Beef Insulin Research Articles

Forensic Hypoglycaemia & Neuroglycopenia — A Clinical Legal Social Endocrinology Challenge for 2024, Forensic Law in Hypoglycaemia 4

Forensic Hypoglycaemia & Neuroglycopenia — A Clinical Legal Social Endocrinology Challenge for 2024, Forensic Law in Hypoglycaemia 4

In vitro Study of Antioxidant, Antidiabetic and Antimicrobial Activities of <i>Solanum Torvum</i> Root

Diabetes mellitus is a metabolic disorder with characteristics of multiple aetiology of chronic hyperglycaemia. These effects include dysfunction and long-term degeneration of various organs. Characteristics of diabetes mellitus include polyuria, blurred vision, thirst, and weight loss. Many types of insulin are available, for example porcine insulin, beef insulin, and human insulin. Weight gain and hypoglycaemia are the most adverse effects of using insulin. Insulin binds to sulfonylurea receptors on β-cell plasma membranes. In this study, ethanolic extract of Solanum torvum root was used to study ATP-sensitive potassium channels, which lead to depolarized cell membranes. The ethanolic extract was also analysed for antioxidant activities by free radical 1,1-diphenyl-2-picryl hydrazyl (DPPH) scavenging method. The different concentration of extracts exhibited varying result of radical scavenging activity using DPPH as substrate. The root of Solanum torvum exhibited significant α-amylase (79.56%) and α-glucosidase (80.58%) inhibitory activities at the concentration 100 μg/ml respectively when compared with standard acarbose drug. It also showed a good antioxidant activity with 65.47% when compared with standard ascorbic acid. The ethanolic extract of Solanum torvum root has a potential antimicrobial activity against Staphylococcus aureus and Candida tropicalis. Thus, Solanum torvum is a better source for natural antioxidant, anti-diabetic and anti-microbial activities due to presence of natural compounds in it.

Determination of the Potency of Extracted, Purified and Formulated Insulin from the Pancreatic Organs of the Sudanese Beef Cattle

The treatment of type 1 diabetes is mainly dependent on insulin therapy and current formulated insulin formulations are used for its control all over the world. The presented study was designed to evaluate the potency of extracted, purified and formulated insulin from the pancreatic organs of the Sudanese beef cattle. Twenty healthy rabbits were used to conduct the study following subcutaneous administration of the sample insulin, to determine the hypoglycemic effect and to analyze the potency of the testing insulin by the hypoglycemic seizure method, blood sugar method and glucose enzymatic colorimetric test (GOD-PAP) respectively. The potency of the injected insulin samples was estimated by comparing the variation in blood glucose levels produced in the treated animals with that produced by a standard insulin preparation under the suitable conditions of the blood sugar method. The results revealed that the potency of the testing beef insulin samples was slightly higher (i.e., 2.2 USP units/ml, 9%) compared to the standard and assumed potency of the prepared insulin preparations (i.e., 1-2 USP units/ml) which indicated that the solvents and diluents used to prepare the assay dilution might be of higher potency and must be diluted to such an extent that the testing insulin potency must be compatible with the standard dilutions. Furthermore, to determine the choice of an assay to analyze the potency of insulin preparations, not only the accuracy of the result but also the purpose for which the test is to be used and the time limit must be taken into consideration.

Circulating insulin antibodies during therapy with highly purified neutrally buffered insulins including desphe insulins. Report of a prospective study with 40 cases.

Circulating insulin binding capacity was studied for 10 to 24 months in forty diabetic patients who were being treated with insulin for the first time. They received either neutral highly purified pork insulin, bovine or porcine desphe insulin containing formulations, semisynthetic human insulin, or "conventional" acid chromatographed bovine surfen insulin over the entire duration of the study. In only 6 of the 23 patients on pork insulin, porcine desphe insulin, or human insulin were minimal antibody concentrations (less than or equal to 2 IU/l) observed. The bovine desphe insulin containing beef insulin, was markedly more immunogenic: only 3 out of 12 patients did not produce any antibodies. All five patients treated with bovine surfen insulin reacted with a strong antibody formation, which was, on average, six times as high as that in the patients on bovine desphe insulin (approximately 90 vs 14 IU/l). According to these results, bovine desphe insulins should be considered more valuable in insulin therapy than formulations of unmodified beef or of mixed beef/pork insulins.

