Characterization of factor-independent variants derived from TF-1 hematopoietic progenitor cells: the role of the Raf/MAP kinase pathway in the anti-apoptotic effect of GM-CSF.

Human hematopoietic progenitor cells (TF-1) undergo apoptosis upon deprivation of their dependent cytokine. In this report, we have isolated and characterized some spontaneously derived cytokine-independent variants from TF-1 cells. Analysis of several signaling molecules known to be activated by the GM-CSF pathway revealed that two non-autocrine variants were still responsive to GM-CSF stimulation. However, both variants, without ligand stimulation, already had some activated forms of Raf and MAP kinases. Given current knowledge, the activated Raf/MAP kinase pathway was likely to be responsible for the survival of both variants in the cytokine-free medium. However, the growth of hybrids between wild type and either variant was unexpectedly dependent on GM-CSF. Both variants like the wild type cells were still susceptible to apoptosis induced by other stimuli. These results suggest that either the activated Raf/MAP kinase pathway in both variants is not sufficient to repress the 'two-fold' death signals generated from the hybrids or that there is another mechanism that is responsible for the factor-independent growth of both variants.

Human T-lymphotropic Virus Type II Infection inVietnamese Thalassemic Patients.

Anti-human T-lymphotropic virus type I/II (HTLV-I/II) antibodies were screened by particle agglutination test in a total of 66 patients with thalassemia major who received multiple transfusion from paid donors at the Blood Transfusion Hematology Center of Ho Chi Minh City in South Vietnam. HTLV-II infection was confirmed in 6 patients (9.1%) by Western blot analysis and/or polymerase chain reaction. Phylogenetic analysis revealed that long terminal repeat sequences of HTLV-II proviruses from 5 thalassemic patients in Vietnam belonged to the same phylogenetic subgroup of HTLV-IIb as those from intravenous drug abusers in North America and Europe. These data shed light on the route of introducing HTLV-II into Vietnam.

Norplant subdermal contraceptive system: experience in Taiwan.

From November 1988 to December 1994, a total of 567 female volunteers were enrolled in Norplant implant studies at the National Taiwan University Hospital. After a median follow-up of 29 months, only 3 of the 529 available cases became pregnant (a cumulative rate of 1.2 pregnancies per 100 users over 5 years). Chromosome analysis of 2 of the 3 abortuses revealed 46,XX/46,XX,inv(3) and 46,XX. Menstrual problems were the most common adverse effects and were also the main reason for discontinuation (65%, 108/166). The continuation rate was 90%, 78%, 70%, 61%, and 42% at the end of 1, 2, 3, 4, and 5 years after insertion, respectively. In the 21 patients who wished to become pregnant, fertility recurred soon after removal of the Norplant implants. The data suggested that the Norplant implants system is a highly effective, safe, and long-acting method of reversible contraception. It would be worthwhile to introduce this contraceptive system to Taiwan's family planning program.

PIP: Between November 1988 and December 1994 health providers at the National Taiwan University Hospital enrolled 567 women aged 17-47 in the Norplant implant studies that aimed to evaluate the benefits and side effects of Norplant as another contraceptive method choice. They followed the women for a median of 29 months. 38 cases were lost to follow-up. The 5-year contraceptive effectiveness rate was 98.8%. The 3 pregnancies (2 intrauterine and 1 ectopic) occurred during the 24th, 45th, and 47th months of use. None of the women who became pregnant weighed more than 70 kg. Two of the aborted fetuses had chromosomal abnormalities (46,XX/46,XX,inv(3) and 46,XX). 29.9% of the Norplant users had menstrual problems. Even though irregular bleeding occurred to 19.7% of Norplant users, hemoglobin levels increased after Norplant use (p 0.05). Triglycerides and total cholesterol levels decreased (p 0.05). None of the women developed thromboembolism. The Norplant continuation rate was 89.7% at 1 year, 78% at 2 years, 70% at 3 years, 61% at 4 years, and 42.4% at 5 years. The leading reason for implant removal was menstrual problems (108). The implants were removed during one sitting without x-ray or ultrasound identification in all 166 women who stopped using Norplant before 5 years and the 16 women who stopped using it at the end of 5 years. 78% of women who discontinued Norplant to become pregnant were pregnant within one year following removal. All their infants were normal. These findings suggest that Norplant is a safe, effective, and acceptable contraceptive method and that the family planning program should introduce Norplant to its contraceptive mix.

Fluorescence microsatellite analysis to study the parental origin of the supernumerary chromosome in Down's syndrome.

