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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. ...
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The o~/13 T-cell antigen receptor is formed by the combination of the o~ and 13 polypeptide chains which both consist of a constant and a variable portion. The genetic information for the variable portion on each chain is generated by... more
The o~/13 T-cell antigen receptor is formed by the combination of the o~ and 13 polypeptide chains which both consist of a constant and a variable portion. The genetic information for the variable portion on each chain is generated by rearrangements of variable (Tcr-V), diversity (Tcr-D, only for 13 chains), and joining (TCR-J) gene segments.The variable gene segments together determine the antigen specificity of a T cell. Characterization of the structure of the mouse and human Tcr gene segments, therefore, has expanded our understanding of T-cell repertoire selection and has helped to clarify the role of T cells in immune protection and T-cell-dependent pathologic processes. Although many rat major histocompatibility complex haplotypes are well defined and the rat is used as a model for a variety of inflammatory, infectious, and autoimmune diseases (Gill et al. 1989), only partial information is available about the structure and repertoire of rat Tcr genes (Kabat et al. 1991). While the structure of the rat Tcrb-D, Tcrb-J, Tcrb-C (Williams et al. 1989, 1991; Blankenhom et al. 1992), and 24 Tcrb-V (Smith et al. 1991) genes has recently been described, only two Tcra-V, -J, -C cDNA sequences are available (Morris et al. 1988; Bums et al. 1989). In this study, we have sequenced 23 rat Tcra -V, -J, -C cDNAs from the popliteal lymph node of a Lewis rat with adjuvant arthritis to build a basis for studies of Tcra gene segment usage in rats with autoimmune diseases . Five-hundred micrograms of heat-killed Mycobacterium tuberculosis H37Ra (Difco, Detroit, MI) suspended in 0.1 ml mineral oil was injected subcutaneously into the pad of the left hind foot of a Lewis rat (Versuchstierzucht CIBA, Werk Steine, Germany), as described (Yang et al. 1992). The clinical onset of
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Rheumatoid arthritis (RA) is a disease characterized by chronic inflammation affecting the synovial membranes of articulating joints. T lymphocytes, as regulators of immune responses, are thought to be involved in the initiation and... more
Rheumatoid arthritis (RA) is a disease characterized by chronic inflammation affecting the synovial membranes of articulating joints. T lymphocytes, as regulators of immune responses, are thought to be involved in the initiation and perpetuation of RA (Feldmann et al. 1988).
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Research Interests: Chemotherapy, Adolescent, Medicine, Biological Sciences, Humans, and 15 moreChild, Benin, Female, Male, Buruli Ulcer, Clarithromycin, Aged, Middle Aged, Adult, Combination drug therapy, Mycobacterium Ulcerans, Pilot Projects, Clinical Infectious Diseases, Child preschool, and Medical and Health Sciences
Glycerol selectively deuterated at various positions was synthesized and supplied to the growth medium of Escherichia coli strain T131 GP, which is defective in endogenous glycerol synthesis as well as glycerol degradation and lacks the... more
Glycerol selectively deuterated at various positions was synthesized and supplied to the growth medium of Escherichia coli strain T131 GP, which is defective in endogenous glycerol synthesis as well as glycerol degradation and lacks the ability to synthesize cardiolipin. The procedure enables the stereospecific labeling of the membrane phospholipids (approximately 80% phosphatidylethanolamine, approximately 20% phosphatdylglycerol). Deuterium magnetic resonance spectra were obtained for cell membranes and lipid dispersions either from total lipid extractions or from purified phosphitidylglycerol or -ethanolamine. When glycerol deuterated at various positions was used, all resonances of the phospholipid glycerol backbone and the terminal glycerol moiety in phosphatidylglycerol could be assigned. The results indicate that the molecular conformation of the glycerol backbone is independent of the phospholipid species investigated and is also not altered by the presence of high amounts of membrane proteins. For the quantitative interpretation of the deuterium magnetic resonance splittings, a model is proposed which assumes essentially free rotation around the glycerol C(2)-C(3) bond combined with an asymmetric and restricted jump process around the C(1)-C(2) bond. This model is compatible with known X-ray structures of phospholipids molecules. The two deuterons of both the glycerol backbone C(1) and C(3) segments were found to be magnetically inequivalent. Stereoselective monodeuteration eliminated one set of quadrupole splittings in both cases.
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... All rights reserved CLO AL ANALYSIS OF DESCENT AND VIRULENCE AMONG SELECTED ESCHERICHIA COLI Mark Achtman Max-Planck-Institut flir Molckularc Genetik, D lOOO Berlin 33, Federal Republic of Germany Gerd Pluschke ...
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Plasmodium falciparum cysteine-rich protective antigen (PfCyRPA) has been identified as a promising blood-stage candidate antigen to include in a broadly cross-reactive malaria vaccine. In the last couple of decades, substantial effort... more
Plasmodium falciparum cysteine-rich protective antigen (PfCyRPA) has been identified as a promising blood-stage candidate antigen to include in a broadly cross-reactive malaria vaccine. In the last couple of decades, substantial effort has been committed to the development of scalable cost-effective, robust, and high-yield PfCyRPA production processes. Despite insect cells being a suitable expression system due to their track record for protein production (including vaccine antigens), these are yet to be explored to produce this antigen. In this study, different insect cell lines, culture conditions (baculovirus infection strategy, supplementation schemes, culture temperature modulation), and purification strategies (affinity tags) were explored aiming to develop a scalable, high-yield, and high-quality PfCyRPA for inclusion in a virosome-based malaria vaccine candidate. Supplements with antioxidants improved PfCyRPA volumetric titers by 50% when added at the time of infection. In a...
Research Interests: Malaria, Virology, Antibody, Plasmodium falciparum, Malaria Vaccine, and 3 moreAntigen, sf, and Titer
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Background: Bacterial contamination is common to all wounds. The bacterial burden of wounds has been found to have an inverse relationship with chronic wound healing. In seeking to develop a better understanding of the evolution of Buruli... more
Background: Bacterial contamination is common to all wounds. The bacterial burden of wounds has been found to have an inverse relationship with chronic wound healing. In seeking to develop a better understanding of the evolution of Buruli ulcer (BU) wounds, we performed a longitudinal study to quantify the bacterial burden of BU wounds during the course of streptomycin/rifampicin (SR) treatment. Methods: Twenty-one IS2404 polymerase chain reaction confirmed patients were longitudinally followed during the course of their treatment. Swab or tissue samples obtained from the lesions were quantitatively analyzed to determine the bacterial burden pre-, during, and post-SR treatment. Furthermore, the species of bacterial isolates obtained at these time points were also identified. Results: Based on the determination of the bacterial burden, 18/22 (81.8%) pretreatment, 15/25 (57.7%) during treatment, and 36/48 (75.0%) posttreatment samples were classified as superinfected, respectively. Thirty bacterial species including two species of anaerobic Clostridia (Clostridium perfringens and Clostridium sporogenes) were identified among 114 isolates. While Enterococcus faecalis, Pseudomonas aeruginosa, and Chryseomonas luteola dominated pretreatment, P. aeruginosa dominated during and posttreatment. Conclusions: Most BU patients presented with lesions with a high bacterial load which increased significantly posttreatment. Therefore, good wound care is necessary to control the microbial burden of BU wounds, especially posttreatment to minimize complications.
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Research Interests: Molecular Biology, Biology, Medicine, Biological Sciences, Infection and immunity, and 15 moreInsecticide Resistance, Humans, Escherichia coli, Mice, Animals, Infection, Monoclonal Antibodies, In vitro culture, Membrane Integrity, Amino Acid Profile, Molecular Mass, Amino Acid Sequence, Erythrocytes, Control Method, and Molecular Sequence Data
Mycolactones A/B (1a/b) are exotoxins of Mycobacterium ulcerans that are the molecular cause of Buruli ulcer. 1a/b represent a rapidly equilibrating mixture of Z/E isomers about the C4'═C5' double bond of the C5-side chain. Here,... more
Mycolactones A/B (1a/b) are exotoxins of Mycobacterium ulcerans that are the molecular cause of Buruli ulcer. 1a/b represent a rapidly equilibrating mixture of Z/E isomers about the C4'═C5' double bond of the C5-side chain. Here, we describe the syntheses of mycolactone analogs with configurationally stable C5-side chains (2a, E mimetic; 2b/c, Z mimetics). Based on the cytotoxicity of 2a-c, the Δ4',5'-trans isomer of mycolactones A/B (1b) appears to be the major virulence factor.
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Research Interests: Medical Microbiology, Biology, Medicine, Emerging Infectious Diseases, Humans, and 13 moreQueensland, Animals, Polymerase Chain Reaction, Endemic Diseases, Buruli Ulcer, Soil Microbiology, Clinical Sciences, Marsupialia, Public health systems and services research, Feces, Culicidae, Mycobacterium Ulcerans, and Insect Vectors
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Clinical diagnosis of Mycobacterium ulcerans infection is currently accepted as sufficient basis for treating the disease. Inadequate laboratory resources in the highly endemic areas of Africa often limit possibilities for in-country... more
Clinical diagnosis of Mycobacterium ulcerans infection is currently accepted as sufficient basis for treating the disease. Inadequate laboratory resources in the highly endemic areas of Africa often limit possibilities for in-country confirmation of clinical judgement. We analysed records of 99 Buruli ulcer (BU) patients diagnosed clinically and treated surgically at Amasaman Health Centre in Ghana, for whom post-treatment diagnostic laboratory tests were performed. Comparison of clinical diagnoses with test results obtained by an in-country laboratory on samples of excised tissue showed a high specificity of clinical judgement. Among lesions with three laboratory tests (microscopy for acid fast bacilli, culture and IS2404 polymerase chain reaction) done, 94% tested positive at least once and 83% twice. Thus correct clinical diagnosis of BU by well trained health workers is achievable, although the quality of clinical diagnosis should be monitored by intermittent testing in national reference laboratories. However, being retrospective, this study did not permit sensitivity and negative predictive value analysis.
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Buruli ulcer (BU) is a chronic necrotizing skin disease caused by Mycobacterium ulcerans. Historically, the disease was treated by surgical excision of the skin lesions, until an 8-week combination therapy of rifampicin and streptomycin... more
Buruli ulcer (BU) is a chronic necrotizing skin disease caused by Mycobacterium ulcerans. Historically, the disease was treated by surgical excision of the skin lesions, until an 8-week combination therapy of rifampicin and streptomycin was introduced in 2004. This treatment modality was effective and reduced recurrence rates. Rifampicin is the most efficacious antibiotic for the treatment of BU and, should rifampicin-resistant M. ulcerans strains emerge, there is currently no replacement for it. As for mycobacterial diseases in general, there is a pressing need for the development of novel, fast-acting drugs. Under market economy conditions, repurposing of new tuberculosis drug candidates is the most promising avenue for alternative BU treatments. Our drug repurposing activities have led to the identification of several actives against M. ulcerans. In particular, the cytochrome bc1 complex inhibitor telacebec (Q203) is a promising drug candidate for the treatment of BU in Africa an...
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An approach to synthetic vaccine design is illustrated, focusing on the immunodominant (NPNA)n repeat region of the circumsporozoite (CS) protein of the malaria parasite Plasmodium falciparum. Modelling suggests that the NPNAN motif may... more
An approach to synthetic vaccine design is illustrated, focusing on the immunodominant (NPNA)n repeat region of the circumsporozoite (CS) protein of the malaria parasite Plasmodium falciparum. Modelling suggests that the NPNAN motif may adopt a helical β-turn, which is tandemly repeated in the CS protein to generate a novel supersecondary structure. Cyclic peptidomimetics of this NPNAN motif were synthesized and shown by NMR to adopt helical turns in aqueous solution. When incorporated into Immunopotentiating Reconstituted Influenza Virosomes (IRIVs), humoral immune responses were generated in mice that crossreact with native CS protein on sporozoites. IRIVs are a human-compatible delivery system that appear generally suitable for inducing antibody responses against conformational epitopes using constrained peptidomimetics. This approach may offer great potential for the design of molecularly defined synthetic vaccines, including those targeted against multiple antigens and developm...
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Buruli ulcer (BU) is a neglected tropical disease caused by subcutaneous infection with Mycobacterium ulcerans and its exotoxin mycolactone. BU displays coagulative necrosis and widespread fibrin deposition in affected skin tissues.... more
Buruli ulcer (BU) is a neglected tropical disease caused by subcutaneous infection with Mycobacterium ulcerans and its exotoxin mycolactone. BU displays coagulative necrosis and widespread fibrin deposition in affected skin tissues. Despite this, the role of the vasculature in BU pathogenesis remains almost completely unexplored. We hypothesise that fibrin-driven ischemia can be an ‘indirect’ route to mycolactone-dependent tissue necrosis by a mechanism involving vascular dysfunction. Here, we tracked >900 vessels within contiguous tissue sections from eight BU patient biopsies. Our aim was to evaluate their vascular and coagulation biomarker phenotype and explore potential links to fibrin deposition. We also integrated this with our understanding of mycolactone’s mechanism of action at Sec61 and its impact on proteins involved in maintaining normal vascular function. Our findings showed that endothelial cell dysfunction is common in skin tissue adjacent to necrotic regions. Ther...
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<p>A: In plaque lesions mainly large clusters of extracellular bacteria were found, whereas in ulcerated lesions single AFB and small clusters were more common. In both types of lesions AFB were primarily found in the subcutis. B:... more
<p>A: In plaque lesions mainly large clusters of extracellular bacteria were found, whereas in ulcerated lesions single AFB and small clusters were more common. In both types of lesions AFB were primarily found in the subcutis. B: Examples for dispersed single bacteria (1) and small clusters of AFB (2) in histological sections stained with ZN (counterstain methylenblue).</p
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For the treatment of chronic wounds, acid-oxidising solutions (AOSs) with broad-spectrum microbicidal activity without disturbing granulation tissue formation, have been developed. We found AOSs to efficiently kill Mycobacterium ulcerans... more
For the treatment of chronic wounds, acid-oxidising solutions (AOSs) with broad-spectrum microbicidal activity without disturbing granulation tissue formation, have been developed. We found AOSs to efficiently kill Mycobacterium ulcerans , the causative agent of Buruli ulcer, which is able to survive harsh decontamination treatments. Topical AOS treatment of Buruli ulcer lesions may support the recommended antibiotic therapy (oral rifampicin and clarithromycin), prevent contamination of the environment by the mycobacteria, and control secondary infections, which are a prevalent wound management problem in resource-poor Buruli ulcer endemic settings.
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Screening of a collection of temperature-sensitive mutants of Escherichia coli for defects in phospholipid metabolism led to the isolation of a mutant deficient in cardiolipin synthesis. The defective gene, named cls, is closely linked to... more
Screening of a collection of temperature-sensitive mutants of Escherichia coli for defects in phospholipid metabolism led to the isolation of a mutant deficient in cardiolipin synthesis. The defective gene, named cls, is closely linked to the trp marker and maps at about Minute 27 on the E. coli chromosome. After transfer of cls to a defined genetic background by transduction, the mutant has the following properties as compared to an isogenic wild type. Exponentially growing cells show a reduction in cardiolipin content by a factor of at least 15 (less than 0.2 mol % of the total phospholipids). A crude membrane fraction derived from the mutant is unable to synthesize cardiolipin from phosphatidylglycerol in vitro. The mutant has no distinctive phenotype regarding its growth properties, membrane-associated respiratory functions, or the ability to insert bacteriophage M13 coat protein into the cell envelope. The cls mutation confers a 5-times reduction in the turnover of the phosphate moiety of phosphatidylglycerol.
