Aliskiren, a novel renin inhibitor, is renoprotective in a model of advanced diabetic nephropathy in rats.

AIMS/HYPOTHESIS: Blockade of the renin-angiotensin system (RAS) with either ACE inhibitors or angiotensin receptor blocker is a key therapeutic strategy in slowing progression of diabetic nephropathy. Interruption of the RAS may also be achieved by blocking the activity of renin, the rate-limiting step in angiotensin II biosynthesis. However, it is not known whether drugs in this class also reduce the structural and functional manifestations of diabetic nephropathy. METHODS: Using diabetic transgenic (mRen-2)27 rats, a rodent model of advanced diabetic nephropathy, we compared the efficacy of the renin inhibitor, aliskiren (10 mg kg(-1) day(-1) by osmotic mini-pump), with the ACE inhibitor, perindopril (0.2 mg kg(-1) day(-1) in drinking water), over a 16 week period. RESULTS: Both perindopril and aliskiren reduced blood pressure, albuminuria and structural injury in experimental diabetic nephropathy, although not to the same extent. Aliskiren, at the dose used, did not reduce systemic blood pressure as much as perindopril, but both compounds were equally effective in reducing albuminuria and glomerulosclerosis in diabetic animals. The magnitude of interstitial fibrosis was attenuated to a greater degree by aliskiren than by perindopril. CONCLUSIONS/INTERPRETATION: These findings suggest that therapies aimed at different targets within the RAS may not have identical effects in attenuating structural injury in experimental diabetic nephropathy.

A novel mimetic antigen eliciting protective antibody to Neisseria meningitidis.

Molecular mimetic Ags are of considerable interest as vaccine candidates. Yet there are few examples of mimetic Ags that elicit protective Ab against a pathogen, and the functional activity of anti-mimetic Abs has not been studied in detail. As part of the Neisseria meningitidis serogroup B genome sequencing project, a large number of novel proteins were identified. Herein, we provide evidence that genome-derived Ag 33 (GNA33), a lipoprotein with homology to Escherichia coli murein transglycosylase, elicits protective Ab to meningococci as a result of mimicking an epitope on loop 4 of porin A (PorA) in strains with serosubtype P1.2. Epitope mapping of a bactericidal anti-GNA33 mAb using overlapping peptides shows that the mAb recognizes peptides from GNA33 and PorA that share a QTP sequence that is necessary but not sufficient for binding. By flow cytometry, mouse antisera prepared against rGNA33 and the anti-GNA33 mAb bind as well as an anti-PorA P1.2 mAb to the surface of eight of nine N. meningitidis serogroup B strains tested with the P1.2 serosubtype. Anti-GNA33 Abs also are bactericidal for most P1.2 strains and, for susceptible strains, the activity of an anti-GNA33 mAb is similar to that of an anticapsular mAb but less active than an anti-P1.2 mAb. Anti-GNA Abs also confer passive protection against bacteremia in infant rats challenged with P1.2 strains. Thus, GNA33 represents one of the most effective immunogenic mimetics yet described. These results demonstrate that molecular mimetics have potential as meningococcal vaccine candidates.

Abnormal cardiac and skeletal muscle mitochondrial function in pacing-induced cardiac failure.

