Improving the quality of data from EFNEP participants with low literacy skills: a participant-driven model.

Low literacy skills and poor evaluation tool readability combined with the stresses of the classroom environment create a high cognitive load for Expanded Food and Nutrition Education Program (EFNEP) participants, resulting in lower quality data. The authors advocate for 9 strategies for improving the participant cognitive load for the evaluation process using the EFNEP Family Record as an example.

Effects of silymarin on hepatitis C virus and haem oxygenase-1 gene expression in human hepatoma cells.

BACKGROUND/AIMS: Hepatitis C virus (HCV) infection is a global medical problem. The current standard treatment of chronic hepatitis C (CHC), pegylated interferon plus ribavirin, is prolonged, expensive, has serious side effects and, at best, is only 50% effective. Silymarin (SI) is a natural antioxidant often used by patients with CHC, although its efficacy for decreasing HCV levels or ameliorating CHC remains uncertain. HCV infection is associated with increased hepatic oxidative stress, and one of the antioxidant enzymes that protect cells against this stress is haem oxygenase-1 (HO-1). METHODS: We investigated effects of SI on HCV and HO-1 gene expression in Huh-7 cells, CNS3 and 9-13 cells (the latter two stably expressing HCV-proteins). RESULTS: Silymarin significantly downregulated HCV core mRNA (by 20%-36%) and protein (by 30%-60%) in CNS3 cells. In contrast, SI did not decrease HCV NS5A mRNA or protein expression in 9-13 cells. HO-1 mRNA was upregulated (60%-400%) by SI in Huh-7, CNS3 and 9-13 cells, whereas BTB and CNC homology 1 and nuclear factor erythroid related factor 2 mRNA levels were not affected. The effect of SI to downregulate HCV core was not related to changes in the Janus-activated tyrosine kinases-signal transducer and activators of transcription signalling pathway. CONCLUSIONS: Silymarin may be of benefit in CHC, although prospective, randomized, controlled trials are needed to be certain.

Differential regulation of human ALAS1 mRNA and protein levels by heme and cobalt protoporphyrin.

5-Aminolevulinic acid synthase 1 (ALAS1) is the first and rate-controlling enzyme of heme biosynthesis. This study was to determine the effects of heme and selected nonheme metalloporphyrins on human ALAS1 gene expression in hepatocytes. We found that, upon heme and cobalt protoporphyrin (CoPP) treatments, ALAS1 mRNA levels were down-regulated significantly by ca. 50% or more. Measurement of mRNA in the presence of actinomycin D showed that these down-regulations were due to the decreases in mRNA half-lives. Furthermore, the levels of mitochondrial mature ALAS1 protein were down-regulated by 60-70%, but those of the cytosolic precursor protein were up-regulated by 2-5-fold. Measurement of protein in the presence of cycloheximide (CHX) suggests that elevation of the precursor form is due to the increase in protein half-lives. These results provide novel insights into the mechanisms of heme repressional effects on ALAS1 and provide a rationale for further investigation of CoPP as a therapeutic agent for acute porphyric syndromes.

Zinc mesoporphyrin induces rapid and marked degradation of the transcription factor Bach1 and up-regulates HO-1.

Heme oxygenase 1 (HO-1) is the first and rate-controlling enzyme in heme degradation. Bach1 is a mammalian transcriptional repressor of HO-1. To understand how zinc mesoporphyrin (ZnMP) induces the expression of HO-1, we investigated the effects of ZnMP on Bach1 mRNA and protein levels in human hepatoma Huh-7 cells by quantitative RT-PCR and Western blots. We found that ZnMP markedly up-regulated HO-1 mRNA and protein levels, and rapidly and significantly decreased Bach1 protein levels by increasing degradation of Bach1 protein [half life (t(1/2)) from 19 h to 45 min], whereas ZnMP did not influence Bach1 mRNA levels. The proteasome inhibitors, epoxomicin and MG132, significantly inhibited degradation of Bach1 by ZnMP in a dose-dependent fashion, indicating that the degradation of Bach1 by ZnMP is proteasome-dependent. Purified Bach1 C-terminal fragment bound heme, but there was no evidence for binding of ZnMP to the heme-binding region of Bach1. In conclusion, ZnMP produces profound post-transcriptional down-regulation of Bach1 protein levels and transcriptional up-regulation of HO-1. Our results indicate that ZnMP up-regulates HO-1 gene expression by markedly increasing Bach1 protein degradation in a proteasome-dependent manner.

