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Biomedical subjects

Sergio Ferrari

Publications and source records attributed to Sergio Ferrari.

At least 19 recordsLinked to original sources
PubMed2006

Identification of new p63 targets in human keratinocytes.

p63 is a transcription factor involved in the development of ectodermal tissues, including limb, skin and, in general, multilayered epithelia. We identified both activated and repressed genes in human keratinocytes via gene expression profiling of p63-depleted cells and validated 21 new primary targets by RT-PCR and ChIP location analysis. The p63 isoforms differentially activate or repress selected promoters. ChIPs in primary keratinocytes indicate that p63 targets are generally shared with p53, but some are p63-specific. Several growth suppressors are among repressed genes. The newly identified genes belong to pathways of growth and differentiation and are regulated in HaCaT differentiation and in stratification of human skin.

Cell Differentiation↗
PubMed2006

The extracellular nucleotide UTP is a potent inducer of hematopoietic stem cell migration.

Homing and engraftment of hematopoietic stem cells (HSCs) to the bone marrow (BM) involve a complex interplay between chemokines, cytokines, and nonpeptide molecules. Extracellular nucleotides and their cognate P2 receptors are emerging as key factors of inflammation and related chemotactic responses. In this study, we investigated the activity of extracellular adenosine triphosphate (ATP) and uridine triphosphate (UTP) on CXCL12-stimulated CD34+ HSC chemotaxis. In vitro, UTP significantly improved HSC migration, inhibited cell membrane CXCR4 down-regulation by migrating CD34+ cells, and increased cell adhesion to fibronectin. In vivo, preincubation with UTP significantly enhanced the BM homing efficiency of human CD34+ cells in immunodeficient mice. Pertussis toxin blocked CXCL12- and UTP-dependent chemotactic responses, suggesting that G-protein alpha-subunits (Galphai) may provide a converging signal for CXCR4- and P2Y-activated transduction pathways. In addition, gene expression profiling of UTP- and CXCL12-treated CD34+ cells and in vitro inhibition assays demonstrated that Rho guanosine 5'-triphosphatase (GTPase) Rac2 and downstream effectors Rho GTPase-activated kinases 1 and 2 (ROCK1/2) are involved in UTP-promoted/CXCL12-dependent HSC migration. Our data suggest that UTP may physiologically modulate the homing of HSCs to the BM, in concert with CXCL12, via the activation of converging signaling pathways between CXCR4 and P2Y receptors, involving Galphai proteins and RhoGTPases.

Adenosine Triphosphate↗
PubMed2006

Tfe3 expression is closely associated to macrophage terminal differentiation of human hematopoietic myeloid precursors.

The MItf-Tfe family of basic helix-loop-helix leucine zipper (bHLH-Zip) transcription factors encodes four family members: MItf, Tfe3, TfeB and TfeC. In vitro, each protein of the family binds DNA in a homo- or heterodimeric form with other family members. Tfe3 is involved in chromosomal translocations recurrent in different tumors and it has been demonstrated, by in vivo studies, that it plays, redundantly with MItf, an important role in the process of osteoclast formation, in particular during the transition from mono-nucleated to multi-nucleated osteoclasts. Since mono-nucleated osteoclasts derive from macrophages we investigated whether Tfe3 might play a role upstream during hematopoietic differentiation. Here we show that Tfe3 is able to induce mono-macrophagic differentiation of U937 cells, in association with a decrease of cell proliferation and an increase of apoptosis. We also show that Tfe3 does not act physiologically during commitment of CD34+ hematopoietic stem cells (HSCs), since it is not able to direct HSCs toward a specific lineage as observed by clonogenic assay, but is a strong actor of terminal differentiation since it allows human primary myeloblasts' maturation toward the macrophage lineage.

Antigens, Differentiation↗
PubMed2006

Molecular profiling of CD34+ cells in idiopathic myelofibrosis identifies a set of disease-associated genes and reveals the clinical significance of Wilms' tumor gene 1 (WT1).

