Determination of IgG subclass antibodies to Pseudomonas aeruginosa outer membrane proteins in cystic fibrosis lung infection using immunoblotting and enzyme-linked immunosorbent assay

IgG subclass antibodies to Pseudomonas aemginosa outer membrane proteins (OMP) were investigated in serum from cystic fibrosis (CF) patients by immunoblotting and enzyme-linked immunosorbent assay (ELISA). Fifteen patients (eight in good and seven in poor clinical condition) have been followed for an average of 13 years with multiple serum samples covering the preinfection, and early and late stages of chronic infection. Laser-scanning densitometry of photographs taken from immunoblots was used to quantify antibody level and compare with ELISA titres. The earliest anti-OMP antibodies to appear were of the IgG1 subclass. There was no significant difference in IgG subclass antibody levels to OMPs between patients in relatively good and poor clinical condition. Data presented indicate a high positive correlation among measurements of IgG subclass antibodies to P. aeruginosa OMPs using ELISA and immunoblotting.     

Characterization of the antibody response to a Haemophilus influenzae type b conjugate vaccine in children with recurrent lower respiratory tract infection

Children with recurrent lower respiratory tract infection (RLRI) may respond poorly to polysaccharide antigens. To examine how such children respond to a polysaccharide coupled to a protein carrier, we immunized 15 children with RLRI aged 8–69 months and 15 carefully age-matched healthy controls once with a Haemophilus influenzae type b (Hib) conjugate vaccine. Total IgG subclasses, total antipolysaccharide Hib antibodies, and antipolysaccharide Hib antibodies of IgM, IgG, IgA, and IgG 1–4 specificity were determined by ELISA. There were no significant differences between the two groups in any single total IgG subclass, but total IgG measured as the sum of all four subclasses was significantly lower in the children with RLRI than in the controls ( P = 0.036). Before vaccination, the children with RLRI had significantly less IgG antipolysaccharide Hib antibody than the controls ( P = 0.005), whereas 1 month later they had significantly more IgM antibody (P = 0.038). No other significant differences were found between the groups before or after immunization with respect to antipolysaccharide Hib antibodies. Since naturally occurring IgG antibodies are thought to be aquired partly as a consequence of antigenic stimulation on mucosal surfaces, we hypothesize that the low level of specific IgG found before immunization, as well as the low total IgG in the children with RLRI, may reflect an impaired ability to prime through mucosal surfaces. This is supported by our finding of an increased IgM response to Hib conjugate vaccine in these children, since this isotype predominates in the primary immune response, i.e., in the absence of immunologic memory. In conclusion, children with RLRI can be protected against invasive Hib infection as well as healthy children, but may have an immunodeficiency characterized by defective ability to respond to antigenic stimulation on mucosal surfaces.     

Methodologic problems in establishing normal values for IgG subclass concentrations in a pediatric population; comparison of radial immunodiffusion and ELISA methods

The aim of this study was to establish an enzyme-linked immunosorbent assay (ELISA) to measure IgG subclasses by means of monoclonal antibodies. The distribution of IgG subclass protein concentrations in sera from 227 healthy Danish children and 90 adults was measured. Furthermore, this newly established ELISA was compared with different assay systems for determination of IgG subclasses: two radial immunodiffusion methods (RID), one using polyclonal and one using monoclonal antibodies, as well as a commercially available ELISA kit. There was good agreement of results obtained by the different methods of measuring IgG3 and IgG4 concentrations. There was good correlation between results obtained by both RID methods. Despite good correlation between the assays, the ELISA kit showed higher levels of IgG1 in all investigated sera, and the ELISA kit showed no correlation with the other methods, when IgG2 was measured. Analysis of the normal ranges measured by ELISA developed in our laboratory and by RID with polyclonal antibodies showed that the levels obtained by RID were higher than those obtained by our ELISA in sera with low levels of both IgG1 and IgG2, and lower in sera with high concentrations of these two immunoglobulins. Our results emphasize the importance of establishing age-related normal limits for any novel assay measuring IgG subclass concentrations.     

