The acute inflammatory response to copper(II)-doped biphasic calcium phosphates.

Infection and inflammation are two key features to consider to avoid septic or aseptic loosening of bone-implanted biomaterials. In this context, various approaches to fine-tune the biomaterial's properties have been studied in order to modulate the crosstalk between immune and skeletal cells. Cation-doping strategies for tuning of calcium phosphates properties has been evidenced as a promising way to control the biomaterial-induced inflammatory process, and thus improving their osteoimmunomodulatory properties. Copper(II) ions are recognized for their antibacterial potential, but the literature on their impact on particulate material-induced acute inflammation is scarce. We synthesized copper(II) ions-doped biphasic calcium phosphate (BCP), intended to exhibit osteoimmunomodulatory properties. We addressed in vitro, for the first time, the inflammatory response of human primary polymorphonuclear neutrophils (PMNs) to copper(II) ions-doped or undoped (BCP) powders, synthesized by an original and robust wet method, in the presence or absence of LPS as a costimulant to mimic an infectious environment. ELISA and zymography allowed us to evidence, in vitro, a specific increase in IL-8 and GRO-α secretion but not MIP-1ß, TNF-α, or MMP-9, by PMNs. To assess in vivo relevance of these findings, we used a mouse air pouch model. Thanks to flow cytometry analysis, we highlighted an increased PMN recruitment with the copper(II) ions-doped samples compared to undoped samples. The immunomodulatory effect of copper(II) ions-doped BCP powders and the consequent induced moderate level of inflammation may promote bacterial clearance by PMNs in addition to the antimicrobial potential of the material. Copper(II) doping provides new insights into calcium phosphate (CaP)-based biomaterials for prosthesis coating or bone reconstruction by effectively modulating the inflammatory environment.

Decellularization of Wharton's Jelly Increases Its Bioactivity and Antibacterial Properties.

The field of regenerative medicine has recently seen an emerging trend toward decellularized extracellular matrix (ECM) as a biological scaffold for stem cell-delivery. Human umbilical cord represents a valuable opportunity from both technical and ethical point of view to obtain allogenic ECM. Herein, we established a protocol, allowing the full removal of cell membranes and nuclei moieties from Wharton's jelly (WJ) tissue. No alterations in the ECM components (i.e., collagen, GAG content, and growth factors), physical (i.e., porosity and swelling) and mechanical (i.e., linear tensile modulus) properties were noticed following WJ processing. Furthermore, no effect of the tissue processing on macromolecules and growth factors retention was observed, assuring thus a suitable bioactive matrix for cell maintenance upon recellularization. Based on the in vitro and in vivo biodegradability and stromal cell homing capabilities, decellularized WJ could provide an ideal substrate for stromal cells adhesion and colonization. Interestingly, the tissue processing increased the antibacterial and antiadhesive properties of WJ against Staphylococcus aureus and Staphylococcus epidermidis pathogens. Altogether, our results indicate that decellularized WJ matrix is able to limit Staphylococcus-related infections and to promote stromal cell homing, thus offering a versatile scaffold for tissue regenerative medicine.

Bone marrow mesenchymal stem cells offer an immune-privileged niche to Cutibacterium acnes in case of implant-associated osteomyelitis.

Considered as some of the most devastating complications, Cutibacterium acnes (C. acnes)-related osteomyelitis are among the hardest infections to diagnose and treat. Mesenchymal stem cells (MSCs) secrete number of immunomodulatory and antimicrobial soluble factors, making them an attractive treatment for bacterial infection. In this study, we examined MSCs/C. acnes interaction and analyzed the subsequent MSCs and bacteria's behaviors. Human bone marrow-derived MSCs were infected by C. acnes clinical strain harvested from non-infected bone site. Following 3 h of interaction, around 4% of bacteria were found in the intracellular compartment. Infected MSCs increased the secretion of prostaglandin E2 and indolamine 2,3 dioxygenase immunomodulatory mediators. Viable intracellular bacteria analyzed by infrared spectroscopy and atomic force microscopy revealed deep modifications in the wall features. In comparison with unchallenged bacteria, the viable intracellular bacteria showed (i) an increase in biofilm formation on orthopaedical-based materials, (ii) an increase in the invasiveness of osteoblasts and (iii) persistence in macrophage, suggesting the acquisition of virulence factors. Overall, these results showed a direct impact of C. acnes on bone marrow-derived MSCs, suggesting that blocking the C. acnes/MSCs interactions may represent an important new approach to manage chronic osteomyelitis infections. STATEMENT OF SIGNIFICANCE: The interaction of bone commensal C. acnes with bone marrow mesenchymal stem cells induces modifications in C. acnes wall characteristics. These bacteria increased (i) the biofilm formation on orthopaedical-based materials, (ii) the invasiveness of bone forming cells and (iii) the resistance to macrophage clearance through the modification of the wall nano-features and/or the increase in catalase production.

