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1.
Sci Rep ; 9(1): 17012, 2019 Nov 18.
Artigo em Inglês | MEDLINE | ID: mdl-31740685

RESUMO

Antimicrobial resistance is a major threat to human health, hence there is an urgent need to discover antibacterial molecule(s). Previously, we hypothesized that microbial gut flora of animals are a potential source of antibacterial molecules. Among various animals, Cuora amboinensis (turtle) represents an important reptile species living in diverse ecological environments and feed on organic waste and terrestrial organisms and have been used in folk medicine. The purpose of this study was to mine turtle's gut bacteria for potential antibacterial molecule(s). Several bacteria were isolated from the turtle gut and their conditioned media were prepared. Conditioned media showed potent antibacterial activity against several Gram-positive (Bacillus cereus, Streptococcus pyogenes and methicillin-resistant Staphylococcus aureus) and Gram-negative (neuropathogenic Escherichia coli K1, Serratia marcescens, Pseudomonas aeruginosa, Salmonella enterica and Klebsiella pneumoniae) pathogenic bacteria. Conditioned media-mediated bactericidal activity was heat-resistant when treated at 95°C for 10 min. By measuring Lactate dehydrogenase release, the results showed that conditioned media had no effect on human cell viability. Tandem Mass Spectrometric analysis revealed the presence of various secondary metabolites, i.e., a series of known as well as novel N-acyl-homoserine lactones, several homologues of 4-hydroxy-2-alkylquinolines, and rhamnolipids, which are the signature metabolites of Pseudomonas species. These findings are significant and provide the basis for rational development of therapeutic interventions against bacterial infections.

2.
Plant Physiol Biochem ; 139: 459-469, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-30999133

RESUMO

Salinity stress can severely affect the growth and production of the crop plants. Cheap and reliable actions are needed to enable the crop plants to grow normal under saline conditions. Modification at the molecular level to produce resistant cultivars is one of the promising, yet highly expensive techniques, whereas application of endophytes on the other hand are very cheap. In this regard, the role of Cochliobolus sp. in alleviating NaCl-induced stress in okra has been investigated. The growth and biomass yield, relative water content, chlorophyll content and IAA were decreased, whereas malondialdehyde (MDA) and proline content were increased in okra plants treated with 100 mM NaCl. On the contrary, okra plants inoculated with C. lunatus had higher shoot length, root length, plant dry weight, chlorophyll, carotenoids, xanthophyll, phenolicss, flavonoids, IAA, total soluble sugar and relative water content, while lower MDA. LC-MS/MS analysis of the Cochliobolus sp. extract revealed the presence of pinocembrin, chlorogenic acids, wogonin, calycosin and diadzein as a salinity stress reliever. From the results, it can be concluded that colonization of Cochliobolus sp. improves the NaCl tolerance by ameliorating the physicochemical attributes of the host plants.


Assuntos
Abelmoschus/efeitos dos fármacos , Abelmoschus/microbiologia , Ascomicetos/metabolismo , Ascomicetos/fisiologia , Cloreto de Sódio/farmacologia , Abelmoschus/metabolismo , Malondialdeído/metabolismo , Prolina/metabolismo , Salinidade , Espectrometria de Massas em Tandem
3.
Plant Physiol Biochem ; 135: 61-68, 2019 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-30504088

RESUMO

Cinnamic acid (CA) is an allelochemical that inhibits the growth of root promoting soil microorganisms. To prevent the growth of soil microbes, CA modulates several metabolic pathways in host plants and soil microbes. The aim of the current study was to investigate the effect of CA on maize root growth, exudation of secondary metabolites and its interaction with beneficial endophyte Pz11. The endophyte Pz11 was isolated from the roots of drought stressed Asphodelus tenuifolius (wild onion). The Pz11 strain was identified as Fusarium culmorum by homology of the internal transcribed spacer (ITS) region of 18 S rDNA sequence. The F. culmorum Pz11 produced phytostimulants and signaling compounds, such as indole-3-acetic acid (IAA), flavonoids and sugars. Moreover, the strain have effectively colonized the roots of maize and subsequently enhanced the growth of its host plants. On the contrary, application of CA has reduced root growth in maize seedlings as well as root colonization ability of F. culmorum Pz11. Also, maize seedlings exposed to CA exude low quantities of flavonoids and polyphenols. In conclusion, CA reduces the maize root growth and exudation of secondary metabolites, which may affects its ability to attract plant growth promoting endophytic fungi.


