Effects of ex vivo γ-tocopherol on airway macrophage function in healthy and mild allergic asthmatics

Elevated inflammation and altered immune responses are features found in atopic asthmatic airways. Recent studies indicate γ-tocopherol (GT) supplementation can suppress airway inflammation in allergic asthma. We studied the effects of in vitro GT supplementation on receptor-mediated phagocytosis and expression of cell surface molecules associated with innate and adaptive immunity on sputum-derived macrophages. Cells from nonsmoking healthy (n = 6) and mild house dust mite-sensitive allergic asthmatics (n = 6) were treated ex vivo with GT (300 µM) or saline (control). Phagocytosis of opsonized zymosan A bioparticles (Saccharomyces cerevisiae) and expression of surface molecules associated with innate and adaptive immunity were assessed using flow cytometry. GT caused significantly decreased (p < 0.05) internalization of attached zymosan bioparticles and decreased (p < 0.05) macrophage expression of CD206, CD36 and CD86 in allergic asthmatics but not in controls. Overall, GT caused downregulation of both innate and adaptive immune response elements, and atopic status appears to be an important factor.

Increased expression of putative cancer stem cell markers in the bone marrow of prostate cancer patients is associated with bone metastasis progression.

BACKGROUND The number of cells positive for the α-6 and α-2 integrin subunits and the c-Met receptor in primary tumors and bone biopsies from prostate cancer patients has been correlated with metastasis and disease progression. The objective of this study was to quantify disseminated tumour cells present in bone marrow in prostate cancer patients using specific markers and determine their correlation with metastasis and survival. METHODS Patients were included at different stage of prostate cancer disease, from localised to metastatic castration-resistant prostate cancer. Healthy men were used as a control group. Bone marrow samples were collected and nucleated cells separated. These were stained for CD45, α-2, α-6 integrin subunits and c-Met and samples were processed for analysis and quantification of CD45-/α2+/α6+/c-met + cells using flow cytometry. Clinical and pathological parameters were assessed and survival measured. Statistical analyses were made of associations between disease specific parameters, bone marrow flow cytometry data, prostate-specific antigen (PSA) progression free survival and bone metastases progression free survival. RESULTS For all markers, the presence of more than 0.1% positive cells in bone marrow aspirates was significantly associated with the risk of biochemical progression, the risk of developing metastasis and death from prostate cancer. CONCLUSIONS Quantification of cells carrying putative stem cell markers in bone marrow is a potential indicator of disease progression. Functional studies on isolated cells are needed to show more specifically their property for metastatic spread in prostate cancer.

Effect of synthetic agonists of toll-like receptor 9 on canine lymphocyte proliferation and cytokine production in vitro.

Synthetic agonists of TLR9 containing novel DNA structures and R'pG (wherein R=1-(2'-deoxy-beta-d-ribofuranosyl)-2-oxo-7-deaza-8-methyl-purine) motifs, referred to as immune modulatory oligonucleotides (IMOs), have been shown to stimulate T(H)-1-type-immune responses and potently reverse allergen-induced T(H)-2 responses to T(H)-1 responses in vitro and in vivo in mice. In order to investigate the immunomodulatory potential of IMOs in dogs, canine peripheral blood mononuclear cells (PBMC) from healthy dogs were stimulated with three different IMOs and a control IMO, alone or in combination with concanavalin A (ConA). Lipopolysaccharide (LPS) was used as a positive control for B lymphocyte activation. Carboxyfluorescein diacetate succinimidyl ester and phenotype staining was used to tag proliferating T and B lymphocytes (CD5(+) and CD21(+)) by flow cytometry. Real-time PCR and ELISA were processed to assay cytokine production of IFN-gamma, IL-10, TGF-beta, IL-6 and IL-10. Like LPS, IMOs alone induced neither proliferation of CD5(+) T cells nor CD21(+) B cells, but both LPS and IMO had the capacity to co-stimulate ConA and induced proliferation of B cells. In combination with ConA, one of the IMOs (IMO1) also induced proliferation of T cells. IMO1 also significantly enhanced the expression of IFN-gamma on the mRNA and protein level in canine PBMC, whereas expression of IL-10, TGF-beta and IL-4 mRNAs was not induced by any of the IMOs. These results indicate that in canine PBMC from healthy dogs, IMO1 was able to induce a T(H)-1 immune response including T- and B-cell proliferation.

