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Allogeneic hematopoietic stem cell transplantation (alloHSCT) is a curative treatment for hematologic malignancies. Acute and chronic graft-versus-host disease (GvHD) are the major immune-mediated complications after alloHSCT. However, there is controversy whether neurologic complications after alloHSCT might represent manifestations of GvHD. We report three patients who acquired distinct, severe immune-mediated peripheral or central nervous system diseases after alloHSCT without other, concomitant GvHD manifestations. One patient had been diagnosed with B-cell chronic lymphocytic leukemia and two patients with high risk myelodysplastic syndrome. Patient #1 presented as LGI1- and GAD-IgG positive immune-mediated encephalitis, patient #2 was diagnosed with MOG-IgG positive encephalomyelitis, and patient #3 had chronic inflammatory polyneuropathy associated with SSA(Ro)-IgG positive Sjögren’s syndrome. 100% donor chimerism was detectable in the peripheral blood in all three. The specific antibodies were undetectable in donors’ and patients’ blood before alloHSCT suggesting that the antibodies had arisen from the transplanted donor immune system. Early intensive immunotherapy led to improvement of clinical symptoms and stability of the neurological disease, however, at the cost of losing the graft-versus-malignancy effect in one patient. In conclusion, we provide evidence of isolated, severe allo-immune diseases of the peripheral and central nervous system as complications of alloHSCT (“neuro-GvHD”). Interdisciplinary surveillance and thorough diagnostic work-up are needed for early diagnosis and treatment of neuro-immunologic complications after alloHSCT to improve the otherwise poor outcome.

The present study discusses about the effects of a combination of probiotics able to stimulate the immune system of patients affected by Chronic Fatigue Syndrome/Myalgic Encephalomyelitis (CFS/ME). To this purpose, patients diagnosed according to Fukuda’s criteria and treated with probiotics were analyzed by means of clinical and laboratory evaluations, before and after probiotic administrations. Probiotics were selected considering the possible pathogenic mechanisms of ME/CFS syndrome, which has been associated with an impaired immune response, dysregulation of Th1/Th2 ratio, and high oxidative stress with exhaustion of antioxidant reserve due to severe mitochondrial dysfunction. Immune and oxidative dysfunction could be related with the gastrointestinal (GI) chronic low-grade inflammation in the lamina propria and intestinal mucosal surface associated with dysbiosis, leaky gut, bacterial translocation, and immune and oxidative dysfunction. Literature data demonstrate that bacterial species are able to modulate the functions of the immune and oxidative systems and that the administration of some probiotics can improve mucosal barrier function, modulating the release of proinflammatory cytokines, in CFS/ME patients. This study represents a preliminary investigation to verifying the safety and efficacy of a certain combination of probiotics in CFS/ME patients. The results suggest that probiotics can modify the well-being status as well as inflammatory and oxidative indexes in CFS/ME patients. No adverse effects were observed except for one patient, which displayed a flare-up of symptoms, although all inflammatory parameters (i.e., cytokines, fecal calprotectin, ESR, and immunoglobulins) were reduced after probiotic intake. The reactivation of fatigue symptoms in this patient, whose clinical history reported the onset of CFS/ME following mononucleosis, could be related to an abnormal stimulation of the immune system as suggested by a recent study describing an exaggerated immune activation associated with chronic fatigue.

Human cytomegalovirus (HCMV) reactivation remains a relevant complication after hematopoietic stem cell transplantation (HSCT) despite the great progress made in prophylaxis and treatment. Adaptive Natural Killer (NK) cells undergo a persistent reconfiguration in response to HCMV reactivation however, the exact role of adaptive NK cells in HCMV surveillance is currently unknown. We studied the relationship between HCMV reactivation and adaptive NK cells in 70 patients monitored weekly until day +100 after HSCT. Absolute cell counts of adaptive NK cells increased significantly after resolution of HCMV-reactivation compared to patients without reactivation. Patients with HCMV-reactivation had an early reconstitution of adaptive NK cells (“Responders”) and had mainly a single reactivation (75% Responders vs 48% Non-Responders). Adaptive NK cells eliminated HCMV-infected human foreskin fibroblasts (HFF) in vitro and recruited T cells in an in vitro transwell migration assay. An extensive cytokine/chemokine panel demonstrated strongly increased secretion of CXCL10/IP-10, IFN-α, IL-1α, IL-1β, IL-5, IL-7 and CCL4. Thus, adaptive NK cells may control viral spread and T cell expansion and survival during HCMV-reactivation. Taken together, we have demonstrated the potential of adaptive NK cells in the control of HCMV reactivation both by direct cytotoxicity and by recruitment of other immune cells.

Introduction The 2-year survival for AML patients relapsing after allogeneic hematopoietic cell transplantation (alloHCT) is Methods We identified mutations present at AML relapse after alloHCT by Illumina myeloid panel sequencing covering 48 AML associated genes. Peripheral whole blood samples were retrospectively collected before hematological relapse, with a minimum of one sample per patient at three months prior to relapse and if available, additional monthly samples. Amplicon-based NGS and bioinformatics error-correction were performed on those samples as described in Thol et al. 2018. Positive MRD was defined as MRD detectable above the limit of detection. In the last step, we performed polynomic curve interpolation to model relapse dynamics. Results MRD was assessed in 75 AML patients after alloHCT using 203 AML-related mutations present at the time of relapse, corresponding to a median of 2.7 trackable mutations per patient (range 1-7). In total, 305 MRD analyses were performed from peripheral blood (median 1.5 per mutation, range 1-5) prior to relapse. VAFs measured above the limit of detection (median LOD across all targets 0.0315) ranged from 0.0048-26% (median 1.3%). In 45 of 75 patients (60%), we detected MRD in at least one sample and one marker before relapse. Of those, 23 patients (51%) were MRD positive in all markers before relapse and 22 patients (49%) were MRD positive in some, but not all markers before relapse. The majority of MRD-positive patients (30 of 45) were first detected three or fewer months before relapse, whereas 15 (33%) of 45 patients were MRD positive more than 3 months before relapse. The median time to relapse from the first MRD-positive sample to relapse was 2.9 months (range 0.6-10.2). Among the 203 mutations found in relapse, 93 (46%) were detectable by MRD monitoring before relapse while the remaining 110 markers (54%) remained undetectable prior to relapse. Of note, 88 of those 110 markers (80%) were measured only once before relapse, indicating that frequent sampling increases the likelihood of MRD detection. Genes in which mutations were found mostly MRD-positive were TET2 (6 out of 6), ASXL2 (4 out of 5), SF3B1 (4 out of 5), and RUNX1 (7 out of 9). Mutations in WT1 (1 out of 13), NRAS (1 out of 8), FLT3-ITD (9 out of 29), and PTPN11 (1 out of 5) were among the most common MRD negative mutations before relapse. To assess clonal relapse dynamics, pre-relapse samples were assigned to the monthly interval that best matched the sampling time. If MRD was measured positive at one time point, all the following monthly intervals were considered MRD-positive, whether a sample was available for that interval or not. The fraction of positive samples from all samples per time point was plotted against time to relapse and the function was approximated by fifth-order polynomials. The percentage of patients being MRD positive increased markedly with shortened distance to relapse. Thus, 29% of patients were MRD positive at 3 months, 44% at 2 months and 66% 1 month prior to relapse. Summarized by functional gene classes, mutations in tumor suppressor genes and especially signaling genes showed a higher slope and thus a shorter lead-time to relapse than mutations in epigenetic modifier genes (Figure 2). Conclusion In summary, hematologic relapse can be detected in peripheral blood in 29, 44, and 66% of patients at 3, 2, and 1 months before relapse by NGS-MRD analysis, respectively. Mutations in epigenetic modifier genes show a higher fraction of MRD positivity before relapse than other mutations. In contrast, mutations in signaling genes show a shorter lead-time to relapse. Figure 1 Figure 1. Disclosures Ganser: Celgene: Honoraria; Novartis: Honoraria; Jazz Pharmaceuticals: Honoraria. Thol: Abbvie: Honoraria; Astellas: Honoraria; Novartis: Honoraria; Pfizer: Honoraria; Jazz: Honoraria; BMS/Celgene: Honoraria, Research Funding. Heuser: BergenBio: Research Funding; Bayer Pharma AG: Research Funding; AbbVie: Membership on an entity's Board of Directors or advisory committees, Research Funding; BMS/Celgene: Membership on an entity's Board of Directors or advisory committees, Research Funding; Janssen: Honoraria; Novartis: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Pfizer: Membership on an entity's Board of Directors or advisory committees, Research Funding; Daiichi Sankyo: Membership on an entity's Board of Directors or advisory committees, Research Funding; Jazz: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Astellas: Research Funding; Tolremo: Membership on an entity's Board of Directors or advisory committees; Karyopharm: Research Funding; Roche: Membership on an entity's Board of Directors or advisory committees, Research Funding.

