Preclinical lentiviral vector-mediated hematopoietic stem and progenitor cell gene therapy corrects Pompe disease-related muscle and neurological manifestations.

Pompe disease, a rare genetic neuromuscular disorder, is caused by a deficiency of acid alpha-glucosidase (GAA), leading to an accumulation of glycogen in lysosomes, and resulting in the progressive development of muscle weakness. The current standard treatment, enzyme replacement therapy (ERT), is not curative and has limitations such as poor penetration into skeletal muscle and both the central and peripheral nervous systems, a risk of immune responses against the recombinant enzyme, and the requirement for high doses and frequent infusions. To overcome these limitations, lentiviral vector-mediated hematopoietic stem and progenitor cell (HSPC) gene therapy has been proposed as a next-generation approach for treating Pompe disease. This study demonstrates the potential of lentiviral HSPC gene therapy to reverse the pathological effects of Pompe disease in a preclinical mouse model. It includes a comprehensive safety assessment via integration site analysis, along with single-cell RNA sequencing analysis of central nervous tissue samples to gain insights into the underlying mechanisms of phenotype correction.

High-throughput analysis of hematopoietic stem cell engraftment after intravenous and intracerebroventricular dosing.

Hematopoietic stem/progenitor cell gene therapy (HSPC-GT) has shown clear neurological benefit in rare diseases, which is achieved through the engraftment of genetically modified microglia-like cells (MLCs) in the brain. Still, the engraftment dynamics and the nature of engineered MLCs, as well as their potential use in common neurogenerative diseases, have remained largely unexplored. Here, we comprehensively characterized how different routes of administration affect the biodistribution of genetically engineered MLCs and other HSPC derivatives in mice. We generated a high-resolution single-cell transcriptional map of MLCs and discovered that they could clearly be distinguished from macrophages as well as from resident microglia by the expression of a specific gene signature that is reflective of their HSPC ontogeny and irrespective of their long-term engraftment history. Lastly, using murine models of Parkinson's disease and frontotemporal dementia, we demonstrated that MLCs can deliver therapeutically relevant levels of transgenic protein to the brain, thereby opening avenues for the clinical translation of HSPC-GT to the treatment of major neurological diseases.

Screening chimeric GAA variants in preclinical study results in hematopoietic stem cell gene therapy candidate vectors for Pompe disease.

Pompe disease is a rare genetic neuromuscular disorder caused by acid α-glucosidase (GAA) deficiency resulting in lysosomal glycogen accumulation and progressive myopathy. Enzyme replacement therapy, the current standard of care, penetrates poorly into the skeletal muscles and the peripheral and central nervous system (CNS), risks recombinant enzyme immunogenicity, and requires high doses and frequent infusions. Lentiviral vector-mediated hematopoietic stem and progenitor cell (HSPC) gene therapy was investigated in a Pompe mouse model using a clinically relevant promoter driving nine engineered GAA coding sequences incorporating distinct peptide tags and codon optimizations. Vectors solely including glycosylation-independent lysosomal targeting tags enhanced secretion and improved reduction of glycogen, myofiber, and CNS vacuolation in key tissues, although GAA enzyme activity and protein was consistently lower compared with native GAA. Genetically modified microglial cells in brains were detected at low levels but provided robust phenotypic correction. Furthermore, an amino acid substitution introduced in the tag reduced insulin receptor-mediated signaling with no evidence of an effect on blood glucose levels in Pompe mice. This study demonstrated the therapeutic potential of lentiviral HSPC gene therapy exploiting optimized GAA tagged coding sequences to reverse Pompe disease pathology in a preclinical mouse model, providing promising vector candidates for further investigation.

Assessment of the mode of anger expression in alcohol dependent male inpatients.

