CRISPR-mediated base editing in mice using cytosine deaminase base editor 4

BACKGROUND: Many human genetic diseases arise from point mutations. These genetic diseases can theoretically be corrected through gene therapy. However, gene therapy in clinical application is still far from mature. Nearly half of the pathogenic single-nucleotide polymorphisms (SNPs) are caused by G:C>A:T or T:A>C:G base changes and the ideal approaches to correct these mutations are base editing. These CRISPR-Cas9-mediated base editing does not leave any footprint in genome and does not require donor DNA sequences for homologous recombination. These base editing methods have been successfully applied to cultured mammalian cells with high precision and efficiency, but BE4 has not been confirmed in mice. Animal models are important for dissecting pathogenic mechanism of human genetic diseases and testing of base correction efficacy in vivo. Cytidine base editor BE4 is a newly developed version of cytidine base editing system that converts cytidine (C) to uridine (U). RESULTS: In this study, BE4 system was tested in cells to inactivate GFP gene and in mice to introduce single-base substitution that would lead to a stop codon in tyrosinase gene. High percentage albino coat-colored mice were obtained from black coat-colored donor zygotes after pronuclei microinjection. Sequencing results showed that expected base changes were obtained with high precision and efficiency (56.25%). There are no off-targeting events identified in predicted potential off-target sites. CONCLUSIONS: Results confirm BE4 system can work in vivo with high precision and efficacy, and has great potentials in clinic to repair human genetic mutations.

Does Oral Hygiene Influence Salivary pH, Lactate, and IL-1β of Basketball Players During Intense Exercise? / La Higiene Oral Influye en el pH Salival, el Lactato y la IL-1β de los Jugadores de Baloncesto Durante el Ejercicio Intenso?

ABSTRACT: This study examined the salivary pH, salivary lactate, and salivary IL-1 β responses from a high-intensity intermittent running test, and the influence of hygiene oral status on these biomarkers in elite adolescent basketball players. Forty-six adolescent players participated. Saliva sampling was taken before and 3 min after a high-intensity exercise (Yo-Yo Intermittent Recovery Test Level 1; Yo-Yo IR1). In order to quantify and classify the oral hygiene level, the athletes were submitted to a dental examination, and an adapted Simplified Oral Hygiene Index was applied. After the dental examination, the whole group was divided into good oral hygiene group (GHG) and poor oral hygiene group (PHG). The results of a two- way analysis of variance showed a significant interaction effect (P = 0.0003), group effect (P < 0.0001), and time effect (pre to post Yo-yo IR1; P < 0.0001) for salivary pH and for salivary lactate (interaction effect, P = 0.008; group effect, P < 0.000 1; time effect, P < 0.0001) with a lower salivary pH and a higher salivary lactate at pre and post-Yo-Yo IR1 for PHG, but no difference was observed for IL-1β. The data demonstrated that the high-intensity exercise led to a significant change in salivary pH and salivary lactate concentration of the basketball players, and that the oral hygiene status influenced these responses, with a greater change for those players showing a poor oral hygiene.

RESUMEN: Este estudio examinó las respuestas de pH salival, lactato salival e IL-1β salival de una prueba de carrera intermitente de alta intensidad, y la influencia del estado de higiene oral en los biomarcadores en jugadores adolescentes de baloncesto de élite. En el análisis participaron 46 jugadores adolescentes. Se tomó una muestra de saliva antes y 3 minutos después de un ejercicio de alta intensidad (Prueba de recuperación intermitente Yo-Yo Nivel 1; Yo-Yo IR1). Para cuantificar y clasificar el nivel de higiene oral, los atletas fueron sometidos a un examen dental y se aplicó un índice adaptado de higiene oral simplificado. Después del examen dental, el grupo se dividió en un grupo de buena higiene oral (GHG) y un grupo de mala higiene oral (PHG). Los resultados de un análisis de varianza mostraron un efecto de interacción significativo (P = 0.0003), efecto de grupo (P<0.0001) y efecto de tiempo (antes y después de Yo-yo IR1; P <0.0001) para el pH salival y para lactato salival (efecto de interacción, P = 0.008; efecto de grupo, P <0.0001; efecto de tiempo, P <0.0001) con pH salival más bajo y lactato salival más alto en IR1 pre y post YoY para PHG, pero no se observó una diferencia para IL-1β. Los datos demostraron que el ejercicio de alta intensidad genera un cambio significativo en el pH salival y el lactato de los jugadores de baloncesto, y que el estado de higiene oral influyó en estas respuestas, con un cambio mayor para aquellos jugadores que mostraron una mala higiene oral.

Avaliação dos níveis solúveis de sTIM-3, s4-1BB e de citocinas inflamatórias em mulheres com câncer de mama triplo negativo localmente avançado submetidas a quimioterapia neoadjuvante / Evaluation of soluble levels of sTIM-3, s4-1BB and inflammatory cytokines in women with locally advanced triple negative breast cancer submitted to neoadjuvant chemotherapy

