The spleen of patients with myelofibrosis harbors defective mesenchymal stromal cells.

Splenic hematopoiesis is a major feature in the course of myelofibrosis (MF). In fact, the spleen of patients with MF contains malignant hematopoietic stem cells retaining a complete differentiation program, suggesting both a pivotal role of the spleen in maintaining the disease and a tight regulation of hematopoiesis by the splenic microenvironment, in particular by mesenchymal stromal cells (MSCs). Little is known about splenic MSCs (Sp-MSCs), both in normal and in pathological context. In this work, we have in vitro expanded and characterized Sp-MSCs from 25 patients with MF and 13 healthy subjects (HS). They shared similar phenotype, growth kinetics, and differentiation capacity. However, MF Sp-MSCs expressed significant lower levels of nestin, and favored megakaryocyte (Mk) differentiation in vitro at a larger extent than their normal counterpart. Moreover, they showed a significant upregulation of matrix metalloprotease 2 (MMP2) and fibronectin 1 (FN1) genes both at mRNA expression and at protein level, and, finally, developed genetic abnormalities which were never detected in HS-derived Sp-MSCs. Our data point toward the existence of a defective splenic niche in patients with MF that could be responsible of some pathological features of the disease, including the increased trafficking of CD34+ cells and the expansion of the megakaryocytic lineage.

Therapeutically targeting SELF-reinforcing leukemic niches in acute myeloid leukemia: A worthy endeavor?

A tight relationship between the acute myeloid leukemia (AML) population and the bone marrow (BM) microenvironment has been convincingly established. The AML clone contains leukemic stem cells (LSCs) that compete with normal hematopoietic stem cells (HSCs) for niche occupancy and remodel the niche; whereas, the BM microenvironment might promote AML development and progression not only through hypoxia and homing/adhesion molecules, but also through genetic defects. Although it is still unknown whether the niche influences treatment results or contains any potential target for treatment, this dynamic AML-niche interaction might be a promising therapeutic objective to significantly improve the AML cure rate.

Comprehensive characterization of mesenchymal stromal cells from patients with Fanconi anaemia.

Fanconi anaemia (FA) is an inherited disorder characterized by pancytopenia, congenital malformations and a predisposition to develop malignancies. Alterations in the haematopoietic microenvironment of FA patients have been reported, but little is known regarding the components of their bone marrow (BM) stroma. We characterized mesenchymal stromal cells (MSCs) isolated from BM of 18 FA patients both before and after allogeneic haematopoietic stem cell transplantation (HSCT). Morphology, fibroblast colony-forming unit (CFU-F) ability, proliferative capacity, immunophenotype, differentiation potential, ability to support long-term haematopoiesis and immunomodulatory properties of FA-MSCs were analysed and compared with those of MSCs expanded from 15 age-matched healthy donors (HD-MSCs). FA-MSCs were genetically characterized through conventional karyotyping, diepoxybutane-test and array-comparative genomic hybridization. FA-MSCs generated before and after HSCT were compared. Morphology, immunophenotype, differentiation potential, ability in vitro to inhibit mitogen-induced T-cell proliferation and to support long-term haematopoiesis did not differ between FA-MSCs and HD-MSCs. CFU-F ability and proliferative capacity of FA-MSCs isolated after HSCT were significantly lower than those of HD-MSCs. FA-MSCs reached senescence significantly earlier than HD-MSCs and showed spontaneous chromosome fragility. Our findings indicate that FA-MSCs are defective in their ability to survive in vitro and display spontaneous chromosome breakages; whether these defects are involved in pathophysiology of BM failure syndromes deserves further investigation.

Chronic lymphocytic leukemia with del13q14 as the sole abnormality: dynamic prognostic estimate by interphase-FISH.

