Determinant spreading associated with clinical response in dendritic cell-based immunotherapy for malignant melanoma.

PURPOSE: The purpose of this study was to determine the toxicity and immunological effects of three different doses and two routes of administration of autologous dendritic cells (DCs) pulsed with the MART-1(27-35) immunodominant epitope. EXPERIMENTAL DESIGN: Eighteen HLA-A*0201-positive subjects with stage III-IV melanoma received three biweekly i.v. or intradermal injections of ex vivo generated myeloid DCs pulsed with MART-1(27-35) epitope. Repeated blood samples were processed to obtain peripheral blood mononuclear cells for immunological analysis using IFN-gamma ELISPOT, MHC class I tetramer, intracellular cytokine staining, and microcytotoxicity assays. RESULTS: The frequency of MART-1/Melan-A (MART-1) antigen-specific T cells in peripheral blood increased in all dose levels as assessed by ELISPOT and MHC class I tetramer assays, but without a clear dose-response effect. The intradermal route generated stronger MART-1 immunity compared with the i.v. route. MART-1-specific immunity did not correlate with clinical outcome in any of the four immunological assays used. However, analysis of determinant spreading to other melanoma antigens was noted in the only subject with complete response to this single-epitope immunization. CONCLUSIONS: Intradermal immunization with MART-1 peptide-pulsed DCs results in an increase in circulating IFN-gamma-producing, antigen-specific T cells. The frequency of these cells did not correlate with response. In contrast, spreading of immune reactivity to other melanoma antigens was only evident in a subject with a complete response, suggesting that determinant spreading may be an important factor of clinical response to this form of immunotherapy.     

Autophagy protein 16-mediated autophagy is required for the encystation of Acanthamoeba castellanii

Autophagy, an evolutionarily conserved protein degradation pathway in eukaryotes, plays essential roles during starvation and cellular differentiation by eliminating unwanted and/or unnecessary cell material including organelles. Autophagy protein 16 (Atg16) is an essential component of the autophagic machinery. The present study identified and characterized an Atg16 homologue (AcAtg16) in Acanthamoeba, an opportunistic pathogen responsible for several distinct diseases in humans. AcAtg16 was highly expressed during encystation and was found to be associated with small or large vesicular structures that partially colocalized with autophagolysosomes. Small interfering RNA against AcAtg16 inhibited autophagosome formation and reduced the encystation efficiency of Acanthamoeba. Moreover, most mitochondria remained undigested in these knockdown cells. Taken together, these results indicate that AcAtg16 is involved in autophagosome formation and plays an essential role in the encystation of Acanthamoeba.     

Pharmacogenomics of bortezomib test-dosing identifies hyperexpression of proteasome genes, especially PSMD4, as novel high-risk feature in myeloma treated with Total Therapy 3

Gene expression profiling (GEP) of purified plasma cells 48 hours after thalidomide and dexamethasone test doses showed these agents' mechanisms of action and provided prognostic information for untreated myeloma patients on Total Therapy 2 (TT2). Bortezomib was added in Total Therapy 3 (TT3), and 48 hours after bortezomib GEP analysis identified 80 highly survival-discriminatory genes in a training set of 142 TT3A patients that were validated in 128 patients receiving TT3B. The 80-gene GEP model (GEP80) also distinguished outcomes when applied at baseline in both TT3 and TT2 protocols. In context of our validated 70-gene model (GEP70), the GEP80 model identified 9% of patients with a grave prognosis among those with GEP70-defined low-risk disease and 41% of patients with favorable prognosis among those with GEP70-defined high-risk disease. PMSD4 was 1 of 3 genes common to both models. Residing on chromosome 1q21, PSMD4 expression is highly sensitive to copy number. Both higher PSMD4 expression levels and higher 1q21 copy numbers affected clinical outcome adversely. GEP80 baseline-defined high risk, high lactate dehydrogenase, and low albumin were the only independent adverse variables surviving multivariate survival model. We are investigating whether second-generation proteasome inhibitors (eg, carfilzomib) can overcome resistance associated with high PSMD4 levels.     

Development and validation of RP HPLC method to determine nandrolone phenylpropionate in different pharmaceutical formulations

This study describes development and subsequent validation of a reversed phase high performance liquid chromatographic (RP-HPLC) method for the estimation of nandrolone phenylpropionate, an anabolic steroid, in bulk drug, in conventional parenteral dosage formulation and in prepared nanoparticle dosage form. The chromatographic system consisted of a Luna Phenomenex, CN (250 mm x 4.6 mm, 5 microm) column, an isocratic mobile phase comprising 10 mM phosphate buffer and acetonitrile (50:50, v/v) and UV detection at 240 nm. Nandrolone phenylpropionate was eluted about 6.3 min with no interfering peaks of excipients used for the preparation of dosage forms. The method was linear over the range from 0.050 to 25 microg/mL in raw drug (r2 = 0.9994). The intra-day and inter-day precision values were in the range of 0.219-0.609% and 0.441-0.875%, respectively. Limits of detection and quantitation were 0.010 microg/mL and 0.050 microg/mL, respectively. The results were validated according to International Conference on Harmonization (ICH) guidelines in parenteral and prepared nanoparticle formulation. The validated HPLC method is simple, sensitive, precise, accurate and reproducible.     

Miconazole induces autophagic death in glioblastoma cells via reactive oxygen species-mediated endoplasmic reticulum stress

Miconazole is an antifungal agent that is used for the treatment of superficial mycosis. However, recent studies have indicated that miconazole also exhibits potent anticancer effects in various types of cancer via the activation of apoptosis. The main aim of the present study was to observe the effect of miconazole on autophagic cell death of cancer cells. Cytotoxicity was measured by viable cell counting after miconazole treatment in glioblastoma cell lines (U343MG, U87MG and U251MG). Induction of autophagy was analyzed by examining microtubule-associated protein light chain 3 (LC3)-II expression levels using western blotting and by detecting GFP-LC3 translocation using a fluorescence microscope. Intracellular ROS production was measured using a fluorescent probe, 2′,7′-dichlorodihydrofluorescein diacetate. It was found that miconazole induced autophagic cell death in the U251MG glioblastoma cell line via the generation of reactive oxygen species (ROS) and endoplasmic reticulum (ER) stress response. An association between miconazole-induced ROS production and autophagy was also identified; in particular, pretreatment of the cells with a ROS scavenger resulted in a reduction in the levels of LC3-II. Miconazole-induced ER stress was associated with increases in binding immunoglobulin protein (BiP), inositol-requiring enzyme 1α (IRE1α) and CHOP expression, and phospho-eIF2α levels. The inhibition of ER stress via treatment with 4-phenylbutyric acid or BiP knockdown reduced miconazole-induced autophagy and cell death. These findings suggest that miconazole induces autophagic cell death by inducing an ROS-dependent ER stress response in U251MG glioma cancer cells and provide new insights into the potential antiproliferative effects of miconazole.