These proteases cleave PARs at established sites with the extracellular N-terminal domains, and expose tethered ligands that stabilize conformations of the cleaved receptors that activate the canonical pathways of G protein- and/or β-arrestin-dependent signaling. However, a growing number of proteases have been identified that cleave PARs at divergent sites to activate distinct patterns of receptor signaling and trafficking. The capacity of these proteases to trigger distinct signaling pathways is referred to as biased signaling, and can lead to unique patho-physiological outcomes. Given that a different repertoire of proteases are activated in various patho-physiological conditions that may activate PARs by different mechanisms, signaling bias may account for the divergent actions of proteases and PARs. Moreover, therapies that target disease-relevant biased signaling pathways may be more effective and selective approaches for the treatment of protease- and PAR-driven diseases. Thus, rather than mediating the actions of a few proteases, PARs may integrate the biological actions of a wide spectrum of proteases in different patho-physiological conditions.In addition to their role in protein degradation and digestion, proteases can also function as hormone-like signaling molecules that regulate vital patho-physiological processes, including inflammation, hemostasis, pain, and repair mechanisms. Certain proteases can signal to cells by cleaving protease-activated receptors (PARs), a family of four G protein-coupled receptors. PARs are expressed by almost all cell types, control important physiological and disease-relevant processes, and are an emerging therapeutic target for major diseases. Most information about PAR activation and function derives from studies of a few proteases, for example thrombin in the case of PAR With over 800 members in mammals, G protein-coupled receptors (GPCRs) are the largest family of cell-surface signaling proteins. They are receptors for an extraordinary range of structurally diverse agonists in the extracellular fluid, including endogenous hormones, neurotransmitters, and paracrine regulators, as well as multiple exogenous ligands , 2. Due The early two-state model of receptor function suggested that a receptor adopts active conformation upon ligand binding. This model considered only one active state, leading to a single functional readout. However, increased understanding of receptor signaling has revealed that different ligands can initiate distinct signaling events through the same GPCR. The heterogeneity of signaling events by a single GPCR can include different maximum responses from a single pathway  or activation of distinctly different signaling pathways by different agonists. The capacity of different agonists to initiate signaling of the same GPCR by distinct mechanisms is referred to as biased agonism or signaling , 11, and1–4) that belong to group A rhodopsin-like GPCR subfamily. The first family member, PAR1, was identified as a receptor for thrombin, a serine protease coagulation factor . The PARs are a family of four GPCRs , that mimic the tethered ligand domain can also activate certain PARs directly, without the requirement for proteolysis Figure C. Peptid of PAR4 , 18, 20.and PAR4 , 30.1 triggers endothelial barrier permeability, whereas PAR1-AP (SFLLRN-NH2) has no significant effect , many other factors also affect patho-physiological outcome of PAR activation. These factors include the availability of activated proteases as well as the existence of regulatory and accessory proteins in different tissues and cell types.The availability of active, functional proteases is a key requirement of PAR signaling, and the predominant endogenous proteases that activate PARs may vary in different patho-physiological states. For instance, the compliment of available active proteases varies markedly during the course of inflammation and healing, depending on the presence of immune cells, which are the source of many proteases, and on the existence of endogenous inhibitors. The compliment of proteases of mast cells, neutrophils, eosinophil, and macrophages, which participate in different phases of inflammation, varies considerably. For example, as the first responders to microbial infection, neutrophil produce elastase, cathepsin G, and proteinase-3 , whereas1, and PAR2  and β-arrestin-dependent pathways. Although in many instances a particular protease or synthetic agonist can activate more than one of these pathways, in some cases proteases and synthetic agonists activate a single pathway. By comparing and categorizing the signaling pathways that are initiated by different proteases and synthetic agonists with the overall outcome of receptor activation, it is possible to identify the primary signaling pathways responsible for PAR-mediated patho-physiological responses  increases the affinity of thrombin for the receptor and facilitates cleavage  mimics the protective effects of APC both in vitro and in vivo. Cleavage of PAR1 at R46/N47 by APC leads to β-arrestin 2-mediated Rac1-activation independent of G protein 1/2. Thus, APC and thrombin cleave PAR1 at different sites leading to the exposure of distinct tethered ligand agonists that activate different signaling pathways.Activated protein C is a natural anticoagulant with powerful anti-inflammatory and cytoprotective activities . In manying PAR1 . Differeveolin-1 , 86. Bese effect , 52  signaling and platelet shape changes  induce Gαi-mediated MAPK activation, regardless of their distinct cleavage positions. Although proteinase-3 cleaves prior to canonical activation site , proteinase-3 fails to induce Ca2+ signaling, suggesting the possibility that the extra 5 residues (TLDPR) may has an inhibitory role in coupling activated PAR1 to Gαq and Ca2+ mobilization , neutrophil elastase (RNPNDKYEPF-NH2), neutrophil proteinase-3 (TLDPRSF-NH2), MMP1 (PRSFLLRN-NH2), and APC (NPNDKYEPF-NH2)    or SLIGR (human)   . Trypsinlization , Gαs-deplization , Gα12/13lization , recruitlization , ERK1/2 lization , 98, andlization , 99.2 at the canonical site  fails to mimic the action of elastase and to activate ERK or to stimulate Ca2+ signals in KNRK-PAR2 cells, suggesting that elastase could activate PAR2 by a mechanism that does not require tethered ligand binding to the cleaved receptor. Indeed, the elastase cleavage site is close to the first transmembrane domain of PAR2, which would suggest that a tethered ligand mechanism is unlikely. Presumably, cleavage per se may allow PAR2 to adopt an active conformation that favors activation of certain signaling pathways. Although the functional relevance of elastase activation of PAR2 is uncertain, it may contribute to inflammatory diseases in which this protease-receptor pair is involved. For example, neutrophil elastase activity is elevated in patients with ulcerative colitis as well as dextran sulfate sodium-induced colitis in mice     from the AP are critical for PAR2 activation. Both mutated PAR2 receptor with substitution of these two residues to alanine and its corresponding soluble peptide ligand (AAIGRL-NH2) exhibit little or no activity  may interact with the basic arginine residue at position 5 of the AP. This residue is required for activity since mutation of either receptor or the peptide results in loss of Ca2+ signal , but with a lower EC50 than AC-55541, another small molecule agonist for PAR2   (3 (50% peptide cleavage reached after ~5 h), suggesting that other modulators may facilitate APC cleaving PAR3 in cells; indeed, the efficiency of this cleavage increased proportional to the expression of EPCR  is able to prevent thrombin-induced endothelial barrier disruption. Interestingly, an extended peptide generated from thrombin cleavage site (40TFGAPPNSFEEFPFS54-NH2) fails to do so. This suggests that APC and thrombin activation of PAR3 mediates different signaling profile, and thus is involved in different cellular response, respectively. However, similar to thrombin activation of PAR3, existence of another receptor, such as PAR1 is necessary for APC/PAR3-mediated signaling , and the EC50 of thrombin toward PAR4 was much higher compare to other thrombin sensitive receptors PAR1 and PAR3   , 19, 20.1 and PAR2, little is known about biased signaling of PAR4, and to date no additional activating cleavage sites have been identified. However, although proteases such as plasmin can activate PAR4 by cleaving the receptor at the canonical site, thrombin and plasmin cleave PAR4 with different kinetics, possibility due to different mechanisms of action or the affinity of the proteases for the receptor  approach suggest dynamic regulation of PARkinetics , 132. Fukinetics . GPCRs lkinetics . It woul2) or -independent (PAR1) manner  manner , 134. Re1 and PAR2 are rapidly phosphorylated by protein kinases, including GRKs, and second messenger kinases such as PKA and PKC (1 is phosphorylated by GRK3 in Xenopus laevis oocytes and GRK5 in human endothelial cells (2 in native systems have not been identified, it is clear that PAR2 activation by trypsin recruits multiple GRKs in overexpression system (1 (2+ signaling whereas GRK3 and GRK6 have no such effect (Most information about the regulation of PARs has been derived from studies of the canonical mechanisms of PAR activation. After cleavage by thrombin or trypsin, both PAR and PKC –137. Phoal cells , 137. Aln system . In termh effect  and opioh effect , suggesth effect . Whether375, which is sufficient for receptor desensitization but not β-arrestin recruitment or receptor internalization. On the other hand, DAMGO, another agonist for the same receptor, leads to phosphorylation at both Ser375 and Thr370, which leads to both receptor desensitization and internalization (Phosphorylation of GPCRs often leads to β-arrestin recruitment and receptor internalization. However, this is not always the case. The morphine-activated μ-opioid receptor is phosphorylated by GRK5 at Serlization . Thus, b1-mediated Akt activation, and PAR1 and PAR2-dependent ERK1/2 activation (2 stably couples to β-arrestin and together they co-translocate to early endosomes where they generate a second wave of intracellular signals (2-Raf1 and activated ERK. This complex will ensure the appropriate subcellular localization of PAR2-mediated ERK activity. Thus, the stability between activated receptor and β-arrestin is essential for determining the duration of this activation. It has been pointed out that PAR2 may induce distinct cellular response from Gαq pathway via a β-arrestin-mediated mechanism (2, where both β-arrestins have similar effects, PAR1-mediated Akt signaling is differentially mediated by different β-arrestins, depending on the mechanism of proteolytic activation. For example, β-arrestin 1 is required for rapid activation of Akt induced by thrombin, whereas APC cleavage leads to β-arrestin 2-dependent Akt activation (β-Arrestins not only act as chaperone proteins that direct receptor trafficking, but also are active participants of signaling by internalized receptors. β-arrestins mediate multiple steps of PAR signaling, including PARtivation . After a signals . As an iechanism , 143. Hotivation , 144. Al1 and PAR2, activation by trypsin or thrombin leads to receptor trafficking to endosomes, followed by lysosome sorting and receptor down-regulation. (1 signals require localization of the signalosome to caveolae, a specific lipid rich plasma membrane microdomain. Caveolae has also been suggested to be involved in TF-mediated PAR2 signaling. In breast carcinoma cells, both TF and PAR2 are observed co-localized in cholesterol-rich caveolae, and depletion or sequestration of plasma membrane cholesterol significantly impaired TF-VII1 induced cell signaling (Besides desensitization, receptor trafficking to different subcellular compartments also plays an important part in regulation of GPCR signaling. For both PARulation. . Howeverignaling . It woulThe contribution of PARs to important patho-physiological processes, including hemostasis, inflammation, pain, and proliferation, has been extensively investigated through studies of PAR-deficient mice and by use of proteases and synthetic agonists/antagonists of the canonical signaling pathways , 22. The2 deletion is protective, suggesting that PAR2 contributes to the development of immunity and to allergic inflammation of the airway (2 shows a protective effect (1 and PAR2. On