IntroductionThe infiltration of FOXP3+ regulatory T-cells into invasive tumors has been reported to be associated with survival in a variety of cancers. The prognostic significance of FOXP3+ tumor infiltrating lymphocytes (TILs) in breast cancer, however, remains controversial.MethodsFOXP3+ TILs were assessed by immunohistochemistry on tissue microarrays constructed from a well-defined cohort of 3,992 breast cancer patients linked to detailed demographic, biomarker, treatment and outcome data. Survival analyses were performed using the Kaplan-Meier function and Cox proportional hazards regression models to evaluate the association of FOXP3+ TILs with breast cancer specific survival, stratified by intrinsic subtype and cytotoxic T-cell infiltration status (as defined by CD8 immunohistochemistry).ResultsThe presence of high numbers of FOXP3+ TILs was significantly associated with young age, high grade, estrogen receptor (ER) negativity, concurrent CD8+ cytotoxic T-cell infiltration, and human epidermal growth factor receptor-2 positive (HER2+)/ER¿ and core basal subtypes. On multivariate survival analysis, a high level of FOXP3+ TILs was significantly associated with poor survival in ER+ breast cancers that lacked CD8+ T-cell infiltrates (Hazard ratio (HR)¿=¿1.30, 95% confidence interval (CI)¿=¿1.02 to 1.66). However, in ER¿ breast cancers, FOXP3+ TILs were strongly associated with improved survival in the HER2+/ER¿ subgroup, particularly in those with co-existent CD8+ T-cell infiltrates (HR¿=¿0.48, 95% CI¿=¿0.23 to 0.98), for which the presence of high levels of FOXP3+ TILs was independent of standard clinical prognostic factors.ConclusionsFOXP3+ regulatory TILs are a poor prognostic indicator in ER+ breast cancer, but a favorable prognostic factor in the HER2+/ER¿ subtype. The prognostic value of FOXP3+ TILs in breast cancer differs depending on ER and HER2 expression status and CD8+ T-cell infiltration.
SNAIL and SLUG are zinc-finger transcription factors that participate in the regulation of cell division, cell survival, mesoderm formation and epithelial-to-mesenchymal transition. We investigate the expression of SNAIL and SLUG during follicular maturation, ovulation and luteinization in the ovaries of both neonatal mice and gonadotropin-induced immature mice. Furthermore, we examine the expression and localization of these transcription factors during early embryonic cleavage. Our data demonstrate that both SNAIL and SLUG are present in the epithelial cells of the ovarian surface in immature mice. SNAIL is first evident in the interstitial cells and theca cells by postnatal day (PD) 6 and then appears in the oocytes by PD 8, remaining at a constant expression level for all stages studied thereafter. SLUG is expressed in oocytes as early as PD 1. Its expression also increases with the development of the follicles in theca and interstitial cells but not in granulosa cells. In gonadotropin-induced immature mice, both SNAIL and SLUG are expressed in the corpora lutea. During early embryo cleavage, SNAIL occurs in the nucleus and cytoplasm of the majority of the embryo, excluding the nucleolus from the germinal vesicle breakdown (GVBD) to the 8-cell stage and is then localized in the cytoplasm during the morula stage and in the nucleus during the blastocyst stage. SLUG has an identical expression pattern as SNAIL from GVBD until the morula stage, except that it is localized in the cytoplasm during the blastocyst stage. Taken together, these different localization patterns suggest that SNAIL and SLUG probably play important roles during follicular development, luteinization and early embryonic development.
