The prevalence of human T-cell lymphotropic virus type 1 (HTLV-1) and hepatitis B virus (HBV) coinfection is high in certain Indigenous Australian populations, but its impact on HTLV-1 has not been described. We compared 2 groups of Indigenous adults infected with HTLV-1, either alone or coinfected with HBV. The 2 groups had a similar HTLV-1 proviral load, but there was a significant increase in clonal expansion of HTLV-1-infected lymphocytes in coinfected asymptomatic individuals. The degree of clonal expansion was correlated with the titer of HBV surface antigen. We conclude that HTLV-1/HBV coinfection may predispose to HTLV-1-associated malignant disease.

Chromatin looping controls gene expression by regulating promoter-enhancer contacts, the spread of epigenetic modifications, and the segregation of the genome into transcriptionally active and inactive compartments. We studied the impact on the structure and expression of host chromatin by the human retrovirus HTLV-1. We show that HTLV-1 disrupts host chromatin structure by forming loops between the provirus and the host genome; certain loops depend on the critical chromatin architectural protein CTCF, which we recently discovered binds to the HTLV-1 provirus. We show that the provirus causes two distinct patterns of abnormal transcription of the host genome in cis: bidirectional transcription in the host genome immediately flanking the provirus, and clone-specific transcription in cis at non-contiguous loci up to >300 kb from the integration site. We conclude that HTLV-1 causes insertional mutagenesis up to the megabase range in the host genome in >104 persistently-maintained HTLV-1+ T-cell clones in vivo.

Spontaneous sympathetic baroreflex sensitivity (BRS) is a valuable tool for assessing how well the baroreflex buffers beat-to-beat changes in blood pressure. However, there has yet to be a study involving appropriate statistical tests to examine the stability of sympathetic BRS within an experimental session and the repeatability between separate sessions. The aim of this study was to use intra-class correlations, ordinary least products regression, and Bland-Altman analyses to examine the stability and repeatability of spontaneous sympathetic BRS assessment. In addition, the influence of recording duration on values of BRS was assessed. In eighty-four healthy young individuals (49 males, 35 females), continuous measurements of blood pressure, heart rate and muscle sympathetic nerve activity (MSNA) were recorded for 10 min. In a subgroup of 13 participants (11 male, 2 female) the measurements were repeated on a separate day. Sympathetic BRS was quantified using MSNA burst incidence (BRSinc) and total MSNA (BRStotal) for the first 5-min period, the second 5-min period, and a 2-min segment taken from the second 5-min period. Intra-class correlation coefficients indicated moderate stability in sympathetic BRSinc and BRStotal between the first and second 5-min periods in males (BRSincr = 0.63, BRStotalr = 0.78) and females (BRSincr = 0.61, BRStotalr = 0.47) with no proportional bias, but with fixed bias for BRSinc in females. When comparing the first 5-min with the 2-min period (n = 76), the intra-class correlation coefficient indicated poor to moderate repeatability in sympathetic BRSinc and BRStotal for males (BRSincr = -0.01, BRStotalr = 0.70) and females (BRSincr = 0.46, BRStotalr = 0.39). However, Bland-Altman analysis revealed a fixed bias for BRStotal in males and proportional bias for BRStotal in females, with lower BRS values for 5-min recordings. In the subgroup, intra-class correlations indicated moderate repeatability for measures of BRSinc (9 male, 2 female, r = 0.63) and BRStotal (6 male, 2 female, r = 0.68) assessed using 5-min periods recorded on separate days. However, Bland-Altman analysis indicated proportional bias for BRSinc and fixed bias for BRStotal. In conclusion, measures of spontaneous sympathetic BRS are moderately stable and repeatable within and between testing sessions in healthy young adults, provided that the same length of recording is used when making comparisons.

