RESUMO
Accurately measuring the translations of objects between images is essential in many fields, including biology, medicine, chemistry, and physics. One important application is tracking one or more particles by measuring their apparent displacements in a series of images. Popular methods, such as the center of mass, often require idealized scenarios to reach the shot noise limit of particle tracking and, therefore, are not generally applicable to multiple image types. More general methods, such as maximum likelihood estimation, reliably approach the shot noise limit, but are too computationally intense for use in real-time applications. These limitations are significant, as real-time, shot-noise-limited particle tracking is of paramount importance for feedback control systems. To fill this gap, we introduce a new cross-correlation-based algorithm that approaches shot-noise-limited displacement detection and a graphics processing unit-based implementation for real-time image analysis of a single particle.
RESUMO
Interferon (IFN)-γ is a cytokine with immunomodulatory properties, which has been shown previously to enhance the generation of tolerogenic dendritic cells (DC) when administered early ex vivo in 7-day monocyte-derived DC culture. To generate tolerogenic DC rapidly within 48 h, human monocytes were cultured for 24 h with interleukin (IL)-4 and granulocyte-macrophage colony-stimulating factor (GM-CSF) in the presence (IFN-γ-DC) or absence of IFN-γ (500 U/ml) (UT-DC). DC were matured for 24 h with TNF-α and prostaglandin E(2) (PGE(2) ). DC phenotype, signal transducer and activator of transcription-6 (STAT-6) phosphorylation and promotion of CD4(+) CD25(+) CD127(neg/low) forkhead box P3 (FoxP3)(hi) T cells were analysed by flow cytometry. DC nuclear factor (NF)-κB transcription factor reticuloendotheliosis viral oncogene homologue B (RELB) and IL-12p70 protein expression were also determined. Phenotypically, IFN-γ-DC displayed reduced DC maturation marker CD83 by 62% and co-stimulation molecules CD80 (26%) and CD86 (8%). IFN-γ treatment of monocytes inhibited intracellular STAT6, RELB nuclear translocation and IL-12p70 production. IFN-γ-DC increased the proportion of CD4(+) CD25(+) CD127(neg/low) foxp3(hi) T cells compared to UT-DC from 12 to 23%. IFN-γ-DC primed T cells inhibited antigen-specific, autologous naive T cell proliferation by 70% at a 1:1 naive T cells to IFN-γ-DC primed T cell ratio in suppression assays. In addition, we examined the reported paradoxical proinflammatory effects of IFN-γ and confirmed in this system that late IFN-γ exposure does not inhibit DC maturation marker expression. Early IFN-γ exposure is critical in promoting the generation of regulatory DC. Early IFN-γ modulated DC generated in 48 h are maturation arrested and promote the generation of antigen-specific regulatory T cells, which may be clinically applicable as a novel cellular therapy for allograft rejection.
Assuntos
Células Dendríticas/efeitos dos fármacos , Células Dendríticas/imunologia , Interferon gama/administração & dosagem , NF-kappa B/antagonistas & inibidores , Fator de Transcrição STAT6/antagonistas & inibidores , Diferenciação Celular/efeitos dos fármacos , Diferenciação Celular/imunologia , Células Cultivadas , Células Dendríticas/citologia , Células Dendríticas/metabolismo , Expressão Gênica/efeitos dos fármacos , Fator Estimulador de Colônias de Granulócitos e Macrófagos/administração & dosagem , Humanos , Tolerância Imunológica/efeitos dos fármacos , Interferon gama/metabolismo , Interleucina-12/metabolismo , Interleucina-2/metabolismo , Interleucina-4/administração & dosagem , NF-kappa B/metabolismo , Fator de Transcrição STAT6/metabolismo , Transdução de Sinais/efeitos dos fármacos , Linfócitos T Reguladores/imunologia , Fator de Transcrição RelB/genética , Fator de Transcrição RelB/metabolismo , Tolerância ao Transplante/efeitos dos fármacosRESUMO
BACKGROUND: Enterochromaffin (EC) cells within the gastrointestinal (GI) tract provide almost all body serotonin (5-hydroxytryptamine [5-HT]). Peripheral 5-HT, released from EC cells lining the gut wall, serves diverse physiological roles. These include modulating GI motility, bone formation, hepatic gluconeogenesis, thermogenesis, insulin resistance, and regulation of fat mass. Enterochromaffin cells are nutrient sensors, but which nutrients they are responsive to and how this changes in different parts of the GI tract are poorly understood. METHODS: To accurately undertake such an examination, we undertook the first isolation and purification of primary mouse EC cells from both the duodenum and colon in the same animal. This allowed us to compare, in an internally controlled manner, regional differences in the expression of nutrient sensors in EC cells using real-time PCR. KEY RESULTS: Both colonic and duodenal EC cells expressed G protein-coupled receptors and facilitative transporters for sugars, free fatty acids, amino acids, and lipid amides. We find differential expression of nutrient receptor and transporters in EC cells obtained from duodenal and colonic EC cells. Duodenal EC cells have higher expression of tryptophan hydroxylase-1, sugar transporters GLUT2, GLUT5, and free fatty acid receptors 1 and 3 (FFAR1 and FFAR3). Colonic EC cells express higher levels of GLUT1, FFAR2, and FFAR4. CONCLUSIONS & INFERENCES: We highlight the diversity of EC cell physiology and identify differences in the regional sensing repertoire of EC cells to an assortment of nutrients. These data indicate that not all EC cells are similar and that differences in their physiological responses are likely dependent on their location within the GI tract.