This study analyzed 140 patients with isolated del13q14 on interphase FISH (I-FISH), to identify subsets with a different progression risk and to assess the acquisition of additional chromosomal abnormalities (clonal evolution) in treatment-naïve del13q14 patients. A monoallelic deletion (del13qx1) was detected in 123 cases (88%), a biallelic deletion (del13qx2) in eight and a mosaic of monoallelic and biallelic deletions (del13qx1/del13qx2) in nine. In 33% of cases, deletion encompassed the Rb1 locus The median percentage of abnormal nuclei was 50% (15%-96%), and it was higher in patients with a biallelic/mosaic pattern in comparison with patients with monoallelic deletion. Sixty two patients (44%) have been treated; 5-year treatment free survival rate was 56% and the median treatment free survival was 65 months. The baseline percentage of deleted nuclei, as a continuous variable, was related to progression (HR: 1.02; p = 0.001). According to deletion burden, three groups were identified: 64 cases (46%) had <50% deleted nuclei, 47 (33%) had 50-69% deleted nuclei, and 29 (21%) had ≥70% deleted nuclei. The 5-year untreated rate was 70.5% , 52.6% and 28.7% (p < 0.0001), respectively. In multivariate analysis using IGHV mutational status, presence of a nullisomic clone, CD38 expression and percentage of deleted nuclei as covariates, only IGHV mutational status and the percentage of deleted nuclei were independent risk factors for treatment. In 103 patients serially monitored by I-FISH before starting any treatment, we observed a significant increase in the proportion of del13q14 cells, and this increase affected the risk of subsequent treatment requirement (HR 2.54, p = 0.001). The appearance of a new clone was detected in 16 patients (15.5%) and chromosome 13 was involved in 14 of them. I-FISH monitoring proves worthwhile for a dynamic risk stratification and for planning clinical surveillance.

Validation of the new comprehensive cytogenetic scoring system (NCCSS) on 630 consecutive de novo MDS patients from a single institution.

This study evaluated whether the NCCSS truly improves the prognostic stratification of 630 consecutive de novo MDS patients and established which cytogenetic grouping [NCCSS or International Prognostic Scoring System (IPSS)], when combined with the WHO classification, best predicted the clinical outcome of myelodysplastic syndromes (MDS). The frequency of chromosomal defects was 53.8%. Clinical parameters, including number of cytopenias, WHO classification, IPSS cytogenetic categories and scores, NCCSS were all relevant for overall survival (OS) and leukemia-free survival (LFS) and were included in six distinct multivariate models compared by the Akaike Information Criterion (AIC). The most effective model to predict OS included the number of cytopenias, the WHO classification and the NCCSS, whereas the model including the number of cytopenias, blast cell percentage and the NCCSS and the model including the number of cytopenias the WHO classification and the NCCSS were almost equally effective to predict LFS. In conclusion, the NCCS (i) improves the prognostic stratification of the good and poor IPSS cytogenetic categories by introducing the very good and the very poor categories; (ii) is still incomplete in establishing the prognostic relevance of rare/double defects, (ii) applied to patients who receive supportive treatment only identifies five different prognostic subgroups, but applied to patients treated with specific therapies reveals only a trend toward a significantly different OS and LFS when patients of the poor and intermediate cytogenetic categories are compared, (iii) combined with the WHO classification is much more effective than the IPSS in predicting MDS clinical outcome.

A phase II multicenter randomized study to evaluate the safety and efficacy of combining thermotherapy and a short course of miltefosine for the treatment of uncomplicated cutaneous leishmaniasis in the New World.

BACKGROUND: Systemic pentavalent antimonials, mainly meglumine antimoniate, continue to be the first-choice drugs for treatment of cutaneous leishmaniasis (CL) despite their toxicity, difficulty of administration and high cost. In the search for therapeutic alternatives, combining two treatment interventions has emerged as a potential alternative to either reduce the use of antimonials with the associated toxicities, or to increase efficacy. Here, we report the results of a recently completed trial assessing the efficacy and safety of a combination of thermotherapy (TT) plus a short course of miltefosine (MLT) for the treatment of uncomplicated CL in Colombia and Peru. METHODS: A multicenter, randomized, evaluator-blinded, phase II, controled clinical trial was conducted. Adult volunteers with a parasitologically confirmed diagnosis of uncomplicated CL were randomly allocated to receive either a single session of TT or a combination of TT plus a short course of MLT (3 weeks). Therapeutic response outcomes and safety were assessed. RESULTS: 130 subjects were included in the study, of whom 64 were randomly assigned to the TT arm and 66 to the TT + MLT arm. Cure at 3 months' follow-up was achieved in 57.8% (n = 37) and 80.3% (n = 53) in the TT and TT + MLT groups, respectively, in the intention to treat analysis. The TT + MLT regimen was better that TT alone (p = 0.0055). The presence of vesicles at the site of heat application was the most common adverse event reported associated with the use of TT; while vomiting (31.8%) and elevation of liver enzymes (28.8%) were the most frequent adverse events reported associated with the use of MLT. CONCLUSION: The combination of TT plus a short course of MLT was shown to be significantly better than TT alone for the treatment of uncomplicated CL in the New World. TRIAL REGISTRATION: Registered in clinicaltrials.gov NCT02687971.