The study of human cytomegalovirus (HCMV) has for long been challenging due to the inability of clinical strains to efficiently proliferate in vitro until adaptive mutations occur. These mutations ...

Dementia is common in the elderly, but there are currently no effective therapies available to prevent or treat this syndrome. In the last decade, polyphenols (particularly curcumin, resveratrol and tea catechins) have been under very close scrutiny as potential therapeutic agents for neurodegenerative diseases, diabetes, inflammatory diseases and aging. Data were collected from Web of Science (ISI Web of Knowledge), Pubmed and Medline (from 2000 to 2015), by searching for the keywords "dementia" AND "curcumin", "resveratrol", "EGCG", "tea catechins". The same keywords were used to investigate the current state of clinical trials recorded in the NIH clinicaltrials.gov registry. Starting from the intrinsic properties of the compounds, we explain their specific action in patients with AD and the most common types of dementia. The pharmacological actions of curcumin, resveratrol and tea catechins have mainly been attributed to their antioxidant activity, interaction with cell signaling pathways, anti-inflammatory effect, chelation of metal ions, and neuroprotection. Evidence from in vitro and in vivo studies on polyphenols have demonstrated that they may play an integral role in preventing and treating diseases associated with neurodegeneration. Furthermore, we critically analyze the clinical trials that we found, which investigate the real pharmacological actions and the possible side effects of these compounds. This review highlights the potential role of polyphenols in the prevention/treatment of dementia and describes the current limitations of research in this field.

This study aims to investigate the capability of pre-exposure to 1950 MHz to protect from the amyloid beta (Abeta)-induced cytotoxicity in human neuroblastoma SH-SY5Y cells. Pre-exposure to radiofrequency (RF) reduced of about 30% the Abeta-induced DNA damage, while RF exposure alone did not affect DNA integrity, as assessed by the alkaline comet assay. Moreover, pre-exposure to RF increased microRNA 107, affecting the enzyme responsible for the Abeta plaques deposition BACE1 and decreased the expression of microRNAs related to long term potentiation and excito-toxicity (miRNA335 and miRNA26b). On the woole, RF seems to induce a protective effect against the damage induced by Abeta in human neuroblastoma cells.

In this manuscript we report on the design, synthesis and evaluation of dual Sigma 1 Receptor (S1R) modulators/Acetylcholinesterase (AChE) inhibitors endowed with antioxidant and neurotrophic properties, potentially able to counteract neurodegeneration. The compounds based on arylalkylaminoketone scaffold integrate the pharmacophoric elements of RRC-33, a S1R modulator developed by us, donepezil, a well-known AChE inhibitor, and curcumin, a natural antioxidant compound with neuroprotective properties. A small library of compounds was synthesized and preliminary in vitro screening performed. Some compounds showed good S1R binding affinity, selectivity towards S2R and N-Methyl- d -Aspartate (NMDA) receptor, AChE relevant inhibiting activity and are potentially able to bypass the BBB, as predicted by the in silico study. For the hits 10 and 20, the antioxidant profile was assessed in SH-SY5Y human neuroblastoma cell lines by evaluating their protective effect against H2O2 cytotoxicity and reactive oxygen species (ROS) production. Tested compounds resulted effective in decreasing ROS production, thus ameliorating the cellular survival. Moreover, compounds 10 and 20 showed to be effective in promoting the neurite elongation of Dorsal Root Ganglia (DRG), thus demonstrating a promising neurotrophic activity. Of note, the tested compounds did not show any cytotoxic effect at the concentration assayed. Relying on these encouraging results, both compounds will undergo a structure optimization program for the development of therapeutic candidates for neurodegenerative diseases treatment.

This paper presents a step toward the development of a data-centric approach to prevention of Mild Cognitive Impairment and frailty in the elderly population. The scientific literature provides a large number of “indicators” for assessing the quality of behavior for aged individuals, in order to predict possible decaying. On the opposite side, a large variety of sensors and datasets today allows the effective collection of elementary data about actions performed by individuals. This paper proposes to build a bridge between these two sides. In a bottom-up vision, data from sensors and smart cities' datasets are aggregated and interpreted in a way that leads to reliable assessment of the indicators. In a top-down vision, indicators are translated into data analysis. The work described in this paper is part of City4age, a project partially funded by the EU within the H2020 Programme.

The aim of the present study was to investigate on the effects of a low-frequency pulsed electromagnetic field (LF-PEMF) in an experimental cell model of Alzheimer’s disease (AD) to assess new therapies that counteract neurodegeneration. In recent scientific literature, it is documented that the deep brain stimulation via electromagnetic fields (EMFs) modulates the neurophysiological activity of the pathological circuits and produces clinical benefits in AD patients. EMFs are applied for tissue regeneration because of their ability to stimulate cell proliferation and immune functions via the HSP70 protein family. However, the effects of EMFs are still controversial and further investigations are required. Our results demonstrate the ability of our LF-PEMF to modulate gene expression in cell functions that are dysregulated in AD (i.e., BACE1) and that these effects can be modulated with different treatment conditions. Of relevance, we will focus on miRNAs regulating the pathways involved in brain degenerative disorders.

