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In 2021, the Israel Ministry of Health began a national hepatitis C elimination program. Implementing a World Health Organization goal, Israel’s program involved targeted screening, barrier minimization, workup simplification, awareness campaigns, and a patient registry. We evaluated program costs for testing and treatment. By May 15, 2023, the program had identified 865,382 at-risk persons, of whom 555,083 (64.3%) were serologically screened for hepatitis C virus (HCV), which was detected in 24,361 (4.4%). Among 20,928 serologically positive patients, viremia was detected in 13,379 (63.9%), of whom 10,711 (80%) were treated, and 4,618 (96.5%) of 4,786 persons receiving posttreatment HCV RNA testing had sustained virologic response. We estimated costs of ₪14,426 (new Israel shekel; ≈$3,606 USD) per person whose HCV infection was diagnosed and successfully treated. The program yielded screening and treatment in almost two thirds of the identified at-risk population. Although not eliminated, HCV prevalence will likely decrease substantially by the 2030 target.

Abstract Background High prostate eicosapentaenoic fatty acid (EPA) levels were associated with a significant reduction of upgrading to grade group (GG) ≥ 2 prostate cancer in men under active surveillance. We aimed to evaluate the effect of MAG-EPA long-chain omega-3 fatty acid dietary supplement on prostate cancer proliferation. Methods A phase II double-blind randomized placebo-controlled trial was conducted in 130 men diagnosed with GG ≥ 2 prostate cancer and undergoing radical prostatectomy between 2015–2017 (Clinicaltrials.gov: NCT02333435). Participants were randomized to receive 3 g daily of either MAG-EPA (n = 65) or placebo (n = 65) for 7 weeks (range 4–10) prior to radical prostatectomy. The primary outcome was the cancer proliferation index quantified by automated image analysis of tumor nuclear Ki-67 expression using standardized prostatectomy tissue microarrays. Additional planned outcomes at surgery are reported including plasma levels of 27 inflammatory cytokines and fatty acid profiles in circulating red blood cells membranes and prostate tissue. Results Cancer proliferation index measured by Ki-67 expression was not statistically different between the intervention (3.10%) and placebo (2.85%) groups (p = 0.64). In the per protocol analyses, the adjusted estimated effect of MAG-EPA was greater but remained non-significant. Secondary outcome was the changes in plasma levels of 27 cytokines, of which only IL-7 was higher in MAG-EPA group compared to placebo (p = 0.026). Men randomized to MAG-EPA prior to surgery had four-fold higher EPA levels in prostate tissue compared to those on placebo. Conclusions This MAG-EPA intervention did not affect the primary outcome of prostate cancer proliferation according to nuclear Ki-67 expression. More studies are needed to decipher the effects of long-chain omega-3 fatty acid dietary supplementation in men with prostate cancer.

Little is known about co-occurring tuberculosis (TB) and COVID-19 in low TB incidence settings. We obtained a cross-section of 333 persons in the United States co-diagnosed with TB and COVID-19 within 180 days and compared them to 4,433 persons with TB only in 2020 and 18,898 persons with TB during 2017‒2019. Across both comparison groups, a higher proportion of persons with TB–COVID-19 were Hispanic, were long-term care facility residents, and had diabetes. When adjusted for age, underlying conditions, and TB severity, COVID-19 co-infection was not statistically associated with death compared with TB infection only in 2020 (adjusted prevalence ratio 1.0 [95% CI 0.8‒1.4]). Among TB–COVID-19 patients, death was associated with a shorter interval between TB and COVID-19 diagnoses, older age, and being immunocompromised (non-HIV). TB–COVID-19 deaths in the United States appear to be concentrated in subgroups sharing characteristics known to increase risk for death from either disease alone.

