Thyroid disturbances after COVID-19 and the effect of vaccination in children: a prospective tri-center registry analysis.

Rapidly evolving clinical data suggest that the novel coronavirus (SARS-CoV-2) and vaccination against COVID-19 might be associated with thyroid disturbances. However, studies remain limited among the pediatric population. Our aim was to assess the prevalence and permanence of thyroid autoimmunity (TA) and dysfunction in children after an acute infection and its potential association with vaccination. A prospective, multicenter registry analysis was performed among 458 children (mean age: 12.4 ± 3,8 years, 45.4% male) with preceding COVID-19. Patient inclusion lasted from 24th March, 2021 to 23rd March, 2022 at three pediatric outpatient facilities at Semmelweis University, Budapest. Primary outcomes were the rate of thyroid disturbances assessed by laboratory parameters (thyroid function tests, antithyroglobulin [ATG] and anti-thyroid peroxidase [ATPO] antibodies) and thyroid ultrasound. TA rate among vaccinated and unvaccinated children was determined. Children with newly diagnosed thyroid alterations were followed up for 12.7 ± 4.3 months. Six children had previous thyroid disease. Out of 452 children, 30 cases (6.6%) of newly diagnosed TA (six of them had abnormal thyroid-stimulating hormone [TSH] levels) and eight cases (1.8%) of isolated TSH elevation were observed. Ultrasound-proven autoimmune thyroiditis (AIT) was 4.0%. No association was found between COVID-19 vaccination and thyroid autoimmunity (χ2(1,N = 452) = 0.138, p = 0.815). Among children with TA, 73.3% had long-lasting alterations.  Conclusion: Vaccination had no effect on the prevalence of TA. Until further controlled studies state otherwise, children with preceding COVID-19 might benefit from thyroid screening. What is Known: • Numerous case reports implicate that coronavirus disease-2019 (COVID-19) and vaccination against SARS-CoV-2 can be responsible for thyroid disturbances. • Thyroid alterations discovered during acute COVID-19 tend to cease by time and only incidental thyroid autoimmunity (TA) is diagnosed after COVID-19. In adults, no increase in vaccine-related hyper- or hypothyroidism was found. What is New: • TA rate after COVID-19 vaccination among children was not increased. TA had no role in long COVID syndrome. • We discovered a considerable rate of TA (6.6%) and ultrasound-proven autoimmune thyroiditis (AIT) (4.0%) after SARS-CoV-2 infection, and the majority of these alterations remained positive after 6 months.

Seroconversion after anti-SARS-CoV-2 mRNA vaccinations among moderate-to-severe psoriatic patients receiving systemic biologicals-Prospective observational cohort study.

It is unclear whether biological antipsoriatic therapies affect seroconversion after messenger ribonucleic acid (mRNA)-based antisevere acute respiratory syndrome coronavirus 2 (anti-SARS-CoV-2) vaccinations. To assess antibody formation and the incidence of side effects after anti-SARS-CoV-2 mRNA vaccinations in psoriatic patients receiving different biologicals compared to healthy controls. 102 moderate-to-severe psoriatic patients (56.2 [±13.5] years) and 55 age-matched healthy (56.4 ± 13.6 years) volunteers were included in our study. Ten to 21 days after the administration of the second dosage of BNT162b2 or mRNA-1273 vaccine, antibody levels specific to the SARS-CoV-2 spike (S) protein receptor binding domain were monitored. The incidence of postvaccination side effects was recorded and compared to real-life data in the literature. Of the 102 patients, 57 (55.88%) received tumor necrosis factor (TNF), 28 (27.45%) received interleukin (IL)-12/23, 16 (15.68%) received IL-17, and 1 (0.99%) received IL-23 inhibitors. No significant differences in the median serum level of anti-SARS-CoV-2S antibody were observed between the study population and the control group (median IQR range: 1681.0 U/mL (600.0-4844.0) versus 1984.0 U/mL (1000.0-3136.0; p = 0.82). The most frequent side effects of the mRNA vaccines within 7 days after the administration of both dosages were arm pain on the side of injection (23.53% and 23.53%), fatigue (9.80% and 13.72%), headache (4.9% and 5.88%), and chills or shivering (4.9% and 8.82%). Detectable antibodies against SARS-CoV-2S protein appear 10-21 days after the administration of the second dosage of BNT162b2 or mRNA-1273 vaccines in moderate-to-severe psoriatic patients receiving biologicals, similar to those of healthy controls.

