Michela Sabbatucci

Experimental studies of susceptibility of Italian Aedes albopictus to Zika virus.

We report a study on vector competence of an Italian population of Aedes albopictus for Zika virus (ZIKV). Ae. albopictus was susceptible to ZIKV infection (infection rate: 10%), and the virus could disseminate and was secreted in the mosquitos saliva (dissemination rate: 29%; transmission rate: 29%) after an extrinsic incubation period of 11 days. The observed vector competence was lower than that of an Ae. aegypti colony tested in parallel.

Clinical course of classic Kaposi's sarcoma in HIV-negative patients treated with the HIV protease inhibitor indinavir

HIV protease inhibitors have been shown to exert antiangiogenic and antitumor actions independently from their antiretroviral effect. Based on these studies, HIV-seronegative patients with classic Kaposis sarcoma were treated with indinavir and followed for clinical evolution, drug pharmacokinetics and Kaposis sarcoma biomarkers. A favorable clinical course was associated with high drug plasma levels, reduced production of basic fibroblast growth factor, lower numbers of circulating endothelial cells, and a decrease in antibody titers against human herpesvirus 8.

Toll-like receptor cross-talk in human monocytes regulates CC-chemokine production, antigen uptake and immune cell recruitment

Chemokines production in monocytes/macrophages is crucial in modulating immune responses generated through Toll-like receptor (TLR)-mediated recognition of microbes. During microbial onset, multiple pathogen-associated structures can be present at infection sites, and simultaneously trigger different TLRs. We report here that TLR3, TLR4 and TLR8 engagement induce CCL1, CCL2 and CCL4 production in freshly isolated monocytes. While differentiating cells maintain the capacity to secrete CCL2 and CCL4, CCL1 is no longer induced at later differentiation stages. Although different pairs of TLR agonists have been described to synergistically induce cytokine production in different cell types, agonist combinations cooperate in reducing CCL1 and CCL2, but not CCL4 secretion in freshly isolated monocytes, and fail to rescue CCL1 production at later differentiation stages. The effects of single, but not combined, TLR engagement on chemokine expression mostly occur at transcriptional level, and are IL-10 independent. Conversely, inhibition of CCL1 secretion upon combined TLR engagement is partially rescued by blocking IL-23. A different chemotactic activity of monocyte-conditioned medium on blood mononuclear cells as well as antigen uptake capacity of TLR agonist activated monocytes parallel the regulated production of chemokines. Overall, these findings indicate that simultaneous engagement of TLRs may lead to different patterns of chemokine expression depending on cellular differentiation state, chemokine, and TLR agonist combination. These different responses may be relevant for the distinct but complementary functions of monocytes and macrophages in the immune response, and may have important implications for the therapeutic manipulation of the innate immune system.

Nuclear Phosphoinositide-Specific Phospholipase C β1 Controls Cytoplasmic CCL2 mRNA Levels in HIV-1 gp120-Stimulated Primary Human Macrophages

HIV-1 envelope glycoprotein gp120 induces, independently of infection, the release of CCL2 from macrophages. In turn, this chemokine acts as an autocrine factor enhancing viral replication. In this study, we show for the first time that phosphoinositide-specific phospholipase C (PI-PLC) is required for the production of CCL2 triggered by gp120 in macrophages. Using a combination of confocal laser-scanner microscopy, pharmacologic inhibition, western blotting and fluorescence-activated cell sorter analysis, we demonstrate that gp120 interaction with CCR5 leads to nuclear localization of the PI-PLC β1 isozyme mediated by mitogen-activated protein kinase ERK-1/2. Notably, phosphatidylcholine-specific phospholipase C (PC-PLC), previously reported to be required for NF-kB-mediated CCL2 production induced by gp120 in macrophages, drives both ERK1/2 activation and PI-PLC β1 nuclear localization induced by gp120. PI-PLC β1 activation through CCR5 is also triggered by the natural chemokine ligand CCL4, but independently of ERK1/2. Finally, PI-PLC inhibition neither blocks gp120-mediated NF-kB activation nor overall accumulation of CCL2 mRNA, whereas it decreases CCL2 transcript level in the cytoplasm. These results identify nuclear PI-PLC β1 as a new intermediate in the gp120-triggered PC-PLC-driven signal transduction pathway leading to CCL2 secretion in macrophages. The finding that a concerted gp120-mediated signaling involving both PC- and PI-specific PLCs is required for the expression of CCL2 in macrophages suggests that this signal transduction pathway may also be relevant for the modulation of viral replication in these cells. Thus, this study may contribute to identify novel targets for therapeutic intervention in HIV-1 infection.

