Repairs of bone defects caused by osteoporosis have always relied on bone tissue engineering. However, the preparation of composite tissue engineering scaffolds with a three-dimensional (3D) macroporous structure poses huge challenges in achieving osteoconduction and osteoinduction for repairing bone defects caused by osteoporosis. In the current study, a three-dimensional macroporous (150–300 μm) reduced graphene oxide/polypyrrole composite scaffold modified by strontium (Sr) (3D rGO/PPY/Sr) was successfully prepared using the oxygen plasma technology-assisted method, which is simple, safe, and inexpensive. The findings of the MTT assay and AO/EB fluorescence double staining showed that 3D rGO/PPY/Sr has a good biocompatibility and effectively promoted MC3T3-E1 cell proliferation. Furthermore, the ALP assay and alizarin red staining showed that 3D rGO/PPY/Sr increased the expression levels of ALP activity and the formation of calcified nodules. The desirable biocompatibility, osteoconduction, and osteoinduction abilities, assure that the 3D macroporous rGO/PPY/Sr composite scaffold offers promising potential for use in the repair of bone defects caused by osteoporosis in bone tissue engineering. Previous studies have reported a gradual increase in the incidence of bone defects caused by osteoporosis due to the aging of the population. More than 200 million people are estimated to suffer from osteoporosis worldwide . FurtherStrontium (Sr), an essential trace element, has been shown to effectively stimulate bone formation and inhibit osteoclast activity ,11,12. PGraphene exhibits several unique properties, including high electron mobility at room temperature, exceptional thermal conductivity, the highest theoretical specific surface area, and superior mechanical properties with a Young’s modulus of 1 TPa. These properties make graphene an ideal material for several applications. In addition, graphene is the material of choice for future applications in the electronics and composites industry ,20. FurtPlasma etching is the most common approach used in semiconductor processing for the fabrication of electronic devices. Plasma etching with oxygen is a particularly common tool used to modify the surface properties of materials, such as surface energy, morphology, and resistivity . In addiIn the current study, oxygen plasma technology was used to fabricate Sr-modified 3D rGO/PPY composite scaffolds. In addition, the morphology, structure, and chemical composition of Sr-modified 3D rGO/PPY composite scaffolds (3D rGO/PPY/Sr) were characterized by SEM, EDS, FTIR, and XRD. Biocompatibility was evaluated by MTT and acridine orange/ethidium bromide (AO/EB) double staining assays in vitro. Osteogenic activity was evaluated by alkaline phosphatase (ALP) activity assays and alizarin red staining experiments in vitro, respectively.Scaffold material for tissue engineering with regular, three-dimensional macroporous structure morphology is beneficial for osteogenesis. The SEM results of the 3D rGO/PPY composite scaffold are shown in −1, 1543 cm−1 and 1032 cm−1 indicate that -OH, C=C, and C-O bonds. Vibrational stretching at 2815 cm−1 indicates the C–H stretching of the aliphatic alkyl group. These are attributed to graphene oxide [−1 indicated the presence of the carboxylate group. The peaks at 894cm−1, 660 cm−1 and 606 cm−1 corresponding to the carbonates (CO3−2) of the reacted SrCO3 were observed [The chemical construction of the scaffolds was characterized by XRD and FTIR. As shown in ne oxide . The vibobserved ,38. It wobserved .p < 0.05). Moreover, the OD value of the 3D rGO/PPY/Sr scaffold group was significantly higher compared with that of the 3D rGO/PPY group at the same co-culture time point (p < 0.05). The findings of the MTT assay also show that both the 3D rGO/PPY and 3D rGO/PPY/Sr composite scaffolds were not cytotoxic and could promote the proliferation of MC3T3-E1 cells. Notably, the 3D rGO/PPY/Sr composite scaffold had a stronger capacity to promote MC3T3-E1 cell proliferation compared with the 3D rGO/PPY scaffold. This finding might be related to the modification of Sr, which was also reported in previous studies, an indication that Sr promoted MC3T3-E1 cell proliferation [The cytotoxicity of the 3D rGO/PPY and 3D rGO/PPY/Sr composite scaffolds were evaluated using MC3T3-E1 cells with MTT assays at 24, 48, and 96 h . MTT assferation .The apoptosis, adhesion, and proliferation of MC3T3-E1 cells on 3D rGO/PPY and 3D rGO/PPY/Sr composite scaffolds were further evaluated using the AO/EB double staining experiment. Representative live/dead fluorescent images showed that almost all MC3T3-E1 cells, both on the backbone of the 3D rGO/PPY and 3D rGO/PPY/Sr composite scaffolds, glowed green throughout the culture cycle and no red-stained cells were detected . This fip > 0.05) (p < 0.05). Furthermore, the findings of the alizarin red staining experiment showed that areas of red dye and numbers of calcified nodules gradually increased both in the 3D rGO/PPY and 3D rGO/PPY/Sr group as the co-culture time gradually increased  . Howeverncreased . In cont2+ can activate calcium sensing receptors (CaR) and increase osteoprotegerin (OPG) production [The findings of the current study established that the 3D rGO/PPY composite scaffold modified by Sr promotes the expression of ALP activity levels and the formation of calcified nodules. In addition, the current study findings showed that 3D rGO/PPY modified by Sr can potentially improve the osteogenic properties of biomaterials, which may be attributed to the possibility that Sroduction ,43.SEM images indicated that MC3T3-E1 had a good morphology and spreading on scaffolds . After 4The findings of the current study indicated that the 3D rGO/PPY/Sr scaffold was loaded with a compression strain up to 90% a; the st2+ gradually released from the 3D rGO/PPY/Sr scaffold to almost constant and slow release after 24 h. This is preferable for bone regeneration, as the scaffold can maintain the moderate concentration of Sr2+ ions for a long time. The 0.0015g 3D rGO/PPY/Sr scaffolds were immersed in 5 mL of PBS at 37 °C, and PBS was collected after 1, 2, 4, 8, 12, 24, 48, 72, 96, and 120 h, respectively. PBS containing released Sr was analyzed using ICP−OES. As shown in −1. The release property of Sr ions from the scaffolds was analyzed using inductively coupled plasma optical emission spectrometry .The 3D rGO/PPY scaffold was prepared based on the previously reported method . Three-dOsteoblast-like MC3T3-E1 cells  were cultured in Dulbecco′s Modified Eagle Medium  containing 10% fetal bovine serum  at 37 °C, with the medium being changed every two days. The cytotoxicity of the 3D rGO/PPY and 3D rGO/PPY/Sr composite scaffolds were examined using a 3--2,5-diphenyl-2-H-tetrazolium bromide  assay, and the absorbance of the solution in each well was determined at a wavelength of 490 nm using a microplate reader .6 cell/mL concentration in a 24-well plate incubated with the 3D rGO/PPY and 3D rGO/PPY/Sr composite scaffolds. After 24, 48, and 96 h, all cultured scaffolds were harvested, washed thrice with PBS, and dehydrated with 4% paraformaldehyde. The scaffolds were then stained with AO/EB solution (1:1 mixture of 1 mg/mL ethidium bromide (EB) and 1 mg/mL acridine orange (AO)) in the dark for 3 min. Cellular morphology was evaluated using a fluorescence microscope .Reagents AO and EB were purchased from Sigma . Living and apoptotic cells were determined morphologically after AO/EB staining, followed by fluorescence microscopy inspection. Briefly, MC3T3-E1 cells were seeded at a density of 104 cell/mL concentration. After co-culturing for 7 and 14 days, scaffolds were washed thrice with PBS and lysed with 0.01% Triton-X 100 to dissolve cells. The mixture was centrifuged at 2000 rpm for 15 min. The supernatant was transferred to an EP tube and the ALP working solution was added. After incubation at 37 °C for 15 min, 1 M NaOH was added to terminate the reaction. Absorbance at 520 nm was determined using a microplate absorbance reader (Bio-Rad iMark). All experiments were undertaken in triplicate.The differentiation of MC3T3-E1 cells was determined by ALP activity, which was assayed using an Alkaline Phosphatase Assay Kit  based on the manufacturer’s protocol. Briefly, MC3T3-E1 cells were seeded onto samples in 6-well plates at a density of 104 cell/mL concentration. After 3 days, the cultured solution was replaced with the osteogenic medium . Cells were rinsed twice with PBS after co-culturing for 7, 14, and 21 days and fixed in 4% paraformaldehyde for 10 to 20 min at room temperature. Cells were stained with alizarin red for 30 min and washed thrice with PBS. The formation of calcium nodules was observed using an inverted microscope.The ECM mineralization of MC3T3-E1 co-cultured with the 3D rGO/PPY and 3D rGO/PPY/Sr composite scaffolds were tested through alizarin red staining. MC3T3-E1 cells were seeded onto samples in 6-well plates at a density of 10MC3T3-E1 were cultured, grown on scaffolds for 24 and 48 h, and fixed  for 15 min. Samples were then rinsed with PBS and dehydrated, followed by drying for 1 min and gold sputtering, before observation using a scanning electron microscope p < 0.05 were considered statistically significant.Statistical analyses were performed using SPSS version 12.0. One-way analysis of variance (ANOVA) with Bonferroni post hoc tests was undertaken to examine differences among groups. Values of The current study developed a simple and effective modification method to fabricate a 3D rGO/PPY/Sr scaffold using oxygen plasma-assisted strategy. The 3D rGO/PPY/Sr scaffold showed an outstanding biocompatibility and a prominent ability to promote the proliferation of MC3T3-E1 cells. By co-culturing with MC3T3-E1 cells, the 3D rGO/PPY/Sr scaffold showed higher levels of ALP activity and an ability to promote the formation of calcified nodules. To the best of our knowledge, 3D rGO/PPY/Sr composite scaffolds are potential materials for the repair of bone defects caused by osteoporosis in the future.