Relationship between leukocyte counts and large vessel occlusion in acute ischemic stroke.

BACKGROUND: Neuroinflammation plays an important role in the pathogenesis of acute ischemic stroke (AIS) and peripheral leukocyte counts have proved to be independent predictors of stroke severity and outcomes. Clinical significance of large vessel occlusion (LVO) in AIS is increasing, as these patients are potential candidates for endovascular thrombectomy and likely to have worse outcomes if not treated urgently. The aim of our study was to assess the relationship between on admission leukocyte counts and the presence of LVO in the early phase of AIS. METHODS: We have conducted a cross-sectional, observational study based on a registry of consecutive AIS patients admitted up to 4.5 h after stroke onset. Blood samples were taken at admission and leukocyte counts were measured immediately. The presence of LVO was verified based on the computed tomography angiography scan on admission. RESULTS: Total white blood cell (WBC) and neutrophil counts were significantly higher in patients with LVO than those without LVO (P < 0.001 respectively). After adjustment for potential confounders total WBC counts (adjusted OR: 1.405 per 1 × 109/L increase, 95% CI: 1.209 to 1.632) and neutrophil counts (adjusted OR: 1.344 per 1 × 109/L increase, 95% CI: 1.155 to 1.564) were found to have the strongest associations with the presence of LVO. Total WBC and neutrophil counts had moderate ability to discriminate an LVO in AIS (AUC: 0.667 and 0.655 respectively). No differences were recorded in leukocyte counts according to the size of the occluded vessel and the status of collateral circulation in the anterior vascular territory. However, total WBC and neutrophil counts tended to be higher in patients with LVO in the posterior circulation (p = 0.005 and 0.010 respectively). CONCLUSION: Higher admission total WBC and neutrophil counts are strongly associated with the presence of LVO and has moderate ability to discriminate an LVO in AIS. Detailed evaluation of stroke-evoked inflammatory mechanisms and changes according to the presence of LVO demands further investigation.

Escalating SARS-CoV-2 specific humoral immune response in rheumatoid arthritis patients and healthy controls.

Background: Immunocompromised patients are at particular risk of Severe Acute Respiratory Syndrome Corona Virus 2 (SARS-CoV-2) infection and previous findings suggest that the infection or vaccination induced immune response decreases over time. Our main goal was to investigate the SARS-CoV-2-specific immune response in rheumatoid arthritis patients and healthy controls over prolonged time. Methods: The SARS-CoV-2-specific humoral immune response was measured by Elecsys Anti-SARS-CoV-2 Spike (S) immunoassay, and antibodies against SARS-CoV-2 nucleocapsid protein (NCP) were also evaluated by Euroimmun enzyme-linked immunosorbent assay (ELISA) test. The SARS-CoV-2-specific T-cell response was detected by an IFN- γ release assay. Results: We prospectively enrolled 84 patients diagnosed with rheumatoid arthritis (RA) and 43 healthy controls in our longitudinal study. Our findings demonstrate that RA patients had significantly lower anti-S antibody response and reduced SARS-CoV-2-specific T-cell response compared to healthy controls (p<0.01 for healthy controls, p<0.001 for RA patients). Furthermore, our results present evidence of a notable increase in the SARS-CoV-2-specific humoral immune response during the follow-up period in both study groups (p<0.05 for healthy volunteers, p<0.0001 for RA patients, rank-sum test). Participants who were vaccinated against Coronavirus disease-19 (COVID-19) during the interim period had 2.72 (CI 95%: 1.25-5.95, p<0.05) times higher anti-S levels compared to those who were not vaccinated during this period. Additionally, individuals with a confirmed SARS-CoV-2 infection exhibited 2.1 times higher (CI 95%: 1.31-3.37, p<0.01) anti-S levels compared to those who were not infected during the interim period. It is worth noting that patients treated with targeted therapy had 52% (CI 95%: 0.25-0.94, p<0.05) lower anti-S levels compared to matched patients who did not receive targeted therapy. Concerning the SARS-CoV-2-specific T-cell response, our findings revealed that its level had not changed substantially in the study groups. Conclusion: Our present data revealed that the level of SARS-CoV-2-specific humoral immune response is actually higher, and the SARS-CoV-2-specific T-cell response remained at the same level over time in both study groups. This heightened humoral response, the nearly permanent SARS-CoV-2-specific T-cell response and the coexistence of different SARS-CoV-2 variants within the population, might be contributing to the decline in severe COVID-19 cases.

