Haptoglobin-haemoglobin complex in human plasma inhibits endothelium dependent relaxation: evidence that endothelium derived relaxing factor acts as a local autocoid.

Endothelium dependent relaxation of isometrically mounted rabbit aortic strip preparations was rapidly inhibited by human plasma at dilutions down to 1:1000. Gel filtration and ion exchange chromatography were used to demonstrate that this inhibitory activity was present in fractions containing haptoglobin. Purified haptoglobin itself possessed no inhibitory action against endothelium dependent relaxation, but the haptoglobin-haemoglobin complex did, consistent with the documented ability of haemoglobin to inhibit this phenomenon. The concentration of haemoglobin normally bound to haptoglobin is sufficient to account for the inhibitory properties of human plasma. This suggests that endothelium derived relaxing factor exerts no downstream intravascular effect in vivo and thus that its physiological dilator role is that of a local autocoid acting on subjacent smooth muscle.

Distribution of non-plasma protein components in meconium from healthy and cystic fibrosis neonates.

The distribution of four antigens of alimentary tract origin was studied in meconium collected from healthy and cystic fibrosis (CF) neonates. Two antigens, carcinoembryonic antigen and a beta glycoprotein designated Mec 6, had significantly lower concentrations in the meconium from CF neonates compared with that from healthy full-term infants. As their concentrations were unrelated to whether or not the infants had had pancreatic insufficiency at birth, their measurement may be of value for screening. Their concentrations also depended on the gestational age of the newborn, and it is speculated that the CF lesion may affect the maturation of intestinal secretions.

Quantitative immunoelectrophoretic analysis of the plasma proteins in the sol phase of sputum from patients with chronic bronchitis.

An analysis of the plasma proteins in the sol phase of sputum was carried out using quantitative cross immunoelectrophoresis. The average concentrations of nine plasma proteins were estimated in the sol phase of sputum specimens from 30 patients with chronic bronchitis and the values were compared with the concentrations of these proteins in saliva and serum specimens from the same group of patients. The results showed that alpha(1) antichymotrypsin and IgA concentrations were higher in the sol phase of sputum than would be expected if their presence were due entirely to passive transudation.

Assay of serum immunoreactive trypsin in dried blood spots and the early detection of cystic fibrosis.

Immunoreactive trypsin (IRT) content in blood spots from 17 cystic fibrosis (CF) children born between 1975-80 were assayed with reagents from two commercial radioimmunoassay kits. The manufacturers' procedures were modified to allow assay of blood spot IRT. Half of the CF samples had a significantly raised IRT content compared to normal blood spots. Of the blood spots from five children whose meconium screening gave negative results, three or five (depending on the kit used) had a raised IRT content. On the other hand, less than 50% of children initially detected by meconium screening had raised blood spot IRT values. In our hands, analysis of blood spots for IRT does not consistently identify the newborn with CF and offers no advantage over other screening methods.

Meningococcal infection and proteolytic control.

Cascade enzyme inhibitors (C1-esterase inhibitor, C3b inactivator, antithrombin III) and other major proteolytic enzyme inhibitors (alpha 1 trypsin inhibitor, alpha 1 chymotrypsin inhibitor, inter-alpha-trypsin inhibitor, alpha 2 macroglobulin) as well as C3 and alpha 1 acid glycoprotein, have been examined in the sera of Nigerian patients suffering from meningococcal infection of varied severity. Patients with meningococcaemia had lower serum concentrations of important inhibitors than did patients with localised meningitic infection. Within the coccaemic group, those who died had the lowest values, notably of antithrombin III and alpha 2 macroglobulin (and also of C3). The clinical end-result of meningococcal infection may be related to the degree of disequilibrium of the linked system of proteolytic control induced by the meningococcal endotoxin.

Neonatal screening for cystic fibrosis in Wales and the West Midlands: 1. Evaluation of immunoreactive trypsin test.

A study programme was set up in Wales and the West Midlands to evaluate serum immunoreactive trypsin screening for cystic fibrosis in neonates using blood spots collected for metabolic screening. By screening half the blood spots from each area, it was hoped to generate two comparable groups of fibrocystic children; those detected by screening and those not screened who would be diagnosed clinically. Over almost three years, more than 120,000 specimens were screened and 37 infants detected with cystic fibrosis. Four additional fibrocystic patients were missed on screening: two had negative immunoreactive trypsin values, of which one had meconium ileus, and two, although giving initial positive tests, were negative on follow up. Excluding infants known to be at risk, comparison of the numbers of children detected in the screened and unscreened groups showed more than a two-fold difference in favour of the screened group. There may be a large number of undiagnosed fibrocystic patients in the general population.

Characterisation of Burkholderia cepacia from cystic fibrosis patients living in Wales by PCR ribotyping.

