Gene polymorphism and frequencies of the NPC1L1 gene (rs2072183, rs217434 and rs217428) in Japanese patients with dyslipidemia.

WHAT IS KNOWN AND OBJECTIVE: Niemann-Pick C1-Like 1 (NPC1L1) plays a pivotal role in intestinal cholesterol absorption. Ezetimibe is known as an inhibitor for NPC1L1 and decreases concentration of low-density lipoprotein cholesterol (LDL-C) in blood. Responses of the decrease of serum LDL-C levels to ezetimibe have been reported to be different among NPC1L1 variants. However, there are still limited data concerning the genetic variation in the NPC1L1 gene, specifically, in Japanese patients with dyslipidemia. The purpose of this study is to elucidate genotype and allele frequencies of the NPC1L1 gene in Japanese patients with dyslipidemia. METHODS: Written informed consent was obtained from all participants. All patients were administered ezetimibe at the dose of 10 mg for once a day either alone or coadministered with statins. Patient's data were retrospectively obtained from their medical records. Genomic DNA was extracted from whole blood samples and analysed three NPC1L1 SNPs (rs2072183, rs217428 and rs217434) by the direct sequencing method. RESULTS AND DISCUSSION: We found that there is a significant difference of genotype frequencies between healthy Japanese and dyslipidemic subjects in rs2072183. No significant differences were observed in rs217428 and rs217434; however, comparison of our data with literature reports suggests that there are significant differences in the frequencies of rs217428 and rs217434 between Canadian and Japanese dyslipidemic patients. WHAT IS NEW AND CONCLUSION: Our study is the first report concerning the genotype and allele frequencies of the gene coding for NPC1L1 in Japanese patients with dyslipidemia. The most notable result was to demonstrate that there exists a significant difference in rs2072183 variant between healthy Japanese and dyslipidemic subjects and also found that there exists genetic variation of rs2072183 between Japanese and Canadian patients with dyslipidemia. Our results are expected to facilitate research in the proper use of ezetimibe-based mono- or combination therapies. Further studies will be required to evaluate the effects of rs2072183 on the efficacy of LDL cholesterol reduction by ezetimibe.

Clinical and endoscopic characteristics of acute haemorrhagic rectal ulcer, and endoscopic haemostatic treatment: a retrospective study of 95 patients.

AIM: Acute haemorrhagic rectal ulcer (AHRU) is characterized by sudden onset of painless and massive rectal bleeding in elderly bedridden patients who have serious illness. Endoscopic diagnosis and management of AHRU is, however, still controversial. We retrospectively investigated 95 AHRU patients to elucidate the clinical characteristics, endoscopic findings and haemostatic strategies. METHOD: Between January 1999 and March 2007, 95 patients were diagnosed with AHRU in our hospital. Medical records and colonoscopy files were reviewed. Clinical features, colonoscopic findings, haemostatic treatment and outcome of the patients were evaluated. RESULTS: Eighty per cent of the patients were bedridden at the onset. The most frequent underlying disorder was cerebrovascular disease (36.8%). Hypoalbuminaemia (< 3.5 g/dl) was seen in 92.6% of the patients. Endoscopic findings of AHRU were classified as circumferential ulcer (41.1%), linear or nearly round small ulcer(s) (44.2%), circumferential and small ulcer(s) (7.4%) and Dieulafoy-like ulcer (7.4%). Primary endoscopic haemostatic treatment was performed in 45.3% of cases. Recurrent bleeding occurred in 24.2% of patients. Permanent haemostasis was achieved by secondary endoscopic treatment in 82.6% of re-bleeding patients. CONCLUSION: Understanding the typical clinical and endoscopic findings and careful endoscopic examination are important for the accurate diagnosis of AHRU, and endoscopic haemostatic therapy may be effective for bleeding patients.

Multidrug resistant Mycobacterium leprae from patients with leprosy.

Sequences of the folP1, rpoB, and gyrA genes were analyzed for 88 isolates of Mycobacterium leprae from leprosy patients in Japan, Haiti, Indonesia, Pakistan, and the Philippines. Thirteen isolates (14.8%) showed representative mutations in more than two genes, suggesting the emergence of multidrug-resistant M. leprae.

Pro- and anti-inflammatory gene expression in the murine small intestine and liver after chronic exposure to alcohol.