the other hand, ovalbumin-induced inflammation is characterized by infiltration of eosinophil and macrophages, leading to activation of distinct proteases (2 may be different in these two models, which could potentially activate different signaling pathways that lead to opposite responses. The contrasting pro-inflammatory and cytoprotective actions of thrombin and APC, respectively, may also be attributed to PAR1 biased signaling. In this instance, the relative concentration of the proteases as well as the occupancy of EPCR by its ligand are critical in determining the PAR1 signaling pathways (Although the patho-physiological importance of biased signaling of PARs is not fully understood, biased agonism could explain certain paradoxes about the patho-physiological contribution of PARs. For example, PARs can have both pro-inflammatory and anti-inflammatory roles, which may depend on the animal models, species, tissues, or the protease that drive the response. In an ovalbumin-induced model of allergic inflammation of the mouse airway, PARe airway . Howevere effect , 150. Throteases . Thus, tpathways .Considerable progress has been made in defining the mechanisms by which proteases and synthetic agonists activate PARs. Proteases, peptides, and small molecules have been identified that can activate PARs by distinct mechanisms, leading to the stimulation of divergent pathways of receptor signaling and trafficking. The information derived from these studies has provided insights into the signaling pathways that are responsible for certain patho-physiological processes.per se can activate signaling is unknown. There is tantalizing evidence to suggest that biased signaling may underlie contrasting patho-physiological consequences of PAR activation, depending on the available proteases and the nature of PAR signaling. However, the proteases that are responsible for PAR activation in particular cell types in different conditions remain to be identified, and the signaling pathways that give rise to particular patho-physiological outcomes are not fully defined. Finally, very little is known about the mechanisms that regulate protease-biased signaling of PARs, particularly by those proteases that fail to promote the recruitment of β-arrestins and endocytosis of the activated receptors.However, there are many unanswered questions about biased signaling of PARs. The ability of a PAR cleaved by different proteases or bound to various synthetic agonists to differentially signal probably arises from distinct receptor conformations. However, the structures of PARs in these different stabilized states remain to be determined. Although some proteases activate PARs by exposure of a tethered ligand, this is not always the case and the mechanism by which proteolysis Whether protease-biased signaling of PARs can be exploited therapeutically remains an open question. The development of receptor antagonists or agonists that target disease-relevant PAR signaling pathways without affecting beneficial signaling events could provide a route for enhanced selectivity, with fewer on target side-effects. Future challenges will be to identify the primary pathways that mediate PAR-dependent physiological and patho-physiological events, and to develop receptor agonists and antagonists that selectively target these pathways. A deeper understanding of the mechanisms of initiation, regulation, and termination of protease-signaling will have profound implication in developing therapeutics for many critical conditions, including sepsis, thrombosis, inflammation, and pain processes.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.


We report on the epidemiology of HTLV in England and Wales (E&W) over the past decade. HTLV diagnoses in E&W are reported to PHE by clinicians, laboratories and NHS Blood and Transplant. Confirmatory testing is done by the VRD. Reports received up to the end of January 2013 were included in these analyses. Of the 892 persons diagnosed with HTLV in E&W between 2003-2012, the majority had HTLV-1 . Two-thirds were women  and 87% were aged ≥35 years (768/887). Where ethnicity was reported (568), black Caribbeans accounted for 59% (337) of diagnoses, followed by whites , black Africans  and persons of Indian-subcontinent ethnicity . 43% (142/338) of people diagnosed probably acquired HTLV infection in the UK and 40% (135/338) in Latin America/ Caribbean. The majority of diagnoses were made among those exposed through mother-to-child transmission and/or heterosexual contact . For persons presenting with HTLV-related symptoms (559), the median age was 57 years and 45 years for those without symptoms (331). Median age for persons reported with adult T cell lymphoma (117) was 59 years and 55 years for HTLV-I-associated myelopathy (74). Of 283 symptomatic infections, 87% (247) were among black African/Caribbeans. 75 (8.4%) persons died over the decade, 84% (63) within one year of HTLV diagnosis. Though rare in E&W, HTLV infection is life-long and can result in debilitating disease. Enhanced surveillance remains of public health importance to continue building an epidemiological picture of HTLV and to better understand clinical disease progression.


Neonatal rats born to mother rats pre-infused with LAT-27 were shown to have acquired a large quantity of LAT-27, and these newborns showed complete resistance against intraperitoneal infection with HTLV-I. On the other hand, when humanized immunodeficient mice were pre-infused intravenously with humanized LAT-27 (hu-LAT-27), all the mice completely resisted HTLV-I infection. These results indicate that hu-LAT-27 may have a potential for passive immunization against both horizontal and mother-to-child vertical infection with HTLV-I.Although the number of human T-cell leukemia virus type-I (HTLV-I)-infected individuals in the world has been estimated at over 10 million, no prophylaxis vaccines against HTLV-I infection are available. In this study, we took a new approach for establishing the basis of protective vaccines against HTLV-I. We show here the potential of a passively administered HTLV-I neutralizing monoclonal antibody of rat origin (LAT-27) that recognizes epitopes consisting of the HTLV-I gp46 amino acids 191–196. LAT-27 completely blocked HTLV-I infection  Human T-cell leukemia virus type I (HTLV-I) ,2 causesin vitro  × 104. All samples were performed in triplicate.Genomic DNA was extracted by QIAamp kit  according to the manufacturer’s instructions. To examine the HTLV-I PVL, we carried out a quantitative PCR method using StepOnePlus (Applied Biosystems) with 100 ng of genomic DNA  for 20 min. Negative control cells were stained with HiLyte Fluor® 647 Lt-4 in the presence of 50 μg/mL of unlabelled Lt-4. Finally, the cells were washed twice and analyzed by standard flow cytometry using a FACSCalibur™ flow cytometer (BD) and FlowJo software (Tree Star).Before staining, live cells were Fc-blocked with 2 mg/mL pooled normal human IgG in FACS buffer  for 10 min on ice, and then incubated for 15 min at room temperature. After washing with FACS buffer, the cells were fixed in PBS containing 4% paraformaldehyde (Sigma) for 20 min at room temperature followed by permeabilization and washing in 0.5% saponin + 1% BSA (Sigma) containing FACS buffer. The cells were incubated with 0.1 μg/mL of HiLyte Fluorin vivo infection with HTLV-I. Although rat T-cells transformed with HTLV-I produce no or little infectious HTLV-I virion, rats can be used to evaluate the efficacy of prophylactic vaccines. Thus, we examined whether LAT-27 could be transferred from mother to neonates without loss of protecting capacity against HTLV-I infection. Our preliminary studies showed that spleen cells of SD strain rats were more sensitive to HTLV-I infection in vitro than those of WKA/H and F344 strain rats. Thus, we chose SD rats for this in vivo study.It has been demonstrated that rats are relatively permissive for n = 2). Two days after the birth, blood samples were collected from both mothers and newborns, and LAT-27 concentrations of their sera were quantitated using ELISA  as shown in in vitro neutralization test showed that the minimum concentration of hu-LAT-27 for complete inhibition of syncytium formation was 5 μg/mL, which was comparable to the original rat LAT-27 . Sera were collected daily for five days and the concentration of hu-LAT-27 was quantitated by ELISA as described in the Materials and Methods. The data presented in hu-LAT-27 was produced in CHO cells. Due to a patent matter, the gene sequence of hu-LAT-27 will be reported elsewhere. The t LAT-27 B, demonsin vivo infection with HTLV-I using our humanized mouse model as reported previously [+ T-cells from all mice immunized with the hu-LAT-27 before HTLV-I infection were negative for Tax antigen, suggesting complete protection against HTLV-I infection (lower panels). PCR testing of HTLV-I proviral loads supported this conclusion. The protective effect was hu-LAT-27-specific since the control anti-CEA did not confer protection against the infection (upper panels). When hu-LAT-27 was administered one day after HTLV-I infection, it did not protect mice against HTLV-I infection. These results suggested that hu-LAT-27 could be used for prophylaxis against HTLV-I infection in vivo when a sufficient amount of it was infused before infection with HTLV-I.Then, we tested the protective efficacy of hu-LAT-27 mAbs against eviously . PBMCs fin vivo is most likely neutralization via inducing conformational changes in the gp46 structure. In addition, as we have previously reported, LAT-27 may also inhibit HTLV-I infection by eliminating HTLV-I-producing cells via HTLV-1-specific ADCC in the presence of NK cells [in vitro is as low as 5 μg/mL, further studies are required to determine the minimum dose of LAT-27 for perfect in vivo protection against HTLV-I in each animal system.The present study suggested a potential of monoclonal anti-gp46 neutralizing LAT-27 for passive immunization for prophylaxis against HTLV-I infection. The main mechanism for LAT-27-mediated protection of animals against HTLV-I NK cells . In the Using a rat model, the present study first showed that LAT-27 could be transferred from dams to infant rats, and that the transferred LAT-27 was active for HTLV-I neutralization and protected the infants against intraperitoneal HTLV-I infection. It remains to be determined how long the antibodies were sustained in the littermates born to pregnant rats infused with LAT-27. In rats, it is known that maternal antibodies are transferred equally effectively to infants either via placenta or from the gut by breastfeeding . It shouin vitro and in vivo. Similar to rat LAT-27, hu-LAT27 neutralized HTLV-I in vitro at a minimum concentration of 5 μg/mL . In collaborative investigation with IBL Co., Ltd., we have succeeded in the generation of hu-LAT-27, and examined its function  5 μg/mL , showing 5 μg/mL . The halNOG mice  will be The present data suggest that hu-LAT-27 mAb will be useful in passive immunization against HTLV-I infection. The rationale of using these monoclonal antibodies instead of polyclonal antibodies obtained from healthy HTLV-I carriers for the prevention of HTLV-I infection is that, in contrast to HIV-1, the genome of HTLV-I is stable , and thuin vivo. Thus, it will be worthy to validate hu-LAT-27 in an animal model in more detail before starting a clinical study. In addition to this passive vaccine, in order to control HTLV-I infection all over the world, it is clear that an active vaccine which can elicit or boost anti-HTLV-I gp46 neutralizing antibody titer should be developed.We anticipate that hu-LAT-27 mAb will be used as a safe passive vaccine against both horizontal and vertical infection with HTLV-I for people at high risk of HTLV-I infection, including adults and babies born to HTLV-I carriers. In addition, since organ transplantation to HTLV-I-negative recipients from HTLV-I-positive donors is another risk factor of HTLV-I infection and the development of HAM/TSP , pretreaThis study describes experimental results indicating a possible concept that passive immunization with our humanized anti-gp46 neutralizing mAb (hu-LAT-27) reduces the chance of HTLV-I infection of adults and babies at high risk. A combination of hu-LAT-27-based passive vaccination and yet-to-be-developed active vaccine might be potent in preventing the spread of HTLV-I infection around the world.


