Osteopontin expression and the effect of anti-VLA-4 mAb treatment in experimental autoimmune encephalomyelitis, a mouse model of multiple sclerosis.

INTRODUCTION: Osteopontin (OPN) is involved in the pathogenesis of multiple sclerosis and its animal model, experimental autoimmune encephalomyelitis (EAE). The aim of this study was to investigate the expression of OPN in spinal cords of mice in the successive phases of EAE, to compare it with the density of inflammatory cells, oligodendrocytes and with the expression of interleukin (IL)-17A and to assess the effect of anti-α4ß1 integrin (VLA-4) treatment. MATERIAL AND METHODS: Experimental autoimmune encephalomyelitis (EAE) mice were injected with anti-VLA-4 antibodies or, as treatment control, with immunoglobulin G (IgG). Spinal cords were sectioned and immunostained for OPN, CD45 (overall leukocytes), CD3 (T cells), Iba1 (activated macrophages/microglia), IL-17A, and CNP1 (oligodendrocytes). Microscopic images were analysed and the percentage of immunopositive areas encompassing the whole spinal cord cross-sectional area were assessed in images for each antigen. RESULTS: Osteopontin was expressed by inflammatory cells and by a minority of neurons and blood vessels. Most of the studied parameters followed the temporal pattern of clinical scores: increase in the peak phase and decrease in the chronic phase. Only OPN and IL-17A remained at a high level in the chronic phase, while CNP1 expression gradually decreased in the successive phases. Anti-VLA-4 treatment lowered the expression of the studied antigens in the peak and chronic phases with the exception of oligodendrocyte marker CNP1 which in both phases showed an increased expression. CONCLUSIONS: Involvement of OPN is particularly significant in advanced EAE. Anti-VLA-4 treatment not only inhibits migration of myelin-reactive T cells, but also downregulates OPN and inhibits loss of oligodendrocytes.

Adhesion Molecule Profile and the Effect of Anti-VLA-4 mAb Treatment in Experimental Autoimmune Encephalomyelitis, a Mouse Model of Multiple Sclerosis.

In the course of multiple sclerosis (MS) and its animal model, experimental autoimmune encephalomyelitis (EAE), the infiltration of lymphocytes and other inflammatory cells across the blood-brain barrier is associated with interactions between adhesion molecules expressed by infiltrating cells and vascular endothelium. Monoclonal antibodies (mAb) against the α4 subunit of α4-ß1 integrin (VLA-4) show beneficial effects in both MS and EAE. (1) Background: The aim of this study was to examine the expression of selected adhesion molecules: VLA-4, VCAM-1, LFA-1, ICAM-1 and PECAM-1 in the successive phases of EAE and the effect of anti-VLA-4 mAb treatment on that expression. (2) Methods: EAE was induced in C57BL/6 mice by immunization with MOG35-55 peptide. The animals were killed in three successive phases of the disease: onset (day 13), peak (day 18) and chronic (day 28). Frozen sections of the lumbar spinal cord were examined by quantitative immunofluorescence microscopy. The expression of the studied molecules was quantified as the percentage of the cross-sectioned spinal cord lesion area occupied by immunopositive structures. (3) Results: The expression of the studied molecules showed two temporal patterns: (1) an increase in the onset phase, a maximum in the peak phase and a decrease in the chronic phase, which corresponded to the temporal pattern of the clinical score, the number of lesions and the inflammation level (ICAM-1, LFA-1 and PECAM-1), and (2) an increase in the peak phase and no significant change or further increase in the chronic phase (VCAM-1, VLA-4). Among the molecules studied, ICAM-1 and LFA-1 exhibited the highest expression levels in the peak phase of EAE. Anti-VLA-4 mAb inhibited the expression of not only VLA-4 but also other adhesion molecules. (4) Conclusions: The interactions of adhesion molecules governing the migration of leukocytes across the blood-brain barrier change in the successive phases of EAE. The therapeutic mechanism of anti-VLA-4 mAb treatment seems to include a complex influence on a variety of adhesion molecules expressed by infiltrating cells and vascular endothelium.

