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.

Blockade of tumour necrosis factor {alpha} significantly alters the serum level of IgG- and IgA-rheumatoid factor in patients with rheumatoid arthritis.

OBJECTIVE: To determine the effect on the humoral immune system of long term treatment of patients with RA with etanercept. METHODS: 12 consecutive patients with seropositive RA treated with etanercept were studied and followed up for 9 months. Clinical efficacy of treatment was evaluated using the 28 joint count Disease Activity Score (DAS28). Serum samples were collected at baseline and after 9 months and serum immunoglobulin, RF isotypes, and anti-cyclic citrullinated peptide (aCCP), antinuclear, nucleosome, and dsDNA antibodies determined. For comparison 7 patients with seropositive RA treated with adalimumab were studied. RESULTS: DAS28 decreased significantly after the first month and then was constant for the whole study (5.7 (0.3) v 3.8 (0.2), p< or=0.000). Serum IgA-RF and IgG-RF increased significantly after 9 months' etanercept treatment (mean (SEM) IgA-RF rose from 19.5 (4.8) to 30.5 (5.9) IU/ml, p< or=0.01; IgG-RF from 20.6 (8.1) to 33.8 (11.5) IU/ml, p< or=0.04). Serum levels of total immunoglobulin and specific autoantibodies remained unchanged during the study. In patients treated with adalimumab, no significant changes in serum levels of RF isotypes and aCCP antibodies were seen. CONCLUSION: Etanercept, although effective in treating the clinical symptoms of RA, seems to have a pivotal effect on RF-producing B cells either directly or indirectly.

[Sense and nonsense in antibiotic therapy for respiratory tract infections]. / Sinn und Unsinn der Antibiotikatherapie respiratorischer Infekte.

Whether an antibiotic successfully eradicates pathogens depends on the pathogens involved, on pharmacokinetics and bioavailability in the target tissue, and on the antimicrobial resistance of the pathogen. Other determinants are drug interactions, individual risk factors, age and compliance with respect to correct dosage and duration of therapy. In many cases, antimicrobial therapy is begun on an empirical basis, because the responsible pathogen can be identified in only half of all respiratory infections. The eradication of the pathogen has to be the first aim if treatment is to be curative and the development of resistance prevented. Long-term prevention of antimicrobial resistance will require a more critical prospective evaluation of the prescription of antibiotics. This paper considers rational and irrational measures in the antimicrobial therapy of respiratory infections.

Clinical interpretation of antineutrophil cytoplasmic antibodies: parvovirus B19 infection as a pitfall.

BACKGROUND: While antibodies directed against proteinase 3 (PR3-ANCA) and myeloperoxidase (MPO-ANCA) have a high specificity for the diagnosis of systemic vasculitis, they may also be found as an epiphenomenon of acute viral infection. OBJECTIVE: To investigate whether positive ANCA test results may be a common feature of acute parvovirus B19 infection. METHODS: Sera were analysed from 1242 patients from a rheumatology outpatient clinic for reactivity with parvovirus B19 and EBV antibodies. They were tested for the presence of PR3-ANCA and MPO-ANCA, along with sera known to contain IgM antibodies to these viruses obtained from among 41,366 samples submitted for virological screening. RESULTS: ANCA were found in 10% (5/50) of the sera positive for IgM antibodies to parvovirus and in 3/51 sera containing IgM antibodies to EBV. Three of six patients with arthritis and concomitant parvovirus infection were found positive for PR3-ANCA and two were found positive for MPO-ANCA. All six patients tested negative for ANCA after six months of follow up. CONCLUSIONS: PR3-ANCA and MPO-ANCA may occur transiently in patients with acute B19 infection or infectious mononucleosis, highlighting the importance of repeated antibody tests in oligosymptomatic clinical conditions in which systemic autoimmune disease is suspected.

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.

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.

Detection and quantification of small numbers of circulating tumour cells in peripheral blood using laser scanning cytometer (LSC).

