Cytokine profile, proliferation and phosphorylation of ERK1/2 and Akt in circulating mononuclear cells from individuals during the chronic intestinal phase of Schistosomiasis mansoni infection.

BACKGROUND: The immune response to Schistosoma mansoni is characterized by a granulomatous reaction around the parasite eggs that are trapped in the host liver, and this reaction modulates the immune response during the chronic phase of the disease. The typical peripheral blood mononuclear cell (PBMC) response of patients during the chronic intestinal phase of infection is characterized by a decreased response to an S. mansoni soluble egg antigen. To obtain a greater understanding of Schistosoma infections, this study investigated the effects of the soluble egg antigen (SEA) and soluble adult worm antigen (SWAP) of S. mansoni on cellular proliferation, cytokine production, and ERK1/2 and Akt phosphorylation in PBMCs from infected (XTO) and egg-negative (NI) individuals living in the same endemic area. METHODS: The activation status was evaluated by cell immunophenotypic staining (cytometry). The cell proliferation assay was by CFSE method. Cytokine detection assay (Th1 and Th2) was by Cytometric Bead and Array phosphorylation status was by ELISA. RESULTS: The XTO, NI and BD (blood donor) individuals from an area not endemic for schistosomiasis were compared. The CD4(+) T lymphocyte proliferation rate was lower in the XTO group, but not the NI group, after SEA stimulation compared to the BD group. The CD8(+) T cell proliferation rate was lower in the XTO group in the unstimulated cultures and after both SEA and SWAP stimulation compared to the BD group. Cytokine analysis after either SEA or SWAP stimulation showed a balanced cytokine pattern in the XTO and NI groups. ERK1/2 and Akt phosphorylation were only marginally detected in all groups; however, a decrease in ERK 1/2 phosphorylation was observed in the SWAP-stimulated XTO group compared to both the NI and BD groups. CONCLUSIONS: The data indicate that SEA-stimulated CD4(+) T cells from infected patients have a lower proliferation rate than the same cells from the NI group. Furthermore, we observed that SWAP stimulation influences ERK1/2 phosphorylation in the XTO group.

Short-Lived Immunity After 17DD Yellow Fever Single Dose Indicates That Booster Vaccination May Be Required to Guarantee Protective Immunity in Children.

The Yellow Fever (YF) vaccination is recommended for people living in endemic areas and represents the most effective strategy to reduce the risk of infection. Previous studies have warned that booster regimens should be considered to guarantee the long-term persistence of 17DD-YF-specific memory components in adults living in areas with YF-virus circulation. Considering the lower seroconversion rates observed in children (9-12 months of age) as compared to adults, this study was designed in order to access the duration of immunity in single-dose vaccinated children in a 10-years cross-sectional time-span. The levels of neutralizing antibodies (PRNT) and the phenotypic/functional memory status of T and B-cells were measured at a baseline, 30-45 days, 1, 2, 4, 7, and 10 years following primary vaccination. The results revealed that a single dose induced 85% of seropositivity at 30-45 days and a progressive time-dependent decrease was observed as early as 2 years and declines toward critical values (below 60%) at time-spans of ≥4-years. Moreover, short-lived YF-specific cellular immunity, mediated by memory T and B-cells was also observed after 4-years. Predicted probability and resultant memory analysis emphasize that correlates of protection (PRNT; effector memory CD8+ T-cells; non-classical memory B-cells) wane to critical values within ≥4-years after primary vaccination. Together, these results clearly demonstrate the decline of 17DD-YF-specific memory response along time in children primarily vaccinated at 9-12 months of age and support the need of booster regimen to guarantee the long-term persistence of memory components for children living in areas with high risk of YF transmission.

Multi-parameter approach to evaluate the timing of memory status after 17DD-YF primary vaccination.

