Immunisation with live attenuated Salmonella dublin expressing a sporozoite protein confers partial protection against Theileria parva

Cattle immunised with a recombinant form of p67, the major surface antigen of Theileria parva sporozoites, have been shown to be protected against parasite challenge. In an attempt to simplify the immunisation procedure live attenuated Salmonella strains expressing p67 have been constructed and used to induce anti-p67 immune responses in cattle. All animals immunised with these strains developed strong antibody responses to p67. Specific T cell responses could be detected in the majority of immunised cattle. Challenge with T. parva sporozoites revealed a significant level of protection in immunised calves compared to naive control animals or animals inoculated with non-recombinant attenuated Salmonella.

A novel fusion toxin derived from an EpCAM-specific designed ankyrin repeat protein has potent antitumor activity

Designed ankyrin repeat proteins (DARPins) hold great promise as a new class of binding molecules to overcome the limitations of antibodies for biomedical applications. Here, we assessed the potential of an epithelial cell adhesion molecule (EpCAM)-specific DARPin (Ec4) for tumor targeting as a fusion toxin with Pseudomonas aeruginosa exotoxin A.

Beta2-adrenergic receptor agonists reduce proliferation but not protein synthesis of periodontal fibroblasts stimulated with platelet-derived growth factor-BB

OBJECTIVE Catecholamines released from β-adrenergic neurons upon stress can interfere with periodontal regeneration. The cellular mechanisms, however, are unclear. Here, we assessed the effect of catecholamines on proliferation of periodontal fibroblasts. METHODS Fibroblasts from the gingiva and the periodontal ligament were exposed to agonists of the β-adrenergic receptors; isoproterenol (ISO, non-selective β-adrenergic agonist), salbutamol (SAL, selective β2-adrenergic receptor agonist) and BRL 37344 (BRL selective β3-receptor agonist). Proliferation was stimulated with platelet-derived growth factor-BB (PDGF-BB). Pharmacological inhibitors and gene expression analysis further revealed β-adrenergic signalling. RESULTS Gingiva and periodontal ligament fibroblast express the β2-adrenergic receptor. ISO and SAL but not BRL decreased proliferation of fibroblasts in the presence of PDGF-BB. The inhibitory effect of β-adrenergic signalling on proliferation but not protein synthesis in response to PDGF-BB was reduced by propranolol, a non-selective β-adrenergic antagonist. CONCLUSIONS These results suggest that β2-receptor agonists can reduce the mitogenic response of periodontal fibroblasts. These data add to the compelling concept that blocking of β2-receptor signalling can support tissue maintenance and regeneration.

Bone marrow-derived cells in palatal wound healing

OBJECTIVE: Myofibroblasts are responsible for contraction and scarring after cleft palate repair. This leads to growth disturbances in the upper jaw. We hypothesized that cells from the bone marrow are recruited to palatal wounds and differentiate into myofibroblasts. METHODS: We transplanted bone marrow from green fluorescent protein (GFP)-transgenic rats into lethally irradiated wild-type rats. After recovery, experimental wounds were made in the palatal mucoperiosteum, and harvested 2 weeks later. GFP-expressing cells were identified using immunostaining. Myofibroblasts, activated fibroblasts, endothelial cells, and myeloid cells were quantified with specific markers. RESULTS: After transplantation, 89 ± 8.9% of mononuclear cells in the blood expressed the GFP and about 50% of adherent cells in the bone marrow. Tissue obtained during initial wounding contained only minor numbers of GFP-positive cells, like adjacent control tissue. Following wound healing, 8.1 ± 5.1% of all cells in the wound area were positive, and 5.0 ± 4.0% of the myofibroblasts, which was significantly higher than in adjacent tissue. Similar percentages were found for activated fibroblasts and endothelial cells, but for myeloid cells it was considerably higher (22 ± 9%). CONCLUSIONS: Bone marrow-derived cells contribute to palatal wound healing, but are not the main source of myofibroblasts. In small wounds, the local precursor cells are probably sufficient to replenish the defect.

