Automated image analysis for quantification of filamentous bacteria.

BACKGROUND: Antibiotics of the ß-lactam group are able to alter the shape of the bacterial cell wall, e.g. filamentation or a spheroplast formation. Early determination of antimicrobial susceptibility may be complicated by filamentation of bacteria as this can be falsely interpreted as growth in systems relying on colorimetry or turbidometry (such as Vitek-2, Phoenix, MicroScan WalkAway). The objective was to examine an automated image analysis algorithm for quantification of filamentous bacteria using the 3D digital microscopy imaging system, oCelloScope. RESULTS: Three E. coli strains displaying different resistant profiles and differences in filamentation kinetics were used to study a novel image analysis algorithm to quantify length of bacteria and bacterial filamentation. A total of 12 ß-lactam antibiotics or ß-lactam-ß-lactamase inhibitor combinations were analyzed for their ability to induce filamentation. Filamentation peaked at approximately 120 min with an average cell length of 30 µm. CONCLUSION: The automated image analysis algorithm showed a clear ability to rapidly detect and quantify ß-lactam-induced filamentation in E. coli. This rapid determination of ß-lactam-mediated morphological alterations may facilitate future development of fast and accurate AST systems, which in turn will enable early targeted antimicrobial therapy. Therefore, rapid detection of ß-lactam-mediated morphological changes may have important clinical implications.

The impact of structural integrity and route of administration on the antibody specificity against three cow's milk allergens - a study in Brown Norway rats.

BACKGROUND: Characterisation of the specific antibody response, including the epitope binding pattern, is an essential task for understanding the molecular mechanisms of food allergy. Examination of antibody formation in a controlled environment requires animal models. The purpose of this study was to examine the amount and types of antibodies raised against three cow's milk allergens; ß-lactoglobulin (BLG), α-lactalbumin (ALA) and ß-casein upon oral or intraperitoneal (i.p.) administration. A special focus was given to the relative amount of antibodies raised against linear versus conformational epitopes. METHODS: Specific antibodies were raised in Brown Norway (BN) rats. BN rats were dosed either (1) i.p. with the purified native cow's milk allergens or (2) orally with skimmed milk powder (SMP) alone or together with gluten, without the use of adjuvants. The allergens were denatured by reduction and alkylation, resulting in unfolding of the primary structure and a consequential loss of conformational epitopes. The specific IgG1 and IgE responses were analysed against both the native and denatured form of the three cow's milk allergens, thus allowing examination of the relative amount of linear versus conformational epitopes. RESULTS: The inherent capacity to induce specific IgG1 and IgE antibodies were rather similar upon i.p. administration for the three cow's milk allergens, with BLG = ALA > ß-casein. Larger differences were found between the allergens upon oral administration, with BLG > ALA > ß-casein. Co-administration of SMP and gluten had a great impact on the specific antibody response, resulting in a significant reduced amount of antibodies. Together results indicated that most antibodies were raised against conformational epitopes irrespectively of the administration route, though the relative proportions between linear and conformational epitopes differed remarkably between the allergens. CONCLUSIONS: This study showed that the three-dimensional (3D) structure has a significant impact on the antibodies raised for both systemic and orally administered allergens. A remarkable difference in the antibody binding patterns against linear and conformational epitope was seen between the allergens, indicating that the structural characteristics of proteins may heavily affect the induced antibody response.

Acid hydrolysis of wheat gluten induces formation of new epitopes but does not enhance sensitizing capacity by the oral route: a study in "gluten free" Brown Norway rats.

