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The Byblos writing system (ca. 1500 BC) comprises 15 inscriptions, with the largest one containing 461 clearly discernible signs. Some further 28 unassigned fragments found in the Levante and in Italy are tentatively assigned to the Byblos corpus too. We have created a Unicode letter for each sign variant found in these inscriptions and transcribed the corpus. Feeding the corpus into our RegEx- based deciphering tool, we established a preliminary (dynamic) syllabary laying a foundation for computer-assisted deciphering efforts of the Byblos script. A first sequence analysis allows to detect morphemes and word boundaries, so that even without knowing the sound values, we gain some insight into the syntax of the language depicted by these beautiful graphemes.

Hygro-mechanical phenomena induced by moisture loading of a historical wooden panel painting from the 16th century are investigated in this study. Due to swelling and shrinking strains resulting from an inappropriate and restraining support frame, damage in terms of ruptures on the painting itself and large curvature have developed. A brief history of the painting and its conservation status are summarised. A two-phase multi-Fickian diffusion model for moisture transport in wood and a consistently coupled hygro-mechanical simulation are applied to explore the influence of different realistic scenarios of climatic change on the panel painting itself and its supporting frame. The hereby gained results can be used to predict the general mechanical behaviour, and help to identify critical parts of the structure and risky climatic situations, which could increase damage. New methods of conservation in terms of innovative stabilising wood frames and adjusted climatic conditions can efficiently be evaluated with the help of the presented approach.

Date : Around 2200 BC. Location : Western, southern and eastern Iran. Type : Syllabic Script. Text Corpus : 22 (known a long time), plus 15 (known since 2015). Sign Corpus : 110 sign type, 1340 sign tokens. Status : Principally undeciphered, except the sound values for in , su , us, si , na , and k , drawn from the divine name Insusinak found in the only bilingual inscription. Several further sound values were proposed. In our paper, some of them are being corroborated, and a new one is presented. Language behind the signs : Based on graphotactical patterns found in the texts, this paper claims that it must be Elamite or a language closely related to it.

The Linear Elamite writing system was used in the late 3rd millennium in ancient Iran.The underlying language is supposed to be Elamite – an isolate language otherwise known from cuneiform sources. 40 to 60% of the Elamite words and morphemes are decoded.In early 2016, about ten new inscriptions and fragments were presented at the University of Hamedan, Iran. They are now in the Mahboubian Gallery. Some of these new texts are the longest ones ever found, depicting up to 200 signs.In the past months, the Deciphering Crew at the Institut für Sprachwissenschaft, Universität Bern, has made drawings of the so far unpublished inscriptions and compiled a sign catalogue.Preliminary results show that fragments from Gonur and Altyn Depe formerly tagged as “Linear Elamite” do not belong to the Linear Elamite text corpus.The Deciphering Project is hoping to collaborate with scholars of different fields. The web page http://elamicon.org is an open source project.

The rapidly increasing amount of plant genomic sequences allows for the detection of cis-elements through comparative methods. In addition, large-scale gene expression data for Arabidopsis (Arabidopsis thaliana) have recently become available. Coexpression and evolutionarily conserved sequences are criteria widely used to identify shared cis-regulatory elements. In our study, we employ an integrated approach to combine two sources of information, coexpression and sequence conservation. Best-candidate orthologous promoter sequences were identified by a bidirectional best blast hit strategy in genome survey sequences from Brassica oleracea. The analysis of 779 microarrays from 81 different experiments provided detailed expression information for Arabidopsis genes coexpressed in multiple tissues and under various conditions and developmental stages. We discovered candidate transcription factor binding sites in 64% of the Arabidopsis genes analyzed. Among them, we detected experimentally verified binding sites and showed strong enrichment of shared cis-elements within functionally related genes. This study demonstrates the value of partially shotgun sequenced genomes and their combinatorial use with functional genomics data to address complex questions in comparative genomics.

