Banking placental tissue: an optimized collection procedure for genome-wide analysis of nucleic acids.

INTRODUCTION: Banking of high-quality placental tissue specimens will enable biomarker discovery and molecular studies on diseases involving placental dysfunction. Systematic studies aimed at developing feasible standardized methodology for placental collection in a typical clinical setting are lacking. METHODS: To determine the acceptable timeframe for placental collection, we collected multiple samples from first and third trimester placentas at serial timepoints in a 2-h window after delivery, simultaneously comparing the traditional snap-freeze technique to commercial solutions designed to preserve RNA (RNAlater™), and DNA (DNAgard(®)). The performance of RNAlater for preserving DNA was also tested. Nucleic acid quality was assessed by determining the RNA integrity number (RIN) and genome-wide microarray profiling for gene expression and DNA methylation. RESULTS: We found that samples collected in RNAlater had higher and more consistent RINs compared to snap-frozen tissue. Similar RINs were obtained for tissue collected in RNAlater as large (1 cm(3)) and small (∼0.1 cm(3)) pieces. RNAlater appeared to better stabilize the time zero gene expression profile compared to snap-freezing for first trimester placenta. DNA methylation profiles remained quite stable over a 2 h time period after removal of the placenta from the uterus, with DNAgard being superior to other treatments. DISCUSSION AND CONCLUSION: The collection of placental samples in RNAlater and DNAgard is simple, and eliminates the need for liquid nitrogen or a freezer on-site. Moreover, the quality of the nucleic acids and the resulting data from samples collected in these preservation solutions is higher than samples collected using the snap-freeze method and easier to implement in busy clinical environments.

Phaeochromocytoma in the Hong Kong Chinese population.

OBJECTIVE: To review the clinical manifestations of phaeochromocytoma in a Hong Kong Chinese population. DESIGN: Retrospective review. SETTING. Five public hospitals in Hong Kong. PATIENTS: Seventeen patients with operated phaeochromocytoma between 1994 and 2003 were reviewed retrospectively. RESULTS: Six patients (35%) were men, 11 (65%) were women. The mean age at presentation was 47 (range, 17-72) years. The diagnosis post-presentation was delayed by 1 to 132 months. Over 70% of the patients had hypertension. The most frequent symptoms were headache (53%), palpitations (53%), and sweating (41%); all these symptoms were present in 24% of the patients. Four (24%) had hereditary phaeochromocytoma/paraganglioma syndrome. The sensitivity of 24-hour urinary catecholamine measurements was 82%. Mean urinary adrenaline and noradrenaline concentrations were respectively 7- and 8-fold greater than the upper reference limits. Computed tomography and metaiodobenzylguanidine scintigraphy were the most widely used means for tumour localisation (sensitivity, 100% and 87% respectively). Approximately 65% of the patients had intra-adrenal tumours; 53% were on right side, 18% were bilateral. All the patients were prescribed phenoxybenzamine (dosage range, 20-120 mg/day) preoperatively. Two thirds of the patients had improved blood pressure 1 year after the operation. No malignancy was reported after a mean follow-up period of 7 years. CONCLUSION: Our series of patients with phaeochromocytomas commonly had a high frequency of normotension and extra-adrenal tumours. A high index of clinical suspicion and appropriate biochemical investigations are necessary to make the diagnosis, especially for patients manifesting adrenal incidentaloma and extra-adrenal lesion.

Usage of a fixed dose of radioactive iodine for the treatment of hyperthyroidism: one-year outcome in a regional hospital in Hong Kong.

OBJECTIVES: To evaluate the efficacy of a fixed dose of radioactive iodine (131-I) in the treatment of thyrotoxicosis, and to identify risk factors associated with treatment failure. DESIGN: Retrospective study. SETTING: Thyroid Clinic of a regional hospital in Hong Kong. PATIENTS: Patients receiving their first dose of radioactive iodine for the treatment of thyrotoxicosis during the inclusive period September 1999 to August 2004. MAIN OUTCOME MEASURES: Relapse rate and time to relapse. RESULTS: A total of 113 patients received a fixed dose of 5 mCi (185 MBq), 6 mCi (222 MBq), 8 mCi (296 MBq), and 10 mCi (370 MBq) 131-I in a proportion of 1:6:71:35. At 1 year, 42 (37%) of the patients had relapsed, of which 69% received a second 131-I dose. The median time to relapse after first receiving 131-I was 4 months. At 1 year, the remaining 71 (63%) of the patients were successfully treated; 46 (41%) were euthyroid, and 25 (22%) had became permanently hypothyroid. Basal free thyroxine level and goitre size were significantly associated with a relapse rate after a single dose of 131-I; larger goitres showed a trend towards high rates of relapse. Patients pretreated with propylthiouracil had a higher rate of relapse during the first year after radioactive iodine than those pretreated with carbimazole, but the difference was not significant when combined with other pretreatment variables. CONCLUSIONS: A single fixed dose of radioactive iodine is a simple, safe, and effective treatment for hyperthyroidism. High basal free thyroxine concentration and large goitre size are associated with higher chance of relapse. Higher radioiodine doses may be considered to improve the cure rate.

