Carolina Alvarez

Ascorbic Acid Has Superior Ex Vivo Antiproliferative, Cell Death-Inducing and Immunomodulatory Effects over IFN-α in HTLV-1-Associated Myelopathy

Background Clear therapeutic guidelines for HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP) are missing due to the lack of randomized double-blind controlled clinical trials. Moderate yet similar clinical benefit has been demonstrated for IFN-α and high-dose ascorbic acid (AA) monotherapy in a large open clinical trial. However, there is a lack of in vivo and in vitro studies exploring and comparing the effects of high-dose AA and IFN-α treatment in the context of HAM/TSP. Therefore, we performed the first comparative analysis of the ex vivo and in vitro molecular and cellular mechanisms of action of IFN-α and high-dose AA in HAM/TSP. Principal Findings Through thymidine incorporation and quantification of Th1/Th2/Th17 cytokines, we demonstrate that high-dose AA displays differential and superior antiproliferative and immunomodulatory effects over IFN-α in HAM/TSP PBMCs ex vivo. In addition, high-dose AA, but not IFN-α, induced cell death in both HAM/TSP PBMCs and HTLV-1-infected T-cell lines MT-2 and MT-4. Microarray data combined with pathway analysis of MT-2 cells revealed AA-induced regulation of genes associated with cell death, including miR-155. Since miR-155 has recently been demonstrated to up-regulate IFN-γ, this microRNA might represent a novel therapeutic target in HAM/TSP, as recently demonstrated in multiple sclerosis, another neuroinflammatory disease. On the other hand, IFN-α selectively up-regulated antiviral and immune-related genes. Conclusions In comparison to IFN-α, high-dose AA treatment has superior ex vivo and in vitro cell death-inducing, antiproliferative and immunomodulatory anti-HTLV-1 effects. Differential pathway activation by both drugs opens up avenues for targeted treatment in specific patient subsets.

CD80+ and CD86+ B cells as biomarkers and possible therapeutic targets in HTLV-1 associated myelopathy/tropical spastic paraparesis and multiple sclerosis

BackgroundHuman T-cell lymphotropic virus (HTLV-1) is the causative agent of the incapacitating, neuroinflammatory disease HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP). Currently, there are no disease-modifying therapies with long-term clinical benefits or validated biomarkers for clinical follow-up in HAM/TSP. Although CD80 and CD86 costimulatory molecules play prominent roles in immune regulation and reflect disease status in multiple sclerosis (MS), data in HAM/TSP are lacking.MethodsUsing flow cytometry, we quantified ex vivo and in vitro expression of CD80 and CD86 in PBMCs of healthy controls, HTLV-1-infected individuals with and without HAM/TSP, and MS patients. We hypothesized ex vivo CD80 and CD86 expressions and their in vitro regulation by interferon (IFN)-α/β mirror similarities between HAM/TSP and MS and hence might reveal clinically useful biomarkers in HAM/TSP.ResultsEx vivo expression of CD80 and CD86 in T and B cells increased in all HTLV-1 infected individuals, but with a selective defect for B cell CD86 upregulation in HAM/TSP. Despite decreased total B cells with increasing disease duration (p = 0.0003, r = −0.72), CD80+ B cells positively correlated with disease severity (p = 0.0017, r = 0.69) in HAM/TSP. B cell CD80 expression was higher in women with HAM/TSP, underscoring that immune markers can reflect the female predominance observed in most autoimmune diseases. In contrast to MS patients, CD80+ (p = 0.0001) and CD86+ (p = 0.0054) lymphocytes expanded upon in vitro culture in HAM/TSP patients. The expansion of CD80+ and CD86+ T cells but not B cells was associated with increased proliferation in HTLV-1 infection. In vitro treatment with IFN-β but not IFN-α resulted in a pronounced increase of B cell CD86 expression in healthy controls, as well as in patients with neuroinflammatory disease (HAM/TSP and MS), similar to in vivo treatment in MS.ConclusionsWe propose two novel biomarkers, ex vivo CD80+ B cells positively correlating to disease severity and CD86+ B cells preferentially induced by IFN-β, which restores defective upregulation in HAM/TSP. This study suggests a role for B cells in HAM/TSP pathogenesis and opens avenues to B cell targeting (with proven clinical benefit in MS) in HAM/TSP but also CD80-directed immunotherapy, unprecedented in both HAM/TSP and MS.

