RESUMEN
Dietary ribonucleotides have been shown to augment type 1 T-helper cell (Th1) responses to a protein antigen (Ag) in Th1-prone C57BL/6 mice, but their effects on type 2 Th (Th2)-prone mice are unknown. BALB/cJ mice have skewed Th2 responses against ovalbumin (OVA), characterized by augmented production of Th2 cytokines and immunoglobulin (Ig)G1/IgE antibodies (Ab); Th1 responses augment IgG2a Ab production, whereas Th2 responses augment IgG1/IgE Ab production. In this study, we determined the effects of dietary ribonucleotides obtained from yeast on the balance of Th1/Th2 responses against OVA in young BALB/cJ mice. Mice were fed a ribonucleotide-free (NF) or ribonucleotide-supplemented (NS) diet (4.74 g nucleotides/kg diet) and given OVA (10 microg/dose) with incomplete Freund's adjuvant (IFA) at 3 and 6 wk. We assessed T-cell responses in the regional draining lymph nodes (LN) by measuring production and expression of Th1/Th2 cytokines, interferon-gamma (IFN-gamma) and interleukin-5 (IL-5), respectively. Anti-OVA IgG subclass and IgE Ab levels were determined 3 wk after the first OVA challenge and 5 d and 2 wk after the second OVA challenge. Dietary ribonucleotides significantly augmented OVA-specific IFN-gamma production by the regional draining LN cells after the first and second OVA challenges. The NS diet increased anti-OVA IgG2a Ab levels after the first OVA challenge and both anti-OVA IgG2a and anti-OVA IgG2b after the second challenge. OVA-specific IgG1 and IgE Ab levels were lower (P < 0.05) after the second OVA challenge in mice fed the NS diet. Dietary ribonucleotides did not affect production or expression of IL-5. Our findings thus indicate that in Th2-prone BALB/c J mice, dietary ribonucleotides modulated skewed Th2 responses against OVA toward Th1 as measured by production of IFN-gamma, a Th1 cytokine, and changes in anti-OVA Ab isotype levels.
Asunto(s)
Interferón gamma/biosíntesis , Interleucina-5/biosíntesis , Ovalbúmina/farmacología , Ribonucleótidos/administración & dosificación , Células TH1/metabolismo , Células Th2/metabolismo , Animales , Anticuerpos/análisis , Dieta , Femenino , Inmunoglobulina E/análisis , Inmunoglobulina G/análisis , Interferón gamma/genética , Interleucina-5/genética , Ratones , Ratones Endogámicos BALB C , Ovalbúmina/inmunología , ARN Mensajero/metabolismo , Ribonucleótidos/farmacología , Células TH1/efectos de los fármacos , Células TH1/inmunología , Células Th2/efectos de los fármacos , Células Th2/inmunologíaRESUMEN
Mice fed a nucleotide-free (NF) diet have impaired antibody (Ab) responses. The mechanisms responsible for this effect are not understood but may be related to specific changes in T-cell functions. The objective of this study was to examine the effects of dietary nucleotides on serum immunoglobulin-G (IgG) subclass Ab levels and T-cell cytokine production by cells from the lymph nodes draining the site of antigen challenge. C57BL/6 (B6) mice were fed an NF diet or the same diet supplemented with nucleotides (NS diet; 4.74 g nucleotides/kg). Keyhole limpet hemocyanin (KLH; 25 microg/dose), a T-dependent protein neoantigen, was given with incomplete Freund's adjuvant. We administered KLH at 3, 6, and 9 wk to determine primary and secondary responses. Anti-KLH IgG subclass Ab levels were measured 3 wk after the first KLH challenge and 2 wk after the last KLH challenge. T-cell responses in lymph nodes draining the site of KLH challenge were assessed 5 d after the primary and 14 d after the final KLH challenge. We measured mRNA expression and production of interferon-gamma and interleukin-5, type-1 and type-2 T-cell cytokines, respectively. Anti-KLH IgG2a and IgG2b Ab levels were higher in the NS diet group than in the NF diet group after the last KLH challenge. The NS diet group had higher interferon-gamma production and mRNA expression than did the NF diet group after the first KLH challenge. Because increased levels of interferon-gamma and IgG2a/IgG2b Ab reflect a shift toward type-1 responses to antigen stimuli, our results show that dietary nucleotides preferentially enhance type-1 responses to KLH given with incomplete Freund's adjuvant.
