In vitro priming of human lymphocytes to heterologous insulins.

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.

Light scattering and absorption--developments in immunology.

Fluid-phase immunoprecipitation analysis has undergone two distinct paths of development. One path utilizes existing absorptiometric equipment while the other relies on apparatus constructed specifically for the measurement of scattered light. The principles and instrumentation employed in light scattering and absorption techniques are reviewed with special reference to their applications in immunology. The main features of available analysers are summarised in the table. Specialised equipment is becoming increasingly sophisticated and expensive and it is important to assess critically the benefits, if any, that such apparatus may offer compared with more conventional devices. The latter are often capable of performing the same function with greater versatility. A good quality bench spectrophotometer is a more appropriate multi-purpose laboratory instrument than a specialised nephelometer with its more limited application. The equipment of choice for automated immunochemical protein quantitation, however, is the centrifugal analyser. The capital outlay is compensated for by high throughput, development potential and reduced consumption of reagents (i.e. antisera).

An immunochemical method for the quantitation of insulin antibodies.

A 125I-labelled insulin binding assay is described in which IgG antibody is precipitated by the addition of an optimal concentration of second antibody. Other features include the removal of unlabelled insulin from test sera prior to assay and the use of 22Na as a volume marker. This approach overcomes problems associated with previous assays for insulin antibodies. Clear differences are seen in the IgG insulin binding capacity (IBC) of sera from patients with insulin resistance and injection site lipo-atrophy when compared with insulin-treated diabetics who lack such complications. The precision and flexibility of this technique make it particularly suitable for studies of the immune response to different species and forms of insulin.

Quantitation of human total IgG, kappa IgG and lambda IgG in serum using monoclonal antibodies.

Monoclonal antibodies to human IgG have been applied, in parallel with polyclonal antisera, to the routine quantitation of human IgG. Two monoclonal antibodies directed against spatially distinct epitopes have been used in combination to form insoluble complexes exhibiting turbidity. Quantitation was performed using a centrifugal fast analyser and a correlation coefficient of 0.979 was obtained for the two estimations. The technique has been further developed to allow the separate quantitation of kappa IgG and lambda IgG in whole serum.

A flow cytometric method for the measurement of phagocytosis by polymorphonuclear leucocytes.

A new method for the measurement of phagocytosis of Candida albicans by human polymorphonuclear leucocytes (PMN) is described using a fluorescence activated cell sorter. We have used acridine orange to discriminate between PMN which have internalised yeast particles and those which have not. This method allows accurate measurement of particle phagocytosis as an event distinct from particle adherence. It also permits detailed examination of the kinetics of phagocytosis, the study of which is likely to be of value in the investigation of diseases where abnormalities of PMN function are suspected.

Transplantation, immunosuppression and plasmapheresis in Goodpasture's syndrome.

Renal transplantation in the face of circulating anti-glomerular basement membrane (GBM) antibody can lead to recurrent glomerulonephritis. The severity of the recurrence may be related to the level of antibody present at the time of transplantation. We describe a patient with Goodpasture's Syndrome and moderate circulating anti-GBM antibody activity who received a renal transplant, followed by plasmapheresis and immunosuppression. The graft has functioned well for almost two years associated with a continued reduction in levels of circulating anti-GBM antibody.

Restorative margin placement and periodontal health.

Subgingival restorative margins are associated with the development of plaque-related inflammatory periodontal disease, primarily because of a shift in the subgingival microflora from a profile associated with health to one associated with disease. The degree and extent of the marginal inflammation is influenced by four factors: failure to maintain proper emergence profile, inability to adequately finish and/or close subgingival margins, placement of subgingival margins in an area with minimum to no attached gingiva, and violation of the biologic width. Supragingival margin placement is the location of choice for all restorative margins to avoid iatrogenic periodontal disease. However, consideration of these four factors will help reduce the adverse impact of restorative margins that must be carried subgingivally.

Development of clinical immunology and allergy in the UK--common problems and solutions.

Despite major advances in immunological science there is comparatively little expertise in clinical immunology in British medical schools and the health service. There is no logical reason why clinical immunology and allergy should be considered separately and a joint approach toward the correction of these deficiencies is required. The most immediate service needs are for the appointment of more laboratory-based consultant immunologists to develop and coordinate clinical services in each centre in conjunction with organ-based specialists who should receive specific training in immunology. Key developments in the science and technology of immunology point toward novel ways of diagnosing, treating, and preventing immunological disorders. Academic units have an important role in exploiting these basic advances as speedily and effectively as possible and teaching contemporary immunology at undergraduate and postgraduate levels. A reduction in the morbidity of any of the common immunologically-mediated diseases would produce considerably savings for the health service.

Insulin antibodies induced by bovine insulin therapy.

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.

C-peptide antibodies induced by bovine insulin therapy.

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.

Neutrophil phagocytosis and killing in insulin-dependent diabetes.

