Gene targeting in adult rhesus macaque fibroblasts

<p>Abstract</p> <p>Background</p> <p>Gene targeting in nonhuman primates has the potential to produce critical animal models for translational studies related to human diseases. Successful gene targeting in fibroblasts followed by somatic cell nuclear transfer (SCNT) has been achieved in several species of large mammals but not yet in primates. Our goal was to establish the protocols necessary to achieve gene targeting in primary culture of adult rhesus macaque fibroblasts as a first step in creating nonhuman primate models of genetic disease using nuclear transfer technology.</p> <p>Results</p> <p>A primary culture of adult male fibroblasts was transfected with hTERT to overcome senescence and allow long term <it>in vitro </it>manipulations. Successful gene targeting of the HPRT locus in rhesus macaques was achieved by electroporating S-phase synchronized cells with a construct containing a SV40 enhancer.</p> <p>Conclusion</p> <p>The cell lines reported here could be used for the production of null mutant rhesus macaque models of human genetic disease using SCNT technology. In addition, given the close evolutionary relationship and biological similarity between rhesus macaques and humans, the protocols described here may prove useful in the genetic engineering of human somatic cells.</p

Gene-enhanced tissue engineering for dental hard tissue regeneration: (1) overview and practical considerations

Gene-based therapies for tissue regeneration involve delivering a specific gene to a target tissue with the goal of changing the phenotype or protein expression profile of the recipient cell; the ultimate goal being to form specific tissues required for regeneration. One of the principal advantages of this approach is that it provides for a sustained delivery of physiologic levels of the growth factor of interest. This manuscript will review the principals of gene-enhanced tissue engineering and the techniques of introducing DNA into cells. Part 2 will review recent advances in gene-based therapies for dental hard tissue regeneration, specifically as it pertains to dentin regeneration/pulp capping and periodontal regeneration

P1 receptors and cytokine secretion

Evidence has accumulated in the last three decades to suggest tissue protection and regeneration by adenosine in multiple different cell types. Adenosine produced in hypoxic or inflamed environments reduces tissue injury and promotes repair by receptor-mediated mechanisms. Among other actions, regulation of cytokine production and secretion by immune cells, astrocytes and microglia (the brain immunocytes) has emerged as a main mechanism at the basis of adenosine effects in diseases characterized by a marked inflammatory component. Many recent studies have highlighted that signalling through A1 and A2A adenosine receptors can powerfully prevent the release of pro-inflammatory cytokines, thus inhibiting inflammation and reperfusion injury. However, the activation of adenosine receptors is not invariably protective of tissues, as signalling through the A2B adenosine receptor has been linked to pro-inflammatory actions which are, at least in part, mediated by increased release of pro-inflammatory cytokines from epithelial cells, astrocytes and fibroblasts. Here, we discuss the multiple actions of P1 receptors on cytokine secretion, by analyzing, in particular, the role of the various adenosine receptor subtypes, the complex reciprocal interplay between the adenosine and the cytokine systems, their pathophysiological significance and the potential of adenosine receptor ligands as new anti-inflammatory agents

Surveillance for severe acute respiratory infections in the 2019/2020 season in Tuscany, Italy

Influenza is a major public health burden. In Italy there were 7.6 million symptomatic case of influenza in the 2019/2020 influenza season. In Italy, the influenza season lasts from October to April of the following year. We analysed influenza A and B viruses from hospitalized patients with Severe Acute Respiratory Infections (SARI) to carry out epidemiological and virological surveillance

Homocysteine and Raynaud’s phenomenon: a review.

Raynaud's phenomenon, categorized as primary and secondary when occurring isolated or in association with an underlying disease, respectively, is a paroxysmal and recurrent acral ischemia resulting from an abnormal arterial vasospastic response to cold or emotional stress. The key issue in the pathogenesis of Raynaud's phenomenon is presumed to be a dysregulation in the mechanisms of vascular motility resulting in an imbalance between vasodilatation and vasoconstriction. Homocysteine, a non-protein forming sulphured amino acid proposed as an independent risk factor for atherothrombosis in the general population, clearly demonstrated to produce vascular damage through mechanisms also including endothelial injury and modifications in circulating mediators of vasomotion. The rationale for homocysteine involvement in the pathogenesis of Raynaud's phenomenon led some authors to investigate the possible association between mild hyperhomocysteinemia and such a vascular disturbance, particularly in the course of connective tissue disease. Here we review data regarding this putative association and the supposed mechanisms involved, also discussing the emblematic case of a patient with new-onset severe Raynaud's phenomenon and markedly elevated homocysteinemia

