Mannose-binding lectin (MBL2) polymorphisms and inflammation in hypertensive patients.

We investigated the possible role of Mannose binding lectin 2 (MBL2) functional polymorphisms in the prevalence of hypertension and hypertensive end-organ damage in 300 hypertensive patients and 313 normotensive individuals from Southern Brazil. Hypertensive subjects with MBL2 AO/OO genotypes presented lower C-reactive protein levels than AA individuals and consequently lower inflammatory status.

A torque removal study on the primary stability of orthodontic titanium screw mini-implants in the cortical bone of dog femurs.

The aim of this study was to biomechanically evaluate the primary stability of pure titanium orthodontic mini-implants, inserted into pre-drilled cavities of differing diameters. Mini-implants (1.2 mm diameter) were placed into 1.0 mm and 1.2 mm diameter cavities prepared in the mid-region of the bilateral hind leg femurs of anesthetized beagles. Removal torque strengths were measured immediately, 1, 3, 6, 9 and 12 weeks post-insertion of the implant. For mini-implants placed into 1-mm cavities, removal torque values decrease over the first 6 weeks (p<0.01), after which values remained static. Average values obtained immediately, 1, 3 and 6 weeks post-insertion were 10.98, 8.83, 7.20 and 5.12 Ncm, respectively . Immediately post-insertion, removal torque values of mini-implants placed in a 1.2-mm cavity, were 11-fold lower than those placed in 1.0-mm cavities, which then demonstrated a significant increase in strength from 3 weeks (1.35 Ncm) to 6 weeks (5.17 Ncm) post-insertion (p<0.01). Measurements 6, 9 and 12 weeks post-insertion were similar to those in the 1.0-mm cavity. Initial stability of titanium mini-implants is considered necessary for immediate and early use in orthodontics, and an implant without this initial stability should be replaced or isolated until it develops the appropriate stability supported by osseointegration.

Multicenter study of QuantiFERON®-TB Gold Plus in patients with active tuberculosis.

SETTING: QuantiFERON®-TB Gold Plus (QFT-Plus), recently approved for use in the United States, is a new-generation QuantiFERON assay that differs from its predecessors in that it uses an additional antigen tube containing peptides to elicit both CD8+ and CD4+ T-lymphocyte responses. OBJECTIVE: To assess the sensitivity of QFT-Plus compared with QuantiFERON®-TB Gold In-Tube (QFT-GIT) in participants with active TB. DESIGN: Adult patients with active TB at three US and two Japanese sites were eligible for this study if they had culture-confirmed TB and were either untreated or had received 14 days of anti-tuberculosis treatment. RESULTS: We enrolled 164 participants, nine of whom had indeterminate results. Excluding indeterminate values, there were 150 QFT-GIT-positive results among 159 tests and 146 QFT-Plus-positive results among 157 tests, with sensitivities of respectively 94.3% (95%CI 89.5-97.4) and 93.02% (95%CI 87.8-96.5%). The estimated sensitivities for the two tests were not significantly different (P = 0.16). Overall test agreement was 98.7%, with a κ statistic of 0.89 (95%CI 0.75-1.00). CONCLUSION: In this multisite study, we found that QFT-Plus had similar sensitivity to QFT-GIT in adult patients with active TB.

Steroid-resistant asthma. Cellular mechanisms contributing to inadequate response to glucocorticoid therapy.

The current study examined whether alterations in glucocorticoid receptor (GR) binding contribute to poor response to glucocorticoid therapy in asthma. 29 asthma patients with forced expiratory volume in 1 s (FEV1) < 70% predicted were studied. Patients were classified as steroid sensitive (SS) if their morning FEV1 increased > 30% after a 1-wk course of oral prednisone 20 mg twice daily and steroid resistant (SR) if they failed to increase > 15%. PBMC obtained from these two groups, 17 SR and 12 SS, as well as 12 normal controls were analyzed. SR patients had two distinguishable GR binding abnormalities: 15 of the 17 SR patients demonstrated a significantly reduced GR binding affinity, as compared with SS patients (P = 0.0001) and normal controls (P = 0.0001). This defect was localized to T cells and reverted to normal after 48 h in culture media. However, incubation with a combination of IL-2 and IL-4 sustained this abnormality. The other two SR patients had an abnormally low GR number with normal binding affinity that was not limited to T cells. Furthermore, GR number failed to normalize after incubation in media alone or IL-2 and IL-4. Therefore, SR asthma may be due to more than one abnormality, the majority related to a reversible cytokine-induced reduction in GR binding affinity and the second related to an irreversible reduction in GR number. These findings may have important implications for the design of alternative treatment approaches for recalcitrant asthma.

