Atazanavir-induced unconjugated hyperbilirubinemia prevents vascular hyporeactivity during experimental human endotoxemia.

Objective: Inflammation-induced free radical release is important in the pathogenesis of several diseases, including atherosclerosis and sepsis. Heme oxygenase (HO) breaks down heme into carbon monoxide, iron, and biliverdin. Biliverdin IXα is directly converted to bilirubin by biliverdin reductase. Unconjugated bilirubin is a powerful antioxidant, and elevated levels have beneficial effects in preclinical models and human cardiovascular disease. However, its impact during acute inflammation in humans is unknown. In the present study, we investigated the impact of atazanavir-induced (unconjugated) hyperbilirubinemia on antioxidant capacity, inflammation, and vascular dysfunction in human experimental endotoxemia. Approach and results: Following double-blinded four-day treatment with atazanavir 2dd300 mg (or placebo), twenty healthy male volunteers received 2 ng/kg Escherichia coli lipopolysaccharide intravenously. Blood was drawn to determine the bilirubin levels, antioxidant capacity, and cytokine response. It was demonstrated that following atazanavir treatment, total bilirubin concentrations increased to maximum values of 4.67 (95%CI 3.91-5.59) compared to 0.82 (95%CI 0.64-1.07) mg/dL in the control group (p<0.01). Furthermore, the anti-oxidant capacity, as measured by the ferric-reducing ability of plasma (FRAP), was significantly increased with 36% in hyperbilirubinemia subjects (p<0.0001), and FRAP concentrations correlated strongly to bilirubin concentrations (R2 = 0.77, p<0.001). Hyperbilirubinemia attenuated the release of interleukin-10 from 377 (95%CI 233-609) to 219 (95%CI 152-318) pg/mL (p=0.01), whereas the release of pro-inflammatory cytokines remained unaltered. In vitro, in the absence of hyperbilirubinemia, atazanavir did not influence lipopolysaccharide-induced cytokine release in a whole blood assay. Vascular function was assessed using forearm venous occlusion plethysmography after intra-arterial infusion of acetylcholine and nitroglycerin. Hyperbilirubinemia completely prevented the LPS-associated blunted vascular response to acetylcholine and nitroglycerin. Conclusions: Atazanavir-induced hyperbilirubinemia increases antioxidant capacity, attenuates interleukin-10 release, and prevents vascular hyporesponsiveness during human systemic inflammation elicited by experimental endotoxemia. Clinical trial registration: http://clinicaltrials.gov, identifier NCT00916448.

The Antiseptic and Antineoplastic Agent Taurolidine Modulates Key Leukocyte Functions.

BACKGROUND/AIM: Although taurolidine is known to exert a wide spectrum of biological actions, its effects on immune cells have not been characterized in detail. In this study, we investigated the ex vivo effects of taurolidine on relevant innate and adaptive immune cell functions. MATERIALS AND METHODS: Leukocyte functions in whole blood were assessed following treatment with various taurolidine concentrations. Viability of peripheral blood mononuclear cells (PBMCs) and granulocytes was measured using the WST-1 assay. PBMC function was assessed by measuring TNFα and IFNγ production after stimulation with lipopolysaccharide (LPS) or Candida, respectively. Reactive oxygen species (ROS) production by granulocytes was measured in whole blood using luminol-enhanced chemiluminescence. Granulocyte degranulation and activation were evaluated by membrane expression of degranulation (CD63, CD66B) and adhesion markers (CD62L, CD11b) using immunofluorescent staining followed by flow-cytometric analysis. RESULTS: Taurolidine decreased viability of PBMCs and granulocytes: after 2 h, IC50 concentrations were 500 and 520 µg/ml, respectively. Following prolonged exposure (≥24 h) of PBMCs, the IC50 concentrations declined to 40 µg/ml. PBMC cytokine production significantly decreased at taurolidine concentrations below the cytotoxic threshold, whereas no changes in ROS production were observed. The expression of all granulocyte adhesion and degranulation markers increased at concentrations higher than 500 µg/ml (the cytotoxic level of taurolidine). CONCLUSION: Taurolidine exhibits a dose- and time-dependent cytotoxicity toward PBMCs and granulocytes. The effects on PBMCs, as exemplified by a decrease in cytokine production, occurred below the toxic threshold, whereas granulocyte function (ROS production) remained unchanged at these taurolidine concentrations. Granulocyte activation and degranulation markers only increased at cytotoxic taurolidine concentrations.

