Blood stasis syndrome (BSS) has been considered to be the major type of syndromes in unstable angina (UA) patients. The aim of this study was to find the systems biology-based microRNA (miRNA) and mRNA expression biomarkers for BSS of UA. We identified 1081 mRNAs and 25 miRNAs differentially expressed between BSS of UA patients and healthy controls by microarrays. We used DAVID, miRTrail, and the protein-protein interactions method to explore the related pathways and networks of differentially expressed miRNAs and mRNAs. By combining the results of pathways and networks, we found that the upregulation of miR-146b-5p may induce the downregulation of CALR to attenuate inflammation and the upregulation of miR-199a-5p may induce the downregulation of TP53 to inhibit apoptosis in BSS of UA patients. The expression patterns of miR-146b-5p, miR-199a-5p, CALR, and TP53 were confirmed by qRT-PCR in an independent validation cohort including BBS of UA patients, non-BBS of UA patients, and healthy controls. miR-146b-5p, miR-199a-5p, CALR, and TP53 could be significant biomarkers of BSS of UA patients. The systems biology-based miRNA and mRNA expression biomarkers for the BSS of UA may be helpful for the further stratification of UA patients when deciding on interventions or clinical trials. Unstable angina (UA) constitutes a clinical syndrome subset of the acute coronary syndrome (ACS) and is associated with an increased risk of cardiac death and subsequent myocardial infarction (MI) . UA is dXueyu” in Chinese Mandarin) is one of the key pathogenesis of UA. This has been confirmed by a binary logistic regression analysis of a multicenter prospective research on TCM Syndromes in 815 cases of UA [Traditional Chinese medicine (TCM) is rapidly gaining attention in the world as sources for discovering new cardiovascular drugs –9. Howeves of UA –21. The es of UA , 22. TheAmong the classic teachings of TCM, it is stated that “when blockage is opened, pain is relieved; when blockage is not opened, pain persists.” If a UA patient has BSS, the treatment should be promoting blood circulation and removing blood stasis (PBCRBS). The concept of PBCRBS is similar to the modern revascularization method, but it is a kind of macroscopic thinking achieved by drugs rather than microscopic mechanism. The benefit of PBCRBS in UA patients with BSS has been demonstrated by increasing clinical evidences in recent years , 24. It in vitro and in vivo models have revealed pathogenic and protective functions of miRNAs; therefore they emerge as interesting novel candidates for the development of miRNA-based therapeutic strategies in cardiovascular disease [MicroRNAs (miRNAs) are endogenous, nonprotein-coding, single-stranded, small RNAs that are generally regarded as negative regulators of gene expression by inhibiting translation and/or promoting messenger RNA (mRNA) degradation . In vari disease . Besides disease . The disThe purpose of this study was to investigate circulating miRNAs' patterns of BSS of UA patients by a systems biology approach. MiRNA and mRNA expression profilings of peripheral blood mononuclear cells (PBMCs) of BSS of UA patients were compared to ones of PBMCs of healthy controls to identify the differentially expressed miRNAs and mRNAs by using a gene expression oligonucleotide microarray and a microRNA microarray. Bioinformatics analysis was used to find critically deregulated miRNAs and mRNAs involved in pathogenesis of BSS of UA and potential biomarkers. The expression patterns of miRNA and mRNA biomarkers in BSS of UA patients were independently validated by real-time quantitative polymerase chain reaction (qRT-PCR) in an independent validation cohort including BBS of UA patients, non-BBS of UA patients, and healthy controls. n = 65) had experienced ischemic chest pain within the preceding 48 h, including angina pectoris with an accelerating pattern, or prolonged duration (>20 min), or recurrent episodes at rest or within minimal effort, but with no evidence of enzymatic criteria. Transient ST-T segment depression and/or T-wave inversion were present in all cases. In patients undergoing percutaneous coronary intervention, all blood samples were taken before this procedure.Patients with UA undergoing clinically indicated coronary angiography were consecutively recruited into the study in Guang, Anmen Hospital, Beijing, China. The diagnosis of coronary artery disease (CAD) was confirmed in all patients by coronary angiography showing at least one vessel disease . UA patients were eligible to participate if they had met the American College of Cardiology/American Heart Association (ACC/AHA) criteria for UA . All UA UA patients were diagnosed as BSS or non-BSS according to the grading system in quantifying BSS diagnosis standards. It had 33 items for diagnosis, including symptoms, signs, and laboratory tests . Each itn = 20) were healthy volunteers, recruited from the same population and the same area of China as the patients' group. Total RNAs isolated from 5 BSS of UA patients and 5 healthy volunteers were used for gene expression oligonucleotide microarray and microRNA microarray. The results obtained from bioinformatics analysis of microarray were then prospectively tested in a validation cohort of 30 BSS of UA patients, 30 non-BSS of UA patients, and 15 healthy volunteers. The study complied with the Declaration of Helsinki and was approved by the local ethics committee. All individuals gave their written informed consent to participate in the study.Patients who had received thrombolytic therapy in the previous month were excluded from the study. Patients with stable angina, MI, heart failure, valvular heart disease, dilated cardiomyopathy, malignant tumor, advanced liver disease, renal failure, autoimmune diseases, and other inflammatory diseases and women who were pregnant and breast-feeding were excluded from the study. Control subjects (Whole blood samples (10 mL) were drawn from each of the 85 participants  with 19-gauge needle for clean venipuncture of an antecubital vein on the following morning after arrival. Timing of phlebotomy of UA patients compared with onset of chest pain was <24 h. Blood was drawn into EDTA-containing tubes and PBMCs were isolated by performing density gradient centrifugation with Ficoll . Total RNAs were extracted from PBMCs using using Trizol reagent (Invitrogen) according to the manufacturer's instruction. RNA quantity and purity were assessed using NanoDrop ND-1000 . Pass criteria for absorbance ratios were established at A260/A280 ≥ 1.8 and A260/A230 ≥ 1.5 indicating acceptable RNA purity. RNA Integrity Number (RIN) values were ascertained using Agilent RNA 6000 Nano assay . Pass criteria for RIN value were established at ≥6 indicating acceptable RNA integrity. Genomic DNA contamination was evaluated by gel electrophoresis. The RNA samples were stored at −80°C until analysis.μg total RNA samples using OneArray Amino Allyl aRNA Amplification Kit  and Cy5 dyes . Fluorescent targets were hybridized to the Human Whole Genome OneArray with Phalanx hybridization buffer using Phalanx Hybridization System. After 16 hrs hybridization at 50°C, nonspecific binding targets were washed away by three different washing steps , and the slides were dried by centrifugation and scanned by Axon 4000B scanner . The intensities of each probe were obtained by GenePix 4.1 software (Molecular Devices). The raw intensity of each spot was loaded into Rosetta Resolver System  to process data analysis. The error model of Rosetta Resolver System could remove both systematic and random errors form the data. Those probes with background signals were filtered out. Probes that passed the criteria were normalized by 50% median scaling normalization method. Normalized spot intensities were transformed to mRNA expression log2 ratios between the UA patients with BSS and healthy controls. Total RNAs of 10 participants  were used for mRNA expression profiling by the Human Whole Genome OneArray v5 . It contained 30275 DNA oligonucleotide probes, and each probe was a 60-mer designed in the sense direction. Among the probes, 29187 probes corresponded to the annotated genes in RefSeq v38 and Ensembl v56 database. Besides, 1088 control probes were also included. Fluorescent aRNA targets were prepared from 1 or 2.5 μg total RNA samples and labeled with miRNA ULS Labeling Kit . Labeled miRNA targets were hybridized to the Human miRNA OneArray v4 with OneArray Hybridization System. After 16 hrs hybridization at 37°C, nonspecific binding targets were washed away by three different washing steps , and the slides were dried by centrifugation and scanned by an Axon 4000B scanner (Molecular Devices). The Cy5 fluorescent intensities of each probe were analyzed by GenePix 4.1 software (Molecular Devices). The raw intensity of each probe was processed by R program. Probes that passed the criteria were normalized by 75% median scaling normalization method. Normalized spot intensities were transformed to miRNA expression log2 ratios between the UA patients with BSS and healthy controls. We performed miRNA expression profiling in the same set of samples  that were used in the analysis of mRNA microarray. MiRNA microarray analysis was performed by using the Human miRNA OneArray v4 . It contained triplicated 1884 unique miRNA probes from Human (miRBase Release v18) each printed in technical triplicate, and 144 experimental control probes. Small RNA was preenriched by Nanoseplook  from 2.5 2 ratio ≥ 1 or log2 ratio ≤ −1 and P value < 0.05 were defined as differential mRNAs. The expressions of miRNAs with log2 ratio ≥ 0.8 or log2 ratio ≤ −0.8 and P value < 0.05 were defined as differential miRNAs. Hierarchical clustering analysis combined with a heatmap was applied to evaluate the overall reproducibility and variation of  5 samples within each group and the differences between the 2 groups. An average linkage hierarchical clustering was performed with clustering software Cluster3.0 and Java TreeView-1.1.6r2 was applied to generate the heatmap [The expressions of mRNAs with log heatmap , 29. P Value, was used to measure the gene enrichment in annotation pathways and Benjamini-Hochberg False Discovery Rate was used as testing correction. The threshold of EASE Score was set 0.1. Because miRNAs were usually negatively correlated with target mRNAs and differentially expressed miRNAs were mainly upregulated, pathways enriched of downregulated mRNAs were chose for further analysis and downregulated mRNAs were marked in green on these pathways by Search & Color Pathway in KEGG [There were thousands of deregulated genes; we cannot analyze the function of all these deregulated genes one by one, so we could only focus on the main functions of deregulated genes. We used DAVID Bioinformatics Resources 6.7  , a compr in KEGG . MiRNAs  in KEGG , 33. If MiRNAs were usually negatively correlated with target mRNAs and differentially expressed miRNAs in BBS with UA group were mainly upregulated, so upregulated miRNAs and downregulated mRNAs were uploaded into the miRTrail, a knowledge-based tool for integrative network analysis that allowed for studying the interactions between microRNAs and their target mRNAs . For eacin vivo [In the integrated miRNA/mRNA network, there were hundreds of upregulated mRNAs. To further understand the function of these miRNAs, we use a method of combing the protein-protein interactions (PPIs) network and hubs. Protein-Protein Interactions (PPIs) were commonly understood as physical contacts with molecular docking between proteins that occur in a cell or in a living organism in vivo . Each ofin vivo . This plin vivo .Hubs were highly connected nodes in a network and vital for the proper function of a network . CytoHubμg of purified total RNA generated cDNA using the QuantiMir RT Kit, following the manufacturer's instructions . qRT-PCR was performed using 2X SYBR Green qPCR Mastermix  7900HT Fast Real-Time PCR System . The mature sequences of hsa-miR-146b-5p and has-miR-199a-5p were used as the forward primers, and the 3′ universal reverse primer provided from the QuantiMir RT