Roland Pálffy

On the physiology and pathophysiology of antimicrobial peptides.

Antimicrobial peptides (AMP) are a heterogeneous group of molecules involved in the nonspecific immune responses of a variety of organisms ranging from prokaryotes to mammals, including humans. AMP have various physical and biological properties, yet the most common feature is their antimicrobial effect. The majority of AMP disrupt the integrity of microbial cells by 1 of 3 known mechanisms—the barrel-stave pore model, the thoroidal pore model, or the carpet model. Results of growing numbers of descriptive and experimental studies show that altered expression of AMP in various tissues is important in the pathogenesis of several gastrointestinal, respiratory, and other diseases. We discuss novel approaches and strategies to further improve the promising future of therapeutic applications of AMP. The spread of antibiotic resistance increases the importance of developing a clinical role for AMP.

Bacteria in gene therapy: bactofection versus alternative gene therapy

Recent advances in gene therapy can be attributed to improvements of gene delivery vectors. New viral and nonviral transport vehicles that considerably increase the efficiency of transfection have been prepared. However, these vectors still have many disadvantages that are difficult to overcome, thus, a new approach is needed. The approach of bacterial delivery could in the future be important for gene therapy applications. In this article we try to summarize the most important modifications that are used for the preparation of applied strains, difficulties that are related with bacterial gene delivery and the current use of bactofection in animal experiments and clinical trials. Important differences to the alternative gene therapy (AGT) are discussed. AGT resembles bacteria-mediated protein delivery, as the therapeutical proteins are produced not by host cells but by the bacteria in situ and the expression can be regulated exogenously. Although the procedure of bacterial gene delivery is far from being definitely solved, bactofection remains a promising technique for transfection in human gene therapy.

Gene therapy for cancer: bacteria-mediated anti-angiogenesis therapy

Several bacterial species have inherent ability to colonize solid tumors in vivo. However, their natural anti-tumor activity can be enhanced by genetic engineering that enables these bacteria express or transfer therapeutic molecules into target cells. In this review, we summarize latest research on cancer therapy using genetically modified bacteria with particular emphasis on blocking tumor angiogenesis. Despite recent progress, only a few recent studies on bacterial tumor therapy have focused on anti-angiogenesis. Bacteria-mediated anti-angiogenesis therapy for cancer, however, is an attractive approach given that solid tumors are often characterized by increased vascularization. Here, we discuss four different approaches for using modified bacteria as anti-cancer therapeutics—bactofection, DNA vaccination, alternative gene therapy and transkingdom RNA interference—with a specific focus on angiogenesis suppression. Critical areas and future directions for this field are also outlined.

Continuous light and L-NAME-induced left ventricular remodelling: different protection with melatonin and captopril.

Objective Blood pressure enhancement induced by continuous light exposure represents an attractive but rarely investigated model of experimental hypertension. Design and methods The aim of this study was to show whether the combination of continuous light (24 h/day) exposure and chronic NG-nitro-L-arginine-methyl ester (L-NAME) treatment induces remodelling of the left ventricle and whether captopril or melatonin can modify these potential alterations. Six groups of 3-month-old Wistar rats (nine per group) were treated for 6 weeks: control (untreated), L-NAME (40 mg/kg per day), exposed to continuous light, L-NAME treated and exposed to continuous light (L24), L24 rats treated with either captopril 100 mg/kg per day, or melatonin (10 mg/kg/24 h). Systolic blood pressure (SBP), relative weights of the left ventricle, endothelial nitric oxide synthase (eNOS) and angiotensin-converting enzyme (ACE) expression in tissues, malondialdehyde and advanced oxidation protein product concentrations in the plasma and hydroxyproline levels in collagenous protein fractions were measured. Results The continuous light and L-NAME treatment led to hypertension, left ventricular hypertrophy (LVH) and fibrosis. An increase in SBP was completely prevented by captopril and partly by melatonin in the L24 group. Both drugs reduced oxidative damage and attenuated enhanced expression of ACE in the myocardium. Neither of the drugs prevented the attenuation of eNOS expression in the combined hypertensive model. Only captopril reduced LVH development in L24, whereas captopril and melatonin reduced left ventricular hydroxyproline concentrations in soluble and insoluble collagen, respectively. The total hydroxyproline concentration was reduced only by melatonin. Conclusion In hypertension induced by a combination of continuous light and L-NAME treatment, melatonin and captopril protect the heart against pathological left ventricular remodelling differently.

