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The Norwegian Food Allergy Register was established at the Norwegian Institute of Public Health in 2000. The purpose of the register is to gain information about severe allergic reactions to food in Norway and to survey food products in relation to allergen labelling and contamination. Cases are reported on a voluntary basis by first line doctors, and submitted together with a serum sample for specific IgE analysis. The register has received a total of 877 reports from 1 July, 2000 to 31 December, 2010. Two age groups, small children and young adults are over-represented, and the overall gender distribution is 40:60 males-females. The legumes lupine and fenugreek have been identified as two "new" allergens in processed foods and cases of contamination and faults in production of processed foods have been revealed. The highest frequency of food specific IgE is to hazelnuts and peanuts, with a marked increase in reactions to hazelnuts during the last three years. The Food Allergy Register has improved our knowledge about causes and severity of food allergic reactions in Norway. The results show the usefulness of population based national food allergy registers in providing information for health authorities and to secure safe food for individuals with food allergies.

The birth cohort BraMat (n = 205; a sub-cohort of the Norwegian Mother and Child Cohort Study (MoBa) conducted by the Norwegian Institute of Public Health) was established to study whether prenatal exposure to toxicants from the maternal diet affects immunological health outcomes in children. We here report on the environmental pollutants polychlorinated biphenyls (PCBs) and dioxins, as well as acrylamide generated in food during heat treatment. The frequency of common infections, eczema or itchiness, and periods of more than 10 days of dry cough, chest tightness or wheeze (called wheeze) in the children during the first year of life was assessed by questionnaire data (n = 195). Prenatal dietary exposure to the toxicants was estimated using a validated food frequency questionnaire from MoBa. Prenatal exposure to PCBs and dioxins was found to be associated with increased risk of wheeze and exanthema subitum, and also with increased frequency of upper respiratory tract infections. We found no associations between prenatal exposure to acrylamide and the health outcomes investigated. Our results suggest that prenatal dietary exposure to dioxins and PCBs may increase the risk of wheeze and infectious diseases during the first year of life., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

Investigating the immunotoxic effects of exposure to chemicals usually comprises evaluation of weight and histopathology of lymphoid tissues, various lymphocyte parameters in the circulation, and immune function. Immunotoxicity assessment is time consuming in humans or requires a high number of animals, making it expensive. Furthermore, reducing the use of animals in research is an important ethical and political issue. Immunotoxicogenomics represents a novel approach to investigate immunotoxicity able of overcoming these limitations. The current research, embedded in the European Union project NewGeneris, aimed to retrieve gene expression profiles that are indicative of exposure to immunotoxicants. To this end, whole-genome gene expression was investigated in human peripheral blood mononuclear cells in response to in vitro exposure to a range of immunotoxic chemicals (4-hydroxy-2-nonenal, aflatoxin B1, benzo[a]pyrene, deoxynivalenol, ethanol, malondialdehyde, polychlorinated biphenyl 153, and 2,3,7,8-tetrachlorodibenzo-p-dioxin) and nonimmunotoxic chemicals (acrylamide, dimethylnitrosamine, 2-amino-3-methyl-3H-imidazo[4,5-F]quinoline, and 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine). Using Agilent oligonucleotide microarrays, whole-genome gene expression profiles were generated, which were analyzed using Genedata's Expressionist software. Using Recursive Feature Elimination and Support Vector Machine, a set of 48 genes was identified that distinguishes the immunotoxic from the nonimmunotoxic compounds. Analysis for enrichment of biological processes showed the gene set to be highly biologically and immunologically relevant. We conclude that we have identified a promising transcriptomic profile indicative of immunotoxic exposure.

Complex exposure to xenobiotics is one of the reasons for the reported increase of respiratory diseases, cancer and immunological disturbances. Among such xenobiotics there are food mutagens whose effects on human health in the low level and/or chronic exposure still remains unknown. In the present manuscript, the compounds ethanol (EtOH), 4-hydroxynonenal (4-HNE), malondialdehyde (MDA), 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), 3,3',4,4'-tetrachlorobiphenyl (PCB 153), benzo[a]pyrene (BaP), 2-amino-3-methylimidazol[4,5-f]quinoline (IQ), 2-amino-1-methyl-6-phenylimidazol[4,5-b]pyridine (PhIP), N-Nitrosodimethylamine (NDMA) and acrylamide (AA) were evaluated in an interlaboratory comparison in the in vitro cytokinesis-block micronucleus assay (CBMN) with objective of assessing the induction of micronuclei, buds and nucleoplasmic bridges in dose responses. Statistically significant increase in MNBN frequency in binucleated cells was recorded by both laboratories for the compound PhIP (2.5μM). The compounds PCB (250 microM) and AA (500 microM) induced statistically significant increase of MNBN although it was recorded by one of the two laboratories. Induction of buds and nucleoplasmic bridges was only observed for BaP (100 microM) and AA (500 microM) by one of the laboratories. Data generated in this study may assist in the interpretation of the mother/newborn biomonitoring study being carried out within project NewGeneris and will contribute to overall knowledge on the genotoxic potential of dietary/environmental toxicants., (Copyright (c) 2010 Elsevier Ltd. All rights reserved.)