Assuntos
Colo/metabolismo , Duodeno/metabolismo , Células Enterocromafins/metabolismo , Animais , Expressão Gênica , Masculino , Camundongos Endogâmicos CBA , Receptores Acoplados a Proteínas G/metabolismoRESUMO
BACKGROUND/AIMS: Replication deficient adenovirus is an efficient vector for gene transfer to the cornea. The aim was to optimise the transduction of human corneal endothelium with adenoviral vectors and to measure transgene production from transduced corneas. METHODS: Adenoviral vectors (AdV) encoding enhanced green fluorescent protein (eGFP) or a transgenic protein (scFv) were used to transfect 34 human corneas. Reporter gene expression was assessed after 72-96 hours of organ culture. The kinetics of scFv production was monitored in vitro for 1 month by flow cytometric analysis of corneal supernatants. RESULTS: Transduction of human corneas with high doses (5 x 10(7)-3 x 10(8) pfu) of AdV caused eGFP expression in 12-100% of corneal endothelial cells. Corneas were efficiently transduced following up to 28 days in cold storage. Very high AdV doses (2 x 10(9) pfu) reduced endothelial cell densities to 98 (SD 129) nuclei/mm(2) (compared to 2114 (716) nuclei/mm(2) for all other groups). Transgenic protein production peaked at 2.4 (0.9) microg/cornea/day at 2 weeks post-transduction, and decreased to 1.2 (0.4) microg/cornea/day by 33 days, at which time endothelial cell density had decreased to 431 (685) nuclei/mm(2). CONCLUSION: Human corneas can be efficiently transduced by AdV following extended periods of cold storage, and transgene expression is maintained for at least 1 month in vitro.
Assuntos
Adenoviridae/genética , Endotélio Corneano/metabolismo , Técnicas de Transferência de Genes , Vetores Genéticos/administração & dosagem , Idoso , Idoso de 80 Anos ou mais , Temperatura Baixa , Endotélio Corneano/virologia , Expressão Gênica , Genes Reporter , Terapia Genética/métodos , Proteínas de Fluorescência Verde/biossíntese , Proteínas de Fluorescência Verde/genética , Humanos , Região Variável de Imunoglobulina/biossíntese , Região Variável de Imunoglobulina/genética , Microscopia de Fluorescência , Pessoa de Meia-Idade , Técnicas de Cultura de Órgãos , Transdução Genética , TransgenesRESUMO
The production of murine monoclonal antibodies against specific antigens by hybridomas is a well utilised technique. The production of hybridomas secreting specific human antibodies would have many advantages in therapeutic applications of monoclonal antibodies. The immortalised human lymphocytes themselves would also provide valuable tools in research on lymphocyte development. Preparation of human-human hybridomas has been limited by a lack of suitable fusion partners. This protocol paper describes the production of human-mouse heterohybridomas by two independent laboratories. The purpose of this protocol is to provide a basis for the development of heterohybridoma technology in laboratories with limited hybridoma experience.