Assuntos
Cinamatos/farmacologia , Endófitos/metabolismo , Flavonoides/metabolismo , Fusarium/metabolismo , Reguladores de Crescimento de Planta/farmacologia , Raízes de Plantas/efeitos dos fármacos , Zea mays/efeitos dos fármacos , Endófitos/efeitos dos fármacos , Fusarium/efeitos dos fármacos , Fusarium/genética , Ácidos Indolacéticos/metabolismo , Raízes de Plantas/metabolismo , Raízes de Plantas/microbiologia , Plântula/efeitos dos fármacos , Plântula/crescimento & desenvolvimento , Zea mays/crescimento & desenvolvimento , Zea mays/metabolismo , Zea mays/microbiologia
4.
BMC Complement Altern Med ; 18(1): 140, 2018 May 02.
Artigo em Inglês | MEDLINE | ID: mdl-29720152

RESUMO

BACKGROUND: Medicinal plants have been founded as traditional herbal medicine worldwide. Most of the plant's therapeutic properties are due to the presence of secondary metabolites such as alkaloids, glycosides, tannins and volatile oil. METHODS: The present investigation analyzed the High-Pressure Liquid Chromatography (HPLC) fractions of Glycyrrhiza glabra (Aqueous, Chloroform, Ethanol and Hexane) against multidrug resistant human bacterial pathogens (Escherichia coli, Acinetobacter baumannii, Staphylococcus aureus and Pseudomonas aeruginosa). All the fractions showed antibacterial activity, were subjected to LC MS/MS analysis for identification of bioactive compounds. RESULTS: Among total HPLC fractions of G. glabra (n = 20), three HPLC fractions showed potential activity against multidrug resistant (MDR) bacterial isolates. Fraction 1 (F1) of aqueous extracts, showed activity against A. baumannii (15 ± 0.5 mm). F4 from hexane extract of G. glabra showed activity against S. aureus (10 ± 0.2 mm). However, F2 from ethanol extract exhibited activity against S. aureus (10 ± 0.3 mm). These active fractions were further processed by LC MS/MS analysis for the identification of compounds. Ellagic acid was identified in the F1 of aqueous extract while 6-aldehydo-isoophiopogonone was present in F4 of hexane extract. Similarly, Liquirtigenin was identified in F2 of ethanol. CONCLUSIONS: Glycyrrhiza glabra extracts HPLC fractions showed anti-MDR activity. Three bioactive compounds were identified in the study. 6-aldehydo-isoophiopogonone and Liquirtigenin were for the first time reported in G. glabra. Further characterization of the identified compounds will be helpful for possible therapeutic uses against infectious diseases caused by multidrug resistant bacteria.


Assuntos
Antibacterianos/farmacologia , Bactérias/efeitos dos fármacos , Benzodioxóis/farmacologia , Cromatografia Líquida de Alta Pressão/métodos , Flavanonas/farmacologia , Glycyrrhiza/química , Isoflavonas/farmacologia , Antibacterianos/análise , Antibacterianos/química , Benzodioxóis/análise , Benzodioxóis/química , Farmacorresistência Bacteriana Múltipla , Flavanonas/análise , Flavanonas/química , Isoflavonas/análise , Isoflavonas/química , Extratos Vegetais/química , Espectrometria de Massas em Tandem
5.
Artigo em Inglês | MEDLINE | ID: mdl-29648964

RESUMO

A simple, reliable and sensitive liquid chromatography-tandem mass spectrometry-based confirmatory method was redeveloped and validated for the simultaneous determination of chloramphenicol, thiamphenicol, florfenicol and florfenicol amine in chicken muscles. The analytes were extracted from minced chicken muscle with acetonitrile and ammoniated water mixture. A second extraction with ethyl acetate was followed by evaporation and dissolution of the residue in ammoniated methanol before defatting with n-hexane. Finally, the extract was further cleaned up by dispersive solid phase extraction using C-18 end-capped dispersive material. The validation protocol was adapted from the European Commission Decision 2002/657/EC and all the performance characteristics were successfully satisfied. The recoveries of all the analytes were found to be in the range of 86.4-108.1% and the precision values, within day and between days, ranged from 2.7% to 11% and 4.4% to 16.3%, respectively. The method was tested in various incurred samples and applied to analyse a wide range of random poultry market samples (n = 120) collected from three cities of the Punjab, Pakistan. Chloramphenicol and florfenicol residues were detected at low levels in less than 11% of the samples. Chloramphenicol was detected only in 4 samples with the concentration range of 0.17-0.477 µg kg-1, whereas the levels of florfenicol/florfenicol amine residues detected in 9 samples ranged from 8.7 to 32.8 µg kg-1. Moreover, most of the florfenicol residues were identified as tissue bound, extractable only after strong acid hydrolysis.


Assuntos
Antibacterianos/análise , Cloranfenicol/análise , Carne/análise , Tianfenicol/análogos & derivados , Tianfenicol/análise , Animais , Galinhas , Cromatografia Líquida de Alta Pressão , Resíduos de Drogas/análise , Paquistão , Espectrometria de Massas em Tandem
6.
Toxins (Basel) ; 10(2)2018 02 10.
Artigo em Inglês | MEDLINE | ID: mdl-29439433

