In vitro and in vivo assessment of dermatophyte acquired resistance to efinaconazole, a novel triazole antifungal.

Efinaconazole is a novel triazole antifungal drug for the topical treatment of onychomycosis, a nail infection caused mainly by dermatophytes. We assessed the potential of efinaconazole to induce resistance in dermatophytes by continuous exposure of Trichophyton rubrum strains to efinaconazole in vitro (12 passages) and in a guinea pig onychomycosis model (8 weeks). There was no evidence of efinaconazole resistance development in the tested strains under the experimental conditions used.

The low keratin affinity of efinaconazole contributes to its nail penetration and fungicidal activity in topical onychomycosis treatment.

Onychomycosis is a common fungal nail disease that is difficult to treat topically due to the deep location of the infection under the densely keratinized nail plate. Keratin affinity of topical drugs is an important physicochemical property impacting therapeutic efficacy. To be effective, topical drugs must penetrate the nail bed and retain their antifungal activity within the nail matrix, both of which are adversely affected by keratin binding. We investigated these properties for efinaconazole, a new topical antifungal for onychomycosis, compared with those of the existing topical drugs ciclopirox and amorolfine. The efinaconazole free-drug concentration in keratin suspensions was 14.3%, significantly higher than the concentrations of ciclopirox and amorolfine, which were 0.7% and 1.9%, respectively (P < 0.001). Efinaconazole was released from keratin at a higher proportion than in the reference drugs, with about half of the remaining keratin-bound efinaconazole removed after washing. In single-dose in vitro studies, efinaconazole penetrated full-thickness human nails into the receptor phase and also inhibited the growth of Trichophyton rubrum under the nail. In the presence of keratin, efinaconazole exhibited fungicidal activity against Trichophyton mentagrophytes comparable to that of amorolfine and superior to that of ciclopirox. In a guinea pig onychomycosis model with T. mentagrophytes infection, an efinaconazole solution significantly decreased nail fungal burden compared to that of ciclopirox and amorolfine lacquers (P < 0.01). These results suggest that the high nail permeability of efinaconazole and its potent fungicidal activity in the presence of keratin are related to its low keratin affinity, which may contribute to its efficacy in onychomycosis.

Mechanism of action of efinaconazole, a novel triazole antifungal agent.

The mechanism of action of efinaconazole, a new triazole antifungal, was investigated with Trichophyton mentagrophytes and Candida albicans. Efinaconazole dose-dependently decreased ergosterol production and accumulated 4,4-dimethylsterols and 4α-methylsterols at concentrations below its MICs. Efinaconazole induced morphological and ultrastructural changes in T. mentagrophytes hyphae that became more prominent with increasing drug concentrations. In conclusion, the primary mechanism of action of efinaconazole is blockage of ergosterol biosynthesis, presumably through sterol 14α-demethylase inhibition, leading to secondary degenerative changes.

A Novel Gene Delivery Vector of Agonistic Anti-Radioprotective 105 Expressed on Cell Membranes Shows Adjuvant Effect for DNA Immunization Against Influenza.

Radioprotective 105 (RP105) (also termed CD180) is an orphan and unconventional Toll-like receptor (TLR) that lacks an intracellular signaling domain. The agonistic anti-RP105 monoclonal antibody (mAb) can cross-link RP105 on B cells, resulting in the proliferation and activation of B cells. Anti-RP105 mAb also has a potent adjuvant effect, providing higher levels of antigen-specific antibodies compared to alum. However, adjuvanticity is required for the covalent link between anti-RP105 mAb and the antigen. This is a possible obstacle to immunization due to the link between anti-RP105 mAb and some antigens, especially multi-transmembrane proteins. We have previously succeeded in inducing rapid and potent recombinant mAbs in mice using antibody gene-based delivery. To simplify the covalent link between anti-RP105 mAb and antigens, we generated genetic constructs of recombinant anti-RP105 mAb (αRP105) bound to the transmembrane domain of the IgG-B cell receptor (TM) (αRP105-TM), which could enable the anti-RP105 mAb to link the antigen via the cell membrane. We confirmed the expression of αRP105-TM and the antigen hemagglutinin, which is a membrane protein of the influenza virus, on the same cell. We also found that αRP105-TM could activate splenic B cells, including both mature and immature cells, depending on the cell surface RP105 in vitro. To evaluate the adjuvanticity of αRP105-TM, we conducted DNA immunization in mice with the plasmids encoding αRP105-TM and hemagglutinin, followed by challenge with an infection of a lethal dose of an influenza virus. We then obtained partially but significantly hemagglutinin-specific antibodies and observed protective effects against a lethal dose of influenza virus infection. The current αRP105-TM might provide adjuvanticity for a vaccine via a simple preparation of the expression plasmids encoding αRP105-TM and of that encoding the target antigen.