T cell immune response predicts survival in severely ill COVID-19 patients requiring venovenous extracorporeal membrane oxygenation support.

Introduction: There is a critical gap in understanding which SARS-CoV-2 patients would benefit most from venovenous extracorporeal membrane oxygenation (VV-ECMO) support. The potential role of a dysregulated immune response is still unclear in this patient population. Objectives: To assess the potential predictive value of SARS-CoV-2 specific cellular and humoral immune responses for survival in critically ill COVID-19 patients requiring VV-ECMO. Methods: We conducted a prospective single-center observational study of unvaccinated patients requiring VV-ECMO support treated at the intensive care unit of Semmelweis University Heart and Vascular Center between March and December 2021. Peripheral blood samples were collected to measure the humoral and cellular immune statuses of the patients at the VV-ECMO cannulation. Patients were followed until hospital discharge. Results: Overall, 35 COVID-19 patients (63% men, median age 37 years) on VV-ECMO support were included in our study. The time from COVID-19 verification to ECMO support was a median (IQR) of 10 (7-14) days. Of the patients, 9 (26%) were discharged alive and 26 (74%) died during their hospital stay. Immune tests confirmed ongoing SARS-CoV-2 infection in all the patients, showing an increased humoral immune response. SARS-CoV-2-specific cellular immune response was significantly higher among survivors compared to the deceased patients. A higher probability of survival was observed in patients with markers indicating a higher T cell response detected by both QuantiFeron (QF) and flow cytometry (Flow) assays. (Flow S1 CD8+ ≥ 0.15%, Flow S1 CD4+ ≥ 0.02%, QF CD4 ≥ 0.07, QF whole genome ≥ 0.59). In univariate Cox proportional hazard regression analysis BMI, right ventricular (RV) failure, QF whole genome T cell level, and Flow S1 CD8+ T cell level were associated with mortality, and we found that an increased T cell response showed a significant negative association with mortality, independent of BMI and RV failure. Conclusion: Evaluation of SARS-CoV-2 specific T cell response before the cannulation can aid the risk stratification and evaluation of seriously ill COVID-19 patients undergoing VV-ECMO support by predicting survival, potentially changing our clinical practice in the future.

Factors influencing the SARS-CoV-2 infection and vaccination induced immune response in rheumatoid arthritis.

Background: To investigate the factors that have significant impact on the Severe Acute Respiratory Syndrome Corona Virus 2 (SARS-CoV-2) infection and vaccination induced immune response in rheumatoid arthritis (RA). Methods: Serological response was measured by quantifying anti-SARS-CoV-2 specific antibodies, while the cell-mediated response was measured by a whole-blood test quantifying the interferon (IFN)-γ response to different SARS-CoV-2-specific domains. Results: We prospectively enrolled 109 RA patients and 43 healthy controls. The median time (IQR) between the confirmed infection or the last vaccination dose and the day when samples were taken ("sampling interval") was 3.67 (2.03, 5.50) months in the RA group. Anti-Spike (anti-S) specific antibodies were detected in 94% of RA patients. Among the investigated patient related variables, age (p<0.004), sampling interval (p<0.001), the brand of the vaccine (p<0.001) and targeted RA therapy (TNF-inhibitor, IL-6 inhibitor, anti-CD20 therapy) had significant effect on the anti-S levels. After covariate adjustment TNF-inhibitor therapy decreased the anti-S antibody concentrations by 80% (p<0.001). The same figures for IL-6 inhibitor and anti-CD20 therapy were 74% (p=0.049) and 97% (p=0.002), respectively. Compared to subjects who were infected but were not vaccinated, the RNA COVID-19 vaccines increased the anti-S antibody levels to 71.1 (mRNA-1273) and 36.0 (BNT162b2) fold (p<0.001). The corresponding figure for the ChAdOx1s vaccine is 18.1(p=0.037). Anti-CCP (anti-cyclic citrullinated peptides) positive patients had 6.28 times (p= 0.00165) higher anti-S levels, than the anti-CCP negative patients. Positive T-cell response was observed in 87% of the healthy volunteer group and in 52% of the RA patient group. Following vaccination or infection it declined significantly (p= 0.044) but more slowly than that of anti-S titer (6%/month versus 25%). Specific T-cell responses were decreased by 65% in patients treated with anti-CD20 therapy (p=0.055). Conclusion: Our study showed that the SARS-CoV-2-specific antibody levels were substantially reduced in RA patients treated with TNF-α-inhibitors (N=51) and IL-6-inhibitor (N=15). In addition, anti-CD20 therapy (N=4) inhibited both SARS-CoV-2-induced humoral and cellular immune responses. Furthermore, the magnitude of humoral and cellular immune response was dependent on the age and decreased over time. The RNA vaccines and ChAdOx1s vaccine effectively increased the level of anti-S antibodies.