Background: Fenugreek is an ingredient in Indian-style spiced foods. Reports of adverse reactions reflect a trend toward a more international cuisine. Fenugreek allergy has not been systematically investigated so far., Objective: Study the allergenicity and antigenicity of fenugreek proteins using patient sera and a newly developed polyclonal antifenugreek antibody., Methods: Allergenic fenugreek proteins were identified by immunoblotting, using sera from 29 patients with specific IgE to peanut and other legumes. In addition, 2 patients were evaluated by skin prick test and open food challenge with native fenugreek powder. Spiced and flavored food products were analyzed for fenugreek by semiquantitative IgE and IgG immunoblotting., Results: High levels of specific IgE to both peanut and fenugreek were seen in most sera. Fenugreek sensitization is believed to be a consequence of cross-reactivity in patients with peanut allergy. Primary fenugreek allergy was suspected in only 1 case. The fenugreek dose eliciting objective symptoms was about 2 mg in the open food challenge. Major fenugreek allergens were identified at 50, 52, and 74 kd and peanut proteins at 22, 36, and 40 kd. A specific polyclonal antifenugreek antibody was found suitable for food analysis. In a food survey, about 1/3 of the fenugreek-containing products were labeled correctly., Conclusion: Fenugreek seed powder, an ingredient in spiced foods, contains several potential allergens. There is evidence for a high rate of cross-reactivity to peanut.

Lung inflammation is an important process in host defence to inhaled particulate matter. To what extent physicochemical properties of particles from different sources influence their inflammatory potential has not been fully clarified. The aim of this study was to investigate the potential of particles from wood smoke and traffic to induce a release of pro-inflammatory cytokines in the monocytic cell line THP-1. The influence of endotoxin on cytokine release was investigated using the inhibitor polymyxin B sulphate, whereas the responses to native particles, washed particles and their organic extracts were compared to determine the role of the organic fraction. Particles from the two sources showed a similar inflammatory potential, but the response was mediated by different particle characteristics. The organic fraction of wood smoke accounted for the majority of the cytokine release, whereas the response to the traffic-derived particles was in addition influenced by endotoxin and the particle core. The sum of the cytokine release induced by the organic extract and washed particles was lower than that induced by native particles, suggesting that the organic fraction must be adsorbed to the particles to exert biological activity. The results also indicated that different particle characteristics may activate different signalling pathways, since inhibition of endotoxin reduced release of TNF-alpha, IL-1beta and IL-8, whereas organic extraction only affected release of TNF-alpha and IL-8. Together, these data illustrate that a similar inflammatory response may be mediated by different particle characteristics and possibly through different signalling pathways.

The concentration of mould-specific IgG antibodies in serum may objectively indicate mould exposure and can help identifying exposed individuals. Although inhaled spores probably are the most important source of mould exposure, the commonly used methods for detecting mould-specific IgG antibodies are based on extracts from all mould components, with only low contribution from spores. We have developed a flow cytometric method using surface antigens on mould spores for quantifying mould-specific IgG antibodies in serum. Flow cytometric results were evaluated by comparison with ImmunoCap and ELISA measurements. The flow cytometric assay showed a broad linear dose-dependency and correlated moderately to strongly (r=0.41-0.97) with ImmunoCap and ELISA measurements. The IgG antibody binding was studied in detail by immunolabelling in scanning electron microscopy (SEM), revealing that morphology and IgG antibody binding differed among spores, both within and between mould strains. Germination studies by flow cytometry and SEM showed that IgG antibody binding to mould spores was altered during germination due to loss of coat. The present spore based antibody assay are simple and suitable for quantification of mould-specific IgG antibodies in serum, and includes specificity to other and possibly more relevant antigens than existing methods.

Understanding how mineral particles trigger cellular responses is crucial in order to elucidate what characteristics determine their harmful effects. It is not clear whether cellular effects are triggered through the cell membrane or require particle uptake. However, studies with asbestos suggest that activation of the epidermal growth factor receptor (EGFR) may be important. We have previously reported that crystalline silica-induced interleukin (IL)-8 release from human lung epithelial cells (A549) was regulated through Src family kinases (SFKs) and the mitogen-activated protein kinases (MAPKs) p38 and extracellular signal-regulated kinase (ERK)-1 and -2. The present study shows that SFK and p38 phosphorylation increased almost immediately upon crystalline silica exposure, whereas ERK1/2 phosphorylation increased after 10 min of exposure. The p38 inhibitor SB202190 increased the silica-induced ERK1/2 phosphorylation suggesting that p38 activity may attenuate activation of ERK1/2. Scanning electron microscopy showed that some silica particles were phagocytosed between 1 and 4h of exposure, but that the majority remained bound by microvilli on the cell surface. The EGFR inhibitor AG1478 attenuated both silica-induced IL-8 release and phosphorylation of SFKs and ERK1/2. However, AG1478 also inhibited the respective background levels, and the EGFR was not phosphorylated at the onset of silica exposure. The results suggest that crystalline silica triggers p38 and SFK-ERK1/2 signaling through interactions with membrane components as both pathways were rapidly activated prior to particle internalization. However, the silica-induced up-regulation of IL-8 release through the SFK-ERK1/2 pathway does not appear to be initiated through activation of the EGFR, although basal EGFR activity may affect the magnitude of the responses.