Introdução: O câncer de mama triplo negativo (CMTN) corresponde de 10 a 20% dos carcinomas invasivos de mama, é altamente agressivo e na maioria das vezes tem apenas a quimioterapia padrão como tratamento. As citocinas inflamatórias são um grupo heterogêneo de proteínas solúveis produzidas por diferentes tipos de células, que medeiam e regulam o sistema imune. A resposta imune celular é regulada por proteínas acessórias denominadas receptores co-estimuladores e co-inibidores, como 4-1BB e TIM-3, respectivamente. A procura por biomarcadores que possam predizer resposta à quimioterapia neoadjuvante (QT neo) ainda é um desafio na medicina. Objetivo: Avaliar os níveis solúveis de sTIM-3 e s4-1BB, e de citocinas inflamatórias no sangue de mulheres com câncer de mama triplo negativo localmente avançado e associá-los à sobrevida livre de doença e com o tipo de resposta patológica à QT neo. Métodos: O estudo foi realizado entre os anos de 2015 e 2017 no Hospital de Câncer de Pernambuco (HCP) e Laboratório de Pesquisa Translacional do Instituto de Medicina Integral Prof. Fernando Figueira (IMIP). Foram incluídas 29 mulheres, entre 18 e 60 anos de idade, com diagnóstico de CMTN localmente avançado e submetidas à QT neo, e um grupo de 30 mulheres saudáveis. Coletas de sangue periférico foram realizadas antes e após a QT neo. A dosagem dos níveis solúveis de s4-1BB e sTIM-3 foi realizada por enzyme linked immunonoSorbent assay (ELISA). As dosagens de IL-1ß, IL-6, IL-8, TNF-α e IL-10 foram realizadas pela técnica de Cytometric Bead Array por citometria de fluxo. O nível de significância estatística foi de p<0,05. Resultados: Níveis mais elevados de IL-6 e de IL-10 foram observados no grupo de pacientes com tumor T4 com relação as grupo T2-T3 (p<0.05). Níveis elevados de IL-6, IL-8, IL-10 e TNF-α no grupo CMTN com status linfonodal N2 versus N0 e N1 (p<0,05). Os níveis de IL-1ß, IL-6, IL-10 e TNF-α foram elevados no grupo com resposta patológica parcial (RP) quando comparado aos grupos com resposta patológica completa (RC) e controles (p<0,05). Com relação aos níveis de IL-8, os grupos de pacientes RC e RP apresentaram níveis elevados quando comparados aos controles (p<0,05). Não foi observada diferença significativa de IL-8 entre os grupos RC e RP. Não foram observadas diferenças significativas em níveis de s4-1BB E sTIM-3 de acordo com o tumor primário e status linfonodal. Elevados níveis de s4-1BB foram observados no grupo RC comparado aos grupos RP e de controles (p<0,0004 e p<0,0001, respectivamente). Da mesma forma, os níveis de sTIM-3 foram mais elevados nas pacientes com RC e RP em relação aos controles (p<0,0001 e p<0,0003, respectivamente). Para análise da sobrevida livre de doença (SLD) em 24 meses de seguimento, utilizamos como ponto de corte o valor da mediana (< ou ≥ percentil 50) dos níveis solúveis de sTIM-3 e s4-1BB. Não houve diferenças significativas na SLD entre os grupos com níveis de s4-1BB ≥ 122 e < 122 pg/mL. A SLD foi de 93,33% no grupo com níveis de sTIM-3 < 2874 pg/mL e de 60% no grupo com níveis ≥ 2874 pg/mL (p=0,03). Conclusão: O s4-1BB foi um bom indicador preditivo de resposta à QT neo, enquanto elevação de sTIM-3 mostrou estar associado ao risco de recidiva loco-regional e metástases à distância. Ambos, TIM-3 e 4-1BB, parecem ser potenciais alvos terapêuticos no CMTN localmente avançado por estarem associadas ao desfecho clínico da doença

Introduction: Triple negative breast cancer (TNBC) corresponds to 10 to 20% of invasive breast carcinoma with high aggressiveness and in most cases has only standard chemotherapy as treatment. Inflammatory cytokines are heterogeneous group of soluble proteins produced by different types of cell, that mediate and regulate the immune system. The cellular immune response is regulated by accessory proteins called co-stimulatory and co-inhibitory receptors, such as 4-1BB and TIM-3, respectively. The search for biomarkers that can predict response to neoadjuvant chemotherapy (NAC) is still a challenge to medicine. Objective: To evaluate the soluble levels of sTIM-3 and s4-1BB, and inflammatory cytokines in the blood of women with locally advanced triple negative breast cancer, and to associate them with disease-free survival and the type of pathological response to NAC. Methods: The study was carried out between the years 2015 and 2017 at the Hospital de Cancer de Pernanbuco (HCP) and Translational Research Laboratory of the Instituto de Medicina Integral Prof. Fernando Figueira (IMIP). Twenty-nine women, between 18 and 60 years old, diagnosed with locally advanced TNBC, and submitted to NAC, and 30 healthy women were included. Peripheral blood samples were taken before and after neo QT. The determination of the soluble levels of s4-1BB and sTIM-3 was performed by enzyme linked immunonosorbent assay (ELISA). The measurements of IL-1ß, IL-6, IL-8, TNF-α and IL-10 were performed using the Cytometric Bead Array by flow cytometry. The level of statistical significance was p <0.05. Results: There were higher levels of IL-6 and IL-10 in the group of patients with tumor size T4 compared to groups T2-T3 (p <0.05). High levels of IL-6, IL8, IL-10 and TNF-α were found in the TNBC group with lymph node status in N2 in relation to N0 and N1 (p <0.05). High levels of IL1ß, IL-6, IL-10 and TNF-α were observed in the group with partial pathological response (PR) when compared to groups with complete pathologial response (RC) and controls (p <0.05). With regard to IL-8 levels, the groups of CR and RP patients showed high levels when compared to controls (p <0.05). There was no significant difference in IL-8 between the CR and PR groups. No significant differences were observed in the levels of s4-1BB and sTIM-3 in accordance with primary tumor and lymph node status. High levels of s4-1BB were observed in the RC group compared to the PR and control groups (p <0.0004 and p <0.0001, respectively). As well, the levels of sTIM-3 were higher in patients with CR and PR compared to controls (p <0.0001 and p <0.0003, respectively). In the analyses of disease-free survival (DFS) with 24 months of follow-up, we used as a cut-off point the median value (< or ≥ 50th percentile) of the soluble levels of sTIM-3 and s4-1BB. There were no significant differences in DFS at 24 months between groups with levels of s4-1BB ≥ 122 and <122 pg/mL. The DFS was 93.33% in the group with sTIM-3 levels <2874 pg/mL and 60% in the group with levels ≥ 2874 pg/mL (p = 0.03). Conclusion: s4-1BB was a good predictive indicator of response to NAC, while elevation of sTIM-3 levels was shown to be associated with the risk of locoregional recurrence and distant metastases. Both TIM-3 and 4-1BB appear to be potential therapeutic targets in locally advanced TNBC because they were associated with the clinical outcome of the disease

Comparison of the phenolic contents and epigenetic and genetic variability of wild and cultivated watercress (Rorippa nasturtium var. aquaticum L.)