BACKGROUND: Acid hydrolyzed wheat proteins (HWPs) are used in the food and cosmetic industry as emulsifiers. Cases of severe food allergic reactions caused by HWPs have been reported. Recent data suggest that these reactions are caused by HWPs produced by acid hydrolysis. OBJECTIVES: To examine the sensitizing capacity of gluten proteins per se when altered by acid or enzymatic hydrolysis relative to unmodified gluten in rats naïve to gluten. METHODS: High IgE-responder Brown Norway (BN) rats bred on a gluten-free diet were sensitized without the use of adjuvant to three different gluten products (unmodified, acid hydrolyzed and enzymatic hydrolyzed). Rats were sensitized by intraperitoneal (i.p.) immunization three times with 200 µg gluten protein/rat or by oral dosing for 35 days with 0.2, 2 or 20 mg gluten protein/rat/day. Sera were analyzed for specific IgG and IgE and IgG-binding capacity by ELISA. IgE functionality was measured by rat basophilic leukemia (RBL) assay. RESULTS: Regardless of the route of dosing, all products had sensitizing capacity. When sensitized i.p., all three gluten products induced a strong IgG1 response in all animals. Acid hydrolyzed gluten induced the highest level of specific IgE but with a low functionality. Orally all three gluten products induced specific IgG1 and IgE but with different dose-response relations. Sensitizing rats i.p. or orally with unmodified or enzymatic hydrolyzed gluten induced specific IgG1 responses with similar binding capacity which was different from that of acid hydrolyzed gluten indicating that acid hydrolysis of gluten proteins induces formation of 'new' epitopes. CONCLUSIONS: In rats not tolerant to gluten acid hydrolysis of gluten enhances the sensitizing capacity by the i.p. but not by the oral route. In addition, acid hydrolysis induces formation of new epitopes. This is in contrast to the enzymatic hydrolyzed gluten having an epitope pattern similar to unmodified gluten.

Assessment of the sensitizing potential of processed peanut proteins in Brown Norway rats: roasting does not enhance allergenicity.

BACKGROUND: IgE-binding of process-modified foods or proteins is the most common method for examination of how food processing affects allergenicity of food allergens. How processing affects sensitization capacity is generally studied by administration of purified food proteins or food extracts and not allergens present in their natural food matrix. OBJECTIVES: The aim was to investigate if thermal processing increases sensitization potential of whole peanuts via the oral route. In parallel, the effect of heating on sensitization potential of the major peanut allergen Ara h 1 was assessed via the intraperitoneal route. METHODS: Sensitization potential of processed peanut products and Ara h 1 was examined in Brown Norway (BN) rats by oral administration of blanched or oil-roasted peanuts or peanut butter or by intraperitoneal immunization of purified native (N-), heated (H-) or heat glycated (G-)Ara h 1. Levels of specific IgG and IgE were determined by ELISA and IgE functionality was examined by rat basophilic leukemia (RBL) cell assay. RESULTS: In rats dosed orally, roasted peanuts induced significant higher levels of specific IgE to NAra h 1 and 2 than blanched peanuts or peanut butter but with the lowest level of RBL degranulation. However, extract from roasted peanuts was found to be a superior elicitor of RBL degranulation. Process-modified Ara h 1 had similar sensitizing capacity as NAra h 1 but specific IgE reacted more readily with process-modified Ara h 1 than with native. CONCLUSIONS: Peanut products induce functional specific IgE when dosed orally to BN rats. Roasted peanuts do not have a higher sensitizing capacity than blanched peanuts. In spite of this, extract from roasted peanuts is a superior elicitor of RBL cell degranulation irrespectively of the peanut product used for sensitization. The results also suggest that new epitopes are formed or disclosed by heating Ara h 1 without glucose.

Sensitization with 7S globulins from peanut, hazelnut, soy or pea induces IgE with different biological activities which are modified by soy tolerance.

BACKGROUND: It is not known why some foods sensitizing via the gastrointestinal tract are prevalent allergenic foods and others are not. Eating habits, processing, and the food matrix have been suggested to influence the allergenicity of a given food. Factors related to protein structure, such as stability to digestion, have also been suggested. 7S globulins from peanut, hazelnut, soy, and pea were studied to determine whether related proteins would induce a similar sensitization when removed from their 'normal' matrix. METHODS: Brown Norway rats (soy tolerant or nontolerant) were immunized i.p. 3 times with 100 µg purified peanut, hazelnut, soy, or pea 7S without adjuvant. Sera were analyzed for specific antibodies by different ELISAs (IgG1, IgG2a, and IgE), inhibition ELISA, and rat basophilic leukemia cell assay. RESULTS: The 4 related 7S globulins induced a response with an almost identical level of specific antibodies, but peanut 7S induced IgE of higher avidity than hazelnut and pea 7S which, again, had a higher avidity than IgE induced by soy 7S. Soy tolerance reduced the functionality of IgE without influencing antibody titers. CONCLUSIONS: Although the 4 7S globulins are structurally related allergens, they induce antibodies with different antigen-binding characteristics. Peanut 7S induces IgE of a higher avidity than hazelnut and pea 7S which, again, has a higher avidity than IgE induced by soy 7S. We also show that soy tolerance influences the function of antibodies to peanut 7S. These findings may help explain how antibodies of different clinical significances can develop in different individuals sensitized to the same allergen.

