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1.
Mol Hum Reprod ; 25(10): 614-624, 2019 10 28.
Artículo en Inglés | MEDLINE | ID: mdl-31323669

RESUMEN

Antral follicle size might be a valuable additive predictive marker for IVF outcome. To better understand consequences of antral follicle size as a marker for reproductive outcome, we aimed to obtain insight in follicle size-related granulosa cell processes, as granulosa cells play an essential role in follicular development via the production of growth factors, steroids and metabolic intermediates. Using the pig as a model, we compared gene expression in granulosa cells of smaller and larger follicles in the healthy antral follicle pool of sows, which had a high variation versus low variation in follicle size. Selected gene expression was confirmed at the protein level. Granulosa cells of smaller antral follicles showed increased cell proliferation, which was accompanied by a metabolic shift towards aerobic glycolysis (i.e. the Warburg effect), similar to other highly proliferating cells. High granulosa cell proliferation rates in smaller follicles might be regulated via increased granulosa cell expression of the androgen receptor and the epidermal growth factor receptor, which are activated in response to locally produced mitogens. While granulosa cells of smaller follicles in the pool are more proliferative, granulosa cells of larger follicles express more maturation markers such as insulin-like growth factor-1 (IGF1) and angiopoietin 1 (ANGPT1) and are therefore more differentiated. As both higher IGF1 and ANGPT1 have been associated with better IVF outcomes, the results of our study imply that including smaller follicles for oocyte aspiration might have negative consequences for IVF outcome.


Asunto(s)
Procesos de Crecimiento Celular/genética , Células de la Granulosa/citología , Células de la Granulosa/fisiología , Folículo Ovárico/citología , Folículo Ovárico/crecimiento & desarrollo , Ovario/citología , Animales , Diferenciación Celular/genética , Tamaño de la Célula , Femenino , Perfilación de la Expresión Génica , Folículo Ovárico/fisiología , Ovario/crecimiento & desarrollo , Ovario/metabolismo , Porcinos , Transcriptoma
2.
Genes Nutr ; 9(1): 366, 2014 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-24343050

RESUMEN

Moderate maternal calorie restriction during lactation protects rat offspring against obesity development in adulthood, due to an improved ability to handle and store excess dietary fuel. We used this model to identify early transcriptome-based biomarkers of metabolic health using peripheral blood mononuclear cells (PBMCs), an easily accessible surrogate tissue, by focusing on molecular markers of lipid handling. Male and female offspring of control and 20 % calorie-restricted lactating dams (CR) were studied. At weaning, a set of pups was killed, and PBMCs were isolated for whole-genome microarray analysis. The remaining pups were killed at 6 months of age. CR gave lower body weight, food intake and fat accumulation, and improved levels of insulin and leptin throughout life, particularly in females. Microarray analysis of weaned rat PBMCs identified 278 genes significantly differentially expressed between control and CR. Among lipid metabolism-related genes, expression of Cpt1a, Lipe and Star was increased and Fasn, Lrp1 and Rxrb decreased in CR versus control, with changes fully confirmed by qPCR. Among them, Cpt1a, Fasn and Star emerged as particularly interesting. Transcript levels of Cpt1a in PBMCs correlated with their levels in WAT and liver at both ages examined; Fasn expression levels in PBMCs at an early age correlated with their expression levels in WAT; and early changes in Star expression levels in PBMCs correlated with their expression levels in liver and were sustained in adulthood. These findings reveal the possibility of using transcript levels of lipid metabolism-related genes in PBMCs as early biomarkers of metabolic health status.

3.
Bioconjug Chem ; 24(12): 1971-89, 2013 Dec 18.
Artículo en Inglés | MEDLINE | ID: mdl-24200346

RESUMEN

Bacterial infections have always been, and still are, a major global healthcare problem. For accurate treatment it is of upmost importance that the location(s), severity, type of bacteria, and therapeutic response can be accurately staged. Similar to the recent successes in oncology, tracers specific for molecular imaging of the disease may help advance patient management. Chemical design and bacterial targeting mechanisms are the basis for the specificity of such tracers. The aim of this review is to provide a comprehensive overview of the molecular imaging tracers developed for optical and nuclear identification of bacteria and bacterial infections. Hereby we envision that such tracers can be used to diagnose infections and aid their clinical management. From these compounds we have set out to identify promising targeting mechanisms and select the most promising candidates for further development.


