An Integrated Analytical Approach for the Characterization of Probiotic Strains in Food Supplements

Research surrounding health benefits from probiotics is becoming popular because of the increasing demand for safer products with protective and therapeutic effects. Proven benefits are species- or genus-specific; however, no certified assays are available for their characterization and quantification at the strain level in the food supplement industry. The objective of this study was to develop a strain-specific Real-time quantitative polymerase chain reaction (RT-qPCR)-based method to be implemented in routine tests for the identification and quantification of Bifidobacterium longum, Bifidobacterium animalis spp. lactis, Lactobacillus paracasei, Lactobacillus rhamnosus, Lactobacillus casei, Bifidobacterium breve, Lactobacillus acidophilus, Lactobacillus plantarum, and Lactobacillus helveticus, starting from a powder mixture of food supplements. The method optimization was carried out in combination with flow cytometry to compare results between the two strategies and implement the analytical workflow with the information also regarding cell viability. These assays were validated in accordance with the International Council for Harmonization of Technical Requirements for Pharmaceuticals for Human Use (ICH) criteria using the plate count enumeration as the gold standard reference. Briefly, probiotic DNAs were extracted from two powder food supplements. Strain-specific primers targeting unique sequence regions of 16S RNA were identified and amplified by RT-qPCR. Primers were tested for specificity, sensitivity, and efficiency. Both RT-qPCR and flow-cytometry methods described in our work for the quantification and identification of Lactobacillus and Bifidobacterium strains were specific, sensitive, and precise, showing better performances with respect to the morphological colony identification. This work demonstrated that RT-qPCR can be implemented in the quality control workflow of commercial probiotic products giving more standardized and effective results regarding species discrimination

Neonatal respiratory distress syndrome: are risk factors the same in preterm and term infants?

Objective: To analyze respiratory distress syndrome (RDS) incidence and risk factors at different gestational age. Methods: We considered data from 321 327 infants born in Lombardy, a Northern Italian Region. We computed multivariate analysis to identify risk factors for RDS by dividing infants in early- and moderate-preterm, late-preterm and term infants. Results: Low-birth weight is the main risk factor for RDS, with higher odds ratio in term births. The risk was higher in infants delivered by cesarean section and in male, for all gestational age. Pathological course of pregnancy resulted in increased risk only in late-preterm and term infants. Maternal age and multiple birth were not associated with increased risk in any group. Babies born at term after assisted conception were at higher risk of RDS. Conclusion: Our analysis suggests as some risk factors do not influence RDS incidence in the same way at different gestational age

Incidence and Risk Factors for Venous Thromboembolism in Female Patients Undergoing Breast Surgery

Albeit it does not have the highest venous thromboembolism (VTE) incidence compared to other neoplasms, breast cancer contributes to many VTE events because it is the most diagnosed tumor in women. We aim to analyze the occurrence and timing of VTE during the follow-up of patients who underwent breast surgery, the possible correlated factors, and the overall survival. This retrospective study included all female patients diagnosed with mammary pathology and surgically treated in our clinic between January 2002 and January 2012. Of 5039 women who underwent breast surgery, 1056 were found to have no evidence of malignancy, whereas 3983 were diagnosed with breast cancer. VTE rate resulted significantly higher in patients with invasive breast cancer than in women with benign breast disease or carcinoma in situ. Invasive cancers other than lobular or ductal were associated with a higher VTE rate. In addition, chronic hypertension, high BMI, cancer type, and evidence of metastasis turned out to be the most significant risk factors for VTE in women who underwent breast surgery. Moreover, VTE occurrence significantly impacted survival in invasive breast cancer patients. Compared to women with benign mammary pathology, VTE prevalence in women with breast cancer is significantly higher. The knowledge about the risk factors of VTE could be helpful as prognostic information, but also to eventually target preventive treatment strategies for VTE, as far as the co-existence of invasive breast cancer and VTE has a significantly negative impact on survival

Human Adipose-Derived Stem Cells in Madelung's Disease: Morphological and Functional Characterization

Madelung Disease (MD) is a syndrome characterized by the accumulation of aberrant symmetric adipose tissue deposits. The etiology of this disease is yet to be elucidated, even though the presence of comorbidities, either genetic or environmental, has been reported. For this reason, establishing an in vitro model for MD is considered crucial to get insights into its physiopathology. We previously established a protocol for isolation and culture of stem cells from diseased tissues. Therefore, we isolated human adipose-derived stem cells (ASC) from MD patients and compared these cells with those isolated from healthy subjects in terms of surface phenotype, growth kinetic, adipogenic differentiation potential, and molecular alterations. Moreover, we evaluated the ability of the MD-ASC secretome to affect healthy ASC. The results reported a difference in the growth kinetic and surface markers of MD-ASC compared to healthy ASC but not in adipogenic differentiation. The most commonly described mitochondrial mutations were not observed. Still, MD-ASC secretome was able to shift the healthy ASC phenotype to an MD phenotype. This work provides evidence of the possibility of exploiting a patient-based in vitro model for better understanding MD pathophysiology, possibly favoring the development of novel target therapies