Does cytogenetic evolution have any prognostic relevance in myelodysplastic syndromes? A study on 153 patients from a single institution.

The present study was designed to establish the incidence of cytogenetic evolution (CE), defined as the acquisition of chromosomal defects during the course of MDS, in order to correlate it with the WHO classification and IPSS score, and to assess its impact on overall survival (OS) and risk of MDS/AML evolution (progression-free interval, PFI) by means of Cox models for time-dependent covariates. Adjustments for known risk factors were achieved by performing a bivariable analysis. The study was carried out in 153 MDS patients who were followed for a median period of 45.2 months. Disease progression occurred in 42.4% of patients after a 65.2-month median PFI, while CE occurred in 30.7% of patients. Our study shows that (1) CE was more common in advanced than in early MDS, and advanced MDS presented secondary chromosomal defects distinct from those of early MDS; (2) CE significantly affected OS and PFI independently of other prognostic variables; (3) del(7)(q31q34) was the only secondary chromosomal defect which significantly affected PFI; trisomy 8 had only a moderate influence.

Generation of the human induced pluripotent stem cell (hiPSC) line PSMi006-A from a patient affected by an autosomal recessive form of long QT syndrome type 1.

We generated human induced pluripotent stem cells (hiPSCs) from dermal fibroblasts of a 40 years old female patient homozygous for the mutation c.535 G > A p.G179S on the KCNQ1 gene, causing a severe form of autosomal recessive Long QT Syndrome type 1 (AR-LQT1). The hiPSCs, generated using classical approach of the four retroviruses enconding the reprogramming factors OCT4, SOX2, cMYC and KLF4, display pluripotent stem cell characteristics, and differentiate into cell lineages of all three germ layers: endoderm, mesoderm and ectoderm.

Generation of two human induced pluripotent stem cell (hiPSC) lines from a long QT syndrome South African founder population.

We generated PSMi001-A and PSMi008-A hiPSC lines from two individuals belonging to a South African (SA) founder population in which the malignant KCNQ1-A341V mutation cosegregates with the Long QT Syndrome (LQTS) phenotype. PSMi001-A was derived from an asymptomatic KCNQ1-A341V mutation carrier, whereas PSMi008-A was derived from a healthy non-mutation carrier, heterozygous for the minor variant rs16847548 on the NOS1AP gene, associated with QT prolongation in the general population, and with a greater risk for cardiac arrest in the affected members of the SA founder population. The hiPSCs, generated using the Yamanaka's retroviruses, display pluripotent stem cell features and trilineage differentiation potential.

Generation of the human induced pluripotent stem cell (hiPSC) line PSMi004-A from a carrier of the KCNQ1-R594Q mutation.

We generated human induced pluripotent stem cells (hiPSCs) from dermal fibroblasts of a male carrier of the heterozygous mutation c.1781 G > A p.R594Q on the KCNQ1 gene. hiPSCs, generated using four retroviruses each encoding for OCT4, SOX2, KLF4 and cMYC, display pluripotent stem cell characteristics, and can be differentiated into spontaneously beating cardiomyocytes (hiPSC-CMs).

Generation of the human induced pluripotent stem cell (hiPSC) line PSMi007-A from a Long QT Syndrome type 1 patient carrier of two common variants in the NOS1AP gene.

We generated human induced pluripotent stem cells (hiPSCs) from a symptomatic Long QT Syndrome (LQTS) type 1 patient, belonging to a South African (SA) founder population segregating the heterozygous mutation c.1022C > T p.A341V on the KCNQ1 gene. The patient is also homozygous for the two minor variants rs4657139 and rs16847548 on the NOS1AP gene, associated with greater risk for cardiac arrest and sudden death in LQTS mutation carriers of the founder population. hiPSCs, obtained using four retroviruses encoding the reprogramming factors OCT4, SOX2, cMYC and KLF4, display pluripotent stem cell characteristics, and can be differentiated into spontaneously beating cardiomyocytes (hiPSC-CMs).

Generation of the human induced pluripotent stem cell (hiPSC) line PSMi005-A from a patient carrying the KCNQ1-R190W mutation.

