Non-invasive evaluation techniques to quantify the efficacy of cosmetic anti-cellulite products.

BACKGROUND: The majority of women suffer from the unattractive sight of dimpling skin on the thighs and buttocks, globally known as cellulite. Cellulite can be regarded as the most investigated non-disease, because, from the cosmetic viewpoint, most women desire a reduction in cellulite severity. Despite investigations made, cellulite is still not well understood at the cellular level, which leads to controversy regarding the investigative methods for cellulite reduction as well as the development of products to treat cellulite skin. OBJECTIVE: The aim of our work was to improve the set up of macrophotography for making images of dimpled skin and to automatize image analysis of 20 MHz ultrasound imaging - these two methods being just two of a variety of available methods for investigating cellulite skin. METHODS: Macrophotography was standardized on the aspects of volunteer's positioning, skin illumination, background used, and camera position. It was performed before, during and after a 3-month-treatment of a cosmetic product. Scoring assessments of the generated images were made by the volunteers themselves as well as by six trained experts. Ultrasound imaging was performed at the baseline visit in order to correlate the newly developed analysis with the visually rated cellulite score. A second study is also presented showing a variety of parameters that can be used for cosmetic testing of cellulite products: skin firmness, blood circulation and circumferential thigh measurements. RESULTS: Standardization of macrophotography minimized differences in image features between assessment times, therefore, enabling follow-up rating assessments of the images. A custom-made rating program simplified the scoring procedure by presenting images as blind and randomized, and by implementing computer-based analysis using an online rating scale. Volunteers and experts scored significant improvement of skin appearance over the course of a 3-month cosmetic treatment. Image analysis of ultrasound imaging was automatized, and a modification of the commonly known roughness parameter Ra was implemented to characterize cellulite severity. Comparison with the visually rated cellulite score showed an existing correlation between the score and the modified parameter Ra(m). Further parameters investigated in an exemplary study, as mentioned above, demonstrated a significant improvement of skin appearance after treatment with a cosmetic product. CONCLUSIONS: Macrophotography and ultrasound imaging can be regarded as important tools for determining and quantifying the aspects of cellulite. With a gold standard missing for investigating cellulite severity, these two methods may not determine cellulite at the cellular level, but they do characterize the skin appearance so typical for cellulite. Combined with a variety of other methods, macrophotography and ultrasound imaging can very well define cellulite-reducing efficacy from the cosmetic point of view.

Minimal size MIDGE vectors improve transgene expression in vivo.

Viral and plasmid vectors may cause immunological side-effects resulting from the expression of therapeutically unwanted genes and from CpG motifs contained in their sequence. A new vector type for minimalistic, immunological-defined gene expression (MIDGE) may overcome these problems. MIDGE is a minimal size gene transfer unit consisting of the expression cassette, including promotor, gene and RNA-stabilizing sequences, flanked by two short hairpin oligonucleotide sequences. DNA not encoding the desired gene is reduced to a minimum. To compare transfection efficiencies in vivo hydrodynamics-based, systemic transfection was performed in BALB/c mice with MIDGE vectors and corresponding plasmids. The transfection efficiencies of the MIDGE vectors as measured by luciferase expression were significantly higher in liver (2.5-fold), lung (3.5-fold), kidneys (3.9-fold) and heart (17-fold) as compared to plasmids. The mean numbers of MIDGE vector molecules per cell as measured by quantitative PCR were also significantly higher. These advantages suggest the preferential use of this new vector type for clinical gene therapy studies.

Effects of recombinant adenovirus-mediated expression of IL-2 and IL-12 in human B lymphoma cells on co-cultured PBMC.

