Therapeutic Effect of an Antibody-Derived Peptide in a Galleria mellonella Model of Systemic Candidiasis.

The synthetic peptide T11F (TCRVDHRGLTF), with sequence identical to a fragment of the constant region of human IgM, and most of its alanine-substituted derivatives proved to possess a significant candidacidal activity in vitro. In this study, the therapeutic efficacy of T11F, D5A, the derivative most active in vitro, and F11A, characterized by a different conformation, was investigated in Galleria mellonella larvae infected with Candida albicans. A single injection of F11A and D5A derivatives, in contrast with T11F, led to a significant increase in survival of larvae injected with a lethal inoculum of C. albicans cells, in comparison with infected animals treated with saline. Peptide modulation of host immunity upon C. albicans infection was determined by hemocyte analysis and larval histology, highlighting a different immune stimulation by the studied peptides. F11A, particularly, was the most active in eliciting nodule formation, melanization and fat body activation, leading to a better control of yeast infection. Overall, the obtained data suggest a double role for F11A, able to simultaneously target the fungus and the host immune system, resulting in a more efficient pathogen clearance.

Dissection of the Structural Features of a Fungicidal Antibody-Derived Peptide.

The synthetic peptide T11F (TCRVDHRGLTF), derived from the constant region of human IgM antibodies, proved to exert a significant activity in vitro against yeast strains, including multidrug resistant isolates. Alanine substitution of positively charged residues led to a decrease in candidacidal activity. A more dramatic reduction in activity resulted from cysteine replacement. Here, we investigated the conformational properties of T11F and its alanine-substituted derivatives by circular dichroism (CD) and nuclear magnetic resonance (NMR) spectroscopy. Peptide interaction with Candida albicans cells was studied by confocal and scanning electron microscopy. T11F and most of its derivatives exhibited CD spectra with a negative band around 200 nm and a weaker positive band around 218 nm suggesting, together with NMR coupling constants, the presence of a polyproline II (PPII) helix, a conformational motif involved in a number of biological functions. Analysis of CD spectra revealed a critical role for phenylalanine in preserving the PPII helix. In fact, only the F11A derivative presented a random coil conformation. Interestingly, the loss of secondary structure influenced the rate of killing, which turned out to be significantly reduced. Overall, the obtained results suggest that the PPII conformation contributes in characterising the cell penetrating and fungicidal properties of the investigated peptides.

Antiviral Activity of Synthetic Peptides Derived from Physiological Proteins.

BACKGROUND/AIMS: New antivirals are needed to supplement or replace currently used drugs. The aim of this study was to evaluate the antiviral activity of synthetic peptides derived from physiological proteins. METHODS: Vero cell monolayers were infected with herpes simplex virus 1, vesicular stomatitis virus, adenovirus, and coxsackievirus B5 strains in the presence of different concentrations of the selected peptides and viral yield was determined by plaque reduction assays to evaluate the antiviral activity of the peptides. Virucidal activity was evaluated by determining the residual infectivity of viral suspensions treated for 1 h with the peptides at the same concentrations as in the viral yield assays. RESULTS: Among the investigated peptides, the killer peptide proved to exert a considerable antiviral activity against herpes simplex virus, attributable to a direct effect on virus particles, while its derivative K10S showed to be effective against the four investigated virus strains only at the highest concentration tested, yet, the inhibitory effects were only partial. CONCLUSION: Overall, initial evidence is provided on the antiviral activity of several peptides, as well as of their derivatives. Further investigation is warranted to ascertain the mechanism of action in order to develop new potential antiviral drugs.

Fungicidal activity of peptides encoded by immunoglobulin genes.