RESUMO

Mycotoxin contamination in rice can create a health risk for the consumers. In this study, the measurement of 23 mycotoxins in rice samples (n = 180) was performed using a validated LC-MS/MS method. A food frequency questionnaire was used to get rice consumption data for the assessment of mycotoxin dietary exposure, before calculating the health risk in adults and children of north and south regions of the Pakistani Punjab province. The prevalence of aflatoxin B1 (56%), aflatoxin B2 (48%), nivalenol (28%), diacetoxyscirpenol (23%), fumonisin B1 (42%), zearalenone (15%), HT-2 toxin (10%), deoxynivalenol (8%), and ochratoxin A (6%) was estimated in samples with a mean concentration range between 0.61 and 22.98 µg/kg. Aflatoxin degradation by traditional Pakistani cooking recipes was evaluated and observed to be 41-63%. The dietary exposure to aflatoxins exceeded the tolerable daily intake at all levels, and ochratoxin A and zearalenone posed health risk at high contamination and high consumption levels. The margin of aflatoxin B1 exposure ranged between 10 and 69 in adults and 10 and 62 in children. The mean cancer risk by aflatoxin B1 exposure was 0.070 (adults) and 0.071 (children) cases/year/100,000 people in South Punjab population, and 0.122 (adults) and 0.127 (children) cases/year/100,000 people in North Punjab population. This study will provide new insights for the planning and management of mycotoxins in Pakistan.


Assuntos
Exposição Ambiental/análise , Contaminação de Alimentos/análise , Micotoxinas/análise , Oryza/química , Adulto , Criança , Humanos , Neoplasias , Paquistão , Medição de Risco
7.
Bioresour Technol ; 253: 297-303, 2018 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-29413995

RESUMO

This study evaluated the bioenergy potential of Wolffia arrhiza via pyrolysis. The biomass was collected from the pond receiving city wastewater. Oven dried powdered biomass was exposed to thermal degradation at three heating rates (10, 30 and 50°â€¯C min-1) using Thermogravimetry-Differential Scanning Calorimetry analyzer in an inert environment. Data obtained were subjected to the isoconversional models of Kissenger-Akahira-Sunose (KSA) and Flynn-Wall-Ozawa (FWO) to elucidate the reaction chemistry. Kinetic parameters including, Ea (136-172 kJmol-1) and Gibb's free energy (171 kJmol-1) showed the remarkable bioenergy potential of the biomass. The average enthalpies indicated that the product formation is favored during pyrolysis. Advanced coupled TG-FTIR-MS analyses showed the evolved gases to contain the compounds containing CO functional groups (aldehydes, ketones), aromatic and aliphatic hydrocarbons as major pyrolytic products. This low-cost abundant biomass may be used to produce energy and chemicals in a cost-efficient and environmentally friendly way.


Assuntos
Gases , Espectroscopia de Infravermelho com Transformada de Fourier , Biomassa , Cinética , Termodinâmica , Termogravimetria
8.
Biochem Genet ; 56(1-2): 7-21, 2018 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-29094226

RESUMO

Inborn errors of metabolism (IEMs) are a group of inherited metabolic disorders which are caused by mutations in the specific genes that lead to impaired proteins or enzymes production. Different metabolic pathways are perturbed due to the deficiency or lack of enzymes. To date, more than 500 IEMs have been reported with most of them being untreatable. However, fortunately 91 such disorders are potentially treatable, if diagnosed at an earlier stage of life. IEMs have been classified into different categories and one class of IEMs, characterized by the physiological disturbances of amino acids is called as aminoacidopathies. Out of 91 treatable IEM, thirteen disorders are amino acid related. Aminoacidopathies can be detected by chromatography and mass spectrometry based analytical techniques (e.g., HPLC, GC-MS, LC-MS/MS) for amino acid level changes, and through genetic assays (e.g., PCR, TaqMan Genotyping, DNA sequencing) at the mutation level in the corresponding genes. Hence, this review is focused to describe thirteen common aminoacidopathies namely: Phenylketonuria (PKU), Maple Syrup Urine Disease (MSUD), Homocystinuria/Methylene Tetrahydrofolate Reductase (MTHFR) deficiency, Tyrosinemia type II, Citrullinemia type I and type II, Argininosuccinic aciduria, Carbamoyl Phosphate Synthetase I (CPS) deficiency, Argininemia (arginase deficiency), Hyperornithinemia-Hyperammonemia-Homocitrullinuria (HHH) syndrome, N-Acetylglutamate Synthase (NAGS) deficiency, Ornithine Transcarbamylase (OTC) deficiency, and Pyruvate Dehydrogenase (PDH) complex deficiency. Furthermore, the etiology, prevalence and commonly used analytical techniques for screening of aminoacidopathies are briefly described. This information would be helpful to researchers and clinicians especially from developing countries to initiate newborn screening programs for aminoacidopathies.


