Calelectrin in human blood cells.

Calelectrin is a new calcium-binding protein isolated from the cholinergic nerve terminals of the electric organ of Torpedo marmorata, which is widely distributed in nervous tissues and selectively binds to membranes, self-aggregates, and promotes calcium-induced membrane aggregation as a function of calcium concentration. We now show by immunofluorescence and immune blotting procedures that this protein is also present in human blood cells. Immunofluorescence demonstrates calelectrin in all human leucocytes, including mononuclear cells, but not in platelets or in erythrocytes. The immunofluorescence indicates an exclusively cytoplasmic location of calelectrin with a diffuse distribution and no primary association with the cytoskeleton or the cell membranes. SDS-polyacrylamide gel electrophoresis with immune blotting of fractionated blood cells (thrombocytes, mononuclear cells, granulocytes and erythrocytes) reveals the presence of a single protein crossreactive with calelectrin from Torpedo marmorata in the granulocyte and mononuclear cell fractions only. Human calelectrin has a molecular weight similar to Torpedo calelectrin (approximately 34-35 kD) and also binds to membranes in a Ca(2+)-dependent manner. Our results have several implications: (1) Calelectrin is conserved during evolution between the fish Torpedo marmorata and humans; (2) its expression in neural and mesenchymal cells points to an important functional role of the protein; (3) its absence from platelets excludes the hypothesis that it is a necessary participant in exocytosis per se and suggests some other function in Ca(2+)-triggered processes.

Antibodies not directed against the acetylcholine receptor in myasthenia gravis. An immunoblot study.

Sera of 45 patients with myasthenia gravis (MG) and of 30 healthy controls were screened for antibodies against muscle antigens in an immunoblotting solid-phase assay using a preparation of human amputated muscle as the substrate. In each group, the sera showed several bands on the blots. The findings are thought to indicate that antibodies against muscle components are normally present in human sera. Sera from patients or controls could be distinguished by differences in the band staining patterns. It is suggested that antibodies which are not directed against the acetylcholine receptor may nonetheless play an important role in the pathogenesis and clinical course of myasthenia gravis.

Antibody pattern analysis in the Guillain-Barré syndrome and pathologic controls.

The Guillain-Barré Syndrome (GBS) is an acute inflammatory polyneuroradiculopathy, which is considered to be caused by autoimmune processes. A number of single antigenic structures has been suggested to be targeted by the immune system, but a conclusive etiological concept has not been evolved yet. We compared reactions of sera from GBS patients (N=28) and from both two pathological control groups, 25 Multiple sclerosis (MS) patients and 32 patients with other non-inflammatory peripheral polyneuropathies (ONP) and from sex- and age-matched healthy controls (N=30). Porcine peripheral nerve proteins were used as antigens in a Western blot procedure. The blots were analysed by densitometry, and a multivariate statistical comparison of the antibody repertoires was carried out. Antibody patterns of GBS patients differed significantly (p<0.001) from each of the control groups. Discriminant analysis indicated that the discrimination resulted from pattern differences of specific regions of the blots containing proteins with estimated molecular weights of 58-64 and 28-29 kDa. We conclude that statistical analysis of antibody patterns may be helpful both in clinical diagnosis and in further research concerning the pathogenesis of GBS.

Additional biochemical criteria in the differential diagnosis of myositis.

Thirty-six biopsy specimens of human biceps and vastus lateralis muscles were examined by histometric analysis and determination of enzyme activities (phosphorylase, triosephosphate dehydrogenase, 3-hydroxacyl-CoA-dehydrogenase, lactate dehydrogenase, hexose isomerase, citrate synthetase, 6-phosphogluconate dehydrogenase). The series included 13 specimens from patients suffering from a benign form of muscular dystrophy (limb girdle and Becker type of muscular dystrophy) and 12 specimens from patients with an acute (n = 5) or chronic (n = 7) form of myositis. Muscle fibres were atrophic in myositis and hypertrophic (with an increased variation of fibre diameters) in muscular dystrophies, as has been shown previously. When myositis samples were compared with either normal or dystrophic muscles, a highly significant lowering of glycolytic enzyme activity was found in chronic myositis, while the activity of 6-phosphogluconate dehydrogenase was elevated to highly significant levels. Measurements of the latter enzyme's activity might be of additional value in differentiating chronic forms of myositis from benign muscular dystrophies.

Age distribution of latent herpes simplex virus 1 and varicella-zoster virus genome in human nervous tissue.