In this study, we evaluated the prognostic value of tumour markers for predicting response , Changes in cancer antigen 15–3 (CA 15-3) and carcinoembryonic antigen (CEA) were prospectively monitored (monthly) and were also evaluated for the 3 months preceding secondary PD. Data from 67 patients with previously treated MBC participating in a Compassionate Use Programme were analysed.n = 7 [10.4%]), a non-significant increase in CA 15-3 occurred during the first 6 months of treatment; CEA was significantly reduced (P = 0.0165). In patients with SD ≥ 6 months (n = 28 [41.8%]), both CA 15-3 (P < 0.0001) and CEA (P = 0.0399) levels increased significantly after 6 months treatment. In those experiencing de novo PD (n = 32 [47.8%]), CA 15-3 increased significantly (P < 0.0001) after 4 months; CEA also increased significantly (P = 0.0002) during the same time period. Both CA 15-3 (P < 0.0001) and CEA (P < 0.0001) increased significantly in the 3 months preceding secondary PD.In patients with a PR (CA 15-3 increases in patients progressing on fulvestrant but may also increase in those experiencing clinical benefit; this should not be taken as a sign of PD without verification. Overall, both CA 15-3 and CEA appear to be poor prognostic markers for determining progression in patients receiving fulvestrant. Tumour markers are often used to detect disease recurrence after primary treatment for cancer ,2 and tode novo disease progression (PD), thus signalling that a change in therapy may be necessary  and SD ≥ 9 months [n = 14]). Between baseline and 4 months a significant increase in CA 15-3 levels was observed both in the SD ≥ 6 but < 9 months (P = 0.0017) and the SD ≥ 9 months (P = 0.0405) subgroups, both of which were maintained at 6 months. In the group of patients experiencing SD ≥ 6 months overall, CEA levels also increased significantly between baseline and 6 months (P = 0.0399).In the 28 patients with SD ≥ 6 months CA 15–3 serial levels increased significantly between baseline and 4 months (de novo PD a significant increase (P < 0.0001) in CA 15-3 levels was noted between baseline and 4 months (Table P = 0.0002) over the same period.In the 32 patients experiencing n = 28), a significant increase in CA 15-3 levels was observed during the last 3 months of treatment (P < 0.0001)  during this time.In patients experiencing CB followed by secondary progression The author(s) declare that they have no competing interests.RB contributed the idea for this trial, participated in the design and drafted the manuscript, CW participated in the statistical analysis and helped drafting the manuscript, UP participated in the design of the study, DH and US both participated in the collection of patient data, GA helped in the design and coordination of the study, GL revised the manuscript critically, RM assisted in the statistical analysis, CZ and GS were important in the design of the study, the coordination and helped to draft the manuscript.All authors have read and approved the final manuscript.The pre-publication history for this paper can be accessed here:


APOBEC3G  has antiretroviral activity associated with the hypermutation of viral DNA through cytosine deamination. APOBEC3G has two cytosine deaminase (CDA) domains; the catalytically inactive amino-terminal domain of APOBEC3G (N-CDA) carries the Vif interaction domain. There is no 3-D structure of APOBEC3G solved by X-ray or nuclear magnetic resonance.We predicted the structure of human APOBEC3G based on the crystal structure of APOBEC2. To assess the model structure, we evaluated 48 mutants of APOBEC3G N-CDA that identify novel variants altering ΔVif HIV-1 infectivity and packaging of APOBEC3G. Results indicated that the key residue D128 is exposed at the surface of the model, with a negative local electrostatic potential. Mutation D128K changes the sign of that local potential. In addition, two novel functionally relevant residues that result in defective APOBEC3G encapsidation, R122 and W127, cluster at the surface.The structure model identifies a cluster of residues important for packaging of APOBEC3G into virions, and may serve to guide functional analysis of APOBEC3G. Primate APOBEC3G has antiretroviral activity associated with the hypermutation of viral DNA through cytosine deamination (for recent review see There is no 3-D structure solved by X-ray or NMR nor an accurate model of APOBEC3G available. APOBEC3G relates to APOBEC family and AID (activation-induced deaminase) at the sequence level. Recent comparative modeling work for APOBEC-1 and AID In the present work, we model human APOBEC3G, with particular emphasis on the N-terminal domain. Using mutant data of the N-CDA, we mapped critical residues for packaging of APOBEC3G into viral particles on the new structure model of the huAPOBEC3G N-CDA. We completed the analysis by mapping N-CDA residues that are under positive evolutionary pressure in primate APOBEC3G.While this work was concluded, Huthoff and Malim provided a detailed molecular genetic analysis of the N-CDA region spanning amino acids residues 119 to 146 Ref . This anhttp://www.ncbi.nih.gov/) was defined as target sequence. The newly crystallized Human APOBEC2 dimer http://www.rcsb.org/pdb/Welcome.do) served as template.The huAPOBEC3G sequence  http://bioweb.pasteur.fr/seqanal/interfaces/dssp-simple.html) http://bioinf.cs.ucl.ac.uk/psipred/) http://www.jalview.org/) We used the align2d function of MODELLER program (http://swissmodel.expasy.org/anolea/) The target-template alignment was used to build the model by satisfaction of spatial restraints. The ANOLEA program  Generation of 100 models allowed selection of the best model candidate based on the global ANOLEA score. Final refinement for alignment of gap regions was performed by generating 100 additional models, followed by selection of the best model on the basis of the ANOLEA score. The final model was energy-minimized using the CHARMM program (http://foldx.embl.de/) http://www.cgl.ucsf.edu/chimera/) The CHARMM program was used to calculate the solvent accessible surface area of the final model. Additionally, using the FoldX program (The plasmid expressing a hemagglutinin (HA)-tagged form of APOBEC3G was a kind gift from M. Malim. A series of APOBEC3G alanine and specific mutants was constructed with the QuickChange Mutagenesis kit (Stratagene). The collection was already constituted, and it was not defined on the basis of the new structural data. HIV-1 particles were produced by transient transfection of 293T cells with Fugene (Roche) of a wild-type or of a Vif-defective HIV-1 proviral clone. Viral titers were determined in single-round infectivity assays by applying filtered supernatant from producer cells on HeLa-CD4-LTRLacZ indicator cells. Virion infectivity was derived by dividing the infectious titer by the amount of physical particles.HIV-1 particles were produced by transient transfection of 293T cells with Fugene (Roche). 1 ml of virus was then spun in Eppendorf tubes at 13'000rpm in a microfuge at 4° for 90 minutes, without sucrose cushion. Pellets were resuspended in PBS 1% Triton, and the virion amount was measured by a standard RT assay. Normalized amount of virions were then loaded on standard Laemmli protein gels to perform Western Blots. Cell extracts were obtained through a standard RIPA extraction procedure. The HA tag was detected with the mouse hrp-coupled anti-HA 3F10 antibody (Roche). PCNA (proliferating cell nuclear antigen) was detected with the mouse Ab-1 antibody (Oncogene Science). The HIV-1 capsid was detected with the murine anti-p24 antibody produced from the AIDS Research and Reference Reagent Program #183-H12-5C.2+2+ with E60, a residue not present at the corresponding position in huAPOBEC3G N-CDA. The corresponding region of huAPOBEC3G C-CDA was modeled ab initio. The target-template alignment generated by MODELLER agreed with the secondary structure alignment  residues of the huAPOBEC3G N-CDA Amino acids substitutions at positions Y13, F17, F21, R29, T32, Y37, K40, S45, L49, L62, E85, Y86, W90, I92, S93, P96, M104, F107, L108, L116, T117, I118, L123, Y125, F126, D130, Y131, E133, L135, L138, M152, C160 and F164 did not result in changes in anti-viral activity. Mutations S28A, F74L, W94L, Y124A, Y154A resulted in reduced inhibition of ΔVif HIV1, while mutations R122A and W127L completely abolished this activity Table 1.Prior evolutionary analysis of primate APOBEC3G identified a number of residues under diversifying (positive) selection α4-β5-α5-α6 configuration, while CDA structures such as human cytidine deaminase have the pattern α1-β1-β2-α2-β3-α3-β4-β5-α4-α5. In addition, the direction of β4 and β5 are different: parallel in APOBEC2 but anti-parallel in cytidine deaminase. We had previously used the yeast Cytosine deaminase  to address the position of the extra helix and the correct direction of β4 and β5.This work presents a model structure of huAPOBEC3G that captures information from the recently published APOBEC2 structure vif due to lack of packaging of APOBEC3G into viral particles. Interaction of APOBEC3G with the NC-domain of HIV-1 Gag and non-specific RNA binding leads to its encapsidation into progeny virions The model emerging from this analysis allows speculation on various functionally relevant structural details of interest for the understanding of the Vif-APOBEC3G interaction and the process of APOBEC3G encapsidation into HIV-1 virions. In contrast with previous secondary and tertiary structure models, the current model locates the distinctive extra alpha helix on the same planar surface as the pseudo-active site. We evaluated extensive mutation data on this surface, that characterized two functionally relevant residues R122 and W127 that resulted in failure to inhibit infection by HIV-1/ΔOur results are consistent with the work of Huthoff and Malim Inspection of surface modifications conferred by various mutations highlights the structural and/or charge differences in this region as the molecular basis for disruption of the APOBEC3G packaging into HIV-1 virions, and modification in the interaction with Vif. The detailed model structure presented here could serve to advance rational drug design. The quality of a homology model is strongly related to the sequence identity with the structural template. The current model, based on a sequence identity of 27%, should be satisfactory in its global fold, as supported by the good ANOLEA energy score profile  monomer of APOBEC2, and the C-CDA to the middle (M) monomer. Panel B, ANOLEA scores. The three ANOLEA profiles and the secondary structure were aligned according to the target-template alignment. The ANOLEA profile excludes the uncharacterized linker (residues 195-214)(2.86 MB EPS)Click here for additional data file.


