[Clinical consequences of intranasal insulin therapy in insulin-dependent diabetes mellitus]. / Kliniske konsekvenser af intranasal insulinbehandling ved insulinkraevende diabetes mellitus.

Metabolic control, hypoglycaemia frequency and nasal mucosal physiology were evaluated in 31 insulin-dependent diabetics treated with intranasal insulin at mealtimes for one month and with subcutaneous fast-acting insulin for another month in a randomized crossover trial. During both periods the patients were treated with intermediate-acting insulin at bedtime. Six of the patients were withdrawn from the study during intranasal insulin therapy due to metabolic dysregulation. Insulin concentrations increased more rapidly and decreased more quickly during intranasal as compared with subcutaneous insulin administration. Metabolic control, assessed by haemoglobin A1c concentrations, deteriorated after intranasal as compared with subcutaneous insulin therapy. The bioavailability of intranasally applied insulin was low, since intranasal insulin doses were approximately 20 times higher than subcutaneous doses. The frequency of hypoglycemia was similar during intranasal and subcutaneous insulin therapy, and nasal mucosal physiology was unaffected after intranasal insulin. We conclude that due to low bioavailability and to a high rate of therapeutic failure, intranasal insulin treatment is not a realistic alternative to subcutaneous insulin injections at the present time.

Intranasal insulin therapy: the clinical realities.

To evaluate metabolic control and safety parameters (hypoglycaemia frequency and nasal mucosa physiology), 31 insulin-dependent diabetic patients were treated with intranasal insulin at mealtimes for 1 month and with subcutaneous fast-acting insulin at meals for another month in an open, crossover randomized trial. During both treatment periods the patients were treated with intermediate-acting insulin at bedtime. Six of the patients were withdrawn from the study during intranasal insulin therapy due to metabolic dysregulation. Serum insulin concentrations increased more rapidly and decreased more quickly during intranasal as compared with subcutaneous insulin administration. Metabolic control deteriorated, as assessed by haemoglobin A1c concentrations, slightly but significantly after intranasal as compared with subcutaneous insulin therapy. The bioavailability of intranasally applied insulin was low, since intranasal insulin doses were approximately 20 times higher than subcutaneous doses. The frequency of hypoglycaemia was similar during intranasal and subcutaneous insulin therapy, and nasal mucosa physiology was unaffected after intranasal insulin. We conclude that due to low bioavailability and to a high rate of therapeutic failure, intranasal insulin treatment is not a realistic alternative to subcutaneous insulin injections at the present time.

Urinary excretion of thromboxane and markers for renal injury in patients undergoing cardiopulmonary bypass.

Urinary excretion of selected markers for renal injury, as well as urinary excretion rates of the thromboxane metabolite, 11-keto-thromboxane B2 (11k-TXB2), was studied in 36 male patients undergoing coronary bypass surgery using cardiopulmonary bypass (CPB). In all patients, excretion of both tubular (N-acetyl-beta-D-glucosaminidase [beta NAG]; alpha 1-microglobulin [alpha 1-MG]) and glomerular markers (albumin [Alb]; transferrin [Trf]; immunoglobulin G [IgG]) sharply increased on Day 1 after CPB, and they remained elevated throughout the observation period of 5 days. Urinary excretion rates of 11k-TXB2 markedly increased on Day 1 after surgery, and they rapidly decreased thereafter. In 12 of the 36 patients, a temporary increase of serum creatinine levels (> 1.30 mg/dl) was noted following surgery. A positive correlation was found between serum creatinine levels and excretion of the tubular enzyme beta NAG (r = 0.36; p < 0.05), but not between creatinine levels and alpha 1-MG or the glomerular markers. Furthermore, no correlation between urinary excretion of 11k-TXB2 and any of the urinary markers for renal injury could be detected. Our data do not strengthen the hypothesis that acute renal injury observed during CPB is related to exaggerated thromboxane biosynthesis in these patients. Monitoring of urinary markers for incipient renal damage, particularly excretion of beta NAG, might be of additional diagnostic value for detection of otherwise subclinical renal injury in patients undergoing CPB.

