Woven bone formation and mineralization by rat mesenchymal stromal cells imply increased expression of the intermediate filament desmin

BackgroundDisordered and hypomineralized woven bone formation by dysfunctional mesenchymal stromal cells (MSCs) characterize delayed fracture healing and endocrine –metabolic bone disorders like fibrous dysplasia and Paget disease of bone. To shed light on molecular players in osteoblast differentiation, woven bone formation, and mineralization by MSCs we looked at the intermediate filament desmin (DES) during the skeletogenic commitment of rat bone marrow MSCs (rBMSCs), where its bone-related action remains elusive.ResultsMonolayer cultures of immunophenotypically- and morphologically - characterized, adult male rBMSCs showed co-localization of desmin (DES) with vimentin, F-actin, and runx2 in all cell morphotypes, each contributing to sparse and dense colonies. Proteomic analysis of these cells revealed a topologically-relevant interactome, focused on cytoskeletal and related enzymes//chaperone/signalling molecules linking DES to runx2 and alkaline phosphatase (ALP). Osteogenic differentiation led to mineralized woven bone nodules confined to dense colonies, significantly smaller and more circular with respect to controls. It significantly increased also colony-forming efficiency and the number of DES-immunoreactive dense colonies, and immunostaining of co-localized DES/runx-2 and DES/ALP. These data confirmed pre-osteoblastic and osteoblastic differentiation, woven bone formation, and mineralization, supporting DES as a player in the molecular pathway leading to the osteogenic fate of rBMSCs.ConclusionImmunocytochemical and morphometric studies coupled with proteomic and bioinformatic analysis support the concept that DES may act as an upstream signal for the skeletogenic commitment of rBMSCs. Thus, we suggest that altered metabolism of osteoblasts, woven bone, and mineralization by dysfunctional BMSCs might early be revealed by changes in DES expression//levels. Non-union fractures and endocrine – metabolic bone disorders like fibrous dysplasia and Paget disease of bone might take advantage of this molecular evidence for their early diagnosis and follow-up

Presence of <i>Legionella</i> and Free-Living Amoebae in Composts and Bioaerosols from Composting Facilities

<div><p>Several species of <i>Legionella</i> cause Legionnaires’ disease (LD). Infection may occur through inhalation of <i>Legionella</i> or amoebal vesicles. The reservoirs of <i>Legionella</i> are water, soil, potting soil and compost. Some species of free-living amoebae (FLA) that are naturally present in water and soil were described as hosts for <i>Legionella</i>. This study aimed to understand whether or not the composting facilities could be sources of community-acquired <i>Legionella</i> infections after development of bioaerosols containing <i>Legionella</i> or FLA. We looked for the presence of <i>Legionella</i> (by co-culture) and FLA (by culture) in composts and bioaerosols collected at four composting facilities located in southern Switzerland. We investigated the association between the presence of <i>Legionella</i> and compost and air parameters and presence of FLA. <i>Legionella</i> spp. (including <i>L. pneumophila</i>) were detected in 69.3% (61/88) of the composts and FLA (mainly <i>Acanthamoeba</i>, <i>Vermamoeba</i>, <i>Naegleria</i> and <i>Stenamoeba</i>) in 92.0% (81/88). <i>L. pneumophila</i> and <i>L. bozemanii</i> were most frequently isolated. FLA as potential host for <i>Legionella</i> spp. were isolated from 40.9% (36/88) of the composts in all facilities. In <i>Legionella</i>-positive samples the temperature of compost was significantly lower (<i>P</i> = 0.012) than in <i>Legionella</i>-negative samples. Of 47 bioaerosol samples, 19.1% (9/47) were positive for FLA and 10.6% (5/47) for <i>L. pneumophila</i>. Composts (62.8%) were positive for <i>Legionella</i> and FLA contemporaneously, but both microorganisms were never detected simultaneously in bioaerosols. Compost can release bioaerosol containing FLA or <i>Legionella</i> and could represent a source of infection of community-acquired <i>Legionella</i> infections for workers and nearby residents.</p></div

Frequency of presence of FLA and temperature (<50°C and >50°C) of compost heaps in samples positive or negative for <i>Legionella</i>.

*<p>Chi-square test with Yates correction; <i>P</i> values <0.05 were considered statistically significant.</p

Number and percentage (in parentheses) of compost and bioaerosol pools containing <i>Legionella</i> species analysed by co-culture in the four composting facilities (CF).

<p><i>L. pneumophila</i> sg 1: <i>L. pneumophila</i> serogroup 1; <i>L. pneumophila</i> other than sg 1: <i>L. pneumophila</i> other than serogroup 1.</p

Physico-chemical characteristic of compost samples positive and negative for <i>Legionella</i> or FLA [mean and 95% confidence intervals (95% Cl)].

†<p>Student t test; <i>P</i> values <0.05 were considered statistically significant.</p

Number and percentage (in parentheses) of samples containing free-living amoebae (FLA) and <i>Legionella</i> spp. in the composting facilities (CF) studied.

<p>Number and percentage (in parentheses) of samples containing free-living amoebae (FLA) and <i>Legionella</i> spp. in the composting facilities (CF) studied.</p

Number and percentage (in parentheses) of composts and bioaerosol pools containing free-living amoebae (FLA) species recovered by culture in the four composting facilities (CF).

<p>0: FLA not detected;</p>1<p>FLA supporting intracellular growth of <i>Legionella</i><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0068244#pone.0068244-Dey1" target="_blank">[9]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0068244#pone.0068244-RodriguezZaragoza1" target="_blank">[33]</a>;</p>2<p>opportunistic pathogenic FLA <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0068244#pone.0068244-RodriguezZaragoza1" target="_blank">[33]</a>; * <i>Echinamoeba thermatum</i> has a nucleotide identity of 75% with a corresponding database entry, which presumably is even not sufficient for a reliable identification at the genus level.</p