The present study examined deposition fluxes of aerosol particles and 15 polycyclic aromatic hydrocarbons (PAHs) associated with the particles in the North China Plain. The annual mean deposition fluxes of aerosol particles and 15 PAHs were 0.69 ± 0.46 g/(m(2) ×d) and 8.5 ± 6.2 ?g/(m(2) ×d), respectively. Phenanthrene, fluoranthene, pyrene, chrysene, benzo[b]fluoranthene, and benzo[k]fluoranthene were the dominant PAHs bound to deposited aerosol particles throughout the year. The total concentration of 15 PAHs in the deposited aerosol particles was the highest in winter but lowest in spring. The highest PAH concentration in the deposited aerosol particles in winter was because the heating processes highly increased the concentration in atmospheric aerosol particles. Low temperature and weak sunshine in winter reduced the degradation rate of deposited aerosol particle-bound PAHs, especially for those with low molecular weight. The lowest PAH concentration in deposited aerosol particles in spring resulted from the frequently occurring dust storms, which diluted PAH concentrations. The mean deposition flux of PAHs with aerosol particles in winter (16 ?g/[m(2) ×d]) reached 3 times to 5 times that in other seasons (3.5-5.0 ?g/[m(2) ×d]). The spatial variation of the deposition flux of PAHs with high molecular weight (e.g., benzo[a]pyrene) was consistent with their concentrations in the atmospheric aerosol particles, whereas such a phenomenon was not observed for those with low molecular weight (e.g., phenanthrene) because of their distinct hydrophobicity, Henry's law constant, and the spatially heterogeneous meteorological conditions.
NanoString's Prosigna™ Breast Cancer Prognostic Gene Signature Assay is based on the PAM50 gene expression signature. The test outputs a risk of recurrence (ROR) score, risk category, and intrinsic subtype (Luminal A/B, HER2-enriched, Basal-like). The studies described here were designed to validate the analytical performance of the test on the nCounter Analysis System across multiple laboratories.
To assess the prognostic value of the PAM50 risk-of-recurrence (ROR) score on late distant recurrence (beyond 5 years after diagnosis and treatment) in a large cohort of postmenopausal, endocrine-responsive breast cancer patients.
Respiratory syncytial virus (RSV) is an important viral pathogen that causes life-threatening respiratory infections in both infants and the elderly; no vaccines are at present available. In this report, we examined the use of influenza virus as a vehicle for production of an experimental RSV vaccine. We used reverse genetics to generate a recombinant influenza A virus with epitopes from the RSV fusion (F) and attachment (G) proteins (rFlu/RSV/F+G) in the influenza virus nonstructural (NS1) protein gene. Expression of RSV F+G epitope proteins was confirmed by Western blotting, and no changes in viral morphology were evident following examination by electron microscopy. BALB/c mice immunized intranasally with rFlu/RSV/F+G showed viral-specific antibody responses against both influenza and RSV. Total IgG, IgG1, IgG2a and IgA were measured in mice immunized with rFlu/RSV/F+G, revealing robust cellular and mucosal immune responses. Furthermore, we found that rFlu/RSV/F+G conferred protection against subsequent influenza and RSV challenges, showing significant decreases in viral replication and obvious attenuation of histopathological changes associated with viral infections. These findings suggest that rFlu/RSV/F+G is a promising vaccine candidate, which should be further assessed using cotton rat and primate models.
Polychlorinated biphenyls (PCBs) are stable, lipophilic compounds that accumulate in the environment and in the food chain. Though some studies provided evidence that PCBs had adverse effects on reproductive function, most of these results were from in vitro models. Therefore we investigated the effect of Aroclor 1254 (a commercial PCBs mixture) treatments on in vivo maturation and developmental potential of mouse oocytes. In the present study, female ICR mice were treated with different doses (12.5, 25 and 50 mg/kg) of Aroclor 1254 (a commercial PCB mixture) once every 72 hours by intraperitoneal injection for 9 days. After three treatments of Aroclor 1254, the mice were superovulated to collect oocytes one day after the last exposure. The effects of Aroclor 1254 on oocyte maturation, fertilization, and preimplantation embryonic development were investigated. Immunofluorescence-stained oocytes were observed under a confocal microscope to assess the effects of Aroclor 1254 on spindle morphology. Parthenogenic activation and the incidence of cumulus apoptosis in cumulus-oocyte complexes were observed as well. Oocytes exposed to different doses of Aroclor 1254 in vivo were associated with a significant decrease in outgrowth potential, abnormal spindle configurations, and the inhibition of parthenogenetic activation of ovulated oocytes. Furthermore, the incidence of apoptosis in cumulus cells was increased after exposed to Aroclor 1254. These results may provide reference for the treatment of reproductive diseases such as infertility or miscarriage caused by environmental contaminants.
