Browsing by Author "Sousa, Ana E."
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- Acute HIV-1 and SARS-CoV-2 infections Share Slan+ Monocyte Depletion - evidence from an hyperacute HIV-1 case reportPublication . Farias, Guilherme B.; Badura, Robert; Conceição, Carolina M.; Gomes, André; Godinho-Santos, Ana; Laia, Joel; Rosmaninho, Pedro; Santos, Diana; Mota, Catarina; Almeida, Afonso; Fernandes, Susana M.; Trombetta, Amelia Chiara; Sousa, Ana E.Monocytes are key modulators in acute viral infections, determining both inflammation and development of specific B- and T-cell responses. Recently, these cells were shown to be associated to different SARS-CoV-2 infection outcome. However, their role in acute HIV-1 infection remains unclear. We had the opportunity to evaluate the mononuclear cell compartment in an early hyper-acute HIV-1 patient in comparison with an untreated chronic HIV-1 and a cohort of SARS-CoV-2 infected patients, by high dimensional flow cytometry using an unsupervised approach. A distinct polarization of the monocyte phenotype was observed in the two viral infections, with maintenance of pro-inflammatory M1-like profile in HIV-1, in contrast to the M2-like immunosuppressive shift in SARS-CoV-2. Noticeably, both acute infections had reduced CD14low/-CD16+ non-classical monocytes, with depletion of the population expressing Slan (6-sulfo LacNac), which is thought to contribute to immune surveillance through pro-inflammatory properties. This depletion indicates a potential role of these cells in acute viral infection, which has not previously been explored. The inflammatory state accompanied by the depletion of Slan+ monocytes may provide new insights on the critical events that determine the rate of viral set-point in acute HIV-1 infection and subsequent impact on transmission and reservoir establishment.
- Age-associated distribution of normal B-cell and plasma cell subsets in peripheral bloodPublication . Blanco, Elena; Pérez-Andrés, Martín; Arriba-Méndez, Sonia; Contreras-Sanfeliciano, Teresa; Criado, Ignacio; Pelak, Ondrej; Serra-Caetano, Ana; Romero, Alfonso; Puig, Noemí; Remesal, Ana; Torres Canizales, Juan; López-Granados, Eduardo; Kalina, Tomas; Sousa, Ana E.; van Zelm, Menno; van der Burg, Mirjam; van Dongen, Jacques J. M.; Orfao, AlbertoBackground: Humoral immunocompetence develops stepwise throughout life and contributes to individual susceptibility to infection, immunodeficiency, autoimmunity, and neoplasia. Immunoglobulin heavy chain (IgH) isotype serum levels can partly explain such age-related differences, but their relationship with the IgH isotype distribution within memory B-cell (MBC) and plasma cell (PCs) compartments remains to be investigated. Objective: We studied the age-related distribution of MBCs and PCs expressing different IgH isotypes in addition to the immature/transitional and naive B-cell compartments. Methods: B-cell and PC subsets and plasma IgH isotype levels were studied in cord blood (n = 19) and peripheral blood (n = 215) from healthy donors aged 0 to 90 years by using flow cytometry and nephelometry, respectively. Results: IgH-switched MBCs expressing IgG1, IgG2, IgG3, IgA1, and IgA2 were already detected in cord blood and newborns at very low counts, whereas CD27+IgM++IgD+ MBCs only became detectable at 1 to 5 months and remained stable until 2 to 4 years, and IgD MBCs peaked at 2 to 4 years, with both populations decreasing thereafter. MBCs expressing IgH isotypes of the second immunoglobulin heavy chain constant region (IGHC) gene block (IgG1, IgG3, and IgA1) peaked later during childhood (2-4 years), whereas MBCs expressing third IGHC gene block immunoglobulin isotypes (IgG2, IgG4, and IgA2) reached maximum values during adulthood. PCs were already detected in newborns, increasing in number until 6 to 11 months for IgM, IgG1, IgG2, IgG3, IgA1, and IgA2; until 2 to 4 years for IgD; and until 5 to 9 years for IgG4 and decreasing thereafter. For most IgH isotypes (except IgD and IgG4), maximum plasma levels were reached after PC and MBC counts peaked. Conclusions: PC counts reach maximum values early in life, followed by MBC counts and plasma IgH isotypes. Importantly, IgH isotypes from different IGHC gene blocks show different patterns, probably reflecting consecutive cycles of IgH isotype switch recombination through life.
