibiting a variety of viral infection symptoms  were collected from plants belonging to the D. tortuosum plant sample that tested positive for CpMMV by ELISA was used to establish a culture and obtain inoculum for transmission and host range determination experiments. The sample of D. tortuosum was mechanically inoculated to Chenopodium quinoa L., which is an established local lesion host for CpMMV 2HPO4, 100 mM Na2HPO4, 10 mM Na2SO3, pH 7.4). Eight days later, chlorotic local lesions were observed on inoculated leaves of C. quinoa. Four of these lesions were removed and individually mechanically inoculated to primary leaves of common bean (Phaseolus vulgaris L. ‘Topcrop’) which is a known systemic host of some CpMMV isolates One B. tabaci . For this purpose, infected beans were placed in separate cages in different growth rooms for acquisition and transmission. Transmissions were performed at different times throughout the day to prevent contamination through whitefly carryover between rooms. Non-viruliferous whiteflies were placed on each infected bean and given an acquisition access period of 20 min. Whiteflies were then transferred to three healthy beans and given an inoculation access period of 4 hrs. Transmission was terminated using insecticidal soap (20 ml/L Safer Soap®) and Imidacloprid . The presence of CpMMV was confirmed in all three whitefly-inoculated beans by RT-PCR. The CpMMV genome was sequenced from each of these bean plants through PCR using the same primers used to sequence the CpMMV genome from whiteflies , the majority of which shared >88% nucleotide identity with their top match in the database  in the DNA library were dominated by begomoviruses , three of which  have not been detected in whiteflies previously  compared to the DNA library, the RNA sequences encompassed a broader viral diversity at the family level. The viral sequences identified in the RNA library had similarities to viruses from at least five different families (eviously . Most ofA. hypogea (n = 71), 36% of I. hirsuta (n = 14), and 100% of D. tortuosum (n = 5) plants tested positive for CpMMV. Note that infection symptoms observed in the field may not have been caused by CpMMV. Since all of the samples of D. tortuosum tested positive for CpMMV, an infected seedling from this species was used to establish a culture and obtain virus inoculum. This plant was used to mechanically infect C. quinoa and local lesions were subsequently inoculated into common bean plants (P. vulgaris). To confirm infection by CpMMV, the common bean plants were tested by ELISA and a degenerate carlavirus RT-PCR assay. Once the presence of CpMMV was confirmed, the CpMMV Florida isolate was successfully transmitted to three common bean plants using whiteflies. All three plants exposed to CpMMV-bearing whiteflies exhibited mild mottling symptoms and were verified as infected with CpMMV based on ELISA and RT-PCR.Since CpMMV-like sequences were abundant in whiteflies and this virus had never been reported in North America, a survey of 90 symptomatic plants from three different species using ELISA with a CpMMV antibody was conducted in the same crop field four years after the whitefly collection. Thirty-eight percent of Amaranthaceae, Chenopodeaceae, Cucurbitaceae, Fabaceae, Solanaceae). Ten of the 18 species tested were successfully infected by the CpMMV Florida isolate, all of which belonged to the Chenopodeaceae and Fabaceae families  encoding the following proteins from 5′ to 3′: replication polyprotein, movement proteins , capsid protein (CP) and nucleic acid binding (NB) protein  2CX12CX4C Searches in the Pfam database using predicted amino acid sequences for each of the six ORFs present in the CpMMV Florida genomes revealed significant matches  to conserved motifs observed in carlaviruses. The replication polyprotein contains four different domains characterized by viral methyltransferase a priori knowledge of the viral types present Begomovirus with high nucleotide identities (>88%) to known viral species. Although the short reads (maximum fragment size of 71 nt) hindered our ability to conclusively identify these begomoviruses to the species level even after assembly, results indicated that DNA viruses present in whiteflies from this site are dominated by members of a single genus. On the other hand, a diversity of RNA viral sequences from various families was detected and the VEM approach ultimately led to the discovery of the whitefly-transmitted carlavirus CpMMV in Florida, USA.The VEM approach has been introduced as a strategy to survey viruses carried by insect vectors in a given region without Since the VEM approach does not rely on sequence-specific primers/probes, this method should have recovered any type of DNA virus present in the whiteflies whose virions are resistant to chloroform and nuclease treatment. However, the use of a 0.22 µm filter during virus particle