Potential of endogenous peptides to induce immunological reactions as observed in type I diabetes, a study in mice.

The initiation of the immunological processes leading to type I diabetes is still not understood. The potential of endogenous peptides to induce autoimmune reactions was investigated. Peptides generated in the beta-cell by proteolysis could bypass antigen processing by binding to MHC molecules. Selfreactive T and B lymphocytes could be activated by these MHC peptide complexes. The antibody production of peptide-induced B lymphocytes was investigated in mice. Insulin A chain, B chain, C-peptide or amylin were tested for potential induction of antibodies to antigens other than the immunizing peptide. Lymph node B lymphocytes were characterized with an avidin at solid phase ELISA-spot assay. In BALB/c mice insulin A chain induced more spots to B29biotin- and to B1biotinDOP insulin than to A chain itself (P < 0.01, each). Spots to insulin were not inhibited by insulin A chain. Spots to B1DOP insulin were not inhibited by A chain or insulin, excluding crossreaction. Inbred strains of mice with H-2d but not with H-2k or H-2b showed the effect. Application of A chain without adjuvant produced the effect. The antigens recognized by A chain-induced B lymphocytes had to be included in the natural IgM antibody repertoire of the spleen. The study supports the hypothesis that endogenous breakdown peptides can bypass antigen processing resulting in an autoreactive T-B cell interaction. A potential to induce type I diabetes could exist.

Insulin as a target antigen in autoimmune diabetes: a natural repertoire as the source of antibody response.

A solid-phase immunoenzymatic technique with B1- or B29-biotinylated insulin coupled to avidin-coated wells was used to characterize serum anti-insulin antibodies and to locate insulin antibody-producing B lymphocytes in different organs of mice. Low natural serum anti-insulin IgM and IgG antibodies were found in ten different healthy inbred strains of mice. Prediabetic non-obese diabetic (NOD) mice had significantly higher measurements than BALB/c mice (P < 0.05). Anti-insulin IgM antibody-producing B lymphocytes were found in bone marrow and spleen of NOD mice and healthy strains of mice, but not in peripheral lymph nodes, thymus, blood or pancreas. B29-fixed insulin was more frequently recognized than B1-fixed insulin. There was no relationship to the MHC or to other immune markers. IgG insulin antibody-producing cells were not detected. IgG insulin antibody-producing cells appeared in the draining lymph node and in the blood 10 days after immunization with insulin. IgM insulin-recognizing cells in the spleen were reduced in number during the same period (P < 0.05-0.01 for BALB/c, DBA2, B10.D2 and NOD), suggesting migration of these cells. This was tested by in vivo staining of spleens with the red-fluorescent membrane linker PKH-26 on day 7 after immunization. Cells from immunized lymph nodes were FACS-sorted on day 10. Insulin antibody-producing B lymphocytes with red-fluorescence were found, indicating a splenic origin. Examination of IgG subclasses showed preferential production of complement-fixing IgG2b in sera and lymph node cells of immunized NOD mice (P < 0.05 vs BALB/c).(ABSTRACT TRUNCATED AT 250 WORDS)

Influence of 6-week, 6 days per week, training on pituitary function in recreational athletes.

The influence on pituitary function of 6 weeks of training on 6 days a week was examined in six recreational athletes. Endurance training on a bicycle ergometer for 31-33 min was performed on 4 days each week at 90-96% (weeks 1-3) and 89-92% (weeks 4-6) of the 4 mmol lactate thresholds determined on day 0 and day 21, respectively, with interval training of 3-5 x 3-5 min in addition on 2 days a week at 117-127% and 115-110%, respectively. Determination of the serum hormone levels and a combined pituitary function test (200 micrograms thyrotropin releasing hormone (TRH), 100 micrograms gonadotrophin-releasing hormone (GnRH), 100 micrograms corticotrophin releasing hormone (CRH), 50 micrograms growth hormone releasing hormone (GHRH)) were made before training, after 6 weeks of training and after another 3 weeks of recovery. Training increased performance at 2 mmol lactate by 25%, at 4 mmol by 12%, and maximum performance by approximately 12%. The releasing hormone-stimulable prolactin, thyroid stimulating hormone (TSH) and somatotrophic hormone (STH) synthesis-secretion capacity remained unchanged, the adrenocorticotrophic hormone (ACTH) was increased after training. Cortisol release was reduced, follicle-stimulating hormone (FSH)-synthesis-secretion capacity was increased after training, and the luteinizing hormone (LH)-synthesis-secretion capacity reduced. This had no influence on base or exercise-induced serum hormone levels (cortisol, aldosterone, insulin, prolactin, FSH, LH, TSH, ACTH, ADH and STH), which showed no dependence on training, except for free testosterone which showed a decreasing trend (P < 0.10) of 19-25% and post-exercise ACTH which showed an increasing trend of 33% (P < 0.10). Conditioning (cortisol sensitivity and ACTH response) or adaptation (FSH and LH responses) to changed testosterone serum levels and altered spermatogenesis is discussed.

