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Biomedical subjects

M L Failla

Publications and source records attributed to M L Failla.

At least 19 recordsLinked to original sources
PubMed2004

Metabolomics in the opening decade of the 21st century: building the roads to individualized health.

It is rapidly becoming possible to measure hundreds or thousands of metabolites in small samples of biological fluids or tissues. This makes it possible to assess the metabolic component of nutritional phenotypes and will allow individualized dietary recommendations. ASNS has to take action to ensure that appropriate technologies are developed and that metabolic databases are constructed with the right inputs and organization. The relations between diet and metabolomic profiles and between those profiles and health and disease must be established. ASNS also should consider the social implications of these advances and plan for their appropriate utilization.

Animals↗
PubMed2001

Assessment of degradation and intestinal cell uptake of carotenoids and chlorophyll derivatives from spinach puree using an in vitro digestion and Caco-2 human cell model.

Although numerous studies have demonstrated the health benefits of chlorophyll derivatives, information regarding the digestion, absorption, and metabolism of these phytochemicals is quite limited. To better understand the digestion of these pigments, green vegetables including fresh spinach puree (FSP), heat- and acid-treated spinach puree (HASP), and ZnCl(2)-treated spinach puree (ZnSP) were subjected to an in vitro digestion method which simulates both the gastric and small intestinal phases of the process. Native chlorophylls were converted to Mg-free pheophytin derivatives during digestion. Conversely, Zn-pheophytins were completely stable during the digestive process. Transfer of lipophilic chlorophyll derivatives, as well as the carotenoids lutein and beta-carotene, into the aqueous micellar fraction from the food matrix was quantified. Micellarization of total chlorophyll derivatives differed significantly (p < 0.05) for FSP (37.6%), HASP (17.2%), and ZnSP (8.7%). Micellarization of chlorophyll a derivatives was determined to be significantly more efficient than chlorophyll b derivatives in FSP and HASP (p < 0.01), but not in ZnSP (p > 0.05). Intestinal cell uptake of micellarized pigments was investigated using HTB-37 (parent) and clonal TC7 lines of human Caco-2 cells. Medium containing the pigment-enriched fraction generated during digestion was added to the apical surface of fully differentiated monolayers for 4 h. Pigments were then extracted from cells and analyzed by C18 HPLC with photodiode array detection. Both Caco-2 HTB-37 and TC7 clone cells accumulated 20-40% and 5-10% of micellarized carotenoid and chlorophyll derivatives, respectively. These results are the first to demonstrate uptake of chlorophyll derivatives by human intestinal cells and to support the potential importance of chlorophylls as health-promoting phytochemicals.

Biological Availability↗
PubMed2001

Nutrition: a reservoir for integrative science.

In the last twenty years, powerful new molecular techniques were introduced that made it possible to advance knowledge in human biology using a reductionist approach. Now, the need for scientists to deal with complexity should drive a movement toward an integrationist approach to science. We propose that nutritional science is one of the best reservoirs for this approach. The American Society for Nutritional Sciences can play an important role by developing and delivering a cogent message that convinces the scientific establishment that nutrition fills this valuable niche. The society must develop a comprehensive strategy to develop our image as the reservoir for life sciences integration. Our efforts can start with our national meeting and publications, with the research initiatives for which we advocate, with our graduate training programs and with the public relations image we project for ourselves. Defining the image and future directions of nutrition as the discipline that can integrate scientific knowledge from the cell and molecule to the whole body and beyond to populations can be the most important task that our society undertakes. If we do not effectively meet this challenge, a golden opportunity will pass to others and nutritional scientists will be left to follow them.

Biological Science Disciplines↗
PubMed2001

Elevated iron status increases bacterial invasion and survival and alters cytokine/chemokine mRNA expression in Caco-2 human intestinal cells.

