Pigmentation and Sporulation Are Alternative Cell Fates in Bacillus pumilus SF214

Bacillus pumilus SF214 is a spore forming bacterium, isolated from a marine sample, able to produce a matrix and a orange-red, water soluble pigment. Pigmentation is strictly regulated and high pigment production was observed during the late stationary growth phase in a minimal medium and at growth temperatures lower than the optimum. Only a subpopulation of stationary phase cells produced the pigment, indicating that the stationary culture contains a heterogeneous cell population and that pigment synthesis is a bimodal phenomenon. The fraction of cells producing the pigment varied in the different growth conditions and occured only in cells not devoted to sporulation. Only some of the pigmented cells were also able to produce a matrix. Pigment and matrix production in SF214 appear then as two developmental fates both alternative to sporulation. Since the pigment had an essential role in the cell resistance to oxidative stress conditions, we propose that within the heterogeneous population different survival strategies can be followed by the different cells.     

Targeting of viral interleukin-10 with an antibody fragment specific to damaged arthritic cartilage improves its therapeutic potency

Introduction

We previously demonstrated that a single-chain fragment variable (scFv) specific to collagen type II (CII) posttranslationally modified by reactive oxygen species (ROS) can be used to target anti-inflammatory therapeutics specifically to inflamed arthritic joints. The objective of the present study was to demonstrate the superior efficacy of anti-inflammatory cytokines when targeted to inflamed arthritic joints by the anti-ROS modified CII (anti-ROS-CII) scFv in a mouse model of arthritis.

Methods

Viral interleukin-10 (vIL-10) was fused to anti-ROS-CII scFv (1-11E) with a matrix-metalloproteinase (MMP) cleavable linker to create 1-11E/vIL-10 fusion. Binding of 1-11E/vIL-10 to ROS-CII was determined by enzyme-linked immunosorbent assay (ELISA), Western blotting, and immune-staining of arthritic cartilage, whereas vIL-10 bioactivity was evaluated in vitro by using an MC-9 cell-proliferation assay. Specific in vivo localization and therapeutic efficacy of 1-11E/vIL-10 was tested in the mouse model of antigen-induced arthritis.

Results

1-11E/vIL-10 bound specifically to ROS-CII and to damaged arthritic cartilage. Interestingly, the in vitro vIL-10 activity in the fusion protein was observed only after cleavage with MMP-1. When systemically administered to arthritic mice, 1-11E/vIL-10 localized specifically to the arthritic knee, with peak accumulation observed after 3 days. Moreover, 1-11E/vIL-10 reduced inflammation significantly quicker than vIL-10 fused to the control anti-hen egg lysozyme scFv (C7/vIL10).

Conclusions

Targeted delivery of anti-inflammatory cytokines potentiates their anti-arthritic action in a mouse model of arthritis. Our results further support the hypothesis that targeting biotherapeutics to arthritic joints may be extended to include anti-inflammatory cytokines that lack efficacy when administered systemically.     

Family with Sequence Similarity 5, Member C (FAM5C) Increases Leukocyte Adhesion Molecules in Vascular Endothelial Cells: Implication in Vascular Inflammation

Identification of the regulators of vascular inflammation is important if we are to understand the molecular mechanisms leading to atherosclerosis and consequent ischemic heart disease, including acute myocardial infarction. Gene polymorphisms in family with sequence similarity 5, member C (FAM5C) are associated with an increased risk of acute myocardial infarction, but little is known about the function of this gene product in blood vessels. Here, we report that the regulation of the expression and function of FAM5C in endothelial cells. We show here that FAM5C is expressed in endothelial cells in vitro and in vivo. Immunofluorescence microcopy showed localization of FAM5C in the Golgi in cultured human endothelial cells. Immunohistochemistry on serial sections of human coronary artery showed that FAM5C-positive endothelium expressed intercellular adhesion molecule-1 (ICAM-1) or vascular cell adhesion molecule-1 (VCAM-1). In cultured human endothelial cells, the overexpression of FAM5C increased the reactive oxygen species (ROS) production, nuclear factor-κB (NF-κB) activity and the expression of ICAM-1, VCAM-1 and E-selectin mRNAs, resulting in enhanced monocyte adhesion. FAM5C was upregulated in response to inflammatory stimuli, such as TNF-α, in an NF-κB- and JNK-dependent manner. Knockdown of FAM5C by small interfering RNA inhibited the increase in the TNF-α-induced production of ROS, NF-κB activity and expression of these leukocyte adhesion molecule mRNAs, resulting in reduced monocyte adhesion. These results suggest that in endothelial cells, when FAM5C is upregulated in response to inflammatory stimuli, it increases the expression of leukocyte adhesion molecules by increasing ROS production and NF-κB activity.     