HIV-1 integrates more frequently into transcribed genes, however the biological significance of HIV-1 integration targeting has remained elusive. Using a selective high-throughput chemical screen, we discovered that the cardiac glycoside digoxin inhibits wild-type HIV-1 infection more potently than HIV-1 bearing a single point mutation (N74D) in the capsid protein. We confirmed that digoxin repressed viral gene expression by targeting the cellular Na+/K+ ATPase, but this did not explain its selectivity. Parallel RNAseq and integration mapping in infected cells demonstrated that digoxin inhibited expression of genes involved in T-cell activation and cell metabolism. Analysis of >400,000 unique integration sites showed that WT virus integrated more frequently than N74D mutant within or near genes susceptible to repression by digoxin and involved in T-cell activation and cell metabolism. Two main gene networks down-regulated by the drug were CD40L and CD38. Blocking CD40L by neutralizing antibodies selectively inhibited WT virus infection, phenocopying digoxin. Thus the selectivity of digoxin depends on a combination of integration targeting and repression of specific gene networks. The drug unmasked a functional connection between HIV-1 integration and T-cell activation. Our results suggest that HIV-1 evolved integration site selection to couple its early gene expression with the status of target CD4+ T-cells, which may affect latency and viral reactivation.

Continuous intramuscular stimulation of tibialis anterior (TA) was used to test the hypothesis that irregular trains of stimuli can increase force production and offset the magnitude of fatigue when compared with a continuous train of regular stimuli at an identical mean frequency (19 or 24 Hz). To achieve this, tungsten microelectrodes were inserted into the muscle belly into the motor point of the tibialis anterior muscle of able-bodied individuals (aged 19-50) and stimulated at current intensities ranging from 5 to 7 mA. The motor point was stimulated with a continuous train of regular stimulation at either 19 or 24 Hz (n = 11) or until the force declined below 25% of the peak force at the onset of stimulation. For the first seven subjects, no fatigue was exhibited, and thus, we simply compared the forces generated by the regular and irregular segments of the continuous train (120 sec for each segment). For four additional subjects, we delivered a higher frequency train (24 Hz) that elicited some fatigue. Once the force had declined below 25% of the initial peak force (which took between 140 and 210 sec), the continuous irregular train was integrated. Interestingly, for those subjects who exhibited muscular fatigue, force always began to rise again once the irregularity was incorporated into the continuous regular train of stimulation at the identical mean frequency (24 Hz). We conclude that incorporating irregularity into continuous trains of stimuli offers a significant advantage to the human neuromuscular system during both fatigued and nonfatigued states and could offer benefits to therapies such as functional electrical stimulation (FES).

The regulation of proviral latency is a central problem in retrovirology. We postulate that the genomic integration site of human T lymphotropic virus type 1 (HTLV-1) determines the pattern of expression of the provirus, which in turn determines the abundance and pathogenic potential of infected T cell clones in vivo. We recently developed a high-throughput method for the genome-wide amplification, identification and quantification of proviral integration sites. Here, we used this protocol to test two hypotheses. First, that binding sites for transcription factors and chromatin remodelling factors in the genome flanking the proviral integration site of HTLV-1 are associated with integration targeting, spontaneous proviral expression, and in vivo clonal abundance. Second, that the transcriptional orientation of the HTLV-1 provirus relative to that of the nearest host gene determines spontaneous proviral expression and in vivo clonal abundance. Integration targeting was strongly associated with the presence of a binding site for specific host transcription factors, especially STAT1 and p53. The presence of the chromatin remodelling factors BRG1 and INI1 and certain host transcription factors either upstream or downstream of the provirus was associated respectively with silencing or spontaneous expression of the provirus. Cells expressing HTLV-1 Tax protein were significantly more frequent in clones of low abundance in vivo. We conclude that transcriptional interference and chromatin remodelling are critical determinants of proviral latency in natural HTLV-1 infection.

