Ionice Felipe

Brazilian Propolis Antileishmanial and Immunomodulatory Effects

The antileishmanial and immunomodulatory effects of propolis collected in Botucatu, São Paulo State, Brazil, were evaluated in Leishmania (Viannia) braziliensis experimental infection. The antileishmanial effect of propolis on promastigote forms was verified by reducing growth and by promoting morphologic alterations observed by scanning electron microscopy. In in vitro immunomodulatory assays, macrophages were pretreated with propolis and then infected with L. (V.) braziliensis. In vivo, supernatants from liver cells and peritoneal exudate of BALB/c mice pretreated with propolis and infected with Leishmania (107/mL promastigotes) were collected, and TNF-α and IL-12 were measured by ELISA. Macrophages incubated with propolis showed a significant increase in interiorization and further killing of parasites. An increased TNF-α production was seen in mice pretreated with propolis, whereas IL-12 was downregulated during the infection. In conclusion, Brazilian propolis showed a direct action on the parasite and displayed immunomodulatory effects on murine macrophages, even though the parasite has been reported to affect the activation pathways of the cell. The observed effects could be associated with the presence of phenolic compounds (flavonoids, aromatic acids, and benzopyranes), di- and triterpenes, and essential oils found in our propolis sample.

Protective effect of Artin M from extract of Artocarpus integrifolia seeds by Th1 and Th17 immune response on the course of infection by Candida albicans.

The immunoregulatory effect of Artin M and jacalin from extract of Artocarpus integrifolia seeds (jack extract) against infection with Candida albicans was investigated. Swiss mice received jack extract containing 500 μg protein/ml PBS intraperitoneally (i.p.) or PBS alone and after 72 h were infected i.p. with C. albicans CR15 (10(7)) and sacrificed after 30 min, 2, 6, 24, and 72 h. ELISA analysis revealed that in jack extract-treated mice IFN-γ was predominantly produced versus IL-10 in control mice. These results suggest that jack extract induced a protective immune response, since C. albicans clearance was complete at 72 h postinfection. Jack extract presents two lectins (Artin M and jacalin) with distinct biological properties. Artin M was able to induce IL-12 production by macrophages. Also, Artin M in different concentrations, associated with jacalin or in jack extract induced both IFN-γ and IL-17 production. As a consequence, phagocytic and candidacidal activity increased significantly. Alanine aminotransferase activity (ALT) was used as parameter for damage of the liver. The activity of ALT correlated with inoculum size that increased significantly in control group, however, mice pretreated with jack extract 3 days before infection presented normal ALT. Mice pretreated with jack extract that received a lethal inoculum of Candida presented 90% survival versus 20% among controls or mice pretreated with jacalin. Thus, the results suggest that Artin M by itself, associated with jacalin or present in jack extract is able to induce protective Th1 and Th17 immune responses against Candida albicans infection.

Early membrane exposure of phosphatidylserine followed by late necrosis in murine macrophages induced by Candida albicans from an HIV-infected individual

The hypothesis that Candida albicans isolate (CR1) from an HIV-infected individual induced apoptosis of macrophages was examined by optical microscopy, binding of annexin V-FITC and analyses of DNA degradation (TUNEL tests and agarose gel electrophoresis). Resident murine peritoneal macrophages co-incubated for 5-15 min with C. albicans CR1 bound annexin V, whereas macrophages incubated with either heat-inactivated strain CR1, C. albicans 577 (isolated from a patient with mucocutaneous candidiasis) or C. albicans FCF14 (a mutant that did not produce proteases and phospholipases) did not bind annexin for up to 2 h of observation. However, macrophages exposed to C. albicans CR1 did not present the pattern of DNA degradation typical of apoptosis. Macrophages became increasingly permeable to propidium iodide from 30 min to 2 h after their exposure to C. albicans CR1. Most of the phagocytosed C. albicans CR1 yeast cells switched to germ-tubes inside the macrophages after incubation for 1-2 h. These results show that macrophages exposed to C. albicans CR1 presented early signs of apoptosis but progressed to necrosis, and suggest that Candida strains that readily switch to germ-tubes inside those apoptotic cells might have a competitive advantage in vivo because released germ-tubes resist further attack by macrophages.

