Meningococcal omp85 in detergent-extracted outer membrane vesicle vaccines induces high levels of non-functional antibodies in mice.

The vaccine potential of meningococcal Omp85 was studied by comparing the immune responses of genetically modified deoxycholate-extracted outer membrane vesicles, expressing five-fold higher levels of Omp85, with wild-type vesicles. Groups (n = 6-12) of inbred and outbred mouse strains (Balb/c, C57BL/6, OFI and NMRI) were immunized with the two vaccines, and the induced antibody levels and bactericidal and opsonic activities measured. Except for Balb/c mice, which were low responders, the genetically modified vaccine raised high Omp85 antibody levels in all mouse strains. In comparison, the wild-type vaccine gave lower antibody levels, but NMRI mice responded to this vaccine with the same high levels as the modified vaccine in the other strains. Although the vaccines induced strain-dependent Omp85 antibody responses, the mouse strains showed high and similar serum bactericidal titres. Titres were negligible with heterologous or PorA-negative meningococcal target strains, demonstrating the presence of the dominant bactericidal PorA antibodies. The two vaccines induced the same opsonic titres. Thus, the genetically modified vaccine with high Omp85 antibody levels and the wild-type vaccine induced the same levels of functional activities related to protection against meningococcal disease, suggesting that meningococcal Omp85 is a less attractive vaccine antigen.

Pandemic influenza vaccination elicits influenza-specific CD4+ Th1-cell responses in hypogammaglobulinaemic patients: four case reports.

In these case reports, we investigated pandemic influenza 2009 vaccination of primary hypogammaglobulinaemic patients. Three combined variable immunodeficiency (CVID) patients and one X-linked agammaglobulinaemia (XLA) patient were vaccinated with the pandemic vaccine A/California/7/2009 (H1N1)-like split virus (X179a) adjuvanted with the oil-in-water emulsion AS03. Subsequently, serum and peripheral blood mononuclear cells were sampled and used to measure the haemagglutination inhibition (HI) and antibody-secreting cell (ASC) responses. In addition, the IFN-γ, IL-2 and TNF-α producing CD4(+) Th1-cell response was determined as these cytokines are important indicators of cell-mediated immunity. Two of the CVID patients responded to vaccination as determined by a >4-fold rise in HI antibodies. These subjects also had influenza-specific ASC numbers, which, albeit low, were higher than prevaccination levels. In addition, vaccination induced CD4(+) Th1-cell responses in both the XLA patient and the CVID patients, although the frequency of influenza-responsive cells varied amongst the patients. These results suggest that hypogammaglobulinaemia patients can mount a CD4(+) Th1 cell-mediated response to influenza vaccination and, additionally, that influenza vaccination of some hypogammaglobulinaemia patients can produce an influenza-specific humoral immune response. The findings should be confirmed in larger clinical studies.

Determinants of microbial exposure in grain farming.

OBJECTIVES: Exposure to organic dust containing high concentrations of microorganisms is common in grain farming, although the farmers have practices to counteract microbial growth to obtain optimal grain yields. We investigated the influence of weather and production practices on personal microbial exposure during grain work. METHODS: Airborne dust was collected by personal sampling during threshing and storage work on 92 Norwegian farms. The personal exposure for bacteria, endotoxin, fungal spores and hyphae, beta-(1-->3)-glucans and actinomycetes was quantified and compared with climatic data expressed as fungal forecasts from the grain growth season and production practices as reported by farmers. RESULTS: Farmers were exposed to a geometrical mean of 4.4 mg m(-3) inhalable dust [geometrical standard deviation (GSD) = 4.0], 4 x 10(6) m(-3) bacteria and fungal spores (GSD = 5.2 and 5.9, respectively), 5.9 x 10(3) EU m(-3) of endotoxins (GSD = 8.6), 2 x 10(5) m(-3) actinomycetes (GSD = 15.3), 120 mug m(-3) beta-(1-->3)-glucans (GSD = 4.7) and 5 x 10(5) AU m(-3) of hyphae (GSD = 4.4). Univariate associations were found between one or several of these microbial factors and work operation, visible fungal damage, grain species, lodging of grain, storage technology or harvester type. As assessed by general linear models, storage work was the main predictive determinant for microbial exposure, although grain species and visible fungal damage also were also important. Wet and warm weather throughout the grain growth season were associated with elevated exposure for inhalable dust, beta-(1-->3)-glucans, endotoxins and hyphae during threshing. The beta-(1-->3)-glucan exposure could biologically be explained by the fungal spore and hyphal exposure, both variables contributing equally. However, spores were most important during storage work, whereas only hyphae were predictive during threshing. CONCLUSIONS: Farmers were exposed to high levels of microorganisms and their components during dusty grain work. Dust prevention and protection may reduce microbial exposure, and may be particularly important in areas with frequent fungal forecasts, when fungal damage has been observed, during storage work or when handling barley.

