RESUMEN

INTRODUCTION: Molecular targets are emerging rapidly and the development of clinical tests that simultaneously screen for multiple targets has become especially important. We assessed the gene expression levels of three known targets in advanced gastric cancer, epidermal growth factor receptor (EGFR), human epidermal growth factor 2 (HER2), and N-methyl-N-nitrosoguanidine human osteosarcoma transforming gene (MET), using the nCounter® assay (NanoString Technologies, Seattle, WA, USA) and compared these results with protein overexpression, detected by immunohistochemistry, to evaluate the performance of this new technology. METHODS: We investigated 42 formalin-fixed, paraffin-embedded tumor samples from patients with gastric cancer. A NanoString-based assay containing a 522 kinase gene panel was investigated. We analyzed the correlations between immunohistochemical findings and kinase gene expression levels of EGFR, HER2 and MET to validate this assay. RESULTS: EGFR, HER2, and MET overexpression were observed in 7 (16.6 %), 5 (11.9 %), and 3 (7.1 %) cases, respectively. For EGFR, HER2, and MET, the concordance rates between the NanoString-based assay results and the immunohistochemistry methods were 83.3, 97.6, and 100 %, respectively. Relative to immunohistochemistry findings, the NanoString-based assay sensitivities and specificities were 85.7 and 82.8 % for EGFR, 100 and 97.2 % for HER2, and 100 and 100 % for MET, respectively. CONCLUSIONS: We found a high concordance between immunohistochemistry- and nCounter-based assessments of EGFR, HER2, and MET in advanced gastric cancer. Judged against immunohistochemistry results, the NanoString assay had high sensitivities and high specificities. These results suggest that the nCounter assay provides a reliable, high-throughput assay to simultaneously screen for the overexpression of several target proteins.

Asunto(s)

Genes erbB-1/genética , Genes erbB-2/genética , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Proteínas Proto-Oncogénicas c-met/genética , Neoplasias Gástricas/genética , Adulto , Anciano , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Área Bajo la Curva , Capecitabina/administración & dosificación , Everolimus/administración & dosificación , Femenino , Humanos , Inmunohistoquímica , Hibridación Fluorescente in Situ , Masculino , Persona de Mediana Edad , Curva ROC , Terapia Recuperativa/métodos , Sensibilidad y Especificidad , Análisis de Secuencia de ARN/métodos , Neoplasias Gástricas/tratamiento farmacológico

RESUMEN

Following a 19-year hiatus, Venezuelan equine encephalitis (VEE) reemerged in western Venezuela in December 1992. This outbreak is important in understanding VEE emergence because phylogenetic studies imply that sympatric, enzootic, subtype ID VEE viruses mutated to generate the epizootic/epidemic. Although the 1992-1993 strains belong to subtype IC, a serotype implicated in extensive outbreaks during the 1960s and in 1995, relatively small numbers of human and equine cases occurred in 1992-1993. We, therefore, evaluated the pathogenicity of these Venezuelan enzootic ID and epizootic IC viruses to determine 1) if they exhibit phenotypes like those described previously for more distantly related enzootic and epizootic strains, and 2) if the 1992-1993 outbreak was limited by the inability of these IC viruses to exploit equines as amplification hosts. All strains were virulent in mice and guinea pigs, but were benign for cotton rats, natural hosts of enzootic viruses. However, only the IC strains produced equine disease, with mean peak viremias of 10(5) suckling mouse 50% lethal doses per mL serum, and some titers exceeding 10(7). These viremias approximate those observed previously with VEE strains isolated during more extensive epizootics, suggesting that efficient equine amplification did not limit the scope and duration of the 1992-1993 outbreak. Enzootic ID virus infection protected all horses from challenge with epizootic strain P676, supporting the hypothesis that epizootics bypass regions of enzootic transmission due to natural immunization of equines by enzootic VEE viruses.