Immunotoxicological studies of genetically modified rice expressing PHA-E lectin or Bt toxin in Wistar rats.

As part of the SAFOTEST project the immunmodulating effect of Cry1Ab protein from Bacillus thuringiensis (Bt) and PHA-E lectin from kidney bean (Phaseolus vulgaris erythroagglutinin) was examined in 28- and 90-day feeding studies in Wistar rats. PHA-E lectin was chosen as positive control. Rats were fed control rice, transgenic rice expressing Cry1Ab protein or PHA-E lectin, or transgenic rice spiked with the purified recombinant protein. Total immunoglobulin levels, mitogen-induced cell proliferation, T-dependent antibody response to sheep red blood cells and the antigen-specific antibody response in serum were examined at the end of the studies. A dose-dependent increase in mesenteric lymph node weight and total immunoglobulin A was seen when feeding PHA-E transgenic rice alone or spiked with 0.1% purified PHA-E lectin for 90 days indicating a local effect of PHA-E in the intestine. No adverse effects of Cry1Ab protein were found. An anti-PHA-E and anti-Cry1Ab antibody response was induced both after inhalation (control groups) and after inhalation/ingestion (groups fed recombinant protein alone or together with transgenic rice). In conclusion, only PHA-E lectin was found to have an immunomodulating effect when feeding rats for 90 days with approximately 70 mg PHA-E/kg bodyweight per day. As both PHA-E lectin and Cry1Ab protein were capable of inducing an antigen-specific antibody response it is important to make careful considerations when designing future animal studies to avoid intake of proteins from the other groups by inhalation as well as to examine the sensitization and elicitation potential of 'foreign' proteins before introduction to the world market.

A 90-day safety study in Wistar rats fed genetically modified rice expressing snowdrop lectin Galanthus nivalis (GNA).

Genetically modified plants expressing insecticidal traits offer a new strategy for crop protection, but at the same time present a challenge in terms of food safety assessment. The present 90-day feeding study was designed to assess the safety of a rice variety expressing the snowdrop Galanthus nivalis lectin (GNA lectin), and forms part of a EU-funded project where the objective has been to develop and validate sensitive and specific methods to assess the safety of genetically modified foods. Male and female Wistar rats were given a purified diet containing either 60% genetically modified or parental rice for 90 days. This corresponds to a mean daily GNA lectin intake of approximately 58 and 67mg/kg body weight for males and females, respectively. Prior to the animal study comprehensive analytical characterization of both rice materials was performed. The chemical analyses showed a number of statistically significant differences, with the majority being within the ranges reported in the literature. In the animal study a range of clinical, biological, immunological, microbiological and pathological parameters were examined. A number of significant differences were seen between groups fed the two diets, but none of them were considered to be adverse. In conclusion, the design of the present animal study did not enable us to conclude on the safety of the GM food. Additional group(s) where the expressed gene products have been spiked to the diet should be included in order to be able to distinguish whether the observed effects were due to the GNA lectin per se or to secondary changes in the GM rice.

Safety testing of GM-rice expressing PHA-E lectin using a new animal test design.

The 90-day animal study is the core study for the safety assessment of genetically modified foods in the SAFOTEST project. The model compound tested in the 90-day study was a rice variety expressing the kidney bean Phaseolus vulgaris lectin agglutinin E-form (PHA-E lectin). Female Wistar rats were given a nutritionally balanced purified diet with 60% parental rice, 60% PHA-E rice or 60% PHA-E rice spiked with 0.1% recombinant PHA-E lectin for 90 days. This corresponded to a mean daily PHA-E lectin intake of approximately 0, 30 and 100mg/kg body weight for each group, respectively. The spiking was used to increase the specificity and to demonstrate the sensitivity of the study. A range of biological, biochemical, microbiological and pathological parameters were examined and significant differences in weight of small intestine, stomach and pancreas and plasma biochemistry were seen between groups. Included in this paper are also data from the molecular characterisation and chemical analysis of the PHA-E rice, from the construction and production of the PHA-E lectin, and from the preceding 28-day in vivo study where the toxicity of the pure PHA-E lectin was determined. In conclusion, the combined use of information from the compositional analysis, the 28-day study and the characterisation of the PHA-E rice and the PHA-E lectin has improved the design of the 90-day study. The spiking procedure has facilitated the interpretation of the results of the study and transferred it into a valuable tool for the future safety testing of genetically modified foods.

