The Notch signaling is an evolutionarily conserved cell-cell communication pathway that plays critical roles in the proliferation, survival, apoptosis, and fate determination of mammalian cells. Retinal pigment epithelial (RPE) cells are responsible for supporting the function of the neural retina and maintaining vision. This study investigated the function of Notch signaling in RPE cells. We found that the members of the Notch signaling pathway components were differentially expressed in RPE cells. Furthermore, blockage of Notch signaling inhibited the migration and proliferation of RPE cells and reduced the expression levels of certain Notch signaling target genes, including HES1, MYC, HEY2, and SOX9. Our data reveal a critical role of Notch signaling in RPE cells, suggesting that targeting Notch signaling may provide a novel approach for the treatment of ophthalmic diseases related to RPE cells. The retinal pigment epithelium (RPE), a polarized monolayer of specialized epithelium and a glial cell layer located between the neural retina and choroid, plays a pivotal role in supporting photoreceptor function , 2. RPE Notch signaling is an evolutionarily conserved cell-cell communication mechanism that regulates the proliferation, survival, apoptosis, and fate decision-making of mammalian cells through local cell interactions . Notch sNotch signaling is activated by a ligand binding to the receptor between two neighboring cells . Upon liTo date, few reports clarify the role of Notch signaling in human RPE cells. In this study, we examined the effect of Notch signaling on the migration and proliferation of RPE cells and the expression levels of Notch target genes after inhibiting Notch signaling by knocking down NOTCH1. Our study revealed a role of Notch signaling in regulating the proliferation and migration of human RPE cells, which is important in understanding the pathogenesis of certain ophthalmic diseases.The following small hairpin RNA (shRNA) lentiviral constructs targeting the human NOTCH1 gene were obtained from Thermo Scientific and have been previously described . The haiThe following antibodies were used in Western blotting: NOTCH1 , GAPDH  [μg/mL) at 37°C in a 5% CO2 atmosphere.The ARPE-19 cells (ATCC) were cultured in DMEM with 10% inactivated fetal calf serum (FCS), penicillin (100 units/mL), and streptomycin (50 μg/mL Polybrene (Sigma-Aldrich). Finally, the RPE cells were selected with puromycin  for one week.Lentiviral transduction was performed as previously described . In brie4) were cultured in 24-well plates. Cells were then trypsinized, and the number of cells was determined by trypan blue assays on the following 4 days. For the scratch assay, RPE cells were seeded into a 6-well plate to reach 80–90% confluence. A scratch was made using a white tip and the cells were cultured in DMEM medium without fetal bovine serum. Migration of the cells into the scratch area was observed at 12, 24, and 36 hours after the scratch had been made.Proliferation and scratch assays were performed as previously described . For thehttp://dx.doi.org/10.1155/2013/178708.For real-time RT-PCR, total RNA was extracted from RPE cells using the Trizol reagent (Invitrogen), and cDNA was reverse transcribed using the PrimeScript II 1st Strand cDNA Synthesis Kit (TaKaRa). Real-time RT-PCR analysis was performed on an ABI PRISM 7000 Sequence Detector using the SYBR Premix Ex TaqTM Kit (TaKaRa). The sequences of the 14 pairs of primers used are listed in Supplementary Tables 1 and 2 of the Supplementary Material availbale online at For Western blotting, total protein was extracted using lysis buffer. Ninety micrograms of protein was separated by electrophoresis using 7.5% SDS-polyacrylamide gels. The details of Western blotting were as described previously .We performed real-time RT-PCR to determine the expression levels of the Notch signaling pathway components, including five Notch ligands  and three MAML transcriptional coactivators . We found that JAG1 had a higher expression level than any of the other ligands . MAML1 and MAML2 were expressed at significant levels, with MAML3 displaying the lowest expression level .To determine the role of Notch signaling in RPE cells, we knocked down the expression of NOTCH1 using several NOTCH1-shRNAs. We chose three lentiviral vectors targeting NOTCH1 . After the RPE cells were selected with puromycin, we obtained four stable RPE cell lines with different levels of NOTCH1 knockdown. The shNOTCH1- was the cell line that was infected with a mixture of the three lentiviruses, including shN1-1, shN1-2, and shN1-4 . Next, win vitro scratch assay. The control cells displayed high migratory ability, as the scratch wound was almost recovered after 36 h of incubation. However, the migration of the three NOTCH1-knockdown RPE cell lines was significantly inhibited  cells . TherefoTo investigate the mechanism of the effects of blocking the Notch signaling on the migration and proliferation of RPE cells, we performed real-time RT-PCR to follow the expression levels of Notch signaling target genes. Among the twelve target genes we selected, four genes, including HES1, SOX9, HEY2, and MYC, exhibited significant downregulation after NOTCH1 was knocked down .The RPE is a single layer of epithelial cells located at the posterior segment of the eye and is crucial for photoreceptor function. The RPE is relevant to several photoreceptor diseases, such as proliferative vitreoretinopathy (PVR) , prolifeDifferent activation levels of Notch signaling induce distinct dose-dependent phenotypes. More specifically, high levels of Notch signaling activation cause a suppression of cell proliferation and downregulation of the expression of certain matrix-adhesion molecules. However, lower levels of Notch signaling activation lead to a proliferative response and maintain matrix adhesion in mammary epithelial cells . In thisin vitro reduced the migration and proliferation of RPE cells. In ophthalmic diseases, such as PVR and AMD, the RPE can initiate disordered migration and proliferation [To establish the stable RPE cell line with inhibited Notch signaling, we knocked down NOTCH1 using several shRNAs. The efficiency of knockdowns was different, and the most effective knockdown was achieved with the mixture of viruses  Figures . Consistferation . TherefoTo explore the mechanism by which a blockage in Notch signaling reduced migration and proliferation in RPE cells, we performed real-time RT-PCR to evaluate the expression levels of several critical transcription factors involved in Notch signaling. The HES family is a family of transcriptional repressors that act as Notch effectors , and itsTo the best of our knowledge, few reports clarify the role of Notch signaling in human RPE cells. We showed that RPE cell lines expressed different levels of Notch signaling components. Our results also highlighted the role of Notch signaling in regulating the migration and proliferation of RPE cells. In addition, the change in Notch signaling altered the expression levels of Notch signaling target genes that might participate in the regulation of migration and proliferation in RPE cells. Our results provide a rational background for understanding Notch signaling and RPE cells more comprehensively and suggest that focusing on the Notch signaling and the Notch target genes may provide a novel approach for the treatment of ophthalmic diseases related to RPE cells.Table 1:The primers of Notch signaling components used in real time RT-PCR.Table 2: The primers of Notch signaling target genes used in real-time RT-PCR.Click here for additional data file.