Thymic stromal lymphoproetin (TSLP) is a cytokine secreted by the airway epithelium in response to respiratory viruses and it is known to promote allergic Th2 responses in asthma. This study investigated whether virally-induced secretion of TSLP is directional in nature  and/or if there are TSLP-mediated effects occurring at both sides of the bronchial epithelial barrier in the asthmatic state.Primary human bronchial epithelial cells (HBEC) from control (n = 3) and asthmatic (n = 3) donors were differentiated into polarized respiratory tract epithelium under air-liquid interface (ALI) conditions and treated apically with dsRNA  or TSLP. Sub-epithelial effects of TSLP were examined in human airway smooth muscle cells (HASMC) from normal (n = 3) and asthmatic (n = 3) donors. Clinical experiments examined nasal airway secretions obtained from asthmatic children during naturally occurring rhinovirus-induced exacerbations (n = 20) vs. non-asthmatic uninfected controls (n = 20). Protein levels of TSLP, CCL11/eotaxin-1, CCL17/TARC, CCL22/MDC, TNF-α and CXCL8 were determined with a multiplex magnetic bead assay.in vivo airway secretion of TSLP and CCL11/eotaxin-1.Our data demonstrate that: 1) Asthmatic HBEC exhibit an exaggerated apical, but not basal, secretion of TSLP after dsRNA exposure; 2) TSLP exposure induces unidirectional  secretion of CCL11/eotaxin-1 in asthmatic HBEC and enhanced CCL11/eotaxin-1 secretion in asthmatic HASMC; 3) Rhinovirus-induced asthma exacerbations in children are associated with There are virally-induced TSLP-driven secretory immune responses at both sides of the bronchial epithelial barrier characterized by enhanced CCL11/eotaxin-1 secretion in asthmatic airways. These results suggest a new model of TSLP-mediated eosinophilic responses in the asthmatic airway during viral-induced exacerbations. The conducting airway epithelium in humans is organized as a pseudostratified columnar structure with a functional polarity and well-defined apical and basolateral compartments Respiratory viruses, the most common apical environmental challenges in asthma in vivo airway secretion of TSLP and CCL11/eotaxin-1. Collectively, our findings suggest a potential TSLP-driven autocrine mechanism occurring at the apical surface of the asthmatic respiratory epithelia that may amplify atopic responses via unidirectional  secretion of CCL11/eotaxin-1 in the lumen of the airways of asthmatic subjects during viral respiratory infections.Notwithstanding the compelling evidence supporting the pivotal role of TSLP in regulating the balance of antiviral Th1/allergic Th2 responses in the airways Human bronchial epithelial cells (HBEC) were purchased from Lonza, Walkersville, MD. Donors were three disease-free and non-smokers  and three adult asthmatic subjects . HBEC were amplified on collagen-coated T-75 flasks as previously described Human ASM cells (HASMC) were purchased from Lonza, Walkersville, MD. Donors were three adult asthmatic subjects  and three disease-free and non-smokers . HASMC were cultured in vitro as previously described rhinovirus-induced asthma exacerbation group, patients were eligible for the study if they had all of the following criteria: 1) history of physician-diagnosed asthma; 2) acute respiratory symptoms such as cough or shortness of breath; 3) documented wheezing; and 4) PCR (+) for rhinovirus. Subjects included in the control group were age-matched (2–15 years old), non-asthmatic children without clinical wheezing or detectable respiratory viral infection by PCR. Nasal samples were analyzed by a viral multiplex PCR panel for 14 targets used for clinical purposes in our institution  according to the microbiology laboratory protocol. All clinical and demographic variables were obtained by reviewing electronic medical records at Children's National Medical Center. The Institutional Review Board (IRB) of Children's National Medical Center, Washington D.C. approved the study and granted a waiver of informed consent given that this research involved materials  collected solely for non-research purposes .Nasal airway secretions were collected from hospitalized children aged 2–15 years seen in our medical center at the onset of their acute respiratory illnesses. Nasal washings were obtained by standard nasal lavage technique using saline (3–5 ml) and gentle suctioning of each nostril. For the The supernatant from HASMC and HBEC  experiments and nasal washings were analyzed for protein levels of human TSLP and CCL11 (eotaxin-1). Cell supernatants were also assayed for CCL22 (macrophage-derived chemokine [MDC]), CCL17 (TARC), TNF-alpha and CXCL8 (IL-8). Cytokine levels were determined using a commercially available multiplex magnetic bead immunoassay  according to the manufacturers' instructions.Data were analyzed using Minitab 16 software package for Windows . All data are reported as mean +/− standard errors (SE), 95% confidence intervals (CI) or as fold changes relative to control values. Data within each group (asthma and control) and between asthma and control groups were analyzed with two sample t-test or non-parametric Mann Whitney U test when appropriate. For multiple comparisons one way ANOVA followed by post- test Bonferroni correction was used. Linear regression and Pearson correlation were used to evaluate the link between TSLP and CCL1/eotaxin-1 adjusting by covariates. A probability of <0.05 was considered statistically significant.Prior studies have reported that the TLR-3 agonist, dsRNA (poly I:C) induces TSLP secretion in submerged HBEC There is increasing evidence linking TSLP to eosinophilic responses in the airways We next investigated additional immune modulatory effects of the apical exposure of TSLP in HBEC. To examine the potential immune modulatory role of TSLP in the sub-epithelial compartment, we next evaluated the effect of TSLP exposure in HASMC. Initial experiments investigated whether dsRNA exposure elicits enhanced TSLP secretion in asthmatic HASMC as a potential source of sub-epithelial TSLP in the asthmatic state. As shown in in vitro pertain to the in vivo state, we conducted a clinical study contrasting TSLP and CCL11/eotaxin-1 nasal airway protein levels in children with rhinovirus-induced asthma exacerbation, defined as hospitalization of a known asthmatic child due to active wheezing and (+) rhinovirus PCR testing (n = 20) vs. age-matched, non-asthmatic controls without wheezing or detectable respiratory virus by PCR (n = 20). To investigate if the TSLP and CCL11/eotaxin-1 responses of asthmatic airways in vitro model of polarized, primary differentiated HBEC at air-liquid interface (ALI), human airway smooth muscle cells (HASMC) and clinical experiments in nasal airway secretions obtained during naturally occurring rhinovirus-induced asthma exacerbations. Our results identified that: 1) Apical dsRNA exposure induces bidirectional  secretion of TSLP in HBEC; 2) Asthmatic HBEC exhibit an exaggerated apical, but not basal, secretion of TSLP after dsRNA exposure; 3) TSLP exposure induces unidirectional  secretion of CCL11/eotaxin-1 in asthmatic HBEC and enhanced CCL11/eotaxin-1 secretion in asthmatic HASMC; and 4) Rhinovirus-induced asthma exacerbations in children are associated with in vivo airway secretion of TSLP and CCL11/eotaxin-1. These data provide new insights about the nature of the TSLP-induced secretion of CCL11/eotaxin-1 at both sides of the bronchial epithelial barrier and suggest a new model of bilateral  TSLP-driven eosinophilic responses in the asthmatic airway during viral-induced exacerbations , a master Th2 cytokine that promotes allergic responses in different cell systems rbations .in vivo TSLP release in the airways of animal models of allergic asthma in vitro model of a human asthmatic bronchial epithelium (ALI-differentiated HBEC), we now demonstrate that dsRNA exposure leads to time-dependent increases in unilateral  TSLP secretion in asthmatic HBEC relative to control. DsRNA induces comparable levels of TSLP basolateral secretion in control and asthmatic ALI-differentiated HBEC. These data suggest that bilateral  TSLP secretion is part of the normal innate antiviral immunity of the human bronchial epithelia, however, there is a distinct immune secretory signature in asthmatic individuals, consistent on dysfunctional apical TSLP hyper-secretion in response to dsRNA exposure. Future studies are necessary to confirm that our observations using dsRNA as a viral surrogate are also pertinent to infection with rhinovirus and other respiratory viruses.One the most important findings of our current study is that dsRNA induces a disproportionate amount of apical vs. basolateral TSLP secretion in ALI-differentiated asthmatic HBEC . DsRNA iTSLP is an epithelial-derived cytokine that contributes to the generation of allergic airway inflammation in asthma in vitro conditions. These findings are in agreement with the recent notion that there is a complex regulation of TSLP bioavailability in epithelial tissues. Indeed, TSLP protein is post-translationally activated by endogenous proteases in nasal polipoid tissue in vitro results suggest that there might be additional factors that determine TSLP activity in the in vivo microenvironment of the