Modulation of Borrelia burgdorferi stringent response and gene expression during extracellular growth with tick cells

Borrelia burgdorferi N40 multiplied extracellularly when it was cocultured with tick cells in L15BS medium, a medium which by itself did not support B. burgdorferi N40 growth. Growth of B. burgdorferi N40 in the presence of tick cells was associated with decreased production of (p)ppGpp, the stringent response global regulator, a fourfold decrease in relA/spoT mRNA, an eightfold net decrease in bmpD mRNA, and a fourfold increase in rpsL-bmpD mRNA compared to growth of B. burgdorferi in BSK-H medium. As a result, the polycistronic rpsL-bmpD mRNA level increased from 3 to 100% of the total bmpD message. These observations demonstrate that there are reciprocal interactions between B. burgdorferi and tick cells in vitro and indicate that the starvation-associated stringent response mediated by (p)ppGpp present in B. burgdorferi growing in BSK-H medium is ameliorated in B. burgdorferi growing in coculture with tick cell lines. These results suggest that this system can provide a useful model for identifying genes controlling interactions of B. burgdorferi with tick cells in vitro when it is coupled with genetic methods to isolate and complement B. burgdorferi mutants.     

Production of herpes B virus recombinant glycoproteins and evaluation of their diagnostic potential

B virus (cercopithecine herpesvirus 1) is the only deadly alphaherpesvirus that is zoonotically transmissible from macaques to humans. The detection of humoral immune responses is the method of choice for the rapid identification of B virus-infected animals. We evaluated the diagnostic potential of recombinant B virus glycoproteins for the detection of immunoglobulin G (IgG) antibodies in monkey and human sera. Glycoproteins B, C, and E and secreted (sgG) and membrane-associated (mgG) segments of glycoprotein G (gG) were expressed in the baculovirus expression system, while gD was expressed in CHO cells. We developed recombinant protein-based IgG enzyme-linked immunosorbent assays (ELISAs) and compared their diagnostic efficacies by using B virus antibody-negative (n = 40) and -positive (n = 75) macaque sera identified by a whole antigen-based ELISA and Western blotting. The diagnostic sensitivities of the gB-, gC-, gD-, and mgG-ELISAs were 100, 97.3, 88.0, and 80.0%, respectively. The specificities of the gB-, gC-, and gD-ELISAs and of the mgG-ELISA were 100 and 97.5%, respectively. In contrast, the sensitivities and specificities of sgG- and gE-ELISAs were low, suggesting that sgG and gE are less effective diagnostic antigens. Sera from nonmacaque monkeys cross-reacted with gB, gC, and gD, and only baboon sera reacted weakly with mgG. Human herpes simplex virus type 1 (HSV-1)- and HSV-2-positive sera pools reacted with gB and gD, whereas sera from B virus-infected individuals reacted with all four antigens. These data indicate that gB, gC, gD, and mgG have a high diagnostic potential for B virus serodiagnosis in macaques, whereas mgG may be a valuable antigen for discrimination between antibodies induced by B virus and those induced by other, closely related alphaherpesviruses, including HSV-1 and -2.     

The prion protein in human neuromuscular diseases

The basis of human prion diseases affecting the nervous system is accumulation of a disease-associated conformer (PrPSc) of the normal cellular prion protein (PrPC). Earlier studies demonstrated increased expression of PrPC in inclusion body myositis (IBM), dermato-, and polymyositis, as well as neurogenic muscle atrophy. To define the spectrum and reliability of PrPC immunoreactivity, its expression was examined systematically in a series of pathologically characterized muscular disorders by means of immunohistochemistry, confocal laser microscopy, and immunogold electron microscopy. Anti-PrPC immunolabelling of rimmed vacuoles was observed in IBM, inclusions of myofibrillary myopathy, targets, regenerating, and atrophic fibres, mononuclear cells, in addition to ragged red fibres in mitochondrial myopathies, and focal sarcolemmal immunostaining in non-diseased controls. Quantitative analysis demonstrated that, in neurogenic muscle lesions, anti-PrPC staining detects a significantly broader spectrum of fibres than anti-vimentin or anti-NCAM. In dystrophic muscle, PrPC expression was mainly restricted to regenerating fibres. In IBM, PrPC expression was not confined to rimmed vacuoles or vacuolated fibres and only a small percentage (7.1%) of rimmed vacuoles were PrPC positive. Ultrastructurally, PrPC was observed in the cytoplasm of lymphocytes, in the myofibrillar network of targets, and in rimmed vacuoles. Knowledge of disease circumstances with altered expression of PrPC is important in the setting of a potentially increased chance for extraneural PrPC-PrPSc conversion. In addition, our observations suggest that PrPC may have a general stress-response effect in various neuromuscular disorders.     