Air pollution has been implicated as one of the factors responsible for the increased incidence of allergic diseases observed in recent years. High concentrations of air pollutants may promote airway sensitization by acting as adjuvants. Ambient particles as carriers of adsorbed allergens are, therefore, of special interest since they may act as mediators of inflammatory as well as allergic responses. Ambient air particles from four cities in Europe were collected, in three different seasons, to examine the variation of allergens and their possible binding to the pollution particles. The particle fraction, PM10, was collected on polycarbonate filters using a low-volume sampling regime. The presence of pollen allergens, latex and beta-glucans was investigated using an immunogold labelling method directly on the collection filters. Scanning electron microscopy revealed mainly the classical carbon particles and aggregates determined to originate from vehicle exhaust. The immunogold labelling visualised in the backscatter electron imaging mode, showed that allergens from pollens, latex and also beta-glucans were bound to and, hence, transported by the combustion particles in ambient air. Thus, combustion particles in ambient air are carriers of allergens and act as depots of allergens inhaled into the airways.

Background: Exposure to ambient particulate matter has been associated with a number of adverse health effects. Particle characteristics such as size, surface area and chemistry seem to influence the negative effects of particles. In this study, combustion particles from vehicle exhaust and wood smoke, currently used in biological experiments, were analysed with respect to microstructure and chemistry., Methods: Vehicle exhaust particles were collected in a road tunnel during two seasons, with and without use of studded tires, whereas wood smoke was collected from a stove with single-stage combustion. Additionally, a reference diesel sample (SRM 2975) was analysed. The samples were characterised using transmission electron microscopy techniques (TEM/HRTEM, EELS and SAED). Furthermore, the elemental and organic carbon fractions were quantified using thermal optical transmission analysis and the content of selected PAHs was determined by gas chromatography-mass spectrometry., Results: Carbon aggregates, consisting of tens to thousands of spherical primary particles, were the only combustion particles identified in all samples using TEM. The tunnel samples also contained mineral particles originating from road abrasion. The geometric diameters of primary carbon particles from vehicle exhaust were found to be significantly smaller (24 +/- 6 nm) than for wood smoke (31 +/- 7 nm). Furthermore, HRTEM showed that primary particles from both sources exhibited a turbostratic microstructure, consisting of concentric carbon layers surrounding several nuclei in vehicle exhaust or a single nucleus in wood smoke. However, no differences were detected in the graphitic character of primary particles from the two sources using SAED and EELS. The total PAH content was higher for combustion particles from wood smoke as compared to vehicle exhaust, whereas no source difference was found for the ratio of organic to total carbon., Conclusion: Combustion particles from vehicle exhaust and residential wood smoke differ in primary particle diameter, microstructure, and PAH content. Furthermore, the analysed samples seem suitable for assessing the influence of physicochemical characteristics of particles on biological responses.

The surface morphology of late colonic lesions in F344 rats treated with 1,2-dimethylhydrazine was studied by scanning electron microscopy. At week 31 after carcinogen treatment, the surface epithelial characteristics of different types of lesions observed in the colonic mucosa were compared, namely classic elevated aberrant crypt foci (ACF), flat lesion and gross tumour. Classic elevated ACF were easily observed as structures with enlarged crypts elevated from the background mucosa. When the various ACF were compared, or when the ACF were compared with the background mucosa, no ultrastructural differences, or differences in the density of goblet cells were found. The flat lesion showed an epithelium without goblet cells and crypts with small openings harbouring a large number of loose, undefined, dysplastic epithelial cells. These changes appeared to be linked to the malignant development since they were also characteristic of the examined tumour.

The authors collected ambient air along two highways in Oslo to investigate the annual variations in particulate matter (PM10) and the presence of latex as an outdoor allergen. PMI, was monitored for a period of five years, during which time the use of studded winter tires was reduced. The presence of latex and of common aeroallergens was examined directly on the collection filters with immunoelectron microscopy visualized in a scanning electron microscope. The annual variation in PM10 was similar over the five years of sampling, with increased mass concentrations in winter. Statistical analysis indicated no major effect from the change to nonstudded tires. The most important factors influencing the PM10 concentration were meteorological parameters like wind and rain. Immnunolabeling of the filters showed latex as an outdoor allergen that adhered to carbon aggregates from vehicle emission. The results also indicated cross-reactive epitopes among the common allergens investigated, which for sensitized subjects may add to the risk of developing latex allergy.