Kit (System Biosciences) was used as the reverse primer. The human U6 RNA was amplified in parallel as the internal control. All the miRNA forward primer sequences were listed in μL 2X SYBR Green qPCR Mastermix buffer, 2 μL miRNA-specific forward primer, 1 μL universal reverse primer, 1 μL diluted QuantiMir cDNA and 6 μL RNase-free water. The thermal cycling conditions were at 95°C for 5 minutes, followed by 40 cycles of at 95°C for 30 seconds, at 55°C for 30 seconds, 72°C for 50 seconds, and a final extension at 72°C for 8 minutes. For mRNA expression analysis, 2 μg of purified total RNA generated cDNA using RevertAid First Strand cDNA Synthesis Kit, following the manufacturer's instructions (Thermo Scientific). qRT-PCR was performed using 2X SYBR Green qPCR Mastermix (Roche Applied Science) 7900HT Fast Real-Time PCR System (Applied Biosystems). GAPDH was used as the internal control. All the mRNA primer sequences were listed in μL 2X SYBR Green qPCR Mastermix buffer, 1 μL mRNA-specific forward primer, 1 μL mRNA-specific reverse primer, 1 μL cDNA, and 7 μL RNase-free water. The thermal cycling conditions were at 95°C for 5 minutes, followed by 35 cycles of at 95°C for 35 seconds, at 54°C for 35 seconds, 72°C for 50 seconds and a final extension at 72°C for 8 minutes. The reaction products were analyzed by electrophoresis in 3% agarose gels to confirm specificity. Analysis was performed by relative standard curve method for quantification [Combining pathway analysis with network analysis, hsa-miR-146b-5p and has-miR-199a-5p seemed to be the most involved miRNAs in UA patients with BSS. Thus, to support the robustness of our analysis, differences in expression of the 2 miRNAs and 2 downregulated target mRNAs (CALR and TP53) were validated in an independent cohort of 30 UA patients with BSS, 30 non-BSS of UA patients, and 15 healthy controls by qRT-PCR. To assay for miRNAs, 1 fication . n groups), or Student's t-test (2 groups) were used as appropriate. For categorical variables, the Chi-square test or Fischer's exact test was used. All tests were performed 2-sided and a significance level of P < 0.05 was considered to indicate statistical significance. For all statistical analyses, the statistical software SPSS 16.0  for Windows was used. GraphPad Prism 5  was used to draw bar and box chars.All results for continuous variables were expressed as means ± SEM, if not stated otherwise. For group-wise comparisons, Man-Whitney test (2 groups), ANOVA, Kruskal-Wallis test (P < 0.05). There were no significant differences in age, percentage of males, BMI (body mass index), percentage of active smoker, total cholesterol, LDL cholesterol and CRP among UA patients with BSS, 30 UA patients with non-BSS and healthy control group in qRT-PCR analysis. There were significant differences in other clinical parameters among the 3 groups in qRT-PCR analysis (P < 0.05). There were no significant differences in HDL cholesterol, triglycerides (TG), hypertension, concurrent medication, and number of vessels between UA patients with BSS and UA patients with non-BSS in qRT-PCR analysis. There was significant difference in BSS grades between UA patients with BSS and UA patients with non-BSS in qRT-PCR analysis (P < 0.05).A total of 85 subjects were studied. 35 UA patients with BSS and 30 UA patients with non-BSS had angiographically documented CAD. 20 healthy volunteers were selected as the healthy controls. 5 UA patients with BSS and 5 healthy volunteers were used for mRNA and miRNA microarray analysis. The clinical characteristics of the 2 groups of microarray analysis were summarized in A list of 1081 mRNAs was identified as differentially expressed between UA patients with BSS and the healthy controls : 673 56, Table 6The KEGG pathway analysis of upregulated and downregulated mRNAs between UA patients with BSS and the healthy controls using DAVID was shown in Tables 23 upregulated miRNAs and 408 downregulated mRNAs were uploaded into miRTrail. 4250 target mRNAs were predicted by microCosm. 115 mRNAs were found to be the overlap between the predicted target mRNAs and the actually downregulated mRNAs. 6 upregulated miRNAs were selected according to the top 5% of the rank based on the number of deregulated target mRNAs. The interactive network of 115 downregulated mRNAs and 6 upregulated miRNAs was visualized by BiNA. The 6 upregulated miRNAs were miR-146b-5p, miR-199a-3p, miR-199a-5p, miR-326, miR-423-3p and miR-484 . Based on the integrated miRNA/mRNA network analysis results, a PPIs network for 115 downregulated mRNAs was built by Reactome FI plugin . It inclP < 0.05). These results confirmed our bioinformatics analysis. Representative qRT-PCR results for hsa-miR-146b-5p, has-miR-199a-5p, CALR, and TP53 were reported in In this study, using a systems biology approach we exploited critically deregulated miRNAs and mRNAs involved in pathogenesis of BSS of UA and potential biomarkers for BSS of UA. Our integration strategy started by extracting differentially expressed mRNAs and miRNAs between UA patients with BSS and the healthy control from the microarray analysis and then took them to integrative bioinformatics analysis. The robustness of our analysis was confirmed by qRT-PCR.Regarding thousands of deregulated genes, we cannot analyze the function of all these deregulated genes one by one, so we only focused on the function of deregulated genes enriched in KEGG pathways. The function of deregulated genes in the pathways related with UA would be discussed in the following part to understand the biomedical mechanisms of BSS of UA. κB mediated chronic inflammation [+ T cells within human coronary plaques [Among upregulated genes of NOD-like receptor signaling pathway , RIPK2 wammation . Althougammation . BIRC2 aammation . BCL2 waammation . The uprammation . The uprammation . CCR1 anammation , 64. Theammation . CXCL1 aammation . CXCL5 wammation . CCL20 wammation . IFNG wa plaques . Monocyt plaques . OSM was plaques . Taken t2+-buffering chaperone, and involved in regulation of intracellular Ca2+ homoeostasis and ER Ca2+ capacity [In the antigen processing and presentation pathway, CALR  was an ER (endoplasmic reticulum) luminal Cacapacity . Recent capacity . It was capacity . In addicapacity . The dowcapacity . There wcapacity . The dowcapacity . NK cellcapacity . The dowcapacity . The inccapacity . The dowcapacity . Prolifecapacity . There wcapacity . The dowcapacity . SHISA5 capacity . SESN2 pcapacity . There wPutting the functional analysis of pathways together, most of the upregulated genes in BSS of UA were implicated in inflammation, apoptosis, and innate and adaptive immunity, and most of the downregulated genes in BSS of UA were implicated in apoptosis, angiogenesis, inflammation, immune response, and cell cycle arrest. The inflammation and immune response in BSS of UA was in accordance with previous reports demonstrating inflammatory and immune related genes were related to BSS of CAD by microarray analysis . In addiin vitro and in vivo [Compared with healthy controls, the miRNA expression of BSS of UA was mainly upregulated. Considering the negative regulation of miRNA on gene, it indicated that miRNAs may mainly play an inhibitory role in BSS of UA. Among the upregulated miRNAs, miR-126, miR-129, miR-146, miR-150, miR-151a-3p, miR-199a-5p, miR-221, miR-223, miR-30d, miR-326 and miR-92a were reported to be upregulated in PBMCs of CAD patients compared with healthy controls –91. Ther in vivo . UpregulAmong upregulated miRNAs, miR-146b-5p, miR-199a-3p, miR-199a-5p, miR-326, miR-423-3p, and miR-484 were found to be key deregulated miRNAs in miRNA/mRNA network. MAPK14, AKT1, EP300, HDAC1, TP53, E2F1, SMAD3, GNB1, MYC, and SRC were found to be hubs of the PPIs network of downregulated target genes of the 6 miRNAs. Among the hubs, EP300, HDAC1, TP53, E2F1, SMAD3, and MYC were involved in the regulation of cell cycle , 97–101.α, IL-1B, and IL-6 in THP-1 monocytes [Compared the network analysis with pathway analysis, we chose miR-146b-5p and miR-199a-5p and their target genes for further analysis for 2 reasons. First, miR-146b-5p and miR-199a-5p were proved to be key upregulated miRNAs in both pathway and network analysis. Second, CALR and TP53 were the key downregulated genes in BSS of UA, while CALR was the target gene of miR-146a-5p and TP53 was the target gene of miR-199a-5p. Although there was lack of research about the role of miR-146b-5p in UA, miR-146b was demonstrated to be rapidly induced in human monocytes in response to a variety of microbial components and proinflammatory cytokines . It was onocytes , 107. Otonocytes . Moreoveonocytes . Furtheronocytes . Collectonocytes . AccordiBased on functional enrichment analysis of upregulated genes, there was active inflammation, apoptosis, and immune response in BSS of UA. Intriguingly, upregulation of miR-146b-5p and miR-199a-5p may extenuate inflammation and apoptosis in BSS of UA. Analysis of miR-146b-5p and miR-199a-5p expression unveiled a pattern of induction in response to inflammation and apoptosis in BSS of UA. The expression pattern of miR-146b-5p and miR-199a-5p in BSS of UA compared with healthy control were more like consequences than causes. Such a pattern was partly supported by the studies about the role of miR-146b in inflammation. There were evidences that pro-inflammatory cytokines could induce the upregulation of miR-146b which in return extenuate inflammation as part of a negative feedback regulation loop . In addiThe present study revealed that microRNAs and mRNAs of PBMCs might be used as biomarkers for BSS of UA patients. Compared with healthy controls, levels of miR-146b-5p and miR-199a-5p were significantly higher, while levels of CALR and TP53 were significantly lower in BSS of UA patients. These miRNAs and mRNAs were closely related with BSS of UA patients. They might serve as biomarkers for distinguishing BSS of UA patients from healthy controls. To confirm this result, miR-146b-5p and miR-199a-5p and their downregulated target genes (CALR and TP53) were selected to validate the expression level in an independent cohort of 30 BSS of UA patients, 30 non-BSS of UA patients and 15 healthy controls by qRT-PCR. Compared with healthy controls and non-BSS of UA patients, significant upregulation of miR-146b-5p and miR-199a-5p and downregulation of CALR and TP53 were proven. It confirmed the robustness of the expression pattern of miR-146b-5p, miR-199a-5p, CALR and TP53 in BSS of UA patients.However, research limitations existed in our study. First, the sample size was small, and clinical studies with larger cohorts of BSS of UA patients and healthy controls were definitely required to extensively evaluate the miRNAs and mRNAs as practical biomarkers in comparison with the diagnostic criteria and scale of BSS, as well as the false-positive rate. Second, there was lack of validation by Western blot. Because TCM syndrome differentiation was based on a collection of multiple symptoms and signs, the related biomedical mechanisms were likely quite complex. Thus, various miRNAs, genes, and intricate interactions were contained in the results, which made short time validation of results difficult. Nonetheless, the present study laid the groundwork for future efforts to identify and develop miR-146b-5p, miR-199a-5p, CALR and TP53 as a novel class of blood-based biomarkers for BSS of UA patients. According to these results, larger cohort investigations were designed to validate the diagnostic sensitivity and specificity of miR-146b-5p and miR-199a-5p as biomarkers. In addition, future studies will be performed to clarify the pathophysiological role of circulating miRNAs during pathogenesis of BSS of UA patients. In general, the present study revealed that miR-146b-5p, miR-199a-5p, CALR, and TP53, which were related to the negative feedback regulation loop to attenuate inflammation and apoptosis, were significant biomarkers of BSS of UA patients. The systems biology-based miRNA and mRNA expression biomarkers for BSS of UA may be useful for further stratification of UA patients when making decisions on treatments.


