Assessing the immunomodulatory potential of high-molecular-weight extracts from mushrooms; an assay based on THP-1 macrophages

BACKGROUND Food is a potential source of immunomodulating compounds that may be used to steer immune responses towards a desired status such as reducing inflammatory disorders. However, to identify and characterize such bioactive compounds, biologically relevant and standardized assays are required. Macrophages play an important role in immunomodulation and are suited for developing cell-based assays. An assay was developed based on macrophages, in a homogeneous differentiation state, using the human monocytic cell line THP-1 previously used to assess immunomodulatory properties of low-molecular-weight allergens, hormones, dietary supplements and therapeutic drugs. RESULTS Zymosan and mushroom polysaccharide extracts lead to a heterogeneous differentiation state of THP-1 monocytes, and these cells secrete low levels of cytokines upon stimulation. Differentiation into macrophages using a low concentration of phorbol 12-myristate 13-acetate improved responsiveness. Elevated levels of cytokines were secreted by cells in a homogenous differentiation state. In addition, it was determined that the assay performs best when using cells at a concentration of (2.5–5)¿×¿105 cells mL-1. CONCLUSION An assay was developed suitable to distinguish the immunomodulatory properties of food compounds in a reproducible manner. It was evaluated using eight mushroom species by measuring the secretion of relevant cytokines TNF-a, IL-1ß, IL-6 and IL-10. © 2014 Society of Chemical Industr

Assessing the immunomodulatory potential of high-molecular-weight extracts from mushrooms; an assay based on THP-1 macrophages

BACKGROUND Food is a potential source of immunomodulating compounds that may be used to steer immune responses towards a desired status such as reducing inflammatory disorders. However, to identify and characterize such bioactive compounds, biologically relevant and standardized assays are required. Macrophages play an important role in immunomodulation and are suited for developing cell-based assays. An assay was developed based on macrophages, in a homogeneous differentiation state, using the human monocytic cell line THP-1 previously used to assess immunomodulatory properties of low-molecular-weight allergens, hormones, dietary supplements and therapeutic drugs. RESULTS Zymosan and mushroom polysaccharide extracts lead to a heterogeneous differentiation state of THP-1 monocytes, and these cells secrete low levels of cytokines upon stimulation. Differentiation into macrophages using a low concentration of phorbol 12-myristate 13-acetate improved responsiveness. Elevated levels of cytokines were secreted by cells in a homogenous differentiation state. In addition, it was determined that the assay performs best when using cells at a concentration of (2.5–5)¿×¿105 cells mL-1. CONCLUSION An assay was developed suitable to distinguish the immunomodulatory properties of food compounds in a reproducible manner. It was evaluated using eight mushroom species by measuring the secretion of relevant cytokines TNF-a, IL-1ß, IL-6 and IL-10. © 2014 Society of Chemical Industr

Uso de esterco bovino e húmus de minhoca na produção de repolho híbrido Utilization of cattle manure and earthworm compost on hybrid cabbage production

