Volume 23, Issue 1

New spectrum of side effects has emerged with the introduction of targeted therapies, including changes in parameters determining cardiovascular risk. Among other laboratory parameters, hyperuricemia and elevated Creactive protein (CRP) are associated with increased risk of cardiovascular disease. The aim of the present study was to investigate the changes of serum uric acid and CRP during the therapy with cetuximab, combined with 5- fluorouracil and high-dose folinic acid, in patients with metastatic colorectal carcinoma and the association of these changes with the outcome of the therapy. CRP and uric acid were determined using particle-enhanced immunoturbidimetric assay and uricase method, respectively. Measurements before and during the treatment were compared by Wilcoxon signed rank test, and comparison between survivors and non-survivors was performed by Mann-Whitney U test. Compared to baseline (pre-treatment) concentrations, serum uric acid was decreased significantly throughout the course of the combination treatment with irinotecan, 5-fluorouracil, highdose folinic acid and cetuximab. The decrease was evident early in the course of treatment. Serum CRP was also decreased significantly throughout the most of the course of therapy. The significant decrease of serum uric acid and CRP was evident already one week after the start of treatment. Serum CRP was significantly lower in patients who survived 12 months, but no difference of uric acid concentrations between survivors and non-survivors was found. A marked decrease of serum uric acid was observed during the combination therapy. High CRP was associated with poor prognosis, but no association between uric acid and prognosis was noted.

The C677T variant of methylenetetrahydrofolate reductase (MTHFR), a key enzyme in the remethylation of homocysteine (HCY) to methionine, is a frequent genetic cause of moderate hyperhomocysteinemia (HHCY) among individuals with cardiovascular disease (CVD), and particularly when combined with other factors such as hyperlipidaemia. However, in Algeria the influence of nutrient-gene interactions is not known. The aim of the present study was to explore the influence of age and gender, together with folate status, on the association between the C677T MTHFR polymorphism and plasma total HCY (tHCY) concentrations. This research was carried out as a prospective study on 98 patients hospitalized in the Cardiology Section, University of Sétif, Algeria. Mean age of participants was 57 y (range 20-96 y).The genetic analysis of the MTHFR C677T polymorphism was performed by real-time polymerase chain reaction (PCR) performed on Light Cycler in borosilicate capillaries with MTHFR 677CT polymorphism detection kit. The concentrations of tHCY, folic acid vitamin B 12 levels were determined using a competitive immunoassay on the IMMULITE 1000 Analyzers. Plasma total cholesterol, triglycerides, glucose, creatinine and urea concentrations were measured by colorimetric methods. Assays were conducted according to the manufacturers' instructions. Plasma tHCY was significantly higher in the patients with CVD, and HHCY was associated with the presence of mildly elevated serum urea and creatinine (p <0.05). MTHFR gene mutation does not seem to be associated with elevation of plasma tHCY in the studied patients and this lack of correlation could be influenced by the higher folate concentrations in our study. CVD patients with 677CT/TT genotypes had a higher concentration of total cholesterol than those with 677CC genotype (p <0.05). Although, the presence of 677T variant together with hypofolatemia (<15.4 ng/ml) had a more detrimental effect on the level of total cholesterol (p <0.05). Folatemia and vitamin B 12 were much higher in 677CC genotype compared to 677CT/TT genotype in CVD subjects without hyperlipidemia (p <0.05). However in patients with hyperlipidemia these values became lower also with 677CC genotype. In conclusion, hyperlipidemia affects the levels of plasma folate and vitamin B 12 concentrations independent of mutated MTHFR genotype. The effect of 677T variant on total cholesterol, folate and vitamin B 12 concentrations may relate to possible adverse effects of elevated tHCY on lipid profiles and on plasma folate and vitamin B 12 .

Increased concentrations of neopterin have been described in patients with tumors of different primary locations, but reports on neopterin in patients with carcinoma of the esophagus are still very limited. We have studied serum neopterin in 45 patients with histologically verified carcinoma of the esophagus. Serum neopterin was determined with radioimmunoassay. Neopterin concentrations above median (6.5 nmol/L) were associated with inferior prognosis in patients with early esophageal carcinoma. In multivariate analysis, tumor stage and serum neopterin were predictors of survival in patients without distant metastases. In conclusion, serum neopterin is an independent prognostic factor in patients with esophageal carcinoma.

Survival of neurons is dependent on the presence of trophic and non-trophic factors. Previous findings showed that purine nucleosides have the potential to rescue cells from hypoxia-induced cell death. Here we demonstrate that purine nucleoside guanosine can efficiently rescue neuronal PC12 cells from serum deprivation-induced cell death. By means of Hoechst/propidium iodide staining and fluorescence analysis a 31.1% decrease of cell death was detected. These data support current efforts to propagate purine nucleosides as neuroprotective and neuroregenerative substances.

