Acid suppression does not change respiratory symptoms in children with asthma and gastro-oesophageal reflux disease.

BACKGROUND: Epidemiological studies have shown an association between gastro-oesophageal reflux disease (GORD) and asthma, and oesophageal acid perfusion may cause bronchial constriction. However, no causative relation has been proven. AIM: To assess whether acid suppression would lead to reduced asthma symptoms in children with concomitant asthma and GORD. METHODS: Thirty eight children (mean age 10.8 years, range 7.2-16.8; 29 males) with asthma and a reflux index > or =5.0 assessed by 24 hour oesophageal pH monitoring were randomised to 12 weeks of treatment with omeprazole 20 mg daily or placebo. The groups were similar in age, gender, mean reflux index, and asthma severity. Primary endpoints were asthma symptoms (daytime wheeze, symptoms at night, in the morning, and during exercise) and quality of life (PAQLQ). Secondary endpoints were changes in lung function and the use of short acting bronchodilators. At the end of the study a repeated pH study was performed to confirm the efficacy of acid suppression. RESULTS: The change in total symptom score did not differ significantly between the omeprazole and the placebo group, and decreased by 1.28 (95% CI -0.1 to 2.65) and 1.28 (95% CI -0.72 to 3.27) respectively. The PAQLQ score increased by 0.62 (95% CI 0.29 to 0.95) in the omeprazole group compared to 0.50 (95% CI 0.29 to 0.70) in the placebo group. Change in lung function and use of short acting bronchodilators were similar in the groups. The acid suppression was adequate (reflux index <5.0) under omeprazole treatment. CONCLUSION: Omeprazole treatment did not improve asthma symptoms or lung function in children with asthma and GORD.

Effect of aluminium hydroxide and meningococcal serogroup C capsular polysaccharide on the immunogenicity and reactogenicity of a group B Neisseria meningitidis outer membrane vesicle vaccine.

Three different formulations of an outer membrane vesicle (OMV) vaccine against group B meningococcal disease have been prepared and tested for immunogenicity and reactogenicity in adult volunteers. The vaccines were prepared with or without aluminium hydroxide and serogroup C-polysaccharide (C-ps). Doses from 12.5 to 100 micrograms protein were given twice at a six weeks' interval. All three formulations were well tolerated and highly immunogenic, inducing bactericidal and opsonizing antibodies in humans. Adsorption of OMVs to aluminium hydroxide reduced the pyrogenicity in rabbits. The differences in immunogenicity between the formulations were relatively small, but after the second dose a stronger booster response was observed when the vaccines were adsorbed. Thus, a formulation with OMVs and C-ps represents a safe and highly immunogenic vaccine, even without aluminium hydroxide.

Human antibody responses after vaccination with the Norwegian group B meningococcal outer membrane vesicle vaccine: results from ELISA studies.

Antibody responses after vaccination with three different formulations of a new meningococcal group B outer membrane vesicle (OMV) vaccine have been studied with the ELISA technique using four different antigens. Sera from about 1200 vaccinees participating in steps 1, 2, 3 and 6 of the phase II clinical trials in Norway were analysed. The effects of non-covalently complexing the OMV antigen to group C polysaccharide (C-PS) and of adsorbing OMV (with and without C-PS) to aluminium hydroxide (AH) were studied. All three vaccine formulations were highly immunogenic in humans. Adsorption of the vaccine to AH had a relatively small effect on the immune response, but the results indicated that the booster response was stronger with the adsorbed than with the unadsorbed vaccines. Some increase in the immune response against OMV was also observed by non-covalent complexing OMV with C-PS, particularly after the second dose. In most of the vaccinees the antibody levels were significantly reduced 6 to 12 months after vaccination. Adsorption of the vaccine to AH had no effect on the antibody response against C-PS. Comparison with bactericidal activity of the same sera was done. A highly significant correlation was observed between the bactericidal titres and the levels of IgG antibodies against OMV and class 5C protein, whereas the correlation between antibody levels against lipopolysaccharide and the bactericidal activity was poor.

Results of an efficacy trial with an outer membrane vesicle vaccine against systemic serogroup B meningococcal disease in Norway.