Research Interests: Biochemistry, Chemistry, Phospholipids, Biological Chemistry, Medicine, and 13 moreBiological Sciences, Mutation, Escherichia coli, Temperature, Membrane Lipids, Genes, CHEMICAL SCIENCES, Polar, Species Specificity, Structure activity Relationship, Second Language Composition, Membrane Fluidity, and Medical and Health Sciences
Hyper-virulent Streptococcus pneumoniae serotype 1 strains are endemic in Sub-Saharan Africa and frequently cause lethal meningitis outbreaks. It remains unknown whether genetic variation in serotype 1 strains modulates tropism into... more
Hyper-virulent Streptococcus pneumoniae serotype 1 strains are endemic in Sub-Saharan Africa and frequently cause lethal meningitis outbreaks. It remains unknown whether genetic variation in serotype 1 strains modulates tropism into cerebrospinal fluid to cause central nervous system (CNS) infections, particularly meningitis. Here, we address this question through a large-scale linear mixed model genome-wide association study of 909 African pneumococcal serotype 1 isolates collected from CNS and non-CNS human samples. By controlling for host age, geography, and strain population structure, we identify genome-wide statistically significant genotype-phenotype associations in surface-exposed choline-binding (P = 5.00 × 10−08) and helicase proteins (P = 1.32 × 10−06) important for invasion, immune evasion and pneumococcal tropism to CNS. The small effect sizes and negligible heritability indicated that causation of CNS infection requires multiple genetic and other factors reflecting a c...
Research Interests: Biology, Virology, Medicine, Virulence, Meningitis, and 3 moreTropism, Serotype, and Communications biology
A single dose of Q203 (Telacebec), a phase 2 clinical candidate for tuberculosis, eradicates Mycobacterium ulcerans in a mouse model of Buruli ulcer infection without relapse up to 19 weeks posttreatment. Clinical use of Q203 may... more
A single dose of Q203 (Telacebec), a phase 2 clinical candidate for tuberculosis, eradicates Mycobacterium ulcerans in a mouse model of Buruli ulcer infection without relapse up to 19 weeks posttreatment. Clinical use of Q203 may dramatically simplify the clinical management of Buruli ulcer, a neglected mycobacterial disease.
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The Plasmodium falciparum (Pf) cysteine-rich protective antigen (PfCyRPA) has emerged as a promising blood-stage candidate antigen for inclusion into a broadly cross-reactive malaria vaccine. This highly conserved protein among various... more
The Plasmodium falciparum (Pf) cysteine-rich protective antigen (PfCyRPA) has emerged as a promising blood-stage candidate antigen for inclusion into a broadly cross-reactive malaria vaccine. This highly conserved protein among various geographical strains plays a key role in the red blood cell invasion process by P. falciparum merozoites, and antibodies against PfCyRPA can efficiently prevent the entry of the malaria parasites into red blood cells. The aim of the present study was to develop a human-compatible formulation of the PfCyRPA vaccine candidate and confirming its activity in preclinical studies. Recombinant PfCyRPA expressed in HEK 293 cells was chemically coupled to phosphoethanolamine and then incorporated into the membrane of unadjuvanted influenza virosomes approved as antigen delivery system for humans. Laboratory animals were immunised with the virosome-based PfCyRPA vaccine to determine its immunogenic properties and in particular, its capacity to elicit parasite b...
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The role of phagocytes and the complement system as potential host defense mechanisms against bacterial infection were studied in mice with two isogenic strains of Yersinia enterocolitica serotype O8 differing in pathogenicity because of... more
The role of phagocytes and the complement system as potential host defense mechanisms against bacterial infection were studied in mice with two isogenic strains of Yersinia enterocolitica serotype O8 differing in pathogenicity because of differences in plasmid content. Complement depletion in mice by intraperitoneal injection of cobra venom factor did not affect the course of colonization of the intestinal tissue by each strain, indicating that in mice complement is not essential for the elimination of these bacteria. This conclusion is supported by the fact that fresh murine serum had no bactericidal effect in vitro either on the pathogenic or on the nonpathogenic strain. However, in the intestinal tissue as well as in the peritoneal cavity, only the pathogenic, plasmid-bearing Y. enterocolitica strain survived, while the nonpathogenic, plasmidless strain was rapidly eliminated. Since elimination from the peritoneal cavity is due to phagocytosis by polymorphonuclear leukocytes and ...
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Publisher Summary This chapter presents an analysis of two-dimensional gel electrophoretic protein patterns upon in vivo labeling of mice. The two-dimensional (2D) gel electrophoresis system allows simultaneous qualitative and... more
Publisher Summary This chapter presents an analysis of two-dimensional gel electrophoretic protein patterns upon in vivo labeling of mice. The two-dimensional (2D) gel electrophoresis system allows simultaneous qualitative and quantitative analyses of numerous proteins. The ISODALT apparatus was developed for running multiple samples simultaneously, which facilitates visual comparison of protein patterns. Together with computerized image analysis systems, the ISODALT system allows the development of standard protein maps. The radiolabel incorporated into proteins of tissue and body fluids of mice injected with 1 mCi of [ 35 S] methionine is sufficient to allow radiofluorographic detection of de novo synthesized methionine-containing proteins after 2D gel electrophoresis. The chapter presents a study in which Escherichia coli bacteria, which are a common cause of neonatal sepsis and meningitis, were injected into 10-day-old mice. Haptoglobin synthesis was analyzed at time zero and at 5 and 20 h after bacterial challenge by 2D gel electrophoresis of the de novo synthesized serum proteins.
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INTRODUCTIONExtraction of genomic DNA from mycobacteria requires special consideration because (i) many mycobacterial species exhibit extremely slow growth, and thus produce only small amounts of starting material, and (ii) a robust and... more
INTRODUCTIONExtraction of genomic DNA from mycobacteria requires special consideration because (i) many mycobacterial species exhibit extremely slow growth, and thus produce only small amounts of starting material, and (ii) a robust and waxy cell wall renders mycobacteria difficult to lyse. Hence, mycobacterial DNA extraction often results in low DNA yields of unsuitable quality. Published protocols for mycobacterial DNA preparations and commercially available extraction kits are mainly designed for the isolation of small amounts of genomic material suitable for polymerase chain reaction (PCR)-based applications like species identification. However, such DNA quantities and qualities are usually not sufficient for contemporary genomic analyses such as whole genome sequence analysis, single nucleotide polymorphism (SNP) detection, or DNA microarrays, or for investigations of bacterial evolution, virulence, or epidemiology on a world-wide population level. Moreover, most protocols that...
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Use of synthetic peptides as vaccine components is hampered by their susceptibility to enzymatic degradation and rapid clearance from biological fluids. Introduction of non-natural structural modifications can render peptides more... more
Use of synthetic peptides as vaccine components is hampered by their susceptibility to enzymatic degradation and rapid clearance from biological fluids. Introduction of non-natural structural modifications can render peptides more resistant to enzymatic degradation, encouraging attempts to profile such non-natural ligands as components of synthetic sub-unit vaccines. We have compared the antigenic and immunogenic properties of a series of non-natural peptide analogues derived from a promiscuous T cell epitope of the major Plasmodium falciparum malaria vaccine candidate merozoite surface protein 1 (MSP-1). A series of HLA class II restricted MSP-1(38-58)-specific TCC established from three volunteers were characterized for their minimal epitope and fine specificity. T cell stimulatory activities of a series of pseudo-peptide analogues with single reduced peptide bond Psi-[CH2-NH] modifications were compared with those of single d-amino acid replacement analogues. Compared to reduced peptide bond analogues the single d-amino acid replacement analogues turned out to be less suitable for stimulation of TCC. In particular, the reduced peptide analogue carrying a Psi-[CH2-NH] backbone modification between positions V52 and L53 of MSP-1(38-58) demonstrated properties that would make it a more suitable vaccine component than the unmodified parent peptide. First, the pseudo-peptide stimulated a number of TCC restricted by a range of HLA class II alleles. Second, trypsin treatment in combination with T cell stimulation assays provided evidence for increased resistance to proteolytic digestion. Third, the parasite-binding anti-MSP-1 mAb 7.27 recognized best this particular pseudo-peptide in competition ELISA experiments and its immunogenicity in out-bred Aotus monkeys was superior to that of the parent peptide eliciting antibodies cross-reactive with native MSP-1.
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Buruli ulcer (BU), caused by Mycobacterium ulcerans, is the third most common mycobacterial infection in immunocompetent humans besides tuberculosis and leprosy. We have compared by ex vivo enzyme-linked immunospot analysis interferon-γ... more
Buruli ulcer (BU), caused by Mycobacterium ulcerans, is the third most common mycobacterial infection in immunocompetent humans besides tuberculosis and leprosy. We have compared by ex vivo enzyme-linked immunospot analysis interferon-γ (IFN-γ) responses in peripheral blood mononuclear cells (PBMC) from BU patients, household contacts, and individuals living in an adjacent M. ulcerans nonendemic region. PBMC were stimulated with purified protein derivative (PPD) and nonmycobacterial antigens such as reconstituted influenza virus particles and isopentenyl-pyrophosphate. With all three antigens, the number of IFN-γ spot-forming units was reduced significantly in BU patients compared with the controls from a nonendemic area. This demonstrates for the first time that M. ulcerans infection-associated systemic reduction in IFN-γ responses is not confined to stimulation with live or dead mycobacteria and their products but extends to other antigens. Interleukin (IL)-12 secretion by PPD-sti...
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Research Interests: Microbiology, Biology, Infectious Diseases, Medicine, Biological Sciences, and 15 moreDisease Outbreaks, Humans, Bacteria, Cluster Analysis, Rs, Infectious, Disease Outbreak, Genotype, Pneumolysin, Outbreak, Hemolysis, Molecular Sequence Data, Sequence homology, alleles, and Medical and Health Sciences
Research Interests: Immunology, Biology, Animals, Immune Evasion, Fluorescent Antibody Technique, and 15 moreImmune system, Analytical Method, Antibody, Malaria Vaccine, Isothermal Titration Calorimetry, Calorimetry, Amino Acid Sequence, Base Sequence, Epitopes, Enzyme Linked Immunosorbent Assay, Antigen, Antigenic Variation, Antibody Response, epitope, and avidity
During immune responses the initial activation of B cells takes place in T cell zones of periarteriolar lymphoid sheaths (PALS) of the splenic white pulp. After initial activation, B cells migrate into the primary follicles and, in... more
During immune responses the initial activation of B cells takes place in T cell zones of periarteriolar lymphoid sheaths (PALS) of the splenic white pulp. After initial activation, B cells migrate into the primary follicles and, in association with follicular dendritic cells (FDCs), undergo clonal expansion and differentiation giving rise to germinal centers (GCs). Peanut agglutinin binding (PNA+) cells of the GC differentiate further into memory or plasma cells. Here we report that in tumor necrosis factor receptor 1–deficient mice (TNFR1−/−), the location of B cells was altered and that plasma cells were abnormally distributed in the splenic PALS. In contrast to lymphotoxin α–deficient mice (LTα−/−), bone marrow or fetal liver transplantation did not correct the abnormal organization of the spleen, location of B cells, the lack of an FDC network, nor the antibody response in TNFR1−/− mice. These results argue for a crucial role of TNFR1 expression on nonhematopoietic cells for the...
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A library of 35 cyclic peptidomimetics has been prepared, by a combination of solid- and solution-phase methods, which, together, scan amino acid residues 444–489 in a protruding loop in the subdomain-III of the apical membrane antigen-I... more
A library of 35 cyclic peptidomimetics has been prepared, by a combination of solid- and solution-phase methods, which, together, scan amino acid residues 444–489 in a protruding loop in the subdomain-III of the apical membrane antigen-I (AMA-I), an integral membrane protein found on the surface of Plasmodium falciparum merozoites. The mimetics each contain twelve residues from AMA-I linked through the N- and C-termini to a β-hairpin-inducing template, comprising the dipeptide D-proline-L-Apro (Apro=cis-4-amino-L-proline). These peptidomimetics were each coupled via the 4-amino group to a phospholipid, which allows their incorporation into reconstituted influenza virus-like particles (virosomes) for immunization of mice, as well as their use in ELISA to characterize epitopes recognized by anti-AMA-I growth-inhibitory monoclonal antibodies.
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An ideal malaria vaccine would prevent disease and reduce transmission by targeting several developmental stages of human malaria parasites. To be cost-effective, a modular antigen delivery technology is required for the development of... more
An ideal malaria vaccine would prevent disease and reduce transmission by targeting several developmental stages of human malaria parasites. To be cost-effective, a modular antigen delivery technology is required for the development of such a multivalent subunit vaccine. In this review, we summarize and discuss a strategy to develop synthetic peptidomimetics of key malaria target antigens for inclusion in a multivalent malaria subunit vaccine based on immunopotentiating reconstituted influenza virosomes. Clinical testing of a bivalent virosomal formulation incorporating two structurally optimized peptidomimetics has demonstrated safety, immunogenicity and pilot efficacy. While this clinical validation supports the concept of using peptide-loaded virosomes for vaccination in humans, it is assumed that additional antigens will have to be added to the bivalent formulation to generate a highly effective malaria vaccine.
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The term ‘neglected tropical diseases’ predominantly refers to single-entity, mostly parasitic diseases. However, a considerable morbidity and mortality burden is carried by patients infected with Gram-positive cocci and Gram-negative... more
The term ‘neglected tropical diseases’ predominantly refers to single-entity, mostly parasitic diseases. However, a considerable morbidity and mortality burden is carried by patients infected with Gram-positive cocci and Gram-negative bacilli that are prevalent all over the world, yet have impact in tropical and developing countries, particularly in children, with much higher incidence rates than those reported from developed countries. Staphylococcus aureus is among these pathogens. The African–German StaphNet consortium uses microbiological characterization of African S. aureus isolates, including identification of virulence factors, alongside the gathering of epidemiological and clinical data in an innovative research network between a European country (Germany) and several African partners. By creating an accessible strain repository and by implementing personnel training and capacity building, this network aims to put staphylococcal disease on the international agenda as a trul...