Metabolic control of diabetes in a diabetes centre.

OBJECTIVE: To examine the effectiveness of a diabetes centre in restoring metabolic control in patients with poorly controlled diabetes. DESIGN: Retrospective review of medical records. SETTING: Diabetes centre of a district hospital, Hong Kong. PARTICIPANTS: Patients with poorly controlled diabetes referred to a diabetes centre. MAIN OUTCOME MEASURES: Primary endpoints were mean change in glycated haemoglobin levels and the number of patients who achieved glycated haemoglobin levels of 7.0% or lower, 7.5% or lower, and 8.0% or lower, respectively. Complementary endpoints were serial changes in body weight, blood pressure, and lipids. RESULTS: One hundred and eighty-five patients, predominantly with type 2 diabetes (94.6%), were reviewed. Median duration since diagnosis of diabetes was 8 years (interquartile range, 4.3-11.8 years). Seventy-three patients had a body mass index of 25 kg/m(2) or higher. The baseline and latest glycated haemoglobin levels were 10.4% (standard deviation, 2%) and 8.2% (1.4%), respectively; mean reduction was 2.2% (95% confidence interval, 1.9-2.5; P<0.0005). Eighty-one patients were discharged after a median 32 weeks of follow-up. Their mean glycated haemoglobin level on discharge was 7.5% (0.8%), and the mean reduction was 2.8% (95% confidence interval, 2.4-3.3; P<0.0005). The cumulative percentages of discharged patients who achieved glycated haemoglobin levels of less than 7.0%, 7.5%, and 8.0% were 30.9%, 53.1%, and 77.8%, respectively. Newly diagnosed diabetes (P=0.006) was the only factor which predicted a favourable glycaemic response. CONCLUSION. The Diabetes Centre provided effective management for a heterogeneous group of patients referred with poorly controlled diabetes.

Altered G protein activity in a desensitization-resistant mutant of the Y1 adrenocortical tumor cell line.

Mutant isolates [designated desensitization resistant (DR)] from the Y1 mouse adrenocortical tumor cell line resist agonist-induced desensitization of adenylyl cyclase by preventing the uncoupling of receptors from their guanyl nucleotide-binding regulatory G proteins. In this study, we tested the hypothesis that an underlying G protein defect is associated with the DR phenotype. We found that the G protein reagent guanyl-5'-yl imidodiphosphate [Gpp(NH)p] shifted beta2-adrenergic receptors from a high affinity state to a low affinity state 4-fold more effectively in mutant DR cells than in parent Y1 cells. In the DR mutant, Gpp(NH)p was able to shift receptors to a low affinity state in the absence of NaCl, whereas the effect of Gpp(NH)p in parent Y1 cells was dependent upon the presence of NaCl. Moreover, these differences in sensitivity to Gpp(NH)p and NaCl were transferred to Gs alpha-deficient S49(CYC-) lymphoma cell membranes in G protein reconstitution assays. These observations suggested that the DR mutation was associated with altered activity of the stimulatory G protein, Gs. Cloning and sequence analysis demonstrated that Gs alpha transcripts in the DR mutant were normal, suggesting that another factor involved in guanyl nucleotide exchange is responsible for the altered G protein activity in DR mutant cells.

Nasopharyngeal carcinoma---time lapse before diagnosis and treatment.