A placebo controlled, double blind efficacy trial with a new outer membrane vesicle vaccine against systemic meningococcal disease of serogroup B, has been conducted in Norwegian secondary schools. The study was randomized at school level (1335 schools) and 171,800 students volunteered. The study started in October 1988 and the code was opened in June 1991. Out of the thirty-six proven cases of acute, severe, systemic disease caused by serogroup B meningococci among the participants, twelve occurred in eleven schools given vaccine, twenty-four in twenty-four schools given placebo. twenty-four cases were recorded among secondary school students who did not participate in the study. The protection rate was calculated to 57.4% with a p-value of 1.2% and lower limit of confidence (95%) to 27.7%. The results have initiated research towards an improved outer membrane vesicle vaccine against this disease.

Design of clinical trials with an outer membrane vesicle vaccine against systemic serogroup B meningococcal disease in Norway.

An outer membrane vesicle vaccine against acute, systemic disease caused by meningococci of serogroup B has been developed. The vaccine has been tested consecutively in phase I and phase II clinical trials including more than 5000 volunteers. These trials provided data on safety, immunogenicity and reactogenicity and possible effect on carriage of meningococci in the throat, and consequently formed the basis for two major protection trials; one in secondary school students and one among military recruits. The aims, design and major results of phase I and phase II studies are described as well as the design and organization of the protection trials.

Local reactivity, local resistance and systemic dissemination in Mycobacterium lepraemurium (MLM) infection.

Local reactivity measured as swelling of the infected footpad, local resistance to bacterial multiplication, and capacity to limit systemic dissemination were studied in C57BL, C3H/Bom, C3H/HeJ, and A/Sn mice inoculated with Mycobacterium lepraemurium. C57BL mice developed a strong local reaction with a sudden onset, and effectively limited local multiplication as well as systemic dissemination of bacteria to the liver and spleen as determined 19 weeks after the inoculation. C3H/Bom mice showed no local reaction, had high numbers of bacteria locally, and had extensive systemic dissemination of the infection. C3H/HeJ mice, on the other hand, developed a small local reaction and had less systemic dissemination of bacteria than C3H/Bom mice. In C57BL mice and in the two C3H substrains local reactivity, local resistance to infection, and resistance to systemic spread of the infection paralleled each other. In contrast, A/Sn mice showed a small local reaction, had the most extensive bacterial multiplication at the site of inoculation of the four mouse strains tested, and at the same time were the mice that most effectively restricted systemic dissemination of the infection. Thus, the mechanisms restricting local bacterial multiplication may be different from the mechanisms limiting bacterial dissemination. Neither bacterial growth locally at the site of subcutaneous inoculation in the footpad, nor systemic dissemination of the infection, followed a mouse strain pattern consistent with the Ity/Lsh/Bcg gene model. In experimental mycobacterial infection both local bacterial growth at the site of inoculation and systemic dissemination should be determined.

The antigens of Mycobacterium tuberculosis, H37Rv, studied by crossed immunoelectrophoresis. Comparison with a reference system for Mycobacterium bovis, BCG.

On the basis of a previously developed reference system for Mycobacterium bovis, BCG, in crossed immunoelectrophoresis (CIE), antigens of Mycobacterium tuberculosis were identified in an analogous system. A majority of the numbered lines in the BCG system were also present and identified in the M. tuberculosis system. The corresponding antigens in the two systems were identified by dual dilution in CIE, and using monospecific antisera and monoclonal antibodies. Some of the antigens were specifically identified by the demonstration of enzyme activity and by means of hydroxyapatite, concanavalin A (Con A), EDTA, and blue-Sepharose. Three antigens (nos 10, 78, and 81), which were found in high concentrations in M. tuberculosis culture fluid, were not identified or were present in low concentrations in BCG culture fluid. The high percentage of corresponding antigens confirms that there is a very close taxonomic relationship between BCG and M. tuberculosis. Corresponding antigens in BCG and M. tuberculosis did not differ in electrophoretic mobility in the antigenic preparations studied.

Characterization of antibody-reactive epitopes on the 65-kilodalton protein of Mycobacterium leprae.