High case-fatality rates of meningococcal disease in Western Norway caused by serogroup C strains belonging to both sequence type (ST)-32 and ST-11 complexes, 1985-2002.

A total of 293 meningococcal disease (McD) patients from Western Norway hospitalized during 1985-2002 were examined for risk factors related to death. The case-fatality rate (CFR) increased from 4% during 1985-1993 to 17% during 1994-2002. We analysed the phenotypic and genotypic characteristics of the meningococcal patient isolates, with the aim of identifying whether highly virulent meningococcal strains contributed to the increased CFR. The Norwegian epidemic strain B:15:P1.7,16/ST-32 complex was overall the most common phenotype/genotype (n=75) and caused most deaths (n=9; CFR 12.0%). However, fatality was significantly associated with disease caused by serogroup C meningococcal strains; C:15:P1.7,16/ST-32 and C:2a/ST-11 complex strains, which had the highest CFRs of 21.1% and 18.2% respectively. Serogroup B strains of the ST-32 complex differing by serotype and/or serosubtype from the epidemic strain had a CFR of 5.1%, while the CFR of disease caused by other strains (all phenotypes and genotypes pooled) was 2.2%. The distribution of phenotypes/clonal complexes varied significantly between 1985-1993 and 1994-2002 (P<0.001); B:15/ST-32 complex strains decreased whereas both C:15:P1.7,16/ST-32 complex strains and strains with other phenotypes/clonal complexes increased. Our results indicate that C:15:P1.7,16/ST-32 and C:2a/ST-11 complex strains were highly virulent strains and contributed to the high CFR of McD in patients from Western Norway. To reduce fatality, rapid identification of such virulent strains is necessary. In addition, early and specific measures should include public information, vaccination of populations at risk of disease and carriage eradication, when clustering of patients occurs.

Variations in case fatality and fatality risk factors of meningococcal disease in Western Norway, 1985-2002.

In a retrospective epidemiological study, 293 meningococcal disease patients hospitalized during 1985-2002, were examined for fatality and risk factors related to death. The overall case fatality rate (CFR) was 8.2%, but increased from 4% during 1985-1993 to 17% during 1994-2002. The latter 9-year period was characterized by more serogroup C infections and more patients with thrombocytopenia on admission to hospital. All patients categorized as meningitis on admission survived. Of the 24 patients who died, 21 had meningococcal skin rash on admission, 23 had an onset to admission time of < or =24 h, and 16 had severe septicaemia with hypotension and/or ecchymoses without meningitis on admission. By multivariate analyses, a short onset to admission time, >50 petechiae, thrombocytopenia and severe septicaemia on admission were associated with fatality. More lives could be saved through earlier admission to hospital. This can be achieved through more information to the public about the early signs of meningococcal septicaemia, with the recommendation to look for skin rash in patients with acute fever during the first day and night.

Upper airway inflammation in waste handlers exposed to bioaerosols.