Research Interests: Epidemiology, Africa, MRSA, Medical Microbiology, International Cooperation, and 15 morePublic Health, Medicine, Germany, Children, Humans, Resistance, Disease, Staphylococcus aureus, Tropical Disease, Bacterial infections, Pathogenicity, Panton Valentine leukocidin, Neglected diseases, high prevalence, and Blood stream infections
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The use of sequential sublining in combination with highly specific and sensitive enzyme-linked immunosorbent assays for the isolation of spontaneous rat Ig heavy chain class switch variants is described. These methods allowed us to... more
The use of sequential sublining in combination with highly specific and sensitive enzyme-linked immunosorbent assays for the isolation of spontaneous rat Ig heavy chain class switch variants is described. These methods allowed us to isolate switch variants from mouse-rat hybridoma lines secreting monoclonal rat antibodies. Switch variants from IgM to IgG2a, from IgG2a or IgG2b to IgE and from IgE to IgA were obtained. Members of the BA1.2 family, which consists of IgG2b, IgE and IgA antibodies are shown to exhibit identical rhamnose-inhibitable binding to the O18A antigen of Escherichia coli and to the paratope-associated anti-idiotypic antibody BA114.
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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 &amp; Sons, Inc.
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Glycosylphosphatidylinositol (GPI) glycolipids abound on the cell surface at the merozoite stage of Plasmodium falciparum life cycle are a central toxin in malaria. The contribution of GPI specific humoral immune responses to protection... more
Glycosylphosphatidylinositol (GPI) glycolipids abound on the cell surface at the merozoite stage of Plasmodium falciparum life cycle are a central toxin in malaria. The contribution of GPI specific humoral immune responses to protection against malaria pathology is not clear, since studies on the correlation between anti-GPI antibody titers and disease severity have yielded contradictory results. Here, we present the application of a carbohydrate microarray based on synthetic PfGPI glycans to assess levels and fine specificities of anti-GPI antibody responses in healthy and malaria diseased individuals. Furthermore, the age dependent development of humoral immune responses against GPI in malaria-exposed children was investigated. Anti-GPI antibodies were only rarely found in children under the age of 18 months. Sera from subjects with severe malaria and healthy children contained antibodies that recognized predominantly synthetic Man(3)-GPI and Man(4)-GPIs. In contrast, antibodies in sera of children with mild malaria also showed substantial reactivity with truncated glycans comprising glucosamine-inositol moieties without mannose or with only one or two mannose residues.
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ABSTRACT
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Research Interests: Genetics, Biology, Malaria, Molecular Genetics, Medicine, and 15 moreGenetic Diversity, Biological Sciences, Humans, Animals, Field Trial, Genetic variation, Amino Acid Profile, Malaria Vaccine, Amino Acid Sequence, Antigens, Antigenic Variation, Molecular Sequence Data, alleles, Medical and Health Sciences, and building block
Serotype 1 is one of the most common causes of pneumococcal disease worldwide. Pneumococcal protein vaccines are currently being developed as an alternate intervention strategy to pneumococcal conjugate vaccines. Pre-requisites for an... more
Serotype 1 is one of the most common causes of pneumococcal disease worldwide. Pneumococcal protein vaccines are currently being developed as an alternate intervention strategy to pneumococcal conjugate vaccines. Pre-requisites for an efficacious pneumococcal protein vaccine are universal presence and minimal variation of the target antigen in the pneumococcal population, and the capability to induce a robust human immune response. We used in silico analysis to assess the prevalence of seven protein vaccine candidates (CbpA, PcpA, PhtD, PspA, SP0148, SP1912, SP2108) among 445 serotype 1 pneumococci from 26 different countries, across four continents. CbpA (76%), PspA (68%), PhtD (28%), PcpA (11%) were not universally encoded in the study population, and would not provide full coverage against serotype 1. PcpA was widely present in the European (82%), but not in the African (2%) population. A multi-valent vaccine incorporating CbpA, PcpA, PhtD and PspA was predicted to provide covera...
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The development of a malaria vaccine represents one of the most important scientific public health challenges of our time. One possible approach is based on subunit vaccines that use distinct malarial antigens for which there is some... more
The development of a malaria vaccine represents one of the most important scientific public health challenges of our time. One possible approach is based on subunit vaccines that use distinct malarial antigens for which there is some evidence of protective immunity from epidemiological data in the field or animal challenge models. It is generally accepted that an effective malaria subunit vaccine will target antigens of several developmental stages of the parasite. At present, the development of peptide-based vaccines is hampered by their poor immunogenicity and lack of in vivo stability of synthetic peptides and suitable antigen delivery systems driving appropriate immune responses in humans. Most importantly, the synthetic structures delivered have to mimic closely the corresponding native malaria protein to induce effective antibody responses. Review of recent publications highlighting the design as well as preclinical and clinical development of conformationally constrained synthetic peptides of two malaria proteins delivered on the surface of influenza virosomes. The great potential of influenza virosomes as a flexible, human-compatible antigen delivery platform for the development of a multivalent malaria subunit vaccine is described.
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Research Interests: Immunology, Clinical Trial, Biology, Cell line, Humans, and 15 moreAnimals, Antigen Processing, Antigen Presentation, Major histocompatibility complex, Clone, Cell Proliferation, Amino Acid Sequence, Antigen, Malaria Vaccines, Interleukin, Interferon gamma, Dilution, epitope, Expression pattern, and B cell
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Research Interests: Immunology, Immune response, Clinical Trial, Biology, Malaria, and 15 moreAdolescent, Clinical immunology, Humans, Female, Animals, Immune system, Cellular Immunity, Adult, Delivery System, Amino Acid Sequence, Enzyme Linked Immunosorbent Assay, Immunogenicity, Antigen, Antibody Response, and Malaria Falciparum
The Plasmodium falciparum merozoite surface protein-1 19 kDa fragment (MSP-1(19)) comprises two closely packed EGF-like domains (EGF=epidermal growth factor), each stabilized by three disulfide bonds. The native conformation of this... more
The Plasmodium falciparum merozoite surface protein-1 19 kDa fragment (MSP-1(19)) comprises two closely packed EGF-like domains (EGF=epidermal growth factor), each stabilized by three disulfide bonds. The native conformation of this protein is important for eliciting P. falciparum growth inhibitory antibodies. Here we show that the N-terminal EGF domain alone can be chemically synthesized and efficiently refolded to a native-like state, as shown by its solution structure as determined by NMR spectroscopy. In order to study its immunogenicity, the domain was coupled through its N terminus to a phospholipid and incorporated into reconstituted influenza virus-like particles (virosomes). When used to immunize mice, the peptide-loaded virosomes elicited potent humoral immune responses that were shown by Western blots and immunofluorescence assays to cross-react with native MSP-1 on the surfaces of P. falciparum blood stage parasites. This opens the way for a medicinal chemistry-oriented approach to the study and optimization of the antigenicity of the protein as a potential malaria vaccine candidate, whilst exploiting the immunopotentiating properties of influenza virosomes as a delivery vehicle.
Research Interests: Biology, Magnetic Resonance Spectroscopy, Medicine, Mice, Animals, and 12 moreEpidermal Growth Factor, Solutions, Plasmodium falciparum, Protozoan Proteins, Amino Acid Sequence, Immunogenicity, Solution Structure, Biochemistry and cell biology, Antigenicity, Merozoite surface protein 1, Molecular Sequence Data, and Protein subunits
Engineered nanoparticles have been designed based on the self-assembling properties of synthetic coiled-coil lipopeptide building blocks. The presence of an isoleucine zipper within the lipopeptide together with the aggregating effects of... more
Engineered nanoparticles have been designed based on the self-assembling properties of synthetic coiled-coil lipopeptide building blocks. The presence of an isoleucine zipper within the lipopeptide together with the aggregating effects of an N-terminal lipid drives formation of 20-25 nm nanoparticles in solution. Biophysical studies support a model in which the lipid is buried in the centre of the nanoparticle, with 20-30 trimeric helical coiled-coil bundles radiating out into solution. A promiscuous T-helper epitope and a synthetic B-cell epitope mimetic derived from the circumsporozoite protein of Plasmodium falciparum have been linked to each lipopeptide chain, with the result that 60-90 copies of each antigen are displayed over the surface of the nanoparticle. These nanoparticles elicit strong humoral immune responses in mice and rabbits, including antibodies able to cross-react with the parasite, thereby, supporting the potential value of this delivery system in synthetic vaccine design.
Research Interests: Small angle X-ray and neutron scattering, Biology, Transmission Electron Microscopy, Neutron Diffraction, Medicine, and 14 moreCircular Dichroism, Mice, Animals, Viruses, Drug Design, X ray diffraction, Plasmodium falciparum, Protein Conformation, Rabbits, Amino Acid Sequence, Biochemistry and cell biology, Lipopeptides, Molecular Sequence Data, and epitope
Methods for the selection of transfectoma cells that express large quantities of mouse-human chimeric antibodies have been develped. SP2/0 mouse myeloma cells were transfected with pSV2-gpt and pSV2-neo based immunoglobulin expression... more
Methods for the selection of transfectoma cells that express large quantities of mouse-human chimeric antibodies have been develped. SP2/0 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. ELISA-based screening of transfectoma clones resulted in the isolation of IgG-producing transfectomas. Introduction of the kappa light-chain 3&amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;#39;-enhancer into the light-chain expression vector significantly increased immunoglobulin expression, but only when the enhancer was located at its physiological site, 9 kb downstream of the kappa constant region exon. With some of the transfectomas, final yields of up to 80 mg/L of chimeric IgG were obtained in conventional flask cultures using serum-free growth medium. A pilot-scale AcuSyst Maximizer hollow fiber cell culture system was used for the production of gram amounts of chimeric IgG. Results obtained with different transfectoma clones in conventional culture were not fully predictive for yields in the hollow fiber system. In contrast, differences in productivity between individual clones in the laboratory-scale Tecnomouse cell culture unit were comparable with those in the Maximizer system. Up to 200 mg of chimeric IgG were produced per day in one Maximizer bioreactor. (c) 1995 John Wiley &amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp; Sons, Inc.
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ABSTRACT
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Defense againstHaemophilus influenzaetype b (Hib) is dependent on antibodies and complement, which mediate both serum bactericidal activity (SBA) and opsonophagocytosis. Here we evaluated the influence of capsule-specific antibodies and... more
Defense againstHaemophilus influenzaetype b (Hib) is dependent on antibodies and complement, which mediate both serum bactericidal activity (SBA) and opsonophagocytosis. Here we evaluated the influence of capsule-specific antibodies and complement inhibitors targeting the central component C3, the alternative pathway (AP; fB, fD), the lectin pathway (LP; MASP-2) and the terminal pathway (C5) on both effector functions. Findings may be relevant for the treatment of certain diseases caused by dysregulation of the complement system, where inhibitors of complement factors C3 or C5 are used. Inhibitors against other complement components are being evaluated as potential alternative treatment options that may carry a reduced risk of infection by encapsulated bacteria. Serum and reconstituted blood of healthy adults were tested for bactericidal activity before and after vaccination with the Hib capsule-conjugate vaccine ActHIB. Most sera had bactericidal activity prior to vaccination, but ...
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To assess the relative contribution of opsonisation by antibodies, classical and alternative complement pathways to pneumococcal phagocytosis, we analyzed killing of pneumococci by human blood leukocytes collected from vaccine-naïve and... more
To assess the relative contribution of opsonisation by antibodies, classical and alternative complement pathways to pneumococcal phagocytosis, we analyzed killing of pneumococci by human blood leukocytes collected from vaccine-naïve and PCV13-vaccinated subjects. With serotype 4 pneumococci as model, two different physiologic opsonophagocytosis assays based on either hirudin-anticoagulated whole blood or on washed cells from EDTA-anticoagulated blood reconstituted with active serum, were compared. Pneumococcal killing was measured in the presence of inhibitors targeting the complement components C3, C5, MASP-2, factor B or factor D. The two assay formats yielded highly consistent and comparable results. They highlighted the importance of alternative complement pathway activation for efficient opsonophagocytic killing in blood of vaccine-naïve subjects. In contrast, alternative complement pathway inhibition did not affect pneumococcal killing in PCV13-vaccinated individuals. Independ...
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Dysregulation of complement activation causes a number of diseases, including paroxysmal nocturnal hemoglobinuria and atypical hemolytic uremic syndrome. These conditions can be treated with monoclonal antibodies (mAbs) that bind to the... more
Dysregulation of complement activation causes a number of diseases, including paroxysmal nocturnal hemoglobinuria and atypical hemolytic uremic syndrome. These conditions can be treated with monoclonal antibodies (mAbs) that bind to the complement component C5 and prevent formation of the membrane attack complex (MAC). While MAC is involved in uncontrolled lysis of erythrocytes in these patients, it is also required for serum bactericidal activity (SBA), i.e. clearance of encapsulated bacteria. Therefore, terminal complement blockage in these patients increases the risk of invasive disease by Neisseria meningitidis more than 1000-fold compared to the general population, despite obligatory vaccination. It is assumed that alternative instead of terminal pathway inhibition reduces the risk of meningococcal disease in vaccinated individuals. To address this, we investigated the SBA with alternative pathway inhibitors. Serum was collected from adults before and after vaccination with a m...
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Background The generation of monoclonal antibodies specific for protein antigens usually depends on purified recombinant protein for both immunisation and hybridoma screening. Purification of recombinant protein in sufficient yield and... more
Background The generation of monoclonal antibodies specific for protein antigens usually depends on purified recombinant protein for both immunisation and hybridoma screening. Purification of recombinant protein in sufficient yield and purity is a tedious undertaking and can be demanding especially in the case of membrane proteins. Furthermore, antibodies generated against a purified recombinant protein are frequently incapable of binding to the endogenous protein in its native context. Results We describe a strategy to generate monoclonal antibodies against membrane or membrane-associated proteins that completely bypasses any need for purified recombinant antigen. This approach utilises stably transfected mammalian cells expressing recombinant antigens on their cell surface for immunisation of mice. The transfected cells are also used for measuring seroconversion, hybridoma selection and antibody characterisation. By presenting the antigen in its native conformation for immunisatio...
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Background Data on the burden and clinical epidemiology of skin wounds in rural sub-Saharan Africa is scant. The scale of the problem including preventable progression to chronic wounds, disability and systemic complications is largely... more
Background Data on the burden and clinical epidemiology of skin wounds in rural sub-Saharan Africa is scant. The scale of the problem including preventable progression to chronic wounds, disability and systemic complications is largely unaddressed. Methods We conducted a cross-sectional study combining active (household-based survey) and passive case finding (health services-based survey) to determine the burden and clinical epidemiology of wounds within the Taabo Health and Demographic Surveillance System (HDSS) in rural Côte d’Ivoire. Patients identified with wounds received free care and were invited to participate in the wound management study simultaneously carried out in the survey area. The data were analysed for wound prevalence, stratified by wound and patient characteristics. Results 3842 HDSS-registered persons were surveyed. Overall wound prevalence derived from combined active and passive case finding was 13.0%. 74.1% (403/544) of patients were below the age of 15 years...
