HTLV-1 and HTLV-2 share broad similarities in their overall genetic organization and expression pattern, but they differ substantially in their pathogenic properties. This review outlines distinctive features of HTLV-1 and HTLV-2 that might provide clues to explain their distinct clinical outcomes. Differences in the kinetics of viral mRNA expression, functional properties of the regulatory and accessory proteins, and interactions with cellular factors and signal transduction pathways are discussed. Four types of HTLV have been described so far, the most prevalent types being HTLV-1 and HTLV-2 .HTLV-1 and HTLV-2 show considerable homology in terms of genome structure, replication pattern, and properties of the structural, regulatory, and accessory proteins. Their transmission follows the same route, by transfer of infected lymphocytes by perinatal transmission and breastfeeding and through blood transfusion, sexual contact, and use of intravenous drugs . Both viDespite these important analogies, HTLV-1 and HTLV-2 are strikingly different in terms of clinical impact, as only HTLV-1 is conclusively associated with neoplasia, namely adult T-cell leukemia/lymphoma (ATLL), which develops in up to 5% of infected individuals. A similar percentage of HTLV-1-infected persons develop a neurological disease termed HTLV-associated myeolopathy/tropical spastic paraparesis HAM/TSP; . In contin vitro, they exhibit distinct cellular tropisms in vivo: HTLV-1 is mainly found in CD4+ T lymphocytes, whereas CD8+ T cells are the preferred target for HTLV-2  and in enhancing the expression of partially spliced plus-strand mRNAs (Rex). The Tax and Rex ORFs  are expressed from the same doubly spliced mRNA. In HTLV-1, all the other X region proteins are coded by individual singly or doubly spliced transcripts, while some HTLV-2 transcripts express more than one ORF.Recent studies of the temporal sequence of HTLV-1 gene expression in peripheral blood mononuclear cells (PBMCs) isolated from infected patients  revealedTable 1 and commented in the following sections.The major differences between HTLV-1 and HTLV-2 gene products are summarized in The principal role of Tax during viral replication is to activate transcription of the LTR promoter through a process that involves the recruitment of CREB/ATF complexes on binding sites in the U3 region. Tax-1 and Tax-2 share this mechanism and are able to cross-activate each others’ LTRs .Experiments performed in the early 1990s established a key role for Tax as the main viral oncoprotein necessary for the initial steps of T-cell transformation by HTLV-1 . Transgein vivo. Although overall highly homologous, Tax-1 and Tax-2 present some distinct structural features. Tax-1 contains a PDZ binding motif (PBM) and leucine zipper domains that are absent in Tax-2. The C-terminal PBM domain is relevant for interaction of Tax-1 with host factors that regulate cell cycle progression and proliferation, such as the human homologue of the Drosophila melanogaster disk large tumor suppressor protein  directs the IKK complex to lipid rafts associated with autophagic molecules such as Beclin 1 and Bif-1 .HTLV-1 Rex (Rex-1) and HTLV-2 Rex (Rex-2) share 60% homology at the amino acid level. Rex-1 and Rex-2 are phosphorylated proteins that actively shuttle between the nucleus and the cytoplasm  and accuin vitro, it is necessary for infectivity and viral persistence in vivo (Rex-1 and Rex-2 share a similar domain structure which includes: (i) a nuclear localization signal NLS; , which m in vivo , as it icis-acting elements have been described: (i) the Rex responsive element (RXRE) which, besides binding Rex, acts as an inhibitory sequence in the absence of Rex, and (ii) cis-acting repressive sequences (CRS) that determine poor stability and/or inefficient nucleo-cytoplasmic export. HTLV-1 contains a CRS which maps at the 5′ end of the unspliced mRNA but is spliced out of the other transcripts (cis-acting inhibitory elements (CIEs) were mapped within the gag-pol and env regions of HTLV-1  are considered to be homologous based on their structure and functional properties, while p13 and p8 appear to be unique to HTLV-1, and p11 is peculiar to HTLV-2.The “accessory” proteins were labeled as such because their ablation does not have apparent consequences on viral replication N-terminal arginine-rich NLS and are therefore incapable of binding the RXRE. HTLV-2 tRex is detected as four main isoforms of 22, 20, 18, and 17 kDa which differ in the initiation codon usage and phosphorylation status  production  and in the Golgi apparatus, where it reduces the expression of the β and γr IL-2R,  and of Mr IL-2R, . p12 alsr IL-2R,  and inter IL-2R, , resultim the ER  and actim the ER . Within m the ER ,b. In anm the ER . No homoin vivo propagation in macaques but not in rabbits.A recent study from The x-V ORF of HTLV-2 codes for p11 from a doubly spliced transcript that also codes for p10 . Aside fN-terminal region that mediates its interaction with the KIX domain of p300/CBP, thus competing for its binding to Tax-1  while the truncated forms of Rex-2 were shown to inhibit Rex function, the HTLV-1 homologue p21Rex might lack this activity; (v) HTLV-1 expresses p13 and p8, which have not been described in HTLV-2; (vi) HTLV-2 expresses p11, which does not seem to have a homologue in HTLV-1; (vii) unlike APH-2, HBZ presents a basic zipper domain, as well a transcriptional activation domain, which mediate its capacity to enhance its own expression and deregulate several cellular pathways; (viii) HTLV-2 shows in vivo tropism for CD8+ T cells and induces expansion of a relatively small number of highly abundant clones.From the comparative analysis on the functional properties of HTLV-1 and HTLV-2 reported above, the following major differences can be outlined: (i) HTLV-2 is characterized by a more abundant expression of gene products that may favor viral latency ; (ii) Tax-1 presents a PDZ binding motif, which is absent from Tax-2 and allows interaction with host factors regulating the cell cycle and proliferation; in addition, Tax-2 is unable to activate the non-canonical NF-κB pathway; (iii) in vivo transforming activities of Tax-2 and APH-2 have not been investigated. Aside from the NF-κB pathway, not much information is available regarding the interactions of Tax-2 with cellular pathways known to be engaged by Tax-1. The fact that HTLV-2, contrary to HTLV-1, is characterized by an oligoclonal proliferative distribution in asymptomatic hosts clearly indicates that the etiology of malignant transformation by HTLV-1 cannot be uniquely attributed to this phenomenon.In spite of the evidence accumulated so far on the similarities and differences between HTLV-1 and HTLV-2, it is not yet clear why only HTLV-1 causes a T-cell malignancy. While Tax-1 and HBZ induce T-cell lymphomas when expressed as transgenes in animal models, the 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.


Autoantibodies contribute significantly to the pathogenesis of systemic lupus erythematosus (SLE). Unfortunately, the long-lived plasma cells (LLPCs) secreting such autoantibodies are refractory to conventional immunosuppressive treatments. Although generated long before the disease becomes clinically apparent, it remains rather unclear whether LLPC generation continues in the established disease. Here, we analyzed the generation of LLPCs, including autoreactive LLPCs, in SLE-prone New Zealand Black/New Zealand White F1 (NZB/W F1) mice over their lifetime, and their regeneration after depletion.Bromodeoxyuridine pulse-chase experiments in mice of different ages were performed in order to analyze the generation of LLPCs during the development of SLE. LLPCs were enumerated by flow cytometry and autoreactive anti-double-stranded DNA (anti-dsDNA) plasma cells by enzyme-linked immunospot (ELISPOT). For analyzing the regeneration of LLPCs after depletion, mice were treated with bortezomib alone or in combination with cyclophosphamide and plasma cells were enumerated 12 hours, 3, 7, 11 and 15 days after the end of the bortezomib cycle.Autoreactive LLPCs are established in the spleen and bone marrow of SLE-prone mice very early in ontogeny, before week 4 and before the onset of symptoms. The generation of LLPCs then continues throughout life. LLPC counts in the spleen plateau by week 10, but continue to increase in the bone marrow and inflamed kidney. When LLPCs are depleted by the proteasome inhibitor bortezomib, their numbers regenerate within two weeks. Persistent depletion of LLPCs was achieved only by combining a cycle of bortezomib with maintenance therapy, for example cyclophosphamide, depleting the precursors of LLPCs or preventing their differentiation into LLPCs.In SLE-prone NZB/W F1 mice, autoreactive LLPCs are generated throughout life. Their sustained therapeutic elimination requires both the depletion of LLPCs and the inhibition of their regeneration. Systemic lupus erythematosus (SLE), the prototype of a systemic autoimmune disease, is characterized by the production of pathogenic autoantibodies that directly or indirectly contribute to the pathogenesis of SLE, resulting in cell destruction and inflammation ,2. NZB/WAutoantibodies are detectable years before the clinical onset of SLE in humans , and by Here, we show that LLPC generation starts very early in NZB/W F1 mice, long before clinical onset of disease. Then, LLPC counts in the spleen plateau after about 10 weeks, but those in the bone marrow and inflamed kidney increase over lifetime. When PCs are eliminated by bortezomib , LLPC coFemale NZB/W F1 mice were bred and maintained under specific pathogen-free (SPF) conditions at the mouse facility of German Rheumatism Research Centre, Berlin. All experiments were performed according to German guidelines for the performance of animal experiments and approved by the according authority .low plasma cells were regarded as LLPCs. The expression of MHC-II on B220-positive cells was used to determine the cutoff between high (same MHC-II expression of B220+ cells) and low (lower MHC-II expression as compared to B220+ cells) MHC-II-expressing plasma cells. BrdU staining was performed with anti-BrdU antibody using the BD Pharmingen BrdU FlowKit™ according to the manufacturer’s recommendations.Flow cytometric analysis of spleen and bone marrow cells was performed after two weeks of bromodeoxyuridine (BrdU) feeding (see below). Staining was performed as described previously . The fol2. After extensive washing, plates were incubated with biotin-labeled goat anti-mouse immunoglobulin G (IgG) and immunoglobulin M (IgM)  for 1 h and followed by ExtrAvidin™-alkaline phosphatase (Sigma-Aldrich) for 20 min. The spots were developed with NBT/BCIP  and enumerated using an automated ELISPOT reader and software .Enzyme-linked immunospot (ELISPOT) analysis to determine the absolute numbers of anti-double-stranded DNA (anti-dsDNA)-specific antibody-secreting cells (ASCs) was performed as described previously ,18. BrieAbsolute cell numbers of spleen, bone marrow and kidneys were assessed by automatic cell counting either by Schaerfe CASY Cell Counter  or by MACSQuant Analyzer . Absolute plasma cell numbers were calculated based on total cell numbers (by automatic cell counting) and frequencies of cell types in each organ measured by flow cytometry. For ELISPOT assay, the absolute numbers of ASCs per organ were calculated by the number of spots/well multiplied by the dilution factor of the cell suspension used for the assay. The total number of bone marrow