Real-time detection of DNA hybridization and melting on oligonucleotide arrays by using optical wave guides.

The challenge of the Human Genome Project is to increase the rate of DNA sequence acquisition by two orders of magnitude to complete sequencing of the human genome by the year 2000. The present work describes a rapid detection method using a two-dimensional optical wave guide that allows measurement of real-time binding or melting of a light-scattering label on a DNA array. A particulate label on the target DNA acts as a light-scattering source when illuminated by the evanescent wave of the wave guide and only the label bound to the surface generates a signal. Imaging/visual examination of the scattered light permits interrogation of the entire array simultaneously. Hybridization specificity is equivalent to that obtained with a conventional system using autoradiography. Wave guide melting curves are consistent with those obtained in the liquid phase and single-base discrimination is facile. Dilution experiments showed an apparent lower limit of detection at 0.4 nM oligonucleotide. This performance is comparable to the best currently known fluorescence-based systems. In addition, wave guide detection allows manipulation of hybridization stringency during detection and thereby reduces DNA chip complexity. It is anticipated that this methodology will provide a powerful tool for diagnostic applications that require rapid cost-effective detection of variations from known sequences.     

Cytidine methylation of regulatory sequences near the pi-class glutathione S-transferase gene accompanies human prostatic carcinogenesis.

Hypermethylation of regulatory sequences at the locus of the pi-class glutathione S-transferase gene GSTP1 was detected in 20 of 20 human prostatic carcinoma tissue specimens studied but not in normal tissues or prostatic tissues exhibiting benign hyperplasia. In addition, a striking decrease in GSTP1 expression was found to accompany human prostatic carcinogenesis. Immunohistochemical staining with anti-GSTP1 antibodies failed to detect the enzyme in 88 of 91 prostatic carcinomas analyzed. In vitro, GSTP1 expression was limited to human prostatic cancer cell lines containing GSTP1 alleles with hypomethylated promoter sequences; a human prostatic cancer cell line containing only hypermethylated GSTP1 promoter sequences did not express GSTP1 mRNA or polypeptides. Methylation of cytidine nucleotides in GSTP1 regulatory sequences constitutes the most common genomic alteration yet described for human prostate cancer.     

Coexistence of focal nodular hyperplasia and pseudotumor caused by focal spared lesion in the liver of a young man

We report a case of 19-year-old male with an underlying case of chronic hepatitis C infection who suffered from two types of benign liver tumor: focal nodular hyperplasia (FNH) and pseudotumor due to focal fatty spared area in a fatty liver. These two kinds of lesions rarely occur simultaneously. The spectral Doppler ultrasound (US) images of these lesions were also provided. We suggest that spectral Doppler US provides an alternative diagnostic tool for the differentiation of liver tumors regarding their vascular pattern, which might help ensure a correct diagnosis.     

Relative impact of low-density lipoprotein-cholesterol concentration and insulin resistance on carotid wall thickening in nondiabetic, normotensive volunteers

The relative effect of an increase in low-density lipoprotein-cholesterol (LDL-C) concentration, as compared with insulin resistance and its manifestations, on intimal medial thickening (IMT) of the common carotid artery was defined in 72 healthy men and women. Insulin-mediated glucose disposal was quantified by the insulin suppression tests, in which the height of the steady-state plasma glucose (SSPG) concentration during the last 30 minutes of a 180-minute infusion of octreotide, insulin, and glucose provides an estimate of insulin resistance. IMT was determined by high-resolution B-mode ultrasonography. Univariate analyses defined statistically significant correlation coefficients between IMT and LDL-C concentration (r =.25, P <.05), SSPG concentration (r =.32, P <.01), triglycerides (TG) (r =.25, P <.05), and high-density lipoprotein-cholesterol (HDL-C) (r = -.28, P <.05) concentrations (changes associated with insulin resistance) and ratio of waist-to-hip girth (r =.29, P <.05). When forward step-wise linear regression analysis was used, concentrations of SSPG, LDL-C and HDL-C all emerged as independent predictors of IMT (P <.05). Furthermore, the magnitude of their relationship to IMT values was comparable. These results provide evidence that insulin resistance is as significant a predictor of degree of atherogenesis (estimated by IMT) of the common carotid artery as a high LDL-C concentration.     

