Skewing effect of sulprostone on dendritic cell maturation compared with dinoprostone.

Abstract Background Dendritic cells (DCs) are the most efficient antigen-presenting cells and act at the center of the immune system owing to their ability to control both immune tolerance and immunity. In cancer immunotherapy, DCs play a key role in the regulation of the immune response against tumors and can be generated ex vivo with different cytokine cocktails. Methods . We evaluated the feasibility of dinoprostone (PGE 2 ) replacement with the molecular analog sulprostone, in our good manufacturing practice (GMP) protocol for the generation of DC-based cancer vaccine. We characterized the phenotype and the function of DCs matured in the presence of sulprostone as a potential substitute of dinoprostone in the pro-inflammatory maturation cocktail consisting of tumor necrosis factor alpha (TNF-α), interleukin-1 beta (IL-1β) and IL-6. Results . We found that sulprostone invariably reduces the recovery, but does not significantly modify the viability and the purity of DCs. The presence of sulprostone in the maturation cocktail increases the adhesion of single cells and of clusters of DCs to the flask, making them more similar to their immature counterpart in terms of adhesion and spreading proprieties. Moreover, we observed that sulprostone impairs the expression of co-stimulatory molecules and the spontaneous as well as the directed migration capacity of DCs. Discussion These findings underscore that the synthetic analog sulprostone strongly reduces the functional quality of DCs, thus cannot replace dinoprostone in the maturation cocktail of monocyte-derived DCs

FRET microscopy autologous tumor lysate processing in mature dendritic cell vaccine therapy

Abstract Background: Antigen processing by dendritic cells (DC) exposed to specific stimuli has been well characterized in biological studies. Nonetheless, the question of whether autologous whole tumor lysates (as used in clinical trials) are similarly processed by these cells has not yet been resolved. Methods: In this study, we examined the transfer of peptides from whole tumor lysates to major histocompatibility complex class II molecules (MHC II) in mature dendritic cells (mDC) from a patient with advanced melanoma. Tumor antigenic peptides-MHC II proximity was revealed by F\uf6rster Resonance Energy Transfer (FRET) measurements, which effectively extends the application of fluorescence microscopy to the molecular level (<100?). Tumor lysates were labelled with Alexa-488, as the donor, and mDC MHC II HLA-DR molecules were labelled with Alexa-546-conjugated IgG, as the acceptor. Results: We detected significant energy transfer between donor and acceptor-labelled antibodies against HLA-DR at the membrane surface of mDC. FRET data indicated that fluorescent peptide-loaded MHC II molecules start to accumulate on mDC membranes at 16 hr from the maturation stimulus, steeply increasing at 22 hr with sustained higher FRET detected up to 46 hr. Conclusions: The results obtained imply that the patient mDC correctly processed the tumor specific antigens and their display on the mDC surface may be effective for several days. These observations support the rationale for immunogenic efficacy of autologous tumor lysates

Matrix metalloproteinases and their inhibitors in canine mammary tumors

BACKGROUND: Malignant canine mammary tumors represent 50% of all neoplasms in female dogs. Matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs) are thought to be involved in tumor progression, and they are also associated with the reactive stroma, which provides structural and vascular support for tumor growth. RESULTS: MMP-2, MMP-9 and MT1-MMP were expressed at both the mRNA and protein levels in tumor samples. MMP-2 and MMP-9 immunohistochemical reactions were evident both in the epithelial tumor cells and in the stromal compartment to varying degrees; in particular, the intensity of the MMP-2 staining was stronger in the stromal fibroblasts close to epithelial tumor cells in simple carcinomas than in adenomas. These data were supported by gelatin-zymography; bands for the active form of MMP-2 were found in 94% of carcinoma samples, compared with 17% of benign tumor samples. The gene expression and immunohistochemical results for MT1-MMP were comparable to those for MMP-2. The immunoreactivity for MMP-13 and TIMP-2 was lower in carcinomas than in adenomas, confirming the mRNA data for MMP-13 and the other MMP inhibitors that were evaluated. The active form of MMP-9, but not the active form of MMP-2, was identified in the plasma of all of the tested dogs. CONCLUSIONS: Our findings suggest that MMP-9, MMP-2 and MT1-MMP, which are synthesized by epithelial cancer cells and cancer-associated fibroblasts, play an important role in malignant canine mammary tumors. The reduction of MMP-13 and TIMP-2 could also be a significant step in malignant transformation. MMP-2 and MT1-MMP could be further evaluated as future biomarkers for predicting the progression and prognosis of canine mammary tumors

Baltic herring hydrolysates: Identification of peptides, in silico DPP-4 prediction, and their effects on an in vivo mice model of obesity