cells was calculated based on the assumption that both femoral bones contain approximately 8% of total bone marrow cells .In the experiments analysing the generation of LLPC over the lifetime mice of different ages 4 to 32 weeks) were given BrdU in their drinking water  for two weeks in order to determine the time point of formation of the LLPC compartment. This made it possible to discriminate between cells generated during and before the feeding period. As shown previously, this two-week feeding interval is sufficient for discriminating between short- and long-lived plasma cells [2 weeks wIn the experiments dissecting the capacity to generate LLPC at different age, mice were continuously fed BrdU for six weeks from the ages of 4 to 10 weeks (young) and 20 to 26 weeks (old). Six weeks after the end of the feeding period, the number of surviving BrdU-positive plasma cells was measured. These cells are LLPCs that were generated during the feeding period and survived the next six weeks.To characterize the establishment kinetics of autoreactive (anti-dsDNA) LLPCs, plasmablasts and SLPCs were depleted in NZB/W F1 mice at different ages  by administering four injections of cyclophosphamide (35 mg/kg bw) within two weeks [high) and mature LLPCs (MHC-IIlow) in these experiments [low cells are included, which may be newly generated LLPCs.Twelve- to 16-week-old NZB/W F1 mice with an established plasma cell pool received a single bortezomib cycle consisting of two intravenous (i.v.) injections (0.75 mg/kg bw) of bortezomib over a 36-hour interval. For the combination treatment, mice were additionally treated with 35 mg/kg bw cyclophosphamide alone or in combination with the starting of the bortezomib cycle. Cyclophosphamide was re-administered every four days until the end of the experiment. Twelve hours and 3, 7 and 15 days after the last bortezomib injection, the mice were sacrificed and their spleen and bone marrow cells were characterized and quantified by flow cytometry and ELISPOT. MHC-II expression on the plasma cells population was analyzed to discriminate between plasmablasts and newly generated plasma cells . If not otherwise indicated, the indicated numbers are means with standard error of the mean (SEM). Statistical significance was calculated using the unpaired two-tailed Student To analyze the generation of LLPCs in SLE-prone NZB/W F1 mice over their lifetime, young  and old  mice received BrdU (1 mg/ml) in their drinking water for two weeks. BrdU-negative CD138+ plasma cells (those which had not performed DNA synthesis during the two weeks of BrdU feeding) were classified as long-lived [In six-week-old mice, 17% (±3%) of all plasma cells in the spleen were LLPCs, which were initially generated before the age of 4 weeks  and 20-week-old NZB/W F1 mice  were fed BrdU for six weeks . BrdU feeding was then stopped and animals were kept for another six weeks without further BrdU feeding. The spleen and bone marrow of the mice were then analyzed for persistent, BrdU-labeled cells. These BrdU-positive cells, which must have been generated during the preceding BrdU feeding period, can thus be regarded as LLPCs that were generated in the feeding period. Twenty-one ± 3%  and 14 ± 2%  of the plasma cells in spleen and bone marrow of 16-week-old mice, respectively, were LLPCs that had been generated between weeks 4 and 10 of age  and from 881,886 to 332,620 in the bone marrow (74.1 ± 30% decrease) (P = 0.0014)  . Twelve Twelve- to 16-week-old NZB/W F1 mice were treated with a cycle of bortezomib, cyclophosphamide alone or with a combination of the two. Cyclophosphamide was injected intraperitoneally (i .p.) on day 0, 3, 7 and 11 after the bortezomib cycle in order to deplete plasma cells and, on the other hand, to block their regeneration Figure . We haveHere, we showed for the first time that the generation of autoreactive LLPCs in SLE-prone NZB/W mice starts very early in life, long before the onset of disease. Already mice at the age of 4 weeks had anti-dsDNA-secreting plasma cells resistant to cyclophosphamide in both bone marrow and spleen. In the bone marrow, autoreactive LLPC counts increased at a stable rate over life without reaching a plateau, even in six-month-old mice. In the spleen, LLPC numbers increased only in the first 12 weeks and then remained stable over time. This demonstrates that autoreactive LLPCs are constantly generated in NZB/W mice and that, later in life, they accumulate in the bone marrow and inflamed kidney but not in the spleen. These findings pose a therapeutic challenge since LLPCs are resistant to conventional immunosuppression  or B cell-depletion strategies ,5,6. TheAre these findings of any relevance for human autoimmune diseases?SLE autoantibodies are considered to be pathogenic in human SLE , and som9 and 106 LLPCs can be hosted in the bone marrow of healthy humans or healthy mice, respectively [Notably, the dynamics of generation and maintenance of autoreactive LLPCs is not only determined by the rate of generation, but also by the capacity of the body to support LLPCs in the long run. It has been shown that the number of plasma cells in human and mouse bone marrow is determined by the number of chemokine (C-X-C motif) ligand 12 (CXCL12)-expressing stromal cells, which organize survival niches for individual LLPCs . The freectively . Here weectively . In the ectively ,18,20, iectively  or, mostectively ,18,20.et al.). Moreover, the administration of cyclophosphamide for immunosuppression, as used here, is only a proof of principle. Other more fitting options are available for patients . In particular, combined LLPCs targeting and anti-BAFF therapy  might be a first efficient way to eliminate LLPCs and on the other hand to interfere with their regeneration and persistence, with the advantage to contrast the increased level of BAFF described after B cell depletion [Our findings have relevant clinical implications. As discussed above, the existence of autoreactive LLPCs is a therapeutic challenge. Considering the very early accumulation of autoreactive LLPCs in the bone marrow and spleen of NZB/W mice, our data suggest that a ‘clinically relevant window of opportunity’ for preventing the accumulation of autoreactive LLPCs would exist only for the kidney. However, persistent activation and accumulation of autoreactive LLPCs in the bone marrow may cause relapses in patient with SLE even after long periods of clinical inactivity ,27. Therepletion ,33. Notaepletion -36. Convepletion . Consistepletion ,38-40. TThis study explored the generation and maintenance of autoreactive LLPCs in SLE-prone NZB/W mice and the kinetic of regeneration of these cells after depletion. We showed that autoreactive LLPC generation starts very early in life, long before the onset of disease and that these cells are constantly generated during disease. Later in life, LLPCs accumulate in the bone marrow and inflamed kidney but not in the spleen. When LLPCs are depleted by the proteasome inhibitor bortezomib, their numbers regenerate within two weeks, due to constant B cell hyperactivity. Persistent depletion of LLPCs was achieved only by combining a cycle of bortezomib with maintenance therapy, for example cyclophosphamide, depleting the precursors of LLPCs or preventing their differentiation into LLPCs. Our results provide clear evidences that targeting LLPCs in isolation would have limited efficacy in autoimmune disease. This study enters into the recent debate concerning which B cell subset should be targeted in SLE ,27,32,41


Thymic stromal lymphopoietin (TSLP) is an epithelial cytokine expressed at barrier surfaces of the skin, gut, nose, lung, and the maternal/fetal interphase. At these sites, it is important for the generation and maintenance of non-inflammatory, tissue-resident dendritic cell responses. We show here that TSLP is also expressed in the central nervous system (CNS) where it is produced by choroid plexus epithelial cells and astrocytes in the spinal cord. Under conditions of low-grade myelin degeneration, the numbers of TSLP-expressing astrocytes increase, and microglia express transcripts for the functional TSLP receptor dimer indicating that these cells are targets for TSLP in the myelin-degenerative CNS. Epithelial cells at barrier surfaces like skin, gut, nose, and lung express the cytokine thymic stromal lymphopoietin (TSLP) -overexpressing transgenic and wildtype Lewis rats were obtained from the Decentral Facilities of the Institute for Biomedical Research, Medical University, Vienna. The genotype of the transgenic animals was determined by PCR analysis as described  prior to the dissection of brains and spinal cords. The tissue was post-fixed in 4% PFA for additional 18–24 hours and subsequently embedded in paraffin.For histology, archival CNS material from 10 to 12 months old PLP-transgenic Lewis rats and age-matched wildtype Lewis rats , or after antigen retrieval for 1 hour in a commercial food steamer in 10 mM EDTA buffer pH 8.5 (anti-GFAP). The sections were rinsed in 0.05 M Tris-buffered saline (TBS) and incubated in Dako buffer  containing 10% fetal calf serum (DB/FCS) for 20 min. The slides were incubated in blocking buffer at 4°C overnight with the following primary antibodies: polyclonal rabbit anti-mouse/rat TSLP , polyclonal rabbit anti-Iba1 , or mouse anti-rat glial fibrillary acidic protein . The sections were then washed three times in TBS and incubated with biotinylated secondary antibodies  in DB/FCS for 1 hour at room temperature, followed by three times washing in TBS and incubation with avidin–peroxidase complex  for 1 hour. After another washing step, labeling was visualized with 3,3′-diaminobenzidine-tetra-hydrochloride  containing 0.01% hydrogen peroxide. All sections were counterstained with Meyer's hematoxylin, dehydrated, and mounted in Eukitt (Sigma).l-lysine-coated culture flasks. Confluent mixed glia cultures (after 7–8 days in vitro) were placed on a shaking platform to detach microglial and oligodendrocyte lineage cells . The supernatant containing the detached cells was used for microglia cultures as described below. The adherent, astrocyte-enriched cells were detached by incubation with accutase , subsequently washed with RPMI/10% FCS and reacted with NMO-IgG  was added to each sample. The samples were incubated first at RT for 10 min, then at 40°C for 50 min, and finally at 70°C for 15 min.Cellular RNA was isolated using the RNeasy Mini Kit  according to the manufacturer's instructions. To avoid contamination of the RNA sample with genomic DNA, the following procedures were carried out: 50 µL RNA sample was mixed with 7 µL 10× Dnase buffer, 2 µL RiboLock RNase inhibitor, 7 µL DNase 1 , and 4 µL RNase-free water  and incubated for 30 min at 37°C, then 7 µL 25 mM EDTA (Fermentas) were added, and the samples were further incubated for 10 min at 65°C. After RNA clean-up, the RNA was eluted in 30 µL RNase-free water and subsequently annealed with a T7-N7 primer , supplemented with 10 µL 5× M-MLV RT Buffer (Promega), 3 µL dTNPs (Roche), 3 µL RiboLock RNase I Inhibitor (Fermentas), and 5 µL RNase/DNase-free H−ΔCt = 2−[Ct(GOI)−Ct(HKG)] .Quantitative real-time polymerase chain reaction (qPCR) was performed using the QuantiFast™ SYBR® Green PCR Kit (Qiagen) in a StepOnePlus RT-PCR system (Applied Biosystems), according to the manufacturer's instructions. The following QuantiTect Primer Assays (Qiagen) were used for gene amplification: Rn_Crfl2_1_SG for Crlf2 (Tslpr), Rn_Gapdh_1_SG for Gapdh, Rn_Gfap_1_SG for Gfap, Rn_Il7r_2_SG