The Expression of CD154 by Kaposi's Sarcoma Cells Mediates the Anti-Apoptotic and Migratory Effects of HIV-1-Tat Protein:

Kaposi's sarcoma (KS) is a malignancy associated to conditions of immune system impairment such as HIV-1 infection and post-transplantation therapy. Here we report that HIV-1-Tat protein, at concentrations well below those detected in AIDS patients, up-regulates the expression of both CD40 and CD154 on KS cells. This occurred also in the presence of vincristine, that at doses shown to induce apoptosis decreased the expression of both CD40 and CD154 on KS cells. The treatment with a soluble CD40-muIg fusion protein (CD40 fp) that prevents the binding of CD154 with cell surface CD40, as well as the transfection with a vector for soluble CD40 (KS sCD40), decreased the anti-apoptotic effect of Tat. Moreover, Tat-induced motility of KS cells was inhibited by soluble CD40 fp. Tat also enhanced the expression of intracellular proteins known to transduce signals triggered by CD40 engagement, in particular TRAF-3. Tat as well as soluble CD154 (sCD154) prevented vincristine-induced reduction of TRAF-3 in KS cells t..

Bull spermatozoa uptake of extracellular vesicles from bovine seminal plasma

Extracellular vesicles (EVs) are important mediators of intercellular communication by transferring microRNAs (miRNAs) that are able to repress translation of messenger RNA. Their presence in seminal plasma suggests a role on sperm fertility. It is already known that bull seminal plasma contains fertility-associated proteins that are predictive of high and low fertility [1]. In addition, in bull, a difference in miRNA content between high and low spermatozoa motility has been observed, which highlight the importance of the role played by EVs on fertility [2]. We hypothesize that co-incubation of sperm of low fertility bulls with EVs isolated from the seminal plasma of high fertility bulls could improve their fertility. Before testing this hypothesis, a preliminary work was carried out with the aim to investigate the presence and type of EVs in bovine seminal plasma and their interaction with spermatozoa. Ejaculates of eight Holstein bulls (4\u20136 years age) were collected weekly by artificial vagina. The ejaculates were centrifuged at 1600 g for 10 min to pellet spermatozoa and then centrifuged again at 2400 g for 30 min to eliminate cell debris and large vesicles. After centrifugation, the supernatants were collected, filtered twice (0.45 and 0.22 \ub5m) and stored at -80\ub0C. A double ultra-centrifugation at 100.000 g for 1 h was performed. Finally, the pellet was re-suspended in a small volume of TRIS buffer and kept at -80\ub0C until used. Three ejaculates of the same animal were pooled to detect concentration and size of EVs by Nanosight Instruments. To trace interaction with spermatozoa by fluorescence microscopy, EVs were labeled with PKH26 dye and a dose\u2013response curve in three replicates was performed. A suspension of 1x106 sp/ml was co-incubated with 200 or 400 x106 EVs labelled with pKH26 for 30, 60, 90, 120, 150 and 180 minutes at 38.5\ub0C. The end point of incubation was at 24h. Internalization of EVs was assessed by confocal microscopy. Our results showed that the size of EVs of all samples ranged from 145.1 to 187.7 nm, with an average of 166\ub129 nm. For all seminal plasma samples, the number of EVs ranged from 3.62 to 6.08x1013 particles/ml, with an average of 4.37\ub10.61x1013. Based on size, these EVs can be categorized as shedding vesicles. Confocal microscopy was set to take fluorescent images at different plans scanned every 0.12 \ub5m from top to bottom of the spermatozoa. Our results showed that no fluorescence signal was detectable after coincubation with 200x106 EVs. At the concentration of 400 x106 EVs, up to 60 minutes no signal was detectable while at 90\u2019 spermatozoa showed a fine granular fluorescent pattern within the intermediate portion. At 120\u2019 signal was within the acrosome and at 180\u2019 spermatozoa were stained for the whole length supposing a distribution of incorporated EVs throughout all the cell. At 24 h, the fluorescence signal decreased. In conclusion, this is the first report demonstrating that bull spermatozoa incorporate EVs from bull semen. We hypothesize a transfer of molecules, like miRNAs and other non coding RNA molecules, from EVs to spermatozoa probably involved in sperm fertility. [1] Killian et al. Biol Reprod 1993,49:1202. [2] Capra et al. BMC Genomics 2017,18:14

Human liver stem cell-derived microvesicles inhibit hepatoma growth in SCID mice by delivering antitumor microRNAs.

