Novel adamantyl retinoid-related molecules with POLA1 inhibitory activity.

Atypical retinoids (AR) or retinoid-related molecules (RRMs) represent a promising class of antitumor compounds. Among AR, E-3-(3'-adamantan-1-yl-4'-hydroxybiphenyl-4-yl)acrylic acid (adarotene), has been extensively investigated. In the present work we report the results of our efforts to develop new adarotene-related atypical retinoids endowed also with POLA1 inhibitory activity. The effects of the synthesized compounds on cell growth were determined on a panel of human and hematological cancer cell lines. The most promising compounds showed antitumor activity against several tumor histotypes and increased cytotoxic activity against an adarotene-resistant cell line, compared to the parent molecule. The antitumor activity of a selected compound was evaluated on HT-29 human colon carcinoma and human mesothelioma (MM487) xenografts. Particularly significant was the in vivo activity of the compound as a single agent compared to adarotene and cisplatin, against pleural mesothelioma MM487. No reduction of mice body weight was observed, thus suggesting a higher tolerability with respect to the parent compound adarotene.

A Herbal Mixture from Propolis, Pomegranate, and Grape Pomace Endowed with Anti-Inflammatory Activity in an In Vivo Rheumatoid Arthritis Model.

Rheumatoid arthritis (RA) is a chronic inflammatory autoimmune disease characterized by the production of inflammatory factors. In order to overcome the side effects of currently used anti-inflammatory drugs, several attempts have been made to identify natural products capable of relieving RA symptoms. In this work, a herbal preparation consisting of propolis, pomegranate peel, and Aglianico grape pomace (PPP) extracts (4:1:1) was designed and evaluated for its effect on a murine collagen-induced arthritis (CIA) model. Firstly, the chemical contents of four different Italian propolis collected in the Campania region (Italy) were here reported for the first time. LC-MS analyses showed the presence of 38 constituents, identified in all propolis extracts, belonging to flavonoids and phenolic acids classes. The Pietradefusi extract was the richest one and thus was selected to design the PPP preparation for the in vivo assay. Our results highlight the impact of PPP on RA onset and progression. By using in vivo CIA models, the treatment with PPP resulted in a delayed onset of the disease and alleviated the severity of the clinical symptoms. Furthermore, we demonstrated that early PPP treatment was associated with a reduction in serum levels of IL-17, IL-1b, and IL-17-triggering cytokines.

Role of Toll-like receptor 13 in innate immune recognition of group B streptococci.

Murine Toll-like receptor 13 (TLR13), an endosomal receptor that is not present in humans, is activated by an unmethylated motif present in the large ribosomal subunit of bacterial RNA (23S rRNA). Little is known, however, of the impact of TLR13 on antibacterial host defenses. Here we examined the role of this receptor in the context of infection induced by the model pathogen group B streptococcus (GBS). To this end, we used bacterial strains masked from TLR13 recognition by virtue of constitutive expression of the ErmC methyltransferase, which results in dimethylation of the 23S rRNA motif at a critical adenine residue. We found that TLR13-mediated rRNA recognition was required for optimal induction of tumor necrosis factor alpha and nitrous oxide in dendritic cell and macrophage cultures stimulated with heat-killed bacteria or purified bacterial RNA. However, TLR13-dependent recognition was redundant when live bacteria were used as a stimulus. Moreover, masking bacterial rRNA from TLR13 recognition did not increase the ability of GBS to avoid host defenses and replicate in vivo. In contrast, increased susceptibility to infection was observed under conditions in which signaling by all endosomal TLRs was abolished, i.e., in mice with a loss-of-function mutation in the chaperone protein UNC93B1. Our data lend support to the conclusion that TLR13 participates in GBS recognition, although blockade of the function of this receptor can be compensated for by other endosomal TLRs. Lack of selective pressure by bacterial infections might explain the evolutionary loss of TLR13 in humans. However, further studies using different bacterial species are needed to prove this hypothesis.

Activation of the NLRP3 inflammasome by group B streptococci.

Group B Streptococcus (GBS) is a frequent agent of life-threatening sepsis and meningitis in neonates and adults with predisposing conditions. We tested the hypothesis that activation of the inflammasome, an inflammatory signaling complex, is involved in host defenses against this pathogen. We show in this study that murine bone marrow-derived conventional dendritic cells responded to GBS by secreting IL-1ß and IL-18. IL-1ß release required both pro-IL-1ß transcription and caspase-1-dependent proteolytic cleavage of intracellular pro-IL-1ß. Dendritic cells lacking the TLR adaptor MyD88, but not those lacking TLR2, were unable to produce pro-IL-1ß mRNA in response to GBS. Pro-IL-1ß cleavage and secretion of the mature IL-1ß form depended on the NOD-like receptor family, pyrin domain containing 3 (NLRP3) sensor and the apoptosis-associated speck-like protein containing a caspase activation and recruitment domain adaptor. Moreover, activation of the NLRP3 inflammasome required GBS expression of ß-hemolysin, an important virulence factor. We further found that mice lacking NLRP3, apoptosis-associated speck-like protein, or caspase-1 were considerably more susceptible to infection than wild-type mice. Our data link the production of a major virulence factor by GBS with the activation of a highly effective anti-GBS response triggered by the NLRP3 inflammasome.

