Search

Antibodies targeting Receptor Binding Domain (RBD) of SARS-CoV-2 have been suggested to account for the majority of neutralizing activity in COVID-19 convalescent sera and several neutralizing antibodies (nAbs) have been isolated, characterized and proposed as emergency therapeutics in the form of monoclonal antibodies (mAbs). However, SARS-CoV-2 variants are rapidly spreading worldwide from the sites of initial identification. The variants of concern (VOC) B.1.1.7 (Alpha), B.1.351 (Beta), P.1 (Gamma) and B.1.167.2 (Delta) showed mutations in the SARS-CoV-2 spike protein potentially able to cause escape from nAb responses with a consequent reduction of efficacy of vaccines and mAbs-based therapy. We produced the recombinant RBD (rRBD) of SARS-CoV-2 spike glycoprotein from the Wuhan-Hu 1 reference sequence in a mammalian system, for mice immunization to isolate new mAbs with neutralizing activity. Here we describe four mAbs that were able to bind the rRBD in Enzyme-Linked Immunosorbent Assay and the transmembrane full-length spike protein expressed in HEK293T cells by flow cytometry assay. Moreover, the mAbs recognized the RBD in supernatants of SARS-CoV-2 infected VERO E6 cells by Western Blot under non-reducing condition or in supernatants of cells infected with lentivirus pseudotyped for spike protein, by immunoprecipitation assay. Three out of four mAbs lost their binding efficiency to completely N-deglycosylated rRBD and none was able to bind the same recombinant protein expressed in Escherichia coli, suggesting that the epitopes recognized by three mAbs are generated by the conformational structure of the glycosylated native protein. Of particular relevance, three mAbs were able to inhibit Wuhan SARS-CoV-2 infection of VERO E6 cells in a plaque-reduction neutralization test and the Wuhan SARS-CoV-2 as well as the Alpha, Beta, Gamma and Delta VOC in a pseudoviruses-based neutralization test. These mAbs represent important additional tools for diagnosis and therapy of COVID-19 and may contribute to the understanding of the functional structure of SARS-CoV-2 RBD.

The Istituto Superiore di Sanità (ISS) has been asked for rapid technical and scientific advice to the State and Regions during Sars-CoV-2 pandemic preparedness.An ad hoc Working Group on Scientific Literature updates (WG SL) was set up at ISS (March-May 2020) to screen pre-prints and peer reviewed papers from arXiv, medRxiv, bioRxiv, and Pubmed to provide a real time knowledge and empirical evidence addressed to health-workers.The WG SL screened a total of 4,568 pre-prints and 15,590 peer reviewed papers, delivered as daily summary report of pre-print selection for ISS President activity in the National Scientific Technical Committee framework and a weekly open access publication (COVID Contents) on peer-reviewed papers of interest for health professionals, monitored by a satisfaction questionnaire.Promoting heath literacy, with a cross-cutting approach is a powerful heritage of Public Health Institutes and a proven effective non pharmacological intervention.

Norovirus (NoV) is now recognized as a major cause of gastroenteritis outbreaks, worldwide. Norovirus replication mechanisms are still poorly understood, mainly because a reliable cell culture system is still lacking. The present study aims at understanding some aspects of the immune response against norovirus, and particularly the capacity of virus like particles (VLPs) from an Italian strain, belonging to the GII.4 genotype predominating worldwide, to interact with target cells via Toll Like Receptors (TLRs). The capacity of GII.4 NoV VLPs to interact and cause the activation of TLR2, 4 and 5 was studied in recombinant HEK cells. The results obtained show the ability of GII.4 NoV VLPs to induce activation of TLR2 and 5. The results on TLRs activation confirm that GII.4 NoV VLPs interact with TLR2 and 5, that may represent putative receptors and play a role in NoV infection of intestinal cells.

The effective discovery of clinically relevant tumor antigens holds a fundamental role for the development of new diagnostic tools and anticancer immunotherapies. D393-CD20 mRNA is absent from normal resting B cells but present in various malignant or transformed B cells. CD8+T lymphocytes play a central role in immunity to cancer. In this study, we want use from T CD8+ against D393-CD20 for effect in RAMOS cell line. After isolation and expanding of specific TCD8 + Lymphocyte against D393-CD20 antigen, for examining the effect of specialized T lymphocyte clone of D393-CD20 antigen on RAMOS cell line, we co-cultured them together, and the rate of apoptosis were examined by flow cytometry and cytotoxicity techniques by using MTT technique. We observed that specialized TCD8+ lymphocyte of D393-CD20 antigen can induce apoptosis in malignant B-lymphocytes, and this antigen can be a proper target for immunotherapy.