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<p>A 27-year-old male presented to an integrated health centre in a BU-endemic area in Cameroon with multiple lesions on the neck and upper chest. After acid-fast bacilli were observed in the wound exudates, the patient was... more
<p>A 27-year-old male presented to an integrated health centre in a BU-endemic area in Cameroon with multiple lesions on the neck and upper chest. After acid-fast bacilli were observed in the wound exudates, the patient was diagnosed with BU and treated according to WHO guidelines. <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0001751#pntd-0001751-g001" target="_blank">Figure 1A</a> shows the patient on day 17 of BU treatment. The lesions healed following eight weeks of rifampicin/streptomycin combination therapy. <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0001751#pntd-0001751-g001" target="_blank">Figure 1B and C</a> show the patient 86 and 178 days after completion of BU treatment, respectively. Further laboratory analysis on the original wound exudates showed that the patient was suffering from TB as opposed to BU, and the appropriate long-term TB treatment was administered.</p
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A total of 95 K1 Escherichia coli strains of the O (lipopolysaccharide) serotypes O1, O7, or O18 had been analyzed previously for the ability to cause bacteremia after colonizing the gut of newborn rats. In this study, these strains were... more
A total of 95 K1 Escherichia coli strains of the O (lipopolysaccharide) serotypes O1, O7, or O18 had been analyzed previously for the ability to cause bacteremia after colonizing the gut of newborn rats. In this study, these strains were tested for their resistance to the bactericidal activity of rat serum. All strains that had caused bacteremia in a high percentage of the inoculated rats were able to survive for several hours in 90% adult rat serum. With only a few exceptions, O7:K1 and O18:K1 strains were serum resistant and virulent, whereas O1:K1 strains were serum sensitive and avirulent. Serum sensitivity was due to the classical complement pathway. K1 strains of all three O serotypes were resistant to the alternative complement pathway. O7:K1 and O18:K1 cells were killed efficiently after the classical pathway was triggered by specific antilipopolysaccharide antibodies. However, killing of O1:K1 bacteria by the classical pathway system did not require antibodies. Isolated O1-...
Research Interests: Microbiology, Complement activation, Biology, Medicine, Complement System, and 15 moreBiological Sciences, Lipopolysaccharide, Infection and immunity, Virulence, Humans, Escherichia coli, Enterobacteriaceae, Newborn Infant, Lipopolysaccharides, Antibody, Meningitis, Bacteremia, Antigen, Serotype, and Medical and Health Sciences
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Invasion of erythrocytes by Plasmodial merozoites is a composite process involving the interplay of several proteins. Among them, the Plasmodium falciparum Cysteine-Rich Protective Antigen (PfCyRPA) is a crucial component of a ternary... more
Invasion of erythrocytes by Plasmodial merozoites is a composite process involving the interplay of several proteins. Among them, the Plasmodium falciparum Cysteine-Rich Protective Antigen (PfCyRPA) is a crucial component of a ternary complex, including Reticulocyte binding-like Homologous protein 5 (PfRH5) and the RH5-interacting protein (PfRipr), essential for erythrocyte invasion. Here we present the crystal structure of PfCyRPA and of its complex with the antigen-binding fragment of a parasite growth inhibitory antibody. While PfCyRPA adopts a 6-bladed β-propeller structure with similarity to the classic sialidase fold, it possesses no sialidase activity, indicating that it fulfills a non-enzymatic function. Characterization of the epitope recognized by protective antibodies will facilitate design of peptidomimetics that focus vaccine responses on protective epitopes. Both in vitro and in vivo anti-PfCyRPA and anti-PfRH5 antibodies showed more potent parasite growth inhibitory a...
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Research Interests: Microbiology, Biology, Medicine, Biological Sciences, Lipopolysaccharide, and 15 moreHumans, Animals, Brucella, Brucella melitensis, Monoclonal Antibodies, Brucellosis, Lipopolysaccharides, Antibody, Immunoassay, Hybridoma Technology, Monoclonal Antibody, Sensitivity and Specificity, Biological Warfare Agents, Antigen, and Medical and Health Sciences
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Background Visceral leishmaniasis (VL) remains as one of the most neglected tropical diseases with over 60% of the world’s total VL cases occurring in the Indian subcontinent. Due to the invasive risky procedure and technical expertise... more
Background Visceral leishmaniasis (VL) remains as one of the most neglected tropical diseases with over 60% of the world’s total VL cases occurring in the Indian subcontinent. Due to the invasive risky procedure and technical expertise required in the classical parasitological diagnosis, the goal of the VL experts has been to develop noninvasive procedure(s) applicable in the field settings. Several serological and molecular biological approaches have been developed over the last decades, but only a few are applicable in field settings that can be performed with relative ease. Recently, loop-mediated isothermal amplification (LAMP) has emerged as a novel nucleic acid amplification method for diagnosis of VL. In this study, we have evaluated the LAMP assay using buffy coat DNA samples from VL patients in Bangladesh and compared its performance with leishmania nested PCR (Ln-PCR), an established molecular method with very high diagnostic indices. Methods Seventy five (75) parasitologi...
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Background Clinical profiling of two components for a synthetic peptide-based virosomal malaria vaccine has yielded promising results, encouraging the search for additional components for inclusion in a final multi-valent vaccine... more
Background Clinical profiling of two components for a synthetic peptide-based virosomal malaria vaccine has yielded promising results, encouraging the search for additional components for inclusion in a final multi-valent vaccine formulation. This report describes the immunological characterization of linear and cyclized synthetic peptides comprising amino acids 211-237 of Plasmodium falciparum merozoite surface protein (MSP-3). Methods These peptides were coupled to phosphatidylethanolamine (PE); the conjugates were intercalated into immunopotentiating reconstituted influenza virosomes (IRIVs) and then used for immunizations in mice to evaluate their capacity to elicit P. falciparum cross-reactive antibodies. Results While all MSP-3-derived peptides were able to elicit parasite-binding antibodies, stabilization of turn structures by cyclization had no immune-enhancing effect. Therefore, further pre-clinical profiling was focused on FB-12, a PE conjugate of the linear peptide. Consi...
Research Interests: Medical Microbiology, Biology, Malaria, Virology, Research, and 15 moreMedicine, Humans, Mice, Animals, Cyclic peptides, Antibody, Plasmodium falciparum, Malaria Vaccine, Protozoan Proteins, Monoclonal Antibody, Rabbits, Enzyme Linked Immunosorbent Assay, Immunogenicity, Orthomyxoviridae, and Malaria Falciparum
γδ T cells are implicated to play crucial roles during early immune responses to pathogens. A subset of human γδ T cells carrying the Vγ9Vδ2 TCR recognize small, phosphorylated nonpeptidic Ags. However, the precise role of these cells and... more
γδ T cells are implicated to play crucial roles during early immune responses to pathogens. A subset of human γδ T cells carrying the Vγ9Vδ2 TCR recognize small, phosphorylated nonpeptidic Ags. However, the precise role of these cells and the ligands recognized in human immune responses against pathogens remains unclear because of the lack of suitable animal models. We have analyzed the reactivity of spleen cells of the New World monkey Aotus nancymaae against isopentenyl pyrophosphate (IPP), a phosphorylated microbial metabolite selectively activating Vγ9Vδ2 T cells. Spleen cells were stimulated by IPP and the expanding cell population expressed the Vγ9 TCR. TRGV-J and TRDV-D-J rearrangements expressed by IPP-stimulated cells of Aotus were analyzed by RT-PCR and DNA sequencing. The TRGV-J and TRDV-D-J rearrangements expressed by IPP-stimulated Aotus and human γδ T cells were similar with respect to 1) TCR gene segment usage, 2) a high degree of germline sequence homology of the TCR...
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An effective malaria vaccine could prove to be the most cost-effective and efficacious means of preventing severe disease and death from malaria. In an endeavor to identify novel vaccine targets, we tested predicted Plasmodium falciparum... more
An effective malaria vaccine could prove to be the most cost-effective and efficacious means of preventing severe disease and death from malaria. In an endeavor to identify novel vaccine targets, we tested predicted Plasmodium falciparum open reading frames for proteins that elicit parasite-inhibitory Abs. This has led to the identification of the cysteine-rich protective Ag (CyRPA). CyRPA is a cysteine-rich protein harboring a predicted signal sequence. The stage-specific expression of CyRPA in late schizonts resembles that of proteins known to be involved in merozoite invasion. Immunofluorescence staining localized CyRPA at the apex of merozoites. The entire protein is conserved as shown by sequencing of the CyRPA encoding gene from a diverse range of P. falciparum isolates. CyRPA-specific mAbs substantially inhibited parasite growth in vitro as well as in a P. falciparum animal model based on NOD-scid IL2Rγnull mice engrafted with human erythrocytes. In contrast to other P. falci...
Research Interests: Immunology, Flow Cytometry, Biology, Malaria, Medicine, and 15 moreIn Vitro, Humans, Sequence alignment, Mice, Animals, Fluorescent Antibody Technique, Plasmodium falciparum, Malaria Vaccine, Amino Acid Sequence, Erythrocytes, Enzyme Linked Immunosorbent Assay, Antigen, Malaria Vaccines, Molecular Sequence Data, and Malaria Falciparum
Polymyositis mediated by gamma/delta T cells is a unique disease in which autoaggressive T lymphocytes surround, invade, and destroy muscle fibers. Histochemically, the vast majority of muscle-infiltrating T cells in a patient with... more
Polymyositis mediated by gamma/delta T cells is a unique disease in which autoaggressive T lymphocytes surround, invade, and destroy muscle fibers. Histochemically, the vast majority of muscle-infiltrating T cells in a patient with polymyositis were reactive with a pan-gamma/delta T cell receptor (TCR)-specific monoclonal antibody (TCR-delta 1+), but unlike > 90% of peripheral blood gamma/delta T cells, these lymphocytes did not react with V delta 1- or V gamma 9-specific antibodies (A13- and Ti gamma A-, respectively). Differential reactivity with two different V delta 2-specific monoclonal antibodies (BB3-/TiV-delta 2+) indicated that the infiltrating T cells express a V delta 2-containing TCR with unusual additional structural features. Using conventional and anchored polymerase chain reaction for the analysis of TCR transcripts, we found a striking predominance of one unusual V delta 2-J delta 3 recombination and one V gamma 3-J gamma 1 recombination. Both the unusual phenoty...
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Monoclonal antibodies (MAbs) specific for Plasmodium falciparum rhoptry-associated protein 1 (RAP-1) were generated and tested for inhibition of parasite growth in vitro. The majority of indirect immunofluorescence assay (IFA)-positive... more
Monoclonal antibodies (MAbs) specific for Plasmodium falciparum rhoptry-associated protein 1 (RAP-1) were generated and tested for inhibition of parasite growth in vitro. The majority of indirect immunofluorescence assay (IFA)-positive MAbs raised against recombinant RAP-1 positions 23 to 711 (rRAP-1 23–711 ) recognized epitopes located in the immunodominant N-terminal third of RAP-1. MAbs specific for the building block 35.1 of the synthetic peptide malaria vaccine SPf66 also yielded an IFA staining pattern characteristic for rhoptry-associated proteins and reacted specifically with rRAP-1 and parasite-derived RAP-1 molecules p67 and p82. Cross-reactivity with RAP-1 was blocked by the 35.1 peptide. Epitope mapping with truncated rRAP-1 molecules and overlapping peptides identified the linear RAP-1 sequence Y 218 KYSL 222 as a target of the anti-35.1 MAbs. This sequence lacks primary sequence similarity with the 35.1 peptide (YGGPANKKNAG). Cross-reactivity of the anti-35.1 MAbs thus...
Research Interests: Molecular Biology, Biology, Medicine, Biological Sciences, Infection and immunity, and 15 moreMice, Animals, Epitope mapping, Infection, Monoclonal Antibodies, Peptide, Plasmodium falciparum, Protozoan Proteins, Monoclonal Antibody, Amino Acid Sequence, Recombinant Proteins, Molecular Sequence Data, epitope, Medical and Health Sciences, and building block
The similarity of endospore surface antigens between bacteria of the Bacillus cereus group complicates the development of selective antibody-based anthrax detection systems. The surface of B. anthracis endospores exposes a tetrasaccharide... more
The similarity of endospore surface antigens between bacteria of the Bacillus cereus group complicates the development of selective antibody-based anthrax detection systems. The surface of B. anthracis endospores exposes a tetrasaccharide containing the monosaccharide anthrose. Anti-tetrasaccharide monoclonal antibodies (MAbs) and anti-anthrose-rhamnose disaccharide MAbs were produced and tested for their fine specificities in a direct spore enzyme-linked immunosorbent assay (ELISA) with inactivated spores of a broad spectrum of B. anthracis strains and related species of the Bacillus genus. Although the two sets of MAbs had different fine specificities, all of them recognized the tested B. anthracis strains and showed only a limited cross-reactivity with two B. cereus strains. The MAbs were further tested for their ability to be implemented in a highly sensitive and specific bead-based Luminex assay. This assay detected spores from different B. anthracis strains and two cross-react...
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Research Interests: Biology, Magnetic Resonance Spectroscopy, Medicine, Influenza virus, Microbes, and 15 moreMice, Animals, Drug Design, Fluorescent Antibody Technique, Antibody, Delivery System, Amino Acid Sequence, Chemistry Biology, Aqueous Solution, Enzyme Linked Immunosorbent Assay, Antigen, Biochemistry and cell biology, Hybridomas, Malaria Vaccines, and Circumsporozoite protein
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<p>Maximum-likelihood phylogenetic tree based on 11,194 variable nucleotide positions across <i>M</i>. <i>ulcerans</i> isolates by RAxML. <i>M</i>. <i>ulcerans</i> isolates from the... more
<p>Maximum-likelihood phylogenetic tree based on 11,194 variable nucleotide positions across <i>M</i>. <i>ulcerans</i> isolates by RAxML. <i>M</i>. <i>ulcerans</i> isolates from the Offin and Densu river (one representative each of the previously identified SNP haplotypes [<a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0004856#pntd.0004856.ref008" target="_blank">8</a>]) valleys were placed in a broader genomic context. The tree was rooted using <i>M</i>. <i>ulcerans</i> strain Mu 06–3844 (isolate from a fish farm in Belgium) as an out-group. Bootstrap values are shown along the branches. 0.02 = scale for genetic distance.</p
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<p>Maximum-likelihood phylogenetic tree based on 776 variable nucleotide positions showing relations between the strains at a higher resolution than in <a... more
<p>Maximum-likelihood phylogenetic tree based on 776 variable nucleotide positions showing relations between the strains at a higher resolution than in <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0004856#pntd.0004856.g002" target="_blank">Fig 2</a>. Bootstrap values are shown along the branches. 0.002 = scale for genetic distance.</p
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Mycobacterium ulcerans is the causative agent of the chronic, necrotizing skin disease Buruli ulcer. Modes of transmission and molecular mechanisms involved in the establishment of M. ulcerans infections are poorly understood.... more
Mycobacterium ulcerans is the causative agent of the chronic, necrotizing skin disease Buruli ulcer. Modes of transmission and molecular mechanisms involved in the establishment of M. ulcerans infections are poorly understood. Interactions with host glycans are often crucial in bacterial pathogenesis and the 22 kDa M. ulcerans protein MUL_3720 has a putative role in host cell attachment. It has a predicted N-terminal lectin domain and a C-terminal peptidoglycan-binding domain and is highly expressed on the surface of the bacilli. Here we report the glycan-binding repertoire of whole, fixed M. ulcerans bacteria and of purified, recombinant MUL_3720. On an array comprising 368 diverse biologically relevant glycan structures, M. ulcerans cells showed binding to 64 glycan structures, representing several distinct classes of glycans, including sulfated structures. MUL_3720 bound only to glycans containing sulfated galactose and GalNAc, such as glycans known to be associated with keratins...
