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951.
Gianluca Storci Pasquale Sansone Sara Mari Gabriele D'Uva Simona Tavolari Tiziana Guarnieri Mario Taffurelli Claudio Ceccarelli Donatella Santini Pasquale Chieco Kenneth B. Marcu Massimiliano Bonafè 《Journal of cellular physiology》2010,225(3):682-691
Extracellular and intracellular mediators of inflammation, such as tumor necrosis factor alpha (TNFα) and NF‐kappaB (NF‐κB), play major roles in breast cancer pathogenesis, progression and relapse. SLUG, a mediator of the epithelial–mesenchymal transition process, is over‐expressed in CD44+/CD24? tumor initiating breast cancer cells and in basal‐like carcinoma, a subtype of aggressive breast cancer endowed with a stem cell‐like gene expression profile. Cancer stem cells also over‐express members of the pro‐inflammatory NF‐κB network, but their functional relationship with SLUG expression in breast cancer cells remains unclear. Here, we show that TNFα treatment of human breast cancer cells up‐regulates SLUG with a dependency on canonical NF‐κB/HIF1α signaling, which is strongly enhanced by p53 inactivation. Moreover, SLUG up‐regulation engenders breast cancer cells with stem cell‐like properties including enhanced expression of CD44 and Jagged‐1 in conjunction with estrogen receptor alpha down‐regulation, growth as mammospheres, and extracellular matrix invasiveness. Our results reveal a molecular mechanism whereby TNFα, a major pro‐inflammatory cytokine, imparts breast cancer cells with stem cell‐like features, which are connected to increased tumor aggressiveness. J. Cell. Physiol. 225: 682–691, 2010. © 2010 Wiley‐Liss, Inc. 相似文献
952.
Fabrizio Maggi Daniele Focosi Melania Albani Letizia Lanini Maria Linda Vatteroni Mario Petrini Luca Ceccherini-Nelli Mauro Pistello Mauro Bendinelli 《Journal of virology》2010,84(13):6891-6893
Many aspects of the life cycle of torquetenoviruses (TTVs) are essentially unexplored. In particular, it is still a matter of speculation which cell type(s) replicates the viruses and maintains the generally high viral loads found in the blood of infected hosts. In this study, we sequentially measured the TTV loads in the plasma of four TTV-positive leukemia patients who were strongly myelosuppressed and then transplanted with haploidentical hematopoietic stem cells. The findings provide clear quantitative evidence for an extremely important role of hematopoietic cells in the maintenance of TTV viremia.Torquetenoviruses (TTVs) are small naked DNA viruses distinguished by a circular single-stranded DNA genome of only 3.8 kb, classified within the newly established family Anelloviridae (7). TTVs have been found in several animal species but do not appear capable of interspecies transmission. Due to their extensive genetic heterogeneity, human TTVs have been operatively subdivided into 5 genogroups and more than 40 genotypes (4). A remarkable feature of these TTVs is their presence in the plasma of nearly all people, regardless of geographical origin, age, and health status, raising many questions about their life cycle and possible pathological implications (2, 5). Plasma loads of TTVs vary extensively in both healthy and diseased individuals, usually ranging between 103 and 107 DNA copies per ml of plasma. However, some patients, including those with selected inflammatory or neoplastic disorders, transplant recipients, and human immunodeficiency virus-infected individuals, have a tendency to carry especially high burdens of TTVs (1, 6, 13, 22-24).By studying the dynamics of TTV viremia in individuals treated with alpha interferon for hepatitis C, the kinetics of virus replication was found to be quite high, with numbers of virions released into plasma and cleared from it daily on the same order of magnitude as other chronic plasma viremia-inducing viruses, such as the hepatitis B, hepatitis C, and human immunodeficiency viruses (16). Yet, due to considerable difficulties encountered in propagating TTVs in culture and in distinguishing the virions passively adsorbed onto the cells from the ones replicating inside cells, the tissue or tissues where these large numbers of TTV virions originate have yet to be established. Given that the amino acid compositions of the capsid protein believed to mediate viral adsorption to cells are quite diverse in different TTVs (2, 3, 