Subversion of human intestinal mucosa innate immunity by a Crohn's disease-associated E. coli.

Adherent-invasive Escherichia coli (AIEC), associated with Crohn's disease, are likely candidate contributory factors in the disease. However, signaling pathways involved in human intestinal mucosa innate host response to AIEC remain unknown. Here we use a 3D model of human intestinal mucosa explant culture to explore the effects of the AIEC strain LF82 on two innate immunity platforms, i.e., the inflammasome through evaluation of caspase-1 status, and NFκB signaling. We showed that LF82 bacteria enter and survive within a few intestinal epithelial cells and macrophages, without altering the mucosa overall architecture. Although 4-h infection with a Salmonella strain caused crypt disorganization, caspase-1 activation, and mature IL-18 production, LF82 bacteria were unable to activate caspase-1 and induce IL-18 production. In parallel, LF82 bacteria activated NFκB signaling in epithelial cells through IκBα phosphorylation, NFκBp65 nuclear translocation, and TNFα secretion. In addition, NFκB activation was crucial for the maintenance of epithelial homeostasis upon LF82 infection. In conclusion, here we decipher at the whole-mucosa level the mechanisms of the LF82-induced subversion of innate immunity that, by maintaining host cell integrity, ensure intracellular bacteria survival.

[Maternal virilization due to mucinous ovarian tumour: a case report]. / Tumeur ovarienne mucineuse virilisante et grossesse: à propos d'un cas.

Virilization in pregnancy is rare and mostly due to luteoma or to hyper-reactio luteinalis. We present a rare case of a virilization borderline mucinous ovarian tumour on a gravida 1 patient. The tumour was responsible for a clinical hyperandrogenism and for an increased level of testosterone. This patient was treated by ovariectomy at 31 weeks of gestation. The surgery was completed one month after delivery. There was no fetal consequence and the clinical and biological signs of virilization totally disappeared after surgery.

[Extra-uterine pelvic leiomyoma: diagnosis and practical management]. / Léiomyome pelvien extra-utérin: diagnostic et prise en charge.

OBJECTIVES: To analyze the possibilities of setting up a therapy for extra-uterine pelvic leiomyomas. METHODS: Three cases of leiomyomas of the broad ligament, of the round ligament and of the ovary, and literature review. RESULTS: Little is known about physiopathology of extra-uterine leiomyoma. The diagnosis of extra-uterine leiomyoma is based on histopathological analysis, using standard histology, and immunohistochemistry with anti-desmin and anti smooth muscle actin antibodies. The main differential diagnoses are fibroma, fibrothecoma, ovarian fibrosarcoma, and gastrointestinal stromal tumors. To define criteria of malignancy, we use Bell's classification without being sure that the uterine and extra-uterine models are comparable. So there is a risk of ignoring a low grade leiomyosarcoma. Providing therapy depends on the clinicopathologic features: the so called "parasitic leiomyoma", a tumor developed at the expense of local smooth muscle cells, metastasis of a benign metastasizing leiomyoma or leiomyomatosis peritonealis disseminata. CONCLUSION: The extra-uterine leiomyoma has no precise nosologic status and no specific criteria of benignity; thus no precise evolution can be predicted. We must be extremely careful, and the issue of the monitoring and long-term therapy of patients must come up.

Altered Calreticulin expression in human colon cancer: maintenance of Calreticulin expression is associated with mucinous differentiation.

Calreticulin is an endoplasmic reticulum luminal calcium-binding chaperone involved in various cellular functions and is a ligand for the scavenger receptor CD91. Recent studies, based on proteomic approaches on whole tissue samples containing both neoplastic and non-neoplastic cells, have shown alterations of Calreticulin expression in colon carcinomas, albeit with divergent results. The aims of this study were: 1) to assess the expression of Calreticulin and its receptor CD91 in 58 human colon adenocarcinomas, compared with paired normal mucosa, using a semi-quantitative immunohistochemical analysis, and 2) to examine associations between the tumour phenotypic features, and Calreticulin and/or CD91 expressions. Calreticulin expression was down-regulated in 51.7% human colon adenocarcinomas. Accordingly, quantitative immunoblot analysis showed that Calreticulin expression was significantly lower in human colonic cancer cell lines than in preparations of isolated human normal colonic epithelial cells. CD91 was co-expressed with Calreticulin in both normal colonic epithelial cells and pericryptic myofibroblasts. Calreticulin and CD91, that characterize the 'amateur phagocyte' function of epithelial cells, were both down-regulated in 48% of adenocarcinomas. Finally, Calreticulin expression was significantly associated with the mucinous differentiation of the tumour. Collectively, these results show that Calreticulin is likely to play a pivotal role in the differentiation of human colonic adenocarcinomas.

