RESUMO
25-Hydroxyvitamin D3-3ß-glucuronic acid (25OHD-Gluc) is produced in the liver and is a constituent of human blood and bile. Bacterial glucuronidases (GUS) in mammalian digestive microbiota cleave glucuronide conjugates, such as 25OHD-Gluc, and release the free aglycone (i.e., 25OHD) inside the intestinal lumen. We hypothesized that 25OHD-Gluc would elicit a VDR-dependent mRNA response in the colon after cleavage by gut microbiota. The activity of 25OHD-Gluc was investigated by measuring expression of cytochrome P450 24A1 (Cyp24) mRNA both in vitro and in vivo. In cell culture, Caco2 cells responded to 25OHD-Gluc, whereas HT29 cells did not. When coincubated with GUS, both cell lines elicited a robust response as indicated by a 5 Ct (32-fold) increase in Cyp24 mRNA. In vitamin D-sufficient mice, we found that both oral and subcutaneous administration of 1 nmol 25OHD-Gluc induced expression of Cyp24 mRNA in the colon whereas 25OHD did not. In contrast, 25OHD, but not 25OHD-Gluc, was active in the duodenum. When the jejunum was surgically ligated to block flow of digesta to the colon, neither oral nor subcutaneous administration of 2 nmol 25OHD-Gluc was able to induce expression of Cyp24 in the colon. Our findings suggest that 25OHD-Gluc, a vitamin D metabolite found in bile, induces VDR-mediated responses in the colon by crossing the apical membrane of the colon epithelium.NEW & NOTEWORTHY We found that 25OHD-Gluc, an endogenously produced metabolite, is delivered to the colon via bile to induce vitamin D-mediated responses in the colon.
Assuntos
Colo/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Vitamina D/análogos & derivados , Animais , Células CACO-2 , Glucuronídeos , Células HT29 , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Vitamina D/química , Vitamina D/metabolismo , Vitamina D/farmacologiaRESUMO
BACKGROUND: 25-Hydroxycholecalciferol [25(OH)D] is the predominant circulating metabolite of vitamin D and serves as the precursor for 1α,25-dihydroxycholecalciferol [1,25(OH)2D], the hormonally active form. The presence of 1α-hydroxylase (1α-OHase) in the intestine suggests that 1,25(OH)2D can be produced from 25(OH)D, but the effects of oral 25(OH)D on the intestine have not been determined. OBJECTIVES: We investigated the acute intestinal response to orally consumed 25(OH)D in mice by assessing mRNA induction of cytochrome p450 family 24 subfamily A member 1 (Cyp24), a vitamin D-dependent gene. The mechanism of action then was determined through in vitro analyses with Caco2 and HT-29 cells. METHODS: Adult male C57BL6 mice were given a single oral dose of 40, 80, 200, or 400 ng 25(OH)D (n = 4 per dose) or vehicle (n = 3), and then killed 4 h later to evaluate the duodenal expression of Cyp24 mRNA by qPCR and RNA in situ hybridization. The 25(OH)D-mediated response was also evaluated with Caco2 and HT-29 cells by inhibition assay and dose-response analysis. A cytochrome p450 family 27 subfamily B member 1 (CYP27B1) knockdown of HT-29 was created to compare the dose-response parameters with wild-type HT-29 cells. RESULTS: Oral 25(OH)D induced expression of Cyp24 mRNA in the duodenum of mice with 80 ng 25(OH)D by 3.3 ± 0.8 ΔΔCt compared with controls (P < 0.05). In vitro, both Caco2 and HT-29 cells responded to 25(OH)D treatment with 200-fold and 175-fold greater effective concentration at 50% maximal response than 1,25(OH)2D, yet inhibition of 1α-OHase and knockdown of CYP27B1 had no effect on the responses. CONCLUSIONS: In mice, orally consumed 25(OH)D elicits a vitamin D-mediated response in the duodenum. In vitro assessments suggest that the response from 25(OH)D does not require activation by 1α-OHase and that 25(OH)D within the intestinal lumen acts as a vitamin D receptor agonist.