Iron status affects both microbial growth and immune function. Mammalian iron homeostasis is maintained primarily by regulating the absorption of the micronutrient in the proximal small intestine. The iron concentration of the enterocyte can fluctuate widely in response to both dietary and whole body iron status, as well as in response to infections. The possibility that an enterocyte with an elevated iron concentration is more susceptible to invasion by enteric pathogens is not known. Therefore, we examined the impact of enterocyte iron status on the invasion and survival of an enteric pathogen, as well as on the levels of several cytokine and chemokine mRNAs by the host cell. The enterocyte-like Caco-2 human intestinal cell line and Salmonella enteritidis served as the models to examine the effect of iron on the host-parasite interaction. Iron status of Caco-2 cells was altered by incubation in serum-free medium supplemented with varying levels of iron. Elevated iron status of Caco-2 cells increased the efficiency of the invasion and the number of bacteria surviving in the intracellular environment. Caco-2 cells constitutively expressed transforming growth factor-beta1, interleukin-8, monocyte chemotactic protein-1, tumor necrosis factor-alpha and interleukin-1beta, and infection with S. enteritidis increased the relative quantities of all cytokine/chemokine mRNAs except interleukin-1beta. Elevated iron status of Caco-2 cells decreased the levels of cytokine/chemokine mRNAs by 25-45% in uninfected cells. In contrast, bacterial infection was associated with a 21-95% increase in cytokine/chemokine mRNAs levels in Caco-2 cells with higher iron concentration compared with infected cells with lower iron concentration. These data support the hypothesis that elevated enterocyte iron status increases susceptibility to infection and exacerbates the mucosal inflammatory response initiated by microbial invasion by increasing cytokine/chemokine expression.

Analysis of Variance↗
PubMed2001

Differentiation of human U937 promonocytic cells is impaired by moderate copper deficiency.

Copper (Cu) deficiency suppresses macrophage activities in animals and humans. Our previous studies indicated that the induction of Cu deficiency in differentiated U937 monocytic cells impairs respiratory burst and bactericidal activities and lipopolysaccharide-mediated secretion of inflammatory mediators. The current investigation examined the roles of Cu in the monocytic differentiation process. Human U937 promonocytic cells were exposed to a high affinity Cu chelator (5 microM 2,3,2-tetraamine [tet]) for 24 hr before inducing differentiation by treatment with 1,25-dihydroxyvitamin D3 plus interferon-gamma (DI). This procedure decreased cell Cu by 55% without compromising cellular Zn, Fe, or general metabolic activities. Lower Cu status significantly attenuated the expression of maturation markers Mac-1 (CD11b), ICAM-1 (CD54), and LPS-R (CD14). This change was associated with a marked suppression in respiratory burst activity and killing of Salmonella. To examine if the adverse effect of inadequate Cu on the DI-induced differentiation represented a more general defect, U937 cells were treated with phorbol 12-myristate 13-acetate (PMA). Lower Cu status also suppressed PMA-mediated differentiation of U937 cells. Supplemental Cu, but not Zn or Fe, blocked the tet-induced declines in cell Cu, expression of maturation markers, and respiratory burst and bactericidal activities. These results demonstrate that Cu is essential for the monocytic differentiation process that contributes to the competency of the host's defense system.

Cell Differentiation↗
PubMed2000

Copper deficiency suppresses effector activities of differentiated U937 cells.

Dietary copper (Cu) deficiency impairs both innate and acquired branches of immunity. Specific roles of Cu in the activation and effector activities of host-defense cells remain largely unknown. The effects of Cu status on effector activities of a monocytic cell line were investigated as an initial step in the elucidation of specific functions of Cu in phagocytic cells. Exposure of differentiating U937 human promonocytic cells to 5 micromol/L 2,3, 2-tetraamine (tet), a high affinity Cu chelator, for 4 d decreased cellular Cu by 62% without altering cellular Cu,Zn-superoxide dismutase (SOD) activity, Zn content, mitochondrial activity and protein synthesis. In contrast, Cu deficiency suppressed the respiratory burst activity and markedly compromised the ability of U937 cells to kill Salmonella. Similarly, treatment of RAW264.7 murine macrophages with 5 micromol/L tet decreased cell Cu by 78% and Cu,Zn-SOD activity by 15% and increased bacterial survival by 180%. The tet-induced impairment of respiratory burst and bactericidal activities was blocked in cultures supplemented with Cu, but not Zn or Fe. In addition, lipopolysaccharide (LPS)-induced secretion of the inflammatory mediators, tumor necrosis factor-alpha, interleukin (IL)-1beta, IL-6 and prostaglandin E(2) (PGE(2)), was decreased by 30-60% in tet-treated U937 cells. Flow cytometric analysis of the surface antigens CD11b and CD71 showed that the suppressed activities of Cu-deficient cells were not due to an attenuation in the degree of differentiation or secondary iron deficiency. These data demonstrate that U937 cells provide a useful model for examining the biochemical roles of Cu in monocyte activity.

Analysis of Variance↗
PubMed1999

Active lifestyle offsets HRT-induced suppression of T cell reactivity to mitogens.