This is a descriptive study of 168 patients with nasopharyngeal carcinoma who were referred to public oncology departments for primary treatment between July and September 1996. The mean duration from the onset of the symptoms to histological diagnosis was 5.0 months; the duration ranged from 6.1 months (for patients presenting with nasal symptoms) to 1.8 months (for those with cranial nerve dysfunction). The mean period between the onset of symptoms and the seeking of medical advice was 2.9months. For 54% of the patients, there was a further delay of up to 2.4 months between the initial medical consultation and referral to the appropriate specialist. The majority (84%) of patients attended public institutions for histological confirmation. The mean total time taken from the onset of symptoms to the commencement of radiotherapy was 6.5 months (range, 1.3-74.0 months)---45% of the delay was attributed to the patient, 20% to initial consultations, 14% to diagnostic arrangement, and 21% to preparation for radiotherapy. Concerted efforts are needed to minimise further the time between the onset of symptoms and treatment. A substantial reduction in this delay can be achieved if both public and primary care doctors were made more aware of the significance of relevant symptoms.

Endoscopic removal of leiomyoma of the colon.

Colonic leiomyoma is a rare condition. Smooth muscle tumours arising from the colon constitute only 3% of gastrointestinal leiomyomas. Complete endoscopic removal of the tumour is a problem because it is often submucosal in origin. We report a patient with a 5 mm leiomyoma of the colon that was successfully removed by conventional colonoscopic snare electrocauterisation, without complications.

Cholestatic hepatitis: a rare hepatic manifestation of systemic lupus erythematosus.

Systemic lupus erythematosus is a multi-system inflammatory disease. The clinical manifestations are diverse. Hepatic manifestation is a rarely seen complication of systemic lupus erythematosus. We report a case of complication of systemic lupus erythematosus presenting as cholestatic hepatitis in a 56-year-old Chinese woman. The cholestatic hepatitis progressed as part of the lupus activity and responded to steroid therapy.

Mutations to forskolin resistance result in loss of adrenocorticotropin receptors and consequent reductions in levels of G protein alpha-subunits.

A family of mutants isolated from the Y1 mouse adrenal cell line on the basis of their resistance to the growth inhibitory effects of forskolin have an underlying mutation that affects the activity of adenylyl cyclase. As part of the mutant phenotype, adenylyl cyclase is partially resistant to activation by forskolin, completely insensitive to ACTH, and fully responsive to NaF; the levels of Gs alpha and G1 alpha in plasma membrane fractions are decreased; and the activity of G beta/gamma is impaired. In the present study, we examine the basis for the complex phenotype associated with forskolin resistance to better understand the factors that contribute to the regulation of adenylyl cyclase activity. We demonstrate that the resistance of these mutants to ACTH results from the failure to express ACTH receptor transcripts. Transfection of these mutants with a gene encoding the mouse beta 2-adrenergic receptor led to the recovery of transformants with normal receptor-G protein coupling and with increased levels of Gs alpha and G1 alpha that approached those in parental Y1 cells. These beta 2-adrenergic receptor transformants, nonetheless, remained resistant to forskolin and ACTH. Two spontaneous Y1 mutants, Y6 and OS3, previously characterized as ACTH-resistant clones that failed to accumulate ACTH receptor transcripts, were shown to be forskolin resistant and to contain less Gs alpha in membrane fractions, indicating that forskolin resistance, failure to express the ACTH receptor, and the consequent reduction in Gs alpha are closely linked. Expression of the human ACTH receptor in Y6 and OS3 cells restored ACTH-responsive adenylyl cyclase activity and increased the level of Gs alpha, but did not otherwise reverse the forskolin-resistant phenotype. Together, these results demonstrate that mutations to forskolin resistance have downstream consequences that result in the loss of ACTH receptor expression and the consequent reduction in levels of membrane-associated G alpha subunits. The results further suggest that G protein-coupled receptors may have a stabilizing influence on G alpha subunits associated with the cell membrane. According to current models, forskolin activates adenylyl cyclase by forming a ternary complex with adenylyl cyclase and Gs alpha. Our results suggest that this model may be incomplete and that an additional component, acting directly or indirectly, is required for optimal activation of adenylyl cyclase by forskolin.

Adrenocorticotropin-resistant mutants of the Y1 adrenal cell line fail to express the adrenocorticotropin receptor.

