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J Ruppert

Publications and source records attributed to J Ruppert.

At least 19 recordsLinked to original sources
PubMed2006

Phase I clinical trial of a TGF-beta antisense-modified tumor cell vaccine in patients with advanced glioma.

We performed a phase I clinical trial in grade IV astrocytoma to assess the safety of a whole-cell vaccine comprising autologous tumor cells genetically modified by a transforming growth factor-beta2 (TGF-beta2) antisense vector. Blocking secretion of the immunosuppressive molecule TGF-beta in this manner should inhibit one of the major mechanisms by which tumor cells evade immune surveillance and should lead to clinically effective antitumor immunity. Six patients with progressive WHO grade IV astrocytoma were enrolled in the trial. Patients received 2-7 subcutaneous injections of 5 x 10(6)-2 x 10(7) autologous tumor cells per injection. TGF-beta2 secretion by the tumor cells used to vaccinate patients was inhibited by 53-98%. Treatment was well tolerated with only low-grade, transient treatment-related toxicities reported. Two patients had partial regressions and two had stable disease following therapy. The overall median survival was 68 weeks. Median survival of the responding patients was 78 weeks, compared to a historic value of 47 weeks for glioma patients treated conventionally. There were indications of humoral and cellular immunity induced by the vaccine. These findings support further clinical evaluation of vaccines comprised of TGF-beta antisense-modified tumor cells.

Adult↗
PubMed2006

Directional atherectomy facilitates the interventional procedure and leads to a low rate of recurrent stenosis in left anterior descending and left circumflex artery ostium stenoses: subgroup analysis of the FLEXI-CUT study.

OBJECTIVES: To examine by retrospective analysis of data from the FLEXI-CUT monocentre registry whether atherectomy can effectively simplify complex stent implantation in ostial bifurcation lesions by reducing the procedure to stenting of the left anterior descending (LAD) or left circumflex (LCX) artery ostium alone. PATIENTS AND METHODS: All patients who had been enrolled in the prospective FLEXI-CUT study (directional atherectomy with adjunctive balloon angioplasty) were retrospectively analysed on the basis of significant LAD or LCX ostial stenosis (>or= 70% stenosis) deriving from an undiseased left main stem. The primary combined end point was the rate of target lesion revascularisation (TLR) and binary restenosis; secondary end points were procedural success and major adverse cardiac events (MACE) at the six-month follow up. RESULTS: Of 30 patients enrolled with significant LAD or LCX ostium stenosis, 29 were effectively treated with directional atherectomy (96.7% procedural success). All patients underwent single-vessel stenting procedures of solely the LAD or LCX ostium. At follow up, binary stenosis was 25% (6 of 24), TLR (angiographic plus clinical) 10.3% (3 of 29) and total MACE 6.9% (2 of 29). CONCLUSIONS: Directional atherectomy with single-vessel stenting procedures facilitates the interventional treatment of LAD and LCX ostium stenosis, and leads to remarkably low TLR and binary stenosis at follow up.

Aged↗
PubMed2004

Two cases of severe sepsis due to Vibrio vulnificus wound infection acquired in the Baltic Sea.

Two severe cases of Vibrio vulnificus wound infection with secondary septicemia occurred during 1 week in August 2003 on the German island of Usedom in the southwestern Baltic Sea. In both cases, pre-existing wounds were inoculated by wading in contaminated sea water. One of the patients died from septic multiorgan failure. To the best of our knowledge, this is the first fatality due to a V. vulnificus infection to have occurred in Germany. Microbiological analysis revealed high concentrations of V. vulnificus in sea water along the coastline, following a period when water temperature exceeded 20 degrees C for more than 2 weeks.

Female↗
PubMed2003

Prediction of pathology and survival by FDG PET in gliomas.

