Cells sense external concentrations and, via biochemical signaling, respond by regulating the expression of target proteins. Both in signaling networks and gene regulation there are two main mechanisms by which the concentration can be encoded internally: amplitude modulation (AM), where the absolute concentration of an internal signaling molecule encodes the stimulus, and frequency modulation (FM), where the period between successive bursts represents the stimulus. Although both mechanisms have been observed in biological systems, the question of when it is beneficial for cells to use either AM or FM is largely unanswered. Here, we first consider a simple model for a single receptor (or ion channel), which can either signal continuously whenever a ligand is bound, or produce a burst in signaling molecule upon receptor binding. We find that bursty signaling is more accurate than continuous signaling only for sufficiently fast dynamics. This suggests that modulation based on bursts may be more common in signaling networks than in gene regulation. We then extend our model to multiple receptors, where continuous and bursty signaling are equivalent to AM and FM respectively, finding that AM is always more accurate. This implies that the reason some cells use FM is related to factors other than accuracy, such as the ability to coordinate expression of multiple genes or to implement threshold crossing mechanisms. Signals, and hence information, can generally be transmitted either by amplitude (AM) or frequency (FM) modulation, as used, for example, in the transmission of radio waves since the 1930s. Both types of modulation are known to play a role in biology with AM conventionally associated with signaling and gene expression, and FM used to reliably transmit electrical signals over large distances between neurons. Surprisingly, FM was recently also observed in gene regulation, making their roles less distinct than previously thought. Although the engineering advantages and disadvantages of AM and FM are well understood, the equivalent question in biological systems is still largely unsolved. Here, we propose a simple model of signaling by receptors (or ion channels) with subsequent gene regulation, thus implementing both AM and FM in different types of biological pathways. We then compare the accuracy in the production of target proteins. We find that FM can be more accurate than AM only for a single receptor with fast signaling, whereas AM is more accurate in slow gene regulation and with signaling by multiple receptors. Finally, we propose possible reasons that cells use FM despite the potential decrease in accuracy. Cells are exposed to changing environmental conditions and need to respond to external stimuli with high accuracy, e.g. to utilize nutrients and to avoid lethal stresses , 2. To rSaccharomyces cerevisiae. Under oxidative stress the nuclear concentration of transcription factor Msn2 is proportional to the H2O2 concentration, suggesting an AM mechanism  and bursty (BM) modulation . In CM, et al. found biologically relevant parameter regimes of promoter switching in gene regulation in which an oscillating input can produce a more constant and hence less noisy protein output level than a constant input with noise T we can rewrite these equations asM and the constants w and z are defined in M is time-independent. As shown in The following method applies to both the incoherent feedforward and the integral feedback loop. To solve the ordinary differential Eqs –12 we liS1 Text(PDF)Click here for additional data file.S1 Figf = k+c0/(1+k+c0/k−). (Insets) Magnification of small-noise approximation region (fast switching). Analytical results for CM (blue) and numerical results for BM (red) and intermediate modulation IM (green) as function of the frequency of binding events . Note that this figure is similar to k− = 10 k+c0  (left column) and k− = 0.1 k+c0  (right column). Averages from CM, BM and IM are constrained to be equal, i.e. ζ (BM) α′ τb (IM). Variances of CM, BM and IM exhibit two different regimes for fast switching: for k+c0 < k− BM is the most accurate mechanism and CM the worst (inset in C) while for k+c0 > k− CM is generally the most accurate (except for ζ = 1) and IM the worst (inset in D). Third moments show that, for large noise, the probability distributions become asymmetric.Averages , variance , and skewness  as a function of the frequency of binding events, (EPS)Click here for additional data file.S2 Figk+c0 < k− with k+c = 0.1 k− . (Bottom) Regime k+c0 > k− with k+c = 10 k− . (Left) Slow switching with ζ = 400. (Right) Fast switching with ζ = 7. Receptor activity r and protein copy numbers n(t) for CM, BM and IM are shown in black, blue, red and green, respectively.(Top) Regime (EPS)Click here for additional data file.S3 Figk+c0 < k− with k+c = 0.1 k− . (Left) ODE model. (Right) Stochastic protein production during τb in CM and IM. (Top) Examples of time traces. (Bottom) Histograms of number of proteins produced after 100s with standard deviation in legend based on 1000 simulations. (B) Analogous to (A) but for regime k+c > k− with k+c = 10 k− . CM, BM and IM are shown in blue, red and green, respectively.(A) Regime (EPS)Click here for additional data file.S4 Figu (left), species y from Eq. (S42) (middle) and species x from (S41) (right) as a function of time. Analytic solutions Eqs. (S32), (S43) and (12) are shown for BM in red, while a (time averaged) time-trace from a stochastic simulation using the Euler method is shown in orange . (B) Corresponding variances as a function of time for k+c0 > k− . Analytic results are shown in blue for CM and in red for BM; average over time (1s) from numerical simulations are shown in light blue for CM and in orange for BM. (C) Corresponding variances as a function of time for k+c0 < k− . Colors same as in (B). Remaining parameters: k+c1 = 104s−2, kx = 10s−1 and ky = 50s−1.(A) Averages of signaling rate (EPS)Click here for additional data file.S5 Figu (left), species y from Eq. (S60) (middle) and species x from (S59) (right) as a function of time. Analytic solutions Eqs. (S32), (S66) and (S65) are shown for BM in red, while a (time averaged) time-trace from a stochastic simulation using the Euler method is shown in orange . (B) Corresponding variances as a function of time for k+c0 > k− . Analytic results are shown in blue for CM and in red for BM; numerical simulations are shown in light blue for CM and in orange for BM. (C) Corresponding variances as a function of time for k+c0 < k− . Colors same as in (B). Remaining parameters: k+c1 = 104s−2, kx = 10s−1 and ky = 50s−1.(A) Averages of signaling rate (EPS)Click here for additional data file.S6 FigN unsynchronized receptors or ion channels in CM mode. (B) N synchronized CM receptors lead to a hybrid mechanism with information encoded in the frequency of broad bursts of variable duration. (C) N unsynchronized BM receptors provide a dense series of bursts. For large N, bursts may start overlapping, leading to variable amplitudes. (D) FM emerges from N synchronized receptors in BM mode. (E) Relative variance for a system of 8 receptors with ρN synchronized and (1−ρ)N unsynchronized receptors, plotted for fast dynamics in the k+c < k− regime (CM in blue and BM in red). Letters refer to panel labels (A-D). Dotted red line indicates uncertainty from FM for comparison. (Inset) Same for a system of two receptors only.(A-D) Schematic of receptor activity in time. (A) AM emerges from (PDF)Click here for additional data file.


MBL2 gene. The aim of the study was to determine the influence of MBL2 polymorphisms in susceptibility to HTLV-1 infection. A total of 43 HTLV-1 infected subjects and 127 healthy controls were evaluated for polymorphisms in the coding region of MBL2 gene. The point mutations in exon 1 at codon 54  and codon 52  and the wild type allele A were detected by PCR-RFLP. The frequency of the allele A, B and D was 66%, 29% and 5% among HTLV-1 infected subjects and 79%, 18% and 3% among healthy controls, respectively. Genotype and allele frequencies were statistically different between both groups, being the allele B more frequent among HTLV-1 infected subjects than in controls . Moreover, the homozygous genotype BB was observed in 14% of HTLV-1 patients and only 3% of controls (p=0.016), and it was associated with an almost five-fold higher risk of HTLV-1 infection . Our results suggest that carriers of the MBL2 allele B are more susceptible to HTLV-1 infection. Further studies with a large number of individuals are ongoing to confirm the impact of MBL polymorphisms as genetic determinant of HTLV-1 susceptibility.Mannose-binding lectin (MBL) plays an important role in the innate immune defense against invading microorganisms. Deficiency of functional MBL is linked to polymorphisms in the


Epidemiological studies have shown that an elevated uric acid (UA) level predicts the development of metabolic syndrome and diabetes; however, there is no direct evidence of this, and the underlying mechanism remains unclear. Here, we showed that a high-UA diet triggered the expression of pro-inflammatory cytokines, activated the NF-κB pathway, and increased gliosis in the hypothalamus. Intracerebroventricular injection of UA induced hypothalamic inflammation and reactive gliosis, whereas these effects were markedly ameliorated by the inhibition of NF-κB. Moreover, magnetic resonance imaging confirmed that hyperuricemia in rodents and humans was associated with gliosis in the mediobasal hypothalamus. Importantly, the rats administered UA exhibited dyslipidemia and glucose intolerance, which were probably mediated by hypothalamic inflammation and hypothalamic neuroendocrine alterations. These results suggest that UA can cause hypothalamic inflammation via NF-κB signaling. Our findings provide a potential therapeutic strategy for UA-induced metabolic disorders. Hyperuricemia has emerged as a major health problem in industrialized nations. Epidemiological evidence has indicated that the prevalence of hyperuricemia is increasing worldwideGrowing evidence has suggested that immune-cell-mediated tissue inflammation acts as a key mechanism of metabolic disorders in metabolically active organs13Crp, a marker of inflammation, in human vascular smooth muscle cells and endothelial cells17UA has been shown to have pro-inflammatory activity. It induces NADP-oxidase activation by activating redox-dependent pro-inflammatory signaling in cultured adipocytesNF-κB is a pro-inflammatory master switch that controls the production of inflammatory markers and mediators. NF-κB activation induced by UA has been reported in several diseases. For example, UA induces renal inflammation in gouty nephropathy via the activation of tubular NF-κB signaling12In the present study, we found that serum UA can pass through the blood-brain barrier (BBB) and act as a potent inflammatory stimulus, leading to NF-κB activation as well as the accumulation of gliosis in the hypothalamus, both in animal models and in humans.To investigate the effect of UA on the hypothalamus, we applied a previously reported strategy to generate a hyperuricemia model. Rats were fed a high-UA diet (HUAD) containing 2% oxonic acid (OA) and 2% UA for 3 months23Il1b, Tnfa, and Ikbkb mRNA within the first 7 days of HUAD administration, followed by a decline to baseline or to a slightly elevated level from days 14 to 28  and in hypothalamic tissue using liquid chromatography tandem mass spectrometry. We found that the average UA concentration in the CSF of HUAD-fed rats was 644.25 ng/ml, which was 1.34-fold higher than the concentrations of chow-fed control rats . The amoTo determine the direct effect of UA in inducing pro-inflammatory cytokines in the hypothalamus, rats were intracerebroventricularly (icv) cannulated and perfused continuously with UA. It has been reported that the average volume of ventricular CSF  in the rat is 7–34 μl, the total cranial CSF volume is 200–440 μl, and the CSF flow rate within the ventricular system is 2.5–3.7 μl /min28Gfap in the hypothalamus was assessed. We found that hypothalamic Gfap mRNA levels increased by 47% after UA icv perfusion for 3 days and were elevated by 96% at day 7. At day 14, a slight elevation of 59% was observed ; however, laterality (right vs. left) had no effect (P = 0.13). Using diffusion tensor imaging (DTI), values were compared for fractional anisotropy and tensor trace assessments. The tensor trace showed a pattern similar to the T2 relaxation times, confirming that inflammation occurred (P = 0.41). Nevertheless, there was no significant difference in fractional anisotropy (Regions of interest (ROIs) and example images from the 2-dimensional sequence, T2 parametric map, and DTI-EPI sequence are shown . There woccurred ; howeverisotropy . These rWe then investigated whether MBH gliosis could also occur in hyperuricemia subjects. We analyzed the data obtained from 15 hyperuricemia subjects with MRI examinations and from 16 normal subjects. The concentration of serum UA in these hyperuricemia subjects ranged from 424 to 659 μM for men and from 397 to 518 μM for women, whereas the average concentration was 293.1 μM in normal subjects. We performed an initial inspection of the MBH for hyperintense signal in T2 coronal sections, as performed in the rodent study described above. Hyperintense signal in the MBH is a characteristic finding of gliosis in numerous inflammatory, ischemic, and degenerative neural disorders in clinicalThe hypothalamus is the nerve center responsible for metabolism. To track UA in relation to lipid and glucose metabolism, we measured several biochemical parameters in serum collected from the HUAD-fed rats  and UA i7Moreover, glucose intolerance also occurred in the mice receiving icv UA perfusion, raising the possibility of a common phenomenon of UA-induced glucose metabolic disorders across species . ImportaAgrp, Pomc, Crh, Trh, Ghrh, and Gnrh were elevated in the hypothalamus within the first 7 days of HUAD administration, followed by a decline to almost 50% of the control levels from days 14 to 28  and male C57BL/6J mice (7–8 weeks old) were purchased from Beijing Animal Center . All of the animals were housed individually in a specific pathogen-free environment, maintained in a temperature-controlled room with a 12-hour-light/12-hour-dark cycle, and provided with To establish the hyperuricemia model, the rats were provided with a HUAD containing 2% OA and 2% UA for periods ranging from 1 day to 3 months2350Wistar rats and C57BL/6J mice were placed in a stereotaxic apparatus  according to a previously described methodThe coordinates (relative to bregma) for icv cannula implantation in mice were 1.0 mm lateral, -0.42 mm anteroposterior, and 2.2 mm deepWe performed a retrospective cohort study to search for radiological evidence of MBH gliosis and to correlate our findings with hyperuricemia. Magnetic resonance brain examinations that used pituitary or epilepsy imaging protocols with coronal T2 FES FLAIR sequences and that were performed between 3/1/2014 and 6/20/2014 at West China Hospital of Sichuan University were reviewed for 2 inclusion criteria: the availability of high quality coronal views of the hypothalamus and the absence of clinical abnormalities that might confound interpretation . For the 51 subjects that met our inclusion criteria, we reviewed electronic medical records to determine serum UA concentration, gender, age, body weight, and final diagnosis. Exclusion criteria were as follows: absence of serum biochemical analysis for UA; age of less than 16 or more than 70 years; diagnosis of pituitary, hypothalamic, or neurodegenerative disorder ; metabolic disease ; serum biochemical parameter abnormalities without definite metabolic disease ; cerebral atrophy; and history of bariatric disease. Twenty subjects were excluded , yielding a total of 31 study participants. All subjects were classified into 2 groups: the hyperuricemia group  and the normal group. The basal characteristics of the study population are provided in P values less than 0.05 were considered statistically significant.Data are presented as the mean ± SEM. Differences between two means were assessed by unpaired, two-tailed Student’s t test. Data involving more than two means were evaluated by one-way ANOVA followed by Tukey’s post-hoc tests . How to cite this article: Lu, W. et al. Uric Acid Produces an Inflammatory Response through Activation of NF-κB in the Hypothalamus: Implications for the Pathogenesis of Metabolic Disorders. Sci. Rep.5, 12144; doi: 10.1038/srep12144 (2015).


The classic neurotrophins Nerve Growth Factor (NGF), Brain Derived Neurotrophic Factor (BDNF) and Neurotrophins NT-3 and NT-4 are well known to regulate various aspects of neuronal differentiation, survival and growth. They do this by binding to their cognate receptors, members of the Tropomyosin-related kinase (Trk) receptor tyrosine kinase family, namely TrkA, TrkB, and TrkC. These receptors are then internalized and localized to different cellular compartments, where signal transduction occurs. Conversely, members of the suppressor of cytokine signaling (SOCS) family are best known as negative regulators of signaling via the JAK/STAT pathway. Some members of the family, and in particular SOCS2, have roles in the nervous system that at least partially overlap with that of neurotrophins, namely neuronal differentiation and neurite outgrowth. Recent evidence suggests that SOCS2 is a novel regulator of NGF signaling, altering TrkA cellular localization and downstream signaling to affect neurite growth but not neuronal survival. This review first discusses regulation of Trk receptor signaling, followed by the role of SOCS2 in the nervous system and finishes with a discussion of possible mechanisms by which SOCS2 may regulate TrkA function. A great deal of interest has focused on the therapeutic potential of neurotrophins to prevent neurodegeneration and promote neural regeneration in a range of neurological diseases and neurotrauma   signaling cascades to effect changes in transcription. The neurotrophins are also known to activate members of the Rho GTPase family such as Cdc42 and Rac that are regulators of the cytoskeletal rearrangements required for cell motility and the formation of growth cones  and phospholipase C- γ1 (PLC-γ1) Figure  Obermei. These sA less established pathway downstream of the Trk receptors is the activation of the signal transducer and activator of transcription-3 (STAT3) transcription factor. Early studies demonstrated that dominant negative STAT3 can promote PC12 neurite outgrowth in the absence of NGF  has been shown to have a transient association with the Trk receptors upon neurotrophin binding  Chao, . The juxThe retrograde transport of neurotrophic stimuli has been most extensively studied in the developing peripheral nervous system and is critical for the formation and refinement of neuronal circuits during development and for the regulation of synaptic plasticity and neuronal survival in the mature nervous system  family have recently emerged as ubiquitin ligases regulating expression of a variety of receptors and their signaling pathways. One member of this family in particular, SOCS2, has a variety of functions in the nervous system, many of which overlap with the biological effects of TrkA signaling, suggesting a possible regulatory role for SOCS2 in TrkA function.The SOCS proteins are a family of intracellular proteins implicated in the negative regulation of a variety of cytokine, growth factor and hormone signals, particularly those mediated by the Janus kinase/signal transducer and activator of transcription (JAK/STAT) signaling pathway   comprise a variable N-terminal region, a central Src Homology 2 (SH2) domain and a highly conserved C-terminal SOCS box Figure . SH2 domThe SOCS proteins couple specific phosphotyrosine-motif recognition with ubiquitin-ligase activity and are thus potent inhibitors of a variety of cytokine signaling pathways. As previously mentioned, the SOCS proteins can participate in “negative feedback” inhibition or “cross-talk” suppression of independent cytokine stimuli. This cross-talk has been shown to extend to the cross regulation amongst the SOCS proteins. SOCS2, SOCS6, and SOCS7 appear to form a subgroup of the SOCS family that have been shown to interact with the SOCS box of all members of the SOCS family and may be required to regulate the stability of other SOCS proteins   receptor was used as bait to screen a cDNA library of proteins expressed in the human fetal brain  signaling  in vitro  neuroepithelial cells and E17 cortical neurons but not cortical astrocytes or adult multipotent stem cells , SOCS2TG (hypo-responsive) and GH receptor null (GHRKO) (non-responsive) mice, exhibit a variety of alterations in neural architecture  contain a large population of TrkA+ neurons that require NGF for survival and promotion of neurite outgrowth. Analysis of DRG neuron cultures derived from SOCS2TG and SOCS2KO mice showed that increased or no expression SOCS2 respectively, regulated NGF-mediated neurite outgrowth, with SOCS2 overexpression increasing and lack of SOCS2 decreasing neurite length and complexity (Uren et al., Co-immunoprecipitation of SOCS2 and TrkA mutant proteins in 293T cells showed that the juxtamembrane region of TrkA was required for binding of SOCS2, with possible involvement of the kinase domain as well. Overexpression of SOCS2 increased total TrkA levels in transfected 293T cells and PC12 cells and increased cell surface expression of TrkA in PC12 cells and SOCS2TG DRG neurons (Uren et al., While the mechanism by which SOCS2 regulates TrkA signaling and functional outcome remains to be determined, it most likely revolves around altered TrkA localization induced by SOCS2-mediated ubiquitination. This could regulate the amount of TrkA receptor at the cell surface and influence the rate at which it is internalized, recycled, or transported to different intracellular compartments, thereby modulating sensitivity of a cell to NGF and altering downstream signal transduction pathways. Ubiquitination of the lysine in the TrkA juxtamembrane KFG motif regulates TrkA expression levels and localization, with deletion of this domain leading to decreased ubiquitination, increased TrkA levels and increased phosphorylation of the AKT and MAP kinase pathways (Kiris et al., Neurotrophins and their receptors are expressed in specific areas throughout the nervous system. Their expression levels, signal transduction pathways and localization are tightly regulated by a range of different mechanisms, including dephosphorylation and degradation. SOCS proteins are best known for their role as negative feedback inhibitors of cytokine signaling but have been shown to have a range of other roles, depending on cell type and receptor under study. Their structure, which includes an SH2 interaction domain and the SOCS box ubiquitin ligase interaction domain, suggests that they may interact with many more proteins. This certainly appears to be the case for the TrkA receptor and SOCS2. However, given that SOCS2 has also been shown to bind to the BDNF and NT-3 neurotrophin receptors (Uren et al., The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Phosphorylation waves drive the propagation of signals generated in response to hormones and growth factors in target cells. cAMP is an ancient second messenger implicated in key biological functions. In mammals, most of the effects elicited by cAMP are mediated by protein kinase A (PKA). Activation of the kinase by cAMP results in the phosphorylation of a variety of cellular substrates, leading to differentiation, proliferation, survival, metabolism. The identification of scaffold proteins, namely A-Kinase Anchor proteins (AKAPs), that localize PKA in specific cellular districts, provided critical cues for our understanding of the role played by cAMP in cell biology. Multivalent complexes are assembled by AKAPs and include signaling enzymes, mRNAs, adapter molecules, receptors and ion channels. A