Quantitative imaging and semiotic phenotyping of mitochondrial network morphology in live human cells.

The importance of mitochondria in tissue homeostasis, stress responses and human diseases, combined to their ability to transition between various structural and functional states, makes them excellent organelles for monitoring cell health. There is therefore a need for technologies to accurately analyze and quantify changes in mitochondrial organization in a variety of cells and cellular contexts. Here we present an innovative computerized method that enables accurate, multiscale, fast and cost-effective analysis of mitochondrial shape and network architecture from confocal fluorescence images by providing more than thirty features. In order to facilitate interpretation of the quantitative results, we introduced two innovations: the use of Kiviat-graphs (herein named MitoSpider plots) to present highly multidimensional data and visualization of the various mito-cellular configurations in the form of morphospace diagrams (called MitoSigils). We tested our fully automated image analysis tool on rich datasets gathered from live normal human skin cells cultured under basal conditions or exposed to specific stress including UVB irradiation and pesticide exposure. We demonstrated the ability of our proprietary software (named MitoTouch) to sensitively discriminate between control and stressed dermal fibroblasts, and between normal fibroblasts and other cell types (including cancer tissue-derived fibroblasts and primary keratinocytes), showing that our automated analysis captures subtle differences in morphology. Based on this novel algorithm, we report the identification of a protective natural ingredient that mitigates the deleterious impact of hydrogen peroxide (H2O2) on mitochondrial organization. Hence we conceived a novel wet-plus-dry pipeline combining cell cultures, quantitative imaging and semiotic analysis for exhaustive analysis of mitochondrial morphology in living adherent cells. Our tool has potential for broader applications in other research areas such as cell biology and medicine, high-throughput drug screening as well as predictive and environmental toxicology.

GTL001, a bivalent therapeutic vaccine against human papillomavirus 16 and 18, induces antigen-specific CD8+ T cell responses leading to tumor regression.

BACKGROUND: Prophylactic vaccines are available for women and girls not yet infected with HPV, but women already infected with HPV need a treatment to prevent progression to high-grade cervical lesions and cancer. GTL001 is a bivalent therapeutic vaccine for eradicating HPV-infected cells that contains HPV16 E7 and HPV18 E7 both fused to detoxified adenylate cyclase from Bordetella pertussis, which binds specifically to CD11b+ antigen-presenting cells. This study examined the ability of therapeutic vaccination with GTL001 adjuvanted with topical imiquimod cream to induce functional HPV16 E7- and HPV18 E7-specific CD8+ T cell responses. METHODS: Binding of GTL001 to human CD11b was assessed by a cell-based competition binding assay. Cellular immunogenicity of intradermal vaccination with GTL001 was assessed in C57BL/6 mice by enzyme-linked immunospot assay and in vivo killing assays. In vivo efficacy of GTL001 vaccination was investigated in the TC-1 murine HPV16 E7-expressing tumor model. RESULTS: GTL001 bound specifically to the human CD11b/CD18 receptor. GTL001 adjuvanted with topical 5% imiquimod cream induced HPV16 E7 and HPV18 E7-specific CD8+ T cell responses. This CD8+ T-cell response mediated in vivo killing of HPV E7-expressing cells. In the HPV16 E7-expressing tumor model, GTL001 adjuvanted with imiquimod but not imiquimod alone or a combination of unconjugated HPV16 E7 and HPV18 E7 caused complete tumor regression. CONCLUSIONS: GTL001 adjuvanted with topical 5% imiquimod is immunogenic and induces HPV16 E7 and HPV18 E7-specific CD8+ T cell responses that can kill HPV E7-expressing cells and eliminate HPV E7-expressing tumors.

GTL001 and bivalent CyaA-based therapeutic vaccine strategies against human papillomavirus and other tumor-associated antigens induce effector and memory T-cell responses that inhibit tumor growth.

