The anti-microbial peptide (Lin-SB056-1)2-K reduces pro-inflammatory cytokine release through interaction with Pseudomonas aeruginosa lipopolysaccharide

The ability of many anti-microbial peptides (AMPs) to modulate the host immune response has highlighted their possible therapeutic use to reduce uncontrolled inflammation during chronic infections. In the present study, we examined the anti-inflammatory potential of the semi-synthetic peptide lin-SB056-1 and its dendrimeric derivative (lin-SB056-1)2-K, which were previously found to have anti-microbial activity against Pseudomonas aeruginosa in in vivo-like models mimicking the challenging environment of chronically infected lungs (i.e., artificial sputum medium and 3-D lung mucosa model). The dendrimeric derivative exerted a stronger anti-inflammatory activity than its monomeric counterpart towards lung epithelial-and macrophage-cell lines stimulated with P. aeruginosa lipopolysaccharide (LPS), based on a marked decrease (up to 80%) in the LPS-induced production of different pro-inflammatory cytokines (i.e., IL-1β, IL-6 and IL-8). Accordingly, (lin-SB056-1)2-K exhibited a stronger LPS-binding affinity than its monomeric counterpart, thereby suggesting a role of peptide/LPS neutralizing interactions in the observed anti-inflammatory effect. Along with the anti-bacterial and anti-biofilm properties, the anti-inflammatory activity of (lin-SB056-1)2-K broadens its therapeutic potential in the context of chronic (biofilm-associated) infections

Escape of HIV-1-Infected Dendritic Cells from TRAIL-Mediated NK Cell Cytotoxicity during NK-DC Cross-Talk—A Pivotal Role of HMGB1

Early stages of Human Immunodeficiency Virus-1 (HIV-1) infection are associated with local recruitment and activation of important effectors of innate immunity, i.e. natural killer (NK) cells and dendritic cells (DCs). Immature DCs (iDCs) capture HIV-1 through specific receptors and can disseminate the infection to lymphoid tissues following their migration, which is associated to a maturation process. This process is dependent on NK cells, whose role is to keep in check the quality and the quantity of DCs undergoing maturation. If DC maturation is inappropriate, NK cells will kill them (“editing process”) at sites of tissue inflammation, thus optimizing the adaptive immunity. In the context of a viral infection, NK-dependent killing of infected-DCs is a crucial event required for early elimination of infected target cells. Here, we report that NK-mediated editing of iDCs is impaired if DCs are infected with HIV-1. We first addressed the question of the mechanisms involved in iDC editing, and we show that cognate NK-iDC interaction triggers apoptosis via the TNF-related apoptosis-inducing ligand (TRAIL)-Death Receptor 4 (DR4) pathway and not via the perforin pathway. Nevertheless, once infected with HIV-1, DCHIV become resistant to NK-induced TRAIL-mediated apoptosis. This resistance occurs despite normal amounts of TRAIL released by NK cells and comparable DR4 expression on DCHIV. The escape of DCHIV from NK killing is due to the upregulation of two anti-apoptotic molecules, the cellular-Flice like inhibitory protein (c-FLIP) and the cellular inhibitor of apoptosis 2 (c-IAP2), induced by NK-DCHIV cognate interaction. High-mobility group box 1 (HMGB1), an alarmin and a key mediator of NK-DC cross-talk, was found to play a pivotal role in NK-dependent upregulation of c-FLIP and c-IAP2 in DCHIV. Finally, we demonstrate that restoration of DCHIV susceptibility to NK-induced TRAIL killing can be obtained either by silencing c-FLIP and c-IAP2 by specific siRNA, or by inhibiting HMGB1 with blocking antibodies or glycyrrhizin, arguing for a key role of HMGB1 in TRAIL resistance and DCHIV survival. These findings provide evidence for a new strategy developed by HIV to escape immune attack, they challenge the question of the involvement of HMGB1 in the establishment of viral reservoirs in DCs, and they identify potential therapeutic targets to eliminate infected DCs

Lung-Directed Bacteriotherapy in Cystic Fibrosis: Could It Be an Option?

Due to the alarming spread of bacterial resistance to conventional drugs, the sole use of antibiotics to fight lung infections in cystic fibrosis (CF) is not resolutive, and novel strategies to replace or complement the use of antibiotics are highly desirable. Among these strategies, the use of probiotics is emerging as a particularly attractive approach. Probiotic administration via the oral route has demonstrated an ability to improve lung function and to reduce infection and exacerbation rates in CF patients through mechanisms mainly attributable to the gut–lung axis. Nevertheless, some studies reported no beneficial effect of probiotic intake suggesting that there is margin for improvement of such innovative intervention in CF. The present review aims to address the rationale behind probiotic use in CF and discuss the hypothesis that nasal/aerosol administration of appropriate probiotic strains may help to exert a direct beneficial effect on the respiratory tract, increasing the effectiveness of probiotic interventions in CF patients

Planktonic and biofilm-associated Pseudomonas aeruginosa and Staphylococcus epidermidis elicit differential human peripheral blood cell responses