Latency in nervous tissue caused by herpes simplex virus 1 (HSV-1) and by varicella-zoster virus (VZV) is an intriguing feature of herpes-virus' neurotropism. HSV-1 and VZV latency are the causes of ophthalmic zoster and recurrent HSV infections in the distributions of the trigeminal branches. HSV-1 neuronal latency may play a role in the etiopathogenesis of HSV encephalitis. We attempted to determine the prevalence and age distribution of VZV and HSV latency. We applied nested polymerase chain reaction (PCR) assays to detect HSV-1 and VZV genome in trigeminal ganglia and olfactory bulbs which were obtained from 109 human corpses at forensic postmortems. HSV-1 latency was found in 72.5% of trigeminal ganglia and in 15.5% of olfactory bulbs. VZV latency was 63.3% in trigeminal ganglia and 1% in olfactory bulbs. Simultaneous latency of VZV and HSV genome occurs in 48.8% of trigeminal ganglia. The age-group specific prevalence of HSV neuronal latency increases from 18.2% in 0-20 years to reach finally 100% in persons older than 60 years. Age specific prevalences of VZV peaked for a first time with 82% between 21-30 years, fell to 50% for 40-50 years, and rose to 89% for 71-80 years. If the latent trigeminal ganglion HSV-1 genome were the source of endogenously acquired encephalitis, the peak incidence of HSV encephalitis in older subjects correlates with our findings. Increased VZV latency prevalence in nervous tissue of younger people without subsequent disease indicates sufficient immune surveillance.

A standardized protocol for flow cytometric analysis of cells isolated from cerebrospinal fluid.

Flow cytometry (FC) is an useful tool for the analysis of subpopulations in complex cell suspensions. When applying this method to the cerebrospinal fluid (CSF), some characteristic properties of this cell type must be taken into consideration: there are only few cells which decay rapidly in their native medium and during centrifugation. One aim of the immunostaining procedure preceding flow cytometric analysis must be to minimize cell loss in order to get an undistorted picture of 'true' CSF cell populations. Consequently, morphological flow cytometric plots of high resolution are an indispensable precondition for reliable determination of subpopulations defined by monoclonal antibody (Mab) binding. We describe a standardized protocol for the flow cytometric examination of CSF cells which minimizes undesired cell loss. By the use of a 'quality control' the extent of cell loss could be monitored. Examples of morphological flow cytometric plots are given. The subsequent determination of Mab binding subpopulations is critical when fluorescence intensities of antigen positive and negative cells are non-disjunct. A statistical test was developed for these cases often seen when cell surface determinants are expressed at low levels only.

Repertoires of natural autoantibodies against muscle tissue are independent of age or gender in normal human adults. A western blot study.

Sera from 69 healthy blood donors were screened for autoantibodies using Western blots of human muscle fractions to assess whether prevalence and repertoires of autoantibodies against muscle tissue would change with age. Each serum contained immunoglobulins binding to a broad spectrum of antigens. Their molecular masses ranged from 20 to more than 200 kDa. Although each staining pattern appeared to be a singular combination of detected bands and staining intensities, the patterns were not different in men and women. When sera were grouped according to age (decades between 10 and over 70 years) age-dependent changes were not found. Individual repertoires of natural autoantibodies against muscle tissue are complex when examined by Western blotting. Mature normal repertoires of low-affinity autoantibodies are found at the age of 10 years which is the lower cut-off in this study. The repertoires remain stable throughout a normal lifespan.

[Multiple sclerosis--disclosing the diagnosis. Attitude of patients: when? How? Disclose at all?]. / Multiple Sklerose--Eröffnung der Diagnose. Stellungnahmen Betroffener: Wann?, Wie?, Uberhaupt?

MS patient members of the German Multiple Sclerosis Society were asked about their opinions concerning time and circumstances of getting to know the diagnosis, based on their personal experiences. Citations from 126 answers to a questionnaire, which had been published in the newsletter of the society, are presented to illustrate typical statements. Two thirds of the patients insisted on being informed about the diagnosis as early and as extensively as possible. One third, however, was more reluctant and stated that extensive information about the diagnosis should be timed individually depending on expected development of the disease and disabilities. Information should be extensive, constructive, and tailored to the individual needs of each patient so that he could deal more effectively with his disease. Most contributions voiced severe criticism as to the practice of information experienced. The study was initiated to prepare a more systematic approach to a field which has not been investigated to a degree matching its importance and impact in everyday neurological practice.

Repertoires of autoantibodies against homologous eye muscle in ocular and generalized myasthenia gravis differ.

If weakness of the eye muscles remains the only symptom of myasthenia gravis (MG) for more than 2 years, the condition is operationally defined as ocular MG (OMG). A number of clinical, genetic, and immunological differences between this variant and generalized MG (GMG) have been described. We analyzed repertoires of autoantibodies against proteins of skeletal and extraocular muscle in sex- and age-matched groups of patients with either GMG or OMG (n = 10 in each group). All GMG sera detected a group of three proteins larger than 200 kDa which were not detected by any of the OMG sera. Two components with apparent molecular weights of 50 and 60 kDa were stained by seven of the ten OMG sera but by none of the GMG group. These antigens are probably soluble, cytoplasmatic proteins of the eye muscle. OMG sera, furthermore, detected a protein of about 45 kDa in the pellet fraction of eye muscle but failed to do so after adsorption with skeletal muscle fractions. We conclude that OMG and GMG sera contain autoantibodies of different specificities. Our findings further support immunological heterogeneity in MG.