Contribution of Genetic and Clinical Risk Factors to Development of Candidemia in Patients Receiving Home Parenteral Nutrition.

BACKGROUND: Patients receiving home parenteral nutrition (HPN) have an increased risk for central line-associated bloodstream infections (CLABSIs), including candidemia. Recently, 7 single-nucleotide polymorphisms (SNPs) in TLR1, CD58, LCE4A-Clorf68, and TAGAP have been associated with the development of candidemia. Identification of host-genetic as well as clinical risk factors may help to identify patients who have an increased susceptibility to such infections. The aim of this study was to investigate the relevance of the reported SNPs in patients receiving HPN, and to explore clinical risk factors associated with candidemia. METHODS: We analyzed blood samples of adult patients who started HPN between 1976 and 2017 at our referral center for intestinal failure. Primary outcome was the association between TLR1, CD58, LCE4A-Clorf68, or TAGAP SNPs and candidemia. Secondary outcomes included the relation between severity of infection and these SNPs, and clinical risk factors for candidemia. RESULTS: Of 341 included patients, 42 (12%) experienced a candidemia (range 1-6). None of the 7 SNPs were associated with candidemia or the severity of infection. The rate of non-Candida-related CLABSIs was significantly associated with candidemia (rate ratio, 1.29; 95% CI, 1.14-1.46; P < 0.001). CONCLUSIONS: None of 7 known SNPs in TLR1, CD58, LCE4A-Clorf68, or TAGAP were associated with candidemia or severity of infection in patients receiving HPN. The rate of non-Candida-related CLABSIs was significantly associated with the development of candidemia. The latter supports the key role of aseptic catheter handling with respect to Candida susceptibility in patients receiving HPN.

Droplet digital polymerase chain reaction for rapid broad-spectrum detection of bloodstream infections.

The droplet digital polymerase chain reaction (ddPCR) is a novel molecular technique that allows rapid quantification of rare target DNA sequences. Aim of this study was to explore the feasibility of the ddPCR technique to detect pathogen DNA in whole blood and to assess the diagnostic accuracy of ddPCR to detect bloodstream infections (BSIs), benchmarked against blood cultures. Broad-range primers and probes were designed to detect bacterial 16S rRNA (and Gram stain for differentiation) and fungal 28S rRNA. To determine the detection limit of ddPCR, 10-fold serial dilutions of E. coli and C. albicans were spiked in both PBS and whole blood. The diagnostic accuracy of ddPCR was tested in historically collected frozen blood samples from adult patients suspected of a BSI and compared with blood cultures. Analyses were independently performed by two research analysts. Outcomes included sensitivity and specificity of ddPCR. Within 4 h, blood samples were drawn, and DNA was isolated and analysed. The ddPCR detection limit was approximately 1-2 bacteria or fungi per ddPCR reaction. In total, 45 blood samples were collected from patients, of which 15 (33%) presented with positive blood cultures. The overall sensitivity of ddPCR was 80% (95% CI 52-96) and specificity 87% (95% CI 69-96). In conclusion, the ddPCR technique has considerable potential and is able to detect very low amounts of pathogen DNA in whole blood within 4 h. Currently, ddPCR has a reasonable sensitivity and specificity, but requires further optimization to make it more useful for clinical practice.

Iron handling by the human kidney: glomerular filtration and tubular reabsorption both contribute to urinary iron excretion.

In physiological conditions, circulating iron can be filtered by the glomerulus and is almost completely reabsorbed by the tubular epithelium to prevent urinary iron wasting. Increased urinary iron concentrations have been associated with renal injury. However, it is not clear whether increased urinary iron concentrations in patients are the result of increased glomerular iron filtration and/or insufficient tubular iron reabsorption and if these processes contribute to renal injury. We measured plasma and urine iron parameters and urinary tubular injury markers in healthy human subjects ( n = 20), patients with systemic iron overload ( n = 20), and patients with renal tubular dysfunction ( n = 18). Urinary iron excretion parameters were increased in both patients with systemic iron overload and tubular dysfunction, whereas plasma iron parameters were only increased in patients with systemic iron overload. In patients with systemic iron overload, increased urinary iron levels were associated with elevated circulating iron, as indicated by transferrin saturation (TSAT), and increased body iron, as suggested by plasma ferritin concentrations. In patients with tubular dysfunction, enhanced urinary iron and transferrin excretion were associated with distal tubular injury as indicated by increased urinary glutathione S-transferase pi 1-1 (GSTP1-1) excretion. In systemic iron overload, elevated urinary iron and transferrin levels were associated with increased injury to proximal tubules, indicated by increased urinary kidney injury marker 1 (KIM-1) excretion. Our explorative study demonstrates that both glomerular filtration of elevated plasma iron levels and insufficient tubular iron reabsorption could increase urinary iron excretion and cause renal injury.