BACKGROUND: Previous studies have shown that marked changes in myocardial mitochondrial structure and function occur in human cardiac failure. To further understand the cellular events and to clarify their role in the pathology of cardiac failure, we have examined mitochondrial enzymatic function and peptide content, and mitochondrial DNA (mtDNA) integrity in a canine model of pacing-induced cardiac failure. METHODS: Myocardium and skeletal muscle tissues were evaluated for levels of respiratory complex I-V and citrate synthase activities, large-scale mtDNA deletions as well as peptide content of specific mitochondrial enzyme subunits. Levels of circulating and cardiac tumor necrosis factor-alpha (TNF-alpha), and of total aldehyde content in left ventricle were also assessed. RESULTS: Specific activity levels of complex III and V were significantly lower in both myocardial and skeletal muscle tissues of paced animals compared to controls. In contrast, activity levels of complex I, II, IV and citrate synthase were unchanged, as was the peptide content of specific mitochondrial enzyme subunits. Large-scale mtDNA deletions were found to be more likely present in myocardial tissue of paced as compared to control animals, albeit at a relatively low proportion of mtDNA molecules (<0.01% of wild-type). In addition, the reduction in complex III and V activities was correlated with elevated plasma and cardiac TNF-alpha levels. Significant increases in left ventricle aldehyde levels were also found. CONCLUSIONS: Our data show reductions in specific mitochondrial respiratory enzyme activities in pacing-induced heart failure which is not likely due to overall decreases in mitochondrial number, or necrosis. Our findings suggest a role for mitochondrial dysfunction in the pathogenesis of cardiac failure and may indicate a commonality in the signaling for pacing-induced mitochondrial dysfunction in myocardial and skeletal muscle. Increased levels of TNF-alpha and oxidative stress appear to play a contributory role.

Carvedilol in the treatment of chronic heart failure.

Along with the angiotensin-converting enzyme inhibitors (ACEIs), the beta-adrenergic receptor blockers have gradually emerged to be standard in the therapy of heart failure. Individual beta-blockers that have been shown to reduce all-cause mortality in patients with heart failure include bisoprolol, metoprolol and carvedilol. Carvedilol distinguishes from the other beta-blockers as being a non-selective beta(1)- and beta(2)-receptor blocker with (1)-receptor blockade effect and anti-oxidant properties. The drug does not have sympathomimetic activity and has vasodilatory effects attributable to its (1)-receptor blockade property. Experimental and clinical studies have confirmed carvedilol's vasodilator, anti-oxidant and anti-apoptotic properties, which may contribute to its effect in reversing cardiac remodelling in animal models and patients with heart failure. These pharmacological properties render carvedilol a potentially useful agent in the treatment of patients with heart failure. Early studies of carvedilol in heart failure have reported beneficial haemodynamic effects but variable effects on exercise tolerance and clinical well being. The large-scale US Carvedilol Heart Failure Program and the Australian/New Zealand Heart Failure Collaborative Research Group reported beneficial effects of carvedilol on mortality, morbidity and clinical well being in patients with mild-to-moderate heart failure. The recently reported but yet unpublished preliminary results of the COPERNICUS study suggest that carvedilol improves mortality and morbidity in patients with advanced heart failure and severe symptoms. At this time, it is unclear whether the ancillary pharmacological properties of carvedilol can be translated to more superior clinical benefit compared to the other beta-blockers. Preliminary studies examining surrogate end points suggest that carvedilol may improve left ventricular ejection fraction (LVEF) more than metoprolol. More conclusive information regarding their relative effects of clinical outcomes will await the completion of the COMET study, which compares the effect of metoprolol and carvedilol on mortality and morbidity, expected at the end of the year 2002.

Functional activity of anti-Neisserial surface protein A monoclonal antibodies against strains of Neisseria meningitidis serogroup B.

Neisserial surface protein A (NspA) is currently being investigated with humans as a candidate vaccine for the prevention of meningococcal disease. Although NspA is highly conserved, the ability of anti-NspA antibodies to bind to or elicit complement-mediated bactericidal activity against diverse Neisseria meningitidis serogroup B strains is controversial. To evaluate strain differences in NspA surface accessibility and susceptibility to bactericidal activity, we prepared murine immunoglobulin G2a anti-NspA monoclonal antibodies (MAbs) and evaluated their functional activity against 10 genetically diverse N. meningitidis serogroup B strains. By colony Western blot, all 10 strains expressed NspA as detected by one or more MAbs. By flow cytometry, two MAbs were found to bind to the bacterial surface of 6 of the 10 strains. In addition, two strains showed variable NspA surface accessibility for the MAbs despite being uniformly positive for NspA expression by colony Western blotting. Only 4 of the 10 strains were susceptible to anti-NspA complement-mediated bacteriolysis. Passively administered MAb protected infant rats from developing bacteremia after challenge with N. meningitidis serogroup B strain 8047 (surface binding positive, susceptible to anti-NspA bacteriolysis), was poorly protective against strain BZ232 (surface binding variable, resistant to bacteriolysis), and did not protect against strain M986 (surface binding negative, resistant to bacteriolysis). Finally, NspA does not appear to be critical for causing bacteremia, as an NspA knockout from strain 8047 was highly virulent in infant rats. Taken together, these findings suggest that an NspA-based vaccine will need to incorporate additional antigens to elicit broad protection against N. meningitidis serogroup B.

