Genetic interaction between DNA polymerase beta and DNA-PKcs in embryogenesis and neurogenesis.

DNA polymerase beta (Polbeta) has been implicated in base excision repair. Polbeta knockout mice exhibit apoptosis in postmitotic neuronal cells and die at birth. Also, mice deficient in nonhomologous end-joining (NHEJ), a major pathway for DNA double-strand break repair, cause massive neuronal apoptosis. Severe combined immunodeficiency (SCID) mice have a mutation in the gene encoding DNA-dependent protein kinase catalytic subunit (DNA-PKcs), the component of NHEJ, and exhibit defective lymphogenesis. To study the interaction between Polbeta and DNA-PKcs, we generated mice doubly deficient in Polbeta and DNA-PKcs. Polbeta(-/-)DNA-PKcs(scid/scid) embryos displayed greater developmental delay, more extensive neuronal apoptosis, and earlier lethality than Polbeta(-/-) and DNA-PKcs(scid/scid) embryos. Furthermore, to study the involvement of p53 in the phenotype, we generated Polbeta(-/-)DNA-PKcs(scid/scid)p53(-/-) triple-mutant mice. The mutants did not exhibit apoptosis but were lethal with defective neurulation at midgestation. These results suggest a genetic interaction between Polbeta and DNA-PKcs in embryogenesis and neurogenesis.

Extracorporeal membrane oxygenation for newborns with gastric rupture.

Extracorporeal membrane oxygenation (ECMO) has been recognized to be beneficial to overcome not only persistent pulmonary hypertension of the newborn, but also cardiopulmonary distress due to neonatal sepsis. However, few papers have reported on the efficacy of ECMO for surgical sepsis in neonates with underlying diseases. This paper reports our experience with ECMO in three newborns with gastric rupture, one of the most serious causes of surgical sepsis in the neonatal period. Over the past 12 years, 14 newborns had gastric rupture; 3 developed lethal cardiopulmonary distress that conservative strategies, including aggressive intensive care, failed to manage, and were selected for ECMO. The clinical data of these patients were retrospectively analyzed. The onset time and duration of ECMO varied from 23 to 143 h of age and 72 to 294 h, respectively. In case 3, complicated by massive intra-abdominal hemorrhage during ECMO, anticoagulants were changed from heparin alone to combined use with nafamostat mesilate, a thrombin inhibitor with a very short half-life. Ultrafiltration or hemodialysis was added in two cases to regulate massive volume overload associated with renal failure. Despite major hemorrhagic complications in two cases, all patients survived. Thus, ECMO may be beneficial in managing neonates with therapy-resistant gastric rupture.

Lordosis of lumbar vertebrae in omphalocele: an important factor in regulating abdominal cavity capacity.

PURPOSE: Although it is well known that the tiny abdominal cavity associated with larger omphalocele enlarges rapidly after surgery, the responsible mechanism remains obscure. The authors have studied the curve of lumbar vertebrae and established a hypothesis in which lordosis of lumbar vertebrae plays a major role in changing the capacity of the abdominal cavity. METHODS: Fifty-three patients with intact omphalocele and 10 normal newborns as the control were studied. The size of omphalocele was determined from the mean value of the length and breadth of the sac. The curve of lumbar vertebrae (lordotic angle) was measured using Cobb's technique on the lateral radiograph serially taken in the supine position. RESULTS: The mean lordotic angle of 53 patients was 22.2 degrees (range, 0 to 65), which was significantly greater than 11.0 degrees of the control (P <.012). The individual lordotic angle before surgery was significantly correlated with the size of omphalocele (r = 0.668, P <.0001). In serial studies carried out in 15 patients with larger omphalocele, the mean lordotic angle was decreased significantly from 30.5 degrees to 20.0 degrees (P <.009) on the first postoperative day and returned to the control level within a few weeks. CONCLUSIONS: These results suggest that severe lumbar lordosis may well become an alternative factor to produce visceroabdominal disproportion observed before surgery, and its correction after surgery may well induce the abdominal cavity to rapidly enlarge. It may be speculated that the dehiscence of abdominal rectus muscles associated with omphalocele causes the tension of ventral muscles to decline, and, then, imbalance in tension between ventral (abdominal) and dorsal (back) muscles causes the lumbar lordosis to increase. Accordingly, at the staged procedure, the prosthetic sheet should be attached under moderate tension, and its plication should be carried out at regular intervals to maintain the tension of abdominal rectus muscles.