for Il7r, and Rn_Ttr_1_SG for Ttr. For the detection of Tslp, transcript self-designed primers (Tspl-forward: 5′-TCCTGAAAATCGACCACCAT-3′ and Tslp-reverse: 5′-GATTGTGGCTTTCCTGCATT-3′) were used, with a final concentration of 0.5 μM for each primer. The following cycling conditions were applied: initial heat activation , followed by 40 cycles of denaturation , and annealing/extension . The absence of non-specific amplification was determined by a melt curve analysis. All reactions were run in duplicates or triplicates. Normalized gene expression levels were calculated using the equation: 22), 1 µL 10 mM dNTP mix, 1 µL forward primer (100 pmol/µL), 1 µL reverse primer (100 pmol/µL), 0.4 µL polymerase (5 U/µL), 1 µL cDNA, and 40.6 µL H2O. The following primer pairs were used: Gfap ; GAPDH ; TSLP ; Transthyretin ; TSLPR , and IL-7Rα . The reaction mixture was subjected to an initial denaturation step , and then to 30, 35, and 40 cycles of denaturation , annealing , and elongation . The reaction was terminated with final extension for 10 min at 72°C, and PCR products were detected by agarose gel electrophoresis. Sequencing of the PCR products  confirmed their identity.Further PCR analyses were done using the FastStart Taq Polymerase kit . One reaction consisted of: 5 µL 10× PCR buffer , with 50% scores  centered around median values . Analyses were performed using PASW Statistics 18 software (SPSS Inc.).Statistical analyses were conducted using Kruskal–Wallis test, followed by pair-wise Mann–Whitney + astrocytes at the superficial glia limitans displayed cytoplasmatic and often also nuclear reactivity for TSLP. The numbers of TSLP+ astrocytes were highly variable, independent of the location of the spinal cord sections along the neuraxis . Instead, choroid plexus cells were positive for TSLP . We alsoTo further confirm the expression of TSLP in astrocytes and choroid plexus epithelium, we established cultures of these cells and searched for the presence of TSLP transcripts by qPCR. TSLP mRNA was detectable in both types of cells indicating that they are able to produce TSLP .+ DCs and spinal cord microglia produced transcripts encoding both the IL-7Rα chain and the TSLPR and were hence able to form a functional receptor for TSLP recognition , interferon-γ, interleukin-16, and interleukin-1 family cytokines (for review see Luheshi et al., TSLP reactivity of astrocytes is highly variable. This may reflect astrocyte heterogeneity (Black et al., + cells in the meningeal/perivascular DC network.In the intact CNS, TSLP expression by choroid plexus epithelial cells in the brain, and by astrocytes at the glia limitans in the spinal cord might regulate the survival and the effector functions of TSLP receptor dimer-expressing MHC class IIHowever, in the myelin-degenerative spinal cords of PLP-transgenic Lewis rats, both the extent of TSLP expression and the cellular targets of TSLP change. Then, additional astrocytes become TSLP-positive. Interestingly, these astrocytes were predominantly located in the spinal cord gray matter, a compartment characterized by low-grade myelin degeneration, oligodendrocyte stress/death and microglia activation (Aboul-Enein et al., 2004; Bradl et al., in vivo can only partially differentiate along the DC lineage in vivo, and are arrested at an immature stage (Fischer et al., Microglial cells of PLP-transgenic rats do not show all features of TSLP-instructed DCs, since they upregulate transcripts for MHC class II products, CD40, CD86, and OX40L, but not for the chemokines CCL17 or CCL22. The most likely explanation for this finding is the fact that microglial cells


Although many studies have examined the relationship between uric acid (UA) and coronary artery disease (CAD), whether UA is an independent risk factor contributing to progression of CAD is still controversial. Whether UA plays a different role in different sexes is also unclear.A total of 1116 individuals with suspected CAD were stratified into four groups according to their serum UA quartiles in total (men and women combined), in men, and in women. The association of UA with coronary atherosclerosis was assessed by univariable and multivariable logistic regression.In total and in women, the prevalence of any plaques and significant/severe stenosis was significantly increased with an increase in quartiles of UA . The proportion of triple-vessel disease and left main artery lesion was highest in the fourth quartile (both p < 0.05). Increasing quartiles of UA were significantly associated with a coronary artery calcium score (CACS) >10 . As UA levels increased in women, the incidence of double-vessel lesions (p = 0.017) and the proportion of mixed plaques (p = 0.022) were significantly increased. The proportion of a CACS of 0 in total, in men and women was highest in the first quartile . UA was the strongest predictor of significant stenosis, multivessel disease, and mixed plaques in women . UA was the only risk factor for mixed plaques in total (P = 0.046).The level of UA was significantly associated with coronary atherosclerosis in women, but not men. Uric acid (UA) is the main end product of purine catabolism . High UASex might be an important factor involved in the relationship between UA and CAD. In a subgroup analysis of LIFE , the relTherefore, in this study, we assessed the association between UA and coronary atherosclerosis in patients with suspected CAD who underwent 256-detector-row coronary computed tomographic angiography (CCTA). We further assessed these associations in sex subgroups.This study included 5150 consecutive individuals (≥18 years) in China undergoing CCTA and coronary artery calcium score (CACS) measurements in our institution from September 2011 to February 2013. CCTA and measurement of the CACS were performed for the suspicion of CAD after clinical assessment . Finally, 1116 individuals were enrolled , triglycerides (TG), high-density lipoprotein cholesterol (HDL-C), low-density lipoprotein cholesterol (LDL-C), creatinine and UA levels were measured after at least a 12-h fasting period within 7 days of CCTA. The contents of UA were measured with enzyme kinetics in all enrolled patients, and it was uniform throughout the study period.Computed tomography scans were performed using 256-detector-row CCTA . Individuals with a heart rate ≥75 beats per minute were treated orally with up to 100 mg metoprolol for several hours (except for contraindications of beta-blockers) before CCTA imaging to achieve a higher image quality. First, after a scout radiograph of the chest , a noncontrast CACS scan was performed, and the image section thickness was 2.5 mm by triggering at a heart rate depending on the percentage of the R-R interval. The sections were collected from the level of the carina and proceeded to the level of the diaphragm. Thereafter, retrospective electrocardiogram-gated contrast-enhanced CCTA was performed. The CCTA scan was initiated 20 mm above the level of the left main artery to 20 mm below the inferior myocardial apex during a single breath-hold. Depending on the individual’s weight, a bolus of 50–80 ml of iopamidol or iohexol was intravenously injected at 4–5.5 ml/s into the antecubital vein, followed by 50 ml of saline. A standard scan protocol was applied, with section collimation of 256 × 0.625 mm, 0.27 s for rotation time, 120 KV tube voltage, and 800–1100 mA tube current. In all scans, electrocardiogram-gate dose modulation was used. The electrocardiograms of individuals were simultaneously collected to allow for retrospective segmental data reconstruction. The images of all individuals were initially reconstructed at 75% of the R-R interval of the cardiac cycle. If motion artefacts were found, reconstruction of additional phases was performed. The best R-R interval image quality was chosen for interpretation. The dose range of radiation for CCTA was estimated to be 10–18 mSv.All images were analysed separately by two experienced radiologists and one cardiologist who were blinded to the patients’ characteristics. Consensus on interpretation was performed to achieve a final CCTA diagnosis. All scans were evaluated by a three-dimensional workstation . The CACS was measured using the scoring system previously described by Agatston et al. .2, which existed either within the coronary artery lumen or was adjacent to the coronary artery lumen, and could be discriminated from surrounding pericardial tissue, epicardial fat, or the vessel lumen itself. For evaluating the degree of stenosis, the coronary lumen was semi-automatically traced at the maximal stenosis site and was compared with the mean value of a proximal and distal reference site. The image quality was evaluated and classified as follows: good, with no artifact; adequate, with the presence of artifacts but feasible for evaluating the degree of stenosis and plaque characteristics; or poor, with the presence of artifacts and not feasible for evaluating the degree of stenosis and plaque characteristics. If an image was graded as poor, the image was not included.The three reads were permitted to use any/all available post-processing image reconstruction algorithms, including two-dimensional axial or three-dimensional maximal intensity projection, multiplanar reformat, cross-sectional analysis, or the volume-rendered technique. A 16-segment coronary artery tree model [Coronary lesions ≥50 and 70% were defined as significant and severe, respectively. Multivessel disease was defined as the presence of stenosis of more than 50% in at least two vessels. The stenosed coronary vessel of individuals was further categorized as having one-, two-, and three-vessel/left main disease. All detected plaques were classified as calcified, non-calcified, or mixed. The calcified component of a stenosis was defined as a lesion with radiodensity greater than the luminal contrast. The non-calcified component of a stenosis was defined as a lesion with radiodensity greater than that of neighbouring soft tissue and lower than the luminal contrast. Plaques that contained calcified tissue greater than 75% of the plaque area were classified as calcified plaques, less than 25% as non-calcified plaques, and 25–75% as mixed plaques .