Role of Exosomes in the Transfer of Viral Nucleic Acids to Recipients Cells: in Vitro Studies with Cell Line Supernatant and Patient-Derived Exosomes

Human PapillomaVirus (HPV) is a main cause of cervical cancer in which HPV DNA is frequently found integrated into human genome at fragile sites. Meta analytic studies reported association between HPV and Breast Cancer (Bae JM, 2016). We found 30% of HPV DNA in breast cancer tissues, confirmed by ISH assay. HPV DNA is also found in other extra-genital tissues, including oro-pharingeal, anal, colon and skin (Kim SM, 2016; Pérez LO, 2010). Owing to the lack of viremia, HPV DNA presence in other districts, is still a conundrum. We hypothesized that the presence of HPV DNA in extra-genital tissues could be in relation with exosomes. Exosomes are extracellular vesicles, involved in cellular communication, tumor progression and biological molecules carriers. We isolated exosomes from 59 serum specimens from breast cancer patients by differential ultracentrifugation and we identified 5 HPV DNA positive samples. Subsequently we isolated exosomes, from HeLa and Caski supernatants (HPV18 and HPV16 positive, respectively) and from HPV DNA positive serum, urine and liquor patient by CD9-immunobeads isolation kit. Exosomes obtained were used to perform viral transfer in different cell lines. Finally, the literature reports association between SV40 and certain types of cancer, including bone tumors (Vilchez RA, 2003). In line with literature, we found 7/70 SV40 DNA in bone tumors (unpublished). We isolated exosomes, from COS7 supernatant (SV40 positive cell line) and we tested acid nucleic viral transfer to osteosarcoma cell lines. Exposed cells were analyzed by PCR, Real-time PCR and Digital PCR, demonstrating the viral nucleic acids transfer in receiving cells. The viral nucleic acids discovery in receiving cells, confirm the role of exosomes as biological molecules carriers. The mechanism through which the viral content is released, is yet to be clarified, as well as if it is sufficient to trigger infection

Putative role of circulating human papillomavirus DNA in the development of primary squamous cell carcinoma of the middle rectum: a case report

Here we present the case of a patient affected by rectal squamous cell carcinoma in which we demonstrated the presence of Human Papillomavirus (HPV) by a variety of techniques. Collectively, the virus was detected not only in the tumor but also in some regional lymph nodes and in non-neoplastic mucosa of the upper tract of large bowel. By contrast, it was not identifiable in its common sites of entry, namely oral and ano-genital region. We also found HPV DNA in the plasma-derived exosome. Next, by in vitro studies, we confirmed the capability of HPV DNA-positive exosomes, isolated from the supernatant of a HPV DNA positive cell line (CaSki), to transfer its DNA to human colon cancer and normal cell lines. In the stroma nearby the tumor mass we were able to demonstrate the presence of virus DNA in the stromal compartment, supporting its potential to be transferred from epithelial cells to the stromal ones. Thus, this case report favors the notion that human papillomavirus DNA can be vehiculated by exosomes in the blood of neoplastic patients and that it can be transferred, at least in vitro, to normal and neoplastic cells. Furthermore, we showed the presence of viral DNA and RNA in pluripotent stem cells of non-tumor tissue, suggesting that after viral integration (as demonstrated by p16 and RNA in situ hybridization positivity), stem cells might have been activated into cancer stem cells inducing neoplastic transformation of normal tissue through the inactivation of p53, p21, and Rb. It is conceivable that the virus has elicited its oncogenic effect in this specific site and not elsewhere, despite its wide anatomical distribution in the patient, for a local condition of immune suppression, as demonstrated by the increase of T-regulatory (CD4/CD25/FOXP3 positive) and T-exhausted (CD8/PD-1positive) lymphocytes and the M2 polarization (high CD163/CD68 ratio) of macrophages in the neoplastic microenvironment. It is noteworthy that our findings depicted a static picture of a long-lasting dynamic process that might evolve in the development of tumors in other anatomical sites. Copyright © 2019 Ambrosio, Vernillo, De Carolis, Carducci, Mundo, Ginori, Rocca, Nardone, Lucenti Fei, Carfagno, Lazzi, Cricca and Tosi

Intrabone transplant provides full stemness of cord blood stem cells with fast hematopoietic recovery and low GVHD rate: results from a prospective study

