Enhancement of the antidermatophytic activity of silver nanoparticles by Q-switched Nd:YAG laser and monoclonal antibody conjugation.

The objective of this research was to investigate the effect of silver nanoparticles (AgNPs), free or conjugated with monoclonal antibody and mediated by Q-switched Nd:YAG laser on five dermatophytes. The laser was applied for 45 s at 532 nm and 0.8 J/cm2. The application of AgNPs combined with laser caused an increase in fungal susceptibility compared to application of AgNPs alone. The MIC50 and MIC100 recorded 3 and 9 µg/ml in the case of E. floccosum (the most susceptible species), 10 and 19 µg/ml for T. rubrum (the most tolerant species), respectively. A decrease in keratinase activity reaching 76.1, 67.1, and 62.4% was attained in the case of M. gypseum, T. rubrum, and T. mentagrophyte, respectively, on application of 10 µg/ml AgNPs combined with Nd:YAG laser. Under the same conditions of application, a steady increase in leaked materials coupled with reduction in ergosterol synthesis was reached. The structural alterations occurred to the fungus were more observed on the application of AgNPs in combination with laser where the conidia and hyphae lost their cellular integrity, become flaccid, permanently destructed, and completely killed. The monoclonal antibody conjugated AgNPs did not result in significant variation in in vitro experiments compared with that produced by nonconjugated nanoparticles. However, the conjugates achieved significantly more curing of M. canis-inoculated guinea pigs compared with nonconjugated nanoparticles.

Potential of anti-Candida antibodies in immunoprophylaxis.

The need for new options for the treatment of invasive candidiasis has fuelled the use of antibodies in combination with conventional antifungal therapy. After a long period of time in which antibodies were considered irrelevant in the resistance against invasive candidiasis, it was demonstrated that a number of antibodies or their engineered derivatives directed against Candida albicans cell-wall polysaccharides and glycopeptides, as well as against some protein epitopes, confer protection against invasive candidiasis. This has confirmed this approach as a new strategy for the prophylaxis of invasive candidiasis. Of particular interest is Mycograb, a human recombinant monoclonal antibody that inhibits heat shock protein 90, and has been administrated in combination with lipid-associated amphotericin B to patients with invasive candidiasis, and the fungicidal anti-beta-glucan antibodies induced by the glycoconjugate vaccine composed of a beta-glucan polysaccharide conjugated with the diphtheria toxoid CRM 197. However, despite the promising data obtained in vitro and in animal models, at present there is very little clinical experience on the use of antibodies in Candida immunoprophylaxis.

Effects of antifungal interventions on the outcome of experimental infections with phenotypic switch variants of Cryptococcus neoformans.

In cryptococcal infection, phenotypic switching from a smooth to a mucoid variant can occur in vivo, producing variants with enhanced virulence that are subsequently selected and affect the outcome of infection. Here, we demonstrate that antifungal treatment of the chronically infected host can promote this phenomenon. Amphotericin B treatment reduces fungal burden less effectively in mucoid variant-infected than in smooth variant-infected mice. Consequently, amphotericin B treatment resulted in a more pronounced prolongation of survival in smooth variant-infected than in mucoid variant-infected mice (20 versus 42 days; P < 0.05). Administration of anticapsular monoclonal antibody mediated better protection in smooth variant-infected than in mucoid variant-infected mice, although a protective effect was not consistently observed at all doses. Most interestingly, both antifungal drug therapy and administration of anticapsular monoclonal antibody promoted the selection of mucoid variants in smooth variant-infected mice, a phenomenon manifested by a statistically higher percentage of mucoid colonies in smooth variant-infected mice than in nontreated control mice. This finding suggests that both chemotherapeutic and immunological antifungal interventions may promote the selection of the more virulent mucoid variant, which could affect the outcome of infection in chronically infected hosts.

Genetically recombinant antibodies: new therapeutics against candidiasis.

Historically, the therapy of serious fungal infection has been dominated by monotherapy with the polyene antibiotic amphotericin B. Clinical failures, side effects, the lack of alternatives and the toxicity of this drug have heightened the need to produce alternative therapies, which have included fluconazole, voriconazole and caspofungin. The observation that recovery from disseminated candidiasis was associated with an antibody response to the 47 kDa Candida heat-shock protein (HSP)90 homologue, coupled with the ability to sequence all the antibodies from patients who have recovered from the infection and to re-express the dominant ones as fragments in Escherichia coli, has opened the possibility of immunotherapy. The first recombinant antibody fragment, Mycograb (Neu Tec Pharma plc), against Candida HSP90 is now in clinical trials in patients with disseminated candidiasis in Europe and the US. Laboratory and early clinical data support the concept of synergy between Mycograb and amphotericin B. This should improve outcome and diminish the risk of resistance occurring to either drug, without an increase in toxicity, as this should be minimal in a human antibody fragment representing the natural antibody that a patient produces on recovery.

Fungal prophylaxis by reduction of fungal colonization by oral administration of bovine anti-Candida antibodies in bone marrow transplant recipients.

Candida overgrowth and invasion constitute a serious threat with a high mortality in BMT recipients. Currently available topical antifungal prophylaxis is largely ineffective, and as resistance to existing, absorbable drugs for systemic use is rapidly developing, new forms of therapy are needed. We investigated the effect of oral treatment of BMT recipients with a bovine immunoglobulin product derived from animals immunized against several Candida species. The natural Candida colonization was first followed in 19 patients to establish the colonization pattern. Half of the patients were found to be colonized prior to transplantation and altogether 72% were colonized at some point during follow-up. Those with a high pre-transplant concentration of Candida in saliva (>100 CFU/ml) remained colonized throughout the BMT treatment period. The therapeutic effect was monitored in two other patient groups. The first group consisted of nine patients, where, due to a low number of primary colonized patients, response in colonized patients was suggestive of a therapeutic effect. In the second group, 10 patients with a high level of colonization (>100 CFU/ml) were given 10 g daily of the product in three divided doses. The results suggest a treatment-related reduction in Candida colonization in a majority (7/10) of patients and one patient became completely negative. As no adverse effects were noted, our findings encourage additional studies in immunocompromised, transplant patients.

Monoclonal antibodies against the 43,000 Da glycoprotein from Paracoccidioides brasiliensis modulate laminin-mediated fungal adhesion to epithelial cells and pathogenesis.

The surface glycoprotein gp43, a highly immunogenic component of Paracoccidioides brasiliensis, is used in the serodiagnosis of paracoccidioidomycosis (PCM) and has recently been shown to specifically bind the extracellular matrix protein laminin. Binding to laminin induces the increased adhesion of the fungus to epithelial cells; a hamster testicle infection model has shown that the gp43-dependent binding of fungal cells to laminin enhances their pathogenicity in vivo. We report on the production and characterization of 12 monoclonal antibodies against the gp43 that recognize peptide sequences in the molecule detecting at least three different epitopes as well as different isoforms of this antigen. MAbs interfered in the fungal pathogenicity in vivo either by inhibiting or enhancing granuloma formation and tissue destruction. Results suggest that P. brasiliensis propagules may start infection in man by strongly adhering to human lung cells. Thus, laminin-mediated fungal adhesion to human lung carcinoma (A549) cells was much more intense than to Madin-Darby canine kidney cells (MDCK), indicating differences in binding affinity. Subsequent growth of fungi bound to the lung cells could induce the granulomatous inflammatory reaction characteristic of PCM. Both steps are greatly stimulated by laminin binding in infective cells expressing gp43.