Cervical spondylodiscitis following cricopharyngeal botulinium toxin injection.

BACKGROUND: Iatrogenic cervical spondylodiscitis is rare, but may occur after various medical interventions. METHODS: We report a case of a diabetic 70-years-old female with C5-C6 spondylodiscitis and symptomatic epidural abscess with neck pain and upper limb paresis after endoscopic botulinum toxin injection for the treatment of dysphagia. Treatment included antibiotic therapy with amoxicillin and later on benzylpenicillin for the next ten weeks and corporectomy with spondylodesis. RESULT: The patient made an excellent recovery, with complete resolution of paresis and only minor residual hypoesthesia at one year after operation. CONCLUSION: Cervical spondylodiscitis should be considered early, in patients with neck pain after endoscopic cricopharyngeal injection, as timely diagnosis and treatment can prevent serious and irreversible neurological deficit.

Tonic GABAA Receptors as Potential Target for the Treatment of Temporal Lobe Epilepsy.

Tonic GABAA receptors are a subpopulation of receptors that generate long-lasting inhibition and thereby control network excitability. In recent years, these receptors have been implicated in various neurological and psychiatric disorders, including Parkinson's disease, schizophrenia, and epilepsy. Their distinct subunit composition and function, compared to phasic GABAA receptors, opens the possibility to specifically modulate network properties. In this review, the role of tonic GABAA receptors in epilepsy and as potential antiepileptic target will be discussed.

The effects of vagus nerve stimulation on tryptophan metabolites in children with intractable epilepsy.

BACKGROUND: The mechanism of action of vagus nerve stimulation (VNS) in intractable epilepsy is not entirely clarified. It is believed that VNS causes alterations in cytokines, which can lead to rebalancing the release of neurotoxic and neuroprotective tryptophan metabolites. We aimed to evaluate VNS effects on tryptophan metabolites and on epileptic seizures and investigated whether the antiepileptic effectiveness correlated with changes in tryptophan metabolism. METHODS: Forty-one children with intractable epilepsy were included in a randomized, active-controlled, double-blind study. After a baseline period of 12 weeks, all children underwent implantation of a vagus nerve stimulator and entered a blinded active-controlled phase of 20 weeks. Half of the children received high-output (therapeutic) stimulation (n=21), while the other half received low-output (active control) stimulation (n=20). Subsequently, all children received high-output stimulation for another 19 weeks (add-on phase). Tryptophan metabolites were assessed in plasma and cerebrospinal fluid (CSF) by use of liquid chromatography-tandem mass spectrometry (LC-MS/MS) and compared between high- and low-output groups and between the end of both study phases and baseline. Seizure frequency was recorded using seizure diaries. Mood was assessed using Profile of Mood States (POMS) questionnaires. RESULTS: Regarding tryptophan metabolites, anthranilic acid (AA) levels were significantly higher at the end of the add-on phase compared with baseline (p=0.002) and correlated significantly with improvement of mood (τ=-0.39, p=0.037) and seizure frequency reduction (τ=-0.33, p<0.01). No significant changes were found between high- and low-output groups regarding seizure frequency. CONCLUSION: Vagus nerve stimulation induces a consistent increase in AA, a neuroprotective and anticonvulsant tryptophan metabolite. Moreover, increased AA levels are associated with improvement in mood and reduction of seizure frequency.

The influence of neuropathology on brain inflammation in human and experimental temporal lobe epilepsy.

It is unclear to what extent neuropathological changes contribute to brain inflammation observed in temporal lobe epilepsy (TLE). Here, we compared cytokine levels between histopathologically-confirmed sclerotic hippocampi and histopathologically-confirmed normal hippocampi from TLE patients. We analyzed a similar cytokine panel in the hippocampi of amygdala-kindled rats and we evaluated neuropathological changes by immunohistochemistry. In TLE patients, cytokine levels were not significantly different between sclerotic and non-sclerotic hippocampi. Though kindling resulted in increased astrocyte activation, cytokine levels and microglia activation were unchanged. These results suggest that the chronic epileptic state in TLE can also occur in the absence of intracerebral inflammation. Highlights.

Rat vagus nerve stimulation model of seizure suppression: nNOS and ΔFos B changes in the brainstem.