In vivo labelling of the spleen with a red-fluorescent cell dye.

A method for labelling mouse spleen cells in situ is described. Spleen vessels were clamped before intrasplenic injection of a red-fluorescent cell dye (PKH-26). The labelling rate was 11.8% of all cells and 13.9% of B lymphocytes 30 min after injection as determined by FACS. 3 days later, the proportions of labelled cells were reduced to 7.4% (P < 0.01) and to 10.7% (P < 0.05), respectively. Only 10% of cells detected by FACS could be detected by fluorescence microscopy. Labelled cells were not found in peripheral lymph nodes 30 min after spleen injection, but were present 3 days later (FACS: 2.8% of all cells and 5.4% of B lymphocytes, P < 0.05 each), indicating migration of stained cells. Neither adverse nor toxic effects of in situ staining were observed. Isolated stained B lymphocytes from spleens of naive mice and from lymph nodes after immunisation with insulin showed proper function when tested in an ELISA-spot assay. The labelling procedure was used to follow splenic B lymphocytes producing natural anti-insulin antibody. These cells were found to be recruited for the early immune response in lymph nodes immunised with insulin.

Glucose-induced water movement from the intracellular to the extracellular space and its influence on calculations of glucose metabolism.

The osmotic effect of intravenous glucose was investigated in eight healthy volunteers. Increases in plasma glucose can induce water movement from the intracellular to the extracellular space. Serum choline esterase was used as an endogenous marker of serum dilution. Intravenous tests with 5, 15, 30 and 35 g of glucose showed that the water shift was proportional to the amount infused. The respective dilutions of choline esterase were 1.3 +/- 0.7%, 3.3 +/- 0.9%, 6.3 +/- 0.8% and 7.8 +/- 0.5%. The effect on extracellular water was maintained when plasma glucose remained elevated (inhibition of insulin secretion with a somatostatin analogue). In comparison to glucose, infusion of 10 g of a mixture of amino acids produced a less pronounced effect than expected. The acute water shift after intravenous glucose dilutes serum components including glucose (8% of total extracellular glucose at 35 g). This can be misinterpreted as glucose clearance when calculating metabolic rates. For estimated amounts a proportional correction should be made (3.5% per 5 mmol l-1 increase). A measured plasma glucose of 22.2 mmol l-1 should be corrected to 24.8 mmol l-1, while a plasma glucose value of 5.0 mmol l-1 needs no correction.

[Postmortem diagnosis of exogenous insulin administration]. / Zur postmortalen Diagnostik der exogenen Insulin-Applikation.

Toxicological analyses are presented in connection with the decrease of a 51-year-old female medical practitioner. The woman was found dead in a woodland about 250 metres far from her motor car. According to her relatives the woman had expressed her intention to commit suicide. Two single use syringes, two empty (300 units) cartridges intermediate type human insulin, and one empty ampoule of 10 mg diazepam were found near the corpse. The toxicological analyses of the heart-blood resulted in 210 ng diazepam/ml, 382 mu units/ml insulin and 0.58 ng/ml C-peptide. After extraction of a tissue sample from the injection area (cubital region) 113 milliunits insulin could be detected by radioimmunoassay. The insulin concentration in relation to the C-peptide concentration in the blood of the corpse indicated an exogenous supply of insulin. The HbA1c and fructosamine data in the blood and the histological examination of the pancreas showed that the woman had not suffered from diabetes.

