Association between Adverse Maternal Clinical Outcomes and Imbalance of Cytokines and Angiogenic Factors in Preterm Preeclampsia / Associação entre desfechos maternos adversos e desbalanço de citocinas e fatores angiogênicos em pré-eclâmpsia pré-termo

Abstract Objective Preeclampsia (PE) is a pregnancy-specific syndrome characterized by abnormal levels of cytokines and angiogenic factors, playing a role in the disease development. The present study evaluated whether immunological markers are associated with the gestational age and with the disease severity in preeclamptic women. Methods Ninety-five women who developed PE were stratified for gestational age as preterm PE (< 37 weeks) and term PE (≥ 37 weeks of gestation) and compared for disease severity as well as plasma concentration of angiogenic factors and cytokines. The concentrations of placental growth factor (PlGF), vascular endothelial growth factor (VEGF), Fms-like soluble tyrosine kinase (sFlt-1) and soluble endoglin (sEng), as well as the cytokines, tumor necrosis factor-α (TNF-α) and interleukin 10 (IL-10), were determined by enzyme-linked immunosorbent assay (ELISA). Results The comparison between preeclamptic groups showed a higher percentage of severe cases in preterm PE (82.1%) than in term PE (35.9%). Similarly, the concentrations of TNF-α, sFlt-1, and sEng, as well as TNF-α/IL-10 and sFlt-1/PlGF ratios were significantly higher in the preterm PE group. In contrast, concentrations of PlGF, VEGF, and IL-10 were significantly lower in women with preterm PE. Negative correlations between TNF-α and IL-10 (r = 0.5232) and between PlGF and sFlt1 (r = 0.4158) were detected in the preterm PE. Conclusion In pregnant women with preterm PE, there is an imbalance between immunological markers, with the predominance of anti-angiogenic factors and TNF-α, associated with adverse maternal clinical outcomes.

Resumo Objetivo A pré-eclâmpsia (PE) é uma síndrome específica da gravidez caracterizada por níveis anormais de citocinas e fatores angiogênicos, que desempenham um papel no desenvolvimento da doença. Este estudo avaliou se os marcadores imunológicos estão associados à idade gestacional e à gravidade da doença em mulheres com pré-eclâmpsia. Métodos Noventa e cinco mulheres que desenvolveram PE foram estratificadas pela idade gestacional em PE pré-termo (< 37 semanas) e PE a termo (≥ 37 semanas de gestação) e comparadas quanto à gravidade da doença, bem como à concentração plasmática de fatores angiogênicos e citocinas. As concentrações de fator de crescimento placentário (PlGF), fator de crescimento endotelial vascular (VEGF), tirosina quinase solúvel semelhante a Fms (sFlt-1) e endoglina solúvel (sEng), bem como as citocinas, fator de necrose tumoral alfa (TNF- α) e interleucina 10 (IL-10), foram determinados porensaio de imunoabsorção enzimática (ELISA, na sigla em inglês). Resultados A comparação entre os grupos com pré-eclâmpsia mostrou maior porcentagem de casos graves em PE pré-termo (82,1%) do que em PE a termo (35,9%). Da mesma forma, as concentrações de TNF-α, sFlt-1 e sEng, bem como as razões TNF-α/IL-10 e sFlt-1/PlGF foram significativamente maiores no grupo de PE pré-termo. Em contraste, as concentrações de PlGF, VEGF e IL-10 foram significativamente menores em mulheres com PE pré-termo. Correlações negativas entre TNF-α e IL-10 (r = 0.5232) e entre PlGF e sFlt1 (r = 0.4158) foram detectadas no grupo de PE pré-termo. Conclusão Em gestantes com PE pré-termo, ocorre um desequilíbrio entre os marcadores imunológicos, com predomínio de fatores antiangiogênicos e TNF-α, associados a desfechos clínicos maternos adversos.

