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Mouse myeloma cells were transfected with pSV2-gpt and pSV2-neo based immunoglobulin expression vectors. Double transfectants were selected using the xanthine-guanine phosphoribosyl transferase (gpt) and the neomycin (neo) selection... more
Mouse myeloma cells were transfected with pSV2-gpt and pSV2-neo based immunoglobulin expression vectors. Double transfectants were selected using the xanthine-guanine phosphoribosyl transferase (gpt) and the neomycin (neo) selection marker genes. A broad distribution in the level of mouse-human chimeric IgG expression was observed with series of independently isolated transfectoma clones. The relative amounts of secreted to membrane-bound antibodies correlated closely, which suggested, that fluorescence-activated cell sorting could be a valuable tool for the selection of high-yielding production cell lines. However, a single cycle of cell sorting did not steer the cloning process significantly toward cells that produce enhanced amounts of recombinant IgG. Only in cases in which the polyclonal transfectoma population contained a large percentage of nonproducing cells, these were successfully separated from the IgG-producing cell population. (c) 1996 John Wiley & Sons, Inc.
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<p>Histological sections were stained with ZN (counterstain methylenblue). A: Cross sections through the excised tissue specimens from BU plaque lesions (A2-A4, A7-A8) or ulcerated BU lesions (A1, A5-A6) revealing large clusters of... more
<p>Histological sections were stained with ZN (counterstain methylenblue). A: Cross sections through the excised tissue specimens from BU plaque lesions (A2-A4, A7-A8) or ulcerated BU lesions (A1, A5-A6) revealing large clusters of AFB located in the subcutis in different tissue depths (3 mm—10 mm). Boxed areas are shown in a higher magnification in (B). B: Large clusters of AFB. C: High magnification of AFB with typical rod shaped appearance.</p
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<p>Increase in the probability of obtaining one or more positive smear microscopy results with the number of swabs analyzed.</p
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<p>Groups of six immunized, female BALB/c mice were infected by s.c. injection of <i>M</i>. <i>ulcerans</i> suspension (30 μl) into the left hind foot pad. (A) Development of the infection was followed by... more
<p>Groups of six immunized, female BALB/c mice were infected by s.c. injection of <i>M</i>. <i>ulcerans</i> suspension (30 μl) into the left hind foot pad. (A) Development of the infection was followed by weekly measurements of the foot pad thickness with a caliper. The mean foot pad thickness (dot) ± standard deviation is shown for each animal group. (B) At day 60 after infection, mice were sacrificed and the number of <i>M</i>. <i>ulcerans</i> bacilli in foot pads determined by classical CFU plating. (C) Quantification of <i>M</i>. <i>ulcerans</i> in foot pad lysates by IS2404 specific qPCR. Genome equivalents are shown. (D) Determination of <i>M</i>. <i>ulcerans</i> genome equivalents in immunized and infected mice. * <i>p</i> ≤ 0.05 (Mann-Whitney test).</p
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The acquisition by a Mycobacterium marinum-like progenitor of a plasmid encoding enzymes for the biosynthesis of the highly potent macrolide toxin mycolactone has set off the evolution of M. ulcerans toward a new mycobacterial species.... more
The acquisition by a Mycobacterium marinum-like progenitor of a plasmid encoding enzymes for the biosynthesis of the highly potent macrolide toxin mycolactone has set off the evolution of M. ulcerans toward a new mycobacterial species. While the selective advantage of producing mycolactone for survival in environmental niche(s) of the pathogen is unclear, there is no doubt that the cytotoxic, immunomodulatory, and analgesic properties of mycolactone are key for the establishment and progression of M. ulcerans infections in the host. Improved procedures for the isolation, handling, and detection of the amphiphilic and light-sensitive toxin have facilitated studies to unravel molecular mechanisms of mycolactone action on host cells in vitro and on cellular and immune responses in animal models. The pivotal role of mycolactone in the pathology of Buruli ulcer and the fact that the toxin has not been associated with other pathogens make it an ideal target for therapeutics/vaccines aiming at mycolactone neutralization and for the development of assays for the diagnosis of the disease.