Role of Bach1 and Nrf2 in up-regulation of the heme oxygenase-1 gene by cobalt protoporphyrin.

Heme oxygenase (HO) catalyzes the conversion of heme to biliverdin with the release of iron and carbon monoxide. HO-1 is highly inducible by a large number of physical and chemical factors. CoPP is known to be a potent and effective inducer of HO-1 activity in many tissues. Here we report that CoPP up-regulates HO-1 via Bach1 and Nrf2 in human liver cells. CoPP did not influence hepatic Bach1 or Nrf2 mRNA levels, but markedly reduced Bach1 protein levels by increasing degradation of Bach1 protein (t(1/2) from 19 h to 2.8 h), and increased Nrf2 by decreasing degradation of Nrf2 protein (t(1/2) from 2.5 h to 9 h). Silencing Bach1 by Bach1-siRNA significantly increased levels of HO-1 mRNA and protein, and addition of CoPP up-regulated HO-1 mRNA and protein further. However, silencing Nrf2 mRNA by Nrf2-siRNA did not significantly change baseline HO-1 mRNA or protein levels, but significantly decreased 5-10 microM CoPP-mediated up-regulation of HO-1 mRNA levels compared with CoPP alone. Transfection with equal amounts of non-Bach1 or non-Nrf2 related control siRNA did not reduce Bach1 or Nrf2 mRNA or protein, confirming the specificity of Bach1- and Nrf2-siRNA in Huh-7 cells. We conclude that the pathway of CoPP-mediated induction of HO-1 involves the repression of Bach1 and up-regulation of the Nrf2 protein by post-transcriptional site(s) of action. Because CoPP, unlike heme, is neither a prooxidant nor a substrate for HO-1, it might be considered as a potential therapeutic agent in situations where up-regulation of HO-1 is desired.

HCV proteins increase expression of heme oxygenase-1 (HO-1) and decrease expression of Bach1 in human hepatoma cells.

BACKGROUND/AIMS: Hepatitis C infection induces hepatic oxidative stress. Heme oxygenase (HO), the rate-controlling enzyme of heme catabolism, plays a key role as a protector against oxidative, and other stresses. Other recent work has implicated Bach1, a heme binding protein that represses gene expression, in the regulation of HO-1 gene expression. METHODS: We investigated the effects of HCV polyprotein expression on expression of HO-1 and Bach1 genes in human hepatoma cells (Huh-7 cells). RESULTS: HO-1 was up-regulated in the cell line expressing HCV proteins from core up to the aminoterminal domain of NS3. Addition of increasing concentrations of N-acetylcysteine (NAC) led to down-regulation of HO-1 in cells expressing HCV proteins. In contrast, Bach1 was significantly down-regulated in these cells. Sodium arsenite, a strong inducer of oxidative stress and HO-1, reduced Bach1 expression in wild type Huh-7 cells, and NAC partially abrogated this decrease. CONCLUSIONS: Huh-7 cells expressing HCV proteins show significant up-regulation of the HO-1 gene, and reciprocal down-regulation of the Bach1 gene. Exogenous oxidative stressors and anti-oxidants can modulate expression of these genes. These and other results suggest a key role of down-regulation of Bach1 and up-regulation of HO-1 in diminishing cytotoxic effects of HCV proteins in human hepatocytes.

A "contract for change" increases produce consumption in low-income women: a pilot study.