Background: Epigenetic modifications are key factors modulating the expression of genes involved in the synthesis of phytochemicals. The knowledge of plant epigenetic and genetic variations can contribute to enhance the production of bioactive compounds. These issues have been little explored thus far in Rorippa nasturtium var. aquaticum L. (watercress), an edible and medicinal plant. The aim of the current study was to determine and compare the phenolic composition and epigenetic and genetic variations between wild and cultivated watercress. Results: Significant differences were found in the quantitative phenolic composition between wild and cultivated watercress. The eight primer combinations used in the methylation-sensitive amplification polymorphism (MSAP) method revealed different epigenetic status for each watercress type, the cultivated one being the most epigenetically variable. The genetic variability revealed by the EcoRI/MspI amplification profile and also by eight inter-simple sequence repeat (ISSR) primers was different between the two types of watercress. The results of the Mantel test showed that the correlation between genetic and epigenetic variations has diminished in the cultivated type. Cluster analyses showed that the epigenetic and genetic characterizations clearly discriminated between wild and cultivated watercress. Conclusions: Relevant chemical, epigenetic, and genetic differences have emerged between wild and cultivated watercress. These differences can contribute to fingerprint and develop quality control tools for the integral and safety use and the commercialization of watercress. The richness of epialleles could support the development of tools to manipulate the watercress epigenome to develop high bioproduct­producing cultivars

El caseinato de sodio incrementa número de linfocitos B en ratones / Sodium caseinate increases the number of B lymphocytes in mouse

Introducción. El caseinato de sodio, una sal de la caseína utilizada como agente proinflamatorio en ratones, es capaz de inducir granulopoyesis en vivo e incrementar la producción de citocinas esenciales en dicho evento. Objetivo. Evaluar si el caseinato de sodio es capaz de inducir un efecto biológico en células de origen linfoide y la producción de citocinas involucradas con este linaje. Materiales y métodos: Se utilizaron ratones hembra BALB/c de 8 a 12 semanas de edad. Los animales se inyectaron cuatro veces, con intervalos de 48 horas, por vía intraperitoneal con 1 ml de caseinato de sodio (10 % de SFB p/v). La población de linfocitos B y la incorporación de bromodesoxiuridina (BrdU) se analizaron mediante citometría de flujo. La detección de la interleucina 7 se evaluó mediante la técnica de ELISA. Resultados. Tras la inyección por vía intraperitoneal, el número de linfocitos B 220+ provenientes del bazo de ratones tratados con caseinato de sodio aumentó comparados con los que solo recibieron el vehículo como tratamiento (89,01±1,03 Vs. 75,66±2,08), así como la incorporación de BrdU en células B220+ (38,59±4,48 Vs. 11,82±1,04). Se evidenció, asimismo, el incremento en la concentración de la interleucina 7 (IL-7) en el suero de los ratones tratados con caseinato de sodio, comparados con los que solo recibieron el vehículo (62,1±17,5 Vs. 26,9±4,4 pg/ml). Conclusión. El caseinato de sodio fue capaz de aumentar el número de linfocitos B en bazo de ratones, así como inducir la producción de IL-7, citocina clave para la linfopoyesis B.

Introduction: Sodium caseinate, a casein salt, is a proinflammatory agent in mice, and it is able to induce granulopoiesis in vivo and to increase the production of cytokines, which is key for this biological process. Objective: To assess whether sodium caseinate is able to induce a biological effect on cells from lymphoid origin and the production of cytokines involved in this lineage in vivo. Materials and methods: We used female BALB /c mice from 8 to 12 weeks old. The animals were injected intraperitoneally (IP) with 1 ml of sodium caseinate (10% PBS w/v) four times every 48 hours. The B cell populations and the incorporation of BrdU were analyzed by flow cytometry. Detection of interleukin-7 was assessed by ELISA (Enzyme-Linked ImmunoSorbent Assay). Results: We established that after intraperitoneal injection, the number of B lymphocytes 220+ from the spleen of mice treated with sodium caseinate increased compared to those that only received the vehicle (89.01±1.03 vs 75.66 ± 2.08), and the same was observed with the incorporation of BrdU in B220 + cells (38.59±4.48 vs 11.82±1.04 respectively). We also established that the concentration of interleukin-7 (IL-7) in the serum of mice treated with sodium caseinate increased compared to those that only received the vehicle (62.1 ± 17.5 vs 26.9 ± 4.4 pg/ml). Conclusion: Sodium caseinate was able to increase the number of B lymphocytes in the spleen; it also induced IL-7 production, a cytokine that is key for the B cell lymphopoiesis.