The proposed method was successfully applied to real-time monitoring of UA excretion in human 24 h urine with different food intake, showing that meats, vegetables, and porridge intake caused differential UA excretions of 879, 798, and 742 mg, respectively. It was also found that UA concentrations in urine under the three kinds of food intake simultaneously reached maximum at 2 h after meals with the values of 417, 318, and 288 μg mL−1, respectively. The UA concentration in human serum was also determined by this approach, and the possible mechanism of luminol-Co2+-UA CL reaction was discussed in detail.Based on the inhibitory effect of uric acid (UA) on luminol-Co Uric acid . The relative standard deviation (RSD) was 2.5% for determining 10.0 nmol L−1 UA. At a flow rate of 2.0 mL min−1, a single UA determination cycle could be accomplished within 36 s, offering a sample throughput (ST) of 100 h−1. UA excretion in urine is known to be closely associated with food intake, and the normal excretion of UA is 250–740 μg mL−1 per day [ Flow injection (FI) analysis combined with CL, FI-CL, which possess merits of low limit of detection (LOD), fast response time, less reagent consumption, and simple instrumentation as well as automation, has become a very useful analytical tool in different scientific fields from food safety –35 to en per day . Herein,All chemical reagents in this work were of analytical reagent grade. Doubly deionized water purified in a Milli-Q system  with the resistivity of 18.2 MΩ cm was used for the preparation of solutions in the whole procedure. Luminol  and UA (Sigma) were obtained from Xi'an Medicine Purchasing and Supply Station, China.−2 mol L−1 was prepared in 1.0 × 10−1 mol L−1 NaOH solution and kept in the dark. The Co2+ stock standard solution of 1.0 × 10−3 mol L−1 was prepared by dissolving 11.90 mg of CoCl2·6H2O (99%) in 50 mL calibrated flask with doubly deionized water. The UA stock standard solution of 1.0 × 10−3 mol L−1 was prepared in 1.0 × 10−2 mol L−1 NaOH solution and stored at 4°C. All the daily used working standard solutions were freshly prepared from the above stock standard solutions by appropriate dilution as required.The luminol stock standard solution of 2.5 × 10μL to quantitatively inject luminol. The CL detector, which was placed inside a black box for precluding the interference from ambient light, contained a flow cell and a photomultiplier tube (PMT). The flow cell, tightly positioned on the window of PMT, was a colorless glass tube  with a spiral disk shape (2.0 cm i.d.). The CL signal from the flow cell was directly detected by the PMT without any wavelength discrimination and the output was recorded by the computer. The polytetrafluoroethylene (PTFE) tubing (1.0 mm i.d.) was used to deliver the reagents.The schematic profile of equipment in the flow system  was depicted in 2+ solutions, respectively. The pump was started with the stream of doubly deionized water at a constant flow rate of 2.0 mL min−1 to wash the whole system. Then 100 μL of luminol solution was injected into the flow system by the six-way valve and, thereafter, merged with the premixed, homogeneous stream of UA and Co2+. The entire mixed solution was then delivered into the CL cell, producing CL emission. The CL signal was detected by PMT, and the PMT negative voltage was set as 700 V. The CL intensity decrement (ΔICL = I0 − Is), where I0 and Is were defined as CL intensity in the absence and presence of UA, respectively, was used as the quantitative parameter for UA concentration.As shown in The urine samples, which came from volunteers who have prehydrated, were collected in beakers with volumes recorded. The UA concentrations in urine samples were measured directly after an appropriate dilution. When necessary, urine samples were refrigerated at 4°C and were determined as soon as possible to guarantee the sample integrity.The human serum samples were obtained from the Hospital of Northwest University. After centrifugation, 0.1 mL supernatant was diluted with doubly deionized water and mixed thoroughly for CL determination after dilution.−1 accordingly. Whereas by using the manifold of injecting UA, the same analytical process needed 120 s, yielding the ST of 30 h−1. It was clear that the ST of the manifold of injecting luminol shown in  Two manifolds of quantitatively injecting luminol or UA sample into the carrier stream by six-way valve were investigated. It was found that two significantly different results were obtained. Using the manifold of quantitatively injecting luminol by six-way valve, a whole analytical process including sampling and washing could be accomplished within 36 s, offering a ST of 100 h−7~5.0 × 10−5 mol L−1) and Co2+ concentration (7.0 × 10−10~7.0 × 10−8 mol L−1) on the CL intensity was investigated, respectively. As shown in −6 mol L−1, above which the CL signal tended to be stable. Thus, 5.0 × 10−6 mol L−1 luminol was chosen as the optimal. It is also clear in 2+ concentration increased to 1.0 × 10−8 mol L−1 and then increased slowly from a higher concentration. Therefore, 1.0 × 10−8 mol L−1 was chosen as the optimum concentration of Co2+. Owing to the nature of luminol CL reaction, which was favorable in alkaline medium, NaOH was added into luminol solution to increase the sensitivity of this CL system. A series of NaOH solutions ranging from 1.0 × 10−4 to 1.0 × 10−1 mol L−1 was tested. At 2.5 × 10−2 mol L−1 NaOH, the CL signal could reach a maximum value. Hence, 2.5 × 10−2 mol L−1 of NaOH was the optimal concentration for this CL system. The effect of luminol concentration , the precision of this method decreased. Considering a good sensitivity, a better precision, and a lower reagent consumption, a flow rate of 2.0 mL minTmax) for reaching maximum CL intensity (Imax) of luminol-Co2+ reaction (curve 1) and luminol-dissolved oxygen reaction (curve 5) were 4.0 and 4.4 s with the Imax of 939 and 59, respectively; the Tmax for luminol-Co2+ reaction in the presence of UA (curves 2–4) was 4.0 s, while the Imax decreased from 939 to 750 (by 20.1%), 550 (by 41.4%), and 310 (by 66.9%) at 5.0, 30.0, and 300.0 nmol L−1 UA, correspondingly. These experiments were carried out by the optimized concentrations of luminol and Co2+, which were 5.0 × 10−6 mol L−1 and 1.0 × 10−8 mol L−1. The relative CL intensity-time profiles of different reactions were given in μL of luminol solution (5.0 × 10−6 mol L−1) was injected into the carrier stream and thereafter merged with the premixing stream of Co2+ (1.0 × 10−8 mol L−1) and UA . The relative CL intensity was recorded to test the stability and reproducibility of the luminol-Co2+ system. The FI system was regularly used over 8 h per day and the experiment lasted for 5 days. The results were listed in 100 2+ system was obviously inhibited, and the decrements of CL intensity were linear with the logarithm of UA concentrations in the range of 3.0–500.0 nmol L−1, giving the calibration equation of ΔICL = 106.8lnCUA + 33.1 (R = 0.9982) with the LOD of 1.0 nmol L−1 (3σ). The RSDs (n = 5) at 5.0, 50.0, and 300.0 nmol L−1 UA were 2.9%, 2.3%, and 1.9%, respectively. At a flow rate of 2.0 mL min−1, a complete analytical process including sampling and washing could be accomplished within 36 s, offering a ST of 100 h−1. Under the optimal experimental conditions, a series of UA standard solutions was determined. It was found that the CL intensity of luminol-Co−1 UA were over 4.0 × 104 for SO42−, NO3−, CO32−, oxalate, PO43−, glucose, and ethanol; 1.0 × 104 for Ba2+, Zn2+ and HCO3−; 9.0 × 103 for urea; 5.0 × 103 for Mg2+ and Ca2+; 5.0 × 102 for Ni2+ and Cr3+; 4.0 × 102 for Cu2+, Mn2+, and ascorbic acid; 1.0 × 102 for creatine and Fe3+/Fe2+; 20 for vitamin B12, respectively. Compounds abundant in human serum and urine such as lipids, proteins, and salts had no obvious interference for the determination of UA.To test for the interference of foreign substances on UA quantification, a potential interfering analyte was added in increasing amounts to the UA standard solution, and the relative CL intensity was examined with a relative error controlled at 5% level. The tolerable concentration ratios of foreign substances with respect to 10 ng mLImax of luminol-dissolved oxygen reaction in the presence of Co2+ increased from 59 to 939 and the Tmax changed from 4.4 to 4.0 s; it also can be seen that the Imax of luminol-Co2+ system in the presence of UA decreased obviously at increasing UA concentration with the identical Tmax of 4.0 s. The CL intensities generated by online ultrasonically degassed solutions and general solutions were compared, and results showed that the CL intensity from the degassed solutions decreased obviously with regard to the CL intensity from general solutions, suggesting the oxygen dissolved in solution with solubility of 0.26 mM [2+ can catalyze luminol-dissolved oxygen CL reaction, with O2−· being increased which led to the CL intensity from luminol being remarkably enhanced [2−· would react with UA and oxidize UA to allantoin [2+ CL reaction can be explained as the decreased amount of O2−·, which was consumed by UA oxidation reaction, causing the CL intensity of luminol-Co2+ system to be inhibited.  From  0.26 mM  was invoenhanced . While illantoin . Thus thμg mL−1, corresponding to porridge, vegetables, and meats intake, respectively.Three volunteers refrained from vegetables and meats intake for 1 day prior to the experiment. In the following 3 consecutive days, volunteers ingested diets at 12:30 am and 6:30 pm daily, with porridge, vegetables, and meats, respectively. On the 3rd day, the urine samples were periodically collected from volunteers and detected by the proposed CL method, with results being summarized in μg mL−1, which were in the normal range of 22–77 μg mL−1 [−1) were added. The results were listed in The proposed method was also applied to determining UA in samples of human serum. The serum samples were collected from two volunteers, with no. 1–3 from volunteer 1 and no. 4–6 from volunteer 2. It was found that the serum UA concentrations were 38.0 ± 1.5–51.4 ± 0.6  μg mL−1 . To vali2+ system for quantitative monitoring of urine UA excretion in 24 h at nanomolar level was described for the first time. In comparison with other reported methods for determining UA (−1, confirming the promise of this method for quantitative monitoring and real-time UA analysis.A rapid and cost-effective FI-CL method with luminol-Coining UA , this pr


