Monocytes from male patients with ankylosing spondylitis display decreased osteoclastogenesis and decreased RANKL/OPG ratio.

The present study investigates the osteoclastogenic capacity of peripheral blood mononuclear cells (PBMCs) in male patients with ankylosing spondylitis (AS). We demonstrated that monocytes from these patients display a lower capacity to generate osteoclasts compared to cells from healthy controls, and osteoclastogenesis was negatively correlated with disease duration. INTRODUCTION: Ankylosing spondylitis (AS) is a disease characterized by new bone growth that leads to syndesmophyte formation but AS patients frequently present with low bone mineral density/fractures. Osteoclastogenesis in AS patients is poorly studied and controversial. The aim of this study is to determine if the osteoclastogenic capacity of PBMCs is different in AS patients compared to controls and the relationship between osteoclastogenesis and clinical/laboratory parameters. METHODS: PBMCs from 85 male AS patients and 59 controls were tested for CD16+ cells and induced to differentiate into osteoclasts over 3 weeks in vitro. Serum levels of RANKL, osteoprotegerin (OPG), C-terminal telopeptide of type I collagen (CTX), and amino-terminal pro-peptide of type I collagen (P1NP) were also evaluated. RESULTS: PBMCs from AS patients had fewer CD16+ cells and produced fewer osteoclasts compared to controls. Apoptosis occurred less frequently in osteoclasts obtained from AS patients than in osteoclasts from the controls. A lower RANKL/OPG and CTX/P1NP were observed in AS patients compared to controls. AS patients taking NSAIDs presented no difference regarding the number of OCs produced and the percentage of CD16+ cells compared to controls. However, patients taking TNF inhibitors (TNFi) presented lower OC numbers than controls. A negative correlation was demonstrated between the number of osteoclasts generated from PBMCs of AS patients and disease duration. CONCLUSION: Monocytes from male AS patients display a lower capacity to generate osteoclasts in vitro compared to cells from controls. Osteoclastogenesis was negatively correlated with disease duration. This finding supports the idea that osteoclasts play a role in the physiopathology of bone disease in AS patients.

Processing strategies for smart electroconductive carbon nanotube-based bioceramic bone grafts.

Electroconductive bone grafts have been designed to control bone regeneration. Contrary to polymeric matrices, the translation of the carbon nanotube (CNT) electroconductivity into oxide ceramics is challenging due to the CNT oxidation during sintering. Sintering strategies involving reactive-bed pressureless sintering (RB + P) and hot-pressing (HP) were optimized towards prevention of CNT oxidation in glass/hydroxyapatite (HA) matrices. Both showed CNT retentions up to 80%, even at 1300 °C, yielding an increase of the electroconductivity in ten orders of magnitude relative to the matrix. The RB + P CNT compacts showed higher electroconductivity by ∼170% than the HP ones due to the lower damage to CNTs of the former route. Even so, highly reproducible conductivities with statistical variation below 5% and dense compacts up to 96% were only obtained by HP. The hot-pressed CNT compacts possessed no acute toxicity in a human osteoblastic cell line. A normal cellular adhesion and a marked orientation of the cell growth were observed over the CNT composites, with a proliferation/differentiation relationship favouring osteoblastic functional activity. These sintering strategies offer new insights into the sintering of electroconductive CNT containing bioactive ceramics with unlimited geometries for electrotherapy of the bone tissue.

Electrochemical and photoluminescence response of laser-induced graphene/electrodeposited ZnO composites.

The inherent scalability, low production cost and mechanical flexibility of laser-induced graphene (LIG) combined with its high electrical conductivity, hierarchical porosity and large surface area are appealing characteristics for many applications. Still, other materials can be combined with LIG to provide added functionalities and enhanced performance. This work exploits the most adequate electrodeposition parameters to produce LIG/ZnO nanocomposites. Low-temperature pulsed electrodeposition allowed the conformal and controlled deposition of ZnO rods deep inside the LIG pores whilst maintaining its inherent porosity, which constitute fundamental advances regarding other methods for LIG/ZnO composite production. Compared to bare LIG, the composites more than doubled electrode capacitance up to 1.41 mF cm-2 in 1 M KCl, while maintaining long-term cycle stability, low ohmic losses and swift electron transfer. The composites also display a luminescence band peaked at the orange/red spectral region, with the main excitation maxima at ~ 3.33 eV matching the expected for the ZnO bandgap at room temperature. A pronounced sub-bandgap tail of states with an onset absorption near 3.07 eV indicates a high amount of defect states, namely surface-related defects. This work shows that these environmentally sustainable multifunctional nanocomposites are valid alternatives for supercapacitors, electrochemical/optical biosensors and photocatalytic/photoelectrochemical devices.

