Elisabetta Straface

Leptin promotes differentiation and survival of human dendritic cells and licenses them for Th1 priming

Leptin is an adipocyte-derived hormone/cytokine that links nutrition, metabolism, and immune homeostasis. Leptin is capable of modulating several immune responses. However, the effect of leptin on dendritic cells (DCs) has not yet been recognized. Because DCs are instrumental in the development of immune responses, in this study, we evaluated the impact of leptin on DC activation. We demonstrated the presence of leptin receptor in human immature and mature DCs both at mRNA and protein level and its capacity to transduce leptin signaling leading to STAT-3 phosphorylation. We found no consistent modulation of DC surface molecules known to be critical for their APC function in response to leptin. In contrast, we found that leptin induces rearrangement of actin microfilaments, leading to uropod and ruffle formation. At a functional level, leptin up-regulates the IL-1β, IL-6, IL-12, TNF-α, and MIP-1α production. Coincident with this, leptin-treated DCs stimulate stronger heterologous T cell responses. Furthermore, we found that leptin down-regulates IL-10 production by DCs and drives naive T cell polarization toward Th1 phenotype. Finally, we found that leptin partly protects DCs from spontaneous and UVB-induced apoptosis. Consistent with the antiapoptotic effect of leptin, we observed the activation of NF-κB and a parallel up-regulation of bcl-2 and bcl-xL gene expression. These results provide new insights on the immunoregulatory function of leptin demonstrating its ability to improve DC functions and to promote DC survival. This is of relevance considering a potential application of leptin in immunotherapeutic approaches and its possible use as adjuvant in vaccination protocols.

Peroxynitrite induces senescence and apoptosis of red blood cells through the activation of aspartyl and cysteinyl proteases

Changes in the oxidative status of erythrocytes can reduce cell lifetime, oxygen transport, and delivery capacity to peripheral tissues and have been associated with a plethora of human diseases. Among reactive oxygen and nitrogen species of importance in red blood cell (RBC) homeostasis, superoxide and nitric oxide radicals play a key role. In the present work, we evaluated subcellular effects induced by peroxynitrite, the product of the fast reaction between superoxide and nitric oxide. Peroxynitrite induced 1) oxidation of oxyhemoglobin to methemoglobin, 2) cytoskeleton rearrangement, 3) ultrastructural alterations, and 4) altered expression of band‐3 and decreased expression of glycophorin A. With respect to control cells, this occurred in a significantly higher percentage of human RBC (∼40%). The presence of antioxidants inhibited these modifications. Furthermore, besides these senescence‐associated changes, other important modifications, absent in control RBC and usually associated with apoptotic cell death, were detected in a small but significant subset of peroxynitrite‐exposed RBC (∼7%). Active protease cathepsin E and μ‐calpain increased; activation of caspase 2 and caspase 3 was detected; and phosphatidylserine externalization, an early marker of apoptosis, was observed. Conversely, inhibition of cathepsin E, μ‐calpain, as well as caspase 2 and 3 by specific inhibitors resulted in a significant impairment of erythrocyte “apoptosis.” Altogether, these results indicate that peroxynitrite, a milestone of redox‐mediated damage in human pathology, can hijack human RBC toward senescence and apoptosis by a mechanism involving both cysteinyl and aspartyl proteases.

C3-Fullero-tris-Methanodicarboxylic Acid Protects Cerebellar Granule Cells from Apoptosis

Buckminsterfullerenols were recently investigated for their protective properties in different models of acute and chronic neurodegeneration. We tested C3‐fullero‐tris‐methanodicarboxylic acid in our in vitro model of apoptotic neuronal death, which consists of shifting the culture K+ concentration from 25 to 5 mM for rat cerebellar granule cells. The impairment of mitochondrial respiratory function as well as chromatin derangement and fragmentation of DNA in apoptotic oligonucleosomes that occur in these conditions were protected by this compound in a concentration‐dependent way. To assess whether antioxidant activity could account for the rescue of cerebellar granule cells from apoptosis, we tested the fullerene derivative under FeSO4‐induced oxidative stress and found significant protection. Thus, we visualized membrane and cytoplasmic peroxides and reactive oxygen species and found a significant reduction of the species after 24 h in 5 mM K+ with the fullerene derivative. Such evidence suggests that this compound exerts a protective role in cerebellar granule cell apoptosis, likely reducing the oxidative stress.

