Michael Szardenings- Fraunhofer Institute for Cell Therapy and Immunology
Michael Szardenings
- Fraunhofer Institute for Cell Therapy and Immunology
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79
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Introduction
Skills and Expertise
Publications
Publications (79)
Hazelnuts are frequently involved in IgE-mediated reactions and are the main cause of nut allergies in Europe. Most food products are processed before human consumption. Food processing can modify the structure, properties, and function of proteins, and as a result, the IgE-binding capacity of allergens can be affected. In this study, we aimed to i...
This study presents the effects of treating polystyrene (PS) cell culture plastic with oxidoreductase enzyme laccase and the catechol substrates caffeic acid (CA), L-DOPA, and dopamine on the culturing of normal human epidermal melanocytes (NHEMs) and human embryonal carcinoma cells (NTERA-2). The laccase–substrate treatment improved PS hydrophilic...
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The global impact of SARS-CoV-2 infection has raised concerns about secondary diseases beyond acute illness. This review explores the significance and potential underlying mechanisms of how SARS-CoV-2 infection might elicit an immune response targeting N-methyl-D-aspartate (NMDA) receptors, and its implications for autoimmune-driven neuropsychiatri...
Introduction
Unlike glycosylation of proteins expressed in mammalian systems, bacterial glycosylation is often neglected in the development of recombinant vaccines.
Methods
Here, we compared the effects of glycosylation of YghJ, an Escherichia coli protein important for mucus attachment of bacteria causing in urinary tract infections (UTIs). A nov...
(1) Background: Coronavirus proteins are quite conserved amongst endemic strains (eCoV) and SARS-CoV-2. We aimed to evaluate whether peptide epitopes might serve as useful diagnostic biomarkers to stratify previous infections and COVID-19. (2) Methods: Peptide epitopes were identified at an amino acid resolution that applied a novel statistical app...
Background:
There are no diagnostic and/or prognostic markers of the treatment outcome in patients receiving allergen immunotherapy (AIT). Although numerous allergen epitopes are known, their value in this context has not been investigated. This paper deals with re-evaluation of sera from patients who underwent AIT against rBet v 1 for treatment o...
Combinations of enzymatic hydrolysis using different proteolytic enzymes (papain, Esperase®, trypsin) and lactic fermentation with Lactobacillus plantarum were used to alter potential pea allergens, the functional properties and sensory profile of pea protein isolate (PPI). The order in which the treatments were performed had a major impact on the...
Thrombotic microangiopathies are hallmarked by attacks of disseminated microvascular thrombosis. In thrombotic thrombocytopenic purpura (TTP), this is caused by a rise in thrombogenic ultra-large von Willebrand factor (VWF) multimers because of ADAMTS13 deficiency. We previously reported that systemic plasminogen activation is therapeutic in a TTP...
Background:
In pollinosis patients, allergen-specific antibody titers show seasonal variations. Little is known about these variations at the epitope level.
Objectives:
We aimed at investigating seasonal variations on the level of allergen epitope recognition in patients with Bet v 1-related food allergy using a peptide phage display approach....
Dupilumab is a fully humanized Immunoglobulin‐(Ig)‐G4 antibody against the interleukin (IL)‐4α subunit of the IL‐4‐ and the IL‐13‐receptor.1 In patients with atopic eczema (AE), most frequent adverse reactions include injection site reactions and conjunctivitis/blepharitis.1,2 Recently, a case with suspicion of an IgE‐mediated hypersensitivity was...
Recombinant hepatitis B virus vaccines confer protection by eliciting specific antibodies against the hepatitis B surface antigen (HBsAg), known as anti-HBs. However, the performance of rapid anti-HBs diagnostic tests generates concerns regarding consistency. Novel indicators of protection might be developed by monitoring changes in targeted HBsAg-...
Background
The precise mapping of multiple antibody epitopes recognized by patients’ sera allows a more detailed and differentiated understanding of immunologic diseases. It may lead to the development of novel therapies and diagnostic tools.
Objective
Mapping soybean specific epitopes relevant for soybean allergy patients and persons sensitized t...
Objective
The development of tyrosine kinase inhibitors (TKIs) has significantly improved the treatment of chronic myeloid leukemia (CML). However, approximately one third of patients are resistant to TKI and/or progress to advanced disease stages. TKI therapy failure has a well-known association with ABL1 kinase domain (KD) mutations, but only aro...
Stem cells display remarkably high levels of 5-hydroxymethylcytosine (5hmC). Both TET2 and IDH1/2 mutations can impair the production of 5hmC, thus decreasing 5hmC levels. TET2 or IDH1/2 mutations are commonly observed in acute myeloid leukemia (AML). However, the implications of 5hmC on survival in normal karyotype AML patients have not been fully...