AIMS: To examine work associated upper airway inflammation in 31 waste handlers, and to correlate these findings with personally monitored exposure to different bioaerosol components. METHODS: Cell differentials, interleukin 8 (IL-8), myeloperoxidase (MPO), and eosinophilic cationic protein (ECP) were examined in NAL (nasal lavage), and swelling of the nasal mucosa was determined by acoustic rhinometry before work start on Monday and the following Thursday. Bioaerosol exposure was determined by personal full shift exposure measurements on Monday, Tuesday, and Wednesday and analysed for total bacteria, fungal spores, endotoxin, and beta(1-->3)-glucans. RESULTS: The increased percentage of neutrophils from Monday (28%) to Thursday (46%) correlated with increases in ECP (r(S) = 0.71, p < 0.001) and MPO (r(S) = 0.38, p < 0.05), and showed a close to significant correlation with nasal swelling (r(S) = -0.55, p = 0.07). The Thursday levels of neutrophils, MPO, and IL-8 were associated with the exposure to fungal spores (range 0-2.0 x 10(6)/m(3)) and endotoxin (range 4-183 EU/m(3)) measured the day before, and the median exposure to beta(1-->3)-glucans (range 3-217 ng/m(3)), respectively (r(S) = 0.47-0.54, p < 0.01). Swelling of the nasal mucosa was associated with the fungal spore and beta(1-->3)-glucan exposure (r(S) = 0.58-0.59, p < 0.05). CONCLUSION: These results are based on a relatively small population, and conclusions must be drawn with care. The results suggested that a moderate exposure to fungal spores, endotoxins, and beta(1-->3)-glucans during waste handling induced upper airway inflammation dominated by neutrophil infiltration and swelling of the nasal mucosa.

Airway inflammation in waste handlers exposed to bioaerosols assessed by induced sputum.

Work-associated lower airway inflammation in waste collectors was examined by induced sputum and correlated with the bioaerosol exposure. Organic waste collectors (n=25) underwent induced sputum collection and spirometry before work on Monday and the following Thursday. Total cells, cell differentials, interleukin (IL)-8 and eosinophilic cationic protein were determined. Personal full-shift exposure measurements were performed Monday, Tuesday and Wednesday and analysed for total bacteria, fungal spores, endotoxins and beta(1-3)-glucans. The percentage of neutrophils (46-58%) and the IL-8 concentration (1.1-1.4 ng x mL(-1)) increased from Monday to Thursday. Forced expiratory volume in one second (FEV1) was significantly reduced on Thursday, and the decrease in FEV1/forced vital capacity correlated with the increase in the percentage of neutrophils. The median exposure to endotoxin (range 7-180 EU x m(-3)) and beta(1-3)-glucan (range 5-220 ng x m(-3)) was correlated with the increase in IL-8. Bioaerosol exposure during waste collection induced an inflammatory response in the lower airways, characterised by neutrophils and interleukin-8 secretion, that influenced the lung function. The inflammatory response was related to microbial components in the bioaerosol and was more pronounced for endotoxin than beta(1-3)-glucan exposure. No associations were found for mould spores or bacteria.

FcgammaRIIa and FcgammaRIIIb polymorphisms were not associated with meningococcal disease in Western Norway.

Fcgamma-receptor (FcyR) polymorphisms have been associated with acquisition and severity of invasive meningococcal disease. We studied FcgammaR polymorphisms in a population with a high incidence of meningococcal disease. Fifty meningococcal disease patients aged 14-60 years, with bacteriologically confirmed disease and without detected complement deficiency, together with 100 healthy adult controls were included in the study. Clinical and bacteriological data were collected prior to FcgammaRIIa and FcgammaRIIb genotyping, which was performed by polymerase chain reaction. The distribution of the FcgammaRIIa and FcgammaRIIIb allotypes and their allele frequencies were not significantly different amongst the patients and the controls. The combination FcgammaRIIa-R/R and FcgammaRIIb-Na2/Na2 was less common among patients than controls (OR = 0.11, Fisher's exact P = 0.05). No significant association was found between the two FcgammaRs and severity of disease, meningococcal serogroup, age groups or gender. In contrast to previous findings, our study indicates that in Norwegian teenagers and adults, the FcgammaRIIa and FcgammaRIIIb allotypes are not decisive for the acquisition or for the severity of meningococcal disease.

[Streptococcus group A infections of skin, soft tissue and blood]. / Infeksjoner med gruppe A-streptokokker i hud, bløtdeler og blod.