This report examines the basis for adrenocorticotropin (ACTH) resistance in two mutant clones (Y6 and OS3) derived from the ACTH-responsive Y1 mouse adrenocortical tumor cell line. These two mutants were originally characterized by their failure to respond to ACTH with increased adenylyl cyclase activity and as a consequence were resistant to the steroidogenic effects of the hormone. We now demonstrate that ACTH resistance in the Y6 and OS3 mutants results from the failure to express the gene encoding the ACTH receptor. Whereas parental Y1 cells express ACTH receptor transcripts at low levels and are stimulated by ACTH or 8-bromo-cAMP to increase the accumulation of ACTH receptor transcripts approximately twofold, the Y6 and OS3 mutants do not express receptor transcripts either in the presence or absence of 8-bromo-cAMP. The gene encoding the ACTH receptor appears to be present in the Y6 and OS3 mutants, as determined by Southern blot hybridization analysis. Moreover, in the Y6 mutant the ACTH receptor gene appears to be silenced by a modification that is reversed following the growth of the cells as tumors in mice. Clonal isolates of Y6 cells grown as tumors recover the ability to express ACTH receptor transcripts at low but detectable levels and acquire the ability to respond to ACTH with increased adenylyl cyclase activity. Finally, Y6 and OS3 cells transformed with a gene encoding the mouse beta 2-adrenergic receptor respond to the beta-adrenergic agonist, isoproterenol, in a manner that is indistinguishable from the similarly transformed parent Y1 cell line. These latter results demonstrate the functional integrity of the adenylyl cyclase system in the ACTH-resistant mutants and indicate that the failure to express ACTH receptor transcripts limits the responsiveness of these clones.

ACTH-receptor deficient mutants of the Y1 mouse adrenocortical tumor cell line.

Two mutant clones (Y6 and OS3) derived from the ACTH-responsive Y1 mouse adrenocortical tumor cell line fail to respond to ACTH with increased adenylyl cyclase activity and, as a consequence, are resistant to the steroidogenic effects of the hormone. As determined from Northern blot and RNase protection assays, ACTH resistance in these mutants results from the failure to accumulate ACTH receptor transcripts. The ACTH receptor gene appears to be present in these mutants as determined by Southern blot hybridization analysis and can be activated following the growth of the mutant cells as tumors in mice, suggesting that the ACTH receptor gene is modified in a reversible manner. When mutant cells are transformed with a gene encoding the mouse beta 2-adrenergic receptor they respond to beta-adrenergic agonists with increased adenylyl cyclase activity in a manner that is indistinguishable from a similarly transformed parent Y1 cell line. These results suggest that the adenylyl cyclase system in the mutants is otherwise intact and that the failure to express ACTH receptor transcripts limits the responsiveness of these clones to the hormone.

Seroepidemiological survey of hepatitis E in Hong Kong by recombinant-based enzyme immunoassays.

The agent that causes the enterally transmitted form of non-A, non-B hepatitis has been cloned and called hepatitis E virus (HEV). We have carried out a seroepidemiological survey on the prevalence of hepatitis E in Hong Kong. In a retrospective study, serum from 394 patients with acute viral hepatitis and 355 healthy subjects was tested for antibodies to HEV (anti-HEV) with a recombinant-based enzyme immunoassay. 65 (16.5%) patients with hepatitis were positive for IgM anti-HEV and 23 (5.8%) were also positive for IgM anti-HEV. Of 18 patients diagnosed as having acute non-A, non-B, non-C hepatitis, 6 were IgM anti-HEV positive. 17 (6%) patients in whom acute hepatitis A was diagnosed were also infected with HEV. None of 70 patients with acute hepatitis B or C or exacerbation of chronic hepatitis B was IgM anti-HEV positive. 57 (16.1%) of the healthy subjects were positive for IgG anti-HEV. The prevalence of IgG anti-HEV was higher in subjects over 20 years old than in younger subjects (24% vs 4%, p < 0.0001). IgG anti-HEV was detected in 26% of subjects who were positive for IgG antibody to HAV and in 7% of those negative for that antibody (p < 0.0001). We demonstrated the validity of the recombinant-based enzyme immunoassays for the diagnosis of hepatitis E. Our results suggest that hepatitis E accounts for a third of non-A, non-B, non-C hepatitis in Hong Kong and that coinfection of hepatitis A and E can occur.

Use of atracurium in patients with or without renal failure.

Nine patients with normal renal function, and eight renal failure patients scheduled for renal transplantation, were anesthetized under nitrous oxide and isoflurane to determine the influence of renal function on the effect of atracurium. Atracurium 0.5 mg/kg, was given as an iv bolus, followed by 0.2 mg/kg as maintenance dose. The onset times, duration of action, duration of maintenance dose and recovery times of neuromuscular blockade were recorded and compared. There were no differences in the pharmacodynamics except that the uremic patients had slightly longer recovery time. It is concluded that atracurium is an ideal drug for uremic patients.