CD8+ T cell response is important in the response to viral infections; this response though is regulated by inhibitory receptors. Expression of inhibitory receptors has been positively correlated with CD8+ T cell exhaustion; the consequent effect of simultaneous blockade of these inhibitory receptors on CD8+ T cell response in viral infections have been studied, however, the role of individual blockade of receptor-ligand pair is unclear. 2B4/CD48 interaction is involved in CD8+T cell regulation, its signal transducer SAP -associated protein) is required for stimulatory function of 2B4/CD244 on lymphocytes hence, we analyzed 2B4/CD244  and SAP -associated protein) on total CD8+ and HTLV-1 specific CD8+T cells in HTLV-1 infection and the effect of blockade of interaction with ligand CD48 on HTLV-1 specific CD8+ T cell function. We observed a high expression of 2B4/CD244 on CD8+ T cells relative to uninfected and further upregulation on HTLV-1 specific CD8+ T cells. 2B4+ CD8+ T cells exhibited more of an effector and terminally differentiated memory phenotype. Blockade of 2B4/CD48 interaction resulted in improvement in function via perforin expression and degranulation as measured by CD107a surface mobilization on HTLV-1 specific CD8+ T cells. In the light of these findings, we thus propose an inhibitory role for 2B4/CD48 interaction on CD8+T cell function. The Human T-lymphotropic virus type 1 (HTLV-1) is implicated in the highly aggressive malignancy, adult T-cell leukemia/lymphoma (ATLL). HTLV-1 infection has a worldwide distribution with endemic areas in Japan, Africa, Caribbean, Central and South America, where majority of infected individuals remain asymptomatic carriers (ACs) and a minority develop a hematologic or neurologic manifestation, ATLL or HTLV-1 associated myelopathy/tropical spastic paraparesis (HAM/TSP) respectivelyIn viral infections, however, CD8+ cytotoxic T lymphocyte (CTL) function is central to immune response, mediating effective clearance of infected and transformed  cells; virus-specific CD8+ T cells also play a role in immune surveillance in HTLV-1 leukemogenesis 2B4/CD244, a member of the signaling lymphocyte activation molecule (SLAM) family of CD2 related receptors is upregulated in chronic viral infections 2B4-CD48 interaction has been variably shown to either activate or inhibit effector function; this however depends on levels of SAP expression; in the presence of insufficient SAP or its absence, inhibitory and stimulatory if high. Increased 2B4 receptor expression or CD48 ligand density could also render SAP limiting Existing studies tend to focus on 2B4 expression on NK cells with less emphasis on the role of 2B4 on CTL function. Previous results from our laboratory have demonstrated an impaired CTL response in HTLV-1 infection resulting in viral persistence, partly due to increased expression of the PD1 receptor (programmed cell death-1); blockade of interaction with its ligand PDL-1 resulted in improved effector function In the present study, we compare 2B4 expression among healthy donors (HDs), clinically asymptomatic carriers (ACs), patients with ATLL and describe its role as a co-inhibitory receptor and marker of CTL exhaustion in ATLL and AC via measurement of CD107a degranulation activity and perforin expression.The medical Ethics Committee of Kagoshima University, Kagoshima, Japan, approved this study involving human participants. All subjects gave written informed consent. All investigations were in line with the principles contained in the Declaration of Helsinki.Blood samples from 52 HTLV-1 seropositive patients (21 adult T-cell leukemia/lymphoma (ATLL), 31 asymptomatic carriers (AC)) and 12 HTLV-1 individuals, all residents of Kagoshima, Southern Kyushu were collected . They al3)) and then stained for antibodies to the following: CD8, CD4 , 2B4 , CD48  and appropriate isotypes incorporated as controls. Surface 2B4 expression was then estimated by subtracting the percentage of isotype control positive cells from the percentage of anti 2B4 positive cells Cryopreserved PBMCs were thawed, washed with staining buffer  and cultured in a 96 well plate in a 5% CO2 incubator at 37°C for 4 hours with or without 0.02 µM HTLV-1 Tax peptide  in combination with CD107a/IgG1 isotype MAbs-FITC, secretion inhibitor monensin . Anti-CD48 antibodies (5 ug/ml) were also added to the culture. Harvested cells were washed and further stained with HLA- tetramer CD8PerCP . About 1×10PBMCs were cultured in 96 well plate in 5% CO2 incubator at 37°C for 6 hours in RPMI 1640 medium with or without 0.02 µm HTLV-1 tax peptide  and brefeldin A  at a final concentration of 10 µg/ml. CD48-blocking antibodies were added at a final well concentration of 5 µg/ml 5 CD8+ T cells were acquired.Cryopreserved PBMCS were thawed and washed twice with staining buffer. Surface staining was done with antibodies to HLA-A*0201, HLA-A*2402 tetramers and incubated for 15 minutes at room temperature; further staining was done with antibodies to CD8 and incubated for another 15 minutes. Cells were washed and then permeabilized using FACS Permeabilizing Solution 2 (BD) according to manufacturer’s instructions, washed twice with staining buffer, incubated with unconjugated rat anti-SH2D1A monoclonal antibody ((1D12), Santa Cruz Biotechnology, CA,USA) or rat IgG2a K Isotype control  for 20 minutes at room temperature and then stained with FITC- labeled goat anti rat- IgG  for 20 mins at room temperature as described Differences were analyzed by Mann-Whitney U test and Wilcoxon matched pairs test using GraphPad Prism 6 (v6.01c). p-values less than 0.05 were considered significant.To determine the expression of 2B4 on total CD8+ T cells, we analyzed PBMCs  from HTLV-1 infected patients (ATLL (adult T cell lymphoma/leukemia) and ACs (asymptomatic carriers)) relative to samples from uninfected individuals. 2B4 was detected on total CD8+ T cells and significantly higher in HTLV-1 infected compared to uninfected. To determine the pattern of expression of 2B4 on CD8+T cells differentiation in CD8+ T cells from HTLV-1 infected, we examined 2B4 expression relative to CD45RA and CCR7 coexpression. 2B4 was present mainly in the naïve, effector and terminally differentiated memory groups, however, with a preference for terminally differentiated (CD45RA+CCR7− mean 87% ±9.9%) and effector memory (CD45RA-CCR7-mean 7.8% ±5.4) compartments. In the naïve compartment, 2B4+CD8+T cells were significantly higher in uninfected healthy donors (HD) compared to ATLL and AC  possibly suggesting ongoing activation in response to exposure to viral infection. Comparing terminally differentiated cells (CD45RA+CCR7−) in 2B4+ CD8+ T cell population to 2B4-CD8+ T-cell population revealed a higher frequency in 2B4+CD8+ T cells population (p<0.05); in contrast a higher proportion of naïve cells (CD45RA+CCR7+) was noted in 2B4− CD8+ T cells relative to 2B4+ CD8+ T cells (P<0.05)  A, B.2B4+CD8+ T cells also revealed CD57 (61.25% ±12.9%) and CD62L (22.3% ±12.3%) expression. Comparison of 2B4+ and 2B4− CD8+ T cells revealed a higher frequency of CD57 (p<0.05) and lower frequency of CD62L expression (p<0.05) on 2B4+CD8+ T cells in paired samples from ATLL subjects as shown in HLA-A*0201 and *2402 are the more commonly detected epitopes in ACs and ATLLs in the study region Similarly, in other latent infections  2B4 was also present on virus-specific CD8+ T cells. 2B4 expression was significantly higher on HTLV-1 infected group compared to virus-specific CD8+ T cells from healthy individuals. No difference in 2B4 expression was detected between ATLL and AC in both CMV− and EBV− specific CD8+ T cells .Comparing 2B4 expression on HTLV-1 specific CD8+ T cells relative to total CD8+ T cells in matched ATLL and ACs subjects, we observed a significant up-regulation of 2B4 expression in both groups as shown in CD48, the known 2B4 ligand, a SLAM family receptor is also involved in signal transduction and co-stimulation of immune response. Previous works have shown a variation of CD48 expression with viral infections; downmodulation on HIV-infected and upregulation on EBV infected cells in-vitro CTL function measured in HTLV-1 infected (12 independent experiments in 8 asymptomatic carrier (AC) samples and 11 experiments in 7 samples with ATLL). As shown in To further determine the effect of addition of CD48 antibodies on effector function in terms of CD107a degranulation activity, PBMCs from HTLV-1 infected patients were incubated with and without HTLV-1 Tax peptide in the presence of anti CD48 blocking antibodies and the effect on Interference in ligand receptor interaction has been shown to modulate effector function; hence, we evaluated the effect of CD48 ligand blockade on effector function via measurement of intracellular perforin production on HTLV-1 specific CD8+T cells. PBMCs from HTLV-1 infected patients were incubated with and without HTLV-1 Tax peptide in the presence of anti CD48 antibodies and the effect on in-vitro CTL response in terms of perforin production measured in 6 ATLL and 5 AC subjects in 7 independent experiments. We observed a weak response with little perforin production on short-term peptide stimulation, however, additional CD48− blockade resulted in enhanced perforin expression (p<0.05) .Levels of SAP expression determine the eventual outcome of 2B4-CD48 interaction in preference to other adaptor proteins. A previous study attributed an activating role to 2B4 in HTLV-1 infection due to high SAP levels, the study demonstrated a significantly higher SAP expression in HAM/TSP individuals compared to asymptomatic carriers although data was only shown for HAM/TSP (a subgroup of HTLV-1 manifestation) and AC subjects.Data for SAP levels in ATLL is non-existent; hence we quantified SAP expression on CD8+ and virus specific CD8+T cells in HTLV-1 infected (ATLL and AC) to determine the possible contribution of SAP to the inhibitory role observed on HTLV-1 specific CD8+T cells.No difference in SAP expression on CD8+ T cells was observed across all groups, however, levels of SAP on HTLV-1 specific CD8+ T cells were significantly increased in comparison to total CD8+T cells in matched ATLL (9) and AC (8) subjects .Statistical analyses were performed using Mann-Whitney test for unpaired samples and Wilcoxon matched pairs test for paired samples.Upregulation of the co-inhibitory marker 2B4 is associated with a decrease in virus-specific CD8+ cytotoxic T lymphocyte (CTL) function in lymphocytic choriomeningitis virus (LCMV) infection in mouse models, HIV, Hepatitis B and Hepatitis C virus infections in humans, chronic HTLV-1 infection is associated with an expansion of CD8+ effector memory T-cells but which are dysfunctional.We demonstrate here that 2B4 is increased in total CD8+ T cells and further upregulated on HTLV-1 specific CD8+ CTLs in chronic HTLV-1 infection compared to healthy uninfected donors; there is generalized immune dysfunction as indicated by upregulation of 2B4 in CMV− and EBV-specific CTLs in HTLV-1 infected individuals compared to healthy donors, lymphoid homing marker CD62L in 2B4-expressing compared with 2B4-negative CD8+ CTLs in keeping with expansion of the CD8+ terminal effector memory (TEM) T-cell compartment and an increase in activation marker CD57 in 2B4-expressing CD8+ T-cells compared with 2B4-negative CTLs. Altogether, these findings suggest a co-inhibitory function of 2B4, and its upregulation in HTLV-1 infection is associated with CTL dysfunction.CTLs expressing 2B4 tended to have terminally differentiated and effector memory phenotypes based on flow cytometric analyses of CD45RA and CCR7 expression. These phenotypes have been shown to be associated with impaired CTL function in chronic viral infections, despite upregulation of activation markers in the TEM  compartment as shown in our study Also, we observed a higher frequency of 2B4 expression on HTLV-1 tetramer specific CD8+ T cells. Several studies have reported on the immune exhaustion phenotype of HTLV-1 specific CD8+T cells, in keeping with our hypothesis of a possible co-inhibitory function of 2B4 on interaction with its ligand, as well as the expansion of the TEM subset. A similar upregulation of 2B4 expression was observed on CMV− and EBV-specific CD8+ T cells from HTLV-1 infected compared to uninfected individuals.The observation that most persons with HTLV-1 infection do not progress to disease, more strongly indicates that HTLV-1 infection is mediated by the host immune response to infection, therefore the upregulation of 2B4 in CD8 CTLs may be understood in the context of a homeostatic response to chronic immune activation. This implicates 2B4 in the regulation of CD8+ T cell response in HTLV-1 infection and a possible role in HTLV-1 pathology.In addition, CTL response in a majority of patients with ATLL is deficient or dysfunctional, this dysfunction is not relegated to patients with ATLL but extends to asymptomatic carriers (AC) and patients with HAM/TSP Our study hereby shows an inhibitory role for 2B4 on CTLs in HTLV-1 infection in both ATLL and AC. However, another study Thus, we demonstrate here, 2B4 up-regulation on HTLV-1 specific T cells, a downregulation of CD48 and an improvement in CD8+ T-cell function on ligand blockade.Our study hereby supports an inhibitory role for the 2B4 receptor on HTLV-1 specific CD8+ T cells in HTLV-1 infection. The role of other co-inhibitory markers was not analyzed in the current study. Our findings show an association of 2B4 with functional impairment on virus specific CD8+ T cells in HTLV-1 infection. Immune therapies interfering with the 2B4-CD48 pathway with the aim of enhancing weak CTL responses and improving CTL function could be investigated as a novel treatment strategy for HTLV-1 infection.


