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BACKGROUND We previously showed that small interfering RNAs (siRNAs) targeting the Zaire Ebola virus (ZEBOV) RNA polymerase L protein formulated in stable nucleic acid-lipid particles (SNALPs) completely protected guineapigs when administered shortly after a lethal ZEBOV challenge. Although rodent models of ZEBOV infection are useful for screening prospective countermeasures,they are frequently not useful for prediction of efficacy in the more stringent non-human primate models. We therefore assessed the efficacy of modified non-immunostimulatory siRNAs in a uniformly lethal non-human primate model of ZEBOV haemorrhagic fever. METHODS A combination of modified siRNAs targeting the ZEBOV L polymerase (EK-1 mod),viral protein (VP) 24 (VP24-1160 mod),and VP35 (VP35-855 mod) were formulated in SNALPs. A group of macaques (n=3) was given these pooled anti-ZEBOV siRNAs (2 mg/kg per dose,bolus intravenous infusion) after 30 min,and on days 1,3,and 5 after challenge with ZEBOV. A second group of macaques (n=4) was given the pooled anti-ZEBOV siRNAs after 30 min,and on days 1,2,3,4,5,and 6 after challenge with ZEBOV. FINDINGS Two (66%) of three rhesus monkeys given four postexposure treatments of the pooled anti-ZEBOV siRNAs were protected from lethal ZEBOV infection,whereas all macaques given seven postexposure treatments were protected. The treatment regimen in the second study was well tolerated with minor changes in liver enzymes that might have been related to viral infection. INTERPRETATION This complete postexposure protection against ZEBOV in non-human primates provides a model for the treatment of ZEBOV-induced haemorrhagic fever. These data show the potential of RNA interference as an effective postexposure treatment strategy for people infected with Ebola virus,and suggest that this strategy might also be useful for treatment of other emerging viral infections. FUNDING Defense Threat Reduction Agency. View Publication

The molecular basis for the unique proliferative and self-renewal properties that hierarchically distinguish human stem cells from progenitors and terminally differentiated cells remains largely unknown. We report a role for the Bcl-2 family member myeloid cell leukemia-1 (Mcl-1) as an indispensable regulator of self-renewal in human stem cells and show that a functional dependence on Mcl-1 defines the human stem cell hierarchy. In vivo pharmacologic targeting of the Bcl-2 family members in human hematopoietic stem cells (HSCs) and human leukemic stem cells reduced stem cell regenerative and self-renewal function. Subsequent protein expression studies showed that,among the Bcl-2 family members,only Mcl-1 was up-regulated exclusively in the human HSC fraction on in vivo regeneration of hematopoiesis. Short hairpin RNA-knockdown of Mcl-1 in human cord blood cells did not affect survival in the HSC or hematopoietic progenitor cell fractions in vitro but specifically reduced the in vivo self-renewal function of human HSCs. Moreover,knockdown of Mcl-1 in ontogenetically primitive human pluripotent stem cells resulted in almost complete ablation of stem cell self-renewal function. Our findings show that Mcl-1 is an essential regulator of stem cell self-renewal in humans and therefore represents an axis for therapeutic interventions. View Publication

To advance T cell-based HIV vaccine development,it is necessary to evaluate the immune correlates of a protective CD8(+) T-cell response. We have developed an assay that assesses the capacity ex vivo of HIV-specific CD8(+) T cells to suppress HIV-1 infection of autologous CD4(+) T cells. This assay directly reflects the ultimate effector function of CD8(+) T cells,the elimination of infected cells,and accurately differentiates the effective CD8(+) T-cell response in spontaneous HIV controllers from ineffective responses in other patients. In this article,we describe all the steps from cell purification to assessment of viral replication by HIV-p24 ELISA and analysis,along with conditions for cell culturing,and how to choose the viral infectious dose that gives the most reliable results. We also depict the conditions of a rapid assay on the basis of flow cytometry analysis of intracellular HIV-Gag products. These procedures take 14-17 d when the p24 ELISA assay is used,or 6 d with the intracellular Gag assay. View Publication