Twenty-three monoclonal antibodies (MAbs) prepared in seven different laboratories were studied, all of which recognized the 65-kilodalton (kDa) protein of Mycobacterium leprae as determined by Western blotting or gel radioimmunoassay or both. Fourteen of the MAbs recognized different epitopes, as evaluated by cross-competition studies using radiolabeled MAb and unlabeled inhibitors; the species specificity of these epitopes was defined by nitrocellulose dot blot immunoassays with bacterial sonic extract antigen preparations from 23 species of mycobacteria. Each of the 14 distinct MAbs recognized a 65-kDa protein produced by a lysogenized Escherichia coli Y1089 host containing cloned rDNA which included the gene for the M. leprae 65-kDa protein. Of the 14 distinct MAbs, 1 recognized an epitope found only on M. leprae, and the others recognized epitopes present on as few as 8 or as many as all 23 of the mycobacterial species studied. Identification of these distinct 65-kDa protein epitopes and use of the MAbs which recognize them should assist future structural studies of this protein and characterization of the T-cell reactive and serodiagnostically useful portions of the molecule.

Catalases, peroxidases, and superoxide dismutases in Mycobacterium leprae and other mycobacteria studied by crossed immunoelectrophoresis and polyacrylamide gel electrophoresis.

The five mycobacteria Mycobacterium lepraemurium, M. leprae, M. bovis BCG, M. smegmatis, and M. intracellulare were studied. Catalase and peroxidase activities were demonstrated in polyacrylamide and crossed immunoelectrophoresis gels for M. lepraemurium, M. intracellulare, and BCG, but not for M. leprae. Peroxidase and catalase activities were associated with the same precipitate line in crossed immunoelectrophoresis for M. lepraemurium, M. intracellulare, and BCG, showing that in these mycobacteria the two enzyme activities resided in the same molecule. M. smegmatis peroxidase and catalase activities were closely associated on polyacrylamide gel electrophoresis, but on the crossed immunoelectrophoresis catalase and peroxidase activities were associated with two different precipitate lines. Catalases without peroxidase activity were demonstrated in crossed immunoelectrophoresis and polyacrylamide gel electrophoresis in M. intracellulare and M. smegmatis. The catalase without peroxidase activity in M. intracellulare was heat resistant and therefore classified as an m-catalase. In M. smegmatis the catalase without peroxidase activity was only partially heat resistant. All of the catalases with peroxidase activity were heat-sensitive t-catalases. Superoxide dismutase activity in the crossed immunoelectrophoresis was associated with the M. leprae antigen no. 4 and with cross-reacting antigens in the other mycobacteria studied. Several superoxide dismutases were demonstrated in Mycobacterium duvalii. They were antigenically different from the other superoxide dismutases in this study, as shown by lack of reactivity with a monospecific antibody to M. lepraemurium superoxide dismutase. Molecular weights were estimated for all the enzymes in this study by sodium dodecyl sulfate-polyacrylamide gels.

Characterization of the immune response to an epitope on Mycobacterium leprae antigen 7 defined by a monoclonal antibody.

A mouse monoclonal antibody (038D-C6) was shown by crossed immunoelectrophoresis and radioimmunoassay to react with an epitope on the Mycobacterium leprae antigen 7. This epitope was highly crossreactive with BCG/M. tuberculosis and of a non-arabinogalactan-arabinomannan nature. A solid-phase radioimmunoassay (SPRIA) was applied, based on competitive inhibition by human sera of antigen binding by this anti-M. leprae monoclonal antibody. Inhibitory activity determined by this assay decreased markedly upon treatment in both lepromatous and tuberculoid leprosy patients. A correlation was found between the bacterial load of the patient and the inhibitory activity measured in the SPRIA assay. Serum-inhibitory activity could therefore perhaps be used as a follow-up test for patients on treatment or as a screening method to detect infective cases. A dot enzyme-linked immunosorbent assay based, like the SPRIA assay, on competitive inhibition by human sera, was explored as an inexpensive and technically simple alternative also applicable under field conditions.

Further characterization including preliminary chemical analysis of antigen MLW1 from Mycobacterium leprae.