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This study revealed that the prevalence of HBV and HDV in a rural area of Cameroon is extremely high, underlining the pressing need for the improvement of control strategies. Systematic serological and phylogenetic analyses of HBV... more
This study revealed that the prevalence of HBV and HDV in a rural area of Cameroon is extremely high, underlining the pressing need for the improvement of control strategies. Systematic serological and phylogenetic analyses of HBV sequences turned out to be useful tools to identify networks of virus transmission within and between households. The high HBsAg carriage rate found among children demonstrates that implementation of the HBV birth dose vaccine and improvement of vaccine coverage will be key elements in preventing both HBV and HDV infections. In addition, the high HBsAg carriage rate in adolescents and adults emphasizes the need for identification of chronically infected individuals and linkage to WHO-recommended treatment to prevent progression to liver cirrhosis and hepatocellular carcinoma.
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Although leprosy is one of the oldest diseases known to humanity, it remains largely misunderstood. Misconceptions about leprosy lead to stigma towards people with the disease. This study aimed at exploring the knowledge, perceptions and... more
Although leprosy is one of the oldest diseases known to humanity, it remains largely misunderstood. Misconceptions about leprosy lead to stigma towards people with the disease. This study aimed at exploring the knowledge, perceptions and attitudes regarding leprosy in rural Cameroon. We carried out a cross-sectional community survey of 233 respondents aged 15-75 years, free from leprosy, and living in two rural health districts of the South-west Region of Cameroon. A questionnaire designed to evaluate knowledge, perceptions and attitudes about leprosy was used. Binary logistic regression was used to determine independent predictors of negative attitudes. About 82% of respondents had heard about, and 64.4% knew someone with leprosy. Information on leprosy was mainly from community volunteers (40.6%), friends (38.0%), and the media (24%). Only 19.7% of respondents knew the cause of leprosy, and a considerable proportion linked it to a spell (25.3%), unclean blood (15.5%) and heredity ...
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A library of compounds covering a broad chemical space was selected from a tuberculosis drug development program and was screened in a whole-cell assay againstMycobacterium ulcerans, the causative agent of the necrotizing skin disease... more
A library of compounds covering a broad chemical space was selected from a tuberculosis drug development program and was screened in a whole-cell assay againstMycobacterium ulcerans, the causative agent of the necrotizing skin disease Buruli ulcer. While a number of potent antitubercular agents were only weakly active or inactive againstM. ulcerans, five compounds showed high activity (90% inhibitory concentration [IC90], ≤1 μM), making screening of focused antitubercular libraries a good starting point for lead generation againstM. ulcerans.
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Research Interests: Traditional Medicine, Wound Healing, Treatment Outcome, Medicinal Plants, Medicine, and 14 moreBiological Sciences, Phytotherapy, Humans, Child, Male, Arm, Skin, Buruli Ulcer, Plant extracts, medicinal & Aromatic plants, Lesion, Mycobacterium Ulcerans, Family Characteristics, and Medical and Health Sciences
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Research Interests: Polymorphism, Biology, Uganda, Medicine, Multidisciplinary, and 15 morePopulation structure, Tuberculosis, Mycobacterium tuberculosis, Mycobacterium, Drug Resistance, Polymerase Chain Reaction, Research article, TYPING, PLoS one, Genotype, Genetic Polymorphism, Mycobacterium tuberculosis complex, Streptomycin, Multilocus sequence typing, and Genotypic diversity
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We evaluated four decontamination methods and one nondecontamination procedure in combination with four egg-based media for the primary isolation of Mycobacterium ulcerans from tissue specimens. With mycobacterial recovery and... more
We evaluated four decontamination methods and one nondecontamination procedure in combination with four egg-based media for the primary isolation of Mycobacterium ulcerans from tissue specimens. With mycobacterial recovery and contamination rates of 75.6 and 2.4%, respectively, the combination of the oxalic acid decontamination method with Lowenstein-Jensen medium supplemented with glycerol yielded the best results.
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ABSTRACTCombination chemotherapy with rifampin and streptomycin (RIF-STR) for 8 weeks is currently recommended by the WHO as the first-line treatment forMycobacterium ulceransinfection (Buruli ulcer). To gain better insight into the mode... more
ABSTRACTCombination chemotherapy with rifampin and streptomycin (RIF-STR) for 8 weeks is currently recommended by the WHO as the first-line treatment forMycobacterium ulceransinfection (Buruli ulcer). To gain better insight into the mode of action of these antibiotics against establishedM. ulceransinfection foci and to characterize recovery of local immune responses during chemotherapy, we conducted a detailed histopathological study ofM. ulcerans-infected and RIF-STR-treated mice. Mice were inoculated withM. ulceransin the footpad and 11 weeks later treated with RIF-STR. Development of lesions during the first 11 weeks after infection and subsequent differences in disease progression between RIF-STR-treated and untreated mice were studied. Changes in histopathological features, footpad swelling, and number of CFU were analyzed. After inoculation withM. ulcerans, massive infiltrates dominated by polymorphonuclear leukocytes developed at the inoculation site but did not prevent bacte...
Research Interests: Microbiology, Medical Microbiology, Biology, Treatment Outcome, Medicine, and 14 moreHumans, Mice, Female, Animals, Buruli Ulcer, Anti-Bacterial Agents, Foot, Antimicrobial agents, Combination drug therapy, Streptomycin, Mycobacterium Ulcerans, Rifampicin, Pharmacology and pharmaceutical sciences, and Rifampin
ABSTRACTAn alamarBlue-based growth inhibition assay has been adapted for the thermosensitive and slow-growing pathogenMycobacterium ulcerans. The standardized test procedure enables medium-throughput screening of preselected compound... more
ABSTRACTAn alamarBlue-based growth inhibition assay has been adapted for the thermosensitive and slow-growing pathogenMycobacterium ulcerans. The standardized test procedure enables medium-throughput screening of preselected compound libraries. Testing of a set of 48 azoles with known antifungal activity led to the identification of an imidazole antifungal displaying an inhibitory dose (ID) of 9 μM forM. ulcerans.
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SUMMARY Immunopotentiating reconstituted influenza virosomes (IRIV) were used as a delivery system for the synthetic peptide-based malaria vaccine SPf66. The reduced SPf66 peptide molecules containing terminal cysteine residues were... more
SUMMARY Immunopotentiating reconstituted influenza virosomes (IRIV) were used as a delivery system for the synthetic peptide-based malaria vaccine SPf66. The reduced SPf66 peptide molecules containing terminal cysteine residues were covalently attached to phosphatidylethanolamine with the heterobifunctional crosslinker γ-maleimidobutyric acid N-hydroxysuccinimide ester. The SPf66-phosphatidylethanolamine was incorporated into IRIV and BALB/c mice were immunized twice by intramuscular injection with peptide-loaded virosomes. Titres of elicited anti-SPf66 IgG were determined by ELISA. These titres were significantly higher and the required doses of antigen were lower, when mice had been preimmunized with a commercial whole virus influenza vaccine. After preimmunization with the influenza vaccine, SPf66-IRIV elicited far more consistently anti-SPf66 antibody responses than SPf(66)n adsorbed to alum. MoAb produced by four B cell hybridoma clones derived from a SPf66-IRIV-immunized mouse...
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Buruli ulcer (BU) is a chronic necrotizing disease of the skin and subcutaneous fat tissue. The causative agent, Mycobacterium ulcerans, produces mycolactone, a macrolide toxin, which causes apoptosis of mammalian cells. Only a small... more
Buruli ulcer (BU) is a chronic necrotizing disease of the skin and subcutaneous fat tissue. The causative agent, Mycobacterium ulcerans, produces mycolactone, a macrolide toxin, which causes apoptosis of mammalian cells. Only a small proportion of individuals exposed to M. ulcerans develop clinical disease, as surrounding macrophages may control the infection by bacterial killing at an early stage, while mycolactone concentration is still low. Otherwise, bacterial multiplication leads to in higher concentrations of mycolactone, with formation of necrotizing lesions that are no more accessible to immune cells. By typing a cohort of 96 Ghanaian BU patients and 384 endemic controls without BU, we show an association between BU and single nucleotide polymorphisms (SNPs) in iNOS (rs9282799) and IFNG (rs2069705). Both polymorphisms influence promoter activity in vitro. A previously reported SNP in SLC11A1 (NRAMP, rs17235409) tended to be associated with BU. Altogether, these data reflect ...
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Mycolactone, the macrolide exotoxin produced by Mycobacterium ulcerans, is central to the pathogenesis of the chronic necrotizing skin disease Buruli ulcer (BU). Here we show that mycolactone acts as an inhibitor of the mechanistic Target... more
Mycolactone, the macrolide exotoxin produced by Mycobacterium ulcerans, is central to the pathogenesis of the chronic necrotizing skin disease Buruli ulcer (BU). Here we show that mycolactone acts as an inhibitor of the mechanistic Target of Rapamycin (mTOR) signaling pathway by interfering with the assembly of the two distinct mTOR protein complexes mTORC1 and mTORC2, which regulate different cellular processes. Inhibition of the assembly of the rictor containing mTORC2 complex by mycolactone prevents phosphorylation of the serine/threonine protein kinase Akt. The associated inactivation of Akt leads to the dephosphorylation and activation of the Akt-targeted transcription factor FoxO3. Subsequent up-regulation of the FoxO3 target gene BCL2L11 (Bim) increases expression of the pro-apoptotic regulator Bim, driving mycolactone treated mammalian cells into apoptosis. The central role of Bim-dependent apoptosis in BU pathogenesis deduced from our experiments with cultured mammalian cel...
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The synergy between Mycobacterium tuberculosis infection and human immunodeficiency virus (HIV)/acquired immunodeficiency syndrome is well established but not so in Buruli ulcer (BU). We screened confirmed BU cases for HIV infection and... more
The synergy between Mycobacterium tuberculosis infection and human immunodeficiency virus (HIV)/acquired immunodeficiency syndrome is well established but not so in Buruli ulcer (BU). We screened confirmed BU cases for HIV infection and followed seven BU/HIV-coinfected patients. Management of BU/HIV was based on the World Health Organization guidelines and patient condition. The HIV positivity among BU patients (8.2%; 11/134) was higher compared with that of general patients attending the facility (4.8%; 718/14,863; P = 0.07) and that of pregnant women alone (2.5%; 279/11,125; P = 0.001). All seven BU/HIV-coinfected cases enrolled in the study presented with very large (category III) lesions with four having multiple lesions compared with 54.5% of category III lesions among HIV-negative BU patients. During the recommended BU treatment with rifampicin and streptomycin (SR) all patients developed immune infiltrates including CD4 T cells in their lesions. However, one patient who recei...
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Research Interests: Microbiology, Genetic Epidemiology, Immunology, Molecular Evolution, Medical Microbiology, and 15 moreBiology, Ghana, Medicine, Next generation sequencing, Genetic Diversity, Molecular Epidemiology, Humans, Genome, Buruli Ulcer, High Resolution, Linkage Disequilibrium, Genetic Polymorphism, Genetic variation, Mycobacterium Ulcerans, and Epidemiologic Studies
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Research Interests: Bioinformatics, Entomology, Epidemiology, Genomics, Biotechnology, and 14 moreCommunicable Diseases, Research, Biological Sciences, Latin America, Humans, Biomedical Research, Infectious Disease, Neglected tropical diseases, Latin America and the Caribbean, Caribbean region, Research Priorities, Neglected diseases, Medical and Health Sciences, and Latin Americans
Research Interests: Immune response, Ghana, Immunohistochemistry, Medicine, Antibiotic Resistance, and 15 moreBiological Sciences, Humans, Child, Mycobacterium, Patient Compliance, Buruli Ulcer, Granuloma, Streptomycin, Process Development, Lymphocytes, Observational Study, Inflammatory response, Antibiotic treatment, Medical and Health Sciences, and Rifampin
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The genomic fine-typing of strains of Mycobacterium ulcerans , the causative agent of the emerging human disease Buruli ulcer, is difficult due to the clonal population structure of geographical lineages. Although large sequence... more
The genomic fine-typing of strains of Mycobacterium ulcerans , the causative agent of the emerging human disease Buruli ulcer, is difficult due to the clonal population structure of geographical lineages. Although large sequence polymorphisms (LSPs) resulted in the clustering of patient isolates originating from across the globe, differentiation of strains within continents using conventional typing methods is very limited. In this study, we analyzed M. ulcerans LSP haplotype-specific insertion sequence elements among 83 M. ulcerans strains and identified single nucleotide polymorphisms (SNPs) that differentiate between regional strains. This is the first genetic discrimination based on SNPs of M. ulcerans strains from African countries where Buruli ulcer is endemic, resulting in the highest geographic resolution of genotyping so far. The findings support the concept of genome-wide SNP analyses as tools to study the epidemiology and evolution of M. ulcerans at a local level.
Research Interests: Microbiology, Africa, Biology, Medicine, Molecular Epidemiology, and 15 moreClinical Microbiology, Biological Sciences, Phylogeny, Humans, Haplotypes, Bacteria, Cluster Analysis, DNA fingerprinting, Endemic Diseases, Buruli Ulcer, Genotype, Mycobacterium Ulcerans, Base Sequence, Molecular Sequence Data, and Medical and Health Sciences
A specific and sensitive serodiagnostic test for Mycobacterium ulcerans infection would greatly assist the diagnosis of Buruli ulcer and would also facilitate seroepidemiological surveys. By comparative genomics, we identified 45... more
A specific and sensitive serodiagnostic test for Mycobacterium ulcerans infection would greatly assist the diagnosis of Buruli ulcer and would also facilitate seroepidemiological surveys. By comparative genomics, we identified 45 potential M. ulcerans specific proteins, of which we were able to express and purify 33 in E. coli. Sera from 30 confirmed Buruli ulcer patients, 24 healthy controls from the same endemic region and 30 healthy controls from a non-endemic region in Benin were screened for antibody responses to these specific proteins by ELISA. Serum IgG responses of Buruli ulcer patients were highly variable, however, seven proteins (MUP045, MUP057, MUL_0513, Hsp65, and the polyketide synthase domains ER, AT propionate, and KR A) showed a significant difference between patient and non-endemic control antibody responses. However, when sera from the healthy control subjects living in the same Buruli ulcer endemic area as the patients were examined, none of the proteins were ab...
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While it is well established that proximity to wetlands is a risk factor for contracting Buruli ulcer, it is not clear what proportion of a population living in an area where the etiologic agent, Mycobacterium ulcerans, is endemic is... more
While it is well established that proximity to wetlands is a risk factor for contracting Buruli ulcer, it is not clear what proportion of a population living in an area where the etiologic agent, Mycobacterium ulcerans, is endemic is actually exposed to this disease. Immunological cross-reactivity among mycobacterial species complicates the development of a specific serological test. Among immunodominant proteins recognized by a panel of anti-M. ulcerans monoclonal antibodies, the M. ulcerans homologue of the M. leprae 18-kDa small heat shock protein (shsp) was identified. Since this shsp has no homologues in M. bovis and M. tuberculosis, we evaluated its use as a target antigen for a serological test. Anti-18-kDa shsp antibodies were frequently found in the sera of Buruli ulcer patients and of healthy household contacts but rarely found in controls from regions where the infection is not endemic. The results indicate that only a small proportion of M. ulcerans-infected individuals ...
