PubMed Health⌕ Search

Biomedical subjects

L Hamann

Publications and source records attributed to L Hamann.

16 recordsLinked to original sources
PubMed2004

A coding mutation within the first exon of the human MD-2 gene results in decreased lipopolysaccharide-induced signaling.

MD-2 is an accessory protein of the Toll-like receptor (TLR)-4, necessary for assembling a receptor complex to sense low quantities of lipopolysaccharide in order to subsequently trigger innate immune responses. MD-2 and TLR-4 are expressed on a variety of immunocompetent cells. Mutations within the TLR-4 gene have been shown to attenuate immune responses against lipopolysaccharide in mice. In humans, a TLR-4 polymorphism has been associated with a higher risk for developing severe Gram-negative sepsis and with a lower risk for atherosclerosis. Since MD-2 is an essential part of the lipopolysaccharide receptor complex, we screened 20 patients that underwent surgical cancer therapy for novel MD-2 mutations by a single-strand conformation polymorphism technique. In one patient we found an A --> G substitution at position 103, resulting in an amino-acid exchange from Thr 35 to Ala. Reporter gene assays revealed that this mutation resulted in a reduced lipopolysaccharide-induced signaling. The patient displayed an uneventful postoperative course, with the exception of slightly decreased TNF-alpha levels after in vitro stimulation with LPS as compared to wt patients. Genotyping of a further 41 patients by a newly developed Lightcycler/FRET method failed to detect any additional polymorphism carriers, indicating that this is a rare mutation.

Alanine↗
PubMed2001

The interaction of human peripheral blood eosinophils with bacterial lipopolysaccharide is CD14 dependent.

Bacterial lipopolysaccharide (LPS, endotoxin) is a ubiquitous component of dust and air pollution and is suspected to contribute after inhalation to an activation of eosinophils in bronchial tissues of asthmatic patients, provoking inflammatory and allergic processes. We were therefore interested in the interaction of eosinophil granulocytes with LPS and have examined the activation of and uptake to human peripheral blood eosinophils by LPS. Eosinophils were stimulated by LPS and the endotoxic component lipid A and the release of tumor necrosis factor alpha (TNF-alpha) and of the eosinophil-specific granule protein eosinophil cationic protein (ECP) was estimated. The results show induction of TNF-alpha and ECP-release by LPS and lipid A in a dose-dependent manner. Anti-CD14 monoclonal antibody (moAb) (clone MEM-18) and the synthetic lipid A partial structure 406 blocked the release of TNF-alpha and ECP by LPS-stimulated eosinophils. Studies with radioactively labeled LPS showed dose-dependent uptake of (3)H-LPS to eosinophils. The (3)H-LPS uptake was found to be specific because preincubation with unlabeled LPS, compound 406 and also anti-CD14 antibodies inhibited uptake of (3)H-LPS to eosinophil granulocytes. By flow cytometry using anti-CD14 moAb and by reverse transcriptase-polymerase chain reaction (RT-PCR) technique, CD14 expression was detectable. Furthermore, messenger RNA (mRNA) expression of Toll-like receptors (TLR) 2 and TLR 4 was detected, indicating the presence of these CD14 coreceptors. The results indicate that eosinophils can take up LPS and can be stimulated by LPS in a CD14-dependent manner. Hence, in addition to allergens, eosinophils interact with endotoxin, a process that possibly exacerbates ongoing inflammatory and allergic processes.

Antibodies, Monoclonal↗
PubMed2001

Ventilation-induced chemokine and cytokine release is associated with activation of nuclear factor-kappaB and is blocked by steroids.

Recent clinical trials have shown that the survival of patients with acute respiratory distress syndrome (ARDS) is improved by ventilation with reduced volumes. These studies suggested that overinflation of the lungs causes overactivation of the immune system. The present study investigated the hypothesis that ventilation with increased tidal volumes results in early responses similar to those caused by stimulation with one of the major risk factors for ARDS: bacterial lipopolysaccharide (LPS). We therefore compared the effects of ventilation (-10 cm H2O or -25 cm H2O end-inspiratory pressure) and LPS (50 microg/ml) on nuclear factor (NF)-kappaB activation, chemokine release, and cytokine release in isolated perfused lungs obtained from BALB/C mice. We found that both LPS and ventilation with -25 cm H2O (overventilation; OV) caused translocation of NF-kappaB, which was abolished by pretreatment with the steroid dexamethasone. Furthermore, both treatments resulted in similar increases in perfusate levels of alpha-chemokines (macrophage inflammatory protein; [MIP]-2; KC), beta-chemokines (macrophage chemotactic protein-1; MIP-1alpha), and cytokines (tumor necrosis factor-alpha, interleukin-6), which were largely prevented by dexamethasone pretreatment. In LPS-resistant C3H/HeJ mice, only OV, and not LPS, caused translocation of NF-kappaB and release of MIP-2. We conclude that OV evokes early inflammatory responses similar to those evoked by LPS (i.e., NF-kappaB translocation and release of proinflammatory mediators). The NF-kappaB translocation elicited by OV appears to be independent of Toll-like receptor 4 and not due to LPS contamination introduced by the ventilator. Our data further suggest that steroids might be considered as a subsidiary treatment during artificial mechanical ventilation.

