[Blocking secretion of exocrine glands in the head-neck area by administration of botulinum toxin A. Therapy of a rare disease picture]. / Sekretionshemmung exokriner Drüsen des Kopf-Hals-Bereiches durch Applikation von Botulinum Toxin A. Therapie seltener Krankheitsbilder.

BACKGROUND: Hypersecretion disorders of the exocrine glands of the head and neck area are a therapeutic problem in the field of otorhinolaryngology. In the present study, we demonstrate the effectiveness of local injections of botulinum toxin A to block secretions of exocrine glands of the head and neck area. PATIENTS AND METHODS: Four patients suffering from hypersecretion disorders received local injections of botulinum toxin A. Two patients suffered from disorders of the salivary glands: one presented an idiopathic hypersialorrhea and another a salivary fistula after parotidectomy. A third patient suffered from epiphora and a further patient presented severe hyperhidrosis on the pilose head region. In a retrospective clinical study, the outcome of therapy was evaluated by clinical examination and chemical parameters. RESULTS: Clear blocking of secretion in the treated glands could be demonstrated in all four cases. Possible side effects of the treatment could not be observed. CONCLUSIONS: The present study was able to demonstrate a clear blocking of secretion of the exocrine glands of the head and neck region through botulinum toxin A, offering an improvement in therapy especially for the innovative indication of blocking the salivary glands of the head.

Development of an ultrasensitive enzyme immunoassay for the determination of matrix metalloproteinase-9 (MMP-9) levels in normal human cerebrospinal fluid.

Determination of matrix metalloproteinase-9 (MMP-9) in human cerebrospinal fluid (CSF) to study blood-brain barrier impairment and immune cell migration in inflammatory neurological diseases recently became a matter of major interest. Regularly, MMP-9 was determined qualitatively or semi-quantitatively by zymography (gelatin gel electrophoresis) or quantitatively by enzyme immunoassay (EIA). As yet, it was not possible by either method to detect MMP-9 in CSF of controls (patients without pathologically increased CSF parameters). We developed an ultrasensitive two-side enzyme-linked immunosorbent assay (ELISA) which allows for the first time to measure reliably MMP-9 concentrations in CSF of controls. This ELISA uses a monoclonal as capture and a polyclonal as detector antibody. The detection limit of the assay is below 10 pg/ml and the assay range is 15-2000 pg/ml. Intra-assay precision is 2.5% for low and 3.7% for high, inter-assay precision is 11% for low and 10.7% for high values, respectively. The determination of the MMP-9 concentration in 50 control CSF gave the following results: range, 22-146 pg/ml; median, 76 pg/ml. The measurement of native and recombinant MMP-9 was carried out with three commercially available ELISAs, most widely employed in MMP-9 research, and compared to the newly developed one. All ELISAs recognize recombinant MMP-9 by factors of 5-20 less sensitively than native MMP-9.

Migration of human granulocytes through reconstituted basement membrane is not dependent on matrix metalloproteinase-9 (MMP-9).

Transmigration of human granulocytes across a basal lamina equivalent was studied in vitro. Transwell inserts were coated with Matrigel, a reconstituted basement membrane. Granulocytes (2x10(6)) were applied to the upper chamber. As chemoattractant interleukin-8 (IL-8; 25 ng/ml) was added to the lower chamber. After 1 h of migration, cells were counted in the lower chamber. Specific hydroxamate inhibitors of MMPs (BB-3103, Ro 31-9790) or of serine proteases (Pefabloc, leupeptin) were added at various concentrations to both chambers before the start of migration. Additional experiments were performed with alpha(2)-macroglobulin, a natural inhibitor of MMPs and a monoclonal antibody which specifically blocks the activity of MMP-9. Migration of granulocytes through Matrigel could not be reduced significantly by any of the MMP inhibitors. A dose-dependent impairment of transmigration was only found with Pefabloc, however, this substance also induced severe morphological changes of the cells. The other inhibitor of serine proteases, leupeptin, did not influence migration at all.