This study was aimed at the characterization of a gene expression signature of the pluripotent hematopoietic CD34(+) stem cell in idiopathic myelofibrosis (IM), which would eventually provide novel pathogenetic insights and/or diagnostic/prognostic information. Aberrantly regulated genes were revealed by transcriptome comparative microarray analysis of normal and IM CD34(+) cells; selected genes were also assayed in granulocytes. One-hundred seventy four differentially expressed genes were identified and in part validated by quantitative polymerase chain reaction. Altered gene expression was corroborated by the detection of abnormally high CD9 or CD164, and low CXCR4, membrane protein expression in IM CD34(+) cells. According to class prediction analysis, a set of eight genes (CD9, GAS2, DLK1, CDH1, WT1, NFE2, HMGA2, and CXCR4) properly recognized IM from normal CD34(+) cells. These genes were aberrantly regulated also in IM granulocytes that could be reliably differentiated from control polycythemia vera and essential thrombocythemia granulocytes in 100% and 81% of cases, respectively. Abnormal expression of HMGA2 and CXCR4 in IM granulocytes was dependent on the presence and the mutational status of JAK2(V617F) mutation. The expression levels of both CD9 and DLK1 were associated with the platelet count, whereas higher WT1 expression levels identified IM patients with more active disease, as revealed by elevated CD34(+) cell count and higher severity score. In conclusion, molecular profiling of IM CD34(+) cells uncovered a limited number of genes with altered expression that, beyond their putative role in disease pathogenesis, are associated with patients' clinical characteristics and may have potential prognostic application.

Algorithms↗
PubMed2006

Endothelial adhesion molecule expression is unaltered in the peripheral nerve from patients with AIDS and distal sensory polyneuropathy.

Some evidence suggests that endothelial dysfunction, including altered expression of cell adhesion molecules contributes to pathophysiology of nervous system disorders in the course of HIV infection. In this immunohistochemical study we investigated and compared the expression of E-selectin, intercellular adhesion molecule-1 (ICAM-1), platelet endothelial cell adhesion molecule-1 (PECAM-1) in peripheral nerve blood vessels from patients with distal sensory polyneuropathy (DSP) and control patients with other axonal neuropathies. Similar quantitative pattern of immunoreactivity was found in patients with DSP and controls. E-selectin and PECAM-1 immunostained vessels tended to increase in number only in patients with major CD4 cell depletion. The vascular endothelium of the peripheral nerve in HIV-infected subjects with DSP shows no changes facilitating the migration of infected or activated monocytes/macrophages into the nerve. These phenomena probably do not play a critical role in the development of axonal damage in DSP.

Acquired Immunodeficiency Syndrome↗
PubMed2006

Hepatocyte growth factor favors monocyte differentiation into regulatory interleukin (IL)-10++IL-12low/neg accessory cells with dendritic-cell features.

Several hematopoietic growth factors, including interleukin-10 (IL-10) and transforming growth factor-beta1 (TGF-beta1), promote the differentiation of tolerogenic dendritic cells (DCs). Hepatocyte growth factor (HGF) is a pleiotropic cytokine whose effects on human DC differentiation and function have not been investigated. Monocytes cultured with HGF (HGFMo) differentiated into accessory cells with DC-like morphology, released low amounts of IL-12p70 and up-regulated IL-10 both at the mRNA and at the protein level. Upon activation with HGFMo, allogeneic CD4+CD25- T cells expressed the T regulatory (Treg)-associated transcription factor FoxP3, proliferated poorly, and released high levels of IL-10. Interestingly, blockade of surface immunoglobulin-like transcript 3 (ILT3) on HGFMo or neutralization of secreted IL-10 translated into partial restoration of T-cell proliferation. Secondary stimulation of HGFMo-primed CD4+ T cells with immunogenic DCs differentiated with granulocyte-macrophage colony-stimulating factor (GM-CSF) and IL-4 from monocytes of the same donor resulted in measurable T-cell proliferation. HGFMo-primed CD4+ T cells significantly inhibited the proliferation of naive CD4+CD25- T cells in a cell-contact-dependent manner. Finally, DNA microarray analysis revealed a unique gene-expression profile of HGF-activated monocytes. Collectively, our findings point to a novel role for HGF in the regulation of monocyte/DC functions that might be exploited therapeutically.

CD4-Positive T-Lymphocytes↗
PubMed2006

Human immunodeficiency virus-associated peripheral neuropathies.

Peripheral neuropathy has emerged as the most common neurologic complication of human immunodeficiency virus (HIV) infection. It will continue to play an Important role in HIV Infection given the fact that HIV-infected Individuals are living longer, are at risk of long-term metabolic complications, and face an Increasing exposure to potentially neurotoxic antiretroviral drugs. We review the various types of peripheral neuropathy that have been associated with HIV infection, including distal symmetrical polyneuropathy, toxic neuropathy from antiretroviral drugs, diffuse infiltrative lymphocytosis syndrome, inflammatory demyelinating polyneuropathies, multifocal mononeuropathies, and progressive polyradiculopathy.

Antiretroviral Therapy, Highly Active↗
PubMed2005

Embryonic stem-derived versus somatic neural stem cells: a comparative analysis of their developmental potential and molecular phenotype.

