RESUMO
It is known that growth hormone (GH) is expressed in immune cells, where it exerts immunomodulatory effects. However, the mechanisms of expression and release of GH in the immune system remain unclear. We analyzed the effect of growth hormone-releasing hormone (GHRH), thyrotropin-releasing hormone (TRH), ghrelin (GHRL), and somatostatin (SST) upon GH mRNA expression, intracellular and released GH, Ser133-phosphorylation of CREB (pCREBS133), intracellular Ca2+ levels, as well as B-cell activating factor (BAFF) mRNA expression in bursal B-lymphocytes (BBLs) cell cultures since several GH secretagogues, as well as their corresponding receptors (-R), are expressed in B-lymphocytes of several species. The expression of TRH/TRH-R, ghrelin/GHS-R1a, and SST/SST-Rs (Subtypes 1 to 5) was observed in BBLs by RT-PCR and immunocytochemistry (ICC), whereas GHRH/GHRH-R were absent in these cells. We found that TRH treatment significantly increased local GH mRNA expression and CREB phosphorylation. Conversely, SST decreased GH mRNA expression. Additionally, when added together, SST prevented TRH-induced GH mRNA expression, but no changes were observed in pCREBS133 levels. Furthermore, TRH stimulated GH release to the culture media, while SST increased the intracellular content of this hormone. Interestingly, SST inhibited TRH-induced GH release in a dose-dependent manner. The coaddition of TRH and SST decreased the intracellular content of GH. After 10 min. of incubation with either TRH or SST, the intracellular calcium levels significantly decreased, but they were increased at 60 min. However, the combined treatment with both peptides maintained the Ca2+ levels reduced up to 60-min. of incubation. On the other hand, BAFF cytokine mRNA expression was significantly increased by TRH administration. Altogether, our results suggest that TRH and SST are implicated in the regulation of GH expression and release in BBL cultures, which also involve changes in pCREBS133 and intracellular Ca2+ concentration. It is likely that TRH, SST, and GH exert autocrine/paracrine immunomodulatory actions and participate in the maturation of chicken BBLs.
Assuntos
Proteínas Aviárias/imunologia , Linfócitos B/imunologia , Bolsa de Fabricius/imunologia , Galinhas/imunologia , Grelina/imunologia , Hormônio Liberador de Hormônio do Crescimento/imunologia , Hormônio do Crescimento/imunologia , Somatostatina/imunologia , Hormônio Liberador de Tireotropina/imunologia , Animais , Linfócitos B/citologia , Bolsa de Fabricius/citologia , Técnicas de Cultura de Células , Células CultivadasRESUMO
The knowledge related to the fate of probiotics in the complex environment of the intestinal microbiota in broilers is just beginning to be elucidated; however, it is not yet well understood. A good method to investigate the mechanisms by which probiotics mediate their effects is to mark probiotic bacteria and trace them. The aim of this research was to develop a new method to estimate in vivo fluorescein isothiocyanate (FITC)-labelled Lactobacillus salivarius DSPV 001P counts during passage through the gastrointestinal tract (GIT) of broilers. Forty-five, 1 d old Cobb broilers were used in this trial. Programmed necropsies were performed 30 min, 6 h, and 12 h after the administration of the probiotic bacterium, and samples of liver, crop, duodenum, caecum, and bursa of fabricius were collected. To determine the spatial and temporal transit of L. salivarius DSPV 001P in broilers, the number of bacteria as well as its respective fluorescent signal produced by FITC were measured. In order to observe the relationship between the variables, a logistic regression analysis was applied. The amount of fluorescence could be used as an indicator of fluorescent probiotic bacteria in the crop and duodenum 30 min after probiotic bacterium supplementation. In addition, the fluorescent signal could be used to estimate bacterial counts in caecum 6 and 12 h after L. salivarius DSPV 001P administration. To the best of our knowledge, this research is the first in vivo trial to employ the bacterial FITC-labelling technique in order to enumerate probiotic bacteria during gastrointestinal transit in broilers.