Decreased susceptibility to oxidative stress-induced apoptosis of peripheral blood mononuclear cells from healthy elderly and centenarians

The susceptibility to undergo apoptosis of fresh human peripheral blood mononuclear cells (PBMCs) from three groups of healthy donors of different ages: young people (19-40 years), old people (65-85 years) and centenarians was assessed. Apoptosis was induced by 2-deoxy-D-ribose (dRib), an agent which induces apoptosis in quiescent PBMCs by interfering with cell redox status and mitochondrial membrane potential (MMP). Our major finding is that an inverse correlation emerged between the age of the donors and the propensity of their PBMCs to undergo dRib-induced apoptosis. PBMCs from old people and centenarians also showed an increased resistance to dRib-induced glutathione depletion and a decreased tendency to lose MMP. The anti-apoptotic molecule Bcl-2 was similarly expressed in PBMCs from the three age groups. Moreover, the plasma level of the stable product of transglutaminase, epsilon(gamma-glutamyl)lysine isodipeptide, a marker of total body apoptotic rate, was decreased in centenarians compared to young and elderly people. On the whole, these findings suggest that physiological aging is characterised by a decreased tendency to undergo apoptosis, a phenomenon likely resulting from adaptation to lifelong exposure to damaging agents, such as reactive oxygen species, and may contribute to one of the major phenomena of immunosenescence, i.e. the progressive accumulation of memory/effector T cells.

HIV-1 Nef triggers Vav-mediated signaling pathway leading to functional and morphological differentiation of dendritic cells.

The accessory HIV‐1 Nef protein plays a key role in AIDS pathogenesis. We recently demonstrated that exogenous Nef triggers phenotypic and functional differentiation of immature dendritic cells (DCs). Here we investigated whether the Nef‐induced DC differentiation occurs with morphological remodeling and have focused on the interference of Nef in the signaling pathways that regulates DC maturation. We found that exogenous Nef enters immature DCs, promoting their functional and morphological differentiation. Specifically, Nef promotes interleukin (IL) ‐12 release, which closely fits with nuclear factor (NF) ‐κB activation. Nef induces rearrangement of actin microfilaments, leading to uropod and ruffle formation. Moreover, Nef increases the capacity of DCs to form clusters with allogeneic CD4+ T cells, improving immunological synapse formation. Searching for molecules involved in Nef‐triggered signaling pathways driving the DC maturation, we found that Nef targets Vav and promotes its tyrosine phosphorylation, associated with its nucleus‐to‐cytoplasm redistribution. This has a direct effect on Vav guanine nucleotide exchange factor activity for the small GTPase Rac1. We hypothesize that targeting Vav, Nef modulates both early signaling events (such as cytoskeletal rearrangement) and delayed responses (such as transcriptional regulation), promoting DC differentiation. Our results highlight how Nef may enhance T lymphocyte activation, thus fostering virus dissemination, manipulating the DC arm of the immune response.—Quaranta, M. G., Mattioli, B., Spadaro, F., Straface, E., Giordani, L., Ramoni, C., Malorni, W., Viora, M. HIV‐1 Nef triggers Vav‐mediated signaling pathway leading to functional and morphological differentiation of dendritic cells. FASEB J. 17, 2025–2036 (2003)

Wheat gliadin induces apoptosis of intestinal cells via an autocrine mechanism involving Fas^Fas ligand pathway

Wheat gliadin and other cereal prolamins have been said to be involved in the pathogenic damage of the small intestine in celiac disease via the apoptosis of epithelial cells. In the present work we investigated the mechanisms underlying the pro‐apoptotic activity exerted by gliadin‐derived peptides in Caco‐2 intestinal cells, a cell line which retains many morphological and enzymatic features typical of normal human enterocytes. We found that digested peptides from wheat gliadins (i) induce apoptosis by the CD95/Fas apoptotic pathway, (ii) induce increased Fas and FasL mRNA levels, (iii) determine increased FasL release in the medium, and (iv) that gliadin digest‐induced apoptosis can be blocked by Fas cascade blocking agents, i.e. targeted neutralizing antibodies. This favors the hypothesis that gliadin could activate an autocrine/paracrine Fas‐mediated cell death pathway. Finally, we found that (v) a small peptide (1157 Da) from durum wheat, previously proposed for clinical practice, exerted a powerful protective activity against gliadin digest cytotoxicity.

