Characterization of the CD14++CD16+ Monocyte Population in Human Bone Marrow

Numerous studies have divided blood monocytes according to their expression of the surface markers CD14 and CD16 into following subsets: classical CD14⁺⁺CD16⁻, intermediate CD14⁺⁺CD16⁺ and nonclassical CD14⁺CD16⁺⁺ monocytes. These subsets differ in phenotype and function and are further correlated to cardiovascular disease, inflammation and cancer. However, the CD14/CD16 nature of resident monocytes in human bone marrow remains largely unknown. In the present study, we identified a major population of CD14⁺⁺CD16⁺ monocytes by using cryopreserved bone marrow mononuclear cells from healthy donors. These cells express essential monocyte-related antigens and chemokine receptors such as CD11a, CD18, CD44, HLA-DR, Ccr2, Ccr5, Cx3cr1, Cxcr2 and Cxcr4. Notably, the expression of Ccr2 was inducible during culture. Furthermore, sorted CD14⁺⁺CD16⁺ bone marrow cells show typical macrophage morphology, phagocytic activity, angiogenic features and generation of intracellular oxygen species. Side-by-side comparison of the chemokine receptor profile with unpaired blood samples also demonstrated that these rather premature medullar monocytes mainly match the phenotype of intermediate and partially of (non)classical monocytes. Together, human monocytes obviously acquire their definitive CD14/CD16 signature in the bloodstream and the medullar monocytes probably transform into CD14⁺⁺CD16⁻ and CD14⁺CD16⁺⁺ subsets which appear enriched in the periphery.     

Harnessing the biodiversity value of Central and Eastern European farmland

A large proportion of European biodiversity today depends on habitat provided by low‐intensity farming practices, yet this resource is declining as European agriculture intensifies. Within the European Union, particularly the central and eastern new member states have retained relatively large areas of species‐rich farmland, but despite increased investment in nature conservation here in recent years, farmland biodiversity trends appear to be worsening. Although the high biodiversity value of Central and Eastern European farmland has long been reported, the amount of research in the international literature focused on farmland biodiversity in this region remains comparatively tiny, and measures within the EU Common Agricultural Policy are relatively poorly adapted to support it. In this opinion study, we argue that, 10 years after the accession of the first eastern EU new member states, the continued under‐representation of the low‐intensity farmland in Central and Eastern Europe in the international literature and EU policy is impeding the development of sound, evidence‐based conservation interventions. The biodiversity benefits for Europe of existing low‐intensity farmland, particularly in the central and eastern states, should be harnessed before they are lost. Instead of waiting for species‐rich farmland to further decline, targeted research and monitoring to create locally appropriate conservation strategies for these habitats is needed now.     

l-2-oxothiazolidine-4-carboxylate influence on age- and heat exposure-dependent peroxidation in rat's liver and kidney

To investigate the impact of acute heat exposure on maintenance of redox homeostasis and antioxidant balance related to aging, we have determined the GSH levels in the liver and kidney, and the activity of antioxidant enzymes in the same organs from Wistar rats at two different ages, 35 days and 18 months. The animals were housed individually in a special heated chamber maintaining a constant temperature of 40±0.5 °C. The results showed that the level of endogenous GSH was signi?cantly lower in aged than in young animals. In general, the activity of antioxidant enzymes in investigated tissues displayed an age-dependent decline. Indeed, we found unchanged CAT activity and decreased GPx activity with age. On the other hand acute heat exposure led to disproportion between peroxide metabolizing enzymes (CAT, GPx) and GR, thus promoting H₂O₂ accumulation and prooxidative state in the liver of young animals. The results for the impact of l-2-oxothiazolidine-4-carboxylate in combined stress model suggested that in spite of restore levels of GSH, the restoration of oxido-reductive balance might have only been partial due to irreversible alterations in antioxidant enzymes set by acute heat exposure and aging. Interestingly, young animals appeared to be more sensitive to the supplementation of the l-2-oxothiazolidine-4-carboxylate, likely because of the more extensive increase of GSH observed in young l-2-oxothiazolidine-4-carboxylate treated animals.     

Do β3-adrenergic receptors play a role in guinea pig detrusor smooth muscle excitability and contractility?

