The inhibition of proton-gated current induced by WIN55,212-2 was almost completely blocked by the selective CB1 receptor antagonist AM 281, but not by the CB2 receptor antagonist AM630. Pretreatment of forskolin, an AC activator, and the addition of cAMP also reversed the inhibition of WIN55,212-2. Moreover, WIN55,212-2 altered acid-evoked excitability of rat DRG neurons and decreased the number of action potentials induced by acid stimuli. Finally, WIN55,212-2 attenuated nociceptive responses to injection of acetic acid in rats. These results suggest that WIN55,212-2 inhibits the activity of ASICs via CB1 receptor and cAMP dependent pathway in rat primary sensory neurons. Thus, cannabinoids can exert their analgesic action by interaction with ASICs in the primary afferent neurons, which was novel analgesic mechanism of cannabinoids.Local acidosis has been found in various pain-generating conditions such as inflammation and tissue injury. Cannabinoids exert a powerful inhibitory control over pain initiation via peripheral cognate receptors. However, the peripheral molecular targets responsible for the antinociceptive effects of cannabinoids are still poorly understood. Here, we have found that WIN55,212-2, a cannabinoid receptor agonist, inhibits the activity of native acid-sensing ion channels (ASICs) in rat dorsal root ganglion (DRG) neurons. WIN55,212-2 dose-dependently inhibited proton-gated currents mediated by ASICs. WIN55,212-2 shifted the proton concentration–response curve downwards, with an decrease of 48.6±3.7% in the maximum current response but with no significant change in the EC Tissue acidosis is a common factor found in various pain-generating conditions such as inflammation, ischemia, infection, tissue injury and tumor development Cannabinoids have been used for thousands of years to provide relief from suffering. Cannabinoids modulate nociceptive processing via their cognate receptors, cannabinoid receptor 1 and 2 (CB1 and CB2). CB1 receptors are constitutionally active and abundantly expressed in the nociceptive primary sensory neurons Genetic deletion of CB1 receptors further confirmed their role in cannabinoid-induced analgesia pH5.5) was evoked by application of a pH 5.5 solution for 5 s in the whole-cell patch-clamp configuration. Under our experimental conditions, three main types of acid-induced currents were observed on the basis of their amplitudes and inactivation time courses. A slow-inactivating transient inward current followed by a small sustained component was present in 56.3% (49/87) of neurons , an inward current , suggesting no effect WIN55, 212-3 on proton-evoked currents.In the majority of the neurons sensitive to acid stimuli , we observed that all three types of proton-evoked currents were inhibited by the pre-application of 10 agonist . The amppH5.5 further decreased when the concentration of WIN55,212-2 increased from 10−8 M to 10−7 M. pH5.5 decreased stepwise with an increase of WIN55,212-2 from 10−10 M to 10−6 M. The WIN55,212-2 caused the maximum effect  at concentration of 10−6 M. The results indicated that WIN55,212-2 inhibited the proton-gated current in a dose-dependent manner.We next investigated whether the inhibition of proton-gated currents was dependent on the concentrations of WIN55,212-2. −7 M). Seven to ten neurons were detected for each point of curves. It can be seen that (i) the concentration- response curve for proton with pretreatment of WIN55,212-2 was shifted downwards as compared with the control; (ii) the EC50 value in both curves was no statistical difference ; (iii) the maximal amplitude of proton-gated currents at pH 4.5 after pretreatment with WIN55,212-2 decreased to 48.6±3.7% as compared with the control ; and (iv) the threshold pH values of both the curves were basically the same.pH5.5 by pretreatment with WIN 55, 212-2 (10−7 M) was almost completely reversed by the addition of 10−6 M AM 281 . However, the suppression of WIN 55, 212-2 was not effected by the addition of the CB2 receptor antagonist AM630. WIN 55, 212-2 (10−7 M) had a 57.5±7.0% inhibiting effect on proton-gated currents, while WIN 55,212-2 caused also a 54.1±7.9% inhibition in the presence of the AM630 (10−6 M) .To verify whether the suppression of proton-gated currents by WIN55,212-2 was mediated by the CB receptor, we examined the effect of AM 281, a selective CB1 receptor antagonist, on the WIN 55,212-2-induced inhibition of proton-gated currents. As shown in −5 M) or 8-Br-cAMP(10−3 M). Suppression of IpH5.5 induced by WIN55,212-2 was 56.5±7.0% (n = 10) in the control experiment. In contrast, the suppression of IpH5.5 induced by WIN55,212-2 was 3.3±4.7% after treatment of forskolin , and 2.8±3.0% after treatment of 8-Br-cAMP .CB1 receptors belong to Gi/o protein-coupled receptor family, and the activation of the receptors leads to a cascade of events that inhibit adenylyl cyclase (AC) and therefore decrease the intracellular cAMP level Taken together, these results suggest that WIN 55,212-2-induced inhibition of proton-gated currents is mediated through the CB1 receptor and cAMP signaling pathway.−7 M, 60 s) produced an inhibition of the acid-induced action potentials. The mean number of action potentials was 3.6±0.4 during exposure to pH 5.5 for 5 s in eight neurons tested. In contrast, the mean number of action potentials decreased to 1.7±0.3 after the pre-treatment of WIN55,212-2   could trigger bursts of action potentials in rat DRG neuron, whereas it also induced an inward current with voltage-clamp recording in the same cell . The pre t-test) . After 3 t-test) . These rIntraplantar injection of acetic acid elicited an intense flinch/shaking response in rats −9 M–10–7 M) in eight rats/each group . The effect of WIN55,212-2 on acetic acid-induced pain behaviour was blocked by AM281 (10−6 M), a selective CB1 receptor antagonist. The number of acid-evoked flinches was 14.3±1.8 with co-treatment of both 10−6 M AM281 and 10−7 M WIN55,212-2, and there was a significant difference compared with treatment of 10−7 M WIN55,212-2 alone   . These rIn this study we first report that CB receptor agonist WIN55,212-2 inhibited the activity of ASICs in the primary sensory neurons. WIN55,212-2 decreased the amplitude of ASIC currents, the number of action potentials induced by acid stimuli and nociceptive responses to injection of acetic acid. We further showed that CB1 receptor and cAMP-dependent signal pathway likely underlie intracellular mechanism in inhibiting ASIC activity by WIN55,212-2.50 and threshold values.In agreement with previous studies, application of a pH 5.5 acid solution was found to evoke a slow-inactivating, rapid-inactivating or sustained inward current in native DRG neurons + and Ca2+ channels Unlike inhibiting effect of the CB receptor agonist WIN55,212-2 on ASIC currents, the CB-inactive enantiomer WIN 55, 212-3 failed to modulate proton-evoke currents, suggesting the involvement of CB receptor. In the present study, we concluded that WIN55,212-2 inhibitory effect was mediated by CB1 receptors, since the inhibition of proton-gated currents was blocked by the selective CB1 receptor antagonist AM 281, but not by the CB2 receptor antagonist AM630. It is been shown that CB1 receptors are abundantly expressed in the majority (70–80%) of nociceptive primary sensory neurons ASICs are a family of cation channels and extracellar pH sensors. Activation of ASICs by proton can depolarize the neurons and generate action potentials ASICs have been proposed to be involved in the perception of pain in conditions associated with tissue acidosis such as inflammation, ischemia, infection, tissue injury and tumor development In summary, we showed that the activity of ASICs can be inhibited by CB receptor agonist WIN55,212-2 in the primary sensory neurons. WIN55,212-2 decreased ASIC currents, action potentials and pain induced by acid stimuli. The inhibition was blocked by selective CB1 receptor antagonist, but not by CB2 receptor antagonist. Our results reveal a novel analgesic mechanism of cannabinoids by modulating native ASICs.All experiments were approved by the Institutional Animal Care and Use Committee of Hubei University of Science and Technology  and were carried out in strict accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals. Two- to three-week old Sprague-Dawley male rats were anesthetized with ether and then decapitated. The DRGs were taken out and transferred immediately into Dulbecco’s modified Eagle’s medium  at pH 7.4. After the removal of the surrounding connective tissues, the DRGs were minced with fine spring scissors and the ganglion fragments were placed in a flask containing 5 ml of DMEM in which trypsin  0.5 mg/ml, collagenase  1.0 mg/ml and DNase  0.1 mg/ml had been dissolved, and incubated at 35°C in a shaking water bath for 25–30 min. Soybean trypsin inhibitor  1.25 mg/ml was then added to stop trypsin digestion. After isolation, dissociated neurons were placed into a 35-mm Petri dish and incubated for >1 h in normal external solution before the start of electrophysiological experiments. The maximal incubated time was not more 5 h. The neurons selected for electrophysiological experiment were 15–35 µm in diameter.2 2.5, HEPES 10, EGTA 11 and Na2ATP 5; its pH was adjusted to 7.2 with KOH and osmolarity was adjusted to 310 mOsm/L with sucrose. Cells were bathed in an external solution containing (mM): NaCl 150, KCl 5, CaCl2 2.5, MgCl2 2, HEPES 10, d-glucose 10; its osmolarity was adjusted to 330 mOsm/L with sucrose and pH was adjusted to 7.4 with NaOH. DRG neurons were maintained in the external solution before they were used for electrophysiological experiments. The resistance of the recording pipette was in the range of 3–6 MΩ. A small patch of membrane underneath the tip of the pipette was aspirated to form a gigaseal and then a negative pressure was applied to rupture it, thus establishing a whole-cell configuration. The series resistance was compensated for by 70–80%. The adjustment of capacitance compensation was also done before recording the membrane currents. The membrane voltage was maintained at −60 mV in all voltage-clamp experiments unless otherwise specified. Current-clamp recordings were obtained by switching to current-clamp mode after a stable whole-cell configuration was formed in voltage-clamp mode. Only cells with a stable resting membrane potential (more negative than − 50 mV) were used in this study. Signals were sampled at 10 to 50 kHz and filtered at 2 to 10 kHz, and the data were stored in compatible PC computer for off-online analysis using the pCLAMP 10 acquisition software .Whole-cell patch clamp and voltage-clamp recordings were carried out at room temperature (22–25°C) using a MultiClamp-700 B amplifier and Digidata-1440 A A/D converter . Recording pipettes were pulled using a Sutter P-97 puller . The micropipettes were filled with internal solution containing (mM): KCl 140, MgClDrugs used in the experiments were purchased from Sigma Chemical Co. and include hydrochloric acid, WIN55,212-2, WIN55,212-3,AM281, forskolin, 8-Br-cAMP, (E)-3-(4-t-butylphenyl)-N- into the same hind paw and observed nociceptive responses. The pH of the solution was 2.55 Sprague-Dawley male rats (3–4 weeks) were kept with a 12-h light/dark cycle and with post hoc test. Statistical analysis of concentration–response data was performed using nonlinear curve-fitting program ALLFIT. Data are expressed as mean ± SEM.Data were statistically compared using the Student’s t-test or analysis of variance (ANOVA), followed by Bonferroni’s