Glucose recovery after intranasal glucagon during hypoglycaemia in man.

We compared the hyperglycaemic effect of intranasal and intramuscular (i.m.) administration of glucagon after insulin-induced hypoglycaemia. Twelve healthy subjects were examined twice, receiving on both occasions an intravenous insulin bolus. Somatostatin and propranolol were administered to block endogenous glucose counterregulation, and glucose turnover was estimated by a 3-[3H]-glucose infusion. When hypoglycaemia was reached, the subjects received either i.m. glucagon of pancreatic extraction (1 mg) or intranasal genetically engineered glucagon (2 mg). The incremental values for plasma glucose concentrations 15 min after intranasal and i.m. administration of glucagon differed marginally. However, after 5 min the glucose appearance rate, as well as the incremental values for plasma glucose, were significantly higher for the i.m. glucagon treatment. The mean time taken for incremental plasma glucose to exceed 3 mmol.l-1 was significantly shorter for i.m. glucagon. The mean plasma glucagon level increased faster after i.m. glucagon than after intranasal glucagon, and the levels remained higher throughout the study period. We conclude that glucose recovery was significantly better after i.m. administration of glucagon than after intranasal administration. However, the differences between the incremental plasma glucose and the time for incremental plasma glucose to exceed 3 mmol.l-1 were not considered of major clinical importance.

Rapid, direct enzyme immunoassay of 11-keto-thromboxane B2 in urine, validated by immunoaffinity/gas chromatography-mass spectrometry.

We have developed a direct enzyme immunoassay (EIA) for quantifying immunoreactive 11-keto-thromboxane B2 (iKTXB) in unprocessed human urine. Cross-reactivity with other thromboxane metabolites and prostanoids was negligible. Analytical recovery of 11-keto-TXB2 in urine specimens was 97.4% to 99.8%. Total imprecision for two clinical specimens was 8.5% and 12.2%. Intake of acetylsalicylic acid decreased the measured concentration of iKTXB. Cardiopulmonary bypass, a procedure known to activate platelets, increased the mean excretion rate of iKTXB 10-fold. Simultaneous gas chromatography-mass spectrometry analysis of 11-keto-TXB2 and 11-keto-2,3-dinor TXB2 in urine specimens (n = 17) from healthy subjects indicated that urinary iKTXB concentrations measured by EIA represented a sum of the two 11-keto metabolites. We conclude that the direct EIA is sufficiently sensitive, rapid, simple, and specific to allow screening for alterations in thromboxane biosynthesis in patients.

Urinary excretion and origin of 11-dehydro-2,3-dinor-thromboxane B2 in man.

The in vivo biosynthesis of thromboxane B2 (TXB2) in man is currently evaluated by measuring urinary excretion of its major urinary metabolites, 11-dehydro-TXB2 and 2,3-dinor-TXB2. 11-Dehydro-2,3-dinor-TXB2, another prominent metabolite of exogenous TXB2 in man, has never been measured in human urine. We measured urinary 11-dehydro-2,3-dinor-TXB2 in parallel with 11-dehydro-TXB2 and 2,3-dinor-TXB2 by immunoaffinity extraction/gas chromatography-mass spectrometry in healthy non-smokers (n = 12) and age-matched smokers (n = 11). In non-smokers, urinary excretion of 11-dehydro-2,3-dinor-TXB2, 11-dehydro-TXB2 and 2,3-dinor-TXB2 was 29.7 +/- 11.1, 53.6 +/- 15.0 and 13.5 +/- 2.8 ng/h (mean +/- SD), respectively. In smokers, only urinary excretion of 2,3-dinor-TXB2 was significantly different (19.7 +/- 6.7 ng/h, p < 0.01). Selective inhibition of platelet thromboxane biosynthesis by chronic low-dose aspirin (30 mg/day for 8 days, 4 subjects) comparably reduced platelet-derived metabolites and 11-dehydro-2,3-dinor-TXB2, suggesting that the latter also derives from platelets in healthy subjects.