Randomized double-blind study of cyclosporin in chronic 'idiopathic' urticaria.

BACKGROUND: Histamine-releasing activity (HRA) is detectable in up to 50% of patients with chronic ordinary urticaria. OBJECTIVES: To determine the effect of cyclosporin on clinical features and HRA in patients with chronic urticaria. METHODS: Thirty patients with severe unremitting disease, responding poorly to antihistamines and showing a positive autologous serum skin test (ASST) as a marker of HRA, were randomized to 4 mg kg-1 daily of cyclosporin (Sandimmun, n = 20) or placebo (n = 10) for 4 weeks. Non-responders were offered open-label cyclosporin for 4 weeks. All were followed for up to 20 weeks or until clinical relapse; all took cetirizine 20 mg daily throughout the study. The primary measure of efficacy was a daily urticaria activity score (UAS) of weal numbers and itch (maximum score 42 per week). A positive response was defined as a reduction to < 25% of baseline weekly UAS and relapse as a return to > 75%. The effect of cyclosporin on serum HRA was assessed by in vitro basophil histamine release assays and ASSTs before and after treatment. RESULTS: Twenty-nine patients (19 active, 10 controls) completed the randomized trial medication. Eight of 19 on active treatment but none on placebo had responded at 4 weeks (P < 0.05). Three others on active drug met the criterion for response at 2 weeks but not at 4 weeks. Mean reduction in UAS between weeks 0 and 4 was 12.7 (95% confidence interval, CI 6.6-18.8) for active and 2.3 (95% CI - 3.3-7.9) for placebo (P = 0.005). Seventeen non-responders (seven randomized to active and 10 to placebo) chose open-label cyclosporin and 11 responded after 4 weeks. Six of the eight randomized active drug responders relapsed within 6 weeks. Of the 19 responders to randomized and open-label cyclosporin, five (26%) had not relapsed by the study end-point. Mean in vitro serum HRA fell from 36% (95% CI 22-49%) to 5% (95% CI 1-8%) after cyclosporin treatment (n = 11, P < 0.0001). The ASST response to post-treatment serum was also reduced (P < 0.05). CONCLUSIONS: This study shows that cyclosporin is effective for chronic urticaria and provides further evidence for a role of histamine-releasing autoantibodies in the pathogenesis of this chronic 'idiopathic' disease.

Secondary excision of choledochal cysts after previous cyst-enterostomies.

BACKGROUND/AIMS: Patients with a choledochal cyst previously treated by internal drainage should undergo secondary cyst excision. The results of such secondary excisions have not yet been reported. METHODOLOGY: Over a 27-year period, 121 patients underwent excision of a choledochal cyst at our hospitals. Of these, 14 patients underwent secondary cyst excision following internal drainage. These patients were compared retrospectively with the remaining 107 patients who underwent primary cyst excision. RESULTS: Blood loss and operative time were greater, and early and late post-operative complications were significantly more frequent in the secondary excision group. Wound infection (early complication), pancreatic stones (late complication), and hepatolithiasis (late complication) were significantly more common in the secondary excision group. Histologically, inflammation and biliary glands were more frequently seen in the resected bile ducts in the secondary excision group. CONCLUSIONS: The long-term results of secondary excision of choledochal cysts are worse than after primary excision. The frequent late complications may be related to the development of biliary glands as a consequence of cyst-enterostomy.

Human leucocyte antigen class II associations in chronic idiopathic urticaria.

