The α-Cyclodextrin/Moringin Complex: A New Promising Antimicrobial Agent against Staphylococcus aureus.

Antimicrobial resistance is one of the major clinical concerns, making the discovery of new antimicrobial drugs desirable. Moringin (MOR), the major isothiocyanate produced from Moringa oleifera seeds, could represent an alternative therapeutic strategy to commonly used antibiotics. The aim of our study was to investigate the antimicrobial effect of MOR conjugated with α-cyclodextrin (MOR/α-CD), a complex with an improved solubility and stability in aqueous solutions. Our data demonstrated that MOR/α-CD was able to exert antimicrobial activity against the S. aureus reference strains (ATCC 25923, ATCC 6538, and ATCC BAA-977). Moreover, MOR/α-CD showed bacteriostatic effects (MIC = minimum inhibitory concentration = 0.5 mg/mL) and bactericidal properties (MBC = minimum bactericidal concentration = 1 mg/mL) against the overall assessed strains. In addition, MOR/α-CD showed bactericidal activity against the S. aureus strain ATCC BAA-977 after treatment with erythromycin (Ery), which induced clindamycin-resistance on the erm (A) gene. This evidence led us to assume that MOR/α-CD could be a promising antimicrobial agent against strains with the clindamycin-resistant phenotype (CC-resistant).

Treatment of Periodontal Ligament Stem Cells with MOR and CBD Promotes Cell Survival and Neuronal Differentiation via the PI3K/Akt/mTOR Pathway.

Periodontal ligament mesenchymal stem cells (hPDLSCs), as well as all mesenchymal stem cells, show self-renewal, clonogenicity, and multi-tissue differentiation proprieties and can represent a valid support for regenerative medicine. We treated hPDLSCs with a combination of Moringin (MOR) and Cannabidiol (CBD), in order to understand if treatment could improve their survival and their in vitro differentiation capacity. Stem cells survival is fundamental to achieve a successful therapy outcome in the re-implanted tissue of patients. Through NGS transcriptome analysis, we found that combined treatment increased hPDLSCs survival, by inhibition of apoptosis as demonstrated by enhanced expression of anti-apoptotic genes and reduction of pro-apoptotic ones. Moreover, we investigated the possible involvement of PI3K/Akt/mTOR pathway, emphasizing a differential gene expression between treated and untreated cells. Furthermore, hPDLSCs were cultured for 48 h in the presence or absence of CBD and MOR and, after confirming the cellular viability through MTT (3-(4,5-dimethylthiazolyl-2)-2,5-diphenyltetrazoliumbromide) assay, we examined the presence of neuronal markers, through immunofluorescence analysis. We found an increased expression of Nestin and GAP43 (growth associated protein 43) in treated cells. In conclusion, hPDLSCs treated with Moringin and Cannabidiol showed an improved survival capacity and neuronal differentiation potential.

Moringin Induces Neural Differentiation in the Stem Cell of the Human Periodontal Ligament.

The therapeutic strategies for neurodegenerative diseases still represent a vast research field because of the lack of targeted, effective and resolutive treatment for neurodegenerative diseases. The use of stem cell-based therapy is an alternative approach that could lead to the replacement of damaged neuronal tissue. For this purpose, adult mesenchymal stem cells (MSC), including periodontal ligament stem cells (PDLSCs), could be very useful for their differentiation capacity, easy isolation and the ability to perform an autologous implant. The aim of this work was to test whether the Moringin [4-(α-L-rhamnosyloxy) benzyl isothiocyanate; GMG-ITC], an isothiocyanate extracted from Moringa oleifera seeds, was able to induce PDLSCs toward neural progenitor differentiation. Next-generation transcriptomics sequencing showed that moringin treatment increased the expression of genes involved in neuron cortical development and in particular in neuron belonging to upper and deep cortical layers. Moreover, moringin treatment upregulated genes involved in osteogenesis and adipogenesis although with a lower fold change compared to upregulated genes involved in neuronal differentiation. Finally, moringin did not induce the expression of oncogenes resulting in a safe treatment.

Biomarkers identification for PML monitoring, during Natalizumab (Tysabri®) treatment in Relapsing-Remitting Multiple Sclerosis.

Natalizumab (NTZ, Tysabri®; Biogen-Idec, Cambridge, MA, USA) is a humanized anti-α4 integrin monoclonal antibody, largely used in the treatment of Relapsing-Remitting Multiple Sclerosis (RRMS). Although the drug has shown great efficacy in clinical trials (AFFIRM and SENTINEL) and in post-marketing observational studies (TYGRIS), by reducing clinical signs as disability status progression, brain lesions and annual relapse rate, there are numerous papers concerning the associated risk of progressive multifocal leukoencephalopathy (PML). PML is a brain infection caused by John Cunningham virus (JCV) and its incidence is related to intrinsic and extrinsic risk factors, such as long-term natalizumab therapy (more than 24 infusion doses), previous pharmacological immunosuppressive treatment and JVC antibody-positive status. The identification of risk factors provides an instrument to improve treatment decisions and to obtain an accurate benefit-risk evaluation. In order to evaluate the most appropriate natalizumab-MS therapy and to obtain minor incidence of PML, an accurate description of risk factors and a biological markers identification are needed. This article review aims to list some biomarkers that have been proposed to evaluate the safety of natalizumab therapy.