We generated human induced pluripotent stem cells (hiPSCs) from dermal fibroblasts of a woman carrier of the heterozygous mutation c.568C > T p.R190W on the KCNQ1 gene. hiPSCs, obtained using four retroviruses enconding the reprogramming factors OCT4, SOX2, cMYC and KLF4, display pluripotent stem cell characteristics, and can be differentiated into spontaneously beating cardiomyocytes (hiPSC-CMs).

Mindfulness and avoidance mediate the relationship between yoga practice and anxiety.

OBJECTIVES: There is accumulating evidence that yoga and mindfulness meditation can alleviate symptoms of anxiety, although the mechanisms by which this occurs remain unclear. The purpose of this study was to examine the relationship between yoga practice and self-reported anxiety as well as the potential mediating roles of mindfulness and emotional avoidance. METHODS: Using a cross-sectional design, 367 participants were recruited online and completed measures of anxiety, avoidance, and mindfulness. RESULTS: Results showed that length of yoga practice was significantly correlated with lower anxiety in yoga practitioners. Avoidance and mindfulness mediated the relationship between length of yoga practice and anxiety, shedding light on possible mechanisms by which these practices reduce anxiety. CONCLUSIONS: Future experimental and longitudinal research is needed to examine the causal role of mindfulness and avoidance in the relationship between yoga practice and anxiety, and whether yoga is a useful adjunct to cognitive behaviour therapy for anxiety disorders.

Alternative splicing of hTERT: a further mechanism for the control of active hTERT in acute myeloid leukemia.

hTERT component is the key regulator of telomerase. Alternatively spliced variants of hTERT generate different telomerase activity. The goal of the study was to determine the role of different hTERT isoforms in the regulation of telomerase expression in AML patients. Among the 97 studied patients, 45 had a complex karyotype and 52 a normal karyotype. hTERT isoforms expression was determined in bone marrow samples by q-RT-PCR, using SYBR Green I. hTERT expression was lower in AML patients than controls (median 2.5 vs. 10.1, p = .003), though no difference was observed between the complex and normal karyotype (median 3.2 vs. 2.3, p = .37). High trans-dominant negative isoform expression increased the response rate by two. High expression of inactive product (-α - ß) was shown to increase the risk of relapse by about three times. In conclusion, our data suggest an intriguing link between the control of hTERT isoforms expression and AML outcome.

Generation of the human induced pluripotent stem cell (hiPSC) line PSMi003-A from a patient affected by an autosomal recessive form of Long QT Syndrome type 1.

We generated human induced pluripotent stem cells (hiPSCs) from dermal fibroblasts of a 51years old female patient homozygous for the mutation c.535 G>A p.G179S on the KCNQ1 gene, causing a severe form of autosomal recessive Long QT Syndrome type 1 (AR-LQT1), not associated with deafness. The hiPSCs, generated using four retroviruses each encoding for a reprogramming factor OCT4, SOX2, KLF4, cMYC, are pluripotent and can differentiate into spontaneously beating cardiomyocytes (hiPSC-CMs).

Generation of the human induced pluripotent stem cell (hiPSC) line PSMi002-A from a patient affected by the Jervell and Lange-Nielsen syndrome and carrier of two compound heterozygous mutations on the KCNQ1 gene.

We report the generation of human induced pluripotent stem cells (hiPSCs) from dermal fibroblasts of a female patient carrier of the two compound heterozygous mutations c.568 C>T p.R190W (maternal allele), and c.1781 G>A p.R594Q (paternal allele) on the KCNQ1 gene, causing Jervell and Lange-Nielsen Syndrome (JLNS). To obtain hiPSCs, we used the classical approach of the four retroviruses each encoding for a reprogramming factor OCT4, SOX2, KLF4, cMYC. The obtained hiPSC clones display pluripotent stem cell characteristics, and differentiate into spontaneously beating cardiomyocytes (hiPSC-CMs).

Harmonized clinical trial methodologies for localized cutaneous leishmaniasis and potential for extensive network with capacities for clinical evaluation.

