L-Arginine supplementation prevents allodynia and hyperalgesia in painful diabetic neuropathic rats by normalizing plasma nitric oxide concentration and increasing plasma agmatine concentration.

PURPOSE: Neuropathic pain is a common diabetic complication. It is characterized by symptoms of spontaneous and stimulus-evoked pain including hyperalgesia and allodynia. L-Arginine is a common precursor of many metabolites of biological interest, in particular, nitric oxide (NO), ornithine, and hence polyamines. In central nervous system, NO, glutamate, and polyamines share an N-methyl-D-aspartate (NMDA) receptor-mediated effect. We hypothesized that a variation in arginine metabolism caused by diabetes may contribute to development and maintenance of neuropathic pain and to the worsening of clinical and biological signs of diabetes. METHODS: We examined whether oral L-arginine supplementation (2.58 ± 0.13 g/l in drinking water for 3 weeks) could improve the development of neuropathic pain and the clinical, biological, and metabolic complications of diabetes in streptozocin (STZ)-induced diabetic (D) rats. RESULTS: STZ administration induced classical symptoms of type 1 diabetes. Diabetic rats also displayed mechanical hypersensitivity, tactile, and thermal allodynia. Plasma citrulline and NO levels were increased in diabetic hyperalgesic/allodynic rats. L-Arginine supplementation failed to reduce hyperglycaemia, polyphagia, and weight loss. Moreover, it abolished hyperalgesia and allodynia by normalizing NO plasma concentration and increasing plasma agmatine concentration. CONCLUSIONS: L-Arginine supplementation prevented the development of mechanical hyperalgesia, tactile, and thermal allodynia in painful diabetic neuropathy with concomitant reduction of NO and increased agmatine production, offering new therapeutic opportunities for the management of diabetic neuropathic pain.

Neutrophils are immune cells preferentially targeted by retinoic acid in elderly subjects.

BACKGROUND: The immune system gradually deteriorates with age and nutritional status is a major factor in immunosenescence. Of the many nutritional factors implicated in age-related immune dysfunction, vitamin A may be a good candidate, since vitamin A concentrations classically decrease during aging whereas it may possess important immunomodulatory properties via its active metabolites, the retinoic acids. This prompted us to investigate the immune response induced by retinoids in adults and elderly healthy subjects. Before and after oral supplementation with 13cis retinoic acid (0.5 mg/kg/day during 28 days), whole blood cells were phenotyped, and functions of peripheral blood mononuclear cells (PBMC) and polymorphonuclear cells (PMN) were investigated by flow cytometry and ELISA tests. RESULTS: In both young adults (n = 20, 25 ± 4 years) and older subjects (n = 20, 65 ± 4 years), retinoic acid supplementation had no effect on the distribution of leukocyte subpopulations or on the functions of PBMC (Il-2 and sIl-2R production, membrane expression of CD25). Concerning PMN, retinoic acid induced an increase in both spontaneous migration and cell surface expression of CD11b in the two different age populations, whereas bactericidal activity and phagocytosis remained unchanged. CONCLUSIONS: We demonstrated that retinoic acid induces the same intensity of immune response between adult and older subjects, and more specifically affects PMN functions, i.e. adhesion and migration, than PBMC functions.

Age-related change in the retinoid X receptor beta gene expression in peripheral blood mononuclear cells of healthy volunteers: effect of 13-cis retinoic acid supplementation.

The regulation of cell growth and differentiation and also expression of a number of genes by retinoids are mediated by nuclear retinoid receptors (RARs and/or RXRs). In this study we investigated age-related alteration in both RAR and RXR receptor subtypes gene expression and tissue transglutaminase (tTG) activity before and after supplementation with 13-cis retinoic acid (13cRA) in human peripheral blood mononuclear cells (PBMCs). Healthy men (40) were divided in two groups according to their age (young group: 26.1+/-4.1 years and old group: 65.4+/-3.8 years). Each volunteer received 13cRA (Curacné), 0.5mg/(kgday)) during a period of 4 weeks. We have shown that RXRbeta expression was decreased significantly (p=0.0108) in PBMCs of elderly men when compared to that of young volunteers. Distribution of retinoic acid receptor subtype expression in PBMCs was found in the order: RXRbeta>RARgamma>RXRalpha>RARalpha. The tTG activity in PBMCs reflected a trend to be enhanced after 13-cis retinoic acid supplementation. In conclusion, we demonstrate a significant decrease in the expression of RXRbeta subtype of rexinoid receptors in PBMCs of healthy elderly men. Our data suggest that in healthy elderly men reduction of RXRbeta expression in PBMCs might be a common feature of physiological senescence.