Epstein–Barr Virus (EBV) MicroRNAs: Involvement in Cancer Pathogenesis and Immunopathology

The Epstein–Barr virus (EBV), which infects over 90% of adults, appears to have evolved to exploit the normal biology of B-cell development in order to persist as a life-long asymptomatic infection. However, EBV can contribute to oncogenesis. It has become evident that alterations in the expression of microRNAs (miRNAs) from the host cell and EBV can also contribute to cancer pathogenesis. MicroRNAs function by inhibiting translation of select groups of mRNA transcripts containing imperfect annealing sequences in their 3′ untranslated regions (3′ UTRs) and less frequently through other regions of the transcript. A number of studies have demonstrated that profiles of miRNA expression could establish phenotypic signatures of different cancer types where viruses have been evolved with highly sophisticated gene silencing machinery to disturb the host–immune response. Based on current review, it is possible that a specific virus miRNA may be involved in cancer pathogenesis.

Increased tumour necrosis factor-α production, higher mannose receptor activity and ability to kill Candida by concanavalin-A-activated macrophages

In a previous study, our group verified that mice pretreated with concanavalin-A (Con-A) produced more tumour necrosis factor (TNF)-alpha and presented greater Candida clearance from the peritoneal cavity, liver and spleen, which yielded a higher survival rate than control animals. In this work, the hypothesis that macrophages were of crucial importance in overcoming the infection was tested. Thus, peritoneal macrophages from mice pretreated for 3 days with Con-A or phosphate-buffered saline (PBS) were coincubated with CR1, CR15 and 577 isolates of Candida albicans for 0.5, 1 and 2 h. The ability of Con-activated macrophages to produce TNF-alpha, ingest via mannose receptors and kill all the isolates was significantly greater compared with PBS-treated macrophages, and activated macrophages exhibited a lower incidence of apoptosis, verified by binding to annexin V-fluorescein isothiocyanate. The transition of yeast cells to filamentous forms during coincubation for 2 h with control macrophages was about 73-80%, whereas in the presence of Con-A-activated macrophages, it was 35-40%. Our results suggest that a greater clearance of C. albicans infection through treatment with Con-A is probably due to the activation of macrophages, which produce more TNF-alpha, express more mannose receptors and are better endowed to kill ingested C. albicans.

Artin M enhances TNF-α production and phagocytosis of Candida albicans mediated by dectin-1 and mannose receptors.

The activities of dectin-1 and mannose receptors on phagocytosis of Candida albicans and the production of TNF-α by macrophages from mice pretreated for 3 days with extract of Artocarpus intergrifolia seeds (jack extract), Artin M or jacalin were studied. Macrophages from these mice were coincubated with C. albicans CR15 (yeast), in the presence of mannose (50mM) plus mannan (100 μg) or laminarin (1mg). Phagocytosis was significantly enhanced to 52% in macrophages from mice pretreated intraperitoneally for 3 days with jack extract (500 μg/250 μl PBS). Reduction in phagocytosis from 52% to 34% (P<0.05) occurred in the presence of mannose receptor inhibitors and from 52% to 16% (P<0.01) in the presence of dectin-1 inhibitor laminarin, whereas only 20% of control macrophages phagocytosed blastoconidia. Similar results were verified for pretreatment of mice with Artin M (2.5 μg/250 μl PBS), but not for jacalin (25 μg/250 μl PBS). Macrophages from mice pretreated 3 days previously with jack extract or Artin M and then coincubated for 2h with C. albicans presented a significant increase in TNF-α production, correlating with significantly less transition of yeast to filamentous forms compared to pretreatment with jacalin. These results suggest that Artin M, but not jacalin present in jack extract significantly increased TNF-α production and the activity of mannose and dectin-1 receptors.

Pre-treatment with Concanavalin-A increases resistance of mice to peritoneal infection by Serratia marcescens

Mice pre-treated with Concanavalin-A largely survived an intra-peritoneal inoculum of 2 x 10(7) Serratia marcescens, whereas all control mice died within 15 h of inoculation. A subpopulation of peritoneal macrophages from Con-A pre-treated mice was able to phagocytose the bacteria in vitro (6.7 SEM 1.2% phagocytosing cells) and in vivo (16.9 SEM 2.1%), whereas control phagocytes did not phagocytose S. marcescens. The survival of Con-A pre-treated mice allowed their immunisation with living bacteria, and the antiserum thus produced increased the phagocytosis of S. marcescens in vitro. Control mice largely survived an inoculum of S. marcescens suspended in 50% immune serum, although the bacteria were resistant to the bactericidal activity of that serum. These results suggest that, in contrast to the delayed humoral protection afforded by immunisation, phagocytosis by phagocytes activated by Con-A conferred early protection to mice against experimental infection by S. marcescens.