Serogroup A Neisseria meningitidis organisms of the subgroup III have caused epidemics of meningitis in sub-Saharan Africa since their introduction into the continent in 1987. The population structure of these bacteria is basically clonal, and these meningococci are strikingly similar in their major outer membrane antigens PorA and PorB. Protein-based vaccines might be an alternative to prevent epidemics caused by these meningococci; thus, we developed an outer membrane vesicle (OMV) vaccine from a serogroup A meningococcal strain of subgroup III. The serogroup A OMV vaccine was highly immunogenic in mice and elicited significant bactericidal activity towards several other serogroup A meningococci of subgroup III. The IgG antibodies generated were in immunoblot shown to be mainly directed towards the PorA outer membrane protein. The results presented demonstrate the potential of an OMV vaccine as an optional strategy to protect against meningococcal disease caused by serogroup A in Africa.

Uptake of zymosan A particles by human umbilical vein endothelial cells (HUVEC) and its effect on cellular cytokine and oxygen radical production was examined. HUVEC took up more serum-opsonized than -unopsonized zymosan as demonstrated by flow cytometry with fluorescence-labeled particles. The former uptake was inhibited in the presence of anti-C3c antibodies and thus complement-mediated. It probably occurred via CR1 (CD35), although participation of other receptors cannot be ruled out. Scanning electron microscopy indicated that HUVEC with fully internalized zymosan particles were damaged. Prolonged incubation of both serum-opsonized and -unopsonized zymosan particles with HUVEC induced increased secretion of the proinflammatory cytokines IL-6 and IL-8 to the cell culture supernatants, but had no effect on production of oxygen radicals. The results confirm previous reports that EC can internalize yeast and other pathogens and points to complement as a mechanism of uptake, but illustrates that the cells may be damaged in the process. Moreover, EC may participate in the anti-infection defense effort by secreting proinflammatory and chemotactic cytokines in response to the contact with pathogens.

We examined the age-dependent susceptibility to azoxymethane (AOM)-induced and spontaneous tumorigenesis in the Min/+ mouse, a murine FAP model. The colon carcinogen AOM was given to pups (weeks 1 and 2) and young adults (weeks 4 and 5). In the colon, AOM exposure of pups and young adults caused a 17-fold and 10-fold increase (p < 0.001) in the number of dysplastic aberrant crypt foci (ACF) compared with vehicle-treated controls, respectively; the pups were 1.7 times mores susceptible than young adults (p = 0.002). AOM-specific dysplastic ACF grew significantly faster (p < 0.001) in pups than in young adults. In the small intestine of AOM-exposed pups, the number of adenomas was increased 1.5-fold compared with controls (p < 0.001), while a similar exposure of young adults did not affect the tumorigenesis. Dysplastic ACF in the colon were morphologically equivalent with nascent adenomas in the small intestine. When comparing the size distributions of AOM-specific and spontaneous lesions the majority of the spontaneous lesions was apparently initiated before week 2. In conclusion, pups were more susceptible than young adults to AOM-induced and spontaneous tumorigenesis, and neonatal exposure was not as critical for AOM-induced tumorigenesis in the colon as in the small intestine.

Culturing elutriation-purified and cryopreserved human monocytes gives a cell loss of about 60% after a week. The main loss is during the first 24 h when one cell population dies by apoptosis and secondary necrosis, while another survives with minimal signs of apoptosis and necrosis. We have studied this initial cell loss using flow cytometry (FCM) and electron microscopy (EM) in parallel. Thawed cells were cultured in ultra low attachment wells and studied by FCM using Annexin V, Propidium iodide (PI), JC-1, APO2.7 and APO-BrDU. The EM studies comprised both transmission EM (TEM) and scanning EM (SEM), the latter employing cells labelled with antiCD14/gold and Annexin V/gold. Cells were counted by light microscopy to provide cell recoveries. DNA ladder patterns were investigated by electrophoresis. Camptothecin (CAM) was used as an apoptosis inducer. In the first 6 h of culture, there was an apoptotic phase with Annexin V(+)/PI(-) positive cells in FCM, chromatin condensation in TEM, a rapid and short phase with Annexin V/gold positively labelled cells in SEM and the cells disappeared by 6 h. All of these effects were enhanced by CAM. The necrotic phase (6-24 h) was associated with Annexin V(+)/PI(+) in FCM, and the data at 24 h was in agreement with the semiquantitatvive results from TEM. Discrepancies in the results for CD14 and Annexin V between FCM and SEM indicated phagocytosis. APO2.7 and APO-BrDU increases also indicated an accumulation of ingested material in vital cells. Centrifugation of supernatants, labelling pellets with Annexin V/FITC and examination by flow cytometry revealed no Annexin V positive cell fragments. We found evidence of rapid and efficient phagocytosis. CAM not only induced apoptosis, but also appeared to stabilise the cell membrane and increase both cell recovery and phagocytosis.

