T-cell differentiative capacity of haematopoietic stem cells immortalized in vitro with radiation leukemia virus.

The differentiative capacity of a unique haematopoietic cell type has been investigated. These cells have been found to be a common target in vitro to infection and immortalization by radiation leukemia viruses (RadLVs). Many continuous lines of these cells have been generated. Since RadLV retroviruses are known to be strictly T-cell-tropic in vivo, we have questioned whether these cells are precursors of T cells. To this end, the RadLV-induced C1-V13D cell line has been inoculated into thymus of sublethally irradiated syngeneic CBA/H mice and tested for capacity to proliferate and differentiate, i.e. express T-cell markers. When inoculated in high number, C1-V13D cells can induce a thymic tumour within 14 to 21 days. Expression of T-cell markers on these cells was determined by fluorescence-activated cell sorting (FACS) analysis, after gating out C1-V13D cells on the basis of their high 90 degree scatter and their high forward scatter. They can also be distinguished by their unique expression of RadLVgp70 envelope (env) protein, B220, CD44, and aberrant expression of a class I epitope. Explanted primary thymomas from many animals showed no evidence of T-cell marker expression on C1-V13D cells upon reisolation. However, when C1-V13D was further passaged intrathymically, there was clear expression of Thy-1, CD4, CD8, and TCR-alpha beta on C1-V13D cells reisolated from these tumours. Two-colour FACS analysis and fluorescent antibody staining have confirmed the acquisition of T-cell surface markers by C1-V13D cells growing in this environment. Northern analysis confirmed endogenous expression of T-cell receptor beta chain genes in C1-V13D cells isolated after the third passage. All data indicate that RadLV preferentially infects a unique haematopoietic precursor cell in spleen which can differentiate along the T lineage once located within the thymic environment. The cell lines described here represent valuable models for studying T-cell differentiation from lymphoid stem cells, and for dissecting the early events in leukemogenesis.

Analysis of oncogenic progression in a radiation leukemia virus model.

The mechanism by which non-oncogene-bearing, slowly transforming retroviruses induce leukemia is not well understood, but appears to represent a multi-step process. Cell lines have been isolated following in vitro infection of lymphoid cells with radiation leukemia virus (RadLV) and they have been used to develop a two-step model for leukemia development. Thymic tumors were induced when one of the cell lines, C1-V13D, was inoculated into CBA/H mouse thymus. Upon reisolation of C1-V13D cells after one, two and three passages through thymus, individual cloned cell lines displayed increased tumorigenic potential compared with the non-tumorigenic parental line. Southern analysis has been used to track any genetic changes occurring while cells undergo further transformation and become increasingly tumorigenic. Specifically, retrovirus integration has been monitored in clones derived from C1-V13D at the primary, secondary and tertiary passage through thymus using probes specific for long terminal repeat (LTR), gag, pol and env genes of RadLV. The data indicate multiple ecotropic retrovirus integration sites in C1-V13D cells. Primary thymic tumors also showed the integration of a new recombinant or defective virus. There was no evidence that new ecotropic retrovirus integration had occurred during subsequent passage of primary tumors through the thymus, i.e. during the progression to oncogenesis. All data indicate an important role for the thymic environment in the development of a fully transformed cell.

Induction of leukemia in mice using a radiation leukemia virus-induced cell line: a model system for studying oncogenic progression.

Leukemogenesis induced by slowly transforming retroviruses is a multistep process which is difficult to dissect because of its long latency and the problem of distinguishing oncogenic from differentiative events. A method for leukemia induction in mice has been developed using a cell line isolated following in vitro infection with the slowly transforming murine radiation leukemia virus (RadLV). The CI-V13D cell line represents a lymphoid precursor cell type at an early stage in cell transformation and can develop subcutaneous tumors in irradiated syngeneic hosts but not in allogeneic mice even after sublethal irradiation. Selective growth in allogeneic (CBA/H) mouse thymus has been demonstrated, but this requires preirradiation of the recipient. Upon reisolation from CBA/H thymus, C1-V13D progeny clones displayed increased tumorigenic potential in comparison to the 'parental' CI-V13D cell line. Tumorigenicity was shown to increase with serial passage through thymus and electron micrographs of clones also revealed increased production of C-type retroviruses. This new model for oncogenic progression should be more amenable to analysis of early genetic changes occurring during replication of leukemia in the thymus.