Identification of a fibronectin interaction site in the extracellular matrix protein ameloblastin

Mammalian teeth are composed of hydroxyapatite crystals that are embedded in a rich extracellular matrix. This matrix is produced by only two cell types, the mesenchymal odontoblasts and the ectodermal ameloblasts. Ameloblasts secrete the enamel proteins amelogenin, ameloblastin, enamelin and amelotin. Odontoblasts secrete collagen type I and several calcium-binding phosphoproteins including dentin sialophosphoprotein, dentin matrix protein, bone sialoprotein and osteopontin. The latter four proteins have recently been grouped in the family of the SIBLINGs (small integrin-binding ligand, N-linked glycoproteins) because they display similar gene structures and because they contain an RGD tripeptide sequence that binds to integrin receptors and thus mediates cell adhesion. We have prepared all the other tooth-specific proteins in recombinant form and examined whether they might also promote cell adhesion similar to the SIBLINGs. We found that only ameloblastin consistently mediated adhesion of osteoblastic and fibroblastic cells to plastic or titanium surfaces. The activity was dependent on the intact three-dimensional structure of ameloblastin and required de novo protein synthesis of the adhering cells. By deletion analysis and in vitro mutagenesis, the active site could be narrowed down to a sequence of 13 amino acid residues (VPIMDFADPQFPT) derived from exon 7 of the rat ameloblastin gene or exons 7-9 of the human gene. Kinetic studies and RNA interference experiments further demonstrated that this sequence does not directly bind to a cell surface receptor but that it interacts with cellular fibronectin, which in turn binds to integrin receptors. The identification of a fibronectin-binding domain in ameloblastin might permit interesting applications for dental implantology. Implants could be coated with peptides containing the active sequence, which in turn would recruit fibronectin from the patient's blood. The recruited fibronectin should then promote cell adhesion on the implant surface, thereby accelerating osseointegration of the implant.

Generation of an osteogenic graft from human placenta and placenta-derived mesenchymal stem cells

The objective of the study was to determine the feasibility of generating a biodegradable, stem cell-loaded osteogenic composite graft from human placenta. Initially, a scaffold from human chorion membrane was produced. Human placenta mesenchymal stem cells (MSCs) derived from either first-trimester chorionic villi or term chorion membrane were differentiated osteogenically on this scaffold. Outgrowth, adherence, and osteogenic differentiation of cells were assessed by immunohistochemistry (IHC), scanning electron microscopy, protein expression, and real-time polymerase chain reaction (RT-PCR). Our results showed that a cell-free extracellular matrix scaffold can be generated from human chorion. Seeded MSCs densely adhered to that scaffold and were osteogenically differentiated. Calcium and alkaline phosphatase were detected in the cell-scaffold constructs as a proof of mineralization and findings were confirmed by IHC and RT-PCR results. This study shows for the first time that generation of an osteogenic composite graft using placental tissue is feasible. It might allow therapeutic application of autologous or allogeneic grafts in congenital skeletal defects by means of a composite graft.

The recruitment of bone marrow-derived cells to skin wounds is independent of wound size

Wounded skin recruits progenitor cells, which repair the tissue defect. These cells are derived from stem cells in several niches in the skin. In addition, bone marrow-derived cells (BMDCs) are recruited and contribute to wound repair. We hypothesized that larger wounds recruit more cells from the bone marrow. Wild-type rats were lethally irradiated and transplanted with bone marrow cells from green fluorescent protein (GFP)-transgenic rats. Seven weeks later, 4, 10, and 20 mm wounds were created. The wound tissue was harvested after 14 days. The density of GFP-positive cells in the wounds and the adjacent tissues was determined, as well as in normal skin from the flank. Bone marrow-derived myofibroblasts, activated fibroblasts, and macrophages were also quantified. After correction for cell density, the recruitment of BMDCs (23±11%) was found to be independent of wound size. Similar fractions of GFP-positive cells were also detected in nonwounded adjacent tissue (29±11%), and in normal skin (26±19%). The data indicate that BMDCs are not preferentially recruited to skin wounds. Furthermore, wound size does not seem to affect the recruitment of BMDCs.

Monomeric and dimeric IgG fractions show differential reactivity against pathogen-derived antigens

