Effects of nitric oxide on neutrophil influx depends on the tissue: role of leukotriene B(4) and adhesion molecules

Background and purpose: We investigated the effect of nitric oxide synthase (NOS) inhibition on polymorphonuclear cell (PMN) influx in zymosan or lipopolysaccharide (LPS)-induced arthritis and peritonitis. Experimental approach: Wistar rats received intra-articular (i.art.) zymosan (30-1000 mu g) or LPS (1-10 mu g). Swiss C57/Bl6 mice genetically deficient in intercellular adhesion molecule-1 (ICAM-1(-/-)) or in beta(2)-integrin (beta(2)-integrin(-/-)) received zymosan either i.art. or i.p. PMN counts, leukotriene B(4) (LTB(4)), tumour necrosis factor-alpha (TNF-alpha) and interleukin-10 (IL-10) levels were measured in joint and peritoneal exudates. Groups received the NOS inhibitors N(G)-nitro-L-arginine methyl ester (LN), nitro-L-arginine, N-[3-(aminomemethyl) benzyl] acetamide or aminoguanidine, prior to zymosan or LPS, given i.p. or s.c. in the arthritis and peritonitis experiments respectively. A group of rats received LN locally (i.art. or i.p.), 30 min prior to 1 mg zymosan i.art. Key results: Systemic or local NOS inhibition significantly prevented PMN migration in arthritis while increasing it in peritonitis, regardless of stimuli, concentration of NOS inhibitors and species. NOS inhibition did not alter TNF-alpha and IL-10 but decreased LTB(4) in zymosan-induced arthritis. LN administration significantly inhibited PMN influx into the joints of ICAM-1(-/-) and beta(2)-integrin(-/-) mice with zymosan-arthritis, while not altering PMN influx into the peritoneum of mice with zymosan-peritonitis. Conclusions and implications: Nitric oxide has a dual modulatory role on PMN influx into joint and peritoneal cavities that is stimulus-and species-independent. Differences in local release of LTB(4) and in expression of ICAM-1 and beta(2)-integrin account for this dual role of NO on PMN migration.

Reduction of ICAM-1 expression by carbon monoxide via soluble guanylate cyclase activation accounts for modulation of neutrophil migration

Previously, it was demonstrated that the heme/heme oxygenase (HO)/carbon monoxide (CO) pathway inhibits neutrophil recruitment during the inflammatory response. Herein, we addressed whether the inhibitory effect of the HO pathway on neutrophil adhesion and migration involves the reduction of intracellular adhesion molecule type (ICAM)-1 and beta(2)-integrin expression. Mice pretreated with a specific inhibitor of inducible HO (HO-1), zinc protoporphyrin (ZnPP) IX, exhibit enhanced neutrophil adhesion and migration induced by intraperitoneal injection of Escherichia coli lipopolysaccharide (LPS). These findings are associated with an increase in ICAM-1 expression on mesentery venular endothelium. In accordance, HO-1 inhibition did not enhance LPS-induced neutrophil migration and adhesion in ICAM-1-deficient mice. Furthermore, the treatment with a CO donor (dimanganese decacarbonyl, DMDC) that inhibits adhesion and migration of the neutrophils, reduced LPS-induced ICAM-1 expression. Moreover, neither DMDC nor ZnPP IX treatments changed LPS-induced beta(2)-integrin expression on neutrophils. The effect of CO on ICAM-1 expression seems to be dependent on soluble guanylate cyclase (sGC) activation, since 1H-(1,2,4)oxadiazolo (4,3-a)quinoxalin-1-one (sGC inhibitor) prevented the observed CO effects. Finally, it was observed that the nitric oxide (NO) anti-inflammatory effects on ICAM-1 expression appear to be indirectly mediated by HO-1 activation, since the inhibition of HO-1 prevented the inhibitory effect of the NO donor (S-nitroso-N-acetylpenicillamine) on LPS-induced ICAM-1 expression. Taken together, these results suggest that CO inhibits ICAM-1 expression on endothelium by a mechanism dependent on sGC activation. Thus, our findings identify the HO-1/CO/guanosine 3`5`-cyclic monophosphate pathway as a potential target for the development of novel pharmacotherapy to control neutrophil migration in inflammatory diseases.

