Subfertility and risk of testicular cancer in the EPSAM case-control study

It has been suggested that subfertility and testicular cancer share genetic and environmental risk factors. We studied both subfertility and the strongest known testicular cancer susceptibility gene, the c-KIT ligand (KITLG), whose pathway is involved in spermatogenesis.The EPSAM case-control study is comprised of testicular cancer patients from the Province of Turin, Italy, diagnosed between 1997 and 2008. The present analysis included 245 cases and 436 controls from EPSAM, who were aged 20 years or older at diagnosis/recruitment. The EPSAM questionnaire collected information on factors such as number of children, age at first attempt to conceive, duration of attempt to conceive, use of assisted reproduction techniques, physician-assigned diagnosis of infertility, number of siblings, and self-reported cryptorchidism. Genotyping of the KITLG single nucleotide polymorphism (SNP) rs995030 was performed on the saliva samples of 202 cases and 329 controls.Testicular cancer was associated with the number of children fathered 5 years before diagnosis (odds ratio (OR) per additional child: 0.78, 95% confidence interval (CI): 0.58-1.04) and sibship size (OR per additional sibling: 0.76, 95% CI: 0.66-0.88). When considering the reproductive history until 1 year before diagnosis, attempting to conceive for at least 12 months or fathering a child using assisted reproduction techniques was not associated with the risk of testicular cancer, nor was age at first attempt to conceive or physician-assigned diagnosis of infertility. The SNP rs995030 was strongly associated with risk of testicular cancer (per allele OR: 1.83; 95%CI: 1.26-2.64), but it did not modify the association between number of children and the risk of testicular cancer.This study supports the repeatedly reported inverse association between number of children and risk of testicular cancer, but it does not find evidence of an association for other indicators of subfertility

Aflibercept Plus FOLFIRI in the Real-life Setting: Safety and Quality of Life Data From the Italian Patient Cohort of the Aflibercept Safety and Quality-of-Life Program Study

Abstract Background Aflibercept combined with FOLFIRI (folinic acid, 5-fluorouracil, irinotecan) as second-line treatment of metastatic colorectal cancer (mCRC) significantly improved survival compared with FOLFIRI alone in the pivotal VELOUR (aflibercept vs. placebo in combination with irinotecan and 5-fluorouracil in the treatment of patients with metastatic colorectal cancer after failure of an oxaliplatin-based regimen) trial. No quality-of-life assessment was performed in VELOUR; therefore, the ASQoP (Aflibercept Safety and Quality-of-Life Program) trial was designed to capture the safety and health-related quality of life (HRQL). Patients and Methods ASQoP was an international, open-label, single-arm trial evaluating the safety and HRQL of aflibercept combined with FOLFIRI administered in a real-life setting to 781 patients with mCRC, pretreated with an oxaliplatin-based regimen with or without bevacizumab. The Italian subset of ASQoP enrolled 200 patients from 28 institutions. The primary endpoint was safety; HRQL was a secondary endpoint, assessed by validated questionnaires (European quality of life 5-dimension instrument 3-level; European Organization for Research and Treatment for Cancer Quality of Life Questionnaire Core 30, version 3; and EORTC-CR29) at baseline, during treatment, and at the end of treatment. Results The median age of the Italian ASQoP population was 63 years; the median number of aflibercept and FOLFIRI cycles was 7. Treatment-emergent adverse events were reported in 97.5% of patients. Hypertension (28.5%), neutropenia (27.5%; from laboratory data), asthenic conditions (20.0%), diarrhea (17.0%), and stomatitis (13.0%) were the most frequent (incidence, ≥ 5%) grade 3/4 toxicities. One toxic death occurred during the study period due to sepsis, without neutropenic complications. No significant worsening of HRQL was shown during treatment. Conclusion Aflibercept combined with FOLFIRI was well tolerated when administered as second-line treatment for patients with mCRC in a real-life setting. It did not affect HRQL and showed similar rates of treatment-emergent adverse events as those observed in the VELOUR trial. No new safety signals were identified

Quality of life, compliance, safety and effectiveness in fit older metastatic colorectal patients with cancer treated in first-line with chemotherapy plus cetuximab: A restrospective analysis from the ObservEr study