Methionine dependency and cancer treatment.

Conventional chemotherapies have showed their limits, notably for patients with advanced cancer. New therapeutic strategies must be identified, and the metabolic abnormalities of cancer cells offer such opportunities. Many human cancer cell lines and primary tumors have absolute requirements for methionine, an essential amino acid. In contrast, normal cells are relatively resistant to exogenous methionine restriction. The biochemical mechanism for methionine dependency has been studied extensively, but the fundamental mechanism remains unclear. A number of investigators have attempted to exploit the methionine dependence of tumors for therapeutic effects in vivo. To reduce in vivo methionine in plasma and tumours, dietary and pharmacological treatments have been used. Methionine-free diet or methionine-deprived total parenteral nutrition causes regression of a variety of animal tumours. Alternatively, methionine depletion was achieved by the use of methioninase. This enzyme specifically degrades methionine and inhibits tumour growth in preclinical models. Because of potential toxicity and quality of life problems, prolonged methionine restriction with diet or with methioninase is not suitable for clinical use. Methionine restriction may find greater application in association with various chemotherapeutic agents. Several preclinical studies have demonstrated synergy between methionine restriction and various cytotoxic chemotherapy drugs. The experimental results accumulated during the last three decades suggest that methionine restriction can become an additional cancer therapeutic strategy, notably in association with chemotherapy.

Aging: a barrier to renutrition? Nutritional and immunologic evidence in rats.

BACKGROUND: Previous reports suggest that correcting the malnourished state is more difficult in elderly people than in younger ones and that protein requirements may be higher in elderly than in younger adults. OBJECTIVE: The aim of this study was to establish whether malnourished old rats respond to protein-supplemented nutritional repletion as do young adult rats. DESIGN: Adult (3 mo old) and old (22 mo old) rats were submitted to dietary restriction programs that induced similar metabolic and nutritional alterations. Malnourished adult and old rats were then killed (R groups) or refed for 1 wk with a high-protein diet (HPD; 23% protein) or a very-high-protein diet (VHPD; 27% protein). Control groups at both ages were fed ad libitum throughout the experiment. Effects of food repletion were evaluated in terms of protein metabolism, intestinal histomorphometry, and nonspecific immune status. RESULTS: In adult rats, HPD sufficed to increase body weight and restore basal values of liver weight and protein content (P: < 0.01 compared with the R adult group), nitrogen balance (P: < 0.01 compared with the R adult group), and hydrogen peroxide production by polymorphonuclear neutrophils and monocytes (P: < 0.01 compared with the R group); VHPD had no supplementary effect except on nitrogen balance. In old rats, HPD was less effective and greater benefit was observed with VHPD in terms of body weight gain (10%; P: < 0.01 compared with the old group fed HPD), albuminemia, muscle weight and protein content, plasma arginine concentration, and hydrogen peroxide production by stimulated polymorphonuclear neutrophils and monocytes compared with the old R group (P: < 0.01). CONCLUSION: Aging is a significant variable affecting the response to nutritional support.

Can a glutamate-enriched diet counteract glutamine depletion in endotoxemic rats?

The study evaluated whether a glutamate-enriched diet would restore glutamine tissue pools and maintain tissue trophicity in endotoxemic rats. For this purpose, young male Sprague-Dawley rats received an intraperitoneal injection of lipopolysaccharide (LPS) from Escherichia coli at 3 mg/kg body weight. After 24 hours of food deprivation, the rats were enterally refed for 48 hours using Osmolite enriched with glutamate at 4 g/kg/d (LPS-Glu group, n = 7) or glycine isonitrogenous to glutamate (LPS-Gly group, n = 7). A control group (healthy group, n = 7) had free access to a standard rodent diet. Tissue weights and protein contents were significantly lower in both LPS-treated groups than in the healthy group. No plasma or tissue accumulation of glutamate was observed except in the liver. Glutamine concentrations were increased in the jejunum, liver, and plasma in the LPS-Glu group versus the other two groups (P < 0.05). Conversely, they were depleted in muscles of the endotoxemic groups versus the healthy group (P < 0.05). Villus height was significantly greater in the LPS-Glu group than in the LPS-Gly group in the jejunum (P < 0.05), but not in the ileum. In conclusion, a glutamate-enriched diet administered enterally to endotoxemic rats can counteract glutamine depletion in the splanchnic area but not in muscles. In addition, glutamate displayed a trophic effect restricted to the jejunum.