Role of nitrogen additive and temperature on growth of diamond films from nanocrystalline to polycrystalline.

In this work, the coupled effect of nitrogen addition into CH4/H2 mixtures and surface temperature on diamond growth ranging from large grained polycrystalline to fine-grained nanocrystalline were investigated. Moreover a new growth parameter window for simultaneous growth of nanocrystalline diamond (NCD) and {100} textured large-grained diamond films was developed by using a high power high pressure 5 kW microwave plasma assisted chemical vapor deposition (MPCVD) reactor. Scanning electron microscope (SEM), Raman spectroscopy, and X-ray diffraction (XRD) are employed to characterize the morphology, crystalline quality and texture of the diamond samples. Our results can be grouped by two catalogs: First, deposition run without and with 0.24% N2 addition, while keeping all the other parameters constant, resulted in a high quality transparent large-grained polycrystalline diamond film and a NCD film, respectively. This result clearly evidences nitrogen induced nanocrystallinity. Then, two different substrate surface temperatures were obtained by overlapping a small silicon slice on the top centre of a large silicon wafer of 5.08 cm in diameter in only one deposition run using 0.24% N2 addition and the same set of parameters as the previous runs. From this growth run, a NCD film of growth rate around 2.3 microm/h was obtained at low temperature, while a {100} textured large-grained diamond film of much higher growth rate about 10.4 microm/h was grown at high temperature. These results not only confirm the reproducibility of NCD by N2 addition, but also indicate that distinct growth modes were involved at different substrate temperatures with 0.24% nitrogen addition, or coupled effect of nitrogen addition and temperature on the growth of CVD diamond films happened. Finite element method (FEM) analysis was employed to simulate the temperature gradient and distribution on these two samples, and based on this simulation and other simulation results in the literature, the growth mechanism is briefly discussed.

Osteoclasts and their circulating precursors in rheumatoid arthritis: Relationships with disease activity and bone erosions.

Patients with rheumatoid arthritis (RA) have very different outcomes, particularly with regard to bone erosions. Since osteoclasts are responsible for bone destruction adjacent to rheumatoid synovium, profiling osteoclasts from circulating precursors in RA could help identify patients at risk for bone destruction. In this study, we sought to determine whether the functional characteristics of osteoclasts generated from their blood precursors were modified by RA activity or were intrinsic to osteoclasts and associated with the RA phenotype (erosive or not). Osteoclasts were generated in vitro from peripheral blood mononuclear cells (PBMCs) of subjects with RA (n = 140), as well as sex- and age-matched healthy controls (n = 101). Osteoclastic parameters were analyzed at baseline and during the follow-up for up to 4 years, with regular assessment of RA activity, bone erosions, and bone mineral density (BMD). As a validation cohort, we examined RA patients from the Early Undifferentiated PolyArthritis (EUPA) study (n = 163). The proportion of CD14+ PBMC was higher in RA than in control subjects, but inversely correlated with the 28-joint disease activity score (DAS28). Also surprisingly, in osteoclast cultures from PBMCs, active RA was associated with lower osteoclastogenic capacity, while in vitro bone resorption per osteoclast and resistance to apoptosis were similar in both active and quiescent RA. In a small subgroup analysis, osteoclasts from subjects with recent RA that had progressed at four years to an erosive RA exhibited at baseline greater resistance to apoptosis than those from patients remaining non-erosive. Our findings establish that when RA is active, circulating monocytes have a reduced potential to generate osteoclasts from PBMCs in vitro. In addition, osteoclasts associated with erosive disease had resistance to apoptosis from the start of RA.

Adhesion and wear behaviour of NCD coatings on Si3N4 by micro-abrasion tests.

