Mirna reference genes in extracellular vesicles released from amniotic membrane-derived mesenchymal stromal cells

Human amniotic membrane and amniotic membrane-derived mesenchymal stromal cells (hAMSCs) have produced promising results in regenerative medicine, especially for the treatment of inflammatory-based diseases and for different injuries including those in the orthopedic field such as tendon disorders. hAMSCs have been proposed to exert their anti-inflammatory and healing potential via secreted factors, both free and conveyed within extracellular vesicles (EVs). In particular, EV miRNAs are considered privileged players due to their impact on target cells and tissues, and their future use as therapeutic molecules is being intensely investigated. In this view, EV-miRNA quantification in either research or future clinical products has emerged as a crucial paradigm, although, to date, largely unsolved due to lack of reliable reference genes (RGs). In this study, a panel of thirteen putative miRNA RGs (let-7a-5p, miR-16-5p, miR-22-5p, miR-23a-3p, miR-26a-5p, miR-29a-5p, miR-101-3p, miR-103a-3p, miR-221-3p, miR-423-5p, miR-425-5p, miR-660-5p and U6 snRNA) that were identified in different EV types was assessed in hAMSC-EVs. A validated experimental pipeline was followed, sifting the output of four largely accepted algorithms for RG prediction (geNorm, NormFinder, BestKeeper and \u394Ct method). Out of nine RGs constitutively expressed across all EV isolates, miR-101-3p and miR-22-5p resulted in the most stable RGs, whereas miR-423-5p and U6 snRNA performed poorly. miR-22-5p was also previously reported to be a reliable RG in adipose-derived MSC-EVs, suggesting its suitability across samples isolated from different MSC types. Further, to shed light on the impact of incorrect RG choice, the level of five tendon-related miRNAs (miR-29a-3p, miR-135a-5p, miR-146a-5p, miR-337-3p, let-7d-5p) was compared among hAMSC-EVs isolates. The use of miR-423-5p and U6 snRNA did not allow a correct quantification of miRNA incorporation in EVs, leading to less accurate fingerprinting and, if used for potency prediction, misleading indication of the most appropriate clinical batch. These results emphasize the crucial importance of RG choice for EV-miRNAs in hAMSCs studies and contribute to the identification of reliable RGs such as miR-101-3p and miR-22-5p to be validated in other MSC-EVs related fields

B Lymphocytes as Targets of the Immunomodulatory Properties of Human Amniotic Mesenchymal Stromal Cells

Mesenchymal stromal cells (MSC) from the amniotic membrane of human term placenta (hAMSC), and the conditioned medium generated from their culture (CM-hAMSC) offer significant tools for their use in regenerative medicine mainly due to their immunomodulatory properties. Interestingly, hAMSC and their CM have been successfully exploited in preclinical disease models of inflammatory and autoimmune diseases where depletion or modulation of B cells have been indicated as an effective treatment, such as inflammatory bowel disease, lung fibrosis, would healing, collagen-induced arthritis, and multiple sclerosis. While the interactions between hAMSC or CM-hAMSC and T lymphocytes, monocytes, dendritic cells, and macrophages has been extensively explored, how they affect B lymphocytes remains unclear. Considering that B cells are key players in the adaptive immune response and are a central component of different diseases, in this study we investigated the in vitro properties of hAMSC and CM-hAMSC on B cells. We provide evidence that both hAMSC and CM-hAMSC strongly suppressed CpG-activated B-cell proliferation. Moreover, CM-hAMSC blocked B-cell differentiation, with an increase of the proportion of mature B cells, and a reduction of antibody secreting cell formation. We observed the strong inhibition of B cell terminal differentiation into CD138+ plasma cells, as further shown by a significant decrease of the expression of interferon regulatory factor 4 (IRF-4), PR/SET domain 1(PRDM1), and X-box binding protein 1 (XBP-1) genes. Our results point out that the mechanism by which CM-hAMSC impacts B cell proliferation and differentiation is mediated by secreted factors, and prostanoids are partially involved in these actions. Factors contained in the CM-hAMSC decreased the CpG-uptake sensors (CD205, CD14, and TLR9), suggesting that B cell stimulation was affected early on. CM-hAMSC also decreased the expression of interleukin-1 receptor-associated kinase (IRAK)-4, consequently inhibiting the entire CpG-induced downstream signaling pathway. Overall, these findings add insight into the mechanism of action of hAMSC and CM-hAMSC and are useful to better design their potential therapeutic application in B-cell mediated diseases

HCV genome-wide genetic analyses in context of disease progression and hepatocellular carcinoma

<div><p>Hepatitis C virus (HCV) is a major cause of hepatitis and hepatocellular carcinoma (HCC) world-wide. Most HCV patients have relatively stable disease, but approximately 25% have progressive disease that often terminates in liver failure or HCC. HCV is highly variable genetically, with seven genotypes and multiple subtypes per genotype. This variation affects HCV’s sensitivity to antiviral therapy and has been implicated to contribute to differences in disease. We sequenced the complete viral coding capacity for 107 HCV genotype 1 isolates to determine whether genetic variation between independent HCV isolates is associated with the rate of disease progression or development of HCC. Consensus sequences were determined by sequencing RT-PCR products from serum or plasma. Positions of amino acid conservation, amino acid diversity patterns, selection pressures, and genome-wide patterns of amino acid covariance were assessed in context of the clinical phenotypes. A few positions were found where the amino acid distributions or degree of positive selection differed between in the HCC and cirrhotic sequences. All other assessments of viral genetic variation and HCC failed to yield significant associations. Sequences from patients with slow disease progression were under a greater degree of positive selection than sequences from rapid progressors, but all other analyses comparing HCV from rapid and slow disease progressors were statistically insignificant. The failure to observe distinct sequence differences associated with disease progression or HCC employing methods that previously revealed strong associations with the outcome of interferon α-based therapy implies that variable ability of HCV to modulate interferon responses is not a dominant cause for differential pathology among HCV patients. This lack of significant associations also implies that host and/or environmental factors are the major causes of differential disease presentation in HCV patients.</p></div