Molecular basis of antigen recognition by insulin specific T cell receptor

The TCR α/β chains recognize antigen peptides bound to the groove of the MHC class II molecule. The crystal structure analyses of the TCR/peptide/MHC class II complexes have revealed that the Vα chains play a significant role in antigen recognition. However, molecular details which amino acid residues of the Vα chain are able to contribute to fine antigen specificity are not clearly understood. Previously, we have classified a panel of T hybrids specific for insulin isotypes from different species of animals into four groups based on response profiles to these antigens. In particular, the group III (pork insulin ≥ beef insulin hierarchy of responsiveness) and IV (pork insulin ⪢ beef insulin hierarchy of responsiveness) T hybrids are interesting, since these TCR α/β chains with marked different antigen specificities demonstrate identical gene usages and very similar sequences. To specifically address the molecular requirements for insulin recognition by TCR, the TCR α and β chain genes from these group III and IV T hybrids were transfected into 58 α−β− T hybrid. The experiments suggested that CDR3α dictates the fine antigen specificity. Then, we have introduced a series of mutations into position 95 of CDR3α. The mutation experiments clearly indicated that position 95α determines the antigen specificity of the group III and IV T hybrids.

T-cell tolerance induction is normal in the (NZB x NZW)F1 murine model of systemic lupus erythematosus.

The (New Zealand black (NZB) x New Zealand white (NZW))F1 (NZB/W) mouse strain spontaneously develops an autoimmune disease characterized by anti-dsDNA antibody production and glomerulonephritis. Although evidence suggests that production of pathogenic autoantibodies is T-cell dependent, the immunological defects that lead to activation of these autoreactive T cells are unknown. In particular, it has not been resolved whether autoreactive T cells become activated in these mice because of a generalized defect in T-cell tolerance induction. Previous work has demonstrated that thymic and peripheral tolerance to strongly deleting antigens are intact in NZB/W mice. In this study we investigate whether these mice possess a more subtle T-cell tolerance defect. To this end, we have produced NZB/W mice carrying a transgene encoding beef insulin (BI) which is expressed at levels close to the threshold for T-cell tolerance induction. In BALB/c mice this transgene produces a profound but incomplete state of BI-specific T-cell tolerance, mediated predominantly by clonal anergy. Comparison of BI-specific tolerance in NZB/W, major histocompatibility complex (MHC)-matched (BALB/c x NZW)F1, and BALB/c BI-transgenic mice clearly demonstrates that T-cell tolerance induction is normal in NZB/W mice. The data suggest that the loss of T-cell tolerance that ultimately supports nephritogenic autoantibody production in NZB/W mice does not result from a generalized defect in T-cell tolerance, and by extension likely results from aberrant activation of specific autoreactive T cells.

Assessment of the Role of Determinant Selection in Genetic Control of the Immune Response to Insulin in H-2b Mice

The immune response to insulin is regulated by MHC class II genes. Immune response (Ir) gene-linked low responsiveness to protein Ags can be mediated by the low affinity of potential antigenic determinants for MHC molecules (determinant selection) or by the influence of MHC on the functional T cell repertoire. Strong evidence exists that determinant selection plays a key role in epitope immunodominance and Ir gene-linked unresponsiveness. However, the actual measurement of relative MHC-binding affinities of all potential peptides derived from well-characterized model Ags under Ir gene regulation has been very limited. We chose to take advantage of the simplicity of the structure of insulin to study the mechanism of Ir gene control in H-2b mice, which respond to beef insulin (BINS) but not pork insulin (PINS). Peptides from these proteins, including the immunodominant A(1-14) determinant, were observed to have similar affinities for purified IAb in binding experiments. Functional and biochemical experiments suggested that PINS and BINS are processed with similar efficiency. The T cell response to synthetic pork A(1-14) was considerably weaker than the response to the BINS peptide. We conclude that the poor immunogenicity of PINS in H-2b mice is a consequence of the T cell repertoire rather than differences in processing and presentation.

The Function of TGF-β-Mediated Innocent Bystander Suppression Associated with Physiological Self-tolerancein Vivo