In neurogenerative diseases, comprising Alzheimer's (AD), functional alteration in autophagy is considered one of the pathological hallmarks and a promising therapeutic target. Epidemiological investigations on the possible causes undergoing these diseases have suggested that electromagnetic fields (EMF) exposition can contribute to their etiology. On the other hand, EMF have therapeutic implications in reactivating neuronal functionality. To partly clarify this dualism, the effect of low-frequency EMF (LF-EMF) on the modulation of autophagy was investigated in human neuroblastoma SH-SY5Y cells, which were also subsequently exposed to Aβ peptides, key players in AD. The results primarily point that LF-EMF induce a significant reduction of microRNA 30a (miR-30a) expression with a concomitant increase of Beclin1 transcript (BECN1) and its corresponding protein. Furthermore, LF-EMF counteract the induced miR-30a up-regulation in the same cells transfected with miR-30a mimic precursor molecules and, on the other side, rescue Beclin1 expression after BECN1 siRNA treatment. The expression of autophagy-related markers (ATG7 and LC3B-II) as well as the dynamics of autophagosome formation were also visualized after LF-EMF exposition. Finally, different protocols of repeated LF-EMF treatments were assayed to contrast the effects of Aβ peptides in vitro administration. Overall, this research demonstrates, for the first time, that specific LF-EMF treatments can modulate in vitro the expression of a microRNA sequence, which in turn affects autophagy via Beclin1 expression. Taking into account the pivotal role of autophagy in the clearance of protein aggregates within the cells, our results indicate a potential cytoprotective effect exerted by LF-EMF in neurodegenerative diseases such as AD. J. Cell. Physiol. 229: 1776-1786, 2014. © 2014 Wiley Periodicals, Inc.

The ApoE gene is associated with the risk of Alzheimer or cardiovascular disease but its influence on exceptional longevity (EL) is uncertain. Our primary purpose was to determine, using a case-control design, if the ApoE gene is associated with EL. We compared ApoE allele/genotype frequencies among the following cohorts: cases (centenarians, most with 1+ major disease condition; n=163, 100-111years) and healthy controls (n=1039, 20-85years) from Spain; disease-free cases (centenarians; n=79, 100-104years) and healthy controls (n=597, age 27-81years) from Italy; and cases (centenarians and semi-supercentenarians, most with 1+ major disease condition; n=729, 100-116years) and healthy controls (n=498, 23-59years) from Japan. Our main findings were twofold. First, the ε4-allele was negatively associated with EL in the three cohorts, with the following odds ratio (OR) values (adjusted by sex) having been found: 0.55 (95% confidence interval (CI): 0.33, 0.94), P=0.030 (Spain); 0.41 (95%CI: 0.18, 0.99), P=0.05 (Italy); and 0.35 (95%CI: 0.26, 0.57), P

Despite advances in allogeneic stem cell transplantation, BCR-ABL-positive acute lymphoblastic leukaemia (ALL) remains a high-risk disease, necessitating the development of novel treatment strategies. As the known oncomir, miR-17~92, is regulated by BCR-ABL fusion in chronic myeloid leukaemia, we investigated its role in BCR-ABL translocated ALL. miR-17~92-encoded miRNAs were significantly less abundant in BCR-ABL-positive as compared to -negative ALL-cells and overexpression of miR-17~19b triggered apoptosis in a BCR-ABL-dependent manner. Stable isotope labelling of amino acids in culture (SILAC) followed by liquid chromatography and mass spectroscopy (LC-MS) identified several apoptosis-related proteins including Bcl2 as potential targets of miR-17~19b. We validated Bcl2 as a direct target of this miRNA cluster in mice and humans, and, similar to miR-17~19b overexpression, Bcl2-specific RNAi strongly induced apoptosis in BCR-ABL-positive cells. Furthermore, BCR-ABL-positive human ALL cell lines were more sensitive to pharmacological BCL2 inhibition than negative ones. Finally, in a xenograft model using patient-derived leukaemic blasts, real-time, in vivo imaging confirmed pharmacological inhibition of BCL2 as a new therapeutic strategy in BCR-ABL-positive ALL. These data demonstrate the role of miR-17~92 in regulation of apoptosis, and identify BCL2 as a therapeutic target of particular relevance in BCR-ABL-positive ALL.

The myostatin (MSTN) gene is a candidate to influence extreme longevity owing to its role in modulating muscle mass and sarcopenia and especially in inhibiting the main nutrient-sensing pathway involved in longevity, i.e. mammalian target of rapamycin. We compared allele/genotype distributions of the exonic MSTN variants K153R (rs1805086), E164K (rs35781413), I225T and P198A, in Spanish centenarians (cases, n = 156; 132 women, age range 100–111 years) and younger adults (controls, n = 384; 167 women, age

Previous studies have indicated that the rs2802292 polymorphism in the human forkhead box O3A (FOXO3A) gene might be associated with exceptional longevity (EL, i.e., living 100+ years), although the results are conflicting. Using a case-control design, we investigated the distribution of the rs2802292 polymorphism in two ethnically distinct cohorts of centenarians (cases) and younger adults (controls). The first cohort included Japanese individuals (733 centenarians and 820 controls) and the second was from Northern Italy (79 disease-free centenarians and 316 controls). No statistically significant association was found between the rs2802292 polymorphism and EL in either cohort (either examined in their entirety or in a sex-based analysis). In light of our negative findings, further research and resequencing efforts are needed to shed more light on the potential association between EL and FOXO3A polymorphisms. PI12/00914 3.255 JCR (2016) Q1, 11/80 Obstetrics and Ginecology; Q2, 13/49 Geriatrics and Gerontology 1.148 SJR (2016) Q1, 51/259 Biochemistry, Genetics and Molecular Biology (miscellaneous), 37/186 Obstetrics and Gynecology No data IDR 2016 UEM

Alzheimer's disease involves the formation of plaques of amyloid beta (A?), that induce cellular damage, including oxidative DNA damage. This study aims to investigate the capability of RF exposure to reduce the A?-induced DNA damage in SH-SY5Y cells. To this purpose a dose-response curve of A? has been set up in terms of DNA migration, and preliminary results on the effect of RF are reported.

Chronic fatigue syndrome (CFS) is a disease that can seriously impair one's quality of life; patients complain of excessive fatigue and myalgia following physical exertion. This disease may be associated with abnormalities in genes affecting exercise tolerance and physical performance. Adenosine monophosphate deaminase (AMPD1), carnitine palmitoyltransferase II (CPT2), and the muscle isoform of glycogen phosphorylase (PYGM) genes provide instructions for producing enzymes that play major roles in energy production during work. The aim of this study was to look for evidence of genotype-associated excessive muscle fatigue. Three metabolic genes (AMPD1, CPT2, and PYGM) were therefore fully sequenced in 17 Italian patients with CFS. We examined polymorphisms known to alter the function of these metabolic genes, and compared their genotypic distributions in CFS patients and 50 healthy controls using chi-square tests and odds ratios. One-way analysis of variance with F-ratio was carried out to determine the associations between genotypes and disease severity using CF scores. No major genetic variations between patients and controls were found in the three genes studied, and we did not find any association between these genes and CFS. In conclusion, variations in AMPD1, CPT2, and PGYM genes are not associated with the onset, susceptibility, or severity of CFS.