OBJECTIVE: Down's syndrome (DS) is an important cause of mental retardation. This study investigated the parental origin of the extra chromosome 21 in DS patients. METHODS: Fourteen families each with a DS patient were recruited for analysis of nine microsatellite markers on chromosome 21. We collected DNA from both parents and the patient and used polymerase chain reaction to amplify nine segments on chromosome 21: D21S1435, D21S1436, D21S1437, D21S1446, D21S156, D21S258, D21S263, D21S265 and D21S270. One of each pair of DNA primers was labeled with a fluorescence dye. The amplified products were subjected to electrophoresis in a semi-automated DNA sequencer and then analyzed with Genescan software to determine the origin of the extra chromosome 21. RESULTS: The extra chromosome 21 originated from the mother in 13 (93%) patients and from the father in one (7%) patient. CONCLUSIONS: Our findings were compatible with those from Caucasian patients. A great majority of Down's syndrome cases resulted from meiotic errors in the eggs.

Congenital adrenal hyperplasia. Molecular characterization.

OBJECTIVE: To study the molecular defects of congenital adrenal hyperplasia (CAH). STUDY DESIGN: Twenty Chinese patients, including 8 with salt-wasting (SW) type CAH, 11 with simple virilizing (SV) type CAH and 1 with nonclassical (NC) type CAH, were recruited. Two rounds of the polymerase chain reaction (PCR) were used to study the 21-hydroxylase gene (CYP21). The primary PCR amplified CYP21-specific DNA fragments, and the secondary PCR used products from the primary PCR for analysis of amplification-created restriction sites (ACRS) and direct DNA sequencing. In all patients, ACRS analysis was done at 12 possible mutation sites, and then direct DNA sequencing was performed to confirm or define the molecular defects. RESULTS: Ten different mutations, including nine point mutations and gross gene deletion or conversion, were found in this study. Of the nine point mutations, eight could be easily detected by ACRS analysis. The three most common mutations were codon (CD)172 t-->a (I172N), IVS-II 656 c/a-->g, and gross gene deletion or conversion, accounting for 27.5% (11/40 alleles), 25% (10/40) and 20% (8/40) of all identified mutations, respectively. All SW patients were compound heterozygotes of IVS-II 656, gross gene deletion or conversion, or other severe defects, including CDs236 (t-->a) (I236N)+ 237 (t-->a) (V237E)+ 239 (t-->a) (M239K), CD306 (+t), CD318 (c-->t) (Q318X) and CD356 (c-->t) (R356W) mutations. All SV patients had one allele with a CD172 (I172N) mutation. One allele of an NC patient had a CD183 (c-->g) (D183E) mutation, and the other allele was not defined. In the whole series, four alleles (10%) had more than one mutation. CONCLUSION: We found 10 different mutations in this study. The correlation between genotypes and phenotypes was compatible with the reported data. Two rounds of PCR and ACRS analysis may provide important information for genetic counseling, prenatal diagnosis and management of families at risk for CAH.

Dystrophin gene deletion in Chinese Duchenne/Becker muscular dystrophy patients via multiplex DNA amplification.

Duchenne/Becker muscular dystrophy (DMD/BMD) is a progressive muscle-wasting disease. The dystrophin gene responsible for the disease is the largest human gene ever cloned and is prone to gross gene deletion in two "hot spot" regions. Using nine pairs of oligonucleotide primers deduced from the two regions, we have screened 23 unrelated Chinese DMD/BMD patients by multiplex polymerase chain reaction. Nine (39%) patients were noted to have gene deletion, one in the 5' terminus and eight in the distal half of the gene. The incidence is similar to that reported in other large series mainly on Caucasian patients. The "hot-spot" regions also seem to be present in Chinese patients. Multiplex gene amplification for deletion analysis is useful in the diagnosis of patients with neuromuscular diseases and is an important aid in the prenatal diagnosis and genetic counseling of at-risk families.

Rapid prenatal determination of fetal sex by polymerase chain reaction on amniocyte DNA.

Sex determination in early gestation is important for fetuses at risk for X-linked disorders or congenital adrenal hyperplasia. One hundred and seventy consecutive samples of amniocytes were collected between the 12th and 31st gestational week. Seven women received early amniocentesis before the 14th week. Fetal sex was determined by amplification of Y-specific DNA fragments within five hours. All results of the polymerase chain reaction, except for one false-negative, were compatible with cytogenetic analyses. Polymerase chain reaction of amniocyte DNA provides a rapid technique for sex determination in early gestation with high specificity and sensitivity.

Cytogenetic study of spontaneous abortions with the Giemsa banding method.

