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Next-generation risk assessment relies on mechanistic data from new approach methods, including transcriptome data. Various technologies, such as high-throughput targeted sequencing methods and microarray technologies based on hybridization with complementary probes, are used to determine differentially expressed genes (DEGs). The integration of data from different technologies requires a good understanding of the differences arising from the use of various technologies.To better understand the differences between the TempO-Seq platform and Affymetrix chip technology, whole-genome data for the volatile compound dimethylamine were compared. Selected DEGs were also confirmed using RTqPCR validation. Although the overlap of DEGs between TempO-Seq and Affymetrix was no higher than 37%, a comparison of the gene regulation in terms of log2fold changes revealed a very high concordance. RTqPCR confirmed the majority of DEGs from either platform in the examined dataset. Only a few conflicts were found (11%), while 22% were not confirmed, and 3% were not detected.Despite the observed differences between the two platforms, both can be validated using RTqPCR. Here we highlight some of the differences between the two platforms and discuss their applications in toxicology.
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Perfilación de la Expresión Génica , Análisis de Secuencia por Matrices de Oligonucleótidos , Análisis de Secuencia por Matrices de Oligonucleótidos/métodos , Perfilación de la Expresión Génica/métodos , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Humanos , Reacción en Cadena en Tiempo Real de la Polimerasa/métodosRESUMEN
OBJECTIVES: An integrated in vitro inhalation approach was outlined to estimate potential adverse acute inhalation effects of aerosols from commercial nebulizer applications used for purposeful room conditioning such as disinfection, scenting or others. Aerosol characterization, exposure estimation and evaluation of acute biological effects by in vitro inhalation were included to generate dose-response data, allowing for determination of in vitro lowest observable adverse effect levels (LOAELs). Correlation of these to estimates of human lung deposition was included for quantitative in vitro to in vivo extrapolation approach (QIVIVE) for acute effects during human exposure. METHODS: To test the proposed approach, a case study was undertaken using two realistic test materials. An acute in vitro inhalation setup with air-liquid interface A549-cells in an optimized exposure situation (P.R.I.T.® ExpoCube®) was used to expose cells and analysis of relevant biological effects (viability, mitochondrial membrane potential, stress, IL-8 release) was carried out. RESULTS: The observed dose-responsive effects in a sub-toxic dose-range could be attributed to the main component of one test material and its presence in the aerosol phase of the nebulized material. QIVIVE resulted in a factor of at least 256 between the in vitro LOAEL and the estimated acute human lung exposure for this test material. CONCLUSIONS: The case-study shows the value of the non-target in vitro inhalation testing approach especially in case of a lack of knowledge on complex product composition. It is expected that approaches like this will be of high value for product safety and environmental health in the future.
Design of a routine in vitro inhalation approach to estimate biological effects of nebulized products.Application in a case study on a potential real product for purposeful room conditioning by use of a commercial nebulizer.Combining results from aerosol characterization and in vitro inhalation experiments allowed for comprehensive correlation of product composition, aerosol properties and biological effects.Assignment of sub-toxic biological effects to a specific product component enabled identification of a product composition with potentially even less biological effect.Combined in vivo exposure estimation and in vitro LOAEL determination enabled a QIVIVE approach.
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Pulmón , Nebulizadores y Vaporizadores , Humanos , Aerosoles , Administración por InhalaciónRESUMEN
This case study explores the applicability of transcriptome data to characterize a common mechanism of action within groups of short-chain aliphatic α-, ß-, and γ-diketones. Human reference in vivo data indicate that the α-diketone diacetyl induces bronchiolitis obliterans in workers involved in the preparation of microwave popcorn. The other three α-diketones induced inflammatory responses in preclinical in vivo animal studies, whereas beta and gamma diketones in addition caused neuronal effects. We investigated early transcriptional responses in primary human bronchiolar (PBEC) cell cultures after 24 h and 72 h of air-liquid exposure. Differentially expressed genes (DEGs) were assessed based on transcriptome data generated with the EUToxRisk gene panel of Temp-O-Seq®. For each individual substance, genes were identified displaying a consistent differential expression across dose and exposure duration. The log fold change values of the DEG profiles indicate that α- and ß-diketones are more active compared to γ-diketones. α-diketones in particular showed a highly concordant expression pattern, which may serve as a first indication of the shared mode of action. In order to gain a better mechanistic understanding, the resultant DEGs were submitted to a pathway analysis using ConsensusPathDB. The four α-diketones showed very similar results with regard to the number of activated and shared pathways. Overall, the number of signaling pathways decreased from α-to ß-to γ-diketones. Additionally, we reconstructed networks of genes that interact with one another and are associated with different adverse outcomes such as fibrosis, inflammation or apoptosis using the TRANSPATH-database. Transcription factor enrichment and upstream analyses with the geneXplain platform revealed highly interacting gene products (called master regulators, MRs) per case study compound. The mapping of the resultant MRs on the reconstructed networks, visualized similar gene regulation with regard to fibrosis, inflammation and apoptosis. This analysis showed that transcriptome data can strengthen the similarity assessment of compounds, which is of particular importance, e.g., in read-across approaches. It is one important step towards grouping of compounds based on biological profiles.