purification may have excluded larger non-plant DNA viruses which are commonly associated with insects . The fact that only begomoviruses were identified suggests that this group indeed dominates the whitefly-transmitted DNA plant viruses, and perhaps exclusively occupies this niche. Future studies investigating plant DNA viruses in whiteflies from different locations using sequence-independent methods are needed to confirm whether or not whiteflies have evolved an exclusive relationship with begomoviruses.Potyviridae), Crinivirus (Closteoviridae) and Carlavirus (Betaflexiviridae) genera, and icosahedral viruses from the Torradovirus genus (Secoviridae) In contrast to DNA viruses, the RNA library indicated that whiteflies from a single site can carry RNA viruses from disparate families. Currently, whiteflies are known to transmit four different groups of RNA viruses, including filamentous viruses from the Ipomovirus  in North America. This virus has been reported in a wide range of geographical areas including Africa, India, Asia, the Middle East, and South America, where it can infect and negatively impact important food crops Fabaceae. However, two of the isolates (CpMMV∶BR∶BA∶02 and CpMMV∶BR∶GO∶01∶1) were able to infect a member of the Solanaceae (Nicotiana glutinosa) which CpMMV Florida fail to infect. CpMMV∶BR∶BA∶02 and CpMMV∶BR∶GO∶01∶1 share 98% and 93% genome-wide pairwise identity with CpMMV Florida, respectively. Therefore CpMMV isolates may exhibit different host ranges despite high nucleotide identities. Further research examining both the full genomes and experimental host range of all available CpMMV isolates will provide insight into which genetic differences explain differences in host range.A recent report published during the review process of this manuscript described six novel CpMMV isolates from Brazil that are closely related to CpMMV Florida (see below) Betaflexiviridae (formerly known as Flexiviridae), distinct species share <72% nucleotide or <80% amino acid identity between the entire CP or replication genes The CpMMV genome recovered from whiteflies collected in 2007 is 99% identical to the genome isolated from vegetation in the same field four years later. The genome organization of the CpMMV Florida isolate is similar to other carlaviruses. Full genome comparisons between CpMMV isolates from Florida and Ghana, which were the only genomes available at the time when the analyses were performed, clearly show that the CP shares a much greater degree of identity (95%) than non-structural proteins in the genomes (∼62%) . This coUnfortunately, most of the available CpMMV sequences only encompass the 3′ end of the genomes, containing the CP and/or NB since available carlavirus-specific degenerate PCR assays target this region The biological significance of the highly conserved CP in CpMMV isolates is largely unknown; however, it may result from selective pressure of transmission driven by the whitefly vector. CpMMV isolates have been reported to be transmitted both non-persistently and semi-persistently as there are no latent periods for virus transmission in whiteflies but retention times vary from minutes to hours B. tabaci whiteflies is not nearly as complete as that of DNA viruses, which appear to be restricted to the genus Begomovirus. Our findings indicate that the range of RNA viruses found in whiteflies may not be limited to the four groups that have been described since viral sequences with low amino acid identities likely represent novel groups. In addition to expanding current knowledge regarding viruses that can be captured by whiteflies, the VEM approach allowed us to expand the geographical range of CpMMV by documenting its presence in North America. Genomic comparisons among CpMMV genomes suggest that the classification criteria for carlaviruses need to be reevaluated, especially when considering variants that cannot be serologically distinguished. Future studies need to establish criteria to classify CpMMV variants and pathotypes by comparing genomic features, symptoms, infectivity and host range.The results of this study demonstrate that current understanding of RNA viruses found in Table S1Primers used to amplify the CpMMV genome.(PDF)Click here for additional data file.Table S2Plant DNA viruses  and associated satellite DNAs identified in whiteflies.(PDF)Click here for additional data file.Table S3Amino acid pairwise comparisons among available Cowpea mild mottle virus (CpMMV) capsid proteins (CP).(PDF)Click here for additional data file.Table S4Amino acid pairwise comparisons among available Cowpea mild mottle virus (CpMMV) nucleic acid binding (NB) proteins.(PDF)Click here for additional data file.