The stem cell zinc finger 1 (SZF1)/ZNF589 protein belongs to the large family of Kruppel-associated box domain–zinc finger (KRAB-ZNF) transcription factors, which are present only in higher vertebrates and epigenetically repress transcription by recruiting chromatin-modifying complexes to the promoter regions of their respective target genes. Although the distinct biological functions of most KRAB–ZNF proteins remain unknown, recent publications indicate their implication in fundamental processes, such as cell proliferation, apoptosis, differentiation, development, and tumorigenesis. SZF1/ZNF589 was first identified as a gene with SZF1-1 isoform specifically expressed in CD34 + hematopoietic cells, strongly suggesting a role in epigenetic control of gene expression in hematopoietic stem/progenitor cells (HSPCs). However, the function of SZF1/ZNF589 in hematopoiesis has not yet been elucidated. Our study reveals SZF1/ZNF589 as a gene with a human-specific nucleotide DNA-change, conferring potential species-specific functional properties. Through shRNA-mediated loss-of-function experiments, we found that changes in expression of fundamental apoptosis-controlling genes are induced on SZF1/ZNF589 knockdown, resulting in inhibited growth of hematopoietic cell lines and decreased progenitor potential of primary human bone marrow CD34 + cells. Moreover, we found that the SZF1/ZNF589 gene is differentially regulated during hypoxia in CD34 + HSPCs in a cytokine-dependent manner, implicating its possible involvement in the maintenance of the hypoxic physiologic status of hematopoietic stem cells. Our results establish the role of SZF1/ZNF589 as a new functional regulator of the hematopoietic system.

Alzheimer's disease (AD) has been recognized as the most common cause of sporadic dementia. It represents both a medical and social problem, as it affects 10% of over-65 population. Even if the elderly are the most involved population, aging alone cannot be considered as the only cause of this disease. In this review we wanted to focus on the last hypotheses on the possible causes of this neuronal affection. We focused in particular on the role of inflammation and alteration of the inflammatory status that is typical of the elderly and may lead to chronic inflammation. The inflammation seems to be a cause of neuronal impairment and loss. Some studies have proposed a protective role of antiinflammatory drugs. Then we analyzed the role of genetic polymorphisms of some pro-inflammatory substances that seem to be linked to some cases of dementia. The complement system seems to have a role too, as some factors have been found in senile plaques, representing a possible involvement of classical complement pathway. One of the latest hypotheses is about the role of blood-brain barrier (BBB), as its loss of integrity may lead to a passage of proteins in cerebro spinal fluid (CSF), causing a compromised role of BBB in preserving the brain as an "immune sanctuary".

It is well known that physiological functions and pathological conditions of cells and tissues can be influenced not only by chemical molecules, but also by physical stimuli such as electromagnetic waves. In particular, epidemiological studies suggest possible associations between exposure to electromagnetic fields and an increased risk of tumors and neurodegenerative disorders, such as Alzheimer's disease. However, depending on the dose and on the length of treatment, the electromagnetic stimuli can be harmful or induce a cytoprotective cellular response, suggesting a possible application in medical therapy. In this study, under a tissue engineering viewpoint, we investigated the effects of an electromagnetic wave (magnetic field intensity, 2 mT; frequency, 75 Hz) on a neuronal cellular model characterized by the overexpression of the amyloid precursor protein (APP). After a prolonged electromagnetic treatment, lower mitochondrial activity and proliferation rate, resulting in a higher cellular quiescence, were observed. Focusing on the stress and oxidative pathways, we detected an overall increase of two fundamental proteins, the chaperone heat shock protein HSP70 and the free radical scavenger superoxide dismutase-1 enzyme (SOD-1). Interestingly, we found that the electromagnetic stimulation promotes the nonamyloidogenic processing of APP through an increased expression of the α-secretase ADAM10 and an enhanced release of the soluble neurotrophic factor sAPPα (a product of the ADAM10-mediated cleavage of APP). In conclusion, these findings suggest that the electromagnetic stimulus, if properly administered in terms of dose and timing, is able to induce a cytoprotective response in the cell. Moreover, these results suggest a possible use of this particular physical stimulation to improve the functional capability of the cells to face noxae.

In T-cell acute lymphoblasic leukemia (T-ALL), neoplastic chromosomal rearrangements are known to deregulate members of the homeobox gene families NKL and HOXA. Here, analysis of T-ALL cell lines and primary cells identified aberrant expression of a third homeobox gene group, the Paired (PRD) class. LOUCY cells revealed chromosomal deletion at 5q31, which targets the downstream regulatory region of the PRD homeobox gene PITX1, removing a STAT1 binding site. STAT1 mediates repressive interleukin 2 (IL2)-STAT1 signaling, implicating IL2 pathway avoidance as a possible activation mechanism. Among primary T-ALL samples, 2/22 (9%) aberrantly expressed PITX1, highlighting the importance of this gene. Forced expression of PITX1 in JURKAT cells and subsequent target gene analysis prompted deregulation of genes involved in T-cell development including HES1, JUN, NKX3-1, RUNX1, RUNX2, and TRIB2. Taken together, our data show leukemic activation of PITX1, a novice PRD-class homeobox gene in a subset of early-staged T-ALL, which may promote leukemogenesis by inhibiting T-cell development.

Myocyte enhancer factor 2C (MEF2C) encodes a transcription factor which is ectopically expressed in T-cell acute lymphoblastic leukemia (T-ALL) cell lines, deregulated directly by ectopically expressed homeodomain protein NKX2-5 or by loss of promoter regions via del(5)(q14). Here, we analyzed the MEF2C 5'-region, thus identifying potential regulatory binding sites for GFI1B, basic helix-loop-helix proteins, STAT5, and HOXA9/HOXA10. Chromatin immunoprecipitation and overexpression analyses demonstrated direct activation by GFI1B and LYL1 and inhibition by STAT5. HOXA9/HOXA10 activated expression of NMYC which in turn mediated MEF2C repression, indicating an indirect mode of regulation via NMYC interactor (NMI) and STAT5. Lacking comma: Chromosomal deletion of the STAT5 binding site in LOUCY cells reduced protein levels of STAT5 in some MEF2C-positve T-ALL cell lines, and the presence of inhibitory IL7-JAK-STAT5 signaling highlighted the repressive impact of this factor in MEF2C regulation. Taken together, our results indicate that the expression of MEF2C in T-ALL cells is principally deregulated via activating leukemic transcription factors GFI1B or NKX2-5 and by escaping inhibitory developmental STAT5 signaling.

