Vanessa Peruhype-Magalhães

HTLV-1-Associated Myelopathy/Tropical Spastic Paraparesis (HAM/TSP) Inflammatory Network

HTLV-1 associated myelopathy/ tropical spastic paraparesis (HAM/TSP) is a systemic immune-mediated inflammatory disease and tissues other than nervous can be damaged, mainly ocular, rheumatic and dermatologic. Over 90% of HTLV-1-infected individuals remain lifelong asymptomatic and this retrovirus persists indefinitely in their CD4+ T-lymphocytes. The infection is maintained due to the proliferation of lymphocytes that harbor a provirus and express HTLV-1 proteins, particularly Tax, promoting an active and selective expansion of infected T cells. High proviral load is related to disease progression, which is correlated to disequilibrium between host and virus. Cytotoxic T lymphocytes are abundant and chronically activated in asymptomatic carriers and in HAM/TSP patients. The asymptomatic carriers were shown to have a high frequency of pro-inflammatory monocytes and anti-inflammatory IL-10+CD4+ and IL-10+CD8+ T-cells, as an immunoregulatory mechanism to counterbalance the monocyte-derived TNF-alpha. A putative immunomodulatory event would be the key to control their overall immunological status. In HAM/TSP, a pro-inflammatory microenvironment is the hallmark of the immunological profile. Enhanced frequency of activated CD8+ T-cells (HLA-DR+) in combination with high CD18 surface expression has been seen. In blood and cerebrospinal fluid, increased levels of Type-1 cytokines, as interferon-(IFN)-gamma, Tumor Necrosis Factor (TNF)-alpha, Interleukin (IL)-2, and pro-inflammatory IL-6, can be found. Concerning the progression, HLA polymorphisms may influence HAM/TSP and the allele HLA-A*2 has been associated with protection. The authors showed that HAM/TSP is strongly associated with a decreased percentage of B-cells, with enhanced T/B-cell ratio and activated CD8+ T-cells. These immunological parameters have been proposed as a prognostic biomarker for HAM/TSP.

Activation/modulation of adaptive immunity emerges simultaneously after 17DD yellow fever first-time vaccination: is this the key to prevent severe adverse reactions following immunization?

Over past decades the 17DD yellow fever vaccine has proved to be effective in controlling yellow fever and promises to be a vaccine vector for other diseases, but the cellular and molecular mechanisms by which it elicits such broad‐based immunity are still unclear. In this study we describe a detailed phenotypic investigation of major and minor peripheral blood lymphocyte subpopulations aimed at characterizing the kinetics of the adaptive immune response following primary 17DD vaccination. Our major finding is a decreased frequency of circulating CD19+ cells at day 7 followed by emerging activation/modulation phenotypic features (CD19+interleukin(IL)10R+/CD19+CD32+) at day 15. Increased frequency of CD4+human leucocyte antigen D‐related(HLA‐DR+) at day 7 and CD8+HLA‐DR+ at day 30 suggest distinct kinetics of T cell activation, with CD4+ T cells being activated early and CD8+ T cells representing a later event following 17DD vaccination. Up‐regulation of modulatory features on CD4+ and CD8+ cells at day 15 seems to be the key event leading to lower frequency of CD38+ T cells at day 30. Taken together, our findings demonstrate the co‐existence of phenotypic features associated with activation events and modulatory pathways. Positive correlations between CD4+HLA‐DR+ cells and CD4+CD25high regulatory T cells and the association between the type 0 chemokine receptor CCR2 and the activation status of CD4+ and CD8+ cells further support this hypothesis. We hypothesize that this controlled microenviroment seems to be the key to prevent the development of serious adverse events, and even deaths, associated with the 17DD vaccine reported in the literature.

