ATRA resolves the differentiation block in t(15;17) acute myeloid leukemia by restoring PU.1 expression

Tightly regulated expression of the transcription factor PU.1 is crucial for normal hematopoiesis. PU.1 knockdown mice develop acute myeloid leukemia (AML), and PU.1 mutations have been observed in some populations of patients with AML. Here we found that conditional expression of promyelocytic leukemia-retinoic acid receptor alpha (PML-RARA), the protein encoded by the t(15;17) translocation found in acute promyelocytic leukemia (APL), suppressed PU.1 expression, while treatment of APL cell lines and primary cells with all-trans retinoic acid (ATRA) restored PU.1 expression and induced neutrophil differentiation. ATRA-induced activation was mediated by a region in the PU.1 promoter to which CEBPB and OCT-1 binding were induced. Finally, conditional expression of PU.1 in human APL cells was sufficient to trigger neutrophil differentiation, whereas reduction of PU.1 by small interfering RNA (siRNA) blocked ATRA-induced neutrophil differentiation. This is the first report to show that PU.1 is suppressed in acute promyelocytic leukemia, and that ATRA restores PU.1 expression in cells harboring t(15;17).

Mechanisms of airway obliteration after lung transplantation

Post-transplant bronchiolitis obliterans, also called bronchiolitis obliterans syndrome, affects up to 50-60% of patients who survive 5 yr after surgery according to its clinical definition, which is based on the degree of obstructive airway disease. Alloimmune-independent and -dependent mechanisms produce injuries and inflammation of epithelial cells and subepithelial structures, leading to aberrant tissue repair. The triggering of innate immunity by various infections or chemical injuries after, for example, gastroesophageal reflux, may lead to the release of danger signals that are able to activate dendritic cells, a crucial link with adaptive immunity. Inflammation can also increase the expression and display of major histocompatibility alloantigens and thus favor the initiation of rejection episodes. These phenomena may be limited in time and location or may be protracted. Reducing the risk of alloimmune-independent factors may be as important as treating acute episodes of lung rejection. Excessive immunosuppression may be deleterious by increasing the risk of infection, thereby triggering innate and adaptive immunity. New potential therapeutic targets are emerging from the research performed on leukotriene receptors, chemokine receptors, and growth factors. Neutralizing these molecules reduces the initial mononuclear and polynuclear infiltrates or the subsequent fibroproliferative process and the neovascular changes, feeding this process.

Association between sleep apnea severity and blood coagulability: Treatment effects of nasal continuous positive airway pressure

A prothrombotic state may contribute to the elevated cardiovascular risk in patients with obstructive sleep apnea (OSA). We investigated the relationship between apnea severity and hemostasis factors and effect of continuous positive airway pressure (CPAP) treatment on hemostatic activity. We performed full overnight polysomnography in 44 OSA patients (mean age 47+/-10 years), yielding apnea-hypopnea index (AHI) and mean nighttime oxyhemoglobin saturation (SpO2) as indices of apnea severity. For treatment, subjects were double-blind randomized to 2 weeks of either therapeutic CPAP (n = 18), 3 l/min supplemental nocturnal oxygen (n = 16) or placebo-CPAP (<1 cm H2O) (n = 10). Levels of von Willebrand factor antigen (VWF:Ag), soluble tissue factor (sTF), D-dimer, and plasminogen activator inhibitor (PAI)-1 antigen were measured in plasma pre- and posttreatment. Before treatment, PAI-1 was significantly correlated with AHI (r = 0.47, p = 0.001) and mean nighttime SpO2 (r = -0.32, p = 0.035), but these OSA measures were not significantly related with VWF:Ag, sTF, and D-dimer. AHI was a significant predictor of PAI-1 (R2 = 0.219, standardized beta = 0.47, p = 0.001), independent of mean nighttime SpO2, body mass index (BMI), and age. A weak time-by-treatment interaction for PAI-1 was observed (p = 0.041), even after adjusting for age, BMI, pre-treatment AHI, and mean SpO2 (p = 0.046). Post hoc analyses suggested that only CPAP treatment was associated with a decrease in PAI-1 (p = 0.039); there were no changes in VWF:Ag, sTF, and D-dimer associated with treatment with placebo-CPAP or with nocturnal oxygen. Apnea severity may be associated with impairment in the fibrinolytic capacity. To the extent that our sample size was limited, the observation that CPAP treatment led to a decrease in PAI-1 in OSA must be regarded as tentative.

