A new physiological medium uncovers biochemical and cellular alterations in Lesch-Nyhan disease fibroblasts.

BACKGROUND: Lesch-Nyhan disease (LND) is a severe neurological disorder caused by the genetic deficiency of hypoxanthine-guanine phosphoribosyltransferase (HGprt), an enzyme involved in the salvage synthesis of purines. To compensate this deficiency, there is an acceleration of the de novo purine biosynthetic pathway. Most studies have failed to find any consistent abnormalities of purine nucleotides in cultured cells obtained from the patients. Recently, it has been shown that 5-aminoimidazole-4-carboxamide riboside 5'-monophosphate (ZMP), an intermediate of the de novo pathway, accumulates in LND fibroblasts maintained with RPMI containing physiological levels (25 nM) of folic acid (FA), which strongly differs from FA levels of regular cell culture media (2200 nM). However, RPMI and other standard media contain non-physiological levels of many nutrients, having a great impact in cell metabolism that does not precisely recapitulate the in vivo behavior of cells. METHODS: We prepared a new culture medium containing physiological levels of all nutrients, including vitamins (Plasmax-PV), to study the potential alterations of LND fibroblasts that may have been masked by the usage of non-physiological media. We quantified ZMP accumulation under different culture conditions and evaluated the activity of two known ZMP-target proteins (AMPK and ADSL), the mRNA expression of the folate carrier SLC19A1, possible mitochondrial alterations and functional consequences in LND fibroblasts. RESULTS: LND fibroblasts maintained with Plasmax-PV show metabolic adaptations such a higher glycolytic capacity, increased expression of the folate carrier SCL19A1, and functional alterations such a decreased mitochondrial potential and reduced cell migration compared to controls. These alterations can be reverted with high levels of folic acid, suggesting that folic acid supplements might be a potential treatment for LND. CONCLUSIONS: A complete physiological cell culture medium reveals new alterations in Lesch-Nyhan disease. This work emphasizes the importance of using physiological cell culture conditions when studying a metabolic disorder.

Identification of Optimal Reference Genes for qRT-PCR Normalization for Physical Activity Intervention and Omega-3 Fatty Acids Supplementation in Humans.

The quantitative polymerase chain reaction (qRT-PCR) technique gives promising opportunities to detect and quantify RNA targets and is commonly used in many research fields. This study aimed to identify suitable reference genes for physical exercise and omega-3 fatty acids supplementation intervention. Forty healthy, physically active men were exposed to a 12-week eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) supplementation and standardized endurance training protocol. Blood samples were collected before and after the intervention and mRNA levels of six potential reference genes were tested in the leukocytes of 18 eligible participants using the qRT-PCR method: GAPDH (Glyceraldehyde-3-phosphate dehydrogenase), ACTB (Beta actin), TUBB (Tubulin Beta Class I), RPS18 (Ribosomal Protein S18), UBE2D2 (Ubiquitin-conjugating enzyme E2 D2), and HPRT1 (Hypoxanthine Phosphoribosyltransferase 1). The raw quantification cycle (Cq) values were then analyzed using RefFinder, an online tool that incorporates four different algorithms: NormFinder, geNorm, BestKeeper, and the comparative delta-Ct method. Delta-Ct, NormFinder, BestKeeper, and RefFinder comprehensive ranking have found GAPDH to be the most stably expressed gene. geNorm has identified TUBB and HPRT as the most stable genes. All algorithms have found ACTB to be the least stably expressed gene. A combination of the three most stably expressed genes, namely GAPDH, TUBB, and HPRT, is suggested for obtaining the most reliable results.

Prolonged cultured human hepatocytes as an in vitro experimental system for the evaluation of potency and duration of activity of RNA therapeutics: Demonstration of prolonged duration of gene silencing effects of a GalNAc-conjugated human hypoxanthine phosphoribosyl transferase (HPRT1) siRNA.