Asunto(s)
Antígenos/inmunología , Dieta , Nucleótidos/administración & dosificación , Linfocitos T/inmunología , Animales , Antígenos/administración & dosificación , Femenino , Hemocianinas/administración & dosificación , Hemocianinas/inmunología , Inmunoglobulina G/sangre , Interferón gamma/biosíntesis , Interferón gamma/genética , Interleucina-5/biosíntesis , Interleucina-5/genética , Ganglios Linfáticos/citología , Ganglios Linfáticos/inmunología , Ratones , Ratones Endogámicos C57BL , ARN Mensajero/análisis , Aumento de PesoRESUMEN
OBJECTIVE: These studies tested the hypothesis that increasing intake of purines, delivered as RNA from soy protein-based infant formula, would increase urinary uric acid excretion in infants. METHODS: Study One examined the influence of feeding on serum uric acid in a total of 178 infants from four separate trials with infants fed commercial and experimental soy-based and milk-based infant formulas or human milk. Studies Two and Three compared the effect of a standard purine soy formula (STD Purine; 180 mg purines/L from RNA) and a reduced purine soy formula (Reduced Purine; 65 mg purines/L; 26 mg/L from RNA and 39 mg/L from ribonucleotides) on urinary uric acid excretion in infants. In Study Two, 11 infants ranging in age from 16 to 128 days of age were fed both formulas in a random crossover design. Complete 72-hour urine collections were done at the end of each 11-day feeding period. Urinary uric acid excretion was expressed as mmol/day. In Study Three, 33 infants were enrolled before eight days of age and randomized to one of the formulas one week later. Spot urine samples were collected at 28 and/or 56 days of age and urinary uric acid concentration was expressed as mmol/mmol creatinine. RESULTS: In Study One, each of the feedings resulted in mean serum uric acid levels within normal reference ranges. Soy formula led to higher serum uric acid levels than human milk, and human milk to levels indistinguishable from cow milk-based formulas. In Study Two, infants excreted significantly more uric acid in the urine when fed the STD Purine formula compared to the Reduced Purine formula (0.86+/-.04 vs. 0.57+/-.04 mmol/d) (p = 0.006). In Study Three, infants fed the STD Purine formula had a significantly higher concentration of uric acid in their urine compared to those fed the Reduced Purine formula (2.1+/-0.2 vs. 1.4+/-0.1 mmol uric acid/mmol creatinine) (p = 0.0001). CONCLUSION: These data indicate that healthy infants can digest RNA and subsequently absorb the liberated purine ribonucleotides as determined by urinary uric acid concentration.
Asunto(s)
Alimentos Infantiles , Purinas/administración & dosificación , Proteínas de Soja/administración & dosificación , Ácido Úrico/orina , Estudios Cruzados , Femenino , Humanos , Lactante , Recién Nacido , Masculino , Proteínas de la Leche/administración & dosificación , Ácido Úrico/sangreRESUMEN
OBJECTIVE: Perceived intolerance to infant formula is a frequently reported reason for formula switching. Formula intolerance may be related to perceived symptoms of constipation, fussiness, abdominal cramps, and excessive spit-up or vomit. Commercially available formulas differ from each other in processing and in sources and levels of protein, lipids, and micronutrients. These differences may affect tolerance. The objective of this article was to compare the tolerance of two commercially available powder infant formulas that differ in composition. Measures of tolerance in exclusively breastfed infants weaned to an infant formula and exclusively formula-fed infants were evaluated. METHODS: Two clinical studies were conducted. In study 1, 82 healthy, full-term infants who were exclusively breastfed at the time of enrollment were randomized at weaning to formula A (commercially available Similac With Iron Powder) or formula B (previously available Enfamil With Iron Powder). Parents completed daily records of tolerance during exclusive breast milk feeding, during the weaning period, and for a 2-week exclusive formula-feeding period. In study 2, 87 healthy, full-term infants who were exclusively formula-fed at the time of study enrollment (by 2 weeks of age) were fed a standard cow milk-based formula (previously commercially available Similac With Iron Powder) and then randomized to receive formula A or B for a 2-week period. Parents completed daily records of tolerance throughout the study. Formula A was a cow milk-based formula with a whey:casein ratio of 48:52 and a fat blend of 42% high-oleic safflower, 30% coconut, and 28% soy oils. Formula B was a cow milk-based formula with a whey:casein ratio of 60:40 and a fat blend of 45% palm olein, 20% soy, 20% coconut, and 15% high-oleic sunflower oils. Both formulas had lactose as the source of carbohydrate and contained 12 mg of iron per liter. Only formula A contained nucleotides at the time of the study. Measures of tolerance included volume of each formula feeding, occurrences of spit-up and/or vomit, and the color (yellow, green, brown, or black) and consistency (water, loose/mushy, soft, formed, or hard) of each stool. RESULTS: In both studies, volume of formula intake, weight gain, and incidence of spit-up or