Nanocrystalline diamond (NCD) coatings offer an excellent alternative for tribological applications, preserving most of the intrinsic mechanical properties of polycrystalline CVD diamond and adding to it an extreme surface smoothness. Silicon nitride (Si3N4) ceramics are reported to guarantee high adhesion levels to CVD microcrystalline diamond coatings, but the NCD adhesion to Si3N4 is not yet well established. Micro-abrasion tests are appropriate for evaluating the abrasive wear resistance of a given surface, but they also provide information on thin film/substrate interfacial resistance, i.e., film adhesion. In this study, a comparison is made between the behaviour of NCD films deposited by hot-filament chemical vapour deposition (HFCVD) and microwave plasma assisted chemical vapour deposition (MPCVD) techniques. Silicon nitride (Si3N4) ceramic discs were selected as substrates. The NCD depositions by HFCVD and MPCVD were carried out using H2-CH4 and H2-CH4-N2 gas mixtures, respectively. An adequate set of growth parameters was chosen for each CVD technique, resulting in NCD films having a final thickness of 5 microm. A micro-abrasion tribometer was used, with 3 microm diamond grit as the abrasive slurry element. Experiments were carried out at a constant rotational speed (80 r.p.m.) and by varying the applied load in the range of 0.25-0.75 N. The wear rate for MPCVD NCD (3.7 +/- 0.8 x 10(-5) mm3 N(-1) m(-1)) is compatible with those reported for microcrystalline CVD diamond. The HFCVD films displayed poorer adhesion to the Si3N4 ceramic substrates than the MPCVD ones. However, the HFCVD films show better wear resistance as a result of their higher crystallinity according to the UV Raman data, despite evidencing premature adhesion failure.

Diamond-Graphite Nanoplatelet Surfaces as Conductive Substrates for the Electrical Stimulation of Cell Functions.

The nanocarbon allotropes constitute valid alternatives when designing control and actuation devices for electrically assisted tissue regeneration purposes, gathering among them important characteristics such as chemical inertness, biocompatibility, extreme mechanical properties, and, importantly, low and tailorable electrical resistivity. In this work, coatings of thin (100 nm) vertically aligned nanoplatelets composed of diamond (5 nm) and graphite were produced via a microwave plasma chemical vapor deposition (MPCVD) technique and used as substrates for electrical stimulation of MC3T3-E1 preosteoblasts. Increasing the amount of N2 up to 14.5 vol % during growth lowers the coatings' electrical resistivity by over 1 order of magnitude, triggers the nanoplatelet vertical growth, and leads to the higher crystalline quality of the nanographite phase. When preosteoblasts were cultured on these substrates and subjected to two consecutive daily cycles of 3 µA direct current stimulation, enhanced cell proliferation and metabolism were observed accompanied by high cell viability. Furthermore, in the absence of DC stimulation, alkaline phosphatase (ALP) activity is increased significantly, denoting an up-regulating effect of preosteoblastic maturation intrinsically exerted by the nanoplatelet substrates.

Diels-Alder functionalized carbon nanotubes for bone tissue engineering: in vitro/in vivo biocompatibility and biodegradability.

The risk-benefit balance for carbon nanotubes (CNTs) dictates their clinical fate. To take a step forward at this crossroad it is compulsory to modulate the CNT in vivo biocompatibility and biodegradability via e.g. chemical functionalization. CNT membranes were functionalised combining a Diels-Alder cycloaddition reaction to generate cyclohexene (-C6H10) followed by a mild oxidisation to yield carboxylic acid groups (-COOH). In vitro proliferation and osteogenic differentiation of human osteoblastic cells were maximized on functionalized CNT membranes (p,f-CNTs). The in vivo subcutaneously implanted materials showed a higher biological reactivity, thus inducing a slighter intense inflammatory response compared to non-functionalized CNT membranes (p-CNTs), but still showing a reduced cytotoxicity profile. Moreover, the in vivo biodegradation of CNTs was superior for p,f-CNT membranes, likely mediated by the oxidation-induced myeloperoxidase (MPO) in neutrophil and macrophage inflammatory milieus. This proves the biodegradability faculty of functionalized CNTs, which potentially avoids long-term tissue accumulation and triggering of acute toxicity. On the whole, the proposed Diels-Alder functionalization accounts for the improved CNT biological response in terms of the biocompatibility and biodegradability profiles. Therefore, CNTs can be considered for use in bone tissue engineering without notable toxicological threats.

Heat Dissipation Interfaces Based on Vertically Aligned Diamond/Graphite Nanoplatelets.