BACKGROUND: Group A streptococcus is one of the most common bacterial pathogens causing infections in tissue and organs, most frequently throat and skin. Since the late 1980s there have been reports from Scandinavia and many other countries documenting a resurgence of highly invasive infections such as puerperal fever, necrotizing fasciitis, myositis and sepsis. MATERIAL AND METHODS: On the basis of relevant studies and reviews and a clinical study of 61 patients between 1992 and 1999 with necrotizing fasciitis and myositis at Haukeland University Hospital, Bergen, Norway, we present an overview of the prevalence, pathogenesis, clinical features and treatment of group A streptococcal infections in skin, soft tissue and blood. RESULTS AND INTERPRETATION: The reason for the increase in severe group A streptococcal infections is unclear. The clinical features depend on the level of infection (superficial skin, subcutis, fascia and muscle): the deeper the initial infection, the more frequent development of bacteraemia and life-threatening disease. Serious infections are associated with shock and multiorgan failure, i.e. streptococcal toxic shock syndrome. Early surgical debridement is essential in necrotizing fasciitis and myositis. Penicillin is still the drug of choice for milder infections. The addition of clindamycin is recommended in cases of more invasive infection.

Phagocytosis: measurement by flow cytometry.

Defects in phagocyte function or in the interactions between phagocytes, microorganisms and serum factors are associated with increased susceptibility to infection. Flow cytometry (FCM) offers rapid and reproducible measurements of single cells in suspension and, following staining with one or more fluorochromes, simultaneous biochemical and functional examinations of the complex process of phagocytosis. FCM techniques have been used for more than two decades to evaluate phagocyte cellular defects, as well as species-specific serum opsonic activities during disease and after vaccination. Recently, multiparameter assays have been developed to reveal the antigen-specificity of opsonophagocytic responses. This review presents basic methodological principles of FCM quantitation of phagocytosis and intracellular oxidative burst, and assays to evaluate species-specific and antigen-specific opsonophagocytosis. The calculations performed to present opsonophagocytosis results, as well as technical and methodological challenges are discussed, and examples of applications are presented.

Eradication of non-typhoid salmonellae in acute enteritis after therapy with ofloxacin for 5 or 10 days.

Eradication of non-typhoid salmonellae was evaluated in a randomized, double-blinded study of 49 patients with acute enteritis after therapy with ofloxacin 400 mg once daily for 5 or 10 days. Early eradication of salmonellae was found in 57% of patients in the 5 day therapy group and in 74% of patients in the 10 day therapy group. This difference was larger among severely ill patients. Together with our previous study of ofloxacin therapy for 3 days or placebo, this shows that early eradication of non-typhoid salmonellae increases with duration of ofloxacin therapy without an increase in persistence of salmonellae in stools or development of resistant strains.

Concurrent measurement of antigen- and antibody-dependent oxidative burst and phagocytosis in monocytes and neutrophils.

The current study aims to review flow cytometric (FCM) parameters for the quantification of phagocytosis. A limitation of existing methods is their difficulty with accurate quantification of the phagocytic index, i.e., number of beads per phagocyte, in individual cell lines in mixed cell suspensions. We have quantified phagocytosis and the oxidative burst simultaneously using fluorescent beads coated with meningococcal outer membrane vesicles (OMV beads) by the conversion of dihydrorhodamine 123 (DHR-123) to rhodamine 123 (R-123). Both these processes depend on specific serum opsonins. After the incubation, staining with a fluorescent anti-CD14 monoclonal antibody succeeded in discriminating phagocytosing monocytes from neutrophils. The spectral overlaps between OMV beads, R-123, and anti-CD14 could be completely compensated. Percentage of phagocytosis and the phagocytic index were similar in monocytes and neutrophils, but the oxidative burst behaved differently. Two monocyte subpopulations were observed. Both subpopulations spontaneously converted some DHR-123 into R-123, whereas the reaction was triggered by phagocytosis in neutrophils. The total oxidative response increased with increasing phagocytic index in both cell types, but the oxidative burst in monocytes was about twice that of neutrophils. The oxidative ratio (mean R-123 fluorescence value divided by the phagocytic index) declined with time in monocytes, but increased in neutrophils. Our results demonstrate the need for careful attention to technical details. This single-laser, three-color FCM method facilitates the comparative research of phagocytosis and the oxidative burst in monocytes and neutrophils and provides a basis for a number of applications in hematology, infectious medicine, and immunology.