AIMS: To assess the relationships between trait anger (T-Anger) and anger expression styles and emotional states-suicide probability, depression, state and trait anxiety and self-esteem--in alcohol-dependent inpatients. METHODS: The patients included in this study were 142 male inpatients with alcohol dependence according to the Diagnostic and Statistical Manual of Mental Disorders (DSM-IV) criteria. The Suicide Probability Scale, the Coopersmith Self-Esteem Inventory, the Beck Depression Inventory, the Beck Hopelessness Scale, the Spielberger State-Trait Anxiety Scales, and the T-Anger and Anger Expressions Scales were used for the assessment of the emotional states of the patients. Pearson correlation, analysis of variance and linear regression were used in the statistical analysis. RESULTS: There were significant correlations between suicide probability, depression, state and the trait anxiety, and the T-Anger and all of the anger expression subscales. The presence of high probability for suicide was related to a high level of T-Anger, Anger-out and Anger-in. Finally, a low level of hopelessness was associated with a high level of T-Anger, and a high level of the trait anxiety was associated with a low level of the Anger Control (AEX-Con). CONCLUSION: The findings indicated that suicide probability, hopelessness and trait anxiety predict T-Anger levels and anger expression styles. Therefore, anxiety, hopelessness and suicide probability must be considered as risk for anger and anger expressions in alcohol-dependent patients. Furthermore, alcohol treatment programmes should attach importance to anger management and AEX-Con training.

CSF1/CSF1R Signaling Inhibitor Pexidartinib (PLX3397) Reprograms Tumor-Associated Macrophages and Stimulates T-cell Infiltration in the Sarcoma Microenvironment.

Colony-stimulating factor 1 (CSF1) is a primary regulator of the survival, proliferation, and differentiation of monocyte/macrophage that sustains the protumorigenic functions of tumor-associated macrophages (TAMs). Considering current advances in understanding the role of the inflammatory tumor microenvironment, targeting the components of the sarcoma microenvironment, such as TAMs, is a viable strategy. Here, we investigated the effect of PLX3397 (pexidartinib) as a potent inhibitor of the CSF1 receptor (CSF1R). PLX3397 was recently approved by the Food and Drug Administration (FDA) to treat tenosynovial giant cell tumor and reprogram TAMs whose infiltration correlates with unfavorable prognosis of sarcomas. First, we confirmed by cytokine arrays of tumor-conditioned media (TCM) that cytokines including CSF1 are secreted from LM8 osteosarcoma cells and NFSa fibrosarcoma cells. The TCM, like CSF1, stimulated ERK1/2 phosphorylation in bone marrow-derived macrophages (BMDMs), polarized BMDMs toward an M2 (TAM-like) phenotype, and strikingly promoted BMDM chemotaxis. In vitro administration of PLX3397 suppressed pERK1/2 stimulation by CSF1 or TCM, and reduced M2 polarization, survival, and chemotaxis in BMDMs. Systemic administration of PLX3397 to the osteosarcoma orthotopic xenograft model significantly suppressed the primary tumor growth and lung metastasis, and thus improved metastasis-free survival. PLX3397 treatment concurrently depleted TAMs and FOXP3+ regulatory T cells and, surprisingly, enhanced infiltration of CD8+ T cells into the microenvironments of both primary and metastatic osteosarcoma sites. Our preclinical results show that PLX3397 has strong macrophage- and T-cell-modulating effects that may translate into cancer immunotherapy for bone and soft-tissue sarcomas.

Validation study of the Turkish version of the Yale - Brown obsessive compulsive scale for heavy drinking in a group of male patients.

Psychometric properties of Turkish version of the Yale Brown Obsessive Compulsive Scale for heavy drinking (YBOCS-hd) were examined in alcohol-dependent male patients. Factor structure, internal consistency and discriminant validity of the YBOCS-hd were analysed in a sample of 117 male patients diagnosed with alcohol dependence. To study its convergent validity, the YBOCS-hd was correlated with the Addiction Severity Index in 34 of the patients. A test - retest reliability study was performed on the data from 34 patients. Correlation between the YBOCS-hd total score and the ASI Alcohol Use score was moderate (r = 0.51). One factor explained 50.2% of the variance. The YBOCS-hd was able to discriminate the groups abstinent for less than 1 month and a second group with at least 1 month of abstinence. Test - retest correlation was high (r = 0.81, ICC = 0.81). The Turkish version of the YBOCS-hd proved to be a reliable and valid instrument measuring craving in alcohol-dependent male individuals.