Despite the considerable progress made in recent years, our understanding of the human immune response to microbial biofilms is still poor. The aim of the present study was to compare the in vitro response of human peripheral blood mononuclear cells (PBMC) to biofilms and planktonic cells of Pseudomonas aeruginosa and Staphylococcus epidermidis, two bacterial species particularly relevant in patients with cystic fibrosis or undergoing endovascular catheterization, respectively. PBMC isolated from healthy donors were co-cultured with 24 h-old biofilms or with exponentially growing cells of both species. Following 24 h of co-culture, the expression of early activation markers and the levels of cytokines in the culture supernatants were assessed by flow cytometry, while biofilm biomass and architecture were evaluated by crystal violet staining, CFU count, and confocal microscopy. Around 20% of PBMC was activated in response to both biofilms and planktonic cells of P. aeruginosa. In contrast, planktonic cells of S. epidermidis induced a statistically higher degree of activation than their biofilm counterpart (25% versus 15%; p < 0.01). P. aeruginosa biofilms stimulated pro-inflammatory (TNF-α, IL-1β, IFN-γ, and IL-6) and anti-inflammatory (IL-10) cytokine production at statistically significant levels higher than its planktonic counterpart, while an opposite trend was observed with S. epidermidis. Differences in the architecture of the biofilms and in the number of PBMC infiltrating the biofilms between the two bacterial species may at least partially explain these findings. Collectively, the results obtained highlighted marked differences in the host–cell response depending on the species and the mode of growth (biofilms versus planktonic cultures), allowing speculations on the different strategies adopted by P. aeruginosa and S. epidermidis to persist in the host during the course of chronic infections

3 CASES OF HUMAN HERPESVIRUS-6 LATENT INFECTION - INTEGRATION OF VIRAL GENOME IN PERIPHERAL-BLOOD MONONUCLEAR CELL-DNA

Saliva and peripheral blood mononuclear cells from three patients, two with lymphoproliferative disorders and one suffering from multiple sclerosis, were examined for the presence of human herpesvirus-6 (HHV-6) genome by using the polymerase chain reaction and Southern blot analysis. The search for anti-HHV-6 antibodies, carried out in the sera of the same cases by an immunofluorescence assay, was negative in two cases at the lowest dilution used (1:40). These three patients had a high number of HHV-6 specific sequences in uncultured peripheral blood mononuclear cells, which are thought to be a normal site of viral latency although, in healthy individuals, the infected cells are extremely rare. In order to gain some insight into the state of the viral genome in this latent HHV-6 infection, we used pulsed field gel electrophoresis to separate HHV-6 DNA directly from HHV-6 (strain GS) infected HSB-2 cells and from the peripheral blood mononuclear cells of these three patients. Our study showed the presence of intact viral genome, of the expected length of 170 kb, persisting as free extrachromosomal element in the HSB-2 cells but not in patients' peripheral blood mononuclear cells. On the other hand, in strong contrast with the results obtained in infected HSB-2 DNA, the restriction analysis of the three patients' peripheral blood mononuclear cell DNA showed fragments of molecular weight constantly higher than the 170 kb segment, indicating that the viral sequences are linked to high molecular weight cellular DNA. Our findings are consistent only with a latent infection in which HHV-6 is integrated in vivo and suggest that pulsed field gel electrophoresis analysis is well worth using to evaluate the presence of integrated, intact, or fragmented viral genomes in HHV-6 associated lymphoproliferative diseases and immune disorders

Three cases of human herpesvirus-6 latent infection: integration of viral genome in peripheral blood mononuclear cell DNA.

Saliva and peripheral blood mononuclear cells from three patients, two with lymphoproliferative disorders and one suffering from multiple sclerosis, were examined for the presence of human herpesvirus-6 (HHV-6) genome by using the polymerase chain reaction and Southern blot analysis. The search for anti-HHV-6 antibodies, carried out in the sera of the same cases by an immunofluorescence assay, was negative in two cases at the lowest dilution used (1:40). These three patients had a high number of HHV-6 specific sequences in uncultured peripheral blood mononuclear cells, which are thought to be a normal site of viral latency although, in healthy individuals, the infected cells are extremely rare. In order to gain some insight into the state of the viral genome in this latent HHV-6 infection, we used pulsed field gel electrophoresis to separate HHV-6 DNA directly from HHV-6 (strain GS) infected HSB-2 cells and from the peripheral blood mononuclear cells of these three patients. Our study showed the presence of intact viral genome, of the expected length of 170 kb, persisting as free extrachromosomal element in the HSB-2 cells but not in patients' peripheral blood mononuclear cells. On the other hand, in strong contrast with the results obtained in infected HSB-2 DNA, the restriction analysis of the three patients' peripheral blood mononuclear cell DNA showed fragments of molecular weight constantly higher than the 170 kb segment, indicating that the viral sequences are linked to high molecular weight cellular DNA.(ABSTRACT TRUNCATED AT 250 WORDS

PURINE METABOLISM AND B-LYMPHOCYTE DEVELOPMENT IN THE CHICKEN BURSA OF FABRICIUS

The activity of three enzymes involved in the salvage pathway of purine nucleosides-purine nucleoside phosphorylase (PNP), xanthine dehydrogenase (XDH), an hypoxanthine-guanine phosphoribosyl transferase (HGPRT)-was investigated in cellular fractions of the chicken bursa of Fabricius differentially enriched in epithelial cells or lymphocytes. Markedly increasing levels of PNP and XDH were observed along with the enrichment in epithelial cells together with a slight, though significant, decrease in HGPRT activity. By contrast, a dramatic fall in PNP and XDH activities was detected along with the enrichment in lymphocytes together with a slight, though significant, increase in HGPRT activity. This sharply different distribution of the three enzymes, all sharing hypoxanthine as a substrate, clearly indicates that lymphocytes preferentially channel hypoxanthine into the salvage and interconversion pathways, phosphorylating it to IMP, while epithelial cells rapidly catabolize such a purine base to uric acid. Moreover, epithelial cells, unlike lymphocytes, are able to retain high intracellular levels of both hypoxanthine and inosine. These results support the possibility that epithelial cells contribute to the normal development of bursal lymphocytes by supplying such actively proliferating cells with purine rings and at the same time by preventing them from accumulating potentially toxic high levels of purine nucleotides being able to rapidly eliminate excess hypoxanthine as uric acid from the bursa environment into the bloodstream