The human T-cell leukemia virus type 1 (HTLV-1) transactivator protein Tax has pleiotropic functions in the host cell affecting cell-cycle regulation, DNA damage response pathways and apoptosis. These actions of Tax have been implicated in the persistence and pathogenesis of HTLV-1-infected cells. It is now known that tax expression occurs in transcriptional bursts of the proviral plus-strand, but the effects of the burst on host transcription are not fully understood. We carried out RNA sequencing of two naturally-infected T-cell clones transduced with a Tax-responsive Timer protein, which undergoes a time-dependent shift in fluorescence emission, to study transcriptional changes during successive phases of the HTLV-1 plus-strand burst. We found that the transcriptional regulation of genes involved in the NF-κB pathway, cell-cycle regulation, DNA damage response and apoptosis inhibition were immediate effects accompanying the plus-strand burst, and are limited to the duration of the burst. The results distinguish between the immediate and delayed effects of HTLV-1 reactivation on host transcription, and between clone-specific effects and those observed in both clones. The major transcriptional changes in the infected host T-cells observed here, including NF-κB, are transient, suggesting that these pathways are not persistently activated at high levels in HTLV-1-infected cells. The two clones diverged strongly in their expression of genes regulating the cell cycle. Up-regulation of senescence markers was a delayed effect of the proviral plus-strand burst and the up-regulation of some pro-apoptotic genes outlasted the burst. We found that activation of the aryl hydrocarbon receptor (AhR) pathway enhanced and prolonged the proviral burst, but did not increase the rate of reactivation. Our results also suggest that sustained plus-strand expression is detrimental to the survival of infected cells.

There is growing evidence that CD8+ cytotoxic T lymphocyte (CTL) responses can contribute to long-term remission of many malignancies. The etiological agent of adult T-cell leukemia/lymphoma (ATL), human T lymphotropic virus type-1 (HTLV-1), contains highly immunogenic CTL epitopes, but ATL patients typically have low frequencies of cytokine-producing HTLV-1-specific CD8+ cells in the circulation. It remains unclear whether patients with ATL possess CTLs that can kill the malignant HTLV-1 infected clone. Here we used flow cytometric staining of TCRVβ and cell adhesion molecule-1 (CADM1) to identify monoclonal populations of HTLV-1-infected T cells in the peripheral blood of patients with ATL. Thus, we quantified the rate of CD8+-mediated killing of the putative malignant clone in ex vivo blood samples. We observed that CD8+ cells from ATL patients were unable to lyse autologous ATL clones when tested directly ex vivo. However, short in vitro culture restored the ability of CD8+ cells to kill ex vivo ATL clones in some donors. The capacity of CD8+ cells to lyse HTLV-1 infected cells which expressed the viral sense strand gene products was significantly enhanced after in vitro culture, and donors with an ATL clone that expressed the HTLV-1 Tax gene were most likely to make a detectable lytic CD8+ response to the ATL cells. We conclude that some patients with ATL possess functional tumour-specific CTLs which could be exploited to contribute to control of the disease.

Human T-cell leukemia virus type 1 (HTLV-1) infects mainly CD4+CCR4+ effector/memory T cells in vivo. However, it remains unknown whether HTLV-1 preferentially infects these T cells or this virus converts infected precursor cells to specialized T cells. Expression of viral genes in vivo is critical to study viral replication and proliferation of infected cells. Therefore, we first analyzed viral gene expression in non-human primates naturally infected with simian T-cell leukemia virus type 1 (STLV-1), whose virological attributes closely resemble those of HTLV-1. Although the tax transcript was detected only in certain tissues, Tax expression was much higher in the bone marrow, indicating the possibility of de novo infection. Furthermore, Tax expression of non-T cells was suspected in bone marrow. These data suggest that HTLV-1 infects hematopoietic cells in the bone marrow. To explore the possibility that HTLV-1 infects hematopoietic stem cells (HSCs), we analyzed integration sites of HTLV-1 provirus in various lineages of hematopoietic cells in patients with HTLV-1 associated myelopathy/tropical spastic paraparesis (HAM/TSP) and a HTLV-1 carrier using the high-throughput sequencing method. Identical integration sites were detected in neutrophils, monocytes, B cells, CD8+ T cells and CD4+ T cells, indicating that HTLV-1 infects HSCs in vivo. We also detected Tax protein in myeloperoxidase positive neutrophils. Furthermore, dendritic cells differentiated from HTLV-1 infected monocytes caused de novo infection to T cells, indicating that infected monocytes are implicated in viral spreading in vivo. Certain integration sites were re-detected in neutrophils from HAM/TSP patients at different time points, indicating that infected HSCs persist and differentiate in vivo. This study demonstrates that HTLV-1 infects HSCs, and infected stem cells differentiate into diverse cell lineages. These data indicate that infection of HSCs can contribute to the persistence and spread of HTLV-1 in vivo.