Assuntos
Calcifediol/administração & dosagem , Duodeno/efeitos dos fármacos , 25-Hidroxivitamina D3 1-alfa-Hidroxilase/genética , Administração Oral , Animais , Células CACO-2 , Calcifediol/farmacologia , Família 24 do Citocromo P450/genética , Relação Dose-Resposta a Droga , Técnicas de Silenciamento de Genes , Células HT29 , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BLRESUMO
Several minerals are required for life to exist. In animals, 7 elements (Ca, P, Mg, Na, K, Cl, and S) are required to be present in the diet in fairly large amounts (grams to tens of grams each day for the dairy cow) and are termed macrominerals. Several other elements are termed microminerals or trace minerals because they are required in much smaller amounts (milligrams to micrograms each day). In most cases the mineral in the diet must be absorbed across the gastrointestinal mucosa and enter the blood if it is to be of value to the animal. The bulk of this review discusses the paracellular and transcellular mechanisms used by the gastrointestinal tract to absorb each of the various minerals needed. Unfortunately, particularly in ruminants, interactions between minerals and other substances within the diet can occur within the digestive tract that impair mineral absorption. The attributes of organic or chelated minerals that might permit diet minerals to circumvent factors that inhibit absorption of more traditional inorganic forms of these minerals are discussed. Once absorbed, minerals are used in many ways. One focus of this review is the effect macrominerals have on the acid-base status of the animal. Manipulation of dietary cation and anion content is commonly used as a tool in the dry period and during lactation to improve performance. A section on how the strong ion theory can be used to understand these effects is included. Many microminerals play a role in the body as cofactors of enzymes involved in controlling free radicals within the body and are vital to antioxidant capabilities. Those same minerals, when consumed in excess, can become pro-oxidants in the body, generating destructive free radicals. Complex interactions between minerals can compromise the effectiveness of a diet in promoting health and productivity of the cow. The objective of this review is to provide insight into some of these mechanisms.
Assuntos
Equilíbrio Ácido-Base , Antioxidantes/metabolismo , Dieta/veterinária , Minerais/metabolismo , Animais , Bovinos , Humanos , Mucosa Intestinal/metabolismo , Ruminantes/metabolismoRESUMO
Most studies demonstrating that diets with low dietary cation-anion difference (DCAD) reduce hypocalcemia in cows add enough anions to the diet to reduce urine pH below 7.0. One objective of these experiments was to determine whether there is any benefit to periparturient plasma Ca concentration if diet anion addition results in a lesser degree of acidification of the cow and urine pH does not go below 7.0. Another method for reducing hypocalcemia involves feeding a prepartal diet that is Ca deficient. This places the cow in negative Ca balance before calving, stimulating parathyroid hormone (PTH) and 1,25-dihydroxyvitamin D secretion before calving and thus promoting Ca homeostasis at calving. As practiced in the field, low-Ca diets are often about 0.5% Ca. Our second objective was to determine whether a 0.46% Ca diet would be sufficiently low in Ca to stimulate PTH secretion before calving. A meta-analysis of the literature suggests that a 0.5% Ca, low-DCAD diet will reduce hypocalcemia better than a 0.7% Ca diet. A third objective was to compare periparturient plasma Ca in cows fed 0.46 or 0.72% Ca diets with similar DCAD. In experiment 1, anions (primarily chloride) or anions plus Ca were added to a 1.4% K basal diet to create the following diets: 0.46% Ca and +167 mEq/kg of DCAD, 0.46% Ca and -13 mEq/kg of DCAD, and 0.72% Ca and -17 mEq/kg of DCAD. In experiment 2, the same amounts of anion were added to a 2.05% K basal diet to create the following diets: 0.46% Ca and +327 mEq/kg of DCAD, 0.46% Ca and +146 mEq/kg of DCAD, and 0.72% Ca and +140 mEq/kg of DCAD. In experiment 1, cows fed the diet with 0.46% Ca and +167 mEq/kg of DCAD had significantly lower plasma Ca concentration after calving than cows fed the 0.46 or 0.72% Ca diets with anions. Periparturient plasma Ca concentrations did not differ in cows fed the low-DCAD diets with 0.46 or 0.72% Ca. Urine pH was reduced from 8.27 in the diet with 0.46% Ca and +167 mEq/kg of DCAD to 7.07 and 7.41 in the 0.46 and 0.72% Ca anion diets, respectively. Precalving plasma PTH and 1,25-dihydroxyvitamin D concentrations were similar in cows fed the 0.46% Ca diets and the 0.72% Ca diets, suggesting that the 0.46% Ca diets were not low enough in Ca to place the cow in negative Ca balance before calving. In experiment 2, adding the anion supplements to a 2.05% K diet did not reduce urine pH below 8.0. Periparturient plasma Ca concentrations did not differ in cows in any group in experiment 2. Precalving diets that are 0.46% Ca fed ad libitum are too high in Ca to stimulate Ca homeostasis before calving. Adding anions to a diet can benefit periparturient cow plasma Ca concentration, but only if it alters acid-base status enough to reduce urine pH below 7.5.