Reliable procedures to induce neural commitment of totipotent undifferentiated embryonic stem (ES) cells have provided new tools for investigating the molecular mechanisms underlying cell fate choices. We extensively characterized the developmental potential of ES-induced neural cells obtained using an adaptation of the multistep induction protocol. We provided evidence that ES-derived neural proliferating cells are endowed with stem cell properties such as extensive self-renewal capacity and single-cell multipotency. In differentiating conditions, cells matured exclusively into neurons, astrocytes, and oligodendrocytes. All these features have been previously described in only somatic neural stem cells (NSCs). Therefore, we consider it more appropriate to rename our cells ES-derived NSCs. These similarities between the two NSC populations induced us to carefully compare their proliferation ability and differentiation potential. Although they were very similar in overall behavior, we scored specific differences. For instance, ES-derived NSCs proliferated at higher rate and consistently generated a higher number of neurons compared with somatic NSCs. To further investigate their relationships, we carried out a molecular analysis comparing their transcriptional profiles during proliferation. We observed a large fraction of shared expressed transcripts, including genes previously described to be critical in defining somatic NSC traits. Among the genes differently expressed, candidate genes possibly responsible for divergences between the two cell types were selected and further investigated. In particular, we showed that an enhanced MAPK (mitogen-activated protein kinase) signaling is acting in ES-induced NSCs, probably triggered by insulin-like growth factor-II. This may contribute to the high proliferation rate exhibited by these cells in culture.

Animals↗
PubMed2005

Analysis of mammalian scrapie protein by novel monoclonal antibodies recognizing distinct prion protein glycoforms: an immunoblot and immunohistochemical study at the light and electron microscopic levels.

The availability of specific monoclonal antibodies (mAbs) recognizing the aberrant form (PrP(Sc)) of the cellular prion protein (PrP(C)) in different mammalian species is important for molecular diagnostics, PrP(Sc) typing and future immunotherapy. We obtained a panel of anti-PrP monoclonal antibodies in PrP(0/0) knock-out mice immunized with recombinant human PrP(23-231). Two mAbs, recognizing PrP epitopes in the alpha-helix 1 (mAb SA65) and alpha-helix 2 (mAb SA21) regions, immunoreacted with PrP(C) and PrP(Sc) and its proteolytic product, PrP27-30, from human, murine, bovine, caprine and ovine brains by Western blot. Remarkably, mAb SA21 recognized unglycosylated and monoglycosylated PrP with the second site occupied by glycan moieties, but not monoglycosylated PrP with the first consensus site occupied or highly glycosylated species. Immunoblots with mAb SA21 disclosed that PrP glycosylated at the second site accounted for the slower migrating form of the customary monoglycosylated PrP doublet. mAb SA65 immunolabelled all PrP glycoforms by Western blot and was highly efficient in detecting tissue PrP by immunohistochemistry in light microscopy and in immunoelectron microscopy. These novel anti-PrP mAbs provide tools to investigate the subcellular site of PrP deposition in mammalian prion diseases and may also contribute to assess the role of different PrP glycoforms in human and animal prion diseases.

Animals↗
PubMed2005

The kinetic status of hematopoietic stem cell subpopulations underlies a differential expression of genes involved in self-renewal, commitment, and engraftment.

The gene expression profile of CD34(-) hematopoietic stem cells (HSCs) and the correlations with their biological properties are still poorly understood. To address this issue, we used the DNA microarray technology to compare the expression profiles of different peripheral blood hemopoietic stem/progenitor cell subsets, lineage-negative (Lin(-)) CD34(-), Lin(-)CD34(+), and Lin(+)CD34(+) cells. The analysis of gene categories differentially expressed shows that the expression of CD34 is associated with cell cycle entry and metabolic activation, such as DNA, RNA, and protein synthesis. Moreover, the significant upregulation in CD34(-) cells of pathways inhibiting HSC proliferation induces a strong differential expression of cyclins, cyclin-dependent kinases (CDKs), CDK inhibitors, and growth-arrest genes. According to the expression of their receptors and transducers, interleukin (IL)-10 and IL-17 showed an inhibitory effect on the clonogenic activity of CD34(-) cells. Conversely, CD34(+) cells were sensitive to the mitogenic stimulus of thrombopoietin. Furthermore, CD34(-) cells express preferentially genes related to neural, epithelial, and muscle differentiation. The analysis of transcription factor expression shows that the CD34 induction results in the upregulation of genes related to self-renewal and lineage commitment. The preferential expression in CD34(+) cells of genes supporting the HSC mobilization and homing to the bone marrow, such as chemokine receptors and integrins, gives the molecular basis for the higher engraftment capacity of CD34(+) cells. Thus, the different kinetic status of CD34(-) and CD34(+) cells, detailed by molecular and functional analysis, significantly influences their biological behavior.

