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ABSTRACT: We have performed freeze-fracture replica immunogold labelling of endotoxin preparations (lipid A and deep rough mutant LPS Re from Salmonella enterica sv. Minnesota), i.e. adding the endotoxins to human monocytes, labelling with monoclonal Abs recognizing either lipid A or LPS Re (A6 and A20 respectively), and fixing with immunogold secondary Ab. We have found that the endotoxins intercalated into the cell membranes with subsequent internalization by the cells. Surprisingly, membrane uptake took place only in the inner, plasmic leaflet of the plasma membrane, but there was no uptake of the outer leaflet for both compounds. Remarkable labelling could be also found for the two membranes of the nuclear envelope-in the case of lipid A only at the plasmic leaflet, but in the case of LPS Re on both leaflets. Isothermal calorimetric titration of the AB A20 with LPS and phospholipids showed that the Ab may bind not only to LPS but also to negatively charged phosphatidylserine. These results are discussed in the frame of the published concepts of cell activation induced by the endotoxins, i.e. how they are able to cause a conformational change of signalling proteins, such as the TLR4/MD2 complex.
Innate Immunity 02/2013; · 4.00 Impact Factor
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Lena Heinbockel,
Susana Sánchez-Gómez,
Guillermo Martinez de Tejada,
Sabine Dömming,
Julius Brandenburg,
Yani Kaconis,
Mathias Hornef,
Aline Dupont,
Sebastian Marwitz,
Torsten Goldmann,
Martin Ernst,
Thomas Gutsmann,
Tobias Schürholz,
Klaus Brandenburg
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ABSTRACT: Bacterial infections are known to cause severe health threatening conditions like sepsis. All attempts to get the disease under control failed in the past and especially in times of increasing antibiotic resistance, this leads to one of the most urgent medical challenges of our times. We designed a peptide to bind with high affinity to endotoxins, one of the most potent pathogenicity factors involved in triggering sepsis. The peptide Pep19-2.5 reveals an endotoxin neutralization efficiency in vitro and here we demonstrate the anti-septic/anti-inflammatory effect in vivo in the mouse models of endotoxemia, bacteremia and cecal ligation and puncture as well as in an ex vivo model of human tissue. Furthermore, we show that Pep19-2.5 can bind and neutralize not only endotoxins but also other bacterial pathogenicity factors such as from Gram-positive bacteria Staphylococcus aureus. This broad neutralization efficiency and the additive action of the peptide with common antibiotics makes it an exceptionally appropriate drug candidate against bacterial sepsis, but offers also multiple other medication opportunities.
Antimicrobial Agents and Chemotherapy 01/2013; · 4.84 Impact Factor
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ABSTRACT: INTRODUCTION: Increasing rates of multi-resistant bacteria are a major problem in the treatment of critically ill patients. Furthermore, conventional antibiotics lead to the release of bacterial derived membrane parts initiating pro-inflammatory cascades with potential harm to the patient. Antimicrobial peptides (AMP) may kill bacteria without releasing pro-inflammatory factors. Thus, we compared three newly developed synthetic anti-lipopolysaccharide peptides (SALPs) with a broader range of efficacy to suppress cytokine release in plasma and CD14 mRNA expression in organ tissue in a murine, polymicrobial sepsis model. METHODS: Randomized, experimental trial in an animal research facility. Male NMRI mice (n=90; 8-12 weeks) were randomized to the following six groups: (i) sham operation and parenteral vehicle (NaCl 0.9%) administration (sham); (ii) cecal ligation and puncture (CLP) and vehicle infusion (sepsis-control), (iii) CLP and polymyxin B infusion (polyB), or (iv-vi) CLP and infusion of three different synthetic antimicrobial peptides Pep19-2.5 (Pep2.5), Pep19-4 (Pep4) or Pep19-8 (Pep8). All animals underwent arterial and venous catheterization for hemodynamic monitoring 48 hours prior to CLP or sham-operation. Physical appearance and behavior (activity), plasma cytokine levels, and CD14 mRNA expression in heart, lung, liver, spleen and kidney tissue were determined 24 hours after CLP or sham operation. RESULTS: Only Pep2.5 significantly enhanced the activity after CLP, whereas none of the therapeutic regimen elevated the mean arterial pressure or heart rate. The strongly elevated IL-6, IL-10 and MCP serum levels in septic animals were significantly reduced after Pep2.5 administration (p<0.001, p<0.001, and p<0.001, respectively). Similarly, Pep2.5 significantly reduced the sepsis-induced CD14 mRNA expression in heart (p=0.003), lung (p=0.008), and spleen tissue (p=0.009) but not in kidney and liver. CONCLUSIONS: Structurally variable SALPs exhibit major differences in their anti-inflammatory effect in vivo. Continuous parenteral administration of Pep2.5 is able to reduce sepsis-induced cytokine release and tissue inflammation.