Among 102 spontaneous abortuses karyotyped, 29 were from parents with a history of unexplained recurrent spontaneous abortion (group A), and 73 were from parents without a history of recurrent spontaneous abortion (group B). The mean maternal ages of these two groups were not significantly different. The chromosomal aberration rate in group A was 48.3% and in group B was 53.4%. The difference was not significant. The most common chromosomal anomaly was autosomal trisomies in both groups, while monosomy X was more common in group B. The subsequent spontaneous abortion rate was also not significantly different between the two groups.

Rapid detection of human hemoglobin Quong Sze by polymerase chain reaction.

Human hemoglobin Quong Size is a nondeletional defect involving codon 125 of the alpha 2-globin gene. The molecular defect is a T to C mutation, therefore, a new MspI site (CCGG) is created. Using a pair of flanking DNA primers and polymerase chain reaction, a DNA fragment of 339 basepairs (bp) was amplified. After MspI restriction and gel electrophoresis, the mutation could be clearly identified by the presence of two fragments, 108 and 123 bp. This method is rapid and simple and will be very useful in the genetic counseling of Southeast Asians, including the Chinese, in which nondeletional alpha-thalassemia defects are common.

Selective reduction of multiple pregnancies in the first trimester or early second trimester.

Nine cases of multiple pregnancies, including 7 sets of triplets and 2 sets of quadruplets, were enrolled for selective fetal reduction between March 1989 and July 1990 at the National Taiwan University Hospital. Under transabdominal or transvaginal ultrasound guidance, potassium chloride was injected into the fetal cardiac chamber to kill the selected fetus during the 10-14th gestational week. The number of fetuses was reduced to two in all cases. No maternal complications occurred during the procedure. Six gravidas have given birth to 6 sets of healthy twins; two pregnancies are progressing smoothly at 26 and 23 weeks of gestation, respectively, and one case underwent spontaneous abortion 4 weeks after the procedure. Selective fetal reduction under ultrasound guidance is an acceptable method of preventing high perinatal mortality and morbidity of immature fetuses in grand multiple pregnancies.

Prenatal cytogenetic diagnosis in amniocentesis.

From 1982 to 1990, cytogenetic studies were successfully conducted in 2,975 (96.19%) of the 3,096 pregnant women who underwent amniocentesis. The average maternal age was 33.7 years and the average gestational age was 18.1 weeks. Common indications of amniocentesis included advanced maternal age (AMA) (54.76%), previous fetus with chromosomal aberrations (6.82%) or gross anomalies (5.01%), intrauterine gross anomaly (4.97%) and maternal exposure to drugs or radiation (5.28%). Among the 89 cases (2.99%) with detected chromosomal aberrations, 53 were numeric (31 trisomies, 21 sex chromosome aberrations and one tripoidy) and 36 were structural (six de novo and 30 hereditary structural rearrangement). The incidence of chromosomal aberrations was 2.03% in cases with AMA. While only four of the 143 cases with previous fetal trisomy 21 had recurrence, the recurrent rate was 90.91% in 11 cases with previous fetal chromosomal translocation. Thirty (20.27%) of the 148 cases with abnormal sonograms showed chromosomal aberrations. Certain congenital anomalies are closely associated with cytogenetic changes: duodenal atresia and trisomy 21; cystic hygroma and 45,X; and polyhydramnios and trisomy 18. Only two of the 157 cases with indications of drug or radiation exposure had abnormal cytogenetic studies. Two of the 53 cases with detected numerical aberrations (47,XXY and 47,XXX) and 27 cases with hereditary structural rearrangement elected to continue their pregnancies. All of these babies were delivered without gross anomalies. This study suggests that for prenatal diagnosis. However, complementary measures, such as routine antenatal ultrasound and maternal serum alphafetoprotein, should be added to increase the efficacy of genetic amniocentesis.

Norplant-2 subdermal contraceptive system: experience in Taiwan.

Subdermal Norplant-2 implantation, developed for contraception based on sustained release of levonorgestrel, has already been conducted in many countries around the world. From October 1986 to November 1988, a total of 267 female volunteers were enrolled in Norplant studies at the National Taiwan University Hospital. After a follow-up of 36 months, only one of the 231 evaluable cases (0.4%) became pregnant. The continuation rate was 84.5%, 61.3%, and 52.0% at the end of 12, 24 and 36 months after insertion, respectively. Menstrual problems were the most common adverse effects and were also the main reason for discontinuation. In the 23 patients who wished to become pregnant, fertility occurred soon after removal of the Norplant implant. After insertion, serum total cholesterol and triglyceride levels significantly decreased and the hemoglobin concentrations were increased. No liver or renal toxicities were detected. The data suggest that Norplant-2 is a highly effective, safe and long-acting method of reversible contraception. It would be worthwhile to introduce this contraceptive system into Taiwan's family planning program.

