Improving the in vitro antigen specific T cell proliferation assay: the use of interferon-alpha to elicit antigen specific stimulation and decrease bystander proliferation.

The measurement of the proliferative response of primed T cells to an antigenic stimulus (lymphocyte transformation assay: LTT) is commonly used for determining T cell immune responsiveness. However, the ratio between the spontaneous and the antigen-triggered response (stimulation index) is frequently quite low (<3-5) making the interpretation difficult. We modified the assay by the addition of interferon-alpha and the use of fresh autologous serum instead of human AB pool serum. These measures significantly enhanced the stimulation index following stimulation with tetanus toxoid, Candida albicans and tick-borne encephalitis (TBE) viral antigen in studies of sensitized patients. There was no concomitant increase in false positive results. Kinetic studies showed a reduced nonspecific background proliferation of non-stimulated cultures particularly between days 4 and 6 of culture. Furthermore, the positive effect of interferon-alpha were confirmed in studies of patients with contact allergy to nickel and gold. We conclude that this modified form of proliferation assay significantly increases the signal to noise ratio which can be attained. This may be of particular value when looking at T cell responses in immunocompromised patients or in diagnostic attempts to detect very low frequencies of antigen-specific T cells.

A case of severe chronic active infection with Epstein-Barr virus: immunologic deficiencies associated with a lytic virus strain.

Infectious mononucleosis (IM) is a self-limiting, lymphoproliferative disease induced by primary infection with the Epstein-Barr virus (EBV). Infection with EBV leads in general to lifelong asymptomatic persistence of the virus. We report the case of a woman who acquired IM at the age of 15 years and then suffered from recurrent high fever, fatigue, and signs of immunologic disorder for more than 12 years until she died of liver failure. In an attempt to describe and to define the course of chronic active infection with EBV, we performed immunologic and molecular assays that demonstrated lytic replication of EBV in the B and T cells of the peripheral blood. In addition to signs of humoral and cellular immune deficiency, we detected an EBV strain with an impaired capability to immortalize B cells and a tendency to lytic replication, thus contributing to the pathogenesis of this chronic active infection.

Continuous mannose infusion in carbohydrate-deficient glycoprotein syndrome type I.

The effects on isoelectrofocusing patterns of serum glycoproteins were studied in a patient with CDG syndrome type I and phosphomannomutase deficiency during 3 weeks of continuous intravenous mannose infusion. Doses of 5.7 g/kg/day led to stable serum mannose levels up to 2.0 mmol/l and were well tolerated without signs of liver or renal toxicity. While most of the pathological glycoprotein patterns, including alpha1-antitrypsin, typical for CDG syndrome type I remained unchanged, mannose infusion led to a unique change of the isoelectrofocusing pattern of serum sialotransferrins with appearance of two extra bands after 3 weeks of treatment.

Molecular characterization of Duarte-1 and Duarte-2 variants of galactose-1-phosphate uridyltransferase.

The N314D polymorphism was found in two different alleles of the galactose-1-phosphate uridyltransferase (GALT) gene, Duarte-1 (D1) and Duarte-2 (D2). Although both variants have identical electrophoretic mobility and isoelectro-focusing points, the galactose-1-phosphate uridyltransferase (GALT) activity varies: D1 alleles showed 110-130% of the normal RBC activity, but D2 alleles only 40-50%. We found that D1 alleles also carried a silent mutation in exon 7 (L218L) in addition to N314D. In contrast, besides N314D, D2 alleles carried two regulatory mutations, G1105C and G1391A, in introns D and E, respectively. In normal and Q188R alleles none of the above four mutations coexisted. However, some galactosaemia alleles with mutations other than Q188R, such as W316X and E340X of exon 10, also carried the N314D mutation. The W316X alleles existed in cis with the intron mutations (G1105C and G1391A), whereas those with E340X are in cis with L218L. In all cases examined, the intron mutations were not found in D1 alleles and no D2 alleles had the silent mutation of L218L. These results suggest that the decrease in the GALT activity in D2 may be due to regulation of the GALT gene expression. The G1105C site may be critical to the function of erythroid transcription factor NF-E1, since it flanks the core consensus sequence for one of its binding sites. The G1391A mutation may affect another cis-acting regulatory sequence. Alternatively, both mutations may be involved in an aberrant splice processing, which possibly results in a low level of correctly spliced mRNA.