Specific antibacterial activity of copper alloy touch surfaces in five long-term care facilities for older adults.

BACKGROUND: Pathogens involved in healthcare-associated infections can quickly spread in the environment, particularly to frequently touched surfaces, which can be reservoirs for pathogens. AIM: The purpose of this study was to investigate naturally occurring bacterial contamination on touch surfaces in five French long-term care facilities and to compare bacterial populations recovered from copper and control surfaces. METHODS: More than 1300 surfaces were sampled. The collected bacteria were identified to obtain a global view of the cultivable bacterial populations colonizing touch surfaces. Haemolytic colonies and putative pathogens were also screened using specific agar plates and then identified with matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry. In total, more than 3400 colonies were analysed. FINDINGS: Staphylococcus and Micrococcus were the two predominant genera present on touch surfaces, respectively occurring on 51.8% and 48.0% of control surfaces. In these facilities with relatively low bioburden, copper surfaces efficiently reduced the occurrence frequencies of three genera: Staphylococcus, Streptococcus and Roseomonas. Pathogenic species such as Staphylococcus aureus, Enterococcus faecalis and E. faecium were observed in very few samples. In addition, meticillin-resistant S. aureus was observed on five control surfaces and one copper surface. CONCLUSION: Contamination of healthcare facilities touch surfaces can be the source for the spread of bacteria through the institution. This in situ study shows that the frequency of the contamination as well as the specific bacterial population bioburden is reduced on copper alloy surfaces.

Interaction of Cutibacterium acnes with human bone marrow derived mesenchymal stem cells: a step toward understanding bone implant- associated infection development.

Crosstalk between mesenchymal stem cells (MSCs) and bacteria plays an important role in regulating the regenerative capacities of MSCs, fighting infections, modulating immune responses and maintaining tissue homeostasis. Commensal Cutibacterium acnes (C. acnes) bacterium becomes an opportunistic pathogen causing implant-associated infections. Herein, we examined MSCs/C. acnes interaction and analysed the subsequent bacteria and MSCs behaviours following infection. Human bone marrow derived MSCs were infected by two clinical and one laboratory C. acnes strains. Following 3h of interaction, all bacterial strains were able to invade MSCs. Viable intracellular bacteria acquired virulence factors by increasing biofilm formation and/or by affecting macrophage phagocytosis. Although the direct and indirect (through neutrophil stimulation) antibacterial effects of the MSCs secretome were not enhanced following C. acnes infection, ELISA analysis revealed that C. acnes clinical strains are able to license MSCs to become immunosuppressive cell-like by increasing the secretion of IL-6, IL-8, PGE-2, VEGF, TGF-ß and HGF. Overall, these results showed a direct impact of C. acnes on bone marrow derived MSCs, providing new insights into the development of C. acnes during implant-associated infections. STATEMENT OF SIGNIFICANCE: The originality of this work relies on the study of relationship between human bone marrow derived mesenchymal stem cells (MSCs) phenotype and C. acnes clinical strains virulence following cell infection. Our major results showed that C. acnes are able to invade MSCs, inducing a transition of commensal to an opportunistic pathogen behaviour. Although the direct and indirect antibacterial effects were not enhanced following C. acnes infection, secretome analysis revealed that C. acnes clinical strains were able to license MSCs to become immunosuppressive and anti-fibrotic cell-like. These results showed a direct impact of C. acnes on bone marrow derived MSCs, providing new insights into the development of C. acnes during associated implant infections.

Cowries derived aragonite as raw biomaterials for bone regenerative medicine.

