Human T lymphotropic virus type I (HTLV-I) is a retrovirus which is associated with adult T cells leukaemia (ATL) and HTLV-I-associated myelopathy/tropical spastic paraparesis (HAM/TSP) in a minority of HTLV-I-infected individuals. It is not clear why a minority of HTLV-I-infected individuals develop HAM/TSP and majority remains lifelong carriers. It seems that the interaction between the virus and the immune response plays an important role in HTLV-I-associated diseases. Although the role of the immune response in HTLV-I pathogenesis is not fully understood, however it seems that the efficacy of the immune response which is involved in controlling or limiting of viral persistence determines the outcome of HTLV-I-associated diseases. Here we discuss the role of innate and adaptive immune response and also the risk factors contribute to the observed differences between HAM/TSP patients and asymptomatic HTLV-I carriers. Human T-cell leukaemia virus type I (HTLV-I) is a type C retrovirus, which belongs to the Deltaretrovirus genera of the Orthoretrovirinae subfamily, first identified 30 years ago from peripheral blood samples of a patients with cutaneous T-cell lymphoma . The virAlthough, it is estimated 10-20 million individuals worldwide are infected with HTLV-I, the prevalence of the virus in the general population remains unknown -6. HTLV-et al showed that mutation in the Tax gene was linked to HTLV-I subtype rather than the risk of HAM/TSP  are critical in controlling or limiting and also the efficacy of the host response to HTLV-I.In this study, we review and update insights relevant to the immune response to HTLV-I in HAM/TSP patients. The role of HTLV-I in ATL will not be discussed here. Immune response to HTLV-IInnate immune responseIn vivo studies have shown that the HTLV-I provirus is mainly detected in CD4+ T lymphocytes  are also shown to be infected in vitro , 24-27. in vitro by cell-free HTLV virions and then the infected cells might transmit the virus to T cells  act as critical regulators of innate and adaptive immunity which contribute to susceptibility and/or health protection . The roled lysis . ex vivo. The spontaneous NK cell proliferation correlates with HTLV-I proviral load  or particle agglutination. The reactive results should be confirmed by confirmatory methods such as Western blot (WB) or polymerase chain reaction (PCR). In Western blot, the reactive samples are tested against the products of HTLV-I such as gag (p19 or p24) and env (gp21 or gp46) gene products -41. After HTLV-I infection, antibodies to core, envelope and tax protein in serum appear within 30 to 60 days after primary HTLV-I infection; antibody to gag proteins predominant with anti-p24 generally appears before anti-p19 antibodies. Antibody to p-21 envelope protein frequently appears before gp46 antibodies. Anti tax antibodies are the latest antibodies that appear in the time course of seroconversion . Higher Real time polymerase chain reaction (PCR) also allows quantifying HTLV-I proviral load which is expressed as the number of HTLV-I DNA copies per fixed number of peripheral blood mononuclear cells (PBMC). Proviruses of HTLV-I are clonally integrated in ATL patients, but randomly integrated in HAM/TSP patients. The efficiency of HTLV-I replication is defined as the level of proviral DNA integrated in random PBMCs population or the proportion of PBMCs that carry a HTLV-I provirus , 46. HTLHigh proviral load and the presence of IgM antibodies and high titres of IgG and IgA antibodies to HTLV-I proteins appear to distinguish HAM/TSP patients from HTLV-I carriers . More reHTLV-I specific CD8+ CTL responseCytotoxic T lymphocytes are a subset of T cells that recognise and kill their targets through the major histocompatibility complex (MHC). The majority of CTLs express CD8 molecules, whereas they rarely express CD4 molecule. Both CD4+ and CD8+ CTLs are able to eliminate infected cells and are important for recovery from viral infection .in vitro generates HTLV-I specific CD4+ T cell lines. Most of these cells are HLA class II restricted and exhibit cytotoxic activity. Although these CTLs recognise the HTLV-I envelope protein between amino acids 196 and 209, their frequency in the circulation is low (Studies have shown that both HAM/TSP patients and asymptomatic carriers have circulatory HTLV-I specific CTLs. These CTLs are human leukocyte antigen (HLA) class I or class II restricted which recognise varieties of HTLV-I epitopes such as Tax, Rex and env proteins -53. Antiet al found that there is no difference between HAM/TSP patients and healthy carriers in the frequency of specific CD8+ CTLs , CCR5 and the IL-2R β-chain (Most HTLV-I-specific CD8+ CTLs recognise the Tax protein (Tax11-19) in association with HLA-A2 allele. The tetrameric MHC-peptide complex technique identified that HAM/TSP patients carried a significantly higher number of CD8+ T cells specific for the HTLV-I Tax11-19 peptide in PBMC and CSF compared with the carriers . Biegano β-chain . The fre β-chain . The high frequency of HTLV-I-specific CD8+ CTLs in PBL and CSF is responsible for the production of proinflammatory cytokines. Patients with HAM/TSP have high levels of peripheral blood CD8+ T cells that produce intracellular IFN-γ. It has been demonstrated that HTLV-I-specific CD8+ clones derived from HAM/TSP patients secret IFN-γ, TNF-α, IL-16, macrophage-inflammatory protein-1α (MIP-1α) and 1β (MIP-1β), and matrix metalloproteinase-9 (MMP-9) . et al shows that in HAM/TSP patients circulating HTLV-I-specific CD8+ lymphocytes produce proinflammatory cytokines such as IL-2, TNF-α and IFN-γ. The proportion of these cytokine expressing HTLV-I-specific CD8+ cells in total CD8+ T cells is extraordinary high (4.9% in HAM/TSP and 0.4% in HTLV-I carriers). These specific CD8+ T cells could promote inflammatory responses to HTLV-I in HAM/TSP . Furthermore, it has been suggested that there is a higher frequency of IFN-γ, TNF-α, and IL-2 production by CD4+ T cells in HAM/TSP patients than the carriers with a similar proviral load , 68. Fur. ImmunogRole of cytokines in HTLV-I infectionIn vivo studies by Tendler identifyalteration in host gene expression and cytokine production accompanies HTLV-I infection. The mRNA expression of TNF-α, IFN-γ and IL-1β is up regulated in HAM/TSP and HTLV-I carriers. This up-regulation is accompanied by an elevation of TNF-α and IL-1β exclusively in the sera of HAM/TSP patients . These specific CD8+ T lymphocytes may promote inflammatory responses to HTLV-I in HAM/TSP . Effector T cells induce activation of immune responses by secreting pro-inflammatory cytokines whereas regulatory T cells, which express the transcription factor Foxp3, play a critical role in maintaining immune system homeostasis by both cell-contact dependent and independent mechanisms -77. In v3+ Tregs -79. It has been suggested that the percentage of Foxp3+ Tregs in CD4+CD25+ cells in HTLV-I carriers and healthy controls is higher than this percentage in patients with HAM/TSP , 80; whiin vivo studies suggest that overexpression of HTLV-I Tax decreases the expression of Foxp3 and prevents Treg cells suppressive function. The frequency of HTLV-I negative Foxp3+CD4+ cells positively correlates with proviral load, while there is a negative association between CTLs’ activity and the frequency of these cells. This suggests that an increase in the number of HTLV-I negative Foxp3+CD4+ Tregs has a pivotal role in the efficiency of T cell mediated immune control of the virus in HTLV-I infected individuals (Lately, it has been reported that patients with HAM/TSP exhibit significant reductions in Foxp3 expression and Treg cell function in CD4+CD25+ T cells . Reducedividuals .et al called this IFN-γ+Foxp3–CD4+CD25+CCR4+ T cell subsets THAM cells (It has been discovered that the chemokine receptor CCR4 is expressed on HTLV-I-infected leukemia cells in patients with ATL . Both TrAM cells .  In HAM/TSP and ATL patients, most of CD4+CD25+CCR4+ T cells are infected with HTLV-I -88 and t+CD25+CCR4+ T cells might also determine the outcome of different immune responses in HTLV-I infection.Accumulated data suggests that the vast majority of HTLV-I-infected individuals remain carriers and HTLV-I is not sufficient to cause HAM/TSP. Therefore, the differences in the efficiency of the immune response to HTLV-I infection and the genetic background among HTLV-I carriers could be crucial for determining the pathogenesis of HTLV-I infectious diseases. Both CTL response and regulatory T cells have critical roles in the outcome of HTLV-I-associated diseases. The CTL response in asymptomatic carriers is more efficient than CTL response in HAM/TSP patients, leading to more powerful predictive role in viral replication and spread. Furthermore, infection of regulatory CD4


Infection with the human T-cell leukemia virus-1 (HTLV-1) is associated with a range of outcomes ranging from asymptomatic infection, to the development of HAM/TSP and adult T cell leukemia/lymphoma. Pathogenesis of these disorders involves complex interactions with the host immune system. Our previous studies showed that phorbol ester (PMA) and T cell receptor (TCR)-mediated activation of chronically infected CD4 T cells increased expression of HTLV-1 gene products . TCR stiWe also investigated mechanisms of HTLV-1 activation following PMA stimulation. Surprisingly, PMA treatment was associated with a rapid, marked stabilization of HTLV-1 tax/rex mRNA. Increased RNA stability represents a novel mechanism for increasing HTLV-1 gene expression in chronically infected cells.


Human T cell leukemia virus type 1 (HTLV-1) causes a fatal hematopoietic malignancy, adult T cell leukemia (ATL), and a viral oncoprotein Tax is considered to play the important roles in leukemogenesis through its potent activation of NF-κB. Protein ubiquitination is crucial for the proper regulation of NF-κB pathway. It is also known that the function of Tax is modified by ubiquitination, indicating that ubiquitination machineries contribute to the oncogenic mechanisms of ATL. We report that two ubiquitin-specific peptidases, USP20 and USP33, deubiquitinate TRAF6 and suppress IL-1β-induced NF-κB activation. We find that USP20 deubiquitinates Tax and inhibits Tax-induced NF-κB activation, consistent with Tax being a substrate of USP20. In HTLV-1-transformed cells, the transcription of USP20 is reduced compared with HTLV-1-negative T cells, and ectopic USP20 expression was found to inhibit the proliferation of an HTLV-1-transformed cell line, MT4. Our findings suggest USP20 is a key negative regulator of NF-κB signaling and can influence HTLV-1-induced leukemogenesis.