BACKGROUND: Modulation of the immune system by genetically modified lymphoma cell vaccines is of potential therapeutic value in the treatment of B cell lymphoma. However, the anti-tumor effect of any single immunogene transfer has so far been limited. Combination treatment of recombinant IL-2 and IL-12 has been reported to be synergistic for inducing anti-tumor responses in solid tumors but the potential of IL-2/IL-12 gene modified B cell lymphoma cells has not been explored yet. METHODS: Using three different human B cell lymphoma cell lines and primary samples from patients with B cell neoplasms, expression levels of the coxsackie B-adenovirus receptor (CAR) and alpha (v) integrins were analyzed by fluorescence-activated cell sorter (FACS). Adenoviral transduction efficiencies were determined by GFP expression analysis and IL-2 and IL-12 cytokine production was quantified by enzyme-linked immunosorbent (ELISA) assays. Proliferative activities of peripheral blood mononuclear cells (PBMC) stimulated with either cytokine derived from supernatants of transduced lymphoma cells were measured by cell proliferation (MTT) assays. An EuTDA cytotoxicity assay was used to compare cytotoxic activities of IL-2 and/or IL-12 stimulated PBMC against unmodified lymphoma cells. RESULTS: We found that B cell lymphoma cell lines could be transduced with much higher efficiency than primary tumor samples, which appeared to correlate with the expression of CAR. Adenoviral-expressed IL-2 and IL-12 similarly led to dose-dependent increases in proliferation rates of PBMC obtained from healthy donors. IL-2 and/or IL-12 transduced lymphoma cells were co-cultured with PBMC, which were assayed for their cytolytic activity against unmodified lymphoma cells. We found that IL-2 stimulated PBMC elicited a significant anti-tumor effect but not the combined effect of IL-2/IL-12 or IL-12 alone. CONCLUSION: This study demonstrates that the generation of recombinant adenovirus modified lymphoma cell vaccines based on lymphoma cell lines expressing IL-2 and IL-12 cytokine genes is technically feasible, induces increases in proliferation rates and cytotoxic activity of co-cultured PBMC, and warrants further development for the treatment of lymphoma patients in the future.

Novel non-viral method for transfection of primary leukemia cells and cell lines.

BACKGROUND: Tumor cells such as leukemia and lymphoma cells are possible targets for gene therapy. However, previously leukemia and lymphoma cells have been demonstrated to be resistant to most of non-viral gene transfer methods. METHODS: The aim of this study was to analyze various methods for transfection of primary leukemia cells and leukemia cell lines and to improve the efficiency of gene delivery. Here, we evaluated a novel electroporation based technique called nucleofection. This novel technique uses a combination of special electrical parameters and specific solutions to deliver the DNA directly to the cell nucleus under mild conditions. RESULTS: Using this technique for gene transfer up to 75% of primary cells derived from three acute myeloid leukemia (AML) patients and K562 cells were transfected with the green flourescent protein (GFP) reporter gene with low cytotoxicity. In addition, 49(+/- 9.7%) of HL60 leukemia cells showed expression of GFP. CONCLUSION: The non-viral transfection method described here may have an impact on the use of primary leukemia cells and leukemia cell lines in cancer gene therapy.

Gene therapy of lymphoma.

Gene therapy offers new and promising treatment for patients with hematological malignancies. Tumor cells--lymphoma cells, for example--are possible targets for gene therapy. In general, gene therapeutic approaches require efficient gene transfer into host cells and sufficient transgene expression. Although many methods of gene transfer into mammalian cells exist, most do not allow efficient DNA transfer into primary lymphocytes. In contrast to gene transfer into tumor cells and many other cell types, which can be successfully performed using a variety of methods, the efficient expression of foreign DNA in lymphoma cells presents unique problems and challenges, requiring a careful selection of the mode of gene transfer. In this review, we discuss the current strategies for gene therapy in the treatment of lymphoma. We also summarize the current gene transfer methods for lymphoma cells and efficiency of transgene expression.

Generation of activated and antigen-specific T cells with cytotoxic activity after co-culture with dendritic cells.

Co-culturing of immunological effector cells with antigen-pulsed DC leads to an increase of cytotoxic activity against antigen-expressing tumour cells. Using this approach, we could detect up to 2.8% antigen-specific CTLs after co-culture with antigen-pulsed DC. However, the required high effector cell numbers remain a major obstacle in immunotherapy. In this study, we show an approach for generating activated and antigen-specific effector cells that enables us to decrease effector to target cell ratios. We used an interferon-gamma secretion assay to enrich activated effector cells after co-culture with antigen-pulsed dendritic cells (DC). Purified immunological effector cells lysed 58.3% of antigen-expressing tumour cells at an effector to target ratio of 1:1. Furthermore, using MHC-IgG complexes, we enriched effector cells expressing antigen-specific T-cell receptor after co-culture with DC. Performing ELISpot, flow cytometry and TCR analysis, we could show a significant increase of activated and specific TCR-expressing effector cells after co-culture with DC.