Maria Luiza Silva

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.

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.

Further evidence that the expression of CD38 and HLA-DR(+) in CD8(+) lymphocytes does not correlate to disease progression in HIV-1 vertically infected children.

Background: In adults, an increase in CD8+CD38+ T cell levels is a strong indicator of disease progression in HIV infection. However, in children, data are conflicting. Slow-progressing children (SPC) provide an exceptional resource for the investigation and clarification of the immunological and virological mechanisms of natural control of HIV infection and can be used to investigate prognostic indicators of disease progression. Objectives: To investigate the immune activation status and T regulatory (Treg) cell levels in SPC. Study design: A cross-sectional study was carried out on 28 children 8 years old and older who were vertically infected with HIV. The children were stratified into 3 groups according to their clinical outcome: SPC (anti-retroviral-therapy-naïve; ≥8 years-old; CD4 ≥20%; viral load <25,000 copies), IF/VF (anti-retroviral-therapy but with no therapeutic response), and IS/VS (anti-retroviral therapy with good therapeutic response). Uninfected children (NI) were assessed as healthy control group. Results: A higher percentage of activated CD8+ T cells were found in all HIV infected children, regardless of the evolution of disease. The activation of CD8+ T cells was not associated with either viral load or the percentage of CD4+ T cells. In addition, Treg cell levels did not show any correlation with the clinical outcome or the activation status of CD8+ T cells. Conclusions: HIV-1-infected children presented an increased percentage of activated CD8+ T cells and an unaltered percentage of Treg cells, regardless of their clinical evolution. Thus, these immunological parameters should not be used for prognostic evaluation.

Associação entre a carga viral e os linfócitos T CD4+ com as lesões intra-epiteliais do colo uterino em mulheres infectadas pelo vírus da imunodeficiência humana

OBJETIVOS: verificar se a contagem de linfocitos T CD4+ e a carga viral do HIV tem influencia na presenca de lesoes intra-epiteliais cervicais (SIL). METODOS: estudo transversal, no qual foram selecionadas 134 mulheres HIV-positivas, todas submetidas a biopsia do colo uterino, quantificacao da carga viral do HIV e contagem de linfocitos T CD4+. Os valores laboratoriais da quantificacao da carga viral e da contagem de linfocitos T CD4+ foram obtidos antes da realizacao da biopsia, tendo sido estabelecidos cortes para o estudo da carga viral ( 50.000 copias/mL) e contagem de linfocitos T CD4+ ( 350 celulas/mm3). Foram realizados os testes c2, c2 de tendencia linear, c2 de Mantel-Haenszel e analise de variância. Estabeleceu-se significância estatistica para p<0,05 e intervalo de confianca a 95%. RESULTADOS: nao houve tendencia de risco para as mulheres HIV-positivas apresentarem SIL com o aumento da carga viral ou diminuicao dos linfocitos T CD4+. Comparando-se a carga viral com a presenca ou ausencia de SIL, estratificada pelo tempo em que foi quantificada, houve diferenca significante para valores acima de 400 copias/mL (OR: 3,17; IC 95%: 1,02-9,93; p=0,048). Nenhuma associacao foi encontrada para a contagem de linfocitos T CD4+ com a presenca da SIL. CONCLUSAO: as pacientes com carga viral do HIV maior que 400 copias/mL, quantificada antes da biopsia do colo uterino, apresentaram chance 3,17 vezes maior de desencadear SIL. A contagem de linfocitos T CD4+ nao influenciou no aparecimento da SIL.

Central nervous system involvement in acute lymphoblastic leukemia: diagnosis by immunophenotyping

The central nervous system is the most commonly affected extramedullary site in acute lymphoblastic leukemia. Although morphologic evaluation of the cerebrospinal fluid has been traditionally used for diagnosing central nervous system involvement, it is a method of low sensitivity. The present study aimed at evaluating the use of immunophenotyping in the detection of blasts in the cerebrospinal fluid from children and adolescents with acute lymphoblastic leukemia.

Social and immunological differences among uninfected Brazilians exposed or unexposed to human immunodeficiency virus-infected partners