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In the United States, rural populations comprise 60 million individuals and suffered from high COVID-19 disease burdens. Despite this, surveillance efforts are biased toward urban centers. Consequently, how rurally circulating SARS-CoV-2 viruses contribute toward emerging variants remains poorly understood. In this study, we aim to investigate the role of rural communities in the evolution and transmission of SARS-CoV-2 during the early pandemic. We collected 544 urban and 435 rural COVID-19-positive respiratory specimens from an overall vaccine-naïve population in Southwest Missouri between July and December 2020. Genomic analyses revealed 53 SARS-CoV-2 Pango lineages in our study samples, with 14 of these lineages identified only in rural samples. Phylodynamic analyses showed that frequent bi-directional diffusions occurred between rural and urban communities in Southwest Missouri, and that four out of seven Missouri rural-origin lineages spread globally. Further analyses revealed that the nucleocapsid protein (N):R203K/G204R paired substitutions, which were detected disproportionately across multiple Pango lineages, were more associated with urban than rural sequences. Positive selection was detected at N:204 among rural samples but was not evident in urban samples, suggesting that viruses may encounter distinct selection pressures in rural versus urban communities. This study demonstrates that rural communities may be a crucial source of SARS-CoV-2 evolution and transmission, highlighting the need to expand surveillance and resources to rural populations for COVID-19 mitigation.
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As SARS-CoV-2 and influenza viruses co-circulate, co-infections with these viruses generate an increasing concern to public health. To evaluate the prevalence and clinical impacts of SARS-CoV-2 and influenza A virus co-infections during the 2021-2022 influenza season, SARS-CoV-2-positive samples from 462 individuals were collected from October 2021 to January 2022. Of these individuals, 152 tested positive for influenza, and the monthly co-infection rate ranged from 7.1% to 48%. Compared to the Delta variant, individuals infected with Omicron were less likely to be co-infected and hospitalized, and individuals who received influenza vaccines were less likely to become co-infected. Three individuals had two samples collected on different dates, and all three developed a co-infection after their initial SARS-CoV-2 infection. This study demonstrates high prevalence of co-infections in central Missouri during the 2021-2022 influenza season, differences in co-infection prevalence between the Delta and the Omicron waves, and the importance of influenza vaccinations against co-infections.
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COVID-19 , Coinfección , Virus de la Influenza A , Vacunas contra la Influenza , Gripe Humana , Humanos , Gripe Humana/epidemiología , SARS-CoV-2 , Coinfección/epidemiología , Estudios Transversales , Estaciones del Año , Missouri/epidemiología , COVID-19/epidemiología , Virus de la Influenza A/genéticaAsunto(s)
COVID-19/virología , Reinfección/virología , SARS-CoV-2/genética , COVID-19/diagnóstico , Femenino , Genes Virales/genética , Variación Genética , Humanos , Filogenia , ARN Viral/genética , Reinfección/diagnóstico , SARS-CoV-2/clasificación , SARS-CoV-2/aislamiento & purificación , Adulto JovenRESUMEN
Inpatient coronavirus disease 2019 (COVID-19) cases present enormous costs to patients and health systems in the United States. Many hospitalized patients may continue testing COVID-19 positive even after the resolution of symptoms. Thus, a pressing concern for clinicians is the safety of discharging these asymptomatic patients if they have any remaining infectivity. This case report explores the viral viability in a patient with persistent COVID-19 over the course of a 2-month hospitalization. Positive nasopharyngeal swab samples were collected and isolated in the laboratory and analyzed by quantitative reverse-transcription polymerase chain reactions (qRT-PCR), and serology was tested for neutralizing antibodies throughout the hospitalization period. The patient experienced waning symptoms by hospital day 40 and had no viable virus growth by hospital day 41, suggesting no risk of infectivity, despite positive RT-PCR results which prolonged his hospital stay. Notably, this case showed infectivity for at least 24 days after disease onset, which is longer than the discontinuation of transmission-based precautions recommended by the Center for Disease Control and Prevention. Thus, our findings suggest that the timeline for discontinuing transmission-based precautions may need to be extended for patients with severe and prolonged COVID-19 disease. Additional large-scale studies are needed to draw definitive conclusions on the appropriate clinical management for these patients. â.