Immunological response and temporal associations in myocarditis after COVID-19 vaccination using cardiac magnetic resonance imaging: An amplified T-cell response at the heart of it?

Introduction: Although myocarditis after anti-SARS-CoV-2 vaccination is increasingly recognized, we have little data regarding the course of the disease and, consequently, the imaging findings, including the tissue-specific features. The purpose of this study is to describe the clinical, immunological, and cardiac magnetic resonance (CMR) features of myocarditis after COVID-19 immunization in the acute phase and during follow-up. We aimed to compare the trajectory of the disease to myocarditis cases unrelated to COVID-19. Methods: We assembled a CMR-based registry of potentially COVID-19 vaccination-related myocarditis cases. All patients who experienced new-onset chest pain and troponin elevation after COVID-19 vaccination and imaging confirming the clinical suspicion of acute myocarditis were enrolled in our study. Participants underwent routine laboratory testing and testing of their humoral and cellular immune response to COVID-19 vaccination. Clinical and CMR follow-up was performed after 3-6 months. We included two separate, sex- and age-matched control groups: (1) individuals with myocarditis unrelated to COVID-19 infection or vaccination confirmed by CMR and (2) volunteers with similar immunological exposure to SARS-CoV-2 compared to our group of interest (no difference in the number of doses, types and the time since anti-SARS-CoV-2 vaccination and no difference in anti-nucleocapsid levels). Results: We report 16 CMR-confirmed cases of myocarditis presenting (mean ± SD) 4 ± 2 days after administration of the anti-SARS-CoV-2 vaccine (male patients, 22 ± 7 years), frequently with predisposing factors such as immune-mediated disease and previous myocarditis. We found that 75% received mRNA vaccines, and 25% received vector vaccines. During follow-up, CMR metrics depicting myocardial injury, including oedema and necrosis, decreased or completely disappeared. There was no difference regarding the CMR metrics between myocarditis after immunization and myocarditis unrelated to COVID-19. We found an increased T-cell response among myocarditis patients compared to matched controls (p < 0.01), while there was no difference in the humoral immune response. Conclusion: In our cohort, myocarditis occurred after both mRNA and vector anti-SARS-CoV-2 vaccination, frequently in individuals with predisposing factors. Upon follow-up, the myocardial injury had healed. Notably, an amplified cellular immune response was found in acute myocarditis cases occurring 4 days after COVID-19 vaccination.

Comparison of virus neutralization activity and results of 10 different anti-SARS-CoV-2 serological tests in COVID-19 recovered plasma donors.