Early postoperative echocardiographic studies of atrial septal defects

Fifteen consecutive adult patients with uncomplicated atrial septal defects (ASD) underwent echocardiographic examinations both before and 3-8 days (mean 7 days) after surgery to study the early postoperative changes in cardiac dimensions and interventricular septal motion. Echocardiographic analyses included patterns of interventricular septal motion, right and left ventricular dimensions at end-diastole (RVDd & LVDd), aortic root dimension at end-diastole (ARDd) and left atrial dimension at end-systole (LADs). The results showed that the septal motion was abnormal in 87% (13/15) before and 40% (6/15) after operation. RVDd decreased from 36 +/- 7 mm to 27 +/- 7 mm (p less than 0.01) while LVDd increased from 33 +/- 6 mm to 39 +/- 4 mm (p less than 0.01). There were no significant changes in LADs and ARDd after surgery. These observations suggest that in patients with ASD the ventricular dimensions and patterns of interventricular septal motion are changed significantly shortly after surgical repair.     

Eradication of Helicobacter pylori significantly reduced gastric damage in nonsteroidal anti-inflammatory drug-treated Mongolian gerbils

AIM: To examine the effect of eradication of Hellcobacter pylori prior to usage of NSAIDs, by investigating gastric inflammatory activity, myeloperoxidase (MPO) activity, prostaglandin (PG) E_2 synthesis in H Pylori-infected, and H pylori-eradicated gerbils followed by administration of indomethacin and rofecoxib. METHODS: Six-week-old male gerbils were orally inoculated with H pylori. Seven weeks later, anti-H pylori triple therapy and vehicle were given to gerbils respectively and followed by oral indomethacin (2 mg/kg·d) or rofecoxib (10 mg/kg·d) for 2 wk. We examined the area of lesions, gastric inflammatory activity, PGE_2 synthesis and MPO activity in the stomach. RESULTS: In indomethacin and rofecoxib-treated gerbils, the following results were obtained in H pylori-infected group vs H pylori-eradicated group respectively: hyperplasia area of the stomach (mm~2): 82.4±9.2 vs 13.9±3.5 (P＜0.05), 30.5±5.1 vs 1.3±0.6 (P＜0.05); erosion and ulcer area (mm~2): 14.4±4.9 vs 0.86±0.5 (P＜0.05), 1.3±0.6 vs0.4±0.3 (P＜0.05); score of gastritis: 7.0±0.0 vs3.6±0.5 (P＜0.05), 7.0±0.0 vs 2.7±0.5 (P＜0.05); MPO activity (μmol H_2O_2/min/g tissue): 104.7±9.2 vs9.0±2.3(P＜0.05), 133.5±15.0 vs2.9±0.7 (P＜0.05); PGE_2 synthesis (pg/mg wet weight/min): 299.2±81.5 vs102.8±26.2 (P＜0.05), 321.4±30.3 vs 11.9±4.8(P＜0.05). CONCLUSION: Eradication of H pylori reduced gastric damage of NSAID-treated Mongolian gerbils. Rofecoxib caused less severe gastric damage than indomethacin in H pylori-eradicated gerbils.     

Prevalence of Childhood Asthma in Taipei, Taiwan, and Other Asian Pacific Countries

Two epidemiological surveys of the prevalence of bronchial asthma in schoolchildren in Taipei, Taiwan, were conducted in 1974 and 1985. The same questionnaire and school, and schoolchildren of the same age (7-15 years), were studied in these two surveys. Bronchial asthma was defined as at least three paroxysmal, recurrent attacks of wheezing and dyspnea in the previous 12 months. A total of 23,678 children were studied in 1974 and 147,373 children in 1985. Data from other Asian Pacific countries were also reviewed.

The results: 1) The questionnaire was able to differentiate asthmatics from nonasthmatics on the basis of differences in methacholine challenge, intracutaneous skin testing, total eosinophil count, total serum IgE, and RAST between the two groups. 2) The prevalence of childhood asthma increased from 1.30% in 1974 to 5.07% in 1985, with boys dominating in both studies. 3) The increase in asthma prevalence could not be explained by air pollution or exposure to new allergens. 4) The prevalence of childhood asthma in the Asian Pacific countries are generally comparable to those of Western countries, and the present study and studies from Japan and New Guinea showed an increasing tendency.

Thus childhood asthma is a major problem in the Asian Pacific countries as well as in Western countries.     

