Marta Granström

The standardization of an assay for pertussis toxin and antitoxin in microplate culture of Chinese hamster ovary cells

A microplate assay, based on the clustering effect induced by pertussis toxin (PT) in Chinese hamster ovary (CHO) cells, has been developed and standardized. Toxin titration is done directly in the culture microplate by twofold dilutions of 25 microliters of test material to which are added 10 000 freshly trypsinized cells in 200 microliters of culture medium per well. The dilution causing the clustering effect is determined by direct microscopic observation after 48 h of incubation. The method allows detection of 50-100 pg toxin per millilitre. For determination of neutralizing antibodies (antitoxin), twofold dilutions of 25 microliters of antiserum are first made directly in the culture microplate. Thereafter 25 microliters of toxin, containing four times the minimal clustering concentration, is added to each well. After three hours for neutralization at +37 degrees C, cells are added, incubated and examined as above. The assay has been found to be simple and reproducible for measuring the antibody response to PT in human and different animal sera. For titration of bacteria associated toxin, the CHO cells are seeded and incubated for 24 h before the addition of bacteria. Incubation and examination are done as described for toxin titration.

Concordance of Helicobacter pylori Strains within Families

ABSTRACT Helicobacter pylori infection is typically acquired in early childhood, and a predominantly intrafamilial transmission has been postulated. To what extent family members share the same strains is poorly documented. Our aim was to explore patterns of shared strains within families by using molecular typing. Family members of H. pylori-infected 10- to 12-year-old index children identified in a school survey were invited to undergo gastroscopy. Bacterial isolates were typed with random amplified polymorphic DNA and PCR-restriction fragment length polymorphism of the genes ureA-B, glmM, or flaA. The presence or absence of the cag pathogenicity island, a bacterial virulence factor, was determined by PCR. GelCompar II software, supplemented with visual inspection, was used in the cluster analysis. In 39 families, 104 individuals contributed 208 bacterial isolates from the antrum and corpus. A large proportion, 29 of 36 (81%) of the offspring in a sibship, harbored the same strain as at least one sibling. Mother-offspring strain concordance was detected in 10 of 18 (56%) of the families. Of 17 investigated father-offspring relations in eight families, none were strain concordant. Spouses were infected with the same strains in 5 of 23 (22%) of the couples. Different strains in the antrum and corpus were found in 8 of 104 (8%) of the subjects. Our family-based fingerprinting study demonstrates a high proportion of shared strains among siblings. Transmission between spouses seems to be appreciable. The data support mother-child and sib-sib transmission as the primary transmission pathways of H. pylori.

Serum antibody response to clostridium difficile toxins in patients with clostridium difficile diarrhoea

SummaryConsecutive serum samples from 61 patients withClostridium difficile diarrhoea were investigated for antibody response toC. difficile toxins A and B in an indirect enzyme immunoassay (ELISA) and in a neutralization assay againstC. difficile cytotoxin. Sera from 64 blood donors, elderly healthy females and patients with other known intestinal enteropathogens served as controls. An immune response was detected by ELISA in approximately half of the patients withC. difficile diarrhoea. The specificity of the ELISA was 94% or 97%, depending on the control material used. Furthermore, a correlation was found between clinical recovery without relapse ofC. difficile diarrhoea and high IgG titers to toxin B in the ELISA, and/or appearance of neutralizing antibodies. It is concluded that the ELISA for detection of serum antibodies toC. difficile toxins may be of diagnostic value in combination with the conventional tissue culture assay for cytotoxin in stool. High ELISA IgG titres to toxin B and/or the appearance of neutralizing antibodies may also be a positive prognostic sign in patients withC. difficile diarrhoea.ZusammenfassungVon 61 Patienten mitClostridium difficile-Diarrhoe wurden konsekutive Serumproben mit einem indirekten Enzymimmunassay (ELISA) und einem Neutralisationstest fürC. difficile Cytotoxin auf die Antikörperantwort gegen die Toxine A und B vonC. difficile untersucht. Als Kontrollen wurden Seren von 64 Blutspendern, älteren gesunden Frauen und Patienten, die an Krankheiten durch andere bekannte enteropathogene Erreger litten, verwendet. Bei annähernd der Hälfte der Patienten mitC. difficile-Diarrhoe wurde mittels ELISA eine Immunantwort entdeckt. In Abhängigkeit vom Kontrollmaterial ergab sich für den ELISA-Test eine Spezifität von 94% oder 97%. Bei Patienten, die sich ohne Rezidiv von derC. difficile-Diarrhoe erholten, bestand eine Korrelation zu hohen IgG-Titern gegen Toxin B im ELISA-Test und/oder Auftreten von neutralisierenden Antikörpern. Daraus läßt sich schließen, daß die Bestimmung der Antikörper gegenC. difficile-Toxine im Serum mit ELISA in Kombination mit dem herkömmlichen Gewebekultur-Test auf Cytotoxin von diagnostischem Wert ist. Hohe ELISA IgG-Titer gegen Toxin B und/oder das Auftreten neutralisierender Antikörper im Serum können bei Patienten mitC. difficile-Diarrhoe als positives prognostisches Zeichen gewertet werden.