IL-6 Amplifies TLR Mediated Cytokine and Chemokine Production: Implications for the Pathogenesis of Rheumatic Inflammatory Diseases

The role of Interleukin(IL)-6 in the pathogenesis of joint and systemic inflammation in rheumatoid arthritis (RA) and systemic juvenile idiopathic arthritis (s-JIA) has been clearly demonstrated. However, the mechanisms by which IL-6 contributes to the pathogenesis are not completely understood. This study investigates whether IL-6 affects, alone or upon toll like receptor (TLR) ligand stimulation, the production of inflammatory cytokines and chemokines in human peripheral blood mononuclear cells (PBMCs), synovial fluid mononuclear cells from JIA patients (SFMCs) and fibroblast-like synoviocytes from rheumatoid arthritis patients (RA synoviocytes) and signalling pathways involved. PBMCs were pre-treated with IL-6 and soluble IL-6 Receptor (sIL-6R). SFMCs and RA synoviocytes were pre-treated with IL-6/sIL-6R or sIL-6R, alone or in combination with Tocilizumab (TCZ). Cells were stimulated with LPS, S100A8-9, poly(I-C), CpG, Pam2CSK4, MDP, IL-1β. Treatment of PBMCs with IL-6 induced production of TNF-α, CXCL8, and CCL2, but not IL-1β. Addition of IL-6 to the same cells after stimulation with poly(I-C), CpG, Pam2CSK4, and MDP induced a significant increase in IL-1β and CXCL8, but not TNF-α production compared with TLR ligands alone. This enhanced production of IL-1β and CXCL8 paralleled increased p65 NF-κB activation. In contrast, addition of IL-6 to PBMCs stimulated with LPS or S100A8-9 (TLR-4 ligands) led to reduction of IL-1β, TNF-α and CXCL8 with reduced p65 NF-κB activation. IL-6/IL-1β co-stimulation increased CXCL8, CCL2 and IL-6 production. Addition of IL-6 to SFMCs stimulated with LPS or S100A8 increased CXCL8, CCL2 and IL-1β production. Treatment of RA synoviocytes with sIL-6R increased IL-6, CXCL8 and CCL2 production, with increased STAT3 and p65 NF-κB phosphorylation. Our results suggest that IL-6 amplifies TLR-induced inflammatory response. This effect may be relevant in the presence of high IL-6 and sIL-6R levels, such as in arthritic joints in the context of stimulation by endogenous TLR ligands.     

Time-, concentration-, and age-dependent inhibition of muscarinic receptor-stimulated phosphoinositide metabolism by ethanol in the developing rat brain

We have previously reported that administration of ethanol (EtOH; 4 g/Kg/day) to rats from postnatal day 4 to day 10 causes microencephaly and decreases muscarinic receptor-stimulated inositol metabolism on days 7 and 10 (1). An identical exposure to EtOH of adult rats, which resulted in similar blood EtOH concentrations, did not have any effect on the same system. Initial in vitro studies have shown the presence of a differential sensitivity to EtOH of the phosphoinositide system coupled to muscarinic receptors during development (2). In the present study we have expanded these findings by investigating the concentration-, time-, and age-dependent effects of EtOH on accumulation of [³H]inositol phosphates ([³H]InsPs) in brain slices. EtOH caused a dose-dependent inhibition of carbachol-stimulated phosphoinositide metabolism in cerebral cortex slices from 7 day-old rats. When the time of incubation with EtOH was increased to 90 minutes, concentrations as low as 50 mM, which are reached following in vivo administration of EtOH, significantly inhibited the muscarinic response. The effect of EtOH was rather specific for the muscarinic receptors, since, even with longer incubation times, the accumulation of [³H]InsPs induced by norepinephrine or serotonin was inhibited only at concentrations of 150–500 mM. The effect of EtOH was more pronounced in cerebral cortex, hippocampus and cerebellum, and less in the brainstem. The potency of EtOH in inhibiting carbachol-stimulated phosphoinositide metabolism was also dependent on the age of the animals. Its effect was maximal in the 7 day-old rat and less pronounced in younger and older animals. These results confirm that the phosphoinositide system coupled to muscarinic receptors might represent a relevant target for the developmental neurotoxicity of EtOH.Presented in part at the 29th Annual Meeting of the Society of Toxicology (Toxicologist 1990; 10: 273).     

