Neonatal screening for cystic fibrosis in the Trent region (UK): two-stage immunoreactive trypsin screening compared with a three-stage protocol with DNA analysis as an intermediate step.

OBJECTIVES: To assess neonatal screening for cystic fibrosis using immunoreactive trypsin, either alone or in conjunction with DNA analysis for the delta F508 mutation. A novel three-stage screening protocol was compared with the previously introduced two-stage immunoreactive trypsin-DNA protocol. DESIGN: (a) Collection of data from a 4 1/2 year period (phase 1) of two-stage immunoreactive trypsin screening. The initial dried blood samples were obtained at 6 days of age and repeat samples at 27 days of age from babies with results above the 99.5th centile. Babies with persistent hypertrypsinaemia were referred for a diagnostic sweat test. (b) Retrospective DNA analysis: patients with cystic fibrosis diagnosed in phase 1 were genotyped and most samples from babies with increased initial immunoreactive trypsin but normal results in the second sample were analysed for the delta F508 mutation. (c) Phase 2, a prospective study of a three-stage neonatal screening protocol, in which only babies heterozygous for the delta F508 cystic fibrosis mutation progressed to the second immunoreactive trypsin test. SETTING: The Trent neonatal screening programme. SUBJECTS: 437 859 babies born between August 1989 and March 1996. MAIN OUTCOME MEASURES: Proportions of unaffected babies requiring a second blood sample or a sweat test. Overall sensitivity for the detection of cystic fibrosis. RESULTS: The two-stage screen failed to identify six out of 94 cases of cystic fibrosis (without meconium ileus). The introduction of the DNA analysis step would have resulted in one additional case being missed. With the three-stage screen there was a 92% reduction in babies requiring a second blood sample and an 80% reduction in negative sweat tests, results close to the predictions of the retrospective study. CONCLUSIONS: The three-stage screening protocol is a marked improvement on the two-stage immunoreactive trypsin strategy and on the two-stage immunoreactive trypsin-DNA strategy recently introduced in some other screening programmes.

Polymorphism in human IL-1 receptor antagonist gene intron 2 is caused by variable numbers of an 86-bp tandem repeat.

We have investigated the polymorphism in intron 2 of the interleukin-1 receptor antagonist gene and identified two new alleles of the system. We have shown that the polymorphism is caused by the variable copy number of an 86-bp sequence, by using the polymerase chain reaction and primers immediately flanking the repeat region, and by direct sequencing. The repeat region contains three potential protein-binding sites and therefore the variable copy number may have functional significance.

The ocular pathology of Norrie disease in a fetus of 11 weeks' gestational age.

The ocular pathology of Norrie disease was studied for the first time in a fetus of 11 weeks' gestation, following prenatal diagnosis using genetic markers for Norrie disease and elective abortion. The eyes were histologically normal, with no evidence of primary neuroectodermal maldevelopment of the retina, previously postulated to be the cause of the ocular changes. We believe that the retinal and other manifestations of Norrie disease are the result of a primary abnormality of vascular proliferation, probably in relation to persistent hyperplastic primary vitreous after approximately 14 weeks' gestation. We postulate that the ocular and otological effects of Norrie disease may be due to a genetically mediated abnormality of secretion of, or sensitivity to, angiogenic growth factors at endodermal-neuroectodermal interfaces during fetal and postnatal development.

Carrier detection and prenatal diagnosis in Norrie disease.

We report the use of DNA probes to determine carrier status in three young women from a large kindred with Norrie disease. One of the women requested prenatal diagnosis during pregnancy. In this pedigree, Norrie disease was not characterized by a deletion at DXS7.

Protection of mice against mixed fission neutron-gamma (n: gamma = 1:1) irradiation by WR-2721, 16,16-dimethyl PGE2, and the combination of both agents.

The survival of mice after whole-body exposure to a modified fission neutron-gamma field (n: gamma = 1:1) was used to examine radiation protection by WR-2721, 16,16-dimethyl PGE2(DiPGE2), and the combination of both agents. Administration of WR-2721 (453 mg/kg) increased the LD50/30 from 5.24 to 7.17 Gy (DMF = 1.37), whereas pretreatment with DiPGE2 (1.6 mg/kg) increased the LD50/30 to 5.77 Gy (dose modification factor (DMF) = 1.10). The combination of 453 mg/kg WR-2721 and 0.4 mg/kg DiPGE2 resulted in an LD50/30 of 7.33 Gy, yielding a DMF of 1.39. However, no significant difference in protection was obtained with the combination of the two agents compared to that seen with WR-2721 alone.

