RESUMO
A pathogen inactivation (PI) process has been developed using the frangible anchor linker effector (FRALE) compound S-303. A series of experiments were performed in whole blood (WB) to measure the level of viral and bacterial inactivation. The results showed that 0.2mM S-303 and 2mM glutathione (GSH) inactivated >6.5 logs of HIV, >5.7 logs of Bluetongue virus, >7.0 logs of Yersinia enterocolitica, 4.2 logs of Serratia marcescens, and 7.5 logs of Staphylococcus epidermidis. Recent development for S-303 is focused on optimization of the PI process for red blood cell concentrates (RBC). A series of studies in RBC showed that 0.2mM S-303 and 20mM GSH inactivated approximately 5 logs or greater of Y. enterocolitica, E. coli, S. marcescens, S. aureus, HIV, bovine viral diarrhoea virus, bluetongue virus and human adenovirus 5. In both applications of the S-303 process, in vitro parameters of RBC function and physiology were retained compared to conventional RBC. Results from these studies indicate that S-303 can be applicable for PI of RBC and WB.
Assuntos
Acridinas/farmacologia , Preservação de Sangue/métodos , Patógenos Transmitidos pelo Sangue , Sangue/efeitos dos fármacos , Eritrócitos/efeitos dos fármacos , Compostos de Mostarda Nitrogenada/farmacologia , Alquilantes/farmacologia , Animais , Sangue/microbiologia , Sangue/virologia , Patógenos Transmitidos pelo Sangue/isolamento & purificação , Bovinos , Células Cultivadas , Contagem de Colônia Microbiana , Desinfetantes/farmacologia , Desinfecção/métodos , Eritrócitos/microbiologia , Eritrócitos/virologia , Escherichia coli/efeitos dos fármacos , Escherichia coli/fisiologia , Estudos de Viabilidade , Humanos , Análise por Pareamento , Controle de Qualidade , Staphylococcaceae/efeitos dos fármacos , Staphylococcaceae/fisiologia , Yersinia enterocolitica/efeitos dos fármacos , Yersinia enterocolitica/fisiologiaRESUMO
Immunochromatographic assays have become popular diagnostic tools in a variety of settings because they are sensitive, fast, and easy to use. Here, we describe the use of a novel reporter, upconverting phosphors (UCP), in this assay format. UCP are submicron-sized, inorganic crystals that are excited with infrared light and that emit photons in the visible range depending on the ion composition of the crystal. Using human chorionic gonadotropin (hCG) as a model analyte to describe the properties of phosphors in immunochromatographic assays, a detection limit of 10 pg hCG in a 100-microl sample has been achieved on a regular basis, with occasional detection of 1 pg hCG. This represents at least a 10-fold improvement over conventional reporter systems such as colloidal gold or colored latex beads. Quantitation of analytes is possible over at least 3 orders of magnitude. Furthermore, an example is given of how UCP can be used for analyte multiplexing using a two-plexed wick for the detection of mouse IgG and ovalbumin. Thus, UCP lateral flow assays can be used for applications that are currently limited by assay sensitivity, and they can increase the probability of a diagnosis by verifying the presence of several analytes in the same sample.