Abstract The clinical yield and benefit of performing bone marrow cultures for various clinical indications has been challenged and their clinical necessity remains debatable. We sought to assess the clinical yield and benefit of performing routine bone marrow cultures and determine whether various clinical, laboratory, and imaging parameters were predictive of a diagnostic bone marrow culture. This was a single center retrospective analysis of all patients who underwent a bone marrow study comprising bone marrow cultures from January 1, 2012, through March 1, 2018. Baseline clinical data were extracted from the institution's electronic medical records system. The analyzed cohort consisted of 139 patients with a median age of 46 years (range 4 months to 85 years). The most common indication for a bone marrow study was workup of a fever of unknown origin (105 patients, 76%) while investigation for infection in immunocompromised patients accounted for 22 cases (16%) and suspected tuberculosis was the reason for acquisition of bone marrow cultures in 6 patients (4%). Only 3 patients had positive bone marrow cultures, yielding in 2 patients a diagnosis of Mycobacterium avium and in one patient a microbiologically unclassifiable fungal infection. A univariate analysis revealed that mean age, hemoglobin level, platelet count, c-reactive protein levels, gender, indication for bone marrow study, yield of blood cultures, and contribution of imaging studies and bone marrow pathology results were not significantly different between patients with diagnostic and non-diagnostic bone marrow cultures. Mean white blood cell count was found to be significantly lower in patients with diagnostic bone marrow cultures (2.4 × 103/µL versus 8.7 × 103/µL; P = 0.038). We conclude that for most patients, performance of bone marrow cultures holds limited clinical value.

Abstract Mitochondria are cellular organelles critical for numerous cellular processes and harboring their own circular mitochondrial DNA (mtDNA). Most mtDNA associated disorders (either deletions, mutations, or depletion) lead to multisystemic disease, often severe at a young age, with no disease-modifying therapies. Mitochondria have a capacity to enter eukaryotic cells and to be transported between cells. We describe a method of ex vivo augmentation of hematopoietic stem and progenitor cells (HSPCs) with normal exogenous mitochondria, termed mitochondrial augmentation therapy (MAT). Here, we show that MAT is feasible and dose dependent, and improves mitochondrial content and oxygen consumption of healthy and diseased HSPCs. Ex vivo mitochondrial augmentation of HSPCs from a patient with a mtDNA disorder leads to superior human engraftment in a non-conditioned NSGS mouse model. Using a syngeneic mouse model of accumulating mitochondrial dysfunction (Polg), we show durable engraftment in non-conditioned animals, with in vivo transfer of mitochondria to recipient hematopoietic cells. Taken together, this study supports MAT as a potential disease-modifying therapy for mtDNA disorders.

Abstract Tissue fibrosis is a major driver of pathology in aging and is involved in numerous age-related diseases. The lungs are particularly susceptible to fibrotic pathology which is currently difficult to treat. The mouse bleomycin-induced fibrosis model was developed to investigate lung fibrosis and widely used over the years. However, a systematic analysis of the accumulated results has not been performed. We undertook a comprehensive data mining and subsequent manual curation, resulting in a collection of 213 genes (available at the TiRe database, www.tiredb.org ), which when manipulated had a clear impact on bleomycin-induced lung fibrosis. Our meta-analysis highlights the age component in pulmonary fibrosis and strong links of related genes with longevity. The results support the validity of the bleomycin model to human pathology and suggest the importance of a multi-target therapeutic strategy for pulmonary fibrosis treatment.

Abstract Within the prostate tumor microenvironment (TME) there are complex multi-faceted and dynamic communication occurring between cancer cells and immune cells. Macrophages are key cells which infiltrate and surround tumor cells and are recognized to significantly contribute to tumor resistance and metastases. Our understanding of their function in the TME is commonly based on in vitro and in vivo models, with limited research to confirm these model observations in human prostates. Macrophage infiltration was evaluated within the TME of human prostates after 72 h culture of fresh biopsies samples in the presence of control or enzalutamide. In addition to immunohistochemistry, an optimized protocol for multi-parametric evaluation of cellular surface markers was developed using flow cytometry. Flow cytometry parameters were compared to clinicopathological features. Immunohistochemistry staining for 19 patients with paired samples suggested enzalutamide increased the expression of CD163 relative to CD68 staining. Techniques to validate these results using flow cytometry of dissociated biopsies after 72 h of culture are described. In a second cohort of patients with Gleason grade group ≥ 3 prostate cancer, global macrophage expression of CD163 was unchanged with enzalutamide treatment. However, exploratory analyses of our results using multi-parametric flow cytometry for multiple immunosuppressive macrophage markers suggest subgroup changes as well as novel associations between circulating biomarkers like the neutrophil to lymphocyte ratio (NLR) and immune cell phenotype composition in the prostate TME. Further, we observed an association between B7–H3 expressing tumor-associated macrophages and the presence of intraductal carcinoma. The use of flow cytometry to evaluate ex vivo cultured prostate biopsies fills an important gap in our ability to understand the immune cell composition of the prostate TME. Our results highlight novel associations for further investigation.