The therapeutic success of BRAF inhibitors (BRAFi) and MEK inhibitors (MEKi) in BRAF-mutant melanoma is limited by the emergence of drug resistance, and several lines of evidence suggest that changes in the tumor microenvironment can play a pivotal role in acquired resistance. The present study focused on secretome profiling of melanoma cells sensitive or resistant to the BRAFi vemurafenib. Proteomic and cytokine/chemokine secretion analyses were performed in order to better understand the interplay between vemurafenib-resistant melanoma cells and the tumor microenvironment. We found that vemurafenib-resistant melanoma cells can influence dendritic cell (DC) maturation by modulating their activation and cytokine production. In particular, human DCs exposed to conditioned medium (CM) from vemurafenib-resistant melanoma cells produced higher levels of pro-inflammatory cytokines&mdash, that potentially facilitate melanoma growth&mdash, than DCs exposed to CM derived from parental drug-sensitive cells. Bioinformatic analysis performed on proteins identified by mass spectrometry in the culture medium from vemurafenib-sensitive and vemurafenib-resistant melanoma cells suggests a possible involvement of the proteasome pathway. Moreover, our data confirm that BRAFi-resistant cells display a more aggressive phenotype compared to parental ones, with a significantly increased production of interferon-&gamma, interleukin-8, vascular-endothelial growth factor, CD147/basigin, and metalloproteinase 2 (MMP-2). Plasma levels of CD147/basigin and MMP-2 were also measured before the start of therapy and at disease progression in a small group of melanoma patients treated with vemurafenib or vemurafenib plus cobimetinib. A significant increment in CD147/basigin and MMP-2 was observed in all patients at the time of treatment failure, strengthening the hypothesis that CD147/basigin might play a role in BRAFi resistance.

Despite intensive antimicrobial and anti-inflammatory therapies, cystic fibrosis (CF) patients are subjected to chronic infections due to opportunistic pathogens, including multidrug resistant (MDR) Pseudomonas aeruginosa. Macrophages from CF patients show many evidences of reduced phagocytosis in terms of internalization capability, phagosome maturation, and intracellular bacterial killing. In this study, we investigated if apoptotic body-like liposomes (ABLs) loaded with phosphatidylinositol 5-phosphate (PI5P), known to regulate actin dynamics and vesicular trafficking, could restore phagocytic machinery while limiting inflammatory response in in vitro and in vivo models of MDR P. aeruginosa infection. Our results show that the in vitro treatment with ABL carrying PI5P (ABL/PI5P) enhances bacterial uptake, ROS production, phagosome acidification, and intracellular bacterial killing in human monocyte-derived macrophages (MDMs) with pharmacologically inhibited cystic fibrosis transmembrane conductance regulator channel (CFTR), and improve uptake and intracellular killing of MDR P. aeruginosa in CF macrophages with impaired bactericidal activity. Moreover, ABL/PI5P stimulation of CFTR-inhibited MDM infected with MDR P. aeruginosa significantly reduces NF-κB activation and the production of TNF-α, IL-1β, and IL-6, while increasing IL-10 and TGF-β levels. The therapeutic efficacy of ABL/PI5P given by pulmonary administration was evaluated in a murine model of chronic infection with MDR P. aeruginosa. The treatment with ABL/PI5P significantly reduces pulmonary neutrophil infiltrate and the levels of KC and MCP-2 cytokines in the lungs, without affecting pulmonary bacterial load. Altogether, these results show that the ABL/PI5P treatment may represent a promising host-directed therapeutic approach to improve the impaired phagocytosis and to limit the potentially tissue-damaging inflammatory response in CF.