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Background Plasmodium falciparum cysteine-rich protective antigen (PfCyRPA) is an invasion complex protein essential for erythrocyte invasion. In contrast to several previously clinically tested merozoite vaccine candidate antigens,... more
Background Plasmodium falciparum cysteine-rich protective antigen (PfCyRPA) is an invasion complex protein essential for erythrocyte invasion. In contrast to several previously clinically tested merozoite vaccine candidate antigens, PfCyRPA is not polymorphic, making it a promising candidate antigen for blood stage vaccine development. Methods Mice and rabbits were immunized with vaccine formulations of recombinantly expressed PfCyRPA adjuvanted either with the glucopyranosyl lipid A (GLA) containing adjuvants GLA-LSQ, GLA-SE, GLA-Alum or with Nanoalum. ELISA and indirect immunofluorescence assays (IFA) were used to analyse elicited IgG titers and the P. falciparum growth inhibitory activity was determined with a standardized in vitro [3H]-hypoxanthine incorporation assay. Results In the mouse experiments, the GLA adjuvanted formulations were superior to the Nanoalum formulation with respect to antibody titer development, IFA sero-conversion rates and in vitro parasite growth-inhibi...
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IntroductionSeveral diseases caused by the dysregulation of complement activation can be treated with inhibitors of the complement components C5 and/or C3. However, complement is required for serum bactericidal activity (SBA) against... more
IntroductionSeveral diseases caused by the dysregulation of complement activation can be treated with inhibitors of the complement components C5 and/or C3. However, complement is required for serum bactericidal activity (SBA) against encapsulated Gram-negative bacteria. Therefore, C3 and C5 inhibition increases the risk of invasive disease, in particular by Neisseria meningitidis. As inhibitors against complement components other than C3 and C5 may carry a reduced risk of infection, we compared the effect of inhibitors targeting the terminal pathway (C5), the central complement component C3, the alternative pathway (FB and FD), and the lectin pathway (MASP-2) on SBA against serogroup B meningococci.MethodsSerum from adults was collected before and after vaccination with the meningococcal serogroup B vaccine 4CMenB and tested for meningococcal killing. Since the B capsular polysaccharide is structurally similar to certain human polysaccharides, 4CMenB was designed to elicit antibodie...
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<p>(A) Genome maps of recombinant VSV: The genome of VSV encodes for the nucleoprotein N, phosphoprotein P, matrix protein M, glycoprotein G, and the large RNA polymerase L. In VSV*ΔG, the glycoprotein G coding region was replaced... more
<p>(A) Genome maps of recombinant VSV: The genome of VSV encodes for the nucleoprotein N, phosphoprotein P, matrix protein M, glycoprotein G, and the large RNA polymerase L. In VSV*ΔG, the glycoprotein G coding region was replaced by the coding sequence for eGFP. In VSV*ΔG(MUL22332) and VSV*ΔG(MUL3720), the VSV G gene was replaced by the <i>M</i>. <i>ulcerans</i> genes MUL2232 and MUL3720, respectively. The reporter protein eGFP is encoded by an additional transcription cassette located downstream of the <i>M</i>. <i>ulcerans</i> genes. (B) BHK-21 cells were infected with the indicated replicons and indirect immunofluorescence analysis performed with mAbs specific for MUL2232 (B1) or MUL3720 (B2) (red fluorescence). The infection of cells with VRPs is indicated by eGFP expression (green fluorescence). Scale bar equals 30 μm. (C) Equal numbers of L929 fibroblasts were infected with the indicated VRPs at a MOI of 10. At 6 hours post infection, the cells were harvested and protein lysates of the soluble (s) and insoluble (i) fraction analysed by Western blotting for expression of <i>M</i>. <i>ulcerans</i> antigens using mAbs specific for MUL2232 (C1) and MUL3720 (C2), respectively.</p
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Many pathogenic bacteria utilize glycan-based interactions to bind to host cells. Glycan array analysis and surface plasmon resonance are glycobioanalytical techniques that have been used to investigate the glycointeractions of a range of... more
Many pathogenic bacteria utilize glycan-based interactions to bind to host cells. Glycan array analysis and surface plasmon resonance are glycobioanalytical techniques that have been used to investigate the glycointeractions of a range of pathogens. The analysis of the glycointeractome, particularly the binding of host glycans by Mycobacteria, has been limited. In this chapter, we outline methodologies that have been successfully implemented for studying Mycobacterium ulcerans glycointeractions.