Vibrio cholerae, a human pathogen that causes the disease cholera. The major V. cholerae autoinducer is (S)-3-hydroxytridecan-4-one (CAI-1). A membrane bound two-component sensor histidine kinase called CqsS detects CAI-1, and the CqsS → LuxU → LuxO phosphorelay cascade transduces the information encoded in CAI-1 into the cell. Because the CAI-1 ligand is known and because the signalling circuit is simple, consisting of only three proteins, this system is ideal for analysing ligand regulation of a sensor histidine kinase. Here we reconstitute the CqsS → LuxU → LuxO phosphorylation cascade in vitro. We find that CAI-1 inhibits the initial auto-phosphorylation of CqsS whereas subsequent phosphotransfer steps and CqsS phosphatase activity are not CAI-1-controlled. CAI-1 binding to CqsS causes a conformational change that renders His194 in CqsS inaccessible to the CqsS catalytic domain. CqsS mutants with altered ligand detection specificities are faithfully controlled by their corresponding modified ligands in vitro. Likewise, pairing of agonists and antagonists allows in vitro assessment of their opposing activities. Our data are consistent with a two-state model for ligand control of histidine kinases.Quorum sensing, a bacterial cell–cell communication process, controls biofilm formation and virulence factor production in  Two-component systems (TCS) are ubiquitous in bacteria and are used to adapt to environmental changes . In phosVibrio cholerae, two QS autoinducer/phosphorelay pathways, CAI-1/CqsS and AI-2/LuxPQ converge to regulate gene expression (V. cholerae. The autoinducer CAI-1 is (S)-3-hydroxytridecan-4-one . QS is a bacterial cell–cell communication process that relies on the production, release and detection of extracellular signalling molecules called autoinducers . This pran-4-one , b. Phosectively , left. Thaviours , right. oduction .in vitro. Second, structural studies of membrane bound proteins are notoriously difficult and therefore structural analyses of TCS have been limited to sensors with well-defined periplasmic domains  using inverted membrane vesicles and [γ-32P] ATP. To verify the prediction that His194 in the DHp domain is the site of phosphorylation, we constructed a CqsS H194Q mutant . In our assay, wild-type CqsS exhibits auto-phosphorylation; however, CqsS -His does not (-Cat) was constructed and it too is incapable of auto-phosphorylation (-Asp) remains capable of auto-phosphorylation  eliminated phosphotransfer to LuxO  and phosphorylated LuxO (LuxO∼P). To examine if this switch can occur dy state . Howeverdy state , indicatdy state .Vibrio harveyi autoinducer HAI-1, and AI-2 for the ability to convert CqsS to a phosphatase. Decanoic acid resembles the fatty acid tail of CAI-1 but lacks the α-hydroxy ketone head group. HAI-1 is the major V. harveyi autoinducer and AI-2 is the autoinducer of the V. cholerae LuxPQ QS pathway    , a synthdetected , left. TC170Y and CqsSF162A receptors compared with the wild-type receptor, their ligand specificities can nonetheless be examined. In the case of CqsSC170Y, CAI-1 did not significantly alter CqsSC170Y phosphorylation and LuxU phosphorylation. However, when C8-CAI-1 was added, CqsSC170Y phosphorylation and LuxU phosphorylation decreased  is controlled by CAI-1, we first tested if His194 auto-phosphorylation is regulated by CAI-1 using the CqsS at 1 min . Additioat 1 min . These r-Asp construct with that of LuxU phosphorylation by the wild-type CqsS receptor. When different concentrations of CAI-1 were added, both His194∼P on the CqsS -Asp construct and phosphotransfer to His58∼P on LuxU from wild-type CqsS were inhibited to the same extent  could also be inhibited by CAI-1 binding. We reason that if His194 auto-phosphorylation is the only step that is controlled by CAI-1, then CAI-1 inhibition of LuxU His58 phosphorylation should track with inhibition of CqsS His194 auto-phosphorylation. By contrast, if either or both of the subsequent phosphotransfer steps, His194∼P to Asp618 in CqsS or CqsS Asp618∼P to LuxU His58, is CAI-1 regulated, following CAI-1 addition, inhibition of LuxU His58 phosphorylation should be more severe than inhibition of CqsS His194 auto-phosphorylation. To assess this, we compared the extent of CqsS His194 auto-phosphorylation in the CqsS e extent . These r-His, CqsS -Cat and CqsS -Asp. Both CqsS -His and CqsS -Cat could dephosphorylate LuxU∼P, indicating that His194 or the CqsS catalytic site is not required for CqsS phosphatase activity  are not histidine kinases; rather, these receptors regulate the activity of the soluble histidine kinase CheA . Therefore, modulation of receptor levels potentially regulates phospho-flow in both directions. Feed-back regulation of QS receptor levels has been demonstrated in V. harveyi  and an attached receiver domain (D1) , multiple HPt proteins called APHs (H2), and multiple response regulators called ARRs (D2)  . While mRRs (D2) . It is sOur work shows that while histidine auto-phosphorylation is inhibited by CAI-1, the CqsS phosphatase activity is not regulated by the ligand. Having a constant CqsS phosphatase activity could be crucial for properly timed QS transitions. During the low cell density to high cell density transition, although CAI-1 inhibition of the CqsS kinase ensures that little new LuxO∼P is generated, existing LuxO∼P could still activate transcription, due to its high stability . HoweverHis194 auto-phosphorylation is inhibited by CAI-1. This result is consistent with previously proposed models suggesting that TCS binding of ligands triggers downstream conformational changes in sensor histidine kinases that affect interactions between the catalytic domains and the histidines in the DHp domains . Competition between a weak agonist and a strong agonist for the ligand binding pocket results in an intermediate effect rather than a synergistic effect. Antagonism can also be replicated in vitro with the CqsSC170Y mutant receptor, which possesses altered specificity for ligands. In vitro, C8-CAI-1 is a strong agonist and CAI-1, which does not affect CqsSC170Y when acting alone, is an antagonist of the CqsSC170Y receptor. Therefore, CAI-1 likely binds and stabilizes the ‘kinase on’ and ‘kinase off’ states of CqsSC170Y receptor with equal preference, and thus appears neutral in the absence of other molecules. Alternatively, to function as an antagonist, CAI-1 could prefer to bind and stabilize the ‘kinase on’ state of CqsSC170Y. In either case, CAI-1 certainly competes for the binding site with C8-CAI-1 and therefore acts functionally as an antagonist. Thus, predictions of the two-state theoretical model are consistent with the results of the experiments using our in vitro phosphorylation system.In our current study, P-CAI-1, a CqsS antagonist, also antagonizes CqsS in the presence of CAI-1 Bacteria exist in niches containing complex microbial-species compositions. Thus, the chemical environments they encounter contain numerous signalling cues, including many classes of autoinducers, the concentrations of which change in time and in space. In a simplified system, we have examined receptor histidine kinase regulation by a set of agonists and antagonists. To the best of our knowledge, this study shows, for the first time, that histidine auto-phosphorylation of the sensor is the only step that is regulated by the ligands. Structural studies of apo-CqsS and the CqsS receptor bound to agonists and antagonists should reveal the conformational changes induced by these ligands. Additionally, future work with reconstituted circuits that contain both the CqsS and LuxPQ sensors will be useful to understand QS signal integration.V. cholerae genomic DNA and cloned into the pET21b vector. Plasmids encoding CqsS H194Q (CqsS -His), CqsS D618N (CqsS -Asp), CqsS G379A/G381A (CqsS -Cat), CqsSC170Y and CqsSF162A were constructed from the plasmid containing wild-type CqsS using the Quikchange II XL Site-Directed Mutagenesis Kit (Stratagene). The genes encoding LuxU and LuxO were PCR amplified from V. cholerae genomic DNA and cloned into the pET28b vector. We call the LuxO site of phosphorylation D47 to be consistent with earlier publications and to match reported nomenclature of mutants. As part of the present work, we discovered the in vivo LuxO translational start site and found it is 14 codons 5′ to the previously reported start site. Thus, the phosphorylated Asp residue is amino acid 61 in the LuxO protein.DNA manipulations were performed using standard methods (Escherichia coli BL21 (DE3) harbouring plasmids encoding wild type or mutant CqsS constructs were grown with shaking in Luria–Bertani (LB) with 100 µg ml−1 kanamycin at 37°C. Overnight bacterial cultures were diluted 1:100 into fresh LB medium with kanamycin. After growth with aeration for 3 h at 37°C, protein production was induced with 1 mM IPTG. The cultures were shifted to room temperature and grown for an additional 5 h with shaking. Cells were harvested by centrifugation, resuspended in lysis buffer , and lysed under 15 000 psi. Cell lysates were centrifuged at 9300 g for 30 min and the cleared fluids were harvested and subjected to ultra-centrifugation at 180 000 g for 1 h. Membrane pellets were resuspended in kinase buffer  and quantified by SDS-PAGE and Western blot using CqsS purified in detergent as the standard.Escherichia coli BL21 (DE3) containing plasmids encoding LuxU or LuxO were grown and lysed using the above conditions. Cell lysates were centrifuged at 21 000 g for 30 min and the cleared fluids were harvested and passed through 5 ml Hi-Trap Chelating columns  pre-charged with Ni2+ ion. Following a wash with 30 ml lysis buffer, proteins were eluted with lysis buffer except that imidazole was included at 500 mM. Proteins were examined by SDS-PAGE followed by Coomassie Brilliant Blue staining and found to be ∼ 95% pure. Purified proteins were dialysed against lysis buffer lacking imidazole and concentrated. Protein concentrations were determined using the Bio-rad protein assay.2, and 10% (v/v) glycerol], and were initiated with the addition of 100 µM ATP and 2 µCi [γ-32P]-ATP (from a stock of 3000 Ci mmol−1: Perkin Elmer). For experiments with CAI-1 and analogues, compounds were added 10 min before the initiation of the reactions. For the reconstitution of the complete CAI-1/CqsS, LuxU, LuxO circuit, CAI-1 was supplied at 500 µM. Regulation of histidine phosphorylation by CAI-1 was assayed with 500 µM CAI-1. Agonism was tested at 100 µM. Antagonism was tested with 10 µM agonist and 100 µM (for P-CAI-1 antagonism of CqsSWT and CAI-1 antagonism of CqsSC170Y) or 500 µM (for C8-CAI-1 antagonism of CqsSWT) antagonist as specified. Reactions were incubated at room temperature and terminated with SDS-PAGE loading buffer. Reaction products were separated using SDS-PAGE. Gels were dried at 80°C on filter paper under vacuum, exposed to a phosphoscreen overnight, and subsequently analysed using a Typhoon 9400 scanner and ImageQuant software.Phosphorylation assays were performed with inverted membranes containing 2 µM wild-type CqsS or CqsS mutant proteins. In assays containing LuxU and/or LuxO, LuxU and LuxO were supplied at 10 µM. Reactions were carried out in phosphorylation buffer -ATP. Subsequently, the membrane vesicles were removed by ultracentrifugation at 180 000 g for 40 min. The supernatants containing LuxU∼P were applied to gel filtration spin columns  to remove ATP. Dephosphorylation reactions were initiated by adding inverted membranes containing 2 µM CqsS receptor and either 500 µM CAI-1 or DMSO. Aliquots were taken at the indicated time points and analysed as described above.LuxU was phosphorylated for 5 min in reactions with membrane vesicles containing 4 µM CqsS, 125 µM LuxU, 100 µM ATP and 10 µCi [γ--Asp mutant construct was carried out under the conditions described above for 2 min. His∼P dephosphorylation reactions were initiated by the addition of ADP at 100 µM final concentration and either 500 µM CAI-1 or DMSO. ATP was also added at 1 mM together with DMSO or CAI-1 to terminate new labelling of CqsS -Asp. In experiments with EDTA, 100 µM EDTA was added together with ligand and nucleotides. Aliquots were taken at the indicated time points and analysed as described above.Phosphorylation of the CqsS Chemical syntheses of CAI-1, C8-CAI-1, P-CAI-1, HAI-1 and AI-2 have been described (


