CRISPR-on system for the activation of the endogenous human INS gene.

Advances in the field of epigenetics have allowed the design of new therapeutic strategies to address complex diseases such as type 1 diabetes (T1D). Clustered regularly interspaced short palindromic repeats (CRISPR)-on is a novel and powerful RNA-guided transcriptional activator system that can turn on specific gene expression; however, it remains unclear whether this system can be widely used or whether its use will be restricted depending on cell types, methylation promoter statuses or the capacity to modulate chromatin state. Our results revealed that the CRISPR-on system fused with transcriptional activators (dCas9-VP160) activated endogenous human INS, which is a silenced gene with a fully methylated promoter. Similarly, we observed a synergistic effect on gene activation when multiple single guide RNAs were used, and the transcriptional activation was maintained until day 21. Regarding the epigenetic profile, the targeted promoter gene did not exhibit alteration in its methylation status but rather exhibited altered levels of H3K9ac following treatment. Importantly, we showed that dCas9-VP160 acts on patients' cells in vitro, particularly the fibroblasts of patients with T1D.

Prevalence of CYP2B6 polymorphisms in Argentinians: the role of genetic testing.

CYP2B6 is a highly polymorphic isoenzyme involved in the metabolism of many drugs including cyclophosphamide, bupropion, and efavirenz. A single nucleotide polymorphism (SNP) in CYP2B6 (516G>T) resulted in decreased expression and function associated with the CYP2B6*6 haplotype. Among the clinical implications of this phenotype, decreased activation of cyclophosphamide and increased plasma levels of efavirenz associated with increased central nervous system toxicity have been reported. The frequency of the CYP2B6 (516G>T) SNP has been studied in several different populations, but there is no data regarding distribution among Argentinians. In this study, 102 DNA samples from healthy volunteers were analyzed using a polymerase chain reaction-restriction fragment length polymorphism reaction specific for the CYP2B6 (516G>T) SNP. Our results showed a prevalence of 71.08% for the G allele and 28.92% for the T allele. This was distributed as 52.9% for the GG genotype (reduced dosage required), 36.6% for the GT genotype (normal dosage range), and 10.8% for the TT genotype (high drug toxicity). There was no preferential gender distribution observed. The relatively high prevalence of the TT genotype in our population supports the clinical use of genotyping as an additional tool in personalized medicine.

Prevalence of CYP2C9 and VKORC1 alleles in the Argentine population and implications for prescribing dosages of anticoagulants.

Dicumarinic oral anticoagulants have a narrow therapeutic range and a great individual variability in response, which makes calculation of the correct dose difficult and critical. Genetic factors involved in this variability include polymorphisms of genes that encode the metabolic enzyme CYP2C9 and the target enzyme vitamin K epoxide reductase complex 1 (VKORC1); these polymorphisms can be associated with reduced enzymatic expression. We examined the frequency of the most relevant variants encoding CYP2C9 (alleles *1, *2 and *3) and VKORC1 (SNP -1639A>G) in the Argentinian population. Molecular typing was performed by PCR-RFLP on a randomly selected sample of 101 healthy volunteers from the Hospital Italiano de Buenos Aires gene bank. Fifty-seven subjects were identified as homozygous for CYP2C9*1 and 14 for *2, while 24 and 5 were heterozygous for *2 and *3 alleles; one individual was a composite heterozygote (*2/*3). When we examined VKORC1, 21 subjects were AA homozygous, 60 were AG heterozygotes and 20 were GG homozygotes. This is the first analysis of genotypic frequencies for CYP2C9 and VKORC1 performed in an Argentinian population. These allele prevalences are similar to what is known for Caucasian population, reflecting the European ancestor of our patient population, coming mostly from Buenos Aires city and surroundings. Knowledge of this prevalence information is instrumental for cost-effective pharmacogenomic testing in patients undergoing oral anticoagulation treatment.

CD24 as a genetic modifier of disease progression in multiple sclerosis in Argentinean patients.