Crystalline carbon-based materials are intrinsically chemically inert and good heat conductors, allowing their applications in a great variety of devices. A technological step forward in heat dissipators production can be given by tailoring the carbon phase microstructure, tuning the CVD synthesis conditions. In this work, a rapid bottom-up synthesis of vertically aligned hybrid material comprising diamond thin platelets covered by a crystalline graphite layer was developed. A single run was designed in order to produce a high aspect ratio nanostructured carbon material favoring the thermal dissipation under convection-governed conditions. The produced material was characterized by multiwavelength Raman spectroscopy and electron microscopy (scanning and transmission), and the macroscopic heat flux was evaluated. The results obtained confirm the enhancement of heat dissipation rate in the developed hybrid structures, when compared to smooth nanocrystalline diamond films.

Characterization of the PGE2 receptor subtype in bovine chondrocytes in culture.

1. Prostaglandin E2 (PGE2) is an autacoid that decreases proteoglycan synthesis, increases metalloprotease production by cultured chondrocytes, and can modulate some of the actions of interleukin-1 on cartilage. The objective of the present study was to characterize the subtype of prostaglandin E2 receptor present in bovine chondrocytes in culture. 2. Primary cultures of articular chondrocytes were prepared from slices of bovine carpal cartilage by sequential digestion with type III hyaluronidase, trypsin, type II collagenase, followed by overnight incubation in Dulbecco's Modified Eagle's Medium (DMEM) with type II collagenase, washing, and seeding at a density of 2 x 10(5) cells cm-2 in DMEM with 10% foetal bovine serum. 3. PGE2 and carbaprostacyclin induced dose-dependent increases in intracellular cyclic AMP in bovine chondrocytes in culture. The potencies of these compounds were different, and maximal doses of PGE2 and carbaprostacyclin had an additive effect. PGD2 induced a small increase in intracellular cyclic AMP only at a high concentration (10(-5) M). 4. PGE2 was more potent that the EP2 agonist 11-deoxy-PGE1 at inducing increases in intracellular cyclic AMP. The EP2 agonist butaprost, however, induced only a small increase at a concentration of 10(-5)M. 17-Phenyl-PGE2 (EP1 agonist), sulprostone and MB 28767 (15S-hydroxy-9-oxo-16-phenoxy-omega-tetranorprost-13E-enoic acid) (EP3 agonists) did not induce an increase in intracellular cyclic AMP at concentrations up to 10(-5)M. 5. The EP4 antagonist AH 23848B ([1 alpha(Z),2 beta, 5 alpha]-(+/-) -7-[5-[[(1,1'-biphenyl)-4-yl]methoxyl-2-(4-morpholinyl) -3-oxocyclopentyl]-5-heptenoic acid) antagonized PGE2 but not carbaprostacyclin effects on intracellular cyclic AMP. The Schild plot slope was different from 1 but this could be due to an interaction of PGE2 with IP receptors in high doses. The exact nature of the antagonism by compound AH 23848B could not be definitely established in these experimental conditions. 6. Neither PGE2 nor any of its analogues inhibited the increase in intracellular cyclic AMP induced by forskolin, and pertussis toxin did not alter the response to PGE2, suggesting that no Gi-coupled PGE2 receptors are present in these cells. Stimulation with PGE2 did not induce significant increases in intracellular inositol-trisphosphate levels nor increases in intracellular free calcium as determined by confocal microscopy, suggesting the absence of phospholipase-C-coupled or of calcium channel-coupled PGE2 receptors in bovine chondrocytes in these experimental conditions. 7. These results show for the first time that bovine chondrocytes in culture present a functional PGE2 receptor that has some pharmacological characteristics of an EP4 subtype, as well as an IP receptor.

Effect of PAF on rat lung vascular permeability: role of platelets and polymorphonuclear leucocytes.

