Search

Antibodies reactive with the C-peptide portion of proinsulin can be detected in the sera of patients on conventional bovine insulin regimes which include the soluble form. Direct and competitive binding studies demonstrate that C-peptide antibodies show species specificity but do not recognize free homologous C-peptide. This is likely to be due to conformational change following cleavage of C-peptide from the proinsulin molecule. Examination of sera from a large group of patients established on a standard insulin regime shows that C-peptide and insulin antibody levels do not correlate with each other and are likely to show different patterns of genetic control.

This is an interim report of a long term single-blind study of the effects of changing diabetic patients treated with highly purified porcine insulin to semi-synthetic human insulins of identical formulation. Twenty four insulin dependent diabetics were randomly allocated to continue with porcine insulin (n = 11) or human insulin (n = 13). There were no significant changes within the groups nor differences between the groups in mean preprandial capillary blood glucose, glycosylated haemoglobin or insulin dose during the first 24 weeks of the study. Insulin antibody levels remained low and did not differ between the groups. No local or systemic adverse reactions were observed. In this group of patients conversion to human insulin did not result in a change in diabetic control or insulin dose.

In a single blind randomised cross-over study, 40 patients were changed from ordinary bovine to highly purified porcine insulins for a period of 6 months. Half were later rechallenged with bovine insulin. Sequential determinations of IgG insulin binding capacity for bovine insulin were correlated with insulin dose and diabetic control. After changing to highly purified insulins the following correlations were observed between percentage change in insulin dose and change in insulin binding capacity: at 2 months r = 0.35 (p less than 0.05), at 4 months r = 0.38 (p less than 0.02) and at 6 months r = 0.37 (p less than 0.02). When the patients who showed substantial changes in HbA1 were removed from the analysis, the remaining 29 demonstrated a clearer relationship between these two variables (r = 0.56, p less than 0.01). Removal of patients with a low initial insulin binding capacity left 18 patients with stable diabetes, and changes in insulin binding capacity and insulin dose showed an even closer correlation for this group (r = 0.77, p less than 0.001). A similar degree of positive correlation was observed after rechallenge with bovine insulin. We conclude that the level of circulating insulin antibody affects the dose of insulin required to maintain stable diabetic control.

Seventy-nine patients were observed prospectively during their initial period of treatment with conventional bovine insulins. Insulin antibody levels 6 months after starting insulin therapy did not correlate with age, gender or beta cell function at onset of treatment. Patients who required soluble insulin in addition to isophane insulin developed higher levels of insulin antibody. Patients bearing the HLA-B8, DR3 and C4AQO alleles had lower levels of insulin antibody, whereas those bearing DR7 produced significantly higher levels. Other alleles at the C4A, C4B, C2, factor B or Gm loci did not appear to have a significant effect on insulin antibody production. The hyporesponsiveness of B8/DR3/ C4AQO -positive individuals probably reflects a non-specific abnormality of immunity whereas the enhanced responsiveness of those positive for DR7 suggests the presence of a specific immune response gene for insulin.

We have developed an in vitro priming assay in which peripheral blood lymphocytes from normal subjects are primed with insulin for 14 days prior to challenge with insulin in conjunction with autologous antigen-presenting cells for a further 5 days. Sheep, beef and pork insulins possess, respectively, four, three and one amino acid differences from the human molecule (out of a total of 51 residues) and the magnitude of the response to priming correlates with the degree of sequence variation. Although human insulin produces little response, priming with heterologous insulins readily induces auto-immunization on secondary challenge. The response to porcine priming was enhanced if the secondary cultures were challenged with bovine or ovine insulin, i.e., a heteroclitic response was observed. Individual donors differ in their response to priming and high responders possess the HLA-DR7 glycoprotein more frequently than low responders. This is in keeping with previous studies on antibody production in vivo and probably relates to the ease with which individual class II glycoproteins complex with processed antigen and stimulate T cells. This method has considerable potential for screening novel insulin molecules and formulations and should facilitate the mapping of helper and suppressor epitopes as well as the identification of agretopes involved in the presentation of insulin to T cells.

