RESUMEN
PURPOSE: [18F]3F4AP is a novel PET radiotracer that targets voltage-gated potassium (K+) channels and has shown promise for imaging demyelinated lesions in animal models of neurological diseases. This study aimed to evaluate the biodistribution, safety, and radiation dosimetry of [18F]3F4AP in healthy human volunteers. METHODS: Four healthy volunteers (2 females) underwent a 4-h dynamic PET scan from the cranial vertex to mid-thigh using multiple bed positions after administration of 368 ± 17.9 MBq (9.94 ± 0.48 mCi) of [18F]3F4AP. Volumes of interest for relevant organs were manually drawn guided by the CT, and PET images and time-activity curves (TACs) were extracted. Radiation dosimetry was estimated from the integrated TACs using OLINDA software. Safety assessments included measuring vital signs immediately before and after the scan, monitoring for adverse events, and obtaining a comprehensive metabolic panel and electrocardiogram within 30 days before and after the scan. RESULTS: [18F]3F4AP distributed throughout the body with the highest levels of activity in the kidneys, urinary bladder, stomach, liver, spleen, and brain and with low accumulation in muscle and fat. The tracer cleared quickly from circulation and from most organs. The clearance of the tracer was noticeably faster than previously reported in nonhuman primates (NHPs). The average effective dose (ED) across all subjects was 12.1 ± 2.2 µSv/MBq, which is lower than the estimated ED from the NHP studies (21.6 ± 0.6 µSv/MBq) as well as the ED of other fluorine-18 radiotracers such as [18F]FDG (~ 20 µSv/MBq). No differences in ED between males and females were observed. No substantial changes in safety assessments or adverse events were recorded. CONCLUSION: The biodistribution and radiation dosimetry of [18F]3F4AP in humans are reported for the first time. The average total ED across four subjects was lower than most 18F-labeled PET tracers. The tracer and study procedures were well tolerated, and no adverse events occurred.
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Enfermedades Desmielinizantes , Radiometría , Masculino , Femenino , Animales , Humanos , Distribución Tisular , Radiometría/métodos , Tomografía de Emisión de Positrones/efectos adversos , Tomografía de Emisión de Positrones/métodos , RadiofármacosRESUMEN
BACKGROUND: Obtaining DNA samples through saliva samples is becoming more common, and more companies are responding to this demand with saliva collection/DNA extraction kits. METHODS: The University of Pittsburgh's School of Dental Medicine maintains a DNA bank, which our lab helps direct. While we have always used Oragene (DNAGenotek, Kanata, Canada) kits to collect samples in the past, we recently compared 5 alternative kits/methods in an effort to reduce costs while maintaining quality. The kits/ methods were: Norgen (Norgen Biotek Corp., Thorold, Canada), Stratec (Stratec Biomedical, Birkenfeld, Germany), DNAgard (Biomatrica, San Diego, CA, USA), Oasis (Oasis Diagnostics, Vancouver, WA, USA), and an inhouse protocol for DNA extraction from whole saliva. We compared 7 protocols for extracting DNA from saliva using 5 commercially available kits. We primarily looked at total DNA yield, but also considered cost, ease of sample collection, and complexity of extraction protocol. RESULTS: When compared to the Oragene kits, only Norgen and Startec had comparable DNA yields (30 µg or more). Oasis was the easiest to use in terms of sample collection. CONCLUSIONS: When compared to our whole saliva DNA extraction protocol, all kits had higher yields, shorter extraction time, and easier protocols.