Vagus nerve stimulation (VNS) is a moderately effective treatment for intractable epilepsy. However, the mechanism of action is poorly understood. The effect of left VNS in amygdala kindled rats was investigated by studying changes in nNOS and ΔFos B expression in primary and secondary vagus nerve projection nuclei: the nucleus of the solitary tract (NTS), dorsal motor nucleus of the vagus nerve (DMV), parabrachial nucleus (PBN) and locus coeruleus (LC). Rats were fully kindled by stimulation of the amygdala. Subsequently, when the fully kindled state was reached and then maintained for ten days, rats received a single 3-min train of VNS starting 1min prior to the kindling stimulus and lasting for 2min afterwards. In control animals the vagus nerve was not stimulated. Animals were sacrificed 48h later. The brainstems were stained for neuronal nitric oxide synthase (nNOS) and ΔFos B. VNS decreased seizure duration with more than 25% in 21% of rats. No VNS associated changes in nNOS immunoreactivity were observed in the NTS and no changes in ΔFos B were observed in the NTS, PBN, or LC. High nNOS immunopositive cell densities of >300cells/mm(2) were significantly more frequent in the left DMV than in the right (χ(2)(1)=26.2, p<0.01), independent of whether the vagus nerve was stimulated. We conclude that the observed nNOS immunoreactivity in the DMV suggests surgery-induced axonal damage. A 3-min train of VNS in fully kindled rats does not affect ΔFos B expression in primary and secondary projection nuclei of the vagus nerve.

Horner's syndrome: a complication of experimental carotid artery surgery in rats.

PURPOSE: To report on the occurrence of iatrogenic Horner's syndrome (HS) in epileptic rats after implantation of an electrode for vagus nerve stimulation and to describe the possible consequences of this new complication of carotid artery surgery in rats. METHODS: A bipolar circular electrode was placed around the left carotid artery and vagus nerve of 31 rats. The incidence of HS was evaluated by visual inspection within 24 h after surgery. RESULTS: 68% of rats suffered from HS immediately after surgery. This complication did not affect epileptogenesis. CONCLUSION: The occurrence of HS in the rat is a frequent complication of vagus nerve electrode implantation, which does not affect epileptogenesis in this study. However, rats affected by HS may suffer from damage to the sympathetic innervation of the gut, due to rat-specific neuroanatomy. Therefore, caution towards other research questions is warranted.

The CREATE project: development of certified reference materials for allergenic products and validation of methods for their quantification.

Allergen extracts have been used for diagnosis and treatment of allergy for around 100 years. During the second half of 20th century, the notion increasingly gained foothold that accurate standardization of such extracts is of great importance for improvement of their quality. As a consequence, manufacturers have implemented extensive protocols for standardization and quality control. These protocols have overall IgE-binding potencies as their focus. Unfortunately, each company is using their own in-house reference materials and their own unique units to express potencies. This does not facilitate comparison of different products. During the last decades, most major allergens of relevant allergen sources have been identified and it has been established that effective immunotherapy requires certain minimum quantities of these allergens to be present in the administered maintenance dose. Therefore, the idea developed to introduce major allergens measurements into standardization protocols. Such protocols based on mass units of major allergen, quantify the active ingredients of the treatment and will at the same time allow comparison of competitor products. In 2001, an EU funded project, the CREATE project, was started to support introduction of major allergen based standardization. The aim of the project was to evaluate the use of recombinant allergens as reference materials and of ELISA assays for major allergen measurements. This paper gives an overview of the achievements of the CREATE project.

A multi-laboratory evaluation of a common in vitro pepsin digestion assay protocol used in assessing the safety of novel proteins.