Rabdomiólisis grave secundaria a deshidratación hipernatrémica / Severe rhabdomyolysis secondary to severe hypernatraemic dehydration

Introducción: La rabdomiólisis es una enfermedad poco frecuente en pediatría. El objetivo es presentar un paciente en el que se desarrolló secundario a una deshidratación hipernatrémica grave tras una diarrea aguda. Caso clínico: Lactante de 11 meses que consultó por fiebre, vómitos, diarrea y anuria. Presentó convulsión tónico-clónica autolimitada. Ingresó en mal estado general, severamente deshidratado, con escasa reactividad. En las pruebas complementarias destacó acidosis metabólica grave, hipernatremia e insuficiencia renal prerrenal. Al tercer día apreció leve hipotonía axial y elevación de creatín fosfokinasa 75.076 UI/l, interpretado como rabdomiólisis. Se inició hiperhidratación y alcalinización sistémica, con buena respuesta clínica y bioquímica, siendo dado de alta sin secuelas motoras. Conclusiones: La hipernatremia grave está descrita como causa rara de rabdomiólisis e insuficiencia renal. En pacientes críticos es importante un alto índice de sospecha de rabdomiólisis y determinación seriada de la creatín fosfokinasa para su detección y tratamiento precoz.

Introduction: Rhabdomyolysis is a rare paediatric condition. The case is presented of a patient in whom this developed secondary to severe hypernatraemic dehydration following acute diarrhoea. Case report: Infant 11 months of age who presented with vomiting, fever, diarrhoea and anuria for 15 hours. Parents reported adequate preparation of artificial formula and oral rehydration solution. He was admitted with malaise, severe dehydration signs and symptoms, cyanosis, and low reactivity. The laboratory tests highlighted severe metabolic acidosis, hypernatraemia and pre-renal kidney failure (Sodium [Na] plasma 181 mEq/L, urine density> 1030). He was managed in Intensive Care Unit with gradual clinical and renal function improvement. On the third day, slight axial hypotonia and elevated cell lysis enzymes (creatine phosphokinase 75,076 IU/L) were observed, interpreted as rhabdomyolysis. He was treated with intravenous rehydration up to 1.5 times the basal requirements, and he showed a good clinical and biochemical response, being discharged 12 days after admission without motor sequelae. Conclusions: Severe hypernatraemia is described as a rare cause of rhabdomyolysis and renal failure. In critically ill patients, it is important to have a high index of suspicion for rhabdomyolysis and performing serial determinations of creatine phosphokinase for early detection and treatment.

Insulin-like growth factor 1 gene (CA)n repeats and a variable number of tandem repeats of the insulin gene in Brazilian children born small for gestational age

OBJECTIVE: To investigate the influence of (CA)n repeats in the insulin-like growth factor 1 gene and a variable number of tandem repeats of the insulin gene on birth size in children who are small or adequate-sized for gestational age and to correlate these polymorphisms with serum insulin-like growth factor 1 levels and insulin sensitivity in children who are small for gestational age, with and without catch-up growth. PATIENTS AND METHODS: We evaluated 439 infants: 297 that were adequate-sized for gestational age and 142 that were small for gestational age (66 with and 76 without catch-up). The number of (CA)n repeat in the insulin-like growth factor 1 gene and a variable number of tandem repeats in the insulin gene were analyzed using GENESCAN software and polymerase chain reaction followed by enzymatic digestion, respectively. Clinical and laboratory data were obtained from all patients. RESULTS: The height, body mass index, paternal height, target height and insulin-like growth factor 1 serum levels were higher in children who were small for gestational age with catch-up. There was no difference in the allelic and genotypic distributions of both polymorphisms between the adequate-sized and small infants or among small infants with and without catch-up. Similarly, the polymorphisms were not associated with clinical or laboratory variables. CONCLUSION: Polymorphisms of the (CA)n repeats of the insulin-like growth factor 1 gene and a variable number of tandem repeats of the insulin gene, separately or in combination, did not influence pre- or postnatal growth, insulin-like growth factor 1 serum levels or insulin resistance. .

Participação de fatores de crescimento e de citosinas nas alterações observadas na prole de ratas tratadas com o veneno do escorpião Tityus Bahiensis no período gestacional / Participation of growth factors and citokynes in alterations observed in offspring of rats treated with Tityus bahiensis scorpion venom during the pregnancy

No Brasil, o escorpionismo é um problema de saúde pública. O escorpião T. serrulatus é considerado o mais perigoso, mas um grande número de acidentes também acontece com o T. bahiensis. O objetivo do estudo foi verificar possíveis efeitos do veneno do escorpião T. bahiensis no desempenho reprodutivo materno e nos níveis de citocinas e fatores de crescimento em embriões de mães tratadas durante a gestação. Para os parâmetros reprodutivos foram utilizadas fêmeas prenhes injetadas com uma dose de 2,5mg/Kg (s.c.) do veneno no 5º (GD5) ou no 10º (GD10) dia gestacional. O grupo controle foi injetado com salina a 1,46% (1ml/Kg) em ambos os dias. No 21º dia gestacional, os filhotes foram retirados por laparotomia e divididos em dois grupos que receberam tratamento específico para a análise visceral e esquelética. Para avaliação dos níveis de citocinas e de fatores de crescimento, as fêmeas prenhes foram injetadas com salina (1ml/Kg), LPS (100μg/kg) ou veneno (2,5mg/Kg) no 10º (GD10) ou 16º (GD16) dia gestacional. Os filhotes foram removidos por laparotomia 6, 12 ou 24 horas após o tratamento materno. As amostras foram maceradas em um homogenizador de tecido e centrifugadas. Os níveis de citocinas e fatores de crescimento foram determinados por ensaios imunoenzimáticos. Nos parâmetros reprodutivos não houve alterações no peso materno durante a gestação, no número de corpos lúteos, número de filhotes, peso do útero e dos filhotes. Houve diminuição no número de implantações e reabsorções no grupo GD5. Houve aumento no peso das placentas em GD5 e GD10. No desenvolvimento dos filhotes foram observadas aumento no peso do coração e pulmão em GD5 e GD10 e no peso do fígado em GD10. Não foram observadas anomalias e malformações internas ou externas nos filhotes de ambos os grupos experimentais. Em GD10 não foram observadas alterações nos níveis de citocinas 6 horas após a aplicação do veneno, mas verificou-se diminuição do nível de INF-γ 24 horas depois...