BACKGROUND: Endotoxin has been proposed to play a primary role in ALD, by initiating an inflammatory cascade within the liver. Although the source of these cytokines has been presumed to be circulating monocytes or tissue macrophages, ethanol-induced, nonhepatic sources of soluble mediators recently have been identified. One potential, but not clearly defined, extrahepatic source of cytokines in ALD is the intestine. In the current study, we hypothesized that alcohol would alter cytokine expression within the small intestine of mice exposed to ethanol and that LPS would alter levels of cytokine expression even more dramatically. METHODS: Mice were fed a modified Lieber-DeCarli liquid ethanol or control diet for up to 14 days prior to injecting either saline or LPS. Plasma alanine aminotransferase (ALT) and cytokine levels, histology, and RT-PCR of pro- and anti-inflammatory cytokine gene expression were determined from distal ileum and liver samples. Translocation of intestinal bacterial flora also was assessed. RESULTS: Ethanol exposure upregulated basal gene expression of IL-1 beta, TNF-alpha, IL-6, and iNOS in the distal ileum, but similar effects of ethanol on the liver were not observed. In contrast, LPS challenge of ethanol-exposed mice increased intestinal gene expression of some cytokines, but decreased expression of others. These effects were not associated with bacterial translocation. Also, ethanol alone induced a modest increase in both ICAM-1 and TLR4 mRNA expression in the intestine, but expression of both molecules was inhibited in mice that received both ethanol and LPS. Finally, whereas basal levels of hepatic IL-11 mRNA were not elevated by exposure to ethanol, intestinal IL-11 mRNA levels were increased more than 100-fold. CONCLUSIONS: These studies are the first to show that ethanol affects cytokine gene expression in the ileum and identifies the ileum as a potential target for ethanol effects. In addition, our results suggest that IL-11 expression may be enhanced in the intestine to help repair or protect this organ from alcohol-induced damage. Collectively, these studies suggest that both pro- and anti-inflammatory soluble mediators in the intestine maintain and exacerbate the local hepatic response to ethanol.

Simultaneous detection of Mycobacterium leprae and its susceptibility to dapsone using DNA heteroduplex analysis.

Currently recommended control measures for treating leprosy with multidrug therapy should control the spread of drug-resistant strains; however, dapsone (DDS) resistance continues to be reported. Comprehensive estimates of drug-resistant leprosy are difficult to obtain due to the cumbersome nature of the conventional drug susceptibility testing method using mouse footpad inoculation, which requires at least 6 months to obtain results. Recently, it has been determined that DDS-resistant strains contain missense mutations in codon 53 or 55 of the folP1 gene of Mycobacterium leprae, and definitive evidence linking these mutations with DDS resistance in M. leprae has been obtained. Based on these mutations, a heteroduplex DDS M. leprae (HD-DDS-ML) assay was developed for the simultaneous detection of M. leprae and of its susceptibility to DDS. The assay relies on the PCR amplification of an M. leprae-specific 231-bp fragment of folP1 containing codons 53 and 55. The PCR products are allowed to anneal to a universal heteroduplex generator, and the separation of the resultant DNA duplexes is accomplished by polyacrylamide gel electrophoresis. M. leprae was detected in crude cell lysates of skin biopsy specimen homogenates from eight leprosy patients and from M. leprae-infected mouse or armadillo tissues infected with 14 separate strains using the HD-DDS-ML assay. The assay was specific for M. leprae in a comparison with results obtained from 14 species of mycobacteria other than M. leprae and four bacterial species known to colonize human skin. The HD-DDS-ML assay detected as few as 100 M. leprae organisms present in homogenates of human skin and demonstrated a 93% correlation with DDS susceptibility as determined by both DNA sequencing of folP1 and mouse footpad susceptibility testing. The HD-DDS-ML assay provides a new tool for the simultaneous detection of M. leprae and of its susceptibility to DDS from a single specimen. The assay should prove useful for drug resistance surveillance in leprosy control programs when combined with similar molecular tests developed for other drug resistance markers.

Mycobacterium leprae typing by genomic diversity and global distribution of genotypes.