PIP: Between October 1986 and November 1988, physicians enrolled 267 20-41 year old women in a clinical trial of Norplant-2 (2 Silastic rods with 70 mg levonorgestrel each) at the National Taiwan University Hospital to evaluate the advantages and side effects of Norplant-2. 36 women were lost to follow up. 63% suffered from menstrual problems during the first 3 months. 73.6% of these women still had menstrual problems at 6 months. 6 women then chose to discontinue using Norplant-2. 51.8% of the remaining women had persistent menstrual problems at 1 year. 62% of women who discontinued Norplant-2 before the end of 3 years discontinued it due to menstrual problems. The continuation rate was 84.5% at 1 year, 61.3% at 2 years, and 52% at 3 years. 78.6% of women who discontinued Norplant-2 to become pregnant conceived within 1 year after its removal. Just 1 woman (.04%) became pregnant while using Norplant-2. She was thin and smoked heavily. Her aborted fetus had an abnormal fetus had an abnormal karyotype (47,XY,+22). The mean serum total cholesterol and triglyceride levels dropped considerably after Norplant-2 implantation from 164 to 147 mg/dl and from 81 to 63 mg/dl at 12 months, respectively; p .05). Mean hemoglobin levels increased from 12.6 g/dl to 13 g/dl at 12 months (p .05). Levonorgestrel did not affect liver or renal functions. Mean body weight and blood pressure remained the same. Observed psychophysiologic symptoms included depression, acne, headaches, nervousness, and insomnia. These findings indicated Norplant-2 to be safe, reversible, long-acting, and very effective, therefore the family planning program in Taiwan should add it to its contraceptive menu.

Familial transmission of human T-lymphotropic virus type 1 (HTLV-1) in patients with adult T-cell leukemia/lymphoma or HTLV-1-associated myelopathy.

The seroprevalence of human T-lymphotropic virus type 1 (HTLV-1) in Taiwan is 0.48%. In this study, we investigated the patterns of intrafamilial transmission of HTLV-1 in Taiwanese patients with adult T-cell leukemia/lymphoma (ATL/L) or tropical spastic paraparesis/HTLV-1-associated myelopathy (TSP/HAM). Fifteen index patients (9 men, 6 women, aged 31-71 yr), 13 with ATL/L, and two with TSP/HAM, and 98 relatives were included. Of the 98 relatives, 23 were seropositive for HTLV-1. Spouses of 11 patients were studied. Seven of eight wives of male patients but none of the three husbands of female patients were HTLV-1 carriers. Mother-to-child transmission was found in seven of 13 families and in 15 of 75 children tested. The correlation of breast-feeding with seropositivity in two families with seropositive mothers indicates its important role in vertical transmission of HTLV-1. Our findings suggest that husband-to-wife and mother-to-child transmission are the main forms of intrafamilial transmission of HTLV-1 in Taiwan, a nonendemic area. Screening for HTLV-1 in family members of patients with ATL/L or TSP/HAM, and seropositive blood donors, may be warranted. Seropositive individuals should be educated to prevent the spread of the virus through sexual contact and breast feeding.

Prevalence and molecular studies of thalassemia in five aboriginal groups in Taiwan.

A total of 1,342 blood samples from five aboriginal groups in Taiwan, comprising 522 of the Ami, 246 of the Bunum, 227 of the Atayal, 214 of the Paiwan and 133 of the Yami group, were collected. A complete blood count was performed in each case. In subjects with a mean corpuscular volume < 85 fl or hemoglobin (Hb) < 12 gm% (female) or 13 gm% (male), quantitation of Hb A2 and DNA analysis of alpha- and beta-globin genes were performed. Alpha-thalassemia was diagnosed by Southern hybridization of subject's DNA to alpha-, and zeta-globin gene fragments, and to Lo probe if needed. DNA from beta-thalassemia carriers was studied by polymerase chain reaction and direct sequencing. In the Ami, 42 (8.2%) were alpha-thalassemia 1 carriers, 42 (8.2%) were alpha-thalassemia 2 carriers, one had Hb H disease, and four (0.8%) were beta-thalassemia carriers. In the Bunun, one (0.2%) was an alpha-thalassemia 1 carrier, and two (0.4%) were alpha-thalassemia 2 carriers. In the Atayal, one (0.2%) was an alpha-thalassemia 1 carrier. In the Paiwan, seven (3.3%) were alpha-thalassemia 1 carriers, and one (0.5%) was an alpha-thalassemia 2 carrier. In the Yami, none were either alpha- or beta-thalassemia carriers. Diverse genetic origin, intragroup breeding and malarial selection may play a role in the significant differences of thalassemia prevalences both between the Chinese and the aborigines, and among different groups of aborigines.