Animals↗
PubMed2001

Decay-accelerating factor (DAF/CD55) is a functional active element of the LPS receptor complex.

Previously, we identified an 80 kDa membrane protein (LMP80) that is capable of binding to LPS and lipid A in the presence of LBP and sCD14. LMP80 could also be detected after immuno-coprecipitation of cell membranes with LPS and lipid A, indicating a physical contact of LMP80 and LPS/lipid A. Further analysis and peptide sequencing revealed that LMP80 is identical to CD55 (decay accelerating factor, DAF), a regulatory molecule of the complement cascade. Transfection of LPS-hyporesponsive Chinese hamster ovary (CHO) cells with human CD55 resulted in the translocation of NF-B upon stimulation with LPS or lipid A. Our results demonstrate a new functional role of CD55 as a molecule able to mediate LPS-induced activation of cells that may be part of a multimeric LPS receptor complex.

Animals↗
PubMed2000

Binding of lipopolysaccharide (LPS) to CHO cells does not correlate with LPS-induced NF-kappaB activation.

Activation of myeloid cells by lipopolysaccharide (LPS) is a key event in the development of gram-negative sepsis. One crucial step within this process is the binding of LPS to CD14. CD14 is a glycosylphosphatidylinositol (GPI)-anchored membrane protein requiring at least one additional membrane-spanning molecule for signal transduction. It is not clear whether the function of CD14 is to merely catalyze LPS binding, followed by the interaction of LPS with the signal transducer, or whether CD14 has a more specific function and may be a part of the signaling complex. To address this question we generated Chinese hamster ovary (CHO) cells expressing a human GPI-anchored form of LPS-binding protein (mLBP) to substitute for CD14 as LPS acceptor molecule. By comparison of CHO / mLBP with CHO / vector and CHO / CD14 cells we found that expression of GPI-linked LBP results in an enhanced binding of LPS but not in an increase in cell activation as determined by translocation of NF-kappaB. Furthermore, excess of recombinant soluble LBP resulted also in increased LPS binding without affecting NF-kappaB translocation. These data show that LPS binding alone is not sufficient to induce signaling. We conclude that CD14 is more than a catalyst for LPS binding: it seems to be directly involved in LPS signaling and thus appears to be an essential part of the signaling complex.

Acute-Phase Proteins↗
PubMed1999

Identification of the 80-kDa LPS-binding protein (LMP80) as decay-accelerating factor (DAF, CD55).

The activation of immunocompetent cells by lipopolysaccharide (LPS) during severe Gram-negative infections is responsible for the pathophysiological reactions, possibly resulting in the clinical picture of sepsis. Monocytes recognize LPS mainly through the LPS receptor CD14, however, other cellular binding structures have been assumed to exist. In previous studies, we have described an 80-kDa LPS-binding membrane protein (LMP80), which is present on human monocytes as well as endothelial cells. Here we demonstrate that LMP80 is widely distributed and that it forms complexes together with LPS and sCD14. Furthermore, we report on the biochemical purification of LMP80 and its identification as decay-accelerating factor, CD55, by amino acid sequencing and cloning techniques. Our results imply a new feature of CD55 as a molecule which interacts with LPS/sCD14 complexes. However, the involvement of CD55 in LPS-induced signaling remains to be elucidated.

Animals↗
PubMed1999

A DNA sequence corresponding to the gene encoding cysteine proteinase 5 in Entamoeba histolytica is present and positionally conserved but highly degenerated in Entamoeba dispar.