HTLV-1 associated infective dermatitis (IDH) is a chronic dermatitis that affects a proportion of HTLV-1 infected children. IDH serves as an early clinical marker for HTLV-1 and may be associated with an increased risk of other HTLV-1 associated conditions such as adult T cell leukaemia/lymphoma (ATLL) and HTLV-1-associated myelopathy/transient spastic paraparesis (HAM/TSP). Since a high proviral load is found in all 3 diseases, we wished to characterise HTLV-1 replication in children with IDH. We studied a cohort of 10 cases with IDH and two asymptomatic carrier (AC) individuals from South Africa. We found high levels of the HTLV-1 provirus in the skin: mean value was 47.09 proviral copies per 1000 cells, compared with 137 proviral copies per 1000 cells in blood. Staining sections from IDH skin lesions revealed strong intracytoplasmic staining of HTLV-1 Gag protein but not Tax protein. To study the clonality of HTLV-1 replication in IDH, we used a recently developed high throughput Illumina sequencing protocol [


Beside their role in primary metabolism, plant mitochondria also act as “signaling organelles”, able to influence processes such as nuclear gene expression. AOX activity can control the level of potential mitochondrial signaling molecules such as superoxide, nitric oxide and important redox couples. In this way, AOX also provides a degree of signaling homeostasis to the organelle. Evidence suggests that AOX function in metabolic and signaling homeostasis is particularly important during stress. These include abiotic stresses such as low temperature, drought, and nutrient deficiency, as well as biotic stresses such as bacterial infection. This review provides an introduction to the genetic and biochemical control of AOX respiration, as well as providing generalized examples of how AOX activity can provide metabolic and signaling homeostasis. This review also examines abiotic and biotic stresses in which AOX respiration has been critically evaluated, and considers the overall role of AOX in growth and stress tolerance.Alternative oxidase (AOX) is a non-energy conserving terminal oxidase in the plant mitochondrial electron transport chain. While respiratory carbon oxidation pathways, electron transport, and ATP turnover are tightly coupled processes, AOX provides a means to relax this coupling, thus providing a degree of  Respiration then uses these carbohydrates to support growth and maintenance through the provision of carbon intermediates, reducing equivalents and ATP. These processes, in turn, release CO2 and convert O2 back to H2O.Photosynthesis and respiration are the primary pathways of carbon and energy metabolism in plants. Photosynthesis uses light energy, CO2 to H2O. Importantly, electron transport at complexes I, III and IV is coupled with proton translocation from the mitochondrial matrix to inner membrane space (IMS) and the resulting proton motive force is used by complex V (ATP synthase) to generate ATP from ADP and Pi)et al..Several studies have shown that respiration and mitochondrial activity are enhanced following infection with plant pathogenic fungi –229. FurThis section highlights studies that have shown an effect of AOX respiration on plant performance  or plant tolerance to different biotic or abiotic stress.2 assimilation and/or oxygen evolution [Arabidopsis plants defective in components that contribute to control of the chloroplast redox state have increased AOX amount. These include plants defective in cyclic electron transport [Arabidopsis localizes to thylakoid membranes rather than the mitochondrion, or perhaps that it is dual-targeted to both organelles, revealing a potential unexpectedly close interaction between AOX and photosynthetic electron transport [Photosynthesis and respiration are interdependent processes. This interaction in the light depends upon a direct or indirect exchange of common metabolites between chloroplast and mitochondrion via the cytosol –237. Sinvolution ,238–241.volution ,243, AOXvolution ,222, andvolution . Furthervolution . Isotopevolution , and alsvolution . Also ofransport  or lackiransport . In thesransport .Arabidopsis plants. Microarray analyses showed that transcripts encoding chloroplast proteins, particularly those associated with the light reactions of photosynthesis and stress are amongst the most perturbed genes in plants lacking AOX [A role for AOX during photosynthesis has also been investigated in transgenic and mutant king AOX ,129,250.king AOX ,129,251.Many plants store cyanogenic glycosides in their vacuole as a defense against herbivory. If released to the cytosol by mechanical damage, these cyanogens are rapidly degraded to release CN. In the cyanogenic crop plant cassava, harvesting of the tuberous roots has been shown to result in a rapid burst of ROS that strongly curtails post-harvest shelf-life. It was recently shown that this ROS burst is dependent upon CN release, caused by the mechanical damage associated with harvesting . This suO-acetylserine(thiol)lyase C (OAS-C) can catalyze the conversion of the hydrogen sulfide and O-acetylserine back to cysteine. It has recently been shown that such a cycle in the mitochondrion is critical to managing cellular levels of CN and hydrogen sulfide. An Arabidopsis T-DNA mutant lacking the mitochondrial CAS isoform was shown to accumulate CN [2. Similarly, mutation of the mitochondrial OAS-C isoform resulted in accumulation of hydrogen sulfide and CN, accompanied again by increased AOX and higher total respiration rate [Arabidopsis were co-cultivated with CN-producing bacterial strains, plant growth was more strongly curtailed in the transgenic plants lacking AOX [In non-cyanogenic plants, the major source of CN in the cell occurs during ethylene biosynthesis. Conversion of 1-aminocyclopropane-1-carboxylic acid (ACC) to ethylene by ACC oxidase generates an equimolar amount of CN. β-cyanoalanine synthase (CAS) can catalyze the conversion of the CN and cysteine to β-cyanoalanine and hydrogen sulfide. Similar to CN, hydrogen sulfide is also a potent inhibitor of cyt oxidase. ulate CN . Interesion rate . Interesion rate , an intrking AOX .As noted earlier, ozone treatment was found to rapidly reduce cyt pathway capacity and increase AOX capacity in tobacco plants . Hence, Arabidopsis, improving growth and decreasing shoot sodium level [2O2 than the WT during stress and the authors suggested that this change may have influenced sodium transport, allowing the plants to better exclude sodium from the shoot [As discussed earlier, salt stress has been shown to disrupt mitochondrial function and increase AOX respiration. Recently, constitutive overexpression of AOX was reported to increase the salt tolerance of um level . The AOXhe shoot .versus salt) or may indicate that expression of the ROS-scavenging network in response to AOX manipulation differs between the two species. In tobacco, AOX over-expression perturbed expression of the ROS-scavenging network during the stress [Arabidopsis study did not report on the expression of the ROS-scavenging network [In summary, the ability of AOX overexpression to increase tolerance toward abiotic conditions associated with oxidative stress such as ozone or salt has met with mixed results. On the one hand, increased AOX during the stress may increase tolerance by reducing ROS generation by the respiratory chain, as suggested by the salt stress study. On the other hand, AOX overexpression might interfere with the normal signaling pathways required to engage acclimation responses to stress, as suggested by the ozone study. In this case, while the increased AOX might still dampen ROS generation by the respiratory chain, it could also paradoxically result in an increased total cellular ROS level. The different outcome in these two studies may relate to the different stresses applied  conditions.The relationship between AOX and growth has been examined in a simple tobacco suspension cell system, and with growth of the cells in macronutrient-sufficient or macronutrient (N or P) deficient conditions . Under mChlamydomonas reinhardtii[Chlamydomonas lacking AOX displayed a large increase in biomass accumulation compared to WT cultures. This was associated with a large increase in cell volume, also similar to that described for tobacco cells under P limitation [Chlamydomonas was consistent with a proteomic analysis that showed an up-regulation of proteins associated with anabolism and attendant down-regulation of catabolism-related genes [Another single cell system in which the relation between AOX and growth has been critically examined is in the green alga inhardtii. Similarmitation . The inced genes . Since cIn general, while the single cell systems discussed above have shown that removing AOX had a positive impact on growth, the relation between AOX and growth will undoubtedly be much more complex in whole plants. A recent example that illustrates this complexity comes from experiments in which AOX was overexpressed in cassava tubers . Under oArabidopsis plants growing at low temperature, AOX was reported to beneficially impact early vegetative growth since antisense knockdown of Aox1a slowed early growth while constitutive overexpression accelerated early growth [Aox1a T-DNA mutant of Arabidopsis. In this case, neither the AOX mutant nor a mutant in cyclic electron transport showed any growth defect at high light. However, the double mutant showed strongly curtailed growth [Arabidopsis leaf [et al.[Aox2b gene in soybean was shown to compromise both vegetative growth and seed yield under typical greenhouse growth conditions.In y growth . A benefd growth . At presd growth . Howeverd growth  and non-sis leaf . Recentlf [et al. were theArabidopsis plants with altered AOX amount, it was reported that knockdown and overexpression plants displayed similar relative growth rate as WT plants under optimal growth conditions. However, under stress (drought) conditions, plants overexpressing AOX displayed a higher relative growth rate than WT plants [In another study comparing growth of T plants . FurtherT plants . The aboStress can perturb metabolism, such as by disruption of enzymes and membrane processes. Stress likely similarly disrupts signal transduction pathways, as well as engaging signal pathways associated with stress acclimation. Over the past decade, AOX has emerged as an important mitochondrial component of the plant stress response. Such work emphasizes that numerous abiotic and biotic stresses impact on mitochondrial bioenergetics and function. Stress alters the cellular demands on mitochondrial metabolism and changes in AOX activity represent a means to rapidly meet these changing demands. AOX activity can also directly impact the level of potential important signaling molecules, thus providing an important link between mitochondrial function, signal transduction, and acclimation to stress.


It’s estimated that 10 to 20 million people are infected with the HTLV-1 virus worldwide. Two principal diseases are associated with this virus: the Adult T-cell Lymphoma/Leukemia (ATLL), and the HTLV-1 Associated Myelopathy/ Tropical Spastic Paraparesis (HAM/TSP). The last one is a debilitating neurological disease characterized by a chronic progressive paraparesis, with autonomic and mild sensorial complains. There are also incomplete forms of HAM/TSP, sometimes called subclinical or oligosymptomatic. Those mild and moderate neurological presentations are found in almost one fourth of the HTLV-1 patients and they include neurogenic bladder syndromes, erectile dysfunction, isolated pyramidal signs and peripheral neuropathy.To describe the evolution of neurological disability in HTLV-1 infected individuals.Cohort study with 419 HTLV-1 infected individuals, divided in three groups according to the main clinical picture in to:1. asymptomatic, defined as absence of any neurological symptoms or signs;2. oligosymptomatic, defined by presence of neurogenic bladder, erectile dysfunction, minor pyramidal signs and neuropathy (isolated or in combination);3. HAM/TSP, as defined by WHO criteria. Those patients had an annual follow up, including neurological examination. EDSS was utilized to measure neurological disability in various domains. Failure was considered as any worse on EDSS scale or death due to HTLV-1. Censored was considered as lost of follow up or death due other causes.In 419 analyzed patients 67 were diagnosed with HAM/TSP, 96 as oligosymptomatics, 194 as asymptomatics. There were 10 deaths on the cohort and the annual death rate was 0,59/100 patients. In survival analyses there were 71 failures with an incidence rate of 0,09 individuals/year. Kaplan-Meyer survival estimate curves with log-rank test showed a significant difference between the three curves (p<0.01), with incidence rates and time at risk of 0,2 and 125 in HAM/TSP 0,08 and 242 in neurological symptoms and 0,05 and 445 in asymptomatic. Cox regression showed that TNF-α higher than 541 pg/ml , female gender , and history of hypotiroidism  had a strong association with neurological decreased function.Neurological disability in HTLV-1 infected patient increase with time, and it is worse in groups with symptoms or HAM/TSP. Some factors proved to be associated with neurological disability and two of them (diabetes and hypotiroidism) is life treating and one (TNF-α) can be a focus of future pharmacotherapy interventions studies.


