Applicability of random primer R143 for determination of Aspergillus fumigatus DNA.

The specificity of random primer R143 for Aspergillus fumigatus DNA was determined in order to test its usefulness in establishing the presence of A. fumigatus DNA in fungal cultures. When PCR reaction products of these cultures were compared with those of 21 other bacterial and fungal DNA samples, R143 proved to produce a 1346 bp band with only A. fumigatus. This band has been sequenced completely and the EcoRI restriction site was used for subsequent confirmation of PCR products. The specificity for A. fumigatus DNA was also confirmed by Southern blotting. Comparison of morphological typing of Aspergillus species in cultures with PCR using R143 on DNA isolated from these cultures showed concordance in 22 of 24 cases. In two cases there was discordance: both times PCR results showed correctly the presence of A. fumigatus, initially not detected by culture. R143 is an A. fumigatus specific random primer, with potential for use in detection of A. fumigatus DNA in clinical specimens.

Host-microflora interaction in systemic lupus erythematosus (SLE): circulating antibodies to the indigenous bacteria of the intestinal tract.

Experimental data suggest a role for the microflora in the disease expression of systemic lupus erythematosus (SLE). In active SLE anti-ds-DNA antibodies are supposed to be pathogenic by forming immune complexes with DNA. Bacteria might induce the production of anti-ds-DNA antibodies. To explore the relation between the host and his microflora in SLE in comparison with healthy controls we studied the prevalence of systemic antibodies to faecal bacteria that were discriminated by their morphology by indirect immunofluorescence. IgM titres against their own faecal microflora were found to be lower both in active and inactive SLE when compared to healthy individuals. IgG-class antibacterial antibodies were increased in inactive SLE but decreased in active SLE compared to inactive SLE and healthy controls, although plasma levels of total IgG were almost doubled in active SLE. The lower IgG antibacterial antibody titres in active SLE might possibly result from sequestration of these IgG antibodies in immune complexes, indicating a possible role for antibacterial antibodies in exacerbations of SLE.

The role of systemic antibodies against intestinal Escherichia coli in the prevention of bacterial translocation of Escherichia coli in a burn model in mice.

Bacterial translocation (BT) from the gastrointestinal (GI) tract has been proposed to play a role in the pathogenesis of septic complications in severely burned patients. It is well known that severely ill patients such as thermally injured patients may acquire new potential pathogenic microorganisms in the GI tract. Because these patients have no antibodies directed against these acquired microorganisms, BT may be facilitated in these patients. To investigate this hypothesis in a burn model, a study was performed in which two groups of C3H-HeN mice underwent a different period of intestinal overgrowth by a single neomycin-resistant (NR) Escherichia coli strain after oral neomycin-bacitracin treatment. Group I underwent a short period (5 days) and group II experienced a long period (44 days) of intestinal overgrowth before a thermal injury was executed. Two days postburn, plasma antibody titers of IgA, IgG, and IgM isotype against NR E. coli were measured by indirect immunofluorescence (IIF) and BT to various organs was determined by culturing. Although there were no significant differences of BT to organs between the groups, the IgG antibody titer against the NR E. coli strain was significantly increased in group II. Antibody titers of IgA and IgM were not significantly different between the groups. Titers of plasma antibodies of IgG isotype against the intestinal NR E. coli did not correlate with BT. We conclude that increased IgG titers against the NR E. coli used are the result of a longer intestinal overgrowth period and are not associated with prevented or decreased BT.

Host-microflora interaction in systemic lupus erythematosus (SLE): colonization resistance of the indigenous bacteria of the intestinal tract.

Experimental data suggest a role for the microflora in Systemic Lupus Erythematosus (SLE). Anti-ds-DNA antibodies may be pathogenic in SLE by forming immune complexes with DNA. Foreign bacteria in the intestines could constitute the stimulus for anti-ds-DNA antibody production in SLE. Colonization Resistance (CR) is the defence capacity of the indigenous microflora against colonization of the intestines by foreign bacteria. A low CR implies increase of translocation of bacteria and a higher chance of subsequent, possibly DNA-cross-reacting antibacterial antibody production. We measured CR by a comprehensive biotyping technique in healthy individuals and patients with inactive and active SLE. CR tended to be lower in active SLE patients than in healthy individuals (P = 0.09, Wilcoxon one sided, with correction for ties). This could indicate that in SLE more and different bacteria translocate across the gut wall due to a lower CR. Some of these may serve as polyclonal B cell activators or as antigens cross-reacting with DNA.