Asunto(s)
Infecciones Bacterianas/diagnóstico , Imagen Molecular/métodos , Trazadores Radiactivos , Animales , Bacterias/citología , Bacterias/efectos de los fármacos , Bacterias/metabolismo , Bacterias/virología , Infecciones Bacterianas/tratamiento farmacológico , Humanos , Marcaje Isotópico
4.
Obes Rev ; 12(8): 637-52, 2011 Aug.
Artículo en Inglés | MEDLINE | ID: mdl-21426479

RESUMEN

Excess weight in early life is believed to increase susceptibility to obesity, and in support of such theory, excess weight and fast weight gain in early childhood have been related to overweight later in life. The aim of this study was to review the literature on body size and growth in 0- to 4-year-old children and the association with body size at age 5-13 years. In total, 43 observational studies on body size and/or growth were included, of which 24 studies had been published in 2005 or later. Twenty-one studies considered body size at baseline, and 31 studies considered growth which all included assessment of weight gain. Eight (38%) studies on body size, and 15 (48%) on weight gain were evaluated as high-quality studies. Our results support conclusions in previous reviews of a positive association between body size and weight gain in early childhood, and subsequent body size. Body size at 5-6 months of age and later and weight gain at 0-2 years of age were consistently positively associated with high subsequent body size. Results in this review were mainly based on studies from developed Western countries, but seven studies from developing countries showed similar results to those from developed countries.


Asunto(s)
Tamaño Corporal , Aumento de Peso , Adolescente , Estudios de Casos y Controles , Causalidad , Niño , Preescolar , Estudios de Cohortes , Países en Desarrollo , Crecimiento , Humanos , Lactante , Recién Nacido , Obesidad/epidemiología , Sobrepeso/epidemiología
5.
Diabetologia ; 50(3): 654-65, 2007 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-17245590

RESUMEN

AIMS/HYPOTHESIS: The thiazolidinedione (TZD) rosiglitazone is a peroxisome proliferator-activated receptor-gamma agonist that induces adipocyte differentiation and, hence, lipid accumulation. This is in apparent contrast to the long-term glucose-lowering, insulin-sensitising effect of rosiglitazone. We tested whether the action of rosiglitazone involves specific effects on mature adipocytes, which are different from those on preadipocytes. MATERIALS AND METHODS: Differentiated mature 3T3-L1 adipocytes were used as an in vitro model. Transcriptomics, proteomics and assays of metabolism were applied to assess the effect of rosiglitazone in different insulin and glucose conditions. RESULTS: Rosiglitazone does not induce an increase, but rather a decrease in the lipid content of mature adipocytes. Analysis of transcriptome data, confirmed by quantitative RT-PCR and measurements of lipolysis, indicates that an altered energy metabolism may underlie this change. The pathway analysis shows a consistent picture dominated by lipid catabolism. In addition, we confirmed at both mRNA level and protein level that rosiglitazone represses adipokine expression and production, except for genes encoding adiponectin and apolipoprotein E. Moreover, transcriptome changes indicate that a general repression of genes encoding secreted proteins occurs. CONCLUSIONS/INTERPRETATION: Our findings suggest that the change of adiposity as seen in vivo reflects a shift in balance between the different effects of TZDs on preadipocytes and on mature adipocytes, while the changes in circulating adipokine levels primarily result from an effect on mature adipocytes.


Asunto(s)
Adipocitos/fisiología , Quimiocinas/metabolismo , Lípidos/fisiología , Tiazolidinedionas/farmacología , Células 3T3 , Adipocitos/efectos de los fármacos , Animales , Ciclo del Ácido Cítrico/efectos de los fármacos , Regulación de la Expresión Génica/efectos de los fármacos , Ratones , Análisis de Secuencia por Matrices de Oligonucleótidos , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Rosiglitazona
6.
Diabetologia ; 49(10): 2453-62, 2006 Oct.
Artículo en Inglés | MEDLINE | ID: mdl-16896944

