Biomedical subjects

Thomas Grunwald

Publications and source records attributed to Thomas Grunwald.

At least 19 recordsLinked to original sources

PubMed2006

beta-Arrestin2 mediates nephrin endocytosis and impairs slit diaphragm integrity.

beta-Arrestins mediate internalization of plasma membrane receptors. Nephrin, a structural component of the glomerular slit diaphragm, is a single transmembrane spanning receptor and belongs to the family of adhesion molecules. Its mutation causes a hereditary nephrotic syndrome. We report the previously undescribed interaction of beta-arrestin2 with the nephrin C terminus. The phosphorylation status of nephrin Y1193 regulates inversely the binding of beta-arrestin2 and podocin. The Src-family member Yes, known to enhance podocin-nephrin interaction by nephrin phosphorylation, diminishes beta-arrestin2-nephrin interaction. beta-Arrestin2 induces nephrin endocytosis and attenuates nephrin signaling. This finding suggests that nephrin Y1193 serves as a molecular switch that determines the integrity of the slit diaphragm by functional competition between beta-arrestin2 and podocin. This concept offers a molecular pathomechanism of slit diaphragm distortion and opens therapeutic avenues for glomerular diseases.

Amino Acid Sequence↗

PubMed2006

Hippocampus proper distinguishes between identified and unidentified real-life visual objects: an intracranial ERP study.

Converging evidence indicates that the medial temporal lobe participates not only in memory but also in visual object processing. We investigated hippocampal contributions to visual object identification by recording event-related potentials directly from within the hippocampus during a visual object identification task with spatially filtered pictures of real objects presented at different levels of filtering. Hippocampal responses differentiated between identified and unidentified visual objects within a time window of 200-900 ms after stimulus presentation: identified objects elicited a small negative component peaking around 300 ms (hippocampal-N300) and a large positive component, around 650 ms (hippocampal-P600), while the N300 was increased and the P600 was reduced in amplitude in response to unidentified objects. These findings demonstrate that the hippocampus proper contributes to the identification of visual objects discriminating from the very early between identified and unidentified meaningful visual objects.

Adult↗

PubMed2006

Molecular cloning and expression in insect cells of honeybee venom allergen acid phosphatase (Api m 3).

BACKGROUND: Acid phosphatase (Api m 3) is a major allergen in honeybee (Apis mellifera) venom, and its availability as a recombinant protein may facilitate the development of improved diagnostic tests and immunotherapies. OBJECTIVE: One objective is the determination of the complete primary structure of Api m 3 and to obtain recombinant Api m 3 on the basis of expression in insect cells. Another objective is the quantitative analysis of patient serum IgE antibody reactive to recombinant Api m 3. METHODS: The cloning of Api m 3 from venom gland cDNA and its expression as a full-length protein in eukaryotic insect cells is described. The immunoreactivity of serum IgE antibodies of honeybee venom-sensitized patients to recombinant Api m 3 was determined in an enzyme immunoassay. RESULTS: PCR amplification generated a 1122-bp DNA fragment whose identity as the coding sequence of Api m 3 was verified by several means. Recombinant Api m 3, expressed in Trichoplusia ni cells, showed an expected molecular weight and enzymatic activity at pH 4.5. Analysis of tryptic fragments of purified recombinant Api m 3 by mass spectrometry confirmed its identity. In immunoassays, recombinant Api m 3 is specifically recognized by IgE antibodies of pooled serum in Western blots and by 37% of the individual sera of honeybee venom-sensitized patients in ELISA analysis. CONCLUSION: The availability of recombinant Api m 3 provides a tool for both the development of improved diagnostic tests and the design of safer and more effective immunotherapeutic approaches for honeybee venom allergy. CLINICAL IMPLICATIONS: The recombinant venom allergen Api m 3 is a key element in the search for an optimized component-resolved approach to honeybee venom allergy with regard to both the development of superior diagnostic tests and the improvement of allergen immunotherapy.

Acid Phosphatase↗

PubMed2006

Bivalent monoclonal IgY antibody formats by conversion of recombinant antibody fragments.