OBJECTIVES: Despite being in use for nearly two decades, the utility of [18F]2-fluoro-2deoxy-D-glucose positron emission tomography (FDG PET) in the evaluation and treatment of brain tumors remains controversial. We retrospectively analyzed all patients with histologically proven gliomas, between the years 1990 and 2000, who underwent FDG PET studies at various stages of their treatment and who were followed till either death or for a minimum period of 1 year in an attempt to bring resolution to this controversy. METHODS: All PET scans prior to 1997 were acquired on an ECAT 951/31 scanner in 2D. Scans since 1997 were obtained on a Siemens HR+ scanner in 3D mode. The majority of FDG PET scans were co-registered with the magnetic resonance imaging (MRI) scans to aid in diagnosis and therapy. Based on independent visual inspection, two board certified nuclear medicine physicians graded the highest activity level of the tumor using the metabolic grading: 0 = no uptake; 1 = uptake less or equal to normal white matter; 2 = uptake greater than normal white matter and less than gray matter; 3 = uptake equal to or greater than gray mater. The measure of association of lambda (lambda) was used to measure the strength of predictive ability of FDG PET for pathological grading of the gliomas. The Cox proportional hazards regression model was used to assess the significance of grade of uptake on survival. RESULTS: A total of 331 patients were analyzed of which 137 had a PET scan prior to histological diagnosis and therapeutic intervention (mean age = 46.5 years; M:F = 1.7:1). Eighty six percent (143/166) of the patients with low uptake (metabolic scores 0,1) had low-grade gliomas (grade I,II) and 14% (23/166) high-grade gliomas (grade III,IV) on histologic examination. Ninety four percent (154/165) of the patients with high uptake (metabolic scores 2,3) on PET had high-grade gliomas and 7% (11/165) had low-grade gliomas on histologic examination. The grade of uptake had increasing significance on survival as the level increased from 'low' to 'high' (P = 0.0009). Ninety four percent (156/166) of the patients with low uptake survived for > 1 year (median survival of 28 months) and 19% survived for > 5 years. Only 29% (48/165) of patients with high uptake survived for > 1 year, (median survival of 11 months) and none survived for > 5 years. Irrespective of when the scan showed a high uptake of FDG, before or after intervention, the prognosis following that scan was poor. CONCLUSIONS: Our observations confirm the utility of FDG PET as a prognostic tool for the histological grading and survival in patients with gliomas and appears to more than complement pathological grading.

Adolescent↗
PubMed1999

Lipopeptide particles as the immunologically active component of CTL inducing vaccines.

Using a bipalmitoylated lipopeptide consisting of an ovalbumin helper T-cell epitope covalently linked to an influenza virus cytotoxic T-lymphocyte (CTL) epitope, we addressed possible factors that may be critical for CTL induction. Antigen processing of lipopeptide appears to be required for T-cell induction since there was virtually no in vitro binding of lipopeptide to purified MHC molecules. A major portion of lipopeptide immunogenicity was due to its particulate nature inasmuch as CTL induction in mice correlated with insoluble lipopeptide constructs, whereas more soluble analogs were significantly less immunogenic. Immunohistological analysis of tissue from immunized animals revealed that lipopeptide migration from the s.c. injection site to the spleen could be detected as early as 1 h after immunization and cell-associated lipopeptide was observed on macrophages and dendritic cells, implicating both cell populations in the processing and presentation of lipopeptide particles to CTLs.

Amino Acid Sequence↗
PubMed1999

IcePick: a flexible surface-based system for molecular diversity.

IcePick is a system for computationally selecting diverse sets of molecules. It computes the dissimilarity of the surface-accessible features of two molecules, taking into account conformational flexibility. Then, the intrinsic diversity of an entire set of molecules is calculated from a spanning tree over the pairwise dissimilarities. IcePick's dissimilarity measure is compared against traditional 2D topological approaches, and the spanning tree diversity measure is compared against commonly used variance techniques. The method has proven easy to implement and is fast enough to be used in selection of reactants for numerous production-sized combinatorial libraries.

Drug Design↗
PubMed1997

Automatic identification and representation of protein binding sites for molecular docking.