Serological testing is a tool to predict protection against later infection. This potential heavily relies on antibody levels showing acceptable agreement with gold standard virus neutralization tests. The aim of our study was to investigate diagnostic value of the available serological tests in terms of predicting virus neutralizing activity of serum samples drawn 5-7 weeks after onset of symptoms from 101 donors with a history of COVID-19. Immune responses against Receptor Binding Domain (RBD), Spike1 and 2 proteins and Nucleocapsid antigens were measured by various ELISA tests. Neutralizing antibody activity in serum samples was assessed by a cell-based virus neutralization test. Spearman correlation coefficients between serological and neutralization results ranged from 0.41 to 0.91 indicating moderate to strong correlation between ELISA test results and virus neutralization. The sensitivity and specificity of ELISA tests in the prediction of neutralization were 35-100% and 35-90% respectively. No clear cut off levels can be established that would reliably indicate neutralization activity. For some tests, however, a value below which the sample is not expected to neutralize can be established. Our data suggests that several of the ELISA kits tested may be suitable for epidemiological surveys 1-2 months after the infection, estimating whether a person may have recently exposed to the virus. Sensitivities considerably superseding specificity at the cut-off values proposed by the manufacturers suggest greater potential in the identification of insufficient antibody responses than in confirming protection. Nevertheless, the former might be important in assessing response to vaccination and characterizing therapeutic plasma preparations.

Diagnosis and treatment of paediatric multisystem inflammatory syndrome / A gyermekkori koronavírus-fertozést követo sokszervi gyulladás diagnosztikája és kezelése.

Összefoglaló. A SARS-CoV-2-fertozés ritka gyermekkori szövodménye a sokszervi gyulladás, angol terminológiával paediatric inflammatory multisystem syndrome (PIMS). Két vagy több szerv érintettségével járó, súlyos tünetekkel induló betegségrol van szó, amelynek tünetei átfedést mutatnak a Kawasaki-betegséggel, a toxikus sokk szindrómával és a makrofágaktivációs szindrómával. A PIMS-betegek intenzív terápiás osztályon vagy intenzív terápiás háttérrel rendelkezo intézményben kezelendok, ahol biztosítottak a kardiológiai ellátás feltételei is. A szükséges immunterápia a klinikai prezentációtól függ. A jelen közleményben a szerzok a releváns nemzetközi irodalom áttekintését követoen ajánlást tesznek a PIMS diagnosztikai és terápiás algoritmusára. Orv Hetil. 2021; 162(17): 652-667. Summary. Pediatric inflammatory multisystem syndrome (PIMS) is a rare complication of SARS-CoV-2 infection in children. PIMS is a severe condition, involving two or more organ systems. The symptoms overlap with Kawasaki disease, toxic shock syndrome and macrophage activation syndrome. PIMS patients should be treated in an intensive care unit or in an institution with an intensive care background, where cardiological care is also provided. The required specific immunotherapy depends on the clinical presentation. In this paper, after reviewing the relevant international literature, the authors make a recommendation for the diagnostic and therapeutic algorithm for PIMS. Orv Hetil. 2021; 162(17): 652-667.

Interaction of photosensitizers with liposomes containing unsaturated lipid.

Small unilamellar liposomes were made of dipalmitoyl-phosphatidylcholine and dioleoyl-phosphatidylcholine, and photosensitized by a symmetrically or an asymmetrically substituted glycosilated tetraphenyl-porphyrin derivative. As differential scanning calorimetry and electron paramagnetic resonance spectroscopy (EPR) revealed these porphyrin derivatives were localized in different depth within the lipid bilayer. Both porphyrin derivatives were able to induce photoreaction and consequent structural changes in the membrane. 5-, 12-, or 16-doxyl stearic acid labeled lipid bilayers were applied and the efficiency of photoinduced reaction was followed by the decay of their EPR signal amplitude. Light dose-dependent destruction of nitroxide radical proved to be dependent on the position of spin label. In this process the porphyrin localized in closer connection with the double bond of unsaturated fatty acid was more effective. EPR signal decay was also dependent on the unsaturated fatty acid content of the liposome and the oxygen saturation of the solvent.

Use of JAR-Based Analysis for Improvement of Product Acceptance: A Case Study on Flavored Kefirs.