Association of repeat sequences with integrated retroviruses in a murine leukaemia cell line.

An analysis was made of the retroviral integration sites for retroviruses in a murine lymphoid precursor cell line, C1-V13D, derived following in vitro infection with RadLV, an ecotropic murine retrovirus. A genomic library was constructed and lambda clones were selected for their capacity to hybridize with the specific RadLV gp70 ecotropic env probe. Analysis of these clones by a combination of approaches, including subcloning, partial restriction mapping and sequencing, has confirmed the existence of multiple recombinant and defective viruses in C1-V13D. To check for the presence of coding sequences in flanking genomic DNA, 32P-labelled cDNA from C1-V13D was used to probe HindIII- and Psti-digested virus-positive lambda clones by Southern analysis. Regions hybridizing specifically with 32P-labelled C1-V13D cDNA were subcloned and analysed. A notable feature of these cDNA+ regions was the frequent presence of B1, B2 and simple repeats. These repeat elements were found to be present in high frequency in the genomic regions flanking the proviruses, in numbers higher than expected for the genome as a whole. All full-length viruses isolated appeared to represent integration events into regions rich in repeat elements. Some B1 and B2 repeats have been shown to code for functional proteins and to play regulatory roles. Viral integration in the vicinity of these genetic elements could contribute to oncogenesis if the integration event were to disrupt normal gene function.

Psychosocial adjustment of victims of occupational hand injuries.

A group of hand injury patients was followed up 2-3 years after their injury. Compared to a non-patient control group hand injury patients were more externally oriented, and reported more psychological symptoms. Based on their General Health Questionnaire scores, the patients were divided into a low score and a high score group. High GHQ scores were associated with external locus of control, less social activities, and dissatisfaction with social life. The medical rating of loss of earning capacity was not predictive of overall occupational or psychosocial adjustment. Suggestions are made for future research into determinants of occupational adjustment of these patients.

Termination of pathological pregnancy in second and early third trimesters with extraamniotic instillation of 16-phenoxy-omega-tetranor prostaglandin E2 methylsulfonylamide.

OBJECTIVES: To evaluate the efficacy, safety and influence on subsequent fertility of sulprostone, a prostaglandin E2 analog, in terminating pathological pregnancies via the extraamniotic route. METHODS: Forty pregnant women with intrauterine fetal death or major congenital anomalies were enrolled. Sulprostone was instilled into the extraamniotic space through a silicon Foley catheter. The instillation rate was 0.5-1 microgram/min. Instillation was discontinued when the catheter was expelled or when rupture of the membranes occurred. The duration of instillation and the time interval to completion of abortion was recorded. Information about subsequent fertility was collected by telephone or at outpatient clinic visits. RESULTS: The mean duration of instillation was 7.0 h and the mean dose of sulprostone was 314.8 micrograms. The mean induction-to-abortion interval (IAI) was 17.0 h. In two of the 40 patients, the cervix was not adequately ripened after 48 h and these pregnancies were ultimately terminated by alternative methods. The success rate of termination in 48 h was 92.5% (37/40). No severe side effects were encountered. CONCLUSION: To the best of our knowledge, this is the first report in the English literature of administration of sulprostone by extraamniotic instillation for termination of pathological pregnancies. The method is effective and safe and has an insignificant influence on subsequent fertility.

Modified pelvic anterior exenteration.

During a 20-year time period, 117 patients with various histologic types of cancer underwent pelvic exentereation. Six of the 107 patients who needed anterior exenteration with urinary diversion received partial excision of the urinary bladder and modification surgery, of which three had uretero-cystostomy, two had mobilization of bladder and uretero-cystostomy and one had uretero-uterostomy. These six patients, except for two, died of disease in 1 year. Two patients are alive and happy, with quality of life because they can urinate normally. We suggest that anterior exenteration should be modified whenever possible to preserve the bladder and not to do an urinary diversion.

Analysis of two sling procedures using polypropylene mesh for treatment of stress urinary incontinence.