As the most frequent pathway of vertical transmission of HTLV-1 is breast-feeding, and considering the higher prevalence in women, it is very important to perform screening examinations for anti-HTLV-1 antibodies as part of routine prenatal care. So far, no studies of HTLV-1 seroprevalence in pregnant women in the Southern region of Bahia, Brazil, have been described.Pregnant women were selected at the two regional reference centers for health care from Southern Bahia. A total of 2766 pregnant women attending the antenatal unit between November 2008 and May 2010 have been analyzed. An extra blood sample was drawn during their routine antenatal testing. A standardized questionnaire was applied and all positive plasma samples were tested by ELISA and were confirmed by Western Blot and PCR. Besides that, positive women were contacted and visited. The family members that were present during the visit were asked to be serologically screened to the virus. A prospective study was also carried out and newborns were followed up to two years for evaluation of vertical transmission.HTLV prevalence was 1.05% (CI 95%: 0.70-1.50). There was no association of HTLV-1 infection with age, education, income and ethnic differences. The association with marital status was borderline . In addition, 43 family members of the HTLV-1 seropositive women have been analyzed and specific reactivity was observed in 32.56%, including two children from previous pregnancy.Conclusion: It is very important to emphasize that the lack of HTLV-1 screening in pregnant women can promote HTLV transmission especially in endemic areas. HTLV screening in this vulnerable population and the promotion of bottle-feeding for children of seropositive mothers could be important cost-effective methods to limit the vertical transmission. Besides that, our data reinforce the need to establish strategies of active surveillance in household and family contacts as important epidemiological surveillance actions for the early detection of virus infection and the prevention of transmission by sexual or and parenteral contact. It has recently been estimated that about 5 to 10 million people can be infected worldwide with HTLV  dependinHTLV-1 transmission occurs from mother to child, predominantly through breastfeeding, via sexual intercourse, or through transfusion of cellular blood components . The effThe higher prevalence in women and the possibility of mother-to-child transmission reinforce that it is very important to perform screening for anti-HTLV-1 antibodies during prenatal care and take measures to avoid or, at least, to decrease the risks of transmission. In fact, postnatal infection by breastfeeding seems to play the most important role in vertical transmission; thus, seropostive mothers have been counseled to avoid breastfeeding ,14. In JIn relation to the rate of HTLV-1 infection in pregnant women from Brazil, the prevalence is diverse and heterogenous. In Salvador, Bahia state, it was found 0.84% and 0.88% ,19, and Ilhéus and Itabuna are the biggest cities in Southern Bahia and are references in health services, treating people from different cities in this region. So far, no studies concerning HTLV-1 seroprevalence amongst pregnant/puerperal women from Southern Bahia have been described. Also considering the importance of these two cities and the high prevalence of HTLV both in Salvador as in other small and mid-sized cities from Bahia, we have decided to evaluate the frequency of this infection amongst women treated at the antenatal units of the two of the largest regional hospitals–one located in Ilheus and the other in Itabuna. In addition, the clinical and epidemiological data of the HTLV-1 positive women were compared with data from a group of HTLV-1 seronegative women. We have also tested HTLV infection in family members of the seropositive women so as to evaluate the possible routes of transmission.Treponema pallidum. Their general prevalences were, respectively, 0.22% (CI 95%: 0.08-0.48) and 0.47% (CI 95%: 0.52-0.80).A total of 2766 pregnant women treated at the antenatal unit between November 2008 and May 2010 were analyzed. Twenty nine pregnant women  were HTLV-1 positive, as confirmed by Western blot and PCR. Five of 2766 pregnant women assessed were positive by ELISA and negative after performing the Western Blot, giving a rate of 0.18% of false positive. It was verified one co-infection with HIV and no co-infection with All of the 29 HTLV-1 positives were found to be asymptomatic. As regards the place of residence, the city with more cases was Ilheus (n = 14), followed by Itabuna (n = 05). Besides, there have been two cases in Itacaré and one case in the following cities: Coaraci, Wenceslau Guimarães, Uruçuca, Itororó, Camamu, Iguaí, Una and Canavieiras. Amongst the HTLV-positive women, 83.3% reported to be brown, 70.8% were illiterate, and 69% said to receive less than 1 minimum Brazilian wage per month. Only one had received a blood transfusion and, importantly, all HTLV-1 positive women who had another child breastfed him/her. Despite the fact that these women were in the pre-partum room, only two of them have been informed to be HTLV-seropositive during the prenatal care. In these cases, women were advised to have a cesarean delivery and not to breastfeed. Table We have been able to visit 21 women, one of which refused to continue in the study. In this opportunity, samples from 43 family members of the HTLV-1 seropositive women have been collected, including: partner (n = 10), mother (n = 8), father (n = 2), sister (n = 2), brother (n = 2), children of previous pregnancies (n = 15) and others (n = 4). Specific reactivity was observed in 14/43 (≈32.6%) individuals. Among these cases, two were children  of previous pregnancies from two HTLV-positive mothers. The other HTLV-seropositives were: 5 mothers, 1 father, 4 partners, 2 sisters  in the analyzed population. It is noteworthy that prevalence rates for these two microorganisms can still be overestimated, since they were calculated from the results of rapid test for HIV and Venereal Disease Research Laboratory (VDRL) for Treponema pallidum. In the case of HTLV, all samples were subjected to confirmatory tests.In this study, the overall analyses of 2766 pregnant women revealed a prevalence of 1.05%. Previous studies in endemic areas of Brazil have found a similar prevalence ,24 demonAs we are studying a specific group, the results probably do not represent the overall population. However, in a study comparing data from specific populations , Hlela et al., have suggested that blood donors and pregnant women in Southern America and the Caribbean may be more representative of the general population and can therefore be suitable for estimating prevalence in these regions . In factAccordingly, it has been suggested that the detection of HTLV infection through prenatal or neonatal screening can be fundamental in sub-areas with high seropositivity rates, permitting to take preventive measures to reduce vertical transmission . A classIn relation of the route of transmission, it was suggested that, in Salvador, the infections have been acquired via breastfeeding, and, in second place, sexually. In this study, the analysis of HTLV-1 serology in relatives, partners and children of previous pregnancies of the index case (pregnant) has revealed HTLV-1 positive cases in different family members, highlighting partners, mothers and children . Therefore, it can be assumed that the virus infection in Southern Bahia can be spread both sexually and vertically. In fact, both routes of transmission have been related to HTLV in endemic areas . In addiStill on the familial transmission, it should be emphasized that half the evaluated families had at least a HTLV-seropositive member. Besides that, the analysis of infection rates in family members indicated a seropositive rate of 32.55% (14/43). This number is higher than that recently found in a survey evaluating familial transmission  in Pará et al. [This study has detected an association with marital status, but it was not precise. Besides, there was no association of HTLV-1 infection with age, education, and income according to what was found by Magalhães et al. , in the et al. ,17. In tet al. . In factIn summary, these results are very relevant because: (1) no studies of HTLV-1 seroprevalence in pregnant/puerperal women from the Southern region of Bahia had been described so far; (2) Southern Bahia must be another endemic area for the virus, presenting a high prevalence in pregnant women that is much higher than the national average; (3) the HTLV-1 interfamilial transmission is important and to carry out an active case search can be an important strategy in the epidemiological surveillance of this infection; (4) there is no effective treatment to HTLV infection and interventions to prevent vertical transmission in geographic areas with high prevalence would likely reduce the incidence of mother-to-child transmission and HTLV-related diseases and; (5) last but not least, despite the high prevalence observed in Southern Bahia, it seems that a number of pregnant women treated in the public health system have not been tested to HTLV during the prenatal routine in this region and may have breastfed their babies and could have infected them and spread the infection that reinforce the need for mandatory serological screening in the routine prenatal care of Bahia state.Maternidade Santa Helena, from Ilhéus, and Hospital Manoel Novaes Santa Casa de Misericórdia, from Itabuna, between November 2008 and May 2010. This macroregion covers 99 cities, totaling more than 1,025,000 women in childbearing age, and more than 25,000 live births in 2011. The mentioned hospitals treat women from these cities as well as different neighboring cities.A cross-sectional study involving pregnant women treated at the antenatal units of the two reference health centers from Southern Bahia has been conducted: Universidade Estadual de Santa Cruz (UESC).For the study, an extra blood sample was drawn during their routine antenatal  appointments. A standardized questionnaire has been applied after informed consent to collect the following data: age, formal education, history of smoking, alcohol consumption, blood transfusion, past medical history, current medication, and income level. Furthermore, the medical records were analysed to know the results of two tests: (1) rapid test for HIV and (2) VDRL. It was included in the analyses all women who fulfilled the following criteria: (1) collected blood sample in the antenatal unit; (2) answered the standardized questionnaire; (3) signed the consent informed . The project was approved by the Ethic Committee of HTLV-1 infection was assessed by ELISA (Ortho HTLV-I/HTLV-II Ab-Capture ELISE Test System). The positive plasma samples were tested repeatedly in duplicate and confirmed by Western blot (HTLV BLOT 2.4–Genelab Diagnostic). Besides, the DNA of 29 HTLV-1 positive samples was extracted by QIAgen Kit (QIAamp DNA Blood Kit), followed by a nested-PCR for the Long Terminal Repeat (LTR) region on HTLV-1. Two HTLV-1 overlapping fragments were amplified: a LTR-gag 473 bp and a LTR-tax 479 bp, as previously described . HTLV-poFrequency distributions were determined for each variable. Age was examined in 3 different strata to analyze trends in prevalence . The other variables (and no exposure condition) were: education (illiterate or literate), income (<1.0 minimum Brazilian wages per month (mw); 1-2 mw and >2 mw), ethnic classification, and marital status (married/with partner or single/Divorced/Widow). Known risk factors  and lifestyle habits have been analyzed . A bivariate analysis was carried out.Odds Ratio (ORs) and 95% confidence intervals (95%CIs) were calculated to measure the association of selected variables with HTLV-1 infection. The statistical package STATA version 10.0 was used for statistical analyses. P-values < 0.05 were considered significant.The authors declare that they have no competing interests.MAGM: participated in the design of the study, carried out the PCR, carried out the maintenance of the database and helped to draft the manuscript. LPSS, TCG, RGB, AFC: applied the standardized questionnaire, the informed consent and processed the blood samples to perform serology. NBS, MCCA: participated in the design of the study, performed the statistical analysis and helped to draft the manuscript. LCA: participated in the design of the study and helped in the realization of molecular analyzes. BGC: participated in the design of the study, helped to draft the manuscript. LJM, SMBS: participated in the design of the study, helped in identifying positive women, made contact and scheduled the visits, and helped to draft the manuscript. NL: carried out the ELISA and Westen Blot and carried out the blood collection during the visits. MRSR: participated in the design of the study and provided orientation concerning the result of the tests and about breastfeeding during the project. SRG: conceived of the study, coordinated it and drafted the manuscript. All authors read and approved the final manuscript.