Sulfoconjugated catecholamines: lack of beta-adrenoceptor binding and adenylate cyclase stimulation in human mononuclear leukocytes.

The racemic 3-O-sulfates of epinephrine and norepinephrine as well as 4-O-sulfoconjugated dopamine were synthesized, highly purified and investigated with respect to their beta-adrenoceptor affinities and relative potencies in the receptor-coupled adenylate cyclase system in isolated human mononuclear leukocytes. The receptor affinities of all catecholamine sulfates were reduced at least 1,000-fold when compared to those of the free catecholamines. Furthermore, catecholamine sulfoconjugates did not produce intracellular cAMP signals. In contrast to the sulfated catecholamine metabolites, the 3-O-methylated catecholamines metanephrine and normetanephrine were found to behave as endogenous beta-adrenoceptor-competing agents with lower beta-receptor affinities than the corresponding free catecholamines. No beta-receptor agonist activity in the adenylate cyclase system was found with metanephrine and normetanephrine. Our data provide direct evidence that sulfoconjugation renders catecholamines inactive as beta-receptor ligands and must thus be regarded as a mechanism to control adrenergic action at the prereceptor level by a buffering of the concentration of free catecholamines. The physiological significance of a potential role of 3-O-methylated catecholamines as endogenous beta-receptor antagonists has to be further clarified.

Human fat cell lipolysis is primarily regulated by inhibitory modulators acting through distinct mechanisms.

The effects of adenosine deaminase and of pertussis toxin on hormonal regulation of lipolysis were investigated in isolated human fat cells. Adenosine deaminase (1.6 micrograms/ml) caused a two-to threefold increase in cyclic AMP, which was associated with an increase in glycerol release averaging 150-200% above basal levels. Clonidine, N6-phenylisopropyladenosine, prostaglandin E2, and insulin caused a dose-dependent inhibition of glycerol release in the presence of adenosine deaminase. Pretreatment of adipocytes with pertussis toxin (5 micrograms/ml) for 180 min resulted in a five- to sevenfold increase in cyclic AMP. Glycerol release was almost maximal and isoproterenol caused either no further increase or only a marginal additional increase of lipolysis after pretreatment with pertussis toxin, whereas cyclic AMP levels were 500 times higher than in controls. The effects of antilipolytic agents known to affect lipolysis by inhibition of adenylate cyclase activity, i.e., clonidine, N6-phenylisopropyladenosine, and prostaglandin E2, were impaired. In contrast, the antilipolytic action of insulin was preserved in adipocytes pretreated with pertussis toxin. As in controls, the peptide hormone had no detectable effect on cyclic AMP after pertussis toxin treatment. The findings support the view that the antilipolytic effect of insulin does not require adenylate cyclase or phosphodiesterase action. In addition, the results demonstrate that, upon relief of endogenous inhibition, human fat cell lipolysis proceeds at considerable (adenosine deaminase) or almost maximal (pertussis toxin) rates. A certain degree of inhibition, therefore, appears to be necessary for human fat cell lipolysis to be susceptible for hormonal activation.

[Pseudohypertriglyceridemia in glycerokinase deficiency]. / Pseudo-Hypertriglyceridämie bei Glycerokinase-Mangel.

Falsely high serum triglyceride concentrations (410-850 mg/dl) were measured in four members of a family of five from Franconia in Germany. The cause was hyperglyceridaemia on the basis of glycerol kinase deficiency. None had any symptoms and no other metabolic anomaly was demonstrated. The possibility of glycerol kinase deficiency should be considered in any case of elevated serum triglyceride concentration but with clear serum, normal lipid electrophoresis and lack of response to lipid-lowering measures.