The initial step in retroviral infection involves specific interactions between viral envelope proteins (Env) and specific receptors on the surface of target cells. For many years, little was known about the entry receptors for HTLV-1. During this time, however, functional domains of the HTLV-1 Env were identified by analyzing the effects of neutralizing antibodies and specific mutations in Env on HTLV-1 infectivity. More recent studies have revealed that HTLV-1 infectivity involves interactions with three different molecules: heparan sulfate proteoglycans (HSPG), the VEGF-165 receptor Neuropilin 1 (NRP-1) and glucose transporter type 1 (GLUT1). Here, we revisit previously published data on the functional domains of Env in regard to the recent knowledge acquired about this multi-receptor complex. We also discuss the similarities and differences between HTLV-1 and other deltaretroviruses in regards to receptor usage. This process is initiated by interaction of the surface subunit (SU) of the virally-encoded envelope glycoprotein (Env) with host cell receptors. Data from several independent groups have determined that three cell surface proteins are involved in HTLV-1 entry: glucose transporter 1 (GLUT1), neuropilin-1 (NRP-1) and heparan sulfate proteoglycans (HSPG). This knowledge provides a new perspective on HTLV-1 tropism and associated pathologies. This also raises many questions about the molecular events that occur during entry, in particular how the HTLV-1 SU interacts with each of the receptor molecules. The impact of the receptors on the selectivity and modulation of HTLV-1 infection n detail . In this2.Here, we will only briefly summarize the evidence that identified the HTLV-1 receptor molecules and led to the proposal of a multi-receptor model for HTLV-1 entry.2.1.+ T cells [The identification of GLUT1 started with the observation that expression of a fragment of the HTLV-1 SU in cells prevents medium acidification . Since i T cells .Drosophila genome, overexpression in transformed cells [In parallel, the role in HTLV-1 entry of another protein, Neuropilin 1 (NRP-1), was investigated. NRP-1 is a cell surface protein known to function as a co-receptor for certain heparin-binding pro-angiogenic cytokines, principally members of the vascular endothelial growth factor (VEGF) family, and for class 3 semaphorins (reviewed in ). It wased cells  and upreed cells . It was ed cells .The role of the third player of HTLV-1 entry, HSPGs, was the first of the three molecules identified to be important for HTLV-1 entry, through experiments showing that removal of HSPGs from cell surface abolished binding of the HTLV-1 SU as well as HTLV-1-Env mediated infection of target cells . Later s2.2.The fact that NRP-1 and GLUT1 can form a complex in the presence of HTLV-1 Env suggested that these two molecules work together to promote HTLV-1 entry. Such cooperation was also recently functionally demonstrated by data showing that inhibition of HTLV-1 entry into primary astrocytes required the blocking of the interactions with both NRP-1 and GLUT1 .Previously, HSPGs and NRP-1 have been shown to cooperate while functioning as co-receptors for the pro-angiogenic factor VEGF-165. Initial binding to cells is believed to involve interactions of VEGF-165 with both NRP-1 and heparin sulfate (HS) chains, followed by interaction of VEGF-165 with its signaling receptor VEGF-R . Structuin vitro and was sufficient to block HTLV-1 entry into primary T or dendritic cells [Recently, it has been shown that HSPG and NRP-1 also work together to promote HTLV-1 Env binding. Indeed, like VEGF-165, HTLV-1 SU binds via a HS-dependent manner to the same domain of NRP-1 important for binding VEGF-165  Figure. Virus a+ T cells by HTLV-1 [The notion that HSPG/NRP-1 are important for the initial binding step is supported by previous observations that HTLV-1 SU and particle binding are dramatically reduced following removal of HS chains or upon blocking NRP-1 interactions by incubation of target cells with either VEGF-165 exon 7 or exon 8-like peptides ,17. Blocy HTLV-1 . Previouy HTLV-1 . Howevery HTLV-1 , it leavy HTLV-1 ,13,14,193.3.1.Retroviral envelope glycoproteins (Env) are type-I transmembrane proteins synthesized as a precursor that is co-translationally imported into the endoplasmic reticulum, where it subsequently undergoes a number of maturation steps, including folding, oligomerization and N-glycosylations. Upon completion of these processes, the Env precursor passes through the Golgi apparatus to the trans golgi network, where it is cleaved by a cellular protease of the furin family into SU (Surface) and TM (Transmembrane) subunits . SU-TM cper se occurs through an initial binding step mediated by the SU and a subsequent fusion step mediated by the TM. Accordingly, the SU contains the determinants for receptor binding and the TM has the hydrophobic fusogenic peptide in its N-terminal region. During the intracellular journey of SU/TM complexes and prior to contact with the receptor, the TM is maintained by close association with the SU in a fusogenic-inactive metastable conformation in which the fusion peptide is buried. This prevents premature membrane fusion that could lead to Env inactivation and cell toxicity. The key event for TM activation is alteration of the SU/TM interactions, which allows the TM to acquire its fusogenic state. In the case of murine leukemia virus (MLV) Env, SU/TM dissociation is triggered upon binding of the SU to its receptor mCAT-1 and relies on the disruption of an intersubunit disulfite bond between cysteines in a CxxC motif in the SU and a CX6CC motif in the TM [Retrovirus entry occurs by fusion of the viral envelope either with the cell plasma membrane , or withn the TM ,27. For n the TM . The conn the TM .For all retroviruses, final activation of the fusion process occurs through successive refolding events of the TM resulting in the projection of the N-terminal fusion peptide. This involves the formation of a six-helix coiled-coil bundle that brings the viral and target membranes in close proximity and triggers membrane fusion. Since this review is focused on the interactions between the SU subunit and the cell receptors, details of these processes will not be described here but can be found in recent reviews ,30.3.2.env gene encodes a 488 amino acid precursor protein, which generates a 62 kDa protein (gp62) after addition of five N-glycan chains, four of which are in the SU  and a C-terminal domain (CTD) separated by a short proline-rich region (PRR) –34. AligIn gammaretroviruses, the PRR domain is believed to represent a hinge region that facilitates the conformational changes induced by receptor binding . The HTL3.3.One approach to identifying functional domains of retroviral envelope proteins, notably the receptor binding determinants, is to identify the epitopes of neutralizing antibodies which block Env-mediated functions. In the case of HTLV-1, analysis of the specificities of anti-HTLV-1 neutralizing antibodies identified four functional regions in the SU: two N-terminal regions located between residues 53–75 and 86–107 ,45, a ceThe 86–107 region was initially mapped from antibodies raised against HTLV-1 peptides corresponding to regions of the SU predicted to be hydrophilic. Adsorption of the neutralizing activities using a set of shorter peptides identified six residues (KKPNRN) at position 90 to 95 as the minimal neutralizing epitope in this domain  Figure . Previou3.4.Of the four neutralizing regions of HTLV-1 SU described above, the two N-terminal regions spanning residues 53–75 and 86–107 are located within the predicted HTLV-1 RBD. The second of these contains residues that were recently shown to be involved in interactions with NRP-1 and GLUT1  and 3. LHTLV-1 is a member of the deltaretrovirus family that includes the closely related primate T-lymphotropic viruses  and the more distantly related bovine leukemia virus (BLV). A structural model for the SU of bovine leukemia virus, which shares 36% identity in amino acid sequence with the HTLV-1 SU, was previously generated. This model predicted that the aa 97–106 region of the BLV SU, which is the homologous to the aa 92–103 region of the HTLV-1 SU, is exposed at the surface of the globular structure of the protein . The BLVThe C-terminal neutralizing region, aa 287–311, also includes a region previously reported to interact with a component of the proposed HTLV-1 receptor complex. Studies with chimera generated between the HTLV-1 SU and the SU of HTLV-2, which does not efficiently bind HSPG, indicate that the residues involved in binding HSPGs map to the CTD (aa 215–313). It would be of interest to investigate whether deletion in the 287–311 domain reduces HSPG binding, which would explain the neutralizing activity of antibodies directed to this part of the SU.3.5.As mentioned above, attachment to HSPGs is mediated by the C-terminal region of the SU while NRP-1 and GLUT1 binding occur via the N-terminal region. This distance between the HSPGs and NRP-1/GLUT1 interacting domains are compatible with successive binding events. In contrast, the interacting domains for NRP-1 aa 90–94) or GLUT1  are only separated by 16 residues . This is0–94 or G4.HTLV-1 is one of a group of deltaretroviruses that infect old world primates and humans, referred to as primate T-lymphotropic viruses (PTLVs). The viruses in this group that infect humans (HTLVs) are believed to have originated from interspecies transmission of simian T-lymphotropic viruses (STLVs). To date, the PTLV group includes three human viruses for which a simian counterpart is known  as well as two viruses (HTLV-4 and STLV-5) for which no counterpart has yet been identified –55.4.1.in vivo tropism and receptor usage of HTLV-1 and/or HTLV-2. The nucleotide sequence homology of the genomes of these two viruses is approximately 60% [in vivo tropism of HTLV-1 and HTLV-2 is not identical. HTLV-1 is found primarily in CD4+ T cells, while HTLV-2 is found primarily in CD8+ T cells [in vitro parallel the in vivo observations: HTLV-1 preferentially transforms CD4+ T cells and HTLV-2 preferentially transforms CD8+ T cells [+ cells, while HTLV-1 viruses with HTLV-2 Env preferentially transform CD8+ T cells [+ and CD8+ T cells up-regulate NRP-1 upon activation [+ or CD8+ T lymphocytes have a reciprocal phenotype in regards to the cell surface expression of HSPG and GLUT1, with a high HSPG/low GLUT1 phenotype for activated CD4+ T cells and a low HSPG/high GLUT1 for activated CD8+ T cells. Moreover, an increase in HTLV-1 but not HTLV-2 particle internalization was observed upon overexpression of HSPG in CD8+ T cells while transfection of GLUT1 in a CD4+ T cell line only enhanced the internalization of the HTLV-2 particle [Among the PTLV group, nearly all of the studies performed to date have examined the tely 60%  and theitely 60%   and the two residues involved in GLUT1 binding (D106 and Y114) are conserved between the HTLV-1 and HTLV-2 SU ,63 Figu. Moreove4.2.HTLV-1 and its simian counterpart STLV-1 are highly related viruses which can share up to 98% nucleotide sequence homology . The ent+ T cells that do not express detectable levels of HSPG, NRP-1 or GLUT1 [The HTLV-3 genome has been sequenced  and the or GLUT1 . Blockinor GLUT1 . These fenv gene is not yet available for STLV-5 but the sequence for one HTLV-4 isolate has been reported (accession NC_011800.1). Examination of this sequence reveals that both the KPxR motif and D106 and Y114 of the HTLV-1 SU are conserved in the amino acid sequence of the HTLV-4 SU  and the C-terminal domain (HSPG binding) of the HTLV-1 SU as critical determinants for HTLV-1 entry. Analysis of the primary sequence of other members of the PTLV family predicts that some members may also use NRP-1 and GLUT1 as entry receptors, but may differ from HTLV-1 in HSPG usage. Further studies are clearly needed to better define the receptor complex used by each PTLV member and understand the impact of virus/receptor interactions on the


