Screening for cystic fibrosis.

During a 12 month period all New Zealand newborns were screened for cystic fibrosis using the Crossley test. Of 49 056 babies tested 522 had a raised blood immunoreactive trypsin level and follow-up testing indicated 19 persistent elevations. Clinical and laboratory studies indicated 14 cases of cystic fibrosis giving an apparent incidence of 1:3500.

Reduced Coxsackie antibody titres in type 1 (insulin-dependent) diabetic patients presenting during an outbreak of Coxsackie B3 and B4 infection.

The potential role of antecedent viral infection in the pathogenesis of Type 1 (insulin-dependent) diabetes was investigated by measuring antibody titres to several viruses in serum obtained at the time of diagnosis of diabetes. An outbreak of Coxsackie B4 infection followed by a wave of Coxsackie B3 and B5 infections occurred in Seattle during the time viral serology was obtained in the diabetic patients. Antibody titres to Cocksackie B5 and Influenza A and B viruses were comparable in diabetics and matched control subjects, but antibody titres to Cocksackie B3 and B4 were lower in the diabetics and a low antibody titre to Coxsackie B3/B4 was associated with a significantly increased relative risk of diabetes.

Antibodies to viruses and to pancreatic islets in nondiabetic and insulin-dependent diabetic patients.

Autoimmunity is frequently involved in the pathogenesis of insulin-dependent diabetes, and viral infections have been implicated in some cases. We have investigated the possibility that islet cells and viruses share antigenic determinants with the result that antiviral antibodies would cross-react with islet cells. Antibody titers to Coxsackie B2, B3, B4, and B5, Influenza A and B, and mumps viruses were compared with islet cell antibody (ICA) titers in newly diagnosed insulin-dependent diabetic patients and in some diabetic patients followed prospectively for 1 yr postdiagnosis. Nondiabetic patients, with culture-proven Coxsackie B4 infections and large rises in Coxsackie B4 antibody titers, were evaluated for islet cell antibodies. No relationship between ICA and viral antibody titers was found either in diabetic or nondiabetic patients. We conclude that it is unlikely that islet cells and the viruses tested share antigenic determinants and other mechanisms relating viral infection and autoimmunity in insulin-dependent diabetes must be sought.

Neonatal screening for cystic fibrosis, using immunoreactive trypsin assay in dried blood spots.

Our previous studies suggested that assay of immunoreactive trypsin (IRT) in dried blood spots might be a valuable neonatal screening test for cystic fibrosis (CF). We have developed a convenient, sensitive, human trypsin radioimmunoassay, which uses a 3-mm diameter disc of dried blood. The molecular species assayed in blood is trypsinogen. A retrospective study of 24 known cases of CF and appropriate controls confirmed that an elevated blood level of IRT is characteristic of all newborn CF infants, whether or not they have residual exocrine pancreatic function. IRT levels did, however, decline with time of sample storage. Guthrie cards from 5040 newborns were prospectively assayed: 2% of tests were reassayed because of elevated IRT. Thirty-three second samples (0.67% of the total) were requested, 32 were received, and 31 had normal IRT values. The baby with an elevated result had no clinical symptoms of CF at one month of age, normal stool trypsin activity, but 2 sweat tests gave grossly abnormal results. In contrast, a second infant in whom CF was diagnosed around the same time, had a similar 5-day blood spot IRT value, but severe clinical symptoms of CF and no stool trypsin activity. No false negatives are yet known. We conclude that blood spot IRT assay is a reliable and convenient neonatal screening test for CF.

Lymphocytotoxic antibodies and histocompatibility antigens in juvenile-onset diabetes mellitus.