[Interest of selective lymphadenectomy in patients with vulvar cancer]. / Intérêt de la technique de lymphadénectomie sélective dans les cancers de vulve.

Vulvar cancer represents approximately 4% of all gynecologic malignancies and the most important prognosis factor in this cancer is the status of the regional lymph nodes. The radical inguinal lymphadenectomy, associated or not with radiotherapy, is accompanied by high morbidity, which can affect 50% of the patients. The sentinel node detection appears now to be feasible in patients with vulvar carcinoma, in order to reduce the morbidity of inguinal lymphadenectomy. But contrary to breast cancer, the learning curve is not easy to obtain because of the low number of cases. That is why we have described the procedure of selective lymphadenectomy. The aim of this technique is to remove the blue and/or marked inguinal lymph node and any other palpable lymph node, without a real radical inguinal lymphadenectomy. Thus, since November 2003, 4 procedures have been performed in total. With the lymphoscintigraphy, we identified 17 marked lymph node and we finally obtained 28 lymph nodes after surgery, with only one metastatic lymph node. There was no complication after our procedure. Selective lymphadenectomy appears to be a new procedure which may reduce the morbidity of usual inguinal lymphadenectomy.

Methods for detecting internalized, FM 1-43 stained particles in epithelial cells and monolayers.

The membrane dye FM 1-43 has frequently been used to quantify exocytosis in neurons. In epithelia, intense lateral intracellular space staining and fluctuations in baseline labeling produced inconsistent results. Membrane retrieved in the presence of FM 1-43 retains the dye, however, and cells that undergo compensatory endocytosis during and following evoked exocytosis contain punctate, fluorescent particles after washout of external stain. As an alternative measure of trafficking, we quantified the fluorescent puncta retained after dye washout and tested our method on both coverslip-grown cell clusters and filter-grown intact monolayers. Images for analysis were acquired using serial sectioning with either epifluorescence or confocal microscopy. Tests with an intestinal goblet cell line that exhibits basal and ATP-stimulated granule trafficking confirmed that 1), the algorithm identified the same number of internalized particles with either epifluorescence or confocal microscopy acquired images; 2), low density clusters exhibited significantly more internalized particles per cell than either filter-grown monolayers or high density clusters; 3), ATP stimulation significantly increased the number of internalized particles in all preparations; and 4), the number of particles internalized was comparable to capacitance measurements of exocytosis. This method provides a single technique for quantifying membrane trafficking in both monolayers and unpolarized cells.

Phosphohistone H3 labelling for histoprognostic grading of breast adenocarcinomas and computer-assisted determination of mitotic index.

BACKGROUND: Microscopic evaluation of mitotic figures is a routine procedure in the assessment of the histoprognostic grade of tumours. Nevertheless, their count may be fraught with difficulties. As histone H3 phosphorylation at serine 10 is closely linked to chromosomal condensation, a new monoclonal antibody directed to phosphorylated histone H3 (PPH3) was recently proposed to detect mitotic cells. AIM: To test the reliability of this antibody in detecting and counting mitotic figures in sections of breast adenocarcinomas, because of the importance of mitotic count in histoprognostic grading. METHODS: The pattern of PPH3 staining in formalin-fixed paraffin wax-embedded tissues, including normal tissues and a series of 39 breast adenocarcinomas, was examined. A new computer-assisted method was also developed for determining the mitotic index. RESULTS AND CONCLUSIONS: In all tissues tested, PPH3-labelled mitotic figures were easily detected, allowing a rapid identification of the area of highest mitotic activity. In breast carcinomas, a strong correlation was observed between PPH3-stained and haematoxylin and eosin-stained mitotic counts (r = 0.86, p<0.0001). Counting of prophase nuclei that coexpress cyclin B1, a marker of the G2/M phase, was possible by PPH3 staining; its accuracy led us to reconsider the tumour grade in three cases. Finally, an automatic computer-assisted method was designed for assessing mitotic index with confocal microscopy and image-analysis software.

Antibiotic therapy reduces nitrosative stress and programmed cell death in the rabbit foetal lung.