Evolutionary models of human reproduction argue that variation in fertility can be understood as the local optimization of a life-history trade-off between offspring quantity and ‘quality’. Child survival is a fundamental dimension of quality in these models as early-life mortality represents a crucial selective bottleneck in human evolution. This perspective is well-rehearsed, but current literature presents mixed evidence for a trade-off between fertility and child survival, and little empirical ground to evaluate how socioecological and individual characteristics influence the benefits of fertility limitation. By compiling demographic survey data, we demonstrate robust negative relationships between fertility and child survival across 27 sub-Saharan African countries. Our analyses suggest this relationship is primarily accounted for by offspring competition for parental investment, rather than by reverse causal mechanisms. We also find that the trade-off increases in relative magnitude as national mortality declines and maternal somatic (height) and extrasomatic (education) capital increase. This supports the idea that socioeconomic development, and associated reductions in extrinsic child mortality, favour reduced fertility by increasing the relative returns to parental investment. Observed fertility, however, falls considerably short of predicted optima for maximizing total offspring survivorship, strongly suggesting that additional unmeasured costs of reproduction ultimately constrain the evolution of human family size. Maxim(a)Child survival is a critical component of reproductive success in traditional societies, with the large majority of deaths prior to adulthood occurring in the first 5 years of life . In factThere is strong experimental evidence that increases in fertility have negative consequences for offspring survival in avian and mammalian species where parental investment is substantial . ParalleMethodological limitations make it difficult to draw firm conclusions from this body of research ,10,28. O(b)In this study, we examine the relationship between fertility and <5 years child mortality in data compiled from national demographic surveys for 27 sub-Saharan African countries. Sub-Saharan Africa currently not only has the highest child mortality and fertility rates in the world, but also encompasses considerable socioeconomic and demographic diversity, with most countries at early- to mid-stages of the demographic transition. By using large sample data, including comparative information on birth histories, household socioeconomics and maternal health, we aim to address several limitations of past research. First, we quantify fertility–child survival relationships in each country adjusting for differences in both maternal somatic and extrasomatic capital in an attempt to exclude potential phenotypic confounds. Second, we harness the unique comparative nature of our data to quantify both socioecological and individual-level variation in the benefits of fertility limitation. Specifically, we examine how the magnitude of the trade-off relates to variation in national-level mortality and to individual differences in age at reproduction and maternal somatic and extrasomatic capital. We then consider relationships between fertility and estimated reproductive success, comparing observed fertility rates with predicted optima for maximizing total offspring survivorship. Finally, we conduct supporting analyses to explore the extent to which relationships between fertility and child survival may be explained by reverse causal mechanisms whereby high child mortality causes high fertility.2.(a)www.measuredhs.com). Data collection is designed to facilitate cross-national comparison, with each survey conducted using a standardized questionnaire and protocol. Notably, however, sampling is focused on the household unit, a concept difficult to fully standardize in the face of considerable cultural variation in residence patterns [The Demographic and Health Surveys (DHSs) are national demographic surveys, carried out throughout the developing world at regular intervals since the 1980s, and funded primarily by the United States Agency for International Development (patterns . For sampatterns . In 2006(b)at the time of survey for women across the full reproductive age range (i.e. 15–49 years) and the odds of survival for selected past births (see below). A multi-level approach corrects for statistical biases inherent in hierarchical data and enables direct estimates of residual variation in child survival associated with each level [Past studies of the quantity–quality trade-off have used standard regression techniques and focused analysis at the mother level, estimating the relationship between the completed fertility of post-menopausal women and total offspring survivorship. This method is sensitive to recall bias when based on retrospective birth histories, ultimately underestimating the number of child deaths. Focusing on post-menopausal women also biases the sample towards healthier mothers in high-mortality populations. Our analysis uses multilevel logistic regression  and is focused at the child level; estimating the relationship between fertility ch level . This maTo ensure that maternal and household characteristics correspond as closely as possible to the timing of child deaths, and to limit recall bias of less recent births and deaths, we restrict our analysis to predicting the survival of children born within 5–10 years prior to the survey date . Limiting analysis to recent births also ensures that children of older women relative to those of younger women are not disproportionality represented in the sample, which would bias estimates against the latter . To accoOur approach enables a robust comparative analysis of individual and socioecological variation in the life-history trade-off between fertility and child survival. In particular, multi-level modelling enables the estimation of individual-level coefficients across groups, accounting for group-level variation in the uncertainty of individual-level coefficients, and estimation of regression coefficients for particular groups . Specifi3.In total, our sample includes 163 827 births and 101 195 mothers. Sampled births are evenly split by gender, 22 per cent were firstborns, 34 per cent second or third born, 44 per cent fourth born or later. Of those children second or later born, 14 per cent had a preceding birth interval of 18 months or less, 51 per cent between 18 months and 3 years, and 35 per cent 3 years or more. Ninety-six per cent of births were singletons, with the remaining 4 per cent being multiple births. The overall odds of child survival for sampled births is 5.9 (i.e. one death for every 5.9 children surviving to their fifth birthday), ranging at the country level between 3.6  and 15.9 (Zimbabwe).(a)p < 0.001; electronic supplementary material, table S3). Adjusting for maternal characteristics  and rural versus urban residence, the effect of fertility is substantial and significant; for every additional maternal birth, the odds of survival for each child decrease by 14 per cent : 0.85–0.87, p < 0.001) compared with children born to mothers between the ages of 24–31 years. Child survival also increased with both somatic and extrasomatic capital. Children of mothers in the tallest quartile have 26 per cent  higher odds of survival than children of mothers in the shortest quartile. Secondary relative to no education increases odds of child survival by 39 per cent , and being from the wealthiest rather than poorest quintile of the population increases odds of child survival by 38 per cent . Additionally, children of formerly or never married mothers have 25 per cent  and 26 per cent  lower odds of survival, respectively, compared with children of married mothers. Finally, children born in rural areas have 7 per cent  lower odds of survival compared with children born in urban areas. The fully adjusted effect of fertility is actually slightly smaller than the effect of fertility unadjusted for differences in maternal capital, i.e. 14 per cent versus 16 per cent reduction in odds of child survival per additional birth respectively . Thus, there is little indication that phenotypic correlations mask the costs of reproduction in these data.In addition to fertility, child survival is influenced by maternal characteristics. Children born to younger mothers were less likely to survive. For example, children born to mothers of under 16 years experience 67 per cent lower odds of survival χ21 = 52.32, p < 0.001; electronic supplementary material, table S3). Each additional maternal birth reduces the odds of survival to age 5 years by 11 per cent in Chad  rising to 19 per cent in Lesotho . Model fit is also further improved by including a correlation between the country odds of survival (i.e. the random intercept) and the effect of fertility . This correlation is strongly negative (r = −0.739), implying that as the odds of survival increase, the effect of fertility on survival becomes more negative. Thus, the trade-off between fertility and child survival is of larger relative magnitude in countries where child survival is more likely overall. We find a strong signal of socioecological variation in the effect of fertility on child survival. Including a country-level random effect for fertility substantially improves model fit (p < 0.001), but only 10 per cent for mothers of 40 years or older . Second, despite higher odds of child survival overall, relatively tall and well-educated mothers experience larger relative costs of increasing fertility. For example, assuming mean characteristics on all other traits, each additional birth reduces the odds of child survival by 9 per cent for mothers in the shortest quartile , but by 12 per cent for mothers in the tallest quartile ; while each additional birth reduces the odds of child survival by 11 per cent for mothers with no education , compared with 20 per cent for mothers with a secondary education . As such, the survival advantage of children of tall and well-educated mothers appears conditional on relatively low fertility et al. [Our results illustrate that reproductive success depends not only on the effect of fertility on child survival (i.e. the trade-off function), but also on the child mortality risk for a given woman and local socioecology. For example, the odds of child survival are over four times greater in Zimbabwe than in Mali. This difference is so large that, despite a stronger trade-off function for Zimbabwean mothers , they aret al. ).(d)p < 0.001; electronic supplementary material, table S4). Hypothetical insurance strategies, whereby fertility is increased to compensate for predicted extrinsic mortality (i.e. largely unavoidable or care-independent deaths), also provide poor fit to the data. The costs of high fertility are not weakened by the exclusion of vulnerable neonatal deaths , which are arguably most likely to be caused by extrinsic factors in environments where mothers have restricted ability to seek treatment for birth complications or infant infections. Furthermore, as we demonstrate, fertility–child survival relationships actually increase in magnitude for healthy and well-educated mothers who are less likely to be exposed to extrinsic sources of child mortality. Finally, we show that, independent of fertility, tight birth intervals and twinning hold strong negative associations with child survival , which strongly suggests that replacement births cannot be the primary driving force underlying observed relationships. Adjusting for number of younger siblings, fertility reduces odds of child survival by 10 per cent for each additional birth The idea that variation in fertility can be understood as the optimization of life history is central to evolutionary accounts of human behaviour –3. The aIt is perhaps unsurprising that high fertility accounts for only a small proportion of child deaths. Given high energetic costs of parental investment, natural selection favours well-established physiological mechanisms that prevent conception and birth when maternal condition dictates a low chance of offspring survival . ConsequEven in the highest mortality country in our sample, where almost one in four children die by age 5 years, observed fertility rates fall dramatically short of estimated optima for maximizing reproductive success. This conclusion is shared by studies of both hunter–gatherer and traditional agriculturalist populations, where estimated trade-offs relationships are either weak ,22, or c(b)It is possible that the discrepancy between observed and predicted optimal fertility can be accounted for by an underestimation of the trade-off. Unlike many previous studies, we controlled for differences in maternal extrasomatic and somatic capital in estimating the effect of fertility on child survival. Further unmeasured factors, such as levels of allomaternal support  or genetConsidering only a single generation may also underestimate the full cost of high fertility. For example, high fertility may reduce investment in grandchildren, either because it is traded-off against post-reproductive survival , or beca(c)If not competition for survival, what additional costs of reproduction set the upper limits of high fertility? One possibility is competition between surviving offspring for later reproductive opportunities. For example, several studies have shown that fertility is negatively associated with offspring height ,55, whicFinally, in the absence of intense competition between offspring for post-natal investment, fertility in pre-demographic transition societies may remain primarily determined by availability of somatic reserves required for offspring production in pregnancy. Such costs may be largely masked when studying life-history trade-offs with observational rather than experimental data. As such, ‘natural fertility’ may be better studied in terms of energetic trade-offs between fertility and somatic maintenance and the related physiological constraints on reproduction . Ultimat