PurposeA rapid increase of recreational nitrous oxide use (i.e. laughing gas, N2O) has been reported in several countries, while it has received limited attention in scientific research. We aimed to study the association of socio-demographic characteristics, mental well-being, sickness absence, truancy, and substance use with the frequency of lifetime nitrous oxide use among adolescents.MethodsWe used self-reported questionnaire data of adolescents (N = 555) attending secondary schools to cross-sectionally assess the frequency of nitrous oxide use and potential factors associated with nitrous oxide use, such as gender, mental well-being, and binge drinking. Ordinal logistic regression models were applied with lifetime nitrous oxide use (never, once, ≥ two times) as the outcome variable.ResultsAdolescents were on average 15.6 years old (SD = 0.83, range 14-18), 47.0% were female. In total, 86 (15.6%) adolescents had used nitrous oxide at least once in their life. In the multivariable ordinal regression model, the risk of having a higher category of lifetime nitrous oxide use was associated with a non-Dutch ethnic background (OR = 2.10, 95% CI 1.22; 3.61), attending pre-vocational education (OR = 1.88, 95% CI 1.06; 3.34), a higher score on the scale of externalizing problems (OR = 1.10, 95% CI 1.01; 1.20), binge drinking twice or more in the past four weeks (OR = 2.49, 95% CI 1.25; 4.94), and cannabis use (OR = 1.98, 95% CI 1.03; 3.79).ConclusionsYouth Health Care professionals should be aware of nitrous oxide use in adolescents, especially among adolescents with a non-Dutch ethnic background, lower education levels, externalizing problems, frequent binge drinking, and cannabis use.

Abstract Infection with Mycobacterium tuberculosis (Mtb), the bacterium that causes tuberculosis, remains a global health concern. Both classically and non-classically restricted cytotoxic CD8+ T cells are important to the control of Mtb infection. We and others have demonstrated that the non-classical MHC I molecule HLA-E can present pathogen-derived peptides to CD8+ T cells. In this manuscript, we identified the antigen recognized by an HLA-E-restricted CD8+ T cell clone isolated from an Mtb latently infected individual as a peptide from the Mtb protein, MPT32. Recognition by the CD8+ T cell clone required N-terminal O-linked mannosylation of MPT32 by a mannosyltransferase encoded by the Rv1002c gene. This is the first description of a post-translationally modified Mtb-derived protein antigen presented in the context of an HLA-E specific CD8+ T cell immune response. The identification of an immune response that targets a unique mycobacterial modification is novel and may have practical impact in the development of vaccines and diagnostics.

Introduction The study aim was to examine the association of snoring and nocturnal gastro-oesophageal reflux (nGOR) with respiratory symptoms and lung function, and if snoring and/or nGOR associated with a steeper decline in lung function. Methods Data from the third visit of the European Community Respiratory Health Survey (ECRHS) was used for cross-sectional analysis. Pre- and post-bronchodilator spirometry was performed, and information on sleep, nGOR and respiratory symptoms was collected (n=5715). Habitual snoring and nGOR were assessed by questionnaire reports. Pre-bronchodilator spirometry from ECRHS I, II and III (20 years follow-up) were used to analyse lung function changes by multivariate regression analysis. Results Snoring and nGOR were independently associated with a higher prevalence of wheeze, chest tightness, breathlessness, cough and phlegm. The prevalence of any respiratory symptom was 79% in subjects with both snoring and nGOR versus 56% in those with neither (p

Asthma often remains uncontrolled, despite the fact that the pharmacological treatment has undergone large changes. We studied changes in the treatment of asthma over a 20-year period and identified factors associated with the regular use of inhaled corticosteroid (ICS) treatment. Changes in the use of medication were determined in 4617 randomly selected subjects, while changes in adults with persistent asthma were analysed in 369 participants. The study compares data from three surveys in 24 centres in 11 countries. The use of ICSs increased from 1.7% to 5.9% in the general population and the regular use of ICSs increased from 19% to 34% among persistent asthmatic subjects. The proportion of asthmatic subjects reporting asthma attacks in the last 12 months decreased, while the proportion that had seen a doctor in the last 12 months remained unchanged (42%). Subjects with asthma who had experienced attacks or had seen a doctor were more likely to use ICSs on a regular basis. Although ICS use has increased, only one-third of subjects with persistent asthma take ICSs on a regular basis. Less than half had seen a doctor during the last year. This indicates that underuse of ICSs and lack of regular healthcare contacts remains a problem in the management of asthma.

