Néstor W. Meza

Centro de Investigaciones Energéticas, Medioambientales y Tecnológicas

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José C. Segovia

Centro de Investigaciones Energéticas, Medioambientales y Tecnológicas

Juan A. Bueren

Centro de Investigaciones Energéticas, Medioambientales y Tecnológicas

Oscar Quintana‐Bustamante

Hospital Universitario Fundación Jiménez Díaz

Susana Navarro

Centro de Investigaciones Energéticas, Medioambientales y Tecnológicas

José M. Bautista

Universidad Complutense de Madrid

Paula Rı́o

Centro de Investigaciones Energéticas, Medioambientales y Tecnológicas

Guillermo Güenechea

Centro de Investigaciones Energéticas, Medioambientales y Tecnológicas

Amalia Díez

Universidad Complutense de Madrid

Nineth E. Rossi

University of the Andes

Celso Arango

Hospital General Universitario Gregorio Marañón

Cooperative Institutions

Centro de Investigación Biomédica en Red

Universidad Complutense de Madrid

Centro de Investigaciones Energéticas, Medioambientales y Tecnológicas

Centre for Biomedical Network Research on Rare Diseases

Instituto de Salud Carlos III

Universidad Autónoma de Madrid

Hospital Universitario Fundación Jiménez Díaz

Hospital General Universitario Gregorio Marañón

Research Institute Hospital 12 de Octubre

University of Tübingen

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Functional analysis of gammaretroviral vector transduction by quantitative PCR

The Journal of Gene Medicine (2006)

Néstor W. Meza Antonio Puyet Susana Pérez-Benavente Oscar Quintana‐Bustamante Amalia Díez

Abstract Background In a clinical setting of gene therapy, quantitative methods are required to determine recombinant viral titres and transgene mRNA expression, avoiding the use of reporter genes. Methods We describe procedures based on quantitative polymerase chain reaction (qPCR) designed to assess functional titres of murine leukaemia virus (MLV) vectors, determine proviral copy numbers in transduced cells, and estimate retroviral transgene expression in both target cell lines and mice with transduced chimeric haematopoiesis. Results Compared to EGFP titration, proviral DNA detection by qPCR was more accurate in assessing the number of infective particles in supernatants, such that average viral titres in terms of proviral copies per cell were two‐fold higher. Transgene mRNA expression was directly determined from the vectors used without the need for reporter assays. A new parameter, defined here as the ‘transcription index’ (TI), served to establish the association between transcribed transgenic mRNA and each proviral insertion. The TI represents the potential expression of every vector or insertion in each cell type, and is thus useful as a control parameter for monitoring preclinical or clinical protocols. Conclusions The practical use of qPCR is demonstrated as a valuable alternative to reporter genes for the assessment and surveillance of insertion numbers and transgene expression. In combination with protein expression, this approach should be capable of establishing safer therapeutic gene doses, avoiding the potential side effects of high transduction and expression levels. Copyright © 2006 John Wiley & Sons, Ltd.

Transduction (biophysics)

10.1002/jgm.951

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Citations (11)

The role of the vav promoter in gene therapy: Safer vectors with weaker and stable expression

Blood Cells Molecules and Diseases (2008)

Elena Almarza Montserrat Aldea Paula Rı́o Néstor W. Meza Xabier Agirre

Tree (set theory)

SAFER

10.1016/j.bcmd.2007.10.012

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Terapia génica de la deficiencia de piruvato quinasa

Néstor W. Meza

La deficiencia en Piruvato Kinasa Eritrocitaria humana (hRPK), un desorden autosomico recesivo producido por mutaciones en el gen PKLR, es una de las causas mas comunes de anemia hemolitica cronica no esferocitica. La expresion transgenica de la RPK en precursores eritropoyeticos deficientes puede resultar una terapia alternativa en aquellos pacientes con sintomas severos. En este trabajo hemos construido vectores retrovirales que expresan el cDNA de la hRPK y la eGFP en un unico RNA mensajero, separados por un IRES eucriotico (SF11RPXEG). Lineas celulares murinas 3T3 y MEL (Celulas de eritroleucemia murina ) y humanas HeLA y 293T fueron transducidas con sobrenadantes retrovirales infectivos. Analisis de western-blot y de FACS demostraron la expresion estable de EGFP y hRPK, respectivamente, con expresion estable del transgen durante mas de 20 semanas en cultivo. La expresion de los trasngenes no vario significativamente durante el tiempo del cultivo. Ademas, la induccion a eritrodiferenciacion de celulas MEL no modifico la expresion de hRPK ni de eGFP. Posteriormente, progenitores hematopoyeticos Lin-Sca-1+ fueron transducidos in vitro con SF11RPKXEG y transplantado s en receptores normales letalmente irradiados. Tres meses post-transplante, una elevada expresion de hRPK y eGFP fue observada en leucocitos, plaquetas y eritrocitos maduros. Estos resultados indican que los vectores retrovirales pueden ser una sistema efectivo para abordar la correccion fenotipica de los defectos eritropoyeticos asociados a la deficiencia de Piruvato Kinasa Eritrocitaria humana mediante de protocolos de terapia genica.

