Tecla Ciociola

Peptides of the Constant Region of Antibodies Display Fungicidal Activity

Synthetic peptides with sequences identical to fragments of the constant region of different classes (IgG, IgM, IgA) of antibodies (Fc-peptides) exerted a fungicidal activity in vitro against pathogenic yeasts, such as Candida albicans, Candida glabrata, Cryptococcus neoformans, and Malassezia furfur, including caspofungin and triazole resistant strains. Alanine-substituted derivatives of fungicidal Fc-peptides, tested to evaluate the critical role of each residue, displayed unaltered, increased or decreased candidacidal activity in vitro. An Fc-peptide, included in all human IgGs, displayed a therapeutic effect against experimental mucosal and systemic candidiasis in mouse models. It is intriguing to hypothesize that some Fc-peptides may influence the antifungal immune response and constitute the basis for devising new antifungal agents.

Antibody Complementarity-Determining Regions (CDRs): A Bridge between Adaptive and Innate Immunity

Background It has been documented that, independently from the specificity of the native antibody (Ab) for a given antigen (Ag), complementarity determining regions (CDR)-related peptides may display differential antimicrobial, antiviral and antitumor activities. Methodology/Principal Findings In this study we demonstrate that a synthetic peptide with sequence identical to VHCDR3 of a mouse monoclonal Ab (mAb) specific for difucosyl human blood group A is easily taken up by macrophages with subsequent stimulation of: i) proinflammatory cytokine production; ii) PI3K-Akt pathway and iii) TLR-4 expression. Significantly, VHCDR3 exerts therapeutic effect against systemic candidiasis without possessing direct candidacidal properties. Conclusions/Significance These results open a new scenario about the possibility that, beyond the half life of immunoglobulins, Ab fragments may effectively influence the antiinfective cellular immune response in a way reminiscent of regulatory peptides of innate immunity.

AFM1 in Milk: Physical, Biological, and Prophylactic Methods to Mitigate Contamination

Aflatoxins (AFs) are toxic, carcinogenic, immunosuppressive secondary metabolites produced by some Aspergillus species which colonize crops, including many dietary staple foods and feed components. AFB1 is the prevalent and most toxic among AFs. In the liver, it is biotransformed into AFM1, which is then excreted into the milk of lactating mammals, including dairy animals. AFM1 has been shown to be cause of both acute and chronic toxicoses. The presence of AFM1 in milk and dairy products represents a worldwide concern since even small amounts of this metabolite may be of importance as long-term exposure is concerned. Contamination of milk may be mitigated either directly, decreasing the AFM1 content in contaminated milk, or indirectly, decreasing AFB1 contamination in the feed of dairy animals. Current strategies for AFM1 mitigation include good agricultural practices in pre-harvest and post-harvest management of feed crops (including storage) and physical or chemical decontamination of feed and milk. However, no single strategy offers a complete solution to the issue.

Antibody constant region peptides can display immunomodulatory activity through activation of the Dectin-1 signalling pathway.

We previously reported that a synthetic peptide with sequence identical to a CDR of a mouse monoclonal antibody specific for difucosyl human blood group A exerted an immunomodulatory activity on murine macrophages. It was therapeutic against systemic candidiasis without possessing direct candidacidal properties. Here we demonstrate that a selected peptide, N10K, putatively deriving from the enzymatic cleavage of the constant region (Fc) of human IgG1, is able to induce IL-6 secretion and pIkB-α activation. More importantly, it causes an up-regulation of Dectin-1 expression. This leads to an increased activation of β-glucan-induced pSyk, CARD9 and pIkB-α, and an increase in the production of pro-inflammatory cytokines such as IL-6, IL-12, IL-1β and TNF-α. The increased activation of this pathway coincides with an augmented phagocytosis of non opsonized Candida albicans cells by monocytes. The findings suggest that some Fc-peptides, potentially deriving from the proteolysis of immunoglobulins, may cause an unexpected immunoregulation in a way reminiscent of innate immunity molecules.

Killer peptide: a novel paradigm of antimicrobial, antiviral and immunomodulatory auto-delivering drugs

The incidence of life-threatening viral and microbial infections has dramatically increased over recent decades. Despite significant developments in anti-infective chemotherapy, many issues have increasingly narrowed the therapeutic options, making it imperative to discover new effective molecules. Among them, small peptides are arousing great interest. This review will focus in particular on a killer peptide, engineered from an anti-idiotypic recombinant antibody that mimics the activity of a wide-spectrum antimicrobial yeast killer toxin targeting β-glucan cell-wall receptors. The in vitro and in vivo antimicrobial, antiviral and immunomodulatory activities of killer peptide and its ability to spontaneously and reversibly self-assemble and slowly release its active dimeric form over time will be discussed as a novel paradigm of targeted auto-delivering drugs.

Antibody peptide based antifungal immunotherapy

Fungal infections still represent relevant human illnesses worldwide and some are accompanied by unacceptably high mortality rates. The limited current availability of effective and safe antifungal agents makes the development of new drugs and approaches of antifungal vaccination/immunotherapy every day more needed. Among them, small antibody(Ab)-derived peptides are arousing great expectations as new potential antifungal agents. In this topic, the search path from the study of the yeast killer phenomenon to the production of Ab-derived peptides characterized by in vitro and in vivo fungicidal activity will be focused. In particular, Abs that mimic the antimicrobial activity of a killer toxin (“antibiobodies”) and antifungal peptides derived from antibiobodies (killer peptide) and other unrelated Abs [complementarity determining regions (CDR)-based and constant region (Fc)-based synthetic peptides] are described. Mycological implications in terms of reevaluation of the yeast killer phenomenon, roles of antibiobodies in antifungal immunity, of β-glucans as antifungal targets and vaccines, and of Abs as sources of an unlimited number of sequences potentially active as new immunotherapeutic tools against fungal agents and related mycoses, are discussed.