Clinical relevance of the combined analysis of circulating tumor cells and anti-tumor T-cell immunity in metastatic breast cancer patients

Background: Metastatic breast cancer (mBC) is a heterogeneous disease with varying responses to treatments and clinical outcomes, still requiring the identification of reliable predictive biomarkers. In this context, liquid biopsy has emerged as a powerful tool to assess in real-time the evolving landscape of cancer, which is both orchestrated by the metastatic process and immune-surveillance mechanisms. Thus, we investigated circulating tumor cells (CTCs) coupled with peripheral T-cell immunity to uncover their potential clinical relevance in mBC. Methods: A cohort of 20 mBC patients was evaluated, before and one month after starting therapy, through the following liquid biopsy approaches: CTCs enumerated by a metabolism-based assay, T-cell responses against tumor-associated antigens (TAA) characterized by interferon-γ enzyme-linked immunosorbent spot (ELISpot), and the T-cell receptor (TCR) repertoire investigated by a targeted next-generation sequencing technique. TCR repertoire features were characterized by the Morisita’s overlap and the Productive Simpson Clonality indexes, and the TCR richness. Differences between groups were calculated by Fisher’s, Mann-Whitney or Kruskal-Wallis test, as appropriate. Prognostic data analysis was estimated by Kaplan-Meier method. Results: Stratifying patients for their prognostic level of 6 CTCs before therapy, TAA specific T-cell responses were detected only in patients with a low CTC level. By analyzing the TCR repertoire, the highest TCR clonality was observed in the case of CTCs under the cut-off and a positive ELISpot response (p=0.03). Whereas, at follow-up, patients showing a good clinical response coupled with a low number of CTCs were characterized by the most elevated TCR clonality (p<0.05). The detection of CTCs≥6 in at least one time-point was associated with a lower TCR clonality (p=0.02). Intriguingly, by combining overall survival analysis with TCR repertoire, we highlighted a potential prognostic role of the TCR clonality measured at follow-up (p=0.03). Conclusion: These data, whether validated in a larger cohort of patients, suggest that the combined analysis of CTCs and circulating anti-tumor T-cell immunity could represent a valuable immune-oncological biomarker for the liquid biopsy field. The clinical application of this promising tool could improve the management of mBC patients, especially in the setting of immunotherapy, a rising approach for BC treatment requiring reliable predictive biomarkers

Application of an artificial intelligence algorithm to prognostically stratify grade II gliomas

(1) Background: Recently, it has been shown that the extent of resection (EOR) and molecular classification of low-grade gliomas (LGGs) are endowed with prognostic significance. However, a prognostic stratification of patients able to give specific weight to the single parameters able to predict prognosis is still missing. Here, we adopt classic statistics and an artificial intelligence algorithm to define a multiparametric prognostic stratification of grade II glioma patients. (2) Methods: 241 adults who underwent surgery for a supratentorial LGG were included. Clinical, neuroradiological, surgical, histopathological and molecular data were assessed for their ability to predict overall survival (OS), progression-free survival (PFS), and malignant progression-free survival (MPFS). Finally, a decision-tree algorithm was employed to stratify patients. (3) Results: Classic statistics confirmed EOR, pre-operative-and post-operative tumor volumes, Ki67, and the molecular classification as independent predictors of OS, PFS, and MPFS. The decision tree approach provided an algorithm capable of identifying prognostic factors and defining both the cut-off levels and the hierarchy to be used in order to delineate specific prognostic classes with high positive predictive value. Key results were the superior role of EOR on that of molecular class, the importance of second surgery, and the role of different prognostic factors within the three molecular classes. (4) Conclusions: This study proposes a stratification of LGG patients based on the different combinations of clinical, molecular, and imaging data, adopting a supervised non-parametric learning method. If validated in independent case studies, the clinical utility of this innovative stratification approach might be proved