Perineuronal nets of extracellular matrix have been shown to characterize the microenvironment of individual neurons and the chemoarchitecture of brain regions such as basal forebrain nuclei. Previous work has also demonstrated that neurons in the human cerebral cortex ensheathed by perineuronal nets rarely undergo cytoskeletal changes in Alzheimer’s disease, suggesting a neuroprotective effect of extracellular matrix components. It is not known, however, whether or not perineuronal nets are absent in the microenvironment of the cholinergic basal forebrain neurons that are involved early in the cascade of neurodegeneration in humans. Therefore, the present study was undertaken to examine the distribution patterns of perineuronal nets in the basal forebrain of the higher primates, rhesus monkey and human. Cytochemical staining was performed with the lectin Wisteria floribunda agglutinin and a polyclonal antibody to core proteins of chondroitin sulfate proteoglycans in the perfusion-fixed tissue of rhesus monkeys. In human brains, perineuronal nets were only stained with the immunoreaction for chondroitin sulfate proteoglycans. The results showed similar characteristics in distribution patterns of perineuronal nets in the medial septum, the diagonal band of Broca, the basal nucleus of Meynert (Ch1–Ch4), the lateral septum, the caudate–putamen, and the globus pallidus in both species. Double-labelling revealed that the vast majority of cholinergic neurons, labelled either with antibodies to choline acetyltransferase or the low-affinity neurotrophin receptor p75 NTR , were not ensheathed by perineuronal nets. A small subpopulation of net-associated neurons in close proximity to or intermingled with cholinergic neurons of the Ch1–Ch4 cell groups was found to be immunoreactive for parvalbumin. In the caudate–putamen, a large number of the parvalbumin-positive neurons were surrounded by perineuronal nets, whereas in the external and internal segments of the globus pallidus the coincidence of both markers was nearly complete. The study demonstrates that perineuronal nets of extracellular matrix are associated with different types of non-cholinergic neurons in the primate basal forebrain. The absence of nets around cholinergic basal forebrain neurons may be related to their slow modulatory activity but may also contribute to their susceptibility to degeneration in Alzheimer’s disease.

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.

Although multiple sclerosis (MS) is considered primarily as a demyelinating disease, neuronal damage is abundant and correlates with the neurological deficit. Therefore, we investigated the frequency and characteristics of human T cells specific for synapsin-a neuronal protein highly conserved among species. Synapsin specific T cell responses were detected at a frequency similar to that of MBP specific T cells in MS patients, one patient with acute demyelinating encephalomyelitis (ADEM) and controls. Long-term T cell lines specific for synapsin exhibited a CD3(+), CD4(+), CD8(-) phenotype and produced high amounts of tumor-necrosis-factor-alpha (TNF-alpha) and interferon-gamma (IFN-gamma) after antigen specific stimulation, whereas lymphotoxin (LT), interleukin-4 (IL-4) and interleukin-10 (IL-10) were detectable in smaller quantities.

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

Glatiramer acetate (GA), represents an established treatment of relapsing/remitting multiple sclerosis (MS). The mechanisms responsible for the effect of GA are not fully understood. We generated GA-, myelin basic protein (MBP)- and purified protein derivative (PPD)-specific T cell lines from three MS patients and one healthy donor. The GA-specific lines were CD3+, CD4+, CD8- and produced tumor-necrosis-factor-alpha (TNF-alpha), interferon-gamma (IFN-gamma), interleukin-4 (IL-4), interleukin-6 (IL-6) and interleukin-10 (IL-10) after stimulation with GA in the presence of irradiated peripheral blood mononuclear cells. MBP-specific T cell lines showed an identical phenotype and secreted TNF-alpha, IFN-gamma, IL-4, IL-10, but not IL-6. Co-culture experiments demonstrated, that GA-specific T cell lines have the capability to suppress the proliferation of MBP-specific T cell lines.

Perineuronal nets (PNs) are known as chondroitin sulphate-rich, lattice-like coatings of the extracellular matrix. In the cortex of mammalian species investigated so far, they were mainly found around GABAergic neurons, but to a lesser degree also around pyramidal cells. Previous investigations in the rat revealed similar distribution patterns of fast-firing neurons expressing both the Kv3.1b subunit of voltage-gated potassium channels and the calcium-binding protein parvalbumin. In the present study, triple fluorescence labelling was applied for the simultaneous demonstration of PNs with the N-acetylgalactosamine-specific Wisteria floribunda agglutinin (WFA), parvalbumin-immunoreactivity (ir) with a monoclonal antibody and of Kv3.1b-ir with several rabbit antibodies. Subsets of non-pyramidal neurons - enwrapped by PNs and expressing parvalbumin and Kv3.1b - were detected in the rat and monkey neocortex and hippocampus. In the rat, faintly stained PNs were additionally found around several layer II/III and V pyramidal cells immunonegative for Kv3.1b, but contacted by Kv3.1b-containing boutons. In the monkey, more intensely labelled PNs frequently occurred around pyramidal cells which themselves appeared to be Kv3. 1b-immunopositive. We also observed minor Kv3.1b-ir and parvalbumin-ir cortical cell populations which were devoid of PNs; occasionally, nets were detected around neurons lacking both immunoreactivities. By confocal laser scanning microscopy, Kv3.1b-ir and WFA-binding sites were found adjoining at the soma and proximal dendritic surface, while lectin-binding sites usually extended on more distal dendritic segments and the axon initial segments which failed to express detectable Kv3.1b-ir. This spatial relationship of both markers was also confirmed by combined WFA-gold labelling and Kv3.1b-immunoperoxidase staining at the electron microscopic level. The data are used for a critical examination of current hypotheses concerning the functional role of PNs. We conclude that PNs may serve as rapid local buffers of excess cation changes in the extracellular space. Somatic membranes of fast-spiking neurons seem to be a main, but not the only source of such changes.