Aims:
In rheumatoid arthritis and collagen type II arthritis (CIA), sympathetic nerve fibers get lost in inflamed tissue. The process is probably induced by nerve repellent factors like semaphorin 3F (SEMA3F). Repulsion of sympathetic nerve fibers in inflamed tissue has proinflammatory effects due to the loss of anti-inflammatory neurotransmitters...
Background:
Detailed IgE-binding epitope analysis is a key requirement for the understanding and development of diagnostic and therapeutic agents to address food allergies.
Methods:
We combined an IgE-specific linear peptide microarray with random phage peptide display for the high-resolution mapping of IgE-binding epitopes of the major soybean...
Janus kinase 2 (JAK2) and calreticulin (CALR) constitute the two most frequent mutations in essential thrombocythemia (ET), and both are reported to be mutually exclusive. Hence, we examined a cohort of 123 myeloproliferative neoplasm (MPN) patients without BCR-ABL1 rearrangement and additional ET patients (n=96) for coexistence of JAK2 and CALR mu...
Bacteria-based anticancer therapies aim to overcome the limitations of current cancer therapy by actively targeting and efficiently removing cancer. To achieve this goal, new approaches that target and maintain bacterial drugs at sufficient concentrations during the therapeutic window are essential. Here, we examined the tumor tropism of attenuated...
Supplementary figures S1-S6.
Background
To the best of our knowledge, the association between pediatric AML and mitochondrial aberrations has not been studied. We investigated various mitochondrial aberrations in pediatric AML and evaluated their impact on clinical outcomes.
Methods
Sequencing, mitochondrial DNA (mtDNA) copy number determination, mtDNA 4,977-bp large deletion...
Clinical and laboratory characteristics in pediatric AML cases and individually matched controls
Characteristics in pediatric AML patients and individually matched controls
Distribution of mtDNA minisatellite instability in the HV1 and HV2 regions of pediatric AML patients and controls
Amplified PCR products of the mtDNA genes for direct sequencing. Agarose gel image after electrophoresis showed a clear amplification of the mtDNA control region, the CYTB gene, and the tRNA leucine 1 gene, respectively.
Primer sets for PCR, direct sequencing, gene scan, mtDNA copy number
The mtDNA sequence alterations (polymorphism) found in pediatric AML patients and controls*
Comparison of the frequencies of mtDNA haplogroups in healthy controls and pediatric AML patients
Although strains of attenuated Salmonella typhimurium and wild-type Escherichia coli show similar tumor-targeting capacities, only S. typhimurium significantly suppresses tumor growth in mice. The aim of the present study was to examine bacteria-mediated immune responses by conducting comparative analyses of the cytokine profiles and immune cell po...
The proliferating activity of a single leukemia stem cell and the molecular mechanisms for their quiescent property remain unknown, and also their prognostic value remains a matter of debate. Therefore, this study aimed to demonstrate the quiescence property and molecular signature of leukemia stem cell and their clinicopathological implications.
S...
Mitochondria are important intracellular organelles that produce energy for cellular development, differentiation, and growth. Mitochondrial DNA (mtDNA) presents a 10- to 20-fold higher susceptibility to genetic mutations owing to the lack of introns and histone proteins. The mtDNA repair system is relatively inefficient, rendering it vulnerable to...
The ligand development unit at Fraunhofer IZI offers the development of human Fab and peptide ligands from naïve combinatorial random phage display libraries. Peptides and also Fab developed with these technologies are amongst others useful for:
■ Epitope mapping
■ Development of enzyme inhibitors
■ Development of affinity purification tools
■...
Background
The loss of sympathetic nerve fibers is a general principle in inflammatory diseases. Since sympathetic neurotransmitters exert anti-inflammatory effects at increased concentrations, their loss is reasonable to overcome infection but detrimental in chronic inflammatory autoimmune diseases. Semaphorins are major factors involved in axon g...
A number of recent reports have demonstrated that attenuated Salmonella typhimurium are capable of targeting both primary and metastatic tumors. The use of bacteria as a vehicle for the delivery of anti-cancer drugs requires a mechanism that precisely regulates and visualizes gene expression to ensure the appropriate timing and location of drug pro...
The loss of sympathetic nerve fibers (SNF) is a general principle in inflammatory diseases. Since sympathetic neurotransmitters exert anti-inflammatory effects at increased concentrations, their loss in inflammed and infected tissue is reasonable to overcome infection. However, this mechanism is unfavourable in chronic inflammation like rheumatoid...
The present invention relates to a method for the production of a gene encoded molecule in an arthropod
in vivo, wherein a nucleic acid comprising a gene encoding said molecule is fed to the arthropod. Furthermore, the present invention relates to a method for the delivery of a purified nucleic acid to arthropod cells in vivo, wherein the purified...
