技术资料

To investigate the difference in heart rate (HR) recovery after exercise between children and young adults,we administered a constant load of light exercise intensity and progressive treadmill exercise tests to nine children (aged 9 to 12 y,group A) and eight young adults (six male and two female,aged 17 to 21 y,group B) who had a history of Kawasaki disease without significant coronary arterial lesions. HR after both exercise protocols was analyzed. The low-frequency (LF) and high-frequency (HF) components of HR variability were measured,and LF/HF was calculated (log LF,log HF,log L/H). Arterial baroreflex sensitivity was assessed by the phenylephrine method. There were no differences between groups A and B in resting HR,peak HR,peak oxygen uptake,and decreases in systolic blood pressure during the recovery period. HR 1 and 2 min after peak exercise and 1 min after constant-load exercise was significantly lower in group A than in group B (p {\textless} 0.05),and the changes in HR from peak values after both exercise tests were also greater in group A than in group B (p {\textless} 0.05-0.01). Although no difference in arterial baroreflex sensitivity was observed,log HF was significantly higher in group A than in group B (p {\textless} 0.01),and log L/H was significantly lower in group A than in group B (p {\textless} 0.05). The value of log HF correlated inversely with the decrease in HR immediately after both exercise protocols (p {\textless} 0.05-0.01). Although log L/H correlated with the decrease in HR after peak exercise (p {\textless} 0.05-0.0005),the early decline in HR after constant-load exercise did not correlate with log L/H. Arterial baroreflex sensitivity did not correlate with the decrease in HR at any recovery time. These data suggest that the early phase of HR recovery after light to severe exercise is influenced by the cardiac parasympathetic nervous activity at rest and that the greater central cholinergic modulation of HR in children than in young adults may be responsible in part for children's faster HR recovery after exercise. View Publication

Pulmonary metastasis of breast cancer requires recruitment and expansion of T-regulatory cells (Treg) that promote escape from host protective immune cells. However,it remains unclear precisely how tumors recruit Tregs to support metastatic growth. Here we report the mechanistic involvement of a unique and previously undescribed subset of regulatory B cells. These cells,designated tumor-evoked Bregs (tBreg),phenotypically resemble activated but poorly proliferative mature B2 cells (CD19(+) CD25(High) CD69(High)) that express constitutively active Stat3 and B7-H1(High) CD81(High) CD86(High) CD62L(Low) IgM(Int). Our studies with the mouse 4T1 model of breast cancer indicate that the primary role of tBregs in lung metastases is to induce TGF-$\beta$-dependent conversion of FoxP3(+) Tregs from resting CD4(+) T cells. In the absence of tBregs,4T1 tumors cannot metastasize into the lungs efficiently due to poor Treg conversion. Our findings have important clinical implications,as they suggest that tBregs must be controlled to interrupt the initiation of a key cancer-induced immunosuppressive event that is critical to support cancer metastasis. View Publication

DNA double-strand breaks (DSB) are the most cytotoxic lesions induced by topoisomerase II poisons. Nonhomologous end joining (NHEJ) is a major pathway for DSB repair and requires DNA-dependent protein kinase (DNA-PK) activity. DNA-PK catalytic subunit (DNA-PKcs) is structurally similar to PI-3K,which promotes cell survival and proliferation and is upregulated in many cancers. KU-0060648 is a dual inhibitor of DNA-PK and PI-3K in vitro. KU-0060648 was investigated in a panel of human breast and colon cancer cells. The compound inhibited cellular DNA-PK autophosphorylation with IC(50) values of 0.019 $\mu$mol/L (MCF7 cells) and 0.17 $\mu$mol/L (SW620 cells),and PI-3K-mediated AKT phosphorylation with IC(50) values of 0.039 $\mu$mol/L (MCF7 cells) and more than 10 $\mu$mol/L (SW620 cells). Five-day exposure to 1 $\mu$mol/L KU-0060648 inhibited cell proliferation by more than 95{\%} in MCF7 cells but only by 55{\%} in SW620 cells. In clonogenic survival assays,KU-0060648 increased the cytotoxicity of etoposide and doxorubicin across the panel of DNA-PKcs-proficient cells,but not in DNA-PKcs-deficient cells,thus confirming that enhanced cytotoxicity was due to DNA-PK inhibition. In mice bearing SW620 and MCF7 xenografts,concentrations of KU-0060648 that were sufficient for in vitro growth inhibition and chemosensitization were maintained within the tumor for at least 4 hours at nontoxic doses. KU-0060648 alone delayed the growth of MCF7 xenografts and increased etoposide-induced tumor growth delay in both in SW620 and MCF7 xenografts by up to 4.5-fold,without exacerbating etoposide toxicity to unacceptable levels. The proof-of-principle in vitro and in vivo chemosensitization with KU-0060648 justifies further evaluation of dual DNA-PK and PI-3K inhibitors. View Publication