novel development derived from the molecular analysis of these complexes nucleated by AKAPs is represented by the presence of components of the ubiquitin-proteasome system (UPS). More to it, the AKAP complex can be regulated by the UPS, eliciting relevant effects on downstream cAMP signals. This represents a novel, yet previously unpredicted interface between compartmentalized signaling and the UPS. We anticipate that impairment of these regulatory mechanisms could promote cell dysfunction and disease. Here, we will focus on the reciprocal regulation between cAMP signaling and UPS, and its relevance to human degenerative and proliferative disorders. Since the discovery of cyclic adenosine 3′,5′-monophosphate (cAMP) in the late 1950s, significant advances have been made to better understand the link between the cAMP and the regulation of downstream signaling and cellular homeostasis. The principal elements of the cAMP cascade have been intensively studied, both at functional and structural side, delineating a complex and finely regulated network of signaling scaffolds and regulatory proteins . cAMP le2+, supporting and integrating distinct signal transduction pathways (G protein–coupled receptors (GPCRs) constitute a large family of membrane proteins that transduce signals from the extracellular microenvironment to inside cell . The binpathways .The cAMP-PDEs are enzymes that hydrolize the 3′,5′ phosphodiester bond in the second messenger cAMP, producing 5′-AMP . By reduIn eukaryotes, most of the effects elicited by cAMP depend on the activation of PKA. This kinase consists of a tetramer composed of two regulatory (R) and two catalytic (C) subunits. The binding of cAMP to R subunit dissociates the PKA holoenzyme and releases the active C subunit, which in turn phosphorylates a wide array of cellular substrates, controlling different aspects of cell physiology . The bioin vitro and in vivo . AKAPs belong to a group of structurally different proteins that share the common feature to target the PKA holoenzyme in close proximity of its substrate . Each AK in vivo . By modu in vivo . AKAPs f in vivo .ε-amine of lysine residues of target proteins. This process requires a series of ATP-dependent enzymatic steps catalyzed by E1 (ubiquitin activating), E2 (ubiquitin conjugating), and E3 (ubiquitin ligating) enzymes (The ubiquitin-proteasome system (UPS) is emerging as an important control mechanism of cell growth, survival and metabolism. Degradation of a protein via UPS involves modification of the substrate protein by the covalent attachment of multiple ubiquitin molecules. The ubiquitin-tagged protein is eventually degraded through the proteasome . Defects enzymes . The res enzymes . Poly-ub enzymes . By modu enzymes . This me enzymes . In this enzymes .The cAMP-PKA signaling is regulated by- and can regulate the UPS at different steps, giving rise to a complex interactive and regulatory network that controls different aspects of cell fate Figure .At cell membrane, the ubiquitylation and consequent proteolysis of receptors by the UPS contributes to post-stimulus receptor desensitization . As examIt emerged that PDEs can be regulated by the UPS. Ubiquitin conjugation and proteasomal degradation of PDE4D by a cullin 1-containing E(3) ubiquitin ligase complex is induced through concomitant phosphorylation of PDE4D by casein kinase 1 (CK1) and glycogen synthase kinase 3β (GSK3β). A phospho-degron motif within the PDE4D was identified as responsible of ubiquitin-mediated proteolysis of the enzyme. Interestingly, protein PPs calcineurin (CaN) counteracts the effects of the SCF complex on PDE4D stability , unveiliThe UPS can also regulate the PKA stability and signaling. praja2 is a widely expressed mammalian RING-H2 protein with intrinsic E3 ligase activity . praja2 Caenorhabditis elegans, inhibiting Siah2 reduces life span, highlighting a role of the UPS-AKAP-PKA axis in the control of essential aspects of nematode aging , inhibits the elongation phase of translation by phosphorylating eukaryotic elongation factor-2  that cells adopt to regulate gene transcription . Histoneiability . Loss ofiability . A link iability .In neurons, cAMP-PKA signaling controls a variety of biological cues, as neurite outgrowth, morphogenesis and synaptic transmission and plasticity . Most ofDephosphorylation of cellular substrates is mediated by distinct families of protein PPs, . Among PcAMP signaling is involved in a variety of different biological responses . The comIn neurons, cAMP balance is crucial for physiological events underlying learning, memory and loco-motor activity . Severalin vivo  connecting the UPS to cAMP-PKA signaling, but also to construct a network that is able to predict and quantify the biological outcome  of human genetic mutations affecting key elements of these transduction pathways. Understanding the complexity of such regulatory mechanisms and exploring further the biological significance of this kinase-ligase network will help to design novel tools and drugs that selectively restore a perturbed cAMP cascade in various human phenotypes.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Human multipotent mesenchymal stromal cells (MSC) display immunoregulatory functions that can modulate innate and adaptive cellular immune responses. The suppressive and immunomodulatory activities of MSC occur by the action of soluble factors that are constitutively produced and released by these cells or alternatively, after induction of MSC stimuli in inflammatory microenvironments. During the infection by the human T cell lymphotropic virus-type 1 (HTLV-1) around 1-5% of the infected individuals will develop a chronic inflammatory disease known as HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP). However, up to date it is unknown the contribution of the MSC in the inflammatory microenvironment resulting from this chronic disease. In this study, we evaluated the MSC immunosuppressive effect on HTLV-1 infected T lymphocytes. Assays using co-culture of MSC and HTLV-1+ T lymphocyte lineages resulted in a decrease of both tax gene expression and HTLV-1 p19 antigen. The reduction of the tax gene expression and the HTLV-1 p19 were at the same time associated with 1.7 fold increase of IL-6 secretion and a higher PGE2, IDO and VCAM-1 gene expression (p≤0.05). To evaluate if MSC immunoregulation can influence the proliferation of HTLV-1 infected T lymphocytes, we compared the proliferation of lymphocytes obtained from HTLV-1+ and healthy individuals co-cultured in the presence of MSC. It was observed that the lymphoproliferative inhibition by MSC on infected lymphocytes was similar compared to the cells obtained from healthy individuals. Additionally, this suppressive effect was related to a significant increase of IDO and PGE2 gene expression (p≤0.05). Furthermore, the HTLV-1 pol gene and p19 protein were less expressed after co-culturing with MSC, suggesting that the MSC immunoregulation is effective on HTLV-1 infected T cells. In conclusion, this study suggests that MSC could be involved in the immunomodulation of the HTLV-1 infected T lymphocytes. Financial Support: FAPESP, CNPq and FUNDHERP. Keywords: MSC, HTLV-1, immunoregulation, gene expression.


Rho GTPases are crucial signaling molecules that regulate a plethora of biological functions. Traditional biochemical, cell biological, and genetic approaches have founded the basis of Rho GTPase biology. The development of biosensors then allowed measuring Rho GTPase activity with unprecedented spatio-temporal resolution. This revealed that Rho GTPase activity fluctuates on time and length scales of tens of seconds and micrometers, respectively. In this review, we describe Rho GTPase activity patterns observed in different cell systems. We then discuss the growing body of evidence that upstream regulators such as guanine nucleotide exchange factors and GTPase-activating proteins shape these patterns by precisely controlling the spatio-temporal flux of Rho GTPase activity. Finally, we comment on additional mechanisms that might feed into the regulation of these signaling patterns and on novel technologies required to dissect this spatio-temporal complexity. The combination of experimental approaches, including genetics in model organisms, cell biology, and biochemistry, was key to establish the basics of Rho GTPase signaling. These techniques revealed the principles of GTPase regulation by guanine nucleotide exchange factors (GEFs), GTPase-activating proteins (GAPs), and Rho guanine nucleotide dissociation inhibitors (RhoGDIs), and identified effectors that exert specific biological functions downstream of Rho GTPases. This uncovered a surprisingly intertwined network of mutual regulatory protein complexes in which Rho GTPases are vastly outnumbered by GEFs, GAPs, and effectors10.Since the seminal articles from Allan Hall’s lab back in the early 1990s11. Visualizing Rho GTPase activity drastically changed our perception of Rho GTPase signaling and implies a higher degree of complexity than the classic ON-OFF schemes typically depicted in feed-forward, linear signaling networks. This fresh view emphasizes the importance of analyzing Rho GTPase activity dynamics by microscopy instead of analyzing steady states of limited information content by biochemistry.In the last 15 years, an additional layer of complexity was superimposed on Rho GTPase biology. Fluorescence resonance energy transfer (FRET)-based and other biosensors enabled investigators to capture the spatio-temporal dimensions of Rho GTPase signaling in living cells with unprecedented resolutionUnderstanding that Rho GTPase signaling is organized in spatio-temporal patterns poses important novel questions: How are these signaling activity patterns generated? What forms their structural basis? And which technologies do we need to dissect the mechanisms of pattern formation in the future? In this review, we will survey the spatio-temporal activity patterns that have been documented to date and highlight possible answers to these intriguing questions. Then we discuss important players that might feed into this spatio-temporal regulation, and we comment on novel technologies to analyze the latter.12. However, the use of FRET biosensors proved this view to be too simplistic. Accordingly, Rac1, Cdc42, RhoA, and RhoC activity has been found at the leading edge in randomly migrating fibroblasts , which broadly inhibits RhoA at the leading edge and simultaneously restricts RhoA activity to a sharp zone at the lamellipodium tip (27 (The RhoA activity pattern considerably changes if fibroblasts are stimulated with platelet-derived growth factor (PDGF). On timescales of 10 to 20 minutes, PDGF stimulation leads to increased edge protrusion that correlates with immediate decrease of RhoA activityium tip . Here, tip , showing that a signaling treadmill generates a GTPase activity flux by proximal RhoA activation and distal RhoA inactivation within the zone -mediated DLC1 knockdown leads to widening of the RhoA activity domain, suggesting that DLC1 acts by shaping the focused RhoA activity zone at filopodial tips. Together, these data clearly suggest that fine spatial regulation of Rho GTPase activation/deactivation cycles enables the formation of a signaling pattern.Similar mechanisms have been documented in mammalian cells. In the case of invadopodia formation, RhoC activity is spatially restricted in a concentric zone surrounding the invadopodium core through the interplay of p190RhoGEF and p190RhoGAPlopodia . A recen2 s−1)39 from a zone preferentially occupied by a GEF to a zone preferentially occupied by a GAP, (3) local inactivation by the GAP, and (4) membrane extraction by RhoGDI. Once in the cytoplasm, the Rho GTPase-RhoGDI complex can quickly diffuse (10 to 100 µm2 s−1)40 and reach an equilibrium within the cytosol before being used for subsequent activation cycles  local activation by a GEF, (2) slow plasma membrane (PM) diffusion , a DOCK homology region 1 (DHR-1), or a Bin-Amphiphysin-Rvs (BAR) domain43. Many GAPs also contain a variety of lipid-binding domains44. Since PH and DHR-1 domains vary in their binding specificity and affinity for phospholipids such as phosphatidylinositol 4,5-bisphosphate (PIP2) and phosphatidylinositol (3-5)-triphosphate46, GEFs and GAPs might be directed to specific PM subdomains  play a paramount role in Rho GTPase activationylation . For ins71. Shank positions β-Pix in the postsynaptic density region to locally control Rac1-dependent dendritic spine formation73, whereas Scribble recruits β-Pix to the PM to regulate thyrotropin receptor trafficking75. Interestingly, the Scribble/β-Pix interaction is modulated by TIP-1, which competes for β-Pix binding and affects its subcellular localization76.A striking feature in approximately 40% of GEFs is the presence of a PSD95-Dlg1-ZO1 (PDZ) domain-binding motif at the C-terminus. These GEFs interact with various scaffold proteins such as Shank and Scribble77. At adhesions, focal adhesion kinase (FAK) phosphorylates β-Pix to strengthen the β-Pix-Rac1 interaction and thus enhance Rac1 recruitment to adhesions79. Notably, the balance of β-Pix distribution between FAs and endosomes is regulated by the PDZ domain-containing sorting nexin 2780. Another GEF that activates Rac at FAs is DOCK180. Its polarized localization to the leading edge is mediated through interaction with the Paxillin-p130Cas-CrkII complex following integrin engagement83. Furthermore, the Rac GEF Tiam1 was found to bind to talin in FAs and to activate Rac1 in a PAR complex-dependent manner84. There are also several Rho-specific GEFs enriched at FAs in a FAK-dependent manner. Net1 is present in FA complexes at the leading and the trailing edge85, whereas PDZ-RhoGEF localizes to the trailing edge only87. LARG and p115RhoGEF also interact with FAK at adhesions88. These four Rho-specific GEFs seem to regulate similar processes, although it is not fully understood yet whether their functions are redundant or whether they depend on different upstream signals.Adhesion complexes are further important hubs for spatial regulation of GEFs and GAPs . β-Pix i90. Thus, MII orchestrates adhesion formation and maturation by adsorption and release of β-Pix. Notably, MII sequesters and inhibits GEFs containing a Dbl homology domain such as FGD1, Kalirin, LARG, DOCK180, Tiam1, Trio, GEF-H1, and Dbl89. F-actin also traps GAPs as FilGAP, which binds to the F-actin cross-linker filamin A. After mechanical deformation of F-actin branches, FilGAP is released and translocates to the PM to inhibit Rac at the leading edge91. Finally, microtubules tie and inactivate GEF-H1, which is released after the depolymerization of microtubules to stimulate RhoA activity and contractility at the leading edge93.The cytoskeleton also tethers GEFs and GAPs. Active myosin II (MII), which generates actomyosin-based contractility, sequesters and inactivates β-Pix at actin fibers and thus confers an MII- and β-Pix-dependent front-back polarity in migrating cellsIn summary, growth factors, mechanosensation, membrane topology/composition, and the actomyosin and tubulin cytoskeletons regulate the spatio-temporal aspects of Rho GTPase activity patterns, which in turn feedback on these different organizational and signaling levels. It is now time to explore how this plethora of different GEF/GAP regulatory mechanisms impact on spatio-temporal Rho GTPase activation.98. Dissection of the complexity of spatio-temporal Rho GTPase signaling patterns will also require obtaining biophysical parameters with subcellular resolution, which can for example be inferred from fluorescence correlation spectroscopy99. Ultimately, such multidisciplinary approaches will inform mathematical models that can describe the network properties required to generate robust spatial signaling patterns100. In addition toin vitro experiments, analyzing Rho GTPase activity by FRET reportersin vivo101 will guarantee new insights, provided that RhoGDI-responsive sensors are used. Beyond the goal of understanding how Rho GTPase signaling is spatio-temporally regulated, these approaches will also unveil how Rho GTPase coordinately regulate different cytoskeletal polymers to fine-tune the highly complex and dynamic processes required for cell morphogenesis. We foresee that a limited number of conserved spatio-temporal Rho GTPase networks will emerge from systematic perturbation approaches. Tuning of a limited number of parameters might then allow the cell to repurpose such networks to regulate edge protrusion, growth cone motility, macropinocytosis, sealing of a cell wound, or other morphodynamic processes.The technological progress in the last 15 years has empowered us with the ability to monitor Rho GTPase signaling with high spatio-temporal resolution. With respect to initial models, this has revealed an unexpected spatio-temporal signaling complexity, which now needs to be systematically analyzed by perturbation of the different players we have discussed in this review. Because spatio-temporal Rho GTPase signaling patterns are constantly regulated on timescales of tens of seconds, novel technologies are required to perturb cell systems at that exact timescale. This can take advantage of existing techniques such as optogenetics or small-molecule dimerizers to control GEF/GAP targeting and activity2, phosphatidylinositol 4,5-bisphosphate; PLS, podosome-like structure; PM, plasma membrane; RhoGDI, Rho guanine nucleotide dissociation inhibitor; RTK, receptor tyrosine kinase.BAR, Bin-Amphiphysin-Rvs; DHR-1, DOCK homology region 1; FA, focal adhesion; F-actin, filamentous actin; FAK, focal adhesion kinase; FRET, fluorescence resonance energy transfer; GAP, GTPase-activating protein; GEF, guanosine triphosphate exchange factor; GTP, guanosine triphosphate; PAK, p21-activated kinase; PDGF, platelet-derived growth factor; PDZ, PSD95-Dlg1-ZO1; PH, pleckstrin homology; PIP


Although recombination is a major source of genetic variability in retroviruses, no recombinant strain had been observed for HTLV-1, the first isolated human-pathogenic retrovirus. Different genotypes exist for HTLV-1: Genotypes b and d to g are restricted to central Africa, while genotype c is only endemic in Australo-Melanesia. In contrast, the cosmopolitan genotype A is widely distributed. We applied a combination of phylogenetics and recombination analysis approaches to a set of new HTLV-1 sequences, including the complete LTR and a 522-bp fragment of the env gene, which we collected from 19 countries throughout North, West and Central Africa, the continent where the virus has the largest endemic presence. The samples were obtained from 41 HTLV-1 infected individuals with different clinical statuses including ATL, TSP/HAM and asymptomatic carriers. The recombinant search and breakpoint detection were performed by boot scanning in Simplot to compare inferred clusters of sequences to each other. Finally, molecular clock analyses were performed to date the recombinant event observed. This led us to demonstrate the presence of recombinants in HTLV-1. Indeed, the HTLV-1 strains currently present in North Africa have originated from a recombinant event between strains from Senegal and West Africa. This recombination is estimated to have occurred around 4,0 years ago. This recombination seems to have been generated during reverse transcription. In conclusion, we demonstrate that, albeit rare, recombination can occur in HTLV-1 and may play a role in the evolution of this retrovirus . In order to precise the geographical distribution of this recombinant within the African Continent, we studied a new set of samples from HTLV-1 infected patients of diverse African origin. The data on this on-going study will be presented.


Osteoclasts, multinucleated giant cells derived from the monocyte/macrophage lineage, are the exclusive specialized bone-resorbing cells that are indispensable for skeletal development, bone remodeling and repair and mineral homeostasis. Increased osteoclastogenesis leads to excessive bone resorption in osteoporosis and in inflammatory diseases with bone destruction, such as rheumatoid arthritis (RA), psoriatic arthritis (PsA), juvenile chronic arthritis (JCA) and periodontitis . As boneκ, site  functions as a central transcription factor that receives inputs from several signaling pathways, such as the canonical Notch pathway, Wnt-β-catenin, NF-κB pathway, TLR and TNF signaling pathways. We recently identified RBP-J as a key negative regulator that imposes the requirement for ITAM-mediated costimulation of osteoclastogenesis, homeostatic bone remodeling and inflammatory bone resorption, and discovered the signaling pathways and mechanisms by which RBP-J regulates ITAM-mediated costimulation [The immunoreceptor tyrosine-based activation motif (ITAM)-mediated signaling pathways play important roles in various cellular activities, including immune response and cancer activation. The main ITAM-containing adaptors expressed by myeloid osteoclast precursors are DNAX-activating protein 12 (DAP12) and Fc receptor common γ subunit (FcRγ). These adaptors associate with and mediate signaling by various receptors, including DAP12-associated triggering receptor expressed in myeloid cells 2 (TREM2) and signal-regulatory protein β 1 (SIRPβ1), FcRγ-associated osteoclast-associated receptor (OSCAR), paired immunoglobulin-like receptor-A (PIR-A) and FcRs. Osteoclasts require ITAM-mediated costimulation of RANK signaling for their appropriate differentiation during bone homeostasis . Osteoclmulation , 5.Dap12 KO or Dap12/Fcrg double knockout (DKO) mice by bypassing the requirement for costimulation of osteoclastogenesis during bone homeostasis. In inflammatory settings, RBP-J deficiency enabled TNF-α to induce osteoclast formation and bone resorption in Dap12 KO mice. These data show that RBP-J deficiency allows osteoclast differentiation to occur independently of ITAM-mediated costimulation during homeostatic bone remodeling and inflammatory bone destruction. Thus, RBP-J imposes the requirement for ITAM-mediated costimulation. During osteoclastogenesis, ITAM-mediated PLCγ2-calcium signaling is constitutively active at basal level, which provides essential costimulatory signals for RANK signaling to induce osteoclast differentiation. We show that RBP-J restrains ITAM signaling and limits crosstalk between RANK and ITAM associated receptors by suppressing PLCγ2 expression and keeping PLCγ2-calcium signaling in a repressed basal state. This is the first study showing the negative regulation of ITAM signaling by RBP-J. The regulatory mechanisms of PLCγ2 expression were unclear. Our results show that the mechanism of PLCγ2 regulation is transcriptional in osteoclast differentiation. Furthermore, built upon RNAseq experiments, we identified a novel regulatory loop whereby RBP-J restrains the expression of TGFβR1 and thereby modulates cell responsiveness to TGF-β, which in turn regulates transcription of PLCγ2 [Loss of DAP12 or both DAP12 and FcRγ in mice leads to severe osteopetrosis due to impaired osteoclastogenesis and resorptive function. To investigate the role of RBP-J in ITAM-mediated bone remodeling, we generated DAP12 and RBP-J double knockout (KO) mice (R-DKO) and DAP12, FcRγ and RBP-J triple KO mice (TKO) . Strikinof PLCγ2 .Our findings provide evidence for a cross talk between RBP-J and ITAM-mediated signaling pathways in osteoclast precursors and suggest a conceptually new model where ITAMs positively while RBP-J negatively regulates PLCγ2-calcium signaling. The balance between these opposing pathways fine tunes osteoclastogenesis and determines the strength of activating signals required for osteoclastogenesis in physiological and various pathological settings. For example, we observed that RBPJ expression level was significantly suppressed in synovial fluid macrophages derived from RA patients, indicating that RBP-J expression can be altered in response to environmental cues in pathological states . As RBPJ