human airway epithelium.Although TSLP and CCL11/eotaxin-1 play a critical role in the pathogenesis of asthma, it is noteworthy that TSLP alone was not enough to induce CCL11/eotaxin-1 in control HBEC. The latter indicates that there are intrinsic molecular differences between control and asthmatic HBEC that are collectively necessary for the development of the asthmatic condition. This concept is supported by genome-wide expression analysis of baseline and rhinovirus-infected HBEC from normal and asthmatic donors that have identified a unique transcriptomic pattern at baseline and during rhinovirus infection. in vitro experiments identified that TSLP also induces unidirectional  secretion of TNF-alpha in asthmatic HBEC . Since TNF-alpha heightens the development of airway TSLP/Th2 pro-asthmatic responses after dsRNA exposure Extended tic HBEC . Since TOur study found that cytokines and chemokines are produced differentially in apical and basal sides of the human bronchial epithelium, which is in agreement with our prior proteomic analysis that identified directional  airway secretomes In concert with effects in the bronchial epithelium, TSLP also directly targets the sub-epithelial human airway smooth muscle cells (HASMC) via activation of the TSLP receptor-(R), which has constitutive and inducible expression in this airway cell type in vivo airway secretion of TSLP and CCL11/eotaxin-1. In this “proof of concept” cross-sectional study we collected nasal secretions of asthmatic children with acute rhinovirus-induced wheezing leading to hospitalization  and compared them with age-matched, non-asthmatic children without wheezing or detectable virus (control). We identified that TSLP and CCL11/eotaxin-1 levels were not only significantly elevated in the group with rhinovirus-induced asthma exacerbation (To investigate the clinical relevance of the proposed model of TSLP-driven eosinophilic responses via CCL11/eotaxin-1 secretion in the asthmatic airways, we conducted a clinical study to see if virally-induced asthma exacerbations are associated with erbation , but thaerbation . It is iIn summary, this study is the first to identify intrinsic differences in the directional secretion of TSLP and CCL11/eotaxin-1 between normal and asthmatic differentiated airway epithelium. It is also the first to evaluate TSLP/CCL11/eotaxin-1 responses in asthmatic airway smooth muscle, a sub-epithelial tissue implicated in the pathologic bronchoconstrictive response seen in the asthmatics during viral respiratory illnesses. In addition, we provide new evidence of the in parallel airway secretion of TSLP/CCL11/eotaxin-1 during rhinovirus-induced asthma exacerbations in children. Our findings set the stage for future mechanistic studies to define the role of TSLP/CCL11/eotaxin-1 in virally mediated Th2 immune responses, as well as potential therapeutic studies designed to detect and attenuate virally induced changes in the bronchial epithelium and airway smooth muscle of asthmatic individuals.


Rupicapra spp. are the most abundant mountain ungulates in Europe, no data are available on the presence of Giardia duodenalis infecting these species.Although chamois Rupicapra rupicaprarupicapra and Apennine Rupicapra pyrenaicaornata chamois were tested for the presence of G. duodenalis by immunofluorescence test, quantitative Real Time PCR and end-point PCR for genotype characterization.A total of 157 fecal samples from Alpine G. duodenalis was detected in R. r. rupicapra and R. p. ornata, with a percentage value of 4.45 , and a cyst burden of up to 31,800 cysts/g of feces. Assemblages A/AI and E were identified in R. r. rupicapra and assemblage A/AIII in R. p. ornata.Giardia duodenalis in Rupicapra spp., suggesting that these wild bovids can play an epidemiological role in environmental contamination and transmission of both zoonotic and non-zoonotic genotypes.The present study represents the first record of  Giardia duodenalis is one of the most common intestinal parasites in humans and several animal species worldwide [The flagellate orldwide , 2. At porldwide , and assorldwide . It is norldwide , 6.viceversa [G. duodenalis epidemiology has been thoroughly studied, wild animals have only recently been considered as having a potential role. In addition to being a possible source of infection for humans, wild animals can be endangered by the spill-over of parasites from domestic animals and even people [Given this great genetic heterogeneity, it is hard to determine the role of animals as a source for human infection, and iceversa ; this isiceversa , 9, evenn people , especian people .G. duodenalis has been recorded in wild ungulates wordwide [wordwide , 12–15. wordwide ).Rupicapra: the Northern chamois, Rupicapra rupicapra, with seven subspecies, including the Alpine chamois R. r. rupicapra, and the Southern chamois, Rupicapra pyrenaica, with three subspecies, including the Apennine chamois R. p. ornata [The chamois (Artiodactyla: Bovidae) is the most abundant mountain ungulate in Europe and the Near East. Two species are recognised in the genus . ornata , 17.Rupicapra species in Europe, no data are available on the presence of G. duodenalis infecting chamois. This study aimed to determine the presence of G. duodenalis, and to quantify and characterize isolates from two subspecies of chamois: R. r. rupicapra living in northern Italy (Alps) and R. p. ornata, living in central Italy (Apennines).Despite the geographical abundance of 2 , ranging from 300 to > 2000 m a.s.l. Here the R. r. rupicapra population in 2014 was estimated as 2077 individuals, giving an average population density of 8.2 chamois/km2 . The second area is in the Lepontine Alps, in the hunting district of Piedmont region (VCO2-Ossola Nord), with an extent of 72,740 ha , ranging from 700 to 2400 m a.s.l. Here the chamois population was estimated as 1328 individuals in 2014, with an average density of 6.7 subjects/km2 [2, 41°44’N, 13°54’E), where samples were collected in Val di Rose, Mt. Meta, and Mt. Amaro sub-areas, ranging from 1650 to 2242 m a.s.l. In ALMNP, about 600 individuals of R. p.ornata were counted in 2014 [2 [The study took place in three areas of Italy. The first is in the Lecchesi Alps and Pre-Alps, a hunting territory in Lombardy region, with an area of 253 kmects/km2 . The thi in 2014 , with lo 2014 [2 .R. r. rupicapra chamois harvested during the hunting season, whereas the 54 fecal samples from R. p. ornata were collected from the ground soon after defecation. To avoid the risk of collecting feces from the same individuals, sampling was carried out on different slope sites and took into account, as far as possible, the animals’ sex and age. Fresh fecal specimens were collected and put into plastic bags, which were labeled and immediately packed in an insulated container with ice or cold packs. Specimens were then transported to the laboratory and processed within 1–3 days after collection.Between August 2013 and January 2014, 103 fresh fecal samples were collected from G. duodenalis .All 157 faecal samples were examined using an immunofluorescence (IF) test for the detection of qPCR) using the SSU-rDNA gene, and to end-point PCR for genotyping using two genes i.e. SSU-rDNA and gdh.The positive samples were frozen and subjected to Real-Time PCR for quantitative analysis . DNA extraction was automatically performed by EZ1 BioRobot  following the manufacturer’s instructions. To obtain a high quality DNA, samples were purified by Amicon Ultra-0.5 Centrifugal Filter Unit (Millipore) following the manufacturer’s instructions.G. duodenalis SSU-rDNA gene (KJ888984) [G. duodenalis SSU-rDNA gene.A sequence of J888984)  was seleG. duodenalis plasmid was measured using a fluorometer, and the corresponding copy number was calculated using the following equation:The concentration of the pEX-A2 G. duodenalis (copy numbers) = 6.02 × 1023 (copy/mol) × pEX-A2 G. duodenalis amount (0.31 x 10-5 g/ml)/pEX-A2 G. duodenalis length (293 bp + 2450 bp) × 660 (g/mol/bp) [pEX-A2 G. duodenalis plasmid (from 1.03 x 107 to 1.03 x 10-3 copies/μl) were used to assess the sensitivity, repeatability and reproducibility parameters of the assay, and to determine the quantity of the unknown samples based on linear regression calculations of the standard curve.Ten-fold serial dilutions of the pEX-A2 G. duodenalis ssRNA primers were GiarF (5’- GAC GCT CTC CCC AAG GAC-3’) and GiarR (5’- CTG CGT CAC GCT GCT CG-3’) [Amplifications and melting analysis were performed in the CFX-96 Real Time Instrument . T CG-3’) .The PCR mixture  contained 1 μl of the plasmid-based control (or 5 μl of genomic DNA sample from 1 to 5 ng), 5X EvaGreen® Reagent  and 0.5 μM final concentration of each forward and reverse primer. Samples without genomic DNA (negative controls) were included in each PCR run. The cycling conditions in a CFX-96 thermocycler (BioRad) were as follows: initial denaturation at 98 °C for 2 min, followed by amplification for 35 cycles of 98 °C for 5 s and 55.6 ° C for 15 s.F/dT) and plotted against changes in temperature. The resolution melting curve (MC) profile was then analyzed using Precision Melt Analysis™ software version 1.2, with fluorescence (MC) normalization by selecting the linear