Abstract Objectives The influence of age ( KRAS wild type (WT) metastatic colorectal cancer (mCRC). Methods 225 patients of the Observed study (PS 0-1) were retrieved based on age ( Results The two patient groups (141  p  = 0.002), which is likely due to higher proportions of metastatic resection (27.0% vs 8.3%; p  = 0.001) and utilization of second-line therapy in younger group (58.9% vs 42.9%; p  = 0.028). Conclusion The current data suggest that fit older patients with mCRC can be safely treated with a cetuximab-based therapy, as QoL and safety profile do not seem to be affected by age. In addition, age did not impact the choice of chemotherapy to be associated to cetuximab and treatment compliance

Paracrine mechanisms of mesenchymal stem cells in tissue repair

Tissue regeneration from transplanted mesenchymal stromal cells (MSC) either through transdifferentiation or cell fusion was originally proposed as the principal mechanism underlying their therapeutic action. However, several studies have now shown that both these mechanisms are very inefficient. The low MSC engraftment rate documented in injured areas also refutes the hypothesis that MSC repair tissue damage by replacing cell loss with newly differentiated cells. Indeed, despite evidence of preferential homing of MSC to the site of myocardial ischemia, exogenously administered MSC show poor survival and do not persist in the infarcted area. Therefore, it has been proposed that the functional benefits observed after MSC transplantation in experimental models of tissue injury might be related to the secretion of soluble factors acting in a paracrine fashion. This hypothesis is supported by pre-clinical studies demonstrating equal or even improved organ function upon infusion of MSC-derived conditioned medium (MSC-CM) compared with MSC transplantation. Identifying key MSC-secreted factors and their functional role seems a reasonable approach for a rational design of nextgeneration MSC-based therapeutics. Here, we summarize the major findings regarding both different MSC-mediated paracrine actions and the identification of paracrine mediators

Testing the paracrine properties of human mesenchymal stem cells using conditioned medium

Mesenchymal stem cells (MSC) produce and secrete a great variety of cytokines and chemokines that play beneficial paracrine actions when MSC are used for tissue repair. The conditioned medium (CM) derived from MSC can be used both in vitro and in vivo to test specific paracrine effects or to screen putative paracrine/autocrine mediators by proteomics.In this chapter, we describe a straightforward method to prepare MSC-derived CM. Furthermore, we summarize some in vitro assays useful for testing the cytoprotective, angiogenic, and regenerative activity of CM. These assays are very helpful when studying the role of MSC in cardiac repair and regeneration

Optimized lentiviral transduction of human amniotic mesenchymal stromal cells

Mesenchymal stromal cells are excellent candidates for regenerative medicine since they are multipotent, easy to isolate, can be expanded to obtain clinically relevant numbers and are immunoprivileged. Stable genetic modification with viral vectors can improve mesenchymal stromal cell function and enhance their therapeutic potential. However, standard viral vectors achieve sub-optimal transduction efficiency with a single infection. On the other hand, multiple transduction cycles or antibiotic-based selection methods may alter the stem cell phenotype. We hypothesized that the use of lentiviral vectors containing specific regulatory sequences may result in improved transduction efficiency. Thus, we compared two types of third generation lentiviral vectors, one of which, the pLenti7.3 vector, contains the optimized sequences for Polypurine Tract and Woodchuck Post-transcriptional Regulatory Element. We demonstrated that with the pLenti7.3 it is possible to efficiently transduce human mesenchymal stromal cells with a single transduction cycle. Additionally, we successfully showed that by using the pLenti7.3 vector it is possible to efficiently over-express different growth factors, particularly relevant for cardiac protection and differentiation, in human mesenchymal stromal cells

Improved transduction efficiency of human amniotic mesenchymal stem cells using optimized lentiviral vectors