Assuntos
Ânions/administração & dosagem , Cálcio/administração & dosagem , Doenças dos Bovinos/prevenção & controle , Suplementos Nutricionais/análise , Hipocalcemia/veterinária , Parto/efeitos dos fármacos , Ração Animal/análise , Animais , Ânions/metabolismo , Cálcio/análise , Cálcio/metabolismo , Bovinos , Doenças dos Bovinos/metabolismo , Cloretos/administração & dosagem , Cloretos/análise , Cloretos/metabolismo , Dieta/veterinária , Feminino , Homeostase , Concentração de Íons de Hidrogênio , Hipocalcemia/metabolismo , Hipocalcemia/prevenção & controle , Hormônio Paratireóideo/metabolismo , Parto/metabolismoRESUMO
Dairy cows often experience decreased immune function around the time of calving, typified by impaired polymorphonuclear neutrophil (PMN) function and a transient neutropenia. This is associated with increased disease incidence, including mastitis, retained placenta, and metritis. In an attempt to improve PMN functional capacity during the periparturient period, we injected cows with recombinant bovine granulocyte colony-stimulating factor covalently bound to polyethylene glycol (PEG rbG-CSF) twice subcutaneously, about 6d before calving and within 24h after calving. Twenty-one cows in their second pregnancy were enrolled in this study and divided into 2 groups: PEG rbG-CSF treated (n=11) and saline-treated controls (n=10). The PMN numbers quickly and dramatically increased after PEG rbG-CSF administration and remained elevated through the end of the experiment (13d after calving). Exocytosis of myeloperoxidase by stimulated PMN, which is generally decreased in periparturient cows, was markedly increased by PEG rbG-CSF after injection. Higher myeloperoxidase exocytosis persisted for at least 10d after calving. The PMN superoxide anion release and phagocytosis activity did not differ between groups. Injection of PEG rbG-CSF was safe for cows, with no significant negative effects observed. The greatest single effect of PEG rbG-CSF administration was a dramatic increase in circulating numbers of PMN. The increased numbers of PMN ready to move to a site of infection early in the course of an infection may improve the ability of the cow to ward off clinical disease in the periparturient period.
Assuntos
Fator Estimulador de Colônias de Granulócitos/farmacologia , Mastite Bovina/epidemiologia , Placenta Retida/epidemiologia , Polietilenoglicóis/farmacologia , Ácido 3-Hidroxibutírico/sangue , Animais , Cálcio/sangue , Bovinos , Doenças dos Bovinos/diagnóstico , Doenças dos Bovinos/epidemiologia , Doenças dos Bovinos/patologia , Ácidos Graxos não Esterificados/sangue , Feminino , Fator Estimulador de Colônias de Granulócitos/administração & dosagem , Injeções Subcutâneas , Contagem de Leucócitos , Mastite Bovina/diagnóstico , Mastite Bovina/patologia , Neutrófilos/efeitos dos fármacos , Neutrófilos/metabolismo , Peroxidase/metabolismo , Fagocitose/efeitos dos fármacos , Placenta Retida/diagnóstico , Placenta Retida/patologia , Polietilenoglicóis/administração & dosagem , Gravidez , Proteínas Recombinantes/administração & dosagem , Proteínas Recombinantes/farmacologiaRESUMO
Synthetic conjugation of a glucuronide to 1,25-dihydroxyvitamin D3 (1,25D3) to produce ß-25-monoglucuronide-1,25D3 (ßGluc-1,25D3) renders the hormone biologically inactive and resistant to mammalian digestive enzymes. However, ß-glucuronidase produced by bacteria in the lower intestinal tract can cleave off the glucuronide, releasing the active hormone. In mice given a single oral dose of 1,25D3, 24-hydroxylase (Cyp24a1) gene expression was strongly enhanced in the duodenum, but not in the colon, despite circulating concentrations of 1,25D3 that peaked at â¼3.0 nmol/l. In contrast, in mice treated with an equimolar dose of ßGluc-1,25D3, Cyp24a1 gene expression increased 700-fold in the colon but was significantly weaker in the duodenum compared with mice treated with 1,25D3. Similar results were observed with another vitamin D-dependent gene. When administered subcutaneously, 1,25D3 weakly stimulated colon Cyp24a1 gene expression while ßGluc-1,25D3 again resulted in strong enhancement. Surgical ligation to block passage of ingesta beyond the upper intestinal tract abolished upregulation of colon Cyp24a1 gene expression by orally and subcutaneously administered ßGluc-1,25D3. Feeding ßGluc-1,25D3 for 5 days revealed a linear, dose-dependent increase in colon Cyp24a1 gene expression but did not significantly increase plasma 1,25D3 or calcium concentrations. This study indicates that the colon is relatively insensitive to circulating concentrations of 1,25D3 and that the strongest gene enhancement occurs when the hormone reaches the colon via the lumen of the intestinal tract. These findings have broad implications for the use of vitamin D compounds in colon disorders and set the stage for future therapeutic studies utilizing ßGluc-1,25D3 in their treatment.