Monitoring land change at multiple spatial scales is essential for identifying hotspots of change, and for developing and implementing policies for conserving biodiversity and habitats. In the high diversity country of Colombia, these types of analyses are difficult because there is no consistent wall-to-wall, multi-temporal dataset for land-use and land-cover change.2) while 264 had a net loss , which resulted in a net gain of 16,963 km2 in woody vegetation at the national scale. Woody regrowth mainly occurred in areas previously classified as mixed woody/plantation rather than agriculture/herbaceous. The majority of this gain occurred in the Moist Forest biome, within the montane forest ecoregions, while the greatest loss of woody vegetation occurred in the Llanos and Apure-Villavicencio ecoregions.To address this problem, we mapped annual land-use and land-cover from 2001 to 2010 in Colombia using MODIS (250 m) products coupled with reference data from high spatial resolution imagery (QuickBird) in Google Earth. We used QuickBird imagery to visually interpret percent cover of eight land cover classes used for classifier training and accuracy assessment. Based on these maps we evaluated land cover change at four spatial scales country, biome, ecoregion, and municipality. Of the 1,117 municipalities, 820 had a net gain in woody vegetation  and ecoregions under-represented in the protected area network  should be considered for new protected areas. Land cover change is the main cause of deterioration in ecological systems at local to global scales Monitoring forest cover at the national level is essential for developing and implementing appropriate biodiversity conservation and carbon emission reduction policies Colombia is one of the most biodiverse countries on Earth Land transformation is not homogeneous in Colombia, but rather varies greatly among its different ecological and political regions The purpose of the present study is to assess land change from 2001 to 2010 in Colombia, with a focus on three objectives: (1) determine how land change varies at the country, biome, ecoregion, and municipality scales; (2) identify and analyze the spatial distribution of areas experiencing significant land change; and, (3) discuss the implications of our findings for land use and conservation planning in Colombia. This research was based on a novel method for mapping LULC annually, which coupled MODIS (250 m) products with reference data interpreted from high spatial resolution imagery (QuickBird) in Google Earth that allows us to quantify land change at multiple spatial scales.2. Colombia has about 45.4 million people and an average population density of 40.1 people per km2 (see http://www.dane.gov.co). Differences in elevation and latitude produce large climatic variation across the country. For example, there are dramatic differences in annual precipitation, ranging from 350 mm (Guajira peninsula) to 12,000 mm (Pacific lowlands). Consequently, the combination of different climates, elevation ranges, and geographic location have allowed the development of a high diversity of habitats and species richness in Colombia, as well as an array of land uses.Colombia is located in the northwestern part of South America, bordered by the Caribbean Sea to the north and the Pacific Ocean to the west, and occupying an area of 1.1 million kmColombia can be divided into five continental regions  because they occupied a large area  in the southern portion of the country . All analyses were thus performed on 1,117 municipalities or study units.In this study, municipalities (second administrative level) were the main unit of analysis. According to the National Administrative Department of Statistics (DANE) data, the number of municipalities in Colombia was 1,100 in 2007. However, we included 1,097 municipalities instead of 1,100 because three municipalities were created after the last census in 2005. We also included 20 Our LULC classification methodology generally follows those first outlined by http://earth.google.com) mainly from Digital Globe’s QuickBird satellite  spanning 2001 to 2010 woody (woody vegetation including trees and shrubs); herb (herbaceous vegetation); ag (agriculture); plant (plantations); built (built-up areas); bare (bare areas); and water. If two interpreters agreed on the majority cover and GE image year of a sample, then their percent cover estimates were averaged. If the interpreters disagreed on the majority cover or year (mostly cover), then an “expert” (author) estimated the final class cover and recorded the year woody, with a bare, herb or ag component <80% were assigned to a mixed woody class.Google Earth reference data collection : reference data for classifier training and accuracy assessment were collected with human interpretation of high-resolution imagery in Google Earth  for LULC classification randomForest package  as number of variables randomly sampled as candidates at each split, where p is number of variables (mtry = default). Predictor variables were MODIS-based 4-, 6- and 12-month statistics for EVI, red, NIR and MIR, and were extracted for the year corresponding to the QuickBird image year  samples for training the final RF , with non-water average producer’s accuracies ranging from 36.3% (mixed woody/plant) to 96.9% (woody) and user’s accuracies ranging from 72.5% (mixed woody/plant) to 89.4% (woody) (Mapping LULC with the Random Forest classifier: we mapped LULC with the Random Forests (RF) tree-based classifier final RF . We used (woody) . The fivwoody, mixed woody/plant, and ag/herb) within each municipality, we analyzed the trends performing a linear regression of cover area (dependent variable) against time . If more than 1% of the total municipality area had pixels mapped as No Data for a given year, then the land cover data for that year were removed from the regression. To determine the strength of this linear relationship we used Pearson’s correlation coefficient (R), where positive values of R represent an increase in a LULC cover and negative values of R represent a decrease. We used this approach to standardize land change through time due to outliers or missing data in any given year, and the use of R for trends allows us to compare municipalities, which can vary in size from 17,6 km2 to 65,568 km2. In addition, this trend analysis takes advantage of the ten years of data, and it is not based on just two points in time. Municipalities with significant changes in any cover had p≤0.05. All analyses incorporating absolute area were performed using estimates based on the each municipality’s regression model, rather than the raw area data used to fit the model.To describe the patterns of land change, for the three most important vegetation classes (2) of the three classes between 2001 and 2010 considering four scales: country, biome, ecoregion, and municipality. Biome and ecoregion scales were established following the World Wildlife Fund biome and ecoregion framework woody vegetation.We calculated the net change in cover , while it increased in the other five biomes, from 16,077 km2 in the Moist Forest, to 57 km2 in the Montane Grasslands , while there was a large decrease in the Moist Forest biome . The ag/herb cover only decreased slightly in Mangroves (21 km2), while increasing in the rest of biomes from 10,652 km2 in the Moist Forest to 58 km2 in the Dry Forest.At the biome level, asslands . The mixwoody vegetation was the dominant cover in four ecoregions, while ag/herb vegetation was the dominant cover in nine ecoregions  also had a small increase in woody cover (144 km2) when compared with the rest of the ecoregions. Mixed woody/plant class increased only in the Guajira Xeric ecoregion (552 km2), and decreased in more than 1,500 km2 in eight ecoregions. Mixed woody/plant net loss varied between 7,147 km2 in the Magdalena-Urabá Moist forest and 9 km2 in the Magdalena Valley Dry forest ecoregions. The ag/herb vegetation mainly decreased in the Sinú-Valley Dry forest , the Northwestern Andean (969 km2), and the Cauca-Valley Montane forest (310 km2). The net gain in ag/herb vegetation varied between 5,256 km2 in the Caquetá Moist forest and 25 km2 in the Magdalena-Valley Dry forest.In 2001 and 2010, oregions . Land cowoody vegetation increased in 73% (820) of the municipalities with a net gain of 28,092 km2, and decreased in 24% (264) of the municipalities with a net loss of 11,129 km2 (mixed woody/plant class increased in 31% (347) of the municipalities with a net gain of 5,199 km2, while it decreased in 68% (762) of municipalities with a net loss of 34,481 km2. For the ag/herb class, the number of municipalities gaining  and losing  cover was similar, but the area gained was almost double  of that lost . We also found that 21% (232) of the municipalities showed significant change in woody vegetation during the last decade. This percentage was similar for mixed woody/plant  and for ag/herb . If we only considered municipalities with significant changes during the last decade, we detected a close correspondence between loss and gain of woody vegetation, mixed woody/plant, and ag/herb classes  transitions from ag/herb vegetation to woody vegetation were located in western Cundiboyacense highplain, and between Nudo de los Pastos and the Macizo Colombiano  transitions from ag/herb vegetation to mixed woody/plant were located only in the north of the Cundiboyacense highplain  transitions from mixed woody/plant to woody vegetation were concentrated in the Catatumbo and to the north of the Magdalena Medio, as well as to the north of the central and western Andean mountain ranges .At the municipality level, ,129 km2 . In cont classes . Examplelombiano ; iii) trighplain ; v) trann ranges . The avewoody vegetation change we selected the top 10 municipalities with the greatest net gain or loss in woody cover  and in the Llanos regions , where 3,000 km2 of woody vegetation were converted to croplands and pastures. Deforestation in these areas is related to gold mining and oil exploitation activities, and agricultural expansion. For example, in the Magdalena Medio region woody vegetation has been cleared for small-scale agriculture and timber extraction by miners since the 1990s 2 in 2001 to 1,800 km2 in 2009 woody vegetation in these regions affects areas of global importance for biodiversity such as Serranía de San Lucas located to the south of Magdalena Medio Our results show that during the last decade, land change in Colombia has been characterized by an unexpected net gain in 2 in 2001 which is lower than the 617,328 km2 and 615,090 km2 (for the year 2000) reported by IDEAM and FAO, respectively (2 from 2001 to 2010) which is much more conservative than the gains  estimated by MOD44B (VCF). These differences can be attributed to the use of different MODIS image inputs, spatial resolution (ours 250 m vs. VCF 500 m) and processing methods, as well as differences in forest (woody vegetation) definition. For example, VCF includes plantations in forest cover. FAO defines “forest” as lands of more than 0.5 ha covered by tress over 5-m height, 10% crown cover, and include areas with native and plantation trees woody” vegetation differs from the former definitions because we include trees and shrubs,  with ≥80% cover. Consequently, our definition of mixed woody vegetation (20–80% woody) combined with plantations is more comparable to FAO’s and IDEAM’s definition of “forest”. In addition, FAO and IDEAM estimates showed the same deforestation trend because their definitions of forest are somewhat similar and FAO results are typically based on existing maps provided by IDEAM woody vegetation.Overall, we report a forest area of 580,420 kmectively . Additioectively  results In general, our methodology to map annual LULC in Colombia, which combined MODIS products, Google Earth reference data, and Random Forest classifier ag and herb samples for which an expert determined the final class label. Additionally, spatial error can be the result of terrain distortions especially in QuickBird images that have not been orthorectified. However, it has been shown that QuickBird scenes are very accurate with an average error of 10 m of disagreement between ground control points and GE QuickBird images We acknowledge that there are two potential caveats to our study. First, MODIS pixels will not detect small-scale changes  due its lower spatial resolution. However, the