Basic helix-loop-helix (bHLH) transcription factors are essential for lymphocytic differentiation. Here, we have analyzed the complete bHLH family in T-cell acute lymphoblastic leukemia cell lines by expression profiling. Differential expression was detected for BHLHB2, HES1, HES4, HEY1, ID1, ID2, ID3, LYL1 and TAL1, highlighting dysregulation of family members with inhibitory activity. Subsequently we focused on the mechanisms responsible for aberrant expression of LYL1 in comparison to TAL1. Quantitative genomic PCR indicated microdeletions upstream of both, TAL1 and LYL1, targeting STIL/SIL and TRMT1, respectively. Additionally, one LYL1-expressing cell line exhibited amplification of TRMT1. While deletion of STIL correlated with expression of the STIL-TAL1 fusion transcript, no TRMT-LYL1 fusion transcripts were detected in parallel with genomic rearrangements thereof. Sequence analysis of the LYL1 promoter region revealed potential binding sites for transcription factors HOXA10, LMO2 and NKX2-5. Overexpression analysis, reporter gene assays and chromatin immuno-precipitation confirmed their activating impact on LYL1 expression. In conclusion, we identified multiple mechanisms which activate LYL1 in leukemic cells, including structural genomic alterations, namely microdeletion or amplification, together with the involvement of prominent oncogenic transcription factors.

In Alzheimer’s disease (AD), the presence of amyloid-β peptide may impair cell energy formation by altering both anaerobic and aerobic metabolism. This study aimed to estimate possible alterations in circulating energy substrates. In 54 community-dwelling AD subjects, fasting peripheral venous blood samples were drawn in the morning to determine the energy substrates lactate, pyruvate and ketone bodies (KBs, β-hydroxybutyrate and acetoacetate). Plasma lactate levels in the entire AD population were significantly lower than in healthy controls ( P 5 years. Moreover, both plasma lactate and pyruvate were lower in subjects with AD >5 years than in subjects with AD ⩽5 years ( P

The NK-like family of homeobox genes includes TLX1, TLX3 and NKX2-5, which are ectopically activated in distinct subsets of T-cell acute lymphoblastic leukemia (T-ALL) cells. Here we analysed their effect on the miR-17-92 cluster overexpressed in several types of cancer, including T-ALL. The pri-miR-17-92 polycistron encodes micro-RNAs (miRNAs), which decrease E2F1 protein expression, regulating proliferation and/or apoptosis. Quantification of pri-miR-17-92 in T-ALL cell lines suggested an implication of the NK-like homeodomain proteins in transcriptional regulation. Lentiviral-mediated overexpression of NKX2-5 in the T-ALL cell line MOLT-4 consistently resulted in increased miR-17-92 pri-miRNA levels and decreased amounts of E2F1 protein. Induction of apoptosis by treating miR17-92 or E2F1 transduced T-ALL cells with etoposide led to reduced or enhanced cell viability, respectively. Furthermore, analysis of pri-miR-17-92 in T-ALL patients indicated elevated expression in those bearing TLX1/3 positive cells. These data support an activatory effect of NK-like homeodomain proteins on pri-miR-17-92 expression and concomitantly reduced E2F1 protein levels, thereby enhancing survival of leukemic T-cells.

Aberrant micro RNA (miRNA) expression has been described in human malignancies including B-cell lymphomas. We here report BCR-ABL– and c-MYC–dependent regulation of miRNA expression in chronic myeloid leukemia (CML) using microarray analysis (miCHIP) and miRNA-specific quantitative real-time reverse transcriptase–polymerase chain reaction (miR-qRT-PCR). In 3 bcr-abl–positive cell lines, expression of miRNAs encoded within the polycistronic miR-17-92 cluster is specifically down-regulated (2- to 5-fold) by both imatinib treatment and anti–BCR-ABL RNA interference (RNAi). In addition, anti–c-MYC RNAi reduces miR-17-92 expression in K562 cells in which miRNAs can specifically repress reporter gene expression, as demonstrated by specific miRNA inhibition with antagomirs. Furthermore, lentivirus-mediated overexpression of polycistronic miRNAs in K562 cells confers increased proliferation, partial resistance against anti–c-MYC RNAi, and enhanced sensitivity to imatinib-induced cell death. Finally, we determined miR-17-92 expression in purified normal (n = 4), early chronic-phase (CP) (n = 24), and blast-crisis (BC) (n = 7) CML CD34+ cells and found up-regulation of polycistronic pri-miRNA transcripts in CML and mature miRNAs in CP but not in BC CML. These data are in accordance with a BCR-ABL–c-MYC–miR-17-92 pathway that mediates enhanced miRNA expression in CP but not BC CML CD34+ cells. Altered miRNA expression may contribute to the pathophysiology of the disease and may provide potential targets for therapeutic intervention.

In this study we analyzed the clinical features of a population of Italian patients with chronic fatigue syndrome (CFS) diagnosed according to the CDC-1994 criteria. The aim was to investigate CFS patients and their relatives, in order to search for events related to the onset of the disease and to identify correlations with other diseases. The analysis was carried out by examining medical records belonging to 82 patients suffering from the syndrome. The documentation was collected between 2008 and 2011 and provided by the non-profit Italian organization AMCFS (Associazione Malati di CFS). The influence of gender on the age of onset and association with potential risk factors were investigated in patients and in their relatives. From the results a significant correlation between the age of onset and autoimmunity was observed.

Although the number of centenarians is growing worldwide, the potential factors influencing the aging process remain only partially elucidated. Researchers are increasingly focusing toward biomarkers as tools to shed more light on the pathophysiology of complex phenotypes, including the ability to reach successful aging, i.e., free of major chronic diseases. We therefore conducted a case-control study examining the potential associations of multiple candidate biomarkers in healthy centenarians and sex-matched healthy elderly controls. Using a case-control study of 81 centenarians (aged ≥ 100 years) selected based on the fact that they were disease-free and 46 healthy elderly controls (aged 70-80 years), serum levels of 15 different candidate biomarkers involved in the regulation of metabolism, angiogenesis, inflammation, and bone formation were measured. Of the 15 biomarkers tested, four molecules (chemerin, fetuin-A, and fibroblast growth factors [FGF] 19 and 21) were found to be independently associated with successful aging regardless of sex. Logistic regression analysis confirmed that chemerin, fetuin-A, FGF19, and FGF21 were independently associated with successful aging [predicted probability (PP) = 1 / [1 + 1 / exp (11.832 - 0.027 × (chemerin) - 0.009 × (fetuin-A) + 0.014 × (FGF19) - 0.007 × (FGF21)]. The area under the curve (AUC) of predicted probability values for the four-biomarker panel revealed that it can discriminate between centenarians and elderly controls with excellent accuracy (AUC > 0.94, P

Generating cellularized 3D constructs with clinical relevant dimensions is challenged by nutrition supply. This is of utmost importance for cardiac tissue engineering, since cardiomyocytes are extremely sensitive to malnutrition and hypoxia in vitro and after implantation. To develop a perfusable myocardial patch, we have focused on seeding a decellularized biological vascularized matrix (BioVaM) with endothelial cells. BioVaM is produced by decellularization of porcine small intestinal segments with preserved arterial and venous pedicles, which can be connected to a perfusion system in vitro or the host vasculature in vivo. The BioVaM vessel bed was re-seeded with porcine primary endothelial cells (pCEC). Seeding efficiency was influenced by detergent composition used for decellularization (sodium dodecyl sulfate (SDS) and/or Triton X-100) and the medium composition used for re-seeding. After decellularization, residual SDS was detected in the matrix affecting the survival of pCEC which showed a low tolerance to SDS and Triton X-100. Sensitivity to detergents was attenuated by supplementation of the medium with bovine serum albumin (BSA) or fetal calf serum (FCS). Pre-conditioning of the BioVaM with 20 % FCS was not sufficient to attain pCEC survival in the vascular bed. However, re-cellularization was achieved by prolonged FCS supplementation during cultivation, resulting in a perfusable, re-endothelialized matrix of 11 cm2 in size. This achievement represents a promising step towards engineering of perfusable, 3D cardiac constructs with clinically relevant dimensions.