Combined diagnostic methods identify a remarkable proportion of asymptomatic Leishmania (Leishmania) chagasi carriers who present modulated cytokine profiles

Peripheral blood samples of 138 co-habitants from 25 families with recently diagnosed cases of visceral leishmaniasis in the Metropolitan Region of Belo Horizonte, Minas Gerais, Brazil, were analyzed by indirect fluorescent antibody test (IFAT), rK39 and Leishmania chagasi Enzyme Linked Immunosorbent Assay (ELISA), intradermal skin-test and Polymerase Chain Reaction (PCR) over a 12-month period. The cumulative positivity was significantly higher by PCR (29.7%) than by IFAT, rK39 ELISA, L. chagasi ELISA and intradermal skin-test (5.1%, 6.5%, 14.5% and 2.9%, respectively). In addition, the cytokine profile was measured in 16 of the 138 volunteers, of whom eight were asymptomatic carriers and eight were non-infected co-habitants. The innate immunity cells from asymptomatic carriers displayed, upon in vitro antigenic stimulation, a modulated increase in cytokine synthesis that was distinct from that observed in non-infected volunteers. This study suggests that the identification of a large proportion of asymptomatic carriers is facilitated when more than one diagnostic method is applied and that a mixed pattern of immune response is correlated with clinical status of asymptomatic individuals. These observations suggest also that asymptomatic infection by L. chagasi is a frequent event and that control programs could benefit by including this indicator in their interventions.

Variation Rhythms of Lymphocyte Subsets during Healthy Aging

Immunological alterations associated with aging (immunosenescence) do not represent a simple unidirectional decline in all functions but develop as a complex remodeling of the immune system, involving multiple reorganization and developmentally regulated changes. In general, most data available about aging were obtained at particular age intervals and most of them come from Caucasian individuals from either Europe or the United States. Here, we report the frequencies of major lymphocyte subsets in healthy Brazilian individuals from 2 distinct geographic regions (Southeast and South) at several age intervals spanning a lifetime period (0–86 years). Overall, we demonstrated that changes in the frequencies of cells related to both innate and adaptive immunity clearly occur with aging in these individuals. These changes were not progressive and equally steady for all cell populations tested but instead showed an oscillatory or rhythmic behavior that was distinctive of each population at different age intervals. We also observed that abrupt changes in the frequencies of immune cells may occur in healthy individuals over 75 years old, suggesting there is an impaired flexibility of the immune system at late stages of life to sustain homeostasis via immune mechanisms. We presented reference ranges for healthy Brazilian individuals at all ages. The knowledge of these parameters in further detail will allow interventions to optimize immune function in advanced age and to improve the quality of life in the elderly.

Innate immunity phenotypic features point toward simultaneous raise of activation and modulation events following 17DD live attenuated yellow fever first-time vaccination.

Detailed multiparametric phenotypic investigation aiming to characterize the kinetics of the innate immune response in the peripheral blood following 17DD yellow fever (17DD-YF) first-time vaccination was performed. Results showed increased frequency of monocytes and NK cell subpopulations besides unexpected up-regulation of granulocytes activation status (CD28+/CD23+ and CD28+/HLA-DR+, respectively). Up-regulation of Fcgamma-R and IL-10-R expression emerge as putative events underlying the mixed pattern of phenotypic features triggered by the 17DD yellow fever (17DD-YF) vaccination. Mixed pattern of chemokine receptors expression further support our hypothesis that a parallel establishment of activation/modulation microenvironment plays a pivotal role in the protective immunity triggered by the 17DD-YF vaccine.

Clinical and Immunological Insights on Severe, Adverse Neurotropic and Viscerotropic Disease following 17D Yellow Fever Vaccination