[A study on alcohol use among working youth who are continuing apprenticeship training]. / Ciraklik egitimine devam eden çalisan gençlerde alkol kullanimi üzerine bir çalisma.

OBJECTIVE: Considering both burden of entering working life and engaging in adult roles in terms of being employed, working youth forms a specific group to be handled regarding alcohol use problems. In this study it was aimed to investigate prevalence and patterns of alcohol use among youth in apprenticeship schools in central Ankara. METHOD: A survey was done to investigate presence of alcohol use problems among students of five apprenticeship schools in Ankara using CAGE Questionnaire and a questionnaire form including more detailed questions about alcohol use. RESULTS: Prevalence of life-time use of alcohol was 37.3% and prevalence of alcohol use more than once was 24.3%. Ten per cent of students with a history of alcohol use told that they went on drinking during last year. Proportion of students who continued drinking during the last year and getting at least 2+ score from CAGE was found as 4.5%. CONCLUSION: Prevalence of clinically significant alcohol use is quite high among working youth. Thus, in order to plan preventive measures, risk factors for alcohol use problems should be considered in detail in working youth.

Investigation of alcohol use disorders in a group of driving-while-intoxicated offenders.

This preliminary study was carried out to investigate alcohol use disorders and personality profiles in a group of driving-while-intoxicated offenders. Thirty nine volunteer offenders were assessed by CAGE, while 21 of them were assessed by SCID-I Alcohol and Drug Use Disorders module and 14 drivers completed MMPI test. According to CAGE scores, 11 was found to have an indication of alcohol problem and 7 had clinically significant alcohol use disorder. Within 21 drivers, 4 had a DSM-IV diagnosis of alcohol abuse. Independent of their diagnosis, MMPI profiles revealed the psychopathic personality characteristics which might explain drinking while driving as a risky behaviour in this group. These results suggest that, besides legal applications, referring offenders to associated centers, in order to be evaluated and informed about alcohol use disorders, would be an important step in the prevention of recurrent alcohol impaired driving as well as alcohol related incidents.

Reliability and Validity of the Turkish Version of the Addiction Severity Index in Male Alcohol Dependents.

INTRODUCTION: We aimed to evaluate the psychometric characteristics of the Turkish translation of the Addiction Severity Index (ASI) in 115 male alcohol-dependent patients. METHOD: The reliability of the instrument was assessed by measuring test-retest, interrater and internal reliabilities. In the validity analysis, the correlation coefficients between corresponding severity ratings and composite scores of each subscale and concurrent validity were assessed. Moreover, the discriminant validity and concurrent validity scores were calculated. RESULTS: The test-retest reliability of the ASI scores ranged from .79 to .91. The interrater reliability assigned by three raters was high (.74 to .99). Cronbach's alpha coefficient for internal consistency was .85 for all scales, and it varied between .64 and .77 for the subscales. The Beck Depression Inventory moderately correlated with the Psychatric status, and the MacAndrew Alcoholism Scale correlated with the Alcohol and Drug Use subscales of the Addiction Severity Index (ASI). The correlation coefficient was .91 for the alcohol use subscale. CONCLUSION: The results obtained in this study suggest that the Turkish version of the ASI could be used as a reliable and valid instrument in alcohol-dependent patients.

Multifaceted regulation of somatic cell reprogramming by mRNA translational control.

Translational control plays a pivotal role in the regulation of the pluripotency network in embryonic stem cells, but its effect on reprogramming somatic cells to pluripotency has not been explored. Here, we show that eukaryotic translation initiation factor 4E (eIF4E) binding proteins (4E-BPs), which are translational repressors, have a multifaceted effect on the reprogramming of mouse embryonic fibroblasts (MEFs) into induced pluripotent stem cells (iPSCs). Loss of 4E-BP expression attenuates the induction of iPSCs at least in part through increased translation of p21, a known inhibitor of somatic cell reprogramming. However, MEFs lacking both p53 and 4E-BPs show greatly enhanced reprogramming resulting from a combination of reduced p21 transcription and enhanced translation of endogenous mRNAs such as Sox2 and Myc and can be reprogrammed through the expression of only exogenous Oct4. Thus, 4E-BPs exert both positive and negative effects on reprogramming, highlighting the key role that translational control plays in regulating this process.

