DNA damage and apoptosis in blood neutrophils of inflammatory bowel disease patients and in Caco-2 cells in vitro exposed to betanin.

Inflammatory bowel diseases (IBD) are chronic, relapsing, inflammatory disorders of the gastrointestinal tract, and continuing colonic inflammation is considered an important risk factor in the development of colorectal cancer. Our previous studies showed that beetroot (Beta vulgaris var. rubra) products and their major component betanin modulate the reactive oxygen species (ROS) production and DNA damage in 12-O-tetradecanoylphorbol 13-acetate (TPA) stimulated human polymorphonuclear neutrophils of healthy volunteers. The aim of the present study was to evaluate the effects of betanin on the oxidative DNA damage and apoptosis in neutrophils isolated from blood of patients with inflammatory bowel disease--ulcerative colitis (UC) and Crohn's disease (CD). The results were compared with those obtained in colon carcinoma-derived Caco-2 cells. Betanin treatment at the concentration of 100 µM for 24 h increased DNA damage assessed by comet assay in IBD patients' neutrophils. A similar effect although less pronounced was observed in Caco-2 cells. Treatment of Caco-2 cells with H2O2 caused a 4-fold increase of DNA strand breaks in comparison to untreated cells, but pre-treatment with betanin reduced DNA damage in these cells. Betanin also induced procaspase-3 cleavage and caspase-3 activity accompanied by the loss of mitochondrial transmembrane potential, indicating its pro-apoptotic activity. These results suggest that betanin may support mechanisms that lead to the release of ROS and apoptotic cell death. In this way betanin may exert anti-inflammatory and potentially cancer preventive activity.

The microbiome and dermatological diseases.

The human skin harbours hundreds of species of commensal organisms, collectively known as the skin microbiota. The composition of the microbiota can be modified by various factors, such as host genotype, diet, antibiotics, hygiene, and pathogen infections, among others. Changes in these factors can cause microbiome disruption known as dysbiosis, leading to the outgrowth of potential pathogenic bacteria or a decrease in the number of beneficial bacteria. Dysbiosis has been implicated in some dermatological diseases. This mini-review aims to discuss the topic of the skin microbiota and its potential effects on various skin diseases.

High-resolution melting analysis of the TPMT gene: a study in the Polish population.

The thiopurine S-methyltransferase (TPMT) gene encoding thiopurine methyltransferase is a crucial enzyme in metabolism of thiopurine drugs: azathioprine and 6-mercoptopurine, which are used in the treatment of leukemia or inflammatory bowel diseases. Genetic polymorphism of the TPMT gene correlates with activity of this enzyme, individual reaction, and dosing of thiopurines. Thirty-one variants of the TPMT gene with low enzymatic activity have been described with three major alleles: TPMT*2 (c.238G>C), *3A (c.460 G>A, c.719A>G), and *3C (c.719A>G), accounting for 80% to 95% of inherited TPMT deficiency in different populations in the world. The aim of the study was to establish a rapid and highly sensitive method of analysis for the complete coding sequence of the TPMT gene and to determine the spectrum and prevalence of the TPMT gene sequence variations in the Polish population. Recently, high-resolution melting analysis (HRMA) has become a highly sensitive, automated, and economical technique for mutation screening or genotyping. We applied HRMA for the first time to TPMT gene scanning. In total, we analyzed 548 alleles of the Polish population. We found 11 different sequence variations, where two are novel changes: c.200T>C (p.P67S, TPMT*30) and c.595G>A (p.V199I, TPMT*31). Detection of these new rare alleles TPMT*30 and *31 in the Polish population suggests the need to analyze the whole TPMT gene and maybe also the extension of routinely used tests containing three major alleles, TPMT*2, *3A, and *3C. Identification of sequence variants using HRMA is highly sensitive and less time consuming compared to standard sequencing. We conclude that HRMA can be easy integrated into genetic testing of the TPMT gene in patients treated with thiopurines.

In vitro effects of beetroot juice and chips on oxidative metabolism and apoptosis in neutrophils from obese individuals.