US Patent Application Number 61/308,143
This study evaluates the binding the melanocyte stimulating hormone peptide analogue [125I]NDP-MSH to melanocortin receptors MC1, MC3, MC4 and MC5 in insect cell membranes produced by baculovirus expression systems. The presence of Ca2+ was found to be mandatory to achieve specific [125I]NDP-MSH binding to the melanocortin receptors. Although assoc...
INTRODUCCIÓN La tecnología de presentación de péptidos en la superficie de un fago, fue descrita por primera vez por George P Smith en 1985 [1] como una vía para presentar, en la superficie del virus, un producto génico que está siempre asociado al gen que lo codifica, y este gen, a su vez, es empaquetado dentro de la misma partícula viral. Desde e...
The identification of ligands from large biological libraries by phage display has now been used for almost 15 years. Most of the successful reports on high-affinity ligand identification originated from work with different antibody libraries. In contrast, the progress of applying phage display to random peptide libraries was relatively slow. Howev...
Trichoderma reesei cellobiohydrolase Cel6A is an inverting glycosidase. Structural studies have established that the tunnel-shaped active site of Cel6A contains two aspartic acids, D221 and D175, that are close to the glycosidic oxygen of the scissile bond and at hydrogen-bonding distance from each other. Here, site-directed mutagenesis, X-ray crys...
The efficiency of existing combinatorial biological library methods has been moderate in terms of the success rates, the affinities of the ligands selected and the time and effort involved in trying to optimize the initial leads. Although mimicking natural evolution, existing strategies take little notice of the importance of recombination within a...
We have investigated the ability of our earlier identified MS04–MS05 MSH-peptide analogues to bind to chimeric MC1–MC3 receptors. While the MS04 and MS05 peptides bind with nanomolar and sub-nanomolar affinities to the wild type MC1 receptor, they bind only with micromolar affinities for the wild type MC3 receptor, thus being the hitherto most MC1...
This work describes the Cosmix-PlexingÒ procedure. It provides a novel way to use and generate combinatorial libraries of peptides displayed on the surface of filamentous phages. Using the Cosmix-Plexing approach, the left and right sections of the variable domain can be induced by the use of type II restriction enzymes. Thus, a population with opt...
A peptide with very high specificity for the human melanocortin MC(1) receptor identified by phage display was used as a lead for the design of new peptides. Two new peptides, MS05 and MS09, were synthesized and found to bind with sub-nanomolar affinities to the MC(1) receptor. Both these peptides showed strong agonistic activity at the MC(1) recep...
The melanocortin receptors MC1 and MC3 are G protein-coupled receptors that have substantial structural similarities and bind melanocyte peptides but with different affinity profiles. We constructed a series of chimeric MC1/MC3 receptors to identify the epitopes that determine their selectivities for natural melanocyte peptides and synthetic analog...
A phage display system for the selection of peptides binding to heterologously expressed human melanocortin receptor 1 on the surface of insect cells has been established. It could be shown that phage particles displaying the natural ligand alpha-melanocyte-stimulating hormone bind selectively to cells expressing this receptor and that these phages...
The non-homologous N-terminal regions of four human melanocortin (MC) receptors were truncated in order to investigate their putative participation in ligand binding. Eleven constructs were made, where different numbers of residues from the N terminus were deleted. These constructs were used for transient expression experiments in COS cells and ana...
Recent site directed mutagenesis studies on the melanocortin 1 (MC1) receptor have indicated the importance of D117 and H260 amino acid residues for the binding of alpha-MSH (melanocyte stimulating hormone). Here, we report the testing of 12 cyclic and linear MSH peptides on the D117A and H260A mutant receptors. Moreover, we constructed a double mu...
We report here the binding of 5-, 6- and 7-amino-acid-long linear and cyclic core peptides of MSH (melanocyte-stimulating hormone) to cells transiently expressing the human melanocortin MC1, MC3, MC4 and MC5 receptors. The results show that, in contrast to the natural peptides, the core peptides did not differentiate between the melanocortin MC3 an...
The TM4, EL2 and TM5 show low amino acid homology within the MC receptor family. Three mutants of the human MC3 receptor were created in order to investigate the participation of these regions in ligand binding. The TM4, EL2 and TM5 were separately changed by multiple mutagenesis so that their amino acid sequences became identical with the human MC...
The genomic DNA for the human melanocortin MC3 receptor indicates an unusually long N-terminus. Two possible translation initiation sites, the one originally proposed and one alternate 111 bp downstream, were mutated. For a third mutant the DNA between these initiation sites was deleted. All mutants were expressed in COS (CV-1 Origin, SV40) cells i...