PLZF confers effector functions to donor T cells that preserve graft-versus-tumor effects while attenuating GVHD.

Efforts to limit GVHD mediated by alloreactive donor T cells after allogeneic bone marrow transplantation are limited by a concomitant decrease in graft-versus-tumor (GVT) activity and increased possibilities of tumor relapse. Using a novel approach, we adoptively transferred conventional T cells expressing the transcription factor promyelocytic leukemia zinc finger (PLZF), which confers effector properties resembling invariant natural killer T cells, such as copious production of cytokines under suboptimal stimulation. PLZF expression in T-cell allografts attenuates expansion of alloreactive T cells, leading to lower GVHD. Intact alloreactivity-driven antitumor cytokine responses result in preserved GVT effects, leading to improved survival. Our findings suggest that therapy with PLZF-overexpressing T cells would result in overall improved outcomes due to less GVHD and intact GVT effects.

Adoptively transferred TRAIL+ T cells suppress GVHD and augment antitumor activity.

Current strategies to suppress graft-versus-host disease (GVHD) also compromise graft-versus-tumor (GVT) responses. Furthermore, most experimental strategies to separate GVHD and GVT responses merely spare GVT function without actually enhancing it. We have previously shown that endogenously expressed TNF-related apoptosis-inducing ligand (TRAIL) is required for optimal GVT activity against certain malignancies in recipients of allogeneic hematopoietic stem cell transplantation (allo-HSCT). In order to model a donor-derived cellular therapy, we genetically engineered T cells to overexpress TRAIL and adoptively transferred donor-type unsorted TRAIL+ T cells into mouse models of allo-HSCT. We found that murine TRAIL+ T cells induced apoptosis of alloreactive T cells, thereby reducing GVHD in a DR5-dependent manner. Furthermore, murine TRAIL+ T cells mediated enhanced in vitro and in vivo antilymphoma GVT response. Moreover, human TRAIL+ T cells mediated enhanced in vitro cytotoxicity against both human leukemia cell lines and against freshly isolated chronic lymphocytic leukemia (CLL) cells. Finally, as a model of off-the-shelf, donor-unrestricted antitumor cellular therapy, in vitro-generated TRAIL+ precursor T cells from third-party donors also mediated enhanced GVT response in the absence of GVHD. These data indicate that TRAIL-overexpressing donor T cells could potentially enhance the curative potential of allo-HSCT by increasing GVT response and suppressing GVHD.

Ways of problem solving as predictors of relapse in alcohol dependent male inpatients.

AIM: The purpose of this study was to identify how remitters and relapsers view their everyday problem solving strategies. METHOD: A total of 128 male alcohol dependent male inpatients who were hospitalized at the Ankara University Psychiatry Clinic, Alcohol and Substance Abuse Treatment Unit were recruited for the study. Subjects demographic status and alcohol use histories were assessed by a self-report questionnaire. Also, patients were evaluated with The Coopersmith Self-esteem Inventory (CSI), The Spielberger State-Trait Anxiety Scale (STAI-I-II), and The Problem Solving Inventory (PSI). Patients were followed for six months with monthly intervals after hospital discharge. Drinking status was assessed in terms of abstinence and relapse. Data were assessed with Student t-test, and univariate and multivariate analyses. In the logistic regression analysis, age, marital status, employment status and PSI subscores were taken as the independent variables and drinking state at the end of six months as the dependent variable. RESULTS: There were significant differences in reflective and avoidant styles, and monitoring style of problem solving between abstainers and relapses. It was found that subjects who perceived their problem solving style as less avoidant and less reflective were at greater risk to relapse. CONCLUSIONS: The findings demonstrated that active engagement in problem solving like utilizing avoidant and reflective styles of problem solving enhances abstinence. In treatment, expanding the behavior repertoire and increasing the variety of ways of problem solving ways that can be utilized in daily life should be one of the major goals of the treatment program.