Complement activation is closely associated with plasma endotoxin levels in patients with meningococcal infections. This study assessed complement activation induced by purified Neisseria meningitidis lipopolysaccharide (Nm-LPS), native outer membrane vesicles (nOMVs), LPS-depleted outer membrane vesicles (dOMVs), wild-type meningococci, and an LPS-free mutant (lpxA(-)) from the same strain (44/76) in whole blood anticoagulated with the recombinant hirudin analogue. Complement activation products (C1rs-C1 inhibitor complexes, C4d, C3bBbP, and terminal SC5b-9 complex) were measured by double-antibody EIAs. Nm-LPS was a weak complement activator. Complement activation increased with preparations containing nOMVs, dOMVs, and wild-type bacteria at constant LPS concentrations. With the same protein concentration, complement activation induced by nOMVs, dOMVs, and the LPS-free mutant was equal. The massive complement activation observed in patients with fulminant meningococcal septicemia is, presumably, an indirect effect of the massive endotoxemia. Outer membrane proteins may be more potent complement activators than meningococcal LPSs.

The multiple intestinal neoplasia (Min)/+ mouse, which harbors only one functional allele of the Apc gene, is susceptible to environmental factors that disrupt this gene and subsequently trigger Apc-driven tumorigenesis in the colon. Aberrant crypt foci (ACF) are assumed to be preneoplastic lesions in colon carcinogenesis. Recently, we reported the absence of "classical" ACF in the colon of untreated Min/+ mice. Instead we identified flat dysplastic lesions, which we denoted ACF(Min) (J. E. Paulsen et al., Scand. J. Gastroenterol., 35: 534-539, 2000). In contrast to the classical type, ACF(Min) are not elevated above the surrounding mucosa, and their detection is totally dependent on methylene blue staining and transillumination. In the present study, we treated Min/+ mice with 5 mg/kg body weight azoxymethane (AOM) at weeks 1 and 2 and demonstrated induction of both types of lesions. However, only ACF(Min) appeared to be associated with the development of adenomas. Monocryptal ACF(Min), large ACF(Min), and adenomas showed a uniform histopathological picture of dysplasia and cytoplasmic overexpression of beta-catenin, indicating a qualitative relationship between these lesions. Also a quantitative relationship was suggested because the dramatic decrease in ACF(Min) number from week 7 to 11 was paralleled by a reciprocal increase in tumor number, indicating fast-crypt multiplication of ACF(Min). In AOM-treated +/+ (wild-type) littermates, a low number of ACF(Min) and tumors with the same characteristics as in Min/+ mice was seen. In contrast to ACF(Min), histopathological and immunohistochemical examination of classical ACF showed normal or hyperplastic crypts with normal levels of beta-catenin expression. In AOM-treated Min/+ mice, the number of classical ACF was virtually constant from week 7 to 11, and only a modest increase of crypt multiplicity was observed. The number of AOM-induced classical ACF at week 11 was not different in Min/+ mice and +/+ mice. In conclusion, we identified two distinct populations of altered crypts in the colon of Min/+ mice after AOM treatment. The ACF(Min), which resemble the dysplastic ACF described previously, clearly showed a continuous development from the monocryptal stage to adenoma, and they were characterized by fast-growing crypts with altered control of beta-catenin. In contrast, the classical ACF, which resemble the hyperplastic ACF described previously, were characterized by slow-growing crypts with normal beta-catenin expression, and they were probably not related to tumorigenesis.

Whole killed meningococci (Nm) and pertussis bacteria (Bp) were tested for mucosal immunogenicity and as mucosal adjuvants for an inactivated influenza virus vaccine given intranasally to unanaesthetized mice. Virus was given alone, or simply mixed with one of the bacterial preparations, in four doses at weekly intervals. The virus alone induced low but significant increases of influenza-specific IgG antibodies in serum measured by ELISA, whereas IgA responses in serum and saliva were insignificant compared to non-immunized controls. With Bp or Nm admixed, serum IgG and IgA and salivary IgA responses to the influenza virus were substantially augmented (P<0.005). However, this adjuvant effect of the bacterial preparations was not significant for responses in the intestine as measured by antibodies in faeces. Antibody responses to Bp itself, but not to Nm, were inhibited by the admixture of the virus vaccine. Moreover, the pertussis preparation induced salivary antibodies which cross-reacted with Nm. Whole-cell bacteria with inherent strong mucosal immunogenicity may also possess mucosal adjuvanticity for admixed particulate antigens which are weakly immunogenic by the nasal route.

Neisseria meningitidis, the cause of epidemic meningitis and acute lethal sepsis, synthesizes surplus lipopolysaccharides (LPSs) during growth, which are released as outer membrane vesicles (OMV) or "blebs". Meningococcal disease severity is related to plasma LPS levels. We have compared the biological activities of native outer membrane vesicles (nOMV) to those of purified Nm-LPS (Nm-LPS) and LPS-depleted OMV (dOMV) prepared from N. meningitidis. The LPS content of nOMV was determined spectrophotometrically by quantifying KDO and by silver-stained SDS-PAGE gels. The morphology of the preparations was studied by transmission electron microscopy. The Limulus amoebocyte lysate (LAL) assay was used to quantify LPS in the plasma solutions. The preparations were diluted in endotoxin-free heparin plasma to equal amounts of LPS (w/w) in the range 50-5000 pg/ml. The biological reactivity was tested by: (i) a monocyte target-assay (monocyte purity > or =96%); and (ii) a whole blood model, measuring the secretion of TNF-alpha and IL-6 induction of procoagulant activity in monocytes (PCA). In both models, nOMV induced dose-dependent cell responses (TNF-alpha, IL-6, PCA) similar to purified Nm-LPS, whereas dOMV induced minimal responses. However, LAL activity was significantly higher for nOMV than for purified Nm-LPS and dOMV. The cellular responses of purified Nm-LPS and nOMV were reduced (>95%) by a specific anti-CD14-antibody.

