L-FABP is exclusively expressed in alveolar macrophages within the myeloid lineage: evidence for a PPARalpha-independent expression

Peroxisome proliferator-activated receptors (PPARs) play a role in inflammation and, in particular, PPARgamma is involved in monocyte/macrophage differentiation. Members of the fatty acid-binding protein (FABP) family have been reported to function as transactivators for PPARs. Therefore, the expression of PPARs and FABPs in the myeloid lineage was investigated by real-time PCR and immunofluorescence analysis. We found adipocyte-, epidermal-, and heart-type FABP to be ubiquitously expressed within the myeloid lineage. In contrast, liver-type FABP was exclusively detected in murine alveolar macrophages (AM), confirmed on protein level by double fluorescence analysis. The PPAR subtypes also showed a temporally and spatially regulated expression pattern in myeloid cells: the beta-subtype was expressed in bone marrow, peritoneal, and alveolar macrophages, whereas it was not detected in dendritic cells (DCs). The gamma1-isoform was present in all cells, however, at different levels, whereas the gamma2-isoform was expressed in alveolar macrophages and dendritic cells. A low level PPARalpha mRNA could be detected in peritoneal macrophages and immature dendritic cells but not in mature dendritic cells and bone marrow macrophages. Interestingly, PPARalpha mRNA was also absent in the alveolar macrophages although liver-type FABP was expressed, indicating that gene expression of liver-type FABP was independent of PPARalpha. Since liver-type FABP is known as transactivator of PPARgamma the simultaneous expression of both proteins may have general implications for the activation of PPARgamma in alveolar macrophages.     

Alpha-melanocyte-stimulating hormone inhibits allergic airway inflammation

Alpha-melanocyte-stimulating hormone (alpha-MSH) is a neuropeptide controlling melanogenesis in pigmentary cells. In addition, its potent immunomodulatory and immunosuppressive activity has been recently described in cutaneous inflammatory disorders. Whether alpha-MSH is also produced in the lung and might play a role in the pathogenesis of inflammatory lung conditions, including allergic bronchial asthma, is unknown. Production and functional role of alpha-MSH were investigated in a murine model of allergic airway inflammation. alpha-MSH production was detected in bronchoalveolar lavage fluids. Although aerosol challenges stimulate alpha-MSH production in nonsensitized mice, this rapid and marked stimulation was absent in allergic animals. Treatment of allergic mice with alpha-MSH resulted in suppression of airway inflammation. These effects were mediated via IL-10 production, because IL-10 knockout mice were resistant to alpha-MSH treatment. This study provides evidence for a novel function of alpha-MSH linking neuroimmune functions in allergic airway inflammation.     

Crystal structures of nucleosome core particles in complex with minor groove DNA-binding ligands

We determined the crystal structures of three nucleosome core particles in complex with site-specific DNA-binding ligands, the pyrrole-imidazole polyamides. While the structure of the histone octamer and its interaction with the DNA remain unaffected by ligand binding, nucleosomal DNA undergoes significant structural changes at the ligand-binding sites and in adjacent regions to accommodate the ligands. Our findings suggest that twist diffusion occurs over long distances through tightly bound nucleosomal DNA. This may be relevant to the mechanism of ATP-dependent and spontaneous nucleosome translocation, and to the effect of bound factors on nucleosome dynamics.     

Mycophenolate mofetil impairs the maturation and function of murine dendritic cells

The immunosuppressive drug, mycophenolate mofetil (MMF), has been successfully introduced in allogeneic transplantation medicine and, more recently, in the treatment of autoimmune skin disorders. MMF inhibits lymphocyte proliferation via a blockade of the enzyme inosine 5'-monophosphate dehydrogenase, an enzyme on which lymphocytes solely depend to generate the purines necessary for DNA/RNA synthesis. To investigate the effects of MMF on cutaneous immune responses, a murine model of contact hypersensitivity (CHS) was used, with oxazolone or trinitrochlorobenzene as a contact allergen. Compared with the respective vehicle, i.p. applied MMF significantly inhibited the elicitation and, surprisingly, the induction of CHS responses. This prompted further studies into the effects of MMF on Ag presentation. Bone marrow-derived dendritic cells (DC) were cultured with GM-CSF and IL-4 in the presence of MMF and were tested for their Ag-presenting capacity. Sensitization and elicitation of CHS and delayed-type hypersensitivity responses by s. c. injected haptenated DC were reduced upon preincubation of DC with MMF. CHS responses were not impaired upon resensitization, indicating that MMF does not induce hapten-specific immunotolerance. In addition, MMF decreased the ability of DC to stimulate allogeneic T cells in MLR assays. Accordingly, flow cytometric analyses revealed a dose-dependent reduction of the expression of CD40, CD80, CD86, I-A, and ICAM-1 on DC with a concurrent reduction of IL-12 production. These data suggest that MMF, in addition to affecting T lymphocytes, directly affects APC, resulting in an impairment of immune responses. They furthermore point to a possible role of inosine 5'-monophosphate dehydrogenase in the maturation of DC.     