Despite recent improvements in patient outcomes using newer androgen receptor (AR) pathway inhibitors, treatment resistance in castrate resistant prostate cancer (CRPC) continues to remain a clinical problem. Co-targeting alternate resistance pathways are of significant interest to treat CRPC and delay the onset of resistance. Both the AKT and MEK signaling pathways become activated as prostate cancer develops resistance to AR-targeted therapies. This pre-clinical study explores co-targeting these pathways in AR-positive prostate cancer models. Using various in vitro models of prostate cancer disease states including androgen dependent (LNCaP), CRPC (V16D and 22RV1) and ENZ-resistant prostate cancer (MR49C and MR49F), we evaluate the relevance of targeting both AKT and MEK pathways. Our data reveal that AKT inhibition induces apoptosis and inhibits cell growth in PTEN null cell lines independently of their sensitivity to hormone therapy; however, AKT inhibition had no effect on the PTEN positive 22RV1 cell line. Interestingly, we found that MEK inhibition had greater effect on 22RV1 cells compared to LNCaP, V16D or ENZ-resistant cells MR49C and MR49F cells. In vitro, combination AKT and MEK blockade had evidence of synergy observed in some cell lines and assays, but this was not consistent across all results. In vivo, the combination of AKT and MEK inhibition resulted in more consistent tumor growth inhibition of MR49F xenografts and longer disease specific survival compared to AKT inhibitor monotherapy. As in our in vitro study, 22RV1 xenografts were more resistant to AKT inhibition while they were more sensitive to MEK inhibition. Our results suggest that targeting AKT and MEK in combination may be a valuable strategy in prostate cancer when both pathways are activated and further support the importance of characterizing the dominant oncogenic pathway in each patient's tumor in order to select optimal therapy.

[This corrects the article DOI: 10.1371/journal.pone.0155711.].

Inhibitors of poly[ADP-ribose] polymerase 1 (PARPis) show promise for treatment of cancers which lack capacity for homologous recombination repair (HRR). However, new therapeutic strategies are required in order to overcome innate and acquired resistance to these drugs and thus expand the array of cancers that could benefit from them. We show that human cancer cell lines which respond poorly to ABT-888 (a PARPi), become sensitive to it when co-treated with vorinostat (a histone deacetylase inhibitor (HDACi)). Vorinostat also sensitized PARPis insensitive cancer cell lines to 6-thioguanine (6-TG)-a drug that targets PARPis sensitive cells. The sensitizing effect of vorinostat was associated with increased phosphorylation of eukaryotic initiation factor (eIF) 2α which in and of itself increases the sensitivity of cancer cells to ABT-888. Importantly, these drug combinations did not affect survival of normal fibroblasts and breast cells, and significantly increased the inhibition of xenograft tumor growth relative to each drug alone, without affecting the mice weight or their liver and kidney function. Our results show that combination of vorinostat and ABT-888 could potentially prove useful for treatment of cancer with innate resistance to PARPis due to active HRR machinery, while the combination of vorinostat and 6-TG could potentially overcome innate or acquired resistance to PARPis due to secondary or reversal BRCA mutations, to decreased PARP-1 level or to increased expression of multiple drug resistant proteins. Importantly, drugs which increase phosphorylation of eIF2α may mimic the sensitizing effect of vorinostat on cellular response to PARPis or to 6-TG, without activating all of its downstream effectors.

In-vitro expansion of insulin-producing cells from adult human pancreatic islets could provide an abundant cell source for diabetes therapy. However, proliferation of β-cell-derived (BCD) cells is associated with loss of phenotype and epithelial-mesenchymal transition (EMT). Nevertheless, BCD cells maintain open chromatin structure at β-cell genes, suggesting that they could be readily redifferentiated. The transforming growth factor β (TGFβ) pathway has been implicated in EMT in a range of cell types. Here we show that human islet cell expansion in vitro involves upregulation of the TGFβ pathway. Blocking TGFβ pathway activation using short hairpin RNA (shRNA) against TGFβ Receptor 1 (TGFBR1, ALK5) transcripts inhibits BCD cell proliferation and dedifferentiation. Treatment of expanded BCD cells with ALK5 shRNA results in their redifferentiation, as judged by expression of β-cell genes and decreased cell proliferation. These effects, which are reproducible in cells from multiple human donors, are mediated, at least in part, by AKT-FOXO1 signaling. ALK5 inhibition synergizes with a soluble factor cocktail to promote BCD cell redifferentiation. The combined treatment may offer a therapeutically applicable way for generating an abundant source of functional insulin-producing cells following ex-vivo expansion.