Tunneling nanotubes provide a unique conduit for intercellular transfer of cellular contents in human malignant pleural mesothelioma.

Tunneling nanotubes are long, non-adherent F-actin-based cytoplasmic extensions which connect proximal or distant cells and facilitate intercellular transfer. The identification of nanotubes has been limited to cell lines, and their role in cancer remains unclear. We detected tunneling nanotubes in mesothelioma cell lines and primary human mesothelioma cells. Using a low serum, hyperglycemic, acidic growth medium, we stimulated nanotube formation and bidirectional transfer of vesicles, proteins, and mitochondria between cells. Notably, nanotubes developed between malignant cells or between normal mesothelial cells, but not between malignant and normal cells. Immunofluorescent staining revealed their actin-based assembly and structure. Metformin and an mTor inhibitor, Everolimus, effectively suppressed nanotube formation. Confocal microscopy with 3-dimensional reconstructions of sectioned surgical specimens demonstrated for the first time the presence of nanotubes in human mesothelioma and lung adenocarcinoma tumor specimens. We provide the first evidence of tunneling nanotubes in human primary tumors and cancer cells and propose that these structures play an important role in cancer cell pathogenesis and invasion.

Quantification of transforming capacity and cooperation of defined genetic alterations in myeloid malignancies.

OBJECTIVE: Mutations found in myeloid malignancies are qualitatively classified as conferring proliferative and survival advantages or impairing cellular differentiation. However, no suitable experimental model to quantify transforming potential of individual mutations and functional cooperation between defined genetic/epigenetic alterations has been established so far. MATERIALS AND METHODS: Based on cytokine-independent proliferation as a marker for cellular transformation, we used limiting dilution and clonal expansion of retrovirally transduced cells in the presence or absence of cytokines to quantify the transformation potential of constitutively active receptor mutants and short hairpin RNAs (shRNA) targeting transcription factors by RNA interference. Interleukin-3-dependent 32D cells were transduced with betaGMR-I374N, c-KitV558D, or c-MplS368C, and cloning efficiencies were normalized to viral integration numbers as determined by quantitative polymerase chain reaction. RESULTS: In this assay, c-KitV558D and c-MplS368C were about 25-fold more effective than betaGMR-I374N. To study cooperation of defined genetic/epigenetic aberrations, receptor mutants were coexpressed with shRNAs targeting PU.1 and p53. In p53-hypomorphic, but not in 32D wild-type cells, RNA interference against PU.1 significantly enhances transformation efficacy by c-KitV558D, but not by c-MplS368C, as compared to control shRNA. These data demonstrate nonredundant, receptor-specific and p53-dependent responses to reduced PU.1 expression in 32D cells. CONCLUSION: This cell culture model represents a useful tool to quantify hematopoietic cell transformation by defined genetic and epigenetic alterations.

Benign mesenchymal stromal cells in human sarcomas.

PURPOSE: Recent evidence suggests that at least some sarcomas arise through aberrant differentiation of mesenchymal stromal cells (MSCs), but MSCs have never been isolated directly from human sarcoma specimens. EXPERIMENTAL DESIGN: We examined human sarcoma cell lines and primary adherent cultures derived from human sarcoma surgical samples for features of MSCs. We further characterized primary cultures as either benign or malignant by the presence of tumor-defining genetic lesions and tumor formation in immunocompromised mice. RESULTS: We show that a dedifferentiated liposarcoma cell line DDLS8817 posesses fat, bone, and cartilage trilineage differentiation potential characteristic of MSCs. Primary sarcoma cultures have the morphology, surface immunophenotype, and differentiation potential characteristic of MSCs. Surprisingly, many of these cultures are benign, as they do not form tumors in mice and lack sarcoma-defining genetic lesions. Consistent with the recently proposed pericyte origin of MSCs in normal human tissues, sarcoma-derived benign MSCs (SDBMSCs) express markers of pericytes and cooperate with endothelial cells in tube formation assays. In human sarcoma specimens, a subset of CD146-positive microvascular pericytes expresses CD105, an MSC marker, whereas malignant cells largely do not. In an in vitro coculture model, SDBMSCs as well as normal human pericytes markedly stimulate the growth of sarcoma cell lines. CONCLUSIONS: SDBMSCs/pericytes represent a previously undescribed stromal cell type in sarcoma that may contribute to tumor formation.