Are the available experimental models of type 2 diabetes appropriate for a gender perspective

Several experimental models have so far been developed to improve our knowledge of the pathogenetic mechanisms of type 2 diabetes mellitus (T2D), to determine the possible pharmacological targets of this disease and to better evaluate diabetes-associated complications, e.g. the cardiovascular disease. In particular, the study of T2D gained the attention of several groups working with different animal species: rodents, cats or pigs, as well as other non-human primate species. Each of these species provided useful and different clues. However, T2D has to be considered as a gender-associated disease: sex differences play in fact a key role in the onset as well as in the progression of the disease and a higher mortality for cardiovascular diseases is detected in diabetic women with respect to men. The results obtained from all the available animal models appear to only partially address this issue so that the search for more precise information in this respect appears to be mandatory. In this review we summarize these concepts and literature in the field and propose a reappraisal of the various animal models for a study of T2D that would take into consideration a gender perspective.

Type I Interferon Gene Transfer Sensitizes Melanoma Cells to Apoptosis via a Target Activity on Mitochondrial Function

Our previous article reported that retroviral transduction of human type I consensus interferon-coding sequence into two human melanoma cells increased their susceptibility to cisplatin-induced apoptosis. Importantly, primary melanoma cells were significantly more sensitive to cisplatin-induced apoptosis with respect to metastatic melanoma cells. The aim of this study was to elucidate the subcellular mechanisms involved in this interferon-induced apoptotic proneness. Our results indicate that 1) cisplatin-induced apoptosis can be referred to as the type II apoptosis, ie, to the mitochondrially driven cascade; 2) treatment of interferon-producing melanoma cells with other type II apoptotic stimuli, such as radiation or staurosporine, also resulted in massive apoptosis, whereas type I stimuli, ie, anti-Fas, were ineffective; 3) interferon sensitization involved the caspase cascade in primary melanoma cells and the alternative pathway represented by cathepsin-mediated apoptosis in metastatic melanoma cells; 4) interferon production sensitizes cells to apoptosis by inducing, as the earliest event, mitochondrial membrane hyperpolarization. These results suggest that constitutive production of type I interferon by melanoma cells can act as an intracellular booster capable of increasing cell proneness to apoptosis by specifically modifying mitochondrial homeostasis and independently from the apoptotic cascade involved.

Rho-dependent cell spreading activated by E.coli cytotoxic necrotizing factor 1 hinders apoptosis in epithelial cells

Cell-cell and cell-matrix interactions play a pivotal role in numerous cell functions including cell survival and death. In this work, we report evidence that the Rho-dependent cell spreading activated by a protein toxin from E. coli, the cytotoxic necrotizing factor 1 (CNF1), is capable of hindering apoptosis in HEp-2 cells. In addition to the promotion of cell spreading, CNF1 protects cells from the experimentally-induced rounding up and detachment and improves the ability of cells to adhere to each other and to the extracellular matrix by modulating the expression of proteins related to cell adhesion. In particular, the expression of integrins such as α5, α6 and αv, as well as of some heterotypic and homotypic adhesion-related proteins such as the Focal Adhesion Kinase, E-cadherin, α and β catenins were significantly increased in cells exposed to CNF1. Our results suggest, however, that the promotion of Rho-dependent cell spreading is the key mechanism in protecting cells against apoptosis rather than cell adhesion per se. A toxin inducing cell spreading without activating Rho, such as Cytochalasin B, was in fact ineffective in favouring cell survival. These data are of relevance (i) for the understanding of the role of the actin-dependent and especially Rho-dependent cellular activities involved in apoptosis regulation and (ii) in providing some clues to understanding the mechanisms by which bacteria, by controlling cell fate, might exert their pathogenic activity.

Oxidative imbalance and cathepsin D changes as peripheral blood biomarkers of Alzheimer disease: A pilot study

Markers of oxidative stress in peripheral blood from patients with Alzheimer disease (AD) were analyzed. Thirty‐three AD patients were recruited. Plasma antioxidant power (AOP), plasma Cystatin C as well as Cathepsin D in PBL were evaluated. We found that the AOP levels were significantly decreased in AD patients if compared to healthy donors, while the plasma level of Cystatin C was significantly higher. Importantly, a significantly decreased expression of Cathepsin D in PBL was also observed. These results suggest that oxidative imbalance in the peripheral blood of AD patients could mirror oxidative changes previously described in the central nervous system.