A rise in blood glucose concentration at the end of the night, and consequent morning hyperglycaemia, are well recognized events in some diabetic patients. In 94 patients on twice daily insulin injections we have examined the prevalence and extent of morning hyperglycaemia, and its relation to control, insulin therapy, and insulin antibody levels. Blood glucose reached the highest level of the day before or after breakfast in 83% of patients, and in 50% this value was 2 mmol/l greater than any other time of day. Patients with higher fasting concentrations did not have worse blood glucose control over the rest of the day. No correlation was found between fasting blood glucose concentrations and the evening dose of intermediate acting insulin or the level of insulin antibodies. No consistent change in fasting blood glucose concentrations occurred with changes in antibody levels in patients switched between pork and beef insulin. Morning hyperglycaemia was as common with both insulin species. Pre- and post-breakfast hyperglycaemia is common and significant in insulin-treated diabetic patients. It is not directly related to diabetic control at other times of the day, and is independent of insulin species and insulin antibody levels.

24 diabetic patients stabilised on conventional bovine insulins and possessing insulin antibodies underwent a study of the immunological and clinical consequences of changes in both purity and species of their insulin preparations. After a 2-month run-in period patients were treated for 3 consecutive 4-month periods with (a) purified bovine insulin (20-40 ppm proinsulin), (b) highly purified porcine insulin, and (c) semisynthetic human insulin, without elective dose changes. Mean insulin antibody levels changed little on purified bovine insulin (22.2 leads to 23.4 micrograms/l) but fell on highly purified porcine (23.4 leads to 12.9 micrograms/l) and remained much the same on Semi-Synthetic human insulin. In contrast, C-peptide antibodies fell significantly and continuously throughout the study. The slower rate of fall in C-peptide antibody levels is likely to be due to the prolonged half-life of circulating exogenous proinsulin in the presence of insulin antibody. Although insulin dose remained constant the incidence of hypoglycaemic episodes did not increase and glycosylated haemoglobin levels rose significantly when patients were on porcine insulin. The deterioration in diabetic control may have been due to greater temporal mismatch between insulin needs and insulin availability with pork or human insulin than with beef insulins, and to reduced insulin antibody levels. The use of purer insulins which more closely resemble the human form can cause a significant reduction in levels of insulin and C-peptide antibodies. Although these changes may have other benefits they do not necessarily produce better diabetic control. Subcutaneous insulin regimens need to be tailored to the individual patient and, indirectly, to his antibody status.

Twenty-six Type 1 diabetic patients previously treated for 10-20 months with twice daily conventional bovine isophane insulin (containing at least 1000 ppm proinsulin) were changed to highly purified (less than 1 ppm proinsulin) bovine isophane for 6 months (Switch group). Insulin antibody levels fell significantly from a geometric mean of 14.9 to 9.1 micrograms/l. Thirty-two patients with newly diagnosed Type 1 diabetes were treated with the same highly purified bovine isophane insulin twice daily for 6 months (Starter group). Their insulin antibody levels rose from a geometric mean of 1.9 to 8.2 micrograms/l in contrast to values of 1.4 rising to 16.3 micrograms/l in an age and sex matched historical control group treated from diagnosis only with twice daily conventional bovine isophane insulin. Lipoatrophy at injection sites developed in three (9%) in the Starter group treated with highly purified bovine isophane compared to 7 (22%) of those on conventional bovine isophane. Insulin dose and diabetic control did not differ between the groups. Starter and Switch groups were subsequently treated with semi-synthetic human isophane insulin for 6 months during which insulin antibody levels fell significantly from a geometric mean of 8.5 to 4.4 micrograms/l (p less than 0.001). We conclude that bovine insulin purified to less than 1 ppm proinsulin is significantly less immunogenic than its conventional proinsulin contaminated counterpart but even at this level of purity is still more immunogenic than human insulin of equivalent purity.

Insulin antibodies are detectable in the sera of most patients within 3-4 months of starting treatment with conventional bovine insulins. Various factors determine the immunogenicity of insulin preparations including the genetic background of the recipient. This causes wide variation in response. Solid phase absorption studies as well as competitive binding in fluid phase indicate that the antibodies formed are almost always specific for determinants shared by the endogenous human molecule. This has important implications for the metabolic effect of insulin antibodies in vivo as well as for mechanisms of autoantibody production in man.