Recognition of fungal RNA by TLR7 has a nonredundant role in host defense against experimental candidiasis.

Despite convincing evidence for involvement of members of the Toll-like receptor (TLR) family in fungal recognition, little is known of the functional role of individual TLRs in antifungal defenses. We found here that TLR7 was partially required for the induction of IL-12 (IL-12p70) by Candida albicans or Saccharomyces cerevisiae. Moreover, the IL-12p70 response was completely abrogated in cells from 3d mice, which are unable to mobilize TLRs to endosomal compartments, as well as in cells from mice lacking either the TLR adaptor MyD88 or the IRF1 transcription factor. Notably, purified fungal RNA recapitulated IL-12p70 induction by whole yeast. Although RNA could also induce moderate TLR7-dependent IL-23 and tumor necrosis factor-alpha (TNF-α) secretion, TLR7 and other endosomal TLRs were redundant for IL-23 or TNF-α induction by whole fungi. Importantly, mice lacking TLR7 or IRF1 were hypersusceptible to systemic C. albicans infection. Our data suggest that IRF1 is downstream of a novel, nonredundant fungal recognition pathway that has RNA as a major target and requires phagosomal recruitment of intracellular TLRs. This pathway differs from those involved in IL-23 or TNF-α responses, which we show here to be independent from translocation of intracellular TLRs, phagocytosis, or phagosomal acidification.

Recognition of yeast nucleic acids triggers a host-protective type I interferon response.

Although type I interferons (IFN-α/ß) have been traditionally associated with antiviral responses, their importance in host defense against bacterial pathogens is being increasingly appreciated. Little is known, however, about the occurrence and functional role of IFN-α/ß production in response to pathogenic yeasts. Here, we found that conventional DCs, but not macrophages nor plasmacytoid DCs, mounted IFN-ß responses after in vitro stimulation with Candida spp. or Saccharomyces cerevisiae. These responses absolutely required MyD88, a Toll-like receptor (TLR) adaptor molecule, and were partially dependent on TLR9 and TLR7. Moreover, Candida DNA, as well as RNA, could recapitulate the IFN-ß response. After intravenous challenge with Candida albicans, most mice lacking the IFN-α/ß receptor died from their inability to control fungal growth, whereas all WT controls survived. These data suggest that recognition of yeast nucleic acids by TLR7 and TLR9 triggers a host-protective IFN-α/ß response.

Antitumor activity of novel POLA1-HDAC11 dual inhibitors.

Hybrid molecules targeting simultaneously DNA polymerase α (POLA1) and histone deacetylases (HDACs) were designed and synthesized to exploit a potential synergy of action. Among a library of screened molecules, MIR002 and GEM144 showed antiproliferative activity at nanomolar concentrations on a panel of human solid and haematological cancer cell lines. In vitro functional assays confirmed that these molecules inhibited POLA1 primer extension activity, as well as HDAC11. Molecular docking studies also supported these findings. Mechanistically, MIR002 and GEM144 induced acetylation of p53, activation of p21, G1/S cell cycle arrest, and apoptosis. Oral administration of these inhibitors confirmed their antitumor activity in in vivo models. In human non-small cancer cell (H460) xenografted in nude mice MIR002 at 50 mg/kg, Bid (qd × 5 × 3w) inhibited tumor growth (TGI = 61%). More interestingly, in POLA1 inhibitor resistant cells (H460-R9A), the in vivo combination of MIR002 with cisplatin showed an additive antitumor effect with complete disappearance of tumor masses in two animals at the end of the treatment. Moreover, in two human orthotopic malignant pleural mesothelioma xenografts (MM473 and MM487), oral treatments with MIR002 and GEM144 confirmed their significant antitumor activity (TGI = 72-77%). Consistently with recent results that have shown an inverse correlation between POLA1 expression and type I interferon levels, MIR002 significantly upregulated interferon-α in immunocompetent mice.

Plasminogen- and fibronectin-binding protein B is involved in the adherence of Streptococcus pneumoniae to human epithelial cells.