The natural history of COVID-19 caused by SARS-CoV-2 is extremely variable, ranging from asymptomatic or mild infection, mainly in children, to multi-organ failure, eventually fatal, mainly in the eldest. We propose here the first model explaining how the outcome of first, crucial 10-15 days after infection, depends on the balance between the cumulative dose of viral exposure and the efficacy of the local innate immune response (natural IgA and IgM antibodies, mannose-binding lectin). If SARS-CoV-2 runs the blockade of this innate immunity and spreads from the upper airways to the alveoli in the early phases of the infections, it can replicate with no local resistance, causing pneumonia and releasing high amounts of antigens. The delayed and strong adaptive immune response (high-affinity IgM and IgG antibodies) that follows, causes severe inflammation and triggers mediator cascades (complement, coagulation, and cytokine storm), leading to complications often requiring intensive therapy and being, in some patients, fatal. Low-moderate physical activity can still be recommended. However, extreme physical activity and oral breathing with hyperventilation during the incubation days and early stages of COVID-19 facilitates re-inhalation and early direct penetration of high numbers of own virus particles in the lower airways and the alveoli, without impacting on the airway's mucosae covered by neutralizing antibodies ("viral auto-inhalation" phenomenon). This allows the virus to bypass the efficient immune barrier of the upper airway mucosa in already infected, young, and otherwise healthy athletes. In conclusion, whether the virus or the adaptive immune response reaches the lungs first is a crucial factor deciding the fate of the patient. This "quantitative and time-/sequence-dependent" model has several implications for prevention, diagnosis, and therapy of COVID-19 at all ages.

The Coronavirus disease 2019 (COVID-19) pandemic has represented an unprecedented challenge for humankind from health, economic, and social viewpoints. In February 2020, Italy was the first western country to be deeply hit by the pandemic and suffered the highest case/fatality rate among western countries. Brand new anti-COVID-19 vaccines have been developed and made available in

Annexin-V/propidium iodide method (A-V/PI) is a common flow cytometric method for the multiparametric analysis of cells in apoptosis. However, A-V/PI does not permit fixation and/or permeabilization of cells making impossible evaluation of intracellular markers, restricting the analysis in a narrow time frame after staining and excluding the possibility to study pathogen-infected cells. We developed a method permitting fixation and permeabilization of stained cells: Fixed Apoptotic/Necrotic (FAN) cells test. FAN relies on the same principle of A-V/PI, but uses reagents that maintain their binding and fluorescence characteristics after fixation/permeabilization: a fluorochrome-labeled anti-phosphatidylserine antibody and fluorescent amine-binding dyes. FAN was tested to discriminate apoptotic and necrotic cells using different stimuli on several cell types and results were always comparable to those obtained using A-V/PI. FAN, unlike A-V/PI, permitted to correlate cell death with intracellular and surface markers expression and to perform cytometry even two weeks after sample preparation. As fixation of stained cells inactivates infective pathogens, we used FAN in an in vitro model of Mycobacterium tuberculosis (Mtb) infection of macrophages to monitor cellular infection and cell death induction. Using a red-fluorescent Mtb, fluorochrome labeled anti-TNF-α and anti-MHC class II monoclonal antibodies, FAN permitted to establish that the extent of macrophage death correlates with intracellular Mtb content and that dying cells accumulate TNF-α and down-modulate MHC class II molecules. Results suggest that FAN may represent an additional tool to study programmed cell death particularly useful when fixation procedures are required for a safe infected sample analysis or to comparatively analyze multiple samples. © 2017 International Society for Advancement of Cytometry.

In the current study, the possible association of positivity (POS), recently defined as general disposition to view life under positive outlook, with immune markers and post-stress modifications, was analyzed. Circulating lymphocyte subsets and serum cytokine levels were evaluated before and after a standard mild acute stress test, in 41 healthy students, previously selected by a questionnaire for their level of POS (high [POS-H] and low [POS-L]). The CD3+ and CD4+ cell frequency was higher in the POS-H students before and after acute stress. CD4+ subpopulation analysis revealed baseline higher terminally differentiated frequency in the POS-H, whereas higher effector memory frequency was present in the POS-L students. Moreover, the frequency of post-stress B cells was higher in the POS-H students. The mild-stress test was associated to an increase of the IL-10 mean values, while mean values of the other cytokines tested did not change significantly. It is tempting to speculate that IL-10 may work as biomarker of response to acute mild stress and that POS-H may be associated to a better capacity of the immune system to contrast the disturbing effects of mild acute stress. Yet further studies on lymphocyte subset absolute number and function of larger and different populations are needed to definitively prove these preliminary observations.