Cysteine proteinases of Entamoeba histolytica are considered to be one of the most important classes of molecules responsible for the parasite's ability to destroy human tissues. Interestingly, one particular cysteine proteinase, located on the surface of E. histolytica trophozoites and designated cysteine proteinase 5 (CP5), is not expressed in the closely related but nonpathogenic species Entamoeba dispar. By comparing the E. histolytica and E. dispar genomic loci containing the gene for CP5 (cp5), it was found that the position of cp5 within the genomic context is conserved between the two organisms, but that the gene is highly degenerated in E. dispar, as it contains numerous nucleotide exchanges, insertions, and deletions, resulting in multiple stop codons within the cp5 reading frame. An alignment of all available orthologous E. histolytica and E. dispar DNA sequences suggested that cp5 started to degenerate in E. dispar coincidently when the two organisms began to diverge from a common ancestor.

Animals↗
PubMed1998

Components of gut bacteria as immunomodulators.

In 1885 Louis Pasteur was the first to propose that the human immune system may be influenced by microorganisms. A large body of data has since been accumulated proving this assumption to be correct. Bacteria constitute the main constituents of the microbial flora of the human digestive tract and compounds of the bacterial cell wall have been shown to play an important role in the interaction of microbes with higher organisms. These components include peptidoglycan (PG) and lipopolysaccharide (LPS) of gram-negative bacteria. Both types of molecules are potent activators of the human immune system and exert their activity through the induction of endogenous mediators which are endowed with biological activity. This review focuses on the structure and activity of LPS and PG and illustrates how these bacterial factors stimulate the immune cells resulting in desired physiological or dramatic pathophysiological responses of the host organism.

Adjuvants, Immunologic↗
PubMed1998

Molecular mechanisms of sepsis.

Today a great number of problems in the field of bacterial sepsis remain to be solved. Understanding the molecular mechanisms of one of the most important bacterial products in the pathogenesis of sepsis - endotoxin may contribute to innovative and more effective therapies. Therefore, this review focuses on the structural and functional elements of endotoxin, its interaction with immune cells, and its biological activity. Finally, other bacterial components and their impact on sepsis are discussed.

Endotoxins↗
PubMed1997

Tetracycline-controlled gene expression in Entamoeba histolytica.

To provide further tools for functional genetics of the protozoan parasite Entamoeba histolytica, we have tested the suitability of the bacterial TN10-encoded tet-repressor/tet-operator system for gene regulation in ameba trophozoites. Expression of the tet-repressor within the ameba was driven by the wild-type endogenous lectin gene promoter from episomal transfected plasmids. Tetracycline-inducible expression of a reporter gene driven by a modified tet-operator-bearing lectin gene promotor was monitored by transient and episomal transfection. Promotor activity was dependent on the position of the tet-operator insertion. Under appropriate conditions, expression of the reporter gene in tet-repressor expressing cells revealed only background levels but was inducible up to 240-fold by the addition of non-toxic amounts of tetracycline reaching full activity within 36 to 48 h. Because of the tight and rapid control by tetracycline, the tet-repressor controlled lectin gene promotor should be a usefull tool for reverse genetic approaches in E. histolytica as well as for recombinant protein expression within this anaerobic organism.

Animals↗
PubMed1995

Transfection and continuous expression of heterologous genes in the protozoan parasite Entamoeba histolytica.

To provide tools for functional molecular genetics of the protozoan parasite Entamoeba histolytica, we investigated the use of the prokaryotic neomycin phosphotransferase (NEO) gene as a selectable marker for the transfection of the parasite. An Escherichia coli-derived plasmid vector was constructed (pA5'A3'NEO) containing the NEO coding region flanked by untranslated 5' and 3' sequences of an Ent. histolytica actin gene. Preceding experiments had revealed that amoebae are highly sensitive to the neomycin analogue G418 and do not survive in the presence of as little as 2 micrograms/ml. Transfection of circular pA5'A3'NEO via electroporation resulted in Ent. histolytica trophozoites resistant to G418 up to 100 micrograms/ml. DNA and RNA analyses of resistant cells indicated that (i) the transfected DNA was not integrated into the amoeba genome but was segregated episomally, (ii) in the amoebae, the plasmid replicated autonomously, (iii) the copy number of the plasmid and the expression of NEO-specific RNA were proportional to the amount of G418 used for selection, and (iv) under continuous selection, the plasmid was propagated over an observation period of 6 months. Moreover, the plasmid could be recloned into E. coli and was found to be unrearranged. To investigate the use of pA5'A3'NEO to coexpress other genes in Ent. histolytica, a second marker, the prokaryotic chloramphenicol acetyltransferase (CAT) gene under control of an Ent. histolytica lectin gene promoter was introduced into the plasmid. Transfection of the amoebae with this construct also conferred G418 resistance and, in addition, allowed continuous expression of CAT activity in quantities corresponding to the amount of G418 used for selection. When selection was discontinued, transfected plasmids were lost as indicated by an exponential decline of CAT activity in trophozoite extracts.