Background. In vitro studies suggest basal breast cancers are more sensitive to gemcitabine relative to other intrinsic subtypes. The main objective of this study was to use specimens from a randomized clinical trial to evaluate whether the basal-like subtype identifies patients with advanced breast cancer who benefit from gemcitabine plus docetaxel (GD) compared to single agent docetaxel (D). Material and methods. From patients randomly assigned to GD or D, RNA was isolated from archival formalin-fixed, paraffin-embedded primary breast tumor tissue and used for PAM50 intrinsic subtyping by NanoString nCounter. Statistical analyses were prespecified as a formal prospective-retrospective clinical trial correlative study. Using time to progression (TTP) as primary endpoint, overall survival (OS) and response rate as secondary endpoints, relationships between subtypes and outcome after chemotherapy were analyzed by the Kaplan-Meier method, and Cox proportional hazards regression models. Data analysis was performed independently by the Danish Breast Cancer Cooperative Group (DBCG) statistical core and all statistical tests were two-sided. Results. RNA from 270 patients was evaluable; 84 patients (31%) were classified as luminal A, 97 (36%) luminal B, 43 (16%) basal-like, and 46 (17%) as HER2-enriched. PAM50 intrinsic subtype was a significant independent prognostic factor for both TTP (p = 0.014) and OS (p = 0.0003). Response rate was not different by subtype, and PAM50 was not a predictor of TTP by treatment arm. PAM50 was however a highly significant predictor of OS following GD compared to D (pinteraction = 0.0016). Patients with a basal-like subtype had a significant reduction in OS events [hazard ratio (HR) = 0.29, 95% confidence interval (CI) = 0.15-0.57; pinteraction = 0.0006]. Conclusion. A significantly improved and clinically important prolongation of survival was seen from the addition of gemcitabine to docetaxel in advanced basal-like breast cancer patients.
The preparative-scale separation of chiral compounds is vitally important for the pharmaceutical industry and related fields. Herein we report a simple approach for rapid preparative separation of enantiomers using functional nucleic acids modified gold nanoparticles (AuNPs). The separation of DL-tryptophan (DL-Trp) is demonstrated as an example to show the feasibility of the approach. AuNPs modified with enantioselective aptamers were added into a racemic mixture of DL -Trp. The aptamer-specific enantiomer (L-Trp) binds to the AuNPs surface through aptamer-L-Trp interaction. The separation of DL-Trp is then simply accomplished by centrifugation: the precipitate containing L-Trp bounded AuNPs is separated from the solution, while the D-Trp remains in the supernatant. The precipitate is then redispersed in water. The aptamer is denatured under 95 °C and a second centrifugation is then performed, resulting in the separation of AuNPs and L-Trp. The supernatant is finally collected to obtain pure L-Trp in water. The results show that the racemic mixture of DL-Trp is completely separated into D-Trp and L-Trp, respectively, after 5 rounds of repeated addition of fresh aptamer-modified AuNPs to the DL-Trp mixture solution. Additionally, the aptamer-modified AuNPs can be repeatedly used for at least eight times without significant loss of its binding ability because the aptamer can be easily denatured and renatured in relatively mild conditions. The proposed approach could be scaled up and extended to the separation of other enantiomers by the adoption of other enantioselective aptamers.
From field harvest to the consumers table, fresh citrus fruit spends a considerable amount of time in shipment and storage. During these processes, physiological disorders and pathological diseases are the main causes of fruit loss. Heat treatment (HT) has been widely used to maintain fruit quality during postharvest storage; however, limited molecular information related to this treatment is currently available at a systemic biological level.