Spotlight on sirukumab for the treatment of rheumatoid arthritis: the evidence to date

Pietro Enea Lazzerini,1 Pier Leopoldo Capecchi,1 Giacomo Maria Guidelli,1 Enrico Selvi,1 Maurizio Acampa,2 Franco Laghi-Pasini1 1Department of Medical Sciences, Surgery and Neurosciences, University of Siena, 2Stroke Unit, University Hospital of&nbsp;Siena, Siena, Italy Abstract: Rheumatoid arthritis (RA) is a chronic autoimmune inflammatory disease primarily affecting synovial joints and is characterized by persistent high-grade systemic inflammation. Proinflammatory cytokines, particularly interleukin-6 (IL-6), are of crucial importance in the pathogenesis of the disease, driving both joint inflammation and extra-articular comorbidities. Tocilizumab, a humanized IL-6 receptor-inhibiting monoclonal antibody, has been the first, and, to date, the only, IL-6 inhibitor approved for the treatment of RA. Many studies have demonstrated the potency and effectiveness of tocilizumab in controlling disease activity and radiological progression of RA. These successful results have encouraged the development of novel IL-6 inhibitors, among which a promising agent is sirukumab (SRK), a human anti-IL-6 monoclonal antibody currently under evaluation in Phase II/III studies in patients with RA, systemic lupus erythematosus, giant-cell arteritis, and major depressive disorder. The evidence to date indicates SRK as an effective and well-tolerated new therapeutic tool for patients with active RA, with some preliminary data suggesting a specific beneficial impact on relevant systemic complications associated with the disease, such as depression and cardiovascular disease. Conversely, although pathophysiological considerations make plausible the hypothesis that IL-6 blockade with SRK may also be beneficial in the treatment of many diseases other than RA (either autoimmune or not), available clinical data in patients with systemic lupus erythematosus do not seem to support this view, also giving rise to potentially relevant concerns about drug safety. If large Phase III clinical trials currently in progress in patients with RA confirm the efficacy and tolerability of SRK, then in the long term, this drug could, in the near future, occupy a place in the treatment of the disease, potentially also opening the doors to a more extended use of SRK in a wide range of disorders in which IL-6 plays a key pathogenic role. Keywords: sirukumab, rheumatoid arthritis, interleukin-6, tocilizumab, systemic lupus erythematosus, cardiovascular disease, interleukin-

Enhanced apoptosis of peripheral blood mononuclear cells (PBMC) in cardiac transplanted patients undergoing chronic immunosuppressive treatment

Apoptosis plays a major role in tissue transplantation because intact T-cell-apoptosis pathways are required for the induction of tolerance to allografts. Moreover, immunosuppressive agents commonly used in clinical transplantation medicine promote lymphocyte apoptosis inhibiting the expression and production of cytokines involved in lymphocyte survival. The aim of our study was to evaluate peripheral blood mononuclear cells (PBMC) spontaneous apoptosis in patients undergoing chronic immunosuppressive treatment after cardiac transplantation. PBMC obtained from patients (n = 31) and controls matched for age and sex (n = 25) were cultured for 72 h and apoptosis was evaluated by quantification of fragmented DNA, staining with Hoechst 33258 dye and annexin V binding. We also investigated Fas expression and FasL mRNA expression as well as the ability of an IgM anti-Fas antibody to induce apoptosis. Finally, we evaluated IL2 production induced by PHA and the ability of IL2 to prevent apoptosis. In patients, PBMC underwent enhanced spontaneous apoptosis in comparison with controls. However, we could not find any difference between patients and normals as regards the expression of Fas and of FasL mRNA, even if the cross-linking of the Fas molecule induced apoptosis in PBMC from patients, whereas it failed to induce cell death in normals. We also found that IL2 production was significantly decreased in patients and that the addition of IL2 to the culture medium reduced PBMC spontaneous apoptosis. Our findings suggest that in cardiac transplanted patients PBMC undergo enhanced spontaneous apoptosis, which may contribute to prevent allograft rejection