Evidence from previous works disclosed the antimicrobial, antiviral, anti-tumour and/or immunomodulatory activity exerted, through different mechanisms of action, by peptides expressed in the complementarity-determining regions or even in the constant region of antibodies, independently from their specificity and isotype. Presently, we report the selection, from available databases, of peptide sequences encoded by immunoglobulin genes for the evaluation of their potential biological activities. Synthetic peptides representing the translated products of J lambda and J heavy genes proved to act in vitro against pathogenic fungi, entering yeast cells and causing their death, and exerted a therapeutic effect in a Galleria mellonella model of infection by Candida albicans. No haemolytic, cytotoxic and genotoxic effects were observed on mammalian cells. These findings raise the hypothesis that antibodies could be the evolutionary result of the adaptive combination of gene products ancestrally devoted to innate antimicrobial immunity.

The synthetic killer peptide KP impairs Candida albicans biofilm in vitro.

Candida albicans is a commensal organism, commonly inhabiting mucosal surfaces of healthy individuals, as a part of the resident microbiota. However, in susceptible hosts, especially hospitalized and/or immunocompromised patients, it may cause a wide range of infections. The presence of abiotic substrates, such as central venous or urinary catheters, provides an additional niche for Candida attachment and persistence, particularly via biofilm development. Furthermore, Candida biofilm is poorly susceptible to most antifungals, including azoles. Here we investigated the effects of a synthetic killer peptide (KP), known to be active in vitro, ex vivo and/or in vivo against different pathogens, on C. albicans biofilm. Together with a scrambled peptide used as a negative control, KP was tested against Candida biofilm at different stages of development. A reference strain, two fluconazole-resistant and two fluconazole-susceptible C. albicans clinical isolates were used. KP-induced C. albicans oxidative stress response and membrane permeability were also analysed. Moreover, the effect of KP on transcriptional profiles of C. albicans genes involved in different stages of biofilm development, such as cell adhesion, hyphal development and extracellular matrix production, was evaluated. Our results clearly show that the treatment with KP strongly affected the capacity of C. albicans to form biofilm and significantly impairs preformed mature biofilm. KP treatment resulted in an increase in C. albicans oxidative stress response and membrane permeability; also, biofilm-related genes expression was significantly reduced. Comparable inhibitory effects were observed in all the strains employed, irrespective of their resistance or susceptibility to fluconazole. Finally, KP-mediated inhibitory effects were observed also against a catheter-associated C. albicans biofilm. This study provides the first evidence on the KP effectiveness against C. albicans biofilm, suggesting that KP may be considered as a potential novel tool for treatment and prevention of biofilm-related C. albicans infections.

Effect of different wavelengths and dyes on Candida albicans: In vivo study using Galleria mellonella as an experimental model.

BACKGROUND: Studies on photodynamic inactivation against microorganisms had a great development in recent years. The aim of this work was to test the application of different laser wavelengths with or without different photosensitizing dyes on Candida albicans cells in vitro and in photodynamic therapy protocols in vivo in larvae of Galleria mellonella. METHODS: Laser application was realized on C. albicans cells suspended in saline solution or cultured on solid medium for the in vitro study, and in a model of G. mellonella candidal infection for the in vivo study. Three wavelengths (650, 405, and 532nm) were used in continuous mode with different values of applied fluences: 10, 20 and 30J/cm2 for the in vitro study and 10J/cm2 for the in vivo study, without and with photosensitizing dyes. RESULTS: No growth inhibition was obtained on yeast cells in saline solution without photosensitizers. The maximum inhibition of growth (100%) was obtained with 405nm diode laser and curcumin at any used fluence. No growth inhibition was observed for yeast cells cultured on solid medium after laser application without dyes. An inhibition was observed after laser application when curcumin and erythrosine were added to the medium. The survival curves of G. mellonella larvae infected with C. albicans with or without the different dyes and after laser application showed a statistically significant difference (p<0.001) in comparison with the proper control groups. CONCLUSIONS: These results show the efficacy of photodynamic inactivation exploiting a suitable combination of light and dyes against C. albicans and the potential of photodynamic therapy for the treatment of candidal infections.

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.

Dissecting the Structure-Function Relationship of a Fungicidal Peptide Derived from the Constant Region of Human Immunoglobulins.