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The highly immunogenic mycobacterial proteins ESAT-6, CFP-10, and HspX represent potential target antigens for the development of subunit vaccines and immunodiagnostic tests. Recently, the complete genome sequence revealed the absence of... more
The highly immunogenic mycobacterial proteins ESAT-6, CFP-10, and HspX represent potential target antigens for the development of subunit vaccines and immunodiagnostic tests. Recently, the complete genome sequence revealed the absence of these coding sequences in Mycobacterium ulcerans, the causative agent of the emerging human disease Buruli ulcer. Genome reduction and the acquisition of a cytopathic and immunosuppressive macrolide toxin plasmid are regarded as crucial for the emergence of this pathogen from its environmental progenitor, Mycobacterium marinum. Earlier, we have shown the evolution of M. ulcerans into two distinct lineages. Here, we show that while the genome of M. marinum M contains two copies of the esxB-esxA gene cluster at different loci (designated MURD4 and MURD152), both copies are deleted from the genome of M. ulcerans strains belonging to the classical lineage. Members of the ancestral lineage instead retained some but disrupted most functional MURD4 or MURD...
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Peptide and protein mimetics are potentially of great value in synthetic vaccine design. The mimetics should function by stimulating the immune system to produce antibodies that recognize the intact parasite. Also the mimetics should be... more
Peptide and protein mimetics are potentially of great value in synthetic vaccine design. The mimetics should function by stimulating the immune system to produce antibodies that recognize the intact parasite. Also the mimetics should be presented to the immune system in a way that leads to efficient antibody production. Here we investigate the application of cyclic peptidomimetics presented on immunopotentiating reconstituted influenza virosomes (IRIVs), a form of antigen delivery that is licensed already for human clinical use, in synthetic vaccine design. We focus on the central (NPNA)(n) repeat region of the circumsporozoite (CS) protein of the malaria parasite Plasmodium falciparum as a model system. Cyclic peptidomimetics of the NPNA repeats were incorporated into both an IRIV and (for comparison) a multiple-antigen peptide (MAP). Both IRIV and MAP delivery forms induced mimetic-specific humoral immune responses in mice, but only with the mimetic-IRIV preparations did a significant fraction of the elicited antibodies cross-react with sporozoites. The results demonstrate that IRIVs are a delivery system suitable for the efficient induction of antibody responses against conformational epitopes by use of cyclic template-bound peptidomimetics. Combined with combinatorial chemistry, this approach may have great potential for the rapid optimization of molecularly defined synthetic vaccine candidates against a wide variety of infectious agents.
Research Interests: Biology, Pharmaceutical Chemistry, Medicine, Humans, Mice, and 13 moreAnimals, Drug Design, Monoclonal Antibodies, Peptides, Drug Delivery Systems, Fluorescent Antibody Technique, Molecular Mimicry, Delivery System, Protein Conformation, Enzyme Linked Immunosorbent Assay, Antigens, Biochemistry and cell biology, and immunoglobulin G
Research Interests: Microbiology, Medical Microbiology, Molecular Microbiology, Molecular Epidemiology, Population structure, and 15 moreHumans, Genetic Structure, Research article, Infant, Gambia, Clinical Sciences, Genotype, Microbial Sensitivity Tests, Antibiotic Susceptibility, Pneumococcal Vaccines, Serotyping, Clinical Trials as Topic, Child preschool, alleles, and Clonal complex
... The lack of convergence to a single structure reflects a low number of long range NOE restraints, which we presume is a result of significant backbone flexibility, compounded by problems of signal overlap. ... 7 F. Zavala, JP Tam, MR... more
... The lack of convergence to a single structure reflects a low number of long range NOE restraints, which we presume is a result of significant backbone flexibility, compounded by problems of signal overlap. ... 7 F. Zavala, JP Tam, MR Hollingdale, AH Cochrane, I. Quakyi, RS ...
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Mycobacterial pathogens can be categorized into three broad groups: Mycobacterium tuberculosis complex causing tuberculosis, M. leprae and M. lepromatosis causing leprosy, and atypical mycobacteria, or non-tuberculous mycobacteria (NTM),... more
Mycobacterial pathogens can be categorized into three broad groups: Mycobacterium tuberculosis complex causing tuberculosis, M. leprae and M. lepromatosis causing leprosy, and atypical mycobacteria, or non-tuberculous mycobacteria (NTM), responsible for a wide range of diseases. Among the NTMs, M. ulcerans is responsible for the neglected tropical skin disease Buruli ulcer (BU). Most pathogenic mycobacteria, including M. leprae, evade effector mechanisms of the humoral immune system by hiding and replicating inside host cells and are furthermore excellent modulators of host immune responses. In contrast, M. ulcerans replicates predominantly extracellularly, sheltered from host immune responses through the cytotoxic and immunosuppressive effects of mycolactone, a macrolide produced by the bacteria. In the year 2018, 208,613 new cases of leprosy and 2713 new cases of BU were reported to WHO, figures which are notoriously skewed by vast underreporting of these diseases.
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The emergence and evolution of community-acquired methicillin resistant Staphylococcus aureus (CA-MRSA) strains in Africa is poorly understood. However, one particular MRSA lineage called ST88, appears to be rapidly establishing itself as... more
The emergence and evolution of community-acquired methicillin resistant Staphylococcus aureus (CA-MRSA) strains in Africa is poorly understood. However, one particular MRSA lineage called ST88, appears to be rapidly establishing itself as an "African" CA-MRSA clone. In this study, we employed whole genome sequencing to provide more information on the genetic background of ST88 CA-MRSA isolates from Ghana and to describe in detail ST88 CA-MRSA isolates in comparison with other MRSA lineages worldwide. We first established a complete ST88 reference genome (AUS0325) using PacBio SMRT sequencing. We then used comparative genomics to assess relatedness among 17 ST88 CA-MRSA isolates recovered from patients attending Buruli ulcer treatment centres in Ghana, three non-African ST88s and 15 other MRSA lineages. We show that Ghanaian ST88 forms a discrete MRSA lineage (harbouring SCCmec-IV [2B]). Gene content analysis identified five distinct genomic regions enriched among ST88 isol...
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In recent years, comparative genome sequence analysis of African Mycobacterium ulcerans strains isolated from Buruli ulcer (BU) lesion specimen has revealed a very limited genetic diversity of closely related isolates and a striking... more
In recent years, comparative genome sequence analysis of African Mycobacterium ulcerans strains isolated from Buruli ulcer (BU) lesion specimen has revealed a very limited genetic diversity of closely related isolates and a striking association between genotype and geographical origin of the patients. Here, we compared whole genome sequences of five M. ulcerans strains isolated in 2004 or 2013 from BU lesions of four residents of the Offin river valley with 48 strains isolated between 2002 and 2005 from BU lesions of individuals residing in the Densu river valley of Ghana. While all M. ulcerans isolates from the Densu river valley belonged to the same clonal complex, members of two distinct clonal complexes were found in the Offin river valley over space and time. The Offin strains were closely related to genotypes from either the Densu region or from the Asante Akim North district of Ghana. These results point towards an occasional involvement of a mobile reservoir in the transmiss...
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Buruli ulcer, caused by infection with Mycobacterium ulcerans, is a chronic ulcerative neglected tropical disease of the skin and subcutaneous tissue that is most prevalent in West African countries. M. ulcerans produces a cytotoxic... more
Buruli ulcer, caused by infection with Mycobacterium ulcerans, is a chronic ulcerative neglected tropical disease of the skin and subcutaneous tissue that is most prevalent in West African countries. M. ulcerans produces a cytotoxic macrolide exotoxin called mycolactone, which causes extensive necrosis of infected subcutaneous tissue and the development of characteristic ulcerative lesions with undermined edges. While cellular immune responses are expected to play a key role against early intracellular stages of M. ulcerans in macrophages, antibody mediated protection might be of major relevance against advanced stages, where bacilli are predominantly found as extracellular clusters. To assess whether vaccine induced antibodies against surface antigens of M. ulcerans can protect against Buruli ulcer we formulated two surface vaccine candidate antigens, MUL_2232 and MUL_3720, as recombinant proteins with the synthetic Toll-like receptor 4 agonist glucopyranosyl lipid adjuvant-stable ...
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Although Neisseria meningitidis is a highly variable organism, most invasive disease is caused by a minority of genotypes. Hypervirulent lineages have been identified and their pandemic spread has been traced. During a longitudinal... more
Although Neisseria meningitidis is a highly variable organism, most invasive disease is caused by a minority of genotypes. Hypervirulent lineages have been identified and their pandemic spread has been traced. During a longitudinal meningococcal colonization study in a district of northern Ghana clonal waves of carriage and disease were observed. Genetic diversification of genoclouds was analysed by pulsed field gel electrophoretic (PFGE) analysis of isolates from healthy carriers and from meningitis patients. Even during the limited time of persistence in the district, microevolution of the dominating genoclouds took place. Population genomic analyses are required to understand the genetic basis for the emergence of new lineages with epidemic potential, which is of crucial importance for the development of long-term global vaccination strategies against meningococcal disease.
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The virulence of the malaria parasite Plasmodium falciparum is due, in part, to its ability to cytoadhere in deep vascular beds. Our inability to quantify the load of sequestered parasites hampers our understanding of the... more
The virulence of the malaria parasite Plasmodium falciparum is due, in part, to its ability to cytoadhere in deep vascular beds. Our inability to quantify the load of sequestered parasites hampers our understanding of the pathophysiological mechanisms involved in disease progression and complicates diagnosis. In this study we evaluate potential biochemical markers of sequestered load by comparing them with estimates of the sequestered load from a statistical model fitted to longitudinal patterns of peripheral parasite densities in a series of 22 patients with severe Plasmodium falciparum malaria. The markers comprised the host factors: haematocrit, circulating host DNA, sTNF-R75 and parasite derived products HRP2, pLDH, pigments and circulating parasite DNA. We investigated the suitability of these markers in determining sequestered loads in patients on quinine treatment. Observed peripheral parasitaemia, plasma levels of sTNF-R75 and circulating parasite DNA were most strongly correlated with estimates of sequestered loads on admission. However the dynamics of both sTNF-R75 and circulating parasite DNA during follow-up were very different from those of the estimated sequestered mass. These analyses suggest that none of the markers gave reliable estimates of the current sequestered load, though they may reflect the history of infection. Longitudinal analyses are needed that allow for the clearance rates of the marker molecules and for variations between hosts in the history of parasitaemia.
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PLoS ONE: an inclusive, peer-reviewed, open-access resource from the PUBLIC LIBRARY OF SCIENCE. Reports of well-performed scientific studies from all disciplines freely available to the whole world.
Research Interests: Functional Analysis, Clinical Trial, Multidisciplinary, Western blotting, Humans, and 9 moreAnimals, Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis, PLoS one, Adult, Plasmodium falciparum, Enzyme Linked Immunosorbent Assay, Malaria Vaccines, Apical Membrane Antigen -1, and Antibody Response
Mycolactone is an immunosuppressive cytotoxin responsible for the clinical manifestation of Buruli ulcer in humans. It was believed to be confined to its etiologic agent, Mycobacterium ulcerans. However, the identification of other... more
Mycolactone is an immunosuppressive cytotoxin responsible for the clinical manifestation of Buruli ulcer in humans. It was believed to be confined to its etiologic agent, Mycobacterium ulcerans. However, the identification of other mycolactone-producing mycobacteria ...
Research Interests: Environmental microbiology, Polymorphism, Biology, Medicine, Multidisciplinary, and 15 morePhylogeny, Bacterial Toxins, Mycobacterium, Applied, Applied Environmental Microbiology, Bacteria, Gene Order, Cluster Analysis, DNA fingerprinting, Molecular Phylogenetics and Evolution, Buruli Ulcer, Genotype, Genetic Polymorphism, Macrolides, and Molecular Sequence Data
Research Interests: Molecular Evolution, Medical Microbiology, Biology, Medicine, Emerging Infectious Diseases, and 11 moreVirulence, Humans, Molecular Phylogenetics and Evolution, Buruli Ulcer, Clinical Sciences, Mycobacterium marinum, Public health systems and services research, Genetic variation, Species Specificity, Mycobacterium Ulcerans, and Molecular Sequence Data
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While it is well established that proximity to wetlands is a risk factor for contracting Buruli ulcer, it is not clear what proportion of a population living in an area where the etiologic agent, Mycobacterium ulcerans, is endemic is... more
While it is well established that proximity to wetlands is a risk factor for contracting Buruli ulcer, it is not clear what proportion of a population living in an area where the etiologic agent, Mycobacterium ulcerans, is endemic is actually exposed to this disease. Immunological cross-reactivity among mycobacterial species complicates the development of a specific serological test. Among immunodominant proteins recognized by a panel of anti-M. ulcerans monoclonal antibodies, the M. ulcerans homologue of the M. leprae 18-kDa small heat shock protein (shsp) was identified. Since this shsp has no homologues in M. bovis and M. tuberculosis, we evaluated its use as a target antigen for a serological test. Anti-18-kDa shsp antibodies were frequently found in the sera of Buruli ulcer patients and of healthy household contacts but rarely found in controls from regions where the infection is not endemic. The results indicate that only a small proportion of M. ulcerans-infected individuals ...
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We have identified a novel conserved protein of Plasmodium falciparum, designated D13, that is stage-specifically expressed in asexual blood stages of the parasite. The predicted open reading frame (ORF) D13 contains 863 amino acids with... more
We have identified a novel conserved protein of Plasmodium falciparum, designated D13, that is stage-specifically expressed in asexual blood stages of the parasite. The predicted open reading frame (ORF) D13 contains 863 amino acids with a calculated molecular mass of 99.7 kDa and displays a repeat region composed of pentapeptide motives. Northern blot analysis with lysates of synchronized blood stage parasites showed that D13 is highly expressed at the mRNA level during schizogony. The first N'-terminal 138 amino acids of D13 were expressed in Escherichia coli and the purified protein was used to generate anti-D13 monoclonal antibodies (MAbs). Using total lysates of blood stage parasites and Western blot analysis, these MAbs stained one single band of approximately 100 kDa, corresponding to the predicted molecular mass of ORF D13. Western blot analysis demonstrated further that D13 is expressed during schizogony, declines rapidly in early ring stages and is undetectable in trop...
Research Interests: Microbiology, Biology, Medicine, Biological Sciences, Infection and immunity, and 15 moreInsecticide Resistance, Humans, Escherichia coli, Mice, Animals, Infection, In vitro culture, Membrane Integrity, Amino Acid Profile, Molecular Mass, Amino Acid Sequence, Erythrocytes, Control Method, Molecular Sequence Data, and Medical and Health Sciences
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Clinical diagnosis of Mycobacterium ulcerans infection is currently accepted as sufficient basis for treating the disease. Inadequate laboratory resources in the highly endemic areas of Africa often limit possibilities for in-country... more
Clinical diagnosis of Mycobacterium ulcerans infection is currently accepted as sufficient basis for treating the disease. Inadequate laboratory resources in the highly endemic areas of Africa often limit possibilities for in-country confirmation of clinical judgement. We ...