OBJECTIVES: To evaluate and compare the surgical outcome between the innovative tension-free vaginal tape (TVT) and conventional pubovaginal sling (PVS) procedures using polypropylene mesh. METHODS: Eighty consecutive women with urodynamic stress urinary incontinence (SUI), who chose to undergo either a TVT (n=23) or a PVS (n=57) procedure using polypropylene mesh based on financial consideration, were recruited for this study. The surgical results were analyzed and compared subjectively and objectively. RESULTS: The mean follow-up interval was 23 months for the TVT and 20 months for the PVS procedure (P=0.062). Postoperatively, SUI (91.3% vs. 93.0%), concomitant urge symptoms (85.0% vs. 85.3%) and the negative impact of incontinence and urogenital distress on patients' quality of life (79.8% vs. 77.8%) (77.4% vs. 68.8%) had improved markedly. After a multivariable logistic regression analysis, the treatment outcome of SUI was found to be independent of the main effects of patient age, parity, concurrent gynecological surgeries, intrinsic sphincter deficiency, previous failed incontinence surgeries, and concomitant urge symptoms. However, it was significantly related to treatment procedures (TVT vs. PVS) and their interaction with patient body mass index (BMI). Based on the fitted logistic model, we see that TVT performs better than PVS when BMI is less than 27.27 kg/m2, and the advantage of TVT decreases as BMI increases. CONCLUSION: Both TVT and PVS procedures using polypropylene mesh are effective treatment modalities for female SUI. However, TVT was not as effective in treating overweight or obese women as PVS.

Human renal organic anion transporter 1 (hOAT1) and its role in the nephrotoxicity of antiviral nucleotide analogs.

hOAT1 is a renal membrane protein able to efficiently transport acyclic nucleoside phosphonates (ANPs). When expressed in CHO cells, hOAT1 mediates the uptake and cytotoxicity of ANPs suggesting that it plays an active role in the nephrotoxicity associated with cidofovir CMV therapy and high-dose adefovir HIV therapy. Although efficiently transported by hOAT1, tenofovir did not show any significant cytotoxicity in isolated human proximal tubular cells, which correlates with the lack of nephrotoxicity observed in HIV-infected patients on prolonged tenofovir therapy.

Continuous epidural morphine and lidocaine for postoperative pain control in obstetric and gynecologic operation.

BACKGROUND: Postoperative wound pain is a remarkable distress and may precipitate major complication. We attempted to develop a reliable and effective technique for postoperative analgesia that is safe and simple enough to be provided in an ordinary ward of a rural hospital. MATERIALS AND METHODS: A retrospective study of continuous epidural infusion of morphine 7.5 mg in 60 mL diluted lidocaine for postoperative pain control in 156 patients in the past year was undertaken. Among the patients, one hundred and twelve patients had undergone. Cesarean Section, and 44 patients had uterine or adnexal operations. All patients received epidural anesthesia. RESULTS: Effective analgesia was achieved in all cases throughout the postoperative period. Pain relief was long lasting and there were only 5 patients (3.2%) who required some additional narcotics. The mean interval between completion of the operation and passage of flatus was 33.2 h. Only 48 patients (30.8%) complained of skin itching, 39 patients (25.0%) reported nausea and vomiting and 62 cases (39.7%) experienced constipation. Urinary retention after Foley catheter removal was noted in 9 cases (5.8%). No major complication pertaining to the technique was encountered. CONCLUSIONS: We concluded that continuous epidural infusion of morphine and diluted lidocaine is an effective, simple and safe technique for postoperative pain control in obstetric and gynecologic operation and it can be administered routinely even in a rural hospital.

Fluorescence-based assay for the interaction of small molecules with the human renal organic anion transporter 1.

Secretion of small molecules from the systemic blood circulation into urine is one of the physiologically essential functions of the kidney. The human organic anion transporter (hOAT1) is a key component in the renal tubular secretion of negatively charged molecules including a variety of important therapeutics. In some cases, compounds interacting with hOAT1 may induce pharmacokinetic drug-drug interactions or cause nephrotoxicity. We developed a fluorescence-based, 96-well format assay using CHO cells stably expressing hOAT1, which allows for the evaluation of interactions between small molecules and hOAT1. The assay is based on the inhibition of the transport of 6-carboxyfluorescein, a high-affinity hOAT1 substrate (Km = 3.9 microM), which was identified as one of several fluorescent organic anions. The relative inhibition potency of various known hOAT1 substrates determined using the 6-carboxyfluorescein-based inhibition assay correlated well with their Km values, indicating that the fluorescent assay exhibits a proper specificity. This in vitro assay can be employed to evaluate the mechanism of renal clearance of organic anions, to assess potential drug-drug interactions and/or nephrotoxic effects of various therapeutics, and to screen for novel hOAT1 inhibitors that could serve as efficient nephroprotectants.