Alzheimer's disease (AD) is widely identified as the most common cause of sporadic dementia. Its aetiology is still debated, as despite several hypotheses, different factors seem to play a role in its establishment and development. Recent studies have proposed a possible preventing role of nutrition. The weight loss typical of earlier phase of disease and the finding of malnutrition as a common trait between patients leads to hypothesize that a supplementation of specific nutrients seems to be useful and effective in terms of improvement of cognitive functions. Malnourished patients show also altered parameters when investigating inflammation markers: for example, hyperhomocysteinemia is a typical finding in elderly affected by dementia, and it can be prevented and corrected by using a proper nutrients supplementation. Pro-inflammatory state can be reduced with supplementation of polyunsaturated fatty acids, vitamins of the group B and phosphatidylserine, that can act reducing IL-1β (pro-inflammatory cytokine) and improving IL-10 (anti-inflammatory cytokine) synthesis. While investigating the role of nutrition, it seems to be deeply linked with genetic; a genetic onset AD-related could be latent and can be influenced by nutritional attitude. AD can be considered a sort of latent clinical condition that would disclose or not, depending also on micro-environment and nutritional parameters. The genetic expression can be influenced by assumptions or not of specific nutrients, with the promotion of different pro- or anti-inflammatory settings. The specific role of each micronutrient (in particular vitamins) and trace elements still needs to be punctuated, as they are involved in a pool of different reactions. Also genes acts not independently but in an interconnected pattern, in which the role of a single gene needs to be cleared, depending on others. This complex system of predisposing conditions and a possible role of nutrition as modulator of the inflammatory state is the object of this review.

In neurogenerative diseases, comprising Alzheimer's (AD), functional alteration in autophagy is considered one of the pathological hallmarks and a promising therapeutic target. Epidemiological investigations on the possible causes undergoing these diseases have suggested that electromagnetic fields (EMF) exposition can contribute to their etiology. On the other hand, EMF have therapeutic implications in reactivating neuronal functionality. To partly clarify this dualism, the effect of low-frequency EMF (LF-EMF) on the modulation of autophagy was investigated in human neuroblastoma SH-SY5Y cells, which were also subsequently exposed to Aβ peptides, key players in AD. The results primarily point that LF-EMF induce a significant reduction of microRNA 30a (miR-30a) expression with a concomitant increase of Beclin1 transcript (BECN1) and its corresponding protein. Furthermore, LF-EMF counteract the induced miR-30a up-regulation in the same cells transfected with miR-30a mimic precursor molecules and, on the other side, rescue Beclin1 expression after BECN1 siRNA treatment. The expression of autophagy-related markers (ATG7 and LC3B-II) as well as the dynamics of autophagosome formation were also visualized after LF-EMF exposition. Finally, different protocols of repeated LF-EMF treatments were assayed to contrast the effects of Aβ peptides in vitro administration. Overall, this research demonstrates, for the first time, that specific LF-EMF treatments can modulate in vitro the expression of a microRNA sequence, which in turn affects autophagy via Beclin1 expression. Taking into account the pivotal role of autophagy in the clearance of protein aggregates within the cells, our results indicate a potential cytoprotective effect exerted by LF-EMF in neurodegenerative diseases such as AD. J. Cell. Physiol. 229: 1776-1786, 2014. © 2014 Wiley Periodicals, Inc.

Focal adhesion is critical for cell survival. The focal adhesion kinase (FAK, or PTK2) is an important component of the human interactome and thus is a potential longevity-related protein. Here we studied the association between two PTK2 gene single-nucleotide polymorphisms (SNPs) (rs7843014, rs7460) and exceptional longevity (EL). In addition to gaining insight into their functionality by determining luciferase gene reporter activity, we studied the genotype/allele frequency of these two SNPs among three different cohorts: (1) Spanish centenarians (n=175, 100–111 years, 144 women) and healthy controls (n=355, 20–50 years, 284 women); (2) Italian centenarians (n=79, 100–104 years, 40 women) and controls (n=316, 29–50 years, 156 women); and (3) Japanese centenarians (n=742, 100–116 years, 623 women) and healthy controls (n=499, 23–59 years, 356 women). Both SNPs had functional significance, with the A allele up-regulating luciferase activity compared to the other allele (rs7460 T allele and rs7843014 C allele, respectively). The A allele of both SNPs was negatively associated with EL in the Spanish cohort (rs7460, odds ratio [OR] adjusted by sex=0.40, 95% confidence intervals [CI] 0.3, 0.6, p

Irisin might play an important role in reducing the risk of obesity, insulin resistance, or several related diseases, and high irisin levels may contribute to successful aging. Thus, the irisin precursor (FNDC5) gene is a candidate to influence exceptional longevity (EL), i.e., being a centenarian. It has been recently shown that two single-nucleotide polymorphisms (SNPs) in the FNDC5 gene, rs16835198 and rs726344, are associated with in vivo insulin sensitivity in adults. We determined luciferase gene reporter activity in the two above-mentioned SNPs and studied genotype distributions among centenarians (n = 175, 144 women) and healthy controls (n = 347, 142 women) from Spain. We also studied an Italian [79 healthy centenarians (40 women) and 316 healthy controls (156 women)] and a Japanese cohort [742 centenarians (623 women) and 499 healthy controls (356 women)]. The rs726344 SNP had functional significance, as shown by differences in luciferase activity between the constructs of this SNP (all P ≤ 0.05), with the variant A-allele having higher luciferase activity compared with the G-allele (P = 0.04). For the rs16835198 SNP, the variant T-allele tended to show higher luciferase activity compared with the G-allele (P = 0.07). However, we found no differences between genotype/allele frequencies of the two SNPs in centenarians versus controls in any cohort, and no significant association (using logistic regression adjusted by sex) between the two SNPs and EL. Further research is needed with different cohorts as well as with additional variants in the FNDC5 gene or in other genes involved in irisin signaling. 3.390 JCR (2014) Q2, 14/50 Geriatrics & gerontology UEM

Alzheimer's disease (AD) is a neurodegenerative disorder involving deposition of misfolded proteins in vulnerable brain regions leading to inexorable and progressive neuronal loss and deterioration of cognitive functions. The AD brain displays features typical of chronic inflammation as defined by the presence of activated microglia and by an excessive amount of neuroinflammatory components such as cytokines and acute-phase proteins. This review aims to shed light on the role of the immune processes involved in AD, focusing on a family of inflammatory modulators belonging to the acute-phase proteins and crucial components of the humoral arm of innate immunity: pentraxins. In particular we analyze function of the pentraxins in AD, their upregulation in the brain and their contribution to neurodegeneration. Additionally, we highlight the role of pentraxins as putative AD biomarkers and as pharmacological therapeutic targets.