Molecular docking is a popular way to screen for novel drug compounds. The method involves aligning small molecules to a protein structure and estimating their binding affinity. To do this rapidly for tens of thousands of molecules requires an effective representation of the binding region of the target protein. This paper presents an algorithm for representing a protein's binding site in a way that is specifically suited to molecular docking applications. Initially the protein's surface is coated with a collection of molecular fragments that could potentially interact with the protein. Each fragment, or probe, serves as a potential alignment point for atoms in a ligand, and is scored to represent that probe's affinity for the protein. Probes are then clustered by accumulating their affinities, where high affinity clusters are identified as being the "stickiest" portions of the protein surface. The stickiest cluster is used as a computational binding "pocket" for docking. This method of site identification was tested on a number of ligand-protein complexes; in each case the pocket constructed by the algorithm coincided with the known ligand binding site. Successful docking experiments demonstrated the effectiveness of the probe representation.

Algorithms↗
PubMed1996

Hammerhead: fast, fully automated docking of flexible ligands to protein binding sites.

BACKGROUND: Molecular docking seeks to predict the geometry and affinity of the binding of a small molecule to a given protein of known structure. Rigid docking has long been used to screen databases of small molecules, because docking techniques that account for ligand flexibility have either been too slow or have required significant human intervention. Here we describe a docking algorithm, Hammerhead, which is a fast, automated tool to screen for the binding of flexible molecules to protein binding sites. RESULTS: We used Hammerhead to successfully dock a variety of positive control ligands into their cognate proteins. The empirically tuned scoring function of the algorithm predicted binding affinities within 1.3 log units of the known affinities for these ligands. Conformations and alignments close to those determined crystallographically received the highest scores. We screened 80 000 compounds for binding to streptavidin, and biotin was predicted as the top-scoring ligand, with other known ligands included among the highest-scoring dockings. The screen ran in a few days on commonly available hardware. CONCLUSIONS: Hammerhead is suitable for screening large databases of flexible molecules for binding to a protein of known structure. It correctly docks a variety of known flexible ligands, and it spends an average of only a few seconds on each compound during a screen. The approach is completely automated, from the elucidation of protein binding sites, through the docking of molecules, to the final selection of compounds for assay.

Algorithms↗
PubMed1996

Epitope mapping of the human TSH receptor; structure function studies.

With the aid of recombinant DNA technology (PCR/site directed mutagenesis, sequencing) the full length coding region of the human TSH receptor was manipulated to place a specific epitope peptide tag (FLAG epitope sequence) at the carboxyl end of the protein. The resulting construct was cloned into a eukaryotic expression vector and stably transfected into HeLa cells. The expression/translation of the tagged TSH receptor molecule was monitored by immune-precipitation and western blotting of protein lysates, and was found to be expressed at considerable levels using the commercially available antibodies directed towards the FLAG epitope. This analysis revealed two discrete specific bands 90-120 KDa representing, presumably, differently glycosylated forms of the receptor. TSH radio receptor assays demonstrated that the FLAG tagged TSH receptor bound TSH comparable with the wild type receptor. Furthermore TSH stimulated cAMP response in these transfected cells were comparable to the wild type receptor, thus demonstrating that the tagged receptor was functionally identical to the transfected wild type receptor. These cell lines will be of great value when analysing TSH/receptor or receptor/autoantibody interactions considering the availability of well characterized experimental anti-TSH receptor sera.

Blotting, Western↗
PubMed1995

Human CTL epitopes encoded by human papillomavirus type 16 E6 and E7 identified through in vivo and in vitro immunogenicity studies of HLA-A*0201-binding peptides.