Compound 48/80 induces endothelium-dependent and histamine release-independent relaxation in rabbit aorta

Compound 48/80 (C48/80) is a synthetic condensation product of N-methyl-p-methoxyphenethyl am me with formaldehyde and is an experimental drug used since the 1950s to induce anaphylactic shock through histamine release. This study was carried out to further elucidate the mechanism by which this drug induces nitric oxide (NO) release. Our specific goals were: (a) to verify if C48/80`s relaxation occurs through the stimulation of histamine receptors; (b) to evaluate the endothelium-dependent relaxation induced by C48/80; (c) to identify NO as the endothelium-relaxing factor released by C48/80; (d) to identify the NO synthase (NOS) responsible for NO release; and (e) to verify if the relaxation induced by C48/80 is calcium and cyclic guanidine monophosphate (cGMP) dependent. Rabbit aorta segments, with and without endothelium, were suspended in organ chambers (25 ml) filled with Krebs solution maintained at 37 degrees C, bubbled with 95% O-2/5% CO2 (pH 7.4). Phenylephrine was used to contract the segments. Other protocol drugs included H-1- and H-2-receptor antagonists, cyclooxygenase, NOS, guanylyl cyclase and phospholipase C (PLC) inhibitors. Endothelium-dependent relaxation induced by C48/80 was also studied in calcium-free Krebs solution associated with a calcium chelator. In summary, our investigation demonstrated that the C48/80 vasodilating action: (a) does not depend on H-1 and H-2 histamine receptors; (b) is NO endothelium-dependent; (c) is dependent on the endothelial constitutive NOS (NOS-3) isoform activation; (d) is cGMP-dependent; and that NOS-3 activation by C48/80: (a) is independent of PLC up to 25 mu g/ml and (b) is partially dependent of this lipase in higher doses. (C) 2007 Elsevier Inc. All rights reserved.

Amitriptyline and Acute Inflammation: A Study Using Intravital Microscopy and the Carrageenan-Induced Paw Edema Model

Background/Aims: Antidepressants are reported to exhibit antiinflammatory effects. However, mechanisms involved in this action have not been elucidated. Thus, the objectives of the present study were (a) to evaluate the effects of amitriptyline on the acute inflammatory process, and (b) to investigate the participation of alpha(1)-adrenergic receptors and glucocorticoids as possible mechanisms implicated in the amitriptyline action on inflammation. Methods and Results: Single and multiple doses of amitriptyline were administered to rats submitted to the carrageenan-induced paw edema model. The results showed a significant antiedematous reaction to amitriptyline, mainly when administered at each elimination half-life. The next step was to evaluate its effects on leukocyte behavior, using intravital microscopy. Amitriptyline produced a significant effect on leukocyte behavior. To investigate possible mechanisms involved, a glucocorticoid receptor antagonist (RU-486) and an alpha(1)-adrenergic receptor antagonist (prazosin) were used. RU-486 administration lacked the ability to decrease the amitriptyline antiinflammatory effects in the carrageenan-induced paw edema model. Prazosin pretreatment potentiated the amitriptyline antiinflammatory effect without presenting an effect per se. Conclusion:The present study shows the ability of amitriptyline to decrease edema and affect leukocyte behavior in an acute inflammatory process; and, for the first time to our knowledge, we suggest the involvement of alpha(1)-adrenoceptors in the antiinflammatory effects of amitriptyline. Copyright (C) 2010 S. Karger AG, Basel

Cariogenic Microorganisms and Oral Conditions in Asthmatic Children

This study evaluated the caries risk of asthmatic patients on the basis of mutans streptococci (MS) and lactobacilli levels in saliva samples as well as the index of oral hygiene and dental caries (DMFT index). The study population was composed of 80 asthmatic children, aged 3-15 years, who use specific medication, and 80 matched, healthy control children. The parents were interviewed about oral health-related factors. The World Health Organization criteria were used for dental examinations. The Kohler and Bratthal methodology was used to detect salivary MS levels and dilutions of saliva were done for lactobacilli counting. No differences between asthma and control groups were observed for caries prevalence in children aged 3-6 and 7-10 years, except in severe cases in the younger group. However, higher caries prevalence for permanent dentition was observed in 11- to 15-year-old asthmatic children. An increased dental biofilm was observed in the asthma group, as well as salivary levels of MS. No differences were observed in levels of lactobacilli. No statistical correlations were found between medication, frequency of treatment, method of consumption and caries experience, dental biofilm and salivary levels of MS or lactobacilli. However, there was a correlation between MS levels and treatment duration. The logistic regression revealed that MS level is an important risk factor for increased caries experience. Asthma should be evaluated as a risk factor for caries experience because it can increase the levels of MS and the dental biofilm. Copyright (C) 2011 S. Karger AG, Basel

Dental caries status and salivary properties of asthmatic children and adolescents