A common question of dairy product developments is the possible success of the new product. Several publications reported successful results using just-about-right (JAR) scales; although there is some debate about their advantages/disadvantages. This study highlights the limitations and opportunities of JAR scales and penalty analysis of fruit flavored kefirs. The first question is whether penalty analysis results help to improve the product and thus its overall liking (OAL)? The second question is what happens to those who rated the products "ideal" (JAR) before product development when evaluating the new products? Fruit flavored live-flora stirred-type kefir samples were formulated and evaluated by 92 consumers before and after the JAR-based product development. The OAL of two products significantly increased after product development. A new visualization tool is introduced, which shows what happens to those who rated the attribute as JAR but the attribute has been modified. A general product development scheme is also introduced for JAR-based kefir product development.

The relationship between hemorheological parameters and mortality in critically ill patients with and without sepsis.

PURPOSE: The prognostic scoring systems for mortality of intensive care patients estimate clinical outcome using several physiological and biochemical parameters. In altered hemodynamic conditions of critically ill patients, hemorheological variables may play a significant role in appropriate tissue perfusion. We investigated if hemorheological parameters are altered in critical status and if they could be markers of mortality. METHODS: 112 patients (67.8 ± 12 years, 58 males, 54 females) treated in intensive care unit with different non-surgical diseases were investigated. Routine laboratory parameters and prognostic scores were determined and hemorheological variables (hematocrit, plasma and whole blood viscosity, red blood cell aggregation and deformability) were measured on the 1st and the 2nd day after admission. RESULTS: ICU scores predicted 35.2-41.3% mortality rate, real mortality in intensive care unit was 37.5%, while 30-day mortality was 46.6%. Whole blood viscosity (WBV) and red blood cell (RBC) deformability were lower, red blood cell aggregation was higher in septic than in nonseptic patients (p < 0.05). In septic patients calcium was increased, osmolality was decreased, while in nonseptic patients WBV and RBC aggregation were higher in nonsurvivors compared to survivors (p < 0.05). Worsening of RBC deformability from day 1 to day 2 predicted higher mortality (p < 0.05). CONCLUSION: Calcium and osmolality level were associated with outcome in sepsis. Whole blood viscosity, red blood cell aggregation and change in red blood cell deformability could predict mortality in nonseptic patients and they may add prognostic information over the ICU scores. Further investigations are needed to evaluate the benefit of our findings in clinical practice.

The effects of training and detraining on memory, neurotrophins and oxidative stress markers in rat brain.

In the current investigation we tested how swimming training (T) (8 week, 5 times/week, 2 h/day), and detraining (DT) affects brain functions and oxidative stress markers in rat brain. The free radical concentration, measured by electron paramagnetic resonance, decreased in brain of T and DT rats compared to controls (C). The level of brain-derived neurotrophic factor (BDNF) increased as a result of training, but decreased below the control level after 6 weeks of detraining. In addition, the concentration of nerve growth factor (NGF) also declined with DT. The passive avoidance test was used to assess the memory of rats, and training-induced improvement was observed but the enhancement disappeared with detraining. When the content of mitochondrial electron transport complexes, as a potent free radical generator, was evaluated by the blue native gel method, no significant alterations were observed. The repair of nuclear and mitochondrial 8-oxodeoxyguanosine, as measured by the activity of OGG1, showed no significant difference. Therefore, the results suggest that regular exercise training improves memory, decreases the level of reactive oxygen species, and increase the production of BDNF and NGF. On the other hand, it appears that the beneficial effects of training are reversible in the brain, since detraining down-regulates the neurotrophin level, and memory. It is suggested that exercise training is more likely to beneficially effect the production of reactive oxygen species and the related oxidative damage.

Physical and psychological benefits of once-a-week Pilates exercises in young sedentary women: A 10-week longitudinal study.

Pilates exercises have several demonstrated physical and psychological benefits. To date, most research in this context was conducted with symptomatic or elderly people with few dependent measures. The current study examined the chronic or longitudinal effects of very low frequency, once a week, Pilates training on several physical and psychological measures, over a 10-week intervention, in young, healthy, and sedentary women. Further, the study gauged the acute effects of Pilates exercises on positive- and negative affect in 10 exercise sessions. Compared to a control group, the Pilates group exhibited significant improvements in skeletal muscle mass, flexibility, balance, core- and abdominal muscle strength, body awareness, and negative affect. This group also showed favorable changes in positive (22.5% increase) and negative affect (12.2% decrease) in nine out of ten exercise sessions. This work clearly demonstrates the acute and chronic benefits of Pilates training on both physical and psychological measures. It also reveals that even only once a week Pilates training is enough to trigger detectable benefits in young sedentary women. While this frequency is below the required levels of exercise for health, it may overcome the 'lack of time' excuse for not exercising and subsequently its tangible benefits may positively influence one's engagement in more physical activity.

