The diagnosis of tuberculous meningitis (TBM) remains a complex issue because the most widely used conventional diagnostic tools, such as culture and PCR assay for cerebrospinal fluid (CSF) samples, are unable to rapidly detect Mycobacterium tuberculosis with sufficient sensitivity in the acute phase of TBM. Based on TaqMan PCR, we designed a novel technique consisting of an internally controlled quantitative nested real-time (QNRT) PCR assay that provided a marked improvement in detection sensitivity and quantification. We applied this novel technique to quantitatively detect M. tuberculosis DNA in CSF samples from patients with suspected TBM. For use as the internal control in the measurement of the M. tuberculosis DNA copy numbers in the QNRT-PCR assay, the original mutation (M) plasmid, which included an artificial random 22-nucleotide sequence within an inserted DNA fragment of the MPB64 gene of M. tuberculosis, was prepared. The QNRT-PCR assay showed high sensitivity and specificity that were approximately equivalent to those of the conventional nested PCR assay. Moreover, the QNRT-PCR assay made it possible to precisely and quantitatively detect the initial copy number of M. tuberculosis DNA in CSF samples. Therefore, compared to the conventional PCR assay, the QNRT-PCR assay can be considered a more useful and advanced technique for the rapid and accurate diagnosis of TBM. To establish the superiority of this novel technique in TBM diagnosis, it will be necessary to accumulate data from a larger number of patients with suspected TBM.

-The glucose-dependent secretion of the insulinotropic hormone glucagon-like peptide-1 (GLP-1) is a critical step in the regulation of glucose homeostasis. Two molecular mechanisms have separately been suggested as the primary mediator of intestinal glucose-stimulated GLP-1 secretion (GSGS): one is a metabotropic mechanism requiring the sweet taste receptor type 2 (T1R2) ϩ type 3 (T1R3) while the second is a metabolic mechanism requiring ATP-sensitive K ϩ (KATP) channels. By quantifying sugar-stimulated hormone secretion in receptor knockout mice and in rats receiving Roux-en-Y gastric bypass (RYGB), we found that both of these mechanisms contribute to GSGS; however, the mechanisms exhibit different selectivity, regulation, and localization. T1R3Ϫ/Ϫ mice showed impaired glucose and insulin homeostasis during an oral glucose challenge as well as slowed insulin granule exocytosis from isolated pancreatic islets. Glucose, fructose, and sucralose evoked GLP-1 secretion from T1R3 ϩ/ϩ , but not T1R3 Ϫ/Ϫ , ileum explants; this secretion was not mimicked by the K ATP channel blocker glibenclamide. T1R2 Ϫ/Ϫ mice showed normal glycemic control and partial small intestine GSGS, suggesting that T1R3 can mediate GSGS without T1R2. Robust GSGS that was K ATP channeldependent and glucose-specific emerged in the large intestine of T1R3 Ϫ/Ϫ mice and RYGB rats in association with elevated fecal carbohydrate throughout the distal gut. Our results demonstrate that the small and large intestines utilize distinct mechanisms for GSGS and suggest novel large intestine targets that could mimic the improved glycemic control seen after RYGB.glucagon-like peptide-1; insulin; T1R3; glucose-stimulated potassium ion channel; enteroendocrine l cells THE BODY TIGHTLY REGULATES blood glucose levels, and disruption of the homeostatic mechanisms that underlie normal glycemic control can have significant deleterious effects. For example, the prolonged hyperglycemia associated with type 2 diabetes mellitus (T2DM) increases the risk of cardiovascular disease, neuropathy, retinopathy, kidney disease, and death (66). Hormonal signals arising in the gastrointestinal tract are key components of the homeostatic mechanisms controlling blood glucose levels after a meal. Ingestion of carbohydrate and other nutrients promotes the secretion of insulinotropic hormones such as glucagon-like peptide-1 (GLP-1) from the gut, resulting in a surge of insulin production before blood glucose levels rise (11,32). This early response contributes to increased glucose disposal during absorption and helps to prevent hyperglycemia. GLP-1 mimetics and inhibitors of GLP-1 degradation help increase insulin biosynthesis and secretion from pancreatic ␤-cells and are valuable additions to previous treatment regimens for T2DM patients (11,32).Despite the importance of intestinal glucose sensing and glucose-stimulated gut hormone secretion, the mechanisms underlying these processes have remained elusive. The distinct glucose-sensing mechanisms found in the pancreas and in the gustat...