Inflammatory lesions in the bone marrow of rheumatoid arthritis patients: a morphological perspective

The synovial tissue stands at the epicenter of joint pathology in rheumatoid arthritis (RA). As a primary target of the disease, studies on the synovium have provided invaluable insights into the mechanisms involved in disease pathogenesis. Recent work has, however, revealed the importance of a previously unseen anatomic compartment in direct contact with the joint space, namely the subchondral bone marrow. Bone marrow edema (BME) visible on magnetic resonance imaging (MRI) is clinically meaningful in both early and late RA as it associates with future development of bone erosions and poor functional outcomes. Although the histopathologic correlates of MRI-based BME in early RA remain obscure, studies in advanced disease are consistent in describing lymphocytic inflammatory infiltrates within the subchondral marrow cavity of affected joints. In this review, we discuss the nature of bone marrow lesions in patients with RA, analyze their relationship with synovitis, and explore their potential contribution to the pathological processes of the disease.Rheumatoid arthritis (RA) is traditionally regarded as an archetypal disease of the synovial tissue. The synovial membrane indeed undergoes early inflammatory changes, which include increased vascularity as well as intimal lining layer hyperplasia and accumulation of macrophages, plasma cells, T cells, B cells, dendritic cells, natural killer cells and mast cells in the sublining, among other changes [1]. Collectively, localization of inflammation to the synovium is primarily responsible for the dysregulated cellular and molecular mechanisms that ultimately lead to the typical signs and symptoms of RA, including joint pain, stiffness, swelling and structural changes. Although the pathogenic and clinical importance of synovitis is beyond question, it is becoming increasingly apparent that a ''synovio-centric'' model of RA could be limiting. Indeed, other anatomic compartments appear to be involved at all stages of the disease. The most important example comes from the recognition that clinical arthritis and subclinical synovitis are anticipated by a pre-articular immunologic phase possibly developing in lymphoid tissues as well as in the lungs [2,3]. Full-blown joint disease itself spreads well beyond synovial tissue inflammation. Established arthritis can be characterized by the involvement of at least two other compartments that are in direct contact with the joint space. These include the draining lymph nodes (LNs) [4,5] and, of special relevance, the subchondral bone marrow (BM) [6].As dysregulated B cell responses are central pathogenic events in RA and B-cell autoreactivity originates, at least in part, from defective checkpoints within the BM (a primary lymphoid organ) [7], the role of this compartment in the immunopathogenesis of RA has attracted great attention over the years. Data supporting the existence of BM abnormalities were mainly derived from studies on marrow aspirates/biopsies from the sternum and the iliac crest, which are primary hematopoietic and immunologic sites in adult life. The role that the ''red marrow'' might play in the immunologic disturbances of RA thus appears interesting but not completely unexpected. Less predictably, evidence has accumulated supporting the additional involvement of the BM adjacent to inflamed joints at peripheral sites. This is normally a fat-rich tissue (''yellow marrow'') where hematopoiesis is not prominent. In RA, the fat can be replaced by a vascularized, cell-rich inflammatory tissue. Combined with imaging data coming from magnetic resonance imaging (MRI) studies, histopathologic changes described within the subchondral BM have been postulated to be intimately involved in the pathological processes producing local inflammation and tissue remodeling in RA joints [6,8-10]. Here we will discuss the most relevant findings related to BM alterations in RA, focusing on their morphological characteristics and pathologic significance. In view of the clinical relevance of MRI findings, special emphasis is given to the subchondral BM.     

Direct demonstration and quantification of long-range DNA looping by the λ bacteriophage repressor

Recently, it was proposed that DNA looping by the λ repressor (CI protein) strengthens repression of lytic genes during lysogeny and simultaneously ensures efficient switching to lysis. To investigate this hypothesis, tethered particle motion experiments were performed and dynamic CI-mediated looping of single DNA molecules containing the λ repressor binding sites separated by 2317 bp (the wild-type distance) was quantitatively analyzed. DNA containing all three intact operators or with mutated o3 operators were compared. Modeling the thermodynamic data established the free energy of CI octamer-mediated loop formation as 1.7 kcal/mol, which decreased to –0.7 kcal/mol when supplemented by a tetramer (octamer+tetramer-mediated loop). These results support the idea that loops secured by an octamer of CI bound at oL1, oL2, oR1 and oR2 operators must be augmented by a tetramer of CI bound at the oL3 and oR3 to be spontaneous and stable. Thus the o3 sites are critical for loops secured by the CI protein that attenuate cI expression.     

WeaselMustela nivalis spatial ranging behaviour and habitat selection in agricultural landscape

This paper reports the findings of a study on the spacing pattern of a population of weaselsMustela nivalis Linnaeus, 1758 in central Italy. We describe this pattern at spatial and habitat level, meant as a set of vegetational and environmental features. The study area is a typical agricultural landscape, consisting of highly fragmented natural habitat modified by thousands of years of human presence and work. The results indicate that individual home ranges vary greatly from non breeding to breeding season: during spring and summer, weasels increase their movements and the number of tracts they cross, which results in home range areas up to 10 times larger. Moreover this study demonstrates a strong preference of weasels for habitats formed by natural residual edges between fields and meadows. The shapes of the individual home ranges follow these edges, producing a typical linear spatial home range pattern.