BACKGROUND: CBL missense mutations have recently been associated with juvenile myelomonocytic leukaemia (JMML),an aggressive myeloproliferative and myelodysplastic neoplasm of early childhood characterised by excessive macrophage/monocyte proliferation. CBL,an E3 ubiquitin ligase and a multi-adaptor protein,controls proliferative signalling networks by downregulating the growth factor receptor signalling cascades in various cell types. METHODS AND RESULTS: CBL mutations were screened in 65 patients with JMML. A homozygous mutation of CBL was found in leukaemic cells of 4/65 (6%) patients. In all cases,copy neutral loss of heterozygosity of the 11q23 chromosomal region,encompassing the CBL locus,was demonstrated. Three of these four patients displayed additional features suggestive of an underlying developmental condition. A heterozygous germline CBL p.Y371H substitution was found in each of them and was inherited from the father in one patient. The germline mutation represents the first hit,with somatic loss of heterozygosity being the second hit positively selected in JMML cells. The three patients display a variable combination of dysmorphic features,hyperpigmented skin lesions and microcephaly that enable a 'CBL syndrome' to be tentatively delineated. Learning difficulties and postnatal growth retardation may be part of the phenotype. CONCLUSION: A report of germline mutations of CBL in three patients with JMML is presented here,confirming the existence of an unreported inheritable condition associated with a predisposition to JMML. View Publication

Reactive oxygen species (ROS) damage brain lipids,carbohydrates,proteins,as well as DNA and may contribute to neurodegeneration. We previously reported that ER- and oxidative stress cause neuronal apoptosis in infantile neuronal ceroid lipofuscinosis (INCL),a lethal neurodegenerative storage disease,caused by palmitoyl-protein thioesterase-1 (PPT1) deficiency. Polyunsaturated fatty acids (PUFA) are essential components of cell membrane phospholipids in the brain and excessive ROS may cause oxidative damage of PUFA leading to neuronal death. Using cultured neurons and neuroprogenitor cells from mice lacking Ppt1,which mimic INCL,we demonstrate that Ppt1-deficient neurons and neuroprogenitor cells contain high levels of ROS,which may cause peroxidation of PUFA and render them incapable of providing protection against oxidative stress. We tested whether treatment of these cells with omega-3 or omega-6 PUFA protects the neurons and neuroprogenitor cells from oxidative stress and suppress apoptosis. We report here that both omega-3 and omega-6 fatty acids protect the Ppt1-deficient cells from ER- as well as oxidative stress and suppress apoptosis. Our results suggest that PUFA supplementation may have neuroprotective effects in INCL. View Publication

Patients with mantle cell lymphoma (MCL) typically respond to initial treatment but subsequently relapse. This pattern suggests that a population of MCL cells is both drug resistant and capable of clonogenic growth. The intracellular enzyme retinaldehyde dehydrogenase (ALDH) provides resistance to several toxic agents. ALDH can also identify stem cells in normal adult tissues and tumorigenic cancer stem cells in several human malignancies. We studied ALDH expression in MCL and found small populations of ALDH(+) cells that were highly clonogenic. Moreover,ALDH(+) MCL cells were relatively quiescent and resistant to a wide range of agents. Normal B cells can be activated by specific unmethylated cytosine-phosphate-guanosine (CpG) DNA motifs through toll-like receptor 9,and we found that the synthetic CpG oligonucleotide 2006 (CpG) reduced the frequency of quiescent ALDH(+) MCL cells,induced terminal plasma cell differentiation,and limited tumor formation in vitro and in vivo. Treatment with CpG also significantly enhanced the activity of the proteasome inhibitor bortezomib that was associated with induction of the unfolded protein response. Our data suggest that CpG may target clonogenic and resistant ALDH(+) cells as well as improve the activity of proteasome inhibitors in MCL. View Publication

CD44+/CD24-/low tumor cells or aldehyde dehydrogenase 1 (ALDH1) positive tumor cells are considered cancer stem cells (CSCs) that possess the properties of self-renewal and tumorigenicity. However,their clinical value and significance in breast cancer remain controversial. A meta-analysis based on published studies was performed with the aim of obtaining an accurate evaluation of the association between the presence of CSCs in clinical samples and clinical outcome. A total of 12 eligible studies with 898 cases and 1,853 controls were included. CSC positive breast cancers,in particular those positive for ALDH1,were significantly associated with high histological grade,estrogen receptor (ER) negativity,progesterone receptor (PR) negativity,and human epidermal growth factor receptor type 2 (HER2) positivity. However,the presence of cancer stem cells was not associated with tumor size or nodal status. ALDH1 positive (RR = 2.83,95% CI: 2.16-3.67,P textless 0.001) and CD44+/CD24-/low tumor cells (RR = 2.32,95% CI: 1.51-3.60,P textless 0.001) were significantly associated with poor overall survival (OS). The stem cell markers are prognostic factors in breast cancer. Larger clinical studies are required to further evaluate the role of these markers in clinical practice. View Publication