Background: Amniotic mesenchymal stem cells (A-MSC) are excellent candidates for regenerative medicine since they are multipotent, easy to isolate, expandable and seem to be immunoprivileged. In rodents, stable genetic modification with viral vectors improves A-MSC function. Unfortunately, a single cycle transduction with standard viral vectors does not achieve high efficiency in human A-MSC. On the other hand, multiple transduction cycles or antibiotic-based selection methods may alter the cell phenotype. We hypothesized that the use of lentiviral vectors containing specific regulatory sequences may result in improved transduction efficiency in human A-MSC. Accordingly, we compared two types of third generation lentiviral vectors, one of which containing the optimized sequences for Polypurine Tract (cPPT) and Woodchuck Posttranscriptional Regulatory Element (WPRE). Methods: Human A-MSC were isolated from amniotic membranes of human term placenta. The immunophenotype of the cells was determined by fluorescence-activated cell sorting (FACS). To confirm their mesenchymal origin, the ability of A-MSC to differentiate into adipocytes and osteocytes was tested by RT-PCR and cytochemistry. The production of both lentiviral vectors was carried out following standard protocols. The cPPT and WPRE sequences were cloned between the LTR sequences. It is known that the cPPT enhances the lentivirus production, whereas the WPRE stabilizes the unspliced RNAs during the transfection. In both vectors, a fluorescent tag (green fluorescent protein – GFP) and a gene encoding for a protein of interest (Insulin-like growth factor I – IGF-I) were also cloned. The number of GFP positive cells was quantified by FACS following a single cycle of transduction. Finally, the expression of IGF-I was assessed with Western blot analysis. Results: A-MSC were successfully isolated and displayed the antigen profile typical of bone marrow-derived MSC. Specifically, they were positive for CD90, CD105, CD73, HLA-ABC e CD117 and negative for HLA-DR, CD34, CD45, CD14, CD80, CD31 and CD133. Furthermore, A-MSC efficiently differentiated into osteocytes and adipocytes. In particular, at the end of the differentiation protocol into osteocytes, the A-MSC expressed the typical osteogenic genes secreted phosphoprotein 10, cathepsin K and bone sialoprotein and stained positive for Alkaline Phosphatase activity and Von Kossa. After the differentiation protocol into adipocytes, the A-MSC expressed the typical adipogenic genes adipocyte differentiation related protein and peroxisome proliferator-activated receptor gamma and stained positive for Oil Red-O. The transduction efficiency with the standard lentivirus was only 15%. Conversely, with the optimized vector we obtained transduction efficiency as high as 50%. Accordingly, the A-MSC transduced with the optimized vector expressed higher levels of IGF-I protein compared with the A-MSC genetically modified with the standard vector. Conclusions: We have demonstrated for the first time that, using lentiviral vectors containing the cPPT and WPRE sequences, it is possible to efficiently transduce human A-MSC with a single cycle of transduction

Synthetic extracellular matrix mimic hydrogel improves efficacy of mesenchymal stromal cell therapy for ischemic cardiomyopathy.

BACKGROUND: Mesenchymal stromal cells (MSC) repair infarcted hearts mainly through paracrine mechanisms. Low cell engraftment limits the release of soluble paracrine factors (SF) over time and, consequently, MSC efficacy. We tested whether a synthetic extracellular matrix mimic, a hydrogel containing heparin (H-HG), could ameliorate MSC engraftment and binding/release of SF, thus improving MSC therapy efficacy. METHODS AND RESULTS: In vitro, rat bone-marrow MSC (rBM-MSC) were seeded and grown into H-HG. Under normoxia, the hydrogel did not affect cell survival (rBM-MSC survival >90% at each time point tested); vice versa, under hypoxia the biomaterial resulted to be protective for the cells (p < .001 vs rBM-MSC alone). H-HG or control PEG hydrogels (HG) were incubated with VEGF or bFGF for binding/release quantification. Data showed significantly higher amount of VEGF and bFGF bound by H-HG compared with HG (p < .05) and a constant release over time. In vivo, myocardial infarction (MI) was induced in female Sprague Dawley rats by permanent coronary ligation. One week later, saline, rBM-MSC, H-HG or rBM-MSC/H-HG were injected in the infarct zone. The co-injection of rBM-MSC/H-HG into infarcted hearts significantly increased cardiac function. Importantly, we observed a significant gain in MSC engraftment, reduction of ventricular remodeling and stimulation of neo-vasculogenesis. We also documented higher amounts of several pro-angiogenic factors in hearts treated with rBM-MSC/H-HG. CONCLUSIONS: Our data show that H-HG increases MSC engraftment, efficiently fine tunes the paracrine MSC actions and improves cardiac function in infarcted rat hearts. STATEMENT OF SIGNIFICANCE: Transplantation of MSC is a promising treatment for ischemic heart disease, but low cell engraftment has so far limited its efficacy. The enzymatically degradable H-HG that we developed is able to increase MSC retention/engraftment and, at the same time, to fine-tune the paracrine effects mediated by the cells. Most importantly, the co-transplantation of MSC and H-HG in a rat model of ischemic cardiomyopathy improved heart function through a significant reduction in ventricular remodeling/scarring and amelioration in neo-vasculogenesis/endogenous cardiac regeneration. These beneficial effects are comparable to those obtained by others using a much greater number of cells, strengthening the efficacy of the biomaterial used in increasing the therapeutic effects of MSC. Given its efficacy and safety, documented by the absence of immunoreaction, our strategy appears readily translatable to clinical scenarios