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.

Targeting bacterial endotoxin: two sides of a coin.

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.

Lipopolysaccharide-mediated cell activation without rapid mobilization of cytosolic free calcium.

The involvement of cytosolic free calcium (Ca2+) as a second messenger in lipopolysaccharide (LPS)-activated cellular pathways remains controversial. Using human monocyte-like THP-1 cells stably transfected with glycosylphosphatidylinositol-anchored human CD14, we have monitored the concentration of intracellular Ca2+ after sequential addition of LPS, ATP, and ionomycin. Whereas ATP and ionomycin induced rapid elevations of cytosolic Ca2+, no such changes were observed after LPS addition. These data argue against the participation of cytosolic Ca2+ in the CD14-dependent LPS activation pathway.

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.

Human neutrophil-derived CAP37 inhibits lipopolysaccharide-induced activation in murine peritoneal macrophages.

Human cationic antimicrobial protein, CAP37, is released from neutrophil granules during infection. CAP37 attracts monocytes, binds Gram-negative endotoxin (lipopolysaccharide, LPS), is bactericidal for a range of Gram-negative bacteria, and reduces mortality in murine polymicrobial sepsis. Here, we report that recombinant CAP37 specifically targets murine peritoneal macrophages. Under steady-state conditions, the bulk of cell-associated CAP37 was localized at the plasma membrane. However, a fraction of CAP37 gained access to the endocytic system, but did not accumulate in recycling endosomes or in the trans-Golgi network (TGN). Instead, CAP37 was internalized by fluid phase endocytosis and accumulated in a prelysosomal compartment. Macrophages that were preexposed to CAP37 exhibited diminished LPS responsiveness, as determined by analysis of c-Jun phosphorylation. Further examination showed that pretreatment with CAP37 reduced the ability of macrophages to bind LPS. Taken together, these observations demonstrate that prolonged exposure to CAP37 desensitizes macrophages to LPS and suggest that this protein plays a novel anti-inflammatory role in polymicrobial sepsis.

Fondaparinux sodium lacks immunomodulatory effects of heparin.

BACKGROUND: Venous thromboembolism (VTE) remains a frequent complication in orthopedic and trauma patients, despite the use of prophylactic antithrombotic drugs like standard or low molecular weight heparin. A new synthetic pentasaccharide with specific anti-Xa activity (fondaparinux sodium) has been shown to reduce the overall risk of VTE in major orthopedic surgery by 50% without increasing the risk of clinically relevant bleeding, when compared with enoxaparin. Beyond their traditional role as anticoagulants, heparin and low molecular weight heparin possess potent immunomodulatory effects. For example, heparin and enoxaparin enhance endotoxin (lipopolysaccharide)-induced cytokine production in isolated human monocytes and in whole blood. In this study, we investigated the immunomodulatory effects of heparin and fondaparinux sodium. METHODS: Treatment of whole blood from 5 healthy volunteers ex vivo with heparin, fondaparinux sodium, and LPS (10 ng/mL, 4 hours). Determination of plasma interleukin (IL)-8 levels, thrombin clotting times, and anti-Xa activity with enzyme-linked immunosorbent assay and the AMAX CS190 device, respectively. Statistical analysis by analysis of variance and Fisher's probable least-squares difference posthoc test. RESULTS: Heparin concentrations > or =20 IU/mL significantly increased LPS-induced IL-8 production. Fondaparinux sodium, however, did not increase the LPS response. Heparin and fondaparinux sodium exhibited the expected anticoagulatory activities: heparin increased both thrombin clotting time and anti-Xa activity, fondaparinux sodium increased anti-Xa activity only. CONCLUSIONS: Fondaparinux sodium is not an immunomodulator like heparin, suggesting that this novel compound may lack adverse effects in endotoxemic patients. Further studies will establish whether fondaparinux sodium can safely be administered to patients with endotoxemia or sepsis.

Factors predisposing to bacterial invasion and infection.

BACKGROUND: Bacterial infections remain important causes of morbidity and mortality in surgical patients. Our understanding of the effects of bacteria on the host, and also the defense mechanisms available to the host, is improving all the time. Modern tools in biochemistry, immunology, and molecular biology have provided powerful methods to further our understanding of the complex interactions that contribute to our host defense response. This review reflects current thinking regarding the factors that contribute to bacterial infection and host defense response. DATA SOURCES: This review was compiled after an extensive review of the current and historical literature, and highlights a number of areas involved in the pathogenesis of bacterial infection. CONCLUSIONS: Bacteria can have a wide-ranging and deleterious effect on the host. Many different therapeutic approaches have been attempted to modulate the host response and limit the deleterious effects of bacteria. As our understanding of the underlying processes improves, these therapies should improve accordingly.

Spontaneous decrease of CD14 cell surface expression in human peripheral blood monocytes ex vivo.

In the continuous presence of bacterial lipopolysaccharide (LPS), the membrane-bound LPS receptor, CD14, exhibits a biphasic pattern of surface expression in monocytes ex vivo; an initial increase is followed by a later decrease. In the presence of LPS, ex vivo changes in monocytic CD14 cell surface expression have been consistently interpreted as the direct result of LPS exposure. There has been little consideration for the possibility of additional cell culture effects. Here, an experiment is presented to dissect the differences between LPS effects and cell culture effects on monocytic CD14 cell surface expression ex vivo. The results show that in monocytes from diluted whole blood cultures, CD14 surface expression is induced in LPS-treated samples but decreased in untreated samples. These observations suggest that the previously observed biphasic surface expression pattern of CD14 in long-term LPS-treated monocytes may be the result of a superposition of LPS-induced expression and spontaneous disappearance of CD14 from the plasma membrane. Further, these results illustrate the importance of taking cell culture conditions into account when analyzing monocyte expression of CD14.

Wound conditioning by vacuum assisted closure (V.A.C.) in postoperative infections after dorsal spine surgery.

The use of vacuum assisted closure (V.A.C.) therapy in postoperative infections after dorsal spinal surgery was studied retrospectively. Successful treatment was defined as a stable healed wound that showed no signs of acute or chronic infection. The treatment of the infected back wounds consisted of repeated debridement, irrigation and open wound treatment with temporary closure by V.A.C. The instrumentation was exchanged or removed if necessary. Fifteen patients with deep subfascial infections after posterior spinal surgery were treated. The implants were exchanged in seven cases, removed completely in five cases and left without changing in one case. In two cases spinal surgery consisted of laminectomy without instrumentation. In two cases only the wound defects were closed by muscle flap, the remaining ones were closed by delayed suturing. Antibiotic treatment was necessary in all cases. Follow up was possible in 14 patients. One patient showed a new infection after treatment. The study illustrates the usefulness of V.A.C. therapy as a new alternative management for wound conditioning of complex back wounds after deep subfascial infection.

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.

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

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-alpha) 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.