The level of T cell activation in untreated HIV disease is strongly and independently associated with risk of immunologic and clinical progression. The factors that influence the level of activation, however, are not fully defined. Since endogenous glucocorticoids are important in regulating inflammation, we sought to determine whether less optimal diurnal cortisol patterns are associated with greater T cell activation.+ T cell count above 250 cells/µl. We assessed T cell activation by CD38 expression using flow cytometry, and diurnal cortisol was assessed with salivary measurements.We studied 128 HIV-infected adults who were not on treatment and had a CD4+ T cells . Participants with lower waking cortisol also showed a trend toward greater activation on CD8+ T cells . A greater diurnal decline in cortisol, usually considered a healthy pattern, correlated with less CD4+  and CD8+  activation.Lower waking cortisol levels correlated with greater T cell immune activation, measured by CD38 mean fluorescent intensity, on CD4These data suggest that the hypothalamic-pituitary-adrenal (HPA) axis contributes to the regulation of T cell activation in HIV. This may represent an important pathway through which psychological states and the HPA axis influence progression of HIV. T cell activation increases cell turnover, and the high level of T cell activation is thought to be one of the factors leading to depletion of CD4+ T cells The factors influencing T cell activation in HIV infection have not been fully identified. While some factors that may contribute to the level of activation have been identified, such as infection with co-pathogens and microbial translocation through damaged gut mucosa The hypothalamic-pituitary-adrenal (HPA) axis regulates secretion of glucocorticoids, endogenous hormones with potent anti-inflammatory properties. While increased secretion of glucocorticoids in periods of acute stress may prevent excessive immune activation chronic stress, may lead to decreased glucocorticoid sensitivity and impairment in the ability of the HPA axis to regulate the immune system Normal HPA physiology results in a diurnal rhythm of cortisol secretion characterized by a peak shortly after waking, a gradual decline over the course of the day, and a nadir one hour after the usual time of sleep A potential role of the HPA axis in HIV pathogenesis is supported by emerging observations linking stress and mood with disease outcome in lentiviral infections. For example, severe stressful life events, lack of social support, and chronic depressive symptoms are associated with accelerated progression of HIV The association between psychological stress and mood and HIV progression is thought to be mediated by molecular messengers of the HPA axis and autonomic nervous system (ANS), and prior research has identified pathways linking the sympathetic nervous system to HIV pathogenesis While there is evidence linking the sympathetic nervous system to accelerated progression of HIV, a clear mechanism linking the HPA axis to disease progression has not been established The study was reviewed and approved by the UCSF Committee on Human Research. We obtained written informed consent from all of the participants.+ T lymphocyte count >250 cells/µl, (3) plasma HIV-1 RNA>100 copies/ml, (4) antiretroviral therapy naive or untreated for at least four months, and (5) 18 or more years of age. Exclusion criteria included a plan to initiate antiretroviral therapy within 12 months from the time of enrollment and current use or use in the past 6 months of immunomodulator drugs.We performed a cross-sectional study of the relationship between glucocorticoids and T cell activation in HIV by analyzing baseline data from HIV-infected individuals in the University of California San Francisco Staying Well study. While participants went on to an intervention stage, the data presented here were all obtained prior to any study intervention. Inclusion criteria included (1) HIV-1 seropositivity, (2) CD4Subjects were recruited in the San Francisco area through an extensive recruitment network that included referrals from physicians, community based organizations, other research studies, and self referral generated by word of mouth, advertising, and community outreach. The study received approval from the UCSF Committee on Human Research, and all subjects gave written informed consent.We measured salivary cortisol by providing participants with a home saliva collection kit and instructions to collect three saliva samples each day over three days. In addition to providing a practical means of measuring cortisol while subjects perform normal daily activities, measurements of salivary cortisol more accurately reflect serum free cortisol concentrations than do measurements of serum total cortisol. The salivary cortisol concentration is independent of salivary flow Saliva samples were collected by having participants drool into collection tubes, which were frozen and stored at −20 C until analysis. Before biochemical analysis, samples were centrifuged at 3000 rpm for 5 minutes, resulting in a clear supernatant of low viscosity. Free salivary cortisol was analyzed with a commercially available chemi-luminescence-assay (CLIA) with a high sensitivity of 0.16 ng/ml .Blood was drawn between 8 am and noon, in acid citrate dextrose venous vacuum collection tubes. Peripheral blood mononuclear cells (PBMCs) were isolated from whole blood by Ficoll-Hypaque density gradient centrifugation within 6 hours of blood drawing, cryopreserved, and stored at the UCSF Biological Specimen Bank.