Human T-lymphotropic virus type 1 (HTLV-1), a retrovirus, is the causative agent of HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP) and adult T-cell leukaemia/lymphoma (ATLL). The reported association with pulmonary disease such as bronchiectasis is less certain.A retrospective case review of a HTLV-1 seropositive cohort attending a national referral centre. The cohort was categorised into HTLV-1 symptomatic patients (SPs) ) and HTLV-1 asymptomatic carriers (ACs). The cohort was reviewed for diagnosis of bronchiectasis.34/246 ACs and 30/167 SPs had been investigated for respiratory symptoms by computer tomography (CT) with productive cough +/- recurrent chest infections the predominant indications. Bronchiectasis was diagnosed in one AC (1/246) and 13 SPs   with high resolution CT. In the multivariate analysis ethnicity (p = 0.02) and disease state (p < 0.001) were independent predictors for bronchiectasis. The relative risk of bronchiectasis in SPs was 19.2  and in HAM/TSP patients compared with all other categories 8.4 . Subjects not of African/Afro-Caribbean ethnicity had an increased prevalence of bronchiectasis .Bronchiectasis was common in the cohort (3.4 %). Risk factors were a prior diagnosis of HAM/TSP and ethnicity but not HTLV-1 viral load, age and gender. The spectrum of HTLV-associated disease should now include bronchiectasis and HTLV serology should be considered in patients with unexplained bronchiectasis. The spectrum of disease associated with HTLV-1 has not been completely defined. The association between HTLV-1 and pulmonary conditions is less well documented and clinically recognised. Bronchiectasis has been reported amongst Australian aborigines infected with HTLV-1. We have observed a high rate of bronchiectasis in a UK HTLV-1 infected cohort. We have shown that patients with non-pulmonary HTLV-1-associated disease, particularly inflammatory disease, are more likely to have an additional diagnosis of bronchiectasis than asymptomatic carriers and postulate that the same dysregulated hyper-inflammatory response seen in HTLV-associated myelopathy/tropical spastic paraparesis may play a role in this setting. Our findings highlight the importance of screening HTLV-1 infected patients, particularly those with inflammatory disease, for pulmonary disease.The human T-lymphotropic virus type-1 (HTLV-1), a retrovirus, has been estimated to infect at least 5-10 million people worldwide with the highest prevalence reported in Japan, Africa, The Caribbean and South America , with a Although less recognised associations between HTLV-1 and pulmonary conditions have been reported: patients with HTLV-1 have increased lymphocytes in bronchoalveolar lavage fluid (BAL) which correlates with HTLV-1 VL in both the BAL and peripheral blood mononuclear cells (PBMC) ; patientet al. [To date the most comprehensive report of pulmonary imaging in HTLV-1 infection is that of Okado et al.  who undeet al. . Two addet al. , 27. A cet al. . Furtheret al. . A causaet al. . It is tAlthough HTLV-1 associated diseases are rare in the UK the National Centre for Human Retrovirology manages a relatively large HTLV cohort, the only one of its kind in Europe. The cohort was initially established in 1993 to gain an understanding of the breadth and pathogenesis of HTLV disease through longitudinal observation. Despite the aforementioned reports HTLV-1 infected patients are not generally considered to be at greater risk of bronchiectasis than the general population and therefore chest imaging in our cohort has only been conducted if there was a clinical suspicion of lung disease.A single national centre retrospective case review of all 464 HTLV-1 seropositive patients attending the National Centre for Human Retrovirology, St Mary’s Hospital, London, UK between 1993 and 2012. Subjects were characterised in regard to HTLV-1 infection as either symptomatic patients (SP) or as ACs. SPs had an underlying disease with documented association with HTLV-1: ATLL; strongyloidiasis; HAM/TSP; or other HTLV associated inflammatory disease (HAID) namely polymyositis, uveitis and arthritis. Twenty-three patients were excluded as they were co-infected with human immunodeficiency virus type 1. A further 28 patients were excluded as they had neurological features that could not be classified as either HAM/TSP or polymyositis and it was difficult to establish whether HTLV-1 infection was causal or co-incidental and thus whether they had underlying HTLV-1-associated disease.The clinic database, case notes, laboratory and radiology reports were examined. In accordance with the guidance produced by the UK National Research Ethics Service, research ethics approval was not required for this study. Once the data were collated we used the clinical code, which is separate from the hospital number, to ensure confidentiality. Bronchiectasis was correlated with HTLV-1 symptoms and with concurrent HTLV-1 VL (this being the number of HTLV-1 DNA copies/100 PBMCs and reported as VL %). Data were collated on the number of patients that had CT i.e. CT chest or high resolution CT (HRCT), the clinical indication for the imaging and the radiological findings particularly the presence or absence of bronchiectasis. A multi-detector CT scanner was used. The CT criteria that were used for diagnosis of bronchiectasis were bronchial dilatation as evidence by bronchoarterial ratio greater than 1 to 1.5 (signet ring sign) and/or non-tapering airways . Indirect signs such as thickening of the bronchial wall, loss of lobe volume, mosaic perfusion pattern, a tree-in- bud pattern, and mucus plugs were also used.t-test, relative risk (RR), Fisher’s exact test and backwards multivariate logistic regression using commercial statistics software . A p-value of 0.05 or less was considered significant.Data was also collected on risk factors for bronchiectasis i.e. congenital conditions, tuberculosis, childhood infection and exposure to chemical irritant. We also collected data on the presence of other chronic conditions i.e. chronic liver disease, myocardial disease, renal and cardiac failure. Data were analysed by Student Four hundred and thirteen patients infected with HTLV-1 were included comprising 246 ACs and 167 SPs. Of the 167 SPs: 54 had ATLL; 95 HAM/TSP; 11 HAID and 7 strongyloidiasis , SPs were significantly more likely to be of African/Afro-Caribbean ethnicity  than the ACs   compared with ACs . HTLV-1 VL among non-HAM/TSP SPs was higher still . The median VLs in the different SP groups were: HAM/TSP 14 %, HAID 8.5 %, ATLL 29.3 % and Strongyloidiasis 20.6 %  in the AC group and 10/30 (33.3 %) in the SP group were high resolution CTs. (HRCTs). The reasons for CT imaging were productive cough +/- recurrent infection (50 % ACs vs. 50 % SPs), weight loss (26.5 % ACs vs. 23.3 % SPs), suspected malignancy (23.5 % ACs vs. 6.7 % SPs) or an abnormal chest X-ray (SPs 16.7 %). The indication for CT was unclear in 3.3 % SPs. The indications for HRCT was productive cough +/- recurrent infection. The time from HTLV-1 diagnosis to imaging was on average 2.5 years in ACs and 2 years in SPs  and ethnicity (p = 0.02) but not age, gender or HTLV-1 VL were independent predictors for bronchiectasis.Our data suggest that bronchiectasis is more common among HTLV-1 infected subjects in the UK than among the general population, that the inflammatory process of HAM/TSP and other HAID is implicated in pathogenesis and that Caucasians infected with HTLV-1 may be more susceptible to bronchiectasis than African/Afro-Caribbeans.Mass chest radiography during the 1950s indicated a prevalence of 100 per 100,000 adult UK population of bronchiectasis . The 3.4In the general population the prevalence of bronchiectasis increases with age. A retrospective cohort study in the US found the prevalence of bronchiectasis to be 4.2 per 100,000 population aged 18-34 years and 271.8 per 100,000 population aged >75 . HoweverAnother consideration is the ethnic bias of the cohort which is predominantly of African/Afro-Caribbean ethnicity. However not being African/Afro-Caribbean carried a greater than 3 fold increased risk for diagnosed bronchiectasis. Cohort data from the two Caucasian populations with highest HTLV-1 prevalence, Iran and Romania have not been reported. Whilst the lower risk of bronchiectasis observed in African/Afro-Caribbean subjects merits further investigation this may account for the paucity reports of association between HTLV-1 and bronchiectasis from outside of Japan. Genetics, timing of primary infection and environmental factors may all contribute to these different rates. Difference in bronchiectasis rates amongst different ethnicities have been reported from New Zealand and Fiji between Polynesian and Non-Polynesian and Melanesian and Indian children respectively , 33. WhiWe hypothesised that HTLV-1 VL would be associated with bronchiectasis and as previously reported for this cohort, and consistent with other studies, HTLV-1 VL was significantly higher in patients with symptomatic HTLV-1 infection than in the ACs . HoweverOur HTLV-1 cohort is comprised of patients from the three main categories of infection, namely asymptomatic carriage, ATLL and HAM/TSP and, as reflects the demographics of HTLV-1 infection in the UK, was predominantly of African/Afro-Caribbean ethnicity. The high number of patients that were Caucasian in the AC group is secondary to universal blood donor screening. The M: F ratio between ACs and SPs was similar. A major cause for bronchiectasis is early childhood infections. This can be difficult to exclude however there was no significant childhood infections reported by the patient or in their health records. Further to this even though we did not test for autoimmune conditions or alpha 1 antitrypsin deficiency our patients had no clinical symptoms or signs of these conditions.Strongyloides stercoralis, with a higher incidence HTLV-1 amongst S. stercoralis carriers [The pathogenesis of bronchiectasis associated with HTLV-1 has not been studied. The association here with a prior diagnosis of another inflammatory disease and the prominent lymphocytosis in BAL reported by others , 21 poincarriers , and a hcarriers .The major limitation of this study is is that CT imaging had only been performed if the patient had clinical or radiological features that raised the suspicion of underlying pulmonary disease with no lower threshold for imaging in this cohort compared to the general population. Thus only 15 % of the cohort had CT imaging (13.8 % ACs and 18.0 % SPs) and the prevalence of bronchiectasis, particularly early/mild or asymptomatic, may have been underestimated. Second, the number of cases of bronchiectasis is relatively few. Despite this the associations reported are statistically robust, suggesting that these are not chance findings. Third, we have no data on the effect of HTLV-1 or associate inflammatory cells in the lung and can only speculate regarding pathogenesis. The relative strengths are studying a population that is socially and ethnically distinct and the median seven years follow-up of each patient group. A prospective study is now justified to confirm and extend these findings on the frequency of bronchiectasis in patients with HTLV particularly amongst those with pre-existing HTLV-associated disease and to explore pathogenesis.In the UK bronchiectasis is more common amongst HTLV-1 infected subjects (3.4 %) than reported for the general population (0.1 %). Non-African/Afro-Caribbean patients with pre-existing HTLV-1-associated disease are most at risk. Our data support the view that the spectrum of HTLV-1-associated disease is broader than previously thought and that disseminated mild inflammation may be common, albeit asymptomatic. Our results emphasise the importance of screening symptomatic HTLV-1 patients for pulmonary disease  early following diagnosis of HTLV-1 and that CT imaging should be requested even in the presence of minimal symptoms. Furthermore HTLV serology should be considered in all patients with bronchiectasis in whom no other cause is apparent.