Immunoglobulin E responses to diphtheria and tetanus toxoids after booster with aluminium-adsorbed and fluid DT-vaccines

Immunoglobulin E responses to diphtheria and tetanus toxoids were investigated in pre- and postbooster samples of 104 children given, at 10 years of age, a DT booster with either an adsorbed (n = 51) or a non-adsorbed, fluid vaccine (n = 53). Vaccination with adsorbed DT is part of a national immunisation programme and represents the first regular booster given to these children, primarily immunised with three doses of adsorbed DT at age 3-6 months. The vaccines, with a content of 30 Lf of diphtheria toxoid and 7.5 Lf of tetanus toxoid per millilitre, were given for booster in a dose of 0.25 ml as a deep subcutaneous injection. In the prebooster samples, 3 and 14% had measurable IgE to diphtheria and tetanus, respectively. These rates rose to 94 and 92% in the postbooster samples, respectively. The median (range) of IgE to tetanus among recipients of the adsorbed vaccine was 0.59 PRU ml-1 (< 0.1-17.5), as compared to 0.27 (< 0.1-8.22) in the recipients of the fluid vaccine (p = 0.027). The median IgE response to diphtheria toxoid in the whole group was 1.8 (< 0.1-17.5), with no significant difference between the two vaccines (p = 0.76). There was no correlation between the IgE and IgG responses (r < 0.07, p > 0.5) but the two IgE responses as well as the ratios of IgG/IgE showed strong correlations (r > 0.7, p < 0.001). The study thus revealed unexpectedly high rates of IgE responses to diphtheria and tetanus toxoids in a regular DT booster vaccination programme, which were associated to high rates of local side effects.(ABSTRACT TRUNCATED AT 250 WORDS)

Acellular pertussis vaccine in adults: Adverse reactions and immune response

An acellular pertussis vaccine JNIH-6 containing pertussis toxin and filamentous hemagglutinin was evaluated in adult volunteers with regard to adverse reactions and antibody response. Adverse reactions were few and mild. A late onset local reaction was seen in 22 of the 47 vaccinees (47 %) as compared to none of the 20 subjects receiving a placebo, the carrier solution of aluminium phosphate of the vaccine. The reaction, which manifested itself on the 6th to 8th day after vaccination, consisted in all cases of an induration and/or swelling considered insignificant by the majority of the subjects. The reaction was only found in vaccinees receiving a first dose of vaccine and was independent of the prevaccination antitoxin level. The vaccine induced a highly satisfactory antibody response to both filamentous hemagglutinin and pertussis toxin.

Subcutaneous versus intramuscular injection for booster DT vaccination of adolescents

The importance of the injection technique in booster vaccination was investigated in an open randomized study with 252 10-year-old Swedish school-children receiving routine DT vaccination either by subcutaneous or by intramuscular route in the upper arm. The adolescents had previously been primed with DT vaccine at 3, 5 and 12 months of age. Adverse reactions, monitored for 2 weeks, showed the same low rates for systemic reactions in both groups, while the intramuscular administration gave significantly less redness (p < 0.001), swelling (p < 0.001), itching (p < 0.01) and pain (p < 0.05). These reactions were also of shorter duration (p < 0.01 to p < 0.001). Girls were found to have more pain and itching than boys (p < 0.001). No significant differences in antibody responses between the two administration routes were found in the 99 samples drawn 2 weeks after the booster. However, girls were found to have a lower response to diphtheria toxoid than boys (p = 0.009). Local reactions to a booster can thus be significantly reduced by choice of injection technique, which may be necessary if increased dosages and/or further valences are to be given to adolescents and adults.

Transmission Studies of Babesia microti in Ixodes ricinus Ticks and Gerbils

ABSTRACT In order to investigate the possible role of Ixodes ricinus as a vector of zoonotic Babesia microti infection in Europe, a European rodent isolate (HK) and a zoonotic American isolate (GI) were studied in transmission experiments. PCR detected B. microti in the blood and spleens of infected gerbils (Meriones unguiculatus) and also in laboratory-induced infections of I. ricinus ticks. B. microti DNA was detected by PCR in all pooled samples of nymphs and the majority of adults that had fed as larvae and nymphs, respectively, on gerbils with acute infection of the European isolate, confirming that I. ricinus could serve as a vector in Europe. The American isolate, GI, proved to be equally infective for larval and nymphal I. ricinus as the HK strain, despite a very different appearance in gerbil erythrocytes. Nymphs infected with the HK and GI strains readily infected gerbils. In contrast to the finding in acute infections, ticks that fed on gerbils with chronic infections of HK and GI did not become infected. It was also found that the HK strain was not transmitted transovarially. The finding that a B. microti strain (GI) from a distant geographical region (United States) can infect and be transmitted by I. ricinus suggests that other European B. microti strains, in addition to the HK strain used here, are probably infective for I. ricinus, supporting the view that infection of humans with European B. microti may be a regular occurrence.