Understanding the social conditions and immunological characteristics that allow some human immunodeficiency virus (HIV)-exposed patients to remain uninfected represents an on-going challenge. In this study, the socio-demographic and sexual behaviour characteristics and immune activation profiles of uninfected individuals exposed to HIV-infected partners were investigated. A confidential and detailed questionnaire was administered and venous blood was tested using HIV-1/enzyme immunoassays, plasma HIV-1 RNA levels/bDNA and immunophenotyping/flow cytometry to determine the frequencies of CD4 and CD8 T cells expressing activation markers. The data analysis showed significant differences (p < 0.05) for immune parameters in individuals who were uninfected, albeit exposed to HIV-infected partners, compared with unexposed individuals. In particular, the exposed, uninfected individuals had a higher frequency (median, minimum-maximum) of CD4+HLA-DR+ (4.2, 1.8-6.1), CD8+HLA-DR+ (4.6, 0.9-13.7), CD4+CD45RO+ (27.5, 14.2-46.6), CD4+CD45RO+CD62L+ (46.7, 33.9-67.1), CD8+CD45RA+HLA-DR+ (12.1, 3.4-35.8) and CD8+CD45RO+HLA-DR+ (9.0, 3.2-14.8) cells, a decreased percentage of CD8+CD28+ cells (11.7, 4.5-24.0) and a lower cell-surface expression of Fcγ-R/CD16 on monocytes (56.5, 22.0-130.0). The plasma HIV-1 RNA levels demonstrated detectable RNA virus loads in 57% of the HIV-1+ female partners. These findings demonstrate an activation profile in both CD4 and CD8 peripheral T cells from HIV-1 exposed seronegative individuals of serodiscordant couples from a referral centre in Belo Horizonte, state of Minas Gerais.

Detection of PNH cells by flow cytometry, using multiparameter analysis

Introducao:O diagnostico laboratorial da hemoglobinuria paroxistica noturna (HPN), doenca caracterizada por deficiencia de sintese da molecula de ancoragem glicosilfosfatidilinositol (GPI), baseia-se na deteccao de celulas sanguineas deficientes em proteinas ancoradas ao GPI, por citometria de fluxo. Clones de celulas com fenotipo HPN podem ser detectados em pacientes com anemia aplasica (AA) e sindrome mielodisplasica (SMD), com implicacoes terapeuticas.Objetivos:Validar tecnica sensivel para deteccao de celulas HPN, por citometria de fluxo, e avaliar a frequencia dos clones em pacientes com AA e SMD.Metodos:Amostras de 20 pacientes com AA, 30 pacientes com SMD e 20 voluntarios (controles) foram analisadas, utilizando anticorpos monoclonais anti-CD16, CD24, CD55 e CD59 (neutrofilos); CD14 e CD55 (monocitos); e CD55 e CD59 (hemacias); alem do reagente de aerolisina fluorescente (FLAER) (neutrofilos e monocitos) e marcadores de linhagem celular. A comparacao do tamanho dos clones HPN detectados em neutrofilos e monocitos, pelas diferentes combinacoes de reagentes, foi realizada por analise de variância (ANOVA) e correlacao de Pearson.Resultados:Em cinco (25%) pacientes com AA foram identificadas celulas HPN, em proporcoes entre 0,14% e 94,84% dos eventos analisados. O clone nao foi detectado nos pacientes com SMD. Contudo, a analise dessas amostras permitiu evidenciar as dificuldades tecnicas secundarias a presenca de celulas imaturas e displasicas circulantes. O reagente FLAER propiciou separacao precisa das celulas alteradas.Conclusao:A analise multiparametrica por citometria de fluxo apresenta sensibilidade e acuracia na deteccao de clones HPN subclinicos. O reagente FLAER apresenta excelente desempenho na deteccao do clone HPN.

Quantification of CD8+CD38+ T lymphocytes by flow cytometry does not represent a good biomarker to monitor the reactivation of cytomegalovirus infection after allogeneic hematopoietic stem cell transplantation

Background Infection/reactivation of cytomegalovirus is a major cause of morbidity and mortality in immunocompromised transplant patients. It has already been observed in kidney and liver transplantation patients that cytomegalovirus disease is accompanied by significant increases in circulating CD8+CD38+ T lymphocytes. There are no reports that study CD8+CD38+ T lymphocytes to monitor/diagnose cytomegalovirus disease in hematopoietic stem cell transplantation patients. Objective The aim of this study was to evaluate some cellular activation markers on circulating mononuclear cells (CD38 and HLA-DR) in patients submitted to hematopoietic stem cell transplantation and to establish any correlation with cytomegalovirus disease as diagnosed by antigenemia. Methods Blood samples of 15 transplant patients were analyzed by flow cytometry using anti-CD3, anti-CD4, anti-CD8, anti-CD38, CD16, CD56 and anti-HLA-DR monoclonal antibodies and the results were evaluated in respect to cytomegalovirus antigenemia measured by indirect immunofluorescence. Minitab for Windows was used for statistical analysis and a p-value < 0.05 was considered significant. Results Patients with positive antigenemia did not show any significant increase in the percentages of cells expressing the CD38 or HLADR activation markers when compared to patients with negative antigenemia. On the contrary, all patients showed high percentages of these cells independent of the presence of cytomegalovirus disease. Conclusions This study suggests that the investigation of these lymphocyte sub-populations in patients submitted to hematopoietic stem cell transplantation does not seem to contribute to the early identification of cytomegalovirus disease.