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.

Analogues of polyamine alkaloids and their synthetic advantages

Several polyamine derivatives were synthesized in order to produce novel antagonists of muscular nicotinic acetylcholine receptors. Their affinities were compared with those of philanthotoxin PhTX-343.

Maxillary sinus grafting with a synthetic, nanocrystalline hydroxyapatite-silica gel in humans: histologic and histomorphometric results.

The aim of this study was to evaluate in humans the amount of new bone after sinus floor elevation with a synthetic bone substitute material consisting of nanocrystalline hydroxyapatite embedded in a highly porous silica gel matrix. The lateral approach was applied in eight patients requiring sinus floor elevation to place dental implants. After elevation of the sinus membrane, the cavities were filled with 0.6-mm granules of nanocrystalline hydroxyapatite mixed with the patient's blood. A collagen membrane (group 1) or a platelet-rich fibrin (PRF) membrane (group 2) was placed over the bony window. After healing periods between 7 and 11 months (in one case after 24 months), 16 biopsy specimens were harvested with a trephine bur during implant bed preparation. The percentage of new bone, residual filler material, and soft tissue was determined histomorphometrically. Four specimens were excluded from the analysis because of incomplete biopsy removal. In all other specimens, new bone was observed in the augmented region. For group 1, the amount of new bone, residual graft material, and soft tissue was 28.7% ± 5.4%, 25.5% ± 7.6%, and 45.8% ± 3.2%, respectively. For group 2, the values were 28.6% ± 6.90%, 25.7% ± 8.8%, and 45.7% ± 9.3%, respectively. All differences between groups 1 and 2 were not statistically significant. The lowest and highest values of new bone were 21.2% and 34.1% for group 1 and 17.4% and 37.8% for group 2, respectively. The amount of new bone after the use of nanocrystalline hydroxyapatite for sinus floor elevation in humans is comparable to values found in the literature for other synthetic or xenogeneic bone substitute materials. There was no additional beneficial effect of the PRF membrane over the non-cross-linked collagen membrane.

In vitro characterization of a synthetic calcium phosphate bone graft on periodontal ligament cell and osteoblast behavior and its combination with an enamel matrix derivative.

OBJECTIVES Recent studies suggest that a combination of enamel matrix derivative (EMD) with grafting material may improve periodontal wound healing/regeneration. Newly developed calcium phosphate (CaP) ceramics have been demonstrated a viable synthetic replacement option for bone grafting filler materials. AIMS This study aims to test the ability for EMD to adsorb to the surface of CaP particles and to determine the effect of EMD on downstream cellular pathways such as adhesion, proliferation, and differentiation of primary human osteoblasts and periodontal ligament (PDL) cells. MATERIALS AND METHODS EMD was adsorbed onto CaP particles and analyzed for protein adsorption patterns via scanning electron microscopy and high-resolution immunocytochemistry with an anti-EMD antibody. Cell attachment and cell proliferation were quantified using CellTiter 96 One Solution Cell Assay (MTS). Cell differentiation was analyzed using real-time PCR for genes encoding Runx2, alkaline phosphatase, osteocalcin, and collagen1α1, and mineralization was assessed using alizarin red staining. RESULTS Analysis of cell attachment revealed significantly higher number of cells attached to EMD-adsorbed CaP particles when compared to control and blood-adsorbed samples. EMD also significantly increased cell proliferation at 3 and 5 days post-seeding. Moreover, there were significantly higher mRNA levels of osteoblast differentiation markers including collagen1α1, alkaline phosphatase, and osteocalcin in osteoblasts and PDL cells cultured on EMD-adsorbed CaP particles at various time points. CONCLUSION The present study suggests that the addition of EMD to CaP grafting particles may influence periodontal regeneration by stimulating PDL cell and osteoblast attachment, proliferation, and differentiation. Future in vivo and clinical studies are required to confirm these findings. CLINICAL RELEVANCE The combination of EMD and CaP may represent an option for regenerative periodontal therapy in advanced intrabony defects.

Endogenous and synthetic MMP inhibitors in CNS physiopathology.