Airway hyperresponsiveness (AHR) is one of the cardinal features of asthma. Contraction of airway smooth muscle (ASM) cells that line the airway wall is thought to influence aspects of AHR, resulting in excessive narrowing or occlusion of the airway. ASM contraction is primarily controlled by agonists that bind G protein-coupled receptor (GPCR), which are expressed on ASM. Integrins also play a role in regulating ASM contraction signaling. As therapies for asthma are based on symptom relief, better understanding of the crosstalk between GPCRs and integrins holds good promise for the design of more effective therapies that target the underlying cellular and molecular mechanism that governs AHR. In this paper, we will review current knowledge about integrins and GPCRs in their regulation of ASM contraction signaling and discuss the emerging concept of crosstalk between the two and the implication of this crosstalk on the development of agents that target AHR. Contracpasmogen , loss ofpasmogen , 7, as wpasmogen . Moreovepasmogen , 10. Evepasmogen . Thus, tWe and others have shown that integrins may influence signaling events that contribute to AHR –14. Howeα and β chain. The expression of different integrins in ASM, their potential ligands and change in expression in asthma are detailed in α-actinin, and paxillin [2+ [Integrins are heterodimeric transmembrane proteins comprising one paxillin –21. Intepaxillin . It is tllin [2+ . 2+ concentration causing the phosphorylation of myosin light chain kinase (MLCK) and activation of myosin ATPase activity, and crossbridge cycling [Activation of integrins by either contractile or mechanical stimuli can result in two signaling events to cause ASM cell contraction. Firstly, integrin activation causes the phosphorylation of focal adhesion kinase (FAK) and association with paxillin, leading to reorganization of the cytoskeleton –26. Seco cycling –26. α, Gβ, and Gγ subunits [q- and Gs-coupled signaling, with Gq being linked to ASM contraction signaling and Gs being linked to relaxation signaling [q, which promotes GTP binding and dissociation of Gα from Gβγ subunits. The dissociated Gq will then bind to effector phospholipase C, which then hydrolyses phosphoinositol 4,5-bisphosphate (PIP2) into 1,2-diacylglycerol (DAG) and inositol 1,4,5-triphosphate (IP3). The net effect of these events is to increase the levels of intracellular Ca2+ as well as to activate cell contractile machinery through Ca2+ and protein kinase C-(PKC-) dependent mechanisms [GPCR spans the cell membrane seven times and transduces extracellular stimuli from the binding of cell surface ligands into intracellular second messengers. These second messengers are known as the heterotrimeric guanine nucleotide-binding protein (G proteins), which consists of Gsubunits . G protesubunits . GPCRs aignaling –43. Agonchanisms . Activatchanisms . Phosphochanisms . 12/13 protein can induce FAK activation and autophosphorylation in Swiss 3T3 cells, a fibroblast cell line, which is associated with integrin engagement signaling [β1 integrin plays a crucial role in negating the apoptotic effects of β-adrenergic receptor stimulation in cardiac myocytes via the involvement of FAK and PI3K/Akt pathways [q/11-coupled receptors in human endothelial cells may involve activation of calcium and PKC [There is emerging interest in crosstalk between integrins and GPCRs . For exaignaling . Arg-Glyignaling . Similarignaling , 53–56. ignaling . Integriignaling . Lysophoignaling . This dapathways . Furtherpathways . In anot and PKC . Collect12/13 and Rho A activation can increase the expression of the ECM protein Cyr61 (CCN1) in fibroblast, smooth muscle cells, and prostatic epithelial cells, respectively [The expression of ECM proteins can be regulated by GPCR ligands. For example, thrombin, sphingosine-1-phosphate, and LPA that signal through Gectively . Cyr61 sαVβ3 integrin and activates Gi signaling [o signaling by the P2Y2 receptor [μ opioid receptor, perhaps by altering interactions with G proteins [2+ influx and intracellular Ca2+ store release [Activated integrins organize supramolecular complexes consisting of cytoskeletal domains and associated receptors and signaling molecules that may contribute to the formation of specialized lipid microdomains, which are referred to as “lipid rafts” . Until nignaling . Integrireceptor . Recentlproteins . Moreove release . Taken tβ1 integrins in association with the actin cytoskeleton are able to attenuate adenylate cyclase (AC) activity. This in turn inhibits cholinergic regulation of L-type Ca2+ current in cardiac muscle contraction [β1 integrins in conjunction with the actin cytoskeleton have the ability to reduce β1-adrenergic receptor-induced L-type Ca2+ and enhance β2-adrenergic receptor-induced L-type Ca2+ current in the same cell [β1-integrin-induced activation of the FAK/PI3K/Akt pathway can inhibit β1-adrenergic receptor-mediated stimulation of L-type Ca2+ current in cardiac muscle contraction [β2-adrenergic receptor signaling to β1-adrenergic receptor signaling, and this may be mediated in part via β1 integrin-induced FAK/PI3K/Akt pathway.There are currently limited studies regarding the involvement of both integrins and GPCRs in the regulation of ASM cell contraction in healthy and asthmatic condition . Howevertraction . Subsequame cell . Recentltraction . This stβ2-adrenergic receptor stimulation of Ca2+ current is shown to be enhanced by β1 integrin via inhibition of cAMP/PKA and activation of Gi/ERK/cPLA2/AA signaling [β2-adrenergic signaling, which depends more on Gi/ERK/cPLA2/AA signaling (contraction) instead of Gs/AC/cAMP/PKA signaling (relaxation). Cheng and coworkers also elegantly show the relationship between β1 integrin and β-adrenergic receptor regulation of L-type Ca2+ current in neonatal rat ventricular myocytes [β1 integrin impedes β-adrenergic receptor-induced Ca2+ current via inhibition of AC/cAMP activity [β-adrenergic receptor crosstalk in a diseased heart in which it is associated with chronic overload of pressure, increased ECM proteins and integrin receptors. Remodeling of GPCR receptor functions in asthma may occur too as there is increased deposition of ECM proteins and altered expression of integrins in the asthmatic airways. Collectively, these studies suggest that integrin activation might play a role in GPCR-induced muscle contraction of the airways. As for atrial myocytes, ignaling . This stmyocytes . Overexpactivity . Similaractivity . These fβ2-adrenergic receptor leads to the activation of Gi rather than Gs. The activation of Gi and decreased Gs signaling translate into low AC activity and thus decreased cAMP accumulation [β2-adrenergic receptor may be the underlying cause of G protein switching [2+ and Gi signaling but stimulated by PKC [2+ release and influx, it suggests that integrins may modulate AC activity. This would explain the decrease in AC activity of human ASM cells cultured on laminin [In the context of ASM cell physiology there is only one study that links ECM proteins to GPCR-induced relaxation signaling . Exposurmulation . Alteredwitching . Since iwitching . Human Ad by PKC , 70. As  laminin , 72. Thiα protein, members of the Rho small GTPase family, and PKC [3 and histamine H1 receptors have been found within the caveolae enriched membrane fraction of human ASM [3 receptors and Gq protein cofractionate in caveolin-1 enriched ASM cell membranes [α-subunits and has been shown to regulate GPCR-mediated signaling [α-subunit to tyrosine kinase Fyn which then recruits Shc and Grb2. This sequence of events couples integrins to downstream signaling pathways such as Ras-ERK pathway. Caveolae function as negative regulators of cAMP accumulation. This suggests that integrin signaling regulated by caveolin-1 may serve as important modifier of GPCR signaling such as cAMP signaling in asthma. Caveolae are found in close proximity to peripheral sarcoplasmic reticulum and mitochondria, suggesting that caveolae may play a role in the spacial coordination of Ca2+-handling channels and organelles, which are implicated in ASM contraction signaling [GPCR signaling has been shown to be highly compartmentalized and disruption of this subcellular organization may affect GPCR function . Integri and PKC . Dependi and PKC –78. Muscuman ASM . Moreoveembranes . Caveoliignaling , 81. Cavignaling . In addiignaling , 83. Col2+. The addition of RGD peptide at millimolar range elicited increased levels of intracellular Ca2+ concentration [2+ store and lysosome-like organelles by RGD peptide [2+ signaling in regulating smooth muscle contraction. In support, α7β1 integrin has been implicated to regulate transient elevation of intracellular-free Ca2+ concentration from both IP3 evoked Ca2+ release from intracellular stores and extracellular Ca2+ influx through voltage-gated L-type Ca2+ channels in skeletal muscle cell [Integrins have also been implicated to regulate vascular smooth muscle cell contraction by mobilizing intracellular Cantration . This ac peptide , 84 suggcle cell . β release. TGFβ is linked to thickening of ASM layer and deposition of collagen. Tatler and colleagues show that GPCR agonists, LPA and methacholine, induced TGFβ activation via integrin αvβ5 by ASM cells [β immunoreactivity within the airway epithelium and increases the thickness of the subepithelial collagen layer, which is indicative of an acute alteration in airway collagen dynamics [Lastly, it is worth noting that GPCR agonists may promote ECM protein production, either directly, or indirectly by promoting autocrine TGFSM cells . In suppdynamics . These sIn summary, integrins may play a role in regulating GPCR-induced ASM cell contraction signaling in asthma. This finding may offer explanations for increased contractility of ASM cells in asthma in which ECM proteins and their binding receptor integrins are highly expressed. Thus, integrins may be an interesting therapeutic target to inhibit ASM contraction signaling in asthma. However, the development of integrin antagonists has proven to be challenging. The role of integrins in asthma is complex as multiple integrins may participate to exert asthma symptoms, making it difficult to specifically target integrins that are involved in ASM contraction signaling. Perhaps targeting “linker proteins” that link the crosstalk between integrins and GPCRs in ASM contraction signaling is a possible therapeutic strategy for the treatment of AHR in asthma. One such possible target is caveolin-1 that may regulate integrins and GPCRs crosstalk. Other possible targets may be those which participate in G protein switching that are induced by integrin activation. Nonetheless, further understanding of the mechanisms behind integrin and GPCR crosstalk in ASM cell contraction signaling will enhance the development of more tailored therapy in the future for asthma treatment where AHR is a feature.