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To obtain the complete sequence and analyze the diversity of the MSP 1 molecule from the Chinese isolates of Plasmodium falciparum. Genomic DNA was prepared directly from blood samples spotted on filter papers from 2 malaria patients from... more
To obtain the complete sequence and analyze the diversity of the MSP 1 molecule from the Chinese isolates of Plasmodium falciparum. Genomic DNA was prepared directly from blood samples spotted on filter papers from 2 malaria patients from Baoting County, Hainan Province. PCR amplification of the target gene was carried out using 5 pairs of oligonucleotides specific for the MSP 1 gene. Direct sequencing of the target gene fragments was performed using ABI PRISM Dye Terminator Cycle Sequencing Ready Reaction Kit (Perkin Elmer) in a automatic ABI PRISM 310 Genetic Analyzer. For the first time, two complete sequences of the MSP1 gene from two Chinese isolates of Plasmodium falciparum were obtained. A comparison with the previously reported sequences identified them as members of the MAD20 allelic family. The deduced amino acid sequences of the MSP1 from this two Chinese isolates were identical with each other except for Blocks 2, 4 and 8. The sequences of the MSP 1 from two Chinese isol...
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Research Interests: Africa, Drug Discovery, Surveillance, Medicine, Senegal, and 15 moreBiological Sciences, Humans, Biomedical Research, Burkina Faso, Vaccination, Meningococcal disease, Meningitis, Elsevier, Outbreak, Meningococcal Vaccines, Meningococcal meningitis, Carriage, Carrier state, Neisseria meningitidis, and Medical and Health Sciences
Mice were injected intravenously with 1 mCi of [35S]methionine, and serum and tissue were sampled 4 h later. Serum and lymphoid and non-lymphoid tissues had all incorporated enough labeled methionine to allow radiofluorographic detection... more
Mice were injected intravenously with 1 mCi of [35S]methionine, and serum and tissue were sampled 4 h later. Serum and lymphoid and non-lymphoid tissues had all incorporated enough labeled methionine to allow radiofluorographic detection on two-dimensional gels of proteins synthesized de novo and, by comparing radiofluorographs, we could distinguish spots peculiar to a given tissue from others more ubiquitous. We selected a protein of 17-kDa apparent molecular mass and pl about 4 to demonstrate tissue specificity. All patterns obtained for thymus samples yielded this spot; in all other immunologically relevant sites it was missing or weak. It also was not detected on gels previously obtained from lymphocyte subpopulations biosynthetically labeled in vitro. The labeling method described here will be especially helpful for characterizing cell populations that cannot be radiolabeled under cell-culture conditions. In contrast to detection methods that detect all proteins of a sample, in...