In the pachymeninx (dura mater) from human spinal cord and brain, expression of mRNA for the beta-trace protein (beta-trace) was studied by in situ hybridization with digoxigenin-labeled cRNA probes. The localization of the protein was investigated using monoclonal and polyclonal antibodies, respectively. Very strong hybridization signals were observed in the fibroblasts of the pachymeninx. The results obtained by in situ hybridization were essentially confirmed by immunocytochemistry. By immunoblotting of proteins solubilized from the dura mater, strong immunoreactions were found with a polyclonal beta-trace antibody. Furthermore, beta-trace was quantified in human ventricular and lumbar cerebrospinal fluid (CSF) by use of an immunonephelometric assay. The beta-trace concentration in human lumbar CSF was elevated 11-fold as compared with ventricular CSF. Beta-trace determined in lumbar CSF most probably originates from fibroblasts of the pachymeninx. According to the bi- or probably multifunctional features of beta-trace, various sites of mRNA expression and protein synthesis exist in the human central nervous system (CNS). The major ones are the fibroblasts of the pachymeninx, and as previously shown, the epithelial layer of the choroid plexus.

In the rabbit model of Streptococcus pneumoniae meningitis, treatment with rifabutin, quinupristin-dalfopristin, moxifloxacin and trovafloxacin led to smaller increases of the CSF concentrations of the pro-inflammatory cell wall components lipoteichoic and teichoic acids (LTA and TA) than did treatment with ceftriaxone. Low doses of moxifloxacin were associated with higher LTA and TA concentrations in CSF than were high doses.

Two-site enzyme-linked immunosorbent assays (ELISA) have been established for the specific and sensitive determination of two membrane proteins of the small synaptic vesicles (SSV), namely: peripheral synapsin I and integral synaptophysin. The ELISA used highly specific capture monoclonal antibodies (mAB) and polyclonal antibodies (pAB) as detectors. For synapsin I, the mAB were newly generated, whereas for synaptophysin, the commercially available mAB SY38 was applied. In order to calibrate the ELISA and to raise pAB, both proteins were purified in the mg-range. Synapsin I was purified by conventional means from human and porcine brain and synaptophysin was purified by immunoaffinity chromatography from porcine brain. Using the ELISA, neither synapsin I nor synaptophysin could be determined in serum or cerebrospinal fluid (CSF) from healthy donors or patients suffering various neurological disorders or pheochromocytomas. For this reason, the degradation of both proteins in serum and CSF was investigated. With the exception of synaptophysin measured in serum, both proteins exhibited fast rates of degradation. Despite the negative results in human body fluids, the two ELISA are appropriate for the quantification of these membrane proteins in neuronal or neuroendocrine cell extracts or preparations of SSV.

Although meninges represent a major site of biosynthesis, beta-trace protein (beta-trace) has not been studied in the cerebrospinal fluid (CSF) of meningitis patients. We measured beta-trace in lumbar CSF of normal controls (n = 27) and in patients with various neurological diseases (n = 92) by an immunonephelometric assay. The mean concentration of beta-trace in CSF of control patients was 16.6+/-3.6 mg/l. In bacterial meningitis (n = 41), CSF beta-trace was significantly decreased (8.7+/-3.9 mg/l; P< 0.001), whereas in spinal canal stenosis it was elevated (29.2+/-10.3 mg/l; P= 0.002). In viral meningoencephalitis (n = 12), beta-trace CSF concentrations were normal. Beta-trace concentrations remained below the normal range even after curing of bacterial meningitis, and normalisation of CSF leucocytes and blood-CSF barrier function. Beta-trace may be a useful tool for studying the pathophysiology of bacterial meningitis.

Choline acetyltransferase (ChAT) was partially purified from human placenta and brain. In order to raise monoclonal antibodies, Balb/c mice were immunized with a preparation from placenta or with a mixture of eight synthetic peptides that were deduced from the primary structures of porcine and human ChAT. Polyclonal antibodies were raised in rabbits against five synthetic peptides deduced from the amino acid sequence of human ChAT. The monoclonal and polyclonal antibodies were characterized by their ability to recognize ChAT in various immunoassays: immunoblot, enzyme-linked immunosorbent assay (ELISA), two-side ELISA and immunohistochemistry. With one exception all monoclonal antibodies recognized ChAT on immunoblots, some were particularly sensitive; one bound active ChAT in ELISA when used as capture reagent; most antibodies recognized immobilized ChAT in ELISA. Two monoclonal antibodies out of nine gave particularly excellent results in staining cholinergic neurons and fibers on sections from rat and primate brain. With the help of nine synthetic peptides it was possible to evaluate two major binding sites for the monoclonal antibodies on the ChAT molecule, comprising amino acids 167-189 and 57-76, respectively.