In this investigation, machine-enhanced techniques were applied to bring about scientific insights to identify a minimum set of phenotypic/functional memory-related biomarkers for post-vaccination follow-up upon yellow fever (YF) vaccination. For this purpose, memory status of circulating T-cells (Naïve/early-effector/Central-Memory/Effector-Memory) and B-cells (Naïve/non-Classical-Memory/Classical-Memory) along with the cytokine profile (IFN/TNF/IL-5/IL-10) were monitored before-NV(day0) and at distinct time-points after 17DD-YF primary vaccination-PV(day30-45); PV(year1-9) and PV(year10-11). A set of biomarkers (eEfCD4; EMCD4; CMCD19; EMCD8; IFNCD4; IL-5CD8; TNFCD4; IFNCD8; TNFCD8; IL-5CD19; IL-5CD4) were observed in PV(day30-45), but not in NV(day0), with most of them still observed in PV(year1-9). Deficiencies of phenotypic/functional biomarkers were observed in NV(day0), while total lack of memory-related attributes was observed in PV(year10-11), regardless of the age at primary vaccination. Venn-diagram analysis pre-selected 10 attributes (eEfCD4, EMCD4, CMCD19, EMCD8, IFNCD4, IL-5CD8, TNFCD4, IFNCD8, TNFCD8 and IL-5CD4), of which the overall mean presented moderate accuracy to discriminate PV(day30-45)&PV(year1-9) from NV(day0)&PV(year10-11). Multi-parameter approaches and decision-tree algorithms defined the EMCD8 and IL-5CD4 attributes as the top-two predictors with moderated performance. Together with the PRNT titers, the top-two biomarkers led to a resultant memory status observed in 80% and 51% of volunteers in PV(day30-45) and PV(year1-9), contrasting with 0% and 29% found in NV(day0) and PV(year10-11), respectively. The deficiency of memory-related attributes observed at PV(year10-11) underscores the conspicuous time-dependent decrease of resultant memory following17DD-YF primary vaccination that could be useful to monitor potential correlates of protection in areas under risk of YF transmission.

In vitro cultured peripheral blood mononuclear cells from patients with chronic schistosomiasis mansoni show immunomodulation of cyclin D1,2,3 in the presence of soluble egg antigens.

Infection with Schistosoma mansoni induces a wide range of effects on the immune responses of the host. In the present study we investigated the influence of soluble egg antigens (SEA) on the cell cycle of peripheral blood mononuclear cells (PBMC) from infected and non-infected individuals with S. mansoni resident in an endemic area and blood donors from non-endemic area. The cell cycle, the expression of activation markers and cyclin D(+)(1,2,3) CD3(+) frequency was assessed by flow cytometry. Stimulation of PBMC from infected patients with SEA resulted in a lower frequency of CD3(+) T cells in S phase when compared with the non-infected group. In addition, infected patients presented a decrease of activation marker expression (CD69(+), HLA-DR(+) and CD28(-) on CD4(+) cells and CD25(+), HLA-DR(+) on CD8(+) cells). A reduced frequency was observed of cyclin D(1,2,3) expression in SEA-stimulated T cells from infected individuals when compared with those from the non-infected group. The decreased expression of activation markers and frequency of cyclin D(1,2,3) in T cells may result in arrest of T cells in the G(0)/G(1) phase of the cell cycle, thus explaining the down-regulation observed in chronic schistosomiasis.

Occurrence of Autoimmune Diseases Related to the Vaccine against Yellow Fever.

Yellow fever is an infectious disease, endemic in South America and Africa. This is a potentially serious illness, with lethality between 5 and 40% of cases. The most effective preventive vaccine is constituted by the attenuated virus strain 17D, developed in 1937. It is considered safe and effective, conferring protection in more than 90% in 10 years. Adverse effects are known as mild reactions (allergies, transaminases transient elevation, fever, headache) and severe (visceral and neurotropic disease related to vaccine). However, little is known about its potential to induce autoimmune responses. This systematic review aims to identify the occurrence of autoinflammatory diseases related to 17D vaccine administration. Six studies were identified describing 13 possible cases. The diseases were Guillain-Barré syndrome, multiple sclerosis, multiple points evanescent syndrome, acute disseminated encephalomyelitis, autoimmune hepatitis, and Kawasaki disease. The data suggest that 17D vaccination may play a role in the mechanism of loss of self-tolerance.

Short-lived immunity after 17DD Yellow Fever single dose indicates that booster vaccination may be required to guarantee protective immunity in children