P2X3 receptors are present in the spinal dorsal horn (SDH) and play an essential role in the regulation of nociception and pain. Pregabalin (PGB) has been used as a new antiepileptic drug in the treatment of neuropathic pain. However, it is unclear whether PGB-induced analgesia was associated with the P2X3 receptor in SDH. Here, rats were randomly divided into four groups (n?=?12 per group), including 2 sham operation groups, which was treated by normal saline (Sham?+?NS group) or PGB (Sham?+?PGB group), other 2 groups with chronic compression of the dorsal root ganglion, a normal saline-treated CCD group (CCD+NS group), and a PGB-treated CCD group (CCD?+?PGB group). A rat model of neuropathic pain was used by compressing the right L4 and L5 dorsal root ganglia. Each group was evaluated using the mechanical withdrawal threshold (MWT). The mRNA and protein levels of the P2X3 receptor in the ipsilateral SDH were measured by RT-PCR, western blot, and immunofluorescence on 14 day after CCD operation. CCD rats showed the highest mechanical hyperalgesia and the lowest pain threshold in the four groups. Simultaneously, CCD rats showed higher P2X3 mRNA and protein expression in ipsilateral side of the SDH than the sham operation rats. However, the MWT was increased and expression of P2X3 mRNA and protein in the ipsilateral SDH in CCD rats was decreased 3 days after PGB treatment. Thus, PGB may partially reverse mechanical hyperalgesia in CCD rats by inhibiting P2X3 receptor expression in the ipsilateral SDH.
Although the role of the erythropoietin (EPO) receptor (EpoR) in erythropoiesis has been known for decades, its role in nonhematopoietic tissues is still not well defined. Klotho has been shown and EPo has been suggested to protect against acute ischemia-reperfusion injury in the kidney. Here we found in rat kidney and in a rat renal tubular epithelial cell line (NRK cells) EpoR transcript and antigen, and EpoR activity signified as EPo-induced phosphorylation of Jak2, ErK, Akt, and Stat5 indicating the presence of functional EpoR. Transgenic overexpression of Klotho or addition of exogenous recombinant Klotho increased kidney EpoR protein and transcript. In NRK cells, Klotho increased EpoR protein, enhanced EPo-triggered phosphorylation of Jak2 and Stat5, the nuclear translocation of phospho-Stat5, and protected NRK cells from hydrogen peroxide cytotoxicity. Knockdown of endogenous EpoR rendered NRK cells more vulnerable, and overexpression of EpoR more resistant to peroxide-induced cytotoxicity, indicating that EpoR mitigates oxidative damage. Knockdown of EpoR by siRNA abolished Epo-induced Jak2, and Stat5 phosphorylation, and blunted the protective effect of Klotho against peroxide-induced cytotoxicity. Thus in the kidney, EpoR and its activity are downstream effectors of Klotho enabling it to function as a cytoprotective protein against oxidative injury.
Citric acid plays an important role in fresh fruit flavor and its adaptability to post-harvest storage conditions. In order to explore organic acid regulatory mechanisms in post-harvest citrus fruit, systematic biological analyses were conducted on stored Hirado Buntan Pummelo (HBP; Citrus grandis) fruits. High-performance capillary electrophoresis, subcellular organelle expression microarray, real-time quantitative reverse transcription polymerase chain reaction, gas chromatography mass spectrometry (GC-MS), and conventional physiological and biochemical analyses were undertaken. The results showed that the concentration of organic acids in HBP underwent a regular fluctuation. GC-MS-based metabolic profiling indicated that succinic acid, ?-aminobutyric acid (GABA), and glutamine contents increased, but 2-oxoglutaric acid content declined, which further confirmed that the GABA shunt may have some regulatory roles in organic acid catabolism processes. In addition, the concentration of organic acids was significantly correlated with senescence-related physiological processes, such as hydrogen peroxide content as well as superoxide dismutase and peroxidase activities, which showed that organic acids could be regarded as important parameters for measuring citrus fruit post-harvest senescence processes.