INTRODUCTION: Progress with the treatment of cutaneous leishmaniasis (CL) has been hampered by inconsistent methodologies used to assess treatment effects. A sizable number of trials conducted over the years has generated only weak evidence backing current treatment recommendations, as shown by systematic reviews on old-world and new-world CL (OWCL and NWCL). MATERIALS AND METHODS: Using a previously published guidance paper on CL treatment trial methodology as the reference, consensus was sought on key parameters including core eligibility and outcome measures, among OWCL (7 countries, 10 trial sites) and NWCL (7 countries, 11 trial sites) during two separate meetings. RESULTS: Findings and level of consensus within and between OWCL and NWCL sites are presented and discussed. In addition, CL trial site characteristics and capacities are summarized. CONCLUSIONS: The consensus reached allows standardization of future clinical research across OWCL and NWCL sites. We encourage CL researchers to adopt and adapt as required the proposed parameters and outcomes in their future trials and provide feedback on their experience. The expertise afforded between the two sets of clinical sites provides the basis for a powerful consortium with potential for extensive, standardized assessment of interventions for CL and faster approval of candidate treatments.

CPAM type 2-derived mesenchymal stem cells: Malignancy risk study in a 14-month-old boy.

INTRODUCTION: The association between congenital pulmonary airway malformations (CPAM) and malignancy is reported in the literature. Interactions between the tumor, immune, and mesenchymal stromal/stem cells (MSCs) have been recognized as crucial for understanding tumorigenesis. We characterized MSCs isolated from CPAM lesions in order to define potential malignancy risks. METHODS: CPAM II pulmonary tissue was used for MSC expansion; a "healthy" lung section from the same child was used as a comparator. Morphology, immunophenotype, differentiation and immunological capacity, proliferative growth, gene signature telomerase activity, and in vivo tumorigenicity in nude mice were evaluated. RESULTS: MSCs were successfully isolated and propagated from CPAM tissue. CPAM-MSCs presented the typical MSC morphology and phenotype, while exhibiting high proliferative capacity, reaching confluence at a median time of 5 days as well as differentiation capabilities. CPAM-MSCs at early passages were not neoplastic and chromosomally normal, even though unbalanced chromosomal rearrangements were noted by molecular karyotype. CONCLUSIONS: CPAM-MSCs exhibited specific features similar to tumor derived MSCs. Whilst there was no evidence of malignant transformation in the cystic tissue, our results provide evidence that this abnormal tissue has malignant potential. MSCs are considered important players in the tumor microenvironment and they have been closely linked to regulation of tumor survival, growth, and progression. Thus, early lesion resection also in asymptomatic patients might be indicated to exclude that the microenvironment may be potentially permissive to cancer development.

MDS/AML del(11)(q14) Share Common Morphological Features Despite Different Chromosomal Breakpoints.

In myelodysplatic syndromes and acute myeloid leukemia (MDS/AML) deletion of the 11q14 region is a rare chromosomal defect (incidence: 0.6-1.0%), included within the intermediate risk criteria by the International Prognostic Scoring System. No fluorescence in situ hybridization (FISH) study has yet been performed to identify a common breakpoint region (CBR). In our study through FISH with bacterial artificial chromosomes and commercial probes, we analyzed seven patients with MDS/AML harboring 11q14 deletion on conventional cytogenetic analysis. FISH revealed deletions in five patients and amplifications in two. Three patients with deletion carried a CBR, two had a deletion involving a more centromeric breakpoint. These five patients exhibited multilineage dysplasia, blast cells with large round nuclei, loose chromatin, small and abundant nucleoli, and vacuolated cytoplasm with very thin Auer bodies. In conclusion, the morphological features which occur independently of the extent of the deletion are of multilineage dysplasia in MDS and leukemic blasts strongly reactive to peroxidase in AML; despite the variable size of the deleted area, some patients harbor a CBR.

Design, synthesis and evaluation of novel feruloyl-donepezil hybrids as potential multitarget drugs for the treatment of Alzheimer's disease.

A novel series of feruloyl-donepezil hybrid compounds were designed, synthesized and evaluated as multitarget drug candidates for the treatment of Alzheimer's Disease (AD). In vitro results revealed potent acetylcholinesterase (AChE) inhibitory activity for some of these compounds and all of them showed moderate antioxidant properties. Compounds 12a, 12b and 12c were the most potent AChE inhibitors, highlighting 12a with IC50 = 0.46 µM. In addition, these three most promising compounds exhibited significant in vivo anti-inflammatory activity in the mice paw edema, pleurisy and formalin-induced hyperalgesy models, in vitro metal chelator activity for Cu2+ and Fe2+, and neuroprotection of human neuronal cells against oxidative damage. Molecular docking studies corroborated the in vitro inhibitory mode of interaction of these active compounds on AChE. Based on these data, compound 12a was identified as a novel promising drug prototype candidate for the treatment of AD with innovative structural feature and multitarget effects.