Epitope Mapping of a Monoclonal Antibody Directed against Neisserial Heparin Binding Antigen Using Next Generation Sequencing of Antigen-Specific Libraries.

We explore here the potential of a newly described technology, which is named PROFILER and is based on next generation sequencing of gene-specific lambda phage-displayed libraries, to rapidly and accurately map monoclonal antibody (mAb) epitopes. For this purpose, we used a novel mAb (designated 31E10/E7) directed against Neisserial Heparin-Binding Antigen (NHBA), a component of the anti-group B meningococcus Bexsero® vaccine. An NHBA phage-displayed library was affinity-selected with mAb 31E10/E7, followed by massive sequencing of the inserts present in antibody-selected phage pools. Insert analysis identified an amino acid stretch (D91-A128) in the N-terminal domain, which was shared by all of the mAb-enriched fragments. Moreover, a recombinant fragment encompassing this sequence could recapitulate the immunoreactivity of the entire NHBA molecule against mAb 31E10/E7. These results were confirmed using a panel of overlapping recombinant fragments derived from the NHBA vaccine variant and a set of chemically synthetized peptides covering the 10 most frequent antigenic variants. Furthermore, hydrogen-deuterium exchange mass-spectrometry analysis of the NHBA-mAb 31E10/E7 complex was also compatible with mapping of the epitope to the D91-A128 region. Collectively, these results indicate that the PROFILER technology can reliably identify epitope-containing antigenic fragments and requires considerably less work, time and reagents than other epitope mapping methods.

Functional characterization of a monoclonal antibody epitope using a lambda phage display-deep sequencing platform.

We have recently described a method, named PROFILER, for the identification of antigenic regions preferentially targeted by polyclonal antibody responses after vaccination. To test the ability of the technique to provide insights into the functional properties of monoclonal antibody (mAb) epitopes, we used here a well-characterized epitope of meningococcal factor H binding protein (fHbp), which is recognized by mAb 12C1. An fHbp library, engineered on a lambda phage vector enabling surface expression of polypeptides of widely different length, was subjected to massive parallel sequencing of the phage inserts after affinity selection with the 12C1 mAb. We detected dozens of unique antibody-selected sequences, the most enriched of which (designated as FrC) could largely recapitulate the ability of fHbp to bind mAb 12C1. Computational analysis of the cumulative enrichment of single amino acids in the antibody-selected fragments identified two overrepresented stretches of residues (H248-K254 and S140-G154), whose presence was subsequently found to be required for binding of FrC to mAb 12C1. Collectively, these results suggest that the PROFILER technology can rapidly and reliably identify, in the context of complex conformational epitopes, discrete "hot spots" with a crucial role in antigen-antibody interactions, thereby providing useful clues for the functional characterization of the epitope.

Rapid profiling of the antigen regions recognized by serum antibodies using massively parallel sequencing of antigen-specific libraries.

There is a need for techniques capable of identifying the antigenic epitopes targeted by polyclonal antibody responses during deliberate or natural immunization. Although successful, traditional phage library screening is laborious and can map only some of the epitopes. To accelerate and improve epitope identification, we have employed massive sequencing of phage-displayed antigen-specific libraries using the Illumina MiSeq platform. This enabled us to precisely identify the regions of a model antigen, the meningococcal NadA virulence factor, targeted by serum antibodies in vaccinated individuals and to rank hundreds of antigenic fragments according to their immunoreactivity. We found that next generation sequencing can significantly empower the analysis of antigen-specific libraries by allowing simultaneous processing of dozens of library/serum combinations in less than two days, including the time required for antibody-mediated library selection. Moreover, compared with traditional plaque picking, the new technology (named Phage-based Representation OF Immuno-Ligand Epitope Repertoire or PROFILER) provides superior resolution in epitope identification. PROFILER seems ideally suited to streamline and guide rational antigen design, adjuvant selection, and quality control of newly produced vaccines. Furthermore, this method is also susceptible to find important applications in other fields covered by traditional quantitative serology.

Essential role of interleukin-1 signaling in host defenses against group B streptococcus.