Aging is related to a number of functional and morphological changes leading to progressive decline of the biological functions of an organism. Reactive Oxygen Species (ROS), released by several endogenous and exogenous processes, may cause important oxidative damage to DNA, proteins, and lipids, leading to important cellular dysfunctions. The imbalance between ROS production and antioxidant defenses brings to oxidative stress conditions and, related to accumulation of ROS, aging-associated diseases. The purpose of this review is to provide an overview of the most relevant data reported in literature on the natural compounds, mainly phytochemicals, with antioxidant activity and their potential protective effects on age-related diseases such as metabolic syndrome, diabetes, cardiovascular disease, cancer, neurodegenerative disease, and chronic inflammation, and possibly lower side effects, when compared to other drugs.

In nature, many plants or their extracted compounds have been found to possess anti-inflammatory features and therapeutic properties against infectious as well as non-infectious diseases, including cancer. In this study, we analysed the immunomodulatory effects on innate immune cells of hydroalcoholic extract from Origanum vulgare L. ssp. hirtum (HyE-Ov), a plant traditionally known for its anti-oxidative properties. The effects of HyE-Ov were tested on human monocyte derived dendritic cells (DC), type-1 (M1) and type-2 macrophages (M2) infected with M. bovis Bacille Calmette-Guérin (BCG), used as a model of persistent intracellular bacterium. DC, M1 and M2 treated with HyE-Ov significantly enhanced their mycobactericidal activity, which was associated with phagosomal acidification in M1 and M2 and increase of phagosomal, but not mitochondrial ROS production in M1, M2, and DC. Treatment of BCG-infected DC with HyE-Ov significantly reduced TNF-α and IL-12 production and increased TGF-β synthesis. Finally, experiments were repeated using eight different HPLC fractions of HyE-Ov. Results showed that the capability to activate anti-microbial and anti-inflammatory response is shared by different fractions, suggesting that diverse bioactive molecules are present within the hydroalcoholic extract. Altogether, these results show that HyE-Ov promotes anti-mycobacterial innate immunity and limits inflammatory response in vitro and suggest that this plant extract may be exploitable as phytocomplex or nutraceutical for novel host-directed therapeutic approaches.

Military personnel of all armed forces receive multiple vaccinations and have been doing so since long ago, but relatively few studies have investigated the possible negative or positive interference of simultaneous vaccinations. As a contribution to fill this gap, we analyzed the response to the live trivalent measles/mumps/rubella (MMR), the inactivated hepatitis A virus (HAV), the inactivated trivalent polio, and the trivalent subunits influenza vaccines in two cohorts of Italian military personnel. The first cohort was represented by 108 students from military schools and the second by 72 soldiers engaged in a nine-month mission abroad. MMR and HAV vaccines had never been administered before, whereas inactivated polio was administered to adults primed at infancy with a live trivalent oral polio vaccine. Accordingly, nearly all subjects had baseline antibodies to polio types 1 and 3, but unexpectedly, anti-measles/-mumps/-rubella antibodies were present in 82%, 82%, and 73.5% of subjects, respectively (43% for all of the antigens). Finally, anti-HAV antibodies were detectable in 14% and anti-influenza (H1/H3/B) in 18% of the study population. At mine months post-vaccination, 92% of subjects had protective antibody levels for all MMR antigens, 96% for HAV, 69% for the three influenza antigens, and 100% for polio types 1 and 3. An inverse relationship between baseline and post-vaccination antibody levels was noticed with all the vaccines. An excellent vaccine immunogenicity, a calculated long antibody persistence, and apparent lack of vaccine interference were observed.

