Jaw and long bone marrow derived osteoclasts differ in shape and their response to bone and dentin

Increasing evidence suggests the existence of osteoclast diversity. Here we investigated whether precursors obtained from marrow of the mandibula or long bone could give rise to phenotypically different osteoclasts. Formation of multinucleated cells was assessed after culturing mouse marrow cells of the two bone types with macrophage colony stimulating factor (M-CSF) and receptor activator of NFκB ligand (RANKL) for up to 10 days on plastic, bone or dentin. Two times more osteoclasts formed from long bone marrow cells on bone compared to dentin, whereas higher numbers of jaw osteoclasts formed on dentin. Resorption of dentin or bone was similar for osteoclasts formed from both types of precursors. In contrast to jaw marrow derived osteoclasts, long bone osteoclasts predominantly had a multi-compartmented shape, with at least two nuclei containing compartments per cell. Osteoclasts on bone contained two times more actin rings than osteoclasts on dentin, regardless of their precursor origin. However, the area per osteoclast covered by actin rings was similar (20%) for both substrates. This study suggests that marrow cells obtained from different bones give rise to different osteoclasts. The substrate on which the osteoclasts are generated plays a role in steering their formation rather than their resorption.     

Differential effects of testosterone,dihydrotestosterone and estradiol on carotenoid deposition in an avian sexually selected signal

Recent studies have demonstrated that carotenoid-based traits are under the control of testosterone (T) by up-regulation of carotenoid carriers (lipoproteins) and/or tissue-specific uptake of carotenoids. T can be converted to dihydrotestosterone (DHT) and estradiol (E2), and variation in conversion rate may partly explain some contradictory findings in the literature. Moreover, most studies on the effect of T on sexual signals have focused on the male sex only, while in many species females show the same signal, albeit to a lesser extent. We studied the effects of T, DHT, and E2 treatment in male and female diamond doves Geopelia cuneata in which both sexes have an enlarged red eye ring, which is more pronounced in males. We first showed that this periorbital ring contains very high concentration of carotenoids, of which most are lutein esters. Both T and DHT were effective in enhancing hue, UV-chroma and size in both sexes, while E2 was ineffective. However, E2 dramatically increased the concentration of circulating lipoproteins. We conclude that in both sexes both color and size of the secondary sexual trait are androgen dependent. The action of androgens is independent of lipoproteins regulation. Potential mechanisms and their consequences for trade-off are discussed.     

Coronin 1-mediated naive T cell survival is essential for the development of autoimmune encephalomyelitis

Autoimmune encephalomyelitis is a disease of the CNS that can develop when an initial peripheral inflammatory stimulus is followed by infiltration and reactivation of T lymphocytes in the CNS. We report a crucial role for coronin 1, which is essential for maintenance of the naive T cell pool, for the development of murine experimental autoimmune encephalomyelitis (EAE), a model for multiple sclerosis. In the absence of coronin 1, immunization with myelin oligoglycoprotein (MOG(35-55)) peptide largely failed to induce EAE symptoms, despite normal mobilization of leukocyte subsets in the blood, as well as effector cytokine expression comparable with wild-type T cells on polyclonal stimulation. Susceptibility of coronin 1-deficient mice to EAE induction was restored by transfer of wild-type CD4(+) T cells, suggesting that the observed resistance of coronin 1-deficient mice to EAE development is T cell intrinsic. Importantly, although coronin 1-deficient regulatory T cells (Tregs) showed a suppressor activity comparable with wild-type Tregs, Treg depletion failed to restore EAE development in coronin 1-deficient animals. These results suggest a hitherto unrecognized role of naive T cells in the development of autoimmune encephalomyelitis and reveal coronin 1 as a crucial modulator of EAE induction.     

Single Nucleus Genome Sequencing Reveals High Similarity among Nuclei of an Endomycorrhizal Fungus

Nuclei of arbuscular endomycorrhizal fungi have been described as highly diverse due to their asexual nature and absence of a single cell stage with only one nucleus. This has raised fundamental questions concerning speciation, selection and transmission of the genetic make-up to next generations. Although this concept has become textbook knowledge, it is only based on studying a few loci, including 45S rDNA. To provide a more comprehensive insight into the genetic makeup of arbuscular endomycorrhizal fungi, we applied de novo genome sequencing of individual nuclei of Rhizophagus irregularis. This revealed a surprisingly low level of polymorphism between nuclei. In contrast, within a nucleus, the 45S rDNA repeat unit turned out to be highly diverged. This finding demystifies a long-lasting hypothesis on the complex genetic makeup of arbuscular endomycorrhizal fungi. Subsequent genome assembly resulted in the first draft reference genome sequence of an arbuscular endomycorrhizal fungus. Its length is 141 Mbps, representing over 27,000 protein-coding gene models. We used the genomic sequence to reinvestigate the phylogenetic relationships of Rhizophagus irregularis with other fungal phyla. This unambiguously demonstrated that Glomeromycota are more closely related to Mucoromycotina than to its postulated sister Dikarya.     