Cellular differentiation determines the expression of the hypoxia-inducible protein NDRG1 in pancreatic cancer

N-myc downstream-regulated gene-1 (NDRG1) is a recently described hypoxia-inducible protein that is upregulated in various human cancers. Pancreatic ductal adenocarcinoma, called pancreatic cancer, is a highly aggressive cancer that is characterised by its avascular structure, which results in a severe hypoxic environment. In this study, we investigated whether NDRG1 is upregulated in these tumours, thus providing a novel marker for malignant cells in the pancreas. By immunohistochemistry, we observed that NDRG1 was highly expressed in well-differentiated cells of pancreatic cancer, whereas the poorly differentiated tumour cells were negative. In addition, hyperplastic islets and ducts of nonquiescent pancreatic tissue were positive. To further explore its selective expression in tumours, two well-established pancreatic cancer cell lines of unequal differentiation status were exposed to 2% oxygen. NDRG1 mRNA and protein were upregulated by hypoxia in the moderately differentiated Capan-1 cells; however, its levels remained unchanged in the poorly differentiated Panc-1 cell line. Taken together, our data suggest that NDRG1 will not serve as a reliable marker of tumour cells in the pancreas, but may serve as a marker of differentiation. Furthermore, we present the novel finding that cellular differentiation may be an important factor that determines the hypoxia-induced regulation of NDRG1.

Important excess morbidity due to upper airway anomalies in the perioperative course in infant cardiac surgery

BACKGROUND: The study aimed at defining the excess morbidity or mortality caused by an additional airway malformation in children with congenital heart disease requiring surgery. METHODS: All patients requiring surgery for heart disease during an 8-year period ending in 2003 who had an associated upper airway malformation were retrospectively studied. All patients were seen in 2004 for a prospective follow-up examination. RESULTS: Eleven patients with upper airway anomalies were identified (tracheobronchial malacia in 6 patients, long-segment tracheal stenosis in 3, and bilateral vocal cord paralysis and tracheal hemangioma in 1 patient each). They accounted for 1.5% of the entire cardiac surgical load of 764 patients. In 5 infants, the airway anomaly was diagnosed before cardiac repair, in 6 patients thereafter. Diagnosis was made by bronchoscopy in all patients, by additional bronchography in 2. Failure of rapid postoperative extubation was the most common finding. Airway management was surgical in 2 and conservative in 8 patients, 1 newborn having been denied therapy because of the severity of airway hypoplasia. Compared with patients with isolated cardiac disease, those with additional airway anomalies had significantly longer duration of postoperative mechanical ventilation (median, 24 days versus 3), perioperative hospitalization (median, 72 days versus 11) and total number of days of hospitalization during the first year of life (median, 104 days versus 14). After a maximum follow-up of 8 years (median, 37 months) only 3 of 10 surviving patients remained symptomatic owing to the airway malformation. CONCLUSIONS: Upper airway anomalies accompanying heart disease in infancy resulted in a significant prolongation of perioperative intensive care and hospital stay, as well as duration of mechanical ventilation. Failure of early postoperative extubation was the leading symptom.

A Mitogen-activated protein kinase controls differentiation of bloodstream forms of Trypanosoma brucei

African trypanosomes undergo differentiation in order to adapt to the mammalian host and the tsetse fly vector. To characterize the role of a mitogen-activated protein (MAP) kinase homologue, TbMAPK5, in the differentiation of Trypanosoma brucei, we constructed a knockout in procyclic (insect) forms from a differentiation-competent (pleomorphic) stock. Two independent knockout clones proliferated normally in culture and were not essential for other life cycle stages in the fly. They were also able to infect immunosuppressed mice, but the peak parasitemia was 16-fold lower than that of the wild type. Differentiation of the proliferating long slender to the nonproliferating short stumpy bloodstream form is triggered by an autocrine factor, stumpy induction factor (SIF). The knockout differentiated prematurely in mice and in culture, suggestive of increased sensitivity to SIF. In contrast, a null mutant of a cell line refractory to SIF was able to proliferate normally. The differentiation phenotype was partially rescued by complementation with wild-type TbMAPK5 but exacerbated by introduction of a nonactivatable mutant form. Our results indicate a regulatory function for TbMAPK5 in the differentiation of bloodstream forms of T. brucei that might be exploitable as a target for chemotherapy against human sleeping sickness.