Enhanced Heterosexual Transmission Hypothesis for the Origin of Pandemic HIV-1

HIV-1 M originated from SIVcpz endemic in chimpanzees from southeast Cameroon or neighboring areas, and it started to spread in the early 20th century. Here we examine the factors that may have contributed to simian-to-human transmission, local transmission between humans, and export to a city. The region had intense ape hunting, social disruption, commercial sex work, STDs, and traffic to/from Kinshasa in the period 1899–1923. Injection treatments increased sharply around 1930; however, their frequency among local patients was far lower than among modern groups experiencing parenteral HIV-1 outbreaks. Recent molecular datings of HIV-1 M fit better the period of maximal resource exploitation and trade links than the period of high injection intensity. We conclude that although local parenteral outbreaks might have occurred, these are unlikely to have caused massive transmission. World War I led to additional, and hitherto unrecognized, risks of HIV-1 emergence. We propose an Enhanced Heterosexual Transmission Hypothesis for the origin of HIV-1 M, featuring at the time and place of its origin a coincidence of favorable co-factors (ape hunting, social disruption, STDs, and mobility) for both cross-species transmission and heterosexual spread. Our hypothesis does not exclude a role for parenteral transmission in the initial viral adaptation.

Family Aggregation of Human T-Lymphotropic Virus 1-Associated Diseases: A Systematic Review

Human T-lymphotropic virus 1 (HTLV-1) is a retrovirus that produces a persistent infection. Two transmission routes (from mother to child and via sexual intercourse) favor familial clustering of HTLV-1. It is yet unknown why most HTLV-1 carriers remain asymptomatic while about 10% of them develop complications. HTLV-1 associated diseases were originally described as sporadic entities, but familial presentations have been reported. To explore what is known about family aggregation of HTLV-1-associated diseases we undertook a systematic review. We aimed at answering whether, when, and where family aggregation of HTLV-1-associated diseases was reported, which relatives were affected and which hypotheses were proposed to explain aggregation. We searched MEDLINE, abstract books of HTLV conferences and reference lists of selected papers. Search terms used referred to HTLV-1 infection, and HTLV-1-associated diseases, and family studies. HTLV-1-associated diseases considered are adult T-cell leukemia/lymphoma (ATLL), HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP), HTLV-1-associated uveitis, and infective dermatitis. Seventy-four records reported HTLV-1-associated diseases in more than one member of the same family and were included. Most reports came from HTLV-1-endemic countries, mainly Japan (n = 30) and Brazil (n = 10). These reports described a total of 270 families in which more than one relative had HTLV-1-associated diseases. In most families, different family members suffered from the same disease (n = 223). The diseases most frequently reported were ATLL (115 families) and HAM/TSP (102 families). Most families (n = 144) included two to four affected individuals. The proportion of ATLL patients with family history of ATLL ranged from 2 to 26%. The proportion of HAM/TSP patients with family history of HAM/TSP ranged from 1 to 48%. The predominant cluster types for ATLL were clusters of siblings and parent-child pairs and for HAM/TSP, an affected parent with one or more affected children. The evidence in the literature, although weak, does suggest that HTLV-1-associated diseases sometimes cluster in families. Whether familial transmission of HTLV-1 is the only determining factor, or whether other factors are also involved, needs further research.

A Fashi Lymphoproliferative Phenotype Reveals Non-Apoptotic Fas Signaling in HTLV-1-Associated Neuroinflammation

Human T-cell lymphotropic virus (HTLV)-1 was the first human retrovirus to be associated to cancer, namely adult T-cell leukemia (ATL), but its pathogenesis remains enigmatic, since only a minority of infected individuals develops either ATL or the neuroinflammatory disorder HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP). A functional FAS -670 polymorphism in an interferon (IFN)-regulated STAT1-binding site has been associated to both ATL and HAM/TSP susceptibility. Fashi T stem cell memory (Tscm) cells have been identified as the hierarchical apex of ATL, but have not been investigated in HAM/TSP. In addition, both FAS and STAT1 have been identified in an IFN-inducible HAM/TSP gene signature, but its pathobiological significance remains unclear. We comprehensively explored Fas expression (protein/mRNA) and function in lymphocyte activation, apoptosis, proliferation, and transcriptome, in PBMC from a total of 47 HAM/TSP patients, 40 asymptomatic HTLV-1-infected individuals (AC), and 58 HTLV-1 -uninfected healthy controls. Fas surface expression followed a two-step increase from HC to AC and from AC to HAM/TSP. In HAM/TSP, Fas levels correlated positively to lymphocyte activation markers, but negatively to age of onset, linking Fashi cells to earlier, more aggressive disease. Surprisingly, increased lymphocyte Fas expression in HAM/TSP was linked to decreased apoptosis and increased lymphoproliferation upon in vitro culture, but not to proviral load. This Fashi phenotype is HAM/TSP-specific, since both ex vivo and in vitro Fas expression was increased as compared to multiple sclerosis (MS), another neuroinflammatory disorder. To elucidate the molecular mechanism underlying non-apoptotic Fas signaling in HAM/TSP, we combined transcriptome analysis with functional assays, i.e., blocking vs. triggering Fas receptor in vitro with antagonist and agonist-, anti-Fas mAb, respectively. Treatment with agonist anti-Fas mAb restored apoptosis, indicating biased, but not defective Fas signaling in HAM/TSP. In silico analysis revealed biased Fas signaling toward proliferation and inflammation, driven by RelA/NF-κB. Correlation of Fas transcript levels with proliferation (but not apoptosis) was confirmed in HAM/TSP ex vivo transcriptomes. In conclusion, we demonstrated a two-step increase in Fas expression, revealing a unique Fashi lymphocyte phenotype in HAM/TSP, distinguishable from MS. Non-apoptotic Fas signaling might fuel HAM/TSP pathogenesis, through increased lymphoproliferation, inflammation, and early age of onset.