Rationale. Evaluation of the potential allergenicity of proteins derived from genetically modified foods has involved a weight of evidence approach that incorporates an evaluation of protein digestibility in pepsin. Currently, there is no standardized protocol to assess the digestibility of proteins using simulated gastric fluid. Potential variations in assay parameters include: pH, pepsin purity, pepsin to target protein ratio, target protein purity, and method of detection. The objective was to assess the digestibility of a common set of proteins in nine independent laboratories to determine the reproducibility of the assay when performed using a common protocol. Methods. A single lot of each test protein and pepsin was obtained and distributed to each laboratory. The test proteins consisted of Ara h 2 (a peanut conglutin-like protein), beta-lactoglobulin, bovine serum albumin, concanavalin A, horseradish peroxidase, ovalbumin, ovomucoid, phosphinothricin acetyltransferase, ribulose diphosphate carboxylase, and soybean trypsin inhibitor. A ratio of 10U of pepsin activity/microg test protein was selected for all tests (3:1 pepsin to protein, w:w). Digestions were performed at pH 1.2 and 2.0, with sampling at 0.5, 2, 5, 10, 20, 30, and 60min. Protein digestibility was assessed from stained gels following SDS-PAGE of digestion samples and controls. Results. Results were relatively consistent across laboratories for the full-length proteins. The identification of proteolytic fragments was less consistent, being affected by different fixation and staining methods. Overall, assay pH did not influence the time to disappearance of the full-length protein or protein fragments, however, results across laboratories were more consistent at pH 1.2 (91% agreement) than pH 2.0 (77%). Conclusions. These data demonstrate that this common protocol for evaluating the in vitro digestibility of proteins is reproducible and yields consistent results when performed using the same proteins at different laboratories.

Reactivity to IgE-dependent histamine-releasing factor is due to monomeric IgE.

BACKGROUND: IgE-dependent histamine-releasing factor (HRF) can distinguish between IgE+ and IgE-. In contrast to IgE-, IgE+ sensitizes basophils to release histamine in response to HRF. But we do not know what particular feature distinguishes IgE+ from IgE-. The objective was to investigate the hypothesis that IgE+ is polymeric IgE. METHODS: IgE+ plasma was separated by size-exclusion chromatography. The basophil-sensitizing capacity of the fractions was analyzed in response to HRF produced by mononuclear cells. RESULTS: We showed that monomeric IgE sensitized basophils to release histamine in response to HRF and to house-dust mite, whereas no enhanced reactivity was found in the fractions containing polymeric IgE. CONCLUSIONS: HRF reacts with monomeric IgE, and not (exclusively) with polymeric IgE.

A sensitive monoclonal antibody sandwich ELISA for the measurement of the major olive pollen allergen Ole e 1.

BACKGROUND: Olive pollen is a major cause of inhalant allergy in countries around the Mediterranean sea. The major allergen of olive pollen is Ole e 1. Measurement of the major allergen content of allergen products for diagnosis and therapy is becoming an essential element of standardization protocols. This study aimed at the development of a monoclonal antibody (mAb) sandwich ELISA for Ole e 1. METHODS: Balb/c mice immunized with Ole e 1 were used for the production of mAbs. Screening of mice and hybridomas was performed in a RIA with radiolabeled purified Ole e 1. Purified mAbs were used as catching and/or (biotinylated) detecting antibodies in sandwich ELISA. RESULTS: Four mAbs (IgG1kappa) directed to nonoverlapping epitopes on Ole e 1 were obtained: 1A12, 5C1, 10A12 and 3H8. Both 1A12 and 10A12 were successfully used for affinity purification of Ole e 1 from olive pollen extract. Two sandwich ELISAs were developed, with 1A12 and 10A12 as catching, and 5C1 and 3H8 as detecting antibodies, respectively. Both catching and detecting antibodies were used in similar concentrations, ranging from 60 to 100 ng/well. For both ELISAs, the sensitivity was approximately 1 ng/ml of Ole e 1. The measuring range was from 1 to 25 ng/ml. No significant differences were observed, when the performance of both ELISAs in standardization of olive pollen extracts was compared. CONCLUSIONS: Two sensitive sandwich ELISAs for the major olive pollen allergen Ole e 1 were developed. They will prove to be useful tools in allergen standardization protocols.

Histamine-releasing activity in supernatants of mononuclear cells: contribution of monocyte chemotactic protein-1 activity compared with IgE-dependent activity.