Lysophosphatidic acid positively regulates the fluid flow-induced local Ca(2+) influx in bovine aortic endothelial cells.

Using real-time confocal microscopy, we have demonstrated that lysophosphatidic acid (LPA), a bioactive phospholipid existing in plasma, positively regulates fluid flow-induced [Ca(2+)](i) response in fluo 4-loaded, cultured, bovine aortic endothelial cells. The initial increase in [Ca(2+)](i) was localized to a circular area with a diameter of <4 microm and spread concentrically, resulting in a mean global increase in [Ca(2+)](i). The local increase often occurred in a stepwise manner or repetitively during constant flow. The percentage of cells that responded and the averaged level of increase in [Ca(2+)](i) were dependent on both the concentration of LPA (0.1 to 10 micromol/L) and the flow rate (25 to 250 mm/s). The response was inhibited by removing extracellular Ca(2+) or by the application of Gd(3+), an inhibitor of mechanosensitive (MS) channels, but not by thapsigargin, an inhibitor of the endoplasmic reticular Ca(2+)-ATPASE: It was also inhibited by 8-bromo-cGMP, and the inhibition was completely reversed by KT5823, an inhibitor of protein kinase G (PKG). These results suggest that the [Ca(2+)](i) response arises from Ca(2+) influx through Gd(3+)-sensitive MS channels, which are negatively regulated by the activation of PKG. The spatiotemporal properties of the [Ca(2+)](i) response were completely different from those of a Ca(2+) wave induced by ATP, a Ca(2+)-mobilizing agonist. Therefore, we called the phenomenon Ca(2+) spots. We conclude that LPA positively regulates fluid flow-induced local and oscillatory [Ca(2+)](i) increase, ie, the Ca(2+) spots, in endothelial cells via the activation of elementary Ca(2+) influx through PKG-regulating MS channels. This indicates an important role for LPA as an endogenous factor in fluid flow-induced endothelial function.

Enhanced intestinal absorption of insulin in rats in the presence of sodium 5-methoxysalicylate.

Sodium 5-methoxysalicylate, previously shown to enhance the rectal absorption of several drugs, facilitates the absorption of insulin from the upper gastrointestinal tract, resulting in significantly elevated insulin levels and lowered glucose concentrations in the plasma of rats. Restricting the movement of insulin and adjuvant down the intestine by either ligation or use of a more viscous vehicle further increased the absorption of insulin.

Adsorption of glycosaminoglycans onto hydroxyapatite using chromatography.

Proteoglycans are known to play an important role in the mineralization process, acting either as promoters or inhibitors. In this study the binding affinity of a variety of constituent glycosaminoglycan to hydroxyapatite was studied. Glycosaminoglycans (10-1000 microg ml(-1) in 0.02 M sodium acetate (pH 6.8) were constantly circulated through a hydroxyapatite column for 1 h. The total amount of glycosaminoglycan bound was determined by dimethylmethylene blue assay. The relative affinities of the different glycosaminoglycans remaining bound to hydroxyapatite was investigated by examining their release in a 0-1 M sodium phosphate gradient. Differences were noted between the desorption profiles of dermatan sulfate with two elution peaks and chondroitin 4-sulfate and chondroitin 6-sulfate each with a single peak. Dermatan sulfate and chondroitin 6-sulfate had a higher affinity for hydroxyapatite than chondroitin 4-sulfate possibly due to the presence of differing di-sulfated disaccharide ratios in the glycosaminoglycan chains. These findings suggest the presence of a variety of binding forms of each glycosaminoglycan or the differing orientation of these forms to yield different complexes with hydroxyapatite. The Ca2+ co-ordinates of the glycosaminoglycans are known to vary and may in part explain these findings.