Although the detection of HTLV antibodies became mandatory in Argentinean blood banks since 2005, there is no recommendation on how to perform the screening. As a consequence, there is a variable number of HTLV false positives depending on the kit used. The aim of the present study was to evaluate the performance of six commercial screening tests available currently for the initial diagnosis of HTLV-1/2 infection in South America. The positive panel included 14 HTLV-1 and 13 HTLV-2 samples confirmed by WB (HTLV-1/2 MP Diagnostics). The negative panel included 233 samples analysed with Architect (n=47), Diapro (n=46), MP (n=45), Murex (n=47) and Serodia (n=48), collected in 5 different institutions in a period of one up to 10 consecutive days. All of them were confirmed by n-PCR. The sensitivity for all HTLV diagnostic kits was 100.0%. The most specific test was Serodia (PA) , followed by the ELISAs: Celquest , Murex , MP and Architect , and Diapro . According to this data, 2 to 16 blood units out of 233 should have been discarded due to false positive results in a period of 10 days. This data must be considered when choosing the assays, not only to obtain an optimal efficiency on HTLV-1/2 diagnosis, to increase the number of potential blood units and to decrease the circulation of anxious individuals without a final result, but also to lower the overall cost-benefits of the diagnosis in the health care system.


Adult T-cell leukemia/lymphoma (ATL) is a peripheral T-cell malignancy that is caused by human T-lymphotropic virus type 1 (HTLV-1) with poor prognosis. We have reported the feasibility and efficacy of allogeneic hematopoietic cell transplantation with reduced intensity conditioning (RIC-HCT) for elderly patients with ATL, and have also shown that in more than half of patients the HTLV-1 proviral load (HTLV-PL) decreased to an undetectable level after RIC-SCT, suggested presence of anti HTLV-1 effects. In this study, we try to compare the kinetics of HTLV-PL after RIC-SCT from HTLV-1 antibody negative sibling donor (SD) with that from unrelated donors (UD), all of whom were HTLV-1 antibody negative. Fourteen patients were included in the SD group, and fifteen patients were in UD group. The HTLV-PL decreased to an undetectable revel in 11 of 14 patients (78.6%) in the SD group and 13 of 15 (86.7%) in the UD group with in 120 days after RIC-SCT. In short term, the kinetics of the HTLV-PL after RIC-SCT from UD was almost similar to that from HTLV-1 negative SD. Looking at the long term follow-up results, the HTLV-PL remains to be an undetectable level in only 2 of 11 (18.2%) patients in the SD group (median follow up 111 months [101-122])), on the other hand, 7 of 13 (53.8%) patient in the UD group (24 month [20-36]). RIC-HCT from UD tends to have better anti-HTLV-1 effect, compared with that from SD, but not conclusive because of the short follow-up period.


Human T-cell leukemia virus type 1 (HTLV-1) was the first human retrovirus discovered. Studies on HTLV-1 have been instrumental for our understanding of the molecular pathology of virus-induced cancers. HTLV-1 is the etiological agent of an adult T-cell leukemia (ATL) and can lead to a variety of neurological pathologies, including HTLV-1-associated-myelopathy/tropical spastic paraparesis (HAM/TSP). The ability to treat the aggressive ATL subtypes remains inadequate. HTLV-1 replicates by (1) an infectious cycle involving virus budding and infection of new permissive target cells and (2) mitotic division of cells harboring an integrated provirus. Virus replication initiates host antiviral immunity and the checkpoint control of cell proliferation, but HTLV-1 has evolved elegant strategies to counteract these host defense mechanisms to allow for virus persistence. The study of the molecular biology of HTLV-1 replication has provided crucial information for understanding HTLV-1 replication as well as aspects of viral replication that are shared between HTLV-1 and human immunodeficiency virus type 1 (HIV-1). Here in this review, we discuss the various stages of the virus replication cycle—both foundational knowledge as well as current updates of ongoing research that is important for understanding HTLV-1 molecular pathogenesis as well as in developing novel therapeutic strategies. Human T-cell leukemia virus type 1 (HTLV-1) was independently discovered in 1980 by two research groups and identified as the etiological agent of an adult T-cell leukemia (ATL) . As the Shortly after the discovery of HTLV-1, another human retrovirus was discovered—human T-cell leukemia virus type 2, HTLV-2—which closely resembled HTLV-1 in genome structure and nucleotide sequence . Unlike HTLV-1 is the etiological agent of ATL as well as a variety of neurological pathologies, primarily HTLV-1-associated-myelopathy/tropical spastic paraparesis (HAM/TSP) . Both ATATL is an aggressive malignancy of the peripheral T-cells and can be divided into four subtypes—acute, lymphomatous, chronic, or smoldering. Patients with the acute form of ATL have a prognosis of approximately 6 months—an estimate that has not significantly changed since the discovery of the disease, despite advances in treatments . CurrentHAM/TSP is characterized by spasticity and weakness of the legs along with urinary disturbances . The priResearch into the HTLV-1 life cycle to date has been essential in the discovery and development of better therapeutic strategies. Here in this review, we highlight what is currently known as well as recent advances in the study of HTLV-1 replication. The recent advances help to provide further reason for hope in effective therapeutic options for HTLV-1-infected individuals.+ T-cells but has the potential to infect a wide variety of cells, including CD8+ T-cells, B-lymphocytes, endothelial cells, myeloid cells, fibroblasts, as well as other mammalian cells ,3837,38]in vivo . G. G94]. G2 . HTLV-1 ng sites . Cellulang sites ,106,107.ng sites . MA remang sites .i.e. from mother-to-child through breastfeeding [in vitro that cell-free HTLV-1 may cross the epithelial barrier via transcytosis before infecting subepithelial dendritic cells [There are generally three modes of inter-host HTLV-1 transmission described: (1) blood and blood products, (2) vertical or (3) sexual transmission , but thetfeeding . Mother-tfeeding ,113. Whitfeeding . Once inic cells . The preic cells . It is pic cells . The rolZoonotic transmission events of simian T-cell leukemia virus type 1 (STLV-1) to humans after contact with nonhuman primates through bites or bushmeat slaughtering still occur in Africa, establishing the emergence of new HTLV-1 infections in humans. A recent study found that more than 8% of individuals bitten by nonhuman primates in Africa are infected with HTLV-1, and virus transmission cannot be attributed to mother-to-child transmission . The strIn general, there are two distinct methods of virus transmission between cells: virus infection of cells in the absence of cell-to-cell contacts and virus infection involving cell-to-cell contacts. Most retroviruses can efficiently infect target cells in the absence of cell-to-cell contacts—in which the virus buds from the cell and infects a target cell through diffusion. HTLV-1 is notorious for being poorly infectious in the absence of direct cell-to-cell transmission, and co-cultivation of permissive target cells with virus-producing cells are the most effective means of virus transmission .Immunofluorescence and confocal microscopy were used previously to demonstrate that Gag and Env proteins are more evenly distributed in isolated T-cells, but once the cell comes into contact with another cell, cell polarization occurs—impacting the localization of HTLV-1 Gag, Env and the genomic RNA towards the cell-cell junction. This cell-to-cell junction, termed the virological synapse (VS), shares many features with the previously described immunological synapse, which includes features such as ordered talin domains and microtubule organizing center (MTOC) polarization . CryoeleIt has been reported that the formation of the VS is triggered by HTLV-1 infection and is not dependent on signaling through the T-cell receptor as is seen in immunological synapses . The VS in vivo. A recent study helps to highlight this through the observation that granulocytes  may actually capture HIV-1 and contribute to virus transmission [When comparing HTLV-1 cell-cell transmission to HIV-1 cell-cell transmission, HTLV-1 is more dependent on cell contact for infectivity. Coculturing of virus-producing cells with permissive target cells significantly enhances HTLV-1 transmission by several thousand-fold, but only increases HIV-1 transmission 10–100 hundred-fold ,131. Fursmission .In addition to transmission via the VS, HTLV-1 particles have been reported to have the ability to form a biofilm-like, carbohydrate-rich extracellular structure on the surface of cells. These structures are composed of collagen, agrin, tetherin, and galectin-3 and may function as a way to concentrate HTLV-1 particles in a single location to increase the likelihood of infection of a permissive target cell . A recenin vitro [While it has been observed that dendritic cells may be infected by cell-free HTLV-1 in vitro , a recenin vitro .in vivo is through mitotic division of host cells and subsequent propagation of the provirus by clonal expansion. DNA analysis of HTLV-1 sequences from patients in geographically distinct locations shows very little genetic variation among HTLV-1 isolates [−7–1.38 × 10−5 substitutions per site per year in the LTR and env regions [−6 mutations per target base pair per replication cycle for HTLV-1 as compared to 3.4 × 10−5 for HIV-1), the fourfold difference is likely not sufficient alone to explain the relative difference in genetic diversity between the two viruses [While HTLV-1 cell-cell transmission is likely a critical determinant of virus transmission from an infected individual to a susceptible individual, many studies have established that the primary route of replication for HTLV-1 isolates . This is regions . While t viruses ,139. Als viruses ,141.The most compelling evidence in favor of a clonal expansion of HTLV-1 proviruses is the clonality of T-cells in infected individuals. HTLV-1 integrates randomly into the genome ,143. WheSince HTLV-1 persistence depends to some extent on the clonal expansion of infected T-cells, it is not surprising that HTLV-1 encodes for gene products that have been found to increase cell proliferation. The oncogenic potential of HTLV-1 is likely a byproduct of the induction of these cellular proliferative pathways. In fact, expression of the HTLV-1 basic leucine zipper (HBZ) and Tax proteins in double transgenic mice was recently shown to be sufficient for the development of lymphoma in the absence of any other viral genes . Since sTax has been established to have many roles in the HTLV-1 proliferative cycle, including essential roles in mediating transcription of HTLV-1 genes as well as the recruitment of the MTOC to sites of cell-cell contact. Tax can also interfere with host cell cycle regulation, apoptotic pathways, and proliferative pathways through a variety of mechanisms. The most commonly studied pathway through which Tax increases cellular proliferation is via the NF-κB/Rel family of proteins.The NF-κB/Rel proteins are transcription factors that share a Rel homology domain responsible for DNA binding and dimerization. There are five proteins in this family: RelA (p65), RelB, c-Rel, p105 (processed to p50), and