Tubular Injury Biomarkers to Detect Gentamicin-Induced Acute Kidney Injury in the Neonatal Intensive Care Unit.

OBJECTIVE: We evaluated whether urinary excretion of tubular injury markers could be useful for early detection of gentamicin (GM)-induced renal damage in neonates. STUDY DESIGN: We conducted a prospective, observational trial in neonates admitted to the neonatal intensive care unit (26 GM treated, 20 control). Kidney injury molecule-1 (KIM-1), neutrophil gelatinase-associated lipocalin (NGAL), N-acetyl-ß-D-glucosaminidase (NAG), and π- and α-glutathione-S-transferase (GSTP1-1 and GSTA1-1) were measured every 2 hours during admission and compared with serum creatinine (sCr) and urine output. RESULTS: Nine neonates developed AKI during the course of the study. The peak in excretion of urinary biomarkers preceded the peak in sCr (p < 0.0001). GM administration resulted in a more pronounced increase of sCr compared with control (13 [12-28] vs. 10 µmol/L [8.5-17]; p < 0.05). The urinary excretion of NAG (178 [104-698] vs. 32 ng/mol Cr [9-82]; p < 0.001) and NGAL (569 [168-1,681] vs. 222 ng/mol Cr [90-497]; p < 0.05) was higher in the GM group compared with control and preceded the peak of sCr and urine output decrease. CONCLUSION: GM administration to neonates is associated with renal damage reflected by a more pronounced increase in sCr preceded by urinary excretion of biomarkers. Urinary biomarkers may be useful for earlier identification of renal injury in neonates.

No Clinical or Biochemical Evidence for Essential Fatty Acid Deficiency in Home Patients Who Depend on Long-Term Mixed Olive Oil- and Soybean Oil-Based Parenteral Nutrition.

BACKGROUND: Home parenteral nutrition (HPN) patients depend on lipid emulsions as part of their parenteral nutrition regimen to provide essential fatty acids (EFAs). Mixed-oil sources are used in modern lipid emulsions to decrease the amount of proinflammatory EFAs, mainly linoleic acid, which is present in large amounts in soybean oil. It is unknown whether patients who fully depend on such mixed lipids have adequate EFA supply. We therefore evaluated whether HPN patients who depend on mixed olive oil- and soybean oil-based HPN show clinical or biochemical evidence of EFA deficiency. MATERIALS AND METHODS: Fatty acid status was assessed in plasma phosphatidylcholine (PC) and peripheral blood mononuclear cells from 30 patients receiving mixed olive oil- and soybean oil-based HPN (>3 months, ≥5 times per week) and 30 healthy controls. Innate immune cell functions were evaluated by assessing expression of surface membrane molecules, and reactive oxygen species, and cytokine production. RESULTS: None of the patients or controls showed clinical evidence (skin rash) or biochemical evidence (increased Holman index [>0.2]) for EFA deficiency. The Holman index in plasma PC (median [25th-75th percentile]) was significantly higher in patients (0.019 [0.015-0.028]) compared with controls (0.015 [0.011-0.017]). No differences were found in innate immune cell functions between groups, except for a 3.6-fold higher tumor necrosis factor-α production in patients. CONCLUSION: We found no clinical or biochemical evidence that HPN patients who fully and long-term depend on mixed olive oil- and soybean oil-based lipids have an increased risk for EFA deficiency.

Genetic polymorphisms in UDP-glucuronosyltransferase 1A6 and 1A7 and the risk for benign Warthin's tumors of the parotid gland.

