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No effect on the inhibition of IFN-γ and IL-17 production by responder cells was noted following Th1 or Th2 cell exposure to UCB (Supplemental Figure 1G), indicating that UCB immunoregulatory properties were predominantly impacting Th17 cell function.

Evaluation of ectoenzymatic activity showed that UCB increased phosphohydrolysis of extracellular nucleotides in the culture supernatant of Th17 cells from healthy subjects but did not have any effect on Th17 cells obtained from patients (Figure 3J). TLC analysis showed that in both healthy subjects and patients UCB reduced Th17 cell adenosine deaminase (ADA) activity, as demonstrated by lower conversion of adenosine into inosine (Supplemental Figure 2A). UCB exposure, however, appeared to impact Th17 cell ADA activity (Supplemental Figure 2A), rather than expression (Supplemental Figure 2B).

The importance of CD39 in the modulation of experimental colitis by UCB was corroborated by in vivo data and strengthened by the observation that treatment of WT mice with UCB increased the proportion of CD4+CD39+ cells in the colon.

There is evidence that Th17 cells acquire immunoregulatory features that are associated with attenuation of the pathogenic potential (18, 45). Our findings indicate that UCB enhances control of Th17 cell regulatory properties, as clearly highlighted by our in vitro data, and boosts immunoregulation in vivo by decreasing IL-17 production and by increasing CD39 and IL-10 expression levels by CD4+ cells in the colonic compartment. These in vitro and in vivo studies support a role for UCB in modulating pathogenicity while boosting immunoregulation.

It remains unclear whether the beneficial effects of UCB on the Th17 cell immune responsiveness depend solely on immunosuppressant properties effected by AHR or whether these can also be linked to UCB antioxidant effects, as previously shown for macrophages (46). Further studies will explore this possibility and further dissect out whether UCB antioxidant effects might also impact the interaction with AHR as well as derived immunoregulatory effects.

Although the importance of the hematopoietic transcription factor PU.1 in acute myeloid leukemia (AML) has been demonstrated, the expression of PU.1 in acute promyelocytic leukemia (APL) patient samples awaits further investigation. The current study used APL patient samples to assess the expression pattern of PU.1 in the initiation and progression of APL.

We used real-time RT-PCR to compare PU.1 expression between de novo APL patient samples and normal blood specimens, and the results indicated that PU.1 expression was significantly lower in newly diagnosed APL patient samples as compared to normal hematopoietic cells. Further evidence showed a significant inverse correlation between the expression level of PML-RARα and that of PU.1. In addition, we analyzed the correlation between PML-RARα and PU.1 expression in a large population of AML patients retrieved from the expression profiles. The results showed that PU.1 expression was lower in patients with APL than other AML subtypes and there was also a trend towards increasing PU.1 expression from AML-M0 to AML-M5, with the exception of AML-M3 (APL). These observations suggested that PU.1 expression was reduced by PML-RARα in APL patients. Furthermore, we measured PU.1 expression in APL-initiating cells isolated from de novo APL patients by side population cell analysis and found that suppression of PU.1 expression occurred concurrently with PML-RARα expression, indicating the pivotal role of PU.1 in APL initiation.

Acute promyelocytic leukemia (APL) is typified by the t(15;17) translocation, which generates the PML-RARα fusion protein and produces a beneficial response to all-trans retinoic acid (ATRA) and arsenic trioxide [1]. At the molecular level, PML-RARα affects the normal functions of wild-type PML and RARα signaling. However, PML- or RARα-deficient mice display few obvious defects [2, 3]. Furthermore, in PML-RARα transgenic mice, on average, only 30% develop APL after a long latent period of observation [4], suggesting that APL development may require additional genetic events that are indispensable for myeloid differentiation.

Recently, we demonstrated that PML-RARα interferes with the function of PU.1, which results in a block of downstream PU.1 signaling [5]. In addition, the study by Mueller BU et al. also reported that PU.1 is suppressed by PML-RARα and that ATRA treatment is capable of restoring PU.1 expression [6]. Although these findings have been confirmed using cell lines, there is a growing evidence to suggest that cell lines do not fully recapitulate the biology of human disease. Therefore, to most accurately examine the role of PU.1 in APL, investigation into the expression profile of PU.1 in APL patient samples is required.