Although the "gold standard" for diagnosis of tuberculous meningitis (TBM) is bacterial isolation of Mycobacterium tuberculosis, there are still several complex issues. Recently, we developed an internally controlled novel wide-range quantitative nested real-time PCR (WR-QNRT-PCR) assay for M. tuberculosis DNA in order to rapidly diagnose TBM. For use as an internal control calibrator to measure the copy number of M. tuberculosis DNA, an original new-mutation plasmid (NM-plasmid) was developed. Due to the development of the NM-plasmid, the WR-QNRT-PCR assay demonstrated statistically significant accuracy over a wide detection range (1 to 10 5 copies). In clinical applications, the WR-QNRT-PCR assay revealed sufficiently high sensitivity (95.8%) and specificity (100%) for 24 clinically suspected TBM patients. In conditional logistic regression analysis, a copy number of M. tuberculosis DNA (per 1 ml of cerebrospinal fluid) of >8,000 was an independent risk factor for poor prognosis for TBM (i.e., death) (odds ratio, 16.142; 95% confidence interval, 1.191 to 218.79; P value, 0.0365). In addition, the copy numbers demonstrated by analysis of variance statistically significant alterations (P < 0.01) during the clinical treatment course for 10 suspected TBM patients. In simple regression analysis, the significant correlation (R 2 ‫؍‬ 0.597; P < 0.0001) was demonstrated between copy number and clinical stage of TBM. We consider the WR-QNRT-PCR assay to be a useful and advanced assay technique for assessing the clinical treatment course of TBM.Tuberculous meningitis (TBM) is the severest form of infection of Mycobacterium tuberculosis, causing death or severe neurological defects in more than half of those affected in spite of antituberculosis treatment (ATT) (1,2,8,18). The diagnosis of TBM remains a complex issue, because the most widely used conventional bacteriological detection methods, such as direct smear for acid-fast bacilli (AFB) and culture for M. tuberculosis, are unable to rapidly detect M. tuberculosis with sufficient sensitivity in the acute phase of TBM (3-13, 18, 19). In 2006, we designed a novel internally controlled quantitative nested real-time PCR (QNRT-PCR) assay based on TaqMan PCR (Applied Biosystems) (15). Moreover, based on this original QNRT-PCR (OR-QNRT-PCR) assay, an improved wide-range QNRT-PCR (WR-QNRT-PCR) assay was developed (17). For use as a "calibrator" in WR-QNRT-PCR assay, a new internal control was constructed (17).In the preliminary experiments, the WR-QNRT-PCR assay demonstrated significantly improved quantitative accuracy and had a wide detection range (1 to 10 5 copies) compared to what was seen for the OR-QNRT-PCR assay (17).In this study, we tried to quantitatively detect M. tuberculosis DNA in actual cerebrospinal fluid (CSF) samples by using the WR-QNRT-PCR assay. In addition, the clinical usefulness of this novel assay technique for the rapid and accurate diagnosis of TBM and for assessing the clinical course of TBM was examined. MATERIALS AND METHODSThis study was approv...

The authors examined the usefulness of nested PCR (N-PCR) to detect Mycobacterium tuberculosis (MTB) DNA in CSF for assessing the clinical course of tuberculous meningitis (TBM). N-PCR successfully detected MTB DNA in all nine CSF samples from patients with suspected TBM. During anti-tuberculosis treatments, N-PCR results converted from positive to negative, correlating with the improvement of the patient's clinical condition.

Previously, we designed an internally controlled quantitative nested real-time (QNRT) PCR assay forTuberculous meningitis (TBM) is the severest form of infection of Mycobacterium tuberculosis, causing death or severe neurological defects in more than half of those affected in spite of antituberculosis treatment (25). The diagnosis of TBM remains a complex issue because the most widely used conventional bacteriological detection methods, such as direct smear for acid-fast bacilli and culture for M. tuberculosis, are unable to rapidly detect M. tuberculosis with sufficient sensitivity in the acute phase of TBM (7,8,11,12,18,19,21,22,23,25). At present, the detection of M. tuberculosis DNA in cerebrospinal fluid (CSF) by use of PCR is widely used as a more rapid, sensitive, and specific diagnostic method (1,7,8,10,11,12,15,17,18,19,21,22,23,26). Recently, we designed a novel internally controlled quantitative nested real-time PCR (QNRT-PCR) assay based on TaqMan PCR (Applied Biosystems) (22). This novel assay technique combines the high sensitivity of nested PCR with the accurate quantification of real-time PCR (22, 23). However, this original QNRT-PCR (OR-QNRT-PCR) assay is still unstable and has many points that should be improved (22,23).In this study, to reliably detect M. tuberculosis DNA in CSF samples with a wider detection range, we attempted to improve on the OR-QNRT-PCR technique; therefore, a new internal control for use as a "calibrator" was prepared. We named this improved assay technique wide-range QNRT-PCR (WR-QNRT-PCR) and examined its ability to quantitatively detect M. tuberculosis DNA in samples. In this paper, the development and methodology of the WR-QNRT-PCR assay are stated. MATERIALS AND METHODSThis study was approved by the Nihon University Institutional Review Board. Preparation of the new internal control (plasmid) for use as a calibrator. For the WR-QNRT-PCR assay, two types of the original plasmid, wild plasmids (W-plasmids) and new-mutation plasmids (NM-plasmids), were prepared for a quantitative detection of M. tuberculosis DNA, and this was done as well for the OR-QNRT-PCR assay (22, 23).W-plasmid, which was inserted into a 239-bp DNA fragment of the gene sequence encoding the MPB64 protein of M. tuberculosis (MPT64; GenBank accession no. NC_000962) (22, 23) into pCR 2.1 vector (Invitrogen Corp., San Diego, CA) was constructed for use as the standard template by the previously reported procedure (22,23).NM-plasmid was developed based on the previously reported M-plasmid (22, 23) for use as a new internal-control "calibrator" in the WR-QNRT-PCR assay. In NM-plasmid, a total of four regions, where two pairs of (outer and inner) forward and reverse primers annealed, were replaced with the artificial random nucleotides added to the TaqMan probe annealing region in the M-plasmid (Fig. 1). The sequences of the artificial random nucleotides were set to have the same nucleotide composition as MPT64 of wild M. tuberculosis. Replacing procedures