9), it is also possible that permissive cells vary depending on the TTV considered. Relevant studies are limited. Short-term cultures of phytohemagglutinin-stimulated peripheral lymphocytes, but not resting lymphocytes were found to permit a measurable level of TTV replication (15, 18), indicative of at least a moderate degree of lymphotropism. On the other hand, the detection of replicative forms of TTV DNA in several tissues, including bone marrow, peripheral blood mononuclear cells, and liver, has suggested that TTVs might be polytropic in nature (2, 21).In 1999, Kanda et al. (10), researching TTV plasma of bone marrow transplant recipients with a qualitative PCR, noticed that 5 out of 6 previously positive patients tested negative in a sampling collected during the myelosuppressed period and became positive again after graft reconstitution, leading them to suggest that TTV might replicate mainly in hematopoietic cells. In the present study, we further developed this observation by measuring the TTV load in sequential plasma samples obtained from four TTV-positive leukemia patients undergoing hematopoietic stem cell transplantation. This procedure basically consists of a myeloablative conditioning regimen (chemotherapy plus radiotherapy) followed by reinfusion of a positively selected CD34+ stem cell population. The findings are of interest because, in addition to confirming the decrease of TTV load observed by Kanda et al., they shed light on the kinetics of the effect, thus providing a better insight onto the role of hematopoietic cells in the maintenance of TTV viremia and on the life cycle of TTV in general.Table Table11 summarizes the main characteristics of the patients selected for the study. They were treated with 10 Gy total-body irradiation (TBI) on day 0 and received 5 mg/kg/day thiotepa on days 2 and 3, 40 mg/m2/day fludarabine on days 3 to 7, and 1.2 mg/kg/day antithymocyte globulin on days 4 to 8, and then, on day 10, they received the indicated numbers of positively selected CD34+ hematopoietic stem cells from HLA-haploidentical donors. Peripheral blood samples were collected for TTV studies immediately before TBI and at selected times for the next 30 days, and plasma was stored in aliquots at −80°C until DNA extraction. The assay used for TTV quantification was a previously described highly sensitive TaqMan real-time PCR having the potential to detect and quantitate all hitherto recognized genetic forms of the virus (15, 16). All samples from each patient were assayed in a single run and in triplicate, and at least two independent DNA extractions for each sample were examined. The DNA extracts obtained at time zero were also typed with a previously described panel of five distinct PCR assays (12), each specific for one of the genogroups into which TTVs are subdivided. At the start of the study, the patients had viral loads ranging from 4.7 to 6.8 log copies per ml of plasma and harbored between 1 and 3 TTV genogroups (Table (Table1).1). In particular, all carried genogroup 1, which is highly represented in our area (12), and two carried one or two further genogroups. Consistent with previous findings (12), the patient who harbored three genogroups was the one with the highest viral load. As shown by Fig. Fig.1,1, in all four patients, TBI was followed by a steady decline of TTV viremia that continued for at least 22 days and progressively brought the virus to levels very close to the detection limit of the detection/quantitation method used, corresponding to values ranging between 0.003 (patient 3) and 0.00009 (patient 1) of the loads present prior to TBI. However, in no instance did the viral loads go below the limit of sensitivity of the assay (2 × 102 TTV DNA copies per ml of plasma). Although the size of the study does not permit firm conclusions on this aspect, it is noteworthy that the extent of decline was unrelated to the type and number of infecting TTV genogroup(s) originally present in the patients.Open in a separate windowFIG. 1.Plasma TTV loads and WBC counts in the peripheral blood of the 4 patients (Pt. 1 to 4) enrolled in the study. The arrow indicates the day the patients were infused with CD34+ hematopoietic stem cells from HLA-haploidentical donors. The horizontal broken line represents the lower limit of sensitivity of the TTV detection method used.