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ADN/aislamiento & purificación , Juego de Reactivos para Diagnóstico , Saliva/química , Alemania , Humanos , Manejo de EspecímenesRESUMEN
BACKGROUND: There is evidence for a genetic contribution to chronic periodontitis. In this study, we conducted a genome wide association study among 866 participants of the University of Pittsburgh Dental Registry and DNA Repository, whose periodontal diagnosis ranged from healthy (N = 767) to severe chronic periodontitis (N = 99). METHODS: Genotypingi of over half-million single nucleotide polymorphisms was determined. Analyses were done twice, first in the complete dataset of all ethnicities, and second including only samples defined as self-reported Whites. From the top 100 results, twenty single nucleotide polymorphisms had consistent results in both analyses (borderline p-values ranging from 1E-05 to 1E-6) and were selected to be tested in two independent datasets derived from 1,460 individuals from Porto Alegre, and 359 from Rio de Janeiro, Brazil. Meta-analyses of the Single nucleotide polymorphisms showing a trend for association in the independent dataset were performed. RESULTS: The rs1477403 marker located on 16q22.3 showed suggestive association in the discovery phase and in the Porto Alegre dataset (p = 0.05). The meta-analysis suggested the less common allele decreases the risk of chronic periodontitis. CONCLUSIONS: Our data offer a clear hypothesis to be independently tested regarding the contribution of the 16q22.3 locus to chronic periodontitis.
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Mapeo Cromosómico , Cromosomas Humanos Par 16/genética , Periodontitis Crónica/genética , Adulto , Anciano , Anciano de 80 o más Años , Alelos , Cromosomas Humanos Par 21/genética , Periodontitis Crónica/etnología , Complicaciones de la Diabetes , Etnicidad/genética , Femenino , Frecuencia de los Genes/genética , Marcadores Genéticos/genética , Predisposición Genética a la Enfermedad/genética , Variación Genética/genética , Estudio de Asociación del Genoma Completo , Genotipo , Humanos , Masculino , Persona de Mediana Edad , Polimorfismo de Nucleótido Simple/genética , Factores de Riesgo , Fumar , Población Blanca/genéticaRESUMEN
OBJECTIVE: The purpose of this study was to fine map the locus Xq25.1-27-2 in order to identify genetic contributors involved in low caries experience. DESIGN: Seventy-two families from the Philippines were studied. Caries experience was recorded and genomic DNA extracted from peripheral blood was obtained from all subjects. One hundred and twenty-eight polymorphisms in the locus Xq25.1-27-2, a region that contains 24 genes, were genotyped. Association between caries experience and alleles was tested using the transmission disequilibrium test (TDT). This initial analysis was followed by experiments with DNA samples from 1481 subjects from Pittsburgh, 918 children from Brazil, and 275 children from Turkey in order to follow up the results found in the Filipino families. Chi-square or Fisher's exact tests were used. Sequencing of the coding regions and exon-intron boundaries of MST4 and FGF13 were also performed on 91 women from Pittsburgh. RESULTS: Statistically significant association with low caries experience was found for 11 markers in Xq25.1-27-2 in the Filipino families. One marker was in MST4, another marker was in FGF13, and the remaining markers were in intergenic regions. Haplotype analysis also confirmed these results, but the follow up studies with DNA samples from Pittsburgh, Brazil, and Turkey showed associations for a subset of the 11 markers. No coding mutations were identified by sequencing. CONCLUSIONS: Our study failed to conclusively demonstrate that genetic factors in Xq25.1-27-2 contribute to caries experience in multiple populations.
