Introduction: Liquid biopsy using circulating microvesicles and exosomes is emerging as a new diagnostic tool that could improve hepatocellular carcinoma (HCC) early diagnosis and screening protocols. Our study aimed to investigate the utility of plasma exosomal miR-21-5p and miR-92-3p for HCC diagnosis during screening protocols. Methods: The study group included 106 subjects: 48 patients diagnosed with HCC during screening, who underwent a potentially curative treatment (surgical resection or liver transplantation), 38 patients with liver cirrhosis (LC) on the waiting list for liver transplantation, and 20 healthy volunteers. The exosomes were isolated by precipitation with a reagent based on polyethylene glycol and were characterized based on morphological aspects (i.e., diameter); molecular weight; CD63, CD9, and CD81 protein markers; and exosomal miR-21-5p and miR-92a-3p expression levels. Results: We first demonstrate that the exosome population isolated with the commercially available Total Exosome Isolation kit respects the same size ranging, morphological, and protein expression aspects compared to the traditional ultracentrifugation technique. The analysis of the expression profile indicates that miR-21-5p was upregulated (p = 0.017), and miR-92a-3p was downregulated (p = 0.0005) in plasma-derived exosomes from HCC subjects, independently from the patient's characteristics. AUROC for HCC diagnosis based on AFP (alpha-fetoprotein) was 0.72. By integrating AFP and the relative expression of exosomal miR-21-5p and miR-92a-3p in a logistic regression equation for HCC diagnosis, the combined AUROC of the new exosomal miR HCC score was 0.85-significantly better than serum AFP alone (p = 0.0007).

We characterized human amniotic fluid stem cells (AFSC) in senescent cultures (6 weeks) versus cryopreserved cells using whole-cell patch-clamp, immunophenotyping, and differential gene expression profiling for senescence genes. We evidenced five ion current components (outward rectifier, A-type, inward rectifier, and big conductance Ca-dependent K currents, fast voltage-dependent Na currents). Senescent AFSC showed reduced expression of CD90, CD44, CD133, over 500-fold increase of interferon gamma and telomerase reverse transcriptase genes, increased cycle-dependent kinase 4 inhibitors, p53-binding protein 1, and decreased calreticulin and CD44. HLA-ABC immune expression was similar, and HLA-DR expression very low in both cell types. A subset of cryopreserved AFSC featured large inward rectifier K currents, voltage-dependent Na currents, and neural progenitor markers evidenced by immunophenotyping and RT-PCR. In all AFSC, in both culture conditions, at patch rupture the outward currents were very low, and they increased progressively over several minutes upon cytoplasm dialysis with pipette solution.

The aim of the study was to evidence replicative senescence‐induced changes in human amniocytes via flow cytometry, quantitative reverse‐transcription‐polymerase chain reaction (qRT‐PCR) and automated/manual patch‐clamp. Both cryopreserved and senescent amniocytes cultured in BIO‐AMF‐2 medium featured high percentages of pluripotency cell surface antigens SSEA‐1, SSEA‐4, TRA1‐60, TRA1‐81 (assessed by flow cytometry) and expression of pluripotency markers Oct4 (Pou5f1) and Nanog (by qRT‐PCR). We demonstrated in senescent vs cryopreserved amniocytes decreases in mesenchymal stem cell surface markers. Senescence‐associated β‐galactosidase stained only senescent amniocytes, and they showed no deoxyuridine incorporation. The gene expression profile revealed a secretory phenotype of senescent amniocytes (increased interleukin (IL)‐1α, IL‐6, IL‐8, transforming growth factor β, nuclear factor κB p65 expression), increases for cell cycle‐regulating genes (p16INK4A), cytoskeletal elements (β‐actin); HMGB1, c‐Myc, Bcl‐2 showed reduced changes and p21, MDM2 decreased. Via patch‐clamp we identified five ion current components: outward rectifier K+ current, an inactivatable component, big conductance Ca2+‐dependent K+ channels (BK) current fluctuations, Na+ current, and inward rectifier K+ current. Iberiotoxin 100 nmol/L blocked 71% of BK fluctuations, and lidocaine 200 μmol/L exerted use‐dependent Na+ current block. Transient receptor potential (TRP)M7‐like current density at −120 mV was significantly increased in senescent amniocytes. The proinflammatory profile acquired by senescent amniocytes in vitro may prevent their use in clinical therapies for immunosuppression, antiapoptotic and healing effects.