The genetic diversity and related global distribution of 51 Mycobacterium leprae isolates were studied. Isolates were obtained from leprosy patients from 12 geographically distinct regions of the world and two were obtained from nonhuman sources. Polymerase chain reaction (PCR) followed by DNA sequencing was performed targeting the rpoT gene of M. leprae. Isolates were classified into two groups based on the number of tandem repeats composed of 6 base pairs in the rpoT gene. Isolates from Japan (except Okinawa) and Korea belonged to one group, while those from Southeast Asian countries, Brazil, Haiti and Okinawa in Japan belonged to a second genotype. M. leprae obtained from two nonhuman sources (an armadillo and a mangabey monkey) revealed the latter genotype. These results demonstrate the genetic diversity of M. leprae and the related genotype-specific distribution in the world.

Molecular basis of clarithromycin-resistance in Mycobacterium avium intracellulare complex.

Nucleotide sequences of domain V and domain II regions of the 23S rRNA gene were determined in both in vitro-made mutants and clinical isolates of Mycobacterium avium and M. intracellulare conferring clarithromycin-resistance. All laboratory-made mutants showed high level resistance to clarithromycin (> 150 micrograms ml-1) and mutation at position 2058 (cognate with Escherichia coli base) in domain V region. In the clinical isolates, while the susceptible ones had no mutation in domain V, the resistant strains showed mutation at 2058 or 2059. Six isolates with low level of resistance exhibited no mutation in domain V. All strains tested had no mutation in domain II region. These results suggested that most of the resistance arose from the mutation in domain V of the 23S rRNA gene, but other unknown mechanisms evidently exist in mycobacteria.

Comparison of polymerase chain reaction, immunohistochemistry and conventional histopathology in the diagnosis of early leprosy in Sichuan Province of China.

46 formalin-fixed, paraffin-embedded skin biopsy specimens, which were clinically suspected or diagnosed as early leprosy, were retrieved from the files of Sichuan, China from 1997 to 1999. All of them were examined by polymerase chain reaction (PCR) using the primers amplifying the 130 base-pair fragment of the gene from the 16S ribosomal RNA of Mycobacterium leprae, hematoxylin and eosin (H&E) staining, modified Fite-Faraco technique for M. leprae and immunostaining with the antiserum against the PGL-1, LAM-B, S-100 protein using ABC method. PCR was positive for 27 (58.7%) of 46 specimens. In 13 (28.3%) among them, only PCR signals were positive for M. leprae and all other test were negative. AFB was positive for 7 (15.2%) of 46, PGL-1 was positive for 17 (36.9%) of 46, LAM-B was positive for 10 (21.7%) of 46. Early epithelioid cells granuloma was detected in 4 (8.7%) patients (TT 3, BT 1), macrophage granuloma was detected in 1 (2.2%) patient (BL), S-100 protein staining showed early nerve granuloma for 4 (8.7%) of 46, peripheral nerve inflammatory infiltration for 11 (23.9%) of 46. Comparison PCR with other method showed statistically significant difference. PCR have an advantage over microscopic examination in detecting M. leprae in biopsy specimens negative for acid-fast bacilli.

Detection of Mycobacterium leprae by Polymerase Chain Reaction.

The improved procedure based on Polymerase Chain Reaction (PCR) for detection of M. leprae has been developed. The sensitivity and specificity of this method were tested using different concentration of genomic DNA of M. leprae Thai 53 and genomic DNAs from mycobacterial species and related microorganisms respectively. Application of this method to biopsy samples obtained from Bangladesh was conducted and detected M. leprae DNA in 7 of the 10 clinical specimens. Acid fast bacilli were not detected in four of the seven positive cases under the microscopic observation. It was concluded that this method was sensitive and specific for detection of M. leprae in clinical specimens and also simple to detect in only one step of PCR.

A Mycobacterium leprae isolate resistant to dapsone, rifampin, ofloxacin and sparfloxacin.

Mycobacterium leprae were isolated from a Japanese patient, and susceptibility to antileprosy drugs was examined by the mouse foot pad method. The isolate was susceptible to clofazimine and clarithromycin, and resistant to dapsone, rifampin, ofloxacin and sparfloxacin. Mutations were identified in the genes associated with resistance to these drugs. The risk of the emergence of leprosy with multidrug resistance is emphasized.

Diaminodiphenylsulfone resistance of Mycobacterium leprae due to mutations in the dihydropteroate synthase gene.

The nucleotide sequence analysis of the dihydropteroate synthase (DHPS) gene of six diaminodiphenylsulfone-resistant Mycobacterium leprae strains revealed that the mutation was limited at highly conserved amino acid residues 53 or 55. Though the mutation at amino acid residue 55 or its homologous site has been reported in other bacteria, the mutation at residue 53 is the first case in bacteria. This is the first paper which links the mutations in DHPS and sulfonamide resistance in M. leprae. This finding is medically and socially relevant, since leprosy is still a big problem in certain regions.

