Early treatment with Lactobacillus delbrueckii strain induces rise in intestinal T cells and granulocytes and modulates immune related genes of larval Dicentrarchus labrax (L.)

Lactobacillus delbrueckii ssp. delbrueckii (AS13B), isolated from the gut of adult Dicentrarchus labrax, was administered live to developing sea bass using rotifers and Artemia as live carriers. Immune-related gene transcripts were quantified in post-larvae at day 70 post-hatch (ph) and histology, electron microscopy and immunocytochemistry of the intestinal tissue were performed at day 74 ph. Since the probiotic was orally administered the studies were focused on intestinal immunity. In treated fish gut integrity was unaffected, while the density of T-cells and acidophilic granulocytes in the intestinal mucosa was significantly higher than in controls. Probiotic-induced increases in intestinal T-cells and total body TcR-beta transcripts are first reported in fish. Significantly lower IL-1beta transcripts and a trend towards lower IL-10, Cox-2 and TGF-beta transcription were found in the treated group. Evidence is provided that early feeding with probiotic-supplemented diet stimulated the larval gut immune system and lowered transcription of key pro-inflammatory genes.L'articolo è disponibile sul sito dell'editore http://www.sciencedirect.com

Chemical investigation and screening of anti-proliferative activity on human cell lines of pure and nano-formulated lavandin essential oil

Lavandin essential oil (LEO), a natural sterile hybrid obtained by crossbreeding L. angustifolia × L. latifolia, is mainly composed by active components belonging to the family of terpenes endowed with relevant anti-proliferative activity, which can be enhanced by proper application of nanotechnology. In particular, this study reports the chemical characterization and the screening of the anti-proliferative activity on different human cell lines of pure and nano-formulated lavandin essential oil (EO). LEO and its formulation (NanoLEO) were analyzed by HS/GC-MS (Headspace/Gas Chromatography-Mass Spectrometry) to describe and compare their chemical volatile composition. The most abundant compounds were linalool and 1,8-cineole (LEO: 28.6%; 27.4%) (NanoLEO: 60.4%; 12.6%) followed by α-pinene (LEO: 9.6%; NanoLEO: 4.5%), camphor (LEO: 6.5%; NanoLEO: 7.0%) and linalyl acetate (LEO: 6.5%; NanoLEO: 3.6%). The cytotoxic effects of LEO and NanoLEO were investigated on human neuroblastoma cells (SHSY5Y), human breast adenocarcinoma cells (MCF-7), human lymphoblastic leukemia cells (CCRF CEM), human colorectal adenocarcinoma cells (Caco-2) and one normal breast epithelial cell (MCF10A) by the MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide)-assay. Caco-2, MCF7 and MCF10A normal cells resulted more resistant to the treatment with LEO, while CCRF-CEM and SHSY5Y cells were more sensitive. The antiproliferative effect of LEO resulted amplified when the essential oil was supplied as nanoformulation, mainly in Caco-2 cells. Scanning and transmission electron microscopy investigations were carried out on Caco-2 cells to outline at ultrastructural level possible affections induced by LEO and NanoLEO treatments

Antibody drug conjugates (ADCs) charged with HDAC inhibitor for targeted epigenetic modulation

We describe here two novel antibody-drug conjugates loaded with the HDAC inhibitor ST7612AA1 (IC50equal to 0.07 μM on NCI-H460 cells), a thiol-based molecule with a moderate toxicity in vivo. Two payloads were prepared using cleavable and non-cleavable linkers. After anchoring to cetuximab through amide bond with lysines, the resulting HDAC inhibitor-antibody conjugates showed ability to recognize EGFR and efficient internalization in tumor cells. Both ADCs induced sensible increment of histones 3 and 4 and alpha-tubulin acetylation. Animal models of human solid tumors showed high anti-tumor efficacy of the conjugates without the toxicity generally observed with traditional ADCs delivering highly potent cytotoxic drugs. These compounds, the first ADCs charged with not highly cytotoxic warheads, are potentially suitable for epigenetic modulation, extending the ADC strategy to the targeted delivery of HDAC inhibitors with many possible therapeutic applications beyond cancer

Proteomics Characterization of Outer Membrane Vesicles from the Extraintestinal Pathogenic Escherichia coli ΔtolR IHE3034 Mutant