Deregulation of Ras/ERK signaling in myeloid leukemias makes this pathway an interesting target for drug development. Myeloid leukemia cell lines were screened for idarubicin-induced apoptosis, cell-cycle progression, cell-cycle-dependent MAP kinase kinase (MEK-1/2) activation, and Top2 expression. Cell-cycle-dependent activation of MEK/ERK signaling was blocked using farnesyltransferase inhibitor (FTI) BMS-214,662 and dual prenyltransferase inhibitor (DPI) L-778,123 to disrupt Ras signaling. Idarubicin caused a G2/M cell-cycle arrest characterized by elevated diphosphorylated MEK-1/2 and Top2α expression levels. The FTI/DPIs elicited distinct effects on Ras signaling, protein prenylation, cell cycling and apoptosis. Combining these FTI/DPIs with idarubicin synergistically inhibited proliferation of leukemia cell lines, but the L-778,123+idarubicin combination exhibited synergistic growth inhibition over a greater range of drug concentrations. Interestingly, combined FTI/DPI treatment synergistically inhibited cell proliferation, induced apoptosis and nearly completely blocked protein prenylation. Inhibition of K-Ras expression by RNA interference or blockade of its post-translational prenylation led to increased BMS-214,662-induced apoptosis. Our results suggest that nearly complete inhibition of protein prenylation using an FTI + DPI combination is the most effective method to induce apoptosis and to block anthracycline-induced activation of ERK signaling.

Aims Mice with a cardiomyocyte (CM)-restricted knockout of signal transducer and activator of transcription 3 (STAT3-KO) develop spontaneous heart failure. We investigated the impact of STAT3-mediated regulation of microRNAs for pathophysiological alterations in the heart. Methods and results MicroRNAchip and qRT–PCR analysis revealed elevated cardiac expression of miR-199a in STAT3-KO mice. Lentiviral shRNA-mediated STAT3-knock-down in neonatal rat CMs markedly increased miR-199a promoter activity and miR-199a levels indicative of a suppressive effect of STAT3 on miR-199a transcription. Up-regulated miR-199a in CM by pre-miR-199a transfection (pre-miR-199a-CM) reduced expression of components of the ubiquitin-proteasome system (UPS), i.e. the ubiquitin-conjugating enzymes Ube2g1 (mRNA and protein) and Ube2i (protein). Pre-miR-199a-CM or CM with siRNA-mediated down-regulation of Ube2i and Ube2g1 (siRNA-Ube2i/2g1-CM) displayed massive down-regulation of α- and β-myosin heavy chain expression associated with disrupted sarcomere structures. In addition, protein arginine methyltransferase I (PRMT-I) expression and asymmetric dimethylarginine (ADMA) synthesis were increased in pre-miR-199a-CM or in siRNA-Ube2i/2g1-CM. Increased ADMA in cell culture supernatant (SN) from pre-miR-199a-CM or siRNA-Ube2i/2g1-CM lowered nitric oxide (NO) bioavailability of rat cardiac endothelial cells while lowering ADMA concentration in CM SNs by the PRMT inhibitor arginine methyltransferase inhibitor 1 (AMI-1) (100 µM) improved NO bioavailability. In STAT3-KO hearts Ube2i and Ube2g1 expression were markedly reduced. Human terminal failing hearts harbouring low STAT3 protein levels displayed increased miR-199a levels and decreased Ube2g1 expression. Conclusion This study identifies a novel pathophysiological circuit in the heart between reduced STAT3 protein levels, increased miR-199a expression, and subsequent impairment of the UPS that disrupts CM sarcomere structure and impairs via the release of ADMA endothelial cell function.

Abstract 1281 Poster Board I-303 Many oncogenes code for transcription factors involved in regulation of developmental pathways. The activity of these pathways is tissue specific and restricted to certain developmental stages. Here, we searched for T-cell acute lymphoblastic leukemia (T-ALL) oncogenes which physiologically regulate differentiation of natural killer (NK) cells. NK- and T-cells are closely related lymphocytes, sharing the same early progenitors which can differentiate into either lineage. We compared expression profiles of malignant NK- and T-cell lines to identify aberrantly expressed genes in T-ALL. This analysis revealed high expression of HOXA9, HOXA10 and ID2 in NK-cell lines and in one T-ALL line, LOUCY, suggesting leukemic deregulation therein. Subsequently, we analyzed mechanisms underlying their regulation. Overexpression and chromatin immuno-precipitation experiments demonstrated that HOXA9 and HOXA10 directly activate ID2 expression. Analysis of other ALL and acute myeloid leukemia cell lines with and without mixed lineage leukemia (MLL) gene translocations demonstrated a correlated expression of HOXA9/10 and ID2, highlighting ID2 as an indirect target of MLL fusion proteins which deregulate HOXA genes. Furthermore, profiling data of genes coding for chromatin regulators of homeobox genes, including the components EZH2 and HOP of polycomb repressor complex 2 (PRC2), showed downregulation of EZH2 in LOUCY and limited expression of HOP to NK-cell lines. Subsequent treatment of T-ALL cell lines JURKAT and LOUCY with DZNep, an inhibitor of EZH2/PRC2, resulted in elevated and unchanged HOXA9/10 expression levels, respectively, confirming repressive activity of EZH2 in T-cells. Additionally, profiling data and overexpression analysis indicated that reduced expression of E2F cofactor TFDP1 contributed to the lack of EZH2 in LOUCY. Forced expression of HOP in JURKAT cells resulted in reduced HOXA10 and ID2 expression levels, suggesting enhancement of PRC2 repression. Taken together, our results show that major differentiation factors of the NK-cell lineage, including HOXA9, HOXA10 and ID2, were (de)regulated via PRC2 and may contribute to T-cell leukemogenesis. Disclosures No relevant conflicts of interest to declare.

Micro (mi)RNAs are highly conserved small regulatory RNAs, which regulate gene expression by hybridization to specific binding sites in the 3'untranslated region (UTR) of many mRNAs. Upon miRNA-guided recruitment of a multiprotein complex, target mRNAs are either degraded or their translation is blocked depending on the complementarity between the miRNAs and their binding sites in target mRNAs. Individual miRNAs have been shown to regulate the expression of hundreds of genes with corresponding miRNA binding sites in the 3'UTR in a dose-dependent manner. Although miRNA-target genes may be predicted by bioinformatic tools, each potential target needs to be confirmed experimentally. We describe here the expression of individual miRNAs or miRNA-specific antagomiRs by lentiviral gene transfer to induce stable gain- and loss-of-function phenotypes. These techniques provide some tools to analyze miRNA function in cell culture or animal models.