Microvesicles (MVs) play a pivotal role in cell-to-cell communication. Recent studies demonstrated that MVs may transfer genetic information between cells. Here, we show that MVs derived from human adult liver stem cells (HLSC) may reprogram in vitro HepG2 hepatoma and primary hepatocellular carcinoma cells by inhibiting their growth and survival. In vivo intratumor administration of MVs induced regression of ectopic tumors developed in SCID mice. We suggest that the mechanism of action is related to the delivery of microRNAs (miRNAs) from HLSC-derived MVs (MV-HLSC) to tumor cells on the basis of the following evidence: (a) the rapid, CD29-mediated internalization of MV-HLSC in HepG2 and the inhibition of tumor cell growth after MV uptake; (b) the transfer by MV-HLSC of miRNAs with potential antitumor activity that was downregulated in HepG2 cells with respect to normal hepatocytes; (c) the abrogation of the MV-HLSC antitumor effect after MV pretreatment with RNase or generation of MVs depleted of miRNAs; (d) the relevance of selected miRNAs was proven by transfecting HepG2 with miRNA mimics. The antitumor effect of MV-HLSC was also observed in tumors other than liver such as lymphoblastoma and glioblastoma. These results suggest that the delivery of selected miRNAs by MVs derived from stem cells may inhibit tumor growth and stimulate apoptosis

Distinct profile of CD34+ cells and plasma-derived extracellular vesicles from triple-negative patients with Myelofibrosis reveals potential markers of aggressive disease

Background: Myelofibrosis (MF) is a clonal disorder of hemopoietic stem/progenitor cells (HSPCs) with high prevalence in elderly patients and mutations in three driver genes (JAK2, MPL, or CALR). Around 10\u201315% of patients are triple-negative (TN) for the three driver mutations and display significantly worse survival. Circulating extracellular vesicles (EVs) play a role in intercellular signaling and are increased in inflammation and cancer. To identify a biomolecular signature of TN patients, we comparatively evaluated the circulating HSPCs and their functional interplay with the microenvironment focusing on EV analysis. Methods: Peripheral blood was collected from MF patients (n = 29; JAK2V617F mutation, n = 23; TN, n = 6) and healthy donors (HD, n = 10). Immunomagnetically isolated CD34+ cells were characterized by gene expression profiling analysis (GEP), survival, migration, and clonogenic ability. EVs were purified from platelet-poor plasma by ultracentrifugation, quantified using the Nanosight technology and phenotypically characterized by flow cytometry together with microRNA expression. Migration and survival of CD34+ cells from patients were also analyzed after in vitro treatments with selected inflammatory factors, i.e. (Interleukin (IL)-1\u3b2, Tumor Necrosis Factor (TNF)-\u3b1, IL6) or after co-culture with EVs from MF patients/HD. Results: The absolute numbers of circulating CD34+ cells were massively increased in TN patients. We found that TN CD34+ cells show in vitro defective functions and are unresponsive to the inflammatory microenvironment. Of note, the plasma levels of crucial inflammatory cytokines are mostly within the normal range in TN patients. Compared to JAK2V617F-mutated patients, the GEP of TN CD34+ cells revealed distinct signatures in key pathways such as survival, cell adhesion, and inflammation. Importantly, we observed the presence of mitochondrial components within plasma EVs and a distinct phenotype in TN-derived EVs compared to the JAK2V617F-mutated MF patients and HD counterparts. Notably, TN EVs promoted the survival of TN CD34+ cells. Along with a specific microRNA signature, the circulating EVs from TN patients are enriched with miR-361-5p. Conclusions: Distinct EV-driven signals from the microenvironment are capable to promote the TN malignant hemopoiesis and their further investigation paves the way toward novel therapeutic approaches for rare MF