Breakdown of these signaling systems results in pathological hyperpermeability and/or genetic vascular abnormalities such as vascular malformations, ultimately progressing to hemorrhage and edema. Hence, blood vessel maintenance is fundamental to controlling vascular homeostasis and tissue functions. This paper discusses signaling pathways essential for vascular maintenance and clinical conditions caused by deterioration of vessel integrity.As our understanding of molecular mechanisms leading to vascular formation increases, vessel maintenance including stabilization of new vessels and prevention of vessel regression began to be considered as an active process that requires specific cellular signaling. While signaling pathways such as VEGF, FGF, and angiopoietin-Tie2 are important for endothelial cell survival and junction stabilization, PDGF and TGF- The vascular system is not only essential for the maintenance of tissue homeostasis, but also important for managing a wide variety of physiological processes. Blood vessels are actively remodeled and reorganized depending on the tissue oxygen demand, suggesting that the maintenance of blood vessels is an active process achieved by a specific biological program. While the biology leading to new vessel formation has been extensively investigated in the last couple of decades , the staThe notion of active signaling required to maintain the vasculature is supported by several lines of evidence: (1) as angiogenic growth factors can promote new vessel formation, inhibition of those growth factors in tumor or tissue has been demonstrated to cause vessel regression with variable sensitivity –6, (2) dThe discoveries of angiogenic growth factors considerably advanced our understanding on biology underlying new vessel formation. This subsequently laid the foundation for new possibilities in the treatment of ischemic diseases over the conventional medical management and invasive procedures such as catheter-based angioplasty and coronary bypass surgery. The new approach to facilitating the endogenous revascularization process has been termed therapeutic angiogenesis. The potential impact of therapeutic angiogenesis in clinical medicine is significant because it will enable us to achieve tissue perfusion by manipulating intrinsic blood vessel growth. However, over the past decades, many attempts to develop biological interventions for therapeutic angiogenesis have not produced significant clinical benefits despite the initial success in preclinical studies. Double-blind randomized clinical trials aiming at biological revascularization in patients with ischemic heart diseases have failed to demonstrate therapeutic efficacy of any single growth factor delivery –12. Thesβ1 (TGF-β1) to stabilize the nascent blood vessels. In contrast with the initial phase of vessel sprouting, endothelial cells stop migration and proliferation in the maturation step and restore the barrier function of vessels. It is, therefore, reasonably assumed that to counteract the action of proangiogenic factors, vessel stabilization requires activation of distinct cellular signaling pathways different from those that initiate vascular cell mobilization. In fact, VEGF-A, one of the most potent growth factors that initiates strong vascular growth, is known to increase vascular permeability [As currently understood, new vessel formation is triggered by endothelial cell activation and sprouting coordinated with controlled detachment of the surrounding mural cells, and proteolytic remodeling of the basement membrane and the extracellular matrix. This initial step is followed by assembly into a nascent vascular structure, lumen formation, and, finally, maturation of the newly formed endothelial tube and expression of endothelial cell survival factors. Throughout the process, proangiogenic factors such as vascular endothelial growth factor (VEGF) and fibroblast growth factor (FGF) differentially regulate defined subpopulations of endothelial cells in the angiogenic sprout, promoting endothelial migration at specialized tip cells and proliferation of the stalk cells. Mural cells are then recruited by platelet-derived growth factor (PDGF) and transforming growth factor-eability , which, eability . This steability . In viewEndothelial intercellular junctions, critically important for the maintenance of vascular integrity, are comprised of distinct adhesion structures including adherens, tight, and gap junctions . Unlike Cdh5, encoding VE-cadherin, die at E9.5 due to defects in vessel remodeling [In endothelial cells, VE-cadherin, a calcium-dependent adhesion protein mediating transhomophilic interactions, localizes at cell-cell contacts, regulating the formation of adherens junctions, and linking the site of the junction to the actin cytoskeleton. In the quiescent endothelium, VE-cadherin-based junctions are subjected to continuous reorganization, which renders endothelial junctions highly dynamic and sensitive to extracellular stimuli. In fact, during the process of new vessel formation, endothelial cells undergo dynamic rearrangement upon angiogenic stimuli while continuously reorganizing cell-cell junctions and maintaining barrier function at the same time. This coordination is largely regulated by adhesion molecules at intercellular junctions and is particularly important for tube stabilization and restoration of the full barrier function. Thus, junction proteins play a critical role in controlling vascular integrity both in developing and existing vessels. Among these, VE-cadherin is crucial for this regulation for its capability to remodel actin cytoskeleton via modifying the function of small GTPases . Disruptmodeling .β, sphingosine-1-phosphate, Tie-2, and Notch, which are required for pericyte differentiation, recruitment, and expansion. On the other hand, pericyte coverage promotes vessel maturation by resolving angiogenic signals and reducing endothelial proliferation. Alterations in pericyte density or the stable attachment of pericytes to endothelial cells are associated with human pathological conditions such as diabetic microangiopathy, venous malformation, and hereditary stroke and dementia syndrome CADASIL. Moreover, pericytes of tumor vessels present multiple abnormalities, including an abnormal shape and altered gene expression of more immature and less-contractile markers [Pericytes are mesenchymal-derived cells which are positioned around microvessels and cover gaps between endothelial cells , 21. Alt markers . These p markers . SignaliAs a major structural component of resistant vessels, the ECM plays a substantial role in the maintenance of vessel integrity. The ECM of the blood vessel wall exists mainly in three forms: the interstitial matrix which fills intercellular spaces of the media and the adventitia, elastic laminae of artery, and the basement membrane which is a sheet-like structure that localizes underneath the endothelium and supports both endothelial cells and pericytes , 26. RedAs ECM degradation by MMPs activates quiescent endothelial cells and thus triggers angiogenesis, constitutive endothelial-matrix interaction is thought to maintain vascular homeostasis and thereby prevent angiogenic vascular growth . TherefoThe importance of the ECM in embryonic vascular development has also been shown by an observation of mice lacking histone deacetylase 7 (HDAC7). HDAC7, exclusively expressed in the vascular endothelium in the early embryonic stage, maintains vascular integrity by repressing MMP-10 expression. HDAC7 null mice show embryonic lethality after E11.0 due to a failure of endothelial cell adhesion, leading to vessel dilatation and rupture .−) and H2O2. These ROS can increase tyrosine nitration (nY), cysteine (Cys), and zinc thiolate (ZnS4) oxidation, affecting important cardiovascular proteins such as eNOS, prostacycline synthase, MnSOD, and sarcoendoplasmic reticulum calcium ATPase (SERCA) [Although physiological levels of ROS, serving as a signaling mediator in the vascular system, are important for the maintenance of vascular homeostasis and vascular integrity, oxidative stress induced by elevated levels of ROS may contribute to the initiation and development of vascular dysfunction associated with hypertension and diabetes . The mec (SERCA) . Protein oxidation may also lead to junction instability by targeting important components of the junction structure. Increased vascular permeability is usually reversible and does not deteriorate vascular integrity because endothelial cells can quickly restore the barrier function by reestablishing VE-cadherin-based junctions; however, prolonged permeability-inducing stimuli may cause a more profound effect such as accumulation of ROS. ROS can irreversibly inactivate protein tyrosine phosphatases (PTPs) by oxidizing a Cys residue in the catalytic center, thereby affecting the function of PTPs . Given tβ-catenin, respectively. Dissociation of catenins especially p120 increases endothelial permeability by dismantling VE-cadherin-based junctions, which is sufficient to maintain endothelial cells in a mesenchymal-like state with a promigratory phenotype [The VEGF gene family consisting of VEGF-A, B, C, D, and placenta growth factor (PlGF) in humans is essential for a wide variety of vascular functions . Multiplhenotype , 45. Whihenotype , 47. 2) production in endothelial cells [Although VEGF-A disrupts endothelial junctions, which can potentially compromise vascular integrity when the action is prolonged, VEGF signaling is indispensable for physiological endothelial functions and vascular homeostasis. Apart from the angiogenic property, VEGF is widely recognized as a vascular protective factor with its ability to increase nitric oxide (NO) and prostacyclin , tissue plasminogen activator (tPA), P-selectin, IL-8, and endothelin with the plasma membrane. This process transfers these molecules to the cell surface where they promote the recruitment of leukocytes and platelets . NO actsMoreover, a recent study demonstrated that autocrine VEGF signaling is required for endothelial cell survival in a cell-autonomous manner under nonpathological conditions. Endothelial-specific deletion of the VEGF-A gene in mice leads to progressive endothelial degeneration and sudden death by 25 weeks of age due to vascular insufficiency, suggesting that endogenous VEGF produced by endothelial cells is crucial for vascular homeostasis . Precise The family of Roundabout (Robo) transmembrane receptors is the canonical receptor for the signaling molecule Slit, regulating commissural axon guidance . Robo4 iα activation in the tumor milieu. Unregulated VEGF expression causes vascular hyperpermeability that leads to leakage of plasma proteins into the stroma, further stimulating persistent angiogenesis and creating a self-reinforcing vicious cycle [Observations of tumor vasculature provided insightful information with regard to mechanisms of vessel fragility and leakiness, which is potentially applicable to anticancer therapy . VEGF isus cycle . As a reus cycle , 61. VEGus cycle , 63.FGFs comprise one of the most versatile and complex signaling families in vertebrates, playing critical roles in a wide variety of biological processes . FGFs arFgfr1 or Fgfr2 in mouse embryos results in embryonic lethality at very early stages of development, precluding further evaluation of their contribution to vascular development [Despite the recognition of FGF as a strong in vivo angiogenic promoter, deciphering its precise functions in the vascular system has suffered from the lack of information obtained from mouse knockout studies , 68. Diselopment . On the elopment . One of elopment . Furtherelopment . In cleaelopment . It has elopment . TogetheThe angiopoietin- (Angpt-)Tie2 signaling system has crucial roles in vascular functions including angiogenesis and vessel maintenance . Angpt-1−/− embryos showed that the earliest detectable defect seen at E9.5 is loss of myocardial trabeculations while the vascular system appears normal. Thereafter, these embryos become markedly growth retarded with generalized disorganization of blood vessels. When Angpt-1 is specifically excised from cardiomyocytes using the Nkx-2.5 Cre-driver line, the vascular defects reported in the global knockout are recapitulated, indicating that vascular phenotypes are dependent on the cardiac defect and resulting from impaired blood circulation. Moreover, deletion of Angpt-1 in the later stage of vascular development does not affect pericyte number or vascular mural cell recruitment. Together, authors concluded that Angpt-1 is not required for embryonic vascular development or maintenance of vascular quiescence; however, it functions as a protective factor, modulating responses to tissue injury and microvascular abnormality in diabetes [Mice lacking Angpt-1 or Tie2 have similar cardiovascular defects, indicating the importance of the Angpt-1-Tie2 signaling system in cardiovascular development. These mice die mid gestation (E10.5–E12.5) due to the absence of hierarchical vascular development and perturbed vascular integrity, manifested by reduced pericyte