BACKGROUND: Supernatants of cultured human mononuclear cells contain factors that induce histamine release from basophils. Some of the histamine-releasing factors present are IgE-independent, but an IgE-dependent form has also been described. The IgE that does respond to the IgE-dependent histamine-releasing factor was defined as IgE+. OBJECTIVE: This study was conducted to analyze the contribution of chemokines, such as monocyte chemotactic protein-1 (IgE-independent), to improve the detection of IgE-dependent histamine-releasing factor. METHODS: Supernatants were prepared from mononuclear cells of three subjects. Monocyte chemotactic protein-1 content and histamine-releasing activity (HRA) were measured. Depletion of chemokine activity was carried out with heparin-Sepharose (Pharmacia, Uppasala, Sweden). RESULTS: Replacing IgE- on basophils with IgE+ serum decreased the correlation between monocyte chemotactic protein-1 and histamine release (rho = 0.80, n = 280 vs p = 0.12, n = 18; p for difference between p values; < 0.05). After depletion of chemokines in three supernatants derived from mononuclear cells, IgE-dependent HRA was still present. CONCLUSION: We conclude that in supernatants derived from mononuclear cells, the IgE-independent HRA masks the IgE-dependent HRA. The latter can be more clearly detected after depletion of chemokine activity with heparin-Sepharose.

Reactivity to IgE-dependent histamine-releasing activity in asthma or rhinitis.

We investigated the relation between IgE reactive with histamine-releasing factor (HRF) and clinical status in patients with asthma or rhinitis. Sera were used to passively sensitize purified, lactic-acid treated basophils. IgE-independent HRF due to chemokines was removed from mononuclear cell supernatants with heparin-Sepharose. IgE-dependent HRF was determined by measuring the increase in histamine release between 1 min and 60 min, which was designated delta HRF. HRF-reactive IgE was demonstrable in nine of 18 patients with allergic asthma, three of 19 patients with nonallergic asthma, five of 17 patients with allergic rhinitis, and none of 19 control patients. The presence of HRF-reactive IgE was associated with: (1) IgE to inhalant allergens; 40% of radioallergosorbent test (RAST)-positive individuals versus 8% of RAST-negative individuals were positive (OR = 7.8, p < 0.005); (2) bronchial sensitivity to histamine in all asthmatic patients (geometric mean PC20: 1.50 versus 0.51 mg/ml; p < 0.005); and (3) bronchial sensitivity to histamine in allergic asthmatic patients (geometric mean PC20: 1.27 versus 0.37 mg/ml, p < 0.02). These findings support the hypothesis that IgE-dependent HRF might contribute to the chronic allergic reaction.

Multiplicity of cross-reactive epitopes on Bet v I as detected with monoclonal antibodies and human IgE.

Six monoclonal antibodies against Bet v I, the major cross-reactive allergen of birch pollen (Betula verrucosa), were obtained. Four did not react with fruits, but two monoclonal antibodies (mAbs) (5H8 and 9C11) were reactive with apple and other fruits. These two cross-reactive antibodies reacted with identical or overlapping sites, but differed in their relative degree of cross-reactivity toward various fruits and hazelnut. Cross-reactive human IgE antibodies reacted with a nonoverlapping epitope, as indicated by results of a two-site radioimmunoassay (RIA) with the fruit-reactive mAb 9C11. By isoelectric focusing (IEF) in conjunction with immunoblotting, a maximum of seven isoforms could be distinguished. Depletion of birch-pollen extract for Bet v I with the most reactive mAb (7F7) removed approximately 95% of the IgE cross-reactivity between birch pollen and apple extract. The remaining 5% cross-reactive material was still capable of inhibiting the binding of IgE to apple allergen completely, and was reactive with mAbs 5H8 and 3C4. By means of IEF/immunoblot, it was shown that these mAbs recognize an isoform of Bet v I that is poorly, if at all, recognized by mAb 7F7. These results illustrate the heterogeneity of Bet v I, both with respect to the cross-reactive sites as well as to the backbone structure. This type of heterogeneity has possible implications for the use of monoclonal antibodies in allergen standardization.

Variability of IgE-dependent histamine-releasing activity in supernatants of human mononuclear cells.