Antigens, CD34↗
PubMed2005

Stem cell plasticity: time for a reappraisal?

In recent years an increasing number of publications have claimed that adult mammalian stem cells (SC) may be capable of differentiating across tissue lineage boundaries and that this plasticity may represent a novel therapeutic strategy for tissue regeneration. However, after a first phase of excitement, the issue of somatic SC plasticity remains controversial and the therapeutic perspectives are still elusive. In this review, we examine the general mechanisms which govern the function of SC, the identification and functional characterization of adult SC of different tissues and their putative capacity to transdifferentiate into mature cells of different origin. The potential clinical applications of adult SC for regenerative medicine are also discussed in each chapter. The method employed for preparing this review was the informal consensus development. Members of the Working Group on SC met four times and discussed the single points, previously assigned by the Chairman (S.T.), in order to achieve an agreement on different opinions and approve the final manuscript. All the authors of the present review have been working in the field of SC and have contributed original papers to peer-reviewed journals. In addition to the authors' own work, the present review examines articles published in journals covered by the Science Citation Index and Medline.

Adult↗
PubMed2004

TGFbeta/BMP activate the smooth muscle/bone differentiation programs in mesoangioblasts.

Mesoangioblasts are vessel-derived stem cells that can be induced to differentiate into different cell types of the mesoderm such as muscle and bone. The gene expression profile of four clonal derived lines of mesoangioblasts was determined by DNA micro-array analysis: it was similar in the four lines but different from 10T1/2 embryonic fibroblasts, used as comparison. Many known genes expressed by mesoangioblasts belong to response pathways to developmental signalling molecules, such as Wnt or TGFbeta/BMP. Interestingly, mesoangioblasts express receptors of the TGFbeta/BMP family and several Smads and, accordingly, differentiate very efficiently into smooth muscle cells in response to TGFbeta and into osteoblasts in response to BMP. In addition, insulin signalling promotes adipogenic differentiation, possibly through the activation of IGF-R. Several Wnts and Frizzled, Dishevelled and Tcfs are expressed, suggesting the existence of an autocrine loop for proliferation and indeed, forced expression of Frzb-1 inhibits cell division. Mesoangioblasts also express many neuro-ectodermal genes and yet undergo only abortive neurogenesis, even after forced expression of neurogenin 1 or 2, MASH or NeuroD. Finally, mesoangioblasts express several pro-inflammatory genes, cytokines and cytokine receptors, which may explain their ability to be recruited by tissue inflammation. Our data define a unique phenotype for mesoangioblasts, explain several of their biological features and set the basis for future functional studies on the role of these cells in tissue histogenesis and repair.

Animals↗
PubMed2004

Gene expression profiling of normal and malignant CD34-derived megakaryocytic cells.

Gene expression profiles of bone marrow (BM) CD34-derived megakaryocytic cells (MKs) were compared in patients with essential thrombocythemia (ET) and healthy subjects using oligonucleotide microarray analysis to identify differentially expressed genes and disease-specific transcripts. We found that proapoptotic genes such as BAX, BNIP3, and BNIP3L were down-regulated in ET MKs together with genes that are components of the mitochondrial permeability transition pore complex, a system with a pivotal role in apoptosis. Conversely, antiapoptotic genes such as IGF1-R and CFLAR were up-regulated in the malignant cells, as was the SDF1 gene, which favors cell survival. On the basis of the array results, we characterized apoptosis of normal and ET MKs by time-course evaluation of annexin-V and sub-G1 peak DNA stainings of immature and mature MKs after culture in serum-free medium with an optimal thrombopoietin concentration, and annexin-V-positive MKs only, with decreasing thrombopoietin concentrations. ET MKs were more resistant to apoptosis than their normal counterparts. We conclude that imbalance between proliferation and apoptosis seems to be an important step in malignant ET megakaryocytopoiesis.

Adult↗
PubMed2004

Identification of distinct N-terminal truncated forms of prion protein in different Creutzfeldt-Jakob disease subtypes.