Background Diesel exhaust (DE) emissions are a major contributor to ambient air pollution and are strongly associated with cardiovascular morbidity and mortality. Exposure to traffic-related particulate matter is linked with acute adverse cardiovascular events; however, the mechanisms are not fully understood. We examined the role of the autonomic nervous system during exposure to DE that has previously only been indirectly investigated. Methods and Results Using microneurography, we measured muscle sympathetic nerve activity (MSNA) directly in the peroneal nerve of 16 healthy individuals. MSNA, heart rate, and respiration were recorded while subjects rested breathing filtered air, filtered air with an exposure mask, and standardized diluted DE (300 µg/m3) through the exposure mask. Heart rate variability was assessed from an ECG. DE inhalation rapidly causes an increase in number of MSNA bursts as well as the size of bursts within 10 minutes, peaking by 30 minutes (P<0.001), compared with baseline filtered air with an exposure mask. No significant changes occurred in heart rate variability indices during DE exposure; however, MSNA frequency correlated negatively with total power (r2=0.294, P=0.03) and low frequency (r2=0.258, P=0.045). Heart rate correlated positively with MSNA frequency (r2=0.268, P=0.04) and the change in percentage of larger bursts (burst amplitude, height >50% of the maximum burst) from filtered air with an exposure mask (r2=0.368, P=0.013). Conclusions Our study provides direct evidence for the rapid modulation of the autonomic nervous system after exposure to DE, with an increase in MSNA. The quick increase in sympathetic outflow may explain the strong epidemiological data associating traffic-related particulate matter to acute adverse cardiovascular events such as myocardial infarction. Registration URL: https://www.clinicaltrials.gov; Unique identifier: NCT02892279.

Human T-cell leukemia virus type 1 (HTLV-1) spreads through cell contact. Therefore, this virus persists and propagates within the host by two routes: clonal proliferation of infected cells and de novo infection. The proliferation is influenced by the host immune responses and expression of viral genes. However, the detailed mechanisms that control clonal expansion of infected cells remain to be elucidated. In this study, we show that newly infected clones were strongly suppressed, and then stable clones were selected, in a patient who was infected by live liver transplantation from a seropositive donor. Conversely, most HTLV-1+ clones persisted in patients who received hematopoietic stem cell transplantation from seropositive donors. To clarify the role of cell-mediated immunity in this clonal selection, we suppressed CD8+ or CD16+ cells in simian T-cell leukemia virus type 1 (STLV-1)-infected Japanese macaques. Decreasing CD8+ T cells had marginal effects on proviral load (PVL). However, the clonality of infected cells changed after depletion of CD8+ T cells. Consistent with this, PVL at 24 hours in vitro culture increased, suggesting that infected cells with higher proliferative ability increased. Analyses of provirus in a patient who received Tax-peptide pulsed dendritic cells indicate that enhanced anti-Tax immunity did not result in a decreased PVL although it inhibited recurrence of ATL. We postulate that in vivo selection, due to the immune response, cytopathic effects of HTLV-1 and intrinsic attributes of infected cells, results in the emergence of clones of HTLV-1-infected T cells that proliferate with minimized HTLV-1 antigen expression.

HTLV-1 causes proliferation of clonal populations of infected T cells in vivo, each clone defined by a unique proviral integration site in the host genome. The proviral load is strongly correlated with odds of the inflammatory disease HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP). There is evidence that asymptomatic HTLV-1 carriers (ACs) have a more effective CD8 + T cell response, including a higher frequency of HLA class I alleles able to present peptides from a regulatory protein of HTLV-1, HBZ. We have previously shown that specific features of the host genome flanking the proviral integration site favour clone survival and spontaneous expression of the viral transactivator protein Tax in naturally infected PBMCs ex vivo. However, the previous studies were not designed or powered to detect differences in integration site characteristics between ACs and HAM/TSP patients. Here, we tested the hypothesis that the genomic environment of the provirus differs systematically between ACs and HAM/TSP patients, and between individuals with strong or weak HBZ presentation.