Open in a separate windowaT-ALL, T-cell acute lymphoblastic leukemia; B-ALL, B-cell acute lymphoblastic leukemia; AML, acute myeloid leukemia.bThe day post-TBI when TTV loads and genogroups were determined is shown in parentheses.cND, not determined.The viral loads observed during the phase of maximum decline (days 0 to 12) were then exploited to investigate the dynamics of TTV infection in the patients by using the mathematical model originally developed by Neumann et al. (20). The results of this analysis are shown in Table Table2.2. The mean clearance rate of circulating TTVs was 3.8 days−1. The half-life of plasma TTVs ranged between 3.6 and 4.8 h, with a mean of 4.3 h, which is a little shorter than previously calculated in patients treated with alpha interferon (16), possibly due to the fact that TBI may have led to a more complete block of viral replication. Overall, however, these values coupled with the calculated numbers of virions produced per day (Table (Table2)2) are a further demonstration that TTV infection is highly dynamic.
Open in a separate windowaCalculated by the equation ln (2)/c.bDaily production of plasma virions was calculated from c multiplied by the pre-TBI viremia load value and by extracellular body fluid volume, which was arbitrarily set at 3.0 × 103 ml.One patient died of multiorgan failure a few h after the 30-day sampling point without noticeable changes in either TTV viremia and white blood cell (WBC) counts. The other patients, starting from day 26, showed a generally moderate but consistent increase of TTV viremia, so that by the end of the 30-day observation period their viral loads were still somewhat to considerably lower than at baseline (Fig. (Fig.1).1). Interestingly, the increase paralleled the reappearance of WBCs in peripheral blood, a clear indicator of substantial engraftment. For two patients, we could also examine plasma samples collected at days 50, 80, and 110. As shown by the inserts in Fig. Fig.1,1, at these times both patients exhibited plasma TTV loads higher than at baseline, indicating that TTV shedding into plasma had resumed and was as abundant as or even more abundant than that at the start of the study. Interestingly, the spectrum of TTV genogroups detected in plasma at this time differed substantially from pre-TBI (Table (Table1),1), indicating that the patients were now replicating newly acquired TTVs, most likely transmitted by the graft or blood component transfusions required to sustain the procedure.Collectively, these findings provide solid quantitative evidence that hematopoietic stem cells represent by far the most important, if not the only source of the generally high TTV burdens found in the blood of infected individuals. The alternative explanation that hematopoietic cells or cytokines produced by them might stimulate other cells to replicate TTV seems less likely. Not only did plasma TTV loads fall dramatically during the myelosuppressed period, but also graft reconstitution was accompanied by a parallel return to high TTV loads. That TTVs have a preference for a highly cycling cell compartment is consistent with the well-established notion that single-stranded DNA viruses, including parvoviruses and circoviruses, have a marked preference for or replicate exclusively in DNA-synthesizing cells (14). The minimal levels of viremia that persisted during myelosuppression might suggest that some TTV replication takes place as well outside the hematopoietic compartment. However, since posttransplant the viral genogroups harbored by the patients were at least partly different from the ones harbored pretransplant, it is also possible that such low viral loads were generated by the hematopoietic cells infused into the patients.The viruses that lack an external lipid envelope are usually cytolytic for the cells in which they replicate. Future studies should therefore focus on clarifying which specific cell type or types within the hematopoietic cell compartment support TTV replication. A preferential replication within the lymphoid cell lineage might explain some of the immunomodulating properties attributed to the TTVs (6, 14, 17), while a preference for the erythroid lineage might explain the cases of aplastic anemia that have been associated with TTV infection (8, 11, 19). On the other hand, the circumstance that the great majority of TTV infections do not emerge clinically is most likely explained by the large regenerative potential of the hematopoietic compartment. 相似文献
TABLE 1.
Relevant parameters of the patients enrolledPatient | Age in yr (sex) | Clinical diagnosisa | No. of CD34 cells grafted (106 cells/kg) | Survival (days) | TTV in plasma | |||
---|---|---|---|---|---|---|---|---|
Pre-TBI | Post-TBI | |||||||
No. of copies/ml | Genogroup(s) | No. of copies/mlb | Genogroup(s) | |||||
1 | 54 (male) | T-ALL | 23.60 | 30 | 6.8 | 1, 3, 5 | NDc | ND |
2 | 47 (female) | ALL | 9.41 | 174 | 4.7 | 1, 4 | 5.4 (day 80) | 1, 3, 4, 5 |
3 | 41 (female) | B-ALL | 11.70 | 111 | 5.3 | 1 | 4.2 (day 30) | 3 |
4 | 58 (female) | AML | 5.90 | 267 | 5.0 | 1 | 7.0 (day 110) | 1, 3, 4, 5 |
TABLE 2.
