Regulations of IRF5 activity in the TLR7 and type I IFN pathways

IRF5 is a member of the Interferon Regulatory Factor (IRF) family of transcription factors activated downstream of the Toll-Like receptors (TLRs). Following activation, IRF5 translocates into the nucleus where it regulates transcription of proinflammatory cytokines and type I IFN. Polymorphisms in IRF5 have been shown to be associated with the autoimmune disease Systemic Lupus Erythematosus (SLE) and other autoimmune conditions, suggesting a central role for IRF5 in the regulation of the immune response. Four different IRF5 isoforms are generated by alternative splicing and by the presence or absence of a 30 nucleotide insertion in IRF5 exon 6 tha t is included in the risk haplotype for SLE. Since the polymorphic region disturbs a PEST domain, we hypothesized that the isoforms bearing the insertion could have increased stability, thus explaining the association of IRF5 with SLE. IRF family members IRF3, IRF7 and IRF8 have been shown to be targeted for proteasomal-mediated degradation by the E3 ubiquitin ligase TRIM21 following activation. In addition, two separate studies have shown that IRF5 and TRIM21 interact. In this study we therefore investigated whether IRF5 is subjected to regulation by TRIM21 and whether dysregulation of this mechanism could explain the association of IRF5 with SLE. We have thus been able to show that IRF5 is degraded following TLR7 activation and that TRIM21 is involved in this process, while confocal analysis has shown colocalization of IRF5 isoforms and TRIM21 in cytoplasmic vesicular and filamentous structures. Comparison of the individual IRF5 variants has shown that isoforms generated by alternative splicing are resistant to TRIM21-mediated degradation following TLR7 stimulation, while the presence of the 30 nucleotide insertion does not influence stability. Similarly, alternately spliced (thus more stable) isoforms present a lower degree of colocalization in TRIM21- containing vesicles following TLR7 stimulation, suggesting a possible role for these structures in protein degradation. Interestingly however, we have been able to show a possible role for TRIM21 in inhibition of TLR7-mediated nuclear translocation of these alternatively spliced isoforms, thus providing an additional mechanism for negative regulation of IRF5-mediated signaling in addition to regulation of stability. Taken together, these results indicate that alteration of splicing mechanisms in SLE, which would result in expression of IRF5 isoforms with increased stability, coupled to impaired TRIM21 function may account fo r increased activity of IRF5 in SLE patients, resulting in increased expression of proinflammatory cytokines and type I IFN characteristic of the disease. We next focused on investigating novel mechanisms of regulation of IRF5 transcriptional activity and we have been able to show that IRF5 is phosphorylated on tyrosine residues following TLR7 and IFNa stimulation. In the TLR7 pathway we could identify Src as the effector tyrosine kinase involved in IRF5 phosphorylation. In the type I IFN pathway we investigated in detail the possible role fo r the IFNARassociated kinase TYK2, since polymorphisms in TYK2 have been shown to be associated with SLE and to have additive effects with polymorphisms in IRF5. We have thus been able to show that IRF5 is a target of TYK2 and that TYK2-mediated phosphorylation of IRF5 can influence its transcriptional activity with opposite effects on different promoters. In conclusion, we have shown in this work novel mechanisms for regulation of IRF5 activity in the TLR7 pathway. In particular, by showing that IRF5 is negatively regulated by TRIM21, and since TRIM21 activity is dysregulated in SLE, we have provided a functional, albeit indirect, link between IRF5 and lupus. We have also provided the first evidence of a possible molecular link between IRF5 and TYK2, thus far only suggested by genetic interaction, and demonstrated a possible role fo r IRF5 in the type I IFN response. Given the pathogenic role of type I IFN in SLE, the evidence of IRF5 activation in this pathway may therefore provide additional insight on the role of IRF5 in lupus

Extensive Characterization of Platelet Gel Releasate From Cord Blood in Regenerative Medicine.