Research Interests: Molecular Biology, Biology, Medicine, Clinical Chemistry, In Vitro, and 15 moreMedical Biotechnology, Mice, Female, Animals, Muscles, Clinical Sciences, In Vivo, Electrophoresis, Myocardium, Methionine, Lymphoid Tissue, Protein Biosynthesis, Lymph nodes, Thymus Gland, and Medical biochemistry and metabolomics
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Expression of D2NS1 and D3NS1 by transfected HEK cells. While aqua staining of lysates prepared from HEK-derived cell lines expressing D2NS1 (D2) and D3NS1 (D3) showed no specific band for the NS1 protein as compared to the lysates of... more
Expression of D2NS1 and D3NS1 by transfected HEK cells. While aqua staining of lysates prepared from HEK-derived cell lines expressing D2NS1 (D2) and D3NS1 (D3) showed no specific band for the NS1 protein as compared to the lysates of untransfected HEK cells (−) (a), Western blot analysis using anti-hexa-His tag antibodies confirmed the expression of D2NS1 and D3NS1 by the HEK cells (b). Western blotting using anti-tubulin antibodies was performed as a control for the amount of untransfected and transfected HEK cell lysates loaded on the gels (c). M = molecular weight marker in kDa. (TIF 3371 kb)
Research Interests: Biochemistry, Genetics, Marine Biology, Hematology, Developmental Biology, and 15 moreImmunology, Molecular Biology, Mental Health, Biotechnology, Cancer, Biology, Cell Biology, Infectious Diseases, Dengue Virus, Immunization, Monoclonal Antibodies, Antibody, Hybridoma Technology, Monoclonal Antibody, and Antigen
The neglected tropical disease Buruli ulcer, caused byMycobacterium ulceransinfection, displays coagulative necrosis in affected skin tissues. We previously demonstrated that exposure to theM. ulceransexotoxin mycolactone depletes the... more
The neglected tropical disease Buruli ulcer, caused byMycobacterium ulceransinfection, displays coagulative necrosis in affected skin tissues. We previously demonstrated that exposure to theM. ulceransexotoxin mycolactone depletes the expression of thrombomodulin and impacts anticoagulation at the endothelial cell surface. Moreover, while widespread fibrin deposition is a common feature of BU lesions, the cause of this phenotype is not clear. Here, we performed sequential staining of serial tissue sections of BU patient biopsies and unbiased analysis of up to 908 individual non-necrotic vessels of eight BU lesions to investigate its origins. Most vessels showed evidence of endothelial dysfunction being thrombomodulin-negative, von Willebrand factor-negative and/or had endothelium that stained positively for tissue factor (TF). Primary haemostasis was rarely evident by platelet glycoprotein CD61 staining. Localisation of TF in these lesions was complex and aberrant, including diffuse...
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<p>Histological sections of foot pads from <i>M</i>. <i>ulcerans</i>-infected mice were stained with Haematoxylin/Eosin (A1-A3, B1-B4) or Ziehl-Neelsen/Methylene blue (ZN) (C1-C5, D1-D3). The right,... more
<p>Histological sections of foot pads from <i>M</i>. <i>ulcerans</i>-infected mice were stained with Haematoxylin/Eosin (A1-A3, B1-B4) or Ziehl-Neelsen/Methylene blue (ZN) (C1-C5, D1-D3). The right, non-infected foot pad showed no swelling (A1), intact muscle tissue (A2) and absence of oedema in the upper part of the foot (A3). As opposed to this, the left foot of a mouse immunized with VSV*ΔG/rMUL2232 (prime-boost control) was strongly swollen 60 days post infection (B1). Cellular infiltration and beginning necrosis of the tissue was explicitly strong at the base of the foot pad, where bacteria were injected (B2) and muscle fibres started to become destroyed (B3). The upper part of the foot is highly oedematous (B4). Staining of the same foot pad for AFB revealed large amounts of bacteria in close contact to infiltrating immune cells at the base of the foot (C3). Appearance of AFB in the destroyed muscle tissue (C4). Big clumps of bacteria were also found towards the ankle of the foot (C2) and in the oedematous tissue in the upper half of the foot pad (C5). The foot pad of a mouse immunized with VSV*ΔG(MUL2232)/rMUL2232 (prime-boost) contained large amounts of AFB (D1) which were less spread in the whole foot pad. Intracellular AFB were frequently observed (D3).</p
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For many years, wide margin surgical excision of Buruli ulcer lesions has been the main approach for the treatment of Mycobacterium ulcerans disease. The WHO now recommends an eight-week course of oral antibiotics with a combination of... more
For many years, wide margin surgical excision of Buruli ulcer lesions has been the main approach for the treatment of Mycobacterium ulcerans disease. The WHO now recommends an eight-week course of oral antibiotics with a combination of rifampicin and clarithromycin in Africa. However, disease management is complicated by social stigma, lack of awareness, and limited access to healthcare facilities, resulting in underreporting and frequently late initiation of medical treatment. Inadequate initial treatment can drive permanent disabilities and also limited compliance to the eight-week therapy is a limitation. Therefore, search for a faster and more simple treatment modality is ongoing, focusing primarily on the testing of new tuberculosis drug candidates for the treatment of M. ulcerans disease.
