Denaturing HPLC coupled with multiplex PCR for rapid detection of large deletions in Duchenne muscular dystrophy carriers.
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Chromatography, High Pressure Liquid↗
Biomedical subjects
Chien-Nan Lee
Publications and source records attributed to Chien-Nan Lee.
Characterization of DNA vaccines encoding the domains of calreticulin for their ability to elicit tumor-specific immunity and antiangiogenesis.
Antigen-specific cancer immunotherapy and antiangiogenesis are feasible strategies for cancer therapy because they can potentially treat systemic tumors at multiple sites in the body while discriminating between neoplastic and non-neoplastic cells. We have previously developed a DNA vaccine encoding calreticulin (CRT) linked to human papillomavirus-16 E7 and have found that this vaccine generates strong E7-specific antitumor immunity and antiangiogenic effects in vaccinated mice. In this study, we characterized the domains of CRT to produce E7-specific antitumor immunity and antiangiogenic effects by generating DNA vaccines encoding each of the three domains of CRT (N, P, and C domains) linked to the HPV-16 E7 antigen. We found that C57BL/6 mice vaccinated intradermally with DNA encoding the N domain of CRT (NCRT), the P domain of CRT (PCRT), or the C domain of CRT (CCRT) linked with E7 exhibited significant increases in E7-specific CD8(+) T cell precursors and impressive antitumor effects against E7-expressing tumors compared to mice vaccinated with wild-type E7 DNA. In addition, the N domain of CRT also showed antiangiogenic properties that might have contributed to the antitumor effect of NCRT/E7. Thus, the N domain of CRT can be linked to a tumor antigen in a DNA vaccine to generate both antigen-specific immunity and antiangiogenic effects for cancer therapy.
Angiogenesis Inhibitors↗
Rapid detection of FGFR3 gene mutation in achondroplasia by DHPLC system-coupling heteroduplex and fluorescence-enhanced primer-extension analysis.
Achondroplasia is a common form of human dwarfism with characteristically rhizomelic shortening of extremities and relative macrocephaly. It is transmitted as an autosomally dominant inheritance, and about 80% of affected individuals result from sporadic mutations without positive family histories. Achondroplasia comes from the genetic point mutations in the fibroblastic growth factor receptor 3 gene (FGFR3), which enables abnormal cartilage growth-plate differentiation and insufficient bony development. The most common genetic mutations in this receptor are G to A at position 1138 (G1138A), which result in the substitution of glycine to arginine at codon 380. Based on genetic information, molecular genetic testing can provide an exact diagnosis comparing to radiological and prenatal ultrasound evaluations. Here we introduce denaturing high-performance liquid chromatography (DHPLC) for the detection of 17 cases of achondroplasia and 120 unaffected cases. After coupling heteroduplex and fluorescence-enhanced primer-extension analysis, all affected patients with G1138A were identified successfully. In conclusion, we demonstrated that DHPLC is an efficient, accurate, and sensitive technique to detect the single gene mutation of achondroplasia in clinical applications.
Achondroplasia↗
Naked RNA vaccine controls tumors with down-regulated MHC class I expression through NK cells and perforin-dependent pathways.
One of the major issues facing cancer immunotherapy is that many human cancers down-regulate expression of MHC class I molecules. The understanding of the mechanisms of antitumor effects against tumors with down-regulated MHC class I will facilitate rational design of vaccines and immunotherapeutic strategies to control such tumors. A naked Sindbis RNA replicon vector (SINrep5) encoding the herpes simplex virus type 1 protein VP22 linked to E7 (SINrep5-VP22/E7) generated significant antitumor effects against TC-1 and TC-1 P3(A15), tumors with down-regulated MHC class I expression. Naked SINrep5 RNA without the insert or an E7 vaccine also produced antitumor effects against TC-1 P3(A15) but not TC-1. Mice vaccinated with any of these naked RNA vaccines generated higher percentages of NK cells. In vivo Ab depletion experiments revealed that NK cells were important for the antitumor effects of naked RNA vaccines against TC-1 P3(A15) and that the antitumor effects were perforin-dependent. Poly I:C also increased the percentage of NK cells and generated antitumor effects against the tumors with down-regulated MHC class I. Thus, the SINrep5-VP22/E7 naked RNA vaccine controls MHC class I-positive and MHC class I-down-regulated tumor cells via different mechanisms, and NK cells play an important role in the antitumor effects generated by naked RNA replicon vaccines.
Adoptive Transfer↗
Meconium peritonitis presenting as isolated massive fetal ascites.
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Adult↗
Postnatal outcome of fetal bradycardia without significant cardiac abnormalities.