Editorial: MSC-Derived Extracellular Vesicles and Secreted Factors as “Cell-Free” Therapeutic Alternatives in Regenerative Medicine

No abstract availabl

Emergence of a HER2-amplified clone during disease progression in an ALK-rearranged NSCLC patient treated with ALK-inhibitors: A case report

Anaplastic lymphoma kinase tyrosine kinase inhibitors (ALK-TKIs) are the standard treatment for advanced ALK-positive non-small cell lung cancer (NSCLC) allowing survivals up to 5 years. However, duration of responses is limited by the almost certain occurrence of drug resistance. Here, we report a case of a never smoker, 59-year-old female with metastatic ALK-positive adenocarcinoma, solid and signet ring patterns, who developed resistance to alectinib, a second-generation ALK-TKI, mediated by HER2 gene amplification. The patient received 22 months of crizotinib as first-line and subsequently 1-year of alectinib therapy. A study of resistance mechanism was performed with next generation sequencing (NGS) on tissue re-biopsy. A HER2-amplified emerging clone was identified by NGS in a liver metastasis and confirmed by fluorescent in situ hybridization (FISH) analysis. The resistant clone was detectable 2 months before disease progression in plasma cell-free DNA (cfDNA) using digital droplet PCR (ddPCR) copy number variation (CNV) assay and it was retrospectively traced in rare cells of the lung primary by FISH. To our best knowledge, this is first evidence of HER2 gene amplification as a resistance mechanism to ALK-TKI in a NSCLC. Future strategies against oncogene-addicted NSCLC might benefit of combined drug treatments, such as ALK and HER2 inhibition

Correlations between tumor-infiltrating and circulating lymphocyte subpopulations in advanced renal cancer patients treated with nivolumab

Background: In clinical trials with immunotherapy, histological features such as tumor-infiltrating lymphocytes (TILs) are investigated as potential predictive biomarkers, with the limit of an outdated parameter for a typically dynamic element. Methods: This explorative study compared, in metastatic renal cell carcinoma (mRCC) patients, basal pathological data about TILs on diagnostic histological specimens with circulating lymphocyte subpopulations measured before and during therapy with nivolumab. Results: Of 11 mRCC patients, 5 had low presence of TILs (L-TILs), 3 moderate amount (M-TILs) and 3 high number (H-TILs). Overall, 8 patients had low intratumoral pathological CD4+/CD8+ ratio (LIPR) ≤1 and 3 cases high intratumoral pathological ratio (HIPR) ≥2. Of 8 patients with LIPR, only 2 matched with low circulating CD4+/CD8+ ratio (LCR) ≤1; 5 had high circulating ratio (HCR) ≥2. All 3 cases with HIPR (≥2) conversely had LCR (≤1). Circulating CD4+/CD8+ ratio remained unchanged during therapy (mean-0.12 in 8 weeks). The respective percentage values of CD4+ and CD8+ circulating T cells also remained stable (variation 0%); the absolute value of CD4+ was more likely to increase (mean +46.3/mm3); the level of CD8+ tended to slightly decrease (mean-6.5/mm3). No correlation of lymphocyte subpopulations with treatment outcome was found. Of note, we did not evidence correspondence between histopathological and circulating findings in terms of T-lymphocyte subpopulations, also suggesting the inconsistency of circulating data in terms of relative variations. Conclusions: Considering the likely high dynamism of TILs, rebiopsy before therapy might be proposed to assess the utility of TILs characterization for predictive purpose. (www.actabiomedica.it)

Amniotic MSCs reduce pulmonary fibrosis by hampering lung B-cell recruitment, retention, and maturation

Growing evidence suggests a mechanistic link between inflammation and the development and progression of fibrotic processes. Mesenchymal stromal cells derived from the human amniotic membrane (hAMSCs), which display marked immunomodulatory properties, have been shown to reduce bleomycin-induced lung fibrosis in mice, possibly by creating a microenvironment able to limit the evolution of chronic inflammation to fibrosis. However, the ability of hAMSCs to modulate immune cells involved in bleomycin-induced pulmonary inflammation has yet to be elucidated. Herein, we conducted a longitudinal study of the effects of hAMSCs on alveolar and lung immune cell populations upon bleomycin challenge. Immune cells collected through bronchoalveolar lavage were examined by flow cytometry, and lung tissues were used to study gene expression of markers associated with different immune cell types. We observed that hAMSCs increased lung expression of T regulatory cell marker Foxp3, increased macrophage polarization toward an anti-inflammatory phenotype (M2), and reduced the antigen-presentation potential of macrophages and dendritic cells. For the first time, we demonstrate that hAMSCs markedly reduce pulmonary B-cell recruitment, retention, and maturation, and counteract the formation and expansion of intrapulmonary lymphoid aggregates. Thus, hAMSCs may hamper the self-maintaining inflammatory condition promoted by B cells that continuously act as antigen presenting cells for proximal T lymphocytes in injured lungs. By modulating B-cell response, hAMSCs may contribute to blunting of the chronicization of lung inflammatory processes with a consequent reduction of the progression of the fibrotic lesion