Capromorelin: a ghrelin receptor agonist and novel therapy for stimulation of appetite in dogs.

Ghrelin is a hormone, secreted from cells in the stomach, which is important in the regulation of appetite and food intake in mammals. It exerts its action by binding to a specific G-protein-coupled receptor, the growth hormone secretagogue receptor 1a (GHS-R1a) which is found in areas of the brain associated with the regulation of food intake. Ghrelin causes a release of growth hormone (GH) through binding to GHS-R1a in the hypothalamus and pituitary gland. A class of compounds known as growth hormone secretagogues, or ghrelin receptor agonists, were developed for therapeutic use in humans for the stimulation of GH in the frail elderly, and have subsequently been studied for their effects on increasing appetite and food intake, increasing body weight, building lean muscle mass, and treating cachexia. Subsequent research has shown that ghrelin has anti-inflammatory and immunomodulatory effects. This article reviews the basic physiology of ghrelin and the ghrelin receptor agonists, including the available evidence of these effects in vitro and in vivo in rodent models, humans, dogs and cats. One of these compounds, capromorelin, has been FDA-approved for the stimulation of appetite in dogs (ENTYCE ®). The data available on the safety and effectiveness of capromorelin is reviewed, along with a discussion of the potential clinical applications for ghrelin receptor agonists in both human and veterinary medicine.

In vitro approach to study the synergistic effects of tobramycin and clarithromycin against Pseudomonas aeruginosa biofilms using prokaryotic or eukaryotic culture media.

Recurrent Pseudomonas aeruginosa infections involving biofilm formation are frequent in cystic fibrosis, aggravating the respiratory distress. Co-administration of clarithromycin and classical tobramycin could improve the health status of patients. Antibiotic toxicity was assessed on epithelial (CFBE41o(-)) and macrophagic (THP-1) cell lines. Non-toxic concentrations of antibiotics alone or in combination were applied twice daily for 12 days on mature (12-day-old) biofilms of three P. aeruginosa strains, developed either in prokaryotic culture broth [tryptic soy broth (TSB)] or in a eukaryotic cell culture medium (RPMI-FCS) more similar to an in vivo environment. The antibiofilm and bactericidal effects of antibiotics were assessed. No toxicity of tobramycin was observed on eukaryotic cell lines at concentrations up to 500µg/mL, whilst 100µg/mL was selected as the clarithromycin upper safe limit. The amount of biofilm was strongly reduced by 100µg/mL and 500µg/mL tobramycin for each strain in both media, whilst clarithromycin was only effective in RPMI-FBS, with synergistic (PAO1 strain) and additive (PYO2 strain) effects detected when combining tobramycin 4µg/mL and clarithromycin 100µg/mL. Finally, tobramycin at ≥100µg/mL exerted strong bactericidal effects on each strain in both media. Clarithromycin also exerted bactericidal effects on each strain in both media; its effect was weaker than tobramycin in TSB but was similar in RPMI-FBS. Synergistic effects were observed on PAO1 and MUCO biofilms, e.g. when combining tobramycin 4µg/mL and clarithromycin 100µg/mL. These in vitro data show that co-administration of clarithromycin and tobramycin acts synergistically against in vitro P. aeruginosa biofilms.

Preliminary characterization of jejunocyte and colonocyte cell lines isolated by enzymatic digestion from adult and young cattle.

In the present study we developed an enzymatic approach (through the use of collagenase and dispase) to isolate bovine intestinal epithelial cells. Using this method, freshly isolated jejunocytes could be distinguished from simultaneously isolated colonocytes, as the jejunocytes specifically exhibited the small intestinal peptidase gene transcript, as well as an active alkaline phosphatase. The transformation of both types of cell suspension was performed by retroviral infection, using reproduction-defective viruses bearing the gene coding for the large T antigen of the leukaemia simian virus (SV40). The success of the transfection was demonstrated by (1) a significant increase in cell passage numbers (52-53 vs. 7 passages for non-transfected cells), (2) the detection of both the large T transcript and the large T antigen in transformed cells. Possible contamination and progressive substitution of bovine primocultures by non-bovine lineages available in the laboratory was excluded, as the transformed cells presented a bovine typical karyotype. Most transfected cells kept an epithelial morphology after transformation. They also maintained the expression of FABP and enterocyte specific enzymes (brush-border associated maltase and IAP). However, levels of specific activity of these enzymes were low, suggesting that cell differentiation is not completely achieved under the applied culture conditions.

Follicular dendritic cells control engulfment of apoptotic bodies by secreting Mfge8.