region before and after the melting transition. The melting temperature (Tm) was interpolated from the normalized data as the temperature at 50 % fluorescence. Samples’ melting curves were distinguished by plotting the fluorescence difference between normalized melting curves. Tm and standard deviation (SD) were recorded for each positive control.Fluorescence data were collected at the end of each cycle as a single acquisition. After amplification, the PCR products were melted by raising the temperature from 70 to 95 °C, with an increment of 0.5 °C/5 s, in order to denature and re-anneal before the high resolution melting; changes in fluorescence were recorded with changes in temperature . Finally, since the number of cysts in each sample was obtained in 200 mg of fecal sample, the results were transformed for cysts per gram (CPG) with the formula: number of cysts obtained in 200 mg of fecal sample X 5.The copy number for each positive sample was calculated by relating the  nucleus , we consSSU-rDNA gene, using the primers RH4 and RH11 for the first step, and the primers GiarR and GiarF in the second amplification round, as used for qPCR [Giardia DNA) and negative (no template added) controls were added. All PCRs were carried out in a 25 μL volume containing 12.5 μL PCR master mix 2X (Promega), 5 μL template DNA, 0.6 mM of each primer and 0.1 mM BSA, 4 % dimethyl sulfoxide (DMSO), and were performed in a TProfessional Basic Thermocycler . PCR products were visualized by electrophoresis on 1 % agarose gel stained by SYBR Safe DNA gel stain (Invitrogen). Amplicons were purified using the mi-PCR Purification Kit (Metabion GmbH). Both strands were sequenced by Bio-Fab Research s.r.l. . Sequences were edited with FinchTV 1.4 software . To assign Giardia isolates to the correct assemblage, a comparison of the SSU-rDNA sequences by multiple alignments was performed using ClustalW2 software against known sequences available in GenBank. To test the significance of the results and to identify the sub-assemblage, a phylogenetic analysis was performed using MEGA6 software to compare the SSU-rDNA and gdh sequences with those of reference strains from different hosts. The best-fit model and parameters for tree construction were selected using the jModeltest software by the Akaike Information Criterion (AIC).A nested PCR was performed to amplify a 130 bp region from the for qPCR . An addifor qPCR . In all G. duodenalis was detected by microscopy in seven out of the 157 fecal samples examined, . In R. r. rupicapra and R. p. ornata, a percentage of 5.82 % (6/103) (CI = 2.2–12.5) and 1.85 % (1/54) (CI = 0.5–9.8) were registered, respectively. All samples which tested positive to microscopy were also positive to qPCR and end-point PCR to one or both genes . Overall, two assemblages i.e. A (with sub-assemblages AI and AIII) and E were identified  were identical to those from different hosts, including white-tailed deer in the USA (Genbank accession number KJ867494) previously reported by [At the orted by , dairy corted by  and Dutcorted by  Fig. 1)SSU-rDNA gdh sequences showed that isolate no. 71 from R. r. rupicapra clustered within sub-assemblage A/AI, sharing the same sequences as those from a large number of isolates from humans (L40509) [R. p.ornata clustered within the sub-assemblage AIII, together with a Giardia isolate from roe deer in the Netherlands (DQ100288) [gdh locus failed for the other Giardia isolates. The nucleotide sequences obtained in this study have been deposited in EMBL/GenBank database under accession number from LN875379 to LN875384 for the SSU-rDNA gene and KT270858-KT270859 for the gdh gene.Phylogenetic analysis of L40509) , 30, sev(L40509) , and als(L40509) , whereasQ100288)  and red  , 30, seGiardia cysts in test-positive samples were predicted to range from 263 to 31,800 per gram of feces  which implies that hunters have to carry culled wild ungulates to the control centres where, for each subject, age, sex, the shooting area and morpho-biometric measures are registered. Thus, no animals were killed specifically for this study.G. duodenalis in Rupicapra spp. Noticeably, the protist was found both in R. r. rupicapra and R. p. ornata, two different chamois subspecies living in quite distinct geographical areas, with an overall percentage value of 4.45 % , and a cyst burden of up to 31,800 cysts/g of feces. Assemblages A (AI and AIII) and E were detected.This is the first report of G. duodenalis genetic groups, it is known that assemblage A recognizes four sub-assemblages  [Dama dama) in Italy [Cervus elaphus) and roe deer (Capreolus capreolus) in Croatia [Regarding the and AIV) . Sub-assand AIV) ; in Euroin Italy , and red Croatia . Sub-ass Croatia ; it has  Croatia  and Pola Croatia , but als Croatia , 33, and Croatia . AIV is  Croatia .Assemblage E is relatively host-specific, or rather ‘group-specific’, since it is limited to ‘hoofed livestock’ i.e. cattle, sheep, goats and pigs , 34–36, R. r. rupicapra and R. p. ornata chamois harbor assemblages/sub-assemblages A/AI/AIII, and confirms that assemblage E is associated to wild hoofed mammals, not only cervids [Rupicapra r. rupicapra. In view of this, the term ‘livestock genotype’ commonly used to classify genetic group E may be considered outdated and could possibly be replaced with the term ‘hoofed animal genotype’.In the present study, the detection of assemblages A and E in chamois living in the Italian Alps and Apennines was not unexpected. It shows that also  cervids  but alsoGiardia in Croatia [Giardia trasmission, but also alpine ibex (Capra ibex), a bovid species as yet uninvestigated for the presence of Giardia.The presence of both assemblages A/AI and E in Alpine chamois can be related to their sharing of pastures with cattle and/or sheep and/or goats, as well as with cervids. In summer, farmers move their livestock up to high altitude alpine pastures, thus facilitating interaction with wild mountain ungulates . Moreove Croatia - which mGiardia may be considered possible [R. r. rupicapra sampling areas, due to the remoteness of this territory accessible only to a few shepherds in summer and hunters in autumn. This seems to confirm that domestic and wild animals play a greater role in the dissemination of sub-assemblage AI than humans [Furthermore, unlike other Alpine areas (i.e. the Dolomites), which attract thousands of human visitors involved in trekking and mountaineering, and where “tourist-borne” arrival of possible , the posn humans .R. p. ornata population is totally isolated from domestic ruminants and never shares pastures with them [Conversely, in the Apennine area investigated where one positive chamois was detected , the ith them ; more imith them ; howeverith them , 38. Basith them  and in Pith them , detectiith them  in the AGiardia cyst burden was up to 31,800 cysts/g of feces, the pathogenic role of Giardia in wildlife remains unclear.Finally, although none of the investigated positive subjects showed signs of diarrhea, since only formed feces were collected, and the Rupicapra spp. chamois harbor G. duodenalis. This is the first report of assemblage A/AI and assemblage E in R. r. rupicapra and AIII in R. p. ornata. The epidemiological roles that these wild bovids play in environmental contamination (including watercourses and watersheds) and transmission to other wild and domestic mammals or even humans, of zoonotic (A/AI) and/or non-zoonotic assemblages/sub-assemblages , require further investigation, as does the impact of Giardia on the health and sustainability of chamois populations, together with the possible cumulative effects of other pathogens [The findings of the present study indicate that athogens .


Antibody screening tests are critical tools to identify individuals infected with HTLV-1 and HTLV-2. However, the lack of reference panels hinders the ability to evaluate existing tests and new technologies that aid in the screening for these agents. For example, some HTLV-2 subtypes may escape detection by currently available technology. The World Health Organization, through the support of the WHO Expert Committee on Biological Standardization, is in the process of generating such a reference panel.This reference panel will best function as a tool for test assessment only if it consists of specimens representing the diversity of HTLV-1 and HTLV-2, especially those subtypes known to present challenges to detection using serological tests.There have been challenges sourcing suitable material of sufficient titre and volume. Initial screening tests carried out by CBER/FDA and NIBSC, using a range of EIA’s, have shown a variation in titre of donated material. Two candidates, HTLV-1a and HTLV-2, have been identified and are in the process of being formulated into lyophilised preparations. Such preparations will shortly be distributed to participating laboratories for testing in a small international collaborative study. It is envisaged that additional panel members will be added as and when appropriate material is sourced.This first WHO HTLV antibody reference panel is expected to be used by blood screening and diagnostic test developers, blood banks, hospitals and other establishments performing HTLV serology testing.