Assuntos
Antígenos/imunologia , Hibridomas/imunologia , Vacinas Pneumocócicas/imunologia , Toxoide Tetânico/imunologia , Adulto , Animais , Anticorpos Monoclonais/biossíntese , Anticorpos Monoclonais/imunologia , Divisão Celular/fisiologia , Fusão Celular/métodos , Meios de Cultura , Humanos , Hibridomas/citologia , Hibridomas/metabolismo , Lactente , Linfócitos/citologia , Linfócitos/imunologia , Camundongos , Mieloma Múltiplo/imunologia , Mieloma Múltiplo/patologia , PolietilenoglicóisRESUMO
B cells express an Fc receptor for IgG (FcgammaRII; CD32) which is involved in feedback inhibition of antibody production. Engagement of FcgammaRII during ligation of the antigen receptor provides an inhibitory signal. FcgammaRII exists as several isoforms, with FcgammaRIIb (which carries an immunoreceptor tyrosine-based inhibition motif; ITIM) being predominant form on adult B cells. The inhibitory role of FcgammaRIIb may be unhelpful to the infant, since primary exposure to infectious agents is likely to be in the presence of maternal IgG. We hypothesized that neonatal B cells would be less susceptible to feedback inhibition by antibody, either through the expression of activation-competent FcgammaRII isoforms (FcgammaRIIa and FcgammaRIIc) or through reduced expression of the inhibitory FcgammaRIIb isoforms. Cord and adult B cells were examined for expression of FcgammaRII isoforms using monoclonal antibodies and RT-PCR. In vitro assays were performed to assess susceptibility of cord and adult cells to FcgammaRII-mediated suppression. Although there is no phenotypic difference in FcgammaRII expression (FcgammaRIIb predominating on both adult and cord B cells), FcgammaRIIb is expressed at lower levels on cord cells. This quantitative difference in FcgammaRIIb expression may explain the reduced susceptibility of cord B cells to antibody-mediated inhibition observed in these experiments.
Assuntos
Subpopulações de Linfócitos B/metabolismo , Sangue Fetal/imunologia , Sangue Fetal/metabolismo , Receptores de IgG/biossíntese , Adulto , Anticorpos Anti-Idiotípicos/fisiologia , Anticorpos Monoclonais/fisiologia , Subpopulações de Linfócitos B/citologia , Subpopulações de Linfócitos B/imunologia , Inibidores do Crescimento/fisiologia , Humanos , Fragmentos Fab das Imunoglobulinas/fisiologia , Imunoglobulina M/imunologia , Imunossupressores/farmacologia , Recém-Nascido , Ativação Linfocitária/imunologia , Isoformas de Proteínas/biossíntese , Isoformas de Proteínas/genética , Receptores de IgG/genética , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
Little is known of the antifungal susceptibility patterns and molecular epidemiology of Cryptococcus neoformans from tropical regions. We studied 164 clinical isolates of C. neofomans from 120 Ugandan AIDS patients with cryptococcal meningitis by analyzing their electrophoretic karyotypes and antifungal susceptibility profiles. Computer-assisted analysis of karyotype patterns was performed to generate dendrograms. MICs of fluconazole and flucytosine were determined by reference methods. A total of 43 distinguishable DNA types were identified among the 164 isolates. Only 30 patients (25%) were infected with their own unique strain of c. neoformans, whereas 75% of the patients shared their infecting strain with at least one other patient. Among 17 patients with more than one CSF isolate of C. neoformans, sequential isolates were identical or highly related in 12 (71%) and were different in five patients (29%). The isolates were susceptible to both fluconazole and flucytosine and there were no instances in which a stepwise increase in either fluconazole or flucytosine MICs was observed among serial isolates. These findings suggest that the epidemiology of cryptococcal disease in AIDS patients from tropical regions may be somewhat different from that observed in more temperate climates.