Matrix metalloproteinase-9 is elevated in serum of patients with amyotrophic lateral sclerosis.

Matrix metalloproteinase-9 (MMP-9) and its specific inhibitor, tissue inhibitor of matrix metalloproteinase-1 (TIMP-1), were analysed by enzyme-linked immunosorbent assay (ELISA) and by zymography in serum and cerebrospinal fluid (CSF) of patients with amyotrophic lateral sclerosis (ALS). In contrast to patients with inflammatory diseases, MMP-9 levels were not elevated in CSF of ALS patients. In serum, however, compared to healthy donors, MMP-9 was significantly (p = 0.0003) increased up to levels as high as those of viral meningoencephalitis (VM) or bacterial meningitis (BM) patients. MMP-9 levels remained elevated during long-term observation of ALS patients. In the absence of an inflammatory response, the results indicate that the increase of MMP-9 in serum of ALS patients might be caused by upregulation of MMP-9 in denervated muscles or in degenerating peripheral nerves following motor neurone loss.

Matrix metalloproteinase-9 (MMP-9) in human cerebrospinal fluid (CSF): elevated levels are primarily related to CSF cell count.

Matrix metalloproteinase-9 (MMP-9) was investigated by enzyme-linked immunosorbent assay (ELISA) and zymography in 111 paired CSF and serum samples from patients with various neurological disorders. In 20 patients with blood-brain barrier (BBB) impairment but normal CSF cell count, elevated levels of MMP-9 were not observed by ELISA measurement. Another 11 patients characterized in the same way, exhibited only slightly increased MMP-9 levels. In contrast, in 12 patients with intact BBB but elevated CSF cell count, MMP-9 was increased too. It was shown by the more sensitive zymography that MMP-9 increased if CSF cell count exceeded five cells per microl. Spearman rank statistics revealed that MMP-9 concentration in CSF correlated with CSF cell count (r=0.755; P<0.0001), but not with CSF/serum albumin ratio (Q(Alb)) (r=0.212; P=0.057), a measure for BBB impairment. Moreover, the CSF/serum MMP-9 ratio (Q(MMP-9)) did not correlate with Q(Alb)(r=0.192; P=0.100). By use of a Boyden chamber, in which granulocytes migrated through a reconstituted basement membrane, it was demonstrated that the MMP-9 concentration in the lower chamber correlated very significantly with the number of accumulated cells (r(2)=0.7692; P<0.0001). The meaning of the increase of MMP-9 in CSF is critically discussed.

Riluzole does not have an acute effect on motor thresholds and the intracortical excitability in amyotrophic lateral sclerosis.

Intracortical excitability in amyotrophic lateral sclerosis (ALS) is impaired. The effectiveness of the glutamate antagonist riluzole (Rilutek, Rhône-Poulenc Rorer) in ALS has been shown in clinical studies. In healthy subjects it modifies intracortical excitability in a frequently used double-stimulus paradigm of transcranial magnetic stimulation (TMS). Under riluzole intracortical inhibition is enhanced in healthy individuals, although not always significantly, whereas intracortical facilitation has been described as reduced [10, 11]. We wanted to find out whether riluzole affects and potentially rebalances impaired intracortical excitability in ALS. We, therefore, enrolled 13 patients with clinically and electromyographically confirmed ALS into this study. Five patients had to be excluded because motor thresholds were too high to get reliable motor evoked potentials (MEPs). In the remaining 8 patients, mean age was 59.9 +/- 11.9 years (+/- standard deviation) and mean symptom duration 9.6 +/- 2.5 months. Intracortical excitability was assessed before and 1.5 hours after the first intake of a loading dose of 100 mg of riluzole using a conventional paired-pulse TMS paradigm with interstimulus intervals (ISI) ranging from 1-30 ms and intensities adjusted to yield MEPs of 1.0 mV for test pulses and of 90% active motor threshold for conditioning pulses. Patients' baseline results were compared to those of 9 age-matched, healthy control subjects. Before drug intake, motor thresholds did not differ between groups, but there was significantly less intracortical inhibition in the ALS patient group. Riluzole intake did not significantly alter motor thresholds or intracortical excitability in the ALS patients. We conclude that riluzole does not immediately influence intracortical excitability in ALS. Our results are in contrast to the findings of Stefan et al (1998) [14] where a partial normalization of intracortical inhibition in ALS was observed after at least 5 days of drug intake. The difference between that study and our result may indicate a delayed onset of riluzole's influence on intracortical excitability.