Human T-cell leukemia virus type 1 (HTLV-1) causes both neoplastic and inflammatory diseases, including adult T-cell leukemia and HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP). Because these life-threatening and disabling diseases are not yet curable, it is important to prevent new HTLV-1 infections.In this study, we have established a simple humanized mouse model of HTLV-1 infection for evaluating prophylactic and therapeutic interventions. In this model, HTLV-1-negative normal human peripheral blood mononuclear cells (PBMCs) are transplanted directly into the spleens of severely immunodeficient NOD-SCID/γcnull (NOG) mice, together with mitomycin-treated HTLV-1-producing T cells. Using this model, we tested the efficacy of monoclonal antibodies (mAbs) specific to HTLV-1 as well as human IgG isolated from HAM/TSP patients (HAM-IgG) in preventing HTLV-1-infection. One hour before and 24 h after transplantation of the human cells, each antibody sample was inoculated intraperitoneally. On day 14, human PBMCs isolated from the mouse spleens were tested for HTLV-1 infection. Whereas fresh CD4-positive and CD8-positive T cells isolated from untreated mice or mice treated with isotype control mAb, HTLV-1 non-neutralizing mAbs to envelope gp46, gag p19, and normal human IgG were all infected with HTLV-1; the mice treated with either HTLV-1 neutralizing anti-gp46 mAb or HAM-IgG did not become infected.in vivo transmission of HTLV-1. The present animal model will also be useful for the in vivo evaluation of the efficacy of candidate molecules to be used as prophylactic and therapeutic intervention against HTLV-1 infection.Our data indicate that the neutralizing function of the antibody, but not the antigen specificity, is essential for preventing the The online version of this article (doi:10.1186/s12977-014-0074-z) contains supplementary material, which is available to authorized users. Human T cell leukemia virus type-1 (HTLV-1) has been linked to the development of adult T-cell leukemia (ATL) and a chronic inflammatory disease called HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP) ,3, some in vivo. In this model, HTLV-1-negative healthy human peripheral blood mononuclear cells (PBMCs) (2 × 106/mouse) were transplanted directly into the spleens of severely immunodeficient NOD-SCID/γcnull (NOG) mice, together with cells from the mitomycin C (MMC)-treated HTLV-1-infected cell line ILT-M1 (1 × 106/mouse), which is an IL-2-dependent CD8+ T cell line derived from a HAM/TSP patient . Cell suspensions in a final volume of 50 μl were administered by intrasplenic injection . As previously reported [Here we established a novel mouse model to evaluate primary HTLV-1 infection of human lymphocytes reported , the sev7 (Donor #1), 1.29 × 107 (Donor #2) and 1.92 × 107 (Donor #3), respectively, which are much higher than the numbers of inoculated human cells, suggesting successful engraftment. The increased numbers of human T cells in the mouse spleens within two weeks after inoculation may have been caused by xenoreactive lymphocyte proliferation, since recent report by Søndergaard et al. suggested that injection of human PBMCs into NOG mice cause polyclonal expansion and activation of functional human T cells [4 of both the human CD4 and human CD8 cells recovered from three hu-PBMC-NOG-spl mice, each inoculated with human PBMCs from different donors, were 9,533 and 4,546, respectively , suggesting highly efficient cell-to-cell transmission of HTLV-1 from infected to uninfected human lymphocytes in vivo. Although a previous study also showed the successful engraftment of an HTLV-1-transformed cell line and uninfected PBMCs in NOG mice, the HTLV-1 PVL in spleen was very low and less than 1% of cells were infected with HTLV-1 [7 uninfected human PBMCs were injected intraperitoneally, and those PBMCs were infected with HTLV-1 by intraperitoneal inoculation of MMC-treated HTLV-1-infected MT-2 cells (103 or 104 cells/mouse) [in vivo could be achieved by this route of inoculation. As shown in the present study, an intrasplenic transfer of human PBMCs can reduce the number of PBMCs required for the initial inoculation by approximately 1 log unit for the generation of more than 107 human T cells within two weeks, probably because human lymphocytes directly inoculated into the mouse spleen are efficiently activated, and thus HTLV-1 could efficiently infect human T cells in vivo. The microanatomic environment of the secondary lymphoid organs, such as the spleen, might also play an important role in the efficient cell-to-cell transmission of HTLV-1.First, we isolated the bulk spleen cells from hu-PBMC-NOG-spl mice sacrificed 14 days post inoculation/infection. Using flow cytometry (FCM), live cells were gated on their forward and side light scatter characteristics, and cell surface markers within the HLA-class I-positive population  were analyzed  were completely protected against HTLV-1 infection whereas other non-neutralizing antibodies such as anti-gp46 mAb (clone LAT-25), anti-Gag (clone GIN-7), anti-HCV (clone MO-8), and anti-OX40 mAb (clone B-7B5) did not protect against infection  or mouse spleens immunized with NC-IgG  activity in vitro in our previous study [in vivo. Indeed, titers of existing neutralizing and ADCC Abs did not correlate with HTLV-1 PVL  . Moreover, HAM/TSP patients also showed high titers of such Abs, indicating that these Abs are not potent in preventing the onset of HAM/TSP in infected individuals. These data also indicate the importance of passive immunization before infection.It is well established that the HTLV-1 virions are not very infectious, and thus cell-to-cell transmission is more efficient both in vitro ,21. The in vitro ,23. Indemacaques ,24 and rmacaques ,26 modelin vivo. As shown in Figure in vivo infection.Recently, we reported that both LAT-27 and HAM-IgG, but not non-neutralizing LAT-25 and NC-IgG, are capable of depleting and/or eliminating HTLV-1-infected cells in the presence of autologous PBMCs in vitro. This occurs in part via ADCC, preventing the spontaneous immortalization of T cells , and LATin vivo evaluation of the efficacy of drug candidates could also be investigated in future studies.In conclusion, we have established a novel and simple small animal model to study primary HTLV-1 infection in vivo. Although our mouse model is not the animal models of HAM/TSP or ATL, the present study has demonstrated an important rational basis for passive immunization against HTLV-1 infection in humans. Using our mouse model,


Uric acid (UA) levels correlate positively with the prevalence of chronic kidney disease (CKD) and/or hypertension. We tested the hypothesis that UA may also have a link to a new incidence of CKD and hypertension.Study design is a cohort study and the predictor is UA levels. Of the 15,470 screened cases, 8223 participants without CKD were eligible for the analysis of the incidence of CKD. Among these CKD candidates, 7569 participants were eligible for the analysis of the new development of hypertension. The observation period was 4 years.p < 0.001 by the Chi-square test). Cox proportional hazard analysis showed that the estimates of the CKD development were eGFR  and male gender . UA levels and new development of hypertension. Higher UA levels had a closer association with the new development of hypertension; 5.0 % (UA < 5 mg/dL), 8.9 % (5.0–5.9 mg/dL), 10.6 % (6.0–6.9 mg/dL), and 11.8 % (≧7 mg/dL), respectively (p < 0.001 by the Chi-square test). Cox proportional hazard analysis showed that the estimates of the hypertension development were BMI , age , HDL-cholesterol , male gender , UA level , and eGFR . Furthermore, the logistic analysis showed that the odds ratio (OR) to estimate hypertension in the high UA group  was greater than that in the low UA group (UA < 5 mg/dL). Kaplan–Meier analysis also confirmed the finding that the higher the UA levels the greater the hypertension development .Relationship of UA with new cases of CKD. Higher UA levels had a closer association with the new development of CKD; 1.1 % (UA < 5 mg/dL), 1.5 % (5.0–5.9 mg/dL), 1.7 % (6.0–6.9 mg/dL), and 3.4 % (≧7 mg/dL), respectively (High UA levels are associated with the new development of hypertension, but not with the incidence of CKD. Previous studies showed that circulating high uric acid (UA) levels were associated with increased prevalence of hypertension and a high risk status of cardiovascular complications which frequently leads to poor patient prognosis –6. IncreIn the present study, with the above-mentioned background, we carried out a longitudinal retrospective survey to look into the associations of UA level with the development of hypertension and the new onset of CKD in a screened population at large. We applied strict exclusion criteria in order to choose eligible participants who did not have a concurrent or a past history of CKD and/or hypertension.The study design is a retrospective population-based cohort of Japanese office workers aged 25–60 years, living in the vicinity of Tokyo. The original number of the participants was 15,470 who had an annual medical check-up every year from 2008 to 2012. They were seen at least once a year by well-trained physicians or public nurses.2. The present and past history of proteinuria was also another rule-out factor. This exclusion process finally leaves 8223 for the analysis of the new onset of CKD. Finally, the additional application of exclusion criterion of a blood pressure (BP) rise to systolic of 140 mmHg or greater, and diastolic of 90 mmHg or greater at the beginning leaves 7569 individuals for the analyses of the new development of hypertension.The basic criteria excludes individuals receiving medications for diabetes, hyperuricemia, hypertension, and dyslipidemia throughout the follow-up period of 4 years; those having insufficient data; those with past history of incident major cardiovascular events (MACE) such as cerebral apoplexy or myocardial infarction; those with any disease requiring hospitalization; those with current cancer or other life-threatening diseases; those with current pregnancy, and those with estimated glomerular filtration rate (eGFR) less than 60 mL/min/1.73 m2 calculated each year. Similarly, the new onset of hypertension was defined as a rise of systolic blood pressure (SBP) to 140 mmHg or greater, and/or diastolic blood pressure (DBP) of 90 mmHg or greater evaluated at each year. Renal function expressed as eGFR for Japanese was calculated based on the equation; eGFR = 194 × Cr−1.094 × Age−0.287 (if women × 0.739), reported elsewhere  and male gender , suggesting that UA is not chosen as an estimate to predict the incident CKD in this multivariate analysis.Table 2, respectively. In contrast, mean eGFR of individuals without CKD incidence at baseline and closeout were 91.6 ± 17.5 and 84.4 ± 13.7 mL/min/1.73 m2 (n = 8084), respectively.In 139 individuals with CKD at year 4, mean eGFR at baseline and closeout were 67.7 ± 7.2 and 57.2 ± 2.5 mL/min/1.73 mParticipants with hypertension at the start were excluded from the population for CKD analysis, eliminating 654 participants from 8223, leaving 7569 participants for the hypertension analysis. This study group had an average age 39 ± 10 years, UA level 5.6 ± 1.3 mg/dL at the start. Greater UA levels were associated with older age, males, greater BMI, waist circumference, SBP, DBP, serum Cr concentration, TC, TG, HDLC, LDLC, PG, HbA1c, and lesser eGFR (Table p < 0.001 by the Chi-square test). The greater incidence remained linearly associated with increasing UA level. A total of 633 individuals developed hypertension . Among them, the rate of proteinuria at year 4 was 1.6 % . In addition, among 633 individuals who became hypertensives, there were 13 individuals who developed both CKD and hypertension at year 4 (data not shown).Figure Table 2, respectively. In contrast, mean eGFR of individuals without developing hypertension within 4 years (n = 6936) at baseline and closeout were 91.5 ± 17.8 and 84.1 ± 14.1 mL/min/1.73 