A vacinação contra a febre amarela (YF) é recomendada para pessoas que vivem em áreas endêmicas e representa a estratégia mais eficaz para reduzir o risco de infecção. Estudos anteriores alertaram que os regimes de reforço devem ser considerados para garantir a persistência a longo prazo dos componentes de memória específicos da 17DD-YF em adultos que vivem em áreas com circulação do vírus da YF. Considerando as menores taxas de soroconversão observadas em crianças (9 a 12 meses de idade) em comparação aos adultos, este estudo foi desenvolvido para acessar a duração da imunidade em crianças vacinadas em dose única em um período de 10 anos de seção transversal . Os níveis de anticorpos neutralizantes (PRNT) e o status fenotípico / de memória funcional das células T e B foram medidos no início, 30 a 45 dias, 1, 2, 4, 7 e 10 anos após a vacinação primária. Os resultados revelaram que uma dose única induziu 85% de soropositividade entre 30 e 45 dias e uma diminuição progressiva dependente do tempo foi observada em apenas 2 anos e diminui para valores críticos (abaixo de 60%) em períodos de tempo ≥ 4 anos . Além disso, a imunidade celular específica de YF de curta duração, mediada pelas células T e B de memória, também foi observada após 4 anos. A análise de probabilidade prevista e a memória resultante enfatizam que os correlatos de proteção (PRNT; células T CD8 + com memória efetiva; células B com memória não clássica) diminuem para valores críticos dentro de ≥4 anos após a vacinação primária. Juntos, esses resultados demonstram claramente o declínio da resposta da memória específica da 17DD-YF ao longo do tempo em crianças vacinadas principalmente entre 9 e 12 meses de idade e suportam a necessidade de um regime de reforço para garantir a persistência a longo prazo dos componentes da memória para crianças que vivem em áreas com alto risco de transmissão YF.

Multi-parameter approach to evaluate the timing of memory status after 17DD-YF primary vaccination

Nesta investigação, as técnicas melhoradas por máquina foram aplicadas para trazer insights científicos para identificar um conjunto mínimo de biomarcadores relacionados à memória fenotípica / funcional para o acompanhamento pós-vacinação da vacinação contra a febre amarela (FA). Para este propósito, o estado de memória das células T circulantes (Naïve / efetor-precoce / Memória-Central / Memória Efetiva) e células B (Naïve / memória não clássica / memória clássica) juntamente com o perfil de citocinas (IFN / TNF / IL-5 / IL-10) foram monitorizados antes do NV (dia 0) e em pontos de tempo distintos após a vacinação primária com 17DD-YF - VP (dia30-45); PV (ano1-9) e PV (ano10-11). Um conjunto de biomarcadores (eEfCD4; EMCD4; CMCD19; EMCD8; IFNCD4; IL-5CD8; TNFCD4; IFNCD8; TNFCD8; IL-5CD19; IL-5CD4) foi observado em PV (dia30-45), mas não em NV (dia0) , com a maioria deles ainda observada em VP (ano1-9). Deficiências de biomarcadores fenotípicos / funcionais foram observadas em NV (dia 0), enquanto a falta total de atributos relacionados à memória foi observada na PV (ano10-11), independentemente da idade na vacinação primária. Análise de diagrama de Venn pré-selecionada 10 atributos (eEfCD4, EMCD4, CMCD19, EMCD8, IFNCD4, IL-5CD8, TNFCD4, IFNCD8, TNFCD8 e IL-5CD4), dos quais a média geral apresentou moderada precisão para discriminar PV (dia30-45) e PV (year1-9) de NV (day0) e PV (year10-11). Abordagens multi-parâmetro e algoritmos de árvore de decisão definiram os atributos EMCD8 e IL-5CD4 como os dois principais preditores com desempenho moderado. Juntamente com os títulos PRNT, os dois principais biomarcadores levaram a um status de memória resultante observado em 80% e 51% dos voluntários em PV (dia30-45) e PV (ano1-9), contrastando com 0% e 29% encontrados em NV ( day0) e PV (year10-11), respectivamente. A deficiência de atributos relacionados à memória observada na PV (year10-11) ressalta a diminuição conspícua dependente do tempo da memória resultante após a vacinação primária com 17DD-YF, que pode ser útil para monitorar potenciais correlatos de proteção em áreas sob risco de transmissão da FA.

Antitumor effectiveness and toxicity of cisplatin-loaded long-circulating and pH-sensitive liposomes against Ehrlich ascitic tumor.

Cisplatin (CDDP) is one of the most active cytotoxic agents commonly used in the treatment of peritoneal carcinomatosis. The disadvantages of its clinical use are systemic side-effects, such as nephrotoxicity and myelotoxicity. Long-circulating and pH-sensitive liposomes containing CDDP (SpHL-CDDP) were developed by our research group aiming to promote the release of CDDP near the tumor as well as decreasing toxicity. The aim of this study was to evaluate the antitumor efficacy and toxicity of SpHL-CDDP after intraperitoneal administration in initial or disseminated tumor-bearing mice, at a dose of 12 mg/kg. The survival was monitored and blood samples were collected for biochemical and hematological analysis. Kidneys, liver and spleen were removed for histopathological examination. Tumor cells were evaluated for cellular viability and cell cycle. The survival of animals treated with SpHL-CDDP was higher than those treated with free CDDP. The cell death caused by treatment with SpHL-CDDP occurred through induction of apoptosis, with a cell cycle arrest at the G0/G1 phase. The treatment of mice presenting initial cancer with both formulations provoked a suppression of granulocytes. Mice treated with free CDDP also showed a decrease in platelet count, which suggests a high myelotoxicity. In an advanced cancer model, SpHL-CDDP treatment allowed an improvement of the immune response. Mice affected by cancer at an early stage and treated with free CDDP or SpHL-CDDP showed a lower urea/creatinine index compared with the saline control group. These findings indicate that both treatments were able to reduce the renal damage caused by peritoneal carcinomatosis. Microscopic analysis of kidneys from mice treated with SpHL-CDDP showed a discrete morphological alteration, while tubular necrosis was observed for free CDDP-treated mice. Concerning hepatotoxicity, no alteration in clinical chemistry parameters was observed. These findings reveal that SpHL-CDDP can improve the antitumor efficacy and decrease renal and bone marrow toxicity.