Effect of low-dose troleandomycin on theophylline clearance: implications for therapeutic drug monitoring.

Troleandomycin (TAO) is an alternative agent used in the treatment of severe, steroid-requiring asthma. Its mechanism of action, once thought to be inhibition of theophylline clearance, remains unclear. Twenty-four-hour theophylline profiles were obtained in 11 children with severe asthma prior to and after 2 and 12 weeks of low-dose TAO therapy. Theophylline dosages were adjusted by blinded investigators to maintain serum theophylline concentrations (STCs) between 10 and 20 micrograms/ml. Dosages were decreased from 877 +/- 60 mg/day (mean +/- SEM) before TAO to 811 +/- 56 mg/day (NS) after 2 weeks and 764 +/- 56 mg/day (p less than 0.05) after 12 weeks. Because of the dosage adjustments, STCs did not increase significantly. Theophylline clearance was reduced from 65.7 +/- 9.8 ml/kg/hour at baseline to 50.2 +/- 4.1 ml/kg/hour (p less than 0.05) after 2 weeks and 50.1 +/- 6.2 ml/kg/hour (p less than 0.05) after 12 weeks of TAO therapy. We conclude that TAO can significantly reduce theophylline clearance, resulting in increased STCs if dosages are not titrated. We recommend an empiric 25% reduction of daily theophylline dose with the initiation of TAO. We also recommend monitoring STCs 4 hours after the morning dose (with twice-daily dosing of sustained-release products) after 3, 7, 14, and 30 days of TAO therapy, then periodically as indicated.

A pharmacokinetic comparison of two oral liquid glucocorticoid formulations.

To compare pharmacokinetics of liquid prednisolone and prednisone solutions and to assess relative bioavailability, six healthy adult men were administered 15 mg of each formulation. Blood samples were obtained and assayed for plasma prednisolone concentrations by high-performance liquid chromatography. Peak concentration was significantly higher with liquid prednisolone (mean +/- SD 430.3 +/- 62.5 vs 333.0 +/- 27.8 ng/ml, p = 0.013), with similar times to peak concentration. Prednisolone liquid gave higher concentrations at every time point (statistically significant for all except 0.25 hrs after the dose), resulting in a significantly greater total area under the curve (2029.8 +/- 246.9 vs 1633.3 +/- 221.1 ng/ml.hour, respectively, p = 0.002). Clearance was slower for prednisolone (128.3 +/- 15.1 vs 149.1 +/- 17.6 ml/min/1.73 m2, p = 0.01), and the relative bioavailability of the prednisolone liquid using prednisone liquid as the reference standard was 116 +/- 14%. Thus, prednisolone liquid has similar pharmacokinetic characteristics as prednisone liquid, with improved bioavailability.

Salicylate-promoted permeation of cefoxitin, insulin and phenylalanine across red cell membrane. Possible mechanism.

Uptake of sodium cefoxitin, D-phenylalanine and insulin into human red blood cells was significantly enhanced by the presence of salicylate and 5-methoxysalicylate in the medium. The mechanism of adjuvant action appeared to depend on an affinity between the adjuvant and the protein fraction in the erythrocyte membrane. The inhibitory effect of DIDS and phlorizin on the salicylate-enhanced uptake of these compounds strongly suggests that the ability of salicylate to permeate the membrane may be essential for it to act as an adjuvant.

Possible mechanism of uptake for several compounds in ionized form through human erythrocyte membrane.

The mechanism underlying uptake of certain compounds in ionized form across human red blood cell membrane was examined. The ionized forms of salicylate, 5-methoxy-salicylate and phenylalanylphenylalanine were significantly taken up into the interior space of human red blood cells instead of remaining in the membrane. Inhibition of the uptake of these compounds by 4,4'-diisothiocyano-2,2'-disulfonate stilbene and phlorizin indicates that their permeation of the erythrocyte membrane may involve the membrane protein fraction. Chelation at the protein site does not appear to occur. Instead, an amino group in the protein structure may mediate the transport of these ionized compounds.

Steroid resistance in asthma: our current understanding.

