Ann Maria Clemente

Low molecular weight, non-peptidic agonists of TrkA receptor with NGF-mimetic activity

Exploitation of the biologic activity of neurotrophins is desirable for medical purposes, but their protein nature intrinsically bears adverse pharmacokinetic properties. Here, we report synthesis and biologic characterization of a novel class of low molecular weight, non-peptidic compounds with NGF (nerve growth factor)-mimetic properties. MT2, a representative compound, bound to Trk (tropomyosin kinase receptor)A chain on NGF-sensitive cells, as well as in cell-free assays, at nanomolar concentrations and induced TrkA autophosphorylation and receptor-mediated internalization. MT2 binding involved at least two amino-acid residues within TrkA molecule. Like NGF, MT2 increased phosphorylation of extracellular signal-regulated kinase1/2 and Akt proteins and production of MKP-1 phosphatase (dual specificity phosphatase 1), modulated p38 mitogen-activated protein kinase activation, sustained survival of serum-starved PC12 or RDG cells, and promoted their differentiation. However, the intensity of such responses was heterogenous, as the ability of maintaining survival was equally possessed by NGF and MT2, whereas the induction of differentiation was expressed at definitely lower levels by the mimetic. Analysis of TrkA autophosphorylation patterns induced by MT2 revealed a strong tyrosine (Tyr)490 and a limited Tyr785 and Tyr674/675 activation, findings coherent with the observed functional divarication. Consistently, in an NGF-deprived rat hippocampal neuronal model of Alzheimer Disease, MT2 could correct the biochemical abnormalities and sustain cell survival. Thus, NGF mimetics may reveal interesting investigational tools in neurobiology, as well as promising drug candidates.

Sex differences in the response to viral infections: TLR8 and TLR9 ligand stimulation induce higher IL10 production in males.

Background Susceptibility to viral infections as well as their severity are higher in men than in women. Heightened antiviral responses typical of women are effective for rapid virus clearance, but if excessively high or prolonged, can result in chronic/inflammatory pathologies. We investigated whether this variability could be in part attributable to differences in the response to the Toll-Like Receptors (TLR) more involved in the virus recognition. Methods Cytokine production by peripheral blood mononuclear cells (PBMCs) from male and female healthy donors after stimulation with Toll-like receptors (TLR) 3, 7, 8, 9 ligands or with viruses (influenza and Herpes-simplex-1) was evaluated. Results Compared to females, PBMCs from males produced not only lower amounts of IFN-α in response to TLR7 ligands but also higher amounts of the immunosuppressive cytokine IL10 after stimulation with TLR8 and TLR9 ligands or viruses. IL10 production after TLR9 ligands or HSV-1 stimulation was significantly related with plasma levels of sex hormones in both groups, whereas no correlation was found in cytokines produced following TLR7 and TLR8 stimulation. Conclusions Given the role of an early production of IL10 by cells of innate immunity in modulating innate and adaptive immune response to viruses, we suggest that sex-related difference in its production following viral nucleic acid stimulation of TLRs may be involved in the sex-related variability in response to viral infections.

Modulation of the Immune and Inflammatory Responses by Plasmodium falciparum Schizont Extracts: Role of Myeloid Dendritic Cells in Effector and Regulatory Functions of CD4+ Lymphocytes

ABSTRACT The optimal immune response to malaria infection comprises rapid induction of inflammatory responses promptly counteracted by regulatory mechanisms to prevent immunopathology. To evaluate the role of dendritic cells (DC) in the balance of parasite-induced inflammatory/anti-inflammatory mechanisms, we studied the activity of monocyte-derived dendritic cells (MDDC), previously exposed to soluble extracts of Plasmodium falciparum-infected red blood cells (PfSE), in the differentiation of CD4 cells isolated from donors never exposed to malaria infection. We show that MDDC exposed to PfSE are extremely efficient to induce a contemporary differentiation of TH1 effector cells and T regulatory (Treg) cells in CD4 T cells even when exposed to low concentrations of parasitic extracts. Treg cells induced by MDDC infected with PfSE (MDDC-PfSE) produce transforming growth factor beta (TGF-β) and interleukin 10 (IL-10) and are endowed with strong suppressive properties. They also show phenotypical and functional peculiarities, such as the contemporary expression of markers of Treg and TH1 differentiation and higher sensitivity to TLR4 ligands both inducing an increasing production of suppressive cytokines. On the whole, our data indicate that MDDC exposed to PfSE orchestrate a well-balanced immune response with timely differentiation of TH1 and Treg cells in CD4 cells from nonimmune donors and suggest that, during the infection, the role of MDCC could be particularly relevant in low-parasitemia conditions.