vomit did not differ between feeding groups. In study 1, stool frequency decreased significantly from the exclusive breast milk period to weaning. Stools also became firmer as infants moved from breast milk to weaning and to exclusive formula feeding. When formula was introduced into the diet, stools became less yellow and more green. Infants weaned to formula B had less frequent stools, fewer brown stools, and more yellow stools than did infants fed formula A. In both studies, infants fed formula B experienced significantly firmer stools than did those fed formula A. CONCLUSIONS: The present clinical studies indicate that the composition and/or processing of milk-based powder iron-fortified infant formulas affect stool characteristics experienced by infants. The inclusion of palm olein oil in formula B may be the reason for the observed differences in stool characteristics. Palm olein is used in infant formulas to provide palmitic acid at a level similar to that found in breast milk. However, palmitic acid from palm olein is arranged differently from that in breast milk triglyceride and is poorly absorbed. Unabsorbed palmitic acid tends to react with calcium to form insoluble soaps, and the level of these soaps is correlated with stool hardness. The pattern of softer stools and greater frequency of stooling associated with formula A is similar to the stool pattern in the exclusively breastfed infant. Thus, the use of formula A may ease the transition from breast milk to formula feeding and ameliorate parents' perception that constipation is associated with iron-fortified formula.
Asunto(s)
Heces , Alimentos Infantiles/efectos adversos , Destete , Animales , Lactancia Materna , Estreñimiento/etiología , Defecación , Grasas de la Dieta/administración & dosificación , Humanos , Lactante , Leche/efectos adversos , Leche/químicaRESUMEN
Exposure of cultured endothelial cells to shear stress resulting from well-defined fluid flow stimulates the production of nitric oxide (NO). We have established that an initial burst in production is followed by sustained steady-state NO production. The signal transduction events leading to this stimulation are not well understood. In the present study, we examined the role of regulatory guanine nucleotide binding proteins (G proteins) in shear stress-mediated NO production. In endothelial cells not exposed to shear stress, AIF4-, a general activator of G proteins, markedly elevated the production of guanosine 3',5'-cyclic monophosphate (cGMP). Pretreatment with NO synthase inhibitor N omega-nitro-L-arginine completely blocked this stimulation. Incubation with guanosine 5'-O-(2-thiodiphosphate) (GDP beta S), a general G protein inhibitor, blocked the flow-mediated burst in cGMP production in a dose-dependent manner. Likewise, GDP beta S inhibited NOx (NO2 + NO3) production for the 1st h. However, inhibition was not detectable between 1 and 3 h. Pertussis toxin (PTx) had no effect on the shear response at any time point. The burst in NO production caused by a change in shear stress appears to be dependent on a PTx-refractory G protein. Sustained shear-mediated production is independent of G protein activation.
Asunto(s)
Endotelio Vascular/metabolismo , Proteínas de Unión al GTP/fisiología , Óxido Nítrico/biosíntesis , Compuestos de Aluminio/farmacología , Células Cultivadas , GMP Cíclico/metabolismo , Endotelio Vascular/citología , Fluoruros/farmacología , Proteínas de Unión al GTP/antagonistas & inhibidores , Guanosina Difosfato/análogos & derivados , Guanosina Difosfato/farmacología , Humanos , Concentración Osmolar , Toxina del Pertussis , Estrés Mecánico , Tionucleótidos/farmacología , Factores de Virulencia de Bordetella/farmacologíaRESUMEN
These experiments demonstrate that exposure of cultured endothelial cells (EC) to well-defined laminar fluid flow results in an elevated rate of NO production. NO production was monitored by release of NOx (NO2- + NO3(2-) and by cellular guanosine 3',5'-cyclic monophosphate (cGMP) concentration. NO synthase (NOS) inhibitor blocked the flow-mediated stimulation of both NOx and cGMP, indicating that both measurements reflect NO production. Exposure to laminar flow increased NO release in a biphasic manner, with an initial rapid production consequent to the onset of flow followed by a less rapid, sustained production. A similar rapid increase in NO production resulted from an increase in flow above a preexisting level. The rapid initial production of NO was not dependent on shear stress within a physiological range (6-25 dyn/cm2) but may be dependent on the rate of change in shear stress. The sustained release of NO was dependent on physiological levels of shear stress. The calcium (Ca2+) or calmodulin (CaM) dependence of the initial and sustained production of NO was compared with bradykinin (BK)-mediated NO production. Both BK and the initial production were inhibited by Ca2+ and CaM antagonists. In contrast, the sustained shear stress-mediated NO production was not affected, despite the continued functional presence of the antagonists. Dexamethasone had no effect on either the initial or the sustained shear stress-mediated NO production. An inducible NOS does not, therefore, explain the apparent Ca2+/CaM independence of the sustained shear stress-mediated NO production.(ABSTRACT TRUNCATED AT 250 WORDS)