Carbonate apatites are sought as a bone substitute due to their biocompatibility and excellent resorbability. The present study deals with Cowrie's shell derived powder (CSDP) as natural biomaterial for bone regenerative medicine. Structural and physicochemical analysis showed that Cowrie's shells, presenting brick and mortar microstructures, were mainly composed of aragonite crystals, which were converted into poorly crystalline B-type carbonate apatite once soaked, at 37 °C, in simulated body fluid for 7 days, reflecting bioactive features. Cytotoxic assays showed that CSDP boosted human stem cell proliferation over the study time compared to nacre derived powder (NDP), used as positive control. Human stem cells adopted a flattened morphology and established physical contact with CSDP, signature of a good biocompatibility. Thus, these results suggested that CSDP presents a great interest for bone regenerative medicine, and could be a useful and versatile carrier/scaffold for bone tissue engineering or a raw material for 3D printed orthopedic devices.

Bioinspired Nanofeatured Substrates: Suitable Environment for Bone Regeneration.

Bone mimicking coatings provide a complex microenvironment in which material, through its inherent properties (such as nanostructure and composition), affects the commitment of stem cells into bone lineage and the production of bone tissue regulating factors required for bone healing and regeneration. Herein, a bioactive mineral/biopolymer composite made of calcium phosphate/chitosan and hyaluronic acid (CaP-CHI-HA) was elaborated using a versatile simultaneous spray coating of interacting species. The resulting CaP-CHI-HA coating was mainly constituted of bioactive, carbonated and crystalline hydroxyapatite with 277 ± 98 nm of roughness, 1 µm of thickness, and 2.3 ± 1 GPa of stiffness. After five days of culture, CaP-CHI-HA suggested a synergistic effect of intrinsic biophysical features and biopolymers on stem cell mechanobiology and nuclear organization, leading to the expression of an early osteoblast-like phenotype and the production of bone tissue regulating factors such as osteoprotegerin and vascular endothelial growth factor. More interestingly, amalgamation with biopolymers conferred to the mineral a bacterial antiadhesive property. These significant data shed light on the potential regenerative application of CaP-CHI-HA bioinspired coating in providing a suitable environment for stem cell bone regeneration and an ideal strategy to prevent implant-associated infections.

Harnessing Wharton's jelly stem cell differentiation into bone-like nodule on calcium phosphate substrate without osteoinductive factors.

An important aim of bone regenerative medicine is to design biomaterials with controlled chemical and topographical features to guide stem cell fate towards osteoblasts without addition of specific osteogenic factors. Herein, we find that sprayed bioactive and biocompatible calcium phosphate substrates (CaP) with controlled topography induce, in a well-orchestrated manner, Wharton's jelly stem cells (WJ-SCs) differentiation into osteoblastic lineage without any osteogenic supplements. The resulting WJ-SCs commitment exhibits features of native bone, through the formation of three-dimensional bone-like nodule with osteocyte-like cells embedded into a mineralized type I collagen. To our knowledge, these results present the first observation of a whole differentiation process from stem cell to osteocytes-like on a synthetic material. This suggests a great potential of sprayed CaP and WJ-SCs in bone tissue engineering. These unique features may facilitate the transition from bench to bedside and the development of successful engineered bone. STATEMENT OF SIGNIFICANCE: Designing materials to direct stem cell fate has a relevant impact on stem cell biology and provides insights facilitating their clinical application in regenerative medicine. Inspired by natural bone compositions, a friendly automated spray-assisted system was used to build calcium phosphate substrate (CaP). Sprayed biomimetic solutions using mild conditions led to the formation of CaP with controlled physical properties, good bioactivity and biocompatibility. Herein, we show that via optimization of physical properties, CaP substrate induce osteogenic differentiation of Wharton's jelly stem cells (WJ-SCs) without adding osteogenic supplement factors. These results suggest a great potential of sprayed CaP and WJ-SCs in bone tissue engineering and may facilitate the transition from bench to beside and the development of clinically successful engineered bone.

Biocompatibility of sol-gel hydroxyapatite-titania composite and bilayer coatings.