Role of the loop containing residue 115 in the induced-fit mechanism of the bacterial cell wall biosynthetic enzyme MurA

The induced-fit mechanism in Enterobacter cloacae MurA has been investigated by kinetic studies and X-ray crystallography. The antibiotic fosfomycin, an irreversible inhibitor of MurA, induced a structural change in UDP-N-acetylglucosamine (UDPGlcNAc)-liganded enzyme with a time dependence similar to that observed for the inactivation progress. The mechanism of action of fosfomycin on MurA appeared to be of the bimolecular type, the overall rate constants of inactivation and structural change being = 104 M(-1) s(-1) and = 85 M(-1) s(-1), respectively. Fosfomycin as well as the second MurA substrate, phosphoenolpyruvate (PEP), are known to interact with the side chain of Cys115. Like wild-type MurA, the catalytically inactive single-site mutant protein Cys115Ser structurally interacted with UDPGlcNAc in a rapidly reversible reaction. However, in contrast to wild-type enzyme, binding of PEP to mutant protein induced a rate-limited, biphasic structural change. Fosfomycin did not affect the structure of the mutant protein. The crystal structure of unliganded Cys115Ser MurA at 1.9 A resolution revealed that the overall conformation of the loop comprising residues 112-121 is not influenced by the mutation. However, other than Cys115 in wild-type MurA, Ser115 exhibits two distinct side-chain conformations. A detailed view on the loop revealed the existence of an elaborate hydrogen-bonding network mainly supplied by water molecules, presumably stabilizing its conformation in the unliganded state. The comparison between the known crystal structures of MurA, together with the kinetic data obtained, suggest intermediate conformational states in the MurA reaction, in which the loop undergoes multiple structural changes upon ligand binding.     

Osteoprotegerin blocks bone cancer-induced skeletal destruction, skeletal pain and pain-related neurochemical reorganization of the spinal cord

Bone cancer pain is common among cancer patients and can have a devastating effect on their quality of life. A chief problem in designing new therapies for bone cancer pain is that it is unclear what mechanisms drive this distinct pain condition. Here we show that osteoprotegerin, a secreted 'decoy' receptor that inhibits osteoclast activity, also blocks behaviors indicative of pain in mice with bone cancer. A substantial part of the actions of osteoprotegerin seems to result from inhibition of tumor-induced bone destruction that in turn inhibits the neurochemical changes in the spinal cord that are thought to be involved in the generation and maintenance of cancer pain. These results demonstrate that excessive tumor-induced bone destruction is involved in the generation of bone cancer pain and that osteoprotegerin may provide an effective treatment for this common human condition.     

Hang on or run? Copepod mating versus predation risk in contrasting environments

Mating durations of copepods were found to differ significantly between fishless high-altitude waters and lowland lakes containing fish. In lowland species the whole mating process was completed within a few minutes, but it averaged over an hour in high-altitude species. Alpine copepods showed a prolonged post-copulatory association between mates, during which the male clasped the female for an extended period after spermatophore transfer, while in lowland species males abandoned their partner immediately after copulation. Prolonged associations also occurred after transfer of spermatophores to heterospecific females with shorter conspecific mating duration, suggesting that male interests largely dictate the time spent in tandem. The differences observed may be adaptations to environments with different predation pressure, as pairs in tandem are more conspicuous and less reactive than single animals. We argue that differences in mating behavior and mating duration evolved under sexual versus natural selection, reflecting trade-offs between enhancement of fertilization success and reduction of vulnerability to visual predation. In fishless mountain lakes with high intrasexual competition, guarding males can reduce the risk of spermatophore displacement or the risk that the female will accept sperm from rival males without increased risk of being eaten, thereby maximizing paternity. Populations from fishless alpine lakes further differed from lowland species by exhibiting higher female/male size dimorphism and more intense pigmentation. While these traits vary between populations according to predation pressure, mating duration appears to be more species-specific.     

Stem cell transplantation for metastatic breast cancer: analysis of tumor contamination

The purpose of this study was to determine the efficacy, engraftment kinetics, effect of bone marrow tumor contamination, and safety of high-dose therapy and granulocyte-colony stimulating factor (G-CSF) mobilized peripheral blood progenitor cell (PBPC) support for patients with responding metastatic breast cancer. Forty two patients underwent G-CSF (10 μg/kg) stimulated PBPC harvest. PBPC and bone marrow aspirates were analyzed by histologic and immunocytochemical methods for tumor contamination. Thirty-seven patients received high-dose therapy consisting of cyclophosphamide 6 g/m², thiotepa 500 mg/m², and carboplatin 800 mg/m² (CTCb) given as an infusion over 4 d followed by PBPC reinfusion and G-CSF (5 μg/kg) support. No transplant related deaths or grade 4 toxicity was recorded. CD34+ cells/kg infused was predictive of neutrophil and platelet recovery. With a median follow-up of 38 months, three year survival was 44% with relapse-free survival of 19%. Histological bone marrow involvement, found in 10 patients, was a negative prognostic factor and was associated with a median relapse-free survival of 3.5 months. Tumor contamination of PBPC by immunohistochemical staining was present in 22.5% of patients and found not to be correlated with decreased survival. G-CSF stimulated PBPC collection followed by a single course of high dose chemotherapy and stem cell infusion with G-CSF stimulated marrow recovery leads to rapid, reliable engraftment with low toxicity and promising outcome in women with responding metastatic breast cancer.