The correlation of clinical and epidemiological data suggests that intrauterine infection/inflammation can promote foetal lung injury. The aim of this study was: 1) to characterise the early inflammatory response elicited in infected foetal lungs, in terms of nitric oxide-derived oxidative stress and programmed cell death; and 2) to investigate the effects of antibiotic therapy on these parameters. A previously described rabbit experimental model of materno-foetal infection was used. Animals were divided into three groups: controls; Escherichia coli infected (12 h); and E. Coli infected (12 h) and treated (24 h gentamicin+ceftriaxone). Foetal lungs were examined in terms of histology, nitric oxide synthase (NOS) activity, immunohistochemical detection of 3-nitrotyrosine, and detection of apoptotic cells by the TUNEL assay and Hoechst staining. In the infected group, a moderate inflammatory response was observed, associated with a significant increase in inducible NOS activity, the formation of 3-nitrotyrosine residues in epithelial and immune cells, the down-regulation of constitutive NOS activity and clusters of apoptotic cells, as compared with the control group. Early antibiotic therapy, initiated at 12 h post-inoculation, elicited a significant decrease in the infection-induced nitrosative stress. Levels of 3-nitrotyrosine and of apoptotic cells were decreased in the infected-and-treated group compared with the infected group, mainly by the re-expression of constitutive NOS and of the basal level of inducible NOS. Altogether, these findings indicate that early antibiotic therapy can curb the inflammatory reaction and help avert antenatal lung injury, which is known to be involved in the onset of bronchopulmonary dysplasia.

Butyrate specifically modulates MUC gene expression in intestinal epithelial goblet cells deprived of glucose.

The mucus layer covering the gastrointestinal mucosa is considered the first line of defense against aggressions arising from the luminal content. It is mainly composed of high molecular weight glycoproteins called mucins. Butyrate, a short-chain fatty acid produced during carbohydrate fermentation, has been shown to increase mucin secretion. The aim of this study was to test 1) whether butyrate regulates the expression of various MUC genes, which are coding for protein backbones of mucins, and 2) whether this effect depends on butyrate status as the major energy source of colonocytes. Butyrate was provided at the apical side of human polarized colonic goblet cell line HT29-Cl.16E in glucose-rich or glucose-deprived medium. In glucose-rich medium, butyrate significantly increased MUC3 and MUC5B expression (1.6-fold basal level for both genes), tended to decrease MUC5AC expression, and had no effect on MUC2 expression. In glucose-deprived medium, i.e., when butyrate was the only energy source available, MUC3 and MUC5B increase persisted, whereas MUC5AC expression was significantly enhanced (3.7-fold basal level) and MUC2 expression was strikingly increased (23-fold basal level). Together, our findings show that butyrate is able to upregulate colonic mucins at the transcriptional level and even better when it is the major energy source of the cells. Thus the metabolism of butyrate in colonocytes is closely linked to some of its gene-regulating effects. The distinct effects of butyrate according to the different MUC genes could influence the composition and properties of the mucus gel and thus its protective function.

Loss of NOS1 expression in high-grade renal cell carcinoma associated with a shift of NO signalling.

In normal human kidney, NOS1 and soluble guanylate cyclase (sGC) are expressed in tubular epithelial cells, suggesting a physiological autocrine NO signalling pathway. Therefore, we investigated both NOS1 and sGC expressions in benign and malignant renal tumours. In addition, we examined the pattern of protein tyrosine nitration in normal and tumour tissue. NOS1 expression and activity were found to be downregulated, correlating with the tumour grade, as shown by immunohistochemistry, quantitative RT-PCR analysis, and histochemical detection of the NADPH-diaphorase activity of nitric oxide synthases (NOS). These results show that the autocrine NO signalling pathway is maintained in benign tumours and lost in malignant tumours. In contrast, sGC expression was maintained in renal tumours whatever the tumour type, a finding showing that tumour cells remain sensitive to the bioregulatory role of exogeneous NO(*). Finally, the staining pattern of protein tyrosine nitration, assessed by immunohistochemistry, parallelled that of NOS1 expression in normal renal parenchyma and benign tumours, supporting the concept that protein nitration was accounted for by NOS1 activity. In contrast, in malignant tumours, protein tyrosine nitration was accounted for by the production of reactive nitrogen oxide species by the inflammatory infiltrate. Altogether, these findings argue for a pattern of NO signalling similar in normal kidney and benign renal tumours, whereas it is completely different in malignant renal tumours.