coverage and detachment of pericytes from the endothelium . Althougdiabetes .Recent studies began to reveal molecular mechanisms of Angpt-1 modulating vascular functions. Angpt-1 inhibits VEGF-induced Src activation through RhoA activation which leads to Src association with mDia (a RhoA downstream target) and sequestration of Src from VEGFR2 . Moreove1–S1P5) to induce a variety of biological responses [1 receptor (Edg1) signaling by promoting Rac1 activation and adherens junction assembly [1 receptor is essential for normal vascular function since systemic antagonism of S1P1 receptor under basal physiological conditions causes S1P1 receptor internalization and degradation through receptor phosphorylation, leading to enhanced pulmonary vascular leakage [Sphingosine-1-phosphate (S1P), a sphingolipid metabolite found in high concentrations in platelets and blood, is a lipid mediator that interacts with GPCRs  produced by the angiogenic endothelium. Paracrine PDGF signaling is thus required for mural cell recruitment and expansion as has been demonstrated that PDGF-B expression is particularly abundant in tip cells of angiogenic vessels and in the endothelium of growing arteries [β null mutant mice die perinatally, displaying lethal hemorrhage and edema caused by a pericyte loss in microvessels [β-deficient mice revealed that vessel instability observed in these mice is modified by systemic upregulation of VEGF. The endothelial junction structure is slightly altered in PDGFRβ-deficient mice, and this is attributed to the VEGF effect, since the onset of endothelial hyperplasia precedes the endothelial junction abnormality [The role of platelet-derived growth factor (PDGF) signaling in the vascular system is established as an important player to mediate pericyte-endothelial interaction . PDGFs aarteries . The sigovessels . The resovessels . The absormality . β. Pericyte populations in different tissues are affected in varying degrees by the loss of PDGF signaling in developing PDGFRβ−/− embryos. Therefore, PDGF signaling is thought to be important in the subsequent maturation process in the angiogenic vessels where PDGF-BB released from endothelial cells drives pericyte migration and expansion [It appears that initial induction of pericyte differentiation from mesenchymal progenitors is independent of PDGF signaling and is most likely mediated by other factors such as TGF-xpansion . This enxpansion , 99.β family includes TGF-βs, activins, inhibins, nodal, bone morphogenetic proteins (BMP), and growth differentiation factors (GDF), comprising one of the largest growth factor/cytokine families in vertebrates. Although TGF-β family members are known to play crucial roles in the vascular development, their precise role especially in endothelial functions is still controversial [β effects are highly context-dependent with in vitro observations suggesting that TGF-β is stimulatory to endothelial cell functions at low concentrations whereas it can be inhibitory at high concentrations [β activation, which, in turn, inhibits endothelial proliferation and migration through VEGFR2 downregulation [β signaling in mural cells is to attenuate the endothelial cell response to angiogenic stimuli, thereby limiting vessel overgrowth and resolving the active angiogenic process. This appears to form a negative feedback loop by which endothelial cells promotes pericyte differentiation and expansion that lead to vessel stabilization and, consequently, vessel maturation.TGF-oversial . This istrations . Moreovegulation , 102. Thβ in pericyte functions and vascular development. Disruption of the TGF-β1 gene in mice leads to extraembryonic vascular defects exemplified by the failure of endothelial differentiation accompanied with fragile yolk sac vasculature [Tgfbr2 in the smooth muscle cell lineage results in vascular defects in the yolk sac and embryonic lethality between E12.5 and E16.5, suggesting impairment of mural cell recruitment [β signaling in mural cell function is further endorsed by the observation that mice with neural crest-specific deletion of Tgfbr2 develop a phenotype similar to that of DiGeorge syndrome due to the failure of neural crest derivative differentiation into smooth muscle cells in the cardiac outflow tract [βRII, endoglin or activin receptor-like kinase 1(ALK1) are highly reminiscent of TGF-β1 null mice, showing vascular abnormalities characterized by systemic vascular dysplasia and recurrent hemorrhage caused by telangiectases and arteriovenous malformations [Genetic studies in mice have indicated the essential role of TGF-culature . Inactivruitment . The impow tract . Moreovermations . These sγ, MAPK, and EphrinB2/EphB4 signaling. EphrinB2, which is an arterial endothelial marker, is a direct transcriptional target of Notch signaling [The Notch signaling pathway is an evolutionarily conserved, intercellular signaling system that plays important roles in a wide variety of biological processes. During vascular development, Notch signaling plays essential roles in endothelial cell specification including angioblast specification, arteriovenous differentiation, and tip/stalk cell formation , 107. Thignaling . As signignaling . γ-secretase and translocates to the nucleus where it regulates gene transcription. In the vascular sprout of the retina, while the VEGF-A gradient generated by astrocytes in the ischemic area induces endothelial cells at the leading edge to adopt a tip cell phenotype, Dll4 prominently expressed in the tip cell signals to following stalk cells through Notch1 to downregulate VEGFR2 and inhibit filopodia formation [When Dll4 binds Notch1, the intracellular domain of Notch1 (NICD) is cleaved by ormation , 112. Thormation .Notch signaling appears to be required for vessel stabilization and homeostasis through multiple mechanisms. Notch signaling inhibits endothelial cell proliferation and motility, thus preventing excessive vessel sprouting and stabilizing newly formed vessels. Although increased Notch signaling does not necessarily induce vessel instability, recent study revealed that coordination of Notch and Wnt signaling plays a role in stabilization of nascent vessels. Notch-regulated ankyrin repeat protein (Nrarp), directly induced by Notch signaling, promotes canonical Wnt signaling and controls the stability of newly formed vessel anastomosis . Notch sThe semipermeable compartmentalization achieved by blood vessels especially by the endothelium is critically important for normal organ functions and tissue homeostasis . The proVenous malformations, the most common type of vascular malformation, comprise either superficial or deep veins that are abnormally formed and dilated in the skin, mucous membrane, or in any organ system characterized by lesions composed of enlarged, tortuous venous channels . Althougβ family signaling pathways. HHT type I results from mutations in ENG, coding for endoglin, a coreceptor of TGF-β receptors. HHT type II causes by mutations in ACVRL1 which encodes ALK1, a type I receptor for the TGF-β superfamily ligands. While mutations of either of the two genes account for most clinical cases, a small subset of patients carry mutations in MADH4 encoding Smad4, a transcription factor that mediates TGF-β signaling. Hereditary hemorrhagic telangiectasia (HHT), also known as Osler-Weber-Rendu syndrome, is a genetic disorder that is inherited as an autosomal dominant trait and affects 1-2 in 10,000 individuals , 125. HHβ signaling pathway such as TGF-β1, TGF-βRII, endoglin, or ALK1 are all leading to severe vascular abnormality and embryonic lethality at mid gestation, in some part recapitulating HHT phenotypes [Mouse phenotypes resulting from targeted disruption of components of TGF-enotypes . β signaling such as wound healing and angiogenesis might play as an extra trigger. While endothelial ALK1 is essential for the establishment of proper arteriovenous (AV) connections during vascular development, in the adult endothelium lacking ALK1, AVM forms following activation of the quiescent endothelium by wound that elicits angiogenesis [β1 or reduced TGF-β1-induced responses. In HHT type I patients, circulating levels of TGF-β1 are reduced [β signaling is defective in mice with endothelial-specific deletion of TGF-βRII or ALK5 as evidenced by reduced phosphorylation of Smad2 in the adjacent mesothelial cell layer of the yolk sac. Phosphorylation of Smad2 and differentiation of smooth muscle cells can be rescued by exogenous TGF-β1 in the yolk sac culture, consistent with the idea that lower levels of TGF-β or reduced responsiveness required for receptor activation play a role in HHT pathogenesis [Detailed analysis of HHT gene functions using mouse models of tissue-specific or haploinsufficient gene inactivation has begun to reveal the mechanism of HHT development , 126. Cuogenesis . In HHT, reduced . Moreoveogenesis . In this regard, the recent discovery of thalidomide as a potential therapeutic agent for HHT confirmed the importance of mural cell dysfunction responsible for the formation of fragile vessels. In mice heterozygous for a null mutation in the endoglin gene, thalidomide treatment stimulates mural cell recruitment by increasing PDGF-B expression in endothelial cells and thus rescues vessel defects .KRIT1 (CCM1), CCM2 , and PDCD10 (CCM3) [Cerebral cavernous malformations (CCMs) are sporadic or inherited vascular malformations in the central nervous system characterized by dilated, thin-walled capillary-like channels without intervening brain parenchyma. They are one of the commonest vascular malformations in the brain, affecting roughly 0.1–0.5% of the general population . Genetic0 (CCM3) . Althoug0 (CCM3) . KRIT1 o0 (CCM3) . Since R0 (CCM3) . Ccm2 in mice, a genotype equivalent to that in human CCM, results in impaired endothelial barrier function. Interestingly, loss of CCM2 leads to activation of RhoA, and the impaired barrier function in heterozygous mice is restored by simvastatin, a drug known to inhibit Rho GTPases [Ccm3 show defects in embryonic angiogenesis and die at an early embryonic stage. In response to VEGF stimulation, CCM3 is recruited to and stabilizes VEGFR2, thereby playing a pivotal role in VEGFR2 signaling [Recent mouse genetic studies advanced our understanding of pathogenesis leading to CCM. Disruption of heart of glass (HEG1), a transmembrane receptor previously implicated in CCM functions in zebrafish, results in defective integrity of the heart and the vasculature in mice. HEG1 coupled with CCM proteins through KRIT1 is required for vascular development and endothelial junction formation . Haploin GTPases . Moreoveignaling .Capillary malformation-arteriovenous malformation (CM-AVM) is a recently discovered hereditary disorder characterized with atypical cutaneous multifocal capillary malformations often in association with high-flow lesions such as arteriovenous malformations of the cerebrospinal and visceral organs or arteriovenous fistulas. Genetic studies identified loss of function mutations in RASA1, which encodes p120-RasGAP , 139. AsNOTCH3, which causes degeneration of smooth muscle cells of cerebral small vessels and accumulation of the Notch extracellular domain (NECD) at the surface of residual smooth muscle cells. These changes lead to thickening of the vessel wall and a reduction of cerebral blood flow [The clinical relevance of Delta-Notch signaling is manifested by the cerebral small vessel disease: cerebral autosomal-dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL), now recognized as the most common cause of inherited stroke and vascular cognitive impairment in adults , 142. CAood flow . GeneticNotch3 in mice causes structural defects of small arteries because of impaired differentiation and maturation of arterial smooth-muscle cells; however, total loss of Notch3 does not cause CADASIL pathology [κ transcription comparable to wild-type levels and do not seem to affect Notch signaling per se [Genetic disruption of athology . Furtherg per se , 145. ThAs discussed earlier, Notch pathway is important for arteriovenous differentiation and vessel patterning during embryonic vascular development, and deficiency of Notch signaling can cause arteriovenous malformations , 147. ExRegulation of the vascular barrier function is crucial for tissue homeostasis. Recently, our understanding of molecular mechanisms leading to vessel stabilization has significantly expanded owing to the advance of basic research followed by critical appraisal of therapeutic angiogenesis trials. It has also become apparent that the maintenance of existing vessels requires active cellular signaling which share common features with signaling mechanisms involved in the vessel maturation process of new vessel formation. The signaling systems controlling vascular maintenance is summarized in As regulation of vessel maintenance is a fundamental vascular function associated with a wide variety of vascular disorders and disease conditions, elucidating the precise mechanisms will benefit the development of new approaches for therapeutic angiogenesis and vascular malformations as well as cancer treatment.