Folic acid, or folates are available from a large variety of sources, such as green leafy vegetables (spinach, beans) and liver. The importance of folates is based on their requirement as coenzymes in the transfer and utilization of one-carbon units in a variety of biosynthetic reactions. Folate is an essential cofactor for the de novo biosynthesis of purine nucleotides and thymine, thus influencing RNA and DNA synthesis. Therefore, during periods of rapid cell regeneration and growth, such as pregnancy and infancy, increased amounts of folate are required. Folate has the roles in the prevention of neural tube defects, an important cause of infant mortality and disability. The periconceptional folic acid intake reduces the risk of neural tube defects. Arginase, as part of the urea cycle, detoxifies human body from ammonia. Its activity is important in the production of ornithine, subsequently leading to the production of polyamines (spermine and spermidine), glutamate or proline, and nitric oxide. Arginase is normally present in mother's milk. In the present study, the effect of folic acid supplementation during pregnancy on the dynamics of arginase activity and malondialdehyde concentration in human colostrum and mature milk was studied. The obtained results suggest that in breastfeeding mothers' colostrum and mature human milk samples, supplemented with folic acid during pregnancy (400mg/daily periconceptionaly), the arginase activity increases compared with the enzyme activity in milk samples taken from mothers who did not take folic acid. At the same time, the supplementation with folic acid causes malondialdehyde decrease in colostrum and mature milk.

Fluorescence labeled oligonucleotides have a long history of being used to monitor nucleic acid transport and uptake. However, it is not known if the fluorescent moiety itself physically limits the number of pathways that can be used by the cell due to steric, hydrophobic, or other chemical characteristics. Here, we report a method for comparing the uptake kinetics of oligonucleotides labeled either with the fluorescent pteridine, 3-methyl-8-(2- deoxy-b-D-ribofuranosyl) isoxanthopterin (3MI), or the common fluorophore 5-carboxyfluorescein (5-FAM). We use a multiphoton microscopic technique to monitor nucleic acid uptake LLC-PK 1 , a pig renal tubular cell line that is known to have multiple uptake pathways. We find that the two fluorophores enter the cells at different rates, suggesting that choice of fluorescent moiety influences the uptake pathway used by a cell. Finally, we reconstituted an LLC-PK 1 membrane channel that is selective for nucleic acids in planar lipid bilayers, and tested the ability of the labeled nucleic acids to permeate the channel. We find that 3MI, and not 5-FAM labeled oligonucleotides can traverse the plasma membrane through the channel. These results have implications for future studies aimed at delivering pteridine moieties to cells and for tracking nucleic acid transport into tissues.

Neopterin, as a non-conjugated pteridine, is synthesized from guanosine triphosphate and its production is upregulated upon the activation of cellular immune response. Alterations of pteridines in body fluids are known to correlate well with existing diseases and stages, prognosis, clinical outcomes and survival data. It is of advantage to have a pteridine database of healthy volunteers to determine normal values. Thereby, especially in children there is no detailed study on pteridine levels. The aim of this study is to initiate the establishment of pteridine database of healthy children in our country. Serum neopterin levels were analysed by enzyme-linked immunosorbent assay. Urinary neopterin and biopterin levels and serum kynurenine, tryptophan levels and kynurenine/tryptophan ratio as an estimate of tryptophan breakdown were assessed with high-pressure liquid chromatography in serum and urine samples of 55 children aged between 3 months and 10 years. The results were evaluated within the subgroups of different ages and sex. Pteridine pathway components were found to be higher in children compared to adults. Higher levels of pteridine pathway components observed within the first years of life may reflect the rapid maturation of the immune system, and environmental adaptation and/or insufficiency of defence systems. On the other hand, it may also relate to a higher frequency of infections not (yet) manifested clinically.