χ2 test.Baseline characteristics are expressed as absolute counts and proportions for categorical variables, and as means ± standard deviations for continuous variables. Continuous variables were analysed by analysis of variance and categorical variables were analysed by χ2 test. Univariable and multivariable logistic regression were used to assess the association of UA with significant stenosis, multivessel disease, high CACS, and plaque characteristics. Variables that showed a P value <0.1 with univariable analysis were applied to multivariate analysis. All analyses were performed using SPSS 15.0. The level of significance was set at P < 0.05.The association of UA with coronary atherosclerosis was assessed by the P < 0.01). Body mass index (BMI) and TC level were significantly higher  and HDL-C level was significantly lower ; the second quartile: 259–309 μmmol/l (n = 274); the third quartile: 310-373 μmmol/l (n = 283) and the fourth quartile: >373 μmmol/l (n = 281). With increasing UA quartiles, the prevalence of male sex, smoking, and hypertension was significantly increased ; the second quartile: <296-349 μmmol/l (n = 140); the third quartile: 350-406 μmmol/(n = 144) and the fourth quartile: >406 μmmol/l (n = 142). The quartiles of UA in women were the first quartile: <238 μmmol/l (n = 136); the second quartile: 238-273 μmmol/l (n = 137); the third quartile: 274-326 μmmol/l(n = 138) and the fourth quartile > 326 μmmol/l (n = 139). With increasing UA quartiles in total (men and women combined) and in women, the prevalence of plaques were significantly increased  and in women . UA was the strongest predictor for significant stenosis, multivessel disease, and a high CACS in univariate analysis , but this association was not apparent after adjustment  were significantly associated with significant stenosis, multivessel disease, and a high CACS . HDL-C level was only significantly associated with significant stenosis and multivessel disease (both P < 0.05). The strongest risk factors for a high CACS were age and hypertension after adjustment . Significant predictors of multivessel disease were age, hypertension, DM, and UA after adjustment . The strongest risk factors for a high CACS were age, hypertension, DM, and HDL-C levels after adjustment . Significant predictors of non-calcified plaques were age, smoking, hypertension, DM, and HDL-C levels after adjustment . After adjustment, the strongest risk factors for mixed plaques were age, male sex, smoking, hypertension, DM, and UA . The strongest risk factors for non-calcified plaques were age, smoking, DM, and HDL-C in univariate and multivariate analysis . The strongest risk factors for a high CACS were age, smoking, and HDL-C levels after adjustment . Significant predictors of non-calcified plaques were age, hypertension, and HDL-C after adjustment . Age, hypertension, DM, and UA were significantly associated with mixed plaques in univariate and multivariate analysis  Overall and in women, the prevalence of coronary atherosclerosis, severe stenosis, and triple-vessel/left main artery lesions of plaques were significantly increased with increasing quartiles of UA. UA was significantly associated with double-vessel lesions in women. 2) UA was significantly negatively correlated with the proportion of a CACS of 0, but was positively correlated with the proportion of a CACS >10 overall and in women. The incidence of mixed plaques was significantly increased only in women with elevated UA levels. 3) After adjustment, only UA was a significant predictor of significant stenosis, multivessel disease, and mixed plaques in women. UA was not significantly associated with coronary atherosclerosis in men.Coronary artery angiography (CAG) is the gold standard for diagnosing CAD, but CAG cannot determine the characteristics of plaques. CAG would be difficult to use as a general screening tool in China because of its highly invasive nature, radiation exposure, and cost. The technique of 256-detector-row CCTA with a shorter scanning time can improve image quality and it has a minimal radiation dose . Use of Many studies have assessed the association of UA with CAD and clinical outcome, but the results were still controversial. Kocaman et al. found that UA was an independent predictor of CAD in individuals undergoing CAG . HoweverPrevious reports have proven the highly predictive value of the CACS on cardiovascular outcomes ,31. SomeUA is a general antioxidant in the body, and a high UA level is suggestive of oxidative stress, endothelial dysfunction, and slow coronary artery flow ,40. UA pThe incidence of cardiovascular events is still high after controlling for traditional risk factors. Our study suggests that UA should be considered as an additional risk factor beyond traditional factors for CAD in women. However, a randomized study still needs to be performed to determine the effect of UA-lowering therapies on cardiovascular prevention. In the GREACE study, decreased UA levels by atorvastatin were independently correlated with a reduced risk of cardiovascular outcomes . The LIFThere were several limitations to our study. All individuals were suspected of having CAD, we mainly want to explore the relationship between UA and early CAD in the study. Patients with known CAD may implement lifestyle modifications and drugs intervention to some extent, the real relationship between UA and CAD may be influenced by those confounding factors, So, we exclude the patients who had known CAD. There was a possible selection bias and this could restrict generalizability of our results to similar care settings. Unmeasured confounders may have affected our results. In our study, almost 90% of women were post-menopausal and few women received oestrogen-replacement therapy. Therefore, we did not analyse the potential effects of oestrogen on the relationship between UA and CAD. In cross-sectional analysis, we did not find a longitudinal relationship between UA and cardiovascular outcomes. In spite of these limitations, the strong association of UA with early CAD in women had an important clinical significance in prevention and treatment of CAD. Longitudinal studies are required to confirm the association of UA with CAD and cardiovascular outcome.UA is a significant predictor of significant stenosis, multivessel disease, and mixed plaques in women. Importantly, our study suggests that UA level may play an important role in the occurrence and development of coronary atherosclerosis in women but not men.UA: Uric acid; CAD: Coronary artery disease; CCTA: Coronary computed tomography angiography; CACS: Calcium score; BMI: Body mass index; TC: Total cholesterol; TG: Triglyceride; HDL-C: High-density lipoprotein cholesterol; LDL-C: Low-density lipoprotein cholesterol; CAG: Coronary artery angiography.The authors declare that they have no competing interest.SYJ participated in the design of the study, collected and organized datas, analysed images, performed the statistical analysis and drafted the manuscript. YX collected, organized and analysed datas. ZY, GS and LH collected and organized datas. LT and XK analysed images. CL and WCW collected datas. QGX conceived of the study, and participated in its design and coordination and helped to draft the manuscript. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2261/14/101/prepub


Brazil is considered the country with the largest absolute number of individuals infected with HTLV-1/2, close to 2,500,000, and is also considered epidemic for HIV/Aids with a prevalence rate of 0.3%-0.6%. The routes of such retroviruses transmission could vary according to the region, population and period of study. This study aimed to characterize HTLV-1 and HTLV-2 that circulate in HIV-coinfected patients from the southern (n=34) and southeastern (n=20) Brazil. For analysis, sequencing of LTR, env and tax regions of HTLV-1 and HTLV-2 and bioinformatic tools were employed, and the results obtained analyzed according to geographic regions and risk factors. The results obtined confirmed HTLV-1a subtype, Transcontinental subgroup A as the prevalent, grouping into two Latin America clusters. The most frequent nucleotide substitution V1981I in the env and the Brazilian molecular signature in TaxA were detected. The HTLV-2a subtype, variant 2c (characteristic of the long Tax found in Brazilian strains) was detected in all except one isolate, as well the amino acid change S1909P in the env region. The HTLV-2 clustered according to geographic region and risk factors . All sequences are in GenBank. This study confirmed the double entrance of HTLV-1 in Brazil, and the presence of the Amerindian HTLV-2c in HIV-coinfected patients. The lack, in the present study, of the prototypic North American HTLV-2a and HTLV-2b subtypes circulating worldwide suggest the introduction of HTLV-2 before HIV-1 in such vulnerable population of Brazil. Support: MCT/CNPq # 481040/2007-2 and # 303545/2012-7, CAPES, IAL # 33/07 and # 39/07), Brazil.


Plants have evolved effective defense strategies to protect themselves from various pathogens. Salicylic acid (SA) is an essential signaling molecule that mediates pathogen-triggered signals perceived by different immune receptors to induce downstream defense responses. While many proteins play essential roles in regulating SA signaling, increasing evidence also supports important roles for signaling phospholipids in this process. In this review, we collate the experimental evidence in support of the regulatory roles of two phospholipids, phosphatidic acid (PA), and phosphatidylinositol 4-phosphate (PI4P), and their metabolizing enzymes in plant defense, and examine the possible mechanistic interaction between phospholipid signaling and SA-dependent immunity with a particular focus on the immunity-stimulated biphasic PA production that is reminiscent of and perhaps mechanistically connected to the biphasic reactive oxygen species (ROS) generation and SA accumulation during defense activation. PR gene expression, reactive oxygen species (ROS) production and callose deposition via conserved interwoven signaling pathways that are regulated by salicylic acid (SA), jasmonic acid (JA), and ethylene (C2H4)  and phosphatidylinositol 4-phosphate (PI4P), and the related enzymes  are implicated in SA signaling during PTI and ETI. For more detailed information on the biochemistry of these phospholipids and their metabolizing enzymes, and their roles in plant stress responses, we recommend several excellent reviews Wang, .Being the simplest phospholipid class, PA has rather versatile functions: it is not only a central intermediate in glycerolipid biosynthesis but also a signaling molecule involved in regulating cellular processes such as lipid metabolism, signal transduction, cytoskeletal rearrangements, and vesicular trafficking. The concentration of PA is normally very low in plant tissues and can be induced rapidly by various stimuli. This signal-induced PA is mainly produced via two distinct enzymatic pathways. The first route is accomplished in a two-step enzymatic process that involves generation of diacylglycerol (DAG) from inositol phospholipids by PLC, followed by production of PA through phosphorylation of DAG by DGK. The other route engages PLD to produce PA through direct hydrolysis of phospholipids such as phosphatidylcholine and phosphatidylethanolamine by removing their head groups     plants  resistance to non-adapted powdery mildew pathogens and RPM1(an NB-LRR)/AvrRpm1(the cognate effector)-triggered immunity     was detected within a few minutes after application of elicitors N,N,N,N-tetraacetylchitotetraose, xylanase, and flg22, which coincided with H2O2 production  before the biphasic ROS production was anticipated. It seems clear that PAMPs could trigger the first phase of PA production, but whether or not they can also induce the second phase remains to be determined.In tomato suspension-cultured cells, formation of PA  started in ~60 min which was followed by a second wave of PA production (via PLD route) occurred around 3~4 h after application of dexamethasone to induce expression of 2H4 Ppi4kIIIβ1β2 double mutant plants accumulated high levels of SA and ROS, constitutively expressed the PR-1 gene and showed enhanced resistance to P. syringae cellular levels of target lipid molecules, results from many individual studies are either descriptive in nature or fragmental. Future studies will be directed to defining the roles of signaling phospholipids and their metabolizing phospholipases in plant immunity by (i) using higher-order genetic mutants to circumvent functional redundancy, (ii) using novel tools and technologies to investigate the spatiotemporal dynamics of target molecules at the subcellular level, and (iii) studying of multiple defense signaling molecules in the same pathosystem.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.