The detection of circulating tumour cells disseminated from solid tumours requires extremely sensitive methods. Molecular genetic methods, which are most sensitive, are not applicable to solid tumours because no tumour-specific genetic markers are available. Detection of disseminated tumour cells by immunocytochemistry is time-consuming, whereas fluorimetry is fast and quantitative. The laser scanning cytometer (LSC) provides an automated microscopic procedure for screening up to 5x10(4) cells in suitable time. Using this system together with an enrichment procedure which allows up to ten thousand-fold enrichment, we have quantified minimal numbers of tumour cells. In a model system, breast cancer cell line cells diluted into peripheral blood mimicked seeding of tumour cells into the periphery. After staining with fluorochrome-conjugated anti-epithelial antibody, slides were screened for positive events directly or after enrichment with antibody-coated magnetic beads. One positive cell was unequivocally detectable in 10(4) cells and 50 out of 60 tumour cells were reliably recovered from a 20 ml blood volume, equal to 1-2 cells per 10(7), after magnetic bead enrichment. This method allows quantitation of tumour cells in peripheral blood and bone marrow in reasonable time and will, for the first time, enable extensive investigation of the seeding behaviour of tumours.

[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.

Ultrasensitive detection and identification of fluorescent molecules by FCS: impact for immunobiology.

An experimental application of fluorescence correlation spectroscopy is presented for the detection and identification of fluorophores and auto-Abs in solution. The recording time is between 2 and 60 sec. Because the actual number of molecules in the unit volume (confocal detection volume of about 1 fl) is integer or zero, the fluorescence generated by the molecules is discontinuous when single-molecule sensitivity is achieved. We first show that the observable probability, N, to find a single fluorescent molecule in the very tiny space element of the unit volume is Poisson-distributed below a critical bulk concentration c*. The measured probability means we have traced, for example, 5 x 10(10) fluorophore molecules per ml of bulk solution. The probability is related to the average frequency, C, that the volume of detection contains a single fluorescent molecule and to the concentration, c, of the bulk solution. The analytical sensitivity of an assay is calculated from the average frequency C. In the Goodpasture experiment, we determined as analytical sensitivity a probability of 99.1% of identifying one single immune complex. Under these conditions, a single molecule event is proven. There exist no instrumental assumptions of our approach on which the experiment itself, the theoretical background, or the conclusion are based. Our results open up a broad field for analytics and diagnostics in solution, especially in immunology.

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.

Detection and Identification of Single Molecules in Immunology and Immunohematology: Single (solution)-phase Single-molecule FCS.

In the Goodpasture experiment, we determined a probability of 99.1% of identifying one single immune complex. Under these conditions, a single molecule event is proven. There exist no instrumental assumptions of our approach on which the experiments themselves, the theoretical background or the conclusion are based on. Our results open up a broad field for analytics and diagnostics in solution, particularly in immunology and immunohematology.

[Neopterin: new immunological marker of autoimmune rheumatic disease]. / Neopterin: novyi immunologicheskii marker autoimmunnykh revmaticheskikh zabolevanii.

Serum neopterin (SN), concentration of soluble (s) TNF-receptors (R) with molecular mass 55 kD and sIL-2R, C-reactive protein (CRP), Willebrand factor antigen (WF Ag) were measured in enzyme immunoassay (EIA) or radioimmunoassay (BRAHMS, Berlin, Germany) in 189 patients with autoimmune rheumatic diseases: 52 patients with systemic lupus erythematosus (SLE), 67 patients with rheumatoid arthritis (RA), 44 patients with polymyositis/dermatomyositis and 26 patients with Wegener granulomatosis. SN appeared elevated in autoimmune rheumatic diseases correlating with the disease activity and concentrations of sTNF R and sIL 2R. Assay for neopterin is clinically essential for examination of the immunopathological process activity, prediction of the outcomes, better knowledge about cytokine synthesis profile in autoimmune rheumatic diseases.