Characterization of cervico-vaginal microbiota in women developing persistent high-risk Human Papillomavirus infection

Changes in cervico-vaginal microbiota with Lactobacillus depletion and increased microbial diversity facilitate human papillomavirus (HPV) infection and might be involved in viral persistence and cancer development. To define the microbial Community State Types (CSTs) associated with high-risk HPV−persistence, we analysed 55 cervico-vaginal samples from HPV positive (HPV+) women out of 1029 screened women and performed pyrosequencing of 16S rDNA. A total of 17 samples from age-matched HPV negative (HPV−) women were used as control. Clearance or Persistence groups were defined by recalling women after one year for HPV screening and genotyping. A CST IV subgroup, with bacterial genera such as Gardnerella, Prevotella, Megasphoera, Atopobium, frequently associated with anaerobic consortium in bacterial vaginosis (BV), was present at baseline sampling in 43% of women in Persistence group, and only in 7.4% of women in Clearance group. Atopobium genus was significantly enriched in Persistence group compared to the other groups. Sialidase-encoding gene from Gardnerella vaginalis, involved in biofilm formation, was significantly more represented in Persistence group compared to the other groups. Based on these data, we consider the CST IV-BV as a risk factor for HPV persistence and we propose Atopobium spp and sialidase gene from G. vaginalis as microbial markers of HPV−persistence.

Effects of soluble extracts from Leishmania infantum promastigotes, Toxoplasma gondii tachyzoites on TGF-β mediated pathways in activated CD4+ T lymphocytes

Interference with transforming growth factor-β-mediated pathways helps several parasites to survive for long periods in immunocompetent hosts. Macrophages and dendritic cells infected by Toxoplasma, Leishmania and Plasmodium spp. produce large amounts of transforming growth factor-β and induce the differentiation of antigen-specific T-regulatory cells. Mechanisms not mediated by antigen-presentation could also account for the expansion of T-regulatory cells in parasitic diseases and they also might be mediated through transforming growth factor-β-receptor activated pathways. We explored the properties of soluble extracts from Leishmania infantum promastigotes, Toxoplasma gondii tachyzoites, Trichinella spiralis muscle larvae to expand the pool of T-regulatory cells in a population of polyclonally activated T cells in the absence of accessory cells, and compared their effects to those induced by Plasmodium falciparum extracts. Similarly to P. falciparum, L. infantum extracts activate the latent soluble form of transforming growth factor-β and that bound to the membrane of activated T lymphocytes. The interaction of the active cytokine with transforming growth factor-β receptor induces Foxp3 expression by activated lymphocytes, favoring their conversion through the T-regulatory phenotype. Both Toxoplasma gondii and L. infantum extracts are able to induce transforming growth factor-β production by activated T cells in the absence of accessory cells.

Stimulatory interactions between human coronary smooth muscle cells and dendritic cells.

Despite inflammatory and immune mechanisms participating to atherogenesis and dendritic cells (DCs) driving immune and non-immune tissue injury response, the interactions between DCs and vascular smooth muscle cells (VSMCs) possibly relevant to vascular pathology including atherogenesis are still unclear. To address this issue, immature DCs (iDCs) generated from CD14+ cells isolated from healthy donors were matured either with cytokines (mDCs), or co-cultured (ccDCs) with human coronary artery VSMCs (CASMCs) using transwell chambers. Co-culture induced DC immunophenotypical and functional maturation similar to cytokines, as demonstrated by flow cytometry and mixed lymphocyte reaction. In turn, factors from mDCs and ccDCs induced CASMC migration. MCP-1 and TNFα, secreted from DCs, and IL-6 and MCP-1, secreted from CASMCs, were primarily involved. mDCs adhesion to CASMCs was enhanced by CASMC pre-treatment with IFNγ and TNFα ICAM-1 and VCAM-1 were involved, since the expression of specific mRNAs for these molecules increased and adhesion was inhibited by neutralizing antibodies to the counter-receptors CD11c and CD18. Adhesion was also inhibited by CASMC pre-treatment with the HMG-CoA-reductase inhibitor atorvastatin and the PPARγ agonist rosiglitazone, which suggests a further mechanism for the anti-inflammatory action of these drugs. Adhesion of DCs to VSMCs was shown also in vivo in rat carotid 7 to 21 days after crush and incision injury. The findings indicate that DCs and VSMCs can interact with reciprocal stimulation, possibly leading to perpetuate inflammation and vascular wall remodelling, and that the interaction is enhanced by a cytokine-rich inflammatory environment and down-regulated by HMGCoA-reductase inhibitors and PPARγ agonists.