Extraintestinal pathogenic Escherichia coli are the cause of a diverse spectrum of invasive infections in humans and animals, leading to urinary tract infections, meningitis, or septicemia. In this study, we focused our attention on the identification of the outer membrane proteins of the pathogen in consideration of their important biological role and of their use as potential targets for prophylactic and therapeutic interventions. To this aim, we generated a DeltatolR mutant of the pathogenic IHE3034 strain that spontaneously released a large quantity of outer membrane vesicles in the culture supernatant. The vesicles were analyzed by two-dimensional electrophoresis coupled to mass spectrometry. The analysis led to the identification of 100 proteins, most of which are localized to the outer membrane and periplasmic compartments. Interestingly based on the genome sequences available in the current public database, seven of the identified proteins appear to be specific for pathogenic E. coli and enteric bacteria and therefore are potential targets for vaccine and drug development. Finally we demonstrated that the cytolethal distending toxin, a toxin exclusively produced by pathogenic bacteria, is released in association with the vesicles, supporting the recently proposed role of bacterial vesicles in toxin delivery to host cells. Overall, our data demonstrated that outer membrane vesicles represent an ideal tool to study Gram-negative periplasm and outer membrane compartments and to shed light on new mechanisms of bacterial pathogenesis

The meningococcal vaccine antigen GNA2091 is an analogue of YraP and plays key roles in outer membrane stability and virulence

K.L.S. was supported by the Australian National Health and Medical Research Council (NHMRC) C. J. Martin Fellowship and Career Development Fellowship. A.F.H. was supported by a Marie Curie Fellowship (PIEF-GA-2012-328377). F.O., L.F., and S.B. were recipients of Novartis fellowships from the Ph.D. program of the University of Siena (Siena, Italy) and University of Bologna (Bologna, Italy), respectively.GNA2091 is one of the components of the 4-component meningococcal serogroup B vaccine (4CMenB) vaccine and is highly conserved in all meningococcal strains. However, its functional role has not been fully characterized. Here we show that nmb2091 is part of an operon and is cotranscribed with the nmb2089, nmb2090, and nmb2092 adjacent genes, and a similar but reduced operon arrangement is conserved in many other gram-negative bacteria. Deletion of the nmb2091 gene causes an aggregative phenotype with a mild defect in cell separation; differences in the outer membrane composition and phospholipid profile, in particular in the phosphoethanolamine levels; an increased level of outer membrane vesicles; and deregulation of the zinc-responsive genes such as znuD. Finally, the Δ2091 strain is attenuated with respect to the wild-type strain in competitive index experiments in the infant rat model of meningococcal infection. Altogether these data suggest that GNA2091 plays important roles in outer membrane architecture, biogenesis, homeostasis, and in meningococcal survival in vivo, and amodel for its role is discussed. These findings highlight the importance of GNA2091 as a vaccine component.PostprintPeer reviewe

Antibody drug conjugates with hydroxamic acid cargos for histone deacetylase (HDAC) inhibition.

The bioconjugation of hydroxamic acids to antibodies has been made possible through a non-cleavable linker based on the p-mercaptobenzyl alcohol structure that releases hydroxamates in the cells

HadA is an atypical new multifunctional trimeric coiled-coil adhesin of Haemophilus influenzae biogroup aegyptius, which promotes entry into host cells.

Summary The Oca (Oligomeric coiled-coil adhesin) family is a subgroup of the bacterial trimeric autotrans- porter adhesins, which includes structurally related proteins, such as YadA of Yersinia entero- colitica and NadA of Neisseria meningitidis. In this study, we searched in silico for novel members of this family in bacterial genomes and identified HadA (Haemophilus adhesin A), a trimeric autotransporter expressed only by Haemophilus influenzae biogroup aegyptius causing Brazilian purpuric fever (BPF), a fulminant septicemic disease of children. By comparative genomics and sequence analysis we predicted that the hadA gene is harboured on a mobile genetic element unique to BPF isolates. Biological analysis of HadA in the native background was limited because this organism is not amenable to genetic manipulation. Alternatively, we demonstrated that expression of HadA confers to a non-invasive Escherichia coli strain the ability to adhere to human cells and to extracellular matrix proteins and to induce in vitro bacterial aggregation and microcolony formation. Intriguingly, HadA is pre- dicted to lack the typical N-terminal head domain of Oca proteins generally associated with cellular receptor binding. We propose here a structural model of the HadA coiled-coil stalk and show that the N-terminal region is still responsible of the binding activity and a KGD motif plays a role. Interestingly, HadA promotes bacterial entry into mammalian cells. Our results show a cytoskeleton re-arrangement and an involvement of clathrin in the HadA-mediated internalization. These data give new insights on the structure-function relationship of oligomeric coiled-coil adhesins and suggest a potential role of this protein in the pathogenesis of BPF

Coordination polymers for gas storage and cation exchange

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