Josep M. Aran

Mesenchymal Stem Cells in Solid Organ Transplantation (MiSOT) Fourth Meeting: Lessons Learned from First Clinical Trials

The Fourth Expert Meeting of the Mesenchymal Stem Cells in Solid Organ Transplantation (MiSOT) Consortium took place in Barcelona on October 19 and 20, 2012. This meeting focused on the translation of preclinical data into early clinical settings. This position paper highlights the main topics explored on the safety and efficacy of mesenchymal stem cells as a therapeutic agent in solid organ transplantation and emphasizes the issues (proper timing, concomitant immunossupression, source and immunogenicity of mesenchymal stem cells, and oncogenicity) that have been addressed and will be followed up by the MiSOT Consortium in future studies.

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.

Intramuscular SP1017-formulated DNA electrotransfer enhances transgene expression and distributes hHGF to different rat tissues

The systemic administration of a therapeutic protein is a common approach for the treatment of multiple disorders. Intramuscular (i.m.) injection of plasmids, followed by electroporation, has been shown to facilitate naked DNA gene transfer in skeletal muscle allowing proteins to be produced and secreted at therapeutically relevant levels.

CD40: an upstream master switch for endothelial cell activation uncovered by RNAi-coupled transcriptional profiling

The CD40-CD154 dyad seems to play a prominent role fostering the immune-inflammatory response triggered by endothelial cell (EC)-T-cell communication. To delineate comprehensively the involvement of CD40 (TNFRSF5) in EC activation, we combined RNAi-mediated CD40 knockdown with comparative genome-wide transcriptional profiling of ECs interacting with (CD154+) T cells. We report the initiation of a profound stress response in ECs upon CD40-CD154 engagement through early up-regulation of, among others, the major proinflammatory NF-kappaB and MAPK/SAPK pathways and their associated transcription factors. Moreover, we have identified novel genes regulated through the CD40-CD154 interaction, and pathways previously unrecognized to be induced by CD40 signaling in ECs. Thus, we document a significant down-regulation of endothelial APLN by CD40-CD154 interaction, TNFalpha/IFNgamma exposure, and in immune-inflammatory pathologies, which could lead to hemodynamic dysfunction. Conversely, CD40-mediated up-regulation of the viral immune surveillance system, notably TLR3, IFIH1, RIG-I, and RNASEL, establishes a reverse link from adaptive to innate immunity in ECs. Moreover, systematic enrichment analysis substantiates endothelial CD40 involvement in the transcriptional regulation of gene networks associated with adhesion and motility, immunity, cell fate control, hemostasis, and metabolism. Our study also highlights the anti-inflammatory potential of RNAi-mediated CD40 inhibition, and the relevance of CD40 signaling for therapeutic intervention.

Targeted gene repair : The ups and downs of a promising gene therapy approach

As a novel form of molecular medicine based on direct actions over the genes, targeted gene repair has raised consideration recently above classical gene therapy strategies based on genetic augmentation or complementation. Targeted gene repair relies on the local induction of the cells endogenous DNA repair mechanisms to attain a therapeutic gene conversion event within the genome of the diseased cell. Successful repair has been achieved both in vitro and in vivo with a variety of corrective molecules ranging from oligonucleotides (chimeraplasts, modified single-stranded oligonucleotides, triplex-forming oligonucleotides), to small DNA fragments (small fragment homologous replacement (SFHR)), and even viral vectors (AAV-based). However, controversy on the consistency and lack of reproducibility of early experiments regarding frequencies and persistence of targeted gene repair, particularly for chimeraplasty, has flecked the field. Nevertheless, several hurdles such as inefficient nuclear uptake of the corrective molecules, and misleading assessment of targeted repair frequencies have been identified and are being addressed. One of the key bottlenecks for exploiting the overall potential of the different targeted gene repair modalities is the lack of a detailed knowledge of their mechanisms of action at the molecular level. Several studies are now focusing on the assessment of the specific repair pathway(s) involved (homologous recombination, mismatch repair, etc.), devising additional strategies to increase their activity (using chemotherapeutic drugs, chimeric nucleases, etc.), and assessing the influence of the cell cycle in the regulation of the repair process. Until therapeutic correction frequencies for single gene disorders are reached both in cellular and animal models, precision and undesired side effects of this promising gene therapy approach will not be thoroughly evaluated.