Assuntos
Calcitriol/análogos & derivados , Colo/metabolismo , Expressão Gênica/efeitos dos fármacos , Esteroide Hidroxilases , Administração Oral , Animais , Disponibilidade Biológica , Calcitriol/biossíntese , Calcitriol/metabolismo , Calcitriol/farmacocinética , Calcitriol/farmacologia , Cálcio/metabolismo , Relação Dose-Resposta a Droga , Glucuronídeos/metabolismo , Injeções Subcutâneas , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Ratos , Ratos Sprague-Dawley , Esteroide Hidroxilases/genética , Esteroide Hidroxilases/metabolismo , Esteroide Hidroxilases/farmacologia , Vitamina D3 24-Hidroxilase , Vitaminas/metabolismo , Vitaminas/farmacocinéticaRESUMO
1,25-Dihydroxyvitamin D(3) [1,25(OH)(2)D] has been shown to inhibit development of dextran sodium sulfate (DSS)-induced colitis in mice but can also cause hypercalcemia. The aim of this study was to evaluate whether ß-glucuronides of vitamin D could deliver 1,25(OH)(2)D to the colon to ameliorate colitis while reducing the risk of hypercalcemia. Initial studies demonstrated that bacteria residing in the lower intestinal tract were capable of liberating 1,25(OH)(2)D from 1,25-dihydroxyvitamin D(3)-25-ß-glucuronide [ß-gluc-1,25(OH)(2)D]. We also determined that a much greater upregulation of the vitamin D-dependent 24-hydroxylase gene (Cyp24) was induced in the colon by treatment of mice with an oral dose of ß-gluc-1,25(OH)(2)D than 1,25(OH)(2)D, demonstrating targeted delivery of 1,25(OH)(2)D to the colon. We then tested ß-glucuronides of vitamin D in the mouse DSS colitis model in two studies. In mice receiving DSS dissolved in distilled water and treated with 1,25(OH)(2)D or ß-gluc-1,25(OH)(2)D, severity of colitis was reduced. Combination of ß-gluc-1,25(OH)(2)D with 25-hydroxyvitamin D(3)-25-ß-glucuronide [ß-gluc-25(OH)D] resulted in the greatest reduction of colitis lesions and symptoms in DSS-treated mice. Plasma calcium concentrations were lower in mice treated with ß-gluc-1,25(OH)(2)D alone or in combination with ß-gluc-25(OH)D than in mice treated with 1,25(OH)(2)D, which were hypercalcemic at the time of death. ß-Glucuronides of vitamin D compounds can deliver 1,25(OH)(2)D to the lower intestine and can reduce symptoms and lesions of acute colitis in this model.
Assuntos
Calcitriol/análogos & derivados , Colite/tratamento farmacológico , Colo/efeitos dos fármacos , Doenças Inflamatórias Intestinais/tratamento farmacológico , Vitamina D/análogos & derivados , Animais , Calcitriol/administração & dosagem , Calcitriol/química , Calcitriol/uso terapêutico , Cálcio/sangue , Colite/sangue , Colite/patologia , Colo/patologia , Modelos Animais de Doenças , Portadores de Fármacos , Doenças Inflamatórias Intestinais/sangue , Doenças Inflamatórias Intestinais/patologia , Masculino , Camundongos , Resultado do Tratamento , Vitamina D/administração & dosagem , Vitamina D/uso terapêuticoRESUMO
BACKGROUND: Drugs currently used for controlling onchocerciasis and lymphatic filariasis (LF) are mainly microfilaricidal, with minimal or no effect on the adult worms. For efficient management of these diseases, it is necessary to search for new drugs with macrofilaricidal activities that can be used singly or in combination with existing ones. Daniellia oliveri and Psorospermum febrifugum are two plants commonly used in the local management of these infections in Bambui, a township in the North West Region of Cameroon, but there is currently no documented scientific evidence to support their claimed anthelmintic efficacy and safety. The aim of this study was to provide evidence in support of the search for means to eliminate these diseases by screening extracts and chromatographic fractions isolated from these plants for efficacy against the parasitic roundworms Onchocerca ochengi and Brugia pahangi. METHODS: The viability of O. ochengi adult worms was assessed using the MTT/formazan assay. Fully confluent monkey kidney epithelial cells (LLC-MK2) served as the feeder layer for the O. ochengi microfilariae (mfs) assays. Viability of the mfs was assessed by microscopic examination for mean motility scoring (relative to the negative control) every 24 h post addition of an extract. The Worminator system was used to test the effects of the extracts on adult B. pahangi motility, and mean motility units were determined for each worm. Cytotoxicity of the active extracts on N27 cells was assessed using the MTS assay. RESULTS: Extracts from D. oliveri and P. febrifugum were effective against the adult roundworms O. ochengi and B. pahangi. Interestingly, extracts showing macrofilaricidal activities against O. ochengi also showed activity against O. ochengi mfs. The hexane stem bark extract of D. oliveri (DOBHEX) was more selective for adult O. ochengi than for mfs, with a half maximal and 100% inhibitory concentration (IC50 and IC100, respectively) against adult O. ochengi of 13.9 and 31.3 µg/ml, respectively. The in vitro cytotoxicity of all active extracts on N27 cells showed selective toxicity for parasites (selectivity index > 1). Bioassay-guided fractionation of the extracts yielded fractions with activity against adult B. pahangi, thus confirming the presence of bioactive principles in the plant extracts. CONCLUSIONS: Our study supports the use of D. oliveri and P. febrifugum in the traditional treatment of onchocerciasis and LF. The further purification of active extracts from these plants could yield lead compounds for filarial drug discovery and development.