Yeast Killer Toxin-Like Candidacidal Ab6 Antibodies Elicited through the Manipulation of the Idiotypic Cascade

A mouse anti-anti-anti-idiotypic (Id) IgM monoclonal antibody (mAb K20, Ab4), functionally mimicking a Wyckerhamomyces anomalus (Pichia anomala) killer toxin (KT) characterized by fungicidal activity against yeasts presenting specific cell wall receptors (KTR) mainly constituted by β-1,3-glucan, was produced from animals presenting anti-KT Abs (Ab3) following immunization with a rat IgM anti-Id KT-like mAb (mAb K10, Ab2). MAb K10 was produced by immunization with a KT-neutralizing mAb (mAb KT4, Ab1) bearing the internal image of KTR. MAb K20, likewise mAb K10, proved to be fungicidal in vitro against KT-sensitive Candida albicans cells, an activity neutralized by mAb KT4, and was capable of binding to β-1,3-glucan. MAb K20 and mAb K10 competed with each other and with KT for binding to C. albicans KTR. MAb K20 was used to identify peptide mimics of KTR by the selection of phage clones from random peptide phage display libraries. Using this strategy, four peptides (TK 1-4) were selected and used as immunogen in mice in the form of either keyhole limpet hemocyanin (KLH) conjugates or peptide-encoding minigenes. Peptide and DNA immunization could induce serum Abs characterized by candidacidal activity, which was inhibited by laminarin, a soluble β-1,3-glucan, but not by pustulan, a β-1,6-glucan. These findings show that the idiotypic cascade can not only overcome the barrier of animal species but also the nature of immunogens and the type of technology adopted.

Structural and functional studies on a proline-rich peptide isolated from swine saliva endowed with antifungal activity towards Cryptococcus neoformans.

A proline-rich peptide of 2733Da, isolated from pig parotid granule preparations was tested against different pathogenic fungi. It showed interesting antifungal activity towards a clinical isolate of Cryptococcus neoformans, with an EC(50) of 2.2μM. Neither cytotoxic nor haemolytic effects were observed towards mammalian cells. Circular dichroism and infrared spectroscopic studies showed that the peptide adopted a combination of polyproline type-II, β-turn and unordered conformations at physiological temperatures. Temperature dependent experiments evidenced a tendency to adopt a polyproline-II helix conformation. From experiments with lipid vesicles, Neutral Red Uptake (NRU), haemolytic assays, and confocal microscopy studies, it could be hypothesized that the peptide may exert its antifungal effect by interacting with an intracellular target rather than through membrane damage.

Natural and synthetic peptides with antifungal activity.

In recent years, the increase of invasive fungal infections and the emergence of antifungal resistance stressed the need for new antifungal drugs. Peptides have shown to be good candidates for the development of alternative antimicrobial agents through high-throughput screening, and subsequent optimization according to a rational approach. This review presents a brief overview on antifungal natural peptides of different sources (animals, plants, micro-organisms), peptide fragments derived by proteolytic cleavage of precursor physiological proteins (cryptides), synthetic unnatural peptides and peptide derivatives. Antifungal peptides are schematically reported based on their structure, antifungal spectrum and reported effects. Natural or synthetic peptides and their modified derivatives may represent the basis for new compounds active against fungal infections.

In vitro and in vivo activity of a killer peptide against Malassezia pachydermatis causing otitis in dogs

In order to overcome the limitations inherent in current pharmacological treatments for Malassezia pachydermatis, the cause of otitis externa in dogs, the efficacy of a killer decapeptide (KP) was evaluated in vitro and in vivo. Sixteen dogs with naturally occurring M. pachydermatis otitis externa were enrolled, and the in vitro fungicidal activity of KP was evaluated using yeasts recovered from these animals. The therapeutic activity was evaluated in four groups of four animals each. The dogs were topically treated with KP (150 μl, 2 mg/ml) three times per week (group A) or every day (group B), treated with a scramble peptide every day (group C), or left untreated (group D). Assessment of clinical signs (pruritus, erythema, and lichenification and/or hyperpigmentation), expressed as mean of the total clinical index score (mTCIS), the population size of M. pachydermatis at the cytological examination (mean number of yeast cells at 40× magnification [mYC]), and culture testing (mean number of log10 CFU/swab [mCFU]), were conducted daily from the first day of treatment (T0) until two consecutive negative cultures (mCFU ≤ 2). KP showed an in vitro fungicidal effect against M. pachydermatis isolates, with an MFC90 value of 1 μg/ml. The mTCIS, mYC and mCFU were negative only in animals in group B after T8. Daily administration of KP for 8 days was safe and effective in controlling both clinical signs and the population size of M. pachydermatis causing otitis externa, thus offering an alternative to the currently available therapeutic or prophylactic protocols for recurrent cases of Malassezia otitis in dogs.