The major histocompatibility complex (MHC) acts as a marker for self during T-cell ontogeny and is associated with the pathogenesis of many autoimmune diseases. Recent investigations have shown about 30% of patients with chronic idiopathic urticaria (CIU) have IgG autoantibodies against the high-affinity IgE receptor, FcepsilonRI, or IgE. A link between MHC class II alleles and CIU has not been reported previously. DNA was extracted from blood of 100 Caucasian patients with CIU, and the MHC class II type determined using the polymerase chain reaction with sequence-specific primers, testing for DRB and DQB1 alleles. The frequency of alleles in CIU patients was compared with that found in 603 controls. Further human leucocyte antigen (HLA) typing on patient subsets, classified by the patients' responses to intradermal injection of autologous serum and their serum-induced histamine release from basophil leucocytes of healthy donors, was undertaken. HLA DRB1*04 (DR4) and its associated allele, DQB1*0302 (DQ8), are raised in CIU patients compared with a control population (P = 2 x 10-5 and P = 2 x 10-4, respectively). HLA DRB1*15 (DR15) and its associated allele, DQB1*06 (DQ6), are significantly less frequently associated with CIU. The HLA DRB1*04 association is particularly strong (corrected P = 3.6 x 10-6) for patients whose serum has in vivo and in vitro histamine-releasing activity. HLA class II typing is consistent with the concept that CIU is a heterogeneous disease, and supports an autoimmune pathogenesis in a subset of patients.

Comparison of venoarterial versus venovenous access in the cerebral circulation of newborns undergoing extracorporeal membrane oxygenation.

This study was designed to compare venoarterial (VA) with venovenous (VV) access in the cerebral circulation of newborn infants during extracorporeal membrane oxygenation (ECMO). Among 14 infants with VA ECMO, 7 had no intracranial complications (group 1), while the others (group 2) developed intracranial hemorrhage (ICH). In contrast, among 19 infants with VV ECMO, only 1 developed ICH. Serial echocardiograms were performed before and after 1, 6, 12, and 24 h and 2 and 3 days of ECMO. The mean cerebral blood flow (CBF) velocities were measured in the anterior cerebral artery (ACA), right and left internal carotid arteries (Rt, Lt-ICA), basilar artery (BA), and right and left middle cerebral arteries (Rt, Lt-MCA). Ejection fraction (EF), cardiac output (CO), and stroke volume (SV) were also measured using standard echography. The velocity levels in the ACA, Rt-MCA, and Lt-MCA in VA ECMO were lower than those in VV ECMO, while those in the Lt-ICA and BA in VA ECMO were higher than those in VV ECMO. The EF, CO, and SV were lower in cases of VA ECMO than in VV ECMO. In cases of VA ECMO, there were no differences between groups 1 and 2 in velocities in the ACA, Rt-ICA, or Lt-ICA. However the velocities in group 2 in the BA, Rt-MCA, and Lt-MCA were lower than those in group 1 before and during ECMO. Similarly, the EF, CO, and SV were lower in group 2 (12.0%-31.0%, 0. 10-0.32 l/min, and 0.66-1.55 ml, respectively) than in group 1 (29. 5%-49.3%, 0.25-0.63 l/min, and 2.15-3.85 ml) during ECMO. However, in the infants on VV ECMO the CBF was either maintained or gradually increased before and during ECMO. Their cardiac parameters were: EF 46.1%-53.0%, CO 0.43-0.52 l/min, and SV 2.72-3.84 ml during ECMO. It is concluded that in VA ECMO CBF velocities, particularly in infants who developed ICH, decreased after the onset of ECMO in association with poor cardiac function, while in VV ECMO they were stable, probably due to normal systemic hemodynamics and cardiac function.

Meiotic contraction of CAG repeats in Saccharomyces cerevisiae.

Several human neurodegenerative disorders are caused by expansion of CAG repeats that occurs during meiosis or gametogenesis. We anticipated that the CAG repeats cloned in a plasmid of Saccharomyces cerevisiae might undergo a change in the number of repeats during meiosis and sporulation. To test this possibility, we devised a new method to change in vitro the number of CAG repeats and constructed plasmids carrying (CAG)39, (CAG)65 or (CAG)123 from a plasmid carrying (CAG)18. We monitored the number of colonies showing an altered length of the repeat tracts during mitosis and meiotic growth. Contraction of long CAG repeat was found to occur frequently, whereas a few cases of expansion were observed. The contraction was equally enhanced in both orientations when the host cells grew through meiosis. Thus, our results suggest that long CAG repeats are destabilized during meiosis or gametogenesis in S. cerevisiae.