Molecular dynamics of the cyclic lipodepsipeptides' action on model membranes: effects of syringopeptin22A, syringomycin E, and syringotoxin studied by EPR technique.

Interaction of pore-forming toxins, syringopeptin22A (SP22A), syringomycin E (SRE) and syringotoxin (ST), with model membranes were investigated. Liposomes were prepared from saturated phospholipids (DPPC or DMPC) or from binary mixtures of DPPC with varying amount of DOPC or cholesterol. The effects of the three toxins on the molecular order and dynamics of the lipids were studied using electron paramagnetic resonance (EPR) techniques. SP22A was the most-, SRE less-, and ST the least effective to increase the ordering and to decrease the rotational correlation time of the lipid molecules. The effects were more pronounced: (a) on small unilamellar vesicles (SUVs) than on multilamellar vesicles (MUVs); (b) on pure DPPC than on DPPC-cholesterol or DPPC-DOPC mixtures. Fluidity changes, determined from EPR spectra at different concentrations of the toxin, suggested the shell structure of the lipid molecules in pore formation. EPR spectra observed at different depth of the hydrocarbon chain of the lipid molecules implied an active role of the lipid molecules in the architecture of the pores created in the presence of the three toxins. Temperature dependence of the fluidity of the SUVs treated with toxins has shown an abrupt and irreversible change in the molecular dynamics of the lipid molecules at a temperature close to the pretransition, depending on the toxin species and the lipid composition. Coalescence and aggregation of the SUVs were proposed as the origin of this irreversible change.

Syringotoxin pore formation and inactivation in human red blood cell and model bilayer lipid membranes.

The effect of syringotoxin (ST), a member of the cyclic lipodepsipeptides family (CLPs) produced by Pseudomonas syringae pv. syringae on the membrane permeability of human red blood cells (RBCs) and model bilayer lipid membranes (BLMs) was studied and compared to that of two recently investigated CLPs, syringomycin E (SRE) and syringopeptin 22A (SP22A) [Biochim. Biophys. Acta 1466 (2000) 79 and Bioelectrochemistry 52 (2000) 161]. The permeability-increasing effect of ST on RBCs was the least among the three CLPs. A time-dependent ST pore inactivation was observed on RBCs at 20 and 37 degrees C but not at 8 degrees C. From the kinetic model worked out parameters as permeability coefficient of RBC membrane for 86Rb(+) and pores mean lifetime were calculated. A shorter pores mean lifetime was calculated at 37 degrees C then at 20 degrees C, which gave us an explanation for the unusual slower rate of tracer efflux measured at 37 degrees C then that at 20 degrees C. The results obtained on BLM showed that the pore inactivation was due to a decrease in the number of pores but not to a change of their dwell time or conductance.

Interaction of tetraphenyl-porphyrin derivatives with DPPC-liposomes: an EPR study.

The effect of the symmetry and polarity of the porphyrin molecules on their membrane localization and interaction with membrane lipids were investigated by electron paramagnetic resonance (EPR). For this purpose, two glycoconjugated tetraphenyl porphyrin derivatives were selected, respectively, symmetrically and asymmetrically substituted. Small unilamellar liposomes composed of dipalmitoylphosphatidylcholine (DPPC) and spin labeled stearic acids were prepared. The spin probe was located at the 5th or 7th or 12th or 16th position of the hydrocarbon chain in order to monitor various regions of the lipid bilayer. EPR spectra of porphyrin-free and porphyrin-bound liposomes were recorded at various temperatures below and above the phase transition temperature of DPPC. The effect on membrane fluidity proved to be stronger with the asymmetrical porphyrin derivative than with the symmetrical one. The rigidity increased when the spin label was near lipid head groups. The difference observed between control and porphyrin-treated samples when measured below the main lipid transition temperature disappeared at higher temperature. When the spin label was near the end of the hydrophobic tails, the symmetrical porphyrin derivative caused increase in fluidity, while the asymmetrical one slightly decreased it. To explain this phenomenon we propose that the asymmetrical derivative exerts a stronger ordering effect caused by its fluorophenyl group located at the level of the lipid heads, which is attenuated to the hydrophobic tails. The perturbing effect of the symmetric derivative could not lead to similar extent of ordering at the head groups and looses the hydrocarbon chains deeper in the membrane.