accumulative change from the small-scale conversion can be captured by our 10-year trend analysis based on the aggregation of all pixels within a municipality. Although Landsat provides higher spatial resolution which facilitates detection of small-changes, there are major gaps due to clouds cover that make it difficult to map the whole country using Landsat imagery. Second, using reference data from QuickBird imagery in Google Earth could include interpretation and spatial error. For example, visual interpretation of some land cover classes in Google Earth is difficult, and therefore, our cover classes were very general. Even though our classes were relatively easy to identify, interpreters sometimes disagreed on woody vegetation observed in our LULC maps. The first possible explanation could be an increase in oil palm plantations. Oil palm plantations have expanded rapidly since the 1990s when Colombia initiated its economic liberalization model 2 in the 1960s to almost 3,600 km2 in 2010– mainly in the Meta, Casanare, Cesar, Magdalena, Bolívar, Cundinamarca, Santander, Norte de Santander, and Nariño departments. Nevertheless, we do not believe that oil palm plantations are an important component of the woody recovery we described. First, we classified plantations separate from woody vegetation. Second, municipalities identified by IGAC woody vegetation . However, during the same period of time, only 7% of the municipalities had a net gain in palm oil plantations  and Riohacha (net gain of 81 km2) have oil palm plantations  following the eradication of these illicit crops.A second factor that could explain 189 km2; . By 2010woody cover, particularly in seasonal forests (i.e. dry forests), is related to the inter-annual variation in precipitation. In these areas, anomalous rainfall years (e.g. La Niña events) could change the vegetation greenness trends detected by MODIS sensors. This phenomenon could change woody vegetation to mixed woody and vice versa, especially for pixels near the 80% decision threshold. However, we attempted to minimize this effect using regression models by municipality, capturing 10-years of real trends in vegetation dynamics. It would be desirable to use high resolution data such as Landsat to evaluate the accuracy in our LULC maps and to verify observed land change; however, on an annual basis it is difficult to obtain the necessary temporal data for the entire country for detecting any climatic anomalies.The third potential factor that could explain the increase in woody vegetation gains occur in small municipalities and exceed woody vegetation losses occurring in large municipalities. This scale of analysis gives a valuable general overview of current land change, but it can mask woody vegetation losses in some areas. National scale analysis does not take into account intrinsic differences  among regions which can promote different land cover patterns and dynamics. Therefore, by examining data at the biome and ecoregion scales it is possible to decipher where and to what extent changes in woody vegetation are occurring, and to better understand the underlying environmental and social drivers of this change.In summary, the national assessment of land cover change in Colombia indicates that woody cover. It is the largest biome in Colombia  and in the tropical forest in the Amazon and the Pacific regions (30%). Other studies have quantified forest regrowth in Colombia from secondary vegetation and abandoned pastures, particularly in the Amazon This biome accounts for 86% of the total increase in Colombia  consistigetation . The netwoody vegetation in Colombia, particularly in the Northern Andean (27%), the Northwestern Andean (22%), and Cauca-Valley (11%) Montane Forest ecoregions. Woody vegetation increases are likely explained by human causes such as the abandonment of traditional productive systems in the 1990s due to harsh environmental conditions (e.g. arid areas) and globalization processes which have promoted strong rural migration in many municipalities http://www.dane.gov.co), and the area is now experiencing significant change from mixed woody/plant class to woody vegetation  of large expanses of montane forest to the south of the Northwestern Andean (between the Macizo Colombiano and Nudo de los Pastos) and to the north of the Cauca-Valley Montane forest ecoregions as well. This reforestation trend has been seen in both developed and developing countries, supporting the idea that the abandonment of less productive lands and the globalization of markets may lead to the regrowth of secondary forest The four montane forest ecoregions (in the Moist Forest biome) located in the Andes mountain ranges contributed 65% of the total net gain in woody and ag/herb vegetation, which transitioned from the mixed woody/plant vegetation class. For instance, the Magdalena-Urabá Moist Forest ecoregion showed a remarkable decrease in mixed woody/plant class, particularly in the Magdalena Medio region where 46% transitioned to woody vegetation and 44% transitioned to ag/herb vegetation, the remaining area transitioned to other classes (i.e. built-up and bare soil). Woody vegetation in the Caquetá Moist forest ecoregion seems to be stable, contributing only 0.8% of the total net gain in woody vegetation in Colombia. These results coincide with data from the Amazon forest, which showed forest regrowth transitioned primarily from previous croplands woody vegetation and mixed woody/plant classes, which is more comparable with the definition of forest used by IDEAM, we detect a loss of >5,000 km2. Virtually all of this change is from mixed woody/plant to ag/herb. These losses in the Caquetá Moist forest are the result of small-scale subsistence agriculture . Large woody vegetation losses were located mainly in the central area of Casanare and the eastern area of Meta departments where an intense land conversion is associated with human population change and investments in infrastructure to support an important oil exploration activity and agricultural intensification. High rates of land conversion (towards mechanized agriculture and cattle grazing) corresponding with urban population growth and migration have been registered since the 1980s http://www.dane.gov.co) and 85% gained ag/herb vegetation, implying that agriculture (e.g. rice) and pastures are still expanding, as in many other regions ag/herb class. Changes are only detected when the change is to perennial plantations.This is the second largest biome in Colombia and includes only the Llanos ecoregion. The Llanos is increasingly being considered as the new agricultural frontier of Colombia . This expansion was located particularly in the foothills of the Arauca department where the majority of the population is located. According to the DANE censuses, 93% of the municipalities in the Apure-Villavicencio Dry Forest ecoregion gained people between 1993 and 2005 and 87% gained ag/herb vegetation. We believe that the expansion of intensive agriculture and cattle pasture will continue as a major driver of deforestation in this region.This is the third largest biome and includes three ecoregions: Magdalena Valley, Sinú Valley, and Apure-Villavicencio Dry forest. This biome accounts for 4% of the total increase in woody vegetation. These biomes account for 9% and 0.7% of the national increase in woody vegetation, respectively. The increase in woody vegetation in the Desert biome was concentrated in three municipalities that alone account for 73% of the total increase in this biome. The gain in woody in these municipalities could be related to a precipitation anomaly (e.g. 2009) or perhaps a problem in the classification given that the Desert biome was classified as part of the Dry Forest biome (Woody regrowth (mostly shrubland cover) in the Deserts have been reported in Mexico and U.S as a result of the increase in annual precipitation and the decrease of fire and grazing, respectively woody gains are likely the result of the implementation of conservation and management strategies of mangrove ecosystems across the country woody vegetation was located in two municipalities  that contain the Vía Parque Isla de Salamanca protected area, which was declared a Ramsar Site in 1998 and Reserve for Humankind and the Biosphere by UNESCO in 2000 .These biomes only include the Guajira Xeric ecoregion which had a net gain in st biome . Woody rwoody vegetation, accounting for only 0.3% of the national increase in woody vegetation. The gain could be the result of regrowth in areas previously occupied by Papaver somniferum (poppy) plantations. Between 1993 and 2008, the area in poppy fields decreased from 75 km2 to 4 km2 across Colombia ag/herb vegetation. The gains in ag/herb cover were located mainly in municipalities in the Santander and Boyacá departments where potato farming and cattle grazing are important activities. In these departments, the cultivated area of potatoes increased from 380 km2 to 482 km2 between 2006 and 2008 This biome includes the Northern Andean Páramo ecoregion which had a slight increase in http://www.parquesnacionales.gov. co), and this is where we detected the highest rates of woody vegetation loss. These areas have had extensive areas of natural savanna vegetation transformed to crops and pastures during the past 20 years http://www.worldwildlife.org/wildworld/profiles/terrestrial_nt.html# tropgrass). We also documented a large decrease in mixed woody/plant in the Magdalena-Urabá Moist forest ecoregion, particularly in the Magdalena Medio region. The increase in agriculture and pastures combined with ongoing illegal logging activities Libidibia ebano, Cariniana pyriformis). This region should be considered for the protected area network given that there is only one reserve  in this region.The implementation of protected areas is an important mechanism to reduce forest conversion and subsequent loss of species woody vegetation in the Andes Mountain Ranges is an excellent opportunity to complement, expand and interconnect the protected areas to create a conservation network across the rural landscape mosaics in the region. A relevant area for conservation is the Northern Andean Montane Forest ecoregion, which is also included in the Global 200 woody vegetation between 2001 and 2010. Other authors have stressed the importance of this region as a priority area for conservation due to its large areas of land transformation and large number of species at risk woody vegetation, the gains in the ag/herb class were almost three times higher than woody cover gains, and therefore, the Andean Páramos remains a threatened ecosystem.On the other hand, the recovery of woody vegetation gains exceed losses between 2001 and 2010. The majority of woody gains occurred in the Moist Forest biome. Analysis at the ecoregion scale showed that montane forest ecoregions contributed substantially to woody vegetation regrowth in Colombia, while the Llanos and Apure-Villavicencio ecoregions experienced the largest woody losses. The gain of woody vegetation does not necessarily imply the recovery of the high biodiversity characteristic of the original forests in many of these regions. If these “new forests” are allowed to grow, they are likely to recover a large proportion of their biodiversity in the next 40–50 years Overall, the present study indicates that at the national scale, Figure S1Distribution of reference data points collected from Google Earth within each of the three biomes which covered Colombia and neighboring countries. Biome description: 1. Tropical and Subtropical Moist Broadleaf Forest (Amazon basin section) 2. Tropical and Subtropical Moist Broadleaf Forest  3. Tropical and Subtropical Dry Broadleaf Forest 4. Tropical and Subtropical Grasslands, Savannas and Shrublands(TIF)Click here for additional data file.Table S1Major biomes and ecoregions in Colombia. The names and area of the 6 major biomes and the 25 ecoregions in Colombia according to Olson et al. (2001). Note that the original 25 ecoregions were grouped into 13 ecoregions because some ecoregions only include one or a few municipalities. The name of the largest ecoregion was used as the name of the aggregation.(DOCX)Click here for additional data file.Table S2Woody vegetation net gain and loss for all municipalities in Colombia. Thirty three municipalities were not included because they did not have any woody vegetation.(DOCX)Click here for additional data file.