Granulocyte macrophage colony-stimulating factor enhances the modulatory effect of cytokines on monocyte-derived multinucleated giant cell formation and fungicidal activity against Paracoccidioides brasiliensis

Multinucleated giant cells (MGC) are cells present in characteristic granulomatous inflammation induced by intracellular infectious agents or foreign materials. The present study evaluated the modulatory effect of granulocyte macrophage colony-stimulating factor (GM-CSF) in association with other cytokines such as interferon-gamma (IFN-γ), tumour necrosis factor-alpha, interleukin (IL)-10 or transforming growth factor beta (TGF-β1) on the formation of MGC from human peripheral blood monocytes stimulated with Paracoccidioides brasiliensis antigen (PbAg). The generation of MGC was determined by fusion index (FI) and the fungicidal activity of these cells was evaluated after 4 h of MGC co-cultured with viable yeast cells of P. brasiliensis strain 18 (Pb18). The results showed that monocytes incubated with PbAg and GM-CSF plus IFN-γ had a significantly higher FI than in all the other cultures, while the addition of IL-10 or TGF-β1 had a suppressive effect on MGC generation. Monocytes incubated with both pro and anti-inflammatory cytokines had a higher induction of foreign body-type MGC rather than Langhans-type MGC. MGC stimulated with PbAg and GM-CSF in association with the other cytokines had increased fungicidal activity and the presence of GM-CSF also partially inhibited the suppressive effects of IL-10 and TGF-β1. Together, these results suggest that GM-CSF is a positive modulator of PbAg-stimulated MGC generation and on the fungicidal activity against Pb18.

Interleukin-15 augments oxidative metabolism and fungicidal activity of human monocytes against Paracoccidioides brasiliensis

Interleukin (IL)-15 is a pleiotropic cytokine that regulates the proliferation and survival of many cell types. IL-15 is produced by monocytes and macrophages against infectious agents and plays a pivotal role in innate and adaptive immune responses. This study analyzed the effect of IL-15 on fungicidal activity, oxidative metabolism and cytokine production by human monocytes challenged in vitro with Paracoccidioides brasiliensis (Pb18), the agent of paracoccidioidomycosis. Peripheral blood monocytes were pre-incubated with IL-15 and then challenged with Pb18. Fungicidal activity was assessed by viable fungi recovery from cultures after plating on brain-heart infusion-agar. Superoxide anion (O2-), hydrogen peroxide (H2O2), tumour necrosis factor-alpha (TNF-α), IL-6, IL-15 and IL-10 production by monocytes were also determined. IL-15 enhanced fungicidal activity against Pb18 in a dose-dependent pattern. This effect was abrogated by addition of anti-IL-15 monoclonal antibody. A significant stimulatory effect of IL-15 on O2- and H2O2 release suggests that fungicidal activity was dependent on the activation of oxidative metabolism. Pre-treatment of monocytes with IL-15 induced significantly higher levels of TNF-α, IL-10 and IL-15 production by cells challenged with the fungus. These results suggest a modulatory effect of IL-15 on pro and anti-inflammatory cytokine production, oxidative metabolism and fungicidal activity of monocytes during Pb18 infection.

Experimental infections with Paracoccidioides brasiliensis obtained from armadillos: comparison to clinical isolates

Paracoccidioides brasiliensis causes paracoccidioidomycosis (PCM) that is one of the most prevalent systemic human mycoses in Latin America. Armadillos show a high incidence of PCM infection and could, therefore, be a natural reservoir for this fungus. In this study were compared the virulence profiles of isolates obtained from nine-banded armadillos (Dasypus novemcinctus) (PbT1 and PbT4) and isolates from PCM patients (Pb265 and Bt83). Pathogenicity was evaluated by fungal load and analysis of colony morphology. Immunity against the fungus was tested by delayed type hypersensitivity test (DTH) and antibody quantification by ELISA. The higher virulence of PbT1 and PbT4 was suggested by higher fungal load in spleen and lungs. Armadillo isolates and Bt83 presented a cotton-like surface contrasting with the cerebriform appearance of Pb265. All isolates induced cellular and humoral immune responses in infected BALB/c mice. DTH reactions were similarly induced by the four isolates, however, a great variability was observed in specific antibody levels, being the highest ones induced by Bt83 and PbT4. The present work confirms that armadillos harbor P. brasiliensis, whose multiplication and induced immunity in experimentally infected mice are heterogeneous, resembling the behavior of isolates from human PCM. This study reinforces the possibility that armadillos play an important role in the biological cycle of this pathogen.