Prenatal diagnosis of placenta previa accreta with power amplitude ultrasonic angiography.

A case of placenta previa accreta was diagnosed at 28 weeks' gestation with use of power amplitude ultrasonic angiography, which showed an unusually intense blood flow within the placental sonolucent spaces and hypervascularity within the placental and surrounding tissues. High-velocity (> or = 15 cm/sec) turbulent venous-type flow was detected within the placental blood lakes, and prominent low-impedance (resistance index 0.24) arterial blood flow was detected within the uterine serosa-posterior bladder wall boundary zone. An uneventful cesarean hysterectomy was performed at 32 weeks' gestation, and histopathologic examination revealed placenta previa increta.

Operative laparoscopy in benign cystic teratoma of ovary.

Eighteen women of reproductive age underwent laparoscopic management of unilateral ovarian benign cystic teratoma from 3 to 7 cm in diameter. Nine patients received pelviscopic enucleation of ovarian cysts and nine patients received unilateral oophorectomy or salpingo-oophorectomy. No major surgical complication was encountered and the hospital stay was short. Our series suggest that operative laparoscopy is a safe and effective method in the management of benign cystic teratoma.

[Clinical experience of GDM screening program].

From Jan. 1985 to Jun. 1990, 2561 pregnant women attending our prenatal clinic were screened for gestational diabetes. A 50-gm, 1-hour glucose challenge test was given and 470 patients (18.4%) showed plasma glucose values greater than or equal to 140 mg/dl. These women were subjected to a further 3-hr 100-gm OGTT to diagnose diabetes using O'Sullivans' criteria. Of these 470 cases, 80 (3.1%) of total cases were shown to be diabetic. Strict glycemic control with a fasting plasma sugar under 105 mg/dl and postprandial sugar under 120 mg/dl was conducted using diet and insulin in all cases. We compared the perinatal outcome of GDM pregnant women with normal pregnant women. Statistically, there was no significant difference. This study clearly demonstrates that a GDM screening program should be recommended for every pregnant woman. Prompt glycemic control should be given for all diabetic mothers to optimize the perinatal outcome.

Incidence of hypotension induced by spinal anesthesia with xylocaine for cesarean section and postpartum tubal ligation.

The incidence of hypotension induced by spinal anesthesia in 154 ASA class I or II patients having Cesarean section and postpartum bilateral tubal ligation was studied. Xylocaine (2%) 60-65 mg was used for spinal anesthesia. There was 41.0% of patients in Cesarean section group developed hypotension induced by spinal anesthesia, and 13.5% in tubal ligation group. We found no correlation between the time after delivery and the occurrence of hypotension induced by spinal anesthesia. In addition, 20.3% of the patients in tubal ligation group required intravenous narcotics because of inadequate sensory blockade during skin incision, but only 9.0% in Cesarean section needed the same supplement which was due to visceral pain during intra-abdominal manipulation. We conclude that spinal anesthesia with 2% xylocaine is safe and effective in both Cesarean section and postpartum tubal ligation although a significant difference of spinal hypotension existed.

Nonsteroidal anti-inflammatory drugs efficiently reduce the transport and cytotoxicity of adefovir mediated by the human renal organic anion transporter 1.

Adefovir is a nucleotide analog with anti-human immunodeficiency virus (HIV) activity that has been extensively studied in clinical trials. While on prolonged anti-HIV therapy with adefovir, some patients may develop drug-associated nephrotoxicity manifested by changes in laboratory markers of renal tubular functions that are reversible upon drug discontinuation. It has been recently shown that adefovir is efficiently transported by the human renal organic anion transporter 1 (hOAT1), a membrane transport protein localized in the kidney, that presumably mediates the accumulation of adefovir in renal proximal tubules. In an effort to look for novel inhibitors of this transport process, we used a cell line stably expressing hOAT1 to demonstrate that nonsteroidal anti-inflammatory drugs (NSAIDs) efficiently inhibit hOAT1-specific transport of adefovir at clinically relevant concentrations. Diflunisal, ketoprofen, flurbiprofen, indomethacin, naproxen, and ibuprofen were equally or more effective (IC(50) = 0.85-8 microM) than probenecid or betamipron, two known potent inhibitors of hOAT1 (IC(50) = 8 and 6 microM, respectively) with in vivo nephroprotective effects. Importantly, NSAIDs significantly reduced the shift in adefovir cytotoxicity observed upon hOAT1 expression with ketoprofen and naproxen being 2- to 3-times more effective than probenecid. Transport experiments with [(3)H]ketoprofen and [(3)H]ibuprofen revealed that NSAIDs themselves were not efficiently transported by hOAT1. None of the NSAIDs tested showed any interference with the anti-HIV activity of adefovir. In conclusion, these observations suggest that NSAIDs may reduce or delay the emergence of adefovir nephrotoxicity.