Prevalence and sociodemographic correlates of substance use in a university-student sample in Turkey.

BACKGROUND: Limited data is available on substance use among university students in Turkey. This study aims to determine the prevalence of substance use among this target group. METHODS: A total of 1,720 students were surveyed to assess substance use, and relationships between sociodemographic variables and substance use were assessed using both univariate and multivariate analyses. RESULTS: Of the recorded student sample, 6.4 % reported having used a substance; 2.8 % used one within the past year. Prevalence of cannabis use at least once during life-time was 5.9 %. Males living alone, or students with families residing abroad increased the risk of substance use. CONCLUSION: Preventive interventions for substance use problems should consider factors related with family relations of the youth.

Alcohol use prevalence and sociodemographic correlates of alcohol use in a university student sample in Turkey.

BACKGROUND: This study is a survey to determine prevalence and sociodemographic correlates of drinking problems among students from five university centres in Turkey. METHOD: Using an anonymous self-administered questionnaire and the CAGE Questionnaire for alcohol use problems, 1,720 students were surveyed. RESULTS: Of the whole student sample 63.3% reported that they had ever tried drinking alcohol, and 48.5% had used alcohol in the past year. Sixty five percent of the students had been drinking once a month or more frequently. The overall prevalence of alcohol use problems according to CAGE2+ was 9.7% (19.9% among the students who used alcohol in the past year). In multivariate analysis, male students tended to have problems with alcohol about three times more than females. Living in the dormitory seemed to be protective in terms of frequent drinking, and as educational level of the parents increased, the odds of drinking at least once a month increased. Students whose mothers were illiterate or primary school graduate tended to give more positive answers to the Cut-down, Annoyed and Guilty items. The odds of giving a positive answer to the Cut-down item among those living alone was greater than the other residence groups. Predictors of positive answer to the Eye-opener item were male gender, living alone at home, and residence of the family being in a foreign country. Paternal educational level being in the illiterate/primary school category was significantly related with more positive answers to the Guilty item. CONCLUSIONS: Drinking problems among university students in Turkey are more prevalent when compared with prevalence rates shown in other surveys in Turkey. Alternative ways of socialization should be provided for the university youth in order to prevent alcohol use problems in the future.

Psychosocial factors in alcohol use-related problems of working youth.

This study investigated the psychosocial correlates of alcohol use related problems in a sample of 581 working adolescents (N = 4405), recruited from five vocational schools in Ankara in June 2004 with the CAGE questionnaire, The Beck Depression Inventory, the Beck Hopelessness Scale, the Spielberger State Anxiety Scale, and the Coopersmith Self-Esteem Inventory. Using a multivariate analysis, the anxiety and hopelessness scores, and the length of stay in Ankara were found to be related to alcohol-use problems of the working youth. The study's limitations were noted and future research was suggested.

Alcoholic case of central pontine myelinolysis with mainly cerebellar signs.

The etiology of central pontine myelinolysis (CPM) is usually related to rapid correction of hyponatremia and alcoholism. Here a case with CPM predominated by cerebellar signs is described, and the neuropsychological assessment of the case is presented as well. Blood biochemistry revealed a normal sodium level and neuropsychological examination revealed impairment in attention and concentration, reduced immediate memory span, and impaired delayed recall. Further studies are needed to discover whether these neuropsychological signs are specific for CPM or due to alcoholism in general.