The physiology of dormant Mycobacterium tuberculosis was studied in detail by examining the gene expression of 51 genes using quantitative Reverse-Transcription Polymerase Chain Reaction. A forty-day period of dormancy in the Wayne culture model depicted four major transcription patterns. Some sigma factors and many metabolic genes were constant, whereas genes belonging to the dormancy regulon were activated on day 9. In particular, alpha-crystallin mRNA showed more than a 1,000-fold increase compared to replicating bacilli. Genes belonging to the enduring hypoxic response were up-regulated at day 16, notably, transcription factors sigma B and E. Early genes typical of log-phase bacilli, esat-6 and fbpB, were uniformly down-regulated during dormancy. Late stages of dormancy showed a drop in gene expression likely due to a lack of substrates in anaerobic respiration as demonstrated by the transcriptional activation observed following nitrates addition. Among genes involved in nitrate metabolism, narG was strongly up-regulated by nitrates addition. Dormant bacilli responded very rapidly when exposed to oxygen and fresh medium, showing a transcriptional activation of many genes, including resuscitation-promoting factors, within one hour. Our observations extend the current knowledge on dormant M. tuberculosis gene expression and its response to nutrients and to aerobic and anaerobic respiration.

Cellular and humoral immune responses to tetanus-diphtheria vaccine (Td) were assessed in human leukocyte antigen (HLA)-typed Italian military personnel who received multiple concomitant vaccines. Td-specific antibodies and T-lymphocytes were measured in individuals with one (group-1) and more than one (group-2) Td boosters. A third group (group-3), who received several vaccines, but not Td, was studied to verify the hypothesis of the polyclonal B-cell activation as mechanism for antibody persistence. The antibody response to Td toxoids was higher in group-1, who showed lower baseline antibody levels, than in group-2 subjects. The antibody response to tetanus was higher than to diphtheria toxoid in both groups. No correlation between antibody and cellular response, and no interference in the response to Td by co-administration of different vaccines were observed. HLA-DRB1∗01 allele was detected at significant higher frequency in subjects unable to double the baseline anti-diphtheria antibody levels after the vaccination. Anti-tetanus and diphtheria antibodies half-lives were assessed and the long-lasting persistence above the threshold for protection (0.1 IU/ml) was estimated in over 65 and 20 years, respectively. No significant increase of anti-diphtheria antibodies was observed in consequence of polyclonal B-cell activation. This study emphasizes the duration of Td vaccination-induced seroprotection, suggesting that re-vaccination should probably be performed at intervals longer than 10 years. No reciprocal interference by concomitantly administered vaccines has been observed. HLA-DRB1∗01 allele was significantly associated with anti-diphtheria defective response. Finally, this study does not confirm that anti-diphtheria antibody levels are maintained by polyclonal B-cell activation. Clinical trial registry: The study was registered with NCT01807780.

Liposomes are closed bilayer structures spontaneously formed by hydrated phospholipids that are widely used as efficient delivery systems for drugs or antigens, due to their capability to encapsulate bioactive hydrophilic, amphipathic, and lipophilic molecules into inner water phase or within lipid leaflets. The efficacy of liposomes as drug or antigen carriers has been improved in the last years to ameliorate pharmacokinetics and capacity to release their cargo in selected target organs or cells. Moreover, different formulations and variations in liposome composition have been often proposed to include immunostimulatory molecules, ligands for specific receptors, or stimuli responsive compounds. Intriguingly, independent research has unveiled the capacity of several phospholipids to play critical roles as intracellular messengers in modulating both innate and adaptive immune responses through various mechanisms, including (i) activation of different antimicrobial enzymatic pathways, (ii) driving the fusion-fission events between endosomes with direct consequences to phagosome maturation and/or to antigen presentation pathway, and (iii) modulation of the inflammatory response. These features can be exploited by including selected bioactive phospholipids in the bilayer scaffold of liposomes. This would represent an important step forward since drug or antigen carrying liposomes could be engineered to simultaneously activate different signal transduction pathways and target specific cells or tissues to induce antigen-specific T and/or B cell response. This lipid-based host-directed strategy can provide a focused antimicrobial innate and adaptive immune response against specific pathogens and offer a novel prophylactic or therapeutic option against chronic, recurrent, or drug-resistant infections.