This study determined whether a "Contract for Change" goal-setting exercise enhanced the effectiveness of the Expanded Food and Nutrition Education/Food Stamp Nutrition Education programs to increase produce consumption in low-income (<130% of poverty) women after 4 weeks. Thirty-eight participants were randomized in this three-group parallel arm study: (a) control group participants received life-skills lessons, (b) the education group received the Expanded Food and Nutrition Education/Food Stamp Nutrition Education "Food Guide Pyramid" lessons, and (c) the contract group also received the "Food Guide Pyramid" series and completed a "Contract for Change." It was hypothesized that the contract group would have the greatest increases in advancement toward dietary change and produce consumption. Compared with controls, the contract group significantly moved toward acceptance of vegetable consumption (P < or = .05). Compared with the education group, the contract group significantly increased fruit consumption. Results suggest that nutrition professionals can effectively use goal-setting to assist low-income populations with dietary change.

Role of Bach-1 in regulation of heme oxygenase-1 in human liver cells: insights from studies with small interfering RNAS.

Heme oxygenase-1 is an antioxidant defense enzyme that converts heme to biliverdin, iron, and carbon monoxide. Bach-1 is a bZip protein that forms heterodimers with small Maf proteins and was reported recently to down-regulate the HO-1 gene in mice. Using small interfering RNAs targeted to human Bach-1 mRNA, we investigated whether modulation of human hepatic Bach-1 expression by small interfering (si)RNA technology influences heme oxygenase-1 gene expression. We found that Bach-1 siRNAs transfected into Huh-7 cells significantly reduced Bach-1 mRNA and protein levels approximately 80%, compared with non siRNA-treated cells. In contrast, transfection with the same amounts of nonspecific control duplexes or LaminB2-duplex did not reduce Bach-1 mRNA or protein levels, confirming the specificity of Bach-1 siRNA. Expression of the heme oxygenase-1 gene in Bach-1 siRNA-transfected cells was up-regulated 7-fold, compared with cells without Bach-1 siRNA. The effect of increasing concentrations of heme to up-regulate levels of heme oxygenase-1 was more pronounced when Bach-1 siRNA was present. Taken together, these results indicated that Bach-1 has a specific and selective ability to repress expression of human hepatic heme oxygenase-1. Silencing of Bach-1 by siRNAs is a useful method for up-regulating HO-1 gene expression. Exogenous heme produces additional up-regulation, beyond that produced by Bach-1 siRNAs, suggesting that heme does not act solely through its effects on Bach-1.

Toxicity of a quinocarmycin analog, DX-52-1, in rats and dogs in relation to clinical outcome.

PURPOSE: Quinocarmycin analog DX-52-1 is a cyanated derivative of quinocarmycin, a compound isolated from cultures of Streptomyces melanovinaceus. DX-52-1 was selected for preclinical development because it showed efficacy against melanoma cell lines in the NCI human tumor cell screen and melanoma xenografts in mice. This report describes studies in rats and dogs to determine the maximum tolerated dose (MTD) and identify dose-limiting toxicities (DLT) in each species in different regimens to establish a safe starting dose and potential target organs of DX-52-1 for phase I clinical trials. METHODS: DX-52-1 was administered to Fischer 344 rats using repeated intravenous (i.v.) slow bolus injections following q3hx3 and q3hx3,q7dx3 regimens, and to beagle dogs using a single injection, 6-h continuous i.v. infusion (c.i.v.) and weekly 6-h c.i.v. for 3 weeks. Endpoints evaluated included clinical observations, body weights, hematology, serum clinical chemistry, and microscopic pathology of tissues. RESULTS: The MTD of DX-52-1 was a total dose of 18 mg/m(2) body surface area for q3hx3 administration in rats and 30 mg/m(2) for a single c.i.v. administration in dogs. The total dose MTD for rats on a weekly (q3hx3,q7dx3) regimen was 54 mg/m(2), and for dogs on the weekly x3 (6-h c.i.v.) infusion was 60 mg/m(2). In rats, significant elevations in blood urea nitrogen and creatinine were observed together with acute renal tubular necrosis histologically. Modest increases in liver enzymes were also observed, as were decreases in reticulocytes that were unaccompanied by histologic changes in liver and bone marrow. In dogs, adverse signs included vomiting/retching, diarrhea, and transient hypothermia; also red blood cells, hemoglobin, hematocrit, and lymphocytes were decreased. Histologic evaluation of tissues from dogs revealed necrosis and cellular depletion of the bone marrow, and extensive damage to the entire gastrointestinal tract, including marked cellular necrosis of the mucosa and lymphoid necrosis of the gastrointestinal associated lymphoid tissue. Destruction of the mucosal lining of the intestinal tract was likely responsible for dehydration, toxemia, septicemia, and shock seen in moribund dogs. CONCLUSIONS: The MTD values were comparable between rats and dogs given roughly similar dose regimens (single dose or weekly) and both species tolerated a higher total dose with weekly administration. However, the principal target organ responsible for DLT in rats was the kidney, whereas in dogs, the most severe effects were on the gastrointestinal tract and bone marrow. Both renal and gastrointestinal toxicities were reported in patients after 6-h c.i.v. infusions in a limited phase I clinical trial, indicating that neither animal model alone was predictive of DX-52-1-induced toxicity in humans, and that both species were required to define human toxicity.