BACKGROUND: Warthin's tumors of the parotid gland are associated with smoking, whereas pleomorphic adenomas are not. Genetic polymorphisms in biotransformation enzymes, involved in detoxification of toxins and carcinogens in cigarette smoke, might modify the corresponding enzyme activity and influence detoxifying capacity. We hypothesize that these genetic polymorphisms may influence the individual risk for Warthin's tumor, but not for pleomorphic adenomas. METHODS: Blood from 146 patients with benign parotid gland tumors and 437 controls were investigated for polymorphisms in several biotransformation enzymes. Based on these polymorphisms, patients and controls were divided according to predicted enzyme activity (low, intermediate, and high). RESULTS: Prevalence of predicted intermediate and high activity UGT1A7 and UGT1A6 genotypes was significantly higher in the patients with Warthin's tumors, but not in patients with pleomorphic adenomas, compared with healthy controls. CONCLUSION: Predicted intermediate and high activity UGT1A7 and UGT1A6 genotypes are associated with an increased risk for Warthin's tumor. © 2015 Wiley Periodicals, Inc. Head Neck 38: E717-E723, 2016.

LPS-Stimulated Whole Blood Cytokine Production Is Not Related to Disease Behavior in Patients with Quiescent Crohn's Disease.

INTRODUCTION: Crohn's disease (CD) is a chronic inflammatory disease in which cytokines play a pivotal role in the induction and maintenance of inflammation. Innate cytokine production is genetically determined and varies largely between individuals; this might impact the severity of inflammation. We aimed to assess whether ex-vivo endotoxin-stimulated levels of cytokines could be associated with disease phenotype. METHODS: Patients with quiescent CD (Harvey-Bradshaw Index ≤ 4 and negative inflammation markers) who were not using immunomodulating drugs or biologicals were eligible. Historical disease characteristics (localization, behavior, number of bowel resections, drug history, extra-intestinal symptoms) were extracted from medical records. We measured cytokine levels (tumor necrosis factor (TNF)-α, interleukin (IL)-1ß, IL-6 and IL-10) in supernatants of lipopolysaccharide (LPS) -stimulated whole blood cultures and correlated these with disease characteristics and age- and sex-matched healthy controls. In addition, we analyzed whether single nucleotide polymorphisms (SNPs) in the promoter region of the TNF-α gene were related to TNF-α levels. RESULTS: We included 75 patients with CD and 24 healthy controls. Six patients were excluded because of increased inflammation markers resulting in a total of 69 patients. The mean age (SD) of patients with CD was 51.2 (12.3) years with a mean (SD) disease duration of 24.1 (11.5) years. Disease localization, peri-anal involvement and behavior were not related to LPS-stimulated TNF-α, IL-1ß, IL-6 or IL-10 levels. In addition, combination of localization with behavior to differentiate mild from severe disease type showed no significant differences. TNF-α levels were higher in patients with CD (428 pg/ml IQR [267-468]) compared to healthy controls (459 pg/ml IQR [364-570], p=0.02). We found no associations between SNPs in the promoter region and TNF-α levels. CONCLUSION: In this study, innate cytokine production of TNF-α, IL-1ß, IL-6 and IL-10 was not related to historical disease characteristics or disease severity in patients with quiescent CD. These findings suggest that genetically determined levels of these cytokines obtained from LPS-stimulated whole blood cultures are not linked with disease behavior or severity.

Immune activation by medium-chain triglyceride-containing lipid emulsions is not modulated by n-3 lipids or toll-like receptor 4.

BACKGROUND: Saturated medium-chain triglycerides (MCT) as part of the parenteral lipid regimen (50% MCT and 50% long chain triglycerides (LCT)) activate the immune system in vitro. Fish oil (FO)-derived n-3 fatty acids (FA) inhibit saturated FA-induced immune activation via a toll-like receptor (TLR)-4 mediated mechanism. We hypothesized that effects of parenteral MCTs on immune cells involve TLR-4 signaling and that these effects are modulated by n-3 FA that are present in FO. MATERIALS AND METHODS: To test this hypothesis we assessed effects of addition of various commercially available mixed parenteral lipid emulsions, n-3 FA and of TLR-4 inhibition on MCT-induced human immune cell activation by evaluation of the expression of leukocyte membrane activation markers and reactive oxygen species (ROS) production. RESULTS: All MCT-containing lipid emulsions activated leukocytes by inducing changes in expression of membrane markers and stimulus induced ROS production, whereas MCT-free lipid emulsions lacked this effect. Moreover, addition of n-3 FA to LCT/MCT did not prevent MCT-induced immune activation. TLR-4 inhibitors did not distinctly modulate MCT-induced changes in immune function. CONCLUSION: Taken together, these findings suggest that leukocyte activation by parenteral MCTs does not involve TLR-4 signaling and is not modulated by n-3 FA in FO-, but is exerted via different signaling pathways.

Allopurinol and 5-aminosalicylic acid influence thiopurine-induced hepatotoxicity in vitro.