In vitro expansion of adult human islet β cells is an attractive solution for the shortage of tissue for cell replacement therapy of type 1 diabetes. Using a lineage tracing approach we have demonstrated that β-cell-derived (BCD) cells rapidly dedifferentiate in culture and can proliferate for up to 16 population doublings. Dedifferentiation is associated with changes resembling epithelial-mesenchymal transition (EMT). The WNT pathway has been shown to induce EMT and plays key roles in regulating replication and differentiation in many cell types. Here we show that BCD cell dedifferentiation is associated with β-catenin translocation into the nucleus and activation of the WNT pathway. Inhibition of β-catenin expression in expanded BCD cells using short hairpin RNA resulted in growth arrest, mesenchymal-epithelial transition, and redifferentiation, as judged by activation of β-cell gene expression. Furthermore, inhibition of β-catenin expression synergized with redifferentiation induced by a combination of soluble factors, as judged by an increase in the number of C-peptide-positive cells. Simultaneous inhibition of the WNT and NOTCH pathways also resulted in a synergistic effect on redifferentiation. These findings, which were reproducible in cells derived from multiple human donors, suggest that inhibition of the WNT pathway may contribute to a therapeutically applicable way for generation of functional insulin-producing cells following ex-vivo expansion.

Mycobacterium tuberculosis (Mtb) is transmitted via inhalation of aerosolized particles. While alveolar macrophages are thought to play a central role in the acquisition and control of this infection, Mtb also has ample opportunity to interact with the airway epithelium. In this regard, we have recently shown that the upper airways are enriched with a population of non-classical, MR1-restricted, Mtb-reactive CD8⁺ T cells (MAIT cells). Additionally, we have demonstrated that Mtb-infected epithelial cells lining the upper airways are capable of stimulating IFNγ production by MAIT cells. In this study, we demonstrate that airway epithelial cells efficiently stimulate IFNγ release by MAIT cells as well as HLA-B45 and HLA-E restricted T cell clones. Characterization of the intracellular localization of Mtb in epithelial cells indicates that the vacuole occupied by Mtb in epithelial cells is distinct from DC in that it acquires Rab7 molecules and does not retain markers of early endosomes such as Rab5. The Mtb vacuole is also heterogeneous as there is a varying degree of association with Lamp1 and HLA-I. Although the Mtb vacuole shares markers associated with the late endosome, it does not acidify, and the bacteria are able to replicate within the cell. This work demonstrates that Mtb infected lung epithelial cells are surprisingly efficient at stimulating IFNγ release by CD8⁺ T cells.

The development of amphetamine dependence largely depends on the effects of amphetamine in the brain reward systems. Ghrelin, an orexigenic peptide, activates the reward systems and is required for reward induced by alcohol, nicotine, cocaine and amphetamine in mice. Human genetic studies have shown that polymorphisms in the pre-proghrelin (GHRL) as well as GHS-R1A (GHSR) genes are associated with high alcohol consumption, increased weight and smoking in males. Since the heritability factor underlying drug dependence is shared between different drugs of abuse, we here examine the association between single nucleotide polymorphisms (SNPs) and haplotypes in the GHRL and GHSR, and amphetamine dependence. GHRL and GHSR SNPs were genotyped in Swedish amphetamine dependent individuals (n = 104) and controls from the general population (n = 310). A case-control analysis was performed and SNPs and haplotypes were additionally tested for association against Addiction Severity Interview (ASI) composite score of drug use. The minor G-allele of the GHSR SNP rs2948694, was more common among amphetamine dependent individuals when compared to controls (pc = 0.02). A significant association between the GHRL SNP rs4684677 and ASI composite score of drug use was also reported (pc = 0.03). The haplotype analysis did not add to the information given by the individual polymorphisms. Although genetic variability of the ghrelin signalling system is not a diagnostic marker for amphetamine dependence and problem severity of drug use, the present results strengthen the notion that ghrelin and its receptor may be involved in the development of addictive behaviours and may thus serve as suitable targets for new treatments of such disorders.

