New "light" for one-world approach toward safe and effective control of animal diseases and insect vectors from leishmaniac perspectives

Light is known to excite photosensitizers (PS) to produce cytotoxic reactive oxygen species (ROS) in the presence of oxygen. This modality is attractive for designing control measures against animal diseases and pests. Many PS have a proven safety record. Also, the ROS cytotoxicity selects no resistant mutants, unlike other drugs and pesticides. Photodynamic therapy (PDT) refers to the use of PS as light activable tumoricides, microbicides and pesticides in medicine and agriculture.Here we describe "photodynamic vaccination" (PDV) that uses PDT-inactivation of parasites, i.e. Leishmania as whole-cell vaccines against leishmaniasis, and as a universal carrier to deliver transgenic add-on vaccines against other infectious and malignant diseases. The efficacy of Leishmania for vaccine delivery makes use of their inherent attributes to parasitize antigen (vaccine)-presenting cells. Inactivation of Leishmania by PDT provides safety for their use. This is accomplished in two different ways: (i) chemical engineering of PS to enhance their uptake, e.g. Si-phthalocyanines; and (ii) transgenic approach to render Leishmania inducible for porphyrinogenesis. Three different schemes of Leishmania-based PDV are presented diagrammatically to depict the cellular events resulting in cell-mediated immunity, as seen experimentally against leishmaniasis and Leishmania-delivered antigen in vitro and in vivo. Safety versus efficacy evaluations are under way for PDT-inactivated Leishmania, including those further processed to facilitate their storage and transport. Leishmania transfected to express cancer and viral vaccine candidates are being prepared accordingly for experimental trials.We have begun to examine PS-mediated photodynamic insecticides (PDI). Mosquito cells take up rose bengal/cyanosine, rendering them light-sensitive to undergo disintegration in vitro, thereby providing a cellular basis for the larvicidal activity seen by the same treatments. Ineffectiveness of phthalocyanines and porphyrins for PDI underscores its requirement for different PS. Differential uptake of PS by insect versus other cells to account for this difference is under study.The ongoing work is patterned after the one-world approach by enlisting the participation of experts in medicinal chemistry, cell/molecular biology, immunology, parasitology, entomology, cancer research, tropical medicine and veterinary medicine. The availability of multidisciplinary expertise is indispensable for implementation of the necessary studies to move the project toward product development

Sox2 Is Essential for Formation of Trophectoderm in the Preimplantation Embryo

In preimplantation mammalian development the transcription factor Sox2 (SRY-related HMG-box gene 2) forms a complex with Oct4 and functions in maintenance of self-renewal of the pluripotent inner cell mass (ICM). Previously it was shown that Sox2-/- embryos die soon after implantation. However, maternal Sox2 transcripts may mask an earlier phenotype. We investigated whether Sox2 is involved in controlling cell fate decisions at an earlier stage.We addressed the question of an earlier role for Sox2 using RNAi, which removes both maternal and embryonic Sox2 mRNA present during the preimplantation period. By depleting both maternal and embryonic Sox2 mRNA at the 2-cell stage and monitoring embryo development in vitro we show that, in the absence of Sox2, embryos arrest at the morula stage and fail to form trophectoderm (TE) or cavitate. Following knock-down of Sox2 via three different short interfering RNA (siRNA) constructs in 2-cell stage mouse embryos, we have shown that the majority of embryos (76%) arrest at the morula stage or slightly earlier and only 18.7-21% form blastocysts compared to 76.2-83% in control groups. In Sox2 siRNA-treated embryos expression of pluripotency associated markers Oct4 and Nanog remained unaffected, whereas TE associated markers Tead4, Yap, Cdx2, Eomes, Fgfr2, as well as Fgf4, were downregulated in the absence of Sox2. Apoptosis was also increased in Sox2 knock-down embryos. Rescue experiments using cell-permeant Sox2 protein resulted in increased blastocyst formation from 18.7% to 62.6% and restoration of Sox2, Oct4, Cdx2 and Yap protein levels in the rescued Sox2-siRNA blastocysts.We conclude that the first essential function of Sox2 in the preimplantation mouse embryo is to facilitate establishment of the trophectoderm lineage. Our findings provide a novel insight into the first differentiation event within the preimplantation embryo, namely the segregation of the ICM and TE lineages

Photodynamic vaccination: Novel strategies of vaccine delivery by intraphagolysosomal parasites and their oxidative inactivation for safe and effective applications.

Leishmania (trypanosomatid protozoa) have a number of advantages, which distinguish them favorably from other microbes/particles to serve as a universal vaccine carrier. Biotechnology is well-established to produce transgenic Leishmania for expressing peptide vaccines. Multiple vaccines can be expressed episomally at high capacity via its eukaryotic mechanisms of protein translation and post-translational modifications, e. g. N-glycosylation. Technology is also available to grow Leishmania in serum-free/autoclavable or chemically defined media that is expandable to industrial scale cost-effectively. The major attributes of Leishmania for vaccine delivery are their surface glyco-conjugates responsible for the following properties: Protection of Leishmania, hence their expressed vaccines from losses to the activities of humoral factors in the mammalian hosts; Targeting the vaccines to the phagosome-lysosome vacuolar system of macrophages and dendritic cells - a desirable destination for effective vaccine processing and presentation; and Serving as potential adjuvants for the natural vaccines to elicit the lasting immunity seen after spontaneous/therapeutic cure of human leishmaniasis. Significantly, the surface glycoconjugates retain these functional activities when Leishmania are photo-inactivated with reactive oxygen species (ROS). Photo-inactivation of Leishmania with ROS eliminates their immunosuppressive activities and releases vaccines in macrophages. A complete loss of Leishmania viability in vitro and in vivo ensues, especially when doubly photo-inactivated for cytosolic accumulation of uroporphyrin and endosomal uptake of exogenous cationic phthalocyanines. The delivery of ovalbumin (OVA) as a surrogate vaccine by such photo-inactivated Leishmania is more effective than by conventional methods in activation of OVA peptide-specific CD4+ and CD8+ T cells. Previously, evidence has been presented, showing that photodynamic vaccination of animals with Leishmania so inactivated produced effective immunity that is adaptively transferable to naïve individuals against experimental leishmaniasis. Immunotherapy of canine leishmaniasis with photo-inactivated Leishmania produced encouraging preliminary results. Work is also underway to use such carrier to deliver add-on vaccines against other infectious and malignant disease