Specific and non-specific measurements of tissue angiotensin II cascade members.

Although angiotensin II (ANG II) has been the focal regulatory peptide of the renin-angiotensin system, its proteolytic fragments have recently been demonstrated to have biological effects. Conventional measurement of angiotensins involves radioimmunoassay (RIA), which is a sensitive binding technique capable of measuring low physiological concentrations. However, ANG II antibody cross-reacts with ANG II and its fragments (ANG II cascade), rendering RIA measurement alone to be a non-specific measure of immunoreactive ANG II (ir-ANG II). On the other hand, high-performance liquid chromatography (HPLC) is capable of separating immunoreactive ANG II cascade members, but may not be sensitive enough to detect these low peptide concentrations often present in biological samples. Consequently, a reverse-phase HPLC method, with triethylammoniun formate as an ion-pair reagent, was developed to separate ANG II and its fragments, ANG III, ANG IV and ANG V. This HPLC separation was applied to extracts from normal canine hearts and ANG II cascade immunoreactive fractions were collected. Collected fractions were quantified by RIA, with the use of separate standard curves. The isocratic HPLC separation of ANG II, ANG III, ANG IV and ANG V was achieved in less than 5 min with adjacent peaks having baseline resolution. Measured cardiac left ventricle ANG III, ANG IV and ANG V concentrations (mean+/-SD) were 5.3+/-2.2,4.0+/-1.0 and 3.1+/-1.0 fmol/g (n=9), respectively. There was a significant difference (P=0.003, n=9) between left ventricular immunoreactive ANG II and 'true' ANG II, corrected for recovery rates of 86.2+/-22.5 and 53.5+/-16.2 fmol/g, respectively. We conclude that the combination of HPLC with RIA ensures the specific measurement of the ANG II cascade family members while non-chromatographic processing of tissue renders ANG II measurement non-specific. In addition, the use of triethylammonium formate as mobile phase additive is superior in the HPLC separation of the angiotensins.

Molecular mimetics of Neisseria meningitidis serogroup B polysaccharide.

Strains of Neisseria meningitidis serogroup B (NmB) are an important cause of meningitis and sepsis. Efforts to develop a NmB vaccine have been hampered by poor immunogenicity of the polysaccharide capsule, which cross-reacts with host polysialic acid, and the danger of eliciting autoantibodies. To investigate the potential of molecular mimetics to circumvent these problems, we prepared murine monoclonal antibodies (mAbs) against the N-propionyl derivative (N-Pr) of NmB polysaccharide. Several mAbs were found that reacted with capsular polysaccharide epitopes, which were distinct from host polysialic acid. These mAbs also passively conferred protection against experimental bacteremia. We used these mAbs to screen novel independently folding peptide phage display libraries, and pools of combinatorial small molecules, each consisting of approximately 30 to approximately 700 small molecules of diverse composition. To date, several mimetic candidates have been identified. One is a peptide selected from a library of independently folding alphabeta peptides, and others are peptoid dimers or trimers selected from the small molecule pools. The peptoids contain an indan-type of ring system, and some of them also contain a large hydrophobic group such as oleyl amine or dehydroabietyl amine, and a positively charged group at the amino-terminus. Both the alphabeta peptide from the phage library, and the peptoids from the small molecule pools, inhibit binding of the mAbs to N-Pr NmB polysaccharide. Future studies will focus on the structure/activity relationship of these mimetics, and the development of immunogens that may be capable of eliciting anticapsular antibody without autoantibody activity.