Healthy individuals possess circulating antibodies against their indigenous faecal microflora as well as against allogenous faecal microflora: an immunomorphometrical study.

Healthy persons were shown to possess circulating antibodies of both IgA, IgG and IgM isotype directed against the bacteria of their faecal microflora, assessed by immunomorphometry. After removal, by absorption, of the fraction of antibodies directed against the autochthonous faecal bacteria or cross-reacting with allogenous faecal bacteria, there were still antibodies left directed against allogenous faecal bacteria of both the IgA, IgG and IgM isotype. However, relatively more antibodies of the IgA isotype appeared to be directed against allogenous bacteria than against indigenous faecal bacteria. Persons who reacted with specific antibodies to many bacteria of their own flora also tended to react specifically to bacteria in the allogenous microflora of the other volunteers. The patterns of antibodies directed to faecal bacteria of different morphologies (morphotypes) were unique for each individual.

Circulating antibodies against faecal bacteria assessed by immunomorphometry: combining quantitative immunofluorescence and image analysis.

A new technique to study the prevalence of circulating antibodies directed against different morphological groups ('morphotypes') of bacteria in fresh faeces is presented. The technique combines quantitative indirect immunofluorescence with digital image analysis. Plasma antibody titres and patterns of IgA, IgG and IgM isotype against morphotypes of faecal bacteria were determined in ten healthy individuals.

Study of colonization resistance for Enterobacteriaceae in man by experimental contamination and biotyping as well as the possible role of antibodies in the clearance of these bacteria from the intestines.

The colonization resistance (CR) of the digestive tract was determined in 10 healthy volunteers by oral contamination with a neomycin resistant Escherichia coli (NR-E. coli) strain and measurement of the faecal concentration of this strain during 14 days after the contamination. This 'gold standard' was compared with another parameter of CR; the determination of the mean number of different biotypes of Enterobacteriaceae isolated from four faecal samples per volunteer. Both measures are significantly correlated (P less than 0.01). The NR-E. coli strain could be cultured from faecal samples of 4/10 volunteers as long as 300 days after contamination. Serum antibody titres against endogenous E. coli strains and the NR-E. coli strain used for experimental oral contamination were measured by an indirect immunofluorescence (IIF) assay. The assay was read by a video camera connected to an image processing system. The 95% confidence limits of antibody titres (log2) against endogenous E. coli strains ranged between less than 3 and 7.1 for IgA, between less than 3 and 8.7 for IgG and between less than 3 and 7.4 for IgM. Antibody titres against the NR-E. coli4 strain were within this (normal) range. The serum antibody titres against the NR-E. coli strain increased slowly after oral contamination, especially IgG and IgM. Little increase in IgA titres could be observed. An increase of serum antibody titres did not correlate with the elimination of the oral contaminant from the intestines. Therefore, we conclude that the CR is not IgG nor IgM antibody mediated.

Objective quantitation of serum antibody titres against Enterobacteriaceae using indirect immunofluorescence, read by videocamera and image processing system.

A new way of measuring indirect immunofluorescence (IIF) of microscopic bacterial slide preparations by videocamera and an image processing system is presented. This method is compared with the conventional method of reading the slides by eye. The advantages of this new approach are objective reading, greater accuracy and easier performance. We have applied the method to measure serum antibody titres against endogenous Enterobacteriaceae. The method offers the opportunity to combine IIF with automatic morphological analysis, thereby maximally exploiting the possibilities of the immunofluorescence technique.

Determination of colonization resistance of the digestive tract by biotyping of Enterobacteriaceae.

In studies concerning the effect of antibiotics on faecal microflora, Colonization Resistance is an important parameter. Colonization Resistance correlates inversely with the number of different biotypes of Enterobacteriaceae isolated from faecal samples. Nine healthy volunteers were studied during 6 weeks, in order to determine the natural variation in the number of different biotypes of Enterobacteriaceae per faecal sample. The numbers of biotypes ranged from 1-15 per faecal sample, the mean number of biotypes varied between 2.6 and 7.3 different biotypes per faecal sample per healthy volunteer. Inter-individual variations of five biotypes in the mean number of biotypes per faecal sample are normal. We assessed the minimal number of faecal samples that should be taken for comprehensive biotyping so as to determine reliably the mean number of different biotypes representative for the Colonization Resistance of an individual. It was found that a minimum of four faecal samples was required.