INTRODUCTION: The use of thiopurines is frequently accompanied by hepatotoxicity. Studies on hepatocyte cultures showed a time- and dose-dependent increase of thiopurine toxicity. 5-Aminosalicylic acid (5-ASA) and allopurinol can influence thiopurine metabolism; however, it is unknown whether this affects in vitro cytotoxicity. METHODS: Human hepatoma cells (Huh7, HepG2 and HepaRG) were incubated with increasing concentrations of thiopurines, 5-ASA or allopurinol. Water-soluble tetrazolium salt-1 (WST-1) cytotoxicity assays were used to calculate cell survival curves and half maximal inhibitory concentrations (IC50). Combination experiments with thiopurines with a fixed dose of 200 µM 5-ASA or 100 µM allopurinol were conducted in HepaRG cells. Caspase-3/7 activation was evaluated, and single cell electrophoresis analysis was performed. RESULTS: A time- and dose-related cytotoxic effect was seen with azathioprine (AZA) in all hepatoma cells, whereas Huh7 and HepG2 cells did not show toxicity to 6-mercaptopurine (6-MP). HepaRG cells expressed the highest levels of drug metabolising enzymes, and therefore, combination experiments were conducted in HepaRG cells. Addition of a non-toxic dose of allopurinol resulted in a twofold to threefold increased cytotoxicity of all thiopurines, which seemed to be mediated by apoptosis/DNA damage. CONCLUSION: The addition of allopurinol to thiopurines leads to a two-threefold increased cytotoxicity in HepaRG cells.

GST Theta null genotype is associated with an increased risk for ulcerative colitis: a case-control study and meta-analysis of GST Mu and GST Theta polymorphisms in inflammatory bowel disease.

Glutathione S-transferases (GSTs) are important in the detoxification of many compounds, including reactive oxygen species. Polymorphisms in GSTs resulting in a decreased enzyme activity might enhance the risk for inflammatory bowel disease by eliciting a state of oxidative stress. Previous case-control studies showed divergent results and were frequently limited in sample size; therefore we conducted a meta-analysis including results from our case-control study. For the case-control study, we genotyped 552 patients with Crohn's disease (CD), 223 patients with ulcerative colitis (UC) and 972 healthy controls by PCR for functional deletions in GST Mu and GST Theta. Both were not analyzed in recent genome-wide association studies. For the meta-analysis, PubMed, EMBASE and Web of Science were searched. In this meta-analysis, we show an enhanced susceptibility for UC in individuals with the GSTT1null genotype (odds ratio (OR) 2.27, 95% confidence interval (CI) 1.31-3.92). In our case-control study, a reduced risk for CD was seen with the GSTT1null genotype (OR 0.58, 95% CI 0.43-0.77); however, pooled analysis showed an OR of 1.67, 95% CI 0.81-3.45. In this meta-analysis, we showed an increased risk for UC in individuals with the GSTT1null genotype.

Genetic polymorphism 609C>T in NAD(P)H:quinone oxidoreductase 1 enhances the risk of proximal colon cancer.

Gastrointestinal (GI) cancer is responsible for the majority of deaths among all types of cancer. Lifestyle factors may not only be the main risk factor for GI cancer but reactive oxygen species (ROS) may also be involved. The single-nucleotide polymorphisms (SNPs) 609C>T (rs1800566) and 465C>T (rs1131341) in the NAD(P)H: quinone oxidoreductase 1 (NQO1) gene lead to a decline in NQO1 enzyme activity. NQO1 catalyzes the two-electron reduction of quinones to hydroquinones, thereby preventing the formation of ROS. Such polymorphisms in NQO1 may increase the risk of GI cancer. The aim of this study was to evaluate the influence of the SNPs rs1800566 and rs1131341 in the NQO1 gene on the risk of GI cancer in the Netherlands. Real-time polymerase chain reaction techniques were conducted to determine the NQO1 genotypes of 1457 patients with GI cancer and 1457 age- and gender-matched controls in a case-control study. Binary logistic regression analyses showed no statistically significant difference in genotype distributions between patients and controls: odds ratios (ORs) with 95% confidence interval (CI) for rs1800566 were 1.09 (0.93-1.28) and 1.17 (0.77-1.77) for the CT and TT genotypes, respectively. ORs for rs1131341 CT and TT genotypes were 1.21 (0.90-1.63) and 0.54 (0.05-5.94), respectively. For rs1800566, a significant association between the CT genotype and proximal colon cancer was detected (OR=1.60; 95% CI=1.09-2.35). The NQO1*2 T allele of SNP rs1800566 was found associated with an increased risk for proximal colorectal cancer, whereas SNP rs1131341 was rare in our Dutch population and was not associated with GI cancer.