IL-4 orchestrates STAT6-mediated DNA demethylation leading to dendritic cell differentiation

BackgroundThe role of cytokines in establishing specific transcriptional programmes in innate immune cells has long been recognized. However, little is known about how these extracellular factors instruct innate immune cell epigenomes to engage specific differentiation states. Human monocytes differentiate under inflammatory conditions into effector cells with non-redundant functions, such as dendritic cells and macrophages. In this context, interleukin 4 (IL-4) and granulocyte macrophage colony-stimulating factor (GM-CSF) drive dendritic cell differentiation, whereas GM-CSF alone leads to macrophage differentiation.ResultsHere, we investigate the role of IL-4 in directing functionally relevant dendritic-cell-specific DNA methylation changes. A comparison of DNA methylome dynamics during differentiation from human monocytes to dendritic cells and macrophages identified gene sets undergoing dendritic-cell-specific or macrophage-specific demethylation. Demethylation is TET2-dependent and is essential for acquiring proper dendritic cell and macrophage identity. Most importantly, activation of the JAK3-STAT6 pathway, downstream of IL-4, is required for the acquisition of the dendritic-cell-specific demethylation and expression signature, following STAT6 binding. A constitutively activated form of STAT6 is able to bypass IL-4 upstream signalling and instruct dendritic-cell-specific functional DNA methylation changes.ConclusionsOur study is the first description of a cytokine-mediated sequence of events leading to direct gene-specific demethylation in innate immune cell differentiation.

In vivo therapeutic efficacy of intra-renal CD40 silencing in a model of humoral acute rejection

The humoral branch of the immune response has an important role in acute and chronic allograft dysfunction. The CD40/CD40L costimulatory pathway is crucial in B- and T- alloresponse. Our group has developed a new small interfering RNA (siRNA) molecule against CD40 that effectively inhibits its expression. The aim of the present study was to prevent rejection in an acute vascular rejection model of kidney transplant by intra-graft gene silencing with anti-CD40 siRNA (siCD40), associated or not with sub-therapeutic rapamycin. Four groups were designed: unspecific siRNA as control; sub-therapeutic rapamycin; siCD40; and combination therapy. Long-surviving rats were found only in both siCD40-treated groups. The CD40 mRNA was overexpressed in control grafts but treatment with siCD40 decreased its expression. Recipient spleen CD40+ B-lymphocytes were reduced in both siCD40-treated groups. Moreover, CD40 silencing reduced donor-specific antibodies, graft complement deposition and immune-inflammatory mediators. The characteristic histological features of humoral rejection were not found in siCD40-treated grafts, which showed a more cellular histological pattern. Therefore, the intra-renal effective blockade of the CD40/CD40L signal reduces the graft inflammation as well as the incidence of humoral vascular acute rejection, finally changing the type of rejection from humoral to cellular.

The alpha(7)beta(0) Isoform of the Complement Regulator C4b-Binding Protein Induces a Semimature, Anti-Inflammatory State in Dendritic Cells