Titania-Hydroxyapatite (TiO2/HAP) reinforced coatings are proposed to enhance the bioactivity and corrosion resistance of 316L stainless steel (316L SS). Herein, spin- and dip-coating sol-gel processes were investigated to construct two kinds of coatings: TiO2/HAP composite and TiO2/HAP bilayer. Physicochemical characterization highlighted the bioactivity response of the TiO2/HAP composite once incubated in physiological conditions for 7days whereas the TiO2/HAP bilayer showed instability and dissolution. Biological analysis revealed a failure in human stem cells adhesion on TiO2/HAP bilayer whereas on TiO2/HAP composite the presence of polygonal shaped cells, possessing good behaviour attested a good biocompatibility of the composite coating. Finally, TiO2/HAP composite with hardness up to 0.6GPa and elastic modulus up to 18GPa, showed an increased corrosion resistance of 316L SS. In conclusion, the user-friendly sol-gel processes led to bioactive TiO2/HAP composite buildup suitable for biomedical applications.

Gene screening of Wharton's jelly derived stem cells.

Stem cells are the most powerful candidate for the treatment of various diseases. Suitable stem cell source should be harvested with minimal invasive procedure, found in great quantity, and transplanted with no risk of immune response and tumor formation. Fetal derived stem cells have been introduced as an excellent alternative to adult and embryonic stem cells use, but unfortunately, their degree of "stemness" and molecular characterization is still unclear. Several studies have been performed deciphering whether fetal stem cells meet the needs of regenerative medicine. We believe that a transcriptomic screening of Wharton's jelly stem cells will bring insights on cell population features.

Chitosan/hydroxyapatite hybrid scaffold for bone tissue engineering.

BACKGROUND: To favor regeneration following critical bone defect, a combination of autologous bone graft and biomaterials is currently used. Major drawbacks of such techniques remain the availability of the autologous material and the second surgical site, inducing pain and morbidity. OBJECTIVE: Our aim was to investigate the biocompatibility in vitro of three dimensions hybrid biodegradable scaffolds combining osteoconductive properties of hydroxyapatite and anti-inflammatory properties of chitosan. METHODS: Hybrid scaffolds were characterized by microscopic observations, equilibrium swelling ratio and overtime weight loss measurements. In vitro studies were performed using primary human bone cells cultured for 7, 14 and 21 days. Cell viability, proliferation, morphology and differentiation through alkaline phosphatase (ALP) activity measurement were assessed. RESULTS: Characterization of our scaffolds demonstrated porous, hydrophilic and biodegradable characteristics. In vitro studies showed that these scaffolds have induced slight decrease in cell death and proliferation comparing to the culture plastic substrate control condition, as well as increased short term osteoinductive properties. CONCLUSIONS: In this study, we have provided evidence that our hybrid hydroxyapatite/chitosan scaffolds could be suitable for bone filling.

Soft tissue adhesion of polished versus glazed lithium disilicate ceramic for dental applications.

OBJECTIVE: Ceramics are widely used materials for prosthesis, especially in dental fields. Despite multiple biomedical applications, little is known about ceramic surface modifications and the resulting cell behavior at its contact. The aim of this study is to evaluate the biological response of polished versus glazed surface treatments on lithium disilicate dental ceramic. METHODS: We studied a lithium disilicate ceramic (IPS e.max(®) Press, Ivoclar Vivadent) with 3 different surface treatments: raw surface treatment, hand polished surface treatment, and glazed surface treatment (control samples are Thermanox(®), Nunc). In order to evaluate the possible modulation of cell response at the surface of ceramic, we compared polished versus glazed ceramics using an organotypic culture model of chicken epithelium. RESULTS: Our results show that the surface roughness is not modified as demonstrated by equivalent Ra measurements. On the contrary, the contact angle θ in water is very different between polished (84°) and glazed (33°) samples. The culture of epithelial tissues allowed a very precise assessment of histocompatibility of these interfaces and showed that polished samples increased cell adhesion and proliferation as compared to glazed samples. SIGNIFICANCE: Lithium disilicate polished ceramic provided better adhesion and proliferation than lithium disilicate glazed ceramic. Taken together, our results demonstrate for the first time, how it is possible to use simple surface modifications to finely modulate the adhesion of tissues. Our results will help dental surgeons to choose the most appropriate surface treatment for a specific clinical application, in particular for the ceramic implant collar.

CXCL10 reduces melanoma proliferation and invasiveness in vitro and in vivo.