In Brazil scorpionism is a public health problem. The scorpion T. serrulatus is considered the most dangerous, but a large number of accidents also occur with T. bahiensis. The objective of this work was to verify the possible effects of the T. bahiensis scorpion venom on the maternal reproductive parameters and on the cytokines levels and growth factors in embryos after the treatment of pregnancy females. To the reproductive parameters it was used pregnant females injected with a dose of 2.5mg/Kg (s.c.) of the venom. The experimental groups were injected with venom on the 5th (GD5) or on the 10th (GD10) gestational day. The control group was injected with NaCl 1.46% on both days. On the 21st gestational day, the pups were taken out by laparotomy and were divided into two groups that received specific treatments for skeletal or visceral analyses. To evaluate the cytokines levels, pregnant females were injected with saline (1ml/kg), LPS (100μg/kg) or crude venom (2.5mg/kg) on the 10th (GD10) or 16th (GD16) gestational day. The pups were removed by laparotomy 6, 12 or 24 hours after the mother’s treatment. The samples (embryo/placenta) were macerated by a tissue homogenizer and centrifuged. The cytokine levels were determined by enzyme immunoassays. In the reproductive parameters no changes were observed in the maternal weight during the gestational period, corpora lutea, number of pups, uterus weight and pups weight. There was a decrease on the number of implantation and resorption in GD5 There was alteration on the placenta’s weight in GD5 and GD10. In pups development there were observed alterations in the heart and lung weight on GD5 and GD10 and on the liver weight on GD10. There were not observed external or internal anomalies and malformations in the offspring of both experimental groups. The cytokines levels were not alterated after 6 hours in GD10. In GD16 there was an increase in the IL-1α levels...

Methylation-specific PCR analysis in Col8A1 promoter in Creole cattle carrier of rob(1; 29)

Robertsonian translocation (rob(1;29)) is the most frequent structural chromosomal abnormality in cattle. Heterozygous carriers have a normal phenotype but show a 3-5 percent decrease in fertility. Chromatin decondensation was evaluated similar to the inactive X chromosome when submitted to demethylating agent. Based on this result, and the concept that imprinted genes are essential in embryonic development, we decided to query genes located on BTA1 and BTA29 that could undergo genome imprinting. The collagen typeVIII- alpha 1 (Col8A1) acted on extracellular matrix structural proteins. DNA bisulfite conversion and sequentiation methods were used to compare its differential methylation patterns. It was performed on eight Creole cattle DNA blood samples from normal and rob(1;29) carriers. An in silico screening for CpG islands in its promoter uncovered a single region of 454 bp prone to methylation. BiQ-Analizer software was used to show the selective conversion of unmethylated cytosines to uracils obtaining the following results: unmethylated CpGs: 0.000 (0 cases), methylated CpGs: 0.802 (77 cases) and CpGs not present: 0.198 (19 cases). No differences between samples were observed in this highly methylated region. This technique was successfully applied so it is a straightforward methodology that can be utilized to evaluate different tissue associated to specific gene expression.

Hypomethylation of tumor suppressor genes in odontogenic myxoma

Odontogenic myxoma (OM) is an ectomesenchymal benign odontogenic tumor characterized by spindle or stellate-shaped cells embedded in an abundant myxoid or mucoid extracellular matrix. DNA methylation is characterized by the addition of methyl groups in cytosines within CpG islands in the promoter gene. DNA methylation can decrease the expression of tumor suppressor genes and contribute to the development of neoplastic lesions. The aim of study was to evaluate the methylation pattern of the tumor suppressor genes P16 (CDKN2A), P21 (CDKN1A), P27 (CDKN1B), P53 (TP53) and RB1 in OM and dental pulp. Methylation was evaluated using methylation-specific polymerase chain reaction (PCR). The transcription was studied in some cases by using reverse transcription quantitative PCR. A higher frequency of unmethylated P27, P53, and RB1 samples was observed in the OM when compared with the dental pulp. OM expressed mRNA of all the genes evaluated. Considering all the samples together, the expression of Rb was higher in the unmethylated samples compared with the partially methylated samples. This investigation revealed hypomethylation of the genes P27, P53, and RB1 in OM. In addition, methylation of tumor suppressor genes was found to be an usual event in normal dental pulp.

O mixoma odontogênico (MO) é um tumor odontogênico benigno de origem mesenquimal caracterizado pela presença de células fusiformes ou estreladas dispostas em abundante matriz extracelular mucóide. A metilação do DNA é caracterizada pela adição de grupos metil em citosinas constituintes de ilhas CpG na região promotora do gene. A metilação pode diminuir a expressão de genes supressores de tumor e contribuir para o desenvolvimento de lesões neoplásicas. O objetivo deste trabalho foi avaliar o padrão de metilação nos genes P16 (CDKN2A), P21 (CDKN1A), P27 (CDKN1B), P53 (TP53), RB1 nos MO e na polpa dental. A metilação foi avaliada pela reação em cadeia da polimerase específica para a metilação. A transcrição dos genes foi estudada em alguns casos pela reação da transcriptase reversa (PCR quantitativa). Uma maior frequência de amostras não metiladas para os genes P27, P53 e RB1 foi observada nos MO quando comparados à polpa dental. Os MO expressaram RNAm de todos os genes avaliados. Considerando todas as amostras juntas, a expressão de Rb foi maior em amostras não metiladas comparadas as amostras parcialmente metiladas. Esta investigação mostrou a hipometilação dos genes P27, P53 e RB1 nos MO. Adicionalmente, a metilação nos genes supressores de tumor é um evento frequente em polpa dental normal.