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Neisseria meningitidis is the leading cause of epidemic meningitis in the "meningitis belt" of Africa, where clonal waves of colonization and disease are observed. Point mutations and horizontal gene exchange lead to constant... more
Neisseria meningitidis is the leading cause of epidemic meningitis in the "meningitis belt" of Africa, where clonal waves of colonization and disease are observed. Point mutations and horizontal gene exchange lead to constant diversification of meningococcal populations during clonal spread. Maintaining a high genomic diversity may be an evolutionary strategy of meningococci that increases chances of fixing occasionally new highly successful "fit genotypes". We have performed a longitudinal study of meningococcal carriage and disease in northern Ghana by analyzing cerebrospinal fluid samples from all suspected meningitis cases and monitoring carriage of meningococci by twice yearly colonization surveys. In the framework of this study, we observed complete replacement of an A: sequence types (ST)-2859 clone by a W: ST-2881 clone. However, after a gap of 1 year, A: ST-2859 meningococci re-emerged both as colonizer and meningitis causing agent. Our whole genome sequ...
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Mycolactone, the macrolide exotoxin produced by Mycobacterium ulcerans, causes extensive tissue destruction by inducing apoptosis of host cells. In this study, we aimed at the production of antibodies that could neutralize the cytotoxic... more
Mycolactone, the macrolide exotoxin produced by Mycobacterium ulcerans, causes extensive tissue destruction by inducing apoptosis of host cells. In this study, we aimed at the production of antibodies that could neutralize the cytotoxic activities of mycolactone. Using the B cell hybridoma technology, we generated a series of monoclonal antibodies with specificity for mycolactone from spleen cells of mice immunized with the protein conjugate of a truncated synthetic mycolactone derivative. L929 fibroblasts were used as a model system to investigate whether these antibodies can inhibit the biological effects of mycolactone. By measuring the metabolic activity of the fibroblasts, we found that anti-mycolactone mAbs can completely neutralize the cytotoxic activity of mycolactone. The toxin neutralizing capacity of anti-mycolactone mAbs supports the concept of evaluating the macrolide toxin as vaccine target.
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In the African "meningitis belt," outbreaks of meningococcal meningitis occur in cycles, representing a model for the role of host-pathogen interactions in epidemic processes. The periodicity of the epidemics is not well... more
In the African "meningitis belt," outbreaks of meningococcal meningitis occur in cycles, representing a model for the role of host-pathogen interactions in epidemic processes. The periodicity of the epidemics is not well understood, nor is it currently possible to predict them. In our longitudinal colonization and disease surveys, we have observed waves of clonal replacement with the same serogroup, suggesting that immunity to noncapsular antigens plays a significant role in natural herd immunity. Here, through comparative genomic analysis of 100 meningococcal isolates, we provide a high-resolution view of the evolutionary changes that occurred during clonal replacement of a hypervirulent meningococcal clone (ST-7) by a descendant clone (ST-2859). We show that the majority of genetic changes are due to homologous recombination of laterally acquired DNA, with more than 20% of these events involving acquisition of DNA from other species. Signals of adaptation to evade herd i...
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Background. The brain’s inflammatory response to the infecting pathogen determines the outcome of bacterial meningitis (BM), for example, the associated mortality and the extent of brain injury. The inflammatory cascade is initiated by... more
Background. The brain’s inflammatory response to the infecting pathogen determines the outcome of bacterial meningitis (BM), for example, the associated mortality and the extent of brain injury. The inflammatory cascade is initiated by the presence of bacteria in the cerebrospinal fluid (CSF) activating resident immune cells and leading to the influx of blood derived leukocytes. To elucidate the pathomechanisms behind the observed difference in outcome between different pathogens, we compared the inflammatory profile in the CSF of patients with BM caused byStreptococcus pneumonia(n=14),Neisseria meningitidis(n=22), andHaemophilus influenza(n=9).Methods. CSF inflammatory parameters, including cytokines and chemokines, MMP-9, and nitric oxide synthase activity, were assessed in a cohort of patients with BM from Burkina Faso.Results. Pneumococcal meningitis was associated with significantly higher CSF concentrations of IFN-γ, MCP-1, and the matrix-metalloproteinase (MMP-) 9. In patient...
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Buruli ulcer (BU) caused by Mycobacterium ulcerans is a chronic necrotizing disease of the skin and the underlying soft tissue. Fat tissue necrosis accompanied by minimal inflammation is considered the most reliable histopathologic... more
Buruli ulcer (BU) caused by Mycobacterium ulcerans is a chronic necrotizing disease of the skin and the underlying soft tissue. Fat tissue necrosis accompanied by minimal inflammation is considered the most reliable histopathologic feature of BU. There may be a constant influx of inflammatory cells to the sites of active infection but these are thought to be killed by mycolactone, a polyketide toxin produced by M. ulcerans, through apoptosis and necrosis. Here we describe the spatial correlations between mycobacterial load and the expression of dendritic cell (DC) surface markers (cluster of differentiation (CD)83, CD11c, and CD123), the Toll-like receptor (TLR) 9 and pro- and anti-inflammatory cytokines (IL-8, IL-6, tumor necrosis factor-alpha (TNF-alpha), IFN-alpha, IL-12p40, IL-10, and IFN-gamma) within BU lesions. Although IL-8, IL-6, and TNF-alpha messenger RNA (mRNA) was detectable by real-time PCR in all lesions, the expression of the other cytokines was only found as small foci in some lesions. Correlations of the distribution of mRNA encoding the activation marker CD83 and the DC subset markers CD123 and CD11c indicate that both activated plasmacytoid and myeloid dendritic cells were present in the lesions. Results suggest that M. ulcerans specific immune responses may develop once therapeutic interventions have limited the production of mycolactone.
Research Interests: Immune response, Biology, Cytokines, Inflammation, Immunohistochemistry, and 15 moreMedicine, Dendritic Cells, Signal Transduction, real time PCR, Humans, Dendritic cell, Epidermis, Buruli Ulcer, Clinical Sciences, Immune system, Necrosis, Soft Tissue, Spatial Correlation, Mycobacterium Ulcerans, and Innate Immune System
Mycobacterium ulcerans causes the devastating infectious skin disease Buruli ulcer and has a monomorphic population structure. The resolution of conventional genetic fingerprinting methods is therefore not sufficient for... more
Mycobacterium ulcerans causes the devastating infectious skin disease Buruli ulcer and has a monomorphic population structure. The resolution of conventional genetic fingerprinting methods is therefore not sufficient for microepidemiological studies aiming to characterize transmission pathways. In a previous comparative genomic hybridization analysis with a microarray covering part of the M. ulcerans genome, we have found extensive insertional-deletional sequence polymorphisms among M. ulcerans isolates of diverse geographic origins that allowed us to distinguish between strains coming from different continents. Since large numbers of insertion sequences are spread over the genome of African M. ulcerans strains, we reasoned that these may drive large sequence polymorphisms in otherwise clonal local mycobacterial populations. In this study, we used a printed DNA microarray covering the whole genome of the Ghanaian M. ulcerans reference strain Agy99 for comparative genomic hybridizati...
Research Interests: Microbiology, Polymorphism, Ghana, Clinical Microbiology, Biological Sciences, and 15 moreIdentification, Humans, Clinical, Bacteria, Endemic Diseases, Risk factors, Buruli Ulcer, Genetic Polymorphism, Tandem Repeats, Risk Factors, Mycobacterium Ulcerans, Genetic Variability, Comparative Genomic Hybridization, Strains, and Medical and Health Sciences
Research Interests: Immunology, Malaria, Immunogenetics, Phylogeny, Humans, and 15 moreAnimals, New World, Animal Model, MHC class II, Amino Acids, Molecular Characterization, Major histocompatibility complex, Plasmodium falciparum, Genetic variation, Amino Acid Sequence, Base Sequence, Plasmodium Vivax, Molecular Sequence Data, alleles, and Nucleotide sequence
Buruli ulcer is a necrotizing skin disease caused by Mycobacterium ulcerans. Major necrosis with abundant clusters of extracellularly replicating mycobacteria and only minor leukocyte infiltration are characteristic histopathologic... more
Buruli ulcer is a necrotizing skin disease caused by Mycobacterium ulcerans. Major necrosis with abundant clusters of extracellularly replicating mycobacteria and only minor leukocyte infiltration are characteristic histopathologic features of the disease. Mycolactone, a cytotoxic macrolide exotoxin of M. ulcerans, plays a key role in the development of this pathology. Antimicrobial therapy, such as rifampicin/streptomycin that was recently introduced, seems to lead to phagocytosis of mycobacteria and massive leukocyte infiltration, which culminates in the development of ectopic lymphoid structures in the lesions. Whereas the curative effect of the antibiotic treatment may be supported by immune defense mechanisms, persisting mycobacterial antigens and immunostimulators occasionally also seem to cause apparent reactivation of the disease. This seems to be related to excessive immunostimulation rather than to incomplete killing of the pathogen.
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PLoS Neglected Tropical Diseases is an open-access journal publishing peer-reviewed research on the world\'s most neglected tropical diseases, such as elephantiasis, river blindness, leprosy, hookworm, schistosomiasis, and African... more
PLoS Neglected Tropical Diseases is an open-access journal publishing peer-reviewed research on the world\'s most neglected tropical diseases, such as elephantiasis, river blindness, leprosy, hookworm, schistosomiasis, and African sleeping sickness.
Research Interests: Immune response, Immunohistochemistry, Biological Sciences, Inflammatory Immune Response, Humans, and 15 moreChild, Male, Skin, Buruli Ulcer, Degeneration, Necrosis, Streptomycin, Mycobacterium Ulcerans, Leukocytes, Neglected diseases, Inflammatory response, Antibiotic treatment, Medical and Health Sciences, Antibiotic Therapy, and Rifampin
Research Interests: Evolutionary Biology, Genetics, Polymorphism, Molecular Evolution, Comparative Genomics, and 15 moreBiology, Medicine, Sequence Analysis, Molecular Epidemiology, Phylogeny, Humans, Mycobacterium tuberculosis, Haplotypes, Buruli Ulcer, Microarray Analysis, Adaptive evolution, Genome Rearrangement, Mycobacterium Ulcerans, Gene expression profiling, and DNA sequence
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We analysed cerebrospinal fluid samples from suspected meningitis cases in Nouna Health District, Burkina Faso, during the meningitis seasons of 2004-2006. Serogroup A ST2859 meningococci belonging to the ST5 clonal complex of subgroup... more
We analysed cerebrospinal fluid samples from suspected meningitis cases in Nouna Health District, Burkina Faso, during the meningitis seasons of 2004-2006. Serogroup A ST2859 meningococci belonging to the ST5 clonal complex of subgroup III meningococci were the predominant causative agent. ST2859 bacteria were associated with focal outbreaks in the north of the district. While &amp;amp;gt;10% of the population of an outbreak village carried ST2859, the population in the south of the district was predominantly colonised by serogroup Y ST4375 meningococci, which were associated with only sporadic cases of meningitis. Colonisation with the less virulent Y meningococci may interfere with the spread of the ST2859 to the south of the district, but there are concerns that this serogroup A clone may cause a third wave of subgroup III meningococcal disease in the African Meningitis Belt.
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Research Interests: Geography, Polymorphism, Comparative Genomics, Biological Sciences, Humans, and 13 moreHaplotypes, Animals, Fishes, Polymerase Chain Reaction, Gene Conversion, Buruli Ulcer, Spectrum, Hot Spot, Genetic variation, Genome sequence, Mycobacterium Ulcerans, Base Sequence, and Medical and Health Sciences
The age-dependent increase in resistance of infant rats to K1 E. coli infection was studied by analysing the clearance of intravenously injected radiolabelled O18:K1 E. coli bacteria. In susceptible 7-day-old rats, the rate of... more
The age-dependent increase in resistance of infant rats to K1 E. coli infection was studied by analysing the clearance of intravenously injected radiolabelled O18:K1 E. coli bacteria. In susceptible 7-day-old rats, the rate of reticuloendothelial clearance could not compete with bacterial multiplication, while the resistance of 18-day-old rats was attributed to the increased sequestration of bacteria in the spleen. After passive immunization with rat monoclonal anti-O18 IgG2b, the O18:K1 E. coli were rapidly cleared by the liver in both age groups. O1:K1, O18:K5 and O18:K- E. coli, which activate complement in an antibody-independent manner and are apathogenic for 7-day-old rats, were also rapidly sequestered in the liver. 7-day-old rats developed a fulminant bacteraemia after receiving 100 O18:K1 E. coli intravenously. After several hours a transient decrease in the level of bacteraemia was observed. However, the clearance remained incomplete, resulting in persistent bacteraemia and death. Endotoxin-responsive infant mice are able to clear the bacteraemia completely after a similar initial phase of bacterial multiplication while no clearance is observed in endotoxin-hypo-responsive mice. The marked host specificity of K1 E. coli infection thus appears to be related to differences in a clearance mechanism which may be sustained by an endotoxin-induced inflammatory response.
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Synovial T lymphocytes seem to contribute to the pathogenesis of rheumatoid arthritis (RA). Since very little is known about the structural heterogeneity of their T cell antigen receptors (TcR), we analyzed TcR alpha chain mRNA of... more
Synovial T lymphocytes seem to contribute to the pathogenesis of rheumatoid arthritis (RA). Since very little is known about the structural heterogeneity of their T cell antigen receptors (TcR), we analyzed TcR alpha chain mRNA of synovial fluid T cells from two RA patients. TcR alpha chain cDNA was amplified by the polymerase chain reaction with single-sided specificity for the alpha chain constant (C alpha) gene segment, and the nucleotide sequences of 51 functionally rearranged cDNA clones were determined. Twenty different V alpha genes and 26 different J alpha gene segments were utilized in these cDNA clones. Three of the V alpha gene segments which are frequently (8%-17% total) expressed in synovial fluid T cells have rarely been found in the TcR repertoire of peripheral blood T cells from healthy individuals. The T cell responses in the rheumatic synovia analyzed here are not oligoclonal, but the usage of TcR V alpha genes is biased.
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The synergy between Mycobacterium tuberculosis infection and human immunodeficiency virus (HIV)/acquired immunodeficiency syndrome is well established but not so in Buruli ulcer (BU). We screened confirmed BU cases for HIV infection and... more
The synergy between Mycobacterium tuberculosis infection and human immunodeficiency virus (HIV)/acquired immunodeficiency syndrome is well established but not so in Buruli ulcer (BU). We screened confirmed BU cases for HIV infection and followed seven BU/HIV-coinfected patients. Management of BU/HIV was based on the World Health Organization guidelines and patient condition. The HIV positivity among BU patients (8.2%; 11/134) was higher compared with that of general patients attending the facility (4.8%; 718/14,863; P = 0.07) and that of pregnant women alone (2.5%; 279/11,125; P = 0.001). All seven BU/HIV-coinfected cases enrolled in the study presented with very large (category III) lesions with four having multiple lesions compared with 54.5% of category III lesions among HIV-negative BU patients. During the recommended BU treatment with rifampicin and streptomycin (SR) all patients developed immune infiltrates including CD4 T cells in their lesions. However, one patient who recei...