Synthetic peptides encompassing sequences related to the complementarity-determining regions of antibodies or derived from their constant region (Fc peptides) were proven to exert differential antimicrobial, antiviral, antitumor, and/or immunomodulatory activitiesin vitroand/orin vivo, regardless of the specificity and isotype of the parental antibody. Alanine substitution derivatives of these peptides exhibited unaltered, increased, or decreased candidacidal activitiesin vitro The bioactive IgG-derived Fc N10K peptide (NQVSLTCLVK) spontaneously self-assembles, a feature previously recognized as relevant for the therapeutic activity of another antibody-derived peptide. We evaluated the contribution of each residue to the peptide self-assembling capability by circular-dichroism spectroscopy. The interaction of the N10K peptide and its derivatives withCandida albicanscells was studied by confocal, transmission, and scanning electron microscopy. The apoptosis and autophagy induction profiles in yeast cells treated with the peptides were evaluated by flow cytometry, and the therapeutic efficacy against candidal infection was studied in aGalleria mellonellamodel. Overall, the results indicate a critical role for some residues in the self-assembly process and a correlation of that capability with the candidacidal activities of the peptidesin vitroand their therapeutic effectsin vivo.

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.

Antibodies as a source of anti-infective peptides: an update.

This review focuses on antibodies (Abs) and their function in immune protection, with particular emphasis on microbicidal Abs. Some aspects of Abs and Ab-drug conjugates as targeting therapeutic agents are also discussed. The main aim, however, is devoted to Ab-derived peptides modulating functions of the immune system and to the latest experimental evidence of Abs as a source of anti-infective and antitumor peptides derived from their complementarity determining regions and constant regions.

Peptides from the inside of the antibodies are active against infectious agents and tumours.

Synthetic peptides, representative of sequences related to the complementarity determining regions and constant region of antibodies, proved to exert in vitro, ex vivo and/or in vivo antimicrobial, antiviral, anti-tumour and/or immunomodulatory activities, conceivably mediated by different mechanisms of action and regardless of the specificity and isotype of the belonging immunoglobulin. Antibody-derived peptides can show intrinsic properties of self-aggregation in ß structures, able to assemble on molecular targets and dissociate spontaneously, leading to the formation of hydrogels. Whilst the self-assembled state may provide protection against proteases and the slow kinetic of dissociation assures a release of the active form over time, the receptor affinity is responsible for targeted delivery. Peptides derived from single amino acid substitution of bioactive antibody fragments, adopted as surrogates of natural point mutations, displayed further differential biological activities. Overall, these observations allow to envisage that antibodies could represent an unlimited source of new anti-infective and anti-tumour peptides.

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 ß-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 ß-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 ß-1,3-glucan, but not by pustulan, a ß-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.

Preliminary evaluation of probiotic properties of Lactobacillus strains isolated from Sardinian dairy products.

Twenty-three Lactobacillus strains of dairy origin were evaluated for some functional properties relevant to their use as probiotics. A preliminary subtractive screening based on the abilities to inhibit the growth of microbial pathogens and hydrolyze conjugated bile salts was applied, and six strains were selected for further characterization including survival under gastrointestinal environmental conditions, adhesion to gut epithelial tissue, enzymatic activity, and some safety properties. All selected strains maintained elevated cell numbers under conditions simulating passage through the human gastrointestinal tract, well comparable to the values obtained for the probiotic strain Lactobacillus rhamnosus GG, and were able to adhere to Caco-2 cells to various extents (from 3 to 20%). All strains exhibited high aminopeptidase, and absent or very low proteolytic and strong ß-galactosidase activities; none was found to be haemolytic or to produce biogenic amines and all were susceptible to tetracycline, chloramphenicol, erythromycin, ampicillin, and amoxicillin/clavulanic acid. Our results indicate that the Lactobacillus strains analyzed could be considered appropriate probiotic candidates, due to resistance to GIT simulated conditions, antimicrobial activity, adhesion to Caco-2 cell-line, and absence of undesirable properties. They could be used as adjunct cultures for contributing to the quality and health related functional properties of dairy products.

Antibodies as an unlimited source of anti-infective, anti-tumour and immunomodulatory peptides.