Histamine-releasing factors (HRF) that release mediators from human basophils by interacting with IgE have been identified from different cell sources, including lymphocytes, monocytes, thrombocytes and endothelial cells. These factors are studied in view of their potential importance as a stimulus in chronic inflammation. In this report we investigated the qualitative variability of the histamine-releasing activity in the supernatants of activated mononuclear cells. Purified human mononuclear cells of 8 donors were activated with streptokinase/streptodornase (SK/SD) and the supernatants (HRF-MN) were tested for histamine-releasing activity (HRA) in both allergic (RAST positive for inhalant allergens) and nonallergic individuals. Four of the eight HRF-MN supernatants were discriminating, i.e. showing no histamine-release response with nonallergic individuals, whereas four supernatants were not. Two of the HRF-MN supernatants that exhibited discriminating properties were studied in more detail. The response to HRF-MN was tested (1) in a direct bioassay on basophils of allergic (RAST positive for inhalant allergens) and nonallergic individuals and (2) in an indirect bioassay with 70% pure basophils of RAST-negative donors after passive sensitization with sera of allergic donors. An association was found between the response to HRF-MN and the RAST for inhalant allergens: none (0/12) of the RAST-negative but 15/22 of the RAST-positive individuals were HRF-MN responders. The IgE dependency of HRF-MN was shown e.g. by inhibition of passive sensitization by preincubating a responder serum with monoclonal antibody (moAb) anti-IgE MH25-1. Our results are in contrast with findings of other investigators who use pooled supernatants and demonstrated HRF-MN responsiveness with both allergic and nonallergic donors.(ABSTRACT TRUNCATED AT 250 WORDS)

Food intolerance in rheumatoid arthritis. II. Clinical and histological aspects.

Six patients with rheumatoid factor positive rheumatoid arthritis who had shown a marked symptomatic improvement during four weeks of hypoallergic, artificial diet were studied in greater detail. Placebo controlled rechallenges showed intolerance for specific foodstuffs in four patients. In three of these patients biopsies of both the synovial membrane and of the proximal small intestine were carried out before and during allergen free feeding. In two patients, both with raised serum IgE concentrations and specific IgE antibodies to certain foods, a marked reduction of mast cells in the synovial membrane and proximal small intestine was demonstrated. Although the number of food intolerant patients with RA remains limited and markers of allergic activity are scanty, our observations suggest an underlying immunoallergological mechanism.

The potent IgG4-inducing antigen in banana is a mannose-binding lectin, BanLec-I.

IgG4 antibodies to banana were found to occur far more frequently than expected. The most important antigen involved proved to be a lectin, BanLec-I. Because of the lectin nature of the antigen, it was important to establish the antibody nature of the lectin-IgG4 interaction and to exclude an interaction between the sugar-binding site of the lectin and glycosidic chains on IgG4. Three arguments in support of immune binding are: (1) the binding of BanLec-I to IgG4 is mannoside resistant, whereas the binding to all other glycoproteins tested is mannoside inhibitable; (2) only a minor fraction of the IgG4 in serum and none of five IgG4 myelomas tested was bound, and (3) the lectin binds to the Fab fragment of the IgG4 molecule. A curious finding was that in the presence of high-molecular-weight glycoproteins the interaction between IgG4 and BanLec-I was enhanced by alpha-methyl mannoside. The probable explanation of this phenomenon is that complexes of the lectin with high-molecular-weight glycoproteins by sterical interference inhibit the interaction with human IgG4 antibodies (or with rabbit antibodies to the lectin). This inhibition is prevented in the presence of alpha-methyl mannoside. These results support the earlier suggestion that some lectins are particularly prone to induce an immune response upon oral feeding. This banana lectin might be a potentially useful carrier protein for oral antihapten immunization in humans.

Newly generated IgE antibodies to Dermatophagoides pteronyssinus in children are directed against components distinct from Der p I and Der p II.

The specificity of newly generated IgE antibodies (Abs) to the house dust mite, Dermatophagoides pteronyssinus, in longitudinal serum samples from 18 young children with an increased risk for IgE-mediated allergy was studied. The first IgE Ab response to house dust mite was detected early in life (mean age, 32 months; range, 11 to 60 months). For 83% of the children, more than half of the newly generated IgE Ab response to house dust mite was directed against components distinct from the major allergens, Der p I (Pl) and Der p II (DpX). These results suggest that the early IgE Ab response to house dust mite is induced by components distinct from the major allergens, Der p I and Der p II.