Background and Objectives: The published sequence of the weak. A subgroup Ael gene from Swedish individuals showed a G insertion in exon VII, causing a frameshift at codon 268 (the A1 gene has 353 codons). We wished to sequence exons VI and VII of two Norwegian Ael individuals and compare the expression of A substance on RBC from different Ael individuals., Materials and Methods: Exon VI and VII were amplified by PCR, cloned in M13 and sequenced. A structure expression on Ael RBC was studied by the immunogold technique., Results: In contrast to the Swedish Ael individuals, the two Norwegians had consensus A1 sequences in exon VI and VII. However, the patterns of A expression were indiscernible from the Swedish cases as visualized by immunogold labeling in SEM. In both cases, a few (1-2%) RBC were very strongly labeled, some were weakly labeled and the majority (95%) were unlabeled., Conclusion: Although some Ael individuals have an inserted nucleotide in exon VII of the ABO gene, others have consensus A1 sequence in exon VI and VII. However, we could not find any differences in phenotype by immunogold labeling in SEM.

A Norwegian outer membrane vesicle (OMV) vaccine against group B meningococcal disease proved to be strongly immunogenic when administered intranasally in mice. The OMV preparation, made from Neisseria meningitidis and intended for parenteral use, was therefore given without adjuvant to human volunteers (n = 12) in the form of nose drops or nasal spray. Such immunizations, which were carried out at weekly intervals during a three-week period, were able to induce systemic antibodies with bactericidal activity in more than half of the individuals. In addition, all vaccinees developed marked increases in OMV-specific IgA antibodies in nasal secretions. The potential of the OMV particles as carriers for other less immunogenic antigens were elucidated in mice with use of whole inactivated influenza virus. Even though influenza virus alone did induce some systemic and salivary antibody responses after being administered intranasally, these responses were greatly augmented when the virus was presented together with OMVs. Thus, it is possible that a nasal OMV vaccine may induce protection against invasive meningococcal disease, and also that it might be used as a vehicle for nasal vaccines against other diseases.

We wanted to compare the potential protective capacity of antibodies to meningococcal lipopolysaccharides (LPS). The frequency of occurrence and degree of expression of the epitopes recognized by murine monoclonal antibodies (MAbs) to immunotypes L3,7,9 (9-2-L379) and L8 (2-1-L8) and to the LPS inner core (216-Lc and 217-Lc), were determined among 77 consecutive Norwegian meningococcal patient isolates from 1995. The immunotype L3,7,9 was strongly expressed by 95% of the isolates, whereas L8 was weakly to moderately expressed by 9%. The inner core epitopes, were widely distributed among the serogroup B organisms, but were proved weakly expressed. The bactericidal activity of the four MAbs to various selected strains, was found to correlate positively with the quantity of the LPS epitopes recognized by these four MAbs in the bacteria. When tested in the serum bactericidal assay (SBA), often a few percent of the colonies of the inocula survived high concentrations of the MAbs. The results indicate that escape from the bactericidal action could be achieved through: (i) selection of variants not expressing the LPS-epitope of the actual MAb, (ii) a relative reduction in the density of the LPS-epitope achieved by dilution with another LPS structure or (iii) other factors, not yet understood. In conclusion, antibodies to the L3,7,9 epitope seem to be of importance for protection, whereas antibodies to the epitopes of the LPS inner core or immunotype L8, are not likely to offer protection alone. However, in order to prevent escape through alteration of the LPS pattern of the microbes, various LPS structures should probably be present in the OMV vaccine.

Although quite common in the eukaryotic cell, bacterial proteins with an extensive coiled-coil domain are still relatively rare. One of the few thus far documented examples, TlpA from Salmonella typhimurium, is characterized by a remarkably long (250 amino acids) alpha-helical coiled-coil domain. Herein, we demonstrate that TlpA is a novel sequence-specific DNA-binding protein. Several tlpA deletion mutants have been constructed, and their corresponding protein products were purified and tested for DNA binding. Two of the mutant proteins were shown to be deficient in DNA binding. Both mutants were analyzed by circular dichroism and electron microscopy, supporting the notion that mutant proteins wre shown to be deficient in DNA binding. Both mutants were analyzed by circular dichroism and electron microscopy, supporting the notion that mutant proteins were largely intact despite lacking the amino acid residues necessary for DNA binding. In vivo studies with transcriptional tlpA-lacZ fusions demonstrated that TlpA acts as a repressor. Using the repressor phenotype as a readout, the chain exchange previously described in vitro could also be confirmed in vivo. We believe the coiled-coil domain acts not only as a dimerization interface but could also serve a role as a flexible modulator of the protein-DNA interaction.