Platelet gel derived from peripheral blood is widely applied in many clinical fields of surgery as biomaterial containing growth factors with high proliferative properties. In 2010, we studied and patented a platelet gel derived from cord blood. In this study, due to the crucial role of the factors released by the platelet gel, we first extended the characterization of its releasate. Using a wide proteomic array and splitting the two components of the releasate, that is, platelets and plasma, we have been able to study their growth factor content. Interestingly, we discovered high levels of hormones and molecules able to support tissue growth in the cord blood platelet gel releasate and, in addition, higher concentrations of several angiogenic factors if compared with the peripheral blood counterpart. On the contrary, the latter was much richer in inflammatory factors. The second aim of our work was to study the effects on cell culture, immunophenotype, and function of mesenchymal stem cells exposed to these two platelet gel releasates as substitute for the animal serum. Since our findings nicely show that the use of the peripheral versus the cord blood platelet gel releasate can differently influence the mesenchymal stem cell commitment, we can suggest that in addition to its peculiar angiogenic properties cord blood platelet gel releasate shows excellent proliferative properties as cell culture supplement

A Survey of Telecardiology Projects in Italy.

It is estimated that in Italy there are about three million people affected by chronic heart failure. Cardiology is the health care field currently getting the largest benefits from telemedicine. Transmission, using wireless devices, makes possible to achieve virtual hospitalization: it is possible to anticipate the time of discharging and the patient can be remotely controlled by the central station in the ICU of the department of Medicine. Teleconsulting (i.e. a distance consulting between physicians) is applied in telecardiology, it allows the realization of a consulting between cardiology departments and remote services in the same hospital or among far-away hospitals. In this paper some of the most significant cardiac telemonitoring projects in Italy are described. Also reported, the projects involving the applications of implantable cardiac devices which can be controlled remotely. In conclusion, we sketch out the future prospects of telecardiology research and its applications in Italy

Tracking of Normal and Malignant Progenitor Cell Cycle Transit in a Defined Niche.

While implicated in therapeutic resistance, malignant progenitor cell cycle kinetics have been difficult to quantify in real-time. We developed an efficient lentiviral bicistronic fluorescent, ubiquitination-based cell cycle indicator reporter (Fucci2BL) to image live single progenitors on a defined niche coupled with cell cycle gene expression analysis. We have identified key differences in cell cycle regulatory gene expression and transit times between normal and chronic myeloid leukemia progenitors that may inform cancer stem cell eradication strategies

A Survey of Telecardiology Projects in Italy

It is estimated that in Italy there are about three million people affected by chronic heart failure. Cardiology is the health care field currently getting the largest benefits from telemedicine. Transmission, using wireless devices, makes possible to achieve virtual hospitalization: it is possible to anticipate the time of discharging and the patient can be remotely controlled by the central station in the ICU of the department of Medicine. Teleconsulting (i.e. a distance consulting between physicians) is applied in telecardiology, it allows the realization of a consulting between cardiology departments and remote services in the same hospital or among far-away hospitals. In this paper some of the most significant cardiac telemonitoring projects in Italy are described. Also reported, the projects involving the applications of implantable cardiac devices which can be controlled remotely. In conclusion, we sketch out the future prospects of telecardiology research and its applications in Italy

A Chemically Defined Medium-Based Strategy to Efficiently Generate Clinically Relevant Cord Blood Mesenchymal Stromal Colonies.

During the last decade it has been demonstrated that mesenchymal progenitors are present and can be isolated also from cord blood (CB). Recently, we managed to set up a standard protocol allowing the isolation of mesenchymal stromal cells (MSCs) with high proliferative potential and multiple differentiation capabilities, whereas the generation rate of MSC-initiating colonies could still be further improved. Herein, we strikingly succeeded in defining some simple and basic culture conditions based on the use of a chemically defined medium that increased the colony isolation efficiency up to almost 80% of processed CB units. Importantly, this result was achieved irrespective of CB unit white blood cell content and time elapsed from delivery, two limiting parameters involved with processing CB units. Thus, this high efficiency is guaranteed without strict selection of the starting material. In addition, since we are profoundly concerned about how different culture conditions can influence cell behavior, we devoted part of this study to in-depth characterization of the established CB-MSC populations to confirm their stemness features in this novel isolation and culture system. Therefore, an extended study of their immunophenotype, including classical pericytic markers, and a detailed molecular analysis addressing telomere length and also stemness-related microRNA contribution were performed. In summary, we propose a straightforward, extremely efficient, and reliable approach to isolate and expand thoroughly characterized CB-MSCs, even when poor-quality CB units are the only available source, or there is no space for an isolation to fail

Avoidable blindness and value based healthcare: more value with a population approach

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