Michael Heinzelmann

Targeting bacterial endotoxin: two sides of a coin.

Abstract:  The term sepsis describes a potentially lethal clinical condition that develops as a result of a dysregulated host response to bacterial infection. The most common bacterial component implicated in initiating the septic syndrome is a cell wall molecule derived from Gram‐negative bacteria, known as lipopolysaccharide (LPS) or endotoxin. Like all mammals, humans are equipped with an LPS‐sensing machinery consisting, primarily, of LPS‐binding protein (LBP), CD14, a glycosylphosphatidylinositol (GPI)‐anchored monocyte differentiation antigen, and toll‐like receptor 4 (TLR4), a signal‐transducing integral membrane protein. Modest stimulation of TLR4 facilitates the elimination of invading microorganisms. Potent TLR4 stimulation, however, produces severe reactions in the host, often leading to multiple organ failure and death. The search for pharmaceuticals that reduce mortality in septic patients has been a painstaking process. Thus far, only a few compounds have been found to significantly reduce mortality rates. Perhaps one of the more promising therapeutic strategies currently pursued is based on the identification of synthetic or naturally occurring substances that neutralize LPS or inhibit LPS‐mediated activation of host immune cells, such as monocytes and macrophages. Here, we describe a number of diverse molecular structures with a capacity to either enhance or blunt LPS‐induced monocyte activation. The underlying molecular mechanisms are discussed.

Heparin Binds to Lipopolysaccharide (LPS)-Binding Protein, Facilitates the Transfer of LPS to CD14, and Enhances LPS-Induced Activation of Peripheral Blood Monocytes

Heparin is one of the most effective drugs for preventing and treating thromboembolic complications in surgical patients. Recent evidence suggests that heparin enhances the proinflammatory responses of human peripheral blood monocytes to Gram-negative endotoxin (LPS). We have identified LPS-binding protein (LBP) as a novel heparin-binding plasma protein. The affinity of LPB to heparin was KD = 55 ± 8 nM, as measured by surface plasmon resonance. Using a fluorescence-based assay, we showed that clinically used heparin preparations significantly enhance the ability of LBP to catalytically disaggregate and transfer LPS to CD14, the LPS receptor. The presence of clinically relevant heparin concentrations in human whole blood increased LPS-induced production of the proinflammatory cytokine IL-8. Fondaparinux, which is identical with the antithrombin III-binding pentasaccharide in heparin, did not bind to LBP or alter LBP function. Thus, this novel anticoagulant drug is a potential candidate for safe administration to patients who have endotoxemia and require anticoagulation.

Measurement of the decay distribution of η′ → π+π−γ and evidence for the box anomaly

Abstract The distribution of m ( π + π − ) in the decay η ′ → π + π − γ has been measured with the Crystal Barrel detector. The results are based on a total of 7392 observed η′ decays. The box anomaly constant is extracted from this and its value is found to agree well with theoretical expectations. The pseudoscalar nonet parameters ( f 1 , f 8 and θ PS ) are determined. Finally, we find that there is a problem of consistency between QCD and the standard VDM assumption.

Endotoxin‐neutralizing effects of histidine‐rich peptides

Inflammatory responses of human peripheral blood monocytes to the Gram‐negative endotoxin lipopolysaccharide (LPS) are enhanced by structurally diverse substances, such as anionic polysaccharides or cationic polypeptides. Only a few substances are known to effectively blunt LPS‐induced monocyte activation. We now show that synthetic poly‐L‐histidine (Hn) binds to LPS and abrogates the release of the proinflammatory cytokine interleukin‐8 (IL‐8) in LPS‐stimulated human whole blood. LPS‐induced stimulation of monocytes was strictly pH‐dependent with only minor amounts of IL‐8 secreted in acidic blood. Maximum levels of IL‐8 secretion occurred at a strongly basic pH. Hn inhibition of the release of IL‐8 from LPS‐stimulated monocytes was observed under acidic, neutral and physiological conditions. With increasing alkalosis, the effectiveness of Hn was gradually lost, suggesting that protonated, but not deprotonated, Hn was effective in inhibiting LPS‐induced monocyte responses. Histidine‐rich protein 2 from the malaria parasite, Plasmodium falciparum, inhibited the ability of LPS to evoke an inflammatory response in CD14‐transfected THP‐1 cells. Further, a short synthetic peptide derived from human histidine‐ and proline‐rich glycoprotein also exhibited LPS‐inhibitory effects in CD14 transfectants. Taken together, these observations demonstrate the capacity of histidine‐rich peptides, irrespective of their origin, to neutralize LPS‐induced proinflammatory host responses.

Arginine-rich cationic polypeptides amplify lipopolysaccharide-induced monocyte activation.