Reconstituted immunity against persistent parvovirus B19 infection in a patient with acquired immunodeficiency syndrome after highly active antiretroviral therapy.

We discovered a patient with AIDS with persistent B19 infection who had slow resolution of anemia after he commenced receiving HAART without intravenous immunoglobulin. The patient's anemia recurred when the initial course of HAART failed, but it remitted slowly after salvage therapy was instituted. However, circulating B19 was still detectable by nested polymerase chain reaction 1 year after commencement of salvage therapy. Immunoglobulin G and immunoglobulin M antibodies against B19 were not detected by means of enzyme-linked immunosorbent assay when the anemia initially resolved, but they were detected after the patient commenced receiving salvage therapy. The absence of antibody response after the initial remission of parvovirus B19 infection suggested that cellular immunity was an important component of reconstituted immune function against B19 after the patient received HAART. The humoral response that was restored later was abnormal; it had strong reactivity to nonstructural protein NS-1 and poor generation of neutralizing antibodies against linear epitopes unique to minor capsid protein VP1.     

Cardiac troponin I: a reliable marker and early myocardial involvement with meningoencephalitis after fatal enterovirus-71 infection

OBJECTIVES: A major outbreak of enterovirus 71 (EV71) in Taiwan in 1998 caused many severe cases and 78 deaths. Our purpose was to find reliable markers and early indicators of fatal EV71 central nervous system (CNS) infection. METHODS: From June 2000 to November 2001, 21 patients with hand foot mouth disease or herpangina with CNS infection were admitted to Kaohsiung Veterans General Hospital. All 21 had culture-confirmed EV71 infection or were EV71 IgM positive. Patients were divided into two groups: group I included the five fatalities at our institution and group II, the 16 surviving patients. RESULTS: Of the 21 infants and children with EV71 infection with CNS involvement, MR imaging studies were completed on 17, and 15 showed hyperintensity in the posterior portions of brain stem. All patients received intravenous immunoglobulin (IVIG) 1 g/day for two days and supportive care. Five patients rapidly deteriorated owing to irreversible hypotension and died. The other 16 patients recovered completely without sequel. In group I patients, the decrease of cardiac ejection function is significant and laboratory findings showed lower platelet count (P=0.0192). The mean of initial cTnI level for groups I and II was 10.6+/-11.6 and 0.48+/-0.55 ng/dl, respectively, higher in group I than in II (P=0.0019). CONCLUSION: We hypothesized that like patients with severe burns, those with severe EV-71 CNS meningoencephalitis have varying degrees of non-ischemic cardiac injury, manifesting as leakage of cTnI from myocytes into the circulation. EV-71 CNS meningoencephalitis likely to die with an early myocardial involvement evidenced by reduced ejection fraction and release of cTnI. We conclude that fatal EV71 CNS infection quickly leads to death due to severe encephalopathy associated with cardiomyopathy.     

Donor demographic and laboratory predictors of allogeneic peripheral blood stem cell mobilization in an ethnically diverse population

A reliable estimate of peripheral blood stem cell (PBSC) mobilization response to granulocyte colony-stimulating factor (G-CSF) may identify donors at risk for poor mobilization and help optimize transplantation approaches. We studied 639 allogeneic PBSC collections performed in 412 white, 75 black, 116 Hispanic, and 36 Asian/Pacific adult donors who were prescribed G-CSF dosed at either 10 or 16 microg/kg per day for 5 days followed by large-volume leukapheresis (LVL). Additional LVL (mean, 11 L) to collect lymphocytes for donor lymphocyte infusion (DLI) and other therapies was performed before G-CSF administration in 299 of these donors. Day 5 preapheresis blood CD34(+) cell counts after mobilization were significantly lower in whites compared with blacks, Hispanics, and Asian/Pacific donors (79 vs 104, 94, and 101 cells/microL, P < .001). In addition, donors who underwent lymphapheresis before mobilization had higher CD34(+) cell counts than donors who did not (94 vs 79 cells/microL, P < .001). In multivariate analysis, higher post-G-CSF CD34(+) cell counts were most strongly associated with the total amount of G-CSF received, followed by the pre-G-CSF platelet count, pre-G-CSF mononuclear count, and performance of prior LVL for DLI collection. Age, white ethnicity, and female gender were associated with significantly lower post-G-CSF CD34(+) cell counts.