Venovenous extracorporeal membrane oxygenation in newborn infants using the umbilical vein as a reinfusion route.

PURPOSE: The authors report on four neonates treated with venovenous (VV) extracorporeal membrane oxygenation (ECMO) using the umbilical vein as a reinfusion route. METHODS: From 1994 to 1997, 26 instances VV-ECMO in neonates have been carried out at our neonatal center for the treatment of severe respiratory and cardiac failure. Among them, 22 patients could be treated with VV-ECMO mainly using 15F double-lumen catheter (DLC), adding the cephalic drainage using another catheter. In the remaining four cases, however, attempts to insert the DLC into the right internal jugular vein failed because the vein was too small or technical problems. For such instances, two catheters were cannulated into the right atrium and the cephalic portion of the right internal jugular vein, respectively. These two venous catheters were connected to the drainage route of ECMO circuit with a "Y" connector. Then, the umbilical vein was cannulated with 10F or 8F catheter, which was connected to the reinfusion route of ECMO to return the oxygenated blood to the infant. RESULTS: The median age at which ECMO was initiated was 18 hours, and the median ECMO course was 72 hours. The liver function tests were slightly and transiently worsened in two patients during VV perfusion, (in one patient serum glutamic-oxaloacetic transaminase [SGOT] elevated to 76 IU/L and serum glutamic-pyruvic transaminase [SGPT] to 49 IU/L, and in another patient SGOT elevated to 56 IU/L and SGPT remained in normal range). Preumbilical cannula pressures were measured in two patients. In a patient who used 10F umbilical cannula, the preumbilical maximum pressure was 43 mm Hg at 250 mL/min of ECMO flow. In another with an 8F catheter, it was 72 mm Hg at 180 mL/min of ECMO flow. All of the patients survived without any neurological complications. CONCLUSIONS: If the right internal jugular vein would not accommodate the DLC, VV-ECMO using the umbilical vein as a infusion route could be selected.

Isolated cavernous hemangioma of the stomach in a neonate.

This is a report of an extremely rare case of isolated cavernous hemangioma of the stomach found in a neonate. Hematemesis developed in a 7-day-old baby boy weighing 2.8 kg at birth. This was followed by melena. Endoscopic examination findings showed a large hemorrhagic mass on the lesser curvature of the stomach. During laparotomy, a large vascular tumor was identified and subtotal gastrectomy was carried out. On section, the tumor extended through the submucosa to the serosal surface. Histological diagnosis was cavernous hemangioma. The authors could collect only 10 cases of hemangioma of the stomach diagnosed in childhood from the world literature.

Intravenous immunoglobulin in autoimmune chronic urticaria.

Histamine releasing autoantibodies play a central role in the pathogenesis of chronic urticaria (CU) in approximately 30% of affected patients. We investigated the therapeutic effect of high-dose intravenous immunoglobulin (IVIG) on disease activity in patients with severe CU of autoimmune aetiology. Autoimmune urticaria was diagnosed by the development of a weal-and-flare reaction to the intradermal injection of autologous serum and by serum-induced histamine release from the basophil leucocytes of healthy donors in vitro. Ten patients with severe, autoimmune CU, poorly responsive to conventional treatment, were treated with IVIG 0.4 g/kg per day for 5 days. The outcome on cutaneous wealing and itch was monitored using urticaria activity scores, visual analogue scales and autologous intradermal serum tests. Clinical benefit was noted in nine of 10 patients: three patients continue in prolonged complete remissions (3 years follow-up), two had temporary complete remissions, and symptoms in four patients improved subsequent to treatment. There was significant improvement in the urticaria activity scores and visual analogue scores at 2 (P < 0.01) and 6 weeks (P < 0.01) post-IVIG compared with the baseline values (Wilcoxon matched pairs). The diminution in urticarial activity in the majority of patients corresponded with a reduced weal-and-flare response to the intradermal injection of autologous post-treatment serum compared with the pretreatment serum. Minor side-effects were common, but there were no serious or long-term adverse effects. IVIG represents a novel therapeutic option in selected patients with recalcitrant CU associated with histamine releasing autoantibodies.