Polymorphisms in the insulin-like growth factor axis are associated with gastrointestinal cancer.

INTRODUCTION: Numerous factors influence the development of gastrointestinal (GI) cancer. The insulin-like growth factor (IGF) axis plays a role in embryonic and postnatal growth and tissue repair. Elevated levels of IGFs, low levels of IGF binding proteins (IGFBPs) and over-expression of IGF receptor (IGFR-I) were associated with several stages of cancer. Here, the prevalence of the single nucleotide polymorphisms (SNPs) rs6214 in the IGF type I (IGF-I) gene and rs6898743 in the growth hormone receptor (GHR) gene in patients with GI cancer and controls was studied. MATERIALS & METHODS: In this Dutch case-control study, DNA isolated from blood of 1,457 GI cancer patients; 438 patients with head and neck cancer (HNC), 475 with esophageal cancer (EC) and 544 with colorectal cancer (CRC) and 1,457 matched controls, was used to determine the rs6214 and rs6898743 genotypes by polymerase chain reaction. The association between these SNPs and GI cancer, HNC, esophageal adenocarcinoma (EAC), esophageal squamous-cell carcinoma (ESCC) and proximal or distal CRC was studied. Odds ratios (ORs) with 95% confidence interval (95% CI) were calculated via unconditional logistic regression. RESULTS: Overall for GI cancer, the ORs for SNPs rs6214 and rs6898743 were approximately 1.0 (p-value>0.05), using the most common genotypes GG as reference. An OR of 1.54 (95% CI, 1.05-2.27) was found for EC for genotype AA of rs6214. The ORs for EAC were 1.45 (95% CI, 1.04-2.01) and 1.71 (95% CI, 1.10-2.68), for genotypes GA and AA, respectively. Genotype GC of rs6898743 showed an OR of 0.47 (95% CI, 0.26-0.86) for ESCC. CONCLUSION: The A allele of SNP rs6214 in the IGF-I gene was associated with EAC, and with HNC in women. The GC genotype of rs6898743 in the GHR gene was negatively associated with ESCC.

Over-expression of COX-2 mRNA in colorectal cancer.

BACKGROUND: Cyclooxygenase-2 (COX-2, PTGS2) is an enzyme involved in the synthesis of prostaglandins and thromboxanes, which are regulators of biologic processes such as inflammation, cell proliferation and angiogenesis. COX-2 over-expression was reported in many (pre) malignant tissues, but data strongly vary and seem to depend on the methodology used. METHODS: Normal colorectal mucosa and paired cancerous tissue from 60 patients with colorectal cancer was investigated for the levels of COX-2 mRNA by real-time quantitative Polymerase Chain Reaction (qPCR). COX-2 levels were expressed relative to either: tissue weight or levels of the housekeeping genes beta-2 microglobulin (B2M) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH). RESULTS: COX-2 mRNA levels, normalized with respect to tissue weight or mRNA levels of the housekeeping genes B2M or GAPDH, were over-expressed in 80%, 70% and 40% of the colorectal tumor tissues, as compared to the paired adjacent normal colorectal mucosa samples, respectively. Highest mRNA COX-2 ratios tumor/normal were measured when expressed per mg tissue (mean ratio 21.6). When normalized with respect to the housekeeping genes B2M or GAPDH, mean tumor/normal ratios were 16.1 and 7.5, respectively. CONCLUSION: Expression of COX-2 mRNA levels per mg tissue is most simple in comparison to normalization with respect to the housekeeping genes B2M or GAPDH. Levels of COX-2 mRNA are found over-expressed in almost 80% of the colorectal tumors, compared to paired adjacent normal colorectal mucosa, suggesting a role of COX-2 as a potential biomarker for cancer risk, whereas inhibitors of COX-2 could be of value in chemoprevention of colon cancer.

Duodenal mucosal risk markers in patients with familial adenomatous polyposis: effects of celecoxib/ursodeoxycholic acid co-treatment and comparison with patient controls.