Exercise-induced regulation of insulin receptor affinity: role of circulating metabolites.

We have described before that different forms of physical exercise induce bidirectional changes of insulin binding of monocytes (Michel G., Vocke T., Fiehn W., Weicker H., Schwarz W., Bieger W.P.: Am J Physiol 246: E 156-E 159, 1984). In vitro experiments suggested these changes to be due to dialyzable serum components. In this study, we investigated several hormones and metabolites as to their capacity to alter insulin binding in vitro. Somatostatin (100 pg/ml) and prostaglandin B1 (10 nmol/l) were the only hormonal agents producing a small and reversible (somatostatin) increase in monocyte insulin binding. Ketones were only effective at concentrations unphysiologically high. Acidosis diminished insulin binding to monocytes to about 35% of that found at pH 7.6. Lactate (10 mmol/l) induced a 28% drop in cellular insulin binding at low pH. The effect persisted after removal of the agent and may hence account for some of the decrease in cellular insulin binding observed after exhaustive exercise. Although the effect of acidosis was reversible in vitro, it may add considerably to the effect of lactate under in vivo conditions. The dialyzable serum factors responsible for the enhancement of binding affinity after long-term moderate exertion remain unknown. Free fatty acids proved effective in increasing monocyte insulin binding (14% with 1 mmol/l oleic acid) in vitro.

Diminished insulin receptors on monocytes and erythrocytes in hypertriglyceridemia.

Carbohydrate intolerance is a common observation in endogenous hypertriglyceridemia (HL). So far the nature of this metabolic defect, which accompanies postprandial hyperinsulinemia and a reduced hypoglycemic action of insulin, has not been elucidated. We have examined cellular insulin binding in 20 subjects affected with HL (average plasma triglyceride level, 437 +/- 311 mg/dL) to test the possibility that a receptor defect is involved in peripheral insulin resistance. Monocytes from the HL subjects bound, on the average, 34% less insulin than cells from eight normotriglyceridemic controls of comparable age and body weight (average plasma TG level, 169 +/- 34 mg/dL). Likewise, erythrocytes from the HL group bound 29.6% less insulin than did those from control subjects. Scatchard analysis of the binding data revealed that the number of insulin receptors was reduced for both types of cells. To test if the abnormality in cellular insulin-binding capacity in these subjects is an inherent defect or secondary to the hypertriglyceridemia, 11 of the subjects participated in a 4-month training program (120 minutes weekly of moderate exercise at 60% VO2 max), while the remaining nine persons served as controls. Training reduced the average plasma TG level from 373 +/- 270 to 277 +/- 139 mg/dL (P less than 0.01), but cellular insulin binding was not significantly affected. In addition, no correlation was found between the individual TG plasma concentration and cellular insulin binding. Thus, training itself also proved ineffective in enhancing insulin binding, most probably due to exertion of insufficient intensity.(ABSTRACT TRUNCATED AT 250 WORDS)

Trial of sulfonylurea in combination with insulin in the therapy of diabetes type I and II. Evidence against a primary extrapancreatic receptor effect.