Polyvalent Ig preparations, derived from the pooled plasma of thousands of healthy donors, contain a complex mix of both 'acquired' and natural antibodies directed against pathogens as well as foreign and self/auto antigens (Ag). Depending on their formulation, donor pool size, etc., liquid Ig preparations contain monomeric and dimeric IgG. The dimeric IgG fraction is thought to represent mainly idiotype-antiidiotype Ab pairs. Treatment of all IgG fractions at pH 4 effectively monomerizes the IgG dimers resulting in separated idiotype-antiidiotype Ab pairs and thus in a comparable F(ab')(2) binding site availability of the different IgG fractions. Previously, we identified an increased anti-self-reactivity within the monomerized dimer fraction. This study addressed if, among the different IgG fractions, an analogous preferential reactivity was evident in the response against different pathogen-derived protein and carbohydrate antigens. Therefore, we assessed the activity of total unseparated IgG, the monomeric and dimeric IgG fractions against antigenic structures of bacterial and viral antigens/virulence factors. All fractions showed similar reactivity to protein antigens except for exotoxin A of Pseudomonas aeruginosa, where the dimeric fraction, especially when monomerized, showed a marked increase in reactivity. This suggests that the production of antiidiotypic IgG antibodies contributes to controlling the immune response to certain categories of pathogens. In contrast, the monomeric IgG fractions showed increased reactivity towards pathogen-associated polysaccharides, classically regarded as T-independent antigens. Taken together, the differential reactivity of the IgG fractions seems to indicate a preferential segregation of antibody reactivities according to the nature of the antigen.

TNFα inhibits the development of osteoclasts through osteoblast-derived GM-CSF

Inflammatory cytokines such as tumor necrosis factor-alpha (TNFα) are potent stimulators of osteoclast formation and bone resorption and are frequently associated with pathologic bone metabolism. The cytokine exerts specific effects on its target cells and constitutes a part of the cellular microenvironment. Previously, TNFα was demonstrated to inhibit the development of osteoclasts in vitro via an osteoblast-mediated pathway. In the present study, the molecular mechanisms of the inhibition of osteoclastogenesis were investigated in co-cultures of osteoblasts and bone marrow cells (BMC) and in cultures of macrophage-colony stimulating factor (M-CSF) dependent, non-adherent osteoclast progenitor cells (OPC) grown with M-CSF and receptor activator of NF-κB ligand (RANKL). Granulocyte-macrophage colony stimulating factor (GM-CSF), a known inhibitor of osteoclastogenesis was found to be induced in osteoblasts treated with TNFα and the secreted protein accumulated in the supernatant. Dexamethasone (Dex), an anti-inflammatory steroid, caused a decrease in GM-CSF expression, leading to partial recovery of osteoclast formation. Flow cytometry analysis revealed that in cultures of OPC, supplemented with 10% conditioned medium (CM) from osteoblasts treated with TNFα/1,25(OH)(2)D(3), expression of RANK and CD11c was suppressed. The decrease in RANK expression may be explained by the finding, that GM-CSF and the CM from wt osteoblasts were found to suppress the expression of c-Fos, Fra-1, and Nfatc-1. The failure of OPC to develop into CD11c(+) dendritic cells suggests that cell development is not deviated to an alternative differentiation pathway, but rather, that the monocytes are maintained in an undifferentiated, F4/80(+), state. The data further implies possible interactions among inflammatory cytokines. GM-CSF induced by TNFα acts on early hematopoietic precursors, inhibiting osteoclastogenesis while acting as the growth factor for M-CSF independent inflammatory macrophages. These in turn may condition a microenvironment enhancing osteoclast differentiation and bone resorption upon migration of the OPC from circulation to the bone/bone marrow compartment.

Elevated systemic monocyte chemoattractrant protein-1 in hepatic steatosis without significant hepatic inflammation

Non-alcoholic fatty liver disease (NAFLD) is strongly associated with obesity and the metabolic syndrome. It encompasses a clinico-pathologic spectrum of conditions ranging from simple steatosis to nonalcoholic steatohepatitis (NASH). The latter develops upon pro-inflammatory cell infiltration and is widely considered as the first relevant pathophysiological step in NAFLD-progression. The chemokine monocyte chemoattractant protein 1 (MCP-1) plays an important role in the progression of hepatic inflammation and fibrosis, and both increased hepatic expression and circulating serum levels have been described in NASH. Here, we aimed to investigate MCP-1 expression in simple hepatic steatosis. Upon feeding a high-fat diet mice developed hepatic steatosis in the absence of significant hepatic inflammation, but elevated hepatic MCP-1 expression compared to control mice fed a standard chow. Interestingly, high-fat diet fed mice had significantly higher MCP-1 serum levels, and MCP-1 mRNA expression was significantly increased in visceral adipose tissue. Furthermore, MCP-1 serum levels were also elevated in patients with ultrasound-diagnosed NAFLD and correlated with the body-mass index and fasting glucose. In conclusion, our data indicate both the liver and adipose tissue as cellular sources of elevated circulating MCP-1 levels already in the early phase of hepatic steatosis. Since MCP-1 derived from visceral adipose tissue reaches the liver via portal circulation at high concentrations it may significantly contribute to the progression of simple steatosis to NASH.