Monoclonal IgY have the potential to become unique tools for diagnostic research and therapeutic purposes since avian antibodies provide several advantages due to their phylogenetic difference when compared to mammalian antibodies. The mechanism of avian immunoglobulin gene diversification renders chicken an excellent source for the generation of recombinant scFv as well as Fab antibody libraries of high diversity. One major limitation of these antibody fragments, however, is their monovalent format, impairing the functional affinity of the molecules and, thereby, their applicability in prevalent laboratory methods. In this study, we generated vectors for conversion of avian recombinant antibody fragments into different types of bivalent IgY antibody formats. To combine the properties of established mammalian monoclonal antibodies with those of IgY constant domains, we additionally generated bivalent murine/avian chimeric antibody constructs. When expressed in HEK-293 cells, all constructs yielded bivalent disulfide-linked antibodies, which exhibit a glycosylation pattern similar to that of native IgY as assessed by lectin blot analysis. After purification by one step procedures, the chimeric and the entire avian bivalent antibody formats were analyzed for antigen binding and interaction with secondary reagents. The data demonstrate that all antibody formats provide comparable antigen binding characteristics and the well established properties of avian constant domains.

Animals↗

PubMed2006

Functional replacement of the R region of simian immunodeficiency virus-based vectors by heterologous elements.

Substitution of lentiviral cis-acting elements by heterologous sequences might allow the safety of lentiviral vectors to be enhanced by reducing the risk of homologous recombination and vector mobilization. Therefore, a substitution and deletion analysis of the R region of simian immunodeficiency virus (SIV)-based vectors was performed and the effect of the modifications on packaging and transfer by SIV and human immunodeficiency virus type 1 (HIV-1) particles was analysed. Deletion of the first 7 nt of R reduced vector titres by 10- to 20-fold, whilst deletion of the entire R region led to vector titres that were 1500-fold lower. Replacement of the R region of SIV-based vectors by HIV-1 or Moloney murine sarcoma virus R regions partially restored vector titres. A non-retroviral cellular sequence was also functional, although to a lesser extent. In the absence of tat, modification of the R region had only minor effects on cytoplasmic RNA stability, steady-state levels of vector RNA and packaging, consistent with the known primary function of R during reverse transcription. Although the SIV R region of SIV-based vectors could be replaced functionally by heterologous sequences, the same modifications of R led to a severe replication defect in the context of a replication-competent SIV. As SIV-based vectors containing the HIV-1 R region were transferred less efficiently by HIV-1 particles than wild-type SIV vectors, a match between R and cis-acting elements of the vector construct seems to be more important than a match between R and the Gag or Pol proteins of the vector particle.

DNA Mutational Analysis↗

PubMed2006

Mesial temporal lobe epilepsy impairs advanced social cognition.

PURPOSE: Although memory, language, and executive functions have been extensively studied in patients with mesial temporal lobe epilepsy (MTLE), investigations into advanced social cognitive abilities have been neglected. In the present study, we investigated the ability to detect social faux pas and studied possible mediating clinical and demographic variables in patients with MTLE compared with patients with an epilepsy not originating within the MTLE and healthy controls. METHODS: The 27 MTLE patients (16 were investigated pre- and 11 postoperatively), 27 patients with an extramesiotemporal epilepsy (except frontal lobe epilepsy), and 12 healthy controls performed a shortened version of the faux-pas test. Additionally, we used standardized tests to measure intelligence. Only patients with intact reading-comprehension abilities were included in the study. RESULTS: MTLE patients, both pre- and postoperative, performed the faux-pas test significantly worse than patients with extramesiotemporal lobe epilepsy and healthy controls. The latter two groups showed comparable performance. No statistical association was found between the MTLE patients' deficit in recognizing a faux pas and the variables IQ, age, age at seizure onset, and duration of epilepsy. CONCLUSIONS: We report for the first time that patients with MTLE are specifically impaired in recognizing faux pas, suggesting that MTLE as such is a specific etiology of deficits in higher-order social cognition.

Adult↗

PubMed2006

Replication properties of human adenovirus in vivo and in cultures of primary cells from different animal species.

Oncolytic adenoviruses have emerged as a promising approach for the treatment of tumors resistant to other treatment modalities. However, preclinical safety studies are hampered by the lack of a permissive nonhuman host. Screening of a panel of primary cell cultures from seven different animal species revealed that porcine cells support productive replication of human adenovirus type 5 (Ad5) nearly as efficiently as human A549 cells, while release of infectious virus by cells from other animal species tested was diminished by several orders of magnitude. Restriction of productive Ad5 replication in rodent and rabbit cells seems to act primarily at a postentry step. Replication efficiency of adenoviral vectors harboring different E1 deletions or mutations in porcine cells was similar to that in A549 cells. Side-by-side comparison of the viral load kinetics in blood of swine and mice injected with Ad5 or a replication-deficient adenoviral vector failed to provide clear evidence for virus replication in mice. In contrast, evidence suggests that adenovirus replication occurs in swine, since adenoviral late gene expression produced a 13.5-fold increase in viral load in an individual swine from day 3 to day 7 and 100-fold increase in viral DNA levels in the Ad5-infected swine compared to the animal receiving a replication-deficient adenovirus. Lung histology of Ad5-infected swine revealed a severe interstitial pneumonia. Although the results in swine are based on a small number of animals and need to be confirmed, our data strongly suggest that infection of swine with human adenovirus or oncolytic adenoviral vectors is a more appropriate animal model to study adenoviral pathogenicity or pharmacodynamic and toxicity profiles of adenoviral vectors than infection of mice.