Homocysteine (HCY) has been identified as a risk factor for vascular disease in the general population. Diabetic retinopathy (DR) itself rather than hyperhomocysteinemia is the leading cause of blindness among patients with type 2 diabetes mellitus (T2DM). Our study was conducted with 60 healthy control subjects and 178 subjects with T2DM. They were enrolled in the Diabetes Prevention Program from September 2007 to December 2008. Of the 178 patients, 121 cases (68%) had DR Mean plasma total HCY (tHCY) levels were found to be higher in T2DM patients compared to controls (p <0.001), and were also higher than that of the DR group (p <0.001). Plasma folic acid levels were lower in the DR group compared with T2DM without DR and the control group (p <0.001), but there were no significant differences between the latter and the controls. Moderate hyperhomocysteinemia was significantly associated with lower vitamin B12 and folic acid concentrations and older age. Concentrations of serum total cholesterol, LDL-cholesterol (LDL-C), and triglycerides (TG) were significantly raised (p <0.001) whereas the level of HDL-cholesterol (HDL-C) was decreased (p <0.001) in diabetic subjects as compared to controls. Logistic regression analysis showed that DR after adjustment was significantly associated with the following factors: cholesterol, HDL-C and TG. The analysis in DR patients after controlling for cholesterol and TG was independent of plasma tHCY concentrations (OR = 28.5 and OR = 11.9; respectively). In conclusion, results suggest a possible association between moderate hyperhomocysteinemia, traditional risk factors and folic acid deficiency could be an independent risk factor for DR.

Fractional exhaled nitric oxide (FeNO) is a useful marker of airway inflammation in asthmatics. Nitric oxide synthase (NOS) requires tetrahydrobiopterin as a cofactor and produces superoxide during NO generation. Therefore, we investigated the relationship of FeNO to pteridine biosynthesis and oxidative stress in pediatric asthma patients. We recruited 67 asthmatic children for FeNO measurement and examined neopterin, biopterin, and Diacron-reactive oxygen metabolites (d-ROMs) as an oxidative stress marker in both summer and winter. Although d-ROMs levels did not show significant correlation with FeNO levels in summer, d-ROMs and FeNO were positively correlated in winter (p <0.05). Both neopterin and biopterin levels in the blood tended to be lower in patients who showed higher FeNO. Multivariate analysis revealed that increased IgE levels correlated with increased FeNO (p <0.01) and decreased neopterin (p <0.05) levels. This data supports a mechanism by which decreased levels of pteridines promote reactive oxygen species production upon NO generation, resulting in airway injury in asthmatic patients. Correlation with IgE level indicates that Th2-mediated allergic inflammation is involved in this process.

Titanium is extensively used for a wide range of implanted medical devices due to its advantageous combination of physico-chemical and biological properties. Nano-size titanium dioxide (TiO 2 ) is also used in a variety of consumer products. Such widespread use and its potential entry through various routes of the body suggest that TiO 2 could pose an exposure risk to humans. Nano-size particles (NP) enter systemic circulation, accumulate and damage tissues that are especially sensitive to oxidative stress. We hypothesized that TiO 2 NPs can exert diverse cytotoxic effects on various human cell type especially neural cell lines. In order to test our hypothesis, putative cytotoxic effects of oxidative stress due to TiO 2 NPs exposure on IM9, U937 and SHSY5Y (human neuroblastoma cells) were investigated in N-acetyl cysteine (NAC), neopterin and dexamethasone pre-treated cell cultures. IM9, U937 and SHSY5Y cells were exposed to ten different concentrations of 25 and 10 nm diameter TiO 2 NPs in three different time periods before and after treatment with NAC, neopterin and dexamethasone. To determine toxicity levels of NPs, cell viability was estimated by MTT test. Concentration of cells was assessed by counting trypan blue stained cells with a heamo-cytometer. Toxicity of 25nm TiO 2 particles was significantly increased (p<0.05) by adding fetal bovine serum (FBS) in SHYS5Y and U937 cell lines culture medium. Concentrationdependent toxicity of 10 nm TiO 2 NP was weakly increased by adding FBS to SHYS5Y, IM9 and U937 cell culture media. NAC pre-treatment provided significant protection for only SHYS5Y cell exposed to 25 nm and 10 nm of TiO 2 NPs after a 24-hour incubation period. Neopterin pre-treated SHYS5Y cells displayed significant increases in viability after 24-hour exposure to 10 nm and 25 nm TiO 2 NPs. While exposure of dexamethasone pre-treated U937 cells to 25 nm of TiO 2 NPs induced a significant increase in cell survival at only 100 mg/ml particle concentration, increase in viability of SHYS5Y cells were observed at all concentrations against 10 nm particle challenge. Our study demonstrated that exposure of SHYS5Y to TiO 2 NPs for 24 hours regularly induced reduction of cell viability. We also found similar dose-related effects of TiO 2 NPs in reducing cell survival in IM9 cells. This study clearly indicated that FBS is an effective dispersing agent for TiO 2 NPs and increased TiO 2 toxicity in all cell lines. NAC and neopterin significantly protected the SHYS5Y cell against the putative cytotoxic effects of TiO 2 NPs.