TTV dynamics in the patients enrolledPatient | Viral parameter | ||
---|---|---|---|
Clearance rate (c [days−1]) | Virion half-life (days)a | Minimal input and clearance of plasma virions/dayb | |
1 | 3.8 | 0.18 | 7.8 × 1010 |
2 | 4.5 | 0.15 | 6.7 × 109 |
3 | 3.7 | 0.19 | 2.1 × 109 |
4 | 3.5 | 0.20 | 9.6 × 108 |
Mean ± SE | 3.8 ± 0.2 | 0.18 ± 0.01 | 2.0 × 1010 ± 1.0 × 1010 |
953.
Thomas W. Geisbert Michael Bailey Joan B. Geisbert Clement Asiedu Mario Roederer Maria Grazia-Pau Jerome Custers Peter Jahrling Jaap Goudsmit Richard Koup Nancy J. Sullivan 《Journal of virology》2010,84(19):10386-10394
The immunogenicity and durability of genetic vaccines are influenced by the composition of gene inserts and choice of delivery vector. DNA vectors are a promising vaccine approach showing efficacy when combined in prime-boost regimens with recombinant protein or viral vectors, but they have shown limited comparative efficacy as a stand-alone platform in primates, due possibly to suboptimal gene expression or cell targeting. Here, regimens using DNA plasmids modified for optimal antigen expression and recombinant adenovirus (rAd) vectors, all encoding the glycoprotein (GP) gene from Angola Marburg virus (MARV), were compared for their ability to provide immune protection against lethal MARV Angola infection. Heterologous DNA-GP/rAd5-GP prime-boost and single-modality rAd5-GP, as well as the DNA-GP-only vaccine, prevented death in all vaccinated subjects after challenge with a lethal dose of MARV Angola. The DNA/DNA vaccine induced humoral responses comparable to those induced by a single inoculation with rAd5-GP, as well as CD4+ and CD8+ cellular immune responses, with skewing toward CD4+ T-cell activity against MARV GP. Vaccine regimens containing rAd-GP, alone or as a boost, exhibited cellular responses with CD8+ T-cell dominance. Across vaccine groups, CD8+ T-cell subset dominance comprising cells exhibiting a tumor necrosis factor alpha (TNF-α) and gamma interferon (IFN-γ) double-positive functional phenotype was associated with an absence or low frequency of clinical symptoms, suggesting that both the magnitude and functional phenotype of CD8+ T cells may determine vaccine efficacy against infection by MARV Angola.The filoviruses Marburgvirus (MARV) and Ebolavirus (EBOV) are endemic primarily to central Africa and cause a severe form of viral hemorrhagic fever. Of all the filovirus strains or species, the Angola strain of MARV is associated with the highest mortality rate (90%) in humans observed to date (26). An increase in natural filovirus outbreak frequency over the past decade and the potential for use to cause deliberate human mortality have focused attention on the need for therapeutics and vaccines against filoviruses. While regulatory pathways have been proposed to facilitate licensing of a preventive vaccine against potently lethal pathogens such as these, there is as yet no licensed vaccine for use in humans, and efforts remain targeted to the optimization of vaccine performance in nonhuman primates (NHP) since this animal model recapitulates many aspects of disease pathogenesis observed in humans.Genetic vaccines are a promising approach for immunization against pathogens that are rapidly changing due to natural evolution, cross-species transmission, or intentional modification. Gene-based vaccines are produced rapidly and can be delivered by a variety of vectors. DNA vectors are advantageous because they are inherently safe and stable and can be used repeatedly without inducing antivector immune responses. However, while filovirus DNA vaccines have demonstrated efficacy in small animal models, efforts to induce protective immunity by injection of plasmid DNA alone into NHP have yielded less encouraging results. EBOV DNA vectors generate immune protection in mice and guinea pigs, but this has not been demonstrated in NHP unless DNA immunization is boosted with a viral vector vaccine (23). MARV DNA fully protects mice and guinea pigs but provides only partial protection in NHP (17). The discordant results between rodent and primate species may be due to the use of slightly modified infectious challenge viruses in rodent models or may reflect underlying differences in vaccine performance and the mechanisms of immune protection between rodents and NHP.In the current study, we examined whether DNA plasmid-based vaccines could be improved to increase potency in NHP and compared immunogenicity of this vaccine modality with those of viral vector and prime-boost approaches. DNA-vectored vaccines were modified by codon optimizing gene target inserts for enhanced expression in primates. These vectors induced antigen-specific cellular and humoral immune responses similar to immunization using a recombinant adenoviral vector and provided protection after lethal challenge with MARV Angola. However, macaques vaccinated with DNA vectors exhibited clinical symptoms associated with MARV hemorrhagic fever (MHF) that were absent in NHP receiving a single inoculation with recombinant adenovirus (rAd) vectors, suggesting qualitative differences in the immune responses elicited by the different modalities. 相似文献
954.