INTRODUCTION: Previous reports have shown that CD24 gene polymorphisms have an important role in the risk of development and progression of multiple sclerosis (MS). OBJECTIVE: To investigate the association between P226 polymorphisms (T/C), P1056 (A/G), P1527 (TG/del) and P1626 (A/G) of the CD24 gene and MS, comparing allele and genotype frequencies of patients versus controls. MATERIALS AND METHODS: We analyzed DNA samples from 102 MS patients and from 205 unrelated healthy individuals. DNA was extracted from peripheral blood and polymorphic regions were amplified by nested PCR. Genotyping was performed by restriction fragments length polymorphisms. Time from disease onset to reach EDSS 6 and time to conversion to secondary progressive phase (SP) were used as variables of survival as well as percentage of patients that reached those endpoints. We used the log Rank test for data comparison (significant p≤0.05). RESULTS: We found no differences between cases and controls in frequency of polymorphisms at the CD24 gene. 44.6% of patients with the AA genotype (P1626) reached an EDSS 6 vs 16% of patients with other genotypes (p<0.001, HR 3.2, 95% CI 1.4 to 7.4). 45.8% of patients with the AA genotype reached SPMS vs 16.7% without this genotype (p<0.001, HR 3.4, 95% CI 1.5 to 7.8). CONCLUSIONS: This study showed a strong association between the presence of AA genotype in the 1626 polymorphism of the CD24 gene and the risk of disease progression in MS patients.

Dietary factors, genetic and epigenetic influences in colorectal cancer.

Genetic influences, together with epigenetic components and dietary factors, play a fundamental role in the initiation and progression of cancer by causing a number of deregulations. Colorectal cancer (CRC) is a disease influenced by dietary factors, for which established genetic and epigenetic alterations have been identified. Within CRC, there are hereditary syndromes that present mutations in the germ-line hMLH1, and also alterations in the methylation of the promoters. Epigenetics has also been established as a pathway of carcinogenesis. In the present review, we analyzed studies conducted to discern the different pathways leading to established CRC, stressing the importance of identifying factors that may predict CRC at an early stage, since it is mostly a silent disease observed at the clinical level in advanced stages.

HLA-DRB1 and multiple sclerosis in Argentina.

BACKGROUND: The association of multiple sclerosis (MS) with HLA DR subtypes, and particularly human leukocyte antigen (HLA)-DRB1*15 has been a consistent finding across nearly all Caucasian MS populations. In South America, scarce data exist about this issue. As the complete characterization of the HLA association range around the world is important to understand the role of this locus in MS susceptibility, we analyzed the frequency of HLA-DRB1* allelic groups in an MS population in Argentina. METHODS: HLA-DRB1 locus was genotyped using PCR and sequence-specific primer amplification in 61 MS patients born in Buenos Aires, Argentina and 1216 healthy controls. Allele frequencies were compared between groups. RESULTS: The HLA-DRB1*15 allele frequency significantly differed between controls and patients (13.5% and 33.9% respectively, P < 0.001, OR 2.51, 95% CI: 1.7-3.0). The other allele frequencies did not show significant differences between patients and controls. CONCLUSIONS: The present HLA class II population study is in accordance with other Caucasian MS surveys which have found that HLA-DRB1*15 allele is strongly associated with MS disease. In Argentina, this is the first study performed to analyze the association of HLA-DRB1*15 and MS susceptibility in a Caucasian population and therefore contributes with new data to the immunogenomic global MS map.

Human reconsolidation does not always occur when a memory is retrieved: the relevance of the reminder structure.

Memory reconsolidation is defined as a process in which the retrieval of a previously consolidated memory returns to a labile state which is then subject to stabilization. The reminder is the event that begins with the presentation of the learned cue and triggers the labilization-reconsolidation process. Since the early formulation of the hypothesis, several controversial items have arisen concerning the conditions that define reconsolidation. It is herein proposed that two diagnostic features characterize reconsolidation, namely: the labilization of the reactivated memory and the specificity of the reminder structure. To study this proposal, subjects received two different training sessions on verbal material on Day 1 and Day 2, respectively. Finally, they were tested for the first and second acquired memories on Day 3. It is demonstrated that the human declarative memory fulfills the two requirements that define the process. First, the reactivated memory is impaired by a new learning only when it is given closely after the reminder, revealing that the memory is labilized. Second, the omission of at least one of the reminder's components prevents labilization. Therefore, results show that the new learning fails to produce an amnesic effect on the target memory either when the reminder omits the learned cue or includes the beginning of the reinforcement.