Asunto(s)

Brotes de Enfermedades/veterinaria , Virus de la Encefalitis Equina Venezolana/patogenicidad , Encefalomielitis Equina Venezolana/epidemiología , Encefalomielitis Equina Venezolana/virología , Enfermedades de los Caballos/virología , Viremia/virología , Animales , Anopheles , Chlorocebus aethiops , Cricetinae , Virus de la Encefalitis Equina Venezolana/clasificación , Encefalomielitis Equina Venezolana/sangre , Femenino , Cobayas , Enfermedades de los Caballos/sangre , Enfermedades de los Caballos/epidemiología , Caballos , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C3H , Ratones Endogámicos C57BL , Distribución Aleatoria , Ratas , Enfermedades de los Roedores/virología , Sigmodontinae , Venezuela/epidemiología , Células Vero , Virulencia

RESUMEN

Venezuelan equine encephalitis viruses (VEEV) belonging to subtype IC have caused three (1962-1964, 1992-1993 and 1995) major equine epizootics and epidemics. Previous sequence analyses of a portion of the envelope glycoprotein gene demonstrated a high degree of conservation among isolates from the 1962-1964 and the 1995 outbreaks, as well as a 1983 interepizootic mosquito isolate from Panaquire, Venezuela. However, unlike subtype IAB VEEV that were used to prepare inactivated vaccines that probably initiated several outbreaks, subtype IC viruses have not been used for vaccine production and their conservation cannot be explained in this way. To characterize further subtype IC VEEV conservation and to evaluate potential sources of the 1995 outbreak, we sequenced the complete genomes of three isolates from the 1962-1964 outbreak, the 1983 Panaquire interepizootic isolate, and two isolates from 1995. The sequence of the Panaquire isolate, and that of virus isolated from a mouse brain antigen prepared from subtype IC strain P676 and used in the same laboratory, suggested that the Panaquire isolate represents a laboratory contaminant. Some authentic epizootic IC strains isolated 32 years apart showed a greater degree of sequence identity than did isolates from the same (1962-1964 or 1995) outbreak. If these viruses were circulating and replicating between 1964 and 1995, their rate of sequence evolution was at least 10-fold lower than that estimated during outbreaks or that of closely related enzootic VEEV strains that circulate continuously. Current understanding of alphavirus evolution is inconsistent with this conservation. This subtype IC VEEV conservation, combined with phylogenetic relationships, suggests the possibility that the 1995 outbreak was initiated by a laboratory strain.

Asunto(s)

Brotes de Enfermedades , Virus de la Encefalitis Equina Venezolana/clasificación , Encefalomielitis Equina Venezolana/epidemiología , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Línea Celular , Chlorocebus aethiops , Cricetinae , Encefalomielitis Equina Venezolana/virología , Humanos , Datos de Secuencia Molecular , Filogenia , Factores de Tiempo , Venezuela

RESUMEN

Pirital-like virus isolates from rodents collected in a variety of habitats within a six-state area of central Venezuela were analyzed genetically by amplifying a portion of the nucleocapsid protein gene using RT-PCR. Comparisons of the sequences from 30 selected Pirital-like virus isolates demonstrated up to 26% divergence in nucleotide sequences and up to 16% divergence in deduced amino acid sequences. Within the Pirital monophyletic group, 14 distinct lineages or genotypes, differing by at least 6% in nucleotide sequences, were identified. Although sample sizes were small for some lineages, many of the different genotypes were sampled in only one region or locality, suggesting allopatric divergence. Complement fixation tests with representatives of the most divergent Pirital virus lineages failed to delineate multiple species or subtypes within the Pirital clade. These results indicate that the previously proposed 12% nucleocapsid protein amino acid sequence divergence cutoff value for delineating arenavirus species is not appropriate for the entire family. When individual clones were examined from PCR amplicons, a mean of 0.17% sequence diversity vs the consensus sequences was detected, suggesting diverse quasispecies populations within infected rodent hosts. Possible explanations for the extreme genetic diversity within and among Pirital virus populations in infected rodents are discussed.