Matrix metalloproteinases (MMPs, including the membrane-type MMPs (MT-MMPs)), a disintegrin and metalloproteinase (ADAM), and ADAM with thrombospondin motifs belong to the metzincins, a subclass of metalloproteinases that contain a Met residue and a Zn(2+) ion at the catalytic site necessary for enzymatic reaction. MMP proteolytic activity is mainly controlled by their natural tissue inhibitors of metalloproteinase (TIMP). A number of synthetic inhibitors have been developed to control deleterious MMP activity. The roles of MMPs and some of their ECM substrates in CNS physiology and pathology are covered by other chapters of the present volume and will thus not be addressed in depth. This chapter will focus (i) on the endogenous MMP inhibitors in the CNS, (ii) on MMP and TIMP regulations in three large classes of neuropathologic processes (inflammatory, neurodegenerative, and infectious), and (iii) on synthetic inhibitors of MMPs and the perspective of their use in different brain diseases.

A novel blood-sparing agent in cardiac surgery? First in-patient experience with the synthetic serine protease inhibitor MDCO-2010: a phase II, randomized, double-blind, placebo-controlled study in patients undergoing coronary artery bypass grafting with cardiopulmonary bypass

BACKGROUND Antifibrinolytics have been used for 2 decades to reduce bleeding in cardiac surgery. MDCO-2010 is a novel, synthetic, serine protease inhibitor. We describe the first experience with this drug in patients. METHODS In this phase II, double-blind, placebo-controlled study, 32 patients undergoing isolated primary coronary artery bypass grafting with cardiopulmonary bypass were randomly assigned to 1 of 5 increasing dosage groups of MDCO-2010. The primary aim was to evaluate pharmacokinetics (PK) with assessment of plasmatic concentrations of the drug, short-term safety, and tolerance of MDCO-2010. Secondary end points were influence on coagulation, chest tube drainage, and transfusion requirements. RESULTS PK analysis showed linear dosage-proportional correlation between MDCO-2010 infusion rate and PK parameters. Blood loss was significantly reduced in the 3 highest dosage groups compared with control (P = 0.002, 0.004 and 0.011, respectively). The incidence of allogeneic blood product transfusions was lower with MDCO-2010 4/24 (17%) vs 4/8 (50%) in the control group. MDCO-2010 exhibited dosage-dependent antifibrinolytic effects through suppression of D-dimer generation and inhibition of tissue plasminogen activator-induced lysis in ROTEM analysis as well as anticoagulant effects demonstrated by prolongation of activated clotting time and activated partial thromboplastin time. No systematic differences in markers of end organ function were observed among treatment groups. Three patients in the MDCO-2010 groups experienced serious adverse events. One patient experienced intraoperative thrombosis of venous grafts considered possibly related to the study drug. No reexploration for mediastinal bleeding was required, and there were no deaths. CONCLUSIONS This first-in-patient study demonstrated dosage-proportional PK for MDCO-2010 and reduction of chest tube drainage and transfusions in patients undergoing primary coronary artery bypass grafting. Antifibrinolytic and anticoagulant effects were demonstrated using various markers of coagulation. MDCO-2010 was well tolerated and showed an acceptable initial safety profile. Larger multi-institutional studies are warranted to further investigate the safety and efficacy of this compound.

Iron-Promoted Nucleophilic Additions to Diimine-Type Ligands: A Synthetic and Structural Study

