No abstract

Recent in vivo studies indicate that mesenchymal stem cells (MSCs) may have beneficial effects in the treatment of sepsis induced by bacterial infection. Administration of MSCs in these studies improved survival and enhanced bacterial clearance. The primary objective of this study was to test the hypothesis that human MSCs possessed intrinsic antimicrobial properties. We studied the effect of human MSCs derived from bone marrow on the bacterial growth of Gram-negative (Escherichia coli and Pseudomonas aeruginosa) and Gram-positive (Staphylococcus aureus) bacteria. MSCs as well as their conditioned medium (CM) demonstrated marked inhibition of bacterial growth in comparison with control medium or normal human lung fibroblasts (NHLF). Analysis of expression of major antimicrobial peptides indicated that one of the factors responsible for the antimicrobial activity of MSC CM against Gram-negative bacteria was the human cathelicidin antimicrobial peptide, hCAP-18/LL-37. Both m-RNA and protein expression data showed that the expression of LL-37 in MSCs increased after bacterial challenge. Using an in vivo mouse model of E. coli pneumonia, intratracheal administration of MSCs reduced bacterial growth (in colony-forming unit) in the lung homogenates and in the bronchoalveolar lavage (BAL) fluid, and administration of MSCs simultaneously with a neutralizing antibody to LL-37 resulted in a decrease in bacterial clearance. In addition, the BAL itself from MSC-treated mice had a greater antimicrobial activity in comparison with the BAL of phosphate buffered saline (PBS)-treated mice. Human bone marrow-derived MSCs possess direct antimicrobial activity, which is mediated in part by the secretion of human cathelicidin hCAP-18/LL-37.

Staphylococcus aureus produces hospital-and community-acquired infections, with methicillinresistant S. aureus posing a serious public health threat. The golden carotenoid pigment of S. aureus, staphyloxanthin, promotes resistance to reactive oxygen species and host neutrophil-based killing, and early enzymatic steps in staphyloxanthin production resemble those for cholesterol biosynthesis. We determined the crystal structures of S. aureus dehydrosqualene synthase (CrtM) at 1.58 angstrom resolution, finding structural similarity to human squalene synthase (SQS). We screened nine SQS inhibitors and determined the structures of three, bound to CrtM. One, previously tested for cholesterol-lowering activity in humans, blocked staphyloxanthin biosynthesis in vitro (median inhibitory concentration ~100 nM), resulting in colorless bacteria with increased susceptibility to killing by human blood and to innate immune clearance in a mouse infection model. This finding represents proof of principle for a virulence factor-based therapy against S. aureus.Over the past 20 years, there has been an explosion in the prevalence of antibiotic-resistant bacterial infections, both in the hospital and in the general community; in the United States,

Persistent mucosal inflammation and microbial infection are characteristics of chronic rhinosinusitis (CRS). Mucosal microbiota dysbiosis is found in other chronic inflammatory diseases; however, the relationship between sinus microbiota composition and CRS is unknown. Using comparative microbiome profiling of a cohort of CRS patients and healthy subjects, we demonstrate that the sinus microbiota of CRS patients exhibits significantly reduced bacterial diversity compared with that of healthy controls. In our cohort of CRS patients, multiple, phylogenetically distinct lactic acid bacteria were depleted concomitant with an increase in the relative abundance of a single species, Corynebacterium tuberculostearicum. We recapitulated the conditions observed in our human cohort in a murine model and confirmed the pathogenic potential of C. tuberculostearicum and the critical necessity for a replete mucosal microbiota to protect against this species. Moreover, Lactobacillus sakei, which was identified from our comparative microbiome analyses as a potentially protective species, defended against C. tuberculostearicum sinus infection, even in the context of a depleted sinus bacterial community. These studies demonstrate that sinus mucosal health is highly dependent on the composition of the resident microbiota as well as identify both a new sino-pathogen and a strong bacterial candidate for therapeutic intervention.

