A Histoplasma capsulatum-Specific IgG1 Isotype Monoclonal Antibody, H1C, to a 70-Kilodalton Cell Surface Protein Is Not Protective in Murine Histoplasmosis ▿

Monoclonal antibodies to Histoplasma capsulatum can modify pathogenesis. We now show that monoclonal antibody H1C to a 70-kDa antigen increases intracellular fungal growth and reduces macrophage nitric oxide release but has no effect on fungal burden or survival in murine infection. This further demonstrates the complexities of host-pathogen interactions

Characterization of Scedosporium apiospermum glucosylceramides and their involvement in fungal development and macrophage functions.

Scedosporium apiospermum is an emerging fungal pathogen that causes both localized and disseminated infections in immunocompromised patients. Glucosylceramides (CMH, GlcCer) are the main neutral glycosphingolipids expressed in fungal cells. In this study, glucosylceramides (GlcCer) were extracted and purified in several chromatographic steps. Using high-performance thin layer chromatography (HPTLC) and electrospray ionization mass spectrometry (ESI-MS), N-2'-hydroxyhexadecanoyl-1-β-D-glucopyranosyl-9-methyl-4,8-sphingadienine was identified as the main GlcCer in S. apiospermum. A monoclonal antibody (Mab) against this molecule was used for indirect immunofluorescence experiments, which revealed that this CMH is present on the surface of the mycelial and conidial forms of S. apiospermum. Treatment of S. apiospermum conidia with the Mab significantly reduced fungal growth. In addition, the Mab also enhanced the phagocytosis and killing of S. apiospermum by murine cells. In vitro assays were performed to evaluate the CMHs for their cytotoxic activities against the mammalian cell lines L.929 and RAW, and an inhibitory effect on cell proliferation was observed. Synergistic in vitro interactions were observed between the Mab against GlcCer and both amphotericin B (AmB) and itraconazole. Because Scedosporium species develop drug resistance, the number of available antifungal drugs is limited; our data indicate that combining immunotherapy with the available drugs might be a viable treatment option. These results suggest that in S. apiospermum, GlcCer are most likely cell wall components that are targeted by antifungal antibodies, which directly inhibit fungal development and enhance macrophage function; furthermore, these results suggest the combined use of monoclonal antibodies against GlcCer and antifungal drugs for antifungal immunotherapy

(Re)Conhecendo a importância da água residual proveniente de fossas sépticas para os pequenos proprietários rurais

As regiões mais afastadas dos pólos industriais, sobretudo na zona rural, apresentam tratamento de esgoto precário, não sendo servidas por redes de coleta pública. Essa inexistência cria um ambiente insalubre, com a eliminação de esgoto sanitário sem tratamento prévio e construção de fossas rudimentares, que, sem o isolamento adequado, acarretam na infiltração de resíduos patogênicos no solo, o que propicia o desenvolvimento de doenças, algumas de alta morbidade. Visando melhorar as condições de higiene da população, e reduzir a incidência de doenças infecciosas e parasitárias, as prefeituras dos municípios de Guapimirim e Magé, juntamente com a EMATER RIO, iniciaram o projeto de implantação de fossas sépticas desenvolvido pela Embrapa. Esse modelo de fossa consiste em três tanques enterrados que recebem os dejetos exclusivamente do vaso sanitário, retêm a parte sólida e realizam o processo biológico de purificação do efluente, que é filtrado por plantas enraizadoras, diminuindo assim o risco de contaminação. Diante deste quadro,os laboratórios da Faculdade de Farmácia associados ao desenvolvimento do projeto Uso e Cultivo Racionais de Plantas Medicinais e Plantas Alimentícias Não Convencionais (PANC) em Magé e Guapimirim, por solicitação da EMATER, iniciaram a análise dos sistemas implantados para validação de sua segurança. Para tanto, estão sendo realizadas coletas periódicas para análise parasitológica, microbiológica e micológica da água residual proveniente do sistema. Tais análises permitirão inferências sobre a possibilidade de reuso destas águas para irrigação do solo e das plantações, contribuindo para melhorias no saneamento rural, que pode refletir na renda dos agricultores e no desenvolvimento da agricultura orgânica. Para repasse das informações obtidas nas análises, serão realizadas oficinas em Magé para orientação dos agricultores e seus familiares, esclarecendo os benefícios das fossas sépticas e as consequências que más condições de higiene e uso de água contaminada por patógenos podem ocasionar. De acordo com os resultados obtidos, novos encontros orientarão os agricultores sobre o manejo adequado das águas residuais. Cada encontro poderá contemplar 40 pessoas. Diante dos dados iniciais coletados, está sendo elaborada uma cartilha a ser finalizada em novembro, contendo as seguintes informações: Ciclo da água e fontes de contaminação; Agentes poluidores e patógenos transmitidos pela água contaminada, com respectivos ciclos de desenvolvimento , sintomas de doenças e medidas profiláticas; Qualidade da água na cadeia produtiva e no consumo doméstico. Após a finalização da edição, a cartilha será disponibilizada não apenas para os moradores locais, mas também para os membros das comunidades adjacentes, em formato digital, através dos sítios eletrônicos da EMATER e SMDA-Magé.