Our study suggests a need for detecting the carriers. This is the first step for the construction of a national database and provides information for health planners and policy makers to help them in planning programs and allocation resources. The molecular testing was well received by pregnant women and appears to be feasible and highly acceptable.

The present study focuses on the application of a therapeutic strategy in patients with chronic severe lower limb ischaemia using a plasmid vector encoding the vascular endothelial growth factor (phVEGF165). It has been shown that VEGF promotes neo-vascularization and blood vessel network formation and thus might have the ability to improve blood-flow at the level of the affected limbs. However, little information is available regarding the necessary level of expression of VEGF and its possible related adverse effects. We have subcloned VEGF 165isoform into pCMV-Script expression vector (Stratagene) under the control of the CMV promoter. Three patients with chronic ischaemia of the lower limb, considered as not suitable for surgical re-vascularization, received intramuscular injection with 0.5 ml saline solution containing 1011 copies of VEGF 165 plasmid. The clinical evolution has been monitored by angiography and estimated by walking time on the rolling carpet (Gardner protocol). Two months after therapy, all three patients showed complete relief of rest pain, improvement of ischaemic ulcer lesions and increased walking distance on the rolling carpet most probably due to appearance of newly formed collateral vessels.

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Fas ligand (First apoptosis signal ligand, FasL, also known as CD95L) is the common executioner of apoptosis within the tumor necrosis factor (TNF) superfamily. We aimed to induce functional FasL expression in transduced cells using an adenovirus vector, which has the advantage of strong and transient induction of the gene included in the adenoviral genome. Here, we report that the adenovirus carrying a truncated FasL gene, named FasL minigene, encoding the full-length FasL protein (Ad-gFasL) is more efficient than the adenovirus carrying FasL cDNA (Ad-cFasL) in the induction of FasL expression in transduced cells. FasL minigene (2887 bp) lacking the second intron and a part of the 3′-UTR was created to reduce the gene length due to the size limitation of the adenoviral genome. The results show that, in transduced hepatocytes, strong expression of mRNA FasL appeared after 10 h for Ad-gFasL, while for Ad-cFasL, a faint expression appeared after 16 h. For Ad-gFasL, the protein expression was noticed starting with 0.5 transfection units (TU)/cell, while for Ad-cFasL, it could not be revealed. FasL-expressing endothelial cells induced apoptosis of A20 cells in co-culture experiments. FasL-expressing cells may be exploitable in various autoimmune diseases such as graft-versus-host disease, chronic colitis, and type I diabetes.

Three bacterial species pertaining to the red complex (Porphyromonas gingivalis, Treponema denticola, and Tannerella forsythia) and Aggregatibacter actinomycetemcomitans were investigated in relation to the incidence and severity of periodontal disease. A total of 259 patients were included in this study, 179 being diagnosed with periodontal disease. The gingival crevicular fluid samples were obtained from periodontal pockets and the presence and levels of target bacteria were assessed following DNA extraction and real-time quantitative PCR.Our results account for significant positive associations between the number of bacterial species from the red complex coexisting within a patient and several clinical signs (gingival bleeding, inflammation and bone deterioration). A similar positive association was found between bacterial load of the red complex species and the clinical Case Type diagnostic of the periodontal disease, as well as the probing depth with the most evident results for T. denticola. In conclusion, our study, a first for the Romanian population, confirms previous results found elsewhere and finds a possible regional pathogenic specificity for T. denticola as a major factor for periodontitis severity.