RESUMEN

AIMS/HYPOTHESIS: Under conditions of insulin resistance and type 2 diabetes, fat cells are subjected to increased levels of insulin, which may have a major impact on the secretion of adipokines. MATERIALS AND METHODS: Using transcriptomics and proteomics, we investigated how insulin affects the transcription and protein secretion profile of mature 3T3-L1 adipocytes. RESULTS: We found that insulin has a significant impact on protein secretion of 3T3-L1 adipocytes. However, transcription is not the major regulation point for these secreted proteins. For extracellular matrix components, our data suggest that the mRNA level of processing enzymes, but not of target proteins, is the regulating point at which insulin stimulates secretion and function of the relevant proteins. Among these enzymes, we report a novel finding, namely that sulfatase 2 gene is regulated by insulin, which may induce a functional change in cultured adipocytes. CONCLUSIONS/INTERPRETATION: We propose that enhancement of protein processing and secretion rather than transcription of the secreted protein genes is part of the strategic role of insulin in the induction of cellular responses.


Asunto(s)
Adipocitos/fisiología , Regulación de la Expresión Génica , Insulina/farmacología , Proteínas/metabolismo , Transcripción Genética/efectos de los fármacos , Células 3T3 , Adipocitos/efectos de los fármacos , Animales , Electroforesis en Gel Bidimensional , Enzimas/genética , Glucosa/farmacología , Ratones , ARN Mensajero/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa
8.
Acta Paediatr ; 90(9): 1047-53, 2001 Sep.
Artículo en Inglés | MEDLINE | ID: mdl-11683194

RESUMEN

UNLABELLED: A prospective cohort study of breastfeeding practice (0-4 mo) was carried out in well-baby clinics. The cohort included 4438 newborns brought to a clinic for the first time between 1 April and 1 July 1998. The odds ratios of demographic and gestational variables connected with the start and duration of breastfeeding were measured. The frequency of the reasons why breastfeeding was interrupted was determined. At birth 71% of the infants included in this study were exclusively breastfed. After 4 mo the percentage had dropped to 21%. Breast milk was replaced directly by formula (23%) or by a mixture of breast milk and formula (77%). Exclusive breastfeeding was given irrespective of the mother's cultural background. Higher education appeared to be the most decisive factor for the initiation of exclusive breastfeeding; higher parity was found to be the most decisive factor for continuation. In 46% of cases the infant's health and behaviour caused mothers to stop exclusive breastfeeding; in 38% the reasons were mother related; in 17% both mother- and infant-related motives were mentioned. CONCLUSION: Mothers' perception of hunger and crying colics were the main infant-related reasons for cessation of breastfeeding, whereas physical problems, return to work, doubt about the sufficiency of breast milk and the feeling of being restricted by breastfeeding were the main mother-related reasons. The decision to abandon exclusive breastfeeding was made primarily by mothers (71%). In The Netherlands more babies are breastfed at birth than was the case 10 y ago, but the duration of the breastfeeding period has become shorter.


Asunto(s)
Lactancia Materna/psicología , Lactancia Materna/estadística & datos numéricos , Adolescente , Adulto , Escolaridad , Femenino , Humanos , Lactante , Recién Nacido , Masculino , Edad Materna , Persona de Mediana Edad , Países Bajos/epidemiología , Oportunidad Relativa , Estudios Prospectivos , Destete
9.
Cancer Epidemiol Biomarkers Prev ; 10(7): 785-91, 2001 Jul.
Artículo en Inglés | MEDLINE | ID: mdl-11440964

RESUMEN

Exposure to aflatoxins is a risk factor for hepatocellular carcinoma (HCC). Aflatoxins occur in peanut butter and are metabolized by genetically polymorphic enzymes such as glutathione-S-transferases encoded by glutathione-S-transferase mu 1 gene (GSTM1) and glutathione-S-transferase theta 1 gene (GSTT1) and microsomal epoxide hydrolase encoded by epoxide hydrolase gene (EPHX). The rate at which aflatoxins become activated or detoxified may depend on polymorphisms in the encoding genes. GSTM1 homozygous deletion was indeed found to modify the association between peanut butter consumption and HCC. In this study, we investigate possible roles of GSTT1 and EPHX polymorphisms in this relationship. From a Sudanese case-control study on HCC, we analyzed data of 112 incident cases and 194 controls. All participants were interviewed using a standardized questionnaire inquiring about social and demographic factors, peanut butter consumption, and other known HCC risk factors. Univariate analysis showed that GSTT1 polymorphism was not associated with HCC, whereas EPHX 113HH and 139HH genotypes increased the risk of HCC (Odds ratio, 3.10; 95% Confidence interval, 1.18-8.12). Adjustment for age and region of origin slightly attenuated this association (Odds ratio, 2.56; 95% Confidence interval, 0.83-7.95). Interestingly, unlike GSTM1, both GSTT1 and EPHX polymorphism did not modify the association between peanut butter consumption and HCC. In conclusion, these epidemiological findings do not suggest significant roles of GSTT1 and EPHX in aflatoxin metabolism, although EPHX polymorphism is possibly related to the increased risk of HCC. Further studies are needed to investigate mechanisms by which the EPHX polymorphism potentially modifies cancer risk.