Summary Meningococcal polysaccharide (Men-Ps) vaccine immunogenicity following either primary immunization or revaccination in adults was evaluated. The study population consisted of subjects who have received tetravalent Men-Ps vaccine once (group 1) or at least twice, with a 2–6 dose range (group 2). Human leucocyte antigen (HLA)-typing was performed by polymerase chain reaction and specific immunoglobulin (Ig)G was measured by enzyme-linked immunosorbent assay. Nine months post-immunization, the percentages of individuals with levels of anti-Men-Ps IgG ≥ 2 µg/ml were comparable in both groups, with the exception of anti-Men-PsW135 IgG, which were significantly higher in group 2. The percentage of subjects doubling IgG levels at 9 months was significantly higher in group 1. The high baseline anti-Men-Ps antibody levels negatively influenced the response to revaccination, suggesting a feedback control of specific IgG. The calculated durability of anti-Men-Ps IgG was 2·5–4·5 years, depending on the Men-Ps, following a single vaccine dose. No interference by other vaccinations nor HLA alleles association with immune response were observed. This study confirms that Men-Ps vaccine in adults is immunogenic, even when administered repeatedly, and underlines the vaccine suitability for large-scale adult immunization programmes that the higher costs of conjugate vaccines may limit in developing countries.

Immunogenicity of 13-valent pneumococcal polysaccharide (PnPS) conjugate vaccine (PCV13) was evaluated in 38 rheumatoid arthritis patients under immunosuppressive treatment and 20 healthy controls (HC). Antibodies to all PnPS and diphtheria-toxin analogue conjugate protein were measured pre- (T0), 1 (T1), 6 (T2), 12 (T3) months post-immunization. Patients and HC had similar response to individual PnPS. Mean antibody levels to all PnPS but one doubled at T1 compared with T0, with T3 persistence for only 8-7/13 PnPS. Baseline antibody levels was inversely associated with the rate of responders at T1 (T1/T0≥2) to 11/13 PnPS. Few subjects reached protective IgG levels against some serotypes frequently isolated in Italian patients with invasive pneumococcal disease. Antibody response was not influenced by therapy, except the one to PS7F, which was reduced by tumor necrosis factor-α-inhibitors. Vaccination increased also anti-diphtheria IgG. Despite this study substantially confirmed the PCV13 immunogenicity in immunocompromised patients, it also revealed some limitations.

Aim of this study was to extend the knowledge on the antineoplastic effect of aloe-emodin (AE), a natural hydroxyanthraquinone compound, both in metastatic human melanoma cell lines and in primary stem-like cells (melanospheres). Treatment with AE caused reduction of cell proliferation and induction of SK-MEL-28 and A375 cells differentiation, characterized by a marked increase of transamidating activity of transglutaminase whose expression remained unmodified. In vitro antimetastatic property of AE was evaluated by adhesion and Boyden chamber invasion assays. The effect of AE on melanoma cytokines/chemokines production was determined by a multiplex assay: interestingly AE showed an immunomodulatory activity through GM-CSF and IFN-γ production. We report also that AE significantly reduced the proliferation, stemness and invasive potential of melanospheres. Moreover, AE treatment significantly enhanced dabrafenib (a BRAF inhibitor) antiproliferative activity in BRAF mutant cell lines. Our results confirm that AE possesses remarkable antineoplastic properties against melanoma cells, indicating this anthraquinone as a promising agent for differentiation therapy of cancer, or as adjuvant in chemotherapy and targeted therapy. Further, its mechanisms of action support a potential efficacy of AE treatment to counteract resistance of BRAF-mutated melanoma cells to target therapy.

Anecdotal case reports, amplified by mass media and internet-based opinion groups, have recently indicated vaccinations as possibly responsible for autoimmunity/lymphoproliferation development. Multiply vaccinated Italian military personnel (group 1, operating in Italy, group 2, operating in Lebanon) were followed-up for nine months to monitor possible post-vaccine autoimmunity/lymphoproliferation onset. No serious adverse event was noticed in both groups. Multivariate analysis of intergroup differences only showed a significant association between lymphocyte increase and tetanus/diphtheria vaccine administration. A significant post-vaccine decrease in autoantibody positivity was observed. Autoantibodies were also studied by microarray analysis of self-proteins in subjects exposed to ≥4 concurrent vaccinations, without observing significant difference among baseline and one and nine months post-vaccine. Moreover, HLA-A2 subjects have been analyzed for the possible CD8T-cell response to apoptotic self-epitopes, without observing significant difference between baseline and one month post-vaccine. Multiple vaccinations in young adults are safe and not associated to autoimmunity/lymphoproliferation onset during a nine-month-long follow-up.