High mobility group box 1 (HMGB1) functions as a chromatin-associated nuclear protein and an extracellular signaling molecule. The concentration of HMGB1 protein and the expression of HMGB1 mRNA were analyzed by ELISA and polymerase chain reaction (PCR), respectively. The present study reports high plasma HMGB1 levels in patients with adult T-cell leukemia  compared with normal controls. In addition, HMGB1 was highly expressed in HTLV-I-infected T-cell lines compared with uninfected T-cell lines. The HTLV-I oncoprotein, Tax, induced extracellular release of HMGB1 in T cells. The results suggest that HMGB1 is a potential biomarker and a therapeutic target for ATL. Infecti(HTLV-I) . The maj(HTLV-I) . Tax, th(HTLV-I) ,4.High mobility group box 1 (HMGB1) is an abundant and ubiquitous nuclear protein that binds to DNA and nucleosomes and induces structural changes in the chromatin fiber. It regulates numerous cellular activities, including transcription, replication and repair . In addiet al  are T-cell lines of leukemic cell origin established from patients with ATL. HuT-102 was also established from a patient with ATL and constitutively expresses viral genes, but its clonal origin is not clear. In JPX-9 cells, Tax is under the transcriptional control of the metallothionein gene promoter and can be induced by the addition of CdCl2 to the medium  prior to chemotherapy and stored at −80°C until use. Informed consent was obtained from all blood donors.μg total cellular RNA using a PrimeScript RT-PCR kit  with random primers. The primer sequences for HMGB1, receptor for advanced glycation end products (RAGE), Tax and GAPDH are listed in Total RNA was prepared from various cell cultures using TRIzol  according to the instructions provided by the manufacturer. First-strand cDNA was synthesized from 1 The concentrations of HMGB1 were measured in cultured supernatants from JPX-9 cells and plasma samples from patients and healthy donors using a commercially available ELISA kit II  according to the manufacturer’s instructions. The minimum detection level for HMGB1 was 1 ng/ml.Differences between groups were examined for statistical significance using the unpaired Student’s t-test. P<0.05 was considered to indicate a statistically significant difference.First, the expression levels of HMGB1, RAGE (HMGB1 receptor) and Tax were analyzed by PCR in eight HTLV-I-infected T-cell lines . HTLV-I-transformed T-cell lines  and HuT-102 constitutively expressed Tax mRNA, but ATL-derived T-cell lines  did not . PCR als2 rapidly induced Tax expression  with CdClpression . ELISA rssion  tended to be higher than those in healthy donors (n=5), albeit without statistical significance (P=0.051) . The meain vitro. Thus, it plays an important role in cellular transformation (The HTLV-I transactivator protein, Tax, is a protein of significant interest in HTLV-I pathogenesis, as it is a potent activator of a variety of transcription pathways and is in itself sufficient to immortalize T cells ormation ,4. Recenormation . The preormation . Tax expWhile the exact function of HMGB1 protein is not clear at present, it is reported to play important roles in sustained angiogenesis, evasion of apoptosis, growth signal self-sufficiency, insensitivity to antigrowth signals, the inflammatory microenvironment, immortalization, tissue invasion and metastasis in cancer cells . In addiIn conclusion, the present study demonstrated that there are elevated plasma HMGB1 levels in patients with ATL and an abundant HMGB1 expression in HTLV-I-infected T-cell lines.


A variety of animal models have contributed to the fundamental knowledge of HTLV-1 transmission, pathogenesis, and to the design of novel therapies to treat HTLV-1-associated diseases. Small animal models  as well as large animal models (monkeys) have been utilized to significantly advance characterization of the viral proteins and of virus-infected cells in the early steps of infection, as well as in the development of leukemogenic and immunopathogenic processes. Over the past two decades, the creation of new immunocompromised mouse strains that are robustly reconstituted with a functional human immune system (HIS) after being transplanted with human tissues or progenitor cells has revolutionized the in vivo investigation of viral infection and pathogenesis. Recent observations obtained in HTLV-1-infected humanized HIS mice that develop lymphomas provide the opportunity to study the evolution of the proviral clonality in human T cells present in different lymphoid organs. Current progress in the improvement of those humanized models will favor the testing of drugs and the development of targeted therapies against HTLV-1-associated diseases.Isolated and identified more than 30 years ago, human T cell leukemia virus type 1 (HTLV-1) is the etiological agent of adult T cell leukemia/lymphoma, an aggressive lymphoproliferative disease of activated CD4 While the great majority of animals are used in the field of pharmaceutical research and toxicology testing, they are also crucial in the understanding of basic and important pathophysiological processes. In particular, mechanisms sustaining the stepwise progression of viral pathogenesis from virus entry to the development of disease has greatly benefited from the use of animal models.Since antiquity, animals have been part of the human scientific and biomedical landscape. Observational studies of animals as well as experimental investigations using animals have greatly contributed to the advancement of scientific knowledge. Thus, during the last two centuries, biomedical research has largely progressed through observations and experiments performed with a variety of animals, some of which having obtained the status of animal models in biomedical research during the last five decades. Data obtained from +CD25+ T cells  in the peripheral blood. This lymphoproliferative disease in its most aggressive form follows an acute and frequently fatal course with a median 1-year of survival from the time of diagnosis. Patients eventually succumb to clinical complications, in part due to immunodeficiency-induced opportunistic infections.Human T cell leukemia virus type 1 (HTLV-1), the first retrovirus linked to a cancer in humans, was isolated from fresh peripheral blood lymphocytes from patients with cutaneous T cell lymphoma more than 30 years ago. Approximately 20 million people are infected worldwide primarily in endemic regions in Southern Japan, the Caribbean, Western Africa, and Central and South America. Most infected individuals remain asymptomatic for life, but approximately 5% develop adult T cell leukemia/lymphoma (ATL) after decades of protracted viral latency, whereas 1–3% develops inflammatory diseases, such as the neurologic disorder known as HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP). Clinical features of ATL include monoclonal expansion of leukemic CD4Figure 1).Human T cell leukemia virus type 1 is a single-stranded diploid RNA virus that carries genetic information for structural proteins and enzymes  and via alternatively spliced mRNAs encodes proteins from open reading frames (ORFs) I–IV  protein, encoded by the ORF IV in the pX proviral region and that behaves as a potent viral and cellular transactivator. Tax promotes proviral transcription and is critically involved in the proliferation of infected T cells, by favoring cell-cycle progression and cell survival and by promoting the activity of cellular transcription factors including NF-κB, AP-1, SRF, and CREB. Interestingly, Tax provokes genomic instability and suppresses host damage response, indicating that Tax harbors oncogenic properties. However, clonal proliferation of ATL cells is frequently associated with a Tax-low or Tax-negative phenotype, as the result of genetic or epigenetic events such as deletion of the 5′ LTR or methylation of this region. Consequently, it is currently accepted that Tax is critically involved in ATL induction and appears to be dispensable in the late stages of this disease. Such an assumption raises the possibility that ATL development and maintenance are dependent on the intervention of another viral product. A few years ago, another regulatory protein named HBZ (HTLV-1 bZip factor) encoded by an ORF in the antisense strand of the HTLV-1 provirus was identified . Since tin vitro studies have underlined that another auxiliary p12 protein (product of ORF I) that is also promoting viral persistence and immune evasion, has the ability of promote T cell proliferation. Finally the p13 protein (product of ORF II) was found to interact with Tax and suppress its transactivation function and to control the turnover of HTLV-1-infected T cells. In addition, p13, a mitochondria localized protein, influences cell survival  acting at the transcriptional and post-transcriptional levels have been demonstrated to play a crucial role in the initial stages of infection, mainly by modulating the level of produced viral particles, allowing in turn the promotion of viral persistence and evasion of the host immune surveillance. Likewise, It is therefore not surprising that soon after the isolation and characterization of HTLV-1 in 1980, animals were used with the main purpose to uncover the natural history of HTLV-1 infection and of associated diseases in humans. Thus, the use of both small animals  and large animals  has contributed to an understanding of transmission, dissemination, and persistence of HTLV-1 infections. Likewise, induced animal models created by genetic manipulations have been used to delineate the involvement of specific viral genes on the initiation and development of pathophysiological processes in the initiation and the development of HTLV-1-induced pathogenic processes. Furthermore, immunodeficient mice arisen through spontaneous mutations have paved the way not only to the xenograft of leukemic cells, but also to the development of humanized mouse models that have allowed new approaches in translational research.Macaca fascicularis) and squirrel monkeys (Saimiri sciureus) can be experimentally infected with HTLV-1 and provide useful primate models of HTLV-1 infection .Rabbits have been used extensively as a model of HTLV-1 infection due to the consistency of transmission, ease of handling, and low cost of maintenance  strains. The first studies in the 1980s, showed the ability to infect rabbits using the intravenous route and the MT-2 cell line  and macaques . In thismacaques . This stin vivo, as well as host cell activation and regulation of viral gene transcription. Limitations of the rabbit model include the lack of consistent disease that mimic those observed in humans infected with HTLV-1 and the lack of a wide-range of immunologic reagents to assess the model.Collectively, using these animal models we now know that the non-structural genes encoded from ORF I and II are vital for viral infectivity, maintenance of the virus life cycle and proviral loads Mouse models have the distinct advantage of being economical and readily available to test the growth of tumors. They permit studies to test the development of tumors and to test preclinical efficacy of potential therapeutic agents. Multi-centric growth of tumor cells transplanted into immunocompetent mice is concurrent with elevated biomarkers such as PTHrP expression and increasing levels of serum interleukin (IL)-2Rα and β-2 microglobulin that correspond to increasing tumor burden. Even if immunocompetent mice are not efficiently infected with HTLV-1, they have been extensively used to produce animal models exhibiting specific symptoms and pathology of diseases through selective breeding and genetic modification. Thus, transgenic mice have been very useful to test the role of HTLV-1 gene products in the outgrowth of tumors  . In thisSpecifically, targeting Tax expression using bitransgenic and doxycycline-inducible mice (Tg(EmuSR-tTa)83Bop) resulted in activation of NF-κB and a fatal dermatologic disease characterized by infiltration of Tax positive T cells into the dermis and epidermis . FinallyCollectively, transgenic mice have provided reproducible models to test specific gene products role in tumor formation and in targeting models have provided insights into the pathogenesis of leukemia and paraneoplastic lesions associated with HTLV-1 gene products. A primary disadvantage of these models is the lack of viral gene expression in context to an intact proviral genome and lesion development that clearly mimics the pathogenesis of HTLV-1-associated disease of humans.Prkdcscid) bearing the severe combined immunodeficiency the Prkdc  mutation  mice . Later omutation . Engraftmutation . These oPrkdcscid/Lystbg) have defective B and T cell function, NK cell activity, and defective