Diversity and abundance of terrestrial molluscs and their associated nematode fauna in urban kitchen gardens in the city of Rio de Janeiro, Brazil.

Terrestrial molluscs can become agricultural pests and transmit parasites, playing an important role in different biological communities. In the present study, we evaluated the diversity and abundance of this group in two horticultural areas in Rio de Janeiro (Manguinhos and Jacarepaguá neighborhoods), as well the presence of parasitic nematodes associated with these molluscs. We collected specimens in the austral spring and summer, with four sites being sampled within each study area, including malabar spinach, sweet potato, chicory greens, and cassava plantations, and one site in an adjacent, non-cultivated area. We collected a total of 522 live mollusc specimens and identified 16 species from 10 different families. The greatest abundance of molluscs was recorded in summer (363) and at Jacarepaguá (309). Overall, 174 (57%) of the 303 specimens analyzed parasitologically were positive for nematodes. Larvae of superfamily Metastrongyloidea, which includes nematodes that are a concern for public health and veterinary medicine, were found parasitizing the slug Sarasinula linguaeformis, in Manguinhos. Our results contribute to understanding of the diversity of terrestrial molluscs occurring in kitchen gardens in the city of Rio de Janeiro, and provide important insights for generating subsidies for health education actions and control of parasitic diseases transmitted by them.

A genetic IFN/STAT1/FAS axis determines CD4 T stem cell memory levels and apoptosis in healthy controls and Adult T-cell Leukemia patients.

Adult T-cell leukemia (ATL) is an aggressive, chemotherapy-resistant CD4+CD25+ leukemia caused by HTLV-1 infection, which usually develops in a minority of patients several decades after infection. IFN + AZT combination therapy has shown clinical benefit in ATL, although its mechanism of action remains unclear. We have previously shown that an IFN-responsive FAS promoter polymorphism in a STAT1 binding site (rs1800682) is associated to ATL susceptibility and survival. Recently, CD4 T stem cell memory (TSCM) Fashi cells have been identified as the hierarchical cellular apex of ATL, but a possible link between FAS, apoptosis, proliferation and IFN response in ATL has not been studied. In this study, we found significant ex vivo antiproliferative, antiviral and immunomodulatory effects of IFN-α treatment in short-term culture of primary mononuclear cells from ATL patients (n = 25). Bayesian Network analysis allowed us to integrate ex vivo IFN-α response with clinical, genetic and immunological data from ATL patients, thereby revealing a central role for FAS -670 polymorphism and apoptosis in the coordinated mechanism of action of IFN-α. FAS genotype-dependence of IFN-induced apoptosis was experimentally validated in an independent cohort of healthy controls (n = 20). The same FAS -670 polymorphism also determined CD4 TSCM levels in a genome-wide twin study (p = 7 × 10-11, n = 460), confirming a genetic link between apoptosis and TSCM levels. Transcriptomic analysis and cell type deconvolution confirmed the FAS genotype/TSCM link and IFN-α-induced downregulation of CD4 TSCM-specific genes in ATL patient cells. In conclusion, ex vivo IFN-α treatment exerts a pleiotropic effect on primary ATL cells, with a genetic IFN/STAT1/Fas axis determining apoptosis vs. proliferation and underscoring the CD4 TSCM model of ATL leukemogenesis.

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.

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

BACKGROUND: Human 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. METHODS: Using 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/ß mirror similarities between HAM/TSP and MS and hence might reveal clinically useful biomarkers in HAM/TSP. RESULTS: Ex 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-ß 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. CONCLUSIONS: We propose two novel biomarkers, ex vivo CD80+ B cells positively correlating to disease severity and CD86+ B cells preferentially induced by IFN-ß, 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.

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.

Estudo da regulação molecular da apoptose ex vivo e in vitro em pacientes com mielopatia associada ao HTLV-I/Paraparesia espástica tropical / Study ex vivo and in vitro of molecular regulation of apoptosis in HTL-I-associated myelopathy/tropical spastic paraparesis patients

O vírus linfotrópico de células T humano - tipo I (HTLV-I) é o agente etiológico da Leucemia/Linfoma de células T no adulto (ATL), da Mielopatia Associada ao HTLV-I/Paraparesia Espástica Tropical (HAM/TSP), e de outras patologias. Os IFN-α e IFN-β foram recentemente introduzidos no tratamento de ATL e HAM/TSP, embora seus mecanismos de ação ainda estejam pouco esclarecidos. A presença da iNOS e seu recém-descoberto papel anti-apoptótico na ATL iniciou novas perspectivas terapêuticas. Considerando a correlação entre a linfoproliferação induzida pelo HTLV-I e as patologias associadas, buscamos estudar a regulação molecular da linfoproliferação e da apoptose ex vivo e in vitro em indivíduos soropositivos assintomáticos e pacientes com HAM/TSP. A expressão ex vivo do mRNA das moléculas pró-apoptóticas Fas, FasL e pró-caspase-3 foi maior nos indivíduos assintomáticos quando comparados aos pacientes com HAM/TSP, sugerindo que uma diminuição da morte celular programada poderia ter influência no processo patológico. O aumento do mRNA da iNOS nos assintomáticos foi correlacionado a inibição da linfoproliferação in vitro pelo L-NMMA (inibidor da iNOS). Por outro lado, apenas o IFN-β mostrou atividade anti-proliferativa significante, mas não pró-apoptótica in vitro em células mononucleares de pacientes com HAM/TSP. Porém, as células mostraram-se sensíveis ao estímulo com anti-CD3 na indução da apoptose. A estimulação com o IFN-β ou anti-CD3 não diminuiu a expressão do mRNA da proteína viral Tax, sugerindo que os efeitos antiproliferativos e pró-apoptóticos ocorrem independente da transcrição viral. De acordo com nossos resultados, o uso combinado de IFN-β_e outras drogas antivirais ou pró-apoptóticas poderia ser considerado em futuros ensaios terapêuticos.