Effect of UVA radiation on membrane fluidity and radical decay in human fibroblasts as detected by spin labeled stearic acids.

The ultraviolet A (UVA) radiation component of sunlight (320-400 nm) has been shown to be a source of oxidative stress to cells via generation of reactive oxygen species. We report here some consequences of the UVA irradiation on cell membranes detected by electron paramagnetic resonance (EPR) spectroscopy. Paramagnetic nitroxide derivatives of stearic acid bearing the monitoring group at different depths in the hydrocarbon chain were incorporated into human fibroblasts membranes to analyze two main characteristics: kinetics of the nitroxide reduction and membrane fluidity. These two characteristics were compared for control and UVA-irradiated (0-250 kJ/m(2)) cells. The term relative redox capacity (RRC) was introduced to characterize and to compare free radical reduction measured by EPR with some well-known viability/clonogenicity tests. Our results showed that UVA-irradiation produces a more rigid membrane structure, especially at higher doses. Furthermore, we found that trends agree in survival measured by neutral red (NR), trypan blue (TB), and clonogenic efficiency compared with RRC values measured by EPR for low and medium exposure doses. Above 100 kJ/m(2), differences between these tests were observed. Antioxidant effect was modeled by alpha-tocopherol-acetate treatment of the cells before UVA irradiation. While NR, TB and clonogenicity tests showed protection at the highest UVA doses (>100 kJ/m(2)), results obtained with EPR measurements, both membrane fluidity and kinetics, or using MTT test did not exhibit this protective effect.

[Investigation of the mechanism of cell membrane-active cyclic lipodepsipeptides compounds]. / Sejtmembránra ható ciklusos lipodepszipeptid vegyületek hatásmódjának vizsgálata.

The cyclic lipodepsipeptides produced by Pseudomonas syringae pv. syringae possess fungicide properties. They inhibit much of the cell functions, presumably on the basis of their pore-forming activity. The syringomycin E, studied earlier by our research group, formed pores on human red blood cells (RBC), and caused also partially hemolysis. To reach selective toxicity, knowledge of the relationship between the structure and function of the CLPs is required. To fulfill this requirement, the pore forming properties of two other CLPs, the syringopeptin22A (SP22A) and the syringotoxin (ST), were investigated on RBCs. On the basis of our transport kinetic measurements on RBC, the SP22A and the ST formed pores built up by few monomers. ST pores inactivated at 37 degrees C, and at 20 degrees C, however, they remained stable at 8 degrees C, similarly to the SRE. Inactivation of the SRE and ST pores was faster at 37 degrees C than at 20 degrees C. SP22A pores did not inactivate even at 37 degrees C. On the basis of our results, we concluded that fluidity has a crucial role in the process and the dimension of the hydrophobic part and the number of the positive charges of the polar headgroup influence the stability of the pores. To study the interactions of CLPs with membranes on molecular level we applied EPR spectroscopy and spectral simulation methods for liposomes of different composition. CLPs decreased the fluidity at all depths of the membrane: the motional and rotational freedom of the lipid molecules decreased, while their ordering increased. These observations in conjunction with the concentration dependence of the CLP's action suggested that CLPs form pores involving the lipids, too. Investigation of the temperature dependence of the CLPs' action showed that to get their complete effect a given temperature range is required. It corroborated that the fluidity has a crucial role in the interactions between CLPs and membrane lipids. From the results, obtained with DPPC-DOPC or DPPC-cholesterol liposomes we concluded that the membrane-inhomogeneity decreases the effectiveness of the CLPs. Dynamic light scattering measurements were used to determine the size-distribution of the liposomes. According to our experiences CLPs provoke fusion and/or aggregation, which can have a role in the cell-killing properties of the CLPs.