Since respiratory epithelial cells are the primary target cell for replication of influenza viruses, it is pertinent to investigate the cytokine induction profile of H5N1 viruses in these cells.Fatal human respiratory disease associated with influenza A subtype H5N1 has been documented in Hong Kong, and more recently in Vietnam, Thailand and Cambodia. We previously demonstrated that patients with H5N1 disease had unusually high serum levels of IP-10 (interferon-gamma-inducible protein-10). Furthermore, when compared with human influenza virus subtype H1N1, the H5N1 viruses in 1997 (A/Hong Kong/483/97) (H5N1/97) were more potent inducers of pro-inflammatory cytokines (e.g. tumor necrosis factor-a) and chemokines (e.g. IP-10) from primary human macrophages in vitro.We used quantitative RT-PCR and ELISA to compare the profile of cytokine and chemokine gene expression induced by H5N1 viruses A/HK/483/97 (H5N1/97), A/Vietnam/1194/04 and A/Vietnam/3046/04 (both H5N1/04) with that of human H1N1 virus in human primary alveolar and bronchial epithelial cells in vitro. Recent H5N1 viruses from Vietnam (H5N1/04) appeared to be even more potent at inducing IP-10 than H5N1/97 virus.We demonstrated that in comparison to human H1N1 viruses, H5N1/97 and H5N1/04 viruses were more potent inducers of IP-10, interferon beta, RANTES  and interleukin 6 (IL-6) in primary human alveolar and bronchial epithelial cells The H5N1/97 and H5N1/04 subtype influenza A viruses are more potent inducers of proinflammatory cytokines and chemokines in primary human respiratory epithelial cells than subtype H1N1 virus. We suggest that this hyper-induction of cytokines may be relevant to the pathogenesis of human H5N1 disease. Influenza pandemics arise from genetic reassortment between avian and human influenza viruses or alternatively by the direct adaptation of a avian influenza viruses to efficient human-to-human transmission . Avian iWhile dissemination outside the respiratory tract was not demonstrated in human H5N1 disease in 1997 and 2003 ,7, therein vitro [Influenza virus infection of blood-monocyte-derived murine and human ,13 macroin vitro . We alsoin vitro . We havein vitro ,12.in vivo [While macrophages are a key sentinel cell of the immune system and are permissive to influenza virus replication, the primary target cell for the virus are respiratory epithelial cells . In primin vivo . Expressin vivo ,21. The in vitro from the transformed bronchial epithelial cell line (NCI-H292) [in vitro to test the hypothesis that H5N1/97 and H5N1/04 viruses differentially hyper-induce pro-inflammatory cytokines in respiratory epithelial cells.Human influenza A viruses have been previously reported to induce interleukin 6 (IL-6), interleukin 8 (IL-8) and RANTES  CI-H292) . HoweverCI-H292) . Here, w50) in MDCK cells. The H5N1 influenza viruses used in this study were handled in a BL3 biocontainment facility.An influenza virus isolated from a patient with fatal influenza A H5N1 disease in Hong Kong in 1997, A/Hong Kong/483/97 (H5N1/97), viruses from patients with H5N1 disease in Vietnam in 2004, A/Vietnam/1194/04 and A/Vietnam/3046/04 (both abbreviated as H5N1/04) and a human H1N1 virus A/Hong Kong/54/98 (H1N1) were studied. Viruses were initially isolated in Madin-Darby canine kidney (MDCK) cells. They were cloned by limiting dilution, and seed virus stocks were prepared in MDCK cells. Virus infectivity was assessed by titration of tissue culture infection dose 50%  under liquid-covered conditions.Primary human bronchial epithelial cells (NHBE) were obtained from Cambrex Bio Science . NHBE cells were grown according to the suppliers instructions in serum-free and hormone supplemented bronchial epithelial growth media (BEGM) which included supplements of 13 g/l bovine pituitary extract, 0.5 g/l hydrocortisone, 0.5 mg/l human recombinant epidermal growth factor, 0.5 g/l epinephrine, 10 g/l transferrin, 5 g/l insulin, 0.1 mg/l retinoic acid, 6.5 mg/l 3,3',5-triiodo-L-thryonine, 50 g/l gentamicin, and 50 mg/l amphotericin B . Medium was changed daily starting from the day after seeding. Cells reached confluency in approximately 9 to 10 days, and nearly confluent cells were subcultured using trypsin/EDTA (Cambrex) at a ratio of 1:5. Experiments were carried out on the same batch of cells at passage 3 to 4. The cells were incubated in a humidified atmosphere  for 30 min at 37°C three times to partially remove macrophages and blood cells. The tissue was digested using a combination of trypsin  and elastase  twice for 15 min at 37°C in a shaking water-bath. The partially digested tissue was minced in the presence of 40% fetal bovine serum (FBS) in DMEM/F12 medium and DNase I (350 units/ml) , and cell clumps dispersed by repeatedly pipetting the cell suspension for 10 minutes. After filtration through gauze and a 40 μm cell strainer to ensure a single cell suspension, the cells were incubated with a 1:1 mixture of DMEM/F12 medium and small airway growth medium  containing 5% FBS and 350 units/ml DNase I, on tissue-culture treated plastic Petri dishes in a humidified incubator  for 2 hours in order to let macrophage attach on the plastic surface. The non-adherent cells were layered on a discontinuous Percoll density gradient (densities 1.089 and 1.040 g/ml) and centrifuged at 25 × g for 20 min. The cell layer at the interface of the two gradients was collected and washed four times with BSS to remove the Percoll. To remove remaining alveolar macrophages, the cell suspension was incubated with magnetic beads coated with anti-CD-14 antibodies at room temperature for 20 min under constant mixing. After the removal of the beads using a magnet and assessment of cell viability by trypan-blue exclusion, the purified type II pneumocyte suspension was suspended in SAGM supplemented with 1% FBS, 100 units/ml penicillin and 100 μg/ml streptomycin, and plated at a cell density of 300,000 cells/cm2. The cells were maintained in a humidified atmosphere  under liquid-covered conditions, and growth medium was changed daily starting from 60 hours after plating the cells.Primary human alveolar epithelial cells (type II pneumocytes) were isolated from human non-tumor lung tissue obtained from 13 patients  undergoing lung resection in Grantham Hospital, Hong Kong. The research protocol was approved by the ethics committee of the University of Hong Kong and Hospital Authority Hong Kong West Cluster. Human type II pneumocytes were isolated using a modification of the methods previously described ,23. BrieHuman type II pneumocytes were identified by staining for alkaline phosphatase. Freshly isolated cells were spun down on glass slides, air-dried, and stained for 20 min at room temperature. The stain was prepared by dissolving 10 mg naphthol AS bi-phosphate (Sigma) in 40 μl DMSO and was diluted in 10 ml of 0.125 M 2-amino-2-methyl propanol buffer  containing 10 mg fast red (Sigma). The slide was washed and counterstained in 1% methylene green (Sigma) for 30 seconds and was mounted in aqueous medium .Cells were fixed in 2% glutaraldehyde , washed three times in phosphate buffered saline and serially dehydrated in acetone. The tissue was post-fixed in 1% osmium tetroxide and embedded in an Araldite resin . Semi-thin sections (1 μm) were cut using an ultra-microtome  with a diamond knife and were stained with toluidine blue for light microscopic examination. Ultra-thin sections (80 nm) mounted on copper grids were electron contrasted with uranyl acetate  and lead citrate , and were examined with a transmission electron microscope .The expression of cell surface antigen was measured by staining purified type II pneumocytes with optimal dilution of rabbit anti-human surfactant protein-C (SP-C)  monoclonal antibodies  followed by a fluorescein isothiocyanate . Each cell preparation was also stained with antibody specific for monocyte/macrophage surface antigen . The cells were examined by the flow cytometry , and the FITC-stained cells were detected by measuring green light emitted at 530 nm (FL1 channel). The percentage of cells expressing the epithelial and macrophage makers were determined.6 cells per well in 24-well tissue-culture plates) were infected at a multiplicity of infection (MOI) of two unless otherwise indicated. After 60 min of virus adsorption, the virus inoculum was removed, and the cells were washed with warm culture medium  and incubated in medium supplemented with 0.6 mg/L penicillin, 60 mg/L streptomycin, and 2 mg/L N-p-tosyl-L-phenylalanine chloromethyl ketone-treated-trypsin . Aliquots of culture supernatant were collected and frozen at -80°C for subsequent virus titration and cytokine analysis. The supernatants were titrated on MDCK cells and the viral titre was quantitated as log10TCID50/ml. RNA was extracted from cells for analysis of cytokine gene expression. Ten hours after infection, replicate cell monolayers were fixed and analyzed by immuno-fluorescent staining specific for influenza virus nucleoprotein  to determine the proportion of cells infected.Human type II pneumocytes and bronchial epithelial cells . The cDNA was synthesized from mRNA with poly(dT) primers and Superscript II reverse transcriptase  and quantified by real-time PCR analysis with a LightCycler . The mRNA for IP-10, interferon beta, IL-6, RANTES and tumor necrosis factor (TNF) alpha were quantitated using real-time RT-PCR. The oligonucleotide primers and methods used for real-time quantification of cytokines, viral matrix gene and the housekeeping gene product γ-actin mRNA have been described previously ,24.The concentrations of IP-10, RANTES, interleukin 6 and interferon beta proteins in the primary human bronchial and alveolar epithelial cell supernatants were measured by a specific ELISA assay . Samples of culture supernatant were irradiated with ultraviolet light (CL-100 Ultra Violet Cross linker) for 15 min to inactivate any infectious virus before the ELISA assays were done. Previous experiments had confirmed that the dose of ultraviolet light used did not affect cytokine concentration as measured by ELISA (data not shown).p < 0.05.The quantitative cytokine and chemokine mRNA and protein expression profile were compared using one-way ANOVA, followed by Bonferroni multiple-comparison test. Differences were considered significant at 6 cells/g lung tissue at 92 ± 5% cell purity as demonstrated by the expression of the type II pneumocyte specific marker surfactant protein C (SP-C), lack of the monocyte/macrophage cell surface antigen (CD14)  Fig.  and 1B, in vitro. The cells were infected with influenza A subtypes H5N1  and H1N1 (54/98) at a MOI of 2 and the proportion of cells expressing influenza A virus protein was analyzed at 10 hours post-infection by immunofluorescent staining using an antibody specific for the virus nucleoprotein . Similar proportions (93–100%) of type II pneumocytes infected with H5N1 and H1N1 virus had evidence of viral antigen (nucleoprotein)  Figure . The qua) Figure . Similar) Figure .We investigated the cytokine induction profile induced by H1N1 and H5N1 viruses in primary human type II pneumocytes. Specifically, we also wanted to determine if the two viruses differed qualitatively or quantitatively in the profile of cytokines induced. The mRNA of several cytokines and chemokines were quantified using quantitative RT-PCR at 3 hr and 6 hr post-infection (Table p < 0.001)  . None of the viruses induced TNF alpha in these cells.The cytokine and chemokine profiles induced by H1N1, H5N1/97 and H5N1/04 viruses in primary human bronchial epithelial cells were similarly investigated. The identity of the cells in culture as human bronchial epithelial cells was confirmed by thin section electron microscopy Figure . The ovep < 0.01) than that secreted by cells infected with H1N1 virus    Figure . RANTES We found that the replication efficiency of the H5N1 and H1N1 viruses was similar in both primary human alveolar Figure  and bronThis differential hyper-induction of cytokines was not explained by differences in the replication kinetics between the two virus subtypes. H5N1 viruses isolated from patients with H5N1 disease in Hong Kong in 1997, Vietnam in 2004 and human influenza viruses of the H1N1 subtype all replicate with similar efficiency. Increase in the MOI of the H1N1 virus did not result in an increase of cytokine responses to levels comparable to that of the H5N1 viruses. The cellular mechanisms underlying this differential cytokine hyper-induction by H5N1 viruses are presently poorly understood. Studies on the transformed bronchial epithelial cell line A549 previously demonstrated that toll-like receptor 3 (TLR-3) is involved in the influenza virus A initiated cytokine responses . It remain vivo result from autocrine and paracrine interactions involving many cell types. Chemokines such as IP-10 and MCP-1 are macrophage chemo-attractants and mediate the inflammatory response by further recruitment of circulating leukocytes into the inflamed tissue. We have previously demonstrated that IP-10 and MCP-1 are up-regulated in primary human macrophage by SARS-CoV [Cytokine and chemokine responses SARS-CoV . The strSARS-CoV  as well SARS-CoV .RANTES attracts monocytes, eosinophils, basophils and T cells, and selectively CD4+ T cells. Its production from the bronchial epithelial cells contributes to the infiltration of the inflammatory cells in airway viral infection . IL-6 isin vivo data that parallels our present findings in vitro. Studies on recombinant viruses bearing the HA and NA of the 1918 "Spanish flu" pandemic virus showed that these viruses have enhanced virulence for mice and induce higher levels of macrophage-derived chemokines in vivo in mice [in vivo may simply reflect more extensive replication of the respective virus. The studies in vitro with H5N1 viruses exclude such potential confounding factors and it would be relevant to study the cytokine profiles of the 1918 recombinant viruses in in vitro models similar to those described here.The H5N1 viruses have continued to reassort, acquiring different internal genes from other influenza viruses of avian origin ,34. The  in mice . Howeverin vitro. Previous findings showed that H5N1/97 viruses also hyper-induce cytokines from macrophages and that patients with H5N1 disease have high levels of IP-10 and other chemokines in the serum. These findings may be relevant to the pathogenesis of H5N1 disease. The recent re-emergence of H5N1 disease in humans is a cause for renewed pandemic concern and highlights the need for a better understanding of the pathogenesis of human H5N1 disease. Such understanding will lead to new strategies for managing human H5N1 disease and enhance our preparedness to confront pandemic influenza, whether from H5N1 or other influenza A subtypes.H5N1 subtype influenza A viruses associated with human disease are more potent than human H1N1 virus at inducing proinflammatory cytokines and chemokines, including IP-10, interferon beta, IL-6 and RANTES, from human primary alveolar and bronchial epithelial cells infected The author(s) declare that they have no competing interests.JSM Peiris, MCW Chan and CY Cheung conceived the study, planned the overall experimental design and wrote the manuscript. MCW Chan carried out the experiments; MCW Chan, CY Cheung and YO Chan carried out experiments in the BL3 laboratory and RWY Chan assisted in experiments in the BL2 laboratory. WH Chui provided the lung biopsy specimens, SW Tsao helped to develop the methods for primary culture of the human alveolar epithelial cells, JM Nicholls advised on morphogical studies, and LLM Poon and Y Guan advised in experimental design. All authors critically reviewed the manuscript.