Thromboxane B2 urinary metabolites in patients undergoing cardiopulmonary bypass.

The urinary excretion of selected markers for renal injury and thromboxane metabolites was studied in 16 patients undergoing cardiopulmonary bypass (CPB). Excretion of both tubular and glomerular markers sharply increased on day 1 after CPB and remained elevated throughout the observation period (five days). Immunoreactive thromboxane B2 (i-TXB2, mainly reflecting 2,3-dinor-TXB2) and immunoreactive 11-keto-thromboxane B2 (i-11-keto-TXB2) were measured by direct enzyme immunoassays. TXB2, 2,3-dinor-TXB2 and 11-keto-TXB2 were also measured in selected samples by GC-MS. Urinary excretion rates of both i-TXB2 and i-11-keto-TXB2 markedly increased on day 1 after surgery and decreased thereafter. Following CPB, excretion rates of 2,3-dinor-TXB2 and TXB2 displayed parallel changes, suggesting that in these patients most urinary TXB2 derives from blood platelets rather than the kidney. Taken together, our observations do not support the hypothesis that acute renal injury observed after CPB is caused by exaggerated thromboxane biosynthesis in the kidney.

Hyposensitization in asthmatics with mPEG-modified and unmodified house dust mite extract. III. Effect on mite-specific immunological parameters and correlation to changes in mite-sensitivity and symptoms.

Forty-six adult asthmatics allergic to D. pteronyssinus (Dp) participated in a 2-year study. Thirty-one underwent hyposensitization (HS-group). Fifteen were treated with Dp-extract (Dp-group), and 16 with a similar extract modified by monomethoxypolyethylene glycol with reduced allergenicity (mPEG-Dp-group). Fifteen patients served as controls. Dp-specific antibodies and histamine release from blood basophils were determined and compared with Dp-sensitivity in lungs and skin. In addition, IgG and IgE against the major allergen Der p I were followed in a subgroup. Dp-specific IgG, IgG1, and IgG4 increased significantly in both HS-treated groups after 1 and 2 years (median: 2.5- to 11.6-fold). IgG4 was not induced if maintenance dose during the first year was less than 20,000 BU. Median skin sensitivity decreased 4.4- to 8.2-fold after 1 year and 7.4- to 21.4-fold after 2 years. Der p I specific IgG response was unrelated to the occurrence or change in IgE with the same specificity. The mPEG-Dp-extract tended to have less effect on skin sensitivity and immunological parameters, differences reaching statistical significance for skin sensitivity only. In the HS-group, the decrease in bronchial sensitivity was significantly correlated to a decrease in IgE (r = 0.36), IgG1/IgG4 (r = 0.49), Dp-specific histamine release (r = 0.58), and to an increase in Dp-specific IgG4 (r = -0.36) and IgG4/IgE (r = -0.48). In patients improving clinically, Dp-specific IgG4/IgE increased, and median Dp-specific IgE was reduced to 80% compared with an increase to 150-160% seen in the unchanged or deteriorated group (P less than 0.05). Findings indicate an improvement of effect, if the allergen dose is sufficient to reduce specific IgE and/or induce an IgG and especially IgG4 response.

IgG subclass concentrations in sera from 200 normal adults and IgG subclass determination of 106 myeloma proteins: an interlaboratory study.