ABSTRACT Yellow fever (YF) vaccines (17D-204 and 17DD) are well tolerated and cause very low rates of severe adverse events (YEL-SAE), such as serious allergic reactions, neurotropic adverse diseases (YEL-AND), and viscerotropic diseases (YEL-AVD). Viral and host factors have been postulated to explain the basis of YEL-SAE. However, the mechanisms underlying the occurrence of YEL-SAE remain unknown. The present report provides a detailed immunological analysis of a 23-year-old female patient. The patient developed a suspected case of severe YEL-AVD with encephalitis, as well as with pancreatitis and myositis, following receipt of a 17D-204 YF vaccination. The patient exhibited a decreased level of expression of Fc-γR in monocytes (CD16, CD32, and CD64), along with increased levels of NK T cells (an increased CD3+ CD16+/− CD56+/−/CD3+ ratio), activated T cells (CD4+ and CD8+ cells), and B lymphocytes. Enhanced levels of plasmatic cytokines (interleukin-6 [IL-6], IL-17, IL-4, IL-5, and IL-10) as well as an exacerbated ex vivo intracytoplasmic cytokine pattern, mainly observed within NK cells (gamma interferon positive [IFN-γ+], tumor necrosis factor alpha positive [TNF-α+], and IL-4 positive [IL-4+]), CD8+ T cells (IL-4+ and IL-5+), and B lymphocytes (TNF-α+, IL-4+, and IL-10+). The analysis of CD4+ T cells revealed a complex profile that consisted of an increased frequency of IL-12+ and IFN-γ+ cells and a decreased percentage of TNF-α+, IL-4+, and IL-5+ cells. Depressed cytokine synthesis was observed in monocytes (TNF-α+) following the provision of antigenic stimuli in vitro. These results support the hypothesis that a strong adaptive response and abnormalities in the innate immune system may be involved in the establishment of YEL-AND and YEL-AVD.

Cytokines, chemokine receptors, CD4+CD25HIGH+ T-cells and clinical forms of human schistosomiasis.

Previous studies have demonstrated that distinct immune response profiles can be correlated with the development/maintenance of different clinical forms of human schistosomiasis. We have previously shown that individuals with the more severe clinical forms of the disease such as those presenting different levels of fibrosis or with the hepatosplenic (HS) clinical form of the disease show significantly different immune response when compared with those with the intestinal clinical form (INT). To better understand the immune mechanisms associated with the clinical form of the schistosomiasis, in this study, we present the results of the evaluation, at a single cell level, of the cytokine patterns as well as the chemokine receptors expression by T-cell subsets after in vitro short-term stimulation with soluble egg antigens as well as the ex vivo frequency analysis of putative regulatory CD4+CD25HIGH+ T-cell subset in the peripheral blood mononuclear cells. We observed an increase on IL-4+, IL-5+ and IL-10+ cells both in the CD4+ and CD8+ lymphocytes in INT and a significant decrease on the number of IL-4+, IL-5+ and IL-10+ T-lymphocytes for HS. However, patients with detectable fibrosis presented decrease on IL-10+ (both CD4+ and CD8+ lymphocytes) and basal levels of IL-4 and IL-5. These data suggested that although INT group is under the influence of an effective immunoregulated immune response, mainly due to the high percentage of IL-10+ cells, it presents a mixed type (Type1/Type-2) immune profile. Moreover, the chemokine receptors expression demonstrated that CXCR3 and CXCR4 by CD4+ T-cells in INT may dictate the selective profile of IL-10 associated with the immunomodulatory events in human schistosomiasis. Additionally, the ex vivo analysis also suggests that higher levels of CD4+CD25HIGH+ T-cells may play a role in controlling morbidity in chronic human schistosomiasis. Taken together, these data suggest a major role of IL-10-producing CXCR4+ CD4+ T-cell subset for the asymptomatic outcome of the disease.

IL-10 produced by CD4+ and CD8+T cells emerge as a putative immunoregulatory mechanism to counterbalance the monocyte-derived TNF-α and guarantee asymptomatic clinical status during chronic HTLV-I infection