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To assess the magnitude of the Buruli ulcer (BU) problem in Cameroon, we conducted a cross-sectional survey in the Nyong River basin and identified on clinical grounds a total of 436 cases of active or inactive BU (202 and 234,... more
To assess the magnitude of the Buruli ulcer (BU) problem in Cameroon, we conducted a cross-sectional survey in the Nyong River basin and identified on clinical grounds a total of 436 cases of active or inactive BU (202 and 234, respectively). Swab specimens were taken from 162 active cases with ulcerative lesions and in 135 of these (83.3%) the clinical
Research Interests: Adolescent, Humans, Child, Female, Male, and 16 morePolymerase Chain Reaction, Infant, Differential Diagnosis, Buruli Ulcer, Newborn Infant, Aged, River Basin, Middle Aged, Adult, Cross Section, Lower limb, Mycobacterium Ulcerans, Upper Limb, Cross Sectional Studies, Clinical Diagnosis, and BCG vaccine
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The distribution of M. ulcerans in Buruli ulcer lesions was analysed by IS2404 real-time PCR quantification of M. ulcerans DNA and by semi-quantitative microscopic assessment of the number of acid-fast bacilli (AFB). Mycobacterial burden... more
The distribution of M. ulcerans in Buruli ulcer lesions was analysed by IS2404 real-time PCR quantification of M. ulcerans DNA and by semi-quantitative microscopic assessment of the number of acid-fast bacilli (AFB). Mycobacterial burden was compared with histopathological changes. Focal distribution of tissue destruction extending into areas with high and low mycobacterial burden was a feature in all lesions analysed. Even where most of the mycobacteria were washed out of ulcerative lesions, peaks of mycobacterial DNA and AFB in the necrotic base of the ulcers still marked the position of the primary infection focus. Significant amounts of mycobacterial DNA and microcolonies were also present in samples from more peripheral regions and occasionally in excised margins of macroscopically and histologically healthy-appearing excised tissue margins. Additional peaks of mycobacterial DNA clearly marked sites where satellite lesions were developing. Even when granulomas provided evidence for the development of cell-mediated immunity, development of satellite lesions by contiguous spreading was not completely prevented. Areas free of mycobacterial DNA were found between primary and secondary infection foci and around scarring tissue of healing lesions. These results demonstrate that IS2404 real-time PCR analysis is a better tool than the less sensitive and only semi-quantitative microscopic enumeration of AFB for studying the dynamics of M. ulcerans infection in situ.
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Research Interests: Tropical Medicine, Ghana, Adolescent, Tropical medicine (Health Sciences), Disease Outbreaks, and 13 moreHumans, Child, Female, Male, Polymerase Chain Reaction, Infant, Phenotype, Middle Aged, Adult, Public health systems and services research, Microbial Sensitivity Tests, Enzyme Linked Immunosorbent Assay, and Serotyping
Meningococcal meningitis is a major cause of morbidity and mortality in the meningitis belt of sub-Saharan Africa where it occurs in epidemics every 8-12 years. Risk factors for the disease in this setting remain largely unknown. We... more
Meningococcal meningitis is a major cause of morbidity and mortality in the meningitis belt of sub-Saharan Africa where it occurs in epidemics every 8-12 years. Risk factors for the disease in this setting remain largely unknown. We carried out a case-control study to investigate possible risk factors among survivors of a meningitis epidemic occurring in 1997 in northern Ghana. A structured questionnaire on socio-economic factors, housing and household overcrowding, smoking and exposure to smoke and close contact with a case was administered to 505 of the survivors and 505 of age-, sex- and location-matched controls. Cooking in kitchens with firewood stoves (OR 9.00, CI 1.25-395) and sharing a bedroom with a case (OR 2.18 CI 1.43-3.4) were found to be risk factors for disease. Socio-economic factors, overcrowding, smoking and passive exposure to tobacco smoke were not found to be risk factors. Exposure to smoke from cooking fires or close contact with a case puts people at risk of contracting meningococcal meningitis. In the hot dry months, exposure to smoke from cooking fires should be minimized by encouraging alternatives to cooking over wood fires, or cooking outside. If wood-burning stoves cannot be avoided, kitchens should be made larger with improved ventilation. Meningitis cases should be nursed in well-ventilated rooms and the number of people sharing a room with a case kept at a minimum.
Research Interests: Microbiology, Medical Microbiology, Housing, Adolescent, Disease Outbreaks, and 17 moreHumans, Child, Smoking, Female, Male, Infant, Risk factors, Smoke, Newborn Infant, Indoor air pollution, Public health systems and services research, Sub Saharan Africa, Risk Factors, Tobacco Smoke, Case Control Study, *Hot Temperature, and Case Control Studies
A human melanoma-associated chondroitin sulfate proteoglycan (MCSP), recognized by mAb 9.2.27, plays a role in stabilizing cell-substratum interactions during early events of melanoma cell spreading on endothelial basement membranes. We... more
A human melanoma-associated chondroitin sulfate proteoglycan (MCSP), recognized by mAb 9.2.27, plays a role in stabilizing cell-substratum interactions during early events of melanoma cell spreading on endothelial basement membranes. We report here the molecular cloning and nucleotide sequencing of cDNA encoding the entire core protein of human MCSP and provide its deduced amino acid sequence. This core protein contains an open reading frame of 2322 aa, encompassing a large extracellular domain, a hydrophobic transmembrane region, and a relatively short cytoplasmic tail. Northern blot analysis indicated that MCSP cDNA probes detect a single 8.0-kb RNA species expressed in human melanoma cell lines. In situ hybridization experiments with a segment of the MCSP coding sequence localized MCSP mRNA in biopsies prepared from melanoma skin metastases. Multiple human Northern blots with an MCSP-specific probe revealed a strong hybridization signal only with melanoma cells and not with other human cancer cells or a variety of human fetal and adult tissues. These data indicate that MCSP represents an integral membrane chondroitin sulfate proteoglycan expressed by human malignant melanoma cells. The availability of cDNAs encoding MCSP should facilitate studies designed to establish correlations between structure and function of this molecule and help to establish its role in the progression of human malignant melanoma.
Research Interests: Membrane Proteins, Multidisciplinary, In Situ Hybridization, Protein Structure and Function, Cell line, and 14 moreHumans, Basement Membrane, Melanoma, Animals, Polymerase Chain Reaction, Molecular cloning, Malignant Melanoma, Rats, Adult, Study design, Amino Acid Sequence, Base Sequence, Open Reading Frame, and Skin Neoplasms
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Research Interests: Ghana, Adolescent, Molecular Epidemiology, Biological Sciences, real time PCR, and 17 moreHumans, Child, Polymerase Chain Reaction, DNA sequence design, Young Adult, Endemic Diseases, Risk factors, Buruli Ulcer, Aged, River Basin, Middle Aged, Genotype, Adult, Single Nucleotide Polymorphism, Very high throughput, Risk Factors, and Mycobacterium Ulcerans
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Buruli ulcer (BU), a neglected tropical disease of the skin and subcutaneous tissue, is caused by Mycobacterium ulcerans and is the third most common mycobacterial disease after tuberculosis and leprosy. While there is a strong... more
Buruli ulcer (BU), a neglected tropical disease of the skin and subcutaneous tissue, is caused by Mycobacterium ulcerans and is the third most common mycobacterial disease after tuberculosis and leprosy. While there is a strong association of the occurrence of the disease with stagnant or slow flowing water bodies, the exact mode of transmission of BU is not clear. M. ulcerans has emerged from the environmental fish pathogen M. marinum by acquisition of a virulence plasmid encoding the enzymes required for the production of the cytotoxic macrolide toxin mycolactone, which is a key factor in the pathogenesis of BU. Comparative genomic studies have further shown extensive pseudogene formation and downsizing of the M. ulcerans genome, indicative for an adaptation to a more stable ecological niche. This has raised the question whether this pathogen is still present in water-associated environmental reservoirs. Here we show persistence of M. ulcerans specific DNA sequences over a period of more than two years at a water contact location of BU patients in an endemic village of Cameroon. At defined positions in a shallow water hole used by the villagers for washing and bathing, detritus remained consistently positive for M. ulcerans DNA. The observed mean real-time PCR Ct difference of 1.45 between the insertion sequences IS2606 and IS2404 indicated that lineage 3 M. ulcerans, which cause human disease, persisted in this environment after successful treatment of all local patients. Underwater decaying organic matter may therefore represent a reservoir of M. ulcerans for direct infection of skin lesions or vector-associated transmission.
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Serine repeat antigen-5 (SERA5) is a candidate antigen for inclusion into a malaria subunit vaccine. During merozoite release and reinvasion the 120 kDa SERA5 precursor protein (P120) is processed, and a complex consisting of an... more
Serine repeat antigen-5 (SERA5) is a candidate antigen for inclusion into a malaria subunit vaccine. During merozoite release and reinvasion the 120 kDa SERA5 precursor protein (P120) is processed, and a complex consisting of an N-terminal 47 kDa (P47) and a C-terminal 18kDa (P18) processing product associates with the surface of merozoites. This complex is thought to be involved in merozoite invasion of and/or egress from host erythrocytes. Here we describe the synthesis and immunogenic properties of virosomally formulated synthetic phosphatidylethanolamine (PE)-peptide conjugates, incorporating amino acid sequence stretches from the N-terminus of Plasmodium falciparum SERA5. Choosing an appropriate sequence was crucial for the development of a peptide that elicited high titers of parasite cross-reactive antibodies in mice. Monoclonal antibodies (mAbs) raised against the optimized peptide FB-23 incorporating amino acids 57-94 of SERA5 bound to both P120 and to P47. Western blotting analysis proved for the first time the presence of SERA5 P47 in sporozoites. In immunofluorescence assays, the mAbs stained SERA5 in all its predicted localizations. The virosomal formulation of peptide FB-23 is suitable for use in humans and represents a candidate component for a multi-valent malaria subunit vaccine targeting both sporozoites and blood stage parasites.
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Parasite glycosylphosphatidylinositol (GPI) is an important toxin in malaria disease, and people living in malaria-endemic regions often produce high levels of anti-GPI antibodies. The natural anti-GPI antibody response needs to be... more
Parasite glycosylphosphatidylinositol (GPI) is an important toxin in malaria disease, and people living in malaria-endemic regions often produce high levels of anti-GPI antibodies. The natural anti-GPI antibody response needs to be understood to aid the design of an efficient carbohydrate-based antitoxin vaccine. We present a versatile approach based on a synthetic GPI glycan array to correlate anti-GPI antibody levels and protection from severe malaria.
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After an epidemic of serogroup A meningococcal meningitis in northern Ghana, a gradual disappearance of the epidemic strain was observed in a series of five 6-month carriage surveys of 37 randomly selected households. As serogroup A... more
After an epidemic of serogroup A meningococcal meningitis in northern Ghana, a gradual disappearance of the epidemic strain was observed in a series of five 6-month carriage surveys of 37 randomly selected households. As serogroup A Neisseria meningitidis carriage decreased, an epidemic of serogroup X meningococcal carriage occurred, which reached 18% (53/298) of the people sampled during the dry season of 2000, coinciding with an outbreak of serogroup X disease. These carriage patterns were unrelated to that of Neisseria lactamica. Multilocus sequence typing and pulsed-field gel electrophoresis of the serogroup X bacteria revealed strong similarity with other strains isolated in Africa during recent decades. Three closely related clusters with distinct patterns of spread were identified among the Ghanian isolates, and further microevolution occurred after they arrived in the district. The occurrence of serogroup X outbreaks argues for the inclusion of this serogroup into a multivalent conjugate vaccine against N. meningitidis.
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Plasmodium sporozoites are transmitted through the bite of infected mosquitoes and invade hepatocytes as a first and obligatory step of the parasite life cycle in man. Hepatocyte invasion involves proteins secreted from parasite vesicles... more
Plasmodium sporozoites are transmitted through the bite of infected mosquitoes and invade hepatocytes as a first and obligatory step of the parasite life cycle in man. Hepatocyte invasion involves proteins secreted from parasite vesicles called micronemes, the most characterized being the thrombospondin-related adhesive protein (TRAP). Here we investigated the expression and function of another microneme protein recently identified in Plasmodium falciparum sporozoites, apical membrane antigen 1 (AMA-1). P. falciparum AMA-1 is expressed in sporozoites and is lost after invasion of hepatocytes, and anti-AMA-1 antibodies inhibit sporozoite invasion, suggesting that the protein is involved during invasion of hepatocytes. As observed with TRAP, AMA-1 is initially mostly sequestered within the sporozoite. Upon microneme exocytosis, AMA-1 and TRAP relocate to the sporozoite surface, where they are proteolytically cleaved, resulting in the shedding of soluble fragments. A subset of serine protease inhibitors blocks the processing and shedding of both AMA-1 and TRAP and inhibits sporozoite infectivity, suggesting that interfering with sporozoite proteolytic processing may constitute a valuable strategy to prevent hepatocyte infection.
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Research Interests: Medical Microbiology, NMR Spectroscopy, Humans, Mice, Animals, and 13 moreMultiple Myeloma, P-glycoprotein, Monoclonal Antibodies, Structure Analysis, Enzyme, Genetic variation, Transfection, Monoclonal Antibody, Neurosciences, Binding Site, Light chain, Biochemistry and cell biology, and immunoglobulin G
A specific and sensitive serodiagnostic test for Mycobacterium ulcerans infection would greatly assist the diagnosis of Buruli ulcer and would also facilitate seroepidemiological surveys. By comparative genomics, we identified 45... more
A specific and sensitive serodiagnostic test for Mycobacterium ulcerans infection would greatly assist the diagnosis of Buruli ulcer and would also facilitate seroepidemiological surveys. By comparative genomics, we identified 45 potential M. ulcerans specific proteins, of which we were able to express and purify 33 in E. coli. Sera from 30 confirmed Buruli ulcer patients, 24 healthy controls from the same endemic region and 30 healthy controls from a non-endemic region in Benin were screened for antibody responses to these specific proteins by ELISA. Serum IgG responses of Buruli ulcer patients were highly variable, however, seven proteins (MUP045, MUP057, MUL_0513, Hsp65, and the polyketide synthase domains ER, AT propionate, and KR A) showed a significant difference between patient and non-endemic control antibody responses. However, when sera from the healthy control subjects living in the same Buruli ulcer endemic area as the patients were examined, none of the proteins were ab...
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Research Interests: Microbiology, Medical Microbiology, Molecular Microbiology, Streptococcus pneumoniae, Molecular Epidemiology, and 11 morePopulation structure, Humans, Genetic Structure, Infant, Gambia, Clinical Sciences, Genotype, Microbial Sensitivity Tests, Antibiotic Susceptibility, Pneumococcal Vaccines, and Serotyping
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ABSTRACT