The effect of epidural analgesia on postpartum urinary retention in women who deliver vaginally.

There have been several investigations reporting on urinary retention in postpartum women who delivered vaginally with epidural blockade. The mechanism and incidence of urinary retention in relation to epidural analgesia, however, are not established. The objectives of this study were to investigate the association between various obstetric parameters and urinary retention and to determine whether those women with postpartum urinary retention subsequently develop urinary problems. From December 1999 to September 2000, 110 primiparas who delivered vaginally with epidural analgesia for labor pain relief were recruited prospectively. One hundred primiparas delivering under similar conditions without epidural analgesia were selected as the control group. Residual urine volume was calculated by trans-abdominal sonogram. A computerized obstetric database was analysed to compare the two groups. Women with epidural analgesia, especially those with residual volume exceeding 500 ml, had significantly longer labor course, a higher percentage of instrumental deliveries and more extensive vaginal or perineal lacerations than the control group. Only a few women had persistent problems with micturition six months after delivery. Epidural analgesia provides valuable pain relief but may be associated with greater residual urine. Postpartum urinary retention is, however, more related to prolonged labor than to the effect of epidural analgesia itself. Close monitoring of the progress of labor and avoiding urine retention are essential.

Correlation of interleukin-10 gene haplotype with hepatocellular carcinoma in Taiwan.

Polymorphisms in cytokine genes can influence immune responses, inflammation and tissue injury, and may affect the outcome of hepatitis B virus (HBV) infection. We analyzed single nucleotide polymorphisms (SNP) in the interleukin (IL)-10 gene among 344 HBV carriers and 208 patients with hepatocellular carcinoma (HCC). Genotypes and haplotypes were tested for association with HCC. IL-10/-592 C/C genotype was associated with a higher risk for HCC compared with IL-10/-592 A/C and A/A genotypes [odds ratio (OR): 2.1, 95% confidence interval (CI): 1.2-3.6]. IL-10/1927 A/A genotype was also associated with a higher risk for HCC compared with IL-10/1927 A/C and C/C genotypes (OR: 1.5, 95% CI: 1.0-2.2). Haplotype analysis revealed that the homozygosity of the C-A haplotype (defined by SNPs at positions -592 and 1927) of IL-10 gene conveys the highest risk for HCC among HBV carriers compared with the homozygosity for the A-C haplotype (OR: 2.6, 95% CI: 1.3-4.9). The results demonstrate that IL-10 gene polymorphism can affect the outcome of chronic HBV infection. Further studies are necessary to clarify how variation in the IL-10 gene affects IL-10 function and risk of HCC.

Glutaraldehyde preparations and pulpotomy in primary molars.

The study clinically and radiographically evaluated the long-term success rate of pulpotomy treatment in pulp-exposed primary molars. Five clinicians participated in this study and four glutaraldehyde preparations included 2% buffered, 2% unbuffered, 5% buffered, and 5% unbuffered glutaraldehyde solutions were used. There were 201 children, 108 boys and 93 girls, ranging in age from 4 to 7 years with 258 primary molars treated. After 36 months, 150 teeth with complete clinical records and radiographs were available for evaluation. The treatment of 98% of the patients was clinically successful, but when evaluated radiographically the overall success rate was 78.7%. The group treated with 5% buffered glutaraldehyde showed the highest success rate (87.5%) and group treated with the 5% unbuffered solution the lowest (74.1%), but no significant difference was found among the four groups. Canal obliteration was noted in 22 teeth successfully treated. Four of the teeth that were not successfully treated had canal obliteration before other pathoses became evident. The relative high failure rate in this long-term follow-up indicated that clinicians should be cautious before extensively using glutaraldehyde as a pulpotomy agent.

Recurrent Down's syndrome due to maternal ovarian trisomy 21 mosaicism.

A family with three children who had Down's syndrome and one healthy child is reported. Cytogenetic studies of the peripheral blood revealed trisomy 21 in the affected children, and normal karyotypes in both the parents and the healthy child. However, a biopsy of the mother's right ovary showed a mosaic trisomy 21 cell line (8/20 cells). By DNA polymorphism analysis, segregation of trisomy oogonia appeared to be the cause of recurrent trisomy 21.