Expansion on specific substrates regulates the phenotype and differentiation capacity of human articular chondrocytes

In this study, we investigated if monolayer expansion of adult human articular chondrocytes (AHAC) on specific substrates regulates cell phenotype and post-expansion multilineage differentiation ability. AHAC isolated from cartilage biopsies of five donors were expanded on plastic dishes (PL), on dishes coated with collagen type II (COL), or on slides coated with a ceramic material (Osteologic, OS). The phenotype of expanded chondrocytes was assessed by flow cytometry and real-time RT-PCR. Cells were then cultured in previously established conditions promoting differentiation toward the chondrogenic or osteogenic lineage. AHAC differentiation was assessed histologically, biochemically, and by real-time RT-PCR. As compared to PL-expanded AHAC, those expanded on COL did not exhibit major phenotypic changes, whereas OS-expanded cells expressed (i) higher bone sialoprotein (BSP) (22.6-fold) and lower collagen type II (9.3-fold) mRNA levels, and (ii) lower CD26, CD90 and CD140 surface protein levels (1.4-11.1-fold). Following chondrogenic differentiation, COL-expanded AHAC expressed higher mRNA levels of collagen type II (2.3-fold) and formed tissues with higher glycosaminoglycan (GAG) contents (1.7-fold), whereas OS-expanded cells expressed 16.5-fold lower collagen type II and generated pellets with 2.0-fold lower GAG contents. Following osteogenic differentiation, OS-expanded cells expressed higher levels of BSP (3.9-fold) and collagen type I (2.8-fold) mRNA. In summary, AHAC expansion on COL or OS modulated the de-differentiated cell phenotype and improved the cell differentiation capacity respectively toward the chondrogenic or osteogenic lineage. Phenotypic changes induced by AHAC expansion on specific substrates may mimic pathophysiological events occurring at different stages of osteoarthritis and may be relevant for the engineering of osteochondral tissues.

In vivo particle uptake by airway macrophages in healthy volunteers

We combined two techniques, radiolabeled aerosol inhalation delivery and induced sputum, to examine in vivo the time course of particle uptake by airway macrophages in 10 healthy volunteers. On three separate visits, induced sputum was obtained 40, 100, and 160 min after inhalation of radiolabeled sulfur colloid (SC) aerosol (Tc99 m-SC, 0.2 microm colloid size delivered in 6-microm droplets). On a fourth visit (control) with no SC inhalation, induced sputum was obtained and SC particles were incubated (37 degrees C) in vitro with sputum cells for 40, 100, and 160 min (matching the times associated with in vivo sampling). Total and differential cell counts were recorded for each sputum sample. Compared with 40 min (6 +/- 3%), uptake in vivo was significantly elevated at 100 (31 +/- 5%) and 160 min (27 +/- 4%); both were strongly associated with the number of airway macrophages (R = 0.8 and 0.7, respectively); and the number and proportion of macrophages at 40 min were significantly (P < 0.05) elevated compared with control (1,248 +/- 256 versus 555 +/- 114 cells/mg; 76 +/- 6% versus 60 +/- 5%). Uptake in vitro increased in a linear fashion over time and was maximal at 160 min (40 min, 12 +/- 2%; 100 min, 16 +/- 4%; 160 min, 24 +/- 6%). These data suggest that airway surface macrophages in healthy subjects rapidly engulf insoluble particles. Further, macrophage recruitment and phagocytosis-modifying agents are factors in vivo that likely affect particle uptake and its time course.