SUMMARY 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.

SUMMARY Dexamethasone appears to show some adverse side-effects as adjunctive anti-inflammatory agent in bacterial meningitis. For this reason, we tested the anti-inflammatory and neuroprotective effect of pentoxifylline administered 15 min before starting antibiotic treatment with ceftriaxone (n = 10) versus antibiotic therapy alone (n = 9) in the rabbit model of pneumococcal meningitis. Pentoxifylline lowered the medians of leucocyte density, tumour necrosis factor-alpha (TNF-α) and lactate in the cerebrospinal fluid (CSF), but only leucocyte migration into the subarachnoid space was significantly inhibited 8 h after initiation of therapy (P = 0.01). CSF protein, brain water content, and the entry of ceftriaxone into CSF were not influenced by pentoxifylline. The density of neuronal apoptoses in the dentate gyrus was slightly lower in animals receiving pentoxifylline than in those treated with ceftriaxone only. The median concentration of neuron-specific enolase in CSF was lower in the pentoxifylline-treated group, but the difference was not significant. In conclusion, pentoxifylline showed some anti-inflammatory activity in pneumococcal meningitis, but the substance failed significantly to reduce neuronal damage.

Thin sputter deposited layers with a nominal composition of Nd2Fe14B (thickness 120 to 400 nm) on SiSiO2 were analysed by the combined application of RBS and heavy ion ERDA. These methods, based on elastic Coulomb scattering, are advantageous for composition analysis of thin compound layers due to their known cross sections. For high accuracy it is necessary that the spectral contributions of each element can be separated from each other and from the substrate background. This is difficult for light elements with RBS. The light element concentration however can be measured by ERDA with heavy incident ions. With 200 MeV 127I ions from the Munich MP tandem the light elements as well as the Fe recoils were detected using a gas telescope. The Fe peak was used as cross reference to the RBS measurements. With 36 MeV 35Cl ions at the 5 MV Rossendorf tandem too little energy is transferred to the Fe recoils to be identified with the Bragg ionisation chamber (BIC) detector. Therefore a thin aluminium layer was evaporated onto the Nd2Fe14B layer. The aluminium peak is well separated both in the RBS and in the ERDA spectra and is used as cross reference here.

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.

The chemical codings of neurons that project from the small intestine, caecum, proximal colon, distal colon and rectum to the coeliac ganglion of the guinea pig were investigated. The coeliac ganglion was injected with the retrogradely transported dye Fast Blue, and each of the regions was examined 6 days later in wholemounts that had been prepared for immunohistochemical localisation of pairs of antigens. In both the small and large intestines, all intestinofugal neurons were immunoreactive (IR) for choline acetyltransferase (ChAT). In each region of the large intestine, the largest population, representing 50-60% of retrogradely labelled neurons in each region, was immunoreactive for ChAT, bombesin (BN), calbindin (Calb) and nitric oxide synthase (NOS). Most intestinofugal neurons in the small intestine contain bombesin and VIP-IR along with ChAT-IR but none contain either Calb or NOS. Thus, nerve endings of enteric origin in the coeliac ganglion that contain NOS-IR or Calb-IR come from the large intestine and those with bombesin-IR but not NOS-IR are from the small intestine. The gastric wall was injected with Fast Blue in order to label noradrenergic (NA) neurons in the coeliac ganglion and to determine, by localisation of NOS and bombesin-IR, whether they receive inputs from the small and large intestine. Some NA neurons received inputs from the large intestine (and perhaps also from the small intestine) and some received inputs exclusively from the small intestine. Most NA neurons that received intestinofugal inputs had the chemical code NA/-; some were immunoreactive for somatostatin (NA/SOM neurons), but those with IR for neuropeptide Y (NA/NPY) rarely received intestinofugal inputs.