Assuntos
Clusiaceae/química , Fabaceae/química , Filaricidas/farmacologia , Onchocerca/efeitos dos fármacos , Extratos Vegetais/farmacologia , Animais , Camarões , Linhagem Celular , Haplorrinos , Humanos , Onchocerca/crescimento & desenvolvimento , Oncocercose/tratamento farmacológico , Oncocercose/parasitologia , Casca de Planta/químicaRESUMO
1,25-Dihydroxyvitamin D3 (1,25(OH)2D) elicits a transcriptional response in the intestines. Assessments of this response are often derived from crude tissue homogenates and eliminate the ability to discriminate among different cell types. Here, we used an RNA in situ hybridization assay, RNAScope (Advanced Cell Diagnostics, Newark, CA), to identify the cells in the intestine that respond to 1,25(OH)2D with expression of cytochrome P450 family 24 subfamily A member 1 (Cyp24a1) mRNA. Mice were gavaged with a single bolus dose of 1,25(OH)2D to target the duodenum or a glucuronic acid conjugate of 1,25(OH)2D, ß-G-1,25(OH)2D, to target the colon. QRT-PCR analysis of Cyp24a1 mRNA verified that the 1,25(OH)2D-induced responses were present. RNAScope revealed that the mRNA response present after six hours is limited to mature enterocytes exposed to the intestinal lumen in both the duodenum and colon. No detectable expression was observed in goblet cells, lamina propria, muscularis mucosa muscle, submucosa and submucosal lymphoid follicles, or tunica muscularis. Our findings have identified epithelial enterocytes to be the intestinal targets for 1,25(OH)2D in both the duodenum and colon.
Assuntos
Intestinos/efeitos dos fármacos , Regulação para Cima/efeitos dos fármacos , Vitamina D3 24-Hidroxilase/genética , Vitamina D/análogos & derivados , Vitaminas/farmacologia , Animais , Colo/citologia , Colo/efeitos dos fármacos , Colo/metabolismo , Colo/ultraestrutura , Duodeno/citologia , Duodeno/efeitos dos fármacos , Duodeno/metabolismo , Duodeno/ultraestrutura , Mucosa Intestinal/citologia , Mucosa Intestinal/efeitos dos fármacos , Mucosa Intestinal/metabolismo , Mucosa Intestinal/ultraestrutura , Intestinos/citologia , Intestinos/ultraestrutura , Masculino , Camundongos , RNA Mensageiro/genética , Vitamina D/farmacologiaRESUMO
The periparturient cow undergoes a transition from non-lactating to lactating at calving. The animal is tremendously challenged to maintain calcium homeostasis. Those that fail can develop milk fever, a clinical disorder that is life threatening to the cow and predisposes the animal to a variety of other disorders. Guidelines for monitoring the incidence of hypocalcemia and methods for treating milk fever are reviewed. The physiological factors that cause milk fever and strategies for prevention of milk fever are discussed, focusing on the effects diet cation-anion difference can have on tissue sensitivity to parathyroid hormone. Another major risk factor for milk fever is hypomagnesemia, which is observed when animals are fed inadequate amounts of magnesium, or some factor is present in the diet that prevents adequate absorption of magnesium. Moderate hypomagnesemia impairs the ability of the cow to maintain calcium homeostasis and hypocalcemia occurs.