Introduction: Muscle sympathetic nerve activity (MSNA) may play a role in insulin resistance in obesity. However, the direction and nature of the relationship between MSNA and insulin resistance in obesity remain unclear. We hypothesized that resting MSNA would correlate inversely with both muscle and liver insulin sensitivity and that it would be higher in insulin-resistant vs. insulin-sensitive subjects. Materials and methods: Forty-five non-diabetic obese subjects were studied. As no significant relationships were found in women, the data presented in on 22 men aged 48 ± 12 years. Two-step (15 and 80 mU/m2/min) hyperinsulinaemic-euglycaemic clamps were performed using deuterated glucose to determine liver and muscle insulin sensitivity. Clinical and metabolic parameters were assessed. MSNA was measured via a microelectrode inserted percutaneously into the common peroneal nerve. Results: MSNA burst frequency correlated inversely with liver insulin sensitivity (r = -0.53, P = 0.02) and positively with the hepatokines C-reactive protein (CRP) and fibroblast growth factor (FGF)-19 (r = 0.57, P = 0.006, and r = -0.47, P = 0.03, respectively). MSNA burst frequency was lower in Liversen compared to Liverres (27 ± 5 vs. 38 ± 2 bursts per minute; P = 0.03). Muscle insulin sensitivity was unrelated to MSNA. Discussion: Sympathetic neural activation is related to liver insulin sensitivity and circulating hepatokines CRP and FGF-19 in non-diabetic obese men. These results suggest a potential hepato-endocrine-autonomic axis. Future studies are needed to clarify the influence of MSNA on liver insulin sensitivity in men.

Human T-lymphotropic Virus Type I (HTLV-1) is a retrovirus that persistently infects 5-10 million individuals worldwide and causes disabling or fatal inflammatory and malignant diseases. The majority of the HTLV-1 proviral load is found in CD4(+) T cells, and the phenotype of adult T cell leukemia (ATL) is typically CD4(+). HTLV-1 also infects CD8(+) cells in vivo, but the relative abundance and clonal composition of the two infected subpopulations have not been studied. We used a high-throughput DNA sequencing protocol to map and quantify HTLV-1 proviral integration sites in separated populations of CD4(+) cells, CD8(+) cells and unsorted peripheral blood mononuclear cells from 12 HTLV-1-infected individuals.

Human T-lymphotropic virus type 1 (HTLV-1) and type 2 (HTLV-2) both cause lifelong persistent infections, but differ in their clinical outcomes. HTLV-1 infection causes a chronic or acute T-lymphocytic malignancy in up to 5% of infected individuals whereas HTLV-2 has not been unequivocally linked to a T-cell malignancy. Virus-driven clonal proliferation of infected cells both in vitro and in vivo has been demonstrated in HTLV-1 infection. However, T-cell clonality in HTLV-2 infection has not been rigorously characterized. In this study we used a high-throughput approach in conjunction with flow cytometric sorting to identify and quantify HTLV-2-infected T-cell clones in 28 individuals with natural infection. We show that while genome-wide integration site preferences in vivo were similar to those found in HTLV-1 infection, expansion of HTLV-2-infected clones did not demonstrate the same significant association with the genomic environment of the integrated provirus. The proviral load in HTLV-2 is almost confined to CD8+ T-cells and is composed of a small number of often highly expanded clones. The HTLV-2 load correlated significantly with the degree of dispersion of the clone frequency distribution, which was highly stable over ∼8 years. These results suggest that there are significant differences in the selection forces that control the clonal expansion of virus-infected cells in HTLV-1 and HTLV-2 infection. In addition, our data demonstrate that strong virus-driven proliferation per se does not predispose to malignant transformation in oncoretroviral infections.