Beatriz Blanco-Fontao Alberto Fernández-Gil José Ramón Obeso Mario Quevedo 《Journal of Ornithology》2010,151(2):269-277
Cantabrian capercaillie Tetrao urogallus cantabricus is a peripheral population with distinctive phenotypic, biogeographic, and genetic characteristics. Hence, the population
may also show substantial ecological differentiation associated with its habitat in purely deciduous forests. We assessed
seasonal diet selection, small-scale habitat selection, and patterns of trophic niche width in Cantabrian capercaillie over
two years. Diet was found to be a driver of small-scale habitat selection, a result consistent with previous studies of stand-scale
habitat selection. Diet and habitat selection showed the importance of beech Fagus sylvatica, holly Ilex aquifolium, bilberry Vaccinium myrtillus, and ferns in Cantabrian capercaillie’s resource selection. Conversely, the abundant oaks Quercus petraea, birches Betula pubescens, and heaths Erica sp. were used below their availability. The reliance on bilberry appears as a unifying characteristic between central and peripheral
capercaillie populations. Cantabrian capercaillie showed stronger reliance on understory resources than range-central populations.
It also showed wider trophic niche and higher specialization of feeding events. Trophic niche patterns and reliance on ground
resources indicated a marked ecological differentiation, which stresses the need for local data and specific conservation
actions. 相似文献
955.
Roberto Bertoni Cristiana Callieri Gianluca Corno Serena Rasconi Emanuele Caravati Mario Contesini 《Hydrobiologia》2010,644(1):279-287
We analysed the long-term dynamics (1980–2007) of hypolimnetic and epilimnetic bacterial abundances and organic carbon concentrations,
both dissolved (DOC) and particulate (POC), in the deep holo-oligomictic Lake Maggiore, included in the Southern Alpine Lakes
Long-Term Ecological Research (LTER) site. During the 28 years of investigation, bacterial abundance and POC concentrations
did not decrease with declining phosphorus concentrations, while DOC concentrations showed a pronounced decrease in the epi-
and hypolimnion. We used the annual mean total lake heat content and total annual precipitation as climate-related variables,
and in-lake total phosphorus as a proxy for trophic state. The model (forward stepwise regression, FSR) showed that reduced
anthropogenic pressure was more significant than climate change in driving the trend in DOC concentrations. Bacterial dynamics
in the hypolimnion mirrored the fluctuations observed in the epilimnion, but average cell abundance was three times lower.
The FSR model indicates that bacterial number variability was dependent on POC in the epilimnion and DOC in the hypolimnion.
In the hypolimnion, cell biovolumes for rod and coccal morphotypes were significantly larger than in the epilimnion. 相似文献
956.