BACKGROUND: Melanoma is often infiltrated by inflammatory and immune cells that might either maintain chronic inflammation, therefore promoting tumour growth, or mount an antitumour response to control tumour outcome. In this setting, Th1-oriented lymphocyte infiltration is associated with a better outcome in melanoma. Although the interferon-induced protein CXCL10 is expressed by Th1 immune cells, its receptor was also shown to be involved in melanoma progression and metastasis. OBJECTIVES: To investigate the CXCL10-mediated antitumoral response in vivo, and its clinical relevance. Methods C57BL/6 mice bearing B16F1 melanoma were treated intraperitoneally with an adenovirus vector expressing CXCL10. In addition, peripheral blood mononuclear cells (PBMC) from 20 patients, 10 with melanoma in remission and 10 with melanoma in progression, were assessed for their cytokine/chemokine content using a 30-plex assay, and for their ability to modulate melanoma invasion in vitro in Transwell(®) (Sigma-Aldrich) chambers coated with Matrigel(®) (BD Biosciences). RESULTS: Treatment with CXCL10 reduced melanoma tumour growth in C57BL/6 mice compared with controls in vivo, and reduced melanoma invasion in vitro. Screening for expression of 30 cytokine/chemokine proteins showed that only CXCL10 was significantly increased in patients in remission compared with patients in progression. PBMC only from patients in remission significantly reduced melanoma cell invasiveness in an ex vivo Transwell(®) assay. Accordingly, this inhibitory effect was also observed with PBMC culture media from patients with melanoma in remission. CONCLUSIONS: The quantitative increase in CXCL10 production, together with its ability to limit melanoma progression, shows the potential benefit of this chemokine to control melanoma progression or metastasis.

Induction of a novel mechanism of accelerated bacterial clearance by lipopolysaccharide in CD14-deficient and Toll-like receptor 4-deficient mice.

Despite the lack of a proinflammatory response to LPS, CD14-deficient mice clear Gram-negative bacteria (Escherichia coli 0111) at least 10 times more efficiently than normal mice. In this study, we show that this is due to an early and intense recruitment of neutrophils following the injection of Gram-negative bacteria or LPS in CD14-deficient mice; in contrast, neutrophil infiltration is delayed by 24 h in normal mice. Similar results of early LPS-induced PMN infiltration and enhanced clearance of E. coli were seen in Toll-like receptor (TLR) 4-deficient mice. Furthermore, the lipid A moiety of LPS induced early neutrophil infiltration not only in CD14-deficient and TLR-4-deficient mice, but also in normal mice. In conclusion, the lipid A component of LPS stimulates a unique and critical pathway of innate immune responses that is independent of CD14 and TLR4 and results in early neutrophil infiltration and enhanced bacterial clearance.

Lipopolysaccharide structure influences the macrophage response via CD14-independent and CD14-dependent pathways.

CD14, a protein expressed on the surface of monocytes and neutrophils, is a major receptor for lipopolysaccharide (LPS). Studies with normal and CD14-deficient macrophages show that responses to low concentrations of LPS require expression of CD14, whereas responses to high concentrations of LPS are CD14-independent. Since LPS isolated from different bacterial species shows structural variability, studies were performed to determine whether differences in LPS structure influence CD14-dependent and CD14-independent responses. Studies with LPS purified from Escherichia coli, Salmonella abortus subspecies equi, Salmonella minnesota, Pseudomonas aeruginosa, Neisseria meningitidis, Bacteroides fragilis, and Rhodobacter sphaeroides show that the strongest CD14-dependent responses require a typical O-antigen, long carbohydrate chains, at least 6 acyl chains in their lipid A, and 2-phosphorylated Kdo moieties; wild-type LPS lacking a typical O-antigen and containing short carbohydrate chains and 2-phosphorylated Kdo moieties induces the strongest CD14-independent response.

CD14 plays no major role in shock induced by Staphylococcus aureus but down-regulates TNF-alpha production.