In many species, β3-adrenergic receptors (β3-ARs) have been reported to play a primary role in pharmacologically induced detrusor smooth muscle (DSM) relaxation. However, their role in guinea pig DSM remains controversial. The aim of this study was to investigate whether β3-ARs are expressed in guinea pig DSM and to evaluate how BRL37344 and L-755,507, two selective β3-AR agonists, modulate guinea pig DSM excitability and contractility. We used a combined experimental approach including RT-PCR, patch-clamp electrophysiology, and isometric DSM tension recordings. β3-AR mRNA message was detected in freshly isolated guinea pig DSM single cells. BRL37344 but not L-755,507 caused a slight decrease in DSM spontaneous phasic contraction amplitude and frequency in a concentration-dependent manner. In the presence of atropine (1 μM), only the spontaneous phasic contractions frequency was inhibited by BRL37344 at higher concentrations. Both BRL37344 and L-755,507 significantly decreased DSM carbachol-induced phasic and tonic contractions in a concentration-dependent manner. However, only BRL37344 inhibitory effect was partially antagonized by SR59230A (10 μM), a β3-AR antagonist. In the presence of atropine, BRL37344 and L-755,507 had no inhibitory effect on electrical field stimulation-induced contractions. Patch-clamp experiments showed that BRL37344 (100 μM) did not affect the DSM cell resting membrane potential and K(+) conductance. Although β3-ARs are expressed at the mRNA level, they play a minor to no role in guinea pig DSM spontaneous contractility without affecting cell excitability. However, BRL37344 and L-755,507 have pronounced inhibitory effects on guinea pig DSM carbachol-induced contractions. The study outlines important DSM β3-ARs species differences.     

Ghrelin suppression of potassium currents in smooth muscle cells of human mesenteric artery

Ghrelin is a 28-amino acid peptide hormone which modulates many physiological functions including cardiovascular homeostasis. Here we report some novel findings about the action of ghrelin on smooth muscle cells (SMC) freshly isolated from human mesenteric arteries. Ghrelin (10(-7) mol/l) significantly suppressed the iberiotoxin-blockable component of potassium currents (I(K)) and depolarized the cell membrane, while having no effect on Ca(2+) currents. Inhibition of inositol-trisphosphate (IP(3))-activated Ca(2+) release channels, depletion of sarcoplasmic reticulum (SR) Ca(2+) stores, blockade of phospholipase D (PLD) or protein kinase C (PKC) each abolished the effect of ghrelin on I(K), while the inhibition of phospholipase C (PLC) did not. These data imply that in human mesenteric artery SMC ghrelin suppresses I(K) via PLD, PKC and SR Ca(2+)-dependent signaling pathway.     

Hypoxia‐induced endothelial secretion of macrophage migration inhibitory factor and role in endothelial progenitor cell recruitment

Macrophage migration inhibitory factor (MIF) is a pleiotropic inflammatory cytokine that was recently identified as a non‐cognate ligand of the CXC‐family chemokine receptors 2 and 4 (CXCR2 and CXCR4). MIF is expressed and secreted from endothelial cells (ECs) following atherogenic stimulation, exhibits chemokine‐like properties and promotes the recruitment of leucocytes to atherogenic endothelium. CXCR4 expressed on endothelial progenitor cells (EPCs) and EC‐derived CXCL12, the cognate ligand of CXCR4, have been demonstrated to be critical when EPCs are recruited to ischemic tissues. Here we studied whether hypoxic stimulation triggers MIF secretion from ECs and whether the MIF/CXCR4 axis contributes to EPC recruitment. Exposure of human umbilical vein endothelial cells (HUVECs) and human aortic endothelial cells (HAoECs) to 1% hypoxia led to the specific release of substantial amounts of MIF. Hypoxia‐induced MIF release followed a biphasic behaviour. MIF secretion in the first phase peaked at 60 min. and was inhibited by glyburide, indicating that this MIF pool was secreted by a non‐classical mechanism and originated from pre‐formed MIF stores. Early hypoxia‐triggered MIF secretion was not inhibited by cycloheximide and echinomycin, inhibitors of general and hypoxia‐inducible factor (HIF)‐1α‐induced protein synthesis, respectively. A second phase of MIF secretion peaked around 8 hrs and was likely due to HIF‐1α‐induced de novo synthesis of MIF. To functionally investigate the role of hypoxia‐inducible secreted MIF on the recruitment of EPCs, we subjected human AcLDL⁺ KDR⁺ CD31⁺ EPCs to a chemotactic MIF gradient. MIF potently promoted EPC chemotaxis in a dose‐dependent bell‐shaped manner (peak: 10 ng/ml MIF). Importantly, EPC migration was induced by supernatants of hypoxia‐conditioned HUVECs, an effect that was completely abrogated by anti‐MIF‐ or anti‐CXCR4‐antibodies. Thus, hypoxia‐induced MIF secretion from ECs might play an important role in the recruitment and migration of EPCs to hypoxic tissues such as after ischemia‐induced myocardial damage.     