Carcinomatosis peritoneal: resultados quirúrgicos de la cirugía de citorreducción y quimioterapia / Peritoneal carcinomatosis: results of surgical cytoreduction surgery and chemotherapy

Objetivo: Presentar la experiencia preliminar en el tratamiento de la carcinomatosis peritoneal de origen gastrointestinal y ginecológico. Métodos: El estudio incluye 11 pacientes portadores de carcinomatosis, tratados por medio de citorreducción y quimioterapia hipertérmica intraoperatoria y operados entre enero del 2005 y enero del 2008. Resultados: La mediana de edad de los pacientes fue de 50 años (rango = 22-56) y pertenecieron al sexo masculino el 54.5%. La mediana de duración del procedimiento citorreductivo fue de 630 min (rango 205-840). La citorreducción completa fue obtenida en el 54,6% (6/11) de los pacientes. La mediana de órganos resecados por paciente fue 4 (rango = 2 - 5) y la mediana de peritonectomia por paciente fue de 3 (rango = 0-6). Las drogas utilizadas para el baño hipertérmico fueron la Mitomicina C en las carcinomatosis por cancer de colon y del apéndice y el Cisplatino en carcinomatosis por cáncer de ovario. El tiempo d irrigación fue de 90 min y la mediana de la temperatura alcanzada fue de 41º C (39ºC - 43ºC). La mortalidad posoperatoria fue del 9,1% (1/11) y la morbilidad del 63,6% (7/11). La mediana de internación fue de 10 días (4-67), en los pacientes complicados la media de internación fue de 44 días para las severas y de 9 días para las leves. La supervivencia libre de tumor fue del 30% (mediana de seguimiento de 8,3 meses).

Hippocampal upregulation of the cyclooxygenase-2 gene following neonatal clomipramine treatment (a model of depression).

Although a putative role has been attributed to inflammation in the pathogenesis of depressive disorders, the relationship of prostaglandins, known mediators of inflammation, and depression has not been elucidated. Clomipramine is an antidepressive drug with a pro-depressive paradoxical effect in adult rats when administrated neonatally. Using this effect as a model of depression, we investigated the differential expression of the cyclooxygenase (COX-2) gene in rat brains. Rats injected neonatally with clomipramine showed depressive-like symptoms in adulthood, as well as decreased levels of the brain-derived neurotrophic factor (BDNF) and a quantitative differential expression of the COX-2 gene (Real Time PCR) and protein (immunohistochemistry) in the hippocampus. As evidenced, the relationship between a key enzyme in the prostaglandin synthesis and biological and behavioral depression-like changes opens an interesting line of investigation regarding the molecular bases of depression and its potential treatment through immunomodulatory drugs.

Inhibition of allospecific response in pancreatic islet transplantation: the glycan approach.

UNLABELLED: Host resistance has precluded clinical islet transplantation from becoming a consistent therapy for type I diabetic patients, mainly due to both specific and nonspecific processes. O-glycosylated proteins have a primary role in immunologic synapses. Therefore, we investigated the effects of a putative immunomodulatory effect of the cleavage of these molecules on islet allotransplantation. METHODS: Murine islets were treated with O-sialoglycoprotein endopeptidase. Three endpoints were studied: (1) proliferation in allogeneic mixed islet mononuclear cell reactions using treated and control irradiated islets as stimulator cells of mononuclear cells; (2) expression of IA-d on monocytes using 48-hour transplants of treated versus control mouse islets into subcutaneous capsules; (3) posttransplant graft function in an in vivo model of islet allotransplantation. Treated and control islets were transplanted in diabetic mice treated daily with cyclosporine. Glycemia was monitored to determine diabetes reversion. RESULTS: The allogeneic proliferative response was maximal when allogeneic mononuclear cells were mixed with control islets; it was significantly decreased with treated islets. Mean proliferative inhibition rate of treated vs. control was 62%. IA-d expression on monocytes was maximal in control islets. Reversion was significantly different for treated versus control islets with its duration varied from 3 to 7 days. CONCLUSION: These results suggest that treatment of islets with O-sialoglycoprotein endopeptidase may modulate allogeneic immunologic reactions.

