Currently available interferon (IFN)-γ-release assays (IGRA) cannot discriminate active tuberculosis (TB) from latent TB infection (LTBI), and so have limited clinical utility for diagnosing active ...TB. Since numbers of tumour necrosis factor (TNF)-α-producing T cells are highly correlated with active TB, we hypothesized that detecting IFN-γ- and/or TNF-α-producing T cells would overcome this limitation of IGRA. This study evaluated the diagnostic performances of the IFN-γ and TNF-α dual release fluorospot assay for active TB.
Adult patients with suspected TB including recent TB exposers were prospectively enrolled over a 28-month period. In addition to the conventional IGRA test (i.e. QuantiFERON-In-Tube), a fluorospot assay for detecting IFN-γ- and TNF-α-producing T cells was performed. The final diagnoses were classified by clinical category. Patients with confirmed or probable TB were regarded as active TB, and patients with not active TB were further classified as having not active TB with and without LTBI, based on the QuantiFERON-In-Tube results.
A total of 153 patients including 45 with active TB and 108 with not active TB (38 LTBI vs. 70 not LTBI) were finally analysed. The sensitivity and specificity of the QuantiFERON-In-Tube assay for active TB were 84% (95% confidence interval (CI), 70–93) and 70% (95% CI 61–79), respectively. The IFN-γ/TNF-α dual release assay by fluorospot had substantially higher diagnostic specificity (94%) for diagnosing active TB than the IFN-γ single release assay (72%, p < 0.001), without compromising sensitivity (84% vs. 89%, p 0.79).
The fluorospot-based IFN-γ/TNF-α dual release assay appears to be a simple and useful test for diagnosing active TB.
The flavin-containing monooxygenases (FMOs) are a family of xenobiotic-metabolizing enzymes that are expressed in a species- and tissue-specific manner. FMO2 expression has been observed in pulmonary ...tissue from several species, but not human. Two human FMO2 point mutations have been reported: a cytosine to thymidine transition at position 1414 resulting in a premature stop codon and a thymidine insertion at position 1589 resulting in a frameshift. To define the frequency of these sequence variations and explore their significance, unrelated African-American, Caucasian, and Korean individuals were genotyped. In the African-American population tested (n = 180), the 1414C allele occurred at a 13% frequency; however, all of the tested Caucasians (n = 52) and Koreans (n = 100) were homozygous for the 1414T allele. The T1589 allele occurred at frequencies of 6.9 and 13.0% in African-Americans (n = 175) and Caucasians (n = 23), respectively, and appears to segregate with the 1414T allele. Thus, it would have no further impact on FMO2 activity. Western blot analysis of pulmonary microsomes failed to detect immunoreactive protein in 1414T homozygotes. A heterozygotic individual did exhibit a single band of the expected size, but no detectable FMO activity in the corresponding lung microsomes. Sequence analysis, however, was consistent with the 1414C allele encoding an active FMO2 enzyme. FMO2 mRNA expression was observed in most individuals, but failed to correlate with genotype or protein expression. In summary, functional FMO2 is expressed in only a small percentage of the overall population. However, in certain ethnic groups, active pulmonary FMO2 enzyme will be present in a significant number of individuals.
Heme oxygenase-1 (HO-1) is a 32-kDa stress induced enzyme that degrades heme to carbon monoxide (CO) and biliverdin. By employing RT-PCR and Western blotting techniques, we have examined the HO-1 ...induction in C6 glioma cells that were treated with cadmium chloride (CdCl2) or spermine NONOate (SPER/NO). By employing a cell viability assay, we have also examined the cytoprotective effect of HO-1 induction against the cytotoxicity caused by toxic dose of CdCl2. In C6 glioma cells exposed to CdCl2, expression of HO-1 (mRNA and protein) was increased in a dose- and time-dependent manner. Nitric oxide (NO) generated from SPER/NO very rapidly increased HO-1 mRNA expression in the C6 glioma cells. The induction of HO-1 by SPER/NO protected the cells from toxic dose of CdCl2. The up-regulation of HO-1 mRNA expression by CdCl2 was inhibited by a pre-incubation of the cells with actinomycin D, a potent inhibitor of mRNA transcription. Upon the inhibition of elevated HO-1 mRNA expression by the use of zinc protoporphyrin IX (ZnPP), an inhibitor of HO activity, the change of HO-1 mRNA expression by ZnPP was not observed. Thus, the glial cell may respond to CdCl2 toxicity by enhancing the HO-1 expression in its effort to minimize the CdCl2-derived oxidative damage, and to survive. In the glioma cells, when the HO-1 expression was elevated by a prior incubation with SPER/NO, the cell viability against the cytotoxicity of CdCl2 was significantly increased. When the results of our experiment are taken together, we discovered that NO provided a rapid enhancement of HO-1 expression, and it provided a protective effect against CdCl2-derived oxidative injury in the C6 rat glioma cells.