STAT1 germline alleles from otherwise healthy patients with mycobacterial disease. The previously reported L706S, like the novel Q463H and E320Q alleles, are intrinsically deleterious for both interferon gamma (IFNG)–induced gamma-activating factor–mediated immunity and interferon alpha (IFNA)–induced interferon-stimulated genes factor 3–mediated immunity, as shown in STAT1-deficient cells transfected with the corresponding alleles. Their phenotypic effects are however mediated by different molecular mechanisms, L706S affecting STAT1 phosphorylation and Q463H and E320Q affecting STAT1 DNA-binding activity. Heterozygous patients display specifically impaired IFNG-induced gamma-activating factor–mediated immunity, resulting in susceptibility to mycobacteria. Indeed, IFNA-induced interferon-stimulated genes factor 3–mediated immunity is not affected, and these patients are not particularly susceptible to viral disease, unlike patients homozygous for other, equally deleterious STAT1 mutations recessive for both phenotypes. The three STAT1 alleles are therefore dominant for IFNG-mediated antimycobacterial immunity but recessive for IFNA-mediated antiviral immunity at the cellular and clinical levels. These STAT1 alleles define two forms of dominant STAT1 deficiency, depending on whether the mutations impair STAT1 phosphorylation or DNA binding.The transcription factor signal transducer and activator of transcription-1 (STAT1) plays a key role in immunity against mycobacterial and viral infections. Here, we characterize three human  IL12B, IL12RB1, IFNGR1, IFNGR2, and STAT1. The signal transducer and activator of transcription-1 (STAT1) amino acid change L706S was previously shown to cause disease by impairing STAT1 phosphorylation. Here, we report two new STAT1 mutations that impair STAT1 DNA-binding activity. We show, by functional analysis of the three STAT1 mutant alleles, that they are intrinsically deleterious for both interferon gamma–induced antimycobacterial immunity, which is mediated through gamma-activated factor and for interferon alpha–induced antiviral immunity, which is mediated through interferon-stimulated genes factor 3. Interestingly, the three alleles are dominant for interferon gamma–induced gamma-activated factor–mediated antimycobacterial immunity, but recessive for interferon alpha–induced interferon-stimulated genes factor 3–mediated antiviral immunity at the cellular and clinical levels. These two new STAT1 alleles, which affect the binding of STAT1 to DNA, define distinct novel genetic causes of Mendelian susceptibility to mycobacterial disease and provide further insight into the molecular mechanism of disease.Mendelian susceptibility to mycobacterial disease is a rare syndrome. It is defined by the occurrence of severe disease caused by low virulence mycobacteria in otherwise healthy individuals, in whom antiviral immune response is not affected. Eleven known genetic defects, affecting five genes, have been involved in this type of deficient response to infection, involving immune-mediator molecules IL12 and interferon gamma:  IL12B, encoding the p40 subunit of IL12 and IL23, and IL12RB1, encoding the β1 chain of the IL12 and IL23 receptors (IL12RB1). Three genes control the response to IFNG: IFNGR1 and IFNGR2, encoding the IFNG receptor (IFNGR) chains, and STAT1, encoding the signal transducer and activator of transcription-1 (STAT1). Allelic heterogeneity results in a total of 11 inherited disorders  is characterized by the occurrence of clinical disease caused by weakly virulent mycobacteria in otherwise healthy individuals . Thisisorders : recessiThe binding of homodimeric IFNG to its tetrameric receptor leads to the activation of constitutively associated Janus kinases 1 and 2 (JAK1 and JAK2), which then phosphorylate tyrosine residues in the intracellular part of IFNGR1  were sequenced following amplification of the corresponding cDNAs from Epstein-Barr virus (EBV)–transformed B cells from the patients. Mutations were found in STAT1 cDNAs, and were confirmed by sequencing products amplified from the corresponding STAT1 genomic exons in EBV-transformed B cells and fresh blood cells. No mutations were found in the coding regions of the other four genes. A heterozygous nucleotide substitution at position 958 (G→C) was found in STAT1 exon 11 in P1, leading to a Glu→Gln substitution at position 320 (E320Q). A heterozygous nucleotide substitution at position 1389 (G→T) was found in exon 17 in P2, leading to a Gln→His substitution at position 463 (Q463H). These mutations were not found in 75 unrelated European control individuals tested (150 chromosomes), suggesting they are not merely irrelevant polymorphisms. Moreover, P1′s mother (A.III.2) had MSMD and was found to be heterozygous for the E320Q allele. Analysis of genomic DNA extracted from biopsy tissue taken from the deceased maternal grandfather of P1 (A.II.1), who had suffered from bona fide tuberculosis, showed that he too was heterozygous for the E320Q allele, providing further evidence for dominant cosegregation of the STAT1 genotype and clinical phenotype. However, healthy relatives of P2 (B.II.3 and B.III.3) were found to be heterozygous for the Q463H mutation, suggesting that this allele (and possibly E320Q), like the previously reported L706S STAT1 allele ,37STAT1 fections B. The E3 in vivo   of the four possible STAT1  combinations therefore forms phosphodimers in heterozygous cells. Whereas L706S exerts its negative effect in the cytosol, the other two mutations, E320Q and Q463H STAT1, exert their negative effects in the nucleus. Only one (WT/WT) of the four possible STAT1 combinations forms phosphodimers binding GAS elements with normal affinity. In contrast, all three alleles are recessive for the IFNA-induced ISGF3-mediated signaling pathway. There is neither haploinsufficiency nor negative interaction. The lack of negative dominance, in particular, is accounted for by the three STAT1 mutant proteins being no better recruited than WT STAT1 proteins to phospho-STAT2 and the IFNAR. Indeed, L706S STAT1 may not be recruited at all to IFNAR-bound phospho-STAT2. Altogether, the STAT1 alleles studied here are dominant for IFNG-induced GAF-mediated signaling and recessive for IFNA-induced ISGF3-mediated signaling.The three mutant alleles are intrinsically deleterious for both IFNG-induced GAF-mediated and IFNA-induced ISGF3-mediated cellular activations, as shown in STAT1-deficient cells transfected with the corresponding alleles . There iSTAT1 mutations are deleterious via different mechanisms, and they affect both cellular and clinical phenotypes, with a well-defined causal relationship between cellular defects and clinical diseases. Impaired IFNG-mediated immunity is associated with mycobacterial diseases, whereas impaired IFNA-mediated immunity is associated with viral diseases. It is widely accepted that the definition of candidate genes should take into account the observation that mutations in genes involved in multiple pathways in vitro can have an impact on only a few biochemical pathways in vivo, reflecting variable redundancy of the gene product. For example, complete STAT1 deficiency principally affects IFNA and IFNG responses in mice [STAT1 alleles profoundly affect IFNG-mediated immune responses to mycobacteria, but not IFNA-mediated immune responses to viruses, in heterozygous cells and patients, whereas they would affect both responses in homozygous cells and patients [The three  in mice ,47 and h in mice ,37 in vipatients ,37.M. avium infection at the age of 2 y. He was 10 y old as of June 2005 and well. P2 had high titers of serum antibodies against EBV, respiratory syncytial virus, adenovirus, parainfluenza virus (I and III), and influenza virus (A and B). His father was hospitalized at the age of 25 y for a fever of unknown origin. He recovered on antibiotic treatment and was 39 y old as of June 2005 and well. He had been vaccinated with BCG in infancy, with no adverse effect. P2′s paternal grandfather developed tuberculosis at the age of 20 y and recovered only after 5 y of hospitalization and antibiotic treatment in the 1950s. He died at the age of 50 y in unknown circumstances. P2′s half-brother has remained healthy and was 2.5 y old as of June 2005. The medical histories of P3, P4, and P5 have been described elsewhere [STAT1 mutation and developed BCG-osis after vaccination in childhood, but recovered after specific antibiotic therapy. P4 was homozygous for the 1758_1759delAG STAT1 mutation. He developed BCG-osis at the age of 2 mo, which was in remission after antibiotic treatment, followed by recurrent HSV-1 encephalitis from the age of 12 mo until his death at the age of 16 mo. P5 is homozygous for the 1928insA STAT1 mutation. He developed BCG-osis after vaccination. His genetic, immunological, and clinical features have been reported elsewhere [P1 was born in Germany to unrelated German parents . He was lsewhere ,36,37. PModels (3-D) of STAT1 mutants E320Q, Q463H, and L706S were built with Modeller software . The X-r5 IU/ml IFNA or IFNG. The nylon membrane was probed with radiolabeled IRF1, ISG15, STAT1, and GADP probes (sequences available upon request).EBV-transformed B cells and