Interleukin-1 receptor deficiency in the hippocampal formation of (NZB x NZW)F2 mice: genetic and molecular studies relating to autoimmunity.

Interleukin-1 receptor (IL-1R) deficiency has been previously described in the dentate gyrus of autoimmune NZB and (NZB x NZW) F1 (or BWF1) mice. In this study, the genetic and molecular characterization of this defect were investigated in BWF2 mice in relation to anti-DNA antibody production and microsatellite D1Nds4 (near the IL1r1 gene) polymorphism. IL-1R density was quantified in the brain, spleen and pancreas, using in vitro quantitative autoradiography with recombinant human [125I]-IL-1alpha as the ligand. This study of the dentate gyrus of F2 mice revealed three phenotypes: NZW-like, NZB-like and F1-like, which occurred in a ratio of 1:1:2, with IL-1R densities of 100%, 17% and 59%, respectively as compared to control NZW mice (100%). In contrast, IL-1R densities observed in the choroid plexus and peripheral organs were similar. Moreover a high production of IgG2a anti-DNA antibodies was observed in F2 mice, as in their parents, particularly those with the NZB-like phenotype. Microsatellite mapping of D1Nds4 revealed polymorphism in both parents and BWF2 mice in relation to the level of IL-1R density in the dentate gyrus. In spite of the acute defect in IL-1 binding in the dentate gyrus of NZB mice, molecular analysis of IL-1R mRNA (type I, II and accessory protein) showed similar amounts of mRNA, measured following RT-PCR amplification, in the hippocampal formation of both NZB and control C3H/He mice. In conclusion, the transmission of the IL-1R defect in the dentate gyrus of NZB mice is monofactorial and the defect appears to be at the post-transcriptional level of IL-1R synthesis. The lack of IL-1R in the dentate gyrus seems to correlate with some autoimmune characteristics. Correlation of D1Nds4 polymorphism with the level of IL-1R density suggests that it could be a predisposing gene to disease or a marker for other closely linked predisposing genes.

Analysis of brain mRNA by reverse transcription-polymerase chain reaction and hybridization with digoxigenin-labeled DNA probe.

The interleukin-1 family of polypeptides (IL-1 alpha, IL-1 beta and IL-1 receptor antagonist (RA)) induces various centrally mediated host defense responses to infectious pathogens. Considerable interest has focussed on IL-1 as a mediator in disease and in the production of systemic acute phase responses. We have recently studied the effects of a peripheral stimulation by intraperitoneal (i.p.) administration of lipopolysaccharide (LPS) on the mRNAs expressions of IL-1 (alpha, beta, RA) and their receptors (IL-1 receptor type I and type II (IL-1R1, IL-1R2)) in the central nervous system (CNS). The levels of these expressions being very low in the CNS, the reverse transcription-polymerase chain reaction (RT-PCR) techniques are required for these studies. RT-PCR is a developed method of identifying mRNAs in very small amount of nucleic acid. We have previously developed a method to choose specific PCR primers. The detection of specific PCR products is extremely important. Since amplifications with these specific PCR primers can be achieved under the same conditions (buffers and temperatures) reliable results can be obtained. Characterization of a PCR product requires the use of a specific DNA probe that hybridizes to the region of interest. In addition to providing specificity of detection, the use of labeled DNA probes provides increased sensitivity over ethidium bromide staining. We have previously described a method of synthesis of non-radioactive probe labeled with digoxigenin by nested PCR. Moreover the major advantage to the use of non-radioactive label is that it does not have a short half-life and can last for weeks or even months. A quantification of the PCR products can be obtained using a method based on the analysis of photographic negatives of agarose gels.

Choosing highly specific primers for the polymerase chain reaction using the octomer frequency disparity method: application to Chlamydia trachomatis.

The eight-nucleotide sequence (octomer) at the 3' end of PCR primers is important to PCR specificity. We describe a correlation between the specificity of PCR primers used with human DNA and the frequency of the 3' octomer in a human database. We therefore applied a methodology (OFD) based on octomer frequency disparity to identify 16 PCR targets in the chromosome of the intracellular bacterium, Chlamydia trachomatis (Ct). In addition, the 16 sets of primers were tested with a standard procedure. All the primer pairs were highly specific for Ct and did not lead to non-specific amplification when used with human DNA. This work shows that the choice of specific PCR primers is possible using a method based on the statistical representativeness of octomers in genomes.

Interleukin-1 receptor accessory protein transcripts in the brain and spleen: kinetics after peripheral administration of bacterial lipopolysaccharide in mice.