A 90-day safety study of genetically modified rice expressing Cry1Ab protein (Bacillus thuringiensis toxin) in Wistar rats.

An animal model for safety assessment of genetically modified foods was tested as part of the SAFOTEST project. In a 90-day feeding study on Wistar rats, the transgenic KMD1 rice expressing Cry1Ab protein was compared to its non-transgenic parental wild type, Xiushui 11. The KMD1 rice contained 15mg Bt toxin/kg and based on the average feed consumption the daily intake was 0.54mg Bt toxin/kg body weight. No adverse effects on animal behaviour or weight gain were observed during the study. Blood samples collected one week prior to sacrifice were analyzed and compared for standard haematological and biochemical parameters. A few parameters were significantly different, but all within the normal reference intervals for rats of this breed and age and not in relation to any other findings, thus not considered treatment related. Upon sacrifice a large number of organs were weighed, macroscopic and histopathological examinations were performed with only minor changes to report. The aim of the study was to use a known animal model in performance of safety assessment of a GM crop, in this case KMD1 rice. The results show no adverse or toxic effects of KMD1 rice when tested in the design used in this 90-day study. Nevertheless the experiences from this study lead to the overall conclusion that safety assessment for unintended effects of a GM crop cannot be done without additional test group(s).

Experimental parameters differentially affect the humoral response of the cholera-toxin-based murine model of food allergy.

BACKGROUND: Recent studies have developed a murine model of IgE-mediated food allergy based on oral coadministration of antigen and cholera toxin (CT) to establish a maximal response for studying immunopathogenic mechanisms and immunotherapeutic strategies. However, for studying subtle immunomodulating factors or factors effective during response initiation, this maximal response-based model is less suitable due to a lack of sensitivity. Therefore, in attempts to identify essential parameters to fine-tune the immune response towards a submaximal level, potentially more sensitive, we were interested in characterizing the individual effects of the parameters in the CT-based model: CT dose, antigen type and dose, and number of immunizations. METHODS: BALB/c mice were orally sensitized weekly for 3 or 7 weeks with graded doses of CT and various food antigens (soy-trypsin inhibitor, ovalbumin or ovomucoid). Antigen-specific IgG1, IgG2a, IgA and IgE were monitored by ELISA. RESULTS: The CT dose exerted a clear dose-dependent effect on the antigen-specific antibody response whereas the antigen dose tended to affect the kinetics of the developing response. Both the intensity and kinetics of the antibody response depended on the type of antigen and number of immunizations. CONCLUSIONS: The critical parameters of the CT-based murine allergy model differentially control the intensity and kinetics of the developing immune response. Adjustment of these parameters could be a key tool for tailoring the response to submaximal levels rendering the model potentially more sensitive for evaluating the effect of subtle immunomodulating factors that would be lost in the maximal response-based model.

Occurrence of the carcinogenic Bracken constituent ptaquiloside in fronds, topsoils and organic soil layers in Denmark.

Bracken (Pteridium aquilinum (L.) Kuhn) is a common fern found on all continents except Antarctica. It is under suspicion of causing cancer among people who utilizes it as food. The main carcinogenic compound is thought to be the water-soluble compound ptaquiloside. Ptaquiloside-uptake may occur not only through food, but also via drinking water as ptaquiloside might leach from plant material. The purpose of the study was to identify environmental parameters that correlate with the ptaquiloside-content in fronds, and to quantify the amount of ptaquiloside in the soil environment. The ptaquiloside-content in fronds, Oi/Oe-, and Oa/A-horizons was quantified at end of the growth season at 20 sites in Denmark. The fronds had ptaquiloside-contents between 108 and 3795 microgg(-1). The Oi/Oe-horizons had contents between 0.09 and 7.70 microgg(-1), while Oa/A-horizons had contents between 0.01 and 0.09 microgg(-1). The ptaquiloside-content in the standing biomass, which could be transferred to the soil by the end of the growing season, ranged between 10 and 260 mgm(-2), with nine sites having ptaquiloside loads over 100 mgm(-2). The carbon-content in the O-horizon, the precipitation, the amount of Bracken-litter, the turnover rate and the size of Bracken-stands determined the ptaquiloside-content in the soil materials while the content in fronds was found to be a function of the frond-height and the light-exposure in the ecosystem.