Analysis of patients with congenital diaphragmatic hernia requiring pre-operative extracorporeal membrane oxygenation (ECMO).

The purpose of this report was to analyze the patients requiring preoperative extracorporeal membrane oxygenation (ECMO) as the most critical group of patients with congenital diaphragmatic hernia (CDH) and to identify any special features. Over the past 11 years, out of 72 neonates with CDH admitted before 24 h of age, 40 (56%) could be managed with conventional therapies while the other 32 (44%) required ECMO. Seventeen infants requiring preoperative ECMO were classified as group 1, and the 15 with postoperative ECMO as group 2 (controls). The records of patients in both groups were analyzed. Patients in group 1 were not only severely hypoxic, but also significantly hypercapneic on admission, and in 14 (82%) the diaphragmatic defect was so large or totally agenetic that a prosthetic patch was necessary. The average age at onset of ECMO in group 1 was 13.1 h, and the average duration was 159 h. Major hemorrhagic complications including intracranial hemorrhage occurred with a significantly higher frequency in group 1. The survival rate in group 1 was 41%, compared with 73% in group 2 and 85% in non-ECMO patients. Four infants in group 1 with extremely hypoplastic lungs could not be weaned from ECMO, and died without undergoing an operation. Moreover, 4 of the 7 survivors in group 1 required prolonged (105-658 days) ventilator care with a tracheostomy after weaning from ECMO, and were frequently hospitalized thereafter. The pulmonary function of these patients remained severely underdeveloped for a long time; indeed, the average pulmonary perfusion ratio of the affected side remained at only 40% of the contralateral side in group 1, although the volume ratio reached 85%. These findings may suggest that the main pathology of the patients requiring preoperative ECMO was a high degree of pulmonary hypoplasia, and that there will be limitations to management using ECMO.

Application of a new anticoagulant (Nafamostat Mesilate) to control hemorrhagic complications during extracorporeal membrane oxygenation--a preliminary report.

Bleeding related to systemic heparinization has been considered one of the major complications associated with extracorporeal membrane oxygenation (ECMO). Development of the heparin-bonded system will be essential in reducing hemorrhagic complications, but has not yet been clinically proven. The authors chose an alternative approach of making a difference in the activated clotting time (ACT) values between the patient and the ECMO circuit, and decreased only the patient's ACT value, while keeping the value of the ECMO circuit at an ideal level. For this purpose, we have used a very short-life anticoagulant, Nafamostat Mesilate (FUT), while decreasing the dose of heparin. FUT is a synthetic protease inhibitor that has been found to inhibit various kinds of enzyme activities for coagulation. Twelve newborns who had some hemorrhagic complications at various sites before or during ECMO, were selected to receive FUT. The heparin dose was decreased after FUT administration into the drainage route. FUT and heparin doses were regulated to maintain the ACT value at the reinfusion route at 190 to 220 seconds. ACT values at the drainage and the reinfusion routes were simultaneously measured. The average time on FUT was 100.3 +/- 86.3 (SD) hours. The average dose of FUT was 0.48 +/- 0.22 mg/kg/h, and that of heparin was 21.0 +/- 7.5 U/kg/h. The average ACT value at the reinfusion route was 205.7 +/- 14.0 seconds compared with that at the drainage route of 178.5 +/- 11.8. The difference was statistically significant (P < .001). The average difference in ACT values between both routes was 27.1 +/- 7.9 seconds. The bleeding was well controlled by FUT administration in 8 of 12 cases. This report may represent the first clinical use of FUT in neonatal ECMO, and serve as a preliminary study.

Prevention of infection in dental procedures.