The secreted phosphatidylserine-binding protein milk fat globule epidermal growth factor 8 (Mfge8) mediates engulfment of apoptotic germinal center B cells by tingible-body macrophages (TBMphis). Impairment of this process can contribute to autoimmunity. We show that Mfge8 is identical to the mouse follicular dendritic cell (FDC) marker FDC-M1. In bone-marrow chimeras between wild-type and Mfge8(-/-) mice, all splenic Mfge8 was derived from FDCs rather than TBMphis. However, Mfge8(-/-) TBMphis acquired and displayed Mfge8 only when embedded in Mfge8(+/+) stroma, or when situated in lymph nodes draining exogenous recombinant Mfge8. These findings indicate a licensing role for FDCs in TBMphi-mediated removal of excess B cells. Lymphotoxin-deficient mice lacked FDCs and splenic Mfge8, and suffer from autoimmunity similar to Mfge8(-/-) mice. Hence, FDCs facilitate TBMphi-mediated corpse removal, and their malfunction may be involved in autoimmunity.

Protective effect of prion protein via the N-terminal region in mediating a protective effect on paraquat-induced oxidative injury in neuronal cells.

Transmissible spongiform encephalopathies are a group of neurodegenerative disorders caused by a posttranslational, conformational change in the cellular isoform of the prion protein (PrP(C)) into an infectious, disease-associated form (PrP(Sc)). Increasing evidence supports a role for PrP(C) in the cellular response to oxidative stress. We investigated the effect of oxidative stress mediated by paraquat exposure on SH-SY5Y neuroblastoma cells. A loss of mitochondrial membrane potential and subsequent reduction in ATP production were demonstrated in untransfected SH-SY5Y cells, an effect that was ameliorated by the expression of PrP(C). Cells expressing either PrP-DeltaOct, which lacks the octapeptide repeats, or PrP-DA, in which the N-terminus is tethered to the membrane, showed increased sensitivity to paraquat compared with cells expressing wild-type PrP(C) as shown by reduced viability, loss of their membrane integrity, and reduced mitochondrial bioenergetic measurements. Exposure of prion-infected mouse SMB15S cells to paraquat resulted in a reduction in viability to levels similar to those seen in the untransfected SH-SY5Y cells. However, "curing" the cells with pentosan sulfate restored the viability to the level observed in the SH-SY5Y cells expressing PrP(C). These data would indicate that the molecular mechanism promoting cellular resistance to oxidative stress had been compromised in the infected SMB15S cells, which could be reinstated upon curing. Our study supports the hypothesis that PrP(C) expression protects cells against paraquat-induced oxidative injury, demonstrates the significance of the N-terminal region of the protein in mediating this protective effect, and also shows that the biochemical consequences of prion infection may be reversed with therapeutic intervention.

Tilted properties of the 67-78 fragment of alpha-synuclein are responsible for membrane destabilization and neurotoxicity.

Alpha-synuclein is a 140 residue protein associated with Parkinson's disease. Intraneural inclusions called Lewy bodies and Lewy neurites are mainly composed of alpha-synuclein aggregated into amyloid fibrils. Other amyloidogenic proteins, such as the beta amyloid peptide involved in Alzheimer's disease and the prion protein (PrP) associated with Creuztfeldt-Jakob's disease, are known to possess "tilted peptides". These peptides are short protein fragments that adopt an oblique orientation at a hydrophobic/hydrophilic interface, which enables destabilization of the membranes. In this paper, sequence analysis and molecular modelling predict that the 67-78 fragment of alpha-synuclein is a tilted peptide. Its destabilizing properties were tested experimentally. The alpha-synuclein 67-78 peptide is able to induce lipid mixing and leakage of unilamellar liposomes. The neuronal toxicity, studied using human neuroblastoma cells, demonstrated that the alpha-synuclein 67-78 peptide induces neurotoxicity. A mutant designed by molecular modelling to be amphipathic was shown to be significantly less fusogenic and toxic than the wild type. In conclusion, we have identified a tilted peptide in alpha-synuclein, which could be involved in the toxicity induced during amyloidogenesis of alpha-synuclein.

Study on the toxic mechanism of prion protein peptide 106-126 in neuronal and non neuronal cells.

A synthetic peptide corresponding to the 106-126 amyloidogenic region of the cellular human prion protein (PrP(c)) is useful for in vitro study of prion-induced neuronal cell death. The aim of the present work was to examine the implication of the cellular prion protein in the toxicity mechanism induced by PrP 106-126. The effect of PrP 106-126 was investigated both on human neuroblastoma SH-SY5Y cells and on SH-SY5Y overexpressing murine cellular prions (wtPrP). We show by metabolic assay tests and ATP assays that PrP(c) expression does not modulate the toxicity of the prion peptide. Moreover, we investigated the effect of this peptide on an established non neuronal model, rabbit kidney epithelial A74 cells that express a doxycycline-inducible murine PrP(c) gene. We show for the first time that the prion peptide 106-126 does not exert any toxic effect on this cell line in the presence or absence of doxycycline. Our results show that the PrP 106-126-induced cell alteration is independent of PrP(c) expression. Rather, it seems to act via an interaction with lipidic components of the plasma membrane as strengthened by our results showing the differential susceptibility of neuronal and non neuronal cell lines that significantly differ by their membrane fatty acid composition.

