Quantification of bacteria in human feces using 16S rRNA-hybridization, DNA-staining and flow cytometry.

Hybridization of bacteria with fluorescent probes targeting 16S rRNA and inspection of hybridized bacteria with fluorescence microscopy (microscopy-FISH, i.e. fluorescence in situ hybridization) have constituted an accessible method for the analysis of mixed bacterial samples such as feces. However, microscopy-FISH is a slow method and prone to errors. Flow cytometry (FCM) enables analysis of bacteria more rapidly, accurately and reliably than microscopy. In this study, a FCM method for the analysis of 16S rRNA-hybridized and DNA-stained fecal bacteria was developed. The results of FCM-FISH were comparable to those of microscopy-FISH, and the coefficients of variation of the FCM analyses were extraordinarily low. In previous FCM-FISH studies, the Eub 338 probe, which is supposed to hybridize all bacteria, has been used to detect all bacteria present in the sample. We found that Eub 338 did not bind to all bacteria, which could be detected by DNA-staining; while SYTOX Orange DNA-stain detected all bacterial species tested and produced high fluorescence intensities enabling clear separation of bacteria from non-bacterial material. Thus, DNA-staining is a method of choice for the detection of all bacteria in FCM-FISH. We conclude that FCM of 16S rRNA-hybridized and DNA-stained bacteria is a rapid and reliable method for the analysis of mixed bacterial samples including feces.

A novel method to analyze viral antigen-specific cytolytic activity in the chicken utilizing flow cytometry.

In order to overcome some of the main drawbacks that have emerged in the conventional assays for cytotoxic T-lymphocytes (CTLs) in the chicken, a novel approach to analyze viral antigen-specific cytolytic activity utilizing flow cytometry was developed. In this method, the target cells were distinguished from the effector cells by pre-labelling them with a fluorescent dye PKH67. Cell death was detected with propidium iodide which labels the DNA of damaged cells. Flow cytometric assay also enables phenotyping of the effector cells by direct or indirect immunofluorescence staining of lymphocyte surface molecules. The results showed that specific cytotoxic T cells were observed in the blood of chickens primed with fixed avian reticuloendotheliosis virus strain T transformed MHC-compatible B cells. Phenotypic analysis of the effector cells from blood demonstrated CTL activity both in CD8+ and CD4+ T cell populations and the majority CTLs were TCR2+ cells.

Fecal microbiota in early rheumatoid arthritis.

OBJECTIVE: To compare the composition of intestinal microbiota of patients with early rheumatoid arthritis (RA) or fibromyalgia (FM), fecal samples were collected from 51 patients with RA and 50 with FM. METHODS: RA patients fulfilled the RA criteria of the American College of Rheumatology, and duration of their disease was < or = 6 months. Only nonhospitalized patients from outpatient care were included. Patients having extreme diets or previous disease modifying antirheumatic drug or glucocorticoid medication were excluded, as were those taking antibiotics or having gastroenteritis for at least 2 months prior to sampling. Fecal bacterial composition was analyzed with a method based on flow cytometry, 16S rRNA hybridization, and DNA-staining. A set of 8 oligonucleotide probes was used. RESULTS: In comparison to patients with FM, the RA patients had significantly less bifidobacteria and bacteria of the Bacteroides-Porphyromonas-Prevotella group, Bacteroides fragilis subgroup, and Eubacterium rectale--Clostridium coccoides group. Results from the 8 probes showed a significant overall difference between the 2 patient groups, indicating widespread microbial differences. CONCLUSION: These findings support the hypothesis that intestinal microbes participate in the etiopathogenesis of RA.

Bactericidal effects of bioactive glasses on clinically important aerobic bacteria.

Bioactive glasses (BAGs) have been studied for decades for clinical use, and they have found many dental and orthopedic applications. BAGs have also been shown to have an antibacterial effect e.g., on some oral microorganisms. In this extensive work we show that six powdered BAGs and two sol-gel derived materials have a clear antibacterial effect on 29 clinically important bacterial species. We also incorporated a rapid and accurate flow cytometric (FCM) method to calculate and standardize the numbers of viable bacteria inoculated in the suspensions used in the tests for antibacterial activity. In all materials tested growth inhibition could be demonstrated, although the concentration and time needed for the effect varied depending on the BAG. The most effective glass was S53P4, which had a clear growth-inhibitory effect on all pathogens tested. The sol-gel derived materials CaPSiO and CaPSiO II also showed a strong antibacterial effect. In summary, BAGs were found to clearly inhibit the growth of a wide selection of bacterial species causing e.g., infections on the surfaces of prostheses in the body after implantation.

Unique cytokine secretion profile in children with both type I diabetes and asthma distinct from that of solely diabetic or asthmatic children.

Asthma and type I diabetes are major causes of chronic illness in childhood which, according to the current paradigm, have mutually antagonistic immunopathologies. Nonetheless, the disorders appear to preferably coexist both on population and individual levels. To assess whether children with asthma and type I diabetes might have a common immunoregulatory defect. The spontaneous and anti-CD3+ anti-CD28-stimulated cytokine production patterns by peripheral blood mononuclear cells of 13 children with both asthma and diabetes, nine children with diabetes, 11 children with asthma and nine healthy children were assessed using cytometric bead assay. The spontaneous production of IFN-gamma, TNF-alpha and IL-10 by mononuclear cells in children with both asthma and diabetes was elevated compared to the other study groups (p=0.02, p=0.001 and p=0.04, respectively). Stimulation in vitro increased IL-10 secretion in solely diabetic (p=0.008), asthmatic (p=0.008) and healthy children (p=0.01), but not in children with both diseases (p=0.22). Children suffering from both diabetes and asthma display a unique cytokine secretion pattern, distinct from those of solely diabetic, asthmatic and healthy children. In particular, these children appear to have a defect in regulation of IL-10 secretion.