White LED phototherapy as an improved treatment for neonatal jaundice.

With the aim of improving phototherapy for neonatal jaundice (hyperbilirubinemia), this study investigates the degradation of unconjugated bilirubin under irradiance by conventional light and by white, red, green, and blue LED sources in vitro. The absorption spectra of bilirubin under these different light sources are compared. The results demonstrate that white LED phototherapy promotes more efficient bilirubin degradation than conventional blue-light therapy. This study provides a basis for the design of novel phototherapy devices for the treatment of hyperbilirubinemia.

CD11/CD18 and CD14 share a common lipid A signaling pathway.

The activation of phagocytes by the lipid A moiety of LPS has been implicated in the pathogenesis of Gram-negative sepsis. While two LPS receptors, CD14 and CD11/CD18, have been associated with cell signaling, details of the LPS signal transduction cascade remain obscure. CD14, which exists as a GPI-anchored and a soluble protein, lacks cytoplasmic-signaling domains, suggesting that an ancillary molecule is required to activate cells. The CD11/CD18 integrins are transmembrane proteins. Like CD14, they are capable of mediating LPS-induced cellular activation when expressed on the surface of hamster fibroblasts Chinese hamster ovary (CHO)-K1. The observation that a cytoplasmic deletion mutant is still capable of activating transfected CHO-K1 argues that CD11/CD18 also utilizes an associated signal transducer. We sought to identify further similarities between the signaling systems utilized by CD14 and CD11/CD18. LPS-binding protein, which transfers LPS to CD14, enhanced both LPS-induced cellular activation and binding of Gram-negative bacteria in CD11/CD18-transfected CHO-K1, thus implying that LPS-binding protein can also transfer LPS to CD11/CD18. When synthetic lipid A analogues were analyzed for their ability to function as LPS agonists, or antagonists, in the CHO transfectants, we found the effects were identical regardless of which LPS receptor was expressed. This supports the hypothesis that a receptor distinct from CD14 and CD11/CD18 is responsible for discriminating between the lipid A of LPS and the LPS antagonists. We propose that this receptor, which is the target of the LPS antagonists, functions as the true signal transducer in LPS-induced cellular activation for both CD14 and CD11/CD18.

Stimulation of macrophages by lipopolysaccharide alters the phosphorylation state, conformation, and function of PU.1 via activation of casein kinase II.

We previously reported that LPS stimulation of the RAW264.7 murine macrophage cell line rapidly induced a structural change within the N terminus of the transcriptional regulatory factor PU.1. PU.1 is required for the expression of a variety of cytokine, cytokine receptor, and integrin genes. Western blot analysis of nuclear extracts prepared from LPS-stimulated macrophages revealed increased phosphorylation of PU.1 at serine residues relative to that in unstimulated controls. PU.1-DNA complexes prepared using nuclear extracts from LPS-stimulated macrophages were less sensitive to protease digestion compared with PU.1-DNA complexes generated using nuclear extracts prepared from unstimulated cells. This altered protease sensitivity probably reflects a conformational change within PU.1 resulting from LPS-induced phosphorylation. This possibility was supported by the finding that in vitro-phosphorylated PU.1 was similarly resistant to protease digestion. Transient transfection studies suggest that LPS-induced phosphorylation of PU.1 at serine 148, located within a casein kinase II (CKII) consensus motif, increases the transactivation function of PU.1. Other serine/CKII sites located at positions 41, 45, 132, and 133 do not appear to be required for LPS-induced PU.1 function. Lastly, we found that LPS also increased the enzymatic activity of CKII in these cells. To our knowledge, these are the first studies to demonstrate that LPS can stimulate CKII activity, induce PU.1 phosphorylation, and enhance the capacity of PU.1 to activate transcription in macrophages.

Mycobacterial lipoarabinomannan recognition requires a receptor that shares components of the endotoxin signaling system.

