IFNalpha activates dormant haematopoietic stem cells in vivo.

Maintenance of the blood system is dependent on dormant haematopoietic stem cells (HSCs) with long-term self-renewal capacity. After injury these cells are induced to proliferate to quickly re-establish homeostasis. The signalling molecules promoting the exit of HSCs out of the dormant stage remain largely unknown. Here we show that in response to treatment of mice with interferon-alpha (IFNalpha), HSCs efficiently exit G(0) and enter an active cell cycle. HSCs respond to IFNalpha treatment by the increased phosphorylation of STAT1 and PKB/Akt (also known as AKT1), the expression of IFNalpha target genes, and the upregulation of stem cell antigen-1 (Sca-1, also known as LY6A). HSCs lacking the IFNalpha/beta receptor (IFNAR), STAT1 (ref. 3) or Sca-1 (ref. 4) are insensitive to IFNalpha stimulation, demonstrating that STAT1 and Sca-1 mediate IFNalpha-induced HSC proliferation. Although dormant HSCs are resistant to the anti-proliferative chemotherapeutic agent 5-fluoro-uracil, HSCs pre-treated (primed) with IFNalpha and thus induced to proliferate are efficiently eliminated by 5-fluoro-uracil exposure in vivo. Conversely, HSCs chronically activated by IFNalpha are functionally compromised and are rapidly out-competed by non-activatable Ifnar(-/-) cells in competitive repopulation assays. Whereas chronic activation of the IFNalpha pathway in HSCs impairs their function, acute IFNalpha treatment promotes the proliferation of dormant HSCs in vivo. These data may help to clarify the so far unexplained clinical effects of IFNalpha on leukaemic cells, and raise the possibility for new applications of type I interferons to target cancer stem cells.

hShroom1 links a membrane bound protein to the actin cytoskeleton.

hShroom1 (hShrm1) is a member of the Apx/Shroom (Shrm) protein family and was identified from a yeast two-hybrid screen as a protein that interacts with the cytoplasmic domain of melanoma cell adhesion molecule (MCAM). The characteristic signature of the Shrm family is the presence of a unique domain, ASD2 (Apx/Shroom domain 2). mRNA analysis suggests that hShrm1 is expressed in brain, heart, skeletal muscle, colon, small intestine, kidney, placenta and lung tissue, as well a variety of melanoma and other cell lines. Co-immunoprecipitation and bioluminescence resonance energy transfer (BRET) experiments indicate that hShrm1 and MCAM interact in vivo and by immunofluorescence microscopy some co-localization of these proteins is observed. hShrm1 partly co-localises with beta-actin and is found in the Triton X-100 insoluble fraction of melanoma cell extracts. We propose that hShrm1 is involved in linking MCAM to the cytoskeleton.

Treatment of experimental endocarditis due to erythromycin-susceptible or -resistant methicillin-resistant Staphylococcus aureus with RP 59500.

RP 59500 is a new injectable streptogramin composed of two synergistic components (quinupristin and dalfopristin) which are active against erythromycin-susceptible and -resistant gram-positive pathogens. The present experiments compared the therapeutic efficacy of RP 59500 with that of vancomycin against experimental endocarditis due to either of two erythromycin-susceptible or two constitutively erythromycin-resistant isolates of methicillin-resistant Staphylococcus aureus. RP 59500 had low MICs for the four test organisms as well as for 24 additional isolates (the MIC at which 90% of the isolates were inhibited was < 1 mg/liter) which were mostly inducibly (47%) or constitutively (39%) erythromycin resistant. Aortic endocarditis in rats was produced with catheter-induced vegetations. Three-day therapy was initiated 12 h after infection, and the drugs were delivered via a computerized pump, which permitted the mimicking of the drug kinetics produced in human serum by twice-daily intravenous injections of 7 mg of RP 59500 per kg of body weight or 1 g of vancomycin. Both antibiotics reduced vegetation bacterial titers to below detection levels in ca. 70% of animals infected with the erythromycin-susceptible isolates (P < 0.05 compared with titers in controls). Vancomycin was also effective against the constitutively resistant strains, but RP 59500 failed against these isolates. Further experiments proved that RP 59500 failures were related to the very short life span of dalfopristin in serum (< or = 2 h, compared with > or = 6 h for quinupristin), since successful treatment was restored by artificially prolonging the dalfopristin levels for 6 h. Thus, RP 59500 is a promising alternative to vancomycin against methicillin-resistant S. aureus infections, provided that pharmacokinetic parameters are adjusted to afford prolonged levels of both of its constituents in serum. This observation is also relevant to humans, in whom the life span of dalfopristin in serum is also shorter than that of quinupristin.