Recent in vitro studies have suggested that CD14, a major receptor for LPS, may also be a receptor for cell wall components of Gram-positive bacteria and thus play a role in Gram-positive shock. To analyze the in vivo role of CD14 in responses to Gram-positive bacteria, CD14-deficient and control mice were injected with Staphylococcus aureus, and the effects on lethality, bacterial clearance, and production of cytokines were analyzed. Survival of CD14-deficient and control mice did not differ significantly after administration of various doses of either unencapsulated or encapsulated S. aureus; furthermore, mice in both groups displayed similar symptoms of shock. In addition, inflammatory cytokines such as TNF-alpha and IL-6 were readily detectable in the serum of CD14-deficient mice injected with live or antibiotic-killed S. aureus. Surprisingly, the serum concentration of TNF-alpha in CD14-deficient mice was at least threefold higher than in control mice after injection of either unencapsulated or encapsulated S. aureus, suggesting that CD14 down-regulates TNF-alpha. A similar increase in serum TNF-alpha occurred when CD14-deficient animals were injected with gentamicin-killed bacteria even though no symptoms of shock were observed. These studies indicate that CD14, in contrast to its key function in responses to the Gram-negative bacterium, Escherichia coli 0111, does not play a prominent role in septic shock induced by S. aureus, and that the symptoms of S. aureus shock are not due solely to TNF-alpha.

Reduced bacterial dissemination and liver injury in CD14-deficient mice following a chronic abscess-forming peritonitis induced by Bacteroides fragilis.

The CD14 myelomonocytic differentiation antigen plays a major role in acute Gram-negative infections with Escherichia coli; however, its role in chronic infections has not yet been analyzed. To address this question, we studied the role of CD14 in a chronic abscess-forming peritonitis, induced by Bacteroides fragilis. B. fragilis (3x10(8) CFU/ml) were resuspended in a liquid nutrient agar and injected into the peritoneal cavity of CD14-deficient (CD 14-/-) and normal C57BL/6J (CD 14+/+) mice, respectively. After 3 days there was a severe phlegmonous intra-abdominal inflammation in both groups. After 7 days an abscess-forming peritonitis developed and by 14 days the infectious foci were compartimentalized. These observations were indistinguishable between CD14-/- and CD14+/+ mice. Although no differences were seen in abscess formation, CD14-/- mice were able to clear B. fragilis more efficiently from the blood than CD14+/+ mice. After 3, 7, and 14 days blood cultures were B. fragilis positive in 11% (1/9), 20% (2/10), and 0% (0/9) in CD14-/-compared with 90% (9/10), 78% (7/9), and 20% (2/10) in CD14+/+ mice, respectively (P<0.05). Furthermore, although the infection resulted in hepatocellular necrosis and severe hepatitis in both groups, at day 14 the liver cell damage was more severe in CD14+/+ than in CD14-/- mice (P<0.05). These results show that the chronic abscess formation induced by B. fragilis capsular polysaccharides is CD14 independent; however, bacterial clearance and/or dissemination and liver cell damage are at least partially influenced by CD14-dependent mechanisms.

Treponema pallidum and Borrelia burgdorferi lipoproteins and synthetic lipopeptides activate monocytic cells via a CD14-dependent pathway distinct from that used by lipopolysaccharide.

Lipoproteins of Treponema pallidum and Borrelia burgdorferi possess potent proinflammatory properties and, thus, have been implicated as major proinflammatory agonists in syphilis and Lyme disease. Here we used purified B. burgdorferi outer surface protein A (OspA) and synthetic lipopeptides corresponding to the N-termini of OspA and the 47-kDa major lipoprotein immunogen of T. pallidum to clarify the contribution of CD14 to monocytic cell activation by spirochetal lipoproteins and lipopeptides. As with LPS, mouse anti-human CD14 Abs blocked the activation of 1,25-dihydroxyvitamin D3-matured human myelomonocytic THP-1 cells by OspA and the two lipopeptides. The existence of a CD14-dependent pathway was corroborated by using undifferentiated THP-1 cells transfected with CD14 and peritoneal macrophages from CD14-deficient BALB/c mice. Unlike LPS, cell activation by lipoproteins and lipopeptides was serum independent and was not augmented by exogenous LPS-binding protein. Two observations constituted evidence that LPS and lipoprotein/lipopeptide signaling proceed via distinct transducing elements downstream of CD14: 1) CHO cells transfected with CD14 were exquisitely sensitive to LPS but were lipoprotein/lipopeptide nonresponsive; and 2) substoichiometric amounts of deacylated LPS that block LPS signaling at a site distal to CD14 failed to antagonize activation by lipoproteins and lipopeptides. The combined results demonstrate that spirochetal lipoproteins and lipopeptides use a CD14-dependent pathway that differs in at least two fundamental respects from the well-characterized LPS recognition pathway.