RESUMO
The developing world is seeing rapid growth in the availability of biological mass spectrometry (MS), particularly through core facilities. As proteomics and metabolomics becomes locally feasible for investigators in these nations, application areas associated with high burden in these nations, such as infectious disease, will see greatly increased research output. This article evaluates the rapid growth of MS in South Africa (currently approaching 20 laboratories) as a model for establishing MS core facilities in other nations of the developing world. Facilities should emphasize new services rather than new instruments. The reduction of the delays associated with reagent and other supply acquisition would benefit both facilities and the users who make use of their services. Instrument maintenance and repair, often mediated by an in-country business for an international vendor, is also likely to operate on a slower schedule than in the wealthiest nations. A key challenge to facilities in the developing world is educating potential facility users in how best to design experiments for proteomics and metabolomics, what reagents are most likely to introduce problematic artifacts, and how to interpret results from the facility. Here, we summarize the experience of 6 different institutions to raise the level of biological MS available to researchers in South Africa.
Assuntos
Países em Desenvolvimento , Espectrometria de Massas/métodos , Custos e Análise de Custo , Espectrometria de Massas/economia , África do SulRESUMO
PURPOSE: Cancer of the prostate (CaP) is the leading cancer among men in sub-Saharan Africa (SSA). A substantial proportion of these men with CaP are diagnosed at late (usually incurable) stages, yet little is known about the etiology of CaP in SSA. METHODS: We established the Men of African Descent and Carcinoma of the Prostate Network, which includes seven SSA centers partnering with five US centers to study the genetics and epidemiology of CaP in SSA. We developed common data elements and instruments, regulatory infrastructure, and biosample collection, processing, and shipping protocols. We tested this infrastructure by collecting epidemiologic, medical record, and genomic data from a total of 311 patients with CaP and 218 matched controls recruited at the seven SSA centers. We extracted genomic DNA from whole blood, buffy coat, or buccal swabs from 265 participants and shipped it to the Center for Inherited Disease Research (Baltimore, MD) and the Centre for Proteomics and Genomics Research (Cape Town, South Africa), where genotypes were generated using the UK Biobank Axiom Array. RESULTS: We used common instruments for data collection and entered data into the shared database. Double-entered data from pilot participants showed a 95% to 98% concordance rate, suggesting that data can be collected, entered, and stored with a high degree of accuracy. Genotypes were obtained from 95% of tested DNA samples (100% from blood-derived DNA samples) with high concordance across laboratories. CONCLUSION: We provide approaches that can produce high-quality epidemiologic and genomic data in multicenter studies of cancer in SSA.
Assuntos
Carcinoma/epidemiologia , Carcinoma/genética , Neoplasias da Próstata/epidemiologia , Neoplasias da Próstata/genética , Baltimore , População Negra , Carcinoma/patologia , Genômica , Genótipo , Humanos , Masculino , Próstata/patologia , Neoplasias da Próstata/patologia , África do Sul/epidemiologiaRESUMO
In June 2005, the work of the EU Integrated Project EuroPrevall was started. EuroPrevall is the largest research project on food allergy ever performed in Europe. Major aims of the project are to generate for the first time reliable data on the prevalence of food allergies across Europe and on the natural course of food allergy development in infants. Improvement of in vitro diagnosis of food allergies is another important aim of the project. The present review summarizes current knowledge about the clinical presentation of food allergy and critically reviews available diagnostic tools at the beginning of the project period. A major problem in diagnosis is a relatively poor 'clinical specificity', i. e. both positive skin tests and in vitro tests for specific IgE are frequent in sensitized subjects without food allergy symptoms. So far, no in vitro test reliably predicts clinical food allergy. EuroPrevall aims at improving the predictive value of such tests by proceeding from diagnosis based on allergen extracts to purified allergen molecules, taking into account the affinity of the IgE-allergen interaction, and evaluating the potential of biological in vitro tests such as histamine release tests or basophil activation tests including assays performed with permanently growing cell lines.