Immunologic changes after transfusion of autologous or allogeneic buffy coat-poor versus WBC-reduced blood transfusions in patients undergoing arthroplasty. II. Activation of T cells, macrophages, and cell-mediated lympholysis.

BACKGROUND: To estimate the impact of RBC preparations on the status of postoperative immune activation, the soluble cytokine receptors of TNFalpha (sTNF-R) and IL-2 (sIL-2R), as well as neopterin and cell-mediated lympholysis (CML), were measured. STUDY DESIGN AND METHODS: Patients undergoing strictly standardized anesthesiologic management for elective orthopedic surgery were enrolled in a prospective study. The perioperative course (Days 0, 3, 7, and 10) of sTNF-R, sIL-2R, neopterin, and CML was compared after random assignment to allogeneic buffy coat-reduced (Group 2, n = 8) or WBC-reduced (Group 3, n = 11) RBC transfusion regimen. Recipients of autologous buffy coat-reduced RBC transfusions (Group 1, n = 15) served as controls. Patients receiving intraoperatively and postoperatively salvaged blood only (n = 10) were separately analyzed as Group 4. RESULTS: In Group 1, a short-lasting increase in soluble cytokine receptors, a diminished cytolytic response (Day 0 vs. Day 7: sTNF-R, p = 0.0001; sIL-2R, p = 0.0004; CML, p = 0. 0238), and an elevation of neopterin (Day 0 vs. Day 3: p = 0.0064) were observed. In contrast, in allogeneically transfused patients, sTNF-R (Group 2, p = 0.0469: Group 3, p = 0.0039), sIL-2R (Group 3, p = 0.002) and neopterin (Group 3, p = 0.0164) increased further from baseline to Day 10 (Day 0 vs. Day 10), and this increase was accompanied by a diminished cytolytic response (Day 0 vs. Day 10: Group 2, p = 0.05; Group 3, p = 0.0076). Patients in Group 4 showed a short-lasting increase in sIL-2R (Day 0 vs. Day 3: p = 0.0078), neopterin (Day 0 vs. Day 3: p = 0.0156) and sTNF-R (Day 0 vs. Day 7: p = 0.0781). CONCLUSION: Allogeneic transfusions seem to prolong the postoperative status of immune activation, even when WBC-filtered RBCs are used for the transfusion regimen.

[Function and repertoire of the immune system in body's defense]. / Funktion und Repertoire des Immunsystems bei körpereigener Abwehr.

This is a minireview on the organisation and activity of the human immune system with special reference to sport and--more precisely--stress by mountaineering. The activation of the immune system under physical exercise is shown and the immune depression after the sport documented. Hence the conclusion of increased susceptibility to diseases in the post activation phase--a sort of depression after alpine sport.

Tryptophan degradation and immune activation in Alzheimer's disease.

Alzheimer's disease (AD) is likely associated with systemic immune activation. During immune response, interferon-gamma stimulates indoleamine 2,3-dioxygenase (IDO) converting tryptophan to N-formylkynurenine followed by kynurenine in an ensuing step. Thus, IDO activity is estimated by the kynurenine per tryptophan quotient (Kyn/Trp). In 21 patients suffering from AD, in 20 controls of similar age, and in 49 blood donors we measured serum tryptophan and kynurenine concentrations by HPLC. Lower tryptophan concentrations were found in elderly control subjects compared to blood donors (62.1 vs. 73.0 microM, p < 0.005). Tryptophan concentrations tended to be still lower in AD patients (54.4 microM, p = 0.07) compared to elderly controls. Enhanced tryptophan degradation in patients was reflected by significantly increased Kyn/Trp (46.1 vs. 34.1 in elderly controls, p < 0.05). Correlations were found in patients between Kyn/Trp and concentrations of soluble immune markers in serum, i.e., neopterin, interleukin-2 receptor and tumor necrosis factor receptor (all p < 0.001). Increased Kyn/Trp was associated with reduced cognitive performance. Tryptophan degradation due to immune activation may exert impact on the pathogenesis of AD.