Assuntos
Fenômenos Fisiológicos da Nutrição Animal , Doenças dos Bovinos/prevenção & controle , Hipocalcemia/veterinária , Deficiência de Magnésio/veterinária , Paresia Puerperal/prevenção & controle , Animais , Ânions/administração & dosagem , Cálcio/administração & dosagem , Cálcio/sangue , Bovinos , Doenças dos Bovinos/diagnóstico , Doenças dos Bovinos/terapia , Suplementos Nutricionais , Feminino , Hipocalcemia/diagnóstico , Hipocalcemia/prevenção & controle , Hipocalcemia/terapia , Magnésio/administração & dosagem , Magnésio/sangue , Deficiência de Magnésio/diagnóstico , Deficiência de Magnésio/prevenção & controle , Deficiência de Magnésio/terapia , Paresia Puerperal/diagnóstico , Paresia Puerperal/terapia , Gravidez , Fatores de Risco , Sais/administração & dosagemAssuntos
Distúrbios do Metabolismo do Ferro/veterinária , Ferro/metabolismo , Perissodáctilos , Animais , Animais de Zoológico , Infecções Bacterianas/complicações , Infecções Bacterianas/veterinária , Eritrócitos , Eritropoese , Regulação da Expressão Gênica , Ferro/sangue , Distúrbios do Metabolismo do Ferro/genética , Distúrbios do Metabolismo do Ferro/imunologia , Proteínas Reguladoras de Ferro/genética , Proteínas Reguladoras de Ferro/metabolismo , Macrófagos/química , Macrófagos/fisiologia , Perissodáctilos/classificação , Perissodáctilos/genética , Fagocitose , Especificidade da EspécieRESUMO
Neutrophils are the first line of defense in a mammary gland infection. However, the process of neutrophil transmigration across a membrane and ingestion of fat and/or casein when incubated in milk have been shown to inhibit bacterial phagocytosis and oxidative burst functions. Recently, a killing mechanism has been described whereby stimulated neutrophils release nuclear and granule material in fibrous webs that physically trap and kill bacteria. We demonstrate that these neutrophil extracellular traps are also produced by bovine blood neutrophils stimulated with PMA/ionomycin. Importantly, neutrophil extracellular traps can be formed when neutrophils have been incubated for up to 6h in milk prior to stimulation. This contrasts milk's rapid inhibition of bacterial phagocytosis and oxidative burst functions in the neutrophil. Furthermore, stimulation of neutrophils with bacteria common to mammary gland infections leads to neutrophil extracellular traps being formed in milk. Some bacteria tested stimulated enhanced formation of neutrophil extracellular traps in milk compared to culture media. Therefore, being unaffected by incubation in milk may indicate an important role for neutrophil extracellular traps in defense against mastitis.
Assuntos
Bovinos/imunologia , Leite/imunologia , Ativação de Neutrófilo/imunologia , Neutrófilos/imunologia , Animais , Bactérias/imunologia , Bovinos/sangue , Feminino , Corantes Fluorescentes/química , Imunidade Inata/imunologia , Ionomicina/imunologia , Microscopia de Fluorescência/veterinária , Compostos Orgânicos/química , Acetato de Tetradecanoilforbol/imunologiaRESUMO
We wished to determine the expression of trafficking/adhesion molecules on the surface of lymphocytes isolated from infected mammary glands of cows challenged with either Serratia marcescens or Staphylococcus uberis. Healthy Holstein cows in mid lactation were infected by intramammary infusion with S. marcescens or S. uberis. Following infection, milk samples were collected at various time points. Body temperatures of the cows were taken, and milk was analyzed for colony forming units (CFU) of bacteria and somatic cell counts (SCC). Leukocytes were isolated from the milk and analyzed by flow cytometry. Percentages and types of lymphocytes were determined as well as expression of CD62L, CD11a, LPAM-1 and CD44 on these cells. We found that the percentage of lymphocytes expressing either CD62L or CD11a showed a marked increase 12 h post infection (PI) with S. marcescens that was not seen in cows infected with S. uberis. Conversely, the percentage of lymphocytes expressing CD44 increased in cows infected with S. uberis at 12 h PI, but the increase was not seen in cows infected with S. marcescens. Expression of LPAM-1 was low at all time points in both groups of cows. Body temperatures became elevated in both groups of cows, peaking at 24 h PI in S. marcescens-infected cows and dropping thereafter. In contrast, temperatures of S. uberis-infected cows continued to rise and were still elevated 96 h PI. CFU of bacteria isolated from mammary glands of S. marcescens-infected cows dropped precipitously 24 h PI but continued at high levels in S. uberis-infected cows. SCC began falling in S. marcescens-infected cows 48 h PI but continued to increase in S. uberis-infected cows. Thus, a greater percentage of lymphocytes in milk had a phenotype consistent with recruitment from the peripheral pool following infection with S. marcescens than was seen following infection with S. uberis. Concurrent with the increases seen in percentages of this lymphocyte phenotype, clinical signs lessened in the S. marcescens-infected cows.
Assuntos
Linfócitos/imunologia , Mastite Bovina/imunologia , Mastite Bovina/microbiologia , Infecções por Serratia/veterinária , Serratia marcescens/imunologia , Infecções Estreptocócicas/veterinária , Streptococcus/imunologia , Animais , Temperatura Corporal/imunologia , Antígeno CD11a/biossíntese , Bovinos , Feminino , Citometria de Fluxo/veterinária , Receptores de Hialuronatos/biossíntese , Integrinas/biossíntese , Selectina L/biossíntese , Linfócitos/metabolismo , Glândulas Mamárias Animais/imunologia , Glândulas Mamárias Animais/microbiologia , Glândulas Mamárias Animais/patologia , Leite/citologia , Leite/microbiologia , Infecções por Serratia/imunologia , Infecções por Serratia/microbiologia , Infecções Estreptocócicas/imunologia , Infecções Estreptocócicas/microbiologiaRESUMO
OBJECTIVE: To determine the effect of a controlled-release monensin capsule administered at cessation of lactation on incidence of calving-related disorders, fertility, and milk yield in dairy cows. ANIMALS: 290 dairy cows treated with monensin and 290 untreated control cows. PROCEDURE: Treated cows received a capsule that released monensin at 335 mg/d for 95 days. Incidence of calving-related disorders; daily milk yield up to 20 days postpartum; test-day milk yield, fat, protein, and mature-equivalent 305-day milk production; and body condition score at calving were determined. Reproductive variables were conception rate at first service, pregnancy rate, and calving-to-conception interval. RESULTS: Cows treated with monensin were 2.1 times as likely to develop dystocia and 0.8 times as likely to develop metritis as control cows. For milk yield, there was an interaction of treatment X time X parity. In multiparous cows, monensin significantly improved milk yield at test days 4 and 7. In addition, monensin increased body condition score at calving. CONCLUSIONS AND CLINICAL RELEVANCE: Despite increasing the likelihood of developing dystocia and metritis, administration of monensin improved the lactational performance of multiparous cows and may be a promising additive for use at the time of cessation of lactation.