Interaction of the 106-126 prion peptide with lipid membranes and potential implication for neurotoxicity.

Prion diseases are fatal neurodegenerative disorders characterized by the accumulation in the brain of an abnormally misfolded, protease-resistant, and beta-sheet rich pathogenic isoform (PrP(SC)) of the cellular prion protein (PrP(C)). In the present work, we were interested to study the mode of prion protein interaction with the membrane using the 106-126 peptide and small unilamellar lipid vesicles as model. As previously demonstrated, we showed by MTS assay that PrP 106-126 induces alterations in the human neuroblastoma SH-SY5Y cell line. We demonstrated for the first time by lipid-mixing assay and by the liposome vesicle leakage test that PrP 106-126, a non-tilted peptide, induces liposome fusion thus a potential cell membrane destabilization, as supported by membrane integrity assay (LDH). By circular dichroism (CD) analysis we showed that the fusogenic property of PrP 106-126 in the presence of liposome is associated with a predominantly beta-sheet structure. These data suggest that the fusogenic property associated with a predominant beta-sheet structure exhibited by the prion peptides contributes to the neurotoxicity of these peptides by destabilizing cellular membranes. The latter might be attached at the membrane surface in a parallel orientation as shown by molecular modeling.

Lymphotoxin-beta receptor-dependent genes in lymph node and follicular dendritic cell transcriptomes.

Affinity maturation and Ab class switches occur in lymphoid germinal centers (GCs), in which differentiation and maintenance depend on lymphotoxin (LT) signaling and include differentiation of follicular dendritic cells (FDCs). The events leading to FDC and GC maturation are poorly defined. Using several approaches of functional genomics, we enumerated transcripts affected in mice by suppressing LT beta receptor (LTbetaR) signaling and/or overrepresented in FDC-enriched GC isolates. Protein expression analysis of 3 of 12 genes both enriched in FDCs and down-regulated by LTbetaR signaling suppression validated them as FDC markers. Functional analysis of one of these three, clusterin, suggests a role as an FDC-derived trophic factor for GC B cells. Hence, the set of genes presented in this study includes markers emanating from LTbetaR signaling and transcripts relevant to GC and FDC function.

Apoptosis induction in human cancer cells by sungucine from Strychnos icaja root.

Sungucine (SG) and isosungucine (ISG) are bisindole alkaloids characterized by a 5'-23 link between the two parts of the compounds, which are till now specific to Strychnos icaja. In this work, SG and ISG were submitted to the NCI's in vitro 60 human tumor cell line screen, where SG showed interesting selectivity (6X) against the tested leukemia cell lines. In HL60-treated cells, apoptosis was demonstrated by observation of apoptotic bodies formation, and phosphatidylserine exposition at cell surface. In HeLa-treated cells, the analysis of cellular cycle by flow cytometry showed G1 accumulation and a small sub-G1 peak that could be related to DNA fragmentation characteristic of apoptosis. The eventual role of p53 was analyzed using wild-type HCT-116 colon cancer cells. Nevertheless, p53 and Bax expression were not modified in SG-treated cells. The cleavage of PARP by caspase-3 protease proved that apoptosis was also induced in this line. These results demonstrate that SG induces apoptosis, but also necrosis, in human cancer cell lines.

Human immune cells express ppMCH mRNA and functional MCHR1 receptor.

Melanin-concentrating hormone (MCH) is highly expressed in the brain and modulates feeding behavior. It is also expressed in some peripheral tissues where its role remains unknown. We have investigated MCH function in human and mouse immune cells. RT-PCR analysis revealed a low expression of prepro-MCH and MCH receptor 1 (MCHR1) but not of MCHR2 transcript in tissular and peripheral blood immune cells. FACS and in vitro assay studies demonstrated that MCHR1 receptor expression on most cell types can trigger, in the presence of MCH, cAMP synthesis and calcium mobilization in peripheral blood mononuclear cells (PBMCs). Moreover, MCH treatment decreases the CD3-stimulated PBMC proliferation in vitro. Accordingly, our data indicate for the first time that MCH and MCHR1 may exert immunomodulatory functions.