In the rat choline acetyltransferase (ChAT)-like immunoreactivity (ChAT-LI) was demonstrated in the dorsal root ganglion (DRG), in the superficial spinal cord and in the subepithelial layer of the ureter using immunohistochemical techniques. In the L1 DRG, 66% of the neurones were ChAT-LI. They did not express neurofilament immunoreactivity (RT97 negative) but could also contain calcitonin gene-related peptide-like immunoreactivity (CGRP-LI). In the superficial spinal cord and in the subepithelial plexus of the ureter - both areas where high numbers of fine afferent fibres have been demonstrated - CGRP-LI and ChAT-LI were co-distributed, indicating that ChAT can be found in the peripheral and central endings of small afferents. The data provide morphological evidence that a substantial proportion of afferent fibres are cholinergic.

The aim of this study was to investigate the neurochemical coding of myenteric neurons in the guinea pig gastric corpus by using immunohistochemical methods. Antibodies and antisera against calbindin (CALB), calretinin (CALRET), choline acetyltransferase (ChAT), calcitonin gene-related peptide (CGRP), dopamine beta-hydroxylase (DBH), beta-endorphin (ENK), neuropeptide Y (NPY), neuron-specific enolase (NSE), nitric oxide synthase (NOS), protein gene product 9.5 (PGP), parvalbumin (PARV), serotonin (5-HT), somatostatin (SOM), substance P (SP), tyrosine hydroxylase (TH), and vasoactive intestinal peptide (VIP) were used. Double- and triple-labeling studies revealed colocalization of certain transmitters and enabled the identification of distinct subpopulations of gastric enteric neurons. NPY/VIP/NOS/ENK were present in 28% of all neurons, whereas 11% had NPY/VIP/DBH/ChAT; NOS-only neurons made up 2% of the population. The combination SP/ChAT/ENK occurred in 21% of the population, whereas SP/ChAT/ENK/CALRET and SP/CHAT/SOM/ +/- CALRET was identified in 5% and 6% of all cells, respectively. 5-HT-containing neurons comprised 2% of all cells and could be further classified by the presence of additional antigens as 5-HT/SP/(ChAT) or 5-HT/VIP/(ChAT). Approximately 21% of all neurons contained only ChAT with no additional antigen present and are referred to as ChAT/-. Gastric myenteric ganglion cells were not immunoreactive for CALB, PARV, CGRP, or TH. The results of this study indicate that gastric myenteric neurons can be characterized on the basis of different chemical coding. Neurochemical coding of corpus myenteric neurons revealed some similarities and significant differences in comparison with other regions of the gut. These differences might reflect adaptation of enteric nerves according to regional specialization and the distinct functions of the proximal stomach as a gastric reservoir.

SUMMARY 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 lo 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.

A specific, sensitive, and reliable sandwich-ELISA (enzyme-linked immunosorbent assay) has been established for the determination of choline acetyltransferase (CHAT) from porcine brain. The detection limit of the assay was 30 micrograms/l and the assay was linear up to 300 micrograms/l. The within-day and day-to-day coefficients of variation were found to be 3.3% and 4.7% respectively for low CHAT concentrations (30 micrograms/l) and 3.1% and 3.4% respectively for high levels of CHAT (300 micrograms/l). The immunoassay was more sensitive than the radiometric assay of Fonnum which is widely used for the measurement of enzyme activity. In the assay monoclonal antibody was adsorbed to the polystyrene surface of the immunoplate as the capture reagent. Using a standard peroxidase protocol the immobilized antigen was detected with a highly specific anti-CHAT antiserum raised in rabbits. Two monoclonal antibodies were available for antigen binding. One of the two--A10.29B4--reacted preferentially with the active enzyme the other one--B3.9B3--reacted only with a degraded form. The polyclonal antiserum recognized both native and denatured enzyme. The effectiveness of employing the two monoclonal antibodies separately or in combination was demonstrated by measurement of porcine CHAT diluted in human cerebrospinal fluid and serum. After some minor modifications the sandwich-ELISA could be used for the determination of CHAT from the central nervous system of the rat.

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.

Choline acetyltransferase (ChAT) from porcine brain was purified by immunoaffinity chromatography, and the highly purified enzyme was subsequently used for immunization of mice and rabbits. After fusion of mouse spleen cells, 32 cultures producing monoclonal antibodies directed against ChAT were detected by an enzyme-linked immu-nosorbent assay (ELISA) with immunoaffinity-purified ChAT. Of these original 32, the most active 11 cultures were cloned and used for ascites production. The 11 clones generated monoclonal antibodies of the immunoglobulin (Ig) M class (three), the IgGl subclass (seven), and the IgG2b subclass (one). The isoelectric points of the antibodies of the IgG class were different in each case. The monoclonal antibodies exhibited different binding characteristics in the above ELISA and on western blots. Two monoclonal antibodies demonstrated excellent immunohistological results with neurons of rat brain and spinal cord. One of them reacted well immu-nohistochemically with neurons of human brain and also recognized partially purified human placenta ChAT in the ELISA.