Experimental pneumococcal meningitis in rabbits: the increase of matrix metalloproteinase-9 in cerebrospinal fluid correlates with leucocyte invasion.

Gelatinolytic activity of matrix metalloproteinases (MMPs), particularly MMP-9 and MMP-2, was studied by quantitative zymography in a rabbit model of bacterial meningitis during 24 h after inoculation with Streptococcus pneumoniae. In cerebrospinal fluid (CSF), MMP-2 was constitutively present and its level did not change during the experiment. In contrast, MMP-9, hardly detectable in CSF of healthy animals, increased dramatically. The increase of MMP-9 was correlated with both, an increase of CSF cell count and of total protein concentration. Intrathecal production of MMP-9 and MMP-2 was demonstrated by zymography of equal amounts of total protein from CSF and serum. Homogenates, prepared from various cortical regions of infected rabbits did not show increase of MMP activities. On the other hand, leucocytes isolated from CSF expressed high levels of MMP-9 suggesting a significant contribution of these cells to the elevation of MMP-9 activity in this body fluid.

Progressive dementia with parkinsonism in corticobasal and brainstem degeneration with neuronal inclusions.

Pathologic findings are presented in a case of neurodegenerative disease with a 5-year course of progressive presenile dementia and parkinsonism. At autopsy, atrophy of the cerebral cortex, caudate nucleus, and substantia nigra were observed. Homogeneous eosinophilic intracytoplasmic neuronal inclusions were found, predominantly in the brainstem and cerebellar nuclei. These inclusions were nonargyrophilic and did not stain with antibodies against tau and ubiquitin.

Decrease of S100 beta protein in serum of patients with amyotrophic lateral sclerosis.

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease of unknown origin characterized by loss of upper and lower motor neurons and concomitant astrogliosis. We have investigated the S100 beta protein levels in serum as a marker for astroglia of patients with ALS (n = 41) in comparison to a control group (n = 32). Additionally we have investigated 12 patients at different follow-up time points (minimum 6 months). We could not observe a significant difference of S100 beta protein in patients with ALS in comparison to our control group (P = 0.11) but we could clearly see a decrease of S100 beta levels in the further course of the disease. As S100 beta is also seen as a protein with nerve growth factor activity we assume that the fall of serum levels may reflect the loss of nerve growth stimulation in patients with ALS and suppose that repetitive measurements of S100 beta in serum can be used as an objective marker for disease progression.

Detection of T cells reactive to intestinal alkaline phosphatase, an autoantigen in acute bacterial infections, and discrimination between autoantigen-specific CD5+ and CD5- B cells.

Autoantibodies of the IgG isotype, specifically directed against intestinal alkaline phosphatase (IAP), occur transiently in the majority of sera from patients with acute bacterial infections. Sometimes they are observed in autoimmune diseases. Using a T cell proliferation assay, it was found that isolated peripheral blood mononuclear cells (PBMC) from IAP autoantibody (IAPA)-positive patients (n = 18) responded significantly to IAP, whereas proliferation could not be induced in PBMC from healthy donors (n = 11). Significant stimulation of PBMC from patients (n = 11) was not obtained by use of transferrin, a common autoantigen in humans, indicating the specificity of stimulation of IAP-reactive T cells. Furthermore, T cell proliferation was observed when a highly purified IAP fragment (CNBr 21) spanning amino acids 334-462 of the primary structure of IAP was used as antigen. Thus, it was shown that an immunodominant T cell epitope resides within the CNBr 21 fragment which also contains a discontinuous B cell epitope as evaluated previously. Double immunocytochemical staining of T cell-depleted PBMC with IAP and an anti-human CD5 antibody allowed the detection of CD5+ B lymphocytes, which probably produce natural IAPA (nIAPA). These nIAPA-specific CD5+ B cells occurred with approximately the same frequency among T cell-depleted PBMC from healthy donors and those from patients. In contrast, IAPA-producing CD5- B cells were found in B cell-enriched preparations from patients, but not in those from healthy individuals.