granulocyte properties. These mice have a higher rate of engraftment role of multiple arms of the immune system in tumor engraftment and spread. This tenet was further demonstrated in non-obese diabetic (NOD) mice that lack functional B and T cells, have low NK cell activity, absence of complement activity, and impaired macrophage and antigen presenting cell function. Compared to the SCID mice and the SCID/bg mice, the NOD/SCID mice (NOD.CB17-Prkdcscid/NCrCrl) are more susceptible to engraftment with the HTLV-1 transformed cell lines  mice are homozygous for the SCID mutation and a targeted disruption of the IL-2Rγ gene mutation. One significant advantage of NOG mice are efficiency to accept transplanted with human cells compare to other mouse models. These mice, in addition to lacking functional B, T, and NK cells, have a severe reduction in interferon γ (IFN-γ) production which allows these mice to be easily transplanted with ATL cells directly from patients to test factors correlative to known paraneoplastic syndromes associated with HTLV-1 infection, such as HHM (The development of the NOD/SCID mice containing a targeted mutation in the beta-2 microglobulin gene (beta 2-m) resulted in mice that lacked NK cell function . This sth as HHM .Although xenograft animal models displaying multiple organ engraftments with ATL cell lines or patient samples have been extensively studied, they failed to provide a complete understanding of HTLV-1-associated ATL development. Thus, developing a faithful animal model able to recapitulate the HTLV-1 infection, and to understand the mechanisms of early infection up to the development of associated pathologies is one of the critical goal in HTLV-1 research.Despite their advantages , mice are not susceptible to several human-specific pathogens, thus restricting their use in experimental and medical research. This limitation has been overcome with the development of humanized mice susceptible to infection by these pathogens. Here, the “humanized mice” are exclusively restricted to severely immunodeficient mice that have been engrafted with functional human cells and tissues. In those animals, the engrafted human cells and tissues carry the same biological functions as they do in the human body. Consequently, the physiological properties and functions of transplanted human tissues and cells can be readily analyzed in the mouse.in vivo approach of the human immune system (HIS) have been achieved by successful genetic modifications of immunodeficient mice and by improving engraftment procedures. Actually, humanized mouse technology has made rapid progress, and it is now possible to achieve high levels of human chimerism in various organs and tissues, particularly the immune system and the liver  mature human peripheral blood leukocytes (PBL) (hu-PBL-SCID) or transplanted human blood-forming fetal liver cells, fetal bone, fetal thymus (thy), and human fetal liver (liv) in SCID mice  model” has been described in 2006 as a humanized mouse model able to mount specific adaptive and innate immune responses. These mice were obtained after transplantation of autologous human hematopoietic fetal liver CD34 tissues . These m– / – and RAG2– / –, respectively). They do not exhibit leaky production of murine lymphocytes. However, RAG-deficient animals produce normal levels of NK cells, and thus additional mutations are required in order to produce animals better suited for xeno-engraftment studies.Another mouse model was obtained with mice deficient in the recombinase activating genes 1 and 2  gene. The γc is a component of the IL-2, IL-4, IL-7, IL-9, IL-15, and IL-21 receptors and is the gene involved in X-linked SCID  either obtained from fetal liver or from umbilical cord blood has resulted in improved, long-term engraftment of human hematopoietic cell types as well as the ability to generate human immune responses. Combinations of the above mutations so far analyzed for HP/HSC engraftment have included such strains as Balb/c RAG2– / –γc– / – (referred as to BRG), NOD RAG1– / –γc– / – (NRG), NOD–SCID-γc– / – , of the reconstitution of human cells into various organs, of the duration of engraftment, and of the ability to generate primary human adaptive immune responses , two different experimental approaches have led to the demonstration that functional CD47/SIRPα interactions are required for optimal human T and NK cell homeostasis o HP/HSC . The seco HP/HSC . Both prin vivo, an immunodeficient NSG-HLA-A2 strain was created, by backcrossing the HLA class I transgene onto the NSG background  or HIS (NSG) mice supports human αβT and γδT cell development more efficiently than that of adults, probably because of the involution of the thymic rudiment in the latter  CD4+ and CD8+ T cells are present with broad Vβ distribution, together with Foxp3+, CD25 regulatory T cells. Furthermore human CD16+CD56+ NK cells are also observed in the murine thymus.An efficient intrathymic e latter . Whatsoe+ cells. They home to bone marrow, spleen, liver, lungs, and peripheral blood (5–20% of human cells). Human T cells developing in HIS mice are functional as indicated by the proliferation of these T cells after in vitro stimulation with allogeneic human DCs . These mice develop a conjoint organ, which supports human thymocyte differentiation and maturation. Viral infection of thymocytes was detected 4–6 weeks later. Interestingly, thymocytes from two mice with the greatest HTLV-1 proviral burdens showed increased expression of the CD25 marker  and perturbation of distribution profiles of the CD4+ and CD8+ thymocyte subsets. Even if no T cell malignancy was reported, these observations clearly implicate the human thymus as a target reservoir for HTLV-1 infection. They further suggest that the dysregulation of thymopoiesis and lymphoproliferation are predisposing events in the development of the leukemogenic process associated with HTLV-1 infection. It was recently reported that sublethally irradiated 4- to 6-week NOD/SCID mice inoculated with human fetal liver-derived CD34+ cells ex vivo infected by HTLV-1 developed CD4+ T cell lymphomas with characteristics similar to ATL . The high ability of humanized BRG and NSG mice to support maturation and all human hematopoietic lineages should therefore facilitate the evaluation of the effects of HTLV-1 infection. Thus, it has been reported that in NSG mice inoculated with human fetal liver-derived CD34+ cells ex vivo infected by HTLV-1, viral infection was skewing hematopoiesis to the T cell lineage leading to hyperproliferation of CD3+ T cells in the bone marrow, thymus, spleen, and mesenteric lymph nodes . In this in vivo model, genes involved in the anti-apoptotic pathway are deregulated in a manner similar to that observed in in vitro Tax-transduced human thymocytes .To determine in which cells the HTLV-1 provirus is integrated, CD3– thymocytes represent immature thymocytes: the early progenitors, the immature CD4 SP population and the early DP T cells. The CD3+ thymocytes encompass a mature population with the late DP thymocytes along with SP CD4+ or CD8+ cells. The level of proviral load in both populations was evaluated and was found to be about 40-fold higher in the CD3+ than that in the CD3– population. These observations indicate that immature cells are indeed infected. One important consequence of that event may explain that in the thymus of infected HIS mice, the mature T lymphocytes represent the main subset when compared to the DP and immature subsets. Indeed, no modification in the distribution profiles of these populations has been observed in mock-infected HIS mice (Table 2). These observations indicate that the accumulation of mature lymphocytes in the thymus of infected mice is consecutive to their increased capacity of proliferation.The CD3Consequently, the thymopoiesis alterations consecutive to HTLV-1 infection may be caused by the expression of Tax in immature thymocytes. Indeed, that viral protein has been shown to down-regulate the expression of the pTα gene, thus perturbing β-selection, the early and critical checkpoint of human αβT cell development in the thymus , 2009. FUsing a highly sensitive high throughput sequencing system described elsewhere , the numin vivo study of human cells and tissues. Humanized mice could provide valuable information about the cellular and molecular in vivo mechanisms supporting the leukemogenic activity of HTLV-1. In addition, they are potentially very helpful for the evaluation of therapeutic treatments.Thanks to the successive modifications of the model systems and genetic manipulation, humanized mice have acquired the ability to serve as preclinical models for the 2O3 has been tested on several patients and has shown promising results as a first-line therapy for ATL patients , and Aspatients . The molpatients .Intraperitoneal inoculation of HTLV-1-producing cells have been successfully used to infect HIS mice with other human viruses like HIV and represent the easiest way to administrate infected cells into the mice. Oral inoculation is more relevant to the route of natural HTLV-1 infection occurring through breast-feeding, as ATL has been linked to the infection of newborn mostly via breast-feeding by an HTLV-1 infected mother .in vivo infection and pathogenic processes. This model also represents a good opportunity of understanding the leukemogenic potential of HTLV-1 in a comparative study with HTLV-2 and HTLV-3 which are closely related to HTLV-1 but have different pathogenesis. HTLV-2 has not been associated with any malignant diseases. The pathogenesis of HTLV-3 is unknown due to the low number of known infected people around the world. The infection of HIS mice with HTLV-2 could provide an interesting approach to determine what are the critical steps of leukemogenesis that are created by HTLV-1 and not by HTLV-2 or HTLV-3.The use of molecular clones to infect the HIS mouse model would represent a powerful tool to dissect the role of the different viral proteins and particularly the regulatory proteins such as Tax, HBZ, Rex, and p30 in an Humanized mice are mainly instrumental in dissecting the early stages of the infection and the critical initial events leading to the development of ATL-like pathologies. It should be emphasized that the proliferation of infected clones is not under immune control, as the human adaptive immune response in this humanized mouse model directed toward HTLV-1 has been very disappointing thus far . The use of alternative immunosuppressed mice strains, such as the NSG-HLA-A2 mice could resolve this problem and improve this model, providing researchers and clinicians with a valuable tool not only to investigate the early steps of leukemogenesis, but also to unravel what is happening during the long latent period following HTLV-1 infection. Over the past three decades, the technology and applications of humanized mouse model have considerably grown providing new opportunity for human retroviral studies and for testing the efficacy of anti-retroviral therapies. The recent development of optimized humanized mice offer great promise in the field of retrovirology knowledge, and reasonable hopes for the development of therapies against this virus-associated cancer.A better knowledge of viral pathogenesis is required to provide comprehensive novel treatments (therapies and vaccines) to eliminate viral infections in humans. Clearly, progress in understanding the cellular and molecular basis of viral pathogenesis has come from animal models. Like for all modeling approaches that use surrogates, one main limitation concerns the validity of extrapolating data derived from specific experiments using animal models to the general human clinical conditions. Nevertheless, it should be emphasized that animal models have largely participated to the study of viral pathogenesis, and as underlined in this review, they have greatly contributed to delineate main aspects of HTLV-1 pathology. It is not surprising that these models include rabbits, rats, and mice, i.e., three of the main laboratory animals. Their use to study viral pathogenesis has led to complementary data. Indeed, the first two of them – rabbits and rats – can be infected with HTLV-1 and have been used to address essential issues, such as entrance and spread in the host, and the development of the immune response. Conversely, mice that cannot be infected with HTLV-1 have first been instrumental either as immunodeficient strains or as transgenic models. Later on, the generation of humanized mice developing a HIS has generated new hopes in the field of human infectious diseases and represent a physiologically potent system allowing studies of direct interactions of HTLV-1 with human hemato-lymphoid cells. Nevertheless, the present survey of the literature devoted to animal models of HTLV-1 infection and pathogenesis has underlined the specificity as well as the complementarity of each of these models.