Innocent bystander suppression has been demonstrated in experimental models of transplantation tolerance and oral tolerance. This phenomenon is associated with expression of cytokines such as TGF-β or/and type II cytokines (e.g., IL-4, IL-10). However, the mechanism responsible for bystander suppression is poorly understood, as is its role in antigen-specific self-tolerance. Here, we describe a series of investigations using an antigen coimmunization strategy to examine the outcome of bystander suppressionin vivoin a well-characterized physiological model, using beef insulin transgenic (BI-Tg) mice, for self-tolerance. Our results demonstrate that: (1) T-cell-mediated peripheral hyporesponsiveness, or CD4+regulatory type II Th cell-mediated adoptive transfer of peripheral hyporesponsiveness (defined by an ELISA antibody assay), is antigen-specific at induction but effector-nonspecific (bystander suppression) when the self-antigen (BI) and a control antigen (chicken ovalbumin) are coadministered in BI-Tg mice; (2) bystander suppression is manifest as a local and transient, rather than a systemic and long-term, phenomenon; (3) bystander suppression is both time and antigen dose dependent; and (4) anti-TGF-β Mab abolishes the effect of bystander suppressionin vivo.We suggest that TGF-β-mediated innocent bystander suppression associated with physiological self-tolerance thus produces no major biological consequence for general immune responsiveness. It may prevent the activation of auto(or cross)-reactive lymphocytes.

Multiple Levels of Regulation for Self-Tolerance in Beef Insulin Transgenic Mice

To characterize the mechanism(s) of tolerance toward soluble self-antigens (Ags), beef insulin (BI) transgenic (Tg) mice were generated in which the transgene was expressed in pancreatic β-cells. Our previous data showed that: (i) Ag-specific tolerance can be induced and/or maintained in peripheral T cells in thymectomized BI-Tg mice and (ii) CD4+Th2 regulatory T cells are involved in maintaining peripheral tolerance (by anti-BI antibody response). In this paper, we have further characterized the relationship of low levels of BI expression (10−10–10−11M) in Th1/Th2 activation. In addition, we have explored intrathymic events associated with tolerance to self-Ags not expressed in the thymus and/or to circulating self-Ags. Limiting dilution analysis showed that there was a significantly higher frequency of BI-specific Th2 cells in Tg mice with a corresponding higher frequency of Th1 cells in non-Tg mice. While there was no transgene expression in the thymus (by RT–PCR), independent studies showed that BI can be processed and presented in the Tg thymus, which correlated with the Ag-specific hyporesponsiveness of mature thymocyes detectedin vitro.High-dose rIL-2 (150 U/ml) was able to restorein vitroperipheral T cell response of Tg mice to levels comparable to those of the non-Tg control. Collectively, our data suggest that: (i) there is a differential activation of BI-specific Th1/Th2 cellsin vivoin the presence of low Ag concentration; (ii) the thymus may play a role in self-tolerance to Ags whose expression in adults is restricted to the periphery; and (iii) multiple levels of regulation such as thymic selection, peripheral anergy, and active suppression may be involved in tolerance to BI in BI-Tg mice.

Tolerance is overcome in beef insulin‐transgenic mice by activation of low‐affinity autoreactive T cells

To gain insight into the factors controlling the maintenance or loss of T cell self tolerance we produced beef insulin (BI)-transgenic BALB/c mice. Transgenic mice express BI under control of the human insulin promoter and secrete physiological amounts of beef insulin. Although these mice are tolerant to BI, as evidenced by the lack of insulin-specific IgG antibody production following intraperitoneal immunization, tolerance is not complete. Footpad immunization results in a weak antigen-specific T cell proliferative response, indicating the presence of self-reactive BI-specific T cell in the periphery. These T cells are functional in vivo, providing support for IgG1, IgG2a, and IgG2b BI-specific antibody production, but require higher higher concentrations of antigen than nontransgenic T cells (both in vivo and following recall responses in vitro) to become activated. In vitro, BI-specific T cell proliferation in BI-transgenic mice can be largely restored by addition of interleukin-2, indicating that a significant component of T cell tolerance is mediated by anergy. To characterize the autoreactive T cells that become activated when tolerance is broken, BI-specific T cell hybridomas were generated from transgenic mice and compared to a panel of hybridomas previously derived from nontransgenic BALB/c mice. The majority of BI-transgenic hybridomas recognized the immunodominant A1-14 beef insulin peptide but with lower avidity than BALB/c hybridomas. Consistent with this, none of the dominant T cell receptor rearrangements found in the BALB/c BI-specific T cell receptor repertoire were found in the transgenic hybridomas. These results indicate that, despite evidence for clonal inactivation of many BI-specific T cells in BI-transgenic mice, loss of tolerance results from activation of low-affinity antigen-specific T cells that appear to have escaped this process.