Neutrophil phagocytosis and killing of Candida albicans were examined using a radiometric assay in 25 patients with insulin-dependent diabetes and 17 controls under various in vitro metabolic conditions. Glucose was present at 5, 10 and 50 mM, beta-hydroxybutyrate at 1, 5 and 20 mM and glucose with beta-hydroxybutyrate in combinations of 10 with 5 and 50 with 20 mM, respectively. Phagocytosis occurred at similar levels in diabetics and controls at all the glucose and beta-hydroxybutyrate concentrations used. The ability to neutrophils from diabetics to kill candida was inhibited by increased concentrations of glucose and beta-hydroxybutyrate, both independently and in combination. Candida killing (mean +/- s.e.) was 20 +/- 2.4, 19 +/- 2.3 and 13 +/- 2.7% at glucose concentrations of 5, 10 and 50 mM; and 20 +/- 3.4, 20 +/- 3 and 13 +/- 3% at beta-hydroxybutyrate concentrations of 1, 5 and 20 mM, respectively, and in glucose and beta-hydroxybutyrate combinations of 10 with 5 and 50 with 20 mM was 20 +/- 2.8 and 10 +/- 2.8%, respectively. Inhibition was not observed with control neutrophils. These data indicate that although phagocytosis occurs at similar levels in diabetics and controls, killing of candida by the diabetic neutrophil is impaired under conditions of hyperglycaemia and ketosis. The biochemical basis for this effect is discussed.

Neutrophil sorbitol production impairs oxidative killing in diabetes.

The mechanism whereby hyperglycaemia inhibits neutrophil function is unclear. We have examined neutrophil killing of Candida albicans in the presence of increased concentrations of glucose and galactose. Killing was abolished in 50 mmol/l glucose and 10 and 50 mmol/l galactose. The oxidative phase of killing was examined using lucigenin-enhanced chemiluminescence. An increase in glucose concentration from 5 to 50 mmol/l produced a fall in chemiluminescence output from 128.5 +/- 16.8 (mean +/- SE) to 82.2 +/- 9.8 mV min (a reduction of 36%). These data suggest the existence of aldose reductase activity in neutrophils and using gas chromatography we have demonstrated the presence of sorbitol in extracts of diabetic neutrophils. As oxidative killing and sorbitol production are both NADPH-dependent the inhibition of killing is likely to be due to competition for this electron donor. This abnormality of neutrophil function may aggravate various infections in the patient with diabetes.

Insulin-induced lipoatrophy: evidence for an immune pathogenesis.

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.

Human monocyte binding of homologous monomer and complexed IgG.

Human peripheral blood monocyte binding of homologous IgG has been studied using monomer and covalently cross-linked dimer and trimer. A competitive radioassay using enriched monocytes was employed to determine the association constant (Ka) and the number of receptor sites per cell. Monomer and dimer bound with very similar Ka values (7.15 X 10(7) and 7.14 X 10(7)LM-1) whereas a slightly higher figure was obtained with trimer (8.9 X 10(7)LM-1). The number of receptor sites for dimer and trimer (90.5 and 107.5 X 10(3)/cell, respectively) were much greater than for monomer (14.5 X 10(3)/cell). Inhibition of dimer and trimer binding by monomer gave heterogenous Scatchard plots, each with two components. These findings suggest that human peripheral blood monocytes possess two types of Fc receptor, one of high affinity binding monomer and complexes and one of low affinity which predominantly binds complexes.

The stimulation of superoxide anion production in guinea-pig peritoneal macrophages and neutrophils by phorbol myristate acetate, opsonized zymosan and IgG2-containing soluble immune complexes.

The kinetics of superoxide anion production in guinea-pig peritoneal macrophages and neutrophils were determined following in vitro stimulation with phorbol myristate acetate (PMA), opsonized zymosan (OZ) and soluble immune complexes of guinea-pig IgG2 (SIC). Superoxide production was recorded as chemiluminescence (CL) arising from the reductive cleavage of lucigenin. With PMA, both macrophages and neutrophils displayed a two-phase response consisting of a rapid initial burst of CL, which preceded ligand ingestion, followed by a plateau in the CL response which persisted for more than 30 min. By contrast, OZ induced a slow progressive increase in CL in both phagocytes which was consistent with the development of an oxidative burst concomitant with ingestion. The phagocytes differed in their responses to SIC, the macrophages displaying CL kinetics similar to those observed with PMA, whereas the neutrophils responded in the manner observed with OZ. The relationship between disparity in the patterns of macrophage and neutrophil CL responses to SIC and differences in their expression of Fc receptors for IgG2 (Coupland & Leslie, 1983) is discussed.

The effect of insulin antibodies on insulin dose and diabetic control.

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.

Congenital discoid lupus in the newborn.

A female infant, born to a 21-year-old mother with systemic lupus erythematosus, had cutaneous discoid lupus at birth. The lesions resolved spontaneously over the first few months and by the age of 1 year the infant's skin was normal. Other possible complications of this maternal disease are discussed and the need for caution in counselling mothers is recommended.

Smoke from laser surgery: is there a health hazard?

The composition of plume produced during carbon dioxide laser endoscopic treatment for endometriosis was examined to determine whether it represented a hazard to the surgical staff. A total of 32 plume samples were collected from 17 women undergoing laser laparoscopic treatment for endometriosis and/or adhesions. The smoke was found to consist of particles having a median aerodynamic diameter of 0.31 micron with a range of 0.10-0.80 micron. The size range has two consequences: 1) using a human red blood cell as a model for all cells, it can be stated with greater than 99.9999% certainty that no cell-size particles, including cancer cells, are present in the plume; 2) particles in this size range are too small to be effectively filtered by currently available surgical masks.