BACKGROUND: A heart rate <100 beats/min may be present in 5% of fetuses with arrhythmia. In this study, we sought to define the feasibility of in utero diagnosis of the underlying mechanisms and the postnatal outcome. METHODS: The types of fetal bradycardia were defined by fetal echocardiogram. Fetuses with transient sinus bradycardia were excluded. RESULTS: From 1995-2000, a total of 18 fetuses were found to have bradycardia. Postnatal 12-lead electrocardiograms made at least 3 times during follow-up confirmed the cardiac rhythm in all except those terminated during pregnancy. Sick sinus bradycardia was noted in 4 patients (22 %), of whom 1 had a positive family history and 2 received permanent pacemakers at age 4 and 5 years. Frequent nonconducted atrial premature beats or nonconducted atrial bigeminy or trigeminy were noted in 5 (28%). All of them revealed normal sinus rhythm after birth. Atrioventricular (AV) block was noted in 6 (33%), of whom 3 were terminated due to severe heart failure, and another 2 fetuses received permanent pacemaker implantation soon after birth. Only 1 fetus whose mother showed normal levels of C3 and C4 recovered at late gestation. Most importantly, 3 patients (17%) developed intermittent bradycardia (AV block) and ventricular tachycardia during the fetal stage. They were found to have long QT syndrome. Two of them died during early infancy. One received in utero propranolol and postnatal propranolol and nicorandil with satisfactory control. CONCLUSION: The mechanisms responsible for fetal bradycardia were diverse and could be characterized by fetal echocardiography. The prognosis was poor in those with long QT syndrome manifested as intermittent fetal bradycardia and tachycardia with AV dissociation.
Adult↗
Power Doppler vascularity index for predicting the response of neoadjuvant chemotherapy in cervical carcinoma.
BACKGROUND: To evaluate whether the power Doppler vascularity index (PDVI) can predict the response to neoadjuvant chemotherapy (NACT) in cervical carcinoma. METHODS: Twenty-five women with bulky early stage cervical carcinoma treated by NACT followed by surgery were enrolled. Their response to NACT was evaluated. Clinical characteristics and pathologic data were recorded. Transvaginal power Doppler was performed before, during and after NACT. PDVI was detected using power Doppler and a quantitative image processing system. Factors that can potentially correlate with the response to NACT were analyzed. RESULTS: Twelve (48%) patients showed a response (responders) to NACT and 13 (52%) were unchanged or had progressive disease (nonresponders) after NACT. Higher PDVI values were noted in tumors with lymphovascular emboli and pelvic lymph node metastasis both before and after NACT. The mean values of the PDVI of the nonresponders before (19.27 +/- 6.01 vs. 12.28 +/- 7.06, p = 0.014), during (20.2 +/- 1.5 vs. 12.9 +/- 2.1, p = 0.009) and after NACT (18.1 +/- 6.0 vs. 9.3 +/- 5.4, p = 0.001) were significantly higher than those of the responders. When the cutoff point for predicting nonresponders to NACT was set at a PDVI value of 15%, the sensitivity was 92.3% and the specificity 66.7%. CONCLUSIONS: The power Doppler vascularity index can predict the response to neoadjuvant chemotherapy in cervical cancer, and might be useful for the evaluation of response to chemotherapy in cancer patients in the future.
Adenocarcinoma↗
Placenta growth factor elevation in the cord blood of premature neonates predicts poor pulmonary outcome.
OBJECTIVE: To determine whether an elevated placenta growth factor (PlGF) level in cord blood is associated with increased risk for preterm infants to develop bronchopulmonary dysplasia (BPD). METHODS: Sixty-three preterm infants who were born at 34 weeks' gestation or earlier were enrolled. Two infants who died before 28 days' postnatal age could not be assigned a BPD status and were excluded. PlGF levels in cord blood were measured using enzyme-linked immunosorbent assay. Mann-Whitney rank sum test, Spearman correlation coefficients, and multivariable linear or logistic regression analyses were used for statistical analysis. RESULTS: The BPD group had a higher PlGF level, lower gestational age, lower birth weight (BW), higher incidence of endotracheal tube intubation, and longer duration of intubation. The PlGF levels in cord blood correlated negatively with gestational age and BW. However, multivariable logistic regression analyses revealed that only elevated cord blood PlGF levels and BW were associated with BPD after adjusting for all contributing factors. Furthermore, an increased PlGF level in cord blood was significantly correlated with the clinical severity of BPD, as measured by duration of intubation. At 17 mg/dL, the specificity of cord blood PlGF level in predicting BPD was 95%, the sensitivity was 53%, the positive predictive value was 83%, and the negative predictive value was 82%. CONCLUSIONS: Measuring cord blood PlGF level at birth might be a biological marker for predicting the occurrence of BPD and allowing early therapeutic intervention.