Conventionally, a semi-quantitative microscopic nitroblue tetrazolium (NBT) assay is used to determine the production of superoxide anion (O
2
−
) in various phagocytic cells. This microscopic assay ...is conducted by counting the cells containing blue NBT formazan deposits, which are formed by reduction of the membrane permeable, water-soluble, yellow-colored, nitroblue tetrazolium (Y-NBT) by O
2
−
. However, this assay is semi-quantitative and is prone to observer bias. In the present study, we modified the NBT assay by dissolving the blue formazan particles using 2 M potassium hydroxide and dimethylsulfoxide and then measured its absorbance using a microplate reader at 620 nm. The absorbance of dissolved NBT increased in proportion to cell number (r=0.9907), incubation time, and stimulus concentration. To test the usefulness of this modified assay, we compared the abilities of a number of types of phagocytic cells to produce O
2
−
. The cells examined included murine macrophage cell lines (RAW 264.7 and J774), freshly prepared murine peritoneal macrophages and neutrophils, a human myeloid cell line (PLB-985), and freshly prepared human peripheral blood neutrophils. In addition, we demonstrate that nitric oxide produced by RAW 264.7 cells does not interfere with the modified colorimetric NBT assay. Taken together, our results indicate that the modified colorimetric NBT assay is simple, sensitive, and quantitative, and that it can be used to determine the amounts of intracellular O
2
−
produced by phagocytic cells. Thus, this assay is sensitive enough to measure, quantitatively, even the small amounts of O
2
−
produced in monocytes and macrophages that are not detectable by the conventional microscopic NBT assay.
Celotno besedilo
Dostopno za:
BFBNIB, DOBA, GIS, IJS, IZUM, KILJ, KISLJ, NUK, PILJ, PNG, SAZU, UILJ, UKNU, UL, UM, UPUK
Objectives
To assess the effect of gender, age, and smoking habits on the in vivo activities of CYP1A2, flavin‐containing monooxygenase (FMO), and xanthine oxidase in Korean subjects.
Methods
One ...hundred thirty‐three age‐ and gender‐matched healthy Korean volunteers (age range, 21 to 78 years; mean age, 35.3 ± 16.6 years) with and without smoking habits participated. After drinking a cup of coffee (200 mL) that contained 110 mg caffeine, a 1‐hour urine sample (between 4 and 5 hours) was collected and caffeine metabolites were analyzed by HPLC.
Results
There were marked individual variations in CYP1A2 (1,7‐dimethylurate + paraxanthine)/caffeine, FMO (theobromine/caffeine), and xanthine oxidase (1‐methylurate/1‐methylxanthine) activities (14‐, 42‐, and 9‐fold, respectively). However, the mean values of these enzyme activities in the nonsmokers were not different between men and women. In the nonsmoking subjects in their 20s, the mean values of CYP1A2 and FMO activities (13.5 ± 5.9 and 2.1 ± 1.9, respectively) were higher than those (7.9 ± 1.8 and 0.95 ± 0.22) of older decennial age groups. Xanthine oxidase activities were the same for all age groups (subjects in their 20s through their 70s). CYP1A2 activity of the smokers (20.0 ± 9.6) was higher than that of the nonsmokers (10.8 ± 5.8; P < .001). Similarly, the FMO activity in smokers (3.4 ± 2.7) was higher than that of the nonsmokers (1.8 ± 1.7; P < .001). The xanthine oxidase activity (1.3 ± 0.5) was not increased in smokers (1.4 ± 0.5; P = .46).
Conclusions
Results of this caffeine metabolism study conducted with age‐ and gender‐matched healthy Korean volunteers with and without smoking habits provided the baseline and the widely varying interindividual activities of CYP1A2, FMO, and xanthine oxidase in a Korean population. The results also suggested that drugs metabolized by CYP1A2 and FMO may require individualized dose adjustment according to the age and smoking habits of the subjects. (Clin Pharmacol Ther 2000;67:258–66.)