- Autoimmunity and allergy control in adults submitted to complete thymectomy early in infancyPublication . Silva, Susana L.; Albuquerque, Adriana; Amaral, Andreia J.; Li, Quan-Zhen; Mota, Catarina; Cheynier, Rémi; Victorino, Rui M. M.; Santos, M. Conceição Pereira; Sousa, Ana E.The contribution of the decline in thymic activity for the emergence of autoimmunity is still debatable. Immune-competent adults submitted to complete thymectomy early in life provide a unique model to address this question. We applied here strict criteria to identify adults lacking thymic activity based on sjTREC levels, to exclude thymic rebound and/or ectopic thymuses. In agreement, they featured severe naïve CD4 T-cell depletion and contraction of T-cell receptor diversity. Notwithstanding this, there was neither increased incidence of autoimmune disease in comparison with age-matched controls nor significant changes in their IgG/IgA/IgM/IgE autoreactivity profiles, as assessed through extensive arrays. We reasoned that the observed relative preservation of the regulatory T-cell compartment, including maintenance of naïve regulatory CD4 T-cells, may contribute to limit the emergence of autoimmunity upon thymectomy. Our findings have implications in other clinical settings with impaired thymic activity, and are particularly relevant to studies of autoimmunity in ageing.
- Blood biomarkers associated with inflammation predict poor prognosis in cerebral venous thrombosis : a multicenter prospective observational studyPublication . de Sousa, Diana Aguiar; Pereira Santos, M. C.; Serra-Caetano, Ana; Lucas Neto, Lia; Sousa, A. L.; Gabriel, D.; Correia, M.; Gil-Gouveia, Raquel; Oliveira, Renato; Penas, Sara; Carvalho Dias, Mariana; Correia, M. A.; Carvalho, M.; Sousa, Ana E.; Canhão, Patrícia; Ferro, JoséBackground and purpose: Experimental studies suggest inflammation can contribute to blood barrier disruption and brain injury in cerebral venous thrombosis (CVT). We aimed to determine whether blood biomarkers of inflammation were associated with the evolution of brain lesions, persistent venous occlusion or functional outcome in patients with CVT. Methods: Pathophysiology of Venous Infarction-Prediction of Infarction and Recanalization in CVT (PRIORITy-CVT) was a multicenter prospective cohort study of patients with newly diagnosed CVT. Evaluation of neutrophil-to-lymphocyte ratio (NLR) and C-reactive protein (CRP) concentrations in peripheral blood samples was performed at admission in 62 patients. Additional quantification of interleukin (IL)-6 was performed at day 1, 3 and 8 in 35 patients and 22 healthy controls. Standardized magnetic resonance imaging was performed at day 1, 8 and 90. Primary outcomes were early evolution of brain lesion, early recanalization and functional outcome at 90 days. Results: Interleukin-6 levels were increased in patients with CVT with a peak at baseline. IL-6, NLR and CRP levels were not related with brain lesion outcomes or early recanalization but had a significant association with unfavourable functional outcome at 90 days (IL-6: OR = 1.28, 95% CI: 1.05-1.56, P = 0.046; NLR: OR = 1.39, 95% CI: 1.4-1.87, P = 0.014; CRP: OR = 1.756, 95% CI: 1.010-3.051, P = 0.029). Baseline IL-6 had the best discriminative capacity, with an area under the receiver operating characteristic curve to predict unfavourable functional outcome of 0.74 (P = 0.031). Conclusions: Increased baseline levels of NLR, CRP and IL-6 may serve as new predictive markers of worse functional prognosis at 90 days in patients with CVT. No association was found between inflammatory markers and early evolution of brain lesion or venous recanalization.
- Bulk cytokine production versus frequency of cytokine-producing cells in HIV1 infection before and during HAARTPublication . Sousa, Ana E.; Chaves, Ana F.; Doroana, Manuela; Antunes, Francisco; Victorino, Rui M. M.Cytokine imbalances play a major role in HIV immunopathogenesis. This study analyzes simultaneously the frequency of cytokine-producing cells at the single cell level by flow cytometry and the disturbances in cytokine secretion assessed by ELISA in a cohort of asymptomatic HIV1 patients in different stages of CD4 depletion and during antiretroviral therapy (HAART). Early in the disease, there is an increased frequency of IFN-γ lymphocytes and bulk IFN-γ+ production, in parallel with a reduced proportion of IL4+ cells and IL4 secreted. The two IL4 measurements are significantly correlated. No such correlation was found for IFN-γ, which is consistent with a large variation in the amount of IFN-γ released per individual cell. Moreover, HAART was associated with a reduction to normal levels in the bulk IFN-γ secretion concomitant with a persistency of the overexpanded IFN-γ+ cell subset in the peripheral blood. This study emphasizes the importance of using a conjoint approach to assess the cytokine network in trials of antiretroviral and/or immune-based therapies to avoid missing significant effects which are possibly relevant in the clinical setting.