Micro (mi)RNAs are highly conserved small regulatory RNAs, which regulate gene expression by hybridization to specific binding sites in the 3'untranslated region (UTR) of many mRNAs. Upon miRNA-guided recruitment of a multiprotein complex, target mRNAs are either degraded or their translation is blocked depending on the complementarity between the miRNAs and their binding sites in target mRNAs. Individual miRNAs have been shown to regulate the expression of hundreds of genes with corresponding miRNA binding sites in the 3'UTR in a dose-dependent manner. Although miRNA-target genes may be predicted by bioinformatic tools, each potential target needs to be confirmed experimentally. We describe here the expression of individual miRNAs or miRNA-specific antagomiRs by lentiviral gene transfer to induce stable gain- and loss-of-function phenotypes. These techniques provide some tools to analyze miRNA function in cell culture or animal models.

Background Homeodomain proteins control fundamental cellular processes in development and in cancer if deregulated. Three members of the NK-like subfamily of homeobox genes (NKLs), TLX1, TLX3 and NKX2-5, are implicated in T-cell acute lymphoblastic leukemia (T-ALL). They are activated by particular chromosomal aberrations. However, their precise function in leukemogenesis is still unclear. Here we screened further NKLs in 24 T-ALL cell lines and identified the common expression of MSX2. The subsequent aim of this study was to analyze the role of MSX2 in T-cell differentiation which may be disturbed by oncogenic NKLs. Methods Specific gene activity was examined by quantitative real-time PCR, and globally by expression profiling. Proteins were analyzed by western blot, immuno-cytology and immuno-precipitation. For overexpression studies cell lines were transduced by lentiviruses. Results Quantification of MSX2 mRNA in primary hematopoietic cells demonstrated higher levels in CD34+ stem cells as compared to peripheral blood cells and mature CD3+ T-cells. Furthermore, analysis of MSX2 expression levels in T-cell lines after treatment with core thymic factors confirmed their involvement in regulation. These results indicated that MSX2 represents an hematopoietic NKL family member which is downregulated during T-cell development and may functionally substituted by oncogenic NKLs. For functional analysis JURKAT cells were lentivirally transduced, overexpressing either MSX2 or oncogenic TLX1 and NKX2-5, respectively. These cells displayed transcriptional activation of NOTCH3-signaling, including NOTCH3 and HEY1 as analyzed by gene expression profiling and quantitative RT-PCR, and consistently attenuated sensitivity to gamma-secretase inhibitor as analyzed by MTT-assays. Furthermore, in addition to MSX2, both TLX1 and NKX2-5 proteins interacted with NOTCH-pathway repressors, SPEN/MINT/SHARP and TLE1/GRG1, representing a potential mechanism for (de)regulation. Finally, elevated expression of NOTCH3 and HEY1 was detected in primary TLX1/3 positive T-ALL cells corresponding to the cell line data. Conclusion Identification and analysis of MSX2 in hematopoietic cells implicates a modulatory role via NOTCH3-signaling in early T-cell differentiation. Our data suggest that reduction of NOTCH3-signaling by physiological downregulation of MSX2 expression during T-cell development is abrogated by ectopic expression of oncogenic NKLs, substituting MSX2 function.

RNA interference (RNAi) is an evolutionarily conserved sequence-specific post-transcriptional gene silencing mechanism triggered by double-stranded RNA (dsRNA) that results either in degradation of homologues mRNAs or inhibition of mRNA translation. The effector molecules which activate the RNAi pathway are small regulatory RNAs including small interfering RNAs (siRNAs) which are processed from longer dsRNAs by the RNAse III enzyme Dicer, and microRNAs (miRNAs) generated in a regulated multistep process from endogenous primary transcripts (pri-miRNA). Since, in principle, any gene can be silenced, RNAi provides a powerful tool to investigate gene function, and it is therefore a widely used gene silencing method in functional genomics. This chapter provides a collection of protocols for specific gene knock-down in hematopoietic cells by the application of short-hairpin RNAs (shRNAs) transcribed by RNA polymerase III (pol III) promoters or artificial-miRNAs (art-miRNAs) expressed from RNA pol II promoters using lentiviral vectors, respectively.

RNA interference (RNAi) is an evolutionarily conserved sequence-specific post-transcriptional gene silencing mechanism triggered by double-stranded RNA (dsRNA) that results either in degradation of homologues mRNAs or inhibition of mRNA translation. The effector molecules which activate the RNAi pathway are small regulatory RNAs including small interfering RNAs (siRNAs) which are processed from longer dsRNAs by the RNAse III enzyme Dicer, and microRNAs (miRNAs) generated in a regulated multistep process from endogenous primary transcripts (pri-miRNA). Since, in principle, any gene can be silenced, RNAi provides a powerful tool to investigate gene function, and it is therefore a widely used gene silencing method in functional genomics. This chapter provides a collection of protocols for specific gene knock-down in hematopoietic cells by the application of short-hairpin RNAs (shRNAs) transcribed by RNA polymerase III (pol III) promoters or artificial-miRNAs (art-miRNAs) expressed from RNA pol II promoters using lentiviral vectors, respectively.

Micro RNAs (miRNA) regulate gene expression by hybridization and recruitment of multi-protein complexes to complementary mRNA target sequences. miRNA function can transiently be antagonized by antagomirs—chemically modified oligonucleotides complementary to individual miRNAs. Here, we describe the induction of stable loss-of-function phenotypes for specific miRNAs by lentivirus-mediated antagomir expression. Lentivirally expressed antagomirs are transcribed from a H1-promoter located within the lentiviral 3′LTR and were directed against miRNAs encoded on the polycistronic miR17-92 transcript. Functional silencing of miR-18a, miR-19b and miR-20a by the corresponding antagomirs specifically relieves miRNA-mediated reporter gene repression. Inhibition of miRNA function correlates to reduction of ‘miRNA’ amplification by miRNA-specific quantitative RT-PCR. Furthermore, protein expression of E2F-1, a known miR-20 target, is enhanced by lentivirally expressed anti-miR-20 antagomirs in a dose-dependent manner, whereas over-expression of miR-20a reduces E2F-1 levels. Finally, combined over-expression of specific miRNAs and antagomirs reveals individual and complementary functions of miR-18a and miR-20a and demonstrates specific miRNA impact on cell proliferation in a cell culture model.

Many members of the nearly 200-strong homeobox gene family have been implicated in cancer, mostly following ectopic expression. In this study we analyzed homeobox gene expression in Hodgkin lymphoma (HL) cell lines. Both reverse transcription–polymerase chain reaction (RT-PCR) using degenerate primers and microarray profiling identified consistently up-regulated HOXB9 expression. Analysis of HOXB9 regulation in HL cells revealed E2F3A and BMI1 as activator and repressor, respectively. Furthermore, a constitutively active ERK5 pathway was identified in all HL cell lines analyzed as well as primary HL cells. Our data show that ERK5 probably mediates HOXB9 expression by repressing BMI1. In addition, expression analysis of the neighboring microRNA gene mir-196a1 revealed coregulation with HOXB9. Functional analysis of HOXB9 by knockdown and overexpression assays indicated their influence on both proliferation and apoptosis in HL cells. In summary, we identified up-regulation of HOXB9 in HL mediated by constitutively active ERK5 signaling which may represent novel therapeutic targets in HL.