Umbilical Cord Blood (UCB) represents a valid option for patients with hematopoietic malignancies lacking an HLA matched donor. To overcome the limitation of the low stem cell dose of UCB, the intrabone (IB) route has been proposed. We report the results of a prospective study on a poor-prognosis cohort of 23 patients receiving intrabone single UCB transplant (Clinicaltrials.gov NCT00886522). Cumulative incidence of hematological recovery at day 90 was 82 ± 9% (ANC > 0.5 × 109/L) and 70 ± 10% (platelet > 50 × 109/L) and correlated with CD34 + cells in the graft. NRM was 20 ±  9%. No severe aGVHD and only one extensive cGVHD occurred, with fast immune reconstitution. To test the hypothesis that the direct IB injection could affect the expression of stem cells regulatory pathways, CD34 + cells from BM aspirates at day + 10, + 20, + 30, processed in hypoxic conditions mimicking the BM-microenvironment (7%pO2), were studied for the expression of c-Mpl, Notch1 and CXCR4. We found that the expression of c-Mpl in CD34 + cells at day + 10 significantly correlated with hematological recovery. In conclusion, IB-UCB transplant success is associated with low incidence of GVHD and high-speed platelet recovery; intrabone route may preserve full hematopoietic stemness by direct delivery of UCB stem cells into the hypoxic HSC niche

Different fetal-neonatal outcomes in siblings born to a mother with Graves-Basedow disease after total thyroidectomy: a case series

ABSTRACT:INTRODUCTION: We describe three different fetal or neonatal outcomes in the offspring of a mother who had persistent circulating thyrotropin receptor antibodies despite having undergone a total thyroidectomy several years before. CASE PRESENTATION: The three different outcomes were an intrauterine death, a mild and transient fetal and neonatal hyperthyroidism and a severe fetal and neonatal hyperthyroidism that required specific therapy. CONCLUSIONS: The three cases are interesting because of the different outcomes, the absence of a direct correlation between thyrotropin receptor antibody levels and clinical signs, and the persistence of thyrotropin receptor antibodies several years after a total thyroidectomy

Changes in the biochemical taste of cytoplasmic and cell-free DNA are major fuels for inflamm-aging

Inflamm-aging depicts the progressive activation of the innate immune system that accompanies human aging. Its role as a disease-predisposing condition has been proposed, but its molecular basis is still poorly understood. A wealth of literature conveys that, particularly upon stress, nuclear and mitochondrial genomes are released into the cytoplasmic and extracellular compartments. Cytoplasmic (cy) and cell-free (cf) DNA pools trigger inflammation and innate immunity at local and systemic level. In particular, cyDNA plays a crucial role in the phenomenon of cell senescence and in the cognate pro-inflammatory secretome. Here we propose that changes in a variety of biochemical characteristics \u201ctastes\u201d of cy- and cf-DNA (e.g. the amount of 8-oxo-deoxy-guanosine and 5-methyl-deoxy-cytosine, the proportion of DNA hybridized with RNA) potentially affect the capability of these DNA pools to ignite the innate immune system. We also underpin that telomeric sequences are major components of the cy/cfDNA payload. Telomere shortening, a hallmark of aging, causes the depletion of telomeric sequences in cy/cfDNA pool, thus unleashing their potential to exert an age-related activation of the innate immune system. Finally, we posit that various sources of DNA (extracellular vesicles, the commensal metagenome and food) contribute to the cy/cfDNA payloads. We speculate that changes in the biochemical \u201ctaste\u201d of cy/cfDNA are major modifiers of inflamm-aging

Carbonic anhydrase 9 mRNA/microRNA34a interplay in hypoxic human mammospheres

The hypoxic environment is a crucial component of the cancer stem cell niche and it is capable of eliciting stem cell features in cancer cells. We previously reported that SNAI2 up-regulates the expression of Carbonic Anhydrase iso-enzyme 9 (CA9) in hypoxic MCF7 cells. Here we show that SNAI2 down-regulates miR34a expression in hypoxic MCF7 cell-derived mammospheres. Next, we report on the capability of miR34a to decrease CA9 mRNA stability and CA9 protein expression. We also convey that the over-expression of cloned CA9-mRNA-3'UTR increases the mRNA half-life and protein levels of two miR34a targets JAGGED1 and NOTCH3. The data here reported shows that the SNAI2-dependent down-regulation of miR34a substantially contributes to the post-transcriptional up-regulation of CA9, and that CA9-mRNA-3'UTR acts as an endogenous microRNA sponge. We conclude that CA9/miR34 interplay shares in the hypoxic regulation of mammospheres and therefore may play a relevant role in the hypoxic breast cancer stem cell niche. This article is protected by copyright. All rights reserved

Exosome-based immunomodulation during aging: a nano-perspective on inflamm-aging

Exosomes are nanovesicles formed by inward budding of endosomal membranes. They exert complex immunomodulatory effects on target cells, acting both as antigen-presenting vesicles and as shuttles for packets of information like proteins, coding and non-coding RNA, and nuclear and mitochondrial DNA fragments. Albeit different, all such functions seem to be encompassed in the adaptive mechanism mediating the complex interactions of the organism with a variety of stressors, providing both for defense and for the evolution of symbiotic relationships with others organisms (gut microbiota, bacteria, and viruses). Intriguingly, the newly deciphered human virome and exosome biogenesis seem to share some physical-chemical characteristics and molecular mechanisms. Exosomes are involved in immune system recognition of self from non-self throughout life: they are therefore ideal candidate to modulate inflamm-aging, the chronic, systemic, age-related pro-inflammatory status, which influence the development/progression of the most common age-related diseases (ARDs). Not surprisingly, recent evidence has documented exosomal alteration during aging and in association with ARDs, even though data in this field are still limited. Here, we review current knowledge on exosome-based trafficking between immune cells and self/non-self cells (i.e. the virome), sketching a nano-perspective on inflamm-aging and on the mechanisms involved in health maintenance throughout life