Recently in vitro evidence has been presented that sulfonylurea derivatives exert their chronic extrapancreatic effect by increasing the number of cellular insulin receptors. To ascertain if this receptor effect holds in vivo, we performed a randomized double-blind study on 21 type I (0.3 ng/ml residual C-peptide secretory capacity after glucose/glibenclamide stimulation), and on 19 insulin treated type II (2.0 ng/ml C-peptide) diabetics. The patients received for six weeks 10 mg/d of glibenclamide in addition to insulin. Insulin binding was initially lower in type II (4.7 +/- 0.75% per 10(7) monocytes and 6.39 +/- 1.08% per 4.5 X 10(9) erythrocytes) than in type I diabetic patients (5.1 +/- 0.48% and 7.95 +/- 0.88% respectively) and in 12 normal subjects (5.25 +/- 0.48 and 8.1 +/- 0.94% respectively). Glibenclamide normalized the number of monocyte receptors (from 4.14 to 5.49 X 10(4) sites/cell) in type II patients, but was without effect in type I diabetics. Blood glucose was significantly reduced (240 to 182 mg/dl; p = 0.02) in the type II group with a concomitant decrease in glycosylated hemoglobin from 12.4 to 10.5% (p = 0.01). Most of the effect occurred during the first week of treatment. Glibenclamide was the more effective the worse the initial metabolic state (r = -0.93; p = 0.001). Erythrocyte insulin receptors decreased markedly in both groups, perhaps due to a sulfonyl urea-induced change in erythrocyte plasma survival time. It is concluded that sulfonylurea treatment is a valuable adjunct in reducing the insulin resistance in insulin treated type II diabetics. The effect depends on the availability of endogenous insulin, thus exhibiting only partly extrapancreatic character.(ABSTRACT TRUNCATED AT 250 WORDS)

Bidirectional alteration of insulin receptor affinity by different forms of physical exercise.

Insulin binding of monocytes and erythrocytes was studied in untrained male volunteers after 15 min of exhaustive bicycle exercise (EE) and several days later after moderate exercise (ME) for 90 min. Insulin receptor affinity decreased after EE in monocytes (26.4% decrease; P less than 0.01) and in erythrocytes (10.4%; P less than 0.05) with no change in receptor number. After ME, however, binding to monocytes was enhanced by 15.2% (P less than 0.05) due to increased receptor affinity. The number of circulating monocytes was markedly increased after both forms of exercise, averaging 105% after EE and 57% after ME. The bidirectional effect on monocyte insulin binding could be reproduced in vitro by incubation of preexercise cells with post-exercise serum: 12.4% (P less than 0.05) decrease with EE serum and 6.1% (P less than 0.05) increase with ME serum. The effect was prevented by overnight dialysis. These results suggest that physical exercise not only entails adjustment of serum insulin but also of cellular hormone sensitivity, presumably through the mediation of low-molecular-weight serum components.

[Dissociation of serum kinetics of amylase and trypsin following stimulation with secretin and pancreozymin]. / Dissoziation der Serumkinetik von Amylase und Trypsin nach Stimulation mit Sekretin und Pankreozymin.

The diagnostic value of basal serum trypsin and amylase values in the assessment of pancreatic exocrine function is limited. Secretin injection evokes a significantly different response in the serum kinetics of trypsin and amylase, due probably to their difference in molecular weight (21,000 vs 52,000 Daltons). Serum trypsin increases in 70% of people with normal secretin-pancreozymin test after stimulation with secretin, whereas amylase remains unchanged. The post-stimulatory rise in trypsin is lower in mild exocrine insufficiency and almost completely abolished in severe exocrine insufficiency. The diagnosis of severe exocrine insufficiency is confirmed in 93% and that of mild insufficiency in 54% by low basal and post-stimulatory levels of serum trypsin. In diabetics with low basal values the post-stimulatory rise in serum trypsin confirmed normal pancreatic function. The poststimulatory kinetics of serum amylase shows no clear correlation to pancreatic function. From the divergent serum kinetics of trypsin and amylase it may be concluded that trypsin is primarily of ductular and amylase primarily of acinar origin.

[Diagnostic significance of pancreatic serum-enzyme patterns after stimulation with secretin in chronic pancreatitis (author's transl)]. / Diagnostische Bedeutund des pankreatischen Serumenzymmusters nach Stimulation mit Sekretin bei chronischer Pankreatitis.