PIP: Even though Cryptococcus neoformans var. neoformans is a leading cause of life-threatening mycotic infection among AIDS patients worldwide, little is known about its antifungal susceptibility patterns and molecular epidemiology in tropical regions. The authors studied 164 clinical isolates of C. neoformans from 120 Ugandan AIDS patients with cryptococcal meningitis by analyzing their electrophoretic karyotypes and antifungal susceptibility profiles. Computer-assisted analysis of karyotype patterns was performed to generate dendrograms, while the MICs of fluconazole and flucytosine were determined using reference methods. 43 distinguishable C. neoformans DNA types were identified among the 164 isolates. 30 patients (25%) were infected with their own unique strain of C. neoformans, while 75% of the patients shared their infecting strain with at least 1 other patient. Among 17 patients with more than 1 cerebrospinal fluid isolate of C. neoformans, sequential isolates were identical or highly related in 12 (71%) and were different in 5 patients (29%). The isolates were susceptible to both fluconazole and flucytosine, and there was no instance in which a stepwise increase in either fluconazole or flucytosine MICs was observed among serial isolates. These findings suggest that the epidemiology of cryptococcal disease in AIDS patients from tropical regions may be somewhat different from that observed in more temperate climates.
Assuntos
Síndrome da Imunodeficiência Adquirida/complicações , Antifúngicos/farmacologia , Cryptococcus neoformans/efeitos dos fármacos , Meningite Criptocócica/epidemiologia , Antifúngicos/uso terapêutico , Análise por Conglomerados , Cryptococcus neoformans/classificação , Cryptococcus neoformans/genética , DNA Fúngico/líquido cefalorraquidiano , Resistência Microbiana a Medicamentos/genética , Eletroforese em Gel de Campo Pulsado , Fluconazol/farmacologia , Fluconazol/uso terapêutico , Flucitosina/farmacologia , Flucitosina/uso terapêutico , Humanos , Processamento de Imagem Assistida por Computador , Cariotipagem , Meningite Criptocócica/tratamento farmacológico , Meningite Criptocócica/microbiologia , Testes de Sensibilidade Microbiana , Filogenia , Uganda/epidemiologiaRESUMO
ICI 180080 (5(Z)-7-[2,2-dimethyl-4-(2-hydroxyphenyl)-1,3-dioxan-cis-5-yl] heptenoic acid) potently inhibited contractions of rat and rabbit aortae and guinea-pig trachea elicited by 11,9-epoxymethano PGH2 (U-46619). This antagonism was selective because contractions of aortae to noradrenaline and 5-hydroxytryptamine and trachea to histamine were not antagonized by ICI 180080. Schild analysis of data obtained from experiments on rabbit aortae indicated that this thromboxane receptor antagonism was competitive (pA2 = 7.50, slope = 1.07). Addition of ICI 180080 to human platelet-rich plasma caused dose-related inhibition of U-46619-induced platelet aggregation. This modification of platelet aggregation was not associated with inhibition of thromboxane synthetase, cyclo-oxygenase or lipoxygenase. ICI 180080 did not modify the primary phase of ADP-induced aggregation of human platelets neither did it affect the platelet inhibitory activity of prostacyclin. When dosed orally to anaesthetized guinea-pigs, ICI 180080 (5-50 mg kg-1) caused dose-related inhibition of U-46619-evoked bronchoconstriction. We conclude that ICI 180080 is a potent, selective, competitive, orally active thromboxane antagonist.
Assuntos
Dioxanos/farmacologia , Dioxinas/farmacologia , Pulmão/efeitos dos fármacos , Músculo Liso/efeitos dos fármacos , Agregação Plaquetária/efeitos dos fármacos , Receptores de Prostaglandina/efeitos dos fármacos , Ácido 15-Hidroxi-11 alfa,9 alfa-(epoximetano)prosta-5,13-dienoico , Animais , Cobaias , Humanos , Técnicas In Vitro , Masculino , Contração Muscular/efeitos dos fármacos , Músculo Liso Vascular/efeitos dos fármacos , Endoperóxidos Sintéticos de Prostaglandinas/antagonistas & inibidores , Coelhos , Ratos , Receptores de TromboxanosRESUMO