Pathogen contact induces quickly in Mammary Epithelial Cells (MEC) the expression of the proinflammatory cytokine IL-8 and delayed that of the bactericidal ?-defensin LAP. Both genes encoding these factors feature on their proximal promoter a composite NF-?B/CEBP binding site. We compare here in MEC the role of NF-?B and C/EBP factors in regulating basal and pathogen-induced expression of both genes from cattle. Abrogating NF-?B binding to that site by introduction of a single point mutation blocks promoter activity of both genes in reporter gene assays. Chromatin accessibility PCR and Chromatin immunoprecipitation reveal that the chromatin of the resting LAP promoter is tightly packed and NF-?B p50 homodimer binding prevails. Infection results in chromatin decompaction accompanied by predominant recruitment of NF-?B p65 for promoter activation. Overexpression of transcription factors confirms a stimulatory role of NF-?B p65 but also a repressive function of C/EBP? for LAP promoter activity. These factors reverse roles to control IL-8 expression. NF-?B p65 homodimers already reside on the resting IL-8 promoter and induction recruits NF-?B p50. Overexpression of both NF-?B factors represses the promoter in MEC, but not in HEK293 cells. Inhibitors of NF-?B activation and nuclear recruitment both tremendously increase basal and pathogen stimulated IL-8 mRNA concentrations in MEC. Mutation of the C/EBP-binding site blocks and overexpression of C/EBP? stimulates IL-8-promoter activity. Thus, the pathogen-induced fast activation of diverse transcription factors acting through a common promoter binding site is gene specifically differentiated into opposite functional significance for swiftly (IL-8) or slowly (LAP) induced genes in MEC.
Many functional details of the piscine Toll-like receptor (TLR) signal-mediated activation of immune defense are still elusive. We used an established reconstitution system of mammalian TLR signaling to examine if this system would allow for pathogen-dependent promoter activation of the serum amyloid A (SAA)-encoding gene from rainbow trout (Oncorhynchus mykiss) and if the key mediators MyD88 and Tollip from trout can functionally substitute for their mammalian orthologues. Cells of the established human embryonic kidney line HEK-293 were transiently co-transfected with vectors expressing bovine TLR2 or TLR4 factors and a reporter gene driven by the promoter of the trout SAA gene. Escherichia coli stimulation increased reporter gene expression more than 3-fold. Deletion series and point mutations identified in the proximal SAA promoter a composite overlapping binding site for NF-?B and CEBP factors as crucial for promoter activation. Overexpression of NF-?B p65, but not of p50 or different members of the CEBP factor family proved this factor as an essential driver for SAA expression. Overexpression of a transdominant-negative mutant of the trout MyD88 factor reduced TLR-mediated SAA promoter activation confirming functional conservation of its TIR domain. Overexpression of the Tollip factor from trout also quenched TLR-mediated NF-?B and TLR4-mediated SAA promoter activation. The MyD88 mutant and Tollip expression studies confirm the functional homology of both piscine factors and their mammalian counterparts. We provide for the first time evidence that also the Tollip-mediated negative loop of TLR signaling may be conserved in non-mammalian organisms.
Acetyl-CoA carboxylase-alpha (ACC-alpha) is the rate-limiting enzyme for de novo fatty acid synthesis. Among the four promoters expressing the bovine gene, promoter IA (PIA) is dominantly active and nutritionally regulated in lipogenic tissues. CCAAT/enhancer binding proteins are crucially involved in regulating the activity of this promoter. We examine here, which member of this family of transcription factors is most important for promoter activation. To differentiate the individual contribution of different members of the C/EBP family transcription factors controlling the ACC-alpha gene expression in cattle, we established vectors expressing full length (FL) or N-terminally truncated (DeltaN) variants of the C/EBP factors (alpha, -beta, -delta, and -epsilon) in mammalian cells. Using nuclear extracts of cells expressing the DeltaN-C/EBP factors we determined in electrophoretic mobility shift assays that C/EBPalpha, -beta and -epsilon, but not C/EBPdelta may directly bind to the cognate C/EBP-binding site in the immediate ACC-alpha PIA. Co-transfection analyses of the various FL-C/EBP expression vectors together with a reporter gene driven by the ACC-alpha-PIA promoter demonstrated that C/EBPbeta has the strongest activation potential. Hence, activity of this factor may be a key regulator of ACC-alpha-expression in lipogenic tissues.
InP hollow nanospheres with an average size of 550 nm and shell thickness of about 110 nm were solvothermally synthesized in EA (ethanolamine)-H2O binary solution at 190 degrees C for 36 h. The shells of InP hollow nanospheres were composed of small nanoparticles. The similar route has been extended to prepare Cd3P2, Cu3P and Sn4P3 hollow nanospheres in 150-190 degrees C for 24-36 h.