Rheological properties of skeletal muscles in a Duchenne muscular dystrophy murine model before and after autologous cell therapy.

Duchenne muscular dystrophy (DMD) is still an incurable muscle degenerative disease; thus, numerous studies focused on novel therapeutic approaches. However, a simple assay of muscle function restoration remains needed. Herein, we used an oscillatory shear rheometer to evaluate changes in rheological properties of mouse muscles (tibialis anterior, TA) and their restoration upon autologous cell therapy by comparing the viscoelastic properties of normal, diseased and treated muscles. Amplitude sweep tests of muscle samples were performed under 20% compression over a range of shear strain between 0.01 and 2% and frequency of 1 rad/s. The samples were tested in plane-plane geometry and horizontal myofiber alignment. Typical linear viscoelastic region (LVER) patterns were found for each muscle type. For healthy muscles, a broad LVER between shear deformations (γ) of 0.013-0.62% was observed. The LVER of DMD mdx/SCID muscles was found at 0.14% to 0.46% shear deformation, and no shear dependence of storage (G') and loss (G") moduli at γ range changing from 0.034% to 0.26% was found for transplanted tissues. G'LVER and G"LVER moduli of healthy muscles were significantly higher than G'LVER and G"LVER of dystrophic tissues. Additionally, muscle resistance assessment by rheometer indicated that muscles transplanted with stem cells restored elastic properties to levels close to those of healthy muscles. Interestingly, histological staining and rheological data indicate that the loss factor is strongly related to structural changes of examined muscles.

Probing the recognition specificity of αVß1 integrin and syndecan-4 using force spectroscopy.

The knowledge on how cells interact with microenvironment is particularly important in understanding the interaction of cancer cells with surrounding stroma, which affects cell migration, adhesion, and metastasis. The main cell surface receptors responsible for the interaction with extracellular matrix (ECM) are integrins, however, they are not the only ones. Integrins are accompanied to other molecules such as syndecans. The role of the latter has not yet been fully established. In our study, we would like to answer the question of whether integrins and syndecans, possessing similar functions, share also similar unbinding properties. By using single molecule force spectroscopy (SMFS), we conducted measurements of the unbinding properties of αVß1 and syndecan-4 in the interaction with vitronectin (VN), which, as each ECM protein, possesses two binding sites specific to integrins and syndecans. The unbinding force and the kinetic off rate constant derived from SMFS describe the stability of single molecular complex. Obtained data show one barrier transition for each complex. The proposed model shows that the unbinding of αVß1 from VN proceeds before the unbinding of SDC-4. However, despite different unbinding kinetics, the access to both receptors is needed for cell growth and proliferation.

Vasodilatory efficacy and impact of papaverine on endothelium in radial artery predilatation for cabg surgery: in search for optimal concentration

Abstract Objective: The aim of this study was to compare the efficacy of two different papaverine concentrations (0.5 mg/ml and 2 mg/ml) for vasospasm prevention and their impact on endothelium integrity. Methods: We have studied distal segments of radial arteries obtained by no-touch technique from coronary artery bypass graft (CABG) patients (n=10). The vasodilatory effect of papaverine (concentrations of 0.5 mg/ml and 2 mg/ml) was assessed in vitro, in isometric tension studies using ex vivo myography (organ bath technique) and arterial rings precontracted with potassium chloride (KCl) and phenylephrine. The impact of papaverine on endothelial integrity was studied by measurement of the percentage of vessel's circumference revealing CD34 endothelial marker. Results: 2 mg/ml papaverine concentration showed stronger vasodilatatory effect than 0.5 mg/ml, but it caused significantly higher endothelial damage. Response to KCl was 7.35±3.33 mN for vessels protected with papaverine 0.5 mg/ml and 2.66±1.96 mN when papaverine in concentration of 2 mg/ml was used. The histological examination revealed a significant difference in the presence of undamaged endothelium between vessels incubated in papaverine 0.5 mg/ml (72.86±9.3%) and 2 mg/ml (50.23±13.42%), P=0.002. Conclusion: Papaverine 2 mg/ml caused the higher endothelial damage. Concentration of 0.5 mg/ml caused better preservation of the endothelial lining.