Asunto(s)
Aflatoxinas/efectos adversos , Carcinoma Hepatocelular/etiología , Epóxido Hidrolasas/genética , Glutatión Transferasa/genética , Neoplasias Hepáticas/etiología , Polimorfismo Genético , Adulto , Aflatoxinas/metabolismo , Anciano , Carcinoma Hepatocelular/enzimología , Carcinoma Hepatocelular/microbiología , Estudios de Casos y Controles , Epóxido Hidrolasas/metabolismo , Femenino , Glutatión Transferasa/metabolismo , Humanos , Neoplasias Hepáticas/enzimología , Neoplasias Hepáticas/microbiología , Masculino , Microsomas Hepáticos/enzimología , Persona de Mediana Edad
10.
Cancer Causes Control ; 12(1): 23-32, 2001 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-11227922

RESUMEN

OBJECTIVE: Hepatocellular carcinoma (HCC) is one of the major cancers in the world. In Sudan the incidence is thought to be high and increasing. This study aims to assess the association between peanut butter intake, as a source of aflatoxins, and the GSTM1 genotype in the etiology of HCC. METHOD: A case control study was conducted among 150 patients and 205 controls from two regions in Sudan. Food habits with special reference to peanut butter consumption, as well as peanut storage systems, have been investigated, as well as confounders such as hepatitis, drinking and smoking habits, and demographic characteristics. GSTM1 genotype was assessed in DNA extracted from blood samples (110 cases, 189 controls). RESULTS: A positive association was observed for highest vs. lowest quartile of peanut butter intake, humid storage system and HCC, with ORs (95% CI) being 3.0 (1.6-5.5) and 1.6 (1.1-2.5) respectively. The positive association with peanut butter intake was essentially limited to subjects with GSTM1 null genotype with OR for highest vs. lowest quartile 16.7 (2.7-105). CONCLUSION: Peanut butter consumption has been identified as a strong risk factor of HCC in a region with endemic aflatoxin contamination in Sudan and was essentially limited to subjects with the GSTM1 null genotype.


Asunto(s)
Aflatoxinas/efectos adversos , Arachis/efectos adversos , Carcinoma Hepatocelular/etiología , Dieta/efectos adversos , Glutatión Transferasa/genética , Neoplasias Hepáticas/etiología , Adulto , Anciano , Carcinoma Hepatocelular/sangre , Carcinoma Hepatocelular/epidemiología , Estudios de Casos y Controles , Factores de Confusión Epidemiológicos , Conducta Alimentaria , Femenino , Genotipo , Glutatión Transferasa/sangre , Humanos , Neoplasias Hepáticas/sangre , Neoplasias Hepáticas/epidemiología , Masculino , Persona de Mediana Edad , Oportunidad Relativa , Características de la Residencia , Factores de Riesgo , Sudán/epidemiología
11.
Ned Tijdschr Geneeskd ; 145(51): 2461-5, 2001 Dec 22.
Artículo en Holandés | MEDLINE | ID: mdl-11789150

RESUMEN

This year marks the centenary of infant welfare centres in the Netherlands. In 1901, Plantenga opened the first infant welfare centre in The Hague, the Netherlands. Initially, only advice about feeding was given and the growth of the infant was monitored. To support mothers, extra milk was supplied in so-called 'milk kitchens'. Over the years the tasks have been extended to include a wide range of preventive measures. At first the doctors in infant welfare clinics were predominantly paediatricians but later general practitioners and doctors specialised in infant primary health care followed. In their 100-years existence, infant welfare clinics have grown into an intricate network which sees 98% of Dutch infants.