We report here the evaluation of a novel in vitro experimental model, prolonged cultured human hepatocytes (PCHC), as an experimental system to evaluate the potency and duration of effects of oligonucleotide therapeutics. A novel observation was made on the redifferentiation of PCHC upon prolonged culturing based on mRNA profiling of characteristic hepatic differentiation marker genes albumin, transferrin, and transthyretin. Consistent with the known de-differentiation of cultured human hepatocytes, decreases in marker gene expression were observed upon culturing of the hepatocytes for 2 days. A novel observation of re-differentiation was observed on day 7 as demonstrated by an increase in expression of the marker genes to levels similar to that observed on the first day of culture. The expression of the differentiation marker genes was highest on day 7, followed by a gradual decrease but remained higher than that on day 2 for up to the longest culture duration evaluated of 41 days. The redifferentiation phenomenon suggests that PCHC may be useful for the evaluation of the duration of effects of oligonucleotide therapeutics on gene expression in human hepatocytes. A proof of concept study was thereby conducted with PCHC with a GalNAc-conjugated siRNA targeting human hypoxanthine phosphoribosyl transferase1 (HPRT1). HPRT1 mRNA expression in siRNA-treated cultures decreased to 21% of that in untreated hepatocytes on day 1, <10% from days 2 to 12, <20% from days 16 to 33, and eventually recovered to 64% by day 41. Our results suggest that PCHC represent a clinically-relevant cost- and time-efficient experimental tool to aid in the evaluation of GalNAc-siRNA silencing activity, providing information on both efficacy and duration of efficacy. PCHC may be applicable in the drug development setting as a species- and cell type-relevant experimental tool to aid the development of oligonucleotide therapeutics.

Aquaporins Alteration Profiles Revealed Different Actions of Senna, Sennosides, and Sennoside A in Diarrhea-Rats.

Senna and its main components sennosides are well-known effective laxative drugs and are used in the treatment of intestinal constipation in the world. Their potential side effects have attracted more attention in clinics but have little scientific justification. In this study, senna extract (SE), sennosides (SS), and sennoside A (SA) were prepared and used to generate diarrhea rats. The diarrhea rats were investigated with behaviors, clinical signs, organ index, pathological examination, and gene expression on multiple aquaporins (Aqps) including Aqp1, Aqp2, Aqp3, Aqp4, Aqp5, Aqp6, Aqp7, Aqp8, Aqp9, and Aqp11. Using qRT-PCR, the Aqp expression profiles were constructed for six organs including colon, kidney, liver, spleen, lung, and stomach. The Aqp alteration profiles were characterized and was performed with Principle Component Analysis (PCA). The SE treatments on the rats resulted in a significant body weight loss (p < 0.001), significant increases (p < 0.001) on the kidney index (27.72%) and liver index (42.55%), and distinguished changes with up-regulation on Aqps expressions in the kidneys and livers. The SS treatments showed prominent laxative actions and down regulation on Aqps expression in the colons. The study results indicated that the SE had more influence/toxicity on the kidneys and livers. The SS showed more powerful actions on the colons. We suggest that the caution should be particularly exercised in the patients with kidney and liver diseases when chronic using senna-based products.

Mutagenicity monitoring following battlefield exposures: Longitudinal study of HPRT mutations in Gulf War I veterans exposed to depleted uranium.

A total of 70 military Veterans have been monitored for HPRT T-cell mutations in five separate studies at 2-year intervals over an 8-year period. Systemic depleted uranium (DU) levels were measured at the time of each study by determining urinary uranium (uU) excretion. Each HPRT study included 30-40 Veterans, several with retained DU-containing shrapnel. Forty-nine Veterans were evaluated in multiple studies, including 14 who were in all five studies. This permitted a characterization of the HPRT mutation assay over time to assess the effects of age, smoking and non-selected cloning efficiencies, as well as the inter- and intra-individual variability across time points. Molecular analyses identified the HPRT mutation and T-cell receptor (TCR) gene rearrangement in 1,377 mutant isolates. An unexpected finding was that in vivo clones of HPRT mutant T-cells were present in some Veterans, and could persist over several years of the study. The calculated HPRT mutant frequencies (MFs) were repeatedly elevated in replicate studies in three outlier Veterans with elevated urinary uranium excretion levels. However, these three outlier Veterans also harbored large and persistent in vivo HPRT mutant T-cell clones, each of which was represented by a single founder mutation. Correction for in vivo clonality allowed calculation of HPRT T-cell mutation frequencies (MutFs). Despite earlier reports of DU associated increases in HPRT MFs in some Veterans, the results presented here demonstrate that HPRT mutations are not increased by systemic DU exposure. Additional battlefield exposures were also evaluated for associations with HPRT mutations and none were found.