Adenoviruses, Human↗

PubMed2006

Mid-latency auditory-evoked responses and sensory gating in focal epilepsy: a preliminary exploration.

The relationship between epilepsy and psychosis is not well defined. Sensory gating is a possible endophenotype for psychosis, and has not been fully examined in epileptic patients. The authors examined 29 patients with focal epilepsy who were on antiepileptic medications, and 29 age-matched healthy comparison subjects, using a paired-stimulus (S1-S2) paradigm. P50 and N100 amplitudes or gating did not differ between the groups. The P200 was significantly smaller and did not gate as well in epileptic patients. Though alteration of sensory gating can be demonstrated in epileptic patients, it seems to be qualitatively different from alterations reported in association with schizophrenia.

Acoustic Stimulation↗

PubMed2006

Novelty detection and encoding for declarative memory within the human hippocampus.

Intracranial recordings of cognitive potentials within the human hippocampal system have identified N400 potentials in the anterior mesial temporal lobe (AMTL-N400) that correlate with verbal memory performance and are associated with novelty detection. Their amplitudes to "new" but not "old" words in a verbal recognition task correlate with the neuronal density of the hippocampal CA1-region and can be reduced selectively by the NMDA-receptor blocker ketamine. Moreover, it could be shown that NMDA-receptor dependent long-term potentiation (LTP), a form of synaptic plasticity with Hebbian characteristics, can be readily induced in human hippocampal slices but not in patients with hippocampal sclerosis. In these latter patients, we also found a reduction of AMTL-N400 amplitudes similar to the one induced by ketamine. In addition, we could show that hippocampal novelty detection is associated with successful encoding for declarative memory. Together, our findings suggest that successful encoding for declarative memory is at least in part mediated by NMDA-receptor dependent novelty detection within the human hippocampal system.

Animals↗

PubMed2005

Immobilized stem-loop structured probes as conformational switches for enzymatic detection of microbial 16S rRNA.

We have designed and evaluated novel DNA stem-loop structured probes for enzymatic detection of nucleic acid targets. These probes constitute a novel class of conformational switches for enzymatic activity, which in the absence of a target sterically shield an affinity label and upon hybridization of the target to the recognition sequence that forms the loop of the probe restore accessibility of the label for the binding of a reporter enzyme. Analysis of probe characteristics revealed stem stability as the most important parameter governing detection functionality, while other factors such as the length of linker molecules attaching the label to the stem-loop structure and the nature of the solid support proved to be less critical. Apparently, the bulky nature of the reporter enzyme facilitates shielding of the label in the absence of the target, thereby conferring considerable structural tolerance to the conformational switch system. The stem-loop structured probes allow sensitive detection of unlabeled nucleic acid targets. Employing a microtiter assay format, 4 ng of bacterial 16S ribosomal RNA corresponding to 8 fmol could be detected, which can be compared favorably with current immobilized molecular beacon concepts based on fluorescence detection.

Colorimetry↗

PubMed2005

Language processing within the human medial temporal lobe.

Although the hippocampal formation is essential for verbal memory, it is not fully understood how it contributes to language comprehension. We recorded event-related potentials (ERPs) directly from two substructures of the medial temporal lobe (MTL), the rhinal cortex and the hippocampus proper, while epilepsy patients listened to sentences that either were correct or contained semantic or syntactic violations. Semantic violations elicited a large negative ERP response peaking at approximately 400 ms in the rhinal cortex. In contrast, syntactically incorrect sentences elicited a negative deflection of 500-800 ms in the hippocampus proper. The results suggest that functionally distinct aspects of integration in language comprehension are supported by different MTL structures: the rhinal cortex is involved in semantic integration, whereas the hippocampus proper subserves processes of syntactic integration. An analysis of phase synchronization within the gamma band between rhinal and hippocampal recording sites showed that both of the above-mentioned ERP components were preceded by an increase of phase synchronization. In contrast to these short phasic increases of phase synchronization in both violation conditions, correct sentences were associated with a long-lasting synchronization in a late time window, possibly reflecting the integration of semantic and syntactic information as required for normal comprehension.