MLW1, an antigen preparation from Mycobacterium leprae previously shown to have a high content of M. leprae antigen No. 7 (ML7), was found to contain the typical cell wall constituents arabinose, galactose and mannose. The fatty acid composition of MLW1 was largely comparable to that of undisrupted cells. The capacity of MLW1 to stimulate lymphocytes was further studied. Good correlation was obtained between the in vitro lymphocyte responses to MLW1 and human-derived M. leprae, indicating similar specificity of the two antigen preparations in this test. The stimulatory activity of MLW1 was not significantly influenced by batch to batch variations, was well-preserved during storage and most of it was heat-stable. Attempts to remove the ML7 antigen indicate that this component plays a dominant role in inducing in vitro lymphocyte stimulation.

Immunization of highly susceptible C3H mice with ultrasonicated Mycobacterium lepraemurium (MLM) bacilli facilitates the development of increased resistance during MLM infection.

C3H mice were immunized subcutaneously with water soluble antigens of ultrasonicated Mycobacterium lepraemurium bacilli (MLMSon-S) in Freund's incomplete adjuvant (FIA) and challenged with 1 X 10(6)-1.25 X 10(8) live MLM bacilli inoculated into the foot pad. No swelling of the infected foot pad and no differences in bacillary multiplication and dissemination between the immunized mice and the control animals were observed in the first nine weeks. From nine weeks on, a small foot pad swelling developed in the immunized mice. Twenty weeks after inoculation, the number of bacilli in the foot pad, the popliteal lymph node, and the spleen was significantly lower in the immunized mice than in the normal controls after challenge with the lowest bacillary doses. Cyclophosphamide (CY) pretreatment did not increase the effect of immunization. The addition of MLM cell wall fragments to the emulsion used for immunization tended to increase the difference between immunized and normal animals, while no further increase of the immunization effect was obtained by the use of Freund's complete adjuvant (FCA). A tendency toward a plateau phenomenon for the multiplication of MLM bacilli was observed in the normal mice. In nonimmunized mice, CY treatment caused some reduction in bacillary numbers in the foot pads, and reinfection experiments suggested that a small reduction in susceptibility had been induced by the priming infection. Although unable to prevent a progressive course of the infection, genetically low-resistant C3H mice were able to modify the development of the infection by mechanisms that were activated by the infection itself. Similar mechanisms were facilitated by immunization with MLMSon-S.

Delayed-type hypersensitivity after immunization with ultrasonicated Mycobacterium lepraemurium in C3H and C57BL mice.

Subcutaneous immunization with ultrasonicated Mycobacterium lepraemurium (MLMSon) in incomplete Freund's adjuvant (IFA) induced long-lasting skin reactivity with the kinetics of a tuberculin-type delayed hypersensitivity (DTH) reaction in both C3H and C57BL mice. The reactivity generally was stronger in C57BL than in C3H mice, and with increasing doses of MLMSon test antigen the local reaction increased more in C57BL than in C3H mice. Pretreatment of C3H mice with cyclophosphamide before immunization caused a shift in the dose-response curve so that the local reaction increased more with increasing doses of test antigen. Histological examination of the reaction elicited by MLMSon in immunized mice revealed a predominantly mononuclear cell infiltrate, and local reactivity could be transferred by immune cells but not by immune serum. The local reaction elicited by MLMSon exerted an adjuvant effect on the induction of DTH to sheep erythrocytes. Thus, MLMSon in IFA given subcutaneously induced stable DTH that conformed to the criteria for tuberculin-type DTH.

Induction of immunity against live Mycobacterium lepraemurium: a requirement for viable bacilli?

Live Mycobacterium lepraemurium (MLM) bacilli, bacilli killed by irradiation or heat, and the water-soluble components of ultrasonicated bacilli (MLMSon) were compared as immunizing and eliciting antigens in C57BL mice which are high-responders to live MLM. The latency period preceding the development of a local granulomatous reaction in normal mice inoculated subcutaneously in the footpad with live MLM was reduced, and the reaction became larger when the dose of bacilli was increased. Immunization with MLMSon induced only weak initial reactivity against an inoculum with live bacilli, but the development of stronger reactivity was accelerated and the magnitude of the local reaction that then developed was increased. MLMSon-immunized mice showed some reactivity also against heat-killed bacilli. Killed bacilli caused the development of a small, late local reaction in normal mice, but no local reactivity was detected upon challenge with live and killed MLM in mice immunized with killed bacilli. However, a local reaction was elicited by MLMSon, which was thus a more potent eliciting antigen than intact bacilli, and MLMSon and whole killed bacilli appeared to induce immune reactivity with overlapping antigen specificities. Subcutaneous inoculation with live bacilli induced reactivity against live MLM but not against MLMSon and not against whole killed MLM, except for a transient early (24 hr) reaction elicited only with a large dose of killed bacilli. The development of a lasting local reaction against killed bacilli was found to be suppressed in mice immunized with live bacilli. Live bacilli and dead MLM antigen appeared to have largely different specificities as inducing as well as eliciting antigens.