Mutagenicity monitoring following battlefield exposures: Molecular analysis of HPRT mutations in Gulf War I veterans exposed to depleted uranium.

Molecular studies that involved cDNA and genomic DNA sequencing as well as multiplex PCR of the HPRT gene were performed to determine the molecular mutational spectrum for 1,377 HPRT mutant isolates obtained from 61 Veterans of the 1991 Gulf War, most of whom were exposed to depleted uranium (DU). Mutant colonies were isolated from one to four times from each Veteran (in 2003, 2005, 2007, and/or 2009). The relative frequencies of the various types of mutations (point mutations, deletions, insertions, etc.) were compared between high versus low DU exposed groups, (based on their urine U concentration levels), with HPRT mutant frequency (as determined in the companion paper) and with a database of historic controls. The mutational spectrum includes all classes of gene mutations with no significant differences observed in Veterans related to their DU exposures.

Mutagenic potential of the isoflavone irilone in cultured V79 cells.

After consumption of red clover-based dietary supplements, plasma concentrations of the isoflavone irilone (IRI) equal that of the well-investigated daidzein. Since some isoflavones are genotoxic, the potential of IRI to induce mutations was investigated. Gene mutations were determined by hypoxanthine-guanine phosphoribosyltransferase (HPRT) assay and sequencing of mutant cDNA, chromosome and genome mutations by micronucleus assay complemented by immunochemical staining of centromere proteins and microtubules in cultured V79 cells. Cell proliferation was monitored by electronic cell counting, flow cytometry and fluorescence microscopy. IRI did not affect the mutant frequency in the Hprt locus but altered the mutation spectrum by increasing the proportion of deletions and decreasing that of base pair substitutions. Induction of chromosome mutations was supported by a slight but significant increase in the number of micronucleated cells containing chromosomal fragments despite activation of three cell cycle checkpoints possibly interfering with micronuclei formation. Moreover, IRI exhibited a strong aneugenic potential characterized by disrupted mitotic spindles, mitotic arrest, and asymmetrical cell divisions leading to chromosome loss, nuclear fragmentation as well as mitotic catastrophe. Thus, IRI might be another isoflavone to be taken into account in safety assessment of dietary supplements.

Evaluation of the cytotoxic and mutagenic potential of three ginkgolic acids.

Ginkgolic acids (GAs) are alkylphenols which can be found in the fruits and leaves of Ginkgo biloba L. (Ginkgoaceae) used in herbal teas, drugs and food supplements. Standardized leaf extracts of G. biloba are widely used in the therapy of cognitive decline including Alzheimer's diseases. However, GAs are known to have cytotoxic and allergenic potential and are suspected to possess genotoxic properties. Therefore, we examined in this study the cytotoxicity and mutagenicity of three major GAs with different alkyl or alkenyl groups (13:0, 15:1, 17:1). Cytotoxicity was assessed in male Chinese hamster lung fibroblasts (V79 cells) using the resazurin reduction assay. The substances showed concentration dependent cytotoxic effects after 24h of incubation at concentrations of 50µM and higher. Mutagenicity was determined by using the Ames fluctuation assay in different Salmonella typhimurium strains (TA97a, TA98, TA100 and TA102) with and without exogenous metabolic activation (S9 mix). Furthermore, we analyzed the mutagenic potency of the three major GAs in V79 cells by performing the hypoxanthine phosphoribosyl transferase (HPRT) assay which detects gene mutations at the HPRT locus. None of the mutagenic assays showed any increase in mutagenicity above background. Therefore, these data provide evidence that the GAs tested have some cytotoxic potency but are not mutagenic. Thus, our findings contribute to the risk assessment of preparations containing plant extracts from G. biloba.