Assuntos
Erros Inatos do Metabolismo dos Aminoácidos , Aminoácidos , Técnicas de Genotipagem , Programas de Rastreamento , Espectrometria de Massas , Erros Inatos do Metabolismo dos Aminoácidos/diagnóstico , Erros Inatos do Metabolismo dos Aminoácidos/epidemiologia , Erros Inatos do Metabolismo dos Aminoácidos/etiologia , Erros Inatos do Metabolismo dos Aminoácidos/terapia , Aminoácidos/sangue , Aminoácidos/genética , Humanos , Prevalência
9.
Artigo em Inglês | MEDLINE | ID: mdl-28866358

RESUMO

Florfenicol, a broad spectrum bacteriostatic antibiotic belonging to amphenicol class, is widely used in poultry and livestock for the treatment of various infections. The major metabolite of florfenicol in different animal species is florfenicol amine which is exploited as the marker residue for the determination of florfenicol. Analysis of florfenicol merely by solvent extraction cannot determine the accurate amount of the drug present in incurred tissues (muscle, liver and kidney) of treated birds, as indicated by this study. Thus the methods solely based on solvent extraction may lead to false negative results. A reliable LC-MS/MS based confirmatory method for the analysis of florfenicol and its metabolites in chicken muscle was developed and validated according to the European Union Commission Decision 2002/657/EC. The method was based on acid hydrolysis to liberate non-extractable residues having presumably been covalently bound to tissues, and to convert all the florfenicol residues as well as its metabolites into florfenicol amine. The amine was subsequently recovered with ethyl acetate at pH 10.5, defatted and further cleaned up with dispersive solid phase extraction (dSPE). The LC separation was achieved on reverse phase C-18 column with isocratic elution using acetonitrile/water mobile phase and the analysis was performed on linear ion trap mass spectrometer. Calibration curve was obtained over a concentration range of 25-600µg/kg for chicken muscles. The accuracy values ranged from 84 to 101.4% and the precision values for within day and between days ranged from 1.2-11.7%, respectively. Limit of detection (LOD), limit of quantification (LOD), CCα and CCß values were 0.98, 3.2, 113 and 126µg/kg, respectively. The developed method was highly robust and was further applied to estimate the relative distribution of solvent-extractable against solvent-non-extractable florfenicol drug residues in muscle, liver and kidney samples of broiler chicken after 5days of oral dosing.


Assuntos
Antibacterianos/análise , Galinhas/metabolismo , Cromatografia Líquida/métodos , Resíduos de Drogas/análise , Espectrometria de Massas em Tandem/métodos , Tianfenicol/análogos & derivados , Animais , Resíduos de Drogas/farmacocinética , Rim/química , Rim/metabolismo , Limite de Detecção , Modelos Lineares , Fígado/química , Fígado/metabolismo , Carne/análise , Músculos/química , Músculos/metabolismo , Reprodutibilidade dos Testes , Tianfenicol/análise , Tianfenicol/farmacocinética , Distribuição Tecidual
10.
BMC Complement Altern Med ; 17(1): 247, 2017 May 03.
Artigo em Inglês | MEDLINE | ID: mdl-28468660

RESUMO

BACKGROUND: Medicinal plants are rich source of traditional herbal medicine around the globe. Most of the plant's therapeutic properties are due to the presence of secondary bioactive compounds. METHODS: The present study analyzed the High Pressure Liquid Chromatography (HPLC) fractions of Puncia granatum (peel) extracts (aqueous, chloroform, ethanol and hexane) against multidrug resistant bacterial pathogens (Acinetobacter baumannii, Escherichia coli, Pseudomonas aeruginosa and Staphylococcus aureus). All the fractions having antibacterial activity was processed for bioactive compounds identification using LC MS/MS analysis. RESULTS: Among total HPLC fractions (n = 30), 4 HPLC fractions of P. granatum (peel) showed potential activity against MDR pathogens. Fraction 1 (F1) and fraction 4 (F4) collected from aqueous extract showed maximum activity against P. aeruginosa. Fraction 2 (F2) of hexane showed antibacterial activity against three pathogens, while ethanol F4 exhibited antibacterial activity against A. baumannii. The active fractions were processed for LC MS/MS analysis to identify bioactive compounds. Valoneic acid dilactone (aqueous F1 and F4), Hexoside (ethanol F4) and Coumaric acid (hexane F2) were identified as bioactive compounds in HPLC fractions. CONCLUSION: Puncia granatum peel extracts HPLC fractions exhibited potential inhibitory activity against MDR bacterial human pathogens. Several bioactive compounds were identified from the HPLC fractions. Further characterization of these compounds may be helpful to conclude it as therapeutic lead molecules against MDR pathogens.


Assuntos
Antibacterianos/farmacologia , Bactérias/efeitos dos fármacos , Resistência a Múltiplos Medicamentos/efeitos dos fármacos , Lythraceae , Extratos Vegetais/farmacologia , Acinetobacter baumannii/efeitos dos fármacos , Fracionamento Químico , Cromatografia Líquida de Alta Pressão , Escherichia coli/efeitos dos fármacos , Frutas , Testes de Sensibilidade Microbiana , Pseudomonas aeruginosa/efeitos dos fármacos , Staphylococcus aureus/efeitos dos fármacos , Espectrometria de Massas em Tandem
11.
J Immunol ; 191(12): 6241-9, 2013 Dec 15.
Artigo em Inglês | MEDLINE | ID: mdl-24244025