- CD4 T cell depletion is linked directly to immune activation in the pathogenesis of HIV-1 and HIV-2 but only indirectly to the viral loadPublication . Sousa, Ana E.; Carneiro, Jorge; Meier-Schellersheim, Martin; Grossman, Zvi; Victorino, Rui M. M.The causal relationships among CD4 cell depletion, HIV replication, and immune activation are not well understood. HIV-2 infection, “nature’s experiment” with inherently attenuated HIV disease, provides additional insights into this issue. We report the finding that in HIV-2 and HIV-1 patients with a comparable degree of CD4 depletion the imbalance in the relative sizes of the naive and memory T cell populations and the up-regulation of CD4 and CD8 cell activation markers (HLA-DR, CD38, CD69, Fas molecules) are similar, even though the viral load in the plasma of HIV-2-infected patients is two orders of magnitude lower than in HIV-1 patients and HIV-2 patients are known to have slower rates of CD4 T cell decline and a better clinical prognosis. Moreover, we found a similar increase in the frequency of cycling CD4 T cells (Ki67+), which was in strong correlation with the expression of activation markers. Finally, the level of T cell anergy, as assessed by the proliferative responses to CD3 stimulation and to a panel of microbial Ags, proved to be comparable in HIV-1 and HIV-2 patients with a similar degree of CD4 depletion despite large differences in viral load. Our data are consistent with a direct causal relationship between immune activation and CD4 cell depletion in HIV disease and an only indirect relation of these parameters to the virus replication rate. Invoking the concept of proximal immune activation and virus transmission, which links efficient transmission of virus to local cell activation and proliferation in response to Ags and inflammation, we propose an integrative interpretation of the data and suggest that strongly elevated immune activation induces CD4 cell depletion and not vice versa, with potential implications for the choice of treatment strategies.
- CD4+ recent thymic emigrants are infected by HIV in vivo, implication for pathogenesisPublication . Fabre-Mersseman, Véronique; Dutrieux, Jacques; Louise, Anne; Rozlan, Sandra; Lamine, Aurélia; Parker, Raphaëlle; Rancez, Magali; Nunes-Cabaço, Helena; Sousa, Ana E.; Lambotte, Olivier; Cheynier, RémiOBJECTIVE: The contribution of naive CD4+ T cells to the pool of HIV-infected cells remains poorly described. This study aimed at evaluating HIV infection in naive T-cell subsets in viremic and HAART-treated patients, together with various parameters implicated in naive T-cell homeostasis, in order to better understand infection in these subsets. DESIGN AND METHODS: HIV provirus was quantified in various FACS-sorted CD4/CD8 T-cell subsets [recent thymic emigrants (RTEs), non-RTE naives and memory T cells] purified from peripheral blood cells of untreated viremic and HAART-treated aviremic HIV-infected patients. HIV proviral DNA was quantified using a highly sensitive real-time PCR assay allowing detection of one HIV copy in 10⁵ cells. Intrathymic precursor T-cell proliferation and circulating T-cell cycling were, respectively, evaluated through measurement of the sj/βTREC ratio (signal joint T-Cell Receptor Excision Circle frequency divided by DβJβTREC frequency) and Ki-67 expression. Plasma interleukin (IL)-7 concentrations were measured by ELISA. RESULTS: RTEs and non-RTEs were equally HIV infected. Altogether, naive CD4+ T cells represented 0.24%-60% of the infected cells. In contrast, HIV DNA was undetectable in naive CD8+ T cells. RTE infection rate directly correlated with IL-7 plasma levels (r = 0.607, P = 0.0035) but was independent from plasma viral load, peripheral T-cell cycling and intrathymic precursor T-cell proliferation. CONCLUSION: We demonstrated that RTEs are effectively HIV infected. The similar infection rate observed in RTEs and other naive T cells, its relationship with plasma IL-7 levels, together with the lack of correlation between RTE infection and either thymic or peripheral proliferation, strongly suggests that RTE infection occurs either late during thymopoiesis or early on during their extrathymic maturation.