Asunto(s)

Arenaviridae/genética , Roedores/virología , Animales , Arenaviridae/clasificación , Pruebas de Fijación del Complemento , Variación Genética , Datos de Secuencia Molecular , Filogenia , Serotipificación , Venezuela

RESUMEN

Eastern equine encephalitis virus (EEEV), the sole species in the EEE antigenic complex, is divided into North and South American antigenic varieties based on hemagglutination inhibition tests. Here we describe serologic and phylogenetic analyses of representatives of these varieties, spanning the entire temporal and geographic range available. Nucleotide sequencing and phylogenetic analyses revealed additional genetic diversity within the South American variety; 3 major South/Central American lineages were identified including one represented by a single isolate from eastern Brazil, and 2 lineages with more widespread distributions in Central and South America. All North American isolates comprised a single, highly conserved lineage with strains grouped by the time of isolation and to some extent by location. An EEEV strain isolated during a 1996 equine outbreak in Tamaulipas State, Mexico was closely related to recent Texas isolates, suggesting southward EEEV transportation beyond the presumed enzootic range. Plaque reduction neutralization tests with representatives from the 4 major lineages indicated that each represents a distinct antigenic subtype. A taxonomic revision of the EEE complex is proposed.

Asunto(s)

Variación Antigénica/genética , Virus de la Encefalitis Equina del Este/genética , Encefalomielitis Equina/epidemiología , Variación Genética , Filogenia , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Aves , América Central/epidemiología , Cartilla de ADN/química , ADN Viral/química , Brotes de Enfermedades/veterinaria , Virus de la Encefalitis Equina del Este/inmunología , Caballos , Humanos , Pruebas de Neutralización/veterinaria , América del Norte/epidemiología , ARN Viral/aislamiento & purificación , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa/veterinaria , Alineación de Secuencia , Análisis de Secuencia de ARN , Sigmodontinae/virología , América del Sur/epidemiología

RESUMEN

Epizootics of Venezuelan equine encephalitis (VEE) involving subtype IAB viruses occurred sporadically in South, Central and North America from 1938 to 1973. Incompletely inactivated vaccines have long been suspected as a source of the later epizootics. We tested this hypothesis by sequencing the PE2 glycoprotein precursor (1,677 nucleotides) or 26S/nonstructural protein 4 (nsP4) genome regions (4,490 nucleotides) for isolates representing most major outbreaks. Two distinct IAB genotypes were identified: 1) 1940s Peruvian strains and 2) 1938-1973 isolates from South, Central, and North America. Nucleotide sequences of these two genotypes differed by 1.1%, while the latter group showed only 0.6% sequence diversity. Early VEE virus IAB strains that were used for inactivated vaccine preparation had sequences identical to those predicted by phylogenetic analyses to be ancestors of the 1960s-1970s outbreaks. These data support the hypothesis of a vaccine origin for many VEE outbreaks. However, continuous, cryptic circulation of IAB viruses cannot be ruled out as a source of epizootic emergence.

Asunto(s)

Brotes de Enfermedades , Virus de la Encefalitis Equina Venezolana/genética , Encefalomielitis Equina/epidemiología , Secuencia de Aminoácidos , Animales , Secuencia de Bases , América Central/epidemiología , Cartilla de ADN/química , Virus de la Encefalitis Equina Venezolana/clasificación , Encefalomielitis Equina/virología , Datos de Secuencia Molecular , América del Norte/epidemiología , Filogenia , ARN Viral/química , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Alineación de Secuencia , Análisis de Secuencia de ADN , Homología de Secuencia de Aminoácido , Homología de Secuencia de Ácido Nucleico , América del Sur/epidemiología , Vacunas Virales/efectos adversos