Stereospecificity of the siderophore pyochelin outer membrane transporters in fluorescent pseudomonads.

Pyochelin (Pch) and enantio-pyochelin (EPch) are enantiomer siderophores that are produced by Pseudomonas aeruginosa and Pseudomonas fluorescens, respectively, under iron limitation. Pch promotes growth of P. aeruginosa when iron is scarce, and EPch carries out the same biological function in P. fluorescens. However, the two siderophores are unable to promote growth in the heterologous species, indicating that siderophore-mediated iron uptake is highly stereospecific. In the present work, using binding and iron uptake assays, we found that FptA, the Fe-Pch outer membrane transporter of P. aeruginosa, recognized (K(d) = 2.5 +/- 1.1 nm) and transported Fe-Pch but did not interact with Fe-EPch. Likewise, FetA, the Fe-EPch receptor of P. fluorescens, was specific for Fe-EPch (K(d) = 3.7 +/- 2.1 nm) but did not bind and transport Fe-Pch. Growth promotion experiments performed under iron-limiting conditions confirmed that FptA and FetA are highly specific for Pch and EPch, respectively. When fptA and fetA along with adjacent transport genes involved in siderophore uptake were swapped between the two bacterial species, P. aeruginosa became able to utilize Fe-EPch as an iron source, and P. fluorescens was able to grow with Fe-Pch. Docking experiments using the FptA structure and binding assays showed that the stereospecificity of Pch recognition by FptA was mostly due to the configuration of the siderophore chiral centers C4'' and C2'' and was only weakly dependent on the configuration of the C4' carbon atom. Together, these findings increase our understanding of the stereospecific interaction between Pch and its outer membrane receptor FptA.

Genetic deficiency of tartrate-resistant acid phosphatase associated with skeletal dysplasia, cerebral calcifications and autoimmunity.

Vertebral and metaphyseal dysplasia, spasticity with cerebral calcifications, and strong predisposition to autoimmune diseases are the hallmarks of the genetic disorder spondyloenchondrodysplasia. We mapped a locus in five consanguineous families to chromosome 19p13 and identified mutations in ACP5, which encodes tartrate-resistant phosphatase (TRAP), in 14 affected individuals and showed that these mutations abolish enzyme function in the serum and cells of affected individuals. Phosphorylated osteopontin, a protein involved in bone reabsorption and in immune regulation, accumulates in serum, urine and cells cultured from TRAP-deficient individuals. Case-derived dendritic cells exhibit an altered cytokine profile and are more potent than matched control cells in stimulating allogeneic T cell proliferation in mixed lymphocyte reactions. These findings shed new light on the role of osteopontin and its regulation by TRAP in the pathogenesis of common autoimmune disorders.

Secretory component delays the conversion of secretory IgA into antigen-binding competent F(ab')2: a possible implication for mucosal defense.

Secretory component (SC) represents the soluble ectodomain of the polymeric Ig receptor, a membrane protein that transports mucosal Abs across epithelial cells. In the protease-rich environment of the intestine, SC is thought to stabilize the associated IgA by unestablished molecular mechanisms. To address this question, we reconstituted SC-IgA complexes in vitro by incubating dimeric IgA (IgAd) with either recombinant human SC (rSC) or SC isolated from human colostral milk (SCm). Both complexes exhibited an identical degree of covalency when exposed to redox agents, peptidyl disulfide isomerase, and temperature changes. In cross-competition experiments, 50% inhibition of binding to IgAd was achieved at approximately 10 nM SC competitor. Western blot analysis of IgAd digested with intestinal washes indicated that the alpha-chain in IgAd was primarily split into a 40-kDa species, a phenomenon delayed in rSC- or SCm-IgAd complexes. In the same assay, either of the SCs was resistant to degradation only if complexed with IgAd. In contrast, the kappa light chain was not digested at all, suggesting that the F(ab')2 region was left intact. Accordingly, IgAd and SC-IgAd digestion products retained functionality as indicated by Ag reactivity in ELISA. Size exclusion chromatography under native conditions of digested IgAd and rSC-IgAd demonstrates that SC exerts its protective role in secretory IgA by delaying cleavage in the hinge/Fc region of the alpha-chain, not by holding together degraded fragments. The function of integral secretory IgA and F(ab')2 is discussed in terms of mucosal immune defenses.