Adult progenitor cells in vascular remodeling during atherosclerosis

The mobilization and recruitment of bone marrow-derived, circulating or tissue resident progenitor cells giving rise to smooth muscle-like cells have been implicated in neointima hyperplasia after arterial injury and in accelerated forms of arterial lesion formation, e.g., transplant arteriopathy or graft vasculopathy. By contrast, convincing evidence has emerged that the vascular homing of endothelial progenitor cells (EPCs) contributes to endothelial recovery, thus limiting neointima formation after arterial injury. In the chronic context of primary atherosclerosis, plaque progression and destabilization, a more complex picture has become apparent. In patients with coronary artery disease, the number and function of EPCs have been linked with an improved endothelial function or regeneration, but have been inversely correlated with cardiovascular risk. In animal models, however, the injection of bone marrow cells or EPCs, or the application of stem-cell mobilizing factors, have been associated with an exacerbation of atherosclerosis and unstable plaque phenotypes, whereas the contribution of bone marrow-derived smooth muscle progenitors to primary atherosclerosis appears to be rather confined. Here, we discuss crucial biochemical cues, namely chemokines, adhesion molecules, growth factors and pharmacological means that guide and control the context-specific mobilization, recruitment and fate of vascular progenitor cells in arterial remodeling during atherosclerosis.     

The transmembrane protein KpOmpA anchoring the outer membrane of Klebsiella pneumoniae unfolds and refolds in response to tensile load

In Klebsiella pneumoniae the transmembrane β-barrel forming outer membrane protein KpOmpA mediates adhesion to a wide range of immune effector cells, thereby promoting respiratory tract and urinary infections. As major transmembrane protein OmpA stabilizes Gram-negative bacteria by anchoring their outer membrane to the peptidoglycan layer. Adhesion, osmotic pressure, hydrodynamic flow, and structural deformation apply mechanical stress to the bacterium. This stress can generate tensile load to the peptidoglycan-binding domain (PGBD) of KpOmpA. To investigate how KpOmpA reacts to mechanical stress, we applied a tensile load to the PGBD and observed a detailed unfolding pathway of the transmembrane β-barrel. Each step of the unfolding pathway extended the polypeptide connecting the bacterial outer membrane to the peptidoglycan layer and absorbed mechanical energy. After relieving the tensile load, KpOmpA reversibly refolded back into the membrane. These results suggest that bacteria may reversibly unfold transmembrane proteins in response to mechanical stress.     

Selective fengycin production in a modified rotating discs bioreactor

Production of lipopeptides fengycin and surfactin in rotating discs bioreactor was studied. The effects of rotation velocity and the addition of agitators between the discs on volumetric oxygen transfer coefficient k _L a were firstly studied in model media. Then the production of lipopeptides was also studied at different agitation conditions in the modified bioreactor (with agitators). The effect of agitation on dissolved oxygen, on submerged and immobilized biomass, on lipopeptide concentrations and yields and on the selectivity of the bioreaction was elucidated and discussed. The proposed modified rotating discs bioreactor allowed to obtain high fengycin concentrations (up to 787 mg L^?1), but also better selectivity of the bioreaction towards fengycin (up to 88 %) and better yields of fengycin per glucose (up to 62.9 mg g^?1), lipopeptides per glucose (up to 71.5 mg g^?1), fengycin per biomass (up to 309 mg g^?1) and lipopeptides per biomass (up to 396 mg g^?1) than those reported in the literature. Highest fengycin production and selectivity were obtained at agitation velocity of 30 min^?1. The proposed non-foaming fermentation process could contribute to the scale-up of lipopeptide fermentors and promote the industrial production of fengycin. The proposed bioreactor and bioprocess could be very useful also for the production of other molecules using bioprocesses requiring bubbleless oxygen supply.     

A novel secretory poly-cysteine and histidine-tailed metalloprotein (Ts-PCHTP) from Trichinella spiralis (Nematoda)

Background

Trichinella spiralis is an unusual parasitic intracellular nematode causing dedifferentiation of the host myofiber. Trichinella proteomic analyses have identified proteins that act at the interface between the parasite and the host and are probably important for the infection and pathogenesis. Many parasitic proteins, including a number of metalloproteins are unique for the nematodes and trichinellids and therefore present good targets for future therapeutic developments. Furthermore, detailed information on such proteins and their function in the nematode organism would provide better understanding of the parasite - host interactions.

Methodology/Principal Findings

In this study we report the identification, biochemical characterization and localization of a novel poly-cysteine and histidine-tailed metalloprotein (Ts-PCHTP). The native Ts-PCHTP was purified from T. spiralis muscle larvae that were isolated from infected rats as a model system. The sequence analysis showed no homology with other proteins. Two unique poly-cysteine domains were found in the amino acid sequence of Ts-PCHTP. This protein is also the first reported natural histidine tailed protein. It was suggested that Ts-PCHTP has metal binding properties. Total Reflection X-ray Fluorescence (TXRF) assay revealed that it binds significant concentrations of iron, nickel and zinc at protein:metal ratio of about 1∶2. Immunohistochemical analysis showed that the Ts-PCHTP is localized in the cuticle and in all tissues of the larvae, but that it is not excreted outside the parasite.

Conclusions/Significance

Our data suggest that Ts-PCHTP is the first described member of a novel nematode poly-cysteine protein family and its function could be metal storage and/or transport. Since this protein family is unique for parasites from Superfamily Trichinelloidea its potential applications in diagnostics and treatment could be exploited in future.