CTR1, thus providing evidence of an alternative pathway from the ethylene receptors not involving CTR1. Here we highlight the evidence for receptor signaling to an alternative pathway and suggest that receptor signaling is coordinated via the N- and C-termini, as we address the biological significance of the negative regulation of ethylene signaling by the two pathways.The gaseous plant hormone ethylene is perceived by a family of ethylene receptors and mediates an array of ethylene responses. In the absence of ethylene, receptor signaling is conveyed via the C-terminal histidine kinase domain to the N-terminus of the CONSTITUTIVE TRIPLE RESPONSE1 (CTR1) protein kinase, which represses ethylene signaling mediated by ETHYLENE INSENSITIVE2 (EIN2) followed by EIN3. In the presence of ethylene, the receptors are inactivated when ethylene binds to their N-terminal domain, and consequently CTR1 is inactive, allowing EIN2 and EIN3 to activate ethylene signaling. Recent findings have shown that the ethylene receptor N-terminal portion can conditionally mediate the receptor signal output in mutants lacking  Arabidopsis, and a linear signal transduction pathway has been proposed  transcription factor gene  compared to the ctr1-1 mutant  lacking the CTR1 docking site, largely rescued the ctr1-1 and ctr1-2 mutant phenotypes and reduced the ERF1 transcript level. The ctr1-2 allele has a 17-bp deletion and probably encodes a truncated protein with 462 residues  consequently shows the typical constitutive ethylene response phenotype  does not associate with ethylene receptors and is detected in the cellular soluble fraction  that is phosphorylated by ETR1 prior to ethylene binding  and AHP1 is involved in a variety of biological processes  Ethylene binding to the receptors results in the dynamic dissociation of CTR1 from, and association of EIN2 with, the receptors; in this case, the ethylene receptors might directly mediate ethylene signaling to EIN2 , 2011. . That is, with ethylene treatment, seedling hypocotyl elongation of this mutant (with ein2 heterozygous) was inhibited to a lesser extent than that of ethylene-treated wild-type (Col-0) seedlings, with the ctr1-1 ein2 seedling hypocotyl elongation unresponsive to ethylene . Light-grown ctr1-1 EIN2/ein2 seedlings produced a weaker growth-inhibition phenotype than ctr1-1 with or without ethylene treatment, while ctr1-1 ein2 seedling growth was unresponsive to ethylene . The transcript level of the ethylene-dependent ERF1 gene in the ctr1-1 EIN2/ein2 mutant was slightly higher than in the ctr1-1 ein2 double homozygote but much lower than in the ctr1-1 mutant alone, and ethylene induction of ERF1 expression was higher in ctr1-1 EIN2/ein2 than ctr1-1 ein2 but much lower than in the wild type .In a genetic screen for suppressors of EIN2, the EIN2 protein level in ctr1-1 EIN2/ein2 may not be sufficient to fully induce the ethylene response to the same degree as ctr1-1. The mutation could be recessive or partially dominant because the heterozygote but not homozygote was responsive to ethylene. An important finding is that the ctr1-1 EIN2/ein2 mutant appears to be slightly responsive to ethylene . This slight responsiveness to ethylene could reflect a pathway that does not involve CTR1. Without an alternative pathway to activate the wild-type copy of EIN2, the ethylene response would not be expected.Sequence analysis revealed that a G1440A transition mutation caused a W308stop early termination of EIN2 in the newly isolated allele. The allele could conceivably produce a truncated EIN2 protein lacking the C-terminus for ethylene signaling and is likely a loss-of-function mutation. With a single-copy of wild-type We therefore propose that ethylene binding could prevent the receptor N-terminal signaling so that ethylene signaling is relieved from suppression. Alternatively, ethylene signaling could be mediated by the receptors via AHPs to ARRs or directly to EIN2 .ctr1-1 and ctr1-3 are slightly responsive to ethylene  In the receptor quintuple mutants, the default “ethylene signaling” mediated by EIN2 is not affected because of the absence of the receptor C- and N-terminal signaling, and the mutant shows an extremely strong constitutive ethylene response phenotype . (2) ctr1-8 does not dock at the receptors, and the N-terminal signaling is not inhibited; although the EIN2 C-terminus is theoretically released, ethylene signaling is nevertheless inhibited by the receptor N-terminal signaling, so that ctr1-8 shows a relatively weak constitutive ethylene response phenotype . (3) In mutants defective in CTR1 kinase activity , the mutant CTR1 proteins dock at the receptors to inhibit but not fully prevent receptor N-terminal signaling, whereas the EIN2 C-terminus is released for ethylene signaling . Thus, the ctr1 mutants produce a typical ethylene response phenotype that is weaker than the receptor quintuple mutant phenotype. (4) In ctr1-1 EIN2/ein2, receptor N-terminal signaling is inhibited; however, it is still sufficient to repress in part the ethylene signaling that is mediated by the EIN2 C-terminus, which exists at a reduced amount. Thus, the heterozygote shows a weaker phenotype than ctr1-1. Ethylene treatment inhibits the receptor N-terminal signaling, and the mutant is ethylene responsive.A proposed model for the dynamic receptor ethylene signaling via the two termini is described in 1-349 ctr1-1ETR1p:etr1-1 could potentially isolate the involved components. The isolation of several ENHANCED ETHYLENE RESPONSE (EER) genes suggests a resetting mechanism damping ethylene signaling by components involved in various biological processes (Little is known about the components involved in the possible alternative pathway of ethylene perceived by ethylene receptors and mediation of receptor N-terminal signaling; mutants isolated from an ongoing suppressor screen in our laboratory for rocesses . REVERSIrocesses , with itrocesses . The RTErocesses  is presuG1440Aein2 allele partially suppressed ctr1-1; evidence for an interaction of ETR1 N-terminus with EIN2 is required to strengthen this scenario. EIN3 and EIL1 are the prime transcription factors responsible for inducing an array of ethylene response genes (Ethylene receptor signaling via the C- and N-termini could act independently, with distinct signaling components and targets; alternatively, the signaling could be mediated to different components, converged at the same signaling component, and sharing a common downstream pathway. EIN2 might mediate ethylene signaling via the receptor N-terminus because ETR1 and EIN2 physically interact and a single copy of the se genes . The twoThe signaling components may be dynamically present in a pathway for an immediate response. EIN2 could be present in a steady-state interconversion between the phosphorylated and underphosphorylated state in the absence of ethylene treatment; alternatively, a small portion, but not most, of EIN2 could escape the phosphorylation by CTR1 to ensure immediate ethylene signaling. Conceivably, a level of constitutive ethylene signaling could occur, if not repressed, to trigger stronger ethylene signaling. Alternative receptor signaling could have a role in minimizing degrees of the basal-level ethylene signaling. The two-level control may facilitate a dynamic fine-tuning of ethylene signaling in response to a wide range of ethylene concentrations.EIN2/ein2 ctr1-1 mutant, Jingyi Zhang characterized the ethylene response, and Chi-Kuang Wen wrote the paper.Jing Yu isolated the heterozygous The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


We have shown that HTLV-1 p30 is critical for cell-free infection of primary dendritic cells (DCs) and that knockout of p30 in the HTLV-1 molecular clone p30KO-HTLV-1 decreases infectivity in rhesus macaques. In addition, p30 was recently shown to influence TLR signaling and expression of cellular genes involved in apoptosis, cell-cycle and transcription, suggesting that p30 plays a role in innate immune responses. Using the monocytic cell line THP-1 transduced with a p30-expressing lentiviral vector, we found that p30 inhibits type-I interferon (IFN) responses. While p30 did not affect the basal expression of type-1 IFN stimulated genes (ISGs), a significant decrease (50-80%) in mRNA levels of MxA, OAS and A3G was found following stimulation of p30-transduced cells with either polyI:C or LPS. HTLV-1-p30 did not affect ISG induction following Imiquimod stimulation, indicating that p30 affects specific signaling pathways. Importantly, these results were confirmed in primary monocyte-derived DCs. Infection of THP-1 cells with wild type (WT) HTLV-1 inhibits ISGs expression 10-50% lower than infection with p30 knockout virus. Moreover, THP-1 cells infected with p30KO-HTLV-1 produced lower levels of virus than WT-HTLV-1. Co-expression of p30 in p30KO-HTLV-1-infected cells rescued the inhibition of ISGs transcription and viral production. ChIP assays revealed that expression of p30 inhibited transcription of IFNα1, IFNβ and TLR4 following TLR stimulation by inhibiting PU.1 promoter binding. These results demonstrate that p30 protein is important for establishing HTLV-1 infection by dampening the antiviral response elicited by monocytes/macrophages and DCs, allowing persistence, replication and spread of HTLV-1 infection.


Grasslands are threatened globally due to the expansion of woody plants. The few remaining headwater streams within tallgrass prairies are becoming more like typical forested streams due to rapid conversion of riparian zones from grassy to wooded. Forestation can alter stream hydrology and biogeochemistry. We estimated the rate of riparian woody plant expansion within a 30 m buffer zone surrounding the stream bed across whole watersheds at Konza Prairie Biological Station over 25 years from aerial photographs. Watersheds varied with respect to experimentally-controlled fire and bison grazing. Fire frequency, presence or absence of grazing bison, and the historical presence of woody vegetation prior to the study time period (a proxy for proximity of propagule sources) were used as independent variables to predict the rate of riparian woody plant expansion between 1985 and 2010. Water yield was estimated across these years for a subset of watersheds. Riparian woody encroachment rates increased as burning became less frequent than every two years. However, a higher fire frequency (1–2 years) did not reverse riparian woody encroachment regardless of whether woody vegetation was present or not before burning regimes were initiated. Although riparian woody vegetation cover increased over time, annual total precipitation and average annual temperature were variable. So, water yield over 4 watersheds under differing burn frequencies was quite variable and with no statistically significant detected temporal trends. Overall, burning regimes with a frequency of every 1–2 years will slow the conversion of tallgrass prairie stream ecosystems to forested ones, yet over long time periods, riparian woody plant encroachment may not be prevented by fire alone, regardless of fire frequency. However, fire frequency, as well as climatic or edaphic conditions within a region, may tightly control recruitment and subsequent expansion of woody plant species across grasslands Other factors may greatly influence success of woody plant species growth and expansion, especially when coupled with fire interval. Large, ungulate herbivores in mesic grasslands  may reduce the spatial extent of burning or fire intensity via grazing and removal of graminoid species Terrestrial, grassland landscapes are globally subject to woody encroachment. However, North American tallgrass prairie streams are especially endangered because entire intact watersheds are even rarer than are remnant patches of prairie i) across all watersheds, riparian woody cover would increase over time, (ii) watersheds exposed to grazing would exhibit greater increases in woody cover relative to those that are ungrazed due to low spatial extent of burning during controlled fires, (iii) watersheds exposed to a high fire frequency would exhibit little woody expansion relative to those with a 4 or 20 year fire frequency. Lastly, we hypothesized that (iv) water yield would significantly decrease over 25 years due to the increase in cover of deeper rooting woody plant species.In this study, we evaluated the magnitude and direction of riparian forest expansion across tallgrass prairie watersheds exposed to variation in grazing and fire frequency treatments. Water yield was also assessed for 4 watersheds to determine if any trends in the proportion of water import to export differed with temporal changes in riparian woody plant cover. We hypothesized that  is a 3,487 hectare tallgrass prairie preserve and is part of the Flint Hills of northeastern Kansas. KPBS is privately-owned land by both the Natural Conservancy and Kansas State University. It is located ∼10 miles south of Manhattan, KS . KPBS granted an LTER permit (#200) for the work presented in this study. This study did not involve any protected or endangered species or involve collections of vertebrates. Any permission for research conducted at KPBS is approved through the Director of KPBS, John M. Briggs. Prescribed burning frequencies of variable intervals  began in 1972. The site uses individual watersheds as experimental units under variable grazing and fire treatments. In 1987, 50 bison were introduced to a 469 ha portion of Konza Prairie and were allowed to increase through herd reproduction and other introductions until 1992 when the bison-grazed area expanded to an additional 480 ha encompassing 10 watershed units differing in their burn frequencies 4 grasses . In downstream riparian areas, oak gallery forest dominates  U. americana) and honey locust (Gleditsia triacanthos) are prominent, and in ungrazed headwaters woody riparian areas are dominated by bur oak (Q. macrocarpa), and chinkapin oak (Q. muehlengbergii) Konza Prairie receives, on average, slightly more than 800 mm of precipitation annually, and receives 75% of its precipitation in late spring and early summer with high interannual variability http://kansasgis.org/catalog/index.cfm).Aerial images of Konza Prairie were taken during years 1985, 1991, and 2010. The 1985 images were originally flown to collect ∼1 m ground resolution data and were scanned to 200 dpi to avoid data loss. The 1985 aerial images were mosaicked and rectified using tools in ArcGIS . The 1991 aerial consists of 1 m ground resolution, black and white imagery available as part of the USGS digital orthoquad (DOQ). The 2010 aerial is part of the 2010 USDA National Agricultural Inventory Program (NAIP) and is available in color at a resolution of 1 m  was analyzed across the 3 years. While the 30 m width is somewhat arbitrary, it is within the range commonly assumed to have the greatest success in stream conservation A linear regression model was performed for each watershed separately using year  as the independent variable and percentage of wooded vegetation within the buffer as the dependent variable. The regression slope estimate was then used to represent the rate of wooded vegetation increase from 1985–2010. All slopes were used regardless of their statistical significance because we were interested in the direction and rate of change or lack thereof. Only using significant slopes would bias toward watersheds with large amounts of change