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Oscillating micro-cantilever array enables immunoassay for single-step label-free analysis of candidate malaria vaccines. Differential read-out reveals epitope-specific timeline of malaria infection in complex serum samples.
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Hitherto anti-CEA monoclonal antibodies (MAbs), normally of mouse origin, have been used primarily for clinical diagnosis of colorectal cancer, either as a tumor marker in serum to monitor tumor recurrence, or latterly as a means to... more
Hitherto anti-CEA monoclonal antibodies (MAbs), normally of mouse origin, have been used primarily for clinical diagnosis of colorectal cancer, either as a tumor marker in serum to monitor tumor recurrence, or latterly as a means to localize in vivo CEA-bearing tumors and metastases in patients. In vivo diagnosis using mouse anti-CEA MAbs has so far had limited clinical utility because the antibodies elicit a strong anti-mouse immunoglobulin immune response on repeated administration in man. This problem has been addressed by the development of various strategies for “humanization” of mouse anti-CEA MAbs by genetic manipulation of immunoglobulin genes. Such humanized, engineered antibodies markedly attenuate the antigenic response directed against the MAb, such that safe, repeated administration to patients has become feasible. Such humanized anti-CEA antibodies can thus be radioactively-labelled and applied for in vivo monitoring and detection of recurrent malignant disease, or use...
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The objective of the EPIDEMIO project is to provide earth-observation-derived information on the environ- ment to epidemiologists working to study, monitor and predict threats to human health. End-user organizations directly involved in... more
The objective of the EPIDEMIO project is to provide earth-observation-derived information on the environ- ment to epidemiologists working to study, monitor and predict threats to human health. End-user organizations directly involved in the project include the World Health Organization, European research institutes, and several organizations based in Africa. Earth observation pro- vides a broad range of information types: digital ele-
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Das intestinale Immunsystem beherbergt nicht nur ca. 50–70% aller lymphatischen Zellen des Gesamtorganismus und ist somit das groste lymphatische Organ im Korper, sondern verrichtet aufgrund seiner standigen Exposition gegenuber der... more
Das intestinale Immunsystem beherbergt nicht nur ca. 50–70% aller lymphatischen Zellen des Gesamtorganismus und ist somit das groste lymphatische Organ im Korper, sondern verrichtet aufgrund seiner standigen Exposition gegenuber der Umwelt eine komplizierte Doppelrolle. Einerseits hat es die Aufgabe, den Organismus an dieser epithelialen Grenzflache zur Umwelt durch eine protektive Immunantwort gegenuber oral zugefuhrten pathogenen Bakterien, Viren und Parasiten zu schutzen, und andererseits mus es Toleranz gegenuber Nahrungsmittelantigenen und der physiologischen Darmflora bewahren. Letztere stellt gewissermasen ein „Organ“ dar, welches entscheidend an der Versorgung des Organismus mit essentiellen Nahrstoffen beteiligt ist. Daruber hinaus schutzt es den Organismus vor der Besiedlung mit pathogenen Keimen, z. B. durch Produktion antibiotischer Stoffe. Immunologisch gesehen ist dieses „Organ“ ein Xenotransplantat, denn es produziert eine reiche Vielfalt an xenogenen Nominal-und Superantigenen. Insbesondere letztere besitzen die Fahigkeit, aufgrund ihrer V-Gen-selektiven Eigenschaften erheblichen Selektionsdruck auf die Zusammensetzung des Antigenrezeporrepertoires von Darmlymphozyten auszuuben [6, 7, 24]. Die Gewahrleistung einer sinnvollen Symbiose zwischen diesem „Organ“ und dem menschlichen Organismus erfordert daher eine auserst kritische Balance zwischen immunkompetenz-und toleranzinduzierenden Mechanismen.