Trypanosoma cruzi trans-sialidase inhibits human lymphocyte proliferation by nonapoptotic mechanisms: implications in pathogenesis and transplant immunology.

Chiefly an intracellular parasite, Trypanosoma cruzi has a transient blood-borne stage (trypomastigote), the acute phase of Chagas' disease, during which surface trans-sialidase is expressed and shed by the parasite. It's immunosuppressive through the induction of apoptosis. Herein, we investigated the role of trans-sialidase as an immune modulator of allo- and xenoreactions. Trans-sialidase strongly inhibited human lymphocyte proliferation; a role for the interleukin-2 receptor CD25 was suggested by flow cytometry. These results may have implications both for the pathogenesis of Chagas' disease and for transplantation immunology.

Células mesenquimales de médula osea: diferenciación y potencial reemplazo neuronal / Mesenchymal stem cells: differentiation and alternative source of neural tissue

Embryonic stem cells are a population of cells located in the blastocyst, committed to specific differentiation according to spatial and temporal factors such as age and place of final location. Despite the final fate of hematic cells, hemopoietic cells retain a relative degree of plasticity dependent on environmental factors. Mesenchymal cells are a well differentiated population of bone marrow derived non hemopoietic cells with totipotential properties. The medical interest of such totipotentiality rests in the potential of such cells to repair damaged tissues. Particularly neuronal differentiation from progenitors obtained from mesenchymae non hemopoietic cells offers a new possibility in the field of neural transplantation and tissue engineering to repair functional entities in the nervous system.

Las células troncales embrionarias son totipotentes y se encuentran en pequeño número en el blastocisto donde pueden expandirse en forma indiferenciada durante un corto tiempo y de acuerdoal sitio donde se alojan, ellas adquirirán determinados fenotipos de diferenciación. Las células hemopoyéticas troncales se caracterizan por poseer un gran potencial proliferativo, cuyo modelo de regulación es jerárquico. Ellas retienen, a lo largo de su existencia, un cierto grado de plasticidad, lo cual hace que se puedan diferenciar en distintos tipos de células o de tejidos no hemopoyéticos, de acuerdo al microambiente donde se encuentran o bien a la presencia de ciertos factores estimulantes. En trabajos recientes se han podido aislar, por adherencia al plástico, en cultivo in vitro de médula ósea, células no hemopoyéticas que fueron llamadas mesenquimales por su semejanza con el tejido mesenquimal del embrión. Estas células, in vitro, pueden ser inducidas hacia nuevas líneas celulares, que se diferenciarán en nuevos tejidos. Las expectativas del beneficio terapéutico del transplante de médula ósea como el de células mesenquimales en enfermedades no hemopoyéticas son grandes, porque al utilizar tejidos o células autólogas, se evitan los graves problemas del rechazo inmunológico. En el caso del tejido nervioso la problemática de una fuente adecuada de donantes hace el tema especialmente interesante.

Resultados iniciales con criopreservación de glándulas paratiroides y potenciales aplicaciones / Histological integrity and functional viability results of previously cryopreserved human para thyroid tissue

Objetivo: Analizar la integridad histológica y viabilidad funcional de tejido paratiroideo criopreservado, con miras a un potencial trasplante futuro. Material y método: Se criopreservaron muestras de tejido paratiroideo proveniente de 14 pacientes operados por HPT (8 secundarios, 3 terciarios , 1 primario recidivado y dos hiperplasias primarias). Fraccionado en porciones de 2 x 3 mm, el material fue preservado en suero + solución de Hanks + DMSO y congelados hasta -90ºC, conservándolo en nitrógeno líquido. Luego de seis meses, el material de 4 pacientes fue parcialmente descongelado y estimulado con solución de Cloruro de Ca++, midiéndose la secreción de PTH y estudiando la histología con H y E y con el método de Tunne

Resultados iniciales con criopreservación de glándulas paratiroides y potenciales aplicaciones / Histological integrity and functional viability results of previously cryopreserved human para thyroid tissue