Assuntos
Galinhas/microbiologia , Trânsito Gastrointestinal , Ligilactobacillus salivarius/fisiologia , Probióticos , Animais , Bolsa de Fabricius/citologia , Bolsa de Fabricius/microbiologia , Ceco/citologia , Ceco/microbiologia , Digestão , Fluoresceína-5-Isotiocianato , Corantes Fluorescentes , Trato Gastrointestinal/citologia , Trato Gastrointestinal/microbiologia , Distribuição AleatóriaRESUMO
Growth hormone (GH) is expressed in several extra-pituitary tissues, including the primary and secondary lymphoid organs of the immune system. In birds, GH mRNA and protein expression show a specific developmental distribution pattern in the bursa of Fabricius (BF), particularly in epithelial and B cells. Changes in the bursal concentration and distribution of locally produced GH during ontogeny suggest it is involved in B cell differentiation and maturation, as well as in a functional survival role in this organ, which may be mediated by paracrine/autocrine mechanisms. Here, we analyzed the anti-apoptotic effect of GH in BF and the intracellular signaling pathways involved in this activity. Also, we studied if this effect was exerted directly by GH or mediated indirectly by IGF-I. Bursal cell cultures showed an important loss of their viability after 4h of incubation and a significant increase in apoptosis. However, treatment with 10nM GH or 40 nM IGF-I significantly increased B cell viability (16.7 ± 0.67% and 13.4 ± 1.12%, respectively) when compared with the untreated controls. In addition, the presence of apoptotic bodies (TUNEL) dramatically decreased (5.5-fold) after GH and IGF-I treatments, whereas co-incubation with anti-GH or anti-IGF-I, respectively, blocked their anti-apoptotic effect. Likewise, both GH and IGF-I significantly inhibited caspase-3 activity (by 40 ± 2.0%) in these cultures. However, the use of anti-IGF-I could not reverse the GH anti-apoptotic effects, thus indicating that these were exerted directly. The addition of 100 nM wortmannin (a PI3K/Akt inhibitor) blocked the GH protective effects. Also, GH stimulated (3-fold) the phosphorylation of Akt in bursal cells, and adding wortmannin or an anti-GH antibody inhibited this effect. Furthermore, GH was capable to stimulate (7-fold) the expression of Bcl-2. Taken together, these results indicate that the direct anti-apoptotic activity of GH observed in the chicken bursal B cell cultures might be mediated through the PI3K/Akt pathway.
Assuntos
Apoptose/efeitos dos fármacos , Bolsa de Fabricius/metabolismo , Hormônio do Crescimento/farmacologia , Fator de Crescimento Insulin-Like I/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Animais , Linfócitos B/citologia , Linfócitos B/efeitos dos fármacos , Linfócitos B/metabolismo , Western Blotting , Bolsa de Fabricius/citologia , Bolsa de Fabricius/efeitos dos fármacos , Caspase 3/metabolismo , Células Cultivadas , Galinhas/metabolismo , Ensaio de Imunoadsorção Enzimática , Técnicas Imunoenzimáticas , Marcação In Situ das Extremidades Cortadas , Masculino , Fosforilação/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacosRESUMO
Gumboro disease is caused by the infectious bursal disease virus (IBDV) which rapidly destroys immature B-lymphocytes of bursa of Fabricious, and causes immune suppression and high mortality in commercial broiler farms in Bangladesh. To investigate the possible effect of IBDV on lymphocytes and its distribution in the major lymphoid organs, bursa of Fabricious including spleen and thymus of naturally Gumboro-infected broilers, a research was conducted in the Department of Anatomy and Histology, collaboration with the Department of Pathology, Bangladesh Agricultural University, Bangladesh. Bursa of Fabricious, spleen and thymus of 21-days-old Gumboro-infected and non-infected broilers of same age (control) were routinely processed and stained by hematoxylin and eosin to examine the distribution of lymphocytes in the major lymphatic organs as well as quantified the number of lymphocytes under high power magnification field and compared with those of control. The number of lymphocytes in bursa of Fabricious, spleen and thymus of Gumboro-infected broilers were 27.20 ± 1.53, 66.50 ± 2.70 and 79.30 ± 3.92 whereas 121 ± 3.82, 89.90 ± 2.09 and 106.30 ± 4.07 were in non-infected control respectively. The numbers of lymphocytes were significantly (p < 0.05) lower in all lymphatic organs of Gumboro-infected broilers than those of non-infected control. The significant numbers of lymphocytes decrease in spleen and thymus suggest that IBVD not only destroy lymphocytes in bursa of Fabricious, but also in spleen and thymus and thus may severely suppress the immune response of IBVD affected broilers.
La enfermedad de Gumboro es causada por el virus de la bursitis infecciosa (VBI), que destruye rápidamente los linfocitos B inmaduros de la bolsa de Fabricio, y causa supresión inmune y la elevada mortalidad en las granjas comerciales de pollos de engorde en Bangladesh. Para investigar el posible efecto del VBI en los linfocitos y su distribución en los órganos linfoides principales, la bolsa de Fabricio, incluyendo el bazo y el timo de pollos de engorde naturalmente infectados con Gumboro, se realizó una investigación en el Departamento de Anatomía e Histología, y el Departamento de Patología, Universidad Agrícola de Bangladesh, Bangladesh. Tanto la bolsa de Fabricio, bazo y el timo de pollos de engorde con 21 días de edad infectados con Gumboro y no infectados de la misma edad (control) se procesaron de forma rutinaria y se tiñeron con H & E para examinar la distribución de los linfocitos en los órganos linfáticos principales, así cuantificar el número de linfocitos bajo campo de alta magnificación y compararlos con los de control. El número de linfocitos en la bolsa de Fabricio, bazo y timo de pollos infectados con Gumboro fue 27,20 ± 1,53, 66,50 ± 2,70 y 79,30 ± 3,92, respectivamente, mientras que en los controles no infectados fue 121 ± 3,82, 89,90 ± 2,09 y 106,30 ± 4,07 respectivamente. El número de linfocitos fue significativamente (p < 0,05) más bajo en todos los órganos linfáticos de pollos de engorde infectados con Gumboro que los no infectados. La disminuición significativa de linfocitos en el bazo y timo, sugiere que el VBI no sólo destruye linfocitos en la bolsa de Fabricio, sino también en el bazo y el timo y, por tanto, puede suprimir severamente la respuesta inmune de pollos de engorde afectados por VBI.