Niflumic acid inhibits ATP-stimulated exocytosis in a mucin-secreting epithelial cell line.

ATP is an efficacious secretagogue for mucin and chloride in the epithelial cell line HT29-Cl.16E. Mucin release has been measured as [3H]glucosamine-labeled product in extracellular medium and as single-cell membrane capacitance increases indicative of exocytosis-related increases in membrane area. The calcium-activated chloride channel blocker niflumic acid, also reported to modulate secretion, was used to probe for divergence in the purinergic signaling of mucin exocytosis and channel activation. With the use of whole cell patch clamping, ATP stimulated a transient capacitance increase of 15 +/- 4%. Inclusion of niflumic acid significantly reduced the ATP-stimulated capacitance change to 3 +/- 1%, although normalized peak currents were not significantly different. Ratiometric imaging was used to assess intracellular calcium (Cai2+) dynamics during stimulation. In the presence of niflumic acid, the ATP-stimulated peak change in Cai2+ was unaffected, but the initial response and overall time to Cai2+ peak were significantly affected. Excluding external calcium before ATP stimulation or including the capacitative calcium entry blocker LaCl3 during stimulation muted the initial calcium transient similar to that observed with niflumic acid and significantly reduced peak capacitance change, suggesting that a substantial portion of the ATP-stimulated mucin exocytosis in HT29-Cl.16E depends on a rapid, brief calcium influx through the plasma membrane. Niflumic acid interferes with this influx independent of a chloride channel blockade effect.

Human submucosal neurones regulate intestinal epithelial cell proliferation: evidence from a novel co-culture model.

The role of the human enteric nervous system (ENS) in the control of the intestinal epithelium organization and proliferation is unknown. To address this issue, we developed a novel co-culture model, consisting of human submucosa containing the submucosal plexus and a human colonic epithelial monolayer. After 3 days in basal conditions (i.e. in absence of neuronal activation) epithelium disorganization and proliferation occurred. In contrast, electrical activation of submucosal neurones maintained monolayer organization and decreased cell proliferation. These effects were blocked by tetrodotoxin and a vasoactive intestinal peptide (VIP) receptor antagonist, and reproduced by VIP. In conclusion, our study suggests that the human ENS is involved in the control of epithelial cell proliferation.

Generation of heme oxygenase-1-transgenic rats.

Heme oxygenase-1 (HO-1) expression protects cells from a variety of cellular insults and inhibits inflammation. However, its role in the regulation of immune responses has not yet been clearly established. We generated HO-1 transgenic rats to directly test the impact of HO-1 on the different immune mechanisms. To temporally control the expression of HO-1, we used a one-plasmid tetracycline (tet)-inducible system. This plasmid contains the H-2K(b) promoter, which transcribes the tet transactivator (tTA) and expression of a human HO-1 cDNA is obtained in the absence of tetracycline. The DNA construct was microinjected into one-cell rat embryos and mothers and pups were maintained with tetracycline. Eight transgenic founders were obtained. Analysis of transgene expression in the absence of tet showed that 2 lines (12.4 and 12.6) expressed HO-1 mRNA in several organs (as detected by reverse transcription polymerase chain reaction) and at the protein level only in the thymus. Expression levels of transgene-derived HO-1 increased after withdrawal of tet compared with transgenic rats maintained with tet, as detected by analysis of mRNA levels by quantitative real-time reverse transcription polymerase chain reaction. Gross examination and histopathological analysis of several organs in both lines showed no anomalies. Thymocytes and splenocytes of both lines showed normal cell subpopulations and allogeneic proliferation compared with controls. Systemic immune responses against cognate antigens were normal in both lines, as evaluated by the proliferation of lymph node cells and the production of antibodies against keyhole limpet hemocyanin after immunization. Animals from line 12.6 rejected transplanted allogeneic hearts with the same kinetics as controls. In conclusion, short-term induction of HO-1 overexpression did not modify immune responses compared to those of control non-transgenic animals.

Effects of glutamine deprivation on protein synthesis in a model of human enterocytes in culture.