Effects of feeding probiotics during weaning on infections and antibody responses to diphtheria, tetanus and Hib vaccines.

Microbial exposure is necessary for the development of normal immune function, which has driven the idea of using probiotics for treatment and prevention of immune‐mediated diseases in infancy and childhood. Mounting evidence indicates that probiotics have immunomodulatory effects. However, the mechanisms are still poorly understood. Specific antibody response is a valuable proxy for immune system maturation status in infancy. We aimed at determining the impact of Lactobacillus F19 (LF19) during weaning on infections and IgG antibody responses to routine vaccines. In a double‐blind, placebo‐controlled randomized intervention trial, infants were fed cereals with (n = 89) or without LF19 (n = 90) from 4 to 13 months of age. Infants were immunized with DTaP (diphtheria and tetanus toxoid and acellular pertussis), polio and Hib‐conjugate vaccines at (3), 5 and 12 months of age. We assessed the number of days with infections, antibiotic prescriptions and antibody concentrations to Hib capsular polysaccharide (HibPS), diphtheria toxin (D) and tetanus toxoid (T) before and after the second and third doses. Days with infectious symptoms did not differ between the groups. Days with antibiotic prescriptions were fewer in the LF19 group (p = 0.044). LF19 enhanced anti‐D concentrations when adjusting for breastfeeding duration and colonization with LF19 (p = 0.024). There was an interaction of the intervention and colonization with LF19 on anti‐T concentrations during the course of vaccination (p = 0.035). The anti‐HibPS concentrations were higher after the first and second dose of Hib vaccine in infants breastfed <6 months compared with those breastfed ≥6 months (p < 0.05), with no effect by LF19. In conclusion, feeding LF19 did not prevent infections, but increased the capacity to raise immune responses to protein antigens, with more pronounced effects in infants breastfed <6 months.

The Diagnostic Value of Enzyme Immunoassay and Immunoblot in Monitoring Eradication of Helicobacter pylori

55 patients with severe ulcer disease and H. pylori infection, successfully treated with antimicrobials, were followed-up with repeated blood samples for up to 32 months. Sera were analysed by enzyme immunoassay (EIA) for IgG and IgA antibodies and by IgG immunoblot. The EIA for IgG antibodies showed a high sensitivity (100%), while IgA antibodies above the cut-off level were found in 55% of the patients. At a median of 77 days after onset of treatment, approximately 50% of the patients showed a significant decrease (> or = 50%) of IgG or had titres below the cut-off level. All patients but 1 had a significant decrease of IgG after 6-12 months. The decrease was slower for IgA. The H. pylori-specific 116 kDa and 19.5 kDa bands were found in all pre-treatment samples, but the decrease in median intensity of the bands was slower than for the IgG EIA. In the 32-months post-treatment samples, both bonds had an intensity still above 50% of the pre-treatment value. The study showed that the IgG EIA is a useful method for monitoring eradication of H. pylori. Immunoblot can detect previous H. pylori infection in EIA negative Individuals.

Signal Transduction-Mediated Adherence and Entry of Helicobacter pylori Into Cultured Cells

BACKGROUND & AIMS An ability to invade host cells could be a means for Helicobacter pylori to achieve resistance to antibiotic therapy. The aim of this study was to investigate the mechanisms involved in adherence and entry of H. pylori into cultured cells. METHODS Coinfection with Yersinia expressing mutant or wild-type YopH tyrosine phosphatase was used. Genistein and cytochalasin D were used as inhibitors of adherence and entry; entry was monitored by a gentamicin-protection assay. Target cells were AGS cells and a beta1-integrin-deficient cell line with its corresponding beta1-integrin-expressing transfectant. RESULTS H. pylori induced phosphorylation of 125-130-kilodalton proteins, similar in size to the target proteins of Yersinia YopH. Adherence of H. pylori was inhibited by Yersinia organisms expressing enzymatically active YopH but not by inactive YopH. Adherence and entry of H. pylori was considerably higher with beta1-integrin-transfected cells than with beta1-integrin-deficient cells. Antibodies directed against alpha5- and beta1-integrin chains reduced adherence to the alpha5beta1-integrin-expressing gastric cell line AGS. Entry was inhibited by both cytochalasin D and genistein. Entry, but not adherence, was higher for 2 type I strains than for a type II isolate. CONCLUSIONS Invasion of gastric epithelium via an integrin-mediated pathway could contribute to the ability of H. pylori to establish persistent infection.