Differential Redox State Contributes to Sex Disparities in the Response to Influenza Virus Infection in Male and Female Mice

Influenza virus replicates intracellularly exploiting several pathways involved in the regulation of host responses. The outcome and the severity of the infection are thus strongly conditioned by multiple host factors, including age, sex, metabolic, and redox conditions of the target cells. Hormones are also important determinants of host immune responses to influenza and are recently proposed in the prophylaxis and treatment. This study shows that female mice are less susceptible than males to mouse-adapted influenza virus (A/PR8/H1N1). Compared with males, PR8-infected females display higher survival rate (+36%), milder clinical disease, and less weight loss. They also have milder histopathological signs, especially free alveolar area is higher than that in males, even if pro-inflammatory cytokine production shows slight differences between sexes; hormone levels, moreover, do not vary significantly with infection in our model. Importantly, viral loads (both in terms of viral M1 RNA copies and tissue culture infectious dose 50%) are lower in PR8-infected females. An analysis of the mechanisms contributing to sex disparities observed during infection reveals that the female animals have higher total antioxidant power in serum and their lungs are characterized by increase in (i) the content and biosynthesis of glutathione, (ii) the expression and activity of antioxidant enzymes (peroxiredoxin 1, catalase, and glutathione peroxidase), and (iii) the expression of the anti-apoptotic protein Bcl-2. By contrast, infected males are characterized by high expression of NADPH oxidase 4 oxidase and phosphorylation of p38 MAPK, both enzymes promoting viral replication. All these factors are critical for cell homeostasis and susceptibility to infection. Reappraisal of the importance of the host cell redox state and sex-related effects may be useful in the attempt to develop more tailored therapeutic interventions in the fight against influenza.

Adipose-derived stem cells decrease pain in a rat model of oxaliplatin-induced neuropathy: Role of VEGF-A modulation

ABSTRACT Oxaliplatin therapy of colorectal cancer induces a dose‐dependent neuropathic syndrome in 50% of patients. Pharmacological treatments may offer limited relief; scientific efforts are needed for new therapeutic approaches. Therefore we evaluated in a preclinical setting the pain relieving properties of mesenchymal stem cells and its secretome. Rat adipose stem cells (rASCs) were administered in a rat model of oxaliplatin‐induced neuropathy. A single intravenous injection of rASCs reduced oxaliplatin‐dependent mechanical hypersensitivity to noxious and non‐noxious stimuli taking effect 1 h after administration, peaking 6 h thereafter and lasting 5 days. Cell‐conditioned medium was ineffective. Repeated rASCs injections every 5 days relieved pain each time with a comparable effect. Labeled rASCs were detected in the bloodstream 1 and 3 h after administration and found in the liver 24 h thereafter. In oxaliplatin‐treated rats, the plasma concentration of vascular endothelial growth factor (pan VEGF‐A) was increased while the isoform VEGF165b was upregulated in the spinal cord. Both alterations were reverted by rASCs. The anti‐VEGF‐A monoclonal antibody bevacizumab (intraperitoneally) reduced oxaliplatin‐dependent pain. Studying the peripheral and central role of VEGF165b in pain, we determined that the intraplantar and intrathecal injection of the growth factor induced a pro‐algesic effect. In the oxaliplatin neuropathy model, the intrathecal infusion of bevacizumab, anti‐rat VEGF165b antibody and rASCs reduced pain. Adult adipose mesenchymal stem cells could represent a novel approach in the treatment of neuropathic pain. The regulation of VEGF‐A is suggested as an effective mechanism in the complex response orchestrated by stem cells against neuropathy. Graphical abstract Figure. No Caption available. HighlightsAdipose stem cells relieve neuropathic pain induced by chemotherapy.The pain killer effect has a rapid onset and lasts until 5 days.High levels of VEGF are related to oxaliplatin‐dependent neuropathic pain.The pain relieving effects of stem cells are related to a decrease of VEGF levels.