Neutralizing Antibodies Induced by Gene-Based Hydrodynamic Injection Have a Therapeutic Effect in Lethal Influenza Infection.

The influenza virus causes annual epidemics and occasional pandemics and is thus a major public health problem. Development of vaccines and antiviral drugs is essential for controlling influenza virus infection. We previously demonstrated the use of vectored immune-prophylaxis against influenza virus infection. We generated a plasmid encoding neutralizing IgG monoclonal antibodies (mAbs) against A/PR/8/34 influenza virus (IAV) hemagglutinin (HA). We then performed electroporation of the plasmid encoding neutralizing mAbs (EP) in mice muscles and succeeded in inducing the expression of neutralizing antibodies in mouse serum. This therapy has a prophylactic effect against lethal IAV infection in mice. In this study, we established a new method of passive immunotherapy after IAV infection. We performed hydrodynamic injection of the plasmid encoding neutralizing mAbs (HD) involving rapid injection of a large volume of plasmid-DNA solution into mice via the tail vein. HD could induce neutralizing antibodies in the serum and in several mucosal tissues more rapidly than in EP. We also showed that a single HD completely protected the mice even after infection with a lethal dose of IAV. We also established other isotypes of anti-HA antibody (IgA, IgM, IgD, and IgE) and showed that like anti-HA IgG, anti-HA IgA was also effective at combating upper respiratory tract IAV infection. Passive immunotherapy with HD could thus provide a new therapeutic strategy targeting influenza virus infection.

Passive immune-prophylaxis against influenza virus infection by the expression of neutralizing anti-hemagglutinin monoclonal antibodies from plasmids.

The genetic delivery of therapeutic monoclonal antibodies (mAbs) by in vivo production may offer a new solution to the current problems in the mAb therapy for microbial diseases. Herein, plasmids encoding the neutralizing mAb against hemagglutinin (HA) of A/PR/8/34 influenza virus (IFV) were electro-transferred into mouse muscle and the relationship between serum recombinant anti-HA mAb (rHA mAb) levels and the prophylactic efficacy against lethal IFV infection were analyzed. Pretreatment of the muscle with hyaluronidase before electroporation and gene transfer into 3 muscles resulted in a marked enhancement of the mAb expression. After single gene transfer, peak serum concentrations were reached around 20 days after the gene transfer following sustained expression of >10 µg/ml of rHA mAbs. This level of rHA mAb expression was sufficient to protect all mice against a lethal IFV infection. Furthermore, a significant rHA mAb expression level sufficient to protect the host against lethal IFV infection was maintained for at least 130 days. Passive immune-prophylaxis with gene transfer using the plasmid encoding neutralizing mAbs may therefore provide effective protection against viral infections, including IFV.

Avaliação do projeto favela: projeto 6 / Slum valuation project: project 6

O desenvolvimento econômico brasileiro, ao longo de sua história, levou à intensa urbanização do país, porém uma urbanização desordenada em que massas de migrantes se fixaram na periferia das grandes cidades, cem locais sem infra-estrutura social. Hoje, várias áreas do município de São Paulo apresemtam latos índices de crescimento demogra´fico sem o correspondente acompanhamento de benefícios urbanos em saneamento básico, lazer, educação e assistência médica. Conforme o Censo das Favelas do Município de São Paulo - 1987 - Secretaria de Habitação do Município de São Paulo: "Favelas seriam um conjunto de unidades domiciliares construídas de madeira, zinco, lata, papelão ou mesmo alvenaria, em geral distribuidas desorganizadamente em terrenos cuja propriedade individuaal do lote não é legalizada para aqueles que os ocupam...