In prion diseases, the cellular prion protein (PrP(C)) is converted to an insoluble and protease-resistant abnormal isoform termed PrP(Sc). In different prion strains, PrP(Sc) shows distinct sites of endogenous or exogenous proteolysis generating a core fragment named PrP27-30. Sporadic Creutzfeldt-Jakob disease (sCJD), the most frequent human prion disease, clinically presents with a variety of neurological signs. As yet, the clinical variability observed in sCJD has not been fully explained by molecular studies relating two major types of PrP27-30 with unglycosylated peptides of 21 (type 1) and 19 kDa (type 2) and the amino acid methionine or valine at position 129. Recently, smaller C-terminal fragments migrating at 12 and 13 kDa have been detected in different sCJD phenotypes, but their significance remains unclear. By using two-dimensional immunoblot with anti-PrP antibodies, we identified two novel groups of protease-resistant PrP fragments in sCJD brain tissues. All sCJD cases with type 1 PrP27-30, in addition to MM subjects with type 2 PrP27-30, were characterized by the presence of unglycosylated PrP fragments of 16-17 kDa. Conversely, brain homogenates from patients VV and MV with type 2 PrP27-30 contained fully glycosylated PrP fragments, which after deglycosylation migrated at 17.5-18 kDa. Interestingly, PrP species of 17.5-18 kDa matched deglycosylated forms of the C1 PrP(C) fragment and were associated with tissue PrP deposition as plaque-like aggregates or amyloid plaques. These data show the presence of multiple PrP(Sc) conformations in sCJD and, in addition, shed new light on the correlation between sCJD phenotypes and disease-associated PrP molecules.

Adult↗
PubMed2004

Msx2 and necdin combined activities are required for smooth muscle differentiation in mesoangioblast stem cells.

Little is known about the molecular mechanism underlying specification and differentiation of smooth muscle (SM), and this is, at least in part, because of the few cellular systems available to study the acquisition of a SM phenotype in vitro. Mesoangioblasts are vessel-derived stem cells that can be induced to differentiate into different cell types of the mesoderm, including SM. We performed a DNA microarray analysis of a mesoangioblast clone that spontaneously expresses an immature SM phenotype and compared it with a sister clone mainly composed of undifferentiated progenitor cells. This study allowed us to define a gene expression profile for "stem" cells versus smooth muscle cells (SMCs) in the absence of differentiation inducers such as transforming growth factor beta. Two transcription factors, msx2 and necdin, are expressed at least 100 times more in SMCs than in stem cells, are coexpressed in all SMCs and tissues, are induced by transforming growth factor beta, and, when coexpressed, induce a number of SM markers in mesoangioblast, fibroblast, and endothelial cell lines. Conversely, their downregulation through RNA interference results in a decreased expression of SM markers. These data support the hypothesis that Msx2 and necdin act as master genes regulating SM differentiation in at least a subset of SMCs.

Animals↗
PubMed2004

Identification of a second bovine amyloidotic spongiform encephalopathy: molecular similarities with sporadic Creutzfeldt-Jakob disease.

Transmissible spongiform encephalopathies (TSEs), or prion diseases, are mammalian neurodegenerative disorders characterized by a posttranslational conversion and brain accumulation of an insoluble, protease-resistant isoform (PrP(Sc)) of the host-encoded cellular prion protein (PrP(C)). Human and animal TSE agents exist as different phenotypes that can be biochemically differentiated on the basis of the molecular mass of the protease-resistant PrP(Sc) fragments and the degree of glycosylation. Epidemiological, molecular, and transmission studies strongly suggest that the single strain of agent responsible for bovine spongiform encephalopathy (BSE) has infected humans, causing variant Creutzfeldt-Jakob disease. The unprecedented biological properties of the BSE agent, which circumvents the so-called "species barrier" between cattle and humans and adapts to different mammalian species, has raised considerable concern for human health. To date, it is unknown whether more than one strain might be responsible for cattle TSE or whether the BSE agent undergoes phenotypic variation after natural transmission. Here we provide evidence of a second cattle TSE. The disorder was pathologically characterized by the presence of PrP-immunopositive amyloid plaques, as opposed to the lack of amyloid deposition in typical BSE cases, and by a different pattern of regional distribution and topology of brain PrP(Sc) accumulation. In addition, Western blot analysis showed a PrP(Sc) type with predominance of the low molecular mass glycoform and a protease-resistant fragment of lower molecular mass than BSE-PrP(Sc). Strikingly, the molecular signature of this previously undescribed bovine PrP(Sc) was similar to that encountered in a distinct subtype of sporadic Creutzfeldt-Jakob disease.

Animals↗
PubMed2004

Painful neuropathy vasculitis in 2 patients with long-standing human immunodeficiency virus-1 infection.

We describe 2 most unusual cases of distal symmetrical painful polyneuropathy in patients with long-standing HIV-1 infection well controlled by HAART. Sural nerve biopsies revealed vasculitis in both cases and steroid therapy led to resolution of symptoms not influenced by analgesics and anti-inflammatory drugs. These unusual cases outline the importance of nerve biopsies in order to reach a diagnosis.

Adult↗