Definition of a discontinuous immunodominant epitope of intestinal alkaline phosphatase, an autoantigen in acute bacterial infections.

In patients suffering from acute bacterial infections specific autoantibodies of the immunoglobulin class G (IgG), directed against intestinal alkaline phosphatase (IAP), are transiently expressed in high titer. The epitopes on IAP which are recognized by these IAP auto-antibodies were investigated using chemical and enzymatic cleavage, followed by two-dimensional electrophoresis and immunoblotting of the fragments. Immunoreactive and nonreactive cleavage fragments were isolated and N-terminally sequenced. An epitope map was constructed by means of sequencing data, relative molecular mass of the fragments, and specific cleavage sites. To evaluate linear epitopes, the overlapping region of the immunoreactive fragments (amino acids 204-484 of the primary structure of IAP) was further analyzed by simultaneous synthesis of 95 peptides on an activated membrane. Four immunoreactive regions were revealed by immunodetection with IAP autoantibody-positive sera. Nine peptides comprising these regions were synthesized quantitatively and coupled to CH-Sepharose. However, specific IAP autoantibodies could not be isolated. This result indicated the absence of continuous epitopes at least in the analyzed region of the molecule. Binding studies with an IAP cleavage fragment suggested a discontinuous epitope involving amino acids 334-371. The results are indicative of an antigen-driven mechanism for the production of IAP autoantibodies initiated and maintained by self.

A putative enzyme from various secretions specifically inhibits antibody-antigen interactions.

Various human secretions (intestinal secretion, saliva, nasal mucus, lacrimal fluid) have been found to inhibit the binding of antibodies to their antigens. Various characteristics (e.g. time, pH, temperature dependence, affinity and size exclusion chromatography) suggested that the inhibitory activity was attributable to an enzyme. Further investigations revealed that this enzyme reacted with the Fab portion of immunoglobulin G, specifically with the heavy chain. It is assumed that it represents a novel immunoglobulin-specific protease since similar results were not obtained with proteolytic enzymes from human digestive organs e.g. pepsin, trypsin and chymotrypsin. Finally, investigating saliva it was demonstrated that the putative protease was not identical to enzymes from periodontal bacteria which are proteolytic for the Fc portion of immunoglobulins. The findings could be of general importance in the design of immunoassays which are to be applied to human (and possibly animal) secretions.

Origin and immunoregulation of autoantibodies against intestinal alkaline phosphatase.

In the sera of patients with acute bacterial infections specific autoantibodies (sIAPa) of the immunoglobulin class G (IgG) were found which bind to intestinal alkaline phosphatase (IAP) through the Fab portion. This was demonstrated using immunoaffinity (IA) isolation of sIAPa from patients' sera (particularly bacterial meningitis and ventriculitis) digestion with pepsin, purification of F(ab')2 fragments on protein A and subsequently binding on IAP coupled to CNBr (cyanogen bromide)-activated Sepharose. Immunoblots using specific anti-Fc and anti-Fab antibodies showed that the bulk of F(ab')2 fragments had bound. Additionally, binding of native IAP to the F(ab')2 fragments was observed after separation of F(ab')2 fragments using isoelectric focusing (IEF), blotting onto nitrocellulose and incubation with IAP. Moreover, we have demonstrated the occurrence of natural anti-IAP autoantibodies (nIAPa) which were isolated from sera of healthy individuals using IA chromatography. Investigation of isotype distribution revealed that IgG but not IgM or IgA were predominant even among nIAPa. The nIAPa fraction exhibited lower binding efficiencies on IEF blots than the sIAPa fraction, however, in contrast to sIAPa, cross-reactions with other autoantigens were observed for nIAPa. NIAPa and sIAPa did not show subclass restriction. As revealed by IEF the spectrotypes of sIAPa were found to be patient-specific, poly- to oligoclonal and stable during longer periods.