Asunto(s)
Servicios de Salud del Niño/historia , Bienestar del Lactante/historia , Servicios de Salud del Niño/organización & administración , Preescolar , Política de Salud/historia , Historia del Siglo XX , Humanos , Lactante , Bienestar del Lactante/tendencias , Recién Nacido , Países Bajos , Pediatría/historia
12.
Anal Biochem ; 285(1): 156-62, 2000 Oct 01.
Artículo en Inglés | MEDLINE | ID: mdl-10998276

RESUMEN

Polymorphism in N-acetyltransferases NAT1 and NAT2 may contribute to differences in cancer susceptibility of subjects exposed to alkylating compounds. We developed a robust method for simultaneous determination of these NAT polymorphisms: Reverse line blot (RLB) hybridization, based on PCR followed by allele-specific oligo hybridization. On a membrane, allele-specific oligonucleotide probes of the NAT genes (NAT1*4, *3, *10, *11 and NAT2*4, *5, *6, *7, *12) were applied in lines. After separate amplification of the NAT genes, simultaneous hybridization of these products in lines perpendicular to the lines with oligonucleotide probes was performed, followed by nonradioactive detection. This resulted in hybridization patterns, representing the NAT genotype of an individual. RLB hybridizations were conducted on DNA from 240 Dutch Caucasian participants in an ongoing case-control study on colorectal adenoma (including 126 polyp-free control subjects). Results were in complete agreement with those obtained by commonly used methods, i.e., allele-specific PCR and PCR-RFLP. Allele-frequencies in the polyp-free control group were similar to those described in the literature. RLB hybridization is, however, considerably faster and cheaper than the common assays. Moreover, expansion with allelic variants of other genes is relatively easy, which makes RLB hybridization very useful for multiplex analysis of numerous polymorphisms in epidemiological studies.


Asunto(s)
Arilamina N-Acetiltransferasa/genética , Isoenzimas/genética , Hibridación de Ácido Nucleico/métodos , Polimorfismo de Longitud del Fragmento de Restricción , Alelos , Secuencia de Bases , Estudios de Casos y Controles , Neoplasias Colorrectales/genética , Cartilla de ADN , Frecuencia de los Genes , Genotipo , Humanos , Reacción en Cadena de la Polimerasa , Factores de Riesgo
13.
Urol Int ; 64(3): 134-8, 2000.
Artículo en Inglés | MEDLINE | ID: mdl-10859543

RESUMEN

INTRODUCTION AND OBJECTIVES: Numerous studies have shown smoking and specific occupational exposures to be risk factors for bladder cancer. The risk of bladder cancer may be modified by the activity of carcinogen metabolizing enzymes. The glutathione-S-transferase Mu1 enzyme (GSTM1) detoxifies arylepoxides which are formed after exposure to certain polycyclic aromatic hydrocarbons and possibly aromatic amines. Approximately 40% of Caucasians lack GSTM1 activity due to a homozygous deletion of the GSTM1 locus on chromosome 1p13 (GSTM1 0/0 genotype). The aim of this study was to evaluate the combined effect of smoking and GSTM1 genotype on the risk of bladder cancer. MATERIALS AND METHODS: Sixty-one patients with transitional cell carcinoma of the bladder and 69 controls matched for age and sex were enrolled from the outpatient clinic. Lifestyle information was collected with a standardized questionnaire. DNA was extracted from white blood cells. The GSTM1 genotype was determined by a PCR-based method. RESULTS: 92% of the 61 patients had a history of smoking compared with 81% of the controls. There was a significant dose-response relationship for pack-years of smoking (trend test: p = 0.003). The proportion of GSTM1 0/0 genotype among patients was 62% compared with 43% among controls (odds ratio = 2.1; 95% CI 1.1-4. 3). The expected interaction between smoking and GSTM1 genotype was not observed. CONCLUSIONS: This study confirms the findings that a germline homozygous deletion of the GSTM1 gene predisposes to bladder cancer. An interaction with smoking was not found.