Nucleotide sequence of the Mycobacterium leprae katG region.

Synthetic oligonucleotide primers based on the DNA sequence data of the Escherichia coli, Mycobacterium tuberculosis, and Mycobacterium intracellulare katG genes encoding the heme-containing enzyme catalase-peroxidase were used to amplify and analyze the Mycobacterium leprae katG region by PCR. A 1.6-kb DNA fragment, which hybridized to an M. tuberculosis katG probe, was obtained from an M. leprae DNA template. Southern hybridization analysis with a probe derived from the PCR-amplified fragment showed that the M. leprae chromosome contains only one copy of the putative katG sequence in a 3.4-kb EcoRI-BamHI DNA segment. Although the nucleotide sequence of the katG region of M. leprae was approximately 70% identical to that of the M. tuberculosis katG gene, no open reading frame encoding a catalase-peroxidase was detectable in the whole sequence. Moreover, two DNA deletions of approximately 100 and 110 bp were found in the M. leprae katG region, and they seemed to be present in all seven M. leprae isolates tested. These results strongly suggest that M. leprae lacks a functional katG gene and catalase-peroxidase activity.

Dharmendra antigen but not integral M. leprae is an efficient inducer of immunostimulant cytokine production by human monocytes, and M. leprae lipids inhibit the cytokine production.

Killed integral Mycobacterium leprae, Mitsuda antigen, and chloroform-treated M. leprae, Dharmendra antigen (Dh-Ag), have been used for the classification of leprosy patients based on cell-mediated immunity. Heat-killed M. leprae also were used as a component of the Convit vaccine. Human blood monocytes were stimulated with M. leprae or Dh-Ag and their cytokine-inducing ability was compared. Monocytes were cultured in the presence of fresh human serum because of the efficiency of cytokine induction and the phagocytosis of M. leprae have been shown to be optimal in the presence of fresh serum. M. leprae and Dh-Ag were equally phagocytosed by monocytes. Dh-Ag was more potent than M. leprae in the induction of immunostimulatory/proinflammatory cytokines, interleukin-1 (IL-1), IL-6 and tumor necrosis factor (TNF). In contrast, a comparable level of IL-1ra, an immunosuppressive cytokine, was induced by M. leprae and Dh-Ag. The lipids extracted from M. leprae induced none of these cytokines by monocytes. Nevertheless, when monocytes were pretreated with the lipids followed by stimulation with Dh-Ag, productions of IL-1, IL-6 and TNF were all inhibited in a dose-dependent manner. However, the lipids did not inhibit the cytokine production induced by other stimuli including BCG and lipopolysaccharide. Moreover the lipids did not affect the production of IL-1ra. These results suggest that the lipids from M. leprae are responsible for the poor cytokine-inducing ability of M. leprae, thus favoring their infection. These results also suggest that Dh-Ag rather than integral M. leprae may be useful as a vaccine candidate because Dh-Ag is able to induce a large amount of cytokines from monocytes.

Comparison of two dentin adhesives to primary vs. permanent bovine dentin.

The purpose of this study was to compare the shear bond strengths of two adhesive systems to the primary and permanent dentin. Labial surfaces of extracted and frozen bovine mandibular primary incisors and permanent incisors were ground with #600 grit SiC paper to expose dentin. Bisco Dental Products All Bond 2 (Group AB2) or Sunmedical Co. Superbond D Liner (Group SDL) tooth surface conditioner and adhesive were applied and bonded with resin composite. A shear bond strength (SBS) test was performed and the data were analyzed by an ANOVA (P < 0.05). After the SBS test, the test surfaces of the dentin and the resin were observed using SEM. SBS on the primary dentin were significantly higher than those on the permanent dentin, both in the nonthermal cycled groups and the thermal cycled groups with the exception of the thermal cycled group of Group SDL. In the thermal cycled group of Group SDL, there was no significant difference between SBS on the primary dentin and SBS on the permanent dentin. Bond strengths on the primary dentin were found to be significantly higher than those on the permanent dentin, when using All Bond 2 or Superbond D Liner adhesive systems.

Pathological investigation of armadillos infected with Mycobacterium leprae.

