On the occurrence of the diabetes-associated antigen GAD 65 in human sera.

BACKGROUND: Glutamic acid decarboxylase (GAD 65) is involved as an antigen in diabetes mellitus type 1 and is generally considered to be located intracellularly in pancreas ß-cells. In this study we demonstrate its appearance in 64 human sera samples representing a cross-section of a blood bank. METHOD: The proof of GAD 65 in sera was done using an enzyme-linked immunosorbent assay (ELISA) setup where it was detected by interaction with corresponding antibodies labeled with an enzyme. The enzyme catalyzes a substrate reaction, resulting in a change of color, that is used for the quantitative evaluation of the antigen-antibody interaction. RESULT: The measurements showed that GAD 65 exists in various amounts in the sera of blood donors, with an average concentration of 58.00 ng/ml. The correlation analysis of samples stored at -80° C and at room temperature demonstrates the stability of GAD 65 at room temperature. The correlation coefficient between the GAD 65 concentrations of samples stored at room temperature and of the same samples after 1 week shows that the molecule remains stable. CONCLUSION: Our results encourage us to propose antigen GAD 65, due to its frequency in human sera in different concentrations and its stability, as a biomarker for the early diagnosis of type 1 diabetes and related inflammations.

The diabetic antigen glutamic acid decarboxylase (GAD 65) in the human peripheral blood.

BACKGROUND: Glutamic acid decarboxylase (GAD 65) is a diabetes-associated antigen which is generally considered to be strictly intracellular. In order to better understand autoimmunity, this study demonstrates the appearance of GAD 65 in the peripheral human blood and presents implications for the diagnosis and therapy of some autoimmune diseases. METHODS: The GAD 65 molecules are detected by their interaction with monoclonal antibodies labeled with dyes in an experimental setup with fluorescence correlation spectroscopy (FCS). These interactions result in changes in Brownian motion measured as fluorescence fluctuations. Sera from 153 patients with diabetes mellitus type 1 and controls were investigated. To enable the representation of the molecule as a model for further discussions, we present structural visualizations of its hydrophobic properties, leading to possible interactions with the cell membrane lipids and epitope locations. RESULTS: The GAD65 antigen could be measured with a sensitivity of 2.65 microg/ml in 'clean systems' resulting from spiking experiments and human sera. The GAD 65 antigen could be identified in 8 patient sera: 4 children with diabetes mellitus type 1 and 4 adults initially taken as controls but who retrospectively showed signs of autoimmunity. CONCLUSION: We conclude that these findings are of significance for the concept of autoimmunity, i.e. in an initial step the immune system is primed by its accessibility to GAD 65. Our experimental results may also be important for the therapy of diabetes mellitus type 1 and other autoimmune diseases by the passive administration of GAD 65 antibodies.

Increased concentrations of neopterin in carotid atherosclerosis.

Activation of T-cells and macrophages may play a role in the pathogenesis of atherosclerosis. Therefore, serum concentrations of the immune activation markers neopterin and soluble interleukin-2 receptor were compared with routine laboratory parameters, candidate risk variables and degree of carotid atherosclerosis. Study subjects were 561 individuals (293 men and 268 women) aged between 50 and 79 years who were enrolled in a cross-sectional community based study (Ischemic Heart Disease and Stroke Prevention Study, Bruneck, Italy). Extent of carotid atherosclerosis was quantitated by an ultrasound B-mode procedure based scoring system. Detailed physical examination and quantification of laboratory and candidate risk variables were performed. By univariate as well as multivariate statistical analyses, serum concentrations of neopterin but not soluble interleukin-2 receptor were significantly higher in subjects with carotid atherosclerosis (men, 8.5 +/- 2.7 nmol/l neopterin; women, 9.6 +/- 3.3) than in those without (men, 6.7 +/- 2.3, P < 0.0001; women, 7.5 +/- 2.3, P < 0.0001). The data show that the macrophage-derived immune activation marker neopterin is closely correlated with the extent of carotid atherosclerosis. Chronic activation of immune cells, preferentially of macrophages, may play a key role in atherogenesis and/or progression of atherosclerosis.