Normal uricemia in Lesch-Nyhan syndrome and the association with pulmonary embolism in a young child-a case report and literature review.

Deficiency of hypoxanthine phosphoribosyltransferase activity is a rare inborn error of purine metabolism with subsequent uric acid overproduction and neurologic presentations. The diagnosis of Lesch-Nyhan syndrome (LNS) is frequently delayed until self-mutilation becomes evident. We report the case of a boy aged 1 year and 10 months who was diagnosed with profound global developmental delay, persistent chorea, and compulsive self-mutilation since the age of 1 year. Serial serum uric acid levels showed normal uric acid level, and the spot urine uric acid/creatinine ratio was >2. The hypoxanthine phosphoribosyltransferase cDNA showed the deletion of exon 6, and the boy was subsequently diagnosed to have LNS. He also had respiratory distress due to pulmonary embolism documented by chest computed tomography scan. This report highlights the need to determine the uric acid/creatinine ratio caused by increased renal clearance in LNS in young children. The presence of pulmonary embolism is unusual and may be the consequence of prolonged immobilization.

The isoflavone irilone contributes to the estrogenic potential of dietary supplements containing red clover.

A recent intervention study demonstrated the occurrence of irilone as second most abundant isoflavone next to daidzein in human plasma after consumption of a red clover-based dietary supplement (RCDS) containing predominately formononetin ≫ biochanin A > irilone (12 % of these isoflavones). To elucidate the relevance of this finding, in the present study (1) the representativeness of the isoflavone composition of the RCDS and (2) the estrogenic activity of irilone were investigated. Thus, major isoflavones were quantified in eight commercially available RCDS. Furthermore, the estrogenic activities of irilone and other isoflavones were determined by marker gene expression in Ishikawa and cell proliferation in MCF-7 cells. Irilone amounted to 1.8-10.9 mg/g capsule content and 5-18 % of the three major isoflavones, respectively, demonstrating the general occurrence of irilone in RCDS. Moreover, irilone significantly induced the activity of alkaline phosphatase (AlP) as well as AlP, progesterone receptor, and androgen receptor mRNA levels in Ishikawa cells. Furthermore, irilone significantly induced MCF-7 cell proliferation. Neither 17ß-estradiol (E2)-induced AlP activity nor E2-induced MCF-7 cell proliferation was affected by irilone. ICI182,780 antagonized IRI-induced effects on both AlP activity and cell proliferation, suggesting an estrogen receptor agonistic mode of action. Taking into account the estrogenic activity of red clover isoflavones (formononetin, biochanin A, prunetin, glycitein) and their biotransformation products (daidzein, genistein, ethylphenol) as well as published plasma levels of isoflavones after consumption of RCDS, irilone could contribute approximately 50 % of the E2 equivalents estimated for daidzein.

Antioxidant induces DNA damage, cell death and mutagenicity in human lung and skin normal cells.

Clinical trials have shown that antioxidant supplementation increased the risk of lung and skin cancers, but the underlying molecular mechanism is unknown. Here, we show that epigallocatechin gallate (EGCG) as an exemplary antioxidant induced significant death and DNA damage in human lung and skin normal cells through a reductive mechanism. Our results show direct evidence of reductive DNA damage in the cells. We found that EGCG was much more toxic against normal cells than H2O2 and cisplatin as toxic and cancer-causing agents, while EGCG at low concentrations (≤100 µM) increased slightly the lung cancer cell viability. EGCG induced DNA double-strand breaks and apoptosis in normal cells and enhanced the mutation frequency. These results provide a compelling explanation for the clinical results and unravel a new reductive damaging mechanism in cellular processes. This study therefore provides a fresh understanding of aging and diseases, and may lead to effective prevention and therapies.

Detection of in vivo mutation in the Hprt and Pig-a genes of rat lymphocytes.