Evidence for Th2 cell‐mediated suppression of antibody responses in transgenic, beef insulin‐tolerant mice

Clonal deletion, anergy and suppression have all been considered mechanisms of immunological tolerance. Although adoptive transfer of immunosuppression has been shown to occur in the periphery, particularly for transplantation tolerance, it has proven difficult to characterize this phenomenon further, due to the lack of suppressor T cell clones. To characterize tolerance towards a physiological soluble antigen, we constructed beef insulin (BI) transgenic (Tg) BALB/c (H-2d) mice, in which the BI transgene is expressed in pancreatic beta cells. These Tg mice were tolerant to BI immunization at the level of both humoral and cell-mediated immune responses. Adoptive transfer of splenocytes from Tg mice into normal syngeneic BALB/c mice demonstrated that the reduction in antibody production is regulated by transferred T cells. The cytokine profile of T cell clones obtained after selection in vitro demonstrated dominant Th1 clones from normal non-Tg mice and dominant Th2 clones from Tg mice. Some Th2 clones (CD4+) from Tg mice produced significant suppression of antibody production after adoptive transfer into normal syngeneic BALB/c mice. These data confirm the existence of Th2 regulatory T cells in vivo in a model of peripheral tolerance to a physiological soluble antigen as a potential mechanism for self tolerance.

The structure of a complex of hexameric insulin and 4'-hydroxyacetanilide.

X-ray crystallographic studies have been carried out on human insulin crystals grown in the presence of 4'-hydroxyacetanilide (Tylenol) and show that this nontoxic phenolic derivative can induce the T-->R transition, producing a T3R3 hexamer. Two different crystals, grown under different conditions, are rhombohedral, space group R3, with cell constants a = 81.11, c = 37.97 and a = 80.88, c = 37.60 A. The T3R3 hexamer is symmetric, resulting from the presence of a crystallographic threefold axis, and the asymmetric unit consists of a TR dimer. Data to a resolution of 1.9 A were measured on a crystal from each of the two crystallizations and the structures have been refined to residuals of 0.168 and 0.173. The guest molecule is bound by the R-state monomer through the formation of two hydrogen bonds from the hydroxy group of Tylenol to the carbonyl oxygen and the nitrogen of A6 Cys and A11 Cys, respectively. Due to steric constraints of the phenolic binding site, the acetamide group of Tylenol is rotated approximately 50 degrees out of the plane of the phenyl group and the methyl group is cis; no hydrogen bonds exist between the acetamide group and the hexamer. Although the zinc ion, which is bound to the R-state trimer, has tetrahedral coordination in both structures, the T-state zinc is observed to have octahedral coordination in one structure but tetrahedral coordination in the other. The side chain of A10 Ile in the R-state monomer adopts a high-energy conformation as a result of close contact to a residue in an adjacent dimer and may explain in part the differences between therapeutic preparations of beef insulin, for which A10 is a Val residue, and human insulin.

Activation of autoreactive T-cell clones from NZB.H-2bm12 mice.

We have previously established H-2bm12-restricted autoreactive T-cell clones from NZB.H-2bm12 mice which induce in-vitro production of IgG anti-dsDNA antibodies by syngeneic B cells. However, the mechanism underlying the activation of autoreactive T cells is not clear. We have taken advantage of the existence of L cells which were co-transfected with the A alpha b gene and independent A beta genes comprising all the permutations of amino acid residues distinguishing A beta b from A beta bm12. Using this panel of L cells expressing recombinant I-A molecules, 6/6 autoreactive T-cell lines responded significantly to the FT7.2 L cell transfectant expressing wild-type I-Abm12 as well as control APC from NZB.H-2bm12 or B6.C-H-2bm12 mice, but not to the other recombinant L-cell transfectants or to allogeneic APC. The fixation of APC with paraformaldehyde prior to co-culture led to dramatically diminished reactivity of all the autoreactive cloned T-cell lines tested. Interestingly, the inability of FT7.2 L cells to induce T-cell activation after paraformaldehyde fixation could be reconstituted by the addition of B/monocyte cells, but not T cells, from NZB.H-2bm12, NZB.H-2b or NZB(H-2d) mice and to a significantly lesser extent, from C57BL/6 (H-2b) and BALB/c (H-2d) mice. Conversely, when treated with either mitomycin C or cycloheximide, before incubation with autoreactive T cells and fixed FT7.2 cells, the ability of spleen cells from NZB.H-2b mice to reconstitute reactivity was reduced. Controls for these observations included KLH-specific T-cell clones from NZB.H-2bm12 mice, the T-cell hybridoma H66.3.6.54 specific for beef insulin and restricted to I-Ab, the IBM026 hybridoma specific for OVA and restricted to I-Abm12, and the TH2.2 hybridoma, and I-Ab antigen-presenting cell line.