The efficacy of a newly-developed anti-cross-contamination device in dentistry, the Air Flushing Clean System (AFCS), was tested under experimental and clinical conditions. In the experimental situation, a dental air turbine handpiece with or without AFCS was contaminated with two bacterial strains, Staphylococcus aureus FDA209P and Streptococcus mutants ATCC25175. After contamination with these bacteria, the handpieces were subjected to two disinfecting methods. Residual bacteria inside the handpiece or an air/water line were cultured and counted, and compared with controls. In this experiment, with AFCS but no dental vacuum suction, wiping of the handpiece with 70% ethanol gauze reduced the count of S. aureus by 99%. No bacterial contamination in the air/water line was detected after exchanging with an autoclaved handpiece. With AFCS and dental vacuum suction, bacterial contamination in the air/water line, as well as in the interior of the handpiece, was not detected. These results indicate that AFCS could reduce bacterial contamination within the air turbine handpiece more effectively than the conventional handpiece regardless of whether or not the dental vacuum suction was used.

Flow cytometric analysis of basophil numbers in chronic urticaria: basopenia is related to serum histamine releasing activity.

BACKGROUND: Peripheral blood basophils are reduced in some chronic urticaria patients when counted with granule stains. Approximately 30% of patients with severe chronic urticaria have functional autoantibodies which release histamine from healthy donor basophils in vitro but the relationship between basophil numbers in vivo and serum histamine releasing activity has not been studied. OBJECTIVE: To determine the relationship between basophil numbers and serum basophil histamine releasing activity and to assess whether basophils are present, but undetectable, in peripheral blood with granule stains by using a new flow cytometric method based on surface immunophenotype. METHODS: Basophils were counted manually by a chamber method using a granule stain and by flow cytometry using dual staining with anti-IgE and anti-Fc epsilonRI in 25 chronic idiopathic urticaria patients and 25 healthy controls. Serum histamine releasing activity was assessed on healthy donor basophils in vitro and by the weal response to autologous serum skin testing in vivo (patients only). RESULTS: Basophils were significantly reduced in chronic urticaria by manual counting and flow cytometry. A subgroup of seven patients with in vitro histamine releasing activity showed a marked reduction or absence of basophils by both methods. There were no obvious distinguishing clinical characteristics between these patients and the others; six of them showed positive autologous serum skin-test responses. On comparing the two methods, the manual basophil counts were generally lower than flow cytometric counts. Agreement over the full range of values was not strong and therefore counts obtained by the two methods are not directly interchangeable. CONCLUSION: Basopenia in chronic idiopathic urticaria is associated with serum basophil histamine releasing activity in a subgroup of patients. The lack of granule and surface immunophenotype staining suggests a reduction in numbers rather than an inability to detect circulating degranulated cells by conventional counting methods.

[Current status of management of omphalocele and gastroschisis].

Forty-five cases of gastroschisis and 85 of omphalocele were reviewed. The survival of gastroschisis has dramatically improved over the past 20 years, however, that of omphalocele still remained in the lower value, because the size of the defect and the presence of associated anomalies are prognostic factors. The primary fascial closure was first employed for the patients with gastroschisis and a silo chimney was used for limited cases. On the other hand, for the patients with omphalocele, primary closure was possible in 34 cases, silo chimney was used in 17, and 45 cases had nonoperative management with epithelialization. Among them, nonoperative management using painting was the most reliable therapeutic for omphalocele.

CD4 expression is important but not essential for infection with exogenous mouse mammary tumor virus.

We studied local events in the popliteal lymph nodes of CD4-deficient mice following foot pad injection with an MMTV strain which carries the gene for a V beta 14-specific superantigen. Injection of the V beta 14-specific MMTV induced vigorous expansion of V beta 14+ CD4+ T cells and B cells in their lymph nodes of CD4+/- heterozygous control mice. On the other hand, CD4-/- mice injected with the MMTV showed a proliferation of V beta 14+ T cells among the population of TCR alpha beta + CD4-CD8- T cells, although to a lesser extent. This phenomenon was not accompanied by vigorous B cell expansion. A PCR assay revelated that the MMTV definitely infected the lymph nodes cells of the CD4-/- mouse. However, the infectivity of the MMTV in CD4-/- mice was approximately 20 times lower than that in CD4+/- mice. These findings indicate that, in MMTV infection of CD4-deficient mice, the superantigen-reactive T cells among the population of TCR alpha beta +CD4-CD8- T cells substitute for the superantigen-reactive CD4- T cells of normal mice, and that the absence of CD4 molecules decreased the infectivity of MMTV because of insufficient expansion of the superantigen-reactive T cells.