and against watersheds where no change was evident.Using the non-parametric, 2 dimensional Kolmogorov-Smironov test The presence of grazers and whether riparian wooded vegetation was present prior to Konza Prairie were also used as predictor variables. Lastly, as a surrogate for proximity of propagule sources, a 1939 aerial image was used to visually distinguish the historic presence or absence of woody vegetation along streams within each watershed. This image was created in the same manner as the 1985 image. While the image quality was poorer than the more recent images used, the image did allow for determination of areas with high densities of large trees. Since the presence or absence of trees in the riparian zone of each watershed was a categorical variable, high precision in cover was not necessary for this analysis. This approach was taken because preliminary examination revealed some areas were largely void of woody vegetation , but other riparian corridors (primarily those in the northwest corner of current Konza Prairie) were already wooded before the site was established.If riparian wooded vegetation is increasing over time, water yield could also change temporally. Annual water yield was calculated for each of the 4 watersheds  that have long-term continuous discharge data spanning 1987 to 2010  segmented, and stats packages . Temporal autocorrelation for water yield estimates was tested via correlograms computed in the stats package as well  whereas Kolmogorov-Smironov tests were performed using Statistica .A multiple, linear regression model was performed for each watershed separately to determine if water yield changed over time and with average, annual air temperature. Finally, a one-way ANOVA was performed to determine if water yield differed among watersheds. A Tukey’s HSD test in conjunction with Bonferroni corrections was then used to determine watershed specific differences in water yield. All regression analyses and ANOVAs were performed in the R programming language using the Analyses of 30 m riparian buffers revealed an increase in wooded vegetation over time among all watersheds except one . Except for this watershed, all exhibited a positive  rate of woody expansion, regardless of fire frequency or historical presence of woody vegetation .2 = 0.45, F3,18 = 6.61; Linear regression models indicated that the cumulative number of burns between 1980 and 2010, and the historical presence of woody vegetation, significantly predicted the rate of riparian vegetation expansion  burns over the 25 years or at about 1.6 years between burns . However, N02B and N04D had greater water yield on average than N20B P<0.01; , Table 1Nearly all watersheds within this study have experienced riparian woody vegetation expansion since the establishment of Konza Prairie . None ofCornus drummondii, a common, clonal shrub species at Konza Prairie, forms “islands” which exclude grassy species C. drummondiiOur study indicates that the rate of riparian woody vegetation expansion is lessened with greater fire frequency, but even in annually burned watersheds, fires cannot prevent some encroachment of woody vegetation within riparian corridors , 3. SegmThe presence or absence of woody riparian vegetation prior to the beginning of Konza Prairie significantly predicted its rate of expansion . HistoriSurprisingly, bison did not have any effect on riparian woody plant cover over time. This is contrary to other studies that found bison greatly increase woody vegetation cover due to their preferential grazing on graminoid plant species thereby reducing spatial extent of burning and allowing for growth of woody plant species 4 grasses have 2 effects Our data suggest that under current management conditions on site, woody riparian vegetation will potentially continue to expand into riparian zones, regardless of burning regimes implemented. Anthropogenic impacts on the environment, such as increased atmospheric carbon dioxide concentrations, may erase the physiological advantages that CC. drummondii and Rhus glabra, use deeper soil water sources than C4 grass species We did not detect any change in water yield over time for any watersheds . The lowTo our knowledge, this is one of the first long-term experimental manipulations of fire at the watershed level to assess the expansion rates of woody, riparian vegetation in a grassland ecosystem. We found that riparian woody vegetation cover is rapidly increasing at Konza Prairie, and although high fire frequency may slow this process, it does not necessarily cease it from occurring. This study indicates that grassy riparian corridors will be maintained only with a minimum of 2 years between burns in tallgrass prairies. We suspect that similar relationships will occur in other grasslands and fire frequency may control riparian, woody expansion though the exact relationships we found may not hold. Although we could not detect any influence of this landscape alteration on stream water yield, the level of variance may make the effect non-detectable. Long-term data collections are mandatory to effectively link land use modification to stream ecosystem dynamics so perhaps additional hydrologic collections will clarify the relationship of woody expansion to prairie stream hydrology. Conservation and management of grassland streams across the globe may require similar considerations in cases where the native condition is grass-lined stream channels.


HTLV-1 associated myelopathy/tropical spastic paraparesis (HAM/TSP) is associated with a chronic inflammatory central nervous system in which the host immune response against HTLV-1 infection is considered immunopathogenic. We have applied a new unbiased deep sequencing method of TCR repertoire to accurately measure the diversity and degree of clonal expansion of peripheral circulating T-cells in patients with HAM/TSP compared to age, gender, and ethnicity matched healthy controls. TCR libraries were generated using 5'rapid amplification of cDNA ends to amplify the V-D-J genes of unsorted T-cells obtained from peripheral blood. Over 100 million reads were analyzed using MiGec software :653-5), that align and match the human TCR β-chain nucleotide sequences through IMGT database. T-cell clonotypes with high-quality CDR3 sequence were selected by a molecular identifier PCR strategy, which allowed for filtering PCR errors and correct for artificial sequences generated on a HiSeq 2000 Illumina system platform. By calculating the coefficient of variation of our dataset we considered clonal expansion as those unique TCR clonotypes showing reads >8. While we did not observe any significant statistical differences of the number of single clones  between HAM/TSP patients and healthy controls groups, by contrast, a higher clonal expansion of the T-cell repertoire was observed in HAM/TSP patients when compared to healthy controls (unpaired parametric T-test with P=0.026). Interestingly, this clonal expansion of the HAM/TSP TCR repertoire correlated with the HTLV-I provirus load  . Our findings strongly suggest that in this chronic neurological disorder, a strong dysregulated T-cell response may be driven by HTLV-I and that strategies to decrease virus may be of clinical benefit.


Most organisms rely on innate immune receptors to recognize conserved molecular structures from invading microbes. Two essential innate immune receptors, RIG-I and MDA5, detect viral double-stranded RNA in the cytoplasm. The inflammatory response triggered by these RIG-I-like receptors (RLRs) is one of the first and most important lines of defense against infection. RIG-I recognizes short RNA ligands with 5′-triphosphate caps. MDA5 recognizes long kilobase-scale genomic RNA and replication intermediates. Ligand binding induces conformational changes and oligomerization of RLRs that activate the signaling partner MAVS on the mitochondrial and peroxisomal membranes. This signaling process is under tight regulation, dependent on post-translational modifications of RIG-I and MDA5, and on regulatory proteins including unanchored ubiquitin chains and a third RLR, LGP2. Here, we review recent advances that have shifted the paradigm of RLR signaling away from the conventional linear signaling cascade. In the emerging RLR signaling model, large multimeric signaling platforms generate a highly cooperative, self-propagating, and context-dependent signal, which varies with the subcellular localization of the signaling platform. Eukaryotic organisms rely on their innate immune system to detect viruses and other microbes. Innate immune receptors detect chemical patterns or structures that are broadly conserved in microbes, including bacterial cell wall components, microbial nucleic acids, and certain highly conserved proteins. These pathogen-associated molecular patterns (PAMPs) are recognized by pattern recognition receptors that fall into several families, including Toll-like receptors (TLRs), NOD like receptors (NLRs), C-type lectin receptors (CLRs), and RIG-I-like receptors (RLRs). At the cell surface and in endocytic compartments, TLRs are the most important family of molecular sentries for the innate immune recognition of a wide range of microbial patterns outside the cytosol . CLRs, sNucleic acids are the largest, and arguably the most important class of ligands for innate immune receptors. To avoid signaling in response to endogenous nucleic acids, which are ubiquitous in the cytoplasm and nucleus, innate immune sensors must recognize specific patterns in specific subcellular locations.  A subfamMany viruses deliver an RNA genome into the cytoplasm or rely on a replication or transcription step that generates viral RNA in the cytoplasm. Infection by these viruses is primarily detected by RIG-I and MDA5, also referred to as the RLRs. RIG-I and MDA5 sense complementary sets of viral RNA ligands –12, 15. Recent biochemical, biophysical, and cellular studies have greatly advanced our understanding at the molecular level of the mechanisms of pattern recognition and signaling by RIG-I and MDA5. Here, we review these studies and their implications on the current models of microbe-induced inflammation, auto-inflammation, and inflammation-induced cancer.RIG-I preferentially binds to short (<300 bp) dsRNAs that have blunt ends and a 5′ triphosphate (5′-ppp) moiety, facilitating discrimination between host and viral dsRNA –12. CrysUpon the presentation of a viral dsRNA, RIG-I undergoes significant conformational rearrangement. The CTD binds tightly to the 5′-ppp and the helicase domains wrap around dsRNA, adopting a more compact configuration –18 FiguB. RIG-I In contrast to RIG-I, MDA5 preferentially binds internally to long dsRNA  with no end specificity –12 and cA crystal structure of the MDA5 helicase domains and CTD bound to dsRNA revealed how MDA5, despite having a similar domain architecture as RIG-I, recognizes dsRNA in a different manner Figure B. The heThe RLRs are part of the DExD/H-box helicase family based on their domain architecture , but theLGP2, the third RLR, has similar helicase and CTD domains as RIG-I and MDA5, but it lacks the tandem CARDs . LGP2 rein vitro and demonstrated that unanchored K63-linked polyubiquitin chains are required for a full signaling response as measured by IRF3 dimerization . K. K35). KAlthough ubiquitin chains promote RIG-I tetramerization, RIG-I and MDA5 can both assemble into oligomeric filaments and induce MAVS filament formation and signaling in the absence of polyubiquitin chains. Indeed, under certain experimental conditions, namely in the absence of polyubiquitin and as a result of ATP hydrolysis, RIG-I has been observed to form filaments along dsRNA , 39, 44.in vitro . I. I36). IThe inflammatory response resulting from RLR signaling unavoidably occurs at a cost to normal tissue function. Multiple regulatory mechanisms have evolved to allow rapid activation, amplification, and inactivation of RLR signaling, and to achieve the optimal trade-off between the cost and benefit of the inflammatory response . PolyubiIn addition to ubiquitination, phosphorylation is slowly emerging as an important regulatory mechanism for RLR signaling. Phosphorylation of Ser8 and Thr170 in the CARDs of RIG-I antagonizes RIG-I signaling , 54. BasBesides post-translational modification of the RLRs, RLR signaling is also modulated by several different proteins, derived both from the host and from pathogens. One such protein is the third RLR, LGP2. Because it lacks CARDs, LGP2 cannot activate MAVS; however, its ability to recognize dsRNA allows it to modulate the signaling capacities of RIG-I and MDA5. LGP2 downregulates signaling by RIG-I , 33. ThiThe seemingly contradictory roles of LGP2 in RLR signaling remain an open question. The experimental approaches used to study LGP2 in relation to MDA5 and RIG-I have been different, potentially explaining some of the differences. As evidence accumulates for the opposing roles of LGP2 on RLR signaling, however, the emerging perspective is that LGP2 can control the balance between RIG-I and MDA5 responses during viral infection.Pathogen evasion tactics against RLR-mediated immune response are extensive and occur at every level of signaling [reviewed in Ref. ]. A compRIG-I and MDA5 are the principal sensors of viral dsRNA in the cytoplasm. The interferon-dependent inflammatory response triggered by RLR ligand binding is one of the first and most important lines of defense against infection. RIG-I and MDA5 recognize distinct and complementary sets of viral dsRNA ligands. The molecular signaling mechanisms of RIG-I and MDA5 differ in some respects but also share certain key features. Differences include the sequestration of CARDs by RIG-I but not by MDA5 in the absence of ligand, the much greater propensity of MDA5 to form filaments along dsRNA, and the different contribution of K63-linked ubiquitin chains, which remains poorly defined for MDA5. Common features in RLR signaling include proximity-induced assembly of CARD oligomers, which serve as platforms to nucleate MAVS CARD polymerization, and signal amplification through the amyloid-like properties of the MAVS CARD. Together, the recent advances reviewed here shift the paradigm of RLR signaling away from the prototypical linear signaling cascade to a model in which signaling is activated by the cooperative assembly of an oligomeric signaling platform. The signal output depends on the cellular localization of MAVS (mitochondria or perixosome), and signaling is finely regulated by a multitude of cellular and pathogen-derived factors. Key outstanding questions include when, where, and how ubiquitin chains potentiate RIG-I and MDA5 signaling, exactly how RLRs interact with MAVS, and how LGP2 and other factors modulate RLR signaling.○Is the mechanism of action of K63-linked ubiquitin chains the same for RIG-I and MDA5?Do K63-linked ubiquitin chains always participate in RLR signaling, or are they only required under specific physiological conditions that do not favor RLR filament formation?○How do RIG-I CARD tetramers, stabilized by K63-linked ubiquitin, nucleate MAVS filament assembly?○How do MDA5 CARDs nucleate MAVS filament assembly? Does this process require K63-linked ubiquitin chains?What are the molecular and structural bases of MAVS activation by RLR oligomers?What are the underlying molecular mechanisms for the opposite activities of LGP2 on RIG-I and MDA5 signaling?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.