Variability of thiobarbituric acid reacting substances in saliva

Introduction: Salivary TBARS are a potential marker of oxidative stress in the oral cavity. Previous studies have found increased levels of salivary TBARS in various diseases. The aim of this study was to assess the variability of salivary TBARS in both genders. Subjects & Methods: Saliva samples from thirty-eight healthy volunteers (18F & 20M) were collected every day during 30 day period. TBARS levels were measured spectrophotometrically using a high-throughput 96-well plate method. Time series analysis was performed using standard statistical methods. Results: Repeated measures ANOVA showed a significant variation of salivary TBARS within day and subjects (p < 0.001). The dynamics did not differ between genders. Intraindividual variability was very high in both genders with coefficients of variation of more than 60%. Interindividual variability was higher in men than in women (73% vs. 46%; p < 0.01). Discussion: The relatively high intraindividual variability indicates that the use of salivary TBARS will be limited to research on a population level, although some informative value might be gained by repeated samplings. Factors influencing the biological variability of salivary TBARS should be identified in further studies.

Salmonella-mediated gene therapy in experimental colitis in mice.

Bacterial gene therapy – bactofection is a simple and effective method to deliver plasmid DNA into target tissue. We hypothesize that oral in vivo bactofection can be an interesting approach to influence the course of inflammatory bowel diseases. The aim of this study was to prove the effects of antioxidative and anti-inflammatory bactofection in dextran sulfate sodium (DSS)-treated mice. Attenuated bacteria Salmonella Typhimurium SL7207 carrying plasmids with genes encoding Cu-Zn superoxide dismutase and an N-terminal deletion mutant of monocyte chemoattractant protein-1 were prepared. Male Balb/c mice had ad libitum access to 1% DSS solution in drinking water during 10 days (mild model of colitis). The animals were daily fed with 200 Mio bacteria via gastric gavage during the experiment. Fecal consistency, clinical status, food and water intake were monitored. After 10 days samples were taken and markers of oxidative stress and inflammatory cytokine levels were measured. Colonic tissue was scored histologically by a blinded investigator. DSS treatment significantly increased the levels of inflammatory cytokines and malondialdehyde as a marker of lipoperoxidation in the colon. Anti-inflammatory gene therapy improved the total antioxidative capacity. In comparison with the untreated group, bacterial gene therapy lowered the histological colitis score. Salmonella-mediated antioxidative and anti-inflammatory gene therapy alleviated colitis in mice. The effect seems to be mediated by increased antioxidative status. Further studies will show whether recombinant probiotics expressing therapeutic gene might be used for the therapy of inflammatory bowel diseases.

Effect of unexpected sequence interruptions to conventional PCR and repeat primed PCR in myotonic dystrophy type 1 testing.

Myotonic dystrophy type 1 (DM1) is caused by expansion of the CTG trinucleotide repeat in the DMPK gene. Our study focuses on the effect of recently described unusual sequence interruptions inside the CTG tract on conventional polymerase chain reaction (PCR) and triplet repeat primed PCR (TP-PCR) amplifications, which are the methods now widely used in molecular testing for DM1. For molecular characterization of the CTG repeat tract, we used conventional fluorescent PCR with bidirectional labeling and both forward and reverse direction TP-PCR. Though the results of the methods are still unambiguous for most alleles, mistyping and false results may occur in the typing of some unordinary alleles carrying sequence interruptions. The presence of these interruptions may lead not only to altered TP-PCR profiles, as can be expected, but also to abnormal electrophoretic mobility of complementary strands produced by conventional amplification of such alleles. Our findings suggest that the simultaneous combination of bidirectionally labeled conventional PCR with TP-PCR performed in both directions may be necessary for increasing the reliability and accuracy of the TP-PCR-based assay for DM1 testing.