SV40-transformed fibroblast cells were cultured as previously described . The par5 IU/ml IFNA or IFNG. RNA was treated with RNase-free DNase  and cleaned by passage through an RNeasy column . RNA was then reverse transcribed directly with Oligo-dT, using the TaqMan Reverse Transcription kit , for the determination of MX1, ISG15, and IRF1 mRNA levels using the Taqman probes delivered by Applied Biosystems France for these genes. The results were normalized with values for the endogenous GUS cDNA.Total RNA was extracted with Trizol reagent  from EBV-transformed B cells or sarcoma fibroblasts left unstimulated or stimulated with 10STAT1 alleles were inserted into an M2-tagged pcDNA3-STAT1 vector (a gift from M. J. Holtzman [STAT1 M2-tagged pcDNA3 vectors or an insertless M2-tagged pcDNA3 vector (mock) and the pcDNA3 zeocin-resistant vector, using Lipofectamine 2000 reagent (Invitrogen). The cells were diluted (1:10) 24 h after transfection, and selected 48 h after transfection by culture on medium containing 200 μg/mL zeocin for 10 days, followed by five days of culture on medium containing 1,000 μg/mL zeocin. Living cells were picked and screened by immunofluorescence staining with a STAT1-specific antibody. The pmock clone was selected by pcDNA3 PCR amplification. The populations selected were then cloned by the limiting dilution method. After 1 mo of culture, cells were transferred to medium without zeocin.The mutated Holtzman ) using t5 IU/ml IFNG or IFNA. We incubated 5–40 μg of nuclear extract with 32P-labeled (αdATP) GAS (from the FCGR1 promoter) or ISRE (from the ISG15 promoter) probes and subjected the mixture to electrophoresis in a polyacrylamide gel. The additional GAS probes used were taken from the M67 promoter 5′-GATCCATTTCCCGTAAATCATGATC-3′ and from the IRF1 promoter 5′-CTGATTTCCCCGAAATGA-3′.EMSA was carried out as previously described . Briefly5 IU/ml IFNG or IFNA for 30 min. For STAT1 immunoprecipitation, whole-cell protein extracts were prepared as previously described [Cells were left unstimulated or were stimulated by incubation with 10escribed . The celescribed . The folescribed .Immunoflorescence staining was carried out as previously described . The priWhole-blood assays were performed as previously described . Heparin5 IU/mL IFNG for 24 h. They were then infected by incubation with various titers of HSV or VSV for 48 h. Viral titers were then determined by visualizing the lysed wall.Viral assays were performed as previously described . BrieflyFigure S1STAT1 alleles (B and C). The cells were not stimulated (NS) or were stimulated for 30 min with 105 IU/ml IFNA (A–C) or IFNG (C). For (A–C), one experiment representative of two to five independent experiments is shown.EMSAs with the radiolabeled GAS probe of nuclear extracts (5 μg) from patient's EBV-B cells (A) or from a parental fibrosarcoma cell line (2C4) and STAT1-deficient U3C fibrosarcoma cell clones, untransfected (U3C) or stably cotransfected with a zeocin-resistance vector and a vector containing a mock, WT, E320Q, Q463H or L706S (821 KB TIF)Click here for additional data file.http://www.ncbi.nlm.nih.gov/OMIM) accession number for MSMD is MIM 209950. The RefSeq (http://www.ncbi.nlm.nih.gov/RefSeq) accession numbers for the genes discussed in this paper are STAT1 (6772); IFNG (3458); IFNA (3438); STAT2 (6773); STAT3 (6774); ISGF3G (10379); IFNGR1 (3459); IFNGR2 (3460); JAK1 (3716); JAK2 (3717); TYK2 (7297); IL12B (3593); IL12RB1 (3594); IRF1 (3659); MX1 (4599); ISG15 (9636).The Online Mendelian Inheritance in Man database (


We examined loss of heterozygosity (LOH) for two loci on chromosome 17p (D17S5 and TP53), and erbB-2 gene amplification, in primary breast cancers from 67 Brazilian patients. We identified two distinct regions of LOH on chromosome 17p, one spanning TP53 and the other a more telomeric region (D17S5). Based on a short-term follow-up, Kaplan-Meier analyses of patients' disease-free survival showed that patients with LOH for D17S5, but retaining heterozygosity for TP53, were at higher risk of recurrence (P = 0.007) than those who retained heterozygosity for D17S5. Bivariate analyses indicated that patients with LOH for D17S5 alone had an increased risk of recurrence (hazard ratio = 7.2) over patients with erbB-2 amplification (hazard ratio = 3.7), when compared with patients with neither alteration (hazard ratio = 1.0). Further, lymph node-positive patients whose tumours had both LOH for D17S5 and erbB-2 gene amplification had a higher risk of recurrence than patients whose tumours had neither of these genetic alterations. Our data confirm previous reports of a putative tumour-suppressor gene, distinct from TP53, on distal chromosome 17p which is associated with breast cancer. They further suggest that LOH for loci in this region may provide an independent indicator to identify patients with poor prognosis.


The presence of A2A and A2B ARs was assessed by immunoblotting using specific antibodies. Equilibrium A2A and A2B ARs binding parameters were evaluated by radioligand binding assay. Functional studies were conducted to investigate coupling of the A2B AR to the adenylyl cyclase pathway. This is the first report of the use of Western blot analysis to confirm the presence of A2A and A2B ARs in human neutrophils. No significant changes in A2A AR binding parameters or expression levels were detected between SSc patients and healthy control individuals. A significant decrease (65%) in the maximum density of A2B AR binding sites occurred in SSc neutrophils, whereas no changes in the affinity constant values were found. Moreover, a decrease in A2B AR mediated adenylyl cyclase activity was observed in patients with SSc. Our findings demonstrate the occurrence of selective alterations in A2B AR density and signalling in SSc.We conducted the present study to investigate protein expression and functioning of A Systemic sclerosis (SSc), also known as scleroderma, is a connective tissue disease of unknown aetiology. Possibly an autoimmune disorder, it is accompanied in the vast majority of cases by the presence of antinuclear antibodies . SSc mayThe mechanisms leading to endothelial damage, inflammation and fibrosis are unclear. Reactive oxygen species in neutrophils may increase the extent of inflammation and fibrosis during the respiratory burst and could be involved in endothelial damage . The end1, A2A, A2B and A3 have been identified and their functions characterized in neutrophils. Specifically, activation of A1 ARs enhances chemotaxis, phagocytosis and adherence CGS21680 (CGS21680 = [2-p-(2-carbowyethyl)phenylethylamino]-5'-N-ethylcarboxamidoadenosine), [3H]NECA (NECA = 5'-N-ethylcarboxamidoadenosine), and [32P]α-ATP were supplied by NEN Life Sciences . Electrophoresis reagents were purchased from BioRad . A2AAR and A2BAR antibodies were supplied by Alpha Diagnostic . All other chemicals were from standard commercial sources.Bacitracine, benzamidine, trypsin inhibitor, sodium orthovanadate, Nonidet P-40, SDS, phenylsulfonylfluoride, aprotinin and adenosine deaminase (ADA) were purchased from Sigma . Unlabelled AR agonists/antagonists and the anti-β-actin antibody were supplied by RBI/Sigma . CGS21680. Aliquots of neutrophil membranes (0.2–0.3 mg protein) were incubated with different [3H]CGS21680 concentrations (5–30 nmol/l) in a final volume of 250 μl of T2 buffer. Nonspecific binding was determined in the presence of 100 μmol/l NECA. After 90 min incubation at 25°C, the binding reaction was terminated by vacuum filtration through Whatman GF/C glass fibre filters , accompanied by three washes with ice-cold T2 buffer (4 ml). A2A AR specificity was evaluated through competition experiments, using different AR ligands.A2B AR binding assay was performed using 20 nmol/l [3H]NECA in the presence of 50 nmol/l cyclopentyladenosine (CPA) and 100 nmol/l SCH58261  to prevent [3H]NECA binding to A1 and A2A ARs, respectively cAMP  to monitor column recovery. Newly formed ZnCO3 allowed precipitation of residual ATP, discarded through centrifugation at 2700 g for 8 min. Supernatants containing both [32P]α-cAMP and [3H]cAMP were further purified by double-step Dowex-Alumina chromatography and counted by means of a β-counter .Neutrophils were homogenized in buffer solution containing 10 mmol/l Hepes, 1 mmol/l EGTA and 10 mmol/l NaCl Salomon  and John Salomon , with so2B AR mediated cAMP accumulation, the reaction was carried out in the presence of selective A2A antagonist SCH58261 at a concentration (100 nmol/l) able to block A2A receptors completely [To evaluate Ampletely .max) were calculated using the nonlinear multipurpose curve-fitting computer program Graph-Pad Prism The 50% inhibitory concentration values were calculated using the same program and converted to Ki values through the Cheng and Prusoff equation.Affinity constant values (Kd) and maximum number of binding sites (BP < 0.01) was used to determine binding data with the best fit to a one-site or two-site model. Differences in binding parameters between SSc patients and control individuals were evaluated by one-way analysis of variance.A GS-670-BIO-RAD imaging densitometer was used for semiquantitative analysis of immunoblots. Partial F test . Scatchard