Biomarkers↗
Rapid prenatal diagnosis of X-linked chronic granulomatous disease using a denaturing high-performance liquid chromatography (DHPLC) system.
OBJECTIVE: To describe a family on whom it was possible to perform a rapid prenatal diagnosis for chronic granulomatous disease (CGD) using a denaturing high-performance liquid chromatography (DHPLC) system. METHODS: For a family whose first-born, a boy, suffered from X-linked chronic granulomatous disease, fetal DNA was obtained from an ongoing pregnancy by amniocentesis early in the second trimester. Denaturing high-performance liquid chromatography and direct sequencing were used to attempt to detect the previously identified X-linked chronic granulomatous disease mutation. RESULTS: Our studies predicted that the fetus in question was not likely to be affected by chronic granulomatous disease, which was demonstrated to be correct at birth. CONCLUSIONS: Here, we introduce a molecular diagnostic tool (DHPLC) for an effective and exact prenatal diagnosis of normality for the second-born child, as determined from amniocentesis during the second trimester.
Amniocentesis↗
Overexpression of placenta growth factor contributes to the pathogenesis of pulmonary emphysema.
To examine the role of placenta growth factor (PlGF) in the pathogenesis of pulmonary emphysema, we generated PlGF-transgenic (TG) mice using a phosphoglycerate kinase promoter. This resulted in constitutive overexpression of PlGF. In these TG mice, pulmonary emphysema, with enlarged air spaces and enhanced pulmonary compliance, first appeared at 6 months of age and became prominent at 12 months. Increased alveolar septal cell apoptosis was noted in their lungs. Fluorescence-activated cell sorter analysis suggests that these apoptotic septal cells are type II pneumocytes. At the same time, the messenger RNA of vascular endothelial growth factor and platelet-endothelial cell adhesion molecule-1, an endothelial cell marker, were downregulated indicating a reduced number of endothelial cells and its survival factor VEGF. In vitro, exogenous PlGF can inhibit the proliferation and promote the cell death of mouse type II pneumocytes. In normal newborn mice, abundant expression of PlGF messenger RNA was detected in the lungs during saccular division but was rapidly downregulated after alveolarization was complete. Thus, a persistently elevated PlGF was detrimental to the developed lung and causes the emphysematous change seen in our TG mice. Our study suggests that PlGF plays an important role in the pathogenesis of pulmonary emphysema via its action on type II pneumocytes.
Animals↗
Interleukin-6 promotes cervical tumor growth by VEGF-dependent angiogenesis via a STAT3 pathway.
Interleukin-6 (IL-6) has received particular attention in the pathogenesis of cervical cancer, although the underlying mechanism remains elusive. This study revealed that IL-6 promotes in vivo tumor growth of human cervical cancer C33A cells, but does not substantially alter their in vitro growth kinetics. The in vivo angiogenic assays showed that IL-6 increases angiogenic activity in human cervical cancer cells, an effect that is specifically associated with upregulation of vascular endothelial growth factor (VEGF). Also, using anti-VEGF antibody to block VEGF function significantly inhibited IL-6-mediated angiogenesis and tumor growth in nude mice, strongly supporting the critical role of VEGF in the IL-6-mediated cervical tumorigenesis. Accordingly, the signaling pathway downstream of IL-6/IL-6R responsible for the regulation of VEGF was investigated. Notably, pharmacological inhibition of PI3-K or MAPK failed to inhibit IL-6-mediated transcriptional upregulation of VEGF. Meanwhile, blocking STAT3 pathway with dominant-negative mutant STAT3D effectively abolished IL-6-induced VEGF mRNA. In transient transfections, a luciferase reporter construct containing the full-length 1.5-kb VEGF promoter or a 1.2-kb fragment lacking the known hypoxic-response element also exhibited the same degree of response to IL-6. Additionally, transient transfection of STAT3D downregulated the 1.2-kb VEGF promoter luciferase reporter stimulated by IL-6. Based on the above phenomenon combined with the concomitant increased tumor expression of IL-6 and VEGF in cervical cancer tissues, we conclude that IL-6 may promote cervical tumorigenesis by activating VEGF-mediated angiogenesis via a STAT3 pathway.
Animals↗
Rapid detection of beta-globin gene (HBB) mutations coupling heteroduplex and primer-extension analysis by DHPLC.