Expression of type IV pili appears to be a requisite determinant of infectivity for the strict human pathogens Neisseria gonorrhoeae and Neisseria meningitidis. The assembly of these colonization factors is a complex process. This report describes a new pilus-assembly gene, pilG, that immediately precedes the gonococcal (Gc) pilD gene encoding the pre-pilin leader peptidase. The nucleotide sequence of this region revealed a single complete open reading frame whose derived polypeptide displayed significant identities to the pilus-assembly protein PilC of Pseudomonas aeruginosa and other polytopic integral cytoplasmic membrane constituents involved in protein export and competence. A unique polypeptide of M(r) 38 kDa corresponding to the gene product was identified. A highly related gene and flanking sequences were cloned from a group B polysaccharide-producing strain of N. meningitidis (Mc). The results indicate that the pilG genes and genetic organization at these loci in Gc and Mc are extremely conserved. Hybridization studies strongly suggest that pilG-related genes exist in commensal Neisseria species and other species known to express type IV pili. Defined genetic lesions were created by using insertional and transposon mutagenesis and moved into the Gc and Mc chromosomes by allelic replacement. Chromosomal pilG insertion mutants were devoid of pili and displayed dramatically reduced competence for transformation. These findings could not be ascribed to pilin-gene alterations or to polarity exerted on pilD expression. The results indicated that PilG exerts its own independent role in neisserial pilus biogenesis.

The surface of the colon mucosa of 1,2-dimethylhydrazine-treated F344 rats was examined with the scanning electron microscope. A detailed examination of the mucosal topography revealed foci with one to several aberrant crypts. These were seen as structures elevated from the background mucosa. The shape of the luminal openings of the aberrant crypts varied from elongated or tortuous to circular. However, we found no ultrastructural variations between the different aberrant crypt foci (ACF) or between the ACF and the background mucosa. There was no direct relationship between the size of ACF and the number of aberrant crypts per focus, which may be explained by the mechanism of crypt fission; in two aberrant crypts we discovered the formation of a transverse epithelial septum, dividing the large crypt into two smaller crypts. The gross morphology of the ACF observed by scanning electron microscopy and light microscopy was in principle the same.

The majority of mouse monoclonal antibodies reacting with blood group epitopes on erythrocytes are of the IgM class, have equal light chain type, and are available as culture supernatants only. To study the interrelationship of the blood group antigens, a method is presented which allows double labeling applying two unconjugated monoclonal antibodies of the same class and species. The method comprises two indirect, sequential labelings using mouse IgM anti-A and anti-H as primary antibodies and two goat anti-mouse IgM conjugated to 30 and 20 nm colloidal gold particles as secondary antibodies. After labeling for the first antigen, free binding sites on the primary antibody are blocked by incubation with an unconjugated goat anti-mouse antibody. The free anti-species on the secondary antibody, conjugated to 30 nm gold particles, are inactivated by silver enhancement. The silver enhancement also enlarges the gold particle for optimal discrimination between the two particle sizes, which are chosen accordingly. Semiquantitations of double labeled cells from subgroup A2 and A3 were found to be in good agreement with the counts of the corresponding single labelings as well as between experiments, irrespective of which of the two antibodies was applied in the first labeling sequence. The results were in accordance with a reciprocal but nonlinear relationship between the A and H antigens and suggest different affinities of the two antibodies for the epitopes in the subgroups investigated, indicating different biochemistry of the antigen determinants.

Scanning immune electron microscopy using a monoclonal anti-A antibody which reacts with all type A oligosaccharide chains revealed A antigens on less than 5% of Am and Ael cells, some of which showed extremely strong labelling. This explains why Am and Ael cells can absorb significant amounts of anti-A without being agglutinated. A3 may be a heterogeneous subgroup, since A antigens were found on 82 and 58%, respectively, of the cells of 2 A3 individuals. A antigens were found on 75% or more of Ax cells. In many weak A individuals A-positive cells are apparently best detected if an anti-A is used which reacts strongly with other A oligosaccharide chains than type 2. From hyperimmune pregnancy sera Ax, Am and Ael erythrocytes absorbed antibodies which seemed to have other fine specificities than those absorbed by A2 cells. We conclude that weak subgroups of A may deviate from A2 both by number of erythrocytes expressing A antigens and the biochemical nature of the antigens.

A vaccine against serogroup B meningococcal disease has been prepared from a B:15:P1.7,16 meningococcal strain (44/76) by fermentor growth and extraction of the bacteria with the detergent deoxycholate. Outer membrane vesicles (OMV) were purified by ultracentrifugation and adsorbed to aluminium hydroxide adjuvant. OMV contained the major class 1, 3, 4 and 5 proteins and some minor high molecular weight protein components. Relative to protein, the vaccine also contained about 8% phospholipid, 7% lipopolysaccharide and 16% deoxycholate. The product was generally non-pyrogenic to humans in ordinary doses and was highly immunogenic in mice and humans. Production and control steps, physical, chemical and immunological data for the vaccine are described.