Immunological and histopathological characterization of cutaneous candidiasis

Chronic mucocutaneous candidiasis constitutes a heterogeneous group of syndromes, characterized by non-invasive infection of the skin, nails and mucosal membranes by the fungus Candida spp. Although symptoms are heterogeneous, in all cases there is a reduction in protective cytokines, favouring the development of disease. The normal role of cytokines in skin lesions is not well understood. The present study aimed to investigate the progression of disease, understand specific cellular and molecular components involved in immunity to Candida albicans and determine the balance between pro- and anti-inflammatory cytokines over the course of cutaneous infection in immunocompetent mice. BALB/c mice (five per group) were inoculated with 5 × 10(6)C. albicans pseudohyphae in the deep dermis of the paw and analysed over 1-14  days post-infection. The contralateral paws were used for negative controls. Haematoxylin and eosin staining of skin sections during C. albicans infection was performed to analyse structural modifications to the epidermis such as hyperplasia, and infiltration of neutrophils and fibroblasts in the dermis. The cytokine populations were determined by capture ELISA using popliteal lymph node tissue. Pro-inflammatory cytokines (IL-6, TNF-α, IL-12, IFN-γ and IL-17) were detected at significant levels during the initial phase of cutaneous infection and correlated with the rapid elimination of C. albicans. Anti-inflammatory cytokines (IL-13, IL-4, IL-10 and transforming growth factor-β) were detected on day 4 post-infection, and prevented exacerbation of inflammation and participated in healing of lesions. Thus, a balance between pro- and anti-inflammatory cytokines was fundamental for the resolution of infection. Importantly, these findings broaden our understanding of the immune mechanisms involved in chronic cutaneous candidiasis.

Treatment of serum with supernatants from cultures of Candida albicans reduces its serum-dependent phagocytosis

Candida albicans e um potente ativador do sistema complemento, e opsoninas labeis ao calor produzidas por ativacao de C3 (C3b e iC3b) aumentam a fagocitose de C. albicans mediada por receptores de complemento. Neste estudo, tratamos o soro de camundongo com sobrenadante de culturas de uma cepa de C. albicans produtora de proteases e avaliamos a capacidade deste soro reduzir a fagocitose de C. albicans. Sobrenadantes livres de celulas obtidos de cultura de C. albicans foram concentrados 5 vezes e adicionados ao soro de camundongo por 30 minutos a 37oC, antes deste soro ser usado para opsonizacao de C. albicans na forma de levedura e fixadas em glutaraldeido. Nos observamos que soro normal aumentou 3 vezes a fagocitose de C. albicans por macofagos peritoneais, enquanto que o soro tratado com sobrenadante nao aumentou a fagocitose. Este efeito do sobrenadante sobre o soro foi evitado por adicao de pepistatina (5 µg/ml; um inibidor de proteinase acida) ao meio. Soro tratado com sobrenadantes de culturas de um mutante de C. albicans deficiente em producao de proteinase tambem aumentou em 3 vezes a fagocitose da levedura. Estes resultados sugerem que uma proteinase produzida por C. albicans causa proteolise de opsoninas do soro, desta maneira reduzindo a fagocitose da levedura.

Concanavalin‐A induces IL‐17 production during the course of Candida albicans infection

In a previous study, our group verified that 100% of mice survived to a lethal dose of Candida albicans following pretreatment with concanavalin-A (Con-A) for 3 days. This work proposed to investigate whether treatment could mediate an adaptative immune response involving T(H) 17 cells. A significant increase in IL-17 levels at 6 h postinfection was observed and was maintained up to 18 h in the Con-A group, whereas in control mice, a reduction in this cytokine was verified. In addition, T(H) 17 cells develop in the presence of TGF-β, IL-1 β, and IL-6 that were increased significantly 2 h postinfection in Con-A-treated mice. Macrophages were involved in the process, engulfing greater numbers of yeast cells, and were activated through TNF-α and interferon-γ produced at significant levels at 2 h postinfection. A significant increase in IL-12 levels was also observed at 2 h postinfection. Thus, activated macrophages were probably more capable of killing and processing Candida antigens, signalizing an adaptative immune response. Macrophages from controls did not prevent yeast-to-hyphae transition and were partially destroyed, as shown in scanning microscopy. These results suggest that treatment with Con-A facilitated the triggering of T(H) 17 and T(H) 1 responses via IL-17 and IFN-γ production, leading to the resolution of C. albicans infection.