Establishment and characterization of a new cell line derived from a human chondrosarcoma.

A new human cell line, CAL 78, derived from a dedifferentiated chondrosarcoma of the muscle of the thigh has been established in culture. Fibroblastoid morphology, vimentin expression and lack of epithelial antigens are in agreement with mesodermic origin of these cells. The xenograft of CAL 78 cells in nude mouse showed the characteristics of hyaline cartilaginous differentiation. Cytogenetic changes were numerous and complex, all the metaphases were tetraploid and no alterations described by other authors have been found. CAL 78 constitutes an appropriate model to evaluate efficiency of a new therapy for chondrosarcomas. Moreover, this cell line may be used to study some stage of chondrocytic differentiation.

Modulation of immune response with ornithine A-ketoglutarate in burn injury: an arginine or glutamine dependency?

Enterally administered ornithine alpha-ketoglutarate (OKG) is an efficient complement of nutritional support in trauma situations, especially after burn injury. A typical feature observed in this intense catabolic state is insufficient production of glutamine (Gln) and arginine (Arg), two amino acids (AAs) involved in the immune response. As OKG in vivo metabolism generates these two AAs, we investigated, in burned rats, the action of OKG with regard to modulation of immunity. Male Wistar rats were randomly allocated to four groups. On day 0, 12 rats were burned with boiling water (20% body surface area). After a 24-h fast, they were enterally refed for 48 h using Osmolite, as a low-calorie low-nitrogen regimen, supplemented with either 5 g OKG x kg(-1) x d(-1) (n = 6) or an equivalent amount of nitrogen in the form of glycine (n = 6). Non-burned pair-fed controls treated with glycine (n = 6) and healthy rats fed ad libitum (n = 6) were also studied. Nitrogen balance was assessed from daily measurement of total nitrogen excretion. On day 3, thymus, Anterior tibialis muscle and proximal jejunum weights were recorded. Muscle and intestinal AA concentrations were also quantified. OKG counteracted (P<0.01) the thymic involution that occurs with burn injury, and increased the concentrations of Gln and Arg in both the muscle (P<0.01 and P<0.05, respectively) and the jejunum (P<0.01 for Gln). When all groups were taken together, a positive correlation was found between thymus weight, and Gln and Arg muscle concentrations (r = 0.71, P<0.001 and r = 0.58, P<0.01, respectively). Furthermore, as expected, OKG improved nitrogen balance. As it is known that total number of thymocytes parallels thymic weight, and as Gln and Arg are essential nutrients for activated immune cells, our results suggest that Gln and Arg derived from OKG are responsible for the immunomodulating properties of this molecule in burn injury.

Supplementation of oral nutrition with pancreatic enzymes improves the nutritional status of aged endotoxemic rats.

Malnutrition is a common problem in elderly people. The association of malnutrition and physical illness or injury leads to both localized and general complications. In particular, impairment of the adaptive response of pancreatic function to undernutrition and refeeding may adversely affect nutritional status and elicit morbidity and mortality. Aged rats (24 mo old) were treated with lipopolysaccharide (LPS) from E. Coli (3 mg/kg body weight). Six days later, survivors were randomized to receive, for 7 days, an oral chow diet enriched with either a pancreatic extract (PE) (2.4 mg/day) or an isonitrogenous supply of casein (CAS). Endotoxemia induced a catabolic state, with a body weight loss of 7.6 +/- 1.1% on day two after LPS treatment. Mean food intake from day 6 to day 13 was similar in LPS-PE and LPS-CAS groups (19.0 +/- 5.6 versus 19.7 +/- 6.9 g). The metabolic response varied according to the type of muscle studied. In fast (white) muscle, the protein content and the glutamine pool remained markedly depleted in endotoxemic rats receiving casein supplementation. In contrast, enrichment of nutrition with PE significantly limited the LPS-induced muscle wasting and increased the muscle glutamine content. As in previous observations, no significant change occurred in slow (red) muscle. These results could indicate that PE supplementation counteracts pancreatic deficiency caused by aging and worsened by stress and this, in turn, could improve the efficiency of nutrition, to support the hypermetabolism of aged injured rats.