[Creutzfeldt-Jakob disease. Report of a case with an unusually long course and immunohistochemical localization of the prion protein and overview of current information]. / Die Creutzfeldt-Jakob-Krankheit. Bericht über einen Fall mit ungewöhnlich langem Verlauf und immunhistochemischer Lokalisierung des Prionproteins sowie Uberblick über den heutigen Wissensstand.

The human spongiform encephalopathies are a group of neurodegenerative disorders of unknown origin. They comprise a group of horizontally transmissible and genetically determined diseases. We present here a case of Creutzfeldt-Jakob disease with an unusually long clinical course, in which the prion protein was localized immunohistochemically. The generally accepted hypothesis on the pathogenesis of these diseases is the so-called 'prion-hypothesis'. The implications of this hypothesis are discussed and a short review of the literature is given.

Human intestinal alkaline phosphatase-binding IgG in patients with severe bacterial infections.

Patterns of alkaline phosphatase (AP)-binding proteins were observed in the alkaline pH range of 6.5-9.5 upon isoelectric focusing and blotting of serum from patients with inflammatory diseases. After isolation using affinity chromatography on protein A or immunoaffinity chromatography on AP coupled to cyanogen bromide (CNBr)-activated Sepharose, the AP-binding protein was identified as IgG on Western blots and in ELISA using human IgG-specific antibodies. It was shown that this IgG binds to AP from both calf (bovine) and human intestine. However, it binds neither to the human liver-bone-kidney (LBK) isoform nor to bacterial AP. Moderate reaction was observed with human placental AP. Comparing patients with various diagnoses (n = 284), AP-binding antibodies were mainly found in severe bacterial infections. They were not detected in serum from healthy blood donors (n = 300). The presence of AP-binding IgG was independent of the infected organ and the bacterial species causing infection. This antibody may be useful for discriminating bacterial from viral infection and for indicating severe bacterial inflammation.

Reactivity of locally produced CSF antibodies in patients with neurosyphilis against antigens of Treponema pallidum.

The reactivity and specificity of locally produced cerebrospinal fluid (CSF) antibodies against antigens of Treponema pallidum were assessed by Western blotting in patients with clinical signs of parenchymal or meningovascular neurosyphilis. All nine patients showed local production of treponeme-specific antibodies in the central nervous system (CNS). In most of the patients serum and CSF antibodies were bound to the same antigens: the common treponemal 48/45 kDa protein and the putative specific T. pallidum protein in the range of 12-14 kDa. In some patients the intensity of staining obtained by CSF antibodies was higher than that derived from serum, indicating locally produced antibodies. In contrast to other more acute inflammatory CNS diseases, no expanded or different antigen binding of the CSF antibodies compared with serum antibodies was found in neurosyphilic patients. The results presented are discussed with regard to the role of the blood-brain barrier in antibody concentrations of CSF and serum.

Specific antigen binding by activated cerebrospinal fluid B lymphocytes in acute neuroborreliosis.

A recently developed immunocytochemical antigen-binding sandwich test for the identification of specific activated B lymphocytes was applied to cerebrospinal fluid cells and peripheral blood mononuclear cells in patients with acute neuroborreliosis. Discrimination of antigen-binding phagocytes was achieved by double staining with monoclonal antibodies. Specific activated B lymphocytes were much more numerous in cerebrospinal fluid than in blood, showing great interindividual differences. When intrathecal immunoglobulin production was present, the number of specific activated B lymphocytes was also high. The specificity of all activated B lymphocytes ranged from 10% to 60% and was higher in the acute stage than after treatment.