p100 (processed to p52). When the NF-κB pathways are turned off, NF-κB dimers are bound to the inhibitor of κB (IκB) proteins. Activation of NF-κB can occur through a canonical pathway in which nuclear localization of NF-κB is induced by inflammatory stimuli such as tumor necrosis factor (TNF) or through a non-canonical pathway reviewed in . NF-κB is constitutively active in HTLV-1 infected cells, an abnormality due to the tight regulation of NF-κB by its inhibitor, IκB . IκB kinIn addition to its upstream effects on IKK, Tax also binds directly to an IKK subunit, IKKγ (referred to as NEMO) ,155. IKKTax also has the ability to form complexes with NF-κB monomers in the cytoplasm. Tax can bind directly to cytoplasmic RelA (p65) and can induce translocation into the nucleus, leading to high transcriptional activity ,161,162.A role for Tax that has only recently been described is the recognition and inactivation of the ubiquitin-editing enzyme A20. This enzyme is a negative regulator of the canonical NF-κB pathway. It binds with the regulatory protein TAX1BP1 and E3 ligase Itch to form a complex that inactivates essential NF-κB upstream regulators. Tax binds to TAX1BP1, preventing the interaction of TAX1BP1, A20, and Itch ,164.Tax is known to facilitate the non-canonical pathway of NF-κB as well as the canonical pathway traditionally induced by MEKK1 and Tak1. This is an important distinction, as the NF-κB dimers are distinct for each pathway and increase expression of different gene products. For example, when the non-canonical pathway is blocked in the presence of Tax, tumorigenesis is significantly delayed in Tax transgenic mice . IKKα phIn addition to its interaction with NF-κB, HTLV-1 Tax has roles in a variety of other cell proliferation pathways. In particular, Tax interacts directly or indirectly with cyclin-dependent kinases (CDK) ,169, photax mRNA transcripts were only found in 34% of all cases [Tax plays a key role in the proliferation of T-cells but is known to be a highly immunogenic protein, and Tax-expressing cells are targeted by cytotoxic T-cells ,177. Thall cases , indicattax genes become mutated so that non-functional transcripts are produced [tax gene sequences are more likely to replicate due to decreased immune surveillance. Additionally, the tax gene is often methylated, leading to gene silencing [Several mechanisms regulate the downregulation of Tax expression. Many produced ,179,180.ilencing ,181. Finilencing .HBZ gene is an important HTLV-1 gene that is consistently associated with ATL. HBZ mRNA transcripts have been found in virtually all ATL cells, and it was shown that HBZ plays a key role in the proliferation of T-cells [HBZ mRNA appears to play a different role in T-cell proliferation than the HBZ protein [HBZ RNA attenuates apoptosis by promoting the transcription of Survivin, a caspase inhibitor that prevents apoptosis. While HBZ protein promotes S-phase entry, it can also promote apoptosis through its pro-inflammatory effects [The  T-cells . Interes protein . In mous protein . HBZ RNA effects .HBZ has been reported to directly or indirectly interact with the following proteins that are essential in CREB-dependent cellular proliferation: CREB, CBP, ATF-1, and ATF-3 ,187,188.157–176) has been described for use in vaccine development [As the first human retrovirus discovered in the early 1980s, HTLV-1 has been studied extensively, yet there is still no treatment or vaccine for HTLV-1 infection. Additionally, ATL and HAM/TSP treatments are symptom-based and do not directly treat the viral infection. Continued research on the molecular aspects of HTLV-1 replication will enhance opportunities for the discovery of potential antiretroviral targets that can be exploited for the development of effective therapeutic strategies. For example, a recent candidate peptide (HBZelopment . Such ob


Induced tolerance status index (ITSI). Naturally occurring naïve autoreactive B cells can escape the “classical” tolerogenic mechanisms of clonal deletion and receptor editing, but remain peripherally tolerized through B cell receptor (BCR) signaling inhibition . ITSI is a statistical index that numerically determines the level of homology between activation patterns of BCR signaling intermediaries in B cells that are either tolerized or activated by auto antigen exposure, and thus quantifies the level of peripheral immune tolerance. The index is based on the logistic regression analysis of phosphorylation levels in a panel of BCR signaling proteins. Our results demonstrate a new approach to identifying autoreactive B cells based on their BCR signaling features.This study examines the loss of peripherally induced B cell immune tolerance in Rheumatoid arthritis (RA) and establishes a novel signaling-based measure of activation in a subset of autoreactive B cells - the  Induced Tolerance Status Index (ITSI).We recently reported about changes in B cell antigen receptor signaling profiles associated with the loss of postdevelopmentally induced peripheral immune tolerance (anergy) in a subset of autoreactive B cells in patients with Rheumatoid arthritis Significant evidence supports the pathogenic role of autoreactive B cells in Rheumatoid arthritis (RA) BCR signaling regulation mechanisms involved in the control of peripheral immune tolerance have been under investigation from the autoimmunity perspective since the development of suitable transgenic mouse models in the 1990's PIT for short) has been identified in humans only recently +CD27−IgD+IgMlow/− peripheral blood B cells (termed NDBin vitro and produce autoreactive Abs (anti-dsDNA and anti-HEp-2). This autoreactivity appears innate, as variable Ig genes show no evidence of somatic hypermutation, indicating that it was not generated during an adaptive immune response. Two major signaling properties distinguish cells with PIT from other B cell types: reduced amplitude of protein phosphorylation and Ca2+ responses to antigen receptor ligation, and elevated BCR-related baseline protein phosphorylation activity in intact cells. These signaling features, described in both humans and mice Despite the landmark studies in mouse models ND cell subset in humans 2+ levels, and that a continuum of functional PIT persists in the mature naïve B cell repertoire After the recent definitive reports on the postdevelopmantally tolerized autoreactive BND cells as a contributing factor in the development of RA and demonstrated that inhibitory BCR signaling cascades involved in the maintenance of PIT are altered in human RA ND cells in RA are illustrated in ND cells and RA Clinical Disease Activity Index (CDAI) +CD27−IgD+IgMlow/− subset of autoreactive BND cells: nominal logistic fit demonstrated that RA predictive value of the combined phosphorylation profile of several major BCR signaling proteins and Ca2+ levels in response to BCR stimulation ND cells was highly significant , while in mature naïve CD19+IgM+ B cells this predictive value was substantially less evident . This moderate effect in CD19+IgM+ cells may be related to the recently recognized immunoregulatory role of B cells with PIT Studies in our laboratory have established the loss of PIT in autoreactive B2 = 0.12, p = 0.0518). However, RA subjects exhibited substantially increased phosphoprotein responses to BCR stimulation in the CD19+CD27−IgD+IgMllow/− B cell subset, as compared to normal controls ), suggesting that the breach of PIT in RA patients occurs regardless of the age  patterns of major BCR signaling intermediaries +CD27−IgD+IgMlow/− BND cells as described in in vitro for 10 min and baseline and phospho-response amplitude levels of individual BCR signal transduction proteins were compared between RA patients and healthy controls +CD27−IgD+IgMlow/− BND cells of RA patients (red) and healthy control subjects (blue) at baseline and in response to anti-BCR stimulation in vitro . This statistical parameter numerically determines the level of homology between BCR phosphoprotein activation patterns in BND cells (CD19+CD27−IgD+IgMlow/−) with intact PIT  vs. the cells with compromised PIT (RA patients), and thus quantifies the level of PIT. The index is based on the logistic regression analysis of phosphorylation levels in the panel of B cell signaling proteins in individual subjects. Phosphorylation of specific BCR signaling proteins was assessed with BD Phosflow assay as mean fluorescence intensity (MFI) of protein-specific phospho-Ab staining as described ND cells that were unmanipulated or stimulated with anti-BCR, and obtained from either RA patients or healthy control subjects To quantify the BCR signaling patterns  in orderThis type of statistical approach (i.e. logistic regression) has been successfully used in another study in our laboratory to compute the Cytokine Score ND cells with assumed dichotomous type of dependant variable (i.e. RA = Yes/No). Because the logistic regression analysis requires complete datasets, some subjects with missing data points for at least one of the phosphoproteins had to be excluded altogether (15 subjects with RA and 12 controls had complete datasets). We designed an algorithm (Appendix 1) to calculate logistic regression coefficients and to construct a composite index that could discriminate RA from healthy control subjects with sensitivity of 88.7% and specificity of 83.3% based on the phosphorylation pattern changes in response to anti-BCR stimulation, and with sensitivity of 73.3% and specificity of 83.3% based on the baseline protein phosphorylation levels. In both instances, the ITSI index predictably distinguished between RA and healthy control groups based on their BND BCR signaling profiles  and from −3.19 to 0.20 (no RA) for baseline MFI phosphorylation values. The mean value of the index ± standard error was 3.37±0.85 (RA) and −2.81±0.99 (no RA) for anti-BCR-stimulated BND cells, and 2.34±0.85 (RA) and −0.91±0.32 (no RA) for baseline MFI phosphorylation values  in RA patients ND cells exhibit a specific type of reversible immune tolerance – the post-developmentally induced peripheral tolerance (PIT), which is attained through selective BCR signaling inhibition  to ubiquitous autoantigens through continuous receptor tuning and BCR signaling inhibition, and our results demonstrate that this tolerogenic mechanism is compromised in RA.We have established the loss of PIT in autoreactive B2+ influx and total protein phosphorylation activity (pTyr), particularly in human B cells. The novelty of our study is in addressing the signaling pathways involved in the maintenance and pathological alterations of PIT in RA with a quantitative approach. ITSI values provide a scale to measure the level of BCR signaling inhibition which sustains the postdevelopmentally induced peripheral immune tolerance and enables future studies of correlations between ITSI and clinical RA scores in large datasets generated by comprehensive phosphoprotein microarrays. Our preliminary findings in a relatively small 8-phosphoprotein panel are encouraging. Furthermore, although murine models of autoimmunity provided important insights into peripheral tolerogenic mechanisms in B cells and suggested that the loss of PIT has clinical significance in autoimmunity, human autoreactive B cells with PIT have not been well studied due to the difficulty of identifying this subset. Recent discovery of the BND subset in humans PIT is a signaling phenomenon occurring late in the development cycle of B cells. However, the signaling aspects of this tolerogenic BCR signaling inhibition mechanism