Adult↗

PubMed2005

Reduced mobilization of Rev-responsive element-deficient lentiviral vectors.

Infection of cells transduced with a lentiviral vector by human immunodeficiency virus (HIV) could lead to packaging of the lentiviral vector RNA into HIV particles and unintended transfer of the vector. To prevent this, the Rev-responsive element (RRE) of an HIV-1 vector was functionally replaced by a heterologous RNA element (MS2). Providing Rev fused to an MS2 binding protein allowed efficient vector production. Mobilization of the vector from infected target cells was below the level of detection and at least 10(3)- to 10(4)-fold lower than for the RRE-containing vector. Thus, RRE-deficient lentiviral vectors provide a novel approach to reduce the risk of vector mobilization.

Gene Products, rev↗

PubMed2004

Phase-locking characteristics of limbic P3 responses in hippocampal sclerosis.

Amplitudes of the P3 recorded invasively from the medial temporal lobe (MTL-P3) have been reported to be reduced on the side of a mediotemporal epileptogenic focus. This reduction has been attributed to the massive cell loss within the hippocampus associated with hippocampal sclerosis. It has remained unclear how functional connectivity between the hippocampus and rhinal cortex, as well as within the hippocampus, is altered in hippocampal sclerosis. To investigate this issue, we analyzed to what extent stimulus-related phase-locking and power changes within the low-frequency range (2-30 Hz) and within the gamma band (32-48 Hz), as well as rhinal-hippocampal phase synchronization contribute to the averaged MTL-P3 potentials. Event-related responses were recorded via bilateral depth electrodes in epilepsy patients with unilateral hippocampal sclerosis, who performed a visual oddball experiment. On the contralateral (nonsclerotic) side, successful target detection was associated with an increase of power and phase locking of hippocampal activity in both the low-frequency range and in the gamma range. Besides, there were rhinal-hippocampal synchronization enhancements in the theta and gamma range. On the ipsilateral (sclerotic) side, the event-related power increase in the low-frequency range had almost disappeared, a finding likely to be explained by the loss of principle neurons. However, low-frequency phase-locking, rhinal-hippocampal synchronization, as well as event-related power changes in the gamma range persisted ipsilaterally, although there were differences in temporal and spectral characteristics. These findings support the hypothesis that functional connectivity between hippocampus and rhinal cortex, as well as intrahippocampal connectivity, are partially preserved in hippocampal sclerosis.

Adult↗

PubMed2004

Electrical detection of viral DNA using ultramicroelectrode arrays.

A fully electrical array for voltammetric detection of redox molecules produced by enzyme-labeled affinity binding complexes is shown. The electronic detection is based on ultramicroelectrode arrays manufactured in silicon technology. The 200-microm circular array positions have 800-nm-wide interdigitated gold ultramicroelectrodes embedded in silicon dioxide. Immobilization of oligonucleotide capture probes onto the gold electrodes surfaces is accomplished via thiol-gold self-assembling. Spatial separation of probes at different array positions is controlled by polymeric rings around each array position. The affinity bound complexes are labeled with alkaline phosphatase, which converts the electrochemically inactive substrate 4-aminophenyl phosphate into the active 4-hydroxyaniline (HA). The nanoscaled electrodes are used to perform a sensitive detection of enzyme activity by signal enhancing redox recycling of HA resulting in local and position-specific current signals. Multiplexing and serial readout is realized using a CMOS ASIC module and a computer-controlled multichannel potentiostat. The principle of the silicon-based electrical biochip array is shown for different experimental setups and for the detection of virus DNA in real unpurified multiplex PCR samples. The fast and quantitative electronic multicomponent analysis for all kinds of affinity assays is robust and particle tolerant.

DNA, Viral↗

PubMed2004

Reducing mobilization of simian immunodeficiency virus based vectors by primer complementation.