The effect of interferon treatment on the herpes simplex virus type 1 (HSV-1)-specific glycoproteins gC and gE in homologous and heterologous cells has been investigated. In human embryonic fibroblastic cells, human leukocyte interferon inhibited virus multiplication and expression of the HSV-1-specific glycoproteins gC and gE on the cell surface in a dose-dependent manner. In heterologous baby hamster kidney cells, the human interferon had no effect on virus multiplication. However, the surface expression of the HSV-1-specific glycoproteins was reduced, as shown by erythrocyte rosette formation, by attachment of monodisperse polystyrene particles coated with antibodies and by immunogold scanning electron microscopy.

Mica is proposed as a convenient substrate for SEM specimens. Mechanically, mica is rigid and can be cut, punched and cleaved into suitable sizes. Compared to glass substrates, mice seems to be more charge 'resistant' when irradiated with the same beam density in the microscope.

The effect of glow-discharging on naked carbon-filmed grids has been evaluated. The effect of different locations of the grids within a DC discharge, operated in the normal glow, was analyzed by applying various biological specimens to the grids. Two locations were found to give consistent results: (a) in Crookes dark space, particulate specimens, negatively stained, spread evenly--suggesting a new negative surface charge of the support; (b) below the anode, nucleic acids, selectively (positively) stained, appeared as well spread filaments, indicating a net positive surface charge.

Silver enhancement of immunogold-labeled cells was carried out to increase the applicability of colloidal gold probes for visualization in the backscatter electron imaging (BEI) mode of a scanning electron microscope. Optimum conditions were established for single particle discrimination and differential counting of labeling density at low magnifications. Red blood cells double-labeled with 15 + 40 nm and 5 + 20 nm gold probes were silver-enhanced for 6 min and 20 min, respectively, at which times both pairs of labels increased to about 25 + 50 nm. The gold probes still appeared spherical after enhancement and were easily discriminated. Cells were also single-labeled with the above probes and enhanced accordingly. The present method enables visualization of individual particles of any probe size, labeling one, or simultaneously two, antigenic sites on cell surfaces. The silver enhancement procedure thereby allows cells to be labeled with small probes with increased labeling efficiency.

Electron microscopy revealed 2 different types of bacteriophages isolated from Actinobacillus actinomycetemcomitans colonizing exclusively diseased sites in 4 patients with localized juvenile periodontitis (LJP). All sites infected with phage were undergoing periodontal destruction, as judged from consecutive routine radiographs. The phages isolated had a wide host range as assessed from their ability to infect a series of reference strains of A. actinomycetemcomitans. A 5th patient harboured non-infected A. actinomycetemcomitans in a surgically treated site which had undergone no bone destruction during the last 12 months. The present findings suggested that the pathogenic potential of A. actinomycetemcomitans in LJP may increase due to phage infection.

Preparatory procedures and different types of electron microscopical imaging of bacterial specimens are described. Transmission electron microscopy (TEM) was performed on ultra-thin sections of plastic embedded material and negatively stained specimens. Metal coated, whole bacteria were prepared for scanning electron microscopy (SEM). The rapid procedure for TEM observation of negatively stained specimens indicates size, shape and appendages. TEM micrographs of ultra-thin sections show details of the cell wall and internal structures of the cytoplasm. SEM is superior in demonstrating size and gross surface morphology. The three methods are complementary in the study of bacterial cell ultrastructural morphology.

Binding patterns of monoclonal IgM anti-B, anti-H and anti-(Leb + Y) antibodies on erythrocytes were visualized in the backscatter electron imaging (BEI) mode of a scanning electron microscope by indirect immunogold labelling. The results were independent of secretor status, supporting the view that type 2 oligosaccharides predominate on erythrocytes. There were considerable cell-to-cell variations of amounts of the antigens, presumably because of differences of antigen development. The amount of H antigens may have been overestimated in previous studies. B and H antigens seemed linked mostly to mobile structures, probably lipid, on the convex parts of the cells and to immobile structures, probably protein, on the concave parts. Y antigens appeared bound to mobile structures also on the concave part of the cell membrane. The results therefore confirm that B oligosaccharides on erythrocytes develop from both lipid- and protein-bound H, but suggest that Y develops mainly from lipid-bound H.

Actinobacillus actinomycetemcomitans was isolated from periodontal pockets in a patient suffering from prepubertal periodontitis. Electron microscopy revealed 3 different groups of bacteriophages in filtrates of subgingival plaque from all the active periodontal lesions. Phage infected A. actinomycetemcomitans in this patient was restricted to periodontal pockets which, according to standardized roentgenograms, had shown bone destruction during the past 12 months. A follow-up study of 7 months revealed that a "burned out" site which harbored noninfected A. actinomycetemcomitans, turned into an active site at the same time as the A. actinomycetemcomitans of that site became infected with the phages. These findings indicate a relationship between rapid prepubertal periodontal destruction and phage-infected A. actinomycetemcomitans.

In order to differentiate between bacterial adhesiveness and invasiveness in cell culture monolayers, a method is reported which enables the same specimen to be examined by both scanning electron microscopy and Nomarski interference contrast microscopy. The source of error made by statistical variation when the two microscopical techniques are used individually is thus avoided.