Waist-to-hip ratio, weight gain, and dietary and serum selenium are associated with DNA content flow cytometry in Barrett's esophagus.

This cross-sectional study reports associations between anthropometric measures, serum antioxidant concentrations, and present diet with measures of elevated cell proliferation in 51 patients with Barrett's esophagus. Cell proliferation was assessed as fractions of cells in the S and G2 phases, measured in biopsies of Barrett's tissue and analyzed by DNA content flow cytometry. Elevated proportions in the S and G2 phases predict progression to adenocarcinoma. The percentage of cells in the S phase was positively associated with waist-to-hip ratio (r = 0.33, p < 0.05) and negatively associated with serum and dietary selenium (r = -0.34 and -0.32, respectively, p < 0.05). The percentage of cells in the G2 phase was positively associated with weight change from age 25 (r = 0.39, p < 0.01) and negatively associated with dietary selenium (r = -0.31, p < 0.05). Selenium from breads and grains was negatively associated with the percentage of cells in the S phase (r = -0.41, p < 0.01) and the percentage of cells in the G2 phase (r = -0.41, p < 0.01). These results suggest that increasing weight gain in adulthood, increasing waist-to-hip ratio, and decreasing dietary selenium intake and serum levels increase the risk of progression of Barrett's esophagus to adenocarcinoma.

Simultaneous separation of angiotensin and endothelin peptide families by high-performance liquid chromatography: application to the specific radioimmunoassay measurement of angiotensin II or endothelin-1 from tissue.

Currently available measurements of endogenous angiotensin II (ANG II) and endothelin-1 (ET-1) concentrations by radioimmunoassay (RIA) lack specificity to ANG II or ET-1. ANG II and ET-1 antibodies cross-react with immuno-reactive angiotensin and endothelin family members, respectively. We have therefore developed an ion-pair reversed-phase high-performance liquid chromatography (HPLC) for simultaneously separating angiotensin and endothelin peptides and enhancing RIA specificity in the measurement of ANG II and ET-1. The developed HPLC separation was applied to canine myocardium extracts; ANG II or ET-1 fractions were collected and quantified by RIA. Elution times for both peptide families, ANG I, ANG II, ANG III, ANG IV, ANG II(4-8), bET-1, ET-1, ET-2 and ET-3 were within 25 min. In normal canine myocardium from the right atrium, right ventricle, left atrium and left ventricle, ANG II concentrations were 39+/-11, 28+/-21, 31+/-11 and 21+/-8 fmol/g and ET-1 concentrations were 43+/-16, 42+/-19, 55+/-21 and 57+/-34 fmol/g (mean+/-SD, N=7), respectively. The combination of HPLC with RIA renders the measurement of ANG II or ET-1 specific and convenient, and saves time. This HPLC separation may be applied to the specific measurement of other immuno-reactive angiotensin and endothelin peptides.

Neurohormonal activation in severe heart failure: relations to patient death and the effect of treatment with flosequinan.