have not been studied in detail beyond the general features such as BCR-mediated CaND cells and severity of RA clinical manifestations ND cells that either have (control group) or do not have (RA group) the PIT. Authors believe this report will be of interest as it demonstrates a new approach to identify autoreactive B cells based on their BCR signaling features, and introduces a novel signaling-based biomarker associated with the loss of B cell immune tolerance in inflammatory autoimmune diseases such as Rheumatoid arthritis.In our previous studies of RA, the conclusions about specific BCR signaling pathways involved in the alterations of PIT were based on direct comparisons of baseline phosphoprotein activation levels and normalized anti-BCR response amplitudes in the context of signaling cascades in RA vs. control groups and on the empirical analysis of phosphorylation/activation levels of specific BCR signaling proteins based on their known functions in the BCR pathways i.e. , as wellThe association of the HLA region with RA was noted when the frequency of individuals with the HLA-Dw4 serotype was found to be increased among RA patients compared with healthy controls Our initial findings strongly support the prospective use of ITSI as a clinically relevant diagnostic measure of the humoral immunity/B cell component in RA, particularly in light of the recent evidence suggesting that peripherally induced immune tolerance in response to the chronic autoantigen stimulation is present in a major fraction of human naive B cells, and the scale of this tolerogenic mechanism is unique to B cells, and that peripheral tolerogenic mechanisms of selective BCR signaling inhibition are compromised in the onset of autoimmunity +CD27−IgD+IgMlow/− subset of autoreactive BND cells, recent reports suggest that another B cell subset with anergic features – IgM+CD21low may also play a role in autoimmunity. According to recent reports +CD27−IgM+CD21+ peripheral blood B cells, but may expand by a few percentage points in some  RA patients +CD21low B cell subset has not been definitively characterized after the initial reports that indicated its potential involvement in RA and in hepatitis C virus infection-related autoimmunity. IgM+CD21low anergic B cells are phenotypically different from the IgM−/low BND subset. Recent evaluations suggest that RA is best considered as a clinical syndrome spanning several disease subsets +CD21low anergic B cells are a promising target for the investigation of their pathogenic role in RA, however, such studies constitute a separate research direction and are beyond the main focus of this study - the IgM−/low BND cells.In addition to the CD19Our results demonstrate a novel approach for identifying autoreactive B cells based on intrinsic BCR signaling features and introduce the ITSI score as signaling-based biomarker associated the loss of peripheral immune tolerance in the autoreactive B cell subset in RA.ITSI discriminates between control and RA groups based on the inhibitory BCR phosphoprotein signaling patterns in autoreactive B cells, and thus confirms the loss of induced peripheral immune tolerance as a pathogenic factor in RA.ND cells.Lower ITSI values reflect increased levels of peripheral immune tolerance and suppressed responses to autoantigens in BND cells in RA.We recommend ITSI as a potential diagnostic measure of activation for innately autoreactive BAppendix S1Logistic regression algorythm (SAS code).(PDF)Click here for additional data file.Data S1Individual subject datapoins with spreads and basic statistical analysis.(PDF)Click here for additional data file.Data S2Detailed description of the logistic regression analysis with model fit statistics, maximum likelihood estimates for each individual phosphoprotein in the pattern, sensitivity and specificity calculation tables, odds ratio estimates and associations of predicted probabilities with observed responses.(PDF)Click here for additional data file.


Background: Human T-cell Lymphotrophic virus type 1 (HTLV-1) has infected more than 20 million people worldwide. Northeast of Iran, Mashhad, the capital of Razavi Khorasan Province, is endemic for HTLV-1 with a prevalence of 3% among general population. Objective: We evaluated the ICSI outcome in our program for (HTLV-1) serodiscordant couples (SDCs) with the female infected in comparison with control group.Materials and Methods: This study was performed between 2007 and 2011 in Novin Infertility Treatment Center . We examined 32 ICSI cycles of HTLV-1 infected women in comparison with an age matched control group (n=62). ICSI outcome was compared regarding fertilization rate (FR), embryo quality parameters, implantation rate (IR), clinical pregnancy rate (PR), and abortion rate (AR).Results: Fertilization (p=0.15), implantation (p=0.33), and pregnancy rate (p=0.12) were similar between the groups. No difference was found regarding the number of transferred embryos (on day 2 or 3) and cryopreserved embryos, multiple pregnancies, or abortion rates between the groups.Conclusion: Our results suggest that the embryo quality and ICSI outcome are not affected by HTLV-1 infection in serodiscordant couples. The major finding of this study is that the outcome of ICSI in HIV-I-infected patients and seronegative controls is similar. Retroviridae virus family called HTLV-1 has infected more than 20 million people worldwide (Human T-cell lymphotropic virus type 1 (HTLV-1) was the first human retrovirus to be described. It was discovered simultaneously in the United States and in Japan in 1980. As documented for all retroviruses, HTLV-1 produces a permanent cell infection. Therefore, all carriers are potential sources of transmission of the infection , joints (arthritis), muscles (myositis), lung , and skin (dermatitis). These conditions are even less common than ATLL (Adult T-cell Leukaemia/ Lymphoma) and HAM (HTLV-1-associated myelopathy) and the skin condition is usually only seen in tropical climates. There are several arguments indicating that HTLV-1 is a STD  in Latin America. The virus has been found in semen and cervical secretions of infected people and sexual intercourse is an important factor for HTLV-1 transmission . Male-feA non-random sampling and a cross-sectional descriptive-analytic study, was performed between September 2007 and August 2011 at Novin Infertility Treatment Center . We examined 32 ICSI cycles of HTLV-1 infected women in comparison with an age matched control group (n=62). This study was not blind. The study was approved by the ethical committee of Infertility center and patients signed consent to participate in the study. A general viral screening was performed on couples before attempting ART including HIV, HTLV-1, hepatitis B, and hepatitis C viruses. After testing the blood samples by ELISA, Western blot (WB) analysis (Genelab Kit) was used for confirmation of positive HTLV-1 associated ELISA results. Criteria for HTLV-1 seropositivity in PCR test was reactivity to GAG proteins (p19 with or without p24). The age of women ranged from 29-46 years old. The control group (62 couples) was matched with regard to age and oocyte retrieval period. The standard long protocol was adopted for most ovulation stimulation cycles. The short protocol was used for patients who were poor responders. Both groups were compared regarding cycle parameters . Moreover, ICSI outcome was compared regarding fertilization rate (FR), embryo quality parameters, implantation rate (IR), clinical pregnancy rate (PR), and abortion rate (AR) , 13. On Then, all recovered metaphase II oocytes were microinjected. Oocyte fertilization was assessed at 16-20 hours post-ICSI. Embryo morphology was evaluated on days 2 or 3 regarding the number, symmetry, and granularity of blastomeres, percentage of fragmentation, and compaction degree. According to the symmetry of the blastomeres and fragmentation, we defined three types of embryos: 1) equal-size blastomeres with no fragmentation; 2) unequal-size blastomeres with <20% fragmentation; and 3) evidence of >50% fragmentation .Statistical analysisStatical analysis was performed by using the statistical package for the Social Science (SPSS 19) .Either t test and chi-square were used to test differences between HTLV-1 serodiscordant couples and control couples. Significance was defined at p<0.05.In this study, half of our infected population was aged >35 years. In fact, the mean age of our HTLV-1-infected patients was higher than the other patients undergoing ICSI at our facility. Because of this difference, we compared the outcome of ICSI obtained in HTLV-1-infected women (group I) to those obtained in a matched control group (p<0.05). Controls were matched with regard to age and oocyte retrieval period. The patients’ characteristics are summarized in The ICSI outcomes compared in the 32 HTLV-1 serodiscordant and 62 healthy seronegative couples (control group). The fertilization and development of about 1199 oocytes were analyzed in this study, which represented 12.9±6.9 and 12.6±7.6 oocytes retrieved per couple . Of these, 11.31±5.8 and 10.7±6.5 oocyte per cycle were mature . There was no significant difference between two groups regarding to the cycle parameters . The number of transferred embryos , fertilization, implantation, pregnancy, and abortion rate were also similar between two groups. In addition, on day 2 or 3, the parameters of embryo quality  were equal in both groups .HTLV-1 was the first retrovirus linked to human disease. It has been convincingly associated with ATLL (adult T-cell leukemia/ lymphoma), HAM/TSP, uveitis. HTLV-1 has also been linked to cases of polymyositis, synovitis, thyroiditis and bronchio-alveolar pneumonitis, although definitive epidemiologic proof of HTLV-1 association is lacking. The two major HTLV-1-associated diseases, ATLL and HAM/TSP, are present in all endemic areas, although prevalence and incidence rates show significant geographic heterogeneity , 17. ComTo date, no report is found in the specialized literature regarding the ICSI outcome such as embryo quality, fertilization rate, implantation rate, pregnancy rate and etc. in HTLV-1 seropositive female. To date, all studies including cycles on HTLV-1 infection have mainly focused on decreasing the risk of transmission. In addition some papers indicated that HTLV-1 causes no disorders in a majority of healthy asymptomatic virus carriers , 20. So To assess the possible effects of HTLV-1 on the reproduction system and sexual cells of couples we decided to study the relation between HTLV-1 serodiscordant with HTLV-1 seronegative couples in ICSI outcomes. Currently because of the lack of serologic tests to differentiate HTLV-2 from HTLV-1, there is no information available regarding to the seroepidemiology or modes of transmission of HTLV-1,2 in assistant reproductive technology (ART). There was not remarkable number of HTLV-1 and 2 seropositive couples among our patients who voluntaries for intracytoplasmic sperm injection (ICSI) program to dividing them in two groups, so that we just consider on group (HTLV-1 seropositive) study group. As a result in this study, no statistically differences were seen between the fertilization rate and the quality of obtained embryos days 2 or 3 in two groups. In this study, our results suggest that the embryo quality and ICSI outcome are not affected by HTLV-1 infection in serodiscordant couples. The major finding of this study is that the outcome of ICSI in HIV-1-infected patients and seronegative controls is similar.