Dietary supplementation with ursolic acid (UA) prevents monocyte dysfunction in diabetic mice and protects mice against atherosclerosis and loss of renal function. The goal of this study was to determine the molecular mechanism by which UA prevents monocyte dysfunction induced by metabolic stress.S-glutathionylation, a measure of cellular thiol oxidative stress, and normalized actin-S-glutathionylation. UA also restored MAPK phosphatase-1 (MKP1) protein expression and phosphatase activity, decreased by metabolic priming, and normalized p38 MAPK activation. Neither metabolic stress nor UA supplementation altered mRNA or protein levels of glutaredoxin-1, the principal enzyme responsible for the reduction of mixed disulfides between glutathione and protein thiols in these cells. However, the induction of Nox4 by metabolic stress, required for metabolic priming, was inhibited by UA in both THP-1 monocytes and peritoneal macrophages.Metabolic stress sensitizes or “primes” human THP-1 monocytes and murine peritoneal macrophages to the chemoattractant MCP-1, converting these cells into a hyper-chemotactic phenotype. UA protected THP-1 monocytes and peritoneal macrophages against metabolic priming and prevented their hyper-reactivity to MCP-1. UA blocked the metabolic stress-induced increase in global protein-UA protects THP-1 monocytes against dysfunction by suppressing metabolic stress-induced Nox4 expression, thereby preventing the Nox4-dependent dysregulation of redox-sensitive processes, including actin turnover and MAPK-signaling, two key processes that control monocyte migration and adhesion. This study provides a novel mechanism for the anti-inflammatory and athero- and renoprotective properties of UA and suggests that dysfunctional blood monocytes may be primary targets of UA and related compounds. •Ursolic acid protects monocytes from metabolic stress-induced dysfunction or “metabolic priming” by preventing the induction of Nox4.•Ursolic acid restores p38 MAP kinases activation and chemotactic responses to MCP-1 in monocytes exposed to metabolic stress.•S-glutathionylation, including actin-S-glutathionylation, in metabolically stressed monocytes.Ursolic acid prevents increased protein •Ursolic acid prevents MKP-1 degradation and restores MKP-1 activity in metabolically stressed monocytes. Ursolic acid (UA), a cyclic triterpenoid, is an anti-inflammatory phytochemical widely distributed within the plant kingdom and found in medicinal and traditional herbs, as well as a large number of fruits in vivo and accelerated atherosclerotic lesion formation, suggesting that monocyte priming by metabolic stress may be a novel, fundamental mechanism underlying atherosclerosis and other chronic inflammatory diseases Atherosclerosis is characterized by chronic infiltration of inflammatory cells, particularly monocytes, into the subendothelial space in the vascular wall 2O2) 2O2 and activates signaling pathways, such as insulin S-glutathionylated thiols, i.e., mixed disulfide bonds between protein thiols and glutathione S-glutathionylation is an important post-translational modification in redox signaling and can inhibit or activate protein function S-glutathionylation in response to metabolic stress increases actin turnover in monocytes, which appears to contribute to enhanced monocyte adhesion to endothelium and accelerated monocyte migration and tissue infiltration S-glutathionylation results in the hyperactivation of MAPK signaling pathways that control monocyte adhesion and migration Nox4 is one among the seven members of the NAPDH oxidase family whose function is to transport electrons across a membrane to produce reactive oxygen species (ROS) S-glutathionylation, specifically, S-glutathionylation of two important redox signaling proteins essential for monocyte adhesion and migration, actin and MKP-1. Based on these data, we propose a novel mechanism of action that may explain many of the anti-inflammatory properties of UA. Our study highlights the therapeutic potential of UA and related compounds.Current prevention strategies and treatments for metabolic and chronic inflammatory diseases focus mainly on reducing or preventing inflammation and oxidative stress. Due to their relatively low cost and low toxicity, phytochemicals may provide an attractive alternative to current approaches in disease prevention and management. A number of compounds have shown promise for reducing or even reversing symptoms of diseases characterized by chronic inflammation ® Invitrogen, Grand Island, NY, and all primers and supplies for qPCR were purchased from Invitrogen, Grand Island, NY.Unless stated otherwise, chemicals were purchased from Sigma-Aldrich, St. Louis, MO, cell culture reagents from Gibco6 cells/ml were cultured at 37 °C for 20 h in RPMI-1640 (Hyclone and Cellgro®) containing, 10% fetal bovine serum (FBS), 5.5 mM d-glucose, 2% Glutamax, 1% sodium pyruvate (Cellgro®), 1% penicillin/streptomycin (Cellgro®), 1% HEPES, 0.1% β-2-mercaptoethanol, and supplemented with either phosphate buffered saline (PBS) or freshly isolated native human LDL (100 µg/ml in PBS) plus d-glucose . l-glucose does not increase monocyte priming ® mouse pan B (B220) and Dynabeads® mouse pan T (Thy 1.2)). This procedure routinely increased the macrophage content of the isolate from approximately 40% CD68-positive cells to greater than 95% CD68 positive cells. Purified macrophages were cultured in Teflon bags under non-adherent conditions d-glucose Monocyte priming was induced as described previously LDL was isolated by KBr-gradient ultracentrifugation from pooled plasma from healthy blood donors and purified by gel-filtration chromatography, filter-sterilized and characterized as described previously 2. The membrane was washed and cells removed from the upper side of the filter. Transmigrated cells were stained with Diff-Quik® Set  and counted in four–five separate high power fields at 400× magnification under a light microscope.THP-1 monocytes or purified peritoneal macrophages were primed with HG+LDL for 20–24 h in the presence of either vehicle  or UA, then loaded into the upper wells of a 48-well modified Boyden chamber . The lower wells contained either vehicle or 2 nM MCP-1 . A 5 µm polyvinyl pyrrolidone-free polycarbonate filter membrane was layered between the upper and lower chambers, and the chamber was incubated for 2 h for THP-1 monocytes or 3 h for peritoneal macrophages at 37 °C and 5% COCells were washed with ice-cold PBS and lysed on ice in RIPA lysis buffer  with protease inhibitor and/or phosphatase inhibitors. Aliquots with equal amounts of protein were loaded and separated on an 8% or 10% SDS-PAGE gel. Proteins were transferred to polyvinylidene difluoride membranes  and probed using specific antibodies. The following antibodies were used: Nox4 Briefly, total RNA was extracted using the PureLink RNA Mini Kit and quantified using a NanoDrop spectrophotometer . Total RNA (1 μg) was synthesized into cDNA using the Maxima First Strand cDNA Synthesis Kit . Taqman probes were used for all genes  using the cycling conditions described by the manufacturer. No amplification was detected in no-template control wells. Gene expression levels were normalized to GAPDH and mRNA fold-change relative to control wells was calculated using the ΔΔCt method 2PO4.MKP-1 activity was determined with a modification of the commercially available MalachiteGreen-based PTP assay . Briefly, to assess MKP-1-specific PTP activity, lysates were analyzed both in the absence and presence of 40 µM sanguinarine (SG), a specific inhibitor of MKP-1 (34). SG-sensitive PTP activity was attributed to MKP-1. Briefly, assays were initiated by adding 10 µl of phosphotyrosine peptide substrate to cell extracts (2 µg protein) diluted in 20 mM Tris–HCl (pH 7.5), 150 mM NaCl, 1% NP-40 and warmed to 30 °C. The reaction was stopped after 10 min. MKP-1 activity was assayed spectrophotometrically as the amount of inorganic phosphate released using a VersaMax . Phosphate released by MKP-1 was quantified from a standard curve prepared with known amounts of KHP<0.05 level.Data were analyzed using ANOVA . Data were tested for use of parametric or nonparametric post hoc analysis, and multiple comparisons were performed by using the Least Significant Difference method. All data are presented as mean±SE of at least 3 independent experiments unless stated otherwise. Results were considered statistically significant at the 2O2, on monocyte chemotaxis with a median inhibitory concentration (IC50) of 0.45 µM 2O2-treated THP-1 monocytes 50 of 0.4 µM, indicating this inhibition may occur through a similar mechanism. Importantly, UA treatment alone did not affect MCP-1-stimulated chemotaxis in unprimed monocytes  -1 cells B and D. -1 cells D.S-glutathionylation, including the increased S-glutathionylation of actin S-glutathionylation induced by metabolic stress  is mediated by increased cellular protein-c stress A and B. d by 75% C. At the priming D, suggesol cells A.Fig. 2US-glutathionylated proteins in THP-1 monocytes S-glutathionylated proteins and prevents the conversion of monocytes into the proatherogenic primed phenotype P=0.073). The inhibitory effect of UA on protein-S-glutathionylation in metabolically primed monocytes can therefore not be explained by induction of Grx1.Glutaredoxin-1 (Grx1) is the primary cytosolic enzyme that specifically reduces S-glutathionylation, resulting in MKP-1 inactivation and subsequent proteasomal degradation MAPK phosphatase-1 (MKP-1) is a redox sensitive phosphatase that regulates the phosphorylation and activity of p38 and Erk proteins S-glutathionylation, inactivation and degradation.Loss of MKP-1 activity leads to the hyperactivation of p38, as measured by the phosphorylation of p38, both in resting THP-1 monocytes and in response to MCP-1 stimulation 2O2 mediates metabolic stress-induced monocyte priming and the S-glutathionylation of actin and MKP-1 induced by metabolic stress S-glutathionylation induced by metabolic stress and rescued MKP-1 degradation and activity without altering