In vitro stimulation of lymphocytes with an antigen fraction prepared from Mycobacterium leprae and tuberculin PPD in contacts and non-contacts of leprosy patients.

An antigen fraction from Mycobacterium leprae, called MLW1, was used as stimulator in the lymphocyte stimulation test, for comparison with tuberculin PPD. The test was performed in three groups of contacts of leprosy patients with various degree of exposure: (1) close contacts, (2) healthy occupational contacts and (3) non-close contacts and, in addition, in a group of BCG vaccinated and non-exposed controls. The MLW1 preparation induced moderate to strong responses in all three groups of contacts. Although the close contact group showed the highest median responses to all three doses tested, there were no significant differences between the contact groups. At all three doses levels the non-exposed group showed markedly and significantly lower median responses than the contact groups. The responses to tuberculin PPD was markedly and significantly lower in the close contact group than in the other groups. Both when individual responses to the two antigens MLW1 and PPD are compared and when the delta ct/min' estimator is used, the results indicate that the intensity of the specific response increases with the closeness of contact with leprosy patients.

Survival of Mycobacterium lepraemurium in C57BL mice after acquired protective immunity.

The protective immune response against Mycobacterium lepraemurium (MLM) in C57BL mice has been shown to stop the increase in bacillary numbers and the dissemination of bacilli, but the acid fast bacilli are not cleared from the tissues. Persistence of viable bacilli was indicated by a significant increase in the number of acid fast bacilli in the footpad of C57BL mice that were treated with cortisone acetate several weeks after the onset of the immune response. Bacilli harvested 9 and 16 days after inoculation into immune C57BL mice showed only a marginally detectable loss of viability as determined by bacillary multiplication after transfer into susceptible C3H mice. Twenty-six weeks after being inoculated into immune C57BL mice a small proportion of the bacilli was found still to be alive. A similar finding was done 15 weeks after primary inoculation of MLM into mice that developed an apparently effective protective immune response 4 weeks after being inoculated. Sixty-seven weeks after inoculation of immunized C57BL mice with MLM, bacillary numbers in the footpad were as with patent immunity, but the bacilli were found to be fully viable, suggesting incipient reactivation of the infection. When bacillary numbers were followed over a period of 52 weeks in the organs of normal C57BL mice inoculated with a low dose of bacilli it was found that after a plateau phase bacillary numbers started to increase again. Thus, in all experiments part of the bacillary population had survived the protective immune response against MLM in C57BL mice.

Mouse monoclonal anti rabbit IgG coupled to monodisperse polymer particles. Comparison with polyclonal antibodies in radioimmunoassay for thyroid hormones.

A solid phase second antibody was prepared by covalent coupling of a mouse monoclonal anti rabbit IgG to monodisperse particles. This preparation was compared with immunosorbent purified sheep anti rabbit IgG antibodies coupled to the same particles. The monoclonal antibody bound rabbit IgG with a dissociation constant of 3 X 10(-11) L/mol, and the binding was Fc specific. The sheep antibodies had a similar Kd and about 75% of the activity was directed against the Fc portion of IgG. The binding capacity per mol of both solid phase antibodies was 0.7 mol of rabbit IgG. Monoclonal and polyclonal solid phase antibodies were equally effective as separating agents in various radioimmunoassays. Direct coupling of the rabbit antibodies to the solid phase resulted in a marked loss of binding capacity for the respective thyroid hormones. However, when rabbit anti-thyroxine or anti-triiodothyronine were preadsorbent to second-antibody-coated particles the binding capacities of the former antibodies were well preserved.