Baltic herring is the main catch in the Baltic Sea; however, its usage could be improved due to the low processing rate. Previously we have shown that whole Baltic herring hydrolysates (BHH) and herring byproducts hydrolysates (BHBH) by commercial enzymes consisted of bioactive peptides and had moderate bioactivity in in vitro dipeptidyl peptidase (DPP)-4 assay. In this study, we identified the hydrolysate peptides by LC-MS/MS and predicted the potential bioactive DPP-4 inhibitory peptides using in silico tools. Based on abundance, peptide length and stability, 86 peptides from BHBH and 80 peptides from BHH were proposed to be novel DPP-4 inhibitory peptides. BHH was fed to a mice intervention of a high-fat, high-fructose diet to validate the bioactivity. The results of the glucose tolerance and insulin tolerance improved. Plasma DPP-4 activities, C-peptide levels, and HOMA-IR scores significantly decreased, while plasma glucagon-like peptide-1 content increased. In conclusion, BHH is an inexpensive and sustainable source of functional antidiabetic ingredients

The dependence of AGN activity on stellar and halo mass in Semi-Analytic Models

AGN feedback is believed to play an important role in shaping a variety of observed galaxy properties, as well as the evolution of their stellar masses and star formation rates. In particular, in the current theoretical paradigm of galaxy formation, AGN feedback is believed to play a crucial role in regulating the levels of activity in galaxies, in relatively massive halos at low redshift. Only in recent years, however, has detailed statistical information on the dependence of galaxy activity on stellar mass, parent halo mass and hierarchy has become available. In this paper, we compare the fractions of galaxies belonging to different activity classes (star-forming, AGN and radio active) with predictions from four different and independently developed semi-analytical models. We adopt empirical relations to convert physical properties into observables (H_alpha emission lines, OIII line strength and radio power). We demonstrate that all models used in this study reproduce the overall distributions of galaxies belonging to different activity classes as a function of stellar mass and halo mass: star forming galaxies and the strongest radio sources are preferentially associated with low-mass and high-mass galaxies/halos respectively. However, model predictions differ from observational measurements in a number of ways. All models used in our study predict that almost every >1.e12 Msun dark matter halo and/or >1.e11 Msun galaxy should host a bright radio source, while only a small fraction of galaxies belong to this class in the data. In addition, radio brightness is expected to depend strongly on the mass of the parent halo mass in the models, while strong and weak radio galaxies are found in similar environments in data. Our results highlight that the distribution of AGN as a function of stellar mass provides one of the most promising discriminants between different gas accretion schemes.Comment: 15 pages; 8 figures; 1 table; updated to match MNRAS accepted versio

Interplay Between SIRT-3, Metabolism and Its Tumor Suppressor Role in Hepatocellular Carcinoma

Sirtuins (SIRT), first described as nicotinamide adenine dinucleotide (NAD + )-dependent type III histone deacetylases, are produced by cells to support in the defense against chronic stress conditions such as metabolic syndromes, neurodegeneration, and cancer. SIRT-3 is one of the most studied members of the mitochondrial sirtuins family. In particular, its involvement in metabolic diseases and its dual role in cancer have been described. In the present review, based on the evidence of SIRT-3 involvement in metabolic dysfunctions, we aimed to provide an insight into the multifaceted role of SIRT-3 in many solid and hematological tumors with a particular focus on hepatocellular carcinoma (HCC). SIRT-3 regulatory effect and involvement in metabolism dysfunctions may have strong implications in HCC development and treatment. Research literature widely reports the relationship between metabolic disorders and HCC development. This evidence suggests a putative bridge role of SIRT-3 between metabolic diseases and HCC. However, further studies are necessary to demonstrate such interconnection

Unexpected High Response Rate to Traditional Therapy after Dendritic Cell-Based Vaccine in Advanced Melanoma: Update of Clinical Outcome and Subgroup Analysis

We reviewed the clinical results of a dendritic cell-based phase II clinical vaccine trial in stage IV melanoma and analyzed a patient subgroup treated with standard therapies after stopping vaccination. From 2003 to 2009, 24 metastatic melanoma patients were treated with mature dendritic cells pulsed with autologous tumor lysate and keyhole limpet hemocyanin and low-dose interleukin-2. Overall response (OR) to vaccination was 37.5% with a clinical benefit of 54.1%. All 14 responders showed delayed type hypersensitivity positivity. Median overall survival (OS) was 15 months (95% CI, 8–33). Eleven patients underwent other treatments (3 surgery, 2 biotherapy, 2 radiotherapy, 2 chemotherapy, and 4 biochemotherapy) after stopping vaccination. Of these, 2 patients had a complete response and 5 a partial response, with an OR of 63.6%. Median OS was 34 months (range 16–61). Our results suggest that therapeutic DC vaccination could favor clinical response in patients after more than one line of therapy

