Conditioned Medium From Human Amniotic Mesenchymal Stromal Cells Limits Infarct Size and Enhances Angiogenesis

The paracrine properties of human amniotic membrane-derived mesenchymal stromal cells (hAMCs) have not been fully elucidated. The goal of the present study was to elucidate whether hAMCs can exert beneficial paracrine effects on infarcted rat hearts, in particular through cardioprotection and angiogenesis. Moreover, we aimed to identify the putative active paracrine mediators. hAMCs were isolated, expanded, and characterized. In vitro, conditioned medium from hAMC (hAMC-CM) exhibited cytoprotective and proangiogenic properties. In vivo, injection of hAMC-CM into infarcted rat hearts limited the infarct size, reduced cardiomyocyte apoptosis and ventricular remodeling, and strongly promoted capillary formation at the infarct border zone. Gene array analysis led to the identification of 32 genes encoding for the secreted factors overexpressed by hAMCs. Among these, midkine and secreted protein acidic and rich in cysteine were also upregulated at the protein level. Furthermore, high amounts of several proangiogenic factors were detected in hAMC-CM by cytokine array. Our results strongly support the concept that the administration of hAMC-CM favors the repair process after acute myocardial infarction

Human 3D Cultures as Models for Evaluating Magnetic Nanoparticle CNS Cytotoxicity after Short- and Repeated Long-Term Exposure

Since nanoparticles (NPs) can translocate to the brain and impact the highly vulnerable central nervous system (CNS), novel in vitro tools for the assessment of NP-induced neurotoxicity are advocated. In this study, two types of CNS spheroids have been developed from human D384 astrocyte- and SH-SY5Y neuronal-like cells, and optimized in combination with standard assays (viability readout and cell morphology) to test neurotoxic effects caused by Fe3O4NPs, as NP-model, after short- (24–48 h; 1–100µg/ml) and long-term repeated exposure (30days; 0.1–25µg/ml). Short-term exposure of 3D-spheroids to Fe3O4NP induced cytotoxicity at 10 µg/mL in astrocytes and 25 µg/mL neurons. After long-term repeated dose regimen, spheroids showed concentration- and time-dependent cell mortality at 10 µg/mL for D384 and 0.5 µg/mL for SH-SY5Y, indicating a higher susceptibility of neurons than astrocytes. Both spheroid types displayed cell disaggregation after the first week of treatment at ≥0.1 µg/mL and becoming considerably evident at higher concentrations and over time. Recreating the 3D-spatial environment of the CNS allows cells to behave in vitro more closely to the in vivo situations, therefore providing a model that can be used as a stand-alone test or as a part of integrated testing strategies. These models could drive an improvement in the species-relevant predictivity of toxicity testing.JRC.F.3-Chemicals Safety and Alternative Method

Multiple human papillomavirus infection with or without type 16 and risk of cervical intraepithelial neoplasia among women with cervical cytological abnormalities

Purpose: To evaluate the impact of multiple human papillomavirus (HPV) infections on the risk of cervical intraepithelial neoplasia grade 3 or worse (CIN3+) in subjects with cervical cytological abnormalities. Methods: A cross-sectional study of 3,842 women attending a colposcopy service was carried out. Genotyping of 18 high-risk, seven low-risk, and two undefined-risk HPVs was carried out by the INNO-LiPA genotyping system. Results: The final colposcopic/pathological diagnoses were as follows: 1,933 (50.3 %) subjects were negative; 1,041 (27.1 %) CIN1; 280 (7.3 %) CIN2; 520 (13.5 %) CIN3; and 68 (1.8 %) invasive cervical cancer. The prevalence of HPV infection was 75.8 % (2,911/3,842), whereas multiple HPVs were detected in 34.5 % of HPV-positive subjects (2,255/3,842). The adjusted risks of CIN3+ in the group with multiple compared to the group with single infection were 2.31 (95 % CI = 1.54–3.47), among HPV16-positive women, and 3.25 (95 % CI = 2.29–4.61, p = 0.21 compared with HPV16-positive subjects), in HPV16-negative subjects. Out of a total of 1,285 subjects with mild lesions, followed up for a median of 16.1 months (interquartile range = 8.9–36.8), the rate of progression to CIN2–3 was 0.6 % (5/541) among subjects negative or with low-risk HPVs, 1.7 % (8/463) among those with single high-risk HPV, and 5 % (14/281, p < 0.001 compared with HPV-negative/low-risk HPV and p = 0.038 compared with single high-risk HPV) among those with multiple high-risk HPVs. Conclusions: Among women with cervical cytological abnormalities, infection by multiple high-risk HPVs increased the risk of CIN3+ in both HPV16-positive and HPV16-negative subjects. These findings suggest a potential synergistic interaction between high-risk HPVs, favoring the progression of CIN lesions

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

In Vitro Efficient Expansion of Tumor Cells Deriving from Different Types of Human Tumor Samples

Obtaining human tumor cell lines from fresh tumors is essential to advance our understanding of antitumor immune surveillance mechanisms and to develop new ex vivo strategies to generate an efficient anti-tumor response. The present study delineates a simple and rapid method for efficiently establishing primary cultures starting from tumor samples of different types, while maintaining the immuno-histochemical characteristics of the original tumor. We compared two different strategies to disaggregate tumor specimens. After short or long term in vitro expansion, cells analyzed for the presence of malignant cells demonstrated their neoplastic origin. Considering that tumor cells may be isolated in a closed system with high efficiency, we propose this methodology for the ex vivo expansion of tumor cells to be used to evaluate suitable new drugs or to generate tumor-specific cytotoxic T lymphocytes or vaccines

Pathologic Findings at Risk Reducing Surgery in <i>BRCA</i> and Non-<i>BRCA</i> Mutation Carriers: A Single-Center Experience

Risk-reducing surgery (RRS) is recommended in BRCA-mutated carriers because of their increased risk of developing ovarian cancer, while its role is still discussed for women harboring mutations in non-BRCA homologous repair genes. The aim of this study was to retrospectively evaluate the occurrence of pathological findings in a high-risk population undergoing RRS in San Matteo Hospital, Pavia between 2012 and 2022, and correlate their genetic and clinical outcomes, comparing them with a control group. The final cohort of 190 patients included 85 BRCA1, 63 BRCA2, 11 CHEK2, 7 PALB2, 4 ATM, 1 ERCC5, 1 RAD51C, 1 CDH1, 1 MEN1, 1 MLH1 gene mutation carriers and 15 patients with no known mutation but with strong familial risk. Occult invasive serous carcinoma (HGSC) and serous tubal intraepithelial carcinoma (STIC) were diagnosed in 12 (6.3%) women, all of them BRCA carriers. No neoplastic lesion was diagnosed in the non-BRCA group, in women with familial risk, or in the control group. Oral contraceptive use and age ≤45 at surgery were both found to be favorable factors. While p53 signature and serous tubal intraepithelial lesion (STIL) were also seen in the control group and in non-BRCA carriers, STIC and HGSC were only found in BRCA1/2 mutation carriers