The effects of ICI 185,282 (5(Z)-7-([ 2,4,5-cis]-4-O-hydroxyphenyl-2-trifluoromethyl-1, 3-dioxan-5-yl)heptenoic acid) have been studied on guinea-pig platelets and pulmonary smooth muscle in-vitro and in-vivo. When tested on guinea-pig lung parenchyma in-vitro. ICI 185,282 (1 x 10(-7) M) produced a significant shift in U-46619 response curves (concentration ratio of 13:3); the antagonist (1 x 10(-5) M) did not modify histamine responses. When tested on guinea-pig trachea in-vitro ICI 185,282 (1 x 10(-7) M) caused significant inhibition of U-46619 and PGD2 responses (concentration ratios of 8.3 and 14.1, respectively); the antagonist (1 x 10(-5) M proved less effective against contractions of PGF2 alpha, LTD4 and histamine (concentration ratios of 7.0, 1.5 and 1.6). When added to guinea-pig platelet rich plasma in-vitro, ICI 185,282 (x 10(-6), 1 x 10(-5) M) caused concentration-dependent parallel shifts to the right of U-46619 aggregation curves, yielding concentration ratios of 13.6 and 141.9, respectively. In-vitro, addition of ICI 185,282 (x 10(-5) M) to indomethacin-treated pulmonary smooth muscle did not modify resting tone, neither did it induce aggregation or swelling in platelet-rich plasma preparations. When administered orally to guinea-pigs ICI 185,282 (0.1, 0.5 mg kg-1) caused a significant inhibition of U-46619-induced platelet aggregation ex-vivo which persisted greater than or equal to 8 h. In-vivo, a single oral dose of ICI 185,282 (1 mg kg-1) inhibited bronchospasm induced by U-46619, PGD2, PGF2 alpha, arachidonic acid, LTD4 and PAF; responses to histamine were unaffected.(ABSTRACT TRUNCATED AT 250 WORDS)
Assuntos
Dioxanos/farmacologia , Dioxinas/farmacologia , Pulmão/efeitos dos fármacos , Receptores de Prostaglandina/efeitos dos fármacos , Animais , Plaquetas/efeitos dos fármacos , Espasmo Brônquico/induzido quimicamente , Espasmo Brônquico/tratamento farmacológico , Cobaias , Técnicas In Vitro , Masculino , Receptores de Tromboxanos , Respiração/efeitos dos fármacos , Testes de Função Respiratória , Músculos Respiratórios/efeitos dos fármacosRESUMO
A comparative study of conventional amphotericin B, Abelcet and AmBisome was performed using a microdilution format of the NCCLS M27-A methodology for susceptibility testing against 300 fungal isolates (152 yeasts, 148 filamentous fungi) in both RPMI-1640 and antibiotic medium #3 (AB3). The clinical isolates included Candida albicans (n=54), Candida glabrata (n=25), Candida parapsilosis (n=23), Candida krusei (n=19), Candida lusitaniae (n=14), Cryptococcus neoformans (n=5), Candida tropicalis (n=12), Aspergillus flavus (n=34), Aspergillus fumigatus (n=46) and 68 other filamentous fungi encompassing 22 different genera. The minimal inhibitory concentrations (MIC) for all drugs were defined as the lowest concentrations in which there was no visible growth. MICs were determined after 48 h for yeasts and 72 h for filamentous fungi. The mean MICs +/- standard error (microg/ml) for yeasts and filamentous fungi, respectively, were: Abelcet, 0.51+/-0.21, 4.34+/-0.61; AmBisome, 1.28+/-0.24, 5.68+/-0.57; amphotericin B, 0.29+/-0.11, 1.12+/-0.19, respectively. Overall, against both yeasts and filamentous fungi Abelcet proved to have more potent antifungal activity than AmBisome. Using AB3 as opposed to RPMI-1640 generally produced lower MIC values but did not have any effect on the order of relative activity with all of the antifungal agents tested. In conclusion, our data shows that Abelcet is more active than AmBisome against pathogenic yeast and filamentous fungi when assayed in AB3 in vitro. Comparison of the activities of these antifungals in experimental animal models is necessary to determine whether these in vitro findings are correlated with in vivo efficacy.