HAM/TSP in relatives of HAM/TSP cases and in relatives of asymptomatic HTLV-1 carriers

To assess the hypothesis that HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP) runs in families, we compared the frequency of HAM/ TSP among HTLV-1-positive relatives of HAM/TSP patients with the frequency of HAM/TSP among HTLV1-positive relatives of asymptomatic HTLV-1 carriers. We reviewed available information at the Instituto de Medicina Tropical Alexander von Humboldt in Lima (period 1990-2012). Index cases with HAM/TSP were defined as unrelated, HTLV-1-positive patients with a clinical diagnosis of HAM/TSP for whom HAM/TSP was the motive for HTLV-1 testing and who brought ≥1 relative (blood relatives and/or partners) for HTLV-1 testing. Asymptomatic index cases were defined as unrelated, asymptomatic HTLV-1 carriers who were tested for HTLV-1 as candidate blood donors and brought ≥1 relative for screening. In the family studies of 334 index cases with HAM/TSP, 1124 relatives were tested, 318/1124 (28%) were HTLV-1 positive, and 30/318 (9%) had HAM/TSP. In the family studies of 230 asymptomatic index cases, 544 relatives were tested, 204/544 (38%) were HTLV-1 positive, and 15/204 (7%) had HAM/TSP. We classified the relatives in groups based on sex and age. HAM/TSP frequency increased with age and was higher in women than in men. We found no significant differences in HAM/TSP frequency between relatives of HAM/TSP index cases and relatives of asymptomatic index cases. In total, there were 21 families with 2 HAM/TSP cases, 4 families with 3 HAM/ TSP cases, and 1 family with 5 HAM/TSP cases. This analysis approach suggests that HAM/TSP usually affects isolated people, but that in some, particular families, HAM/ TSP clusters can occur.

A Peruvian family with a high burden of HTLV-1-associated myelopathy/tropical spastic paraparesis.

Human T-lymphotropic virus 1 (HTLV-1) is frequent in Peru; an estimated 1–2% of the Peruvian population carry this retrovirus. HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP) is a chronic disabling disease that affects about 1% of the carriers of HTLV-1. It is not yet known why some HTLV-1-infected people develop HAM/TSP while others do not. In this case report, we present a family with an unusually high burden of HAM/TSP: 5 (the 2 parents and 3 of their children) of 7 HTLV-1 carriers developed the same disease. We describe the clinical presentation and discuss the clustering of disease against the current knowledge of the pathogenesis of HAM/TSP. Families such as this may hold the key to discovering which factors trigger the development of HAM/TSP.

Differential miRNA expression profiles in Peruvian HTLV-1 carriers

MicroRNAs (miRNAs) are small non-coding RNAs that regulate protein expression. HTLV-1 is able to promote oncogenesis in T cells by altering the expression of miRNAs involved in the control of cell-cycle. It is not known whether HTLV-1 deregulates miRNAs expression in cells of HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP) patients. To asses if HTLV-1 infection might alter the expression of miRNAs involved in inflammatory response, we evaluated the expression of 84 miRNAs involved in inflammatory process in asymptomatic HTLV-1 carriers (AC) and HAM/TSP patients using the miScript miRNA PCR Array Human Inflammatory Response & Autoimmunity (SABioscience). For this purpose, fourteen HTLV-1-positive individuals were selected and classified into three groups: five asymptomatic carriers (AC), 4 HAM/TSP patients with EDSS score of 1-5 (=mild HAM/TSP), and 5 HAM/TSP patients with EDSS score of 5.5-9 (=severe HAM/TSP). Total RNA was isolated from PBMCs and pooled according to the groups. qBase software was used for normalization, ANOVA was used for comparisons and False Dicosvery Rate to correct for multiple comparisons. We found nine differentially expressed miRNAs between AC and HAM/TSP patients (mild and severe HAM/TSP). Twelve miRNAs were differentially expressed among mild HAM/TSP, severe HAM/TSP and AC groups. These findings support results previously reported in Adult T-cell leukemia/lymphoma (ATLL) cells, in which hsa-miR-145, miR-130a, miR181a and miR101a were found to be down-regulated and miR-30d was found to be up-regulated in comparison to those of healthy donors. Further analysis to confirm these findings are needed.