Adult T cell leukemia/lymphoma (ATL) and HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP) are caused by HTLV-I infection. In these patients, the antibody titer and provirus load are elevated compared to levels in asymptomatic carriers (AC). These methods that determine HTLV-I infection do not differentiate between AC, HAM/TSP patients, and ATL patients. We have reported on a Luciferase Immunoprecipitation System (LIPS), a highly sensitive, quantitative technology that can efficiently detect HTLV-I antibody responses in serum of infected individuals [


Adult T-cell leukemia/lymphoma(ATLL) has so far had a very poor prognosis by chemotherapy. From the long-term observations of our previous clinical trials  for ATLL patients), we suspect that RIC strategy might have a possible curative power for ATLL patients (pts).We evaluated the safety and feasibility of allogeneic hematopoietic stem cell transplantation with RIC from matched sibling donors (MSD) for ATLL pts using a conditioning regimen consisting of fludarabine and busulfan. Low-dose antithymocyte globulin was added in the 1st study (NST-1), while it was omitted for the 2nd study (NST-2). We present the results of long-term follow-up of the two trials as well as the longitudinal patterns of changes in HTLV-1 proviral load in survivors.Between Apr, 2001 and Feb, 2006, 30 pts ranged from 50 to 67 years of age were enrolled in NST-1(16 pts) and NST-2 (14 pts). After undergoing the conditioning regimen, they received G-CSF-mobilized peripheral blood (PB) stem cells from HLA-matched sibling donors (MSD). Half of the donors were HTLV-1 carriers. The primary end points in both studies were achievement of complete donor chimerism before day 90, and absence of early transplant-related mortality (TRM) before day100. The HTLV-1 proviral load was estimated using PB samples serially after RIC. HTLV-1 proviral DNA was measured by the quantitative PCR amplification of HTLV-1 pX DNA. The detection limit of the HTLV-1 proviral load was 0.5 The overall survival (OS) curve was estimated by the Kaplan–Meier method.The results have been already published elsewhere  . Ten of the29 evaluable patients have survived for a median of 115 months  after RIC. All of them maintain their good health (Karnofsky PS score ?90%).The majority of survivors have developed the graft-versus host disease (GVHD) (10/10 pts for acute GVHD and 9/10 pts for chronic GVHD). Overall and progression free survival rates at 5 years for the studies were 36%  and 31% , respectively. Serial changes in the HTLV-1 proviral load after RIC in the pts are heterogeneous but can be roughly classified into 3 patterns. In the first pattern, seen in 3 pts, the proviral load became undetectable after RIC and continued to remain so. In the second pattern, seen in 3 pts who had received RIC from HTLV-1 negative donors, the proviral load had become undetectable but returned to detectable levels thereafter. Lastly, in the third pattern, seen in 4 pts who had received the grafts from HTLV-1-carrier donors, the proviral load had remained at the carrier level. All the 10 survivors continue to show complete donor chimera during the observation period regardless of the HTLV-1 proviral load level.The long-trem follow–up in our study indicates not only that RIC from MSD is a feasible treatment modality for ATL, but also that one third of the pts may be cured with this procedure because all survivors are good for health. As for post-RIC changes in HTLV-1 proviral load in long-term survivors it seems heterogenous, which may be categoryzed into 3 patterns.


The formation of the vertebrate kidney is tightly regulated and relies on multiple evolutionarily conserved inductive events. These are present in the complex metanephric kidney of higher vertebrates, but also in the more primitive pronephric kidney functional in the larval stages of amphibians and fish. Wnts have long been viewed as central in this process. Canonical β-Catenin-dependent Wnt signaling establishes kidney progenitors and non-canonical β-Catenin-independent Wnt signaling participate in the morphogenetic processes that form the highly sophisticated nephron structure. While some individual Wnt signaling components have been studied extensively in the kidney, the overall pathway has not yet been analyzed in depth.Xenopus laevis using in situ hybridization. Out of 19 Wnt ligands, only three, Wnt4, Wnt9a and Wnt11, are specifically expressed in the pronephros. Others such as Wnt8a are present, but in a broader domain comprising adjacent tissues in addition to the kidney. The same paradigm is observed for the Wnt receptors and its downstream signaling components. Fzd1, Fzd4, Fzd6, Fzd7, Fzd8 as well as Celsr1 and Prickle1 show distinct expression domains in the pronephric kidney, whereas the non-traditional Wnt receptors, Ror2 and Ryk, as well as the majority of the effector molecules are rather ubiquitous. In addition to this spatial regulation, the timing of expression is also tightly regulated. In particular, non-canonical Wnt signaling seems to be restricted to later stages of pronephros development.Here we report a detailed expression analysis of all Wnt ligands, receptors and several downstream Wnt effectors during pronephros development in Together these data suggest a complex cross talk between canonical and non-canonical Wnt signaling is required to establish a functional pronephric kidney. In vertebrates the kidney is an essential organ required to excrete nitrogenous waste and maintain salt and water balance. Embryologically, the kidney is derived from the intermediate mesoderm and develops through three increasingly complex forms, the pronephros, the mesonephros and the metanephros. While the mesonephros is the adult kidney of fish and amphibians, the metanephros is found in higher vertebrates such as mammals and avians 2+, modification of the actin cytoskeleton by the small G proteins Rac and Rho and the regulation of planar cell polarity (PCP) by the PCP core proteins Prickle, Van Gogh-like  and Celsr. Importantly, Wnt signaling is no longer restricted to only Frizzled receptors, but non-Frizzled receptors like Ror2 and Ryk have emerged as well Wnt signaling is one of the most critical pathways in many aspects of development Xenopus pronephros as a paradigm. Using in situ hybridization we identified pronephric kidney-specific expression for Wnt4, Wnt9a, Wnt11, Fzd1, Fzd4, Fzd6, Fzd7, Fzd8 as well as the PCP core proteins Celsr1 and Prickle1. Moreover, these expression domains were distinct not only in respect to their spatial domains, but also in their temporal aspect. This suggests that even in the rather primitive pronephros Wnt signaling has to be tightly controlled to assure proper organ growth.The diversity of Wnt signaling is reflected by the fact that 19 different Wnts, 10 Frizzled receptors, three disheveled as well as multiple members of the PCP core proteins, Celsr, Vangl and Prickle have been identified in most vertebrates , S2, S3.Xenopus (see below), their expression was not studied in respect to pronephros formation. Thus, we decided to systematically monitor the expression patterns of the entire Wnt family, its receptors and downstream signaling components by whole mount in situ hybridization  and two non-Frizzled receptors, Ror2 and Ryk  is the key element shared by canonical and non-conical Wnt signaling the tail . Moreove pattern . CuriousXenopus  are rather uniformly expressed, while others (i.e. Prickle and Celsr) are more regionally restricted.Taken together these expression patterns demonstrate that some Wnt signaling components , but does not share its role in nephron induction. This leaves the two possibilities that either the pronephric mesenchyme does not need to be maintained in a similar fashion as its metanephric counterpart or that another Wnt molecule takes its place. Indeed, Wnt8a is expressed throughout the mesoderm from gastrula stage onwards , Vangl and Diego function together to regulate PCP in e.g. bristle orientation and ommatidial rotation Xenopus, Prickle1 or Celsr1 mRNAs are detected in distinct and temporally non-overlapping expression domains in the kidney. While Vangl1/2 are expressed throughout the developing pronephros and could presumably interact with both proteins, a complex of all three molecules is unlikely. Interestingly, the dichotomy in their expression patterns is not only observed in the pronephros, but also in the other expression domains  exhibit diffuse and rather low expression. Together these expression data support the hypothesis that a classical PCP signaling pathway involving all core components is absent in the pronephros and probably in some of the other organ systems.As expected many downstream signaling components in particular the pan-Wnt signaling components  domains : Pricklein Xenopus has so far resulted in embryos completely lacking a pronephros. Nevertheless, Xenopus is an ideal system to explore redundancy issues. As previously shown Our expression analysis supports the notion that pronephros development is regulated by an interplay between canonical and non-canonical Wnt signaling. Our study identified candidates for both processes. Interestingly, at either stage 25 or 35 multiple Wnt ligands, receptors and effector molecules are expressed. This suggests that a high degree of redundancy exists and that single loss-of-function studies may only reveal part of the whole process. Indeed, none of the loss-of-function experiments This study was carried out in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. The protocol was approved by the IACUC committee of the LSU Health Sciences Center (IACUC protocol #2760).Xenopus embryos obtained by in vitro fertilization were maintained in 0.1x modified Barth medium in situ hybridizations and in situ hybridizations on paraplast sections were performed as described previously pBSK(-)-Wnt1-EcoRI/T7 (NIBB #XL020a18), pGEM-T-Easy-Wnt2b-NcoI/Sp6, pGEM-T-Easy-Wnt3a - SalI/T7, pGEM2-Wnt4- Nhe1/T7 pGEM-T-Easy-Wnt5a-ApaI/Sp6, pCMV-SPORT6-Wnt5b - EcoRI/T7 (IMAGE: 5048927), pGEM-T-Easy-Wnt6-ApaI/Sp6, pGEM-T-Easy-Wnt7a-ApaI/Sp6, pCMV-SPORT6.1-Wnt7b-PstI/T7 (IMAGE:7019251), pCS111-Wnt7c - SmaI/T7 (IMAGE: 8532605), pCS2-Wnt8a-XhoI/T3, pBSKS-Wnt8b - NotI/T7 (NIBB #XL040e10), pBSKS-Wnt9a - ClaI/T7, pBSKS-Wnt9b - HindIII/T7, pBSK(-)-Wnt10a-EcoRI/T7 (NIBB #XL100k05), pGEM-T-Easy-Wnt10b - Nco1/Sp6, pBSK(-)-Wnt11NotI/T7, pBSK(-)-Wnt11b - EcoRI/T7 (NIBB #XL092j06), pGEM-T-Easy-Wnt16-NcoI/T7 (GenBank Accession Number pending), pExpress1-Fzd1-SmaI/T7 (IMAGE: 7392077), pCMV-SPORT6-Fzd2-EcoRI/T7 (IMAGE: 3399141), pBSK(-)-Fzd3- ClaI/T7 (NIBB #XL260j03ex), pBSK(-)-Fzd4-EcoRI/T7 (NIBB #XL024e15), pCMV-SPORT6-Fzd5- BamHI/T7 (IMAGE: 3401522), pBSK(-)-Fzd6- XbaI/T7 (NIBB #XL220p06), pCMV-SPORT6-Fzd7-EcoRI/T7 (IMAGE: 5570875), pBSKS-Fzd8-EcoRI/T7, pCS107-Fzd9- EcoRI/T3 (IMAGE: 3200715), pGEM-T-Easy-Fzd10-SalI/T7, pBSK(-)-Ror2- PstI/T7 (NIBB #XL105o06), pBSK(-)-Ryk - XbaI/T7 (NIBB #XL049d20), pCMV-SPORT6-Dvl1- Asp718/T7 (IMAGE: 6956452), pCMV-SPORT6-Dvl2- EcoRI/T7 (IMAGE: 3401885), pCMV-SPORT6-Dvl3- Asp718/T7 (IMAGE: 6317854), pBSK(-)-Prickle1 - BamHI/T7 (NIBB #XL141p03), pGEM-T-Easy-Prickle2- SalI/T7, pCS111-Prickle3-BamHI/T7 (IMAGE: 8824324), pBSK(-)-Celsr1-EcoRI/T7 (NIBB #XL005e01), pCS105-Celsr2-EcoRI/T3 (NIBB #XL513h08ex), pGEM-T-Easy-Celsr3- NcoI/Sp6, pBSK(-)-Vangl1-PstI/T7 (NIBB #XL038m21), and pCMV-SPORT6-Vangl2-EcoRI/T7 (IMAGE: 7010992). The identity of the individual family members was confirmed by multi-sequence alignment with the human, mouse, chick and zebrafish homologues using ClustalW2 or MUSCLE and visualized using the Phylogeny.fr web service Whole mount Figure S1Phylogenetic Tree of Wnt Ligands. Analysis of all Wnt ligands from human, mouse, chick, zebrafish and Xenopus using the MUSCLE algorithm.