Identification and classification of autoantibody repertoires (Western blots) with a pattern recognition algorithm by an artificial neural network.

The screening of sera for autoantibodies with Western blots reveals complex repertoires. the compostion of such repertoires depends on genetic control of autoantibody-producing cells, the individual's history of exposure to its own and to foreign antigens, and also on the presence of autoimmune diseases. Our method shows how staining patterns of Western blots can be recoded as binary or grey-value vectors. Vectors are transferred to artificial neural networks for learning. Artificial neural networks are able to recognize group-specific antibody binding patterns. Staining patterns can be attributed to diagnostic groups. This may support diagnostic procedures.

[Abscess of the thyroid gland caused by Salmonella enteritidis in immunosuppressive treatment of generalized myasthenia gravis with thymoma]. / Schilddrüsenabszess durch Salmonella enteritidis unter immunsuppressiver Behandlung einer generalisierten Myasthenia gravis mit Thymom.

We report the case of a 69-year old male caucasian patient who developed a lateral neck tumor while under immunosuppression with azathioprine. The tumor was diagnosed finally as an abscess caused by Salmonella enteritidis after isolation of the agent from blood, tumor biopsy and feces. This extremely rare manifestation of an infection by Salmonella enteritidis is considered as a complication of immunosuppressive therapy.

Identification of a proteoglycan antigen characteristic of cholinergic synaptic vesicles.

An antiserum to cholinergic synaptic vesicles isolated from the electric organ of Torpedo marmorata was purified by adsorption with fractions containing unwanted antigens. The adsorbed antiserum responds to the proteoglycan core material of the cholinergic synaptic vesicles. The major antigen migrates in an anomalous fashion on sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE), forming a broad band with an apparent molecular weight of approximately 120,000 - 300,000. The distribution of this antigen after sucrose density gradient centrifugation of synaptic vesicles is the same as that of vesicular ATP. The antigen comigrates with a substance that can be stained with Alcian-Blue after SDS-PAGE of highly purified synaptic vesicles. This substance is related to the low-molecular-weight, Alcian-Blue-positive glycosaminoglycan vesiculin, which is formed from the high-molecular-weight proteoglycan by prolonged dialysis against water or by protease treatment. No antibodies were detected against vesiculin itself, indicating that the antigenic determinants are restricted to the proteoglycan.

Multivariate statistical comparison of autoantibody-repertoires (western blots) by discriminant analysis.

A procedure for the quantification and comparison of complex (auto-) antibody repertoires of many individuals is described. It is based on multivariate statistical analyses of densitometric data of individual staining patterns, in this case Western blots. Implementation of algorithms can be based on standard software and hardware components. This procedure allows (i) the quantitative assessment of the reproducibility and reliability of any electrophoretic procedure, such as for the production of the immunological substrate, (ii) the statistical comparison of any group of staining patterns, e.g., derived from patients with autoimmune diseases or normal controls, (iii) the identification of the bands that contribute most to the differences between such groups, and (iv) the determination whether an unknown individual sample belongs to a known group. A statistical analysis of autoantibody repertoires has important possible applications: it detects spontaneous or therapeutically induced changes of repertoires, it identifies regions of interest, it supports the isolation of relevant antigens, and it will also be helpful in the diagnosis of autoimmune diseases.

Computer-supported analysis (MegaBlot) of allopurinol-induced changes in the autoantibody repertoires of rats suffering from experimental lens-induced uveitis.

Using a new quantitative immunoblot technique (MegaBlot and artificial neural network) sera of rats with lens-induced uveitis (LIU) were tested against Western blots (WB) of sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) separations of protein fractions from rat lenses. The blots were scanned using digital image analysis and evaluated by multivariate analysis of discriminance and an artificial neural network. Five groups of LIU rats (male Wistar rats, n = 28) were investigated: no treatment, single doses of methylprednisolone (MPR; 7.5 mg/kg body weight, intravenous), allopurinol (AL; 50 mg/kg body weight, intravenous), a combination of both drugs (AL and MPR), and repeated application of AL (ALFR; 50 mg/kg body weight intravenous every 2 weeks during the immunization period and a daily dose of approximately 25 mg/kg body weight, orally). ALFR, MPR, and MPRAL show statistically significant immunological effects in LIU treatment. Classification of WBs using both discriminance analysis or neural network resulted in 100% correct assignment; and 82% (discriminance analysis) and 86% (neural network) of WBs were correctly assigned using only unknown blots for classification (not studied before). The MegaBlot technique is able to detect therapeutically induced changes in autoantibody repertoires allowing therapeutic control and a prognostic view of treatment.