Oxidative stress and inflammation are involved in the development of obesity. Beetroot (Beta vulgaris var. rubra) is a food ingredient containing betalain pigments that show antioxidant activity. The in vitro effect of beetroot juice and chips on oxidative metabolism and apoptosis in neutrophils from obese individuals has been investigated. Fifteen obese women (aged 45 +/- 9 years, BMI >30 kg/m2) and nine healthy controls (women, aged 29 +/- 11 years, BMI = 22.2 +/- 1.6 kg/m2) were examined. The investigated products were used as concentrates and after transport and digestion in an artificial gastrointestinal tract. Neutrophil oxidant production, in response to phorbol 12-myristate 13-acetate, was characterized by luminol-dependent chemiluminescence and a flow cytometric dichlorofluorescin oxidation assay. Caspase-3 activity, a marker of apoptosis, was measured by cleavage of the fluorogenic substrate Ac-DEVD-AMC. Neutrophils from obese individuals had a significantly higher ROS production compared with the controls (p < 0.05). Beetroot products inhibited neutrophil oxidative metabolism in a concentration-dependent manner. Also observed were the pro-apoptotic effects of beetroot at a concentration range of 0.1-10% in 24 h culture of stimulated neutrophils. These natural products (in both the liquid and solid state) have antioxidant and antiinflammatory capacity, and could be an important adjunct in the treatment of obesity.

[Massive pulmonary embolism in a patient with ulcerative colitis and hyperhomocysteinaemia -- a case report]. / Masywna zatorowosc plucna w przebiegu wrzodziejacego zapalenia jelita grubego i hiperhomocysteinemii.

We describe a case of a 37-year-old man with active ulcerative colitis complicated by proximal deep vein thrombosis of the left lower limb and subsequent massive pulmonary embolism requiring mechanical ventilation and catecholamine infusion. In spiral CT a large thrombus obturating left pulmonary artery as well as bilateral embolic material in lobar and segmental branches were visible. Haemodynamic status improved after infusion of rtPA. Haemoglobin decrease (7.0-5.6 mmol/L) was corrected with erythrocyte mass transfusion. During subsequent therapy with intravenous full dose of unfractionated heparin and further long-term treatment with subcutaneous enoxaparin (1.5 mg/kg and after 3 months 1.0 mg/kg daily) haemoglobin value was relatively stable. Underlying disease was treated with 5-ASA (mesalazine) and steroids. Due to hyperhomocysteinaemia (16.0 micromol/L) coexisting with a low plasma folic acid (2.1 ng/ml) and cyanocobalamin (137 pg/ml) levels, supplementation with these vitamins was prescribed. The screening tests for familial thrombophilia (including 677C-->T MTHFR mutation) were negative. The authors discuss the pathogenesis of increased thromboembolic risk in inflammatory bowel disease and therapeutic dilemmas connected with treatment of such complications.

[Effect of tea polyphenols on oxidative metabolism of polymorphonuclear neutrophils in healthy and obese people]. / Polifenole czerwonej herbaty a aktywnosc metabolizmu tlenowego neutrofili u osób zdrowych i otylych.

UNLABELLED: Obese people are at high risk for developing diabetes, dyslipidemia, hypertension, and cardiovascular diseases, which lead to an increased risk of mortality. Evidence for the potential role of oxidative stress in various diseases and pathophysiological processes suggests that the dietary intake and the therapeutic use of antioxidants may have positive health effects. The aim of the study was: 1) to investigate the ability of the major tea polyphenols: (-)-epigallocatechin gallate (EGCG), theaflavins (TF) and gallic acid (GA) to protect in vitro human neutrophils from oxidative damage induced by phorbol myristate acetate (PMA), 2) estimation the level of reactive oxygen species (ROS) production in obese patient depending on the red tea Pu-Erh drinking, 3) estimation inflammatory marker: CRP. MATERIAL AND METHODS: We tested 14 obese patients (aged 45+/-12 years, women, BMI=34+/-5.1 kg/m2). The inclusion criteria were based on physical examination, BMI, WHR, and the body composition examination based on bioimpedance method. PMA were isolated and oxidant production, in response to 1 microg/ml PMA, was characterized by the production of hydrogen peroxide, nitric oxide and chemiluminescence intensity. CRP level was assayed by the immunoturbidimetric test in serum. Control group consisted of healthy blood donors. RESULTS: Women consuming red tea revealed alteration in reactive oxygen species generation; the relative decrease of RFT was greater after 5 months than that after 1 month of treatment. A decrease in ROS generation after red tea consumption was accompanied by the decrease of ROS in response to tested compounds in normal cells. EGCG and TF showed similar potency in antioxidative activities. Tea polyphenols were not found to modulate CRP level in obese women. CONCLUSIONS: Tea may thus represent an important source of dietary antioxidants; there is need for more detailed studies to improve our understanding of the role of tea in reducing risk of major disease states.

[Oxidative metabolism of neutrophils in obese patients before and during body mass reduction: the in vitro effect of quercetin and rutin]. / Metabolizm tlenowy granulocytów oboj tnochlonnych u pacjentek z otyloscia przed i w trakcie redukcji masy ciala: wplyw kwercetyny i rutyny in vitro.