Assuntos
Hipersensibilidade Alimentar/diagnóstico , Imunoglobulina E/sangue , Alérgenos/imunologia , Hipersensibilidade Alimentar/classificação , Humanos , Testes Cutâneos , Terminologia como AssuntoRESUMO
Over the last few decades, the prevalence of allergic diseases has increased dramatically in developed nations. The resulting burden on health care systems worldwide has provoked a whole series of research initiatives among allergy experts and commercial companies that aim to develop novel tests to improve the diagnostic risk assessment and early preventive treatment of the disease. The advent of protein microarray technology has inspired the development of miniaturized immunological applications that permit the simultaneous analysis of huge numbers of disease-related parameters. Allergen microarrays have been developed for the monitoring of patient-specific antibody profiles to a previously unknown variety of allergens in a single analytical step. This has been accomplished by the successful adaptation of solid-phase antibody assays for the detection of surface-bound allergens to the microarray format, the development of appropriate assay conditions, and the improvement of software-guided microarray image analysis. Here we report a protocol for the development and analysis of food allergen microarrays.
Assuntos
Alérgenos/análise , Ovos/análise , Imunoensaio/métodos , Procedimentos Analíticos em Microchip/métodos , Leite/química , Animais , Bovinos , Galinhas , Humanos , Imunoensaio/instrumentação , Dispositivos Lab-On-A-Chip , SoftwareRESUMO
Type I allergy is an immunoglobulin E (IgE)-mediated hypersensitivity disease affecting more than 25% of the population. Currently, diagnosis of allergy is performed by provocation testing and IgE serology using allergen extracts. This process defines allergen-containing sources but cannot identify the disease-eliciting allergenic molecules. We have applied microarray technology to develop a miniaturized allergy test containing 94 purified allergen molecules that represent the most common allergen sources. The allergen microarray allows the determination and monitoring of allergic patients' IgE reactivity profiles to large numbers of disease-causing allergens by using single measurements and minute amounts of serum. This method may change established practice in allergy diagnosis, prevention, and therapy. In addition, microarrayed antigens may be applied to the diagnosis of autoimmune and infectious diseases.
Assuntos
Alérgenos/imunologia , Hipersensibilidade Imediata/diagnóstico , Análise de Sequência com Séries de Oligonucleotídeos/métodos , Alérgenos/genética , Alérgenos/isolamento & purificação , Humanos , Hipersensibilidade Imediata/imunologia , Hipersensibilidade Imediata/terapia , Imunoglobulina E/imunologia , Proteínas Recombinantes/imunologia , Proteínas Recombinantes/isolamento & purificaçãoRESUMO
Over the last few decades, the prevalence of allergic diseases has increased dramatically in developed nations. The resulting worldwide burden on healthcare systems has provoked a whole series of research initiatives among allergy experts and commercial companies that aim to develop novel tests to improve the diagnostic risk assessment and early preventive treatment of disease. The advent of protein microarray technology has fueled aspirations of multianalyte immunological applications that permit the simultaneous analysis of huge numbers of disease-related parameters that will hopefully become amenable in the near future. Allergen microarrays have been developed for the monitoring of patient-specific antibody profiles to a previously unknown variety of allergens in a single analytical step. This review describes significant discoveries and developments in allergy research against a background of the increasing prevalence of disease and hence the emerging challenges for national healthcare systems. The development of novel protein microarray-based allergy diagnostic tests is portrayed in concert with the recent advances and benefits of this technology, along with the challenges that must be met by manufacturers in order to succeed with innovative allergen microarrays in a highly competitive market.