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Cromosomas Humanos X/genética , Caries Dental/genética , Adolescente , Adulto , Anciano , Alelos , Brasil/epidemiología , Niño , Preescolar , Mapeo Cromosómico , Caries Dental/epidemiología , Exones , Femenino , Marcadores Genéticos , Predisposición Genética a la Enfermedad , Genotipo , Haplotipos , Humanos , Lactante , Recién Nacido , Masculino , Persona de Mediana Edad , Pennsylvania/epidemiología , Fenotipo , Filipinas/epidemiología , Reacción en Cadena de la Polimerasa , Polimorfismo de Nucleótido Simple , Factores Sexuales , Turquía/epidemiologíaRESUMEN
BACKGROUND: Our previous genome-wide linkage scan mapped five loci for caries experience. The purpose of this study was to fine map one of these loci, the locus 13q31.1, in order to identify genetic contributors to caries. METHODS: Seventy-two pedigrees from the Philippines were studied. Caries experience was recorded and DNA was extracted from blood samples obtained from all subjects. Sixty-one single nucleotide polymorphisms (SNPs) in 13q31.1 were genotyped. Association between caries experience and alleles was tested. We also studied 1,481 DNA samples obtained from saliva of subjects from the USA, 918 children from Brazil, and 275 children from Turkey, in order to follow up the results found in the Filipino families. We used the AliBaba2.1 software to determine if the nucleotide changes of the associated SNPs changed the prediction of the presence of transcription-binding site sequences and we also analyzed the gene expression of the genes selected based on binding predictions. Mutation analysis was also performed in 33 Filipino individuals of a segment of 13q31.1 that is highly conserved in mammals. RESULTS: Statistically significant association with high caries experience was found for 11 markers in 13q31.1 in the Filipino families. Haplotype analysis also confirmed these results. In the populations used for follow-up purposes, associations were found between high caries experience and a subset of these markers. Regarding the prediction of the transcription-binding site, the base change of the SNP rs17074565 was found to change the predicted-binding of genes that could be involved in the pathogenesis of caries. When the sequence has the allele C of rs17074565, the potential transcription factors binding the sequence are GR and GATA1. When the subject carries the G allele of rs17074565, the potential transcription factor predicted to bind to the sequence is GATA3. The expression of GR in whole saliva was higher in individuals with low caries experience when compared to individuals with high caries experience (p = 0.046). No mutations were found in the highly conserved sequence. CONCLUSIONS: Genetic factors contributing to caries experience may exist in 13q31.1. The rs17074565 is located in an intergenic region and is predicted to disrupt the binding sites of two different transcription factors that might be involved with caries experience. GR expression in saliva may be a biomarker for caries risk and should be further explored.
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Cromosomas Humanos Par 13 , Adolescente , Adulto , Anciano , Anciano de 80 o más Años , Alelos , Pueblo Asiatico/genética , Sitios de Unión , Niño , Preescolar , Mapeo Cromosómico , Biología Computacional , Análisis Mutacional de ADN , Caries Dental/genética , Femenino , Genoma Humano , Genotipo , Haplotipos , Humanos , Lactante , Masculino , Persona de Mediana Edad , Linaje , Filipinas , Polimorfismo de Nucleótido Simple , Factores de Transcripción/metabolismo , Adulto JovenRESUMEN
BACKGROUND: Over 90% of adults aged 20 years or older with permanent teeth have suffered from dental caries leading to pain, infection, or even tooth loss. Although caries prevalence has decreased over the past decade, there are still about 23% of dentate adults who have untreated carious lesions in the US. Dental caries is a complex disorder affected by both individual susceptibility and environmental factors. Approximately 35-55% of caries phenotypic variation in the permanent dentition is attributable to genes, though few specific caries genes have been identified. Therefore, we conducted the first genome-wide association study (GWAS) to identify genes affecting susceptibility to caries in adults. METHODS: Five independent cohorts were included in this study, totaling more than 7000 participants. For each participant, dental caries was assessed and genetic markers (single nucleotide polymorphisms, SNPs) were genotyped or imputed across the entire genome. Due to the heterogeneity among the five cohorts regarding age, genotyping platform, quality of dental caries assessment, and study design, we first conducted genome-wide association (GWA) analyses on each of the five independent cohorts separately. We then performed three meta-analyses to combine results for: (i) the comparatively younger, Appalachian cohorts (N = 1483) with well-assessed caries phenotype, (ii) the comparatively older, non-Appalachian cohorts (N = 5960) with inferior caries phenotypes, and (iii) all five cohorts (N = 7443). Top ranking genetic loci within and across meta-analyses were scrutinized for biologically plausible roles on caries. RESULTS: Different sets of genes were nominated across the three meta-analyses, especially between the younger and older age cohorts. In general, we identified several suggestive loci (P-value ≤ 10E-05) within or near genes with plausible biological roles for dental caries, including RPS6KA2 and PTK2B, involved in p38-depenedent MAPK signaling, and RHOU and FZD1, involved in the Wnt signaling cascade. Both of these pathways have been implicated in dental caries. ADMTS3 and ISL1 are involved in tooth development, and TLR2 is involved in immune response to oral pathogens. CONCLUSIONS: As the first GWAS for dental caries in adults, this study nominated several novel caries genes for future study, which may lead to better understanding of cariogenesis, and ultimately, to improved disease predictions, prevention, and/or treatment.
