Temperature dependent FCS studies using a long working distance objective: Viscosities of supercooled liquids and particle size.

In this work, we describe new experimental setups for Fluorescence Correlation Spectroscopy (FCS) where a long working distance objective is used. Using these setups, FCS measurements in a broad temperature range for a small sample volume of about 50 µl can be performed. The use of specially designed cells and a dry long working distance objective was essential for avoiding temperature gradients in the sample. The performance of the new setups and a traditional FCS setup with immersion objectives is compared. The FCS data in combination with the Stokes-Einstein (SE) relation were used to obtain the values of the nanoviscosity of a fluid. We show for selected molecular van der Waals supercooled liquids that despite the fact that in these systems, a characteristic length scale can be defined, the nanoviscosity obtained from FCS is in a very good agreement with the macroscopic (rheometric) viscosity of the sample in a broad temperature range. This result corroborates the applicability of the SE relation to supercooled liquids at temperatures above 1.2 Tg. We also show that the temperature dependent size of thermoresponsive microgel particles can be determined by FCS using the designed cells and a long working distance objective in a broader size range without a need to use the correction procedure since the size correction is proportional to the square of the ratio of the hydrodynamic radius to the confocal volume size.

[Acute renal failure associated with Stevens-Johnson syndrome - case study].

Stevens-Johnson syndrome is an acute, life-threatening, necrotic skin and mucosal reaction, most often caused by drugs. This case presents 81-year old Patient with Stevens-Johnson syndrome caused by amoxicillin, complicated by acute renal failure.

Thermographic assessment of skin prick tests in comparison with the routine evaluation methods.

INTRODUCTION: The skin prick test is still the first and basic procedure in the diagnosis of allergic diseases. The possibility of using a sensitive thermographic method supported by the mathematical model for the assessment of skin test results will be highlighted in the studies. AIM: To compare the proposed approach with routine planimetric and thermographic methods. MATERIAL AND METHODS: A mathematical model of allergic reaction was developed. Simplifying assumptions of the IgE-mediated skin reaction is the essence of the model. Investigations were performed in a group of 40 patients. RESULTS: Using the spatio-temporal evolution of temperature distributions, the ratios of the histamine released from mast cells to the control histamine were determined. The obtained values very well correlate with the standard evaluation of skin prick tests (correlation coefficient = 0.98). CONCLUSIONS: The proposed method of skin test evaluation presents several advantages. The continuous acquisition of data provides the monitoring of time course of the allergic response. The transport of mediator and its concentration were distinctly discriminated, which may be diagnostically useful, especially for abnormal cases. The high sensitivity of the method enables studying patients regardless of age and skin sensitivity.

Nicotinamide adenine dinucleotide phosphate (NADPH) oxidase p22phox subunit polymorphisms, systemic oxidative stress, endothelial dysfunction, and atherosclerosis in type 2 diabetes mellitus.

INTRODUCTION: Diabetes mellitus is an important and rapidly increasing problem in public health. It associates with endothelial dysfunction and increased endothelial permeability, which may lead to severe cardiovascular events. OBJECTIVES: We aimed to evaluate the relationship between polymorphisms in the cytochrome b-245 alpha chain (CYBA) gene encoding p22phox, a key subunit of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase, with endothelial function, atherosclerosis and systemic oxidative stress in type 2 diabetes mellitus (T2DM). PATIENTS AND METHODS: Intima-media thickness (IMT), flow- and nitroglycerin-mediated dilatation (FMD and NMD) were measured in 182 patients with T2DM. Assessment of plasma levels of von Willebrand factor (vWF) and malondialdehyde (MDA) levels as well as genotyping of coding sequence C242T (rs4673) and promoter region A-930G (rs9932581) polymorphisms of CYBA were performed using standardized protocols. RESULTS: We observed a significant association of the impaired endothelial function, as measured by FMD, with the C allele of C242T, but not with the A-930G polymorphism. Functional relationship of the C242T polymorphism with endothelial dysfunction remained significant following a multivariable adjustment for major risk factors for atherosclerosis. Mean IMT, NMD, concentrations of MDA or vWF were not related to the specific genotypes of the investigated polymorphisms. CONCLUSIONS: C242T, but not A-930G, polymorphism of CYBA significantly affects endothelial function in T2DM. Thus, it might be a useful marker of endothelial dysfunction in T2DM patients.