BACKGROUND: Human gene therapy vectors based on primate lentiviruses harbour in contrast to oncoretroviruses the risk of vector mobilization by human immunodeficiency viruses (HIV). Infection of cells transduced with a lentiviral vector by HIV could lead to packaging of the lentiviral vector RNA into HIV particles and transfer of the vector. METHODS: A new approach based on primer complementation was developed to reduce the risk of vector mobilization. The primer binding site (PBS) of an SIV-based vector was mutated abolishing tRNA primer binding and thus blocking reverse transcription. This block was efficiently by-passed during vector production by providing an artificial tRNA matching the mutated PBS with titers reaching 10(6) infectious units/ml. RESULTS: Primer-complemented SIV vectors were mobilized from transduced cells by HIV-1 >150-fold less efficiently than vectors with wild-type PBS. Mobilization of the primer-complemented SIV vector by SIV was inhibited to a lesser extent indicating reduced efficacy of the primer complementation approach for preventing mobilization of lentiviral vectors by homologous virus. The analysis of the PBS of the vector DNA in target cells transduced with vectors containing mutated PBS in the absence of a matched tRNA suggests that formation of heterozygous particles followed by priming on the helper RNA and strand switch during reverse transcription can lead to mobilization of the primer-complemented vector by SIV, but not HIV-1. Although self-inactivating vectors were more efficient in preventing vector mobilization by HIV-1 than primer-complemented vectors, mobilization remained undectable only if both approaches were combined. CONCLUSIONS: The primer complementation approach should further reduce the risk of mobilization of self-inactivating SIV-based vectors by HIV-1 and thus increase their safety.

Animals↗

PubMed2004

Persistent severe amnesia due to seizure recurrence after unilateral temporal lobectomy.

Anterograde amnesia is a severely disabling state which has been reported as a consequence of bilateral mesiotemporal lesions in humans. In the present paper, recurrent epileptic seizures after temporal lobectomy are described as a rare cause of severe amnesia in two patients. Diffusion-weighted MRI in one patient showed cytotoxic edema during a nonconvulsive status epilepticus and subsequent progressive hippocampal atrophy within the following month. In the other patient, repeated conventional MRI revealed no structural abnormalities in the contralateral temporal lobe.

Adult↗

PubMed2004

Neural bases of cognitive ERPs: more than phase reset.

Up to now, two conflicting theories have tried to explain the genesis of averaged event-related potentials (ERPs): Whereas one hypothesis claims that ERPs originate from an event-related activation of neural assemblies distinct from background dynamics, the other hypothesis states that ERPs are produced by phase resetting of ongoing oscillatory activity. So far, this question has only been addressed for early ERP components. Late ERP components, however, are generally thought to represent superimposed activities of several anatomically distinct brain areas. Thus, the question of which mechanism underlies the genesis of late ERP components cannot be easily answered based on scalp recordings. In contrast, two well-investigated late ERP components recorded invasively from within the human medial temporal lobe (MTL) in epilepsy patients, the so-called MTL-P300 and the anterior MTL-N400 (AMTL-N400), are based on single source activity. Hence, we investigated whether the MTL-P300 and the AMTL-N400 are based on an event-related activity increase, a phase reset of ongoing oscillatory activity or both. ERPs were recorded from the hippocampus and rhinal cortex in subjects performing a visual oddball paradigm and a visual word recognition paradigm. With wavelet techniques, stimulus-related phase-locking and power changes were analyzed in a frequency range covering 2 to 48 Hz. We found that the MTL-P300 is accompanied by both phase reset and power increase and that both effects overlap partly in time. In contrast, the AMTL-N400 is initially associated with phase locking without power increase and only later during the course of the AMTL-N400 we observed an additional power increase. In conclusion, both aspects, event-related activation of neural assemblies and phase resetting of ongoing activity seem to be involved in the generation of late ERP components as recorded in cognitive tasks. Therefore, separate analysis of event-related power and phase-locking changes might reveal specific insights into the mechanisms underlying different cognitive functions.

Adult↗

PubMed2003

Paratope-based protein identification by antibody and peptide phage display.

In this paper we report a novel application of single-chain antibody fragments (scFv) for protein identification utilizing the inherent information of the paratope for primary structure analysis. Combining the potential of antibody phage display and peptide phage display, selected scFvs are employed to select phage-displayed peptides mimicking an epitope of the protein of interest. Proof of principle is demonstrated by identification of the neuroblastoma protein NB-p260. This protein is recognized by apoptosis-inducing IgM antibodies present in the sera of healthy individuals. Identification of NB-p260 has been hindered by its high molecular weight in the range of 260-280kDa and its instability in purified protein preparations. Employing our approach, we subjected a human synthetic scFv library to selection using sodium dodecyl sulfate-denatured NB-p260. Specific scFvs were further used for selection of a heptapeptide phage display library. From analyzed clones, peptide sequences were identified, two of which could not be related to known proteins by conservative amino acid replacement and one of which, obtained from several clones, could be related to the actin-binding protein ABP278 after two conservative amino acid replacements. The identity of NB-p260 with ABP278 was verified by specific antibodies directed against the N and C termini of ABP278.

Amino Acid Sequence↗