Beta-thalassemia is a common inherited disease, resulting from one or more of a total of more than 200 different mutations in the beta-globin gene (HBB). Efficient and reliable mutation-screening methods are essential in order to establish appropriate prevention programs for at-risk populations based upon a molecular diagnosis. We have developed a rapid and highly-specific mutation screening test for the diagnosis of beta-thalassemia by coupling heteroduplex and primer-extension analysis based on the denaturing high performance liquid chromatography (DHPLC) system. A total of 161 healthy heterozygous Taiwanese carriers featuring 10 different HBB mutations and 30 patients exhibiting 12 different compound heterozygous or homozygous HBB mutations were subjected to DHPLC. The elution profile for the heteroduplex analysis of DHPLC could be successfully used to identify the common disease-causing mutations of HBB. To further confirm the sequence variants, we developed a technique combining multiplex primer-extension analysis coupled with DHPLC for the genotyping of eight common disease-causing mutations in the HBB gene. Overall, by coupling heteroduplex and primer-extension analysis based upon DHPLC, we were able to unambiguously identify the most-common beta-thalassemia mutations corresponding to more than 99% of HBB alleles among the Taiwanese population. In conclusion, compared to classic approaches to mutation screening for this malady, we suggest that DHPLC is an excellent technique to be applied to the genetic screening of prenatal and postnatal individuals as a part of a diagnosis program for beta-thalassemia and provides a more-efficient, economic, and sensitive means to undertake such a screening program.
Base Sequence↗
Prenatal diagnosis and treatment of fetal long QT syndrome: a case report.
We report a case of a fetus presenting with bradycardia, intermittent atrioventricular (AV) block, ventricular tachycardia (VT) and the signs of fetal congestive heart failure (ascites and scrotal hydrocele) during mid-gestation. Prenatal treatment with beta-adrenergic blocker (propranolol) and digitalis glycosides was prescribed because of suspicion of long QT syndrome occurring with fetal congestive heart failure. The male baby was born at 39 weeks of gestation and showed a prolonged QT interval (QTc = 492 ms) and frequent variable AV block or alternating left and right bundle branch block, depending on the atrial rate. Prenatal administration of lidocaine failed to correct the fetal VT. Conversely, propranolol decreased the attack frequency of fetal VT. Postnatal administration of the K(+) channel opener (nicorandil) successfully shortened the QT interval and improved the outcome.
Adult↗
Intrauterine diagnosis of heterotaxy syndrome.
BACKGROUND: Heterotaxy syndrome, including right isomerism and left isomerism, is characterized by an abnormal symmetry of the viscera and veins and is frequently associated with complex cardiac anomalies. We sought to define the feasibility of in utero diagnosis and the postnatal outcome. METHODS: Patients with heterotaxy syndrome were identified from 579 fetal echocardiograms performed from January 1994 to December 1998. The diagnosis was made on the basis of the fetal echocardiographic findings and was confirmed with autopsy or postnatal evaluation. RESULTS: A total of 25 fetuses with right isomerism and 4 with left isomerism constitute the study population. The pregnancies of 7 fetuses (6 right and 1 left isomerism) were terminated before the 24th gestational week and subjected to autopsy. Twelve fetuses (10 right and 2 left isomerism) were lost to follow-up. Nine with right isomerism and 1 with left isomerism were delivered and underwent palliation. Among them, 5 patients (56%) with right isomerism died and more than half of the deaths occurred during infancy. The major cardiac anomalies detected and confirmed with postnatal evaluation or autopsy in fetuses with right isomerism were total anomalous pulmonary venous connection (6/15; 40%), common atrium (15/15; 100%), complete atrioventricular canal (15/15; 100%), double outlet right ventricle (15/15; 100%), and pulmonary stenosis (11/15; 73%). The major cardiac anomalies in fetuses with left isomerism were interruption of inferior vena cava (2/2; 100%), common atrium (1/2; 50%), and complete atrioventricular canal (1/2; 50%). Undetected lesions with fetal echocardiogram were abnormal pulmonary venous return to systemic veins in 1 case (sensitivity, 83%; 5/6; and specificity, 90%; 9/10) and outflow obstruction in 1 case (sensitivity, 91%; 11/12; and specificity, 67%; 2/3). Different patterns of rhythm disturbances were identified: supraventricular tachycardia in 1 case with right isomerism and sinus bradycardia with junctional rhythm in 3 cases with left isomerism (2 of them lost to follow-up). After birth, another 2 patients with right isomerism had supraventricular tachycardia, and 1 with left isomerism had sinus bradycardia develop at age 2 years. CONCLUSION: Heterotaxy syndrome is usually detected in fetuses with the sonographic cardiac abnormalities. Visualization of the pulmonary venous return and outflow obstruction and characterization of the rhythm disturbances are feasible. However, in spite of prenatal diagnosis, the prognosis remains poor.