Asunto(s)
Calcio/fisiología , Calmodulina/fisiología , Endotelio Vascular/metabolismo , Endotelio Vascular/fisiología , Óxido Nítrico/biosíntesis , Células Cultivadas , GMP Cíclico/metabolismo , Endotelio Vascular/citología , Humanos , Iones , Nitratos/metabolismo , Nitritos/metabolismo , Estrés MecánicoRESUMEN
The in vitro responses of canine mammary tumor (CMT-13) cells and normal canine mammary (NCM) cells to 1-cyano-2-hydroxy-3-butene (CHB), a naturally occurring nitrile in cruciferous plants, and selenium (Se) were investigated. CHB at 10 and 20 mM inhibited growth and viability of CMT-13 and NCM cells, respectively. This differential sensitivity was associated with a decreased ability of CMT-13 cells to increase intracellular glutathione (GSH) in comparison to NCM cells. Exposure of both cell types to 3.2 microM Se as sodium selenite alone had no effects, but addition of 3.2 microM Se 24 h after exposure to non-toxic doses of CHB resulted in a substantial decrease in growth of CMT cells, while NCM cells remained unaffected. The synergy noted between CHB and Se in inhibiting growth and viability of neoplastic mammary cells at levels not toxic to normal mammary cells is promising initial evidence that CHB could have a role in chemoprotection or chemotherapy.
Asunto(s)
Alquenos/farmacología , Glándulas Mamarias Animales/efectos de los fármacos , Neoplasias Mamarias Experimentales/metabolismo , Nitrilos/farmacología , Selenio/farmacología , Células Tumorales Cultivadas/efectos de los fármacos , Animales , Supervivencia Celular/efectos de los fármacos , Perros , Glutatión/metabolismo , Glándulas Mamarias Animales/metabolismo , Selenito de Sodio , Células Tumorales Cultivadas/metabolismoRESUMEN
The effect of shear stress on the release of endothelin-1 (ET-1) from endothelial cells is at present controversial with various investigators observing an increase and others observing a decrease. Our data reveal that the release of ET-1 from primary cultures of human umbilical vein endothelial cells varies with the duration and the level of shear. Sustained exposure to low levels of shear (1.8 dyn/cm2) or a brief exposure (< 1 h) to 10 dyn/cm2 caused a sustained stimulation of ET-1 release. Staurosporine (STPN) completely blocked the stimulation in both cases, suggesting that ET-1 release is increased via activation of protein kinase C (PKC). Exposure to 6-25 dyn/cm2 for > or = 6 h dramatically inhibited ET-1 release and led to 0-70% inhibition of cumulative release by 16 h. Pretreatment with N omega-nitro-L-arginine (L-NNA) reversed this suppression in a dose-dependent manner, implicating either nitric oxide (NO) and/or guanosine 3',5'-cyclic monophosphate (cGMP) as a requirement for shear-mediated inhibition of ET-1 release. Treatment of stationary cultures with 8-bromo-cGMP and atrial natriuretic peptide mimicked the inhibition of ET-1 release caused by shear and revealed that cGMP is capable of inhibiting ET-1. Likewise, the inhibitory effects of shear were potentiated and diminished by 3-isobutyl-1-methylxanthine (IBMX) and methylene blue, respectively. Thus cGMP also appears to exert an inhibitory effect in cells exposed to shear.(ABSTRACT TRUNCATED AT 250 WORDS)
Asunto(s)
GMP Cíclico/fisiología , Endotelinas/metabolismo , Endotelio Vascular/metabolismo , Proteína Quinasa C/fisiología , Adenosina Trifosfato/farmacología , Células Cultivadas , Endotelinas/antagonistas & inhibidores , Endotelio Vascular/citología , Humanos , Óxido Nítrico/metabolismo , Inhibidores de Proteínas Quinasas , Proteínas Quinasas/fisiología , Estrés MecánicoRESUMEN
The present studies demonstrate that the ability of supplemental selenite to alter the in vitro growth of canine mammary tumor cell line 13 was dependent on the quantity and duration of selenium exposure and on the culture density. Exposure to 3.2 microM selenite did not significantly alter growth but led to an increase in intracellular glutathione (GSH). The severity of growth inhibition between 3.2 and 9.6 microM selenite was dependent on the duration of exposure and culture density. The toxicity of selenite generally increased as the culture density increased. Likewise, changes in intracellular GSH were dependent on the quantity and duration of selenite exposure and the culture density. Depressing intracellular GSH by increasing the culture density or by incubating with buthionine sulfoximine; a specific inhibitor of gamma-glutamyl cysteine synthetase, increased the severity of growth inhibition caused by selenite and markedly increased cellular retention of selenium. Nevertheless, marked cellular retention of selenium did not occur until growth was inhibited by more than 50%. The present studies revealed that the log of the molar ratio of GSH to selenium correlated negatively with the severity of growth inhibition (P less than 0.0001). These studies suggest that cellular toxicity of selenite is dependent on the regulation of the GSH:selenium ratio. An inability to regulate this ratio likely leads to the accumulation of toxic seleno compounds.