Hypertension and increased endothelial mechanical stretch promote monocyte differentiation and activation: roles of STAT3, interleukin 6 and hydrogen peroxide.

Aims: Monocytes play an important role in hypertension. Circulating monocytes in humans exist as classical, intermediate, and non-classical forms. Monocyte differentiation can be influenced by the endothelium, which in turn is activated in hypertension by mechanical stretch. We sought to examine the role of increased endothelial stretch and hypertension on monocyte phenotype and function. Methods and results: Human monocytes were cultured with confluent human aortic endothelial cells undergoing either 5% or 10% cyclical stretch. We also characterized circulating monocytes in normotensive and hypertensive humans. In addition, we quantified accumulation of activated monocytes and monocyte-derived cells in aortas and kidneys of mice with Angiotensin II-induced hypertension. Increased endothelial stretch enhanced monocyte conversion to CD14++CD16+ intermediate monocytes and monocytes bearing the CD209 marker and markedly stimulated monocyte mRNA expression of interleukin (IL)-6, IL-1ß, IL-23, chemokine (C-C motif) ligand 4, and tumour necrosis factor α. STAT3 in monocytes was activated by increased endothelial stretch. Inhibition of STAT3, neutralization of IL-6 and scavenging of hydrogen peroxide prevented formation of intermediate monocytes in response to increased endothelial stretch. We also found evidence that nitric oxide (NO) inhibits formation of intermediate monocytes and STAT3 activation. In vivo studies demonstrated that humans with hypertension have increased intermediate and non-classical monocytes and that intermediate monocytes demonstrate evidence of STAT3 activation. Mice with experimental hypertension exhibit increased aortic and renal infiltration of monocytes, dendritic cells, and macrophages with activated STAT3. Conclusions: These findings provide insight into how monocytes are activated by the vascular endothelium during hypertension. This is likely in part due to a loss of NO signalling and increased release of IL-6 and hydrogen peroxide by the dysfunctional endothelium and a parallel increase in STAT activation in adjacent monocytes. Interventions to enhance bioavailable NO, reduce IL-6 or hydrogen peroxide production or to inhibit STAT3 may have anti-inflammatory roles in hypertension and related conditions.

CD14+CD16++ "nonclassical" monocytes are associated with endothelial dysfunction in patients with coronary artery disease.

Endothelial dysfunction and inflammation are key mechanisms of vascular disease. We hypothesised that heterogeneity of monocyte subpopulations may be related to the development of vascular dysfunction in coronary artery disease (CAD). Therefore, we examined the relationships between monocyte subsets (CD14++CD16- "classical - Mon1", CD14++CD16+ "intermediate - Mon2" and CD14+CD16++ "nonclassical - Mon3"), endothelial function and risk factor profiles in 130 patients with CAD undergoing coronary artery bypass grafting. This allowed for direct nitric oxide (NO) bioavailability assessment using isometric tension studies ex vivo (acetylcholine; ACh- and sodium-nitropruside; SNP-dependent) in segments of internal mammary arteries. The expression of CD14 and CD16 antigens and activation markers were determined in peripheral blood mononuclear cells using flow cytometry. Patients with high CD14+CD16++ "nonclassical" and low CD14++CD16- "classical" monocytes presented impaired endothelial function. High frequency of CD14+CD16++ "nonclassical" monocytes was associated with increased vascular superoxide production. Furthermore, endothelial dysfunction was associated with higher expression of activation marker CD11c selectively on CD14+CD16++ monocytes. Nonclassical and classical monocyte frequencies remained independent predictors of endothelial dysfunction when major risk factors for atherosclerosis were taken into account (ß=0.18 p=0.04 and ß=-0.19 p=0.03, respectively). In summary, our data indicate that CD14+CD16++ "nonclassical" monocytes are associated with more advanced vascular dysfunction measured as NO- bioavailability and vascular reactive oxygen species production.