Immunohistochemical analysis of spinal cord components in mouse model of experimental autoimmune encephalomyelitis.

INTRODUCTION: Experimental autoimmune encephalomyelitis (EAE) is a widely used animal model for studying immunopathology of multiple sclerosis (MS) because it repeats the hallmarks of the human disease, such as focal inflammation and demyelination of the central nervous system, subsequently leading to axonal and neuronal loss. The interrelationships, timing and sequence of different pathological processes that lead to histologically observed lesions in SM are still incompletely understood. MATERIAL AND METHODS: EAE was induced in female C57Bl/6 mice by active immunization with MOG35-55 antigen. Development of the neurological symptoms in the animals was monitored and on that basis spinal cords were collected in three successive phases of the disease (onset, peak, chronic). Total leukocytes, T cells, macrophages/microglia, oligodendrocytes, damaged axons and surviving neuronal cell bodies were visualized using appropriate immunohistochemical markers and their density was quantitatively assessed using image analysis software. RESULTS: The density of all studied cells except neurons was significantly higher in EAE mice than in the control mice. The density of total leukocytes, T cells, and damaged axons increased from the onset to the peak phase and decreased in the chronic phase to reach values lower than those in the peak phase. The density of macrophages/microglia increased in the peak phase and remained at the elevated level in the chronic phase. Oligodendrocytes showed the highest density in the onset phase and gradually decreased afterwards. The density of neuronal cell bodies decreased only in the chronic phase of the disease. CONCLUSIONS: In mouse model of EAE, inflammatory cells predominate in the early phases of the disease. This study shows for the first time that inflammation precedes oligodendrocyte death and neuronal loss and that macrophages/ microglia are the only cells persisting in large numbers in the chronic phase of the disease, probably because of the switch from proinflammatory to anti-inflammatory phenotype.

AFM-based nanomechanical characterization of bronchoscopic samples in asthma patients.

Asthma is not a single disease, but recently, it is considered as a syndrome characterized through various clinical presentations and different etiopathologies. Large degree of the disease heterogeneity manifests in distinct characteristics that translate into variability of properties at single cell and molecular levels. Here, we conducted measurements of mechanical properties of bronchial tissue samples collected from patients suffering from asthma. The results obtained from different applied protocols for sample preparation may indicate that deep freezing and storage in liquid nitrogen, followed by consecutive unfreezing of tissue samples, preserve tissue mechanical properties as indicated by a parameter referred here as a tissue relative stiffness index. Tissue relative stiffness index quantifies both the degree of heterogeneity and deformability of tissue samples regarding healthy one. These studies demonstrate that the freezing protocol, optimized towards asthma tissue, can facilitate atomic force microscopy use what, together with recent findings on standardization of elasticity measurements, enables the measurements of large group of samples with minimized influence of errors stemming from the applied methodology of tissue stiffness determination.

Vasodilatory Efficacy and Impact of Papaverine on Endothelium in Radial Artery Predilatation for CABG Surgery: in Search for Optimal Concentration.