Assuntos
Animais , Doenças das Aves Domésticas , Linfócitos , Vírus da Doença Infecciosa da Bursa , Tecido Linfoide/citologia , Aves Domésticas , Baço/citologia , Timo/citologia , Bolsa de Fabricius/citologia , Galinhas , Tecido Linfoide/imunologiaRESUMO
Growth hormone (GH) is expressed in the chicken bursa of Fabricius (BF), an organ that undergoes three distinct developmental stages: rapid growth (late embryogenesis until 6-8 weeks of age [w]), plateaued growth (between 10 and 15w), and involution (after 18-20w). The distribution and abundance of GH-immunoreactivity (GH-IR) and GH mRNA expression in stromal and non-stromal bursal cells during development, as well as the potential anti-apoptotic effect of GH in bursal cell survival were the focus of this study. GH mRNA expression was mainly in the epithelial layer and in epithelial buds at embryonic day (ED) 15; at 2w it was widely distributed within the follicle and in the interfollicular epithelium (IFE); at 10w it clearly diminished in the epithelium; whereas at 20w it occurred in only a few cortical cells and in the connective tissue. Parallel changes in the relative proportion of GH mRNA expression (12, 21, 13, 1%) and GH-IR (19, 18, 11, <3%) were observed at ED 15, 2w, 10w, and 20w, respectively. During embryogenesis, GH-IR co-localized considerably with IgM-IR, but scarcely with IgG-IR, whereas the opposite was observed after hatching. Significant differences in bursal cell death occurred during development, with 9.3% of cells being apoptotic at ED 15, 0.4% at 2w, 0.23% at 10w, and 21.1% at 20w. Addition of GH increased cultured cell survival by a mechanism that involved suppression (up to 41%) of caspase-3 activity. Results suggest that autocrine/paracrine actions of bursal GH are involved in the differentiation and proliferation of B lymphocytes and in BF growth and cell survival in embryonic and neonatal chicks, whereas diminished GH expression in adults may result in bursal involution.
Assuntos
Bolsa de Fabricius/embriologia , Galinhas/fisiologia , Hormônio do Crescimento/fisiologia , Animais , Apoptose/fisiologia , Bolsa de Fabricius/citologia , Bolsa de Fabricius/fisiologia , Sobrevivência Celular/fisiologia , Embrião de Galinha , Galinhas/crescimento & desenvolvimento , Galinhas/metabolismo , Hormônio do Crescimento/genética , Imunoglobulina G/fisiologia , Imunoglobulina M/fisiologia , Imuno-Histoquímica/veterinária , Hibridização In Situ/veterinária , Marcação In Situ das Extremidades Cortadas/veterinária , Masculino , RNA Mensageiro/química , RNA Mensageiro/genética , Organismos Livres de Patógenos Específicos , Células Estromais/citologia , Células Estromais/metabolismo , Células Estromais/fisiologiaRESUMO
The estimated volumes of the follicular medulla (x) and cortex (y) from 14-day-old embryos till 28-day-old White Leghorn chicks were associated through the allometric formula y = bxk or log y = log b + k log x. Two successive allometric growth stages (I and II) are discernable, being hatching the transition region between them. The volumetric growth of the cortex is 2.49 times greater than that of the medulla in stage I, whereas cortex and medulla grow isometrically in stage II. The curve fitting procedure analysis of the absolute cortical and medullary growth confirmed these results. The fine structure of the cell types in the follicular medulla revealed that: a) in the allometric stage I pre-existing, bud precursor (Pr) cells appear to give rise to basal (Ba) and medullary epithelial (ME) cells, in both cases showing lucent and dark varieties. A medullary cytoreticulum is established at the onset of this stage. b) The marked lymphocyte proliferation during stage II occurs among the thin and short cytoplasmic processes of BA cells. These processes extend towards the centre of the medulla and also show many lateral interdigitating processes. During this same stage, the cytoplasmic processes of ME cells elongate and become thinner promoting a widening of the cytoreticulum interstices. The fine structural analysis of the cortical cytoarchitectural arrangement showed that: a) before the onset of stage I (14-day-old embryos) the cortex consists mainly of typical fibroblasts (F) and a few blastic (Pr?) cells. Later in this stage I, a poorly defined cortical framework is made up of typical fibroblasts, few cortical branching (CB) cells of the epithelial variety (which seem to be derived from Pr cells) and CB cells of the fibroblast-like variety. These cells are interspersed with mature and immature lymphocytes. b) Allometric stage II of the cortex is characterized by the presence of very thin and long cytoplasmic processes from CB cells of both epithelial and fibroblast-like varieties. The arrangement of CB cell profiles, visualized in electron micrographic montages, is remarkably similar to that of the ME cells profiles which are known to form a cytoreticulum. We thus propose that the mature follicular cortex is endowed with a cellular framework forming wide interstices in which packed mature lymphoid cells are lodged.