Assuntos
Cromatografia Líquida/métodos , Compostos de Fósforo , Gonadotropina Coriônica/análise , Humanos , Microscopia Eletrônica de Varredura , Sensibilidade e EspecificidadeRESUMO
The induction of cytochrome P450IA1 (CYP1A1) activity in human hepatoma cell lines has been used as a model response for exposure to the environmental contaminant, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). CYP1A1 induction is mediated by the intracellular Ah receptor. We have designed a cell culture analogue system to evaluate the effect of low dose continuous exposures to TCDD such that the responses can be compared with traditional static in vitro exposures. The two-compartment model is designed to mimic aspects of time-dependent dose exposure in humans and consists of a 'liver' compartment with HepG2 cells and an 'other tissues' compartment. Exposure of microcarrier-attached HepG2 cells in spinner flask to a single dose of TCDD resulted in an EC(50) for ethoxyresorufin-o-deethylase (EROD) activity induction of 1.49nm. Using a one-compartment reactor with continuous TCDD dosing resulted in an EC(50) for EROD induction of 0.69nm TCDD. Finally, using a two-compartment reactor with continuous TCDD dosing resulted in an EC(50) for EROD induction of about 0.05nm. Parallel studies using (3)H-TCDD were performed to determine the amount of cell associated (3)H-TCDD and subsequently to estimate the amount of bound Ah receptor. CYPIA1 mediated EROD activities in both cell lines correlated with the estimated amount of bound Ah receptor. To illustrate how these systems might alter estimates of risk, the data were evaluated to estimate the TCDD level which would increase CYPIA1 mediated EROD activity to 0.01% of the maximal response. The two-compartment reactor data yielded an estimate of allowable TCDD exposure of 1x10(-5)nm for an acceptable 'risk' level of 0.01%. In contrast, values were estimated as 4x10(-4)nm from the one compartment reactor and 4x10(-3)nm from spinner flasks. This work demonstrates the importance of design of the in vitro system on risk assessment.
RESUMO
We have previously reported that a microcarrier-attached human hepatoma (Hep G2) cell line responds to hydrodynamic shear upon transfer to an agitated, clean, autoclaved spinner flask with a transient increase in cytochrome P450IA1 (CYPIA1) activity. Physiological changes induced by hydrodynamic stress could be problematic in the scaleup of microcarrier cultures. A better understanding of how stress alters cell physiology may assist in reactor scaleup. The induction of CYPIA1 activity was dependent on the agitation level of the cultures, and the level of CYPIA1 induction was comparable to that obtained with exposure to approximately 0.1 nM TCDD (2, 3, 7, 8-tetrachlorodibenzo-p-dioxin). It has been well documented that hydrodynamic shear stress can cause alterations in the metabolism of phospholipid membrane-bound arachidonic acid (AA) in adherent cells in a parallel plate system. The present study was carried out to determine if either AA or a metabolite of AA was involved in the induction of CYPIA1 activity in the microcarrier cultures of Hep G2 cells. Addition of exogenous AA followed by initiation of the stress resulted in an increase in the level of CYPIA1 activity. Pretreatment of the cultures with quinacrine, an inhibitor of phospholipase A2, reduced the stress-induced CYPIA1 activity. Furthermore, addition of propranolol, an inhibitor of phosphatidic acid phosphohydrolase, resulted in an increase in the response in addition to sustaining the induced enzyme activity. Pretreatment with the cyclooxygenase inhibitor, indomethacin, or the lipoxygenase inhibitor, caffeic acid, had no effect on the response, suggesting that the cyclooxygenase and lipoxygenase pathways were not involved in generating AA metabolites that alter CYPIA1 activity. The agent, nordihydroguaiaretic acid, blocks the monooxygenase pathway and blocks CYPIA1 activity increases. These observations suggest a possible mechanism where the stress on the cells induces phospholipase D, resulting in the formation of phosphatidic acid which then activates phospholipase A2, resulting in the release of AA. Further, these results are consistent with a mechanism in which the metabolism of AA, most likely through the monooxygenase pathway, results in a metabolite that by a yet unknown mechanism induced CYPIA1.
Assuntos
Ácido Araquidônico/fisiologia , Citocromo P-450 CYP1A1/metabolismo , Estresse Mecânico , Ácido 5,8,11,14-Eicosatetrainoico/farmacologia , Carcinoma Hepatocelular , Dexametasona/farmacologia , Ativação Enzimática/efeitos dos fármacos , Humanos , Indometacina/farmacologia , Cinética , Neoplasias Hepáticas , Masoprocol/farmacologia , Fosfolipase D/metabolismo , Proteína Quinase C/metabolismo , Quinacrina/farmacologia , Células Tumorais CultivadasRESUMO
Cell damage for cells grown on microcarriers in suspension is a critical problem for scale-up of microcarrier reactors. In order to study cell damage as a mechanistic process, a cellular response that is more sensitive than changes in growth and death rates and would be more closely related to cell regulatory mechanisms would be advantageous. We have observed the induction of a specific cytochrome P-450 monooxygenase, P-450IA1 (CYP1A1), to be a sensitive method for assessing the response of microcarrier-attached Hep G2 cells to stress resulting from hydrodynamic shear and oxygen deprivation. The kinetics of induction and amount of CYP1A1 formed in response to subtle shear stress, moderate shear, and hypoxia are described. Increased stress results in increased CYP1A1 formation.