Rationale: Mesenchymal stem cells secrete paracrine factors that can regulate lung permeability and decrease inflammation, making it a potentially attractive therapy for acute lung injury. However, concerns exist whether mesenchymal stem cells' immunomodulatory properties may have detrimental effects if targeted toward infectious causes of lung injury.Objectives: Therefore, we tested the effect of mesenchymal stem cells on lung fluid balance, acute inflammation, and bacterial clearance. Methods: We developed an Escherichia coli pneumonia model in our ex vivo perfused human lung to test the therapeutic effects of mesenchymal stem cells on bacterial-induced acute lung injury. Measurements and Main Results: Clinical-grade human mesenchymal stem cells restored alveolar fluid clearance to a normal level, decreased inflammation, and were associated with increased bacterial killing and reduced bacteremia, in part through increased alveolar macrophage phagocytosis and secretion of antimicrobial factors. Keratinocyte growth factor, a soluble factor secreted by mesenchymal stem cells, duplicated most of the antimicrobial effects. In subsequent in vitro studies, we discovered that human monocytes expressed the keratinocyte growth factor receptor, and that keratinocyte growth factor decreased apoptosis of human monocytes through AKT phosphorylation, an effect that increased bacterial clearance. Inhibition of keratinocyte growth factor by a neutralizing antibody reduced the antimicrobial effects of mesenchymal stem cells in the ex vivo perfused human lung and monocytes grown in vitro injured with E. coli bacteria. Conclusions: In E. coli-injured human lungs, mesenchymal stem cells restored alveolar fluid clearance, reduced inflammation, and exerted antimicrobial activity, in part through keratinocyte growth factor secretion.Keywords: acute lung injury; bacterial pneumonia; cell-based therapy; keratinocyte growth factor Despite extensive research into the pathogenesis of acute lung injury (ALI) and the acute respiratory distress syndrome, mortality remains high (1-3). Current treatment is supportive with lung protective ventilation and a fluid conservative strategy (4, 5). Innovative therapies are needed. Recent studies have suggested that mesenchymal stem or stromal cells (MSC) may have therapeutic applications in multiple models of lung disease (6-17). Despite initial interest in their multipotent properties, engraftment in the lung does not seem to play a major therapeutic role. The beneficial effect of MSC derives from their capacity to secrete paracrine factors that modulate immune responses and alter the responses of the endothelium or epithelium and inflammatory cells to injury (17-28). Because of the immunosuppressive properties of MSC, one safety concern is a potential deleterious effect on host defense against bacterial infection. Bacterial pneumonia and sepsis from a nonpulmonary cause are the two most common etiologies of ALI and acute respiratory distress syndrome (2). However, recent studies have provided eviden...

No abstract

Bisphosphonate drugs (e.g., Fosamax and Zometa) are thought to act primarily by inhibiting farnesyl diphosphate synthase (FPPS), resulting in decreased prenylation of small GTPases. Here, we show that some bisphosphonates can also inhibit geranylgeranyl diphosphate synthase (GGPPS), as well as undecaprenyl diphosphate synthase (UPPS), a cis-prenyltransferase of interest as a target for antibacterial therapy. Our results on GGPPS (10 structures) show that there are three bisphosphonate-binding sites, consisting of FPP or isopentenyl diphosphate substrate-binding sites together with a GGPP product-or inhibitor-binding site. In UPPS, there are a total of four binding sites (in five structures). These results are of general interest because they provide the first structures of GGPPSand UPPS-inhibitor complexes, potentially important drug targets, in addition to revealing a remarkably broad spectrum of binding modes not seen in FPPS inhibition.cell wall ͉ geranylgeranyl diphosphate synthase ͉ undecaprenyl diphosphate synthase ͉ x-ray structure I soprenoid biosynthesis involves the condensation of C 5 -diphosphates to form a very broad range of compounds used in cell membrane (cholesterol, ergosterol), cell wall (lipid I, II, peptidoglycan) and terpene biosynthesis, electron transfer (quinone, heme a, carotenoid, chlorophyll), and in many eukaryotes, cell signaling pathways (Ras, Rho, Rap, Rac). There has, therefore, been considerable interest in developing specific inhibitors of some of these pathways to modify cell function. For example, the bisphosphonate drugs used to treat bone resorption diseases such as osteoporosis (1) have been thought to function by targeting farnesyl diphosphate synthase (FPPS, EC 2.5.1.10) in osteoclasts, leading to dysregulation of cell-signaling pathways involving small GTPases, and in some parasitic protozoa, leading to inhibition of ergosterol biosynthesis (2). However, in recent work Goffinet et al. (3) proposed that the main biological activity of the most potent bisphosphonate zoledronate (Zometa) in humans cells is directed against protein geranylgeranylation. This opens up the intriguing possibility that it might be possible to enhance potency by developing drugs that work by inhibiting geranylgeranyl diphosphate synthase (GGPPS, EC 2.5.1.30), the enzyme that produces the geranylgeranyl diphosphate (GGPP) used to geranylgeranylate e.g., Rac, Rap, and Rho. Based on the recent observation of a previously uncharacterized (GGPP) inhibitor site in GGPPS (4), we reasoned that larger, more hydrophobic species than those in current use might bind to this site and exhibit enhanced activity, because of increased hydrophobic stabilization and, in cells, enhanced lipophilicity. Here, we thus report structures of a series of five bisphosphonates bound to GGPPS together with, for comparative purposes, the structures of five isoprenoid diphosphate-GGPPS complexes. We find three quite different binding modes, corresponding to FPP/GPP (substrate), IPP (substrate), and GGPP [product/ inhibitor (4)...

MA. Human mesenchymal stem cells reduce mortality and bacteremia in gram-negative sepsis in mice in part by enhancing the phagocytic activity of blood monocytes. Am