Pathogen- and Host-Directed Antileishmanial Effects Mediated by Polyhexanide (PHMB)

BACKGROUND:Cutaneous leishmaniasis (CL) is a neglected tropical disease caused by protozoan parasites of the genus Leishmania. CL causes enormous suffering in many countries worldwide. There is no licensed vaccine against CL, and the chemotherapy options show limited efficacy and high toxicity. Localization of the parasites inside host cells is a barrier to most standard chemo- and immune-based interventions. Hence, novel drugs, which are safe, effective and readily accessible to third-world countries and/or drug delivery technologies for effective CL treatments are desperately needed. METHODOLOGY/PRINCIPAL FINDINGS:Here we evaluated the antileishmanial properties and delivery potential of polyhexamethylene biguanide (PHMB; polyhexanide), a widely used antimicrobial and wound antiseptic, in the Leishmania model. PHMB showed an inherent antileishmanial activity at submicromolar concentrations. Our data revealed that PHMB kills Leishmania major (L. major) via a dual mechanism involving disruption of membrane integrity and selective chromosome condensation and damage. PHMB's DNA binding and host cell entry properties were further exploited to improve the delivery and immunomodulatory activities of unmethylated cytosine-phosphate-guanine oligodeoxynucleotides (CpG ODN). PHMB spontaneously bound CpG ODN, forming stable nanopolyplexes that enhanced uptake of CpG ODN, potentiated antimicrobial killing and reduced host cell toxicity of PHMB. CONCLUSIONS:Given its low cost and long history of safe topical use, PHMB holds promise as a drug for CL therapy and delivery vehicle for nucleic acid immunomodulators

Identification of proliferating cells in chicken embryos using 5-bromo- 2'-deoxyuridine immunohistochemical detection

Chicken embryos were incubated with BrdU, embedded in plastic resin, sectioned and screened immunohistochemically to identify proliferating cells in the neural tube and somites. Fixation in 4% paraformaldehyde for 1 h was essential for detecting specific colorimetric signals of BrdU incorporation into cells during the S phase of the cell cycle. Transverse sections of the neural tube showed that the nuclei of proliferating cells (BrdU positive) had a uniform and centralized distribution, whereas unstained nuclei were found only along the extremities of the neural tube. Transverse sections of differentiated somites showed proliferating cells in the scleratome and dermatome. However, no incorporation of BrdU was observed in myotomic cells, which give rise to axial skeletal muscle. In spite of their proximity, the dermatome and myotome showed marked differences in cell proliferation. The excellent preservation of morphological characteristics in the embryonic tissues facilitated identification of variations in BrdU incorporation.<br>Embriões de frango foram incubados na presença de BrdU e montados em resina plástica. A detecção de células em proliferação nos somitos e tubo neural foi feita através de anticorpos contra BrdU. Um ponto essencial para a otimização do método foi a fixação dos embriões por apenas uma hora em paraformaldeído a 4%. Análise de cortes transversais revelou que no tubo neural os núcleos marcados se posicionavam na região central. Cortes transversais em somitos diferenciados revelaram a presença de células em proliferação no dermátomo e esclerótomo, no entanto não foi observado nenhum sinal no miótomo. A metodologia aqui apresentada permitiu identificar com clareza e boa resolução as células em proliferação presentes em tecidos embrionários