Phagocytosis is a key mechanism of innate immunity, and promotion of phagosome maturation may represent a therapeutic target to enhance antibacterial host response. Phagosome maturation is favored by the timely and coordinated intervention of lipids and may be altered in infections. Here we used apoptotic body-like liposomes (ABL) to selectively deliver bioactive lipids to innate cells, and then tested their function in models of pathogen-inhibited and host-impaired phagosome maturation. Stimulation of macrophages with ABLs carrying phosphatidic acid (PA), phosphatidylinositol 3-phosphate (PI3P) or PI5P increased intracellular killing of BCG, by inducing phagosome acidification and ROS generation. Moreover, ABLs carrying PA or PI5P enhanced ROS-mediated intracellular killing of Pseudomonas aeruginosa, in macrophages expressing a pharmacologically-inhibited or a naturally-mutated cystic fibrosis transmembrane conductance regulator. Finally, we show that bronchoalveolar lavage cells from patients with drug-resistant pulmonary infections increased significantly their capacity to kill in vivo acquired bacterial pathogens when ex vivo stimulated with PA- or PI5P-loaded ABLs. Altogether, these results provide the proof of concept of the efficacy of bioactive lipids delivered by ABL to enhance phagosome maturation dependent antimicrobial response, as an additional host-directed strategy aimed at the control of chronic, recurrent or drug-resistant infections.

The emergence of multidrug resistant Mycobacterium tuberculosis (Mtb) strains, the difficulties in the development of new safe therapies or the production of a protective vaccine against tuberculosis make the study of the strategies developed by Mtb to escape from the host immune system an important source of potential targets of immunointervention in the fight against tuberculosis. A mechanism of immune-evasion specific for the post-primary tuberculosis based on the differentiation of disabled antigen presenting cells is described.

PGE2 is a potent modulator of the T helper (Th)17 immune response that plays a critical role in the host defense against bacterial, fungal and viral infections. We recently showed high serum levels of interleukin (IL)-17 in patients with gonococcal infection and we hypothesized that Neisseria gonorrhoeae could exploit a PGE2 mediated mechanism to promote IL-17 production. Here we show that N. gonorrhoeae induces human dendritic cell (DC) maturation, secretion of prostaglandin E2 and proinflammatory cytokines, including the pro-Th17 cytokine IL-23. Blocking PGE2 endogenous synthesis selectively reduces IL-23 production by DC in response to gonococcal stimulation, confirming recent data on PGE2/IL-23 crosstalk. N. gonorrhoeae stimulated DC induce a robust IL-17 production by memory CD4+ T cells and this function correlates with PGE2 production. Our findings delineate a previously unknown role for PGE2 in the immune response to N. gonorrhoeae, suggesting its contribute via Th17 cell expansion.

Dormant, non-replicating Mycobacterium tuberculosis H37Rv strain cultured in hypoxic conditions was used to infect THP-1 cells. CFUs counting, Kinyoun staining and electron microscopy showed that dormant bacilli infected THP-1 cells at a rate similar to replicating M. tuberculosis, but failed to grow during the first 6 days of infection. The absence of growth was specific to the intracellular compartment, as demonstrated by efficient growth in liquid medium. Quantification of β-actin mRNA recovered from infected cells showed that, in contrast with log-phase bacteria, infection with dormant bacilli determined a reduced THP-1 cell death. Gene expression of intracellular non-replicating bacteria showed a pattern typical of a dormant state. Intracellular dormant bacteria induced the activation of genes associated to a proinflammatory response in THP-1 cells. Though, higher levels of TNFα, IL-1β and IL-8 mRNAs compared to aerobic H37Rv infected cells were not paralleled by increased cytokine accumulation in the supernatants. Moreover, dormant bacilli induced a higher expression of inducible cox-2 gene, accompanied by increased PGE2 secretion. Overall, our data describe a new model of in vitro infection using dormant M. tuberculosis that could provide the basis for understanding how non-replicating bacilli survive intracellularly and influence the maintenance of the hypoxic granuloma.