Characterizing New Fluorescent Tools for Studying 5-HT3 Receptor Pharmacology

The pharmacological characterization of ligands depends upon the ability to accurately measure their binding properties. Fluorescence provides an alternative to more traditional approaches such as radioligand binding. Here we describe the binding and spectroscopic properties of eight fluorescent 5-HT3 receptor ligands. These were tested on purified receptors, expressed receptors on live cells, or in vivo. All compounds had nanomolar affinities with fluorescent properties extending from blue to near infra-red emission. A fluorescein-derivative had the highest affinity as measured by fluorescence polarization (FP; 1.14 nM), flow cytometry (FC; 3.23 nM) and radioligand binding (RB; 1.90 nM). Competition binding with unlabeled 5-HT3 receptor agonists (5-HT, mCPBG, quipazine) and antagonists (granisetron, palonosetron, tropisetron) yielded similar affinities in all three assays. When cysteine substitutions were introduced into the 5-HT3 receptor binding site the same changes in binding affinity were seen for both granisetron and the fluorescein-derivative, suggesting that they both adopt orientations that are consistent with co-crystal structures of granisetron with a homologous protein (5HTBP). As expected, in vivo live imaging in anaesthetized mice revealed staining in the abdominal cavity in intestines, but also in salivary glands. The unexpected presence of 5-HT3 receptors in mouse salivary glands was confirmed by Western blots. Overall, these results demonstrate the wide utility of our new high-affinity fluorescently-labeled 5-HT3 receptor probes, ranging from in vitro receptor pharmacology, including FC and FP ligand competition, to live imaging of 5-HT3 expressing tissues.

Engraftment of autologous bone marrow cells into the injured cranial cruciate ligament in dogs

Current research indicates that exogenous stem cells may accelerate reparative processes in joint disease but, no previous studies have evaluated whether bone marrow cells (BMCs) target the injured cranial cruciate ligament (CCL) in dogs. The objective of this study was to investigate engraftment of BMCs following intra-articular injection in dogs with spontaneous CCL injury. Autologous PKH26-labelled BMCs were injected into the stifle joint of eight client-owned dogs with CCL rupture. The effects of PKH26 staining on cell viability and PKH26 fluorescence intensity were analysed in vitro using a MTT assay and flow cytometry. Labelled BMCs in injured CCL tissue were identified using fluorescence microscopy of biopsies harvested 3 and 13 days after intra-articular BMC injection. The intensity of PKH26 fluorescence declines with cell division but was still detectable after 16 days. Labelling with PKH26 had no detectable effect on cell viability or proliferation. Only rare PKH26-positive cells were present in biopsies of the injured CCL in 3/7 dogs and in synovial fluid in 1/7 dogs. No differences in transforming growth factor-beta1, and interleukin-6 before and after BMC treatment were found and no clinical complications were noted during a 1 year follow-up period. In conclusion, BMCs were shown to engraft to the injured CCL in dogs when injected into the articular cavity. Intra-articular application of PKH26-labelled cultured mesenchymal stem cells is likely to result in higher numbers of engrafted cells that can be tracked using this method in a clinical setting.

Targeting of the MET receptor tyrosine kinase by small molecule inhibitors leads to MET accumulation by impairing the receptor downregulation.

The MET receptor tyrosine kinase is deregulated primarily via overexpression or point mutations in various human cancers and different strategies for MET inhibition are currently evaluated in clinical trials. We observed by Western blot analysis and by Flow cytometry that MET inhibition by different MET small molecule inhibitors surprisingly increases in a dose-dependent manner total MET levels in treated cells. Mechanistically, this inhibition-related MET accumulation was associated with reduced Tyr1003 phosphorylation and MET physical association with the CBL ubiquitin ligase with concomitant decrease in MET ubiquitination. These data may suggest careful consideration for design of anti-MET clinical protocols.