Endogenous CCL2 neutralization restricts HIV-1 replication in primary human macrophages by inhibiting viral DNA accumulation

Background: Macrophages are key targets of HIV-1 infection. We have previously described that the expression of CC chemokine ligand 2 (CCL2) increases during monocyte differentiation to macrophages and it is further up-modulated by HIV-1 exposure. Moreover, CCL2 acts as an autocrine factor that promotes viral replication in infected macrophages. In this study, we dissected the molecular mechanisms by which CCL2 neutralization inhibits HIV-1 replication in monocyte-derived macrophages (MDM), and the potential involvement of the innate restriction factors protein sterile alpha motif (SAM) histidine/aspartic acid (HD) domain containing 1 (SAMHD1) and apolipoprotein B mRNA-editing, enzyme-catalytic, polypeptide-like 3 (APOBEC3) family members. Results: CCL2 neutralization potently reduced the number of p24 Gag + cells during the course of either productive or single cycle infection with HIV-1. In contrast, CCL2 blocking did not modify entry of HIV-1 based Virus Like Particles, thus demonstrating that the restriction involves post-entry steps of the viral life cycle. Notably, the accumulation of viral DNA, both total, integrated and 2-LTR circles, was strongly impaired by neutralization of CCL2. Looking for correlates of HIV-1 DNA accumulation inhibition, we found that the antiviral effect of CCL2 neutralization was independent of the modulation of SAMHD1 expression or function. Conversely, a strong and selective induction of APOBEC3A expression, to levels comparable to those of freshly isolated monocytes, was associated with the inhibition of HIV-1 replication mediated by CCL2 blocking. Interestingly, the CCL2 neutralization mediated increase of APOBEC3A expression was type I IFN independent. Moreover, the transcriptome analysis of the effect of CCL2 blocking on global gene expression revealed that the neutralization of this chemokine resulted in the upmodulation of additional genes involved in the defence response to viruses. Conclusions: Neutralization of endogenous CCL2 determines a profound restriction of HIV-1 replication in primary MDM affecting post-entry steps of the viral life cycle with a mechanism independent of SAMHD1. In addition, CCL2 blocking is associated with induction of APOBEC3A expression, thus unravelling a novel mechanism which might contribute to regulate the expression of innate intracellular viral antagonists in vivo. Thus, our study may potentially lead to the development of new therapeutic strategies for enhancing innate cellular defences against HIV-1 and protecting macrophages from infection.

The CCL2/CCR2 Axis in the Pathogenesis of HIV-1 Infection: A New Cellular Target for Therapy?

The identification of chemokine receptors as necessary co-receptors for HIV entry into target cells represented a breakthrough in the understanding of the pathogenesis of this viral infection. Since this initial discovery, it was unraveled that chemokines, in addition to their role in blocking viral entry by binding to co-receptors, have other functions in HIV pathogenesis. Indeed, chemokines can either inhibit or enhance HIV replication, and these effects may involve both entry and post-entry events of the viral life cycle. Depending on the balance of their negative versus positive effects on HIV replication and spreading, chemokines contribute to different outcomes of HIV pathogenesis. CCL2 is unique among the chemokines in that mostly enhancing effects on viral replication and pathogenesis have been reported. Either HIV infection itself or exposure to viral products can induce the expression of this chemokine and of its receptor CCR2, and high levels of CCL2/CCR2 are indeed found in HIV-infected subjects. The CCL2/CCR2 axis is tightly linked to the high level of immune activation and inflammation that is the hallmark of HIV infection even in patients undergoing antiretroviral therapy. In addition, more direct effects of CCL2 on HIV replication are becoming apparent. Thus, modulation of CCL2/CCR2-driven effects may have significant impact on HIV disease progression. In this review, we will discuss the complex interaction between CCL2/CCR2 and HIV and the emerging therapeutic approaches based on the inhibition of this axis.

Molecular and Epidemiologic Analysis of Reemergent Salmonella enterica Serovar Napoli, Italy, 2011–2015

Human infections with Salmonella enterica serovar Napoli are uncommon in Europe. However, these infections represented 5.9% of salmonellosis cases in Italy during 2014–2015. The source of infection is unknown. We analyzed surveillance data and compared strain genetic similarities and found that contaminated vegetables and surface water are probable sources of human infection.