The IgG subclass protein concentrations in sera from 200 normal subjects were determined independently in two laboratories, using the same technique (radial immunodiffusion), the same subclass-specific antibodies and the same calibrator. The coefficients of correlation (rs) between IgG subclass concentrations determined in the two laboratories were 0.487, 0.883, 0.928 and 0.926 for IgG1, IgG2, IgG3 and IgG4, respectively (p less than 0.0005 in all four cases). By the chi-square test for goodness of fit, the frequency distributions observed in the two laboratories were found to differ significantly for all four subclasses (p less than 0.01). In one laboratory, the distributions of IgG1 and IgG2 were not significantly different from normal distributions, whereas the distributions of IgG3 and IgG4 deviated significantly. In the other laboratory, all four subclass distributions were significantly different from normal distributions. In the first laboratory, the IgG2 concentrations were log-normal distributed, whereas in the second, IgG1, IgG2 and IgG4 concentrations were log-normal distributed. We conclude that use of identical reagents does not ensure identical frequency distributions. This finding emphasizes the need for standardization of the measurement technique too. Furthermore, we argue that at present, intralaboratory reference intervals for IgG subclass protein concentrations are necessary. The reference intervals should be based on non-parametric statistics. The subclass of 106 monoclonal IgG proteins, which were demonstrated by agarose gel electrophoresis, was identified by RID for 89 samples. The subclass of the remaining 16 M-components was readily determined by qualitative immunoelectrophoretic analysis using the same subclass-specific antibodies.

Diagnosis and immunotherapy of mould allergy. VII. IgG subclass response and relation to the clinical efficacy of immunotherapy with Cladosporium.

The IgG subclass response was evaluated by a sensitive allergen- and subclass-specific solid phase immunoradiometric assay during a 1-year placebo-controlled, double-blind study of immunotherapy with Cladosporium herbarum in 22 adult asthmatics. The IgG response was mainly restricted to subclasses 1 and 4 but a few patients were IgG2 and IgG3 responders. An intense and early IgG1 response was observed during the first clusters of injection followed by a levelling down of the titer. The IgG4 response had a later onset and showed slowly increasing levels during the 12 months of immunotherapy. The graded clinical efficacy estimated by symptom-medication score was significantly correlated to the preseasonal IgG1 value, with high values indicating a deleterious response of immunotherapy (deterioration of disease activity). Likewise, the fold increase of IgG1 and IgG4 after two clusters of immunotherapy (i.e. after 4 weeks) was significantly related to the clinical outcome. Little or no increase of IgG1 and IgG4 was associated with improvement, i.e. decrease in symptom-medication score. The magnitude of the IgG1 response during the dose-increase phase was directly correlated to the number of systemic side effects. No relation of IgG1, IgG4 or IgG4/IgG1 ratio to changes in the IgE-mediated parameters (skin prick test, bronchial challenge and circulating specific IgE) was observed. Our data, which are based on few patients and only one allergen system, do not support the hypothesis of IgG acting as blocking antibody being the immunologic mechanism of immunotherapy. The association between high IgG4 values and a deleterious efficacy of immunotherapy might be caused by IgG4 acting as sensitizing antibodies. This explanation, however, is opposed by the lack of relation to systemic side effects.

Glomerular size and charge selectivity in insulin-dependent diabetes mellitus.

The pathogenesis of clinical nephropathy in Type 1 (insulin-dependent) diabetes was investigated by measuring renal fractional clearances of albumin, total IgG, IgG4 and beta 2-microglobulin, four plasma proteins which differ in size and charge. Seventy patients and eleven control subjects were studied. In diabetic patients with normal urinary albumin excretion (less than 30 mg/24 hr), fractional IgG clearance was two to three times higher than in control subjects, whereas fractional clearance of the anionic plasma proteins IgG4 and albumin was similar to that of control subjects. These alterations indicate an increase in anionic pore charge within the glomerular basement membrane concomitant with an increase in either pore size or impairment of tubular reabsorption. Diabetic patients, whose urinary albumin excretion has started to rise (30 to 100 mg/24 hr), had unchanged fractional IgG compared to patients with normal albumin excretion, while fractional IgG4 and albumin clearances were increased three- to fourfold; indicating unchanged glomerular pore size, but a decrease in anionic pore charge. In patients demonstrating urinary albumin excretion of greater than 100 mg/24 hr fractional IgG clearance increased to the same extent as fractional albumin clearance, indicating an increase in large pore area. Fractional beta 2-microglobulin clearances were similar to that of control subjects in the different patient groups indicating unchanged tubular reabsorption of proteins. Thus, the increase in large pore area seen in patients with clinical nephropathy is preceded by loss of anionic charge in the glomerular basement membrane. It is likely that this loss of anionic charge is due to loss of heparan sulphate-proteoglycan.