(PDF)Click here for additional data file.Figure S2Phylogenetic Tree of Frizzled Receptors. Analysis of all Frizzled receptors from human, mouse, chick, zebrafish and Xenopus using the ClustalW2 algorithm.(PDF)Click here for additional data file.Figure S3Phylogenetic Tree of Wnt Signaling Intermediates. Analysis of all disheveled (A), prickle (B), celsr (C) and vangl proteins (D) from human, mouse, chick, zebrafish and Xenopus using the ClustalW2 (C) or MUSCLE  algorithm.(PDF)Click here for additional data file.


Nicotiana benthamiana plants in which alternative respiratory pathway capacity was either increased by constitutive expression of AOX, or decreased by expression of a dominant-negative mutant protein (AOX-E). N. benthamiana was used because it is a natural mutant that does not express a functional RNA-dependent RNA polymerase 1.Salicylic acid (SA) regulates multiple anti-viral mechanisms, including mechanism(s) that may be negatively regulated by the mitochondrial enzyme, alternative oxidase (AOX), the sole component of the alternative respiratory pathway. However, studies of this mechanism can be confounded by SA-mediated induction of RNA-dependent RNA polymerase 1, a component of the antiviral RNA silencing pathway. We made transgenic Aox transgenic plants while SA-induced resistance to this virus appeared to be stronger in Aox-E transgenic plants. These effects, which were limited to directly inoculated leaves, were not affected by the presence or absence of a transgene constitutively expressing a functional RNA-dependent RNA polymerase (MtRDR1). Unexpectedly, Aox-transgenic plants infected with potato virus X (PVX) showed markedly increased susceptibility to systemic disease induction and virus accumulation in inoculated and systemically infected leaves. SA-induced resistance to PVX was compromised in Aox-transgenic plants but plants expressing AOX-E exhibited enhanced SA-induced resistance to this virus.Antimycin A  and SA triggered resistance to tobacco mosaic virus (TMV). Resistance to TMV induced by antimycin A, but not by SA, was inhibited in We conclude that AOX-regulated mechanisms not only play a role in SA-induced resistance but also make an important contribution to basal resistance against certain viruses such as PVX. Salicylic acid (SA) is an important defensive signal in plants that is required for elicitor-triggered immunity and the establishment of systemic acquired resistance (SAR)-8. PlantRNA silencing  is thought likely to contribute to SA-induced virus resistance although it is unlikely to be the only mechanism involved . RNA silThere is evidence that mitochondrial signaling processes regulate some aspects of SA-induced virus resistance . ReacAox1a gene expression  or 1 mM SA dissolved in 0.05% (w/v) ethanol before rub inoculation with PVX or TMV on one or two lower leaves as previously described for tobacco [For whole-plant treatments with SA, five-to-six week old  tobacco ,20. ChemWSL and FSF carried out the experimental procedures, with additional experiments by JVL on PVX accumulation. JPC initiated and directed the research and wrote the manuscript. All authors read and approved the final manuscript.Nicotiana benthamiana plant lines used in this studyDetection of AOX-E protein expression and over-expression of AOX protein in transgenic . Immunoblot detection of AOX (A) or mutant AOX-E (B) present in non-transgenic (NT) N. benthamiana plants and T2 generation transformed plants belonging to various independent lines (numbered) harboring AOX or AOX-E transgenes expressed under the control of the 35S constitutive promoter. Equal amounts of Triton X-100 soluble proteins were denatured in the presence of 0.1 M dithiothreitol and subjected to immunoblot analysis using an anti-AOX monoclonal antibody. Anti-AOX binding was detected using anti-mouse immunoglobulin conjugated to horseradish peroxidase and a chemiluminescent substrate. A protein sample extracted from a plant of the Sn6 transgenic tobacco plant line, which over-expresses AOX , served as the Positive Control. The major cross-reacting band in all cases corresponded in size (apparent Mr c.35kDa) to the reduced form of AOX. Pre-stained Mr markers were not visible on the X-ray film. All lines express AOX or AOX-E at much higher levels than the native AOX protein which is not detectable on this western blot.Click here for fileN. benthamianaImage showing TMV-induced systemic symptoms of TMV on non-transgenic, control transgenic and MtRDR1-transgenic . Non-transgenic (NT) Nicotiana benthamiana plants, plants transformed with an 'empty' transformation vector (Control) and transgenic lines constitutively expressing MtRDR1 (35S:MtRDR1: Reference 22) were inoculated with TMV U1 strain (0.05 ug/ml) and photographed four weeks later. Mock-inoculated NT plants are shown for comparison. The scale bar is 8 cm.Click here for file


The majority of leukemia cells were enriched in the L subpopulation and the same clone was included in the D and L subpopulations, suggesting clonal evolution. In this study, we analyzed patients with indolent-type ATL and human T-cell leukemia virus type I (HTLV-I) asymptomatic carriers (ACs) to see whether the CD3 versus CD7 profile reflected progression in the properties of HTLV-I-infected cells.In a recent study to purify adult T-cell leukemia-lymphoma (ATL) cells from acute-type patients by flow cytometry, three subpopulations were observed in a CD3 versus CD7 plot . In the follow-up of some patients, clinical disease progression correlated well with the CD3 versus CD7 profile. In clonality analysis, we clearly detected a major clone in the D and L subpopulations in ATL cases and, intriguingly, in some ACs in Group 2.In the D(%) versus L(%) plot, patient data could largely be categorized into three groups . Some exceptions, however, were noted (We propose that the CD3 versus CD7 plot reflects progression of disease stage in patients infected with HTLV-I. The CD3 versus CD7 profile will be a new indicator, along with high proviral load, for HTLV-I ACs in forecasting disease progression. Human T-cell leukemia virus type I (HTLV-I) is the agent that causes HTLV-I-associated diseases, such as adult T-cell leukemia-lymphoma (ATL), HTLV-l-associated myelopathy/tropical spastic paraparesis (HAM/TSP), and HTLV-I uveitis (HU) e.g., tax and HBZ), genetic and epigenetic alterations, and the host immune system are considered to cooperatively contribute to leukemogenesis in ATL i.e., it does not directly indicate progression of disease stage in HTLV-I infection. Moreover, the majority of ACs with high VL remained intact during the study period, indicating that a more accurate indicator of progression is needed.Various cellular dysfunctions induced by viral genes , and the majority of ATL cells were enriched in the L subpopulation In our recent study to purify monoclonal ATL cells from acute-type patients by flow cytometry, three subpopulations were observed in a CD3 versus CD7 plot of CD4TL-Om1, an HTLV-I-infected cell line, established Dr. Hinuma's laboratory The present study was approved by the research ethics committee of the institute of medical science, the university of Tokyo. Subjects provided written informed consent.Peripheral blood mononuclear cells (PBMCs) were isolated from heparin-treated whole blood by density gradient centrifugation, as described previously 5′-CGGATACCCAGTCTACGTGTT-3′ and reverse primer 5′-CAGTAGGGCGTGACGATGTA-3′), FAM-labeled probe at 0.1 µM (5′- CTGTGTACAAGGCGACTGGTGCC-3′), and 1× TaqMan Universal PCR master mix (Applied Biosystems), was subjected to 50 cycles of denaturation  and annealing to extension , following an initial Taq polymerase activation step . The RNase P control reagent (Applied Biosystems) was used as an internal control for calculating the input cell number (using VIC reporter dye). DNAs extracted from TL-Om1 and normal human PBMCs were used as positive and negative controls, respectively. The HTLV-I proviral load (%) was calculated as the copy number of the pX region per input cell number. To correct the deviation of data acquired in each experiment, data from TL-Om1 (positive control) were adjusted to 100%, and the sample data were corrected accordingly by a proportional calculation.The HTLV-I proviral load in FACS-sorted PBMCs was quantified by real-time quantitative polymerase chain reaction  using the ABI Prism 7000 sequence detection system  as described previously EcoRI and PstI at 37°C overnight. Purification of DNA fragments was performed using a QIAEX2 gel extraction kit (Qiagen). The purified DNA was self-ligated with T4 DNA ligase  at 16°C overnight. The circular DNA obtained from the EcoRI digestion fragment was then digested with MluI, which cuts the pX region of the HTLV-I genome and prevents amplification of the viral genome. Inverse long PCR was performed using Takara LA Taq polymerase (Takara Bio). For the EcoRI-treated template, the forward primer was 5′-TGCCTGACCCTGCTTGCTCAACTCTACGTCTTTG-3′ and the reverse primer was 5′-AGTCTGGGCCCTGACCTTTTCAGACTTCTGTTTC-3′. For the PstI-treated group, the forward primer was 5′-CAGCCCATTCTATAGCACTCTCCAGGAGAG-3′ and the reverse primer was 5′-CAGTCTCCAAACACGTAGACTGGGTATCCG-3. Each 50-µL reaction mixture contained 0.4 mM of each dNTP, 25 mM MgCl2, 10× LA PCR buffer II containing 20 mM Tris-HCl and 100 mM KCl, 0.5 mM of each primer, 2.5 U LA Taq polymerase, and 50 ng of the processed genomic DNA. The reaction mixture was subjected to 35 cycles of denaturation  and annealing