Cold-reacting serum lymphocytotoxic antibodies (LCAs) were measured in sera from 230 insulin-dependent juvenile-onset diabetes mellitus (IDDM) patients and from 116 control subjects. LCAs were present in only 4% of control sera compared with 19% in IDDM patients. The most significant determinant of LCAs was time since onset of diabetes; within the first 12 mo, 55% of IDDM sera had LCAs, compared with 25% after one year and 15% after five years of diabetes. LCAs were absent in sera from patients with IDDM for 10 yr or more. Genetic factors were also implicated in susceptibility toi occurrence of LCAs. HLA antigen B8 and B18 were associated with an increased risk for LCAs, whereas HLA-B7 was associated with a decreased risk. The relative risk for LCAs in patients positive for HLA-B8 but not B7 was 2.3, compared with 0.0 in HLA-B7/B8 heterozygotes. In contrast, B7 did not provide protection from LCAs in B18/B7 IDDM patients. Properdin factor B (Bf) alleles, which are in linkage disequilibrium with alleles of the HLA-B locus, were also associated with LCAs, IDDM patients with alleles BfS1 or BfF hd a prevalence of LCAs of 7%, significantly less than the 39% in Bf-F1S or -F1 patients. LCAs were not identical or closely correlated to pancreatic islet cell antibodies. Our findings indicate genetic heterogeneity in, yet, another autoimmune process in IDDM.

Coxsackie B virus antibody responses in juvenile-onset diabetes mellitus.

A prospective serological study of patients with recently diagnosed juvenile-onset diabetes mellitus was carried out to determine neutralizing antibody responses to Coxsackie B viruses. The sera were tested with and without staphylococcal protein A absorption, to selectively ascertain whether specific IgM or IgG antibody predominated. Of eleven patients studied, ten had reciprocal antibody titres of sixteen or greater to at least one serotype. Three patients showed no reduction in titre in at least one serum sample after protein A absorption, suggesting a predominant IgM-specific primary response to a recent infection by a Coxsackie B virus. The findings support the possibility that at least some cases of juvenile-onset diabetes mellitus are closely related to Coxsackie B virus infections.

The incidence of juvenile diabetes mellitus in New Zealand.

The incidence of juvenile diabetes in New Zealand over a five year period (1968--1972) was determined from hospital admission data stored at the Department of Health, National Statistics Centre. The average annual incidence for persons under 20 years was 10.4 persons/100,000. There was no sex difference below 16 years, and the increased incidence among females 16--19 years could be attributed to pregnancy. There was a 1.4-fold higher incidence in the South Island than in the North Island. There were no regular seasonal trends. The incidence was constant between 1--9 years increasing to a sustained 2.2-fold higher level from 11 years. The absence of childhood peaks and the sustained higher incidence in adolescence is in contrast to European studies.

Dried-blood spot screening for cystic fibrosis in the newborn.

Serum-immunoreactive-trypsin (I.R.T.) was measured in children with cystic fibrosis (C.F.) and a variety of controls. In the first few months of life all C.F. children had a raised serum-I.R.T. A dried blood-spot assay for I.R.T. was established and has potential as a screening test for C.F. in the newborn.

Cystic-fibrosis screening in the newborn.

In a new method of testing stool samples from newborn babies for cystic fibrosis (C.F.), a colourless substrate, benzoyl-arginine-p-nitroanilide (B.A.P.N.A.), releases yellow p-nitroaniline when hydrolysed by trypsin. Samples from infants with C.F., who lack trypsin, give negligible colour. 2 infants with C.F. were detected among 2500 consecutive newborn babies tested. The incidence of false-positive results was 1.2% after the first specimen and 0.05% after the second specimen. A further refinement has reduced the positive rate to 0.1% after the first specimen (2000 samples). Tests on samples from 5 other older patients with untreated C.F. have yielded no evidence for false-negative results.

The effect of N-acylglucosamines on the biosynthesis and secretion of insulin in the rat.