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Cell Fusion Reprogramming Leads to a Specific Hepatic Expression Pattern during Mouse Bone Marrow Derived Hepatocyte Formation In Vivo

PLoS ONE (2012)

Oscar Quintana‐Bustamante Esther Grueso Ramón García‐Escudero Elvira Arza Alberto Álvarez

The fusion of bone marrow (BM) hematopoietic cells with hepatocytes to generate BM derived hepatocytes (BMDH) is a natural process, which is enhanced in damaged tissues. However, the reprogramming needed to generate BMDH and the identity of the resultant cells is essentially unknown. In a mouse model of chronic liver damage, here we identify a modification in the chromatin structure of the hematopoietic nucleus during BMDH formation, accompanied by the loss of the key hematopoietic transcription factor PU.1/Sfpi1 (SFFV proviral integration 1) and gain of the key hepatic transcriptional regulator HNF-1A homeobox A (HNF-1A/Hnf1a). Through genome-wide expression analysis of laser captured BMDH, a differential gene expression pattern was detected and the chromatin changes observed were confirmed at the level of chromatin regulator genes. Similarly, Tranforming Growth Factor-β1 (TGF-β1) and neurotransmitter (e.g. Prostaglandin E Receptor 4 [Ptger4]) pathway genes were over-expressed. In summary, in vivo BMDH generation is a process in which the hematopoietic cell nucleus changes its identity and acquires hepatic features. These BMDHs have their own cell identity characterized by an expression pattern different from hematopoietic cells or hepatocytes. The role of these BMDHs in the liver requires further investigation.

Reprogramming

Hepatocyte nuclear factors

10.1371/journal.pone.0033945

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Risk factors for respiratory disease in chronic psychiatric in patients

The European Journal of Psychiatry (2007)

Nora Sánchez-Mora Óscar Medina Beatriz Francisconi Néstor W. Meza Nineth E. Rossi

Objective: Individuals with schizophrenia and other chronic mental illnesses present a series of risk factors, that predispose them to extensive medical comorbidity.The aim of this study was to determine the risk factors for respiratory disease in chronic psychiatric inpatients.Methods: All patients at a long-term mental institution were invited to participate in this study.Socio-demographic data, tobacco and alcohol consumption, respiratory symptoms and chest x-rays were collected from 154 patients.Sputum microscopy and cultures for M. tuberculosis were done in specific cases.Results:The symptoms reported were cough (58%); expectoration (44.2%); dyspnea (28.6%); hemoptysis (9.7%), and chest pain (20.1%).There were chest x-ray changes in 57.1% of patients.These were suggestive of chronic obstructive pulmonary disease (COPD) in 31.2% and an interstitial pattern in 19.1%.The prevalence of tuberculosis in this sample was 4.5%.Schizophrenia was the most common mental illness (43.5%), followed by an organic mental disorder (32.5%).We found a positive relationship between schizophrenia and cigarette smoking (odds ratio 2.30, 95% CI 1.11 to 4.76), and schizophrenia and x-ray changes (odds ratio 2.35, 95% CI 1.15 to 4.83).There was no significant relationship with other psychiatric disorders. Conclusions:The high prevalence of respiratory morbidity in patients with chronic mental illness hospitalized in long-term care facilities, is probably due to factors such as extreme poverty, tobacco consumption, overcrowding, and nutritional deficit.

Overcrowding

10.4321/s0213-61632007000300006

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Citations (4)

CYSTICERCOSIS IN CHRONIC PSYCHIATRIC INPATIENTS FROM A VENEZUELAN COMMUNITY

American Journal of Tropical Medicine and Hygiene (2005)

Néstor W. Meza Nineth E. Rossi TATIANA N. GALEAZZI Nery Sánchez FRANCISCO I. COLMENARES

Cysticercosis due to Taenia solium infection is endemic in developing countries of the Americas, Asia, and Africa. This study was designed to establish the prevalence of cysticercosis in 158 inpatients of a psychiatric institution in the state of Tachira (Venezuela) and in 127 healthy control subjects. Positive blood tests for cysticercosis by Western blotting were recorded in 18.35% of the patients and in 1.57% of the controls. Individuals with mental retardation were found to carry an increased risk of cysticercosis (RR: 2.92; 1.22 < 2.92 > 7.0; P < 0.05) compared with patients with other psychiatric disorders. Taeniasis by Taenia spp. was not demonstrated in the patient group, although a high incidence of infection by other helminths (95.1%) was detected. The high prevalence of cysticercosis in the psychiatric inpatient group, compared with healthy individuals, and the lack of a differential diagnosis of neurocysticercosis suggest cerebral cysticercosis in a large proportion of these patients. Cysticercosis could be the origin of the psychiatric disorders of these patients and may also be due to contact with the parasite in an environment with poor hygiene conditions and a deficient health care system.