The ceramide kinase inhibitor NVP-231 inhibits breast and lung cancer cell proliferation by inducing M phase arrest and subsequent cell death

Background and Purpose Ceramide kinase (CerK) catalyzes the generation of ceramide-1-phosphate which may regulate various cellular functions, including inflammatory reactions and cell growth. Here, we studied the effect of a recently developed CerK inhibitor, NVP-231, on cancer cell proliferation and viability and investigated the role of cell cycle regulators implicated in these responses. Experimental Approach The breast and lung cancer cell lines MCF-7 and NCI-H358 were treated with increasing concentrations of NVP-231 and DNA synthesis, colony formation and cell death were determined. Flow cytometry was performed to analyse cell cycle distribution of cells and Western blot analysis was used to detect changes in cell cycle regulator expression and activation. Key Results In both cell lines, NVP-231 concentration-dependently reduced cell viability, DNA synthesis and colony formation. Moreover it induced apoptosis, as measured by increased DNA fragmentation and caspase-3 and caspase-9 cleavage. Cell cycle analysis revealed that NVP-231 decreased the number of cells in S phase and induced M phase arrest with an increased mitotic index, as determined by increased histone H3 phosphorylation. The effect on the cell cycle was even more pronounced when NVP-231 treatment was combined with staurosporine. Finally, overexpression of CerK protected, whereas down-regulation of CerK with siRNA sensitized, cells for staurosporine-induced apoptosis. Conclusions and Implications Our data demonstrate for the first time a crucial role for CerK in the M phase control in cancer cells and suggest its targeted inhibition, using drugs such as NVP-231, in combination with conventional pro-apoptotic chemotherapy.

Circulating endothelial progenitor cells in periodontitis.

BACKGROUND Several biologically plausible mechanisms have been proposed to mediate the association between periodontitis and atherosclerotic vascular disease (AVD), including adverse effects on vascular endothelial function. Circulating endothelial progenitor cells (cEPCs) are known to contribute to vascular repair, but limited data are available regarding the relationship between cEPC levels and periodontitis. The aims of this cross-sectional study are to investigate the levels of hemangioblastic and monocytic cEPCs in patients with periodontitis and periodontally healthy controls and to associate cEPC levels with the extent and severity of periodontitis. METHODS A total of 112 individuals (56 patients with periodontitis and 56 periodontally healthy controls, aged 26 to 65 years; mean age: 43 years) were enrolled. All participants underwent a full-mouth periodontal examination and provided a blood sample. Hemangioblastic cEPCs were assessed using flow cytometry, and monocytic cEPCs were identified using immunohistochemistry in cultured peripheral blood mononuclear cells. cEPC levels were analyzed in the entire sample, as well as in a subset of 50 pairs of patients with periodontitis/periodontally healthy controls, matched with respect to age, sex, and menstrual cycle. RESULTS Levels of hemangioblastic cEPCs were approximately 2.3-fold higher in patients with periodontitis than periodontally healthy controls, after adjustments for age, sex, physical activity, systolic blood pressure, and body mass index (P = 0.001). A non-significant trend for higher levels of monocytic cEPCs in periodontitis was also observed. The levels of hemangioblastic cEPCs were positively associated with the extent of bleeding on probing, probing depth, and clinical attachment loss. Hemangioblastic and monocytic cEPC levels were not correlated (Spearman correlation coefficient 0.03, P = 0.77), suggesting that they represent independent populations of progenitor cells. CONCLUSION These findings further support the notion that oral infections have extraoral effects and document that periodontitis is associated with a mobilization of EPCs from the bone marrow, apparently in response to systemic inflammation and endothelial injury.

Autophagy in Trypanosoma brucei: amino acid requirement and regulation during different growth phases.

Autophagy in the protozoan parasite, Trypanosoma brucei, may be involved in differentiation between different life cycle forms and during growth in culture. We have generated multiple parasite cell lines stably expressing green fluorescent protein- or hemagglutinin-tagged forms of the autophagy marker proteins, TbAtg8.1 and TbAtg8.2, in T. brucei procyclic forms to establish a trypanosome system for quick and reliable determination of autophagy under different culture conditions using flow cytometry. We found that starvation-induced autophagy in T. brucei can be inhibited by addition of a single amino acid, histidine, to the incubation buffer. In addition, we show that autophagy is induced when parasites enter stationary growth phase in culture and that their capacity to undergo starvation-induced autophagy decreases with increasing cell density.