Perinatal management of congenital complete heart block.

BACKGROUND: The perinatal management of congenital complete heart block (CCHB) remains controversial. The purpose of this study was to present a therapeutic modality for CCHB. METHODS: We collected retrospective cases of all pregnant women admitted to our hospital between January 1992 and June 1999 whose babies developed CCHB antenatally. After a series of examinations, maternal, fetal and neonatal data were analyzed. RESULTS: Nine fetuses from six mothers (cases 1-6) in nine different pregnancies were studied. In case 1, both consecutive fetuses had CCHB and in case 2, all three consecutive fetuses had CCHB. The other mothers (cases 3-6) had only one fetus each with CCHB. Of the seven fetuses with isolated CCHB, four underwent observation only due to late-onset, or nonimmunologic CCHB, two received dexamethasone and/or intravenous immunoglobulin therapy because of the presence of hydropic signs, and one received dexamethasone at 23 weeks' gestation due to early-onset CCHB. Shortening fractions of the right ventricle had good compensation in four fetuses, without any treatment, and improving compensation in two of three fetuses receiving dexamethasone therapy. All seven fetuses were delivered smoothly and pacemakers were implanted shortly after birth. Two other fetuses had a poor outcome due to associated ventricular septal defect or hemoglobin Bart's disease. Furthermore, we gave dexamethasone (2 mg/day) instead of prednisolone (10 mg/day) for the next pregnancies of patients 3 to 5, beginning at 12 weeks of gestation. No fetal CCHB developed again. CONCLUSIONS: For pregnant women with previous fetal immunologic CCHB, early initiation of dexamethasone instead of prednisolone might be effective to cross the placenta and avoid recurrences. Dexamethasone is also effective for fetal CCHB of early onset, fetal hydrops or heart failure. Observation only is suggested for nonimmunologic CCHB and remote or late-onset immunologic CCHB. Other modalities were tried for very sick fetuses, but their effectiveness was not predictable.

In utero sonographic diagnosis of a communicating enteric duplication cyst in a giant omphalocele.

Enteric duplications are rare lesions, and relatively few cases have been diagnosed prenatally. We present, to our knowledge, the first case of an associated communicating ileal duplication cyst in a huge omphalocele diagnosed prenatally. The prenatal ultrasound findings revealed four features of the cystic lesion including peristaltic movements of the cystic wall, communication between the cyst and normal bowel lumen, intra-cystic echogenic contents, and echogenic mesenteric tissue (fat) close to the cyst. These distinct characteristics helped us to make a firm in utero diagnosis.

Severe limb reduction defects after uterine lavage at 7-8 weeks' gestation.

A 35-year-old multiparous Chinese woman underwent a uterine lavage procedure for prenatal sex determination at 7-8 weeks' gestation. The baby was delivered by elective Caesarean section because of breech presentation at 38 weeks' gestation. The child weighed 2425 g and had severe reduction defects of all four limbs. This case warrants advising prospective patients of a possible association and encouraging practitioners to mount a careful prenatal ultrasonography and postnatal follow-up of all pregnancies investigated by prenatal diagnostic uterine lavage.

Leukaemogenic progression: the importance of differentiation and associated genetic events.

A feature common to many blood cell cancers is the uncoupling of normal proliferative and differentiative events, both of which are intimately linked in the cell's developmental programme. In some cancers, further differentiative events have been associated with oncogenic progression and, in other cancers, terminal differentiation of cells has been shown to result in reversal of malignancy and death of the cancer cell. Clearly the development of cancer is not the result of a single oncogenic event, but rather a myriad of events which appear to proceed in concert in a step-wise fashion and which are likely to be influenced by the cellular environment. Here we review some of the major genetic changes which occur in leukaemogenesis and discuss the possible role of differentiative events in the development of leukaemia.