Anna Giardina

Obesity and reduction of the response rate to anti–tumor necrosis factor α in rheumatoid arthritis: An approach to a personalized medicine

Obesity is a mild, long‐lasting inflammatory disease and, as such, could increase the inflammatory burden of rheumatoid arthritis (RA). The study aim was to determine whether obesity represents a risk factor for a poor remission rate in RA patients requiring anti–tumor necrosis factor α (anti‐TNFα) therapy for progressive and active disease despite treatment with methotrexate or other disease‐modifying antirheumatic drugs.

A 2-year comparative open label randomized study of efficacy and safety of etanercept and infliximab in patients with ankylosing spondylitis

The signs and symptoms of ankylosing spondylitis (AS) respond inadequately to nonsteroidal antiinflammatory drugs, corticosteroids, and disease modifying antirheumatic drugs in quite a number of patients. Tumor necrosis factor inhibitors have demonstrated to be of value in reducing AS disease activity in clinical trials. The efficacy and safety of both etanercept and infliximab in patients with ankylosing spondylitis were compared in a 2-year open label randomised study. Our results are consistent with a significant more rapid clinical improvement in the infliximab treated group. Treatment with both etanercept and infliximab at the end of the study was effective, safe, and well tolerated.

Phenotype and functional changes of Vγ9/Vδ2 T lymphocytes in Behçet's disease and the effect of infliximab on Vγ9/Vδ2 T cell expansion, activation and cytotoxicity

IntroductionInfliximab is a chimeric monoclonal antibody against tumor necrosis factor alpha (TNF-α) that has been introduced recently for Behçets disease (BD) patients who were resistant to standard treatment. The aim of this study was to analyse the functional changes of Vγ9/Vδ2 T lymphocytes in both active and inactive disease and the effect of infliximab on Vγ9/Vδ2 T cell expansion, activation and cytotoxicity.MethodsWe investigated 1) cell expansion, 2) expression of TNFRII receptor, 3) perforin and gamma interferon (IFN) content, 4) release of granzyme A (GrA) and 5) phenotype changes, in vitro and in vivo, in Vγ9/Vδ2 T lymphocytes by means of fluorescence-activated cell sorter analysis of lymphocyte cultures from patients with active and inactive BD and healthy subjects.ResultsCell expansion, expression of TNFRII, perforin and gamma IFN content and release of granzyme A were significantly higher in active patients. In vitro and ex vivo treatment with infliximab resulted in a significant reduction of all parameters together with changes in the phenotype of Vγ9/Vδ2 T cells.ConclusionsAll together these data indicate that infliximab is capable of interfering with Vγ9/Vδ2 T cell function in BD and although cell culture models cannot reliably predict all potential effects of the drug in vivo, our results present the possibility that this drug may find use in a range of immunological disorders, characterized by dysregulated cell-mediated immunity.

Expression in Streptomyces lividans of Nonomuraea genes cloned in an artificial chromosome

A bacterial artificial chromosomal library of Nonomuraea sp. ATCC39727 was constructed using Escherichia coli–Streptomyces artificial chromosome (ESAC) and screened for the presence of dbv genes known to be involved in the biosynthesis of the glycopeptide A40926. dbv genes were cloned as two large, partially overlapping, fragments and transferred into the host Streptomyces lividans, thus generating strains S. lividans∷NmESAC50 and S. lividans∷NmESAC57. The heterologous expression of Nonomuraea genes in S. lividans was successfully demonstrated by using combined RT–PCR and proteomic approaches. MALDI-TOF analysis revealed that a Nonomuraea ABC transporter is expressed as two isoforms in S. lividans. Moreover, its expression may not require a Nonomuraea positive regulator at all, as it is present at similar levels in both clones even though S. lividans∷NmESAC57 lacks regulatory genes. Considered together, these results show that S. lividans expresses Nonomuraea genes from their own promoters and support the idea that S. lividans can be a good host for genetic analysis of Nonomuraea.

Are Toll-Like Receptors and Decoy Receptors Involved in the Immunopathogenesis of Systemic Lupus Erythematosus and Lupus-Like Syndromes?

In this paper we focus our attention on the role of two families of receptors, Toll-like receptors (TLR) and decoy receptors (DcR) involved in the generation of systemic lupus erythematosus (SLE) and lupus-like syndromes in human and mouse models. To date, these molecules were described in several autoimmune disorders such as rheumatoid arthritis, antiphospholipids syndrome, bowel inflammation, and SLE. Here, we summarize the findings of recent investigations on TLR and DcR and their role in the immunopathogenesis of the SLE.

Tryptophan catabolism via kynurenine production in Streptomyces coelicolor: identification of three genes coding for the enzymes of tryptophan to anthranilate pathway

Most enzymes involved in tryptophan catabolism via kynurenine formation are highly conserved in Prokaryotes and Eukaryotes. In humans, alterations of this pathway have been related to different pathologies mainly involving the central nervous system. In Bacteria, tryptophan and some of its derivates are important antibiotic precursors. Tryptophan degradation via kynurenine formation involves two different pathways: the eukaryotic kynurenine pathway, also recently found in some bacteria, and the tryptophan-to-anthranilate pathway, which is widespread in microorganisms. The latter produces anthranilate using three enzymes also involved in the kynurenine pathway: tryptophan 2,3-dioxygenase (TDO), kynureninase (KYN), and kynurenine formamidase (KFA). In Streptomyces coelicolor, where it had not been demonstrated which genes code for these enzymes, tryptophan seems to be important for the calcium- dependent antibiotic (CDA) production. In this study, we describe three adjacent genes of S. coelicolor (SCO3644, SCO3645, and SCO3646), demonstrating their involvement in the tryptophan-to-anthranilate pathway: SCO3644 codes for a KFA, SCO3645 for a KYN and SCO3646 for a TDO. Therefore, these genes can be considered as homologous respectively to kynB, kynU, and kynA of other microorganisms and belong to a constitutive catabolic pathway in S. coelicolor, which expression increases during the stationary phase of a culture grown in the presence of tryptophan. Moreover, the S. coelicolor ΔkynU strain, in which SCO3645 gene is deleted, produces higher amounts of CDA compared to the wild-type strain. Overall, these results describe a pathway, which is used by S. coelicolor to catabolize tryptophan and that could be inactivated to increase antibiotic production.