UNLABELLED: Signal transduction via MyD88, an adaptor protein engaged by the Toll-like receptor (TLR) and interleukin-1 receptor (IL-1R) family receptors, has a crucial role in host defenses against group B streptococcus (GBS). To examine the contribution of IL-1R signaling to MyD88-dependent host defenses, we analyzed GBS infection in type I IL-1R (IL-1RI)-deficient mice. Most of these animals displayed clinical signs of sepsis and neurological disease and died after a challenge with a bacterial dose that did not cause illness or death in any of the wild-type animals. Moreover, bacterial numbers in the blood and brains of the immunodefective mice were considerably increased. The ability of blood leukocytes or bone marrow-derived macrophages to kill GBS in vitro was not affected by a lack of IL-1RI. However, it was found in a newly developed model of GBS-induced peritoneal inflammation that IL-1 signaling selectively promoted the production of the chemokines KC and MIP-1α and neutrophil recruitment. Moreover, the secretion of KC and MIP-1α, but not tumor necrosis factor alpha, by peritoneal macrophages stimulated with GBS was significantly decreased in the absence of IL-1RI. Accordingly, the number of neutrophils in the blood and the concentration of myeloperoxidase, a neutrophil marker, in infected organs were severely reduced in the immunodefective mice during GBS disease, concomitantly with a reduction in tissue KC and MIP-1α levels. In conclusion, IL-1RI plays a crucial role in host defenses against GBS by inducing the high-level production of chemokines and the subsequent recruitment of neutrophilic polymorphonuclear leukocytes to infection sites. IMPORTANCE: Group B streptococcus (GBS) is a serious and frequent human pathogen. Experimental infection with this bacterium has been widely used to understand the mechanism whereby the body's first line of defense, represented by cells and molecules of the innate immune system, fights infections. In both humans and mice, defective function of the adaptor molecule MyD88 has been associated with extreme susceptibility to infection by GBS and other extracellular bacteria. We show here that lack of signaling by interleukin-1 (IL-1) cytokines can largely, although not completely, explain the increased susceptibility to infection observed in the absence of MyD88 function. We show, in particular, that IL-1 signaling through the IL-1 receptor promotes the production of the leukocyte attractant chemokines KC and MIP-1α and recruitment of neutrophils to GBS infection sites, thereby enabling these leukocytes to clear the infection. Our findings indicate that stimulation of IL-1 signaling may be useful as an alternative therapeutic strategy to treat GBS infections.

Immunogenic properties of Streptococcus agalactiae FbsA fragments.

Several species of Gram-positive bacteria can avidly bind soluble and surface-associated fibrinogen (Fng), a property that is considered important in the pathogenesis of human infections. To gain insights into the mechanism by which group B Streptococcus (GBS), a frequent neonatal pathogen, interacts with Fng, we have screened two phage displayed genomic GBS libraries. All of the Fng-binding phage clones contained inserts encoding fragments of FbsA, a protein displaying multiple repeats. Since the functional role of this protein is only partially understood, representative fragments were recombinantly expressed and analyzed for Fng binding affinity and ability to induce immune protection against GBS infection. Maternal immunization with 6pGST, a fragment containing five repeats, significantly protected mouse pups against lethal GBS challenge and these protective effects could be recapitulated by administration of anti-6pGST serum from adult animals. Notably, a monoclonal antibody that was capable of neutralizing Fng binding by 6pGST, but not a non-neutralizing antibody, could significantly protect pups against lethal GBS challenge. These data suggest that FbsA-Fng interaction promotes GBS pathogenesis and that blocking such interaction is a viable strategy to prevent or treat GBS infections.

Analysis of the Streptococcus agalactiae exoproteome.

The two-component regulatory system CovRS is the main regulator of virulence gene expression in Group B Streptococcus (GBS), the leading cause of invasive infections in neonates. In this study we analyzed by mass spectrometry the GBS extracellular protein complex (i.e. the exoproteome) of NEM316 wild-type (WT) strain and its isogenic covRS deletion mutant (ΔcovRS). A total of 53 proteins, 49 of which had classical secretion signals, were identified: 12 were released by both strains while 21 and 20 were released exclusively by WT and ΔcovRS strains, respectively. In addition to known surface proteins, we detected here unstudied cell-wall associated proteins and/or orthologs of putative virulence factors present in other pathogenic streptococci. While the functional role of these proteins remains to be elucidated, our data suggest that the analysis of the exoproteome of bacterial pathogens under different gene expression conditions may be a powerful tool for the rapid identification of novel virulence factors and vaccine candidates. BIOLOGICAL SIGNIFICANCE: We believe that this manuscript will be of interest to Journal of Proteomics readers since the paper describes the identification of several putative virulence factors and vaccine candidates of the group B streptococcus, an important pathogen, using a simple proteomics strategy involving LC-MS analysis of culture supernatants obtained from two strains with divergent gene expression patterns. This technique provided the most comprehensive inventory of extracellular proteins obtained from a single streptococcal species thus far. The approach described has the added benefit of being easily applicable to a large number of different strains, making it ideal for the identification of conserved vaccine candidates.