Ribosomal DNA instability: An evolutionary conserved fuel for inflammaging

Across eukaryotes, ribosomal DNA (rDNA) loci are characterized by intrinsic genomic instability due to their repetitive nature and their base composition that facilitate DNA double strand breaks and RNA:DNA hybrids formation. In the yeast, ribosomal DNA instability affects lifespan via the formation of extrachromosomal rDNA circles (ERC) that accrue into aged cells. In humans, rDNA instability has long been reported in a variety of progeric syndromes caused by the dysfunction of DNA helicases, but its role in physiological aging and longevity still needs to be clarified. Here we propose that rDNA instability leads to the activation of innate immunity and inflammation via the interaction with the cytoplasmic DNA sensing machinery. Owing to the recent clarified role of cytoplasmic DNA in the pro-inflammatory phenotype of senescent cells, we hypothesize that the accrual of rDNA derived molecules (i.e. ERC and RNA:DNA hybrids) may have a role in aging by contributing to inflammaging i.e. the systemic pro-inflammatory drift that associates with the onset of geriatric syndromes and age related dysfunctions in humans

5FU/Oxaliplatin-induced Jagged1 cleavage counteracts apoptosis induction in colorectal cancer: a novel mechanism of intrinsic drug resistance

Colorectal cancer (CRC) is characterized by early metastasis, resistance to anti-cancer therapy, and high mortality rate. Despite considerable progress in the development of new treatment options that improved survival benefits in patients with early-stage or advanced CRC, many patients relapse due to the activation of intrinsic or acquired chemoresistance mechanisms. Recently, we reported novel findings about the role of Jagged1 in CRC tumors with Kras signatures. We showed that Jagged1 is a novel proteolytic target of Kras signaling, which induces Jagged1 processing/activation resulting in Jag1-ICD release, which favors tumor development in vivo, through a non-canonical mechanism. Herein, we demonstrate that OXP and 5FU cause a strong accumulation of Jag1-ICD oncogene, through ERK1/2 activation, unveiling a surviving subpopulation with an enforced Jag1-ICD expression, presenting the ability to counteract OXP/5FU-induced apoptosis. Remarkably, we also clarify the clinical ineffectiveness of gamma-secretase inhibitors (GSIs) in metastatic CRC (mCRC) patients. Indeed, we show that GSI compounds trigger Jag1-ICD release, which promotes cellular growth and EMT processes, functioning as tumor-promoting agents in CRC cells overexpressing Jagged1. We finally demonstrate that Jagged1 silencing in OXP- or 5FU-resistant subpopulations is enough to restore the sensitivity to chemotherapy, confirming that drug sensitivity/resistance is Jag1-ICD-dependent, suggesting Jagged1 as a molecular predictive marker for the outcome of chemotherapy

DNA VIRAL INFECTIONS ASSAY IN KIDNEY TRANSPLANT RECIPIENTS BY A NEW HIGH THROUGHPUT MASS SPECTROMETRY METHOD

Introduction: Infections represent a major complication after renal transplant with an important impact on allograft survival and outcome. Polyomaviruses (PyVs), a group of small and circular dsDNA viruses, mediate a broad spectrum of diseases in immune-compromised patients. NF-jB (nuclear factor kappalight-chain-enhancer of activated B cells) is a key regulator of immune and inflammatory responses and the -94ins/delATTG (rs28362491) polymorphism in the gene promoter has been widely investigated for clinical associations. To date, rs28362491 has been shown to influence the susceptibility to systemic lupus erythematosus, rheumatoid arthritis, inflammatory bowel disease and recently renal transplant rejection. Materials and Methods: We developed a high throughput mass spectrometry (MS)-based method to detect rs28362491 and 18 Py vs. types. Primer pairs of MS assay were designed within the specific large T antigen genes. Viral and human DNAs were extracted from blood samples of 43 kidney transplant recipients, before and after transplantation. Results: We analysed the correlation among Py vs. infections, rs28362491 genotype and post-transplant follow up. Five out of the 18 viral types tested were found in the specimens analysed: BKV, JCV, Merkel cell PyV, Human PyV6 and SV12. In our cohort, 14 patients showed SV12 infection: 10 cases were -94ins/-94ins, 4 were -94ins/-94del. All the patients with the NF-kB-94del/- 94del genotype were characterized by the absence of SV12 strain. No correlation between genotype and viral infection was observed for the other viral types. Conclusions: Our MS assay improved the Py vs. typing and allowed to drive towards the identification of novel biomarkers for the infective management of transplanted patients. The genetic background might modulate the viral infection susceptibility in renal transplant recipients