Trichoderma reesei cellobiohydrolase II (CBHII) is an exoglucanase cleaving primarily cellobiose units from the non-reducing end of cellulose chains. The beta-1,4 glycosidic bond is cleaved by acid catalysis with an aspartic acid, D221, as the likely proton donor, and another aspartate, D175, probably ensuring its protonation and stabilizing charge...
We expressed epitope-tagged human melanocortin 1 receptor (MC1R) in insect cells using two different recombinant baculovirus constructs; one of which encoded MC1R with an N-terminal Flag epitope and a C-terminal polyHis tag, while the other encoded the MC1R with a C-terminal Myc tag. The constructs were used to infect Sf9 insect cells. For both con...
The results of a protein design project are used to compare different predictive strategies with respect to protein-protein interactions. We have been able to generate variants of human pancreatic secretory trypsin inhibitor (hPSTI) optimized with respect to the affinity and specificity for human leukocyte elastase relative to trypsin and chymotryp...
Several known and some new retinoids were synthesized and their in vivo activity was investigated by an assay, based on induction of alkaline phosphatase in P19 teratocarcinoma cells, human prostate carcinoma cells and primary cultures of neonatal rat heart cells. The assay used in this study was found to be reproducible and useful for rapid screen...
A modified version of the human pancreatic trypsin inhibitor (PSTI), generated in a protein-design project, has been crystallized in spacegroup P4(3) with lattice constants a = 40.15 A, c = 33.91 A. The structure has been solved by molecular replacement. Refinement of the structure by simulated annealing and conventional restrained least-squares yi...
The human retinoic acid receptor alpha was expressed in Escherichia coli. The recombinant protein was found to be very unstable in several E. coli strains, probably due to proteolysis. Conditions were established to obtain reasonable amounts of active protein for ligand and DNA binding studies. The recombinant receptor showed the expected DNA bindi...
Variants of the human pancreatic secretory trypsin inhibitor (PSTI) have been created during a protein design project to generate a high-affinity inhibitor with respect to some serine proteases other than trypsin. Two modified versions of human PSTI with high affinity for chymotrypsin were crystallized as a complex with chymotrypsinogen. Both cryst...
Protein design requires the rapid production of recombinant genes and active recombinant proteins, the latter in sufficient amounts for functional and physical studies. We present here the construction and application of a new phasmid vector system, using the fd phage origin, lambda pL promoter, ompA-leader sequence and pMB1 origin, which allows th...
Variants of human secretory trypsin inhibitor were constructed with the aim of producing inhibitors specific for human leukocyte elastase. Models of the hPSTI/HLE and hPSTI/chymotrypsin complexes were generated by computer aided protein design and used to plan better HLE inhibitors. This resulted in the production of the strongest and most specific...
This paper describes a process for the purification of hPSTI from a recombinant Escherichia coli. The E. coli strain carries the hPSTI-gene on a multicopy plasmid with the ompA-leader-sequence, which results in the secretion of hPSTI into the medium. The separation of the cells and the prepurification were carried out either by ultrafiltration alon...
We report here the binding of 5-, 6- and 7-amino-acid-long linear and cyclic core peptides of MSH melanocyte-stimulating hormone to cells transiently expressing the human melanocortin MC , MC , MC and MC receptors. The results show that, in contrast to the 134 5 natural peptides, the core peptides did not differentiate between the melanocortin MC a...
2nd Nordic Conference on Protein Engineering. Helsingör, 10 - 11 Oct. 1990, 86
Questions
Questions (3)
Hi, thank you for your interest. We are searching for your opinion on software used to process microarray data, e.g. pictures obtained with a scanner. Is there anything out there that you would recommend? We are working with peptide arrays containing several thousand peptide spots of epitopes/mimotopes discovered with phage display selection directly from serum. We detect antibodies bound to these with a secondary/tertiary antibody combinations with fluorphore. Thanks for your input!
Hi, thank you for reading this. We are using peptide arrays on glass slides to determine antibodies in serum. In my eyes there is no really good software that would identify symmetrically applied spots, monitor the spatial distribution of pixels in the spot and provide a reasonable estimate of the signal by taking into account local and global background. Do I ask for too much?
Thank you for any advice
We are trying to transfer an assay based on antibody capture with strep-beads coated with biotin-peptide-antigen into the 96 well format. There are multiple ways to coat the streptavidin and people either use styrene or plasma activated styrene ("high binding") for this procedure. We found at least three possible variables, got various interesting results and still feel we would be able to optimize for ever. Therefore we would like to know, what you would recommend.
The variables are
- plastic surface (styrene, (non)activated, brand)
- coating conditions (pH, salt)
- Which source/quality of the streptavidin, e.g. for the coupling to carboxy surfaces only certain qualities are working
What is your experience. What do you suggest to optimize this process?