The classical pathway complement regulator C4b-binding protein (C4BP) is composed of two polypeptides (α- and β-chains), which form three plasma oligomers with different subunit compositions (α7β1, α7β0, and α6β1). We show in this article that the C4BP α7β0 isoform (hereafter called C4BP[β−] [C4BP lacking the β-chain]), overexpressed under acute-phase conditions, induces a semimature, tolerogenic state on human monocyte-derived dendritic cells (DCs) activated by a proinflammatory stimulus. C4BP isoforms containing β-chain (α7β1 and α6β1; C4BP[β+]) neither interfered with the normal maturation of DCs nor competed with C4BP(β−) activity on these cells. Immature DCs (iDCs) treated with C4BP(β−) retained high endocytic activity, but, upon LPS treatment, they did not upregulate surface expression of CD83, CD80, and CD86. Transcriptional profiling of these semimature DCs revealed that treatment with C4BP(β−) prevented the induction of IDO and BIC-1, whereas TGF-β1 expression was maintained to the level of iDCs. C4BP(β−)–treated DCs were also unable to release proinflammatory Th1 cytokines (IL-12, TNF-α, IFN-γ, IL-6, IL-8) and, conversely, increased IL-10 secretion. They prevented surface CCR7 overexpression and, accordingly, displayed reduced chemotaxis, being morphologically indistinguishable from iDCs. Moreover, C4BP(β−)-treated DCs failed to enhance allogeneic T cell proliferation, impairing IFN-γ production in these cells and, conversely, promoting CD4+CD127low/negCD25highFoxp3+ T cells. Deletion mutant analysis revealed that the complement control protein-6 domain of the α-chain is necessary for the tolerogenic activity of C4BP(β−). Our data demonstrate a novel anti-inflammatory and immunomodulatory function of the complement regulator C4BP, suggesting a relevant role of the acute-phase C4BP(β−) isoform in a number of pathophysiological conditions and potential applications in autoimmunity and transplantation.

Characterization of mesenchymal stem cells isolated from the rabbit fetal liver.

Physiological attributes of mesenchymal stem cells (MSCs) including straightforward manipulation, multilineage differentiation, immunoregulation, and tropism for injury settings render them ideal therapeutic agents for tissue repair/regeneration. Nevertheless, further studies in suitable animal models of disease are needed to translate the potential of MSCs into clinical applications. We report here the isolation and preliminary characterization of MSCs from fetal rabbit liver (fl-MSCs). Compared with MSCs isolated from adult rabbit bone marrow, fl-MSCs had superior growth rate, clonogenic capability, and plastic adherence owing to their developmental immaturity. Both cytochemical staining and mRNA expression analysis of fl-MSCs confirmed mesodermal lineage differentiation into adipocytes, osteocytes, and chondrocytes. Moreover, fl-MSCs were capable to prevent lymphocyte proliferation both in a 2-way MLC and upon phytohemagglutinin (PHA) stimulation. In contrast, fl-MSCs co-cultured with allogeneic lymphocytes induced proliferation of the latter. Relatedly, although freshly isolated fl-MSCs did express neither major histocompatibility complex (MHC) class I/II nor CD80/CD86, all these immune synapse components were induced upon in vitro culture. Furthermore, fl-MSCs became efficiently transduced for long-term transgene expression with a retroviral vector. Thus, the special biological qualities of fl-MSCs endow them as model candidate vehicles/agents for gene/cell therapy strategies applied to a variety of rabbit models of injury, such as osteochondral lesions.

Misleading Gene Conversion Frequencies Due to a PCR Artifact Using Small Fragment Homologous Replacement

Recent studies have reported successful correction of the most common F508del mutation in cystic fibrosis (CF) airway epithelial cells by small fragment homologous replacement (SFHR). We wished to apply the SFHR methodology to our CF bronchial epithelial cells, of compound heterozygous genotype (F508del/W1282X), in which nucleic acid transfer was previously optimized by electroporation. Using a PCR-based detection methodology, with one of the primers located outside the SFHR homology region, we obtained SFHR dose-dependent F508del to wild-type CFTR gene conversion frequencies reaching 30%. However, the increased wild-type/F508del CFTR allele ratio was transient, vanishing at 5 days posttransfection. Furthermore, we have been unable to reproduce the SFHR-mediated repair of the F508del mutation in our cellular model when both detection primers were located outside the SFHR homology region. A thorough reexamination of our initial detection strategy revealed that a false positive result was originated from a PCR artifact created by the SFHR fragment itself. Thus, nonamplifiable detection methods, such as Southern blotting, protein analysis, or functional assays, should be performed, whenever possible, to correctly assess gene conversion frequencies.