Inhibitory effect of deferoxamine on Paracoccidioides brasiliensis survival in human monocytes: reversal by holotransferrin not by apotransferrin

Os mecanismos utilizados pelo Paracoccidioides brasiliensis para sobreviver em células fagocitárias ainda não estão elucidados. O metabolismo celular férrico é muito importante para o crescimento de inúmeros patógenos intracelulares cuja capacidade de se multiplicarem em fagócitos mononucleares é dependente da disponibilidade intracelular do íon ferro. Assim, o objetivo deste trabalho foi investigar o papel do ferro intracelular sobre a capacidade do P. brasiliensis sobreviver em monócitos humanos. O tratamento de monócitos com deferoxamina, uma droga quelante, diminuiu a sobrevivência de leveduras do fungo de forma dose-dependente. O efeito inibidor da deferoxamina sobre a sobrevivência do P. brasiliensis foi revertido por transferrina saturada com ferro (holotransferrina) mas não por transferrina insaturada (apotransferrina). Estes resultados sugerem que a sobrevivência do P. brasiliensis em monócitos humanos é dependente do íon ferro.

Immunologic aspects of West syndrome and evidence of plasma inhibitory effects on T cell function

STUDY OBJECTIVE: The purpose of this study was to assess the extent of immune dysfunction in a well-defined group of epileptic patients: children with diagnosis of West syndrome (WS) or with transitions to another age-related EEG patterns, the multifocal independent spikes (MIS), and the slow spike-wave complexes (Lennox-Gastaut syndrome - LGS). Thus, WS was studied at different points of the natural evolutive history of the disease. METHOD: A group of 50 patients (33 with WS, 10 with LGS and 7 with MIS) and 20 age-matched healthy controls were submitted to enumeration of T lymphocyte subsets: CD1, CD3, CD4, CD8, CD4/CD8 ratio and lymphocyte proliferation assay to phytohaemagglutinin (PHA), in the presence of autologous and AB, homologous plasma. Dinitrochlorobenzene (DNCB) skin test sensitization was performed only in patients. Determinations of IgG, IgA, and IgM serum levels were compared to standard values for Brazilian population in different age ranges. RESULTS: Sensitization to DNCB showed absent or low skin reactions in 76 percent of the patients. High levels of IgG (45.7 percent) and IgM (61.4 percent), and lower levels of IgA (23.9 percent) were detected in the serum of the patients. Enumeration of lymphocyte subsets in peripheral blood showed: low CD3+ (p<0.05), low CD4+ (p<0.05), high CD8+ (p<0.01) and low CD4+ / CD8+ ratio (p<0.001). The proportion of CD1+ cells in the control group was less than 3 percent, while ranged between 6 and 11 percent in 18 percent of the patients. The in vitro PHA-induced T cell proliferation showed significantly low blastogenic indices only when patients, cells were cultured in presence of their own plasma. No differences in blastogenic indices were observed when the cells of patients and controls were cultured with human AB plasma. CONCLUSION: The immunodeficiency in WS was mainly characterized by anergy, impaired cell-mediated immunity, altered levels of immunoglobulins, presence of immature thymocytes in peripheral blood and functional impairment of T lymphocytes induced by plasma inhibitory factors

Familial cancer: depressed NK-cell cytotoxicity in healthy and cancer affected members

Depressed natural killer (NK) cell activity has been showed in family members of patients with different types of cancer. The present work aimed to evaluate T cell subsets and NK cell cytotoxic activity in 15 members of a family with high incidence of tumors, such as glioblastoma, gastric, pancreas and colon rectal carcinoma, chronic myelocitic leukemia, melanoma and osteoblastoma. As controls, 19 healthy subjects with the age range equivalent were studied. The enumeration of CD3+ lymphocytes and their CD4+ and CD8+ subsets were defined by monoclonal antibodies and NK cell cytotoxicity towards K562 target cells were evaluated by single cell-assay. The results showed in family members low percentage of total T cells (CD3+), and their CD4+ subset and impairment of CD4/CD8 ratio in relation to control group. All family members presented percentage of NK-target cell conjugate formation bellow the minimum value observed in control group. Thirteen people were examined and followed up during five years, in order to assure that there was no undiagnosed or unsuspected disease at the moment of evaluation. One of them developed osteoblastoma and other malignant melanoma. Two cancer patients, with glioblastoma and chronic myelocytic leukemia were studied during illness. All the corresponding values were comparable. The persistence of low percentage of conjugate formation may be related to a defect on adhesion molecules expression in the surface of NK cells that was probably responsible for the low activity of these cells presented by the family group. Thus, the inheritance mechanism of low adherence of NK cells should have a prognostic value in determining the risk of developing tumors