1. The objectives of the present experiments were to assess the contribution of polymorphonuclear leucocytes (PMNLs), platelets and their products such as thromboxane A2 (TxA2), histamine and 5-hydroxytryptamine to platelet activating factor (PAF)-mediated protein extravasation in rat lungs. 2. Intravenous injection of PAF (1.0 and 5.0 micrograms kg-1) increased dose-dependently (up to 7.5 fold) the vascular permeability of the trachea, upper and lower bronchi to Evans blue dye (EB), a marker of albumin extravasation. The permeability of the pulmonary parenchyma was not affected significantly by PAF. 3. Thrombocytopenia induced by administration of the IgG fraction of goat anti-rat platelet serum (APS; 15 mg 100 g-1, i.p., 16-18 h) reduced by 55, 58 and 40% the effects of the lower dose of PAF (1.0 microgram kg-1) and by 31, 23 and 15% the effects of the higher dose of PAF (5.0 micrograms kg-1) on the permeability of the trachea, upper and lower bronchi respectively to albumin. 4. PMNL depletion induced by administration of rabbit anti-rat polymorphonuclear serum (ANS; 2 mg kg-1, i.v., 24 h) did not reduce significantly the effects of the lower dose of PAF (1.0 microgram kg-1) on the airways, however the effects of the higher dose of PAF (5.0 micrograms kg-1) on the permeability of the trachea, upper and lower bronchi to albumin were reduced by 43, 25 and 23% respectively. 5. The injection of both the anti-platelet and the anti-PMNL sera reduced by 61, 62 and 96% the effects of the lower dose of PAF (1.0 microg kg-1) and by 44, 39 and 47% the effects of the higher dose of PAF (5.0 microg kg-1) on the permeability of the trachea, upper and lower bronchi respectively.6. The combined injection of the TxA2-mimetic (U-44069; 5.0 microg kg-1) and PAF (1.0 and 5.0 microg kg-1)in thrombocytopenic rats overcame the vascular permeability decrease induced by APS treatment.7. Pretreatment of the animals with a combination of antagonists to histamine (mepyramine;3.0 mg kg-1) and 5-hydroxytryptamine (methysergide; 2.5 mg kg-1) did not cause a significant inhibition of the effect of PAF (1.0 and 5.0 microg kg-1) on EB extravasation in the airways.8. These data show that the effect of intravenous PAF on rat vascular permeability is partly modulated by polymorphonuclear leucocyte and platelet activation. Our results suggest that following its release,TxA2 could increase postcapillary hydrostatic pressure by inducing a venoconstriction and potentiate the extravasation elicited by PAF. These results do not suggest a major role for histamine and/or 5-hydroxytryptamine on PAF-induced albumin extravasation.

A study of experimental reinfection by Trypanosoma cruzi in dogs.

The role of reinfection in the evolution of Chagas' disease was evaluated in dogs alternately infected with the 147 and SC-1 strains of Trypanosoma cruzi. A parasitologic, serologic, clinical, and electrocardiographic follow-up was carried out on the infected and noninfected dogs. The dogs were reinfected five times over a period of 38 months. No deaths were observed during the experiment. They presented a brief oligosymptomatic acute phase. The level of parasitemia decreased progressively with the number of reinfections. Bloodstream parasites were not detectable after the fifth reinfection. All parasite samples isolated during the follow-up were zymodeme B, corresponding to strain 147, irrespective of the strain with which the dogs were first infected and of the triatomine species used for isolation. Conversely, amplification by the polymerase chain reaction of a segment of the T. cruzi mini-exon gene showed the simultaneous presence of both strains in three of the eight reinfected animals. Antibody titers were greater among the dogs successively infected than those infected only once. Neither amastigotes nor T. cruzi DNA were detected in the tissues of the infected dogs. Alterations related to Chagas' disease were identified only in the heart and consisted of chronic focal and discrete myocarditis, compatible with the indeterminate form of Chagas' disease. All infected dogs developed this form of the disease, which was independent of the number of infections.

Expression of recombinant human cyclooxygenase isoenzymes in transfected COS-7 cells in vitro and inhibition by tenoxicam, indomethacin and aspirin.

The recent discovery of cyclooxygenase-2 (COX-2), an isoenzyme associated mainly with inflammation created the need to reevaluate cyclooxygenase inhibitors with reliable screening methods. In the present study we standardized a technique to determine the IC50S of cyclooxygenase inhibitors on recombinant human COX-1 and COX-2 expressed in mammalian cells and used it to study the compounds tenoxicam, aspirin and indomethacin. The IC50S of aspirin, indomethacin and tenoxicam for human COX-1 were 0.41 +/- 0.07 microgram/ml, 0.008 +/- 0.003 microgram/ml, and 7.94 +/- 3.28 micrograms/ml, respectively, and for human COX-20.64 +/- 0.16 microgram/ml, 0.09 +/- 0.05 microgram/ml, and 10.61 +/- 1.50 micrograms/ml, for aspirin, indomethacin, and tenoxicam. Tenoxicam had the lowest IC50hCOX-2/IC50hCOX-1 ratio (1.34), followed by aspirin (1.53) and indomethacin (10.82). The system described in the present study provides a simple and efficient way to determine the specificity of NSAID inhibition for each of the human cyclooxygenase isoenzymes separately.