OBJECTIVE: The purpose of the study was to investigate the combined impact of hormone replacement therapy (HRT) and active lifestyle on the phenotypic profile and in vitro activities of specific immune cells in postmenopausal women. METHODS: Healthy postmenopausal women aged 45-70 were assigned to one of four groups: (a) HRT/sedentary (n = 9); (b) HRT/active (n = 12); (c) no HRT/sedentary (n = 10); and (d) no HRT/active (n = 9). Blood samples were collected from each subject on 3 days within 1 week. The mean value of three samples was used to assess the in vitro response of T lymphocytes to the mitogens phytohemagglutinin and concanavalin A and natural killer cell activity. One of three blood samples was utilized for phenotypic analysis of circulating leukocytes. RESULTS: The mitogenic reactivity of T lymphocytes in whole blood cultures for the groups receiving HRT was lower than from the groups not receiving HRT. There also was a trend for T lymphocyte reactivity to be higher in the active women when compared to the sedentary women. In contrast, the phenotypic profile of leukocytes and natural killer cell activity were not significantly different for samples collected from the four groups. CONCLUSIONS: These data suggest that the suppressive effect of HRT on T cell function in postmenopausal women may be attenuated by a physically active lifestyle.

Aged↗
PubMed1999

Accumulation and retention of micellar beta-carotene and lutein by Caco-2 human intestinal cells.

Despite the interest in the diverse roles of dietary carotenoids in human health, little is known about the transfer of these plant pigments from foods to micelles during digestion and their subsequent transfer across the intestinal epithelium. We conducted this study to characterize the intestinal uptake of micellarized carotenoids using monolayers of differentiated Caco-2 human intestinal cells. Crystalline beta-carotene (BC) and lutein (LUT), solubilized in mixed micelles for delivery to cells, were stable in a tissue culture environment for 20 hours. Cellular accumulation of micellar BC and LUT was proportional to the media content of carotenoids at </=2 micromol/L and the length of exposure. Cellular accumulation of BC routinely exceeded LUT and was due in part to the enhanced efflux or possible metabolism of LUT. Cellular BC content increased in a curvilinear manner when cultures were incubated in micellar medium containing 2 to 27 micromol/L BC prepared from water miscible beadlets; cellular BC content was maximum when medium BC was >/=18 micromol/L. There was no indication that high levels of BC in medium or within cells adversely affected micellar LUT accumulation. These data support the use of the Caco-2 human cell line as a model for studying the intestinal uptake, absorption, and possible interactions of dietary carotenoids.

Journal Article↗
PubMed1999

Considerations for determining 'optimal nutrition' for copper, zinc, manganese and molybdenum.

Defining optimal dietary intakes of Cu and Zn throughout the life cycle continues to present a considerable challenge for nutrition scientists. Although the daily intake of these micronutrients is below that currently recommended for many groups, traditional biochemical indicators of nutritional status for these trace metals largely remain within the normal range. Thus, it is unclear whether the recommended daily intakes may be unnecessarily high, or if the commonly-used markers simply lack the necessary sensitivity and specificity that are required for accurately assessing Cu and Zn status. The increasing number of reports that daily supplements with these trace metals enhance the activities of selective metalloenzymes and specific cellular and organ processes further points out the need to differentiate between meeting the requirement and providing optimal nutriture. Results from recent studies suggesting that alternative molecular and functional markers possess sufficient sensitivity to better assess Cu and Zn status are discussed. Likewise, recent studies evaluating the impact of very low and excessive levels of dietary Mn and Mo on selective biochemical and metabolic indicators are reviewed.

Biomarkers↗
PubMed1999

Development of an in vitro digestion method to assess carotenoid bioavailability from meals.

The objective of this study was to develop a model for assessing the bioavailability of carotenoids from meals using an in vitro digestion procedure. A meal was prepared using baby food carrots, spinach, and a meat, plus tomato paste. The aqueous fraction was isolated from digesta to determine the quantity of carotenoids transferred from the food to micelles. The micellarization of lutein (25-40%) exceeded (p < 0.01) that of alpha- and beta-carotene (12-18%) and lycopene (<0.5%). Micellarization of carotenoids was not affected by elimination of the gastric phase of the digestive process. The absence of bile extract prevented the transfer of carotenoids from foods to micelles, whereas omission of pancreatin only reduced the micellarization of the carotenes. Differentiated cultures of Caco-2 human intestinal cells accumulated 28-46% of micellarized carotenoids from the medium after 6 h. These results support the usefulness of the in vitro digestion process as a rapid and cost-effective model for screening the bioavailability of carotenoids from meals.

