Qualitative and Quantitative Detection of Chlamydophila pneumoniae DNA in Cerebrospinal Fluid from Multiple Sclerosis Patients and Controls

A standardized molecular test for the detection of Chlamydophila pneumoniae DNA in cerebrospinal fluid (CSF) would assist the further assessment of the association of C. pneumoniae with multiple sclerosis (MS). We developed and validated a qualitative colorimetric microtiter plate-based PCR assay (PCR-EIA) and a real-time quantitative PCR assay (TaqMan) for detection of C. pneumoniae DNA in CSF specimens from MS patients and controls. Compared to a touchdown nested-PCR assay, the sensitivity, specificity, and concordance of the PCR-EIA assay were 88.5%, 93.2%, and 90.5%, respectively, on a total of 137 CSF specimens. PCR-EIA presented a significantly higher sensitivity in MS patients (p = 0.008) and a higher specificity in other neurological diseases (p = 0.018). Test reproducibility of the PCR-EIA assay was statistically related to the volumes of extract DNA included in the test (p = 0.033); a high volume, which was equivalent to 100 µl of CSF per reaction, yielded a concordance of 96.8% between two medical technologists running the test at different times. The TaqMan quantitative PCR assay detected 26 of 63 (41.3%) of positive CSF specimens that tested positive by both PCR-EIA and nested-PCR qualitative assays. None of the CSF specimens that were negative by the two qualitative PCR methods were detected by the TaqMan quantitative PCR. The PCR-EIA assay detected a minimum of 25 copies/ml C. pneumoniae DNA in plasmid-spiked CSF, which was at least 10 times more sensitive than TaqMan. These data indicated that the PCR-EIA assay possessed a sensitivity that was equal to the nested-PCR procedures for the detection of C. pneumoniae DNA in CSF. The TaqMan system may not be sensitive enough for diagnostic purposes due to the low C. pneumoniae copies existing in the majority of CSF specimens from MS patients

Anion transport in oocytes of Xenopus laevis induced by expression of mouse erythroid band 3 protein--encoding cRNA and of a cRNA derivative obtained by site-directed mutagenesis at the stilbene disulfonate binding site

A vector was constructed containing a cDNA for mouse band 3 obtained from Demuth et al. (1986, EMBO J., 5, 1205-1214), a synthetic linker (containing 5'-non-translated region, start codon and a coding region for the first 12 N-terminal amino acids), and RNA polymerase promoters suitable for in vitro transcription of cRNA. After injection of the cRNA into the cytoplasm of Xenopus oocytes and incubation for 16 h, expression of mouse band 3 was demonstrated by immunoprecipitation, immunohistochemical methods and influx or efflux measurements with 36Cl-. Antisense cRNA inhibits the expression. Lysines 558 and 561 were replaced by asparagines using oligonucleotide-directed mutagenesis. Like the original band 3, the mutant shows stilbene disulfonate-inhibitable anion exchange. However, in contrast to the original band 3, inhibition by 4,4'-diisothiocyano dihydrostilbene-2,2'-disulfonate (H2DIDS) is no longer irreversible. This indicates that thiourea bond formation between H2DIDS and band 3 involves one of the two modified lysine residues. It also shows that the two lysine residues are not essential for the execution of the anion transport function of band 3. The results described suggest that the cDNA clone of Demuth et al. (1986) encodes a protein with properties that are representative for the properties of the bulk of the band 3 protein in the plasma membrane of the red cell of the mouse