Assuntos
Doenças dos Bovinos/induzido quimicamente , Fertilidade/efeitos dos fármacos , Lactação/efeitos dos fármacos , Leite/efeitos dos fármacos , Monensin/administração & dosagem , Monensin/farmacologia , Animais , Bovinos , Preparações de Ação Retardada , Distocia/induzido quimicamente , Distocia/veterinária , Endometrite/induzido quimicamente , Endometrite/veterinária , Feminino , Fertilidade/fisiologia , Ionóforos/farmacologia , Lactação/fisiologia , Monensin/efeitos adversos , GravidezRESUMO
The four quarters of bovine mammary glands are completely separated and two quarters on each side (right or left) are connected to ipsi lateral supra mammary lymph nodes. It is not clear whether cells infused into the cistern of the mammary gland are capable of migrating to lymph nodes or the general circulation. To examine cell migration, a prescapular lymph node was removed from each of two lactating and three non-lactating dairy cows, and isolated lymphocytes were stained with Hoechst 33342. Autologous stained cells were infused into the mammary gland and then activated by intramammary infusion of zymosan-stimulated serum (source of C5a). After 17 h, Escherichia coli J5 bacterin was infused into the contra lateral mammary gland to mimic infection. After 43 h cows were euthanized and tissue samples (mammary quarters, right and left supra mammary, mesenteric, ileocecal and prescapular lymph nodes, liver and spleen) were collected for microscopic examination as well as flow cytometric analysis. Hoechst stained cells were detected not only in infused quarters, but also in contra lateral quarters as well as in both supra mammary lymph nodes. This indicates that cells infused into the mammary gland migrate to contra lateral tissues and supra mammary lymph nodes.
Assuntos
Bovinos/imunologia , Movimento Celular , Linfonodos/citologia , Linfonodos/imunologia , Linfócitos/citologia , Glândulas Mamárias Animais/citologia , Glândulas Mamárias Animais/imunologia , Animais , Feminino , Linfócitos/imunologia , Leite/citologiaRESUMO
Adhesion molecule and homing receptor expression on blood and milk polymorphonuclear leukocytes (PMN) from periparturient dairy cattle was studied. Both percentages and the mean fluorescence intensity (MFI) of PMN expressing CD11a, CD44, CD62L, and LPAM-1 (alpha4 beta7) were evaluated at seven time points during the twenty-one day period post calving. CD11a and CD62L were expressed on 94-100% of PMN in both blood and milk and there were no significant differences in these percentages at any time point. LPAM-1 was expressed on 3-10% of the PMN in the blood and 13-45% in the milk and the percentage of cells expressing LPAM-1 in milk was significantly (P<0.05) greater than in blood at 0, 4, 10, 14, 18 and 21 days after calving. CD44 was expressed on 11-39% of the PMN in blood and 33-69% in the milk and the percentage of cells expressing CD44 in milk was significantly (P<0.05) greater than in blood at all time points. The MFI of CD11a on milk PMN was consistently higher than that of blood PMN throughout the study period and significantly (P<0.05) higher at days 4, 10 and 18 after calving.