Two heterologously expressed Planobispora rosea proteins cooperatively induce Streptomyces lividans thiostrepton uptake and storage from the extracellular medium

BackgroundA bacterial artificial chromosomal library of Planobispora rosea, a genetically intractable actinomycete strain, was constructed using Escherichia coli-Streptomyces artificial chromosome (ESAC) and screened for the presence of genes known to be involved in the biosynthesis of antibiotics.ResultsOne clone with a 40 kb insert showed antimicrobial activity against Gram positive bacteria. Insert sequence analysis and subcloning experiments revealed that the bioactivity was due to a 3.5 kb DNA fragment containing two open reading frames. These orfs encode two proteins with high similarity to a putative membrane protein of Streptomyces coelicolor and to the nogalamycin resistance protein SnorO of Streptomyces nogalater, respectively. The role of these two Orfs is unknown in Planobispora. Disruption and complementation experiments revealed that both proteins are necessary for the antibacterial activity and chemical analysis demonstrated that the antibiotic activity was due to thiostrepton, antibiotic used as recombinant clone selection marker.ConclusionTwo Planobispora rosea orfs are responsible for increasing intracellular amounts and storage of thiostrepton in Streptomyces lividans.

TrpM, a Small Protein Modulating Tryptophan Biosynthesis and Morpho-Physiological Differentiation in Streptomyces coelicolor A3(2).

In the model actinomycete Streptomyces coelicolor A3(2), small open reading frames encoding proteins with unknown functions were identified in several amino acid biosynthetic gene operons, such as SCO2038 (trpX) in the tryptophan trpCXBA locus. In this study, the role of the corresponding protein in tryptophan biosynthesis was investigated by combining phenotypic and molecular analyses. The 2038KO mutant strain was characterized by delayed growth, smaller aerial hyphae and reduced production of spores and actinorhodin antibiotic, with respect to the WT strain. The capability of this mutant to grow on minimal medium was rescued by tryptophan and tryptophan precursor (serine and/or indole) supplementation on minimal medium and by gene complementation, revealing the essential role of this protein, here named TrpM, as modulator of tryptophan biosynthesis. His-tag pull-down and bacterial adenylate cyclase-based two hybrid assays revealed TrpM interaction with a putative leucyl-aminopeptidase (PepA), highly conserved component among various Streptomyces spp. In silico analyses showed that PepA is involved in the metabolism of serine, glycine and cysteine through a network including GlyA, CysK and CysM enzymes. Proteomic experiments suggested a TrpM-dependent regulation of metabolic pathways and cellular processes that includes enzymes such as GlyA, which is required for the biosynthesis of tryptophan precursors and key proteins participating in the morpho-physiological differentiation program. Altogether, these findings reveal that TrpM controls tryptophan biosynthesis at the level of direct precursor availability and, therefore, it is able to exert a crucial effect on the morpho-physiological differentiation program in S. coelicolor A3(2).

Inorganic phosphate is a trigger factor for Microbispora sp. ATCC-PTA-5024 growth and NAI-107 production

BackgroundNAI-107, produced by the actinomycete Microbispora sp. ATCC-PTA-5024, is a promising lantibiotic active against Gram-positive bacteria and currently in late preclinical-phase. Lantibiotics (lanthionine-containing antibiotics) are ribosomally synthesized and post-translationally modified peptides (RiPPs), encoded by structural genes as precursor peptides.The biosynthesis of biologically active compounds is developmentally controlled and it depends upon a variety of environmental stimuli and conditions. Inorganic phosphate (Pi) usually negatively regulates biologically-active molecule production in Actinomycetes, while it has been reported to have a positive control on lantibiotic production in Firmicutes strains. So far, no information is available concerning the Pi effect on lantibiotic biosynthesis in Actinomycetes.ResultsAfter having developed a suitable defined medium, Pi-limiting conditions were established and confirmed by quantitative analysis of polyphosphate accumulation and of expression of selected Pho regulon genes, involved in the Pi-limitation stress response. Then, the effect of Pi on Microbispora growth and NAI-107 biosynthesis was investigated in a defined medium containing increasing Pi amounts. Altogether, our analyses revealed that phosphate is necessary for growth and positively influences both growth and NAI-107 production up to a concentration of 5 mM. Higher Pi concentrations were not found to further stimulate Microbispora growth and NAI-107 production.ConclusionThese results, on one hand, enlarge the knowledge on Microbispora physiology, and, on the other one, could be helpful to develop a robust and economically feasible production process of NAI-107 as a drug for human use.

Identification of SCP2165, a new SCP2-derived plasmid of Streptomyces coelicolor A3(2)

Aims:  Characterization of SCP2165, a plasmid identified in the Gram‐positive bacterium Streptomyces coelicolor A3(2).