Katerina Vavrova

Enhancement effects of (R) and (S) enantiomers and the racemate of a model enhancer on permeation of theophylline through human skin

Abstract. The conformation of a permeation enhancer, given their mechanism of action, could influence its enhancing properties, since the stratum corneum components form essentially a chiral environment. The racemate and both enantiomers of 6-aminohexanoic acid 2-octylester as model enhancers with one chiral center were synthesized and their ability to enhance in vitro theophylline permeation through human skin was tested. The MTMT concept could not be applied in this study (the melting points of the substances were lower than 20ºC) and we observed no significant difference in enhancement ratios (ERs) of racemic 6-aminohexanoic acid 2-octylester and that of each enantiomer. However, differences in permeation rates between enantiomers and their racemates do not have to be related to stereoselective interactions, since they may also be explained by differences in physicochemical properties. The study also showed that there was no difference in the permeation enhancement ability between the (R)-(–) and (S)-(+) isomers of 6-aminohexanoic acid 2-octylester (the ERs were 2.72±0.42 and 2.79±0.60 for (R) and (S) enantiomers, respectively), suggesting that the enhancing properties of the compounds are not dependent on their spatial arrangement. Although stereoselective interactions between an enhancer and stratum corneum components may exist, they seem not to be important for the enhancer action.

Generation of T cell effectors using tumor cell-loaded dendritic cells for adoptive T cell therapy

Adoptive T cell transfer has been shown to be an effective method used to boost tumor-specific immune responses in several types of malignancies. In this study, we set out to optimize the ACT protocol for the experimental treatment of prostate cancer. The protocol includes a pre-stimulation step whereby T cells were primed with autologous dendritic cells loaded with the high hydrostatic pressure-treated prostate cancer cell line, LNCaP. Primed T cells were further expanded in vitro with anti-CD3/CD28 Dynabeads in the WAVE bioreactor 2/10 system and tested for cytotoxicity. Our data indicates that the combination of pre-stimulation and expansion steps resulted in the induction and enrichment of tumor-responsive CD4+ and CD8+ T cells at clinically relevant numbers. The majority of both CD4+ and CD8+ IFN-γ producing cells were CD62L, CCR7 and CD57 negative but CD28 and CD27 positive, indicating an early antigen experienced phenotype in non-terminal differentiation phase. Expanded T cells showed significantly greater cytotoxicity against LNCaP cells compared to the control SKOV-3, an ovarian cancer line. In summary, our results suggest that the ACT approach together with LNCaP-loaded dendritic cells provides a viable way to generate prostate cancer reactive T cell effectors that are capable of mounting efficient and targeted antitumor responses and can be thus considered for further testing in a clinical setting.

Cellular and Metabolic Basis for the Ichthyotic Phenotype in NIPAL4 (Ichthyin) Deficient Canines

Mutations in several lipid synthetic enzymes that block fatty acid and ceramide production produce autosomal recessive congenital ichthyoses (ARCIs) and associated abnormalities in permeability barrier homeostasis. However, the basis for the phenotype in patients with NIPAL4 (ichthyin) mutations (among the most prevalent ARCIs) remains unknown. Barrier function was abnormal in an index patient and in canines with homozygous NIPAL4 mutations, attributable to extensive membrane stripping, likely from detergent effects of nonesterified free fatty acid. Cytotoxicity compromised not only lamellar body secretion but also formation of the corneocyte lipid envelope (CLE) and attenuation of the cornified envelope (CE), consistent with a previously unrecognized, scaffold function of the CLE. Together, these abnormalities result in failure to form normal lamellar bilayers, accounting for the permeability barrier abnormality and clinical phenotype in NIPA-like domain-containing 4 (NIPAL4) deficiency. Thus, NIPAL4 deficiency represents another lipid synthetic ARCI that converges on the CLE (and CE), compromising their putative scaffold function. However, the clinical phenotype only partially improved after normalization of CLE and CE structure with topical ω-O-acylceramide because of ongoing accumulation of toxic metabolites, further evidence that proximal, cytotoxic metabolites contribute to disease pathogenesis.

Abstract B065: Use of simultaneous detection of externalized CD107a and CD137 for evaluation of specificity and effector functions of polyclonal T-cells produced for adoptive cellular immunotherapy of prostate cancer

The ex vivo production of T-cells that can recognize tumor-associated antigens (TAAs) and through this recognition eliminate cancer cells is the goal of T-cell-based adoptive cellular immunotherapy (ACI). The technological approaches that use whole cancer cells or their pertinent lysates as a source of TAAs for production of these T-cells lead to generation of polyclonal T-cells their antigen specificity of which is often unknown. Evaluation of such polyclonal T-cells is therefore challenging because their target and off-target performance might be difficult to predict. In our study we used human dendritic cells (DCs) pulsed with inactivated prostate cancer cell line LNCaP and matured with polyinosinic:polycytidylic acid (poly(I:C)) to induce expansion of autologous lymphocytes. The expanded lymphocytes were then challenged with live LNCaP cells and analyzed by simultaneous detection of a marker of T-cell cytotoxicity and antigen-induced T-cell stimulation, the surface externalization of, respectively, CD107a and CD137. Our data showed that lymphocytes expanded with LNCaP-pulsed and poly(I:C)-matured DCs contained a notable fraction of CD137 and/or CD107a positive T-cells after their challenge with live LNCaP cells. No such notable fraction was however observed in resting lymphocytes, or when the lymphocytes were challenged with ovarian cancer cell line SKOV-3. Importantly, autologous lymphocytes that were expanded with poly(I:C)-matured DCs not previously pulsed with the inactivated LNCaP cells did not show a notable population of the marker positive cells. Collectively, our data showed that simultaneous detection of externalized CD107a and CD137 on the surface of polyclonal T-cells may allow for evaluation of polyclonal T-cell specificity and effector functions after their challenge with complex antigens. Citation Format: Pavla Taborska, Dmitry Stakheev, Katerina Vavrova, Petra Vrabcova, Jirina Bartunkova, Daniel Smrz. Use of simultaneous detection of externalized CD107a and CD137 for evaluation of specificity and effector functions of polyclonal T-cells produced for adoptive cellular immunotherapy of prostate cancer [abstract]. In: Proceedings of the Second CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; 2016 Sept 25-28; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(11 Suppl):Abstract nr B065.