Biocompatible Anionic Polymeric Microspheres as Priming Delivery System for Effetive HIV/AIDS Tat-Based Vaccines

Here we describe a prime-boost regimen of vaccination in Macaca fascicularis that combines priming with novel anionic microspheres designed to deliver the biologically active HIV-1 Tat protein and boosting with Tat in Alum. This regimen of immunization modulated the IgG subclass profile and elicited a balanced Th1-Th2 type of humoral and cellular responses. Remarkably, following intravenous challenge with SHIV89.6Pcy243, vaccinees significantly blunted acute viremia, as compared to control monkeys, and this control was associated with significantly lower CD4+ T cell depletion rate during the acute phase of infection and higher ability to resume the CD4+ T cell counts in the post-acute and chronic phases of infection. The long lasting control of viremia was associated with the persistence of high titers anti-Tat antibodies whose profile clearly distinguished vaccinees in controllers and viremics. Controllers, as opposed to vaccinated and viremic cynos, exhibited significantly higher pre-challenge antibody responses to peptides spanning the glutamine-rich and the RGD-integrin-binding regions of Tat. Finally, among vaccinees, titers of anti-Tat IgG1, IgG3 and IgG4 subclasses had a significant association with control of viremia in the acute and post-acute phases of infection. Altogether these findings indicate that the Tat/H1D/Alum regimen of immunization holds promise for next generation vaccines with Tat protein or other proteins for which maintenance of the native conformation and activity are critical for optimal immunogenicity. Our results also provide novel information on the role of anti-Tat responses in the prevention of HIV pathogenesis and for the design of new vaccine candidates.

Bovine Lactoferrin-Induced CCL1 Expression Involves Distinct Receptors in Monocyte-Derived Dendritic Cells and Their Monocyte Precursors

Lactoferrin (LF) exhibits a wide range of immunomodulatory activities including modulation of cytokine and chemokine secretion. In this study, we demonstrate that bovine LF (bLF) up-modulates, in a concentration- and time-dependent manner, CCL1 secretion in monocytes (Mo) at the early stage of differentiation toward dendritic cells (DCs), and in fully differentiated immature Mo-derived DCs (MoDCs). In both cell types, up-modulation of CCL1 secretion is an early event following bLF-mediated enhanced accumulation of CCL1 transcripts. Notably, bLF-mediated up-regulation of CCL1 involves the engagement of distinct surface receptors in MoDCs and their Mo precursors. We show that bLF-mediated engagement of CD36 contributes to CCL1 induction in differentiating Mo. Conversely, toll-like receptor (TLR)2 blocking markedly reduces bLF-induced CCL1 production in MoDCs. These findings add further evidence for cell-specific differential responses elicited by bLF through the engagement of distinct TLRs and surface receptors. Furthermore, the different responses observed at early and late stages of Mo differentiation towards DCs may be relevant in mediating bLF effects in specific body districts, where these cell types may be differently represented in physiopathological conditions.

A severe outbreak of listeriosis in central Italy with a rare pulsotype associated with processed pork products

Purpose. From May 2015 to March 2016, an outbreak due to Listeria monocytogenes serotype 1/2a and clinical pulsotype never previously isolated in Europe occurred in central Italy, involving 24 confirmed clinical cases. The article provides a description of the outbreak and the investigation carried out by a multidisciplinary network. Methodology. Epidemiological and microbiological surveillance was conducted to confirm the outbreak and to detect the food vehicle of infection. The origin and destination of the implicated food and its ingredients were investigated by tracing‐back and ‐forward investigation. Results. Next‐generation sequencing confirmed the unique outbreak strain. On 4 January 2016, a L. monocytogenes strain with pulsotype indistinguishable from that isolated from clinical cases in the outbreak was detected in a sample of hog head cheese purchased from a retail supermarket by one of the patients. The hog head cheese was produced by a small meat processing plant in the Marche region, where microbiological investigation confirmed environmental and food contamination by the outbreak strain. Plant production was suspended and all contaminated batches of the hog head cheese were withdrawn from the market by 19 February by local health authority. We subsequently observed a sharp decline in clinical cases, the last being reported on 11 March 2016. Conclusion. The key factor in the timely conclusion of this investigation was intersectoral collaboration among epidemiologists, microbiologists, veterinarians, statisticians and health and food safety authorities at national, regional and local levels.