Virus-like particles (VLPs) are excellent tools for vaccines against pathogens and tumors. They can accommodate foreign polypeptides whose incorporation efficiency and immunogenicity however decrease strongly with the increase of their size. We recently described the CD8(+) T cell immune response against a small foreign antigen (i.e., the 98 amino acid long human papilloma virus E7 protein) incorporated in human immunodeficiency virus (HIV)-1 based VLPs as product of fusion with an HIV-1 Nef mutant (Nef(mut)). Here, we extended our previous investigations by testing the antigenic/immunogenic properties of Nef(mut)-based VLPs incorporating much larger heterologous products, i.e., human hepatitis C virus (HCV) NS3 and influenza virus NP proteins, which are composed of 630 and 498 amino acids, respectively. We observed a remarkable cross-presentation of HCV NS3 in dendritic cells challenged with Nef(mut)-NS3 VLPs, as detected using a NS3 specific CD8(+) T cell clone as well as PBMCs from HCV infected patients. On the other hand, when injected in mice, Nef(mut)-NP VLPs elicited strong anti-NP CD8(+) T cell and CTL immune responses. In addition, we revealed the ability of Nef(mut) incorporated in VLPs to activate and mature primary human immature dendritic cells (iDCs). This phenomenon correlated with the activation of Src tyrosine kinase-related intracellular signaling, and can be transmitted from VLP-challenged to bystander iDCs. Overall, these results prove that Nef(mut)-based VLPs represent a rather flexible platform for the design of innovative CD8(+) T cell vaccines.

Mycobacterium tuberculosis (Mtb) evades the immune response by impairing the functions of different antigen-presenting cells. We have recently shown that Mtb hijacks differentiation of monocytes into dendritic cells (DCs). To further characterize the mechanisms underlying this process, we investigated the consequences of inducing dendritic cell differentiation using interferon-α and granulocyte-macrophage colony-stimulating factor in the presence of supernatants (SNs) obtained from monocyte cultures treated with or without heat-inactivated Mtb. Although the SNs from control cultures do not interfere with the generation of fully differentiated DCs, monocytes stimulated with SNs from Mtb-stimulated cells (SN Mtb) remained CD14(+) and poorly differentiated into CD1a(+) cells. Among cytokines known to affect dendritic cell differentiation, we observed a robust production of interleukin-1β, interleukin-6, interleukin-10 and tumor necrosis factor-α upon Mtb stimulation. However, only interleukin-10 neutralization through the addition of soluble interleukin-10 receptor reversed the inhibitory activity of SN Mtb. Accordingly, the addition of recombinant interleukin-10 was able to significantly reduce CD1a expression. The interaction of Mtb with differentiating monocytes rapidly activates p38 mitogen-activated protein kinase, signal transducer and activator of transcription pathways, which are likely involved in interleukin-10 gene expression. Taken together, our results suggest that Mtb may inhibit the differentiation of bystander non-infected monocytes into DCs through the release of interleukin-10. These results shed light on new aspects of the host-pathogen interaction, which might help to identify innovative immunological strategies to limit Mtb virulence.

Group I CD1 proteins are specialized antigen-presenting molecules that present both microbial and self lipid antigens to CD1-restricted α/β T lymphocytes. The production of high levels of gamma interferon and lysis of infected macrophages by lipid-specific T lymphocytes are believed to play pivotal roles mainly in the defense against mycobacterial infections. We previously demonstrated that Mycobacterium tuberculosis and bacillus Calmette-Guérin ( Mycobacterium bovis BCG) induce human monocytes to differentiate into CD1 − dendritic cells (DC), which cannot present lipid antigens to specific T cells. Here, we show that in human monocytes mycobacteria trigger phosphorylation of p38 mitogen-activated protein kinase to inhibit CD1 expression in DC derived from infected monocytes. Pretreatment with a specific p38 inhibitor renders monocytes insensitive to mycobacterial subversion and allows them to differentiate into CD1 + DC, which are fully capable of presenting lipid antigens to specific T cells. We also report that one of the pathogen recognition receptors triggered by BCG to activate p38 is complement receptor 3 (CR3), as shown by reduced p38 phosphorylation and partial reestablishment of CD1 membrane expression obtained by CR3 blockade before infection. In conclusion, we propose that p38 signaling is a novel pathway exploited by mycobacteria to affect the expression of CD1 antigen-presenting cells and avoid immune recognition.