Summary The peroxidase isoenzyme-groups of Nicotiana tabacum L. (A1 acidic, A2 slightly acidic, C1 basic) were purified from leaves and cell suspension cultures to apparent homogeneity as revealed by SDS-PAGE. The relative molecular masses were 38,000 (A1), 45,000 (A2) and 40,000 (C1). The final and most important purification step was a HPLC with hydrophobic interaction. The RZ-values (A 403nm /A 275nm ) achieved were 2.3 (A1), 2.9 (A2) and 3.4 (C1). Antisera were raised in rabbits against these highly purified peroxidases. By Western blotting and by the Ouchterlony double diffusion method the reactivity of the antisera and the immunological inhibition of the enzymes were studied. By the development of an ELISA the cross reactions of the antisera with the various peroxidase isoenzymes were quantitatively evaluated. The most interesting finding is that A2 reacts hardly with the antisera against the other two isoenzyme-groups. A1 and C1 show higher degrees of cross reaction with the other antisera. The results are discussed in relation to earlier findings on immunology of plant peroxidases.

A newly developed enzyme immunoassay (EIA) was used to detect the presence of pneumococcal teichoic and lipoteichoic acids in cerebrospinal fluid (CSF) from patients with Streptococcus pneumoniae meningitis who were being treated with antibiotics. All initial CSF samples, which on culture grew S. pneumoniae , were positive in the EIA. A total of 14 subsequent culture-negative samples gave clear signals in the EIA up to day 15 after the onset of antibiotic treatment. For 11 CSF specimens, culture, microscopy, and latex agglutination were negative while the EIA detected pneumococcal antigens. The EIA did not react either with CSF of patients with meningitis caused by bacteria other than S. pneumoniae or by viral pathogens. In conclusion, this EIA can be a valuable tool for the diagnosis of S. pneumoniae meningitis from CSF samples in cases in which prior antimicrobial therapy minimizes the usefulness of culture or other antigen detection tests.

Glatiramer acetate (GA) is an approved drug for therapy of relapsing remitting MS that acts as a T cell antigen. Here, we report the cloning of HLA restricted, GA-specific human CD8(+) T cells. In addition, we analyzed the cytokine profile of GA-reactive CD8(+) T cell lines. Unexpectedly, IL-4 was increased in untreated MS patients as compared to healthy individuals (p0.001). In GA-treated patients, however, IL-4 (p0.001), IL-10 (p0.001) and TNF-alpha (p0.001) were decreased. Thus, while GA is known to induce a TH2 bias in CD4(+) T cells, we detected a distinct pattern in GA-reactive CD8(+) T cells.

β-Trace, a protein that represents a major constituent of human cerebrospinal fluid with unknown function has been purified to apparent homogeneity by gel chromatography and electrophoresis. After sodium dodecylsulfate electrophoresis on polyacrylamide gels (SDS-PAGE) and Western blotting, the N-terminal sequence (28 amino acids) has been determined. The high degree of idendity with the corresponding sequences of prostaglandin D synthetase from rat (68%) and human (93%) suggests a strong relationship if not identity with this enzyme. Thus, 30 years after its discovery β-trace might unravel as a well-known protein of the prostaglandin metabolism.

Transmigration of human granulocytes across a basal lamina equivalent was studied in vitro. Transwell® inserts were coated with Matrigel®, a reconstituted basement membrane. Granulocytes (2×10 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 α 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.

Wallerian degeneration is characterized by breakdown of myelin and axons with subsequent macrophage infiltration and removal of the degenerating nerve components. Proteinases of the matrix metalloproteinase (MMP) family seem to play an important role in demyelinating processes, since some of their members have been shown to cleave myelin basic protein. In the present study we investigated the expression of MMP-2 and MMP-9 (gelatinases A and B) during myelin removal after peripheral nerve trauma. After transection of the sciatic nerve an upregulation of MMP-2 and MMP-9 with a first peak 12 h and a second peak 48 h after axotomy was observed by zymography. These peaks correlate with the breakdown of the blood-nerve barrier, the accumulation of granulocytes, and the invasion of macrophages into the damaged nerves, respectively. Furthermore, MMP-2 was found to be upregulated in the contralateral nontransected nerves. Immunocytochemistry for MMP-9 and in situ zymography identified MMP-reactive cells within the distal nerve stump. Chloracetate esterase staining was used to detect granulocytes, which accumulated at the transection site and were colocalized with the in situ zymography signal. Wallerian degeneration of the transected nerve could be delayed either by intraperitoneal injections of hydroxamate (Ro 31-9790), a nonspecific MMP inhibitor, or by local application of an MMP-9-specific antibody. Following these treatment strategies, a decreased number of invading macrophages was seen in the nerves associated with an increased amount of preserved myelin sheaths. These results suggest that the invasion of macrophages into a transected peripheral nerve is accompanied by an increased expression of MMPs, particularly MMP-9. Thus, MMPs may seem to play an important role in the breakdown of the blood-nerve barrier and subsequent cell recruitment from the systemic circulation into the damaged nerve.