OBJECTIVE: The aim of this study was to compare the efficacy of two different papaverine concentrations (0.5 mg/ml and 2 mg/ml) for vasospasm prevention and their impact on endothelium integrity. METHODS: We have studied distal segments of radial arteries obtained by no-touch technique from coronary artery bypass graft (CABG) patients (n=10). The vasodilatory effect of papaverine (concentrations of 0.5 mg/ml and 2 mg/ml) was assessed in vitro, in isometric tension studies using ex vivo myography (organ bath technique) and arterial rings precontracted with potassium chloride (KCl) and phenylephrine. The impact of papaverine on endothelial integrity was studied by measurement of the percentage of vessel's circumference revealing CD34 endothelial marker. RESULTS: 2 mg/ml papaverine concentration showed stronger vasodilatatory effect than 0.5 mg/ml, but it caused significantly higher endothelial damage. Response to KCl was 7.35±3.33 mN for vessels protected with papaverine 0.5 mg/ml and 2.66±1.96 mN when papaverine in concentration of 2 mg/ml was used. The histological examination revealed a significant difference in the presence of undamaged endothelium between vessels incubated in papaverine 0.5 mg/ml (72.86±9.3%) and 2 mg/ml (50.23±13.42%), P=0.002. CONCLUSION: Papaverine 2 mg/ml caused the higher endothelial damage. Concentration of 0.5 mg/ml caused better preservation of the endothelial lining.

Probing fibronectin-antibody interactions using AFM force spectroscopy and lateral force microscopy.

The first experiment showing the effects of specific interaction forces using lateral force microscopy (LFM) was demonstrated for lectin-carbohydrate interactions some years ago. Such measurements are possible under the assumption that specific forces strongly dominate over the non-specific ones. However, obtaining quantitative results requires the complex and tedious calibration of a torsional force. Here, a new and relatively simple method for the calibration of the torsional force is presented. The proposed calibration method is validated through the measurement of the interaction forces between human fibronectin and its monoclonal antibody. The results obtained using LFM and AFM-based classical force spectroscopies showed similar unbinding forces recorded at similar loading rates. Our studies verify that the proposed lateral force calibration method can be applied to study single molecule interactions.

LN-5 antibody against human macrophages cross-reacts with routinely processed human sebaceous glands.

LN-5 monoclonal antibody against human macrophages was found to selectively stain human sebaceous glands in formalin-fixed, paraffin-embedded skin samples. Undifferentiated sebocyte progenitors were negative, and only sebocytes from the onset of their differentiation revealed positive cytoplasmic immunofluorescence. Since there are very few selective and easy-to-use markers of sebaceous glands, LN-5 antibody can offer a simple and relatively specific way to detect human sebocytes from the onset of their differentiation in routinely processed material, both freshly prepared and archival.

Respiratory epithelial adenomatoid hamartoma of the anterior nasal septum a rare localisation of an unusual tumour in a child: a case report.

INTRODUCTION: Hamartomas are non-neoplastic lesions constituted by a mixture of tissues indigenous to the region. Respiratory epithelial adenomatoid hamartomas are characterised by glandular proliferation lined by ciliated airway epithelium. Their localisation in the nasal cavity is rare and most frequent cases described so far were associated with the posterior nasal septum. CASE PRESENTATION: A 9-year-old Caucasian boy presented with long-standing nasal obstruction. A large right nasal mass was evident on physical and CT examinations. It was surgically removed from the anterior nasal septum under general anaesthesia. Histologically, the diagnosis of REAH was established. The tumour lined by stratified squamous and ciliated respiratory epithelium was characterised by prominent glandular proliferation. By immunohistochemistry, the tumour was positive for cytokeratins, smooth muscle actin, vimentin, laminin, collagen type IV, CD8, and CD68. No S-100 immunoreactivity was observed. The patient has been asymptomatic for 12 months with completely healed lining of the nose. CONCLUSION: Respiratory epithelial adenomatoid hamartoma, although rare, must be taken into consideration in differential diagnosis of nasal exophytic lesions.

Germ cell cluster formation and ovariole structure in viviparous and oviparous generations of the aphid Stomaphis quercus.