Renal and metabolic effects of three months of decarbonated cola beverages in rats.

Epidemiological studies have shown an association between the intake of cola beverages and chronic kidney diseases. Experimental evidence for the negative effects of cola intake on kidneys is lacking. Male Wistar rats had ad libitum access to water (control group) or three different sugar-sweetened cola beverages for three months. Despite very high cola intake (daily cca 140 mL), no differences were found in body weight, kidney weight, glomerular morphology, oxidative and carbonyl stress or expression of selected marker genes in the renal cortex. Interestingly, all groups consuming cola beverages had lower blood glucose levels during an oral glucose tolerance test, suggesting improved insulin sensitivity. Despite hyperfiltration (5–6-fold increase in diuresis), cola beverages had no effect on assessed parameters of renal function, histology, gene expression or oxidative stress. Moreover, cola intake seems to increase creatinine clearance and to decrease plasma levels of urea. In our study increased insulin sensitivity and altered renal functional parameters were observed in rats receiving cola beverages for three months. Whether the findings are due to the short duration of the study or interspecies metabolic differences should be uncovered in further studies. Even more interesting might be the analysis of effects of cola intake in animal models of diabetes.

The Effects of Anti-Inflammatory and Anti-Angiogenic DNA Vaccination on Diabetic Nephropathy in Rats

Inflammation and angiogenesis play a crucial role in the pathomechanism of diabetic nephropathy. Monocyte chemoattractant protein 1 (MCP) is a key regulator of the immune system in kidneys, and its inhibition with a dominant-negative mutant lacking the N-terminal amino acids 2-8 (7ND) reduces renal fibrosis. Angiomotin (Amot) is a novel angiogenesis modulator. We studied the effects of inhibition of Amot and MCP using DNA vaccination on incipient diabetic nephropathy in rats. Plasmid DNA (with either 7ND or human Amot) was electroporated twice into hind-limb muscles of rats with streptozotocin-induced diabetes mellitus. Sham-electroporated diabetic rats and healthy animals served as controls. After 4 months, renal histology and biochemical analyses were performed. In sham-electroporated diabetic rats, glomerular histology revealed pathological changes. 7ND and Amot treatments reduced glomerular hypertrophy and periodic acid-Schiff positivity. In both treated groups, the expression of profibrotic (transforming growth factor-β, collagen 1), proinflammatory (interleukin-6, tumor necrosis factor-α), and proangiogenic (vascular endothelial growth factor) genes in the renal cortex was lower than in the diabetic group without treatment. The mentioned renoprotective effects could be mediated via higher total antioxidant capacity and improved glycemic control. Anti-angiogenic and anti-inflammatory DNA vaccination ameliorates the progression of glomerular pathology in an animal model of diabetic nephropathy.

The short-term effects of soybean intake on oxidative and carbonyl stress in men and women.

Beyond other beneficial effects, a soy-rich diet has been shown to reduce the risk of cardiovascular diseases and diabetic complications. Reduction of oxidative and carbonyl stress has been proposed as the underlying mechanism, but the evidence for this is lacking. The aim of our study was to evaluate the effects of short-term increased soy intake on oxidative and carbonyl stress parameters in young volunteers. Young healthy probands (omnivores) of both genders (55 women, 33 men) were given soybeans (2 g/kg bodyweight daily) for one week. Markers of oxidative and carbonyl stress were measured in plasma at the beginning and at the end of one week soybean intake and after another week of a wash-out period. Total antioxidant capacity was increased by soybean intake in both genders. This led to decreased levels of advanced oxidation protein products in women, but not in men. On the contrary, in men, soybean intake increased lipoperoxidation. No effects on carbonyl stress markers (advanced glycation end products-specific fluorescence and fructosamine) were found. Soybean intake has gender-specific effects on oxidative stress in young healthy probands potentially due to divergent action and metabolism of phytoestrogens in men and women. Effects of soybean intake on carbonyl stress should be evaluated in longer studies.