Training-overtraining: performance, and hormone levels, after a defined increase in training volume versus intensity in experienced middle- and long-distance runners.

Performance and hormones were determined in eight middle- and nine long-distance runners after an increase in training volume (ITV, February 1989) or intensity (ITI, February 1990). Seven runners participated in both studies. The objective was to cause an overtraining syndrome. The mean training volume of 85.9 km week-1 increased within 3 weeks to 176.6 km week-1 during ITV and 96-98% of training volume was performed as long-distance runs at mean(s.d.) 67(8)% of maximum capacity. Speed endurance, high-speed and interval runs averaging 9 km week-1 increased within 3 weeks to 22.7 km during ITI, and the total volume increased from 61.6 to 84.7 km. A plateau in endurance performance and decrease in maximum performance occurred during ITV, probably due to overtraining, with performance incompetence over months. Nocturnal catecholamine excretion decreased markedly (47-53%), contrary to exercise-related plasma catecholamine responses, which increased. Resting and exercise-related cortisol and aldosterone levels decreased. Improvement in endurance and maximum performance occurred during ITI indicating a failure to cause an overtraining syndrome in ITI. Decrease in noctural catecholamine excretion was clearly lower (9-26%), exercise-related catecholamine responses showed a significant decrease, cortisol and aldosterone levels remained almost constant, exercise-related prolactin levels decreased slightly. There were no differences in insulin, C-peptide, free testosterone, somatotropic hormone (STH), follicle stimulating hormone (FSH), luteinizing hormone (LH), thyroid stimulating hormone (TSH), tri-iodothyronine (T3) and thyroxine (T4). The decrease in nocturnal catecholamine excretion during ITV might indicate a decrease in intrinsic sympathetic activity in exhausted sportsmen. But it remains open whether this reflected a central nervous system incompetence.

Osmotic stress due to changes in plasma glucose and its regulation in IDDM patients.

Intravenous glucose tolerance tests (30 g, 5 min, constant rate) were performed in 8 IDDM patients and in 8 controls. The consequences of the osmotic pressure, induced by glucose, were investigated. Serum choline esterase was used as an endogenous marker of serum dilution. Five minutes after the end of infusion plasma glucose was raised by 182 +/- 12 mg.dl-1 in patients and by 189 +/- 6 mg.dl-1 in controls. Choline esterase values decreased by 6.6 +/- 0.8% and 6.3 +/- 1.0% respectively, P less than 0.01 each. Calculated water shifts into the extracellular space were 924 +/- 112 ml and 882 +/- 140 ml respectively. Fifteen minutes after the end of infusion glucose decreased by 32 +/- 1 mg.dl-1 in IDDM patients and by 57 +/- 2 mg-1 in controls. Serum choline esterase recovered by 2.6 +/- 0.2% and 2.7 +/- 0.2% respectively, P less than 0.01 each, indicating comparable water correction in spite of the slower fall of glucose in IDDM patients. Water correction was more rapid than glucose fall. Diuresis (46 +/- 4 ml versus 42 +/- 3 ml) or cellular uptake of serum solutes (electrolytes, amino acids, urea, creatinine) could not explain this. It is hypothesized that accumulation of free intracellular glucose reduces the osmotic gradient and facilitates cellular water re-uptake.

Sympathetic autonomic dysfunction. Programmed subcutaneous noradrenaline administration via microdosing pump.

Sympathetic dysfunction is characterized by postural hypotension, decreasing blood pressure without compensatory tachycardia during graded supine ergometric exercise, impaired catecholamine metabolism, and hypersensitivity to catecholamines. We report on eight patients, seven with sympathetic dysfunction, of whom three patients were treated for the first time with programmed subcutaneous noradrenaline administration by means of a microdosing pump over a time period of 1.5-13 months. A clear improvement in blood pressure values, orthostasis tolerance, and exercise capacity has been observed in these three patients which permit them to extend their action radius. Adjustment of the noradrenaline dose to the wide range of everyday stress and the possible occurrence of adrenoreceptor desensitization, however, remains a severe problem.