Quantitative, functional and biochemical alterations in the peritoneal cells of mice exposed to whole-body gamma-irradiation. I. Changes in cellular protein, adherence properties and enzymatic activities associated with platelet-activating factor formation and inactivation, and arachidonate metabolism.

Changes in total number, differentials, cell protein, adherence properties, acetyltransferase and acetylhydrolase activities, prostaglandin E2 and leukotriene C4 production, as well as Ca2+ ionophore A23187 stimulation were examined in resident peritoneal cells isolated from mice 2 h to 10 days postexposure to a single dose (7, 10 or 12 Gy) of gamma-radiation. Radiation dose-related reductions in macrophage and lymphocyte numbers and increases in cellular protein and capacity to adhere to plastic surfaces were evident. In vivo irradiation also elevated the activities of acetyltransferase and acetylhydrolase (catalysing platelet-activating factor biosynthesis and inactivation, respectively) in adherent and nonadherent peritoneal cells, particularly 3-4 days postexposure. Blood plasma from irradiated animals did not reflect the increased cellular acetylhydrolase activity. Prostaglandin E2 and leukotriene C4 synthesis were elevated postexposure, suggesting increased substrate (arachidonate) availability and increased cyclooxygenase and lipoxygenase activities. Ionophore stimulation of enzyme activities and eicosanoid release also differed in irradiated peritoneal cells. While the properties of adherence, platelet-activating factor synthesis/inactivation-associated enzyme activities, and eicosanoid production are generally characterized as those of macrophages, lymphocytes or their products may influence or contribute to the observed radiation-induced changes.

Marginal adaptation of BIS-GMA-based composites containing various diluents.

The aim of this study was to determine how otherwise acceptable diluent monomers affect the marginal adaptation of BIS-GMA-based composites. Based on the results of the investigation, the following conclusions can be drawn: 1. Addition to dimethacrylate diluents containing (CH2) recurring units generally yields composites having better marginal adaptation than do those containing (CH2 CH2 O) groups. Best marginal adaptation for a single diluent is obtained for compositions using 1, 4 and 1, 10-polymethylene glycol dimethacrylate as diluent. 2. Marginal adaptation is improved on lowering the diluent concentration. Optimum adaptation will be obtained for a formulation containing a minimum percentage of diluent with clinically acceptable working properties. 3. Volume changes on temperature cycling resulting from differences in thermal expansion coefficients of composites do not effect the marginal integrity as much as does curing shrinkage.

The effects of hepes buffer on clotting tests, assay of factors V and VIII and on the hydrolysis of esters by thrombin and thrombokinase.

Shorter clotting times were found in the presence of 50 mM Hepes (N-2-hydroxyethylpiperazine-N1-2-ethanesulfonic acid) buffer than of 50mM Imidazole buffer in one-stage assays of factors V and VIII, in modified APTT and PT tests and in tests of the clotting of human plasma by purified human thrombin. All tests were performed at ionic strength 0.155 in the presence of either Hepes. NaOH or Imidazole. HCl buffer, pH 7.4 at 37 degrees. The faster clotting in the presence of Hepes buffer, therefore, is probably due, at least in part, to acceleration by Hepes of thrombin's enzymatic action on fibrinogen and/or of the polymerization of the fibrin monomers. Hepes may also have effects of other blood clotting reactions. Rates of hydrolysis of TAME or BAME (p-toluenesulfonyl- or benzoyl-L-arginine methyl ester) at pH 7.4 37 degrees by purified human bovine thrombin were essentially the same in 200 mM Hepes as in 250 mM Tris. HCl buffer (rates in Hepes. NaOH or Hepes. KOH buffers were compared with those in Tris. HCl plus NaCl for KCl). However, with purified bovine thrombokinase, rates of TAME hydrolysis in Hepes buffer were accelerated and rates of BAME hydrolysis slightly inhibited. Hepes, therefore, reacts with thrombokinase but whether this accelerates (or inhibits) the rate of converting prothrombin to thrombin remains to be determined. In addition, Hepes has an inhibitory effect on clotting since increasing the concentration of Hepes from 50 mM to 200 mM inhibits clotting in the PT, APTT and bovine thrombin-human plasma tests. Hepes buffer is being added to some plasmas and to some reagents used in clotting tests. It is, therefore, important to realize that its concentration must be monitored closely or erroneous results may be obtained in clotting tests and assays of clotting factors. The clotting times were the same in the presence of 50 mM Tris. HCl as in Imidazole. HCl buffers in APTT tests at three ionic strengths but they differed slightly in plasma-thrombin tests. Depending upon the ionic strength, 17 mM Barbital Sodium. HCl buffer inhibited APTT tests but accelerated plasma-thrombin tests. All the buffers tested, therefore, have individual effects on the clotting tests.