BACKGROUND: Flosequinan is a direct-acting vasodilator that exerts beneficial hemodynamic effects and improves the exercise tolerance of patients with heart failure. However, a multicenter trial has demonstrated that long-term administration of flosequinan is associated with increased mortality rate. To explore a possible role of neurohormonal activation on this adverse outcome, we conducted a substudy to examine the plasma levels of 3 neurohormonal systems known to have prognostic implications in heart failure. METHODS: At 20 participating Canadian centers, paired plasma samples at baseline and 1 month after randomization for the measurement of N-terminal atrial natriuretic peptide (N-ANP), angiotensin II, and norepinephrine were obtained in 234 patients (114 receiving flosequinan and 120 receiving placebo). RESULTS: Treatment with flosequinan was associated with a decline in median plasma N-ANP levels (2139 pmol/L at baseline to 1625 pmol/L at 1 month [P =. 0001]), unchanged plasma angiotensin II levels (40 to 50 pmol/L [P =. 2700]), and a modest increase in plasma norepinephrine levels (391 to 439 pg/mL [P =.002]). These changes were not observed in the placebo group. Multivariate analysis of baseline variables revealed that plasma norepinephrine level predicted patients' death whereas analysis incorporating both baseline and 1-month variables indicated that plasma N-ANP level predicted patients' death. Furthermore, in the flosequinan group, a significant decline in plasma N-ANP level was observed in the survivors only. On multivariate analysis of baseline and 1-month data, the increase in plasma norepinephrine level did not predict the increase in heart rate associated with the use of flosequinan, suggesting that the 2 effects might be mediated by separate mechanisms. CONCLUSIONS: Results of our study demonstrate that in patients with severe heart failure, baseline norepinephrine level predicts death. Flosequinan increases plasma norepinephrine level and heart rate in these patients through mechanisms that override its beneficial hemodynamic effects. Our study reinforces the concept that the direct actions of a pharmacologic agent may have a more profound impact on the prognosis of these patients than the hemodynamic effects.

Molecular mimetics of polysaccharide epitopes as vaccine candidates for prevention of Neisseria meningitidis serogroup B disease.

Neisseria meningitidis is a major cause of meningitis and sepsis. Despite nearly 25 years of work, there is no promising vaccine candidate for prevention of disease caused by meningococcal B strains. This review summarizes newer approaches for eliciting protective meningococcal B immune responses, including the use of molecular mimetics of group B polysaccharide and conserved membrane proteins as immunogens. The capsular polysaccharide of this organism is conserved and serum antibody to this capsule confers protection against disease. However, the immunogenicity of meningococcal B polysaccharide-based vaccines is poor. Further, a portion of the antibody elicited has autoantibody activity. Recently, our laboratory produced a panel of murine monoclonal antibodies (Mabs) that react specifically with capsular polysaccharide epitopes on meningococcal B that are distinct from host polysialic acid. These Mabs elicit complement-mediated bactericidal activity and confer passive protection in animal models. The anti-capsular Mabs were used to identify molecular mimetics from phage display peptide libraries. The resulting peptides were antigenic mimetics as defined by binding to the Mabs used to select them but, to date, are poor immunogenic mimetics in failing to elicit anti-capsular antibodies.

ARCTIC: assessment of haemodynamic response in patients with congestive heart failure to telmisartan: a multicentre dose-ranging study in Canada.

BACKGROUND: The aim of this study was to examine the acute hemodynamic and neurohormonal effects of the angiotensin II antagonist telmisartan relative to placebo in patients with chronic symptomatic (New York Heart Association class II to III) congestive heart failure and to explore the dose-response relation for these effects. METHODS AND RESULTS: After baseline hemodynamic and neurohormonal measurements made with the use of a pulmonary artery and radial arterial catheter, 82 patients were randomly assigned to placebo or 10, 20, 40, or 80 mg of telmisartan in a double-blind fashion. Hemodynamic and neurohormonal measurements were carried out over 24 hours. Telmisartan caused significant decreases in systemic arterial, pulmonary arterial, and pulmonary capillary wedge pressures with evidence of a dose-response relation for each of these parameters. The drug had no significant effects on heart rate, cardiac index, or systemic vascular resistance. Telmisartan did not have consistent effects on either plasma norepinephrine or plasma atrial natriuretic peptide levels, although it did cause significant increases in both plasma renin activity and angiotensin II levels at higher doses. CONCLUSIONS: The acute administration of the angiotensin II antagonist telmisartan was associated with significant dose-dependent reductions in systemic arterial blood pressure and pulmonary pressures. Long-term follow-up studies are required to translate changes in hemodynamic parameters into a clinical benefit.

Organic leachables from polymer-based dental filling materials.