Asunto(s)
Carcinoma de Células Transicionales/genética , Eliminación de Gen , Glutatión Transferasa/genética , Fumar/efectos adversos , Neoplasias de la Vejiga Urinaria/genética , Anciano , Femenino , Genotipo , Células Germinativas , Homocigoto , Humanos , Masculino
14.
Vet Microbiol ; 61(1-2): 111-20, 1998 Mar 15.
Artículo en Inglés | MEDLINE | ID: mdl-9646470

RESUMEN

Ninety-two Mycobacterium bovis isolates from cattle, deer and badgers in Northern Ireland and the Republic of Ireland were genotyped by spacer-oligotyping (spoligotyping) and 67 of these were analysed by restriction fragment length polymorphism (RFLP). RFLP analysis was performed using three DNA probes, PGRS, DR and IS6110. Forty-seven of the M. bovis isolates were from 45 different sources; these were typed using both RFLP and spoligotyping. These 47 isolates could be differentiated into 24 different RFLP types and 15 distinct spoligotypes. Although RFLP was found to be more discriminatory compared to the present spoligotyping technique, spoligotyping was able to differentiate 21 RFLP type 'ACA' isolates into three different patterns. The remaining 45 M. bovis isolates were from a small case study, involving infected cattle, deer and badgers from the same geographic region. All these isolates were analysed by spoligotyping and a selection of 20 isolates were RFLP typed. All the isolates in the case study had the same spoligotype pattern with the exception of one cervine isolate. Similarly all the isolates typed by RFLP had the same pattern. Consequently, the predominant strain in the case study was not host restricted. The consistency between the results obtained using the two techniques indicates the potential value of both techniques for epidemiological studies. Spoligotyping was found to be a much more rapid technique and easier to perform, requiring less sophisticated computer software for strain typing. Spoligotyping results were more readily documented and analysed and the technique was also more suitable than RFLP analysis for large-scale screening studies.


Asunto(s)
Técnicas de Tipificación Bacteriana , ADN Ribosómico/análisis , Mycobacterium bovis/clasificación , Mycobacterium bovis/genética , Polimorfismo de Longitud del Fragmento de Restricción , Animales , Carnívoros , Bovinos , Sondas de ADN , ADN Ribosómico/genética , Ciervos , Humanos , Mycobacterium bovis/aislamiento & purificación , Tuberculosis/microbiología , Tuberculosis/veterinaria , Tuberculosis Bovina/microbiología
15.
J Clin Microbiol ; 36(1): 168-78, 1998 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-9431942

RESUMEN

DNA fingerprinting techniques were used to type 273 isolates of Mycobacterium bovis from Australia, Canada, the Republic of Ireland, and Iran. The results of restriction fragment length polymorphism (RFLP) analysis with DNA probes from IS6110, the direct repeat (DR), and the polymorphic GC-rich sequence (PGRS) were compared with those of a new PCR-based method called spacer oligonucleotide typing (spoligotyping) developed for the rapid typing of Mycobacterium tuberculosis (J. Kamerbeek et al., J. Clin. Microbiol. 35:907-914, 1997). Eighty-five percent of the isolates harbored a single copy of IS6110, and 81.5% of these carried IS6110 on the characteristic 1.9-kb restriction fragment. RFLP analysis with IS6110 identified 23 different types, RFLP analysis with the DR probe identified 35 types, RFLP analysis with the PGRS probe identified 77 types, and the spoligotyping method identified 35 types. By combining all results, 99 different strains could be identified. Isolate clusters were frequently associated within herds or were found between herds when epidemiological evidence confirmed animal movements. RFLP analysis with IS6110 was sufficiently sensitive for the typing of isolates with more than three copies of IS6110, but RFLP analysis with the PGRS probe was the most sensitive typing technique for strains with only a single copy of IS6110. Spoligotyping may have advantages for the rapid typing of M. bovis, but it needs to be made more sensitive.