Human papillomavirus type 16 (HPV16) is strongly associated with cervical carcinogenesis. The HPV16 E6 and E7 oncoproteins are constitutively expressed in the majority of cervical tumor cells and are, therefore, attractive targets for CTL-mediated immunotherapy. In mice, the outgrowth of a lethal dose of HPV16-induced tumor cells has been prevented by vaccination with a CTL epitope encoded by HPV16 E7, indicating the feasibility of peptide immunization to obtain antitumor CTL responses. In the present study, the immunogenicity of 9 HLA-A*0201-binding peptides encoded by HPV16 E6 and E7 was analyzed in vivo in HLA-A*0201Kb transgenic mice and in vitro in CTL cultures induced from PBMC of HLA-A*0201+ healthy donors. Four peptides with a good binding affinity were immunogenic in HLA-A*0201Kb transgenic mice, and three of them were also highly immunogenic in CTL induction experiments with PBMC of HLA-A*0201+ healthy donors. Human CTL clones specific for these three peptides were capable of lysing the HPV16 E7-containing HLA-A*0201+ cervical carcinoma cell line CaSki. These E7-derived peptides (11-20, YMLDLQPETT; 82-90, LLMGTLGIV; 86-93, TLGIVCPI), therefore, are likely to represent naturally processed human CTL epitopes of HPV16. Additionally, these three HPV16-encoded peptides have the highest affinity of binding to the HLA-A*0201 molecule. In this study, peptides with a lower binding affinity were less immunogenic. Therefore, our data illustrate that the HLA-binding affinity of a peptide has a major impact on its immunogenicity. In conclusion, we have identified immunogenic peptides encoded by HPV16 E6 and E7 that could be used in vaccines for the prevention and treatment of cervical carcinoma.

Amino Acid Sequence↗
PubMed1995

Class I MHC-peptide interactions: structural requirements and functional implications.

In this chapter, we have defined the structural motifs that dictate the capacity of peptides to bind to five different HLA-A alleles that represent some of the most common alleles found in different ethnic populations. In general, these peptide motifs were very specific for the individual HLA-A alleles, with the exception of HLA-A degree 0301 and HLA-A degree 1101, for which the motifs were very similar. When these motifs were tested against an unbiased and complete set of nonamer peptides derived from human papillomavirus E6 and E7 proteins, it was found that the vast majority of high and intermediate binding peptides contained the appropriate motif. Furthermore, using the dominant anchor residues, together with the amino acid positions that interact with secondary anchor residues, it was possible to predict high and intermediate binders. Finally, the finding that there is a direct correlation between binding affinity for MHC and immunogenicity suggests a practical application of being able to predict those peptides that have a high affinity binding for a particular MHC allele--that is, in the design of peptide based vaccines for prophylactic or therapeutic use.

Alleles↗
PubMed1994

The relationship between class I binding affinity and immunogenicity of potential cytotoxic T cell epitopes.

The relationship between binding affinity for HLA class I molecules and immunogenicity of discrete peptide epitopes has been analyzed in two different experimental approaches. In the first approach, the immunogenicity of potential epitopes ranging in MHC binding affinity over a 10,000-fold range was analyzed in HLA-A*0201 transgenic mice. In the second approach, the antigenicity of approximately 100 different hepatitis B virus (HBV)-derived potential epitopes, all carrying A*0201 binding motifs, was assessed by using PBL of acute hepatitis patients. In both cases, it was found that an affinity threshold of approximately 500 nM (preferably 50 nM or less) apparently determines the capacity of a peptide epitope to elicit a CTL response. These data correlate well with class I binding affinity measurements of either naturally processed peptides or previously described T cell epitopes. Taken together, these data have important implications for the selection of epitopes for peptide-based vaccines, and also formally demonstrate the crucial role of determinant selection in the shaping of T cell responses. Because in most (but not all) cases, high affinity peptides seem to be immunogenic, our data also suggest that holes in the functional T cell repertoire, if they exist, may be relatively rare.

Amino Acid Sequence↗
PubMed1994

Peptide binding to the most frequent HLA-A class I alleles measured by quantitative molecular binding assays.