analysis for SSc neutrophils revealed no significant differences in Kd and Bmax between patients  and healthy control individuals .The selective A2B AR binding sites were identified using [3H]NECA as radioligand in the presence of 50 nmol/l CPA and 100 nmol/l SCH58261, to prevent nonspecific binding to A1 and A2A AR subtypes. We performed competition experiments using a wide range (50 nmol/l to 2 mmol/l) of [3H]NECA concentrations to allow the identification of A2B AR low-affinity binding sites. Data analysis revealed that the one-site model produced a significantly better fit than the two-site model (P < 0.05), both in control and SSc neutrophils. In our experimental conditions, control neutrophils exhibited the presence of low-affinity binding sites with Kd and Bmax values of 476 ± 34 nmol/l and 3696 ± 210 fmol/mg, respectively  as compared with controls  on AC activity. NECA stimulated AC activity in a concentration dependent manner. Dose-response curves revealed significant differences between SSc patients  and controls , suggesting an alteration in A2B AR responsiveness in SSc  was observed, with no significant change in Kd values.A2B ARs are known to be low-affinity adenosine binding sites. Competition experiments using a variety of A2B AR agonists and antagonists revealed a pharmacological profile typical of A2B ARs, which is consistent with studies conducted in transfected cell models. Our findings represent the first characterization of A2B ARs in neutrophils with binding experiments.A2 ARs, SSc patients were randomly divided into two subgroups. No difference was found when the two groups were compared, suggesting that different degrees of disease severity and activity had no impact on the assays, but that the disease per se is required to modulate levels and functioning of A2B receptors.In order to analyze a population of nonhomogenous patients and to evaluate the impact of the disease on A2B ARs was accompanied by alterations in receptor responsiveness. An evaluation of the ability of NECA to increase AC activity revealed functional coupling of A2B receptors to G proteins. In SSc patients a significant reduction (by more than 50%) in NECA potency was observed, without any effect on agonist efficacy.Functional studies were performed to investigate whether the decrease in level of A2B AR levels and responsiveness occurred in SSc. Alterations in the expression and functionality of A2B ARs (low-affinity ARs) in patients with SSc may be responsible for the increase in free oxygen radicals, and consequent oxidative damage, that characterizes SSc. This would account for impaired control of hypoxic and inflammatory processes.Our findings suggest that a selective reduction in A2 - generation, phagocytosis and cell adherence by occupying specific A2 ARs. Because hypoxia, ischaemia and inflammation can stimulate adenosine production, A2 AR regulation has been postulated to be a self-protective mechanism for cells from activated neutrophils pheylethylamino)-5'N-ethylcarboxamidoadenosine; CPA = cyclopentyladenosine; Kd = affinity constant; NECA = 5'-N-ethylcarboxamidoadenosine; R-PIA = R-N6-phenylisopropyladenosine; SCH58261 = 5-amino-7-(phenylethyl)-2-(2-furyl)-pyrazolo-1,2,4-triazolopyrimidine; SSc = systemic sclerosis.AC = adenylyl cyclase; ADA = adenosine deaminase; AR = adenosine receptor; BThe author(s) declare that they have no competing interests.LB organized the study design and recruited the patients. LT carried out the binding experiments and statistical analysis. AR participated in the immunoblotting experiments and helped to draft the manuscript. FdF participated in the collection of human samples. AL participated in the coordination of the study and helped with problem solving. SB participated in the coordination of the study and in planning the manuscript. CM participated in the coordination of the study and designed the AC assay. All authors read and approved the final manuscript.


Additional experiments are necessary to probe the role of double phosphorylation of HDAC1 at S421 and S423.Histone deacetylase (HDAC) proteins are associated with cell proliferation, differentiation, apoptosis, and cancer. Specifically, HDAC1 is linked with cell growth, a hallmark of cancer formation. HDAC1 is a phosphoprotein and phosphorylation at S421 and S423 promotes HDAC1 enzymatic activity and protein association. While single and double point mutants of HDAC1 at S421 and S423 appear functionally similar, the evidence suggests that HDAC1 is phosphorylated simultaneously at both S421 and S423 To characterize HDAC1 phosphorylation at S421 and S423, limited proteolysis of HDAC1 was performed for the first time. HDAC1 degraded without production of discrete fragments. By performing concentration-dependent proteolysis, HDAC1 double point mutants with disrupted phosphorylation at S421 and S423 displayed different trypsin sensitivities compared to wild type HDAC1. Unexpectedly, HDAC1 single point mutants with disrupted phosphorylation at either S421 or S423 demonstrated protease sensitivity similar to the wild type HDAC1.Concentration-dependent proteolysis experiments provide evidence that phosphorylation of S421 and S423 individually contribute to HDAC1 function. In addition, the limited proteolysis experiments support a model where associated proteins promote HDAC1 enzymatic activity, reinforcing the importance of protein interactions in HDAC1 structure and function. Finally, because HDAC1 does not display distinct regions of protease sensitivity, the proteolysis studies suggest that HDAC1 comprises inter-related structural regions. Specifically, HDAC proteins remove the acetyl group of acetyl-lysine residues on histones, which can result in nucleosomal remodelling -acetate incorporated histones (approximately 0.2 μg or 500 dpm) were incubated with the digested protein for 1 hour at 37°C in 50 μL HD buffer  [3H]-acetic acid.HDAC1-F was immunoprecipitated and digested with increasing concentrations of trypsin as described. In this case, to maintain HDAC1 in a native form for subsequent HDAC assays, the reactions were quenched by addition of trypsin inhibitor Type I-S (Sigma) in a 1:1 (w/w) ratio with trypsin enzyme. Lower concentrations of trypsin were used in these reactions (0.00007.8-0.0050 mg/mL or 1.4–90 units/mL) because the trypsin inhibitor did not immediately stop the reaction, as was the case with SDS quenching. The HDAC assays were described previously where [lycerol) . An equaNK performed all proteolysis experiments and data quantification. PK-D contributed the HDAC assay data. MKHP conceived of the study, performed data analysis, and drafted the manuscript. All authors read and approved the final manuscript.The predicted fragments produced from digestion of human HDAC1 with trypsin using Peptide Cutter . Table iClick here for filePercentage of full-length protein remaining after incubation of each wild type or mutant HDAC1 with increasing concentrations of trypsin. Table indicating the percentage full length protein remaining after cleavage, along with standard error, for all trypsin concentrations and all proteinsClick here for fileLimited proteolysis of endogenous and transiently expressed HDAC1. Figure showing all proteolysis experiments with endogenous and transiently expressed HDAC1 used for quantitative analysis, as well as control reactions with anti-Flag-bound solid phase in the presence and absence of trypsinClick here for fileLimited proteolysis of HDAC1 S421A/S423A and E424A/E426A mutants. Figure showing all proteolysis experiments with HDAC1 S421A/S423A and E424A/E426A mutants used for quantitative analysisClick here for fileLimited proteolysis of HDAC1 S421A and S423A mutants. Figure showing all proteolysis experiments with HDAC1 S421A and S423A mutants used for quantitative analysisClick here for fileLimited proteolysis of HDAC1 E424A and E426A mutants. Figure showing all proteolysis experiments with HDAC1 E424A and E426A mutants used for quantitative analysisClick here for fileLimited proteolysis of the HDAC1 H141A mutant. Figure showing all proteolysis experiments with the HDAC1 H141A mutant used for quantitative analysisClick here for file


The correlation between histological subtype  and the prognosis of pure yolk sac tumours (YSTs) of the ovary was investigated. From 1964 to 1989, 35 patients with YSTs were treated with primary surgery and adjuvant chemotherapy. The prevalence of histological subtypes was as follows: 14 patients had a single subtype, either ES (12) or G (2); 12 patients had two subtypes, ES+G (4), ES+PV (3), ES+H (4) or G+H (1); six patients had three subtypes, ES+P+H (4) or ES+G+H (2); and three patients had all four subtypes. Multivariate analysis showed that important predictors were FIGO stage, chemotherapeutic regimen and residual tumour size. However, for stage I, multivariate analysis showed that the histological subtype was a superior predictor to the subclassification of FIGO stage I, age or chemotherapeutic regimen (P = 0.03). Kaplan-Meier analysis showed that YSTs composed of an admixture of three or four subtypes was associated with a better prognosis than those composed of one or two subtypes (P < 0.01), other variables being constant.