to extension . Following PCR, the products were subjected to electrophoresis on 0.8% agarose gels. In samples from which a sufficient amount of DNA was extracted, PCRs were performed in duplicate.For clonality analysis, inverse long PCR was performed + cells in PBMCs of various clinical subtypes from patients infected with HTLV-I and normal controls. The three subpopulations  observed are referred to as the H, D, and L subpopulations, respectively. Representative results for each clinical subtype of HTLV-I infection are shown in The clinical profiles of the 77 cases analyzed in this study are shown in The proportions of D and L subpopulations in all AC cases analyzed are shown in To further characterize each subpopulation  in the CD3 versus CD7 plot, cells in each subpopulation were FACS-sorted and subjected to analysis of VL to determine the percentage of HTLV-I-infected cells in each subpopulation. In HTLV-I infection, progression to ATL requires several pathological steps, including clonal expansion As noted above, the D(%) versus L(%) plot generally represented disease stage in HTLV-I infection. However, we observed one case of chronic-type disease and three cases of smoldering-type disease in Group 1 and three cases of acute-type disease in Group 2. Furthermore, some ACs with VLs of >4% were observed in Group 2. Representative data from these apparently exceptional cases are shown in In several cases, we could obtain time-sequential samples . The patThe patient in highCD7high(H) subpopulation. i.e., less than 55% of the H subpopulation and 80% of the L subpopulation), included indolent-type (smoldering and chronic) cases and some AC cases. These results suggest that the CD3 versus CD7 expression profile reflects disease stage. Initially, both the D and L subpopulations gradually and simultaneously increased. However, at the clinically advanced stage, the increase in the L subpopulation was prominent. The change is considered to reflect the biological difference between the D and L subpopulations, which needs to be clarified.Findings in our previous analysis of acute-type ATL samples prompted our analysis of various clinical subtypes of patients infected with HTLV-I to examine whether the CD3 versus CD7 profile reflects the progression of oncogenesis in HTLV-I-infected cells et al. reported that two cases of HTVL-I AC with oligoclonal bands on Southern blots and high VLs (20%) had progressed to ATL by 4 and 3.5 years later In HTLV-I infection, the small clones of infected cells are considered to coexist from the AC stage et al. reported that high HTLV-I proviral load (>4%) in whole PBMCs was a risk factor for progression to ATL Iwanaga In general, the categorization by flow cytometric profile correlated well with the current classification of clinical subtypes, with some exceptional cases of acute-type and smoldering-type disease . The onlThe VL in each subpopulation indicated that HTLV-I-infected cells were relatively concentrated in the D and L subpopulations . The CD3 versus CD7 plot of CD4+ cells was constructed according to the gating procedure shown in this figure. In the plot, we designated three subpopulations: H (CD3highCD7high), D (CD3dimCD7dim), and L (CD3dimCD7low).(PPTX)Click here for additional data file.Figure S2A two-dimensional plot of 10 normal controls showing the percentage of the D and L subpopulations.(PPTX)Click here for additional data file.Figure S3Southern blot analysis of clonal integration of the HTLV-I provirus. Representative data  are shown. In EcoRI or PstI digestion, a band indicated by a red arrow represents the monoclonal integration of the provirus. The band pattern indicates that two major clones coexist. This analysis was performed by a commercial laboratory .(PPTX)Click here for additional data file.Table S1Clinical data in a case of chronic-type ATL (No. 53). Proportion of abonormal lymphocytes in the preipheral blood WBC were evaluated by morphological exmanination. LDH: Lactate dehydrogenase  sIL-2R: soluble interleukin-2 receptor .(XLSX)Click here for additional data file.


We therefore assayed the sensitivity of HTLV-1 primary isolates to a panel of RT inhibitors. HTLV-1 primary isolates were obtained, pre- and post- NRTI treatment, from patients with HTLV-1-associated myelopathy. Sensitivity to azidothymidine (AZT), lamivudine (3TC), tenofovir (TDF) and three phosphonated carbocyclic 2’-oxa-3’aza nucleosides (PCOANs) was assessed in a RT inhibitor assay. With the exception of 3TC, HTLV RT from primary isolates was less sensitive to all tested inhibitors than HTLV-1 RT from MT-2 cells. HTLV-1 RT from primary isolates and from chronically infected, transformed MT-2 cells was insensitive to 3TC. Sensitivity of primary isolates to RT inhibitors was not reduced following up to 12 months of patient treatment with AZT plus 3TC. The sensitivity of HTLV-1 primary isolates to NRTIs differs from that of cell lines and may vary among patients. Failure of NRTIs to reduce HTLV-1 viral load in vivo was not due to the development of phenotypic NRTI resistance. AZT and the three PCOANs assayed all consistently inhibited primary isolate HTLV-1 RT.Since human T-lymphotropic virus type 1 (HTLV-1)-associated diseases are associated with a high HTLV-1 load, reducing this load may treat or prevent disease. However, despite  In contnfection  has beennfection ,9. Mutatnfection . Whilst in vitro ,10 at muin vitro ,11. The than AZT . A new cin vitro .1.2.AZT has been shown to protect rabbits from challenge with HTLV-1 infection , while tIn vitro data had indicated that tenofovir, which has an established safety profile, inhibits HTLV-1 RT better than other licensed RT inhibitors [hibitors . Thus, sex vivo inhibition of HTLV-1 RT isolates from patients who participated in the Bridge study pre and post therapy. We demonstrate that the NRTI sensitivity of HTLV-1 RT derived from cell lines differs from that of primary isolates, and show differences in drug susceptibility between primary isolates.Here we present data on the 2.2.1.in vitro inhibitory activity of different compounds on HTLV-1 RT from patient isolates, the optimal amount of RT needed to generate cDNA from a known RNA template was first assessed in centrifugation-enriched isolates from patient PBMC culture and, for comparison, from MT-2 cell cultures. Isolates were prepared from PBMC cultures of all of the patients under study collected before and after 48 weeks of therapy with AZT plus 3TC except for patient TBJ, due to insufficient material. The amount of HTLV-1 p19 in the enriched isolates was quantified as a surrogate measure of the amount of RT. Serial dilutions of resuspended isolates, corresponding to 5 to 30 pg of p19, were added to the assay mixture in order to determine the amount of p19-equivalent HTLV-1 RT to be used in the assay. The amount of amplified product plateaued with the addition of isolate preparations corresponding to 20–30 pg of p19 (To test the g of p19 . These r2.2.3 nM but in one patient (TBJ) the inhibitory concentration of 3TC was 400 nM. HTLV-1 RT from MT-2 cells was susceptible to TDF at 0.4 nM but 10–1000 nM of this compound were required to inhibit RT from the primary isolates. The three unlicensed compounds all inhibited MT-2 HTLV-1 RT at 0.8 nM and also inhibited HTLV-1 RT from the primary isolates at 2–10 nM, with PCOAN1 being the most potent. No change in the inhibitory concentration of any RT inhibitor was seen in the primary isolates obtained from patients after 6 to 12 months therapy with AZT plus 3TC.The concentration of AZT, 3TC, TDF and of PCOAN1, PCOAN2, PCOAN3 required to inhibit RT activity in primary isolates prior to and after any antiretroviral therapy was determined. The concentrations used are shown in 2.3.The supernatants utilized for obtaining centrifugation-enriched isolates to assay, were also evaluated for viral particle production by measuring released p19. 2.4.In vivo HTLV-1 DNA loads and the duration of exposure to nucleoside analogues are presented in in vivo after 24–48 weeks of therapy with AZT plus 3TC [pol was sequenced at baseline and at the conclusion of therapy. In three patients the consensus sequence was wild type at both time points. Conversely, duplications, insertions or deletions were observed in the other three patients. However, no relationship was observed between individual proviral load variations and detection of mutations in patients. Thus, apparently the lack of reduction in proviral load was not due to the outgrowth of mutated viruses.plus 3TC . In our 2.5.TDF was well tolerated with no reported side effects. Whilst some patients exhibited a deterioration during therapy those patients who received therapy for longest had measurable improvement in pain and/or gait  and 4. T3.3.1.in vitro HTLV-RT data [In addition to the published ‘Bridge Study’, blood samples were collect during a study of the pathogenesis and treatment of HTLV-1 between April 2004 and May 2005, from five patients with HAM and one diagnosed with HTLV-1-associated encephalitis. The study was approved by the local research ethics committee. These patients had been, after informed consent and on a case-by-case basis, treated with TDF based on -RT data ,17. At t3.2.6 cells/mL in RPMI plus 15% FBS in the presence of 20 U/mL of IL-2, following a two-day stimulation with PHA. PBMCs from patients were maintained in culture for 3–4 weeks. PBMCs were pelleted weekly, supernatants harvested and fresh medium completely replenished. Aliquots of the harvested supernatants were utilized to detect viral particle production by p19 release, while the rest were cleared at 1,000 g and then ultracentrifuged at 30,000 g for 4 h at 4 °C. HTLV-1 p19 viral protein in centrifugation-enriched viral lysates was detected using a commercially available ELISA (see Section 3.6).PBMCs from heparinized blood of patients with HAM were separated by Ficoll/Hypaque density gradient  and then washed three times in phosphate buffered saline (PBS). To use virus isolates from patients as a source of RT for the subsequent assay, paired samples of PBMC at week 0 and week 48 from six patients were cultured at 0.8 × 103.3.HTLV-1 proviral load in the patients treated with TDF was measured by quantitative real-time PCR as previously reported . Samples3.4.Three licensed reverse transcriptase inhibitors  and three experimental compounds prototype PCOANs referred to as, PCOAN1, PCOAN2 and PCOAN3 were used. The acyclic phosphonate TDF was kindly provided by Dr. Jan Balzarini  while AZT and 3TC were provided by Wellcome Research Laboratories . The PCOANs are a family of newly synthesized compounds which belong to a group of cyclic