The pancreatic serum evocation test with secretin has regained importance now that it is possible to determine immunoreactive trypsin and pancreatic isoamylase. After secretin stimulation there was a significant abnormal increase in serum trypsin (p less than 0.01) in 34 patients with proven chronic pancreatitis associated with mild to moderate dysfunction (groups I-II), no rise if there was marked insufficiency (group III). Patients with steatorrhoea and obstruction in the region of the head of the pancreas formed a special group because, contrary to other patients in groups III, they had marked serum enzyme rise after secretin. In 24 control subjects with a normal pancreas there was no significant change in basal pancreatic serum enzyme levels with secretin stimulation. Trypsin and amylase reaction patterns differed during secretin stimulation, with a rise in the amylase occurring at the expense of pancreas isoamylase.

Characterization of human exocrine pancreatic proteins by two-dimensional isoelectric focusing/sodium dodecyl sulfate gel electrophoresis.

Exocrine proteins contained in human pancreatic juice were separated in two dimensions using isoelectric focusing and sodium dodecyl sulfate gel electrophoresis. Nineteen discrete proteins were found. Fifteen of these were identified by actual or potential enzyme activity and include three forms of trypsinogen, two forms each of procarboxypeptidase A, procarboxypeptidase B, proelastase, and colipase, and one form each for amylase, lipase, chymotrypsinogen, and prophospholipase A2. Lipase and four unidentified proteins were found to contain carbohydrate by the periodic acid Schiff staining method. Each pancreatic protein was characterized by isoelectric point and molecular weight. Proteins were quantitated according to relative mass, as measured by the incorporation of a mixture of 15 3H-amino acids into secretory proteins contained in tissue slices, and according to the distribution of Coomassie blue R stain among proteins contained in pancreatic juice, as determined by two-dimensional gel scanning and computer analysis. The second form of pancreatic procarboxypeptidase B (IEPn6.7) was present in only 4 of 10 subjects tested. Trypsinogens 1 and 3 were covalently labeled with 35SO4. Trypsin derived from trypsinogen 2 showed no inhibition with soybean trypsin inhibitor or Trasylol.

Transport and utilization of amino acids and glucose in human monocytes: activation of glucose metabolism by insulin.

The influence of insulin on transport and utilization of amino acids and glucose in purified human peripheral blood monocytes has been studied. Insulin had an immediate stimulating effect on the uptake of 3-O-methylglucose and 2-deoxyglucose; the maximal effects were 55% and 47% increases, respectively, during the first 2 min, in which energy-dependent hexose uptake dominates. Later, with advancing free diffusion, values declined to 16% and 25%. After a lag of 30 min, the rise in glucose uptake was followed by a small rise in glucose oxidation, documented by an 18% increase of 14CO2 production from [1-14C]glucose in the presence of hormone. No effect of insulin on sodium dependent alpha-aminoisobutyric acid or sodium-independent leucine uptake in monocytes could be found. The incorporation of amino acids into monocyte protein remained unchanged as well. Our results prove that the well documented binding of insulin to human monocytes initiates specific cellular reactions. The increased hexose monophosphate shunt activity may result in increased immune reactivity of the monocyte.

Amino acid transport in the exocrine pancreas. IV. Do glucagon or insulin mediate the in vivo effect of caerulein on amino acid transport and incorporation?

The direct in vitro effect of caerulein on pancreatic protein synthesis and amino acid transport has been investigated. In contrast to in vivo conditions we were unable to demonstrate any effect on alpha-aminoisobutyric acid and leucine uptake and on leucine incorporation usin rat pancreatic lobules. Insulin and glucagon were therefore examined as possible mediators for the in vivo effect of caerulein. Insulin (1--5 microM) slightly enhanced AIB uptake (16% but did not change uptake and incorporation of leucine. Glucagon (0.01--1 microM) was ineffective. Both islet hormones had no influence on the formation of cyclic GMP induced by secretagogue either in rat (40% increase) or in guinea pig lobules (500% increase). It seems unlikely that the two islet hormones exert any direct effect on the exocrine pancreas and thus could serve as mediators for the in vivo synthetic effect of caerulein.