Stability Program in Dendritic Cell Vaccines: A “Real-World” Experience in the Immuno-Gene Therapy Factory of Romagna Cancer Center

Advanced therapy medical products (ATMPs) are rapidly growing as innovative medicines for the treatment of several diseases. Hence, the role of quality analytical tests to ensure consistent product safety and quality has become highly relevant. Several clinical trials involving dendritic cell (DC)-based vaccines for cancer treatment are ongoing at our institute. The DC-based vaccine is prepared via CD14+ monocyte differentiation. A fresh dose of 10 million DCs is administered to the patient, while the remaining DCs are aliquoted, frozen, and stored in nitrogen vapor for subsequent treatment doses. To evaluate the maintenance of quality parameters and to establish a shelf life of frozen vaccine aliquots, a stability program was developed. Several parameters of the DC final product at 0, 6, 12, 18, and 24 months were evaluated. Our results reveal that after 24 months of storage in nitrogen vapor, the cell viability is in a range between 82% and 99%, the expression of maturation markers remains inside the criteria for batch release, the sterility tests are compliant, and the cell costimulatory capacity unchanged. Thus, the data collected demonstrate that freezing and thawing do not perturb the DC vaccine product maintaining over time its functional and quality characteristics

Dendritic cell vaccination in metastatic melanoma turns \u201cnon-T cell inflamed\u201d into \u201cT-cell inflamed\u201d tumors

Dendritic cell (DC)-based vaccination effectively induces anti-tumor immunity, although in the majority of cases this does not translate into a durable clinical response. However, DC vaccination is characterized by a robust safety profile, making this treatment a potential candidate for effective combination cancer immunotherapy. To explore this possibility, understanding changes occurring in the tumor microenvironment (TME) upon DC vaccination is required. In this line, quantitative and qualitative changes in tumor-infiltrating T lymphocytes (TILs) induced by vaccination with autologous tumor lysate/homogenate loaded DCs were investigated in a series of 16 patients with metastatic melanoma. Immunohistochemistry for CD4, CD8, Foxp3, Granzyme B (GZMB), PDL1, and HLA class I was performed in tumor biopsies collected before and after DC vaccination. The density of each marker was quantified by automated digital pathology analysis on whole slide images. Co-expression of markers defining functional phenotypes, i.e., Foxp3+ regulatory CD4+ T cells (Treg) and GZMB+ cytotoxic CD8+ T cells, was assessed with sequential immunohistochemistry. A significant increase of CD8+ TILs was found in post-vaccine biopsies of patients who were not previously treated with immune-modulating cytokines or Ipilimumab. Interestingly, along with a maintained tumoral HLA class I expression, after DC vaccination we observed a significant increase of PDL1+ tumor cells, which significantly correlated with intratumoral CD8+ T cell density. This observation might explain the lack of a significant concurrent cytotoxic reactivation of CD8+ T cell, as measured by the numbers of GZMB+ T cells. Altogether these findings indicate that DC vaccination exerts an important role in sustaining or de novo inducing a T cell inflamed TME. However, the strength of the intratumoral T cell activation detected in post-DC therapy lesions is lessened by an occurring phenomenon of adaptive immune resistance, yet the concomitant PDL1 up-regulation. Overall, this study sheds light on DC immunotherapy-induced TME changes, lending the rationale for the design of smarter immune-combination therapies

A model for the cosmological evolution of low frequency radio sources

We present a new evolutionary model that describes the population properties of radio sources at frequencies <5 GHz, thus complementing the De Zotti et al. (2005) model, holding at higher frequencies. We find that simple analytic luminosity evolution is still sufficient to fit the wealth of available data on local luminosity functions, multi-frequency source counts, and redshift distributions. However, the fit requires a luminosity-dependent decline of source luminosities at high redshifts, at least for steep-spectrum sources, thus confirming earlier indications of a "downsizing" also for radio sources. The upturn of source counts at sub-mJy levels is accounted for by a straightforward extrapolation, using the empirical far-IR/radio correlation, of evolutionary models matching the far-IR counts and redshift distributions of star-forming galaxies. We also discuss the implications of the new model for the interpretation of data on large-scale clustering of radio sources and on the Integrated Sachs-Wolfe (ISW) effect, and for the investigation of the contribution of discrete sources to the extragalactic background. As for the ISW effect, a new analysis exploiting a very clean CMB map, yields at a substantially higher significance than reported before.Comment: 14 pages, 11 figures, accepted for publication on MNRA