Exploring the Immunomodulatory Moonlighting Activities of Acute Phase Proteins for Tolerogenic Dendritic Cell Generation

The acute phase response is generated by an overwhelming immune-inflammatory process against infection or tissue damage, and represents the initial response of the organism in an attempt to return to homeostasis. It is mediated by acute phase proteins (APPs), an assortment of highly conserved plasma reactants of seemingly different functions that, however, share a common protective role from injury. Recent studies have suggested a crosstalk between several APPs and the mononuclear phagocyte system (MPS) in the resolution of inflammation, to restore tissue integrity and function. In fact, monocyte-derived dendritic cells (Mo-DCs), an integral component of the MPS, play a fundamental role both in the regulation of antigen-specific adaptive responses and in the development of immunologic memory and tolerance, particularly in inflammatory settings. Due to their high plasticity, Mo-DCs can be modeled in vitro toward a tolerogenic phenotype for the treatment of aberrant immune-inflammatory conditions such as autoimmune diseases and allotransplantation, with the phenotypic outcome of these cells depending on the immunomodulatory agent employed. Yet, recent immunotherapy trials have emphasized the drawbacks and challenges facing tolerogenic Mo-DC generation for clinical use, such as reduced therapeutic efficacy and limited in vivo stability of the tolerogenic activity. In this review, we will underline the potential relevance and advantages of APPs for tolerogenic DC production with respect to currently employed immunomodulatory/immunosuppressant compounds. A further understanding of the mechanisms of action underlying the moonlighting immunomodulatory activities exhibited by several APPs over DCs could lead to more efficacious, safe, and stable protocols for precision tolerogenic immunotherapy

Engraftment Potential of Adipose Tissue-Derived Human Mesenchymal Stem Cells After Transplantation in the Fetal Rabbit

Due to their favorable intrinsic features, including engraftment, differentiation, and immunomodulatory potential, adult mesenchymal stem cells (MSCs) have been proposed for therapeutic in utero intervention. Further improvement of such attributes for particular diseases might merely be achieved by ex vivo MSC genetic engineering previous to transplantation. Here, we evaluated for the first time the feasibility, biodistribution, long-term engraftment, and transgenic enhanced green fluorescent protein (EGFP) expression of genetically engineered human adipose tissue-derived MSCs (EGFP+-ASCs) after intra-amniotic xenotransplantation at E17 of gestation into our validated pregnant rabbit model. Overall, the procedure was safe (86.4% survival rate; absence of anatomical defects). Stable, low-level engraftment of EGFP+-ASCs was confirmed by assessing the presence of the pWT-EGFP lentiviral provirus in the young transplanted rabbit tissues. Accordingly, similar frequencies of provirus-positive animals were found at both 8 weeks (60%) and 16 weeks (66.7%) after in utero intervention. The presence of EGFP+-ASCs was more frequent in respiratory epithelia (lung and trachea), according to the route of administration. However, we were unable to detect EGFP expression, neither by real-time polymerase chain reaction nor by immunohistochemistry, in the provirus-positive tissues, suggesting EGFP transgene silencing mediated by epigenetic events. Moreover, we noticed lack of both host cellular immune responses against xenogeneic ASCs and humoral immune responses against transgenic EGFP. Therefore, the fetal microchimerism achieved by the EGFP+-ASCs in the young rabbit hosts indicates induction of donor-specific tolerance after fetal rabbit xenotransplantation, which should boost postnatal transplantation for the early treatment/prevention of many devastating congenital disorders

Human Mesenchymal Stem Cells Resolve Airway Inflammation, Hyperreactivity, And Histopathology In A Mouse Model Of Occupational Asthma

Occupational asthma (OA) is characterized by allergic airway inflammation and hyperresponsiveness, leading to progressive airway remodeling and a concomitant decline in lung function. The management of OA remains suboptimal in clinical practice. Thus, establishing effective therapies might overcome the natural history of the disease. We evaluated the ability of human adipose-tissue-derived mesenchymal stem cells (hASCs), either unmodified or engineered to secrete the IL-33 decoy receptor sST2, to attenuate the inflammatory and respiratory symptoms in a previously validated mouse model of OA to ammonium persulfate (AP). Twenty-four hours after a dermal AP sensitization and intranasal challenge regimen, the animals received intravenously 1 x 10(6) cells (either hASCs or hASCs overexpressing sST2) or saline and were analyzed at 1, 3, and 6 days after treatment. The infused hASCs induced an anti-inflammatory and restorative program upon reaching the AP-injured, asthmatic lungs, leading to early reduction of neutrophilic inflammation and total IgE production, preserved alveolar architecture with nearly absent lymphoplasmacytic infiltrates, negligible smooth muscle hyperplasia/hypertrophy in the peribronchiolar areas, and baseline airway hyperreactivity (AHR) to methacholine. Local sST2 overexpression barely increased the substantial efficacy displayed by unmodified hASCs. Thus, hASCs may represent a viable multiaction therapeutic capable to adequately respond to the AP-injured lung environment by resolving inflammation, tissue remodeling, and bronchial hyperresponsiveness typical of OA

