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

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

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

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

Aorta and pulmonary trunk - comparison of wall structure in typical and atypical (Ross procedure) blood pressure conditions

The ascending aorta and pulmonary trunk develop from the common truncus arteriosus that later becomes symmetrically divided by the aorticopulmonary septum. Normally, the systolic pressure value and the diastolic pressure gradient in the aorta is much higher than that in the pulmonary artery. In the Ross procedure, patient’s diseased aortic valve is replaced with their own pulmonary valve and as a consequence the pulmonary trunk is placed in the aortic root position. Typically, a reinforcement of transposed pulmonary trunk is necessary to avoid vessel dilation. In order to investigate how the blood flow characteristics determine the vessel wall structure we examined by immunochemistry the wall of normal aorta (NA), normal pulmonary trunk (NPT), transposed pulmonary trunk (trPT) and transposed pulmonary trunk with reinforcement (trPT-R). Throughout the tunica media of NA, elastic fibers form numerous, conspicuous and orderly arranged wavy lamellae that parallel thin layers of smooth muscle cells between the internal and external elastic membrane. In the NPT, smooth muscle cells run amid collagen fibers and form layers that are thicker and irregular, with elastic fibers arranged accordingly. In the trPT, intima denudation and media disruptions were observed. In the tunica media, smooth muscle cells were abundant, but muscle fibers, with irregular profiles and no discernible alignment, were widely spaced with intervening collagen fibers. In the trPA-R, the endothelial lining was preserved and elastic fibers formed a thick and highly organized layer of concentric lamellae in the middle third of tunica media. The structure of normal aorta and pulmonary trunk, both elastic arteries, with common embryological origin, differs significantly in terms of smooth muscle and elastic sheets number and organization. The animal model of Ross procedure with pulmonary trunk in aorta position further underscores the role of blood pressure and mechanical stress in vessel wall modification

Positional memory of fibroblasts may affect efficiency of iPSC reprogramming

Induced Pluripotent Stem cells (iPSC) are pluripotent stem cells reprogrammed from adult somatic cells. Although iPSC hold great potential for applications in regenerative medicine, technical problems, mostly related to the low efficiency of reprogramming, are yet to be solved. Since the most used cells for iPSC reprogramming are skin fibroblasts (FB), and since FB preserve positional memory, we hypothesize that the anatomic origin of FB might influence iPSC reprogramming.We isolated FB from skin of five different sites (neck, arm, thigh, breast, abdomen) of 13 patients undergoing plastic surgery or from heart wall or ascending aorta wall of the explanted heart of 3 patients receiving heart transplantation. FB from different anatomic sites and control FB from neonatal foreskin, were cultured for one week to evaluate morphology, proliferation rate and proneness to apoptosis. Additionally, expression of vimentin, cadherin, smooth muscle actin and Factor VIII was investigated to exclude the presence of other cell types. Transcriptome analysis including genes involved in stemness maintenance, embryogenesis, cell growth, activation and development, was performed by real-time PCR. Despite the similar morphology of FB from different sites, and immunopositivity for vimentin, along with the absence of other cell type markers, FB isolated from abdomen and heart had 1.5-fold higher doubling time, while FB from heart, abdomen and breast were less susceptible to apoptosis. Intriguingly, Real-Time PCR revealed that in abdomen, breast, neck, arm and heart FB genes involved in cell growth, development, proliferation, and migration, as TM4SF1, GPC4, CSPG2, DDIT4, ID1 were up-regulated, while genes regulating embryogenesis and tissue morphogenesis, like VCAN, FN1, HOXA5, CD49a were up-regulated in FB isolated from abdomen, arm and heart. However, all FBs had transcripts of markers of Mesenchymal Stem Cells (MSC), as CD105 and CD90. Our results provide evidence that human adult FB from different sites have different genetic program. Therefore, FB may respond to reprogram technology in different manner, thus affecting reprogramming efficiency. While offering novel perspective of the reprogramming technology, our study also demonstrates that abdomen and breast FB share cardiac genetic signature of cardiac FB while expressing markers of MSC and they might represent the ideal cell for cardiac reprogramming

Bioresorbable reinforcement induces histological rearrangement of pulmonary autograft in an experimental model of Ross operation