The developing ovaries of S. quercus contain a limited number of oogonial cells which undergo a series of incomplete mitotic divisions resulting in the formation of clusters of cystocytes. Ovaries of viviparous generations contain 6 to 9 clusters, containing 32 cystocytes each, whereas ovaries of oviparous generations contain 5 clusters containing 45-60 cystocytes. During further development, clusters become surrounded by a single layer of follicular cells, and within each cluster the cystocytes differentiate into oocytes and trophocytes (nurse cells). Concurrently, cysts transform into ovarioles. The anterior part of the ovariole containing the trophocytes becomes the tropharium, whereas its posterior part containing oocytes transforms into the vitellarium. The vitellaria of viviparous females are composed of one or two oocytes, which develop until previtellogenesis. The nuclei of previtellogenic oocytes enter cycles of mitotic divisions which lead to the formation of the embryo. Ovarioles of oviparous females contain a single oocyte which develops through three stages: previtellogenesis, vitellogenesis and choriogenesis. The ovaries are accompanied by large cells termed bacteriocytes which harbor endosymbiotic microorganisms.

Erythrocyte stiffness in diabetes mellitus studied with atomic force microscope.

The mechanical properties of erythrocyte membrane have been studied using an atomic force microscope. Measurements were carried out on blood samples taken from 7 diabetes mellitus patients and 8 healthy individuals. For each blood sample a distribution of a Young's modulus was constructed. It has been found that both the mean value and the width of the distribution in diabetic patients exceed the corresponding results for healthy persons by a factor greater than 3. The high sensitivity of the atomic force microscopy and the ability to measure the full distribution of the erythrocyte membrane Young's modulus makes it a unique, powerful and promising tool in studies of the membrane stiffness of red blood cells.

Erythrocyte stiffness probed using atomic force microscope.

The stiffness of erythrocytes in patients (N=45) suffering from certain disorders, such as coronary disease, hypertension, and diabetes mellitus has been assessed using the Atomic Force Microscopy (AFM) and compared with that in a group of healthy individuals (N=13). For each blood sample, around 20 erythrocytes were selected at random and the stiffness of each one was probed in 20-30 arbitrarily chosen points. From these results, distributions of the cell Young's modulus (YM) were determined. Average values and widths of YM distributions significantly increased in samples taken from diabetes mellitus patients and cigarette smokers, as compared to those taken from healthy donors. At the same time, the average values of YM were found to increase as a function of the patient's age. We demonstrated that the atomic force microscope is a very sensitive tool for determination of cell stiffness with every prospect of a routine application as a diagnostic tool in quantitative analysis of the physiological and pathological states of red blood cells.

Structure of the ovary in Steingelia (Sternorrhyncha: Coccinea), and its phylogenetic implications.

The paired ovaries of Steingelia gorodetskia are composed of about 100 telotrophic ovarioles devoid of terminal filaments (scale insect autapomorphy). In structure they resemble those of other scale insects, but differ in the following details: (a) all ovarioles develop synchronously, (b) they are suspended to the lateral oviducts by means of long stalks, (c) the tropharium is tubular (unique in scale insects) and (d) consists of 15-35, trophocytes, 2-4 previtellogenic oocytes that further develop, and numerous somatic prefollicular cells, (e) the vitellarium houses 2-4 linearly arranged vitellarial oocytes (versus one in most scale insects). Most of these features must be considered as plesiomorphic corresponding with the conditions in the most primitive Heteroptera. Bacterial endosymbionts have been found in some somatic cells, trophocytes, oocytes and in the nutritive cord. Present results support the opinion, based on external morphology, that the Steingeliidae are closely related to the Ortheziidae, Xylococcidae and Matsucoccidae.

Ultrastructural studies on the formation of egg envelopes in fulgoromorphans (Insecta, Hemiptera, Fulgoromorpha: Dictyopharidae).

Follicular cells in ecuadorian dictyopharids diversify into two subpopulations: the main body cells (MFs) and cells surrounding the anterior pole of the oocyte (AFs). The synthetic activity of both categories of follicular cells is manifested by the presence of numerous cisternae of rough endoplasmic reticulum, Golgi complexes and vacuoles containing electron-dense material in their cytoplasm. The MFs synthesize precursors of the main body chorion, whereas the AFs are responsible for the formation of micropylar apparatus and respiratory tubules. The main body chorion is composed of thin endochorion and exochorion which forms pillar-like projections.