Hepatozoon invade the blood cells of many mammalian species, being transmitted by range of arthropods. In domestic carnivores, three species of Hepatozoon were described to date: H. canis and H. americanum in dogs and H. felis in cats. The classification of Hepatozoon in wild carnivores is still not complete due to lack of field and experimental data as well as phylogenetic studies. The aim of this study is to carry out a survey on the prevalence and diversity of Hepatozoon sp. in red foxes Vulpes vulpes. Samples of tissues were collected from dead foxes in 11 counties of Romania; 91 samples of liver tissue were examined in total. DNA extraction was performed with commercial kit according to the manufacture's protocol. Hepatozoon sp. DNA was detected by PCR using primers amplifying 400-600 bp long part of 18S rRNA gene. These primers are commonly used for diagnostic purposes in dogs and in some studies also for detection of Hepatozoon sp. in wild carnivores. PCR products were sequenced to validate positive results of reaction. DNA of parasite was confirmed in 55% of examined samples. Recent findings classified Hepatozoon sp. in foxes and other wild canids in Europe as H. canis. However, Rhipicephalus sanguineus, the only known vector of H. canis in Europe, is absent in most of our sampling sites. Moreover, this tick is typical for the dogs but rare or even absent in foxes. In order to clarify the identity of the parasite, the next step of our study is to focus on amplification of longer or full segment of 18s rRNA gene to allow more accurate phylogenetic analyses and comparison with H. canis sequences from dogs and Hepatozoon isolates from other carnivores.Apicomplexan parasites of genus This study was supported by IGA UVPS Brno, project 115/2013/FVL.


Among its many effects, the virus is able to stimulate the production of thymic stromal lymphopoietin (TSLP) by infected hepatocytes. In this study, we assessed the systemic levels and tissue distribution of TSLP in 60 chronically HCV-infected patients, 36 with and 24 without CV.Hepatitis C virus (HCV) infection can be detected in virtually all patients with cryoglobulinemic vasculitis Serum TSLP levels were measured by an enzyme-linked immunosorbent assay (ELISA) method. TSLP mRNA was assessed in patient samples by real-time reverse transcriptase-polymerase chain reaction (RT-PCR). TSLP protein in liver and skin biopsy samples was revealed by indirect immunofluorescence. All other methods were carried out according to standardized procedures.+ B-cells (10.3% vs. 15.5% p = 0.04). In addition, TSLP mRNA expression in the liver of CV patients was lower than in their correspondent skin tissue and paralleled specific immune deposits of TSLP protein in keratinocytes.Serum TSLP levels were significantly higher in patients with than in those without CV and in healthy individuals. Higher TSLP levels paralleled specific mRNA expression and the up-regulation of TSLP protein in liver tissue. Compared with non-CV patients, higher TSLP levels in CV were accompanied by a higher frequency of circulating mono/oligoclonal B-cell expansions  and a higher number of peripheral CD20Overall, this study shows that TSLP secreted by hepatocytes and keratinocytes of HCV-infected patients with CV is involved in the pathogenesis of vasculitis and may possibly support the therapeutic use of TSLP-targeted monoclonal antibodies. Thymic stromal lymphopoietin (TSLP) is a four-helix-bundle cytokine and a member of the common γ-chain cytokines, which are able to induce dendritic cells (DCs) and to stimulate naïve T-cell differentiation into T-helper 2  and T-heTSLP is a potent modulator of systemic B-cell development and is capable of promoting humoral autoimmunity. In the skin of a genetically engineered mouse, TSLP released into the systemic circulation by Notch-deficient keratinocytes induced a remarkable expansion of peripheral pre-B cells and immature B lymphocytes, resulting in B-lymphoproliferative disorders and death . In addiIn the Mediterranean basin, over 90% of CV patients are chronically infected with hepatitis C virus (HCV), thus emphasizing the role of this virus in the pathogenesis of cryoglobulin production. However, only a subset of HCV-positive individuals develops mixed cryoglobulins and only a minority of these patients has clinically overt CV . B-cell in vitro by HCV results in a remarkable production of TSLP [Recently, it has been reported that the infection of hepatocytes  of TSLP  through  of TSLP , it can in vivo inducible upregulation of TSLP can be shown in patients with chronic HCV infection and CV. A possible relationship between TSLP and HCV nucleocapsid core protein, devoid of envelope proteins, as a constitutive component of cryoglobulins and potentially able to cause cryoglobulin-mediated tissue injury [Here, we asked whether an e injury  was alsoThirty-six naïve patients with a diagnosis of CV and the classical symptom triad of palpable purpura, arthralgia, and asthenia were enrolled in this study. Eligibility criteria were as follows: no previous therapy with interferon, steroids, or immunosuppressive drugs; serum positivity for anti-HCV antibodies and HCV RNA; liver histology compatible with chronic active hepatitis; negativity for serum HBsAg and anti-HIV antibodies; alcohol intake <40 g/day; and currently not pregnant. The study was approved by the Independent Ethical Committee ; written, informed consent was obtained from all patients, in accordance with the declaration of Helsinki.Serum cryoglobulins were isolated and characterized as described elsewhere . In addiPatients were treated with pegylated interferon alpha/ribavirin (pIFNα/RBV) combination therapy. Those infected with genotype 2 and genotype 3 were treated for 6 months, and those infected with genotype 1 for 12 months. Patients were reassessed 6 months after discontinuation of therapy to establish whether they had achieved a sustained virological response.Virological, epidemiological, histological, and laboratory features as well as major symptomatology are summarized in Table Serum HCV antibodies were detected by second-generation enzyme-linked immunosorbent assay and then confirmed by third-generation enzyme-linked immunosorbent assay  in the first 18 patients enrolled in this study. The last method was consistently employed in the remaining 42 patients. Serum HCV RNA was determined by RT-PCR  and quantified with the Versant HCV RNA quantitative 3.0 assay . HCV genotyping was performed with INNO-LiPA .Immunoreactive TSLP in serum samples was quantified using an enzyme-linked immunosorbent assay with matched antibodies, according to the basic laboratory protocol provided by the manufacturer . TSLP protein was quantified with reference to serial dilutions of the recombinant standards, falling within the linear part of the standard curve for each specific TSLP value measured. The sensitivity threshold of the TSLP assay is 6.7 pg/ml. Each data point represents readings from two independent assays performed in duplicate.Serum nonenveloped HCV core protein was tested using Architect HCV Ag , a fully automated, quantitative, chemiluminescent microparticle immunoassay for the detection of nonenveloped HCV core protein in HCV-infected sera .Total RNA was extracted from liver and skin samples with the RNeasy Mini kit , according to the manufacturer’s protocol, and reverse transcribed to cDNA using the iScript Select cDNA synthesis kit . Five microliters of cDNA were used in a nested PCR for HCV RNA detection together with the AmpliTaq Gold PCR master mix  and the following primer sets targeting the 5′-untranslated region: sense 5′-GGGGGCGACACTCCACCA-3′ (position 15 to 32) and anti-sense 5′-TCGCGACCCAACACTACTC-3′ (position 256 to 274) for the first round of amplification; and the inner primers sense 5′-GAGTGTCGTGCAGCCTCCAG-3′ (position 98 to 117) and anti-sense 5′-CTCGGCTAGCAGTCTCGCGG-3′ (position 239 to 258) for the second round.An additional PCR for TSLP mRNA was carried out using 5 μl cDNA, the AmpliTaq Gold PCR master mix (Applied Biosystems), and the QuantiTect human TSLP primer assay (Qiagen). This primer set targets exons 1/2/3 and amplifies a region of 94 base pairs in the two transcripts .TSLP mRNA was measured in patient samples by absolute real-time RT-PCR quantification. The reaction was performed using the QuantiTect human TSLP primer assay (Qiagen) and FastStart DNA Master SYBR Green I  on a LightCycler 1.5 instrument (Roche Diagnostics), according to the manufacturer’s protocol. The cycling conditions were 95°C for 15 minutes, 45 cycles of 94°C for 15 seconds, 55°C for 20 seconds, and 72°C for 20 seconds. The final concentration of TSLP mRNA was expressed as copies of mRNA per nanogram of total RNA.Real-time RT-PCR for HCV RNA quantitation was performed using the RNA Master HybProbe kit (Roche Diagnostics) and the following primers and probes: 5′-AGCGTCTAGCCATGGCGT (sense), 5′-CAAGCACCCTATCAGGCAGT (antisense), 5′-LC640-CCCGGGAGAGCCATAGTGGTCTG--PH (5′LCRed640) and 5′-GCAGCCTCCAGGACCCCCC--FL (3′HCV-G-Fitch) . To generate a standard curve, serial dilutions of the AcroMetrix HCV Mid Control  were prepared. The HCV RNA concentration was calculated as International Units per nanogram of total RNA; the sensitivity was 20 IU. Each sample and standard curve point was run in duplicate.Escherichia coli competent cells (Promega). The transformants were plated onto duplicate LB/ampicillin/IPTG/X-Gal plates, incubated overnight at 37°C, and then selected and processed for plasmid isolation. Five milliliters of Luria-Bertani broth cultures of single colonies were grown overnight at 37°C with shaking at 200 rpm. The DNA plasmid was purified using the QIAprep spin miniprep kit (Qiagen) and then solubilized in 100 μl of 10 mM Tris–HCl (pH 8.5) buffer. To confirm the sequence of the cloned product, DNA sequencing was performed using the BigDye Terminator v1.1 cycle sequencing kit and ABI Prism 310 genetic analyzer (both Applied Biosystems). The purified plasmid was quantified and the copy number calculated as follows:The TSLP standard was constructed by cloning product of real-time RT-PCR obtained using a bioinformatically