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Anticuerpos Neutralizantes/sangre , Anticuerpos Antivirales/sangre , Prueba de Ácido Nucleico para COVID-19 , COVID-19/diagnóstico , Esparcimiento de Virus/fisiología , Anciano , Infecciones Asintomáticas , Humanos , Masculino , ARN Viral/análisis , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , SARS-CoV-2/genética , SARS-CoV-2/inmunologíaRESUMEN
The long-lasting global COVID-19 pandemic demands timely genomic investigation of SARS-CoV-2 viruses. Here, we report a simple and efficient workflow for whole-genome sequencing utilizing one-step reverse transcription-PCR (RT-PCR) amplification on a microfluidic platform, followed by MiSeq amplicon sequencing. The method uses Fluidigm integrated fluidic circuit (IFC) and instruments to amplify 48 samples with 39 pairs of primers, including 35 custom-designed primer pairs and four additional primer pairs from the ARTIC network protocol v3. Application of this method on RNA samples from both viral isolates and clinical specimens demonstrates robustness and efficiency in obtaining the full genome sequence of SARS-CoV-2.
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Genoma Viral , Secuenciación de Nucleótidos de Alto Rendimiento , Microfluídica , SARS-CoV-2/genética , Secuenciación Completa del Genoma , COVID-19/virología , Cartilla de ADN , Humanos , ARN Viral/genética , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
The severe acute respiratory syndrome coronavirus 2 (SARS-COV-2) Spike glycoprotein is solely responsible for binding to the host cell receptor and facilitating fusion between the viral and host membranes. The ability to generate viral particles pseudotyped with SARS-COV-2 Spike is useful for many types of studies, such as characterization of neutralizing antibodies or development of fusion-inhibiting small molecules. Here, we characterized the use of a codon-optimized SARS-COV-2 Spike glycoprotein for the generation of pseudotyped HIV-1, murine leukemia virus (MLV), and vesicular stomatitis virus (VSV) particles. The full-length Spike protein functioned inefficiently with all three systems but was enhanced over 10-fold by deleting the last 19 amino acids of the cytoplasmic tail. Infection of 293FT target cells was possible only if the cells were engineered to stably express the human angiotensin-converting enzyme 2 (ACE2) receptor, but stably introducing an additional copy of this receptor did not further enhance susceptibility. Stable introduction of the Spike-activating protease TMPRSS2 further enhanced susceptibility to infection by 5- to 10-fold. Replacement of the signal peptide of the Spike protein with an optimal signal peptide did not enhance or reduce infectious particle production. However, modifications D614G and R682Q further enhanced infectious particle production. With all enhancing elements combined, the titer of pseudotyped HIV-1 particles reached almost 106 infectious particles/ml. Finally, HIV-1 particles pseudotyped with SARS-COV-2 Spike were successfully used to detect neutralizing antibodies in plasma from coronavirus disease 2019 (COVID-19) patients, but not in plasma from uninfected individuals.IMPORTANCE In work with pathogenic viruses, it is useful to have rapid quantitative tests for viral infectivity that can be performed without strict biocontainment restrictions. A common way of accomplishing this is to generate viral pseudoparticles that contain the surface glycoprotein from the pathogenic virus incorporated into a replication-defective viral particle that contains a sensitive reporter system. These pseudoparticles enter cells using the glycoprotein from the pathogenic virus, leading to a readout for infection. Conditions that block entry of the pathogenic virus, such as neutralizing antibodies, will also block entry of the viral pseudoparticles. However, viral glycoproteins often are not readily suited for generating pseudoparticles. Here, we describe a series of modifications that result in the production of relatively high-titer SARS-COV-2 pseudoparticles that are suitable for the detection of neutralizing antibodies from COVID-19 patients.