Immunological alterations in patients with primary tumors in central nervous system

Natural Killer (NK) cells play an important role in immune surveillance against tumors. The present work aimed to study the cytotoxic activity of NK cells and T cell subsets in peripheral blood of 13 patients with primary in central nervous system (CNS). As controls 29 healthy subjects with the age range equivalent to the patients were studied. The methods employed were: a) determination of cytotoxic activity of NK cells towards K562 target cells, evaluated by single cell-assay; b) enumeration of CD3+ lymphocytes and their CD4+ and CD8+ subsets defined by monoclonal antibodies; c) the identification of tumors were done by histologic and immunochemistry studies. The results indicated that adults and children with tumor in CNS display reduced percentage of total T cells, helper/inducer subset and low helper/suppressor ratio. The cutotoxic activity of NK cells was decreased in patients with CNS tumors due mainly to a decrease in the proportion of target-binding lymphocytes. These results suggest that cytotoxic activity of NK cells may be affected by the immunoregulatory disturbances observed in patients with primary tumors in CNS.

Increased natural killer activity does not prevent progression of experimental Kala-azar

Kala-azar is the visceral form of leishmaniasis and it is caused by intracellular parasites from the complex Leishmania donovani. Golden hamster (Mesocricetus auratus) infected with Leishmania donovani develop a disease very similar to human Kala-azar. There is conspicuous hipergammaglobulinaemia and their T cells do not respond to stimulation with parasite antigens. We used this experimental model to evaluate the natural killer (NK) activity during the initial phase of the disease. Outbred hamsters infected by intravenous route with 5.106 amastigotes of L. donovani 1S showed a concurrent increase in the spleen weight and in the spleen cell number. Using the single cell assay we detected a significant increase in the percentage of NK effector cells on the 4th day of infection. Imprints from spleen and liver showed at days 14 and 28 a significant increase in the parasite burden . These results show that the increased NK activity in the beginning of the infection was not able to restrain the progression of the disease in this experimental model

Virulence factors in fungi of systemic mycoses

Fungos patogênicos causadores de micoses sistemicas possuem vários fatores que permitem seu crescimento nas condiçöes adversas oferecidas pelo hospedeiro, propiciando o estabelecimento da relaçäo parasitária e contribuindo no processo de doença. Esses fatores säo conhecidos como fatores de virulencia auxiliando no desenvolvimento da infecçäo e interferindo com a patogenese das micoses. O presente trabalho avalia os fatores de virulencia em fungos patogenicos como Blastomyces dermatitis, Coccidioides immitis, Cryptococcus neoformans, Histoplasma capsulatum e Paracoccidioides brasiliensis, em relaçäo a termotolerancia, dimorfismo, componentes da parede celular ou capsula, bem como a producao de enzimas...

Alterations of cell-mediated immune response in children with febrile seizures

The aim of the present investigation was to study the distribution of T-cell subsets in peripheral blood defined monoclonal antibodies and by the lymphocyte proliferative response to phytohemagglutinin (PHA) in 30 children febrile seizures and in 14 age-matched control subjects. Frequent respiratory, urinary and dermatologic infections were observed in 22 patients. The immunologic parameters showed that 64 percent of the patients presented an increases number of CD8+ cells and a low helper/suppressor ratio was observed in 60 percent of the patients. In addition, the proliferative response of lymphocytes to PHA was impared in the patients. It was observed the presence of inhibitory activity on lymphocyte function in the plasma of 33 percent of children with febrile seizures. These results suggest that patients with febrile seizures have an impairment of cellular immunity that may be connected with this epileptic syndrome and explain the infections observed.

Natural killer cell activity in experimental paracoccidioidomycosis of the syrian hamster

Com o objetivo de avaliar a atividade de celulas natural killer na paracoccidioidomicose experimental do hamster, 80 hamsters foram infectados por via intratesticular com Paracoccidioides brasiliensis e sacrificados apos 24h, 48h, 96h, 1, 2, 4, 8 e 11 semanas de infeccao. Como controle foram avaliados 40 hamsters normais, nao infectados. Os animais foram submetidos ao estudo da atividade citotoxica de celulas NK pela tecnica de "single-cell assay" e da resposta imune humoral pelas tecnicas de imunodifusao dupla e Elisa...