New aspects of airway mechanics in pre-term infants

High-frequency respiratory impedance data measured noninvasively by the high-speed interrupter technique (HIT), particularly the first antiresonance frequency (f(ar,1)), is related to airway wall mechanics. The aim of this study was to evaluate the feasibility and repeatability of HIT in unsedated pre-term infants, and to compare values of f(ar,1) from 18 pre-term (post-conceptional age 32-37 weeks, weight 1,730-2,910 g) and 18 full-term infants (42-47 weeks, 3,920-5,340 g). Among the pre-term infants, there was good short-term repeatability of f(ar,1) within a single sleep epoch (mean (sd) coefficient of variance: 8 (1.7)%), but 95% limits of agreement for repeated measures of f(ar,1) after 3-8 h were relatively wide (-41 Hz; 37 Hz). f(ar,1) was significantly lower in pre-term infants (199 versus 257 Hz), indicating that wave propagation characteristics in pre-term airways are different from those of full-term infants. The present authors suggest that this is consistent with developmental differences in airway wall structure and compliance, including the influence of the surrounding tissue. Since flow limitation is determined by wave propagation velocity and airway cross-sectional area, it was hypothesised that the physical ability of the airways to carry large flows is fundamentally different in pre-term than in full-term infants.

alpha1-Antitrypsin inhalation reduces airway inflammation in cystic fibrosis patients

The airways of cystic fibrosis (CF) patients are characterised by neutrophils that release high amounts of elastase overwhelming the local antiprotease shield. Inhalation of alpha(1)-antitrypsin (AAT) may restore the protease-antiprotease balance and attenuate airway inflammation in CF airways. The aims of the present study were: 1) to assess the best deposition region for inhaled AAT by two different inhalation strategies; and 2) to examine the effect of 4 weeks of AAT inhalation on lung function, protease-antiprotease balance and airway inflammation in CF patients. In a prospective, randomised study, 52 CF patients received a daily deposition by inhalation of 25 mg AAT for 4 weeks targeting their peripheral or bronchial compartment. The levels of elastase activity, AAT, pro-inflammatory cytokines, neutrophils, immunoglobulin G fragments and the numbers of Pseudomonas aeruginosa were assessed in induced sputum before and after the inhalation period. Inhalation of AAT increased AAT levels and decreased the levels of elastase activity, neutrophils, pro-inflammatory cytokines and the numbers of P. aeruginosa. However, it had no effect on lung function. No difference was found between the peripheral and bronchial inhalation mode. In conclusion, although no effect on lung function was observed, the clear reduction of airway inflammation after alpha(1)-antitrypsin treatment may precede pulmonary structural changes. The alpha(1)-antitrypsin deposition region may play a minor role for alpha(1)-antitrypsin inhalation in cystic fibrosis patients.

Neuronal differentiation of human mesenchymal stem cells: changes in the expression of the Alzheimer's disease-related gene seladin-1

Seladin-1 (SELective Alzheimer's Disease INdicator-1) is an anti-apoptotic gene, which is down-regulated in brain regions affected by Alzheimer's disease (AD). In addition, seladin-1 catalyzes the conversion of desmosterol into cholesterol. Disruption of cholesterol homeostasis in neurons may increase cell susceptibility to toxic agents. Because the hippocampus and the subventricular zone, which are affected in AD, are the unique regions containing stem cells with neurogenic potential in the adult brain, it might be hypothesized that this multipotent cell compartment is the predominant source of seladin-1 in normal brain. In the present study, we isolated and characterized human mesenchymal stem cells (hMSC) as a model of cells with the ability to differentiate into neurons. hMSC were then differentiated toward a neuronal phenotype (hMSC-n). These cells were thoroughly characterized and proved to be neurons, as assessed by molecular and electrophysiological evaluation. Seladin-1 expression was determined and found to be significantly reduced in hMSC-n compared to undifferentiated cells. Accordingly, the total content of cholesterol was decreased after differentiation. These original results demonstrate for the first time that seladin-1 is abundantly expressed by stem cells and appear to suggest that reduced expression in AD might be due to an altered pool of multipotent cells.