Avaliação da eficácia do cidofovir na papilomatose respiratória recorrente juvenil / Efficacy of cidofovir in recurrent juvenile respiratory papillomatosis

O uso do cidofovir para papilomatose respiratória recorrente juvenil (PRRJ) ainda não tem estudos caso-controle suficientes que comprovem sua eficácia em literatura. OBJETIVO: Avaliar fatores que influenciem o prognóstico da PRRJ, e observar a atuação do cidofovir na erradicação da PRRJ. DESENHO DO ESTUDO: Retrospectivo. MATERIAIS E MÉTODOS: 22 crianças com PRRJ foram avaliadas num centro terciário. Todas as crianças foram submetidas ao tratamento cirúrgico, seguido (Grupo 2) ou não (Grupo 1) pelo uso do cidofovir. A idade ao diagnóstico foi correlacionada ao escore de Derkay e à evolução da doença. Os Grupos 1 e 2 tiveram suas evoluções comparadas entre si. RESULTADOS: Quinze crianças foram consideradas curadas, 8 no Grupo 2 e 7 no Grupo 1. Houve uma correlação negativa entre idade e Escores Total e Clínico (P<0,05). O número médio de cirurgias necessárias para controlar a doença foi semelhante entre os Grupos, mas a duração do tratamento até remissão foi significativamente maior no Grupo 1 quando comparado ao Grupo 2 (P<0,05). CONCLUSÕES: A PRRJ é mais agressiva quanto mais nova a idade do paciente ao diagnóstico. Pacientes tratados com cidofovir apresentaram duração significativamente menor de tratamento até erradicação da PRRJ do que os submetidos apenas ao tratamento cirúrgico.

The efficacy of cidofovir in juvenile recurrent respiratory papillomatosis (JRRP) remains uncertain due to the lack of published case-control studies. AIM: To establish factors affecting the progression of JRRP prognosis, and to evaluate cidofovir for eradicating JRRP. STUDY DESIGN: Retrospective. METHODS: 22 children with JRRP were evaluated at a referral center. All children underwent surgical debulking, followed by cidofovir injection (Group 2) or not (Group 1). Age at diagnosis was correlated with the Derkay score and disease outcome. Disease progression was compared between groups 1 and 2. RESULTS: fifteen children were considered disease-free; 8 were in Group 2 and 7 in Group 1. Age and total and clinical scores (P<0.05) were negatively correlated. The mean number of surgeries required to control the disease was identical in both groups; the duration of treatment until remission was significantly higher in Group 1 (P<0,05). CONCLUSION: JRRP is more aggressive in earlier onset disease. The duration of treatment was significantly lower in patients treated with cidofovir until eradication of JRRP compared to patients treated with surgery only.

Hydrogen peroxide induces a specific DNA base change profile in the presence of the iron chelator 2, 2’ dipyridyl in Escherichia coli

Pretreatment of Escherichia coli cultures with the iron chelator 2,2’-dipyridyl (1 mM) protects against the lethal effects of low concentrations of hydrogen peroxide (<15 mM). However, at H2O2 concentrations equal to or greater than 15 mM, dipyridyl pretreatment increases lethality and mutagenesis, which is attributed to the formation of different types of DNA lesions. We show here that pretreatment with dipyridyl (1 mM) prior to challenge with high H2O2 concentrations (≥15 mM) induced mainly G:C→A:T transitions (more than 100X with 15 mM and more than 250X with 20 mM over the spontaneous mutagenesis rate) in E. coli. In contrast, high H2O2 concentrations in the absence of dipyridyl preferentially induced A:T→T:A transversions (more than 1800X and more than 300X over spontaneous mutagenesis for 15 and 20 mM, respectively). We also show that in the fpg nth double mutant, the rpoB gene mutation (RifS-RifR) induced by 20 mM H2O2 alone (20X higher) was increased in 20 mM H2O2 and dipyridyl-treated cultures (110X higher), suggesting additional and/or different lesions in cells treated with H2O2 under iron deprivation. It is suggested that, upon iron deprivation, cytosine may be the main damaged base and the origin of the pre-mutagenic lesions induced by H2O2.

Aplicação local de cidofovir como tratamento adjuvante na papilomatose laríngea recorrente em crianças / Local application of cidofovir as adjuvant therapy in recurrent laryngeal papillomatosis in children

OBJETIVO: Avaliar a eficácia da aplicação local de cidofovir em associação com o tratamento cirúrgico na papilomatose laríngea recorrente (PLR) em crianças. Desenho do estudo: Prospectivo. MÉTODOS: Quatorze pacientes, com idade média de 4.7 anos e com duas ou mais recidivas após tratamento cirúrgico, foram submetidos à ressecção dos papilomas e injeção de 22.5 mg de cidofovir (7,5 mg/ml) no tecido de onde as lesões foram removidas. Após intervalos de 2-3 semanas, a mesma dose de cidofovir foi repetida duas ou três vezes. Em caso de recidiva, um novo ciclo de cirurgia seguido de aplicações locais de cidofovir era reiniciado. Cinco crianças apresentavam HPV-6 e cinco HPV-11; em quatro casos a tipagem não foi realizada. RESULTADOS: Antes do início do estudo, os pacientes eram submetidos, em média, a duas cirurgias por ano para o controle das recidivas; após o tratamento com cidofovir, a taxa anual de cirurgia diminuiu para 1,1 (p = 0,013). O intervalo médio entre as recidivas antes do início do estudo era de 1.6 meses; ao final do estudo, o intervalo aumentou para 4,4 meses (p = 0,014). Os pacientes com HPV-6 não apresentaram alteração significante nos intervalos entre as recidivas após o tratamento com cidofovir, enquanto 60 por cento das crianças com HPV-11 encontravam-se livres de doença ao final do estudo. CONCLUSÃO: O cidofovir é um adjuvante eficaz no tratamento da PLR em crianças, quando utilizado sob a forma de aplicações locais em associação com a ressecção cirúrgica das lesões. O HPV-11 pode ser mais susceptível aos efeitos benéficos do cidofovir.

