Multi-Step Regulation of the TLR4 Pathway by the miR-125a~99b~let-7e Cluster

An appropriate immune response requires a tight balance between pro- and anti-inflammatory mechanisms. IL-10 is induced at late time-points during acute inflammatory conditions triggered by TLR-dependent recognition of infectious agents and is involved in setting this balance, operating as a negative regulator of the TLR-dependent signaling pathway. We identified miR-125a~99b~let-7e as an evolutionary conserved microRNA cluster late-induced in human monocytes exposed to the TLR4 agonist LPS as an effect of this IL-10-dependent regulatory loop. We demonstrated that microRNAs generated by this cluster perform a pervasive regulation of the TLR signaling pathway by direct targeting receptors (TLR4, CD14), signaling molecules (IRAK1), and effector cytokines (TNFα, IL-6, CCL3, CCL7, CXCL8). Modulation of miR-125a~99b~let-7e cluster influenced the production of proinflammatory cytokines in response to LPS and the IL-10-mediated tolerance to LPS, thus identifying this gene as a previously unrecognized major regulatory element of the inflammatory response and endotoxin tolerance

Multi-Step Regulation of the TLR4 Pathway by the miR-125a~99b~let-7e Cluster

An appropriate immune response requires a tight balance between pro- and anti-inflammatory mechanisms. IL-10 is induced at late time-points during acute inflammatory conditions triggered by TLR-dependent recognition of infectious agents and is involved in setting this balance, operating as a negative regulator of the TLR-dependent signaling pathway. We identified miR-125a~99b~let-7e as an evolutionary conserved microRNA cluster late-induced in human monocytes exposed to the TLR4 agonist LPS as an effect of this IL-10-dependent regulatory loop. We demonstrated that microRNAs generated by this cluster perform a pervasive regulation of the TLR signaling pathway by direct targeting receptors (TLR4, CD14), signaling molecules (IRAK1), and effector cytokines (TNFα, IL-6, CCL3, CCL7, CXCL8). Modulation of miR-125a~99b~let-7e cluster influenced the production of proinflammatory cytokines in response to LPS and the IL-10-mediated tolerance to LPS, thus identifying this gene as a previously unrecognized major regulatory element of the inflammatory response and endotoxin tolerance

Serum micrornas as novel biomarkers for primary sclerosing cholangitis and cholangiocarcinoma

The diagnosis of primary sclerosing cholangitis (PSC) is difficult due to the lack of sensitive and specific biomarkers, as is the early diagnosis of cholangiocarcinoma (CC), a complication of PSC. The aim of this study was to identify specific serum miRNAs as diagnostic biomarkers for PSC and CC

Expression of chemokines and chemokine receptors in human colon cancer

Human colorectal cancer (CRC), the second largest cause of tumor-related death in Western countries, represents a paradigm for the now well-established connections between inflammation and cancer. In this study, we investigated which inflammatory mediators are mostly expressed in the microenvironment of human CRC. The RNA profile of a large panel of inflammatory genes, in particular chemokines and chemokine receptors, was analyzed in eight surgical tumor samples and in paired normal tissues from CRC patients. We employed an "inflammatory gene card" (TaqMan Low Density Array by Applied Biosystem), designed by our group, containing probes for 24 chemokines and 17 chemokine receptors. Several chemokines were strongly upregulated in the tumor microenvironment, most frequently CCL4 and CCL5, chemotactic for monocytes/macrophages and T cells, and the corresponding receptors CCR1 and CCR5; the angiogenic chemokines CXCL1 and CXCL8, and the receptor CXCR2. The antiangiogenic chemokines CXCL9 and CXCL10 were also expressed, but in the absence of the receptor CXCR3. Selected results have been confirmed in a larger number of samples. The levels of mRNA CXCL8 were significantly associated with the levels of osteopontin, a matrix-associated protein that shares with chemokines important functions such as induction of cell migration and survival, and modulation of the neoangiogenesis. Overall these results could be helpful to identify the most relevant inflammatory pathways present in CRC tumors and to build a solid rationale for future therapeutic interventions based on anti-inflammatory strategies

Multilineage hematopoietic reconstitution without clonal selection in ADA-SCID patients treated with stem cell gene therapy

Gene transfer into HSCs is an effective treatment for SCID, although potentially limited by the risk of insertional mutagenesis. We performed a genome-wide analysis of retroviral vector integrations in genetically corrected HSCs and their multilineage progeny before and up to 47 months after transplantation into 5 patients with adenosine deaminase–deficient SCID. Gene-dense regions, promoters, and transcriptionally active genes were preferred retroviral integrations sites (RISs) both in preinfusion transduced CD34+ cells and in vivo after gene therapy. The occurrence of insertion sites proximal to protooncogenes or genes controlling cell growth and self renewal, including LMO2, was not associated with clonal selection or expansion in vivo. Clonal analysis of long-term repopulating cell progeny in vivo revealed highly polyclonal T cell populations and shared RISs among multiple lineages, demonstrating the engraftment of multipotent HSCs. These data have important implications for the biology of retroviral vectors, the dynamics of genetically modified HSCs, and the safety of gene therapy

Gene therapy for immunodeficiency due to adenosine deaminase deficiency.

We investigated the long-term outcome of gene therapy for severe combined immunodeficiency (SCID) due to the lack of adenosine deaminase (ADA), a fatal disorder of purine metabolism and immunodeficiency.Clinical Trial, Phase IClinical Trial, Phase IIJournal ArticleMulticenter StudyResearch Support, Non-U.S. Gov'tSCOPUS: ar.jinfo:eu-repo/semantics/publishe

The mutational spectrum of human malignant autosomal recessive osteopetrosis

Human malignant infantile osteopetrosis (arOP; MIM 259700) is a genetically heterogenous autosomal recessive disorder of bone metabolism, which, if untreated, has a fatal outcome. Our group, as well as others, have recently identified mutations in the ATP6i (TCIRG1) gene, encoding the a3 subunit of the vacuolar proton pump, which mediates the acidification of the bone/osteoclast interface, are responsible for a subset of this condition. By sequencing the ATP6i gene in arOP patients from 44 unrelated families with a worldwide distribution we have now established that ATP6i mutations are responsible for ∼50% of patients affected by this disease. The vast majority of these mutations (40 out of 42 alleles, including seven deletions, two insertions, 10 nonsense substitutions and 21 mutations in splice sites) are predicted to cause severe abnormalities in the protein product and are likely to represent null alleles. In addition, we have also analysed nine unrelated arOP patients from Costa Rica, where this disease is apparently much more frequent than elsewhere. All nine Costa Rican patients bore either or both of two missense mutations (G405R and R444L) in amino acid residues which are evolutionarily conserved from yeast to humans. The identification of ATP6i gene mutations in two families allowed us for the first time to perform prenatal diagnosis: both fetuses were predicted not to be affected and two healthy babies were born. This study contributes to the determination of genetic heterogeneity of arOP and allows further delineation of the other genetic defects causing this severe conditio