BACKGROUND: Familial adenomatous polyposis (FAP) is a disease characterized by the development of hundreds to thousands of adenomatous polyps in the colorectum early in life. Virtually all patients with FAP will develop colorectal cancer before the age of 40 to 50 years, unless prophylactic colectomy is performed, which significantly improves their prognosis. The mortality pattern has changed and duodenal cancer now is one of the main cancer-related causes of death in these patients. Practically all patients with FAP develop premalignant duodenal adenomas, which may develop to duodenal cancer in approximately 3-7% of patients. Duodenal cancer in patients with FAP has a poor prognosis. The clinical challenge is to identify patients at high-risk for duodenal carcinoma. Chemoprevention would be desirable to avoid duodenectomy. The main goal of this study is to identify risk markers in normal duodenal mucosa of patients with FAP, that could help identify patients at increased risk for malignant transformation. METHODS: Messenger RNA (mRNA) levels of glutathione S-transferase A1 (GSTA1), glutathione S-transferase P1 (GSTP1), KIAA1199, E-cadherin, peroxisome proliferative activated receptor δ (PPARδ), caspase-3, cyclin D1, ß-catenin, and cyclooxygenase-2 (COX-2) were measured in duodenal mucosa, using the QuantiGene 2.0 Plex assay. Levels in normal appearing mucosa of patients with FAP (n = 37) were compared with levels in non-FAP patient controls (n = 16). In addition, levels before and after treatment with either celecoxib & ursodeoxycholic acid (UDCA, n = 14) or celecoxib & placebo (n = 13) were evaluated in patients with FAP. RESULTS: mRNA levels of glutathione S-transferase A1 (28.16% vs. 38.24%, p = 0.008) and caspase-3 (3.30% vs. 5.31%, p = 0.001) were significantly lower in patients with FAP vs. non-FAP patient controls, respectively. COX-2 mRNA levels in normal duodenal mucosa of patients with FAP were found to be unexpectedly low. None of the potential risk markers was influenced by celecoxib or celecoxib & UDCA. CONCLUSIONS: Protection against toxins and carcinogens (GSTA1) and apoptosis (caspase-3) is low in patients with FAP, which could contribute to increased susceptibility for malignant transformation of duodenal mucosa. TRIAL REGISTRATION: http://ClinicalTrials.gov number NCT00808743.

Disparities of conjugating protective enzyme activities in the colon of patients with adenomas and carcinomas.

AIM: To investigate the metabolic enzymatic capacity of the colon mucosa to detoxify noxious carcinogenic compounds. METHODS: We investigated the activity of 2 conjugating enzymes-the microsomal uridine glucuronosyltransferase (UGT) and the cytosomal glutathione S-transferase (GST) in the uninvolved mucosa of the colon transversum and sigmoideum in patients with adenomatous polyps and colorectal cancer. Biopsies were taken from the mucosa during colonoscopies which were done for clinical (diagnostic) reasons. After storage, the biopsy material was homogenized and after differential centrifugation the enzyme assays were performed with 4-nitrophenol (UGT) and 1-chloro 2,4-dinitrobenzene (GST) as substrates. RESULTS: About 48 patients were included of which 28 had adenomas and 20 had colorectal carcinomas confirmed by histopathology. Enzyme activities were expressed as nmol/mg per minute protein for the GST and as pmol/mg per minute protein for the UGT. Analysis of variance (F-test) indicated that both enzymes were more widely distributed in adenoma than in cancer patients. The means ± SD were smaller for cancer patients: GST for adenomas 268 ± 152 vs 241 ± 69 for carcinomas and UGT for adenomas 197 ± 200 vs 150 ± 86 for carcinomas. CONCLUSION: Compared to patients with adenomatous colon polyps those with colorectal carcinoma exhibited a lower capacity of detoxifying enzyme metabolism and their activities clustered over a smaller range.

Polymorphisms in alcohol-metabolizing enzymes and esophageal carcinoma susceptibility: a Dutch Caucasian case-control study.

Esophageal cancer (EC), mainly consisting of squamous cell carcinoma (ESCC) in the Eastern world and adenocarcinoma (EAC) in the Western world, is strongly associated with dietary factors such as alcohol use. We aimed to clarify the modifying role in EC etiology in Caucasians of functional genotypes in alcohol-metabolizing enzymes. In all, 351 Caucasian patients with EC and 430 matched controls were included and polymorphisms in CYP2E1, ADH and near ALDH2 genes were determined. In contrast to the results on ESCC in mainly Asian studies, we found that functional genotypes of alcohol-metabolizing enzymes were not significantly associated with EAC or ESCC in an European population.