The diagnostic value of serum autoantibodies in endocrine orbitopathy and idiopathic ocular myositis.

Sera of patients with Graves' orbitopathy (GOP) often contain antibodies against retroorbital tissue components. The presence of such autoantibodies has been considered to indicate an autoimmune pathogenesis of the disease. However, their specificity has not been conclusively tested, because studies demonstrating autoantibodies used controls with no lesion in the extraocular eye muscles. Although ocular myositis (OM) is clinically distinct from GOP, damage to the retroorbital muscles is a common histopathological finding in both conditions. Using an immunoblot technique, reactions of sera from patients suffering from either disease were compared. Sera from both groups contained antibodies against a variety of antigenic determinants recognized by both sera. These autoantibodies may have been induced secondary to tissue damage and should be considered to be nonspecific. Because the role of tissue damage was not accounted for in previous studies, evidence concerning antigens supposedly specific for GOP should be reevaluated. The reaction patterns of OM and GOP sera were slightly different. These differences were specific enough to suggest that sera from patients with GOP contain antibodies against eye muscle components that are not present in the sera of patients with idiopathic OM. These findings support the assumption that GOP is an autoimmune disease. However, the major autoimmune targets remain to be identified and their pathogenic relevance is still unclear.

The Lyapunov exponent of heart rate dynamics as a sensitive marker of central autonomic organization: an exemplary study of early multiple sclerosis.

In a study of central autonomic dynamics in early multiple sclerosis, we measured the temporal oscillations of the momentary heart rate (heart rate dynamics). 11 young patients suffering from relapsing remitting definite multiple sclerosis in relapse-free and early stages of illness and 11 healthy controls were examined under vagotonic and sympathicotonic conditions. The temporal structure of the heart rate dynamics was operationalized phase-space analytically through the estimation of the largest Lyapunov exponent. Positive Lyapunov exponents were found in all participants under all conditions indicating deterministically chaotic heart rate oscillations. The variance analysis of these exponents detected no significant effect of sympathetic or vagal activity (experimental condition) but a significant group difference (p < .02). The multiple sclerosis patients were characterized by significantly lower Lyapunov exponents than the healthy controls. This finding suggests a more stable and thus less adaptive central-autonomic organization in early multiple sclerosis.

T and B cell specific immune responses to purified protein derivative in the cerebrospinal cavity may be maintained and regulated independently of systemic immune control.

In vivo activated T cells could be isolated from cerebrospinal fluid (CSF) of a patient suffering from chronic meningitis of unclear origin. Although the patient's skin reactivity to purified protein derivative (PPD) was negative, and peripheral T cells did not proliferate to this antigen in vitro, the majority of T cell clones from CSF specifically recognized PPD on either autologous or allogeneic HLA class II compatible macrophages. Remarkably, peripheral blood mononuclear cells potently suppressed the PPD-specific proliferative responses of healthy donors. The selective enrichment of oligoclonal IgG in the CSF but not in the patient's serum further indicated T and B cell responses lacking systemic feedback control. Analyses of a persisting immune stimulation in the CSF provide a potent diagnostic tool and may explain neurological complications as observed in a number of autoimmune diseases and chronic infections.

Immunomodulating activity of allopurinol in experimental lens-induced uveitis.

PURPOSE: The aim of this study was to evaluate the immunomodulating activity of allopurinol using a model of lens-induced uveitis (LIU) and to compare these effects to those of steroids. METHODS: We tested the sera of both LIU and control rats against western blots (WB) of SDS-PAGE separations of protein fractions from normal and LIU rat lenses. These blots were scanned using digital image analysis. A newly developed technique was used to compare the complex autoantibody (AAB) repertoires. Five groups of LIU rats were investigated: no treatment; single doses of methylprednisolone (MPR; 7.5 mg/kg body wt.i.v.); allopurinol (AL; 50 mg/kg body wt. i.v.); a combination of both drugs (AL and MPR); repeated application of AL (ALFR; 50 mg/kg body wt.i.v. every 2 weeks during the immunization period and a daily dose of approx. 25 mg/kg body wt. orally). RESULTS: Immunization induced complex antibody repertoires against lens proteins. Antibody repertoires of LIU rats were identical, regardless of whether the proteins were obtained from control, uveitis eyes, or corresponding healthy eyes of the same individual. AL showed a dose-dependent immunological effect in LIU treatment. Given as a single dose, AL revealed no significant change in the AAB repertoire; however, ALFR showed very clear modification of the AAB repertoires compared to both controls and rats receiving steroids. CONCLUSIONS: These results suggest dose-dependent effects of allopurinol in LIU treatment. Repeated application during the immunization period induced a strong immunomodulating effect of AL that was not observed after single doses.