Results are reported from a study on the in vitro separation and identification of leachables from three different polymer-based dental filling materials by using a combined method of gas chromatography and mass spectrometry. The median number of separable organic leachables in these materials was between 14 and 22. Of these organic leachables the following were identified and quantified: DL-camphorquinone, 4-dimethylaminobenzoic acid ethyl ester (DMABEE), drometrizole, 1,7,7-trimethylbicyclo[2,2,1]heptane, 2,2-dimethoxy[1,2] diphenyletanone (DMBZ), ethyleneglycol dimethacrylate (EGDMA), and triethyleneglycol dimethacrylate (TEGDMA). Three of the leachables have previously been shown to provoke allergy. The range of log P(ow) values (representing the lipophilicity of these compounds) varied between 1.09 and 4.20. By multivariate data analysis, selected leachables from the tested materials were shown to separate into characteristic patterns. The results contribute to a characterization of potential hazardous compounds in polymer-based dental filling materials.

Differences in surface expression of NspA among Neisseria meningitidis group B strains.

NspA is a highly conserved membrane protein that is reported to elicit protective antibody responses against Neisseria meningitidis serogroups A, B and C in mice (D. Martin, N. Cadieux, J. Hanel, and B. R. Brodeur, J. Exp. Med. 185:1173-1183, 1997). To investigate the vaccine potential of NspA, we produced mouse anti-recombinant NspA (rNspA) antisera, which were used to evaluate the accessibility of NspA epitopes on the surface of different serogroup B strains by an immunofluorescence flow cytometric assay and by susceptibility to antibody-dependent, complement-mediated bacteriolysis. Among 17 genetically diverse strains tested, 11 (65%) were positive for NspA cell surface epitopes and 6 (35%) were negative. All six negative strains also were resistant to bactericidal activity induced by the anti-rNspA antiserum. In contrast, of the 11 NspA surface-positive strains, 8 (73%; P < 0.05) were killed by the antiserum and complement. In infant rats challenged with one of these eight strains, the anti-rNspA antiserum conferred protection against bacteremia, whereas the antiserum failed to protect rats challenged by one of the six NspA cell surface-negative strains. Neither NspA expression nor protein sequence accounted for differences in NspA surface accessibility, since all six negative strains expressed NspA in outer membrane preparations and since their predicted NspA amino acid sequences were 99 to 100% identical to those of three representative positive strains. However, the six NspA cell surface-negative strains produced, on average, larger amounts of group B polysaccharide than did the 11 positive strains (reciprocal geometric mean titers, 676 and 224, respectively; P < 0.05), which suggests that the capsule may limit the accessibility of NspA surface epitopes. Given these strain differences in NspA surface accessibility, an rNspA-based meningococcal B vaccine may have to be supplemented by additional antigens.

In vitro lymphocyte reactivity to gold compounds in the diagnosis of contact hypersensitivity.

The use of the lymphocyte transformation test (LTT) in the diagnosis of contact hypersensitivity to gold was studied in 8 patients who had positive patch tests to gold salts, and in 8 control subjects who were negative to such patch tests. Gold sodium thiosulfate and gold chloride were added to cultures of lymphocytes, which were labeled by 3H-thymidine after 96 h. The lymphocyte stimulation index was calculated as the beta-counts in stimulated cultures divided by those in control cultures. The index was statistically significantly higher for the patient group (p=0.005-0.04) than for the control group. Levels of interferon-gamma (IFN-gamma) were determined for the supernatants of the lymphocyte cultures. An index IFN-gamma, which is defined as the level of IFN-gamma in stimulated cultures divided by that in control cultures, was statistically significantly higher for the patient group (p=0.01-0.006). The LTT stimulation index showed specificity and sensitivity between 67 and 80%, the respective values for Index IFN-gamma being between 73 and 100% when the patch test was used as a reference method. Evaluation of lymphocyte reactivity might be of future interest in the diagnosis of allergic reactions to gold if the sensitivity and specificity can be improved.