Receptors for IL-1, type I (IL-1R1) and type II (IL-1R2), have been characterized by pharmacological and molecular techniques in the mouse brain. High densities are mainly found in the cortex, dentate gyrus and choroid plexus. It was therefore of interest to investigate the expression of mRNA IL-1 receptor accessory protein (IL-1R AcP), which is a part of the IL-1 receptor complex and has been shown to interact specifically with IL-1R1. IL-1R AcP transcripts were detected under basal conditions following RT-PCR amplification in the mouse brain, as well as in the pituitary, spleen, adrenal and liver. IL-1R AcP transcripts were found in higher amounts than IL-1R1 transcripts in all tissues except the spleen, where their expression was minor. Following bacterial lipopolysaccharide (LPS) stimulation (3-48 h), IL-1R AcP transcripts were not changed in the brain, while IL-1R1 transcripts were increased for 3-6 h. In the spleen, a slight increase in IL-1R AcP and IL-1R1 was observed during the first hours following LPS stimulation. In conclusion, IL-1R AcP mRNA is expressed in the brain and in other tissues where IL-1R1 transcripts are found. However, the regulation of its expression is distinct from IL-1R1. The high level of expression and the lack of regulation of IL-1R AcP transcripts in the brain under inflammatory conditions suggest that the protein might be constitutively expressed in excess.

Interleukin-1 receptors type I and type II in the mouse brain: kinetics of mRNA expressions after peripheral administration of bacterial lipopolysaccharide.

The expression of transcripts for Interleukin-1 (IL-1) type I and type II receptors (IL-1R1, IL-1R2) was investigated in the mouse brain and spleen using reverse transcription-polymerase chain reaction techniques under basal conditions and following injection of endotoxin (LPS, i.p., 4 mg/kg). Under basal conditions, mRNAs for both receptor types were found in various parts of the brain, in pituitary as well as in spleen. Following LPS stimulation, mRNA expressions were increased in all studied tissues. IL-1R1 mRNAs were predominant in the brain and pituitary while, IL-1R2 mRNAs were more abundant in the spleen. The maximal quantity of transcripts (IL-1R1, IL-1R2) was obtained 6 h after LPS injection in all studied tissues. The decrease to basal level was observed within 48 h in the brain. In the spleen, IL-1R1 mRNAs remained elevated 48 h after LPS while IL-1R2 mRNAs had already reached basal level. These results indicate a LPS-induced stimulation of IL-1 receptors mRNAs in the brain and a differential expression of IL-1R1 and IL-1R2 transcripts in brain and immune tissues.

Investigation of animal botulism outbreaks by PCR and standard methods.

A double PCR procedure is proposed for identification of Clostridium botulinum C and D. This method consists of a first PCR amplification with a degenerate primer pair able to amplify a 340 bp common DNA fragment from botulinum neurotoxin (BoNT) C1 and D genes, followed by two subsequent PCR amplifications with two primer pairs specific for BoNT/C1 and D respectively (198 bp DNA fragment). This method was found to be specific for C. botulinum C and D, amongst 81 strains of C. botulinum and 21 different species of other Clostridium and bacteria tested. The detection limit ranged from 10 to 10(3) bacteria in the reaction volume according to the C. botulinum C and D strains. In 160 naturally contaminated animal and food samples submitted to a 48 h enrichment culture, the double PCR showed an 89.4% correlation rate with the standard mouse bioassay. A clear distinction between botulism type C and D was obtained. The double PCR provides a reliable alternative for detection and identification of C. botulinum C and D in clinical and food samples.

Amplification of plasmid and chromosome Chlamydia DNA in synovial fluid of patients with reactive arthritis and undifferentiated seronegative oligoarthropathies.

OBJECTIVE: To investigate the hypothesis that whole bacteria might be found in the joints of patients with Chlamydia-associated reactive arthritis. METHODS: The presence of 2 plasmid- and 2 chromosome-specific sequences of Chlamydia DNA was investigated by amplification with the polymerase chain reaction, in synovial fluid (SF) samples from 71 patients with various arthropathies. RESULTS: Chlamydia DNA was found in SF samples from 22 patients. CONCLUSION: Whole chlamydiae are likely present in the SF of patients with Chlamydia-associated reactive arthritis.

Expression of interleukin 1 alpha, interleukin 1 beta and interleukin 1 receptor antagonist mRNA in mouse brain: regulation by bacterial lipopolysaccharide (LPS) treatment.

Lipopolysaccharide (LPS) stimulation is known to induce interleukin-1 (IL-1) mRNA expression in various immune cell types. Since IL-1 synthesis has been suggested to occur locally in brain tissue, we investigated the expression of IL-1 (alpha and beta) and IL-1 receptor antagonist (IL-1ra) mRNAs in various structures of the central nervous system, as well as in the spleen, following intraperitoneal injection of LPS (100 micrograms/mouse). After RNA extraction and amplification by the reverse transcription-polymerase chain reaction (RT-PCR), the PCR products were separated on an agarose gel, transferred and hybridized with digoxigenin-labeled probes synthetized by nested PCR. Glyceraldehyde phosphate deshydrogenase mRNA was used as an internal control. Under basal conditions the expression of IL-1 alpha, IL-1 beta and IL-1ra mRNAs in the brain was extremely low for the three cytokines; in the spleen these mRNAs were clearly detectable. Following LPS stimulation, mRNAs were strongly increased in all the tested tissues (cortex, hippocampus, hypothalamus, cerebellum, pituitary and spleen). The kinetics of mRNAs expressions in the brain were similar for all the tested regions, with a maximum at 6 h and a decrease up to 24 h after LPS administration. In the spleen the maximum was observed as soon as 1 h following stimulation. In conclusion, peripheral LPS stimulation induces a strong and transient expression of IL-1 alpha and IL-1 beta mRNAs in the brain. IL-1ra mRNA is also stimulated by LPS in various regions of the brain.(ABSTRACT TRUNCATED AT 250 WORDS)

PCR and gene probe identification of botulinum neurotoxin A-, B-, E-, F-, and G-producing Clostridium spp. and evaluation in food samples.