Research Interests: Biology and Gynecology
ABSTRACT Hypertrophic scars and keloids are fibrous overgrowths, which may develop in response to skin injury, but spontaneous forms also exist. It has been reported that tissue extracts from keloids contain elevated levels of activated... more
ABSTRACT Hypertrophic scars and keloids are fibrous overgrowths, which may develop in response to skin injury, but spontaneous forms also exist. It has been reported that tissue extracts from keloids contain elevated levels of activated mTOR (mammalian target of rapamycin) and the downstream mTOR signalling component p70KDa S6 kinase (p70S6K)1. mTOR is a serin/threonine kinase which is involved in the regulation of fundamental cellular processes, such as growth, proliferation, protein synthesis and autophagy, and has emerged as an important target for the treatment of certain cancers and inflammatory diseases2.This article is protected by copyright. All rights reserved.
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Publisher Summary This chapter discusses the isolation of rat immunoglobulin class switch variants from hybridomas by sequential sublining. Monoclonal antibodies have contributed significantly to understanding the relationship between the... more
Publisher Summary This chapter discusses the isolation of rat immunoglobulin class switch variants from hybridomas by sequential sublining. Monoclonal antibodies have contributed significantly to understanding the relationship between the structure and function of immunoglobulins (Igs). Ig effector functions—such as complement activation and interaction with Fc receptors—critically depend on the heavy-chain isotype. Matched sets of Igs that have different heavy-chain constant-region (CH) genes but structurally identical light chains and heavy-chain variable-region (VH) genes would be ideal tools for analysis of the CH-region effector functions. While Ig class switching of B cells in vivo is regulated by the action of T cells, myeloma and hybridoma cells growing in vitro spontaneously produce class switch variants, although at low frequency. Variants that have switched from a constant region proximal to the rearranged VDJ gene to a more distal one can be selected from large populations of cells without complicated equipment by sequential sublining and enzyme-linked immunosorbent assay.
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The ribosomal protein S6 is part of the translation machinery and is activated by phosphorylation via the mammalian target of rapamycin pathway, which is activated in psoriatic skin. To investigate which S6 sites are phosphorylated in... more
The ribosomal protein S6 is part of the translation machinery and is activated by phosphorylation via the mammalian target of rapamycin pathway, which is activated in psoriatic skin. To investigate which S6 sites are phosphorylated in psoriasis and atopic dermatitis (AD), and to study whether S6 phosphorylation is associated with inflammation and/or keratinocyte hyperproliferation. Healthy skin and skin lesions of patients with psoriasis and AD were investigated by immunostaining using antibodies that stain proliferating cells, leucocytes and distinct phosphorylated sites of S6. All psoriasis and AD lesions revealed abnormal S6 phosphorylation in the epidermis. The extent of S6 phosphorylation was diverse, generally stronger in psoriasis and correlated, in both diseases, with inflammation. S6 showed differential phosphorylation in distinct epidermal layers, which was most pronounced in hyperproliferative regions. Differential S6 phosphorylation may have a role in abnormal keratinocy...
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... NMR studies and MD simulations with time-averaged NOE-derived upper distance restraints support the formation of a stable β-I turn conformation in the NPNA motif of this template-bound antigen. ... NOE connectivities with Pro 3 are to... more
... NMR studies and MD simulations with time-averaged NOE-derived upper distance restraints support the formation of a stable β-I turn conformation in the NPNA motif of this template-bound antigen. ... NOE connectivities with Pro 3 are to the H−C(δ)'s in place of NH. ...