An infection experiment with M. leprae was carried out using 20 nine-banded armadillos. As a result, the development of leprous lesions and a marked multiplication of AFB were confirmed in a high rate of 13 out of 15 cases (86.8%) in the inoculated groups. These changes were found to be progressing at post mortem of one case even with the shortest life period for 7.5 months and were very serious in one case with the longest life period for 33 months, suggesting the continuation of symptoms, though it is an expression neglecting the individual difference in susceptibility to leprosy. Among infected viscera with AFB, the most conspicuous lesions were found in the liver and spleen. The developed lesions were found in the lung, stomach and kidney which had been never seen in HD in human cases, and so, which may characterize armadillos' leprosy. The change in the peripheral nerve was not so severe when compared with that in HD in human cases. This difference will remain as a future pathological problem to be solved.

Purification and properties of a membrane-bound phospholipase B from Mycobacterium lepraemurium.

The phospholipid deacylating enzyme was solubilized from the particulate (membrane) fraction of Mycobacterium lepraemurium with Triton X-100 and sodium cholate, and purified 1100-fold to homogeneous state by 5 steps of column chromatography: DE-52, PL-Sepharose (phosphatidylserine-attached sepharose), Mono P, heparin-Agarose and Mono Q column chromatography. The purified enzyme was composed of single polypeptide chain and molecular mass of 37 kDa was estimated for the protein by SDS-PAGE. The isoelectric point was determined about pH 4.6 and the protein was highly resistant to various kinds of proteolytic enzymes. The purified enzyme hydrolyzed both diacyl and monoacyl phospholipids showing that this enzyme was classified to phospholipase B (phospholipase A1/lysophospholipase). This phospholipase B had acidic pH optima and hydrolyzed both neutral phospholipids such as phosphatidylcholine (PC), phosphatidylethanolamine (PE) and acidic phospholipids such as phosphatidylserine (PS), phosphatidylinositol (PI) and phosphatidylglycerol (PG). Various fatty acids such as 12:0, 14:0, 16:0, 18:0 and 18:1 at sn-1 position, and 18:1, 18:2, 18:3 and 16:0 at sn-2 position were liberated from PC, suggesting no strict specificity toward the fatty acyl groups of phospholipids. From the comparison of degradation patterns of phosphatidylcholine with sn-1-[1-14C]- and sn-2-[1-14C]fatty acids, this enzyme was suggested to hydrolyze sn-1 position of phospholipid first and then sn-2 position, as the phospholipase B of M. phlei. This enzyme also attacked 1-acyl- and 2-acyl-lyso-PC at about same rates. The Km values for 1-acyl-2-oleoyl-PC and 2-oleoyl-lyso-PC were estimated 1.6 and 0.75 mM, respectively.

[Rapid detection of acid-fast bacilli with Mycobacteria Growth Indicator Tube (MGIT)].

The BBL MGIT Mycobacteria Growth Indicator Tube is a novel broth based culture system for the detection of mycobacteria from clinical specimens. The tubes consist of a fluorescent indicator embedded in silicone on the bottom of a 16x100 mm round-bottom tube, filled with 4ml of an enriched BBL Middlebrook 7H9 broth base, with 0.25% glycerol. Actively growing mycobacteria consume the oxygen dissolved in the medium and fluorescence will occur when the tubes are observed with a 365nm transilluminator. The purpose of this study is to evaluate comparatively MGIT with 1% Ogawa egg medium by using two hundred and forty-five clinical specimens. The samples were digested, decontaminated and concentrated for culture using N-acetyl-L-cysteine-sodium hydroxide method. Fifty-nine of 245 (24%) clinical samples were culture positive for mycobacteria (43 M. tuberculosis complex, 12 M. avium complex and 4 other species) by one or both test systems. The MGIT detected 4 isolates of M. tuberculosis complex and 6 isolates of M. avium complex not recovered by the Ogawa egg medium, respectively. The mean time of detection of M. tuberculosis complex was 13 days (range: 2-26 days) and 19 days (range: 8-31 days) for MGIT and Ogawa egg medium, respectively, and that of M. avium complex was 5 days (range: 2-8 days) and 16 days (range: 6-22 days) for the MGIT and Ogawa egg medium, respectively. Overall, the MGIT is a sensitive culture system for the detection of mycobacteria from clinical specimens, is easy to use and may be applicable to clinical laboratories.