Immunolocalization of the prostaglandin E2 receptor subtypes in human bone tissue: differences in foetal, adult normal, osteoporotic and pagetic bone.

PGE(2) is an important mediator of bone metabolism, but the precise localization of its receptors in human bone remains unknown. The present study used specific antibodies against EP(1), EP(2), EP(3) and EP(4) receptors for immunolocalization in normal, osteoporotic and pagetic human adult bone and in human foetal bone. No labelling was obtained for the EP(1) and EP(2) receptors. The EP(3) receptor was detected in foetal osteoclasts, osteoblasts and osteocytes, but only in osteoclasts and some osteoblasts from adult bone. The EP(4) receptor was detected in foetal osteoclasts, osteoblasts and osteocytes and in adult osteoclasts and osteoblasts, but not in adult osteocytes. Our results show differences in PGE(2) receptor expression in foetal and adult human bone but no difference in adult normal compared to pathologic bone. Finally, these results show that the distribution of EP receptors in human osteoblasts in bone corresponds in part to what we recently described in human osteoblasts in culture.

Systematic pharmacological approach to the characterization of NSAIDs.

Non-steroidal anti-inflammatory drugs (NSAIDs) are widely used for the treatment of inflammatory diseases. NSAIDs inhibit cyclooxygenase (COX), the rate limiting enzyme responsible for the conversion of arachidonic acid into prostaglandins. Recent studies have shown the existence of two isoforms of cyclooxygenase: COX-1, now often referred to as the constitutive form, and COX-2, an inducible form which is the major isoenzyme involved in prostaglandin synthesis in inflammation and other pathological situations. Since inhibition of prostaglandin production in tissues where they play a physiological role leads to important side effects, a COX-2 preferential inhibitor would present therapeutical advantages. In the present study, we evaluated the inhibitory properties of cyclooxygenase inhibitors on human COX-1 and COX-2 using a heterologous expression system. We investigated instantaneous inhibition and pre-incubation inhibition as well as time recovery of cyclooxygenase activity assays with the aid of four NSAIDs: mefenamic acid, indomethacin, aspirin and NS-398. Our results demonstrate that instantaneous inhibition assays have little correlation with clinical results. Inhibition assays using pre-incubation with the drugs tested, however, more closely resemble the data from in vivo studies. Cyclooxygenase recovery assays enabled better characterization of simple competitive inhibitors, competitive reversible time-dependent inhibitors and irreversible time-dependent inhibitors. The data illustrate the usefulness of our system in allowing a better determination of the pharmacological characteristics of NSAIDs as well as permitting a comparison among different drugs.

Leukotriene receptors in HL-60 cells differentiated into eosinophils, monocytes and neutrophils.

The expression of leukotriene B4 (LTB4) and leukotriene D4 (LTD4) receptors was determined, by binding assay, in HL-60 cells differentiated into the monocyte/macrophage, neutrophil, and eosinophil lineages. Monocyte/ macrophage- and neutrophil-differentiated cells developed specific LTB4 receptors with high affinities (Kd = 1.27 nM and 2.65 nM, respectively) and low affinities (Kd = 26.41 nM and 55.63 nM, respectively). These receptors were functional and specific as indicated by the ability of LTB4 to elicit an increase in intracellular calcium concentration antagonised by specific antagonists. Eosinophil-differentiated cells developed mainly LTD4 receptors (Kd = 41.91 nM), and stimulation with LTD4 induced an increase in intracellular calcium that was antagonised by a specific LTD4 antagonist. These results show, for the first time, that eosinophil-differentiated HL-60 cells express specific functional LTD4 receptors. These cells could be used for the study of the actions of peptidoleukotrienes on eosinophils, and for studies on the molecular mechanisms regulating LTD4 receptor expression.

Immunohistochemical localization of the prostacyclin receptor (IP) human bone.