During the progression of autoimmune (type 1) diabetes,T cells and macrophages infiltrate the pancreas,disrupt islet function,and destroy insulin-producing beta cells. B-lymphocytes,particularly innate like B-cell populations such as marginal zone B cells and B-1 cells,have been implicated in many autoimmune diseases,and non-obese diabetic (NOD) mice that lack B cells do not develop spontaneous autoimmune diabetes. Hence,inhibitors of B cell signaling pathways could be useful for limiting the autoimmune processes that contribute to type 1 diabetes. Signaling via phosphoinositide 3-kinase (PI3K) regulates many cellular processes. The p110$\delta$ isoform of PI3K is expressed primarily in cells of hematopoietic origin and the catalytic activity of p110$\delta$ is important for B cell migration,activation,proliferation,and antigen presentation. Because innate-like B cells are particularly sensitive to inhibition of p110$\delta$ activity,and p110$\delta$ inhibitors also suppress pro-inflammatory functions of other cell types that contribute to autoimmunity,we tested whether a p110$\delta$ inhibitor could delay the onset or reduce the incidence of autoimmune diabetes in NOD mice. We found that long-term preventative treatment of pre-diabetic NOD mice with IC87114,a highly selective small molecule inhibitor of p110$\delta$,reduced the infiltration of inflammatory cells into the pancreatic islets and,accordingly,delayed and reduced the loss of glucose homeostasis. Moreover in a therapeutic treatment mode,IC87114 treatment conferred prolonged protection from progression to overt diabetes in a number of animals. These findings suggest that PI3K$\delta$ inhibitors could be useful for managing type 1 diabetes. View Publication

Characterization of pluripotent stem cells is required for the registration of stem cell lines and allows for an impartial and objective comparison of the results obtained when generating multiple lines. It is therefore crucial to establish specific,fast and reliable protocols to detect the hallmarks of pluripotency. Such protocols should include immunocytochemistry (takes 2 d),identification of the three germ layers in in vitro-derived embryoid bodies by immunocytochemistry (immunodetection takes 3 d) and detection of differentiation markers in in vivo-generated teratomas by immunohistochemistry (differentiation marker detection takes 4 d). Standardization of the immunodetection protocols used ensures minimum variations owing to the source,the animal species,the endogenous fluorescence or the inability to collect large amounts of cells,thereby yielding results as fast as possible without loss of quality. This protocol provides a description of all the immunodetection procedures necessary to characterize mouse and human stem cell lines in different circumstances. View Publication

IMPORTANCE Although leukocyte telomere length is associated with mortality and many chronic diseases thought to be manifestations of age-related functional decline,it is not known whether it relates to acute disease in younger healthy populations. OBJECTIVE To determine whether shorter telomeres in leukocytes,especially CD8CD28- T cells,are associated with decreased resistance to upper respiratory infection and clinical illness in young to midlife adults. DESIGN,SETTING,AND PARTICIPANTS Between 2008 and 2011,telomere length was assessed in peripheral blood mononuclear cells (PBMCs) and T-cell subsets (CD4,CD8CD28+,CD8CD28-) from 152 healthy 18- to 55-year-old residents of Pittsburgh,Pennsylvania. Participants were subsequently quarantined (single rooms),administered nasal drops containing a common cold virus (rhinovirus 39),and monitored for 5 days for development of infection and clinical illness. MAIN OUTCOME MEASURES Infection (virus shedding or 4-fold increase in virus-specific antibody titer) and clinical illness (verified infection plus objective signs of illness). RESULTS Rates of infections and clinical illness were 69{\%} (n = 105) and 22{\%} (n = 33),respectively. Shorter telomeres were associated with greater odds of infection,independent of prechallenge virus-specific antibody,demographics,contraceptive use,season,and body mass index (PBMC: odds ratio [OR] per 1-SD decrease in telomere length,1.71 [95{\%} CI,1.08-2.72]; n = 128 [shortest tertile 77{\%} infected; middle,66{\%}; longest,57{\%}]; CD4: OR,1.76 [95{\%} CI,1.15-2.70]; n = 146 [shortest tertile 80{\%} infected; middle,71{\%}; longest,54{\%}]; CD8CD28+: OR,1.93 [95{\%} CI,1.21-3.09],n = 132 [shortest tertile 84{\%} infected; middle,64{\%}; longest,58{\%}]; CD8CD28-: OR,2.02 [95{\%} CI,1.29-3.16]; n = 144 [shortest tertile 77{\%} infected; middle,75{\%}; longest,50{\%}]). CD8CD28- was the only cell population in which shorter telomeres were associated with greater risk of clinical illness (OR,1.69 [95{\%} CI,1.01-2.84]; n = 144 [shortest tertile,26{\%}; middle,22{\%}; longest,13{\%}]). The association between CD8CD28- telomere length and infection increased with age (CD8CD28- telomere length × age interaction,b = 0.09 [95{\%} CI,0.02-0.16],P = .01,n = 144). CONCLUSION AND RELEVANCE In this preliminary study among a cohort of healthy 18- to 55-year-olds,shorter CD8CD28- T-cell telomere length was associated with increased risk for experimentally induced acute upper respiratory infection and clinical illness. View Publication

This protocol describes a strategy for the generation of 3D prostate organoid cultures from healthy mouse and human prostate cells (either bulk or FACS-sorted single luminal and basal cells),metastatic prostate cancer lesions and circulating tumor cells. Organoids derived from healthy material contain the differentiated luminal and basal cell types,whereas organoids derived from prostate cancer tissue mimic the histology of the tumor. We explain how to establish these cultures in the fully defined serum-free conditioned medium that is required to sustain organoid growth. Starting with the plating of digested tissue material,full-grown organoids can usually be obtained in ∼2 weeks. The culture protocol we describe here is currently the only one that allows the growth of both the luminal and basal prostatic epithelial lineages,as well as the growth of advanced prostate cancers. Organoids established using this protocol can be used to study many different aspects of prostate biology,including homeostasis,tumorigenesis and drug discovery. View Publication