Critical care (London, England) 01/2013; 17(1):R3. · 4.61 Impact Factor
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Guillermo Martínez de Tejada,
Susana Sánchez-Gómez,
Iosu Rázquin-Olazaran,
Ina Kowalski,
Yani Kaconis, Lena Heinbockel,
Jörg Andrä,
Tobias Schürholz,
Mathias Hornef,
Aline Dupont,
Patrick Garidel,
Karl Lohner,
Thomas Gutsmann,
Sunil A David,
Klaus Brandenburg
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ABSTRACT: The first barrier that an antimicrobial agent must overcome when interacting with its target is the microbial cell wall. In the case of Gram-negative bacteria, additional to the cytoplasmic membrane and the peptidoglycan layer, an outer membrane (OM) is the outermost barrier. The OM has an asymmetric distribution of the lipids with phospholipids and lipopolysaccharide (LPS) located in the inner and outer leaflets, respectively. In contrast, Gram-positive bacteria lack OM and possess a much thicker peptidoglycan layer compared to their Gram-negative counterparts. An additional class of amphiphiles exists in Gram-positives, the lipoteichoic acids (LTA), which may represent important structural components. These long molecules cross-bridge the entire cell envelope with their lipid component inserting into the outer leaflet of the cytoplasmic membrane and the teichoic acid portion penetrating into the peptidoglycan layer. Furthermore, both classes of bacteria have other important amphiphiles, such as lipoproteins, whose importance has become evident only recently. It is not known yet whether any of these amphiphilic components are able to stimulate the immune system under physiological conditions as constituents of intact bacteria. However, all of them have a very high pro-inflammatory activity when released from the cell. Such a release may take place through the interaction with the immune system, or with antibiotics (particularly with those targeting cell wall components), or simply by the bacterial division. Therefore, a given antimicrobial agent must ideally have a double character, namely, it must overcome the bacterial cell wall barrier, without inducing the liberation of the pro-inflammatory amphiphiles. Here, new data are presented which describe the development and use of membrane-active antimicrobial agents, in particular antimicrobial peptides (AMPs) and lipopolyamines. In this way, essential progress was achieved, in particular with respect to the inhibition of deleterious consequences of bacterial infections such as severe sepsis and septic shock.
Current drug targets 05/2012; 13(9):1121-30. · 3.93 Impact Factor
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Tobias Schuerholz,
Sabine Dömming,
Mathias Hornef,
Aline Dupont,
Ina Kowalski,
Yani Kaconis, Lena Heinbockel,
Jörg Andrä,
Patrick Garidel,
Thomas Gutsmann,
Sunil David,
Susana Sánchez-Gómez,
Guillermo Martinez de Tejada,
Klaus Brandenburg
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ABSTRACT: The bacterial cell wall represents the primary target for antimicrobial agents. Microbial destruction is accompanied by the release of potent immunostimulatory membrane constituents. Both Gram-positive and Gram-negative bacteria release a variety of lipoproteins and peptidoglycan fragments. Gram-positive bacteria additionally provide lipoteichoic acids, whereas Gram-negative bacteria also release lipopolysaccharide (LPS, endotoxin), essential component of the outer leaflet of the bacterial cell wall and one of the most potent immunostimulatory molecules known. Immune activation therefore can be considered as an adverse effect of antimicrobial destruction and killing during anti-infective treatment. In contrast to antibiotics, the use of cationic amphiphilic antimicrobial peptides allows both effective bacterial killing and inhibition of the immunostimulatory effect of the released bacterial membrane constituents. The administration of antimicrobial peptides alone or in combination with antibiotic agents thus represents a novel strategy in the antiinfective treatment with potentially important beneficial aspects. Here, data are presented which describe immunological and clinical aspects of the use of antimicrobial peptides (AMPs) as therapeutic agents to treat bacterial infection and neutralize the immunostimulatory activity of released cell wall constituents.
Current drug targets 05/2012; 13(9):1131-7. · 3.93 Impact Factor