OBJECTIVE: Evaluate the efficacy of local application of cidofovir in association with surgical treatment of recurrent laryngeal papillomatosis in children. Study design: Prospective. METHODS: Fourteen patients, with an average age of 4.7 years and with two or more relapses after surgical treatment, were submitted to resection of the papillomas and injection of 22.5 mg of cidofovir (7.5 mg/ml) in the tissue where the lesions had been removed. After 2 to 3 week intervals, the same dose of cidofovir was repeated two or three times. In the case of relapse, a new cycle of surgery followed by local applications of cidofovir was repeated. Five children presented HPV-6 and five HPV-11, while in four, the type was not determined. RESULTS: Before beginning of the study, patients were submitted, on the average, to 2 operations a year for control of relapses. After treatment with cidofovir, the annual rate for surgery dropped to 1.1 (p = 0.013). The average interval between relapses before beginning of the study was 1.4 months; at the end of the study, the interval reached 4.4 months (p = 0.014). Patients with HPV-6 did not show a significant change in the intervals between relapses after treatment with cidofovir, while 60 percent of the children with HPV-11 were disease free at the study end. CONCLUSION: Cidofovir was found to be an effective adjuvant in the treatment of recurrent laryngeal papillomatosis in children, when used in the form of local applications in association with surgical resection of the lesions. HPV-11 may be more susceptible to the beneficial effects of cidofovir.

Melanina: implicaciones en la patogénesis de algunas enfermedades y su capacidad de evadir la respuesta inmune del hospedero / Melanin: implications in some disease pathogenesis and its capacity to evade the host immune response

La melanina es uno de los pigmentos más comunes y de mayor distribución en la naturaleza. Es responsable de la coloración de plantas y animales; se encuentra en los ojos, el cabello, la piel, el plumaje, la cáscara de los huevos, la cutícula de los insectos, la tinta de los cefalópodos y en la pared y el citoplasma de muchos microorganismos. En los humanos este pigmento se ha encontrado también fuera de la piel, en las neuronas de la sustancia nigra y en los hepatocitos. Entre los microorganismos que se han reportado como productores de melanina tenemos Vibrio cholerae, Mycobacterium leprae, Bacillus thurigiensis, Pseudomonas aeruginosa, Schistosoma mansoni, Fasciola gigantita Trichuris suis, Alternaria alternata, Aspergillus niger, Blastomyces dermatitidis, Candida albicans, Cladosporium carionii, Coccidioides immitis, Cryptococcus neoformans, Exophiala (Wangiella) dermatitidis, Fonsecaea pedrosoi, Histoplasma capsulatum, Paracoccidioides brasiliensis, Penicillium marneffei, Pneumocystis carinii (jirovecii), Scedosporium prolificans, Scytalidium dimidiatum y Sporothrix schenckii; esto sin tener en cuenta los hongos dematiáceos, entre muchos otros. Esta revisión pretende hacer un compendio de las más recientes publicaciones sobre melanina relacionadas principalmente con su función, su importante contribución a la supervivencia en el ambiente y durante la infección, como factor de virulencia en diversos microorganismos, principalmente en hongos patógenos, y su papel como agente inmunomodulador, así como la reducida susceptibilidad que confiere contra muchos de los antimicóticos usados en la actualidad.

Melanin is one of the common pigments in nature; it is responsible for pigmentation in plants and animals. It is found in skin, eyes, feathers, egg shell, hair, insect cuticle, cuttlefish ink and wall and/or cytoplasm from many microorganisms. Melanin in humans is also present in substantia nigra and hepatocytes. Some microorganisms that have been reported producing melanin are: Vibrio cholerae, Mycobacterium leprae, Bacillus thurigiensis, Pseudomonas aeruginosa, Schistosoma mansoni, Fasciola gigantita, Trichuris suis, Alternaria alternata, Aspergillus niger, Blastomyces dermatitidis, Candida albicans, Cladosporium carionii, Coccidioides immitis, Cryptococcus neoformans, Exophiala (Wangiella) dermatitidis, Fonsecaea pedrosoi, Histoplasma capsulatum, Paracoccidioides brasiliensis, Penicillium marneffei, Pneumocystis carinii (jirovecii), Scedosporium prolificans, Scytalidium dimidiatum, Sporothrix schenckii, and most of the dematiaceous fungi. This review is focused on recent international publications concerning melanin analysing its capacity to survive in nature and during infection inside the host and its evasion of the immune response. Melanin acts as an inmunomodulador particle and it is known that its presence in many of microorganisms could protect them from microbicidal agents presently used.

Uso de cidofovir en la papilomatosis respiratoria recurrente / Use of cidofovir in recurrent respiratory papillomatosis

La Papilomatosis Respiratoria Recurrente (PRR) es una enfermedad causada por el virus papiloma humano (VPH) que se caracteriza por la presencia de tumores epiteliales en la vía aérea. Su principal mecanismo de contagio es por contacto directo, la zona más frecuentemente afectada es la laringe, pero puede comprometer cualquier lugar de la vía aéreo-digestiva. Sus manifestaciones van desde la disfonía a la obstrucción completa de la vía aérea, causando incluso la muerte. En algunas ocasiones los papilomas pueden malignizarse y transformarse en carcinoma epidermoide. El tratamiento existente es sólo paliativo y consiste en la excisión quirúrgica de los papilomas para mantener la vía aérea sin obstrucción y mejorar la calidad de la voz, pero tiene una alta tasa de recidiva y habitualmente se requieren múltiples intervenciones quirúrgicas, resultando un tratamiento de alto costo. En los últimos tiempos se ha usado el cidofovir, un nucleósido fosfanato acíclico, administrado en forma intralesional para el tratamiento de la papilomatosis laríngea obteniéndose promisorios resultados, observándose remisión completa en un alto porcentaje de los casos y disminución de las recidivas.