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Betacoronavirus/fisiología , Infecciones por Coronavirus/virología , Neumonía Viral/virología , Glicoproteína de la Espiga del Coronavirus/fisiología , Enzima Convertidora de Angiotensina 2 , Anticuerpos Neutralizantes/inmunología , Anticuerpos Antivirales/inmunología , Betacoronavirus/genética , Betacoronavirus/inmunología , Betacoronavirus/metabolismo , COVID-19 , Infecciones por Coronavirus/inmunología , Infecciones por Coronavirus/metabolismo , Células HEK293 , VIH-1/genética , VIH-1/metabolismo , Humanos , Virus de la Leucemia Murina , Pandemias , Peptidil-Dipeptidasa A/metabolismo , Neumonía Viral/inmunología , Neumonía Viral/metabolismo , SARS-CoV-2 , Serina Endopeptidasas/metabolismo , Glicoproteína de la Espiga del Coronavirus/genética , Glicoproteína de la Espiga del Coronavirus/inmunología , Glicoproteína de la Espiga del Coronavirus/metabolismo , Virus de la Estomatitis Vesicular Indiana/genética , Virus de la Estomatitis Vesicular Indiana/metabolismo , Virión/genética , Virión/inmunología , Virión/metabolismo , Internalización del VirusRESUMEN
Diagnostic tests for the coronavirus infection 2019 (COVID-19) are critical for prompt diagnosis, treatment and isolation to break the cycle of transmission. A positive real-time reverse-transcriptase polymerase chain reaction (RT-PCR), in conjunction with clinical and epidemiologic data, is the current standard for diagnosis, but several challenges still exist. Serological assays help to understand epidemiology better and to evaluate vaccine responses but they are unreliable for diagnosis in the acute phase of illness or assuming protective immunity. Serology is gaining attention, mainly because of convalescent plasma gaining importance as treatment for clinically worsening COVID-19 patients. We provide a narrative review of peer-reviewed research studies on RT-PCR, serology and antigen immune-assays for COVID-19, briefly describe their lab methods and discuss their limitations for clinical practice.
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Betacoronavirus/aislamiento & purificación , Técnicas de Laboratorio Clínico/normas , Infecciones por Coronavirus/diagnóstico , Neumonía Viral/diagnóstico , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa/normas , COVID-19 , Prueba de COVID-19 , Vacunas contra la COVID-19 , Humanos , Técnicas de Diagnóstico Molecular/normas , Pandemias , SARS-CoV-2RESUMEN
Due to the increasing need of new treatment options against bacterial lung infections, novel antimicrobial peptides (AMPs) are under development. Local bioavailability and less systemic exposure lead to the inhalation route of administration. Combining AMPs with nanocarriers (NCs) into nanosystems (NSs) might be a technique for improved results. An air-liquid interface (ALI) in vitro inhalation model was set up including a human alveolar lung cell line (A549) and an optimized exposure system (P.R.I.T.® ExpoCube®) to predict acute local lung toxicity. The approach including aerosol controls (cupper-II-sulfate and lactose) delivered lowest observable adverse effect levels (LOAELs). Different combinations of AMPs (AA139, M33) and NCs (polymeric nanoparticles (PNPs), micelles and liposomes) were tested under ALI and submerged in vitro conditions. Depending on the nature of AMP and NCs, packing of AMPs into NSs reduced the AMP-related toxicity. Large differences were found between the LOAELs determined by submerged or ALI testing with the ALI approach indicating higher sensitivity of the ALI model. Since aerosol droplet exposure is in vivo relevant, it is assumed that ALI based results represents the more significant source than submerged testing for in vivo prediction of local acute lung toxicity. In accordance with the current state-of-the-art view, this study shows that ALI in vitro inhalation models are promising tools to further develop in vitro methods in the field of inhalation toxicology.