While much information has recently been obtained regarding the features of steroid-resistant asthma, it continues to be a dilemma for practitioners, and investigation into its mechanisms will remain an important part of asthma research. Until a clear marker defining steroid-resistant asthmatics is found, the principle first put forth by Carmichael and colleagues should be adhered to: that is, asthmatics resistant to glucocorticoid therapy need to be identified at an early stage so that unnecessary and perhaps harmful therapy can be discontinued. A 10 day course of high-dose (> or = 30 mg/day) systemic glucocorticoid therapy, as suggested by Kamada and colleagues, may constitute an adequate trial and may sufficiently identify asthmatics who may require alternative treatments. A more rational approach to the selection of alternative asthma treatments will be gained when the mechanisms of steroid resistance are identified.

Combined effect of alcohol and urea on the in vitro transport of indomethacin across rat dorsal skin.

An aqueous gel, prepared with hydrogenated soya phospholipid, increased the in vitro transport of indomethacin across rat dorsal skin. The addition of various alkanols further accelerated the transport, with an increasing effect as the chain length of the alkanol increased. The addition of urea alone did not significantly affect the transport of indomethacin. However, the addition of urea markedly accelerated the transport of indomethacin when included in an aqueous gel containing an alkanol such as 1-octanol, 1-decanol, or 1-dodecanol. Thus, it appears that a combination of urea and these alkanols strongly enhances the transdermal absorption of indomethacin. Urea appears to accelerate enhanced drug transport into the stratum corneum by a mechanism involving the transport of urea enhanced by these alkanols.

Adjuvant effects of glyceryl esters of acetoacetic acid on rectal absorption of insulin and inulin in rabbits.

The promoting effect of glyceryl esters of acetoacetic acid on the rectal absorption of insulin and inulin was studied. A decrease in the serum glucose level was observed in rabbits following the administration of an insulin suppository containing glyceryl-1,3-diacetoacetate (adjuvant II) or 1,2-isopropylideneglycerine-3-acetoacetate (adjuvant IV). The promoting effects of adjuvants II and IV on the rectal absorption of insulin and inulin were suppressed by the addition of calcium and magnesium to the suppository. This indicates that adjuvant interaction with the calcium and magnesium ion located in the rectal membrane is involved in the enhanced absorption of insulin and inulin. Adjuvant release from the suppository formulation in addition to adjuvant lipid solubility were found to be other important factors for enhanced absorption of insulin and inulin.

Disaccharide analysis of chondroitin sulphate in human gingival crevicular fluid using high-performance liquid chromatography.

Gingival crevicular fluid (GCF) was collected into capillary tubes from healthy gingiva and sites of advanced periodontitis. Following digestion with Pronase E, the glycosaminoglycans were isolated by successive precipitation into 5% cetylpyridinium chloride and 95% ethanol. Unsaturated disaccharide isomers of chondroitin sulphate, obtained following chondroitinase ACII digestion, were analysed by high-performance liquid chromatography. Chondroitin sulphate was found in all GCF samples, with greater amounts in patients with periodontal disease than at control sites with a relatively healthy periodontium. The predominant isomer in the periodontal diseased group was delta Di-4S, while that in the control group and serum samples was delta Di-0S. Comparison of the relative proportions of the unsaturated disaccharides in GCF with previously reported values for alveolar bone, cementum, gingiva and periodontal ligament, as well as for serum, indicates that the chondroitin sulphate present in GCF of patients with periodontal disease originated from the mineralized connective tissues of the periodontium, notably alveolar bone, possibly with some contributions from soft connective tissues of gingiva and periodontal ligament and from serum.

Characteristics and localization of rat submandibular gland proteoglycans.

The submandibular gland proteoglycans were investigated biochemically and immunohistochemically in male Sprague-Dawley rats. Proteoglycans were extracted with 4 M guanidine-HCl, followed by ultracentrifugation in a CsCl density gradient, and fractionated by ion-exchange chromatography and gel filtration. The molecular weight of PGs was estimated by SDS-PAGE and immunoblot analysis with monoclonal antibodies (HepSS-1 or 6-B-6). The glycosaminoglycan side-chains in the proteoglycan fractions were identified by electrophoresis on cellulose acetate membrane. Three proteoglycan fractions were obtained. One was a heparan sulphate proteoglycan that migrated as a diffuse band of about 210 kDa. The other two fractions contained at least two dermatan sulphate proteoglycans of 70-85 kDa and 40-50 kDa. Digestion of these two proteoglycans with chondroitinase ABC, but not heparitinase, produced two bands of 50 and 21 kDa, which were core proteins. The smaller dermatan sulphate proteoglycan may be a portion of the other, as the core protein of both bound to 6-B-6 antibody, and sugar chains of both were the same (20-30 kDa). Heparan sulphates recognized by antibody HepSS-1 were observed widely in the basement membrane, fibrous connective tissue, and striated and excretory ductal cells, while dermatan sulphate proteoglycans recognized by antibody 6-B-6 were located in the connective tissue surrounding striated and excretory ducts.