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Susceptibilidad a Caries Dentarias/genética , Caries Dental/genética , Estudio de Asociación del Genoma Completo , Sistema de Señalización de MAP Quinasas/genética , Vía de Señalización Wnt/genética , Adolescente , Adulto , Anciano , Anciano de 80 o más Años , Cromosomas Humanos/genética , Índice CPO , Dentición Permanente , Humanos , Persona de Mediana Edad , Adulto JovenRESUMEN
Caries is a multifactorial disease, and studies aiming to unravel the factors modulating its etiology must consider all known predisposing factors. One major factor is bacterial colonization, and Streptococcus mutans is the main microorganism associated with the initiation of the disease. In our studies, we have access to DNA samples extracted from human saliva and blood. In this report, we tested a real-time PCR assay developed to detect copies of genomic DNA from Streptococcus mutans in 1,424 DNA samples from humans. Our results suggest that we can determine the presence of genomic DNA copies of Streptococcus mutans in both DNA samples from caries-free and caries-affected individuals. However, we were not able to detect the presence of genomic DNA copies of Streptococcus mutans in any DNA samples extracted from peripheral blood, which suggests the assay may not be sensitive enough for this goal. Values of the threshold cycle of the real-time PCR reaction correlate with higher levels of caries experience in children, but this correlation could not be detected for adults.
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The family Caliciviridae is divided into four genera and consists of single-stranded RNA viruses with hosts ranging from humans to a wide variety of animals. Human caliciviruses are the major cause of outbreaks of acute nonbacterial gastroenteritis, whereas animal caliciviruses cause various host-dependent illnesses with a documented potential for zoonoses. To investigate inter- and intragenus structural variations and to provide a better understanding of the structural basis of host specificity and strain diversity, we performed structural studies of the recombinant capsid of Grimsby virus, the recombinant capsid of Parkville virus, and San Miguel sea lion virus serotype 4 (SMSV4), which are representative of the genera Norovirus (genogroup 2), Sapovirus, and Vesivirus, respectively. A comparative analysis of these structures was performed with that of the recombinant capsid of Norwalk virus, a prototype member of Norovirus genogroup 1. Although these capsids share a common architectural framework of 90 dimers of the capsid protein arranged on a T=3 icosahedral lattice with a modular domain organization of the subunit consisting of a shell (S) domain and a protrusion (P) domain, they exhibit distinct differences. The distally located P2 subdomain of P shows the most prominent differences both in shape and in size, in accordance with the observed sequence variability. Another major difference is in the relative orientation between the S and P domains, particularly between those of noroviruses and other caliciviruses. Despite being a human pathogen, the Parkville virus capsid shows more structural similarity to SMSV4, an animal calicivirus, suggesting a closer relationship between sapoviruses and animal caliciviruses. These comparative structural studies of caliciviruses provide a functional rationale for the unique modular domain organization of the capsid protein with an embedded flexibility reminiscent of an antibody structure. The highly conserved S domain functions to provide an icosahedral scaffold; the hypervariable P2 subdomain may function as a replaceable module to confer host specificity and strain diversity; and the P1 subdomain, located between S and P2, provides additional fine-tuning to position the P2 subdomain.