RESUMEN

Venezuelan equine encephalitis (VEE) epidemics and equine epizootics occurred periodically in the Americas from the 1920s until the early 1970s, when the causative viruses, subtypes IAB and IC, were postulated to have become extinct. Recent outbreaks in Columbia and Venezuela have renewed interest in the source of epidemic/epizootic viruses and their mechanism of interepizootic maintenance. We performed phylogenetic analyses of VEE virus isolates spanning the entire temporal and geographic range of strains available, using 857-nucleotide reverse transcription-PCR products including the E3 and E2 genes. Analyses indicated that epidemic/epizootic viruses are closely related to four distinct, enzootic subtype ID-like lineages. One of these lineages, which occurs in Columbia, Peru, and Venezuela, also included all of the epidemic/epizootic isolates; the remaining three ID-like lineages, which occur in Panama, Peru, Florida, coastal Ecuador, and southwestern Columbia, were apparently not associated with epizootic VEE emergence. Within the Columbia/Peru/Venezuela lineage, three distinct monophyletic groups of epidemic/epizootic viruses were delineated, indicating that VEE emergence has occurred independently at least three times (convergent evolution). Representative, complete E2 amino acid sequences were compared to identify potential determinants of equine virulence and epizootic emergence. Amino acids implicated previously in laboratory mouse attenuation generally did not vary among the natural isolates that we examined, indicating that they probably are not involved in equine virulence changes associated with VEE emergence. Most informative amino acids correlated with phylogenetic relationships rather than phenotypic characteristics, suggesting that VEE emergence has resulted from several distinct combinations of mutations that generate viruses with similar antigenic and equine virulence phenotypes.

Asunto(s)

Virus de la Encefalitis Equina Venezolana/genética , Encefalomielitis Equina Venezolana/epidemiología , Encefalomielitis Equina Venezolana/virología , Secuencia de Aminoácidos , Secuencia de Bases , Evolución Biológica , ADN Viral , Brotes de Enfermedades , Virus de la Encefalitis Equina Venezolana/clasificación , Genotipo , Datos de Secuencia Molecular , Filogenia , Homología de Secuencia de Aminoácido , Proteínas del Envoltorio Viral/genética

RESUMEN

Part I of this article (J Am Acad Dermatol 1989; 20:537-63) focused on normal sweat gland function. Part II provides a discussion of hyperhidrosis and hypohidrosis. Hyperhidrotic disorders affect the palms and soles and the axillae and are associated with previous spinal cord injuries, peripheral neuropathies, brain lesions, intrathoracic neoplasms, systemic illness, and gustatory sweating. Hypohidrotic disorders include anhidrotic ectodermal dysplasia, hereditary sensory neuropathy, Holmes-Adie syndrome, and generalized anhidrosis.

Asunto(s)

Humanos , Hiperhidrosis , Hipohidrosis

RESUMEN

The basic mechanisms of sweat gland function and an updated review of some relatively common disorders of sweat secretion, are presented. Although sweat secretion and ductal absorption are basically biophysical and biologic cellular processes, a detailed description of the basic biophysical principles of membrane transport has been avoided to make the discussion more readable. The cited references will, however, help those readers primarily interested in the basic details of sweat gland function. Part I of this article includes a discussion of morphologic characteristics, central and peripheral nervous control of sweat secretion, neurotransmitters, intracellular mediators and stimulus secretion coupling, Na-K-Cl cotransport model for the ionic mechanism of sweat secretion, ingredients of sweat, ductal function, the pathogenesis of abnormal sweat gland function in cystic fibrosis, and the discovery of the apoeccrine sweat gland. Part II, to be published in the May issue of the Journal, reviews reports of all those major disorders of hyperhidrosis and hypohidrosis that have appeared in the literature during the past 10 years. It is hoped that this review will serve as a resource for clinicians who encounter puzzling disorders of sweating in their patients, as well as for investigators who wish to obtain a quick update on sweat gland function.

Asunto(s)

Humanos , Enfermedades de las Glándulas Sudoríparas , Glándulas Sudoríparas