Asunto(s)
Técnicas de Tipificación Bacteriana , ADN Bacteriano/análisis , Mycobacterium bovis/clasificación , Tuberculosis Bovina/microbiología , Animales , Bovinos , Dermatoglifia del ADN , Mycobacterium bovis/genética , Polimorfismo de Longitud del Fragmento de Restricción
17.
J Clin Microbiol ; 35(9): 2210-4, 1997 Sep.
Artículo en Inglés | MEDLINE | ID: mdl-9276389

RESUMEN

Spoligotyping (for spacer oligotyping) is an easy, economical, and rapid way of typing Mycobacterium tuberculosis complex strains with the DR spacer markers (J. Kamerbeek et al., J. Clin. Microbiol. 35:907-914, 1997; D. van Soolingen et al., 33:3234-3248, 1995). The stability of the markers was demonstrated by showing that all the Mycobacterium bovis BCG strains tested gave the same spoligotyping pattern. None of the 42 atypical mycobacterial strains tested gave a spoligotyping signal, indicating the specificity of the technique for M. tuberculosis complex. The utility of the spoligotyping method was demonstrated by analyzing 106 isolates of M. tuberculosis obtained over 1 year in three Paris hospitals. The results obtained by this technique were compared to those obtained by Torrea et al. (G. Torrea et al., J. Clin. Microbiol. 34:1043-1049, 1996) by IS6110-based restriction fragment length polymorphism (RFLP) analysis. Strains from patients with epidemiological relationships that were in the same IS6110-RFLP cluster were also in the same spoligotyping group. Spoligotyping was more discriminative than RFLP analysis for strains with one or two copies of IS6110. RFLP analysis did not discriminate between the nine strains with one or two IS6110 bands with no known epidemiological relation, whereas spoligotyping distinguished between eight different types. IS6I10-RFLP analysis split some of the spoligotyping clusters, particularly when the IS6110 copy number was high. Therefore, we propose a strategy for typing M. tuberculosis strains in which both markers are used.


Asunto(s)
Técnicas de Tipificación Bacteriana , ADN Bacteriano/análisis , Mycobacterium tuberculosis/clasificación , Oligonucleótidos/análisis , Tuberculosis/diagnóstico , Procesamiento Automatizado de Datos , Humanos , Epidemiología Molecular , Mycobacterium bovis/genética , Mycobacterium tuberculosis/genética , Micobacterias no Tuberculosas/genética , Polimorfismo de Longitud del Fragmento de Restricción , Secuencias Repetitivas de Ácidos Nucleicos , Sensibilidad y Especificidad , Factores de Tiempo , Tuberculosis/epidemiología
18.
J Clin Microbiol ; 35(4): 907-14, 1997 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-9157152

RESUMEN

Widespread use of DNA restriction fragment length polymorphism (RFLP) to differentiate strains of Mycobacterium tuberculosis to monitor the transmission of tuberculosis has been hampered by the need to culture this slow-growing organism and by the level of technical sophistication needed for RFLP typing. We have developed a simple method which allows simultaneous detection and typing of M. tuberculosis in clinical specimens and reduces the time between suspicion of the disease and typing from 1 or several months to 1 or 3 days. The method is based on polymorphism of the chromosomal DR locus, which contains a variable number of short direct repeats interspersed with nonrepetitive spacers. The method is referred to as spacer oligotyping or "spoligotyping" because it is based on strain-dependent hybridization patterns of in vitro-amplified DNA with multiple spacer oligonucleotides. Most of the clinical isolates tested showed unique hybridization patterns, whereas outbreak strains shared the same spoligotype. The types obtained from direct examination of clinical samples were identical to those obtained by using DNA from cultured M. tuberculosis. This novel preliminary study shows that the novel method may be a useful tool for rapid disclosure of linked outbreak cases in a community, in hospitals, or in other institutions and for monitoring of transmission of multidrug-resistant M. tuberculosis. Unexpectedly, spoligotyping was found to differentiate M. bovis from M. tuberculosis, a distinction which is often difficult to make by traditional methods.


Asunto(s)
Técnicas de Tipificación Bacteriana , ADN Bacteriano/análisis , Mycobacterium tuberculosis/aislamiento & purificación , Tuberculosis/microbiología , Datos de Secuencia Molecular , Mycobacterium tuberculosis/clasificación , Polimorfismo de Longitud del Fragmento de Restricción , Tuberculosis/diagnóstico , Tuberculosis/epidemiología
19.
J Clin Microbiol ; 34(11): 2734-40, 1996 Nov.
Artículo en Inglés | MEDLINE | ID: mdl-8897175