Streptococcus pneumoniae is a major cause of morbidity and mortality worldwide. The ability of this bacterium to adhere to epithelial cells is considered as an essential early step in colonization and infection. By screening a whole genome phage display library with sera from infected patients, we previously identified three antigenic fragments matching open reading frame spr0075 of the strain R6 genome. This locus encodes for an approximately 120-kDa protein, herein referred to as plasminogen- and fibronectin-binding protein B (PfbB), which displays an LPXTG cell wall anchoring motif and six repetitive domains. In this study, by using isogenic pfbB-deleted mutants of the encapsulated D39 and of the unencapsulated DP1004 type 2 pneumococcal strains, we show that PfbB is involved in S. pneumoniae adherence to various epithelial respiratory tract cell lines. Our data suggest that PfbB directly mediates bacterial adhesion, because fluorescent beads coated with the recombinant PfbB sp17 fragment (encompassing one of the six repetitive domains and the C-terminal region) efficiently bound to epithelial cells. Mutants lacking PfbB bound to fibronectin and plasminogen considerably less efficiently than wild type bacteria, whereas sp17-coated beads specifically bound to both of these substrates. Taken together, our data suggest that, by directly interacting with fibronectin, PfbB significantly increases the ability of S. pneumoniae to adhere to human epithelial cells.

Hybrid topoisomerase I and HDAC inhibitors as dual action anticancer agents.

Recent studies have shown that HDAC inhibitors act synergistically with camptothecin derivatives in combination therapies. To exploit this synergy, new hybrid molecules targeting simultaneously topoisomerase I and HDAC were designed. In particular, a selected multivalent agent containing a camptothecin and a SAHA-like template showed a broad spectrum of antiproliferative activity, with IC50 values in the nanomolar range. Preliminary in vivo results indicated a strong antitumor activity on human mesothelioma primary cell line MM473 orthotopically xenografted in CD-1 nude mice and very high tolerability.

Tumor response of temozolomide in combination with morphine in a xenograft model of human glioblastoma.

Despite multimodal treatments comprising, radiation therapy (RT) and chemotherapy with temozolomide (TMZ), the prognosis of glioblastoma multiforme (GBM) remains dismal and consolidated therapy yields a median survival of 14.6 months. Blood Brain Barrier (BBB) mediated chemoresistance and high dose related toxicity make necessary the development of new therapeutic approach to sensitize GBM to TMZ. The aim of the present study was to investigate the potential of the treatment morphine plus TMZ metronmic doses (1,77 and 0,9 mg/kg) in GBM therapy. The effect of morphine, on tumor cell growth and P-glycoprothein (P-gp) activity, was investigate in in vitro models. The results demonstrated that GBM cells growth is not influenced by morphine treatment and, for the first time, we show that morphine is an inhibitor of the activity of P-gp efflux transporter who is markedly expressed on BBB. In vivo, response to the treatments TMZ plus morphine was investigated in an orthotopic nude mice model of GBM. Animals treated with TMZ metronomic doses showed a significant tumor growth inhibition compared to untreated mice and association with morphine appears to improve TMZ efficacy. Moreover, the combination of morphine with lower dose of TMZ result in a cytostatic effect on tumor growth over the period of the pharmacological treatments. In conclusion this novel approach could be a successful strategy to overcome chemoresistance and side effects TMZ mediated, reducing drug dosage and improving long term response, in GBM therapy.

Identification of OppA2 linear epitopes as serodiagnostic markers for Lyme disease.

Laboratory diagnosis of Lyme disease is based on the serological detection of antibodies against the etiologic agent Borrelia burgdorferi. Current diagnostics are insensitive at detecting early infection, when treatment is most effective. This deficiency results from the limited number of B. burgdorferi antigens expressed in early infection and the use of an insensitive two-tier paradigm, put in place to deal with insufficient specificity associated with the use of whole-protein antigens and/or bacterial lysates as serodiagnostic targets. Whole-protein antigens contain epitopes that are unique to B. burgdorferi as well as cross-reactive epitopes found in other bacteria. One method for overcoming the limitations imposed by cross-reactive epitopes is the use of short peptides containing epitopes unique to B. burgdorferi as antigen targets. This eliminates nonspecific epitopes. Using overlapping peptide libraries, we performed epitope mapping of linear epitopes in oligopeptide permease A2 (OppA2), a member of the oligopeptide permease (Opp) family of peptide transporters, expressed during early B. burgdorferi infection. We identified 9 epitopes, synthesized peptides containing these epitopes, and screened those using panels of blood from patients with early Lyme disease, rheumatoid arthritis (RA), or syphilis or from healthy individuals. Two of the peptides, OppA2 (191-225) (amino acids comprising the peptide are shown in parentheses) and OppA2 (381-400), are highly conserved among the three major pathogenic Borrelia species responsible for most Lyme disease cases in North America and Europe. They detected antibodies in Lyme disease patient sera with sufficient sensitivity and specificity to indicate that they could have value in a serological assay for Lyme disease.

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.

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.

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.