Bone morphogenetic protein-7 is a MYC target with prosurvival functions in childhood medulloblastoma

Medulloblastoma (MB) is the most common malignant brain tumor in children. It is known that overexpression and/or amplification of the MYC oncogene is associated with poor clinical outcome, but the molecular mechanisms and the MYC downstream effectors in MB remain still elusive. Besides contributing to elucidate how progression of MB takes place, most importantly, the identification of novel MYC-target genes will suggest novel candidates for targeted therapy in MB. A group of 209 MYC-responsive genes was obtained from a complementary DNA microarray analysis of a MB-derived cell line, following MYC overexpression and silencing. Among the MYC-responsive genes, we identified the members of the bone morphogenetic protein (BMP) signaling pathway, which have a crucial role during the development of the cerebellum. In particular, the gene BMP7 was identified as a direct target of MYC. A positive correlation between MYC and BMP7 expression was documented by analyzing two distinct sets of primary MB samples. Functional studies in vitro using a small-molecule inhibitor of the BMP/SMAD signaling pathway reproduced the effect of the small interfering RNA-mediated silencing of BMP7. Both approaches led to a block of proliferation in a panel of MB cells and to inhibition of SMAD phosphorylation. Altogether, our findings indicate that high MYC levels drive BMP7 overexpression, promoting cell survival in MB cells. This observation suggests the potential relevance of targeting the BMP/SMAD pathway as a novel therapeutic approach for the treatment of childhood MB.

Gerbu adjuvant modulates the immune response and thus the course of infection in C56BL/6 mice immunised with Echinococcus multilocularis rec14-3-3 protein

Vaccination with Echinococcus multilocularis 14-3-3 protein can protect mice against primary E. multilocularis infection. The present study investigated the efficacy and efficiency of the adjuvant muramyl dipeptide Gerbu, alone or together with recombinant 14-3-3 protein, to modulate the course of secondary E. multilocularis infection in C56BL/6 mice. The application of Gerbu alone already resulted in a parasite weight reduction when compared with infected control mice, while rec14-3-3 did not add to this effect. Immunological parameters were concurrently assessed with a mixed cell reaction including bone marrow-derived dendritic cells (BMDCs) together with lymph node cells from mice with or without immunisation and/or infection. While mice having received Gerbu adjuvant were found to highly proliferate in response to co-cultivation with 14-3-3-stimulated bone marrow dendritic cells, a sensitisation of BMDCs with vesicle fluid (VF) antigen lead to a striking decrease of the lymphoproliferative response in comparison to that of control mice, raising the hypothesis that immunosuppressive components may be part of this VF-antigen. Anti-14-3-3 antibody production was only found in those mice that had been previously 14-3-3-immunised, whereas all other only-infected mice failed to produce such antibodies. Conclusively, Gerbu adjuvant appears to directly generate a non-specific immune response that contributes to the control of the metacestode growth, putatively in association with a BMDC activity suppressed by components of the VF-antigen.

Differences in milk production, glucose metabolism, and carcass composition of 2 Charolais x Holstein F2 families derived from reciprocal paternal and maternal grandsire crosses