Lung disease in cystic fibrosis (CF) patients is dominated by chronic inflammation with an early and inappropriate influx of neutrophils causing airway destruction. Congenic C57BL/6 CF mice develop lung inflammatory disease similar to that of patients. In contrast, lungs of congenic BALB/c CF mice remain unaffected. The basis of the neutrophil influx to the airways of CF patients and C57BL/6 mice, and its precipitating factor(s) (spontaneous or infection induced) remains unclear.The lungs of 20-day old congenic C57BL/6 (before any overt signs of inflammation) and BALB/c CF mouse lines maintained in sterile environments were investigated for distinctions in the neutrophil chemokines S100A8 and S100A9 by quantitative RT-PCR and RNA in situ hybridization, that were then correlated to neutrophil numbers.The lungs of C57BL/6 CF mice had spontaneous and significant elevation of both neutrophil chemokines S100A8 and S100A9 and a corresponding increase in neutrophils, in the absence of detectable pathogens. In contrast, BALB/c CF mouse lungs maintained under identical conditions, had similar elevations of S100A9 expression and resident neutrophil numbers, but diverged in having normal levels of S100A8.The results indicate early and spontaneous lung inflammation in CF mice, whose progression corresponds to increased expression of both S100A8 and S100A9, but not S100A9 alone. Moreover, since both C57BL/6 and BALB/c CF lungs were maintained under identical conditions and had similar elevations in S100A9 and neutrophils, the higher S100A8 expression in the former (or suppression in latter) is a result of secondary genetic influences rather than environment or differential infection. Cystic fibrosis (CF) is an autosomal recessive disease caused by mutations in the Cystic Fibrosis Transmembrane conductance Regulator (CFTR) gene ,2. CliniLung disease is the leading cause of morbidity and mortality among CF patients, and is increasingly regarded as multifactorial, being a combination of abnormalities in inflammatory response and pathogen clearance, in addition to electrolyte transport and airway surface layer composition ,5-16. DuMouse models of cystic fibrosis, containing disruptions of the CFTR gene, show epithelial bioelectric lesions similar to that observed in CF patients ,24(reviePrevious studies by us and others have described a congenic C57BL/6J CF mouse model (B6-CF) that manifests an inflammatory lung phenotype ,27,42 toTo gain further insight into the early pathogenesis of the lung disease in B6-CF mice we previously undertook a study to identify genes having differential expression between 20 day-old lungs  of B6-CF and age- and sex-matched wild-type sibs maintained in a specific pathogen free environment and free of any detectable lung infection, using Affymetrix GeneChip™ analysis . These sThe B6-CF and Bc-CF mice used for this study and their phenotypes have been described in detail elsewhere ,27,52,532, 200 mM dNTPs, 100 nM each primer, 100 ng genomic DNA, and 1 U Taq polymerase. Thermal cycling was carried out for 35 cycles . After electrophoresis the PCR products were visualized on an ethidium bromide stained 1% agarose gel.Genomic DNA was prepared from tail clips using a salting-out extraction procedure . BrieflyCarbacillus and Mycoplasma pulmonis. A second group of sentinels (congenic C57BL/6J CF and C57BL/6J heterozygous CF breeders) housed in open cages adjacent to the heterozygous CF breeders were maintained under the same conditions for an additional 6 weeks. Half of these animals were screened as above, while the remaining mice were sent to the Ontario Veterinary College Department of Pathology, University of Guelph  for detailed histopathological screening for signs of infections of their lungs, kidneys, heart, spleen, pancreas, salivary glands, jejunum, ileum, colon, brain, seminal vesicles, thymus, and lymph nodes. Lung and jejunal tissue were also routinely cultured for bacteria, and found to be negative for conventional CF lung pathogens . In addition, histopathological screens were also performed to detect pathogenic infections of the specific lung samples used for RNA preparation. The studies performed did not identify any obvious signs of lung infection in the CF animals; nevertheless it is not possible to completely rule out the presence of any undetected pathogens.All mice (CF and wild-type controls) were maintained under stringent Specific Pathogen Free (SPF) conditions in microisolator cages at the Hospital for Sick Children Animal Facility, as previously described . Detaile260/A280) between 1.6 and 1.9 was considered acceptable for purity. RNA integrity was assessed by visualization on an ethidium bromide stained 1% agarose gel. One microgram of total cellular RNA from each sample was then treated with 1 unit of amplification grade DNase I (Invitrogen) according to the manufacturer's protocol.Total cellular RNA was extracted from snap-frozen whole right lung lobes dissected from 20-day old CF and wild-type sibs from both the C57BL/6 and BALB/c strains (8 of each genotype/strain) using the Qiagen RNAeasy™ Midi kit according to the manufacturer's protocol. Sample concentration and purity were determined by measuring optical density at 260 nm and the ratio of 260 nm to 280 nm, respectively. A ratio of absorbance 12–18 primer were incubated at 65°C for 5 minutes, added to 5X RT buffer, 0.1 M DTT, 10 mM deoxyribose nucleotide triphosphate (dNTP) mix and incubated at 42°C for 2 minutes. Fifty units of Superscript™ II reverse transcriptase was added and the mixture was incubated at 42°C for 50 minutes, 70°C for 15 minutes, then treated with 2 units of RNase H at 37°C for 20 minutes and stored at -20°C. Oligonucleotide primers to amplify the target S100A8, S100A9 and the β-actin cDNA sequences were designed from published cDNA sequences . The primers were chosen to span at least 1 intron to distinguish products resulting from the amplification of cDNA and potentially contaminating genomic DNA. Primer sequences were as follows: S100A8 sense 5'-CCCGTCTTCAAGACATCGTTTG-3' (position 1–22 in the cDNA), S100A8 antisense 5'-ATATCCAGGGACCCAGCCCTAG-3' (position 347–326 in the cDNA), S100A9 sense 5'-CCCTGACACCCTGAGCAAGAAG-3' (position 120–141 in the cDNA), S100A9 antisense 5'-TTTCCCAGAACAAAGGCCATTGAG-3' (position 453–430 in the cDNA), β-actin sense 5'-GTGGGCCGCCCTAGGCACCAG-3' (position 183–203 in the cDNA), β-actin antisense 5'-CTCTTTGATGTCACGCACGATTTC-3' (position 722–699). The expected size of the PCR products was 347 bp for S100A8, 333 bp for S100A9 and 539 bp for β-actin. Multiplex PCR amplification was performed using 1/20 of the total cDNA reverse transcribed from each sample. A total reaction volume of 20 μL also contained 200 μM dNTP mix, 150 μM MgCl2, 10 mM Tris-HCl (pH 8.3), 50 mM KCl, 2.5 units of Thermus aquaticus (Taq) DNA polymerase (Fermentas) and 2.5 ng/μL of both sense and antisense oligonucleotide primers for the target (either S100A8 or S100A9) and the endogenous standard (β-actin). Four reactions were run in parallel for each sample in a Perkin Elmer Applied Biosystems Geneamp Thermocycler 9700, using a hot-start protocol where Taq polymerase was added to reaction mixtures after an initial denaturation step at 94°C for 3 minutes. The reactions were cycled at 94°C for 30 seconds (denaturation), 65°C for 30 seconds  and 72°C for 30 seconds (extension). Equal volumes of the PCR reaction were removed at cycles 19, 21, 23 and 25. Fifteen microliters from each PCR reaction were loaded unto an ethidium bromide stained 1% agarose gel and documented with a Kodak EDAS 290 electrophoresis documentation system. Band intensities (and thus starting product levels) of the target relative to control were measured using the program NIH Image® . Band intensities of PCR products  were plotted against cycle number in order to determine the exponential phase of amplification. For each sample, the S100A8 and S100A9 multiplex RT-PCR product band intensities after 21 cycles of amplification were normalized to that of the β-actin produced in the same tube and the mean of the four runs was calculated to obtain relative expression levels. All measurements for expression were performed with the investigator blinded to mouse strain and genotype.To determine S100A8 and S100A9 mRNA expression levels, 1 μg of DNase I-treated total cellular RNA from the mouse whole lung was reverse transcribed using the Invitrogen Superscript™ II RNase HTo ascertain relative neutrophils numbers in the lungs of the different mice  the left lung lobes of 7 animals of each strain and genotype were harvested, inflated, and infused with 10% buffered formalin. After overnight fixation in formalin the lobes were cut into 4 separate sections (from top to bottom of the lobe to maximize representation of the specimen), embedded in paraffin blocks and sectioned to a thickness of 4 μm followed by Hematoxylin & Eosin (H&E) staining for visual inspection and counts of neutrophils (recognized by their characteristic multi-lobed nuclei) by an experienced pathologist blinded to strain, genotype and expression status. For each of the 4 lung sections from each animal, the number of neutrophils in 6 distinct and randomly chosen fields was counted and the average of the 6 was calculated for that lung section. Thus, a total of 24 distinct sections of each lung from 7 mice  of each genotype and strain were counted to arrive at a representative measure of neutrophil content for each group of animals.2 for 10 min. The substrate, nitro blue tetrazolium/5-bromo-4-chloro-3-inolyl phosphate (Invitrogen), was added to the samples and the color reaction was allowed to develop overnight. All samples were hybridized to both anti-sense and sense (negative control) probes to ensure specific signal detection. The number of positive-staining neutrophils in 5 independent fields for each section was counted and the average taken as representative of that lung.Left lung lobes (4 of each genotype/strain) were inflated, fixed in paraformaldehyde, OCT-embedded and thin-sliced (5 independent sections for each lung) onto aminoalkylsilane-coated slides (SIGMA) followed by air-drying for 2 hrs. Samples were fixed in 4% paraformaldehyde in PBS for 20 min, protein hydrolyzed in 20 μg/ml proteinase K for 7.5 min, and then post-fixed for 5 min in 4% paraformaldehyde in PBS. Tissues were incubated for 10 min in a 0.1 M triethanolamine, 0.5 % acetic anhydride solution. To dehydrate samples, slides were dipped successively in a graded series of ethanol baths before hybridization. Samples were hybridized overnight at 55°C in 50% formamide, 0.3 M NaCl, 20 mM Tris-HCL (pH 7.6), 5 mM EDTA, 10% dextran sulphate, 1.5 × Denhardts, 0.5 mg/ml yeast tRNA, and digoxigenin-UTP-labeled RNA probes. Antisense and sense probes were prepared by in vitro transcription, using T7 RNA polymerase, from a 347 bp sequence (nucleotides 1–347) of S100A8, and a 333 bp sequence (nucleotides 120–453) of S100A9, of HindIII linearized pCR2.1 (Invitrogen) vector with S100A8 and S100A9 inserted in both orientations into the BamHI/HindIII sites of the multiple cloning region. Following hybridization, slides were soaked for 15 min in 0.1 M maleic acid and 0.15 M NaCl, then for 1 hr in a 1% Boehringer blocking reagent solution in 0.1 M maleic acid and 0.15 M NaCl. Bound probes were detected by exposing samples to alkaline phosphatase-conjugated anti-digoxigenin antibodies for 1.5 hrs and slides were then washed in 0.1 M Tris (pH 9.5), 0.1 M NaCl, 50 mM MgClAll statistical comparisons were performed using non-parametric Mann-Whitney Tests (2-tailed) and Spearman Rank Correlation tests, as appropriate. Data is plotted as the median with interquartile ranges.We had previously reported a roughly 2.5-fold elevation of S100A8 expression in the lungs of 20 day-old B6-CF mice, as ascertained through an Affymetrix GeneChip experiment . To confThese results indicate an early and specific increase of both S100A8 and S100A9 expression levels in lungs of B6-CF mice in contrast to Bc-CF lungs in which only S100A9 expression levels were elevated.To assess the basis of the differential S100A8 and S100A9 levels, the number of resident neutrophils (primary sites of S100A8 and S100A9 expression) between the lungs of 20 day-old B6-CF, Bc-CF and their wild-type sibs were next quantified as described in Materials and Methods. As shown in Fig. in situ hybridization of lung sections taken from 20-day old Bc-CF, B6-CF mice and their wild-type sibs was