Immune activation markers to predict AIDS and survival in HIV-1 seropositives.

Neopterin concentrations in body fluids of HIV-1 seropositives provide predictive information. In 1986, we examined serum and urine neopterin concentrations in 29 HIV-1 seropositives. Serum levels of soluble IL-2 receptor (sIL2R), soluble CD8 (sCD8), tumour necrosis factor alpha (TNF-alpha) and circulating immune complexes (CIC) were retrospectively analysed in 1989. All individuals had increased serum and urine neopterin, sIL2R and CIC concentrations, 27/29 had increased sCD8 concentrations, whereas all had normal TNF-alpha levels. During a 3-year follow-up, high urine and serum neopterin concentrations were significantly associated with progression to AIDS and with the occurrence of AIDS-associated death. Both neopterin variables were of similar predictive value (p less than 0.001, generalized Wilcoxon test). sIL2R concentrations were of borderline significance in predicting the onset of AIDS (p = 0.05). All other parameters lacked predictive information in our study. We conclude, that chronic immune activation is detectable in almost all HIV-1 seropositives. Chronic immune activation may be associated with HIV-1 replication and may contribute to the immunopathology of HIV-1 infection.

Increased plasma concentrations of circulating intercellular adhesion molecule-1 (cICAM-1) in patients with necrotizing pancreatitis.

The intercellular adhesion molecule-1 (ICAM-1), a membrane glycoprotein, is important in the adhesion of cytokine-stimulated leukocytes to the endothelium of microvessels and their transendothelial migration. Circulating isoforms of ICAM-1 (cICAM-1) are known to be elevated in human serum as an indirect consequence of inflammatory responses. The aim of this study was to investigate whether cICAM-1 levels are elevated in patients with acute pancreatitis within 48 h of the onset of abdominal pain and whether cICAM-1 levels correlate with the severity of the tissue damage. Twenty-five consecutive patients admitted to a medical ICU had elevated cCAM-1 concentrations of 548 +/- 68 ng/ml, significantly different when compared to a control group of 18 healthy subjects (343 +/- 29; p = 0.018). According to the findings of contrast-enhanced CT or laparotomy patients were further divided in a group with acute edematous pancreatitis and a group with acute necrotizing pancreatitis. Pancreatic necrosis was associated with cICAM-1 levels of 729 +/- 106 ng/ml, significantly different from patients with mild disease (367 +/- 48) and controls (p < 0.001). Plasma cICAM-1 levels were not significantly different between healthy subjects and patients with mild pancreatitis. A significant correlation was found between cICAM-1 and C-reactive protein, an acute phase reactant and marker of necrotizing pancreatitis (r = 0.62; p < 0.01). The sensitivity and specificity for the detection of edematous or necrotizing pancreatitis of cICAM-1 plasma concentrations (cutoff point at 500 ng/ml) were 75% and 85%, respectively. These results suggest an enhanced release of ICAM-1 into plasma in the early stage of acute necrotizing pancreatitis. Leukocyte-endothelial cell adhesion may be associated with the inflammatory process of necrotizing tissue damage in acute pancreatitis. It could thus serve as a marker or predictor of a severe clinical course of pancreatitis.

Increased immune activation during and after physical exercise.

The present study has been performed to examine the pattern of immune response during and following a long-duration of physical exercise. We have measured plasma concentrations of serum soluble immune activation markers namely soluble interleukin-2 receptor (sIL-2R), soluble CD8 (sCD8), soluble intercellular adhesion molecule 1 (sICAM-1), soluble CD23 (sCD23), soluble tumor necrosis factor receptor (sTNF-R) and neopterin in 18 individuals before, during (ascent: 3 h, descent: 2 h) and after an alpine tour. Compared to baseline levels, all the parameters were significantly increased on top of the mountain and/or after descent. Within 36 hours after the tour sIL-2R, sCD8 and sICAM-1 decreased. In contrast, sTNF-R and neopterin levels remained higher than baseline throughout the study, only partially decreasing 24 and 36 hours from start. These data show immune system activation induced by physical exercise. The increase of parameters sTNF-R and neopterin, reflecting activation of macrophages, was sustained. The data suggest that immune activation phenomena may be involved in the pathogenesis of impaired immune function after exercise and the exercise-induced asthma.