ABSTRACT The human neutrophil-derived cationic protein CAP37, also known as azurocidin or heparin-binding protein, enhances the lipopolysaccharide (LPS)-induced release of tumor necrosis factor alpha (TNF-α) in isolated human monocytes. We measured the release of the proinflammatory cytokine interleukin-8 (IL-8) in human whole blood and found that in addition to CAP37, other arginine-rich cationic polypeptides, such as the small structurally related protamines, enhance LPS-induced monocyte activation. As CAP37 and protamines share high levels of arginine content, we tested different synthetic poly-l-amino acids and found that poly-l-arginine, and to a lesser extent poly-l-lysine, increased IL-8 production in LPS-stimulated human whole blood. Protamine-enhanced LPS responses remained unaffected by the presence of free l-arginine or l-lysine, indicating that basic polypeptides but not basic amino acids act synergistically with LPS. In agreement with observations previously reported for CAP37, the LPS-enhancing effect of poly-l-arginine was completely abolished upon antibody blockade of the human LPS receptor, CD14. Protamines, either immobilized or in solution, bound LPS specifically. Poly-l-arginines, protamines, and CAP37 were equally effective in inhibiting binding of LPS to immobilized l-arginines. Taken together, our results suggest a CD14-dependent mechanism by which arginine-rich cationic proteins modulate LPS-induced monocyte activation and support the prediction that other strongly basic proteins could act as amplifiers of LPS responses.

Thoracolumbar Spinal Injuries

Systematic epidemiologic data on traumatic thoracolumbar fractures are rare and differ depending on the area studied and on the treating center. The studies available from western countries reveal typical and comparable data on incidence, localization, and mechanisms of injury. Thoracolumbar fractures are more frequent in men (2/3) than in women (1/3) and peak between the ages of 20 and 40 years [30,47,65,81,94]. Approximately, 160 000 patients/year sustain an injury of the spinal column in the United States. The majority of these injuries comprise cervical and lumbar (L3–L5) spine fractures. However, between 15% and 20% of traumatic fractures occur at the thoracolumbar junction (T11-L2), whereas 9–16% occur in the thoracic spine (T1–T10) [36,46]. Hu and coworkers [56] studied the total population of a Canadian province over a period of 3 years. The incidence of spine injuries was 64/100 000 inhabitants per year, predominantly younger men and older women. A total of 2063 patients were registered and 944 patients were treated in hospital: 182 patients (20%) with a cervical spine injury, 286 patients (30%) with a thoracic spine injury and 403 patients (50%) with an injury of the lumbosacral spine. Traumatic cross-section spinal cord injury occurred in 40 out of 1 million inhabitants. About 50–60% of thoracolumbar fractures affect the transition T11-L2, 25–40% the thoracic spine and 10–14% the lower lumbar spine and sacrum [80,86].

Decay dynamics of the process η→3π0

Abstract The parameter α =−0.052±0.020 describing the shape of the η →3 π 0 Dalitz plot has been determined using the data from the Crystal Barrel detector at LEAR. The value is compared to predictions of chiral perturbation theory.

Momentum dependence of the decay η→π+π−π0

Abstract The π 0 momentum dependence of the decay η → π + π − π 0 has been measured with the Crystal Barrel detector. The analysis is based on 3230 events. The results of this independent measurement are compared to new chiral perturbation theory calculations and previous measurements.

Antiproton-proton annihilation at rest into KLKSπ0π0

Abstract The annihilation channel p p →K + K − π 0 in liquid hydrogen at rest has been studied with the Crystal Barrel detector at LEAR. The measured branching ratio is: BR ( p p → K + K − π 0 )=(2.37±0.15)×10 −3 . A partial wave analysis shows that this reaction is dominated by the π (K K ) S , K K ∗ (892) and π φ (1020) intermediate states. Weak signals are observed for π a 2 (1320), π f 2 (1270), π f 2 ′(1525) and K(Kπ) S . A satisfactory description of the Dalitz plot requires the introduction of π (K K ) P intermediate states with at least one pole.

Direct transplantation of native pericytes from adipose tissue: A new perspective to stimulate healing in critical size bone defects.

BACKGROUND AIMS Fractures with a critical size bone defect (e.g., open fracture with segmental bone loss) are associated with high rates of delayed union and non-union. The prevention and treatment of these complications remain a serious issue in trauma and orthopaedic surgery. Autologous cancellous bone grafting is a well-established and widely used technique. However, it has drawbacks related to availability, increased morbidity and insufficient efficacy. Mesenchymal stromal cells can potentially be used to improve fracture healing. In particular, human fat tissue has been identified as a good source of multilineage adipose-derived stem cells, which can be differentiated into osteoblasts. The main issue is that mesenchymal stromal cells are a heterogeneous population of progenitors and lineage-committed cells harboring a broad range of regenerative properties. This heterogeneity is also mirrored in the differentiation potential of these cells. In the present study, we sought to test the possibility to enrich defined subpopulations of stem/progenitor cells for direct therapeutic application without requiring an in vitro expansion. METHODS We enriched a CD146+NG2+CD45- population of pericytes from freshly isolated stromal vascular fraction from mouse fat tissue and tested their osteogenic differentiation capacity in vitro and in vivo in a mouse model for critical size bone injury. RESULTS Our results confirm the ability of enriched CD146+NG2+CD45- cells to efficiently generate osteoblasts in vitro, to colonize cancellous bone scaffolds and to successfully contribute to regeneration of large bone defects in vivo. CONCLUSIONS This study represents proof of principle for the direct use of enriched populations of cells with stem/progenitor identity for therapeutic applications.