Protein metabolism in rats during long-term dietary restriction: influence of aging.

BACKGROUND: Protein depletion is frequent in the elderly, but the underlying mechanisms are not yet fully understood. In particular, it is unknown whether there is a defect of adaptation to a restriction of food intake in the elderly. This study was performed to compare the effects of 6-week dietary restriction (DR) on protein metabolism in both adult and aged rats. METHODS: Adult (3-month-old) and aged (22-month-old) rats were acclimatized for 2 weeks and then fed a standard diet for 6 weeks, either ad libitum (control adult [C(Adult)] and aged [C(Aged)] rats) or with only 50% of the average intake of the second week of acclimatization (restricted adult [R(Adult)] and aged [R(Aged)] rats). Protein metabolism, in terms of tissue protein content, nitrogen balance, and 3-methylhistidine (3-MH) urinary excretion, was evaluated. RESULTS: C(Adult) rats gained 30.4% of initial weight, whereas the body weight (BW) of C(Aged) rats was maintained. DR induced a rapid decrease in BW during the first 2 weeks in R(Adult) rats, but afterward BW remained stable. In R(Aged) rats, BW loss was linear during the 6 weeks and significantly higher than for R(Adult) rats (p<.01). In both restricted groups, muscle protein content was moderately affected by DR, whereas DR induced a marked decrease in visceral protein content. Nitrogen balance was decreased by DR but stayed positive in R(Adult) rats, whereas it became null in R(Aged) rats. CONCLUSIONS: In terms of protein metabolism, aged rats adapted less efficiently than adult rats to a long-term dietary restriction.

[Labelling index in breast cancer and in upper respiratory and digestive tract cancer (author's transl)]. / L'index de marquage dans les cancers du sein et des voies aéro-degestives supérieures.

The labelling index (LI) of 96 breast adenocarcinomas and of 43 upper respiratory and digestive tract cancers has been measured. For the two histological groups, the distribution of individual values is found to be log-normal, with a mean value depending on the tumour type. Our data have been compared with other authors results. The dispersion of individual values within a same histological type suggests that the LI is interesting as an individual value depending on the patient and his own cancer. For breast adenocarcinomas the LI values obtained have been separated in two groups depending upon the patient age, the age of 53 has been chosen to separate premenopausal from post-menopausal patients. A test of significance showed that the two corresponding log-normal distributions were different, even at the 0.01 level of significance, a mean LI value thus characterizing each distribution.

In vitro clonogenicity in relation to kinetic and clinicopathological features of breast cancer.

The clonogenicity in soft agar and the labeling index (LI) which represents the percentage of cells in the DNA synthesis phase, were studied in 59 breast cancer patients and these parameters were related to other known clinicopathological features, namely age, histological grading, estrogen and progesterone receptors and the status of axillary lymph nodes. Out of 59 tumors, 49 could be successfully cloned in soft agar and the mean plating efficiency (PE) was 0.1%. Low grade tumors were more frequently encountered in tumors which did not form colonies (P = 0.025). Cloned tumors had a higher mean LI (P = 0.05). A high PE was associated with low estrogen receptors (ER) (P = 0.03). Clonogenicity was not related to patient age, progesterone receptors (PR) or the status of axillary lymph nodes. These results suggest that a successful in vitro cloning and a high PE are associated with unfavorable prognostic factors in breast cancer.

[Characterization of a human cell line from an anaplastic carcinoma of the thyroid gland]. / Caractérisation d'une lignée cellulaire humaine provenant d'un carcinome anaplasique de la thyroïde.