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.

Glutamine synthetase (GS) activity is higher in the neocortex but not in the hippocampal formation of rabbit brain during Streptococcus pneumoniae meningitis compared to the respective brain region of uninfected control animals. One-dimensional polyacrylamide gel electrophoresis (1D-SDS-PAGE) revealed an apparent molecular mass (Mr) of 44 000 Dalton (Da) for GS from rabbit brain. After two-dimensional gel electrophoresis (2D-PAGE), followed by Coomassie-blue staining, GS separated into three distinct spots (S1, S2, S3). One additional spot (S4) occurred on the immunoblot. All four GS spots exhibited the same Mr (44 000 Da), but differed in their isoelectric points. Densitometric evaluation of the two-dimensional maps revealed a strong increase of optical density (OD) of S3 in the frontal cortex of infected animals. The calculated OD ratio S3/S2 in the frontal cortex from rabbits with meningitis was 1.75±0.68 (mean±standard deviation). Compared to controls (0.85±0.39), this value was significantly increased (p=0.0006). In the hippocampal formation, the ratio S3/S2 was nearly unchanged during meningitis. It is suggested that the ratio S3/S2 may indicate a neuroprotective feature of rabbit brain during meningitis since neuronal apoptosis occurs only in the dentate gyrus and not in the frontal cortex.

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.

When in situ hybridization was used for the detection of mRNA for the beta-trace protein (beta-trace; prostaglandin-D-synthase) in sections of rat and porcine brains, unspecific binding reactions of sense and antisense probes to neurons were observed. The beta-trace fragment which served as a template for the synthesis of cRNA probes was blunt end-cloned in the vector pCR-Script SK (+). It was demonstrated that the unspecific signals were caused by artifactual binding of two portions of the cRNA which correspond to sequences of the multicloning site of this vector. These sequences are localized between the SrfI restriction site (or the insert) and the promoter for the T7 RNA polymerase. Thus, artifactual binding could be prevented using riboprobes synthesized by T3 RNA polymerase instead of T7 RNA polymerase. Because of the relatively weak transcription efficiency of T3 RNA polymerase, as compared with T7 RNA polymerase, a blocking procedure was established which allowed successful in situ hybridization with T7 RNA polymerase-synthesized probes. Blocking was performed using synthetic oligonucleotides deduced from the two sequences of the multicloning site which were found to be responsible for artifactual binding.

We evaluated S-100 levels in paired cerebrospinal fluid (CSF) and serum samples in a group of 135 patients referred to the German Creutzfeldt-Jakob disease (CJD) surveillance unit from June 1993 to May 1995. The patients were seen in a prospective case control study. The diagnosis of probable CJD during life was made in any patient presenting with rapidly progressive dementia of less than 2 years' duration, typical periodic sharp wave complexes (PSWCs) in the EEG and at least two of the following findings: myoclonus, visual/or cerebellar symptoms, pyramidal and/or extrapyramidal signs and/or akinetic mutism. Patients presenting with the above clinical signs and symptoms but without PSWCs were classified as possible, while those with a dementia of a duration exceeding 2 years and without PSWCs were classified as other. S-100 was determined in paired CSF and serum samples by a commercially available enzyme-linked immunosorbent assay. In a group of 76 patients with definite and probable CJD, S-100 concentration (median 25 ng/ml, range 2-117) in CSF was significantly higher (P < 0.0001) than in 32 patients diagnosed as other (median 4 ng/ml, range 1-19). Serum levels of S-100 were below 0.5 ng/ml in all groups. At a cut-off of 8 ng/ml an optimum sensitivity of 84.2% with a specificity of 90.6% for the diagnosis of CJD by the determination of S-100 in CSF is obtained. S-100 levels exceeding 8 ng/ml in CSF support the diagnosis of CJD in any patient presenting with rapidly progressive dementia.

A specific and sensitive two-side enzyme-linked immunosorbent assay (sandwich-ELISA) was established for the reliable quantification of human brain and placental choline acetyltransferase (ChAT). In contrast to the radiometric assay developed by Fonnum, which is widely used for the measurement of enzyme activity, the sandwich-ELISA particularly recognized inactivated forms of the antigen. In the assay, affinity-purified polyclonal synthetic peptide antibodies adsorbed to the polystyrene surface of the microtiter plate were employed as capture reagent. Based on standard peroxidase protocols, immobilized ChAT was detected using monoclonal antibodies raised against human placental ChAT. By use of this ELISA, ChAT was determined at various purification stages of the enzyme, in body fluids, during recovery experiments and in sera of patients with severe brain damage.