Patterns of cell death induced by eluates from denture base acrylic resins in U-937 human monoblastoid cells.

The purpose of this study was to investigate in vitro the apoptosis- and necrosis-inducing potential of eluates from three heat-polymerized and four autopolymerized poly(methyl methacrylate)-based denture base resins. Our hypothesis was that the rate of cell death by apoptosis and/or necrosis induced by such denture base resins could be an important indicator of their cytotoxicity degree. U-937 human monoblastoid cells were exposed for 24 h and 48 h to eluates of 0.1 g/ml, 0.2 g/ml, 0.4 g/ml, and 0.8 g/ml extracted for 24 h and 48 h. The characteristics of apoptosis and necrosis were evaluated by flow cytometry and light and electron microscopy. Eluates from all resins enhanced cell death by apoptosis and necrosis in U-937 cells in a dose- and time-dependent fashion. Eluates from autopolymerized resins yielded higher percentages of apoptosis and necrosis than the heat-polymerized ones. The results support our hypothesis that eluates of poly(methyl methacrylate)-based denture base acrylic resins activate death-signaling pathways, and that the extent of this process reflects their biocompatibility degree.

Functional differentiation of acute myeloid leukaemia blast cells.

Little is known of the functional status of blast cells from patients with acute myeloid leukaemia (AML). We have studied phagocytosis and membrane receptors by flow cytometry (FCM), and secretory activities in blast cells from 24 AML patients prior to treatment. Blast cells from 11/16 patients attached N. meningitidis, and internalization occurred in 7/14. The phagocytosis of zymosan particles and N. meningitidis correlated linearly (r = 0.9, p<0.01, n = 11). Surface membrane expression of CD32 and CD11b was sufficient to account for opsonin-dependent attachment in all except one patient. A significant fraction of the blast cells attached, but did not internalize meningococci. CD32 and CD11b were non-functional in all the blasts from five patients, and in a subpopulation from seven additional patients. Significantly more large than small blasts expressed CD32, CD35 and CD11b (p<0.001). Phagocytosis was unrelated to the secretion of IL-1alpha, IL-1beta, and TNFalpha. In conclusion, AML blast cell function is related to receptor expression, cell size and granularity, and to FAB-type.

Human opsonins induced during meningococcal disease recognize transferrin binding protein complexes.

Patient serum opsonins against transferrin binding protein A+B (TbpA+B) complexes from two Neisseria meningitidis strains (K454 and B16B6, with 85- and 68-kDa TbpB, respectively) were quantified by a functional phagocytosis and oxidative burst assay. TbpA+B complexes adsorbed to fluorescent beads were opsonized with individual acute and convalescent sera from 40 patients infected by a variety of meningococcal strains. Flow cytometric quantitation of leukocyte phagocytosis products (PP) demonstrated that disease-induced serum opsonins recognized TbpA+B, and the highest anti-TbpA+B serum opsonic activities were found between admission to hospital and 6 weeks later. The PP values obtained with TbpA+B from strain B16B6 (PP(B16B6)) were higher than those obtained with TbpA+B from strain K454 (PP(K454)), with both acute and convalescent sera (P < 0.0001), and correlated positively with higher immunoglobulin G enzyme-linked immunosorbent assay titers against TbpA+B from strain B16B6 than from strain K454 (P < 0.001). In spite of considerable variations between individuals, significant correlations were found between the PP(B16B6) and PP(K454) values, and the PP values did not depend on the variability of the TbpB proteins of the disease-causing strains. Simultaneously measured oxidative burst activity correlated closely with the PP values. We conclude that highly cross-reactive anti-TbpA+B serum opsonins are produced during meningococcal disease. The anti-TbpA+B opsonic activities were not affected by the variability of the TbpB proteins of the disease-causing strains, which further adds to the evidence for the vaccine potential of meningococcal TbpA+B complexes.