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Caliciviridae/clasificación , Caliciviridae/ultraestructura , Cápside/química , Variación Genética , Secuencia de Aminoácidos , Animales , Caliciviridae/química , Caliciviridae/genética , Cápside/metabolismo , Cápside/ultraestructura , Proteínas de la Cápside/química , Proteínas de la Cápside/genética , Proteínas de la Cápside/metabolismo , Microscopía por Crioelectrón , Cristalización , Cristalografía por Rayos X , Humanos , Modelos Moleculares , Datos de Secuencia Molecular , Norovirus/química , Norovirus/genética , Norovirus/ultraestructura , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Sapovirus/química , Sapovirus/genética , Sapovirus/ultraestructura , Especificidad de la Especie , Vesivirus/química , Vesivirus/genética , Vesivirus/ultraestructura , Virión/química , Virión/metabolismo , Virión/ultraestructuraRESUMEN
Among viral agents causing gastroenteritis, human astroviruses (HAstVs) take second or third place, after rotaviruses and caliciviruses, as the most frequent cause of illness. The aims of this study were to determine the prevalence of HAstV infection and to characterize the circulating HAstV strains in children with diarrhea under 3 years of age treated between 1995 and 1998 at out- or in-patient facilities of the children's hospital in Mendoza, Argentina. Reverse transcription-polymerase chain reaction (RT-PCR) and enzyme immunoassay (EIA) were used to detect HAstVs in stool specimens. Positive specimens were tested further by EIA and/or sequenced to type detected HAstV strains. HAstVs were detected in 40 (3.7%) of 1,070 samples that were rotavirus and calicivirus-negative: 14 (3.5%) of 402 from outpatients and 26 (3.9%) of 668 from inpatients. HAstV infection tended to be more severe in children during their first year of life: 18 (4.7%) of 383 HAstV-positive children 0-11 months old were hospitalized versus 8 (2.8%) of 285 children 1 year of age or older (P = 0.29). Type 1 (HAstV-1) was the most common type (41%), followed by HAstV-4 (25%), HAstV-2 (13%), HAstV-3 (13%), and HAstV-5 (8%). In this first epidemiological study of HAstV infection in this region, we confirmed HAstV to be a cause of severe gastroenteritis in children, more often among children younger than 12 months of age. HastV-4 caused 25% of HastV infections in Mendoza, although it has been detected commonly elsewhere. Distinct genetic lineages were apparent but their epidemiological significance remains to be demonstrated.
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Infecciones por Astroviridae/epidemiología , Gastroenteritis/epidemiología , Mamastrovirus/clasificación , Mamastrovirus/aislamiento & purificación , Enfermedad Aguda , Argentina/epidemiología , Infecciones por Astroviridae/virología , Células CACO-2 , Preescolar , Estudios Transversales , Gastroenteritis/virología , Humanos , Lactante , Recién Nacido , Mamastrovirus/genética , Datos de Secuencia Molecular , Filogenia , Prevalencia , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Análisis de Secuencia de ADN , SerotipificaciónRESUMEN
Between July 1997 and June 2000, fecal specimens from 284 outbreaks of nonbacterial gastroenteritis were submitted to the Centers for Disease Control and Prevention for testing for "Norwalk-like viruses" (NLVs). Specimens were examined by reverse-transcription polymerase chain reaction and direct electron microscopy for the presence of NLVs. Adequate descriptive data were available from 233 of the outbreaks, and, of these, 217 (93%) were positive for NLVs. Restaurants and events with catered food were the most common settings, and contaminated food was the most common mode of transmission. Genogroup II (GII) strains were the predominant type (73%), with genogroup I strains causing 26% of all NLV-positive outbreaks. Certain GII clusters (GII/1,4,j) were more commonly associated with outbreaks in nursing home settings than with outbreaks in other settings. Strain diversity was great: one potential new sequence cluster was implicated in multiple outbreaks, and strains belonging to a tentative new genogroup were identified.