Ľubomíra Tóthová

In vivo phage display — A discovery tool in molecular biomedicine ☆

In vivo phage display is a high-throughput method for identifying target ligands specific for different vascular beds. Targeting is possible due to the heterogeneous expression of receptors and other antigens in a particular vascular bed. Such expression is additionally influenced by the physiological or pathological status of the vasculature. In vivo phage display represents a technique that is usable in both, vascular mapping and targeted drug development. In this review, several important methodological aspects of in vivo phage display experiments are discussed. These include choosing an appropriate phage library, an appropriate animal model and the route of phage library administration. In addition, peptides or antibodies identified by in vivo phage display homing to specific types of vascular beds, including the altered vasculature present in several types of diseases are summarized. Still, confirmation in independent experiments and reproduction of identified sequences are needed for enhancing the clinical applicability of in vivo phage display research.

Markers of oxidative stress in plasma and saliva in patients with multiple sclerosis

BACKROUNDnOxidative stress plays a role in multiple sclerosis. Saliva can be potentially used to study the disease progression or treatment, because of its non-invasiveness and easy collection. But studies on saliva and multiple sclerosis are missing. The aim of this study was to compare the concentrations of salivary oxidative stress markers in patients and healthy controls.nnnOBJECTIVEnWhole saliva and blood samples were collected from 29 patients and 29 healthy controls. Samples were collected during relapse, after corticosteroid therapy, and after three months. Markers of oxidative, carbonyl stress and antioxidant status were measured.nnnRESULTSnIn plasma, thiobarbituric acid reacting substances, advanced oxidation protein products and fructosamine were significantly higher in patients compared to controls (by 271%, 46% and 24%, respectively; p<0.01). Total antioxidant capacity in plasma was lower by 20% (p<0.01) in patients versus controls. In saliva, higher levels of thiobarbituric acid reacting substances and advanced glycation end-products were observed in patients when compared to controls (by 51% and 49% respectively; p<0.01). Ferric reducing ability was reduced by 38% (p<0.05) in patients with multiple sclerosis.nnnCONCLUSIONnAccording to our knowledge, this is the first report showing higher markers of oxidative stress and lower antioxidant status in patients with multiple sclerosis in saliva.

Salivary markers of oxidative stress in patients with oral premalignant lesions

The aetiology of oral premalignant lesions is unknown. Oxidative stress is associated with inflammation and cancerogenesis. The aim of our study was to compare salivary markers of oxidative and carbonyl stress in patients with oral premalignant lesions and age-matched healthy controls. Unstimulated saliva samples were collected from 16 patients with oral premalignant lesions (leukoplakia, lichen planus, erythroplakia) and 16 age-matched healthy controls. Biochemical analysis included measurement of thiobarbituric acid reacting substances (TBARS), advanced oxidation protein products (AOPP), advanced glycation endproducts (AGEs) and total antioxidant capacity (TAC). Salivary RNA was analyzed using real time PCR. Salivary TBARS and AGEs were significantly higher in patients than in controls. No differences were found in AOPP. TAC and expression of superoxide dismutase were lower in patients than in age-matched controls. Other analyzed transcripts (vascular endothelial growth factor, sialotransferase, neuraminidase) did not differ between patients and the control group. Markers of lipoperoxidation and carbonyl stress were increased in patients with oral premalignant lesions. Decreased antioxidant status potentially due to decreased expression of antioxidant enzymes might be responsible for these findings. Our results might point to the aetiology or pathogenesis of oral premalignant lesions as well as to the mechanism of transition to oral carcinoma.

Salivary DNA and markers of oxidative stress in patients with chronic periodontitis