Urine screening in outdoor volunteers: day versus night versus 24 hour collection.

Urine constituents were measured in 12 healthy outdoor volunteers on four occasions within a month. Day, night, and 24 hour collection periods were compared. Measurements made on the four occasions did not differ. The amount of water, creatinine, electrolytes, proteins, and enzymes were higher during the day (up to three fold, p always less than 0.05), while equal amounts of amino acids were excreted in the day and the night period. A comparison of all values from all individuals for all four sampling occasions showed that the variation was lowest in the 24 hour samples. Relating the values to creatinine did not consistently reduce the variation. Twenty-four hour samples correlated better with the day than with the night samples. Day and night samples did not correlate. Twenty-four hour collection is superior to day or night collection in healthy outdoor volunteers. Based on the normal variation of values found in the present study, criteria for suspected kidney damage and therefore for the withdrawal of drugs in pharmacological studies can be defined for each collection period.

The detection of single cells forming antibodies to defined epitopes on insulin.

A solid-phase immunoenzymatic technique was modified to permit the detection and enumeration of antibody-secreting cells recognizing the amino or carboxy terminal of the insulin B chain. The procedure involves coating well surfaces with avidin and binding insulins specifically labelled with biotin at the B1 or B30 residue. On day 15 after immunization with human insulin, 20 outbred NMRI mice had generated cells secreting anti-insulin antibodies. The recognition of B1- or B30-related epitopes differed between individuals, suggesting that there was genetic determination of the epitopes expressed early in the immune response. The method can be used to find strains of mice with a preferential immune response to defined areas on the surface of small peptide molecules. Such strains could then be used to produce specific monoclonal antibodies.

Natural occurrence of IgE antibodies to peptide hormones in man.

IgE antibodies to peptide hormones or their fragments were investigated using specific radioallergosorbent tests. Ninety-six sera were obtained from otherwise healthy individuals with a positive RAST to at least one common allergen and without previous contact to exogenous hormones. Total serum IgE was normal (less than 300 ng ml-1) in 41 and elevated in 55 sera. Only one serum with normal total IgE had reagins to proinsulin. In sera with elevated total IgE positive tests (greater than 8 x background) were observed frequently: hGH 9.1%, GHRH1-44 5.5%, GHRH1-29 12.7%, erythropoietin 10.9%, human calcitonin 14.6%, salmon calcitonin 1.8%, ACTH 5.5%, insulin 14.6%, proinsulin 7.3%, insulin A chain 1.8%, insulin B chain 18.2, C-peptide 9.1%, and IGF 9.1%. The data show natural occurrence of reagins to peptide hormones in atopic persons. A risk at exposure to these peptides may exist in about 1% of the general population.

Insulin abuse in long-standing IDDM.

Two patients with insulin-dependent diabetes mellitus (IDDM) for 42 and 46 years respectively were diagnosed to produce factitious hypoglycemia. They had several properties in common: abnormal personality, refusal to stop incipient hypoglycemia, ideal body weight, good hemoglobin A1c (HbA1c) values, non-specific changes in the EEG, and brain atrophy sparing the periventricular areas. The addiction forced the patients to continue their habit during hospital stay. The dose of surreptitiously injected regular insulin always produced serum insulin concentrations large enough to clarify the diagnosis.

Gamma irradiation as a substitute for H2O2 in peroxidase-labelled enzyme immunoassays.

The ability of gamma irradiation to substitute for H2O2 in peroxidase-labelled enzyme immunoassays was investigated. Colouring of TMB (3,3',5,5'-tetramethylbenzidine), ABTS [2,2'-azino-di(3-ethylbenzthiazoline-6-sulphonic acid)], and OPD (o-phenylenediamine) was produced with doses of 10 and 20 Gy. Addition of superoxide dismutase enhanced TMB and ABTS, and inhibited OPD. The reaction was terminated when the irradiation was stopped. The use of denaturing compounds to stop the reaction was unnecessary and coated antigens could be re-used for a second immunoassay.