Aims. This study aimed to investigate the dental caries status and salivary properties in 3- to 15-year-old children/adolescents. Methods. The sample was split in two groups: asthma group (AG), composed of 65 patients who attended Public Health Service; asthma-free group (AFG), composed of 65 nonasthmatic children/adolescents recruited in two public schools. Stimulated salivary samples were collected for 3 min. Buffering capacity and pH were ascertained in each salivary sample. A single trained and calibrated examiner (kappa = 0.98) performed the dental caries examination according to WHO criteria. Results. The AFG showed salivary flow rate (1.10 +/- 0.63 mL/min) higher (P = 0.002) than AG (0.80 +/- 0.50 mL/min). An inverse relationship was observed between asthma severity and salivary flow rate (Phi coefficient, r phi: 0.79, P = 0.0001). Children with moderate or severe asthma showed an increased risk for reduced salivary flow rate (OR: 17.15, P < 0.001). No association was observed between drug use frequency (P > 0.05) and drug type (P > 0.05) with salivary flow rate. Buffering capacity was similar in both groups. No significant differences were encountered in dental caries experience between AFG and AG groups. Conclusions. Although asthma can cause reduction in flow rate, the illness did not seem to influence dental caries experience in children with access to proper dental care.

Treatment With a Growth Factor-Protein Mixture Inhibits Formation of Mineralized Nodules in Osteogenic Cell Cultures Grown on Titanium

Despite wide clinical application, the efficacy of platelet-rich plasma (PRP) for repairing bone defects and enhancing osseointegration of metal implants is still subject of debate. This study aimed to evaluate the effects of a well-defined PRP-like mixture containing platelet-derived growth factor-BB, transforming growth factor (TGF)-beta 1, TGF-beta 2, albumin, fibronectin, and thrombospondin [growth factors (GFs) + proteins] on the development of the osteogenic phenotype on titanium (Ti) in vitro. Human alveolar bone-derived osteoblastic cells were subcultured on Ti discs and exposed during the first 7 days to osteogenic medium supplemented with GFs + proteins and to osteogenic medium alone thereafter up to 14 days. Control cultures were exposed to only osteogenic medium. Dose-response experiments were carried out using rat primary calvarial cells exposed to GFs + proteins and 1:10 or 1:100 dilutions of the mixture. Treated human-derived cell cultures exhibited a significantly higher number of cycling cells at days 1 and 4 and of total cells at days 4 and 7, significantly reduced alkaline phosphatase (ALP) activity at days 4, 7, and 10, and no Alizarin red-stained areas (calcium deposits) at day 14, indicating an impairment in osteoblast differentiation. Although the 1:10 and 1:100 dilutions of the mixture restored the proliferative activity of rat-derived osteogenic cells to control levels and promoted a significant increase in ALP activity at day 10 compared with GFs + proteins, mineralized nodule formation was only observed with the 1:100 dilution (similar to 50% of the control). These results showed that a PRP-like protein mixture inhibits development of the osteogenic phenotype in both human and rat osteoblastic cell cultures grown on Ti. (J Histochem Cytochem 57:265-276, 2009)

Effects of a mixture of growth factors and proteins on the development of the osteogenic phenotype in human alveolar bone cell cultures

Strategies to promote bone repair have included exposure of cells to growth factor (GF) preparations from blood that generally include proteins as part of a complex mixture. This study aimed to evaluate the effects of such a mixture on different parameters of the development of the osteogenic phenotype in vitro. Osteoblastic cells were obtained by enzymatic digestion of human alveolar bone and cultured under standard osteogenic conditions until subconfluence. They were subcultured on Thermanox coverslips up to 14 days. Treated cultures were exposed during the first 7 days to osteogenic medium supplemented with a GFs + proteins mixture containing the major components found in platelet extracts [plate I et-derived growth factor-BB, transforming growth factor (TGF)-beta 1, TGF-beta 2, albumin, fibronectin, and thrombospondin] and to osteogenic medium alone thereafter. Control cultures were exposed only to the osteogenic medium. Treated cultures exhibited a significantly higher number of adherent cells from day 4 onward and of cycling cells at days 1 and 4, weak alkaline phosphatase (ALP) labeling, and significantly decreased levels of ALP activity and mRNA expression. At day 14, no Alizarin red-stained nodular areas were detected in cultures treated with GFs + proteins. Results were confirmed in the rat calvaria-derived osteogenic cell culture model. The addition of bone morphogenetic protein 7 or growth and differentiation factor 5 to treated cultures upregulated Runx2 and ALP mRNA expression, but surprisingly, ALP activity was not restored. These results showed that a mixture of GFs + proteins affects the development of the osteogenic phenotype both in human and rat cultures, leading to an increase in the number of cells, but expressed a less differentiated state.