m2, respectively. Greater UA levels were associated with greater OR of hypertension development. After adjustment for age, sex, BMI, waist circumference, SBP, DBP, TC, TG, HDLC, LDLC, PG, and HbA1c, multivariate adjusted OR of the logistic regression analysis for new onset of hypertension was 1.00 (reference), 1.30 (95 % CI 1.00–1.69), 1.38 (95 % CI 1.05–1.81), and 1.33 (95 % CI 1.01–1.80) for Group 1 through Group 4, respectively . Furthermore, multivariate Cox proportional hazard analysis shows that the UA levels to be associated with hypertension development were Group 2 , Group 3 , and Group 4  as a reference of Group 1 (UA < 5.0 mg/dL).Figure The present study demonstrates that UA levels were associated with the development of hypertension, but not with the incidence of CKD. The strengths of this study are the large number of individuals who did not have CKD or hypertension at the entry, the complete nature of the data set, and the ability to link demographic and clinical factors with CKD and rise in BP. Other advantages include no new medications for hyperuricemia, diabetes, hypertension, and dyslipidemia throughout the observation period and the ability to adjust for multiple factors that may affect initiation of CKD and hypertension. We also controlled for the currently treated diabetes, hypertension, hyperuricemia, dyslipidemia, and MACE etc., all known as well-established predictors of kidney dysfunction.Increased UA is associated with increased risk for future hypertension, diabetic nephropathy, and CKD, and that hyperuricemia, per se, could be an independent risk factor for poor cardiovascular prognosis , 16, 17.The cause of CKD is multiple. Thus, it is of interest to look into factors that have impact on both the initiation and the progression of CKD in conjunction with UA levels. So far, the role of UA on the incidence of CKD is controversial , 18–21. The present study demonstrates that after the adjustment of confounding factors by the multivariate analysis, UA is no longer an estimate to predict the incident CKD Table , despiteAnimal experiments demonstrate that increased serum UA causes hypertension that is reversible at the early stage but becomes irreversible at the later stage . In humaOur present study confirms previous studies that hyperuricemia, per se, worsened hypertension but also act as a trigger in its first onset. There were a small number of overlapping participants  who developed both CKD and hypertension at year 4, but we feel that this concomitance might not affect the positive link between the development hypertension and UA level.A prospective cohort study recruiting 49,413 Japanese workers with a 7-year follow-up showed hyperuricemia has a strong association with the risks of death in all causes, CHD, stroke, liver disease and CKD suggesting that high UA appeared to be a considerable risk factor for reduced life expectancy .The present trial shows that high UA levels are associated with the new development of hypertension, but not with the incidence of CKD. Whether lowering of UA levels in hyperuricemic patients is beneficial or not remains to be investigated in the future.


We analyze various pathways through which access to electricity affects fertility in Indonesia, using a district difference-in-difference approach. The electrification rate increased by 65 % over the study period, and our results suggest that the subsequent effects on fertility account for about 18 % to 24 % of the overall decline in fertility. A key channel is increased exposure to television. Using in addition several waves of Demographic and Health Surveys, we find suggestive evidence that increased exposure to TV affects, in particular, fertility preferences and increases the effective use of contraception. Reduced child mortality seems to be another important pathway.The online version of this article (doi:10.1007/s13524-015-0420-3) contains supplementary material, which is available to authorized users. In most developing countries, fertility is declining, albeit at different speeds within and across countries. The debate about the causes of that heterogeneity and the very slow progress in some areas is typically also a debate about whether family planning or economic development is more effective in triggering the fertility transition  stood at 5.6 children per women at the end of the 1960s, it declined to 4.1 in 1985, 2.9 in 1993, and then to 2.6 in 2010, albeit with substantial variations in the speed and timing of the decline across regions and urban and rural areas BPS , 2013. TThe effect of electrification on fertility has already received some attention in the literature. Studies looking at this relationship in cross-sectional data have typically found a negative correlation but with some interesting variation  affect norms, (2) provide information, for example about contraception, and (3) affect the allocation of time. We briefly discuss each aspect.Electrification may affect the marginal utility of children through the exposure to TV and other electronic media that broadcast new norms, which may alter fertility preferences. That is, even at constant income and prices, the desired number of children may decrease, conforming to the fertility preferences depicted by modern households in TV shows. For example, La Ferrara et al.  showed tObviously, even if the desired number of children is not affected, the exposure to new media may still affect the actual number of children if more information about family planning services helps to reduce unwanted births by reducing the cost of not having a child ). In addition, we use data from the Indonesian Demographic and Health Surveys (DHS) conducted in 1991, 1994, 1997, 2002/2003, and 2007. Finally, we use a data set retrieved from the Indonesian National Electricity Provider PLN (PT Perusahaan Listrik Negara) containing information on all power plants with a capacity of more than 1 megawatt (MW); their exact location; the date they started operation; and, if applicable, the date the plant was shut down. Between 1993 and 2010, 133 power plants of that size were in operation.In this article, we use four sources of data. The core of our analysis draws on the annual Indonesian national socioeconomic household survey (SUSENAS) from 1993 to 2010, and the village census (PODES) for 1996, 2000, 2003, 2006, and 2008. Both the SUSENAS and PODES are conducted by Statistics Indonesia . A problem with our fertility measure could arise if electrification affects the timing of birth: that is, if electrification leads women to have their children later without altering the total number of children they have. In this case, we would overestimate the effect of electrification on completed fertility. However, we see no particular reason why electrification should affect only the timing of births. Moreover, we also look at fertility preferences , which is not affected by timing effects.The survey further includes an array of socioeconomic variables, including education (highest level completed), use of contraceptives, labor market participation, and detailed household spending. We use these household expenditures as a proxy for income in the analysis. For the years 1993–1998, the questionnaire also included a module on mass media, asking respondents whether they have watched TV, listened to radio, or read a newspaper in the previous week, thus allowing us to test whether electrification affects fertility through the exposure to new media. Except for the most recent years, however, the survey collected no data regarding the use of computers or cell phones.desa) and urban precincts (kelurahan) in Indonesia, collecting information from the village or precinct head. The village census is aggregated at the district level using village population weights and then merged to the SUSENAS district panel. The PODES village census provides information on infrastructure and economic development. We found consistent questions across PODES waves regarding economic structure (agriculture as the main activity), presence of a market with a (semi-) permanent building or larger shopping complex, the quality of roads , source of clean drinking water (pump or piped water), health care providers  and schools in the village.The PODES village census covers all villages , which followed both cost considerations  and equity considerations . Locations that could not be reached through the grid were typically supplied electricity using diesel generators or other off-grid solutions, mostly also by PLN , and those in the poorest quintile nearly caught up (88.0 %).Figure The general trend is similar to what DHS data reveal, although the level is clearly different given that we do not focus on completed fertility but instead on age-specific fertility. Another difference is that the SUSENAS data include all women between 15 and 49 years old, whereas the DHS focuses only on married women. This leads to a significant overestimation of women’s fertility in the DHS data  or a neighboring district (pnit). In this case, the first-stage equation is given byt as instruments, and (2) using only those power plants that have been in operation for at least five years.Finally, to further assess the robustness of our fixed-effects estimates, we take an instrumental variables (IV) approach , in whiGuided by our theoretical framework, we assess the role of three potential transmission channels through which electrification may affect fertility: 1) children’s and women’s labor market participation, (2) media exposure, and (3) child mortality. We apply a fixed-effects specification similar to that in Eq.  to the set of control variables. A summary of the results is presented in Table These results are robust to the specification and choice of control variables, even though some of the PODES variables do appear statistically significant. The results are also not dependent on the inclusion or exclusion of certain subsets of PODES variables, which could be the case if the PODES variables were highly colinear.To further document the robustness of our findings, we conduct a few more sensitivity checks. First, we reestimate the model on either only those districts where in 1993 the electrification rate was below 50 % (roughly the median) or above 50 % to see whether the initial electrification level matters.or neighboring district and alternatively using the presence of a power plant in the same and a neighboring district as an instrument for the electrification rate. The results are presented in Table F statistics suggest that the instruments are strong. The results for our preferred instrument—namely, power plants that are older than five years—are closer to the results in Table Finally, we reestimate the model controlling for the presence of a power plant in the same Guided by our theoretical framework, we now explore in more detail the channels through which electricity may affect fertility.As we noted earlier, access to electricity may influence the shadow price of children by affecting both the direct and indirect cost of raising children. Table Electricity coverage also lowers female labor market participation by roughly the same extent that it lowers child labor. This might seem counterintuitive because the availability of electricity may open up new labor market opportunities. Similar to child labor, we find no effects of female labor participation on fertility. We also control for the district-specific share of men working to account for the complete allocation of time within the household. The share of men working is associated with fertility decline, but these effects are difficult to interpret because they account solely for time allocation and do not capture wage or income effects. The absence of an effect of electrification on female labor market participation is in contrast with the findings by Dinkelman  for SoutMedia exposure is another possible channel linking electricity and fertility given that it may change the fertility preferences through the promotion of specific role models or by providing information on modern contraception. This exposure may reduce fertility through a more efficient prevention of unwanted births by reducing the price of not having a child and conversely increasing the relative price of having a child.The results in Table TV exposure enters negatively and statistically significant in the fertility equation. When it is introduced together with electricity, the effect of TV exposure is still statistically significant, but the effect of electricity is slightly lower and less precise than a regression in which TV exposure is excluded. Thus, TV exposure indeed seems to be a relevant transmission channel linking electricity and fertility. The point estimate implies that an increase of the share of women exposed to TV by 1 standard deviation (SD)  reduces the average number of live births per woman by 0.022 . This means that the TV effect explains about one-quarter of the total reduction in fertility that can be attributed to the increase in electrification. Our point estimate is comparable to the effect identified by La Ferrara et al. . For Bra10ex post tend to rationalize actual fertility: that is, older women may overreport the children that they would have preferred at the beginning of their fertility cycle  reduces the average number of live births per woman by 0.022 . TV explains about one-quarter of the total fertility effect. Further analysis suggests that the effect through TV is partly explained by changes in desired fertility and a more effective use of modern contraception. However, assuming that watching TV simply reduces the time spent on physical intimacy is also plausible. Anecdotic evidence seems to suggest that this effect is important not only in Indonesia but, for example, also in India or China