Abnormalities, Multiple↗
Postnatal outcome of fetal cardiac echogenic foci.
BACKGROUND AND PURPOSE: Cardiac echogenic foci are found frequently during fetal echocardiographic investigations and may be related to increased mineralization of the papillary muscles. However, data from postnatal follow-up are limited. This study investigated the clinical characteristics and postnatal echocardiographic findings in infants with cardiac echogenic foci identified prenatally. METHODS: Between March 1995 and April 1998, 43 fetuses were noted to have cardiac echogenic foci during the second trimester. Postnatal evaluation was completed for 20 of these 43 fetuses. No other congenital malformations were noted during the fetal stage or after birth. Postnatal echocardiography was performed from 17 months to 4 years and 7 months after birth. RESULTS: Seven (35%) infants had persistent cardiac echogenic foci. However, only one had mild mitral valve prolapse without mitral regurgitation. All fetuses had left ventricular (LV) foci and three also had right ventricular (RV) foci. One infant who had a LV focus prenatally was noted to have a RV focus on postnatal follow-up. Among the three infants with prenatal biventricular involvement, only one had biventricular involvement on postnatal follow-up. Other cardiac echogenic foci had disappeared in all infants. The probability of persistence of foci decreased with age and reached 50% at the age of 4 years and 4 months. Thereafter, cardiac echogenic foci tended to regress and only 11% of infants had persistence at the last follow-up. No significant difference was found in the rate of persistence between children with univentricular foci and those with biventricular foci. CONCLUSIONS: Although some fetal cardiac echogenic foci may persist after birth, fetal echogenic foci were not associated with significant intracardiac or extracardiac anomalies.
Child, Preschool↗
Limb-reduction defects and chorion villus sampling.
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Chorionic Villi Sampling↗
Differential analgesic effect of tenoxicam on the wound pain and uterine cramping pain after cesarean section.
BACKGROUND: Nonsteroidal anti-inflammatory drugs (NSAIDs) are widely used to enhance opioid analgesia in the acute pain service. The question, however, of whether NSAIDs produce a similar extent of potentiation among different types of pain, has not been thoroughly investigated. MATERIALS AND METHODS: A randomized, placebo-controlled, double-blind study was performed to characterize the analgesic effect of tenoxicam, a long-acting NSAID, on resting wound pain, evoked wound pain, and uterine cramping pain after cesarean section. Saline (n = 48) or 20 mg tenoxicam (n = 45) was intravenously injected immediately after clamping the umbilical cord. All patients were instructed to obtain maximal postoperative analgesia by intravenous patient-controlled morphine. RESULTS Tenoxicam profoundly reduced the intensity of uterine cramping pain (3.6 [2.0-5.6] versus 5.5 [3.4-6.6]; p < 0.01) but had no additional effect on wound pain at rest, with movement, changing position, sitting, and walking. Intraoperative injection of 20 mg tenoxicam decreased the demand ratio for patient-controlled analgesia (PCA) and 24-hour morphine consumption by approximately 30%. CONCLUSIONS: The data show that tenoxicam potentiates opioid analgesic effect on the somatic and visceral types of pain to different extents, and they suggest that intraoperative injection of 20 mg tenoxicam is sufficient to enhance intravenous PCA morphine on uterine cramping pain for the first 24 hours after cesarean section.
Adolescent↗
Correlation of prenatal ultrasound and postnatal outcome in meconium peritonitis.
OBJECTIVES: To study the relationship between prenatal ultrasound features and postnatal course of meconium peritonitis. METHODS: Meconium peritonitis was diagnosed by prenatal ultrasound. Fetuses were treated by intrauterine paracentesis of ascites when indicated, and symptomatic newborns received surgery. RESULTS: Totally 17 cases were enrolled. Prenatal ultrasound findings include abdominal calcification (16/17), fetal ascites (12/17), hydramnios (9/17), pseudocyst (7/17) and dilated bowel loop (6/17). Persistent ascites, pseudocyst or dilated bowel loop are most sensitive (92%) to predict postnatal surgery (p = 0.022). The survivors have a higher gestational age at birth (36.4 vs. 33.3 weeks, p = 0.008). Persistent ascites and postnatal persistent pulmonary hypertension of the newborns significantly correlate with neonatal mortality (p = 0.029 and 0.022). CONCLUSION: Prenatal ultrasound can predict the neonatal outcome in meconium peritonitis.
Adult↗