Antibodies (Abs) are emerging as an important class of therapeutic agents for the treatment of various human diseases, often conjugated to drugs or toxic substances. In recent years, the incidence of cancer and infectious diseases has increased dramatically making it imperative to discover new effective therapeutic molecules. Among these, small peptides are arousing great interest. Synthetic peptides, representative of variable and constant region fragments of Abs, were proved to exert in vitro, ex vivo and/or in vivo anti-microbial, anti-viral, anti-tumour and/or immunomodulatory activities, mediated by different mechanisms of action and regardless of the specificity and isotype of the Ab. Some of these synthetic peptides possess the ability to spontaneously and reversibly self-assemble in an organised network of fibril-like structure. Ab fragments may represent a novel model of targeted anti-infective and anti-tumour auto-delivering drugs.

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, ß-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.

In vitro bactericidal effect of Nd:YAG laser on Actinomyces israelii.

A bactericidal effect has been reported by the use of near-infrared laser light on both Gram-positive and Gram-negative bacteria. The aim of this study was to evaluate the effect of Nd:YAG laser on Actinomyces israelii, filamentous bacteria causing cervicofacial actinomycosis. Experiments were realized on bacterial cells in saline suspension or streaked on Mueller-Hinton (MH) agar plates with or without India ink. Laser application was performed in Eppendorf tubes with different powers and frequencies for 40 s; bacterial suspensions were then streaked on agar plates and incubated at 35 °C in proper conditions for 5 days before colony enumeration. A reduction of colony number variable from 60.13 to 100 % for powers of 2, 4, and 6 W at 25-50 Hz of frequency was observed in comparison with growth control. For agar plates, laser application was performed with different powers at 50 Hz for 60 s. A growth inhibition was observed after 5 days of incubation on MH plates with powers of 6 W and on MH-ink plates with all applied powers. This preliminary study showed a bactericidal effect caused by Nd:YAG laser application worthy to be evaluated in further experiments in vivo.

Accidental discovery of lung metastases from differentiated thyroid cancer by 99mTc sodium pertechnetate scan in a patient with secondary hyperparathyroidism.

We present a 75-year-old-man affected by chronic renal failure in whom metastases from differentiated thyroid cancer (DTC) were incidentally discovered at TcO4 neck-thorax scan in the setting of a diagnosis of secondary hyperparathyroidism. Ten years before, he had undergone near-total thyroidectomy for a multinodular goiter. Result of the pathological examination was negative for DTC. At our observation, serum thyroglobulin was high in the absence of Tg-Ab, consistent with metastatic disease. Neck ultrasonographynography was negative. The patient underwent I therapy (3700 MBq) after stimulation with recombinant human thyroid-stimulating hormone. Postdose whole-body scan confirmed the metastases already demonstrated by TcO4.

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.

Vaccination of lactating dairy cows for the prevention of aflatoxin B1 carry over in milk.

The potential of anaflatoxin B(1) (AnAFB(1)) conjugated to keyhole limpet hemocyanin (KLH) as a vaccine (AnAFB(1)-KLH) in controlling the carry over of the aflatoxin B(1) (AFB(1)) metabolite aflatoxin M(1) (AFM(1)) in cow milk is reported. AFB(1) is the most carcinogenic compound in food and foodstuffs amongst aflatoxins (AFs). AnAFB(1) is AFB(1) chemically modified as AFB(1)-1(O-carboxymethyl) oxime. In comparison to AFB(1), AnAFB(1) has proven to be non-toxic in vitro to human hepatocarcinoma cells and non mutagenic to Salmonella typhimurium strains. AnAFB(1)-KLH was used for immunization of cows proving to induce a long lasting titer of anti-AFB(1) IgG antibodies (Abs) which were cross reactive with AFB(1), AFG(1), and AFG(2). The elicited anti-AFB(1) Abs were able to hinder the secretion of AFM(1) into the milk of cows continuously fed with AFB(1). Vaccination of lactating animals with conjugated AnAFB(1) may represent a solution to the public hazard constituted by milk and cheese contaminated with AFs.

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 ß-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.