Plant antibodies for human antifungal therapy

There is an increasing interest in the development of therapeutic antibodies (Ab) to improve the control of fungal pathogens, but none of these reagents is available for clinical use. We previously described a murine monoclonal antibody (mAb 2G8) targeting β-glucan, a cell wall polysaccharide common to most pathogenic fungi, which conferred significant protection against Candida albicans, Aspergillus fumigatus and Cryptococcus neoformans in animal models. Transfer of this wide-spectrum, antifungal mAb into the clinical setting would allow the control of most frequent fungal infections in many different categories of patients. To this aim, two chimeric mouse-human Ab derivatives from mAb 2G8, in the format of complete IgG or scFv-Fc, were generated, transiently expressed in Nicotiana benthamiana plants and purified from leaves with high yields (approximately 50 mg Ab/kg of plant tissues). Both recombinant Abs fully retained the β-glucan-binding specificity and the antifungal activities of the cognate murine mAb against C. albicans. In fact, they recognized preferentially β1,3-linked glucan molecules present at the fungal cell surface and directly inhibited the growth of C. albicans and its adhesion to human epithelial cells in vitro. In addition, both the IgG and the scFv-Fc promoted C. albicans killing by isolated, human polymorphonuclear neutrophils in ex vivo assays and conferred significant antifungal protection in animal models of systemic or vulvovaginal C. albicans infection. These recombinant Abs represent valuable molecules for developing novel, plant-derived immunotherapeutics against candidiasis and, possibly, other fungal diseases

<i>Candida albicans</i> Targets a Lipid Raft/Dectin-1 Platform to Enter Human Monocytes and Induce Antigen Specific T Cell Responses

<div><p>Several pathogens have been described to enter host cells via cholesterol-enriched membrane lipid raft microdomains. We found that disruption of lipid rafts by the cholesterol-extracting agent methyl-β-cyclodextrin or by the cholesterol-binding antifungal drug Amphotericin B strongly impairs the uptake of the fungal pathogen <i>Candida albicans</i> by human monocytes, suggesting a role of raft microdomains in the phagocytosis of the fungus. Time lapse confocal imaging indicated that Dectin-1, the C-type lectin receptor that recognizes <i>Candida albicans</i> cell wall-associated β-glucan, is recruited to lipid rafts upon <i>Candida albicans</i> uptake by monocytes, supporting the notion that lipid rafts act as an entry platform. Interestingly disruption of lipid raft integrity and interference with fungus uptake do not alter cytokine production by monocytes in response to <i>Candida albicans</i> but drastically dampen fungus specific T cell response. In conclusion, these data suggest that monocyte lipid rafts play a crucial role in the innate and adaptive immune responses to <i>Candida albicans</i> in humans and highlight a new and unexpected immunomodulatory function of the antifungal drug Amphotericin B.</p></div

Disruption of lipid rafts blunts T cell response <i>to C</i>. <i>albicans</i>.

<p>Monocytes, treated or not with DAmb or MBCD before incubation with HK <i>C</i>. <i>albicans</i> (Ca) or with MP65, were co-cultured with autologous memory CD4⁺ T lymphocytes for 6 days. At day 6 cell proliferation was measured by thymidine incorporation, expressed as count per minutes (cpm), and culture supernatants were harvested for cytokine production analysis by ELISA. <b>A</b>. Part of the monocytes treated with drugs and pulsed with <i>C</i>. <i>albicans</i> were extensively washed or not before incubation with T cells (+/- washing). Drugs removal only partially restored T cell response supporting a major effect on monocytes. <b>B</b>. T cell response to MP65, which does not require phagocytosis for its uptake and processing, was left unmodified by the presence of drugs excluding a non specific effect on antigen processing and/or presentation by monocytes. Asterisks indicate values significantly different from non-treated monocytes: (*p<0.05; **p<0.001). One experiment out of two is shown as means ± SD of triplicate wells.</p

Disruption of lipid rafts inhibits <i>C</i>. <i>albicans</i> phagocytosis by human monocytes.

<p><b>A.</b> Phagocytosis inhibition assessed by flow cytometry. Human monocytes were pretreated with MBCD, DAmB or left untreated (none), and then incubated with HK FITC-conjugated <i>C. albicans</i> or <i>Staphylococcus aureus</i> (<i>St. aureus</i>). The data highlight that drugs specifically impair fungal phagocytosis. Markers were set up to exclude background staining of cells incubated with the microorganisms at 0°C. Numbers indicate the percentage of monocytes with ingested microorganisms. Data shown are from one experiment representative of four. <b>B.</b> Phagocytosis analysis by confocal microscopy. Phagocytosis was performed as in A. Samples were acquired in 3D stacks by DIC and three-color fluorescent images. The triple staining allows to clearly establish if a FITC-conjugated <i>C</i>. <i>albicans</i> (green) was internalized by a monocyte labelled by anti-CD14 (red). DNA was counterstained with DAPI (blue). The first column (DIC overlay) shows the combined signals from the DIC and confocal fluorescent channels of a single xy slice. In the second column (3D rendering), the 3D reconstructions of the 22 slices highlighting the reciprocal spatial information of fungal and human cells are displayed. The third column (orthogonal views) shows the xy, xz and yz single planes at the indicated orthogonal xyz axes (white lines). Scale bar for all panels is 3 μ.</p

Dectin-1 is the predominant PRR involved in phagocytosis of <i>C</i>. <i>albicans</i> by human monocytes.