Monocyte accumulation is an early event in HAM/TSP pathogenesis, while monocyte activation and IFN-regulated gene expression persist in chronic HAM/TSP

Tattermusch et al (2012) identified an IFN-inducible gene signature in whole blood of HAM/TSP patients, with a strong myeloid component, while abortive HTLV-1 infection induces monocyte apoptosis (Sze et al. 2013). We previously demonstrated that B cell CD80 expression correlates to disease severity in HAM/TSP (Menezes et al 2014), whereas B cell CD86 is selectively up-regulated by IFN-beta in both HAM/TSP and multiple sclerosis (MS). In this study, we propose a cell type-and gene-specific, rather than a generalized IFN response in HAM/TSP. Using polychromatic flow cytometry, comprehensive phenotyping of monocytes (CD14, CD64, CD80, CD86, CD95/Fas, HLA-DR) was performed in a total of 53 individuals (HAM/TSP patients, asymptomatic HTLV-1-infected and uninfected controls), and absolute and relative monocyte counts were obtained from >600 HTLV-1-infected individuals with complete clinical follow-up and proviral load. Ex vivo monocyte levels increased in early HAM/TSP (p<0.01), independent of proviral load, and were significantly correlated to age of onset of HAM/TSP in both Brazilian and Peruvian cohorts. On the other hand, monocyte activation measured by systemic soluble CD14 was significantly increased in chronic (p<0.01) but not early HAM/TSP, whereas CD95 and CD86 expression in monocytes correlated negatively to disease progression. Interestingly, membrane expression of CD14 is down-regulated and CD95/CD86 up-regulated by IFN-beta in vitro (controls) and in vivo (MS), suggesting IFN-regulated expression of all three monocyte receptors in HAM/TSP. Transcriptomic analysis of whole blood vs. purified monocytes/B cells confirmed cell-specific expression of CD64/CD80/CD86 ex vivo, whereas a selective decrease of myeloid/monocyte-specific genes was observed upon in vitro culture of HAM/TSP PBMCs, possibly due to apoptosis of specific monocyte subsets. In conclusion, an increase in soluble CD14, as well as monocyte-specific expression of CD64, CD95 and CD86 differentially reflect disease progression in HAM/TSP, in addition to B-cell specific CD80 expression, arguing for a complex and compartmentalized IFN response.

Proteomic profiles among asymptomatic HTLV-1 carriers and HAM/TSP patients in Peru

High proviral loads have been described in HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP) patients in comparison to asymptomatic HTLV-1 carriers (AC). However, biomarkers related to HAM/TSP progression have not been identified. We analyzed differential proteome changes by two-dimensional gel electrophoresis (2D-electrophoresis) to identify spots of proteins in plasma samples of three groups of patients: five AC, nine HAM/TSP patients: five patients with EDSS scores of 1.0-5.0 (=mild HAM/TSP) and 4 patients with EDSS scores of 5.5-9.0 (=severe HAM/TSP). Proteins were extracted from pooled plasma samples from each group of patients. Protein separations were performed by electrofocusing with 11 cm strips at 3-10 pH range, electrophoresis with 12% poliacrilamide gel electrophoresis and silver staining. Analyses were performed in Progenesis Same Spot software; ANOVA was used to compare the profiles of the groups. Six spots of proteins were differentially expressed among these groups: five proteins increased their expression according to HAM/TSP disease progression (highest fold change= 6.1, p=1.671 x10-4; lowest fold change= 1.5, p= 0.014) while one protein was highly expressed in AC and decreased according to HAM/TSP progression (fold change= 5.8, p= 0.002). Further studies to confirm the expression of these proteins in a larger set of samples are still in progress, the identification of these proteins will be performed by mass spectrometry. These results might be promissory to identify biomarkers for HAM/TSP progression.