Objetivo: Analizar la integridad histológica y viabilidad funcional de tejido paratiroideo criopreservado, con miras a un potencial trasplante futuro. Material y método: Se criopreservaron muestras de tejido paratiroideo proveniente de 14 pacientes operados por HPT (8 secundarios, 3 terciarios , 1 primario recidivado y dos hiperplasias primarias). Fraccionado en porciones de 2 x 3 mm, el material fue preservado en suero + solución de Hanks + DMSO y congelados hasta -90°C, conservándolo en nitrógeno líquido. Luego de seis meses, el material de 4 pacientes fue parcialmente descongelado y estimulado con solución de Cloruro de Ca++, midiéndose la secreción de PTH y estudiando la histología con H y E y con el método de Tunnel para apoptosis. Resultados: Se observó en las cuatro muestras un patrón similar de respuesta, con un pico inicial de secreción, un descenso a la hora, y un nuevo ascenso a las dos horas del estímulo. El estudio histológico de las piezas mostró integridad histológica luego de seis meses de criopreservación, pero importante depauperización celular luego del estímulo. Conclusiones: En la muestra analizada, la criopreservación del tejido paratiroideo ha permitido conservar su integridad histológica, así como sus capacidades secretorias básicas. El fenómeno apoptótico que invariablemente acompaña a la estimulación prolongada in vitro, requiere nuevas investigaciones que permitan controlarlo

Células mesenquimales de médula osea: diferenciación y potencial reemplazo neuronal / Mesenchymal stem cells: differentiation and alternative source of neural tissue

Embryonic stem cells are a population of cells located in the blastocyst, committed to specific differentiation according to spatial and temporal factors such as age and place of final location. Despite the final fate of hematic cells, hemopoietic cells retain a relative degree of plasticity dependent on environmental factors. Mesenchymal cells are a well differentiated population of bone marrow derived non hemopoietic cells with totipotential properties. The medical interest of such totipotentiality rests in the potential of such cells to repair damaged tissues. Particularly neuronal differentiation from progenitors obtained from mesenchymae non hemopoietic cells offers a new possibility in the field of neural transplantation and tissue engineering to repair functional entities in the nervous system.(AU)

Las células troncales embrionarias son totipotentes y se encuentran en pequeño número en el blastocisto donde pueden expandirse en forma indiferenciada durante un corto tiempo y de acuerdoal sitio donde se alojan, ellas adquirirán determinados fenotipos de diferenciación. Las células hemopoyéticas troncales se caracterizan por poseer un gran potencial proliferativo, cuyo modelo de regulación es jerárquico. Ellas retienen, a lo largo de su existencia, un cierto grado de plasticidad, lo cual hace que se puedan diferenciar en distintos tipos de células o de tejidos no hemopoyéticos, de acuerdo al microambiente donde se encuentran o bien a la presencia de ciertos factores estimulantes. En trabajos recientes se han podido aislar, por adherencia al plástico, en cultivo in vitro de médula ósea, células no hemopoyéticas que fueron llamadas mesenquimales por su semejanza con el tejido mesenquimal del embrión. Estas células, in vitro, pueden ser inducidas hacia nuevas líneas celulares, que se diferenciarán en nuevos tejidos. Las expectativas del beneficio terapéutico del transplante de médula ósea como el de células mesenquimales en enfermedades no hemopoyéticas son grandes, porque al utilizar tejidos o células autólogas, se evitan los graves problemas del rechazo inmunológico. En el caso del tejido nervioso la problemática de una fuente adecuada de donantes hace el tema especialmente interesante.(AU)

[Characteristics of murine primordial germ cells and their relation with hemopoiesis]. / Características de las células primordiales germinales murinas y su relación con la hemopoyesis.

Primordial germ cells (PGC) are a population of cells characterised by a positive reaction to alkaline phosphatase, usually present in the mouse embryo at 7.5 days post coitus (dpc). These cells migrate through various tissues before they become incorporated into the gonadal ridges. Hematopoiesis is a complex developmental system in which the hemopoietic stem cells (HSC) were experimentally shown to have been derived from a single multipotent stem cell. PGC, as well as HSC are regulated by a range of growth factors that control both proliferative and differentiative processes. Leukemia inhibitory factor (LIF) is a cytokine that regulates the differentiation and the totipotentional phenotype of PGC. Recently, other growth factors, such as stem cell factor (SCF), macrophage growth factor (MGF), and forskolin (FRKL) have been proposed as the possible in vivo and in vitro regulators for PGCs and HSCs. Induction of hematopoiesis in an embryonic germ cell derived from PGCs indicates that germ cells acquire the potentiality to differentiate toward hematopoietic cells. The coincidental presence of both PGCs and HSCs at the sites where early hemopoiesis is established, together with similar growth factor requirements support the hypothesis that PGCs may also be considered hemopoiesis initiating cells.