Studies on orally active cephalosporins. I. Synthesis and structure-activity relationships of new 3-substituted carbamoyloxymethyl cephalosporins.

The synthesis and antibacterial activities of 7 beta-[2-(2-aminothiazol-4-yl)-2-hydroxyiminoacetamido]-3-N,N - dimethylcarbamoyloxymethyl-3-cephem-4-carboxylic acid (E1100) and its analogs are described, as well as oral absorbability and in vivo activities of the 1-(isopropoxycarbonyloxy)ethyl ester (E1101) and its analogous esters. The introduction of acyclic and cyclic lower alkyl groups at the N-position of 3-carbamoyloxymethyl cephems influences antibacterial activities, especially against H. influenzae, and oral absorbability of their prodrug esters. The structure-activity relationships are also discussed.

Studies on orally active cephalosporins. II. Synthesis and structure-activity relations of new [(E) or (Z) 3-substituted carbamoyloxy]-1-propenyl cephalosporins.

In an effort to find a new oral cephalosporin with well-balanced antibacterial spectrum, good oral absorbability and long plasma half-life, a series of oxyimino aminothiazolyl 3-[(E)- or (Z)-N-substituted carbamoyloxy]propenyl cephems was synthesized and evaluated for antibacterial activity and oral absorbability. The substituents of the carbamoyloxy group affected their in vitro activity and bioavailability after oral administration of their pivaloyloxymethyl esters at the C-4 position. The compound possessing an N,N-dimethylcarbamoyloxy moiety at the C-3 position showed good oral absorption and well-balanced antibacterial activity. In this report, the structure-activity relationships and the structure-oral absorbability relationships of 3-(N-substituted carbamoyloxy)-propenyl cephems are described.

Enhancement of nasal absorption of insulin and calcitonin using polyacrylic acid gel.

The effects of polyacrylic acid gel on the nasal absorption of insulin and [Asu1,7]-eel calcitonin were investigated in rats. The nasal administration of insulin (1 IU kg-1) in polyacrylic acid gel at 0.1 and 1% w/v showed maximum hypoglycaemic effects at 30 min and 1 h after administration, respectively. However, the nasal administration of insulin in carboxymethyl cellulose (1% w/v) solution had no hypoglycaemic effect at the same dose. When [Asu1,7]-eel calcitonin (10 U kg-1) was administered nasally in polyacrylic acid gel (0.1% w/v), a prominent hypocalcaemic effect was observed during the first 30 min. Nasal administration of [Asu1,7]-eel calcitonin in saline had no hypocalcaemic effect at the same dose. The results indicate that the polyacrylic acid gel base significantly enhanced the absorption of insulin and [Asu1,7]-eel calcitonin via the nasal cavity.

Effect of enamine derivatives on the rectal absorption of insulin in dogs and rabbits.

Enamine derivatives resulting from the reaction between ethyl acetoacetate and amino acids were found to promote rectal absorption of insulin in normal rabbits, with the enamine of sodium DL-phenylalanate being the most effective adjuvant tested. Results of glucose tolerance tests in fasted alloxan diabetic rabbits showed that after oral administration of glucose, insulin suppositories containing enamines derived from either sodium phenylalanine or leucine were able to lower the serum glucose level to within the normal range for more than 2.5 h. The absorption-promoting effect of the sodium DL-phenylalanate enamine on rectal absorption of insulin in dogs was studied in two different formulations, gelatin microenema and suppository. The gelatin microenema containing the insulin solution caused a greater increase in the plasma insulin level than that obtained after administration of an insulin suppository.