Obese people are at high risk for developing diabetes, dyslipidemia, hypertension, and cardiovascular diseases, which lead to an increased risk of mortality. Activated polymorphonuclear neutrophils (PMN) generate extremely high amounts of reactive oxygen species (ROS), but these are normally targeted at pathogens inside intracellular phagosomes. These same beneficial antimicrobial functions can cause significant local tissue injury and lead to the development of pathologic systemic inflammatory conditions. PMN apoptosis is a major mechanism associated with the resolution of inflammatory reactions. The goals of the present study were: 1) to evaluate the level of reactive oxygen species production in PMN from obese people before and during body mass reduction, 2) to investigate the in vitro effect of flavonoids: quercetin and rutin on oxidative metabolism and apoptosis of stimulated neutrophils in obese patient. We tested 30 obese patients (women) before body mass reduction and 20 patients during low calories diet. The inclusion criteria were based on physical examination, BMI, WHR, the body composition examination based on bioimpedance method and biochemical assessment. PMN were isolated and oxidant production, in response to 1 microg/ml PMA, was characterised by the production of hydrogen peroxide, nitric oxide and chemiluminescence intensity. Caspase-3 activation was assayed by the method of DEVD-AMC cleavage in PMN cultured up to 24 hours. The results of our study showed: 1) the decrease in PMN oxidant production in patient during the mass reduction, 2) the strong antioxidant activity of quercetin and rutin in obese patients before and during the body mass reduction, these effects were dose dependent and rutin was less potent than quercetin, 3) acceleration of PMN apoptosis by rutin is associated with an increase in caspase 3 activity.

[Does smoking influence the course of Crohn's disease in patients with the presence of gene NOD2/CARD15 mutation?]. / Czy palenie papierosów ma wplyw na przebieg choroby Lesniowskiego-Crohna u chorych z obecnym wariantem mutacji genu NOD2/CARD15?

Inflammatory bowel diseases can be divided into two diseases: ulcerative colitis and Crohn's disease. It is well known that there is the influence of smoking on these course of diseases and the number of flares. This influence is different in both diseases and is definitely negative among patients with Crohn's disease. The aim of the study was to establish if there is a difference of smoking influence on the course of disease depending if the patient is the carrier of NOD2/CARD15 mutation or not. 150 patients with CD was examined by careful interview about smoking habits, physically and there was molecular analyze performed of monocytes' DNA. The most often variant of NOD2/CARD15 mutation in Polish population was 802 C>T which causes the conversion of proline into serine in 268 position of protein product. The second most often observed variant was found during the analyze of exon 11 in the temperature of 20 degrees C. There was a frameshift mutation 3020insC present in 14.9% patients with CD which causes the C insertion in 3020 position of protein product. All patients with the frameshift mutation were also carriers of 802C>T mutation. The analysed features were the course of disease, the smoking habit and the difference in group of NOD2/CARD15 carries and patients without the mutation. We took into consideration the fact if the affected person was smoker, ex-smoker or non-smoker. Patients with 802C>T mutation ex-smokers and smokers were older on the onset of the disease (average: 35.8 years), whereas the non-smokers (average: 26.07 years). And what is more interesting, the non-smoking patients were statistically less frequent operated (the partial resection of terminal ileum). Only 31.82% of non-smoking patient with 802C>T mutation were operated.

Beneficial in vitro effect of N-acetyl-cysteine on oxidative stress and apoptosis.

Chronic renal failure (CRF) is usually accompanied by abnormalities of both humoral and cellular immune response. The aim of the study was to investigate the influence of N-acetyl-cysteine (NAC) on intracellular oxidative stress and apoptosis rate of T lymphocytes in children with CRF. Twenty-two children (aged 4-16, mean 7.4) with CRF treated with dialysis were enrolled in the study. Intracellular reactive oxygen species (ROS) production was quantified by mean rhodamine 123 (RHO) fluorescence intensity with flow cytometry. Annexin V FITC was used for identifying apoptotic cells. Mean fluorescence intensity (MFI), which reflected intracellular oxidative stress in T lymphocytes, was increased in patients with CRF compared with the controls (CD3+: 31.58+/-11.58 vs 22.55+/-4.97, p = 0.043; CD3+CD4+: 32.50+/-8.59 vs 27.75+/-12.76, NS; CD3+CD8+: 32.10+/-11.85 vs 20.77+/- 4.89, p =0.012). Apoptotic T lymphocytes occurred more frequently in patients with CRF treated with hemodialysis (HD) (11.36+/-6.96%) than in the controls (6.14%+/-3.36%; p = 0.025). After 24 h incubation with NAC MFI and apoptosis rate decreased significantly in all subpopulations of lymphocytes. NAC, as a strong antioxidant, has a favorable effect on intracellular oxidative stress and apoptosis rate of T lymphocytes in patients with CRF. A decreased apoptosis rate may have positive effect on functional abnormalities of T cells already found in patients with CRF.