Assuntos
Hipersensibilidade Imediata/diagnóstico , Hipersensibilidade/diagnóstico , Imunoglobulina E/imunologia , Análise Serial de Proteínas/métodos , Alérgenos/análise , Humanos , Hipersensibilidade/terapia , Hipersensibilidade Imediata/terapia , Análise Serial de Proteínas/instrumentação , Kit de Reagentes para Diagnóstico/economia , Kit de Reagentes para Diagnóstico/normasRESUMO
In the emerging field of Functional Proteomics, protein microarrays are considered to be one of the most promising tools for the simultaneous analysis of the a) abundance, b) function, and c) interaction of proteins on a system-wide scale. Resting on the technological grounds of widely used DNA biochips, the great power of microarray-based miniature solid-phase immunoassays lies in their potential to investigate in parallel large numbers of analyte pairs in a variety of biological samples. Consequently, this has fueled aspirations that protein microarrays may serve as tools for the high-throughput functional investigation of complete proteomes and, moreover, that they will develop into promising candidates for innovative in-vitro diagnostic (IVD) applications. To date, published examples of protein microarrays for IVD purposes have included tests for allergy, autoimmune and infectious diseases. Here, we discuss recent advancements in the development of protein microarrays for the profiling of IgE antibodies in the diagnosis of Type 1-related allergic diseases.
Assuntos
Doenças Autoimunes/diagnóstico , Hipersensibilidade/diagnóstico , Imunoglobulina E/imunologia , Análise Serial de Proteínas/métodos , Anticorpos/análise , Antígenos/análise , Doenças Autoimunes/fisiopatologia , Humanos , Hipersensibilidade/fisiopatologia , Infecções/diagnóstico , Infecções/fisiopatologia , Miniaturização , Análise Serial de Proteínas/instrumentação , Análise Serial de Proteínas/tendências , Kit de Reagentes para Diagnóstico/normas , Sensibilidade e EspecificidadeRESUMO
A number of antibiotics have been reported to disturb the decoding process in prokaryotic translation and to inhibit the function of various natural ribozymes. We investigated the effect of several antibiotics on in vitro splicing of a eukaryotic nuclear pre-mRNA (beta-globin). Of the eight antibiotics studied, erythromycin, Cl-tetracycline and streptomycin were identified as splicing inhibitors in nuclear HeLa cell extract. The K(i) values were 160, 180 and 230 microm, respectively. Cl-tetracycline-mediated and streptomycin-mediated splicing inhibition were in the molar inhibition range for hammerhead and human hepatitis delta virus ribozyme self-cleavage (tetracycline), of group-I intron self-splicing (streptomycin) and inhibition of RNase P cleavage by some aminoglycosides. Cl-tetracycline and the aminocyclitol glycoside streptomycin were found to have an indirect effect on splicing by unspecific binding to the pre-mRNA, suggesting that the inhibition is the result of disturbance of the correct folding of the pre-mRNA into the splicing-compatible tertiary structure by the charged groups of these antibiotics. The macrolide, erythromycin, the strongest inhibitor, had only a slight effect on formation of the presplicing complexes A and B, but almost completely inhibited formation of the splicing-active C complex by binding to nuclear extract component(s). This results in direct inhibition of the second step of pre-mRNA splicing. To our knowledge, this is the first report on specific inhibition of nuclear splicing by an antibiotic. The functional groups involved in the interaction of erythromycin with snRNAs and/or splicing factors require further investigation.
Assuntos
Antibacterianos/farmacologia , Núcleo Celular/metabolismo , Precursores de RNA/genética , Splicing de RNA/efeitos dos fármacos , Catálise , Clortetraciclina/farmacologia , Eritromicina/farmacologia , Estreptomicina/farmacologiaRESUMO
Diagnosis of type I allergy is based on anamnesis, provocation testing, and serological determination of total and specific IgE. Currently, in vivo and in vitro diagnostic tests employ allergen extracts prepared from various allergen sources (e.g., pollen, mites, animal dander, moulds, foods, venoms, etc.). The application of recombinant DNA technology to the field of allergen characterization has allowed to reveal the molecular nature of the most common allergens. To date a continuously increasing number of allergen sequences has become available and panels of recombinant allergens-assembling the epitope complexity of natural allergens sources-can be produced. The use of recombinant allergens instead of crude, natural extracts for allergy diagnosis allows us to determine the individual IgE reactivity profile of each patient. To enable a comprehensive analysis of the patient's IgE binding pattern to a large number of individual allergens, a new type of serological test is required. In this paper, we applied microarray technology to create a multi-allergen test system, based on microarrayed recombinant allergens.