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Staphylococcus aureus contains an autoinducing quorum-sensing system encoded within the agr operon that coordinates expression of virulence genes required for invasive infection. Allelic variation within agr has generated four agr specific groups, agr I–IV, each of which secretes a distinct autoinducing peptide pheromone (AIP1-4) that drives agr signaling. Because agr signaling mediates a phenotypic change in this pathogen from an adherent colonizing phenotype to one associated with considerable tissue injury and invasiveness, we postulated that a significant contribution to host defense against tissue damaging and invasive infections could be provided by innate immune mechanisms that antagonize agr signaling. We determined whether two host defense factors that inhibit AIP1-induced agrI signaling, Nox2 and apolipoprotein B (apoB), also contribute to innate control of AIP3-induced agrIII signaling. We hypothesized that apoB and Nox2 would function differently against AIP3, which differs from AIP1 in amino acid sequence and length. Here we show that unlike AIP1, AIP3 is resistant to direct oxidant inactivation by Nox2 characteristic ROS. Rather, the contribution of Nox2 to defense against agrIII signaling is through oxidation of LDL. ApoB in the context of oxLDL, and not LDL, provides optimal host defense against S. aureus agrIII infection by binding the secreted signaling peptide, AIP3, and preventing expression of the agr-driven virulence factors which mediate invasive infection. ApoB within the context of oxLDL also binds AIP 1-4 and oxLDL antagonizes agr signaling by all four agr alleles. Our results suggest that Nox2-mediated oxidation of LDL facilitates a conformational change in apoB to one sufficient for binding and sequestration of all four AIPs, demonstrating the interdependence of apoB and Nox2 in host defense against agr signaling. These data reveal a novel role for oxLDL in host defense against S. aureus quorum-sensing signaling. Staphylococcus aureus is a common colonizer of humans but can also cause severe, invasive infection. S. aureus uses a secreted peptide-based communication system, agr, to induce production of virulence factors needed for invasive infection. Allelic variation has generated four agr types, agr I–IV, and each secretes a distinct autoinducing peptide (AIP1-4) that differs in amino acid sequence and length. Understanding host factors that prevent signaling by each of the four agr specific groups (agrI–IV) could provide opportunities for prevention of infection or therapeutic intervention. We previously demonstrated that apolipoprotein B (apoB), the major structural protein of very low and low density lipoproteins , binds to the secreted agrI peptide, AIP1, and prevents agr signaling. In addition, the NADPH oxidase Nox2 produces reactive oxygen species which directly modify and inactive AIP1. Here we examined the role of apoB and Nox2 in defense against agrIII-signaling. We found that apoB in oxidized LDL, but not in native LDL, mediated optimal binding of AIP3. Also, unlike AIP1, Nox2 did not directly inactivate AIP3. Rather Nox2 contributed to defense against agrIII-signaling by oxidizing LDL. Furthermore, we found that oxLDL bound all four AIPs and antagonized agr signaling by each agr allele in vitro. These results expand our understanding of host defense against S. aureus agr signaling. Staphylococcus aureus uses global gene regulation to coordinate gene transcription required for survival within distinct host niches agr, that encodes a quorum sensing system that combines secretion of an autoinducing peptide pheromone (AIP) with a sensor regulator. Activation of this system upregulates genes for toxins, hemolysins, lytic enzymes, and metabolic pathways that are required for a phenotypic change in this pathogen from an adherent colonizing phenotype to one associated with significant tissue injury and invasiveness agr upregulated virulence factors are associated with acute infections particularly at epithelial barriers like the skin and lung agr signaling thus permitting optimal phagocyte and epithelial cell function. While the molecular mechanisms involved in agr sensing and signaling have been extensively studied agr sensing to control tissue damage and cell injury have not been fully elucidated agrI allele agr alleles are represented within the species S. aureus and because all four alleles are associated with significant disease in humans agr types.agr allele encodes a unique secreted AIP that differs in amino acid content and length but contains a common thiolactone bond that creates a 5-membered ring essential for biologic function agr signaling by either direct modification of key amino acids, cleavage of the thiolactone bond, proteolytic degradation, or sequestration to prevent AIP binding to its receptor, AgrC. For AIP1, reactive oxygen species (ROS) generated by the Nox2 NADPH oxidase expressed in phagocytes and other cells directly modify a key C-terminal member of the thiolactone ring, a methionine, to form methionine sulfoxide agrI-mediated invasive infection beyond epithelial and mucosal barriers. To extend these studies to the other agr alleles, we first focused on the contribution of these two barriers to host antagonism of agrIII signaling. AIP3 is shorter and composed of amino acids that are more resistant to oxidant modification as compared to AIP1 and therefore might be less amenable to control by either apoB or ROS. We hypothesized that Nox2 and apoB within the context of its serum lipoproteins would differ in the molecular mechanism by which they antagonize agrIII signaling and that this difference would impact the susceptibility to invasive infection in mouse models that lack these innate barriers.Each agrIII-mediated signaling requires binding and sequestration of AIP3 by apoB, not in the form of LDL but in LDL oxidized by Nox2. Importantly, these studies revealed an important role for oxLDL in binding to and antagonizing signaling by all four agr alleles. In addition, while ROS directly inactivate AIP1 and 4, they do not affect the biologic function of either AIP 2 or 3. Thus, the contribution of Nox2 in antagonizing agr signaling for these alleles is primarily through the production of oxLDL. OxLDL-mediated antagonism of agr signaling inhibits agr driven virulence factor expression by all four alleles, providing a mechanistic basis for its importance in preventing invasive S. aureus infection which we demonstrate in a murine agrIII-mediated skin infection model. Therefore, while best studied for its contribution to atherosclerosis, our data reveal a novel role for oxLDL as a host defense effector that controls S. aureus agr-mediated signaling.Here, we show that optimal host innate defense against agrI-mediated invasive S. aureus infection by early extravasation of activated neutrophils and apoB-containing plasma lipoproteins that act to antagonize agrI signaling agr signaling requires AIP cyclized through a 5-membered thiolactone ring. AIP binds and activates AgrC which generates phosphorylated AgrA. Phosphorylated AgrA activates the agr P3 promoter generating the effector molecule RNAIII. ApoB recognizes the cyclic, active form of AIP1 and prevents binding to and signaling through its cognate AgrC receptor agrIII strains. In addition to binding the agr P3 promoter, phosphorylated AgrA directly enhances transcription of genes encoding phenol soluble modulin virulence factors  psmα in clinical isolates USA300 LAC (agrI) and USA400 MW2 (agrIII) psmα transcription in the agrIII strain MW2  and agrIV (AH1872 – MN TG) clinical isolate  which detects epitopes present in oxLDL but absent in LDL −/−Nox2 mice  compared to controls, confirming that the contribution of Nox2 to apoB-mediated host defense is specific to control of agr-mediated pathogenesis. In contrast, infection of −/−Nox2 mice with MW2 at an inoculum readily controlled by wild-type mice resulted in significant dermal invasion, morbidity and bacterial burden in the spleen as compared to wild-type mice. In addition, infection with the MW2 agr deletion mutant also resulted in increased dissemination to the spleen  agrIII clinical isolates. OxLDL significantly inhibited transcription of both the agr effector molecule RNAIII and of a key agr regulated virulence factor, alpha hemolysin (Hla), by both MRSA and MSSA agrIII clinical isolates  is a strong oxidizing agent released by activated neutrophils and contributes significantly to neutrophil extracellular trap (NET) formation, ozone mediated bacterial killing and ozone formation in atherosclerotic plaques as well as conformational changes of apoB within LDL 1O2 induces lipid oxidation of LDL 1O2 , provide unique barriers against S. aureus agrI-mediated virulence and the loss of either significantly increases susceptibility to invasive infection agrI-mediated virulence. This current work demonstrates that the roles of apoB and Nox2 in defense against agrIII-mediated infections are interdependent. Unlike AIP1, AIP3 is resistant to oxidation and direct inactivation by ROS characteristically produced by Nox2. Instead, the contribution of Nox2 to host innate defense against S. aureus agrIII-dependent quorum sensing is through oxidation of LDL. ApoB in the context of oxLDL, and not LDL, provides optimal host defense against S. aureus agrIII infection by binding the secreted signaling peptide, AIP3, and preventing expression of the agr-driven virulence factors which mediate invasive infection. Our results suggest that oxidation of LDL facilitates a conformational change in apoB to one required for optimal binding of AIP3 and apoB-mediated defense against agrIII-dependent virulence. Intriguingly, agrI-, agrII- and agrIV-signaling was also antagonized by oxLDL in vitro, suggesting that apoB within oxLDL is sufficient for antagonism of signaling by each of the four agr alleles. Each of the four S. aureus agr alleles are associated with invasive human infections which may range in severity from mild skin and soft tissue infections to severe disease such as osteomyelitis, endocarditis, necrotizing pneumonia and bacteremia S. aureus infections of any agr type in which agr-signaling contributes to pathogenesis. In contrast, we would predict that oxLDL would play a minimal role in host defense in chronic or device related infections in which agr signaling may be less relevant Host innate immunity is critical for maintaining a defensive barrier against opportunistic pathogens such as agrIII S. aureus into the underlying dermis. This suggests that other serum proteins which extravasate to the site of infection may also contribute to defense against agr-mediated signaling. Recently, Surewaard et alagr-regulated S. aureus virulence factors. Among these lipoproteins, HDL was attributed with the highest PSM scavenging activity and, using a wide variety of in vitro assays, demonstrated inhibition of all PSMs investigated. However, unlike our data demonstrating apoB binding and antagonism of AIP1-4, lipoprotein-mediated inhibition of PSMs was attributed to the lipid and not the protein components of the lipoprotein particles. Another serum component, hemoglobin, also suppresses agr function when released from red blood cells agr-signaling is initiated and PSMs as well as hemolysins needed to lyse RBCs for release of hemoglobin have begun to be expressed, both lipoprotein-mediated scavenging of PSMs and hemoglobin-mediated suppression of agr-signaling could clearly