Regulatory T cells (Tregs) are a suppressive subset of CD4(+) T lymphocytes implicated in the prevention of acute GVHD (aGVHD) after allo-SCT (ASCT). To determine whether increased frequency of Tregs with a skin-homing (cutaneous lymphocyte Ag,CLA(+)) or a gut-homing (α(4)β(7)(+)) phenotype is associated with reduced risk of skin or gut aGVHD,respectively,we quantified circulating CLA(+) or α(4)β(7)(+) on Tregs at the time of neutrophil engraftment in 43 patients undergoing ASCT. Increased CLA(+) Tregs at engraftment was associated with the prevention of skin aGVHD (2.6 vs 1.7%; P=0.038 (no skin aGVHD vs skin aGVHD)),and increased frequencies of CLA(+) and α(4)β(7)(+) Tregs were negatively correlated with severity of skin aGVHD (odds ratio (OR),0.67; 95% confidence interval (CI),0.46-0.98; P=0.041) or gut aGVHD (OR,0.93; 95% CI,0.88-0.99; P=0.031),respectively. This initial report suggests that Treg tissue-homing subsets help to regulate organ-specific risk and severity of aGVHD after human ASCT. These results need to be validated in a larger,multicenter cohort. View Publication

Osteoclasts are resident cells of the bone that are primarily involved in the physiological and pathological remodeling of this tissue. Mature osteoclasts are multinucleated giant cells that are generated from the fusion of circulating precursors originating from the monocyte/macrophage lineage. During inflammatory bone conditions in vivo,de novo osteoclastogenesis is observed but it is currently unknown whether,besides increased osteoclast differentiation from undifferentiated precursors,other cell types can generate a multinucleated giant cell phenotype with bone resorbing activity. In this study,an animal model of calvaria-induced aseptic osteolysis was used to analyze possible bone resorption capabilities of dendritic cells (DCs). We determined by FACS analysis and confocal microscopy that injected GFP-labeled immature DCs were readily recruited to the site of osteolysis. Upon recruitment,the cathepsin K-positive DCs were observed in bone-resorbing pits. Additionally,chromosomal painting identified nuclei from female DCs,previously injected into a male recipient,among the nuclei of giant cells at sites of osteolysis. Finally,osteolysis was also observed upon recruitment of CD11c-GFP conventional DCs in Csf1r(-/-) mice,which exhibit a severe depletion of resident osteoclasts and tissue macrophages. Altogether,our analysis indicates that DCs may have an important role in bone resorption associated with various inflammatory diseases. View Publication

Aurora kinases are key regulators of cell mitosis and have been implicated in the process of tumorigenesis. In recent years,the Aurora kinases have attracted much interest as promising targets for cancer treatment. Here we report on the roles of Aurora A and Aurora B kinases in clear cell renal cell carcinoma (ccRCC). Using genomewide expression array analysis of 174 patient samples of ccRCC,we found that expression levels of Aurora A and B were significantly elevated in ccRCC compared to normal kidney samples. High expression levels of Aurora A and Aurora B were significantly associated with advanced tumor stage and poor patient survival. Inhibition of Aurora kinase activity with the drug VX680 (also referred to as MK-0457) inhibited ccRCC cell growth in vitro and led to ccRCC cell accumulation in the G2/M phase and apoptosis. Growth of ccRCC xenograft tumors was also inhibited by VX680 treatment,accompanied by a reduction of tumor microvessel density. Analysis of endothelial cell lines demonstrated that VX680 inhibits endothelial cell growth with effects similar to that seen in ccRCC cells. Our findings suggest that VX680 inhibits the growth of ccRCC tumors by targeting the proliferation of both ccRCC tumor cells and tumor-associated endothelial cells. Aurora kinases and their downstream cell cycle proteins have an important role in ccRCC and may be potent prognostic markers and therapy targets for this disease. View Publication

Changes in progenitor cell biology remain at the forefront of many theories of biologic aging,but there are limited studies evaluating this in humans. Aging has been associated with a progressive depletion of circulating progenitor cells,but age-related bone marrow-resident progenitor cell depletion has not been systematically determined in humans. Patients undergoing total hip replacement were consented,and bone marrow and peripheral progenitor cells were enumerated based on aldehyde dehydrogenase activity and CD34 and CD133 expression. Circulating progenitors demonstrated an age-dependent decline. In contrast,marrow-resident progenitor cell content demonstrated no age association with any progenitor cell subtype. In humans,aging is associated with depletion of circulating,but not marrow-resident,progenitors. This finding has impact on the mechanism(s) responsible for age-related changes in circulating stem cells and important implications for the use of autologous marrow for the treatment of age-related diseases. View Publication

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