Apoptosis and antioxidant defense in the nephrotic syndrome.

Nephrotic syndrome (NS) is accompanied, and probably caused by, abnormalities in T lymphocyte function. The aim of this study was to investigate the antioxidant status of children with NS and its influence on the apoptosis of T cells. Fifty-seven children with NS were studied, aged 4-16 years (mean 7.4 years), 34 with a first episode (group I) and 23 in remission (>6 months) of NS (group II). The control group comprised 26 healthy children matched for age. Annexin V-FITC was used as a sensitive probe for identifying cells undergoing apoptosis. We found that apoptotic T lymphocytes occurred more frequently in patients with a first episode of NS than in children in remission and in the controls. In group I, total antioxidant status (TAS, plasma) was significantly reduced compared with controls (0.77+/-0.14 vs. 1.18+/-0.42 mmol/l, P<0.001). In group I children, glutathione reductase (GR, red blood cells) and glutathione peroxidase (GPX, red blood cells) activity was lower than in controls (GR 8.10+/-2.40 vs.10.55+/-3.81 U/g Hb, P<0.001) (GPX 28.65+/-6.99 vs. 33.84+/-13.11 U/g Hb, P=0.010). TAS levels and GR activity in group II were also lower than in the controls. A negative correlation between GR activity and the apoptosis rate of T lymphocytes was found. We conclude that in patients with NS, reduced antioxidant defense may contribute to an increase in the apoptosis rate of circulating lymphocytes.

Decreased antioxidant activity in hypercholesterolemic children with nephrotic syndrome.

BACKGROUND: The aim of the present study was to estimate the total antioxidant status (TAS) and main antioxidant enzyme activity in children with nephrotic syndrome (NS). MATERIAL/METHODS: The study was carried out in a group of 82 children with NS, age 4-16 years (mean 7.4). Of these, 59 with a first episode formed group I, and 23 children in remission (>6 months, with no immunosuppressive treatment) formed group II. TAS was estimated using the two-reagent Randox Total Antioxidant Status test in plasma. RESULTS: TAS was significantly reduced in group I compared to controls (0.77+/-0.14 mmol/l vs. 1.18+/-0.42 mmol/l; p<0.001). The TAS level was more reduced in those patients with higher total cholesterol (TChol) and low density lipoproteins cholesterol (LDL-cholesterol). In children from group I the activity of glutathione reductase (GR) was lower than in the controls (8.10+/-2.40 U/g Hb vs. 10.55+/-3.81 U/g Hb, p<0.001) and similarly the activity of glutathione peroxidase (GPX) was lower in group I compared to controls (28.65+/-6.99 U/g Hb vs. 33.84+/-13.11 U/g Hb, p=0.010). There was no significant difference in superoxide dismutase (SOD) activity between the 3 groups. CONCLUSIONS: Reduced antioxidant activity may be related to lipid abnormalities in NS.

Increased apoptosis of peripheral blood lymphocytes in children with nephrotic syndrome.

Nephrotic syndrome is accompanied by and probably related to abnormal T-lymphocyte function. Decreased stimulation of survival factors and increased levels of "dead signals" may lead to the malfunction of many cells, including lymphocytes. In our study, we investigated the process of apoptosis within T cells in children with a first attack of nephrotic syndrome (NS). We found that the number of apoptotic T cells is greater in these patients than in both children in remission from NS and in controls. The percentage of annexin-V-fluorescein isothiocyanate (FITC)-positive CD3+ cells was 27.30+/- 12.13% in children with a first attack of NS, 19.22+/- 15.16% (P=0.006) in children in remission and 16.20+/- 6.13% (P=0.004) in controls. The percentage of annexin-V-FITC-positive CD3+CD4+ cells was 7.35+/-7.72% in children with a first attack of NS, 3.80+/-3.75% (P=0.0001) in children in remission and 3.82+/-2.01% (P=0.0002) in controls. We conclude that abnormal number and function of T lymphocytes found in NS patients may be related to an increased apoptotic rate of circulating lymphocytes.