contribute to prevention of further agr-mediated virulence and invasion of host tissues. Although these additional mechanisms of controlling quorum-sensing dependent S. aureus virulence may contribute in part to our in vivo results, the following factors support the primary role of apoB-mediated control of agr-dependent virulence in our model: 1) apoB binds immobilized AIP1-4 and apoB and oxLDL antagonize agr-dependent P3 promoter activation, RNAIII, psmα and hla transcription and Hla production in vitro in both laboratory strains and clinical isolates, 2) addition of oxLDL, but not LDL, to MW2 infected air-pouches of LDL-deficient −/−Nox2 mice was sufficient to suppress quorum-sensing independent of other lipoproteins and 3) compared to untreated mice, 4APP-treated wild-type mice with equivalent oxLDL levels were not more susceptible to MW2 invasive infection, suggesting that oxLDL-mediated control of agrIII-dependent virulence factor expression predominated and offset the need for lipoprotein scavenging of PSMs. Therefore, apoB plays a unique role in inhibition of agr signaling upon initiation of infection. All of these findings highlight the host's multi-tiered innate defense system to combat S. aureus agr-mediated virulence and suggest that conditions which result in decreased lipoprotein levels may contribute on more than one level to host susceptibility to S. aureus invasive infection.Our data show that LDL and oxLDL are present at the site of infection and prevent quorum-sensing dependent invasion of agrIII-virulence is supported by the ability of an apoB-specific antibody to block AIP3 binding by both apoB and oxLDL, but not LDL. Further investigations using antibody blocking and competition binding studies should help to elucidate differences in binding of AIP 1-4 by the different apoB-containing lipoprotein particles and to determine whether there is a single site or multiple sites for AIP binding to apoB. At present however, the specific binding site(s) for AIP 1-4 within the three-dimensional structure of this 515 kDa protein and the relevant binding affinities require further investigation.Our data comparing oxLDL and LDL binding of AIP1 versus AIP3 demonstrate that whereas oxLDL provides optimal binding and antagonism of AIP3, both lipoprotein particles are equally efficient at binding AIP1. Such differences in the ability of apoB-containing lipoprotein particles to bind and antagonize these AIPs may result from multiple factors. One explanation could be that although both peptides contain the conserved thiolactone bond, the length and sequence variation between AIP1 and AIP3 may suggest that each has a unique binding site on apoB. Because oxidation of LDL is known to alter the conformation of apoB within the particle, oxLDL may present apoB in the ideal conformation for binding AIP3 −/−Nox2 mice prior to infection indicating that Nox2-derived ROS contribute to constitutive oxidation of LDL in the circulation. This may be explained in part by expression of Nox2 in other cell types including endothelial cells and adventitial fibroblasts, where it participates in cell cycle regulation and apoptosis agrI and agrIII-mediated S. aureus invasive infection.Nox2 activation contributes to phagocyte oxidative responses and in this regard patients with severe sepsis have significantly elevated levels of serum oxLDL suggesting that Nox2 can contribute to oxLDL formation during infection S. aureus have recently been reported. First, Sun and colleagues demonstrated that oxidation of amino acid Cys199 of AgrA disrupted DNA binding and inhibited agrI-dependent expression of RNAIII and psmα in vitroagr system is viewed as a bacterial mechanism to counter oxidative stress. However, oxidation of the AgrA disulfide redox switch may also be considered a form of host defense against S. aureus agr-signaling. This mechanism is distinct from ROS-mediated inactivation of AIP or oxidation of LDL as oxidation of either AgrA, AIP1 or LDL in isolation is sufficient to antagonize agr-signaling. Second, apoB and LDL were shown to inhibit LTA-induced cytokine release from immune cells through direct interaction with LTA S. aureus independent of its role in antagonizing agr-signaling. If this were the primary function of apoB and oxLDL in our current work we would have expected to see a significant role for reduced serum apoB in susceptibility to an agr negative infection in which LTA would be equivalent and we did not. Together, these reports demonstrate multiple distinct and independent roles for both ROS and apoB in both agr-dependent and agr-independent host defense against S. aureus infection.Additional roles for both ROS and apoB in innate defense against in vitroPlasmodium falciparum by binding the scavenger receptor SR-BI which is also utilized by the parasite Streptococcus pneumoniae and Haemophilus influenzaeHelicobacter pylori enhance atherosclerosis by increasing serum oxLDL Chlamydia pneumonia in vascular smooth muscle cells is enhanced by oxLDL S. aureus agr dependent quorum-sensing by each of the four agr alleles provides new insight into the role of oxLDL in host defense.Although to our knowledge this is the first description of oxLDL as an innate defense factor controlling quorum-sensing dependent virulence, oxLDL contributes in other direct and indirect ways to host defense. For example, oxLDL inhibits hepatitis C virus cellular entry Animal work in this study was carried out in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health, the Animal Welfare Act and US federal law. The protocol was approved by the Institutional Animal Care and Use Committee (IACUC) of the Research Service of the New Mexico VA Health Care Service.−80°C. Purified human LDL and oxLDL  and purified apoB  were assayed for apoB content using a commercially available kit . The following reagents were obtained as follows: tributyrin, 4-aminopyrazolo- pyrimidine (4APP), HOCl, Rose Bengal, N-acetymethionine (NAM), ethylthioacetate, ; E06 monoclonal Ab ; Rabbit anti-apoB ; Goat anti-apoB ; Goat IgG .Synthetic AIPs were synthesized by Biopeptide Co., Inc  as described agr I LAC [agr:P3-yfp]), AH1747 (strain agr III MW2 [agr:P3-yfp]), AH430 (strain agrII 502a [agr:P3-yfp]) and AH1872 (strain agrIV MN TG [agr:P3-yfp]) were provided by Dr. Alex Horswill (University of Iowa) agr deletion mutants were provided by Dr. Mike Otto. agrIV clinical isolates (NRS165 and NRS166) were obtained through the Network on Antimicrobial Resistance in Staphylococcus aureus (NARSA) program supported under NIAID, NIH Contract No. HHSN272200700055C. Additional clinical MRSA and MSSA isolates were provided by Dr. Larry Massey, New Mexico VAHCS. To generate synchronized early exponential phase, non-fluorescent bacteria, frozen stocks were cultured in Trypticase soy broth (TSB)  as described The bacterial strains used in this study were as follows: AH1677 (strain 7/ml) were incubated in TSB in polystyrene tubes with shaking  for the indicated times with either broth, synthetic AIP, AIP treated with ROS, or antagonists including lipoprotein particles or apoB. After incubation, bacteria were washed by centrifugation at 3000 rpm for 4 min at 4°C in PBS with 0.1% Triton X-100, sonicated, cultured for CFU where indicated, and then fixed with 1% paraformaldehyde containing 25 mM CaCl2 for analysis by flow cytometry . Promoter activation was demonstrated as fluorescence induction and measured as the mean channel fluorescence (MCF) of YFP-positive bacteria.Early exponential phase, nonfluorescent reporter bacteria  Oxidation of AIP or LDL was performed using HOCl or singlet oxygen. HOCl (Sigma) was diluted and incubated with AIP or antagonist (lipoprotein or apoprotein) at 37°C for 30 min. The HOCl concentration was determined by its absorbance at 292 nm , using a microfiltration apparatus . Slots were washed with additional TBS following sample application. Membranes were blocked for 30 min using SuperBlock , then probed with SuperBlock containing rabbit anti-apoB or E06 anti-oxLDL for 30 min at room temperature (RT). Unbound primary antibody was removed by washing 3× with TBS, 0.1% Tween20 (TBST), followed by incubation with the appropriate alkaline phosphatase (AP) conjugated secondary antibody also in SuperBlock. After 30 min at 25°C. RT, blots were washed 3× with TBST and 1× with TBS. Blots were developed using nitro-blue tetrazolium and 5-bromo-4-chloro-3′-indolyphosphate . Band intensity was quantified using Carestream Molecular Imaging software .Surface plasmon resonance was performed using a Biacore ×100  to analyze the interaction of the analytes  with immobilized ligand (AIP) as previously described Nox2 knockout mice on the C57BL/6 background (−/−Nox2) C57BL/6 mice  from Charles Rivers , and Subcutaneous air pouches were created and infected with early exponential phase bacteria at the indicated dose as previously described S. aureus antibody  conjugated with Alexafluor 488 (Molecular Probes/Invitrogen). Slides were rinsed and mounted in Prolong Gold Antifade (Molecular Probes/Invitrogen). Images were acquired on a Zeiss LSM 510 confocal microscope. Bacterial density as a measure of tissue invasion into the dermis was quantified from LSM images using Slidebook software . Regions of interest were selected using histiologic criteria for the dermis (3000 µm2) and the total area and the portion of that area stained for S. aureus quantified in microns2. Values are displayed as percentage of square microns of staining within the identified area. A minimum of 20 sections were examined for each experimental condition.Basolateral pouch tissue was excised, rinsed in PBS and fixed in 3% paraformaldehye containing 0.1 mM CaCl2 and 0.1 mM MgCl in PBS. Pouch tissue was then rinsed with PBS and stored in 25% sucrose until freezing. Pouches were frozen in O.C.T. compound using liquid nitrogen. Cryo blocks were stored at −80°C until sectioned. Cryosections (10 µm) were cut onto slides, fixed and permeabilized with acetone at −20°C for 5 minutes, rehydrated with PBS, and blocked overnight with 0.5% BSA, 5% normal rabbit serum, 0.1% Triton-X-100 in PBS. Slides were incubated for 1 h at 4°C with anti-280 = 1 mg/ml.LDL was isolated from mouse serum basically as previously described In vitro data were analyzed by the Student's t test and the in vivo results by the Mann-Whitney U test for nonparametrics.Data are displayed as the mean ± SEM. Figure S1OxLDL antagonizes AIP binding and agr::P3 promoter activation mediated by agrI, agrII and agrIV alleles. (A) Quantification of psmα transcript relative to 16S RNA produced by LAC or MW2 cultured 1 h in the presence of 50 nM AIP, plus 50 nM LDL or apoB. Data points represent the mean ± SEM normalized to psmα induction by AIP plus broth alone. Statistics are in reference to AIP plus broth. (B) agrI isolate LAC [agr::P3-yfp] or agrIII MW2 [agr::P3-yfp] was cultured for 2 h with 50 nM AIP plus 10 nM LDL or oxLDL. Data points represent the mean ± SEM normalized to broth control. (C) LDL, apoB or oxLDL binding to immobilized AIP1 was measured by SPR. Data were normalized to the mean ± SEM of LDL binding. (D) Schematic representation of AIP2 and AIP4. (E) OxLDL binding to immobilized AIP2 and AIP4 was measured by SPR. (F) OxLDL antagonizes agrII and agrIV P3 promoter activation. AH430 (agrII) and AH1874 (agrIV) were cultured overnight with broth control or 10 nM oxLDL and agr::P3 promoter activation measured by flow cytometry. ns, not significant; *, p<0.05; **, p<0.01; ***, p<0.001.(TIF)Click here for additional data file.Figure S2Effect of Nox2 deletion on oxidation of LDL and of 4APP on serum cholesterol associated with apoB-containing lipoprotein particles. (A) Lipid oxidation of LDL purified from the serum of −/−Nox2 mice relative to LDL from wild-type mice. (B) Serum was collected from wild-type mice treated with vehicle control or 4APP and the cholesterol content of the LDL/VLDL fraction determined. (C) Lipid oxidation of LDL purified from the serum of 4APP-treated wild-type mice relative to LDL from control treated mice. Data reported as the mean ± SEM. *, p<0.05; **, p<0.01.(TIF)Click here for additional data file.Figure S3OxLDL antagonizes RNAIII transcription and agr-dependent virulence factor transcription and expression by MRSA and MSSA agrI, II and IV clinical isolates. (A) Addition of LDL or oxLDL to (A) purified Hla or (B) supernatant containing lipase has no direct impact on the activity of either virulence factor as measured by functional assay. (C–E) Clinical isolates were cultured 2 h with 50 nM AIP (agrI and II) or overnight without exogenous AIP (agrIV) plus 10 nM oxLDL or media control. Transcription of RNAIII and hla relative to 16S rRNA was measured by qRT-PCR and Hla expression was assessed by functional assay and reported as percent activity relative to media control. *, p<0.05; **, p<0.01; ***, p<0.001; ****, p<0.0001.(TIF)Click here for additional data file.Figure S4Singlet oxygen mediates oxidative inactivation of AIP4 but not AIP2. AIP2 and AIP4 were exposed to singlet oxygen by incubation with rose bengal in the presence or absence of light, prior to culture with (A) AH430 or (B) AH1872, respectively. agr::P3 promoter activation was measured by flow cytometry. Data points represent the mean ± SEM of the mean channel fluorescence. ****, p<0.0001.(TIF)Click here for additional data file.Figure S5Exogenous oxLDL reduces morbidity in S. aureus agrIII infected 4APP-treated −/−Nox2 mice at 4 and 8 h post-infection. Air-pouches were generated on the backs of 8 to 12 week old −/−Nox2 mice treated with 4APP as previously described. At time zero, 4×107 cfu of early exponential phase agrIII MW2 [agr::P3-yfp] were injected into the air-pouch along with 100 nM AIP3 and either 100 nM oxLDL or saline control. Weight loss was measured at the time of infection and at (A) 4 h and (B) 8 h post-infection and is shown as percent body weight lost relative to time zero. *, p<0.05; **, p<0.01.(TIF)Click here for additional data file.