Phagocytic leukocytes respond to a variety of bacterial products including Gram-negative bacterial LPS and mycobacterial lipoarabinomannan (LAM). Anti-CD14 mAbs have been shown to block LPS and LAM activation of myeloid cells, suggesting that CD14 is required for cellular recognition of both ligands. Activation of undifferentiated promonomyelocytic THP-1 cells with LAM or LPS under serum-free conditions was enhanced in the presence of recombinant soluble CD14 (rsCD14). LPS binding protein (LBP), which is present in normal serum, further enhanced the sensitivity of undifferentiated THP-1 cells to both ligands even in the absence of rsCD14. Although CD14-transfected Chinese hamster ovary and human HT1080 fibrosarcoma cell lines can be activated by LPS, neither cell line was activated by LAM. Furthermore, U373 astrocytoma cells, which respond to LPS using sCD14 and LBP, failed to be activated by LAM in the presence of rsCD14 and rLBP. We then tested the effects of lipid IVA and Rhodobacter sphaeroides lipid A, compounds that function as endotoxin inhibitors in human cells by interacting with a molecule thought to be a CD14-dependent LPS signal transducer. Both lipid IVA and R. sphaeroides lipid A inhibited the effects of LPS and LAM in THP-1 cells. Thus, the LPS and LAM receptors share CD14, LBP, and a putative endotoxin antagonist-inhibitable signal transducing component. However, the LAM signaling system appears to require an additional receptor component whose expression is restricted to cells of hemopoietic origin.

CD14 enhances cellular responses to endotoxin without imparting ligand-specific recognition.

Binding of the lipid A portion of bacterial lipopolysaccharide (LPS) to leukocyte CD14 activates phagocytes and initiates the septic shock syndrome. Two lipid A analogs, lipid IVA and Rhodobacter sphaeroides lipid A (RSLA), have been described as LPS-receptor antagonists when tested with human phagocytes. In contrast, lipid IVA activated murine phagocytes, whereas RSLA was an LPS antagonist. Thus, these compounds displayed a species-specific pharmacology. To determine whether the species specificity of these LPS antagonists occurred as a result of interactions with CD14, the effects of lipid IVA and RSLA were examined by using human, mouse, and hamster cell lines transfected with murine or human CD14 cDNA expression vectors. These transfectants displayed sensitivities to lipid IVA and RSLA that reflected the sensitivities of macrophages of similar genotype (species) and were independent of the source of CD14 cDNA. For example, hamster macrophages and hamster fibroblasts transfected with either mouse or human-derived CD14 cDNA responded to lipid IVA and RSLA as LPS mimetics. Similarly, lipid IVA and RSLA acted as LPS antagonists in human phagocytes and human fibrosarcoma cells transfected with either mouse or human-derived CD14 cDNA. Therefore, the target of these LPS antagonists, which is encoded in the genomes of these cells, is distinct from CD14. Although the expression of CD14 is required for macrophage-like sensitivity to LPS, CD14 cannot discriminate between the lipid A moieties of these agents. We hypothesize that the target of the LPS antagonists is a lipid A recognition protein which functions as a signaling receptor that is triggered after interaction with CD14-bound LPS.

CD14-mediated translocation of nuclear factor-kappa B induced by lipopolysaccharide does not require tyrosine kinase activity.

During the course of serious bacterial infections, lipopolysaccharide (LPS) is believed to interact with macrophage receptors, resulting in the generation of inflammatory mediators and systemic symptoms including hemodynamic instability and shock. CD14, a glycosylphosphatidylinositol-linked antigen, functions as an LPS signaling receptor. A critical issue concerns the mechanism by which CD14, which has no transmembrane domain, transduces its signal following LPS binding. Recently, investigators have hypothesized that CD14-mediated signaling is effected through a receptor-associated tyrosine kinase (TK), suggesting a multicomponent receptor model of LPS signaling. Wild-type Chinese hamster ovary (CHO)-K1 cells can be activated by endotoxin to release arachidonate following transfection with human CD14 (CHO/CD14). Nuclear translocation of cytosolic NF-kappa B is correlated with a number of LPS-inducible responses. We sought to determine if this pathway were present in CHO/CD14 cells and to elucidate the relationship of NF-kappa B activation to the CD14 receptor system. LPS-stimulated translocation of NF-kappa B in CHO/CD14 cells resembled the same response in the murine macrophage-like cell line RAW 264.7. Protein synthesis inhibitors and corticosteroids, which suppress arachidonate release and the synthesis of proinflammatory cytokines, had no effect on translocation of NF-kappa B in CHO/CD14 or RAW 264.7 cells, demonstrating that NF-kappa B translocation is an early event. Although TK activity was consistently observed by immunoblotting extracts from activated RAW 264.7 cells, LPS-induced phosphotyrosine residues were not observed from similarly treated CHO/CD14 cells. Furthermore, the TK inhibitors herbimycin A and genistein failed to inhibit translocation of NF-kappa B in CHO/CD14 or RAW 264.7 cells, although both of these agents inhibited LPS-induced TK activity in RAW 264.7 cells. These results imply that TK activity is not obligatory for CD14-mediated signal transduction to occur in response to LPS.