Usefulness of circulating microRNAs for the prediction of early preeclampsia at first-trimester of pregnancy

To assess the usefulness of circulating microRNAs (miRNAs) as non-invasive molecular biomarkers for early prediction of preeclampsia, a differential miRNA profiling analysis was performed in first-trimester pooled sera from 31 early preeclampsia patients, requiring delivery before 34 weeks of gestation, and 44 uncomplicated pregnancies using microfluidic arrays. Among a total of 754 miRNAs analyzed, the presence of 63 miRNAs (8%) was consistently documented in the sera from preeclampsia and control samples. Nevertheless, only 15 amplified miRNAs (2%) seemed to be differentially, although modestly, represented (fold change range: 0.4-1.4). After stem loop RT-qPCR from individual samples, the statistical analysis confirmed that none of the most consistent and differentially represented miRNAs (3 overrepresented and 4 underrepresented) were differentially abundant in serum from preeclamptic pregnancies compared with serum from normal pregnancies. Therefore, maternal serum miRNA assessment at first-trimester of pregnancy does not appear to have any predictive value for early preeclampsia

CD40 gene silencing reduces the progression of experimental lupus nephritis modulating local milieu and systemic mechanisms

Lupus nephritis (LN) is an autoimmune disorder in which co-stimulatory signals have been involved. Here we tested a cholesterol-conjugated-anti-CD40-siRNA in dendritic cells (DC) in vitro and in a model of LPS to check its potency and tissue distribution. Then, we report the effects of Chol-siRNA in an experimental model of mice with established lupus nephritis. Our in vitro studies in DC show a 100%intracellular delivery of Chol-siRNA, with a significant reduction in CD40 after LPS stimuli. In vivo in ICR mice, the CD40-mRNA suppressive effects of our Chol-siRNA on renal tissue were remarkably sustained over a 5 days after a single preliminary dose of Chol-siRNA. The intra-peritoneal administration of Chol-siRNA to NZB/WF1 mice resulted in a reduction of anti-DNA antibody titers, and histopathological renal scores as compared to untreated animals. The higher dose of Chol-siRNA prevented the progression of proteinuria as effectively as cyclophosphamide, whereas the lower dose was as effective as CTLA4. Chol-siRNA markedly reduced insterstitialCD3+ and plasma cell infiltrates as well as glomerular deposits of IgG and C3. Circulating soluble CD40 and activated splenic lymphocyte subsets were also strikingly reduced by Chol-siRNA. Our data show the potency of our compound for the therapeutic use of anti-CD40-siRNA in human LN and other autoimmune disorders

Regression of advanced diabetic nephropathy by hepatocyte growth factor gene therapy in rats

Diabetic nephropathy is the main cause of end-stage renal disease requiring dialysis in developed countries. In this study, we demonstrated the therapeutic effect of hepatocyte growth factor (HGF) on advanced rather than early diabetic nephropathy using a rat model of streptozotocin-induced diabetes. Early diabetic nephropathy (16 weeks after induction of diabetes) was characterized by albuminuria, hyperfiltration, and glomerular hypertrophy, whereas advanced diabetic nephropathy showed prominent transforming growth factor (TGF)-beta1 upregulation, mesangial expansion, and glomerulosclerosis. An SP1017-formulated human HGF (hHGF) plasmid was administered by intramuscular injection combined with electroporation over a 30-day follow-up in rats with early and advanced diabetic nephropathy. hHGF gene therapy upregulated endogenous rat HGF in the diabetic kidney (rat HGF by RT-PCR was threefold higher than in diabetic rats without therapy). hHGF gene therapy did not improve functional or morphologic abnormalities in early diabetic nephropathy. hHGF gene therapy reduced albuminuria and induced strong regression of mesangial expansion and glomerulosclerosis in advanced diabetic nephropathy. These findings were associated with suppression of renal TGF-beta1 and mesangial connective tissue growth factor (CTGF) upregulation, inhibition of renal tissue inhibitor of metalloproteinase (TIMP)-1 expression, and reduction of renal interstitial myofibroblasts. In conclusion, our results suggest that hHGF gene therapy may be considered as an innovative therapeutic strategy to treat advanced diabetic nephropathy

NcRNAs in Therapeutics: Challenges and Limitations in Nucleic Acid-Based Drug Delivery

Non-coding RNAs (ncRNAs) are emerging therapeutic tools but there are barriers to their translation to clinical practice. Key issues concern the specificity of the targets, the delivery of the molecules, and their stability, while avoiding "on-target" and "off-target" side effects. In this "ncRNA in therapeutics" issue, we collect several studies of the differential expression of ncRNAs in cardiovascular diseases, bone metabolism-related disorders, neurology, and oncology, and their potential to be used as biomarkers or therapeutic targets. Moreover, we review recent advances in the use of antisense ncRNAs in targeted therapies with a particular emphasis on their basic biological mechanisms, their translational potential, and future trends