F = 2.76, P = 0.027). These results suggested that combined assessment of UA and hsCRP levels provides incremental information for risk stratification of patients with MetS, independent of other confounding factors in community-dwelling women.Metabolic syndrome (MetS) is associated with an increased risk of major cardiovascular events. In women, increased uric acid (UA) levels are associated with MetS and its components. High-sensitivity C-reactive protein (hsCRP) levels are also associated with MetS, and hsCRP levels could be modulated by UA. We investigated whether combining UA and hsCRP levels are independently associated with MetS and insulin resistance in Japanese community-dwelling women. From a single community, we recruited 1,097 women (63 ± 12 years) during their annual health examination, and examined the cross-sectional relationship between UA, hsCRP, and MetS and insulin resistance, which was evaluated by homeostasis of minimal assessment of insulin resistance. Of these subjects, 218 women (19.9 %) had MetS. Multiple linear regression analysis was performed to evaluate the contribution of each confounding factor for MetS and insulin resistance, both UA and hsCRP as well as age and alcohol consumption, were independently and significantly associated with MetS and insulin resistance. The adjusted-odds ratios  for MetS across tertiles of UA and hsCRP were 1.00, 1.45 (0.95–2.22), and 2.61 (1.74–3.93), and 1.00, 1.80 (1.18–2.74), and 3.23 (2.15–4.85), respectively. In addition, the combination increased UA, and hsCRP was also a significant and independent determinant for MetS and insulin resistance. In direction associations, we also observed a synergistic effect between these two molecules ( Metabolic syndrome (MetS), a clustering of cardiovascular risk factors such as visceral obesity, insulin resistance, hypertension, glucose intolerance, hypertriglyceridemia, and low high-density lipoprotein cholesterol (HDL-C) levels, is a major worldwide public health problem. In Japan, it is 13.3 % in men and 11.5 % in women , and carRecently, results from a meta-analysis of individual participant data from 54 long-term prospective studies demonstrated that high-sensitivity C-reactive protein (hsCRP) is also an inflammatory marker and independent predictor of CVD such as coronary heart disease, ischemic stroke, and vascular and non-vascular mortality .Increased serum uric acid (UA) in humans is also associated with systemic inflammation , endotheThe aim of this study was to determine whether combining UA and hsCRP levels are independently associated with MetS and insulin resistance by examining cross-sectional data from Japanese community-dwelling women.Participants were recruited at the time of their annual health examination in a rural town with a total population of 11,136 (as of April 2002) and located in Ehime prefecture, Japan, in 2002. Among the 4,738 female aged 19–90 years in this population, a random sample of 1,713 (36.2 %) subjects was recruited. For all these individuals, overnight fasting plasma sample were available for measuring hsCRP. The final study sample compromised 1,097 residents. This study was approved by the ethics committee of Ehime University School of Medicine, and written informed consent was obtained from each subjects.Information on demographic characteristics and risk factors was collected using the clinical files. Body mass index was calculated by dividing weight (in kg) by the square of the height (in m). We measured blood pressure with an appropriate-sized cuff on the right upper arm of the subjects in the sedentary position using an automatic oscillometric blood pressure recorder  while they were seated after having rested for at least 5 min. Other characteristics such as smoking, alcohol habit, and medication, were investigated by individual interviews that were conducted using a structured questionnaire. Smoking status was classified into non-current smoker and current smokers. The daily alcohol consumption was measured using the Japanese liquor unit in which a unit corresponds to 22.9 g of ethanol, and the participants were classified into never-drinkers, occasional drinkers (<1 U/day), and light–heavy drinkers (≥1 U/day). Total cholesterol (T-C), triglycerides (TG), HDL-C, fasting plasma glucose (FPG), and UA were measured by the laboratory of health examination center during an overnight fast of more than 11 h. The plasma sample were immediately frozen and stored at −80 °C until measurements were taken by the laboratory of our department. The plasma hsCRP concentration was measured using a Behring BN II nephelometer  and the inter- and intra-assay coefficient variations were 3.2 and 6.7 %, respectively. Low-density lipoprotein cholesterol (LDL-C) level was calculated by the Friedewald formula . Individ2 according to the guidelines of the Japanese Society for the Study of Obesity (waist circumference was not available in this study) [We applied condition-specific cutoff points for MetS based on the modified criteria of the National Cholesterol Education Program’s Adult Treatment Panel (NCEP-ATP) II report . MetS was study) ; (2) raiχ2 test for categorical variables. Multiple logistic regression analyses were used to evaluate the contribution of confounding factors for MetS and insulin resistance (HOMA-IR ≥ 2.6), and further used to also evaluate the contribution of each tertile of UA and hsCRP for MetS, each component of MetS, and insulin resistance. The combining effect of UA and hsCRP was evaluated using multiple logistic regression analyses adjusted for the following parameters: age, current smoking status, alcohol consumption, UA, and hsCRP. The synergistic effect of UA and hsCRP was evaluated using a general linear model  unless otherwise specified, and in the cases of parameters with non-normal distributions , the data are shown as median (interquartile range) values. In all analyses, parameters with non-normal distributions were used after log-transformation. Statistical analysis was performed using PASW Statistics 17.0 . Correlations between UA and hsCRP were determined using Pearson’s correlation. Multiple linear regression analysis was used to evaluate the contribution of confounding factors  for number of MetS components and HOMA-IR. Subjects were divided into three groups based on tertiles of UA and hsCRP, and differences among the groups categorized by each tertile of UA and hsCRP were analyzed by ANOVA for continuous variables or the The characteristics of the subjects categorized according to tertiles of UA and hsCRP are illustrated in Table r = 0.285, P < 0.001; data not shown). As shown in Table UA levels significantly increased with hsCRP , in addition to their direct associations .In addition to their direct associations, we observed a synergistic effect between UA and hsCRP. In Fig. In 1,097 community-dwelling women, we determined the prevalence rate of MetS, as defined by the modified NCEP-ATP III criteria , and exaP < 0.001 for trend). In the highest quartile of CRP levels (>1.50 mg/L), the risk for MetS was substantially higher  compared with that in the lowest quartile of CRP levels (≤0.33 mg/L) after adjustment for age, sex, geographic location, lifestyle factors, level of education attained, and family history of chronic diseases [Elevated CRP was associated with increased odds of MetS after adjusting for potential confounding factors . In a rudiseases . We havediseases . In thisThe mechanisms by which UA reflect the risk for MetS are not completely understood. In the past, most authorities have not considered UA as having a pathogenetic role of CVD and have viewed UA as biologically inert or possibly anti-inflammatory because it could function as an antioxidant . HoweverThere are some limitations to this study. First, our cross-sectional study design does not eliminate potential causal relationships between UA, hsCRP, and MetS. Second, the prevalence rate of MetS, UA, and hsCRP categories is based on a single assessment of blood, which may introduce a misclassification bias. Third, we used BMI ≥25 to classify individuals with visceral obesity because waist circumference measurements were not available, which might have caused an under or over estimation of the effect of visceral obesity on MetS . In factIn conclusion, the present study showed that combining UA and hsCRP levels are significantly associated with MetS, independent of other confounding factors in community-dwelling women. The underlying mechanism behind this relationship is unclear, but seems to be independent of traditional cardiovascular risk factors such as age, current smoking status, and alcohol consumption. For community-dwelling healthy women, prospective population-based studies are needed to investigate the mechanisms underlying this association to determine whether intervention, such as effective lifestyle modifications or medication  that decrease UA and hsCRP in adults , will de