Immunological and Metabolic Responses of Patients with History of Antibody-Induced Beef Insulin Resistance to Treatment With Beef, Pork, Human, and Sulfated Beef Insulin

We evaluated immunological and metabolic responses during therapy with beef (B), pork (P), human (H, rDNA), and sulfated beef (SB) insulins in patients with insulin-antibody-mediated insulin resistance. A randomized double-blind sequential crossover study was performed with each insulin administered for 56 days unless dose reached 200 U/day or allergy developed. Participants were 26 individuals with history of B-P insulin dosage greater than or equal to 200 U/day and insulin binding capacities greater than 0.216 nM (30 mU/ml serum). Twenty-one participants completed the study. Insulin dosage/day, fasting plasma glucose, percentage HbA1, insulin antibody binding capacity (IABC), bound insulin (BI), percentage binding of 125I-labeled B, P, and H insulins, and receptor inhibition factor (RIF) were assessed. Mean insulin dosage (U/day) was significantly greater on B (88.9) than on P (29.2), H (29.4), or SB (29.6). On B, dosage increased in 12 individuals and reached 200 U/day in 6 individuals. Mean fasting plasma glucose (12.1 mM) and HbA1 (11%) were significantly higher on B than on P, H, and SB. Mean IABC, bound insulin, RIF, and percentage of B, P, and H bound were significantly higher on B than on P, H, and SB. Prolonged treatment with SB before entry into the study (greater than 5 wk) resulted in a blunted anamnestic response to B insulin. Rechallenge with B results in anamnestic immunological response and deterioration of metabolic control. SB, H, and P insulins have equivalent effects in patients with insulin antibody-mediated immunologic resistance.

Reduction of disulfide bonds during antigen processing: evidence from a thiol-dependent insulin determinant.

Previous studies have demonstrated that insulin, like other protein antigens, requires processing in metabolically active antigen-presenting cells (APC) before it can be recognized by class II-restricted T lymphocytes. Unlike many other proteins, insulin peptides of minimal size retain the requirement for antigen processing. We demonstrate that this requirement can be bypassed by incubation of insulin with reducing agents in the presence of aldehyde-fixed APC. Fixed APC treated in this way were able to stimulate I-Ab- and I-Ad-restricted T cell hybridomas. Data are presented that demonstrate that cloned and polyclonal T cells recognize a determinant within the NH2-terminal 14 residues of the beef insulin A chain with no requirement for B chain residues. The common feature among peptides capable of stimulating these cells in the presence of live APC is the chemical form of the cysteine thiol groups. Those forms that produce free thiols upon reduction are active, whereas those with irreversibly protected sulfhydryls are not. Functional experiments with fixed APC and competition binding experiments with purified I-Ad indicate that only A chain peptides with free thiols are able to stably associate with the peptide-binding site on class II in a form that is recognized by specific T cells. Our findings indicate that reduction of disulfide bonds is both necessary and sufficient for presentation of insulin to a major population of class II-restricted T cells. The results provide strong support for the hypothesis that protein disulfides can be reduced during physiologic antigen processing.

Successful Treatment of Unusual Case of Brittle Diabetes With Sulfated Beef Insulin

Successful Treatment of Unusual Case of Brittle Diabetes With Sulfated Beef Insulin

Presence of Insulin Autoantibodies as Regular Feature of Nondiabetic Repertoire of Immunity

With an ultrasensitive noncompetitive enzyme-linked immunosorbent assay (ELISA), we tested the hypothesis that the presence of insulin autoantibodies in nondiabetic individuals is a normal event. Plasma and peripheral blood mononuclear cells were obtained from 50 nondiabetic whites for determination of insulin autoantibodies by ELISA and radioimmunoassay (anti-insulin IgG [AI-IgG] and 125I-labeled insulin bound [%]), islet cell antibodies, anti-nuclear antibodies and rheumatoid factor, and HLA class II-type antigens (DR, DRw, and DQ). The range of 125I-insulin binding was significantly less than was seen in pretreatment sera from individuals with diabetes (from -0.4 to 0.4% vs. -0.8 to 7.7%, respectively, P = 0.001). Eighty-eight percent of these nondiabetic individuals had significant levels of AI-IgG with preferential binding to human insulin. The geometric mean of AI-IgG concentrations in individuals with significant levels was 180 pM. Binding to human insulin was seen in 88%, to pork insulin in 42%, and to beef insulin in 24% of individuals (P less than 0.001 overall; P less than 0.05 where more bound to pork than beef insulin). Binding of AI-IgG to human insulin-coated plates was substantially inhibited by preincubation with human insulin (median inhibition 57.6%) with little if any inhibition by glucagon, C-peptide, albumin, or IgG. Four individuals had highly specific human AI-IgG as shown by immunoaffinity studies. AI-IgGs were significantly higher in individuals with the HLA haplotype DR4,DRw53,DQ3 and lower in individuals with DR5,DRw52,DQ1 (P = 0.03 for both).(ABSTRACT TRUNCATED AT 250 WORDS)