High IgG4 antibody level is associated with failure of immunotherapy with inhalant allergens.

We have analysed all available data on the relationship between IgG4 Ab level and clinical effect of immunotherapy (IT) with inhalant allergens. The data from three of the seven independent studies could, without reservations, be analysed by a joint statistical analysis. We found that late high IgG4 Ab level, measured at the end of IT, was strongly associated with treatment failure (P = 6.54 x 10(5); n = 67). The ratio of risks for treatment success in the group with late low IgG4 Ab level was 1.82, whereas the ratio of risks for treatment failure in the group with late high IgG4 Ab level was 11.4. The data from a fourth, presumably comparable, study further supported the existence of an association between high IgG4 Ab level and treatment failure. In contrast, two other studies found that high mean IgG4 Ab level was associated with good clinical response. Possible reasons for this apparent discrepancy are discussed. We also found that early high IgG4 Ab level, measured within 3 months after initiation of IT, was strongly associated with treatment failure after 1-2 years of IT (P = 1.05 x 10(-4); n = 30). The sensitivity and specificity of early high IgG4 Ab level as indicator for treatment failure was 100% and 83%, respectively. At the prevalences found in the present study, the predictive value of early high IgG4 Ab level for treatment failure was 0.6, whereas the predictive value of early low IgG4 Ab level for treatment success was 1.00.

Acetylcholine receptor antibody in myasthenia gravis: predominance of IgG subclasses 1 and 3.

The concentrations of IgG subclass antibodies (Ab) to acetylcholine receptor (AchR) were quantified in 36 patients with myasthenia gravis (MG) treated with pyridostigmine only, and in eight patients who underwent thymectomy, using an IgG subclass-specific immunoprecipitation assay. IgG1, IgG2, IgG3, and IgG4 subclass Ab to AchR were present in 100%, 33%, 64% and 39% of the pyridostigmine-treated patients, respectively. The concentration of IgG1 Ab increased significantly with disease severity as graded by the Osserman-Genkins classification (rs = 0.37, P less than 0.05). IgG1 and IgG3 subclass protein concentrations were significantly higher (P less than 0.0003) in the 36 pyridostigmine-treated MG patients than in 44 age- and sex-matched healthy subjects. Thymectomy induced an appreciable reduction in anti-AchR IgG1 concentration in two patients, whereas six patients showed no changes in Ab to AchR. The results support the hypothesis that binding of anti-AchR IgG1 and IgG3 on AchR in the neuromuscular junction followed by complement-mediated cell lysis or phagocytosis, may play a role in the pathogenesis of MG.

Raised serum IgG4 levels in patients with atopy and filariasis: application of an automated particle-counting immunoassay using monoclonal antibody.

We show here an automated (50 samples/h) assay for serum IgG4 having a throughput time of 40 min per sample and a sensitivity of 10 micrograms/ml. The assay procedure is based on the inhibition by sample of the agglutination reaction between monoclonal anti-IgG4 antibodies and latex particles to which IgG4 myeloma protein has been coupled. Assay reliability was ascertained by testing for linearity, analytical recovery (96.4%), interassay precision (less than or equal to 8%), specificity and correlation between the results obtained with monoclonal and polyclonal anti-IgG4 antibodies (n = 84; rs = 0.97). Application of the assay to sera from various groups of patients indicated significantly (p less than 0.00005) higher geometrical means (Gx) in patients suffering from atopy (n = 87; Gx = 617 micrograms/ml), atopic dermatitis (n = 28; Gx = 1,043 micrograms/ml), filariasis with Onchocerca volvulus (n = 48; Gx = 1,681 micrograms/ml) and Brugia malayi (n = 20; Gx = 1,078 micrograms/ml) as compared to nonatopic subjects (n = 103; Gx = 302 micrograms/ml) and randomized paired maternal/cord sera (n = 41; Gx = 276 and 296 micrograms/ml, respectively). IgG4 in the paired maternal/cord sera correlated (r = 0.98; p less than 0.00005). There was no significant influence of age or sex on the IgG4 levels either among the nonatopics or the atopics even though low IgG4 (less than or equal to 30 micrograms/ml) was more common among women. The results suggest that IgG4 and IgE responses are somehow closely related in atopic and parasite-infested patients at the physiological, pathogenic or genetic level.