Human T-cell lymphotropic virus type 1 (HTLV-1) infection is intractable and endemic in many countries. Although a few individuals have severe symptoms, most patients remain asymptomatic throughout their lives and their infections may be unknown to many health professionals. HTLV-1 can be considered a neglected public health problem and there are not many studies specifically on patients' needs and emotional experiences.To better understand how women and men living with HTLV-1 experience the disease and what issues exist in their healthcare processes.A qualitative study using participant observation and life story interview methods was conducted with 13 symptomatic and asymptomatic patients, at the outpatient clinic of the Emilio Ribas Infectious Diseases Institute, in Sao Paulo, Brazil.The interviewees stated that HTLV-1 is a largely unknown infection to society and health professionals. Counseling is rare, but when it occurs, focuses on the low probability of developing HTLV-1 related diseases without adequately addressing the risk of infection transmission or reproductive decisions. The diagnosis of HTLV-1 can remain a stigmatized secret as patients deny their situations. As a consequence, the disease remains invisible and there are potentially negative implications for patient self-care and the identification of infected relatives. This perception seems to be shared by some health professionals who do not appear to understand the importance of preventing new infections.Patients and medical staff referred that the main focus was the illness risk, but not the identification of infected relatives to prevent new infections. This biomedical model of care makes prevention difficult, contributes to the lack of care in public health for HTLV-1, and further perpetuates the infection among populations. Thus, HTLV-1 patients experience an “invisibility” of their complex demands and feel that their rights as citizens are ignored. Human T-cell lymphotropic virus type 1 (HTLV-1) infection is commonly confounded with Human Immunodeficiency Virus (HIV) infection and it is unknown to many health professionals. It is endemic in many countries and there is no effective treatment available. Although a few individuals have severe symptoms, most patients remain asymptomatic throughout their lives. Further, HTLV-1 is considered a neglected public health problem and limited studies cover specific patients' needs and emotional experiences. To better understand how women and men living with HTLV-1 experience the disease and what issues exist in their healthcare processes, we conducted a qualitative study of both symptomatic and asymptomatic patients at an outpatient clinic at the Emílio Ribas Infectious Diseases Institute in São Paulo, Brazil. We found that the main focus of health staff was on illness risk, but not identifying infected relatives and preventing new infections. This point of view, ultimately neglected patients' complex demands, and overshadows the prevention of new infections and contributes to the lack of care in public health for HTLV-1 infected subjects. Furthermore, this perpetuates the infection among these populations and the patients experience an “invisibility” of their specific needs, such as reproductive rights and feel that their rights as citizens are ignored. The human T-cell lymphotropic type 1 (HTLV-1) The most common diseases associated with HTLV-1 infection are adult T-cell leukemia/lymphoma (ATL) and HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP) Brazil has received worldwide recognition for the country's public health policies, especially with respect to how the nation has addressed the pandemic of HIV/AIDS Few HTLV-1 epidemiological studies have been conducted in the general population, but the average prevalence of HTLV-1/2 among donor blood banks is 0.46% nationwide In 1993, it was recommended by ministry of health the mandatory tests for hepatitis C and HTLV-1/2 in blood banks. In 2002, Brazil also instituted a policy which provided free testing of HIV/AIDS and syphilis during prenatal care visits as an approach to preventing mother-to-child-transmission (PMTCT). However, the same policy was not provided for HTLV-1 infection and the Brazilian Ministry of Health only published the Guide Recommendations on HTLV Management in 2003, without update From a public health perspective, prevention of HTLV-1 is crucial, especially because HTLV-1 is a life-long infection and is currently incurable and untreatable. The health field recognizes that disease prevention requires strategies beyond strictly medical approaches. Thus, the aim of this study was to identify perceptions of HTLV-1 by women and men living with this infection, and to better understand their families, subjective issues, needs and how they cope with health care.The present study was conducted by using participant observation methods Using a thematic script and in parallel an in-depth interviews with 13 individuals (11 women and two men) diagnosed with HTLV-1 infection and without co-infections see . SubjectData were analyzed by using life narratives from each subject and constructed content categories from these narratives The study was approved by the Ethical Research Board Committee - CAPPesq (n. 297/06) and the Emílio Ribas Institute of Infectious Diseases Ethical Committee (n. 34/06). The volunteers signed an informed consent form that faithfully follows the resolution of the national Ministry of Health (CNS 196/96) and the Declaration of Helsinki.While HTLV-1 infection is frequently “invisible” in a literal way, this article aimed to highlight the disease's symbolic invisibility from patients' perspectives and life experiences, as well as patients' relations with healthcare providers.The HTLV-1 diagnosis is frequently a doubts and fear moment in primary medical care settings. While recalling their original diagnosis, subjects reported an initial shock followed by some feelings of denial  or hopefulness for a “cure.” In addition, many participants thought that their illness was HIV infection, which has been similarly reported by Guiltinan (1998) Interestingly, many subjects reported that “it was better not to know about HTLV” and “not to think about HTLV”. These observations indicated the use of denial as a defense mechanism. Subjects also reported fear of disclosure and that their HTLV-1 diagnoses would be disrupted family relations. Consequently, this perception leads subjects to hide their HTLV-1 diagnosis from everyone, as a “secret.” Such difficulties have also been observed in people living with HIV/AIDS Regarding this “secret,” the thoughts and perceptions between subjects and health providers seem connected. Subjects and providers understood the absence of HTLV-1 symptoms as “normal”, in consequence HTLV-1 infection remained “invisible” because they are focused on risk of sickness. This observation has important implications for understanding the lack of diagnosis among partners and family members.“Living with HTLV is normal for me. I feel nothing, do not take any medication. For me, HTLV does not bring any problem, feel anything. How can I bring my mother to be tested, disturbing everyone?” .“I prefer not to think and not bring my children to be tested. I think it is better this way. I'm dealing with HTLV. I do not want to mention it. If I do this, it can mess with my children minds”. Except for Maria, all subjects indicated that someone in their family circle  should be tested for HTLV-1 infection but had not been tested yet. This situation illustrated how poor HTLV-1 disclosure among family members has negative consequences. In Maria's case the lack of disclosure was the possible cause of her infection, because Maria's husband, Walter, was infected by his mother, and hidden his HTLV-1 infection from her. Her husband, Walter, did not reveal his diagnosis to his wife because he thought they were both healthy. Although his wife was pregnant (30 weeks), Walter only sought medical attention when he began to have symptoms of HAM/TSP. Only on this time, his health team requested to his pregnant woman to be tested for HTLV-1 infection.The symptomatic condition further expanded upon these issues, as patients experience changes in their daily activities, self-image and social lives. The progressive disability associated with symptomatic HTLV-1 infection limited these patients' lives, as illustrated by the sentences below.“After I developed this disease my husband told me that I'm not good for anything anymore. As much as we do not want to remember this, this prejudice keeps reminding us of the disease” .“People already have a bias to get close because I was not walking well. If you have a problem, they say that you are no different. They say that you are the same. But it is not true! You have a huge problem because you have a difficulty to walk. Imagine if you say that you have an infectious disease” .The suffering and mobility limitations constitute “narcissistic injuries” Subjects report that their perceptions and experiences changed when their family members are symptomatic. HTLV-1 becomes present in subjects' lives through the symptoms of their family, and the need to care for these members may trigger additional emotional coping mechanisms.In cases where subjects know that they were infected by their parents, HLTV-1 infection is viewed as a type of “family heritage” that the subject must “carry for life.” This knowledge may be very painful emotionally, as demonstrated by Alba and Walter:“I can not accept it. I thought, why my story is so painful? HTLV is one heritage! I kept myself a virgin for my future husband. I did not get anything financially from my family but I inherited a disease” .“HTLV is a painful thing because it's something we will carry for life”. .This lack of disclosure to partners and family on HLTV-1 diagnosis could be explained by poor information regarding infection transmission; however, the speeches revealed an appropriate understanding of transmission modes. On the other hand, some subjects had doubts how they became infected. These subjects were not willing to search for this information, which illustrates their difficulties in dealing with the HLTV-1 diagnoses.Invisibility among persons living with HTLV-1 may have meaning, especially because patients have reported feeling stigmatized and socially disadvantaged  since receiving their diagnoses “Having a child would be a burden of conscience. I would try to do something to no longer generate another discarded person in my family. Another contaminated one in the family.” .According to subjects, primary health teams are generally unaware of HTLV-1 and did not investigate or recognize the health problems associated with the infection and disease. Subjects reported a poor access to pre- and post-test counseling for HTLV-1 infection. In fact, an author showed that a person can be tested and remained without access to specific information and follow up Most interviewers reported they were the only sources of information about HTLV for the health teams, which created uncertainty concerning the teams' medical knowledge. This finding leads to a paradoxical question on healthcare for HTLV: how can a doctor inform and treat patients when himself does not know HTLV? Maria and Maria Rita's speeches, who were both pregnant, illustrated this paradox.“The doctor asked me what HTLV infection is. But I did not know it either. Even doctors do not know what it is, can I imagine? They said ‘I cannot say anything. I will first study and then I will see what I have to do’” .The obstetrician who provided prenatal care explained the following to Maria Rita: “You have a virus like AIDS. I do not know if this disease can be transmitted to the baby. I do not know anything about this virus”. Another medical staff member, during an ultrasound exam, told her: “If the virus has infected the baby, apparently it did not affect anything. But I do not know anything about this virus”.The “invisibility” of HTLV-1 infection should not be a problem for symptomatic patients such as HAM/TSP and ATL cases. However, this study revealed that symptomatic patients had to make “pilgrimages” to several specialists as they searched for a diagnosis. In fact, HAM/TSP patients experienced an average of eight years seeking a diagnosis Interviewers and medical staff were concerned on the risk of disease development, which is considered “low” for both parties. Healthcare providers and HTLV-infected subjects frequently do not realize that asymptomatic individuals may transmit the virus to partners or offspring. Consequently, preventive processes may become ineffective.In conclusion, there appeared to be an underlying logic for the “invisibility” in people living with HTLV-1. Even when individuals had access to necessary information regarding the disease, they frequently denied the situation. In consequence, there were implications for self-care and understanding the need for testing family members for HTLV-1 infection.As people living with HTLV-1 struggle to cope with this health problem, it is possible to observe that these difficulties were logically articulated through the hegemonic healthcare approach. This hegemonic conception of health care is referred to major paradigm in health focused at the biomedical approach. Nowadays, it has been questioned by the researchers at the public health field to contrast this model to the bio-psych-social model which aims the health care in the entire complexity. The biomedical perspective does not encourage prevention activities and health promotion Beyond the “invisibility” of HTLV-1, there is an “invisibility” of the subjects living with the infection or disease, their individual difficulties and idiosyncrasies