The study was approved by the Ethics Committee of Ruijin Hospital affiliated to Shanghai Jiaotong University School of Medicine and was adherent to the regulations of the declaration of Helsinski. Bone marrow specimens were obtained after receiving informed consent from patients at the time of their diagnosis with de novo APL (Table 1). Peripheral blood specimens were obtained from healthy volunteers with informed consent. Umbilical cord blood (UCB) specimens were obtained with informed consent from volunteer donors attending the obstetrics department at Ruijin Hospital.

Total RNA was extracted from cells using an RNeasy Kit from Qiagen (Chatsworth, CA). Reverse transcription was performed using the Superscript II reagent set (Invitrogen, Carlsbad, CA) with random hexamer primers. Quantitative real-time PCR was performed using an ABI Prism 7900HT detection system (Applied Biosystems, Foster City, CA). The relative expression level for each target in comparison to the internal control GAPDH was calculated using the following equation: ΔCt = Ct (target) - Ct (GAPDH), where the relative mRNA expression = 2-ΔCt × 100. Each assay was performed in triplicate.

We first examined PU.1 mRNA expression in mononuclear cells from 10 newly diagnosed APL patients with high percentages of blasts (> 80%, mean 91%) (Table 1). The peripheral blood cells isolated from 7 healthy volunteers and the normal hematopoietic stem cell-enriched CD34+ cells isolated from 8 fresh human UCB specimens served as controls. As shown in Figure 1, PU.1 expression was significantly lower in the primary APL samples in comparison to normal hematopoietic cells, including white blood cells (WBCs), mononuclear cells (MNCs), granulocytes and immature progenitor cells (p = 7.6 × 10-7, 4.6 × 10-4, 1.5 × 10-7 and 0.015, respectively).

Lower level of PU.1 mRNA expression in de novo APL patients in comparison to normal hematopoietic cells. The relative PU.1 expression, as compared to that of the reference gene GAPDH, in leukemic cells freshly isolated from APL samples (Table 1), normal peripheral blood cells (WBCs, MNCs, and granulocytes) and CD34+ cells from human UCB specimens was determined using real-time RT-PCR. Each symbol represents the average value from an individual patient or healthy subject, and the lines indicate the median value.

Furthermore, we evaluated PU.1 expression for each subtype of AML according to the French-American-British (FAB) classification. As shown in Figure 3, low PU.1 expression was observed in the M3 subtype (APL). Interestingly, in addition to the M3 subtype, low PU.1 expression was also observed in the M0, M6 and M7 subtypes. As a low level of PU.1 is a prerequisite for the differentiation of common myeloid progenitors (CMPs) to megakaryocyte/erythroid progenitors (MEPs) [12], it is reasonable that PU.1 levels would be lower in the M6 (acute erythroleukemia) and M7 (acute megakaryocytic leukemia) subtypes. The observation that PU.1 expression appeared low in the M0 subtype (the most immature FAB subtype) may be explained by the fact that PU.1 expression is low in bone marrow cells lacking definitive signs of myeloid differentiation [12]. In addition, Figure 3 shows a trend towards increasing PU.1 expression from M0 to M5 with the exception of M3, which supports the notion that PU.1 expression is specifically repressed by PML-RARα.

Particularly low levels of PU.1 expression in patients with APL as compared to other AML subtypes. AML patients were divided into eight subtypes (M0, M1, M2, M3, M4, M5, M6 and M7) according to FAB classification. The expression levels of PU.1 in the eight subtypes were evaluated. The four profiling data sets of AML patients, including GSE1729, StJude, GSE10358 and GSE1159, were combined. The PU.1 level relative to that of GAPDH was calculated as described in the Materials and methods.

Growing evidence suggests that leukemia-initiating cells are responsible for initiating and sustaining the growth of the disease. Therefore, we evaluated the expression of PU.1 in APL-initiating cells. Unlike other subtypes of AML, most APL cells lack the CD34+ surface marker [

Lower levels of PU.1 expression in APL-initiating cells. (A) SP cell analysis. The SP cells were gated in the R2 box and were detected following Hoechst 33342 and verapamil co-treatment. Hoechst staining of APL mononuclear cells in the absence or presence of verapamil is shown in the upper and lower panels, respectively. The FACS profiles represent the results of the flow cytometry analysis of SP cells from APL samples. (B) Lower PU.1 levels in APL-derived SP cells as compared to UCB-derived SP cells. The PU.1 mRNA expression levels of SP cells from UCB specimens or primary APL samples were measured using real-time RT-PCR and were normalized to GAPDH expression. Each symbol represents the average value from an individual patient or UCB specimen. 781b155fdc