Glioblastomas (GBM), the most common and aggressive type of malignant glioma, are characterized by increased invasion into the surrounding brain tissues. Despite intensive therapeutic strategies, the median survival of GBM patients has remained dismal over the last decades. In this study we examined the expression of miR-145 in glial tumors and its function in glioma cells. Using TCGA analysis and real-time PCR we found that the expression of miR-145/143 cluster was downregulated in astrocytic tumors compared to normal brain specimens and in glioma cells and glioma stem cells (GSCs) compared to normal astrocytes and neural stem cells. Moreover, the low expression of both miR-145 and miR-143 in GBM was correlated with poor patient prognosis. Transfection of glioma cells with miR-145 mimic or transduction with a lentivirus vector expressing pre-miR 145 significantly decreased the migration and invasion of glioma cells. We identified connective tissue growth factor (CTGF) as a novel target of miR-145 in glioma cells; transfection of the cells with this miRNA decreased the expression of CTGF as determined by Western blot analysis and the expression of its 3'-UTR fused to luciferase. Overexpression of a CTGF plasmid lacking the 3'-UTR and administration of recombinant CTGF protein abrogated the inhibitory effect of miR-145 on glioma cell migration. Similarly, we found that silencing of CTGF decreased the migration of glioma cells. CTGF silencing also decreased the expression of SPARC, phospho-FAK and FAK and overexpression of SPARC abrogated the inhibitory effect of CTGF silencing on cell migration. These results demonstrate that miR-145 is downregulated in glial tumors and its low expression in GBM predicts poor patient prognosis. In addition miR-145 regulates glioma cell migration by targeting CTGF which downregulates SPARC expression. Therefore, miR-145 is an attractive therapeutic target for anti-invasive treatment of astrocytic tumors.

In an effort to circumvent resistance to rapamycin--an mTOR inhibitor--we searched for novel rapamycin-downstream-targets that may be key players in the response of cancer cells to therapy. We found that rapamycin, at nM concentrations, increased phosphorylation of eukaryotic initiation factor (eIF) 2α in rapamycin-sensitive and estrogen-dependent MCF-7 cells, but had only a minimal effect on eIF2α phosphorylation in the rapamycin-insensitive triple-negative MDA-MB-231 cells. Addition of salubrinal--an inhibitor of eIF2α dephosphorylation--decreased expression of a surface marker associated with capacity for self renewal, increased senescence and induced clonogenic cell death, suggesting that excessive phosphorylation of eIF2α is detrimental to the cells' survival. Treating cells with salubrinal enhanced radiation-induced increase in eIF2α phosphorylation and clonogenic death and showed that irradiated cells are more sensitive to increased eIF2α phosphorylation than non-irradiated ones. Similar to salubrinal--the phosphomimetic eIF2α variant--S51D--increased sensitivity to radiation, and both abrogated radiation-induced increase in breast cancer type 1 susceptibility gene, thus implicating enhanced phosphorylation of eIF2α in modulation of DNA repair. Indeed, salubrinal inhibited non-homologous end joining as well as homologous recombination repair of double strand breaks that were induced by I-SceI in green fluorescent protein reporter plasmids. In addition to its effect on radiation, salubrinal enhanced eIF2α phosphorylation and clonogenic death in response to the histone deacetylase inhibitor--vorinostat. Finally, the catalytic competitive inhibitor of mTOR--Ku-0063794--increased phosphorylation of eIF2α demonstrating further the involvement of mTOR activity in modulating eIF2α phosphorylation. These experiments suggest that excessive phosphorylation of eIF2α decreases survival of cancer cells; making eIF2α a worthy target for drug development, with the potential to enhance the cytotoxic effects of established anti-neoplastic therapies and circumvent resistance to rapalogues and possibly to other drugs that inhibit upstream components of the mTOR pathway.