17DD and 17D-213/77 yellow fever substrains trigger a balanced cytokine profile in primary vaccinated children.

BACKGROUND: This study aimed to compare the cytokine-mediated immune response in children submitted to primary vaccination with the YF-17D-213/77 or YF-17DD yellow fever (YF) substrains. METHODS: A non-probabilistic sample of eighty healthy primary vaccinated (PV) children was selected on the basis of their previously known humoral immune response to the YF vaccines. The selected children were categorized according to their YF-neutralizing antibody titers (PRNT) and referred to as seroconverters (PV-PRNT(+)) or nonseroconverters (PV-PRNT(-)). Following revaccination with the YF-17DD, the PV-PRNT(-) children (YF-17D-213/77 and YF-17DD groups) seroconverted and were referred as RV-PRNT(+). The cytokine-mediated immune response was investigated after short-term in vitro cultures of whole blood samples. The results are expressed as frequency of high cytokine producers, taking the global median of the cytokine index (YF-Ag/control) as the cut-off. RESULTS: The YF-17D-213/77 and the YF-17DD substrains triggered a balanced overall inflammatory/regulatory cytokine pattern in PV-PRNT(+), with a slight predominance of IL-12 in YF-17DD vaccinees and a modest prevalence of IL-10 in YF-17D-213/77. Prominent frequency of neutrophil-derived TNF-α and neutrophils and monocyte-producing IL-12 were the major features of PV-PRNT(+) in the YF-17DD, whereas relevant inflammatory response, mediated by IL-12(+)CD8(+) T cells, was the hallmark of the YF-17D-213/77 vaccinees. Both substrains were able to elicit particular but relevant inflammatory events, regardless of the anti-YF PRNT antibody levels. PV-PRNT(-) children belonging to the YF-17DD arm presented gaps in the inflammatory cytokine signature, especially in terms of the innate immunity, whereas in the YF-17D-213/77 arm the most relevant gap was the deficiency of IL-12-producing CD8(+)T cells. Revaccination with YF-17DD prompted a balanced cytokine profile in YF-17DD nonresponders and a robust inflammatory profile in YF-17D-213/77 nonresponders. CONCLUSION: Our findings demonstrated that, just like the YF-17DD reference vaccine, the YF-17D-213/77 seed lot induced a mixed pattern of inflammatory and regulatory cytokines, supporting its universal use for immunization.

Human schistosomiasis mansoni: immune responses during acute and chronic phases of the infection.

Schistosoma mansoni infection may occur either as an acute infection in individuals who have recently visited an endemic area, with no previous contact with the parasite, or as a lasting chronic disease, if not interrupted by specific chemotherapy. The acute phase is characterized by symptoms such as fever, cough, diarrhea, anorexia, and arthralgias in combination with leukocytosis and eosinophilia, and a high cellular immune response to schistosome antigens especially those from the parasite's eggs. In the chronic phase, most patients living in endemic areas are asymptomatic, and their immune responses to egg antigens are modulated. A few develop periportal fibrosis of the liver, which may result in the hepatosplenic form of the disease. The humoral response (IgG, IgM and IgE) in acute patients to egg and worm antigens does not differ from the chronic phase. However, a high level of IgG and IgM antibodies to KLH were detected in acute patients. Acute patients express a considerably higher in vitro cellular responsiveness than do chronic patients, especially to egg antigens. They present a mixed profile of Th1 and Th2 cytokines. Ultrasound examinations of endemic population reveal a high heterogeneity between the patients as regards the presence and intensity of periportal fibrosis. Most patients are asymptomatic and their immune responses to schistosoma egg antigens (SEA) are modulated. In contrast, a high percentage of patients with incipient fibrosis (early stage of hepatosplenic) responded strongly to SEA. Patients with advanced hepatosplenic disease were likely to be non-responders to SEA. Most of the chronic patients presented a Th2 profile with low production of interferon-gamma (IFN-gamma). The intensity of infection favors the production of interleukin (IL)-10. After adjusting for age, sex, and intensity of infection, a strong correlation was observed between the production of IL-13 and the degree of fibrosis. Chronic asymptomatic patients and those with incipient fibrosis expressed very high levels of heterogeneity of their antibody responses. IgG response to soluble worm antigen preparation (SWAP) was distinct and significantly higher in hepatosplenic patients than in those asymptomatic or with incipient fibrosis. Levels of IgG4 to SEA were significantly higher in sera from patients with incipient fibrosis as compared to uninfected and hepatosplenic groups. Polyclonal idiotypic antibodies and their fragments F(ab')2, directly stimulate in culture T cells of schistosomiasis patients in presence of IL-1. Polyclonal idiotypic antibodies are able to modulate in vitro granuloma formation around SEA-polyacrylamide. The importance of idiotypes for protection or pathology in schistosomiasis is still not clear.