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Antibacterianos/toxicidad , Nanopartículas/toxicidad , Péptidos/toxicidad , Células A549 , Aerosoles , Antibacterianos/administración & dosificación , Infecciones Bacterianas/tratamiento farmacológico , Supervivencia Celular/efectos de los fármacos , Humanos , Liposomas , Pulmón/efectos de los fármacos , Enfermedades Pulmonares/tratamiento farmacológico , Metacrilatos/administración & dosificación , Metacrilatos/toxicidad , Micelas , Nanopartículas/administración & dosificación , Nylons/toxicidad , Péptidos/administración & dosificaciónRESUMEN
In vitro air-liquid interface (ALI) cell culture models can potentially be used to assess inhalation toxicology endpoints and are usually considered, in terms of relevancy, between classic (i.e., submerged) in vitro models and animal-based models. In some situations that need to be clearly defined, ALI methods may represent a complement or an alternative option to in vivo experimentations or classic in vitro methods. However, it is clear that many different approaches exist and that only very limited validation studies have been carried out to date. This means comparison of data from different methods is difficult and available methods are currently not suitable for use in regulatory assessments. This is despite inhalation toxicology being a priority area for many governmental organizations. In this setting, a 1-day workshop on ALI in vitro models for respiratory toxicology research was organized in Paris in March 2016 to assess the situation and to discuss what might be possible in terms of validation studies. The workshop was attended by major parties in Europe and brought together more than 60 representatives from various academic, commercial, and regulatory organizations. Following plenary, oral, and poster presentations, an expert panel was convened to lead a discussion on possible approaches to validation studies for ALI inhalation models. A series of recommendations were made and the outcomes of the workshop are reported.
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Higher throughput methods, high content analysis and automated screening methods are of highest demand in drug development today. In toxicology, these strategies are becoming increasingly important, as well. Therefore, an integrated higher throughput method for the comet assay is addressed by the development presented here. The sensitivity, specificity and relevance of the comet assay as a method for determination of DNA damage in vivo and in vitro have been highlighted in many studies. Actually, efforts are made to include it in a panel of genotoxicity tests for regulatory purposes. However, the standard comet assay is a time consuming procedure due to the specific methods needed. The improvements presented here lead to a faster and easier slide-production, a smaller amount of cells needed, a higher amount of comets quantified, a fully automated analysis of comets including reanalysis, storing, visualisation and documentation possibilities using standard comet quantification models such as tail length or tail moment, and - by introduction of clearly definable selection criteria based on image analysis algorithms - clearly improve objectivity and standardization of the analysis procedure. Results prove the high reproducibility, flexibility, efficiency and suitability of the procedure as a fully automated analysis method in higher throughput genotoxicity testing in vitro.
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Ensayo Cometa/métodos , Ensayos Analíticos de Alto Rendimiento/métodos , Daño del ADN , Humanos , Procesamiento de Imagen Asistido por ComputadorRESUMEN
Although aspirin is cardioprotective in high-risk populations, many with diabetes mellitus (DM) are unresponsive to these benefits. We questioned whether cardiovascular unresponsiveness might be demonstrated by lack of aspirin sensitivity to in vitro platelet functions especially in subjects with poorly controlled diabetes. Six women and 4 men (48+/-8 years [mean+/-S.D.]), selected for poor control (glycohemoglobin 11.9+/-2.2%) and 10 sex-age (+/-5 years) matched controls received 81 mg aspirin daily. There was a 2-week washout from aspirin and related drugs. After the aspirin dose on day-7, blood for platelet aggregation assays, and 24-h urine for 2,3 dinor thromboxane B2 (TxB2) and 2,3 dinor 6-keto (PGF1alpha) were obtained. Aspirin sensitivity was defined as inhibition (i.e., lower than expected) platelet aggregation after exposure to an agonist. Those with diabetes and controls were sensitive to aspirin inhibition of platelet aggregation induced by 1.6 mM arachidonic acid (9.5+/-3.9% versus 9.1+/-3.1%, normal range 40-100%) and by 0.83 microg/mL collagen (17.4+/-13.9% versus 13.2+/-9.3%, normal range 60-93%), respectively. Aspirin sensitivity to 2 microM ADP was present in five with diabetes and five controls. Urinary prostaglandin metabolites were suppressed below reference ranges, without differences between those with DM or controls for TxB2 (350+/-149 pg/mg versus 348+/-93 pg/mg creatinine) and PGF1alpha (255+/-104 pg/mg versus 222+/-88 pg/mg creatinine). In conclusion, in poorly controlled diabetes, there was no differential lack of aspirin sensitivity to platelet aggregation, or lack of aspirin suppression of urinary TxB2 or PGF1alpha, compared with controls on aspirin. Despite suppression of urinary prostaglandin metabolites, aspirin resistance was most apparent to ADP-mediated platelet aggregation. It is not known what level of inhibition of in vitro tests is necessary for the cardioprotective benefits of aspirin in diabetes mellitus. Thus, the lack of aspirin protection in diabetes may be due to undefined aspects of platelet function.