Th17 responses are not altered by natural exposure to seasonal allergens in pollen-sensitive patients.

BACKGROUND: Allergic rhinitis affects 10-30 % of the global population and this number is likely to increase in the forthcoming years. Moreover, it commonly co-exists with allergic asthma as a chronic allergic respiratory syndrome. While the involvement of Th2 cells in allergy is well understood, alterations of pro-inflammatory Th17 responses remain poorly characterized. The aim of our study was to determine whether natural seasonal allergen exposure causes changes in T cell subset characteristics in patients with allergic rhinitis and asthma. METHODS: Sixteen patients with allergic rhinitis/atopic asthma (9M, 7F; age 31.8 ± 12.1) and 16 healthy controls were recruited into the study (9M, 7F; age 31.2 ± 5.3). Blood samples were collected from the patients 1-3 months before pollen season (visit 1), within 7 days of the appearance of pollen/initiation of allergic symptoms (visit 2) and 2 weeks after visit 2 following the introduction of symptomatic treatment with antihistamines (visit 3). Flow cytometry was used to assess major T cell subsets (naïve, central memory, effector memory and CD45RA+ effector) and key T cell cytokine production (IFNγ, IL-17A, TNF and IL-4) using intracellular staining. Data were analyzed using repeated measures ANOVA and paired t test. RESULTS: As expected, an increase in the percentage of IL-4+ CD4+ cells was observed during natural pollen exposure in patients with allergic respiratory syndrome. No significant changes were observed in the production of other cytokines, including Th17 cells, which tended to be lower than in the control population but unchanged during pollen exposure. Introduction of antihistamine treatment led to only moderate changes in cytokine production from CD4 and CD8 T cells. Selective changes in CD8+ T cells were observed during natural pollen exposure including a decrease in transient cells (with features of CD45RA+ and CD45RO+ cells) and a decrease in the percentage of central memory cells in the peripheral circulation. Within the CD4 cell group the total percentage of CD45RA positive CD4 cells was increased during pollen exposure. CONCLUSIONS: Th1 and Th17 responses are not altered during pollen season but allergen exposure affects T cell activation and memory cell status in patients with allergic respiratory syndrome.

Novel therapeutic approaches in limiting oxidative stress and inflammation.

The interaction between reactive oxygen species (ROS) and inflammation plays an important role in the pathogenesis of endothelial dysfunction and cardiovascular disease, cancer and other diseases. Thus, antioxidant strategies may be important in immune regulation and in limiting inflammation. Surprisingly, large clinical trials have shown that ROS scavenging by antioxidant vitamins is ineffective or even harmful in spite of the fact that reactive oxygen species themselves are pro-inflammatory, regulate immune system and enhance atherosclerosis. Therefore, there is a need of novel, more specific antioxidant and anti-inflammatory approaches aimed on prevention of ROS formation, by targeting specific molecular pathways involved in ROS generation and their activation of pro-inflammatory cascades. Potential targets include the NADPH oxidases (Nox enzymes), xanthine oxidase, endothelial nitric oxide synthase and mitochondrial oxidases. Nox enzymes play central role, as they can stimulate other enzymatic sources of ROS. The interplay between inflammation and oxidative stress is discussed in the context of adipose tissue, perivascular inflammation and role of the central nervous system in immune regulation. All of the above participate in "brain-vessel axis" critical in the pathogenesis of numerous pathologies. Role of cytokines such as TNF-alpha, IL-17 or IL-6 and their links to superoxide and hydrogen peroxide production are discussed. Statins, angiotensin converting enzyme inhibitors and angiotensin II receptor antagonists, block upstream signaling of Nox activation, including MAP kinase signaling or G protein activation, which contribute to their clinical effectiveness. Here, we discuss novel possibilities that drugs directly inhibiting Nox activation could successfully inhibit oxidative stress and inflammation related to cardiovascular disease. Moreover, we describe potential gene therapy approaches in limiting oxidative stress in the vasculature. These approaches can become also useful in cancer immunomodulation.