Asunto(s)
Antioxidantes/metabolismo , Glutatión/metabolismo , Neoplasias Mamarias Animales/metabolismo , Neoplasias Mamarias Animales/patología , Selenio/metabolismo , Animales , Antioxidantes/toxicidad , Butionina Sulfoximina , Recuento de Células , División Celular/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Glutatión/administración & dosificación , Metionina Sulfoximina/análogos & derivados , Metionina Sulfoximina/farmacología , Ácido Selenioso , Selenio/toxicidadRESUMEN
Present studies show the in vitro addition of glutathione (GSH) can significantly alter selenite induced growth inhibition of mammary tumor cell line 13 (CMT-13). Preincubation with 100 microM GSH for 24 h partially prevented the growth inhibition caused by 12.6 microM selenite. Pre-incubation with 100 microM GSH for 48 h completely prevented the growth inhibition caused by 12.6 microM selenite. In marked contrast, simultaneous addition of GSH and selenite dramatically increased the severity of selenite-mediated growth inhibition and resulted in cell death. Exposure to selenite (12.6 microM) increased intracellular GSH throughout the 3 day incubation period. Addition of GSH to the medium also led to an approximate 25% increase in intracellular GSH that persisted for 72 h. Cellular retention of selenium following selenite supplementation was decreased up to 70% by GSH preincubation yet increased markedly (greater than or equal to 240%) by the simultaneous addition of GSH and selenite. The rate of selenite uptake was not consistently altered by GSH pretreatment. However, the simultaneous addition of GSH and selenite resulted in a dramatically increased rate of selenium uptake. These data indicate that extracellular GSH can alter the toxicity of supplemental selenite. The protective effect of GSH pre-incubation against selenite toxicity appears to relate to altered selenium retention.
Asunto(s)
Glutatión/farmacología , Neoplasias Mamarias Experimentales/patología , Selenio/farmacología , Animales , División Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Perros , Interacciones Farmacológicas , Neoplasias Mamarias Experimentales/metabolismo , Ácido Selenioso , Selenio/metabolismo , Células Tumorales Cultivadas/efectos de los fármacos , Células Tumorales Cultivadas/metabolismo , Células Tumorales Cultivadas/patologíaRESUMEN
Previous studies with cultures of canine mammary cells revealed differences in the degree of growth inhibition caused by selenite supplementation, with canine mammary tumor cell line 13 > 11 >> non-neoplastic canine mammary cells. The present studies show this variation in growth retardation cannot be explained by selenium retention. Intracellular glutathione related inversely to the degree of growth inhibition resulting from the addition of selenite. Dimethyl selenide formation by S-9 preparations corresponded to the sensitivity of the culture to supplemental selenite. DL-buthionine-SR-sulfoximine, a specific inhibitor of glutathione biosynthesis, accentuated the growth inhibition and prevented the increase in intracellular glutathione caused by supplemental selenite. Treatment of canine mammary tumor cell line 13 cultures with DL-buthionine-SR-sulfoximine resulted in a persistent depletion of intracellular glutathione without affecting growth. Glutathione reductase activity, before and following selenite, was inversely related to the degree of growth inhibition, with canine mammary tumor cell line 13 > 11 > non-neoplastic canine mammary tumor cell line. Selenite addition increased the activity of gamma-glutamylcysteine synthetase in canine mammary tumor cell line 11 and non-neoplastic canine mammary cells, but not in canine mammary tumor cell line 13 cells. The present data suggest the differences in the growth inhibition caused by selenite among these mammary cells is related to glutathione regulation and ultimately to selenium detoxification.