Synaptophysin (SYP I), an integral membrane protein, was purified on a large scale (0.55 - 2.7 mg) from isolated small synaptic vesicles (SSV) of porcine cortex. In order to achieve this, a conventional purification procedure which consists of size exlusion chromatography, hydrophobic interaction chromatography and chromatofocusing has been developed. This procedure was compared with purification of SYP I by immunoaffinity chromatography. The elution patterns of both procedures were monitored using sodium dodecylsulfate gel electrophoresis (SDS-PAGE) with subsequent Coomassie blue staining of proteins and simultaneous immunoblotting with SYP I-specific antibody. Contaminating proteins with relative molecular masses (M(r)) very similar to SYP I could be removed during the process of purification, demonstrating that the 38 kDa protein found after Triton X-100 lysis of enriched SSV does not exclusively represent SYP I. A specific antiserum was raised in rabbits using a highly purified preparation of SYP I. This antiserum was used in combination with a monoclonal antibody to establish a specific and sensitive enzyme-linked immunosorbent assay (ELISA) which allowed rapid and reliable quantification of this hydrophobic membrane protein in all purification steps, starting with Triton X-100-lysed brain homogenates. Using this ELISA, the concentration of SYP I in highly purified SSV was determined to be 5.8% of solubilized protein.

A two-sided enzyme-linked immunosorbent assay (ELISA) has been established for reliable, specific and sensitive determination of synaptophysin (SYN), an intrinsic membrane protein of the small synaptic vesicles. This ELISA used a highly specific monoclonal antibody (SY 38) as capture reagent and a specific SYN antiserum in combination with a secondary peroxidase-conjugated antibody for detection. Calibration was carried out with immunoaffinity-purified SYN from porcine cortex. The sensitivity was found to be improved substantially when the ELISA was compared with previously used dot-immunobinding assays. This ELISA allowed rapid and reliable determination of SYN from detergent lysed homogenates, partially and highly purified preparations of rat, porcine and human brain. SYN was determined in highly purified small synaptic vesicles, and it was calculated to be 5.8% of total detergent solubilized protein.

Expression of beta-trace protein (beta-trace), recently identified as glutathion-independent prostaglandin D synthase (prostaglandin-H2 D-isomerase; EC 5.3.99.2), was localized in paraffin sections of the human brain by in situ hybridization using digoxigenin-labeled antisense cRNA probes. The mRNA for beta-trace was predominantly found in the epithelial cells of the choroid plexus. Hybridization signals were also obtained in some oligodendrocytes, particularly in the white matter. In the leptomeninges, specific signals were found in meningeal macrophages and in single cells of the arachnoid barrier layer. The cells exhibiting hybridization signals with the antisense cRNA probes for beta-trace were identified by counterstaining with antibodies directed against specific cell markers. Additionally, beta-trace mRNA was localized in tubular epithelial and basal cells of the human epididymis and in different cell types within the seminiferous epithelium of the testis.

In the sera of patients with acute bactetial infections specific autoantibodies (sIAPa) of the imtnuno-globulin class G (IgG) were found which bind to intestinal alkahne 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 nitro cellulose 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.

In half of the sera from patients with acute bacterial infections and 15% of the sera from patients with acute viral infections glycoproteins were found that form a scalariform pattern in the cationic range upon isoelectric focusing. The cationic glycoproteins appeared with the clinical illness. After subsidence of the symptoms they disappeared within 4 to 6 weeks. The proteins were identified as immunoglobulin G (IgG) by determination of the NH2-terminal amino acid sequence. Remarkably, these IgG only contained light chains of the kappa type with high proportions of carbohydrates. Both, N-glycosidic- and O-glycosidic-bound glycans were present. The glycosylated light chains may render the cationic IgG multireactive. Thus, it may be part of an early nonspecific immune defense mechanism.

Choline acetyltransferase (ChAT) was purified by immunoaffinity chromatography using a covalently immobilized monoclonal antibody. In a two-step procedure, 10 kg porcine brain yielded 750 micrograms active enzyme of apparent homogeneity. This amount of ChAT was purified routinely. The purification factor was 18,000 and the yield of activity 4.3%. The affinity resin was stable under the experimental conditions applied and was used many times. The highly purified enzyme was subsequently employed to obtain a specific anti-ChAT antiserum of high titer.