Engraftment Potential of Adipose Tissue-Derived Human Mesenchymal Stem Cells After Transplantation in the Fetal Rabbit

Due to their favorable intrinsic features, including engraftment, differentiation, and immunomodulatory potential, adult mesenchymal stem cells (MSCs) have been proposed for therapeutic in utero intervention. Further improvement of such attributes for particular diseases might merely be achieved by ex vivo MSC genetic engineering previous to transplantation. Here, we evaluated for the first time the feasibility, biodistribution, long-term engraftment, and transgenic enhanced green fluorescent protein (EGFP) expression of genetically engineered human adipose tissue-derived MSCs (EGFP+-ASCs) after intra-amniotic xenotransplantation at E17 of gestation into our validated pregnant rabbit model. Overall, the procedure was safe (86.4% survival rate; absence of anatomical defects). Stable, low-level engraftment of EGFP+-ASCs was confirmed by assessing the presence of the pWT-EGFP lentiviral provirus in the young transplanted rabbit tissues. Accordingly, similar frequencies of provirus-positive animals were found at both 8 weeks (60%) and 16 weeks (66.7%) after in utero intervention. The presence of EGFP+-ASCs was more frequent in respiratory epithelia (lung and trachea), according to the route of administration. However, we were unable to detect EGFP expression, neither by real-time polymerase chain reaction nor by immunohistochemistry, in the provirus-positive tissues, suggesting EGFP transgene silencing mediated by epigenetic events. Moreover, we noticed lack of both host cellular immune responses against xenogeneic ASCs and humoral immune responses against transgenic EGFP. Therefore, the fetal microchimerism achieved by the EGFP+-ASCs in the young rabbit hosts indicates induction of donor-specific tolerance after fetal rabbit xenotransplantation, which should boost postnatal transplantation for the early treatment/prevention of many devastating congenital disorders

Human Mesenchymal Stem Cells Resolve Airway Inflammation, Hyperreactivity, And Histopathology In A Mouse Model Of Occupational Asthma

Occupational asthma (OA) is characterized by allergic airway inflammation and hyperresponsiveness, leading to progressive airway remodeling and a concomitant decline in lung function. The management of OA remains suboptimal in clinical practice. Thus, establishing effective therapies might overcome the natural history of the disease. We evaluated the ability of human adipose-tissue-derived mesenchymal stem cells (hASCs), either unmodified or engineered to secrete the IL-33 decoy receptor sST2, to attenuate the inflammatory and respiratory symptoms in a previously validated mouse model of OA to ammonium persulfate (AP). Twenty-four hours after a dermal AP sensitization and intranasal challenge regimen, the animals received intravenously 1 x 10(6) cells (either hASCs or hASCs overexpressing sST2) or saline and were analyzed at 1, 3, and 6 days after treatment. The infused hASCs induced an anti-inflammatory and restorative program upon reaching the AP-injured, asthmatic lungs, leading to early reduction of neutrophilic inflammation and total IgE production, preserved alveolar architecture with nearly absent lymphoplasmacytic infiltrates, negligible smooth muscle hyperplasia/hypertrophy in the peribronchiolar areas, and baseline airway hyperreactivity (AHR) to methacholine. Local sST2 overexpression barely increased the substantial efficacy displayed by unmodified hASCs. Thus, hASCs may represent a viable multiaction therapeutic capable to adequately respond to the AP-injured lung environment by resolving inflammation, tissue remodeling, and bronchial hyperresponsiveness typical of OA

Usefulness of circulating microRNAs for the prediction of early preeclampsia at first-trimester of pregnancy

To assess the usefulness of circulating microRNAs (miRNAs) as non-invasive molecular biomarkers for early prediction of preeclampsia, a differential miRNA profiling analysis was performed in first-trimester pooled sera from 31 early preeclampsia patients, requiring delivery before 34 weeks of gestation, and 44 uncomplicated pregnancies using microfluidic arrays. Among a total of 754 miRNAs analyzed, the presence of 63 miRNAs (8%) was consistently documented in the sera from preeclampsia and control samples. Nevertheless, only 15 amplified miRNAs (2%) seemed to be differentially, although modestly, represented (fold change range: 0.4-1.4). After stem loop RT-qPCR from individual samples, the statistical analysis confirmed that none of the most consistent and differentially represented miRNAs (3 overrepresented and 4 underrepresented) were differentially abundant in serum from preeclamptic pregnancies compared with serum from normal pregnancies. Therefore, maternal serum miRNA assessment at first-trimester of pregnancy does not appear to have any predictive value for early preeclampsia