Use of long-term suppressive acyclovir after hematopoietic stem-cell transplantation: impact on herpes simplex virus (HSV) disease and drug-resistant HSV disease.

The effect that long-term use of suppressive acyclovir (ACV) has on both overall herpes simplex virus (HSV) disease and ACV-resistant HSV disease was examined in 3 consecutive cohorts of hematopoietic stem-cell transplant (HCT) recipients (n=2049); cohort 1 received ACV for 30 days after HCT, cohort 2 received it for 1 year after HCT, and cohort 3 received it for an extended period (i.e., >1 year) if the patient's immunosuppression continued after 1 year. The 2-year probability of HSV disease was 31.6% (95% confidence interval [CI], 28.0%-35%) in cohort 1, 3.9% (95% CI, 2.7%-5.2%) in cohort 2, and 0% in cohort 3 (P<.001). ACV-resistant HSV disease developed in 10 patients in cohort 1 (2-year probability, 1.3% [95% CI, 0.8%-2.7%]), in 2 patients in cohort 2 (2-year probability, 0.2% [95% CI, 0%-0.8%]; P=.006), and in 0 patients in cohort 3 (cohort 2 vs. cohort 3, P=.3). Long-term use of suppressive prophylactic ACV appears to prevent the emergence of drug-resistant HSV disease in HCT.

High activity of Fosfomycin and Rifampin against methicillin-resistant staphylococcus aureus biofilm in vitro and in an experimental foreign-body infection model.

Increasing antimicrobial resistance reduces treatment options for implant-associated infections caused by methicillin-resistant Staphylococcus aureus (MRSA). We evaluated the activity of fosfomycin alone and in combination with vancomycin, daptomycin, rifampin, and tigecycline against MRSA (ATCC 43300) in a foreign-body (implantable cage) infection model. The MICs of the individual agents were as follows: fosfomycin, 1 μg/ml; daptomycin, 0.125 μg/ml; vancomycin, 1 μg/ml; rifampin, 0.04 μg/ml; and tigecycline, 0.125 μg/ml. Microcalorimetry showed synergistic activity of fosfomycin and rifampin at subinhibitory concentrations against planktonic and biofilm MRSA. In time-kill curves, fosfomycin exhibited time-dependent activity against MRSA with a reduction of 2.5 log10 CFU/ml at 128 × the MIC. In the animal model, planktonic bacteria in cage fluid were reduced by <1 log10 CFU/ml with fosfomycin and tigecycline, 1.7 log10 with daptomycin, 2.2 log10 with fosfomycin-tigecycline and fosfomycin-vancomycin, 3.8 log10 with fosfomycin-daptomycin, and >6.0 log10 with daptomycin-rifampin and fosfomycin-rifampin. Daptomycin-rifampin cured 67% of cage-associated infections and fosfomycin-rifampin cured 83%, whereas all single drugs (fosfomycin, daptomycin, and tigecycline) and rifampin-free fosfomycin combinations showed no cure of MRSA cage-associated infections. No emergence of fosfomycin resistance was observed in animals; however, a 4-fold increase in fosfomycin MIC (from 2 to 16 μg/ml) occurred in the fosfomycin-vancomycin group. In summary, the highest eradication of MRSA cage-associated infections was achieved with fosfomycin in combination with rifampin (83%). Fosfomycin may be used in combination with rifampin against MRSA implant-associated infections, but it cannot replace rifampin as an antibiofilm agent.

Characterization of the staphylococcal cassette chromosome mec insertion site in 108 isolates lacking the mecA gene and identified as methicillin-resistant Staphylococcus aureus by the Xpert MRSA assay.

During a 3-year period, 848 patients were detected as carriers of methicillin-resistant Staphylococcus aureus (MRSA) by the Xpert MRSA assay (Cepheid). Among them, 108 patients (12.7 %) were colonized with strains showing methicillin-susceptible phenotypes and absence of the mecA gene, despite being positive with the rapid polymerase chain reaction (PCR) assay. DNA sequences of the staphylococcal cassette chromosome mec (SCCmec) insertion site of these "false-positive" strains was determined by direct sequencing of the genomic DNA. More than half (53.7 %) of the strains had DNA sequences unrelated to either SCC or SCCmec and one-third had DNA sequences related to non-mec SCC. Only 10.2 % of the strains carried sequences related to SCCmec, suggesting that a sequence containing the mecA gene was lost from an SCCmec. These findings differ from the general idea that all methicillin-susceptible S. aureus having positive Xpert MRSA assay results are essentially MRSA that lost the mecA gene.