Elevated plasma levels of soluble tumor necrosis factor receptor (sTNFRp60) reflect severity of acute pancreatitis.

OBJECTIVE: To investigate the role of activated leukocytes in acute pancreatitis, we measured soluble receptors of tumour necrosis factor alpha (sTNFR, p60 subtype) in plasma and evaluated the association of sTNFR with the clinical severity of the disease. DESIGN: Prospective, descriptive study. SETTING: A medical intensive care unit (ICU) in a university hospital. PATIENTS: 25 consecutive ICU admissions of adult patients with acute pancreatitis. MEASUREMENTS AND RESULTS: The clinical severity of the disease was assessed using weights for the worst 17 physiological abnormalities of the Acute Physiology and Chronic Health Evaluation III score over a 24-h period after admission. According to the sum of these weights (giving the Acute Physiology Score, APS) patients were divided into a group with mild pancreatitis (APS < 25) and into a group with severe pancreatitis (APS > or = 25). Soluble TNFR was determined in plasma using an enzyme-linked immunoadsorbent assay. In patients with clinically severe pancreatitis, plasma sTNFR concentrations of 8.8 (16) ng/ ml (median, interquartile range) were significantly higher when compared to patients with mild disease [2.7 (1.5) ng/ml; p < 0.0001]. The sensitivity and specificity of sTNFR plasma concentrations (cutoff point at 5 ng/ml) for the prediction of severe pancreatitis were 90 and 100%, respectively. A highly positive correlation between sTNFR and deviations of physiological parameters from normal (APS score) was demonstrated (r = 0.81). The development of multiple organ failure (MOF) and death was associated with significantly higher sTNFR levels when compared to patients without MOF and survivors [16.4 (17) vs 3.2 (2) ng/ml, p = 0.0014 and 16.0 (18) vs 3.3 (4) ng/ml, p = 0.016, respectively]. For evidence of necrotizing pancreatitis, plasma C-reactive protein concentrations were measured and a significant exponential regression was found with sTNFR (r = 0.77, p < 0.0001). Patients developing pancreatic necrosis, as demonstrated by contrast-enhanced computed tomography, had significantly higher sTNFR concentrations when compared to patients with edematous pancreatitis [9.1 (17) vs 3.2 (2) ng/ml, p = 0.0018). CONCLUSION: The p60 subtype of soluble TNFR is elevated in the plasma of patients with clinically severe acute pancreatitis. This elevation is positively correlated to abnormalities in physiological parameters, development of MOF, and mortality. The association with pancreatic necrosis suggests that, by mediating the effects of TNF, TNFRp60 reflects inflammatory tissue damage leading to severe systemic complications.

Increased prevalence of IgA-Chlamydia antibodies in NIDDM patients.

Chlamydia trachomatis oculogenital infection is a common disease in western societies. Despite the fact that diabetes is accompanied by increased risk for infections, no data on chlamydial infections in the non-insulin-dependent diabetic (NIDDM) patient exist. In our study Chlamydia antibodies were determined using an immunoperoxidase reaction in NIDDM patients (n = 79) and in a local nondiabetic control population (n = 125) which was randomly invited to a medical control visit without any preselection criteria. In total, 46% of diabetics and 55% of controls were IgG-Chlamydia antibody positive (ns). Using IgA-Chlamydia antibodies to define 'seroactive' chlamydial infection, 22% of NIDDM patients and 14% of controls were positive. Thus seroactive chlamydial infection of all patients with proven contact to Chlamydia (IgG-Chlamydia antibody positive) was 47% in diabetics versus 25% in controls, respectively (P < 0.05). Forming subgroups, significance was reached in females (52% vs. 32%, P < 0.05) only, but a similar trend was observed in males (36% vs. 21%, ns). Seroactivity was neither correlated with HbA1c nor with nephelometrically determined total serum immunoglobulins (IgG, IgA). Additionally we observed significantly elevated total IgM and IgA-levels in NIDDM patients whereas IgG-levels were comparable in both groups. In conclusion, seroactive chlamydial infections in subjects with proven contact to Chlamydia are more frequent in NIDDM patients than in nondiabetic controls. Additionally, higher IgM and IgA serum levels might indicate a higher susceptibility to active surface infections in NIDDM.