RESUMO

Plerixafor (Mozobil) is a CXCR4 antagonist that rapidly mobilizes CD34(+) cells into circulation. Recently, plerixafor has been used as a single agent to mobilize peripheral blood stem cells for allogeneic hematopoietic cell transplantation. Although G-CSF mobilization is known to alter the phenotype and cytokine polarization of transplanted T cells, the effects of plerixafor mobilization on T cells have not been well characterized. In this study, we show that alterations in the T cell phenotype and cytokine gene expression profiles characteristic of G-CSF mobilization do not occur after mobilization with plerixafor. Compared with nonmobilized T cells, plerixafor-mobilized T cells had similar phenotype, mixed lymphocyte reactivity, and Foxp3 gene expression levels in CD4(+) T cells, and did not undergo a change in expression levels of 84 genes associated with Th1/Th2/Th3 pathways. In contrast with plerixafor, G-CSF mobilization decreased CD62L expression on both CD4 and CD8(+) T cells and altered expression levels of 16 cytokine-associated genes in CD3(+) T cells. To assess the clinical relevance of these findings, we explored a murine model of graft-versus-host disease in which transplant recipients received plerixafor or G-CSF mobilized allograft from MHC-matched, minor histocompatibility-mismatched donors; recipients of plerixafor mobilized peripheral blood stem cells had a significantly higher incidence of skin graft-versus-host disease compared with mice receiving G-CSF mobilized transplants (100 versus 50%, respectively, p = 0.02). These preclinical data show plerixafor, in contrast with G-CSF, does not alter the phenotype and cytokine polarization of T cells, which raises the possibility that T cell-mediated immune sequelae of allogeneic transplantation in humans may differ when donor allografts are mobilized with plerixafor compared with G-CSF.


Assuntos
Citocinas/genética , Regulação da Expressão Gênica/imunologia , Fator Estimulador de Colônias de Granulócitos/farmacologia , Mobilização de Células-Tronco Hematopoéticas , Compostos Heterocíclicos/farmacologia , Subpopulações de Linfócitos T/efeitos dos fármacos , Animais , Antígenos CD/biossíntese , Antígenos CD/genética , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD4-Positivos/transplante , Citocinas/biossíntese , Fatores de Transcrição Forkhead/biossíntese , Fatores de Transcrição Forkhead/genética , Doença Enxerto-Hospedeiro/imunologia , Humanos , Imunofenotipagem , Teste de Cultura Mista de Linfócitos , Linfopoese , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Quimera por Radiação , Receptores CXCR4/efeitos dos fármacos , Organismos Livres de Patógenos Específicos , Subpopulações de Linfócitos T/imunologia
12.
BMC Complement Altern Med ; 13: 265, 2013 Oct 14.
Artigo em Inglês | MEDLINE | ID: mdl-24119438

RESUMO

BACKGROUND: The main objective of this study was the phytochemical characterization of four indigenous essential oils obtained from spices and their antibacterial activities against the multidrug resistant clinical and soil isolates prevalent in Pakistan, and ATCC reference strains. METHODS: Chemical composition of essential oils from four Pakistani spices cumin (Cuminum cyminum), cinnamon (Cinnamomum verum), cardamom (Amomum subulatum) and clove (Syzygium aromaticum) were analyzed on GC/MS. Their antibacterial activities were investigated by minimum inhibitory concentration (MIC) and Thin-Layer Chromatography-Bioautographic (TLC-Bioautographic) assays against pathogenic strains Salmonella typhi (D1 Vi-positive), Salmonella typhi (G7 Vi-negative), Salmonella paratyphi A, Escherichia coli (SS1), Staphylococcus aureus, Pseudomonas fluorescens and Bacillus licheniformis (ATCC 14580). The data were statistically analyzed by using Analysis of Variance (ANOVA) and Least Significant Difference (LSD) method to find out significant relationship of essential oils biological activities at p <0.05. RESULTS: Among all the tested essential oils, oil from the bark of C. verum showed best antibacterial activities against all selected bacterial strains in the MIC assay, especially with 2.9 mg/ml concentration against S. typhi G7 Vi-negative and P. fluorescens strains. TLC-bioautography confirmed the presence of biologically active anti-microbial components in all tested essential oils. P. fluorescens was found susceptible to C. verum essential oil while E. coli SS1 and S. aureus were resistant to C. verum and A. subulatum essential oils, respectively, as determined in bioautography assay. The GC/MS analysis revealed that essential oils of C. cyminum, C. verum, A. subulatum, and S. aromaticum contain 17.2% cuminaldehyde, 4.3% t-cinnamaldehyde, 5.2% eucalyptol and 0.73% eugenol, respectively. CONCLUSIONS: Most of the essential oils included in this study possessed good antibacterial activities against selected multi drug resistant clinical and soil bacterial strains. Cinnamaldehyde was identified as the most active antimicrobial component present in the cinnamon essential oil which acted as a strong inhibitory agent in MIC assay against the tested bacteria. The results indicate that essential oils from Pakistani spices can be pursued against multidrug resistant bacteria.