Grx-1 expression, we next investigated whether UA is able to prevent the induction of Nox4 we observed in metabolically-primed THP-1 monocytes. Indeed, we found that at 3 µM, UA inhibited the metabolic stress-induced increase in Nox4 protein levels by 77%  is a structural isomer of UA that differs only in the position of one methyl group. Despite its structural similarities to UA, OA is 3.5-fold less potent than UA in inhibiting accelerated monocyte chemotaxis induced by metabolic stress , members of the O subclass of forkhead transcription factors (FOXO), and SMA/MAD related transcription factor (SMAD) Alternatively, UA may suppress Nox4 expression by inhibiting translational events. Nox4 translation was shown to be regulated by serum in vivo, including its ability to improve kidney function and block atherosclerotic lesion formation in diabetic mice While the exact mechanisms through which UA prevents metabolic stress-induced Nox4 expression remains to be elucidated, the ability of UA to block Nox4 induction, and thus metabolic priming in monocytes, may explain UA’s potent anti-inflammatory properties In summary, we found that UA prevents metabolic stress-induced monocyte priming by blocking Nox4 induction, thereby inhibiting the dysregulation of two important thiol redox-sensitive signaling mechanisms involved in monocyte adhesion and migration, i.e. actin turnover and MAPK signaling NIH (AT-006885) and the Morrison Trust (F065300). S.U. was supported by a fellowship from the Translational Science Training (TST) Across Disciplines program at the University of Texas Health Science Center at San Antonio, with funding provided by the University of Texas System's Graduate Programs Initiative.This work was supported by grants to R.A. from the


Plasmodium falciparum malaria. Recently, ‘parasite-derived’ uric acid (UA) was shown to activate human immune cells in vitro, and plasma UA levels were associated with inflammatory cytokine levels and disease severity in Malian children with malaria. Since UA is associated with endothelial inflammation in non-malaria diseases, we hypothesized that elevated UA levels contribute to the endothelial pathology of P. falciparum malaria.Inflammatory cytokinemia and systemic activation of the microvascular endothelium are central to the pathogenesis of We measured levels of UA and soluble forms of intercellular adhesion molecule-1 (sICAM-1), vascular cell adhesion molecule-1 (sVCAM-1), E-selectin (sE-Selectin), thrombomodulin (sTM), tissue factor (sTF) and vascular endothelial growth factor (VEGF) in the plasma of Malian children aged 0.5–17 years with uncomplicated malaria  and non-cerebral severe malaria . In 69 of these children, we measured these same factors once when they experienced a malaria episode and twice when they were healthy . We found that levels of UA, sICAM-1, sVCAM-1, sE-Selectin and sTM increase during a malaria episode and return to basal levels at the end of the transmission season (p<0.0001). Plasma levels of UA and these four endothelial biomarkers correlate with parasite density and disease severity. In children with UM, UA levels correlate with parasite density , sICAM-1  and sTM  levels. After adjusting for parasite density, UA levels predict sTM levels.Elevated UA levels may contribute to malaria pathogenesis by damaging endothelium and promoting a procoagulant state. The correlation between UA levels and parasite densities suggests that parasitized erythrocytes are one possible source of excess UA. UA-induced shedding of endothelial TM may represent a novel mechanism of malaria pathogenesis, in which activated thrombin induces fibrin deposition and platelet aggregation in microvessels. This protocol is registered at clinicaltrials.gov (NCT00669084). Plasmodium falciparum (Pf) malaria, a major cause of childhood morbidity and mortality in sub-Saharan Africa, is characterized by significant impairment of the microvascular endothelium P. falciparum-infected red blood cells (PfRBCs) is a critical initiating event. The cytoadherence of PfRBCs not only activates MVECs, but also concentrates PfRBCs in microvessels, where mature schizonts release merozoites and parasite-derived virulence factors  et al., submitted). The intravascular dissolution of parasite-derived UA precipitates may also contribute to excess plasma UA levels.Although the mechanisms of endothelial activation, damage and dysfunction in malaria have not been fully elucidated, the systemic microvascular sequestration of in vitroUA is a weak organic acid present mainly as monosodium urate at physiological pH, and is found as microscopic crystals in diseases  associated with elevated UA levels P. falciparum malaria and with disease severity P. falciparum malaria.In Malian children, we recently found that plasma UA levels increase during an acute episode of All protocol activities were approved by the Ethics Committee of the Faculty of Medicine, Pharmacy and Odontostomatology at the University of Bamako, Mali, and the Institutional Review Board of the U.S. National Institute of Allergy and Infectious Diseases. Written informed consent was obtained from the parent or guardian of all children. The protocol is registered at clinicaltrials.gov (NCT00669084).et al., in preparation). Our previous report on UA and cytokines used data collected from the 2008 transmission season This study was conducted within a 4-year (2008–2012) prospective cohort study of malaria incidence in three rural Malian villages   no symptoms or signs of NCSM (see below), and (iv) no other etiology of fever discernible on clinical examination. Children with NCSM had any parasite density and met one or more of the following criteria: prostration, severe anemia (hemoglobin ≤5 g/dl), cessation of eating and drinking, and repetitive vomiting. Children with UM and P. falciparum density <100,000/µl were treated daily with artesunate (4 mg/kg) plus amodiaquine (10 mg/kg) for 3 days, given orally. Children with NCSM were treated intravenously with artesunate, followed by the oral dose regimen for UM. Children with UM and P. falciparum density ≥100,000/µl were treated as for NCSM. All children were confirmed to have undetectable parasitemia 72 h after presentation.UM was defined by (i) fever  with or without other symptoms of malaria , (ii) the presence of any asexual Venous blood (2–10 ml) was obtained from a sub-cohort of 69 healthy children aged 3–11 years before and after the 2009 transmission season, at which times they had undetectable parasitemia. Venous blood was also obtained from all children who presented with malaria. Blood samples were collected by venipuncture in sodium heparin Vacutainers®  and transported for 2 h on ice to the University of Bamako. Plasma was separated from whole blood by centrifugation at 2500 rpm for 10 min at ambient temperature, aliquotted, immediately stored at −80°C, shipped in a liquid nitrogen dry shipper to NIAID, and maintained at −80°C until use.Plasma samples were thawed at room temperature and centrifuged at 14000 rpm for 10 min at 4°C before measuring UA and endothelial biomarker levels. UA levels were quantified in triplicate by a colorimetric method with a linear detection range of 0.22–30 mg/dl using a QuantiChrom™ Uric Acid Assay Kit . Levels of sICAM-1, sVCAM-1 and sE-Selectin were quantified using a Human Adhesion Molecule MultiAnalyte Profiling Base kit ; samples were diluted 30-fold according to the manufacturer’s instructions and analyzed using a Luminex200™ flow-based sorting and detection platform . sTM levels were measured using a Human Thrombomodulin Quantikine ELISA (R&D Systems); samples were diluted 10-fold according to the manufacturer’s instructions and optical densities measured using a microplate reader set to 450 nm with 750 nm wavelength correction. sTF levels were quantified using the Human Coagulation Factor III/Tissue Factor Quantikine ELISA (R&D Systems); samples were diluted 2-fold and assayed according to the manufacturer’s instructions. VEGF levels were measured using the Human VEGF single bead Luminex Kit (Invitrogen); samples were diluted 3-fold, assayed according to the manufacturer’s instructions, and analyzed using a Luminex200™ flow-based sorting and detection platform (Invitrogen).10 biomarker level as a response and log10 UA and parasite density as covariates. Residuals plots were examined to check for large deviations from the normality assumption. Descriptive statistics and univariate analyses were made using GraphPad version 5.01 , and the relationships between variables were measured using R version 2.13.0 . p-values <0.05 were considered significant.Due to technical issues, there is a small number of children with missing UA or endothelial biomarker levels [UM  and NCSM ]. For clarity of presentation, we refer to ‘487’ children with UM and ‘68’ children with NCSM throughout this report. To compare groups of children, Fisher’s exact test was used for categorical variables (sex) and the Mann-Whitney test for continuous variables . The Wilcoxon signed rank test was used to compare the changes in levels of UA and biomarkers in children either from before the transmission season to a malaria episode, or from a malaria episode to after the transmission season. Confidence intervals on the fold-change over time used t-tests on the log-transformed responses. Spearman’s correlation test was used to measure correlations between UA levels, biomarker levels and parasite densities. Spearman’s correlation is based on ranks, so it measures the correlation for the bulk of the data and is not overly influenced by points at the extremes. We sometimes additionally check for continued significance after adjusting p-values with Holm’s multiple comparison correction test because we were testing six biomarkers vs. 52.9% for NCSM, p = 1.0) or median age  , and parasite densities increase with disease severity   or NCSM (n = 68) in the 2009 transmission season. Most children with NCSM had prostration, repetitive vomiting, cessation of eating and drinking or some combination of these criteria. Only 2 