The effects of N-acylglucosamines on insulin release have been studied. N-Acylglucosamines stimulated insulin release from rat islets in vitro only if a sub-stimulatory concentration of glucose was also present, and this secretory response was abolished by mannoheptulose. In perifused islets the rapidity of the secreotry response to N-acetyl-D-glucosamine was similar to that observed with D-glucose. Increasing acyl-chain length from N-acetyl- to N-hexanoyl-D-glucosamine impaired the secretory response; however, N-dichloroacetyl-D-glucosamine was a more potent stimulator of release than was N-acetyl-D-glucosamine. Polymers of N-acetyl-D-glucosamine containing two to six monomers linked alpha1-4 did not stimulate insulin release; glucosamine linked to dextran via a propionyl or hexanoyl spacer group was also without insulin-releasing ability. N-Acylglucosamines were also effective in eliciting insulin release in vivo when injected into conscious rats. At the dose used (86 mumol), N-acetylgucosamine elicited a rapid rise in plasma-insulin concentration; N-butyrylglucosamine was less effective, and there was little or no response to N-hexanoylglucosamine. The response to N-dichloroacetyl-glucosamine was greater than that to N-acetylglucosamine; an increase in plasma insulin concentration could be elicited by N-dichloroacetylglucosamine at a dose (17 mumol) at which neither glucose nor N-acetylglucosamine was effective. The secretory response to acetylglucosamine is not mediated by conversion into glucose. Rates of (pro)-insulin biosynthesis by rat islets have been measured (Pro)-insulin biosynthesis was stimulated by glucose, and this response was abolished by mannoheptulose. N-Acetylglucosamine also stimulated (pro)-insulin biosynthesis; this effect of N-acetylglucosamine did not require the presence of glucose, and was not abolished by mannoheptulose. It is concluded that there are differences in signal reception and/or transduction for the processes of insulin biosynthesis and release.

The effects of glucose, N-acetylglucosamine, glyceraldehyde and other sugars on insulin release in vivo.

The specificity for carbohydrates of insulin secretory responses in vivo was studies. Test sugars were injected via a left femoral vein cannula into conscious rats. Blood samples collected over the ensuing 60 min via a left femoral arterial cannula were assayed for plasma insulin and glucose, and, in some experiments, for N-acetyl glucosamine. Whereas L-glucose or saline produced no significant changes in plasma insulin or glucose concentrations, D-glucose, N-acetylglucosamine, D-glucosamine, fructose, D-glyceraldehyde and DL-glyceraldehyde were potent secretagogues. Simultaneous injection of mannoheptulose abolished the insulinortopic action of glucose and N-acetylglucosamine, but not of DL-glyceraldehyde. Fructose, glucosamine, and DL-glyceraldehyde induced hyperglycaemia, but peak insulin concentraions occurred before any change in plasma glucose concentration. No evidence was obtained for a stimulatory effect of galactose on insulin release. Infusion for 60 min of N-acetyglucosamine produced a sustained elevated plasma insulin concentration and significant hypoglycaemia. The present in vivo results agree with previous in vitro observations and could indicate a role for sugars other than glucose in the regulation of insulin release.

Insulin-like insulinase-resistant material, distinguishable from normal insulin, in juvenile diabetes.

This study compares some properties of the immunoreactive insulin-like material extracted from the urine of children with overt diabetes with that from normal children. Insulin-like species were fractionated by gel filtration and by isoelectric focusing and were tested for sensitivity to an insulin-specific degradative enzyme. Insulin concentration was measured by radioimmunoassay. The major insulin-like component from the urine of ten normal children and fifteen untreated juvenile diabetics and from the urine of four and the serum of one latent diabetics behaved (on gel filtration) as normal insulin, was sensitive to insulinase, and (in all cases studied) had an identical isoelectric point (resolution 0.1 pH units). A proportion of the immunoreactivity extracted from urine (0-4 per cent from normal children, 5-30 per cent from twelve of the thirteen nonobese untreated diabetic children) eluted from the gel filtration column before insulin. This material from diabetic urine was of two size classes, "proinsulin-like" and "mid-insulin," both resistant to degradation by insulinase. Insulinase-resistant immunoreactivity from one patient was analyzed by isoelectric focusing. Urine samples from two obese children with overt diabetes and four children with latent diabetes contained normal proportions (less than 4 per cent) of immunoreactive species larger than insulin. The possible nature and significance of the present insulinase-resistant species are briefly considered.