A triple antibody assay for the quantitation of plasma IgG subclass antibodies to acetylcholine receptors in patients with myasthenia gravis.

Monoclonal anti-human IgG subclass antibodies have been used in an immunoprecipitation assay for the determination of anti-acetylcholine receptor IgG subclasses in plasma from patients with myasthenia gravis. Solubilized acetylcholine receptors labelled with 125I-alpha-bungarotoxin were incubated with patient plasma. Monoclonal mouse antibodies to human IgG subclasses 1-4 were added to the incubation and finally precipitated with anti-mouse IgG antibody. A maximal IgG subclass precipitation of 62-76% was determined with 125I-labelled myeloma IgG subclasses 1-4 added to normal human plasma. The anti-IgG subclass antibodies were added in excess which ensured that the precipitation of IgG2, IgG3 or IgG4 were unchanged, and that of IgG1 was only reduced by 17%, when the plasma IgG concentration was increased by a factor of two. The anti-IgG subclass antibodies were highly specific for their complementary subclasses. Determination of the IgG subclass of the anti-acetylcholine receptor antibodies from 8 patients with myasthenia gravis showed that IgG1 and IgG3 antibodies are predominant. This may support the hypothesis that complement mediated lysis of the neuromuscular end-plate plays a pathogenetic role in myasthenia gravis.

The subclass nature and clinical significance of the IgG antibody response in patients undergoing allergen-specific immunotherapy.

The purpose of this paper is to discuss the methodological difficulties in quantitation of human IgG subclass antibodies to allergens, to describe the subclass nature of the IgG antibody response in patients undergoing allergen-specific immunotherapy, and to discuss the possible immunological functions and clinical significance of allergen-specific IgG antibodies of different subclasses. Based on results obtained by use of assays with documented specificity it is concluded that the IgG antibody response during allergen-specific immunotherapy is IgG1 and IgG4 restricted, although low levels of IgG2 and IgG3 antibodies to some allergens may occur. In most patients the early IgG antibody response is IgG1 dominated and the late IgG4 dominated. A too early or too pronounced IgG4 dominated antibody response seems to indicate a poor clinical outcome of immunotherapy with inhalant allergens, whereas a pronounced early IgG1 antibody production has been found to be associated with a decrease in synthesis of IgE antibodies to an insect venom. It is therefore proposed that an early IgG1 dominated response is necessary to induce suppression of the ongoing IgE antibody production, which in its turn may be a prerequisite for long-lasting clinical effect. The possibility of induction of an early IgG1 dominated response in every patient by use of alternative immunotherapy procedures is discussed.

Oral administration of grass pollen to hay fever patients. An efficacy study in oral hyposensitization.

Oral hyposensitization is still widely used in the treatment of allergic diseases, but controlled studies proving a beneficial effect are lacking. Fifty-eight hay fever patients were admitted to a double-blind placebo efficacy study in oral hyposensitization. An enterosoluble tablet containing timothy whole pollen or placebo was taken daily. Preseasonally, the actively treated patients received 4,315,000 PNU (880,260 AUR) and totally for 6 months 8,915,000 PNU (1,818,660 AUR). Such high doses have never been tried in similar studies. A new principle has been used - "the pollen count interval method" - in the evaluation of symptom and medication score. The study failed to prove any beneficial effect of oral hyposensitization measured by symptom score, medication score, nasal provocation test or skin prick test. There was no change in timothy specific IgE and IgG which could be caused by the treatment. The possibility that oral hyposensitization might be an effective treatment of hay fever in the future is discussed, but it is concluded that the present regimens cannot be recommended.