+ T cell proliferation Several immunophenotypic markers can be used to quantify T cell activation in vivo, but CD38 expression on T cells is the marker that has shown the best characterized ability to predict disease progression in HIV We measured expression of CD38 and HLA-DR on T cells at the UCSF Core Immunology Laboratory using flow cytometry and previously described methods that have been optimized and validated for frozen PBMCs + and CD8+ T cells expressing each of the T cell markers. Combinations of markers were calculated in FlowJo using the Boolean gate function  to determine the proportion of CD4Plasma specimens were used to measure HIV-1 RNA copies per milliliter by polymerase chain reaction using the Amplicor HIV-1 Monitor Test, v1.5 (Roche Diagnostics).Raw cortisol data were screened for outliers thought to be due to contamination (e.g. blood) or poor participant compliance; samples with values ≥100 nmol/L were deleted. Sample 1 (waking sample) was excluded if collected >10 minutes after waking, sample 2 (30 min post-waking sample) if collected <20 minutes or >45 minutes after waking, and sample 3 (bedtime sample) if collected later than 3 am the following day.In addition to the cortisol variables representing specific time points across the day, two derived cortisol variables were calculated: the cortisol awakening response (CAR) and cortisol slope. The CAR, or increase in cortisol from waking to 30 min post-waking, was calculated as the difference score between samples 1 and 2 (sample 2 – sample 1). The CAR typically represents a 50% increase in cortisol levels and is larger in persons experiencing chronic worry and stress + counts were positively skewed and normalized using a logarithmic (log10) transformation. In computing derived variables, raw variables were logarithmically transformed prior to other calculations. Scatterplots were created and pairwise Pearson correlation coefficients calculated to quantify the association between cortisol predictor variables and immunologic outcome variables. Multiple linear regression was used to model T cell activation as a function of diurnal cortisol adjusted for age, viral load, and CD4+ count. All analyses were performed using Stata 10.Averages were computed for each cortisol variable across the three days of sample collection. Data for cortisol, viral load, and CD4+ T cell count at baseline was 469 cells/mm3 (IQR 372–585) and the median plasma HIV RNA level was 4.3 log10 copies/mL (IQR 3.7–4.7)  .+ count was negatively correlated with activation, as measured by CD38 MFI, on both CD4+  and CD8+  cells. Also consistent with previous work, there was a positive correlation between plasma HIV RNA levels and both CD4+ activation  and CD8+ activation .As expected, CD4+ T cell activation  and a trend toward greater CD8+ T cell activation   and CD8+ activation  after adjustment for age, CD4+ count, and viral load  . In the ral load .+ activation . This relationship was statistically significant following adjustment for age, CD4+ count, and viral load .The 30 minute post-waking cortisol is typically the highest cortisol value over the course of the day. In this study, lower 30 minute post-waking cortisol correlated with greater CD4+  and CD8+  cells. In addition, cortisol slope was a statistically significant predictor of CD4+ and CD8+ activation after adjusting for covariates  were correlated with greater CD38 MFI on both CD4variates .+HLA-DR+. +/CD8+ cells with the CD38+HLA-DR+ phenotype. Greater bedtime cortisol was correlated with greater HLA-DR+CD38+CD8+%, but this relationship was not statistically significant in the multivariate regression analysis. There was also a non-statistically significant trend toward flatter cortisol slope predicting greater T cell co-expression of HLA-DR/CD38. Overall, we found few associations between diurnal cortisol and percentage of cells co-expressing activation antigens.Finally, we examined T cell activation as measured by the percentage of T cells that were CD38+ and CD8+ T cell subsets after adjustment for covariates. To our knowledge, this is the first report of the relationship between diurnal cortisol patterns and immune activation in HIV+ subjects, and the results point to the HPA axis as an important regulator of immune activation in persons with HIV. These findings are particularly significant given the importance of immune activation in the pathogenesis of HIV infection, and