Assays for in vivo mutation are used to identify genotoxic hazards and phenotypes prone to genomic instability and cancer. The hypoxanthine guanine phosphoribosyl transferase (Hprt) gene and the phosphatidyl inositol glycan, class A (Pig-a) gene are endogenous X-linked genes that can be used as reporters of mutation in peripheral blood lymphocytes from most mammals. Here we describe methodology for measuring Hprt and Pig-a mutation in rat T-lymphocytes. The identification and selective expansion of mutant lymphocytes are based upon the phenotypic properties of Hprt- and Pig-a-deficient cells, i.e., resistance to the purine analog, 6-thioguanine, or to the bacterial toxin, proaerolysin. Expanded mutants can be further analyzed by sequencing cDNA from the target transcripts for identification of small sequence alterations and by multiplex PCR analysis of genomic DNA for the detection of deletions.

History of gene therapy.

Two decades after the initial gene therapy trials and more than 1700 approved clinical trials worldwide we not only have gained much new information and knowledge regarding gene therapy in general, but also learned to understand the concern that has persisted in society. Despite the setbacks gene therapy has faced, success stories have increasingly emerged. Examples for these are the positive recommendation for a gene therapy product (Glybera) by the EMA for approval in the European Union and the positive trials for the treatment of ADA deficiency, SCID-X1 and adrenoleukodystrophy. Nevertheless, our knowledge continues to grow and during the course of time more safety data has become available that helps us to develop better gene therapy approaches. Also, with the increased understanding of molecular medicine, we have been able to develop more specific and efficient gene transfer vectors which are now producing clinical results. In this review, we will take a historical view and highlight some of the milestones that had an important impact on the development of gene therapy. We will also discuss briefly the safety and ethical aspects of gene therapy and address some concerns that have been connected with gene therapy as an important therapeutic modality.

Induction of the metabolic regulator Txnip in fasting-induced and natural torpor.

Torpor is a physiological state characterized by controlled lowering of metabolic rate and core body temperature, allowing substantial energy savings during periods of reduced food availability or harsh environmental conditions. The hypothalamus coordinates energy homeostasis and thermoregulation and plays a key role in directing torpor. We recently showed that mice lacking the orphan G protein-coupled receptor Gpr50 readily enter torpor in response to fasting and have now used these mice to conduct a microarray analysis of hypothalamic gene expression changes related to the torpor state. This revealed a strong induction of thioredoxin-interacting protein (Txnip) in the hypothalamus of torpid mice, which was confirmed by quantitative RT-PCR and Western blot analyses. In situ hybridization identified the ependyma lining the third ventricle as the principal site of torpor-related expression of Txnip. To characterize further the relationship between Txnip and torpor, we profiled Txnip expression in mice during prolonged fasting, cold exposure, and 2-deoxyglucose-induced hypometabolism, as well as in naturally occurring torpor bouts in the Siberian hamster. Strikingly, pronounced up-regulation of Txnip expression was only observed in wild-type mice when driven into torpor and during torpor in the Siberian hamster. Increase of Txnip was not limited to the hypothalamus, with exaggerated expression in white adipose tissue, brown adipose tissue, and liver also demonstrated in torpid mice. Given the recent identification of Txnip as a molecular nutrient sensor important in the regulation of energy metabolism, our data suggest that elevated Txnip expression is critical to regulating energy expenditure and fuel use during the extreme hypometabolic state of torpor.

Investigation of the mutagenic potential of cold atmospheric plasma at bactericidal dosages.

In the past few years, cold atmospheric plasma (CAP) has evolved into a new tool in the fight against nosocomial infections and antibiotic-resistant microorganisms. The products generated by the plasma-electrons, ions, reactive species and UV light-represent a 'lethal cocktail' for different kinds of pathogen, which opens up possible applications in hygiene and medicine. Nevertheless, to ensure the safe usage of CAP on skin (e.g., to treat wounds or skin diseases) several pre-clinical in vitro studies have to be performed before implementing clinical trials on humans. In the study presented here, inactivation experiments with Escherichia coli were carried out to identify the necessary plasma dosage for a 5 log reduction: with a small hand-held battery-operated CAP device, these disinfection properties were achieved after application during 30s. This and higher plasma dosages were then used to analyze the mutagenicity induced in V79 Chinese hamster cells-to furthermore define a 'safe application window'-with the HPRT (hypoxanthine-guanine phosphoribosyl transferase) mutation assay. The results show that a CAP treatment of up to 240 s and repeated treatments of 30s every 12h did not induce mutagenicity at the Hprt locus beyond naturally occurring spontaneous mutations.