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Aspirina/farmacología , Diabetes Mellitus Tipo 1/fisiopatología , Diabetes Mellitus Tipo 2/fisiopatología , Agregación Plaquetaria/efectos de los fármacos , Cardiotónicos/farmacología , Diabetes Mellitus Tipo 1/sangre , Diabetes Mellitus Tipo 2/sangre , Femenino , Humanos , Masculino , Inhibidores de Agregación Plaquetaria/farmacologíaRESUMEN
Three cigarette types were compared using an experimental approach for quantifying selected toxicological effects of diluted fresh whole cigarette mainstream smoke in vitro. The test procedure involved automatic smoking of cigarettes according to the FTC/ISO standard, online monitoring of generated smoke aerosols with respect to particulate and gas-phase components, and direct exposure of a human type II-like lung cell line (A549) using exposure conditions relevant to human smoking. Test specimens were the K1R4F standard research cigarettes (9.2 mg tar/cigarette) and two commercial European light filter cigarettes (brand 1, brand 2) having the same tar content (7.0 mg/cigarette). As a representative of the toxicological effect of smoke, intracellular reduced glutathione was analyzed directly after exposure of cells. Results revealed statistically significant different quantitative effects with regard to glutathione depletion when comparing whole smoke and filtered smoke from all three cigarettes. ED50 values revealed a depletion of reduced glutathione by brand 1 cigarettes that was more than twice the depletion caused by brand 2 cigarettes on a per cigarette basis. Also, quantitatively different effects were found on a per particle and on a per CO concentration basis using whole or filtered smoke from the cigarettes. We conclude that the methods we employed provide sensitive and reproducible ways of detecting differences in the toxicological action of smoke from various types of cigarettes.
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Nicotiana , Plantas Tóxicas , Alveolos Pulmonares/efectos de los fármacos , Humo/efectos adversos , Breas/toxicidad , Línea Celular Tumoral , Relación Dosis-Respuesta a Droga , Glutatión/metabolismo , Humanos , Exposición por Inhalación , Alveolos Pulmonares/metabolismo , Humo/análisis , Breas/análisis , Nicotiana/químicaRESUMEN
BACKGROUND: Testing for troponin has important clinical value for patients who present with typical symptoms of acute coronary syndromes (ACS) such as chest pain (CP). Much less is known about the value of troponin testing for patients who present with other symptoms of ACS (anginal equivalent symptoms). METHODS: The utilization and prognostic value of cardiac troponin I (cTnI) were evaluated at a Veterans Affairs Acute Care Facility. Clinical charts of 1184 predominantly male patients, who submitted specimens for initial cTnI testing by AxSYM, were evaluated for demographic data, cardiovascular risk factors, major diseases, and complaints at the time of testing. The endpoint was defined as all-cause death during a 200-day period after initial testing. RESULTS: Sixty-one percent of cTnI tests were ordered for patients who did not present with CP. Patients presenting with symptoms other than CP did not have significantly lower plasma cTnI than patients with CP. However, patients with symptoms other than CP were rarely diagnosed with ACS unless cTnI was >/=2 microg/L. The mortality during the follow-up period was severalfold higher among patients presenting with symptoms other than CP (CP, 6%; without CP, 22%; P <0.0001, chi(2) test). cTnI >/=0.2 microg/L provided significant additional predictive information for patients who presented with anginal equivalent symptoms such as shortness of breath or general weakness. CONCLUSION: Patients with anginal equivalent symptoms of ACS and low-positive cTnI are less often diagnosed with ACS and have a higher mortality than patients with CP.