However, HTLV-1 and HTLV-2 exhibit a clearly distinct pathogenic potential in infected patients. To find clues to the possible viral determinants of the biology of these viruses, recent studies investigated the timing of expression and the intracellular compartmentalization of viral transcripts in ex-vivo samples from infected patients. Results of these studies revealed a common overall pattern of expression of HTLV-1 and -2 with a two-phase kinetics of expression and a nuclear accumulation of minus-strand transcripts. Studies in cells transfected with HTLV-1 molecular clones demonstrated the strict Rex-dependency of this “two-phase” kinetics. These studies also highlighted interesting differences in the relative abundance of transcripts encoding the Tax and Rex regulatory proteins, and that of the accessory proteins controlling Rex expression and function, thus suggesting a potential basis for the different pathobiology of the two viruses.Human T-cell leukemia virus types 1 and 2 (HTLV-1 and HTLV-2) are delta retroviruses that share a common overall genetic organization, splicing pattern, and ability to infect and immortalize T-cells  The pX regions of HTLV-1 and HTLV-2 contain, respectively, four and five major open reading frames (ORFs), termed x-I through x-V. The x-III and x-IV ORFs code for Rex and Tax, which are produced from a dicistronic doubly-spliced mRNA containing exons 1, 2, and 3. Tax and Rex play a critical role in regulating viral expression at the transcriptional and post-translational levels, respectively. Other transcripts produced by the X region code for accessory proteins; these transcripts include singly-spliced mRNAs coding for p21rex, p12, and p13 (HTLV-1), truncated isoforms of Rex (tRex) and p28 (HTLV-2) and doubly-spliced mRNAs coding for p30/tof (HTLV-1), p10 and p11 (HTLV-2)   using splice site-specific primers, which allowed measurements of the levels of individual alternatively spliced transcripts. qRT-PCR analysis was also carried out in cells transfected with wild type and Rex-knockout HTLV-1-molecular clones.To gain further insight into these aspects of HTLV-1 regulation, we recently investigated the timing of spontaneous onset of HTLV-1 expression ex vivo viral reactivation model based on the depletion of CD8+ cytotoxic T cells from peripheral blood mononuclear cells (PBMC) of HTLV-1-infected patients, which results in a sharp rise in viral expression , p28/tRex(1-B), and 1-2-B preceding that of the gag/pol, env, and p28/tRex(1-3) mRNAs.To verify these findings, we employed an tax/rex mRNA. Flow cytometry analyses showed a relative accumulation of Rex at later time points; consistent with this finding, Rex revealed a slower rate of degradation compared with Tax, suggesting that the activity of these two regulatory proteins might be controlled at the post-translational level. Similar experiments have not yet been carried out for HTLV-2.To find clues to the possible regulatory mechanism underlying the 2-phase kinetics of HTLV-1 expression, we used the expression data to generate a mathematical model  and HTLV-2 (p28) were shown to inhibit both the tax/rex mRNA and the Rex protein  and Rex (p21rex and tRex) appears to be skewed toward tax/rex in HTLV-1 and toward the inhibitors in HTLV-2 . These differences might provide the molecular basis for the different tropism and pathogenicity of the two viruses.The fact that both HTLV-1 and HTLV-2 show an overall similar two-phase kinetics of expression suggests that this converging expression strategy might reflect important in vitro are, at present, poorly understood phenomena. Further investigations are needed to define the pattern (and levels) of HTLV expression in different lymphoid organs.However, in contrast to the other tumor viruses mentioned above, neither the levels or the timing of expression of different genes has been linked to a different disease outcome, a topic that should be addressed in future studies. In addition, both the “latent” expression detected in peripheral blood HTLV-infected cells and the “reactivation” upon culture Interestingly, also in the case of HIV-1, another complex human retrovirus, viral gene expression is controlled by two key regulatory proteins Tat and Rev, which control, respectively, transcription and post-transcriptional processing of alternatively-spliced viral mRNAs.Early studies suggest that the HIV-1 life cycle is also characterized by a two-phase kinetics with Tat, Rev, and Nef expressed as early genes and Env, Vpu, Vif, Vpr, Gag, Pro, and Pol expressed as late genes. In the context of this expression strategy, Rev acts as a molecular switch controlling the transition between latent and productive infection (Ahmad et al., However, to date, the relative abundance and the time course of expression has not been measured at a single transcript level using qRT-PCR like in the case of HTLV-1 and HTLV-2.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.


Almost 40 years ago, Takatsuki et al. recognized the existence of a peculiar T cell leukemia in Kyoto, Japan that they named Adult T Leukemia (ATL). They reported a series of 13 patients in 1976 (Uchiyama et al., Almost 10 years ago, ours colleagues Kuan Teh Jeang and Mitsuaki Yoshida organized a special issue on HTLV infection in Oncogene. In setting up this issue, we cannot forget the memory our friend Teh.We called upon the expertise of different research groups from Europe, Japan, and USA. However, we regret that the format of this issue prevented us from soliciting many other colleagues. The following reviews will deal with many fascinating aspects of viral cycle, but summarizes also new approaches that should allow a better integrated research.A first group of articles provides information about HTLV-1 epidemiology and associated-pathogenesis. The article from Gessain and Cassar provides an updated view on HTLV-1 distribution, based on data obtained from 1.5 billion individuals originating from endemic areas (Gessain and Cassar, A second group of articles summarizes the interaction between the virus and the host's cells. Before causing diseases, HTLV-1 has to enter the cell. However, the mechanisms of HTLV-1 transmission and cell entry have remained elusive for a long period of time. Pique and Jones have summarized recent insights about those mechanisms both at the cell level but also between individuals (Pique and Jones, in vivo (Bai and Nicot, A third group of articles reports data on individual viral proteins that play important roles in the viral cycle and/or in pathogenesis. Nakano and Watanabe remind us the important role played by Rex, which uses cellular pathways to export unspliced or singly spliced viral mRNAs in the cell cytoplasm, therefore allowing expressing of structural proteins (Nakano and Watanabe, Finally, Duc Dodon and colleagues remind us that studying HTLV-1 pathogenesis requires animal models (Dodon et al.,


Simian T-lymphotropic virus type 1 (STLV-1) strains occasionally infect humans. However, the frequency of such infections is unknown. We show that direct transmission of STLV-1 from nonhuman primates to humans may be responsible for a substantial proportion of human T-lymphotropic virus type 1 infections in rural Côte d’Ivoire, where primate hunting is common. Human T-lymphotropic virus type 1 (HTLV-1) can induce adult T-cell leukemia or lymphoma and HTLV-1–associated myelopathy or tropical spastic paraparesis. These pathologies are a serious threat to the several million persons infected with HTLV-1   strains derived from nonhuman primates  sequences (env and 2–20 LTR sequences (15–40 sequences per person) were determined by Sanger sequencing. No evidence of multiple infections was found.To determine effective exposure to HTLV-1/STLV-1, we used an HTLV-1/2 ELISA to test serum samples for reactivity to HTLV-1/2 antigens (equences . To idenequences . For eacenv and LTR datasets (env tree topology (Cercocebus atys) from Taï National Park  (Piliocolobus badius badius) and chimpanzees (Pan troglodytes verus) from Taï National Park     Table 2,ties, 1)  Table 2 ies, 1) ,. Observe)  .,We investigated the frequency of direct zoonotic transmission of STLV-1 in a rural region of Côte d’Ivoire neighboring Taï National Park and found that only 2 of the STLV-1–related sequences would be compatible with a local human-to-human transmission  ,. Such loThe analysis of behavioral data reveals generalized exposure of local populations to cooked nonhuman primate bushmeat . ExposurDetailed material and methods for a study on the origin of human T-lymphotropic virus type 1 in rural western Africa, 2006–2007, and a table showing Simian T-lymphotropic virus type 1 infection in nonhuman primates from Taï National Park, Côte d'Ivoire.


Bone morphogenetic proteins (BMPs) are members of the transforming growth factor-beta superfamily and were originally identified as proteins that induce a full cascade of endochondral bone formation in vivo and ectopically. We hypothesized that the BMP signaling pathway could also play a role in the process of ankylosis that characterizes ankylosing spondylitis and related spondyloarthritides. By over expression of noggin, a BMP antagonist, in a dedicated mouse model of joint ankylosis, we provided evidence for this hypothesis. Our current studies focus on the relationship between stromal cell activation and inflammation, a link that is essential in ankylosing spondylitis. Emerging functional and genetic evidence further corroborates the essential role of BMPs in these processes. BMPs can trigger different downstream signaling cascades, in particular Smad and p38 signaling. Inhibition of p38 signaling in vivo in the spontaneous ankylosing enthesitis model appears to accelerate ankylosis while inhibiting in vitro chondrogenesis. Genetic analysis of intercrosses between the susceptible DBA/1 strain and the H2 identical Balb/c strain point towards a region containing the BMP type Ib receptor as a factor determining genetic susceptibility. Recent clinical data suggest that levels of BMPs are higher in patients with progressive ankylosis in comparison with patients that show no structural progression of disease. Taken together, these data support our hypothesis that BMP signaling is a therapeutic target in ankylosing spondylitis.