Prostacyclin (PGI(2)) is an important mediator implicated in bone metabolism. Among the natural prostaglandins it is the most potent inhibitor of bone resorption and mediates bone modelling and remodelling induced by strain changes. The effects of prostacyclin depend on its interaction with a specific receptor (IP). Despite its well documented effects on bone the localization and distribution of the IP receptor in human bone remain unknown. The present study used specific antipeptide antibodies to IP receptor for immunolocalization of the IP receptor in normal, osteoporotic and Pagetic human adult bone and in human fetal bone. The IP receptor was detected in fetal and adult osteoclasts and osteoblasts. Fetal osteocytes also expressed IP receptor but not adult osteocytes. Interestingly, the expression of IP receptor in adult osteoblasts was gradually lost as these cells were trapped in the matrix and became osteocytes. The IP receptor showed a perinuclear distribution within the cells, but in multinuclear osteoclasts not all nuclei were positive. Our results showed differences in IP receptor expression in fetal and adult human bone and, in adult bone, with the differentiation of osteoblasts into osteocytes. They also showed that there is no difference on the expression of prostacyclin receptors in Pagetic, osteoporotic and normal human bone, and they confirm the presence of the IP receptor in human osteoblasts as had been demonstrated by our previous study with human osteoblasts in culture.

Characterization of the prostaglandin receptors in human osteoblasts in culture.

Prostaglandins have complex actions on bone metabolism that depend on interactions with different types and subtypes of receptors. Our objective was to characterize the prostaglandins receptors present in primary cultures of human osteoblasts. RT-PCR analysis revealed the presence of DP, EP(4), IP, FP and TP receptor mRNA in primary cultures of human osteoblasts. FP receptor mRNA was detected only after 3 weeks of confluency, all the others were detected at every culture time tested. To verify the functionality of these receptors we challenged the cells with the prostanoids and synthetic analogues and determined the intracellular levels of cAMP. All receptors found by RT-PCR were coupled to second messengers except for the DP subtype. These results clearly show the presence of functional EP(4), IP, FP and TP receptors in human osteoblasts in culture.

Anti-inflammatory effects of the products from Wilbrandia ebracteata on carrageenan-induced pleurisy in mice.

Wilbrandia ebracteata Cogn. (Cucurbitaceae) is commonly known in Brazil as "Taiuia". The roots are employed in folk medicine for the treatment of several diseases, such as rheumatic disease. This study has evaluated the anti-inflammatory action of dicloromethane fraction (F-DCM), purified fraction (PFIII) and Cucurbitacin B extracted from crude extract of W. ebracteata in experimental models in vivo. The F-DCM (0.3 to 10 mg.kg(-1), i.p. or 3 to 30 mg.kg(-1) p.o.) produced significant but not dose-dependent inhibition of the carrageenan-induced cell influx and exsudate leakage in the pleural cavity of mice. The F-DCM 0.01 to 10 mg.kg(-1), i.p. or 0.1 to 10 mg.kg(-1) p.o.) decreased the levels of PGE2 in the exsudate leakage induced by carrageenan in the pleural cavity after 4 h with a calculated ID50 of 0.01 (0.002-0.09, i.p.) and 0.29 (0.05-1.45, p.o.) mg.kg(-1). The PFIII (3 mg.kg(-1), i.p.) inhibited 80% of cell migration (1.50 +/- 0.09 x 10(6) cells/cavity) and exsudate leakage by about 50% (3.09 +/- 0.71 microg/ml) in relation to the control group. Cucurbitacin B (0.1 mg.kg(-1), i.p.), the main compound of PFIII, reduced significantly the levels of PGE2 in the exsudate leakage by 40.7% (10.41 +/- 2.67 ng.ml(-1)). These data show that the active principle(s) present in the F-DCM of W. ebracteata elicited pronounced anti-inflammatory effects when assessed by i.p. or p.o. routes, as well as PFIII. The F-DCM was also able to prevent PGE2 formation in exsudate leakage induced by carrageenan, as well as Cucurbitacin B, its active principle. These results indicate that the anti-inflammatory activity of Wilbrandia ebracteata can be related with the inhibition of the production of PGE2.

Anticardiolipin antibodies in patients with rheumatoid arthritis.

Anticardiolipin antibodies (ACA) were assayed by ELISA in 73 patients with rheumatoid arthritis. Twelve (16.48%) patients showed levels of ACA three standard deviations above the value of the control group and were considered positive; these patients were compared to the group with ACA within the normal levels regarding the following clinical and laboratorial characteristics: spontaneous abortions, central nervous system involvement, systematization and activity of disease, alterations in platelet counts, presence of antinuclear antibodies and rheumatoid factor. Significant statistical association could be demonstrated between systematization and presence of antinuclear antibodies (ANA) and positiveness to ACA (IgG, IgM or both). These findings might indicate that ACA in patients with RA could have relevance to morbidity of disease or perhaps to its pathogenesis.