To assess the effect of glutamine availability on rates of protein synthesis in human enterocytes, Caco-2 cells were grown until differentiation and then submitted to glutamine deprivation produced by exposure to glutamine-free medium or methionine sulfoximine [L-S-[3-amino-3-carboxypropyl]-S-methylsulfoximine (MSO)], a glutamine synthetase inhibitor. Cells were then incubated with (2)H(3)-labeled leucine with or without glutamine, and the fractional synthesis rate (FSR) of total cell protein was determined from (2)H(3)-labeled enrichments in protein-bound and intracellular free leucine measured by gas chromatography-mass spectrometry. Both protein FSR (28 +/- 1.5%/day) and intracellular glutamine concentration (6.1 +/- 0.6 micromol/g protein) remained unaltered when cells were grown in glutamine-free medium. In contrast, MSO treatment resulted in a dramatic reduction in protein synthesis (4.6 +/- 0.6 vs. 20.2 +/- 0.8%/day, P < 0.01). Supplementation with 0.5-2 mM glutamine for 4 h after MSO incubation, but not with glycine nor glutamate, restored protein FSR to control values (24 +/- 1%/day). These results demonstrate that in Caco-2 cells, 1) de novo glutamine synthesis is highly active, since it can maintain intracellular glutamine pool during glutamine deprivation, 2) inhibition of glutamine synthesis is associated with reduced protein synthesis, and 3) when glutamine synthesis is depressed, exogenous glutamine restores normal intestinal FSR. Due to the limitations intrinsic to the use of a cell line as an experimental model, the physiological relevance of these findings for the human intestine in vivo remains to be determined.

Proinflammatory role of leptin in experimental colitis in rats benefit of cholecystokinin-B antagonist and beta3-agonist.

Leptin, a hormone primarily secreted from adipocytes, plays a key role in controlling body weight homeostasis. In vitro studies indicate that it is also implicated in immune responses. Hyperleptinaemia has been reported in acute inflammation, especially during the early stages of intestinal inflammation in rats. The present study investigated the possible role of leptin in the pathogenesis of trinitrobenzene sulfonic acid (TNBS)-induced colitis in rats. Since no specific antagonist of leptin is available, a CCK-B antagonist (YM022) and a beta3 agonist (BRL37344) were used in this study to inhibit leptin secretion. Colitis was induced by intracolonic instillation of TNBS in rats. Five TNBS-groups were subcutaneously implanted with micropumps containing: placebo, YM022, BRL37344, BRL37344 and exogenous leptin simultaneously, or leptin alone. At sacrifices, colitis severity was assessed by macroscopic and histological scoring systems and by determination of tissue myeloperoxidase activity. The TNBS-induced hyperleptinaemia was significantly reduced by YM022 and BRL37344 (p<0.05). Inhibition of leptin secretion markedly reduced colonic inflammation, whatever the criteria considered (i.e. macroscopic, histological or biochemical). In contrast, administration of exogenous leptin completely abolished the beneficial effect of leptin-lowering drugs on colitis severity. These results provide the first direct evidence for an important deleterious role of leptin in the pathogenesis of experimental intestinal inflammation and suggest that a pro-inflammatory activity is attributable to leptin in vivo. Further studies are required to determine if these results have clinical significance.

Overexpression of the CD155 gene in human colorectal carcinoma.

BACKGROUND AND AIMS: The Tage4 gene (tumour associated glycoprotein E4) is overexpressed in rat colon tumours and Min mouse intestinal adenomas. The rat Tage4 protein has approximately 40% identity with human CD155, a member of the immunoglobulin superfamily coding for a transmembrane protein capable of serving as an entry receptor for poliovirus, porcine pseudorabies virus, and bovine herpesvirus 1. Analysis of the rat Tage4 gene has revealed structural and functional similarities with the human CD155 gene. We therefore investigated expression of the CD155 gene in human colorectal carcinomas. METHODS: Overall CD155 expression was assessed by semiquantitative reverse transcription-polymerase chain reaction (RT-PCR) and immunohistochemical analysis using tissue specimens from patients with colorectal adenomas and adenocarcinomas. We also used a qualitative RT-PCR assay to determine relative expression of different splicing variants in each sample. RESULTS: mRNA levels of CD155 were increased in six of six colorectal cancer tissues compared with the tumour free colon mucosa. Immunohistochemical analysis revealed an increased level of CD155 protein in 12 of 12 samples. The qualitative RT-PCR assay revealed that relative expression of the different CD155 variant transcripts was similar in the different normal and cancer samples tested, indicating that this overexpression is not associated with a particular mRNA variant generated by alternative splicing of the CD155 gene. CONCLUSION: We have shown for the first time that the CD155 gene is overexpressed in colorectal carcinoma and that this overexpression begins at an early stage in tumorigenesis and continues to late stages.