imply that improvements in some of the stress-related cortisol patterns could decrease immune activation during HIV infection. Our findings add to an expanding literature supporting a link between stress, the neuroendocrine system, the immune system, and the overall risk of HIV disease progression.Psychosocial stress and mood affect disease outcomes in immune-related disorders such as HIV infection. Although some potential pathways linking these psychological states to disease progression have been identified (23), the mechanisms responsible for this association require further elucidation. Given the strong link between stress, mood, and the neuroendocrine system, we examined whether altered cortisol patterns are associated with greater T cell activation, a known risk factor for accelerated progression of HIV. We found that lower morning cortisol and flatter diurnal rhythms are associated with greater activation of both CD4The link between altered cortisol rhythmicity and greater immune activation may be explained by a state in which immune cells demonstrate impaired sensitivity to glucocorticoid signals, or relative glucocorticoid resistance. Studies have shown that chronic psychological stress correlates with impaired immune cell responses to anti-inflammatory signals, including down-regulation of glucocorticoid response elements in leukocytes Our ability to draw conclusions from this study is limited by its cross-sectional design. While our data show an association between diurnal cortisol and T cell activation, we cannot presently assert a causal relationship between predictor and outcome variables. We suspect that the observed associations are driven by the influence of glucocorticoids on immune function, but it is also conceivable that HPA function is altered in the setting of excessive immune activation. Furthermore, a bidirectional relationship may exist. Data from prospective studies are needed to elucidate the direction of this relationship. In addition, HIV-1 infection itself has also been linked to alterations in the HPA axis Though the statistically significant correlations between diurnal cortisol and immune activation are moderate, they are internally consistent and may be biologically significant. From previous work we know that immune activation in HIV is influenced by multiple factors, including plasma HIV RNA levels, bacterial translocation across altered gut mucosa, age, gender, and infection with co-pathogens such as CMV We chose to examine CD38 expression due to the evidence supporting its importance in predicting HIV disease progression As with most studies of basal cortisol, this study relies on self-report of times when measurements were taken and the relationship between measurement and waking time. To the extent that cortisol patterns truly influence T cell activation, this limitation is likely to cause noise in our predictor variable and underestimation of the true relationship. Some relationships in which we observed trends towards associations, such as increased cortisol awakening response and CD38 MFI on CD4+ and CD8+ T-cells, might not be statistically significant in part due to variability in the collection time of the 30 minute post-waking specimen. We invested considerable effort, however, to minimize errors in the data by screening all cortisol data for participant non-compliance and contacting participants to clarify sampling times when necessary. Given the widths of our confidence intervals and the care with which sampling was performed, we believe the lack of statistical significance in some of the hypothesized relationships does not rule out meaningful associations between particular measures of diurnal cortisol and T cell activation, but suggests that any actual associations are likely to be variable and/or of modest strength.The most unexpected finding in our study was the apparent difference in the associations between cortisol and our two measures of T cell activation (the density of CD38 on T cells versus the frequency of T cells co-expressing CD38/HLA-DR). There was a consistent association between the density of CD38 on T cells and diurnal cortisol rhythms, but similar associations appeared to be absent  between the percentage of cells expressing CD38/HLA-DR and cortisol. This contrast underscores the difference between relative mean fluorescence intensity and percentage measurements for quantifying levels of activation. Glucocorticoids may have more influence on the degree of immune activation than on the percentage of cells in an activated state.In conclusion, we have shown that cortisol patterns reflective of chronic stress  were correlated with greater T cell activation in the setting of HIV-1 infection. If supported by further data, these findings indicate a pathway through which psychological states and the HPA axis influence immune function in HIV and other immune-mediated diseases.