The pathogenesis of HAM remains uncertain: the disease is thought to be caused by the immune response to HTLV-1, possibly by bystander damage to neurons in the spinal cord. The strongest correlate of HAM in HTLV-1-infected individuals is the proviral load of HTLV-1, i.e. the percentage of peripheral blood mononuclear cells that carry the provirus. To aid in the differential diagnosis of HAM, and to search for clues as to the pathogenetic mechanisms of the disease, we carried out SELDI mass spectrometry on plasma samples from 68 HTLV-1-positive individuals, 16 uninfected controls and 11 patients with secondary progressive MS. We identified three plasma protein biomarkers that are specifically associated with HAM, independently of proviral load. The three proteins were identified by tandem mass spectrometry as b2-microglobulin, calgranulin B, and apolipoprotein A2. Using the two most strongly associated biomarkers, b2-microglobulin and calgranulin B, we derive a simple algorithm that correctly classified the disease status (presence or absence of HAM) in 81% of HTLV-1-infected subjects in the cohort.


HTLV-1 infection is endemic among people of Melanesian descent in Papua New Guinea, the Solomon Islands and Vanuatu. Molecular studies reveal that these Melanesian strains belong to the highly divergent HTLV-1c subtype. In Australia, HTLV-1 is also endemic among the Indigenous people of central Australia; however, the molecular epidemiology of HTLV-1 infection in this population remains poorly documented.gag, pol, env, tax) and non-coding (LTR) genomic proviral regions. Four complete HTLV-1 proviral sequences were also characterized. Phylogenetic analyses implemented with both Neighbor-Joining and Maximum Likelihood methods revealed that all proviral strains belong to the HTLV-1c subtype with a high genetic diversity, which varied with the geographic origin of the infected individuals. Two distinct Australians clades were found, the first including strains derived from most patients whose origins are in the North, and the second comprising a majority of those from the South of central Australia. Time divergence estimation suggests that the speciation of these two Australian clades probably occurred 9,120 years ago .Studying a series of 23 HTLV-1 strains from Indigenous residents of central Australia, we analyzed coding  infects at least 5–10 million persons worldwide. In Oceania, previous studies have shown that HTLV-1 is present in a few ancient populations from remote areas of Papua New Guinea, the Solomon Islands, the Vanuatu archipelago and central Australia. The latter comprise one of the most socio-economically disadvantaged groups within any developed country. Characterization of the few available HTLV-1 viruses from Oceania indicates that these belong to a specific HTLV-1 genotype, the Australo-Melanesian c-subtype. In this study, we provide details for 23 HTLV-1 viruses derived from the Indigenous population of central Australia, a vast remote area of 1,000,000 km The Central Australian Human Research Ethics Committee (CAHREC) approved this study (CAHREC Ref: 2011.11.01).The plasma and PBBC samples were transferred to Institut Pasteur, Paris, and stored at −80°C until HTLV-1 analysis. Plasma HTLV-1 antibodies were tested by a particle agglutination (PA) technique  and by an indirect immunofluorescence assay (IFA) using the HTLV-1-transformed human T cell lines MT2. All samples were also tested by Western blot assay (WB) .gag and Px-LTR primers which were designed using highly conserved regions that are common to the major HTLV-1 subtypes . The Px-LTR primers are F6501: 5′-CTTAACTGGGACCTTGGCCTCTCAC-3′  and 3VLTRext: 5′-CGCAGTTCAGGAGGCACCRMA-3′  . Samples were first subjected to PCR using human β-globin specific primers, to ensure that DNA was amplifiable subtypes . The LTRo 8,761) .env gene, 1 µg of DNA from the Aus-GN strain was subjected to 2 series of PCR as previously described To obtain the 522-bp fragment of the gp21 pro-pol and pol-env primers were necessary to obtain the entire genome amplification of the four Australian proviral sequences . The pro-pol primers are F2279: 5′-GGAGCAGACATGACAGTCCTTCC-3′  and R5005: 5′-GGCGGCTATTAAGACCAGGAAGC-3′ . The pol-env primers are the following F4583: 5′-CAGGAGCCATCTCAGCTACCC-3′  and env2c: 5′-TTTATAAGAGAGTAATGGGGGTATCTG-3′   . The siz2,273-bp .gag: 98°C, 1 mn; 40×; 72°C, 2 mn and Px-LTR: 98°C, 1 mn; 40×; 72°C, 2 mn. For pro-pol: 98°C, 1 mn; 40×; 72°C, 2 mn and pol-env: 98°C, 1 mn; 40×; 72°C, 2 mn. Reaction tubes were prepared in a dedicated room outside the laboratory, with a final volume of 50 µl , 40 µM; 5×Phire reaction buffer which contains 1.5 mM MgCl2 at final reaction concentration , 5 µl; Phire hot start DNA polymerase , 2 U and 0.5 µM of each oligonucleotide primer . Five µl of amplified DNA was size fractionated by 1.5% agarose gel electrophoresis, and the PCR products (45 µl) were sent for purification and sequencing reactions to the MilleGen Company   was used with the following amplification conditions. LTR- France) .env gene (522-bp), colinearized gag-tax  and gag-pol-env-tax (7567-bp) genes  that were characterized in the present study together with appropriate sequences of previously characterized strains from PNG , the Solomon Islands (MEL3 to MEL6 and MEL 8 to MEL10), Vanuatu  and Australia (MSHR-1). Additional representative sequences of the HTLV-1 a, b, d-g subtypes available in Genbank were also included.Both strands of each PCR product were sequenced, and the ClustalW algorithm  was implemented to align forward and reverse sequences of each segment to derive a consensus sequence of the full LTR (758-bp) region, a fragment of the gp21 p) genes . PhylogeThe sequences were aligned using the DAMBE program (version 4.2.13) In order to estimate the divergence time between the different clades, we initially performed a typical molecular clock analysis The 23 HTLV-1 infected individuals from Australia included ten women  and 13 men  . The fouenv gene. We therefore compared the gp21 env gene fragments of seven proviral sequences from Australia, including five new proviral strains  and two previously characterized sequences (MSHR-1 and Aus-RDJ)  The primary purpose of our work was to study the molecular relationship between the new Australian HTLV-1 proviral strains and those from HTLV-1 infected individuals in Australia and the neighboring islands whose sequences have been previously published. For most strains, the only available sequences in the Genbank database are the 522-bp fragment of the gp21 gag and Px-LTR fragments. Alignment of the 746-bp LTR fragments for these 27 strains revealed no significant deletion or insertion in comparison to the HTLV-1 ATK-1 reference strain. Within group comparisons of the 23 new Australian HTLV-1 strains indicate that they are closely related to each other , though quite divergent from the LTR strains from Vanuatu  and from the known HTLV-1c subtype prototype strain from the Solomon Islands (MEL5) .Complete LTR proviral sequences were obtained for all 27 samples by PCR amplification of both LTR-Using both NJ and ML methods, the phylogenetic analyses of the LTR region revealed two distinct subgroups within the Australo-Melanesian HTLV-1c subtype. The first group includes strains from the Solomon Islands and Vanuatu (Sol/Van) while the second comprises all the HTLV-1 Australian proviral sequences (Aus) . Unfortuenv and LTR fragments were consistent with the existence of two Australian HTLV-1c clades. The nature of this relationship was further clarified by comparing larger genomic fragments using both ML and NJ methods.Phylogenetic analyses using both gp21 gag-tax genes revealed a high degree of nucleotide homology among the Australian strains  and a comparable degree of divergence of the Australian strains relative to those from both Vanuatu  and the Solomon Islands . Additional phylogenetic analyses using both NJ and ML methods of the colinearized gag-tax  genomic fragment, confirmed the tree topology derived from the gp21 env and LTR analyses and demonstrated the existence of an Australian subgroup that was highly supported phylogenetically   . InteresThese analyses were performed using the complete proviral sequences obtained from four Australian samples: Aus-CS, Aus-NR, Aus-DF and Aus-GM . In addition, two of these complete proviral sequences were selected as the representative prototypes of each Australian clade . The overall range of nucleotide divergence of the first complete Australian strains from the prototypes ATK-1 and MEL5 was 7.8–8% and 3.3–3.4% respectively. The nucleotide homology between these two Australian prototypic sequences was 98.9% .gag-pol-env-tax  genomic fragments, including HTLV-1, HTLV-2 and HTLV-3 representative sequences available in Genbank, confirmed the existence of an Australian subgroup that was highly supported phylogenetically  Finally, we estimated the time of divergence for the various Australian strains using the evolution rate of the HTLV-1 LTR region, which has previously been determined by Lemey The origin of most HTLV-1 subtypes appears to be linked to ancient and multiple episodes of interspecies transmission between STLV-1-infected non-human primates (NHPs) and humans Despite the presence of STLV-1-infected NHP species in Asia, there is no evidence of recent interspecies transmission in this area env, the LTR and the colinearized gag-tax and gag-pol-env-tax genes, which confirm that HTLV-1 strains from the Solomon Islands and Vanuatu belong to the same subgroup. Based on a combination of paleo-anthropological data and genomic DNA analyses, it is believed that the initial human settlement of the Solomon archipelago dates from the Paleolithic period, ca. 30,000 years ago A first wave of migration led to the progressive colonization of the Solomon Islands, followed by the Vanuatu archipelago and finally, New Caledonia and the neighboring Melanesian islands. Consistent with the common origin of these Melanesian populations are our analyses performed on gp21 A second wave of migration is likely to have occurred from PNG to Australia. Indeed, it is thought that the occupation of Sahul, the continent formed when glacio-eustatically lowered sea levels exposed dry land connections between Australia and Papua New Guinea, may have commenced 45,000 years ago and continued until the end of the Pleistocene period 12,000 years ago In the present study, we reveal a high degree of genetic diversity among the HTLV-1c subtype proviral strains that infect the Indigenous people of central Australia. At least two different HTLV-1c genetic clades exist in this Indigenous population and these cluster according to the geographic origin of their human hosts. Thus, it is possible to propose that the common ancestor of the modern Australian HTLV-1 strains arrived in Australia when a group originating from the ancestral HTLV-1 infected population migrated from PNG and settled in Australia. Subsequently, the Australian population split  leading to continued viral evolution among small, isolated clan groups of Indigenous people dwelling in the remote desert regions of central Australia and this resulted in the speciation of the two Australian clades. The broad ethno-geographic distinction between the Indigenous human hosts of these Australian clades is particularly interesting given that considerable movement of Indigenous people has resulted from a century of European dominance in this region. Thus, Northern Ngarrkic speaking clan groups were moved to the South while Western Desert groups moved to the East, in each case toward ration supply centers that were established nearer the major regional center of Alice Springs Further studies, which characterize the HTLV-1c proviral strains that infect other Indigenous populations elsewhere in Australia and Oceania, will provide new insights into the origin of these retroviruses, potentially enhancing our understanding of the pathogenicity, evolution and modes of dissemination of these HTLV-1c variants and their human hosts.