A.P. Shinn C. Collins M. Snow G. Paladini T. Lindenstrøm J.F. Turnbull C. Vázquez Rivera T.A. Mo K. Olstad P.D. Harris D. Graham G.H. Yoon N.G.H. Taylor R. Raynard J.E. Bron 《International journal for parasitology》2010,40(12):1455-1467
Despite routine screening requirements for the notifiable fish pathogen Gyrodactylus salaris, no standard operating procedure exists for its rapid identification and discrimination from other species of Gyrodactylus. This study assessed screening and identification efficiencies under real-world conditions for the most commonly employed identification methodologies: visual, morphometric and molecular analyses. Obtained data were used to design a best-practice processing and decision-making protocol allowing rapid specimen throughput and maximal classification accuracy. True specimen identities were established using a consensus from all three identification methods, coupled with the use of host and location information. The most experienced salmonid gyrodactylid expert correctly identified 95.1% of G. salaris specimens. Statistical methods of classification identified 66.7% of the G. salaris, demonstrating the need for much wider training. Molecular techniques (internal transcribed spacer region-restriction fragment length polymorphism (ITS-RFLP)/cytochrome c oxidase I (COI) sequencing) conducted in the diagnostic laboratory most experienced in the analysis of gyrodactylid material, identified 100% of the true G. salaris specimens. Taking into account causes of potential specimen loss, the probabilities of a specimen being accurately identified were 95%, 87% and 92% for visual, morphometric and molecular techniques, respectively, and the probabilities of correctly identifying a specimen of G. salaris by each method were 81%, 58% and 92%. Inter-analyst agreement for 189 gyrodactylids assessed by all three methods using Fleiss’ Kappa suggested substantial agreement in identification between the methods. During routine surveillance periods when low numbers of specimens are analysed, we recommend that specimens be analysed using the ITS-RFLP approach followed by sequencing of specimens with a “G. salaris-like” (i.e. G. salaris, Gyrodactylus thymalli) banding pattern. During periods of suspected outbreaks, where a high volume of specimens is expected, we recommended that specimens be identified using visual identification, as the fastest processing method, to select “G. salaris-like” specimens, which are subsequently identified by molecular-based techniques. 相似文献
957.
Neungseon Seo Brooke H. Russell Jose J. Rivera Xiaowen Liang Xuejun Xu Vahid Afshar-Kharghan Magnus H??k 《The Journal of biological chemistry》2010,285(40):31046-31054
Collagen is an extracellular matrix structural component that can regulate cellular processes through its interaction with the integrins, α1β1, α2β1, α10β1, and α11β1. Collagen-like proteins have been identified in a number of bacterial species. Here, we used Scl2 from Streptococcus pyogenes serotype M28 strain MGAS6274 as a backbone for the introduction of discrete integrin-binding sequences. The introduced sequences GLPGER, GFPGER, or GFPGEN did not affect triple helix stability of the Scl (Streptococcal collagen-like) protein. Using ELISA and surface plasmon resonance, we determined that Scl2GLPGER and Scl2GFPGER bound to recombinant human α1 and α2 I-domains in a metal ion-dependent manner and without a requirement for hydroxyproline. We predicted a novel and selective integrin-binding sequence, GFPGEN, through the use of computer modeling and demonstrated that Scl2GFPGEN shows specificity toward the α1 I-domain and does not bind the α2 I-domain. Using C2C12 cells, we determined that intact integrins interact with the modified Scl2 proteins with the same selectivity as recombinant I-domains. These modified Scl2 proteins also acted as cell attachment substrates for fibroblast, endothelial, and smooth muscle cells. However, the modified Scl2 proteins were unable to aggregate platelets. These results indicate that Scl2 is a suitable backbone for the introduction of mammalian integrin-binding sequences, and these sequences may be manipulated to individually target α1β1 and α2β1. 相似文献
958.
S. Mario Sousa 《Brittonia》2010,62(4):321-336
The Lonchocarpus cruentus complex, within sect. Lonchocarpus, is described. This complex is characterized by the presence of a keeled vexillary margin on the pod. Five of the six species
described and illustrated are new to science: Lonchocarpus aequatorialis, L. alternifoliolatus, L. guianensis, L. septentrionalis and L. trinitensis. A pronounced flower size difference in the north-south distribution of this complex is shown. 相似文献
959.