children with NCSM developed severe anemia. None of the children died. The groups of children with UM and NCSM did not differ significantly in sex  Table 1.To determine whether plasma UA levels increase during a malaria episode and return to basal levels thereafter, we measured UA levels in paired plasma samples from 69 children before and after the 2009 transmission season, when they were healthy and without parasitemia, and at their first episode of malaria in the interim. We found that geometric mean basal UA levels increase 1.23-fold  during a malaria episode and then decrease by a factor of 1/1.23 = 0.82  to basal levels after the transmission season Table 2.To determine whether plasma levels of UA and endothelial biomarkers increase with disease severity, we compared these levels in 487 and 68 children with UM and NCSM, respectively. We found that levels of UA, sICAM-1, sVCAM-1, sE-Selectin and sTM are higher in children with NCSM compared to those with UM Table 3.P. falciparum densities and found only a small, barely significant, correlation  in a clinically-homogeneous group of 487 children with UM. Because we have no information on the developmental stage of parasites, it is tentative whether we can use P. falciparum density as a surrogate for the biomass of recently ruptured schizonts, making it difficult to interpret this correlation. Nevertheless, in exploring relationships between parasitemia and endothelial pathology, we found much stronger correlations between parasite densities and levels of sE-Selectin , sICAM-1 , sTM  and sVCAM-1 . These latter correlations are still highly significant after using Holm’s correction to adjust for the fact that we tested six biomarkers. No correlations were found for sTF or VEGF.We explored whether plasma UA during a malaria episode is associated with To explore whether parasite-derived UA (in addition to other parasite-derived factors) contribute to the endothelial pathology of malaria, we analyzed correlations between UA and endothelial biomarker levels in 487 children with UM. Interestingly, UA levels correlate only with sICAM-1  and sTM  levels; these correlations remain significant after using Holm’s correction to adjust for testing six biomarkers. We used Spearman correlations, which are driven by the bulk of the data, located in the middle range of UA levels Fig. 2. 10 biomarker as a response, we found that, for a fixed log10 parasite density, there is a 1.29-fold  increase in sTM for each log10 increase in UA. Fold-changes in the other five biomarkers are not significant or the linear model assumptions were not met.To further investigate a specific role for UA in the endothelial pathology of malaria, we investigated whether UA levels predict those of six endothelial biomarkers – independent of the effects of parasite density. Using a series of simple linear models with each logP. falciparum malaria by causing endothelial pathology, as it does in non-malaria diseases. In support of this hypothesis, we found that basal plasma levels of UA and four endothelial biomarkers  increase during episodes of UM and further increase with disease severity. In a cohort of 487 Malian children with UM, the levels of UA and these four endothelial biomarkers correlated with parasite density. This finding suggests that parasitized RBCs, as a possible source of excess UA and other virulence factors, contribute directly to endothelial pathology. In this same cohort, we found that plasma UA levels correlate significantly with sICAM-1 and sTM levels. After adjusting for parasite density, we found that UA levels significantly predict sTM levels. This finding suggests a model of malaria pathogenesis in which excess UA induces shedding of TM from the surface of MVECs, producing a pro-coagulant state. This is because the shedding of TM, which normally binds and keeps thrombin inactivated at the cell surface, leads to an excess of active thrombin, which converts fibrinogen to fibrin and induces platelet activation and aggregation. Such activated platelets induce inflammatory gene expression profiles, apoptosis and blood-brain barrier compromise in human brain endothelial cells in vitroWe hypothesized that UA contributes to the pathogenesis of P. falciparum malaria, on the other hand, offers a model in vivo system in which the role of UA in endovascular pathogenesis can be efficiently studied in humans. This is because the development of acute episodes of malaria and their clinical resolution can be intensively followed over time in a significant number of patients. Also, dermal biopsies from children with P. falciparum malaria can provide ideal histological samples to investigate the relationship between UA levels and the degree of endothelial pathology, for example, by examining MVECs for upregulated expression or shedding of cell surface molecules involved in microvascular inflammation and coagulation. The acute nature of a malaria episode and its rapid resolution with effective antimalarial drugs offers additional opportunities to investigate temporal relationships between UA levels and endothelial dysfunction in vivo. Using non-invasive procedures to repeatedly measure indices of endothelial function  P. falciparum malaria syndromes. Our study also suggests that the endothelial pathology and thrombocytopenia (due to platelet aggregation) of P. vivax malaria may in part result from UA-mediated shedding of TM from the surface of MVECs.Studying the role of UA in the pathogenesis of endovascular diseases such as cardiovascular disease, hypertension and diabetes has been challenging in vitro. How UA may induce shedding of TM from MVECs in patients with malaria has not yet been specifically investigated, but studies of non-malaria diseases provide some initial guidance. One possibility is that UA is transported into MVECs through organic anion transporters (OATs) and then activates the expression and release of proteases, which cleave ICAM-1 and TM from the cell surface To our knowledge, we report the first data implicating UA in the endothelial pathology of human malaria. Our study has several strengths. The major finding of an association between UA and sTM levels is based on data from a large number (n = 487) of Malian children with UM and is adjusted for the effects of parasite density – a surrogate for UA precipitates and other parasite-derived virulence factors that we have not measured. Also, we show that sTM levels increase in UM and further increase in NCSM, thus implicating the shedding of TM in the pathogenesis of malaria in our study population. Finally, our study suggests that parasitized RBCs contribute only modestly to the excess UA in plasma; however, regardless of the source of increased UA levels, their association with increased sTM levels justifies a variety of experiments to evaluate the potential role of soluble UA in modulating the shedding of TM from MVECs. One potential source of excess soluble UA in plasma is the cytosol of schizont-infected RBCs, which lyse during parasite development in microvessels. Whether this parasite-derived UA mediates the shedding of TM from the surface of MVECs can now be tested ex vivo in P. vivax-infected RBCs and short-term-cultured PfRBCs , may also contribute to elevated UA levels by dissolving into plasma after schizont rupture. This possibility is supported by strong correlations between parasite density (a surrogate for parasite-derived UA precipitates) and four biomarkers of endothelial pathology. While our study is obviously unable to quantify the contribution of parasite-derived UA precipitates to the soluble UA level in plasma, we have observed the adherence of UA precipitates to dermal MVECs after schizont rupture in vitro . Future studies are needed to directly visualize parasite-derived UA precipitates in the microvessels of patients with malaria and follow their dissolution over time. Whether these UA precipitates directly activate MVECs to shed sICAM-1, sVCAM-1, sE-Selectin and sTM in vitro can also be investigated. If these or other endothelial responses are observed, determining whether parasite-derived UA precipitates stimulate the same inflammatory response pathways as monosodium urate crystals may have clinical relevance.One significant limitation of our study is that it does not identify the sources of excess UA in our cohort of Malian children with malaria. Possible sources include fasting and the release of free iron, which may upregulate xanthine oxidase activity. Parasite-derived UA precipitates, which have been directly observed


We hypothesized that the cytotoxic T lymphocytes(CTLs) play a pivotal role in the pathogenesis of human T lymphotropic virus type I (HTLV-I)-associated myelopathy/tropical spasticparaparesis(HAM/TSP).One of the most striking features of the cellular immune response in the patients with HTLV-I-associated myelopathy/tropical spastic paraparesis (HAM/TSP) is the highly increased numbers of HTLV-I-specific cytotoxic T lymphocytes (CTLs) in the circulation of the blood and the cerebrospinal fluid, nevertheless HTLV-I proviral load in the PBMC remains high in the patients.To determine the CTL’s association with the pathogenesis of HAM/TSP in the CNS, we developed novel methods of in-situ detection for HTLV-I-specific CTL using HLA/antigen peptide tetramer and for the cells expressing HTLV-I viral protein. We visualized the HTLV-I specific CTLs and HTLV-I-infected cells in autopsied spinal cords of the patients with HAM/TSP.We demonstrated that HTLV-I-specific CTLs expressing cytotoxic molecules accumulated in the spinal cords from three patients with HAM/TSP and that HTLV-I exclusively infected CD4 positive T lymphocytes but neither resident cells nor macrophages. The phenotype of apoptotic cells was HTLV-I infected CD4+ T lymphocytes or HTLV-I non-infected oligodendrocytes.The findings suggest a unique pathogenesis for the neuroinflammatory disease that an inflammation of the central nervous system is attributed to the interaction between HTLV-I-infected CD4+ T cells and HTLV-I-specific CD8+ CTLs from the periphery.