Polymerase chain reaction (PCR) ribotyping detects differences in the intergenic spacer between the 16S and 23SrRNA genes. This method was applied to Burkholderia cepacia isolates from 16 Welsh cystic fibrosis (CF) patients attending three different clinics. Amplification of the intergenic spacer followed by an additional digestion step with TaqI restriction endonuclease identified seven distinct electrophoretic patterns among the patient isolates. Each of the seven patterns was distinct from that of the so called "epidemic strain" commonly isolated from patients attending clinics elsewhere in the UK. Two environmental isolates from the hospital clinics and four NCTC reference strains gave different patterns. The simplicity of the method lends itself to use in a general microbiological laboratory.

A comparative study of Fusobacterium necrophorum strains from human and animal sources by phenotypic reactions, pyrolysis mass spectrometry and SDS-PAGE.

Fusobacterium necrophorum strains from human infection (21) were compared with strains from animals (17 biotype A, 2 biotype AB, 4 biotype B, 1 biotype unknown), and the type strain NCTC 10575 in conventional tests reaction patterns (CTRPs), SDS-PAGE and pyrolysis mass spectrometry (PMS). Classifications from the three approaches showed one major consensus group comprising all human strains, and another comprising animal biotype A strains. Animal biotype B strains and one animal strain, designated with some doubt to biotype A, were outliers of the consensus 'human strain' group. Again, animal biotype AB strains were outliers of the consensus 'animal biotype A group', as was the type strain, which was clearly atypical in conventional tests and PMS. Colonial and microscopic characters showed good discrimination between the major consensus groups. However, only haemagglutination and the API-ZYM leucine arylamidase of the biochemical tests discriminated well between these groups. The 'animal biotype A group' clearly corresponds to F. necrophorum subsp. necrophorum, but synonymy of F. necrophorum subsp. funduliforme with the group of human strains was less certain. The latter subspecies was described solely on the basis of animal strains, all of biotype B, but each of four animal biotype B strains in this study was an outlier of the 'human strain group' in one or more of the characterisation approaches. Strains of F. necrophorum causing human infection were clearly distinct from the biotype A strains commonly found in animal infection. This has implications for the validity of animal models of human necrobacillosis. In view of these differences, it would be useful to have a validated designation for strains causing human infection. However, it would be premature to assume that the definition of F. necrophorum subsp. funduliforme encompasses the human strains in the absence of confirmatory DNA-homology and 16S rRNA-sequencing studies.

Human red blood cells inhibit endothelium-derived relaxing factor (EDRF) activity.

We have used a bioassay-cascade system to investigate the inhibitory effects of human red blood cells on EDRF activity. The vascular smooth muscle relaxant effect of EDRF released from an endothelium-intact pig coronary artery by the calcium ionophore A23187 was inhibited by washed red cells. This inhibition of EDRF activity by red cells was similar to that expected from their haemoglobin content. This study provides further evidence that in vivo EDRF acts as a local autocoid.

The composition of a mucus glycoprotein from meconium of cystic fibrosis, healthy pre-term and full-term neonates.

A high molecular mass mucus glycoprotein fraction (molecular mass greater than 1 million) which is a major component of meconium mucin, has been isolated from individual specimens collected from 20 healthy full-term infants, 19 premature infants and 19 infants with proven cystic fibrosis. The mucus glycoprotein fraction isolated from cystic fibrosis meconium had a significantly lower saccharide content than that isolated from specimens from healthy full-term infants but had a similar composition to that isolated from meconium of premature infants, gestational age 28-32 weeks. The composition of the glycoprotein fraction from the meconium of infants, gestational age 32-36 weeks, lay between that from cystic fibrosis and full-term. There is therefore a change or 'maturation' of epithelial mucin during gestation and it is hypothesised that the cystic fibrosis genetic lesion affects the maturation of epithelial secretion, resulting in the abnormal exocrine secretion associated with the disease.

Screening for cystic fibrosis by analysis of meconium for albumin and protease inhibitors.

A qualitative method of detecting elevated meconium protein concentration was compared with a method of determining meconium albumin concentration by electroimmunoassay since elevated meconium protein levels can indicate pancreatic insufficiency caused by cystic fibrosis. Between 5 and 10 per 1000 healthy infants passed meconium specimens that gave a false positive reaction with the Boehringer Mannheim test strip and contained a greater than expected concentration of albumin. It was possible to exclude pancreatic insufficiency in all of these children by determining the ratio, albumin : alpha1-antitrypsin in meconium and subsequent faecal specimens, since it was found that values of this ratio in excess of 2.0 suggested pancreatic insufficiency of the type associated with cystic fibrosis. Three of 14 neonates with subsequently proven cystic fibrosis yielded meconium specimens giving negative test strip results and low albumin concentrations. In two of these patients, the ratio, albumin : alpha1-antitrypsin in the meconium was within normal limits but, within two months of birth, the albumin : alpha1-antitrypsin ratio in the faeces of both children was greater than 3.0 suggesting that pancreatic insufficiency had developed.