Swine pericardium as dermal substrate for human keratinocyte culture

Background Synthetic skin analogues or living allogeneic or autologous cells are used as dressings for the care of skin wounds, as well as temporary or permanent substitutes for damaged epithelia. Objectives To evaluate if keratinocyte growth on a swine pericardium substrate mimics the natural epithelial layers compared with cultures on allogeneic dermis, which is accepted as having appropriate physical and chemical properties for growth and differentiation. Methods Keratinocytes were cultured on a swine pericardium substrate and allogeneic dermis, either submerged or at the air-liquid interface. At 7, 14 and 21 days postseeding the cultures were evaluated by light microscopy after both haematoxylin and eosin staining and immunohistochemistry. Results Cell-substrate interactions led to growth, stratification and differentiation of cells, with the definition of epithelial layers. The submerged system showed a continuous growth rise on both composites, but this was more prominent with the swine pericardium substrate. An increase in the number of layers at the air-liquid interface with the dermis composites, in contrast to the submerged cultures, occurred only from days 7 to 14. The pattern of keratinocyte growth on swine pericardium substrate was much better in the submerged than in the air-liquid interface cultures. Conclusions The results indicate that swine pericardium is a better substrate than allogeneic dermis for keratinocyte cultures in submerged but not in air-liquid interface cultures. Swine pericardium as a substrate opens one more possibility for skin restoration after trauma or burns

Comparative clinical and ultrasound study of egg-negative and egg-positive individuals from Schistosoma mansoni low morbidity endemic areas, and hospitalized patients with hepatosplenic disease / Estudo clínico comparativo e ultra-sonografia entre indivíduos negativos e positivos para Schistosoma mansoni em áreas endêmicas de baixa morbidade e pacientes hospitalizados com doença hepatosplênica

Duzentos e vinte e três indivíduos de área endêmica de baixa morbidade para esquistossomose e nove pacientes hospitalizados com a forma hepatoesplênica foram submetidos ao exame de fezes e clínico e à ultra-sonografia do abdômen. De acordo com os resultado dos exames de fezes e do ultra-som eles foram agrupados do seguinte modo: G1 - 63 indivíduos sem ovos de Schistosoma mansoni nas fezes; G2 - 141 indivíduos apresentando ovos de Schistosoma mansoni nas fezes, sem ecogenicidade peripttal. G3 ­ 19 indivíduos com ovos de Schistosoma mansoni nas fezes e ecogenicidade peripttal entre 3-6mm.; G4 ­ 9 pacientes hepatesplênicos com ecogenicidade peripttal > 6mm. Pelo exame físico do abdômen, a hepatomegalia na linha hemiclavicular direita foi constatada em G1, G2 E G3, respectivamente, em 11,1, 12,1 e 26,3%. Nos grupos G1, G2 e G3, houve espessamento peripttal somente em esquistossomáticos (8,5%). Alterações patológicas leves em pacientes, as quais não puderam ser detectadas pelo exame clínico, foram evidenciadas no fígado pelo ultra-som e podem ser devidas à fibrose. O grau de fibrose peripttal leve foi diminuído em 57,9% dos pacientes 12 meses após tratamento da esquistossomose com oxamniquine. Na ultra-sonografia, a média da medida do lobo esquerdo do fígado dos indivíduos de G3 foi maior que a de G1 e, a de G4 maior que a de G1 e G2. O tamanho médio do baço de G4 foi significativamente maior que o dos outros grupos e o de G3 foi maior que o de G1 e G2.