Mutations in the human melanocortin-4 receptor gene associated with severe familial obesity disrupts receptor function through multiple molecular mechanisms

Mutations in the melanocortin-4 receptor gene (MC4R) represent the commonest monogenic cause of human obesity. However, information regarding the precise effects of such mutations on receptor function is very limited. We examined the functional properties of 12 different mutations in human MC4R that result in severe, familial, early-onset obesity. Of the nine missense mutants studied, four were completely unable to generate cAMP in response to ligand and five were partially impaired. Four showed evidence of impaired cell surface expression and six of reduced binding affinity for ligand. One mutation in the C-terminal tail, I316S, showed reduced affinity for alpha-MSH but retained normal affinity for the antagonist AgRP. None of the mutations inhibited signaling through co-transfected wild-type receptors. Thus, in the most comprehensive study to date of the functional properties of naturally occurring MC4R mutations we have (1) established that defective expression on the cell surface is a common mechanism impairing receptor function, (2) identified mutations which specifically affect ligand binding affinity thus aiding the definition of receptor structure-function relationships, (3) provided evidence against the notion that these receptor mutants act as dominant-negatives, and (4) identified a potentially novel molecular mechanism of receptor dysfunction whereby a mutation alters the relative affinities of a receptor for its natural agonist versus antagonist.     

Ectopic bone formation associated with mesenchymal stem cells in a resorbable calcium deficient hydroxyapatite carrier

Bone substitute materials can induce bone formation in combination with mesenchymal stem cells (MSC). The aim of the current study was to examine ectopic in vivo bone formation with and without MSC on a new resorbable ceramic, called calcium deficient hydroxyapatite (CDHA). Ceramic blocks characterized by a large surface (48 m2/g) were compared with beta-tricalcium phosphate (beta-TCP), hydroxyapatite (HA) ceramics (both ca. 0.5 m2/g surface) and demineralized bone matrix (DBM). Before implantation in the back of SCID mice carriers were freshly loaded with 2x10(5) expanded human MSC or loaded with cells and kept under osteogenic conditions for two weeks in vitro. Culture conditions were kept free of xenogenic supplements. Deposits of osteoid at the margins of ceramic pores occurred independent of osteogenic pre-induction, contained human cells, and appeared in 416 MSC/CDHA composites compared to 216 MSC/beta-TCP composites. ALP activity was significantly higher in samples with MSC versus empty controls (p<0.001). Furthermore, ALP was significantly (p<0.05) higher for all ceramics when compared to the DBM matrix. Compared to previous studies, overall bone formation appeared to be reduced possibly due to the strict human protocol. Ectopic bone formation in the novel biomaterial CDHA varied considerably with the cell pool and was at least equal to beta-TCP blocks.     

A de novo mutation affecting human TrkB associated with severe obesity and developmental delay

An 8-year-old male with a complex developmental syndrome and severe obesity was heterozygous for a de novo missense mutation resulting in a Y722C substitution in the neurotrophin receptor TrkB. This mutation markedly impaired receptor autophosphorylation and signaling to MAP kinase. Mutation of NTRK2, which encodes TrkB, seems to result in a unique human syndrome of hyperphagic obesity. The associated impairment in memory, learning and nociception seen in the proband reflects the crucial role of TrkB in the human nervous system.