- CD4+ T-cell depletion in HIV infection: are we closer to understanding the cause?Publication . Grossman, Zvi; Meier-Schellersheim, Martin; Sousa, Ana E.; Victorino, Rui M. M.; Paul, William E.The cause of the progressive depletion of CD4+ T cells in HIV-infected people is one of the most fundamental and controversial issues in AIDS research. HIV infects and kills CD4+ T cells. The infection results in high T-cell activation and turnover. An immediately intuitive assumption is that HIV-mediated destruction of CD4+ cells directly reduces the number of these cells and that the high turnover rates of T cells and the slow progression to AIDS reflect a long, but eventually lost struggle of the immune system to replace killed cells in its effort to maintain T-cell homeostasis1–4. However, HIV mainly infects activated CD4+ cells, and activated cells normally follow different dynamics than cells that belong to resting populations whose numbers are controlled by homeostatic mechanisms (Fig. 1a). Upon activation,T cells undergo several rapid rounds of division, and then they stop dividing and most die. Some of the activated cells escape this activation-induced cell death (AICD) and (re-)enter the population of resting memory cells. An alternative explanation for the high turnover rates of T cells in HIV infection may be that a large number of cells stimulated by antigen and/or inflammatory molecules rapidly replicate and subsequently die, as opposed to the immune system responding to high rates of virus-mediated death with high rates of homeostatic proliferation.
- Cell-associated viral burden provides evidence of ongoing viral replication in aviremic HIV-2-infected patientsPublication . Soares, Rui S.; Tendeiro, Rita; Foxall, Russell B.; Baptista, António P.; Cavaleiro, Rita; Gomes, Perpétua; Camacho, Ricardo; Valadas, Emília; Doroana, Manuela; Lucas, Margarida; Antunes, Francisco; Victorino, Rui M. M.; Sousa, Ana E.Viremia is significantly lower in HIV-2 than in HIV-1 infection, irrespective of disease stage. Nevertheless, the comparable proviral DNA burdens observed for these two infections indicate similar numbers of infected cells. Here we investigated this apparent paradox by assessing cell-associated viral replication. We found that untreated HIV-1-positive (HIV-1(+)) and HIV-2(+) individuals, matched for CD4 T cell depletion, exhibited similar gag mRNA levels, indicating that significant viral transcription is occurring in untreated HIV-2(+) patients, despite the reduced viremia (undetectable to 2.6 × 10(4) RNA copies/ml). However, tat mRNA transcripts were observed at significantly lower levels in HIV-2(+) patients, suggesting that the rate of de novo infection is decreased in these patients. Our data also reveal a direct relationship of gag and tat transcripts with CD4 and CD8 T cell activation, respectively. Antiretroviral therapy (ART)-treated HIV-2(+) patients showed persistent viral replication, irrespective of plasma viremia, possibly contributing to the emergence of drug resistance mutations, persistent hyperimmune activation, and poor CD4 T cell recovery that we observed with these individuals. In conclusion, we provide here evidence of significant ongoing viral replication in HIV-2(+) patients, further emphasizing the dichotomy between amount of plasma virus and cell-associated viral burden and stressing the need for antiretroviral trials and the definition of therapeutic guidelines for HIV-2 infection.
- Comparison of the frequency of interleukin (IL)-2-, interferon-γ-, and IL-4-producing T cells in 2 diseases, human immunodeficiency virus types 1 and 2, with distinct clinical outcomesPublication . Sousa, Ana E.; Chaves, Ana F.; Loureiro, Ana; Victorino, Rui M. M.Human immunodeficiency virus (HIV) type 2 infection is associated with a better clinical outcome, slower rates of CD4 T cell decline, and lower viremia than is HIV-1. This study compares HIV-1 and HIV-2 in regard to the percentages of interleukin (IL)–2–, interferon (IFN)–γ–, and IL-4–producing cells at the single-cell level, as determined by flow cytometry. At a given degree of CD4 T cell depletion, the frequency of T cells able to produce IL-2 is better preserved in HIV-2 than in HIV-1 infection, particularly within the CD4 T cell subset. As described for HIV-1 immunodeficiency, HIV-2–positive patients exhibit a marked expansion of terminally differentiated effector CD8 T cells (CD28−CD27−IFN-γ+). However, the proportion of CD8 T cells able to simultaneously produce IL-2 and IFN-γ is higher in HIV-2 disease. Considering the central role of IL-2 as a lymphocyte proliferative and survival factor, these findings provide a possible immunologic basis for the distinct course of HIV-2 immunodeficiency