Assuntos
Anfotericina B/farmacologia , Antifúngicos/farmacologia , Fosfatidilcolinas/farmacologia , Fosfatidilgliceróis/farmacologia , Anfotericina B/administração & dosagem , Antifúngicos/administração & dosagem , Combinação de Medicamentos , Fungos/efeitos dos fármacos , Humanos , Lipossomos , Testes de Sensibilidade Microbiana , Fosfatidilcolinas/administração & dosagem , Fosfatidilgliceróis/administração & dosagemAssuntos
Receptores de IgG/metabolismo , Sequência de Aminoácidos , Animais , Anticorpos Monoclonais , Sistema Hematopoético/citologia , Sistema Hematopoético/imunologia , Humanos , Camundongos , Camundongos Knockout , Dados de Sequência Molecular , Fenótipo , Isoformas de Proteínas/química , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Receptores de IgG/química , Receptores de IgG/genética , Homologia de Sequência de Aminoácidos , Distribuição TecidualRESUMO
Corneal transplantation has not matched the improvements in outcome seen with other clinical transplantation procedures. The therapeutic strategies, which have improved the outcomes of solid vascularised organs are not applicable to corneal transplantation. Corneal transplantation is different with respect to relevant transplantation biology and the clinical context in which it is practiced. New approaches need to be developed which provide regional rather than systemic immunosuppression. The accessibility of the cornea makes it particularly suitable for topical medication and for gene therapy approaches. Engineered antibodies, small enough to pass through the cornea, and directed at key molecules in the allograft response have been developed. Gene therapy had been developed using viral vectors to transfect the corneal endothelium with the genes for immunosuppressive lymphokines. Both approaches show promise.
Assuntos
Transplante de Córnea , Rejeição de Enxerto/imunologia , Terapia de Imunossupressão/métodos , Terapia Genética , Humanos , Tolerância Imunológica , Transplante HomólogoRESUMO
Understanding the relationship between microbial community dynamics and functional instability is an important step towards designing reliable biological water treatment systems. In this study, the community dynamics of two dispersed-growth denitrifying reactors were examined during periods of functional stability and instability. In both reactors during the period of functional instability, the effluent chemistry changed over time, with periods of high nitrate concentrations followed by periods of fluctuating nitrite concentrations. Community structure was examined by clone library analysis of the 16S rRNA gene. Community dynamics were investigated with terminal restriction fragment (T-RF) length polymorphism, and the functional diversity represented by T-RFs was assessed through nitrate reduction assays of representative isolates. During the period of functional instability, the community structure changed considerably, and the dynamics correlated significantly with effluent chemistry. The nitrite concentration was significantly correlated with the relative abundances of the nitrate-reducing Delftia- and Achromobacter-like T-RFs. The isolate representing the Acidovorax-like T-RF reduced nitrate directly to nitrogen in batch assays without the accumulation of any intermediates. The Acidovorax-like T-RF relative abundance was significantly negatively correlated with nitrite concentration, indicating that it was associated with good functional performance. The results of this study reveal a clear relationship between community dynamics and functional instability and the importance of diversity among nitrate-reducing populations within a denitrifying community.
Assuntos
Bactérias/crescimento & desenvolvimento , Bactérias/metabolismo , Reatores Biológicos , Ecossistema , Nitratos/metabolismo , Purificação da Água/métodos , Bactérias/classificação , Bactérias/genética , DNA Bacteriano/análise , DNA Bacteriano/isolamento & purificação , Água Doce/microbiologia , Biblioteca Gênica , Genes de RNAr , Filogenia , Reação em Cadeia da Polimerase/métodos , Polimorfismo de Fragmento de Restrição , RNA Ribossômico 16S/genéticaRESUMO
Gene therapy of the cornea shows promise for modulating corneal transplant rejection but the most appropriate vector for gene transfer has yet to be determined. We investigated a lentiviral vector (LV) for its ability to transduce corneal endothelium. A lentivector expressing enhanced yellow fluorescent protein (eYFP) under the control of the Simian virus type 40 early promoter (LV-SV40-eYFP) transduced 80-90% of rat, ovine and human corneal endothelial cells as detected by fluorescence microscopy. The kinetics of gene expression varied among species, with ovine corneal endothelium showing a relative delay in detectable reporter gene expression compared with the rat or human corneal endothelium. Vectors containing the myeloproliferative sarcoma virus promoter or the phosphoglycerate kinase promoter were not significantly more effective than LV-SV40-eYFP. The stability of eYFP expression in rat and ovine corneas following ex vivo transduction of the donor cornea was assessed following orthotopic corneal transplantation. Following transduction ex vivo, eYFP expression was maintained in corneal endothelial cells for at least 28 days after corneal transplantation in the sheep and >60 days in the rat. Thus, rat, ovine and human corneal endothelial cells were efficiently transduced by the LV, and gene expression appeared stable over weeks in vivo.