Clinical Pharmacology & Therapeutics (2000) 67, 258–266; doi: 10.1067/mcp.2000.104617
Heme oxygenase (HO)-1 is a stress response protein, which confers cytoprotection against oxidative injury and provides a vital function in maintaining tissue homeostasis. Molecular mechanisms ...involved in the inducible transcription of ho-1 occurring in response to numerous and diverse stressful conditions have remained elusive. Since the discovery of E1 and E2, the two upstream enhancers regulating induction of ho-1 transcription in 1989, there have been many studies dealing with molecular mechanisms involved in enhancing HO-1 expression. In this commentary, recent advances in our understanding of the mechanisms involved in the induction of HO-1 expression in mammalian cells are summarized with some supportive results reported by others. Currently available data indicate that activation of ho-1 transcription involves both the heme (native substrate)-dependent selective alleviation of repressor and the oxidative stress-dependent activation of transcriptional activator. The stress-released free-heme (HO-1 substrate) from hemoproteins involved in causing oxidative stress itself appears to act as a molecular switch controlling the repressor- activator antagonism on the enhancer sequences of ho-1. Thus, induction of HO-1 appears to operate in a manner like a simple feedback loop. dox Signal. 7, 1674-1687.
Objective
To investigate the efficacy of double inversion recovery (DIR) sequence for evaluating the synovium of the femoro-patellar joint without contrast enhancement (CE).
Methods
Two radiologists ...independently evaluated the axial DIR and CE T1-weighted fat-saturated (CET1FS) images of 33 knees for agreement; the visualisation and distribution of the synovium were evaluated using a four-point visual scaling system at each of the five levels of the femoro-patellar joint and the location of the thickest synovium. The maximal synovial thickness at each sequence was measured by consensus.
Results
The interobserver agreement was good (
κ
= 0.736) for the four-point scale, and was excellent for the location of the thickest synovium on DIR and CET1FS (
κ
= 0.955 and 0.954). The intersequential agreement for the area with the thickest synovium was also excellent (
κ
= 0.845 and
κ
= 0.828). The synovial thickness on each sequence showed excellent correlation (r = 0.872).
Conclusion
The DIR showed as good a correlation as CET1FS for the evaluation of the synovium at the femoro-patellar joint. DIR may be a useful MR technique for evaluating the synovium without CE.
Key Points
• DIR can be useful for evaluating the synovium of the femoro-patellar joint.
• Interobserver and intersequential agreements between DIR and CET1FS were good.
• Mean thickness of the synovium was significantly different between two sequences.
Methicillin-resistant Staphylococcus aureus (MRSA) bacteria have been responsible for substantial morbidity and mortality in hospitals because they usually have multidrug resistance. Some natural ...products are candidates as new antibiotic substances. In the present study, we investigated the antimicrobial activity of berberine, the main antibacterial substance of Coptidis rhizoma (Coptis chinensis Franch) and Phellodendri cortex (Phellodendron amurense Ruprecht), against clinical isolates of MRSA, and the effects of berberine on the adhesion to MRSA and intracellular invasion into human gingival fibroblasts (HGFs). Berberine showed antimicrobial activity against all tested strains of MRSA. Minimum inhibition concentrations (MICs) of berberine against MRSA ranged from 32 to 128 microg/mL. Ninety percent inhibition of MRSA was obtained with 64 microg/mL or less of berberine. In the checkerboard dilution test, berberine markedly lowered the MICs of ampicillin and oxacillin against MRSA. An additive effect was found between berberine and ampicillin, and a synergistic effect was found between berberine and oxacillin against MRSA. In the presence of 1-50 microg/mL berberine, MRSA adhesion and intracellular invasion were notably decreased compared with the vehicle-treated control group. These results suggest that berberine may have antimicrobial activity and the potential to restore the effectiveness of beta-lactam antibiotics against MRSA, and inhibit the MRSA adhesion and intracellular invasion in HGFs.
Capsaicin (trans-8-methyl-N-vanillyl-6-nonenamide), a major pungent ingredient of red pepper, is reported to have antimutagenic and anticarcinogenic properties. However, the mechanisms underlying its ...chemoprotective effects remain largely unresolved. In the present study, we found that capsaicin induced expression of heme oxygenase-1 (HO-1) in HepG2 cells. Capsaicin treatment resulted in a transient increase in the phosphorylation of Akt and subsequently nuclear translocation of NF-E2-related factor 2 (Nrf2), enhancing its binding to antioxidant response element (ARE). HepG2 cells treated with capsaicin exhibited increased production of reactive oxygen species (ROS). Prior exposure of cells to N-acetyl-L -cysteine blocked not only the ROS production but also the nuclear translocation of Nrf2 and its ARE binding, as well as HO-1 induction by capsaicin. Immunoblot analysis showed that whereas the level of HO-1 protein was elevated, that of NAD(P)H:quinone oxidoreductase (NQO1) was decreased after the treatment with capsaicin or the inhibitor of NQO1, dicumarol. We hypothesize that quinone metabolites or other reactive forms of capsaicin may bind covalently to NQO1 and thereby inhibit its activity, leading to production of ROS. This, in turn, would trigger the activation of Akt via phosphorylation, increase the nuclear translocation and ARE binding of Nrf2, and upregulate the expression of HO-1.
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