verified primer assay . The PCR product was visualized on a 2% agarose gel and the respective band was excised and loaded onto a DNA purification column . The purified PCR product was cloned into a pGEM-T Easy vector using pGEM-T Easy vector system II , according to the manufacturer’s protocol. The recombinant vector was transformed into JM109 23 (molecules/mole) is Avogadro’s number and 660 (g/mole) is the average weight of a single base pair. The standard curve was prepared from five-point serial 10-fold dilutions, starting from 300,000 copies.where 6,023 × 102 fragment  as the secondary reagent. The latter was incubated with the samples for 2 hours at room temperature. To block a positive reaction on selected samples, the primary antibody was preincubated with human recombinant TSLP protein (5 ng/ml).TSLP protein in liver and skin biopsy samples was detected by indirect immunofluorescence, using a goat anti-human TSLP antibody  at a working concentration of 10 μg/ml. As a positive control, human tonsil sections were employed. The samples were incubated with the primary antibody for 3 hours at room temperature. Isotype-matched antibodies were used for control staining and a fluorescein isothiocyanate-conjugated rabbit anti-goat antibody F(ab)A two-stage indirect immunofluorescence procedure was carried out as described previously , using aThe immunoglobulin heavy chain variable-diversity-joining gene segments were amplified in duplicate reactions according to the Fr3 protocol, as described elsewhere ,10. ConsP <0.05. A Spearman rank correlation coefficient (r) test was used to evaluate the relationships between variables. All statistical analyses were performed using IBM SPSS statistics version 19.0 .Descriptive statistics included the mean or median as appropriate for continuous variables, and the frequency (%) for categorical variables. In the univariate analysis, chi-square and Fisher’s exact tests were used as appropriate to compare categorical variables, and the nonparametric Mann–Whitney test to compare continuous variables. The differences were considered significant at P <0.0001). The mean percentage of CD20+ B cells was higher in CV than in non-CV patients (P = 0.04).As shown in Table There was a strikingly higher number of female patients with mixed cryoglobulinemia . This reflects the higher female prevalence in all major cohorts of CV – including our own series of 141 patients, 68% of whom were females . HoweverAmong the clinical symptoms of CV patients, palpable purpura, asthenia, and arthralgia/arthritis were recorded with high frequency. Peri-malleolar and/or pre-tibial cutaneous ulcers were also found in over one-half of the patients. Long-lasting hyperpigmentation affecting the legs to a variable extent, as the result of recurrent purpuric eruptions, was also almost invariably present. Skin histology showed leukocytoclastic vasculitis in 32 patients (89%), whereas pandermal vasculitis characterized by vessel thrombosis and severe endothelial alterations was diagnosed in the remaining four patients (11%). Sensory-motor peripheral neuropathy was demonstrated in one-half of the patients, in whom electromyography prevalently revealed symmetrical, distal polyneuropathy and, less frequently, mononeuritis multiplex. Renal damage was diagnosed in 25% of the patients, with renal biopsy showing membrano-proliferative glomerulonephritis in 78% and membranous nephropathy with segmental glomerulosclerosis in the remaining 22%.TSLP-specific immune reactive material was demonstrated in 31 of 36 (86%) and 19 of 24 (79%) liver tissue specimens from CV and non-CV patients, respectively. As shown in Figure TSLP and HCV core proteins were studied in skin biopsy samples from all CV and non-CV patients and from healthy controls. The results showed that TSLP protein was mostly restricted to the basal and suprabasal layers of the epidermis Figure A,B,C in Notably, although TSLP mRNA expression was demonstrated in the skin samples of CV and non-CV patients and in those of healthy controls, HCV RNA amplification products were present in only two (6%) of 36 skin specimens from CV patients and in none of those from either the non-CV patients or healthy individuals.P <0.0001), but in none of the controls (P <0.0001). HCV core protein was detected in the skin tissue of 29 (81%) CV patients and two (8%) non-CV patients (P <0.0001). Conversely, all of the skin biopsies from healthy subjects tested negative for the immune detection of both TSLP protein and HCV core protein.The relationship between TSLP protein, specific mRNA expression, and the occurrence of HCV core protein or viral RNA is summarized in Table P <0.0001) of CV patients. In sharp contrast, TSLP gene expression was significantly downregulated in the skin compared with the liver in non-CV patients, with a median of 0.29  mRNA copies/ng total RNA and 3.3  mRNA copies/ng total RNA (P <0.0001), respectively. TSLP gene expression was thus strikingly different in the skin from CV patients compared with that from non-CV patients (P <0.0001).Potential differences in TSLP gene regulation between specific biologic compartments were searched for by measuring mRNA expression levels in the skin and in the corresponding liver tissue samples of CV and non-CV patients Figure . A mediaP <0.0001 in both cases).Basal serum levels of TSLP protein were measured in all HCV-positive CV and non-CV patients and in all healthy subjects. At the time of serum TSLP measurement, none of the HCV-infected patients was receiving specific antiviral treatment and/or immunosuppressive drugs. TSLP levels were significantly higher in CV patients  than in either non-CV patients  or healthy subjects  (P = 0.02), C1q (P = 0.003), C3 (P = 0.0001), and number of peripheral CD20+ cells (P = 0.0009). On the contrary, there was no relationship with mean age of the patients, circulating HCV RNA or cryoprotein levels, rheumatoid factor, serum concentrations of IgM and C4 complement component, and alanine aminotransferase activity.Spearman’s rank correlation test was used to assess the strength of the association between basal TSLP concentrations and virologic and laboratory parameters Table . A positChanges in the serum TSLP levels were investigated following therapy in all CV patients. Baseline serum levels of this cytokine were slightly but not significantly lower in 16 responder patients 44.4%) compared with 20 nonresponder patients (55.6%). In the first group, the administration of pIFNα/RBV combination therapy resulted in a dramatic improvement of cryoglobulin-related signs and symptoms, a remarkable decrement of the cryocrit, and the disappearance of serum HCV RNA. This complete response was found to be associated with a significant drop in TSLP serum levels .An important point of our study is the detection of a significant reduction of TSLP serum levels in patients responsive to pIFNα/RBV combination therapy, probably reflecting the appropriate blockade of TSLP release by hepatocytes and keratinocytes. Conversely, the unchanged levels in unresponsive patients suggest persisting HCV-mediated induction of TSLP production that contributes to immune system activation. Although the addition of the anti-CD20 monoclonal antibody rituximab to the pIFNα/RBV combination has been shown to result in higher long-term response rates , rituximIn a proof-of-concept study, the administration of a fully human anti-TSLP monoclonal antibody G2λ to a small cohort of patients with stable allergic asthma resulted in a significant attenuation of allergy-induced early and late asthmatic responses . No therThis study points out a novel biological role for TSLP. Cutaneous expression of TSLP by the keratinocytes of patients with HCV-related CV may be pivotal in perpetuating the activation of tissue-resident immunocytes and in attracting inflammatory cells, thus creating an environment favorable to the onset of vasculitis. The local production of TSLP by hepatocytes as well as keratinocytes causes a significant increase in its systemic concentrations, a step sufficient to promote a break in B-cell homeostasis and likely to induce B-lineage-dependent autoimmunity.+ T cells and of promoting proliferation and T-helper 2 differentiation of naïve CD4+ T cells through the induction of interleukin-4 [Further studies are necessary to evaluate the molecular basis underlying TSLP upregulation and B-cell derangement in patients with HCV-related CV. Although a deeper comprehension of cell-specific signaling pathways awaits to be defined, given that TSLP has been found capable of directly acting on CD4leukin-4 , it seem


Moreover, HBZ-specific antibody inhibited lymphoproliferation in PBMC of HAM/TSP patients. This is the first report demonstrating humoral immune response against HBZ associated with HTLV-I infection. Thus, humoral immune response against HBZ might play a role in HTLV-I infection.Human T cell lymphotropic virus type I (HTLV-I) infection can lead to development of adult T cell leukemia/lymphoma (ATL) or HTLV-I-associated myelopathy/tropical spastic paraparesis (HAM/TSP) in a subset of infected subjects. HTLV-I basic leucine zipper factor (HBZ) gene has a critical role in HTLV-I infectivity and the development of ATL and HAM/TSP. However, little is known about immune response against HBZ in HTLV-I-infected individuals. In this study, we examined antibody responses against HBZ in serum/plasma samples from 436 subjects including HTLV-I seronegative donors, asymptomatic carriers (AC), ATL, and HAM/TSP patients by the luciferase immunoprecipitation system. The immunoreactivity for HBZ was detected in subsets of all HTLV-I-infected individuals but did not discriminate between AC, ATL and HAM/TSP. However, the frequency of detection of HBZ-specific antibodies in the serum of ATL patients with the chronic subtype was higher than in ATL patients with the lymphomatous subtype. Antibody responses against HBZ were also detected in CSF of HAM/TSP patients with anti-HBZ in serum. Antibody responses against HBZ did not correlate with proviral load and HBZ mRNA expression in HAM/TSP patients, but the presence of HBZ-specific response was associated with reduced CD4