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Enfermedad Coronaria/diagnóstico , Troponina I/sangre , Enfermedad Aguda , Angina de Pecho/diagnóstico , Dolor en el Pecho/diagnóstico , Dolor en el Pecho/mortalidad , Enfermedad Coronaria/mortalidad , Femenino , Insuficiencia Cardíaca/diagnóstico , Humanos , Masculino , Persona de Mediana Edad , PronósticoRESUMEN
Numerous approaches have been employed for testing the biological activity of cigarette smoke in vitro. None of them has managed to expose cultured lung cells in a realistic manner to the complex gaseous and particulate mixture that constitutes cigarette smoke. We have devised a system that makes this possible. The system presented here enables the direct exposure of human lung cells to native, unmodified cigarette mainstream smoke. It consists of a smoking machine, a dilution device for the smoke, analytical devices for online monitoring and a specially adapted exposure module based on the Cultex** cell cultivation system that is equipped with a gas-exposure top. Due to the special design of the exposure device and the optimised exposure conditions, this equipment allows cultured human lung cells to be exposed to freshly generated cigarette mainstream smoke. Exploratory experiments revealed that the smoke could be diluted over a wide concentration range in a reproducible way with respect to gas and particulate phases, and also demonstrated reproducible particle deposition depending on smoke concentration. Furthermore, it was shown that the exposed cells maintained their viability. Native cigarette mainstream smoke induced dose-dependent cellular effects in exposed cells with respect to cellular viability (viable cell number monitored by tetrazolium salt cleavage) and intracellular parameters (ATP and glutathione content). Therefore, fresh, physically and chemically unmodified cigarette mainstream smoke can be tested using this novel system.
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Bronquios/efectos de los fármacos , Contaminación por Humo de Tabaco/efectos adversos , Pruebas de Toxicidad/métodos , Aerosoles/administración & dosificación , Bronquios/metabolismo , Bronquios/patología , Línea Celular , Supervivencia Celular/efectos de los fármacos , Células Epiteliales/efectos de los fármacos , Células Epiteliales/metabolismo , Células Epiteliales/patología , Humanos , Técnicas In Vitro , Mucosa Respiratoria/efectos de los fármacos , Mucosa Respiratoria/metabolismo , Mucosa Respiratoria/patología , Método Simple Ciego , Sales de Tetrazolio/metabolismo , Factores de Tiempo , Pruebas de Toxicidad/instrumentaciónRESUMEN
An experimental approach was established for the validation of an in vitro test system for complex environmental test atmospheres consisting of both gaseous substances and particulates. Smoke from two different cigarette types (generated by an automatic cigarette-smoking machine) was employed to assess both the sensitivity and the specificity of the system. The smoke was diluted with synthetic air and used to expose human lung cells grown on microporous membranes. Cells were exposed alternately to diluted cigarette smoke and pure synthetic air. The effect of diluted smoke was assessed without humidification, addition of CO2, or any other physical or chemical modification of the smoke. The experimental setup included online monitoring of the gas phase (by analysis of CO concentration) and particulate phase (by light-scattering photometry). Replicate experiments confirmed a reproducible generation and dilution of the smoke and a smoke age of about 7 s at the time it came into contact with the cells. Experiments using human lung cells revealed that smoke from the two different cigarette types induced different levels of dose-dependent toxicity. A cell exposure of 6 min using 6 alternating smoke and synthetic air periods was sufficient to cause different effects as measured by intracellular glutathione content. The fact that the system could differentiate between two different types of cigarette smoke demonstrated its high sensitivity and specificity. The system offers new ways to test native complex gaseous and aerosol mixtures in vitro using short exposure times and very small amounts of test substances.
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Aire , Exposición por Inhalación/efectos adversos , Pulmón/efectos de los fármacos , Contaminación por Humo de Tabaco/efectos adversos , Aerosoles , Línea Celular , Supervivencia Celular/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Humanos , Pulmón/citología , Sensibilidad y Especificidad , Factores de TiempoRESUMEN
Persistent elevation of aspartate aminotransferase (AST) activity in serum due to the presence of a macroenzyme form of AST (macro-AST) may lead to diagnostic confusion in many clinical conditions, particularly those associated with chronic liver disease. We describe a case of macro-AST arising in an adult female with a false-positive hepatitis C virus (HCV) RNA test result that was not accompanied by other biochemical or histologic evidence of liver disease. The presence of macro-AST in serum was confirmed utilizing size-exclusion, high performance liquid chromatography (HPLC) and Protein G-agarose beads to precipitate immune complexes of AST and immunoglobulin G followed by centrifugation and AST activity measurements in the supernatant. A brief review of the clinical enzymology of AST and methods used to quantify serum macro-AST activity is provided.