Dammar resin, a non-mutagen, induces [corrected] oxidative stress and metabolic enzymes in the liver of gpt delta transgenic mouse which is different from a mutagen, 2-amino-3-methylimidazo[4,5-f]quinoline.

Dammar resin has long been used in foods as either a clouding or a glazing agent. In a recent study, 2% Dammar resin showed significant hepatocarcinogenicity in a rat 2-year bioassay. Therefore, for an accurate estimate of human risk, it is necessary to understand whether Dammar resin induces liver genotoxicity and the underlying mechanisms of its hepatocarcinogenicity. Modifying effects of 2-amino-3-methylimidazo[4,5-f]quinoline (IQ), a typical genotoxic carcinogen produced during cooking of protein-rich foods, was also studied in the present study. Exposure of gpt delta mice to Dammar resin at a dose of 2% for 12 weeks did not induce any obvious mutagenicity in the liver. However, the index of cell proliferation, the level of 8-OHdG, and bax, bcl-2, p53, cyp1a2, cyp2e1, gpx1 and gstm2 gene expression were all significantly increased when compared with the control group. In the IQ treatment group, at a dose of 300ppm, mutagenicity was readily detected, the index of cell proliferation increased, and p53, cyp2e1 and gpx1 gene expression was down-regulated in the liver. Down-regulation of p53, P450s, and gpx1 in the livers of IQ treated mice are consistent with its genotoxic mechanism of carcinogenicity observed in a 675-day study. In contrast, our results using gpt delta mice suggest that Dammar resin is not genotoxic. Instead, the Dammar resin-induced hepatocarcinogenicity seen in our previous 2-year study with rats may have been mediated by non-genotoxic mechanisms, including increased P450 enzyme activity, increased oxidative stress, altered gene expression, and promotion of cell proliferation.

Measures of genotoxicity in Gulf war I veterans exposed to depleted uranium.

Exposure to depleted uranium (DU), an alpha-emitting heavy metal, has prompted the inclusion of markers of genotoxicity in the long-term medical surveillance of a cohort of DU-exposed Gulf War veterans followed since 1994. Using urine U (uU) concentration as the measure of U body burden, the cohort has been stratified into low-u (<0.10 µg U/g creatinine) and high-u groups (≥ 0.10 µg U/g creatinine). Surveillance outcomes for this cohort have historically included markers of mutagenicity and clastogenicity, with past results showing generally nonsignificant differences between low- vs. high-U groups. However, mean hypoxanthine-guanine phosphoribosyl transferase (HPRT) mutant frequencies (MFs) have been almost 50% higher in the high-U group. We report here results of a more comprehensive protocol performed in a 2009 evaluation of a subgroup (N = 35) of this cohort. Four biomarkers of genotoxicity [micronuclei (MN), chromosome aberrations, and MFs of HPRT and PIGA] were examined. There were no statistically significant differences in any outcome measure when results were compared between the low- vs. high-U groups. However, modeling of the HPRT MF results suggests a possible threshold effect for MFs occurring in the highest U exposed cohort members. Mutational spectral analysis of HPRT mutations is underway to clarify a potential clonal vs. a threshold uU effect to explain this observation. This study provides a comprehensive evaluation of a human population chronically exposed to DU and demonstrates a relatively weak genotoxic effect of the DU exposure. These results may explain the lack of clear epidemiologic evidence for U carcinogenicity in humans. Environ. Mol. Mutagen., 2011. © 2011 Wiley-Liss, Inc.

Dietary arginine levels alter markers of arginine utilization in peripheral blood mononuclear cells and thymocytes in young broiler chicks.