The signaling of the plant hormone ethylene has been studied genetically, resulting in the identification of signaling components from membrane receptors to nuclear effectors. Among constituents of the hormone signaling pathway, functional links involving a putative mitogen-activated protein kinase kinase CONSTITUTIVE TRIPLE RESPONSE1 (CTR1) and a membrane transporter-like protein ETHYLENE INSENSITIVE2 (EIN2) have been missing for a long time. We now learn that EIN2 is cleaved and its C-terminal end moves to the nucleus upon ethylene perception at the membrane receptors, and then the C-terminal end of EIN2 in the nucleus supports EIN3-dependent ethylene-response gene expression. CTR1 kinase activity negatively controls the EIN2 cleavage process through direct phosphorylation. Despite the novel connection of CTR1 with EIN2 that explains a large portion of the missing links in ethylene signaling, our understanding still remains far from its completion. This focused review will summarize recent advances in the EIN3-dependent ethylene signaling mechanisms including CTR1–EIN2 functions with respect to EIN3 regulation and ethylene responses. This will also present several emerging issues that need to be addressed for the comprehensive understanding of signaling pathways of the invaluable plant hormone ethylene. Ethylene is a small volatile hydrocarbon gas and mediates diverse physiological responses in plant cells. The plant hormone is synthesized by a simple two-step biochemical pathway involving conversion of S-adenosyl-L-methionine (SAM) to 1-aminocyclopropane-1-carboxylic acid (ACC) and then to ethylene, which occurs in all higher plants . EthylenArabidopsis genetics , ETR2, ETHYLENE RESPONSE SENSOR1 (ERS1), ERS2, and EIN4. The receptors differentially express and act in different tissues and at distinct developmental stages  and EBF2 , containing 21% sequence identity to NRAMP metal ion transporter proteins . Even soein2 . The einasmonate , but hypasmonate , indicatein2 correlates well with the diminished EIN3 protein levels in the mutant  that is the experimentally determined amino acid residue as a cleavage site of EIN2 is the main phosphorylation and dephosphorylation site in the absence and presence of ethylene, respectively (ein2–5 with EIN2S645A (Ser to Ala) preventing phosphorylation at this residue results in induction of the EIN2C cleavage and translocation to the nucleus, and constitutive ethylene responses in the absence of ethylene. This implicates that EIN2S645 is phosphorylated and its phosphorylation prevents the protein cleavage and translocation to the nucleus to suppress the hormone signaling in the absence of ethylene.lication . Proteinectively . ComplemUpon ethylene treatment, two different patterns of EIN2 protein accumulation were reported . EIN2 prin vitro. In another report, CTR1 phosphorylates four amino acids in EIN2 /ACC INSENSITIVE1 (AIN1) activity.Once ethylene signaling is initiated, EBF2 appears to be activated transcriptionally. Unlike ethylene . The mRNEIN5 activity also controls the stability of many other RNAs including small RNAs . Therefobotrytis induced kinase1 (bik1), but this protein accumulation does not result in a triple response  and PIF3 directly to activate their transcription and causes seedling greening and hypocotyl growth under lights, respectively  involved in salicylic acid (SA) biosynthesis (In contrast to the well characterized transcription activator role of EIN3/EIL1, recent studies on EIN3 function have proposed transcriptional repression functions through direct binding to target gene promoters as well. EIN3 binds to the 5ynthesis  to negatynthesis . Thus, eEthylene is a key signaling molecule mediating physiological events underlying plant growth and development. Multifaceted functions of ethylene ensure developmental plasticity of plants in response to diverse environmental stress conditions. Our understanding of ethylene signaling is advanced by recent functional characterization of CTR1 phosphorylation of EIN2 and its phosphorylation status-dependent movement into the nucleus that controls EIN3 stability mediating ethylene signaling Figure . As EIN2Arabidopsis will provide invaluable information to screen and characterize regulatory chemicals to specifically manipulate ethylene signaling in crops and certainly be able to manipulate ethylene-dependent physiology for practical purposes.Since ethylene mediates so many physiological traits important for plant-based biomass productivity and its genetic constituents in signaling pathways are largely conserved in diverse plant genomes, detailed understanding of ethylene signaling in the model plant 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.


Expression of cell phenotypes highly depends on metabolism that supplies matter and energy. To achieve proper utilisation of the different metabolic pathways, metabolism is tightly regulated by a complex regulatory network composed of diverse biological entities . The integrated analysis of both regulatory and metabolic networks appears very insightful but is not straightforward because of the distinct characteristics of both networks. The classical method used for metabolic flux analysis is Flux Balance Analysis (FBA), which is constraint-based and relies on the assumption of steady-state metabolite concentrations throughout the network. Regarding regulatory networks, a broad spectrum of methods are dedicated to their analysis although logical modelling remains the major method to take charge of large-scale networks.We present FlexFlux, an application implementing a new way to combine the analysis of both metabolic and regulatory networks, based on simulations that do not require kinetic parameters and can be applied to genome-scale networks. FlexFlux is based on seeking regulatory network steady-states by performing synchronous updates of multi-state qualitative initial values. FlexFlux is then able to use the calculated steady-state values as constraints for metabolic flux analyses using FBA. As input, FlexFlux uses the standards Systems Biology Markup Language (SBML) and SBML Qualitative Models Package  extension  file formats and provides a set of FBA based functions.http://lipm-bioinfo.toulouse.inra.fr/flexflux/. It can be defined as a research tool that enables a better understanding of both regulatory and metabolic networks based on steady-state simulations. FlexFlux integrates well in the flux analysis ecosystem thanks to the support of standard file formats and can thus be used as a complementary tool to existing software featuring other types of analyses.FlexFlux is an open-source java software with executables and full documentation available online at The online version of this article (doi:10.1186/s12918-015-0238-z) contains supplementary material, which is available to authorized users. Analysis of metabolic networks is extensively used as a direct reflect of the phenotype of living cells. Moreover, the increasing amount of available omics data has encouraged the reconstruction of a significant number of genome-scale metabolic networks . A very Bacillus subtilis 14], in tAs detailed previously, new FBA constraints are set considering the attractor found in the regulatory network steady-state analysis. In the case of a cyclic attractor, each constraint is defined by the mean of the bound values corresponding to each component state in the attractor. We have chosen to use the mean of the states because we consider that it is more suitable to a broad range of studies when multiple cells are not synchronised. Indeed, when considering a whole population with multiple cells at different states, different states of the attractor will be encountered. Since the method is not able to evaluate the importance of the different states, they will all be considered as equally important for the steady-state of the cell. In consequence, the mean of the translated values simulates an average of the attractor states in the population. A different strategy could be to perform a FBA for each state of the attractor. This can still be done in FlexFlux by setting each state of the attractor as a constraint in separate FBA analyses.Lastly, the fact that FlexFlux does not analyse both types of networks in the same optimisation process allows a higher flexibility in the possible regulatory logical rules. For example, negative auto-regulation, which is very common in biological systems , as wellE. coli model, iAF1260, and 12 s for 10,000 RSA with the Tcell model.FlexFlux already offers 13 functions. All of them support a Regulatory Steady-state Analysis (RSA). FlexFlux code is open-source and thus can be used by other developers to create new functions that use the capabilities described in this paper. In addition, as we show in Additional file To facilitate the reader’s understanding of FlexFlux features compared to other applications and methods, we have compiled two comparison Tables.Table Table FlexFlux is a free, open source Java software that joins two types of steady-state based analyses that are usually performed separately: FBA and qualitative multi-state simulations. FlexFlux is the first tool to support both SBML and SBML-qual standards file formats to describe metabolic and regulatory networks respectively. This support ensures compatibility with other FBA software and qualitative network analysis software. Regulatory network simulations are based on synchronous updates of the network state and the result can be translated into continuous intervals used as constraints in the FBA. This allows to easily constrain a flux analysis depending on regulatory network reprogramming. FlexFlux also contains different methods to analyse in detail regulatory and metabolic networks in interaction. The methods implemented in FlexFlux are efficient to deal with genome-scale networks.FlexFlux is designed for researchers looking for an accessible tool capable of performing sophisticated analysis of the relations between metabolic and regulatory networks.Project name: FlexFlux.Project home page:http://lipm-bioinfo.toulouse.inra.fr/flexflux/index.htmlOperating system(s): Linux, Windows.Programming language: Java.Other requirements: Java 1.7 or higher, CPLEX orGLPK.License: GNU LGPL.Any restriction to use by non-academics: none.