Cystic fibrosis-related deaths in infancy and the effect of newborn screening.

Although newborn screening for cystic fibrosis (CF) is widely advocated, hard evidence in its favor is difficult to obtain, partly because of a dramatically improved life expectancy. Between 1985--1989 infants, born in Wales and the West Midlands were randomized to newborn CF screening by heel-prick immunoreactive trypsin (IRT) measurement or diagnosis by clinical presentation. Eligible children with CF who died in the first 5 years of life were identified from the local pediatricians and from the National UK CF Survey. In all, 230,076 infants were randomized to be screened, while 234,510 were unscreened. One hundred seventy-six CF children were identified, of whom 7 died in the first 5 years of life, 3 having presented with meconium ileus. Median age of diagnosis in the screened group was 8 weeks. On an intention to treat analysis, all 4 nonmeconium ileus-related deaths occurred in the unscreened group (Fisher's exact test, P < 0.05). However, the clinical presentation of 2 of these infants led to them being diagnosed prior to 8 weeks, i.e., earlier than would have been likely by screening. In conclusion, newborn screening has the potential to decrease infant CF deaths, but if it is to be successful, identification and treatment must occur as soon as possible after birth.

Transmission and prevalence of Burkholderia cepacia in Welsh cystic fibrosis patients.

From 1987 to 1994, 16 of 162 cystic fibrosis (CF) patients attending CF clinics at three different hospitals in South Wales, U.K. were found to have respiratory secretions colonized with Burkholderia cepacia (B. cepacia). Bacteriological typing by polymerase chain reaction (PCR) ribotyping demonstrated seven strains of B. cepacia among these 16 CF patients. This typing confirmed that cross-infection was the mechanism of colonization in six of the nine patients who were colonized at the paediatric CF clinic at the University Hospital of Wales in Cardiff, and in three of the six patients who were colonized at the adult CF clinic at Llandough Hospital in Cardiff (cross-infection rate nine of 16 patients or 56%). A search was made for a nosocomial source, with screening of wards and clinics. Swabs from fomites produced four positive cultures for B. cepacia. Two isolates had the same PCR ribotype as that of the previous CF room occupant. To establish prevalence of B. cepacia among CF children living throughout Wales, respiratory secretions were cultured from 151 of 186 CF children (age < 16 years). This failed to demonstrate B. cepacia colonization other than in the CF patients already identified.

The effect of secretory immunoglobulin A on the in-vitro adherence of the yeast Candida albicans to human oral epithelial cells.

Secretory immunoglobulin A (s-IgA) isolated from human breast milk inhibited the adherence of C. albicans to human oral epithelial cells. This inhibitory effect of s-IgA was maximal at 1 1/2 hours, it was concentration-dependent and was still detectable at subagglutinating antibody concentrations. The inhibitory action of s-IgA was due to its content of specific candidal antibody. Non-specifically bound s-IgA enhanced adherence of the yeast and presumably tends to impair the immune disposal of candida by specific antibody. The reduced adherence of candida pre-treated with 0.4 per cent formol saline at a concentration which kills the organism but leaves its surface antigens intact suggests that, although dead organisms may form an initial loose attachment to the epithelial surface, only viable organisms bind irreversibly. The specific-s-IgA appears to block surface sites on C. albicans involved in epithelial adherence but this action of s-IgA cannot be attributed solely to its agglutinating properties.

Enzyme immunoassay of immunoreactive trypsin in serum and blood spots.

An enzyme immunoassay method for the assay of serum immunoreactive trypsin (IRT) is described. The method is a two site binding assay carried out on microtitre plates as the solid phase. Wells were coated with affinity purified anti-human trypsin and biotinylated anti-trypsin and avidin-beta-galactosidase were used as the second antibody and detection system respectively. The assay was sensitive enough to determine IRT concentrations in either serum or dried blood spots. A good correlation was obtained when the method was compared with the Hoechst radioimmunoassay method.

Screening for cystic fibrosis.

Practicable methods are now available for whole population screening of neonates for cystic fibrosis. Although diagnosis and treatment of the disease from birth has not yet been unequivocally shown to improve prognosis, existing evidence suggests that this is likely. Further ethical reasons are proposed in support of neonatal diagnosis and early treatment. The development of tests for prenatal diagnosis and carrier detection is under active investigation, and raises ethical problems for heterozygotes and their medical advisers. The heavy financial and emotional burden this disease imposes on the patient and the family should not be underestimated when policy decisions are made.