Arginine is an essential amino acid in Aves and is also an important substrate for the immune system. Dietary Arg in avian diets must be sufficient to not only support growth but also immunity. To better understand Arg needs for immunity, 2 experiments examined markers of Arg use by the immune system in growing broiler chicks. Broiler hatchlings were fed diets containing adequate (1.2%) or high (1.35%) dietary Arg for 21 d. On d 7, the Arg importer cationic amino acid transporter-1 mRNA abundance in peripheral blood mononuclear cells was 2-fold greater in chicks fed 1.35% Arg than in chicks fed 1.2% Arg (P < 0.05). On d 14, chicks fed the diet containing 1.2% Arg had 2.5-fold greater mRNA abundance of the y(+)L type amino acid transporter-2 exporter compared with chicks fed 1.35% Arg (P < 0.05). In experiment 2, broiler hatchlings were fed diets containing low (1.1%), high (1.3%), or excess (1.5%) dietary Arg for 17 d. The percentage of peripheral blood B cells at a given age tended (P = 0.06) to be affected by the dietary Arg level. On d 14, but not on d 10 or 17, the percentage of monocytes from chicks fed 1.5% Arg was higher than from those fed 1.1 and 1.3% Arg (P < 0.05). These studies indicate that the dietary Arg levels in excess of 1.2% increase the mRNA abundance of markers for Arg use by immune cells undergoing development (thymocytes) and at maintenance (peripheral blood mononuclear cells) and also increase the percentage of monocytes within peripheral blood. Understanding Arg use by the immune system will provide a better understanding of how to formulate immunosupportive diets to promote animal health.

alpha-Tocopherol enhances degranulation in RBL-2H3 mast cells.

Based on the observation that 3 months alpha-tocopherol supplementation caused an up-regulation of the mRNA of vesicular transport proteins in livers of mice, the functional relevance was investigated in RBL-2H3 cells, a model for mast cell degranulation. In total, 24 h incubation with 100 muM alpha-tocopherol enhanced the basal and phorbol-12-myristyl-13-acetate/ionomycin-stimulated release of beta-hexosaminidase and cathepsin D as measured by enzymatic analysis as well as Western blotting and immunocytochemistry, respectively. beta-Tocopherol exerted the same effect, whereas alpha-tocopheryl phosphate and trolox were inactive, indicating that both the side chain and the 6-OH group at the chroman ring are essential for activation of degranulation. alpha-Tocopherol did not induce mRNA expression of soluble NSF-attachment protein receptor (soluble N-ethylmaleimide-sensitive factor-attachment protein receptor) proteins, such as N-ethylmaleimide sensitive fusion protein, complexin-2, SNAP23 or syntaxin-3, in the RBL-2H3 cell model. In view of the well known alpha-tocopherol-mediated activation of protein phosphatases, which regulate soluble NSF-attachment protein receptor activities by dephosphorylation, underlying mechanisms are discussed in terms of preventing oxidative inactivation of protein phosphatases and so far unknown functions in certain membrane domains.

Health surveillance of Gulf War I veterans exposed to depleted uranium: updating the cohort.

A cohort of seventy-four 1991 Gulf War soldiers with known exposure to depleted uranium (DU) resulting from their involvement in friendly-fire incidents with DU munitions is being followed by the Baltimore Veterans Affairs Medical Center. Biennial medical surveillance visits designed to identify uranium-related changes in health have been conducted since 1993. On-going systemic exposure to DU in veterans with embedded metal fragments is indicated by elevated urine uranium (U) excretion at concentrations up to 1,000-fold higher than that seen in the normal population. Health outcome results from the subcohort of this group of veterans attending the 2005 surveillance visit were examined based on two measures of U exposure. As in previous years, current U exposure is measured by determining urine U concentration at the time of their surveillance visit. A cumulative measure of U exposure was also calculated based on each veteran's past urine U concentrations since first exposure in 1991. Using either exposure metric, results continued to show no evidence of clinically significant DU-related health effects. Urine concentrations of retinol binding protein (RBP), a biomarker of renal proximal tubule function, were not significantly different between the low vs. high U groups based on either the current or cumulative exposure metric. Continued evidence of a weak genotoxic effect from the on-going DU exposure as measured at the HPRT (hypoxanthine-guanine phosphoribosyl transferase) locus and suggested by the fluorescent in-situ hybridization (FISH) results in peripheral blood recommends the need for continued surveillance of this population.