RESUMEN

The spacer oligonucleotide typing (spoligotyping) method was evaluated for its ability to differentiate Mycobacterium bovis strains. This method detects the presence or absence of spacers of the direct repeat locus of the M. bovis genome. The spacers in the direct repeat locus are amplified by PCR and are detected by hybridization of the biotin-labelled PCR product with a membrane containing oligonucleotides derived from spacer sequences that have previously been bound to a membrane. One hundred eighty-two M. bovis isolates from domestic animals (cattle, goat, sheep, and cats) and wild animals (deer and wild boar) were spoligotyped, and the results were compared with those obtained by IS6110 restriction fragment length polymorphism analysis. Two rather homogeneous clusters of isolates containing 20 and 4 types, respectively, were identified by spoligotyping. The first cluster included isolates from cattle, cats, and feral animals. By spoligotyping, isolates from the Spanish wild boar and deer had the same pattern as some bovine isolates, suggesting transmission between these animals and cattle and highlighting the importance of the study of these reservoirs. The second cluster included all the caprine and ovine isolates. Within each cluster, the patterns of the different strains differed only slightly, suggesting that the spoligotypes may be characteristic of strains from particular animal species. Spoligotyping proved to be useful for studying the epidemiology of bovine M. bovis isolates, especially of those isolates containing only a single copy of IS6110. In view of our results, we suggest fingerprinting all M. bovis strains by the spoligotyping method initially and then by IS6110 restriction fragment length polymorphism typing of the strains belonging to the most common spoligotypes.


Asunto(s)
Técnicas de Tipificación Bacteriana , Mycobacterium bovis/clasificación , Mycobacterium bovis/genética , Tuberculosis Bovina/epidemiología , Tuberculosis/veterinaria , Animales , Secuencia de Bases , Gatos , Bovinos , Elementos Transponibles de ADN , ADN Bacteriano/genética , ADN Ribosómico/genética , Ciervos , Estudios de Evaluación como Asunto , Cabras , Epidemiología Molecular , Mycobacterium bovis/aislamiento & purificación , Reacción en Cadena de la Polimerasa , Ovinos , Tuberculosis/epidemiología , Tuberculosis/microbiología , Tuberculosis Bovina/microbiología
20.
J Clin Microbiol ; 34(7): 1717-21, 1996 Jul.
Artículo en Inglés | MEDLINE | ID: mdl-8784576

RESUMEN

A collection of 399 "Streptococcus milleri" strains were identified to the species level by the use of a line blot assay. Their PCR-amplified partial 16S rRNA gene sequences were hybridized with species-specific 5'-biotinylated oligonucleotide probes homologous to the bp 213 to 231 regions of the 16S rRNA gene sequences of the type strains Streptococcus anginosus ATCC 33397, Streptococcus constellatus ATCC 27823, and Streptococcus intermedius ATCC 27335. The hybridization results were compared with the reference phenotypic identification method data (R. A. Whiley, H. Fraser, J. M. Hardie, and D. Beighton, J. Clin. Microbiol. 28:1497-1501, 1990). Most strains (357 of 399 [89.5%]) reacted unambiguously with only one probe. However, 42 of the 399 strains (10.5%) reacted with both the S. constellatus- and S. intermedius-specific probes; 41 of them were phenotypically identified as S. constellatus. These dually reactive strains hybridized with a 5'-biotinylated probe based on the bp 213 to 231 region of the 16S rRNA gene sequence of one of two species. Analysis of the 5' ends of the 16S rRNA gene sequences (487 bp) demonstrated that the dually reactive strains represent a distinct rRNA population sharing 98.1% sequence similarity with S. constellatus. Phenotypic consistency between the dually reactive strains and the S. constellatus strains was not demonstrated. Line blot hybridization proved to be a simple and inexpensive method to screen large numbers of strains for genetic relatedness, and it allowed the detection of a distinct 16S rRNA type within the "S. milleri" group.


Asunto(s)
ARN Bacteriano/genética , ARN Ribosómico 16S/genética , Streptococcus/clasificación , Streptococcus/genética , Técnicas de Tipificación Bacteriana , Secuencia de Bases , Sondas de ADN/genética , Genes Bacterianos , Humanos , Datos de Secuencia Molecular , Hibridación de Ácido Nucleico , Fenotipo , Reacción en Cadena de la Polimerasa , Especificidad de la Especie , Streptococcus/aislamiento & purificación
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