Animals↗
PubMed1994

Interaction of several related GC-box- and GT-box-binding proteins with the intronic enhancer is required for differential expression of the gb110 gene in embryonal carcinoma cells.

The molecular mechanisms by which expression of a gene is down-regulated after differentiation of F9 embryonal carcinoma cells into parietal endoderm-like cells was studied by characterizing the cis- and trans-regulatory elements of the gb110 gene. This gene encodes a putative RNA helicase, and its expression is down-regulated when F9 cells are differentiated with retinoic acid and cyclic AMP. The 5'-flanking region of the gene has all of the features of a GC-rich island promoter and seems to play only a minor role, if any, in the regulated expression. A 133-bp enhancer in the first intron was identified by transient chloramphenicol acetyltransferase assays that activated expression in undifferentiated F9 cells about 50- to 100-fold. As this enhancer was not active in differentiated F9 cells, it seems to be the prime mediator of the differentiation-specific down-regulation of the gb110 gene. Four different protein-binding sites, three of which contain GC- and GT-box motifs, were identified in the enhancer element. The fourth site, interacting with previously described transcription factor FTZ-F1/ELP, seems to be of minor importance for the activity of the enhancer. Mutational analysis showed that the cooperative interaction of several most likely related proteins with the three GC- and GT-box motifs was required for full enhancer activity. On the basis of their binding properties, at least two of these proteins seem to be identical or closely related to ubiquitous transcription factor Sp1. One of the GT-box-binding proteins was present in undifferentiated F9 cells but not, however, in its differentiated derivatives. The cell specificity of this transcription factor explains why the gb110 gene is not expressed or expressed only at low levels in parietal endoderm-like cells.

Animals↗
PubMed1993

Consecutive inactivation of both alleles of the gb110 gene has no effect on the proliferation and differentiation of mouse embryonic stem cells.

The gene gb110 was originally identified by provirus integration in the Mov10 mouse strain and encodes a 110-kDa protein with potential GTP-binding activity. The gene is evolutionarily conserved, and its expression is controlled in a developmentally and cell-cycle-specific manner, suggesting that it has an important function in differentiation and development. As a first step in studying the functional role of gb110, embryonal stem (ES) cell lines were derived in which both gb110 alleles were inactivated by consecutive gene targeting via homologous recombination. The first allele was interrupted by integration of a neomycin resistance-encoding gene (neo) and, subsequently, the second allele by integration of a hygromycin B resistance-encoding gene (hyg). Selection for homologous recombination was achieved by using promoter and AUG codon-deficient hyg or neo whose expression was dependent on integration into the host genome next to the transcriptional and translational start signals. The efficiency of gb110 gene targeting was very high, with 85-100% of all drug-resistant colonies having undergone homologous recombination. ES cells lacking a functional gb110 were indistinguishable from the wild-type ES cells, indicating that this gene is not required for normal ES cell proliferation and differentiation in vitro.

Alleles↗
PubMed1991

Structure and expression of a gene encoding a putative GTP-binding protein identified by provirus integration in a transgenic mouse strain.

The Mov-10 mouse strain was derived by infection of preimplantation embryos with the Moloney murine leukemia virus and carries one copy of the provirus in its germ line. Here we show that the provirus has integrated into an evolutionarily conserved gene that can code for a protein of 110 kDa containing the three consensus elements characteristic for GTP-binding proteins. The Mov-10 locus was expressed in a variety of cell types, including embryonal carcinoma and embryonic stem cells. Transcription of the gene was down-regulated about 10-fold when F9 embryonal carcinoma cells are differentiated into parietal endodermlike cells and about 2-fold when they are differentiated into visceral endodermlike cells. High levels of Mov-10 transcripts were also found at different stages of embryonal development and in the testes and thymus of adult animals. Expression was cell cycle controlled, with steady-state RNA levels significantly higher in growth-arrested than in growth-stimulated cells. The results suggest that the Mov-10 locus has an important function in development and/or control of cell proliferation. The provirus was shown to have integrated into intron 1 of the gene without disrupting expression, indicating that integration into intronic sequences of a transcription unit does not necessarily affect transcription. This result together with previous results from the Mov-13 mouse strain suggested that proviruses exert their mutagenic effect only by integration in specific sites, such as cis-regulatory DNA elements.

Amino Acid Sequence↗