Quantitative and qualitative influences of tapasin on the class I peptide repertoire

Tapasin is critical for efficient loading and surface expression of most HLA class I molecules. The high level surface expression of HLA-B*2705 on tapasin-deficient 721.220 cells allowed the influence of this chaperone on peptide repertoire to be examined. Comparison of peptides bound to HLA-B*2705 expressed on tapasin-deficient and -proficient cells by mass spectrometry revealed an overall reduction in the recovery of B*2705-bound peptides isolated from tapasin-deficient cells despite similar yields of B27 heavy chain and beta (2)-microglobulin. This indicated that a proportion of suboptimal ligands were associated with B27, and they were lost during the purification process. Notwithstanding this failure to recover these suboptimal peptides, there was substantial overlap in the repertoire and biochemical properties of peptides recovered from B27 complexes derived from tapasin-positive and -negative cells. Although many peptides were preferentially or uniquely isolated from B*2705 in tapasin-positive cells, a number of species were preferentially recovered in the absence of tapasin, and some of these peptide ligands have been sequenced. In general, these ligands did not exhibit exceptional binding affinity, and we invoke an argument based on lumenal availability and affinity to explain their tapasin independence. The differential display of peptides in tapasin-negative and -positive cells was also apparent in the reactivity of peptide-sensitive alloreactive CTL raised against tapasin-positive and -negative targets, demonstrating the functional relevance of the biochemical observation of changes in peptide repertoire in the tapasin-deficient APC. Overall, the data reveal that tapasin quantitatively and qualitatively influences ligand selection by class I molecules.

The oral medicine of tooth wear

This review illustrates, through a series of case histories, how oral medicine insights aid the diagnosis and management of patients with excessive tooth wear. The cases reviewed are drawn from the records of 500 southeast Queensland patients referred to the author over a 12 year period. Patients most at risk of dental erosion have work and sports dehydration, caffeine addiction, gastro-oesophageal reflux, asthma, diabetes mellitus, hypertension or other systemic diseases or syndromes that predispose to xerostomia. Saliva protects the teeth from the extrinsic and intrinsic acids which cause dental erosion. Erosion, exacerbated by attrition and abrasion, is the main cause of tooth wear. These cases illustrate that teeth, oral mucosa, salivary glands, skin and eyes should be examined for evidence of salivary hypofunction and attendant medical conditions. Based on comprehensive oral medicine, dietary analyses and advice, it would seem patients need self-management plans to deal with incipient chronic tooth wear. The alternative is the expensive treatment of pain, occlusal damage and pulp death required to repair the effects of acute severe tooth wear.

Ivermectin, an unconventional agonist of the glycine receptor chloride channel

The effects of the antihelmintic, ivermectin, were investigated in recombinantly expressed human alpha (1) homomeric and alpha (1)beta heteromeric glycine receptors (GlyRs), At low (0.03 muM) concentrations ivermectin potentiated the response to sub-saturating glycine concentrations, and at higher (greater than or equal to0.03 muM) concentrations it irreversibly activated both alpha (1) homomeric and alpha (1)beta heteromeric GlyRs. Relative to glycine-gated currents, ivermectin-gated currents exhibited a dramatically reduced sensitivity to inhibition by strychnine, picrotoxin, and zinc. The insensitivity to strychnine could not be explained by ivermectin preventing the access of strychnine to its binding site. Furthermore, the elimination of a known glycine- and strychnine-binding site by site-directed mutagenesis had little effect on ivermectin sensitivity, demonstrating that the ivermectin- and glycine-binding sites were not identical. Ivermectin strongly and irreversibly activated a fast-desensitizing mutant GlyR after it had been completely desensitized by a saturating concentration of glycine. Finally, a mutation known to impair dramatically the glycine signal transduction mechanism had little effect on the apparent affinity or efficacy of ivermectin, Together, these findings indicate that ivermectin activates the GlyR by a novel mechanism.