Assuntos
Antibacterianos/farmacologia , Óleos Voláteis/farmacologia , Óleos Vegetais/farmacologia , Salmonella/efeitos dos fármacos , Amomum/química , Antibacterianos/química , Bactérias/efeitos dos fármacos , Cinnamomum/química , Cuminum/química , Farmacorresistência Bacteriana Múltipla , Cromatografia Gasosa-Espectrometria de Massas , Testes de Sensibilidade Microbiana , Óleos Voláteis/química , Óleos Vegetais/química , Syzygium/química
13.
Cytotherapy ; 11(3): 341-55, 2009.
Artigo em Inglês | MEDLINE | ID: mdl-19308771

RESUMO

BACKGROUND AIMS: Cancer immunotherapy involving natural killer (NK) cell infusions and administration of therapeutic agents modulating the susceptibility of tumors to NK-cell lysis has been proposed recently. We provide a method for expanding highly cytotoxic clinical-grade NK cells in vitro for adoptive transfer following bortezomib treatment in patients with advanced malignancies. METHODS: NK cells were expanded with irradiated Epstein-Barr virus-transformed lymphoblastoid cells. Expanded cells were evaluated for their phenotype, cytotoxicity, cytokine secretion, dependence on interleukin (IL)-2 and ability to retain function after cryopreservation. RESULTS: A pure population of clinical-grade NK cells expanded 490+/-260-fold over 21 days. Expanded NK cells had increased TRAIL, FasL and NKG2D expression and significantly higher cytotoxicity against bortezomib-treated tumors compared with resting NK cells. Expanded NK cells, co-cultured with K562 and renal cell carcinoma tumor targets, secreted significantly higher levels of soluble Fas ligand 6; fgjhd IFN-gamma, GM-CSF, TNF-alpha, MIP-1alpha and MIP-1beta compared with resting NK cells. Secretion of the above cytokines and NK-cell cytolytic function were IL-2 dose dependent. Cryopreservation of expanded NK cells reduced expression of NKG2D and TRAIL and NK-cell cytotoxicity, although this effect could be reversed by exposure of NK cells to IL-2. CONCLUSIONS: We describe a method for large-scale expansion of NK cells with increased expression of activating receptors and death receptor ligands resulting in superior cytotoxicity against tumor cells. This ex vivo NK-cell expansion technique is currently being utilized in a clinical trial evaluating the anti-tumor activity of adoptively infused NK cells in combination with bortezomib.


Assuntos
Carcinoma de Células Renais/imunologia , Proteína Ligante Fas/metabolismo , Células Matadoras Naturais/metabolismo , Subfamília K de Receptores Semelhantes a Lectina de Células NK/metabolismo , Ligante Indutor de Apoptose Relacionado a TNF/metabolismo , Ácidos Borônicos/farmacologia , Bortezomib , Carcinoma de Células Renais/tratamento farmacológico , Carcinoma de Células Renais/patologia , Técnicas de Cultura de Células , Proliferação de Células , Criopreservação , Citocinas/metabolismo , Citotoxicidade Imunológica/imunologia , Proteína Ligante Fas/genética , Proteína Ligante Fas/imunologia , Humanos , Imunofenotipagem , Imunoterapia/métodos , Células K562 , Células Matadoras Naturais/imunologia , Células Matadoras Naturais/patologia , Ativação Linfocitária , Subfamília K de Receptores Semelhantes a Lectina de Células NK/genética , Subfamília K de Receptores Semelhantes a Lectina de Células NK/imunologia , Pirazinas/farmacologia , Ligante Indutor de Apoptose Relacionado a TNF/genética , Ligante Indutor de Apoptose Relacionado a TNF/imunologia , Regulação para Cima
14.
Transfusion ; 49(3): 536-47, 2009 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-19243546

RESUMO

BACKGROUND: To generate clinical-grade dendritic cells (DCs) ex vivo for immunotherapy trials, peripheral blood monocytes are typically cultured in granulocyte-macrophage-colony-stimulating factor (GM-CSF) and interleukin (IL)-4 and then matured using one or more agents. Duration of the initial DC culture is one important variable that has not been systematically evaluated for its effect on the characteristics of the final mature DC product. STUDY DESIGN: DCs were generated from elutriated peripheral blood monocytes by incubation in medium containing 2000 units per mL each of GM-CSF and IL-4 for 3 to 7 days, followed by maturation with lipopolysaccharide and interferon-gamma (IFN-gamma). DC yield, viability, flow cytometric phenotype, and cytokine production were evaluated. RESULTS: The percentage yield and viability of mature DCs were similar after GM-CSF/IL-4 culture for 3 or 7 days. In either case, mature DCs expressed abundant CD80, CD86, CD83, and CCR7, but 3-day DCs expressed these antigens in a more consistent and homogeneous manner. Mature 3-day DCs produced much more IL-12 and less IL-10 after restimulation with CD40L-LTK than 7-day DCs. The former were also more effective in presenting immunogenic peptides to CD8 T cells. Analogous changes in cytokine production were observed in mature DCs prepared using lower concentrations of GM-CSF/IL-4 or when the alternative maturation cocktails poly(I:C)/IFN-gamma and soluble CD40L/IFN-gamma were used. CONCLUSION: Extended initial culture of DCs in GM-CSF/IL-4 does not affect yield or viability of subsequently matured DCs, but can adversely affect their ability to homogeneously express high levels of functionally important surface molecules such as CD83 and CCR7 and to produce IL-12.