The Ross procedure has emerged as a popular choice for aortic valve replacement in infants and children. However, pulmonary artery (PA) autograft dilation remains the major concern; hence, several modifications of the valve implantation techniques, such as reinforcing the autografts with a tubular synthetic mesh, have been reported. With the aim to prevent dilation and permit the normal growth of the neo-aortic root following pulmonary autograft implantation, we assessed the biological effect and long term performance of an external bioresorbable reinforcement for PA autograft in an experimental Ross model in growing animals. An experimental model of translocation of the pulmonary trunk as autograft in aortic position, funded on the Hook’s law and Laplace equilibrium, has been developed and performed under cardiopulmonary bypass in young lambs. The PA without reinforcement (n=5) was compared to PA reinforced with new scaffold polymer with an external armour of Polytetrafluoroethylene. The PA autograft diameter was measured using transoesophageal echography at day 0 and at 6 months and compared to the distal aortic diameter. Pathological analysis of the PA autograft reinforced was performed at 6 months and the results were compared to those of a control group with no reinforcement (n=5) Animal weight was 25+5 kg at day 0 and 58+10 kg at 6 months and the reference aortic diameter increased from 14+1mm at day 0 to 17+2mm at 6 months. With no reinforcement, an instantaneous PA graft distension (27,4+2mm) was noted followed by an aneurysmal formation at 6 months (38+3mm). Reinforcement with scaffold polymer on polidioxanone allowed maintaining the PA graft diameter close to the reference value (17+2mm at day 0). Immunohistochemistry revealed MMP-9 overexpression indicating the induction of a matrix remodeling process that are not detectable in the control group. Mallory staining revealed elastin deposition in the reinforced PA in comparison to the collagen present in the non-reinforced group, reliably suggesting a shift towards an elastic remodeling and arterialization. PicroSirius red staining reveled in the control group collagen fibers non- homogeneously distributed with a increased cellularity indicating inflammatory infiltrates. The reinforced PA displays more organized and dense collagen fibers in the “elastic zone” of the vessel and less pronounced cellular infiltrate. In conclusion, bioresorbable external polydioxanone-based reinforcement allowed a structural rearrangement of PA autograft consisting of media reorganization with an increase in the elastic wall component. Such histological outcome arguably prevented autograft dilation and conferred enhanced mechanical properties on the PA wal

An international inter-laboratory study on Nosema spp. spore detection and quantification through microscopic examination of crushed honey bee abdomens

Nosemosis is a microsporidian disease causing mortality and weakening of honey bee colonies, especially in the event of co-exposure to other sources of stress. As a result, the disease is regulated in some countries. Reliable and harmonised diagnosis is crucial to ensure the quality of surveillance and research results. For this reason, the first European Interlaboratory Comparison (ILC) was organised in 2017 in order to assess both the methods and the results obtained by National Reference Laboratories (NRLs) in counting Nosema spp. spores by microscopy. Implementing their own routine conditions of analysis, the 23 participants were asked to perform an assay on & nbsp;a & nbsp;panel of ten positive and negative samples of crushed honey bee abdomens. They were asked to report results from a qualitative and quantitative standpoint. The assessment covered specificity, sensitivity, trueness and precision. Quantitative results were analysed in compliance with international standards NF ISO 13528 (2015) and NF ISO 5725-2 (1994). Three results showed a lack of precision and five a lack of trueness. However, overall results indicated a global specificity of 98% and a global sensitivity of 100%, thus demonstrating the advanced performance of the microscopic methods applied to Nosema spores by the NRLs. Therefore, the study concluded that using microscopy to detect and quantify spores of Nosema spp. was reliable and valid.panel of ten positive and negative samples of crushed honey bee abdomens. They were asked to report results from a qualitative and quantitative standpoint. The assessment covered specificity, sensitivity, trueness and precision. Quantitative results were analysed in compliance with international standards NF ISO 13528 (2015) and NF ISO 5725-2 (1994). Three results showed a lack of precision and five a lack of trueness. However, overall results indicated a global specificity of 98% and a global sensitivity of 100%, thus demonstrating the advanced performance of the microscopic methods applied to Nosema spores by the NRLs. Therefore, the study conclude

Basic fibroblast growth factor and vascular endothelial growth factor serum levels in breast cancer patients and healthy women: useful as diagnostic tools?

INTRODUCTION: The aim of the present study was to analyze the relationship between the expression of vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF) in breast cancer cells and the corresponding serum levels in individual patients. The study also evaluated the potential of serum levels of the two growth factors as diagnostic markers in a case–control study. METHODS: VEGF expression and bFGF expression were determined in 62 and 63 tumor samples, respectively. Serum VEGF and bFGF levels were determined in 54 and 65 healthy women and in 69 and 73 breast cancer patients, respectively, using a quantitative sandwich enzyme immunoassay technique. RESULTS: A direct correlation was observed between VEGF expression and bFGF expression in individual tumors (P = 0.001) and between serum levels (P = 0.038) in individual patients, but not between tumor cell expression and the corresponding serum level for either growth factor. Median values of serum levels in healthy women and breast cancer patients were not different for VEGF (P = 0.055), but were significantly different for bFGF (P < 0.001). The receiver operating characteristic curve identified a serum bFGF concentration of 1.0 pg/ml, with 84.9% sensitivity and 63.1% specificity, as the best cut-off value to discriminate between healthy women and breast cancer patients. An age-based subgroup analysis showed that serum values of patients older than 70 years of age mainly contributed to the high accuracy. CONCLUSIONS: Our data repropose bFGF as a noninvasive diagnostic tool for breast cancer