Cardiac myxomas: 24 years of experience in 49 patients.

OBJECTIVES: In this single-center study we reviewed our experience with a significant number of cardiac myxoma cases occurring over the past two decades. PATIENTS AND METHODS: Cardiac myxomas represented 86% of all surgically treated cardiac tumors at our center. Specifically, there were 49 consecutive patients, each with at least one myxoma. A detailed clinical, immunological, and echocardiographic long-term examination of 37 patients revealed one recurrent myxoma. RESULTS: Most myxomas originated from the left atrium (87.7%), but also much less frequently from the mitral valve (6.1%), from the right atrium (4.1%), and from the left and right atria (2.0%). The myxomas produced a prolapse into the left ventricle in 40.8% of the patients, mitral stenosis in 10.2%, and threatened left ventricular outflow tract obstruction in 2.0%. Multiple myxomas were found in 20.4% of the patients. Cardiac signs appeared in 93.9% of the patients. Preoperative embolic events had occurred in 26.5%. Immunologic alterations were present in 87.5%. For resection, a bilateral atriotomy was used. An additional aortotomy was needed to expose one mitral valve myxoma. Postoperatively, 81.1% of the patients remained without cardiac symptoms. The early mortality rate was 2.0% and the late mortality rate was 6.1%. Long-term prognosis was excellent with an actuarial survival rate of 0.74. Specific immunologic alterations were found in 71.4% of the patients. The actuarial freedom from reoperation of the myxoma was 0.96. The rate of reoperations was low with 2.0% after 24 years. CONCLUSIONS: Myxomas were usually detected and operated on in symptomatic patients. A high index of suspicion seems important for early diagnosis. Immunologic findings may play an additional role in confirming the diagnosis and the recurrence of a myxoma. Immediate surgical treatment was indicated because of the high risk of embolization or of sudden cardiac death. Also, a familial genesis must be excluded in myxoma patients.

[Sonography and cytology of cold thyroid nodules]. / Sonographie und Zytologie von kalten Schilddrüsenknoten.

270 patients with a scintigraphically cold thyroid nodule of sonographically increased (n = 34), diminished (n = 72) or neutral (n = 86) echogenity or cystic criteria (n = 78) were subjected to fine needle aspiration biopsy. This revealed unequivocal malignancy in 8 and follicular neoplasia in another 30 patients, 10 of whom proved to have malignomas on further evaluation. A total of 12 papillary and 2 follicular carcinomas, 2 non-Hodgkin lymphomas, 1 sarcoma and the metastasis of a breast carcinoma were diagnosed. The most sensitive criteria for malignancy were diminished echogenity, an inhomogeneous echo pattern and the occurrence of a solitary nodule. The incidence of malignancy was increased among males but not among especially young persons. There was no sonographic feature that would permit omission of fine needle aspiration.

Detection of tumour cells in the peripheral blood of patients with breast cancer. Development of a new sensitive and specific immunomolecular assay.

Malignant cells in the peripheral blood of patients with solid tumours are of considerable importance for the prognosis and therapeutic correlation. Their detection however is difficult due to lack of sensitivity, specificity and technical problems in standardisation. In this original article we show a new sensitive method overcoming the hitherto known difficulties by combining traditional antibody-techniques with a RT-PCR. Due to this method 2 tumour cells within 5 ml of peripheral blood can be detected in spiking experiments.

Degradation of tryptophan in neurodegenerative disorders.