Effect of the anti-CMH Mab on the germination and viability of <i>S. apiospermum</i> conidial cells.

<p>(A) Germinated conidia in the presence of different concentrations of anti-CMH Mab over 24 h were counted by optical microscopy. At least 100 conidia per field were counted, and the mean value of three independent counts was calculated. (B) Viability assays were performed using <i>S. apiospermum</i> conidia and the anti-CMH Mab over 24 h and evaluated using MTT. The absorbance at 570 nm was measured using a spectrophotometer.</p

MAb to CMH affect phagocytosis.

<p>Influence of the Mab to CMH on the phagocytosis of <i>S. apiospermum</i> conidia cells by peritoneal macrophages (A) and on phagocyte antimicrobial activity. Fungal cells treated with the MAb against GlcCer were more efficiently internalized, as demonstrated by the phagocytic indices. (B) Microbial killing by macrophages was enhanced after treatment with antibodies to CMH.</p

Cytotoxic activity of <i>S. apiospermum</i> CMH.

<p>Cytotoxic assay of <i>S. apiospermum</i> CMH on L929 and RAW cells during 48 h of incubation. Cell viability was measured by adding a neutral-red solution and measuring the absorbance at 490 nm using a spectrophotometer. The presence of <i>S. apiospermum</i> CMH in the culture supernatant was analyzed using TLC.</p

ESI-MS (positive ion mode, Li+ adducts) analysis of the GlcCer species of <i>S. apiospermum</i>.

<p>(A) MS1 spectrum. (B) ESI-MS2 of the ions species with <i>m/z</i> 734.9 observed in (A) and proposed structures for the major GlcCer species in <i>S. apiospermum.</i> (C) HPTLC plate of monosaccharides from <i>S. apiospermum</i> CMH. 1. Galactose, glucose, mannose and rhamnose standards; 2. Glucose from CMH. The sugars were detected using the orcinol-sulfuric acid spray reagent.</p

HPTLC analysis of CMH released by <i>S. apiospermum</i> in the culture supernatant.

<p>Lane 1: Purified CMHs from <i>S. apiospermum</i>; Lane 2: CMH released in the supernatant; Lane 3: culture medium supernatant (control). The solvent used was chloroform: methanol: 2 M NH₄OH (40:10:1 v/v). Detection was performed using iodine and the orcinol-sulfuric acid reagent.</p

Reactivity of fungal CMH with the anti-CMH MAb.

<p>(A) ELISA analysis of the binding of Mabs to <i>S. apiospermum</i> CMH. The amount of antibody bound to CMHs was determined by incubation with a rabbit anti-mouse IgG. CMH from <i>S. apiospermum</i> (closed circle); CMH from <i>A. fumigatus</i> (closed square); CMH from <i>S. apiospermum</i> + unrelated antibody (open circle); negative control (open square). Inset: HPTLC of <i>S. apiospermum</i> CMH (spots a,b,c), which was developed in CHCl3: MeOH: 2 M NH4OH (40:10:1 v/v). Lane 1: stained with orcinol/ H2SO4; Lane 2: immunostaining with the anti-CMH MAb. Indirect immunofluorescence analysis of conidial (B and C) and mycelial (D and E) forms of <i>S. apiospermum</i> using the anti-CMH MAb. B and D: phase contrast; C and E: fluorescence signal.</p

Synergistic effect of combining the anti-CMH Mab with antifungal drugs.

<p>The viability of <i>S. apiospermum</i> conidia was analyzed in the presence of the anti-CMH Mab (50 µg/ml) and different concentrations (0.5 -16 µg/ml) of itraconazole (A) or amphotericin B (B). The values shown are means of three independent experiments.</p