Estimation of immunological and microbiological diversity is vital to our understanding of infection and the immune response. For instance, what is the diversity of the T cell repertoire? These questions are partially addressed by high-throughput sequencing techniques that enable identification of immunological and microbiological "species" in a sample. Estimators of the number of unseen species are needed to estimate population diversity from sample diversity. Here we test five widely used non-parametric estimators, and develop and validate a novel method, DivE, to estimate species richness and distribution. We used three independent datasets: (i) viral populations from subjects infected with human T-lymphotropic virus type 1; (ii) T cell antigen receptor clonotype repertoires; and (iii) microbial data from infant faecal samples. When applied to datasets with rarefaction curves that did not plateau, existing estimators systematically increased with sample size. In contrast, DivE consistently and accurately estimated diversity for all datasets. We identify conditions that limit the application of DivE. We also show that DivE can be used to accurately estimate the underlying population frequency distribution. We have developed a novel method that is significantly more accurate than commonly used biodiversity estimators in microbiological and immunological populations.

The human retroviruses HTLV-1 (human T cell leukemia virus type 1) and HIV-1 persist in vivo as a reservoir of latently infected T cell clones. It is poorly understood what determines which clones survive in the reservoir. We compared >160,000 HTLV-1 integration sites (>40,000 HIV-1 sites) from T cells isolated ex vivo from naturally infected individuals with >230,000 HTLV-1 integration sites (>65,000 HIV-1 sites) from in vitro infection to identify genomic features that determine selective clonal survival. Three statistically independent factors together explained >40% of the observed variance in HTLV-1 clonal survival in vivo: the radial intranuclear position of the provirus, its genomic distance from the centromere, and the intensity of local host genome transcription. The radial intranuclear position of the provirus and its distance from the centromere also explained ~7% of clonal persistence of HIV-1 in vivo. Selection for the intranuclear and intrachromosomal location of the provirus and host transcription intensity favors clonal persistence of human retroviruses in vivo.

Mal de Debarquement Syndrome (MdDS) is a condition characterized by a persistent perception of self-motion, in most cases triggered from exposure to passive motion (e.g., boat travel, a car ride, flights). Patients whose onset was triggered in this way are categorized as Motion-Triggered (MT) subtype or onset group. However, the same syndrome can occur spontaneously or after non-motion events, such as childbirth, high stress, surgery, etc. Patients who were triggered in this way are categorized as being of the Spontaneous/Other (SO) subtype or onset group. The underlying pathophysiology of MdDS is unknown and there has been some speculation that the two onset groups are separate entities. However, despite the differences in onset between the subtypes, symptoms are parallel and a significant female predominance has been shown. To date, the role of gonadal hormones in MdDS pathophysiology has not been investigated. This study aimed to evaluate the hormonal profile of MdDS patients, the presence of hormonal conditions, the influence of hormones on symptomatology and to assess possible hormonal differences between onset groups. In addition, the prevalence of migraine and motion sickness and their relation to MdDS were assessed.

Long-lasting experimental muscle pain elicits divergent muscle sympathetic responses, with some individuals exhibiting a persistent increase in muscle sympathetic nerve activity (MSNA), and others a decrease. These divergent responses are thought to result from sustained functional changes in specific brain regions that modulate the cardiovascular responses to pain.

Human T-lymphotropic virus type 1 (HTLV-1) persists by driving clonal proliferation of infected T lymphocytes. A high proviral load predisposes to HTLV-1-associated diseases. Yet the reasons for the variation within and between persons in the abundance of HTLV-1-infected clones remain unknown. We devised a high-throughput protocol to map the genomic location and quantify the abundance of > 91,000 unique insertion sites of the provirus from 61 HTLV-1(+) persons and > 2100 sites from in vitro infection. We show that a typical HTLV-1-infected host carries between 500 and 5000 unique insertion sites. We demonstrate that negative selection dominates during chronic infection, favoring establishment of proviruses integrated in transcriptionally silenced DNA: this selection is significantly stronger in asymptomatic carriers. We define a parameter, the oligoclonality index, to quantify clonality. The high proviral load characteristic of HTLV-1-associated inflammatory disease results from a larger number of unique insertion sites than in asymptomatic carriers and not, as previously thought, from a difference in clonality. The abundance of established HTLV-1 clones is determined by genomic features of the host DNA flanking the provirus. HTLV-1 clonal expansion in vivo is favored by orientation of the provirus in the same sense as the nearest host gene.