A new cell line derived from a thyroid anaplastic carcinoma, CAL 62, has been established in culture. This line is constituted by highly tumorigenic cells. Their epithelial phenotype is stable in culture. Immunochemical staining for human thyroglobulin is negative. Cytogenetic analysis showed a gain of chromosome 20, the translocation i (14q), and breakpoints of centrometric chromatine. These results are similar to those previously reported by other investigators. CAL 62 radiosensibility has been studied. The survival curve of the in vitro irradiated cells has been adjusted with a linear-quadratic model. This cell line is thus showed to be radioresistant. Cell line CAL 62 constitutes an appropriate model for in vitro studies of thyroid anaplastic carcinoma.

[Viability of human cancer cells in culture after exposure to diagnostic ultrasound]. / Viabilité de cellules cancéreuses humaines en culture après exposition aux ultrasons émis par un appareil de diagnostic.

Reports have appeared in the literature regarding biological damage to human cells following exposure to diagnostic ultrasound. We have examined the effects of diagnostic ultrasound (Sonel 400, C.G.R.) on human cell lines established in our laboratory. We report here that exposure to diagnostic ultrasound, at maximum exposure intensity and at exposure time as long as 60 minutes, produces no cell lysis as determined by vital dye exclusion ability, and as confirmed by electron microscopy. However, the exposure to ultrasound produced by an apparatus delivering an acoustic power higher than the diagnostic levels (Sonoscope-Alcatel) can cause the complete lysis of the same cells.

Extracorporeal photochemotherapy induces arginase 1 in patients with graft versus host disease.

The benefits of extracorporeal photochemotherapy (ECP; psoralen and UVA exposure of blood mononuclear cells) in graft-versus-host-disease (GVHD) are well-recognized, but the mechanisms of action remain elusive. As the metabolism of l-arginine in immune cells is known to play a role in immune tolerance, we investigated the effect of ECP on arginine metabolism, and the influence of extracellular l-arginine concentration on the response to ECP in cells from patients on therapy by ECP for a GVHD and healthy donors cultured before and after ECP in the presence of different concentrations of arginine (0, 50, 100, 200 and 1000 µmol/l). At baseline arginine was not metabolized through the same pathway in patients and donors. When cells were exposed to ECP, the production of ornithine but not NO° was enhanced, while mRNA of arginase 1 was up-regulated but not INOS. In GVHD patients, increasing arginine concentration resulted in down-regulation of IFNγ and TNFα mRNA expression, whereas IL10 was up-regulated especially at physiological plasma levels (between 0 and 100 µM). Overall, our study shows that ECP orients the metabolism of arginine toward the arginase pathway together with shifting the cytokine profile toward IL-10, providing new insights into the enigmatic mechanism of action of ECP.

Ornithine alpha-ketoglutarate modulates tissue protein metabolism in burn-injured rats.

Enterally administered ornithine alpha-ketoglutarate (OKG) displays whole body anabolic and anticatabolic properties in trauma situations, especially after burn injury. The aim of this study was to get information about the anabolic effect of OKG at tissue level. Thirty-six male Wistar rats (95 +/- 7 g) were allocated to four groups. Eighteen rats were burned by water (20% body surface area). After a 24-h fast (day 0-day 1), rats were enterally refed for 48 h (day 1-day 3) by use of Osmolite as a low-calorie, low-nitrogen regimen supplemented with either 5 g OKG.kg-1.day-1 (B-OKG) or an equivalent amount of nitrogen in the form of glycine (B-Gly). Nonburned pair-fed controls treated with glycine (C-Gly) and healthy rats fed ad libitum were also studied. On day 3, protein synthesis rates (large dose method), free glutamine concentrations, and total protein content were assessed in tissues. Myofibrillar degradation was assessed by measuring urinary 3-methylhistidine excretion daily from day 0 to day 3. With regard to tissue protein synthesis rates, we demonstrate for the first time that OKG displays anabolic properties in the jejunum [fractional synthesis rate (FSR) in %/day, ad libitum = 101.9 +/- 4.0; C-Gly = 84.7 +/- 3.1, P < 0.01 vs. ad libitum; B-Gly = 84.5 +/- 1.6, P < 0.01 vs. ad libitum; B-OKG = 97.5 +/- 3.2, P < 0.05 vs. C-Gly and B-Gly] as well as in the liver (FSR in %/day, ad libitum = 75.9 +/- 3.7; C-Gly = 53.2 +/- 3.8, P < 0.01 vs. ad libitum; B-Gly = 70.2 +/- 2.0, P < 0.01 vs. C-Gly; B-OKG = 98.7 +/- 4.6, P < 0.01 vs. ad libitum, C-Gly and B-Gly), the latter having previously been observed in vitro. Furthermore, we confirm that OKG inhibits myofibrillar degradation, counteracts the trauma-induced fall of muscle glutamine pool, and induces an increase in glutamine concentration in the jejunum.