Ursodeoxycholic acid counteracts celecoxib in reduction of duodenal polyps in patients with familial adenomatous polyposis: a multicentre, randomized controlled trial.

BACKGROUND: Due to prophylactic colectomy, mortality in patients with familial adenomatous polyposis (FAP) has changed, with duodenal cancer currently being the main cause of death. Although celecoxib reduces duodenal polyp density in patients with FAP, its long-term use may increase the risk of cardiovascular events and alternatives need to be explored. Preclinical studies suggest that the combination of celecoxib with ursodeoxycholic acid (UDCA) is a potentially effective strategy. We performed a randomized, double-blind, placebo-controlled trial to investigate the effect of celecoxib and UDCA co-treatment on duodenal adenomatosis in patients with FAP. METHODS: Patients with FAP received celecoxib (400 mg twice daily) and UDCA (1000-2000 mg daily, ~20-30 mg/kg/day, n=19) or celecoxib and placebo (n=18) orally for 6 months. Primary outcome was drug efficacy, assessed by comparing duodenal polyp density at pre- and post-intervention by blinded review of endoscopic recordings. As secondary outcomes, cell proliferation, apoptosis, and COX-2 levels in normal duodenal mucosa were assessed by immunohistochemistry or real-time quantitative polymerase chain reaction. RESULTS: In intention-to-treat analysis, deceased polyp density was observed after celecoxib/placebo treatment (p=0.029), whereas increased polyp density was observed after celecoxib/UDCA treatment (p=0.014). The difference in change in duodenal polyp density was statistically significant between the groups (p=0.011). No changes in secondary outcomes were observed. Thirty patients (81%) reported one or more adverse events, 16 patients (84%, Common Toxicity Criteria for Adverse Events version 3.0 (CTCAE) grade 1-3) treated with celecoxib/UDCA and 14 patients (78%, CTCAE grade 1-2) treated with celecoxib/placebo. Nine patients (24%) discontinued intervention prematurely, 5 patients (26%) treated with celecoxib/UDCA and 4 patients (22%) treated with celecoxib/placebo. CONCLUSIONS: Celecoxib reduces duodenal polyp density in patients with FAP, and unexpectedly, high dose UDCA co-treatment counteracts this effect. The benefit of long term use of celecoxib for duodenal cancer prevention needs to be weighed against the (risk of) adverse events. TRIAL REGISTRATION: http://ClinicalTrials.gov, identifier NCT00808743.

No role for glutathione S-transferase genotypes in Caucasian esophageal squamous cell or adenocarcinoma etiology: an European case-control study.

BACKGROUND: Identifying and monitoring high-risk patients can aid the prevention of esophageal cancer (EC). The interaction of environmental risk factor exposure and genetic susceptibility may contribute to the etiology of EC. Biotransformation enzymes such as Glutathione S-Transferases (GSTs ) detoxify mutagenic and genotoxic compounds and therefore control the rate of detoxification of carcinogens. Functional polymorphisms in the genes coding for GSTs alter their enzyme activity in vitro, and were reported to modify EC risk in Asians. We hypothesized that altered enzyme activity GST genotypes influence the susceptibility for esophageal adeno- (EAC) and squamous cell carcinoma (ESCC) in Caucasians. METHODS: We performed a case-control study including 440 Caucasian patients with EC and 592 healthy Caucasian controls matched for age and sex. Functional polymorphisms were selected and genotypes were determined in GST classes Alpha, Mu, Theta and Pi by means of polymerase chain reaction. Genotypes were classified into predicted high, intermediate and low enzyme activity categories based on in vitro activity data. The distribution of the activity genotypes were compared between patients with EAC or ESCC, and controls. Odds ratios (OR) with 95% confidence intervals (CI) were calculated by logistic regression analyses. Gene-gene interactions were tested and for comparison purposes, the predicted low and intermediate activity genotypes were combined. Genotypes with similar risks for EAC or ESCC were combined and analyzed for multiplicative effects. RESULTS: Our analyses includes 327 patients with EAC and 106 patients with ESCC. Low or intermediate activity enzyme genotypes for GSTM1, GSTA1, GSTP1 I105V and A114V as well as for GSTT1, did not significantly modify the risk for ESCC or EAC in our Dutch population. CONCLUSION: Functional genotypes in GST genes are not involved in EAC or ESCC susceptibility in Caucasians, in contrast to results on ESCC from Asia or Africa.