Although it is believed widely that distinct patterns of the host immune response are associated with the outcome of chronic human T cell lymphotropic virus type 1 (HTLV‐I) infection toward asymptomatic or symptomatic neurodegenerative myelopathy (HAM/TSP), the exact mechanism underlying these immunological events still remains unknown. In this study, we have evaluated the cytokine pattern [interleukin (IL)‐12, interferon (IFN)‐γ, tumour necrosis factor (TNF)‐α, IL‐4 and IL‐10] of innate and adaptive immunity cells present at the peripheral blood from non‐infected (NI) and HTLV‐I infected individuals [asymptomatic (AS), oligosymptomatic (OL) and HAM/TSP‐HT], following in vitro short‐term incubation in the absence/presence of phorbol myristate acetate (PMA) pan‐leucocyte stimulation. In the absence of PMA stimulation, our data demonstrate that despite the overall immunological profile of AS mimicry that observed for NI, the high frequency of IL‐12+ neutrophils and TNF‐α+ monocytes are also a hallmark of this group of individuals. However, the outstanding positive correlation between the high frequency of TNF‐α+ monocytes and high levels CD4+ IL‐10+ and CD8+ IL‐10+ T cells suggests the establishment of immunoregulatory mechanisms that guarantee their asymptomatic clinical status. On the other hand, OL and HT did not present any association between the high frequency and TNF‐α+ neutrophils and monocytes and this immunoregulatory profile at their adaptive immunity cells. Upon PMA‐index analysis, high levels of type 1 CD4+ T cells, as well as higher IFN‐γ/IL‐10 and TNF‐α/IL‐10 ratios, were observed in HT, and re‐emphasize the role of Th1‐cytokines from CD4+ cells to HTLV‐I immunity and disease. Moreover, increasing frequency of CD8+ IFN‐γ+ and CD8+ TNF‐α+ cells were observed in the HT, which corroborates the marked inflammatory profile underlying this pathological condition and the role of CD8+ T cells in the pathogenesis of HAM/TSP.

Impaired phagocytic capacity driven by downregulation of major phagocytosis-related cell surface molecules elicits an overall modulatory cytokine profile in neutrophils and monocytes from the indeterminate clinical form of Chagas disease

The distinct ability of phagocytes to present antigens, produce cytokines and provide co-stimulatory signals may contribute to the severity of the outcome of Chagas disease. In this paper, we evaluate the phenotypic features of phagocytes along with the cytokine signature of circulating T-cells from Chagas disease patients with indeterminate (IND) and cardiac (CARD) clinical forms of the disease. Our data demonstrated that neutrophils from IND patients displayed an impaired ability to produce cytokines. A lower Trypanosoma cruzi phagocytic index and higher nitric oxide levels were characteristics of monocytes from IND. The impaired phagocytic capacity did not reflect on the levels of anti-T. cruzi IgG, but was detectable in the downregulation of Fc-γR, TLR and CR1 molecules. The monocyte-derived cytokine signature demonstrated that a down-regulated synthesis of IL-12 and a modulatory state were evidenced by a positive correlation between IL-12 and IL-10 with a lower synthesis of TNF-α. The down-regulation of MHC-II and CD86 in monocytes supports the occurrence of particularities in the APC-activation-arm in IND, and may be involved in the T-cell pro-inflammatory pattern counterbalanced by a potent IL-10 response. Our findings support the hypothesis that differential phenotypic features of monocytes from IND may be committed to the induction of a distinct immune response related to low morbidity in chronic Chagas disease.

Booster dose after 10 years is recommended following 17DD-YF primary vaccination

A single vaccination of Yellow Fever vaccines is believed to confer life-long protection. In this study, results of vaccinees who received a single dose of 17DD-YF immunization followed over 10 y challenge this premise. YF-neutralizing antibodies, subsets of memory T and B cells as well as cytokine-producing lymphocytes were evaluated in groups of adults before (NVday0) and after (PVday30-45, PVyear1-4, PVyear5-9, PVyear10-11, PVyear12-13) 17DD-YF primary vaccination. YF-neutralizing antibodies decrease significantly from PVyear1-4 to PVyear12-13 as compared to PVday30-45, and the seropositivity rates (PRNT≥2.9Log10mIU/mL) become critical (lower than 90%) beyond PVyear5-9. YF-specific memory phenotypes (effector T-cells and classical B-cells) significantly increase at PVday30-45 as compared to naïve baseline. Moreover, these phenotypes tend to decrease at PVyear10-11 as compared to PVday30-45. Decreasing levels of TNF-α+ and IFN-γ+ produced by CD4+ and CD8+ T-cells along with increasing levels of IL-10+CD4+T-cells were characteristic of anti-YF response over time. Systems biology profiling represented by hierarchic networks revealed that while the naïve baseline is characterized by independent micro-nets, primary vaccinees displayed an imbricate network with essential role of central and effector CD8+ memory T-cell responses. Any putative limitations of this cross-sectional study will certainly be answered by the ongoing longitudinal population-based investigation. Overall, our data support the current Brazilian national immunization policy guidelines that recommend one booster dose 10 y after primary 17DD-YF vaccination.