Körperliches Training in der Therapie von Herzerkrankungen

Exercise training has been firmly established as an additional therapeutic strategy in addition to pharmacological and interventional treatment in patients with cardiovascular disease. Benefits for quality of life as well as prognosis have been confirmed for cardiovascular risk factors, ischemic heart disease, after myocardial infarction, in heart failure with preserved as well as reduced ejection fraction, in atrial fibrillation and in patients after catheter-assisted aortic valve implantation (TAVI), with an implantable cardioverter defibrillator (ICD) or with left ventricular assist devices (VAD). Training programs have to be tailored according to the disease, stage of disease, comorbidities, age of the patient, medication as well as exercise capacity. For prescribing exercise mode and intensity, a maximum exercise test has to be performed. Ideally, this is accompanied by spirometry to assess maximum values such as maximum oxygen consumption. Training intensity will then be prescribed according to the optimal training range and maximum training intensity.     

High levels of complement-activation capacity in sera from patients with cystic fibrosis correlate with high levels of IgG3 antibodies to Pseudomonas aeruginosa antigens and poor lung function

Heat-stable opsonins from sera of cystic fibrosis (CF) patients were investigated for their ability to activate complement. Complement activation by Pseudomonas aeruginosa after opsonization with patient serum was examined in a complement-consumption assay. Absorption of patients' sera with formalin-treated and boiled bacteria removed specific antibodies and the complement activation decreased. We found a positive correlation between serum complement-activation ability and IgG3 antibody levels to lipopolysaccharide (LPS), alginate, and a crude mixture of P. aeruginosa antigens (sonicate) in a group of patients with high levels of anti-Pseudomonas precipitins. In the same group of patients a significant negative correlation was found between complement activation and lung function. Eighteen patients have been followed longitudinally with serum samples covering the pre-infection, the early, and the late stages of chronic infection. Patients with poor lung function showed significantly higher levels of complement-activation capacity. We conclude that patients with high levels of specific IgG3 antibodies are able to induce a high level of complement activation and then develop more aggressive pulmonary tissue damage, probably secondary to local immune complex formation. Pediatr Pulmonol. 1995; 20:71–77 . © 1995 Wiley-Liss, Inc.     

Associations between Borg’s rating of perceived exertion and physiological measures of exercise intensity

Borg’s rating of perceived exertion (RPE) is a widely used psycho-physical tool to assess subjective perception of effort during exercise. We evaluated the association between Borg’s RPE and physiological exercise parameters in a very large population. In this cohort study, 2,560 Caucasian men and women [median age 28 (IQR 17–44) years] completed incremental exercise tests on treadmills or cycle ergometers. Heart rate, blood lactate concentration, and RPE (Borg scale 6–20) were simultaneously measured at the end of each work load. Rating of perceived exertion was strongly correlated with heart rate (r = 0.74, p < 0.001) and blood lactate (r = 0.83, p < 0.001). The mean values for lactate threshold (LT) and individual anaerobic threshold corresponded to an RPE of 10.8 ± 1.8 and 13.6 ± 1.8, respectively. Fixed lactate thresholds of 3 and 4 mmol/L corresponded to RPEs of 12.8 ± 2.1 and 14.1 ± 2.0. Gender, age, coronary artery disease (CAD), physical activity status and exercise testing modality did not influence this association significantly (all p > 0.05). Borg’s RPE seems to be an affordable, practical and valid tool for monitoring and prescribing exercise intensity, independent of gender, age, exercise modality, physical activity level and CAD status. Exercising at an RPE of 11–13 (“low”) is recommended for less trained individuals, and an RPE of 13–15 may be recommended when more intense but still aerobic training is desired.     