ObjectivesPrevious observational studies have shown that periodontal status is associated with salivary markers of oxidative damage. A direct comparison of periodontitis patients and controls using a wide palette of salivary markers of oxidative stress is lacking. Characteristics of salivary DNA in periodontitis are unknown. The aim of this study was to compare the salivary markers of oxidative stress and characteristics of salivary DNA between patients with chronic periodontitis and periodontitis-free controls.Materials and methodsSaliva was collected from 23 patients with chronic periodontitis and 19 periodontitis-free controls. All participants underwent a clinical periodontal examination. Markers of oxidative and carbonyl stress were measured in saliva. Human and bacterial DNA was quantified, and human DNA integrity was assessed.ResultsSalivary thiobarbituric acid-reacting substances were higher in patients than in controls; at least in men, the difference was significant (pu2009<u20090.01). In women, patients had significantly lower salivary antioxidant status (pu2009<u20090.001). No quantitative differences were found regarding salivary DNA. Tendencies towards reduced DNA integrity were found in periodontitis patients.ConclusionsThe results confirmed the association of salivary thiobarbituric acid-reacting substances with periodontitis. Lipid peroxidation in periodontitis seems to be caused by increased production of reactive oxygen species in men and by decreased antioxidant status in women. Whether lower salivary DNA integrity is involved in the pathogenesis of periodontitis remains to be elucidated.Clinical relevanceSalivary thiobarbituric acid-reacting substances are associated with periodontitis at least on a population level. Sex-specific causes of lipid peroxidation might point towards different pathogenic mechanisms.

Sex Differences in Experimentally Induced Colitis in Mice: a Role for Estrogens

Sex differences have been found in the incidence and progression of inflammatory bowel diseases (IBD) such as ulcerative colitis and Crohn’s disease. The reported differences in observational studies are controversial, and the effects of sex hormones on the pathogenesis of IBD are not clear. The aim of this study was to analyze sex differences in the progression of experimentally induced colitis. Experimental colitis was induced in adult mice by adding 2xa0% dextran sodium sulfate (DSS) into drinking water. Male and female mice were used as intact, gonadectomized, and supplemented with either estradiol or testosterone. In comparison to males, female mice with induced colitis had significantly longer colon (pu2009<u20090.05), lower decrease in body weight (pu2009<u20090.001), and lower stool consistency score (pu2009<u20090.05). Histopathological analysis showed less inflammatory infiltrates (pu2009<u20090.001) and crypt damage (pu2009<u20090.001) in female mice. Female mice with colitis had also lower concentration of TNF-α in colon homogenates (pu2009<u20090.01). Supplementation with estradiol in ovariectomized mice ameliorated the severity of colitis. Female mice are partially protected against chemically induced colitis. This protection seems to be mediated by estradiol.

Salivary markers of oxidative stress are related to age and oral health in adult non‐smokers

BACKGROUNDnSalivary concentrations of thiobarbituric acid-reacting substances (TBARS) are associated with the periodontal status assessed as papillary bleeding index (PBI). Whether this association is age independent is currently unclear. Salivary concentrations of advanced oxidation protein products (AOPPs) and advanced glycation end products (AGEs) have not been assessed in relation to age or oral health yet. The aim of our study was to analyse salivary markers of oxidative stress in dental patients in relation to age, gender and oral health.nnnMETHODSnConsecutive adult non-smoking dental patients were enrolled (n = 204; aged 19-83 years). PBI and the caries index (CI) were assessed. Markers of oxidative stress, such as TBARS, AOPPs and AGEs, and the total antioxidant capacity (TAC) were measured in saliva samples taken before clinical examination.nnnRESULTSnLinear regression showed that salivary TBARS, AGEs and TAC significantly increase with age (r squared = 5.3%, 2.1% and 5%, respectively). PBI is an independent predictor of salivary TBARS (r squared = 5.5%), and the CI negatively affected AOPPs (r = 3.2%) and positively affected TBARS (r = 2.5%). Gender did not affect any of the analysed parameters.nnnCONCLUSIONSnAge as a significant contributor to the variance should be taken into account in studies focusing on salivary markers of oxidative stress. The relationship between PBI and salivary TBARS confirms results from previous studies. In addition, our results show that the association is age independent. Negative association between the CI and AOPPs might be related to recent findings that AOPP might be actually a marker of non-enzymatic antioxidant status.

Blood Contamination in Saliva: Impact on the Measurement of Salivary Oxidative Stress Markers

Salivary oxidative stress markers represent a promising tool for monitoring of oral diseases. Saliva can often be contaminated by blood, especially in patients with periodontitis. The aim of our study was to examine the impact of blood contamination on the measurement of salivary oxidative stress markers. Saliva samples were collected from 10 healthy volunteers and were artificially contaminated with blood (final concentration 0.001–10%). Next, saliva was collected from 12 gingivitis and 10 control patients before and after dental hygiene treatment. Markers of oxidative stress were measured in all collected saliva samples. Advanced oxidation protein products (AOPP), advanced glycation end products (AGEs), and antioxidant status were changed in 1% blood-contaminated saliva. Salivary AOPP were increased in control and patients after dental treatment (by 45.7% and 34.1%, p < 0.01). Salivary AGEs were decreased in patients after microinjury (by 69.3%, p < 0.001). Salivary antioxidant status markers were decreased in both control and patients after dental treatment (p < 0.05 and p < 0.01). One % blood contamination biased concentrations of salivary oxidative stress markers. Saliva samples with 1% blood contamination are visibly discolored and can be excluded from analyses without any specific biochemic detection of blood constituents. Salivary markers of oxidative stress were significantly altered in blood-contaminated saliva in control and patients with gingivitis after dental hygiene treatment.