<p><b>A</b>. Flow cytometry analysis of monocyte surface expression of <i>C</i>. <i>albicans</i>-specific PRRs. Circulating human monocytes express Dectin-1 and CR3 receptors but not MR and DC-SIGN. The appropriate isotype control for each mAb is reported as histogram with dotted lines. One experiment representative of five is shown. <b>B</b>. Inhibition of <i>C</i>. <i>albicans</i> phagocytosis by blocking <i>C</i>. <i>albicans-</i>specific PRRs. Monocytes were pre-treated with laminarin, mannan, acetylglucosamine (GlcNAc) to block Dectin-1, Dectin-2 or CR3, respectively or with a specific antibody anti-CR3, before incubation with FITC-conjugated <i>C</i>. <i>albicans</i>. Only laminarin impairs fungus phagocytosis, supporting the relevant role of Dectin-1 on <i>C</i>. <i>albicans</i> uptake. The asterisk indicates values significantly different from untreated monocytes (<i>p</i><0.05). Results are the mean ± standard error (SE) of three independent experiments performed. <b>C.</b> Inhibition of the uptake of the specific Dectin-1 ligand β-glucan following monocyte lipid rafts disruption. Monocytes were pre-treated or not with DAmb or MBCD and then incubated with HK FITC-conjugated <i>C</i>. <i>albicans</i> or FITC-conjugated β-glucan. The extent of phagocytosis was assessed by flow cytometry. The uptake reduction was similar for both β-glucan and <i>C</i>. <i>albicans</i> suggesting the involvement of lipid rafts in Dectin-1 mediated phagocytosis. The asterisk indicates values significantly different (<i>p<</i>0.05) from untreated monocytes. Results are expressed as mean ± SE of three independent experiments.</p

Involvement of lipid rafts in Dectin-1-mediated phagocytosis of <i>C</i>. <i>albicans</i>.

<p>Confocal time lapse analysis of Dectin-1 (red) and lipid rafts (CTB, magenta) dynamics in control, unstimulated human monocytes (<b>A</b>) and in FITC-<i>C</i>. <i>albicans</i> (green) stimulated human monocytes (<b>B</b>). Merge panels show the combined confocal fluorescent signals. The DIC images showing cell morphology are merged with the fluorescent channels in the last column. <b>A</b>. In absence of external stimuli, Dectin-1 receptor is scattered on the plasma membrane and no signal could be associated with the lipid rafts compartments. Scale bar is 5 μm. <b>B</b>. The timescale on the left indicates recording time. Soon after <i>C</i>. <i>albicans</i> stimulus, CTB signal starts to aggregate, indicating lipid rafts assembly in larger structures. With phagocytosis progression Dectin-1 association with lipid raft domains became stronger and clustering in correspondence of the point of monocyte-fungal cell contact increased and persisted during uptake and internalization. See also <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0142531#pone.0142531.s001" target="_blank">S1 Movie</a>. Scale bars are 5 μm.</p

Effects of lipid raft perturbation on Dectin-1 dynamics during phagocytosis.

<p>Confocal time lapse analysis of Dectin-1 (red) and lipid rafts (CTB, magenta) dynamics in stimulated human monocytes after MBCD or DAmb treatment. Merge panels show the combined confocal fluorescent signals. The DIC images showing cell morphology are merged with the fluorescent channels in the last column. Scale bars are 5 μm. First lane are control cell not treated with drugs. MBCD or DAmb were added to the monocytes before fungi stimulation. Drugs treatment caused a complete disaggregation of lipid rafts (CTB channel). Dectin-1 polarization at the sites of <i>C</i>. <i>albicans</i> contact was strongly impaired. The resulting effect of these events was a severely decreased phagocytosis. See also Supplementary <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0142531#pone.0142531.s002" target="_blank">S2</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0142531#pone.0142531.s003" target="_blank">S3</a> Movies.</p

Disruption of lipid rafts does not modify cytokine production by monocytes in response to <i>C</i>. <i>albicans</i>.

<p>Monocytes, pretreated or not with DAmb or MBCD, were incubated with or without <i>C</i>. <i>albicans</i>. Supernatants were collected and cytokine levels were evaluated by ELISA. Histograms indicate mean values ± SE of three independent experiments. The asterisk indicates values (<i>p<</i>0.05) significantly different from untreated monocytes. <i>ns</i>: not significant.</p

Prosafe: a european endeavor to improve quality of critical care medicine in seven countries

BACKGROUND: long-lasting shared research databases are an important source of epidemiological information and can promote comparison between different healthcare services. Here we present ProsaFe, an advanced international research network in intensive care medicine, with the focus on assessing and improving the quality of care. the project involved 343 icUs in seven countries. all patients admitted to the icU were eligible for data collection. MetHoDs: the ProsaFe network collected data using the same electronic case report form translated into the corresponding languages. a complex, multidimensional validation system was implemented to ensure maximum data quality. individual and aggregate reports by country, region, and icU type were prepared annually. a web-based data-sharing system allowed participants to autonomously perform different analyses on both own data and the entire database. RESULTS: The final analysis was restricted to 262 general ICUs and 432,223 adult patients, mostly admitted to Italian units, where a research network had been active since 1991. organization of critical care medicine in the seven countries was relatively similar, in terms of staffing, case mix and procedures, suggesting a common understanding of the role of critical care medicine. conversely, icU equipment differed, and patient outcomes showed wide variations among countries. coNclUsioNs: ProsaFe is a permanent, stable, open access, multilingual database for clinical benchmarking, icU self-evaluation and research within and across countries, which offers a unique opportunity to improve the quality of critical care. its entry into routine clinical practice on a voluntary basis is testimony to the success and viability of the endeavor