Assuntos
Doenças da Córnea/terapia , Endotélio Corneano/metabolismo , Terapia Genética/métodos , HIV-1/genética , Transdução Genética/métodos , Animais , Transplante de Córnea , Expressão Gênica , Genes Reporter , Humanos , Proteínas Luminescentes/genética , Microscopia de Fluorescência , Ratos , Ovinos , Fatores de Tempo , Transgenes , Transplante Homólogo , Transplante IsogênicoRESUMO
Terbinafine has previously been shown to be highly active against dermatophytes and many other filamentous fungi. However, its activity against yeasts is controversial, with earlier reports suggesting that it has low activity, while more recent studies demonstrated that terbinafine is effective against yeasts. In this study, the in vitro activity of terbinafine was evaluated against a broad range of fungal isolates. We examined the susceptibility of 100 yeast strains (10 species including Candida albicans, non-C. albicans, fluconazole-susceptible and -resistant candidal strains), and 184 strains of filamentous fungi and dermatophytes (29 species including Aspergillus, Fusarium, Sporothrix, Trichophyton rubrum, T. mentagrophytes, T. tonsurans, Microsporum canis and Epidermophyton floccosum), using the NCCLS M27-A microdilution methodology for yeasts and a modified M38-P methodology for moulds. The endpoint for terbinafine was defined as 80% inhibition compared with the growth control well. The mean yeast and filamentous fungi minimum inhibitory concentration values +/- SEM (in microg ml(-1)) for terbinafine were: 6.60 +/- 0.73 and 1.04 +/- 0.28, respectively. In conclusion, our data suggest that terbinafine, in addition to its potent activity against dermatophytes, is considerably effective against a broad range of yeasts and filamentous fungi in vitro. Therefore, investigations concerning its antifungal activity in vivo against such organisms should be pursued.
Assuntos
Antifúngicos/farmacologia , Fungos/efeitos dos fármacos , Naftalenos/farmacologia , Estudos de Avaliação como Assunto , Fluconazol/farmacologia , Testes de Sensibilidade Microbiana , TerbinafinaRESUMO
A standardized reference method for dermatophyte in vitro susceptibility testing is lacking. In a previous study, Norris et al. (H. A. Norris, B. E. Elewski, and M. A. Ghannoum, J. Am. Acad. Dermatol. 40(6, part 2):S9-S13) established the optimal medium and other growth variables. However, the earlier study did not address two issues: (i) selection of an optimal medium for conidial formation by dermatophytes and (ii) validation of the method with a large number of dermatophytes. The present study addresses these two points. To select which agar medium best supported conidial growth, representative isolates of dermatophytes were grown on different agars. Preliminary experiments showed that only oatmeal cereal agar supported the production of conidia by Trichophyton rubrum. We tested the abilities of 251 T. rubrum isolates to form conidia using three different cereal agars and potato dextrose agar. Overall, oatmeal cereal and rice agar media were comparable in their abilities to support T. rubrum conidial growth. Next, we used the oatmeal cereal agar for conidial formation along with the optimal conditions for dermatophyte susceptibility testing proposed by Norris et al. and determined the antifungal susceptibilities of 217 dermatophytes to fluconazole, griseofulvin, itraconazole, and terbinafine. Relative to the other agents tested, terbinafine possessed the highest antifungal activity against all of the dermatophytes. The mean +/- standard error of the mean MICs of fluconazole, itraconazole, terbinafine, and griseofulvin were 2.07 +/- 0.29, 0.13 +/- 0.01, 0.002 +/- 0.0003, and 0.71 +/- 0.05 microgram/ml, respectively. This study is the first step in the identification of optimal conditions that could be used for the standardization of the antifungal susceptibility testing method for dermatophytes. Inter- and intralaboratory agreement as well as clinical correlations need to be established.