Oxidative and carbonyl stress in pregnant women with obstructive sleep apnea

PurposePregnant women are particularly susceptible to sleep-disordered breathing. Obstructive sleep apnea (OSA) in pregnancy is associated with poor pregnancy and fetal outcomes. Oxidative stress caused by intermittent hypoxemia and reoxygenation may impact pregnancy health. We hypothesize that pregnant women with OSA have a pronounced oxidative stress profile.MethodsA case-control study was performed to study oxidative stress markers in the serum of pregnant women with or without OSA. Patients with OSA were identified between 2003 and 2009. Contemporaneous controls were pregnant subjects without apnea, gasping, or snoring around the time of delivery. Serum markers of oxidative and carbonyl stress were measured by spectrophotometric/fluorometric methods. Multiple linear regression analysis was used with a model including age, body mass index at delivery, history of diabetes, and gestational age.ResultsSerum samples from 23 OSA cases and 41 controls were identified. Advanced oxidation protein products, a marker for oxidative stress, and advanced glycation end products (AGEs), a marker for carbonyl stress, were significantly lower in women with OSA than in controls (p value <0.0001). Total antioxidant capacity was higher in women with OSA in comparison to controls (p value <0.0001). The difference in AGEs remained significant even after adjusting for confounders.ConclusionContrary to our hypothesis, the results of this study suggest that pregnant women with OSA have higher antioxidant capacity and lower oxidative and carbonyl stress markers compared to controls, suggesting a possible protective effect of intermittent hypoxia. Whether OSA in pregnancy impacts oxidative stress differently than OSA in the general population remains to be confirmed.

Salivary markers of kidney function — Potentials and limitations

Saliva can be collected non-invasively, repeatedly and without trained personnel. It is a promising diagnostic body fluid with clinical use in endocrinology and dentistry. For decades, it is known that saliva contains also urea, creatinine and other markers of renal function. Clinical studies have shown that the salivary concentrations of these markers could be useful for the assessment of kidney function without the need of blood collection. This article summarizes the clinical and experimental data on the use of saliva as a diagnostic fluid in nephrology and points out the advantages, pitfalls, technical requirements and future perspective for the use of saliva as a novel potential diagnostic biofluid.

Improved Preservation of Warm Ischemia-Damaged Porcine Kidneys after Cold Storage in Ecosol, a Novel Preservation Solution

BACKGROUNDnEcosol, an extracellular-type, colloid-based preservation solution, has recently been introduced for washout, cold storage, and machine perfusion preservation of kidney grafts. Here, we assessed the efficacy of Ecosol compared to the widely used Histidine-Tryptophan-Ketoglutarate solution (HTK) for 24-h cold storage preservation of warm ischemia-damaged kidney grafts.nnnMATERIAL AND METHODSnBefore recovery, warm ischemia was induced by clamping the renal pedicle for 45-min. Thereafter, kidneys were washed-out and cold-stored for 24-h in Ecosol or HTK solution. Kidneys recovered without warm ischemia and cold-stored for 24-h in HTK served as controls (n=5). Renal function and damage parameters were assessed during 1-h normothermic reperfusion using the isolated perfused porcine kidney model.nnnRESULTSnRenal function did not differ between Ecosol and controls and was significantly reduced in HTK compared to controls. Total output of urine was higher in Ecosol compared to HTK. Intrarenal resistance and urine protein concentrations in Ecosol were lower compared to HTK and equal to controls. In the Ecosol group, oxygen consumption during reperfusion was higher and reduced tissue lipid peroxidation products were detected compared to HTK.nnnCONCLUSIONSnThe preservation quality of warm ischemia-damaged, cold-stored porcine kidneys was improved using the recently developed Ecosol preservation solution compared to HTK.