Two F(2) Charolais x German Holstein families comprising full and half sibs share identical but reciprocal paternal and maternal Charolais grandfathers differ in milk production. We hypothesized that differences in milk production were related to differences in nutritional partitioning revealed by glucose metabolism and carcass composition. In 18F(2) cows originating from mating Charolais bulls to German Holstein cows and a following intercross of the F(1) individuals (n=9 each for family Ab and Ba; capital letters indicate the paternal and lowercase letter the maternal grandsire), glucose tolerance tests were performed at 10 d before calving and 30 and 93 d in milk (DIM) during second lactation. Glucose half-time as well as areas under the concentration curve for plasma glucose and insulin were calculated. At 94 DIM cows were infused intravenously with 18.3 micromol of d-[U-(13)C(6)]glucose/kg(0.75) of BW, and blood samples were taken to measure rate of glucose appearance and glucose oxidation as well as plasma concentrations of metabolites and hormones. Cows were slaughtered at 100 DIM and carcass size and composition was evaluated. Liver samples were taken to measure glycogen and fat content, gene expression levels, and enzyme activities of pyruvate carboxylase, phosphoenolpyruvate carboxykinase, and glucose 6-phosphatase as well as gene expression of glucose transporter 2. Milk yield was higher and milk protein content at 30 DIM was lower in Ba than in Ab cows. Glucose half-life was higher but insulin secretion after glucose challenge was lower in Ba than in Ab cows. Cows of Ab showed higher glucose oxidation, and plasma concentrations at 94 DIM were lower for glucose and insulin, whereas beta-hydroxybutyrate was higher in Ba cows. Hepatic gene expression of pyruvate carboxylase, glucose 6-phosphatase, and glucose transporter 2 were higher whereas phosphoenolpyruvate carboxykinase activities were lower in Ba than in Ab cows. Carcass weight as well as fat content of the carcass were higher in Ab than in Ba cows, whereas mammary gland mass was lower in Ab than in Ba cows. Fat classification indicated leaner carcass composition in Ba than in Ab cows. In conclusion, the 2 families showed remarkable differences in milk production that were accompanied by changes in glucose metabolism and body composition, indicating capacity for milk production as main metabolic driving force. Sex chromosomal effects provide an important regulatory mechanism for milk performance and nutrient partitioning that requires further investigation.

Surfactant protein D modulates allergen particle uptake and inflammatory response in a human epithelial airway model

Background Allergen-containing subpollen particles (SPP) are released from whole plant pollen upon contact with water or even high humidity. Because of their size SPP can preferentially reach the lower airways where they come into contact with surfactant protein (SP)-D. The aim of the present study was to investigate the influence of SP-D in a complex three-dimensional human epithelial airway model, which simulates the most important barrier functions of the epithelial airway. The uptake of SPP as well as the secretion of pro-inflammatory cytokines was investigated. Methods SPP were isolated from timothy grass and subsequently fluorescently labeled. A human epithelial airway model was built by using human Type II-pneumocyte like cells (A549 cells), human monocyte derived macrophages as well as human monocyte derived dendritic cells. The epithelial cell model was incubated with SPP in the presence and absence of surfactant protein D. Particle uptake was evaluated by confocal microscopy and advanced computer-controlled analysis. Finally, human primary CD4+ T-Cells were added to the epithelial airway model and soluble mediators were measured by enzyme linked immunosorbent assay or bead array. Results SPP were taken up by epithelial cells, macrophages, and dendritic cells. This uptake coincided with secretion of pro-inflammatory cytokines and chemokines. SP-D modulated the uptake of SPP in a cell type specific way (e.g. increased number of macrophages and epithelial cells, which participated in allergen particle uptake) and led to a decreased secretion of pro-inflammatory cytokines. Conclusion These results display a possible mechanism of how SP-D can modulate the inflammatory response to inhaled allergen.

Prolyl hydroxylase inhibitors increase the production of vascular endothelial growth factor in dental pulp-derived cells

Introduction: Prolyl hydroxylase (PHD) inhibitors can induce a proangiogenic response that stimulates regeneration in soft and hard tissues. However, the effect of PHD inhibitors on the dental pulp is unclear. The purpose of this study was to evaluate the effects of PHD inhibitors on the proangiogenic capacity of human dental pulp–derived cells. Methods: To test the response of dental pulp–derived cells to PHD inhibitors, the cells were exposed to dimethyloxalylglycine, desferrioxamine, L-mimosine, and cobalt chloride. To assess the response of dental pulp cells to a capping material supplemented with PHD inhibitors, the cells were treated with supernatants from calcium hydroxide. Viability, proliferation, and protein synthesis were assessed by formazan formation, 3[H]thymidine, and 3[H]leucine incorporation assays. The effect on the proangiogenic capacity was measured by immunoassays for vascular endothelial growth factor (VEGF). Results: We found that all 4 PHD inhibitors can reduce viability, proliferation, and protein synthesis at high concentrations. At nontoxic concentrations and in the presence of supernatants from calcium hydroxide, PHD inhibitors stimulated the production of VEGF in dental pulp–derived cells. When calcium hydroxide was supplemented with the PHD inhibitors, the supernatants from these preparations did not significantly elevate VEGF levels. Conclusions: These results show that PHD inhibitors can stimulate VEGF production of dental pulp–derived cells, suggesting a corresponding increase in their proangiogenic capacity. Further studies will be required to understand the impact that this might have on pulp regeneration.