Assuntos
Bovinos/imunologia , Moléculas de Adesão Celular/biossíntese , Leite/imunologia , Neutrófilos/imunologia , Período Pós-Parto/imunologia , Receptores de Retorno de Linfócitos/biossíntese , Animais , Antígenos CD/imunologia , Bovinos/sangue , Moléculas de Adesão Celular/sangue , Moléculas de Adesão Celular/imunologia , Feminino , Citometria de Fluxo/veterinária , Período Pós-Parto/sangue , Receptores de Retorno de Linfócitos/sangue , Receptores de Retorno de Linfócitos/imunologiaRESUMO
Leaves of the Solanum glaucophyllum (Sg) plant, indigenous to South America, have long been known for their calcinogenic toxicity in ruminant animals. It was determined the leaves contained glycosidic derivatives of 1,25-dihydroxyvitamin D3 (1,25D3) and liberation of the free hormone by rumen bacterial populations elicited a hypercalcemic response. Our interest in the leaves is predicated on the concept that the glycoside forms of 1,25D3 would target release of the active hormone in the lower gut of non-ruminant mammals. This would provide a means of delivering 1,25D3 directly to the colon, where the hormone has been shown to have beneficial effects in models of inflammatory bowel disease (IBD) and colon cancer. We fed mice for 10 days with variable amounts of Sg leaf. Feeding 7-333µg leaf/day produced no changes in plasma Ca(2+) and 1,25D3 concentrations, and only at ≥1000µg leaf/day did these values become significantly elevated compared to controls. Gene expression studies from colon tissue indicated a linear relationship between the amount of leaf consumed and expression of the Cyp24a1 gene. In contrast, Cyp24a1 gene expression in the duodenums and ileums of these mice was unchanged compared to controls. One of the major 1,25D3-glycosides was isolated from leaves following extraction and purification by Sep-Pak cartridges and HPLC fractionation. Ultraviolet absorbance was consistent with modification of the 1-hydroxyl group, and positive ion ESI mass spectrometry indicated a diglycoside of 1,25D3. 2-Dimensional NMR analyses were carried out and established the C1 proton of the A-ring was interacting with a C1' sugar proton, while the C3 proton of the A-ring was linked with a second C1' sugar proton. The structure of the isolated compound is therefore consistent with a ß-linked 1,3-diglycoside of 1,25D3. Thus, Sg leaf administered to mice at up to 333 ug/day can elicit colon-specific enhancement of Cyp24a1 gene expression without inducing hypercalcemia, and the 1,3-diglycoside is one of the major forms of 1,25D3 found in the leaf. This article is part of a Special Issue entitled '17th Vitamin D Workshop'.
Assuntos
Calcitriol/farmacologia , Colo/efeitos dos fármacos , Sistemas de Liberação de Medicamentos , Glicosídeos/farmacologia , Folhas de Planta/química , Solanum glaucophyllum/química , Animais , Colo/metabolismo , Glicosídeos/isolamento & purificação , Humanos , Camundongos , Vitaminas/farmacologiaRESUMO
Fifteen Holstein dairy cattle were monitored for lymphocyte subsets and expression of adhesion molecules on cells in milk and blood at parturition and at intervals up to 21 days post-partum. Using flow cytometry, we determined percentages of T cells (CD4+, CD8+, gammadelta) and B cells from milk and blood of these cows. We also measured expression of adhesion molecules (CD62L, LFA-1, LPAM-1, and CD44) on lymphocytes in milk and blood. Significantly higher percentages of CD8+ cells were found in milk than in blood at all time points while significantly higher percentages of B cells were found in blood than in milk at all time points. There were minimal to no significant differences in percentages of CD4+ or gammadelta+ cells between milk and blood. Expression of adhesion molecules was consistently higher on all subsets of milk lymphocytes compared with blood lymphocytes. These differences were most pronounced and statistically significant at calving and in the first week following calving. CD62L, LPAM-1 and CD44 were expressed on a significantly higher percentage of lymphocytes in milk at calving than in milk at subsequent sampling times, while LFA-1 expression on lymphocytes in milk was significantly lower at calving than at subsequent times.
Assuntos
Linfócitos B/imunologia , Bovinos/imunologia , Moléculas de Adesão Celular/biossíntese , Subpopulações de Linfócitos/imunologia , Leite/imunologia , Linfócitos T/imunologia , Animais , Moléculas de Adesão Celular/sangue , Moléculas de Adesão Celular/imunologia , Feminino , Citometria de Fluxo/veterinária , Imunofenotipagem/veterinária , Contagem de Linfócitos/veterinária , Leite/química , Período Pós-Parto , GravidezRESUMO
Twelve dairy cows infected with Mycobacterium avium subsp. paratuberculosis were monitored for lymphocyte subsets and expression of adhesion molecules on cells in blood and milk at parturition and at intervals up to 21 days post-partum. Using fluorescent antibody labeling of cells and analysis by flow cytometry, we determined percentages of T cell subsets (CD4+, CD8+, gammadelta+) and expression of adhesion molecules (CD62L, LFA-1, LPAM-1, and CD44) on cells from blood and milk of these cows. Significantly higher percentages of CD8+ cells were found in milk than in blood at all time points; there were no significant differences in percentages of CD4+ or gammadelta+ cells. CD62L, LFA-1, and LPAM-1 were expressed on a significantly higher percentage of all T cell subsets in milk than in blood at various times after parturition. No differences were seen in expression of CD44. Increased percentages of T lymphocytes expressing adhesion molecules in milk compared to blood suggest that a migratory population of cells is being selectively recruited to the mammary gland from the circulation.