Salt-sensitive hypertension and cardiac hypertrophy in mice deficient in the ubiquitin ligase Nedd4-2.

Nedd4-2 has been proposed to play a critical role in regulating epithelial Na+ channel (ENaC) activity. Biochemical and overexpression experiments suggest that Nedd4-2 binds to the PY motifs of ENaC subunits via its WW domains, ubiquitinates them, and decreases their expression on the apical membrane. Phosphorylation of Nedd4-2 (for example by Sgk1) may regulate its binding to ENaC, and thus ENaC ubiquitination. These results suggest that the interaction between Nedd4-2 and ENaC may play a crucial role in Na+ homeostasis and blood pressure (BP) regulation. To test these predictions in vivo, we generated Nedd4-2 null mice. The knockout mice had higher BP on a normal diet and a further increase in BP when on a high-salt diet. The hypertension was probably mediated by ENaC overactivity because 1) Nedd4-2 null mice had higher expression levels of all three ENaC subunits in kidney, but not of other Na+ transporters; 2) the downregulation of ENaC function in colon was impaired; and 3) NaCl-sensitive hypertension was substantially reduced in the presence of amiloride, a specific inhibitor of ENaC. Nedd4-2 null mice on a chronic high-salt diet showed cardiac hypertrophy and markedly depressed cardiac function. Overall, our results demonstrate that in vivo Nedd4-2 is a critical regulator of ENaC activity and BP. The absence of this gene is sufficient to produce salt-sensitive hypertension. This model provides an opportunity to further investigate mechanisms and consequences of this common disorder.

Inner-membrane transporters for the siderophores pyochelin in Pseudomonas aeruginosa and enantio-pyochelin in Pseudomonas fluorescens display different enantioselectivities.

Iron uptake and transcriptional regulation by the enantiomeric siderophores pyochelin (Pch) and enantio-pyochelin (EPch) of Pseudomonas aeruginosa and Pseudomonas fluorescens, respectively, are stereospecific processes. The iron-loaded forms of Pch (ferriPch) and of EPch (ferriEPch) are recognized stereospecifically (i) at the outer membrane by the siderophore receptors FptA in P. aeruginosa and FetA in P. fluorescens and (ii) in the cytoplasm by the two AraC-type regulators PchR, which are activated by their cognate siderophore. Here, stereospecific siderophore recognition is shown to occur at the inner membrane also. In P. aeruginosa, translocation of ferriPch across the inner membrane is carried out by the single-subunit siderophore transporter FptX. In contrast, the uptake of ferriEPch into the cytoplasm of P. fluorescens was found to involve a classical periplasmic binding protein-dependent ABC transporter (FetCDE), which is encoded by the fetABCDEF operon. Expression of a translational fetA-gfp fusion was repressed by ferric ions, and activated by the cognate siderophore bound to PchR, thus resembling the analogous regulation of the P. aeruginosa ferriPch transport operon fptABCX. The inner-membrane transporters FetCDE and FptX were expressed in combination with either of the two siderophore receptors FetA and FptA in a siderophore-negative P. aeruginosa mutant deleted for the fptABCX operon. Growth tests conducted under iron limitation with ferriPch or ferriEPch as the iron source revealed that FptX was able to transport ferriPch as well as ferriEPch, whereas FetCDE specifically transported ferriEPch. Thus, stereospecific siderophore recognition occurs at the inner membrane by the FetCDE transporter.

Identification of a mammalian H(+)-myo-inositol symporter expressed predominantly in the brain.

Inositol and its phosphorylated derivatives play a major role in brain function, either as osmolytes, second messengers or regulators of vesicle endo- and exocytosis. Here we describe the identification and functional characterization of a novel H(+)-myo- inositol co-transporter, HMIT, expressed predominantly in the brain. HMIT cDNA encodes a 618 amino acid polypeptide with 12 predicted transmembrane domains. Functional expression of HMIT in Xenopus oocytes showed that transport activity was specific for myo-inositol and related stereoisomers with a Michaelis-Menten constant of approximately 100 microM, and that transport activity was strongly stimulated by decreasing pH. Electrophysiological measurements revealed that transport was electrogenic with a maximal transport activity reached at pH 5.0. In rat brain membrane preparations, HMIT appeared as a 75-90 kDa protein that could be converted to a 67 kDa band upon enzymatic deglycosylation. Immunofluorescence microscopy analysis showed HMIT expression in glial cells and some neurons. These data provide the first characterization of a mammalian H(+)-coupled myo- inositol transporter. Predominant central expression of HMIT suggests that it has a key role in the control of myo-inositol brain metabolism.