Biological Availability↗
PubMed1999

Transcriptional regulation of interleukin-2 gene expression is impaired by copper deficiency in Jurkat human T lymphocytes.

Copper deficiency reduces secretion of the cytokine interleukin-2 (IL-2) by activated rodent splenocytes, human peripheral blood mononuclear cells and Jurkat cells, a human T lymphocyte cell line. Previous studies showed that low Cu status also decreased the level of IL-2 mRNA in activated Jurkat cells by 50%. Synthesis of this cytokine is regulated by alterations in transcription of the IL-2 gene and the stability of IL-2 mRNA. To determine if Cu status influenced promoter activity of the IL-2 gene, Jurkat cells were transfected with a luciferase reporter gene construct containing the entire 300 bp human IL-2 promoter/enhancer sequence. Cu deficiency was induced by incubating stably transfected cells with the Cu chelator 2,3,2-tetraamine for 35 h prior to activating cells with phytohemagglutinin-P and phorbol myristate acetate. Luciferase activity in lysates of Cu-deficient cells was approximately 50% lower in several multiclonal and clonal cell lines of stably transfected cells than in replicate cultures that were not exposed to chelator. The relative levels of endogenous IL-2 bioactivity and luciferase activity were highly correlated in the transfected cell lines. The chelator-mediated reduction in reporter gene activity was dose-dependent at levels of 5-40 micromol 2,3,2-tetraamine/L. The addition of a slight molar excess of Cu, but not Zn or Fe, to medium containing 2,3,2-tetraamine prevented the decline in luciferase activity. IL-2 mRNA stability in parental Jurkat cells was independent of Cu status. These data indicate that decreased cellular Cu attenuates IL-2 synthesis in T lymphocytes by inhibiting transcription of the IL-2 gene.

Chelating Agents↗
PubMed1999

Hepatocyte-specific localization and copper-dependent trafficking of the Wilson's disease protein in the liver.

Wilson's disease is an inherited disorder of copper metabolism characterized by hepatic cirrhosis and neuronal degeneration. In this current study, a polyclonal antiserum specific for the Wilson's disease ATPase was used to examine the hepatic expression of this protein. Immunoblot analysis of lysates from human and rat liver detected a single 165-kDa protein, which by immunofluorescence was present only in hepatocytes and localized predominantly to the trans-Golgi network and exclusively in this compartment under low hepatic copper concentrations. Although hepatic copper concentration had no effect on the steady-state levels of the Wilson's disease protein, copper administration in vivo resulted in redistribution of this protein to a cytoplasmic vesicular compartment localized toward the hepatocyte canalicular membrane. The relative abundance of the Wilson's disease protein in the liver was found to be greatest in the fetus before the onset of biliary copper excretion. Taken together, these studies reveal a novel posttranslational mechanism of copper homeostasis in vivo consistent with the proposed function of the Wilson's disease protein in holoceruloplasmin biosynthesis and biliary copper excretion and of relevance to the broad clinical heterogeneity observed in this disease.

Adenosine Triphosphatases↗
PubMed1997

Copper deficiency reduces interleukin-2 (IL-2) production and IL-2 mRNA in human T-lymphocytes.

Although dietary copper (Cu) deficiency has been associated with decreased production of interleukin-2 (IL-2) by activated splenic mononuclear cells in rodent models, the basis for this relationship and its relevance for humans remain unknown. To address these matters, we have developed an in vitro model of cellular copper deficiency by treating Jurkat, a human T-lymphocyte cell line, with low concentrations of 2,3,2-tetraamine (2,3,2-tet), a high affinity copper chelator. Exposure to 5-20 micromol/L 2,3,2-tet for 35 h decreased cell copper and the activity of Cu,Zn-superoxide dismutase (Cu,Zn-SOD) by 30-40% and IL-2 production by 60-70% in cultures activated with phytohemagglutinin and phorbol myristate acetate. Similarly, IL-2 mRNA levels were 40-70% lower in chelator-treated cells than in untreated cells at 3-12 h after activation. In contrast, chelator treatment had no significant effect on cell viability, growth, protein synthesis or mitochondrial activity. The presence of a slight molar excess of copper, but not zinc or iron, during exposure to 2,3,2-tet prevented the chelator-induced decrease in Cu,Zn-SOD activity and the reductions in IL-2 mRNA and bioactivity. Moreover, binding of diferric transferrin (Tf) and cellular uptake of Tf-59Fe by Jurkat cells were not increased by 2,3,2-tet, indicating that chelator-treated cells were not iron deficient. Finally, incubation of human peripheral blood mononuclear cells (PBMC) with 2,3,2-tet decreased mitogen-induced IL-2 production by 50% compared with untreated controls. These data indicate that a decline in copper status decreases IL-2 production by activated human T-cells due to reduced synthesis and/or stability of IL-2 mRNA.