Effect of hepatic artery flow on bile secretory function after cold ischemia.

These studies evaluated the influence of hepatic arterial flow on biliary secretion after cold ischemia. Preparation of livers for transplantation or hepatic support impairs biliary secretion. The earliest indication of cold preservation injury during reperfusion is circulatory function. Arterial flow at this time may be critical for bile secretion. Porcine livers were isolated, maintained at 4 degrees for 2 h and connected in an extracorporeal circuit to an anesthetized normal pig. The extracorporeal livers were perfused either by both the hepatic artery and portal vein (dual) or by the portal vein alone (single). Incremental doses of sodium taurocholate were infused into the portal vein of both the dual and single perfused livers, and the bile secretion was compared. Most endogenous bile acids are lost during hepatic isolation. After supplementation, the biliary secretion of phosphatidyl choline and cholesterol was significantly better in the dual than single vessel-perfused livers; however, no difference was seen in bilirubin output. Single perfused livers were completely unable to increase biliary cholesterol in response to bile acid. The dependence of bile cholesterol secretion on arterial flow indicates the importance of this flow to the detoxification of compounds dependent on phosphatidyl choline transport during early transplantation.

IL-7 therapy dramatically alters peripheral T-cell homeostasis in normal and SIV-infected nonhuman primates.

Interleukin-7 (IL-7) is important for thymopoiesis in mice and humans because IL-7 receptor alpha (IL-7Ralpha) mutations result in a severe combined immunodeficiency phenotype with severe thymic hypoplasia. Recent evidence has indicated that IL-7 also plays an important role as a regulator of T-cell homeostasis. Here we report the immunologic effects of recombinant human IL-7 (rhIL-7) therapy in normal and simian immunodeficiency virus (SIV)-infected nonhuman primates. Cynomolgus monkeys receiving 10 days of rhIL-7 showed substantial, reversible increases in T-cell numbers involving a dramatic expansion of both naive and nonnaive phenotype CD4(+) and CD8(+) subsets. Although IL-7 is known to have thymopoietic effects in mice, we observed marked declines in the frequency and absolute number of T-cell receptor excision circle-positive (TREC(+)) cells in the peripheral blood and dramatic increases in the percentage of cycling T cells in the peripheral blood as measured by Ki-67 expression (baseline less than 5% to approximately 50% after 6 days of therapy) and ex vivo bromodeoxyuridine (BrdU) incorporation. Similarly, moderately CD4- depleted SIV-infected macaques treated with rhIL-7 also had significant increases in peripheral blood CD4(+) and CD8(+) T cells following rhIL-7 therapy. Thus, rhIL-7 induces dramatic alterations in peripheral T-cell homeostasis in both T-cell-replete and T-cell-depleted nonhuman primates. These results further implicate IL-7 as a promising immunorestorative agent but illustrate that a major component of its immunorestorative capacity reflects effects on mature cells. These results also raise the possibility that IL-7 therapy could be used to temporarily modulate T-cell cycling in vivo in the context of immunotherapies such as vaccination.

Preclinical evaluation of amino acid prodrugs of novel antitumor 2-(4-amino-3-methylphenyl)benzothiazoles.