Taenia solium

Neurocysticercosis

Taeniasis

Taenia

10.4269/ajtmh.2005.73.504

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Citations (28)

Life-threatening nonspherocytic hemolytic anemia in a patient with a null mutation in the PKLR gene and no compensatory PKM gene expression

Blood (2005)

Amalia Díez Florinda Gilsanz Joaquín Martínez‐López Susana Pérez-Benavente Néstor W. Meza

Pyruvate kinase deficiency

Null allele

10.1182/blood-2005-02-0555

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In VitroandIn VivoExpression of Human Erythrocyte Pyruvate Kinase in Erythroid Cells: A Gene Therapy Approach

Human Gene Therapy (2007)

Néstor W. Meza Oscar Quintana‐Bustamante Antonio Puyet Paula Rı́o Susana Navarro

Human pyruvate kinase deficiency (PKD), an autosomal recessive disorder produced by mutations in the PKLR gene, is the most common cause of chronic nonspherocytic hemolytic anemia. Transduction of wild-type erythroid (R-type) pyruvate kinase (RPK) cDNA into deficient hematopoietic stem cells could be of potential use as rescue therapy in severe clinical cases. In this study, gammaretroviral vectors expressing human RPK were designed as possible gene therapy candidates for this disease. Through real-time quantitative reverse transcriptase-polymerase chain reaction, Western blotting, and flow cytometric analysis, we demonstrate stable RPK expression in both undifferentiated and differentiated murine erythroleukemia cells. In this in vitro assay, the proportion of transduced cells and the intensity of expression of the transgene remained unaltered after 6 months of culture. Moreover, transplanting human RPK-transduced Lin− Sca-1+ mouse cells in myeloablated primary and secondary recipients rendered high proportions of erythroid precursors and mature erythrocytes expressing RPK, without inducing hematopoietic effects. These findings suggest that retroviral vectors could be useful for the delivery and expression of RPK in erythroid cells, and provide evidence of the potential use of gene therapy strategies to phenotypically correct erythroid PKD.

Pyruvate kinase deficiency

10.1089/hum.2006.052

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Differential antibody binding to the surface TCR{middle dot}CD3 complex of CD4+ and CD8+ T lymphocytes is conserved in mammals and associated with differential glycosylation

International Immunology (2008)

Nineth E. Rossi Jesús Reiné Miguel Pineda-Lezamit Marian A Pulgar Néstor W. Meza

We have previously shown that the surface alphabeta T cell antigen receptor (TCR).CD3 complex borne by human CD4(+) and CD8(+) T lymphocytes can be distinguished using mAbs. Using two unrelated sets of antibodies, we have now extended this finding to the surface alphabetaTCR.CD3 of seven additional mammalian species (six non-human primates and the mouse). We have also produced data supporting that differential glycosylation of the two main T cell subsets is involved in the observed TCR.CD3 antibody-binding differences in humans. First, we show differential lectin binding to human CD4(+) versus CD8(+) T lymphocytes, particularly with galectin 7. Second, we show that certain lectins can compete differentially with CD3 mAb binding to human primary CD4(+) and CD8(+) T lymphocytes. Third, N-glycan disruption using swainsonine was shown to increase mAb binding to the alphabetaTCR.CD3. We conclude that the differential antibody binding to the surface alphabetaTCR.CD3 complex of primary CD4(+) and CD8(+) T lymphocytes is phylogenetically conserved and associated with differential glycosylation. The differences may be exploited for therapeutic purposes, such as T cell lineage-specific immunosuppression of graft rejection. Also, the impact of glycosylation on CD3 antibody binding requires a cautious interpretation of CD3 expression levels and T cell numbers in clinical diagnosis.

10.1093/intimm/dxn081

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The gray boundaries of aberrant shortening of the cellular timekeepers’ edges

EMBO Molecular Medicine (2024)

Guillermo Güenechea Néstor W. Meza

Telomeres are supramolecular structures that allow the DNA strand to fold back on itself and protect the linear chromosome end from being sensed as a double-strand DNA break. Telomeric conservation relies on mechanisms that replace terminal DNA sequences, and ensuring structural integrity. Telomere biology disorders (TBDs) are a heterogeneous group of low-prevalence pathologies defined by germline mutations in genes involved in telomere maintenance mechanisms (TMMs). TBDs manifest across a broad clinical spectrum, often with substantial phenotypic and genetic overlap among clinical entities. In this issue of EMBO Molecular Medicine, Tummala and collaborators present clinical and biological data from DC/DCL patients, that enhances the understanding of the natural history of these diseases. In addition, the description of novel TBD-associated genetic variants in POT1 and ZCCHC8 and of the new POLA1 gene advances the understanding of the functional network of genes involved in TBD and highlights new pathogenic mechanisms.

10.1038/s44321-024-00122-1

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