nucleoside phosphonates, with the furanose ring replaced by an N,O-heterocyclic ring ,20. PCOA3.5.ex vivo, cell-free HTLV-1-RT activity assay. This assay is a modified version of a method we have described and used to screen the HTLV-1-RT-inhibitory activity of antiretroviral compounds in vitro [To test the sensitivity of HTLV-1 isolates from patients to RT inhibitors we set up a novel in vitro ,12. The in vitro  and trea6 PBMCs, previously stimulated with PHA (2 μg/mL) and IL-2 (20 U/mL), for 72 h in RPMI plus 20% FCS, were lysed on ice in buffer , and centrifuged at 10000 g. Lysed extracts were incubated with different compound concentrations for 15 minutes on ice followed by 45 minutes at 30 °C and then a 5 minute incubation at 95 °C to inactive the mixture. As an ex vivo source of HTLV-1 RT, viral lysates from different HAM patient sample isolates  were used for each RT-PCR reaction. Total DNase-treated RNA (1 μg) was specifically reverse transcribed using 0.5 μM reverse primer specific for the gD gene US6 (5’ TGTCGTCATAGTGGGCCTCCAT 3’) in a reaction mix containing 1 × RT buffer, 100 U RNase inhibitor, 1 mM dNTP, 10 mM DTT,  plus HTLV-1-RT preparations from patients in a volume of 10 μL. The reactions were performed in the presence or absence of activated substances, for 1 h at 37 °C. After incubation at 95 °C for 5 minutes, DNA PCR (20 cycles) was performed using 5 μL of RT reaction in a mix containing 1X Taq Gold buffer (Promega), 0.5 μM primer pair , 0.2 mM dNTP, 5 mM MgCl2, 1.25 U Taq Gold Polymerase (Promega). To investigate the dose–response, the assayed compounds, AZT, 3TC, TDF or PCOAN1, PCOAN2, PCOAN3 were added at the concentrations shown in Compounds to which sensitivity was assayed were activated by pre-incubating with a crude extract, prepared from PHA plus IL-2 stimulated PBMCs from healthy, HIV/HBV/HCV negative donors. To prepare the crude extract, 1 × 103.6.p19 in supernatants or enriched isolates was assayed using an HTLV-1 p19 Gag antigen capture enzyme-linked immunosorbent assay (ELISA) . Assays were performed according to the manufacturer’s protocols.3.7.pol coding region corresponding to nucleotides 2100–5076 of HTLV-1 reference sequence (NC_001436.1) was performed by sequencing PCR products from DNA of MT-2 cells and of cultured cells from patients enrolled in the study at baseline and at week 48 (post AZT and 3TC therapy). Primer pairs were designed to amplify overlapping products of about 300 nucleotides in length.Genotypic analysis of the entire 4.in vivo treatment with AZT and 3TC was not associated with the development of reduced sensitivity of HTLV-1 RT to these nucleoside analogues or with consistent changes in the sequence of RT. Therefore, we conclude that the lack of viral, and consequent lack of immunological and clinical response seen in this study [pol consensus sequence was identical to the reference sequence in 3/6 patients these results will likely be applicable to the majority of patients infected with HTLV-1.The sensitivity of HTLV-1 primary isolates to AZT, 3TC, TDF and three novel PCOANs has been determined using a novel RT inhibition assay. Using PBMCs from patients with HAM who had been treated for up to 48 weeks with AZT and 3TC, we have demonstrated for the first time, both the sensitivity of HTV-1 RT to AZT and its primary resistance to 3TC in HTLV-1 clinical isolates. Furthermore we have shown that prolonged is study  was not versus RT inhibitor treated patients, and at least one order of magnitude, and in some cases a thousand times, less sensitive to AZT and to TDF than RT from MT-2 cells. This lack of sensitivity to TDF was seen prior to, and not altered by, in vivo NRTI exposure. We have not, at the moment, a clear explanation for the different sensitivity of isolates from patients and MT-2 associated virus to RT-inhibitors. However, we have previously demonstrated variable efficacy of RT inhibitors against HTLV-1 RT from different transformed cell lines. For example, the concentration of PCOAN2 needed to inhibit RT activity from C5/MJ and MT-2 cell supernatants was 10–100 times less than that required to inhibit RT from C91/PL cells. Similarly, the concentration of TDF needed to inhibit RT from MT-2 cell supernatants was 10–100 times less than that required to inhibit RT from C91/PL and C5/MJ cells. Conversely, RT from HUT 102 supernatants was resistant to most of the RT inhibitors used [Unexpectedly, the HTLV-1 RT from patients’ PBMCs displayed considerable variability in TDF sensitivity and consistent sensitivity to PCOANs. In the present study, the phenotypic RT sensitivity of whole virus primary isolates from patients with HAM was quantitatively similar in samples from untreated ors used . Whereasors used . Thus, pin vitro using MT-2 and HTLV-1 donor cells as a source of HTLV-1 as effectively as TDF and AZT [While the data from this study do not suggest that a combination of AZT with TDF might inhibit HTLV-1 RT in some patients, this possibility remains. We therefore promote pre-treatment phenotypic sensitivity testing to avoid unnecessary exposure to these therapies. Conversely, each of the PCOANs assayed had an RT inhibitory concentration similar to that of AZT suggesting that these compounds should be considered in future studies. We have recently demonstrated that these new RT inhibitors, PCOANs, are able to inhibit HTLV-1 cell-to cell transmission  and AZT .in vivo [pol were detected, suggests that the lack of virological effect in the dual therapy clinical trial was not due to the emergence of mutations in RT during therapy. This finding supports the hypothesis that RT only partially explains HTLV-1 viral load maintenance and spread within patients with HAM while mitosis plays a major role in maintaining HTLV-1 viral burden. It is less likely that viral load was maintained by HTLV-1 infection in drug sanctuary sites with migration of infected lymphocytes into the peripheral blood.The presence of defective HTLV-1 proviruses in clinical samples is well known  and our in vivo , suggestOverall, patients treated with TDF showed no clinical improvement. The apparent association between longer duration of TDF therapy and reduced pain and 10m timed walk most likely reflects a bias to continue therapy due to perceived benefit. The CD4+ and CD8+ T lymphocyte count rose significantly from baseline. In HIV infection a rise in CD+ T lymphocyte count is associated with a decreased HIV RNA viral load in patients treated with TDF and other antiretroviral therapies. We could not find previous reports of a change in lymphocyte counts in HIV or HTLV-1 non-infected patients treated with antiretrovirals. In HTLV-1 infected patients AZT did not cause a significant rise of lymphocyte counts. In our earlier study of patients with HAM receiving dual-therapy with AZT and 3TC, the CD4+ T lymphocyte counts rose, but the total lymphocyte count and the CD8+ T lymphocyte count fell. In the same study total lymphocyte and subset counts increased in the placebo group at week 24 but by much less than seen in patients treated with TDF. We hypothesize that, in addition to the antiretroviral effect, TDF therapy is associated with a virus independent increase in lymphocyte and T cell subset counts.in vivo is urgently required.Is there any role for HTLV-1 RT inhibitors in clinical practice? Treatment of adult T-cell leukaemia/lymphoma with AZT together with interferon-α currently induces remission in about 70% of patients. However, there is no evidence that the available RT inhibitors, given as monotherapy or in combination with other RT inhibitors, reduce HTLV viral load. Recently, AZT in combination with a histone deacetylase inhibitor significantly and persistently reduced STLV-1 viral load in naturally infected baboons over three months of treatment  and reprin vivo, due to its reduced sensitivity in treating naïve HTLV-1 primary isolates. Thus, alternative HTLV-1 inhibitors should be tested.If sustained reduction in HTLV-1 viral load in patients with, or at risk of, HAM requires long term combination therapy that includes an RT inhibitor, less toxic alternatives to AZT will be desirable. TDF, which is better tolerated than AZT, would be a suitable candidate based on data using MT-2 cells as donor cells or as a source of RT. However, our data suggest that TDF might not be as effective as AZT ex vivo cell-free assay may be particularly useful in preclinical studies for future clinical trials.Since HTLV-1 is a highly cell-associated virus, RT inhibitors might be useful in clinical trials when used in combination with compounds such as cell signaling modulators . Therefoex vivo data from an additional six patients who were not exposed to TDF, suggests that reduced sensitivity may be common but this needs to be confirmed with more HTLV-1 primary isolates. These data suggest a general lack of RT efficacy when used alone as an anti-HTLV therapy in patients with HAM, justify further studies of novel drug combinations and/or PCOANs to treat and prevent HTLV-1 infection, and provide a tool to test HTLV-1 RT susceptibility to trial drugs ex vivo.While HTLV-1 RT inhibitors are not routinely used clinically to reduce the risk of transmission following occupational exposure or following delivery by an HTLV-1 infected mother, they remain the only potential therapies for pre- or post- exposure prophylaxis. The data presented here suggest that AZT rather than TDF (unless sensitivity is known) would be the preferred therapeutic agent following high-risk exposure. The lack of efficacy in the six patients studied, in conjunction with the


The human T cell leukemia virus type I (HTLV-I) inhibits host antiviral signaling pathways although the underlying mechanisms are unclear. Here we found that the HTLV-I Tax oncoprotein induced the expression of SOCS1, an inhibitor of interferon signaling. Tax required NF-κB, but not CREB, to induce the expression of SOCS1 in T cells. Furthermore, Tax interacted with SOCS1 in both transfected cells and in HTLV-I transformed cell lines. Although SOCS1 is normally a short-lived protein, in the presence of Tax, the stability of SOCS1 was greatly increased. Accordingly, Tax enhanced the replication of a heterologous virus, vesicular stomatitis virus (VSV), in a SOCS1-dependent manner. Surprisingly, Tax required SOCS1 to inhibit RIG-dependent antiviral signaling, but not the interferon-induced JAK/STAT pathway. Inhibition of SOCS1 by RNA-mediated interference in the HTLV-I transformed cell line MT-2 reduced HTLV-I replication and p19Gag levels. Taken together, our results reveal that Tax inhibits antiviral signaling, in part, by hijacking an interferon regulatory protein.