Blotting, Northern↗
PubMed1997

Human lipoproteins as a vehicle for the delivery of beta-carotene and alpha-tocopherol to HepG2 cells.

Highly differentiated human cell lines represent a useful in vitro model for the study of carotenoid uptake, metabolism, and function. Carotenoids are usually introduced into tissue culture media either in organic solvents or as micelles, whereas carotenoids are localized in lipoproteins in vivo. Initially, the stability of beta-carotene and alpha-tocopherol in micelles and human lipoproteins under standard tissue culture conditions was compared. Recovery of beta-carotene and alpha-tocopherol was 27% +/- 2% and 73% +/- 2%, respectively, after overnight incubation of micellar beta-carotene and alpha-tocopherol in serum-free medium without cells. This marked loss of beta-carotene was attenuated by inclusion of alpha-tocopherol in micelles. In contrast, recovery of beta-carotene and alpha-tocopherol was 88%-95% when medium containing the total lipoprotein fraction isolated from beta-carotene supplemented individuals was incubated overnight without cells. Cellular accumulation of beta-carotene and alpha-tocopherol from medium containing total lipoproteins (1 mg/ml) was proportional to their concentrations in the lipoprotein fraction (r = 0.94 for beta-carotene and 0.74 for alpha-tocopherol). Cells exhibited similar capability of acquiring beta-carotene and alpha-tocopherol from medium containing either low- or high-density lipoproteins. These data show that lipoproteins represent a stable vehicle for delivery of beta-carotene and alpha-tocopherol to HepG2 human liver cells.

Adult↗
PubMed1996

Expression of ceruloplasmin gene in human and rat lymphocytes.

The acute phase plasma protein ceruloplasmin (Cp) appears to play some role in host defense. The possibility that production of Cp in extrahepatic sites may also be essential for the activation, effector functions and cytoprotection of immune cells in localized environments has received minimal attention. Here, we have surveyed various types of human and rat immune cells for the presence of Cp mRNA using RT-PCR with primers that span exons 17-19 as an initial step in addressing this possibility. Validated Cp RT-PCR bands were obtained from RNA samples isolated from resting and activated human lymphocytes, CD4 and CD8 T-cells and B-cells. Semiquantitative RT-PCR indicated that Cp mRNA in immune cells is present at about 0.2% the level of Cp mRNA in HepG-2 human liver cell line. Various human cell lines derived from the immune system, rat splenic MNC and purified rat T-lymphocytes also constitutively express Cp gene.

Animals↗
PubMed1996

Beta-carotene and lutein protect HepG2 human liver cells against oxidant-induced damage.

Numerous epidemiological studies support a strong inverse relationship between consumption of carotenoid-rich fruits and vegetables and the incidence of some degenerative diseases. One proposed mechanism of protection by carotenoids centers on their putative antioxidant activity, although direct evidence in support of this contention is limited at the cellular level. The antioxidant potential of beta-carotene (BC) and lutein (LUT), carotenoids with or without provitamin A activity, respectively, was evaluated using the human liver cell line HepG2. Pilot studies showed that a 90-min exposure of confluent cultures to 500 mumol/L tert-butylhydroperoxide (TBHP) at 37 degrees C significantly (P < 0.05) increased lipid peroxidation and cellular leakage of lactate dehydrogenase (LDH), and decreased the uptake of 3H-alpha-aminoisobutyric acid and 3H-2-deoxyglucose. Protein synthesis, mitochondrial activity and glucose oxidation were not affected by TBHP treatment, suggesting that the plasma membrane was the primary site of TBHP-induced damage. Overnight incubation of cultures with > or = 1 mumol/L dl-alpha-tocopherol protected cells against oxidant-induced changes. In parallel studies, overnight incubation of HepG2 in medium containing micelles with either BC or LUT (final concentrations of 1.1 and 10.9 mumol/L, respectively), the cell content of the carotenoids increased from < 0.04 to 0.32 and 3.39 nmol/mg protein, respectively. Carotenoid-loaded cells were partially or completely protected against oxidant-induced changes in lipid peroxidation, LDH release and amino acid and deoxyglucose transport. These data demonstrate that BC and LUT or their metabolites protect HepG2 cells against oxidant-induced damage and that the protective effect is independent of provitamin A activity.

Aminoisobutyric Acids↗