Assuntos
Carcinoma Hepatocelular/enzimologia , Sistema Enzimático do Citocromo P-450/biossíntese , Dextranos , Fígado/enzimologia , Estresse Fisiológico/enzimologia , Biotecnologia/métodos , Carcinoma Hepatocelular/patologia , Adesão Celular , Divisão Celular/fisiologia , Meios de Cultura , Sistema Enzimático do Citocromo P-450/metabolismo , Indução Enzimática , Humanos , Hipóxia/enzimologia , Fígado/patologia , Microesferas , Células Tumorais CultivadasRESUMO
The exposure of two hepatoma cell lines, Hep G2 and Hepa-1, to moderate hydrodynamic shear, in microcarrier-attached suspension cultures, resulted in the transient induction of cytochrome P450IA1 (CYP1A1). Both cell lines have been characterized with respect to their Ah receptor (AhR) concentrations and induce CYP1A1 in response to exposure to xenobiotics such as 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). Using an AhR antagonist, alpha-naphthoflavone (alpha-NF) and a protein kinase C (PKC) inhibitor, staurosporine (ST), in the Hep G2 cell line, the induced CYP1A1 activity was modulated in the same manner as when the cells were coexposed to TCDD and either alpha-NF or ST. Exposure of the Hep G2 cell line to TCDD and shear resulted in both enhancement of the induced CYP1A1 activity in addition to a competitive response. Finally, using the wild type and AhR defective Hepa-1 cell lines, it was demonstrated that a functional AhR was required for shear-induced CYP1A1 expression. The data obtained in the three cell lines indicate a role for the AhR in the induction of CYP1A1 by shear in agitated microcarrier cultures.
Assuntos
Benzoflavonas/farmacologia , Carcinoma Hepatocelular/metabolismo , Sistema Enzimático do Citocromo P-450/biossíntese , Neoplasias Hepáticas/metabolismo , Dibenzodioxinas Policloradas/farmacologia , Receptores de Hidrocarboneto Arílico/fisiologia , Estresse Mecânico , Alcaloides/farmacologia , Linhagem Celular , Técnicas de Cultura/instrumentação , Técnicas de Cultura/métodos , Indução Enzimática , Humanos , Cinética , Microesferas , Proteína Quinase C/antagonistas & inibidores , Receptores de Hidrocarboneto Arílico/antagonistas & inibidores , Estaurosporina , Células Tumorais CultivadasRESUMO
A dose-dependent increase in tyrosine phosphorylation of five hepatic intracellular proteins with approximate molecular weights of 17, 21, 27, 29, and 34 kDa was seen 24 h after administration of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) to C57BL/6J female mice. The ED50 values for tyrosylphosphorylation of these five proteins, respectively, were 0.26, 0.21, 0.26, 0.31, and 0.38 micrograms TCDD/Kg. TCDD induction of 7-ethoxyresorufin O-deethylase activity (EROD) was characterized by an ED50 of 2.5 micrograms/Kg. An eighteen h exposure of a human lymphoblastoma cell line (X3) to TCDD increased tyrosylphosphorylation status of ten proteins with approximate molecular weights of 16, 17, 24, 26, 27, 32, 33, 34, 35, and 36 kDa in a dose-dependent manner. The EC50 values for these TCDD-dependent tyrosylphosphorylation ranged from 0.01 to 0.07 nM TCDD. EROD induction by TCDD in X3 cells exhibited an EC50 of 0.14 nM. These data indicate that TCDD alters intracellular protein tyrosine phosphorylation and these changes are more sensitive biological indicators of TCDD exposure than induction of EROD.