Assuntos
Diferenciação Celular/imunologia , Citocinas/biossíntese , Citocinas/imunologia , Células Dendríticas/citologia , Células Dendríticas/imunologia , Monócitos/citologia , Monócitos/imunologia , Apresentação do Antígeno/efeitos dos fármacos , Apresentação do Antígeno/imunologia , Antígenos CD/imunologia , Linfócitos T CD8-Positivos/imunologia , Técnicas de Cultura de Células , Sobrevivência Celular/imunologia , Células Cultivadas , Células Dendríticas/efeitos dos fármacos , Células Dendríticas/metabolismo , Antígenos de Histocompatibilidade Classe I/imunologia , Humanos , Imunoglobulinas/imunologia , Lipopolissacarídeos/farmacologia , Glicoproteínas de Membrana/imunologia , Fenótipo , Receptores CCR7/imunologia , Fatores de Tempo , Regulação para Cima/imunologia
15.
Blood ; 113(10): 2245-55, 2009 Mar 05.
Artigo em Inglês | MEDLINE | ID: mdl-18988867

RESUMO

Preferentially expressed antigen of melanoma (PRAME) is aberrantly expressed in hematologic malignancies and may be a useful target for immunotherapy in leukemia. To determine whether PRAME is naturally immunogenic, we studied CD8(+) T-cell responses to 4 HLA-A*0201-restricted PRAME-derived epitopes (PRA100, PRA142, PRA300, PRA425) in HLA-A*0201-positive patients with acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), chronic myeloid leukemia (CML), and healthy donors. CD8(+) T cells recognizing PRAME peptides could be detected ex vivo in 4 of 10 ALL, 6 of 10 AML, 3 of 10 CML patients, and 3 of 10 donors by HLA-A2 tetramer analysis and flow cytometry for intracellular interferon-gamma. The frequency of PRAME-specific CD8(+) T cells was greater in patients with AML, CML, and ALL than healthy controls. All peptides were immunogenic in patients, while responses were only detected to PRA300 in donors. High PRAME expression in patient peripheral blood mononuclear cells was associated with responses to greater than or equal to 2 PRAME epitopes compared with low PRAME expression levels (4/7 vs 0/23, P = .001), suggesting a PRAME-driven T-cell response. PRAME-specific T cells were readily expanded in short-term cultures in donors and patients. These results provide evidence for spontaneous T cell reactivity against multiple epitopes of PRAME in ALL, AML, and CML. The potential for developing PRAME as a target for immunotherapy in leukemia deserves further exploration.


Assuntos
Antígenos de Neoplasias/imunologia , Linfócitos T CD8-Positivos/imunologia , Leucemia Mieloide/imunologia , Leucemia-Linfoma Linfoblástico de Células Precursoras/imunologia , Epitopos de Linfócito T/imunologia , Citometria de Fluxo , Antígenos HLA-A/imunologia , Antígeno HLA-A2 , Humanos , Imunofenotipagem , Interferon gama/biossíntese , Peptídeos/imunologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa
16.
J Clin Invest ; 118(3): 1099-109, 2008 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-18292810

RESUMO

Transplanted donor lymphocytes infused during hematopoietic stem cell transplantation (HSCT) have been shown to cure patients with hematological malignancies. However, less is known about the effects of HSCT on metastatic solid tumors. Thus, a better understanding of the immune cells and their target antigens that mediate tumor regression is urgently needed to develop more effective HSCT approaches for solid tumors. Here we report regression of metastatic renal cell carcinoma (RCC) in patients following nonmyeloablative HSCT consistent with a graft-versus-tumor effect. We detected RCC-reactive donor-derived CD8(+) T cells in the blood of patients following nonmyeloablative HSCT. Using cDNA expression cloning, we identified a 10-mer peptide (CT-RCC-1) as a target antigen of RCC-specific CD8(+) T cells. The genes encoding this antigen were found to be derived from human endogenous retrovirus (HERV) type E and were expressed in RCC cell lines and fresh RCC tissue but not in normal kidney or other tissues. We believe this to be the first solid tumor antigen identified using allogeneic T cells from a patient undergoing HSCT. These data suggest that HERV-E is activated in RCC and that it encodes an overexpressed immunogenic antigen, therefore providing a potential target for cellular immunity.