Human opsonins induced during meningococcal disease recognize outer membrane proteins PorA and PorB.

Human opsonins directed against specific meningococcal outer membrane structures in sera obtained during meningococcal disease were quantified with a recently developed antigen-specific, opsonin-dependent phagocytosis and oxidative burst assay. Outer membrane vesicles (OMVs) and PorA (class 1) and PorB (class 3) proteins purified from mutants of the same strain (44/76; B:15:P1.7. 16) were adsorbed to fluorescent beads, opsonized with acute- and convalescent-phase sera from 40 patients with meningococcal disease, and exposed to human leukocytes. Flow cytometric quantitation of the resulting leukocyte phagocytosis products (PPs) demonstrated that disease-induced serum opsonins recognized meningococcal OMV components and both porins. The PPPorA and PPPorB values induced by convalescent-phase sera correlated positively with the PPOMV values. However, the PPPorB values were higher than the PPPorA values in convalescent-phase sera (medians [ranges] of 754 [17 to 1,057] and 107 [4 to 458], respectively) (P < 0.0001) and correlated positively with higher levels of immunoglobulin G against PorB than against PorA as evaluated by enzyme-linked immunosorbent assay. Extensive individual variations in the anti-OMV and antiporin serum opsonic activities between patients infected by serotypes and serosubtypes homologous and heterologous to the target antigens were observed. Simultaneously measured oxidative burst activity correlated with the opsonophagocytosis, an indication that both of these important steps in the in vitro phagocytic elimination of meningococci are initiated by opsonins directed against OMV components, including PorA and PorB. In conclusion, human patient opsonins against meningococcal OMV components and in particular PorB epitopes were identified by this new method, which might facilitate selection of opsonin-inducing meningococcal antigens for inclusion in future vaccines.

Outbreak of meningococcal disease in western Norway due to a new serogroup C variant of the ET-5 clone: effect of vaccination and selective carriage eradication.

A new sulphonamide resistant (SR) C: 15:P1.7,16 meningococcal strain, a variant of the ET-5 clone, dominated in an outbreak of 22 cases in western Norway commencing in 1995. The first eight patients were 15-21 years old from the Nordhordland area, initiating a carrier study in the local high schools. Carriage of SR serogroup C meningococci was detected by routine methods and treated with a single dose of ofloxacin 400 mg. Of 20 treated carriers, 14 harboured the outbreak strain C: 15:P1.7,16. Vaccination of 4000 children, adolescents and close contacts of patients was also performed. After the intervention, 14 additional cases of meningococcal disease occurred, 8 due to the outbreak strain. However, incidence rates dropped from 180 to 30 per 100000 per year in the student population, but increased from 0 to 13 in the rest of the population in Nordhordland. Carriage eradication is not generally recommended in Norway. However, tracing and treating meningococcal carriage may have reduced transmission and disease in this outbreak situation.

Flow cytometric quantitation of human opsonin-dependent phagocytosis and oxidative burst responses to meningococcal antigens.

A one-step flow cytometric (FCM) assay has been developed to quantify both opsonin- and antigen-dependent phagocytosis and intraphagocyte oxidative burst responses. Meningococcal outer membrane structures (OMV) were adsorbed to fluorescent polystyrene beads, opsonized with serum, and exposed to leukocytes. FCM parameters of phagocytosis were evaluated in combinations with oxidative burst indicators. Rhodamine-123 was the most sensitive indicator and was compatible with quantitation of phagocytosis. The phagocytosis and oxidative burst responses induced by OMV beads were dependent on both antigens and opsonins. Increased human opsonic responses against OMV were induced during clinical meningococcal disease. A dissociation was noted between phagocytosis and oxidative burst in individual cells, indicating that functional opsonins against OMV components may differ in their ability to stimulate phagocytosis and oxidative burst responses. The method facilitates evaluation of purified bacterial structures as mediators of opsonin-dependent phagocytosis and intracellular oxidative microbicidal mechanisms, which is of interest in the complex process of selecting bacterial antigens as constituents of certain vaccines.

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.