Free amino acid concentrations in milk: effects of microwave versus conventional heating.

Microwave effects on free amino acid concentrations in milk versus a water bath heating were investigated in view of their importance for infant growth. Concentrations of few amino acids, such as aspartate, serine or lysine, are unchanged whatever the way and the temperature of heating. In contrast, tryptophan concentrations decreased similarly whatever the way of heating (110 +/- 3 mumol/l before heating vs 84 +/- 4 mumol/l after 30 degrees C microwave heating, p < 0.05). On the contrary, concentrations of glutamate and glycine increased more after water bath heating at 90 degrees C (325 +/- 4 and 101 +/- 1 mumol/1, respectively) than after microwave heating (312 +/- 4 and 95 +/- 1 mumol/l, respectively, p < 0.05) suggesting milk proteolysis. Moreover, the accumulation of ammonia observed at 90 degrees C with the water bath together with increase Glu levels might reflect a degradation of glutamine. An ornithine enrichment, more evident with microwave heating, was shown and could be of interest as it is a polyamine precursor. Also, considering few variations of free amino acid concentrations and the time saved, microwave heating appears to be an appropriate method to heat milk.

Thymidine labeling index and estrogen receptor level in 64 human breast cancers.

Concurrent assays of labeling index (LI) and estrogen receptors (ER) have been performed in 64 human breast cancers. The dipping method of autoradiography has been used for measurements of LI. The LI values are given as a number per hundred of cell nuclei labeled by tritiated thymidine. ER levels were determined by dextran-coated charcoal method followed by Scatchard analysis, and expressed in femtomoles per milligram of cytosol protein. Tumours with ER greater than or equal to 28 fmol/mg were demonstrating a low LI (less than 2.3%) and come all from patients of age over 50 years. LI and ER values have been compared with clinical data. The best clinical outcome have been found among patients with low LI values (less than 2.3%). Patients with borderline ER values (3 fmol/mg less than ER less than 28 fmol/mg) showed a satisfactory clinical outcome in 88% of cases when LI less than 2.3% and 57% of cases when LI greater than or equal to 2.3%.

Brain GABA editing by localized in vivo (1)H magnetic resonance spectroscopy.

Editing of GABA by (1)H MRS in a specific brain area is a unique tool for in vivo non-invasive investigation of neurotransmission disorders. Selective GABA detection is achieved using sequences based on double quantum coherence (DQC). Our pulse sequence makes accurate measurements without artefacts due to spatial localization. The sequence was tested on a phantom solution. The effect of vigabatrin, a specific inhibitor of GABA transaminase, was measured in rat brain and GABA detection was performed in vivo in monkey brain using this procedure. Rats were split into two groups. In the control group, the rats had access to water and, in the other group (vigabatrin, VGB, rats), animals were allowed free access to drinking water containing vigabatrin. After 3 weeks of treatment, rats were anesthetized for in vivo NMR spectroscopy investigation. At the end of the experiment, brains were quickly removed, freeze-clamped and extracted with 4% perchloric acid. One part of the acid extract was used for GABA concentrations assessment by ion exchange chromatography with ninhydrin detection. The second was used for high-resolution NMR analysis. By chromatography measurements, the GABA concentration was 1.23+/-0.06 micromol/g for controls, while for vigabatrin-treated rats the GABA concentration was 4.89+/-1.60 micromol/g. The NMR in vivo results were closely correlated with the NMR ex vivo (r=0.99, p<0.01) and chromatography results (r=0.98, p<0.01). The correlation between ex vivo results and chromatography results was also high (r=0.99, p<0.001). This pulse sequence performed GABA editing from a 376 microl voxel located on the right basal ganglia area in a non-human primate brain. This in vivo GABA editing scheme can thus be proposed for accurate measurement of brain GABA concentrations.