In patients with neurodegenerative disorders, namely Alzheimer's disease and Huntington's disease, we compared serum concentrations of tryptophan, kynurenine and the kynurenine per tryptophan ratio with concentrations of soluble immune activation markers. Significantly lower tryptophan concentrations were observed in the patients, and lower tryptophan levels as well as higher kynurenine levels and higher kynurenine per tryptophan ratios correlated with higher concentrations of neopterin, and soluble receptors for TNF and interleukin-2. In both groups of patients tryptophan concentrations correlated inversely with the degree of mental retardation. No such association existed for the duration of the disease. The data show that systemic chronic immune activation in patients with Alzheimer's disease and Huntington's disease is associated with significant degradation of tryptophan, which is most likely due to activation of indoleamine (2,3)-dioxygenase by immunologic stimuli. Further studies will be necessary to investigate a potential role of tryptophan degradation in the pathogenesis of neurodegenerative disorders.

Time course of plasma soluble intercellular adhesion molecule-1 (sICAM-1) is related to severity of acute pancreatitis.

BACKGROUND/AIMS: In severe acute pancreatitis the release of cytokines indicates a key step from local to systemic inflammation. Increased plasma concentrations of circulating soluble intercellular adhesion molecule-1 (sICAM-1), a marker of leukocyte activation, were detected in necrotizing pancreatitis at the time of diagnosis, however, the exact role of sICAM-1 in the development of complications such as shock or organ dysfunction is unclear. Therefore, we investigated in what manner the time course of plasma sICAM-1 is associated with the development of severe pancreatitis and whether these results are of any predictive value for the further course of the disease. METHODOLOGY: In a medical intensive care unit we studied 29 consecutive patients admitted for acute pancreatitis. Plasma levels of sICAM-1 were measured serially over a period of 6 days and the time courses were assigned either to a group of patients with uncomplicated, mild disease or to patients who developed complications including multiple organ failure. RESULTS: In mild pancreatitis, decreasing and peak sICAM-1 concentrations were found in 88% of the patients with a mean maximal level of 574 +/- 59 ng/ml (SE) (upper limit of normal: 400 ng/ml) on day 1. Partial pancreatic necrosis was present in 24% and no deaths were observed. In severe pancreatitis an increase of sICAM-1 levels or an initial fall followed by an increase (relapsing response) was the predominant pattern (92%). Maximal values of 1453 +/- 136 ng/ml occurred on day 6, significantly different when compared to mild disease (p < 0.0001). Necrotizing pancreatitis was diagnosed in 75% and the mortality rate was 58%. The sensitivity in predicting severe pancreatitis using sICAM-1 plasma levels with an increasing or relapsing pattern was much higher (92%) when compared with serial C-reactive protein measurements (42%). CONCLUSIONS: In acute pancreatitis, increasing or relapsing plasma levels of sICAM-1 over 6 days after admission to hospital are associated with a high rate of pancreatic necrosis and a high mortality. Daily measurements of sICAM-1 would allow early recognition of patients prone to develop complications and follow a severe course.

[Bone marrow transplantation for aplastic anaemia in a ten-year-old girl (author's transl)]. / Knochenmarkstransplantation an einem 10jährigen Mädchen mit Panmyelopathie.

A 10-year-old girl suffering from post-hepatitic aplastic anaemia received 21 x 10(10) (5 x 10(8)/kg body weight) nucleated cells from her brother, whose HLA typing, but not his blood group, was identical with the patient's. Conditioning had been carried out with cyclophosphamide according to the Seattle protocol (50 mg/kg body weight on four successive days). On day 9 she showed signs of early engraftment by the appearance of granulocytes and their precursor cells in the peripheral blood. The new blood group of the patient's erythrocytes and the male karyotype of her leucocytes demonstrate successful engraftment. The child is now in excellent health 18 months after transplantation and requires no treatment of any kind. This paper reports the first bone-marrow transplantation in Austria.

[Clinical experiences with Wegener's granulomatosis from the nephrologic viewpoint]. / Klinische Erfahrungen mit der Wegenerschen Granulomatose aus nephrologischer Sicht.