Culture of porcine hepatocytes: the dogma of exogenous matrix revisited.

The use of exogenous matrices has been described as an essential component in securing the viability and functionality of hepatocytes in vitro whether cultured for extracorporeal devices or cell transplantation. Here we report on the in vitro culture of porcine hepatocytes in polystyrene tissue-culture flasks without exogenous matrices showing adequate attachment and viability. Cell proliferation was evidenced by uptake of 5-bromo-2'-deoxyuridine, with peaks at Days 2 (19.7 +/- 8.5%), 15 (20.8 +/- 3.3%), and 35 (21.4 +/- 0.3%). Detoxification capacity was assessed by determination of monoethylglycinexylidide, a product of lidocaine metabolism (highest value 156.5 +/- 10.1 ng/ml at Day 4), and by diazepam clearance (maximum clearance 66.2% at Day 6). Diazepam metabolite levels were highest at Day 4 both for temazepam and oxazepam (6.5 +/- 0.1 and 0.10 +/- 0.01, respectively). These results suggest that the need for an exogenous matrix to achieve sustained proliferative activity and differentiated hepatocyte function should not necessarily be considered a sine qua non condition.

Complicaciones urológicas en el transplante reno-pancreático / Urological complications in kidney/pancreas transplantation

Introducción: El transplante Reno- Pancreático (TRP) es el tratamiento más efectivo para la diabetes tipo 1 en insuficiencia renal crónica, ya que el páncreas transplantado protege al riñón transplantado de sufrir la nefropatía diabética y mejora el estado general evitando las complicaciones crónicas de la diabetes. La dertivación de lña secreción pancreática exócrina es un punto controvertido, en nuestra experiencia la hemos derivado a la vejiga, siendo el objetivo de este trabajo estudiar las complicaciones urológicas ocurridas en nuestros primeros 10 TRP. Material y Métodos: En el Hospital Italiano de Buenos Airesentre octubre de 1994 y mayo de 1999 se realizaron 10 TRP con páncreas vascularizado. El tiempo promedio de edad al transplante fue de 34,1 años, 3 pacientes fueron del sexo masculino y 7 del femenino. El tiempo medio de seguimiento fue de 16,7 meses (2-41 meses). La supervivencia actuarial al año de paciente, riñon y páncreas fue de 79 por ciento, 68 por ciento y 79 por ciento respectivamente. Ambos órganos fueron colocados en situación intraperitoneal, uno en cada fosa ilíaca derivando la secreción exócrina del páncreas a la vejiga anastomodsando el duodeno a la vejiga mediante 2 suturas continuas. Resultados: nueve de los 10 pacientes presentaron complicaciones urológicas y éstas fueron 17 episodios de infección urinaria en 9, pancreatitis por reflujo en 2, 6 episodios de hematuria en 4, pielonefritis abscedada en 1, fístula tardía duodeno- pancreática en 3, fístula uretral en 1, litiasis vesical en 1 paciente en 2 ocaciones y linfocele en 2 pacientes. No hemos tenido complicaciones relacionadas con la uretroneocistostomía, ni hemos tenido que convertir el drenaje exócrino pancreático al intestino. Concluciones: La derivación exócrina del páncreas a lo vejiga condiciona las complicaciones urológicas del TRP, siendo el compromiso del urólogo vital para el manejo de las mismas. Las complicaciones pueden ocurrir en cualquier momento después del transplante, siendo más común la parición de éstas en los primeros meses.La elevación de la presión vesical favorece la pancreatitis por reflujo, la fístula urinaria y los síntomas irritativos. A todo paciente con dolor súbito abdominal i/o elevación de la amilasemia se debe colocar cateter vesical. Las fístulas resultaron ser las complicaciones de peor pronóstico y el síntoma más común fue el dolor abdominal(AU)