We examined if and how mammary epithelial cells (MECs) calibrate and confine the intensity of an inflammatory response elicited by different concentrations of mastitis pathogens. Therefore we quantified in primary bovine MEC the effect of different E. coli pathogen concentrations upon the abundance of mRNA molecules encoding factors of immune defence. Induced synthesis of the mRNAs encoding tumor necrosis factor alpha and interleukin 8 both clearly correlated with the E. coli dose 1h after stimulation. Also the decay rate of those mRNAs reflected the pathogen load. The higher the concentration of E. coli, the faster and stronger was the up regulation and also the subsequent degradation of those particular mRNA species. Modulation of the mRNA concentration of tristetraprolin, a factor crucially involved in the mRNA degradation, followed the same pattern. In contrast, extent and kinetics of increasing the mRNA concentrations of serum amyloid A3 and lingual antimicrobial peptide were almost independent of the pathogen dose. We show that MEC perceive the information about the different pathogen concentrations and convert this signal into a calibrated synthesis of pro-inflammatory cytokines. We suggest that selective degradation of the mRNA molecules encoding those inflammatory cytokines contributes significantly to prevent an overshooting immune response in the udder.
Myxoid liposarcoma displays variably aggressive behavior and responds poorly to available systemic therapies. Expression profiling followed by tissue microarray validation linked to patient outcome is a powerful approach for validating biological mechanisms and identifying prognostic biomarkers. We applied these techniques to independent series of primary myxoid liposarcomas in an effort to assess markers of adipose differentiation in myxoid liposarcoma and to identify prognostic markers that can be efficiently assessed by immunohistochemistry. Candidate genes were selected based on analysis of expression profiles from 9 primary myxoid/round liposarcomas and 45 other soft tissue tumors, and by reference to publicly available data sets. Protein products were validated on an adipose neoplasm tissue microarray, including 32 myxoid liposarcomas linked to patient outcome. Results were scored visually and correlated with clinical outcome by Kaplan-Meier and Cox regression analyses. In the study, by examining expression patterns of several lipogenic regulatory gene products, an immature adipogenic status was verified in myxoid liposarcomas. We also found that expression levels of the ret proto-oncogene, insulin-like growth factor 1 receptor, and insulin-like growth factor 2 correlate with poor metastasis-free survival, supporting a role for ERK/MAPK and PI3K/AKT pathways in clinically aggressive myxoid liposarcomas.
Estrogen receptor status in breast cancer is associated with response to hormonal therapy and clinical outcome. The additional value of progesterone receptor (PR) has remained controversial. We examine the value of PR for prognosis and response to tamoxifen on a population-based series of 4,046 invasive early stage breast cancer patients. Clinical information for age at diagnosis, stage, pathology, treatment and outcome was assembled for the study cohort; the median follow-up was 12.4 years. PR status was determined by immunohistochemistry using a rabbit monoclonal antibody on tissue microarrays built from breast tumor surgical excisions. Survival analyses, Kaplan-Meier functions and Cox proportional hazards regression models were applied to assess the associations between PR and breast cancer specific survival. Progesterone receptor was positive in 51% of all cases and 67% of estrogen receptor positive (ER+) cases. Survival analyses for both the whole cohort and ER+ cases given tamoxifen therapy showed that patients with PR+ tumors had 24% higher relative probability for breast cancer specific survival as compared to PR- patients, adjusted for ER, HER2, age at diagnosis, grade, tumor size, lymph node status and lymphovascular invasion covariates. Higher PR expression showed stronger association with patient survival. Log-likelihood ratio tests of multivariate Cox proportional hazards regression models demonstrated that PR was an independent statistically significant factor for breast cancer specific survival in both the whole cohort and among ER+ cases treated with tamoxifen. PR adds significant prognostic value in breast cancer beyond that obtained with estrogen receptor alone.
Tumor infiltrating lymphocytes may indicate an immune response to cancer development, but their significance remains controversial in breast cancer. We conducted this study to assess CD8+ (cytotoxic T) lymphocyte infiltration in a large cohort of invasive early stage breast cancers, and to evaluate its prognostic effect in different breast cancer intrinsic subtypes.
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