A degenerate primer pair was selected to amplify specifically a 260-bp DNA fragment from Clostridium botulinum types A, B, E, F, and G, and five individual probes allowed identification of each toxinotype by hybridization of the PCR products. The 72 strains of different Clostridium species tested and 11 other bacterial species commonly found in food samples gave an amplification product. This assay was able to detect 1 C. botulinum type A or B and 10 C. botulinum type E strains per reaction. With 184 artificially contaminated food samples, after an 18-h enrichment step, the sensitivity was 10 bacteria per g of sample and the correlation with the mouse bioassay reached 95.6%.

Chemiluminescent and colorimetric detection of a fluorescein-labelled probe and a digoxigenin-labelled probe after a single hybridization step.

The objective of the simple and fast method we describe is the simultaneous hybridization of two non-radioactive probes and their detection from the same blot, using two different systems. These two probes are synthesized by PCR: one is labelled with fluorescein and the other with digoxigenin. The former is detected by chemiluminescence and the latter by colorimetry. We applied this rapid and simple method to the specific detection of multiplex polymerase chain reaction products. We used the human herpes simplex virus HSV1 and HSV2 PCR models studied in our laboratory.

Development of PCR-based assays for the detection of two human mollicute species, Mycoplasma penetrans and M. hominis.

Recently, a 16S rDNA-based polymerase chain reaction (PCR) assay was developed for the selective and sensitive detection of Mycoplasma pirum. In this study, the same procedure was used in order to selectively detect by PCR two human mycoplasmas, M. hominis and M. penetrans, with a high level of sensitivity even in a context of human DNA. For each assay, the specificity was verified by testing DNA from other mollicute species (including those closely related to the corresponding mycoplasma), from bacteria phylogenetically close to mollicutes, from Escherichia coli and from human peripheral blood mononuclear cells (PBMCs). Each assay proved to be highly sensitive since it reliably detected 10 DNA molecules, even in a context of human DNA. The results of this study demonstrate the suitability of our procedure using primers which were designed for the PCR detection of human mollicutes with a high specificity and a low and reproducible threshold of sensitivity.

Development of a selective and sensitive polymerase chain reaction assay for the detection of Mycoplasma pirum.

A new assay using the polymerase chain reaction to amplify a 173-nucleotide DNA fragment within the 16S ribosomal RNA gene of Mycoplasma pirum has been developed. The assay selectively amplified DNA from all strains of M. pirum tested with a high level of sensitivity, even in a context of human DNA. DNA from other mollicute species, including those closely related to M. pirum, from bacteria phylogenetically close to mollicutes (Clostridium innocuum, C. ramosum and Bacillus subtilis), from Escherichia coli and from human peripheral blood mononuclear cells, did not produce the amplified DNA product specific for M. pirum.

Thymic pseudotumorous enlargement due to follicular hyperplasia in a human immunodeficiency virus sero-positive patient. Immunohistochemical and molecular biological study of viral infected cells.

An enlargement of the thymus suggesting a tumor was discovered in a 28-year-old man who had early-stage acquired immune deficiency syndrome. A biopsy was performed. The adipose involuted thymus, with persistence of many Hassall's corpuscles, was judged to be a large lymphoid follicular hyperplasia. This follicular hyperplasia was similar to that previously described for lymph nodes, spleen, and other lymphoid tissues at earlier stages of human immunodeficiency virus infection, before the development of acquired immune deficiency syndrome. Human immunodeficiency virus RNA and p24 human immunodeficiency virus protein were detected in the hyperplastic germinal centers (lymphocytes and follicular dendritic infected cells), and also in many cells that may have been either lymphocytes and/or epithelial cells in the interfollicular areas. The tissue was negative for Epstein-Barr virus DNA sequences, as determined by the polymerase chain reaction. These observations identify the first state of infection of the thymus in a human immune deficiency virus-infected adult, preceding the severe involution with lymphoid depletion observed in all fatal cases of acquired immunodeficiency syndrome in which the thymus has been analyzed.

K-tuple frequency in the human genome and polymerase chain reaction.

The frequency occurrences of K-tuple (overlapping sequences of defined length, K) were computed from the known human genome sequences. The significance of these frequencies for the whole human genome was tested by polymerase chain reaction (PCR). A computer programs based on these results was written to choose primers to amplify DNA target sequences, either of human genes or of human infectious agents. The software also gave nested primer sequences which were used to synthesize non radioactive probes by PCR. We applied these two methods, primer selection and non radioactive probes, to easily and quickly set up very efficient PCR sets to work in the human genome context.