Immunosuppressive treatment with cyclophosphamide and prednisolone has markedly improved the prognosis of Wegener's granulomatosis (WG). In patients with WG the detection of anti-neutrophil-cytoplasmic autoantibodies (ANCA or ACPA) has become an important parameter to confirm or even routinely establish the diagnosis of WG over the past few years. From 1985 to 1990 we observed and treated 12 patients (5 males and 7 females) aged 14-64 years (mean 41.1 years) with WG. In the same time span an analysis of 35 patients with rapidly progressive glomerulonephritis showed that the octiology was idiopathic in the majority of cases (54.3%), but nevertheless 8 cases (22.8%) were caused by WG. In 9 out of 10 cases the ANCA test was positive; whereas in 8 out of 10 cases we found a close correlation between the serum level of ANCA and disease activity. However, extraordinarily high serum levels (titres up to 1:2560) were recorded in one patient with WG without any clinical symptoms, whilst another patient with severe symptoms of WG showed a titre of only 1:5. 10 out of 12 patients were successfully brought to remission under cyclophosphamide-cortisone treatment. 4 out of 10 patients with renal insufficiency have been retained on the chronic haemodialysis regimen. 2 patients, aged 31 and 51 years, died within 2-5 months after the onset of clinical symptoms of WG.

Unilateral high-altitude pulmonary edema (HAPE): a case report and discussion of pathophysiology.

High-altitude pulmonary edema (HAPE), a potentially life-threatening altitude adaptation disorder, is considered to be caused by an exaggerated increase in pulmonary blood pressure and a non-cardiogenic rise in pulmonary vascular permeability subsequent to alveolar hypoxia. A 40-year-old male mountaineer was affected by an advanced stage of HAPE at high altitude (Monte Rosa plateau, 4000 m). The symptoms abated immediately after the patient descended from the altitude. However, six hours after the symptoms had resolved, radiographic signs of pulmonary edema, confined to the right lung, were seen. This rarely described unilateral radiological pattern of HAPE resolved completely within two days. We suggest that aspiration events of nasal secretion, the right sleeping position at night and an elevated right diaphragm reduced the patient's compensatory hyperventilation capacity of the right lung. The resulting increased alveolar hypoxia in the right lung was responsible for unilateral edema. The pathophysiological mechanism underlying unilateral HAPE is discussed.

[Soluble adhesion molecules (P-selectin, ICAM-1 and ICAM-3) in rheumatoid arthritis]. / Rastvorimye molekuly adgezii (P-selektin, ICAM-1 i ICAM-3) pri revmatoidnom artrite.

AIM: Investigation of serum levels and clinical role of soluble intercellular molecules of adhesion (pICAM-1, PICAM-3 and pP-selectin) in rheumatoid arthritis (RA). MATERIALS AND METHODS: Enzyme immunoassay with Bender MedSystem kits (Austria) was employed to measure serum concentration of soluble intercellular molecules of adhesion in 36 RA patients. RESULTS: Elevated levels of serum pICAM-1, pICAM-3 and pP-selectin were registered in 74.2, 28.6 and 25.7% of RA patients, respectively. Content of pP-selectin more strongly correlated with activity and severity indices than that of pICAM-3 (p < 0.001). Content of pICAM-1 and clinical picture of RA were unrelated. CONCLUSION: Levels of pP-selectine can characterize RA activity.

[Soluble receptors of TNF-alpha in rheumatoid arthritis]. / Rastvorimye retseptory faktora nekroza opukholi pri revmatoidnom artrite.

The serum level of soluble TNF-alpha receptors with molecular mass 55 kDa (sTNF-a55R) was measured by enzyme immunoassay with commercial kits in 30 patients with rheumatoid arthritis (RA) and 38 healthy donors. High sTNF-a55R serum levels were registered in 90% of RA patients. These levels correlated with RA activity by DAS. Thus, assay for sTNF-a55R can be used for assessing RA activity.