GTL001 is a bivalent therapeutic vaccine containing human papillomavirus (HPV) 16 and HPV18 E7 proteins inserted in the Bordetella pertussis adenylate cyclase (CyaA) vector intended to prevent cervical cancer in HPV-infected women with normal cervical cytology or mild abnormalities. To be effective, therapeutic cervical cancer vaccines should induce both a T cell-mediated effector response against HPV-infected cells and a robust CD8+ T-cell memory response to prevent potential later infection. We examined the ability of GTL001 and related bivalent CyaA-based vaccines to induce, in parallel, effector and memory CD8+ T-cell responses to both vaccine antigens. Intradermal vaccination of C57BL/6 mice with GTL001 adjuvanted with a TLR3 agonist (polyinosinic-polycytidylic acid) or a TLR7 agonist (topical 5% imiquimod cream) induced strong HPV16 E7-specific T-cell responses capable of eradicating HPV16 E7-expressing tumors. Tumor-free mice also had antigen-specific memory T-cell responses that protected them against a subsequent challenge with HPV18 E7-expressing tumor cells. In addition, vaccination with bivalent vaccines containing CyaA-HPV16 E7 and CyaA fused to a tumor-associated antigen (melanoma-specific antigen A3, MAGEA3) or to a non-viral, non-tumor antigen (ovalbumin) eradicated HPV16 E7-expressing tumors and protected against a later challenge with MAGEA3- and ovalbumin-expressing tumor cells, respectively. These results show that CyaA-based bivalent vaccines such as GTL001 can induce both therapeutic and prophylactic anti-tumor T-cell responses. The CyaA platform can be adapted to different antigens and adjuvants, and therefore may be useful for developing other therapeutic vaccines.

Ligand-regulated internalization of the opioid receptor-like 1: a confocal study.

To better understand opioid receptor-like 1 (ORL1) internalization, we fused the C terminus of ORL1, the nociceptin (noc) receptor, to the N terminus of a green fluorescent protein and used the fusion protein to characterize receptor endocytosis in live human embryonic kidney cells. The fusion altered neither the affinity of the receptor for noc or other ORL1 receptor ligands nor the ability of the receptor to mediate agonist-induced binding of GTPgamma(35)S, i.e. coupling with heterotrimeric G protein. Confocal microscopy showed that the fluorescent receptor was mostly associated (>75%) with the periplasmic membrane. In the presence of 0.1 microm noc, approximately 80% of receptors were internalized, and half-maximum internalization was reached in approximately 12 min at 22 C and approximately 6 min at 37 C. After washing, a normal receptor level was recovered within 70 min at 22 C. The lack of internalization in the presence of 0.45 m sucrose suggests that noc-induced receptor endocytosis mainly occurred via clathrin-coated pits. Coincubation of the recombinant cells with noc and tetramethylrhodamine-transferrin showed that ORL1 was mainly internalized through the endosome compartment. Lofentanil and Ro64-6198 ([(1S,3aS)-8-(2,3,3a,4,5,6-hexahydro-1H-phenalen-1-yl)-1-phenyl-1,3,8-triaza-spiro[4.5]decan-4-one]) promoted endocytosis of the fluorescent receptor as efficiently as noc. Among the two ORL1 receptor antagonists, J-113397 (1-[(3R,4R)-1-cyclooctylmethyl-3-hydroxymethyl-4-piperidyl]-3-ethyl-1,3-dihydro-2H-benzimidazol-2-one), but not III-BTD, blocked the noc-induced internalization of the fluorescent receptor. Two partial agonists were dramatically less efficient than noc to promote ORL1 internalization. They recruited very little (the pseudopeptide [Phe(1)psi(CH(2)-NH)Gly(2)]-noc-(1-13)NH(2)) or no (the hexapeptide Ac-Arg-Tyr-Tyr-Lys-Trp-Arg-NH(2)) G protein receptor kinase type 2 coupled to red fluorescent protein 1 at the membrane, suggesting that subsequent receptor phosphorylation necessary for internalization via coated pits is altered. Thus, partial agonists that induce a prolonged cell response without causing substantial receptor internalization may be good tools for further clinical treatments.