Listeriolysin O-induced stimulation of mucin exocytosis in polarized intestinal mucin-secreting cells: evidence for toxin recognition of membrane-associated lipids and subsequent toxin internalization through caveolae.

Lysteriolysin O (LLO) induces a microtubule-dependent activation of mucin exocytosis in the human mucin-secreting HT29-MTX. Cholesterol inhibits the LLO-induced mucin exocytosis, whereas the oxidized form of cholesterol had no inhibitory effect. LLO-induced mucin exocytosis inhibited by cholesterol can be restored by enzymatic treatment with cholesterol oxidase. Inhibition of cholesterol synthesis in HT29-MTX cells results in a decrease in the LLO-induced mucin exocytosis. Other lipids such as gangliosides are able to inhibit the LLO-induced mucin exocytosis, suggesting that the binding of the toxin occurs at a multiplicity of membrane-associated lipids acting as receptors. Incubation of the toxin with lipids such as cholesterol or gangliosides does not decrease binding of LLO to target membranes. The present work also provides evidence that the LLO-induced mucin exocytosis develops independently of the pore-forming activity of the toxin. Finally, we demonstrated that the toxin associates with detergent-insoluble glycolipid microdomains (DIGs) containing VIP/21 caveolin, allowing internalization of the toxin and subsequent activation of the mucin exocytosis.

[Protective effect of an apoptosis inhibitor in a new model of hepatitis induced by interleukin-4 in the rat]. / Effet protecteur d'un peptide inhibiteur de l'apoptose dans un nouveau modèle d'hépatite induite par l'interleukine-4 chez le rat.

OBJECTIVES: Interleukin-4 is a cytokine with pleiotropic effects on many cells. The effects of its expression on the liver remain unclear. To obtain organ-localized cytokine expression and analyze its effect on the liver, recombinant adenovirus with coding sequences of interleukin-4 were transduced to rat livers. METHODS: Adenovirus with coding sequences of rat interleukin-4 were injected into the portal vein of Wistar rats. Microscopic examination of the liver was performed. The effects of interleukin-4 were confirmed in vitro on primary cultured rat hepatocytes. The same analysis was performed after intraperitoneal injection of l'YVADcmk, an inhibitor of the interleukin 1 converting enzyme. RESULTS: Interleukin-4 expression due to the recombinant adenovirus produced dose-related, potentially lethal, severe hepatitis. This hepatitis was characterized by a leucocyte infiltrate mainly composed of eosinophilic polymorphonuclear and mast cells with numerous apoptotic hepatocytes. Intraperitoneal injection of YVADcmk decreased hepatocyte apoptosis and biological hepatitis and prevented death. CONCLUSION: These results suggested that YVADcmk might be used in fulminant hepatitis in which apoptosis is predominant.

The in vitro manipulation of carbohydrate metabolism: a new strategy for deciphering the cellular defence mechanisms against nitric oxide attack.

This study was aimed at examining the effects of manipulating the carbohydrate source of the culture medium on the cellular sensitivity of epithelial cells to an oxidative attack. Our rationale was that substituting galactose for glucose in culture media would remove the protection afforded by glucose utilization in two major metabolic pathways, i.e. anaerobic glycolysis and/or the pentose phosphate pathway (PPP), which builds up cellular reducing power. Indeed, we show that the polarized human colonic epithelial cell line HT29-Cl.16E was sensitive to the deleterious effects of the NO donor PAPANONOate [3-(2-hydroxy-2-nitroso-1-propylhydrazino)-1-propanamine] only in galactose-containing medium. In such medium NO attack led to cytotoxic and apoptotic cell death, associated with formation of derivatives of NO auto-oxidation (collectively termed NOx) and peroxynitrite, leading to intracellular GSH depletion and nitrotyrosine formation. The addition of 2-deoxyglucose, a non-glycolytic substrate, to galactose-fed cells protected HT29-Cl. 16E cells from NO attack and maintained control GSH levels through its metabolic utilization in the PPP, as shown by (14)CO(2) production from 2-deoxy[1-(14)C]glucose. Therefore, increasing the availability of reducing equivalents without interfering with energy metabolism is able to prevent NO-induced cell injury. Finally, this background provides the conceptual framework for establishing nutritional manipulation of cellular metabolic pathways that could provide new means for (i) deciphering the mechanisms of cell injury by reactive nitrogen species and reactive oxygen species at the whole-cell level and (ii) establishing the hierarchy of intracellular defence mechanisms against these attacks.