Deeper Penetration of Erythrocytes into the Endothelial Glycocalyx Is Associated with Impaired Microvascular Perfusion

Changes in endothelial glycocalyx are one of the earliest changes in development of cardiovascular disease. The endothelial glycocalyx is both an important biological modifier of interactions between flowing blood and the vessel wall, and a determinant of organ perfusion. We hypothesize that deeper penetration of erythrocytes into the glycocalyx is associated with reduced microvascular perfusion. The population-based prospective cohort study (the Netherlands Epidemiology of Obesity [NEO] study) includes 6,673 middle-aged individuals (oversampling of overweight and obese individuals). Within this cohort, we have imaged the sublingual microvasculature of 915 participants using sidestream darkfield (SDF) imaging together with a recently developed automated acquisition and analysis approach. Presence of RBC (as a marker of microvascular perfusion) and perfused boundary region (PBR), a marker for endothelial glycocalyx barrier properties for RBC accessibility, were assessed in vessels between 5 and 25 µm RBC column width. A wide range of variability in PBR measurements, with a mean PBR of 2.14 µm (range: 1.43–2.86 µm), was observed. Linear regression analysis showed a marked association between PBR and microvascular perfusion, reflected by RBC filling percentage (regression coefficient β: −0.034; 95% confidence interval: −0.037 to −0.031). We conclude that microvascular beds with a thick (“healthy”) glycocalyx (low PBR), reflects efficient perfusion of the microvascular bed. In contrast, a thin (“risk”) glycocalyx (high PBR) is associated with a less efficient and defective microvascular perfusion.     

Direct fluorimetric sensing of UV-excited analytes in biological and environmental samples using molecularly imprinted polymer nanoparticles and fluorescence polarization

A rapid, robust, sensitive and economic sensing method, based on a molecularly imprinted polymer (MIP) synthetic antibody mimic, and fluorescence polarization analysis, for the direct detection of UV-excited fluorescent analytes in food and environmental samples was developed. Fluoroquinolone (FQ) antibiotics were used as fluorescent model analytes. Water-compatible MIP nanoparticles were synthesized with enrofloxacin (ENRO) as the imprinting template. Fluorescence polarization measurements then allow the direct determination of the amount of ENRO and other structurally related piperazine-based fluoroquinolones that bind to the MIP. No separation step was required since this technique distinguishes in situ analyte molecules bound to the MIP from the free analyte in solution. This assay was successfully applied for the first time to determine FQs in real samples, i.e. tap water and milk, without any prior concentration step, by simply adding a known amount of MIP. No interference by the sample components was observed even though the excitation was in the UV region. In tap water, a low limit of detection of 0.1 nM for ENRO was achieved with 5 μg mL(-1) of MIP. In milk, ENRO and danofloxacin, whose MRLs have been fixed at 0.28 μM and 0.08 μM, respectively, could be selectively measured and distinguished from other families of antibiotics. The procedure is very easy and practical as it consists of simply precipitating the milk proteins with acetonitrile and adding buffer and MIP to the supernatant before reading the polarization values with a spectrofluorimeter.     

RNA interference targeting programmed death receptor-1 improves immune functions of tumor-specific T cells

Adoptive cell transfer (ACT), either using rapidly expanded tumor infiltrating lymphocytes or T-cell receptor transduced peripheral blood lymphocytes, can be considered one of the most promising approaches in cancer immunotherapy. ACT results in the repopulation of the host with high frequencies of tumor-specific T cells; however, optimal function of these cells within the tumor micro-environment is required to reach long-term tumor clearance. We and others have shown that ongoing anti-tumor immune responses can be impaired by the expression of ligands, such as PD-L1 (B7-H1) on tumor cells. Such inhibitory molecules can affect T cells at the effector phase via their receptor PD-1. PD-L1/PD-1 interaction has indeed been shown crucial in inducing T-cell anergy and maintaining peripheral tolerance. In order to maximize anti-tumor responses, antibodies that target the PD-1/PD-L1 axis are currently in phase I/II trials. Alternatively, a more refined approach could be the selective targeting of PD-1 in tumor-specific T cells to obtain long-term resistance against PD-1-mediated inhibition. We addressed whether this goal could be achieved by means of retroviral siRNA delivery. Effective siRNA sequences resulting in the reduction of surface PD-1 expression led to improved murine as well as human T-cell immune functions in response to PD-L1 expressing melanoma cells. These data suggest that blockade of PD-1-mediated T-cell inhibition through siRNA forms a promising approach to achieve long-lasting enhancement of tumor-specific T-cell function in adoptive T-cell therapy protocols.     

Pegylated interferon-alpha protects type 1 pneumocytes against SARS coronavirus infection in macaques

The primary cause of severe acute respiratory syndrome (SARS) is a newly discovered coronavirus. Replication of this SARS coronavirus (SCV) occurs mainly in the lower respiratory tract, and causes diffuse alveolar damage. Lack of understanding of the pathogenesis of SARS has prevented the rational development of a therapy against this disease. Here we show extensive SCV antigen expression in type 1 pneumocytes of experimentally infected cynomolgus macaques (Macaca fascicularis) at 4 d postinfection (d.p.i.), indicating that this cell type is the primary target for SCV infection early in the disease, and explaining the subsequent pulmonary damage. We also show that prophylactic treatment of SCV-infected macaques with the antiviral agent pegylated interferon-alpha (IFN-alpha) significantly reduces viral replication and excretion, viral antigen expression by type 1 pneumocytes and pulmonary damage, compared with untreated macaques. Postexposure treatment with pegylated IFN-alpha yielded intermediate results. We therefore suggest that pegylated IFN-alpha protects type 1 pneumocytes from SCV infection, and should be considered a candidate drug for SARS therapy.