We report here three examples of the reactivity of protic nucleophiles with diimine-type ligands in the presence of FeII salts. In the first case, the iron-promoted alcoholysis reaction of one nitrile group of the ligand 2,3-dicyano-5,6-bis(2-pyridyl)-pyrazine (L1) permitted the isolation of an stable E-imido−ester, [Fe(L1‘)2](CF3SO3)2 (1), which has been characterized by spectroscopic studies (IR, ES-MS, Mössbauer), elemental analysis, and crystallographically. Compound 1 consists of mononuclear octahedrally coordinated FeII complexes where the FeII ion is in its low-spin state. The iron-mediated nucleophilic attack of water to the asymmetric ligand 2,3-bis(2-pyridyl)pyrido[3,4-b]pyrazine (L2) has also been studied. In this context, the crystal structures of two hydration−oxidation FeIII products, [Fe(L2‘)2](ClO4)3·3CH3CN (2) and trans-[FeL2‘‘Cl2] (3), are described. Compounds 2 and 3 are both mononuclear FeIII complexes where the metals occupy octahedral positions. In principle, L2 is expected to coordinate to metal ions through its bipyridine-type units to form a five-membered ring; however, this is not the case in compounds 2 and 3. In 2, the ligand coordinates through its pyridines and through the hydroxyl group attached to the pyrazine imino carbon after hydration, that is, in an N,O,N tridentate manner. In compound 3, the ligand has suffered further transformations leading to a very stable diamido complex. In this case, the metal ion achieves its octahedral geometry by means of two pyridines, two amido N atoms, and two axial chlorine atoms. Magnetic susceptibility measurements confirmed the spin state of these two FeIII species:  compounds 2 and 3 are low-spin and high-spin, respectively.

Synthetic studies towards a novel, chemical stable, abasic site analogue of DNA

We synthesized the phosphinate 7 via photoaddition of methanol to the alpha, beta unsaturated deoxyribono lactone as the key step, followed by an Arbusov reaction for the introduction of phosphorous. Precursor 7 serves for the synthesis and incorporation into DNA of a novel chemically stable abasic site analogue that might act as an inhibitor for DNA glycosylases

End caps prevent nail migration in elastic stable intramedullary nailing in paediatric femoral fractures: a biomechanical study using synthetic and cadaveric bones.

End caps are intended to prevent nail migration (push-out) in elastic stable intramedullary nailing. The aim of this study was to investigate the force at failure with and without end caps, and whether different insertion angles of nails and end caps would alter that force at failure. Simulated oblique fractures of the diaphysis were created in 15 artificial paediatric femurs. Titanium Elastic Nails with end caps were inserted at angles of 45°, 55° and 65° in five specimens for each angle to create three study groups. Biomechanical testing was performed with axial compression until failure. An identical fracture was created in four small adult cadaveric femurs harvested from two donors (both female, aged 81 and 85 years, height 149 cm and 156 cm, respectively). All femurs were tested without and subsequently with end caps inserted at 45°. In the artificial femurs, maximum force was not significantly different between the three groups (p = 0.613). Push-out force was significantly higher in the cadaveric specimens with the use of end caps by an up to sixfold load increase (830 N, standard deviation (SD) 280 vs 150 N, SD 120, respectively; p = 0.007). These results indicate that the nail and end cap insertion angle can be varied within 20° without altering construct stability and that the risk of elastic stable intramedullary nailing push-out can be effectively reduced by the use of end caps.

Utility of 30 and 60 minute cortisol samples after high-dose synthetic ACTH-1-24 injection in the diagnosis of adrenal insufficiency

BACKGROUND: In clinical practise the high dose ACTH stimulation test (HDT) is frequently used in the assessment of adrenal insufficiency (AI). However, there is uncertainty regarding optimal time-points and number of blood samplings. The present study compared the utility of a single cortisol value taken either 30 or 60 minutes after ACTH stimulation with the traditional interpretation of the HDT. METHODS: Retrospective analysis of 73 HDT performed at a single tertiary endocrine centre. Serum cortisol was measured at baseline, 30 and 60 minutes after intravenous administration of 250 µg synthetic ACTH1-24. Adrenal insufficiency (AI) was defined as a stimulated cortisol level <550 nmol/l. RESULTS: There were twenty patients (27.4%) who showed an insufficient rise in serum cortisol using traditional HDT criteria and were diagnosed to suffer from AI. There were ten individuals who showed insufficient cortisol values after 30 minutes, rising to sufficient levels at 60 minutes. All patients revealing an insufficient cortisol response result after 60 minutes also had an insufficient result after 30 minutes. The cortisol value taken after 30 minutes did not add incremental diagnostic value in any of the cases under investigation compared with the 60 minutes' sample. CONCLUSIONS: Based on the findings of the present analysis the utility of a cortisol measurement 30 minutes after high dose ACTH injection was low and did not add incremental diagnostic value to a single measurement after 60 minutes.