Assuntos
Antifúngicos/farmacologia , Arthrodermataceae/efeitos dos fármacos , Meios de Cultura , Testes de Sensibilidade Microbiana/métodos , Epidermophyton/efeitos dos fármacos , Testes de Sensibilidade Microbiana/normas , Microsporum/efeitos dos fármacos , Esporos Fúngicos , Fatores de Tempo , Trichophyton/efeitos dos fármacosRESUMO
The susceptibility of Aspergillus fumigatus to mulundocandin, an echinocandin-like compound, and other antifungal agents was assessed by the National Committee for Clinical Laboratory Standards (NCCLS) M38-P method, a 2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-5-[(phenyl-amino)carbonyl]-2H-tetrazolium hydroxide (XTT)-based colorimetric assay, and determination of morphologic alterations by microscopy. In contrast to the NCCLS M38-P method, which does not predict the activity in vivo, the XTT-based assay showed that A. fumigatus is susceptible to mulundocandin. Thus, the XTT-based assay might be useful for determination of the susceptibilities of molds to echinocandins. Further evaluation is warranted.
Assuntos
Antifúngicos/farmacologia , Aspergillus fumigatus/efeitos dos fármacos , Peptídeos Cíclicos/farmacologia , Sais de Tetrazólio/farmacologia , Equinocandinas , Humanos , Testes de Sensibilidade Microbiana/métodos , Testes de Sensibilidade Microbiana/normasRESUMO
It has been postulated that phospholipases of fungal origin can affect in vitro susceptibility testing of amphotericin B lipid complex (ABLC). We used specific phospholipase-deficient mutants of Candida albicans and Cryptococcus neoformans in susceptibility testing and demonstrated that extracellular fungal phospholipase activity does not influence the in vitro susceptibilities of these two fungi to ABLC.
Assuntos
Anfotericina B/farmacologia , Antifúngicos/farmacologia , Fungos/efeitos dos fármacos , Fungos/enzimologia , Lisofosfolipase/metabolismo , Anfotericina B/administração & dosagem , Antifúngicos/administração & dosagem , Portadores de Fármacos , Lipossomos , Testes de Sensibilidade MicrobianaRESUMO
MICs for clinical Candida and Cryptococcus isolates were determined by a method incorporating the colorimetric indicator 2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-5-[(phenylamino)carbonyl] -2H-tetrazolium hydroxide (XTT), and the results were compared with MICs obtained by the National Committee for Clinical Laboratory Standards approved standard method (M27-A). One hundred percent of all isolates demonstrated agreement within 2 dilutions between the MICs of amphotericin B, fluconazole, itraconazole, ketoconazole, and flucytosine obtained by the two methods. These data suggest that an XTT-based method could provide a useful means for the determination of antifungal susceptibility of yeasts.
Assuntos
Antifúngicos/farmacologia , Candida/efeitos dos fármacos , Cryptococcus/efeitos dos fármacos , Anfotericina B/farmacologia , Colorimetria/métodos , Estudos de Avaliação como Assunto , Fluconazol/farmacologia , Flucitosina/farmacologia , Humanos , Itraconazol/farmacologia , Cetoconazol/farmacologia , Testes de Sensibilidade Microbiana/métodosRESUMO
1. ICI 170,809 (2-(2-dimethylamino-2-methylpropylthio)-3-phenylquinoline hydrochloride) is a potent 5-hydroxytryptamine (5-HT) type 2 postsynaptic receptor antagonist. 2. Effects of ICI 170,809 as single oral doses (3, 7, 15 and 30 mg) or placebo were studied on the duration of antagonism for the ex vivo platelet aggregatory response to 5-HT and to the pupillary light constrictor response in eight healthy male volunteers. 3. Pupillary dark adapted responses to a 0.5 s light stimulus were measured using a portable infrared pupillometer, for up to 24 h after dosing. 4. The in vitro platelet 5-HT aggregation response was reduced by ICI 170,809, with depression of the dose-response curve to 5-HT at all concentrations of 5-HT and with no evidence for a parallel shift. 5. The ex vivo platelet 5-HT response demonstrated a dose related significant (P less than 0.02) decrease in aggregation reaching a maximum at 2 h after dosing with the effect persisting for at least 8 h after dosing with the 7 and 15 mg doses. 6. Resting pupil diameter (RPD), and light induced pupillary responses in the dark adapted pupil, showed a significant (P less than 0.01) dose related reduction with significant (P less than 0.05) effects still present with the 15 and 30 mg doses at 8 h after dosing. 7. We conclude that, changes in both ex vivo platelet aggregation to 5-HT and dark adapted pupil size, are significantly correlated (P less than 0.0001) with log plasma concentrations (ng ml-1) of ICI 170,809, enabling the assessment of 5-HT2-receptor antagonism in man.