Semisynthetic human insulin and highly purified porcine insulin were compared in a double blind crossover study in 21 diabetic children. Glycosylated haemoglobin values at the end of four month treatment periods were higher after treatment with human insulin than after treatment with porcine insulin (mean 15.7% (SD 2.3%) v 14.2% (2.3%); p less than 0.01). Higher fasting blood glucose concentrations occurred during treatment with human insulin than with porcine insulin (mean 12.0 (SD 2.1) v 11.0 (2.4) mmol/1; mean 216 (SD 38) v 198 (43) mg/100 ml; p less than 0.05), but there were no significant differences at other time points during the day. The incidence of hypoglycaemia was similar for both treatment groups. Concentrations of antibody reactive with porcine and human insulins were similar for the two treatment groups, although greater fluctuation was observed in the amount of antibody reactive with human insulin. Semisynthetic human insulin is safe and effective in diabetic children, although further work is needed to devise regimens which achieve optimal blood glucose control.

Skin biopsy samples from 14 diabetic patients with lipoatrophy at injection sites and from five insulin-treated diabetic patients without such lipoatrophy (controls) were examined by immunofluorescence for the deposition of immunological components. Also sera from 13 of the patients with lipoatrophy and from all of the controls were assayed for insulin-binding capacity. Biopsy samples from the edge of lipoatrophic areas (eight cases) invariably showed abnormal deposition of immunological components in dermal vessel walls, whereas no such deposition was seen in the control samples. Mean serum insulin-binding capacity was 33.1 microgram/l in the patients with lipoatrophy compared with only 4.6 microgram/l in the controls. These findings suggest that insulin-induced lipoatrophy results from the local formation of immune complexes, complement fixation, and release of inflammatory mediators from the cellular infiltrate.

We have evaluated the clinical and immunogenetic features of 100 consecutive patients presenting to an adult diabetic clinic who were judged clinically to need insulin therapy but were not sufficiently ill to be admitted to hospital. Over this same period 15 newly diagnosed patients (aged 13-70 years) were started on insulin as in-patients of whom ten were in ketoacidosis. The 100 out-patients, aged 11-75 years at the time of starting insulin, were followed for at least a year. Fifty-six had islet cell antibodies and/or were heterozygous for HLA DR3 and DR4 (Group A) whereas 44 had neither of these markers (Group B). Islet cell antibodies and/or DR3, DR4 heterozygosity were most common in the 70 patients diagnosed below the age of 40 years but were also found in older patients. Patients in Group A were significantly younger at diagnosis (29 vs. 43 years), had a shorter duration of symptoms (17 vs. 61 weeks), were more likely to have ketonuria, and had a lower random C-peptide level at diagnosis (0.2 vs. 0.31 nmol/l). The two groups could not be distinguished by weight, haemogloblin A1 or blood glucose at diagnosis or by diabetic control or insulin dose after one year. The National Diabetes Data Group (NDDG) definition of insulin dependence stresses the importance of HLA types and islet cell antibodies although we found their prevalence to be low in the 30 patients diagnosed over 40 years who clinically were indistinguishable from the younger patients.(ABSTRACT TRUNCATED AT 250 WORDS)

The antigenicity and efficacy of semi-synthetic human and pancreatic pork insulins have been compared in a double-blind double cross-over study in 96 insulin-treated diabetic patients. Transfer from pork to human insulin was associated with a 1.2 +/- 0.5 mmol/l deterioration (p less than 0.05) in the fasting blood glucose level, while the opposite change caused a 1.1 +/- 0.5 mmol/l improvement (p less than 0.05). After 4 months treatment, glycosylated haemoglobin levels were lower on pork (11.1 +/- 0.3%) than on human (11.7 +/- 0.3% p less than 0.01) insulin. The incidence of hypoglycaemia was similar with the two insulins. IgG insulin antibody levels were identical after human insulin treatment (5.7 +/- 0.4 micrograms/l) compared to pork insulin treatment (5.9 +/- 0.5 micrograms/l). Patients with high levels of antibodies (greater than 10 micrograms/l) showed a similar reduction in level when switched to either species of highly purified insulin. The deterioration in fasting blood glucose control is consistent with similar reports for biosynthetic human insulin and suggests, in the absence of changes in insulin antibody levels, a small but clinically significant pharmacokinetic difference between human and pork insulin.