Quantitative assays to measure the binding of defined synthetic antigenic peptides and purified MHC class I molecules are described for several common human HLA-A alleles (A1, A2.1, A3, A11 and A24). Under appropriate conditions, the binding of radiolabeled peptides to purified MHC class I molecules is very effective, highly specific, and appears to be dependent on the specific sequence motif of the peptide as defined by critical anchor residue positions. Establishment and optimization of the assay reveals that a relatively high fraction of the MHC class I molecules isolated from EBV transformed B cell line sources is capable of binding exogenously added peptide. Scatchard analysis for all alleles yields 5-10% occupancy values. There is a stringent peptide size requirement that is reflected by the direct influence of peptide length on the binding affinity. The peptide-MHC class I interactions demonstrate remarkable similarity to peptide-MHC class II interactions, both in overall affinity and kinetic behavior. The immunological relevance of the peptide-MHC class I binding assay is also demonstrated by measuring the affinity of a panel of previously described HLA restricted peptides for their HLA restriction element. In 91% (10/11) of the cases, the peptides bound with affinities of 50 nM or less, and in the remaining 9% (1/11) of the cases, in the 50 to 500 nM range. Thus, these data provide the first quantitative estimate of what level of HLA-A binding affinity is associated with a diverse panel of immunodominant CTL epitopes in man.

Amino Acid Sequence↗
PubMed1994

Development of high potency universal DR-restricted helper epitopes by modification of high affinity DR-blocking peptides.

Pan DR-binding peptides engineered by introducing anchor residues for different DR motifs within a polyalanine backbone bound 10 of 10 DR molecules tested, with affinities, in most cases, in the nanomolar range. Because of the small methyl group exposed for T cell recognition, these peptides were poor immunogens but effective blockers of DR-restricted antigen presentation. Introduction of bulky and charged residues at positions accessible for T cell recognition yielded extremely powerful Pan DR epitope peptides (PADRE). These peptides elicited powerful responses in vitro from human peripheral blood mononuclear cells (PBMC). Because these cells also cross-react on certain mouse class II alleles, we could also demonstrate that PADRE peptides are active in vivo. In one example of their capacity to elicit T help, they were approximately 1000 times more powerful than natural T cell epitopes. We propose that PADRE peptides may be useful in the development of subunit vaccines.

Alleles↗
PubMed1994

TCR antagonism and T cell tolerance can be independently induced in a DR-restricted, hemagglutinin-specific T cell clone.

The outcome of TCR engagement with peptide-MHC is of central importance for the immune response of the host. TCR antagonism is one phenomenon known which is characterized by selective inhibition of T cell responses by non-stimulatory antigen analogs. T cell anergy is another state resulting in T cell unresponsiveness, generally characterized by lack of proliferation and lymphokine production. In the present study, the relationship between TCR antagonism and T cell anergy was examined by using protocols known to induce either phenomenon. Re-isolation experiments demonstrated that antagonized T cells were not tolerized, in that they were fully capable of responding to a subsequent antigen challenge. Conversely, while high doses of soluble antigen could efficiently induce T cell tolerance, TCR antagonists, either alone or in conjunction with suboptimal antigen doses, could not. Taken together, these data demonstrate that TCR antagonism and T cell tolerance are phenomena independent of each other.

Antigen-Presenting Cells↗
PubMed1994

Antigen analogs/MHC complexes as specific T cell receptor antagonists.

Recent studies demonstrated that antigen analogs can act as powerful and specific inhibitors of T cell activation, leading to the formulation of the concept that antigen analog/MHC complexes may act as antagonists of the T cell receptor (TCR). TCR antagonism appears to be associated with engagement of the TCR below a crucial affinity threshold necessary for full T cell activation. Studies addressing the molecular mechanism of this effect suggest that TCR antagonists could act by interfering with membrane-related events (such as proper receptor clustering) that might precede intracellular signaling. Discovery of the TCR antagonism phenomenon also suggested a possible rational approach to antigen-specific immunointervention in allergies and autoimmune diseases. The feasibility of such an approach is now being actively investigated. Finally, TCR antagonist peptides may provide a useful tool to probe TCR-peptide/MHC interactions involved in the process of thymic education.

Animals↗