Differences in Inflammatory Response Induced by Two Representatives of Clades of the Pandemic ST258 Klebsiella pneumoniae Clonal Lineage Producing KPC-Type Carbapenemases

ST258-K. pneumoniae (ST258-KP) strains, the most widespread multidrug-resistant hospital-acquired pathogens, belong to at least two clades differing in a 215 Kb genomic region that includes the cluster of capsule genes. To investigate the effects of the different capsular phenotype on host-pathogen interactions, we studied representatives of ST258-KP clades, KKBO-1 and KK207-1, for their ability to activate monocytes and myeloid dendritic cells from human immune competent hosts. The two ST258-KP strains strongly induced the production of inflammatory cytokines. Significant differences between the strains were found in their ability to induce the production of IL-1β: KK207-1/clade I was much less effective than KKBO-1/clade II in inducing IL-1β production by monocytes and dendritic cells. The activation of NLRP3 inflammasome pathway by live cells and/or purified capsular polysaccharides was studied in monocytes and dendritic cells. We found that glibenclamide, a NLRP3 inhibitor, inhibits more than 90% of the production of mature IL-1β induced by KKBO1 and KK207-1. KK207-1 was always less efficient compared to KKBO-1 in: a) inducing NLRP3 and pro-IL-1β gene and protein expression; b) in inducing caspase-1 activation and pro-IL-1β cleavage. Capsular composition may play a role in the differential inflammatory response induced by the ST258-KP strains since capsular polysaccharides purified from bacterial cells affect NLRP3 and pro-IL-1β gene expression through p38MAPK- and NF-κB-mediated pathways. In each of these functions, capsular polysaccharides from KK207-1 were significantly less efficient compared to those purified from KKBO-1. On the whole, our data suggest that the change in capsular phenotype may help bacterial cells of clade I to partially escape innate immune recognition and IL-1β-mediated inflammation.

Differential Th17 response induced by the two clades of the pandemic ST258 Klebsiella pneumoniae clonal lineages producing KPC-type carbapenemase

The spread of KPC-type carbapenemases is mainly attributed to the global dissemination of Klebsiella pneumoniae (KP) strains belonging to the clonal group (CG) 258, including sequence type (ST) 258 and other related STs. Two distinct clades of CG258-KP have evolved, which differ mainly for the composition of their capsular polysaccharides, and recent studies indicate that clade 1 evolved from an ancestor of clade 2 by recombination of a genomic fragment carrying the capsular polysaccharide (cps) locus. In this paper, we investigated the ability of two ST258-KP strains, KKBO-1 and KK207-1, selected as representatives of ST258-KP clade 2 and clade 1, respectively, to activate an adaptive immune response using ex vivo-stimulation of PBMC from normal donors as an experimental model. Our data showed that KKBO-1 (clade 2) induces a Th17 response more efficiently than KK207-1 (clade 1): the percentage of CD4+IL17+ cells and the production of IL-17A were significantly higher in cultures with KKBO-1 compared to cultures with KK207-1. While no differences in the rate of bacterial internalization or in the bacteria-induced expression of CD86 and HLA-DR by monocytes and myeloid dendritic cells were revealed, we found that the two strains significantly differ in inducing the production of cytokines involved in the adaptive immune response, as IL-1β, IL-23 and TNF-α, by antigen-presenting cells, with KKBO-1 being a more efficient inducer than KK207-1. The immune responses elicited by KK207-1 were comparable to those elicited by CIP 52.145, a highly virulent K. pneumoniae reference strain known to escape immune-inflammatory responses. Altogether, present results suggest that CG258-KP of the two clades are capable of inducing a different response of adaptive immunity in the human host.