Dental erosion in asthma: A case-control study from south east Queensland

Background: Asthma medication places patients at risk of dental erosion by reducing salivary protection against extrinsic or intrinsic acids. But patterns of lesions in asthmatics may differ from patterns in non-asthmatics, because gastro-oesophageal reflux (GOR) is found in 60 per cent of asthmatics. Methods: The lesions in 44 asthma cases were compared to those of age and sex match controls with no history of asthma or medications drawn from the dental records of 423 patients referred concerning excessive tooth wear. The subjects were 70 males age range 15 to 55 years and 18 females age range 18 to 45. Anamnestic clinical data were compared between the two groups. Models of all 88 subjects were examined by light microscopy, and wear patterns were recorded on permanent central incisor, canine, premolar and first molar teeth. Results: Clinical differences were a higher incidence of tooth hypersensitivity; xerostomia, salivary gland abnormalities, gastric complaints, and self induced vomiting in the cases. No differences were found between the cases and controls on citrus fruit and acid soft drink consumption. More occlusal erosion sites were found in cases, whereas more attrition sites were found in the controls. There were no significant differences in palatal erosion on maxillary anterior teeth found between cases and controls. Lingual erosion of the mandibular incisors, found only in GOR patients, was not observed. Conclusions: A higher incidence of erosion was found in asthmatics. Gastro-oesophageal reflux symptoms were not associated with the sign of lingual mandibular incisor erosion. The clinical significance is that asthmatics are at risk of dental erosion from extrinsic acid, but GOR does not appear to contribute in a site-specific manner.

The therapeutic potential of dopamine modulators on the cardiovascular and renal systems

In the periphery, physiological dopamine increases renal blood flow, decreases renal resistance and acts on the kidney tubule to enhance natriuresis and diuresis. The loss of dopamine function may be involoved in the deterioration in kidney function associated with ageing and may have a role in the pathogenesis of hypertension and diabetes. Intravenous dopamine is used as a positive inotrope in the treatment of acute heart failure and cardiogenic shock and as a diuretic in renal failure. The clinical uses of dopamine are limited, as it must be given intravenously, and also has widespread effects. The levels of peripheral dopamine can be increased by the administration of L-dopa to increase synthesis, prodrugs to release dopamine (docarpamine, glu-dopa) or by inhibiting the breakdown of dopamine (nitecapone). Preliminary clinical trials suggest that docarpamine may be useful in patients with low cardiac output syndrome after cardiac surgery and in refractory cirrhotic ascites. Ibopamine is an agonist at dopamine D1 and D2 receptors, which may retard the progression of chronic renal failure. Gludopa is selective for the kidney thus avoiding widespread side effects. The early clinical studies with ibopamine as a diuretic in heart failure were favourable but the subsequent large mortality study showed that ibopamine increased mortality. Fenoldopam is a selective dopamine D1 receptor agonist. Intravenous fenoldopam may be useful in the treatment of hypertension associated with coronary artery bypass surgery or in hypertensive emergencies. Although our understanding of physiological and pathological roles of peripheral dopamine has been increasing rapidly in recent times, we still need more information to allow the design of clinically useful drugs that modify these roles. One priority is an orally-active selective dopamine D1 receptor agonist.

Quantitation of human brain GABAA receptor isoforms by competitive RT-PCR

We have developed a competitive RT-PCR assay, adapted from Lewohl et al. [Brain Res. Brain Res. Protoc. 1 (1997) 347]. for the quantitation of GABA, receptor beta isoforms in human brain using an internal standard that shares high sequence homology to the targets. The internal standard is identical to the beta(1) sequence except for a 61 bp deletion and the incorporation of a Hind III restriction enzyme site. Unlike traditional competitive RT-PCR, which requires a range of internal standard concentrations to be titrated against a constant amount of unknown, this method relies on a standard curve for quantitation of each sample and thus permits increased sample throughput. This method is suitable for the quantitation of beta(1), beta(2) and beta(3) isoforms of the GABA(A) receptor in human alcoholic and control brain. (C) 2003 Elsevier Science B.V. All rights reserved.

An Extension of Gompertzian Growth Dynamics Weibull and Frechet Models

In this work a new probabilistic and dynamical approach to an extension of the Gompertz law is proposed. A generalized family of probability density functions, designated by Beta* (p, q), which is proportional to the right hand side of the Tsoularis-Wallace model, is studied. In particular, for p = 2, the investigation is extended to the extreme value models of Weibull and Frechet type. These models, described by differential equations, are proportional to the hyper-Gompertz growth model. It is proved that the Beta* (2, q) densities are a power of betas mixture, and that its dynamics are determined by a non-linear coupling of probabilities. The dynamical analysis is performed using techniques of symbolic dynamics and the system complexity is measured using topological entropy. Generally, the natural history of a malignant tumour is reflected through bifurcation diagrams, in which are identified regions of regression, stability, bifurcation, chaos and terminus.