Recurrent respiratory papillomatosis (RRP) is a disease caused by infection with the human papillomavirus (HPV), and is characterized by the presence of epithelial tumors in the airway. It is most commonly spread by direct contact, and it usually affects the larynx, but it may involve any region of the airway. Its symptoms vary from dysphonia to complete obstruction of the airway, and it may even cause death. in some cases, papillomas can become malignant and transform into epidermoid carcinoma. Current treatments are only palliative, and consist of the papillomas surgical resection in order to maintain the airway unobstructed and to improve voice quality; but it has a high recurrency rate, and usually several surgical procedures are needed, resulting in a high-cost treatment. Recently, cidofovir, an acyclic nucleoside phosphonate, has been used for the intralesional treatment of laryngeal papillomatosis with promising results, including complete remission in a high percentage of cases, and decreased recurrence.

Influência da pentoxifilina nos níveis citosinas e parâmetros inflamatórios no modelo de endotoxemia aguda / Influence of pentoxifylline on cytokine levels and inflammatory parameters in acute endotoxemia model

A produção excessiva de espécies reativas do oxigênio pelos polimorfonucleares está associada a dano tecidual durante inflamação. A pentoxifilina, um inibidor da fosfodiesterase, diminui a produção de citosinas pró-inflamatórias e o dano tecidual induzido pelo lipopolissacarídeo (LPS)...

Excessive production of reactive oxygen species by PMN is associated with tissue damage during inflammation. Pentoxifylline, a phosphodiesterase inhibitor, decreases proinflammatory cytokine production and tissue injury after LPS challenge...

Citosinas sericas (IL-4, IL-6 e IFN-y) en embarazadas de la ciudad de Valencia Venezuela / Serum cytokines (IL-4, IL-6 e IFN-y) in pregnant women from Valencia city

A fin de evaluar el comportamiento de algunas citosinas o interleukinas (IL-4, IL-6, IFN-y) durante el embarazo, seestudiaron 25 mujeres embarazadas durante los tres trimestres, 12 adolescentes y 13 adultas. Las determinaciones de las interleukinas se realizaron por el método de ELISA, tipo sándwich, observándose incremento significativo (p<0,05) para la IL-4 entre el 1er y 2do trimestre (8,18±2,43 vs 53,42±17,25 pg/ml) y entre el 1er y el 3er (8,18±2,43 vs 42,47 ± 6,27 pg/ml) pero no entre el 2do y el 3er (53,42 ± 17,25 vs 42,47 ± 6,27 pg/ml) para todas las embarazadas, con un patrón similar en las adolescentes y adultas, pero la comparación entre ellas por edad sólo mostró diferencia significativa para la evaluación del 2do trimestre, correspondiendo a las adultas niveles más altos (60,53± 17,47 vs 45,71±13,85 pg/ml). Con relación a la IL-6, se observó que los valores se incrementan a medida que avanza el embarazo con diferencia significativa (p<0,05) entre los trimestres (2,01±3,46; 9,25±7,22; 11,54±9,40 pg/ml) pero similar para ambos grupos etarios sin diferencia estadística significativa entre ellos en ninguno de los periodos. Para el IFN-y se observó un ligero aumento para todas las embarazadas que no alcanzó diferencia significativa entre los trimestres ni para los grupos etarios considerados. Se concluye que el embarazo induce aumentos significativos de IL-4, de IL-6 y en menor grado de IFN-y y que estos cambios no están asociados a la edad de la embarazada.

In order to assess interleukin levels (IL-4, IL-6, IFN-y) during pregnancy, 25 pregnant women (12 adolescents and13 adults) along the three trimesters were evaluated. An ELISA test was performed to determine serum interleukin. A significant increase (p<0.05) for IL-4 between first and second trimester (8.18±2.43 vs. 53.42±17.25 pg/ml) and between first and third (8.18±2.43 vs. 42.47 ± 6.27 pg/ml) but not between second and third (53.42 ± 17.25 vs. 42.47 ± 6.27 pg/ml) was observed. Similar pattern of increase was found for adolescents and adults, but comparison by age showed significantly higher levels in adults at second trimester (60.53± 17.47 vs. 45.71±13.85 pg/ml). Regarding IL-6, increase during pregnancy was also observed, reaching significant differences between trimesters (2.01±3.46; 9.25±7.22; 11.54±9.40 pg/ml): A similar pattern was found for adolescents and adults, but no differences by age were found at any moment. For IFN-y, a slight increase was observed but it did not reach significant differences either between trimesters or by age groups. It is concluded that pregnancy induced significant increases of IL-4 and IL-6, and in a lesser degree for IFN-y but age of the women was not related to the changes.

Endoftalmitis candidiásica: presentación de dos casos / Candida endophthalmitis: report of two cases

Se denomina endoftalmitis a un proceso inflamatorio que afecta a la cavidad ocular y a las estructuras adyacentes. En su etiología intervienen agentes infecciosos (hongos, virus, bacterias, protozoos y parásitos), neoplasias, sangre intraocular y material retenido de lentes, luego de la cirugía de cataratas. Se presentan dos casos de endoftalmitis candidiásica que afectaron a dos pacientes jóvenes inmunocompetentes. En los dos casos se realizó tratamiento con anfotericina B y 5 fluorcitosina. A uno de ellos se le realizó una vitrectomía bilateral, quedando con una secuela visual permanente. Sólo el diagnóstico precoz y el tratamiento oportuno y efectivo reducen las posibilidades de lesiones retinianas permanentes en estos pacientes