Vertical Sleeve Gastrectomy Restores Glucose Homeostasis in Apolipoprotein A-IV KO Mice

Bariatric surgery is the most successful strategy for treating obesity, yet the mechanisms for this success are not clearly understood. Clinical literature suggests that plasma levels of apolipoprotein A-IV (apoA-IV) rise with Roux-en-Y gastric bypass (RYGB). apoA-IV is secreted from the intestine postprandially and has demonstrated benefits for both glucose and lipid homeostasis. Because of the parallels in the metabolic improvements seen with surgery and the rise in apoA-IV levels, we hypothesized that apoA-IV was necessary for obtaining the metabolic benefits of bariatric surgery. To test this hypothesis, we performed vertical sleeve gastrectomy (VSG), a surgery with clinical efficacy very similar to that for RYGB, in whole-body apoA-IV knockout (KO) mice. We found that VSG reduced body mass and improved both glucose and lipid homeostasis similarly in wild-type mice compared with apoA-IV KO mice. In fact, VSG normalized the impairment in glucose tolerance and caused a significantly greater improvement in hepatic triglyceride storage in the apoA-IV KO mice. Last, independent of surgery, apoA-IV KO mice had a significantly reduced preference for a high-fat diet. Altogether, these data suggest that apoA-IV is not necessary for the metabolic improvements shown with VSG, but also suggest an interesting role for apoA-IV in regulating macronutrient preference and hepatic triglyceride levels. Future studies are necessary to determine whether this is the case for RYGB as well.     

Nitric oxide production by polymorphonuclear leucocytes in infected cystic fibrosis sputum consumes oxygen

Chronic Pseudomonas aeruginosa lung infection in cystic fibrosis (CF) patients is characterized by persisting mucoid biofilms in hypoxic endobronchial mucus. These biofilms are surrounded by numerous polymorphonuclear leucocytes (PMNs), which consume a major part of present molecular oxygen (O₂) due to production of superoxide (O₂⁻). In this study, we show that the PMNs also consume O₂ for production of nitric oxide (NO) by the nitric oxide synthases (NOS) in the infected endobronchial mucus. Fresh expectorated sputum samples (n = 28) from chronically infected CF patients (n = 22) were analysed by quantifying and visualizing the NO production. NO production was detected by optode measurements combined with fluorescence microscopy, flow cytometry and spectrophotometry. Inhibition of nitric oxide synthases (NOS) with N^G-monomethyl-L-arginine (L-NMMA) resulted in reduced O₂ consumption (P < 0·0008, n = 8) and a lower fraction of cells with fluorescence from the NO-indicator 4-amino-5-methylamino-2′,7′-difluorofluorescein diacetate (DAF-FM) (P < 0·002, n = 8). PMNs stained with DAF-FM and the superoxide indicator hydroethidine (HE) and host cells with inducible NOS (iNOS) were identified in the sputum. In addition, the production of the stable end-products of NO in CF sputum was correlated with the concentration of PMNs; NO₃⁻ (P < 0·04, r = 0·66, n = 10) and NO₂⁻ (P< 0·006, r = 0·78, n = 11). The present study suggests that besides consumption of O₂ for production of reactive oxygen species, the PMNs in CF sputum also consume O₂ for production of NO.     

Early treatment with inhaled antibiotics postpones next occurrence of Achromobacter in cystic fibrosis

ObjectivesIn this nationwide retrospective study, we analysed species distribution, antimicrobial susceptibility and time to next occurrence of Achromobacter in Danish cystic fibrosis (CF) patients from 2000 to 2011.MethodsThirty-four primary isolates were identified to species level and subjected to antimicrobial susceptibility testing. Effectiveness of early antimicrobial treatment was assessed by a Kaplan–Meier estimation of time to recurrence.ResultsAchromobacter xylosoxidans accounted for 13 (38%) of the isolates, and an unnamed species accounted for 11 (32%) of the isolates. Meropenem, piperacillin–tazobactam and trimethoprim–sulfamethoxazole were highly active against chemotherapy-naïve Achromobacter, while ceftazidime, colistin and tobramycin were judged adequate for inhalation therapy. Fifty-five percent of 25 patients treated with inhaled ceftazidime, colistin, or tobramycin remained free of Achromobacter three years after acquisition, in contrast to 17% of 22 patients who did not receive inhaled antibiotics (P < 0.01).ConclusionsEarly treatment with inhaled antibiotics may prevent or postpone chronic infection with Achromobacter in CF patients.