Novel 2-(4-aminophenyl)benzothiazoles (e.g., compounds 1 and 2) possess highly selective, potent antitumor properties in vitro and in vivo. Elucidation of the mechanism of action of this structurally simple class of compounds has occurred in parallel with selection of a candidate clinical agent. Antitumor benzothiazoles induce and are biotransformed by cytochrome P 450 1A1 to putative active, as well as inactive metabolites. Metabolic inactivation of the molecule has been thwarted by isosteric replacement of hydrogen with fluorine atoms at positions around the benzothiazole nucleus. Amino acid conjugation to the exocyclic primary amine function of 2-(4-aminophenyl)benzothiazoles has been used to overcome limitations posed by drug lipophilicity. Water soluble, chemically stable prodrugs rapidly and quantitatively revert to their parent amine in mice, rats, and dogs in vivo. Plasma concentrations of 2-(4-amino-3-methylphenyl)-5-fluorobenzothiazole (2) regenerated from the lysylamide prodrug (2b), sufficient to elicit cytocidal activity against ZR-75-1 and T47D human mammary carcinoma cell lines persist > 6 h. The growth of breast (MCF-7) and ovarian (IGROV-1) xenograft tumors is significantly retarded by 2b. Manageable toxic side effects are reported from preclinically efficacious doses of 2b. Cytochrome P 450 1A1 protein expression, selectively induced in sensitive carcinoma cells, was detected in MCF-7 and IGROV-1 tumors 24 h after treatment of mice with 2b (20 mg/kg). The lysyl amide prodrug of 2-(4-amino-3-methylphenyl)-5-fluorobenzothiazole is potentially suitable for clinical evaluation.

Donor hepatic function: a factor in postreperfusion syndrome.

Reperfusion of support livers after cold preservation produces hemodynamic instability (i.e., postreperfusion syndrome) in the recipient during both orthotopic liver transplantation and extracorporeal liver perfusion. We evaluated the effect of the normal porcine cold-preserved support liver on healthy recipient hemodynamics and in situ liver function during extracorporeal liver perfusion. Support livers were harvested from Yorkshire pigs and reperfused in an extracorporeal circuit with a healthy, anesthetized recipient pig. Correlation analyses were performed between support liver variables of function (oxygen consumption, bile flow, and biliary phospholipid and cholesterol output) and both recipient hemodynamic stability (heart rate, blood pressure, urine output, and vasopressor use) and hepatic function (bile flow and biliary phospholipid secretion). The data indicate that optimally functioning support livers are associated with improved recipient hemodynamic stability manifested by decreased recipient heart rate and vasopressor use and increased recipient urine output. Support livers exhibiting poor biliary secretory function (i.e., bile flow and phospholipid output) were associated with similarly diminished recipient liver biliary secretory function. These data indicate that the functional condition of the support liver after harvest and cold preservation may influence both recipient hemodynamic parameters and the endogenous function of the recipient liver.

Porcine hepatic phospholipid efflux during reperfusion after cold ischemia.

BACKGROUND: Cold preservation produces hepatic injury that is difficult to assess during early reperfusion. The value of reperfusion plasma choline phospholipid in predicting subsequent organ function is documented in these studies. MATERIALS AND METHODS: Livers of female Yorkshire pigs were prepared for transplantation. After 2 h of cold ischemia the reperfusion plasma was evaluated for choline phospholipid and cholesterol. These values were correlated with bile secretion, hepatic hemodynamics, oxygen uptake, and plasma sorbitol dehydrogenase levels. RESULTS: The isolated porcine liver demonstrates a rapid efflux of choline phospholipids into plasma during early reperfusion after cold preservation. After this initial efflux no subsequent plasma increment occurred. These choline-phospholipid increments were isolated in plasma higher density (d > 1.063) lipoproteins and were not accompanied by equivalent increases in cholesterol. Neither biliary reflux nor lecithin cholesterol acyl transferase abnormalities contributed appreciably to the phospholipid increments in reperfusion plasma. Livers with the largest efflux of choline phospholipids had the most impaired circulatory and bile secretory function at 4 h of reperfusion. CONCLUSION: The immediate increase of choline phospholipids, particularly lysophosphatidylcholine, in reperfusion plasma after cold ischemia provides an index of the injury occurring during this interval and correlates with early organ function.