Comparou-se a eficácia do esterco bovino e húmus de minhoca na produção de repolho, híbrido Matsukaze, em experimento realizado no Centro de Ciências Agrárias da UFPB, Areia, de 10/12/97 a 05/03/98. Os tratamentos utilizados foram 20; 30; 40; 50 e 60 t/ha de esterco bovino e 10; 15; 20; 25 e 30 t/ha de húmus de minhoca e tratamento testemunha (sem matéria orgânica). O delineamento experimental utilizado foi o de blocos casualizados com onze tratamentos distribuídos em esquema fatorial (5 x 2) + 1, em quatro repetições. Foram avaliados o diâmetro longitudinal, transversal, índice de formato e compacidade da cabeça, peso médio e produção total de cabeças. A dose de 46,0 t/ha de esterco bovino e 29,0 t/ha de húmus de minhoca resultaram em maiores diâmetros longitudinais na cabeça de repolho (13 e 12 cm, respectivamente). A dose de 47,0 t/ha de esterco bovino e 20,0 t/ha de humus de minhoca proporcionaram a formação de cabeças com maiores diâmetros transversais (13 e 11 cm, respectivamente). Todas as doses de esterco bovino induziram a formação de cabeças mais uniformes e compactas, enquanto a dose de 20 t/ha de húmus de minhoca propiciou a formação de cabeças desuniformes de baixa aceitação comercial. A dose de 41,0 t/ha de esterco bovino promoveu máximo peso médio (900 g) e máxima produtividade (47,0 t/ha) de cabeças, enquanto as doses de 27,0 e 29,0 t/ha de húmus foram responsáveis pelo peso médio máximo (700 g) e máxima produtividade (38,0 t/ha), respectivamente.<br>The use of bovine manure and earthworm compost were compared in cabbage production, hybrid Matsukaze, at the Federal University of Paraíba, Brazil, from December 1997 to March, 1998. The treatments consisted of 20; 30; 40; 50 and 60 t/ha of bovine manure and 10; 15; 20; 25 and 30 t/ha of earthworm compost and a treatment without organic matter (control). The experimental design was of randomized blocks, with eleven treatments arranged in a factorial scheme (5 x 2) + 1, with four replications. The longitudinal and transversal diameters, format index and head compactness, average weight and total production of heads were evaluated. The level of 46.0 t/ha of bovine manure and 29.0 t/ha of earthworm compost resulted in larger longitudinal diameters of cabbage heads (13 and 12 cm, respectively). The level of 47.0 t/ha of bovine manure and 20.0 t/ha of earthworm compost provided heads with larger transversal diameters (13 and 11 cm). All bovine manure levels induced the formation of more uniform and compact heads, while the use of 20 t/ha of earthworm compost resulted in not uniform heads with low commercial value. The level of 41.0 t/ha of bovine manure promoted maximum average head weight (900 g) and yield (47.0 t/ha), while the use of 27.0 t/ha of earthworm compost was responsible for the maximum average head weight (700 g) and yield (38.0 t/ha), respectively

Dynamics of the α6β4 Integrin in Keratinocytes

The integrin α6β4 has been implicated in two apparently contrasting processes, i.e., the formation of stable adhesions, and cell migration and invasion. To study the dynamic properties of α6β4 in live cells two different β4-chimeras were stably expressed in β4-deficient PA-JEB keratinocytes. One chimera consisted of full-length β4 fused to EGFP at its carboxy terminus (β4-EGFP). In a second chimera the extracellular part of β4 was replaced by EGFP (EGFP-β4), thereby rendering it incapable of associating with α6 and thus of binding to laminin-5. Both chimeras induce the formation of hemidesmosome-like structures, which contain plectin and often also BP180 and BP230. During cell migration and division, the β4-EGFP and EGFP-β4 hemidesmosomes disappear, and a proportion of the β4-EGFP, but not of the EGFP-β4 molecules, become part of retraction fibers, which are occasionally ripped from the cell membrane, thereby leaving “footprints” of the migrating cell. PA-JEB cells expressing β4-EGFP migrate considerably more slowly than those that express EGFP-β4. Studies with a β4-EGFP mutant that is unable to interact with plectin and thus with the cytoskeleton (β4(R1281W)-EGFP) suggest that the stabilization of the interaction between α6β4 and LN-5, rather than the increased adhesion to LN-5, is responsible for the inhibition of migration. Consistent with this, photobleaching and recovery experiments revealed that the interaction of β4 with plectin renders the bond between α6β4 and laminin-5 more stable, i.e., β4-EGFP is less dynamic than β4(R1281W)-EGFP. On the other hand, when α6β4 is bound to laminin-5, the binding dynamics of β4 to plectin are increased, i.e., β4-EGFP is more dynamic than EGFP-β4. We suggest that the stability of the interaction between α6β4 and laminin-5 is influenced by the clustering of α6β4 through the deposition of laminin-5 underneath the cells. This clustering ultimately determines whether α6β4 will inhibit cell migration or not