Dysmetabolic circulating tumor cells are prognostic in metastatic breast cancer

Circulating tumor cells (CTCs) belong to a heterogeneous pool of rare cells, and a unequivocal phenotypic definition of CTC is lacking. Here, we present a definition of metabolically-altered CTC (MBA-CTCs) as CD45-negative cells with an increased extracellular acidification rate, detected with a single-cell droplet microfluidic technique. We tested the prognostic value of MBA-CTCs in 31 metastatic breast cancer patients before starting a new systemic therapy (T0) and 3\u20134 weeks after (T1), comparing results with a parallel FDA-approved CellSearch (CS) approach. An increased level of MBA-CTCs was associated with: I) a shorter median PFS pre-therapy (123 days vs. 306; p < 0.0001) and during therapy (139 vs. 266 days; p = 0.0009); ii) a worse OS pre-therapy (p = 0.0003, 82% survival vs. 20%) and during therapy (p = 0.0301, 67% survival vs. 38%); iii) good agreement with therapy response (kappa = 0.685). The trend of MBA-CTCs over time (combining data at T0 and T1) added information with respect to separate evaluation of T0 and T1. The combined results of the two assays (MBA and CS) increased stratification accuracy, while correlation between MBA and CS was not significant, suggesting that the two assays are detecting different CTC subsets. In conclusion, this study suggests that MBA allows detection of both EpCAM-negative and EpCAM-positive, viable and label-free CTCs, which provide clinical information apparently equivalent and complementary to CS. A further validation of proposed method and cut-offs is needed in a larger, separate study

Predicting lymphoma development in patients with Sjögren's syndrome

ABSTRACTIntroduction: The issue of predicting lymphoma in primary Sjogren's syndrome (pSS) starts from its clinical and biologic essence, i.e., an autoimmune exocrinopathy with sicca syndrome, infl..

Accurate prediction of response to endocrine therapy in breast cancer patients: current and future biomarkers

WOS: 000390900700001PubMed ID: 27903276Approximately 70% of patients have breast cancers that are oestrogen receptor alpha positive (ER+) and are therefore candidates for endocrine treatment. Many of these patients relapse in the years during or following completion of adjuvant endocrine therapy. Thus, many ER+ cancers have primary resistance or develop resistance to endocrine therapy during treatment. Recent improvements in our understanding of how tumours evolve during treatment with endocrine agents have identified both changes in gene expression and mutational profiles, in the primary cancer as well as in circulating tumour cells. Analysing these changes has the potential to improve the prediction of which specific patients will respond to endocrine treatment. Serially profiled biopsies during treatment in the neoadjuvant setting offer promise for accurate and early prediction of response to both current and novel drugs and allow investigation of mechanisms of resistance. In addition, recent advances in monitoring tumour evolution through non-invasive (liquid) sampling of circulating tumour cells and cell-free tumour DNA may provide a method to detect resistant clones and allow implementation of personalized treatments for metastatic breast cancer patients. This review summarises current and future biomarkers and signatures for predicting response to endocrine treatment, and discusses the potential for using approved drugs and novel agents to improve outcomes. Increased prediction accuracy is likely to require sequential sampling, utilising preoperative or neoadjuvant treatment and/or liquid biopsies and an improved understanding of both the dynamics and heterogeneity of breast cancer.European CommissionEuropean Commission Joint Research Centre [658170]This work was funded by the European Commission H2020 Marie Sklodowska Curie Action Individual Fellowship (H2020-MSCA-IF, 658170) to CS and Breast Cancer Now to JMD and AHS

Exploring the Adsorption Properties of Zeolite in a New Skin Care Formulation

Introduction: Zeolites are natural or synthetic aluminosilicates, characterized by a regular and microporous crystalline structure that plays a particularly active role in neutralizing free radicals, screening UV rays and in the adsorption of toxins and heavy metals. Skin is one of the main areas for the accumulation of toxic substances released by environmental pollutants. The biological scavenger activity of zeolite opens a wide spectrum of applications in cosmetics and dermatology. Up to now, there is little evidence related to the use of natural zeolite in cosmetics. Aim: The purpose of this work was to evaluate the ability of zeolite to retain heavy metals in a new skin care formulation, in order to provide a proof of principle of its employment in the field of cosmetics. Materials and Methods: Taking the advantages of spiked samples, we studied the in vitro adsorption properties of zeolite in a new skin care formulation. The removal capacities of Cadmium, Lead, Chromium, Nickel and Cobalt were studied, using the inductively coupled plasma optical emission spectrometry (ICP-OES). First of all, the better concentration of zeolite was defined, testing two different proportions of zeolite, from 1% to 3%, keeping all other components constant. Then, on the 3% formulation, the adsorption properties of each single metal were measured. Results and Conclusions: Our preliminary study demonstrated the selectivity of zeolite in retaining Cadmium (p < 0.0001), Nickel (p = 0.026), in a 3% zeolite-based formulation. This work provides a proof of principle of zeolite employment in the field of cosmetics. Based on the data collected, our work provides a scientific proof of principle of zeolite employment in the field of cosmetics. New and extensive research will be needed to explore all the potential benefits of zeolite