Assuntos
Antígenos Virais/imunologia , Carcinoma de Células Renais/terapia , Retrovirus Endógenos/imunologia , Transplante de Células-Tronco Hematopoéticas , Neoplasias Renais/terapia , Linfócitos T/imunologia , Adulto , Sequência de Aminoácidos , Sequência de Bases , Carcinoma de Células Renais/imunologia , Linhagem Celular Tumoral , Humanos , Neoplasias Renais/imunologia , Masculino , Pessoa de Meia-Idade , Dados de Sequência Molecular , Linfócitos T Citotóxicos/fisiologia , Transplante Homólogo
17.
Transfusion ; 46(9): 1494-504, 2006 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-16965575

RESUMO

BACKGROUND: Fluorinated ethylene-propylene (FEP) bags have been used instead of polystyrene (PS) flasks for ex vivo clinical-scale production of human dendritic cells (DCs) to facilitate closed-system recovery of these highly adherent cells. To assess the impact of DC culture on this nonadherent surface, the function of DCs generated in FEP and PS was compared. STUDY DESIGN AND METHODS: Cell yield, phenotype, cytokine production, migration, and antigen-presenting activity were measured in DCs prepared from peripheral blood monocytes in FEP bags or PS flasks with medium supplemented with serum, interleukin (IL)-4, and granulocyte-macrophage-colony-stimulating factor for 5 days to induce DC differentiation and CD40L or poly(I:C) plus interferon-gamma to promote maturation. RESULTS: DCs cultured in FEP or PS had comparable cell yield, viability, and CD83 and CCR7 expression. DCs generated in FEP, however, produced significantly less IL-12 and IL-10 during maturation, and differences persisted on rechallenge after harvest. FEP-cultured DCs migrated spontaneously or in response to CCR7 ligand more actively than PS-cultured DCs, but this difference was not significant. Mature DCs prepared in FEP and PS were equipotent in stimulating peptide-specific CD8 T-cell expansion in vitro. CONCLUSION: FEP- and PS-cultured DCs are similar in phenotype and in some functional measures, but FEP markedly reduces DC production of IL-12 and IL-10. This phenomenon presumably reflects intracellular changes linked to the absence of a surface for firm cell adherence. Given the importance of these cytokines in the immune response, these changes could have a significant impact on DC function in vivo.


Assuntos
Células Dendríticas/citologia , Células Dendríticas/imunologia , Poliestirenos/farmacologia , Politetrafluoretileno/análogos & derivados , Células Apresentadoras de Antígenos/efeitos dos fármacos , Células Apresentadoras de Antígenos/imunologia , Ligante de CD40/farmacologia , Linfócitos T CD8-Positivos/imunologia , Contagem de Células , Técnicas de Cultura de Células , Diferenciação Celular , Movimento Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Meios de Cultura/química , Meios de Cultura/farmacologia , Citocinas/biossíntese , Células Dendríticas/efeitos dos fármacos , Fator Estimulador de Colônias de Granulócitos e Macrófagos/farmacologia , Humanos , Interferon gama/farmacologia , Interleucina-10/biossíntese , Interleucina-12/biossíntese , Interleucina-4/farmacologia , Monócitos/citologia , Peptídeos/farmacologia , Politetrafluoretileno/farmacologia , Fatores de Tempo
18.
Int Immunol ; 14(2): 225-32, 2002 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-11809741

RESUMO

Mice infected with Listeria monocytogenes (LM) produce large numbers of H2-M3-restricted CD8 T cells directed against the formylated peptides, f-MIGWII and f-MIVIL. To examine responsiveness to these epitopes in the absence of infection, we inoculated mice with recombinant lemA (r-lemA) containing f-MIGWII or r-vemA (a variant of r-lemA containing f-MIVIL in place of f-MIGWII) without adjuvant. To monitor responses, we measured peptide-specific cytoplasmic IFN-gamma production ex vivo by freshly harvested splenocytes at varying times post-inoculation. B6 mice inoculated with r-lemA produced substantial numbers of epitope-specific CD8 cells with peak levels on day 7 when there were 1.1 x 10(6) f-MIGWII-specific CD8 cells in the spleen (8.2% of total CD8 splenocytes). The r-vemA-treated animals accumulated 0.25 x 10(6) cells (1.8% of total CD8 cells) at this time point. Comparable responses were observed after rechallenge of immunized animals. Other elements in the lemA moiety distinct from the immunogenic peptide were required since mice did not respond to equimolar amounts of synthetic f-MIGWII or f-MIVIL alone. In comparative studies, B6 and C3H/HeJ mice responded to r-lemA much more vigorously than BALB/c animals. When r-lemA- or r-vemA-treated B6 animals were challenged i.v. with LM 7 days later, they suppressed splenic accumulation of bacteria much more effectively than controls. On the other hand, antigen-treated animals were not protected against infection 1 month later. Thus, responsive strains of mice respond vigorously to H2-M3-restricted epitopes, even in the absence of bacterial infection or adjuvant. The resulting effectors acutely enhance antimicrobial resistance but do not confer long-term memory protection.


Assuntos
Proteínas de Bactérias/farmacologia , Linfócitos T CD8-Positivos/imunologia , Antígenos de Histocompatibilidade Classe I/imunologia , Listeriose/prevenção & controle , Sequência de Aminoácidos , Animais , Proteínas de Bactérias/química , Imunização Passiva , Memória Imunológica , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C3H , Camundongos Endogâmicos C57BL , Dados de Sequência Molecular , Proteínas Recombinantes/farmacologia , Especificidade da Espécie
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