Infrequent transmission of HIV-1 drug-resistant variants.

Transmission of drug-resistant variants is influenced by several factors, including the prevalence of drug resistance in the population of HIV-1-infected patients, HIV-1 RNA levels and transmission by recently infected patients. In order to evaluate the impact of these factors on the transmission of drug-resistant variants, we have defined the population of potential transmitters and compared their resistance profiles to those of newly infected patients. Sequencing of pol gene was performed in 220 recently infected patients and in 373 chronically infected patients with HIV-1 RNA >1000 copies/ml. Minimal and maximal drug-resistance profiles of potential transmitters were estimated by weighting resistance profiles of chronically infected patients with estimates of the Swiss HIV-1-infected population, the prevalence of exposure to antiviral drugs and the proportion of infections attributed to primary HIV infections. The drug-resistance prevalence in recently infected patients was 10.5% (one class drug resistance: 9.1%; two classes: 1.4%; three classes: 0%). Phylogenetic analysis revealed significant clustering for 30% of recent infections. The drug-resistance prevalence in chronically infected patients was 72.4% (one class: 29%; two classes: 27.6%; three classes: 15.8%). After adjustment, the risk of transmission relative to wild-type was reduced both for one class drug resistance (minimal and maximal estimates: odds ratio: 0.39, P<0.001; and odds ratio: 0.55, P=0.011, respectively), and for two to three class drug resistance (odds ratios: 0.05 and 0.07, respectively, P<0.001). Neither sexual behaviour nor HIV-1 RNA levels explained the low transmission of drug-resistant variants. These data suggest that drug-resistant variants and in particular multidrug-resistant variants have a substantially reduced transmission capacity.

Plasma membrane H+-ATPase regulation is required for auxin gradient formation preceding phototropic growth.

Phototropism is a growth response allowing plants to align their photosynthetic organs toward incoming light and thereby to optimize photosynthetic activity. Formation of a lateral gradient of the phytohormone auxin is a key step to trigger asymmetric growth of the shoot leading to phototropic reorientation. To identify important regulators of auxin gradient formation, we developed an auxin flux model that enabled us to test in silico the impact of different morphological and biophysical parameters on gradient formation, including the contribution of the extracellular space (cell wall) or apoplast. Our model indicates that cell size, cell distributions, and apoplast thickness are all important factors affecting gradient formation. Among all tested variables, regulation of apoplastic pH was the most important to enable the formation of a lateral auxin gradient. To test this prediction, we interfered with the activity of plasma membrane H(+)-ATPases that are required to control apoplastic pH. Our results show that H(+)-ATPases are indeed important for the establishment of a lateral auxin gradient and phototropism. Moreover, we show that during phototropism, H(+)-ATPase activity is regulated by the phototropin photoreceptors, providing a mechanism by which light influences apoplastic pH.

Discordant increases in CD4+ T cells in human immunodeficiency virus-infected patients experiencing virologic treatment failure: role of changes in thymic output and T cell death.

Some patients infected with human immunodeficiency virus (HIV) who are experiencing antiretroviral treatment failure have persistent improvement in CD4+ T cell counts despite high plasma viremia. To explore the mechanisms responsible for this phenomenon, 2 parameters influencing the dynamics of CD4+ T cells were evaluated: death of mature CD4+ T cells and replenishment of the CD4+ T cell pool by the thymus. The improvement in CD4+ T cells observed in patients with treatment failure was not correlated with spontaneous, Fas ligand-induced, or activation-induced T cell death. In contrast, a significant correlation between the improvement in CD4+ T cell counts and thymic output, as assessed by measurement of T cell receptor excision circles, was observed. These observations suggest that increased thymic output contributes to the dissociation between CD4+ T cell counts and viremia in patients failing antiretroviral therapy and support a model in which drug-resistant HIV strains may have reduced replication rates and pathogenicity in the thymus.