performed. As expected, hybridization of the lung sections with S100A8 and S100A9 sense probes showed no positively staining cells  were negative for S100A8 staining in B6-CF lungs . In contrast, the lungs of 20-day old congenic BALB/cJ CF mice, which do not develop any obvious inflammatory phenotype, housed with the congenic C57BL/6J CF animals, had no increase in S100A8 levels, although resident airway neutrophil numbers and S100A9 levels were similarly elevated.2+, with potentially different function(s). Although an understanding of the complete role(s) of each of S100A8, S100A9 and calprotectin is currently lacking [2+ [S100A8  and S100A9  are small cytoplasmic proteins  that ar lacking ,59 diver lacking , cell di lacking ,62, as w lacking -69. More lacking -46,51,70king [2+ -78 as weking [2+ . S100A8'king [2+ .During chronic inflammatory conditions, including that underlying CF lung disease, S100A8 and S100A9 are coordinately upregulated and secreted into the extracellular milieu ,81, and The results of the present study are important to further understand the basis and pathogenesis of the inflammatory lung phenotype of CF mice, its distinction among different congenic strains and possibly having implications to understanding airway disease of CF patients. Several important points can be drawn from these results. First, these results provide further support for the increasingly prevalent notion of spontaneous inflammation of the CF airways. This conclusion is supported by: 1) the early incidence of elevations in S100A8 and S100A9 expression along with resident neutrophil influx, 2) the fact that the mice were maintained in sterile environments without detectable lung pathogens, and 3) elevated S100A8 levels were detected in the B6-CF lungs but not Bc-CF airways maintained in identical environments.Second, since S100A8 and S100A9 act as potent leukocyte chemokines and their elevation at 20-days of age are the earliest reported signs of a lung inflammatory phenotype in CF mice, this elevation may be directly responsible for eliciting the massive neutrophil influx observed in 4–5 week old B6-CF lungs ,27,42.Third, these results implicate S100A8 alone or both S100A8/S100A9 , but not S100A9 alone, as having a possible role in progression of the inflammatory lung phenotype in CF mice.Finally, since both the B6-CF and Bc-CF mice were maintained in identical environments, the differential levels of S100A8 expression between the two strains is likely influenced by secondary genetic factors acting on neutrophils  to either suppress or upregulate its expression in the Bc or B6 strain, respectively, rather than the effect of differential environmental exposures or infection status. However, since the elevated levels of S100A8 in the B6-CF lungs agrees with the corresponding increased population of neutrophils and no expression was detected in inducible cells , it is more likely that its expression is being suppressed in the Bc strain as opposed to B6-CF, which maintains expression in resident neutrophils. Since S100A8 is normally expressed in circulating but not interstitial neutrophils , a possiThe results of this study extend on two previous reports of S100A8 overexpression in the lungs of distinct CF mouse lines ,38. In tThe results presented here justify additional studies to clarify the role of S100A8 overexpression on the pathogenesis and/or progression of the CF lung inflammatory disease, and, in particular, the possible effect of S100A8 inhibition.Taken together, these results derived from genetically-defined CF mice maintained in strict controlled environments provide further support for an early and spontaneous induction of inflammation in lungs devoid of the cystic fibrosis transmembrane conductance regulator, and suggest that S100A8 may play a prominent role. Moreover, since similar elevations of S100A8/S100A9 are detected in CF patients, these results also provide justification for the application of congenic C57BL/6J CF mice as a potential model to gain insight into the pathogenesis of lung disease of CF patients and potential therapeutic avenues.CF: cystic fibrosisCFTR: cystic fibrosis transmembrane conductance regulatorB6-CF: congenic C57BL/6 CF miceBc-CF: congenic BALB/c CF miceThe author(s) declare that they have no competing interests.in situ hybridization, neutrophil counts and drafting of the manuscript. SN assisted in morphometric analysis, neutrophil and macrophage counts and lung histopathology. VN assisted in RNA preparation, interpretation of quantitative RT-PCR and RNA in situ hybridizations. MK performed mouse colony maintenance and genotyping. CA assisted in lung neutrophil and macrophage analysis, measurements and interpretation. GK provided the mice and pathogen monitoring/status and interpretation. RFR designed and supervised the study, and revised the final manuscript.ST performed the majority of the studies, particularly the lung dissection, quantitative RT-PCR, RNA


Global influenza surveillance is one of the most effective strategies for containing outbreaks and preparing for a possible pandemic influenza. Since the end of 2003, highly pathogenic avian influenza viruses (HPAI) H5N1 have caused many outbreaks in poultries and wild birds from East Asia and have spread to at least 48 countries. For such a fast and wide-spreading virulent pathogen, prediction based on changes of micro- and macro-environment has rarely been evaluated. In this study, we are developing a new climatic approach by investigating the conditions that occurred before the H5N1 avian influenza outbreaks for early predicting future HPAI outbreaks and preventing pandemic disasters. The results show a temperature drop shortly before these outbreaks in birds in each of the Eurasian regions stricken in 2005 and 2006. Dust storms, like those that struck near China's Lake Qinghai around May 4, 2005, exacerbated the spread of this HPAI H5N1 virus, causing the deaths of a record number of wild birds and triggering the subsequent spread of H5N1. Weather monitoring could play an important role in the early warning of outbreaks of this potentially dangerous virus. In most studies, the central factor thought to contribute to the unprecedented spread of H5N1 or novel avian influenza A viruses (AIV) has been the interaction between migrating waterfowl and domestic poultry in Eurasia In this study, we look beyond virology to other factors, namely changes in weather patterns that might contribute to the increase of outbreak occurrence and spread of the fatal pathogen HPAI H5N1 among live birds. We hypothesized that H5N1 viruses, which are carried by live birds, would not be activated randomly, instead, they would be automatically and predictably elicited and imposed by acute environmental health stress like sudden cold air masses.The study method started by searching for each AIV HPAI H5N1 outbreak date first, followed by analyzing the synoptic and local meteorological data collected from the website of the United States National Climate Data Center (NCDC). Since some outbreak areas were not associated with a World Meteorological Organization (WMO) registered weather station, and many outbreaks were happening in a close range within a short period of time, a total of twenty-seven representative cases  are anals of wild birds started.In May and June 2005, the HPAI H5N1 virus killed an unprecedented six thousand bar-headed geese near Lake Qinghai in western China In early May 2005, dust storms struck a wide area stretching from Mongolia to most of northwestern China . These sOutbreaks of avian flu H5N1 in Europe came with similar changes in weather conditions. By tracking the dates and locations of HAPI H5N1 outbreaks in Eurasia between July 2005 and June 2006 and cross-referencing that information with meteorological data from local WMO stations, we observed a striking correlation. All of the 27 AIV H5N1 outbreaks listed in Two of the outbreaks, marked by nearby dust storms, were peculiar to the May 4, 2005 and April 15, 2006 outbreaks at China's Qinghai Lake . The dust storms combined with the quick temperature drops may have contributed to the spread of H5N1 in these areas.After careful investigation of the composite plots of surface pressures, temperatures and wind flows on day 0 of each selected H5N1 outbreaks  and S7, Most of the H5N1 outbreaks were associated with a Siberian cold air mass dominating Eurasia  and S7. The occurrence of each of the lowest minimum temperatures was associated with both an arrival of cold air mass and an efficient radiative cooling under clear night-time skies. These conditions were sometimes followed by bright sunshine and air flows from a warmer region, resulting in a subsequent quick increase in temperature. In the outbreaks V-b and V-e, the temperatures increased by 11°C to 15°C just one day after day 0. H5N1 outbreaks were reported the next day. After examining the distribution of all the HPAI H5N1 outbreaks that occurred across many regions in the three major continents, the outbreaks indeed were associated with the very similar weather patterns as we mentioned above. Apparently, these dramatic weather fluctuations physically challenged the already fatigued migrating birds, just before official reports of avian flu outbreaks. This increased environmental stress is further strongly supported by the facts that HPAI H5N1 virus was detected in dead wild birds in even the more remote regions (outbreak VII-b) without poultry farms or trading centers It should be noted that our findings apply to regions north of the 25°N. How far the minimum temperatures would drop in these regions depends on season, altitude and synoptic weather conditions. Therefore, more studies investigating the optimal weather conditions before the HPAI H5N1 outbreaks in each region are needed before weather can be used to better predict outbreaks of this virus.In conclusion, our findings show that regional weather monitoring may serve as an integral part of a bird flu surveillance system. We strongly advocate using temperature monitoring along with active virological surveillance Figure S1Surface observation of dust storms happened on May 2–4, 2005. Symbols are explained in (0.16 MB PDF)Click here for additional data file.Figure S2Locations of Altay mountains (M1), Tianshan Mountains(M2), Karakorum Mountains(M3), Kunlun Mountains(M4), Arjin Mountains(M5), Qilian Mountains(M6), Gurbantunggut desert(D1), Taklamakan desert(D2), Tsaidam desert(D3), Gobi desert(D4), Tsaidam Basin (B), Gangca city (O) and the Bird Mountain (△) at Qinghai Lake. Square mark (▭) indicates the area in Qinhgai, China where wild birds with H5N1 virus were reported in spring 2006. Solid purple lines indicate the movement of Siberia cold air mass which would trigger the development of dust storms.(0.19 MB PDF)Click here for additional data file.Figure S3Contour plots of sea level pressure, surface temperature and wind flow on selected day 0 of outbreak event I-a (2005/5/4) and event II-a (2005/7/19), II-b (2005/8/14), II-c (2005/9/28), II-d (2005/10/7), II-e (2005/10/10).(0.29 MB PDF)Click here for additional data file.Figure S4Contour plots of sea level pressure, surface temperature and wind flow on selected day 0 of outbreak event III-a (2005/10/13), III-b (2005/10/21), III-c (2005/11/5), III-d (2005/11/8).(0.18 MB PDF)Click here for additional data file.Figure S5Contour plots of sea level pressure, surface temperature and wind flow on selected day 0 of outbreak event IV-a (2005/11/24), IV-b (2005/12/20), IV-c (2005/12/26), IV-d (2006/1/13), IV-e (2006/1/22).(0.27 MB PDF)Click here for additional data file.Figure S6Contour plots of sea level pressure, surface temperature and wind flow on selected day 0 of outbreak event V-a (2006/1/26), V-b (2006/2/6), V-c (2006/2/12), V-d (2006/2/27), V-e (2006/3/8).(0.27 MB PDF)Click here for additional data file.Figure S7http://www.cdc.noaa.gov/Composites/Day/) using NCEP reanalysis data. In each figure, a white dot or a red dot is marked to indicate the area where avian influenza broke out.Contour plots of sea level pressure, surface temperature and wind flow on selected day 0 of outbreak event VI-a (2006/2/18), VI-b,c (2006/3/11) and VII-a (2006/4/15), VII-b (2006/5/21), VII-c (2006/5/26), VII-d (2006/6/15). Each plot is downloaded from NOAA CDC Interactive Plotting and Analysis Pages ((0.30 MB PDF)Click here for additional data file.Table S1Meaning of weather symbols shown in (0.10 MB PDF)Click here for additional data file.