Demonstration of Insulin Transformation Products in Insulin Vials by High-Performance Liquid Chromatography

To assess the importance of temperature, light, agitation, and regular withdrawal of insulin on the rates of appearance of three groups of insulin transformation products (ITPs) in vials of human and beef soluble insulin. Twelve insulin-treated patients (6 receiving human soluble insulin, 6 receiving beef soluble insulin) participated in the study. Reverse-phase high-performance liquid chromatography was used to measure ITP in serial samples from vials stored in various laboratory environments or issued to patients. Vials in the laboratory were analyzed on nine occasions over 26 wk; those issued to patients were analyzed weekly. Results were expressed as rates of change (% total protein/wk). Except for human insulin stored at 4 degrees C, sampling did not accelerate transformation. Storage at higher temperatures caused more rapid transformation to all ITP groups, and human insulin was more susceptible than beef insulin. Exposure to light accelerated transformation mainly to group 3 ITP. Fibrillation occurred in beef but not human insulin carried in a shirt pocket. Results from patients' vials were consistent with reported storage conditions. All three ITP groups form in human and beef soluble insulin. Their rate of production is a function of temperature and light exposure. Differences between the two insulins were probably due to differences in formulation. Patients should be discouraged from hoarding vials and storing insulin in direct sunlight. One or more ITPs could contribute to the residual immunogenicity of modern insulin preparations.

Insulin and C-peptide levels following oral administration of insulin in intestinal-enzyme protected capsules

1. 1. Crystalline beef insulin was administered orally in capsules composed of a methacrylic acid copolymer which prevented breakdown of the insulin by enteric and pancreatic peptidases. 2. 2. In studies performed in 3 individuals blood was sampled before oral ingestion of the insulin (40–144 units), and at 15 or 30 min intervals thereafter for 5.5 hr for measurement of immunoreactive insulin and C-peptide concentrations. 3. 3. Following the administration of oral insulin, plasma immunoreactive insulin concentrations became elevated 4–5 hr after ingestion. 4. 4. The rise in plasma insulin concentrations was associated with a corresponding fall in the concentration of C-peptide. 5. 5. The data suggest that this preparation of oral insulin can produce significant enteric absorption of the peptide, and that further investigation of agents that facilitate insulin absorption from the gut might render the use of methacrylic acid copolymer coated capsules a physiologically sound and a commercially feasible method of oral insulin administration.

Continuous subcutaneous insulin infusion (CSII) and insulin antibodies in rabbits

Using prepubertal male New Zealand White rabbits, continuous subcutaneous insulin infusion (CSII), delivered by either an external or an implantable infusion device, resulted in significantly higher insulin antibody (I-Ab) production than bolus injection (BII). We tested the influence during CSII of (1) the insulin species, (2) the insulin diluent, (3) the materials of which the infusion devices were made and (4) the incubation of insulin in a syringe on the backs of rabbits (‘sham-infusion’), with the following results: (1) beef and sulphated beef insulins produced high levels of I-Abs, while porcine and human insulins produced moderate levels; (2) with all insulins used, 0.9% NaCl and 0.9% NaCl with 24–26 mmol NaHCO 3 added, produced high levels of I-Ab. A buffer containing 0.7% NaCl, 0.136% sodium acetate trihydrate and 0.1% methyl- p-hydroxybenzoate and a buffer containing 16 mg/ml glycerol and 2 mg/ml phenol, produced highly significantly lower I-Abs ( P < 0.001); (3) insulin glass syringes produced much lower I-Ab levels than in standard polypropylene syringes and (4) polypropylene syringes in a ‘sham-infusion’ technique, resulted in intermediate levels of insulin antibodies [( P < 0.02) vs CSII; ( P < 0.005) vs BII]. Our data suggest that insulin immunogenicity is influenced by all four factors tested. We suggest that benefits of CSII therapy may be attenuated unless a best possible control of these factors is achieved.