BACKGROUND/AIMS: MicroRNAs (miRNAs) are short non-coding regulatory RNAs that control gene expression and play an important role in cancer development and progression. However, little is known about the role of miRNAs in chronic myeloid leukemia (CML). Our objective is to decipher a miRNA expression signature associated with CML and to determine potential target genes and signaling pathways affected by these signature miRNAs. RESULTS: Using miRNA microarrays and miRNA real-time PCR we characterized the miRNAs expression profile of CML cell lines and patients in reference to non-CML cell lines and healthy blood. Of all miRNAs tested, miR-31, miR-155, and miR-564 were down-regulated in CML cells. Down-regulation of these miRNAs was dependent on BCR-ABL activity. We next analyzed predicted targets and affected pathways of the deregulated miRNAs. As expected, in K562 cells, the expression of several of these targets was inverted to that of the miRNA putatively regulating them. Reassuringly, the analysis identified CML as the main disease associated with these miRNAs. MAPK, ErbB, mammalian target of rapamycin (mTOR) and vascular endothelial growth factor (VEGF) were the main molecular pathways related with these expression patterns. Utilizing Venn diagrams we found appreciable overlap between the CML-related miRNAs and the signaling pathways-related miRNAs. CONCLUSIONS: The miRNAs identified in this study might offer a pivotal role in CML. Nevertheless, while these data point to a central disease, the precise molecular pathway/s targeted by these miRNAs is variable implying a high level of complexity of miRNA target selection and regulation. These deregulated miRNAs highlight new candidate gene targets allowing for a better understanding of the molecular mechanism underlying the development of CML, and propose possible new avenues for therapeutic treatment.

Expansion of beta cells from the limited number of adult human islet donors is an attractive prospect for increasing cell availability for cell therapy of diabetes. However, attempts at expanding human islet cells in tissue culture result in loss of beta-cell phenotype. Using a lineage-tracing approach we provided evidence for massive proliferation of beta-cell-derived (BCD) cells within these cultures. Expansion involves dedifferentiation resembling epithelial-mesenchymal transition (EMT). Epigenetic analyses indicate that key beta-cell genes maintain open chromatin structure in expanded BCD cells, although they are not transcribed. Here we investigated whether BCD cells can be redifferentiated into beta-like cells.Redifferentiation conditions were screened by following activation of an insulin-DsRed2 reporter gene. Redifferentiated cells were characterized for gene expression, insulin content and secretion assays, and presence of secretory vesicles by electron microscopy. BCD cells were induced to redifferentiate by a combination of soluble factors. The redifferentiated cells expressed beta-cell genes, stored insulin in typical secretory vesicles, and released it in response to glucose. The redifferentiation process involved mesenchymal-epithelial transition, as judged by changes in gene expression. Moreover, inhibition of the EMT effector SLUG (SNAI2) using shRNA resulted in stimulation of redifferentiation. Lineage-traced cells also gave rise at a low rate to cells expressing other islet hormones, suggesting transition of BCD cells through an islet progenitor-like stage during redifferentiation.These findings demonstrate for the first time that expanded dedifferentiated beta cells can be induced to redifferentiate in culture. The findings suggest that ex-vivo expansion of adult human islet cells is a promising approach for generation of insulin-producing cells for transplantation, as well as basic research, toxicology studies, and drug screening.

BackgroundNeural stem cells are currently being investigated as potential therapies for neurodegenerative diseases, stroke, and trauma. However, concerns have been raised over the safety of this experimental therapeutic approach, including, for example, whether there is the potential for tumors to develop from transplanted stem cells.Methods and findingsA boy with ataxia telangiectasia (AT) was treated with intracerebellar and intrathecal injection of human fetal neural stem cells. Four years after the first treatment he was diagnosed with a multifocal brain tumor. The biopsied tumor was diagnosed as a glioneuronal neoplasm. We compared the tumor cells and the patient's peripheral blood cells by fluorescent in situ hybridization using X and Y chromosome probes, by PCR for the amelogenin gene X- and Y-specific alleles, by MassArray for the ATM patient specific mutation and for several SNPs, by PCR for polymorphic microsatellites, and by human leukocyte antigen (HLA) typing. Molecular and cytogenetic studies showed that the tumor was of nonhost origin suggesting it was derived from the transplanted neural stem cells. Microsatellite and HLA analysis demonstrated that the tumor is derived from at least two donors.ConclusionsThis is the first report of a human brain tumor complicating neural stem cell therapy. The findings here suggest that neuronal stem/progenitor cells may be involved in gliomagenesis and provide the first example of a donor-derived brain tumor. Further work is urgently needed to assess the safety of these therapies.